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==== Front BMC GenomicsBMC Genomics1471-2164BioMed Central London 1471-2164-5-621534743110.1186/1471-2164-5-62Research ArticleEnhancer trapping in zebrafish using the Sleeping Beauty transposon Balciunas Darius [email protected] Ann E [email protected] Sridhar [email protected] Spencer B [email protected] Zachary [email protected] Stephen C [email protected] Arnold and Mabel Beckman Center for Transposon Research, Department of Genetics, Cell Biology and Development, University of Minnesota, 6-160 Jackson Hall, 321 Church St SE, Minneapolis, MN-55455, USA2 Molecular, Cellular, Developmental Biology and Genetics Graduate Program, University of Minnesota, USA2004 3 9 2004 5 62 62 5 8 2004 3 9 2004 Copyright © 2004 Balciunas et al; licensee BioMed Central Ltd.2004Balciunas et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Among functional elements of a metazoan gene, enhancers are particularly difficult to find and annotate. Pioneering experiments in Drosophila have demonstrated the value of enhancer "trapping" using an invertebrate to address this functional genomics problem. Results We modulated a Sleeping Beauty transposon-based transgenesis cassette to establish an enhancer trapping technique for use in a vertebrate model system, zebrafish Danio rerio. We established 9 lines of zebrafish with distinct tissue- or organ-specific GFP expression patterns from 90 founders that produced GFP-expressing progeny. We have molecularly characterized these lines and show that in each line, a specific GFP expression pattern is due to a single transposition event. Many of the insertions are into introns of zebrafish genes predicted in the current genome assembly. We have identified both previously characterized as well as novel expression patterns from this screen. For example, the ET7 line harbors a transposon insertion near the mkp3 locus and expresses GFP in the midbrain-hindbrain boundary, forebrain and the ventricle, matching a subset of the known FGF8-dependent mkp3 expression domain. The ET2 line, in contrast, expresses GFP specifically in caudal primary motoneurons due to an insertion into the poly(ADP-ribose) glycohydrolase (PARG) locus. This surprising expression pattern was confirmed using in situ hybridization techniques for the endogenous PARG mRNA, indicating the enhancer trap has replicated this unexpected and highly localized PARG expression with good fidelity. Finally, we show that it is possible to excise a Sleeping Beauty transposon from a genomic location in the zebrafish germline. Conclusions This genomics tool offers the opportunity for large-scale biological approaches combining both expression and genomic-level sequence analysis using as a template an entire vertebrate genome. ==== Body Background Human, mouse and rat genomes likely have less than 40 000 genes each [1-4]. This is only two to three times as many genes as in Caenorhabditis elegans and Drosophila melanogaster, and only six times as many as Saccharomyces cerevisiae [5-7]. The increased complexity of vertebrates therefore can not be simply accounted for by a larger gene number. A part of the increased complexity is thought to be accomplished by alternative splicing, RNA editing and the use of protein modifications to generate a variety of protein products from a single gene, but everything starts with increased complexity at the level of transcriptional regulation. While promoters are relatively simple and short in yeast, their complexity increases in multicellular organisms, making regulatory sequences ever harder to identify. In humans, enhancer elements can be located over a megabase away from the transcriptional start site [8]. Furthermore, current gene prediction programs used to annotate genomes often fail to correctly identify the 5' start site of a transcription unit, making in silico analysis of the regulatory sequences even more complex. To further complicate the matter, enhancer sequences diverge in evolution, co-evolving with their respective transcription factors, and often do not work across large evolutionary distances – worm to fly, for example [9]. This makes information from non-vertebrate model systems sometimes inapplicable to vertebrate sequences. Enhancer detection ("trapping") using insertion site context vectors was popularized as a genomics tool in Drosophila. The first fly enhancer trap vectors were based on the P element transposon and often used the transposase's own promoter fused to the beta-galactosidase reporter gene for enhancer detection [10-12]. Several of the enhancer trap lines were shown to express the LacZ reporter in cells corresponding to the expression patterns of nearby genes, validating the approach [12,13]. In other work, promoters such as engrailed, fushi tarazu and Hsp70 were successfully developed for enhancer trapping in the fruitfly [14-16]. Further modifications to the system included the implementation of a bipartite system with a Gal4 transactivator [17], green fluorescent protein (GFP) [18], and even a GFP-LacZ fusion protein [19] as reporters. In addition to the P element, other transposons such as hobo and piggyBac with insertion site preferences distinct from those of the P element have been used in Drosophila [20,21]. The availability of a variety of transposons, promoters and reporters for enhancer trapping in the fruitfly enabled researchers to obtain enhancer trap insertions into a considerable fraction of Drosophila genes (reviewed by [22]) and allows an investigator to choose vectors most suitable for the problem at hand. The ability to excise from a genomic location has been instrumental to the utility of P element based vectors. For mutation-causing insertions, reversion of the mutant phenotype by P element excision proves that a given insertion causes the mutation. Since the mutagenicity of Drosophila enhancer trap transposons is not significantly higher than the average 15% rate obtained with regular P elements, most insertions do not result in a mutation [22]. In these instances, the P element's ability to induce genomic deletions by "imprecise excision" can be used to obtain a mutation in the neighboring gene(s) [23]. The success of enhancer trapping in Drosophila prompted application of this approach in the mouse [24-27]. As was the case in Drosophila, the lacZ reporter was shown to be expressed in part of the target gene's expression domain [28]. Despite the considerable success of these early experiments, enhancer detection as an experimental approach in mouse was not explored further, giving way to different versions of gene traps (for a review, see [29]). We believe the success of enhancer trapping in Drosophila can be largely attributed to the advantages of this experimental system over mouse. In Drosophila, large numbers of transgenic organisms can be readily generated and screened for gene expression patterns. It is far less practical in the mouse. This is partly due to the availability of efficient and precise transgene delivery tools in the fruitfly: the native P element, hobo and piggyBac transposons. In contrast, early mouse experiments were carried out by non-facilitated DNA transgenesis. This approach is less efficient and prone to induce deletions and other genome rearrangements in the recipient locus, as noted in the first published mouse enhancer trap locus [25,30]. The compact nature of the Drosophila genome also contributed to the success of enhancer trapping, making the path from an enhancer trap insertion to the identification of the affected gene straightforward, especially once the Drosophila genome was sequenced. The zebrafish Danio rerio is a vertebrate model system that provides many of the advantages found in invertebrates. A few hundred transparent, externally developing embryos can be obtained from a single pair of fish per week. The zebrafish genome is about two-fold smaller than the mouse genome, and its sequencing and annotation are nearing completion. Finally, transposon tools for efficient and precise transgene delivery into the zebrafish genome are available. We focused our research on the Sleeping Beauty (SB) transposon system [31,32]. While not as efficient as the highest titer retrovirus used in zebrafish [33,34], the Sleeping Beauty transposon system offers advantages in expression as well as ease of construction and testing of diverse vectors that can be done using basic molecular biology tools. Furthermore, the SB system offers the possibility of transposase-induced excision out of the genome to induce local deletions or to revert possible mutant phenotypes. In this report, we investigated the potential of the SB transposon system for enhancer detection in zebrafish. Our results indicate that zebrafish enhancer trap lines with diverse GFP expression patterns can be readily generated using the SB system. Most of the obtained lines harbor a single transposon insertion event, facilitating the rapid identification of transposon insertion sites responsible for specific GFP expression patterns. We show that two enhancer trap lines exhibit GFP expression patterns matching the expression patterns of the target genes, and that both expected and novel gene expression patterns can be identified using this genomics tool. We conclude that enhancer trapping using the Sleeping Beauty transposon system is a viable experimental approach using as template a vertebrate genome. Results The Sleeping Beauty transposon can detect enhancers in cis We have previously established multiple zebrafish lines using SB transposons with ubiquitous and tissue-specific promoters driving reporter expression [32]. Surprisingly, we did not observe any dependency of the expression pattern on the genomic context of the transposon insertion. Multiple studies describing insertion-site dependent transgene expression in vertebrates have suggested that many of those events are due to the transgene falling under control of nearby enhancers [35-40]. For enhancer detection approaches it is imperative that the reporter gene be sensitive to neighboring transcriptional regulatory elements. At least three explanations can be put forward to explain the absence of expression patterns in our previous work in zebrafish. First, the Sleeping Beauty transposons are flanked by relatively large inverted repeats. These repeats might function as silencer elements and not allow for transcriptional regulation across them. Second, the promoter we used (Xenopus laevis EF1α, [41]) may not be subject to transcriptional regulation by tissue-specific enhancers. Third, the expression level from the selected promoter may be too high to be effectively modulated, as enhancer traps usually contain attenuated promoters. To test these hypotheses, we decided to produce an artificial enhancer trapping event by cloning a tissue-specific promoter / enhancer just outside the inverted repeats and test for an increase in tissue-specific expression in injected embryos (Figure 1). We started with the transposon used in our previous work, pT2/S1EF1α-GM2, which contains a shortened version of the Xenopus laevis EF1α promoter driving the GM2 version of GFP in a pT2 transposon vector [32]. We took advantage of the observation that relatively few pT2/S1EF1α-GM2 injected embryos express GFP in the eye. We added a lens-specific Xenopus laevis γ1-Crystallin promoter [42] to the pT2/S1EF1α-GM2 vector, as we had previously shown that this promoter specifically expresses in the lens of injected (F0) and transgenic (F1) zebrafish [32]. Embryos injected with pT2/S1EF1α-GM2 or γ Cry1/pT2/S1EF1α-GM2 were scored for any GFP fluorescence and for eye-specific GFP fluorescence at 3 dpf (Figure 1). The addition of γ Cry1 to pT2/S1EF1α-GM2 caused a modest (two-fold) increase in injected embryos showing any GFP fluorescence. In contrast, the increase in eye-specific GFP expression was ten-fold (Figure 1). We concluded that at least in this assay, the Sleeping Beauty inverted repeat sequences do not block transcriptional regulation and that the EF1α promoter can be subject to transcriptional regulation from external, tissue-specific regulatory sequence elements. Figure 1 Artificial enhancer trapping with a Sleeping Beauty transposon. Comparison of GFP expression in embryos injected with pT2/S1EF1α or γCry1/pT2/S1EF1α. Plasmids are diagramed as cartoons on the top of the picture. The SB transposon's inverted repeats are shown as boxes with open triangles, and the GFP open reading frame is depicted as a grey arrow. The gamma-Crystallin promoter/enhancer is shown as a hatched box. DNA-injected embryos which survived to 3 dpf were counted and scored for GFP fluorescence anywhere in the embryo (any GFP) and for fluorescence in the eye (eye GFP), even if there was additional fluorescence elsewhere. The average percentage of embryos positive for particular GFP fluorescence in three independent experiments is shown ± standard deviation. Promoter truncations and pilot screens We next tested the hypothesis that the absence of expression patterns in our previous work was due to the fact that S1EF1α is expressed too strongly in transgenic animals. Since most successful enhancer traps in Drosophila and mice were based on truncated or weak promoters, we decided to attenuate the S1EF1α promoter by removing sequences upstream of Bst1107I (S2EF1α) and EcoRI (S3EF1α) restriction enzyme sites (Figure 2). We then co-injected the corresponding transposon constructs with SB10 transposase mRNA to assess germline transmission, expression and enhancer trapping rates. In pilot experiments, progeny from over 20 fish were screened with each construct. While overall germline transmission and expression rates were comparable (Figure 2), there was a difference in the expression patterns of the two constructs in transgenic embryos. Most of the transgenic animals generated with pT2/S3EF1α-GM2 exhibited weak GFP expression, and we could not detect any expression patterns (data not shown). In contrast, when pT2/S2EF1α-GM2 was used, most of the GFP-positive fish exhibited fairly strong, ubiquitous expression. Closer analysis indicated that many of these "ubiquitous" expression patterns were rather unique, with GFP expression often noted to be particularly strong in some tissues, consistent with a tissue-specific expression pattern superimposed on a ubiquitous expression pattern (data not shown). In most cases, GFP expression segregated as a single integration event, indicating that a tissue-specific expression pattern was not likely being masked by a ubiquitous expression pattern from a different insertion event. A similar phenomenon has been observed with Drosophila enhancer traps [11]. We speculate that in those instances, the GFP expression cassette may have fallen under the control of multiple enhancers – some tissue-specific, some ubiquitous. Alternatively, the ubiquitous expression may stem from the ubiquitous activity of the EF1α promoter used in the screen, with tissue-specific enhancers only elevating the expression levels in certain tissues, but not restricting it. We did not consider such expression patterns valuable and did not establish any fish lines with such GFP expression. Importantly, one of the founders in the pT2/S2EF1α-GM2 pilot screen yielded three kinds of GFP expression in its progeny. Some were ubiquitously GFP positive, some showed a hatching gland-specific expression profile, and some exhibited both. When three F1 fish with both ubiquitous and hatching gland specific expression were raised and outcrossed, the two expression patterns exhibited independent segregation: 24% of the embryos were GFP negative, 26% expressed GFP ubiquitously, 25% had hatching gland-specific GFP expression, and 25% had both hatching gland-specific and ubiquitous expression (n = 245). Independent segregation indicates that the two transposon insertions causing the two expression patterns are unlinked. Two independent integration events were confirmed by Southern hybridization and inverse PCR (data not shown). The hatching gland-specific GFP expressing embryos were used to establish our first enhancer trap line, ET1 (Figure 3A). We concluded from our pilot screens that pT2/S2EF1α-GM2 demonstrated the desired properties for potential use in enhancer trapping studies. Figure 2 EF1α promoter truncations and endogenous enhancer trap screening. A diagram of the S1EF1α promoter [32, 41]. Restriction enzyme sites are shown on top as single letters. S is SphI, N is NheI, B is Bst1107I and R is EcoRI. G/C, G and C rich box. Sp1, Sp1-like site. TATA, TATA box. Numbering below is relative to the first T of the TATA box. The table below the diagram shows the results of the pilot and scale-up (*) screens. Transgenesis and expression rates are shown, non-expressing transposon insertions were not scored. Transgenesis and expression rate from scale-up screen (#) is an underestimate since many founders were screened by incross and crosses from doubly transgenic founders were scored as a single transmission event (see text). Figure 3 Enhancer trap lines exhibit a variety of unique GFP expression patterns. (A). Lateral view of GFP expression in Enhancer Trap line 1 (ET1) at 38 hours post fertilization (hpf). (B) ET3 at 5–6 somite stage. (C) ET3 at 36 hpf. (D) ET4 at 26 hpf. (E) ET5 at 30 hpf. (F) ET5 at 48 hpf. (G) ET6 at 26 hpf. (H) ET7 at 32 hpf. (I) Ventral view of ET7 at 5 dpf. (J) Lateral view of ET8 at 26 hpf. (K) Dorsal view of ET9 at 28 hpf. (L) Lateral view of ET9 at 30 hpf. In all panels, anterior is to the left. See text for details. Germline excision of a Sleeping Beauty transposon insertion We have previously demonstrated the excision of a Sleeping Beauty transposon from the genome in somatic tissues of transposase-injected zebrafish embryos [43]. We tested if such an excision event could be inherited by examining transposon excision in the germline. Embryos homozygous for the ET1 insertion were injected with SB10 transposase mRNA, and while some were used for a somatic excision assay the rest were raised to test for germline transmission of an excision event. A PCR reaction on genomic DNA from transposase-injected embryos with primers flanking ET1 insertion point produced two bands. A large band corresponded in size to the transposon insertion allele, and a small band corresponded to a transposon-less allele (data not shown). Both cannonical Sleeping Beauty transposon footprints (ATGTCAT and ATGACAT, [44,45]) were obtained upon cloning and sequencing of the smaller band, indicating a transposase-mediated excision and DNA repair. 26 fish were screened for germline transmission (see Materials and Methods), and one was shown to transmit the expected excision footprint. We conclude that the Sleeping Beauty transposon can be excised from a genomic location in the zebrafish germline. pT2/S2EF1α-GM2 scale-up screening: 10% of GFP-expressing integrations yield tissue-specific patterns One tissue-specific expression pattern was recovered from our pilot screen. We sought to recover more patterns and to test if enhancer detection in zebrafish is amenable to scale-up. To that end, we co-injected 3248 zebrafish embryos with the pT2/S2EF1α-GM2 and SB10 transposase mRNA mix. 2102 embryos survived to day 3 for scoring, of which 848 were mosaic GFP positive and were selected to be raised. 330 survived to adulthood and were screened for germline transmission of GFP expression, primarily by sibling incrossing. This approach provided a lower estimate of the transgenesis and expression rate because it does not distinguish instances were both parents are transgenic. In this screen, at least 80 of the founder fish produced GFP-expressing progeny resulting in a minimum estimate of a 24% transgenesis rate. The actual transgenesis rate is closer to 30% because most of the fish were screened by incross, and if a pair produced GFP-expressing progeny, only one parent was counted as a transmitter. Eight of the GFP-expressing fish displayed distinct GFP expression patterns (Figure 3). Together with the pilot screen, 9 tissue-specific expression lines were obtained from 90 transgenic founder fish (10%) using the pT2/S2EF1α-GM2 transposon. Recovered expression patterns label a diverse array of tissues during embryogenesis GFP expression in ET1 can be first observed in the polster region at 7–8 somite stage (not shown). The expression is very pronounced between 20 and 40 hours post-fertilization (hpf), when it marks the hatching gland (Figure 3A). Expression disappears as the hatching gland is resorbed. Line ET3 represents a pattern with the earliest onset of expression. Anterior localization of GFP in the diencephalon is detected by 5–6 somite stage in this line (Figure 3B). Extremely bright anterior expression persists in the ventral diencephalon (Figure 3C) and by 6 days post-fertilizations (dpf) is restricted slightly more posterior in the midline. The onset of expression for ET4 is 18 hpf with a bilateral expression pattern in cranial sensory ganglia that remains strong until 2 dpf and is undetectable by 5 dpf. This anterior expression in ET4 seems to label the lateral line ganglia both anterior and posterior to the otic vesicle (Figure 3D), however, no expression is detected in the lateral line in the trunk. In ET5 a single bilateral patch of strong GFP expression in the hyoid arch is observed by 24 hpf (Figure 3E), that by 48 hpf marks a more anterior location in the embryo (Figure 3F). Expression in this line is greatly diminished by 3 dpf and is undetectable by 5 dpf. Strong GFP expression is observed in ET6 by 26 hpf as a bilateral expression pattern consisting of two distinct patches in a subset of cranial sensory ganglia/placodes (Figure 3G). The expression weakens by 2 dpf and is undetectable by 3 dpf. GFP expression in ET7 begins weakly in the midbrain-hindbrain boundary (MHB) at 12–14 somites with the most pronounced expression in the anterior side of the MHB detected by 26 hpf (Figure 3H). Robust expression in the heart is first detected at around 32 hpf and remains ventricle-specific through 5 dpf (Figure 3I), even though expression in the brain is no longer restricted to the MHB. GFP expression in ET8 is already localized by 10–12 somites and remains strong in the telencephalon, and posterior side of the MHB through 26 hpf (Figure 3J). By 3 dpf the localized anterior expression is undetectable over autofluorescence, however, caudal expression appears to be enhanced in the dorsal neural tube. The onset of expression in ET9 occurs around 22 hpf and is difficult to detect by 2 dpf. At 28–30 hpf (Figure 3K,3L), three distinct expression domains are apparent in the telencephalon, diencephalon and hindbrain of ET9. The ET2 line expresses GFP specifically in the motoneurons We analyzed the ET2 line in detail because of the highly specific expression of GFP in these fish. GFP expression was first observed at the 16 somite stage, when 2 bilateral cells in the spinal cord of the 10 anterior somites become GFP positive (Figure 4). At later stages, multiple cells per somite become GFP positive, either due to continued expression of GFP mRNA or due to segregation of GFP to daughter cells. GFP expression follows the wave of somitogenesis, with the posterior-most somites lagging in GFP expression. At about 24 hours, ventrally-projecting axons become visible by GFP fluorescence. Later yet a pattern of nodes appears along the axons (Figure 4). Based on the position of neuronal cell body and the axonal trajectory, we conclude that caudal primary motoneurons express GFP in this line [46]. To our knowledge, this is the first gene to be specifically expressed only in this subpopulation of motoneurons. We therefore sought to identify the locus tagged by this transposon insertion. Figure 4 The ET2 transgenic fish line expresses GFP in caudal primary motoneurons. GFP expression in ET2 was visualized in motoneurons using a bandpass GFP filter set at various stages of embryonic development. In all panels anterior is to the left. (A) The onset of GFP expression in ET2 line at 16 somite stage. (B) 26 somite stage. (C) 24 hpf. (D, E) 36 hpf. Axonal trajectories are visible at 24 and 36 hpf. Southern analysis indicated that there is a single transposon insertion in this line, and it is linked to GFP expression (Figure 5). Inverse PCR identified a transposase-mediated insertion into a TA dinucleotide at position 256083 on contig ctg9701 (zebrafish genome assembly Zv3). The insertion occurred into a Genescan-predicted gene. Further analysis indicated that the Genescan-predicted gene actually consists of parts of at least two different genes, myoferlin and poly(ADP-ribose) glycohydrolase (PARG). The insertion located in the PARG part of the predicted transcript, 649 nucleotides from an exon just upstream of the PARG catalytic site. To confirm that the transposon insertion into the PARG gene induced GFP expression in primary motoneurons, we prepared genomic DNA from both GFP positive and GFP negative embryos from an independent outcross, and we conducted a PCR with NeuroIns-F1 and NeuroIns-R1 primers specific to the flanking sequences. In GFP negative embryos, a 0.5 kb band corresponding to wild type locus is noted. In GFP positive embryos, the same band is seen in addition to a larger band of approximately 2.4 kb, corresponding to a locus with transposon insertion (Figure 5). Since the inverse PCR and confirming PCR was performed on DNA from different batches of embryos, we can exclude the possibility of DNA contamination or fish husbandry error and conclude that the enhancer trap transposon insertion into the PARG gene causes GFP expression in caudal primary motoneurons. Figure 5 Identification of the transposition event in the ET2 line. (A) The pT2/S2EF1α transposon insertion into zebrafish genome is shown; restriction enzyme sites and primers used for molecular analysis are indicated. Transposon IR/DR's are shown as solid boxes with open triangles, and the GFP open reading frame is shown as a grey arrow. Genomic DNA is shown as a dotted line. N is NsiI, E is EcoRV. (B) Southern blot on ET2 line outcross embryos. DNA from GFP positive (lanes 1 and 2) and GFP negative (lanes 3 and 4) embryos was digested with NsiI (lanes 1 and 3) or EcoRV (lanes 2 and 4) and probed with a GFP-specific probe. (C) Linkage of the transposon insertion event to GFP expression. Primers flanking the transposon insertion event (arrows) were used to conduct PCR on DNA from GFP positive (lane 2) and GFP negative (lane 3) embryos from an ET2 outcross different from the one used in (B). Lane 1, λ Eco47III Marker (Fermentas Inc). GFP expression in ET2 line matches the expression of the endogenous PARG gene Poly(ADP-ribosyl)ation is a protein modification that is extensively studied at the biochemical level and is associated with changes in DNA replication, recombination, repair and transcription [47], for a review, see [48]. Recently poly(ADP-ribosyl)ation was demonstrated to have a role in long term memory in the sea slug Aplysia [49]. Most organisms have multiple genes for poly (ADP-ribose) polymerases but only a single known gene for poly (ADP-ribose) glycohydrolase [48]. PARG activity is noted to be expressed in many cell lines, among them neuronal [50-53], but the tissue specificity of PARG expression during embryogenesis has not been reported for any organism. To test if the pT2/S2EF1α-GM2 enhancer trap recapitulates the expression pattern of the endogenous PARG gene, we conducted whole mount in situ hybridization on ET2 outcross embryos to compare PARG and GFP reporter expression (Figure 6). In situ hybridization visualizes axonal cell bodies, the position of which appears indistinguishable with both PARG and GFP probes. We therefore conclude that GFP mRNA expression in this enhancer trap line faithfully recapitulates the expression of the zebrafish PARG gene during embryogenesis. Figure 6 GFP expression in ET2 line embryos is indistinguishable from endogenous PARG gene expression. 23 hpf embryos collected from a heterozygous outcross were photographed for GFP fluorescence and sibling embryos were fixed for in situ hybridization. (A) In situ hybridization with PARG antisense probe. (B) In situ with GFP antisense probe. (C) Visualization of GFP expression in living embryos using a bandpass GFP filter set. (D) The same embryo as in (C) photographed using a bandpass GFP filter set with a low level of bright field illumination to visualize GFP expression in relative position to the somites. Molecular analysis of other enhancer trap lines identifies target genes We characterized insertion events in other enhancer trap lines. GFP positive F1 or F2 fish were outcrossed, and embryos were sorted into GFP positive and GFP negative pools. Genomic DNA was prepared from each pool, and Southern analysis was conducted to assess transposon copy number and linkage to the GFP expression pattern. In all lines except ET1 (see above), a single GFP expression-linked transposon insertion event was detected by Southern hybridization. We then conducted inverse PCR analysis on the DNA from GFP positive embryos to identify the insertion locus. For verification, DNA from embryos from an independent outcross was prepared and PCR was run with primers flanking the insertion site to link GFP expression and transposon insertion at a particular locus. Verified enhancer trap loci are presented in Table 1. Notably, seven of the insertions have occurred into introns of Genescan-predicted genes. Four of the tagged genes show significant homologies to previously identified genes: PARG (see above), MAPK upstream kinase-binding inhibitory protein (MBIP) [54], a member of cytochrome P450 superfamily and Nidogen [55]. The other three tagged predicted genes do not have significant homologies to previously identified genes. In the two lines which have insertions into intergenic regions, transposons have integrated less than 25 kb from the nearest predicted transcript. Table 1 pT2/S2EF1α-GM2 transposon insertion events in analyzed enhancer trap lines. Trap line Sequence Insertion location Predicted gene ET1 ATTGTCCtTAGTGTATGTGTTTGTGTGA Chr. 4 none ET2 CAAAAAGACTATATATAGGAGGCTTCAA ctg9701 PARG ET3 AACGCTTACCATGTATGTTAATAAATGT Chr. 17 MBIP ET4 TATATCAAAATTATATATATGAACGTAT Chr. 6 Cyt. P450 ET5 GTACATAcACATGTACAAATCaACATTA ctg13471 novel ET6 ATTTTAAACAAACTAAGTtGAACATTAC ctg13605 Nidogen ET7 ATCACAGAGCATCTAGCTTGGATGTGCT ctg12155 novel/mkp3 ET8 TATACAACAAACTTATCTAACGTGCAAT Chr. 2 none ET9 TATTTAATATATATATTATATTATATTA Chr. 19 novel Left column, line designation. Sequence column, the genomic sequence the transposon has inserted into. The target TA dinucleotide is highlighted in bold. The sequences flanking the left inverted repeat are to the left of the target TA, and sequences flanking the right inverted repeat are to the right of the target TA. Lowercase indicates mismatches between an actual sequence read and the current zebrafish genome sequence (Assembly ZV3). Both left and right transposon/genomic DNA junctions were sequenced for ET1, ET2, ET3, ET5 and ET7. Only the left junction was read for ET8, and only the right junction was read for ET4, ET6 and ET9. Insertion location column, predicted insertion chromosome or contig (Zv3 assembly of the zebrafish genome). Predicted gene column, the gene into which the transposon has inserted as annotated in the zebrafish genome assembly Zv3. Novel indicates no significant homologies. Gene name indicates significant homology to denoted genes. The predicted integration into an intron of the PARG locus for line ET2 was experimentally confirmed (see text). For ET7, a comparison of the observed expression pattern in this line with that of a known nearby gene (mkp3) indicates this insertion has most likely trapped an enhancer for this gene (Fig. 7, see text). Actual sequence reads which were used to determine the genomic location of the transposon insertions were longer than shown in this table and are available upon request. ET7 line has a transposon insertion near mkp3 locus and matches mkp3 expression pattern The transposon insertion in ET7 line has occurred into a predicted novel gene (Table 1). Closer investigation of the target locus revealed the presence of a previously characterized zebrafish mkp3 gene within 30 kb of the insertion site. Our attempts to amplify the predicted novel candidate gene from maternal and post-somitogenesis zebrafish cDNA libraries using 2 different primer pairs failed, while mkp3 cDNA was readily amplified in parallel PCR reactions (data not shown). This suggests that the novel target gene may be a false prediction by Genescan. The mkp3 gene encodes a dual specificity phosphatase which was cloned as a member of fgf8 synexpression group and is a negative feedback regulator of FGF8 signaling. mkp3 is expressed in the midbrain-hindbrain boundary, forebrain, tailbud, branchial arches, developing ear, pectoral fin buds and other tissues [56,57]. GFP expression in ET7 line closely mimics mkp3 mRNA expression pattern in 23 hour embryo (Figure 7). The only significant difference is that GFP expression is stronger in somites and not as bright in the tailbud, even though the tailbud expression becomes brighter at later stages of development (data not shown). We did not observe GFP expression in the pectoral fin buds, even though we reproduced mkp3 expression in the fin buds just after after 24 hpf by in situ (data not shown, [56,57]. An intriguing possibility is that mkp3 expression in pectoral fin buds may be controlled by a different enhancer, one we do not detect in this transgenic line. Additionally, ET7 expresses GFP in the heart after 24 hpf, and the expression clearly localizes to the ventricle at 5 dpf (Figure 3I). Expression of mkp3 in the heart after 24 hpf was not reported, and we did not conduct in situ hybridization on late pharyngula stage embryos to test for it. However, fgf8 is expressed in the ventricle of the zebrafish heart at 36 hpf [58]. Taken together, this suggests that GFP expression in ET7 line mimics a subset of the complete expression pattern of the zebrafish mkp3 gene. Figure 7 GFP expression in ET7 line matches mkp3 mRNA expression. (A) GFP fluorescence photograph of an ET7 embryo at 23 hpf. (B) In situ hybridization on 23 hpf wild type embryo using mkp3 antisense RNA probe. Discussion In this paper, we describe the first use of enhancer trapping, or enhancer detection, as an experimental approach in zebrafish. We show that Sleeping Beauty transposons can trap enhancers by testing an artificial enhancer trapping event in vivo. This approach is likely to also be useful in the construction and testing of other trap vectors: gene (5' exon) and polyA (3' exon) and other related constructs. We then constructed two further truncations to the S1EF1α promoter in the transgenesis cassette [32] and found one to be particularly suitable for enhancer trapping. Ten percent (9 of 90) of GFP-expressing transgenic fish generated lines with unique GFP expression patterns. All reagents described in this paper, including the enhancer trap fish strains, are readily available upon request . Many of the obtained enhancer trap lines express GFP in the nervous system. This was previously observed with both mouse and Drosophila enhancer trap vectors and was speculated to stem from the transcriptional complexity of neural tissue [11,28]. Several of our lines also exhibit some level of GFP expression in the eye. At least two explanations can be put forward to explain this observation. First, many genes are expressed in the developing eye. Thus, the eye expression that we see may reflect expression of the tagged genes in the eye. Second, optical properties of the tissues in the eye may permit detection of GFP expression that is lower that what would be required for detection in other tissues. The ET2 line harbors a transposon insertion into the zebrafish gene for poly(ADP-ribose) glycohydrolase (PARG). We demonstrate that both PARG and GFP in ET2 line are expressed in caudal primary motoneurons of 23 hour old embryos. Thus, GFP expression in the ET2 line mimics that of an endogenous gene (PARG), indicating that transgene expression is under control of an endogenous enhancer. A very intriguing question is what the actual trapped enhancer sequence is, how far away from the genomic enhancer the trap can insert and still detect it, and weather artificial enhancer trap approach (Figure 1) can be used to answer these questions. The ET7 line has a transposon insertion into a predicted novel gene 30 kb downstream of the zebrafish mkp3 locus. GFP expression in that line closely resembles part of the mkp3 expression domain, suggesting that the enhancer trap transposon in that line is under control of a subset of mkp3 enhancer elements. Zebrafish enhancer trap lines will be valuable in future developmental genetics studies, be it classical mutagenesis or morpholino "knockdown" screening [59]. GFP expression can be used as a sensitive marker for certain tissue or cell types. For example, the ET1 line expresses GFP in the hatching gland. The expression of the hgg1 gene is specific to the polster and hatching gland depends on nodal signaling and is absent in one-eyed-pinhead mutants [60]. We phenocopied the one-eyed-pinhead mutation by morpholino injection in ET1 homozygotes and observed a complete loss of hatching gland-specific GFP expression (data not shown). While the ET1 line expresses GFP in an organ that can be readily observed using regular light microscopy techniques, other lines visualize tissues that are not nearly as easily morphologically accessible. In particular, the ET2 line visualizes the position of primary motoneuron cell bodies and axonal trajectory. Morpholinos against known genes or new members of the zebrafish secretome [61] can be screened for effects on neuronal cell body position or axonal pathfinding in the developing embryos by injection into ET2 line embryos. The ET7 line may provide a fluorescent readout of FGF8 signaling, thus facilitating the identification of genes involved in that signaling pathway. A further utility offered by the transposon system is the possibility to revert a mutant phenotype or to generate localized deletions by transposon excision [23]. We successfully excised the transposon in the germline of the ET1 line, resulting in the expected transposon footprint. It has been shown that excision of the Sleeping Beauty transposon from a plasmid results in local deletions with fairly high frequency which is dependent on the cell type or tissue used [45]. Furthermore, the frequency of imprecise excision of Sleeping Beauty transposons significantly increases in cells with a compromised DNA repair pathway [62,63]. It remains to be determined how frequently the excision of a Sleeping Beauty transposon from a genomic location in zebrafish germ line is accompanied by a deletion of flanking genomic DNA, and it should be possible to compromise the embryo's DNA repair machinery to induce such deletions at a high frequency. Our experiments indicate that enhancer detection using Sleeping Beauty transposons is an easily scalable and efficient experimental technique in zebrafish. Obtaining fish with different GFP expression patterns is not the rate limiting step in this process. Preliminary molecular analysis of the insertion site is also straightforward using inverse PCR techniques. Identification of candidate genes should benefit from the progress in zebrafish genome sequencing and annotation. The main bottleneck step is the detailed biological analysis of GFP and the corresponding candidate gene expression profile. In Drosophila, the generation of transposase-expressing lines of flies made enhancer detection and P-element mutagenesis in general a mainstream approach. Even without a similar gain in efficiency, transposase expressing fish lines would make enhancer trapping as well as related gene- and poly(A)-trap methodologies even more accessible for high-throughput functional analysis of the vertebrate genome. Methods Plasmid construction pT2/S1EF1α-GFP (pDB358) was previously published [32]. To make γ Cry/pT2/S1EF1α-GFP (pDB375), a BamHI-HindIII fragment from Cry1-GFP3 [42] containing part of the X. laevis γ-Cry1 promoter was cloned into the Ecl136II site of pDB358. To produce pT2/S2EF1α-GFP (pDB371), a part of the EF1α promoter was deleted from pDB358 by ligation of the Bst1107I-NcoI and NheI-NcoI fragments of pDB358. Similarly, the EcoRI-NcoI and NheI-NcoI fragments of pDB358 were ligated to produce pT2/S3EF1α-GFP (pDB372). Inverse PCR, PCR and primer sequences For inverse PCR experiments, zebrafish genomic DNA was digested and ligated as described [64]. 1 and 2.5 microliters of the ligation reaction were used for the first PCR reaction with RP1/LP1 or RP1/GFP-R1 primers in total volume of 25 μl. 1 μl of the first PCR reaction was used as a template for the second (nested) PCR reaction with primer pairs RP2/LP2 or RP2/GFP-R2, respectively. Expand Hi Fi PCR system (Roche) was used for all PCR reactions. A MJ Research PTC-100 PCR machine was used for PCR with the following program : 92°C 4 min., 92°C 10 sec., 60°C 30 sec., 68°C 6 min., 30 cycles. Starting at cycle 11, 20 sec. per cycle was added to the extension time. The same PCR reaction with an annealing temperature 55°C was used for amplification with primers flanking transposon insertion sites, and for amplification of partial PARG cDNA from a maternal cDNA library. Primer sequences are: LP1 GTGTCATGCACAAAGTAGATGTCC [32]; LP2 ACTGACTTGCCAAAACTATTGTTTG; nRP1 CTAGGATTAAATGTCAGGAATTGTG; RP2 GTGAGTTTAAATGTATTTGGCTAAG; GFP-R1 TTCGGGCATGGCACTCTTG; GFP-R2 TATGATCTGGGTATCTCGCAA; NeuroC1-F1 CGTAAAGATGCCTTGTTCAGAA; NeuroC1-R1 ATTCCGTGACTCTCCTGAAATA; NeuroIns-F1 GGCTTGCATACATGACTAATG; NeuroIns-R1 GAAGACTGAAGTCCTCAAACT; HG1-1 ACATTGAGCCACTAAGCATTG; HG-2 TGTGTGCACTTAAGGGGCGA. Mkp3-F1 AGTGTTGCATTCTCCAGGATA; Mkp3-R1 TGACACAGAACTTCCCTGAAC; EF1a-F2 TTCCTGCAGGTCGACTCT; GFP-R0 GTGTAATCCCAGCAGCTG. Information about other primers is available from the authors upon request. In situ hybridization A partial sequence for the zebrafish poly(ADP-ribose) glycohydrolase cDNA was amplified using primers neuroC1-F1 and neuroC1-R1 and cloned using a Topo TA cloning kit (Invitrogen) to make pDB376. To make antisense RNA probe, pDB376 was digested with SpeI and transcribed with T7 RNA polymerase (Promega) and DIG labeling kit (Roche). GFP probe was made by amplifying GFP with 46 base pairs of EF1α promoter from pT2/S1EF1α-GM2 using primers EF1a-F2 and GFP-R0, and cloning it into Topo TA cloning kit resulting in pSS100. pSS100 was linearized with SpeI and transcribed with T7 RNA polymerase using DIG labeling kit. To make mkp3 antisense probe, mkp3 cDNA was amplified from maternal cDNA library with primers Mkp3-F1 and Mkp3-R1 and cloned into Topo TA cloning kit to produce pDB528. The plasmid was linearized with SpeI and transcribed with T7 polymerase using DIG labeling kit. Screening for germline transmission of Sleeping Beauty transposons Embryos injected with SB10 transposase mRNA and transposon DNA mix were raised as described [32,64]. In pilot screens, adult fish were outcrossed to brass for ease of husbandry. All collected embryos were screened for GFP expression at 1 day post fertilization (dpf) and 3 dpf. We set an arbitrary 200 embryo cutoff for screening, meaning that if less that 200 embryos were obtained from a founder, an additional cross was set up and to obtain additional embryos for screening. Analysis of transgenesis data from pilot screens indicated that 10% of transgenic lines would have been missed if cutoff was set at 100 embryos, and this less stringent coverage protocol was used in scale up screen. Also, we decided to limit screening to 1 dpf since none of the transgenics would have been missed in the pilot screens without the 3 dpf screening. Transposon excision in the germline Homozygous ET1 embryos were injected with SB10 transposase mRNA, raised and screened for loss of hatching gland specific GFP expression, or for a change in the GFP expression pattern. Twenty six fish were screened (R0, for Remobilization), and 2 gave GFP negative embryos, with an additional 2 giving ubiquitously GFP positive embryos, suggesting that germline remobilization events may have occurred in as many as 15% of transposase injected embryos. Ubiquitous GFP positive embryos (one in each of the two R0) did not survive. Of the two R0's that gave GFP negative embryos, one gave mosaic hatching gland expression in the next generation. PCR with transposon specific and flanking primers did not show any changes in the locus. The second R0 produced 19 embryos that were GFP negative from the total of 671 embryos obtainted. An R1 adult was outcrossed, embryo DNA was prepared, and PCR with primers HG1-1 and HG1-2 was conducted. The resulting PCR fragment was cloned using PCR 4 Topo cloning kit (Invitrogen). Plasmids were sequenced using M13 Forward primer, and one clone with a transposon footprint was identified. To confirm that it was not due to PCR contamination, a second clutch of embryos was obtained, the procedure was repeated, and the same footprint was obtained (data not shown). Authors' Contributions The experiments described in this paper were planned, conducted and analyzed as a joint effort between the authors. In particular, DB, AD, SH and ZW contributed to fish screening, line establishment and to scientific descriptions of these lines, DB and SS to molecular analysis, AD and DB to GFP expression and in situ analysis. DB designed and built the transposons used in this study and was responsible for drafting the manuscript for publication. SE conceived and supervised the study and edited the manuscript. All authors read and approved the final manuscript. Acknowledgements We thank Paul Phelps, Sandra Leo, Amanda Mahoney and Tessa Hodapp for help with fish screening, and Aubrey Nielsen, Rachel Bowers, Dan Carlson and Pat Cliff for fish maintenance and Perry Hackett for critical reading of the manuscript. We thank all members of the Arnold and Mabel Beckman Center for Transposon Research for valuable discussions. 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Efficient Gene Delivery Methods in Cell Biology 2004 77 351 364

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==== Front BMC GenomicsBMC Genomics1471-2164BioMed Central London 1471-2164-5-631535554910.1186/1471-2164-5-63Research ArticleIntegrated analysis of metabolic phenotypes in Saccharomyces cerevisiae Duarte Natalie C [email protected] Bernhard Ø [email protected] Pengcheng [email protected] Department of Bioengineering, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0412, USA2 Department of Molecular Biosciences & Bioengineering, University of Hawaii, 1955 East-West Road, Honolulu, HI 96822-2321, USA2004 8 9 2004 5 63 63 13 5 2004 8 9 2004 Copyright © 2004 Duarte et al; licensee BioMed Central Ltd.2004Duarte et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background The yeast Saccharomyces cerevisiae is an important microorganism for both industrial processes and scientific research. Consequently, there have been extensive efforts to characterize its cellular processes. In order to fully understand the relationship between yeast's genome and its physiology, the stockpiles of diverse biological data sets that describe its cellular components and phenotypic behavior must be integrated at the genome-scale. Genome-scale metabolic networks have been reconstructed for several microorganisms, including S. cerevisiae, and the properties of these networks have been successfully analyzed using a variety of constraint-based methods. Phenotypic phase plane analysis is a constraint-based method which provides a global view of how optimal growth rates are affected by changes in two environmental variables such as a carbon and an oxygen uptake rate. Some applications of phenotypic phase plane analysis include the study of optimal growth rates and of network capacity and function. Results In this study, the Saccharomyces cerevisiae genome-scale metabolic network was used to formulate a phenotypic phase plane that displays the maximum allowable growth rate and distinct patterns of metabolic pathway utilization for all combinations of glucose and oxygen uptake rates. In silico predictions of growth rate and secretion rates and in vivo data for three separate growth conditions (aerobic glucose-limited, oxidative-fermentative, and microaerobic) were concordant. Conclusions Taken together, this study examines the function and capacity of yeast's metabolic machinery and shows that the phenotypic phase plane can be used to accurately predict metabolic phenotypes and to interpret experimental data in the context of a genome-scale model. ==== Body Background The development of numerous high-throughput experimental techniques such as DNA microarrays, genome sequencing, and protein chips has revolutionized the analysis of biological systems and generated a catalog of information about a cell's components [1-3]. Efforts are now focused on the integration of this data to enable the systemic understanding of cellular functions [4-6]. This integration is typically in the form of a mathematical model that can be used to simulate complex cellular behaviors based on a limited amount of biological data. Several modeling approaches have been implemented in the study of Saccharomyces cerevisiae. Flux-balance models of yeast have appeared for small-scale network reconstructions. Most of these studies are specific for growth conditions, such as anaerobic, glucose-limited metabolism [7], aerobic growth on galactose [8] or growth on mixtures of glucose and ethanol [9]. In addition to flux-balance models, many dynamic models of simplified central metabolic networks in yeast also have been developed [10,11], along with full-scale kinetic models specific to pathways such as glycolysis [12,13] and the pentose phosphate pathway [14]. These models have been useful to study detailed metabolic events such as concentration changes of individual metabolites and key flux splits. Small-scale reconstructions can be limited in their prediction of cellular functions since these processes are typically dependent on the interaction of components at a whole-cell level. This has motivated the development of genome-scale models, several of which have already appeared for bacterial cells, including Escherichia coli [15,16], Haemophilus influenzae [17], and Helicobacter pylori [18]. We have previously reconstructed a genome-scale metabolic network of Saccharomyces cerevisiae based on its annotated genome sequence and a thorough examination of online pathway databases, biochemistry textbooks, and journal publications [19]. A total of 708 open reading frames, 1175 metabolic reactions, and 733 metabolites are accounted for in this stoichiometric network, which includes both cytosolic and mitochondrial compartments. This genome-scale reconstruction is the most comprehensive model of yeast metabolism to date and has been previously validated through in silico gene deletion studies [20] and the calculation of key physiological parameters [21]. The reconstruction and analysis of genome-scale microbial networks have advanced significantly in recent years [22,23], as has the development of a variety of constraint-based modeling methods that allow for the deduction a cell's phenotype based on its genotype and environmental conditions [24-28]. Phenotypic phase plane (PhPP) analysis is a constraint-based method used to obtain a global perspective of genotype-phenotype relationships in genome-scale metabolic networks. In PhPP analysis, flux balance analysis and linear programming are used to map all of the cellular growth conditions represented by two environmental variables onto a two-dimensional plane and identify phases with distinct metabolic pathway utilization patterns. Some applications of PhPP analysis include the study of optimal growth rates [29], adaptability of microorganisms [30,31], metabolic network functions and capacities [15], and the impact of gene regulations [32]. Thus, PhPP analysis provides a way to guide experiments and analyze phenotypic functions based on genome-scale metabolic networks. The constraint-based modelling approach is based on the assumption that organisms have developed control structures to ensure optimal growth in response to environmental constraints [35]. Numerous experimental observations have been made in support of this hypothesis [36]. The mathematical descriptions for the PhPP have relevant metabolic meaning for the biological systems being studied. For example, any point in the PhPP corresponds to a single solution of the linear programming problem, which metabolically represents a possible growth behavior. A phase or region in the PhPP (where the shadow price is constant) represents a metabolic phenotype with specific pathway utilization. The shadow prices change continuously at the boundary from one phase to the next. Accordingly, the metabolic phenotype will vary. Metabolically, this is interpreted as a different optimal utilization of the metabolic pathways since each basis solution corresponds to a different flux distribution. In this study, we formulate a glucose-oxygen phenotypic phase plane for yeast based on its recent genome-scale metabolic reconstruction [19]. The growth states predicted by the PhPP are then characterized using shadow price analysis, in silico gene deletion simulations, and in vivo growth experiments. Finally, we evaluate the network's predictions for these growth states by comparing in silico biomass formation and by-product secretion rates to in vivo measurements. Results S. cerevisiae phenotypic phase plane (PhPP) The S. cerevisiae genome-scale metabolic network constructed by Forster et al. [19] was used to generate a PhPP [33] that describes yeast's metabolic states at various levels of glucose and oxygen availability (Fig. 1). The surface of the three-dimensional PhPP corresponds to the maximum growth rate allowable for each pair of glucose and oxygen uptake rates in the x-y plane (Fig. 1a). All feasible metabolic flux distributions lie on or below this surface. The two-dimensional projection of the PhPP (Fig. 1b) has been divided into seven regions, or "phases," to allow for qualitative comparisons (P1 - P7). The seven phases represent areas of the PhPP that have distinct metabolic phenotypes as defined by shadow price analysis, which identifies how changes in metabolite levels affect biomass formation [33]. There also are two regions of the PhPP with infeasible steady-state flux distributions: the area along the y-axis and the small square near the origin. Growth is infeasible in the region between the ordinate and P1 since yeast cannot use more than six oxygen molecules per glucose molecule. The two red lines in Fig. 1b are lines of optimality (LO). LOgrowth represents optimal aerobic glucose-limited growth of S. cerevisiae in which substrates are completely oxidized to produce biomass and LOethanol corresponds to maximum ethanol production under microaerobic conditions while growth is maximized. Figure 1 The yeast glucose-oxygen phenotypic phase plane (PhPP). (a) The three-dimensional S. cerevisiae PhPP drawn with Statistica™ (Statsoft, Tulsa, OK). The x and y axes represent the glucose uptake rate and oxygen uptake rate, respectively. The third dimension is the cellular growth rate. (b) A two-dimensional projection of the 3-D polytope in panel (a). The two lines of optimality are shown in red. LOgrowth represents optimal aerobic glucose-limited growth and LOethanol corresponds to maximum ethanol production under microaerobic conditions. P1 - P7 represent phases with various metabolic phenotypes. The hatched regions correspond to infeasible growth conditions. The orange line (glucose uptake flux = 5 mmol/gDCW/hr) represents the conditions which were used for the simulations in Figure 2. Simulation of optimal metabolic phenotypes Computer simulations (Fig. 1b) were used to illustrate the change of metabolic phenotypes described by the yeast phase plane. For the simulations, we arbitrarily set glucose uptake rate to 5 mmol/gDCW/hr and varied the oxygen uptake rate from 0 to 20 mmol/gDCW/hr. This allowed us to study the influence of a single environmental variable on cellular metabolism. Small amounts of NH3, sulfate and phosphate were introduced for the biomass synthesis. During anaerobic conditions (OUR = 0, on the x-axis), the growth rate was low and the respiratory quotient (RQ) was infinite by definition (Fig. 2a). As the oxygen uptake rate increased to 13 mmol/gDCW/hr to reach LOgrowth, the growth rate increased to its maximum value and the respiratory quotient approached 1.06. Further increasing the oxygen uptake rate caused both the growth rate and respiratory quotient to decrease due to futile cycles in which a combination of two or more biochemical reactions resulted only in the hydrolysis of ATP or other high-energy compounds [33]. Figure 2 Simulation of metabolic behavior for optimal cellular growth as a function of oxygen availability, ranging from completely anaerobic fermentation to completely aerobic growth in S. cerevisiae. The range of oxygen uptake rates used in the simulations (orange line, Fig. 1) allows for the characterization of the PhPP's seven phases (P1 - P7) and two lines of optimality (LOgrowth, LOethanol). (a) Growth rate and respiratory quotient (RQ). (b) Secretion profile for acetate, succinate, ethanol, and glycerol. Metabolic by-product secretion profiles also were calculated with increasing oxygen uptake rates. Since alternative optimal solutions exist in the genome-scale metabolic flux models [34], a range of secretion rates can be found amongst all of the equivalent optimal solutions for a fixed point in the PhPP. Remarkably, there was less than 1% difference between the maximum and minimum allowable secretion rates for a fixed maximal growth rate; thus, only the maximum predicted secretion fluxes for ethanol, succinate, glycerol, and acetate are shown (Fig. 2b). During anaerobic fermentation, ethanol, glycerol, and succinate were produced. Maximum ethanol production occurred at an oxygen uptake rate of 0.5 mmol/gDCW/hr, a condition defining LOethanol. Glycerol production ceased at this point. With a slight increase in oxygen uptake rate above LOethanol, acetate began to be secreted but succinate secretion decreased to zero. Ethanol and acetate were no longer secreted once the oxygen uptake rate was equal to or greater than 13 mmol/gDCW/hr, a point on LOgrowth where the metabolic pathway utilization enables complete aerobic growth. Further characterization of oxidative-fermentative phases (P2 - P6) Linear programming simulations generate parameters called shadow prices that can be used to evaluate how changes in metabolite availability affect the biomass formation [33]. Shadow price analysis was used to further characterize the oxidative-fermentative phases. A positive shadow price indicated that a metabolite was available in excess, meaning that a decrease in its availability would increase biomass synthesis, and a negative shadow price indicated that a metabolite was limiting such that increasing its availability would increase the biomass synthesis. In silico gene deletions were also performed in order to determine which reactions were essential in each phase. Essential reactions were defined as those whose deletion resulted in no predicted growth (growth rate equal to zero). This approach was especially useful for interpreting the physiological differences between growth states in phases 2 – 6 since their phenotypes were indistinguishable in terms of their secretion profiles. Phase 2 In phase 2, the ratio of oxygen uptake rate and glucose uptake rate (GUR) is lower than that on the line of optimality. As a result, the cell is oxygen limited and begins to ferment. Mitochondrial NAD+ is available in excess, meaning that the biomass synthesis would improve if its availability decreased. In order to maintain the cell's redox balance, the excess mitochondrial NAD+ must be reduced. This is done through the production of acetate and ethanol, which begin to be secreted in this phase. Thus it is the production of acetate and ethanol that makes the optimal growth rate less than that defined on the line of optimality. Phase 3 As the ratio of oxygen and glucose uptake rates is further decreased, three lower glycolysis reactions (fructose bis-phosphate aldolase, triose phosphate dehydrogenase, and phosphoglycerate kinase) become essential for growth in phase 3. Although these deletions severely hinder growth in phase 2 (reducing the growth rate by 55%, 19%, and 19%, respectively), the simulated growth rate is non-zero so these reactions were not considered to be essential. However, these reactions are essential in subsequent phases as the oxygen uptake rate is further decreased. Due to the limited oxygen, more carbons "overflows" into the fermentation pathway while at the same time oxidative metabolism becomes less effective. Phase 4 Shifting from phase 3 to phase 4, the pentose phosphate pathway is utilized to generate NADPH because not enough NADPH is produced through respiration at the lower oxygen uptake rate. The NADPH is then converted to NADH which is subsequently used for ATP production. Phase 5 Further lowering the ratio of oxygen and glucose uptake rates restricts the cell's ability to produce pyruvate in phase 5. Yeast can no longer utilize the oxidative pathways because an insufficient amount of cytosolic NAD+ is produced. When comparing phases 4 and 5, all of the metabolites with shadow price sign changes were folate intermediates. These are important energy carriers that are directly linked to the availability of both cytosolic and mitochondrial NAD+ and NADP+. Phase 6 As you enter phase 6, the acetate production is completely ceased. Ethanol is secreted as the only metabolic by-product to balance the redox potential of the cell. Growth experiments Three groups of experiments were conducted under different growth conditions in the PhPP (Fig. 3a). These three conditions were: Figure 3 Growth experiments shown on the PhPP. (a) The three groups of experimental data displayed on the S. cerevisiae PhPP were used as an index for the time course profiles in panels (b), (c) and (d). (b) Aerobic glucose-limited growth controlled by fed-batch operation. (c) Oxidative-fermentative growth with unlimited glucose and oxygen availability. (d) Microaerobic growth with unlimited glucose and very low oxygen availability. The AGL (b) and MA (d) data sets are located on lines of optimality and as a result are stable metabolic states with only one degree of freedom (glucose for AGL and oxygen for MA). OF (c) is an unstable metabolic state with two degrees of freedom (glucose and oxygen), making it more difficult to control this type of growth condition. By perturbing the environmental conditions, cells in OF can be shifted to either AGL or MA (unpublished results). • Aerobic, glucose-limited (AGL) growth experiments were conducted with a data acquisition and process control system. The dissolved oxygen (DO) level was maintained above 30% by sparging the compressed air into the CelliGen® Plus bioreactor during the cell cultivation. The system was controlled at the respiratory quotient of 1.06 for optimal S. cerevisiae growth by RQ-stat feeding strategy to maintain the glucose concentration at a low, stable level (Fig. 3b). The cell concentration rose steadily with almost no acetate or ethanol production and most of the carbon was incorporated into the yeast biomass. • Oxidative-fermentative (OF) batch growth experiments were carried out by allowing an essentially unlimited supply of oxygen and glucose. The DO level was maintained above 30% by sparging the compressed air into the shaker's flasks during the cell cultivation. Ethanol and acetate were accumulated in the aerated processes (Fig. 3c). • Microaerobic (MA) batch cultivations with S. cerevisiae were performed at a low dissolved oxygen level. The experiments were performed in side-arm flasks (Fig. 3d) in which a small amount of air was allowed to diffuse into the flasks via the cotton filter on the side arms. The DO level was measured to be less than 5%. The initial glucose concentration and the limited oxygen supply resulted in high levels of ethanol and low levels of acetate. Integration of experimental data and in silico predictions The S. cerevisiae PhPP is a genome-scale model-based visualization platform which allows experimental data and simulation results to be displayed and compared. The three groups of batch and fed-batch experimental data are projected on Fig. 3a using the experimentally measured OUR and glucose uptake rates. These rates were then used as constraints in the computer simulations. Table 1 shows that the experimental observations and the in silico predictions are in good agreement. Table 1 Comparison of In Silico Predictions and Experimental Measurements. Microaerobic fermentation Oxidative-fermentative growth Aerobic, glucose-limited growth OUR = 1, GUR = 14 OUR = 9, GUR = 12 OUR = 8, GUR = 2.5 In silico Experimental In silico Experimental In silico Experimental Growth rate 0.33 0.31 0.53 0.51 0.22 0.20 Ethanol 21.29 20.08 11.98 11.07 0 0.16 Acetate 0.26 0.22 2.62 2.57 0 0.31 Abbreviations: oxygen uptake rate (OUR), glucose uptake rate (GUR) Units: growth rate (1/hr), substrate uptake rates and metabolite production rates (mmol/gDCW/hr) Discussion In this study, the S. cerevisiae genome-scale metabolic network constructed by Forster et al. [13] was used to generate a PhPP [9] that describes yeast's metabolic states at various levels of glucose and oxygen availability (Fig. 1). Examination of the S. cerevisiae PhPP has led to clear interpretation and prediction of its metabolic capabilities. First, only a few distinct optimal S. cerevisiae growth phenotypes were found in silico, and these phenotypes correspond to well-defined phases of the PhPP. Second, two lines of optimality were identified in yeast's PhPP: LOgrowth, which represents optimal biomass production during aerobic, glucose-limited growth, and LOethanol, which corresponds to both maximal ethanol production and optimal growth during microaerobic conditions. The predictions of S. cerevisiae's PhPP and genome-scale model were compared to independent experimental data. The results showed that the agreement between the computed and observed growth rates, uptake rates, and secretion rates was close to the measured values or within the experimental error, and qualitatively the predictions agreed with published literature. Analysis of experimental data within the PhPP formalism suggests that yeast has only a few primary phenotypes, designated by the various phases. In P1, the oxygen supply is sufficient for growth by aerobic respiration, resulting in carbon dioxide as the sole by-product. Phases P2-P6 correspond to states of oxidative-fermentative growth, which is characterized by secretion of oxidative and fermentative metabolic by-products, i.e., acetate and ethanol, respectively. These states are highly similar since the phases are essentially co-planar in the 3-dimensional PhPP (Fig. 1a). The secretion profile (Fig. 2b) does not show any phenotypic differences between phases P2 - P6. However, through the use of shadow price analysis and in silico gene deletions, distinct pathway utilization patterns could be found for each phase. Finally, P7 represents microaerobic conditions. In this environment, yeast grows primarily by fermentation and secretes ethanol, glycerol, and succinate (Fig. 2b). This limited range of metabolic states is strikingly different from that found for E. coli, whose glucose-oxygen PhPP has five distinct optimal in silico phenotypes [32]. Comparison of the simulation results generated by the E. coli [35] and S. cerevisiae [19] models indicates that E. coli's metabolic by-product secretion patterns are more sensitive to the OUR variation than those of S. cerevisiae. Moreover, computer simulations show that when the OUR is lower than 7 mmol/gDCW/hr at a glucose uptake rate of 5 mmol/gDCW/hr, the TCA cycle in E. coli is broken into two branched pathways, one operating as a reductive pathway reversing the usual sequence from succinate to oxaloacetate and the other continuing to operate oxidatively to convert oxaloacetate to α-ketoglutarate. For S. cerevisiae, the pathway still functions as a cycle even when the OUR is as low as 1 mmol/gDCW/hr. Thus, it can be concluded by the comparison of E. coli and S. cerevisiae metabolic networks that yeast appears to be more robust to environmental perturbations. Furthermore, we predict that yeast secretes fewer metabolic by-products under these conditions, suggesting that its metabolism is more efficient than that of E. coli. This may represent a universal difference in how prokaryotes and eukaryotes respond to shifts in environmental parameters. Another feature that distinguishes the S. cerevisiae PhPP from the E. coli PhPP is the existence of two definable lines of optimality. The conditions that define LOgrowth are similar to those that define E. coli's sole line of optimality, i.e. they both represent the relationship between the glucose and oxygen uptake rates that results in optimal growth rate. Analysis of yeast's PhPP suggests that at a specific ratio of glucose and oxygen uptake rates glycerol production is halted and NADH is re-oxidized by maximal ethanol formation. This phenomenon, defined by LOethanol, has been supported by many research reports in the literature [3,17,25]. For example, Cysewski and Wilke [37] found a sharp stimulation of the specific ethanol productivity at a very low but non-zero level of dissolved oxygen. Later studies showed that a value of 10 ppb of dissolved oxygen maximized ethanol production in yeast chemostat cultures [25]. Thus, LOethanol, the second line of optimality predicted by the genome-scale model, is consistent with the experimental observations. A useful application of the S. cerevisiae PhPP is to qualitatively classify yeast's metabolic state based on phenotypic observations made in vivo (Fig. 3). The metabolite concentration profile obtained from all of the experiments qualitatively agrees with the corresponding metabolic state predicted by the PhPP. For example, in growth conditions near LOethanol, cells are expected to grow almost entirely by fermentation, with significant production of ethanol and lesser amounts of glycerol, acetate and succinate secretion. This phenotype is qualitatively similar to experimental observation, in which more ethanol is produced than acetate as shown in Fig. 3d. Points in the PhPP representative of the three data sets also were used to quantitatively predict yeast's metabolic phenotype (Table 1). Overall, the predictions are in good agreement with the experimental measurements. However, the predicted growth rates are slightly higher than the measured values. This difference may result from the model's prediction of optimal performance not reflecting suboptimal growth in vivo. Conclusions The genome-scale metabolic networks developed for other microorganisms, namely Escherichia coli, Haemophilus influenzae, and Helicobacter pylori, have led to useful insights into substrate preferences, the effects of gene deletions, optimal growth patterns, outcomes of adaptive evolution, and shifts in expression profiles [22]. With the recent reconstruction of S. cerevisiae's genome-scale metabolic network [19], these analytical techniques can now be applied to the first genome-scale model of an eukaryotic cell. By developing methods such as the PhPP to explore in silico the metabolic capabilities of microorganisms, we can generate new hypotheses as to how these organisms operate, and, more importantly, we can gain insight into the impact of individual cellular components on the organism as a whole. Methods Experimental methods Strains and media All cultures were grown at 30°C in SD medium [38] and supplemented with glucose (Sigma Chemical Co., St. Louis, MO) as appropriate for each phase of the experiment conducted. The S. cerevisiae strain FY4 MATα [39] was used in this study. Growth and fermentation system For experiments, 5 ml of overnight culture inoculated from single colonies grown on YPD agar was used to seed 50 ml of SD media pre-warmed to 30°C in a 250-ml Erlenmeyer flask, which was placed in a 30°C shaking incubator at 225 rpm for approximately 12 hours. This secondary seed was then used to inoculate either a 1.5-L Erlenmeyer flask with side arms for parallel batch fermentations or a 1.0-L bioreactor (CelliGen® Plus, New Brunswick Scientific Co., Inc., Edison, NJ, USA). Cultures for aerobic, glucose-limited, fed-batch growth were initially grown in a batch mode, and a specific substrate limited after the culture reached particular biomass concentrations in each respective experimental condition. All batch culture experiments were performed in our multiple fermentation system which consists of acrylic enclosures filled with de-ionized water that can accommodate 32 cultures in parallel in batch operation mode at volumes ranging from 100 mL to 1500 mL capacity. We used this setup with either shaker's flasks as reactor vessels. A magnetic agitator (Bellco Glass, Inc., Vineland, NJ, USA) was used to continually mix flask contents at a speed of 225 rpm, and each flask was sealed with a rubber stopper containing apertures for probes, nutrient inlets/outlets, and sample harvesting. Temperature is strictly and uniformly controlled using a water circulator (model C10, Thermo Haake, Portsmouth, NH) with a temperature control module that drives a closed circuit of water to and from the controller to inlet and outlet drains on the water bath. Dissolved oxygen is measured and controlled using a polarographic electrode connected to DO meters/controllers. The fed-batch S. cerevisiae cultivations were automatically controlled in the 1-L bioreactor (CelliGen® Plus, New Brunswick Scientific Co., Inc., Edison, NJ, USA). It has its own controllers for temperature, pH and dissolved oxygen (DO). A Pentium II computer (233 MHz processor, Microsoft Windows 98) equipped with an AT-MIO-16E-10 Analog Input computer interface board (National Instruments Corp., Austin, TX, USA) was used for data acquisition and process control for both the multiple fermentation system and CelliGen® Plus bioreactor. Data from the batch and fed-batch cell cultures, including pH, temperature, and dissolved oxygen concentration were acquired through the interface board. The real-time graphical data acquisition and process control programs was written in LabVIEW 6.0 (National Instrument Corp., Austin TX). Media fed to the bioreactor was controlled by a feeding pump (Masterflex Computerized Drive 7550-90, Cole-Parmer Instrument Co., Chicago, IL, USA), with a RS 232 serial link accepting control signal from the computer, for fed-batch cell cultivation process. Acquisition of dissolved oxygen (DO) data was obtained with the aid of a respirometer [40] using a dissolved oxygen probe (Cole-Parmer Instrument Co., Chicago, IL, USA). For all the experiments, temperature was controlled at 30°C. Determination of respiratory quotient (RQ) The ratio of carbon dioxide evolution rate (CER) to oxygen uptake rate (OUR) has previously proven useful in inferring a lack of substrates in the growth medium and in the calculation of feeding rates [41]. For the fed-batch experiment, compressed air was fed into the bioreactor through a gas flowmeter (Manostat 125, New York, NY, USA), which was manually adjusted to a flow rate of 100 mL/min. The composition of exhaust gas from the bioreactor was measured using a gas analyzer (1440C Gas Analyzer, Servomex Co., Inc., Norwood, MA, USA) connected to the interface board to gauge exiting O2 and CO2 levels. Calculations for CER, OUR, and RQ were performed using the equations: where O2, in and CO2, in and O2, out and CO2, out are the oxygen and carbon dioxide fractions in % v/v in the inlet air and exiting gas measurements, respectively, Qin and Qout are the air flow rates, and Vm is the working volume of the bioreactor. When the estimated RQ reached 1.06, a peristaltic pump (Cole-Parmer) was utilized to begin feeding 10X concentrated growth medium into the bioreactor, and this quotient was maintained by an RQ-stat control strategy to limit the production of by-products or consumption of these by-products as an alternative energy source. Sampling procedures During cultivations, two separate 1-ml aliquot samples were taken at early, mid, and late log-phase from both the bioreactor and flasks. The first aliquot was used to determine cell density by measuring the optical density A600, A420, using a spectrophotometer (Beckman DU640, Beckman Coulter, Inc., Fullerton, CA, USA), and cell counts (Coulter Electronics Inc., Hialeah, FL, USA). The second aliquot was then filtered on a manifold containing a dry, pre-weighed, 0.2 μm pore-size filter to isolate a cell pellet, and was washed three times with 250 ml of sterile deionized water to ensure all salts were removed. After washing, the filters are placed in aluminum foil inside a 65°C incubator for 24 hours and subsequently weighed in an analytical balance to measure cell dry weight. The second aliquot was filtered through a 0.45 μm acrodisc syringe filter to separate cells from supernatant. The concentrations of metabolites in the supernatant such as glucose, acetate, ethanol, and glycerol were determined by using enzyme-based assay kits (glucose and acetate assay kit, Sigma Chemical Co., St. Louis, MO, USA; ethanol and glycerol assay kit, R-Biopharm, Inc., Marshall, MI, USA). Growth rate, specific uptake/production rates and OURflask All specific growth rate curves were obtained by a linear regression of all data points within the exponential growth phase using the following formula X = Xoeμt, where X is the cell concentration (gL-1), Xo is the initial inoculum cell concentration (gL-1), t is the time of inoculation, and μ is the specific growth rate (1 hr-1). A minimum of ten optical density measurements were needed for the growth rate determination for both batch and fed-batch cultures. The specific glucose uptake rate (GUR), ethanol and acetate formation rates, and OURflask (OUR for the batch culture using flasks) were determined by fitting the dynamic mass balance equations for glucose, ethanol, acetate and DO measurements to the data points spanning the time period of the exponential cell growth phase. The specific uptake and production rates were then calculated by solving the dynamic mass balance equation within the culture medium using the following equation: where V (L) is the culture volume, [S] (mM) is the substrate/product or DO concentration in the flask, q (mmole/g-dry weight/hr) is the substrate uptake rate or by-product formation rate or OURflask, and X(t) (g-dry weight/L) is the biomass concentration at time = t (hr). + is for the by-product formation and - is for the substrate consumption. The solution to this equation was fitted to the experimental data points using the nonlinear estimation routine in Statistica (StatSoft Inc, Tulsa OK) or the solver in Microsoft Excel. All data, to be considered valid and included in the analysis, must have correlation coefficients of 0.95 or greater. The data that were generated in this way represented the "pseudo-steady-state" [42] of the batch or fed-batch cell culture, and thus suitable for the calculation of growth rate, specific uptake and production rates, and OURflask. In silico calculations Flux balance analysis and linear programming A genome-scale S. cerevisiae metabolic network has been reconstructed [19]. The network includes 733 metabolites and 1175 metabolic reactions, which are compartmentalized between the cytosol and the mitochondria. In metabolic network analysis, the relationship between metabolite concentrations, x, and reaction activities, v, is described by the dynamic mass balance equation [43,44]: where S is an m × n matrix of stoichiometric coefficients, x is an m × 1 vector of metabolite concentrations, and v is and n × 1 vector of reaction activities. Thus, the rows of S correspond to the internal metabolites and the columns represent the reactions in the network. Under steady-state conditions, the dynamic mass balance equation simplifies to: S • v = 0     (Eq. 6) Since the number of reactions is often greater than the number of metabolites, Eq. 6 is underdetermined and contains multiple solutions. One approach to solving Eq. 6 for microbial networks is to define a set of inputs and outputs that correspond to the growth conditions and use linear optimization to maximize the cell growth [35]. This approach has been successful in capturing the phenotypic behavior of S. cerevisiae for various growth conditions [21]. Phenotypic phase plane (PhPP) formulation The S. cerevisiae PhPP displays optimal growth rates for all possible variations in two constraining environmental variables, such as the carbon substrate and oxygen uptake rates. In this study, the glucose uptake rate (x-axis) was allowed to vary from 0 to 20 mmol/gDCW/hr and the oxygen uptake rate (y-axis) ranged from 0.1 to 20 mmol/gDCW/hr. The oxygen uptake rate was not allowed to reach zero because anaerobic simulations required additional supplements to maintain cell growth (ergosterol and zymosterol). Linear programming was used to calculate the optimal growth rate for all points in the x-y plane. Growth rate values were then plotted as the z-axis to form the surface of a three-dimensional PhPP (Fig. 1a). A two-dimensional PhPP was formed by projecting the 3-D PhPP onto the x-y axis (Fig. 1b). The phases of the PhPP were determined by the calculation of shadow prices [46], which describe the sensitivity of the objective function (Z) to changes in the availability of each metabolite: where bi is the ith metabolite and γi is the ith shadow price. Shadow prices were calculated for each point in the x-y plane during the linear programming simulations. By definition, phases were identified as regions of the PhPP in which all of the points have the same shadow prices. Lines of optimality, which represent the optimal ratio of glucose and oxygen uptake rates for maximal biomass synthesis, were also identified using shadow price analysis [45]. Shadow price analysis and in silico gene deletions To obtain a physiological interpretation of the differences between the oxidative fermentative phases (phases 2–6), we analyzed how the shadow prices of key metabolites changed across the phase boundaries. Simulations were run at a fixed glucose uptake rate of 5 mmol/gDCW/hr and an oxygen uptake rate ranging from 1.5 to 15 mmol/gDCW/hr. The sign of the shadow price was used to identify whether a small change in the metabolite's availability would positively or negatively affect the objective value. According to the convention defined in [45], a negative shadow price indicates that a metabolite is limiting, e.g. the value of the objective function increases if the metabolite's net production increases or its net consumption decreases. Similarly, a positive shadow price indicates that a metabolite is available in excess and a shadow price equal to zero indicates that a change in the availability of the metabolite does not affect the objective value. Phases 2 – 6 were also characterized by performing gene deletions in silico (as described in [20]). Single genes were deleted at a representative point within each phase to determine which reactions were essential for viability in that region. Secretion profile calculations The first step in generating the secretion profile was to calculate the optimal growth rate for a given glucose and oxygen uptake rate. For the simulations in Figures 2a and 2b, the glucose uptake rate was fixed at 5 mmol/gDCW/hr and the oxygen uptake rate varied from 0 to 16 mmol/gDCW/hr (ergosterol and zymosterol uptake rates of 5.92 × 10-5 and 1.27 × 10-4 mmol/gDCW/hr, respectively, were used for the calculation at OUR = 0). The simulations were then re-run with a fixed glucose uptake rate, oxygen uptake rate, and growth rate to determine the maximum and minimum secretion rates of each metabolite with a shadow price equal to zero. Authors' contributions NCD calculated the phenotypic phase plane (Fig. 1), carried out the secretion profile simulations (Fig. 2), characterized the phases, calculated the flux predictions for Table 1, and drafted the manuscript. BOP conceived the study, participated in its design and coordination, and assisted with manuscript preparation. PF designed the study and conducted the growth experiments (Fig. 3, Table 1) and revised the manuscript. All authors have read and approved the final manuscript. Acknowledgements The authors thank Dr. Uwe Sauer, Dr. John Leslie, Mr. Scott McCuine and the anonymous reviewer for their useful comments on the manuscript. They would also like to acknowledge Dr. Radhakrishnan Mahadevan and Dr Markus Herrgard for insightful discussions on phase characterization. Support for this work was provided by the National Science Foundation (MCB98-73384 and BES98-14092) and the National Institutes of Health (GM57089). The authors and the University of California, San Diego, disclose potential financial conflict of interest related to the US Patent Application (Pub. No: US2003/0228567 A1), published on December 11, 2003. ==== Refs Brown PO Botstein D Exploring the new world of the genome with DNA microarrays Nature Genetics 1999 21 33 37 9915498 10.1038/4462 Fraser CM Eisen JA Salzberg SL Microbial genome sequencing Nature 2000 406 799 803 10963611 10.1038/35021244 Zhu H Bilgin M Snyder M Proteomics Annu Rev Biochem 2003 72 783 812 14527327 10.1146/annurev.biochem.72.121801.161511 Ge H Walhout AJ Vidal M Integrating 'omic' information: a bridge between genomics and systems biology Trends Genet 2003 19 551 560 14550629 10.1016/j.tig.2003.08.009 Palsson BO In silico biology through "omics" Nat Biotechnol 2002 20 649 650 12089538 10.1038/nbt0702-649 Kitano H Systems biology: a brief overview Science 2002 295 1662 1664 11872829 10.1126/science.1069492 Nissen TL Schulze U Nielsen J Villadsen J Flux distributions in anaerobic, glucose-limited continuous cultures of Saccharomyces cerevisiae Microbiology 1997 143 203 218 9025295 Ostergaard S Olsson L Nielsen J In vivo dynamics of galactose metabolism in Saccharomyces cerevisiae: metabolic fluxes and metabolite levels Biotechnol Bioeng 2001 73 412 425 11320512 10.1002/bit.1075 Vanrolleghem PA De Jong-Gubbels P Van Gulik WH Pronk JT Van Dijken JP Heijnen S Validation of a metabolic network for Saccharomyces cerevisiae using mixed substrate studies Biotechnology Progress 1996 12 434 448 8987472 10.1021/bp960022i Visser D van der Heijden R Mauch K Reuss M Heijnen S Tendency modeling: a new approach to obtain simplified kinetic models of metabolism applied to Saccharomyces cerevisiae Metab Eng 2000 2 252 275 11056067 10.1006/mben.2000.0150 Lei F Rotboll M Jorgensen SB A biochemically structured model for Saccharomyces cerevisiae J Biotechnol 2001 88 205 221 11434967 10.1016/S0168-1656(01)00269-3 Hynne F Dano S Sorensen PG Full-scale model of glycolysis in Saccharomyces cerevisiae Biophys Chem 2001 94 121 163 11744196 10.1016/S0301-4622(01)00229-0 Rizzi M Baltes M Theobald U Reuss M In vivo analysis of metabolic dynamics in Saccharomyces cerevisiae .2. 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Biotech Prog 1987 3 121 126 Cysewski GR Wilke CR Rapid ethanol fermentations using vacuum and cell recycle Biotechnol Bioeng 1977 19 1125 1144 Sherman F Fink GR Hicks JB Methods in yeast genetics 1986 Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press Winston F Dollard C Ricupero-Hovasse SL Construction of a set of convenient Saccharomyces cerevisiae strains that are isogenic to S288C Yeast 1995 11 53 55 7762301 Ibarra RU Fu P Palsson BO DiTonno JR Edwards JS Quantitative analysis of Escherichia coli metabolic phenotypes within the context of phenotypic phase planes J Mol Microbiol Biotechnol 2003 6 101 108 15044828 10.1159/000076740 Bajpai R Control of bacterial fermentations Annual New York Academy of Science 1987 506 446 456 Horn F Jackson R General mass action kinetics Arch Rational Mech Anal 1972 47 81 116 10.1007/BF00251225 Reich JG Sel'kov EE Energy metabolism of the cell : a theoretical treatise 1981 London ; New York: Academic Press Briggs GE Haldane JBS A note on the kinetics of enzyme action Biochem J 1925 19 338 339 16743508 Edwards JS Covert M Palsson B Metabolic modelling of microbes: the flux-balance approach Environmental Microbiology 2002 4 133 140 12000313 10.1046/j.1462-2920.2002.00282.x

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==== Front BMC NeurosciBMC Neuroscience1471-2202BioMed Central London 1471-2202-5-331536959910.1186/1471-2202-5-33Research ArticleNestin-positive mesenchymal stem cells favour the astroglial lineage in neural progenitors and stem cells by releasing active BMP4 Wislet-Gendebien Sabine [email protected]ère Françoise [email protected] Grégory [email protected] Pierre [email protected] Gustave [email protected] Bernard [email protected] Centre for Cellular and Molecular Neurobiology, University of Liège, Liège, Belgium2 Department of Neurology, C.H.U. of Liège, Sart Tilman, Belgium2004 15 9 2004 5 33 33 11 5 2004 15 9 2004 Copyright © 2004 Wislet-Gendebien et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Spontaneous repair is limited after CNS injury or degeneration because neurogenesis and axonal regrowth rarely occur in the adult brain. As a result, cell transplantation has raised much interest as potential treatment for patients with CNS lesions. Several types of cells have been considered as candidates for such cell transplantation and replacement therapies. Foetal brain tissue has already been shown to have significant effects in patients with Parkinson's disease. Clinical use of the foetal brain tissue is, however, limited by ethical and technical problems as it requires high numbers of grafted foetal cells and immunosuppression. Alternatively, several reports suggested that mesenchymal stem cells, isolated from adult bone marrow, are multipotent cells and could be used in autograft approach for replacement therapies. Results In this study, we addressed the question of the possible influence of mesenchymal stem cells on neural stem cell fate. We have previously reported that adult rat mesenchymal stem cells are able to express nestin in defined culture conditions (in the absence of serum and after 25 cell population doublings) and we report here that nestin-positive (but not nestin-negative) mesenchymal stem cells are able to favour the astroglial lineage in neural progenitors and stem cells cultivated from embryonic striatum. The increase of the number of GFAP-positive cells is associated with a significant decrease of the number of Tuj1- and O4-positive cells. Using quantitative RT-PCR, we demonstrate that mesenchymal stem cells express LIF, CNTF, BMP2 and BMP4 mRNAs, four cytokines known to play a role in astroglial fate decision. In this model, BMP4 is responsible for the astroglial stimulation and oligodendroglial inhibition, as 1) this cytokine is present in a biologically-active form only in nestin-positive mesenchymal stem cells conditioned medium and 2) anti-BMP4 antibodies inhibit the nestin-positive mesenchymal stem cells conditioned medium inducing effect on astrogliogenesis. Conclusions When thinking carefully about mesenchymal stem cells as candidates for cellular therapy in neurological diseases, their effects on resident neural cell fate have to be considered. ==== Body Background During development of the central nervous system (CNS), all types of neuronal and macroglial cells derive from neuroepithelial neural stem cells (NSCs) [1,2]. NSCs self-renew and continue to function as source of new cells in adults [3,4]. The fate determination of neural stem cells is regulated by cell-intrinsic programs as well as extrinsic cues from the surrounding environment [5]. In the adult, the molecular mechanisms that regulate the production of new neurons in the dentate gyrus or in the subventricular zone are still unknown, although extrinsic factors expressed for example by astrocytes could play a role [6]. BMPs (Bone Morphogenetic Proteins) are secreted members of the TGF-β superfamily. Together with their receptors, they are abundantly expressed in the brain both during embryogenesis and in the adult [7-9]. More specifically, an expression of BMP4 was demonstrated in different cell types like ectodermal cells [10], radial glia [11], haematopoietic cells [12], chondrocytes [13] and stromal cells [14]. As for other members of the TGF-β superfamily, BMPs signal transduction is triggered by binding to type 1 and type 2 serine-threonine kinase receptors, inducing their dimerization. In this manner, BMP2 and BMP4 signalling specifically leads to the formation of BMPR-1A/BMPR-II and BMPR-1B/BMPR-II heterodimers [15]. BMPs have been shown to play a role in patterning and cellular fate determination in many tissues [16]. For examples, during development, BMPs are involved in the induction of the neuroectoderm, the patterning of the dorsal roof of neural tube, the development of neural crests and of the peripheral nervous system [10,17-20]. In postnatal animals, they promote the differentiation of neuronal precursors in the spinal cord [21] and in the cortex [22,23]. Finally, they facilitate an astroglial lineage commitment of forebrain subventricular zone progenitor cells [24]. Cellular therapies using stem cells are promising approaches for the treatment of several chronic or acute neurological diseases such as Parkinson's [25] or Huntington's diseases [26] or spinal cord injuries [27]. One main problem relates to the origin and the nature of the cells to be used for such procedures. Foetal brain tissue has already been shown to have significant effects in patients with Parkinson's disease. Clinical use of the foetal brain tissue is, however, limited by ethical and technical problems as it requires high numbers of grafted foetal cells and a possible immunosuppression. Alternatively, somatic stem cells derived from adult tissues seem to be better candidates for cell replacement therapy [28,29]. These observations raise hopes in cell replacement strategies based on an autograft approach. However, the exact mechanism by which mesenchymal stem cells (MSCs) adopt a neural fate is not completely understood as recent in vivo studies demonstrated that MSCs fuse with host neuronal cells [30,31]. These observations impose a better knowledge of the mechanisms underlying the phenotypic plasticity of somatic stem cells and the characterization of their daughter differentiated cells is a prerequisite before considering their use in the treatment of human patients. Recently, we demonstrated that two phenotypes of MSCs could be obtained in culture: nestin-positive MSCs (npMSCs) which are able to integrate some extrinsic signals when co-cultured with neurons leading to a differentiation into astrocyte-like cells and nestin-negative MSCs (nnMSCs) which are unable to adopt a neural phenotype but remain able to differentiate into adipocytes, chondrocytes or osteocytes [32]. When considering the use of MSCs as a source of material for cell replacement protocols in neurological diseases, one has also to be concerned by a possible effect of these MSCs on the host nervous tissue and more particularly on immature resident neural cells. In several models of neurological diseases, grafted MSCs were shown to favour host CNS regeneration rather than to express themselves a neural phenotype [33,34]. This positive effect of MSC grafts could result from the release of factors acting on resident immature cells in the adult brain. Recently, it has been demonstrated that following ischemia in the adult striatum, intra-ventricular EGF injections are able to stimulate neurogenesis from resident neural stem cells, although EGF is devoid of any effect in non-ischemic striatum [35]. These observations emphase the importance of the lesion priming in order to respond to extrinsic factors or cytokines. In this paper, we address the question of the influence of MSCs on neural stem cells in vitro and demonstrate that MSCs favour astroglial lineage. We observe that npMSCs are able to stimulate astroglial fate in striatal progenitor cultures and to repress neuronal and oligodendroglial fate through the release of diffusible factor(s). Using BrdU incorporation, we demonstrate that this npMSC conditioned medium has no effect on the astrocytic or oligodendrocytic progenitor proliferation. Propidium iodide incorporations suggest that the npMSCs conditioned medium protect GFAP-positive cells from cell death in comparison to the effect of nnMSCs conditioned medium or to the control condition. Meanwhile, no increase of cell death is observed in neuronal and oligodendroglial populations. We then demonstrated that BMP4 is present in a biologically-active form in the npMSCs but not in nnMSCs conditioned medium and is responsible for both the increase of astroglial numbers and the inhibition of oligodendrocyte differentiation in striatal NSC cultures. Results Nestin-positive MSCs increase astrocytes number in differentiating neural stem cell cultures Neurosphere-derived cells from GFP-positive E16 green mouse striata include neural stem cells and progenitors which are still proliferating but already committed to a given cell fate. Upon transfer on adherent surfaces (poly-ornithine coated dishes), these cells spread and spontaneously differentiate as follows after 5 days of culture: 44.2 ± 2.5% GFAP-positive cells (astroglial cells), 15 ± 2.1% Tuj1-positive cells (neuronal cells) and 5.86 ± 0.6% 04-positive cells (oligodendroglial cells). When co-cultivated with nestin-positive MSCs (npMSCs), phenotypic allocation of neurosphere-derived cells (identified as GFP-positive cells) become strikingly different: GFAP labelling is increased to 78.5 ± 3.9% (Fig. 1A) (n = 12, ***Student T-test, P < 0.001), while Tuj-1 and O4 are decreased to respectively 3.2 ± 1.1% (Fig. 1B) (n = 12, ***Student T test, P < 0.0001) and 2.9 ± 0.5% (Fig. 1C) (n = 8, ***Student T test, P < 0.0001). On the other hand, if neurospheres are co-cultured with nestin-negative MSCs (nnMSCs), the percentage of labelled cells were 52.1 ± 2.9%, 4.7 ± 0.8% and 7.8 ± 1.5 for respectively GFAP, Tuj-1 and O4. These numbers are not significantly different from the numbers obtained in control cultures for GFAP- and O4-positive cells (n = 8, Student T test, P > 0.05), while significantly lower for Tuj-1-postive cells (n = 8, ***Student T test, P < 0.001, Fig. 1G). MSCs effect on neurospheres-derived astrocytes number is due to a soluble factor To further characterize the mechanism (soluble factor versus membrane-bound factor) responsible for such an increase in astrocytes number in presence of npMSCs, we tried to induce the differentiation of neurospheres with npMSC conditioned medium. The following data were obtained: 77.5 ± 2.5% of the neurosphere-derived cells became GFAP-positive, 4.3 ± 0.9% were Tuj1-positive and 1.5 ± 0.6% were 04-positive (n = 8, Fig. 2A,2B,2C). Conversely, in nnMSCs conditioned medium, differentiated phenotypes were distributed as follows: 56 ± 3.5%, 3.7 ± 1.3% and 5.9 ± 1.3% (n = 7, Fig. 2G) and were similar to results obtained in corresponding co-cultures. Moreover, we confirmed those results by absolute count: 1) in npMSCs conditioned medium 392/504 cells were GFAP-positive, 9/504 cells were O4-positive and 32/504 cells were Tuj1-positive ; 2) in nnMSCs conditioned medium 251/515 cells were GFAP-positive, 29/515 were O4-positive and 23/515 were Tuj1-positive; 3) in the control condition (DEM/F12 + B27), 211/531 cells were GFAP-positive, 27/531 cells were O4-positive and 167/531 cells were Tuj1-positive. Hence, regarding the phenotypic allocation, no significant difference is observed between neurosphere differentiated in co-culture and in conditioned medium. (Student T test, P > 0.05). This clearly suggest that npMSCs-derived soluble factor(s) is (are) responsible for an increase in the astrocytes number and a decrease in the neurons and oligodendrocytes numbers. Nestin-positive MSC conditioned medium act on the GFAP-positve cell death As mentioned above, neurosphere derived-cells include neural stem cells and progenitors which are still proliferating but already committed to a given cell fate. We characterised the proportion of each progenitor cell-type present in our neurospheres. After dissociation, cells were allowed to adhere for one hour in coated dish and then fixed, labelled and counted. In those conditions we observed 14.4 ± 7.1% of GFAP-positive cells, 7.1± 4.1% of Tuj1-positive cells and 3.4 ± 1.6% of O4-positive cells (these data were obtained by absolute counts on 1576 cells, Fig. 3A). In order to define if npMSCs conditioned medium has an instructive or/and a selective effect on neural cells, we analysed the proliferative capacity and the cell death in the differentiating neurospheres. The BrdU incorporation in differentiating neurosphere-derived cells cultivated in npMSCs- or in nnMSCs-conditioned medium or in control (non-conditioned) medium (DEM/F12 + B27) did not show any significant differences (Statistical test ANOVA, P > 0.05, Fig. 3B,3C). These data rule out a proliferative effect on already committed astrocyte progenitors or an inhibition of cell proliferation in oligodendroglial and neuronal progenitors. Furthermore, using propidium iodide incorporations and counting, we quantified the cell death in GFAP-, Tuj1- and O4-positive cell population during the differentiation culture in the three conditioned media (by npMSCs, by nnMSCs and non-conditioned). After 48 hours, we observed a significant decrease of the number of GFAP-positive cells which have incorporated the propidium iodide in npMSCs-conditioned medium (***Student T test, p < 0.0001) in comparison to the two other culture condition. This result suggests that npMSC conditioned medium partially inhibit the cell death in the GFAP-positive cell population. Nevertheless, no significant increase of cell death is observed in O4- and Tuj1-positive cell population (Student T test, p > 0.05). When propidum iodide incorporations are performed after four days of differentiation without renewing the conditioned media, no significant differences in the cell death could be observed in the three lineages, whatever the culture condition (Fig. 3D,3E). BMP4 is present in nestin-positive conditioned medium and is responsible for the increase of GFAP-positive cells in differentiating NSCs Previous reports have shown that several secreted growth factors, including BMP2, BMP4, LIF and CNTF stimulate the differentiation of cultured neural precursors into astrocytes [36-41]. The expression of those cytokines was compared in nnMSCs and in npMSCs using quantitative RT-PCR. Compared to nnMSCs, we found that npMSCs slightly increased the expression level of BMP4 mRNA (190 ± 26%) while the BMP2 (36 ± 7%) and LIF (50 ± 10%) expression is slightly decreased (Fig. 4A). No significant difference was observed concerning the CNTF mRNA expression. Western blotting analyses of npMSCs, nnMSCs and neurosphere-derived cells conditioned media showed that only npMSCs release in their culture medium the mature and biologically-active form of BMP4 (27 kDa), although nnMSCs and neurosphere-derived cells expressed the biologically-inactive precursor form of BMP4 (57 kDa) (Fig. 4B). Finally, we observed that neurospheres-derived cells cultivated in npMSCs conditioned medium supplemented with an anti-BMP4 blocking antibodies, differentiate into 47.1 ± 1.5% GFAP-positive cells, 9.9 ± 0.9% Tuj1-positive cells and 5.7 ± 0.5% O4-positive cells (Fig. 4C). Statistical analyses did not show significant differences in GFAP- or O4-positive cells compared to control culture conditions (student T test, p > 0.05, n = 5). However, the number of Tuj1-positive cells remains significantly lower (***student T test, p < 0.001, n = 5). We thus conclude that the major glial effects (increase of astrocytes and decrease of oligodendrocytes) of the npMSCs on the neural progenitor differentiation is due to the release of biologically-active BMP4. The inhibition of neuronal differentiation should be attributed to another yet uncharacterized soluble factor(s). Discussion During the last few years, a number of studies have addressed the phenotypic plasticity of MSCs. Most of them were performed in vivo and demonstrated that environmental factors play important roles in determining the ability of grafted MSCs to adopt a neural-like phenotype. Grafting of a subset of MSCs into the lateral ventricle of neonatal mice resulted in their migration within the forebrain and cerebellum, and their differentiation into astrocytes [42]. When MSCs were grafted into adult rat cerebellum after an ischemic lesion, they differentiated into cells expressing neuronal markers [43]. Nakano et al. [44] demonstrated that murine bone marrow cells differentiated into three distinct glial phenotypes (oligodendrocytes, astrocytes and microglia) when they were directly injected into the striatum of previously irradiated mice. Similarly, systemic injection of MSCs in lethally irradiated mice allowed their differentiation into neuronal and astroglial cells [45]. Interestingly, the systemic injection of MSCs in non-irradiated but brain-lesioned mice had positive effects on injury repair, but very few MSC-derived cells expressed neural marker in such conditions [22,33,34,46,47]. Given those results, somatic stem cells raise thus hopes in cell replacement strategies based on an autograft approach. Recent in vivo studies [30,31] demonstrated that MSCs adopt neural fate by fusion with host neural cells. At least so far, there is no a clear and conclusive demonstration of a real differentiation of MSCs in neural cells. However, all those data obtained in vivo suggested that somatic stem cells seem to be promising for cellular therapy in neural diseases whatever the mechanism responsible for. It remains that a better knowledge of the molecular and cellular mechanism underlying the neural phenotypic plasticity of MSCs is required before considering those cells in human graft protocols. As we mentioned above, environment is able to modify the cellular differentiation capacity. In this study, we addressed the question of a possible effect of MSCs on neural progenitor cell fate and we choose an in vitro approach. Co-culture experiments demonstrate that MSCs display multiple activities in the regulation of embryonic striatum-derived progenitors and stem cells. Their effect mainly depends of their age in vitro: npMSCs (more than 10 passages in vitro or 25 doubling cell populations) increase the astrocytes numbers while inhibiting neuronal and oligodendroglial numbers. Conversely, nnMSCs (with a maximum of 5 passages in vitro) only decrease the neuronal differentiation. Since similar results were obtained using MSC conditioned media, we concluded that these effects could be mediated by diffusible factor(s). We then analysed the effect of various conditioned media on the cell proliferation (using BrdU incorporation) as well as the cell death (using propidium iodide incorporation) in each neurosphere-derived cell types and at two different differentiation period (48 or 96 hours). We only observed a significant decrease of propidium iodide incorporation in GFAP-positive cell population at 48 hours of differentiation suggesting that soluble factor(s) select astroglial lineage by protecting those cells from cell death. The activity which drives neurosphere-derived cells into astrocytes only occurs in npMSC conditioned medium while the neuronal differentiation-inhibiting activity is present both in np- and nnMSC conditioned media. We tried to identify the astrogliogenetic factor(s) by measuring the expression by nn- and npMSCs of cytokines known to promote an astroglial fate [36-41]. Our results show that only npMSCs express mature and biologically-active BMP4 while a biologically-inactive BMP4 precursor form is expressed and released by nnMSCs and neurosphere-derived cells in culture. Indeed, BMP4 is synthesized and released as an inactive precursor before being proteolytically activated by cleavage at the amino acid motif -Arg-Ser-Lys-Arg- [48]. Relatively little is known about the regulatory mechanisms controlling the susceptibility of individual TGF-β family members to proteolytic cleavage. However, recent studies suggest that members of the subtilisin-like proprotein convertase (SPC) family, SPC1 and SPC4, could enhance the cleavage of BMP4 precursor. The availability of biologically active BMPs may therefore be controlled by the released of their precursor followed by the action of the proprotein convertases [49,50]. The release by npMSCs of biologically-active form of BMP4 which promotes astroglial differentiation and inhibits oligodendroglial differentiation is consistent with previous studies demonstrating that BMPs play multiple roles in development [8]. Other studies have reported that the effects of BMPs are age- and tissue-dependent [51,52] and that BMPs promote astroglial differentiation and inhibit oligodendroglial differentiation when applied to cultures of cortical cells plated at E16 [23]. Likewise, brief treatment with BMPs induces astroglial fate in cultured neural precursors from embryonic mice [37] or in oligodendrocytes from newborn rats [51]. More recently, Rajan et al. [41] demonstrated that BMP4 induces astroglial differentiation of E14 and adult cortical neural stem cells from the subventricular zone when they are placed in high density culture. In this case, BMP4 has a true instructive role as NSCs cultures were clonal. Beside the instructive effect of BMP4 on glial lineages, some studies explained how BMP4 could act on astrocytic and oligodendrocytic precursors. A study realised on cerebellar primitive neurectodermal tumor cell line demonstrated that a high concentration of BMP2 and BMP4 attenuate apoptosis [53]. BMP-mediated inhibition of oligodendrogenesis is controlled through the repression of the former transcription factor olig2 known to be essential for the oligodendrocytic development [54,55]. All of these data suggest that BMP4 released by npMSCs selectively act on astrocytic and oligodendrocytic progenitors. In our experiments, we only demonstrate the anti-apoptotic effect of npMSCs-conditioned medium but we didn't test its possible instructive effect in clonal cultures. As BMP4, present in a biologically-active form in npMSCs conditioned medium, has been identified to be responsible of the increase of astrocytes numbers by the immunoblocking experiment and as it has been already demonstrated to be instructive [41], one could hypothesise that, in our system, the increase of astrocytes in response to npMSCs-derived BMP4 is a consequence of both a anti-apoptotic effect on GFAP-positive cells and also an instructive effect on NSCs. Conclusions When considering the use of MSCs in cell replacement strategies for the treatment of various neurological diseases, it should be taken into account that those cells could also influence the development host neural precursors. Methods Preparation and culture of rat mesenchymal stem cells (MSCs) Adult rat bone marrow was obtained from femoral and tibial bones by aspiration and was resuspended into 5 ml of DEM (Invitrogen, Merelbeke, Belgium) [56]. Between 100 and 200 × 106 marrow cells were plated on 175-cm2 tissue culture flask in DEM/10% foetal bovine serum (Invitrogen). After 24 hours, the non-adherent cells were removed. When the rMSCs became confluent, they were resuspended with 0.25% Trypsin and 1 mM EDTA and then sub-cultured. Nestin expression by MSCs was induced as described in [32]. Preparation and culture of mouse striatal neural progenitor and stem cells Green C57BL/6 mice embryos (Jackson Immunoresearch Laboratory, Inc., West Grove, USA) or NMRI mice were used as a source of striatal neural progenitor and stem cells. Green mouse express GFP under control of the β-actin promoter [57]. The day of conception was determined by the presence of a vaginal plug (embryonic day 0). E16 striata were isolated and triturated in DEM/F12 (Invitrogen) with a sterile Pasteur pipette. The cell suspension was filtered with a 70 μm-mesh and viable cells were estimated by trypan blue exclusion. The cells were plated (1 × 106 cells/75-cm2 tissue culture flask) in DEM/F12 supplemented with EGF (20 ng/ml, Sigma) and B27 (Invitrogen), a multi-component cell culture supplement devoid of any growth factor. When the size of neurospheres reached approximately 50 cells, they were dissociated into a single cell suspension by trituration and replated in fresh culture medium. Neurospheres with a maximum of 3 passages were used in this study. Co-culture of MSCs and neurospheres MSCs and neurospheres were plated on polyornithine coated dishes for 5 days, in DEM/F12 containing only B27 supplement and were then processed for immunocytochemical analysis as described below. Culture of neurospheres in MSCs conditioned medium MSCs were placed in DEM/F12 medium supplemented with B27 (Invitrogen), during 3 days. The conditioned media were then filtered with 0.22 μm-pore filter before being replaced on plated neurospheres during 5 days. Immunocytochemistry The cultures were fixed with 4% (v/v) paraformaldehyde for 15 minutes at room temperature and washed 3 times in TBS buffer. They were then permeabilized in 1% Triton-X100 (v/v) for 15 minutes and washed 3 times in TBS buffer. Non-specific binding was blocked by a 1 hour treatment in TBST (TBS buffer with 0.1% Tween) containing defatted milk powder (30 mg/ml). The cells were then incubated for 1 hour at room temperature with either anti-glial fibrillary acidic protein (GFAP, Dako, mouse IgG, dilution 1:500), or Tuj1 (Molecular Probes, mouse IgG, dilution 1:1000), or O4 (Chemicon, mouse IgM, dilution 1:100) primary antibodies (diluted in blocking buffer). After 3 washes in TBS, cells were then incubated in FITC- or Cy5-conjugated anti-mouse IgG or IgM (Jackson Immunoresearch, 1:500) for 1 hour at room temperature and in the dark. The nuclei were stained with ethidium homodimer (0.2 μM, Sigma). The preparations were then mounted in Fluoprep™ (Biomerieux; France) and observed using a Bio-Rad MRC1024 laser scanning confocal microscope. The fraction of positive cells was determined by counting 10 non-overlapping microscopic fields (±50 cells/field, ±3500 cells/experiments) for each coverslip in at least three separate experiments (n then corresponds to the number of coverslips). BrdU and propidium iodide incorporation After 24 hours or 3 days of culture, BrdU (20 μM, Sigma) which is a S-phase marker, or propidium iodide (400 mg/ml, Sigma) was added to the differentiating neurosphere cultures for 18 hours before fixation and staining. Tuj1, O4 and GFAP immuno-labellings were performed as described above. For BrdU labelling, coverslips were then post-fixed for 10 minutes in 4% (v:v) paraformaldehyde, incubated in HCl 1 N for 20 minutes at 37°C, washed with sodium perborate solution (50 mM, pH 8.5) and finally incubated with an anti-BrdU antibody for 1 hour at room temperature (Oxford, rat IgG, dilution 1:200) and Cy-5-conjugated anti-rat antibody, 1 hour at room temperature. The preparations were analysed as describe above. RNA extraction and quantitative RT-PCR analysis Total RNA was prepared using the RNeasy total RNA purification kit (Qiagen, Westburg). For cDNA synthesis, random hexamer primers (Invitrogen) were used to prime reverse transcriptase reactions. The cDNA synthesis was carried out using Moloney-murine leukemia virus (M-MLV) Superscript II Reverse transcriptase (Invitrogen) following the manufacturer's instructions. Quantitative PCR was carried out using standard protocols with Quantitec SYBR Green PCR Kit (Qiagen). The PCR mix contained SYBR Greeen Mix, 0.5 μM primers (Table 1), 1 ng DNA template and nuclease free water to final volume of 25 μl. PCR were performed on RotorGene RG-3000 (Corbett Research) and analyzed with Rotorgene Software (Corbett Research). The percentage of gene expression by npMSCs was normalized in function of GAPDH gene expression and compared to the gene expression by nestin-negative MSCs that was considered as 100%. Each gene analysis was realised on three different samples with two run/sample (we then provided 6 PCR analyses by target gene) Western Blot After 72 hours of culture, the conditioned medium (1.5 ml) from cultures of neural progenitor cells, npMSCs and nnMSCs were incubated with heparin sepharose CL-6B (Amersham Biosciences, Belgium) at 4°C for 24 hours. After centrifugation (700 × g), the supernatant was removed and bound proteins were eluted in loading buffer (glycerol 10% v/v; Tris 0.05 M pH 6.8; SDS 2%, bromophenol blue and 2.5% v/v – mercaptoethanol) by heating at 70°C for 10 minutes. The protein concentrations of various samples were quantified using the "RC DC Protein Assay" (Bio-Rad, Belgium) and equal protein quantities were loaded in each lane in a 15% sodium dodecyl sulfate polyacrylamide gel and electrophoresed. Then the proteins were transferred to PVDF membrane (Amersham Biosciences, Belgium) using a Trans-Blot Semi-Dry Transfer apparatus (Bio-Rad). The membranes were saturated with 3% gelatin (BioRad) during 1 hour at 37°C, then incubated for 1 hour with a monoclonal antibody against BMP4 (R&D, goat IgG, 0.1 μg/ml) or actin, as control for protein loading (Sigma, mouse IgG 1:5 000) at room temperature and then washed several times with PBS-0.1% Tween. The membrane was then incubated in Cy5-conjugated anti-goat IgG or anti-mouse IgG (Jackson Immunoresearch, 1:2500) for 1 hour at room temperature, in the dark. After several washes in PBS, the membranes were scanned using a Typhoon 9200 Scanner (Amersham Biosciences) and subsequent analyses were performed with ImageQuant Software (Amersham Biosciences). BMP4 neutralization Neurospheres were plated on polyornithine-coated dishes and incubated during 5 days in DEM/F12 medium supplemented with B27 (Invitrogen) and previously conditioned by npMSCs during 3 days or not. Anti-BMP4 antibody (R&D, 2 μg/ml) was added on day 1 of this incubation. Authors' contributions SW performed most of the work and wrote a first draft of the manuscript; FB performed quantitative RT-PCR; GH, GM and PL were involved in the writing of the manuscript; BR conceived the study and participated in its design and coordination. All authors read and approved the final manuscript. Acknowledgements This work was supported by a grant of the Fonds National de la Recherche Scientifique (FNRS) of Belgium, by the Fondation Médical Reine Elisabeth (FMRE), by the Fonds Charcot, and by the Belgian League against Multiple Sclerosis. SW and FB are research fellow of the Télévie (FNRS). BR is senior research associate and PL research associate of the FNRS. We thank Patricia Ernst (CNCM, University of Liège, Belgium) for her competence in neurosphere cultures. Figures and Tables Figure 1 Nestin-positive MSCs increase astrocytes number in differentiating neural stem cell cultures. Neurosphere-derived GFP-expressing cells were co-cultivated with npMSCs or nnMSCs on polyornithine-coated dishes. After 5 days, the cells were fixed and immunostained with antibodies raised against GFAP (A, D, red), Tuj1 (B, E, red) and O4 (C, F, red). Nuclei were counterstained with EtD1 (blue). Co-cultures with npMSCs are illustrated in A (n = 12, ***Student T-test, P < 0.001), B (n = 12, ***Student T test, P < 0.0001) and C (n = 8, ***Student T test, P < 0.0001). Co-cultures with nnMSCs are illustrated in D (n = 8, Student T test, P > 0.05), E (n = 8, ***Student T test, P < 0.001) and F (n = 8, Student T test, P > 0.05). Stained cells counting results are shown in G and include the control condition of neurosphere-derived cells without co-culture. Scale bars = 20 μm in A-F. Figure 2 Humoral effect of nestin-positive MSCs. Neurosphere-derived GFP-expressing cells were cultivated with npMSC or nnMSC conditioned media, on polyornithine-coated dishes. After 5 days, the cells were fixed and immunostained with antibodies raised against GFAP (A, D, green), Tuj1 (B, E, green) and O4 (C, F, green). Nuclei were counterstained with EtD1 (red). Cultures with npMSC conditioned medium are illustrated in A, B and C. As observed in co-culture conditions, there is a significant increase of the number of GFAP-positive cells and significant decreases of the number of Tuj1-positive cells and O4-positive cells (n = 8, ***Student T test, P < 0.0001). Cultures with nnMSC conditioned medium are illustrated in D, E and F (n = 7, ***Student T test, P < 0.0001). Stained cells counting results are shown in G and include the control condition of neurosphere-derived cells without conditioned medium. No significant difference is observed between neurosphere differentiated in co-culture and in conditioned medium. (Student T test, P > 0.05). Scale bars = 20 μm in A-F. Figure 3 Nestin-positive MSC conditioned medium act on the GFAP-positive cell death but not on cell proliferation. (A) Characterization of committed cell types in proliferating neurospheres in presence of EGF. We observed that 14.4 ± 7.1%, 7.1± 4.1% and 3.4 ± 1.6% of cells were respectively GFAP-, Tuj1- and O4-positive (these data were obtained by absolute counts on 1576 cells). (B-C) The proliferative capacity of the differentiating neurosphere-derived cells placed in npMSC conditioned medium, nnMSC conditioned medium and in control medium (DEM/F12 + B27) was compared. BrdU incorporation was performed after 48 hours and 4 days in differentiating conditions. Double labelling GFAP-, Tuj1- and O4 with BrdU were performed. The BrdU incorporation by differentiating neurosphere-derived cells placed in the various conditions did not shown significant differences (Statistical test ANOVA, P > 0.05). (D-E) The cell death quantified by propidium iodide incorporation and counting was analysed after 48 hours and 4 days in differentiating conditions and in GFAP-, Tuj1- and O4-positive cells. After 48 hours, a significant decrease of the number of GFAP-positive cells which have incorporated the propidium iodide is observed (***Student T test, p < 0.0001). However, no significant increase of cell death was observed in O4- and Tuj1-positive cell population (Student T test, p > 0.05). After 4 days of differentiation, no difference in cell death is observed whatever the cell type or the culture condition. Figure 4 Release of an active form of BMP4 by nestin-positive MSCs. (A)RT-PCR and quantitative RT-PCR were performed on npMSCs and nnMSCs. The results of quantitative RT-PCR are expressed as percent of gene expression in npMSCs compared to nnMSCs (arbitrarily as 100%) after normalisation with the GAPDH house-keeping gene expression. (B) Western blotting analysis of conditioned media by npMSCs, nnMSCs and NSC. The mature and biologically-active form of BMP4 (27 kDa) is only observed in medium conditioned by npMSCs and the 57 kDa immature and non biologically-active form can be seen in nnMSC and in neural stem cells conditioned medium. This high MW form is not present in npMSC conditioned medium, indicating a complete cleavage and activation of BMP4. (C) Effect of anti-BMP4 antibodies in differentiating NSCs cultivated in DEM/F12 or npMSCs conditioned medium. Anti-BMP4 antibodies inhibit the npMSCs effect on astroglial and oligodendroglial cell numbers but not the effect on neurons number (47.13 ± 1.53% of GFAP-positive cells, 9.98 ± 0.92% of Tuj1-positive cells and 5.67 ± 0.45% of O4-positive cells with anti-BMP4 compared to respectively 77.5 ± 2.5%, 4.30 ± 0.95% and 1.47± 0.60% in npMSC conditioned-medium). Tuj1-positive cells remains significantly lower (***student T test, p < 0.001, n = 5). Table 1 Primer list used for quantitative RT-PCR Primers Sequence Tm BMP2 Forward CCAAGGTTAGTGACTCAGAACAC 52 BMP2 Reverse TCATCTTGGTGCAAAGACCTGC 52 BMP4 Forward ACTGCCCCACCTTCTCTGAG 50 BMP4 Reverse TTCTCCAGATGTTCTTCGTG 50 LIF Forward GCAACCTCATGAACCAGATC 52 LIF Reverse CAACTTGGTCTTCTCTGTCC 52 CNTF Forward ATTCGTTCAGACCTGACTGC 50 CNTF Reverse CCTGATGGAAGTCACCTTCA 50 GAPDH forward GACCCCTTCATTGACCTCAACTACATG 48–55 GAPDH Reverse GCCTTCTCCATGGTGGTGAACAC 48–55 ==== Refs Gage FH Mammalian neural stem cells Science 2000 287 1433 1438 10688783 10.1126/science.287.5457.1433 McKay R Stem cells in the central nervous system Science 1997 276 66 71 9082987 10.1126/science.276.5309.66 Alvarez-Buylla A Temple S Stem cells in the developing and adult nervous system J Neurobiol 1998 36 105 110 9712298 10.1002/(SICI)1097-4695(199808)36:2<105::AID-NEU1>3.3.CO;2-V van der Kooy D Weiss S Why stem cells? 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43 1 17 10756062 10.1002/(SICI)1097-4695(200004)43:1<1::AID-NEU1>3.3.CO;2-S Mehler MF Mabie PC Zhu G Gokhan S Kessler JA Developmental changes in progenitor cell responsiveness to bone morphogenetic proteins differentially modulate progressive CNS lineage fate Dev Neurosci 2000 22 74 85 10657700 10.1159/000017429 Iantosca MR McPherson CE Ho SY Maxwell GD Bone morphogenetic proteins-2 and -4 attenuate apoptosis in a cerebellar primitive neuroectodermal tumor cell line J Neurosci Res 1999 56 248 258 10336254 10.1002/(SICI)1097-4547(19990501)56:3<248::AID-JNR4>3.3.CO;2-L Agius E Soukkarieh C Danesin C Kan P Takebayashi H Soula C Cochard P Converse control of oligodendrocyte and astrocyte lineage development by Sonic hedgehog in the chick spinal cord Dev Biol 2003 270 308 321 10.1016/j.ydbio.2004.02.015 Mekki-Dauriac S Agius E Kan P Cochard P Bone morphogenetic proteins negatively control oligodendrocyte precursor specification in the chick spinal cord Development 2002 129 5117 5130 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==== Front BMC PharmacolBMC Pharmacology1471-2210BioMed Central London 1471-2210-4-191535300710.1186/1471-2210-4-19Research ArticleThe FK506 binding protein 13 kDa (FKBP13) interacts with the C-chain of complement C1q Neye Holger [email protected] Eugen J [email protected] KV Niedersachsen, Berliner Allee 22, D-30175 Hannover, Germany2 Institute of Medicinal Chemistry, Dept. of Pharmacology, Hittorfstr. 58-62, D-48149 Münster, Germany2004 7 9 2004 4 19 19 2 4 2004 7 9 2004 Copyright © 2004 Neye and Verspohl; licensee BioMed Central Ltd.2004Neye and Verspohl; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background The pharmacological action of specific immunosuppressants is mediated by immunophilins. While cyclosporin A binds to cyclophilins, FK506/tacrolimus, rapamycin, and others bind to FK506 binding proteins (FKBPs). Different physiological actions of immunophilins were described but their genuine function, however, remains elusive and is still under investigation. A yeast two-hybrid screen was performed using the FK506 binding protein 13 kDa (FKBP13) as a bait and a fetal liver expression library as a prey. Results The C-chain of complement C1q (C1q-C) was detected to interact with FKBP13 in the yeast two-hybrid system and in a protein complementation assay. Neither FKBP12, FKBP25, FKBP52 nor the unrelated immunophilin CypA did react with C1q-C in the yeast system stressing the specificity of the interaction. Binding of C1q-C to FKBP13 could not be prevented in the presence of FK506, demonstrating that possibly other regions than the binding pocket of the drug are responsible for the interaction of the two proteins. Conclusion It is concluded that exclusively FKBP13 but no other FKBPs tested so far interact with the C-chain of complement C1q in the two different assays and further work will be initiated to investigate the physiological relevance of the interaction. ==== Body Background The pharmacological action of cyclosporin A and FK506/tacrolimus is mediated by cytosolic immunophilins namely cyclophilin A and the FK506 binding protein 12 kDa (FKBP12). The drug immunophilin complex binds to and inhibits the protein phosphatase calcineurin thus preventing signal transduction in activated T cells [1]. FKBPs are a family of proteins that were found from procaryotes to humans. At least 10 human FKBPs are known and can be found in every tissue so far analyzed. Some FKBPs bind to and "stabilize" intracellular receptors. For example, FKBP12 and FKBP12.6 interact with the ryanodine Ca2+ channels RyR1 and RyR2, respectively [2,3]. FKBP52 is part of the steroid receptor complex [4]. Another FKBP, FKBP13, is localized in the endoplasmic reticulum. FKBP13 mRNA is up-regulated in the presence of unfolded proteins, e.g. after tunicamycin treatment and heat shock and it is regarded to act as a chaperone [5]. On the other hand FKBP13 was shown to interact specifically with single proteins like a homologue of the erythrocyte membrane cytoskeletal protein 4.1 and a FKBP associated protein 48 kDa [6,7]. Finally, FKBP25 was described to be a nuclear protein and the transcription factor YY1 or histone deacetylases (HDAC1 and HDAC2) specifically interact with this FKBP family member [8,9]. The phylogenetically ancient complement system composed of more than 30 proteins is part of the immunogenic system including a cascade of interacting proteins called C1 to C9. Activation finally leads to lysis of marked cells. C1, the first protein in this cascade, is composed of one C1q and two C1r and C1s proteins. While C1q binds the Fc regions of two IgGs or one IgM molecule, C1r and C1s are known to activate other components of the pathway, namely C4 and C2. C1q contains 18 polypeptide chains (six A-, six B-, and six C-chains) and is composed of six globular heads linked via six collagen like stalks to a fibril-like central region [10]. Each of the globular heads is formed by association of the C-terminal region of an A-, B-, and C-chain. In addition C1q seems to play an important role as an immunmodulator in its own and has pathophysiological impact. C1q deficient individuals develop a systemic lupus erythematodes (SLE)-like disease [11] and C1q deficient mice show elevated auto-antibody titres and develop glomerulonephritis and renal damage probably because of accumulation of apoptotic bodies [12]. As C1q can bind to a variety of pathologically relevant targets in an antibody-independent manner it might directly activate cellular functions. Indeed, several receptors for C1q have been described. C1qRp is a putative receptor for phagocytosis enhancement by monocytes [13]. The rodent homologue of C1qRp is tightly regulated during development [14]. C1q was shown to bind to cell lines expressing the complement receptor 1 (CR1) in a saturable manner [15]. A receptor for the collagenous domains of C1q has been purified and was shown to be idenical to calreticulin [16]. Finally, a binding protein for the globular head of C1q (gC1qbp) was isolated [17]. In this work interaction of the C-chain of complement C1q (C1q-C) with a member of the FK506 binding protein (FKBP) family was detected and the specificity of the interaction was further investigated. Results Yeast two-hybrid screen The physiological function of immunophilins in endocrine systems is rarely characterized. A commercially available fetal human liver cDNA expression library was used to identify proteins that interact with the immunophilin FKBP13 which is localized in the endoplasmic reticulum. Plasmids encoding FKBP13 without signal peptide fused to the LexA-DBD (pBTM-FKBP13w/oS) and the liver cDNA library were sequentially transformed into yeast L40. Ten million yeast double transformants (corresponding to 3.5 × 106 independent clones) were screened and selected for histidine prototrophy. Among 216 colonies isolated as His+, 21 were found to display beta-galactosidase (β-gal) activity. Plasmids were isolated from each of these clones and were used for retransformation of yeast L40 pretransformed with pBTM-FKBP13w/oS. Six clones remained positive (clone A3, B10, B31, B55, C8, C30) and turned out to code for five different cDNAs after sequence analysis. One of the clones (A3) representing the cDNA of the C-chain of complement C1 corresponding to amino acids 122 to 217 was selected for the present study. The interaction of a second clone with FKBPs was described elsewhere [18]. Interaction of full length C-chain of C1q with FKBP13 and with other FKBPs FKBPs share a common binding motif for the immunosuppressant FK506. To check the specificity of the FKBP13/C1q-C interaction, yeast L40 was cotransformed with a plasmid coding for the full length C1q-C-Gal4AD hybrid protein lacking a 26 amino acids signal peptide (pGAD-C1q-Cw/oS) and different plasmids each of them coding for FKBP12, FKBP13w/oS, FKBP25, and FKBP52 fused in frame to the DBD of LexA (pBTM-FKBP12, 13w/oS, 25, and 52). Cotransformants were checked for histidine prototrophy and β-gal activity. Only FKBP13 interacted with the C1q-Cw/oS protein in the yeast two-hybrid system whereas FKBP12, 25, and 52 failed in this respect. Interaction of C1q-C with cyclophilin A Cyclophilins are the second family of immunophilins. To check for cross reactivity, binding of C1q-Cw/oS to the prototype of cyclophilins, cyclophilin A (CypA), was investigated in the same system. Yeast L40 was cotransformed with a plasmid expressing LexA-CypA hybrids (pBTM-CypA) and pGAD-C1q-Cw/oS and was tested for β-gal activity and histidine prototrophy. C1q-Cw/oS and CypA failed to interact in the yeast two-hybrid system. Interaction of C1q-C with FKBP13 in a protein complementation assay In order to verify the results obtained with the yeast two-hybrid screen, the GST pull-down approach was used first. As the expression of C1q-Cw/oS either in a coupled reticulocyte lysate system or in bacteria failed, the interaction of C1q-Cw/oS with FKBP13w/oS was analyzed in a protein complementation assay. INS-1 cells were cotransformed with a plasmid coding for FKBP13w/oS fused in frame via a 15 amino acid peptide linker to aa 198 to 287 of β-lactamase (pCDNA-FKBP13w/oS-Bla2) and a plasmid coding for β-lactamase aa 21 to 196 fused in frame via a 15 amino acid peptide linker to C1q-Cw/oS (pCMVSPORT-Bla[1]M182TlinkCENP-A). As shown in Fig. 1, cotransformation of the hybrid-proteins resulted in β-lactamase activity; the interaction of C1q-Cw/oS with FKBP13w/oS was confirmed by using this second assay. Figure 1 Interaction of C1q-C and FKBP13 in the protein complementation assay. INS-1 cells were transfected with pCMVSPORT-Bla[1]M182TlinkBla[2] (control), or were cotransfected with pCDNA3.1Zeo+/F [I]M182T-15-Zip and pCDNA3.1Zeo+/Zip-15-F[2] (Zip+Zip), or were cotransfected with pCMVSPORT-Bla[1]M182TlinkC1q-Cw/oS and with pCDNA-FKBP13linkBla[2] (13+C1q-C). As a negative control cells were transfected with pCDNA-FKBP13linkBla[2] (sham). After lysis of the cells, β-lactamase activity was measured by hydrolysis rates of nitrocefin. The insert resembles the time dependent effect. The absorbances at 490 nm after 90 min of three independent experiments (mean ± S.E.M.) are shown. The control was normalized to 100 %. Interaction of C1q-C with FKBP13 in the presence of FK506 The crystal structure of FK506/FKBP complexes predicts that the recognition site in the endogenous ligand(s) equivalent to FK506 would best be emulated by a Iso-Pro or Leu-Pro motif [19]. FK506 might mimic the binding motif of C1q-C to FKBP13. For this reason, the influence of the drug on the interaction of C1q-Cw/oS with FKBP13w/oS was investigated in the yeast two-hybrid system. Cotransformants of yeast L40 with pGAD-C1q-Cw/oS and pBTM-FKBP13w/oS were incubated in the presence or absence of FK506. Cells were collected and measured for β-gal activity. As shown in figure 2, FK506 does not decrease the interaction of C1q-C with FKBP13, but rather seems to increase the affinity of C1q-C to the FKBP at low micromolar concentrations. The interaction of C1q-C with FKBP13 could not be suppressed, even in the presence of 100 μM FK506. Figure 2 Influence of FK506 on the interaction of C1q-C with FKBP13. Yeast L40 was cotransformed with pBTM-FKBP13w/oS and with pGAD-C1q-Cw/oS. Cotransformants were incubated in the presence or absence of FK506 and analyzed as described in material and methods. Shown are the results of 4–8 independent experiments ± S.E.M.. The control (absence of FK506) was defined as 100 %; the solvent ethanol was without influence. Discussion The C-chain of complement C1q interacts with FKBP13 A yeast two-hybrid screen was carried out using the FK506 binding protein 13 kDa (FKBP13) as a bait and a human liver expression library as a prey. The screen revealed the C-chain of complement C1q (C1q-C) to interact with FKBP13. Three other FKBPs, namely FKBP12, FKBP25 and FKBP52 failed to interfere with C1q-C under same conditions indicating specificity for the positive result in the yeast system. The interaction of C1q-C with FKBP13 was proved by using additionally a protein complementation assay (PCA), demonstrating that the interaction of C1q-C with the FKBP13 was not an artifact in the yeast two-hybrid system. The interaction of C1q-C with FKBP13 in the PCA seems to be comparable to the interaction of the two GCN4 leucine zippers (Zip) which were used as a positive control. The intensity of the interaction might depend on the efficiency of the cell transfection, cell density, cell growth, and other factors but hydrolysis rates of nitrocefin as a marker of the interaction are clearly different from the negative control which can be best seen after 270 min of incubation (Fig. 1, insert). The verification of the interaction in a classical GST pull-down assay was used elsewhere [18] but failed in this work as the rather small C1q-C protein could not be produced and labelled in vitro (data not shown). Immunoprecipitation of the interacting proteins was not planned since a C1q-C antibody is not available. The interaction, therefore, of C1q-C with different FKBPs was demonstrated in two independent approaches albeit the presence of an additional cellular factor necessary for the interaction can not be excluded with these cellular assays. The C-chain of complement C1q does not interact with cyclophilin A Immunophilins belong to the class of peptidyl prolyl isomerases (E.C. 5.2.1.8). They can be divided into FKBPs that bind for example FK506/tacrolimus or rapamycin and cyclophilins that mediate the pharmacological action of cyclosporin A (CsA). Cyclophilin A (CypA) can be regarded as the prototype of the cyclophilins. Unlike the aforementioned FKBP13, the immunophilin CypA does not interfere with C1q-C in the yeast two-hybrid system underlining the specificity of the interaction of C1q-C with FKBP13. FK506 fails to negatively influence the FKBP13 C1q-C interaction The immunosuppressive drug FK506 is not able to decrease the affinity of C1q-C to FKBP13 in the yeast two-hybrid system even when used in a concentration of 100 micromolar. The common binding motif of FKBPs for FK506 is, therefore, less likely to be uniquely involved in the C1q-C FKBP13 interaction. On the other hand, at low micromolar concentrations FK506 seems to „strengthen" the interaction of C1q-C with FKBP13. Possibly, C1q-C, when produced artificially in yeast, binds to yeast FKBP as well and has to be liberated from these interfering positions by FK506, then contributing to the interaction with the FKBP13-AD hybrid protein. An excess of FK506 should further diminish the C1q-C FKBP13 interaction but the drug cannot be tested in the milimolar range because of its toxicity. The influence of FK506 on the interaction of the two proteins was not investigated in a mammalian cell line because the influence in the yeast system is more likely comparable to other investigations [e.g. [7,18]]. Additionally the immunosuppressive agent is expected to be far toxic in the micromolar range when used in a mammalian cell system. Finally, a negative effect of the immunosuppressive drug on the C1q-C FKBP13 complex could not be demonstrated albeit the involvement of the FK506 binding pocket in the interaction cannot be excluded. Conclusions Using two different experimental approaches, the C-chain of complement C1q was shown to interact with FKBP13. FKBP13, initially used as the bait for the yeast two-hybrid screen in this work, is regarded as an endoplasmic reticulum (ER) protein. It might act as a chaperone but specifically interacts with other proteins like a homologue of the erythrocyte membrane cytoskleletal protein 4.1 or the FKBP associated protein 48 (FAP48), too. FKBP13 was detected in membrane preparations from erythrocytes as well, demonstrating that its expression and maintenance is not restricted to the ER [6]. The C-chain of complement C1q is part of the C1q molecule that is composed of six A-, six B-, and six C-chains, respectively. Interaction of FKBP13 with the A- or B-chain or with the entire C1q molecule has not been investigated yet. The carboxy-terminal part of the C-chain is sufficient to interact with FKBP13 in the yeast two-hybrid system. If the entire C1q molecule interacts with FKBP13, interaction of its globular head with the immunophilin will be likely therefore, as the globular head is is formed by association of the C-terminal regions of an A-, B-, and C-chain. On the other hand, FKBP13 might act as a specific chaperone, triggering C1q-C in the endoplasmic reticulum and preventing the complement protein to interact with other intracellular proteins. Further work will be necessary to elucidate the physiological significance of the interaction of the two proteins and to investigate whether FKBP13 plays an important role in the complement system, too. Methods Expression vectors The cDNA encoding FKBP13 amino acids 16–142 was amplified from pBluescript-FKBP13 (a gift from Dr. S. Burakoff, Dana-Faber Cancer Institute, Boston, MA) using specific primers CGCCGGAATTCATGCTGAGCGCCGTG and CGGCTGGATCCGAACAGTCTGGTC. The cDNA encoding full-length FKBP25 was amplified from pBluescript-FKBP25 (a gift from Dr. S. Burakoff, Dana-Faber Cancer Institute, Boston, MA) using the specific primers CGCGCGAATTCAACA GTCTGGTCCCTGATG and GGCGTAGGATCCGGGGTTGACTCCGGGGGC. The cDNA encoding full-length cDNA of CypA was amplified from I.M.A.G.E clone 5264185 (ResGen, Huntsville, AL) by using the specific primers GGTCCGGAATTC ATGGTCAACCCCACCGTGTTC and GGCAGCTGGATCCACAAGTCA AAC TTATTCGAG. After restriction digest, cDNAs were fused to the DNA binding domain of LexA by inserting the EcoRI/BamHI fragments into pBTM116 [20] to give pBTM-FKBP13w/oS, pBTM-FKBP25, and pBTM-CypA, respectively. pBTM-FKBP12 and pBTM-FKBP52 (a gift from Dr. B. Chambraud, INSERM U488, Bicêtre, France) are described elsewhere [7]. Full lenght cDNA of human C1q-C lacking a 26 amino acids signal peptide was amplified from I.M.A.G.E clone AL568589 (ResGen, Huntsville, AL) using the specific primer CACGGAATTCAA GCCAACACAGGCTGCTAC and primer M13 reverse. After restriction digest the cDNA fragment was fused in frame to the GAL4AD by inserting the EcoRI/NotI fragment into pGAD1318 to give pGAD-C1q-Cw/oS. The two vectors pCDNA3.1Zeo+/F [I]M182T-15-Zip and pCDNA3.1Zeo+/Zip-15-F[2], a gift from Dr. S.W. Michnick, Montreal, were used as a positive control in the protein complementation assay and are described elsewhere [21]. The cDNA encoding the TEM-1 β-lactamase gene amino acids 21 to 196 (Bla[1]M182T) was amplified from pGEM11Zf(+) using the primer pairs (i) TTGGGCACCATGGACCCAGAAACG CTGGTGAAAG and GTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACA GGCGACGAGGTGTC and (ii) GCGGCGAAGCTTCAATTGGGCACCATGGAC CCAGAAACG and ACCACCGGATCCGCCAGTTAATAGTTTGCGCAACG. Note that a point mutation leading to M182T was inserted thatway [22]. The cDNA encoding the TEM-1 β-lactamase gene amino acids 198 to 287 and an amino-terminal 15 amino acid linker (linkBla[2]) was amplified from pGEM11Zf(+) sequentially using the primer pairs (i) GGTGGTGGTAGTCGAATTCTACTTACTCTAGCT TCCCGGC and CCAGCTCTCGAGTTACCAATGCTTAATCAGTGAGGCACC (primerBla2XhoI) and (ii) CTGGCGGATCCGGTGGTGGTGGTAGTGGTGGTG GTGGTAGTGGTGGTGGTGGTAGTCGAATTCTAC and primer Bla2XhoI. After restriction digest cDNA of FKBP13 (from pBTMFKBP13w/oS) was fused into the EcoRI/BamHI site of pCDNA3.1 (Invitrogen, Groningen, The Netherlands) and the linkBla[2] fragment was fused into the BamHI/XhoI site of this plasmid to give pCDNA-FKBP13linkBla[2]. After restriction digest Bla[1]M182T and linkBla[2] fragments were sequentially fused into the MunI/BamHI and BamHI/XhoI site of pCMVSPORT6 (Invitrogen, Groningen, The Netherlands) to give pCMVSPORT-Bla[1]M182TlinkBla[2]. After restriction digest cDNA of C1q-C lacking the signal peptide (from pGAD-C1q-Cw/oS) was inserted into the EcoRI/XhoI site of pCMVSPORT-Bla[1]M182TlinkBla[2] to give pCMVSPORT-Bla[1]M182TlinkC1q-Cw/oS. All inserts were verified by sequencing. Two-hybrid screen The human fetal liver MATCHMAKER cDNA library (Clontech, Palo Alto, CA) was a gift from Dr. C. Sorg (Institute of Experimental Dermatology, Münster, Germany). The yeast reporter strain L40, containing two reporter genes, HIS3 and LacZ, was sequentially transformed with pBTM-FKBP13w/oS and the cDNA library using the lithium acetate method [23]. Double transformants were plated on Minimal SD Base containing the -Leu/-Trp/-His DO supplement (Clontech, Palo Alto, CA). The plates were incubated at 30°C for 5 days. His+ colonies were patched and assayed for β-gal activity. Positive clones, inserted into the pACT2 vector, were rescued and tested for specificity by retransformation into yeast L40 with pBTM-FKBP13w/oS. Different clones, that expressed detectable β-gal activity within four hours, were used for further analysis. All assays of β-gal activity and histidine prototrophy were performed according to standard procedures [24]. Protein complementation assay The protein complementation assay (PCA) was performed as described elsewhere [21]. Briefly, 1 × 106 INS-1 cells, an insulinoma cell line [25], were cotransformed with 1 μg of the plasmids pCDNA-FKBP13linkBla[2] and with 1 μg pCMVSPORT-Bla[1]M182TlinkCENP-A, or with pCMVSPORT-Bla[1]M182TlinkBla[2] alone, or with the positive control plasmids using the FuGENE 6 transfection reagent (Roche Diagnostics, Mannheim, Germany) according to the manufacturer's instructions. After two days, cells were washed with PBS and harvested into 100 μl of 100 mM phosphate buffer, pH 7.0 and lysed by three freeze-thaw cycles. After centrifugation at 13,000 g for 4 min, the supernatant was used to check for β-lactamase activity. In a 96-well microtiter plate each well was loaded with 80 μl of the phosphate buffer, 80 μl dejonized water, 20 μl of phosphate buffer containing 1 mM nitrocefin (EMD Biosciences, San Diego, CA, freshly prepared from a 10 mM stock solution in DMSO) and 20 μl of each supernatant. The absorbance at 496 nm was measured time-dependently in a platereader (Spectra Max 340, Molecular Devices, Sunnyvale, CA). Data after 90 min of incubation were used to calculate the absorbance as percent of control. Authors' contributions The authors contributed equally to this work Acknowledgements We thank Dr. B. Chambraud (INSERM U488, Bicêtre, France) for providing pBTM-FKBP12 and pBTM-FKBP52 and Dr. S. Burakoff (Dana-Faber Cancer Institute, Boston, MA) for providing cDNA of FKBP13 and FKBP25. We thank Dr. C. Sorg (Institute of Experimental Dermatology, Münster, Germany) for the gift of the cDNA expression library and Dr. A. Friedrich (Fujisawa GmbH, München, Germany) for the gift of FK506. We thank Dr. Mishnick (Canada) for sending pCDNA3.1Zeo+/F [I]M182T-15-Zip and pCDNA3.1Zeo+/Zip-15-F[2]. ==== Refs Liu J Albers MW Wandless TJ Luan S Alberg DG Belshaw PJ Cohen P MacKintosh C Klee CB Schreiber SL Inhibition of T cell signaling by immunophilin-ligand complexes correlates with loss of calcineurin phosphatase activity Biochemistry 1992 31 3896 3901 1373650 Jayaraman T Brillantes A Timerman AP Fleischer S Erdjument-Bromage H Tempst P Marks AR FK506 binding protein associated with the calcium release channel (ryanodine receptor) J Biol Chem 1992 267 9474 9477 1374404 Timerman AP Onoue H Xin HB Barg S Copello J Wiederrecht G Fleischer S Selective binding of FKBP12.6 by the cardiac ryanodine receptor J Biol Chem 1996 271 20385 91 8702774 10.1074/jbc.271.34.20385 Peattie DA Harding MW Fleming MA DeCenzo MT Lippke JA Livingston DJ Benasutti M Expression and Characterization of Human FKBP52, an Immunophilin That Associates With The 90-kDa Heat Shock Protein and is a Component of the Steroid Receptor Complexes Proc Natl Acad Sci USA 1992 89 10974 10978 1279700 Nigam SK Jin YJ Jin MJ Bush KT Bierer BE Burakoff SJ Localization of the FK506-binding protein, FKBP 13, to the lumen of the endoplasmic reticulum Biochem J 1993 294 511 515 8373365 Walensky LD Gascard P Fields ME Blackshaw S Conboy JG Mohandas N Snyder SH The 13-kD FK506 binding protein, FKBP13, interacts with a novel homologue of the erythrocyte membrane cytoskeletal protein 4.1 J Cell Biol 1998 141 143 153 9531554 10.1083/jcb.141.1.143 Chambraud B Radanyi C Camonis JH Shazand K Rajkowski K Baulieu EE FAP48, a new protein that forms specific complexes with both immunophilins FKBP59 and FKBP12. Prevention by the immunosuppressant drugs FK506 and rapamycin J Biol Chem 1996 271 32923 32929 8955134 10.1074/jbc.271.51.32923 Jin YJ Burakoff SJ The 25-kDa FK506-binding protein is localized in the nucleus and associates with casein kinase II and nucleolin Proc Natl Acad Sci U S A 1993 90 7769 73 7689229 Yang WM Yao YL Seto E The FK506-binding protein 25 functionally associates with histone deacetylases and with transcription factor YY1 Embo J 2001 20 4814 25 11532945 10.1093/emboj/20.17.4814 Sellar GC Blake DJ Reid KB Characterization and organization of the genes encoding the A-, B- and C-chains of human complement subcomponent C1q. The complete derived amino acid sequence of human C1q Biochem J 1991 274 481 90 1706597 Navratil JS Korb LC Ahearn JM Systemic lupus erythematosus and complement deficiency: clues to a novel role for the classical complement pathway in the maintenance of immune tolerance Immunopharmacology 1999 42 47 52 10408365 10.1016/S0162-3109(99)00018-1 Botto M Dell'Agnola C Bygrave AE Thompson EM Cook HT Petry F Loos M Pandolfi PP Walport MJ Homozygous C1q deficiency causes glomerulonephritis associated with multiple apoptotic bodies Nat Genet 1998 19 56 9 9590289 McGreal EP Ikewaki N Akatsu H Morgan BP Gasque P Human C1qRp is identical with CD93 and the mNI-11 antigen but does not bind C1q J Immunol 2002 168 5222 32 11994479 Petrenko O Beavis A Klaine M Kittappa R Godin I Lemischka IR The molecular characterization of the fetal stem cell marker AA4 Immunity 1999 10 691 700 10403644 10.1016/S1074-7613(00)80068-0 Klickstein LB Barbashov SF Liu T Jack RM Nicholson-Weller A Complement receptor type 1 (CR1, CD35) is a receptor for C1q Immunity 1997 7 345 55 9324355 10.1016/S1074-7613(00)80356-8 Malhotra R Willis AC Jensenius JC Jackson J Sim RB Structure and homology of human C1q receptor (collectin receptor) Immunology 1993 78 341 8 8478019 Ghebrehiwet B Lim BL Peerschke EI Willis AC Reid KB Isolation, cDNA cloning, and overexpression of a 33-kD cell surface glycoprotein that binds to the globular "heads" of C1q J Exp Med 1994 179 1809 21 8195709 10.1084/jem.179.6.1809 Feldkämper I Verspohl EJ Neye H The serpins alpha-1-antitrypsin and alpha-1-antichymotrypsin specifically interact with immunophilins Pharmazie 2003 58 811 816 14664338 Clardy J The chemistry of signal transduction Proc Natl Acad Sci U S A 1995 92 56 61 7529414 Bartel PL Chien C-T Sternglanz R Fields S Hartley DA Using the two-hybrid system to detect protein-protein interactions Cellular Interactions in Development: A Practical Approach 1993 Oxford: Oxford Univ Press 153 179 Galarneau A Primeau M Trudeau LE Michnick SW Beta-lactamase protein fragment complementation assays as in vivo and in vitro sensors of protein protein interactions Nat Biotechnol 2002 20 619 22 12042868 10.1038/nbt0602-619 Sideraki V Huang W Palzkill T Gilbert HF A secondary drug resistance mutation of TEM-1 beta-lactamase that suppresses misfolding and aggregation Proc Natl Acad Sci U S A 2001 98 283 8 11114163 10.1073/pnas.011454198 Gietz D S Jean A Woods RA Schiestl RH Improved method for high efficiency transformation of intact yeast cells Nucleic Acids Res 1992 20 1425 1561104 Kaiser C Michaelis S Mitchell A Methods in Yeast Genetics: A Cold Spring Harbor Laboratory Course Manual 1994 Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press Asfari M Janjic D Meda P Li G Halban PA Wollheim CB Establishment of 2-mercaptoethanol-dependent differentiated insulin-secreting cell lines Endocrinology 1992 130 167 78 1370150 10.1210/en.130.1.167

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==== Front BMC CancerBMC Cancer1471-2407BioMed Central London 1471-2407-4-651536733410.1186/1471-2407-4-65Research ArticleMethylation profiles of thirty four promoter-CpG islands and concordant methylation behaviours of sixteen genes that may contribute to carcinogenesis of astrocytoma Yu Jian [email protected] Hongyu [email protected] Jun [email protected] Song [email protected] Junhua [email protected] Wei [email protected] Yifei [email protected] Jingde [email protected] Cancer Epigenetics and Gene Therapy, State-Key Laboratory for Oncogenes and Related Genes, Shanghai Cancer Institute, Shanghai Jiao Tong University, LN 2200/25, Xie-Tu Road, Shanghai 200032, China2 Department of Neurosurgery, Tiantan Hospital of Capital University of Medical Sciences, Beijing Neurosurgical Institute, Beijing 100050, China3 Department of Mathematics, Shanghai University, No. 99, Shangda Road, Shanghai 200436, P. R. China2004 14 9 2004 4 65 65 4 2 2004 14 9 2004 Copyright © 2004 Yu et al; licensee BioMed Central Ltd.2004Yu et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Astrocytoma is a common aggressive intracranial tumor and presents a formidable challenge in the clinic. Association of altered DNA methylation patterns of the promoter CpG islands with the expression profile of cancer-related genes, has been found in many human tumors. Therefore, DNA methylation status as such may serve as an epigenetic biomarker for both diagnosis and prognosis of human tumors, including astrocytoma. Methods We used the methylation specific PCR in conjunction with sequencing verification to establish the methylation profile of the promoter CpG island of thirty four genes in astrocytoma tissues from fifty three patients (The WHO grading:. I: 14, II: 15, III: 12 and IV: 12 cases, respectively). In addition, compatible tissues (normal tissues distant from lesion) from three non-astrocytoma patients were included as the control. Results Seventeen genes (ABL, APC, APAF1, BRCA1, CSPG2, DAPK1, hMLH1, LKB1, PTEN, p14ARF, p15INK4b, p27KIP1, p57KIP2, RASSF1C, RB1, SURVIVIN, and VHL) displayed a uniformly unmethylated pattern in all the astrocytoma and non-astrocytoma tissues examined. However, the MAGEA1 gene that was inactivated and hypermethylated in non-astrocytoma tissues, was partially demethylated in 24.5% of the astrocytoma tissues (co-existence of the hypermethylated and demethylated alleles). Of the astrocytoma associated hypermethylated genes, the methylation pattern of the CDH13, cyclin a1, DBCCR1, EPO, MYOD1, and p16INK4a genes changed in no more than 5.66% (3/53) of astrocytoma tissues compared to non-astrocytoma controls, while the RASSF1A, p73, AR, MGMT, CDH1, OCT6,, MT1A, WT1, and IRF7 genes were more frequently hypermethylated in 69.8%, 47.2%, 41.5%, 35.8%, 32%, 30.2%, 30.2%, 30.2% and 26.4% of astrocytoma tissues, respectively. Demethylation mediated inducible expression of the CDH13, MAGEA1, MGMT, p73 and RASSF1A genes was established in an astrocytoma cell line (U251), demonstrating that expression of these genes is likely regulated by DNA methylation. AR gene hypermethylation was found exclusively in female patients (22/27, 81%, 0/26, 0%, P < 0.001), while the IRF7 gene hypermethylation preferentially occurred in the male counterparts (11/26, 42.3% to 3/27, 11%, P < 0.05). Applying the mathematic method "the Discovery of Association Rules", we have identified groups consisting of up to three genes that more likely display the altered methylation patterns in concert in astrocytoma. Conclusions Of the thirty four genes examined, sixteen genes exhibited astrocytoma associated changes in the methylation profile. In addition to the possible pathological significance, the established concordant methylation profiles of the subsets consisting of two to three target genes may provide useful clues to the development of the useful prognostic as well as diagnostic assays for astrocytoma. ==== Body Background Diffusely infiltrating astrocytoma is a leading group of the primary central nervous system tumors, accounting for more than 60% of all primary brain tumors [1,2]. It may arise aggressively from the normal astrocytes, or evolve stepwise from the less its benign precursors. Owing to the difficulties with its early diagnosis and surgical removal of all residue diseased tissues, rapid progression, and frequent reoccurrence, the most advanced form of astrocytoma, glioblastoma (WHO grading IV) represents an extremely life-threatening intracranial malignant tumor both inside and outside of China [1,2]. Molecular genetic analyses have demonstrated multiple genetic lesions implicating to pathogenesis of astrocytoma, glioblastoma in particular. In addition to the frequent amplification and deletion of the EGF receptor gene (EGFR) [3], the main genetic events affecting the following tumor suppressor genes: the members of the INK4A initiated cell-cycle arrest pathway (the p16INK4a) [4], the p14ARF [5], the RB1 [6] and the p53 [7]), a wide spectrum of the cell surface receptor genes (i.e., CD44, integrin, and receptors for various growth factors), and the PTEN genes [8]. Transcription in eukaryotes is regulated at multiple levels and inversely correlated with the hypermethylated state as well as the chromatin condensation. It has been well established that the methylation status of CpG islands directly affects the DNA-protein interactions by eliminating the otherwise occurring sequence specific binding of the transcription factors whereas inducing the DNA-bindings of members of the methyl-CpG binding protein family (MBD). Histone modifications (deacetylation and methylation) may occur subsequently leading to chromatin condensation and a long-term transcriptional silencing status of the affected DNA segments. Over 40% of the protein coding genes have at least one CpG island within or near to their promoter, an strong indication for transcription of which is likely to be under the control of DNA methylation status. Three DNA methyl transferases are involved in the control of the methylation state of the CpGs in genome. DNA methyl transferase I is mainly responsible for the maintenance of the methylation status of the genome after DNA replication, while IIIA and IIIB act principally in the de novo DNA methylation in the early development of high eukaryotes. DNA methylation patterns in somatic cells are established during the early development and contribute to the allele-specific transcription silencing of the imprinted genes, including the silenced alleles in the X-chromosome and other chromosomes. The epigenetic pattern (the DNA methylation profiles of the genome) in high eukaryotes is integral to the normal execution of the biological activities in cells and needs to be actively maintained. In addition to the changes linked to the cell lineage specific pattern of gene expression, both global hypomethylation and local hypermethylation of the CpG islands occur progressively as cell ages. Aberrant DNA methylation pattern changes gene transcription that has been etiologically linked to cancer formation [9,10]. The genome-wide hypomethylation has been believed to activate transcription of the otherwise silenced transposon like repetitive sequences (such as the Alu and LINE repeats in mammals). As a result, the transposition occurs more prevalently so that the genomic instability in cancer cells will be significantly increased [11-13]. The hypermethylated state of the promoter CpG islands has been etiologically associated with transcription inactivation of a number of tumor suppressor genes in tumors, which are hypomethylated and transcribed in their normal counterparts. Therefore, the hypermethylated CpG island(s) of those genes have been regarded as a defect, reminiscent to the loss of heterozygosity or other types of genetic deletion for total inactivation of the tumor suppressor genes in cancer. The most noticeable example is the p16INK4a gene that has been frequently hypermethylated in almost all types of the tumors examined [14-17] including hepatocellular carcinoma [18]. The loss of the genetic imprinting (changes in DNA methylation status) has been found to reactivate transcription of the otherwise silenced allele of the genes such as the insulin like growth factor 2 gene, which has been well documented in human tumors [19]. On the other hand, the reverse process, i.e., demethylation of the promoter CpG island, has also been found instrumental to the transcription activation of the otherwise inert genes in tumor cells [20]. A prominent example is the gene encoding the melanoma antigen, MAGEA1 that was hypermethylated and transcriptionally silenced in the normal liver tissues, and demethylated prevalently in the hepatocellular carcinoma tissues [18], correlating well with the elevated level of its expression in HCC [21,22]. The over-expressed gene, SURVIVIN, has also been reported to be demethylated in human ovarian cancer [23]. Despite of the fact that the elevated levels of expression of three DNA methyl transferase genes were detected in virtually all cancers, the profiles of the hypermethylated genes vary with both the types and stages of cancers. Therefore, the undefined defects in the epigenetic homeostasis during carcinogenesis, rather than the aberrant expression of any given DNA methyl transferase, are more likely to account for the cancer type specific pattern of DNA methylation at both global and local levels. Methylation profiling of the promoter CpG islands has been an important information gathering process for new insights into our understanding of the role of DNA methylation in both initiation and progression of human carcinogenesis. It would result in development of the DNA methylation based assays for cancer diagnosis as well as identification of the cancer genes suffering from the epigenetic defects . However, as the majority of studies had only targeted one or a few genes in rather small patient groups, the concurrent hypermethylation behavior of multiple genes has only been addressed in a limited number of tumor types, such as colorectal cancer. The majority, if not all, of the previous studies on the astrocytoma associated changes in methylation profiles have only examined a small number of genes for methylation status at the promoter CpG island [25,26]. In this study, we determined the methylation profiles of as many as thirty four genes in a cohort consisting of 53 astrocytoma patients and established the concordant methylation behavior of up to three targets. Our observations should provide new insights into the DNA methylation epigenetic defects in human astrocytoma. Methods All the experiments were performed according to protocols described previously [18]. The primer pairs for the methylation specific PCR were either adopted (APC, BRCA1, CDH1, DAPK1, hMLH1, p14ARF, p15INK4b, RASSF1A, RB1 and VHL) or designed according to the same principle with assistance of the software packages for the CpG islands identification and the primer design [Additional file 1]. Tissue samples Tissue samples and DNA preparation With the informed consent of all patients and approval of the ethics committee, the tumor samples were collected from astrocytoma patients (n = 53) during operation at the Tiantan Neurosurgical Hospital in Beijing. The pathological classification of tumor tissues was carried out and the stage of each astrocytoma patients was determined according to the WHO classification [1]. No significant geographic impart was observed as patients came from different places in China and went to Beijing for treatment. In addition, the compatible tissues (normal tissues distant from the lesions) were surgically obtained from three non-astrocytoma patients [gangliocytoma (21 years old, male), angiocavernoma (49 years old, male) and meningioma (54 years old, female)] as the normal controls, which have been subjected to the proper pathological evaluation. Total genomic DNA was extracted from frozen tissue specimens (50 – 100 mg) according to a standard protocol with some modifications [18,27]. Frozen pulverized powders of the specimens were re-suspended with 2 ml lysis buffer: 50 mM Tris-HCl pH 8.0, 50 mM EDTA, 1% SDS, 10 mM NaCl plus 100 μg/ml boiling-treated RNase A (Sigma). Following one hour of incubation at 37°C, Proteinase K (Roche, USA) was added to the cellular lysates for a final concentration of 100 μg/ml and the digestion was carried out at 55°C for 2 hours. Organic extractions with a half volume of Phenol/Chloroform/Isoamyl alcohol (1:1:0.04) were repeatedly carried out until no visible interphase remained after centrifugation. DNA was precipitated from the aqueous phase in the presence of 0.3 M NaOAc pH 7.0 and two and a half volumes of ethanol and followed by one 70% ethanol-washing and dissolved at 65°C for 30 minutes with 0.2 – 0.4 ml TE (10 mM Tris-HCl pH 7.4 and 1 mM EDTA)and stored at 4°C till use. The DNA concentrations were calculated according to the OD260 nm readings. Bisulphate treatment of DNA and Methylation specific PCR (MSP) The methylation status of the promoter CpG islands of thirty four genes in all DNA samples was analyzed by MSP on the sodium-bisulfite converted DNA [18]. In detail, 10 μg DNA in 50 μl TE was incubated with 5.5 μl of 3 M NaOH at 37°C for 10 minutes, followed by a 16 hour treatment at 50°C after adding 30 μl of freshly prepared 10 mM hydroquinone and 520 μl of freshly prepared 3.6 M sodium-bisulfite at pH 5.0. The DNA was desalted using a home-made dialysis system with 1% agarose (detailed protocol will be provided upon request). The DNA in the desalted sample (approximately 100 μl in volume) was denatured at 37°C for 15 minutes with 5.5 μl of 3 M NaOH followed by ethanol precipitation with 33 μl 8 M NH4OAC and 300 μl ethanol. After washing with 70% ethanol, the gently dried DNA pellet was dissolved with 30 μl TE at 65°C for 10 min. The DNA sample was finally stored at -20°C until further use. PCR reaction was carried out in a volume of 15 μl with 50 ng or less template DNA with FastStart Taq polymerase (Roche, Germany) as follows. After an initial heat denaturing step 4 minutes treatment at 94°C, 30 cycles of 92°C for 15 sec, varying temperatures with primer pairs (Additional file 1) for 15 sec and 72°C for 20 sec, was carried out. The PCR products were separated by 1.2% ethidium bromide containing agarose gel electrophoresis with 1 × TAE and visualized under UV illumination. To verify the PCR results, representative bands from each target were gel-purified and cloned into T-vector (Promega, USA) followed by automatic DNA sequencing provided by BuoCai (Shanghai, China). Only verified results are presented in this report. To optimize the MSP procedure, the M. Sss I treated DNA was used as the methylated control template. In detail, the DNA from a normal liver tissue of the healthy liver donor [18,24] was batch cleaved with EcoR I, followed by M. Sss I treatment according to the manufacture's instruction (New England Biol., Boston, USA) for over night. The purified DNA was bisulphate treated as usual and subjected to MSP with the primer pairs for each of thirty three genes (except for the MAGEA1 gene), and only the verified targets were included for the study of the astrocytoma tissues. Statistical analysis The methylation data were dichotomized as 1 for the co-existence of the methylated and unmethylated alleles, 2 for methylated allele only and 0 for the unmethylated for both alleles to facilitate statistical analysis using contingency tables. The methylation profiles of each individual gene (in percentage) classified by the genders and grading of the patients were presented both in table and in plot. The statistic analyses for the association between the methylation profile of the gene and each of the clinical-pathological parameters were carried out with the statistics package , where both Pearsong's Chi-square test with Upton's adjustment and Fisher exact test were used to examine the tissue samples with the low expected values. The relative frequency with a 95% confidence interval (P < 0.05) for a binomial distribution was calculated for the whole as well as each subtype of astrocytoma patients. The concordant methylation behavior of the genes was established by comparing frequency of co-occurrence of 2 to 3 target subsets with a mathematic method, namely Discovery of Association Rules [28], which is frequently utilized for association analysis. Demethylation of U251 cells with 5-Aza-2'-deoxycytidine U251 cells (an established glioma cell line) were cultured in DEME plus 10% new born calf serum at 37°C in a 5% CO2 atmosphere. When cell culture reached 50% confluence, they were treated with 5-Aza-2'-deoxycytidine (Sigma A3656) at the final concentration 10 and 20 nM, respectively for 3 days. The primer pairs for the RT-PCR (Table 1) was either adopted from published papers or designed with an assistance of the software . The total RNA was extracted with Trizol solution according to manufacturer's instruction (Invitrogen, USA), and cDNA was obtained using the Supertranscript plus reverse transcriptase with the oligo-dT as primers. PCR with single pair of the target primers run for 15 cycles, followed by another 15 cycle PCR reactions in the presence of the beta-actin primers (Table 1) (the parameter of each cycle is 94°C 20", 60°C for 20" and 72°C for 30"). The resulted PCR products were visualized under UV illumination after an electrophoretic separation on a 1.2% agarose. The methylation status of the target was analyzed by MSP. Table 1 The primers for RT-PCR analysis Primer Name sequence PCR Product Length (bp) Accession Number beta-actin L AAGTACTCCGTGTGGATCGG 616 NM_001101 beta-actin R TCAAGTTGGGGGACAAAAAG cdh13f GCTGGACTGGATGTTGGATT 246 NM_001257 cdh13t TTGAGGGTTGGTGTGGATTT magea1rf ACCTGACCCAGGCTCTGT 401 NM_004988 magea1rt CTCACTGGGTTGCCTCTG mgmtrf AAACGCACCACACTGGAC 404 NM_002412 imgmtrt AGGATGGGGACAGGATTG p73f AGATGAGCAGCAGCCACAG 218 NM_005427 p73t GTACTGCTCGGGGATCTTCA rassf1arf GTCTGCCTGGACTGTTGC 401 NM_007182 rassf1art AGCAGGGCCTCAATGACT Results and discussion Clinical-pathological classification To establish the methylation profile of thirty four genes during the process of astrocytoma development, we recruited 53 astrocytoma patients (27 female and 26 male; 49 primary and 4 recurrent) for this study. 14 cases were pathologically classified at the Grade I pilocytic astrocytoma (10–62 years old, mean: 39.1; 9 female, 5 male), 15 cases at the Grade II diffuse astrocytoma (4–50 years old, mean: 33.1; 10 female, 5 male), 12 cases at the Grade III anaplastic astrocytoma (1–72 years old; mean: 40.4; 4 female, 8 male), and 12 cases (including 4 recurrent cases) at the Grade IV glioblastoma (22–66 years old, mean: 44.6; SD = 22–66, 4 female, 8 male) (Table 2). The normal brain tissues distant from the lesions were also obtained from three non-astrocytoma patients who underwent brain surgery as normal controls in this study. Table 2 The clinical and pathological profiles of the patients Astrocytoma Non astrocytoma Gender female 27 1 male 26 2 Age, y <40 27 1 40–60 23 2 >60 3 0 Grade Age Mean Range I 39.1 10 to 62 14 II 33.1 4 to 50 15 III 40.4 1 to 72 12 IV 44.6 22 to 66 12 Recurrent 4 Primary 8 Aberrant Methylation profiling in astrocytoma The technical considerations The methylation-specific PCR (MSP) is widely used for methylation profiling of the genes in human cancers for both its easiness and sensitivity. However, the necessary steps have to be taken to eliminate both false positive and negative results. Comparing the MSP-data with the non-PCR data by Southern analysis of the methylation sensitive restriction enzyme is a valuable choice, as our previous work where the hypomethylated status of both p14ARF and p15INK4b genes shown by MSP was confirmed by Southern analysis [18]. Alternatively, the PCR reaction with the in vitro methylated genomic DNA (by M. Sss I) as template would be an ideal positive control for the absence of methylated targets in tumor tissue samples. By taking extra caution, we carried out MSP of all the targets with the M. Sss I treated normal liver DNA as positive control templates, except for the MAGEA1 gene was unmethylated in the normal liver tissue. While only the PCR reaction designated to the unmethylated template gave rise to the detectable bands with the parental DNA, the PCR bands were evident in both reactions with the M. Sss I treated DNA (Additional File 2). Therefore, failure to detect the methylated alleles with the tissue samples should genuinely reflect the lack of methylated targets. To control the false positive with either pair of primers, the representative PCR products, were T-cloned and sequenced. Only the positive PCR results with the expected sequence profiles were scored and analyzed further. The methylation profiling of thirty four targets in astrocytoma Eleven of the thirty four target genes were previously studied either in astrocytoma or other types of tumors. The published PCR conditions for these genes: APC, BRCA1, CDH1, DAPK1, hMLH1, p14ARF, p15INK4b, p16INK4a RASSF1A, RB1 and VHL (Additional file 1) were adopted to enable the relevant inter-study comparisons if necessary. The remaining twenty three targets were selected from a list of genes displaying the altered pattern of the promoter CpG island in various biological settings including cancers. Their CpG islands were identified via bioinformatical tools and the primer pairs were designed accordingly [18,24]. Some of these thirty four genes have been shown to play a role in carcinogenesis, whereas the others have no obvious association with human carcinogenesis. Since it is still disputed whether DNA methylation mediated the gene silencing is causative in the malignant transformation of cell, we specifically selected both sets of genes in this study. The "cancer unrelated" genes selected encode erythropoiesis (EPO) [29], a ubiquitously expressed transcription factor (OCT6) [30], and the myogenesis lineage-specific transcription factor (MYOD1) [31]. The majority of the cancer associated genes examined were tumor suppressor genes including genes operating in the RB1/p16INK4a pathway (p14ARF, p15INK4b, p16INK4a, and RB1) [32], and two cyclin-dependent kinase inhibitors (p27KIP1 [33] and p57KIP2) [34]. Other genes in this subset were a p53 analogue:(p73) [33,35], two alternative forms of a tumor suppressors in the Ras mediated signal transduction pathway (RASSF1A, and RASSF1C [36]), VHL [37], APC [38], PTEN [6], the deleted in bladder cancer chromosome region candidate 1 (DBCCR1) [39], and the Wilms tumor 1 gene(WT1) [40]. We included the genes encoding the cell membrane proteins or nuclear receptors which act actively in the intercellular interactions: melanoma specific antigen A1 (MAGEA1) [41], caveolin 1 (CAV) [42], chondroitin sulfate proteoglycan 2 (CSPG2) [43], androgen receptor (AR) [44], and cadherins (CDH1 [45] and CDH13) [46]. Three genes implicated in signal transduction were also selected: cyclin a1 [47], the interferon regulatory factor 7 (IRF7), and a serine/threonine kinase 1 (Peutz-Jeghers syndrome) gene (LKB1) [14]. There were the genes encoding the O-6-methylguanine-DNA methyltransferase (MGMT) [14]and metallothionein 1 A gene (MT1A) [48] which play a key role in the cellular response to alkalyting agents and heavy metal stress. The genes acting in DNA repair process were hMLH1 [49], and BRCA1 [50], while four genes are involved in apoptosis (APAF1 [51], DAPK1 [15], and SURVIVIN [23]). Finally, the proto-oncogenes in this group were represented by v-abl homologue 1 (ABL) [52] (Additional files 3,4,5,6,7,8,9). The genes displayed the uniformly unmethylated profiles in astrocytoma Of the unmethylated genes in all samples tested, EPO was a cancer unrelated gene, while "cancer associated" genes included ABL(1), APAF1(2), APC(3), BRCA1(5), CAV(6), CDH13(8), DAPK1(11), hMLH1(14), LKB1(16), p14ARF(22), p15INK4b(23), p27KIP1(25), p57KIP2(26), PTEN (28), RASSF1C(30), RB1(31), SURVIVIN(32), and VHL(33) genes (Additional files 3,4,5,6,7,8,9). Lack of hypermethylation of the RB1 gene in our observation was inconsistent with a recent report that the hypermethylated RB1 gene was detected in 19% of astrocytoma patients (26/136 cases analyzed) [53]. Since the same region was looked at in this work, the discrepancy noticed may simply reflect the inherent difference in the patient cohorts between our work and the published [53]. The genetic defects affecting the PTEN gene contributed to the pathogenesis of astrocytoma [54]. Lack of the hypermethylation of its promoter CpG island in both normal and astrocytoma tissues indicates that the DNA hypermethylation mediated silencing mechanism unlikely plays a significant role in the PTEN inactivation that occurs frequently in astrocytoma. This explanation might also be applicable to the no change type of methylation behavior for both the tumor associated genes (ABL(1), APAF1(2), APC(3), BRCA1(5), CAV(6), CDH13 (8), DAPK1(11), hMLH1(14), LKB1(16), p14ARF(22), p15INK4b(23), p27KIP1(25), p57KIP2(26), PTEN (28), RASSF1C(30), RB1(31), SURVIVIN(32), and VHL(33) genes) and the "cancer unrelated" genes (EPO (14)) (Additional files 3,4,5,6,7,8,9). The genes with the astrocytoma specific alteration in methylation As shown in Additional files 3,4,5,6,7,8,9, thirteen genes (CDH1 (7), CSPG2(9), cyclin a1(10), DBCCR1(12), IRF7(15), MGMT(18), MT1A(19), MYOD1(20), OCT6(21), p16INK4a (24), p73(27), RASSF1A (39) and WT1(34)) were unmethylated in all three normal controls. In contrast, these genes were hypermethylated to various extents in the astrocytoma samples. The following six genes were marginally hypermethylated: p16INK4a, EPO, DBCCR1 and MYOD1 genes were hypermethylated in 1.9% (1/53) of astrocytoma tissues, while both CDH13 and cyclin a1 genes were hypermethylated in 5.7% (3/53) of astrocytoma cases. No significant changes of these six genes shown in here acted against the notion that DNA methylation related mechanisms underline potential inactivation of this set of genes in the pathogenesis of astrocytoma. The infrequent hypermethylation of the p16INK4a gene in astrocytoma was a total surprise, as it was frequently reported hypermethylated in various human tumors tested, including in HCC where we have previously found that the p16INK4a, MYOD1, CDH13 and cyclin a1 genes were frequently methylated [18,24]. To further verify this unexpected observation, we repeated the MSP analysis on five astrocytoma samples (shown unmethylated) along with one HCC sample (previously shown heterozygously methylated). As shown in panel 1, Fig. 1, MSP patterns of the astrocytoma as well as HCC tissues remained the same. The identities of which were also confirmed by sequencing (panel 2, Fig. 1), showing that while the MSP products with the primers specific to the methylated targets in the HCC sample (Z92K) contained CpGs, the unmethylated targets in all the five astrocytoma tissues (21, 22, 26, A11 and B6) contained TpGs. Therefore, lack of hypermethylation of the p16INK4a gene in astrocytoma was unlikely incorrect, which is consistent with a recent report that inactivation of the p16INK4a gene in 48% of astrocytoma cases was genetic [55]. Figure 1 MSP/sequencing analyses of the p16INK4a gene in astrocytoma and hepatocellular carcinoma Both electrophoretic patterns of the PCR products of the p16INK4a in each of five astrocytoma cases (21, 22, 26, A11 and B6) and one HCC case (Z92K) (indicated respectively, at the top of figures) were presented. To indicate the methylation status, the sequenced data are aligned with the wild-type sequence. The remaining 7 targets were hypermethylated more frequently, occurring in 26.4% to 69.8% (14 to 37/53) of astrocytoma cases. The OCT6 gene was hypermethylated in 30.2% of the astrocytoma cases (16/53). Despite of the association of the OCT6 methylation with the aging process reported previously, we found no significant correlation/association of the OCT6 methylation to any clinical-pathological features, including age, gender and clinical grading of the patients. The significance of such a prevalent occurrence of the hypermethylated OCT6 gene remains to be determined. The RASSF1A (hypermethylated in 37/53 cases, 69.8%) is a variant of the recently identified tumor suppressor, the RASSF1 gene that acts at downstream of the Ras mediated apoptotic pathway and is capable of binding to Ras in a GTP dependent manner [36]. The RASSF1A gene has a more extended 5' part and its promoter CpG island displays a tumor specific hypermethylated profile in a variety of tumors, HCC in particular. Furthermore, lack of the RASSF1A expression in nineteen established tumor cell lines correlates with the hypermethylated state of its promoter CpG island [36]. The RASSF1C gene has its own promoter CpG island, but is not methylated in any tumors. The methylation behavior of these two genes was very similar to our previous observation in hepatocellular carcinoma, where 22/29 cases (79%) had the fully methylated 1A along with the unmethylated 1C variants [18]. As shown in Additional file 4,5,6,7,8,9, the RASSF1A promoter-CpG island was methylated in 69.8% (37/53) of astrocytoma tissues, while the C variant was not methylated in any astrocytoma tissues. The hypermethylated state of the RASSF1A promoter CpG island was not correlated with gender, age and clinical grading. Consistent with the hypermethylated status of the RASSF1A gene in U251 cells, no expression at the mRNA level was detected. Partial demethylation of its promoter by the treatment with 5-Aza-2'-deoxycytidine indeed resulted in its transcription (Fig. 2). Figure 2 The methylation state and expression profiles of the CDH13, p73, MAGEA1, MGMT and RASSF1A genes in U251 astrocytoma cells before and after the demethylation treatment with 5-Aza-2'-deoxycytidine U251 cells were subjected to the 10 and 20 nM 5-Aza-2'-deoxycytidine (5-Aza) treatment for 3 days before both DNA and RNA were prepared for either MSP analyses or RT-PCR assessments. Panels; A, the methylation status of the CDH13, p73, MAGEA1, MGMT and RASSF1A genes and B, the expression profiles of each of these five genes, respectively in U251 cells. The p73 gene encodes a homologue to TP53, and loss of its heterozygosity has been observed in up to 90% of oligodendrogliomas and in 10–25% of diffuse astrocytoma [56,57]. In this study, we found that the p73 gene was prevalently methylated (25/53, 47.2%) with no significant association with any clinical-pathological parameters, such as gender and the WHO grading. The occurrence of the hypermethylated p73 gene was more prevalent in our results than a recent report which detected the hypermethylated p73 gene in 18% (5 /28) of the WHO grade IV but not in grade III astrocytoma [35]. Again, even the partially elevated demethylated status of its promoter CpG island in U251 cells resulted in reactivation of p73 transcription (Fig. 2). Both genetic defects and epigenetic abnormalities of the WT1 gene have been etiologically implicated in the formation of the Wilm's tumor [58]. In this study, we also found that the WT1 gene was hypermethylated in 30% (16/53) of cases, implying its possible involvement in the formation of astrocytoma. Tumor resistance to the cytotoxic chemotherapies may result from the disrupted apoptosis programs and remains a major obstacle in cancer treatment. In this study, the interferon regulatory factor 7 (IRF7) gene was analyzed. The analogue (IRF1) of IRF7 has been implicated in the IFN gamma mediated apoptosis with a profound effect on the chemo-sensitivity of tumor cells [59,60]. In consistence with the recent report that the IRF7 expression was negatively regulated by the promoter methylation [61], we found that the IRF7 gene was hypermethylated in astrocytoma (14/53, 26.5%) (Additional file 4,5,6,7,8,9), with a strong male inclination (11/26, 42.3% verse the female group: 3/27, 11%, χ2 = 6.632, P = 0.014). Although the gender difference remains to be understood, such a strong male association with IRF7 hypermethylation may have prognostic value. O(6)-methylguanine-DNA methyltransferase (MGMT), a DNA repair enzyme, removes alkylating adducts from the O(6) position of guanine and protects cells from cytotoxic and mutagenic stress. Silencing of the MGMT gene has been suggested to predispose the neoplastic clones to acquisition of the guanine to adenine point mutations in K-ras and p53 [62] and is associated with low-levels of micro-satellite instability in colorectal cancer [63]. We found that the MGMT gene was prevalently hypermethylated in astrocytoma (35%, 19/53), and its transcription could be reactivated by demethylation with 5-Aza-2'-deoxycytidine in U251 cells (Fig. 2). Hence, the MGMT hypermethylation in astrocytoma may indeed have the pathological significance. In this connection, a recent report suggested that the astrocytoma sensitivity to the alkylating type of chemotherapeutics might be contributed by the hypermethylated MGMT gene [64]. Expression of the metallothionein I A (MT1A) is inducible by a number of adversary agents such as heavy metals and oxidative stress. Both basal and inducible expression of this gene has been impaired in various tumor cell lines and attributed to the hypermethylated state of this gene [48]. In this study, we found that the MT1A gene was hypermethylated in 30% (16/53) of cases, with no significant gender and grading difference. The functional and pathological implications of the MT1A hypermethylation in astrocytoma remain to be established. Cadherins, the calcium-dependent proteins, contribute to various biological processes such as differentiation, migration and extra-cellular signal transduction of cell. Loss of expression of both E-cadherin (CDH1) and H-cadherin (CDH13) has been found in parallel with the hypermethylated promoter CpG islands in various cancers [65,66]. In this study, we found that the CDH1 gene was hypermethylated in 32.8% (17/53) of astrocytoma tissues, while the CDH13 gene was not methylated in all the astrocytoma tissues examined (Additional files 4,5,6,7,8,9). In contrast, in human hepatocellular carcinoma [18], the CDH1 gene was unmethylated, while the CDH13 gene was frequently hypermethylated. Obviously, the molecular basis for tumor type specific methylation patterns of these two genes remains to be determined. Although the hypermethylation mediated gene silencing of the tumor suppressor genes is at the focal point of the epigenetic studies, demethylated status of the promoter CpG islands has been linked to the tumor associated activation of the normally silenced genes [19-23]. Therefore, we also studied both MAGEA1 and SURVIVIN genes. The promoter CpG islands were hypermethylated in normal tissues (for MAGEA1 in HCC [18] and for SURVIVIN in ovarian cancer [23]) and demethylated in parallel with the transcriptional activation in tumor cells. The unmethylated status of the SURVIVIN gene in astrocytoma is consistent with the over-expression of this gene (unpublished observations). However, its unmethylated status in all the non-astrocytoma tissues acts odd with the notion that its demethylation is associated with pathogenesis in human ovarian cancer reported previously [23]. Our previous studies indicated that demethylation of the promoter CpG island was correlated well with the over-expression profile of the MAGEA1 gene [18,21] in HCC. The MAGEA1 gene was fully hypermethylated in all four cases of the normal liver tissues but significantly demethylated in HCC tissues (21/28, 75%). It was found fully hypermethylated in all the three control tissues and in 74.5% (40/53) of the astrocytoma tissues and partially hypermethylated (13/53, 25.5%) in the other astrocytoma tissues. The occurrence of the MAGEA1 demethylation in HCC differed significantly from astrocytoma (75% verse 25.5%, P < 0.001). As it was fully methylated in the normal tissue, the partial hypermethylation (both hypermethylated and demethylated alleles existed) would imply that the event resulting in the loss of the hypermethylation state of the MAGEA1 gene indeed occurred in astrocytoma and should be scored positive for the changes in the methylation pattern in this study. The same principle has been applied for the opposite changes from the unmethylated pattern in the normal control to the partial or full hypermethylated status of all the other genes in astrocytoma tissues. It was also found that the partial demethylated status of the MAGEA1 gene in U251 cells induced by 5-Aza-2'-deoxycytidine occurred co-currently with activation of its transcription (Fig. 2). The gender association of the methylation profiles of the AR and IRF7 gene in astrocytoma By statistic analysis with both Pearson Chi-Square and Fisher's Exact tests, associations of the DNA methylation profiles of the targets displaying no less than 24.5% changes (the RASSF1A, p73, MGMT, CDH1, OCT6, WT1 as well as MAGEA1 genes) with the clinical pathological parameters (age, grading and gender) were assessed. The methylation profiles of the AR and IRF7 genes were found gender-oriented. The AR gene encodes the androgen receptor that plays a key role in the signal transduction pathways in response to the male steroid hormone, androgen and has been reported to be inactivated via the epigenetic mechanism in prostate cancers [67]. Physiologically, the AR gene should express exclusively in the somatic cells in males, while lacking of its expression in females is likely mediated by DNA methylation based mechanisms. Indeed, the hypermethylated along with the unmethylated AR genes were only found in the normal female brain tissue, but not from two male non-astrocytoma samples. The hypermethylation of the AR gene occurred frequently in the female group (81.5%, 22/27) but not in any males (0%, 0/26, χ2 = 36.22, P = 0.000). It may simply be gender associated and do not have any significant relevance to carcinogenesis of astrocytoma. It was also noticed that hypermethylation of the IRF7 gene displayed an opposite gender inclination, detected in 11% of the female patients (3/27), and 42% of male patients (11/26, χ2 = ?6.632, P = 0.014). Despite of the difficulty to offer a mechanistic interpretation, the potential prognostic value of such a gender-associated phenomenon might be worthwhile exploring in future. Demethylation by 5-Aza-2'-deoxycytidine treatment of the astrocytoma cells in culture resulted in partial demethylation and reactivated expression of the genes The hypermethylated status of the promoter CpG island has been linked to gene transcription silencing in a number of biological settings. The effect of the astrocytoma associated changes in the methylated state of the promoter CpG islands detected in this study on gene expression was assessed in U251 astrocytoma cells treated with the a demethylating agent, 5-Aza-2'-deoxycytidine. We used MSP to establish the methylation status of the promoter CpG island of all the genes with the astrocytoma associated methylation changes (Additional files 3,4,5,6,7,8,9) in U251 astrocytoma cells, and analyzed the ability of 5-Aza-2'-deoxycytidine to demethylate five genes, as measured by MSP, and reactivate their expression, as detected by RT-PCR. As shown in panel 1 of Fig. 2, while the CDH13, MAGEA1 and p73 genes were heterozygously methylated, both MGMT and RASSF1A genes were fully hypermethylated in U251 cells. The CDH13 gene was found expressed, while the rest transcriptionally inert as measured by the RT-PCR. Although both methylated and unmethylated alleles for p73 and MGMT genes were evident in U251 cells, no expression was detected, indicating that the unmethylated allele may remain silent by the other mechanisms, including the genetic defects at critical control region. By the 5-Aza-2'-deoxycytidine treatment, both demethylation of the promoter CpG island and activation of transcription of these five genes were achieved (Fig. 2). Despite of the fact that demethylation of the promoter CpG islands was incomplete in samples treated with 20 nM 5-Aza-2'-deoxycytidine (Fig. 2), the expression of this five genes was either induced (the MAGEA1, MGMT, p73 and RASSF1A genes) or elevated (the CDH13 gene). The concordant methylation behavior of the promoter CpG islands of the genes in Astrocytoma The DNA methylation mediated epigenetic changes also display the tumor type specific patterns, which seem to reflect the differentiation and maturation histories of the cell lineages as well as the aging process during which both global hypo- and local hyper-methylation occur. Hypermethylation of the promoter CpG islands in accord with the transcriptional silencing of the tumor suppressor genes, such as the p16INK4a, and RASSF1A genes, has been well established in human tumors [16,68]. However, it remains unclear whether there is a common mechanism for the concurrent methylation changes of multiple tumor suppressor genes in tumors. To address this matter, it is necessary to examine a large number of genes for frequent changes in methylation in any type of human tumors. The concordant methylation behavior of multiple genes was firstly detected in colon cancer [69], based upon a comprehensive methylation profiling of over thirty genes. In this study, we have profiled the methylation status of thirty four genes in a cohort of 53 astrocytoma and 3 non-astrocytoma patients. Twenty three of these genes had not been studied previously in astrocytoma. As far as the number of the genes is concerned, this study is the most extensive in the astrocytoma field to our knowledge. Among thirty four genes, sixteen genes exhibited the astrocytoma associated changes in methylation profiles of the promoter CpG islands and nine genes displayed rather frequent changes (the occurrence ≥ 13/53, frequency ≥ 24.5%) (Additional file 8). Four of 53 cases (7.5%) maintained the same methylation profile as the normal control. The rest 49 cases (92.5%) suffered from the methylation changes as much as no less than one target, an occurrence was significantly lower than in HCC, where all the cases displayed methylation changes affecting no less than three targets in the studies involved with twenty or twenty four targets [18,24], indicating that alterations in DNA methylation \occur more frequently in HCC than in astrocytoma. This may be contributed by the apparent anatomic inaccessibility of the brain to environmental adverse factors in comparison to the liver. The size of the subsets containing various number of the target affected (from one to nine) ranged from 1 to 11 cases, and peaked with 10 cases at three and 11 cases at five target subsets (Additional file 9). To identify the most frequent changes of the target sets (one to three), a mathematic method called "the Discovery of Association Rules" [28] was used. The co-occurrence (case number/the total) and frequency (% of the total) of any subset of the targets that changed in methylation together in astrocytoma were counted and compared. In the entire cohort of patients in this study, the most altered target was the RASSF1A gene, 69.8% (37/53). The two genes that most altered together were the RASSF1A and p73 genes, hypermethylation of which was found in 20 (37.7%). Three genes that changed together were the former two plus CDH1 or OCT6, hypermethylation of which occurred in 20.8% cases (11/53) (Column 2, a, Additional file 10). Furthermore, the occurrence in methylation change in any target in the two gene subsets was 79.3% (42/530 and in three gene subsets was 81.1–83% (43–44/53) (Column 3, a, Additional file 10). Since the hypermethylated AR is associated closely with the female gender of the astrocytoma patients and devoid of any association with the formation of astrocytoma, it was taken out from this analysis. Hypermethylation of the RASSF1A gene occurred in 21 female cases (77.8%, 21/27). Both RASSF1A and WT1 were hypermethylated in 13 (13/27, 48.1%); and the former two plus the hypermethylated p73 or CDH1 or OCT6 were found in 9 female cases (9/27, 33.3%), respectively (Column 2, b, Additional file 10). The subsets in the male patient group showed very different patterns. The single to three target subsets were the RASSF1A (16/26, 61.5%); the RASSF1A and IRF7 (10/26, 38.5%); and the former two plus the p73 or MGMT or MT1A (5/26, 19.2%), respectively (c, Additional file 10). In Grade I astrocytoma, the subsets for one, two and three targets were RASSF1A (10/14, 71.4%), RASSF1A plus p73 (6/14, 42.9%), and the former two plus either WT1 or IRF7 or MAGEA1 as well as RASSF1A plus CDH1 and WT1 (3/14, 21.4%). For Grade II astrocytoma, the corresponding sets consisted of the RASSF1A (12/15, 80%), the RASSF1A and MGMT or IRF7 (5/15, 33.3%), and the RASSF1A and MGMT plus p73 or OCT6, or MT1A, or WT1 as well as the RASSF1A and IRF7 and MT1A (3/15, 20%), respectively. For Grading III astrocytoma, those subsets were composed of the RASSF1A (8/12, 66.7%), the RASSF1A and CDH1 (5/12, 41.7%), and the formal two plus MGMT (4/12, 33.3%), respectively. For Grading IV astrocytoma, the comparative subsets contained the RASSF1A or p73 (7/12, 58.3%), the RASSF1A and p73 (6/12, 50%), and the former two plus MGMT or OCT6 (4/12, 33.3%), respectively. (d-g, Additional file 10). Our methylation profiling efforts described in this report provided the following informative targets: the RASSF1A, p73, WT1, MGMT, CDH1, OCT6, and IRF7 genes. The established concordant methylation profiles of these eight genes (Additional file 10) may provide useful clues for the epigenetic biomarker selection to for the novel diagnostic and prognostic assays of astrocytoma. The hypermethylated status of this lest of genes in the serum, and biopsies of the suspected astrocytoma patients may serve as good diagnostic indicators, if they can be reliably detected. With the death/survival profiles of this cohort of astrocytoma patients available in the future, the methylation profile established in this study may have certain prognostic value. Abbreviations HCC: Hepatocellular carcinoma; PCR: polymerase chain reaction; MSP: methylation specific PCR; ABL: v-abl Abelson murine leukemia viral oncogene homolog 1; APAF1: apoptotic protease activating factor; APC: adenomatosis polyposis coli; AR: androgen receptor; BRCA1: breast cancer 1; CAV: caveolin 1, caveolae protein; CDH1: cadherin type 1, E-cadherin; CDH13: cadherin 13, H-cadherin; CSPG2: chondroitin sulfate proteoglycan 2 (versican); cyclin a1: cyclin A1; DAPK1: death-associated protein kinase 1; DBCCR1: deleted in bladder cancer chromosome region candidate 1; EPO: erythropoietin; hMLH1: mutL homolog 1, colon cancer, nonpolyposis type 2; IRF7: interferon regulatory factor 7; LKB1: serine/threonine kinase 11 (Peutz-Jeghers syndrome); MAGEA1: melanoma antigen, family A, 1 (directs expression of antigen MZ2-E); MGMT: O-6-methylguanine-DNA methyltransferase; MT1A: metallothionein 1A (functional); MYOD1: myogenic factor 3; OCT6: POU domain, class 3, transcription factor 1; p14ARF: the alternative reading frame of the cyclin-dependent kinase inhibitor 4a; p15INK4b: cyclin-dependent kinase inhibitor 4b; p16INK4a: cyclin-dependent kinase inhibitor 4a; p27KIP1: cyclin-dependent kinase inhibitor 1B (p27, KIP1); p57KIP2: cyclin-dependent kinase inhibitor 1C (p57, KIP2); p73: tumor protein p73; PTEN: phosphatase and tensin homolog; RASSF1A: ras association (RalGDS/AF-6) domain family 1 protein isoform 1a; RASSF1C: ras association (RalGDS/AF-6) domain family 1 protein isoform 1c; RB1: retinoblastoma 1; VHL: von Hippel-Lindau syndrome; WT1: Wilms tumor 1. Competing interests None declared. Authors' contributions JY, HYZ, JG, executing the experiments; SL and JHL, providing the patient samples; WL and YFW, carrying out the mathematic analyses of the data JDZ: designing and organizing experiments as well as completing manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: Supplementary Material Additional File 1 The target promoter CpG islands and the primer pairs for methylation specific PCR. This file contains his study. Click here for file Additional File 2 Methylation profiles of thirty three genes on the in vitro methylated genomic DNA by M. Sss I methyl transferase. The Eco RI restricted genomic DNA from the liver tissue of a healthy donor was in vitro methylated overnight with M. Sss I methyl transferase and subjected to the MSP analysis, followed by electrophoresis in a 1.3% agarose gel. *, the DNA size markers, NL, the untreated sample, U and M, MSP with the pair of primers specific to the unmethylated and methylated targets, respectively. Panels: 1, ABL; 2, APAF1; 3, APC; 4, AR; 5, BRCA1; 6, CAV; 7, CDH1; 8, CDH13; 9, CSPG2; 10, cyclin a1; 11, DAPK1; 12, DBCCR1; 13, EPO; 14, hMLH1; 15, IRF7; 16, LKB1; 17, MGMT; 18, MT1A; 19, MYOD1; 20, OCT6; 21, p14ARF; 22, p15INK4b; 23, p16INK4a; 24, p27 KIP1; 25, p57KIP2; 26, p73; 27, PTEN; 28, RASSF1A; 29, RASSF1C; 30, RB1; 31, SURVIVIN; 32, VHL and 33, WT1. Click here for file Additional File 3 Methylation profiles of thirty four genes in astrocytoma (part I). Both electrophoretic patterns of the representative PCR products of each of thirty four targets (indicated respectively, at the top of figures) and the sequencing verification of the one representative PCR product were presented. To indicate the methylation status, the sequenced data are aligned with the wild-type sequence. *, size markers, the bands of 250 bp and 100 bp were shown. U, the unmethylated; M, the hypermethylated. Panels: 1, ABL; 2, APAF1; 3, APC; 4, AR; 5, BRCA1; 6, CAV; 7, CDH1; 8, CDH13; 9, CSPG2; 10, cyclin a1; 11, DAPK1 and 12, DBCCR1. Click here for file Additional File 4 Methylation profiles of the promoter CpG islands of thirty four genes in astrocytoma (part II). Both electrophoretic patterns of the representative PCR products of each of thirty four targets (indicated respectively, at the top of figures) and the sequencing verification of the one representative PCR product were presented. To indicate the methylation status, the sequenced data are aligned with the wild-type sequence. *, size markers, the bands of 250 bp and 100 bp were shown. U, the unmethylated; M, the hypermethylated. Panels: 13, EPO; 14, hMLH1; 15, IRF7; 16, LKB1; 17, MAGEA1; 18, MGMT; 19, MT1A; 20, MYOD1; 21, OCT6 and 22, p14ARF>. Click here for file Additional File 5 Methylation profiles of the promoter CpG islands of thirty four genes in astrocytoma (part III). Both electrophoretic patterns of the representative PCR products of each of thirty four targets (indicated respectively, at the top of figures) and the sequencing verification of the one representative PCR product were presented. To indicate the methylation status, the sequenced data are aligned with the wild-type sequence. *, size markers, the bands of 250 bp and 100 bp were shown. U, the unmethylated; M, the hypermethylated. Panels: 23, p15INK4b; 24, p16INK4a; 25, p27 KIP1; 26, p57KIP2; 27, p73; 28, PTEN; 29, RASSF1A; 30, RASSF1C; 31, RB1; 32, SURVIVIN; 33, VHL and 34, WT1. Click here for file Additional File 6 The summary of the astrocytoma cases displaying no or changes in the methylation profiles (part I). The frequency (%) of the astrocytoma displaying no or the changes in the methylation profile of each target from the normal control were counted and presented in table as well as plotted in the figure below. The filled, shading and empty boxes indicate the cases where only hypermethylated allele, both hypermethylated and unmethylated alleles and only unmethylated alleles were detected, respectively. The frequency (%) of the hypermethylated targets (except for the MAGEA1 gene) among the total cases was scored for positive changes in astrocytoma. The MAGEA1 was fully methylated (3/3, 100%) in the control, and become partially demethylated in some astrocytoma, therefore, demethylation of the MAGEA1 in astrocytoma was scored as positive changes. Sub-tables: a, the female patient group, b, the male patient group, and c, the control. Click here for file Additional File 7 The summary of the astrocytoma cases displaying no or changes in the methylation profiles (part II). The frequency (%) of the astrocytoma displaying no or the changes in the methylation profile of each target from the normal control were counted and presented in table as well as plotted in the figure below. Sub-tables d-h, the WHO grading I to IV, respectively; The filled, shading and empty boxes indicate the cases where only hypermethylated allele, both hypermethylated and unmethylated alleles and only unmethylated alleles were detected, respectively. The frequency (%) of the hypermethylated targets (except for the MAGEA1, where the heterozygously hypermethylated) among the total cases was presented in the plot. Click here for file Additional File 8 The occurrences and frequency of changes in methylation. *, One of three cases was methylated; **, The MAGEA1 gene was fully methylated in the normal tissues and partially demethylated in astrocytoma patients as indicated in the relevant cells. Therefore, the astrocytoma associated changes in methylation of this gene is opposite to the rest, i.e., demethylation rather than hypermethylation. Figure is each cells are the frequency in % and occurrence (case number). Click here for file Additional File 9 The summary of changes in the methylation pattern in subsets. Both occurrence (case number) and frequency (%) for the subsets having no change in methylation and changes in one to nine genes are presented in % and (case number) in the top half of table, which was also plotted. Both occurrence (case number) and frequency (%) for the subsets having no change in methylation and changes in, at least, one to nine genes are presented in % and (case number) in the bottom half of table. Click here for file Additional File 10 The summary of the concordant methylation behavior of the hypermethylated targets in astrocytoma. The co-occurrence (/total case) and frequency (%) of a panel subsets consisting of one to three targets were treated with method "Discovery Association Rules" and presented. Sub-tables: a, the total, b, the female, c, the male, and d-g, the grade I to IV, respectively. Column 1 is the number of target in each subset. Column 2 is the co-occurrence (case number/total) (frequency in %). Column 3 is the occurrence of any single target in each subsets, presented in case number (frequency %). The column 4 is the gene(s) in subset. N.B., In view of the strong female inclination of the AR methylation and lacking of any association with astrocytoma, AR has been taken off from this analyses. Click here for file Acknowledgements This work was supported by the National High Technology Research and Development Program of China (863 Program) (2001AA217011, 2002AA2Z3352), the Major State Basic Research Development Program of China (973 Program) (G1998051004), and the Science Foundation of Shanghai Municipal Government (02DJ14056) to Jingde Zhu. Thanks are due to J. Xin for his assistance in the statistical analysis of the data and D. Niu, J. Li, P. Wang, and C. 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==== Front BMC PsychiatryBMC Psychiatry1471-244XBioMed Central London 1471-244X-4-261535554610.1186/1471-244X-4-26Research ArticleThe efficacy of duloxetine: A comprehensive summary of results from MMRM and LOCF_ANCOVA in eight clinical trials Mallinckrodt Craig H [email protected] Joel [email protected] Madelaine M [email protected] John G [email protected] Michael J [email protected] Eli Lilly & Company, Lilly Corporate Center, Indianapolis, IN, USA2 Departments of Psychiatry, Harvard Medical School, Boston MA; McLean Hospital, Belmont MA; and Indiana University School of Medicine, Indianapolis, IN, USA2004 8 9 2004 4 26 26 23 3 2004 8 9 2004 Copyright © 2004 Mallinckrodt et al; licensee BioMed Central Ltd.2004Mallinckrodt et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background A mixed-effects model repeated measures approach (MMRM) was specified as the primary analysis in the Phase III clinical trials of duloxetine for the treatment of major depressive disorder (MDD). Analysis of covariance using the last observation carried forward approach to impute missing values (LOCF_ANCOVA) was specified as a secondary analysis. Previous research has shown that MMRM and LOCF_ANCOVA yield identical endpoint results when no data are missing, while MMRM is more robust to biases from missing data and thereby provides superior control of Type I and Type II error compared with LOCF_ANCOVA. We compared results from MMRM and LOCF_ANCOVA analyses across eight clinical trials of duloxetine in order to investigate how the choice of primary analysis may influence interpretations of efficacy. Methods Results were obtained from the eight acute-phase clinical trials that formed the basis of duloxetine's New Drug Application for the treatment of MDD. All 202 mean change analyses from the 20 rating scale total scores and subscales specified a priori in the various protocols were included in the comparisons. Results In 166/202 comparisons (82.2%), MMRM and LOCF_ANCOVA agreed with regard to the statistical significance of the differences between duloxetine and placebo. In 25/202 cases (12.4%), MMRM yielded a significant difference when LOCF_ANCOVA did not, while in 11/202 cases (5.4%), LOCF_ANCOVA produced a significant difference when MMRM did not. In 110/202 comparisons (54.4%) the p-value from MMRM was lower than that from LOCF_ANCOVA, while in 69/202 comparisons (34.2%), the p-value from LOCF_ANCOVA was lower than that from MMRM. In the remaining 23 comparisons (11.4%), the p-values from LOCF_ANCOVA and MMRM were equal when rounded to the 3rd decimal place (usually as a result of both p-values being < .001). For the HAMD17 total score, the primary outcome in all studies, MMRM yielded 9/12 (75%) significant contrasts, compared with 6/12 (50%) for LOCF_ANCOVA. The expected success rate was 80%. Conclusions Important differences exist between MMRM and LOCF_ANCOVA. Empirical research has clearly demonstrated the theoretical advantages of MMRM over LOCF_ANCOVA. However, interpretations regarding the efficacy of duloxetine in MDD were unaffected by the choice of analytical technique. ==== Body Background Treatment effects are often evaluated by comparing change over time in outcome measures. However, valid analyses of longitudinal data can be problematic, particularly if some data are missing for reasons related to the outcome measure [1,2]. Since the problem of missing data is almost ever-present in clinical trials, numerous methods for handling missingness have been proposed, examined, and implemented [3]. A common method of analyzing clinical trial data is to use analysis of variance or analysis of covariance (ANOVA or ANCOVA) with missing data imputed by the last observation carried forward approach (LOCF_ANCOVA). The popularity of LOCF_ANCOVA may be due to its simplicity, and also the belief that violations of the restrictive assumptions inherent to LOCF_ANCOVA lead to a conservative analysis [4]. Considerable advances in statistical methodology, and in our ability to implement these methods, have been made in recent years. Thus, methods that require less restrictive assumptions than LOCF_ANCOVA are now readily implemented. For example, likelihood-based repeated measures approaches have a number of theoretical and practical advantages for analysis of longitudinal data with dropout [4]. One such method, termed MMRM (Mixed Model Repeated Measures [5]), has been studied extensively in the context of neuropsychiatric clinical trials [6-9]. In these studies, MMRM was found to be more robust to biases from missing data than LOCF_ANCOVA, and thereby provided superior control of Type I and Type II errors. The LOCF_ANCOVA method was shown to underestimate treatment group differences in some scenarios, while overestimating differences in others. When no data were missing, the two methods yielded identical results. The MMRM approach was specified as the primary analysis in the Phase III clinical trials of duloxetine for the treatment of major depressive disorder (MDD), while LOCF_ANCOVA was specified as a secondary analysis. In the present investigation, we provide a comprehensive summary of results from MMRM and LOCF_ANCOVA in the eight acute-phase clinical trials that formed the basis for duloxetine's New Drug Application (NDA) for MDD. The primary objective of this investigation was to determine whether differences in results between MMRM and LOCF_ANCOVA influenced conclusions regarding the efficacy of duloxetine. Methods Data The data source for this investigation was the eight acute-phase clinical trials in which duloxetine was compared with placebo in the treatment of MDD. Relevant details of these studies are highlighted in Table 1. Table 1 Summary of studies included in the comparisons between MMRM and LOCF_ANCOVA Study Treatment Duration Drug Number of Patients Drug Dose & Design 1 9 weeks Placebo 122 - Duloxetine 123 60 mg/d (QD) 2 9 weeks Placebo 139 - Duloxetine 128 60 mg/d (QD) 3 8 weeks Placebo 70 - Duloxetine 70 40–120 mg/d (20 mg-60 mg BID) Fluoxetine 33 20 mg/d (QD) 4 8 weeks Placebo 75 - Duloxetine 82 40–120 mg/d (20 mg-60 mg BID) Fluoxetine 37 20 mg/d (QD) 5 8 weeks Placebo 90 - Duloxetine 91 40 mg/d (20 mg BID) Duloxetine 84 80 mg/d (40 mg BID) Paroxetine 89 20 mg/d (QD) 6 8 weeks Placebo 89 - Duloxetine 86 40 mg/d (20 mg BID) Duloxetine 91 80 mg/d (40 mg BID) Paroxetine 87 20 mg/d (QD) 7 8 weeks Placebo 93 - Duloxetine 95 80 mg/d (40 mg BID) Duloxetine 93 120 mg/d (60 mg BID) Paroxetine 86 20 mg/d (QD) 8 8 weeks Placebo 99 - Duloxetine 93 80 mg/d (40 mg BID) Duloxetine 103 120 mg/d (60 mg BID) Paroxetine 97 20 mg/d (QD) Results are summarized from all rating scale total scores, subscales, and global assessments that were specified a priori in the various protocols to be analyzed for mean change from baseline to endpoint, and were collected at more than one postbaseline time point (Table 2). Efficacy measures that were assessed only at baseline and endpoint were not included in this summary because repeated measures analyses were not possible for these outcomes. Thus, the present investigation included every rating scale total score and subscale from every clinical trial relevant to duloxetine's NDA for an indication in major depression. In total, 20 efficacy and health outcome variables were included in the summary of MMRM and LOCF_ANCOVA. Some of the eight trials included multiple dose arms; therefore, some outcomes were assessed in as many as 12 comparisons with placebo. Table 2 Outcomes included in the summary of results. 17-item Hamilton Rating Scale for Depression (HAMD17)  Total score [24]  Core subscale (items 1, 2, 3, 7, 8) (not published)  Maier subscale (items 1, 2, 7, 8, 9, 10) [25]  Anxiety/Somatization subscale (items 10, 11, 12, 13, 15, 17) [26]  Retardation subscale (items 1, 7, 8, 14) [27]  Sleep subscale (items 4, 5, 6) [27] Montgomery-Asberg Depression Rating scale [28] Hamilton Anxiety Rating Scale total score [29] Clinical Global Impression of Severity [26] Patient Global Impression of Improvement [26] Visual Analog Scale for pain [30]  Overall pain severity  Headaches  Back pain  Shoulder pain  Time in pain while awake  Interference with daily activities Somatic Symptom Inventory [31]  26 Item total score  28 Item total score (includes 2 additional questions on painful physical symptoms) Quality of Life in Depression Scale total score [32] Comparisons of MMRM and LOCF_ANCOVA focused on contrasts between duloxetine and placebo. However, six of the studies also included known effective antidepressants approved for marketing in the United States and other countries. Contrasts between duloxetine and the active comparators are not included in this summary since these results may draw attention to the drug versus drug results and detract from the primary focus of comparing MMRM with LOCF_ANCOVA. Statistical analysis This summary makes no attempt to provide formal statistical comparisons of results from MMRM and LOCF_ANCOVA. Previous research has demonstrated conclusively that in the absence of missing data the two methods yield identical endpoint contrasts, while differences do exist in the presence of subject dropout [6-9]. Furthermore, formal statistical comparisons are typically applied to random samples obtained from larger populations in order to assess the uncertainty associated with the sampling. However, the eight studies included in this summary are not a sample, but rather represent all of the acute-phase, double-blind, placebo-controlled trials of duloxetine. Thus, there is no uncertainty associated with sampling. Consequently, results from the two methods need only be summarized in order to assess how differences between the methods may influence overall conclusions regarding the efficacy of duloxetine. Three overall summary measures were used to compare results from the two analytic techniques: 1) With regard to statistical significance of the difference between duloxetine and placebo, the proportion of outcomes showing agreement between MMRM and LOCF_ANCOVA was compared with the proportion of outcomes for which MMRM and LOCF_ANCOVA yielded disparate results; 2) The proportion of outcomes for which MMRM yielded the lowest p-value was compared with the corresponding proportion for LOCF_ANCOVA; 3) The number of outcomes for which "substantial evidence of efficacy" was demonstrated. In regulatory settings, the criterion for substantial evidence of efficacy is frequently the demonstration of a statistically significant advantage over placebo in two or more studies. This criterion was utilized here to define substantial evidence of efficacy for a particular outcome. The frequency of lower p-values provides a "fine-tuned" measure of sensitivity of the two analytic methods. However, in certain cases such an assessment may actually be misleading. For example, to distinguish between p= .800 and p = .810, or between p =. 002 and p =. 003, implies that the methods yielded different results when in fact the similarities far outweigh the differences. Hence, it is equally appropriate to simply categorize based upon the presence or absence of a significant difference. Furthermore, given the large number of outcomes assessed across the eight studies, it would not be surprising to see the two methods disagree with regard to statistical significance on at least a small number of outcomes. Therefore, perhaps the most clinically meaningful summary measure is the number of outcomes for which substantial evidence of efficacy was demonstrated. Three outcomes were selected for more detailed presentation of results: the 17-item Hamilton Rating Scale for Depression (HAMD17) total score, the HAMD17 Maier subscale, and the Visual Analog Scale (VAS) for overall pain severity. The HAMD17 total score was an obvious choice as it was the primary outcome in all studies. The other outcomes were selected since they are frequently focal points in manuscripts and presentations regarding duloxetine's efficacy. Finally, we provide case studies to help explain how and why results from MMRM and LOCF_ANCOVA may differ. MMRM and LOCF_ANCOVA analyses were specified in the Phase III duloxetine protocols as follows. In LOCF_ANCOVA analyses, change from baseline to the last observation was the dependent variable. Treatment and investigative site were included as categorical independent variables, and baseline severity was included as a covariate. In MMRM analyses, change from baseline to all postbaseline times was the dependent variable. Independent variables included the fixed, categorical effects of investigative site, treatment, time, and the treatment-by-time interaction, along with the continuous covariates of baseline severity and the baseline severity by time interaction. Parameters were estimated using Restricted Maximum Likelihood with the Newton-Raphson algorithm. The protocols specified an algorithm for choosing the best fitting covariance structure. In all cases an unstructured matrix provided the best fit. Hence, within-patient errors were modeled using an unstructured covariance matrix. Results The protocols for the eight studies in the duloxetine NDA specified a priori a total of 202 mean change analyses for the 20 rating scale total scores or subscales. The frequency of significant outcomes and the frequency of higher/lower p-values for each analytic technique are summarized in Table 3. MMRM and LOCF_ANCOVA agreed with regard to substantial evidence of efficacy for 18 of the 20 outcomes, with each analysis yielding substantial evidence for 15 outcomes. That is, MMRM and LOCF_ANCOVA both found substantial evidence of efficacy for 14 outcomes; both methods did not find substantial evidence for four outcomes; and each method found substantial evidence when the other did not for one outcome (Table 3). Table 3 Summary of results from MMRM and LOCF_ANCOVA in duloxetine clinical trials. Outcome Frequency of significant outcomes1MMRM LOCF_ANCOVA P-value lower with MMRM LOCF_ANCOVA Outcome significant with MMRM but not LOCF_ANCOVA Outcome significant with LOCF_ANCOVA but not MMRM HAMD17  Total score 9\12 6\12 9\12 1\12 3\12 0\12  Core subscale 9\12 8\12 5\12 3\12 2\12 1\12  Maier subscale 10\12 9\12 6\12 2\12 2\12 1\12  Anxiety subscale 6\12 5\12 7\12 3\12 2\12 1\12  Retardation subscale 8\12 6\12 6\12 3\12 4\12 2\12  Sleep subscale 1\12 1\12 10\12 2\12 0\12 0\12 MADRS Total score 5\10 3\10 7\10 2\10 2\10 0\10 HAMA Total score 3\10 3\10 4\10 5\10 0\10 0\10 CGI-Severity 6\12 6\12 8\12 2\12 0\12 0\12 CGI-Improvement 1\2 1\2 2\2 0\2 0\2 0\2 PGI-Improvement 9\12 7\12 8\12 2\12 2\12 0\12 Somatic Symptom Inventory  26-item Average score 2\10 2\10 7\10 3\10 0\10 0\10  28-item Average score 3\10 2\10 7\10 3\10 1\10 0\10 VAS Pain Severity  Overall pain 3\10 4\10 2\10 8\10 1\10 2\10  Headaches 1\10 0\10 5\10 5\10 1\10 0\10  Back pain 2\10 2\10 6\10 4\10 1\10 1\10  Shoulder pain 2\10 1\10 3\10 6\10 2\10 1\10  Daily activities 1\10 0\10 2\10 8\10 1\10 0\10  Pain while awake 2\10 2\10 5\10 5\10 1\10 1\10 QLDS Total score 1\4 2/4 1\4 2\4 0\4 1\4 Totals 84\202 70\202 110\202 69\202 25\202 11\202 1. Some of the eight trials included more than one dose arm. Therefore, an individual outcome could be assessed in as many as 12 comparisons with placebo. In 166/202 outcomes (82.2%), MMRM and LOCF_ANCOVA agreed with regard to the statistical significance of the difference between duloxetine and placebo. In 25 cases (12.4%) MMRM yielded a significant difference whereas LOCF_ANCOVA did not, while in 11 cases (5.4%) LOCF_ANCOVA yielded a significant difference when MMRM did not. Both methods tended to yield significance more frequently in depression rating scales and subscales than in outcomes related to somatic and painful physical symptoms. The studies were generally underpowered for these secondary somatic and pain outcomes owing to the greater variance in changes score for these outcomes. For example, the variance in VAS overall pain severity was approximately nine-fold greater than the variance in HAMD17 total scores, leading to a three-fold greater standard error. In 110 of the 202 outcomes (54.4%) the p-value from MMRM was lower than that from LOCF_ANCOVA, while in 69 cases (34.2%) the p-value from LOCF_ANCOVA was lower than that from MMRM. In the remaining 23 cases (11.4%) the p-values from LOCF_ANCOVA and MMRM were equal when rounded to the 3rd decimal place (usually as a result of both p-values being < .001). More detailed results from the three focus outcomes (HAMD17 total score, HAMD17 Maier subscale, and VAS overall pain) are presented in Table 4. In the case of the HAMD17 total score, the advantage of duloxetine over placebo in mean change from baseline to endpoint from MMRM analyses was greater than the corresponding advantage from LOCF_ANCOVA in 9/12 comparisons (Table 4). In 9/12 comparisons the p-value from MMRM was lower than that from LOCF_ANCOVA, while LOCF_ANCOVA yielded a smaller p-value in one case, and p-values were identical in the two remaining cases. In 3/12 comparisons, MMRM yielded a significant difference when LOCF_ANCOVA did not, but in no instance did LOCF_ANCOVA produce a significant difference when MMRM did not. When averaging results across all eight studies, the advantage of duloxetine over placebo in HAMD17 total score was 2.4 from MMRM analyses compared with 2.0 from LOCF_ANCOVA. Thus, the advantage of duloxetine over placebo, based on LOCF_ANCOVA results, was approximately 83% as large as the advantage from MMRM. Table 4 MMRM and LOCF_ANCOVA analysis of three focus outcomes from duloxetine clinical trials. Study 1 Study 2 Study 3 Study 4 Study 5 Study 6 Study 7 Study 8 Dose 60 mg QD 60 mg QD 60 mg BID 60 mg BID 20 mg BID 40 mg BID 20 mg BID 40 mg BID 40 mg BID 60 mg BID 40 mg BID 60 mg BID Δ p Δ p Δ p Δ p Δ p Δ p Δ p Δ p Δ p Δ p Δ p Δ p HAMD17Total Score MMRM 4.9 < .001 2.2 .024 3.1 .009 0.9 .415 1.4 .143 1.5 .116 2.4 .034 3.6 .002 2.2 .001 3.3 < .001 1.4 .045 1.6 .014 LOCF 3.8 < .001 1.7 .048 2.1 .066 0.4 .681 1.2 .222 1.5 .138 2.4 .022 3.1 .003 2.2 .007 3.0 < .001 0.9 .253 1.5 .054 HAMD17 Maier Subscale MMRM 2.8 < .001 1.6 .003 2.0 .005 0.7 .282 1.2 .037 1.4 .012 1.1 .068 1.7 .005 1.4 < .001 2.0 < .001 0.9 .022 1.2 .002 LOCF 2.3 < .001 1.4 .007 1.0 .030 0.6 .481 1.0 .058 1.3 .012 1.5 .028 1.8 .004 1.4 .001 1.9 < .001 0.7 .090 1.0 .014 VAS Overall Pain Severity MMRM 5.9 .055 4.4 .135 NA NA 0.3 .931 1.2 .731 1.0 .771 7.4 .035 5.5 .085 6.3 .050 5.6 .044 1.3 .625 LOCF 6.9 .019 5.2 .037 NA NA 3.5 .573 3.5 .647 3.2 .710 7.1 .048 6.1 .063 5.6 .086 7.8 .014 5.5 .066 NA = not assessed Similar results were obtained for the Maier subscale. Thus, the advantage of duloxetine over placebo in mean change from baseline to endpoint from MMRM was greater than that from LOCF_ANCOVA in 9/12 comparisons (Table 4). The p-value from MMRM was lower than that from LOCF_ANCOVA in 6/12 comparisons, while LOCF_ANCOVA produced a lower p-value in 2 cases, and p-values were identical in the remaining four cases. In 2/12 comparisons MMRM yielded a significant difference when LOCF_ANCOVA did not, while there was one instance in which LOCF_ANCOVA yielded a significant difference when MMRM did not. Averaged over all eight studies, the advantage of duloxetine over placebo in mean Maier subscale score was 1.5 from MMRM analyses compared with 1.3 from LOCF_ANCOVA. Thus, the advantage of duloxetine over placebo based on LOCF_ANCOVA results was approximately 87% as large as the advantage from MMRM. For VAS overall pain severity, the advantage of duloxetine over placebo from MMRM analyses was greater than the corresponding advantage from LOCF_ANCOVA in 2/10 comparisons (Table 4). The p-value from MMRM was lower than that from LOCF_ANCOVA in 2/10 comparisons, while in the remaining 8 comparisons the p-value from LOCF_ANCOVA was lower than that from MMRM. In 1 comparison MMRM yielded a significant difference when LOCF_ANCOVA did not, while in 2 comparisons LOCF_ANCOVA yielded a significant difference when MMRM did not. Over all eight studies, the average advantage of duloxetine over placebo in VAS overall pain severity was 3.9 from MMRM analyses compared with 5.4 from LOCF_ANCOVA. Thus, the advantage of duloxetine based on LOCF_ANCOVA results was approximately 138% as large as the advantage from MMRM. Case Studies Mean changes in HAMD17 total score and VAS overall pain severity from two studies (Studies 1 and 2) are used to further illustrate MMRM and LOCF_ANCOVA (analysis of variance with missing data imputed via last observation carried forward) results. Results from these studies were originally reported by Detke et al [10,11]. In both studies the advantage of duloxetine over placebo in HAMD17 total score tended to increase over time whereas duloxetine's advantage in VAS overall pain was greatest at intermediate visits (Tables 5 and 6). Table 5 MMRM and LOCF_ANCOVA analyses of HAMD17 total score in Studies 1 and 2. STUDY 1 STUDY 2 MMRM THERAPY Week N Mean change Std.error p-value N Mean change Std.error p-value DULOX 1 121 -2.89 0.36 .435 123 -2.89 0.38 .601 PLACEBO 1 115 -2.50 0.37 136 -2.64 0.36 DULOX 2 112 -5.72 0.49 < .001 109 -5.54 0.48 .071 PLACEBO 2 110 -3.39 0.50 129 -4.43 0.45 DULOX 3 105 -7.37 0.53 < .001 108 -6.82 0.55 .287 PLACEBO 3 103 -4.58 0.54 122 -6.06 0.52 DULOX 5 100 -8.76 0.60 < .001 98 -8.58 0.66 .116 PLACEBO 5 101 -5.74 0.60 111 -7.20 0.62 DULOX 7 91 -9.93 0.64 < .001 89 -10.14 0.69 .008 PLACEBO 7 93 -5.82 0.65 97 -7.69 0.65 DULOX 9 84 -10.91 0.70 < .001 81 -10.46 0.71 .024 PLACEBO 9 89 -6.05 0.69 90 -8.29 0.67 LOCF_ANCOVA DULOX LOCF_ANCOVA 121 -9.47 0.63 < .001 123 -8.75 0.71 .048 PLACEBO LOCF_ANCOVA 115 -5.67 0.66 136 -7.02 0.68 Table 6 MMRM and LOCF_ANCOVA analyses of VAS overall pain severity in Studies 1 and 2. STUDY 1 STUDY 2 MMRM THERAPY Week N Mean change Std.error p-value N Mean change Std.error p-value DULOX 1 120 -2.69 1.97 .181 121 -2.02 1.89 .157 PLACEBO 1 113 1.04 2.04 134 1.38 1.79 DULOX 2 113 -8.20 1.87 .003 108 -6.95 2.04 .003 PLACEBO 2 108 -0.42 1.93 127 0.91 1.90 DULOX 3 105 -10.46 1.95 .005 106 -9.81 1.90 < .001 PLACEBO 3 101 -2.56 2.01 119 -1.59 1.78 DULOX 5 99 -8.68 2.20 .028 95 -9.70 2.05 .011 PLACEBO 5 99 -1.85 2.24 108 -2.92 1.92 DULOX 7 91 -10.25 2.34 .016 87 -8.91 2.12 .254 PLACEBO 7 92 -2.26 2.38 94 -5.76 2.01 DULOX 9 83 -8.68 2.18 .055 80 -10.05 2.24 .135 PLACEBO 9 88 -2.80 2.20 88 -5.65 2.12 LOCF_ANCOVA DULOX LOCF_ANCOVA 121 -8.31 2.09 .019 121 -10.39 2.05 .037 PLACEBO LOCF_ANCOVA 114 -1.40 2.18 134 -5.22 1.94 In the case of the HAMD17 total score, advantages for duloxetine over placebo at endpoint (Week 9) from MMRM in Studies 1 and 2 were 4.86 (p < .001) and 2.17 (p = .024), respectively. The corresponding advantages from LOCF_ANCOVA were 3.80 (p < .001) and 1.73 (p = .048). Although the differences were significant for both methods in both studies, MMRM yielded treatment contrasts that were approximately 25% greater than LOCF_ANCOVA. For VAS overall pain, the advantage of duloxetine over placebo at endpoint from MMRM in Studies 1 and 2 were 5.88 (p = .055) and 4.40 (p = .135), respectively. The corresponding advantages from LOCF_ANCOVA were 6.91 (p = .019) and 5.17 (p = .037). In both studies, the endpoint differences were significant from LOCF_ANCOVA, but not from MMRM. The LOCF_ANCOVA treatment contrasts were approximately 15% greater than those from MMRM. Standard errors from LOCF_ANCOVA were approximately 5% smaller than the Week 9 standard errors from MMRM for both the HAMD17 total score and VAS overall pain. Discussion In many areas of clinical research, the impact of missing data can be profound [2,12-14]. Traditional approaches to analyses of data from clinical trials with dropouts, such as LOCF_ANCOVA, have focused on ease of implementation and interpretation. However, simple methods rely upon assumptions that are often unrealistic. For example, LOCF_ANCOVA assumes that patient dropout is completely random, i.e. it is unrelated to the outcome being analyzed. Hence, in an analysis of efficacy data, LOCF_ANCOVA assumes that patients do not drop out due to lack of efficacy. The LOCF_ANCOVA approach also assumes that, for those patients who drop out, their observations would not have changed had they stayed in the trial. When these assumptions do not hold true, estimates of treatment effects and associated standard errors may be biased [2-4,6,7,15-17]. Considerable advances in statistical methodology, and in our ability to implement these methods, have been made in recent years. Methods such as MMRM, which require less restrictive assumptions regarding missing data, may now be easily implemented with standard software [4,5,18,19]. No universally superior approach to analysis of longitudinal data exists. However, a series of studies [6-9] demonstrated empirically what may have been anticipated from statistical theory – namely that the MMRM approach, while providing no guarantee of immunity from bias due to subject dropout, was a sensible analytic choice for many clinical trial scenarios. MMRM has repeatedly been shown to provide adequate control of Type I (false positive) and Type II (false negative) errors in a wide variety of situations modeled after neuropsychiatric clinical trials. In these head-to-head comparisons involving 456,000 data sets, the LOCF_ANCOVA approach did not perform as well as MMRM. We therefore specified MMRM as the primary analysis and LOCF_ANCOVA as a secondary analysis in the Phase III clinical trials of duloxetine in the treatment of MDD. Similar results regarding control of Type I and Type II error for LOCF_ANCOVA and mixed-effects model analyses have been obtained independently [16,20-22]. Furthermore, following an independent investigation of data from two placebo- and active-comparator controlled duloxetine trials, in which treatments were coded A, B, C, etc. to blind analysts to the treatment names, Molenberghs et al [4] concluded that MMRM analysis was a sensible choice for those data. The theoretical differences between MMRM and LOCF_ANCOVA have been summarized [5,18], established empirically [6-9], and proven mathematically [4]. However, we are unaware of any previous investigations of how these differences manifest themselves in efficacy assessments of a new medicinal product. The VAS pain results highlight a limitation of endpoint analyses of any type, namely that they provide only a snapshot view of the response profile. From LOCF_ANCOVA analysis, one can only conclude that drug was superior to placebo at endpoint. However, MMRM analysis reveals that drug had a significant effect early in the trials, but that advantage was somewhat transitory as the placebo group tended to "catch up" over time. In order to understand the response profile of a drug, the entire longitudinal profile should be considered [2]. From MMRM, the entire profile can be assessed from the same analysis that provided the primary result (the contrast at endpoint). In the duloxetine database, results from MMRM and LOCF_ANCOVA were in general agreement regarding substantial evidence of efficacy and frequency of significant differences. However, MMRM tended to be more sensitive to drug-placebo differences for outcomes related to overall depressive symptoms and core emotional symptoms of depression, with mean advantages over placebo that were 10% to 20% greater than LOCF_ANCOVA. However, MMRM did not universally increase duloxetine's advantage over placebo in comparison to results from LOCF_ANCOVA. For example, in somatic and painful physical symptom outcomes, results from LOCF_ANCOVA showed mean advantages over placebo that were approximately 40% greater than that from MMRM. Therefore, while the overall conclusions regarding the efficacy of duloxetine were unaffected by the choice of analytic method, this should not mask the important differences between MMRM and LOCF_ANCOVA. The advantages of MMRM and similar methods over LOCF_ANCOVA have been conclusively demonstrated in many studies and are evident in the duloxetine data. Khan et al [23] compiled a database from FDA summaries of efficacy for all antidepressants approved between 1985 and 2000. Less than half of the studies – which were analyzed using LOCF_ANCOVA as the primary analysis – found significant advantages for drug over placebo. These studies were generally anticipated to have at least 80% power and, if the analysis worked as expected, the success rate would be 80%. Therefore, LOCF_ANCOVA was less sensitive to the drug effects than anticipated. In the duloxetine database, MMRM yielded a 75% success rate for the primary outcome measure (HAMD17 total score), while LOCF_ANCOVA produced a 50% success rate, in comparison to the expected rate of 80%. While many factors may reduce the success rate in Phase III clinical trials, the use of a statistical method with known inflation of Type II error (false negative results) is an obvious suspect. An unduly high rate of false negative results could be especially problematic in early phases of drug development where only one or two chances exist to make the correct decision regarding the efficacy of a drug. It is noteworthy that one of the instances when MMMRM yielded a significant difference on the primary outcome (HAMD17 total score) when LOCF did not was in the Phase II study (Study 3). Also consider that across all therapeutic areas only about 50% of the molecules that enter Phase III testing receive regulatory approval. Many factors may reduce the success rate of Phase III development. However, the use in Phase II of a statistical method with known inflation of Type I error (false positive results) is an obvious suspect. Thus, the unexpectedly low success rate in Phase III is consistent with the conclusion that LOCF_ANCOVA inflates Type I error (as a result of an unduly high rate of false positive results in Phase II). Hence, results from the duloxetine NDA are consistent with research suggesting a move away from LOCF_ANCOVA and other simple analytic techniques to methods such as MMRM that are more robust to the biases from missing data. Conclusion Important differences exist between MMRM and LOCF_ANCOVA. Research has clearly demonstrated the advantages of MMRM over LOCF_ANCOVA. However, interpretations regarding the efficacy of duloxetine in MDD were unaffected by the choice of analytical technique. Competing Interests Drs. Mallinckrodt, Raskin, Wohlreich, Watkin, and Detke are employees of Eli Lilly and Company. Author's Contributions CHM designed the statistical analyses, and participated in interpretation of data and drafting of the manuscript. JR, MMW, JGW, and MJD participated in interpretation of data and drafting of the manuscript. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: Acknowledgements This work was sponsored by Eli Lilly and Company. ==== Refs Milliken GA Johnson DE The Analysis of Messy Data 2002 New York, NY: CRC Press Gibbons RD Hedeker D Elkin I Waternaux C Kraemer HC Greenhouse JB Shea MT Imber SD Sotsky SM Watkins JT Some conceptual and statistical issues in analysis of longitudinal psychiatric data. Application to the NIMH treatment of Depression Collaborative Research Program dataset Arch Gen Psychiatry 1993 50 739 750 8357299 Verbeke G Molenberghs G Linear Mixed Models for Longitudinal Data 2000 New York, NY: Springer Verlag Molenberghs G Thijs H Jansen I Beunckens C Kenward MG Mallinckrodt C Carroll RJ Analyzing incomplete longitudinal clinical trial data Biostatistics 2004 5 445 464 15208205 Mallinckrodt CH Clark WS Carroll RJ Molenberghs G Assessing response profiles from incomplete longitudinal clinical trial data under regulatory considerations J Biopharm Stat 2003 13 179 190 12729388 10.1081/BIP-120019265 Mallinckrodt CH Clark WS David SR Accounting for dropout bias using mixed-effects models J Biopharm Stat 2001 11 9 21 11459446 10.1081/BIP-100104194 Mallinckrodt CH Clark WS David SR Type I error rates from mixed effects model repeated measures versus fixed effects ANOVA with missing values imputed via Last Observation Carried Forward Drug Information J 2001 35 1215 25 Mallinckrodt CH Kaiser CJ Watkin JG Detke MJ Molenberghs G Carroll RJ Type I error rates from likelihood-based repeated measures analyses of incomplete longitudinal data Pharm Stat Mallinckrodt CH Kaiser CJ Watkin JG Molenberghs G Carroll RJ The effect of correlation structure treatment contrasts estimated from incomplete clinical trial data with likelihood-based repeated measures compared with last observation carried forward Clin Trials Detke MJ Lu Y Goldstein DJ Hayes JR Demitrack MA Duloxetine, 60 mg once daily, for major depressive disorder: A randomized double-blind placebo-controlled trial J Clin Psychiatry 2002 63 308 315 12000204 Detke MJ Lu Y Goldstein DJ McNamara RK Demitrack MA Duloxetine 60 mg once daily dosing versus placebo in the acute treatment of major depression J Psychiatr Res 2002 36 383 12393307 10.1016/S0022-3956(02)00060-2 Laird NM Missing data in longitudinal studies Stat Med 1988 7 305 15 3353609 Little RJ Rubin DB Statistical Analysis with Missing Data 1987 New York, NY: John Wiley and Sons Lavori PW Clinical trials in psychiatry: should protocol deviation censor patient data? Neuropsychopharmacology 1992 6 39 48 1571068 Lavori PW Dawson R Shera D A multiple imputation strategy for clinical trials with truncation of patient data Stat Med 1995 14 1913 25 8532984 Siddiqui O Ali MW A comparison of the random-effects pattern mixture model with last-observation-carried-forward (LOCF) analysis in longitudinal clinical trials with dropouts J Biopharm Stat 1998 8 545 63 9855033 Heyting A Tolboom JT Essers JG Statistical handling of drop-outs in longitudinal clinical trials Stat Med 1992 11 2043 61 1293667 Mallinckrodt CH Sanger TM Dube S DeBrota DJ Molenberghs G Carroll RJ Potter WZ Tollefson GD Assessing and interpreting treatment effects in longitudinal clinical trials with subject dropout Biol Psychiatry 2003 53 754 760 12706959 10.1016/S0006-3223(02)01867-X Gueorguieva R Krystal JH Progress in analyzing repeated-measures data and its reflection in papers published in the Archives of General Psychiatry Arch Gen Psychiatry 2004 61 310 317 14993119 10.1001/archpsyc.61.3.310 Liu G Gould AL Comparison of alternative strategies for analysis of longitudinal trials with dropouts J Biopharm Stat 2002 12 207 2 12413241 10.1081/BIP-120015744 Gadbury GL Coffey CS Allison DB Modern statistical methods for handling missing repeated measurements in obesity trial data: beyond LOCF Obes Rev 2003 4 175 184 12916818 10.1046/j.1467-789X.2003.00109.x Shao J Zhong B Last observation carry-forward and last observation analysis Stat Med 2003 22 2429 2441 12872300 10.1002/sim.1519 Khan A Detke MJ Khan SR Mallinckrodt C Placebo response and antidepressant clinical trial outcome J Nerv Ment Dis 2003 191 211 218 12695731 10.1097/00005053-200304000-00001 Hamilton M A rating scale for depression J Neurol Neurosurg Psychiatry 1960 23 56 61 14399272 Maier W Philipp M Improving the assessment of severity of depressive states: a reduction of the Hamilton Depression Scale Pharmacopsychiatry 1985 18 114 115 Guy W ECDEU assessment manual for psychopharmacology, revised 1976 Rockville, MD: National Institute of Mental Health, Psychopharmacology Research Branch. 217 331 Cleary MA Guy W Factor analysis of the Hamilton Depression Scale Drugs Exp Clin Res 1975 1 115 120 Montgomery SA Asberg MA A new depression scale designed to be sensitive to change Br J Psychiatry 1979 134 382 389 444788 Hamilton M The assessment of anxiety states by rating Br J Psychiatry 1959 32 50 55 DeLoach LJ Higgins MS Caplan AB Stiff JL The visual analog scale in the immediate postoperative period: intrasubject variability and correlation with a numeric scale Anesth Analg 1998 86 102 106 9428860 10.1097/00000539-199801000-00020 Barsky AJ Wyshak G Klerman GL Hypochondriasis: an evaluation of the DSM-III criteria in medical outpatients Arch Gen Psychiatry 1986 43 493 500 3964028 Hunt SM McKenna SP The QLDS: A scale for the measurement of quality of life in depression Health Policy 1992 22 307 319 10122730 10.1016/0168-8510(92)90004-U

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==== Front BMC Musculoskelet DisordBMC Musculoskeletal Disorders1471-2474BioMed Central London 1471-2474-5-321536310410.1186/1471-2474-5-32Case ReportScoliosis treatment using a combination of manipulative and rehabilitative therapy: a retrospective case series Morningstar Mark W [email protected] Dennis [email protected] Gary [email protected] Director of Research, Pettibon Biomechanics Institute 3416-A 57th St Ct. NW; Gig Harbor, WA 98335, USA2 Director, CLEAR Institute; 437 North 33rd Ave; St. Cloud, MN 56303, USA3 Director, Lawrence Chiropractic Clinic, 13961 60th St North; Stillwater MN, 55082, USA2004 14 9 2004 5 32 32 22 4 2004 14 9 2004 Copyright © 2004 Morningstar et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background The combination of spinal manipulation and various physiotherapeutic procedures used to correct the curvatures associated with scoliosis have been largely unsuccessful. Typically, the goals of these procedures are often to relax, strengthen, or stretch musculotendinous and/or ligamentous structures. In this study, we investigate the possible benefits of combining spinal manipulation, positional traction, and neuromuscular reeducation in the treatment of idiopathic scoliosis. Methods A total of 22 patient files were selected to participate in the protocol. Of these, 19 met the study criterion required for analysis of treatment benefits. Anteroposterior radiographs were taken of each subject prior to treatment intervention and 4–6 weeks following the intervention. A Cobb angle was drawn and analyzed on each radiograph, so pre and post comparisons could be made. Results After 4–6 weeks of treatment, the treatment group averaged a 17° reduction in their Cobb angle measurements. None of the patients' Cobb angles increased. A total of 3 subjects were dismissed from the study for noncompliance relating to home care instructions, leaving 19 subjects to be evaluated post-intervention. Conclusions The combined use of spinal manipulation and postural therapy appeared to significantly reduce the severity of the Cobb angle in all 19 subjects. These results warrant further testing of this protocol. ==== Body Background In the MEDLINE- indexed literature, chiropractic treatment has shown to be largely ineffective at significantly reducing scoliotic curvatures. Chiropractic treatment for scoliosis typically consists of spinal manipulation, electric stimulation, some form of isotonic, active exercises, and shoe lifts [1]. However, Lantz et al [2] has shown that these procedures, when applied over a one-year duration, were not sufficient to significantly reduce the Cobb angle of a scoliotic curvature. The treatment in this study focuses on the reduction of scoliosis by manipulative and rehabilitative methods not commonly used by most chiropractors. The major difference in this treatment compared to others is that stimulation of the involuntary postural reflexes is utilized in the clinic setting as well as in home care. Many of the proposed etiologies of idiopathic scoliosis are neurological in origin, including brain asymmetry [3], neural axis deformities [4], and central nervous system processing errors [5]. Additionally, many coexistent neurological alterations are present in scoliosis patients, such as visual deficiency [6] and decreased postural stability [7,8]. Therefore, the goals of the proposed treatment are not only to reduce the scoliotic curvatures, but also to rehabilitate any underlying postural and neurological weaknesses or imbalances. Previous chiropractic authors have investigated the effectiveness of various physiotherapeutic modalities in the treatment of scoliosis, such as Pilates [9], stretching and massage [10], therapeutic exercises [11], orthotics [2], and ultrasound or electric stimulation [1]. The purpose of the present study is to investigate any possible benefits from combining manipulative and rehabilitative techniques from a randomized sample collected from various chiropractic facilities. Preliminary evidence [12] suggests that these procedures may be beneficial for reducing the curvatures associated with scoliosis. Methods A nonrandomized set of 22 patients participated in the study. The age range of the subject group was 15–65 years of age. The patients were selected from 3 different chiropractic facilities in the United States. Patients were evaluated according to their chief complaint at initial presentation. Patients were excluded from the study if neoplasm, malignancy, fracture, scoliosis secondary to genetic disorders, or previous arthrodesis were identified. Each patient was examined radiographically for location and severity of scoliosis with standing anteroposterior full spine imaging. All patients removed their shoes for the imaging. Cobb angles were drawn on each radiograph to identify the degree of curvature present. A specific treatment plan was created based upon the results of each patient's radiographic measurements before and after a sample trial of the proposed clinical procedures. Initially, standing lateral cervical, nasium, lateral lumbar, and anteroposterior lumbopelvic views were taken. These views were taken to quantify forward head posture, cervical lordosis, lumbar lordosis, the sacral base angle, and the Cobb angle of the major lateral curvature. We decided to use the radiographic positioning and analysis outlined by Harrison et al [13-16], due to its previously published reliability. After these images were taken, each patient was fitted with a 4-lb anterior headweight. They were instructed to walk around with the headweight for 10 minutes. After 10 minutes, a follow-up lateral cervical radiograph was taken while wearing the anterior headweight. The purpose of this lateral stress view is to evaluate the potential improvement in cervical lordosis and reduction in forward head posture from using these procedures [17,18]. The basis for this aspect of the protocol is based upon the inherent properties of a curved column. In the spine, lateral spinal displacements may occur when the normal sagittal spinal curves [19-22] are flattened, reversed, or accentuated. These curves are necessary for the overall strength and flexibility of the curved spinal column, according to the Delmas Index [23]. Therefore, the proposed treatment is intended to restore a normal cervical and lumbar lordosis, and reduce forward head posture before the scoliotic curvatures are addressed. The specific manipulative and rehabilitative procedures used in this study are designed to both reduce the scoliotic curvature and theoretically retrain the involuntary neuromuscular, reflexive control of posture and balance. However, the specific neurological effects, if any, remain to be investigated. Some of the procedures have been separately introduced or tested [17,18,24-26]. The manipulative procedures included an upper cervical adjustment designed to mobilize the atlantal-occipital joint with the use of a percussive instrument. This technique is shown in Figure 1. This technique is delivered to patients whose lateral cervical radiographs demonstrated atlanto-occipital flexion. If atlanto-occipital extension was present on the initial lateral cervical radiograph, a -Z drop piece was used to mobilize the occiput into flexion. This is also shown in Figure 1. An anterior thoracic adjustment was administered with the patient's thoracic cage rotated opposite to the rotational displacement. A thoracic drop piece was also used to mobilize and correct the smaller upper thoracic curvature. Side posture lumbopelvic adjustments were delivered bilaterally to correct the rotational component of the pelvic misalignment. These side-posture manipulations were performed on a 30°-incline bench to help pre-stress the spine out of its existing scoliotic curvatures. Certain traction procedures are also employed. These procedures are delivered using high-density foam blocks to pre-stress the spine into specific positions so ligament deformation and stress-relaxation can take place. Supine pelvic blocking was performed on each patient for 15 minutes. The position of the blocks was determined by each patient's pelvic rotation on radiograph and posture analysis. One block is placed under the iliac crest of the posterior ilium, and the other block is placed under the femoral head of the opposite, anteriorly-rotated ilium. Figure 2 illustrates the position of the pelvic blocks. The rehabilitative procedures, demonstrated in Figure 3, included the use of head, shoulder, and hip weighting devices. These devices may be used while simultaneously performing specific balancing exercises. These exercises include the use of a Pettibon Wobble Chair® and a Posturomed® [17]. Tjernstrom et al [27] showed that repeated performance of a postural alteration induces a long-term motor memory for achieving that novel postural position. The position of the body weighting was also determined radiographically for each patient. Initially, hipweights and shoulderweights were applied according to each patient's posture analysis. Anteroposterior cervicothoracic and lumbopelvic views were taken while wearing the head and body weighting. Since changes in spinal position are not reliably seen by visualization [28,29], these stress radiographs were taken to confirm their corrective effects. The attending physician treated each patient 3 times per week for the first 4–6 weeks. A total of 3 physicians performed the treatment intervention for all patients. However, each patient did not receive identical treatment at all visits. The physicians performed only those manipulative procedures that were deemed necessary based upon a visual posture analysis at the beginning of each treatment session. However, the rehabilitative procedures remained constant throughout the study for all patients. Specific home care exercise programs were taught to each patient. These exercises were performed on a daily basis. Each patient was instructed to wear the head and body weighting twice daily for 15 minutes each time. Secondly, each patient was given a set of triangular foam blocks to lie on once daily for 20 minutes, immediately prior to going to bed at night. The foam blocks were positioned under the cervicothoracic and thoracolumbar regions simultaneously. The position of these blocks is shown in Figure 4. Patients participating in any weightlifting activities were required to cease those activities until further notice from the attending physician. Patients who failed to perform the home care more than 3 times were dismissed from the study. A total of 3 subjects were eventually dismissed, leaving 19 subjects to perform post-intervention evaluations. Results At the conclusion of the trial period, a post-intervention radiographic study was conducted. The same anteroposterior full spine view was taken, and Cobb angles were again measured at the same vertebral levels. The average starting Cobb angle was found to be 28°, while the post-intervention Cobb angles measured an average of 11°, for an overall average reduction of 17°. Every patient made at least a 25% improvement. The largest improvement measured 33°, and the smallest improvement measured 8°. Table 1 shows the results of all 19 patients that followed through with the entire treatment plan. Figure 5 is a sample of the improvements made by a few of the patients. It is important to mention that these patients were initially treated prior to this study. Because of this, the pre and post treatment radiographs had previously been analyzed for sagittal curve and Cobb angle measurements. For purposes of this study, however, all of the radiographs were sent to a single chiropractic physician to analyze each of the patient files. This physician did not participate in the treatment process, nor did this physician have contact with any of the patients. This was performed to separate examiner bias from the treatment results. While only radiographic procedures were reported for this study, other physiologic parameters were utilized to document patient progress. Unfortunately, since the patient files were extracted from 3 different spine clinics, a consistent functional or symptomatic measure was not used in all 22 cases. A functional rating index, a visual analog scale, and SF-36 were used on the patients here. As a result, these values are not reported to avoid variability in outcome interpretation. Discussion Scoliosis has recently been associated with a lower quality of life [30-32], lower scores on the SF-36 health questionnaire [33], and makes patients prone to developing chronic pain more often than the general population [34]. Therefore, reducing scoliotic curvatures, even in the absence of symptoms, seems to be a worthy outcome objective for clinical practice. This opinion is further supported by recent evidence of the deleterious effects of abnormal spinal loading [35-37]. Given that the average curvature progression in idiopathic scoliosis is 7.03° per year [38], the traditional method of regular observation without treatment seems to be reactionary rather than corrective or preventive. Spinal manipulation alone does not appear to significantly alter spinal structure when administered as a sole treatment modality [39,40]. Therefore, in the instance of scoliosis, treatment should include the use of both manipulative and rehabilitative procedures, so that structural changes can be attempted. It is important to stress that spinal manipulation was avoided, when possible, in the present study. Unpublished clinical observation by the authors has shown that over-manipulating or adjusting the spine seems to create a certain amount of instability, possibly leading to further buckling of the scoliotic curvature. The significance of home care to the results was not reported here. It is unknown how the omission of home care would have affected the outcome measurements, given that 3 subjects were dropped from the study for noncompliance in performing home care. Future research should account for this potential variable to determine its necessity and relevance. The outcome measures for this study are divided into a series of both short-term and long-term goals. The outcome of the initial stage of care is to reduce forward head posture and improve the sagittal cervical and lumbar curves. As the position of the head migrates forward, or away from the body's vertical axis, increased strain is placed upon the muscles of the head, neck and shoulders. Cailliet and Zohn indicated that an additional 10 inch/lbs of leverage is added to the spinal system in a forward head posture [41,42]. Additionally, this added leverage causes increased isometric contraction of various spinal muscles, such as the splenius capitis, trapezius, SCM, and levator scapula. Sjogaard et al [43] reported that blood flow through a given muscle is decreased as a muscles contraction increases, being virtually cut off at 50–60% contraction. The resultant lack of blood flow forces the muscle to rely on anaerobic metabolism. As anaerobic metabolism progresses, metabolites such as substance P, bradykinin, and histamine build up and excite chemosensitive pain receptors, causing a barrage of nociceptive afferent input [44], resulting in dysafferentation [45]. Being that postural control is largely dependant upon cervical joint mechanoreceptors and afferent input from ligament and musculotendinous sources [46,47], correcting the postural distortions responsible for this pathophysiologic process may be beneficial in patient populations, such as scoliosis, where postural control is significantly altered [48]. The effects of the loss of cervical and lumbar lordosis have been previously reported [19,35-37]. Rhee et al [49] noted that correction of the sagittal curves might be related to the long-term health of the spine in scoliosis management. Harrison et al [35] illustrated how a loss of the sagittal curve alters the mechanical properties of the spinal cord and nerve roots, which may change the firing patterns of involved neurons. Schafer illustrated how an increased demand is placed upon the cervical musculature when the cervical curve is straightened or reversed [50]. It is important that the cervical spine be in a normal structural alignment. A loss of the cervical lordosis and concomitant forward head posture may elicit the pelvo-ocular reflex, which causes an anterior pelvic translation to balance the head's center of gravity [51]. Wu et al [52,53] point out that in postural control, preference is given to the position of the head, neck, and trunk. Therefore, correction of the cervical spine becomes imperative so that the rest of the spine can be rehabilitated in relation to a normal reference point in space. Once the cervical and lumbar lordoses are corrected, coronal reduction of the scoliotic curvatures begins. Here this was accomplished by adding a shoulderweight to the right shoulder and a hipweight to the anterior right ilium and posterior left ilium. Wu and Essien [53] have previously reported the effects of adding external weight to the upper body via a shoulder weight. They identified predictable patterns in which the trunk would compensate for the amount and position of the weight. Wu and MacLeod [52] identified a shift in the center of mass toward the added weight when placed on the side of the pelvis. However, the trunk and head remained in the same position, while the pelvis and lower extremities shifted to counteract the weight while supporting the head and trunk [52]. In this protocol, we created an environment where external weight was added to the head, shoulder, and pelvic regions simultaneously. Knowing the predictable patterns of compensatory shifting to an altered center of gravity, we placed the headweight, shoulderweight, and hipweights in areas designed to reduce each patient's specific spinal distortion patterns. Learning a new motor coordination skill can be divided into 3 phases: cognitive, associative, and autonomous [54]. In the cognitive phase, the patient performs the motor task repetitively to learn until the task requirements are understood. As the patient progresses through the associative and autonomous phases, the task becomes easier to perform, and may ultimately be performed in a variety of practical contexts with decreased repetitions [54]. While Lantz et al [2] have shown that chiropractic management, consisting of a combination of manipulative procedures, electric stimulation, and orthotic inserts did not significantly reduce a scoliosis, this treatment does not incorporate these physiotherapeutic procedures. Instead, this treatment requires the use of specific rehabilitative equipment that theoretically recruits the use of head, neck, trunk, and extremity postural reflexes to create specific adaptation to an altered center of gravity and field of gaze. The study design used here does present specific limitations. Due to the lack of a control group, comparative data and conclusions cannot be made. Additionally, a retrospective design does not blind the practitioners to treatment. Although we attempted to select patient files at random from 3 separate spine clinics, nonrandomized sample populations such as ours do not necessarily reflect the potential outcomes in a general population. Therefore, future studies in this area should incorporate a control group and a randomized patient population. Follow-up studies should also focus on the potential long-term benefits of conservative scoliosis treatment, given the relative scarcity of biomedical literature available on long-term benefits from any scoliosis treatment. Conclusions Within the design limitations of the present study, the combined use of manipulative and neuromuscular rehabilitation seemed to reduce scoliotic curvatures in 19 subjects by an average of 17°. This reduction took place within a 4 to 6-week period. Although this treatment was not tested over the long term, the magnitude of the present results warrants further studies into its effectiveness. This treatment should also be tested on specific types of scoliosis in follow-up trials. A long-term investigation of this protocol is desirable. Competing interests This manuscript was submitted by Spinal Technologies, a BioMed Central institutional member. The rehabilitation equipment used in this study is patented by Burl R Pettibon, DC and Spinal Technologies. MWM is the Director of Research for the Pettibon Biomechanics Institute, and an active postgraduate instructor for Spinal Technologies. MWM does not receive monetary compensation for this position. Rather, he is granted funding from Spinal Technologies to obtain biomedical literature and statistician services. DW is a past postgraduate instructor for Spinal Technologies, founder and director of the CLEAR Institute, and CEO of the Flex Neck Company. GL is an active postgraduate instructor for Spinal Technologies. The authors receive lecture fees for each continuing education seminar conducted. All 3 authors maintain private chiropractic practices from where all of the patient files in this study were taken. None of the above companies donated, funded, or reimbursed any monies or equipment for this study. None of the authors have any ownership in Spinal Technologies or its subsidiary companies, and none will gain any financial interest as a result of this paper. Authors' contributions Each author worked on one-third of the patient population. The first author was responsible for collecting the data and putting our findings into written format. Pre-publication history The pre-publication history for this paper can be accessed here: Acknowledgements The authors would like to thank Darin Weeks and Cassi Little for procedure demonstration. Figures and Tables Figure 1 The picture on the left demonstrates the mechanically assisted manipulation used when a patient's skull is restricted in extension on lateral cervical radiograph. The picture on the right is the procedure used when the skull flexion is restricted. Figure 2 This picture shows the placement of the pelvic blocks for an anterior right ilium. The blocks are placed opposite of the pelvic rotation. Figure 3 The above picture illustrates a sample placement of the Pettibon Bodyweighting System. Here we have an anterior headweight, right shoulderweight, and left-back and right-front hipweights. Figure 4 This figure shows a demonstration of the positional traction procedure. The cervical block is placed under the patient's cervicothoracic junction, allowing the head to extend back over the sloped portion of the block. The low back support is placed under the patient's thoracolumbar junction, posterior to the lowest palpable ribs. The blocks are outlined in white. Figure 5 This figure illustrates some of the pre and post x-rays taken after 4–6 weeks of treatment. Table 1 Cobb Angle Measurements after 4–6 Weeks (Degrees) Subject # Sex/Age Initial Post Reduction 1 F/19 32 24 8 2 M/17 23 8 15 3 F/15 29 11 18 4 F/64 52 19 33 5 F/16 19 4 15 6 F/22 28 11 17 7 F/27 16 2 14 8 F/34 25 11 14 9 F/21 35 20 15 10 F/41 28 13 15 11 F/53 40 22 18 12 F/18 31 9 22 13 F/16 27 14 13 14 F/20 33 7 26 15 F/23 32 18 14 16 F/15 16 4 12 17 M/33 15 4 11 18 M/24 21 6 15 19 F/25 38 11 17 ==== Refs Feise RJ An inquiry into chiropractors' intention to treat adolescent idiopathic scoliosis: A telephone survey J Manipulative Physiol Ther 2001 24 177 182 11313613 10.1067/mmt.2001.113774 Lantz CA Chen J Effect of chiropractic intervention on small scoliotic curves in younger subjects: a time-series cohort design J Manipulative Physiol Ther 2001 24 385 393 11514815 10.1067/mmt.2001.116419 Niesluchowski W Dabrowska A Kedzior K Zagrajek T The potential role of brain asymmetry in the development of adolescent idiopathic scoliosis: a hypothesis J Manipulative Physiol Ther 1999 22 540 544 10543585 Dobbs MB Lenke L Szymanski DA Morcuende JA Weinstein SL Bridwell KH Sponseller PD Prevalence of neural axis abnormalities in patients with infantile idiopathic scoliosis J Bone Joint Surg Am 2002 84-A 2230 2234 12473713 Lowe TG Edgar M Margulies JY Miller NH Raso VJ Reinker KA Rivard CH Etiology of idiopathic scoliosis: current trends in research J Bone Joint Surg Am 2000 82-A 1157 1168 10954107 Catanzariti JF Salomez E Bruandet JM Thevenon A Visual Deficiency and Scoliosis Spine 2001 26 48 52 11148645 10.1097/00007632-200101010-00010 Nault ML Allard P Hinse S Le Blanc R Caron O Labelle H Sadeghi H Relations between standing stability and body posture parameters in adolescent idiopathic scoliosis Spine 2002 27 1911 1917 12221357 10.1097/00007632-200209010-00018 Chen PQ Wang JL Tsuang YH Liao TL Huang PI Hang YS The postural stability control and gait pattern of idiopathic scoliosis adolescents Clin Biomech 1998 13 S52 S58 10.1016/S0268-0033(97)00075-2 Blum CL Chiropractic and pilates therapy for the treatment of adult scoliosis J Manipulative Physiol Ther 2002 25 e3 12021749 10.1067/mmt.2002.123336 Tarola GA Manipulation for the control of back pain and curve progression in patients with skeletally mature idiopathic scoliosis: two cases J Manipulative Physiol Ther 1994 17 253 257 7519232 Golembiewski GV Catanzaro DJ Scoliosis reduction utilizing an exercise J Vertebral Subluxation Res 2001 4 31 36 Morningstar MW Strauchman MN Gilmour G Idiopathic Scoliosis Treatment Using the Pettibon Corrective Procedures: A Case Report J Chiropr Med Harrison DE Harrison DD Colloca CJ Betz J Janik TJ Holland B Repeatability over time of posture, radiograph positioning, and radiograph line drawing: an analysis of six control groups J Manipulative Physiol Ther 2003 26 87 98 12584507 10.1067/mmt.2003.15 Troyanovich SJ Harrison SO Harrison DD Harrison DE Payne MR Janik TJ Holland B Chiropractic biophysics digitized radiographic mensuration analysis of the anteroposterior lumbopelvic view: a reliability study J Manipulative Physiol Ther 1999 22 309 315 10395433 Jackson BL Harrison DD Robertson GA Barker WF Chiropractic biophysics lateral cervical film analysis reliability J Manipulative Physiol Ther 1993 16 384 391 8409786 Troyanovich SJ Harrison DE Harrison DD Harrison SO Janik T Holland B Chiropractic biophysics digitized radiographic mensuration analysis of the anteroposterior cervicothoracic view: a reliability study J Manipulative Physiol Ther 2000 23 476 482 11004652 10.1067/mmt.2000.108818 Saunders ES Woggon D Cohen C Robinson DH Improvement of cervical lordosis and reduction of forward head posture with anterior head weighting and proprioceptive balancing protocols J Vertebral Subluxation Res 2003 4 000 Morningstar MW Strauchman MN Weeks DA Spinal manipulation and anterior headweighting for the correction of forward head posture and cervical hypolordosis: a pilot study J Chiropr Med 2003 2 51 55 Harrison DE Harrison DD Troyanovich SJ Harmon S A normal spinal position: it's time to accept the evidence J Manipulative Physiol Ther 2000 23 623 644 11145804 10.1067/mmt.2000.110941 Harrison DD Janik TJ Troyanovich SJ Harrison DE Colloca CJ Evaluation of the assumptions used to derive an ideal normal cervical spine model J Manipulative Physiol Ther 1997 20 246 254 9168409 Harrison DE Janik TJ Harrison DD Cailliet R Harmon SF Can the thoracic kyphosis be modeled with a simple geometric shape? The results of circular and elliptical modeling in 80 asymptomatic patients J Spinal Disord Tech 2002 15 213 220 12131422 Harrison DD Cailliet R Janik TJ Troyanovich SJ Harrison DE Holland B Elliptical modeling of the sagittal lumbar lordosis and segmental rotation angles as a method to discriminate between normal and low back pain subjects J Spinal Disord 1998 11 430 439 9811104 Kapandji IA The physiology of the joints The trunk and vertebral column 1974 3 5 Churchill Livingstone 235 236 Morningstar MW Cervical hyperlordosis correction: a novel treatment method for mid thoracic pain J Chiropr Med 2003 2 111 115 Morningstar MW Strength gains through lumbar lordosis restoration J Chiropr Med 2003 2 137 141 West DT Mathews RS Miller MR Kent GM Effective management of spinal pain in one hundred seventy-seven patients evaluated for manipulation under anesthesia J Manipulative Physiol Ther 1999 22 299 308 10395432 Tjernstrom F Fransson PA Hafstrom A Magnusson M Adaptation of postural control to perturbations- a process that initiates long-term motor memory Gait Posture 2002 15 75 82 11809583 10.1016/S0966-6362(01)00175-8 Johnson GM The correlation between surface measurements of head and neck posture and the anatomic position of the upper cervical vertebrae Spine 1998 23 921 927 9580960 10.1097/00007632-199804150-00015 Fedorak C Ashworth N Marshall J Paull H Reliability of the visual assessment of cervical and lumbar lordosis: how good are we? Spine 2003 28 1857 1859 12923476 10.1097/01.BRS.0000083281.48923.BD Shapiro GS Taira G Boachie-Adjei O Results of surgical treatment of adult idiopathic scoliosis with low back pain and spinal stenosis: a study of long-term clinical radiographic outcomes Spine 2003 28 358 363 12590210 10.1097/00007632-200302150-00009 Danielsson AJ Nachemson AL Radiologic findings and curve progression 22 years after treatment for adolescent idiopathic scoliosis: comparison of brace and surgical treatment with matching control group of straight individuals Spine 2001 26 516 525 11242379 10.1097/00007632-200103010-00015 Freidel K Petermann F Reichel D Steiner A Warschburger P Weiss HR Quality of life in women with idiopathic scoliosis Spine 2002 27 E87 E91 11840115 10.1097/00007632-200202150-00013 Schwab F Dubey A Pagala M Gamez L Farcy JP Adult scoliosis: a health assessment analysis by SF-36 Spine 2003 28 602 606 12642769 10.1097/00007632-200303150-00016 Weinstein SL Dolan LA Spratt KF Peterson KK Spoonamore MJ Ponseti IV Health and function of patients with untreated idiopathic scoliosis: a 50-year natural history study JAMA 2003 289 559 567 12578488 10.1001/jama.289.5.559 Harrison DE Cailliet R Harrison DD Troyanovich SJ Harrison SO A review of biomechanics of the central nervous system- part III: spinal cord stresses from postural loads and their neurologic effects J Manipulative Physiol Ther 1999 22 399 410 10478773 Harrison DE Cailliet R Harrison DD Troyanovich SJ Harrison SO A review of biomechanics of the central nervous system-part II: spinal cord strains from postural loads J Manipulative Physiol Ther 1999 22 322 332 10395435 Harrison DE Cailliet R Harrison DD Troyanovich SJ Harrison SO A review of biomechanics of the central nervous system-part I: spinal canal deformations resulting from changes in posture J Manipulative Physiol Ther 1999 22 227 234 10367759 Chuah SL Kareem BA Selvakumar K Oh KS Borhan Tan A Harwant S The natural history of scoliosis: curve progression of untreated curves of different aetiology, with early (mean 2 year) follow up in surgically treated curves Med J Malaysia 2001 56 37 40 11814247 Harrison DD Jackson BL Troyanovich SJ Robertson G DeGeorge D Barker WF The efficacy of cervical extension-compression traction combined with diversified manipulation and drop table adjustments in the rehabilitation of cervical lordosis: a pilot study J Manipulative Physiol Ther 1994 17 454 464 7989879 Harrison DD Harrison DE Troyanovich SJ Structural Rehabilitation of the spine and posture: rationale for treatment beyond the resolution of symptoms J Manipulative Physiol Ther 1998 21 37 50 9467100 Cailliet R Davis FA Neck and arm pain 1981 2 Zohn DA Little Brown Musculoskeletal pain diagnosis and treatment 1988 2 Sjogaard G Savard G Juel C Muscle blood flow during isometric activity and its relation to muscle fatigue Eur J Appl Physiol Scand 1988 57 327 335 Travell JG Simons D Myofascial pain and dysfunction: the trigger point manual 1973 Williams & Wilkins Seaman DR Winterstein JF Dysafferentation: a novel term to describe the neuropathophysiological effects of joint complex dysfunction. A look at likely mechanisms of symptom generation J Manipulative Physiol Ther 1998 21 267 280 9608382 Grod JP Diakow PR Effect of neck pain on verticality perception: A cohort study Arch Phys Med Rehabil 2002 83 412 415 11887124 10.1053/apmr.2002.29660 Dietz V Muller R Colombo G Locomotor activity in spinal man: significance of afferent input from joint and load receptors Brain 2002 125 2626 2634 12429590 10.1093/brain/awf273 Gauchard GC Lascombes P Kuhnast M Perrin PP Influence of different types of progressive idiopathic scoliosis on static and dynamic postural control Spine 2001 26 1052 1058 11337624 10.1097/00007632-200105010-00014 Rhee JM Bridwell KH Won DS Lenke LG Chotigavanichaya C Hanson DS Sagittal plane analysis of adolescent idiopathic scoliosis Spine 2002 27 2350 2356 12438983 10.1097/00007632-200211010-00008 Schafer RC Clinical biomechanics: musculoskeletal actions and reactions 1987 2 Williams & Wilkins Lewit K Muscular and articular factors in movement restriction Manual Medicine 1985 1 83 85 Wu G MacLeod M The control of body orientation and center mass location under asymmetrical loading Gait Posture 2001 13 95 101 11240357 10.1016/S0966-6362(00)00102-8 Wu G Essien I The regulation of human orientation and center of mass in the frontal plane during upright stance In Proceedings of North America Congress of Biomechanics Waterloo, Canada 1988 Harbst KB Wilder PA Neurophysiologic, motor control, and motor learning basis of closed kinetic chain exercise Orthop Phys Ther Clin N Am 2000 9 137 149

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BMC Musculoskelet Disord

10.1186/1471-2474-5-32

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==== Front BMC BiotechnolBMC Biotechnology1472-6750BioMed Central London 1472-6750-4-191534166410.1186/1472-6750-4-19Methodology ArticleMulti-line split DNA synthesis: a novel combinatorial method to make high quality peptide libraries Tabuchi Ichiro [email protected] Sayaka [email protected] Shingo [email protected] Yuzuru [email protected] Tokyo Evolution Research Center, 1-1-45-504, Okubo, Shinjuku-ku, Tokyo 169-0072, Japan2 Department of Functional Materials Science, Saitama University,255 Shimo-Okubo, Saitama 338-8570, Japan2004 1 9 2004 4 19 19 31 3 2004 1 9 2004 Copyright © 2004 Tabuch et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background We developed a method to make a various high quality random peptide libraries for evolutionary protein engineering based on a combinatorial DNA synthesis. Results A split synthesis in codon units was performed with mixtures of bases optimally designed by using a Genetic Algorithm program. It required only standard DNA synthetic reagents and standard DNA synthesizers in three lines. This multi-line split DNA synthesis (MLSDS) is simply realized by adding a mix-and-split process to normal DNA synthesis protocol. Superiority of MLSDS method over other methods was shown. We demonstrated the synthesis of oligonucleotide libraries with 1016 diversity, and the construction of a library with random sequence coding 120 amino acids containing few stop codons. Conclusions Owing to the flexibility of the MLSDS method, it will be able to design various "rational" libraries by using bioinformatics databases. ==== Body Background The combinatorial synthesis method has been demonstrating its effectiveness in discovering novel functional molecules. Examples of this method in the field of evolutionary protein engineering are selections of a novel functional peptide from a random library on solid support [1], phage display [2] or in vitro virus (synonym for RNA-peptide fusion or mRNA-display) [3-5]. The efficiency of the methods depends on the screening technique employed and the library quality. In the display methods, a library of polynucleotide templates must be prepared in order to obtain a random peptide library. A primitive random library of such templates is (NNN)n (N = equimolar mixture of A, T, G and C). This library leads to premature short peptides and a particular bias of the amino acid composition, which makes the effective searchable sequence space biased. A slightly improved library NNK or NNS (K / S = equimolar mixture of T and C / G and C) has been conventionally used. Several methods have been developed for a more improved library. Various "rational" libraries in which the nucleotide mixtures were optimized for a target amino acid composition by using a computer calculation have been developed [6-8]. Removal of stop codons to obtain long ORFs is important for the evolutionary design of a novel protein starting from a random library. Several methods based on random block-ligation were reported [9,10]. Two high quality libraries that lead to the successful evolutionary protein design were as follows: the trinucleotide phosphoramidites (3NPs) method using twenty pre-synthesized trimers of nucleotide phosphoramidites [11-14], and the pre-selecting method using an mRNA display with a C-terminus affinity tag in order to remove stop codons [15]. We report in this article on a convenient method for the construction of a high quality library based on combinatorial DNA synthesis. This library has few stop codon and has an optimized amino acid composition for various purposes. A random library based on the split synthesis [1] is made routinely in combinatorial chemistry, but a few methods [16,17] and a few applications [18,19] have been reported for oligonucleotides synthesis. They were used for mutagenesis and the products did not have high quality for evolutionary protein engineering. We applied the split synthesis to oligodeoxyribonucleotide synthesis and developed a new procedure, based on the synthesis of designed codon mixtures using multi-line DNA synthesizers. Our method, Multi-Line Split DNA Synthesis (MLSDS), requires only standard reagents and three or four synthesizers for DNA synthesis. MLSDS can make various "rational" libraries of huge diversity with few stop codons. Results and Discussions Adaptive design to the target amino acid composition Scheme of the MLSDS method is shown in Fig. 1 and Table 1, and described in detail in Methods section. MLSDS is able to remove not only stop codons but also particular codons. It is able to design the codon composition. We incorporated the effect of the single nucleotide deletion during a general oligonucleotide synthesis [20] into the design. Designed biased libraries are useful for creating various novel proteins such as a functional peptide without Cys [21] or an engineered protein without Met [22]. Unnatural codons and unnatural amino acid [23] will be also incorporated in desired composition. It will be able to incorporate various results of analysis of bioinformatics databases in order to make an initial library with higher evolvability in experimental protein evolution. The optimum amino acid composition in the library may be different for each target protein. For example, when we want to explore the global protein sequence space exhaustively, the uniform amino acid composition may be the best. When we want to explore only a proven region in the protein sequence space, the use of the average amino acid composition among natural proteins [24] might be better for many aspects. When we want to design a protein with some specific properties, a library with increased or decreased fraction of specific amino acid should be constructed for each segmental region on the polypeptide chain. Among these wide spectra of requirements, we designed DNA libraries that code peptide libraries having various characteristics and have no stop codons. Examples are: a library with the average amino acid composition of natural proteins [24], which is named "Natural" library in this article, the uniform amino acid composition; and the uniform composition except [Cys] = 0. A library encoding only four kinds of amino acid (a c-Fos mutant library [26]) was also designed. Designed molar mixing ratios of A:T:G:C for some of these libraries are shown in Table 1. Another interesting example was obtained when the target composition was "Uniform except [Met] = 0 and [Term] = 0". The designed molar mixing ratio of A:T:G:C gave the high fitness F value (0.96 on three lines splits) and gave no stop codon even if the effect of a point deletion was included in the GA calculation. A Met-less random library may be the best starting library for global search of the protein sequence space. This speculation is supported by the report [22] stating that a mutant dihydrofolate reductase generated by the replacement of all Met had much higher enzymatic activity than the wild type. Internal deletion problem in the oligonucleotides synthesis process is important. It destroys the codon-based design, leading to stop-codon generation and undesirable amino acid composition. Our program incorporated deletion effects into the GA calculation and succeeded to minimize the deletion problem. Moreover it was reported that contamination of deletion products could be decreased on a denaturing PAGE for DNA of this length [15]. We also investigated the practical number of DNA synthesizers. For this purpose, we calculated the final correlation coefficient between the designed and the various target compositions with up to 6-line DNA synthesizers. As shown in Fig. 2, the final correlation coefficient (= the final fitness) became saturated at about 3- or 4-lines on this program. Our GA program is not the best for obtain best F value but suitable for designing actual synthesizing operations. These results showed MLSDS method gave a high quality library even with three DNA synthesizers. When we took the natural abundance as the target amino acid composition, we got a highest fitness value F = 0.99 (on three lines) in the GA calculations. This is reasonable, because the average amino acid composition among natural proteins highly correlates to the number of synonymous codons in the standard genetic code table [25]. Synthesis of MLSDS libraries We synthesized a "Natural" library and a "Uniform except [Cys] = 0" library mentioned in the previous section. In Table 2 the compositions of the actually synthesized DNA libraries are listed in comparison with the target compositions. They were high quality libraries (F = 0.85 and 0.66, respectively) without stop codons in full-length DNAs. The deletion rate was about 0.3% per coupling. For the total DNAs including deletants, F = 0.90 and 0.60, respectively. We also synthesized MLSDS products composed of limited kinds of amino acid. It has been regarded that such a peptide can be synthesized only by 3NP method. A mutant c-Fos library that contained only four kinds of amino acid was synthesized, which was equivalent to a library synthesized by 3NP method [26]. It was a high quality library (F = 1.00) (Table 2). So far, fifteen libraries with various amino acid compositions were successfully synthesized. In order to make long ORFs, we assembled 8 units of the oligomers. Stem sequences of them did not have any stop codons. A DNA library encoding 120 amino acids plus nine 5'- and 3'-flanking semi-random di-peptides (thus, total 138 amino acids) was constructed (Fig. 3). The diversity of the synthesized library is about 1016 judging from the mass (data of A260) and purity (data of PAGE) of synthesized DNA. With an in vivo selection, there is a diversity limit by the transformation step. But with an in vitro selection, there is no such limitation. Thus exploration of huge sequence space by in vitro virus [3-5] or related techniques [28,29] will become possible, depending on the experimental cost. Comparison of MLSDS with other methods So far, a really random library has been generated by four methods. Other methods do not give a really random library, because they can not provide a library in which all the 20 amino acids are encoded at all sites. A comparison of library quality for three methods is shown in Table 3. An application of 3NPs method to mutagenesis of antibodies [27] or coiled-coils [30] gave good results. Twenty kinds of 3NPs mean one codon per one amino acid, but the codons are degenerate. Thus 3NPs method makes many tRNAs useless. The translation efficiency was calculated based on the codon usage, giving maximum 4-fold decrease in Triticum aestivum. It was reported that the reaction efficiency of 3NPs was far from uniform. The sequence data of synthesized DNA using an equimolar mixture of 19 kinds of 3NPs (without Cys) showed 12-fold (maximum) difference in composition [27] or more [12]. The coupling yield was affected by the mixing ratio of 3NPs and by the context, showing 8-fold (maximum) difference for the same 3NP [27]. Thus it will be difficult to correct reaction efficiencies by adjusting the mixing ratio. The correlation coefficient between the target composition and the actual composition was about 0.4 (for uniform 19 kinds of amino acids) [27] (Table 3). Dimer-phosphoramidites [17] method is a variation of 3NPs method, using pre-synthesized amidites, and had the same problems. In fact, the bias was observed [17]. A pre-selecting method using an mRNA display [15] was fruitful in evolutionary protein design. Novel peptide aptamers were evolved starting from a long ORF random library [31,32]. But this method could not remove all the stop codons. It gave limited library diversity. This method has low flexibility in amino acid composition. For example it is difficult to generate a "Uniform except [Met] = 0" library. The correlation coefficient between the target composition and the actual composition were not so high (Table 3). The Y-Ligation Block Shuffling (YLBS) method [9] has high potentiality in the evolutionary design of peptides. It has problems on deletion and reaction bias of RNA ligase. MLSDS produced libraries with high quality as shown in Table 3. Above-mentioned problems are not so severe for MLSDS method, because it uses only standard phosphoramidites and is free from any biochemical bias such as in mRNA display and in YLBS. It was reported that the difference in the reaction efficiency of equimolar mixture of four kinds of mono-phosphoramidites was only about 1–5 % [33,34]. MLSDS can create any specific amino acid composition as same as 3NP method, and a MLSDS library is made at lower cost than that made with other methods. Conclusions We applied the split synthesis to oligodeoxyribonucleotide synthesis and developed a new procedure, Multi-Line Split DNA Synthesis (MLSDS), based on the synthesis of designed codon mixtures using three-line DNA synthesizers. MLSDS can make various "rational" libraries of huge diversity with few stop codons by using bioinformatics databases. Combination of an MLSDS library with a screening method for huge diversity will accelerate the protein evolution in vitro. Methods A random MLSDS library was synthesized as follows. A standard DNA synthesis method was used in three lines of DNA synthesizer running in parallel. The randomized regions were combinatorialy synthesized in codon units. Triplet codons were synthesized separately in the three synthesizers as an elongation reaction of oligonucleotides on beads made of controlled pore glass (CPG). CPG beads were mixed together manually, and then splitted again into three reaction tubes manually and the next triplet codons were synthesized (Fig. 1). The sequence of a 87 mer library was 5'-GAT GAG GCG AAG ACG NAC TGS (123/456/789)15 NAC TGS GAG GCT GGC TGC CAC-3', where N and S denote A/T/G/C and G/C, respectively. The A:T:G:C mixing ratio in each letter of three codon groups 123, 456, and 789 was shown in Table 1. These values were calculated as described below. Both flanking regions contain the recognition sequences of type-IIs restriction enzymes BbsI and BbvI, respectively. In order to make longer sequences, we ligated 2 to 8 units of oligomers at the cohesive ends (the underlined sequences shown above) generated by the restriction enzyme treatment. The assembly method was as described in Ref. [16]. The italicized sequence shown above represents the assembly unit (random region of 45 bp and flanking semi-random linking region of (6+6)/2 bp). The synthesized DNA libraries were amplified by PCR using KOD Dash polymerase (TOYOBO), inserted into pCR2.1TOPO vector (Invitrogen) and cloned, avoiding cloning bias. The clones were sequenced. Computer calculations to determine the optimum molar mixing ratio of four bases in the codon synthesis step were performed by using Mathematica (Wolfram Research). We made a GA program for this purpose. Firstly, the target amino acid composition, pT = (pT1, pT2, pT3,..., pT21), was established, where normally pT21 = 0 for stop codons. Secondly, we calculated an expected amino acid (plus stop codons) composition, p = (p1, p2, p3,.., p21), from the molar mixing ratio of the bases, x = (x1, x2, x3,....., x12L), where 12L is equal to 4(number of bases) × 3(number of codon letters) × L(number of synthesizer-line). For example, the mixtures for the first letter and the second letter of the first DNA synthesizer have the molar mixing ratio [A]: [T]: [G]: [C] = x1 : x2 : x3 : x4 and x5 : x6 : x7 : x8, respectively. And for example, when L = 3, the expected alanine composition p1 is given by: for the full-length sequence without deletion. We solved an integer-programming problem (6-valued 12L-dimensional optimization problem) having the solution xi as integer (0,1,2,3,4,5). The reason for 6-digits "integer" was to simplify the DNA synthesizer handling and also to simplify the calculation. As the fitness F of x in the GA, we took a correlation coefficient between the expected (or designed) amino acid composition and the target amino acid composition: where N = 21 for our normal case. The optimum x, which gave the maximum fitness F, was calculated using a simple GA program. It was reported the deletion rate during a general oligonucleotide synthesis is about 0.5% per coupling [20], and our data (about 0.3% per coupling) were compatible with this value. We incorporated the effect of the single nucleotide deletion into the GA calculation. We considered only the affect of a point deletion in a synthesized oligonucleotide because the deletion rate is low enough. When a point deletion occurs in the 5' constant region, all the amino acids in the random region are the frame shifted ones. When the event occurs at the i-th site of the random region, it affects the composition in the all downstream from the i-th site, and so on. We incorporated all these effects into the calculation of the composition. Details are described in Additional file 1. Authors' contributions IT conceived of this specific study and participated in its design and coordination. SS carried out GA calculation. SU carried out the sequence analysis and the making of the assembled longer sequence. YH conceived of general background and mathematical detail. Abbreviations MLSDS, multi-line split DNA synthesis; ORF, open reading frame; CPG, controlled pore glass; PAGE, polyacrylamide gel electrophoresis; GA, genetic algorithm; 3NPs, trinucleotide phosphoramidites. Supplementary Material Additional File 1 In the additional WORD file (MLSDS22AdditionalFile.doc), the detail of calculation method is described for the the expected amino acids composition p = (pAla, pArg, ....., pterm) considering the single nucleotide deletion. Click here for file Acknowledgements We thank Dr. T. Aita and Dr. T. Sasaki for useful advise and discussions, and Dr. J. Hattori for critical reading of the manuscript. Figures and Tables Figure 1 Scheme of the MLSDS method. The case of three-lines is shown. Uppermost three boxes indicate the state in the DNA synthesizers after the first three synthetic cycles, that is, partial mixtures of triplet codons attached to the CPGs. 1, 2 and 3 denote 1st, 2nd and 3rd letter mixture in the line-1 DNA synthesizer, respectively. Their A:T:G:C mixing ratios are designed with the GA program so that 123 gives a partial mixture of triplet codons without any stop codons. In the same way, 456 and 789 denote corresponding partial mixtures of triplet codons in the line-2 and the line-3 DNA synthesizers, respectively. The equimolar mixture of 123, 456 and 789, which is obtained after split-and-mix procedure, can be designed to give approximately the target amino acid composition without stop codons. Examples of 1,2,.....,9 are shown in Table 1. Figure 2 Dependence of the final fitness on the number of DNA synthesizers for various libraries. Ordinate: final fitness (=correlation coefficient). Abscissa: line number of DNA synthesizers. Libraries are "Uniform", "Natural", "No Met" and "No Cys". Figure 3 Production of 2×, 4×, and 8× length library. Lane-M: Size marker. 10 base ladder (left) and 100 base ladder (right). Lane-S: Initial synthesized library (87 mer). Lane-1U: Initial single-unit library (94 mer) made by PCR amplification. Lane-2U: Double-unit library (138 mer) made by ligation product of digested 1U. Lane-4U: quadruple-unit library (240 mer) Lane-8U: octuple-unit library (444 mer). PAGE was done with 5 % polyacrylamide, TAE buffer (pH 8.0), 8 M urea, 65°C and stained with SybrGreen I and visualized with a fluorescence imager (Bio-Rad FX). Table 1 Examples of molar mixing ratio of bases for MLSDS libraries Library name No Cys Natural Line Number of Synthesizers Line-1 Line-2 Line-3 Line-1 Line-2 Line-3 ID Number of premix phosphoramidites 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 Molar mixing ratio of A:T:G:C A 2 3 5 5 0 0 2 5 0 5 4 2 2 0 0 3 4 0 T 0 0 3 4 3 0 3 5 2 0 1 1 3 2 3 4 2 5 G 3 0 0 4 3 1 1 0 0 5 0 2 5 5 2 4 0 0 C 3 1 0 0 1 0 1 3 5 3 1 0 2 2 1 2 4 0 Table 2 Comparison of the amino acid compositions of the actually synthesized DNA libraries (the full length library) with the target composition Library name Natural No Cys c-Fos e', g'- Target Actual Target Actual Target Actual Ala 8.9% 11.4% 5.3% 6.7% 0.0% 0.0% Arg 4.7 3.5 5.3 5.9 30.0 29.7 Asn 4.4 3.5 5.3 4.3 0.0 0.0 Asp 5.5 8.6 5.3 5.9 0.0 0.0 Cys 2.8 2.5 0.0 0.0 0.0 0.0 Gln 3.9 2.1 5.3 5.5 20.0 19.6 Glu 6.2 6.9 5.3 5.5 20.0 25.0 Gly 7.8 12.0 5.3 8.2 0.0 0.0 His 2.0 1.2 5.3 2.7 0.0 0.0 Ile 4.6 3.9 5.3 5.9 0.0 0.0 Leu 7.5 3.7 5.3 4.3 0.0 0.0 Lys 7.0 6.2 5.3 4.7 30.0 27.9 Met 1.7 1.1 5.3 3.5 0.0 0.0 Phe 3.5 4.0 5.3 5.1 0.0 0.0 Pro 4.6 2.5 5.3 2.4 0.0 0.0 Ser 7.1 8.1 5.3 1.6 0.0 0.0 Thr 6.0 5.8 5.3 6.7 0.0 0.0 Trp 1.1 1.8 5.3 8.2 0.0 0.0 Tyr 3.5 4.4 5.3 4.7 0.0 0.0 Val 6.9 6.9 5.3 8.2 0.0 0.0 Term 0.0 0.0 0.0 0.0 0.0 0.0 Number of sequenced codon - 568 - 255 - 219 Correlation coefficient - 0.85 - 0.66 - 1.00 Sum of absolute errors - 26.9% - 28.1% - 5.66% "Term" denotes stop codons. In each target composition, [Term] = 0. "Natural": the library with the natural abundance (the average composition in 207 natural proteins) [18]; "No Cys": the library with uniform composition except [Cys] = 0; "c-Fos e', g'-": a c-Fos mutant library containing only 4 kinds of amino acid as same as in Ref.[26]. Table 3 The comparison between the target amino acids composition and the actual composition of various libraries. A. Uniform Type Method 3NPs mRNA display MLSDS Reference, Library [29], 96T λ 6 [18], Random This work, No Cys Target value Equimolar 19 amino acids Equimolar 20 amino acids Equimolar 19 amino acids Correlation coefficient a 0.43 0.56 0.66 Sum of absolute errors b [%] 54.5 23.7 28.1 Percentage of stop codon ND c 0.46 0.00 Percentage of cassettes containing stop codon ND c 8.33 0.00 B. Doping Type Method 3NPs mRNA display MLSDS Reference, Library [29], 100z [18], β-Cassette polar This work, c-Fos, e', g'- Target value Biased Polar amino acid Biased Correlation coefficient a 0.94 0.56 1.00 Sum of absolute errors b [%] 42.4 73.7 5.66 Percentage of stop codon ND c 0.39 0.00 Percentage of cassettes containing stop codon ND c 6.25 0.00 The values were calculated using the data of full-length (without deletion) libraries. Values of 3NPs and mRNA display method were calculated with some assumptions. In mRNA display method, the value of stop codons were estimated from data described in the article and some assumptions; one cassette did not have two or more stop codons and stop codons appeared equally in every position. The target composition of β-Cassette polar is assumed that polar amino acids are equi-molar and the others are 0%. aCorrelation coefficient is calculated using target and actual amio acids composition including stop codons contribution. And it is assumed that stop codons were not appeared in full-length libraries of 3NPs method. b Sum of absolute values of difference between each target and actual amino acid. c ND is abbreviation of no data. ==== Refs Lam KS Salmon SE Hersh EM Hruby VJ Kazmierski WM Knapp RJ A new type of synthetic peptide library for identifying ligand-binding activity Nature 1991 354 82 84 1944576 10.1038/354082a0 Smith GP Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface Science 1985 228 1315 1317 4001944 Roberts RW Szostak JW RNA-peptide fusions for the in vitro selection of peptides and proteins Proc Natl Acad Sci USA 1997 94 12297 12230 9356443 10.1073/pnas.94.23.12297 Tabuchi I Soramoto S Nemoto N Husimi Y An in vitro DNA virus for in vitro protein evolution FEBS Lett 2001 508 309 312 11728441 10.1016/S0014-5793(01)03075-7 Tabuchi I Next-generation protein-handling method: puromycin analogue technology Biochem Biophys Res Commun 2003 305 1 5 12732187 10.1016/S0006-291X(03)00686-7 Arkin AP Youvan DC Optimizing nucleotide mixtures to encode specific subsets of amino acids for semi-random mutagenesis Biotechnology 1992 10 297 300 1368102 10.1038/nbt0392-297 LaBean TH Kauffman SA Design of synthetic gene libraries encoding random sequence proteins with desired ensemble characteristics Protein Sci 1993 2 1249 1254 8401210 Tomandl D Schober A Schwienhorst A Optimizing doped libraries by using genetic algorithms J Comput Aided Mol Des 1997 11 29 38 9139109 10.1023/A:1008071310472 Kitamura K Kinoshita Y Narasaki S Nemoto N Husimi Y Nishigaki K Construction of block-shuffled libraries of DNA for evolutionary protein engineering: Y-ligation-based block shuffling Protein Eng 2002 15 843 853 12468719 10.1093/protein/15.10.843 Shiba K Takahashi Y Noda T Creation of libraries with long ORFs by polymerization of a microgene Proc Natl Acad Sci U S A 1997 94 3805 3810 9108059 10.1073/pnas.94.8.3805 Sondek J Shortle D A general strategy for random insertion and substitution mutagenesis: substoichiometric coupling of trinucleotide phosphor-amidites Proc Natl Acad Sci U S A 1992 89 3581 3585 1565654 Virnekas B Ge L Pluckthun A Schneider KC Wellnhofer G Moroney SE Trinucleotide phosphoramidites: Ideal reagents for the synthesis of mixed oligonucleotides for random mutagenesis Nucleic Acids Res 1994 22 5600 5607 7838712 Kayushin AL Korosteleva MD Miroshnikov AI Kosch W Zubov D Piel N A convenient approach to the synthesis of trinucleotide phosphoramidites – synthons for the generation of oligonucleotide/peptide libraries Nucleic Acids Res 1996 24 3748 3755 8871554 10.1093/nar/24.19.3748 Ono A Matsuda Zhao J Santi DV The synthesis of blocked triplet-phosphoramidites and their use in mutagenesis Nucleic Acids Res 1995 23 4677 4682 8524660 Cho G Keefe AD Liu R Wilson DS Szostak JW Constructing high complexity synthetic libraries of long ORFs using in vitro selection J Mol Biol 2000 297 309 319 10715203 10.1006/jmbi.2000.3571 Glaser SM Yelton DE Huse WD Antibody engineering by codon-based mutagenesis in a filamentous phage vector system J Immunol 1992 149 3903 3913 1460281 Neuner P Cortese R Monaci P Codon-based mutagenesis using dimer-phosphoramidites Nucleic Acids Res 1998 26 1223 1227 9469829 10.1093/nar/26.5.1223 Cormack BP Valdivia RH Falkow S FACS-optimized mutants of the green fluorescent protein (GFP) Gene 1996 173 33 38 8707053 10.1016/0378-1119(95)00685-0 Gaytan P Osuna J Soberon X Novel ceftazidime-resistance beta-lactamases generated by a codon-based mutagenesis method and selection Nucleic Acids Res 2002 30 e84 12177312 10.1093/nar/gnf083 Hecker KH Rill RL Error analysis of chemically synthesized polynucleotides Biotechniques 1998 24 256 260 9494726 Proba K Worn A Honegger A Pluckthun A Antibody scFv fragments without disulfide bonds made by molecular evolution J Mol Biol 1998 275 245 253 9466907 10.1006/jmbi.1997.1457 Aita T Iwakura M Husimi Y A cross-section of the fitness landscape of dihydrofolate reductase Protein Eng 2001 14 633 638 11707608 10.1093/protein/14.9.633 Hirao I Ohtsuki T Fujiwara T Mitsui T Yokogawa T Okuni T Nakayama H Takio K Yabuki T Kigawa T Kodama K Yokogawa T Nishikawa K Yokoyama S. 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==== Front BMC Complement Altern MedBMC Complementary and Alternative Medicine1472-6882BioMed Central London 1472-6882-4-131536959610.1186/1472-6882-4-13Research ArticleHarpgophytum procumbens for osteoarthritis and low back pain: A systematic review Gagnier Joel J [email protected] Sigrun [email protected] Eric [email protected] Department of Health Policy Management and Evaluation, University of Toronto, Faculty of Medicine, Toronto, Canada2 Canadian College of Naturopathic Medicine, Academics, Toronto, Canada3 Department of Forensic Medicine, University of Freiburg, Freiburg, Germany4 Herbal Medicines Research and Education Center, Faculty of Pharmacy, University of Sydney, Sydney, Australia5 Center for Integrative Medicine, University of Maryland, School of Medicine, USA2004 15 9 2004 4 13 13 27 2 2004 15 9 2004 Copyright © 2004 Gagnier et al; licensee BioMed Central Ltd.2004Gagnier et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background The objective of this review is to determine the effectiveness of Harpagophytum procumbens preparations in the treatment of various forms of musculoskeletal pain. Methods Several databases and other sources were searched to identify randomized controlled trials, quasi-randomized controlled trials, and controlled clinical trials testing Harpagophytum preparations in adults suffering from pain due to osteoarthritis or low back pain. Results Given the clinical heterogeneity and insufficient data for statistical pooling, trials were described in a narrative way, taking into consideration methodological quality scores. Twelve trials were included with six investigating osteoarthritis (two were identical trials), four low back pain, and three mixed-pain conditions. Conclusions There is limited evidence for an ethanolic Harpagophytum extract containing less than <30 mg harpagoside per day in the treatment of knee and hip osteoarthritis. There is moderate evidence of effectiveness for (1) the use of a Harpagophytum powder at 60 mg harpagoside in the treatment of osteoarthritis of the spine, hip and knee; (2) the use of an aqueous Harpagophytum extract at a daily dose of 100 mg harpagoside in the treatment of acute exacerbations of chronic non-specific low back pain; and (3) the use of an aqueous extract of Harpagophytum procumbens at 60 mg harpagoside being non-inferior to 12.5 mg rofecoxib per day for chronic non-specific low-back pain (NSLBP) in the short term. Strong evidence exists for the use of an aqueous Harpagophytum extract at a daily dose equivalent of 50 mg harpagoside in the treatment of acute exacerbations of chronic NSLBP. ==== Body Background Natives in the steppes of South and Southwest Africa use the secondary root tubers of Harpagophytum procumbens (H) for the treatment of various diseases, including musculoskeletal complaints. For more than half a century, various preparations from H have been continuously used in Europe and have become an established traditional treatment for rheumatic complaints. The monograph of the European Scientific Cooperative on Phytotherapy (ESCOP) [1] recommends H preparations for painful osteoarthritis and relief of low back pain in a dosage equivalent of up to nine grams of crude plant material and over a treatment period of at least two to three months. It has been suggested that the plant material should contain not less than 1.2% of the constituent harpagoside, an iridoid glycoside. Pharmacological studies indicate that in various animal models (e.g. the writhing test in mice) [2] the extract is more effective than its marker compound harpagoside. However, a number of contradictory findings make it difficult to draw definitive conclusions on the analgesic and anti-inflammatory effect of H preparations [3]. Recent in-vitro studies indicate that preparations from H may interact with the inflammatory cascade, including the cytokines [4-6]. Moreover, a significant decrease in stimulated production of matrix-degrading enzymes has recently been shown in isolated chondrocytes [7] and a dose-dependent weak elastase inhibition [8]. The objective of this review was to determine the effectiveness of Harpagophytum preparations in the treatment of musculoskeletal pain. Methods Searching Two reviewers (JG) and (SC) conducted electronic searches using the following databases: PUBMED (1966 up to September 16, 2003), EMBASE (OVID technologies: 1980 to wk 40 2003), Cochrane Controlled Trials Registry, Cochrane Musculoskeletal specialized register, Dissertation Abstracts, BIDS ISI, and the Cochrane Complementary Medicine Fields Specialized Register. The search strategy was developed by combining a highly sensitive method for isolating controlled clinical trials developed for the Cochrane Collaboration [9] [see appendix 1] with a variety of indexing and text words specific to the intervention (H. procumbens) and musculoskeletal conditions. Search strategies were modified for each database [for PUBMED strategy see appendix 2]. One reviewer (SC) contacted experts and acquired relevant citations. In addition, manufacturers of commercial Harpagophytum preparations and content experts were contacted and asked to contribute published and unpublished material. Reference lists in review articles and the retrieved trials were searched for further trials. Study selection Trials that met the criteria outlined in Table 1 were included. Two individuals (JG & SC) independently reviewed titles and abstracts to determine study inclusion. A consensus method was used to resolve disagreements about inclusion of studies. Table 1 Inclusion criteria for considering studies for this review Types of studies Randomized controlled trials (RTCs), quasi-randomized controlled trials, and controlled clinical trials (CCTs) with no language restriction. Types of participants Adults suffering from pain in the musculoskeletal system due to osteoarthritis or low back pain. Types of interventions Studies utilizing preparations of Harpagophytum procumbens were included. Preparations may differ in the solvent (water, alcohol) used to prepare the extract (if not crude powdered plant material is used), the drug extract ratio, and the galenic aplication form. They also differ in the content of the active principles (the sum of active ingredients) and in the quantity of the co-active marker compound harpagoside (Chrubasik et al. 1996, Sporer and Chrubasik 1999). Types of outcome measures Primary outcome: pain (e.g. visual analogue scale, visual rating scale, pain component of the disease-specific Arhus Low Back Pain Index, component pain of the Western Ontario MacMaster (WOMAC) instrument). Secondary outcomes: number of pain-free patients (defined as being pain-free on at least five days in the last treatment week without taking any rescue medication see above), functional indices (e.g. Lequesne index, finger-ground distance), and generic outcome measures [global assessments, health assessment questionnaire (HAQ)] or the consumption of additional analgesic treatment. Data abstraction Two reviewers (SC, JG) extracted data from each trial using a standardized form. The following data were extracted from each study: authors' names; date of publication; country of origin; type of study, including number of study centers; participants (numbers, disease(s), characteristics of the study population (age, size, weight, gender)); duration of acute exacerbation or chronic disease; baseline values with details on pain and previous treatments; additional treatments; types of outcome measures; summary statistics; timing of outcome assessment; withdrawals and drop-outs; and adverse events. Blinding to authors, institution or journal title was not done, given the contradictory evidence relating blinded reviewing and bias [10]; additionally, two reviewers (JG and SC) were very familiar with the literature. Methodological quality assessment Methodological quality was assessed using the criteria list developed by van Tulder et al (1997; 2003) [10,11]. Specifically, the internal validity criteria A, B, C, E, F, G, H, I, J, K, L1, N, and O were used. Each criterion could be scored as yes (Y), no (N), or don't know (DK). The score of Y reflects the fulfillment of that criterion. The scoring of N reflects lack of fulfillment of that criterion. The scoring of DK reflects the inability to determine whether or not the criterion was fulfilled. High quality studies are defined as those that fulfill more than 50% (>6) of the quality criteria. Sensitivity analyses were carried out to explore the results when the definition of high quality trials was set at 40 % (>5) and 60% (>7) fulfillment of the quality criteria. According to the Van Tulder Scoring [10,11], the levels of evidence were defined as follows: 1. Strong – consistent findings among multiple high quality RCTs 2. Moderate – consistent findings among multiple low quality RCTs and/or CCTs and/or one high quality RCT 3. Limited – one low quality RCT and/or CCT 4. Conflicting – inconsistent findings among multiple trials (RCTs and/or CCTs) 5. No evidence from trials – no RCTs or CCTs Planned subgroup analyses included: (1) pain site, (2) type of pain (acute (≤ 6 weeks duration), sub-acute (6 to 12 weeks duration), and chronic pain (> 12 weeks)), and (3) comparison (botanical medicine (considering preparation form) versus placebo and botanical medicine versus other treatment). Results A total of 130 citations were isolated from electronic searches and all abstracts were retrieved. A total of three additional references were supplied by content experts. A total of 120 papers were excluded because of publication type (reviews or reports) or because of improper trial design. The complete references were retrieved for the remaining 13 trials. All 13 references were included in the current review [12-24]. Of these, two trials were duplicate publications [13,21], leaving 12 trials with unique data. A total of five randomized trials included 385 patients (range 46 to 122) with osteoarthritis of the hip or the knee [12,13,18,21-23]. Three of these trials were placebo controlled [12,18,22], and two were compared to standard pharmaceutical treatment forms [13,23]. The H preparations in these trials included a powder of crude plant material [13,22], a 60% ethanolic extract (solvent 60% ethanol)[12,18], and an aqueous extract [23]. A total of four trials included 505 patients (range 88 to 197) with acute exacerbation's of chronic non-specific low back pain [14-17]. All were randomized controlled trials, with two using placebo control [14,16], one using various conventional treatment controls (e.g. NSAIDs, exercise, massage, nerve blocks, acupuncture, etc.) [15], and one using a Cox-2 inhibitor control (Vioxx) [17]. These trials all used aqueous extracts of H. The last three trials included 215 patients with various forms of musculoskeletal pain (range 50 to 100) [19,20,24]. Schmelz et al (1997)[24] included subjects with acute exacerbations of joint arthrosis, chronic low back pain, and rheumatic muscle pain. Guyader (1984) [20] included subjects with gonarthrosis, poly-arthrosis, coxarthrosis, and arthrosis of the cervical spine, lumbar spine, or the nerve root canal. Gobel et al (2001) [19] included subjects with pain and/or muscle tension in shoulder, neck, and/or back. All three trials were placebo controlled. Schmelz et al (1997) [24] used an aqueous extract, Guyader (1984) [20] dried mother tincture (solvent 45% ethanol), and Gobel et al (2001) [19] an ethanolic extract (solvent 60% ethanol). All trials, except for two [12,24], reported adverse events for the interventions. Methodological quality and sensitivity analysis Ratings for each trial on each quality criterion are reported in Table 2. Of the trials including patients with various forms of arthritis, four were considered high quality [13,18,22,23] and one low quality [12] with 60% and 50% cutoffs for methodological quality fulfillment; all trials were considered high quality if the cutoff was 40% [12,13,18,22,23]. There were very few instances of inadequate reporting (DK scores) in these trials (5/52) criteria assessed across trials); in cases where inadequate reporting was found, these tended not to be the same criteria across trials. The authors of these trials were not contacted for clarification regarding these items. Table 2 Methodological quality of controlled trials of Harpagophytum procumbens Methodological Quality Criteria Gobel et al, 2001 Schmelz et al, 1999 Guyader, 1984 Chrubasik et al, 1996 Chrubasik et al, 1997 Chrubasik et al, 1999 Chrubasik et al, 2003 Chantre et al, 2000 Frerick et al, 2001 Lecomte & Costa, 1992 Biller et al, 2002 Schruffer, 1980 A Were eligibility criteria specified? n n n y y y y y y n y n B Was randomization appropriate? y y y y y y y y y y y y C Was treatment allocation concealed? y y y y n y y y y y y y E Were groups similar at baseline regarding important prognostic indicators? dk dk n y y y y y n dk dk dk F Were outcome measure(s) and the control interventions explicitly described? y y y y y y y y y y y y G Were co-interventions avoided or comparable? dk dk dk y y y y y y dk y y H Were the outcome measures relevant? y y y y y y y y n y n y I Were adverse events described? y dk y y y y y y y y n y J Were drop-outs described? n y y y y y y y n dk n y K Was the sample size based on a priori power calculation? n n n y n y n y n n n n L1 Did the study include intention-to-treat analysis? and/or n y n n y y y y n y n y N Were point estimates and measures of variability presented for the POM? y n n y y y y y y y n n O Was the timing of outcomes appropriate? y y n y y y y y y y y Y Total 7 7 6 12 11 13 12 13 8 8 6 9 Of the trials utilizing subjects with acute exacerbations of chronic non-specific low back pain, all four were considered to be of high methodological quality regardless of the cutoff [14-17]. Methodological aspects that were unclear in the published reports were clarified by the study author (SC). Of those trials including a mixed sample of subjects [19,20,24], two were low quality when the cutoff was 60%, or 50% and all three were high quality when the cutoff was 40%. All trials did not adequately report sufficient information to judge baseline similarity, if co-interventions were avoided or comparable, or if there were any adverse events [24]. Harpagophytum preparations for osteoarthritis Powdered crude plant material compared to placebo Lecomte & Costa (1992) [22] utilized a powder of the secondary roots of H in 89 subjects (44 placebo; 45 H) with 98 locations of arthrosis (with two locations in three subjects in the placebo group and six in the H group). Of those with one location, 31 had osteoarthritis of the spine, 18 of the cervical spine, 14 of the hip, and 30 of the knee. Results favoured the H group. Detailed descriptions of each original study included in this review are provided in Table 3. Table 3 Description of trials included in this review Study Sample Size Condition; mean age (range) Harpagophytum Intervention / control Outcome measures and effects Adverse effects Reviewer's Overall Conclusions Schrüffler 1980 (Germany) 50 Osteoarthritis; 51 years 2500 mg/ day, (harpagoside less than 30 mg per day) / Phenylbutazone for 4 weeks mean pain improvements: H 80%, Phenylbutazone 72%; physical impairment: H n = 1, Phenybutazone n = 5; morning stiffness: H n = 2, Phenybutazone n = 5 0 H vs 4 Phenylbutazone H better than Phenylbutazone Lecomte and Costa 1992 (France) 89 Osteoarthritis; (55–75) years 2000 mg/day (Harpagoside content estimated indirectly as 60 mg per day) / placebo for 60 days mean pain improvement: H 38%, P 25% p < .05; finger-ground distance modified Schober test (cm) mean improvement: H 16%, P 6% p < .05 none for either group H better than placebo Biller 2002 (Germany) 78 Osteoarthritis; not stated 4500 mg/day, (harpagoside content estimated at < 30 mg per day) / placebo for 20 weeks responders: H 90%, P 80% p-value not stated; mean consumption of ibuprofen: H .1, P .5 tablets not stated H better than placebo Chantre et al. 2000 (France) 122 Osteoarthritis; 62 years 4500 mg / day, (57 mg harpagoside per day) / Diacerhein for 16 weeks difference after 16 weeks between groups as measured by Lequesne functional index: less than 10 mm NS (intention-to-treat analysis with not all possible confounders considered) 10 H vs 21 Diacerhein H not worse than diacerhein Frerick et al. 2001 (Germany) 46 Osteoarthritis; 59 years 4500 mg/day, (< 30 mg harpagoside per day) / placebo for 20 weeks responders: H 71%, P 41% p=.041; WOMAC component pain NS (type of statistical analysis not stated) 8 H vs 7 P H better than placebo Chrubasik et al. 1996b (Germany) 118 Back pain; 54 years 4500 mg/day, (50 mg harpagoside per day) / placebo for 4 weeks mean tramadol consumption: H 99 ± 157 mg, P 102 ± 250 mg p =.44; number of pain-free patients at 4th week: H 9 P 1 p=.008; percentage change Arhus component pain: H 34%, P 6% p=.016 (per protocol analysis) 4 H vs 10 P not on primary outcome measure Chrubasik et al. 1997 (Germany) 102 Back pain; 49 years 4500 mg/day, (30 mg harpagoside per day) / conventionally treating physicians administering oral NSAIDs, physical exercises, or paravertaebral injections for 6 weeks number of pain-free patients 4th week: H 16, C12 NS; number of pain free patients 6th week: H 20, C 23 NS; percentage change Arhus component pain after four weeks: H 23%, C 22% p=.95; after 6 weeks H 33%, C 38% p=.38 5 H vs 0 C H not worse than C Chrubasik et al. 1999 (Germany) 197 Back pain; 56 years 4500 and 9000 mg/day, (50 and 100 mg harpagoside per day) / placebo for 4 weeks number of pain-free patients: H-100 18%, H-50 9%, P 5% p=.027); percentage change Arhus component pain: H-100 vs H-50 vs P NS (intention-to-treat analysis) 10 P, 18 H-50, 17 H-100 H better than placebo Chrubasik et al. 2003a (Germany) 88 Back pain; 62 years 4500 mg/day, (60 mg harpagoside per day) / Rofecoxib for 6 weeks number of pain-free patients: H 22%, Rofecoxib 11% NS; percentage change Arhus component pain: H 30%, Rofecoxib 29% (intention-to-treat analysis) 14 H, 14 Rofecoxib H not worse than Rofecoxib Schmelz and Hämmerle 1999 (Germany) 100 Mixed pain; not stated 4500 mg/day, (30 mg harpagoside per day) / placebo for 30 days free of low back pain: H n = 4, P n = 2; free of other pain: H n = 5, P n = 0 (confounders not considered) not stated H better than placebo Guyader 1984 (France) 50 Mixed pain; 64 years Harpagoside content estimated indirectly as <20 mg harpagoside per day / placebo for 1–3 'cycles' of 21 days each mean pain improvements: H 72%, P 65% (confounders not considered) 6 H vs 3 P H better than placebo Goebel et al. 2001 (Germany) 65 Mixed pain; 28 years 4500 mg/day, (< 30 mg harpagoside per day) / placebo for 28 days 4 H vs 2 P H better than placebo Key: NS = not significant; H = harpagophytum; P = placebo; WOMAC = Western Ontario and McMaster Universities Arthritis Index Powdered crude plant material compared to diacerhein Chantre et al (2000) [13] gave H cryodried drug powder (proprietary product HarpadolR) or Diacerhein (D), to 122 subjects with acute exacerbations of hip and knee. Groups did not differ significantly in sponanteous pain or the Lequesne index, though differences from baseline were larger for the H group. Subjects in the H group used less diclofenac (mean = 21 tablets) than those in the D group (60 tablets) and also used less acetominophen-caffeine (H = 40 tablets and D = 60 tablets). H extract (solvent 60% ethanol) compared to placebo Biller (2002) [12] gave knee arthrosis participants a H product named FlexilogesR or placebo in addition to ibuprofen (at 800 mg ibuprofen during weeks 1–8, 400 mg during weeks 9 – 16), and only H or placebo during weeks 17–20. The main outcome measure was the responder rate, which allowed a WOMAC pain score increase of up to 20% and no additional consumption of ibuprofen in weeks 17 to 20. Results favoured the H group. Frerick et al (2001) [18] gave 46 individuals with acute exacerbations of coxarthrosis FlexilogesR or placebo in addition to ibuprofen (at 800 mg ibuprofen during weeks 1–8, 400 mg during weeks 9–16) and only H or placebo during weeks 17–20. The main outcome was the responder rate, which was defined as the number of patients that required fewer than 4000 mg ibuprofen and had a pain score increase of no more than 20% on the WOMAC component pain during weeks 17 to 20. Results indicated more responders in the H group. Aqueous extract compared to NSAID Schruffler (1980) [23] compared H (proprietary product, SalusR,) with phenylbutazone among 40 individuals with acute exacerbations of rheumatic joint and muscle pain and 10 with gouty arthritis. Results favoured the H group. Harpagophytum procumbens for acute exacerbation of chronic non-specific low back pain (NSLBP) Aqueous extract compared to placebo In the Chrubasik study (1996) [14], 128 patients suffering from pseudo-radiating or non-radiating NSLBP were allocated to receive either a proprietary extract, DoloteffinR,, or placebo. Results favoured the H group (see Table 4). Of the 59 patients in the H group, five dropped out, one of these due to tachycardia. Of the 59 patients in the P group, four dropped out for unknown reasons. A total of four adverse effects occurred in the H group. These consisted of two individuals with nausea/emesis due to the tramadol, one patient with repeated tussive irritation, and the patient with tachycardia mentioned above. A total of 10 adverse events occurred in the P group. These included nausea (N = 2), and one patient each of nausea/vertigo due to tramadol, fatigue/vertigo, vertigo alone, diuresis/normalization of constipation (i.e. intractable constipation), constipation (several times), diuresis (several times), and sleep disturbances (permanent). An additional study by Chrubasik et al (1999) [16] randomized subjects suffering from pseudo-radiating or non-radiating NSLBP were allocated to receive the proprietary extract WS1531, at a dose equivalent to either 4500 mg H (with 50 mg harpagoside per day, H50) or 9000 mg H /day (with 100 mg harpagoside per day, H100), or placebo (P; N = 66). Participants had acute exacerbations of non-specific low back pain; current pain that was > 5 on a VAS (0–10). The median durations of chronic pain were P 15 years, H50 15 years, H100 15 years. The number of patients with acute exacerbations of greater than 3 months for each group was P 54 (82%), H50 53 (82%), H100 55 (83%). Current pain and Arhus scores were similar among all groups. The principle outcome measure, the number of patients who were pain-free without the permitted rescue medication for 5 days out of the last treatment week was 3 (P), 6 (H50) and 10 (H100) (p = 0.027, one-tailed Cochrane-Armitage test). The authors found significant improvements in pain in both H groups as compared with the placebo group. A subgroup analysis found differences between groups for those without pain radiating to the legs (H100 40%, H50 43% and P 23%; P = 0.017) and for those without a neurological deficit (H100 40%, H50 60% and P 20%; P = 0.034). Aqueous extract compared to NSAID Chrubasik et al (2003) [17] randomized 88 individuals suffering from pseudo-radiating or non-radiating NSLBP recieved either the proprietary extract DoloteffinR (N = 44) or VioxxR (rofecoxib; R; N = 44). The percentage of subjects with chronic pain of > 6 days for each group was H 84%, R 84% and acute pain of > 90 days in H 91%, R 89%. Results indicated statistically non-significant difference between H and R. Also, a total of 21 (group H) and 13 (group R) patients used tramadol, with the average consumption being 230 mg (H) and 133 mg (R). A total of seven drop-outs occurred due to adverse events (H = 1; R = 6); two others resulted from excessive low back pain (R = 2). There were a total of seven protocol violations. A total of 14 participants in each group had adverse events with gastrointenstinal complaints, equaling eight in the H group and nine in the R group, with more severe events in the R group. Two adverse events were reported to be unrelated to the study medication in the H group. Aqueous extract compared to various conventional treatments In another study by Chrubasik et al (1997) [15], participants suffering from non-radiating NSLBP were randomized to receive either the proprietary extract JurcubaR or conventional treatments (NSAIDs, exercise, massage, nerve blocks, acupuncture). Subjects' pain was > 5 (VAS 0–10) on at least two of the following five scores: pain at rest, while sitting, lying, and walking or at night. The median duration of acute pain was six weeks for both groups. The median duration of chronic pain was 120 months for the H group and 72 months for the control group. There were no statistically significant differences between the groups. Harpagophytum preparations for mixed pain conditions Dried mother tincture compared to placebo In a placebo controlled double-blind study, Guyader (1984) [20] included 50 patients with poly-arthrosis (N = 14), coxarthrosis (N = 2), arthrosis of the cervical spine (N = 11), lumbar spine (N = 2), and arthrosis of the nerve root canal (N = 6). Subjects were given either "Extract G", a dried mother tincture of Devil's claw (H), or placebo (P) for one to three cycles (18 single P, 16 single H, 6 PH, 4 HP, 2 HH, 1 PPP, 1 PHH, 1 PPH, 1 PHP) of 21 days with seven day intervals. Outcome measures included pain at rest, pain during exercise, joint pressure pain, pain while walking (in cases of cox- and gonarthrosis), and pain at night. All outcomes were taken 10 days after each cycle and assessed on a five-point rating scale for amount of pain (no pain = 0, mild = 1, moderate = 2, severe = 3, excrutiating = 4). A total of 70 cycles were analyzed (37 cycles for P and 33 for H) and a mean pain improvement of 72% in the H group and 65% in the placebo group (p < 0.05) was found. Drop-outs included two subjects. During the H cycles, six adverse events occurred, including nausea, gastralgia, diarrhea, severe constipation, pruritic eruptions with erythema, and generalized pruritis. During the P cycles, five adverse events were observed, including gastralgia, sweating, headache, and aerophagy. Aqueous extract compared to placebo In a double-blind, placebo-controlled trial, Schmelz et al (1997) [24] randomized 100 individuals with acute exacerbations of joint arthrosis (N = 29 (H), N = 27 (P)), chronic low back pain (N = 14 H, N = 17 P), and rheumatic muscle pain (N = 7 H, N = 6 P). Subjects were given either the proprietary extract ArthrotabsR containing extract based on 4500 mg crude plant material (equivalent of 30 mg harpagoside) per day or a placebo for 30 days. Outcome measures included a subjective pain scale (no pain, mild pain, moderate pain, severe pain, excrutiating pain) at baseline and after four weeks of treatment. The number of pain-free individuals with low back pain was H n = 4, P n = 2, and for other pain sites H n = 5, P n = 0. Harpagophytum extract (solvent 60% ethanol) compared to placebo Gobel et al (2001) [19] conducted a double-blind study in 65 individuals with pain or muscle tenseness in shoulder, neck, and/or back having lasted for 14 days prior to the study. They were randomized to placebo (N = 32) or a proprietary extract (Rivoltan; N = 31). The daily extract dosage was based on 4500 mg crude plant material (equivalent to <30 mg harpagoside per day). Outcomes included a visual analogue scale for pain (0–50 mm) as well as an experimental test battery for pain and muscle tension before and after treatment. In the per-protocol analysis the Hgroup had less pain than the P group. A total of two individuals dropped out of the trial with four adverse events occurring in the H group versus two in the P group. Discussion Osteoarthritis of the knee, hip, and spine as well as non-specific low back pain may be associated with pain, stiffness, limitation of function, and diminished quality of life [25]. Although treatment guidelines recommend simple analgesics as first-line drugs [26], surveys indicate that NSAIDs are used in preference to simple analgesics despite the lower safety-margin and the higher cost [11,27,28]. Because of the high incidence of NSAID-related adverse events and complications in the gastrointestinal and cardiovascular systems (especially in the elderly), and the high costs related to adverse events (i.e. gastrointestinal bleeding or perforation), additional medical attendances, diagnostic procedures, treatments and admissions to hospital, alternatives to NSAID therapy should be strongly considered [29-33]. This qualitative analysis of the 12 trials suggests that specific preparations and doses of Harpagophytum procumbens may be effective in various types of musculoskeletal pain conditions. Statistical pooling was not possible because of a lack of adequate data and clinical heterogeneity. The sensitivity analysis for methodological quality revealed that the trials on low back pain were of high quality, the trials on osteoarthritis were of high quality except one moderate quality study, and the trials on mixed pain conditions were of moderate quality. The quality of reporting in most of these trials was good. In order to increase transparency, trialists should refer to the CONSORT statement in designing and reporting clinical trials of herbal medicinal products [34]. One high quality trial indicates that there is moderate evidence of effectiveness for powdered H plant material at a dose equivalent to 60 mg of harpagoside per day for osteoarthritis of the spine, hip, and knee. However, because of the clinical heterogeneity of patients in this trial, a confirmatory study is required to firmly establish efficacy for each location of osteoarthritis. In one high quality study, 4500 mg powder containing 57 mg harpagoside in the daily dosage showed moderate evidence for non-inferiority to diacerhein in patients suffering from acute exacerbations of osteoarthritis in the hip and knee. Two trials employed an ethanolic extract (solvent 60% ethanol) containing less than 30 mg harpagoside per day in patients with osteoarthritis of the knee [12] and hip [18]. Both trials showed statisitically significant favourable results for the H group in terms of percentage of responders. However, the definition of responder in these trials may be questioned because of an allowance of pain increase up to 20% and additional rescue medication in one of the studies [18]. Therefore, given the low methodological quality of the trials and the lack of clinically significant differences between groups, we conclude there is limited evidence for the use of an ethanolic H extract based on 4500 mg crude plant material per day in patients with osteoarthritis of the knee and hip. Additional high quality trials must be done to determine the efficacy of Harpagophytum procumbens in osteoathritis. These trials must include homogenous pain conditions and must test H against standard osteoarthitis medications. Additionally, trialists should consider using symptom severity outcome measures that have proven validity and reliability, such as visual analogue scales [35-37], osteoarthritis specific outcome measures (e.g. WOMAC, Lequesne Index) [35-38], and health-related quality of life instruments (e.g. Medical Outcomes Survey Short-Form 36) [35,37,38]. A total of four high quality trials tested various dosages of Hextract in acute exacerbations of chronic non-specific low back pain. Two trials with a total of 325 patients showed that an aqueous extract at the equivalent daily dosage of 50 mg harpagoside appears to reduce pain in patients with acute episodes of chronic NSLBP greater than does placebo [14,16]. Therefore, the 50 mg harpagoside per dose of an aqueous extract of H can be said to have strong evidence for the treatment of acute episodes of chronic NSLBP in the short term. Additionally, a one year survey indicates that the aqueous extract is well tolerated [38]. One trial with 197 patients showed that an aqueous H extract at the equivalent daily dose of 100 mg harpagoside appears to reduce pain in patients with acute episodes of chronic NSLBP greater than does placebo [16]. Therefore, the 100 mg harpagoside per dose of an aqueous H extract has moderate evidence for the treatment of acute episodes of chronic NSLBP in the short term. Superiority of the higher dose was seen in the primary outcome (number of pain-free patients) but not in the secondary outcome measure. Therefore, there is moderate evidence for superiority of the 100 mg H dose to the 50 mg H dose. However, additional trials are required to confirm superiority of 100 mg H over 50 mg H. It is possible that a subgroup of individuals with neurological deficits (e.g. radiation into the leg) may respond well to the 100 mg harpagoside dose, yet more research is required to clarify this. An aqueous extract of H at the equivalent daily dose of 60 mg harpagoside appears to be equivalent to 12.5 mg Rofecoxib in improving pain in individuals with acute episodes of chronicNSLBP [17]. Therefore, a 60 mg daily harpagoside dose in aqueous extract of H has moderate evidence for being not inferior to 12.5 mg rofecoxib per day in the treatment of acute episodes of chronic NSLBP in the short term. Additional high quality trials, especially over longer treatment periods, are mandatory. Furthermore, equivalence trials testing Harpagophytum procumbens against standard treatments will clarify relative efficacy and safety. The final trials (two of moderate [19,24] and of poor quality [20] included heterogeneous musculoskeletal pain conditions. Therefore, it is difficult to reach any conclusions on the basis of these trials. Future trials should attempt to include homogenous pain conditions. The results obtained with the proprietary Harpagophytum products containing aqueous extracts can neither be transferred to an aqueous extract containing less harpagoside in the daily dosage [41] or to a product containing an ethanolic extract. This can only be done if the ethanolic extract was shown to be essentially similar to aqueous extract, and if both extracts have the same qualitative and quantitative composition of co-active constituents, same pharmaceutical form, and bioequivalence in terms of safety and efficacy [40]. With 60% ethanol as solvent, only half the amount of harpagoside (and possibly other co-active constituents) is extracted compared to water as solvent [42]; therefore, it is of great importance that a confirmatory study provide evidence of effectiveness for the ethanolic extract. Since the "active principle" has not yet been identified for Harpagophytum procumbens, the constituent harpagoside is used as a marker for standardization of Harpagophytum preparations. For harpagoside, the dose-dependent absorption into systemic circulation has been shown and may be related to lipoxygenase inhibition [43]. However, it remains to be established if the inhibitory effect on leukotriene production corresponds to therapeutic efficacy. Future research should attempt to identify the active constituent or profile of constituents that relate to therapeutic efficacy in order to make extract dosing transparent. There are several drawbacks to the present study. First, this is a qualitative review and as such it does not provide a quantitative summary of results, thus making it difficult to determine the size of effect of each intervention. Secondly, this review includes a small number of trials, often with small sample sizes. This makes it difficult to state definitive conclusions of efficacy and suggests the need for more trials. On the other hand, the trials reviewed were generally of good methodological quality and have several statistically significant and clinically significant effects. Therefore, these trials help us reach some clear conclusions regarding the use of specific preparations and doses of Harpagophytum procumbens for osteoarthritis and non-specific low back pain. Another strength of this study is the comprehensive search strategy, the methodological quality assessment, and the use of an accepted method for a best evidence synthesis. Future reviews may attempt to statistically combine the results of such trials into a meta-analysis. Conclusions There is limited evidence for an ethanolic Harpagophytum extract containing less than <30 mg harpagoside per day in the treatment of knee and hip osteoarthritis. There is moderate evidence of effectiveness for (1) the use of a Harpagophytum powder at 60 mg harpagoside in the treatment of osteoarthritis of the spine, hip and knee; (2) the use of an aqueous Harpagophytum extract at a daily dose of 100 mg harpagoside in the treatment of acute exacerbations of chronic non-specific low back pain; and (3) the use of an aqueous extract of Harpagophytum procumbens at 60 mg harpagoside being non-inferior to 12.5 mg rofecoxib per day for chronic non-specific low back pain (NSLBP) in the short term. Strong evidence exists for the use of an aqueous Harpagophytum extract at a daily dose equivalent of 50 mg harpagoside in the treatment of acute exacerbations of chronic NSLBP. Competing interests One individual (SC) was an author of several original trials included in this systematic review. This did not appear to influence the content of this paper. Authors' contributions JG developed the initial idea for the manuscript, searched for trials, extracted data and wrote and edited the manuscript. SC developed the initial idea for the manuscript, searched for trials, extracted data, and wrote and edited the manuscript. EM extracted data and wrote and edited the manuscript. Appendix 1 Highly sensitive search strategy for randomized controlled trial searches using PUBMED (randomized controlled trial [pt] OR controlled clinical trial [pt] OR randomized controlled trials [mh] OR random allocation [mh] OR double-blind method [mh] OR single-blind method [mh] OR clinical trial [pt] OR clinical trials [mh] OR (clinical trial [tw]) OR ((singl* [tw] OR doubl* [tw] OR trebl* [tw] OR tripl* [tw]) AND (mask* [tw] OR blind* {tw])) OR (latin square [tw]) OR placebos [mh] OR placebo* [tw] OR random* [tw] OR research design [mh:noexp] OR comparative study [mh] OR evaluation studies [mh] OR follow-up studies [mh] OR prospective studies [mh] OR cross-over studies [mh] OR control* [tw] OR prospectiv* [tw] or volunteer* [tw]) NOT (animal [mh] NOT human [mh]) Appendix 2 PUBMED search strategy (randomized controlled trial [pt] OR controlled clinical trial [pt] OR randomized controlled trials [mh] OR random allocation [mh] OR double-blind method [mh] OR single-blind method [mh] OR clinical trial [pt] OR clinical trials [mh] OR (clinical trial [tw]) OR ((singl* [tw] OR doubl* [tw] OR trebl* [tw] OR tripl* [tw]) AND (mask* [tw] OR blind* {tw])) OR (latin square [tw]) OR placebos [mh] OR placebo* [tw] OR random* [tw] OR research design [mh:noexp] OR comparative study [mh] OR evaluation studies [mh] OR follow-up studies [mh] OR prospective studies [mh] OR cross-over studies [mh] OR control* [tw] OR prospectiv* [tw] or volunteer* [tw]) NOT (animal [mh] NOT human [mh]) AND ("harpagophytum procumbens" OR (devil's AND claw)) AND (pain OR "musculoskeletal pain" OR "muscle pain" OR "skeletal pain" OR "bone pain" OR "joint pain" OR "extremity pain" OR myaligia OR osteoarthritis OR "rheumatoid arthritis" OR arthrosis OR "low back pain" OR lumbago OR "back pain") NOT review Pre-publication history The pre-publication history for this paper can be accessed here: Acknowledgements None declared. ==== Refs ESCOP Monograph. 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Etude historique et pharmacology, et etude clinique du nebulisat d'harpagophytum procumbens d.c. chez 50 patients arthrosiques suivis en service hospitalier These pour le doctorat en medecine diplome d'etat 1984 Leblan D Chantre P Fournie B Harpagophytum procumbens in the treatment of knee and hip osteoarthritis Four-month results of a prospective multicentre, double-blind trial versus diacerhein 2000 76 462 467 Lecomte A Costa JP Harpagophytum dans l'arthrose: Etudes en double insu contre placebo 37°2 Le Magazine 1992 15 27 30 Schruffer H Salus teufelskralle-tabletten Die Medizinische 1980 22 25 Schmelz H Hämmerle HD Springorum Chrubasik S Hrsg Chrubasik S, Wink M Analgetische Wirkung eines Teufelskrallenwurzel-Extrakts bei verschiedenen chronisch-degenerativen Gelenk-erkrankungen Rheumatherapie mit Phytopharmaka 1997 Hippokrates-Verlag Stuttgart 77 85 Sangha O Epidemiology of rheumatic diseases Rheumatology 2000 39 3 12 11276800 Hochberg MC Altman RD Brandt KD Clark BM Dieppe PA Griffin MR Moskowitz RW Schnitzer TJ Guidelines for the medical management of steoarthritis. Parts 1 and 2 Arthritis Rheumatism 1995 38 1535 1546 7488272 Pincus T Swearingen C Cummins P Callahan LF Preference for nonsteroidal anti-inflammatory drugs versus acetaminophen and concomitant use of both types of drugs in patients with osteoarthritis J Rheumatol 2000 27 1020 1027 10782831 Wolfe F Zhao S Lane N Preference for nonsteroidal antiinflammatory drugs over acetaminophen by rheumatic disease patients Arthritis Rheumatism 2000 43 378 385 10693878 10.1002/1529-0131(200002)43:2<378::AID-ANR18>3.0.CO;2-2 Griffin MR Piper JM Daugherty JR Snowden M Ray WA Nonsteroidalanti-inflammatory drug use and increased risk for peptic ulcer disease in elderly patients Ann Int Med 1991 114 257 263 1987872 Mazzuca SA Brandt KD Anderson SL Musick BS Katz BP The therapeutic approaches of community based primary care practitioners to osteoarthritisof the hip in an elderly patient J Rheumatol 1991 18 1593 1600 1765987 MacDonald TM Epidemiology and pharmacoeconomic implications of non-steroidal anti-inflammatory drug-associated gastrointestinal toxicity Rheumatology 2000 39 Suppl 2 3 20 11276800 Page J Consumption of NSAIDs and the development of congestive heart failure in elderly patients Arch Intern Med 2000 160 777 782 10737277 10.1001/archinte.160.6.777 Smalley WE Ray WA Daugherty JR Griffin MR Nonsteroidal anti-inflammatory drugs and the incidence of hospitalizations for peptic ulcer disease in elderly patients Am J Epidemiol 1995 141 539 545 7900721 Moher D Schulz KF Altman DG for the CONSORT Group The CONSORT statement: revised recommendations for improving the quality of reports of parallel-group randomized trials Lancet 2001 357 1191 1194 11323066 10.1016/S0140-6736(00)04337-3 Strand V Kelman A Outcome measures in osteoarthritis: randomized controlled trials Curr Rheumatol Rep 2004 6 20 30 14713399 Dougados M Monitoring osteoarthritis progression and therapy Osteoarthritis and Cartilage 2004 12 S55 S60 14698644 Lequesne MG Maheu E Clinical and radiological evaluation of hip, knee and hand osteoarthritis Aging Clin Exp Res 2003 15 380 390 14703004 Chrubasik S Künzel O Thanner J Conradt C Black A A one year follow-up with aqueous Harpagophytum extract DoloteffinR for low back pain Fact (Focus on Alternative and Complementary Therapies) 2003 8 Bellamy N Buchanan WW Goldsmith CH Campbell J Stitt LW Validation study of WOMAC: a health status instrument for measuring clinically important patient relevant outcomes to antirheumatic drug therapy in patients with osteoarthritis of the hip and kenn J Rheumatol 1988 15 1833 40 3068365 Chrubasik S Sporer F Wink M Zum Harpagosidgehalt in Arzneimitteln aus Harpagophytum procumbens Forsch Komplementärmed 1996 3 57 63 Chrubasik S Roufogalis B Issues in quality and comparability of herbal medicinal products, Australian J Pharmacy 2001 82 444 445 Sporer F Chrubasik S Präparate aus der Teufelskralle (Harpagophytum procumbens) Zschr Phytotherapie 1999 20 235 236 Loew D Möllerfeld J Schroedter A Puttkammer S Kaszkin M Investigations on the pharmacokinetic properties of Harpagophytum extracts and their effects on eicosanoid biosynthesis in vitro and ex viso Clin Pharmacol Ther 2001 69 356 364 11372004 10.1067/mcp.2001.115445

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==== Front BMC Health Serv ResBMC Health Services Research1472-6963BioMed Central London 1472-6963-4-261536125510.1186/1472-6963-4-26Research ArticleGood physicians from the perspective of their patients Schattner Ami [email protected] Dan [email protected] Navah [email protected] Department of Medicine, Kaplan Medical Center, Rehovot, Israel2 Hebrew University and Hadassah Medical School, Jerusalem, Israel3 Division of Biostatistics, Clalit Health Services, Tel Aviv, Israel2004 12 9 2004 4 26 26 25 1 2004 12 9 2004 Copyright © 2004 Schattner et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background It is not currently known what is the patient's viewpoint of a "good" physician. We set out to define patient's priorities regarding different physician's attributes in 3 domains important in medical care. Methods Patients hospitalized or attending clinics at a large teaching hospital selected the 4 attributes that they considered most important out of 21 listed arbitrarily in a questionnaire. The questionnaire included 7 items each in the domains of patient autonomy, professional expertise and humanism. Results Participating patients (n = 445, mean age 57.5 ± 16 years) selected professional expertise (50%), physician's patience and attentiveness (38% and 30%, respectively), and informing the patient, representing the patient's interests, being truthful and respecting patient's preferences (25–36% each) as the most essential attributes. Patient's selections were not significantly influenced by different demographic or clinical background. Selections of attributes in the domain of patient's autonomy were significantly more frequent and this was the preferred domain for 31% and as important as another domain for 16% – significantly more than the domain of professional expertise (P = 0.008), and much more than the domain of humanism and support (P < 0.0005). Conclusions Patients studied want their physicians to be highly professional and expert clinicians and show humaneness and support, but their first priority is for the physician to respect their autonomy. ==== Body Background An expert clinician whose choices are evidence-based; sensitive and dedicated to the patient – not just disease-oriented; who heeds and respects the patient's autonomy, striving at conveying all relevant information to the patient to enable a truly shared decision-making. That can be seen as a prototype or role model of a first class physician. However, these laudable qualities, discussed in many texts [1-3], were purely conceived by physicians, for physicians. How do patients value these different physicians' qualities? What would their priorities be? This unique point of view, little discussed in previous studies, is the subject of our current research. Methods Patients hospitalized at our 600-bed teaching medical center or attending one of its day clinics were randomly approached and asked to fill in a one-page questionnaire. Over a period of one month, patients were approached by one of the authors (DR) and invited to participate. Every third patient on the specific day's ambulatory clinic lists and every fifth patient on the list of patients hospitalized at the Department of Medicine were approached. If the fifth patient was found to be too ill to participate (e.g. ventilated, unstable or confused) the next name on the list was selected. These numbers were arbitrarily chosen. Following a brief explanation they were handed the questionnaire. The questionnaire listed 21 physicians' characteristics or behaviors regarding the care of patients, presented in a varying order (different order for different patients) to prevent bias due to an item's position on the list. Patients were asked to select 4 attributes that they considered the most important and would like the best in their own physician. No grading was required. The questionnaire aimed at the patient's image of an excellent physician in general, and did not specify whether the physician was hospital-based or in primary care. The 21 attributes included 7 characteristics in each one of three domains: reflecting professional expertise and high-quality care; reflecting a humanitarian, patient-centered approach; and reflecting patient's autonomy and attentiveness to the patient's preferences and rights. Then patients supplied basic demographic data and the questionnaire was collected about 20 minutes later. The selection of the 21 different physician characteristics or behaviors started from collecting pertinent articles on patient autonomy; physician's humanism and patient support; and on physicians' expertise and professionalism using the author's (AS) collection and a Medline search. Some of the articles were based on patient-derived data. The next stage involved a series of meetings and discussions at the Department of Ambulatory Care and Prevention, Harvard Medical School, Boston. Three senior researchers (2 clinicians, all with extensive experience in research and medical education) – RH Fletcher, T Peters and AS, selected and categorized the 21 items based on the literature and on personal experience. The third stage was a validation study among thirty residents in various stages of their training. They were presented with a mixed list of the 21 items and requested to categorize each one into one of the 3 domains. To rule out that patients categorized these characteristics differently, we performed an additional post-study validation on 30 outpatients that were representative of our study population. Results were similar to those obtained from the residents and confirmed that the vast majority of participants view each of the items as representative of the corresponding domain (Appendix 1) [see additional file 1]. No changes in classification were necessary following the validation study. We planned to a) quantify and study the most 'popular' physician's attributes selected by patients as well as those selected by only a few patients. b) find-out for each patient, whether any one domain was over-represented in the patient's selections (e.g. 2 out of 4 selections belonging to a single domain and less than 2 for each of the others) or – under-represented (no selections in a domain). The study was approved by our Institutional Review Board. Statistical analysis was done using chi-square tests to examine differences between the domains, and between preference of each domain and demographic variables. T-test or one-way ANOVA were used when appropriate. Results A total of 450 patients received the questionnaire and all but 5 consented to participate and returned the filled questionnaires (n = 445). Patient's ages varied from 18 to 89 years (mean 57.5 ± 16) and two thirds were aged 50–80 years. Other patient's characteristics are summarized in Table 1. The top eight physician's qualities preferred by the patients participating in the study (each selected by >25% of patients) are given in Table 2. (Top) and the five physician's qualities selected by <5% are given in the bottom of the same Table. When we transposed each attribute for the domain it represents, basically 2 types or patterns of responses were generated: the AAPH type, reflecting this patient's preference for domain A; and the AAPP type, reflecting the importance (but not dominance) of domain A (as well as P). When no selection at all was made in one domain (such as domain H in the AAPP example), this was also noted (Table 3). Analysis of patients' preferences of physicians' qualities according to domains (qualities within Professional, Humanitarian or patient's Autonomy domains), yielded highly significant differences. For example, 139/445 patients (31%) selected more answers in the patient's autonomy domain than in any other domain, and 69 more patients gave equal importance to patient's autonomy and to one other domain (mostly professionalism). In contrast, humanistic qualities of physicians were selected as the most important by 76/445 patients (17%) only, and just 48 additional patients gave an equal importance to the humanistic and one other domain. Altogether, 90/445 patients clearly selected more characteristics in the professional domain than in any other domain (20%), and 86 other patients gave equal importance in their choices of qualities to the professional and one other domain. Thus, 69% of participating patients gave clear indication as to their preferred domain of physician's characteristics in administering medical care (Table 3). When these preferences and those of patients who entirely disregarded the domain were analyzed and compared, significant differences were found favoring the domain of patient's autonomy (P = 0.008 vs. professional expertise and P < 0.0005 vs. humane attitude) (Table 3). About 10% (38/445), selected equally between the three domains (i.e. one quality of each domain). The remaining 102/445 (22%) were 'indeterminate' in that their choices gave equal importance to two domains. When we analyzed patient's responses to the most essential questions in each of the domains, a similar pattern emerged. For example, 34% selected 2 or more qualities pertaining to patient's autonomy, and 21% selected none. In comparison, <10% named = 2 'humanistic' qualities and over 50% selected none (P < 0.0005). When patient's preferences of the different physicians' characteristics or domains were further analyzed according to the patient's age, gender, origin, income, being hospitalized or main diagnosis – no statistically significant differences or associations could be identified (not shown). Discussion Our study population of 445 patients was heterogeneous (Table 1), yet no statistically significant relationship could be demonstrated between demographic or clinical variables and patient's choices or priorities. These facts lend more impact to our findings. Patient's preferences of physician's attributes were found to be as unique and individual as the patients themselves. It is remarkable that among 445 patients, only 6 made identical choices (1%). Not surprisingly, patients want their physicians to be experienced and highly professional. This was the physician's characteristic that was chosen by 50% of the patients (Table 2, Top). However, 4 of the other 7 most frequently selected attributes, each selected by 25–38% of the patients, were in the domain of patient's autonomy (523/1172 responses, 45%). The distinct priority accorded by patients to attributes in the realm of patient's autonomy, overrides even the domain of professionalism, and certainly that of physician's humanism and support. Attributes belonging to the domain of patient's autonomy were uncommon among the least demanded attributes (1 of 5, Table 2, Bottom), and were significantly more often selected and less often disregarded than any other domain (Table 3). In contrast, attributes of humanism were not selected at all by almost 30% of participating patients. An overview of the results reveals that patients studied want their physicians to show professional expertise and provide humane personal care (a preferred or important domain for 39% or 28% of the patients, respectively) (Table 3) – however, it is even more important to them to be well informed and participate in decisions (Tables 2, Top, and 3). On the other hand, 'humane' qualities that are traditionally considered important, such as showing empathy or being friendly with the patient were surprisingly found to be among the least selected attributes (3–4%, Table 2, Bottom). Moreover, patients participating in this study seldom selected professional qualities such as research and teaching abilities, although they often go hand in hand with appointments at academic medical centers and high quality medical care [4]. Patients also seemed unaware of the unequivocal power of the prevention of "accidents waiting to happen" [5] that can be offered to them by professional physician's counsel. Only 69 patients (15.5%) selected this option as one of four of their priorities. What patients did value, in addition to clinical experience and being up-to-date (Table 2, Top), was for their physicians not to be impatient (38%) or distracted (30%). These two attributes, perhaps the most vulnerable to current time constraints in clinical practice, may reflect patient's needs of a more relaxed, leisurely communication with their physician, be it at the hospital or in primary care. This issue may be more pertinent with the recent changes in context of the consultation, mandating more informed patients and shared decision-making [6,7]. The reason why patient's autonomy features so highly in our study, is not entirely clear. The patient population was heterogeneous (Table 1) so that factors related to origin, income or education are unlikely to be operative. Our medical center is serving a large (about 250,000) population in central Israel which is mostly urban but also rural, and has nothing unique regarding physicians or health delivery system. It is conceivable that the growing emphasis in recent years on patient's rights and patient's autonomy which is prominently reflected in the media and draws much public attention, affected patient's preferences. Further studies will be needed to determine whether, as we believe, our results reflect a novel worldwide trend in patient's preferences. Several limitations of our study ought to be considered. The study was performed in Israel, on a predominantly Jewish population. Thus, its generalizability is open to question. However, Jewish medical ethics put no special value on patient's autonomy so that the findings are likely to apply to other Western affluent societies as well. Also, intentionally, our methodology does not address the relative weight of respondent opinion. Thus, it is likely – but uncertain – that choosing more attributes in a domain is the crucial measure of the relative importance of a domain. Finally, we cannot be entirely certain that the selection process did not lead to some bias, although we consider this contingency to be unlikely. The age of paternalism in medical care has come to an end and few are sorry for its demise. Most patients want to be informed about their health even if the news are bad [8], and to be involved with their care plans [9]. To do that, patients must have clear information, which takes into account their unique circumstances [10], and there is no better source for that than the patient's physician. Our results strongly suggest that patients expect their physicians to heed these needs and prefer physicians who are sensitive to the varied aspects of patient's autonomy and patient's rights (Appendix 1) [see additional file 1]. The studies of Thom et al. have already indicated that certain physician's behaviors were important for patient's trust. Prominent among them were discussing options with the patient and finding out preferences – essential components of patient autonomy [11]. Similar to the present study, measures of professional competence and humanism were also required. Patient trust was significantly correlated with compliance and with clinical improvement [12]. Since patient trust appears to be such a crucial component of the patient-physician relationship, and since trust is dependent on patient's preferences being met [12] it is mandatory to establish what these preferences are today, and this is where our study comes in. Previous research has already identified the complexity of patient's needs in the modern era. While older studies make no mention of issues of patient autonomy [13], a relatively recent systematic review of the literature on patient's priorities found "humaneness" to be the most highly rated aspect of care, followed by clinical competence and patient's participation in decisions [14]. Another study from Scotland identified physicians' attentiveness and patience at the top of the list, and patients in the Netherlands mostly desired sufficient consultation time [15,16]. However, a strong desire for information and participation in decision-making already features in these and other studies [14-18]. Among our patients it came out for the first time as a top priority, second to none (Table 3). Failure of physicians to provide a patient-centered approach may therefore seriously undermine patient's expectations and satisfaction. Recent data suggest that this may be associated with significant adverse outcomes [19,20]. Physicians can be effectively trained to listen to the patient's narrative [21], recognize the patient's perspective [7,22] and adopt a more patient-centered approach [23]. However, besides skills in communication [21,24,25], this requires time. Time management can also be effectively taught [25] but it is still unclear whether decreasing hospital length of stay and consultation time in primary care will not adversely affect physicians' performance and patient's expectations [3]. Nevertheless, patient's preferences remain integral to modern evidence-based practice [26], and our study should provide a poignant reminder that autonomy is nowadays what the patients want most. Conclusions Our study focused on the selections of several hundred outpatients and inpatients, regarding their preferences for different attributes of their physicians. We found heterogeneous preferences, but attributes in the domain of patient autonomy and physicians' expertise (in that order), headed the list. Competing interests None declared. Authors' contributions AS initiated, designed and supervised the study, analyzed the results and wrote the manuscript. DR performed the actual study. NJ provided statistical advice and analysis. All authors read, discussed, contributed to and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: Supplementary Material Additional File 1 The patient's questionnaire including the 21 attributes in the three domains. Click here for file Acknowledgement The advice of Robert Fletcher, MD, MPH and Toni Peters, PhD of the Harvard Medical School, Boston in the design of this study is greatly appreciated. Figures and Tables Table 1 Characteristics of 445 patients participating in the study (%). Characteristic Percentage Gender Male 46 Female 54 Origin Ashkenazi 31 Sephardic 22 Israeli 47 Family status Married 75 Single 9 Divorced 6 Widower 10 Income High 18 Medium 60 Low 22 Setting Ambulatory 77 Hospitalized 23 Main diagnosis Cardiovascular 24 Gastroenterological 14 Pulmonological 12 Dermatological 11 Endocrinological 10 Rheumatological 8 Neurological 8 Oncological 5 Nephrological 4 Hematological 4 Table 2 The eight 'best' physicians' qualities preferred by more than a quarter of the patients (Top) and the least desired physicians' qualities, each preferred by less than 5% of the patients (Bottom). Physicians' attribute / Domain** Patients' preference (Number, %) Most desired Experienced [Prof.] 223 (50%) Patient [Hum.] 170 (38%) Informs the patient [Auton.] 159 (36%) Attentive [Hum.] 132 (30%) Represents patient's interests [Auton.] 127 (29%) Truthful [Auton.] 125 (28%) Up-to-date [Prof.] 124 (28%) Respects patient's preferences [Auton.] 112 (25%) Least desired Discretion [Auton.] 22 (5%) Empathy [Hum.] 19 (4%) Research [Prof.] 17 (4%) Friendly [Hum.] 12 (3%) Teaching [Prof.] 3 (1%) ** Auton. = patient's autonomy and patient's rights; Prof.= Professional expertise; Hum.= humanism and support (Detailed in the Appendix [see additional file 1]). Table 3 Patient's priorities according to domains (n = 445), number (%). Patient's autonomy Professional expertise Humane attitude Preferred domain # 139 (31) 90 (20) 76 (17) Important domain## 69 (16) 86 (19) 48 (11) Combined ### 47% 40% 28% No choices in the domain 54 (12) 64 (14) 119 (27) Significance P = 0.008 vs. Prof P < 0.0005 vs. Hum. P < 0.0005 vs. Hum. # Preferred domain = at least 2 choices (a majority) of attributes in that domain and more than in any other domain. ## Important domain = domain selected as important, but with equal selections in one other domain. (In an additional 9% of the patients, all 3 domains were equally selected). ### Combined 'preferred' and 'important' domains. ==== Refs Tumulty PA What is a clinician and what does he do? N Engl J Med 1970 283 20 4 5419315 Goldman L Plum F Bennett JC Goldman L, Bennett JC Medicine as a learned and humane profession Cecil Textbook of Medicine 2000 21 Saunders, Philadelphia 1 4 Schattner A Fletcher R Pearls and pitfalls in patient care. 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A model for empathy, reflection, profession, and trust JAMA 2001 286 1897 902 11597295 10.1001/jama.286.15.1897 Coulter A Fitzpatrick R Albrecht GL, Fitzpatrick R, Scrimshaw RC The patient's perspective regarding appropriate health care The handbook of social studies in health and medicine 2000 London, Sage 454 64 Lewin SA Skea ZC Entwistle V Zvarenstein M Dick J Interventions for providers to promote a patient centered approach in clinical consultations Cochrane Database Syst Rev 2001 CD003267 11687181 Maguire P Pitccathly C Key communication skills and how to acquire them BMJ 2002 325 697 700 12351365 10.1136/bmj.325.7366.697 Yedidia MJ Gillespie CC Kachur E Schwartz MD Ockene J Chepaitis AE Snyder CW Lazare A Lipkin M Jr Effect of communications training on medical student performance JAMA 2003 290 1157 65 12952997 10.1001/jama.290.9.1157 Haynes RB Devereaux PJ Guyatt GH Physicians' and patients' choices in evidence based practice BMJ 2002 324 1350 12052789 10.1136/bmj.324.7350.1350

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==== Front BMC BioinformaticsBMC Bioinformatics1471-2105BioMed Central London 1471-2105-5-1251535019710.1186/1471-2105-5-125Research ArticleDetermination of the differentially expressed genes in microarray experiments using local FDR Aubert J [email protected] A [email protected] J-J [email protected] S [email protected] UMR INAPG/INRA/ENGREF 518, 16, rue C. Bernard, 75231 Paris Cedex 05, France2004 6 9 2004 5 125 125 27 5 2004 6 9 2004 Copyright © 2004 Aubert et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Thousands of genes in a genomewide data set are tested against some null hypothesis, for detecting differentially expressed genes in microarray experiments. The expected proportion of false positive genes in a set of genes, called the False Discovery Rate (FDR), has been proposed to measure the statistical significance of this set. Various procedures exist for controlling the FDR. However the threshold (generally 5%) is arbitrary and a specific measure associated with each gene would be worthwhile. Results Using process intensity estimation methods, we define and give estimates of the local FDR, which may be considered as the probability for a gene to be a false positive. After a global assessment rule controlling the false positive error, the local FDR is a valuable guideline for deciding wether a gene is differentially expressed. The interest of the method is illustrated on three well known data sets. A R routine for computing local FDR estimates from p-values is available at . Conclusions The local FDR associated with each gene measures the probability that it is a false positive. It gives the opportunity to compute the FDR of any given group of clones (of the same gene) or genes pertaining to the same regulation network or the same chromosomic region. ==== Body Background Microarrays are part of a new class of biotechnologies that allow the monitoring of the expression level of thousands of genes simultaneously. Among the applications of microarrays, an important task is the identification of differentially expressed genes, i.e genes whose expressions are associated with the status of the patient (treatment/control for example). The biological question of the identification of differentially expressed genes can be restated as a one (for paired data) or two-sample (for unpaired data) hypothesis testing procedure: is the gene differentially expressed between the two situations? However, when thousands of genes in a microarray data set are evaluated simultaneously by fold changes or significance tests approach, multiple testing problems immediately arise and lead to many false positive genes. In this 'one-by-one gene' approach the probability of detecting false positives rises sharply. The False Discovery Rate (FDR), is defined as the expected fraction of false rejections among those hypotheses rejected. In their seminal paper Benjamini & Hochberg [1] provided a distribution free procedure (BH) for choosing a threshold on p-values that guarantees that the FDR is less than a target level α. The same paper demonstrated that the BH procedure is more powerful than the Bonferroni method that controls the familywise error rate. The FDR gives an idea of the expected number of false positive hypotheses that a practitioner can expect if the experiment is done an infinite number of time. As usual with expectation, it gives very little information about the number of false discovery hypotheses in a given experiment. Motivation The value of 1, 5 or 10% for the FDR, which determines the threshold t, is arbitrary. Storey and Tibshirani [2] stressed the importance of assessing to each feature its own measure of significance. They proposed to use the q-value, where Pi is the p-value of the ordered gene i, Ri is the total number of rejected genes whose p-values are less than the threshold t = Pi and is an estimate of the total number of non differentially expressed genes, m0. The q-value is appealing because it gives a measure of significance that can be attached to each gene, but it must be stressed that it is not an estimate of the probability for the gene to be a false positive. The q-value is generally lower than the latter because it is computed using all the genes that are more significant than gene i. Obviously a gene whose p-value is near to the threshold t does not have the same probability to be differentially expressed than a gene whose p-value is close to zero. Therefore the q-value gives a too optimistic view of the probability for the gene to be a false positive. Therefore it is interesting to obtain an estimate of the FDR attached to each gene, called local FDR, from an inferential point of view and without any assumption about the distribution of the p-values under H1. Results Let H0(i) = {gene i is not differentially expressed}. Let the local FDR be the probability that a given gene is not differentially expressed. More specifically, FDR(i) is the probability that a gene, whose p-value is Pi, is not differentially expressed, taking into account the whole set of tests. A raw local FDR estimate is defined in a first step. In a second step the local FDR estimate is defined as a smoothed value based on the raw values. Let P1 < … <Pm denote the ordered p-values for testing H0(i). The raw local FDR estimate for gene i is: where where λ is a tuning parameter and W(λ) = #{i, Pi > λ}, see Storey [3]. Assume that the p-values for the non-differentially expressed genes are independent. The raw local FDR estimate has the following properties: • Under H0(i) and H0(i - 1) and if E() = m0, (i, λ) is unbiased with mean 1. • Let (i, m0) = m0(Pi - Pi-1). Under H0(i) and H0(i - 1) and if m0 is known, V((i, m0)) = /[(m0 + 1)2(m0 + 2)] ≈ 1, for m0 large enough. This value is a lower bound for V((i, λ)) when m0 is unknown. • The variance of the raw local FDR under H1 is generally much smaller than under H0. • where qj is the q-value of gene j. The q-value may thus be viewed as the mean of the local FDR of the genes with p-values lower than Pj. (i, λ) is generally a very variable estimator. Moreover the local FDR should increase with the p-value. This is not the case for the raw local FDR. Therefore it is necessary to use a smoothed estimate. The smoothed local FDR(i) is where fi is a smoothing function of the (j, λ) for j = 1, m, computed at position Pi. (i, λ) gives a very valuable guideline for the choice of a threshold. One may consider the curve of the local FDR versus the index of the gene ordered by their p-values: a good candidate for the threshold should be a point with a high second order derivative, which corresponds to an abrupt change in the slope of the curve (see the examples of the following section). The second order derivative of the smoothed local FDR can be computed numerically using finite differences. As an interesting application of the local FDR, it is possible to compute the FDR associated with a class of genes or clones by summing up the local FDR estimate of each clone or gene: one may consider for example clones corresponding to the same gene, genes known involved in a given regulatory network, or gene from the same chromosomic region, and associate a FDR with the whole class. These genes do not need to have consecutive p-values. The following sections demonstrate how the local FDR can be useful using the data of well known experiments. Local FDR on Golub data set Golub [4] were interested in identifying genes that are differentially expressed in patients with two types of leukemias (ALL, AML). Gene expression levels were measured using Affymetrix high-density chips containing 6817 human genes. The learning set comprises 27 ALL cases and 11 AML cases. Data are available in the R multtest package. We used the preprocessing proposed by the authors and the p-values based on random permutations of the ALL/AML labels on Welch t-statistics for each gene, Dudoit [5], on the 3051 remaining genes. m0 is estimated with bootstrap method as suggested by Storey and Tibshirani and implemented in the library GeneTS of software R. Figure 1(a) presents the (i) for ordered genes and 1(b) presents the smooth curves obtained using lowess with a span of 0.2 and an adaptative moving average method. We can see that there is an abrupt change of the smoothed local FDR around gene number 500 which corresponds to a threshold t = 0.15 for the p-value. This may be an indication about the threshold. The Figure 1(c) presents a zoom of the Figure 1(b) for the first 600 p-values. We can see in Figure 1(c) that if we select the 438 (14%) top genes, we obtain a q-value equal to 0.0078 while the 438th gene has a local FDR equal to 0.027. It must be noticed that there is a big difference between the two measures of FDR because the numerous regulated genes with very small p-values have a great influence on the q-value, which is not the case of the local FDR (see Figure 1(c)). The p-values have been obtained using random permutations. Therefore the p-values are discrete with several genes possessing the same p-value. Therefore the values of (i, λ) may be equal to 0 because the difference between two successive p-values is 0. The discrete structure of the p-values implies a departure from the theoretical continuous uniform distribution. This explains why the moving average smoothing creates discrete jumps which appear in Figure 1(c). If the distribution of the statistics under H0 is correct, the p-values are distributed as a uniform distribution over [0, 1]. The empirical distribution of the high observed p-values (say above 0.5) is far from the uniform distribution. There are several non-exclusive possibilities to explain this: more than 50% of the genes are differentially expressed, the gene results for non-differentially expressed are correlated or there is a technical problem in the random permutations of the Welch t-statistics. Local FDR on Breast Cancer data set Storey and Tibshirani [2], have analysed in detail data from Hedenfalk [6] on 15 microarrays on breast cancer. Using the same p-values, we have computed local FDR estimates. The three genes which have been analysed in detail by Storey and Tibshirani [2] are presented in Table 1. One can see that the smooth local FDR estimate is generally greater than the q-value and gives a better idea of the probability that a gene is a false positive. For example, at the level of 5%, CTGF will be considered as differentially expressed on the basis of the q-value while it will be considered as non differentially expressed using the local FDR. Figure 2(a) presents the (i) for ordered genes and 2(b) presents the smooth curves obtained using lowess with a span of 0.2 and moving average methods. The two smoothing methods give similar results. Setting λ = 0.5, Storey and Tibshirani [2] estimate that 67% of the 3170 genes in the data are not differentially expressed. The asymptote near 1 of the smooth curve supports this estimation. Local FDR on ApoAi data The goal of the study is to identify genes with altered expression in the livers of two lines of mice with very low HDL cholesterol levels compared to inbred control mice. The mouse model is the apolipoprotein AI (ApoAI) knock-out mice. ApoAI is a gene known to play a pivotal role in HDL metabolism. The statistical analysis is described in Dudoit [7]. Height clones are expected to be differentially expressed between the control and the knock-out mices because they are clones of the ApoAI gene or of genes coregulated with ApoAI. The height clones are actually the 8 top clones detected by the statistical tests. However there are other following clones which seem statistically significant if we consider the q-value. We can see on the Figure 3(c) that the local FDR values are much higher than the q-values. Figure 3(a) presents the (i) for ordered clones and Figure 3(b) presents the smooth curves obtained using lowess with a span of 0.2 and moving average methods. The two smoothing methods give different results at the two ends of the [0, 1] interval. The moving average method which uses a special adaptative algorithm for the ends gives a better smoothing. This is particularly important for the clones with a small p-value for which it is crucial to obtain good estimates of the probability of being false positives. The lowess smoothing does not work well for the 50 first clones. In this particular case the default smoothing parameter f = 0.2 is not well suited and should be lower. However if it is chosen too low, the smoothing will not fit well the rest of the curve. There are two clones of the gene Apo-AI. If we want to estimate the FDR of these two clones taken in a whole, we compute the mean of the smoothed local FDR of the two clones (the first and the height top clones) and obtain a local FDR for the gene Apo-AI, which is equal to . This example shows that it is possible to estimate the local FDR of any group of clones. This opportunity provided by the local FDR is certainly one of its major advantage with many potential applications. Discussion The curve of the smoothed local FDR is an efficient tool to summarize the information about the number and the statistical significance of differentially expressed genes, and may also be used to give an indication about the validity of the statistical assumptions. Moreover it is a valuable tool to choose the threshold for separating the differentially expressed genes from the non-differentially expressed one: one can choose a value of t maximizing the second derivative. Alternatively one can use a cost function and choose the threshold that minimizes the mean cost for a given cost function: using cost of the experiment, cost of false positive gene validation and the profit of discovering a differentially expressed gene, it is direct to compute the optimal strategy for choosing the threshold. Note that a decision rule based on the local FDR would lead to a different set of selected genes than the usual one obtained by controlling the FDR. Consider the set of tests for which the local FDR is below 0.05, say. This set is not identical to the set identified by the standard criterion that FDR < 0.05. The local FDR is higher than the q-value. Therefore the first set is strictly included in the second one. The local FDR rule is therefore more conservative than the usual FDR one. Conclusions The p-value gives the probability that a non differentially expressed gene would be as or more extreme than the gene under concern. The q-value indicates the estimated proportion of genes as or more extreme than the gene under concern that are a false positive. The local FDR gives the estimated proportion of genes around the gene under concern which are false positive. The latter may be used as the probability that the gene under concern is a false positive, taking into account the multiplicity of the test. One of the major interest of the local FDR is that it gives the opportunity to compute the FDR of any given group of clones (of the same gene) or genes pertaining to the same regulatory network or the same chromosome. Methods Model Basically, the various procedures proposed in the literature aim to test the null hypothesis H0(i) = {gene i is not differentially expressed}. Let consider a particular experiment. We observed the differential expression of the genes and compute the associated ordered p-values Pi. In the following we will use the classical property: the p-values corresponding to non differentially expressed genes are uniformly distributed over [0, 1]. Furthermore, we will assume, as often, that these p-values are independent. However, the independence of the p-values of differentially expressed genes is not required. Consider a multiple testing situation in which m tests are being performed. Let m0 be the number of non differentially expressed genes. Let I(t) be the set of the genes having a p-value lower than t: I(t) = {i : Pi ≤ t} and R(t) = #I(t), its cardinal. Let V(t) = #[I(t) ∩ (i ∈ H0)] and S(t) = #[I(t) ∩ (i ∈ H1)]. Using a threshold t, the m genes can be classified according to the following 2 × 2 table 2: The Family Wise Error Rate (FWER) is defined to be FWER = P [V(t) ≥ 1]. A classical way to control FWER is given by the Bonferroni inequality. This quantity corresponds to the most direct extension from a test hypothesis procedure but can be very restrictive in a multiple testing procedure. The status of the gene associated with the Pi is an unobserved value. It is the same framework as point process (see for example [8]). In fact we observe R(t) = V(t) + S(t) the sum of two counting processes. The first one V(t) is a counting process associated with non differentially expressed gene. Since the p-values under H0 are uniformly distributed, V(t) has a binomial distribution with parameter m0 and t. The intensity of V(t) is constant and proportional to m0. S(t) is the counting process associated with gene under H1 and very few can be said about its distribution. One may expect the intensity of S(t) to be decreasing with t. The false discovery rate is defined as: It corresponds to the expected proportion of rejections that are incorrect. The BH procedure works as follows. Let P1 < … <Pm denote the ordered p-values. Calculate k = maxi{Pi ≤ αi/m}. The procedure rejects all null hypotheses for which Pi ≤ Pk. If the tests are independent, this procedure ensures that Let FDR(t) be the FDR when rejecting all null hypotheses with Pi ≤ t. Because the p-values of non-differentially expressed genes are uniformly distributed over [0, 1], a natural estimate of FDR(t) is Therefore the problem is to estimate m0. Storey [3], proposed to estimate m0 with where λ is a tuning parameter. In particular the case λ = 0 leads to . This is the most conservative case and corresponds to the BH procedure. Since the practical implementation of Storey method gives reasonably good results, we used it in the examples. FDR is defined as the expectation of the ratio of two counting processes V(t) and R(t): FDR(t) = E[V(t)/max(R(t), 1)]. The expectation of V(t) is m0t and R(t) is observed. Therefore, Storey [3] propose to use the following estimate: The ratio of the expectations differs from the expectation ratio but Storey [3] proved that E((t, λ)) ≥ FDR(t) using a convexity argument. Definition and Estimation of the Local FDR As stated before, V(t) and R(t) are counting (i.e. cumulative) processes. It would be very interesting to estimate the ratio of the local intensities of the two processes at point t. The intensity of process V(t) is equal to m0 and thus is known, provided that we know m0. The intensity of process R(t) is unknown, but R(t) is observed. Therefore, using point process methods it is possible to estimate its intensity at each point t. We first define the cumulative processes from t1 to t2: Let 0 ≤ t1 <t2, I(t1, t2) = {i : t1 <Pi ≤ t2}, R(t1, t2) = #I(t1, t2), V(t1, t2) = #[I(t1, t2) ∩ (i ∈ H0)] and S(t1, t2) = #[I(t1, t2) ∩ (i ∈ H1)]. FDR (t1, t2) is defined as the expected ratio of V (t1, t2) and R(t1, t2): It is a generalization of the usual FDR: if t1 = 0 and t2 = t then FDR(t1, t2) = FDR(t). So, the natural estimate of FDR(t1, t2) is: The substitution of 0 by t1 does not change the proof, so using the same convexity argument as Storey [3], we obtain the following property: E((t1, t2, λ)) ≥ FDR (t1, t2). The local FDR is the FDR(t1, t2) for small intervals [t1, t2]. If we want to estimate the local FDR around the p-value of the gene i, the question can be restated as how to estimate the ratio of the intensities of two processes around a given point Pi. The intensity of process R(t) has to be estimated at each value of t. It is possible to consider small windows of size h, or alternatively, to consider windows of different sizes corresponding to a fixed count for R(t). We have chosen the latter solution, for windows of variable size seem more appealing in the particular context. Let FDR(i) be the local FDR around Pi. To estimate FDR(i) we need to define a neighborhood around Pi. Let Vi = V(Pi-1, Pi). Remarking that R(Pi-1, Pi) = 1, we have FDR(i) = E(Vi). Furthermore E(Vi) = P(Vi = 1) since Vi is a binary variable. Thus FDR(i) provides an unbiased estimation of P(Vi = 1), the probability for gene i to be a false positive. The raw local FDR estimate for gene i is: Assume that H0(i) and H0(i - 1) are true and E() = m0. Therefore this estimate is unbiased with mean 1. Using definition (1), it is direct to obtain: which equals the q-value of gene j. The q-value may thus be viewed as the mean of the raw local FDR of the genes with p-values lower than Pj. Under the hypothesis H0, it is known that the differences between successive ordered values of independent realizations of the uniform([0,1]) distribution have a Beta distribution with parameters 1 and m0 (see Johnson [9] Chap. 26). Therefore the variance of the raw local FDR estimate for non-differentially expressed genes when m0 is known is equal to /[(m0 + 1)2 (m0 + 2)] ≈ 1, for m0 large enough. The variance of estimates (1) under H1 is generally much smaller than under H0 (see Figures 1(a), 2(a) and 3(a) for an illustration). However, one may see on these Figures that (i, λ) is a very variable estimator. This fact is well known in point process literature, [8]. Moreover, the interval ]Pi-1, Pi[ is not symmetric. If we consider the neighborhood interval around Pi defined by t1 = (Pi-1 + Pi)/2, t2 = (Pi+1 + Pi)/2 then we obtain another estimate of the local FDR: Note that (2) is a moving average of order 2 of (1). It is well known that estimates provided by moving average (or kernel estimators) are more stable, see [8]. This smoothing is generally not enough to obtain usable results and we can consider any kind of smoothing. We propose to estimate FDR(i) by where fi is a smoothing function of the (j, λ) for j = 1, m, computed at position Pi. The smoothing method must be suited to the properties of the raw FDR: • its variance is low for low p-values corresponding to highly differentially expressed genes • its variance is very high for p-values corresponding to non differentially expressed genes Therefore the window of smoothing should be short for low p-values and large for p-values corresponding high p-values. The lowess smoothing method has a fixed number of neighbor points. Therefore its window size depends of the density of points around the p-value under concern. The density of points is higher for low p-values which in turn implies a shorter window size, which is a good property. However the adaptation of the window size is not sufficient in some cases such as in the Apo-AI example. Moreover the smoothed FDR should be an increasing function of the p-values, a property which is not satisfied by the lowess smoothing. Therefore we prefer to use an ad hoc moving average smoothing using the following algorithm for computing (i, λ): let 0 <t1 <t2 <t3 be three pre-definite thresholds and m1 <m2 <m3 <m4 four pre-definite integers. • if maxj≤i (j, λ) <t1 use a moving average of order min(2i - 1, m1) • if t1 < maxj≤i (j, λ) <t2 use a moving average of order min(2i - 1, m2) • if t2 < maxj≤i (j, λ) <t3 use a moving average of order min(2i - 1, m3). • if maxj≤i (j, λ) >t3 use a moving average of order min(2i - 1, m4). We have obtained good empirical results on many data sets with t1 = 0.01, t2 = 0.05, t3 = 0.2, m1 = 3, m2 = 5, m3 = 15 and with the constraint that (i, λ) is not decreasing. This adaptative moving average method is quite empirical. This topic deserve some more work to build a well assessed smoothing method. This is one of our ongoing research project. Authors' contributions Avner Bar-Hen, Jean-Jacques Daudin and Stephane Robin equally contributed to the statistical work and the redaction task. Julie Aubert coded the R-program and analyzed the three data sets. Figures and Tables Figure 1 Plots of the local FDR estimate for Golub data x-axis: index of genes ordered along their p-values, y-axis: local FDR estimate. (a): raw values, (b): smooth estimates: moving average (discrete jumps), lowess (smooth curve), (c): zoom on the first 600 genes of (b): moving average (discrete jumps), lowess (upper smooth curve), q-value (lower thick smooth curve). Figure 2 Plots of the local FDR estimate for Hedenfalk data x-axis: index of genes ordered along their p-values, y-axis: local FDR estimate. (a): raw values, (b): smooth estimates: moving average (discrete jumps), lowess (smooth curve), (c): zoom on the first 200 genes of (b): raw values (discrete jumps), moving average and lowess (smooth curves), q-value (lower thick smooth curve). Figure 3 Plots of the local FDR estimate for Apo-AI data x-axis: index of clones ordered along their p-values, y-axis: local FDR estimate. (a): raw values, (b): smooth estimates: moving average (small discrete jumps), lowess (smooth curve), (c): zoom on the 50 first genes of (b): raw values (discrete jumps), moving average (smooth curve) lowess (upper rectangular curve), q-value (lower thick smooth curve). Table 1 p-value, q-value and local FDR estimates for three genes in Hedenfalk data. gene p-value rank q-value raw local FDR smoothed local FDR MSH2 0.00005 8 0.013 0.013 0.010 PDCD5 0.00048 47 0.022 0.013 0.033 CTGF 0.0036 159 0.049 0.176 0.098 Table 2 Classification of m genes using threshold H0 accepted H0 rejected Total H0 true U(t) V(t) m0 H0 false T(t) S(t) m1 Total W(t) R(t) m ==== Refs Benjamini Y Hochberg Y Controlling the false discovery rate: a practical and powerful approach to multiple testing JRSSB 1995 57,1 289 300 Storey JD Tibshirani R Statistical significance for genomewide studies PNAS 2003 100,16 9440 9445 12883005 10.1073/pnas.1530509100 Storey JD Taylor JE Siegmund D Strong control, conservative point estimation, and simultaneous conservative consistency of false discovery rates: A unified approach JRSSB 2004 66 187 205 Golub TR Slonim DK Tamayo P Huard C Gaasenbeek M Mesirov M Coller JP Loh M Downing JR Caligiuri MA Bloomfield CD Lander ES Molecular classification of cancer:class discovery and class prediction by gene expression monitoring Science 1999 286 531 537 10521349 10.1126/science.286.5439.531 Dudoit S Shaffer CBJ Multiple hypothesis testing in microarray experiments Statistical Science 2003 18,1 71 103 10.1214/ss/1056397487 Hedenfalk I Duggan D Chen Y Radmacher M Bittner M Simon R Meltzer P Gusterson B Esteller M Kallioniemi OP N Engl J Med 2001 344 539 548 11207349 10.1056/NEJM200102223440801 Dudoit S Yang YH Callow MJ Speed TP Statistical methods for identifying differentially expressed genes in replicated cdna microarray experiments Statistica Sinica 2002 12 1 Cressie N Statistics for Spatial Data 1993 New York: Wiley Johnson NL Kotz S Balakrishnan N Continuous Univariate Distributions 1995 New York: Wiley

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==== Front BMC Public HealthBMC Public Health1471-2458BioMed Central London 1471-2458-4-341529871310.1186/1471-2458-4-34Research ArticleLogistics of community smallpox control through contact tracing and ring vaccination: a stochastic network model Porco Travis C [email protected] Karen A [email protected] Susan E [email protected] Diane L [email protected] Randy [email protected]ón Tomás J [email protected] San Francisco Department of Public Health, Community Health and Epidemiology Section, Epidemiology and Effectiveness Research Unit, 101 Grove Street Suite 204, San Francisco, California 94102 USA2 Center for Infectious Disease Preparedness, School of Public Health, University of California, Berkeley, USA3 Surveillance and Epidemiology Section, Tuberculosis Control Branch, Division of Communicable Disease Control, California Department of Health Services, Berkeley, California, USA2004 6 8 2004 4 34 34 3 1 2004 6 8 2004 Copyright © 2004 Porco et al; licensee BioMed Central Ltd.2004Porco et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Previous smallpox ring vaccination models based on contact tracing over a network suggest that ring vaccination would be effective, but have not explicitly included response logistics and limited numbers of vaccinators. Methods We developed a continuous-time stochastic simulation of smallpox transmission, including network structure, post-exposure vaccination, vaccination of contacts of contacts, limited response capacity, heterogeneity in symptoms and infectiousness, vaccination prior to the discontinuation of routine vaccination, more rapid diagnosis due to public awareness, surveillance of asymptomatic contacts, and isolation of cases. Results We found that even in cases of very rapidly spreading smallpox, ring vaccination (when coupled with surveillance) is sufficient in most cases to eliminate smallpox quickly, assuming that 95% of household contacts are traced, 80% of workplace or social contacts are traced, and no casual contacts are traced, and that in most cases the ability to trace 1–5 individuals per day per index case is sufficient. If smallpox is assumed to be transmitted very quickly to contacts, it may at times escape containment by ring vaccination, but could be controlled in these circumstances by mass vaccination. Conclusions Small introductions of smallpox are likely to be easily contained by ring vaccination, provided contact tracing is feasible. Uncertainties in the nature of bioterrorist smallpox (infectiousness, vaccine efficacy) support continued planning for ring vaccination as well as mass vaccination. If initiated, ring vaccination should be conducted without delays in vaccination, should include contacts of contacts (whenever there is sufficient capacity) and should be accompanied by increased public awareness and surveillance. ==== Body Background Concerns about intentional releases of smallpox have prompted extensive preparations to improve our ability to detect and respond to an outbreak of smallpox [1,3,4,2]. Many factors contribute to the public health challenge of understanding and preparing for smallpox, including the age and quality of epidemiological data on native smallpox and the smallpox vaccine, the difficulty of extrapolating that data to our current populations, the possible terrorist use of altered smallpox, our ignorance of terrorist methods of release, and the relatively high risk of adverse events caused by the smallpox vaccine. The Centers for Disease Control and Prevention (CDC) established ring vaccination (selective epidemiological control [5]), a strategy in which contacts of cases are identified and vaccinated, as the preferred control measure in the event of a smallpox outbreak (interim plan). The successful use of ring vaccination during the smallpox eradication campaign and its logical emphasis of case-contacts for immediate vaccination support its use (though the attribution of the success of the eradication program to ring vaccination has been challenged [6]). Health Officers should initiate ring vaccination upon identification of the first cases of smallpox. However, there are legitimate concerns regarding the ability of public health practitioners to mount a quick, comprehensive and successful ring vaccination program, particularly in the face of a moderate-sized or large smallpox outbreak. To guide preparation efforts and inform incident decision-making, we attempt to identify outbreak characteristics and response capacities that significantly impact the ability of ring vaccination to control a smallpox outbreak and to determine whether ring vaccination is useful in the presence of a mass vaccination campaign. Our analysis uses a newly developed mathematical model: a continuous-time, event-driven network simulation model of smallpox ring vaccination. Mathematical models can advance our understanding of how a smallpox outbreak might progress. Several mathematical and computer models address the question of smallpox transmission [7-13]. The first model to appear [8] concluded that ring vaccination would be effective, but did not treat response logistics in detail; the model was linear and did not treat the depletion of susceptibles as the epidemic progressed (appropriate, however, for assessing control early in an epidemic, when the number infected is small compared to the number of susceptibles, e.g. [14]). The innovative model by Kaplan et al. [9] emphasized the importance of resource limitation and the logistics of smallpox response, but assumed that full infectiousness began before the onset of symptoms (and the subsequent identification and removal), and did not separately monitor close epidemiological contacts of patients (which are at greatest risk, but also easiest to find and vaccinate); the conclusions were highly critical of ring vaccination. The model by Halloran et al. [11], a stochastic, discrete-time network model omitted the explicit inclusion of response logistics while otherwise used parameter values similar to those in Kaplan et al. [9]; the inclusion of residual immunity from individuals vaccinated prior to the discontinuation of routine vaccination, however, led to a more favorable view of ring vaccination. The model by Bozzette et al. [12] assumed that ring vaccination would reduce the number of transmissions and focused on health care workers (but did not explicitly include the network structure of the population nor the response logistics of ring vaccination). The model by Eichner [15] did not explicitly include the network structure of the population nor the logistics of ring vaccination, but did use parameters based on data from an outbreak in Nigeria, and did distinguish close and casual contacts, case isolation, and surveillance of contacts; it concluded that case isolation and contact tracing could prevent the spread of smallpox. Finally, the individual-based model by Epstein et al. [16] presented scenarios illustrating certain alternatives to pure mass vaccination and ring vaccination of contacts of cases in preventing smallpox transmission in small populations of 800 individuals; this model includes no homogeneity assumptions, but did not analyze tracing of contacts of contacts. Because none of the available models includes both network structure (with explicit contact tracing) and response logistics limited by the number of available disease control investigators [9], we included these features in a continuous-time event-driven network simulation model of smallpox ring vaccination. Specifically, the model we developed includes the following features: Network structure Smallpox was primarily a disease of close contact, especially household contacts [5]. Such contacts are both the most important epidemiologically, and also the easiest to identify. Post-exposure vaccination Some evidence suggests that vaccination soon after exposure may lessen the severity of the resulting case of smallpox or possibly prevent disease entirely [17-20]. Second ring Ring vaccination may involve not only vaccinating contacts of cases, but also contacts of contacts of cases [21,22] – potentially allowing the public health authorities to "outrun" the chain of transmission. Response capacity Limited case-finding and vaccination capabilities lead to the possibility that it may be impossible to find newly exposed individuals and vaccinate them in time, resulting in a "race to trace" [9]. Heterogeneity in natural history Mild, ambulatory cases of smallpox may spread disease because such cases may be harder to recognize. Prior vaccination Vaccination of individuals prior to the discontinuation of routine vaccination may provide some, possibly considerable, protection against infection [11,23,24], although it may also result in more mild cases which may be harder to detect. Public awareness Public awareness may lead to more rapid detection of cases. We use this model to determine what factors promote or hinder the success of ring vaccination during a smallpox outbreak, and whether ring vaccination is useful in the presence of a mass vaccination campaign. In particular, the goal of this paper is to examine the control of smallpox by contact tracing and ring vaccination using a network model which includes response logistics [9]. Methods Model structure Natural history of smallpox We briefly review relevant features of the natural history and epidemiology of smallpox [17,25,8,28]. Following infection by the variola virus, individuals exhibited an incubation period of approximately 7–19 days with 10–14 being most typical. Sudden onset of fever and malaise, often with accompanying headache and backache, began the initial (or pre-eruptive) phase of smallpox. After 2–3 (or perhaps 4) days, individuals with the most common form, ordinary type smallpox, developed the characteristic focal rash, preceded in many cases by oropharyngeal lesions. In fatal cases of ordinary smallpox, death often occurred between the tenth and sixteenth day of symptoms; among survivors, most scabs had separated by day 22–27 of illness [26]. The course of smallpox varied widely between individuals, and several different clinical classifications were developed [29-31,17,26]. Consideration of the clinical features and severity of smallpox is important from the standpoint of mathematical transmission modeling because (1) the clinical features affect the ease of diagnosis (and thus of case identification), (2) more severe forms of smallpox may result in more transmission, (3) vaccinated individuals may develop less severe disease. We utilize a modified or simplified version of the classification system developed by Rao [32,31,26]; for the mathematical model, we will classify smallpox into five categories: early hemorrhagic, flat and late hemorrhagic, ordinary, modified, and mild. However, the clinical features and severity of smallpox in different populations may have been affected by underlying host factors, differences in viral strains, or differences in the infectious dose owing to different prevailing modes of transmission, and thus robust and precise quantitative estimates of the effects of (pre- or post-exposure) vaccination on the resulting smallpox severity, or of the infectivity differences between individuals exhibiting different forms of smallpox, are not available. The significance of such differences will be revealed through sensitivity analysis. Further details are given in Appendix 1 [see Additional file: 1]. Vaccination with vaccinia virus provided substantial protection against infection. Dixon assessed the risk of infection for an individual successfully vaccinated 3 years prior to exposure to be 0.1% the infection risk of an unvaccinated individual [17]. However, smallpox vaccination did not always take when applied, and moreover, in many instances, individuals who experienced a repeated vaccination failure developed severe smallpox upon exposure. The probability of a successful take depended on the vaccination method used; we assume that the take rate is between 95% and 100% [22,28]. In addition to protection against infection, vaccination could in many cases modify the course of infection and reduce the severity. Vaccine protection waned over time, but individuals vaccinated 20 years prior to exposure were believed to still have half the infection probability that an unvaccinated person had [17], and to have some protection against the most severe manifestations of smallpox. Dixon [17] believed that vaccine protection had at least three components, which decayed at different rates; for the purpose of this paper, we will assume that the severity of smallpox in previously any (recently or otherwise) vaccinated individuals follows the same distribution as for the vaccinated subjects seen in the case series observed by Rao in Madras [26], except that anywhere from 0 to 5% of vaccinated subjects develop smallpox too mild to diagnose without special surveillance or awareness. Observe that the vaccinated cases studied by Rao were vaccinated (at some point in their lives) before exposure, rather than after exposure to smallpox. Smallpox was largely a disease of close contacts [17,26,33], spread primarily through face to face contact with an infected person (or occasionally through contaminated clothing). Individuals in the incubation period of smallpox were not infectious, and long term carriers did not exist. Patients were believed to be infectious following the development of oropharyngeal lesions, which could precede the rash by 24 hours [26]. However, patients were believed to be most infectious during the first week of the rash [26]; Dixon (1962) believed that patients could be infectious from the onset of acute viremia, but most evidence suggested that little transmission occurred prior to the development of the rash [26,33]. The more severe the case, the more infectious they appeared to be [34]; mild cases were believed to have very little infectiousness. While scabs contained infectious material and patients were considered to be infectious until the last scab fell off, in practice patients were not highly infectious during the scabbing phase. Importantly, patients who had been vaccinated were found to cause fewer secondary cases [34]. Very severe cases, such as hemorrhagic or flat smallpox, occasionally resulted in considerable transmission, owing to diagnostic difficulties; mild cases, in which the patient remained ambulant during the course of the disease, could cause considerable spread as well [35,36]. Within a household or family dwelling, the secondary attack rate of unvaccinated susceptibles depended on the time and place, occasionally below 50% [29], but often approaching 100% [37]. Drier conditions were often believed to favor transmission [17,27], so that lower rates of transmission derived from tropical regions may not be applicable to the temperate zone [38]. The number of secondary cases resulting from a given importation into Europe varied widely [39], with most importations yielding few cases, but with the occasional large outbreak being seen. Mathematically, we represent the course of smallpox according to Figure 1. We distinguish eight epidemiologically relevant states: (1) just following exposure, during which time vaccination could afford complete protection against disease, (2) a period of several days during which vaccination will not prevent disease, but may still reduce the severity of disease, (3) still prior to the development of symptoms, but too late for vaccination, (4) the beginning of the pre-eruptive period, during which the patient exhibits fever, malaise, and possibly other symptoms, but is not yet infectious, (5) a short period prior to the appearance of the rash, during which the appearance of oropharyngeal lesions will permit variola transmission, (6) the first week of the rash, during which time the patient is most infectious, (7) and (8), succeeding stages of the rash, during which time the patient is less infectious. For each of these states, we assume that conditional on surviving, the waiting time until the next stage is chosen from a uniform distribution as indicated in Appendix 2 [see Additional file: 2], except that the incubation period (the time from infection until Stage 4) is derived from estimates of the incubation distribution of smallpox based on importation cases in Europe [26] (see Appendix 2 [see additional file 2] for details). We chose to sample from a uniform distribution as a simple way to ensure a minimum waiting time in each state; many alternatives to this choice are possible. Figure 1 Smallpox stages used in the simulation model. Flat and ordinary smallpox rashes are indicated with more dots than modified and "mild" smallpox, suggesting potentially greater infectiousness. Hemorrhagic smallpox is indicated by horizontal line shading. Further details are provided in Table 6. Network structure We simulate the transmission of smallpox on a "small-worlds" network (highly clustered, but with short characteristic path lengths) [40]. Specifically, we assume that each person is located in a single household, and that the transmission rates were greatest in the household. We also assume that a fraction of the population are grouped into workplace or social groups, in which transmission may also occur, but with a lower rate per unit time than for household contacts. Finally, we assume that with a still smaller probability, any individual may transmit infection to any other individual in the population (casual contacts). In general, in a network-structured model, the number of secondary cases caused by an index case in a completely susceptible population is not a useful index of epidemic potential [41,42] (for a simple example, see [43]), since (for instance) an individual could infect everyone in his or her household, and not cause a widespread epidemic unless between-household transmission were sufficiently frequent. Rather than constructing the appropriate generalized basic reproduction number for our model (leading to highly cumbersome expressions), we chose an alternative (ad hoc) index of epidemic potential. For any given scenario of interest, we simulated the introduction of 10 index cases at random into a population of size 10000, and operationally defined "containment" to occur whenever the final size of the epidemic was less than 500 cases within 250 days (we showed, in the discussion of Figure 5A below, that in nearly all cases, the 250-day window differs very little from a 1000-day window). Because we simulate a disease with a finite duration on a finite and non-renewing population, epidemic extinction always occurs in finite time. Figure 5 5A - The mean containment probability increases as the number of ring vaccinations per day is increased. For this figure, the 1000 "calibrated" parameter sets were chosen, and for each parameter set, 100 realizations were simulated and the fraction of these for which the epidemic was contained to fewer than 500 cases was determined. The average of these 1000 containment fractions is plotted on the vertical axis. We assumed a household contact finding probability of 95% and that the diagnosis rates double after community awareness of the epidemic. We considered high levels of workplace/social (w/s) contact finding (0.9), as well as moderate levels (0.8). We also considered two levels of diagnosis of smallpox among investigated (alerted) contacts: high levels (corresponding to a 3 hour mean delay, indicated by "high contact isolation"), and moderate levels (corresponding to a one day delay, and indicated by "less contact isolation"). The figure shows four such conditions, a. high workplace/social contact finding probability and high contact isolation, b. moderate workplace/social contact finding probability and high contact isolation, c. high workplace/social contact finding probability and less contact isolation, and d. moderate workplace/social contact finding probability and less contact isolation. All other parameter values were chosen from the uncertainty analysis (the 1000 "calibrated" parameter sets). In this figure, "contact isolation" refers to the monitored diagnosis rate, i.e. the rate at which previously asymptomatic contacts who subsequently develop disease will be diagnosed (φ, Table 1, Table 8). 5B - The minimum containment probability out of the same 1000 scenarios chosen in Figure 5A. Whereas in Figure 5A, we averaged the simulated containment frequency (out of 100 realizations for each scenario), in this figure we determined which of the 1000 scenarios led to the lowest containment frequency, and we plotted this single worst (out of 1000) containment frequency, at different levels of ring vaccination capacity, for the same four conditions as in Figure 5A: a. high workplace/social contact finding probability (0.9) and high contact isolation (effective 3 hour delay following symptoms), b. moderate workplace/social contact finding probability (0.8) and high contact isolation, c. high workplace/social contact finding probability (0.9) and less contact isolation (effective one day delay), and d. moderate workplace/social contact finding probability (0.8) and less contact isolation. All parameters are the same as in Figure 5A (the household contact finding probability is 0.95 for all scenarios, and the diagnosis rates are doubled after the onset of community awareness). In this figure, "contact isolation" refers to the monitored diagnosis rate, i.e. the rate at which previously asymptomatic contacts who subsequently develop disease will be diagnosed (φ, Table 1, Table 8). Medical and public health intervention We assume that even in the absence of specific case investigations, the presence of smallpox symptoms will prompt patients to be diagnosed; we assume, however, a higher diagnosis rate for all forms of ordinary smallpox than for the severe flat and hemorrhagic forms, or for the mildest form. We assume that once an individual is diagnosed, their household and workplace contacts are investigated and detected with some probability; we assume that a high fraction (such as 95%) of household contacts are assumed to be traceable (see below). We assume that the fraction of workplace/social contacts that are traceable is less than the fraction of household contacts that are traceable; we assume that no casual contacts are traceable. High contact-finding rates may be plausible; we examined San Francisco Department of Public Health records of contact investigations for meningococcal disease (like smallpox, a potentially fatal disease for which rapid intervention may prevent mortality and morbidity). Records were available from December 2001 to April 2002; 13 such investigations during this period resulted in identification of 62 household contacts, all of which were contacted; out of 38 workplace/social contacts identified, 32 were contacted (84%). In our model, we assume that identified asymptomatic contacts are vaccinated, quarantined, and monitored for symptom development, while symptomatic patients are isolated and treated as necessary [9]; thus, the modeled interventions include more than ring vaccination alone. Finally, we include the possibility that all contacts (of both symptomatic and asymptomatic) traced and the same procedure applied, so that all contacts of contacts would be investigated. We assume that uninfected or asymptomatic individuals who are visited or traced individuals will be diagnosed more rapidly than if they had not been traced; in fact, such individuals would be isolated and would not be able to continue a chain of transmission. We follow previous models [9] in assuming a limited vaccination capability of Kr per day for ring vaccination. We assumed one of two strategies for contact tracing: (1) tracing only of direct contacts of diagnosed cases, and (2) tracing of contacts of contacts of diagnosed cases as well as direct contacts. The contact structure of the network is illustrated in Figure 2. Observe that individuals b and c are household contacts of individual a, so that if individual a were a smallpox case, an attempt would be made to find and vaccinate individuals b and c as household contacts of a case. If individuals a and b were both cases, then two attempts could be made to find individual c. We have modeled the effect of multiple contact-finding attempts conservatively in the sense that if the first attempt to find an individual as a household contact (of a case or of a contact) is determined to fail, no further attempts will be made. This maintains the failure rate of contact tracing (looked at from the standpoint of finding individuals) even in large households. Similar considerations apply to workplace/social groups. Figure 2 Network structure shown for households (joined by thick lines) of size 3 and workplace/social groups of size 4 (joined by thin lines); a small portion of the network is shown. Individual a has two household contacts (b and c), and three workplace/social contacts (d, e, and f). If individual a were a smallpox case, the household contacts would be at highest risk for acquiring smallpox, followed by workplace/social contacts; all individuals in the population are at a low risk of casual transmission from individual a. Case investigation of individual a would identify the direct contacts b-f with probabilities that depend on whether the contact is household or workplace/social; if such individuals are identified, they will be vaccinated. If contacts of contacts are being traced, the investigation will subsequently identify individuals g-p. Analysis We analyzed the model in three ways. First, we selected a Latin Hypercube sample [44-46] of parameters chosen uniformly from the parameter ranges given in Appendix 2 [see additional file 2], and simulated the transmission and control of smallpox to determine which parameters were most important for contact tracing and ring vaccination to be effective. Second, we used the same Latin Hypercube Sample of input parameters, but assumed that all disease control efforts were inactive. We used these parameters to simulate smallpox transmission, but then iteratively selected transmission parameters so that (1) between 1% and 10% of new infections resulted from casual (random) transmission, and (2) each index case resulted in between two and five secondary cases (thought to be plausible for historic smallpox; [8] suggest three secondary cases). For each of the resulting smallpox parameter sets using 100 stochastic simulations per set, we determined the daily ring vaccination/case tracking capacity needed to contain all simulated smallpox epidemics (i.e., keep the total number of cases below 500 within 250 days). Third and finally, we chose parameter values to yield an moderately large smallpox epidemic (with each index case causing approximately six secondary cases), and present illustrative scenarios for ring vaccination. These scenarios are intended to complement the simulations which were calibrated to historic smallpox, since the characteristics of smallpox that may be used in a deliberate release are not known. It is important to realize that in our model, the case finding time determines the fraction of contacts that will become infected, and that our model parameters have been chosen to yield quite rapid transmission to close contacts; in reality, much transmission of natural smallpox occurred through "sickbed" routes which would not occur in a modern setting [47], so that in this sense our model errs considerably on the side of caution and pessimism. Results Most important parameters (sensitivity analysis) To determine which of the input parameters were most important in determining the total number of smallpox cases, we selected a Latin Hypercube sample of size 1000 from the input parameter ranges indicated in Appendix 2 [see additional file 2] and simulated the mean number of cases within 250 days in a population of 10000. We then computed the partial rank correlation coefficient [46] (PRCC; see Appendix 2 [see additional file 2]) between each input parameter and the number of smallpox cases; when the PRCC is close to zero, the value of the parameter has little relation to the simulation output; when the PRCC is close to +1.0 or -1.0, the value of the parameter is highly important in determining the simulation output. Neglecting the number of index cases (which is directly related to the number of new cases), those parameters whose PRCC exceeds 0.1 are shown in Table 2. Most of these parameters identified as important are related to the density of available contacts (mean household size, prior vaccination fraction, and protection due to prior vaccination) or the transmission rate and infectivity (including the length of the pre-eruptive infectious period (stage 5 in Figure 2)). Note, however, that the speed of ring vaccination (household tracing delay) and faster diagnosis due to awareness of the outbreak are important parameters. Additionally, the infectivity of mild cases appears as an important parameter as well. Table 2 Most important parameters. PRCC: partial rank correlation coefficient (see Appendix 2 [see additional file 2] for definition and references). Parameter PRCC Mean Household Size 0.575 Transmission Rate from Close Contacts 0.520 Infectivity prior to rash 0.309 Ring Vaccination Capacity -0.296 Casual Transmission Probability 0.244 Pre-eruptive infectious period (lower bound) 0.224 Number of Casual Contacts per Day 0.210 Relative Infectiousness of Social/Workplace Contacts 0.200 Fraction of Individuals in Social/Workplace Groups 0.183 Faster Diagnosis due to Awareness of Outbreak -0.175 Household Tracing Delay 0.104 Pre-eruptive Diagnosis Probability -0.103 Diagnosis Probability after Rash -0.103 Illustrative scenarios To explore factors which contribute to the success of ring vaccination, we chose smallpox scenarios which resulted in severe and fast-moving epidemics in the absence of disease control; these simulated epidemics are considerably more severe than is believed likely under present circumstances. Effect of contact tracing and ring vaccination We used these parameters to simulate smallpox epidemics beginning with 10 cases, for a variety of levels of ring vaccination capacity per day (contact tracing capacity per day), as shown in Figure 3A. In this Figure, we assume that the population size is 10000, and that the epidemic began with 10 infected individuals. The mean household size is assumed to be 4, the mean size of the workplace/social contact group is 8, and contacts of contacts are traced. We assume that each day, the number of contacts that can be traced and vaccinated as a result of case investigation is 0, 10, 20, 30 and 40 per day; the probability of finding a workplace/social contact is assumed to be 80%. The Figure shows the average number of infected individuals each day (based on 100 stochastic simulations) for each of these scenarios. Selected parameter values are indicated in the caption for Figure 3A and in Table 1. Figure 3 3A - Expanding severe smallpox epidemic beginning with 10 initial cases, assuming 0, 10, 20, 30, and 40 possible ring vaccinations per day. The household size is 4 and the workplace/social group size is 8; we assume 95% of household contacts are traceable (with a mean delay of 1 day) and 80% of workplace/social contacts are traceable (with a mean delay of 2 days). We also assume that 25% of the population have 50% protection from infection resulting from vaccination prior to the discontinuation of routine vaccination. We assume that infection will be transmitted to close contacts with a mean time of 0.2 days, and that each person while infective causes on average 0.15 casual (untraceable) infections per day. We assume that individuals are 20% as infectious in the day just before the appearance of the rash as they will be during the first week of the rash, and that individuals are 20% as infectious as this (4% as infectious as during the first week of the rash) during the prodromal period. We assume that diagnosis rates will increase by a factor of 50% after smallpox becomes known to the community; we assume that each individual contacted during an investigation has a additional diagnosis or removal rate of 0.75 per day following the onset of symptoms (reflecting enhanced surveillance or contact isolation). Important parameters are summarized in Table 1; the full set of parameter choices is outlined in Tables 8-11 in Appendix 2 [see additional file 2]. Diagnosis times are discussed in Appendix 2 [see additional file 2]. 3B - An expanding severe smallpox epidemic under inadequate ring vaccination is shown for parameters identical to Figure 3A, except that workplace/social group sizes are 12 (instead of 8), and the probability of tracing workplace/social contacts is 0.6 (instead of 0.8). 3C - A severe smallpox epidemic is controlled by ring vaccination despite the large number of initial cases. The parameters are identical to Figure 3A, except that 1000 index cases inaugurate the attack in these scenarios (and ring vaccination capacity is much greater, as indicated). While not recommended, ring vaccination may ultimately halt epidemics beginning with many index cases if sufficient vaccination capacity were available, contact finding feasible, and follow-up sufficient. 3D - Tracing contacts of contacts (red) is beneficial when sufficient contact tracing/ring vaccination capacity exists (dotted lines). In these scenarios, all parameters are the same as in Figure 3A; the number of contact tracings possible per day is either 20 or 40 per day. Contacts of contacts are traced in two scenarios; in the other two, only direct contacts of cases are traced. For low levels of ring vaccination (20 per day), tracing contacts of contacts is harmful; for high levels (40 per day) of ring vaccination, it is beneficial to trace contacts of contacts. When the contact tracing/ring vaccination capacity is too small to adequately cover contacts of the cases themselves, diversion of resources to contacts of contacts is harmful; however, provided that sufficient capacity exists, tracing contacts of contacts helps outrun the chain of transmission. Each line corresponds to the average of 100 realizations. Table 1 Selected parameter values for Figure 3A and other illustrative scenarios. The notations "Other" or "Other factors" in the column "See also" refers to the text section "Other factors". The symbols are defined in Appendix 2 [see additional file 2] and are included for reference. Description Values See also Symbol Number of index cases 10–1000 Figure 3C A Mean household size 4 H Workplace/social group size 8 Figure 3B W Ring vaccinations per day 0–200 Fig. 3A, 3B, Other Kr Monitored diagnosis rate 1–8 day-1 Figure 5A, 5B φ Prob. of finding household contact 0.95 Table 4 υ1 Prob. of finding workplace/social contact 0.8 Fig. 3B, 4AB; Tb. 4 υ2 Delay, tracing household contacts 1–5 days Figure 6 δ1 Delay, tracing workplace/social contacts 2–10 days Figure 6 δ2 Relative diagnosis rate after 1st diagnosed case 1.5 Figure 7 a1 Infectivity, stage 4 relative to stage 5 0.2 Figure 8 k Infectivity, stage 5 relative to stage 6 0.2 Figure 8 k' Infection hazard for close contacts 5 day-1 Table 3 λ Relative hazard for workplace/social contacts 1/3 Table 3 h2 Casual transmission rate 0.15 day-1 β Prior vaccination fraction 0.25 Other factors f Fraction of mild cases 0.03 Other factors Vaccine success rate (for very recent vaccination) 0.667 Other factors α1 Vaccine success rate (vaccination prior to discontinuation of routine vaccination) 0.5 Other factors α2 Vaccine success rate full protection 0.999 Other factors α3 Because we assumed nonzero diagnosis probabilities during the prodromal period for all individuals in Figure 3A, we repeated the simulation assuming no diagnosis in the prodromal period unless individuals were under specific surveillance. The results were nearly identical: assuming 30 contact tracings (ring vaccinations) per day, we found 26% of the scenarios in Figure 3A exhibited decontainment, and 28% assuming no diagnosis during the prodromal period; assuming 40 contact tracings per day, we found 1 out of 100 scenarios showed loss of containment in Figure 3A and when we repeated the scenario of Figure 3A assuming no diagnosis during the prodromal period. In Figure 3B, we illustrate control of an epidemic for which all parameters are identical to Figure 3A, except that the workplace/social group size is 12 (instead of 8, as in Figure 3A), and the probability of finding workplace/social contacts is 60% (instead of 80%, as in Figure 3A). In this case, the larger size of the workplace/social groups and the lower contact finding probability makes it necessary to have a higher ring vaccination capacity to attain a high probability of containing the epidemic, and on average it takes longer for eradication to finally occur. Finally, in Figure 3C, we show control of an epidemic in a population of 100,000, beginning with 1000 initial infectives, keeping all other parameters the same as in Figure 3A. Each curve corresponds to the indicated number of possible ring vaccinations per day. This figure shows that assuming sufficient capacity, ring vaccination is in principle capable of containing even epidemics beginning with very many infected individuals. However, mass vaccination in such cases is justified because of the far larger number of individuals at risk and the inability to perform such extensive contact tracing. In Figure 3D, we compare the effect of tracing contacts of contacts (as described in Appendix 2 [see additional file 2]) at different levels of ring vaccination capacity. Thin lines in red correspond to the presence of tracing contacts of contacts; thick lines in black correspond to tracing direct contacts of cases only. Each simulation was performed 100 times, with 10 initial infectives, and for 20 and 40 ring vaccinations possible per day (as indicated). The average number infected on each day is plotted in the Figure. The figure illustrates that when ring vaccination capacity is low, tracing contacts of contacts (as modeled) yields a more severe average epidemic; when ring vaccination capacity is large, tracing contacts of contacts results in a less severe average epidemic; if the contact tracing/ring vaccination capacity is too low to cover adequately the contacts of contacts in addition to the contacts of cases, extension of tracing to the contacts of contacts (the second ring) is harmful; however, if there is sufficient capacity to cover the contacts of contacts, then the tracing of contacts of contacts is beneficial. Finally, in Figure 4, we illustrate the considerable variability that may be seen from simulation to simulation. This figure shows twenty simulations when contacts of contacts are not traced. Stochastic variability between realizations is considerable, even when all parameters are held constant; this variability is expected to limit the ability to make inferences based on observation of a single realization of the process. Figure 4 Stochastic variability is illustrated by plotting the number of infectives over time over multiple replications. In this example, most simulations exhibit rapid containment of smallpox. The mean number of cases (averaging over simulations) is influenced by a small number of simulations exhibiting an uncontained epidemic. The parameters are the same as in Figure 3A, except that contacts of contacts are not traced in these replications. Because our baseline hazard for infection of individuals may be larger than would be expected for naturally occurring smallpox, we examined the effect of more realistic values of this hazard. In particular, we chose different levels of ring vaccination capacity (10, and 20), and of the relative hazard for workplace/social contacts, and then chose values of the baseline hazard for infection varying from 0.5 per day (for a mean time to infection of 2 days) to 2 per day (for a mean time to infection of one half day), and introduced 10 index cases into a population of 10000. We then repeated this 100 times, and reported the fraction of scenarios in which the number of infections ultimately exceeded 500 (as before, chosen as a cutoff to indicate the ultimate "escape" of containment of the epidemic). These results, shown in Table 3, support the idea that ring vaccination can easily control introduced smallpox provided there is sufficient capacity and efficacy of tracing. Table 3 Estimated decontainment probability for different levels of ring vaccination capacity (Kr) and relative hazard for infection due to workplace/social contacts (h2), for different levels of the baseline hazard for infection from household contacts λ (based on replications of 100 simulations for each level). For each scenario, 10 index cases were introduced into a population of size 10000. All other parameters were the same as for Figure 3A. As before, we define decontainment to mean that the total number of cases from 10 index cases eventually exceeded 500 by day 250. Relative hazard for workplace or social contacts Ring vaccinations per day 10 20 λ λ 1/3 0.5 0 0.5 0 0.75 0.02 0.75 0 1 0.26 1 0 1.25 0.73 1.25 0 1.5 0.96 1.5 0.02 2 1 2 0.16 λ λ 2/3 0.5 0 0.5 0 0.75 0.46 0.75 0 1 0.82 1 0.02 1.25 1 1.25 0.11 1.5 1 1.5 0.26 2 1 2 0.49 λ λ 1 0.5 0.14 0.5 0 0.75 0.86 0.75 0 1 0.99 1 0.07 1.25 1 1.25 0.22 1.5 1 1.5 0.49 2 1 2 0.85 Because of considerable uncertainty in the model parameters, we chose a collection of parameter values, and for each, estimated the containment probability (operationally defined as fewer than 500 total cases as a result of 10 index cases, within 250 days). We estimated this containment probability by simulating the smallpox epidemic 100 times for the same parameter values, and computing the frequency out of these 100 realizations for which fewer than 500 index cases resulted within 250 days. (Using a 1000 day window produces slightly smaller containment estimates; for 3 out of 1000 parameter set choices, this difference was greater than 0.06; the maximum difference seen was 0.23; the mean absolute difference was 0.0029; in only one case out of 1000 did we see containment in all 100 cases for the 250-day window, but not in all 100 cases for the 1000-day window). One thousand scenarios chosen from a Latin Hypercube sample were analyzed, and as indicated before, we chose the hazard for close contact transmission and the hazard for random transmission to guarantee that between 2 and 5 secondary cases per case occur, and that no more than 5% of cases are attributable to random transmission (we refer to this set as the "calibrated" scenarios further in this text). Having chosen this collection of 1000 parameter sets, we considered two levels of two different control parameters which were applied to each (so that each of the 1000 parameter sets were simulated under four different control conditions). The first of the two control parameters was the probability of workplace/social group contact finding; we chose values of 0.8 and 0.9 for this parameter (the household contact finding probability was 0.95 in all cases). The second of the control parameters was the rate of diagnosis (and effective removal) from the community of cases developing among previously identified and traced contacts who were initially asymptomatic (we refer to this as the monitored diagnosis rate); we assumed first a low level corresponding to a mean diagnosis time of one day from the onset of symptoms, and a high level corresponding to a mean time of 3 hours from the onset of symptoms (high levels of the monitored diagnosis rate correspond effectively to isolation of contacts). Finally, we assumed a doubling of the diagnosis rate after the beginning of widespread community awareness of smallpox. We then computed the containment fraction at different levels of ring vaccination capacity (contact tracing capacity per day). Thus, for each of 1000 scenarios (parameter set choices), we assigned the workplace/social group contact tracing success probability (υ2), the monitored diagnosis rate φ (Appendix 2 [see additional file 2]), and the contact tracing/ring vaccination capacity per day (Kr). We then performed 100 realizations beginning with 10 index cases, and computed the containment fraction (fraction showing fewer than 500 cases in 250 days, beginning with 10 index cases). Thus, for each of the two choices each of υ2 and φ, and for each value of Kr we examined, we obtained 1000 values of the containment fraction. We use the resulting distributions in Figure 5A (averaging over these 1000 containment fractions), and Figure 5B (displaying the minimum value of the 1000 containment fractions). In Figure 5A, we plot the mean containment fraction (averaging the containment fraction over all 1000 scenarios), as ring vaccination capacity varies, for the two levels of workplace/social group contact finding probabilities (0.8 and 0.9), and for the two levels of monitored diagnosis rate among initially asymptomatic contacts (1 day-1 and 8 day-1). For low levels of ring vaccination (traceable contacts per day), the epidemic is almost never contained, but for ring vaccination levels near 50–60 per day (5–6 per index case per day), the average containment fraction became close to 1. However, this average conceals the fact that for some scenarios (parameter sets chosen from the calibrated uncertainty analysis), control remains difficult or impossible even at high levels of ring vaccination. Therefore, in Figure 5B, we plotted the single lowest containment fraction seen out of the 1000 computed; focusing on the single worst scenarios reveals a different picture, and shows that isolation of asymptomatic contacts and very high probabilities of finding workplace or social contacts would be needed to control smallpox under these most pessimistic parameter choices. Effect of contact tracing speed Rapid contact tracing in ring vaccination may play an important role in suppressing the epidemic, since the longer it takes to trace a contact, the less likely the vaccine is to be efficacious, and the more opportunities the infected individual may have to transmit disease before they are finally located, isolated, and vaccinated if appropriate. We illustrate this possibility in Figure 6 by examining the same scenario we showed earlier in Figure 3A (e.g. households of size 4, workplace/social groups of size 8, 95% of household contacts traceable, 80% of workplace/social groups traceable, an average time to infection for a household contact of an infective given by 0.2 days). We assume in one case that contacts may be traced quickly (1 day for a household contact, 2 days for a workplace/social contact), and in the other that the contacts are on average found slowly (5 days for a household contact, 10 days for workplace/social contacts); we assumed 30 ring vaccinations (traceable contacts) possible per day. In this scenario, the epidemic is more severe and containment (as we have been defining it) less likely when contact tracing is slow: in the fast scenario, 238 infections occurred on average and the (estimated) containment probability was 99%; for the slow scenario, on average 3587 infections occurred and the (estimated) containment probability was only 1%. Figure 6 Faster contact tracing may improve the efficacy of ring vaccination. We assume the same baseline parameters as in Figure 3A (e.g. households of size 4, workplace/social groups of size 8, 95% of household contacts traceable, 80% of workplace/social contacts traceable), and 30 ring vaccinations available per day (with contacts of contacts not traced). The fast scenario corresponds to an average one day delay for household and two days for workplace/social contacts (as in Figure 3A); the slow scenario corresponds to an average five day delay for household and ten day delay for workplace/social contacts. This figure shows the average of one hundred realizations starting with ten index cases. While Figure 6 illustrates the possibility that rapid contact tracing may be of decisive importance in some scenarios (parameter set choices), this is not always the case. For some parameter sets, the probability of tracing contacts (household or workplace/social) may be too low, or the transmission rate too high, for more rapid contact tracing to make any difference. Conversely, for other parameter sets, the smallpox transmission rate may be so low that smallpox is easily contained even with slow contact tracing. While rapid contact tracing is never harmful, overall, how typical are the results of Figure 6 (in which rapid contact tracing was important in ensuring the efficacy of ring vaccination)? To address this question, we simulated the growth of smallpox for the 1000 "calibrated" scenarios we used in Figure 5A and 5B. As before, we assumed ten initial cases, and (as in Figure 6) that 30 ring vaccinations were possible per day; then we simulated 100 epidemics assuming one day to find a household contact (and 2 days to find a workplace/social contact). We then simulated 100 epidemics assuming that it takes five days to find a household contact and 10 days to find a workplace/social contact (as in Figure 6). For each of these 1000 scenarios, we calculated the fraction of simulations for which the total number of cases is 500 or less within 250 days, i.e. the containment fraction. For nearly all scenarios (parameter set choices), the containment fraction was smaller (sometimes much smaller) when the contact finding time is faster (since faster contact finding, all else being equal, improves smallpox control, as illustrated in Figure 6). However, for 64.5% of the scenarios (parameter set choices) examined, the difference was less than 2.5% in absolute terms (smallpox was either contained or not contained depending on other factors, and rapid contact tracing did not make the difference). On the other hand, for 18.7% of the scenarios examined, the absolute difference in the containment probability was 20% or more; thus, a substantial difference in containment probability is occasionally attributable to the difference between fast and slow contact tracing. Effect of more rapid diagnosis Public awareness of smallpox, leading to more rapid isolation and identification, may play an important role in eliminating the epidemic, as illustrated by the scenarios in Figure 7. We assumed 20 ring vaccinations possible per day, a capacity too small to contain the epidemic in the absence of increased surveillance or diagnosis; the black line in the figure shows the steeply rising average number of cases for the first 100 days. If, however, surveillance or public awareness of the symptoms of smallpox increases the diagnosis rate by 50% (multiplies the baseline diagnosis rates by 1.5), containment becomes possible (blue line); with a doubling of the diagnosis rate (red line) the peak number of cases is lower still. In these scenarios, increased diagnostic rates markedly improve the ability of ring vaccination to control the epidemic, this suggest that any ring vaccination effort be accompanied by increased public awareness and surveillance. Figure 7 More rapid diagnosis due to public awareness or increased surveillance may lead to far more effective epidemic control. We assume the same baseline parameters as in Figure 3A, and averaged 100 realizations of the epidemic beginning with 10 index cases and assumed a ring vaccination capacity of 20 per day (and contacts of contacts not traced). For the black line, the diagnosis rate of cases does not change after the first case is identified (the multiplier is 1.0); for the blue line, the diagnosis rate increases by 50% (multiplier 1.5) after the first case is identified (as in Figure 3A), resulting in substantially fewer cases; and for the red line, the diagnosis rate is doubled (multiplier 2.0) after the first case is identified, resulting in still fewer cases. In many cases, however, more rapid diagnosis was not required for ring vaccination to be effective. As before, we simulated smallpox epidemics for each of 1000 calibrated scenarios, performing 100 realizations each beginning with 10 index cases, and computed the fraction of scenarios for which the epidemic was always contained (as defined earlier), assuming no change in diagnosis rates. We assumed 80 ring vaccinators per day, contact finding probabilities of 0.95 for households and 0.8 for workplace/social contacts (as in Figure 3A). Under these assumptions, for 83.4% of the scenarios, the epidemic was contained within 500 total cases in each of the 100 realizations, even with no change in diagnosis rates. Uncertainty analysis (using the 1000 calibrated scenarios, and based on the fraction of 100 replications showing decontainment) revealed the most important parameters which predict the failure of ring vaccination without more rapid diagnosis were the same as we found in the earlier uncertainty analysis; a higher fraction vaccinated before the epidemic, smaller households or workplace/social groups, less transmissibility, lowered infectivity prior to the rash, more rapid diagnosis, and a higher rate of diagnosis for alerted individuals all contribute to a greater containment probability even without an overall increase in the diagnosis rate. Effect of continued surveillance of contacts We have been assuming that whenever an individual is contacted during an investigation, the individual will be diagnosed more quickly should they subsequently develop symptoms. When transmission is assumed to be very rapid (smallpox is assumed to be highly contagious), most individuals may already be infected when identified through contact tracing from an infective. Using the scenario we examined in Figure 3A, we see that continued surveillance of contacts is an essential component of effective ring vaccination designed to control rapidly spreading smallpox: if smallpox in a contact is not diagnosed any more quickly than for a non-contact, containment by ring vaccination requires over 98% contact finding probabilities for both household and workplace/social contacts – even if unlimited numbers of ring vaccinators are available; containment cannot be guaranteed by adding additional ring vaccination capacity if the contact finding rates are too low and/or the follow-up for contacts is insufficient. Smallpox which is transmitted less rapidly to contacts would, however, be containable with a lower contact finding probability (results not shown). Finally, we used the "calibrated" scenarios (parameter set choices) to explore the levels of contact finding probability needed to contain the epidemic (as before, defined to mean 500 or fewer cases ultimately resulting from ten initial cases) (Table 4). In these scenarios, we assumed that all traceable contacts were followed up very quickly (1/a = 1 hour, so that cases arising in previously contacted persons almost never transmit the infection further). We chose different levels of household and workplace/social contact finding probabilities and different levels of ring vaccination capacity, and performed 100 replications of each of the 1000 different scenarios. In Table 4 we report the fraction of scenarios for which all 100 replications exhibited containment. Scenarios in which smallpox is highly contagious require high contact finding probability to ensure the containment of the epidemic. Table 4 Containment of severe smallpox at different levels of contact finding. The first three columns are assumed levels for the probability of finding a household contact, the probability of finding a workplace/social (W/S) contact, and for the number of contact tracings/ring vaccinations possible per day; the last two columns express (as percentages) the resulting probability of containment given the assumed contact finding probabilities and contact tracing capacities; two containment probabilities are given: the containment probability when only contacts of cases are traced (first column, "Contacts"), and the containment probability when contacts of contacts of cases are traced in addition to the contacts of cases (second column, "Contacts of Contacts"). All other parameters are the same as in Figure 3A. Probability of finding Number of Ring vacc. per day Containment Contacts Probability when Tracing Contacts of contacts Household contacts W/S contacts 0.95 0.85 50 99.1% 97.9% 0.95 0.85 100 99.3% 100.0% 0.95 0.85 200 99.1% 100.0% 0.9 0.8 50 95.7% 95.8% 0.9 0.8 100 95.6% 99.9% 0.9 0.8 200 95.4% 100.0% 0.85 0.75 50 86.0% 93.3% 0.85 0.75 100 86.1% 99.1% 0.85 0.75 200 86.3% 99.2% 0.75 0.6 50 52.1% 72.0% 0.75 0.6 100 51.5% 78.5% 0.75 0.6 200 53.0% 78.6% Transmission prior to rash Transmission prior to the rash makes epidemic control more difficult. In Figure 8, we show an expanding smallpox epidemic assuming differing levels of infectivity prior to the rash (adding increased infectivity prior to the rash, keeping constant the infectivity after the rash). We assume all parameters are the same as in Figure 3A (and that the ring vaccination capacity is 40 per day). Infectivity prior to the rash is modeled as the relative infectivity during the short (1 day) period of oropharyngeal lesions just prior to the rash (compared to the infectivity during the first week of the rash), and as the relative infectivity during the prodromal period (relative to the period just prior to the rash). We consider three scenarios: a relative infectivity during entire period is one (i.e., infectivity during the prodromal period and just prior to the rash is the same as during the first week of the rash), b the relative infectivity just prior to the rash is the same as during the first week of the rash, but during the prodromal period is 4% (as in Figure 3A) of this value, and c the relative infectivity just prior to the rash is 20% of the infectivity during the first week of the rash, and during the prodromal period is 20% of this value. The figure shows that increased infectivity just prior to the rash leads to a larger epidemic (comparing b and c); in case b (high infectivity just prior to onset of rash), loss of containment occurs 36% of the time (but in none of the 100 realizations shown in case c (low infectivity prior to rash)). Scenario a (full infectivity during entire the prodromal period) showed loss of control in every realization. Increasing the ring vaccination capacity from 40 per day to 80 per day (results not shown) led to containment in all of the realizations with high infectivity just prior to the rash and low infectivity during the prodromal period (case b), but made no difference if the infectivity was as high during the prodromal period as during the rash (case a). While intuitively adding additional infectiousness must increase the number of secondary cases and make control more difficult, these results do illustrate that even a small amount of increased infectiousness prior to the rash (when diagnosis is more difficult) may substantially increase the difficulty of smallpox control. Figure 8 Transmission prior to the rash makes epidemic control more difficult. The figure shows a expanding smallpox epidemic assuming differing levels of infectivity prior to the rash. We assume all parameters are the same as in Figure 3A (and that the ring vaccination capacity is 40 per day). Infectivity prior to the rash is modeled as the relative infectivity during the short (1 day) period of oropharyngeal lesions just prior to the rash (compared to the infectivity during the first week of the rash), and as the relative infectivity during the prodromal period (relative to the period just prior to the rash). For scenario a, relative infectivity during the prodromal period and just prior to the rash is the same as during the first week of the rash, for scenario b, the relative infectivity just prior to the rash is the same as during the first week of the rash, but during the prodromal period is 4% (as in Figure 3A) of this value, and for scenario c, the relative infectivity just prior to the rash is 20% of the infectivity during the first week of the rash, and during the prodromal period is 20% of this value (these two parameters are the same as in Figure 3A). Other factors Finally, in Figure 9, we present scenarios in which each of four other parameters are modified from the baseline values of Figure 3A, assuming 40 contact tracings (ring vaccinations) are possible per day (line a in the figure). Specifically, we assume that severe smallpox (hemorrhagic and flat) on average takes four times longer to diagnose and isolate than ordinary smallpox (case b), that no one in the population has prior vaccination protection (from before the discontinuation of routine vaccination, case c), that 10% more smallpox is too mild to diagnose (but still contagious, case d) compared to baseline, and finally that the vaccine is completely ineffective (case e). Each of these scenarios will be discussed further below. Figure 9 Additional scenarios, assuming 40 ring vaccinations or contact tracings possible per day, and that contacts of contacts are traced; all parameters are identical to those in Figure 3A unless otherwise indicated. The figure shows the average of 100 replications of five scenarios (Case a repeats the result from Figure 3A for reference); the numbers in parentheses in the legend are the corresponding fraction of the 100 scenarios for which decontainment occurred. For case b, we assumed that flat and hemorrhagic smallpox cases took four times as long on average to diagnose as ordinary cases; for case c., we assumed that no one in the population had prior protection (as opposed to 25% for Figure 3A); for case d, we assumed that an additional 10% of individuals (13% instead of 3%) would develop mild smallpox (with 75% developing ordinary smallpox instead of 85% as in Figure 3A); and for case e, we assumed that the vaccine is completely ineffective and provides no protection against infection. Scenario b was motivated by the possibility that individuals with severe forms of smallpox may be more difficult to diagnose, and thus remain infectious in the community longer (despite the much greater degree of illness of such patients), or that such patients may be more infectious. In this particular case, quadrupling the mean diagnosis time led to one additional replication out of 100 in which containment was not achieved (2/100, compared to the baseline of 1/100). However, we assumed that community awareness of smallpox leads to the same relative rate of increased diagnosis among severe cases as for ordinary cases, and that the most severe forms are relatively rare. In addition to the scenario shown in the figure, we also replicated the same 1000 "calibrated" simulations, assuming that in each case 40 contact tracings per day are possible and that the diagnosis time for severe cases was four times that of ordinary cases. Finally, we repeated each "calibrated" scenario 100 times assuming long diagnosis times for severe cases, and not making this assumption, and found that the difference in the decontainment fraction was not large (results not shown). Scenario c illustrates that vaccination prior to the discontinuation of routine vaccination does play a role in smallpox control by ring vaccination; there were more decontainment scenarios (5/100) when no prior protection exists in the population. The results suggest that prior vaccination aids in the control of smallpox, but that it is not strictly necessary for control (in this scenario, 95% of the replications exhibited containment). In Figure 3A, we assumed 25% of individuals had protection due to vaccination prior to the discontinuation of routine vaccination; in scenario c of Figure 9, we assumed this fraction was zero. Scenario d demonstrates that if 10% more smallpox infections (in absolute terms, i.e. 13% compared to 3% in Figure 3A) lead to mild cases among individuals with no prior protection, the epidemic is more difficult to contain (13/100 replications showed loss of containment). Finally, scenario e demonstrates that containment is still possible even when the vaccine is completely ineffective in everyone – because of case isolation and isolation of contacts (and of contacts of contacts). Here, with 40 contact tracings possible per day, 55% of the replications nevertheless exhibited containment even with a vaccine which offered no protection whatever. With 90 contact tracings possible per day, all replications exhibited containment even assuming no vaccine protection. Effect of mass vaccination Although less efficient than ring vaccination in the sense that more vaccinations must be delivered to eliminate infection, comprehensive mass vaccination following the introduction of smallpox is sufficient to eliminate the infection. In Figure 10, we show the probability of achieving containment (defined to be fewer than 500 total cases resulting from 10 index cases) for different levels of ring vaccination (0, 5, 10, and 20 vaccinations per day) and mass vaccination (0, 0.5%, 1%, and 2%; compare with the 10%-20% per day many jurisdictions in the United States are planning to vaccinate). Specifically, for each level of ring vaccination and mass vaccination, we used the same 1000 parameter sets used in Figure 5, and performed 100 simulated epidemics for each parameter set. On the vertical axis, we plot the fraction of the 1000 scenarios for which each of the 100 simulated epidemics was contained. We further computed the fraction of scenarios for which none of the 100 simulated epidemics was contained; this is indicated by the colored segment in the small pie chart at each symbol. When the mass vaccination rate was 2% per day, the mean number of deaths (averaging over all scenarios and all simulations within each scenario) was 47.7, 33.7, 26.4, and 20.1 for a ring vaccination level of 0, 5, 10, and 20 per day (respectively) out of a population of 10000. Moreover, when we increased the mass vaccination level to 3%, an average of 28.9 deaths occurred when no ring vaccination was used, but this fell to 22.3 deaths when only 5 ring vaccinations per day were available (again assuming a population of 10000, and 10 index cases). With a mass vaccination level of 5% per day, an average of 18.8 deaths occurred without ring vaccination, and 15.8 deaths occurred when only 5 ring vaccinations per day were possible. (At a mass vaccination rate of 3% per day, containment as defined above was achieved in all 100 replications for 95% of the scenarios even without ring vaccination; at a mass vaccination rate of 5% per day, containment was achieved in all replications for all scenarios.) These results show that over a wide range of simulated epidemics, even seemingly small levels of ring vaccination (coupled with follow-up) may have a substantial effect in preventing epidemic spread and reducing deaths from smallpox, even during a mass vaccination campaign. Note that many jurisdictions in the United States are planning mass vaccination campaigns which could reach 10%-20% of the population per day, far greater than the mass vaccination levels examined here; it is interesting to note that mass vaccination campaigns may be effective in preventing a widespread epidemic even at much lower levels than are being planned for. Where feasible, such rapid mass vaccination rapidly eliminates smallpox transmission in our model; vaccination of contacts is still beneficial, since we are assuming that earlier vaccination yields a greater probability of preventing or ameliorating infection (results not shown). Figure 10 Mass and ring vaccination together. Low-level mass vaccination programs are improved substantially by the addition of ring vaccination. The shaded pie segments represent the fraction of 1000 scenarios for which containment (as defined in the text) was never realized; the vertical position of the pie chart represents the fraction of the 1000 "calibrated" scenarios for which containment was always achieved. As the fraction of the population mass vaccinated increases or the ring vaccination capacity increases, the probability of containment increases. Discussion We constructed a simple network model of smallpox transmission, and addressed the question of what circumstances contribute to the success of a ring vaccination campaign designed to control smallpox. Our analysis focused on the use of contact tracing/ring vaccination to prevent a widespread epidemic following a deliberate release. We conducted a sensitivity analysis based on particular, but reasonable, ranges for the unknown parameters. Our results are consistent with prior vaccination models in identifying prior vaccination and ring vaccination capacity as significant factors in determining the spread of smallpox. Unsurprisingly, we also find that household size and ring vaccination speed are particularly important parameters; these results are intuitively plausible. The contact finding probability did not appear important in this analysis only because a narrow range of values was chosen. We illustrated smallpox control by presenting scenarios based on control of moderately severe smallpox epidemics. We find that swift, aggressive contact tracing and ring vaccination is is usually sufficient to bring the infection under control. Provided that there is sufficient capacity, vaccination of contacts of contacts is beneficial, and results in fewer infected individuals and more rapid elimination of infection; investigating contacts of contacts allows the chain of transmission to be outrun to some extent. When ring vaccination capacity is small, diversion of crucial resources away from contacts is harmful; contacts of contacts should only be traced and vaccinated provided that no resources are diverted away from contacts of cases. The increased surveillance (or isolation) of contacts, together with improved rates of diagnosis due to community awareness, play important roles in smallpox control; we note that in some cases, lowered diagnosis rates among severe cases contributed to a small extent to loss of epidemic control, and suggest that any public awareness campaign include information to help the public be more aware of the full spectrum of the clinical features of smallpox. One limitation of our analysis is that we chose not to explicitly incorporate the specific epidemiology of health care workers (or mortuary workers), who are likely to be exposed to infected individuals during any smallpox epidemic (e.g. [17,22]), and who may then infect further members of the community [22] (as was also seen in the recent outbreak of SARS, e.g. [48]). Transmission to health care workers may be considered to amplify the initial attack or to be simply accounted among the exposures we considered (and thus be approximated by the behavior of our model), since health care workers and their household contacts are in all likelihood traceable contacts, and ring vaccination/contact tracing would identify and halt these chains of transmission as in our model. The disruption of smallpox control and patient care that may occur is not accounted for in our analysis, however, causing our model in this sense to err on the side of optimism. The appropriateness of pre-event vaccination of health care workers or other first responders has been addressed by other analyses [12,49], and is beyond the scope of our model. While we analyzed the effect of contact tracing, case and contact isolation, and ring vaccination (together with mass vaccination), in a real smallpox epidemic, in practice, control efforts are unlikely to be limited strictly to vaccinating contacts (and health care workers, as likely contacts) and isolating cases. Indeed, making vaccine available to individuals who believe they live near cases or to others on a voluntary basis occurred in smallpox control efforts in the past [22]. Vaccination of such individuals can only harm the disease control effort if it hinders or delays the diagnosis of cases or the investigation and vaccination of contacts; our results show that even relatively low levels of vaccination of the general population may have a beneficial effect in preventing the epidemic from escaping control. More serious is the possibility that individuals who should be vaccinated or isolated would be missed; this could occur either because individuals or institutions did not cooperate with the disease control effort, or because the individuals simply could not be found. Our analysis suggests that ring vaccination need not be perfect to successfully contain the epidemic, and yet, under conditions where there is a high rate of infection among contacts, or a relatively high rate of casual transmission, high rates of contact finding (in excess of 90%), together with increased surveillance and contact isolation, are needed to contain the epidemic. Finally, the vaccination of individuals at low risk of contracting smallpox will cause harm due to adverse events of the vaccine; in our model, the assumed death rate due to vaccination was small compared to the probability of death from smallpox, and played essentially no role in the analysis. In practice, individuals suspected to be at high-risk for vaccine complications, but at relatively low risk for contracting smallpox, might simply be isolated or closely monitored even during an outbreak; while the presence of individuals in the population at higher risk for vaccine complications would increase the death rate during an outbreak, such individuals are unlikely to impair the containment of the epidemic (the primary focus of this analysis). Our results support ring vaccination against epidemics of smallpox (even assuming high rates of transmission to close contacts), but do note that stochastically, for severe (rapidly transmissible) smallpox, scenarios of loss of control are seen, with resulting widespread epidemics. In scenarios in which the transmission potential of smallpox is smaller, such loss-of-control scenarios occur less frequently (results not shown). Mass vaccination campaigns, when conducted quickly and with very high coverage, do not result in loss of control in our model. Nevertheless, fewer deaths due to smallpox result when ring vaccination is conducted along with mass vaccination. Conclusion Simulated smallpox epidemics with ring vaccination suggest that aggressive, fast ring vaccination can control epidemics of smallpox. To do so, however, smallpox must be identified quickly and contacts vaccinated promptly. We also identify public awareness of smallpox – leading to prompt identification of cases – as a major factor in smallpox control; in some simulations, it may play a role as significant as ring vaccination itself [15]. However, we also found that uncertainty in (1) transmission from mild cases, (2) the household size, and (3) casual transmission contributed to the overall uncertainty in the epidemic size. Other parameters to which the number of infections were highly sensitive were the prior vaccination fraction, parameters related to infectiousness, and parameters related to transmission prior to the rash. Because our model combines network structure with response logistics, our results support and complement the results of other investigators. Our results support the notion that prior vaccine protection may play an important role in slowing the epidemic [11], despite the possibility that some vaccinated individuals may develop mild cases which are harder to identify, but which nevertheless transmit disease. Likewise, our results provide support for the view that ring vaccination should play a central part in smallpox control. If initiated, ring vaccination should be conducted without delays in vaccination, should include contacts of contacts (whenever there is sufficient capacity to cover all contacts of cases), and should be accompanied by a vigorous campaign of public awareness which can facilitate more rapid identification and isolation of cases. We assumed that ring vaccination could be fast (little delay between identification of a case and vaccination of the contacts), effective (nearly all household contacts can be found, and most of workplace and social contacts), and available (there is sufficient capacity). To be effective, ring vaccination planning must yield a system capable of meeting these benchmarks; we should not only be able to assess the number of contact vaccinations that will be possible per day, but should have a plan in place to (1) identify contacts by working with individuals, employers, schools, community representatives, and authorities or businesses who may have access to information facilitating contact tracing, (2) rapidly investigate and vaccinate such individuals, perhaps using field teams managed by central dispatch. It is important to realize that for high-risk, transient, or unstably housed populations where reliable contact tracing is impossible, the conclusions of the model we present cannot be applied. It is important to note that while our model suggests that ring vaccination together with contact tracing and isolation is likely to be successful, we found that for some scenarios (where smallpox was more transmissible, or was relatively more transmissible before the rash), epidemic containment required not only ring vaccination, but increased public awareness, the isolation of contacts, and tracing of contacts of contacts. For scenarios in which the smallpox was less transmissible, epidemic containment was possible at lower contact finding probabilities. Thus, while our simulations suggest that contact tracing/ring vaccination need not be perfect to succeed, because of uncertainties in our knowledge of the behavior of bioterrorist smallpox, it is impossible to know in advance how good it will have to be. Thus, that high contact finding rates, mass public awareness leading to early identification of cases, isolation of contacts, and investigation of contacts of contacts should all be conducted with maximum effectiveness to reduce the probability of a widespread epidemic. While the possibility of smallpox uncontrollable by ring vaccination has made mass vaccination preparations wise, and while mass vaccination may be unavoidable in the event of a deliberate release of smallpox, we believe that ring vaccination is essential in any case. This is not only because individuals recently exposed to smallpox may be protected if they are vaccinated promptly, but because each contact identified potentially lies in the immediate future of the transmission chain. From the standpoint of epidemic control, it is far more valuable to vaccinate individuals next in the transmission chain than to vaccinate other persons. Our results support the idea that ring vaccination/case isolation may in many, if not most cases, eliminate smallpox even without mass vaccination, but also support planning for mass vaccination (so that the vastly more costly and difficult policy of mass vaccination will be available in the event of an explosive epidemic). When faced with the unknown, multiple redundant preparations are appropriate; case investigation/isolation may control smallpox even if the vaccine does not work at all, but mass vaccination is useful in the event of an explosive epidemic for which case tracking becomes impossible. Competing interests None declared. Authors' contributions TP, KH, SF, TA, RR, and DP performed the literature review (and parameter evaluation), TP developed and implemented the model and simulation, TP performed the analysis of the simulation model and drafted the manuscript, DP performed analysis of contact tracing data, and KH conceived of the study. All authors contributed to, read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: Supplementary Material Additional file 1 For consistency, all references are included in the bibliography of the main text. Click here for file Additional file 2 For consistency, all references are included in the bibliography of the main text. Click here for file Acknowledgments TCP acknowledges the support of NIDA grant DA 5-R01-13510. 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==== Front BMC Public HealthBMC Public Health1471-2458BioMed Central London 1471-2458-4-341529871310.1186/1471-2458-4-34Research ArticleLogistics of community smallpox control through contact tracing and ring vaccination: a stochastic network model Porco Travis C [email protected] Karen A [email protected] Susan E [email protected] Diane L [email protected] Randy [email protected]ón Tomás J [email protected] San Francisco Department of Public Health, Community Health and Epidemiology Section, Epidemiology and Effectiveness Research Unit, 101 Grove Street Suite 204, San Francisco, California 94102 USA2 Center for Infectious Disease Preparedness, School of Public Health, University of California, Berkeley, USA3 Surveillance and Epidemiology Section, Tuberculosis Control Branch, Division of Communicable Disease Control, California Department of Health Services, Berkeley, California, USA2004 6 8 2004 4 34 34 3 1 2004 6 8 2004 Copyright © 2004 Porco et al; licensee BioMed Central Ltd.2004Porco et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Previous smallpox ring vaccination models based on contact tracing over a network suggest that ring vaccination would be effective, but have not explicitly included response logistics and limited numbers of vaccinators. Methods We developed a continuous-time stochastic simulation of smallpox transmission, including network structure, post-exposure vaccination, vaccination of contacts of contacts, limited response capacity, heterogeneity in symptoms and infectiousness, vaccination prior to the discontinuation of routine vaccination, more rapid diagnosis due to public awareness, surveillance of asymptomatic contacts, and isolation of cases. Results We found that even in cases of very rapidly spreading smallpox, ring vaccination (when coupled with surveillance) is sufficient in most cases to eliminate smallpox quickly, assuming that 95% of household contacts are traced, 80% of workplace or social contacts are traced, and no casual contacts are traced, and that in most cases the ability to trace 1–5 individuals per day per index case is sufficient. If smallpox is assumed to be transmitted very quickly to contacts, it may at times escape containment by ring vaccination, but could be controlled in these circumstances by mass vaccination. Conclusions Small introductions of smallpox are likely to be easily contained by ring vaccination, provided contact tracing is feasible. Uncertainties in the nature of bioterrorist smallpox (infectiousness, vaccine efficacy) support continued planning for ring vaccination as well as mass vaccination. If initiated, ring vaccination should be conducted without delays in vaccination, should include contacts of contacts (whenever there is sufficient capacity) and should be accompanied by increased public awareness and surveillance. ==== Body Background Concerns about intentional releases of smallpox have prompted extensive preparations to improve our ability to detect and respond to an outbreak of smallpox [1,3,4,2]. Many factors contribute to the public health challenge of understanding and preparing for smallpox, including the age and quality of epidemiological data on native smallpox and the smallpox vaccine, the difficulty of extrapolating that data to our current populations, the possible terrorist use of altered smallpox, our ignorance of terrorist methods of release, and the relatively high risk of adverse events caused by the smallpox vaccine. The Centers for Disease Control and Prevention (CDC) established ring vaccination (selective epidemiological control [5]), a strategy in which contacts of cases are identified and vaccinated, as the preferred control measure in the event of a smallpox outbreak (interim plan). The successful use of ring vaccination during the smallpox eradication campaign and its logical emphasis of case-contacts for immediate vaccination support its use (though the attribution of the success of the eradication program to ring vaccination has been challenged [6]). Health Officers should initiate ring vaccination upon identification of the first cases of smallpox. However, there are legitimate concerns regarding the ability of public health practitioners to mount a quick, comprehensive and successful ring vaccination program, particularly in the face of a moderate-sized or large smallpox outbreak. To guide preparation efforts and inform incident decision-making, we attempt to identify outbreak characteristics and response capacities that significantly impact the ability of ring vaccination to control a smallpox outbreak and to determine whether ring vaccination is useful in the presence of a mass vaccination campaign. Our analysis uses a newly developed mathematical model: a continuous-time, event-driven network simulation model of smallpox ring vaccination. Mathematical models can advance our understanding of how a smallpox outbreak might progress. Several mathematical and computer models address the question of smallpox transmission [7-13]. The first model to appear [8] concluded that ring vaccination would be effective, but did not treat response logistics in detail; the model was linear and did not treat the depletion of susceptibles as the epidemic progressed (appropriate, however, for assessing control early in an epidemic, when the number infected is small compared to the number of susceptibles, e.g. [14]). The innovative model by Kaplan et al. [9] emphasized the importance of resource limitation and the logistics of smallpox response, but assumed that full infectiousness began before the onset of symptoms (and the subsequent identification and removal), and did not separately monitor close epidemiological contacts of patients (which are at greatest risk, but also easiest to find and vaccinate); the conclusions were highly critical of ring vaccination. The model by Halloran et al. [11], a stochastic, discrete-time network model omitted the explicit inclusion of response logistics while otherwise used parameter values similar to those in Kaplan et al. [9]; the inclusion of residual immunity from individuals vaccinated prior to the discontinuation of routine vaccination, however, led to a more favorable view of ring vaccination. The model by Bozzette et al. [12] assumed that ring vaccination would reduce the number of transmissions and focused on health care workers (but did not explicitly include the network structure of the population nor the response logistics of ring vaccination). The model by Eichner [15] did not explicitly include the network structure of the population nor the logistics of ring vaccination, but did use parameters based on data from an outbreak in Nigeria, and did distinguish close and casual contacts, case isolation, and surveillance of contacts; it concluded that case isolation and contact tracing could prevent the spread of smallpox. Finally, the individual-based model by Epstein et al. [16] presented scenarios illustrating certain alternatives to pure mass vaccination and ring vaccination of contacts of cases in preventing smallpox transmission in small populations of 800 individuals; this model includes no homogeneity assumptions, but did not analyze tracing of contacts of contacts. Because none of the available models includes both network structure (with explicit contact tracing) and response logistics limited by the number of available disease control investigators [9], we included these features in a continuous-time event-driven network simulation model of smallpox ring vaccination. Specifically, the model we developed includes the following features: Network structure Smallpox was primarily a disease of close contact, especially household contacts [5]. Such contacts are both the most important epidemiologically, and also the easiest to identify. Post-exposure vaccination Some evidence suggests that vaccination soon after exposure may lessen the severity of the resulting case of smallpox or possibly prevent disease entirely [17-20]. Second ring Ring vaccination may involve not only vaccinating contacts of cases, but also contacts of contacts of cases [21,22] – potentially allowing the public health authorities to "outrun" the chain of transmission. Response capacity Limited case-finding and vaccination capabilities lead to the possibility that it may be impossible to find newly exposed individuals and vaccinate them in time, resulting in a "race to trace" [9]. Heterogeneity in natural history Mild, ambulatory cases of smallpox may spread disease because such cases may be harder to recognize. Prior vaccination Vaccination of individuals prior to the discontinuation of routine vaccination may provide some, possibly considerable, protection against infection [11,23,24], although it may also result in more mild cases which may be harder to detect. Public awareness Public awareness may lead to more rapid detection of cases. We use this model to determine what factors promote or hinder the success of ring vaccination during a smallpox outbreak, and whether ring vaccination is useful in the presence of a mass vaccination campaign. In particular, the goal of this paper is to examine the control of smallpox by contact tracing and ring vaccination using a network model which includes response logistics [9]. Methods Model structure Natural history of smallpox We briefly review relevant features of the natural history and epidemiology of smallpox [17,25,8,28]. Following infection by the variola virus, individuals exhibited an incubation period of approximately 7–19 days with 10–14 being most typical. Sudden onset of fever and malaise, often with accompanying headache and backache, began the initial (or pre-eruptive) phase of smallpox. After 2–3 (or perhaps 4) days, individuals with the most common form, ordinary type smallpox, developed the characteristic focal rash, preceded in many cases by oropharyngeal lesions. In fatal cases of ordinary smallpox, death often occurred between the tenth and sixteenth day of symptoms; among survivors, most scabs had separated by day 22–27 of illness [26]. The course of smallpox varied widely between individuals, and several different clinical classifications were developed [29-31,17,26]. Consideration of the clinical features and severity of smallpox is important from the standpoint of mathematical transmission modeling because (1) the clinical features affect the ease of diagnosis (and thus of case identification), (2) more severe forms of smallpox may result in more transmission, (3) vaccinated individuals may develop less severe disease. We utilize a modified or simplified version of the classification system developed by Rao [32,31,26]; for the mathematical model, we will classify smallpox into five categories: early hemorrhagic, flat and late hemorrhagic, ordinary, modified, and mild. However, the clinical features and severity of smallpox in different populations may have been affected by underlying host factors, differences in viral strains, or differences in the infectious dose owing to different prevailing modes of transmission, and thus robust and precise quantitative estimates of the effects of (pre- or post-exposure) vaccination on the resulting smallpox severity, or of the infectivity differences between individuals exhibiting different forms of smallpox, are not available. The significance of such differences will be revealed through sensitivity analysis. Further details are given in Appendix 1 [see Additional file: 1]. Vaccination with vaccinia virus provided substantial protection against infection. Dixon assessed the risk of infection for an individual successfully vaccinated 3 years prior to exposure to be 0.1% the infection risk of an unvaccinated individual [17]. However, smallpox vaccination did not always take when applied, and moreover, in many instances, individuals who experienced a repeated vaccination failure developed severe smallpox upon exposure. The probability of a successful take depended on the vaccination method used; we assume that the take rate is between 95% and 100% [22,28]. In addition to protection against infection, vaccination could in many cases modify the course of infection and reduce the severity. Vaccine protection waned over time, but individuals vaccinated 20 years prior to exposure were believed to still have half the infection probability that an unvaccinated person had [17], and to have some protection against the most severe manifestations of smallpox. Dixon [17] believed that vaccine protection had at least three components, which decayed at different rates; for the purpose of this paper, we will assume that the severity of smallpox in previously any (recently or otherwise) vaccinated individuals follows the same distribution as for the vaccinated subjects seen in the case series observed by Rao in Madras [26], except that anywhere from 0 to 5% of vaccinated subjects develop smallpox too mild to diagnose without special surveillance or awareness. Observe that the vaccinated cases studied by Rao were vaccinated (at some point in their lives) before exposure, rather than after exposure to smallpox. Smallpox was largely a disease of close contacts [17,26,33], spread primarily through face to face contact with an infected person (or occasionally through contaminated clothing). Individuals in the incubation period of smallpox were not infectious, and long term carriers did not exist. Patients were believed to be infectious following the development of oropharyngeal lesions, which could precede the rash by 24 hours [26]. However, patients were believed to be most infectious during the first week of the rash [26]; Dixon (1962) believed that patients could be infectious from the onset of acute viremia, but most evidence suggested that little transmission occurred prior to the development of the rash [26,33]. The more severe the case, the more infectious they appeared to be [34]; mild cases were believed to have very little infectiousness. While scabs contained infectious material and patients were considered to be infectious until the last scab fell off, in practice patients were not highly infectious during the scabbing phase. Importantly, patients who had been vaccinated were found to cause fewer secondary cases [34]. Very severe cases, such as hemorrhagic or flat smallpox, occasionally resulted in considerable transmission, owing to diagnostic difficulties; mild cases, in which the patient remained ambulant during the course of the disease, could cause considerable spread as well [35,36]. Within a household or family dwelling, the secondary attack rate of unvaccinated susceptibles depended on the time and place, occasionally below 50% [29], but often approaching 100% [37]. Drier conditions were often believed to favor transmission [17,27], so that lower rates of transmission derived from tropical regions may not be applicable to the temperate zone [38]. The number of secondary cases resulting from a given importation into Europe varied widely [39], with most importations yielding few cases, but with the occasional large outbreak being seen. Mathematically, we represent the course of smallpox according to Figure 1. We distinguish eight epidemiologically relevant states: (1) just following exposure, during which time vaccination could afford complete protection against disease, (2) a period of several days during which vaccination will not prevent disease, but may still reduce the severity of disease, (3) still prior to the development of symptoms, but too late for vaccination, (4) the beginning of the pre-eruptive period, during which the patient exhibits fever, malaise, and possibly other symptoms, but is not yet infectious, (5) a short period prior to the appearance of the rash, during which the appearance of oropharyngeal lesions will permit variola transmission, (6) the first week of the rash, during which time the patient is most infectious, (7) and (8), succeeding stages of the rash, during which time the patient is less infectious. For each of these states, we assume that conditional on surviving, the waiting time until the next stage is chosen from a uniform distribution as indicated in Appendix 2 [see Additional file: 2], except that the incubation period (the time from infection until Stage 4) is derived from estimates of the incubation distribution of smallpox based on importation cases in Europe [26] (see Appendix 2 [see additional file 2] for details). We chose to sample from a uniform distribution as a simple way to ensure a minimum waiting time in each state; many alternatives to this choice are possible. Figure 1 Smallpox stages used in the simulation model. Flat and ordinary smallpox rashes are indicated with more dots than modified and "mild" smallpox, suggesting potentially greater infectiousness. Hemorrhagic smallpox is indicated by horizontal line shading. Further details are provided in Table 6. Network structure We simulate the transmission of smallpox on a "small-worlds" network (highly clustered, but with short characteristic path lengths) [40]. Specifically, we assume that each person is located in a single household, and that the transmission rates were greatest in the household. We also assume that a fraction of the population are grouped into workplace or social groups, in which transmission may also occur, but with a lower rate per unit time than for household contacts. Finally, we assume that with a still smaller probability, any individual may transmit infection to any other individual in the population (casual contacts). In general, in a network-structured model, the number of secondary cases caused by an index case in a completely susceptible population is not a useful index of epidemic potential [41,42] (for a simple example, see [43]), since (for instance) an individual could infect everyone in his or her household, and not cause a widespread epidemic unless between-household transmission were sufficiently frequent. Rather than constructing the appropriate generalized basic reproduction number for our model (leading to highly cumbersome expressions), we chose an alternative (ad hoc) index of epidemic potential. For any given scenario of interest, we simulated the introduction of 10 index cases at random into a population of size 10000, and operationally defined "containment" to occur whenever the final size of the epidemic was less than 500 cases within 250 days (we showed, in the discussion of Figure 5A below, that in nearly all cases, the 250-day window differs very little from a 1000-day window). Because we simulate a disease with a finite duration on a finite and non-renewing population, epidemic extinction always occurs in finite time. Figure 5 5A - The mean containment probability increases as the number of ring vaccinations per day is increased. For this figure, the 1000 "calibrated" parameter sets were chosen, and for each parameter set, 100 realizations were simulated and the fraction of these for which the epidemic was contained to fewer than 500 cases was determined. The average of these 1000 containment fractions is plotted on the vertical axis. We assumed a household contact finding probability of 95% and that the diagnosis rates double after community awareness of the epidemic. We considered high levels of workplace/social (w/s) contact finding (0.9), as well as moderate levels (0.8). We also considered two levels of diagnosis of smallpox among investigated (alerted) contacts: high levels (corresponding to a 3 hour mean delay, indicated by "high contact isolation"), and moderate levels (corresponding to a one day delay, and indicated by "less contact isolation"). The figure shows four such conditions, a. high workplace/social contact finding probability and high contact isolation, b. moderate workplace/social contact finding probability and high contact isolation, c. high workplace/social contact finding probability and less contact isolation, and d. moderate workplace/social contact finding probability and less contact isolation. All other parameter values were chosen from the uncertainty analysis (the 1000 "calibrated" parameter sets). In this figure, "contact isolation" refers to the monitored diagnosis rate, i.e. the rate at which previously asymptomatic contacts who subsequently develop disease will be diagnosed (φ, Table 1, Table 8). 5B - The minimum containment probability out of the same 1000 scenarios chosen in Figure 5A. Whereas in Figure 5A, we averaged the simulated containment frequency (out of 100 realizations for each scenario), in this figure we determined which of the 1000 scenarios led to the lowest containment frequency, and we plotted this single worst (out of 1000) containment frequency, at different levels of ring vaccination capacity, for the same four conditions as in Figure 5A: a. high workplace/social contact finding probability (0.9) and high contact isolation (effective 3 hour delay following symptoms), b. moderate workplace/social contact finding probability (0.8) and high contact isolation, c. high workplace/social contact finding probability (0.9) and less contact isolation (effective one day delay), and d. moderate workplace/social contact finding probability (0.8) and less contact isolation. All parameters are the same as in Figure 5A (the household contact finding probability is 0.95 for all scenarios, and the diagnosis rates are doubled after the onset of community awareness). In this figure, "contact isolation" refers to the monitored diagnosis rate, i.e. the rate at which previously asymptomatic contacts who subsequently develop disease will be diagnosed (φ, Table 1, Table 8). Medical and public health intervention We assume that even in the absence of specific case investigations, the presence of smallpox symptoms will prompt patients to be diagnosed; we assume, however, a higher diagnosis rate for all forms of ordinary smallpox than for the severe flat and hemorrhagic forms, or for the mildest form. We assume that once an individual is diagnosed, their household and workplace contacts are investigated and detected with some probability; we assume that a high fraction (such as 95%) of household contacts are assumed to be traceable (see below). We assume that the fraction of workplace/social contacts that are traceable is less than the fraction of household contacts that are traceable; we assume that no casual contacts are traceable. High contact-finding rates may be plausible; we examined San Francisco Department of Public Health records of contact investigations for meningococcal disease (like smallpox, a potentially fatal disease for which rapid intervention may prevent mortality and morbidity). Records were available from December 2001 to April 2002; 13 such investigations during this period resulted in identification of 62 household contacts, all of which were contacted; out of 38 workplace/social contacts identified, 32 were contacted (84%). In our model, we assume that identified asymptomatic contacts are vaccinated, quarantined, and monitored for symptom development, while symptomatic patients are isolated and treated as necessary [9]; thus, the modeled interventions include more than ring vaccination alone. Finally, we include the possibility that all contacts (of both symptomatic and asymptomatic) traced and the same procedure applied, so that all contacts of contacts would be investigated. We assume that uninfected or asymptomatic individuals who are visited or traced individuals will be diagnosed more rapidly than if they had not been traced; in fact, such individuals would be isolated and would not be able to continue a chain of transmission. We follow previous models [9] in assuming a limited vaccination capability of Kr per day for ring vaccination. We assumed one of two strategies for contact tracing: (1) tracing only of direct contacts of diagnosed cases, and (2) tracing of contacts of contacts of diagnosed cases as well as direct contacts. The contact structure of the network is illustrated in Figure 2. Observe that individuals b and c are household contacts of individual a, so that if individual a were a smallpox case, an attempt would be made to find and vaccinate individuals b and c as household contacts of a case. If individuals a and b were both cases, then two attempts could be made to find individual c. We have modeled the effect of multiple contact-finding attempts conservatively in the sense that if the first attempt to find an individual as a household contact (of a case or of a contact) is determined to fail, no further attempts will be made. This maintains the failure rate of contact tracing (looked at from the standpoint of finding individuals) even in large households. Similar considerations apply to workplace/social groups. Figure 2 Network structure shown for households (joined by thick lines) of size 3 and workplace/social groups of size 4 (joined by thin lines); a small portion of the network is shown. Individual a has two household contacts (b and c), and three workplace/social contacts (d, e, and f). If individual a were a smallpox case, the household contacts would be at highest risk for acquiring smallpox, followed by workplace/social contacts; all individuals in the population are at a low risk of casual transmission from individual a. Case investigation of individual a would identify the direct contacts b-f with probabilities that depend on whether the contact is household or workplace/social; if such individuals are identified, they will be vaccinated. If contacts of contacts are being traced, the investigation will subsequently identify individuals g-p. Analysis We analyzed the model in three ways. First, we selected a Latin Hypercube sample [44-46] of parameters chosen uniformly from the parameter ranges given in Appendix 2 [see additional file 2], and simulated the transmission and control of smallpox to determine which parameters were most important for contact tracing and ring vaccination to be effective. Second, we used the same Latin Hypercube Sample of input parameters, but assumed that all disease control efforts were inactive. We used these parameters to simulate smallpox transmission, but then iteratively selected transmission parameters so that (1) between 1% and 10% of new infections resulted from casual (random) transmission, and (2) each index case resulted in between two and five secondary cases (thought to be plausible for historic smallpox; [8] suggest three secondary cases). For each of the resulting smallpox parameter sets using 100 stochastic simulations per set, we determined the daily ring vaccination/case tracking capacity needed to contain all simulated smallpox epidemics (i.e., keep the total number of cases below 500 within 250 days). Third and finally, we chose parameter values to yield an moderately large smallpox epidemic (with each index case causing approximately six secondary cases), and present illustrative scenarios for ring vaccination. These scenarios are intended to complement the simulations which were calibrated to historic smallpox, since the characteristics of smallpox that may be used in a deliberate release are not known. It is important to realize that in our model, the case finding time determines the fraction of contacts that will become infected, and that our model parameters have been chosen to yield quite rapid transmission to close contacts; in reality, much transmission of natural smallpox occurred through "sickbed" routes which would not occur in a modern setting [47], so that in this sense our model errs considerably on the side of caution and pessimism. Results Most important parameters (sensitivity analysis) To determine which of the input parameters were most important in determining the total number of smallpox cases, we selected a Latin Hypercube sample of size 1000 from the input parameter ranges indicated in Appendix 2 [see additional file 2] and simulated the mean number of cases within 250 days in a population of 10000. We then computed the partial rank correlation coefficient [46] (PRCC; see Appendix 2 [see additional file 2]) between each input parameter and the number of smallpox cases; when the PRCC is close to zero, the value of the parameter has little relation to the simulation output; when the PRCC is close to +1.0 or -1.0, the value of the parameter is highly important in determining the simulation output. Neglecting the number of index cases (which is directly related to the number of new cases), those parameters whose PRCC exceeds 0.1 are shown in Table 2. Most of these parameters identified as important are related to the density of available contacts (mean household size, prior vaccination fraction, and protection due to prior vaccination) or the transmission rate and infectivity (including the length of the pre-eruptive infectious period (stage 5 in Figure 2)). Note, however, that the speed of ring vaccination (household tracing delay) and faster diagnosis due to awareness of the outbreak are important parameters. Additionally, the infectivity of mild cases appears as an important parameter as well. Table 2 Most important parameters. PRCC: partial rank correlation coefficient (see Appendix 2 [see additional file 2] for definition and references). Parameter PRCC Mean Household Size 0.575 Transmission Rate from Close Contacts 0.520 Infectivity prior to rash 0.309 Ring Vaccination Capacity -0.296 Casual Transmission Probability 0.244 Pre-eruptive infectious period (lower bound) 0.224 Number of Casual Contacts per Day 0.210 Relative Infectiousness of Social/Workplace Contacts 0.200 Fraction of Individuals in Social/Workplace Groups 0.183 Faster Diagnosis due to Awareness of Outbreak -0.175 Household Tracing Delay 0.104 Pre-eruptive Diagnosis Probability -0.103 Diagnosis Probability after Rash -0.103 Illustrative scenarios To explore factors which contribute to the success of ring vaccination, we chose smallpox scenarios which resulted in severe and fast-moving epidemics in the absence of disease control; these simulated epidemics are considerably more severe than is believed likely under present circumstances. Effect of contact tracing and ring vaccination We used these parameters to simulate smallpox epidemics beginning with 10 cases, for a variety of levels of ring vaccination capacity per day (contact tracing capacity per day), as shown in Figure 3A. In this Figure, we assume that the population size is 10000, and that the epidemic began with 10 infected individuals. The mean household size is assumed to be 4, the mean size of the workplace/social contact group is 8, and contacts of contacts are traced. We assume that each day, the number of contacts that can be traced and vaccinated as a result of case investigation is 0, 10, 20, 30 and 40 per day; the probability of finding a workplace/social contact is assumed to be 80%. The Figure shows the average number of infected individuals each day (based on 100 stochastic simulations) for each of these scenarios. Selected parameter values are indicated in the caption for Figure 3A and in Table 1. Figure 3 3A - Expanding severe smallpox epidemic beginning with 10 initial cases, assuming 0, 10, 20, 30, and 40 possible ring vaccinations per day. The household size is 4 and the workplace/social group size is 8; we assume 95% of household contacts are traceable (with a mean delay of 1 day) and 80% of workplace/social contacts are traceable (with a mean delay of 2 days). We also assume that 25% of the population have 50% protection from infection resulting from vaccination prior to the discontinuation of routine vaccination. We assume that infection will be transmitted to close contacts with a mean time of 0.2 days, and that each person while infective causes on average 0.15 casual (untraceable) infections per day. We assume that individuals are 20% as infectious in the day just before the appearance of the rash as they will be during the first week of the rash, and that individuals are 20% as infectious as this (4% as infectious as during the first week of the rash) during the prodromal period. We assume that diagnosis rates will increase by a factor of 50% after smallpox becomes known to the community; we assume that each individual contacted during an investigation has a additional diagnosis or removal rate of 0.75 per day following the onset of symptoms (reflecting enhanced surveillance or contact isolation). Important parameters are summarized in Table 1; the full set of parameter choices is outlined in Tables 8-11 in Appendix 2 [see additional file 2]. Diagnosis times are discussed in Appendix 2 [see additional file 2]. 3B - An expanding severe smallpox epidemic under inadequate ring vaccination is shown for parameters identical to Figure 3A, except that workplace/social group sizes are 12 (instead of 8), and the probability of tracing workplace/social contacts is 0.6 (instead of 0.8). 3C - A severe smallpox epidemic is controlled by ring vaccination despite the large number of initial cases. The parameters are identical to Figure 3A, except that 1000 index cases inaugurate the attack in these scenarios (and ring vaccination capacity is much greater, as indicated). While not recommended, ring vaccination may ultimately halt epidemics beginning with many index cases if sufficient vaccination capacity were available, contact finding feasible, and follow-up sufficient. 3D - Tracing contacts of contacts (red) is beneficial when sufficient contact tracing/ring vaccination capacity exists (dotted lines). In these scenarios, all parameters are the same as in Figure 3A; the number of contact tracings possible per day is either 20 or 40 per day. Contacts of contacts are traced in two scenarios; in the other two, only direct contacts of cases are traced. For low levels of ring vaccination (20 per day), tracing contacts of contacts is harmful; for high levels (40 per day) of ring vaccination, it is beneficial to trace contacts of contacts. When the contact tracing/ring vaccination capacity is too small to adequately cover contacts of the cases themselves, diversion of resources to contacts of contacts is harmful; however, provided that sufficient capacity exists, tracing contacts of contacts helps outrun the chain of transmission. Each line corresponds to the average of 100 realizations. Table 1 Selected parameter values for Figure 3A and other illustrative scenarios. The notations "Other" or "Other factors" in the column "See also" refers to the text section "Other factors". The symbols are defined in Appendix 2 [see additional file 2] and are included for reference. Description Values See also Symbol Number of index cases 10–1000 Figure 3C A Mean household size 4 H Workplace/social group size 8 Figure 3B W Ring vaccinations per day 0–200 Fig. 3A, 3B, Other Kr Monitored diagnosis rate 1–8 day-1 Figure 5A, 5B φ Prob. of finding household contact 0.95 Table 4 υ1 Prob. of finding workplace/social contact 0.8 Fig. 3B, 4AB; Tb. 4 υ2 Delay, tracing household contacts 1–5 days Figure 6 δ1 Delay, tracing workplace/social contacts 2–10 days Figure 6 δ2 Relative diagnosis rate after 1st diagnosed case 1.5 Figure 7 a1 Infectivity, stage 4 relative to stage 5 0.2 Figure 8 k Infectivity, stage 5 relative to stage 6 0.2 Figure 8 k' Infection hazard for close contacts 5 day-1 Table 3 λ Relative hazard for workplace/social contacts 1/3 Table 3 h2 Casual transmission rate 0.15 day-1 β Prior vaccination fraction 0.25 Other factors f Fraction of mild cases 0.03 Other factors Vaccine success rate (for very recent vaccination) 0.667 Other factors α1 Vaccine success rate (vaccination prior to discontinuation of routine vaccination) 0.5 Other factors α2 Vaccine success rate full protection 0.999 Other factors α3 Because we assumed nonzero diagnosis probabilities during the prodromal period for all individuals in Figure 3A, we repeated the simulation assuming no diagnosis in the prodromal period unless individuals were under specific surveillance. The results were nearly identical: assuming 30 contact tracings (ring vaccinations) per day, we found 26% of the scenarios in Figure 3A exhibited decontainment, and 28% assuming no diagnosis during the prodromal period; assuming 40 contact tracings per day, we found 1 out of 100 scenarios showed loss of containment in Figure 3A and when we repeated the scenario of Figure 3A assuming no diagnosis during the prodromal period. In Figure 3B, we illustrate control of an epidemic for which all parameters are identical to Figure 3A, except that the workplace/social group size is 12 (instead of 8, as in Figure 3A), and the probability of finding workplace/social contacts is 60% (instead of 80%, as in Figure 3A). In this case, the larger size of the workplace/social groups and the lower contact finding probability makes it necessary to have a higher ring vaccination capacity to attain a high probability of containing the epidemic, and on average it takes longer for eradication to finally occur. Finally, in Figure 3C, we show control of an epidemic in a population of 100,000, beginning with 1000 initial infectives, keeping all other parameters the same as in Figure 3A. Each curve corresponds to the indicated number of possible ring vaccinations per day. This figure shows that assuming sufficient capacity, ring vaccination is in principle capable of containing even epidemics beginning with very many infected individuals. However, mass vaccination in such cases is justified because of the far larger number of individuals at risk and the inability to perform such extensive contact tracing. In Figure 3D, we compare the effect of tracing contacts of contacts (as described in Appendix 2 [see additional file 2]) at different levels of ring vaccination capacity. Thin lines in red correspond to the presence of tracing contacts of contacts; thick lines in black correspond to tracing direct contacts of cases only. Each simulation was performed 100 times, with 10 initial infectives, and for 20 and 40 ring vaccinations possible per day (as indicated). The average number infected on each day is plotted in the Figure. The figure illustrates that when ring vaccination capacity is low, tracing contacts of contacts (as modeled) yields a more severe average epidemic; when ring vaccination capacity is large, tracing contacts of contacts results in a less severe average epidemic; if the contact tracing/ring vaccination capacity is too low to cover adequately the contacts of contacts in addition to the contacts of cases, extension of tracing to the contacts of contacts (the second ring) is harmful; however, if there is sufficient capacity to cover the contacts of contacts, then the tracing of contacts of contacts is beneficial. Finally, in Figure 4, we illustrate the considerable variability that may be seen from simulation to simulation. This figure shows twenty simulations when contacts of contacts are not traced. Stochastic variability between realizations is considerable, even when all parameters are held constant; this variability is expected to limit the ability to make inferences based on observation of a single realization of the process. Figure 4 Stochastic variability is illustrated by plotting the number of infectives over time over multiple replications. In this example, most simulations exhibit rapid containment of smallpox. The mean number of cases (averaging over simulations) is influenced by a small number of simulations exhibiting an uncontained epidemic. The parameters are the same as in Figure 3A, except that contacts of contacts are not traced in these replications. Because our baseline hazard for infection of individuals may be larger than would be expected for naturally occurring smallpox, we examined the effect of more realistic values of this hazard. In particular, we chose different levels of ring vaccination capacity (10, and 20), and of the relative hazard for workplace/social contacts, and then chose values of the baseline hazard for infection varying from 0.5 per day (for a mean time to infection of 2 days) to 2 per day (for a mean time to infection of one half day), and introduced 10 index cases into a population of 10000. We then repeated this 100 times, and reported the fraction of scenarios in which the number of infections ultimately exceeded 500 (as before, chosen as a cutoff to indicate the ultimate "escape" of containment of the epidemic). These results, shown in Table 3, support the idea that ring vaccination can easily control introduced smallpox provided there is sufficient capacity and efficacy of tracing. Table 3 Estimated decontainment probability for different levels of ring vaccination capacity (Kr) and relative hazard for infection due to workplace/social contacts (h2), for different levels of the baseline hazard for infection from household contacts λ (based on replications of 100 simulations for each level). For each scenario, 10 index cases were introduced into a population of size 10000. All other parameters were the same as for Figure 3A. As before, we define decontainment to mean that the total number of cases from 10 index cases eventually exceeded 500 by day 250. Relative hazard for workplace or social contacts Ring vaccinations per day 10 20 λ λ 1/3 0.5 0 0.5 0 0.75 0.02 0.75 0 1 0.26 1 0 1.25 0.73 1.25 0 1.5 0.96 1.5 0.02 2 1 2 0.16 λ λ 2/3 0.5 0 0.5 0 0.75 0.46 0.75 0 1 0.82 1 0.02 1.25 1 1.25 0.11 1.5 1 1.5 0.26 2 1 2 0.49 λ λ 1 0.5 0.14 0.5 0 0.75 0.86 0.75 0 1 0.99 1 0.07 1.25 1 1.25 0.22 1.5 1 1.5 0.49 2 1 2 0.85 Because of considerable uncertainty in the model parameters, we chose a collection of parameter values, and for each, estimated the containment probability (operationally defined as fewer than 500 total cases as a result of 10 index cases, within 250 days). We estimated this containment probability by simulating the smallpox epidemic 100 times for the same parameter values, and computing the frequency out of these 100 realizations for which fewer than 500 index cases resulted within 250 days. (Using a 1000 day window produces slightly smaller containment estimates; for 3 out of 1000 parameter set choices, this difference was greater than 0.06; the maximum difference seen was 0.23; the mean absolute difference was 0.0029; in only one case out of 1000 did we see containment in all 100 cases for the 250-day window, but not in all 100 cases for the 1000-day window). One thousand scenarios chosen from a Latin Hypercube sample were analyzed, and as indicated before, we chose the hazard for close contact transmission and the hazard for random transmission to guarantee that between 2 and 5 secondary cases per case occur, and that no more than 5% of cases are attributable to random transmission (we refer to this set as the "calibrated" scenarios further in this text). Having chosen this collection of 1000 parameter sets, we considered two levels of two different control parameters which were applied to each (so that each of the 1000 parameter sets were simulated under four different control conditions). The first of the two control parameters was the probability of workplace/social group contact finding; we chose values of 0.8 and 0.9 for this parameter (the household contact finding probability was 0.95 in all cases). The second of the control parameters was the rate of diagnosis (and effective removal) from the community of cases developing among previously identified and traced contacts who were initially asymptomatic (we refer to this as the monitored diagnosis rate); we assumed first a low level corresponding to a mean diagnosis time of one day from the onset of symptoms, and a high level corresponding to a mean time of 3 hours from the onset of symptoms (high levels of the monitored diagnosis rate correspond effectively to isolation of contacts). Finally, we assumed a doubling of the diagnosis rate after the beginning of widespread community awareness of smallpox. We then computed the containment fraction at different levels of ring vaccination capacity (contact tracing capacity per day). Thus, for each of 1000 scenarios (parameter set choices), we assigned the workplace/social group contact tracing success probability (υ2), the monitored diagnosis rate φ (Appendix 2 [see additional file 2]), and the contact tracing/ring vaccination capacity per day (Kr). We then performed 100 realizations beginning with 10 index cases, and computed the containment fraction (fraction showing fewer than 500 cases in 250 days, beginning with 10 index cases). Thus, for each of the two choices each of υ2 and φ, and for each value of Kr we examined, we obtained 1000 values of the containment fraction. We use the resulting distributions in Figure 5A (averaging over these 1000 containment fractions), and Figure 5B (displaying the minimum value of the 1000 containment fractions). In Figure 5A, we plot the mean containment fraction (averaging the containment fraction over all 1000 scenarios), as ring vaccination capacity varies, for the two levels of workplace/social group contact finding probabilities (0.8 and 0.9), and for the two levels of monitored diagnosis rate among initially asymptomatic contacts (1 day-1 and 8 day-1). For low levels of ring vaccination (traceable contacts per day), the epidemic is almost never contained, but for ring vaccination levels near 50–60 per day (5–6 per index case per day), the average containment fraction became close to 1. However, this average conceals the fact that for some scenarios (parameter sets chosen from the calibrated uncertainty analysis), control remains difficult or impossible even at high levels of ring vaccination. Therefore, in Figure 5B, we plotted the single lowest containment fraction seen out of the 1000 computed; focusing on the single worst scenarios reveals a different picture, and shows that isolation of asymptomatic contacts and very high probabilities of finding workplace or social contacts would be needed to control smallpox under these most pessimistic parameter choices. Effect of contact tracing speed Rapid contact tracing in ring vaccination may play an important role in suppressing the epidemic, since the longer it takes to trace a contact, the less likely the vaccine is to be efficacious, and the more opportunities the infected individual may have to transmit disease before they are finally located, isolated, and vaccinated if appropriate. We illustrate this possibility in Figure 6 by examining the same scenario we showed earlier in Figure 3A (e.g. households of size 4, workplace/social groups of size 8, 95% of household contacts traceable, 80% of workplace/social groups traceable, an average time to infection for a household contact of an infective given by 0.2 days). We assume in one case that contacts may be traced quickly (1 day for a household contact, 2 days for a workplace/social contact), and in the other that the contacts are on average found slowly (5 days for a household contact, 10 days for workplace/social contacts); we assumed 30 ring vaccinations (traceable contacts) possible per day. In this scenario, the epidemic is more severe and containment (as we have been defining it) less likely when contact tracing is slow: in the fast scenario, 238 infections occurred on average and the (estimated) containment probability was 99%; for the slow scenario, on average 3587 infections occurred and the (estimated) containment probability was only 1%. Figure 6 Faster contact tracing may improve the efficacy of ring vaccination. We assume the same baseline parameters as in Figure 3A (e.g. households of size 4, workplace/social groups of size 8, 95% of household contacts traceable, 80% of workplace/social contacts traceable), and 30 ring vaccinations available per day (with contacts of contacts not traced). The fast scenario corresponds to an average one day delay for household and two days for workplace/social contacts (as in Figure 3A); the slow scenario corresponds to an average five day delay for household and ten day delay for workplace/social contacts. This figure shows the average of one hundred realizations starting with ten index cases. While Figure 6 illustrates the possibility that rapid contact tracing may be of decisive importance in some scenarios (parameter set choices), this is not always the case. For some parameter sets, the probability of tracing contacts (household or workplace/social) may be too low, or the transmission rate too high, for more rapid contact tracing to make any difference. Conversely, for other parameter sets, the smallpox transmission rate may be so low that smallpox is easily contained even with slow contact tracing. While rapid contact tracing is never harmful, overall, how typical are the results of Figure 6 (in which rapid contact tracing was important in ensuring the efficacy of ring vaccination)? To address this question, we simulated the growth of smallpox for the 1000 "calibrated" scenarios we used in Figure 5A and 5B. As before, we assumed ten initial cases, and (as in Figure 6) that 30 ring vaccinations were possible per day; then we simulated 100 epidemics assuming one day to find a household contact (and 2 days to find a workplace/social contact). We then simulated 100 epidemics assuming that it takes five days to find a household contact and 10 days to find a workplace/social contact (as in Figure 6). For each of these 1000 scenarios, we calculated the fraction of simulations for which the total number of cases is 500 or less within 250 days, i.e. the containment fraction. For nearly all scenarios (parameter set choices), the containment fraction was smaller (sometimes much smaller) when the contact finding time is faster (since faster contact finding, all else being equal, improves smallpox control, as illustrated in Figure 6). However, for 64.5% of the scenarios (parameter set choices) examined, the difference was less than 2.5% in absolute terms (smallpox was either contained or not contained depending on other factors, and rapid contact tracing did not make the difference). On the other hand, for 18.7% of the scenarios examined, the absolute difference in the containment probability was 20% or more; thus, a substantial difference in containment probability is occasionally attributable to the difference between fast and slow contact tracing. Effect of more rapid diagnosis Public awareness of smallpox, leading to more rapid isolation and identification, may play an important role in eliminating the epidemic, as illustrated by the scenarios in Figure 7. We assumed 20 ring vaccinations possible per day, a capacity too small to contain the epidemic in the absence of increased surveillance or diagnosis; the black line in the figure shows the steeply rising average number of cases for the first 100 days. If, however, surveillance or public awareness of the symptoms of smallpox increases the diagnosis rate by 50% (multiplies the baseline diagnosis rates by 1.5), containment becomes possible (blue line); with a doubling of the diagnosis rate (red line) the peak number of cases is lower still. In these scenarios, increased diagnostic rates markedly improve the ability of ring vaccination to control the epidemic, this suggest that any ring vaccination effort be accompanied by increased public awareness and surveillance. Figure 7 More rapid diagnosis due to public awareness or increased surveillance may lead to far more effective epidemic control. We assume the same baseline parameters as in Figure 3A, and averaged 100 realizations of the epidemic beginning with 10 index cases and assumed a ring vaccination capacity of 20 per day (and contacts of contacts not traced). For the black line, the diagnosis rate of cases does not change after the first case is identified (the multiplier is 1.0); for the blue line, the diagnosis rate increases by 50% (multiplier 1.5) after the first case is identified (as in Figure 3A), resulting in substantially fewer cases; and for the red line, the diagnosis rate is doubled (multiplier 2.0) after the first case is identified, resulting in still fewer cases. In many cases, however, more rapid diagnosis was not required for ring vaccination to be effective. As before, we simulated smallpox epidemics for each of 1000 calibrated scenarios, performing 100 realizations each beginning with 10 index cases, and computed the fraction of scenarios for which the epidemic was always contained (as defined earlier), assuming no change in diagnosis rates. We assumed 80 ring vaccinators per day, contact finding probabilities of 0.95 for households and 0.8 for workplace/social contacts (as in Figure 3A). Under these assumptions, for 83.4% of the scenarios, the epidemic was contained within 500 total cases in each of the 100 realizations, even with no change in diagnosis rates. Uncertainty analysis (using the 1000 calibrated scenarios, and based on the fraction of 100 replications showing decontainment) revealed the most important parameters which predict the failure of ring vaccination without more rapid diagnosis were the same as we found in the earlier uncertainty analysis; a higher fraction vaccinated before the epidemic, smaller households or workplace/social groups, less transmissibility, lowered infectivity prior to the rash, more rapid diagnosis, and a higher rate of diagnosis for alerted individuals all contribute to a greater containment probability even without an overall increase in the diagnosis rate. Effect of continued surveillance of contacts We have been assuming that whenever an individual is contacted during an investigation, the individual will be diagnosed more quickly should they subsequently develop symptoms. When transmission is assumed to be very rapid (smallpox is assumed to be highly contagious), most individuals may already be infected when identified through contact tracing from an infective. Using the scenario we examined in Figure 3A, we see that continued surveillance of contacts is an essential component of effective ring vaccination designed to control rapidly spreading smallpox: if smallpox in a contact is not diagnosed any more quickly than for a non-contact, containment by ring vaccination requires over 98% contact finding probabilities for both household and workplace/social contacts – even if unlimited numbers of ring vaccinators are available; containment cannot be guaranteed by adding additional ring vaccination capacity if the contact finding rates are too low and/or the follow-up for contacts is insufficient. Smallpox which is transmitted less rapidly to contacts would, however, be containable with a lower contact finding probability (results not shown). Finally, we used the "calibrated" scenarios (parameter set choices) to explore the levels of contact finding probability needed to contain the epidemic (as before, defined to mean 500 or fewer cases ultimately resulting from ten initial cases) (Table 4). In these scenarios, we assumed that all traceable contacts were followed up very quickly (1/a = 1 hour, so that cases arising in previously contacted persons almost never transmit the infection further). We chose different levels of household and workplace/social contact finding probabilities and different levels of ring vaccination capacity, and performed 100 replications of each of the 1000 different scenarios. In Table 4 we report the fraction of scenarios for which all 100 replications exhibited containment. Scenarios in which smallpox is highly contagious require high contact finding probability to ensure the containment of the epidemic. Table 4 Containment of severe smallpox at different levels of contact finding. The first three columns are assumed levels for the probability of finding a household contact, the probability of finding a workplace/social (W/S) contact, and for the number of contact tracings/ring vaccinations possible per day; the last two columns express (as percentages) the resulting probability of containment given the assumed contact finding probabilities and contact tracing capacities; two containment probabilities are given: the containment probability when only contacts of cases are traced (first column, "Contacts"), and the containment probability when contacts of contacts of cases are traced in addition to the contacts of cases (second column, "Contacts of Contacts"). All other parameters are the same as in Figure 3A. Probability of finding Number of Ring vacc. per day Containment Contacts Probability when Tracing Contacts of contacts Household contacts W/S contacts 0.95 0.85 50 99.1% 97.9% 0.95 0.85 100 99.3% 100.0% 0.95 0.85 200 99.1% 100.0% 0.9 0.8 50 95.7% 95.8% 0.9 0.8 100 95.6% 99.9% 0.9 0.8 200 95.4% 100.0% 0.85 0.75 50 86.0% 93.3% 0.85 0.75 100 86.1% 99.1% 0.85 0.75 200 86.3% 99.2% 0.75 0.6 50 52.1% 72.0% 0.75 0.6 100 51.5% 78.5% 0.75 0.6 200 53.0% 78.6% Transmission prior to rash Transmission prior to the rash makes epidemic control more difficult. In Figure 8, we show an expanding smallpox epidemic assuming differing levels of infectivity prior to the rash (adding increased infectivity prior to the rash, keeping constant the infectivity after the rash). We assume all parameters are the same as in Figure 3A (and that the ring vaccination capacity is 40 per day). Infectivity prior to the rash is modeled as the relative infectivity during the short (1 day) period of oropharyngeal lesions just prior to the rash (compared to the infectivity during the first week of the rash), and as the relative infectivity during the prodromal period (relative to the period just prior to the rash). We consider three scenarios: a relative infectivity during entire period is one (i.e., infectivity during the prodromal period and just prior to the rash is the same as during the first week of the rash), b the relative infectivity just prior to the rash is the same as during the first week of the rash, but during the prodromal period is 4% (as in Figure 3A) of this value, and c the relative infectivity just prior to the rash is 20% of the infectivity during the first week of the rash, and during the prodromal period is 20% of this value. The figure shows that increased infectivity just prior to the rash leads to a larger epidemic (comparing b and c); in case b (high infectivity just prior to onset of rash), loss of containment occurs 36% of the time (but in none of the 100 realizations shown in case c (low infectivity prior to rash)). Scenario a (full infectivity during entire the prodromal period) showed loss of control in every realization. Increasing the ring vaccination capacity from 40 per day to 80 per day (results not shown) led to containment in all of the realizations with high infectivity just prior to the rash and low infectivity during the prodromal period (case b), but made no difference if the infectivity was as high during the prodromal period as during the rash (case a). While intuitively adding additional infectiousness must increase the number of secondary cases and make control more difficult, these results do illustrate that even a small amount of increased infectiousness prior to the rash (when diagnosis is more difficult) may substantially increase the difficulty of smallpox control. Figure 8 Transmission prior to the rash makes epidemic control more difficult. The figure shows a expanding smallpox epidemic assuming differing levels of infectivity prior to the rash. We assume all parameters are the same as in Figure 3A (and that the ring vaccination capacity is 40 per day). Infectivity prior to the rash is modeled as the relative infectivity during the short (1 day) period of oropharyngeal lesions just prior to the rash (compared to the infectivity during the first week of the rash), and as the relative infectivity during the prodromal period (relative to the period just prior to the rash). For scenario a, relative infectivity during the prodromal period and just prior to the rash is the same as during the first week of the rash, for scenario b, the relative infectivity just prior to the rash is the same as during the first week of the rash, but during the prodromal period is 4% (as in Figure 3A) of this value, and for scenario c, the relative infectivity just prior to the rash is 20% of the infectivity during the first week of the rash, and during the prodromal period is 20% of this value (these two parameters are the same as in Figure 3A). Other factors Finally, in Figure 9, we present scenarios in which each of four other parameters are modified from the baseline values of Figure 3A, assuming 40 contact tracings (ring vaccinations) are possible per day (line a in the figure). Specifically, we assume that severe smallpox (hemorrhagic and flat) on average takes four times longer to diagnose and isolate than ordinary smallpox (case b), that no one in the population has prior vaccination protection (from before the discontinuation of routine vaccination, case c), that 10% more smallpox is too mild to diagnose (but still contagious, case d) compared to baseline, and finally that the vaccine is completely ineffective (case e). Each of these scenarios will be discussed further below. Figure 9 Additional scenarios, assuming 40 ring vaccinations or contact tracings possible per day, and that contacts of contacts are traced; all parameters are identical to those in Figure 3A unless otherwise indicated. The figure shows the average of 100 replications of five scenarios (Case a repeats the result from Figure 3A for reference); the numbers in parentheses in the legend are the corresponding fraction of the 100 scenarios for which decontainment occurred. For case b, we assumed that flat and hemorrhagic smallpox cases took four times as long on average to diagnose as ordinary cases; for case c., we assumed that no one in the population had prior protection (as opposed to 25% for Figure 3A); for case d, we assumed that an additional 10% of individuals (13% instead of 3%) would develop mild smallpox (with 75% developing ordinary smallpox instead of 85% as in Figure 3A); and for case e, we assumed that the vaccine is completely ineffective and provides no protection against infection. Scenario b was motivated by the possibility that individuals with severe forms of smallpox may be more difficult to diagnose, and thus remain infectious in the community longer (despite the much greater degree of illness of such patients), or that such patients may be more infectious. In this particular case, quadrupling the mean diagnosis time led to one additional replication out of 100 in which containment was not achieved (2/100, compared to the baseline of 1/100). However, we assumed that community awareness of smallpox leads to the same relative rate of increased diagnosis among severe cases as for ordinary cases, and that the most severe forms are relatively rare. In addition to the scenario shown in the figure, we also replicated the same 1000 "calibrated" simulations, assuming that in each case 40 contact tracings per day are possible and that the diagnosis time for severe cases was four times that of ordinary cases. Finally, we repeated each "calibrated" scenario 100 times assuming long diagnosis times for severe cases, and not making this assumption, and found that the difference in the decontainment fraction was not large (results not shown). Scenario c illustrates that vaccination prior to the discontinuation of routine vaccination does play a role in smallpox control by ring vaccination; there were more decontainment scenarios (5/100) when no prior protection exists in the population. The results suggest that prior vaccination aids in the control of smallpox, but that it is not strictly necessary for control (in this scenario, 95% of the replications exhibited containment). In Figure 3A, we assumed 25% of individuals had protection due to vaccination prior to the discontinuation of routine vaccination; in scenario c of Figure 9, we assumed this fraction was zero. Scenario d demonstrates that if 10% more smallpox infections (in absolute terms, i.e. 13% compared to 3% in Figure 3A) lead to mild cases among individuals with no prior protection, the epidemic is more difficult to contain (13/100 replications showed loss of containment). Finally, scenario e demonstrates that containment is still possible even when the vaccine is completely ineffective in everyone – because of case isolation and isolation of contacts (and of contacts of contacts). Here, with 40 contact tracings possible per day, 55% of the replications nevertheless exhibited containment even with a vaccine which offered no protection whatever. With 90 contact tracings possible per day, all replications exhibited containment even assuming no vaccine protection. Effect of mass vaccination Although less efficient than ring vaccination in the sense that more vaccinations must be delivered to eliminate infection, comprehensive mass vaccination following the introduction of smallpox is sufficient to eliminate the infection. In Figure 10, we show the probability of achieving containment (defined to be fewer than 500 total cases resulting from 10 index cases) for different levels of ring vaccination (0, 5, 10, and 20 vaccinations per day) and mass vaccination (0, 0.5%, 1%, and 2%; compare with the 10%-20% per day many jurisdictions in the United States are planning to vaccinate). Specifically, for each level of ring vaccination and mass vaccination, we used the same 1000 parameter sets used in Figure 5, and performed 100 simulated epidemics for each parameter set. On the vertical axis, we plot the fraction of the 1000 scenarios for which each of the 100 simulated epidemics was contained. We further computed the fraction of scenarios for which none of the 100 simulated epidemics was contained; this is indicated by the colored segment in the small pie chart at each symbol. When the mass vaccination rate was 2% per day, the mean number of deaths (averaging over all scenarios and all simulations within each scenario) was 47.7, 33.7, 26.4, and 20.1 for a ring vaccination level of 0, 5, 10, and 20 per day (respectively) out of a population of 10000. Moreover, when we increased the mass vaccination level to 3%, an average of 28.9 deaths occurred when no ring vaccination was used, but this fell to 22.3 deaths when only 5 ring vaccinations per day were available (again assuming a population of 10000, and 10 index cases). With a mass vaccination level of 5% per day, an average of 18.8 deaths occurred without ring vaccination, and 15.8 deaths occurred when only 5 ring vaccinations per day were possible. (At a mass vaccination rate of 3% per day, containment as defined above was achieved in all 100 replications for 95% of the scenarios even without ring vaccination; at a mass vaccination rate of 5% per day, containment was achieved in all replications for all scenarios.) These results show that over a wide range of simulated epidemics, even seemingly small levels of ring vaccination (coupled with follow-up) may have a substantial effect in preventing epidemic spread and reducing deaths from smallpox, even during a mass vaccination campaign. Note that many jurisdictions in the United States are planning mass vaccination campaigns which could reach 10%-20% of the population per day, far greater than the mass vaccination levels examined here; it is interesting to note that mass vaccination campaigns may be effective in preventing a widespread epidemic even at much lower levels than are being planned for. Where feasible, such rapid mass vaccination rapidly eliminates smallpox transmission in our model; vaccination of contacts is still beneficial, since we are assuming that earlier vaccination yields a greater probability of preventing or ameliorating infection (results not shown). Figure 10 Mass and ring vaccination together. Low-level mass vaccination programs are improved substantially by the addition of ring vaccination. The shaded pie segments represent the fraction of 1000 scenarios for which containment (as defined in the text) was never realized; the vertical position of the pie chart represents the fraction of the 1000 "calibrated" scenarios for which containment was always achieved. As the fraction of the population mass vaccinated increases or the ring vaccination capacity increases, the probability of containment increases. Discussion We constructed a simple network model of smallpox transmission, and addressed the question of what circumstances contribute to the success of a ring vaccination campaign designed to control smallpox. Our analysis focused on the use of contact tracing/ring vaccination to prevent a widespread epidemic following a deliberate release. We conducted a sensitivity analysis based on particular, but reasonable, ranges for the unknown parameters. Our results are consistent with prior vaccination models in identifying prior vaccination and ring vaccination capacity as significant factors in determining the spread of smallpox. Unsurprisingly, we also find that household size and ring vaccination speed are particularly important parameters; these results are intuitively plausible. The contact finding probability did not appear important in this analysis only because a narrow range of values was chosen. We illustrated smallpox control by presenting scenarios based on control of moderately severe smallpox epidemics. We find that swift, aggressive contact tracing and ring vaccination is is usually sufficient to bring the infection under control. Provided that there is sufficient capacity, vaccination of contacts of contacts is beneficial, and results in fewer infected individuals and more rapid elimination of infection; investigating contacts of contacts allows the chain of transmission to be outrun to some extent. When ring vaccination capacity is small, diversion of crucial resources away from contacts is harmful; contacts of contacts should only be traced and vaccinated provided that no resources are diverted away from contacts of cases. The increased surveillance (or isolation) of contacts, together with improved rates of diagnosis due to community awareness, play important roles in smallpox control; we note that in some cases, lowered diagnosis rates among severe cases contributed to a small extent to loss of epidemic control, and suggest that any public awareness campaign include information to help the public be more aware of the full spectrum of the clinical features of smallpox. One limitation of our analysis is that we chose not to explicitly incorporate the specific epidemiology of health care workers (or mortuary workers), who are likely to be exposed to infected individuals during any smallpox epidemic (e.g. [17,22]), and who may then infect further members of the community [22] (as was also seen in the recent outbreak of SARS, e.g. [48]). Transmission to health care workers may be considered to amplify the initial attack or to be simply accounted among the exposures we considered (and thus be approximated by the behavior of our model), since health care workers and their household contacts are in all likelihood traceable contacts, and ring vaccination/contact tracing would identify and halt these chains of transmission as in our model. The disruption of smallpox control and patient care that may occur is not accounted for in our analysis, however, causing our model in this sense to err on the side of optimism. The appropriateness of pre-event vaccination of health care workers or other first responders has been addressed by other analyses [12,49], and is beyond the scope of our model. While we analyzed the effect of contact tracing, case and contact isolation, and ring vaccination (together with mass vaccination), in a real smallpox epidemic, in practice, control efforts are unlikely to be limited strictly to vaccinating contacts (and health care workers, as likely contacts) and isolating cases. Indeed, making vaccine available to individuals who believe they live near cases or to others on a voluntary basis occurred in smallpox control efforts in the past [22]. Vaccination of such individuals can only harm the disease control effort if it hinders or delays the diagnosis of cases or the investigation and vaccination of contacts; our results show that even relatively low levels of vaccination of the general population may have a beneficial effect in preventing the epidemic from escaping control. More serious is the possibility that individuals who should be vaccinated or isolated would be missed; this could occur either because individuals or institutions did not cooperate with the disease control effort, or because the individuals simply could not be found. Our analysis suggests that ring vaccination need not be perfect to successfully contain the epidemic, and yet, under conditions where there is a high rate of infection among contacts, or a relatively high rate of casual transmission, high rates of contact finding (in excess of 90%), together with increased surveillance and contact isolation, are needed to contain the epidemic. Finally, the vaccination of individuals at low risk of contracting smallpox will cause harm due to adverse events of the vaccine; in our model, the assumed death rate due to vaccination was small compared to the probability of death from smallpox, and played essentially no role in the analysis. In practice, individuals suspected to be at high-risk for vaccine complications, but at relatively low risk for contracting smallpox, might simply be isolated or closely monitored even during an outbreak; while the presence of individuals in the population at higher risk for vaccine complications would increase the death rate during an outbreak, such individuals are unlikely to impair the containment of the epidemic (the primary focus of this analysis). Our results support ring vaccination against epidemics of smallpox (even assuming high rates of transmission to close contacts), but do note that stochastically, for severe (rapidly transmissible) smallpox, scenarios of loss of control are seen, with resulting widespread epidemics. In scenarios in which the transmission potential of smallpox is smaller, such loss-of-control scenarios occur less frequently (results not shown). Mass vaccination campaigns, when conducted quickly and with very high coverage, do not result in loss of control in our model. Nevertheless, fewer deaths due to smallpox result when ring vaccination is conducted along with mass vaccination. Conclusion Simulated smallpox epidemics with ring vaccination suggest that aggressive, fast ring vaccination can control epidemics of smallpox. To do so, however, smallpox must be identified quickly and contacts vaccinated promptly. We also identify public awareness of smallpox – leading to prompt identification of cases – as a major factor in smallpox control; in some simulations, it may play a role as significant as ring vaccination itself [15]. However, we also found that uncertainty in (1) transmission from mild cases, (2) the household size, and (3) casual transmission contributed to the overall uncertainty in the epidemic size. Other parameters to which the number of infections were highly sensitive were the prior vaccination fraction, parameters related to infectiousness, and parameters related to transmission prior to the rash. Because our model combines network structure with response logistics, our results support and complement the results of other investigators. Our results support the notion that prior vaccine protection may play an important role in slowing the epidemic [11], despite the possibility that some vaccinated individuals may develop mild cases which are harder to identify, but which nevertheless transmit disease. Likewise, our results provide support for the view that ring vaccination should play a central part in smallpox control. If initiated, ring vaccination should be conducted without delays in vaccination, should include contacts of contacts (whenever there is sufficient capacity to cover all contacts of cases), and should be accompanied by a vigorous campaign of public awareness which can facilitate more rapid identification and isolation of cases. We assumed that ring vaccination could be fast (little delay between identification of a case and vaccination of the contacts), effective (nearly all household contacts can be found, and most of workplace and social contacts), and available (there is sufficient capacity). To be effective, ring vaccination planning must yield a system capable of meeting these benchmarks; we should not only be able to assess the number of contact vaccinations that will be possible per day, but should have a plan in place to (1) identify contacts by working with individuals, employers, schools, community representatives, and authorities or businesses who may have access to information facilitating contact tracing, (2) rapidly investigate and vaccinate such individuals, perhaps using field teams managed by central dispatch. It is important to realize that for high-risk, transient, or unstably housed populations where reliable contact tracing is impossible, the conclusions of the model we present cannot be applied. It is important to note that while our model suggests that ring vaccination together with contact tracing and isolation is likely to be successful, we found that for some scenarios (where smallpox was more transmissible, or was relatively more transmissible before the rash), epidemic containment required not only ring vaccination, but increased public awareness, the isolation of contacts, and tracing of contacts of contacts. For scenarios in which the smallpox was less transmissible, epidemic containment was possible at lower contact finding probabilities. Thus, while our simulations suggest that contact tracing/ring vaccination need not be perfect to succeed, because of uncertainties in our knowledge of the behavior of bioterrorist smallpox, it is impossible to know in advance how good it will have to be. Thus, that high contact finding rates, mass public awareness leading to early identification of cases, isolation of contacts, and investigation of contacts of contacts should all be conducted with maximum effectiveness to reduce the probability of a widespread epidemic. While the possibility of smallpox uncontrollable by ring vaccination has made mass vaccination preparations wise, and while mass vaccination may be unavoidable in the event of a deliberate release of smallpox, we believe that ring vaccination is essential in any case. This is not only because individuals recently exposed to smallpox may be protected if they are vaccinated promptly, but because each contact identified potentially lies in the immediate future of the transmission chain. From the standpoint of epidemic control, it is far more valuable to vaccinate individuals next in the transmission chain than to vaccinate other persons. Our results support the idea that ring vaccination/case isolation may in many, if not most cases, eliminate smallpox even without mass vaccination, but also support planning for mass vaccination (so that the vastly more costly and difficult policy of mass vaccination will be available in the event of an explosive epidemic). When faced with the unknown, multiple redundant preparations are appropriate; case investigation/isolation may control smallpox even if the vaccine does not work at all, but mass vaccination is useful in the event of an explosive epidemic for which case tracking becomes impossible. Competing interests None declared. Authors' contributions TP, KH, SF, TA, RR, and DP performed the literature review (and parameter evaluation), TP developed and implemented the model and simulation, TP performed the analysis of the simulation model and drafted the manuscript, DP performed analysis of contact tracing data, and KH conceived of the study. All authors contributed to, read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: Supplementary Material Additional file 1 For consistency, all references are included in the bibliography of the main text. Click here for file Additional file 2 For consistency, all references are included in the bibliography of the main text. Click here for file Acknowledgments TCP acknowledges the support of NIDA grant DA 5-R01-13510. 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BMC Bioinformatics. 2004 Sep 9; 5:128

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==== Front BMC Cell BiolBMC Cell Biology1471-2121BioMed Central London 1471-2121-5-341536960310.1186/1471-2121-5-34Research ArticleEndoplasmic reticulum degradation impedes olfactory G-protein coupled receptor functional expression Lu Min [email protected] Lena [email protected] Fernando [email protected] Hong [email protected] Bryan D [email protected] Senomyx, Inc., 11099 North Torrey Pines Road, La Jolla, CA 92037, USA2 Present Address: Kalypsys, Inc., 10420 Wateridge Circle, San Diego, CA 92121 USA2004 15 9 2004 5 34 34 2 6 2004 15 9 2004 Copyright © 2004 Lu et al; licensee BioMed Central Ltd.2004Lu et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Research on olfactory G-protein coupled receptors (GPCRs) has been severely impeded by poor functional expression in heterologous systems. Previously, we demonstrated that inefficient olfactory receptor (OR) expression at the plasma membrane is attributable, in part, to degradation of endoplasmic reticulum (ER)-retained ORs by the ubiquitin-proteasome system and sequestration of ORs in ER aggregates that are degraded by autophagy. Thus, experiments were performed to test the hypothesis that attenuation of ER degradation improves OR functional expression in heterologous cells. Results To develop means to increase the functional expression of ORs, we devised an approach to measure activation of the mOREG OR (Unigene # Mm.196680; Olfr73) through coupling to an olfactory cyclic nucleotide-gated cation channel (CNG). This system, which utilizes signal transduction machinery coupled to OR activation in native olfactory sensory neurons, was used to demonstrate that degradation, both by the ubiquitin-proteasome system and autophagy, limits mOREG functional expression. The stimulatory effects of proteasome and autophagy inhibitors on mOREG function required export from the ER and trafficking through the biosynthetic pathway. Conclusions These findings demonstrate that poor functional expression of mOREG in heterologous cells is improved by blocking proteolysis. Inhibition of ER degradation may improve the function of other ORs and assist future efforts to elucidate the molecular basis of odor discrimination. ==== Body Background The sense of smell originates in the olfactory epithelium when olfactory receptors (ORs), members of the seven transmembrane domain G-protein coupled receptor (GPCR) superfamily, bind odorant ligands [1,2]. Despite identification of the first constituents of the ~1000 member OR superfamily over a decade ago, efforts to uncover the molecular basis of odor discrimination have been severely limited by the inability to efficiently express ORs at the plasma membrane in heterologous expression systems [2-6]. Recently, we elucidated three specific cellular mechanisms responsible for inefficient OR trafficking to the plasma membrane: ORs are retained within the endoplasmic reticulum (ER) due to inefficient folding and poor coupling to ER export machinery, degraded via the ubiquitin-proteasome system, and sequestered in ER aggregates that are degraded by autophagy [7]. Thus, we have a clearer understanding of the problems associated with OR expression in heterologous cells. To develop rationale means to improve the functional expression of ORs, an approach was devised to quantitate activation of the mouse mOREG OR (Unigene # Mm.196680; Olfr73), which recognizes the odorant eugenol (spicy, cinnamon-like odor) [8], following coupling to an olfactory cyclic nucleotide-gated cation channel (CNG) [9]. Using this assay, we show that degradation by both the ubiquitin-proteasome system and autophagy limits mOREG functional expression. Our results demonstrate for the first time that inhibition of proteolysis can positively modulate OR function. Results and discussion Functional expression of mOREG using a CNG-based assay A cell-based approach was developed to measure functional expression of mOREG in heterologous cells. This system was designed to mimic the signal transduction events involved in OR activation in the olfactory epithelium and utilizes CNG as a cAMP biosensor [10,11]. In olfactory sensory neurons, odorant binding to ORs initiates a signaling cascade involving the heterotrimeric G protein Golf, adenylate cyclase III, and an olfactory CNG, leading ultimately to the sensation of smell [1,12]. Accordingly, we transiently co-expressed mOREG, as an N-terminal fusion protein with the first 20 amino acids of rhodopsin (Rho20-mOREG), with untagged olfactory CNG subunits in HEK293 cells that endogenously express both the heterotrimeric G protein Gs, a functional homologue of Golf [13,14], and adenylate cyclase III [15]. The Rho tag has been shown to facilitate chemosensory GPCR functional expression [8,16,17], possibly by enhancing translocation into the ER during protein synthesis. In this system, odorant binding to Rho-mOREG, which couples to endogenous Gs and elicits increases in the second messenger cAMP in HEK cells [8,18,19], leads to the opening of CNG and the influx of calcium from the extracellular medium. Thus, Rho-mOREG function can be directly correlated with cellular calcium levels. Responses to eugenol, a ligand for mOREG, were detected in cells co-expressing Rho-mOREG and CNG (Fig. 1A and 1B) but not in cells expressing vector only, Rho-mOREG only, or CNG only (Fig. 1B). Eugenol activation of Rho-mOREG became evident at 20 sec and increased to a maximal level at 50 sec following odorant application. The EC50 for eugenol (20.8 +/- 3.4 uM) closely approximated published values (35 uM and 46 uM), thereby validating the utility of our approach [8,19]. Rho-mOREG activation was specific for eugenol as no response was observed when cells were challenged with the control odorants heptanal and octanal, which activate the I7 OR (Fig. 1B) [4,16,20]. Collectively, these data demonstrate that Rho-mOREG can functionally couple to CNG in heterologous cells following odorant stimulation. Figure 1 Functional expression of Rho-mOREG using a CNG-based assay. (A) HEK293 cells transiently transfected with Rho-mOREG and CNG were assayed for increases in intracellular calcium in response to 100 uM eugenol. Images contain ~2000 confluent cells and represent responses at the indicated times following odorant stimulation. Scale bar is 300 um. Responses were observed in ~10% of total cells or ~20% of transfected cells (~50% transfection efficiency). (B) Quantitation of cells expressing vector only (a), Rho-mOREG only (b), CNG only (c), Rho-mOREG and CNG (d and e) following stimulation with 100 uM eugenol (a-d) or 100 uM heptanal plus 100 uM octanal (e). * p < 0.001 compared to cells expressing vector only (a). ER degradation limits mOREG functional expression Previously, we demonstrated that ORs are inefficiently expressed at the plasma membrane of heterologous cells due, in part, to degradation of ER-retained ORs by the ubiquitin-proteasome system and sequestration of ORs in ER aggregates that are degraded by autophagy [7]. Specifically, inhibition of the proteasome using MG-132 or inhibition of autophagy using 3-methyladenine (3-MA) enhanced Rho-mOREG protein expression 2 to 3-fold by biochemical and cellular analyses [7]. Therefore, experiments were performed to test the hypothesis that ER degradation limits OR functional expression. These studies used cells stably expressing CNG and transiently expressing Rho-mOREG to achieve more robust responses. As shown in Figure 2, treatment of cells with 3-MA, a specific inhibitor of autophagy that blocks sequestration of material into autophagosomes [21], enhanced Rho-mOREG activation by eugenol (compare Fig. 2A and 2B). Similar results were obtained when cells were treated with MG-132 (compare Fig. 2A and 2C) or epoxomicin (data not shown), a specific inhibitor of the proteasome [22]. Autophagy and proteasome inhibitors increased Rho-mOREG functional expression 2 to 3-fold in the linear range of eugenol dose response curves (Fig. 2D and 2F). Importantly, 3-MA and MG-132 specifically promoted Rho-mOREG function, since responses of the β-adrenergic receptor (β-AR), a GPCR that utilizes the same signal transduction machinery as Rho-mOREG (GPCR-Gs-adenylate cyclase) [14], following isoproterenol challenge were unaffected (Fig. 2E and 2G). Thus, degradation inhibitors were not modulating the function or trafficking of elements of the signal transduction machinery, including CNG, coupled to Rho-mOREG. Collectively, these data suggest that ER degradation, by autophagy and the ubiquitin-proteasome system, limits Rho-mOREG functional expression. Figure 2 ER degradation limits Rho-mOREG functional expression. CNG cells transiently transfected with Rho-mOREG were assayed for increases in intracellular calcium in response to 10 uM eugenol following treatment with vehicle (control, 0.1% DMSO; A), 10 mM 3-MA (B) or 50 uM MG-132 (C) for 4 h. Images contain ~750–1000 confluent cells and represent responses 60 sec following odorant stimulation. Scale bar is 150 um. Dose-response curves were determined for eugenol (300 nM to 300 uM; D and F) or isoproterenol (100 pM to 100 nM; E and G) in cells treated with vehicle (control), 10 mM 3-MA (D and E) or 50 uM MG-132 (F and G) for 4 h. ~20–25% of total cells or ~40–50% of transfected cells (~50% transfection efficiency) responded to 100 uM eugenol under control conditions. Note that degradation inhibitors specifically increased Rho-mOREG functional responses and had no effect on β-AR function. The EC50 value for eugenol was larger for control (17.1 +/- 3.0 uM) compared to 3-MA (10.8 +/- 2.6 uM) and MG-132 (8.1 +/- 2.6 uM) treatments, but these differences did not achieve statistical significance. The EC50 values for isoproterenol for control (3.7 +/- 1.0 nM), 3-MA (4.2 +/- 2.5 nM), and MG-132 (2.6 +/- 0.9 nM) treatments were not significantly different. To determine if increased functional expression of Rho-mOREG following inhibition of ER degradation was attributable to use of an OR fusion protein, we examined the effect of autophagy and proteasome inhibitors on mOREG lacking the Rho tag. Although eugenol activation of Rho-mOREG (EC50 = 17.1 +/- 3.0 uM; maximum number of responding cells at 300 uM eugenol = 214 +/- 10) was more robust than untagged mOREG (EC50 = 36.5 +/- 12.9 uM; maximum number of responding cells at 300 uM eugenol = 48 +/- 5; p < 0.05 compared to Rho-mOREG for both EC50 and maximal number of responding cells), likely due to an established role of the Rho tag in facilitating chemosensory GPCR functional expression [8,16,17], MG-132 and 3-MA increased untagged mOREG functional expression 2 to 3-fold (Fig. 3), similar to the magnitude observed with Rho-mOREG (Fig. 2). Thus, inhibition of ER degradation increased Rho-mOREG and untagged mOREG functional expression, demonstrating that observed effects were not attributable to use of a non-native OR fusion protein. Figure 3 ER degradation limits untagged mOREG functional expression. CNG cells transiently transfected with untagged mOREG were assayed for increases in intracellular calcium in response to increasing concentrations of eugenol (from 3 uM to 1000 uM) following treatment with vehicle (control), 10 mM 3-MA, or 50 uM MG-132 for 4 h. The EC50 values for eugenol for control (36.5 +/- 12.9 uM), 3-MA (42.6 +/- 6.5 uM), and MG-132 (36.4 +/- 7.5 uM) treatments were not significantly different. CNG cells transfected with vector only (pUC18) and treated with vehicle, 3-MA, or MG-132 did not respond to 1000 uM eugenol. Two seemingly independent mechanisms degrade ORs retained in the ER. First, OR aggregates sequestered in ER subdomains are targeted to lysosomes for degradation by autophagy, and second, misfolded ORs are covalently modified by polyubiquitination and degraded by the proteasome [7]. To determine if simultaneous inhibition of autophagy and the ubiquitin-proteasome system produced additive effects on Rho-mOREG function, we co-treated cells with 3-MA and MG-132. As shown in Figure 4, Rho-mOREG function, measured using 10 uM eugenol, a non-saturating concentration near the EC50 value, was equivalent in cells treated with 3-MA alone, MG-132 alone, or 3-MA plus MG-132. Figure 4 Simultaneous inhibition of autophagy and the proteasome does not produce additive Rho-mOREG functional responses. CNG cells transiently transfected with Rho-mOREG were assayed for increases in intracellular calcium in response to 10 uM eugenol following treatment with vehicle (control), 10 mM 3-MA, 50 uM MG-132, or 10 mM 3-MA plus 50 uM MG-132 for 4 h. Individual or combined treatment with degradation inhibitors yielded similar levels of Rho-mOREG activity. * p < 0.001 compared to cells treated with vehicle. The non-additive effects of 3-MA and MG-132 on Rho-mOREG function suggest one of the following two non-mutually exclusive scenarios: first, OR degradation by autophagy may be linked to OR degradation by the ubiquitin-proteasome system, by a poorly defined mechanism as suggested for other aggregation prone proteins [23-25]; second, in addition to ER degradation, an additional step(s) downstream of proteolysis may limit Rho-mOREG functional expression in heterologous cells. A recent preliminary report described specialized accessory proteins that increase OR surface expression and function [26]. These proteins could serve as chaperones to package OR cargo into COPII vesicles for export from the ER and/or couple ORs to requisite signal transduction machinery at the plasma membrane, steps that are both downstream of ER degradation. In the absence of necessary accessory proteins, functional expression may not exceed a certain level regardless of the quantity of OR that is diverted from the degradative pathways by autophagy and ubiquitin-proteasome inhibitors. The existence of multiple steps limiting OR functional expression in heterologous cells is further supported by our findings that the function of mOREG, lacking the Rho tag, is less robust than Rho-mOREG. Since the Rho tag may facilitate translocation into the ER during protein synthesis [8,16,17], ER translocation could comprise an additional limiting step, upstream of ER degradation, for OR functional expression. ER export and trafficking through the Golgi apparatus are necessary for Rho-mOREG functional expression Inhibition of ER degradation events could permit a pool of Rho-mOREG to achieve an ER export competent conformation and traffic through the Golgi apparatus to the plasma membrane. However, using surface biotinylation, surface immunofluorescence microscopy, flow cytometry, and glycosidase digestion assays, we were unable to demonstrate convincing Rho-mOREG surface expression or visualize a pool of Rho-mOREG containing endoglycosidase H-resistant carbohydrate modifications indicative of transit through the Golgi apparatus following treatment with ER degradation inhibitors (ML and BDM unpublished observations). These results suggested that either a small pool of properly folded Rho-mOREG was expressed at the plasma membrane in quantities below the threshold of the cell biological techniques employed to visualize the receptor, or that a pool of intracellular Rho-mOREG comprised the functionally responsive population in calcium imaging experiments. Notably, numerous studies have documented the functional expression of GPCRs and requisite signal transduction machinery in intracellular compartments, including ER membranes [27-29]. To differentiate between these two possibilities, we adopted a pharmacological approach to selectively and independently block trafficking from compartments in the early secretory pathway, specifically the ER and Golgi apparatus. If inhibition of ER degradation does not increase Rho-mOREG activity under conditions that block export from ER and Golgi compartments, the functionally responsive Rho-mOREG population is likely derived from an internal pool that is required to traffic to the plasma membrane to function. Conversely, if inhibition of ER degradation increases Rho-mOREG activity under conditions that block export from ER and Golgi compartments, the functionally responsive Rho-mOREG population likely resides in an intracellular compartment. To inhibit protein trafficking from the ER, brefeldin A (BFA), which blocks ER export of cargo proteins by inducing collapse of the Golgi stacks into the ER, was used [30]. As shown in Fig. 5A, BFA completely blocked the enhancement of Rho-mOREG function by 3-MA and MG-132; by contrast, BFA had no effect on functional responses of the β-AR, indicating that BFA was not affecting the function or trafficking of signal transduction machinery, including CNG, coupled to Rho-mOREG. By blocking transport of proteins present in the ER that are in route to the plasma membrane, specifically Rho-mOREG following inhibition of ER degradation, BFA inhibited Rho-mOREG function; β-AR and CNG function were unperturbed since these proteins were already present at the plasma membrane prior to BFA treatment. Since β-AR exhibits a long half-life at the plasma membrane [31], inhibiting delivery of newly synthesized β-AR by BFA would not adversely affect isoproterenol responses. Thus, ER export is required for increased Rho-mOREG functional expression by degradation inhibitors. Figure 5 ER export and trafficking through the Golgi apparatus are required for Rho-mOREG functional expression. CNG cells transiently transfected with Rho-mOREG were assayed for increases in intracellular calcium in response to 10 uM eugenol, to gauge Rho-mOREG function, or 1 nM isoproterenol, to gauge β-AR function, following treatment with vehicle (0.1% ethanol), BFA (5 ug/ml), monensin (10 uM), or incubation at 20°C for 4 h (A) or 5 min (B). (C) Cells were treated with vehicle or CHX (75 uM) for 4 hr. In all experiments, cells were also co-treated for 4 h with control (0.1% DMSO), 10 mM 3-MA, or 50 uM MG-132 as indicated. Partial inhibition of Rho-mOREG function with BFA or monensin but not 20°C (A, control columns), a temperature that also attenuates endocytic events, is likely attributable to turnover of cell surface Rho-mOREG when the biosynthetic pathway, which normally replenishes the plasma membrane Rho-mOREG pool, is blocked. By contrast, BFA and monensin do not affect function of the β-AR, a control GPCR that exhibits a long half-life at the plasma membrane [31]. * p < 0.005 compared to cells treated with vehicle in the same group. To inhibit protein trafficking from the Golgi apparatus, monensin, an ionophore that disrupts Golgi structure and inhibits Golgi trafficking events, was used [32]. Monensin, similar to BFA, completely blocked the enhancement of Rho-mOREG function by 3-MA and MG-132 while having no effect on β-AR function (Fig. 5A). Similar effects were observed when cells were incubated at 20°C (Fig. 5A), a temperature that arrests protein transport at trans Golgi cisternae [33]. Importantly, trafficking disrupting agents did not affect Rho-mOREG or β-AR function when acutely applied to cells, further substantiating that results were not attributable to non-specific effects on signal transduction machinery or eugenol binding (Fig. 5B). Thus, Golgi trafficking events are required for increased Rho-mOREG functional expression by degradation inhibitors. Inhibition of Rho-mOREG function by trafficking disrupting agents, specifically BFA, could be due to activation of the unfolded protein response [34] and inhibition of Rho-mOREG protein synthesis. To directly address this possibility, experiments were performed to test the effect of the protein synthesis inhibitor cycloheximide (CHX) on functional expression of Rho-mOREG. As shown in Figure 5C, CHX, used at concentrations previously demonstrated to inhibit Rho-mOREG translation [7], had no effect on increased Rho-mOREG function by 3-MA and MG-132. These data suggest that autophagy and ubiquitin-proteasome inhibitors diverted an existing pool of Rho-mOREG from degradative pathways to the secretory pathway and that effects of degradation inhibitors and trafficking disrupting agents were not attributable to modulation of protein synthesis. Collectively, these data support a model whereby inhibition of ER degradation promotes a small fraction of Rho-mOREG to achieve an ER export competent conformation, thereby satisfying ER quality control processes, and traffic through the secretory pathway to the plasma membrane. Thus, similar to chemical and pharmacological chaperones that promote folding and restore ER export of misfolded GPCR cargo [35-37], agents interfering with ER degradation may promote ER export of GPCR and non-GPCR cargo [38], trafficking through the biosynthetic pathway, and functional expression at the cell surface. We speculate that the pool of Rho-mOREG expressed at the plasma membrane is below the detection limits of cell biological techniques used to visualize the receptor [7] but above the detection threshold for calcium imaging methodology used to examine receptor function. Indeed, following pharmacological treatments, improved ΔF508 cystic fibrosis transmembrane conductance regulator functional expression at the plasma membrane is readily detectable by sensitive electrophysiological analyses but neither by cell surface labeling nor by biochemical approaches measuring carbohydrate modifications indicative of transit through the Golgi apparatus [39-41]. Though we favor a model whereby inhibition of ER degradation promotes OR export from the ER and improves OR surface expression, we were unable to obtain cellular and biochemical evidence to support this proposal. We cannot exclude the possibility that inhibition of ER degradation also stabilizes an otherwise labile cofactor or chaperone protein, endogenously expressed by heterologous cells, that modulates OR trafficking in a post-Golgi compartment, stability at the cell surface, and/or function at the plasma membrane [42,43]. Conclusions We have developed an expression system for ORs that utilizes signal transduction machinery coupled to OR activation in native olfactory sensory neurons. Using CNG as a cAMP biosensor to gauge mOREG function, we demonstrate that inhibition of ER degradation, by both autophagy and the ubiquitin-proteasome system, promotes functional expression of Rho-mOREG as well as untagged mOREG. Thus, proteolysis limits mOREG function in heterologous cells. Inhibition of ER degradation may improve the function of other ORs and assist future efforts to elucidate the molecular basis of odor discrimination. Methods Molecular biology Rho20-mOREG expression vector was generated in pRK5 as previously described [7]. To generate untagged mOREG expression vector, mOREG coding sequence was excised using AscI/NotI and subcloned into a modified pRK5 vector lacking the Rho20 tag. The human CNGA2 and CNGB1b expression constructs encode untagged human CNGA2 and CNGB1b in pEAK10-derived vectors (Edge Biosystems, Gaithersburg, MD) [44]. For the generation of stable transfectants, CNGA2 was subcloned into pCDNA3.1/zeo (Invitrogen, Carlsbad, CA). The CNGA2 clone contains the C458W and E581M mutations, introduced using the QuickChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA), previously shown to increase cAMP sensitivity in rat CNGA2 [11]. Compounds, odorants, and ligands BFA, isoproterenol, monensin, and 3-MA were from Sigma (St. Louis, MO); MG-132 was from Calbiochem (San Diego, CA); eugenol, heptanal and octanal were from Aldrich (Milwaukee, WI). Cell culture and transfections HEK293 cells were maintained and transfected as previously described [7]. For the generation of CNG stable transfectants, cells were transfected with linearized CNGA2 and CNGB1b expression constructs (1:1 ratio) and selected using 50 ug/mL zeocin (Invitrogen) and 0.5 ug/mL puromycin (Calbiochem). Individual colonies were expanded and screened for CNG expression by assaying functional responses to 500 uM eugenol following transient transfection with Rho-mOREG. For functional expression studies, CNG cells were grown in media without selection 72 h prior to experimentation and transiently transfected with mOREG 48 h prior to experimentation. mOREG functional expression Functional expression of mOREG was investigated using calcium imaging methodology as previously described [44]. Cells seeded in 24 well plates were loaded with the calcium dye fluo-4 acetoxymethyl ester (Molecular Probes, Eugene, OR) 2 d post-transfection using the following conditions: 3 uM dye in 0.5 ml Hanks' balanced salt solution containing divalent cations (HBSS, Invitrogen) for 1 h at room temperature in the dark. Cells were subsequently washed once with 0.5 ml HBSS to remove excess fluo-4, supplemented with 0.25 ml HBSS, and then stimulated with an additional 0.25 ml HBSS containing the appropriate ligand at twice the final concentration. A single ligand was applied to each dish of cells. Typically, 3–4 separate dishes of cells were used for each ligand concentration or condition and experiments were repeated 3–4 times. Thus, individual data points represent the average of 9–16 separate measurements. Changes in intracellular calcium were monitored by fluorescence microscopy using an Axiovert S100 TV inverted microscope with a 10× long working distance Plan Fluor objective (numerical aperture 0.5) and a cooled charge-coupled device camera (Princeton Instruments, Trenton, NJ). Images were acquired using a Lambda DG-4 automated wavelength controller (Sutter Instrument Co., Novato, CA) at 480 nm excitation and 535 nm emission and analyzed using Imaging Workbench 4.0 (Axon Instruments, Union City, CA). Counting the number of cells responding to ligands 60 sec following stimulus addition, when cells had achieved a maximal response, quantitated receptor activity. This established methodology has been used to functionally characterize the human T1R1/T1R3 umami receptor, the human T1R2/T1R3 sweet receptor, and the Drosophila Gr5a trehalose receptor [44,45]. To independently validate this method, we determined that the EC50 for isoproterenol activation of the β-AR (3.7 +/- 1.0 nM), measured by counting responding cells, closely matched published values (1.7–3.3 nM), measured either by fluorescent intensity measurements or a cAMP accumulation assay [10]. In addition, the EC50 for eugenol activation of Rho-mOREG (20.8 +/- 3.4 uM), measured by counting responding cells, closely approximated the published value (35 uM and 46 uM), determined by monitoring fluorescent intensity of responding cells [8,19]. Finally, the EC50s for glutamate activation of mGluR4 and cycloheximide activation of mT2R05 were similar when determined by counting cells or by monitoring fluorescent intensity [44]. We estimate ~40–50% of transfected CNG cells express functional cell surface Rho-mOREG based on the following points. First, ~20–25% of total cells in a microscopic field respond to maximal doses of eugenol. Second, ~50% of cells are transfected, measured by either co-transfection with red fluorescent protein or by immunolabelling permeabilized cells expressing Rho-mOREG with an anti-Rho antibody. Thus, ~40–50% of transfected cells express sufficient Rho-mOREG at the plasma membrane to elicit a functional response. Statistics Data represent the mean +/- SEM. Statistical significance was determined using an unpaired, two-tailed Student's t-test. Dose-response curves were plotted and EC50 values were determined using GraphPad Prism v3.02 software. Authors' contributions ML generated the mOREG expression vectors and carried out most of the calcium imaging experiments. LS generated the cells stably transfected with CNG. FE assisted with the calcium imaging experiments. HX generated the CNG expression vectors. BDM coordinated the study and wrote the paper. Acknowledgement We thank Elliot Adler for important contributions with CNG cloning as well as Xiaodong Li, Alexey Pronin, Guy Servant, Mark Zoller, and Lubert Stryer for critical review of the manuscript. ==== Refs Firestein S How the olfactory system makes sense of scents Nature 2001 413 211 218 11557990 10.1038/35093026 Mombaerts P How smell develops Nat Neurosci 2001 4 Suppl 1192 1198 11687829 10.1038/nn751 Buck L Axel R A novel multigene family may encode odorant receptors: a molecular basis for odor recognition Cell 1991 65 175 187 1840504 10.1016/0092-8674(91)90418-X Mombaerts P Genes and ligands for odorant, vomeronasal and taste receptors Nat Rev Neurosci 2004 5 263 278 15034552 10.1038/nrn1365 Gimelbrant AA Stoss TD Landers TM McClintock TS Truncation releases olfactory receptors from the endoplasmic reticulum of heterologous cells J Neurochem 1999 72 2301 2311 10349839 10.1046/j.1471-4159.1999.0722301.x McClintock TS Sammeta N Trafficking prerogatives of olfactory receptors Neuroreport 2003 14 1547 1552 14502073 10.1097/00001756-200308260-00001 Lu M Echeverri F Moyer BD Endoplasmic reticulum retention, degradation, and aggregation of olfactory g-protein coupled receptors Traffic 2003 4 416 433 12753650 10.1034/j.1600-0854.2003.00097.x Kajiya K Inaki K Tanaka M Haga T Kataoka H Touhara K Molecular bases of odor discrimination: Reconstitution of olfactory receptors that recognize overlapping sets of odorants J Neurosci 2001 21 6018 6025 11487625 Kaupp UB Seifert R Cyclic nucleotide-gated ion channels Physiol Rev 2002 82 769 824 12087135 Reinscheid RK Kim J Zeng J Civelli O High-throughput real-time monitoring of G(s)-coupled receptor activation in intact cells using cyclic nucleotide-gated channels Eur J Pharmacol 2003 478 27 34 14555181 10.1016/j.ejphar.2003.08.036 Rich TC Tse TE Rohan JG Schaack J Karpen JW In vivo assessment of local phosphodiesterase activity using tailored cyclic nucleotide-gated channels as cAMP sensors J Gen Physiol 2001 118 63 78 11429444 10.1085/jgp.118.1.63 Ronnett GV Moon C G proteins and olfactory signal transduction Annu Rev Physiol 2002 64 189 222 11826268 10.1146/annurev.physiol.64.082701.102219 Jones DT Reed RR Golf: an olfactory neuron specific-G protein involved in odorant signal transduction Science 1989 244 790 795 2499043 Jones DT Masters SB Bourne HR Reed RR Biochemical characterization of three stimulatory GTP-binding proteins. 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==== Front BMC AnesthesiolBMC Anesthesiology1471-2253BioMed Central London 1471-2253-4-61536732910.1186/1471-2253-4-6Research ArticleComparative effect of intraoperative propacetamol versus placebo on morphine consumption after elective reduction mammoplasty under remifentanil-based anesthesia: a randomized control trial [ISRCTN71723173] Binhas Michèle [email protected] François [email protected] Saïda [email protected] Powen [email protected] Marc [email protected]çois Véronique [email protected] Xavier [email protected] Philippe [email protected] Service d'Anesthésie-Réanimation, Hôpital Henri Mondor, AP-HP, Université Paris XII, 51, avenue du Maréchal de Lattre de Tassigny, 94010 Créteil, France2004 14 9 2004 4 6 6 5 3 2004 14 9 2004 Copyright © 2004 Binhas et al; licensee BioMed Central Ltd.2004Binhas et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Postoperative administration of paracetamol or its prodrug propacetamol has been shown to decrease pain with a morphine sparing effect. However, the effect of propacetamol administered intra-operatively on post-operative pain and early postoperative morphine consumption has not been clearly evaluated. In order to evaluate the effectiveness of analgesic protocols in the management of post-operative pain, a standardized anesthesia protocol without long-acting opioids is crucial. Thus, for ethical reasons, the surgical procedure under general anesthesia with remifentanil as the only intraoperative analgesic must be associated with a moderate predictable postoperative pain. Methods We were interested in determining the postoperative effect of propacetamol administered intraoperatively after intraoperative remifentanil. Thirty-six adult women undergoing mammoplasty with remifentanil-based anesthesia were randomly assigned to receive propacetamol 2 g or placebo one hour before the end of surgery. After remifentanil interruption and tracheal extubation in recovery room, pain was assessed and intravenous titrated morphine was given. The primary end-point was the cumulative dose of morphine administered in the recovery room. The secondary end-points were the pain score after tracheal extubation and one hour after, the delay for obtaining a Simplified Numerical Pain Scale (SNPS) less than 4, and the incidence of morphine side effects in the recovery room. For intergroup comparisons, categorical variables were compared using the chi-squared test and continuous variables were compared using the Student t test or Mann-Whitney U test, as appropriate. A p value less than 0.05 was considered as significant. Results In recovery room, morphine consumption was lower in the propacetamol group than in the placebo group (p = 0.01). Pain scores were similar in both groups after tracheal extubation and lower in the propacetamol group (p = 0.003) one hour after tracheal extubation. The time to reach a SNPS < 4 was significantly shorter in the propacetamol group (p = 0.02). The incidence of morphine related side effects did not differ between the two groups. Conclusions Intraoperative propacetamol administration with remifentanil based-anesthesia improved significantly early postoperative pain by sparing morphine and shortening the delay to achieve pain relief. ==== Body Background Postoperative administration of paracetamol or its prodrug propacetamol has been shown to decrease pain with a morphine sparing effect [1-4]. The effect of propacetamol administered intraoperatively on postoperative pain and early postoperative morphine consumption has not been clearly evaluated. However, for a predictable moderate postoperative pain, intraoperative administration of non-opioid analgesics such as paracetamol and postoperative intravenous administration of morphine are recommended in patients undergoing general anesthesia with remifentanil [5]. Indeed, remifentanil differs from potent mu agonists by its extremely short elimination half-life [6]. The elimination kinetics of remifentanil is so fast that its analgesic effect wears off abruptly, thus making the management of postoperative pain critical. In order to evaluate the effectiveness of intraoperative paracetamol administration in the management of postoperative pain and morphine consumption, a standardized anesthesia protocol without long-acting opioid is necessary. Thus, for ethical reasons, the surgical procedure under general anesthesia with remifentanil as the only intraoperative analgesic must be associated with a moderate predictable postoperative pain. Therefore, the present study was designed to evaluate the effect of intraoperative administration of propacetamol during remifentanil-based anesthesia on postoperative pain in patients undergoing reduction mammoplasty. Methods Patients After approval by the Local Ethical Committee and written informed consent, 36 consecutive female patients who underwent elective reduction mammoplasty were included. Exclusion criteria were the preoperative use of analgesic drugs; a body mass index ≥ 35, an American Society of Anesthesiology physical status ≥ 3 and sensitivity to paracetamol. Pain evaluation using a Simplified Numerical Pain Scale (SNPS) was explained to the patients during the preoperative anesthetic visit and the day before surgery. A standardized surgical technique was used for all patients. Anesthetic protocol Hydroxyzine 2 mg.kg-1 was given orally 12 h and 2 h before anesthesia as premedication. Before induction of anesthesia, patients were randomly allocated to either Placebo group or Propacetamol group. Randomization was based on computer-generated codes maintained in sequentially numbered, opaque envelopes. The preparation of the propacetamol or saline infusion was done by a nurse who was not in charge of the patient. The anesthetist, the patient and the nurse's staff caring for the patients in the recovery room were unaware of the treatment group. In both groups, anesthesia was induced with propofol 2.5 mg.kg-1 followed by a slow bolus (1 min) of remifentanil 1 mcg.kg-1. Tracheal intubation was facilitated by atracurium 0.5 mg.kg-1. Anesthesia was maintained with remifentanil 0.1 mcg.kg-1.min-1 and isoflurane (0.5–1.0% end-tidal) with nitrous oxide (N2O) in 50% oxygen. Remifentanil infusion rate was increased or decreased by 0.05 mcg.kg.min-1 in order to maintain an arterial systolic pressure of 20% more or less than the baseline value. One hour before the end of surgery, which corresponded to the beginning of the skin closure, patients received either propacetamol 2 g in 50 cc saline (Propacetamol group) or 50 cc saline alone (Placebo group) infused over 10 min. At the end of surgery, administration of isoflurane and N2O were withdrawn and the patient was transferred in the recovery room. The anaesthetist, the patient and the nurse's staff caring for the patients in the recovery room were unaware of the treatment group. Remifentanil infusion was interrupted when the patient arrived in recovery room. Tracheal extubation was performed within a few minutes after remifentanil discontinuation. Clinical monitoring included heart rate, blood pressure, pulse oxymetry, respiratory rate, and sedation score (0: awake, 1: drowsy and 2: asleep). From the time of extubation, pain was evaluated on using a SNPS (from 0, no pain to 10 the worse pain) and intravenous 2 mg morphine was administered on request every 5 min until pain relief (SNPS<4). When pain relief was reached, SNPS was subsequently evaluated every 15 min. Morphine was interrupted when the sedation score went up to 1, systemic arterial pressure < 80 mmHg or respiratory rate less than 8/min. During the data collection period, intravenous morphine titration was further administered if SNPS was up to 4. Patients did not receive antiemetic prophylaxis. If post-operative nausea and vomiting (PONV) occurred, metoclopramide 10 mg and ondansetron 4 mg if necessary were intravenously administered. Patients fulfilling Aldrete criteria [7] were discharged from recovery room. Measurements Morphine requirement, pain and sedation scores were measured every 5 min until pain relief was obtained. When SNPS was below 4 during 15 min, parameters were subsequently recorded every 15 min. Morphine side effects (nausea, vomiting, urinary retention, shivering and itching) and need for supplemental medications (e.g., antiemetics) were also recorded. The total dose of morphine in recovery room was the primary end point. Pain scores after extubation and one hour after tracheal extubation, delay for morphine requirement, delay for pain relief and incidence of morphine side effects were recorded. Statistical analysis Data are expressed as mean (± standard deviation) for quantitative variables normally distributed, or otherwise as median (25th – 75th percentiles) when data were not normally distributed, and as percentage for categorical variables. Data were analyzed using Statview 5.0 software (SAS Institute Inc, USA). For intergroup comparisons, categorical variables were compared using the chi-squared test and continuous variables were compared using the Student t test or Mann-Whitney U test, as appropriate. A p value less than 0.05 was considered as significant. Anticipating a standard deviation of 2.49 [8], it was calculated that 15 patients at least were necessary to show a difference between groups in morphine consumption of 4 mg (considered as a clinically relevant difference) with a 80% power and a 5% type 1 error. Results Thirty-six patients were included during a 12 months period: 19 in Propacetamol group and 17 in Placebo group (Table 1). There was no significant difference between the groups concerning clinical characteristics, anesthesia duration and total amount of remifentanil administered. In all patients, extubation was obtained within 7 ± 3 min (Table 1) after remifentanil discontinuation. Table 1 Demographic characteristics and perioperative parameters. Placebo Group (n = 17) Propacetamol Group (n = 19) Age (years) 41(16) 34 (14) Body mass index (kg.m-2) 26 (4) 25 (3) Duration of anesthesia (min) 245 (55) 245 (80) Remifentanil consumption (μg.kg-1.min-1) 0.150 (0.055) 0.155 (0.040) Final intraoperative corporeal temperature (Celsius) 36.8 (0.4) 36.7 (0.5) Delay before extubation after remifentanil interruption (min) 7 (3) 7 (3) Data are expressed as mean (standard deviation) for age, body mass index, final intraoperative corporeal temperature and delay before extubation. Other data are expressed as median (interquartile). In recovery room, cumulative morphine consumption was significantly lower in the Propacetamol group than in the Placebo group (Table 2). Five minutes after extubation, pain scores were similar in both groups (SNPS = 6). Pain scores one hour after tracheal extubation were significantly lower in the Propacetamol group than in the Placebo group (Table 2). Moreover, the time to reach a SNPS score less than 4 was significantly shorter in Propacetamol group compared to the Placebo group. All the patients received intravenous morphine titration in the first hour after extubation and three patients (one in Placebo group and two in Propacetamol group) required a morphine titration over one hour after extubation. Once a SNPS value less than 4 was obtained, pain scores remained stable and similar in both groups except in one patient in Placebo group who required an additional 2 mg intravenous bolus of morphine 80 min after extubation. Table 2 Postoperative intravenous morphine requirement and pain scores in recovery room. Placebo Group (n = 17) Propacetamol Group (n = 19) p value Morphine consumption in recovery room (mg) 16 [8–34] 10 [6–28] 0.01 Delay between extubation and first morphine administration (min) 5 [5–20] 5 [5–15] NS SNPS score five minutes after extubation 6 [0–9] 6 [0–10] NS SNPS score one hour after extubation 3 [2–6] 2 [0–4] 0.003 Delay between extubation and obtaining a SNPS score < 4 (min) 40 [20–85] 30 [15–70] 0.02 Data are expressed as median [25th–75th percentile]. P < 0.05 was considered as statistically significant. Pain score was evaluated on using a Simplified Numerical Pain Scale (SNPS: from 0, no pain to 10 the worse pain). The incidence of morphine adverse effects was similar in both groups: 5 patients had nausea (3 in Propacetamol and 2 in Placebo group, p=NS) and 4 patients had vomiting (2 in each group, p=NS), no other side effects were observed. In one patient receiving placebo, morphine titration was interrupted because of nausea and vomiting. Discussion This study shows that propacetamol administered one hour before end of surgery reduced the morphine dose given over the first four postoperative hours and shortened the elapsed time to obtain a SNPS under 4 in patients undergoing elective reduction mammoplasty. In our study, surgical technique and anesthetic protocol were similar in both groups. Remifentanil was the only analgesic used during anesthesia period and was interrupted before morphine administration. Thus, the beneficial effect on postoperative pain observed is clearly linked to intraoperative administration of propacetamol. In a recent study [9], Verchère and colleagues failed to demonstrate a postoperative analgesic effect of intraoperative propacetamol administration, after remifentanil anesthesia for supratentorial craniotomy. Pain after supratentorial neurosurgery was too severe and paracetamol was insufficient to relief it. In our study mammoplasty was chosen because postoperative pain is moderate [10], and intravenous administration of morphine was used in recovery room. Thus, the morphine sparing effect of intraoperative administration of paracetamol could be really evaluated with respect of ethical requirement. Our results are apparently at variance with those of other previous studies. Paracetamol given rectally immediately after induction of anesthesia [11] or at the end of gynecological surgery [12] and orally before surgery [13-16] failed to improve early postoperative analgesia. The negative results of these studies may be ascribable to several causes such as the low initial pain score in the control group [11,12,15,17], and the difference in the route used for paracetamol administration [11-16]. An other hypothesis to consider is the use of long-acting opioid such as fentanyl that might have contributed to the early post-operative analgesia [14,18]. Cobby and colleagues observed a morphine sparing effect when paracetamol 1.3 g was administered rectally at the end of hysterectomy [19]. One explanation is that plasma concentration after 1.3 g paracetamol was sufficient to achieve opioid sparing effect comparable with that of intravenous propacetamol. This hypothesis is strengthened by the results of another trial showing that after rectal administration of a high dose of paracetamol (40 and 60.mg kg-1) pain score and analgesic demand were significantly reduced in the early postoperative period [20]. The presently observed incidence of postoperative nausea and vomiting was of 25% and was similar to that of a previous study [21]. Despite the reduced dose of postoperative morphine in the Propacetamol group, the incidence of postoperative nausea and vomiting was not diminished. Our results are in agreement with the recent study of Aubrun and co-workers [4] who observed that postoperative intravenous propacetamol allowed a morphine-sparing effect but did not reduce the incidence of morphine-related adverse effects in patients undergoing general surgery. Propacetamol was administered at the beginning of skin closure, which corresponds to one hour before the end of surgery. This delay may be insufficient to achieve pain control immediately after tracheal extubation, as the peak effect of intravenous propacetamol was shown to occur only two hours after its administration [22]. Nevertheless, we considered that it was not feasible to administer propacetamol earlier, as in our practice duration of surgery was unpredictable. In summary, intraoperative propacetamol administration in women undergoing reduction mammoplasty improved significantly early postoperative pain in recovery room, and should be recommended for postoperative pain management. Pre-publication history The pre-publication history for this paper can be accessed here: ==== Refs Delbos A Boccard E The morphine sparing effect of propacetamol in orthopaedic post-operative pain J Pain Symptom Manage 1995 10 279 86 7541435 10.1016/0885-3924(95)00004-I Hyllested M Jones S Pedersen JL Kehlet H Comparative effect of paracetamol, NSAID or their combination in postoperative pain management: a qualitative review Br J Anaesth 2002 88 199 214 11878654 10.1093/bja/88.2.199 Romsing J Moiniche S Dahl JB Rectal and parenteral paracetamol, and paracetamol in combination with NSAIDs, for postoperative analgesia Br J Anaesth 2002 88 215 26 11878655 10.1093/bja/88.2.215 Aubrun F Kalfon F Mottet P Bellanger A Langeron O Coriat P Adjunctive analgesia with intravenous propacetamol does not reduce morphine-related adverse effects Br J Anaesth 2003 90 314 9 12594143 10.1093/bja/aeg076 Albrecht S Schuttler J Yarmush J Postoperative pain management after intra-operative remifentanil Anesth Analg 1999 89 S40 5 10511077 10.1097/00000539-199910001-00008 Glass PS Gan TJ Howell S A review of the pharmacokinetics and pharmacodynamics of remifentanil Anesth Analg 1999 89 S7 14 10511072 10.1097/00000539-199910001-00003 Aldrete JA Kroulik D A post anesthetic recovery score Anesth Analg 1970 49 924 34 5534693 Rosaeg OP Bell M Cicutti NJ Dennehy KC Lui AC Krepski B Pre-incision infiltration with lidocaine reduces pain and opioid consumption after reduction mammoplasty Reg Anesth Pain Med 1998 23 575 9 9840853 10.1016/S1098-7339(98)90084-0 Verchere E Grenier B Mesli A Siao D Sesay M Maurette P Postoperative pain management after supratentorial craniotomy J Neurosurg Anesthesiol 2002 2 96 101 11907388 10.1097/00008506-200204000-00002 Bonica J Bonica J Postoperative pain In The management of pain 1990 Philadelphia: Lea and Febiger 461 80 Bremerich D Neidhart G Heimann K Kessler P Behne M Prophylactically-administered rectal acetaminophen does not reduce post-operative opioids requirements in infants and small children undergoing elective cleft palate repair Anesth Analg 2001 92 907 12 11273923 Hein A Jakobsson J Ryberg G Paracetamol 1 g given rectally at the end of minor gynaecological surgery is not efficacious in reducing post-operative pain Acta Anaesthesiol Scand 1999 43 248 51 10081528 10.1034/j.1399-6576.1999.430302.x Gustafsson I Nyström E Quiding H Effect of preoperative paracetamol on pain after oral surgery Eur J Clin Pharmacol 1983 24 63 5 6832203 Cade L Ashley J Prophylactic paracetamol for analgesia after vaginal termination of pregnancy Anaesth Intensive Care 1993 21 93 6 8447616 Watcha MF Ramirez-Ruiz M White PF Jones MB Lagueruela RG Terkonda RP Perioperative effects of oral ketorolac and acetaminophen in children undergoing bilateral myringotomy Can J Anaesth 1992 39 649 54 1394752 Bennie RE Boeringer LA Mc Mahon S Allen H Dierdorf SF Postoperative analgesia with preoperative oral ibuprofen or acetaminophen in children undergoing myringotomy Paediatr Anaesth 1997 7 399 40 9308064 10.1046/j.1460-9592.1997.d01-87.x Kalso E Better standardisation will improve the quality of analgesic studies Acta Anaesthesiol Scand 1996 40 397 8 8738681 Jackobsson J Rane K Anaesthesia for short outpatient procedures. A comparison between thiopentone and propofol in combination with fentanyl or alfentanil Acta Anaesthesiol Scand 1995 39 503 7 7676787 Cobby TF Crighton IM Kyriakides K Hobbs GJ Rectal paracetamol has a significant morphine-sparing effect after hysterectomy Br J Anaesth 1999 83 253 6 10618939 Korpela R Korvenoja P Meretoja O Morphine-sparing effect of acetaminophen in pediatric day-case surgery Anesthesiology 1999 91 442 7 10443608 10.1097/00000542-199908000-00019 Marley R Postoperative nausea and vomiting: the outpatient enigma J Perianesth nursing 1996 11 147 61 10.1016/S1089-9472(96)90004-0 Piguet V Desmeules J Dayer P Lack of acetaminophen ceiling effect on RIII nociceptive flexion reflex Eur J Clin Pharmacol 1998 53 321 4 9516030 10.1007/s002280050386

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==== Front BMC OphthalmolBMC Ophthalmology1471-2415BioMed Central London 1471-2415-4-121536310710.1186/1471-2415-4-12Research ArticleEvaluation of the prophylactic use of mitomycin-C to inhibit haze formation after photorefractive keratectomy in high myopia: a prospective clinical study Hashemi Hassan [email protected] Seied Mohammad Reza [email protected] Akbar [email protected] Azita [email protected] Farabi Eye Hospital, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran2 Noor Vision Correction Center, Tehran, Iran3 Epidemiology and Biostatistics Department, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran2004 14 9 2004 4 12 12 9 5 2004 14 9 2004 Copyright © 2004 Hashemi et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background To study the effect of prophylactic application of mitomycin-C on haze formation in photorefractive keratectomy (PRK) for high myopia. Methods Fifty-four eyes of 28 myopic patients were enrolled in this prospective study. All eyes were operated by PRK followed by 0.02% mitomycin-C application for two minutes and washed with 20 ml normal saline afterwards. All eyes were examined thoroughly on the first 7 days and one month after surgery; 48 eyes (88.9%) at 3 and 6 months postoperatively. Hanna grading (in the scale of 0 to 4+) was used for assessment of corneal haze. Results The mean spherical equivalent refraction (SE) was -7.08 diopters (D) ± 1.11 (SD) preoperatively. Six months after surgery, 37 eyes (77.1%) achieved an uncorrected visual acuity (UCVA) of 20/20 or better, all eyes had a UCVA of 20/40 or better and 45 (93.7%) eyes had an SE within ± 1.00D. One month postoperatively, 2 eyes (3.7%) had grade 0.5+ of haze, while at 3 and 6 months after surgery no visited eye had haze at all. All eyes had a best corrected visual acuity (BCVA) of 20/40 or better and there were no lost lines in BCVA by 6 months after surgery. In spatial frequencies of 6 and 12 cycles per degree contrast sensitivity had decreased immediately after PRK and it had increased 1.5 lines by the 6th postoperative month compared to the preoperative data. Conclusions The results show the efficacy of mitomycin-C in preventing corneal haze after treatment of high myopia with PRK. This method- PRK + mitomycin-C – can be considered an alternative treatment for myopic patients whose corneal thicknesses are inadequate for laser in situ keratomileusis (LASIK). However, the results should be confirmed in longer follow-ups. ==== Body Background In refractive surgery, patients with -5.00 diopters (D) myopia or more and corneal thicknesses less than 500 μm are not suitable candidates for laser in situ keratomileusis (LASIK) or conventional photorefractive keratectomy (PRK) due to the inadequate corneal thickness and risk of haze.[1-13] There are patients in which LASIK may seem possible, but the low pachymetry reading limits us to a small ablation zone, and as a result some post-LASIK complications such as glare and halos may occur. [14] If haze following PRK can be prevented in myopic eyes with a spherical equivalent (SE) over -5.00 D, obviously there would be less concern about the corneal thickness, ablation zone, and even the flap induced aberrations following Lasik. [15] Haze after PRK may result from the corneal wound healing process. In animal models, it has been shown that this process is probably initiated by keratocyte apoptosis and the subsequent over-proliferation of cells. [16] Collagen type IV alpha 3 is also an important factor in the development of corneal haze after PRK. [16] Mitomycin-C is an antibiotic with anti-metabolite effects that inhibit the proliferation of keratocytes,[17] but it has no effect on normal epithelial cells of the cornea. Mitomycin-C 0.02% has been used in the treatment of post-PRK haze. [18] There are also some reports on the prophylactic use of mitomycin-C to prevent haze following PRK in moderate to high myopia. [14,19] In this prospective non-controlled clinical study we have assessed the prophylactic application of mitomycin-C on regression and haze in PRK performed on patients with high myopia (SE ≥ -5.00 D). Methods Study design and patient selection Fifty-four eyes in 28 patients with a spherical equivalent refraction over -5.00 D were included in this prospective non-controlled clinical study. Since the residual stromal bed thickness under the flap after LASIK would be less than 250 μm, no eye had an appropriate corneal thickness for this procedure. In planning PRK, the post-ablation corneal thickness was calculated to be greater than 350 μm. Exclusion criteria in this study were systemic or ocular disease with the potential to interfere with the healing process of the cornea, such as collagenosis, diabetes, dry-eye syndrome, anterior or posterior uveitis, ectatic diseases such as keratoconus, and also corneal dystrophy or degeneration, glaucoma, retinal diseases, lens opacity, history of severe ocular trauma, and previous ocular surgery. The study was approved by the Research and Ethics Committee of the Noor Vision Correction Center. Before the operation, the nature of the procedures, their results and complications were thoroughly explained to all patients and they were asked to read and sign a formal informed consent in their native language. Procedure All treatments were performed between April and October 2002. All eyes underwent the standard PRK procedure by two surgeons with extensive experiences with PRK. Patients received topical anesthesia without systemic sedation. Pre-incision of the corneal epithelium was made using a microtrephine with an 8 mm diameter and 70 μm deep calibrated blade (Janach, J 2900S). The epithelium was removed mechanically with a hocky knife. Then, the ablation was performed using a Technolas 217-C excimer laser (Bausch and Lomb, CA, USA). In all treatments, the overall ablation diameter was 8.4 to 8.9 mm and consisted of a central optical zone of 5.5 to 6.0 mm respectively by a 2.9 mm transition zone. Considering our previous experiences, eyes receiving mitomycin-C were intentionally under-corrected by 5 % compared with LASIK nomogram. Immediately after laser ablation, a single topical application of mitomycin-C 0.02% (0.2 mg/ml) diluted in balanced salt solution was instilled in each eye with a weck sponge placed over the ablated stroma for 2 minutes. The corneal surface and the entire conjunctiva were then vigorously irrigated with 20 ml cold normal saline to remove the residual mitomycin-C. At the end of the procedure, a bandage contact lens was applied which was removed after three days. After surgery, patients were instructed to take an analgesic (diclofenac sodium) every 8 hours and all eyes received artificial tears and flourometholone eye drop every four hours for 2 weeks, and chloramphenicol eye drop every 6 hours for 3 days. During the next two weeks, all patients were treated with flourometholone and artificial tears every 6 hours. These two eye drops were used every 8 and 12 hours during the second and third postoperative months, respectively, and were then discontinued. All patients were instructed to wear sunglasses in direct sunlight for 3 months. The preoperative visit included uncorrected visual acuity (UCVA), best-corrected visual acuity (BCVA), manifest, subjective, and cycloplegic refractions, slit-lamp exams, applanation tonometry, corneal topography, ultrasonic pachymetry, keratometry, indirect funduscopy, and contrast sensitivity with and without glare. UCVA and BCVA were tested with the Snellen chart. On the first seven days after surgery, all patients were examined with a slit lamp and the area of the epithelial defect was measured with its ruler to identify the time of complete reepithelialization. On the 7th and 14th days after surgery we measured the UCVA, BCVA, and the manifest, subjective, and cycloplegic refraction. At the 1st, 2nd, and 6th postoperative months, exams and measurements included UCVA, BCVA, manifest, subjective, and cycloplegic refraction, slit-lamp exams, applanation tonometry, ultrasonic pachymetry, keratometry, topography, and contrast sensitivity with and without glare. For evaluation of haze we used Hanna's grading scale from 0 (no haze) to 4+ (dense white corneal haze). Contrast sensitivity was tested by Vector Vision CSV-1000 (Vector Vision, Dayton, OH) in spatial frequencies of 6 (B) and 12 (C) cycles per degree. [20] The manufacturer's recommended testing procedures were followed. Absolute values of log contrast sensitivity were obtained for each patient and spatial frequency, and means and standard deviations were calculated. Data were then expressed in the notation of normalized log contrast sensitivity values. [21] Statistical analysis Repeated measures analysis of variance was used to assess changes over time after surgery. For all statistical tests, the significance level was considered 0.05. Results Of the 28 study group patients, 3 did not show up on the 3rd and 6th months visits (48 eyes out of 54 were visited; 88.9%). The mean age was 29.3 years (range, 20 to 45 years). The mean preoperative SE was -7.08 D ± 1.11 (SD) (range, -9.88 to -5.00D). The mean preoperative corneal thickness was 488.6 μm ± 11.9 (SD). At the first, 3rd, and 6th month after surgery, the mean corneal thickness was 357 μm ± 30 (SD), 373 μm ± 27 (SD), and 380 μm ± 28 (SD), respectively. Thirty-three eyes (61.1 %) had a preoperative BCVA of 20/20 or better, and 51 eyes (94.4 %) had a BCVA of 20/25 or better. Complete reepithelialization was seen in no eye by the 2nd postoperative day but in all eyes by the 6th day. The epithelial defect had resolved in 53.7 % of the eyes by the 3rd day, and in 92.6 % by the 4th postoperative day. Figure 1 shows the refractive results in this study group. At the 3rd postoperative month, 33 eyes (68.7 %) were within ± 0.5 D of emmetropia (SE) and 43 eyes (89.6 %) within ± 1.00D (SE). At the 6th postoperative month 39 eyes (81.3 %) were within ± 0.5D of emmetropia and 45 eyes (93.7 %) within ± 1.00D. Figure 2 shows the refractive changes after surgery during these six months. The refraction of patients had already become stable one month after surgery. At the 3rd postoperative month 32 eyes (66.7 %) had a UCVA of 20/20 or better and 47 (97.7 %) of 20/40 or better. All eyes had a BCVA of 20/40 or better and there were no lost lines in BCVA. On the 6th month visit, 37 eyes (77.1 %) had a UCVA of 20/20 or better and 48 (100%) of 20/40 or better. All eyes had a UCVA of 20/40 or better. On the first month follow-up visit, 2 eyes (3.7%) had +0.5 haze, but all had 0 haze on the 3rd and 6th month visits. Figure 3 shows the changes of contrast sensitivity in spatial frequencies of 6 and 12 cycles per degree (CPD) with and without glare over time. In the spatial frequency of 6 CPD, contrast sensitivity had decreased immediately after PRK and it had increased by the 6th postoperative month compared to preoperative data while in the spatial frequency of 12 CPD, it had increased over time after surgery (P < 0.001). In our study, no complications such as eccentric ablation, delayed reepithelialization, persistent epithelial defect, or microbial keratitis have been observed. Discussion PRK in high myopia remains a challenge due to its complications of haze and regression reported in previous experiments, and also the success rate of LASIK in these patients. [19] Yet, LASIK cannot be performed in some patients with an insufficient corneal thickness resulting in an undesirable residual stromal bed, or smaller ablation zones had to be applied to correct the refractive error completely. Such a small ablation zone can lead to visual inconveniences such as impaired night vision when the pupil dilates, halos, blurred vision, and ghost images. [15,22] If PRK plus mitomycin-C safety and predictability can be verified, larger ablation zones can be used, therefore the above complications will practically be avoided. On the other hand, correction of higher refractive errors will be possible. This study was carried out to evaluate the safety and efficacy of PRK along with a 2-minute application of mitomycin-C 0.02% (0.2 mg/ml) on the exposed stromal bed after ablation was performed. Mitomycin-C is an antimetabolite and antibiotic drug. It is mostly used systemically in cancer chemotherapy. It has been used in ophthalmology in cases of intraepithelial neoplasms of the cornea and conjunctiva, ocular pemphygoid, and following surgical treatment of glaucoma and pterygium. Mitomycin-C has cytotoxic effects through inhibiting DNA synthesis. The logic behind using mitomycin-C is that the topical application of the drug on the cornea can inhibit subepithelial fibrosis through preventing the proliferation of stromal keratocytes, while the main causes of regression and haze are overacitvity and proliferation of stromal keratocytes following laser ablation. [19] The effects of mitomycin-C 0.02% in preventing haze has been shown by Talamo et al [22], and Xu et al [23] in experimental models. In a study by Majmudar et al, it was concluded that the application of mitomycin-C can remove haze following PRK and radial keratectomy (RK). [18] The usefulness of PRK with mitomycin-C (0.2 mg/ml) for preventing haze in high myopia was reported by Carones et al. [19] In this study we used the same concentration of 0.2 mg/ml to perform PRK + mitomycin-C in high myopia; considering the different climate and races in this Middle Eastern region. It seems that the problems of haze and regression after PRK are more prominent in people of this region. [24] In addition, the inclusion criteria aimed at patients who were at a relatively high risk of haze formation after PRK. Among factors determining the ablation depth, an important predictor of haze, correction and ablation zone size are more important. A 6.0 mm optical zone and a minimum of 5.00 diopters of correction ethically and practically limited our study to those patients who were at a high risk of developing haze with PRK. No immediate toxic effects such as conjunctival chemosis or any delay or irregularity in re-epithelialization were seen. In only one case, after primary reepithelialization a large epithelial defect was seen on the fourth day (one day after complete epithelialization and contact lens removal), which healed in three days by applying a bandage contact lens. No complications such as corneal edema, melting, and perforation were observed in follow-up visits up to six months. Therefore, the topical application of mitomycin-C of the aforementioned concentration and duration was safe in our series. Yet, a longer follow-up is required to assess the long-term complications of mitomycin-C, and its careful application is recommended before long-term complications can be ruled out. According to our previous experiences, we planned a 5% under-correction compared to our usual nomogram for LASIK. As a result of this modification, patients were close to emmetropia with a low variability on their 6th month visit. In the report by Carones, patients had a hyperopic shift of +0.5D by the sixth month; this is while our patients had a myopic shift of 0.43D. The UCVA at six months after surgery was 20/40 or better in 100% and 20/20 or better in 77.1% of patients. The refractive and visual results of this study are much better compared to reports concerning the same amount of correction with LASIK, [25,26] PRK, or LASEK [27,28]. Considering the fact that haze influences BCVA, patients' BCVA is of great importance. In this study, no eyes lost any lines of BCVA by six months after surgery compared to the preoperative BCVA. None of the eyes had any grade of haze 3 or 6 months after surgery, which shows the efficacy of prophylactic application of mitomycin-C in preventing haze following PRK for patients with myopia over -5.00D. In addition, an average postoperative improvement in contrast sensitivity of 1.5 lines points to the safety and efficacy of the procedure in the quality of vision. In conclusion, using mitomycin-C in PRK for myopia greater than -5.00D seems safe and effective, and can reduce haze formation after surgery; therefore it can be considered a suitable alternative for patients with myopia greater than -5.00 D whose corneas lack an appropriate thickness to perform LASIK with a desirable optical zone. With this method, vision can be corrected with a better quality of vision regarding contrast sensitivity. Competing interests None declared. Authors' contributions HH, SMRT, AF and AK participated in the design of the study and the preparation of the manuscript. HH carried out all operations. SMRT visited patients in the follow up visits. AF and AK participated in the statistical analysis of the study. All authors have read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: Figures and Tables Figure 1 Scatter plots of attempted refractive changes versus the achieved refractive changes at 6 months after surgery (D = diopter). Figure 2 Mean manifest spherical equivalent refraction changes (diopter) over time. Figure 3 The changes of contrast sensitivity in spatial frequencies of 6 and 12 cycles per degree with and without glare over time (CPD = cycles per degree; M = month; D = diopter). ==== Refs Sher NA Barak M Daya S Excimer laser photorefractive keratectomy in high myopia: a multicenter study Arch Ophthalmol 1992 110 935 943 1637278 Dausch D Klein R Schroeder E Dausch B Excimer laser photorefractive keratectomy with tapered transition zones for high myopia: a preliminary report of 6 cases J Cataract Refract Surg 1993 19 590 594 8229712 Heitzman J Binder P Kasser B Nordon L The correction of high myopia using excimer laser Arch Ophthalmol 1993 111 1627 1634 8155032 Ditzen K Anschutz K Schroeder E Dausch B keratectomy to treat low, medium and high photorefractive myopia J Cataract Refract Surg 1994 20 234 236 8006793 Snibson C Carson G Aldred H Taylor H One year of photorefractive keratectomy for myopia evaluation and myopic astigmatism Arch Ophthalmol 1995 113 994 1000 7639676 Carson CA Taylor HR Excimer laser treatment for high and extreme myopia Arch Ophthalmol 1995 113 431 436 7710391 Teal P Breslin C Arshinoff S Edmison D Corneal subepithelial infiltrates following excimer laser photorefractive keratectomy J Cataract Refract Surg 1995 21 516 518 7473111 Kim JH Sah WJ Park CK Hahn TW Kim MS Myopic regression after photorefractive keratectomy Ophthalmic Surg Lasers 1996 27 S435 439 8724148 Probst LE 5thMachat JJ Corneal subepithelial infiltrates following photorefractive keratectomy J Cataract Refract Surg 1996 22 281 8778354 O'Brart DP Corbett MC Verma S Heacock G Oliver KM Lohmann CP Kerr Mulr MG Marshall J Effects of ablation diameter, depth, and edge contour on the outcome of photorefractive keratectomy J Cataract Refract Surg 1996 12 50 60 Pop M Aras M Multizone / Multipass photorefractive keratectomy: 6 month results J Cataract Refract Surg 1995 21 633 643 8551440 Zato MA Matilla A Gomez T Jimenez V Multizone versus monozone in the treatment of high and moderate myopia with an excimer laser Ophthalmic Surg Lasers 1996 27 S466 470 8724153 Maldonado MJ Arnau V Navea A Martinez-Costa R Mico FM Cisneros AL Menezo JL Direct objective quantification of corneal haze after Excimer laser photorefractive keratectomy for high myopia Ophthalmology 1996 103 1970 1978 8942897 McCarty CA Aldred GF Taylor HR Comparison of results of excimer laser correction of all degrees of myopia at 12 months postoperatively. The Melbourne Excimer Laser Group Am J Ophthalmol 1996 121 372 383 8604730 Holladay JT Dudeja DR Chang J Functional vision and corneal changes after laser in situ keratomileusis determined by contrast sensitivity, glare testing, and corneal topography J Cataract Refract Surg 1999 25 663 669 10330642 10.1016/S0886-3350(99)00011-5 Winker Von Mohrenfels C Reischl U Lohmann CP Corneal haze after photorefractive keratectomy for myopia: role of collagen IV mRNA typing as a predictor of haze J Cataract Refract Surg 2002 28 1446 1451 12160818 10.1016/S0886-3350(02)01273-7 Katzung BG Clinical pharmacology 1988 San Mateo, CA: Appleton and Lange Majmudar PA Forstot SL Dennis RF Nirankari VS Damiano RE Brenart R Epstein RJ Topical mitomycin-C for subepithelial fibrosis after refractive corneal surgery Ophthalmology 2000 107 89 94 10647725 10.1016/S0161-6420(99)00019-6 Carones F Vigo L Scandola E Vacchini L Evaluation of the prophylactic use of mitomycin-C to inhibit haze formation after photorefractive keratectomy J Cataract Refract Surg 2002 28 2088 2095 12498842 10.1016/S0886-3350(02)01701-7 Montes-Mico R Charman WN Choice of spatial frequency for contrast sensitivity evaluation after corneal refractive surgery J Refract Surg 2001 17 646 651 11758982 Wachler BS Krueger RR Normalized contrast sensitivity values J Refract Surg 1998 14 463 466 9699172 Talamo JH Gollamudi S Green WR De La Cruz Z Filatov V Stark WJ Modulation of corneal wound healing after excimer laser keratomileusis using topical mitomycin-C and steroids Arch Ophthalmol 1991 109 1141 1146 1907822 Xu H Liu S Xia X Huang P Wang P Wu X Mitomycin-C reduces haze formation in rabbits after excimer laser photorefractive keratectomy J Refract Surg 2001 17 342 349 11383766 Hashemi H Fatehi F Results of photorefractive Keratometry (PRK) for high myopia in Noor Clinic of Tehran The Journal of School of Medicine 2000 58 24 28 Stojanovic A Nitter TA 200 Hz flying-spot technology of the Laser Sight LSX excimer laser in the treatment of myopic astigmatism, six and 12 month outcomes of laser in situ keratomileusis and photorefractive keratectomy J Cataract Refract Surg 2001 27 1263 1277 11524200 10.1016/S0886-3350(01)00996-8 Rashad KM Laser in situ keratomileusis for myopic astigmatism J Refract Surg 1999 15 653 660 10590003 Nagy ZZ Fekete O Suveges I Photorefractive Keratectomy for myopia with the Meditec MEL 70 G-Scan flying spot laser J Refract Surg 2001 17 319 326 11383763 Lee JB Seong GJ Lee JH Seo KY Lee YG Kim EK Comparison of laser epithelial keratomileusis and photorefractive keratectomy for low to moderate myopia J Cataract Refract Surg 2001 27 565 570 11311625 10.1016/S0886-3350(00)00880-4

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==== Front BMC BioinformaticsBMC Bioinformatics1471-2105BioMed Central London 1471-2105-5-1341538314310.1186/1471-2105-5-134Methodology ArticlePATTERNFINDER: combined analysis of DNA regulatory sequences and double-helix stability Huang Yanlin [email protected] David [email protected] Cancer Genetics Department, Roswell Park Cancer Institute, Buffalo, NY 14263, USA2 Microsoft Corp., Redmond, WA 98052, USA2004 21 9 2004 5 134 134 10 5 2004 21 9 2004 Copyright © 2004 Huang and Kowalski; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Regulatory regions that function in DNA replication and gene transcription contain specific sequences that bind proteins as well as less-specific sequences in which the double helix is often easy to unwind. Progress towards predicting and characterizing regulatory regions could be accelerated by computer programs that perform a combined analysis of specific sequences and DNA unwinding properties. Results Here we present PATTERNFINDER, a web server that searches DNA sequences for matches to specific or flexible patterns, and analyzes DNA helical stability. A batch mode of the program generates a tabular map of matches to multiple, different patterns. Regions flanking pattern matches can be targeted for helical stability analysis to identify sequences with a minimum free energy for DNA unwinding. As an example application, we analyzed a regulatory region of the human c-myc proto-oncogene consisting of a single-strand-specific protein binding site within a DNA region that unwindsin vivo. The predicted region of minimal helical stability overlapped both the protein binding site and the unwound DNA region identified experimentally. Conclusions The PATTERNFINDER web server permits localization of known functional elements or landmarks in DNA sequences as well as prediction of potential new elements. Batch analysis of multiple patterns facilitates the annotation of DNA regulatory regions. Identifying specific pattern matches linked to DNA with low helical stability is useful in characterizing regulatory regions for transcription, replication and other processes and may predict functional DNA unwinding elements. PATTERNFINDER can be accessed freely at: ==== Body Background Regulatory regions in DNA are comprised of multiple functional elements that act in cis to control transcription, replication and other biological processes. Types of cis-acting DNA elements include unique sequences that bind specific proteins, spacer elements that provide a proper distance for interaction among protein binding sites, and structural elements that determine the flexibility of the DNA sequence. The structure of DNA is crucial to genetic regulation. The double helix needs to be locally unwound at start sites for DNA replication [1]. Additionally, DNA unwinding, flexibility and topology are important in regulating gene transcription and other processes [2]. Little is known about functional elements in most gene regulatory regions. Annotation of regulatory regions lags far behind the annotation of features of coding regions in DNA sequence databases. Best characterized are specific sequences that bind known proteins in vitro and whose functional importance has been determined by mutational analysis in vivo. Not as well characterized, but also important, are less-specific sequences that play roles in determining DNA structure and flexibility. DNA in certain regulatory sequences has a low helical stability as revealed by hypersensitivity to single-strand-specific nucleases and by stable DNA unwinding seen in plasmid topoisomers [3,4]. Helical stability minima computed using thermodynamic properties of nearest-neighbor nucleotides correctly predict the locations and hierarchy of the nuclease-hypersensitive sites [5]. Mutational analysis has revealed a functional region of low helical stability, called a DNA unwinding element, in replication origins in several species [6-9]. Some regulatory sequences with a low helical stability have been demonstrated to adopt an unwound DNA structure inside cells [10,11]. Low helical stability of DNA is of general importance since it is a feature of regulatory sequences involved in a variety of cellular processes including gene transcription, replication, nuclear matrix attachment and DNA repeat instability [6,10,12-14]. Progress towards characterizing and predicting regulatory regions could be accelerated by the availability of computer programs that perform a combined analysis of DNA sequence patterns and DNA unwinding properties. Here we present PATTERNFINDER, an easy to use web server that combines the search for specific or flexible sequence patterns with an analysis of DNA helical stability. The output of a DNA pattern search can be linked to THERMOCALC, a new program which ranks the helical stabilities of pattern matches, and an enhanced version of WEB-THERMODYN [15], which profiles the helical stability of individual matches and finds the most easily-unwound sequence. Below is information on using the PATTERNFINDER web server as well as an example analysis of the multi-functional regulatory region of a human proto-oncogene, c-myc. Results PATTERNFINDER Web Server Web servers offer advantages to the user including platform-independent software that requires no installation and a friendly browser interface for data entry and display. The PATTERNFINDER web server searches DNA sequences for matches to specific or flexible patterns, and analyzes DNA helical stability within or flanking the matches. Examples of specific patterns include sequences known to be recognized by particular proteins or enzymes, or sequences complementary to oligonucleotide primers. Flexible patterns include, for example, consensus sequences containing ambiguous nucleotides and unspecified nucleotides (N's), or multiple specific patterns separated by a variable number of N's, or simple sequences that are repeated a variable number of times. The DNA pattern used in the search can be comprised of A, G, C, T, ambiguous nucleotides (IUPAC-IUB nomenclature), and N's. Tables listing abbreviations of ambiguous nucleotides (e.g., W = A or T) and annotations for patterns using N's and sequence repeats are provided on the home page of the web server [16]. Mismatches are not permitted. Including N's in a pattern query is useful for retrieving sequences adjacent to a pattern match or known landmark (e.g., N{100}GAATTC = 100 bp sequence 5' to GAATTC) for further analysis. N's can also be used to fix or vary spacing between patterns, as indicated on the home page. The maximum pattern span entry must include the total number of nucleotides in the pattern, including repeats and N's (span = 106 in the example above). The span entry can not exceed the total length of the DNA sequence or region analyzed. Users input the name of the DNA molecule, the shape (linear or circular) and the nucleotide sequence. The sequence can be uploaded from a computer file (≤40 kb) in a various user-selected formats (ASCII text, Genbank, Fasta). Alternatively, the sequence can be pasted (≤30 kb) or typed into the DNA Sequence Query window of the browser. Acceptable characters are A, G, C, and T. Integers used for sequence numbering are permitted. Under the "Analysis Parameters" the user can select to search the whole DNA molecule or a part of the molecule from one position to another. Both DNA strands are searched by default in order to find matches to asymmetric patterns. If desired, the search can be restricted to only the upper (+, input) or lower (-, complementary) strand by selecting the appropriate checkbox. The output displays the molecule name, size in bp, and size of the region analyzed, and the DNA sequence of the upper and lower strands. Note that all DNA sequences in the output are displayed 5'->3' regardless of strand. This is useful for creating sequence alignments. The total number of hits and the pattern query are displayed. Tabulated are the position, strand (+ or -) and sequence of all pattern matches. The data are also output to an ASCII text file for printing, archiving and for any further processing (e.g., in a spreadsheet). A BATCH version of PATTERNFINDER is included to search simultaneously for multiple patterns in a DNA sequence. Entries are pasted or typed into the BATCH query window, one per line. Entries take the form of "Pattern name, Pattern expression, Maximum span". A table of pattern names and expressions that were entered is included in the output. The tabular output of hits is similar to that for PATTERNFINDER (above) except that it also includes the names of patterns that are hit as well as those that are not hit. The ASCII output provides a tabular map of the positions and strands of different pattern matches found in the DNA sequence. In addition to flexible sequence patterns, BATCH lists can include a variety of specific DNA elements and landmarks such as protein binding sequences, restriction sites and oligonucleotide primers. Low helical stability of DNA is often a property of regulatory sequences [3,5,6]. A unique feature of PATTERNFINDER is that the output can serve as input for analysis of double-helix stability within or flanking the pattern matches. Selecting the appropriate checkbox on the input page enables THERMOCALC or WEB-THERMODYN to process the output of the pattern search. THERMOCALC is a new program that analyzes multiple DNA sequences in a user-selected, fixed window and ranks the free energy values. WEB-THERMODYN performs a sliding-window analysis of a DNA sequence to profile the helical stability and to identify the most easily-unwound sequence (free energy minimum). The utility of the published WEB-THERMODYN program [15] has been enhanced. The results of a PATTERNFINDER sequence search are directly output into an new input page for WEB-THERMODYN that accomodates multiple sequence entries, permiting the facile and rapid analysis of multiple pattern hits. The algorithm used to calculate free energy (ΔG) from thermodynamic parameters of nearest-neighbor nucleotides is described on the web server [17]. Briefly, the standard entropy and enthalpy values for each of the ten possible nearest-neighbor nucleotide interactions present in a DNA sequence are individually summed and then used to calculate the free energy using the equations previously described [9]. The WEB-THERMODYN program has also been upgraded to include the unified thermodynamic data set of SantaLucia [18]. This is now the current default data set since it represents a consensus agreement among six independent data sets and has been found to rank the free energies of DNA sequences even more accurately than the previous data set [18]. A drop down menu permits selection of the current default or the previous data set. Default values for temperature and salt concentration [3] are present on the input page and these values can be altered by the user if desired. The input value for the start position is used by THERMOCALC to calculate ΔG from that position to the end of the sequence hit. Input values for Start Position, Step Size, Window Size and Number of Markers at minimum energy windows are used by WEB-THERMODYN, which also provides links to the DNA sequences at energy minima and a graphical profile of helical stability. Enabling WEB-THERMODYN also permits further adjustment or variations of the parameters for helical stability analysis on the PATTERNFINDER output page. PATTERNFINDER was designed to be fast and user friendly. No password restriction or registration is required. All entries are error checked before processing and, if errors exist, the user is prompted with specific suggestions to correct the entries. The output is returned directly to the browser in real time, as opposed to a return via e-mail at a later time. The home page of the web server provides a DEMO link [19] containing sequence and pattern files relevant to the application that follows. PATTERNFINDER analysis of the regulatory region of the human c-myc gene The 5' regulatory region of the c-myc gene resides in the first 2500 bp of an ~11 kb DNA sequence. The sequence contains multiple elements that regulate transcription and replication [10,12], but the locations of the DNA elements are not yet annotated in public databases. The far upstream sequence element (FUSE) acts in cis to stimulate c-myc promoter activity and a 42 bp sequence becomes single-stranded in vivo (HeLa cells) when the gene is actively transcribed [10]. The single-stranded region is located primarily 3' to a specific Ava I restriction enzyme site. PATTERNFINDER was used to search for specific Ava I sites and any ("n") 36 bp sequences in a 42 bp segment (pattern: CCCGAGn{36}; span = 42) with THERMOCALC enabled to rank the DNA helical stabilities of the segments. The search was restricted to the regulatory region from positions 1 to 2500 (entered under the "Analysis Parameters"). The output is shown in Fig. 1A. The locations and strands of three Ava I sites found and the helical stability ranks (free energy, kcal/mol) of the 42 bp segments are displayed. The site with rank 1 has the lowest free energy and begins at position 789 on the + strand. As shown below, this predicted region of low helical stability contains the single-stranded DNA region identified experimentally in the FUSE. To map the locations of other DNA elements in the 5' regulatory region, the BATCH version of PATTERNFINDER was used to search simultaneously for multiple, specific sequences. The patterns included two separate sequences that interact with the FUSE binding protein (FBP) in domains 3 and 4 [20], the Ava I site, and sequences of the -10 regions at the P1 and P2 promoters for c-myc transcription [10]. The BATCH output in Fig. 1B shows the precise locations and strands of the DNA elements in relation to the Ava I sites, providing an informative tabular map of functional elements and landmarks in the regulatory region. In conjunction with PATTERNFINDER, WEB-THERMODYN was used to profile the DNA helical stability of all 42 bp windows in a ~400 bp region containing the binding site for FBP domain 3 at the center. The pattern query used was n{200}TAAAAAATn{200} and the span = 408. This query finds the complementary sequence (+ strand) to the FBP_3 site (ATTTTTTA, - strand) and retrieves the 200 nt sequences 5' and 3' for the WEB-THERMODYN analysis (Step Size = 1, Window Size = 42, Number of Markers = 1[energy minima]). Fig. 1C shows that the sequence with the minimum free energy (ΔG Min) in the 408 bp region analyzed overlaps most of the single-stranded DNA region detected experimentally at FUSE in vivo (FUSE ssDNA), reflecting a reduced helical stability intrinsic to DNA at FUSE. A separate WEB-THERMODYN analysis of the entire 2500 bp regulatory region identified the identical sequence as the ΔG Min, indicating that the low helical stability at FUSE is unique within the entire 5' regulatory region. The location of the ΔG Min sequence (+ strand shown in Fig. 1C) also overlaps the single-stranded DNA binding sites for FBP_3 and FBP_4 present in the -strand. The values of ΔG Min and ΔG FUSE ssDNA are well below the average ΔG (29.44 kcal/mol) for all 42 bp windows for the entire 2500 bp region. The predicted DNA sequence with the lowest helical stability in the 5' regulatory region contains the single-stranded DNA region detected experimentally at FUSE [10]. Discussion The PATTERNFINDER web server provides a convenient means to search DNA regulatory regions for specific or flexible sequence patterns and to identify flanking sequences that are easy to unwind. Specific sequence searches permit localization of known protein binding sites and sequence landmarks such as restriction sites and oligonucleotide primers. The capacity to perform BATCH analysis of multiple patterns and generate accurate tabular maps facilitates the annotation of DNA regulatory regions. The latter lags far behind the annotation of protein coding regions in public sequence databases, and individual laboratories must usually draw on their own annotation resources in order to design experiments. The utility of PATTERNFINDER in annotation of known DNA elements can facilitate the design of experiments to further characterize regulatory regions. New DNA elements with potential functions can be predicted using flexible patterns such as consensus sequences containing ambiguous nucleotides and N's as well as using multiple motifs with fixed or variable spacing. Sequences found are displayed 5'->3' regardless of the DNA strand which is useful for creating sequence alignments for position weight matrices and for identifying evolutionary conserved sequences. More sophisticated pattern match and discovery methods exist ([21], and references therein) and advanced versions of PATTERNFINDER that permit base mismatches and utilize weight matrices are under development. A strength of the current version of PATTERNFINDER is the capacity to identify pattern matches that are linked to DNA sequences of low helical stability. Such a combined search for two different types of DNA elements can lead to predictions of greater specificity than can be obtained by searching for either element alone and may help predict functional DNA unwinding elements associated with specific protein binding sites. The availability of PATTERNFINDER will hopefully stimulate experiments to verify the biological function of predicted DNA elements. The ability to find DNA sequences and to target the flanking regions for helical stability analysis is a unique feature of PATTERNFINDER. This feature is useful in characterizing regulatory regions for gene transcription and DNA replication which require specific protein-binding sequences as well as less-specific flanking sequences in which the double helix is easy to unwind. Helical stability is ranked by the free energy value beginning with the most easily-unwound sequence by enabling a new program, THERMOCALC. Helical stability is profiled to identify free energy minima by enabling an enhanced version of the previously described WEB-THERMODYN program [15]. As shown in the Results for the transcription regulatory region of the human proto-oncogene, c-myc, the predicted region of minimum helical stability overlaps significantly the FUSE single-stranded DNA region identified experimentally in vivo [10]. The predicted region of minimum helical stability also overlaps the protein binding sites for FBP domains 3 and 4 [20]. FUSE is an example of a DNA unwinding element that appears to function through both intrinsic helical instability and binding a protein, FBP, a single-strand-specific DNA binding protein with a non-processive helicase activity [10,22]. Our analysis of helical stability is general and makes no assumptions about the DNA unwinding mechanism. DNA opening at FUSE has been proposed to be induced by torsional stress generated by transcription of the c-myc gene [10]. Consistent with a role for torsional stress, an independent computer analysis predicted a high probability of DNA opening at FUSE at specific levels of negative supercoiling [23]. WEB-THERMODYN analysis predicted an overlapping sequence as the helical stability minimum of the c-myc regulatory region (Fig. 1C). When the DNA opening mechanism is initiated by torsional stress alone, helical stability analysis predicts the site that corresponds to the DNA sequence of lowest helical stability identified experimentally in negatively supercoiled DNA in vitro [3,5]. When the DNA opening mechanism requires prior binding of proteins to duplex DNA, as is the case for replication origins (see below), functional DNA opening in a cell can occur at a low helical stability sequence that does not necessarily correspond to the lowest-stability site that opens in negatively supercoiled DNA in vitro [24]. In the latter case, consideration of specific protein binding sequences in addition to the helical stability is required, as is done in PATTERNFINDER with THERMOCALC or WEB-THERMODYN enabled. Our computer analysis described here and that of He et al. [23] provide useful and complementary information about DNA regulatory sequences, and both analyses could be employed to take full advantage of their individual strengths. PATTERNFINDER will also have direct application in the further characterization of DNA replication origins, which are comprised of origin recognition elements, less-specific sequences including a DNA unwinding element, and additional elements such as transcription factor binding sites [1,6,7,11,25,26]. Interestingly, the transcription regulatory region of the c-myc gene also contains a replication origin, and a low helical stability region in the vicinity of FUSE has been suggested to function as a DNA unwinding element that facilitates replication initiation [12]. A functional role in initiation of c-myc replication for FBP, the single-strand DNA binding protein that binds FUSE and has a non-processive helicase activity [10,22], is not known. The function of several well-characterized replication origins requires an initiator protein complex that recognizes double-stranded DNA [1,11,25]. Also important is a DNA unwinding element [7], which must be properly spaced and oriented relative to the sequences that bind initiator proteins in certain origins [6,9]. PATTERNFINDER is capable of searching DNA sequences for all of the features of replication origins: specific sequences, less-specific sequences including those that are easily-unwound, and proper orientation and spacing between multiple types of sequences. Finally, PATTERNFINDER is likely to have applications in studying processes in addition to replication and transcription, such as nuclear matrix attachment and DNA repeat instability, which also involve specific DNA sequences and a region of low helical stability [13,14]. Conclusions The PATTERNFINDER web server will be useful in annotating, predicting and characterizing regulatory regions for replication, transcription and other processes that require specific or less specific sequences recognized by particular proteins and additional sequences in which the double-helix is unstable and functions in localized DNA unwinding. Methods The PATTERNFINDER program was written in Practical Extraction and Report Language (PERL) and HyperText Markup Language (HTML) and uses the Common Gateway Interface (CGI) for input and output to a web browser. The DNA sequence of the human c-myc gene was obtained from GenBank (accession number: X00364). The average ΔG for all windows was determined by analysis of the WEB-THERMODYN output using spreadsheet software. Authors' Contributions YH wrote the program code for PATTERNFINDER and established a web site. DK served as scientific advisor and participated in the program design and development. Acknowledgments This research was supported by a grant from NIH (GM30614) and in part by shared resources of an NIH Cancer Center Support Grant (P30 CA16056). We thank K. Pandian for technical assistance. This paper is dedicated to the memory of Professor Michael Laskowski, Jr., physical biochemist and mentor to DK. Figures and Tables Figure 1 Examples of analysis of the human c-myc gene regulatory region using PATTERNFINDER in conjunction with THERMOCALC (A), in BATCH mode (B), and with WEB-THERMODYN (C) [see text]. ==== Refs DePamphilis ML DePamphilis ML Origins of DNA Replication DNA Replication in Eukaryotic Cells 1996 Plainview, New York: Cold Spring Harbor Laboratory Press 45 86 Sinden RR DNA Structure and Function 1994 San Diego, CA: Academic Press Kowalski D Natale DA Eddy MJ Stable DNA unwinding, not "breathing," accounts for single-strand-specific nuclease hypersensitivity of specific A+T-rich sequences Proc Natl Acad Sci U S A 1988 85 9464 9468 2849106 Umek RM Kowalski D Thermal energy suppresses mutational defects in DNA unwinding at a yeast replication origin Proc Natl Acad Sci U S A 1990 87 2486 2490 2181439 Natale DA Schubert AE Kowalski D DNA helical stability accounts for mutational defects in a yeast replication origin Proc Natl Acad Sci U S A 1992 89 2654 2658 1557369 Kowalski D Eddy MJ The DNA unwinding element: a novel, cis-acting component that facilitates opening of the Escherichia coli replication origin Embo J 1989 8 4335 4344 2556269 Lin S Kowalski D Functional equivalency and diversity of cis-acting elements among yeast replication origins Mol Cell Biol 1997 17 5473 5484 9271423 Carles-Kinch K Kreuzer KN RNA-DNA hybrid formation at a bacteriophage T4 replication origin J Mol Biol 1997 266 915 926 9086270 Lin S Kowalski D DNA helical instability facilitates initiation at the SV40 replication origin J Mol Biol 1994 235 496 507 8289278 10.1006/jmbi.1994.1009 Michelotti GA Michelotti EF Pullner A Duncan RC Eick D Levens D Multiple single-stranded cis elements are associated with activated chromatin of the human c-myc gene in vivo Mol Cell Biol 1996 16 2656 2669 8649373 Speck C Messer W Mechanism of origin unwinding: sequential binding of DnaA to double- and single-stranded DNA Embo J 2001 20 1469 1476 11250912 10.1093/emboj/20.6.1469 Liu G Malott M Leffak M Multiple functional elements comprise a Mammalian chromosomal replicator Mol Cell Biol 2003 23 1832 1842 12589000 10.1128/MCB.23.5.1832-1842.2003 Kieffer LJ Greally JM Landres I Nag S Nakajima Y Kohwi-Shigematsu T Kavathas PB Identification of a candidate regulatory region in the human CD8 gene complex by colocalization of DNase I hypersensitive sites and matrix attachment regions which bind SATB1 and GATA-3 J Immunol 2002 168 3915 3922 11937547 Potaman VN Bissler JJ Hashem VI Oussatcheva EA Lu L Shlyakhtenko LS Lyubchenko YL Matsuura T Ashizawa T Leffak M Benham CJ Sinden RR Unpaired Structures in SCA10 (ATTCT)(n).(AGAAT)(n) Repeats J Mol Biol 2003 326 1095 1111 12589756 10.1016/S0022-2836(03)00037-8 Huang Y Kowalski D WEB-THERMODYN: sequence analysis software for profiling DNA helical stability Nucl Acids Res 2003 31 3819 3821 12824427 10.1093/nar/gkg562 PATTERNFINDER Web Server README Free Energy SantaLucia J Jr A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics PNAS 1998 95 1460 1465 9465037 10.1073/pnas.95.4.1460 Demonstration Files Braddock DT Louis JM Baber JL Levens D Clore GM Structure and dynamics of KH domains from FBP bound to single-stranded DNA Nature 2002 415 1051 1056 11875576 10.1038/4151051a Stormo GD DNA binding sites: representation and discovery Bioinformatics 2000 16 16 23 10812473 10.1093/bioinformatics/16.1.16 Vindigni A Ochem A Triolo G Falaschi A Identification of human DNA helicase V with the far upstream element-binding protein Nucleic Acids Res 2001 29 1061 1067 11222755 10.1093/nar/29.5.1061 He L Liu J Collins I Sanford S O'Connell B Benham CJ Levens D Loss of FBP function arrests cellular proliferation and extinguishes c-myc expression Embo J 2000 19 1034 1044 10698944 10.1093/emboj/19.5.1034 Umek RM Kowalski D The DNA unwinding element in a yeast replication origin functions independently of easily unwound sequences present elsewhere on a plasmid Nucleic Acids Res 1990 18 6601 6605 2174542 Bell SP The origin recognition complex: from simple origins to complex functions Genes Dev 2002 16 659 672 11914271 10.1101/gad.969602 Wang L Lin C-M Brooks S Cimbora D Groudine M Aladjem MI The Human β-Globin Replication Initiation Region Consists of Two Modular Independent Replicators Mol Cell Biol 2004 24 3373 3386 15060158 10.1128/MCB.24.8.3373-3386.2004

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==== Front BMC GenomicsBMC Genomics1471-2164BioMed Central London 1471-2164-5-701538002810.1186/1471-2164-5-70Research ArticlecDNA array-CGH profiling identifies genomic alterations specific to stage and MYCN-amplification in neuroblastoma Chen Qing-Rong [email protected] Sven [email protected] Jun S [email protected] Craig C [email protected] Nicola [email protected] Alexei L [email protected] Braden T [email protected] Chang-Gue [email protected] Frank [email protected] Frank [email protected] Manfred [email protected] Daniel [email protected] Javed [email protected] Oncogenomics Section, Pediatric Oncology Branch, Advanced Technology Center, National Cancer Institute, 8717 Grovemont Circle, Gaithersburg, MD 20877, USA2 Department of Internal Medicine, College of Oriental Medicine, Daejeon University, Daejeon 301-724, Korea3 Department of Cytogenetics, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany4 Department of Pediatrics, Klinik für Kinderheilkunde der Universität zu Köln, Joseph Stelzmann Straße 9, D-50924 Köln, Germany5 Tumour Bank, The Children's Hospital at Westmead, Locked Bag 4001, Westmead, NSW, 2145, Australia2004 20 9 2004 5 70 70 19 5 2004 20 9 2004 Copyright © 2004 Chen et al; licensee BioMed Central Ltd.2004Chen et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Recurrent non-random genomic alterations are the hallmarks of cancer and the characterization of these imbalances is critical to our understanding of tumorigenesis and cancer progression. Results We performed array-comparative genomic hybridization (A-CGH) on cDNA microarrays containing 42,000 elements in neuroblastoma (NB). We found that only two chromosomes (2p and 12q) had gene amplifications and all were in the MYCN amplified samples. There were 6 independent non-contiguous amplicons (10.4–69.4 Mb) on chromosome 2, and the largest contiguous region was 1.7 Mb bounded by NAG and an EST (clone: 757451); the smallest region was 27 Kb including an EST (clone: 241343), NCYM, and MYCN. Using a probabilistic approach to identify single copy number changes, we systemically investigated the genomic alterations occurring in Stage 1 and Stage 4 NBs with and without MYCN amplification (stage 1-, 4-, and 4+). We have not found genomic alterations universally present in all (100%) three subgroups of NBs. However we identified both common and unique patterns of genomic imbalance in NB including gain of 7q32, 17q21, 17q23-24 and loss of 3p21 were common to all three categories. Finally we confirm that the most frequent specific changes in Stage 4+ tumors were the loss of 1p36 with gain of 2p24-25 and they had fewer genomic alterations compared to either stage 1 or 4-, indicating that for this subgroup of poor risk NB requires a smaller number of genomic changes are required to develop the malignant phenotype. Conclusions cDNA A-CGH analysis is an efficient method for the detection and characterization of amplicons. Furthermore we were able to detect single copy number changes using our probabilistic approach and identified genomic alterations specific to stage and MYCN amplification. ==== Body Background Neuroblastoma (NB) is one of the most common pediatric solid tumors, and accounts for 7–10% of all childhood cancers. The prognosis of patients with NB varies according to the stage, age and MYCN amplification status. Stage 1 disease is essentially curable, whereas patients with stage 4 disease, in particular those with MYCN amplification, remain largely incurable despite advances in cancer therapeutics [1]. Genomic alterations in NB have been investigated by cytogenetic, and molecular methods including spectral karyotyping and metaphase comparative genomic hybridization (M-CGH) [2-6]. Based on these studies several genomic alterations have been reported to correlate with prognosis including amplification of the MYCN oncogene (found in 30% of NB) [1,7], gains of 17q (>50%) and loss of 1p36 (30–35%) [1,8,9]. Other recurrent changes including losses of 3p, 4p, 9p, 11q, and 14q, as well as frequent gain of chromosome 7 have also been suggested to have relevance to the development and progression of these tumors [9]. Recently array-based CGH (A-CGH) on BAC and cDNA microarrays has been used to investigate the genomic alterations with high resolution [10-14]. cDNA A-CGH has been successfully utilized to detect amplification and to investigate the direct effects of genomic changes over gene expression level by using the same microarray for both A-CGH and gene expression analysis [14-16]. In this study, we applied A-CGH, on cDNA microarrays containing 42,000 elements, to systematically identify common aberrant genomic alterations in NB of various stages. We have applied a probabilistic approach to detect single-copy losses and gains of chromosomal regions. Our study has three principal aims: 1) Detection and high resolution mapping of amplicons in NB. 2) Detection of low copy number genomic alterations using a probabilistic approach. 3) Establishing a map of genomic imbalances in NB profiling samples with good (stage 1) and poor (stage 4 with or without MYCN amplification) prognosis. Results Amplicon Mapping by A-CGH Totally around 24,000 qualified array cDNA clones were applied for data analysis in 12 NB cell lines and 32 NB primary tumor samples (see Table 1 for sample information). Fig. 1 shows the number of clones as well as the average spacing for each chromosome. We first determined the sensitivity of A-CGH to detect the copy number of highly amplified genes. We here chose MYCN since it is the most commonly amplified gene in NB and correlates with the biological behavior of these tumors. Fig. 2A shows the linear regression plot of the MYCN amplification results from A-CGH and Quantitative-PCR (Q-PCR). We found that the slope of the fitting line was 0.35, and therefore an observed ratio of 2 by A-CGH corresponds to Q-PCR ratio of ~6. In order to identify the amplified regions, we initially selected genes with A-CGH ratio ≥2 for at least two contiguous clones in genome sequence order. Only two chromosomes (2p and 12q) showed amplifications by this criterion exclusively in the MYCN amplified samples. Focusing on 2p (Fig. 2B), we found 6 independent non-contiguous amplicons (10.4–69.4 Mb). For the MYCN amplicon, the largest contiguous region was 1.7 Mb and bounded by NAG and an EST (clone: 757451) in three tumor samples, whilst the smallest region was 27 Kb including an EST (clone: 241343), NCYM, and MYCN. We identified 9 previously reported co-amplified genes (HPCAL1, ODC1, NSE1, NAG, DDX1, NCYM, POMC, DNMT3A, ALK, MEIS1, TEM8) [16,20-27], and detected the novel amplification of several known genes (NCOA1, ADCY3, PPP1CB, CGI-127, LBH, CAPN13, GalNac-T10, EHD3, XDH, SRD5A2, CGI-27, AMP18) and ESTs. Three of the cell lines (CHP134, IMR-5 and IMR-32) contained two amplicons in 2p13-15. The first (66.6–67.6 Mb) included previously reported amplified gene MEIS1, and the size of the second amplicon was 0.3 Mb (69.1–69.4 Mb), which was bounded by LOC200504 and TEM8. In addition to chromosome 2p, we identified another amplicon on 12q14-q15 in a single tumor (NB21); bounded by PRO2268 (68.9 Mb) and RAB3IP (69.9 Mb) containing one previously reported amplified gene (MDM2) [28] as well as several novel amplifications (CPM, CPSF6, LYZ, GAS41, SNT-1, CCT2, VMD2L3, and RAB3IP) (Fig. 2C). We verified the amplification of NSE1, NAG, DDX1, MYCN and TEM8 by Q-PCR (data not shown). Simultaneous gene expression profiling by using the same cDNA arrays for all samples showed that 47% of the amplified genes correlate with gene expression (using a correlation coefficient cutoff 0.5; data not shown). Table 1 Summary of Neuroblastoma Information Sample No Sample Label Age (yr.mo) Sex Diagnosis MYCN Source C1 CHP-134B 1.1 M 4 + NCI C2 GI-LI-N 1.11 M 4 + ICLC C3 IMR-32 1.1 M 4 + ATCC C4 IMR-5 ND ND ND + ICLC C5 LAN-1 2 M 4 + ICLC C6 LAN-5 0.4 M ND + NCI C7 SK-N-BE(2) 2.2 M 4 + ATCC C8 SK-N-DZ 2 F ND + ATCC C9 SMS-KCNR 1.2 M 4 + NCI C10 SH-SY5Y 4 F 4 - ATCC C11 SK-N-AS 8 F 4 - ATCC C12 SK-N-FI 11 M ND - ATCC C13 SK-N-SH 4 F 4 - ATCC T1 NB19 12.9 F 1 - DZNSG T2 NB20 1.3 M 1 - DZNSG T3 NB229 0.3 M 1 - CHTN T4 NB248 0.6 M 1 - CHW T5 NB29 0.3 M 1 - DZNSG T6 NB33 1.5 F 1 - DZNSG T7 NB34 1.2 M 1 - DZNSG T8 NB43 1.1 F 1 - DZNSG T9 NB44 1.6 M 1 - DZNSG T10 NB5 0.3 M 1 - DZNSG T11 NB7 1.3 F 1 - DZNSG T12 NB9 1.1 M 1 - DZNSG T13 NB16 3.11 F 4 - DZNSG T14 NB205 3.11 F 4 - CHTN T15 NB217 2 M 4 - CHTN T16 NB24 0.7 M 4 - DZNSG T17 NB246 3.7 M 4 - CHW T18 NB247 1.5 M 4 - CHW T19 NB26 1 M 4 - DZNSG T20 NB30 0.11 F 4 - DZNSG T21 NB31 1.4 F 4 - DZNSG T22 NB32 1.2 M 4 - DZNSG T23 NB35 2.7 F 4 - DZNSG T24 NB8 4.7 M 4 - DZNSG T25 NB14 0.11 M 4 + DZNSG T26 NB21 5.3 M 4 + DZNSG T27 NB249 0.8 M 4 + CHW T28 NB251 0.9 F 4 + CHW T29 NB252 0.10 F 4 + CHW T30 NB266 2 F 4 + CHW T31 NB27 10.6 M 4 + DZNSG T32 NB28 1.8 F 4 + DZNSG Abbreviations used are ND: not determined ATCC: American Type Culture Collection, CHTN: Cooperative Human Tissue Network CHW: The Children's Hospital at Westmead, Australia, DZNSG: German Cancer Research Center, Heidelberg, ICLC: Interlab Cell Line Collection, NCI: National Cancer Institute, NIH. 1Naming convention "C" denotes cell lines, "T" tumors, such that C1 is cell line number 1. Figure 1 Distribution of cDNA clones in our microarray. Total 23975 unique UniGene clusters remained from the initial 42591 clones after quality filtering. Number of clones in each chromosome was represented in gray bar on the left side. Average spacing (chromosome size/number of clones in the chromosome) was represented in black dot on the right side. Figure 2 Amplifications in MYCN amplified samples. A. Regression analysis of MYCN ratio (sample vs. normal) obtained from A-CGH and real-time Q-PCR in all of neuroblastoma samples including 13 cell lines and 32 primary tumors. The slope of the regression line is 0.35 indicating that an observed A-CGH ratio of 2 is equivalent to a Q-PCR ratio of 5.7. B. Independent amplicons in chromosome 2p. All amplified genes are listed under each amplicon in genome order. Map position, genome sequence position (Mb) and samples containing the specific amplicon are listed for each amplicon. The percentage of the MYCN amplified samples harboring these amplicons are shown in brackets following the gene name for all clones present in our microarray (gray), the remainder of the clones are predicted genes found in the NCBI database that are mapped between the boundaries of the amplicon. Amplicons were selected based on the criteria of A-CGH ratio ≥2 for at least two contiguous clones in genome sequence order. In cases where a single clone has a ratio <2 but the ratio of its adjacent clones is greater than 2, that single clone was still considered as a part of amplicon. *: previously reported amplification. C. Amplicon in chromosome 12q in tumor NB21. Detection of low-level DNA copy number alterations To test the sensitivity of A-CGH to detect single copy number changes, we performed A-CGH with DNA from cell lines containing different numbers of X chromosomes (1–5 copies) [12] and compared them to a sample with 2 copies of X chromosomes. The observed mean fluorescence ratio of all clones across X chromosome was calculated (Fig. 3A). For single copy deletions we observed an A-CGH ratio 0.9 (expected 0.5). The regression slope was 0.3, similar to that for the MYCN above (Fig 2A). The underestimation of the expected ratio by A-CGH demonstrated that it is difficult to detect single-copy changes using pre-set threshold-based approaches. Figure 3 Sensitivity of A-CGH to detect the low-level copy number alteration. A. Measurement of X-chromosomal copy number. A-CGH was performed to analyze the copy number of genes in the X-chromosome. Female DNA (XX) was used as the reference DNA. Male DNA (XY), female DNA (XX), and DNA samples containing different number of X-chromosome (XXX, XXXX, XXXXX) were used as test DNA, with an expected ratio of test/reference of 0.5, 1.0, 1.5, 2.0, and 2.5 respectively for X-chromosome. Mean fluorescence ratios (±SEM) of autosomal DNAs (blue diamonds) and X-chromosomal DNAs (red circles) from each experiment are shown. The slope of the regression line is 0.3. B. Visualization of p-values derived from the topological statistics as described in the Methods along the X-chromosome from samples containing different X-chromosomal copy numbers. Each column represents a different experiment; and each row represents the p value for the alteration at a given SW-locus (a sliding window of 40 adjacent clones, details in Methods), ordered by genome map position from Xpter to Xqter. Red represents gain and green loss. The intensity of the color shows the level of significance according to the p-value shown in the color scale. In order to increase the sensitivity for detecting low copy number changes, we applied a probabilistic approach utilizing t-statistics and the local genomic sequence mapping information of each of the cDNA clones on our arrays. To validate our method, we re-analyzed the A-CGH data generated from the cell lines containing 1–5 copies of the X chromosome as described above, and we were able to detect a single copy loss and gain of X chromosome where the expected ratio was 0.5 and 1.5 respectively (Fig. 3B). In addition, we used the reported results from the literature as an independent validation. The cell line SK-N-AS is deleted within 1p36.2-p36.3, which has been investigated by FISH and southern blot analysis [29,30]. The proximal SK-N-AS deletion breakpoint was mapped to between NPPA and PLOD, while the distal breakpoint is proximal of CDC2L1. The deletion detected by our method is bordered by KIAA0495 and CTNNBIP1, which is within the region reported. They are in a very good accordance. In addition, we also compared the 17q gain results for four NB cell lines (CHP134, IMR-5, SMS-KCNR, and SK-N-AS) with the results in literature by FISH and Q-PCR [31]. Our results confirmed the gains in 17q for all 4 cell lines and the loss in 17p in SK-N-AS detected by FISH. We next analyzed the A-CGH data using this method to detect genome-wide alterations of DNA copy number in our NB samples. Using this t-statistics, we identified DNA copy number alterations that involved the majority of the chromosomes in both primary tumors and cell lines (Fig. 4). We confirmed previously reported genomic changes, including gains of whole chromosome 1, 2, 6, 7, 8, 12, 13, 17, 18 and 22, and losses of 3, 4, 9, 11, and 14; partial gains of 1q, 2p, 11p, 12q and 17q; partial losses of 1p, 3p, 4p, 9p, 11q and 14q [9,32]. The most common changes were losses on chromosome 1p, 4 and 11q; gains on 2p, 7, and 17q. Figure 4 Genome-wide analysis of DNA copy number alteration by A-CGH. Samples were grouped based on sample type, MYCN amplification status and tumor stage. Each column represents a different sample; and each row represents a p-value of a given SW-locus using a sliding window of 40 adjacent clones, ordered in genome order across the whole genome. Black triangles on the right side of image represent centromere positions. Cell-: cell line without MYCN amplification; Cell+: cell line with MYCN amplification; Stage 4-: tumor in stage 4 without MYCN amplification; Stage 4+: tumor in stage 4 with MYCN amplification. On the right is shown an enlarged view of the region around the MYCN gene (2p24) for the amplified NB samples. Stage specific genomic alterations In order to identify the recurrent regions of genomic alterations that are specific to stage and MYCN amplification status, we partitioned the tumors into three subgroups (stage 1, stage 4 MYCN not amplified (4-) and amplified (4+)), and analyzed the frequency of genomic changes at each SW-locus (as defined in Methods) for each subgroup. Since cell lines may contain tissue culture related genomic alterations, we only used primary NB tumor samples for this analysis. The frequency of alterations for a given SW-locus was estimated using the average probability (P) value as described in the Methods. Fig. 5 shows the graphic depiction of the P associated with each SW-locus for all possible pair-wise comparisons among the three subgroups for gains or losses: Stage 1 vs. 4-, 4+ vs. 4- and 1 vs. 4+. As expected, since the majority of the loci show no change, they were plotted to values of co-ordinates around (0.5, 0.5) (shown in black). Regions altered in both classes with similar frequency are plotted close to the y = x diagonal, whilst off-diagonal points represented regions primarily altered in one or the other of the classes (termed differential imbalance). Loci that plot around (0,0) reflect loci altered in both groups (termed common regions). The colored points were selected by using our criterion for "common" and "specific" SW-locus (see Methods). In summary, we found alterations that were common to all three subgroups, which included gain of 7q32, 17q21, 17q23-24 and loss of 3p21. We also found genomic imbalances that were specific for each of the subgroups and common regions of gain for stage 1 and 4- tumors. Of note there were no shared alterations of 4+ with 1 or 4- besides the regions common to all. A detailed description and map positions for all these recurrent regions are provided in the Table 2, and a graphic representation of these imbalances is shown in Fig. 5, where we will discuss in detail in the discussion section. Figure 5 Genomic alterations specific to stage and MYCN status. Shown is the graphic depiction of the average p-values (P) of genomic alteration of each SW-locus (using a sliding window of 20 adjacent clones) within each tumor subgroup. All possible pair-wise comparisons among the three subgroups for gains or losses (stage 1 vs 4-, 4+ vs 4- and 1 vs. 4+) are shown. The frequency of alteration is estimated by P such that P = 0.15 is equivalent to a frequency of 70% and the lower the P the higher the frequency (details in Methods). Different colors were used to represent different clusters. Magenta: loci common to all groups; cyan: common to 1 and 4-; blue: 1 specific; green: 4- specific; red: 4+ specific, and black: all remaining loci. Colored dots were enlarged for easier visualization. Table 2 Recurrent regions related to MYCN status and stage Imbalance Chr. Cytoband Start (Mb) End (Mb) Clone No. Common to 1, 4-, & 4+ gain 7 7q32 133.19 137.05 26 gain 17 17q21 42.13 53.53 157 gain 17 17q23 55.41 61.8 99 gain 17 17q24 65.59 73.44 70 loss 3 3p21 45.72 46.77 13 Shared by 1 & 4- gain 7 7p14 32.41 32.83 11 gain 7 7q11 69.89 72.34 28 gain 7 7q36 148.74 152.97 39 gain 17 17q12 35.45 36.41 15 gain 17 17q21 39.56 40.03 16 Specific to 1 gain 2 2p22 32.23 33.33 12 gain 17 17p13 2.12 2.59 11 gain 17 17p13 5.67 7.17 22 gain 17 17p13 8.23 9.42 17 loss 8 8p12 38.32 39.84 12 loss 8 8q22-23 109.54 116.64 13 loss 11 11q12 60.87 63.09 29 loss 14 14q12 28.94 31.32 11 loss 14 14q23 58.04 58.76 12 loss 19 19q13 43.4 44.05 13 Specific to 4- gain 7 7p15 24.26 25.89 12 gain 7 7q34 139.53 141.16 22 loss 11 11q21 96.25 96.83 11 loss 11 11q22 108.12 110.64 11 loss 11 11q23 114.31 120.39 51 loss 11 11q24 125.28 126.76 20 loss 11 11q25 131.98 134.08 19 Specific to 4+ gain 2 2p24-25 10.97 20.08 38 loss 1 1p36 6.16 13.42 78 loss 1 1p36 15.22 16.13 20 Discussion Amplicons in Neuroblastoma In our study we found that unlike breast cancers [14] NBs do not have a wide variety of different amplicons or amplified genes. We identified 6 independent amplicons on 2p and one on 12q and precisely defined boundaries for all amplicons. Several genes related to angiogenesis and oncogenesis were in these novel amplified regions including TEM8 (tumor-specific endothelial marker), mapped to 2p13.1, which has been shown to have elevated expression during tumor angiogenesis [33]. Indeed this gene was recently reported to be amplified in the cell line IMR-32 in accord with our data [27]. The gene is expressed in human endothelium and has been implicated in colorectal cancer. Our present study showed TEM8 was amplified and over-expressed (data not shown) in several neuroblastoma cell lines. The significance of amplification of TEM8 in neuroblastoma cell lines but not endothelial cells raises an intriguing possibility that these tumor cells themselves contribute to the angiogenic process and requires further investigation. We also identified amplification of GAS41 (glioma amplified sequence) mapped to 12q14-q15 in one tumor sample. GAS41, a transcription factor ubiquitously expressed with the highest expression in human brain, was previously shown frequently amplified in human gliomas [34]. ALK (anaplastic lymphoma kinase) receptor, an oncogene and reported highly expressed in neuroblastoma [26], was identified to be amplified in two of our tumor samples. In addition to these genes, most of those newly identified amplified genes have not been implicated previously in neuroblastoma tumorigenesis and progression; therefore a further characterization of these genes might provide the biological insights to neuroblastoma biology. Interestingly, all amplicons occurred in MYCN amplified samples, and we have not found a single amplicon in MYCN single copy samples. Additionally, using our search criterion (A-CGH ratio >2 corresponding to copy number >6 see above) we found no evidence of amplifications in other chromosomal regions. This was in conflict with a study by Satito-Ohara et al. who found evidence of high level gains 9 NB cell lines and amplification as evidenced by a homogeneous staining region (HSR) in one line [35]. This difference could be explained by potential artifacts that arise in cell lines in tissue culture or be as a result of under detection by our study because of the relatively small number of tumor samples in our study, and would require confirmation in a larger sample set. Detection of low level of genomic changes In this study, we have applied a t-statistics-based method to explore genomic alterations in cancer from data generated by A-CGH on a cDNA microarray platform. Our method efficiently dealt with the low sensitivity of cDNA microarrays to detect low copy changes. The microarrays we utilized contain 42,000 clones, containing around 24,000 unique UniGene clusters with an average coverage of one cluster every 125 Kb. Underestimation of DNA copy number ratios by cDNA A-CGH data made it difficult to detect low level of gains and losses using ratio threshold based approaches, which was addressed in our study and previous reports [14-16]. The algorithm we have implemented included an efficient noise reduction strategy by combining ratios within a sliding window of clones as has been previously described [13,14]. However the incorporation of t-statistics demonstrated several advantages over the sole reliance of moving average for detecting genomic changes. It provides confidence levels for detecting genomic changes at each DNA location (SW-locus) in terms of p-values. This has theoretical advantages over the original raw ratio, because it incorporates an estimate of possible statistical errors in the analysis by giving a p-value attached to each genomic change within a SW-locus. By this method we were able to detect 1.5 fold changes of gene copy number as shown in our X-chromosome validation experiment. However, all these advantages are traded for a loss of resolution: genomic imbalances much smaller than the window-size cannot be detected and the boundaries of instable regions are blurred. Therefore we should choose the smallest window that has the desired level of statistical significance. The effective resolution can be obtained by analyzing the correlation of overlapping sliding windows. The integrated autocorrelation time is an estimator of the minimal distance for windows to be effectively uncorrelated [36] even when they overlap. For the sliding window t-test in our algorithm this distance can be calculated to be half the window size w, thus the number (N) of total unique Unigene clusters is reduced to 2N/w for the effectively independent measurements of the DNA copy number. Our results indicate that a window size 20 is needed in order to detect the lowest possible DNA change (one copy change) with reasonable statistical significance. According to the discussion above, this window size reduces the approximately 24,000 quality filtered unique Unigene clusters to 2 * 24,000/20 = 2400 independent estimates. This resolution is comparable to typical BAC arrays. For the stronger signals, less noise reduction is required. To detect 2-copy number DNA changes, only a small window size 5 is needed, therefore the resolution will be 4 fold higher. Although cDNA A-CGH is known not as sensitive as BAC A-CGH for the detection of low level of DNA copy number changes, currently we are able to obtain the comparable detection by using the probabilistic approach. In addition, with cDNA array it is possible to identify genomic amplification at the gene level and investigate the direct effect of gene copy number change over gene expression level in parallel, which will be addressed in future studies. Conclusions In this study we explored the genomic alterations in NB from the data generated by A-CGH on a cDNA microarray platform. We have not found genomic alterations universally present in all (100%) three subgroups of NBs, although such a region would be interesting since it may harbor specific genes that are uniquely responsible for NB tumorigenesis. We therefore focused on commonly altered regions where >70% of tumors showed changes in a given region, for our three different subgroups (Fig. 5 and Table 2). We found only a few of imbalances occurring in all three subgroups, of which gain of 17q21-24 and loss of 3p21 have been previously described in NB biology [8,37]. Apart from these regions stage 4+ tumors did not have any other regions that commonly change with the other two stages, whereas stages 1 and 4- had several common alterations. Stage 4- tumors demonstrated several unique changes of which losses in 11q has been previously described in MYCN single copy NB [38] and acts as a possible marker of unfavorable phenotype independent of MYCN amplification [39]. Remarkably stage 4+ disease appears to have very few genomic alterations when compared with Stage 1 and 4- implying that MYCN amplification is sufficient to drive these tumors to an aggressive phenotype, and although other genomic changes occur, including loss of 1p36 as shown by us and others [40], it does not require extensive changes. This is in agreement with the murine MYCN transgenic model of NB where the MYCN transgene itself is enough for tumor development, but these tumors develop additional genomic changes characteristic of NB [41]. Based on these results we found that cDNA A-CGH analysis is an efficient method for the detection and characterization of amplicons. We confirmed the previously reported amplified genes and also identified novel amplifications in neuroblastoma. Furthermore our probabilistic approach allows the detection of single copy number changes from cDNA A-CGH and can be applied to other CGH platforms including BAC or oligonucleotides based arrays. Methods Tumors, cell lines, and genomic DNA Thirty-two snap frozen neuroblastoma specimens were obtained from 12 patients with stage 1, and 20 patients with stage 4 of which 12 were MYCN-amplified and 8 were MYCN single-copy tumors. The original histological diagnoses were made at tertiary hospitals with extensive experience in diagnosis and management of neuroblastoma. Additionally, 12 neuroblastoma cell lines including 8 MYCN-amplified and 4 MYCN single copy samples were used in the study. Details of individual sample are summarized in Table 1. The conditions for cell cultures were done as described previously [17]. High molecular weight genomic DNA was extracted from interphase of a Trizol preparation for RNA extraction according to the manufacturer's instructions (Invitrogen, Gaithersburg, MD). Genomic DNA was treated with RNase A and protease (Qiagen, Valencia, CA), and purified by phenol/chloroform extraction followed by ethanol precipitation. We obtained normal genomic DNA samples (male, female or 1:1 mixture of male and female) from Promega, and genomic DNA samples containing the different numbers of X chromosomes (XXX, XXXX, and XXXXX) from the NIGMS . Microarray experiments Preparation of glass cDNA microarrays was performed according to a previously published protocol [18]. Image analysis was performed using DeArray software [19]. The cDNA library containing 42,000 clones was obtained from Research Genetics (Huntsville, AL) and clones were printed on two microscope glass slides as a set. Approximately 50% of the cDNAs on the microarrays were either known genes or similar to known genes in other organisms, whereas the remainders were anonymous ESTs. For A-CGH experiments on cDNA microarrays, 20 μg of genomic DNA from neuroblastoma tumor or cell line samples were sonicated and purified with QIAquick PCR purification column (Qiagen, Valencia, CA). Three micrograms of sonicated DNA were labeled with aminoallyl-dUTP (Sigma) in a 25-μl reaction, including random hexamer (0.24 μg/μl, Roche), dATP, dCTP and dGTP (125 μM each), dTTP (25 μM), aminoallyl-dUTP (100 μM) and high concentration of Klenow fragment (2.5 U/μl, NEB). The labeling reaction was purified with QIAquick PCR purification column. Cy3 and Cy5 dyes were coupled to the reference DNA (1:1 mixture of normal male and female DNA) and sample DNA respectively. Cy3- and Cy5-labeled probes were then combined along with human Cot-1 DNA (50 μg, Invitrogen) and yeast tRNA (100 μg, Invitrogen). The mixture was concentrated and re-suspended in 32 μl of hybridization buffer (50% formamide, 10% dextran sulfate, 4 × SSC, and 2% SDS). The hybridization mix was first heated at 75°C for 10 min, then at 37°C for an hour, and finally loaded to the pre-hybridized array. The hybridization was performed at 37°C overnight. The washing procedure was performed as described previously [17]. Real time quantitative PCR Real time PCR was carried out using SYBR Green PCR core reagents according to the manufacturer's instructions (Applied Biosystems, Foster City, CA). Each DNA sample was analyzed in triplicate using the ABI PRISM 7000 Sequence Detector. For quantitative PCR, 10 ng of genomic DNA was used for SYBR green PCR assay. Serial dilutions of neuroblastoma cell line CHP134 DNA were used as templates for a standard curve, and the normal genomic DNA was used as a calibrator. The normalization was performed as described using BCMA and SDC4 as reference genes [20]. Data analysis Fluorescence ratios were normalized for each microarray by setting the average log ratio for each subarray elements equal to zero (commonly referred to as "pin-normalization"). The data was quality-filtered by removing those clones with quality lower than 0.5 in more than 20% of all the samples [19]. For the clones that passed this filter, if the quality for a specific sample is lower than 0.5, then its fluorescence ratio is replaced by the average ratio value of all other samples with the good quality. The clones were finally assigned to UniGene Cluster (Build 154 September 2002). For the UniGene clusters represented by multiple clones, mean fluorescence ratios of those clones are used. After these processes we had 23975 unique UniGene clusters remaining from the initial 42591 clones. Map positions for the cluster were assigned by Blat searches against the "Golden Path" genome assembly (; June, 2002 Freeze). Throughout this publication, all genomic coordinates are given with the respect to this assembly. Finally the clusters were sorted according to their starting position of sequence on each individual chromosome. Detection of single copy changes To identify the alterations of copy number along the genome, we compared the distribution of the ratios in a sliding window of 20 clones in genomic order with a "null distribution" using a t-test. The p-value for genomic change (Pgc) obtained in this way is assigned to the center of the sliding window (referred to as the "SW-locus" throughout this manuscript). The t-test is valid in this instance because the observed distribution of Pgc for the loci in random order matches the expected theoretical distribution. The null-distribution used in t-test represents the unaltered part of genome. To identify the cDNA clones for the null-distribution, we start with the whole genome. The SW-loci corresponding to portions of the DNA that are amplified or deleted with a p-value smaller than 0.05 are removed recursively from the null dataset, until the null dataset is stable and there is no more amplified or deleted SW-locus in the null data set. Finally, the confidence of identified genomic alterations is visualized in a pseudo-color map in which color intensity represents the log of p-values (red for gain and green for loss). Estimation of frequency of genomic changes among the samples The probabilistic approach above provides P-values for the presence of genomic alteration in a given sample. In order to estimate the frequency of a genomic alteration we can set a threshold ratio and identify how many samples have a ratio value outside this threshold in the same genomic region. The disadvantage of this method is that different ratio thresholds will give different frequencies. We therefore applied another approach to avoid the use of ratio thresholds. To determine the frequency of loss or gain that correlate with the stage or MYCN amplification status, we first calculated the mean of the Pgc or P, for each group. This value is proportional to the frequency fwith change = Nwith change/Ntotal of their occurrence, where Nwith change is the number of samples in a subgroup with a given genomic imbalance and Ntotal is the overall number of samples in that subgroup. This is valid as follows. For all loci in which there are no genomic imbalances the observed Pgc will follow the flat theoretical distribution with a mean (expectation value) <P> = 0.5. Therefore, for those cases where we are sure of a genomic imbalance Pgc is close to 0 (for example p < 0.01), whereas for the samples in which there are no changes Pgc = 0.5. According to the formula: P = ((Nno change × 0.5) + (Nwith change × 0))/Ntotal = (1 - fwith change) × 0.5, the lower the P the higher the frequency of a genomic change in that SW-locus. Thus the P can be used to determine the frequency of a given change e.g. P = 0.15 corresponds to a frequency of ~70% of the samples with that given change. Determination of recurrent regions We first define a SW-locus with P < 0.15 in a specific subgroup as altered. This threshold corresponds to roughly a fraction of >70% of all tumor samples harboring that alteration in each subgroup. We define an altered SW-locus as common in all tumors, if the SW-locus passes the threshold for each of the three subgroups. A SW-locus is called differential in one subgroup with respect to another subgroup, if the frequency of genomic change is at least 3 times higher in one subgroup as compared to another. A SW-locus is defined as specific if the locus in one subgroup is differential with respect to each of the other subgroups; a SW-locus is defined as shared in two groups, if in both groups it is differential with respect to the third subgroup. Abbreviations NB: neuroblastoma A-CGH: array-comparative genomic hybridization M-CGH: metaphase-comparative genomic hybridization Stage 1-: stage 1 without MYCN amplification Stage 4-: stage 4 without MYCN amplification Stage 4+: stage 4 with MYCN amplification BAC: Bacterial artificial chromosome Author's contributions QC carried out all experiments and participated in data analysis. SB performed array CGH data analysis and statistical analysis. QC and SB drafted the manuscript. JSW, CCW, NC and CS were involved in the microarray production and the manuscript edition. ALK and BG were involved in the data analysis and the manuscript edition. FW, FB, MS and DC provided the tumor samples and patient information and were also involved in the manuscript edition. JK principal investigator of the project, participated in its design and is the final editor of the manuscript. All authors read and approved the final manuscript. Acknowledgements We thank Dr John Maris, Children's Hospital of Philadelphia and Dr Steven Qualman at the Cooperative Human Tissue Network (CHTN) for several of the tumors. ==== Refs Brodeur GM Neuroblastoma: biological insights into a clinical enigma Nat Rev Cancer 2003 3 203 16 12612655 10.1038/nrc1014 Brodeur GM Azar C Brother M Hiemstra J Kaufman B Marshall H Moley J Nakagawara A Saylors R Scavarda N Neuroblastoma. Effect of genetic factors on prognosis and treatment Cancer 1992 70 1685 94 1325279 Brinkschmidt C Christiansen H Terpe HJ Simon R Boecker W Lampert F Stoerkel S Comparative genomic hybridization (CGH) analysis of neuroblastomas--an important methodological approach in paediatric tumour pathology J Pathol 1997 181 394 400 9196436 10.1002/(SICI)1096-9896(199704)181:4<394::AID-PATH800>3.3.CO;2-T Plantaz D Vandesompele J Van Roy N Lastowska M Bown N Combaret V Favrot MC Delattre O Michon J Benard J Comparative genomichybridization (CGH) analysis of stage 4 neuroblastoma reveals high frequency of 11q deletion in tumors lacking MYCN amplification Int J Cancer 2001 91 680 6 11267980 10.1002/1097-0215(200002)9999:9999<::AID-IJC1114>3.0.CO;2-R Cohen N Betts DR Trakhtenbrot L Niggli FK Amariglio N Brok-Simoni F Rechavi G Meitar D Detection of unidentified chromosome abnormalities in human neuroblastoma by spectral karyotyping (SKY) Genes Chromosomes Cancer 2001 31 201 8 11391790 10.1002/gcc.1136 Schleiermacher G Janoueix-Lerosey I Combaret V Derre J Couturier J Aurias A Delattre O Combined 24-color karyotyping and comparative genomic hybridization analysis indicates predominant rearrangements of early replicating chromosome regions in neuroblastoma Cancer Genet Cytogenet 2003 141 32 42 12581896 10.1016/S0165-4608(02)00644-1 Schwab M Varmus HE Bishop JM Human N-myc gene contributes to neoplastic transformation of mammalian cells in culture Nature 1985 316 160 2 4040214 Maris JM Matthay KK Molecular biology of neuroblastoma J Clin Oncol 1999 17 2264 79 10561284 Vandesompele J Speleman F Van Roy N Laureys G Brinskchmidt C Christiansen H Lampert F Lastowska M Bown N Pearson A Multicentre analysis of patterns of DNA gains and losses in 204 neuroblastoma tumors: how many genetic subgroups are there? Med Pediatr Oncol 2001 36 5 10 11464905 10.1002/1096-911X(20010101)36:1<5::AID-MPO1003>3.0.CO;2-E Pinkel D Segraves R Sudar D Clark S Poole I Kowbel D Collins C Kuo WL Chen C Zhai Y High resolution analysis of DNA copy number variation using comparative genomic hybridization to microarrays Nat Genet 1998 20 207 11 9771718 10.1038/2524 Snijders AM Nowak N Segraves R Blackwood S Brown N Conroy J Hamilton G Hindle AK Huey B Kimura K Assembly of microarrays for genome-wide measurement of DNA copy number Nat Genet 2001 29 263 4 11687795 10.1038/ng754 Pollack JR Perou CM Alizadeh AA Eisen MB Pergamenschikov A Williams CF Jeffrey SS Botstein D Brown PO Genome-wide analysis of DNA copy-number changes using cDNA microarrays Nat Genet 1999 23 41 6 10471496 10.1038/14385 Pollack JR Sorlie T Perou CM Rees CA Jeffrey SS Lonning PE Tibshirani R Botstein D Borresen-Dale AL Brown PO Microarray analysis reveals a major direct role of DNA copy number alteration in the transcriptional program of human breast tumors Proc Natl Acad Sci U S A 2002 99 12963 8 12297621 10.1073/pnas.162471999 Hyman E Kauraniemi P Hautaniemi S Wolf M Mousses S Rozenblum E Ringner M Sauter G Monni O Elkahloun A Impact of DNA amplification on gene expression patterns in breast cancer Cancer Res 2002 62 6240 5 12414653 Heiskanen MA Bittner ML Chen Y Khan J Adler KE Trent JM Meltzer PS Detection of gene amplification by genomic hybridization to cDNA microarrays Cancer Res 2000 60 799 802 10706083 Beheshti B Braude I Marrano P Thorner P Zielenska M Squire JA Chromosomal localization of DNA amplifications in neuroblastoma tumors using cDNA microarray comparative genomic hybridization Neoplasia 2003 5 53 62 12659670 Khan J Wei JS Ringner M Saal LH Ladanyi M Westermann F Berthold F Schwab M Antonescu CR Peterson C Classification and diagnostic prediction of cancers using gene expression profiling and artificial neural networks Nat Med 2001 7 673 9 11385503 10.1038/89044 Khan J Saal LH Bittner ML Jiang Y Gooden GC Glatfelter AA Meltzer PS Gene expression profiling in cancer using cDNA microarrays Methods Mol Med 2002 68 205 22 11901504 Chen Y Dougherty ER Bittner ML Ratio-based decisions and the quantitative analysis of cDNA microarray images Biomedical Optics 1997 2 364 374 10.1117/1.429838 De Preter K Speleman F Combaret V Lunec J Laureys G Eussen BH Francotte N Board J Pearson AD De Paepe A Quantification of MYCN, DDX1, and NAG gene copy number in neuroblastoma using a real-time quantitative PCR assay Mod Pathol 2002 15 159 66 11850545 10.1038/modpathol.3880508 Tonin PN Yeger H Stallings RL Srinivasan PR Lewis WH Amplification of N-myc and ornithine decarboxylase genes in human neuroblastoma and hydroxyurea-resistant hamster cell lines Oncogene 1989 4 1117 21 2780050 Kuroda H White PS Sulman EP Manohar CF Reiter JL Cohn SL Brodeur GM Physical mapping of the DDX1 gene to 340 kb 5' of MYCN Oncogene 1996 13 1561 5 8875996 Jones TA Flomen RH Senger G Nizetic D Sheer D The homeobox gene MEIS1 is amplified in IMR-32 and highly expressed in other neuroblastoma cell lines Eur J Cancer 2000 36 2368 74 11094311 10.1016/S0959-8049(00)00332-4 Armstrong BC Krystal GW Isolation and characterization of complementary DNA for N-cym, a gene encoded by the DNA strand opposite to N-myc Cell Growth Differ 1992 3 385 90 1419902 Wimmer K Zhu XX Lamb BJ Kuick R Ambros PF Kovar H Thoraval D Motyka S Alberts JR Hanash SM Co-amplification of a novel gene, NAG, with the N-myc gene in neuroblastoma Oncogene 1999 18 233 8 9926938 10.1038/sj.onc.1202287 Miyake I Hakomori Y Shinohara A Gamou T Saito M Iwamatsu A Sakai R Activation of anaplastic lymphoma kinase is responsible for hyperphosphorylation of ShcC in neuroblastoma cell lines Oncogene 2002 21 5823 34 12185581 10.1038/sj.onc.1205735 De Preter K Pattyn F Berx G Strumane K Menten B Van Roy F De Paepe A Speleman F Vandesompele J Combined subtractive cDNA cloning and array CGH: an efficient approach for identification of overexpressed genes in DNA amplicons BMC Genomics 2004 5 11 15018647 10.1186/1471-2164-5-11 Corvi R Savelyeva L Breit S Wenzel A Handgretinger R Barak J Oren M Amler L Schwab M Non-syntenic amplification of MDM2 and MYCN in human neuroblastoma Oncogene 1995 10 1081 6 7700632 Cheng NC Van Roy N Chan A Beitsma M Westerveld A Speleman F Versteeg R Deletion mapping in neuroblastoma cell lines suggests two distinct tumor suppressor genes in the 1p35-36 region, only one of which is associated with N-myc amplification Oncogene 1995 10 291 7 7838528 White PS Maris JM Beltinger C Sulman E Marshall HN Fujimori M Kaufman BA Biegel JA Allen C Hilliard C A region of consistent deletion in neuroblastoma maps within human chromosome 1p36.2-36.3 Proc Natl Acad Sci U S A 1995 92 5520 4 7777541 Morowitz M Shusterman S Mosse Y Hii G Winter CL Khazi D Wang Q King R Maris JM Detection of single-copy chromosome 17q gain in human neuroblastomas using real-time quantitative polymerase chain reaction Mod Pathol 2003 16 1248 56 14681326 10.1097/01.MP.0000097364.64566.81 Tonini GP Romani M Genetic and epigenetic alterations in neuroblastoma Cancer Lett 2003 197 69 73 12880962 10.1016/S0304-3835(03)00081-8 Carson-Walter EB Watkins DN Nanda A Vogelstein B Kinzler KW St Croix B Cell surface tumor endothelial markers are conserved in mice and humans Cancer Res 2001 61 6649 55 11559528 Munnia A Schutz N Romeike BF Maldener E Glass B Maas R Nastainczyk W Feiden W Fischer U Meese E Expression, cellular distribution and protein binding of the glioma amplified sequence (GAS41), a highly conserved putative transcription factor Oncogene 2001 20 4853 63 11521196 10.1038/sj.onc.1204650 Saito-Ohara F Imoto I Inoue J Hosoi H Nakagawara A Sugimoto T Inazawa J PPM1D is a potential target for 17q gain in neuroblastoma Cancer Res 2003 63 1876 83 12702577 Sokal A CP DeWitt-Morette C, Folacci A Cargese Monte Carlo methods in statistical mechanics: foundations and new algorithms Funtial Integration: Basics and Applicaitons 1996 France: Proceedings of the NATO ASI B-361 Ejeskar K Aburatani H Abrahamsson J Kogner P Martinsson T Loss of heterozygosity of 3p markers in neuroblastoma tumours implicate a tumour-suppressor locus distal to the FHIT gene Br J Cancer 1998 77 1787 91 9667647 Guo C White PS Weiss MJ Hogarty MD Thompson PM Stram DO Gerbing R Matthay KK Seeger RC Brodeur GM Allelic deletion at 11q23 is common in MYCN single copy neuroblastomas Oncogene 1999 18 4948 57 10490829 10.1038/sj.onc.1202887 Luttikhuis ME Powell JE Rees SA Genus T Chughtai S Ramani P Mann JR McConville CM Neuroblastomas with chromosome 11q loss and single copy MYCN comprise a biologically distinct group of tumours with adverse prognosis Br J Cancer 2001 85 531 7 11506492 10.1054/bjoc.2001.1960 Fong CT Dracopoli NC White PS Merrill PT Griffith RC Housman DE Brodeur GM Loss of heterozygosity for the short arm of chromosome 1 in human neuroblastomas: correlation with N-myc amplification Proc Natl Acad Sci U S A 1989 86 3753 7 2566996 Hackett CS Hodgson JG Law ME Fridlyand J Osoegawa K De Jong PJ Nowak NJ Pinkel D Albertson DG Jain A Genome-wide array CGH analysis of murine neuroblastoma reveals distinct genomic aberrations which parallel those in human tumors Cancer Res 2003 63 5266 5273 14500357

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==== Front BMC Evol BiolBMC Evolutionary Biology1471-2148BioMed Central London 1471-2148-4-301535555110.1186/1471-2148-4-30Research ArticleExperimental evaluation of the relationship between lethal or non-lethal virulence and transmission success in malaria parasite infections Paul REL [email protected] T [email protected]üller-Graf CDM [email protected] S [email protected] PT [email protected] JC [email protected] Unité de Biochimie et Biologie Moléculaire des Insectes, Institut Pasteur, 25 rue du Dr. Roux, 75724 Paris Cedex 15, France2 Laboratoire d'Entomologie Médicale, Institut Pasteur de Dakar, 36, Avenue Pasteur BP 220, Dakar, Sénégal3 Laboratoire de Parasitologie Evolutive, CC237, CNRS UMR 7103, Université P. & M. Curie, 7 quai Saint Bernard, 75252 Paris Cedex 05, France4 Veterinary Parasitology, Faculty of Veterinary Science, Chulalongkorn University, Bangkok 10330, Thailand2004 8 9 2004 4 30 30 7 7 2003 8 9 2004 Copyright © 2004 Paul et al; licensee BioMed Central Ltd.2004Paul et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Evolutionary theory suggests that the selection pressure on parasites to maximize their transmission determines their optimal host exploitation strategies and thus their virulence. Establishing the adaptive basis to parasite life history traits has important consequences for predicting parasite responses to public health interventions. In this study we examine the extent to which malaria parasites conform to the predicted adaptive trade-off between transmission and virulence, as defined by mortality. The majority of natural infections, however, result in sub-lethal virulent effects (e.g. anaemia) and are often composed of many strains. Both sub-lethal effects and pathogen population structure have been theoretically shown to have important consequences for virulence evolution. Thus, we additionally examine the relationship between anaemia and transmission in single and mixed clone infections. Results Whereas there was a trade-off between transmission success and virulence as defined by host mortality, contradictory clone-specific patterns occurred when defining virulence by anaemia. A negative relationship between anaemia and transmission success was found for one of the parasite clones, whereas there was no relationship for the other. Notably the two parasite clones also differed in a transmission phenotype (gametocyte sex ratio) that has previously been shown to respond adaptively to a changing blood environment. In addition, as predicted by evolutionary theory, mixed infections resulted in increased anaemia. The increased anaemia was, however, not correlated with any discernable parasite trait (e.g. parasite density) or with increased transmission. Conclusions We found some evidence supporting the hypothesis that there is an adaptive basis correlating virulence (as defined by host mortality) and transmission success in malaria parasites. This confirms the validity of applying evolutionary virulence theory to biomedical research and adds support to the prediction that partially effective vaccines may select for increasingly virulent malaria parasite strains. By contrast, there was no consistent correlation between transmission and sub-lethal anaemia, a more common outcome of malaria infection. However, overall, the data are not inconsistent with the recent proposal that sub-lethal effects may impose an upper limit on virulence. Moreover, clone specific differences in transmission phenotypes linked to anaemia do suggest that there is considerable adaptive potential relating anaemia and transmission that may lead to uncertain consequences following intervention strategies. ==== Body Background Over the last few decades there has been considerable effort to introduce an evolutionary perspective to biomedical research and examine the extent to which adaptionist argumentation can be used to address questions pertinent to disease management, particularly that of pathogen virulence [1,2]. Contrary to the conventional wisdom that pathogens should evolve to become benign to their hosts (i.e. prudent host exploitation), considerable theoretical work has shown that the extent of pathogen virulence depends on the trade-off between the exploitation of the host (virulence) and the benefits accrued by increasing R0, the reproductive rate, either via increasing transmission directly or by reducing host recovery rate [3,4]. Thus, pathogens are expected to evolve a schedule of host exploitation that maximises their transmission and there are an increasing number of experimental studies demonstrating a relationship between transmission and virulence [5-8]. Incorporation of such virulence theory into an epidemiological framework has led to the suggestion that partially effective vaccines targeting pathogen virulence traits (e.g. malaria parasite growth rate) may actually lead to the selection of increased pathogen virulence [9]. Empirical confirmation of such important theoretical predictions requires the examination of how selection may be acting on the virulence factors underlying the pathogenicity through variability in transmission success [10]. Identifying the biological determinants governing the constraint between the transmission/virulence trade-off in turn requires an appreciation of the host-pathogen system in question. This has been recently exemplified by the case of the bacteria Neisseria meningitides, where virulence arises through colonisation of atypical tissues with no apparent direct benefit for transmission [11]. However, such apparently short-sighted evolution has been suggested to be an inadvertent consequence of within-host evolution that enables pathogen survival and transmission in diverse host spp. [12]. In these situations, there is an indirect trade-off between virulence and transmission that is strongly influenced by the time lag occurring between the beneficial and deleterious outcomes of within-host evolution [13]. Theoretical advances in virulence theory have revealed the importance of additional properties of host-pathogen systems, such as pathogen population structure and sub-lethal (significantly deleterious for host fitness but not lethal) effects, which can significantly alter the conclusions arising from the simple virulence/transmission trade-off hypothesis [14,15]. Infections are often composed of many simultaneously co-infecting strains that may interact directly or indirectly through their shared host. When parasites share a host, competition for the limited resources is generally expected to favour the most aggressive parasite – the tragedy of the commons [16]. Many theoretical models incorporating selection into an epidemiological framework show that selection favours increasingly virulent genotypes as the number of co-infecting parasite genotypes increases [14,17-20]. The expected levels of virulence, however, depend critically on how parasites interact with their hosts and how co-infecting genotypes affect each other; specifically, optimal levels of virulence depend on the cooperative or competitive nature of host exploitation by co-infecting parasites [15,21-23]. Hence, in what way such co-infection and sub-lethal effects potentially influence virulence evolution needs to be addressed explicitly for each pathogen system. In this paper we address the virulence/transmission relationship for the malaria parasite system and thereby examine the premises underlying the theoretical predictions on vaccine efficacy [9]. Malaria parasites replicate asexually within their vertebrate hosts, but must produce sexual stages (male and female gametocytes) to be transmitted to mosquitoes. Asexual parasites provide the source for gametocyte production and so increasing asexual parasite density is expected to correlate with transmission success. Therefore, the adaptive trade-off hypothesis would predict that faster asexual replication leads both to higher virulence and to higher transmission success [24]. Although transmission success does broadly increase with gametocyte density [25,26], which largely reflects asexual parasite density, many factors can affect transmission success [27]. I.e. high density does not always guarantee infection success [25,28]. Malaria parasites demonstrate considerable phenotypic plasticity in the production of sexual transmission stages. This plasticity appears to be linked to the quality of the blood environment: as conditions for asexual replication worsen, the parasite alters its sex allocation strategy by changing the sex ratio of gametocytes [29] or the proportion of asexual parasites producing gametocytes [30-32]. Thus transmission traits (e.g. gametocyte density and sex ratio) are highly susceptible to environmental changes and may therefore not conform to the simple trade-off between transmission and mortality. Although infection with malaria parasites can lead to host death, non-lethal anaemia is more commonly the result, where the parasitic infection provokes a reduction in haematocrit (red blood cell density) [33] and thus a reduced quality resource for parasite replication. Thus, anaemia is both a physical manifestation of virulence and a trigger of sex allocation. In this study we examine the trade-offs between transmission and virulence defined by mortality and anaemia in single and mixed clone infections, using the avian malaria system P. gallinaceum in the chicken host. In this specific system, anaemia and parasite transmission are linked physiologically, where the host's response to anaemia (reticulocyte production) triggers an increase in the proportion of gametocytes that are male rather than female [29]. This has been proposed to be an adaptive parasite response to assure mating success in the face of an immune response (transmission-blocking immunity) that develops simultaneously to the anaemia [34-37]. Experimental abnegation of this immune response can be achieved by injection of a high density parasite inoculum, which results in an uncontrolled infection, host death, and importantly, an absence of both the host reticulocyte response to anaemia and the linked parasite sex allocation response [37]. Thus, we are able to generate infections known a priori to result in host death or control by the host, enabling independent study of parasite transmission to mosquitoes with respect to either mortality or anaemia. Under both infection conditions, we examine the impact of mixed clone infections on overall virulence and transmission. Thus we assess to what extent there is an adaptive basis relating virulence and transmission under infection conditions of theoretical significance. Results Course of infection – competition between clones We first considered infection by injection of 106 infected red blood cells (iRBC) (an inoculum that almost always leads to a lethal infection outcome) of either of two clones of P. gallinaceum (the Thai or SL clone) or an equal mix of the two. The peak densities of asexual parasites did not differ between Thai and SL clone infections (χ21 = 0.3 P = 0.629), but were both significantly higher than those in mixed clone infections (combined single clones vs. mixed clone χ21 = 6.3 P = 0.012). The peak gametocyte density was, however, significantly higher in Thai than in SL clone infections (χ21 = 4.3 P = 0.038), and higher in both the single clone groups than in the mixed infections (χ21 = 4.05 P = 0.044) (Fig. 1). The extent of anaemia reflected parasite density, but was greater in the mixed clone group than in either of the two single clones, despite its lower parasite density (parasite density χ21 = 14.1, P < 0.001; Group χ22 = 6.1, P = 0.048). This effect of co-infection was confirmed with a comparison of the mixed clone with the 2 single clone type infections grouped together (parasite density χ21 = 14.2, P < 0.001; combined single clones vs. mixed clone: χ21 = 5.8, P = 0.016). The gametocyte sex ratios (proportion of gametocytes that were male) were found to be different among groups (χ22 = 33.5, P < 0.001), where the Thai clone had a higher sex ratio than the SL clone infections. Closer inspection and analysis of the data revealed similar sex ratios in the SL clone and mixed clone infections (Fig. 2) (Haematocrit χ21 = 16.8, P < 0.001; Group χ21 = 25.1, P < 0.001). As previously observed after infections by injection with high inocula [37], the production of reticulocytes is almost always absent during the growth phase of the infection that is followed either immediately by death or "crisis" when the host destroys all red blood cells (and parasites) and floods its system with reticulocytes. Notably, here one individual displayed a strong reticulocyte response to anaemia during the growth phase of infection, as occurs in natural, low density infections (See Methods); this was predictably associated with a strong increase in sex ratio (see Fig. 2 circled cross) [29]. Figure 1 P. gallinaceum gametocyte (sexual) parasite density (mean ± S.E.) following intra-muscular injection of 106 parasites single clones or 5 × 105 of each clone in mixed infections. Thai clone (square, long dotted line); SL clone (open circles, solid line); mixed clones (cross, dotted line). Figure 2 Gametocyte sex ratios (proportion male) of mixed vs. single clone infections compared with the corresponding haematocrit (red blood cell density) measured on the same day of infection. Each individual infection was measured daily for both these parameters until either the host died or the infection was cleared by the host (thus until the peak of infection). Infections were initiated by injection of 106 parasites for single clone treatments and 5 × 105 of each clone in mixed infections. Thai clone (squares); SL clone (open circles); mixed clones (crosses). Circled cross represents one individual that had a high sex ratio associated with a high density of reticulocytes on one particular day (see text). These data strongly suggest that the clones had a negative impact on each other, thus resulting in lower densities of asexual parasites and hence gametocytes. The anaemia (and hence sub-lethal virulence) was greater in the mixed clone infections, as previously observed in mouse models of malaria [38] and natural infections in humans [39], but was not accompanied by an increase in parasite densities. Thus, such a virulence outcome likely reflects a more intense host response to infection rather than an adaptive response relating pathogenicity and transmission [10]. Secondly we considered the outcome of infections initiated by infected mosquito bites (i.e. as with natural infections). These sporozoite-induced infections were initiated by gorging a fixed number of infected mosquitoes on the individual birds: 8 mosquitoes per bird for the single clone infections and either 8+8 or 4+4 for the mixed clone infections. Thus we controlled for total or clone-specific dose in the mixed infections. The experiment was performed twice, with a different batch of infected mosquitoes used for each experiment. The resulting infections were very consistent and no differences were found between the two experimental replicates for any of the analyses. Parasite densities of the Thai clone were significantly higher (χ21 = 4.73, P = 0.029) than the SL and mixed clone infections (4+4 or 8+8), which did not differ from one another. Gametocyte densities followed the patterns of asexual parasite densities, except for the high mixed clone infection (8+8), whose peak gametocyte production paralleled that of the Thai clone (Fig. 3). Again, the extent of anaemia reflected parasite densities and treatment group (Interaction between parasite density and group χ23 = 7.9, P = 0.048). Data inspection and model simplification regrouping the treatments into single vs. mixed clone infections revealed that mixed clone infections resulted in greater anaemia for a given parasite density (Parasite density χ21 = 52.8, P < 0.001; mixed vs. single clone infections χ21 = 6.8, P = 0.009). This increased anaemia was particularly evident for the 4+4 mixed infection group and was reflected in the significantly stronger reticulocyte response in this group (mixed vs. single clone infections χ21 = 6.3, P = 0.012). Once again, the gametocyte sex ratio varied with group and anaemia independently (group: χ23 = 11.7, P = 0.008; haematocrit: χ21 = 16.2, P < 0.001) (Fig. 4). Data inspection and model simplification showed that Thai/8+8 type infections had a significantly higher sex ratio (χ21 = 10.7, P = 0.001) than SL/4+4 types and both types responded similarly to haematocrit (χ21 = 17.7, P < 0.001) (Fig. 4). Figure 3 P. gallinaceum gametocyte (sexual) parasite density (mean ± S.E.) following infection by either or both parasite clones using a fixed number of mosquitoes. Thai or SL clone alone, 8 infectious mosquitoes for either clone per bird; mixed infections were initiated using 8 infectious mosquitoes of each clone per bird (8+8) or 4 infectious mosquitoes of each clone per bird (4+4). 3 birds were thus infected for each treatment type and the experiment was performed twice using different batches of mosquitoes infectious for each clone. Thai clone (squares, thin line); SL clone (open circles, thick line); 8+8 mixed clones (triangles, long dotted line); 4+4 mixed clones (crosses, short dotted line). Figure 4 Gametocyte sex ratios (proportion male) of mixed vs. single clone infections compared with their corresponding haematocrits (red blood cell density) measured on the same day of infection. Each individual infection was measured daily for both these parameters until either the host died or the infection was cleared by the host (thus until the peak of infection). Thai or SL clone alone, 8 infective mosquitoes per bird; Mixed infections were initiated using 8 or 4 infective mosquitoes from batches of mosquitoes infected with either of the clones. Thai clone (squares); SL clone (open circles); 8+8 mixed clones (triangles); 4+4 mixed clones (crosses). Thus, again the data support the notion that there is competition between the clones. The greater anaemia in mixed infections was again observed. In addition, the level of anaemia and the reticulocyte response to infection were greatest for the lowest inoculation dose. The extent of anaemia did not, however, seem to affect parasite densities in the mixed infections, where the parasitological parameters measured (e.g. asexual parasite / gametocyte densities and the sex ratio) aligned with those of either one of the 2 clones. Although the clones do appear to compete aggressively, sub-lethal virulence was more strongly influenced by infecting dose. Thus the sub-lethal measure of virulence (i.e. anaemia) appears to be more strongly dependent on the host's response to infection than on parasite density per se. Virulence and transmission success to mosquitoes For the iRBC-induced infections, one set of mosquito transmission studies was carried out on individuals with comparable parasite and gametocyte densities 3 days after inoculation, when parasite densities were low. Transmission success to mosquitoes at this early infection time point, as measured by both geometric mean oocyst load and the percentage of mosquitoes that were oocyst positive, was proportional to the subsequent rate of death (oocyst load: χ21 = 5.6, P = 0.017; percentage infected: χ21 = 4.6, P = 0.032) (Figs. 5a &5b). Infection type (Thai vs. SL vs. mixed clone) was not found to correlate with transmission success (oocyst load: χ22 = 2.3, P = 0.339; percentage infected: χ22 = 3.34, P = 0.17). In addition, neither sex ratio (oocyst load: χ21 = 2.5, P = 0.114; percentage infected: χ21 = 2.1, P = 0.15) nor gametocyte density (oocyst load: χ21 = 0.1, P = 0.75; percentage infected: χ21 = 0.3, P = 0.62) was correlated with transmission success. This is evidence that virulence, as defined by mortality, is positively correlated with transmission success. Figure 5 Transmission success as measured by (A) the geometric mean oocyst load per mosquito and (B) the percentage of mosquitoes that were positive for at least one oocyst, related to day of death for the 2 single clone and the mixed clone type infections. Three birds in each group (injected with single or mixed parasite clones as in Figs. 1 & 2) were gorged on mosquitoes on the first day of patent infection. Thai clone (squares); SL clone (open circles); mixed clones (crosses). Line represents least squared residual best fit for the relationship between oocyst load and day of death. (A) R2 = 0.68, P = 0.017; (B) R2 = 0.41, P = 0.032. For the sporozoite-induced infections (Figs. 6a &6b), we first carried out the same analysis as above, comparing transmission success to mosquitoes at the earliest infection time point with the rate of death, using only those 8 individuals that died. The percentage of mosquitoes infected was proportional to the rate of death (χ21 = 4.54, P = 0.032), however the oocyst load was not (χ21 = 3.36, P = 0.067). By contrast, infection type was not found to correlate with the percentage infected (χ23 = 5.56, P = 0.135), but did with the oocyst load (χ23 = 9.71, P = 0.021); this latter result is most likely the consequence of the small sample size (n = 8), three individuals of which were infected with the Thai clone, died on days 4 and 5 and produced very similar oocyst loads in the mosquitoes. The significant correlation between rate of death and the percentage of mosquitoes infected in both iRBC- and sporozoite-induced infections, despite their small sample sizes, provides consistent evidence that virulence, as defined by mortality, is positively correlated with transmission success. Figure 6 Comparison of lifetime transmission success with day of host death. Lifetime transmission success is defined as (A) the mean number of oocysts per mosquito for a given day totalled over the 4 days of acute phase transmission; (B) the percentage of mosquitoes with at least one oocyst. Infections were initiated using infectious mosquitoes as in Figs. 3 & 4. Thai or SL clone alone, 8 infective mosquitoes per bird; Mixed infections were initiated using 8 or 4 infective mosquitoes from batches of mosquitoes infected with either of the clones. Mosquitoes were gorged on all birds from the first day parasites were patent in the blood, (a) Thai clone (square); SL clone (open circles); mixed 8+8 clones (triangle); mixed 4+4 clones (crosses). For the sporozoite-induced infections, we then examined the relationship between mortality and parasite lifetime transmission success, as defined by total mean geometric oocyst load and total percentage of mosquitoes infected. For these analyses, individuals were first grouped according to whether they lived or died, and secondly, if they died, whether it was before or after the 4-day acute transmission period. There was no significant difference in either the percentage of mosquitoes infected (χ21 = 1.91, P = 0.16) or the total oocyst load (χ21 = 0.2, P = 0.63) between those individuals that died at any time and those that lived. However, those individuals that survived for the 4-day acute transmission period, irrespective of whether they subsequently died, infected a higher percentage of mosquitoes (percentage infected: χ21 = 12.9, P < 0.001) and with a higher mean oocyst density mosquitoes (χ21 = 18.2, P < 0.001) than those 3 individuals that died rapidly. Excluding these 3 early deaths, we compared the transmission success of those that lived and died following the acute transmission period. There was no significant effect of host mortality on either the percentage of mosquitoes infected (χ21 = 2.37, P = 0.12) or on mean oocyst loads (χ21 = 1.07, P = 0.33). These results suggest that although parasite-induced mortality may incur a transmission cost by reducing the duration of transmission, there is no additional effect of mortality on transmission success. This absence of a positive relationship between virulence and transmission success contradicts the results in the previous paragraphs where the rate of mortality was correlated with a single day measure of transmission success. This may, however, be the consequence of the small sample size (number of individuals that died after completing the acute transmission period, n = 5) and complexity of infection types. Lifetime transmission success was then compared against the level of anaemia at infection peak (Figs. 7a &7b). Because mixed clone infections led to increased anaemia independent of parasite density, we first examined the transmission/anaemia relationship in the single clone infections. There was a strong interaction between clone type and haematocrit on lifetime transmission success for both the percentage of mosquitoes infected (χ21 = 12.2, P = 0.0005) and the total mean oocyst load (χ21 = 10.21, P = 0.0015). In the Thai clone infections, anaemia at infection peak was negatively correlated to total mean oocyst load (χ21 = 7.28, P = 0.007) and the percentage of mosquitoes that were infected (χ21 = 8.4, P = 0.004); in the SL clone infections, however, there was no correlation between anaemia and either oocyst load (χ21 = 0.85, P = 0.36) or the percentage of infected mosquitoes (χ21 = 0.56, P = 0.46). When examining the data for all infection types, although the 8+8 mixed clone infections aligned with the Thai clone infections when considering the oocyst load, this was lost when considering the percentage of mosquitoes infected (Figs. 7a &7b). Why there is this disparity in mosquito infection parameters for this mixed infection group is unclear, but highlights the importance of considering mixed infections and of careful choice of the measure of transmission success used. Further work on transmission success from mixed infections is clearly required. In conclusion, the data suggest that there are clone-specific effects relating sub-lethal virulence to transmission and importantly that in the case of the Thai clone, that excessive anaemia may be a fitness cost for virulence evolution. Figure 7 Comparison of lifetime transmission success with peak anaemia. Lifetime transmission success is defined as (A) the mean number of oocysts per mosquito for a given day totalled over the 4 days of acute phase transmission; (B) the percentage of mosquitoes with at least one oocyst. Infections were initiated using infectious mosquitoes as in Figs. 3, 4 & 6. Thai or SL clone alone, 8 infective mosquitoes per bird; Mixed infections were initiated using 8 or 4 infective mosquitoes from batches of mosquitoes infected with either of the clones. Mosquitoes were gorged on all birds from the first day parasites were patent in the blood. Individuals that died before completion of the transmission period are excluded (a) Thai clone (square – thin solid line); SL clone (open circles – thick solid line); mixed 8+8 clones (triangle – long dotted line); mixed 4+4 clones (crosses – short dotted line). Lines are least squared residual best fit. Discussion This study examined the validity of some basic assumptions underlying evolutionary models of virulence for the specific case of malaria parasites, and hence the extent to which such models may be relevant to malaria control. One of the fundamental assumptions is that a parasite's virulence is a necessary consequence of the strategy that it uses to exploit its host in its (evolutionary) attempt to achieve maximal transmission. For the case of malaria, the production of transmission stages (gametocytes) depends on the asexual replication of the parasite clone within the host's blood system. Thus, faster asexual replication would be expected to result in more transmission before the host's immune responses control the infection, but also to cause greater host damage and to increase the risk of the host's death. Hitherto, experimental studies have either used gametocyte density as a proxy for transmission success [24], or taken a snap-shot count of successfully transmitted parasite stages at a time of estimated maximal transmission [7,40,41]. Here we measured transmission success directly by gorging mosquitoes on the infected hosts and counted the number of successfully transmitted parasites either at a time independent of the virulence outcome or throughout the period during which the parasite transmits from the vertebrate host to the mosquito vector. We found (i) that in both iRBC and sporozoite-induced infections, transmission success was positively correlated with host mortality rate but (ii) that there was a total transmission cost to host death only if the host died before the completion of the transmission period; if the host died after completing the acute transmission period there was no impact on total transmission. Finally (iii) there was no clear correlation between transmission success and anaemia (sub-lethal virulence): infections with one of the parasite clones resulted in a negative virulence-transmission relationship whereas with the other clone there was no relationship. These observations suggest that although malaria parasites may conform to the predictions of virulence theory based on parasite-induced host mortality, there appears to be no general trade-off between transmission and sub-lethal virulence. Notably, measurable parasitological parameters such as asexual and sexual parasite densities were predictive of neither mortality rate nor transmission success. This is because the relationships between asexual density and host mortality and between transmission success and the densities of asexual stages and gametocytes are not straightforward and are influenced considerably by developing immune responses. Indeed, despite the appealing simplicity of the idea, it is not obvious that there should be simple measurable parameters, such as asexual parasite density, defining parasite virulence or that these should correlate in a straightforward manner with transmission; this has been discussed in depth for the case Neisseria, as noted in the Background section [11,12]. Irrespective, however, of the precise biological details underlying parasite-induced mortality, malaria parasites do generally confirm one of the most robust predictions of virulence theory. Therefore, natural parasite populations might confirm the prediction that, in the simplest case of single clone infections in a homogeneous host population, intervention strategies targeting asexual replication rate could select for increased virulence [9]. Previous work in experimental mouse models found there to be positive genetic correlations between virulence, asexual density and transmission [24]. However, sub-lethal effects, rather than lethal infection outcome, were found to impose an upper threshold to virulence [7] and transmission was maximised at intermediate levels of host morbidity [42]. In our system, host death did reduce total transmission success, but only when the mortality rate was very high; mortality per se incurred no cost to transmission. In addition, more virulent infections were generally more infectious prior to host death. These results conform to standard virulence theory where there is a relationship between transmission and virulence, such that intermediate levels of virulence evolve. By contrast, the relationship between transmission success and sub-lethal virulence (anaemia) was less clear. On the one hand, infections with one of the parasite clones (Thai) confirm the suggestion from the mouse models [42] that sub-lethal effects impose selection against virulence. On the other hand, there was no transmission/virulence relationship for the other clone. How to interpret such results? In the mouse models [42], sub-lethal virulence was measured as a composite parameter including weight loss and anaemia; here we only used anaemia and therefore may have missed additional negative effects of the parasite on the host that might alter our conclusions. Indeed, for a given parasite dose, the level of anaemia induced was the same for the two clones. Thus, there was apparently no between-clone variance in putative parasite traits provoking anaemia, but there was clone-specific variation relating transmission to sub-lethal virulence. Additional clone-specific virulent effects on the host (e.g. weight loss) may therefore be as important as anaemia. An additional consideration is the proximate effect of anaemia on transmission. Previously, clone SL has been shown to adaptively alter its gametocyte sex ratio according to the host response to anaemia [29]. Notably the gametocyte sex ratio of the Thai clone was different from that of the SL clone, though its response to anaemia appeared similar (sex ratio became less female-biased). If the two clones differ in a transmission trait (sex ratio) that is apparently sensitive to host anaemia, they may be expected to vary in other, less evident traits implicated in transmission. Such variation in transmission traits would result in between-clone variability in the anaemia/transmission relationship. Further exploration of the covariance in sex ratio and the anaemia/transmission relationship is clearly required to ascertain whether sex ratio can be used as a marker phenotype for the complex interactions between the parasite and the host that determine parasite transmission success. In conclusion, the data are not inconsistent with the proposal that sub-lethal effects may impose an upper limit on virulence [42], but a more detailed understanding of parasite traits implicated in transmission is required. The final consideration concerns how the complexity of infection may alter the virulence/transmission trade-off. Competition between clones in multiple infections would generally be expected to favour the more virulent clone [20], although sub-lethal effects can considerably reduce the optimum level of virulence [15]. Malaria parasites are confronted with an unpredictable number of co-infecting clones. Under such conditions, parasites may employ facultative strategies of host exploitation according to co-infecting clone number and virulence would be expected to increase in mixed vs. single clone infections [38]. Anaemia was increased in all mixed infections, as previously observed in mouse models [38]. If parasites have evolved transmission strategies correlating with anaemia, such increased anaemia would be expected to result in altered transmission success. Overall transmission success was, however, unaltered and consistent with that observed for the single clone infections. However, the degree of increased anaemia in the mixed clone infections did not seem sufficient to alter the gametocyte sex ratios, previously shown to respond adaptively to changes in anaemia [29]. Thus, the sub-lethal virulence effect of the mixed infection does not seem to be sufficient to reduce the efficacy of the parasite's transmission strategy, of which facultative shifts in sex ratio is but one manifestation. In addition, although molecular data were not available, transmission phenotypes (e.g. gametocyte density and sex ratio) of mixed clone infections seemed to align themselves with either of the two clones: i.e. there seemed to be competitive dominance by one clone of the other, although the genetic identity of the clones abnegated any objective measurement. Interestingly, the competitive outcome was seemingly resolved early on in the infection. This suggests that there are clone-specific biological features determining the outcome at the very early stages of infection. The importance of the early stages of infection in the host-parasite interaction was further highlighted by the positive correlation between transmission success at the start of infection and host mortality rate, which occurred many days later. An additional surprising outcome of the mixed infections was that the identity of the dominant clone depended upon infecting dose. Infecting dose has previously been shown to influence disease severity in mouse models, but importantly there were consistent clone-specific differences in disease severity across a wide-range of doses [43]. Here, it is notable that at high infection doses (iRBC-induced infections) there were no differences between peak parasite densities of the two clones whereas at low infection doses (sporozoite-induced), the Thai clone reached a higher peak density. Thus we found dose-dependent differences that may not only alter the potential competitive outcome of a mixed infection, but may also alter the relative virulent nature of the 2 clones: i.e. relative differences in maximum parasite densities of clones may be more influenced by infecting dose than by clone identity. Our data from two clones are clearly limited, but if infecting dose is important, there may be additional subtle consequences of local heterogeneity in transmission intensity (number of infectious mosquito bites per host per unit time; i.e. dose) in addition to increasing R0 [44,45] and genetic complexity [46]. Conclusions Research using animal models is generating increasing evidence supporting an adaptive basis to life history traits and virulence of malaria parasites [7,24,42]. The studies presented here generally confirm these previous findings from a mammalian model system. Where they are different, probably reflects fundamental life history strategies pertaining to blood use (e.g. type of red blood cell invaded) [47], which include strategies for transmission stage production [30,34,35]. Such life history traits are well-documented (reviewed in [47]) and may be simple phenotypic markers reflecting the suite of alternative strategies available to the parasite. While the relevance of laboratory models to natural systems is easily criticised, the consistency of infection patterns across both avian and mammalian models suggests that there are general features of Plasmodium-host interactions. Therefore, with careful consideration of the biological details of each system, extrapolation from laboratory model to field data may be justified. Where field data fail to fit predictions, it is likely that we are missing important biological processes underlying the system in question [48]. One major deficiency of models is the inability to simulate repeated infections. With rare exceptions [49,50], the emphasis is placed on primary infections. The majority of infected humans, however, are not presenting with their first infections and will have confronted parasites for a considerable length of time. The host immune response to primary vs. re-infections is markedly different and it is likely that the host haematological response alters as well. The proportion of individuals confronting parasites for the first time (i.e. primary infections) depends on the transmission intensity, which also determines the parasite population structure and the parasite dose. Thus, interpreting field patterns with respect to evolutionary predictions necessitates stratification according to the epidemiology of the populations in question. However, despite such complexity, that both laboratory and field populations of malaria parasites generally conform to evolutionary theories concerning life history traits (e.g. sex allocation) and virulence is encouraging, and suggests that evolutionary theory can play an important role in predicting consequences of public health intervention strategies [9]. It remains, however, to be seen whether less conventional, but probably more important, measures of parasite virulence such as anaemia can be considered within an evolutionary framework. Methods Parasite, host and mosquito species Two strains of the chicken malaria parasite P. gallinaceum were used: Strain 8A (originally from Sri Lanka and obtained from D. Kaslow, NIH, Bethesda, USA) and a new Thai strain (obtained from S. Nithiuthai, Chulalongkorn University, Bangkok, Thailand). In this paper, 8A strain is designated as SL and the Thai strain as Thai. Both strains were cloned with limiting dilution in 5-day old chick hosts (Gallus gallus domesticus) (INRA, France). The clones were maintained in vivo by inoculation of 1 ml infected blood (20–45% parasitaemia, percent red blood cells (rbcs) infected) into naïve chicken hosts, with frequent passage through the mosquito vector, Aedes aegypti (Liverpool Blackeye strain). For experimentation, different, healthy 3-week old White Leghorn chickens were used for each experiment. All experimental animals were maintained according to European Union guidelines. Parasitaemias were obtained with Giemsa staining of daily blood smears. Parasitaemias and reticulocytes were calculated as percentages observed in a minimum of 100 rbcs; gametocytaemias observed in 10,000 rbcs. Haematocrit (number of rbcs per unit volume) was measured daily for each host using a haemocytometer. Thus, parasite and gametocyte densities are the number of rbcs infected with any parasites or gametocytes per unit volume. Mature male and female gametocytes are distinguishable after Giemsa staining: males stain a pale rose with no distinct nucleus, females stain blue with a distinct red nucleus [51,52]. Sex ratios based on counts of 50–75 gametocytes were found to be representative. We calculated sex ratios from the lesser of 50,000 rbcs or 100 gametocytes. Sex ratios are given as the proportion of males. Ae. aegypti mosquitoes were used in all transmission studies. Mosquitoes were maintained under standard conditions (80% humidity and 26°C). Transmission success was measured by (i) the percentage of mosquitoes positive for oocyst stage parasites and (ii) the mean oocyst density in gorged mosquitoes. Oocysts are the zygote stages of the parasite found on the mosquito stomach wall and which are those parasites that have developed successfully from fertilised female parasite gametes (gametocytes in the vertebrate host give rise to gametes once inside the mosquito bloodmeal which then undergo fertilisation; only fertilised gametes can continue development). Oocyst stage parasite counts were made 7 days post mosquito infection on midguts dissected from 30 gravid females and then stained with 0.5% mercurochrome in 1× Phosphate Buffer solution. Mean oocyst number per mosquito was chosen as an additional measure of transmission success to the number of infected mosquitoes for several reasons. Firstly, oocyst number is a more sensitive measure and is related to the percentage of infected mosquitoes by a simple negative binomial relationship [53]. Secondly, subsequent transmission from an infected mosquito to a new host may be affected by overall oocyst number through altering the number of sporozoites injected during a bloodmeal (See [47] for relevant literature). Experimental design a) Infection was initiated by injection of parasitized blood to generate an uncontrolled infection (in this paper, these infections are denoted iRBC-induced). Chickens were inoculated by intra-muscular injection of 106 parasites: 6 were inoculated with the SL clone, 6 with the Thai clone and 6 with 5 × 105 of each clone. Ae. aegypti mosquitoes were gorged upon 3 infected chickens from each group, chosen for their matching parasite densities on the day P. gallinaceum was detectable by blood smear (>0.1% parasitaemia). Transmission success (mean number of oocysts per mosquito) on day 3 post inoculation, when parasites were visible in the blood smear (>0.1%), was subsequently related to the day at which that individual died. b) Infection was performed using infectious mosquitoes to mimic natural low intensity infections (sporozoite-induced infections). Experimental infections were induced by gorging infected mosquitoes on the chicken hosts. 4 experimental groups (3 chickens per group) were considered: (i) 8 infective bites per host using the SL clone (ii) 8 infective bites per host using the Thai clone (iii) 4 infective bites from the Thai clone and 4 infective bites from the SL clone per host (iv) 8 infective bites from the Thai clone and 8 infective bites from the SL clone per host. The presence of sporozoites in each of these mosquitoes was verified subsequently by dissection of the salivary glands. (Note: the clones do not differ in the gene sequences of the molecular markers available for this species in Genbank (NCBI), so that the clones could not be differentiated and identified, unpubl. data). This experiment was repeated using different batches of mosquitoes infected with either parasite clone and healthy, uninfected chickens. Within each experiment for each parasite clone, each individual was infected by mosquitoes coming from the same cage. Ae. aegypti mosquitoes were gorged upon all infected chickens on the 1st day P. gallinaceum became patent in the blood (<0.1% parasitaemia), predictably day 7 (following the 7 day pre-erythrocytic developmental period), and for the next 3 days at which point the infection reaches its zenith; this is the acute stage of infection. Post-peak infection the parasite is rapidly cleared by the chicken and resurges only intermittently at very low densities. This chronic stage of infection represents the infectious reservoir yielding irregular low levels of transmission to mosquitoes [54]. The initial acute phase results in very high transmission rates and is here taken to represent the parasite lifetime transmission success (total of the daily mean number of oocysts per mosquito) and is compared against the highest level of anaemia (lowest haematocrit) observed in each individual chicken. Statistical Analyses Statistical analyses were conducted using the statistical package Genstat 6.1. Because each individual chicken was included in the data set many times, we corrected for repeated measures by fitting a generalised linear mixed model (GLMM procedure), nesting day within individual chicken in the random model. Parasite densities and blood cell counts were analysed specifying a Poisson error structure. Transmission success: the percentage of mosquitoes that were infected was analyzed using logistic regression specifying a binomial error structure with a logit link function and the mean oocyst densities were analyzed with a logistic regression specifying a Poisson error structure with a logarithmic link function (which gives the same fit as a negative binomial, [55]). For the iRBC-induced infections, transmission success was analyzed with respect to day of death, sex ratio and gametocyte density. For sporozoite-induced infections, transmission success on the first day of infection was similarly analyzed with respect to day of death for those individuals that died. In addition, we examined whether host death incurred a transmission cost by comparing transmission success of (i) the bird hosts died or not and (ii) whether birds survived the acute transmission period or not. Between-group comparisons of parasite lifetime transmission success for each individual were also analyzed with respect to the level of anaemia at infection peak. For the sex ratio and transmission analyses the data were over-dispersed and so were corrected for by estimating a dispersion parameter for each analysis. Statistical significance was presented as Wald statistics, which have a χ2 distribution. When sex ratio was used as an explanatory variable it was arcsine-transformed. Author's contributions RELP carried out the experimental studies, TL assisted in iRBC-induced studies, CMG assisted in experimental design, NS isolated the Thai strain parasite, PTB assisted in experimental design and edited the manuscript and JCK in elaboration of the manuscript. Acknowledgements RELP & PTB were funded by Institut Pasteur and French Ministère de la Recherche (PRFMMIP program). We thank M. Mackinnon and S. West for comments on earlier drafts on the manuscript. 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parasites Proc R Soc Lond Biol Sci 2001 268 1 6 10.1098/rspb.2000.1322 Mackinnon MJ Read AF The effects of host immunity on virulence-transmissibility relationships in the rodent malaria parasite Plasmodium chabaudi Parasitology 2003 126 103 112 12636347 10.1017/S003118200200272X Garnham PCC Malaria Parasites and Other Haemosporidia 1966 Oxford: Blackwell Scientific Hawking FK Gammage K Worms ME The asexual and sexual circadian rhythms of Plasmodium vinckei chabaudi, of P. berghei and of P. gallinaceum Parasitology 1972 65 189 201 4156397 Medley GF Sinden RE Fleck S Billingsley PF Tirawanchai N Rodriguez MH Heterogeneity in patterns of malarial oocyst infections in the mosquito vector Parasitology 1993 106 441 449 8341579 Paul REL Nu VA Krettli AU Brey PT Interspecific competition during transmission of 2 sympatric malaria parasite species to the mosquito vector Proc R Soc Lond Biol Sci 2002 269 2551 2557 10.1098/rspb.2002.2171 Wilson K Grenfell BT Shaw DJ Analysis of aggregated 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==== Front BMC CancerBMC Cancer1471-2407BioMed Central London 1471-2407-4-611535300510.1186/1471-2407-4-61Research ArticleCharacterization of a novel large deletion and single point mutations in the BRCA1 gene in a Greek cohort of families with suspected hereditary breast cancer Belogianni Ioulia [email protected] Angela [email protected] Markos [email protected] Evangelia [email protected] Stefanos [email protected] Andreas [email protected] Vasiliki [email protected] Antonios [email protected] Nikos [email protected] Kyriacos [email protected] Andreas [email protected] Paris [email protected] Drakoulis [email protected] Georgios [email protected] Molecular Biology Research Center HYGEIA «Antonis Papayiannis», DTCA HYGEIA, 15123 Maroussi, Athens, Greece2 1st Pathology – Oncology Clinic, DTCA HYGEIA, 15123 Maroussi, Athens, Greece3 Breast Cancer Unit, Iaso Women's Hospital, 15123 Maroussi, Greece4 Department of Genetics, "Saint Savas" Anticancer Hospital, 11522 Athens, Greece5 Department of Electron Microscopy/Molecular Pathology, The Cyprus Institute of Neurology and Genetics, 1683 Nicosia, Cyprus6 2nd Pathology – Oncology Clinic, DTCA HYGEIA, 15123 Maroussi, Athens, Greece7 Molecular Diagnostics Lab, I/R-RP, National Center for Scientific Research "Demokritos" 15310 Athens, Greece2004 7 9 2004 4 61 61 8 6 2004 7 9 2004 Copyright © 2004 Belogianni et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Germline mutations in BRCA1 and BRCA2 predispose to breast and ovarian cancer. A multitude of mutations have been described and are found to be scattered throughout these two large genes. We describe analysis of BRCA1 in 25 individuals from 18 families from a Greek cohort. Methods The approach used is based on dHPLC mutation screening of the BRCA1 gene, followed by sequencing of fragments suspected to carry a mutation including intron – exon boundaries. In patients with a strong family history but for whom no mutations were detected, analysis was extended to exons 10 and 11 of the BRCA2 gene, followed by MLPA analysis for screening for large genomic rearrangements. Results A pathogenic mutation in BRCA1 was identified in 5/18 (27.7 %) families, where four distinct mutations have been observed. Single base putative pathogenic mutations were identified by dHPLC and confirmed by sequence analysis in 4 families: 5382insC (in two families), G1738R, and 5586G > A (in one family each). In addition, 18 unclassified variants and silent polymorphisms were detected including a novel silent polymorphism in exon 11 of the BRCA1 gene. Finally, MLPA revealed deletion of exon 20 of the BRCA1 gene in one family, a deletion that encompasses 3.2 kb of the gene starting 21 bases into exon 20 and extending 3.2 kb into intron 20 and leads to skipping of the entire exon 20. The 3' breakpoint lies within an AluSp repeat but there are no recognizable repeat motifs at the 5' breakpoint implicating a mechanism different to Alu-mediated recombination, responsible for the majority of rearrangements in the BRCA1 gene. Conclusions We conclude that a combination of techniques capable of detecting both single base mutations and small insertions / deletions and large genomic rearrangements is necessary in order to accurately analyze the BRCA1 gene in patients at high risk of carrying a germline mutation as determined by their family history. Furthermore, our results suggest that in those families with strong evidence of linkage to the BRCA1 locus in whom no point mutation has been identified re-examination should be carried out searching specifically for genomic rearrangements. ==== Body Background Germ line mutations in the BRCA1 and BRCA2 genes predispose individuals to breast and ovarian cancer. The lifetime risk of breast cancer in female carriers of a BRCA1 mutation is 60–80% while that of ovarian cancer is 20–40%. The median age of diagnosis of breast cancer is 42 years, i.e. 20 years earlier than the median of unselected women in the U.S.A. and Western Europe [1]. BRCA1 is a large gene with 22 coding exons encoding a 220 kD protein [2] that functions in maintaining genomic integrity and in transcriptional regulation [3,4]. A multitude of mutations scattered throughout the 5592 bp coding sequence have been described. In particular, germ line mutations in BRCA1 have been identified in 15–20% of women with a family history of breast cancer and 60–80% of women with a family history of breast and ovarian cancer [5,6]. The percentage of mutations identified is strongly dependent on the population studied, with strong founder effects evident in some populations [7-9]. The vast majority of mutations described to date are point mutations and small insertions and deletions . Such mutations are detected by PCR-based screening methods such as the Protein Truncation Test (PTT), Single Strand Conformational Polymorphism (SSCP), Denaturing Gradient Gel Electrophoresis (DGGE), heteroduplex analysis (HA) and more recently denaturing high performance liquid chromatography (dHPLC) with varying degrees of sensitivity for each method. Direct DNA sequencing is used in order to confirm and characterize mutations detected by any of these approaches [10,11]. While in the early studies for mutation detection only single point or small insertion/deletion mutations were screened for, recent studies have shown that genomic rearrangements are also a common type of mutation in the two genes accounting for 10–30 % of all mutations identified in some populations [12-16]. In this study we document and extend previous work suggesting the necessity of screening for large genomic rearrangements in a complete program for mutation detection of the BRCA1 gene by characterizing a deletion encompassing 3.2 kb of the gene including exon 20. In addition, we add more evidence supporting pathogenicity of a previously described variant, G1738R, which seems to be specific to the Greek population [17]. Methods Patients BRCA1 patients and their families were referred through the Oncology Departments of Hygeia Hospital and other hospitals throughout Greece. Patients were included on the basis of affected family members, types of cancer present in the family and the age at diagnosis of breast cancer in the proband. The families were subdivided into high risk when multiple cases of breast and ovarian cancer were diagnosed, medium risk if there were only 2–3 cases of breast cancer, and low risk in isolated cases of breast cancer with diagnosis before the age of 40 years. The study population consisted therefore of 12 high risk families, 2 with medium risk, and 4 families at low risk as determined by their cancer history. Ethical approval was obtained from the hospital's advisory committee and all patients signed informed consent. Screening has been completed in 25 individuals from 18 families. Testing was initially carried out on DNA from an affected family member and upon detection of an inactivating mutation the rest of the family members were directly tested for this mutation. DNA and RNA isolation Genomic DNA and RNA were purified from peripheral blood leukocytes using standard extraction protocols. PCR amplification The complete coding sequence of BRCA1 including splice junctions was amplified by PCR. Similarly, exons 10 and 11 of the BRCA2 gene were amplified in 3 of the patients. Primers used have been chosen from the BIC database . In addition primers: mBRCAF: GAG TTT GTG TGT GAA CGG ACA CTG and mBRCAR: GTG CCA AGG GTG AAT GAT GAA AGC, designed during the course of this study, were used for amplification of cDNA from the patients found to carry a deletion of exon 20 of the BRCA1 gene. Reactions of 50 μl were heated on a PTC-200 MJ Research Thermocycler (MJ Research Inc., USA) at 95°C for 5 min then cycled 35 times of denaturation at 95°C for 40 sec, annealing at the appropriate temperature for 30 sec and extension at 72°C for 30 sec, followed by a final extension step at 72°C for 6 min. Reaction mixture was 20 mM TrisHCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl2, 200 μM each dNTP, 1.5 U Taq DNA polymerase (Invitrogen, Netherlands) or 2.5 U Optimase polymerase (Transgenomic, Inc., USA) and 12.5 pmol of each primer. dHPLC analysis The WAVE DNA Fragment Analysis System (Transgenomic, Inc., USA) and associated WAVE-Maker™ software were used as previously described [18]. Sequence analysis Purification of the PCR products was performed using the Concert Rapid PCR purification or gel extraction system kits (Marligen Biosciences INC, U.S.A.). Automated cycle sequencing for both strands was performed with the ABI Prism® 310 Genetic Analyzer using the Big-Dye Terminator Cycle Sequencing Kit. Sequences obtained were aligned, using Sequencher® PC software, with normal sequences from Genbank (BRCA1: L78833, BRCA2: U43746) and examined for the presence of mutations. All nucleotide numbers refer to the wild-type cDNA sequence of BRCA1 as reported in GenBank (accession number U14680). Multiplex Ligation – dependent PCR Amplification (MLPA) MLPA was carried out using the P002_BRCA1 kit (MRC-Holland, Netherlands) as instructed by the manufacturer. Fragment analysis was carried out on ABI Prism® 310 Genetic Analyzer (Applied Biosystems, USA) using TAMRA-500 (Applied Biosystems, USA) as size standard. A peak pattern of 34 peaks ranging in size from 127 to 454 nt is obtained [19]. Long PCR The deletion in BRCA1 exon 20 was confirmed by long PCR using the GeneAmp XL PCR System (Applied Biosystems, U.S.A.) according to the manufacturer's instructions. PCR was carried out in 50 μl reactions consisting of 1 × buffer II (supplied with the enzyme), 12 pmole each primer, 0.2 mM each dNTP and 2.4 U rTth DNA polymerase, XL (Applied Biosystems, U.S.A.). After 2.5 min denaturation at 95°C PCR was carried out for 19 cycles of 95°C for 30 sec, 58°C for 30 sec and 68°C for 8 min followed by 15 cycles of 95°C for 30 sec, 58°C for 30 sec and 68°C for 4 min with a time increment of 10 sec per cycle. A final extension step was carried out at 72°C for 10 min. PCR products were separated by agarose gel electrophoresis and visualized by EtBr staining. RT-PCR Total RNA was extracted from whole blood of patients from family D using Trizol (Life Technologies, USA) according to the manufacturer's instructions. First strand synthesis was performed by denaturing approximately 500 – 1000 ng total RNA, random hexamers (5 μM final concentration) for 4 min at 70°C, followed by snap freezing on ice and addition of dNTPs (0.5 mM final concentration), 1 U/μl recombinant RNase inhibitor (Invitrogen, Netherlands) and 200 U MMLV reverse transcriptase (Invitrogen, Netherlands). The mixture was incubated at 37°C for 1 hour followed by denaturation of the enzymes at 95°C for 5 min. 4 μl of cDNA were used for subsequent PCR amplification. Results A total of 18 families, 12 of which were at high risk of having hereditary breast cancer, have been examined for mutations at the BRCA1 locus. A pathogenic mutation was identified in five families, where four distinct mutations have been observed. In addition, 18 polymorphisms, including a novel silent polymorphism in exon 11 of the BRCA1 gene, have been detected (Table 1). Furthermore, in 3 of the families exons 10 and 11 of BRCA2 were analyzed. Table 1 summarizes the results of single nucleotide variants detected in this study. All of the variants have been identified by dHPLC and characterized by sequencing. In family A there were four cases of breast cancer affecting four successive generations. The proband was diagnosed with breast cancer at the age of 45. The most frequently occurring mutation, 5382insC in exon 20, was identified. Analysis of the proband's daughter in whom cancer developed at the age of 38 revealed that she also carried the mutation. The same mutation was also identified in family E where breast cancer was diagnosed in 3 members in three successive generations at 42, 37 and 41 years (data not shown). In family B there were seven cases of breast cancer and one case of colorectal cancer at age 50. Mutation analysis at the BRCA1 locus revealed a missense mutation 5331 G > A. The mutation results in substitution of a Glycine by an Arginine at codon 1738. The mutation was not detected in the patient's unaffected sister. Unfortunately, no other family members were available for analysis. In family C there were 4 cases of breast cancer in addition to cancer of the larynx, ovaries, lung and the genitals. A single base substitution, G > A at nucleotide 5586 was identified. This mutation causes a splicing defect resulting in a protein lacking exon 23. Unfortunately, no DNA was available from other family members in order to test the correlation of this mutation with the other tumor types. In Family D (Figure 1a) there were 5 cases of breast cancer with age at onset ranging from 29 to 50 years. In addition there was one individual who had CRC. Sequencing of the complete coding region of BRCA1, failed to reveal a mutation. This prompted the analysis of other genes, namely BRCA2 and p53 in order to try and characterize the phenotype in this family, but no mutation was identified. For this reason, we decided to use the MLPA technique [19] to screen the proband for genomic rearrangements which have been shown to be responsible for a large proportion of BRCA1 mutations. MLPA analysis of the proband (IV:10 in figure 1a) revealed deletion of exon 20 of the BRCA1 gene. This was confirmed by Long PCR (Figure 1b) and RT – PCR. Sequencing of PCR products generated by RT-PCR confirmed absence of the entire exon 20 in the mRNA of the patient. Subsequently, a number of restriction endonucleases were employed in order to narrow down the deletion breakpoints and facilitate their characterization. A SmaI-generated irregular fragment was finally isolated from agarose gel and sequenced revealing a deletion of 3.2 kb starting 21 bases into exon 20 and extending 3.2 kb into intron 20 (Figure 1c). The 3' breakpoint lies within an AluSp repeat but there are no recognizable repeat motifs at the 5' breakpoint. The deletion was also found in 4 relatives of the proband (Figure 1a) who were tested, two of whom had breast cancer. The other two relatives had not yet developed cancer at the ages of 36 and 55 at the time of testing and are therefore assumed to be pre – symptomatic carriers. Finally, the proband's sister, who at the time of testing had not developed cancer at the age of 33 was found not to carry the mutation. Discussion A total of 18 breast cancer families have been examined for mutations at the BRCA1 locus. In three of the families analysis was extended to exons 10 and 11 of the BRCA2 gene. A pathogenic mutation in BRCA1 was identified in 5/18 (27.7 %) families, where four distinct mutations have been observed. In addition, 18 polymorphisms have been found to be present in more than one family. In family B a missense mutation, 5331 G > A, was identified. The exact effect of this single amino acid change, G1738R, on the protein function is unclear. The altered glycine is located on the surface of the coil structure of the BRCT linker region and the mutation therefore may disrupt the interface or affect protein interaction [20]. This mutation has been previously described in 4 unrelated Greek patients [21]. An alternative mutation affecting the same amino acid, G1738E, has been shown to result in loss of function of the protein in vitro [22,23]. Based on this information, the family history of the individual described here and absence of the mutation in the proband's unaffected sister we hypothesize that the mutation is pathogenic although additional data are needed. Our results indicate, as has been documented by others that family history is the major determinant of the risk of breast cancer. As can be seen in Table 1 in all families where a pathogenic mutation was identified there were at least 3 cases of breast cancer. In addition, the extremely high risk suggested by the family history in some cases prompted a more intense analysis of further genes and approaches in an attempt to characterize the underling reason for such a family history. This was the case in family D where sequencing of the complete coding region of BRCA1, failed to reveal a mutation. This prompted the analysis of other genes, namely BRCA2 and p53, but without finding a mutation. For this reason, we decided to use the MLPA technique [19] to screen the proband for genomic rearrangements which have been shown to be responsible for a large proportion of BRCA1 mutations [12-16]. This led to the identification of deletion of an entire exon of the BRCA1 gene, namely exon 20. Deletion of exon 20 has been previously described in an Italian family [12]. However, the authors of that report have localized the breakpoints of the deletion in the two flanking introns [12]. In the deletion described here the 5' breakpoint is localized inside exon 20 (21 bp upstream of the splice donor site) while the 3' breakpoint is located 3.2 kb into intron 20 within an AluSp repeat. The deletion described here, therefore, is different to the majority of rearrangements described so far for the BRCA1 gene, since they have been shown to result from homologous recombination of Alu repeats [24,25]. In this case, the 5' breakpoint does not correlate with any recognizable repeat motifs suggesting a repeat – independent recombination mechanism at play. In 2/5 pedigrees in whom a pathogenic mutation was identified there was one case each of colorectal cancer (CRC). In particular, in family D (Figure 1a) the disease seems to have originated from a CRC patient. Data on the correlation between BRCA1 mutations and the risk of CRC are not conclusive. Two recent studies carried out on Ashkenazi Jewish patients suggest that there is no correlation [26,27]. Other reports carried out on more diverse populations suggest 2- to 3-fold elevated risks of CRC among first-degree relatives of BRCA1 mutation carriers [28,29]. Unfortunately, there was no DNA available for analysis from the two CRC patients in the two families. A further interesting point observed in this study is the identification of two BRCA1 unaffected mutation carriers at the ages of 39 and 55. The penetrance and age of onset of disease in BRCA1 mutation carriers is variable. Various reports have suggested the existence of modifying genetic and environmental factors on the penetrance of BRCA1 and BRCA2 mutation carriers [30,31]. In this respect it is interesting to examine this large pedigree for such modifying factors. Conclusions To our knowledge this is the first report of a genomic rearrangement identified as the underlying mutation of BRCA1 in a Greek family. Although our sample group is quite small identification of 1 genomic rearrangement in 5 mutations detected, suggests that in yet another population this type of mutations contribute to the BRCA1 mutation spectrum. This therefore warrants use of a combination of techniques capable of identifying both single base mutations in addition to large genomic rearrangements. In this respect, we have found that use of dHPLC for single base mutations and MLPA for large genomic rearrangements is a reliable combination for use as an initial screening step followed by sequencing for characterization of the mutations identified. Furthermore, our results suggest that in those families with strong evidence of linkage to the BRCA1 locus in whom no point mutation has been identified re-examination should be carried out searching specifically for genomic rearrangements. List of abbreviations dHPLC denaturing High Performance Liquid Chromatography MLPA Multiplex Ligation – dependent PCR Amplification PCR Polymerase Chain Reaction RT-PCR Reverse Transcription Polymerase Chain Reaction CRC Colorectal Cancer Competing interests None declared. Authors contributions IB carried out mutation detection by dHPLC and sequencing for the patients analyzed. AA carried out MLPA, the molecular characterization of the deletion, and drafted the manuscript. MM contributed in the molecular studies and preparation of the manuscript and figures, ER, SL, AP, VG, AK NP, PK and DY provided the patient material, diagnosis and management,, KK and AH carried out molecular genetic analysis of the p53 gene, GN conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: Acknowledgements This work is supported by STAVROS NIARCHOS FOUNDATION FOR CHARITY and by the Greek General Secretary for Research and Technology (8057/31-5-2001). Figures and Tables Figure 1 Family D in whom deletion of exon 20 of the BRCA1 gene was detected. a. Pedigree of family D. Black symbols indicate individuals with cancer. The proband is indicated by an arrow. A red circle inside a symbol indicates individuals found to carry the mutation. (CaBr=breast cancer, CRC=colorectal cancer d.=age of death, N=individual tested for the family mutation and was found to be of normal genotype). b. Long PCR of DNA from three individuals in family D. (M=molecular size marker (λ/HindIII, New England Biolabs, U.S.A.), C=normal control, numbers denote the identifier in the pedigree). c. Schematic representation (not to scale) of the fragment of BRCA1 gene amplified by Long PCR showing the position of exons, restriction endonuclease cleavage sites and the deletion. Empty boxes indicate exons. Vertical colored bars indicate restriction sites: blue (S) = SmaI. Red (H) = HindIII. Table 1 Variants identified in the BRCA1 and BRCA2 genes during this study. Mutation Family No Total No of CaBr1 No of CaOv2 cases Exon Nucleotide Effect Other cancers (age at onset) A 4 - 20 5382ins C Fs3 - B 7 - 20 5331 G > A G1738R Colorectal (50) C 4 1 23 5586G > A Splicing Larynx Lungs Genitalia D 5 - 20 Deletion of the entire exon Protein missing 20 a.a. of the BRCT-linker domain Colorectal E 3 - 20 5382ins C Fs3 - Benign polymorphisms Exon Codon Nucleotide Change Consequences No of families BRCA1 IVS 4 IVS4-19 C > A - 1 IVS 7 IVS7-34 C > T - 2 11 693 2196G > A Asp > Asn 2 694 2201C > T Ser > Ser 5 771 2430T > C Leu > Leu 6 871 2731C > T Pro > Leu 3 1038 3232A > G Glu > Gly 7 1040 3238G > A Ser > Asn 1 11454 3544T > C Val > Val 1 1150 3567C > T Pro > Ser 1 1183 3667A > G Lys > Arg 7 13 1436 4427T > C Ser > Ser 4 16 1613 4956A > G Ser > Gly 1 24 1829 5606G > A Glu > Glu 2 BRCA2 10 372 1342A > C Asn > His 1 11 1132 3624A > G Lys > Lys 2 1269 4053T > C Val > Val 1 14 2414 7470A > G Ser > Ser 1 1 CaBr = Breast Cancer 2 CaOv = Ovarian Cancer 3 fs = frame-shift mutation 4 novel polymorphism identified in this study ==== Refs Nathanson KN Wooster R Webber BL Breast cancer genetics: What we know and what we need Nature Medicine 2001 7 552 556 11329055 10.1038/87876 Miki Y Swensen J Shattuck-Eidens D Futreal PA Harshman K Tavtigian S Liu Q Cochran C Bennett LM Ding W Bell R Rosenthal J Hussey C Tran T McClure M Frye C Hattier T Phelps R Haugen-Strano A Katcher H Yakumo K Gholami Z Shaffer D Stone S Bayer S Wray C Bogden R Dayananth P Ward J Tonin P Narod S Bristow PK Norris FH Helvering L Morrison P Rosteck P Lai M Barrett JC Lewis C Neuhausen S Cannon-Albright L Goldgar D Wiseman R Kamb A Skolnick MH A strong candidate for the breast and ovarian cancer susceptibility gene BRCA1 Science 1994 266 66 71 7545954 Venkitaraman AR Functions of BRCA1 and BRCA2 in the biological response to DNA damage Journal of Cell Science 2001 114 3591 3598 11707511 Deng C-X Wang R-H Roles of BRCA1 in DNA damage repair: a link between development and cancer Human Molecular Genetics 2003 12 R113 R123 12668603 10.1093/hmg/ddg082 Gayther SA de Foy KA Harrington P Pharoah P Dunsmuir WD Edwards SM Gillett C Ardern-Jones A Dearnaley DP Easton DF Ford D Shearer RJ Kirby RS Dowe AL Kelly J Stratton MR Ponder BA Barnes D Eeles RA The frequency of germ-line mutations in the breast cancer predisposition genes BRCA1 and BRCA2 in familial prostate cancer. 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Am J Hum Genet 1999 64 300 302 9915971 10.1086/302211 Kirchhoff T Satagopan JM Kauff ND Huang H Kolachana P Palmer C Rapaport H Nafa K Ellis NA Offit K Frequency of BRCA1 and BRCA2 mutations in unselected Ashkenazi Jewish patients with colorectal cancer J Natl Cancer Inst 2004 96 68 70 14709740 10.1093/jnci/djh006 Niell BL Rennert G Bonner JD Almog R Tomsho LP Gruber SB BRCA1 and BRCA2 founder mutations and the risk of colorectal cancer J Natl Cancer Inst 2004 96 15 21 14709734 10.1093/jnci/djh008 Brose MS Rebbeck TR Calzone KA Stopfer JE Nathanson KL Weber BL Cancer risk estimates for BRCA1 mutation carriers identified in a risk evaluation program J Natl Cancer Inst 2002 94 1365 1372 12237282 10.1093/jnci/94.18.1365 Risch HA McLaughlin JR Cole DE Rosen B Bradley L Kwan E Jack E Vesprini DJ Kuperstein G Abrahamson JL Fan I Wong B Narod SA Prevalence and penetrance of germline BRCA1 and BRCA2 mutations in a population series of 649 women with ovarian cancer Am J Hum Genet 2001 68 700 710 11179017 10.1086/318787 Rebbeck TR Kantoff PW Krithivas K Neuhausen S Blackwood MA Godwin AK Daly MB Narod SA Garber JE Lynch HT Weber BL Brown M Modification of BRCA1-associated breast cancer risk by the polymorphic androgen-receptor CAG repeat Am J Hum Genet 1999 64 1371 1377 10205268 10.1086/302366 Ginolhac SM Gad S Corbex M Bressac-De-Paillerets B Chompret A Bignon YJ Peyrat JP Fournier J Lasset C Giraud S Muller D Fricker JP Hardouin A Berthet P Maugard C Nogues C Lidereau R Longy M Olschwang S Toulas C Guimbaud R Yannoukakos D Szabo C Durocher F Moisan AM Simard J Mazoyer S Lynch HT Goldgar D Stoppa-Lyonnet D Lenoir GM Sinilnikova OM BRCA1 wild-type allele modifies risk of ovarian cancer in carriers of BRCA1 germ-line mutations Cancer Epidemiol Biomarkers Prev 2003 12 90 95 12582017

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BMC Cancer. 2004 Sep 7; 4:61

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BMC Cancer

10.1186/1471-2407-4-61

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==== Front BMC Palliat CareBMC Palliative Care1472-684XBioMed Central London 1472-684X-3-41535787110.1186/1472-684X-3-4Research ArticleMitoxantrone pleurodesis to palliate malignant pleural effusion secondary to ovarian cancer Barbetakis Nikolaos [email protected] Michalis [email protected] Konstantinos [email protected] Rosalia [email protected] Christodoulos [email protected] Cardiothoracic Surgery Department, Theagenio Cancer Hospital, A. Simeonidi 2, Thessaloniki, Greece2 Anaesthesiology Department, Theagenio Cancer Hospital, A. Simeonidi 2, Thessaloniki, Greece3 Gynaecologic Oncology Department, Theagenio Cancer Hospital, A. Simeonidi 2, Thessaloniki, Greece4 Cytopathology Department, Theagenio Cancer Hospital, A. Simeonidi 2, Thessaloniki, Greece2004 9 9 2004 3 4 4 18 3 2004 9 9 2004 Copyright © 2004 Barbetakis et al; licensee BioMed Central Ltd.2004Barbetakis et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Advanced ovarian cancer is the leading non-breast gynaecologic cause of malignant pleural effusion. Aim of this study was to assess the efficacy of mitoxantrone sclerotherapy as a palliative treatment of malignant pleural effusions due to ovarian cancer. Methods Sixty women with known ovarian cancer and malignant recurrent symptomatic pleural effusion were treated with chest tube drainage followed by intrapleural mitoxantrone sclerotherapy. Survival, complications and response to pleurodesis were recorded. The data are expressed as the mean ± SEM and the median. Results The mean age of the entire group was 64 ± 11,24 years. The mean interval between diagnosis of ovarian cancer and presentation of the effusion was 10 ± 2,1 months. Eighteen patients (30%) had pleural effusion as the first evidence of recurrence. The mean volume of effusion drained was 1050 ± 105 ml and chest tube was removed within 4 days in 75% of patients. There were no deaths related to the procedure. Side effects of chemical pleurodesis included fever (37–38,5°C) chest pain, nausea and vomiting. At 30 days among 60 treated effusions, there was an 88% overall response rate, including 41 complete responses and 12 partial responses. At 60 days the overall response was 80% (38 complete responses and 10 partial responses). The mean survival of the entire population was 7,5 ± 1,2 months. Conclusions Mitoxantrone is effective in the treatment of malignant pleural effusion secondary to ovarian cancer without causing significant local or systemic toxicity. ==== Body Background Cancer accounts for 40% of all pleural effusions, especially in patients over 50 years old [1]. Bronchogenic and breast cancer account for 75% of malignant pleural effusions, with the remaining 25% represented by a cross-section of other neoplastic diseases [2]. Approximately two thirds of malignant pleural effusions occur in women because of the strong association with breast and ovarian cancer [3]. Advanced ovarian cancer is the leading non-breast gynaecologic cause of malignant pleural effusion. Pleural metastases were found in 48% of women who died from ovarian cancer [4]. The general approach to managing malignant effusions is determined by symptoms, performance status of the patient, expected survival and response of the known primary tumor to systemic treatment. Intervention options range from observation in the case of asymptomatic effusions through simple thoracentesis to more invasive methods such as thoracoscopy, pleuroperitoneal shunting and pleurectomy. In patients with reasonable survival expectancy and good performance status every attempt should be made to prevent recurrence of the effusion. Intercostal tube drainage with instillation of a sclerosing agent, resulting in the obliteration of the pleural space, is the most widely used method to control recurrent symptomatic malignant pleural effusions. Aim of this study was to study 60 patients with ovarian cancer who had a pleural effusion as a direct consequence of metastatic disease and to assess the efficacy of mitoxantrone as a sclerosing agent. Methods Over an 8-year period (1996–2003), all patients with known ovarian malignancy and recurrent symptomatic malignant pleural effusion referred to Thoracic Surgery Department of Theagenio Cancer Hospital for drainage and sclerotherapy, were eligible to participate in this study. This study was approved by the Theagenio Cancer Research Ethics Committee and patients were included after giving their informed consent. All patients satisfied the following eligibility criteria: 1. Known ovarian malignancy. 2. Recurrent symptomatic malignant pleural effusion. The diagnosis established by positive pleural fluid cytology on thoracentesis or positive pleural biopsy. 3. Evidence of expansion of the lung after fluid drainage and abscence of bronchial obstruction and/or fibrosis preventing lung expansion. 4. No previous intrapleural therapy. 5. Predicted survival of >1 month. Patients were ineligible if they had a history of cardiac disease, obstructive jaundice or surgery within the previous month. No patient had systemic chemotherapy immediately prior to or during the first 30-day interval following sclerotherapy. Sixty women fulfilled the above eligibility criteria. Pretreatment assessment was performed during admission and included history and physical examination, full blood count, liver biochemistry, electrocardiogram, a pre-drainage base line posteroanterior and lateral chest radiograph and other imaging as clinically indicated. A chest tube (28–32 F) was inserted into the midaxillary line through the 5th or 6th intercostal space under local anesthesia and in some case additional intravenous benzodiazepines and/or narcotics. The pleural effusion was drained to dryness initially by gravity and followed if necessary by suction from a wall-mounted suction pump using a pressure of 20 cm H2O usually for 12–24 hours to achieve complete drainage of the effusion and lung re-expansion. Daily tube outputs were recorded and when drainage fell below 100 ml in a 24 h period, posteroanterior and lateral chest radiographs were obtained to assure that the fluid had been sufficiently evacuated, there were no loculated collections and the lung had fully re-expanded. Then the patients were eligible for pleurodesis. Fifty ml of normal saline solution containing 2 mg/kg lidocaine were infused through the chest tube. After 15 minutes, a pleurodesis solution containing a mixture of 40 mg mitoxantrone and 20 ml normal saline was infused into the pleural cavity, after which the tube was clamped for 2 hours, while the patients changed position (rotated 90°) every 15 minutes. The tube then was re-opened. If the post-sclerotherapy drainage was <100 ml per day thetube was removed. Complications related to the procedure were recorded. Post-sclerotherapy posteroanterior and lateral chest radiographs were obtained immediately after tube removal in order to be compared with others obtained 30 and 60 days later. The radiographic response was determined on posteroanterior and lateral chest radiographs by observing the level of fluid meniscus overlying the costophrenic or vertebrophrenic angles and was determined as follows: complete response(CR) – no re-accumulation of pleural fluid, partial response (PR) – fluid recurrence less than 50% of the original level without symptoms or not requiring repeat drainage, progressive disease (PD) – re-accumulation to or above the original level with symptoms and requiring repeat drainage. Survival was calculated from the day of diagnosis of pleural effusion to the day of death or to the last day of follow up if alive. The data are expressed as the mean ± SEM and the median. Results Sixty women were included in this study. The mean age of the entire group was 64 ± 11,24 years. The interval between diagnosis of ovarian cancer and the development of a subsequent malignant pleural effusion ranged from 1 to 36 months (mean: 10 ± 2,1 months). Fifty one patients (85%) had unilateral effusion and 9 (15%) bilateral. Histology, degree of differentiation and TNM stage [5] at the time of diagnosis of the primary tumor are shown in Table 1. Table 1 Histology, degree of differentiation and TNM stage at the time of diagnosis of the primary tumor. Histology Number of patients (n:60) Percentage Serous cystadenocarcinoma 38 63,3% Mucinous adenocarcinoma 8 13,4% Mixed 4 6,7% Clear cell 3 5% Endometrioid 2 3,4% Unknown 5 8,4% Degree of differentiation G1 16 26,7% G2 18 30% G3 26 43,3% TNM stage I 8 13,3% II 18 30% III 22 36,7% IV 12 20% Eigtheen patients (30%) had pleural effusion as the first manifestation of recurrent disease, whereas 42 patients (70%) were already diagnosed as having local or distant spread before the onset of pleural effusion. These 42 patients with preexisting metastases showed a variable pattern of secondary spread. Eighteen patients had parenchymal liver metastases and intraabdominal lymph nodes, 11 patients had liver metastases only, 8 had synchronous lung and liver metastases, 2 had lung metastases only, 1 patient had umbilical nodule, 1 had anterior wall abdominal wall infiltration and 1 had brain metastases. The mean volume of effusion drained was 1050 ± 105 ml (range: 450–1500 ml). Chest tube was removed within 4 days in 75% of patients (range: 3 – 10 days). There were no deaths related to the thoracostomy procedure. One patient experienced vasovagal reflex during the procedure with systemic hypotension and intense pleuritic pain. Hypotension was treated with intravenous fluids and the pain was controlled with narcotics. This episode lasted 20 minutes. The patient recovered without incident. The most frequent complications related to pleurodesis were fever (temperature > 37°C), chest pain, nausea and vomiting (Table 2). Table 2 Complications related to chemical pleurodesis with mitoxantrone Complications Number of patients (n:60) None 31 (51,6%) Fever 16 (26,6%) Chest pain 12 (20%) Nausea 11 (18,3%) Vomiting 9 (15%) Diarrhea 4 (6,6%) Alopecia 1 (1,6%) Skin Rash 1 (1,6%) Dyspnea 1 (1,6%) Myelosuppression 1 (1,6%) Three patients died within 1 month of pleurodesis due to rapid progression metastatic disease. At 30 days, 57 patients were alive and 41 out of them had a complete response and 12 had a partial response. The overall response to chemical pleurodesis with mitoxantrone was 88% (53/57 patients). Four patients had progressive disease and revealed reaccumulation of fluid to or above the original level. At 60 days 52 patients were alive and 38 out of them had a complete response and 10 had a partial response. The overall response was 80% (complete response 38/60 patients – 63,4%, partial response 10/60 patients – 16,6%). Follow up ranged from 10 days to 38 months with a mean of 10 ± 1,36 months. Eight patients out of the 41, who initially had complete response developed later recurrent pleural effusion and needed again tube thoracostomy and a second attempt of chemical pleurodesis. The mean survival of the entire study population was 7,5 ± 1,2 months (median: 5,4 months). Discussion Management of malignant pleural effusions depends on the underlying malignancy, extent of disease, potential effectiveness of treatment and performance status. In patients with lymphoma, small cell lung cancer or germ cell neoplasms, pleural effusions may be controlled initially by systemic therapy alone. In patients with metastatic breast or non small cell lung carcinoma, local palliative treatment is often required. Since malignant pleural effusions are frequently a preterminal event with a 30-day mortality rate of 29 to 50%, treatment is directed toward symptomatic relief with minimal discomfort, inconvenience and cost [6-8]. Local treatment options include repeated thoracenteses, chest tube drainage with sclerotherapy, pleuroperitoneal shunt or pleurectomy. Repeated thoracentesis is usually a temporizing measure and carries the risk for pneumothorax and pleural infection [9]. Inpatient drainage with large-bore tubes (28–36 F) is effective, with variable 30-day success rates reported between 55% and 95% [10]. For this reason, large-bore tube thoracostomy with sclerotherapy has become the most common palliative treatment for malignant effusions. It has to be mentioned that recent studies have shown that small drainage catheters (10 to 14 F) are as effective as large bore chest tubes in the treatment of malignant effusions [11]. Using imaging guidance, small tubes can be placed into loculated collections, are well tolerated and have complication rates less than the larger tubes [12]. Pleural effusion due to metastatic ovarian cancer is a frequent phenomenon and as shown in our study it can occur as early as one month or as late as 36 months with a median of 8,5 months. This is in complete accord with the study of Cheng et al who found a median interval of 9 months [13]. When effusion occurs within 1 month of diagnosis of ovarian cancer, one is probably dealing with IV stage and clinical experience has proved that these patients have an especially poor prognosis. Numerous sclerosing agents have been used to treat malignant pleural effusions. Until recently, tetracycline was the most commonly used sclerosing agent with response rates ranging from 25 to 100% [14,15]. Because the intravenous form of tetracycline is no longer available, doxycycline has been proposed as an alternative. Bleomycin has been studied extensively as a sclerosing agent [16,17]. Goff et al succesfully used bleomycin intrapleurally to treat malignant pleural effusions from gynecological cancer with a 71% overall response at 30 days and minimal adverse reactions. Intrapleural instillation is usually well tolerated but a few patients may report mild fever or transient nausea. Pleuritic pain and rigors are rarely reported side effects. This relative lack of systemic toxicity is likely due to limited absorption of bleomycin (approximately 40%) of the pleural cavity [18]. At 30 days bleomycin has been reported to be superior to tetracycline [19]. Talc has proved to be one of the most effective sclerosing agents for treating malignant pleural effusions. Talc causes severe pleuritis resulting in effective pleurodesis but can worsen dyspnea and can result in respiratory failure [20]. Other complications associated with talc pleurodesis include fever, acute pneumonitis, granulomatous pneumonitis and empyema [21]. Talc is instilled either as a slurry via chest tube or insufflated via thoracoscope. Many other chemotherapeutic agents such as doxorubicin, cisplatin and cytarabine combination, etoposide, fluorouracil and mitomycin have been used for sclerotherapy. In addition radioactive isotopes, corynebacterium parvum, interferon and recombinant interleukin-2 have been instilled in the pleural space for treatment of malignant pleural disease. Response rate have been variable and less than optimal. Side effects are not inconsequential and thus none of these agents have gained widespread use [22]. Mitoxantrone is a synthetic anthracenedione which has been demonstrated to be effective in the treatment of peritoneal and pleural effusion. From a pharmacological point of view, mitoxantrone may be an especially appropriate choice due to its higher molecular weight and polarity since this may be factor important in prolonging contact with the pleura. The mechanism of intrapleural action of mitoxantrone has not yet been established. Both the inflammatory and antineoplastic activity of mitoxantrone intrapleurally have been described [23,24]. Our findings are consistent with the findings of others. In a prospective study in 18 patients, Musch et al [25] reported a 30-day success rate of 75%. A comparative study including bleomycin and mitoxantrone showed almost an equal 30-day response of 64% and 67% respectively [26]. Van Belle et al [27] had an overall 30-day response of successful pleurodesis of 67% in patients with ovarian cancer (2/3 patients). Morales et al [28] treated a group of 21 patients with malignant pleural effusions, with instillation of mitoxantrone with a 100% response and no toxic effects. There is only one study which proved mitoxantrone ineffective. Groth et al. [29] presented a prospective randomized trial on the treatment of malignant pleural effusions with intrapleural mitoxantrone versus placebo (pleural tube alone with instillation of isotonic NaCl). Their data suggest no statistically significant difference between the two arms with respect to response and response duration. Our study confirmed the majority of previous reports that mitoxantrone is an effective agent in controlling recurrent malignant pleural effusions. The overall 30-day response rate was 88%. Side effects were mild and rare. To develop new treatment plans for the management of pleural effusions, one must consider several requirements. First, no treatment regimen should exacerbate patients' symptoms, since palliation is the main aim. Second, seriously ill patients should not be subjected to procedures associated with high mortality and morbidity. Third, since about half the patients with pleural effusion will have no other clinically apparent metastases, treatment should be local rather than systemic. To be successful, the local treatment has to be effective and given at the first sign of the effusion, because inadequate or delayed treatment may eliminate the possibility of any subsequent therapy being effective, by producing loculation of the effusion. Conclusions Pleural effusion often occurs during the course of ovarian cancer. Chemical pleurodesis via bedside thoracostomy has been shown to be effective and has become a common therapeutic approach. Using this approach we found mitoxantrone to be highly effective at controlling malignant pleural effusions and decreasing the associated symptoms of dyspnea and pain. Our data justify further studies in a controlled setting to elucidate the biological action and prognostic relevance of mitoxantrone in the treatment of malignant pleural effusions and to compare this agent with other treatment procedures. Competing interests None declared. Authors's contributions NB conceived the study.and performed thestatistical analysis. MV and RV participated in the design of the study. KK and CT conceived of the study and participated in its coordination. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: ==== Refs Hausheer FH Yarbro JW Diagnosis and treatment of malignant pleural effusion Semin Oncol 1985 12 54 75 2579439 Lynch TJ Management of malignant pleural effusions Chest 1993 103 385 389 Saffran L Ost D Fein A Schiff M Outpatient pleurodesis of malignant pleural effusions using a small-bore pig tail catheter Chest 2000 118 417 421 10936134 10.1378/chest.118.2.417 Dvoretsky PM Richards KA Bonfiglio TA The pathology and behavior of ovarian cancer Path Ann 1989 24 1 24 Ovary In American Joint Committee on Cancer: AJCC Cancer Staging Manual 2002 6 New York, NY: Springer 275 284 Statement of the American Thoracic Society: management of malignant pleural effusions Am J Respir Crit Care Med 2000 162 1987 2001 11069845 Sahn SA Pleural effusion in lung cancer Clin Chest Med 1993 14 189 2000 8384963 Johnston WW The malignant pleural effusion: a review of cytopathologic diagnosis of 584 specimens from 472 consecutive patients Cancer 1985 56 905 909 4016683 Antunes G Neville E Duffy J Ali N BTS guidelines for the management of malignant pleural effusions Thorax 2003 58 29 38 10.1136/thorax.58.suppl_2.ii29 Hausheer FH Yarbro JW Diagnosis and treatment of malignant pleural effusion Cancer Metastasis Rev 1987 6 23 40 2439221 Patz E McAdams P Erasmus J Goodman P Culhane D Gilkeson R Herndon J Sclerotherapy for malignant pleural effusions. A prospective randomized trial of bleomycin vs doxycycline with small-bore catheter drainage Chest 1998 113 1305 1311 9596311 Seaton KG Patz EF JrGoodman PC Palliative treatment of malignant pleural effusions: value of small-bore catheter thoracostomy and doxycycline sclerotherapy Am J Roentgen 1995 164 589 591 Cheng D Chan YM Ng T Cheung A Ngan H Wong LC Mitomycin chemotherapeutic pleurodesis to palliate malignant pleural effusions secondary to gynaecological cancer Acta Obstet Gynecol Scand 1999 443 446 10326892 10.1034/j.1600-0412.1999.780518.x Hartman DL Gaither JM Kesler KA Comparison of insufflated talc under thoracoscopic guidance with standard tetracycline and bleomycin pleurodesis for control of malignant pleural effusions J Thorac Cardiovasc Surg 1993 105 743 748 7682268 Gravelyn TR Michelson MK Gross BH Tetracycline pleurodesis for malignant pleural effusions: a 10-year retrospective study Cancer 1987 59 1973 1977 2436744 Goff BA Mueller PR Muntz HG Small chest tube drainage followed by bleomycin sclerosis for malignant pleural effusions Obstet Gynecol 1993 81 993 996 7684517 Moffet MJ Ruckdeschel JC Bleomycin and tetracycline in malignant pleural effusions: a review Semin Oncol 1992 19 59 63 Ostrowski MJ Intracavitary therapy with bleomycin for the treatment of malignant pleural effusions J Surg Oncol 1989 1 7 13 Ruckdeschel JC Moores D Lee JY Einhorn LH Momdelbaum I Koeller J Weiss GR Losada M Koller JH Intrapleural therapy for malignant pleural effusions: a randomised comparison of bleomycin and tetracycline Chest 1991 100 1528 1535 1720370 Rehse DH Aye RW Florence MG Respiratory failure following talc pleurodesis Am J Surg 1999 177 437 440 10365887 10.1016/S0002-9610(99)00075-6 Marom EM Patz EF Erasmus JJ McAdams HP Goodman PC Herndon JE Malignant pleural effusions: treatment with small-bore catheter thoracostomy and talc pleurodesis Radiology 1999 210 277 281 9885620 Fingar BL Sclerosing agents used to control malignant pleural effusions Hosp Pharm 1992 27 622 628 Vargas FS Teixeira LR Antonangelo L Silva lM Strauz CM Light RW Acute and chronic pleural changes after intrapleural instillation of mitoxantrone in rabbits Lung 1998 176 227 236 9617739 Seitzer D Musch E Kuhn W Die locale behandlung maligner pleuraergusse bei gynakologischen tumoren Zent Bl Gynakol 1990 112 757 765 Musch E Loos U Mackes KG Kreidler H, Link KH, Aigner RB Intrapleural mitoxantrone in the treatment of malignant pleural effusions In Advances in Regional Cancer Therapy 1988 1 Basel: Karger 184 189 Maiche AG Virkunnen P Kontkanen T Mokkynen K Porkka K Bleomycin and mitoxantrone in the treatment of malignant pleural effusions Am J Clin Oncol 1993 16 50 53 7678715 Van Belle AF Velde GPM Weuters EFM Chemical pleurodesis with mitoxantrone in the management of malignant effusion Eur J Cancer 1998 34 206 207 9624263 10.1016/S0959-8049(98)00098-7 Morales M Exposito MC Intrapleural mitoxantrone for the palliative care treatment of malignant pleural effusions Support Care Cancer 1995 3 147 149 7539702 Groth G Gatzmeier U Haussingen K Heckmayr M Magnussen H Neuhauss R Pavel JV Intrapleural palliative treatment of malignant pleural effusions with mitoxantrone versus placebo (pleural tube alone) Ann Oncol 1991 2 213 215 1710483

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BMC Palliat Care. 2004 Sep 9; 3:4

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BMC Palliat Care

10.1186/1472-684X-3-4

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==== Front BMC Womens HealthBMC Women's Health1472-6874BioMed Central London 1472-6874-4-61536310210.1186/1472-6874-4-6Research ArticleAdditional collection devices used in conjunction with the SurePath Liquid-Based Pap Test broom device do not enhance diagnostic utility Day Sarah J [email protected]'Shaughnessy Darla L [email protected]'Connor Jason C [email protected] Gregory G [email protected] Departments of Cytopathology, Carle Clinic Association, Urbana, IL, USA2 Obstetrics and Gynecology, Carle Clinic Association, Urbana, IL, USA2004 13 9 2004 4 6 6 28 5 2004 13 9 2004 Copyright © 2004 Day et al; licensee BioMed Central Ltd.2004Day et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background We have previously shown that use of an EC brush device in combination with the Rovers Cervex-Brush (SurePath broom) offered no significant improvement in EC recovery. Here we determine if use of additional collection devices enhance the diagnostic utility of the SurePath Pap for gynecologic cytology. Methods After informed consent, 37 women ages 18–56 receiving their routine cervical examinations were randomized into four experimental groups. Each group was first sampled with the SurePath broom then immediately re-sampled with an additional collection device or devices. Group 1: Rover endocervix brush (n = 8). Group 2: Medscand CytoBrush Plus GT (n = 7). Group 3: Rover spatula + endocervix brush (n = 11). Group 4: Medscand spatula + CytoBrush Plus GT (n = 11). Results Examination of SurePath broom-collected cytology yielded the following abnormal diagnoses: atypia (n = 2), LSIL (n = 5) and HSIL (n = 3). Comparison of these diagnoses to those obtained from paired samples using the additional collection devices showed that use of a second and or third device yielded no additional abnormal diagnoses. Importantly, use of additional devices did not improve upon the abnormal cell recovery of the SurePath broom and in 4/10 cases under-predicted or did not detect the SurePath broom-collected lesion as confirmed by cervical biopsy. Finally, in 36/37 cases, the SurePath broom successfully recovered ECs. Use of additional devices, in Group 3, augmented EC recovery to 37/37. Conclusions Use of additional collection devices in conjunction with the SurePath broom did not enhance diagnostic utility of the SurePath Pap. A potential but not significant improvement in EC recovery might be seen with the use of three devices. ==== Body Background In gynecologic cytology, sampling of both the ecto and endocervix is critical to increasing Pap test sensitivity [1]. Controversy still exists, however, as to whether "all-in-one" broom-type devices appropriately sample the cervix. While agreement has been reached on the importance of sampling the transformation zone [2], concern as to how proximal the transformation zone is to the face of the cervix [3] has lead to lingering doubt over how effective current broom-type sampling devices are compared to a separate spatula and endocervical brush [4]. This debate has been re-energized by the generalized adoption of the liquid-based Pap test in the United States. Currently, two FDA approved liquid-based Pap tests are available, one manufactured by Cytyc (Boxborough, MA) and one manufactured by TriPath Care Technologies (Burlington, NC). Currently, the ThinPrep Pap Test (Cytyc) and the SurePath Pap (TriPath Care Technologies) offer two types of sampling devices a broom-type device or a spatula + cytobrush combination. It has been reported, that the use of the broom-type device for both the ThinPrep Pap Test and the SurePath Pap appears to under-sample the endocervix resulting in increased limited-bys due to lack of an EC component [4,5]. We have not observed this phenomenon and have previously shown that the SurePath Pap reduced by 33% the number of limited-by cases due to lack of an EC component when compared to the traditional Pap test when the SurePath Pap utilized the SurePath broom and the traditional Pap test utilized the spatula + EC brush combination [6]. In that failure to sample the endocervix can coincide with failure to sample the transformation zone we sought, in this study, to determine if additional sampling devices used in conjunction with the SurePath broom improved SurePath Pap EC recovery and/or increased SurePath Pap diagnostic effectiveness. Methods Cervical/endocervical sampling After study design approval from the Carle Clinic Association Institutional Review Board and informed consent, 37 women ages 18–56 receiving their routine cervical examinations were sampled with the SurePath broom. This device, which is packaged with the SurePath Pap, is the Rovers Cervex-Brush (Rovers Medical Devices, Oss, The Netherlands). Its use followed the manufactures recommendations of 5 full clockwise rotations. The same patient was immediately re-sampled with either the Rovers endocervix brush, patient group 1; the Medscand Cytobrush Plus GT (Medscand Medical, Malmö, Sweden), patient group 2: the Rovers endocervix brush + Rovers Spatula, patient group 3; or the Medscand Cytobrush Plus GT + Medscand Pap Perfect Spatula, patient group 4. All devices had "pop-off" heads. The SurePath broom device was collected into CytoRich Preservative vials (TriPath Care Technologies, Burlington, NC) and processed routinely using the PrepStain Slide Processor (TriPath Care Technologies, Burlington, NC). In samples using multiple collection devices, all devices, including the SurePath broom, were placed in a single collection vial and processed as above. Slide diagnosis The diagnostic terminology used was derived from the 1991 revision of the Bethesda System (TBS) [7-9]. The diagnostic categories available were: 1) no intraepithelial lesion (NIL), 2) inflammation/repair (BCC), 3) atypical squamous cells of uncertain significance (ASC-US), 4) low-grade squamous intraepithelial lesion (LSIL), 5) high-grade squamous intraepithelial lesion (HSIL) 6) squamous cancer, 7) atypical glandular cells of uncertain significance (AGUS) and 8) glandular cancer. ECs were considered present if they appeared as a group of 6 or more cells. Slides were reviewed following standard practice. Slide screening was performed blinded to the sample collection device type. Screening of alternate device collections from the same patient were screened by the same cytotechnologist. Screened slides were re-reviewed by a senior cytotechnologist and a pathologist for diagnoses other than NIL. Re-reviewers were blinded to device type and all slides from a particular patient were reviewed by the same senior cytotechnologist and pathologist. Quality control rescreen of slides did not result in revision of a diagnosis. Cytology/cervical biopsy comparison All cases where the cytology diagnosis was ASC-US or more serious underwent colposcopic-guided cervical biopsy (cervical biopsy). Cases were excluded from analysis if the: 1) cytology and corresponding tissue diagnosis were separated in time by more than 6 months; 2) cytology and/or corresponding cervical biopsy were not performed and interpreted within the Carle Clinic Association/Hospital system. In cases where multiple cervical biopsies were performed, the cytology closest in temporal relationship to the cervical biopsy was correlated. No cervical biopsies met the exclusion criteria. Statistical analysis All analyses were performed using SAS statistical software (Cary, NC). Data comparisons were made using the Student's paired t-test, the Sign Test and the Wilcoxon Signed Rank test for analysis of non-parametric data. Results Diagnostic utility of the SurePath broom with supplemental EC sampling We have previously shown that use of the Surgipath C-E brush (Richmond, IL) in combination with the SurePath broom did not increase EC recovery in women undergoing a SurePath Pap [6]. This previous study, however, did not investigate whether use of an EC brush enhanced SurePath Pap diagnostic utility. Therefore, to determine if an EC brush aided recovery of cytologically abnormal cells, EC brushes from Rovers and Medscand were examined. Tables 1 and 2 show results of women who were first sampled with the SurePath broom then immediately re-sampled with either a Rovers (Table 1) or Medscand EC brush device (Table 2). In group 1 patients (Table 1), 1/8 had LSIL identified by cytology after use of the SurePath broom. In addition, EC cells were identified in all 8 cases. Immediate re-sampling of group 1 patients with the Rover brush did not result in increased abnormal diagnoses. In fact, 3/8 cases had less than 5000 squamous cells/slide and required a diagnosis of QNS. In these QNS cases, no EC cells were seen. Importantly, follow-up cervical biopsy confirmed the SurePath broom-identified LSIL. This case was associated with a Rovers brush QNS. Table 1 Broom Followed By Rovers Endocervix Brush Broom Diagnosis EC Rovers Diagnosis EC Cervical Biopsy Diagnosis NIL Yes NIL Yes Ø NIL Yes NIL Yes Ø NIL Yes NIL Yes Ø NIL Yes NIL Yes Ø NIL Yes NIL Yes Ø NIL Yes QNS No Ø NIL Yes QNS No Ø LSIL Yes QNS No LSIL *EC, endocervical cells; NIL, negative for intraepithelial lesion; QNS, quantity not sufficient for diagnosis; Ø, no biopsy performed P value for diagnosis: paired t test = 0.14, Sign Test = 0.25, Wilcoxon = 0.25 P value for presence of ECs: paired t test = 0.08, Sign Test = 0.25, Wilcoxon = 0.25 Table 2 Broom Followed By Medscand CytoBrush Plus GT Broom Diagnosis EC Medscand Diagnosis EC Biopsy Diagnosis NIL Yes NIL Yes Ø NIL Yes NIL Yes Ø NIL Yes NIL Yes Ø NIL Yes NIL No Ø ASC-US Yes ASC-US Yes NIL HSIL Yes HSIL Yes HSIL HSIL Yes HSIL Yes HSIL *EC, endocervical cells; NIL, negative for intraepithelial lesion; QNS, quantity not sufficient for diagnosis; Ø, no biopsy performed P value for diagnosis: paired t test = 1.0, Sign Test = 1.0, Wilcoxon = 1.0 P value for presence of ECs: paired t test = 0.36, Sign Test = 1.0, Wilcoxon = 1.0 When group 2 patients were examined by cytology after using the SurePath broom (Table 2), 1/7 patients had ASC-US and 2/7 had HSIL. As above, adequate EC cells were identified in all SurePath broom cases. Immediate re-sampling of group 2 patients with the Medscand brush did not provide additional diagnostic utility. Importantly, follow-up cervical biopsy confirmed the SurePath broom-identified HSILs. In one of these HSIL cases, the Medscand brush was associated with a QNS. Taken together these findings indicate that addition of a Rover endocervix brush or Medscand CytoBrush Plus GT to the SurePath broom does not improve SurePath Pap abnormal cell or EC recovery. Diagnostic utility of the SurePath broom with supplemental ectocervical and endocervical sampling As shown above, use of an endocervical sampling device in addition to the SurePath broom did not enhance the usefulness of the SurePath Pap. To determine if a spatula plus an EC brush increased recovery of diagnostic cells over the SurePath broom, ectocervical spatulas from Rovers and Medscand were examined. Tables 3 and 4 show results of women who were first sampled with the SurePath broom then immediately re-sampled with either a Rovers spatula + endocervix brush (Table 3) or a Medscand PAP Perfect Spatula + CytoBrush Plus GT (Table 4). In group 3 patients (Table 3), 4/11 had abnormal cytology (1 AGUS, 3 LSIL) identified by use of the SurePath broom. EC cells were identified in 11/11 cases. Immediate re-sampling of group 3 patients using the Rovers devices resulted in 4 abnormal Pap diagnosis (2 ASC-US, 1 LSIL, 1 LSIL + AGUS) in the same four women. Complete diagnosis concordance, however, was seen in only one case. Follow-up cervical biopsy of these abnormals demonstrated 1 chronic cervicitis, 2 LSIL and 1 HSIL. Table 3 Broom Followed By Rovers Endocervix Brush + Rovers Spatula Broom Diagnosis EC Rovers Diagnosis EC Cervical Biopsy Diagnosis NIL Yes NIL Yes Ø NIL Yes NIL Yes Ø NIL Yes NIL Yes Ø NIL Yes NIL Yes Ø NIL Yes NIL Yes Ø NIL Yes NIL Yes Ø NIL Yes NIL Yes Ø AGUS Yes ASC-US Yes Chronic Cervicitis LSIL Yes ASC-US Yes LSIL LSIL Yes LSIL Yes LSIL LSIL Yes LSIL, AGUS Yes HSIL *EC, endocervical cells; NIL, negative for intraepithelial lesion; Ø, no biopsy performed P value for diagnosis: paired t test = 0.34, Sign Test = 1.0, Wilcoxon = 1.0 P value for presence of ECs: paired t test = 1.0, Sign Test = 1.0, Wilcoxon = 1.0 Table 4 Broom Followed By Medscand CytoBrush Plus GT + Medscand Pap Perfect Spatula Broom Diagnosis EC Medscand Diagnosis EC Cervical Biopsy Diagnosis NIL Yes NIL Yes Ø NIL Yes NIL Yes Ø NIL Yes NIL Yes Ø NIL Yes NIL Yes Ø NIL Yes NIL Yes Ø NIL Yes NIL Yes Ø NIL Yes NIL Yes Ø NIL Yes QNS No Ø NIL No NIL Yes Ø LSIL Yes ASC-US Yes LSIL HSIL Yes ASC-US Yes HSIL *EC, endocervical cells; NIL, negative for intraepithelial lesion; QNS, quantity not sufficient for diagnosis; Ø, no biopsy performed P value for diagnosis: paired t test = 0.1, Sign Test = 0.25, Wilcoxon = 0.25 P value for presence of ECs: paired t test = 0.34, Sign Test = 1.0, Wilcoxon = 1.0 When group 4 patients were examined cytologically after sampling with the SurePath broom (Table 4), 1/11 patients had LSIL and 1/8 patients had HSIL. EC cells were identified in 10/11 patients. Immediate re-sampling of group 4 patients with the Medscand spatula + EC brush yielded 2 diagnoses of ASC-US and 1 QNS. EC cells were, again, identified in 10/11 patients, however, the patients lacking EC cells with the SurePath and Medscand devices were not concordant. Follow-up cervical biopsy confirmed the SurePath broom results as LSIL and HSIL. Taken together these findings indicate that addition of both a spatula plus a brush device does not alter SurePath Pap diagnoses but may enhance EC cell detection. Discussion We have previously shown that the majority (88%) of SurePath Pap limited by diagnoses are due to lack of an EC component [6]. To overcome this problem, some clinicians have turned to using additional devices (usually an EC brush) in combination with the SurePath broom to attempt to increase the EC yield. The presumed rationale for use of an EC brush with the SurePath broom is that broom-type instruments do not reach into the cervical os as far as stand-alone brushes nor do they have bristles that are perpendicular to the handle. These concerns appear anecdotal but have concerned clinicians enough that TriPath Imaging sought and received recent approval for expanded labeling claims from the FDA to allow use of a spatula + brush combination with the SurePath Pap [10]. Our study was designed to test whether additional devices when used in combination with the SurePath broom enhanced recover of ECs or added diagnostic value to the SurePath Pap. The reason this study was undertaken was to demonstrate if the SurePath broom device was sufficient for obtaining an appropriate Pap sample. The strategy involved used a sequential testing method to show that added sampling of the cervix with spatula and/or brush devices did not recovery additional abnormal cells or additional ECs that altered the diagnostic results. These non-broom devices have different shapes and tinctoral qualities than the broom device, therefore, it is possible that they may sample portions of the cervix inaccessible to the broom, although no published evidence of such qualities exists. Importantly, this study was not designed to compare the SurePath broom to other devices used in the collection of SurePath Paps, instead, it was to designed to probe whether the SurePath broom alone was an appropriate device. This is important in light of the aforementioned SurePath Pap expanded labeling claim where clinicians might interpret such new FDA labeling as a repudiation of the broom device in favor of the spatula + brush combination. Currently, there is no available data detailing the results of this expanded labeling claim. In this study, we found that 3% of women sampled with just the SurePath broom lacked EC cells. These findings were consistent with but better than our previous report based on 3,994 women in which we found that 6% of SurePath Paps were "satisfactory for evaluation but limited by no EC cells" [6]. Other studies using a broom-type device have reported a range of EC absence from as low as 4.38% to as high as 29.2% [4,11-16]. This considerable variability in EC recovery is not easily understood nor is it clear why in only one [11] of these seven other studies was the absence of ECs lower than our previously observed rate of 6%. The next lowest EC absence rate observed in these seven studies was 10.1% [13]. In our current investigation, the likely reason why our EC absence rate was lower than our previous findings [6] was the use of a single nurse practitioner to collect all samples. Important to the SurePath broom is the flat and rounded sides to each bristle. Counterclockwise rotation brings the rounded bristle edges in contact with the cervix instead of the flat side reducing device effectiveness. In addition, a single experienced collector is more likely to achieve a satisfactory Pap sample than multiple inexperienced Pap collectors [17]. Another important reason why our previous and current studies show a relative low EC absence rate is that these studies utilized liquid-based Pap preparation. Most previous studies focusing on broom-type devices have compared their effectiveness to other sampling devices using traditional preparation. As we have shown, the SurePath Pap reduces by 33% limited bys due to lack of ECs when compared to the traditional Pap [6]. Debate over cervical sampling devices often focuses on EC sampling. Unfortunately, few studies are available that report both the EC absence rate and the abnormal cell detection rate in studies where multiple devices are compared. The retrospective study by Boon et al [11] stands out in that it suggests that there is a correlation between lack of endocervical cell recover with the Rovers Cervex-Brush and reduced detection of CIN III. Most other studies have shown equivalence between spatula + EC brush and broom-type devices. In fact, Buntinx et al in a meta-analysis of 29 trials that included 85,000 patients concluded that there was no significant difference between spatula + cotton swab or EC brush, extended tip spatula or broom-type device in recovery of abnormal cells [18]. This analysis did underscore that use of just an EC brush, cotton swab or Ayre spatula alone is inappropriate. Interestingly, they also found that obtaining a second cervical sample immediately after the first, even with the same device, increased abnormal cell detection by nearly 33%. We, as Tables 1,2,3,4 demonstrate, did not see this benefit when using multiple devices. In the 37 patients we immediately re-sampled after use of the SurePath broom, no additional abnormal diagnoses were rendered nor was additional diagnostic material provided that clarified a SurePath broom collected indeterminate diagnosis. As with any study, the strength of statistical analysis increases as the sample size is increased. However, even with small sample sizes, compelling results can be obtained if the statistical significance is large. Here the data was analyzed using three different nonparametric statistical tests (for non-continuous or non-numeric data) to ensure stringency. In addition, we chose to include the p-values for each of these statistical tests to show that multiple analyses yield the same result and that no single statistical test was chosen to favor a desired outcome. The hypothesis being tested, in this study, is that the use of additional collection devices in conjunction with the SurePath broom device does not enhance diagnostic utility. Normally, p-values <0.05 indicate that one should reject the hypothesis being tested and conclude enhanced utility. In this study, the large p-values generated from analysis of the data indicate a very high probability that the hypothesis be rejected and that no enhanced diagnostic utility is realized with the use of additional collection devices. Since liquid-based Pap testing is relatively new, little work has been done to examine sampling device effectiveness utilizing this technology. Selvaggi et al compared the ThinPrep broom to the ThinPrep spatula + cytobrush and the ThinPrep broom +cytobrush [4]. These authors found that the EC component was missing in 24%, 10% and 13% of cases, respectively. However, no examination of diagnostic utility was included so it is not clear how these findings relate to device effectiveness in a liquid-based setting. In addition, their findings differed significantly from our previous examination of broom + brush combination using liquid-based preparation. When we examined 23 women for EC adequacy using both the SurePath broom and an EC brush, we found that the EC brush provided no additional benefit over the broom in the SurePath Pap [6]. Like the Selvaggi et al study we did not comment on diagnostic differences when a secondary device was added but unlike the Selvaggi et al study we found all broom-only samples to have EC cells present. Importantly, our current study is the first to examine diagnosis differences that may result from adding additional devices to a broom device in the liquid-based setting. Here we found that 10/37 (27%) of cases had abnormal cytology when the SurePath broom was used. Immediate re-sampling with a second or third device did not increase the number of abnormal cytologies found. In addition, cervical biopsy of all abnormal cytologies was performed and as Tables 1,2,3,4 show use of additional devices did not improve cytology/tissue correlation. In conclusion, the SurePath broom appears to be a very effective cervix sampling device when coupled with the SurePath Pap. In 60 patients examined prospectively (37 in this study, 23 in our previous study [6]) only one patient (1.6%) failed to have EC cells recovered with the broom device alone. This is in contrast to the Selvaggi et al study that showed in 432 ThinPrep patients a 10% failure to detect ECs using two devices [4]. We must note, however, that the EC adequacy standard was different between their study and our studies because we defined EC presence as at least one group of 6 or more EC cells and they defined it as 10 or more EC and/or squamous metaplastic cells singly or in groups. Finally, our current work is the first to show in the liquid-based setting that the SurePath broom alone is as effective at identifying abnormal cells as the broom + additional devices. Conclusions Use of additional collection devices in conjunction with the SurePath broom did not enhance diagnostic utility of the SurePath Pap. A potential but not significant improvement in EC recovery might be seen with the use of three devices. Competing interests GGF has served as a speaker for TriPath Care Technologies. Abbreviations Atypical squamous cells (ASC) of uncertain significance (ASC-US), endocervical cell (EC), Food and Drug Administration (FDA), high grade SIL (HSIL), low grade SIL (LSIL), Papanicolaou (Pap), quantity not sufficient for diagnosis (QNS), squamous intraepithelial lesion (SIL), SurePath Liquid-Based Pap Test (SurePath Pap), SurePath Liquid-Based Pap Test Broom Device (SurePath broom), The Bethesda System (TBS). Authors' contributions SJD coordinated the study and analyzed the study data, DLO collected all samples, JCO performed the statistical analysis. GGF constructed the manuscript. Figure 1 Sampling Devices. A, SurePath broom. B, Rover spatula. C, Rover endocervix brush. D, Medscand spatula. E, Medscand CytoBrush Plus GT. Pre-publication history The pre-publication history for this paper can be accessed here: Acknowledgements This research was supported by TriPath Care Technologies, Burlington, NC ==== Refs Garite TJ Feldman MJ An evaluation of cytologic sampling techniques. A comparative study Acta Cytol 1978 22 83 85 276236 Boon ME Alons-van Kordelaar JJ Rietveld-Scheffers PE Consequences of the introduction of combined spatula and Cytobrush sampling for cervical cytology. Improvements in smear quality and detection rates Acta Cytol 1986 30 264 270 3521176 Rome RM Chanen W Ostor AG Preclinical cancer of the cervix: diagnostic pitfalls Gynecol Oncol 1985 22 302 312 4065705 10.1016/0090-8258(85)90044-7 Selvaggi SM Guidos BJ Specimen adequacy and the ThinPrep Pap Test: The endocervical component Diagn Cytopathol 2000 23 23 26 10907927 10.1002/1097-0339(200007)23:1<23::AID-DC5>3.0.CO;2-K Royer MC Smith KL Comparison of AutoCyte PREP Adequacy to the conventional Pap Smear [abstract] Acta Cytol 2001 45 827 Day SJ Deszo EL Freund GG Dual Sampling of the Endocervix and itsImpact on AutoCyte Prep Endocervical Adequacy Am J Clin Pathol 2002 118 41 46 12109854 10.1309/U72E-XD57-NF4J-XVG8 The Bethesda System for reporting cervical/vaginal cytologic diagnoses Acta Cytol 1993 37 115 124 8465628 The 1988 Bethesda System for reporting cervical/vaginal cytologic diagnoses: developed and approved at the National Cancer Institute workshop in Bethesda, MD, December 12–13, 1988 Diagn Cytopathol 1989 5 331 334 2791840 The 1988 Bethsda System for reporting cervical/vaginal cytological diagnoses. National Cancer Institute Workshop JAMA 1989 262 931 4 2754794 Boon ME de Graaff Guilloud JC Rietveld WJ Analysis of five sampling methods for preparation of cervical smears Acta Cytol 1989 33 843 848 2588917 Szarewski A Cuzick J Singer A Cervical smears following laser treatment comparison of Cervix brush versus cytobrush-Ayre spatula sampling 1991 35 76 78 1994639 McCord ML Stovall TG Meric JL Summitt RL Coleman SA Cervical cytology: A randomized comparison of four sampling methods Am J Obstet Gynecol 1992 166 1772 1779 1615986 Cannon JM Blythe JG Comparison of the Cytobrush Plus plastic spatula with the Cervix Brush for obtaining endocervical cells Obstet Gynecol 1993 82 569 572 8377984 Germain M Heaton R Erickson D Henry M Nash J O'Connor D A comparison of the three most common Papanicolaou smear collection techniques Obstet Gynecol 1994 84 168 173 8041524 Risberg B Andersson A Zetterberg C Nordin B. Cervix-Brush vs spatula and cytobrush a cytohistologic evaluation J Reprod Med 1997 42 405 408 9252930 Neinstein LS Church J Akiyoshi T Comparison of cytobrush with Cervex-Brush for endocervical cytologic sampling J Adolesc Health 1992 13 520 523 1390820 Buntinx F Brouwers M Relation between sampling device and detection of abnormality in cervical smears: a meta-analysis of randomised and quasi-randomised studies BMJ 1996 313 1285 1290 8942687

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==== Front BMC Med EducBMC Medical Education1472-6920BioMed Central London 1472-6920-4-151538002310.1186/1472-6920-4-15Research ArticleA preliminary report of an educational intervention in practice management Crites Gerald E [email protected] Richard J [email protected] Department of Internal Medicine, Wright State University School of Medicine, 128 E. Apple Street, 2nd Floor/Fred Weber Building, Dayton, OH, 45409-2902 USA2 Division of Health Systems Management, Department of Community Health, Wright State University School of Medicine, 3139 Research Park Blvd., Kettering Ohio 45420-401 USA2004 20 9 2004 4 15 15 20 5 2004 20 9 2004 Copyright © 2004 Crites and Schuster; licensee BioMed Central Ltd.2004Crites and Schuster; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Practice management education continues to evolve, and little information exists regarding its curriculum design and effectiveness for resident education. We report the results of an exploratory study of a practice management curriculum for primary care residents. Methods After performing a needs assessment with a group of primary care residents at Wright State University, we designed a monthly seminar series covering twelve practice management topics. The curriculum consisted of interactive lectures and practice-based application, whenever possible. We descriptively evaluated two cognitive components (practice management knowledge and skills) and the residents' evaluation of the curriculum. Results The mean correct on the knowledge test for this group of residents was 74% (n = 12) and 91% (n = 12) before and after the curriculum, respectively. The mean scores for the practice management skill assessments were 2.62 before (n = 12), and 3.65 after (n = 12) the curriculum (modified Likert, 1 = strongly disagree, 5 = strongly agree). The residents rated the curriculum consistently high. Conclusions This exploratory study suggests that this curriculum may be useful in developing knowledge and skills in practice management for primary care residents. This study suggests further research into evaluation of this curriculum may be informative for practice-based education. ==== Body Background Practice management education for residents has traditionally included training physicians in management issues related to the practice environment, including fiscal management, leadership skills, business and management skills, and managed care concepts [1]. Managed care concepts include ethics, communication skills, payment systems, population medicine, informatics and disease prevention. Although in existence since the 1970's, most practice management curricula have focused on managed care concepts, with little attention to the other skills [2-7]. In 2001, educators from Tuft's University wrote a report for curriculum development in the evolving practice environment [8]. This report, which was synthesized from nine component reports of national medical educational organizations, recommended future curriculum development beyond the traditional scope of managed care curriculum. It recommended redefining practice management as a curricular domain of fiscal, business, and practice system management skills distinct from traditional managed care topics [8]. The ACGME has recognized the need for residency training in the evolving practice environment, and has recommended training to include practice-based learning and improvement and systems-based practice [9]. The regression of traditional third-party managed care plans also implies an increased value and need for practice management skills [10]. Given this broad support of practice-based learning, physicians will need ongoing practice management and health systems education for the foreseeable future. A few studies on practice management curricula exist for resident education, but much more information is needed on successful curriculum design and evaluation [11-13]. We describe, in detail, a pilot practice management curriculum design using the evolving curricular theme for a group of primary care residents. We also report its initial analysis on improving resident knowledge and skills, and describe the residents' evaluation of the curriculum. Methods Educational setting We developed the curriculum for the University Medicine/Pediatrics Practice (UMP). This practice is a primary care-oriented, faculty-resident practice on the campus of Wright State University. Thirteen internal medicine/pediatrics residents, five general internist faculty, two internal medicine/pediatrics faculty, and one pediatrics faculty practice here. The practice is managed by Premier Healthnet, a 100 physician multi-site primary care group. Although UMP's mission includes addressing the needs of indigent patients in the Dayton area, it is modeled after a community-based, teaching practice model. Therefore, faculty and residents are expected to use effective practice management skills in their individual practices. A typical resident from our program enters a small (1–5 physician) community practice upon graduation and practices both internal medicine and pediatrics. Needs assessment In 2001, two faculty members (GEC and RJS) at the Wright State University Departments of Medicine and Community Health were identified as lead faculty for this curricular project. Primary care faculty in the Departments of Pediatrics and Medicine tasked these two lead faculty members to development a practice management curriculum to reflect the evolving practice theme. By "evolving practice theme," we mean teaching practice management topics similar to the Tuft's curricular theme. One lead faculty member (GEC) performed a needs assessment on the Internal Medicine/Pediatrics residents at UMP in the spring of 2001. The assessment used qualitative analysis via informal interviews of two senior residents, one of whom was chief resident. The interviews included open-ended questions on the need for practice management knowledge (example question: "What do you need to learn this year to help prepare you for running a community practice?"). The needs assessment also included an open feedback session with the residents after discussion of potential topics at the monthly resident education meeting (majority of residents present). The results of the need assessment were uniform; the residents felt inadequately trained in practice management. The lead faculty concluded that these residents had some training in a few specific content areas (i.e., coding), but lacked an overall basic practice management knowledge or skill. Curriculum design The lead faculty met again in mid-2001 to design the curriculum. The goals of the curriculum were to give the residents a basic understanding of practice management concepts and skills in the evolving practice environment. The lead faculty were free to select the most effective methods to meet their goals. They did face some challenges. They were given only 30-minute time slots each month and had 12 months to accomplish these goals. They also had to show some objective evidence of its effectiveness and have support of the residents at the end of the 12 months to continue the curricular project. The design process resulted in a series of seminars covering 12 topics, listed in Table 1, with objectives. The seminars began in July 2001, and concluded in June 2002. The lead faculty assigned teachers to each seminar who were content experts, and included a medical biller, a nurse manager, a health systems researcher, two local HMO medical directors, a financial advisor, a risk manager, and a WSU junior faculty member. The assignment of seminar teachers is listed in Table 1, and one lead faculty member (RJS) led two sessions (referred to as the health systems researcher listed under Revenue Management and Accounts Payable Management in Table 1). Although the seminar teachers were free to utilize any method and media to meet their session objectives, they were encouraged to use as much interactive teaching approach as possible. The sessions were primarily in the form of teacher-centered discussions augmented primarily with handouts, overheads, and slides. The seminar teachers often supplied references and reference materials as tools for the residents in their daily practices. We encouraged ambulatory practice faculty throughout the year to discuss with residents, during resident ambulatory practices, application of principles learned in the seminar series. Table 1 Schedule of topics (in bold), teacher assignments, and objectives for the practice management seminar series Topic, teacher Objectives Basic Coding, Medical Biller Introduction to the Fee Ticket E/M and PT Basics ICD-9 Basics Revenue Management, Health Systems Researcher Health System Overview Payment Systems How Physicians Get Paid Optimizing Coding to Enhance Reimbursement, Medical Biller Reimbursable Diagnoses in Primary Care Using Modifiers Procedures and Medication Coding Physician Personal Finance, Financial Advisor Financial Goals Financial Planning Insurance Systems and Payment Mechanisms, HMO Director #1 Insurance Contracts IPAs and Collective Bargaining Dynamics of Group Practice, HMO Director #2 Partnerships Structures: Solo, Small Group, Multi-specialty Practices Physician Leadership and Consensus Building Getting a Good Job, WSU Faculty Member Finding Positions and Writing CVs The Interview Process Contract Negotiations Accounts Receivable Management, Medical Biller The A/R Sheet Fiscal Targets Collections Management Accounts Payable Management, Health Systems Researcher Minimizing Expenses in Primary Care Economics of Running a Primary Care Practice Human Resources, Nurse Manager Staffing Needs Assessment Hiring/Firing/EEO Payroll & Benefits Conflict Resolution Risk Management, Risk Manager Minimizing Medico-legal Risk in Practice Regulatory Restrictions in Practice, Nurse Manager Understanding CLEA, OSHA, and HIPPA Curriculum evaluation The respondents were a convenience sample of Internal Medicine/Pediatrics residents from all four years of training. We used a pre-experimental (one-group pretest/posttest) design for this exploratory study. We descriptively evaluated the curriculum on two cognitive components: practice management knowledge and skills. We also assessed the residents' evaluation of the curriculum. To evaluate practice management knowledge, we used a knowledge test consisting of identical 12 item (true/false statements), and each question covered one objective from each topical area from Table 1. One example of a test item in the content area of coding is: "An established patient who has an expanded problem focused history and exam may be billed at a 99215 level." We administered the 0-month test to the entire group immediately before the first seminar session. We administered the 12-month test to the entire group immediately after the last seminar session. To evaluate practice management skills, we devised a survey of self-assessed practice management skills. The survey consisted of 12 statements, and each statement queried the residents to respond on their assessment of their own practice management skills. Each statement consisted of one specific skill from an objective from each topical area listed in Table 1. An example of one survey item in the content area of coding is: "I understand how to use modifiers with E/M (evaluation and management) coding." We based the responses to the statements on a modified Likert scale, with 1 being strongly disagree, and 5 being strongly agree. We administered the 0-month self-assessed skill survey to the entire group immediately before the first seminar session. We administered the 12-month self-assessed skill survey to the entire group immediately after the last seminar session. To explore the residents' evaluation of the curriculum, we devised another survey. This survey consisted of four statements querying the residents on their overall assessment of this curriculum and practice management education in general. The statements from the survey are given in Table 2B. The responses were based on the same Likert scale described above. This survey was administered to the entire group immediately after the last session. Table 2 Resident self-assessed practice management skills (A) and curriculum evaluation (B) (modified Likert scale: 1 = strongly disagree and, 5 = strongly agree) Evaluation component 0-month (n = 12) 12-month (n = 12) Mean (95%CI) Mean (95%CI) A: Self-assessed practice management skills: Results from 12 item survey 2.62 (2.27 – 2.97) 3.65 (3.41–4.08) B: Evaluation of practice management curriculum: Mean (1 SD) Mean (1 SD) Practice management series was effective in teaching me basic practice management knowledge NA 4.13 (0.61) I feel more confident in my own practice skills because of this curriculum NA 3.96 (0.45) I feel practice management curriculum should be incorporated into primary care curriculum NA 4.67 (0.65) I would be interested in expanding my primary care curriculum to include more practice management education NA 4.67 (0.49) The process of test instrument development was the same for both the knowledge test and the self-assessed skills survey. One lead faculty member (GEC) would generate a list of candidate items based on each objective in Table 1. The second lead faculty member (RJS) would review the list and select and/or modify items to match the item content to the objectives listed. Thus, both instruments possessed good face validity. Reliability testing was not performed due to the small sample size. Post-hoc item analysis on the 0-month knowledge test showed that only 2 items were answered 100% correct and the lowest item scored was 33% correct for this group. This suggests minimal floor and ceiling effects in the item mix. All other items ranged from 52% to 92% correct. Results The participants were the 13 Internal Medicine/Pediatrics residents, and represented all four years of training (2, 4th-year; 3, 3rd-year; 4, 2nd year; and 4, 1st-year residents). A third year resident failed to complete the 0-month tests and surveys, and a first year resident failed to complete the 12-month tests and surveys. This left 12 responses for both sets (0- and 12-month) tests and surveys. The average attendance for the sessions was 12, with a range of 10–13 attendees. The results from the knowledge test are given in Figure 1. As a group, the residents' mean score was 74% (95% CI, 68%–80%) for the 0-month survey and 91% (95% CI, 85–96%) for the 12-month survey. These confidence intervals do not overlap. This suggests that, if hypothesis testing were done, the results would probably reach statistical significance for the knowledge test. Figure 1 Practice knowledge test results (mean and 95% CI): before (0-month) and after (12-month) the course On follow-up, we performed two post-hoc analyses. First, we were interested if these knowledge scores would decline over time. Therefore, we compared the knowledge test scores on the first six months topics to the scores on the last six months topics. Both sets of scores were derived from the 12-month knowledge test. We found that the mean scores appeared similar (first 6 months mean scores: 92% correct; the last 6 months mean scores: 90% correct). Second, we were interested if the missing data on the 0-month and 12-month data could have impacted the results. Since one third-year resident completed the 12-month but not the 0-month test, we were interested in exploring if his responses on the 12-month test could have caused a larger difference between these two tests. After censoring his data, there appeared to be little impact on the 12-month results (censored mean score = 0.91, censored 95% CI, 85–96%). Additionally, the first year resident who failed to complete the 12-month test may have also impacted the results. Due to loss of identity links, we could identify her data to censor from the 0-month test. However, we censored the lowest score on the 0-month test as representing hers (this assumes that her score lowered the 0-month data the most, and, therefore, had the largest impact on 0-month mean score by skewing it away from the 12-month mean score). After censoring this data, we found no significant change in the 0-month results (censored mean score 0.75, censored 95% CI, 70–80%). The results for the self-assessed skill survey are given in Table 2A. The mean scores on the 12-month survey (3.65) were higher than in the mean scores for the 0-month survey (2.62). The confidence intervals from this data do not overlap. This suggests that, if hypothesis testing were done, the results would probably reach statistical significance for the self-assessed practice management skills survey. The results of the curriculum evaluation survey are given in Table 2B. All statements had a mean rating of greater than 3.90. The two statements assessing the residents' views towards practice management education in general (value of practice management education and the need to expand their education) both had mean scores of 4.67. Discussion The practice environment continues to evolve [8]. Although a traditional term for educating physicians in the practice environment, "managed care curriculum" is a vague terminology and lacks comprehensiveness [8]. The Tufts' report did not use this term for specific curricular terminology, and this may parallel the purported demise of the term for the traditional payer system [10]. The Tufts' report included a comprehensive list of 10 curriculum domains in the evolving practice environment [8]. This report gave the practice management domain, which had lacked emphasis in half of its nine component reports, equal emphasis as the traditional managed care curricular domains [8]. The practice management domain included training on topics such as basic business skills, management skills, financial risk, payment systems, process improvement, and practice systems [8]. With respect to the evolving practice environment, the challenge for educators is devising practice management curricula that cover these topics adequately and relating them to other curricular domains (i.e., health systems, quality improvement, etc.). We were interested in whether a curriculum design with this evolving theme may be useful in primary care education. We describe, in detail, a curriculum design similar to the evolving theme designed for a small group of primary care residents. The advantage of such a program as ours is its detailed design based on general and specific needs assessments and a description of evaluation methodologies. Our data suggests that this intervention may have had an impact on resident knowledge scores and self-assessed skills. Additionally, the residents appeared remarkably positive towards this practice management curriculum and practice management education in general. A few studies have been published on practice management curricular design and evaluation for primary care residents. In a response to the growing need physician-managers, both Zoorob and Taylor and Johnson described curricular designs they proposed would fill this need [12,14]. Lynch and Johnson published a report on the evaluation of business management skills in primary care residents, and found no improvement with a short educational intervention (two day seminar) [11]. Werblun et al. described a proposed curriculum design and evaluation that would meet the needs for business management skills, and like our curriculum, recommended implementation over the course of the term of residency. Our study does have some limitations. Because our small sample size, formal hypothesis testing was not possible and our data remains descriptive only. Stronger conclusions of these results would need to be re-evaluated with more subjects using formal hypothesis testing methods. Our experience suggests that internal motivation was probably one key factor to acceptance and apparent acceptance of this curriculum; the request for developing the curriculum came from our residents themselves. Also, the UMP faculty is uniformly positive towards developing these skills in themselves and in the residents, and this probably influenced residents' motivation to learn the subject matter. Since our faculty-resident practice is based on a primary care, community model, it may be difficult to generalize it to hospital-based practices or specialty residency training. Conclusions We conclude that an extended curriculum in practice management with an evolving practice theme may be useful in primary care education. We also believe that attention to instructional design, including performing a needs assessments, using many teaching methods, and applying the concepts learned in learners' practices, may contribute to its acceptance and success. Future educational designs for this curriculum include its continued expansion, exploring more educational opportunities for implementation, and addressing specific characteristics of success and failure. Future educational research in this area would require a more formal research design to derive stronger conclusions regarding its effectiveness. Competing interests None declared. Author contributions GEC participated in the curricular needs assessments, curricular design, curriculum implementation, and drafting of the manuscript. RJS participated in the curricular design, curriculum implementation, and drafting of the manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: ==== Refs Paller MS Becker T Cantor B Freeman SL Introducing Residents to a Career in Management: The Physician Management Pathway Acad Med 2000 75 761 764 10926031 Leshan LA A Curriculum in Managed Care for Family Practice Residents Wis Med J 1996 95 22 26 8650950 Maclean JR Rahn DW Salazar W Teaching Managed Care Principles to Residents Acad Med 1999 74 603 604 10676229 Zoorob RJ Sidani M A Managed Care Curriculum Developing a Managed Care Curriculum for Primary Care Residents Med Educ 1999 33 854 859 10583795 10.1046/j.1365-2923.1999.00508.x Hakim A Kachur E Santilli V A Comprehensive Curriculum in Managed Care for Residents Acad Med 2001 76 560 11346614 10.1097/00001888-200105000-00107 Williams BC Woolliscraft JO Heindel JE A Managed Care Curriculum Implemented Across Four Academic Departments Using Mandated Evaluation Instruments Acad Med 1999 74 604 605 10676230 Rabinowitz HK Babbott D Bastacky S Pascoe JM Patel KK Pye KL Rodak J Veit KJ, Wood DL Innovative Approaches to Educating Medical Students for Practicing in a Changing Health Care Environment: The National UME-21 Project Acad Med 2001 76 587 597 11401801 Halpern R Lee MY Boulter PR Phillips RR A Synthesis of Nine Major Reports on Physicians' Competencies for the Emerging Practice Environment Acad Med 2001 76 606 615 11401803 Education Accredation Council for Graduate Medical ACGME Outcomes Project: General Competencies Robinson JC The End of Managed Care JAMA 2001 285 2622 2628 11368736 10.1001/jama.285.20.2622 Lynch DC Johnson JJ A Separate Pretest-Posttest Design to Evaluate a Practice Management Seminar for Residents Acad Med 1999 74 446 447 Taylor JM Johnson KG A Residency Program in Primary Care: The Physician as Provider-Manager J Med Educ 1973 48 654 660 4708422 Werblun MN Martin LR Drennan MR Parker CE Berggren RE A Competency-based Curriculum in Business Practice Management J Fam Pract 1977 4 893 897 864414 Zoorob RJ Practice-Management Curriculum for Family Practice Residents Acad Med 1997 72 446 447 10676378

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==== Front BMC GenomicsBMC Genomics1471-2164BioMed Central London 1471-2164-5-651536309610.1186/1471-2164-5-65Research ArticleAn integrated 4249 marker FISH/RH map of the canine genome Breen Matthew [email protected] Christophe [email protected] Travis D [email protected] Rachael [email protected] Edouard [email protected] Leah [email protected] Allyson [email protected] Gwenaelle [email protected] Heidi G [email protected] Ewen F [email protected] Ruth [email protected] Richard [email protected] Gregory G [email protected] Boris [email protected] Claire M [email protected]é Catherine [email protected] Francis [email protected] Elaine A [email protected] Department of Molecular Biomedical Sciences, College of Veterinary Medicine, North Carolina State University, 4700 Hillsborough Street, Raleigh, NC 27606, USA2 UMR 6061 CNRS, Génétique et Développement, Faculté de Médecine, 35043 Rennes Cédex, France3 Clinical and Human Biology Divisions, Fred Hutchinson Cancer Research Center, Seattle WA 98109-1024, USA4 The Institute for Genomic Research, 9712 Medical Center Drive, Rockville, MD, 20850, USA5 Oncology Research, Animal Health Trust, Lanwades Park, Suffolk, CB8 7UU, U.K6 VieVax Corp. 1616 Eastlake Ave. E., Seattle, WA 98102 USA2004 13 9 2004 5 65 65 23 8 2004 13 9 2004 Copyright © 2004 Breen et al; licensee BioMed Central Ltd.2004Breen et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background The 156 breeds of dog recognized by the American Kennel Club offer a unique opportunity to map genes important in genetic variation. Each breed features a defining constellation of morphological and behavioral traits, often generated by deliberate crossing of closely related individuals, leading to a high rate of genetic disease in many breeds. Understanding the genetic basis of both phenotypic variation and disease susceptibility in the dog provides new ways in which to dissect the genetics of human health and biology. Results To facilitate both genetic mapping and cloning efforts, we have constructed an integrated canine genome map that is both dense and accurate. The resulting resource encompasses 4249 markers, and was constructed using the RHDF5000-2 whole genome radiation hybrid panel. The radiation hybrid (RH) map features a density of one marker every 900 Kb and contains 1760 bacterial artificial chromosome clones (BACs) localized to 1423 unique positions, 851 of which have also been mapped by fluorescence in situ hybridization (FISH). The two data sets show excellent concordance. Excluding the Y chromosome, the map features an RH/FISH mapped BAC every 3.5 Mb and an RH mapped BAC-end, on average, every 2 Mb. For 2233 markers, the orthologous human genes have been established, allowing the identification of 79 conserved segments (CS) between the dog and human genomes, dramatically extending the length of most previously described CS. Conclusions These results provide a necessary resource for the canine genome mapping community to undertake positional cloning experiments and provide new insights into the comparative canine-human genome maps. caninedogradiation hybridmicrosatellitesESTsBAC-ends ==== Body Background Three major advances in the development of resources for mapping canine disease genes have been: 1) the development of a radiation hybrid (RH) map composed of large numbers of microsatellite markers and genes that link the canine and human genomes [1], 2) the development of canine specific whole chromosome paints that have allowed preliminary assignment of conserved segments between human and dog [3-5]; and 3) the publication of a 1.5x genome sequence of the dog [2]. The most recently published RH map of the dog comprises 3270 markers including 1596 microsatellite-based markers, 900 canine-specific cloned gene sequences and expressed sequence tags (ESTs), and an initial set of 668 canine-specific BAC-ends [1]. The map was constructed using the RHDF5000-2 whole genome radiation hybrid panel [6] and features markers mapped to 3009 unique positions, defining an average inter-marker distance of one megabase (Mb). The map also defines a minimal screening set of 325 highly informative well-spaced markers, to be used in the initiation of genome-wide scans [1]. A well-defined synteny between the dog and human genomes was established as a function of this work [1] and from extensive reciprocal chromosome painting studies [3,5,7]. The above mapping efforts are complemented by the recent release of a 1.5x sequence of a Standard Poodle genome [2]. The sequence includes 6.2 million sequence reads that span approximately 78% of the genome. More than 650 million base pairs (>25%) align uniquely to the human genome, and the resulting alignment includes fragments of putative orthologs for 18,473 of 24,567 annotated human genes. The current alignment supports most of the proposed comparative segments, but suggests that the final comparative dog-human map will be composed of at least 160 comparative blocks [2]. Using the above resources, canine researchers have undertaken genome-wide screens for linkage to a variety of disease loci as well as morphological traits defining differences between breeds. Many of these studies have met with success. Disease loci have been genetically mapped or otherwise localized in the dog for several disorders including: vision-associated disease such as progressive rod cone degeneration, early retinal degeneration, cone degeneration, and collie eye anomaly [8-11], kidney cancer [12,13], narcolepsy [14], rheumatoid arthritis [15], Severe Combined Immunodeficiency (SCID) [16], hip dysplasia [17], cystinuria [18], bleeding disorders [19,20], ceroid lipofuscinosis [21], and copper toxicosis [22,23]. In addition, quantitative trait loci (QTL) have been identified for principal components defining skeletal variation and hip dysplasia [17,24]. In general, investigators have been able to use the existing resources to localize traits of interest to an interval of 10–20 Mb, but in most cases the causative gene remains to be identified. To facilitate positional cloning efforts in the canine community, we have localized a large set of randomly selected canine-specific BACs onto the canine 5000-rad RH map, a subset of which we have also localized cytogenetically. The resulting map of 4249 markers includes 1760 mapped BAC-ends, 851 of which have been FISH mapped, to generate a dense, accurate and highly integrated map of the canine genome. Results General RH map characteristics This novel RH map of the dog genome contains 4249 markers of three different types: 900 genes, 1589 microsatellites and 1760 BACs. The map was generated by genotyping 1092 new BAC-end markers on the RHDF5000-2 panel, and recomputing the new vectors with those from the previous map [1] using MULTIMAP [25] and TSP/CONCORDE [26]. Table 1 summarizes the key features of the new map. The typing of 4249 markers resulted in an RH map containing 4106 markers that were eventually grouped and assigned to each of the canine chromosomes, leaving only 143 unlinked markers. Human orthologs were identified for 2233 mapped markers. The resolution limit of the RHDF5000 panel has been determined to be 4 cR5000 (600 Kb) [6]. Thus, markers falling within any 4 cR5000 or 600 Kb stretch cannot be ordered relative to one another with high confidence and, consequently, are reported as co-localized on the map. Table 1 Key features of the integrated canine RH/FISH map Number of Markers Unique BACs Unique BACs BACs Chromosome Size (Mb)(1) RH Mapped RH Positions(2) RH Mapped BAC RH Positions FISH Mapped RH/FISHed in Common Human Coordinates(3) Human/ Dog CS(4) CFA01 137 220 169 104 83 60 48 110 4 CFA02 99 140 99 55 43 37 27 78 4 CFA03 105 155 123 74 60 46 35 71 3 CFA04 100 147 106 60 51 37 29 80 3 CFA05 99 158 102 76 53 40 28 74 4 CFA06 87 122 69 53 35 29 19 69 3 CFA07 94 173 107 78 56 44 37 97 2 CFA08 86 135 98 64 50 30 25 77 1 CFA09 77 124 93 39 33 31 26 78 2 CFA10 80 121 91 53 41 28 22 68 3 CFA11 86 134 116 56 49 38 36 78 2 CFA12 85 181 127 71 55 32 29 94 1 CFA13 75 93 65 38 32 21 18 53 2 CFA14 72 114 85 54 42 31 28 62 2 CFA15 75 113 94 42 38 26 22 63 5 CFA16 73 93 79 42 36 23 23 44 3 CFA17 80 130 96 53 41 20 18 69 2 CFA18 66 118 99 50 45 30 27 71 2 CFA19 66 96 66 40 31 18 18 49 2 CFA20 66 139 95 52 41 20 16 74 2 CFA21 61 103 86 34 31 26 22 60 1 CFA22 61 113 76 52 33 17 15 70 1 CFA23 61 79 67 40 36 23 21 45 1 CFA24 73 84 66 41 33 18 16 42 1 CFA25 60 94 75 41 36 27 22 56 4 CFA26 48 85 66 39 33 19 13 44 3 CFA27 57 94 76 29 27 19 16 50 1 CFA28 55 84 70 39 35 26 24 47 1 CFA29 51 81 71 33 31 20 18 42 1 CFA30 47 63 50 26 21 18 16 38 1 CFA31 50 53 47 25 23 16 13 25 2 CFA32 51 56 46 33 28 14 15 27 1 CFA33 41 63 52 30 28 20 20 40 1 CFA34 50 65 49 23 13 15 15 47 2 CFA35 38 49 35 24 16 14 12 30 1 CFA36 41 61 52 26 26 13 13 40 1 CFA37 40 65 45 27 21 20 17 37 1 CFA38 38 32 28 16 14 9 9 16 1 CFAX 139 66 44 26 21 23 22 28 1 CFAY 27 10 10 2 2 2 1 1 1 unlinked 143 TOTAL 2797 4249 3090 1760 1423 1000 851 2233 79 Average Distance (Mb) Between Markers -- 0.66 0.91 1.59 1.97 2.80 3.48 1.25 Legend: Map statistics.(1) Chromosome size in Mb based on bivariate flow cytometry measurements (2) Unique RH positions with one or more markers (3) Human coordinates identified through BLAST analysis (see Methods) (4) Conserved segment identified by RH mapping with 2 or more loci Analysis of the entire dataset of 4249 RH vectors by MULTIMAP [25] at a Lod score of 8.0, and for a subset of regions a Lod of 9.0, resulted in 60 individual linkage groups that could be assigned to the 38 canine autosomes and two sex chromosomes. For chromosomes that were covered by multiple linkage groups, the data were merged and ordered as described in the Methods. FISH data combined with significant Lod scores between selected markers led to the unambiguous assignment and orientation of specific linkage groups to chromosomes and, ultimately, complete coverage of each chromosome. The resulting 4106 linked markers defined 3090 unique positions regularly spaced across all autosomes. The largest chromosome, canine chromosome 1 (CFA 1), contains 220 markers, while CFA 38, the smallest chromosome at 38 Mb, has only 32 markers (Figure 1). Excluding the sex chromosomes, the least dense chromosome, CFA 38, has a marker located every 1.2 Mb, while the most dense, CFA 12 and CFA 20, have markers positioned at an average of every 0.47 Mb. The increase in density is due solely to the addition of 1092 new BAC-ends, bringing the total to 1760 from the 668 reported in the previous version of the map. The mean interval size is now 1.59 Mb. The chromosome with the most BACs mapped to it is CFA 1, featuring 104 localized to 83 unique positions (Figure 1). See all chromosomes in the Supporting Online Material (SOM) at . Again, the chromosome with the fewest is CFA 38, with 16 BACs localized to 14 unique positions. This resource provides a dense scaffold on which to build regional physical maps and search for new genes. Of particular interest to disease gene mappers will be the 26 BACs that are RH mapped to the X chromosome, 13 of which are also localized by FISH. Only two BACs were localized to the Y chromosome, one of which is also FISH mapped. See Table 1 for details. Figure 1 Integrated FISH/RH map and dog/human comparative data for CFA1. BAC-ends also localized by FISH mapping are reported in the left panel, symbolized by a vertical bar along with BAC addresses. Asterisks (**) represent BAC clones that did not have a unique cytogenetic location (multiples hits are listed on SOM). RH mapped markers and their cumulative positions in centiRay 5000 units are reported to the right of the FISH map. Connecting lines between FISH and RH maps indicate integration points between maps. The right panel shows the human evolutionarily conserved segments, represented by colored boxes as determined by RH data. Human coordinates (in Mb) identified from dog/human sequence alignments (see Methods) are reported on the right most vertical bar. RH markers and their corresponding human sites are connected by lines and illustrate the rearrangement within synteny blocks. FISH mapped BACs A total of 1,000 BACs were assigned to a chromosome band, and then ordered along the length of the corresponding chromosome using a combination of metaphase and interphase multi-color FISH. CFA 1 is shown, for example, in Figure 2. The panel of FISH mapped clones included clones representing both ends of each chromosome, with the exception of the centromeric end of CFA 6 (6q11-q12) and the centromeric 20% of CFA 9 (9q11-q12). Nine hundred and eighty-one out of 1,000 BACs had a unique cytogenetic location, and 851 were also ordered on the RH map. This yielded an average of one BAC-end that was both FISH and RH mapped every 3.48 Mb. See SOM material for data on all chromosomes and . Figure 2 Assignment of 60 canine BAC clones to CFA 1. BAC addresses alongside the ideogram of CFA 1 refer to clones from the RPCI-81 canine BAC library. A pair of colored spots spanning an interval of approximately 4 Mb represents the cytogenetic assignment of each clone. The color of the spots identifies the fluorochrome used to label the clone as follows: red = Spectrum Red; orange = Spectrum Orange; green = Spectrum Green; blue = DEAC; purple = Cy5. Multi-color FISH of neighboring clones, using both metaphase and interphase analyses, was used to establish the precise order of the clones along the length of the chromosome. Clones whose assignment is represented by a circular rainbow have been tentatively placed, but not yet co-localized with neighboring clones to establish one equivocal, linear order. BAC addresses followed by ** identify those clones that resulted in fluorescent signal at more than one location. Human orthologous regions (HSA) are reported on the left of the figure by vertical bars. Integration of FISH and RH data Analysis of co-linearity between the two maps was performed by connecting the BAC markers in common between the RH map and FISH data. This approach allowed the identification of markers that serve as anchors to each chromosome for both RH and FISH maps, thus validating their localization and marker order. Early in the assembly process, a small number of discrepancies between the two maps were easily identified through graphical drawings of all chromosomes, leading to a systematic re-examination of experimental data and, in most cases, consensus resolution. However, 19 BACs did not have one unique cytogenetic location. These are indicated with a double asterisk in Figure 1 and 2, as well as the SOM and figures provided on all web sites. For each of these 19, one of their FISH localizations was in agreement with the corresponding RH map position. In addition, seven (7/19) had an identified human ortholog that was in agreement with the RH map localization. For an additional set of 24 BACs, conflict remained after rechecking both the FISH and RH data. These are indicated by "#" in all Figures. Interestingly, the DNA used to obtain FISH data for these clones PCR amplified successfully with the same primers used to obtain RH data, indicating that the DNA samples were the same and had not undergone sample mix-up. Finally, a human ortholog sequence was identified that was in agreement with the RH data for 15 (15/24) of these clones. Although we have no clear explanation for these discrepancies, it is of note that the two mapping methods used different DNA sequences to define the same marker. In the RH method, a pair of 25-mer oligonucleotides defining only one end of the dog insert DNA is used. By comparison, in the FISH analysis, the whole BAC encompassing approximately 150 Kb is hybridized. Since the DNA for the FISH localization was prepared from cultures initiated from single bacterial colonies, these anomalies suggest that at least a subset of the clones may be chimeric. An additional explanation is that the clones are hybridizing to genomic regions containing one member of a multi-gene family. Whatever the explanation for these discrepancies, it is noteworthy that the total number of discrepancies between the FISH and RH data represent 3% of the total data. This is in agreement with reports that the frequency of chimeric clones in this BAC library is very low [27]. Thus, in most cases, the co-linearity of the FISH and RH maps is perfect, i.e. the order of the BACs determined by multi-color FISH is identical to that derived from analysis of genotyping data using primers defining single BAC-ends. In addition to the conflicting results described above, in some cases, we note that the order for two closely positioned markers is inverted between the FISH and the RH data. However, for all of these cases the distance between the two markers is estimated to be less than 50–100 Kb, as these clones overlap in interphase nuclei. Thus, they are well within the 600 Kb resolution limit of the RH5000 panel. Such minor inaccuracies in marker order will not be detrimental to gene mapping, as the integrated map has fixed positions every 3.42 Mb with support from FISH data. The example of CFA 1 is provided (Figure 1 and 2). Please refer to SOM and websites for all other chromosomes. Synteny and conserved segments Two criteria were used to assign dog BAC clones to orthologous locations of the human genome. First, when compared to the complete human genome, each component of the paired end-sequences had greatest similarity to locations that are separated by 50–500 Kb. Although most BAC clones have inserts of 100–200 Kb, minor differences between the two genomes such as local duplication or loss of specific genes should be accommodated by the 50–500 Kb range. If one considers a single aligned end-sequence, the probability of the paired end-sequence aligning spuriously within 500 Kb in the human genome is approximately 0.03%. The second criterion was that each pair of end-sequences should align in a head-to-head orientation. With this additional condition, the probability of scoring pairs that contain spurious alignments falls to less than 0.01%. In the most recent version of the map [1], 75 conserved segments (CS) were detected within the 38 canine autosomes. In addition, two CS corresponding to the sex chromosomes and nine singletons were detected. These 77 CS were identified through the analysis of 820 canine markers having an unambiguous ortholog in the human sequence. In the present map, that number is more than doubled with 2233 markers having an ortholog in the human sequence. These newer data confirm all previously described CS, and incorporate two singletons into two novel CS for a new total of 79 CS. For the sake of clarity, the remaining singletons were not reported in this new version of the map. Interestingly, while the number of CS did not change significantly compared to the previous iteration of the RH map, their nature and composition were altered, as shown for CFA 1 (Figure 1). Four CS were identified for CFA1 that correspond, in order from centromere to telomere, to human chromosomes (HSA) 18, 6, 9 and 19. From the density of markers for which an ortholog can be identified, it is clear that the order of markers is consistent between CFA 1 and HSA 18. Thus, this CS would be considered a "conserved and ordered segment" (CSO). For the CS corresponding to HSA 6, however, two sub-segments are noted which are caused by an inversion. In each of these two sub-segments, the order of markers is comparable to the syntenic portion of HSA 6, and each canine segment would independently be considered a CSO. In addition to identification of some previously unknown CSO, the increased number of markers for which a human ortholog could be identified unambiguously allowed for a substantial increase in the size of many previously identified CS. At present, as shown in Figure 1 and SOM, CS in the dog are highly contiguous with the human genome. Indeed, only a few markers identified as human orthologs fall outside of a CS. This significantly refines our knowledge of evolutionary breakpoints between the dog and human genomes. Discussion The 4106 markers that constitute this new map occupy 3090 distinct positions, with an average of 1.3 markers per position. This reflects the fact that with 4106 genotyped markers, we are approaching the saturation level of 4500 markers predicted on the basis of size of the canine genome, and resolving power of the 5000 rad panel [6]. A detailed analysis of the co-localized markers indicates that in the majority of cases markers are co-positioned with other markers of different types, i.e. BACs and microsatellites, or microsatellite and genes, etc. Thus, all of the data will likely prove useful for mapping and cloning genes of interest, as the information provided at each location is non redundant. In addition, even when markers of the same type are co-localized, they are likely to be of value; any given microsatellite is not informative in every pedigree, and closely localized BACs may represent the beginning of an overlapping contig. A direct consequence of markers mapping to the same positions is that the mean distance between two adjacent positions is now 0.9 Mb, which is very similar to the 1 Mb calculated for the most recent published version of the canine map [1]. Again, this reflects the resolving power of the 5000 rad panel [6], as well as the method of map computation. TSP/CONCORDE considers markers co-localized if they map to a region of 0.9 Mb or less, regardless of RH panel mapping power [26]. The number and identity of conserved segments (CS) between the human and the dog genomes is 79. CS were identified by both FISH localization and RH mapping with four chromosomes showing four human/dog CS, and the remainder showing one to three. The number of markers and the resolving power of the 5000 rad panel allowed us to detect several instances in which CS appear to be split in sub-segments. In several such cases, the order of markers in the two adjacent sub-fragments is conserved, although the two sub-fragments are oriented in opposing directions. Following the previously agreed definitions, these sub-fragments are termed CSO for "conserved segment order" [28]. Such a situation is observed in CFA 1 (Figure 1). In other instances, one of the two adjacent sub-fragments appears to correspond to a CSO, while the other corresponds to a CS. Obviously a better distinction between CS and CSO segments would require genotyping of additional markers, ideally on a panel constructed with a higher dose of radiation, and with concomitantly higher resolution. Such experiments are currently underway using 10,000 genes derived from the available dog genome sequence [2] and a newly constructed 9000 rad panel (data not shown). As shown in Table 1, and as indicated in the Results, 851 BACs were localized both by FISH and RH mapping, with a high level of concordance between the two methods. Thus, the map presented here provides a verified anchor point every 3.48 Mb. Unlike a FISH map that is not altered or modified by the addition of a new dataset, an RH map does change after adding information because different algorithms provide different solutions for local marker order. Each is statistically valid when ordering the same set of markers. However, discrepancies may arise as more markers are added to the map. Moreover, even one algorithm can generate maps with slightly different marker orders within localized regions, even when only minor adjustments are made to the dataset like removing seemingly redundant markers [29]. In the present study, the high degree of anchorage by a set of markers localized in different laboratories with differing technologies yields a high level of confidence for the integrated FISH/RH map. Of interest to many researchers will be the multiple applications that a cytogenetically defined and RH verified set of canine BAC clones can provide. For example, the ordered set of 981 FISH mapped canine BAC clones with a unique cytogenetic location will be welcomed by cancer geneticists as a means to characterize chromosome aberrations in canine tumors. The dense cytogenetic coverage available for most chromosomes allows the use of serial differential labeling of some or all clones in chromosome-specific panels designed to 'tile' structurally aberrant chromosomes. This approach will allow a much more accurate assessment of chromosome rearrangements than is possible with single color whole chromosome paint probes. In addition, the ordered clones will allow expansion of the current canine BAC microarrays, which in turn may be used for a variety of applications including array-based Comparative Genome Hybridization (CGH) analysis of canine tumors [30], array-painting of aberrant canine chromosomes, and investigation of cytogenetically cryptic copy number changes and gene dosage alterations in congenital abnormality syndromes. In addition, ordered arrays of BAC clones will be an important resource for refining the comparative cytogenetic data within the diverse karyotypes that comprise the Canidae. Finally, with the imminent release of the 7x canine genome assembly, the resources described in this paper will provide a long-awaited means to translate canine cytogenetics data into canine DNA sequence data, thus advancing our knowledge of canine and comparative genomics. Conclusions The presentation of a map containing 900 canine specific genes, 1589 microsatellites and 1760 BACs provides the canine genetics community with nearly all the resources it needs to undertake experiments aimed at both mapping and cloning traits of interest. A dense set of microsatellite markers (MSS-2) for undertaking genome wide scans was provided in the previous iteration of the map [1]. These same markers are integrated into the current map. Thus, within 0.7/0.8 Mb of any linked marker on the current map is now an array of anchored BACs for contig building, comparative mapping, and searching for new genes and splice variants. The current map defines some 79 conserved segments between human and dog. While we expect this number to approximately double when the 7x canine genome sequence is completed, we speculate that given the density of markers mapped and the distance between adjacent CS, new CS defined by the 7x sequencing effort will be short in size and harbor a limited number of genes. What remains in canine genome map building is the development of a very high resolution gene map that can assist in the assembly of the 7x canine genome sequence now underway, and provide a mechanism for moving easily between canine and human comparative segments. This is currently being undertaken. In the meantime, canine researchers can move forward with the continued development of the dog model for mapping and cloning genes of interest to both human and companion animal health. Methods Markers and primer selection Canine BACs were randomly selected from the RPCI-81 canine BAC library [27] and were end-sequenced as described previously [1]. DNA was prepared using standard automated approaches [31] and sequenced either at The Institute for Genomic Research (TIGR) or the University of Washington High Throughput Sequencing Center. Average read lengths were in excess of 700 bp. Trace files representing BAC-end sequences were imported from ABI sequencers and examined for sequence homology to cloning vectors, Escherichia coli (E. coli) and repetitive DNA sequences. BAC sequences were also compared using Cross_Match with the complete E. coli genome to remove contaminating sequences of bacterial host origin. Finally, sequences were examined for interspersed repeats and regions of low sequence complexity using RepeatMasker . Primers defining each BAC-end were designed to regions of high quality sequence data using Primer3 software . Primers of 25 bp lengths were preferentially designed in order to minimize problems associated with non-specific amplification, to generate amplicons of 200 to 500 bp, and to work under a single optimal set of PCR conditions. Genotyping Genotyping was performed using the RHDF5000-2 panel, which is comprised of 118 cell lines. The panel was constructed by fusing dog fibroblasts irradiated at 5000 rads with TK-HTK3 hamster cells and has an experimental retention frequency of 22% with a theoretical resolution limit of 600 Kb [32]. PCR reactions were carried out at the University of Rennes and the Fred Hutchinson Cancer Research Center (FHCRC) as described previously in 15 μl volumes [1,33-35] using the following touchdown program: 8 min 95°C, followed by 20 cycles of 30 sec 94°C, 30 sec 63°C decreasing by 0.5°C per cycle, 1 min 72°C and 15 cycles of 30 sec 94°C, 30 sec 53°C, 1 min 72°C and a final extension of 2 min 72°C. Primer pairs yielding either faint or spurious bands were rejected. PCR products were resolved and recorded as described [33,35] through a semi-automated data acquisition software. Quality control Duplicate genotypes were obtained for the 1092 new BACs added to the map. Data were considered consistent when the number of discrepancies between data sets was ≤ 16%, a value determined to correspond to a distance lower than the resolution limit of the RHDF5000-2 panel. In the rare cases where two independent typings yielded >16% discrepancies, a third typing was done and the resulting vector was either integrated into the map construction, or the marker was discarded if no agreement was observed between two of three genotypes. RH map construction RH vectors corresponding to the BAC-end markers and marker vectors of the previous map [1] were computed as a single data set using the MultiMap and TSP/Concorde algorithms [1,25,26,29]. Linkage groups were generated initially at a Lod score of 8.0, and where needed, at 9.0 to ensure strong statistical support. Individual linkage groups were analyzed using the multipoint approach of the rh_tsp_map version 2.0 of TSP/CONCORDE, as described previously [1,29]. Inter-marker distances are expressed in cR5000 units. Framework maps of well-spaced markers, supported by high quality data between adjacent markers, were initially generated for all chromosomes. Distance criteria were set to 4cR, corresponding to the resolution capacity of the RH panel used, and quality criteria were fixed to 4, corresponding to a maximum allowable number of ambiguous data within RH vectors, as determined from previous analyses [1,35]. Distance and ordering of markers within each group was then determined by the TSP/Concorde and rh_tsp_map-2 algorithms [26,29]. Markers that could not be ordered with a high confidence level were submitted for re-analysis by stepwise increases in the Lod score to > 9.0, forcing the linkage group to split into two or more groups, until satisfactory order with a high statistical confidence level was achieved for each resulting group. Groups were then merged and oriented into a unique dataset. The merging step utilized the cytogenetic data obtained as part of this study, as well as the 2-point Lod scores between the markers at the extremes of each linkage group. Lod scores were generated using the pairlod_dist software from the rh_tsp_map package [26]. FISH mapping A total of 851 BAC clones from the RH map were also localized by multi-color FISH analysis. DNA from each clone was prepared from 2.5 ml cultures using a BAC RealPrep (Qiagen, Valencia, CA) protocol. Two hundred nanograms from each sample were labeled using nick translation to incorporate one of five fluorochromes, Spectrum Red/Orange/Green dUTP (Vysis, Downers Grove, IL), diethylaminomethylcoumarin (DEAC)-5-dUTP (NEN/Perkin Elmer Life Sciences, Boston, MA), or Cy5-dUTP (Amersham Biosciences, Piscataway, NJ). Typically, 25 ng of each of five differentially labeled probes were pooled and precipitated in the presence of 15 μg of sonicated genomic dog DNA as competitor. Chromosome preparation, probe hybridization and post hybridization washes were performed as described previously [35,36]. Chromosomes were counterstained in 80 ng/ml 4', 6-diamidino-2-phenylindole (DAPI) and mounted in anti-fade solution (Vectashield, Vector Laboratories, Burlingame, CA). Images were acquired and processed using a multi-color FISH workstation comprising a fluorescence microscope (Axioplan 2ie, Zeiss) equipped with narrow pass filter sets and a cooled CCD camera (CoolSnapHQ, Photometrics, Tuscon, AZ) both driven by dedicated software (SmartCapture 2.3.1 Digital Scientific, Cambridge, U.K.). The digital image of each DAPI stained metaphase spread was processed using a high-pass spatial filter to reveal enhanced DAPI bands. Clones were assigned to a chromosome region according to the DAPI banded nomenclature of Breen et al. [35,36]. Refinement of probe order along the length of each chromosome was made by subsequent rehybridization to elongated canine chromosome preparations and/or by reference to interphase FISH analysis. Additional information may be found at . Alignment of dog BAC clones to orthologous regions of the human genome Nineteen 384-well plates of BAC clones from the RPCI-81 library [27] were selected at random, and end-sequence data were obtained from each clone using previously described methods [1]. Paired end-sequences for 1910 clones were masked for repetitive elements and searched against the human genome (NCBI build 31, November 2002, ). For 648 of the BACs (34%), each of the paired end-sequences gave a best hit to human genomic locations that are separated by 50–500 Kb, and aligned head-to-head (mean span of human genomic DNA = 191 Kb). The remaining 1262 BAC-end sequences were searched against scaffolds of the 1.5x assembly [2] using wu-blastn (matrix = identity, W = 40) to identify scaffold sequences that contained at least short overlaps (40 bases) of identical sequence. For 954 of the 1262, hits were detected for both of the paired end-sequences. The homologous scaffold sequences were trimmed to remove any sequence that extended beyond 5 Kb from the region of alignment. They were then masked for repetitive elements, and searched against the human genome using wu-blastn (E<0.1). Again, only the best hit was considered. For 604 of the BACs (32% of the original sample), the paired scaffolds gave the best hits to genomic locations that are separated by 50–500 Kb (mean = 202 Kb), and aligned with their component BAC-end sequences in a head-to-head orientation. Altogether a total of 1252 (648+ 604) canine paired BAC-end sequences demonstrated significant hits with the human sequence. Accession numbers, PCR conditions, primers and BAC-end sequences are available for all markers at: and . Abbreviations BAC-Bacterial Artificial Chromosome FISH-Fluorescence in situ Hybridization PCR-Polymerase Chain Reaction RH-Radiation Hybrid CGH-Comparative Genomic Hybridization cR-centiRays Mb-megabases Kb-kilobases Bp-base pair Min.-minutes E. Coli-Escherichia coli SOM-Supporting Online Material DAPI-Diamidino-2-phenylindole CS-Conserved Segments CSO-Conserved and Ordered Segments TIGR-The Institute for Genomics Research FHCRC-Fred Hutchinson Cancer Research Center Authors' contributions MB, RT, AS, and RH all contributed to the FISH mapping aspects of the manuscript, including isolation of BAC DNA samples, generation of canine chromosome preparations, probe labeling and purification, fluorescence in situ hybridization, and microscopy. MB performed all the cytogenetic analysis to generate detailed probe ordering. MB and RT completed the merging of RH and FISH data, and prepared the drawing of Figure 2 for both the manuscript and web sites. MB also wrote and edited relevant sections of the manuscript. CH did the statistical analysis for the localization of the 4249 markers on the radiation hybrid map and the integration of the FISH and RH maps. In addition, CH constructed Figure 1 (paper and web sites) and Table 1 and implemented the accompanying web site. GGM and EFK organized and oversaw the BAC-end sequencing, including data production and analysis, with CMF overseeing the ultimate effort at TIGR. Markers were RH-mapped in Rennes, France by EC and GE, and in Seattle, Washington, USA by HGP, GB, LS, and TDL. The Seattle data were checked, duplicated, data-entered and generally overseen by TDL. TDL also drafted portions of the methods section of the manuscript. RG computed the marker sequences using BLAST software against the human sequence to identify orthologoussequences and synteny conservation. RG also drew some of the chromosomes for Figures 1 (web sites). Efforts in Rennes, France were overseen by CA and FG, including analysis, quality control, supervision, and portions of manuscript production. Efforts in Seattle, Washington, USA were overseen by EAO including experimental design, quality control, supervision, and portions of manuscript production. Individual and joint grants funding this work were written by and awarded to EAO, MB, and FG. All authors read and approved the final manuscript. Links FISH Information: RH Map Information: and . Acknowledgements We thank the American Kennel Club-Canine Health Foundation for their long-term support and acknowledge grants AKC-CHF-2214 (M.B.) and AKC-CHF 2215 (E.A.O. and F.G.) We also acknowledge support from U.S. Army grant DAAD19-01-1-0658 (E.A.O. and F.G.) and NIH R01CA-92167 (E.A.O, E.K. and F.G.). In addition, E.A.O is supported by K05 CA90754 and is the recipient of a Burroughs Wellcome Award in Functional Genomics. R.G. is partly supported by an AKC and CNRS fellowship. ==== Refs Guyon R Lorentzen TD Hitte C Kim L Cadieu E Parker HG Quignon P Lowe JK Renier C Gelfenbeyn B A 1 Mb resolution radiation hybrid map of the canine genome Proc Natl Acad Sci USA 2003 100 5296 5301 12700351 10.1073/pnas.0831002100 Kirkness EF Bafna V Halpern AL Levy S Remington K Rusch DB Delcher AL Pop M Wang W Fraser CM The dog genome: survey sequencing and comparative analysis Science 2003 301 1898 903 14512627 10.1126/science.1086432 Breen M Thomas R Binns MM Carter NP Langford CF Reciprocal chromosome painting reveals detailed regions of conserved synteny between the karyotypes of the domestic dog (Canis familiaris) and human Genomics 1999 61 145 155 10534400 10.1006/geno.1999.5947 Yang F O'Brien PC Milne BS Graphodatsky AS Solanky N Trifonov V Rens W Sargan D Ferguson-Smith MA A complete comparative chromosome map for the dog, red fox, and human and its integration with canine genetic maps Genomics 1999 62 189 202 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==== Front Reprod Biol EndocrinolReproductive biology and endocrinology : RB&E1477-7827BioMed Central London 1477-7827-2-621534506110.1186/1477-7827-2-62Research17beta-estradiol induced vitellogenesis is inhibited by cortisol at the post-transcriptional level in Arctic char (Salvelinus alpinus) Berg Hakan [email protected] Carina [email protected] Per-Erik [email protected] Department of Molecular Biology, Umea University, Umea, Sweden2 Department of Marine Science, University of Texas Marine Science Institute, University of Texas, Port Aransas, Texas, USA3 Department of Natural Science, Unit of Molecular Biology, Orebro University, Orebro, Sweden2004 2 9 2004 2 62 62 6 4 2004 2 9 2004 Copyright © 2004 Berg et al; licensee BioMed Central Ltd.2004Berg et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. This study was performed to investigate stress effects on the synthesis of egg yolk precursor, vitellogenin (Vtg) in Arctic char (Salvelinus alpinus). In particular the effect of cortisol (F) was determined since this stress hormone has been suggested to interfere with vitellogenesis and is upregulated during sexual maturation in teleosts. Arctic char Vtg was purified and polyclonal antibodies were produced in order to develop tools to study regulation of vitellogenesis. The Vtg antibodies were used to develop an enzyme-linked immunosorbent assay. The corresponding Vtg cDNA was cloned from a hepatic cDNA library in order to obtain DNA probes to measure Vtg mRNA expression. Analysis of plasma from juvenile Arctic char, of both sexes, exposed to different steroids showed that production of Vtg was induced in a dose dependent fashion by 17β-estradiol (E2), estrone and estriol. Apart from estrogens a high dose of F also upregulated Vtg. In addition, F, progesterone (P) and tamoxifen were tested to determine these compounds ability to modulate E2 induced Vtg synthesis at both the mRNA and protein level. Tamoxifen was found to inhibit E2 induced Vtg mRNA and protein upregulation. P did not alter the Vtg induction while F reduced the Vtg protein levels without affecting the Vtg mRNA levels. Furthermore the inhibition of Vtg protein was found to be dose dependent. Thus, the inhibitory effect of F on Vtg appears to be mediated at the post-transcriptional level. ==== Body Introduction The major proteinaceous egg yolk precursor vitellogenin (Vtg) is a large complex lipoglycophosphoprotein produced under estrogenic control in the liver of sexually maturing female oviparous animals. The estrogenic control of Vtg is mediated by binding of the most potent estrogen, 17-β-estradiol (E2), to the hepatic estrogen receptor (ER) [1]. The ER-E2 complex activates the transcription of the Vtg-genes by binding to estrogen responsive elements [1]. Vtg is transported from the liver as a dimer via the circulation to the oocytes, where it is taken up by receptor mediated endocytosis [2,3] and proteolytically cleaved into the smaller yolk units lipovitelin, phosvitin [4,5] and phosvettes [6], which serve as a nutritional source for the growing embryos [7]. Studies have shown that Vtg bind metal-ions such as zinc, calcium [8,9] and magnesium [10]. It has been suggested that Vtg is involved in the transport of metal-ions, crucial for embryonic development, into the growing oocyte [11]. A number of Vtg genes have been characterized in a wide variety of oviparous species and it has been shown that the Vtg genes are highly conserved [12,13]. The Vtg-genes belong to a small gene family where the number of genes varies depending on species [7,14,15]. The different genes give rise to multiple forms of the protein, which are expressed at different times during oogenesis. This indicates that Vtg isoforms may have different roles during oocyte maturation and embryonic development [5]. Vitellogenin genes are present in both females and males but the lack of estrogens in the males prevents the expression of the protein under normal conditions [16]. In teleosts, cortisol (F) is released from interrenal cells in response to stress. It has been shown that F affects reproduction by decreasing the amount of gonadotropins produced by the pituitary, the amount steroids present in the plasma and by reducing gamete quality [17]. Earlier studies on stress responses on teleost reproduction are ambiguous. In some studies F does not interact with E2 systems [16,18], while other studies indicate that F interferes with the binding of E2 to ER, thereby decreasing hepatic Vtg production [19]. It has been proposed that this ambiguity is due to species-specific responses to F thereby giving rise to different stress responses in different species. Many manmade substances with endocrine disrupting properties (EDS) are present in the environment. It has been observed that stress responses are induced in organisms when exposed to EDS. Numerous EDS have been shown to impair reproductive function in teleost fish [18]. It is therefore important to examine how stress responses interfere with the expression of commonly used biomarkers. Exposure of male or juvenile fish to estrogenic substances results in stimulation of Vtg production [20,21]. Vtg is therefore widely used as a biomarker for estrogenicity [22,23]. In the present study Arctic char Vtg was characterized and the effect of F on E2 induced vitellogenesis was investigated. Materials and methods Experimental animals and rearing conditions Juvenile Arctic char with an average weight of 18.4 ± 10.7 g were obtained from the National Swedish Board of Fisheries Research Station, Kälarne, Sweden. They were kept in indoor 50 l tanks with a continuous flow-through water system with temperature and photoperiods as close to the natural conditions as possible. The fish were allowed to acclimatize for 1 week prior to initiating the experiments. No food was administered to the fish during the experiments. Fish treatment and sampling Vtg synthesis was induced by intraperitoneally (i.p.) injection of Arctic char with 10-6 M E2. Peanut oil was used, as a carrier and control injections were made with carrier alone. The fish were kept for four days prior to sampling. Plasma was collected by centifugation and used to purify Vtg in order to develop polyclonal antibodies. Juvenile Arctic char were injected i.p. with different doses of E2, estriol and estrone (end-concentrations ranging between 10-9 to 10-6 M) and F, corticosterone, cortisone, 11-ketotestosterone and progesterone (P) (end-concentrations ranging between 10-8 to 10-5 M) to determine the effect of these 8 hormones on Vtg expression. Four days after injection the fish were sacrificed, bled and the livers were removed. The obtained blood was centrifuged at 5000 × g for 1 minute in order to separate the blood cells from the plasma. The plasma and livers were immediately frozen in liquid nitrogen and stored at -80°C until analyzed. To further investigate the effects of steroids on Vtg production, different doses of E2 (end-concentration ranging between 10-8 to 10-6 M) was administered i.p. with or without co-injection of F (end-concentration ranging between 10-8 to 10-4 M), P (10-5 M) or tamoxifen (Tam) (10-5 M). After four days the fish were sacrificed, the liver and plasma were collected and stored as described above. Hormone determinations E2 and F plasma levels were determined by radioimmunoassay according to manufacturers instructions (E2-Coat-a-Count, DPC, USA, F-Spectria Cortisol RIA, Orion Diagnostica, Espoo, Finland). The measurements were made in triplicates. Isolation of vitellogenin Prior to chromatography, the Vtg in the plasma was concentrated by selective precipitation as described by [24]. 0.5 ml of plasma were mixed with 2 ml of 20 mM EDTA, and precipitation was obtained by subsequently adding 0.1 ml 0.5 M MgCl2. The precipitate was collected by centrifugation at 5000 × g for 15 minutes at +4°C, and the supernatant was discarded. The obtained precipitate was re-dissolved in 1 ml of 1 M NaCl prior to a second precipitation, performed by lowering the ionic strength of the sample by adding 10 ml of ultrapure deionized water (MQ). The precipitate was collected by centrifugation at 5000 g for 15 minutes +4°C and the pellet was dissolved in 1 ml of 1 M NaCl prior to fast performance liquid chromatography (FPLC). All solutions used for FPLC contained aprotinin (0.5% v/v) and were filtered through 0.22 μm filters and degassed. The column used was a Resource Q (Pharmacia, Sweden), which was equilibrated with five volumes of 20 mM Tris-HCl pH 8.0 (Buffer A). The plasma was diluted 50 times and 0.5 ml of the diluted sample was loaded onto the equilibrated column. Unbound plasma-proteins were eluted with 5 ml of buffer A. The bound proteins were separated by a 15 ml linear gradient from 0.00 M to 0.50 M NaCl. The column was washed with 5 ml of 1.0 M NaCl to ensure that no other proteins remained bound. The flow-rate was 1 ml min-1 and 1 ml fractions were collected. The obtained Vtg was stored in 50% (v/v) glycerol until further analysis. In order to control the efficiency of the different purification steps, 10 μg of total protein from each of the steps were run onto an 8% discontinuous polyacrylamide gel (SDS-PAGE) and stained with Coomassie brilliant blue. The FPLC purified Vtg was used to immunize rabbits (AgriSera, Vindeln, Sweden). Western blot analysis To identify Vtg present in the plasma of sampled fish total protein was loaded onto a discontinuous polyacrylamide gel with a 2 or 4% stacking gel and an 8% separating gel [25]. Following electrophoresis, the proteins were blotted onto nitrocellulose membrane (Hybond-ECL™) or PVDF membrane (Amersham) using either semi-dry or tank transfer system (Bio-Rad Laboratories). To block non-specific antibody binding, the membranes were incubated with fat-free milk powder (5% in Tris-buffered saline, pH 7.4, containing 0.5% Tween 20; TBS-T). The membranes were incubated with primary antibody for 1 hour at room temperature (RT) or over night at 4°C. The primary antibodies were directed against Arctic char Vtg and diluted 1:5000 in TBS-T. The membranes were washed 3 × 5 minutes in TBS-T and incubated for 1 hour with the secondary antibody (Horseradish Peroxidase-conjugated anti-rabbit Ig, DAKO A/S Denmark), diluted 1:5000 in TBS-T. Prior to detection, the membranes were washed 3 × 5 minutes in TBS-T. The detection was performed using the ECL™ detection system (Amersham Pharmacia Biotech, Uppsala, Sweden) Two-dimensional polyacrylamide gel electrophoresis analysis Two-dimensional poloyacrylamide gel electrophoresis (2D-PAGE) of plasma proteins was run on Multiphor II electrophoretic unit (Pharmacia Biotech) according to the manufactures manual. Separation in the first dimension (IEF) was performed using linear pH 4–7 gradient immobiline DryStrips (Amersham Biosciences), 40μg protein was loaded per strip. In the second dimension an 8–18% gradient polyacrylamide gel (ExcelGel SDS, Amersham Biosciences) was used. The gels were either stained with Coomassie Brilliant Blue or the proteins was transferred to PVDF-membrane. The blot was blocked with fat-free milk powder (5%) in TBS, pH 7.4, followed by anti-Vtg (diluted 1:3000) incubation over night at 4°C. After 3 × 10 minutes washes in TBS-T the membrane was incubated for 2 hours with the secondary antibody (HRP-conjugated anti-rabbit Ig, Amersham Biosciences), diluted 1:3000. Prior to detection, the membranes were washed 2 × 15 minutes in TBS-T and 1 × 5 minutes in TBS. For detection of antibody staining ECL™ reagents was used and the chemiluminescent signal was detected on Hyperfilm MP (Amersham Biosciences). ELISA procedure Quantification of plasma Vtg was performed by enzyme-linked immunosorbent assay (ELISA), prepared by coating 96 well microtiter plates (Nunc A/S, Roskilde, Denmark) with plasma-samples diluted in coating buffer (0.1 M Na2CO3, pH 9.6). A standard curve made from purified Arctic char Vtg was also loaded onto each plate as a control. The plates were incubated at RT for 1 hour prior to blocking non-specific binding by adding phosphate buffered saline, pH 7.6 (PBS) containing 1% dry milk to each well. The plates were washed in PBS containing 0.05% Tween 20 (PBS-T) before addition of primary antibody. The polyclonal primary antibodies against Arctic char Vtg were diluted 1:10000 in PBS-T, added to the plates and incubated in RT for 1 hour. After washing the plates with PBS-T, a secondary antibody incubation was performed by adding HRP-conjugated goat-antirabbit polyclonal antibodies (DAKO A/S Denmark) diluted 1:5000 in PBS-T. The plates were incubated for 1 hour at RT prior to PBS-T-wash and detection. The detection was performed using a peroxidase substrate kit (Horseradish peroxidase substrate kit, BIO-RAD, Hercules, CA, USA). The plates were read at 415 nm, using a microplate reader (BIO-RAD microplate reader Model 550). All samples were analyzed in triplicates. To establish the titer of the polyclonal Vtg antibodies an ELISA with the wells loaded with equal amount VTG and various antibody concentrations were used. The detection limit of the ELISA procedure was determined by loading a standard curve of pure Vtg and using a fixed antibody concentration. cDNA cloning A ZAP Express cDNA library (Stratagene, La Jolla, CA, USA) from E2 induced Arctic char liver was used. The library was screened using a probe constructed from the rainbow trout pSG Vg 5.09 cDNA clone [26]. The isolated phage DNA clones were subjected to in vivo excision prior to sequencing. Positive clones from the screening were selected for sequencing by dot blot and Northern blot analysis (data not shown) and sequencing was performed using Thermo Sequenase (Amersham). RNA extraction and slot blot procedure Total RNA was isolated from Arctic char livers according to Chomczynski and Sacci [27]. Slot blot analysis was used to quantify Vtg mRNA levels. Nylon membranes (Hybond N, Amersham) were soaked in 20 × SSC (1 × SSC, 0.15 M NaCl; 15 mM sodium citrate buffer, pH 7.0). RNA samples were prepared by mixing 10 μg of total RNA with 6 × SSC and 7.5% formaldehyde and heating to 68°C for 15 min. The RNA samples were immediately cooled down on ice prior application onto the slot blot. Following slot blot the membranes were washed twice with 2 × SSC and cross-linked on both sides before hybridization against a single stranded digoxigenin (DIG) labeled cRNA Arctic char Vtg probe. Hybridization and detection of Vtg was performed as described previously [28]. Quantification of the mRNA was performed with Quantity One version 4.2.3 (BIO-RAD Laboratories AB, Sundbyberg, Sweden). In order to normalize the amount of total RNA in each slot, a slot blot membrane was hybridized with a DIG-labeled probe complementary to Arctic char 18S rRNA The probe was made as follow: total RNA from liver was used for first-strand cDNA synthesis according to the manual of Amersham. 18S fragments was PCR amplified by 30 cycles of 94°C for 30 seconds, 57°C for 30 seconds and 72°C for 30 seconds, using Quantum RNA classic 18S PCR primer pair (Ambion). The PCR fragment was cloned into pGEM-T vector (Promega). The purified plasmid was used as DNA-template in a PCR reaction (as above) to synthesize the DIG-labeled 18S DNA probe (DIG-11-dUTP was obtained from Roche). The Vtg mRNA levels in liver from control fish was arbitrarily set to 1. Statistics Significance was calculated using one-way ANOVA followed by Bonferroni's multiple comparison test with a P < 0.05. All statistical analysis was performed using GraphPad Prism version 3.02 for Windows (GraphPad Software, San Diego California USA). Results Administration of E2 to juvenile Arctic char led to a rapid increase in plasma protein concentrations from 6.2 ± 0.3 mg/ml in control fish to 21.4 ± 0.5 mg/ml in E2 injected fish. The plasma contained low molecular weight proteins that were excluded from the preparation by sequential precipitations. The final pellet was re-dissolved in 1 M NaCl and subjected to FPLC purification. A single absorbance peak containing Vtg was identified at an ion concentration of 0.37 M (Fig. 1). This peak was not present in plasma from untreated juvenile fish (data not shown). SDS-PAGE analysis showed that the purified Vtg had a molecular mass of 185 kDa. Figure 1 Elution profiles from Resource Q-chromatography of E2 treated Arctic char plasma proteins following selective precipitation. The linear gradient used was between 0.00–0.50 M NaCl. The absorbance was measured at 280 nm. The pure Vtg gave rise to one homogenous absorbance peak at an ion concentration of 0.37 M. The purified Vtg was used to produce polyclonal Vtg antibodies. The specificity of the polyclonal rabbit antiserum against Arctic char Vtg was determined using western blot analysis. A single band with a molecular mass of 185 kDa was detected only in the plasma of sexually mature females or E2 exposed fish (Fig. 2). To determine if the antibodies could be used quantitatively, plasma from E2 injected fish was separated on SDS-PAGE and detected by western blot analysis. The western blot displayed an increase in plasma Vtg from fish injected with increasing E2 concentrations, further confirming the specificity of the antibodies (Fig. 3). In order to develop an ELISA, the antibodies were tested both at increasing concentrations of antibodies with fixed antigen concentrations and at fixed concentrations of antibodies with increasing concentrations of antigen. The results show that the produced antisera have a high titer allowing dilution up to 10.000 fold without increasing the detection limit (Fig. 4). From these experiments the detection limit of the ELISA was determined to be 5 ng Vtg/well. Figure 2 Western blot analyses using a polyclonal antibody against Arctic char Vtg, on plasma from A) untreated juveniles. B) E2 exposed juveniles. C) male fish. D) female fish. Figure 3 Western blot of plasma from Arctic char exposed to different concentrations of E2 using a polyclonal antibody against Arctic char Vtg. Figure 4 ELISA titration curves. A) Titration; A maximum dilution of the antisera was determined to be 10.000×. B) Detection limit; An antibody dilution of 1:10.000 was used and the detection limit was determined to 5 ng Vtg/well. Screening of the Arctic char hepatic cDNA library revealed several positive clones. The longest clones were selected and sequenced to completion (clone 1 and clone 3). Sequencing of clone 1 and clone 3 revealed that the Arctic char Vtg mRNA displayed high homology to rainbow trout Vtg mRNA, both at the nucleotide level (89% and 83% respectively) and at the protein level (85% and 82% respectively). Clone 1 and clone 3 showed high similarity (94% on both nucleotide and protein level). In addition, clone 1 was found to contain a second polyadenlyation site and a 116 bases longer 3'UTR. Even though no full-length clones were obtained, these features imply that the clones are products of different genes. Eight substances were injected into juvenile Arctic char to determine their potency at inducing Vtg synthesis. ELISA analysis of plasma revealed that only the three estrogens and F induced Vtg synthesis (Fig. 5). The most potent estrogen, E2, was found to be 3 times more effective at inducing Vtg synthesis than estrone and 7 times more potent than the weakest estrogen, estriol. All estrogens induced a dose dependent induction of Vtg. The ability of F to induce Vtg was approximately 70 times lower than E2 and was only observed at the highest dose. Slot blot analysis of Vtg mRNA levels revealed a dose dependent induction corresponding to the induction pattern observed with the ELISA. E2 was the strongest inducer, with both estrone and estriol being weaker but equally potent inducers of Vtg mRNA (Fig. 6). In agreement with the ELISA determinations, F induced Vtg mRNA only at the highest dose. None of the other substances tested displayed any effects on Vtg mRNA. Figure 5 Plasma Vtg concentrations in fish exposed to estrogens and cortisol. Control fish (C) were injected i.p. with peanutoil. All values are presented as a mean of 10 fish ± SEM. a denotes P < 0.05 when compared with control and b denotes P < 0.05 when compared to the highest concentration of each substance. Figure 6 Relative Vtg mRNA levels in fish subjected to i.p. administration of estrogens and cortisol. Each bar represents a mean value of three fish ± SEM. Significant differences are marked with a and b. a denotes P < 0.05 when compared with control (C) and b denotes P < 0.05 when compared to the highest concentration of each substance. Arctic char were co-injected with E2 and F, P or tamoxifen in order to determine if other compounds could inhibit Vtg production. Plasma hormone determinations were performed on all groups of fish and the mean plasma levels of E2 and F are shown in table 1. The known antiestrogen tamoxifen was used as a control substance and was found to inhibit the E2 dependent upregulation of both Vtg mRNA and protein levels (Fig. 7). However, while P did not affect the E2 dependent Vtg induction, F co-injection resulted in lowered Vtg protein levels without affecting the Vtg mRNA levels. A second experiment was therefore performed to determine the dose-response effect of co-injection of F with the three different estrogens. ELISA analysis of plasma from co-injected fish reveled dose-dependent inhibition of estrogen induced Vtg levels in plasma (Fig. 8). Western blot of plasma proteins from fish treated with a combination of E2 and F confirmed that F was able to decrease the level of Vtg that are expected in the plasma from an E2-injected fish (Fig. 9). Table 1 Plasma levels of E2 and F following intraperitoneal injections. Treatment Plasma levels* Cortisol control nd Cortisol 10-7 M 42.3 ± 6.4 Cortisol 10-6 M 132.1 ± 51.8 Cortisol 10-5 M 2593 ± 697 Cortisol 10-4 M 15015 ± 3051 17β-estradiol control nd 17β-estradiol 10-8 M 10.1 ± 2.8 17β-estradiol 10-7 M 76.7 ± 12.6 17β-estradiol 10-6 M 687 ± 75 * The plasma levels are presented as mean (ng/ml) ± S.E. nd: non detectable levels, below detection limit Figure 7 Vtg mRNA and protein levels following co-injection of E2 and P, F and tamoxifen. The dark bar indicates the relative hepatic Vtg mRNA levels while the light bars displays Vtg protein levels present in the plasma. All bars represent a mean value from 5 fish ± SEM. a denotes P < 0.05 when compared with control and b denotes P < 0.05 when compared to Vtg protein levels in E2 induced fish. c denotes P < 0.05 when compared with control (C) and d denotes P < 0.05 when compared to Vtg mRNA levels in E2 induced fish. Figure 8 Dose dependent effects of F and E2 on plasma Vtg levels in juvenile Arctic char. All values are presented as a mean value of 5 fish ± SEM. a denotes P < 0.05 when compared with control (C). b denotes P < 0.05 when compared with each E2 concentrations positive control. c denotes P < 0.05 when compared with each E2 + F 10-7 control. Figure 9 Plasma proteins, 20 μg per lane, from Arctic char treated with 17-β-estradiol (E2, 10-7 M) or/and cortisol (F, 10-5 M) separated on 8% SDS-PAGE. Coomassie-stained gel and corresponding Western blot using a polyclonal antibody against Arctic char vitellogenin. Lane 1: control, lane 2: E2, lane 3: F, lane 4: E2 + F. Molecular weight (Da) are shown to the left. The polyclonal antibody directed against a 185 kDa Vtg recognized several high and low molecular weight spots of Vtg and Vtg-derivatives as shown by 2D-PAGE analysis (Fig. 10). Since Vtg is transported in the plasma as a dimmer it migrates as a large complex on 2D-PAGE. There is less of both high and low molecular Vtg-isoforms in the plasma from co-injected fish compared to E2 injected fish. Figure 10 Immunoblots of Arctic char plasma proteins from control, E2- (10-7 M), and E2 + F- (10-7 M and 10-5 M) treated fish separated by two-dimensional electrophoresis. 40 μg total protein was separated by isoelectric focusing in the first dimension using a pH gradient 4–7, followed by SDS-PAGE using 8–18% acrylamide gradient. Polyclonal anti-Arctic char vitellogenin was used. Figure show a part of the PVDF-membrane, spots recognized by the vitellogenin antibody are circled. Discussion In this study Arctic char Vtg was purified and polyclonal antibodies was made in order to use Vtg protein determinations as a marker of F effects on egg yolk formation. The purification was performed following the procedure outlined by Silversand and Haux [24]. The chromatographic profile of the purified Arctic char Vtg displayed large similarities when compared to turbot (Schophthalmus maximus) [24]. Elution of the protein was obtained at a Cl- concentration of 0.37 M, a value comparable to those earlier reported [29]. The purified Vtg was used to obtain polyclonal antisera from rabbits. The antisera displayed a high specificity for the 185 kDa Vtg, and also recognized Vtg dimers and derivatives as observed by 2D PAGE. Vtg was only detected in females or E2 exposed juvenile Arctic char. It has been found that teleost Vtg, even though highly conserved, may differ in size between 120 – 300 kDa, and are present in the blood plasma mainly as a 300 – 600 kDa dimer [30]. It was also found that the E2 induced Vtg production was dose dependent, as described earlier in many species [31-33]. ELISA procedures have been developed for Vtg from many teleost species [34,35]. This method requires a high specificity of the antibody and a low inter-assay variability. During the evaluation of the antibodies it was found that the antisera contained a high titer of specific Vtg antibodies giving the ELISA a low detection limit of 5 ng Vtg. Low intra and inter assay variability (3%, data not shown) was observed. Eight substances were tested for their ability to induce Vtg production in Arctic char. It has earlier been shown that Vtg production in teleost fish is under dose dependent estrogenic control [16] and this was also evident in the Arctic char. Presence of Vtg in plasma was detected by the ELISA procedure revealing that Vtg protein was only present in fish exposed to the three estrogens and F. E2 was found to be the most potent estrogen, followed by estrone and estriol. Estriol was the weakest inducer of Vtg synthesis both at mRNA and protein level. These results are in accordance with earlier studies on different species, including human, mouse and rainbow trout [36,37]. The results reported here demonstrate that cortisol acts as a partial antagonist on Vtg expression. The plasma levels of E2 and F following hormone injections showed that the resulting plasma levels covered the range normally observed for Arctic char and other salmonids [38,39]. Exposure of Arctic char to high F levels (10-5 M) resulted in elevated plasma Vtg levels. While F alone induced a low level of Vtg mRNA expression the co exposure of Arctic char to estrogens and F resulted in a reduction in circulating Vtg levels while the Vtg mRNA levels were not affected. These results suggest that F acts at a post-transcriptional level in Arctic char. Our results are in contrast to earlier in vitro studies that indicate that F can down regulate Vtg mRNA levels in rainbow trout hepatocytes [18,40], but are supported by a study on Xenopus that showed F upregulation of hepatic Vtg production [41]. In Xenopus it was suggested that the C/EBPβ-like protein is involved in upregulation of Vtg by increasing the ER levels [41]. Reduced binding of E2 to ER has been observed following F exposure in rainbow trout liver [17]. F has been suggested to interfere with ER transcription by destabilizing ER mRNA, thereby decreasing the mRNA half-life. ER and the glucocorticoid receptor (GR) interact in the liver through C/EBPβ-like protein, and it has been suggested that GR suppress C/EBPβ-like protein binding to the rainbow trout ER promoter, thereby reducing the ER expression [40]. It is known that stress factors are species specific and it cannot be ruled out at the present time that the differences observed between rainbow trout and Arctic char are due to such species differences. However, it should be noted that the earlier studies were conducted on in vitro systems as opposed to the whole animal model used in the present study, and that no determination of circulating Vtg levels was performed in the previous studies. Adding to the complexity of F involvement in reproduction we have recently shown that F potentiates the E2 mediated expression of eggshell protein in Arctic char [38]. F is upregulated during final oocyte maturation and spawning in teleost fish [42]. Thus, it is conceivable that the increase in circulating F levels in maturing female fish is involved in the regulation of eggshell proteins. However, the present results indicate that this involvement is limited to the eggshell proteins as the circulating Vtg levels are reduced under the same conditions. In the present study the main effect of F was observed at the circulating Vtg level. We hypothesize that the co-treatment of Arctic char with glucocorticoids and estrogens results in upregulation of both stress induced systems, such as metallothionein (MT), and estrogen responsive genes, such as eggshell proteins and vitellogenin. MT has been shown to be upregulated by cortisol [43] in rainbow trout primary cultures and has a main function to sequester zinc [44]. The involvement of MT in fish reproduction has been shown previously for rainbow trout and Arctic char [38,39]. In both species MT is upregulated towards the end of vitellogenesis [38,39] and is believed to sequester Zn from the liver in order to control the Zn homeostasis once vitellogenesis is over [45]. It has also been shown that E2 functions as an antagonist of MT induction in both rainbow trout [28] and Arctic char [46] further supporting the involvement of MT in reproduction. If Vtg requires Zn for proper tertiary folding, then upregulation of MT by cortisol could lead to a redistribution of Zn from Vtg to MT with degradation of Vtg as a consequence. As egg shell proteins do not use Zn as a structural motif the upregulation of MT would not have the same effect on eggshell proteins. This is in part confirmed by our previous study showing that F potentiates estrogenic induction of eggshell proteins. Further studies are underway to determine the cause of the reduction in circulating Vtg levels. Acknowledgements We would like to thank Torleif Andersson and the rest of the staff at the National Board of Fisheries Research Station, Kälarne for helping out with the sampling and sharing information and experience during the course of this study. 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of a C/EBPbeta-like protein Endocrinology 2002 143 2961 2974 12130562 10.1210/en.143.8.2961 Marilley D Robyr D Schild-Poulter C Wahli W Regulation of the vitellogenin gene b1 promoter after transfer into hepatocytes in primary cultures Mol Cell Endocrinol 1998 141 79 93 9723889 10.1016/S0303-7207(98)00090-2 Carruth LL Dores RM Maldonado TA Norris DO Ruth T Jones RE Elevation of plasma cortisol during the spawning migration of landlocked kokanee salmon (Oncorhynchus nerka kennerlyi) Comp Biochem Physiol C Toxicol Pharmacol 2000 127 123 131 11083023 10.1016/S0742-8413(00)00140-7 Hyllner SJ Andersson T Haux C Olsson P-E Cortisol induction of metallothionein in primary culture of rainbow trout hepatocytes J Cell Physiol 1989 139 24 28 2708457 Olsson P-E Hochachka PW, Mommsen TP Metallothionein gene expression and regulation in fish In The Biochemistry and Molecular Biology of Fishes 1993 2 Molecular Biology Frontiers 259 278 Olsson P-E Zafarullah M Gedamu L A role of metallothionein in zinc regulation after estradiol indcuction of vitellogenin synthesis in rainbow trout, Salmo gairdneri Biochem J 1989 257 555 560 2467659 Gerpe M Kling P Berg AH Olsson P-E Arctic char (Salvelinus alpinus) metallothionein: cDNA sequence, expression, and tissue-specific inhibition of cadmium-mediated metallothinein induction by 17β-estradiol, 4-OH-PCB30, and PCB 104 Environ Toxicol Chem 2000 19 638 645

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==== Front Respir ResRespiratory Research1465-99211465-993XBioMed Central 1465-9921-5-111537739610.1186/1465-9921-5-11ResearchProinflammatory role of inducible nitric oxide synthase in acute hyperoxic lung injury Hesse Anne-Karin [email protected]örger Martina [email protected] Christian [email protected] Fritz [email protected] Institute for Surgical Research, University of Munich, Marchioninistr. 15, 81366 Munich, Germany2 Department of Internal Medicine I, University of Munich, Marchioninistr. 15, 81366 Munich, Germany2004 15 9 2004 5 1 11 11 16 7 2004 15 9 2004 Copyright © 2004 Hesse et al; licensee BioMed Central Ltd.2004Hesse et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Hyperoxic exposures are often found in clinical settings of respiratory insufficient patients, although oxygen therapy (>50% O2) can result in the development of acute hyperoxic lung injury within a few days. Upon hyperoxic exposure, the inducible nitric oxide synthase (iNOS) is activated by a variety of proinflammatory cytokines both in vitro and in vivo. In the present study, we used a murine hyperoxic model to evaluate the effects of iNOS deficiency on the inflammatory response. Methods Wild-type and iNOS-deficient mice were exposed to normoxia, 60% O2 or >95% O2 for 72 h. Results Exposure to >95% O2 resulted in an increased iNOS mRNA and protein expression in the lungs from wild-type mice. No significant effects of iNOS deficiency on cell differential in bronchoalveolar lavage fluid were observed. However, hyperoxia induced a significant increase in total cell count, protein concentration, LDH activity, lipid peroxidation, and TNF-α concentration in the bronchoalveolar lavage fluid compared to iNOS knockout mice. Moreover, binding activity of NF-κB and AP-1 appeared to be higher in wild-type than in iNOS-deficient mice. Conclusion Taken together, our results provide evidence to suggest that iNOS plays a proinflammatory role in acute hyperoxic lung injury. ==== Body Background Supplemental oxygen therapy is administered for the treatment of tissue hypoxia, most commonly in an intensive care setting of respiratory insufficient patients, though its potent toxicity is well described [1]. The pathophysiology of oxygen injury is characterized by lung inflammation including activation and recruitment of neutrophils and alveolar macrophages, tissue and alveolar edema, surfactant dysfunction, and excess production of free radicals and inflammatory cytokines [2-4]. Although the exact mechanisms of pulmonary oxygen toxicity are still unknown, compelling evidence suggests that reactive oxygen species such as superoxide anion, hydroxyl radical, and hydrogen peroxide are important mediators of lung injury [5-7]. High oxygen concentrations induce cellular damage by several mechanisms such as oxidation of proteins, peroxidation of membrane lipids, and breakage of DNA strands [8-10]. Moreover, hyperoxia also induces the release of a wide spectrum of proinflammatory cytokines such as tumor necrosis factor-[11-13]. However, the precise molecular mechanisms by which hyperoxia produces acute lung injury remain unclear. Reactive oxygen species can also react with other free radicals such as nitric oxide (NO) to yield more cytotoxic species including peroxynitrite anion [14,15]. Peroxynitrite is a strong oxidizing agent that can also initiate lipid peroxidation [16,17]. Since the discovery of NO as a potent vascular smooth muscle relaxant and regulator of blood pressure, NO generated by the inducible nitric oxide synthase (iNOS) has been identified in many cell types such as alveolar macrophages or epithelial cells and implicated in a variety of biological roles [15,18,19]. NO is synthesized from L-arginine by two main isoforms of the NO synthase: the constitutive and the inducible isoform [20]. The NOS enzymes are complex homodimeric proteins consisting of a N-terminal oxygenase domain, a central calmodulin binding sequence, and a C-terminal reductase domain [21,22]. Inducible NOS is expressed following induction by a variety of inflammatory cytokines such as TNF-α [22] or by lipopolysaccharide (LPS) [23-25]. Constitutive iNOS expression has been reported in the lung [26,27] and several inflammatory processes involving the lung, such as sepsis [23-25], asbestosis-induced lung injury [28,29] and hyperoxia [30,31] are associated with an elevated NO production. However, the effect of hyperoxia on endogenous NO production is a matter of controversial discussion, depending on the experimental conditions [30,32-34]. Inappropriate regulation of nuclear factor-κB (NF-κB)-and activator protein-1 (AP-1)-mediated transcription has also been associated with pathological conditions, including acute inflammation such as hyperoxic exposure [35]. Intracellular signaling pathways leading to an activation of transcriptional regulators such as NF-κB and AP-1 can be affected by reactive oxygen and nitrogen species [36-38]. Both transcription factors are activated in lung cells after short periods of hyperoxic exposure. Binding sites for NF-κB and AP-1 are present in the promotor of the iNOS gene and of proinflammatory cytokines such as TNF-α [35,39,40]. The objective of this study was to investigate the effect of iNOS deficiency on acute hyperoxic lung injury. As indicators of lung hyperpermeability, lavageable lung protein and LDH activity were measured; lung lipid oxidation was assessed based on the levels of thiobarbituric acid reactive substances. To characterize the inflammatory process, lavageable cell counts, cell differential, and TNF- concentration were analyzed. Binding activity of NF-κB and AP-1 was investigated in order to elucidate transcriptional mechanisms for iNOS and TNF-α expression. Methods Animals Inducible NOS-deficient mice (C57BL6/J × 129SvEv) were originally provided by Dr. J. Mudgett (Merck & Co., Rathway, New Jersey, USA), Dr. J. MacMicking, and Dr. C. Nathan (Cornell University Medical College, New York, USA) [41]. As controls, matching wild-type mice were used (C57BL6/J × 129SvEv). Animals were bred in the facilities of the Institute for Surgical Research (Munich, Germany). Protocols used in this study were approved by the appropriate government body. Hyperoxic exposure Male mice (12 – 16 weeks old, body weight between 26.1 g and 27.3 g) were kept in groups of seven in a sealed Plexiglas chamber (27 × 27 × 20 cm3). Animals were randomized and exposed to 60% O2 and >95% O2 with a gas flow rate of 6 l/min at atmospheric pressure for 72 h. Mice exposed to room air in the same chamber served as controls. O2 levels were monitored twice daily with an oxygen analyzer (Drägerwerk AG, Lübeck, Germany). The environmental temperature was maintained at 24°C ± 1, relative humidity was 73% ± 13, and air pressure was 947 mbar ± 5. Oxygen was humidified by bubbling through a water chamber. The Plexiglas chamber bottom was lined with soda lime for CO2 absorption (Mallinkrodt Baker B. V., Deventer, Holland). Exposures were continuous for the time indicated except for 5 – 10 min daily when the chamber was opened for housekeeping purposes. Animals were kept on a 12 h light/dark cycle. Standard rodent food and water were available ad libitum. Bronchoalveolar lavage cell counts and cell differential Immediately following exposure, mice were anaesthetized by intraperitoneal injection of sodium pentobarbital (10 mg/kg body weight, Narcoren®, Merial, Halbergmoos, Germany). Tracheotomy was performed and a 20 G × 32 mm needle (Abbocath®-T, Venisystems, Sligo, Ireland) was inserted and secured. Bronchoalveolar lavage (BAL) was performed five times with 1 ml of sterile non-pyrogenic phosphate-buffered saline solution (PBS; Serva, Heidelberg, Germany) in each animal. After centrifugation at 300 × g for 10 min, the supernatant was collected and stored at -20°C and -80°C for later protein assays. The BAL cell pellet was resuspended in PBS and washed by centrifugation. Cells were stained with May-Grunwald-Giemsa (Varistain 4, Shandon Labortechnik GmbH, Frankfurt, Germany) to identify cellular populations. Total cell counts were assessed with a hemacytometer (Coulter Ac T 8, Coulter Electronics, Krefeld, Germany). Lavageable lung protein assay Cell free BAL fluid was evaluated for total protein content by the bicinchoninic acid assay using bovine serum albumin (PAA Laboratories, Linz, Austria) based on a method of Smith et al. [42]. Lactate dehydrogenase activity assay To evaluate lactate dehydrogenase (LDH) activity in cell free BAL fluids, a commercially available kit was used (LDH Optimiert, Roche Diagnostics, Mannheim, Germany). TNF-α assay Concentration of TNF-α in cell free BAL fluid was measured by an enzyme linked immunosorbent assay using a commercially available kit (EM-TNFA, Endogen, Woburn, Massachusetts, USA). Briefly, 50 ml biotinylated antibody reagent were added to 50 ml-samples in an anti-mouse TNF-α pre-coated strip well plate. After incubation for 2 h at room temperature, the plate was washed, a streptavidin horseradish peroxidase solution and the 3, 3',5, 5'-tetramethylbenzidine substrate solution were added and incubated in the dark. The absorbance was detected at 450 nm in a microplate reader (EAR 400 AT, Salzburger Labortechnik, Salzburg, Austria). A standard curve was used to determine the amount of TNF-α concentration in the samples. Reverse transcriptase-polymerase chain reaction Total RNA was isolated from non-lavaged lung homogenate of each mouse (RNeasy Mini Kit, Quiagen, Hilden, Germany), reverse transcribed into cDNA in a volume of 20 ml, containing 2 μg RNA, 1.5 μM Oligo-p(dT)15-primer, 5 × PCR-buffer, 0.1 M DTT, 10 nM dNTP-mix and 200 U/μl of Moloney murine leukemia virus reverse transcriptase. Reverse transcriptase-polymerase chain reaction (RT-PCR) amplifications were performed with aliquots of cDNA (3 μl) in total volume of 50 μl (5 μl 10 × PCR reaction-buffer, 1 μl dNTP-mix, 1 μl each of forward and reverse single strand DNA primers specific for mouse iNOS, 38.8 μl sterile deionized water, 0.2 μl Taq DNA-polymerase 1 U/ml). Oligonucleotide primers for iNOS were 5'-CAC AAG GCC ACA TCG GAT TTC-3' (sense) and 5'-TGC ATA CCA CTT CAA CCC AG-3' (antisense). Co-amplification of the housekeeping gene β-actin served as an internal control, using the following primers, 5'-GGA CTC CTA TGT GGG TGA CGA GG-3' (sense), 5'-GGG AGA GCA TAG CCC TCG TAG AT-3' (antisense). RT-PCR was started with 1 min incubation at 95°C followed by the steps of denaturation at 95°C for 45 sec, annealing at 55 – 64°C for 45 sec, elongation at 72°C for 1 min. The number of cycles (30 – 35 each) was chosen to ensure that the amplification product did not reach the level of saturation. Reactions were electrophoresed in 1% agarose gel and stained with ethidium bromide. The densitometry of each cDNA band was quantified using BIO-1D.V96 software and the ratio of iNOS cDNA to β-actin cDNA was determined. Electrophoretic mobility shift assay Nuclear protein extracts were prepared from pooled lung tissue as previously described [43]. Briefly, the oligonucleotides were incubated with a binding buffer (0.04 M Tris, 0.2 M NaCl, 2 mM EDTA, 8% glycerine, 2 μm Ficoll 400, 0.2 mM PMSF, 4 mM DTT). After 5 min incubation, 5 μ l of [γ32P]-dATP end-labeled double-stranded oligonucleotides containing an NF-κB-consensus sequence (5'-AGT TGA GGG GAC TTT CCC AGG C-3') or AP-1-consensus sequence (5'-CGC TTG ATG AGT CAG CCG GAA-3') were added to the reaction followed by an incubation for 1 h at 37°C. The mixture was subjected to electrophoresis on a 6% PAA-Gel (75% H2O, 45 mM Tris, 45 mM bore acid, 1 mM EDTA, pH 8. 6% APS, 60 μl TEMED) for 2 h at 250 V. Thiobarbituric acid reactive substances assay Concentration of thiobarbituric acid reactive substances (TBARS) was evaluated with an assay according to Thiery et al. [44]. BAL fluid was prepared to denaturate proteins with 50% trichloroacetic acid. The supernatants were transferred to a clean tube and 75 μl of 1.3% thiobarbituric acid (Sigma Chemie, Deisenhofen, Germany) in 0.3% NaOH were added. After incubation for 1 h at 90°C and subsequent cooling in ice water, samples were centrifuged for 6 min. Finally, 200 μl of sample were transferred to a 96-well plate and the absorbance at 530 nm was read in a microplate reader (Dynex Technologies, Denkendorf, Germany). TBARS were quantified by using a standard curve of malondialdehyde (Sigma Chemie, Germany). Statistical analysis Results are presented as the group mean ± standard error of the mean (SEM). Statistical comparison between values of the three oxygen concentrations was performed by using analysis of variance on ranks and Mann-Whitney rank sum test followed by Bonferroni's correction. Statistical comparison between wild-type and iNOS knockout mice was analyzed by using Mann-Whitney rank sum test. Significance was accepted at p < 0.05. Results General conditions of the animals and body weight Wild-type and iNOS knockout mice all survived hyperoxia the entire 72 h. After hyperoxic exposure >95% O2, animals showed signs of reduced general conditions and reactions. Hyperoxic exposure >95% O2 also caused a significant reduction in body weight of wild-type mice compared to normoxic conditions and 60% oxygen exposure within the 72 h experimental period. In contrast, there was no significant change in body weight of iNOS knockout mice before and after normoxia and hyperoxia, respectively (table 1). Table 1 Body weight (g) of wild-type and iNOS knockout mice after 72 h exposure to 21%, 60%, and >95% O2* 21% O2 60% O2 >95% O2 wild-type mice before exposure 27.1 ± 1.0 26.5 ± 0.8 27.8 ± 0.9 after exposure 28.1 ± 0.9 28.1 ± 0.3 23.2 ± 0.6# iNOS knockout mice before exposure 24.9 ± 0.7 23.3 ± 0.5 25.2 ± 0.9 after exposure 26.1 ± 0.4 24.0 ± 0.5 24.1 ± 0.7 *Each value represents mean ± SEM, n = 7. #p < 0.05 vs. before exposure. Differential and total cell counts BAL in wild-type and iNOS knockout mice was performed to assess cellular infiltration in the alveolar space upon 72 h hyperoxic exposure (60% and >95% O2). Results presented in table 2 demonstrate no differences in baseline cell differentials between wild-type and iNOS knockout mice. Upon 72 h exposure to >95% O2, there was a significant decrease in the percentage of alveolar macrophages as well as a significant increase in the percentage of neutrophils and lymphocytes in both wild-type and iNOS knockout mice compared to normoxic conditions. No significant differences between wild-type- and iNOS knockout mice were found. However, hyperoxic exposure (>95% O2) resulted in a significant increase in total BAL cell counts after 72 h in wild-type (0.54 ± 0.05 × 106/ml) and in iNOS knockout mice (0.38 ± 0.04 × 106/ml) compared to normoxia (0.20 ± 0.03 × 106/ml and 0.16 ± 0.02 × 106/ml, respectively) and 60% O2 exposure (0.24 ± 0.04 × 106/ml and 0.19 ± 0.04 × 106/ml, respectively). This increase in BAL total cell counts under >95% O2 was significantly higher in wild-type than in iNOS knockout mice (figure 1). Table 2 BAL cell differential in wild-type and iNOS knockout mice after 72 h exposure to 21%, 60%, and >95% O2* alveolar macrophages (%) neutrophils (%) lymphocytes (%) wild-type mice 21% O2 98.8 ± 0.5 0.0 ± 0.0 1.2 ± 0.5 60% O2 97.7 ± 1.0 0.3 ± 0.2 2.0 ± 1.0 >95% O2 86.2 ± 1.8#* 4.6 ± 1.0#* 9.2 ± 0.9#* iNOS knockout mice 21% O2 99.0 ± 0.4 0.7 ± 0.2 0.3 ± 0.2 60% O2 98.9 ± 0.5 0.4 ± 0.3 0.7 ± 0.4 >95% O2 85.1 ± 2.0#* 4.9 ± 1.3#* 10.0 ± 1.2#* *Each value represents mean ± SEM, n = 7. #p < 0.05 vs. normoxia; *p < 0.05 vs. 60% O2 Figure 1 Total cell counts in BAL from wild-type and iNOS knockout mice after 72 h exposure to 21%, 60%, and >95% O2. Data are mean ± SEM of seven mice for each group. #p < 0.05 vs. normoxia; *p < 0.05 vs. 60% O2; $p < 0.05 vs. iNOS knockout mice. Lavageable lung protein Total protein concentration in the BAL fluid was determined as an indicator of lung hyperpermeability induced by hyperoxic exposure. Under normoxia and 60% O2, total protein concentration did not differ between wild-type mice (21% O2: 86.4 ± 37.3 μg/ml; 60% O2: 95.5 ± 22.8 μg/ml) and knockout mice (21% O2: 157.1 ± 23.7 μg/ml; 60% O2: 86.0 ± 26.6 μg/ml). Exposure to >95% O2 resulted in a significant increase in protein concentration in wild-type mice (973.8 ± 95.7 μg/ml) and only in a modest increase in iNOS knockout mice (326.8 ± 90.4 μg/ml) that did not reach statistical significance. Lactate dehydrogenase activity As an indicator of cellular damage, LDH activity was measured in BAL fluid. Under normoxic conditions and 60% O2, LDH activity was comparable between wild-type (21% O2: 3.1 ± 1.0 U/min/ml; 60% O2: 1.6 ± 0.4 U/min/ml) and iNOS knockout mice (21% O2: 2.3 ± 0.5 U/min/ml; 60% O2: 6.5 ± 1.7 U/min/ml). Exposure to >95% O2 resulted in a significant enhancement of LDH activity in wild-type mice (41.8 ± 10.8 U/min/ml) compared to iNOS knockout mice (9.6 ± 3.0 U/min/ml) and to normoxia (figure 3). Figure 3 Lactate dehydrogenase activity in BAL from wild-type and iNOS knockout mice after 72 h exposure to 21%, 60%, and >95% O2. Data are mean ± SEM of seven mice for each group. #p < 0.05 vs. normoxia; *p < 0.05 vs. 60% O2; $p < 0.05 vs. iNOS knockout mice. TNF-α concentration TNF-α concentrations were determined in BAL fluid to investigate inflammatory cytokine release. Under normoxic conditions, TNF-α release did not differ between wild-type (28.5 ± 3.8 pg/ml) and iNOS knockout mice (35.0 ± 5.4 pg/ml), the same as upon 60% O2 exposure (29.2 ± 2.6 pg/ml and 25.4 ± 6.0 pg/ml, respectively). However, there was a significantly enhanced TNF-α release measured upon >95% O2 exposure in wild-type (83.0 ± 9.8 pg/ml) and iNOS knockout mice (54.9 ± 9.0 pg/ml) compared to normoxic conditions. TNF-α concentration was significantly higher in wild-type than in iNOS knockout animals (figure 4). Figure 4 TNF-α concentration in BAL from wild-type and iNOS knockout mice after 72 h exposure to 21%, 60%, and >95% O2. Data are mean ± SEM of seven mice for each group. #p < 0.05 vs. normoxia; *p < 0.05 vs. 60% O2; $p < 0.05 vs. iNOS knockout mice. Concentration of thiobarbituric acid reactive substances Lung lipid peroxidation was assessed based on the levels of thiobarbituric acid reactive substances in BAL (figure 5). Wild-type mice exposed to >95% O2 exhibited a pronounced increase in concentration of TBARS (146.0 ± 62.0 nmol/ml) compared to normoxia (35.0 ± 14.0 nmol/ml) and 60% O2 (31.0 ± 17.0 nmol/ml). In iNOS knockout mice, concentrations of TBARS after >95% O2 (52.0 ± 18.0 nmol/ml) did not differ from those after normoxic conditions (26.0 ± 0.0 nmol/ml) and 60% O2 exposure (35.0 ± 26.0 nmol/ml), respectively. Figure 5 Concentration of thiobarbituric acid reactive substances in BAL from wild-type and iNOS knockout mice after 72 h exposure to 21%, 60%, and >95% O2. Data are mean ± SEM of seven mice for each group. $p < 0.05 vs. iNOS knockout mice. Activation of NF-κB and AP-1 In an effort to elucidate transcriptional mechanisms for increased iNOS and TNF-α expression after hyperoxic exposure, electrophoretic mobility shift assays for NF-κB and AP-1 were performed (figure 6). NF-κB and AP-1 were weakly activated under normoxic conditions. Increased activation of both NF-κB and AP-1 was observed after >95% O2 compared to normoxia and 60% O2. This enhancement of binding activity under hyperoxia appeared to be more prominent in the group of wild-type mice in comparison to iNOS knockout animals. Figure 6 Binding activity of NF-κB and AP-1 in lung tissue from wild-type and iNOS knockout mice after 72 h normoxia or hyperoxia. Figure shown is representative for seven experiments. iNOS mRNA expression To investigate the induction of the iNOS gene in lung tissue, expression of iNOS mRNA was analyzed (figure 7). As expected, there was no expression of iNOS mRNA in lung tissues from iNOS knockout mice. In wild-type mice, hyperoxic exposure (60% and >95% O2) induced an increased expression of iNOS mRNA in lung samples compared to normoxic situation. Densitometric analysis was performed by determining the ratio of iNOS cDNA to β-actin cDNA. Results demonstrated a significant increase in iNOS mRNA expression upon >95% O2 (1.2 ± 0.1) compared to 60% O2 (0.8 ± 0.1). Figure 7 Ethidium bromide stained gels of β-actin and iNOS RT-PCR products in lung tissue from wild-type (A) and iNOS knockout mice (B) after 72 h normoxia or hyperoxia. Data shown are representative for seven experiments. Discussion Prolonged exposure to high concentrations of oxygen (>50% O2) during an intensive care setting to maintain arterial pO2 can lead to progressive lung injury. Several cellular systems including alveolar macrophages and leukocytes are involved in this process. Activation of inflammatory cells causes the release of reactive oxygen species and proinflammatory cytokines, resulting in endothelial dysfunction, tissue and alveolar edema formation, and surfactant inactivation. Furthermore, high levels of NO produced by inducible NO synthase may contribute to tissue damage. NO is directly cytotoxic or can combine with superoxide anions to form the more reactive oxidant peroxynitrite. Although a large amount of literature exists concerning the pulmonary response to oxidant exposure, some issues remain unresolved. Our findings confirm previous results showing that hyperoxia is able to upregulate iNOS expression in lung tissue [30,34]. As expected, there was no expression of iNOS mRNA in lungs of iNOS knockout mice. In wild-type mice, exposure to 60% and >95% O2 induced a significant increase in iNOS mRNA expression. This enhanced iNOS mRNA expression during hyperoxic exposure seems to contradict findings reported in a study published by Arkovitz and colleagues, in which hyperoxia did not induce iNOS expression in lungs of mice [32]. This may be explained by the fact that the detection of iNOS mRNA by using northern blot technique is not as sensitive as RT-PCR. In accordance with results from others [28-30], we found little iNOS protein immunostaining under normoxic conditions and 60% oxygen exposure, while hyperoxic exposure >95% O2 induced a prominent expression of iNOS protein in the lungs from wild-type mice (data not shown). The data from the present study demonstrate that in vivo oxygen exposure significantly elevated total BAL cell count after 72 h >95% O2 both in wild-type and in iNOS knockout mice. According to this, oxygen exposure resulted in a significant enhancement in the number of neutrophils and lymphocytes in BAL fluid, combined with a significant reduction in the number of alveolar macrophages both in wild-type and iNOS knockout mice. Dedhia et al. also found elevated numbers of neutrophils and lymphocytes combined with decreased numbers of alveolar macrophages in rat lungs [45]. Recent studies report that, although iNOS deficiency does not affect leukocyte rolling and adhesion following treatment with thrombin [46], iNOS-deficient mice have significantly elevated leukocyte accumulation and enhanced leukocyte-endothelium interactions in endotoxinemia [24]. These results suggest that iNOS expression plays a potent role in regulation of leukocyte recruitment depending on the way of induction. Hyperoxia-induced inflammatory cell influx, particularly of neutrophils, can contribute to oxidant stress through formation of reactive oxygen species. Auten and collaborators demonstrated that DNA damage in hyperoxia-exposed rat lungs may be reduced by blocking neutrophil influx [47]. In our model of oxidant injury, no effect of iNOS deficiency on BAL cell differentials could be made out, whereas total BAL cell counts were significantly elevated in wild-type mice compared to iNOS knockout mice. The increase in the number of neutrophils and lymphocytes in BAL fluid may partially reflect the loss of integrity of the endothelium barrier. This damage is indicated by a significant elevation of total protein concentration and LDH activity after acute hyperoxia in wild-type mice in comparison to iNOS knockout animals. Kleeberger and colleagues previously reported that iNOS expression is involved in ozone-induced lung hyperpermeability showing reduced mean BAL fluid protein and leukocyte accumulation [48]. Recent studies indicate that iNOS also plays a proinflammatory role in the development of asbestosis-related pulmonary disorders, measured as a significantly decreased total protein count, LDH activity, and nitrotyrosine staining in iNOS-deficient mice [27]. In contrast, Kobayashi et al. reported that hyperoxia caused an increased accumulation of leukocytes, elevated LDH activity and albumin concentration, and a higher wet-dry-ratio in lungs from iNOS-deficient mice compared to wild-type animals [31]. Based on their findings, these authors suggest the presence of an iNOS-independent pathway of lung nitration and injury in hyperoxia. In our study, we found that nitrosylation of proteins in the lungs of mice exposed to >95% O2 was attenuated in iNOS-deficient mice (data not shown). Formation of nitrotyrosine was proposed as a relatively specific marker for detecting endogenous generation of peroxynitrite. However, recent evidence indicates that alternate reactions are capable of inducing nitration of tyrosine in proteins, for example the reaction of myeloperoxidase with hydrogen peroxide. Therefore, increased nitrotyrosine staining is considered as an indicator of "increased nitrative stress" rather than a "footprint" for the formation of peroxynitrite [49,50]. Amplified formation of reactive oxygen and nitrogen species can be proved by determination of thiobarbituric acid reactive substances, a secondary product of lipid peroxidation indicating oxidative and/or nitrative stress [9]. In our study, significantly reduced formation of thiobarbituric acid reactive substances following >95% oxygen exposure was found in iNOS knockout mice, again suggesting a beneficial effect of iNOS deficiency on oxidant lung injury. Cytokines may also play a role in oxygen toxicity. Several studies point out that TNF-α is produced during hyperoxic exposure [51,52]. Furthermore, hyperoxia induces sequential formation of pulmonary TNF-α and IL-6, which corresponds to the severity of pathological findings [12]. In our study, iNOS deficiency resulted in a significant decrease in BAL TNF-α concentration during hyperoxic exposure. Findings of Sass et al. also demonstrate that iNOS-derived NO regulates proinflammatory genes in vivo resulting in inflammatory liver injury in mice by stimulation of TNF-α production [53]. To investigate whether hyperoxia-induced TNF-α expression was regulated on the level of protein or mRNA, activation of the redox-sensitive transcription factors NF-κB and AP-1 was analyzed. As recently described, NF-κB was activated following hyperoxia resulting in an increase in TNF-α and IFN-γ gene expression in murine pulmonary lymphocytes [35]. Moreover, we found that the activation of both factors seen in wild-type mice was weaker in iNOS knockout mice suggesting that induction of iNOS upon hyperoxia may in fact activate these transcription factors. These findings contrast the silencing effect of NO on NF-κB demonstrated upon stimulation with LPS or silica [54]. Data from Kupatt et al. [55] also indicate a negative feedback mechanism of eNOS-derived NO on activation of NF-κB following myocardial reoxygenation. In addition to isotype-specific differences in NO forming capacity, the synergistic NF-κB and AP-1 activation upon an reactive oxygen or nitrogen species challenge might diminish the inhibitory effect of NO. Recent studies indicate that exogenously administered NO causes increased c-fos and c-jun gene and protein expression combined with an evident AP-1 binding activity mediated by reactive oxygen and nitrogen species [56]. Conclusions Taken together, our data show that the absence of the iNOS gene does attenuate, but not fully abolish, oxidation, nitration, and cytotoxicity in response to acute hyperoxic exposure. The degree of transcriptional activation, inflammation, and oxidative lung injury caused by hyperoxia is significantly reduced in iNOS knockout mice compared to wild-type animals. In conclusion, these findings provide evidence to suggest that, upon hyperoxic exposure to >95% O2, proinflammatory effects of iNOS may be predominant, thereby contributing to the extent of acute hyperoxic lung injury. Authors' contributions AKH carried out the hyperoxic model, subsequent cytological and biochemical analyses, and writing and preparation of the manuscript. MD and FK participated in the direction of the study as well as in writing and preparation of the manuscript. CK carried out the electrophoretic mobility shift assays. The data presented in this paper are part of the doctoral thesis of AKH. All authors read and approved the final manuscript. List of abbreviations AP-1 activator protein-1 BAL bronchoalveolar lavage iNOS inducible nitric oxide synthase LDH lactate dehydrogenase LPS lipopolysaccharide NF-κB nuclear factor-kappa B NO nitric oxide TBARS thiobarbituric acid reactive substances TNF-α tumor necrosis factor-alpha Figure 2 Protein concentration in BAL from wild-type and iNOS knockout mice after 72 h exposure to 21%, 60%, and >95% O2. Data are mean ± SEM of seven mice for each group. #p < 0.05 vs. normoxia; *p < 0.05 vs. 60% O2; $p < 0.05 vs. iNOS knockout mice. Acknowledgements The authors gratefully acknowledge the excellent technical assistance of Mrs. A.-M. Allmeling, Mrs. A. Schropp, and Mrs. E. 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Nohl H Redl H Bahrami S Organ specific formation of nitrosyl complexes under intestinal ischemia/reperfusion in rats involves NOS-independent mechanism(s) Shock 2001 15 366 371 11336196 Desmarquest P Chadelat K Corroyer S Cazals V Clement A Effect of hyperoxia on human macrophage cytokine response Respir Med 1998 92 951 960 10070569 10.1016/S0954-6111(98)90195-0 Horinouchi H Wang CC Shepherd KE Jones R TNF alpha gene and protein expression in alveolar macrophages in acute and chronic hyperoxia-induced lung injury Am J Respir Cell Mol Biol 1996 14 548 555 8652183 Sass G Koerber K Bang R Guehring H Tiegs G Inducible nitric oxide synthase is critical for immune-mediated liver injury in mice J Clin Invest 2001 107 439 447 11181643 Chen F Kuhn DC Sun SC Gaydos LJ Demers LM Dependence and reversal of nitric oxide production on NF-kappa B in silica and lipopolysaccharide-induced macrophages Biochem Biophys Res Commun 1995 214 839 846 7575553 10.1006/bbrc.1995.2363 Kupatt C Weber C Wolf A Becker BF Smith TW Kelly RA Nitric oxide attenuates reoxygenation-induced ICAM-1 expression in coronary microvascular endothelium: role of NF-kappa B J Mol Cell Cardiol 1997 29 2599 2609 9344755 10.1006/jmcc.1997.0439 Janssen YM Matalon S Mossman BT Differential induction of c-fos, c-jun, and apoptosis in lung epithelial cells exposed to ROS or RNS Am J Physiol 1997 273 L789 L796 9357854

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==== Front Cardiovasc UltrasoundCardiovascular Ultrasound1476-7120BioMed Central London 1476-7120-2-161535787010.1186/1476-7120-2-16ResearchImmunological evaluation of the new stable ultrasound contrast agent LK565: a phase one clinical trial Funke B [email protected] HK [email protected] S [email protected] S [email protected] I [email protected] U [email protected] P [email protected] T [email protected] RJ [email protected] Department of Internal Medicine/Cardiology, Klinikum Schwalmstadt Schwalmstadt, Germany2 Heart Center Leipzig, University of Leipzig, Germany3 Institute of Clinical Immunology and Transfusion Medicine, University of Leipzig, Germany2004 10 9 2004 2 16 16 12 5 2004 10 9 2004 Copyright © 2004 Funke et al; licensee BioMed Central Ltd.2004Funke et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Ultrasound contrast agents (UCAs) allow the enhancement of vascular definition, thereby providing more diagnostic information. LK565 is a new second-generation UCA based on synthetic polymers of aspartic acid which is eliminated from the blood stream via phagocytosis. LK565 forms very stable air-filled microspheres and is capable of repeated passage through the pulmonary capillary bed after peripheral intravenous injection. This characteristic allows examination of the cardiac function or extracardiac vessel abnormalities up to 15 minutes. Methods A phase one clinical study was conducted on 15 healthy volunteers to identify the development of an undesirable immune response. Phagocytosis capacity, TNF-α secretion, and MHC class II upregulation of monocytes was monitored, as well as microsphere specific antibody development (IgM, IgG). Furthermore, the kinetics of the activation surface markers CD69, CD25, CD71, and CD11b on leukocytes were analyzed. Results Due to LK565-metabolism the administration of the UCA led to saturation of phagocytes which was reversible after 24 hrs. Compared to positive controls neither significant TNF-α elevation, neither MHC class II and activation surface markers upregulation, nor specific antibody development was detectable. Conclusion The administration of LK565 provides a comfortable duration of signal enhancement, esp. in echocardiography, without causing a major activation cascade or triggering an adaptive immune response. To minimize the risk of undesirable adverse events such as anaphylactoid reactions, immunological studies should be included in clinical trials for new UCAs. The use of LK565 as another new ultrasound contrast agent should be encouraged as a safe means to provide additional diagnostic information. ==== Body Background Echocardiography allows the analysis of ventricular motion and heart-valve morphology. The discovery that injection of small air bubbles dispensed in saline are capable of causing an opacification of the heart [1,2] started an enormous effort to develop contrast agents to assist in diagnosing coronary artery disease and myocardial infarction [3]. Early developments led to stabilized bubbles in hyperosmolar solutions [4]. Albumin-stabilized bubbles were one of the first contrast agents to allow examination of both ventricles [5], but there are stability problems in patients with elevated pulmonary pressure [6]. Second-generation contrast agents improved the contrast especially in patients with subnormal echo signals [7-9]. LK565 and comparable substances represent a new concept in contrast agents as it consists of small, stable, air-filled particles [10] that provides a good contrast in both ventricles (figure 1, 2). Due to its high stability, LK565 improves examination of cardiac morphology and function as well as extracardiac vessel abnormalities for at least 15 minutes [11] (figure 3). Figure 1 Electron micrograph of LK565 Electron micrograph of the particles of the new contrast agent LK565 Figure 2 Apical 4 chamber view with LK565 Apical 4 chamber view before (a) and 1 minute after (b) intravenous LK565 application (4 mg/kg BW). Device: SONOS 5500, S3 Probe, harmonic Figure 3 Apical 4 chamber view with LK565 Apical 4 chamber view before (a), 1 minute (b), and 30 minutes (c) after intravenous LK565 application (4 mg/kg BW) and intermittent ultrasound exposure. Device: SONOS 5500, S3 Probe, harmonic The particles consist of a synthetic polymer of aspartic acid. Via peptide bonds, the molecules form bold, stable beads filled with air. The average size of LK565 particles is 3 μm in diameter (figure 1). Because of their size and even polymeric surface the beads have similarities to microorganisms. This circumstance implies the induction of an immune response, which needs to be considered. Results from previous studies indicate that phagocytes are responsible for eliminating the contrast agent from the blood stream [11]. In vitro, it has been shown that macrophages and neutrophils quickly devour the LK565 particles. Preliminary studies proved that the LK565 particles are phagocytosed by macrophages, resulting in slight activation with an increase in the intracellular calcium content. However, there were no symptoms of a respiratory burst [12]. The activation of macrophages can lead to different conditions: no activation, normal activation, and overreaction. In the last case, over-expression of tumor necrosis factor alpha (TNF-α) can lead to a septic shock by induction of intravascular blood clots apart from other effects, which may result in disseminated intravascular coagulation [13,14]. Furthermore, macrophages act as antigen-presenting cells which are highly important to induce an adaptive immune response, i.e. specific antibody production. It is probable that the LK565 particles attract antigen-presenting cells to present LK565 fragments as antigens. This may induce the production of specific antibodies against the contrast agent, which in rare cases could result in pathogenic immune reactions. The aim of this study was to examine a possible immune response to LK565 considering phagocytosis, TNF-α secretion by macrophages, lymphocyte activation and specific antibody production. Methods Volunteers The experiment was approved by the local ethics committee (registration number 708/98). After medical examination, 15 healthy male volunteers under the age of 30 were enrolled in this phase one clinical study. Each volunteer was exposed to a single dose of the contrast agent LK565 applied intravenously. Four participants were exposed to the contrast agent LK565 for the second time because they had already been involved in a prior study [11]. The 15 volunteers were divided up into 3 groups (n = 5). The first group received 0.15 mg/kg bodyweight, the second group 0.4 mg/kg bodyweight and finally the third group 0.7 mg/kg LK565. Contrast agent Prior to application, the contrast agent LK565 was dissolved in 10 ml physiological sodium chloride solution (37°C) under sterile conditions. To eliminate aggregations, the solution was filtered using a 20 μm mesh just before intravenous injection. Sampling Heparinized blood samples (2 ml) were taken before application (t = 0), after 2 h, 6 h, 24 h, 48 h, 72 h, 96 h, and 1 ml after 10 d, 12 d and 14 d. For routine analysis, additional blood samples were drawn before and 2 hours after intravenous contrast agent injection. The volunteers were under medical care throughout the whole experiment. Phagocytosis Phagocytosis capacity was determined in vitro before (t = 0) and after 2 h, 6 h and 24 h with Phagotest™ (Becton Dickinson, Germany) and analyzed via flow cytometry (FACScalibur™, BectonDickinson, Germany). FITC-labeled E. coli, which were opsonized with antibodies and complement factors, were incubated with leukocytes. After incubation, the increased fluorescence of the phagocytes was due to the uptake of bacteria. The fluorescence intensity is dependent on the amount of phagocytosed bacteria. A sample chilled on ice was used for control. Tumor necrosis factor alpha (TNF-α) Intracellular TNF-α production in monocytes and macrophages was analyzed before (t = 0) and after 2 h, 6 h, and 24 h. For detection, an anti-human TNF-α Pycoerythrin (PE) monoclonal antibody (mAb) was applied (Pharmingen, Germany). The specificity of the antibody had been validated in blocking experiments. To 40 μl blood, 145 μl RPMI and 15 μl 40 μM Monensin were added (Monensin prevents cytokine release by blocking the golgi apparatus). Cells were incubated for 2 h at T = 37°C and 5% CO2 atmosphere. After washing, monocytes were surface stained by anti-human CD14 TriColor mAb (Medac, Germany). Red blood cells were lysed using FACS™ Lysing Solution (Becton Dickinson, Germany). Cells were fixed and permeabilized by addition of 250 μl Cytofix/Cytoperm™ (Becton Dickinson, Germany). Finally, the retained intracellular TNF-α was stained by the anti-TNF-α PE mAb. For a positive control, cells were stimulated with 0.25 μg Lipopolysaccharide (LPS), following the same procedure as mentioned above. Surface activation markers Surface activation markers were analyzed before (t = 0) and after 2 h, 6 h, 24 h, 48 h, 72 h, and 96 h. For the detection of activation markers, anti-human CD69 PE (Holzel Diagnostika, Germany), CD25 Fluoresceinisothiocyanat (FITC), CD71 FITC, CD11b PE (Diaclone Research, Germany) and HLA-DR PerCP (Becton Dickinson, Germany) mAbs were used. For a positive control, cells were stimulated with 10 ng/ml Phorbol-12-Myristat-13-Acetat (PMA) and 1 μg/ml Ionomycin for 2 h, while macrophages/monocytes were stimulated with 1.25 μg/ml LPS for 2 h. To distinguish between leukocytes, additional surface markers, anti-human CD45 Allophycocyanine (APC), CD19 APC (Caltag Laboratories, Germany), CD3 Tri/Color (DAKO, Germany) and CD14 FITC (Diaclone Research, Germany) mAbs were used for detection. Blood samples (40 μl) were stained for 4-color flow cytometry analysis. After staining, red blood cells were lysed, subsequently leukocytes were analyzed. Antibody specificity was validated by isotype controls (Becton Dickinson, Germany). Specific antibody development Serum samples were analyzed before (t = 0) and after 24 h, 48 h, 72 h, 96 h, 10 d, 12 d, and 14 d. Specific anti-LK565 antibodies were determined via indirect ELISA. Indirect ELISA was performed in a 96-well plate by LK565 immobilization overnight. Optimal concentrations had previously been determined by common cross-testing. Serum was obtained from blood samples after centrifugation at 400 G. After incubation for 2 h at room temperature, detector antibodies (Biozol Diagnostica GmbH, Germany) were added and incubated at 4°C overnight. Washing was carried out with phosphate buffered saline (PBS) and blocking buffer. For detection, alkaline phosphatase (AP)-linked goat-anti-human IgG and IgM antibodies with para-Nitrophenylphosphate (p-NPP) as substrate (Biozol Diagnostica GmbH, Germany) were used. After development, the enzyme reaction was stopped with 0.5 M NaOH and measured at λ = 405 nm. For the functional (positive) control of the indirect ELISA, total immunoglobuline was captured polyclonally. For a negative control, the serum of a person who had not been exposed to the contrast agent was obtained. Results We investigated 15 volunteers in a clinical phase one trial over a period of 14 days. The kinetics of activation surface markers CD69, CD25, CD71 and CD11b on leukocytes, the phagocytosis capacity, and TNF-α production in monocytes were analyzed, as was the specific antibody production after LK565 application. The expression of CD69 [15], CD25 [16], CD71, HLA-DR [17], and CD11b [18] is increased after activation [19]. CD69 will be presented quickly after activation on almost all leukocytes normally after some hours but on neutrophils even after a few minutes due to mobilisation of intracellular storages [20]. The α-chain of the IL-2 receptor CD25, also known as the high-affinity receptor on lymphocytes, is developed after 6 hours at the earliest and up to 1–2 days. Transferrin receptor CD71 correlates directly with cell proliferation and a late marker which can be expected after 3–4 days similar to antigen presentation via MHC-II analyzed as HLA-DR. Integrin expression (CD11b) on monocytes and neutrophils is associated with phagocyte migration into tissue [21]. Independent of dosage or repeated contact, the contrast agent LK565 was well tolerated by all volunteers. After exposure to LK565 no erythema, no drop in blood pressure, neither significant change of the heart frequency nor fever occurred (data not shown). The volunteers did not complain of relevant clinical symptoms such as chest discomfort or asthenia. It exhibited good opacification of both ventricles a few seconds after application (figure 2). While the minimum dosage of 0.15 mg/kg LK565 was sufficient for just one echocardiogram, a suitable duration of echo contrast was obtained at a 0.4 mg/kg dose. A further increase in dosage provided no improvement in quality or contrast duration (data not shown). The uptake of LK565 led to the saturation of phagocytes. The uptake capacity of the phagocytes from blood samples 6 h after injection was exhausted (figure 4). Neither macrophages nor neutrophils phagocytosed any more labeled bacteria after LK565 uptake, the observed effect was reversible. After 24 h the phagocytic capacity reached almost the starting level. Figure 4 Phagocytosis capacity after LK565 exposure Phagocytosis capacity refer to opsonized E. coli of neutrophils (a) and monocytes (b) in vitro illustrated in percent of control after LK565 exposure (n = 15, error bars show standard error). During the first 24 h an increase in intracellular TNF-α production was detected in monocytes and macrophages (figure 5a). However, in relation to the positive control after 2 h of LPS stimulation, the slight increase in intracellular TNF-α was negligible. No connection between repeated exposure and dosage was found. The amount of intracellular TNF-α decreased to starting levels after 24 h. Figure 5 TNF-α and specific antibody production after LK565 application (a) intracellular TNF-α (Monocytes) after LK565 exposure relative to starting value and (b) LK565 specific antibody production (IgM, IgG) relative to positive control (n = 15, error bars show standard error). An increase in integrin expression CD11b was detected on monocytes and macrophages 6 hours after application. Compared to the positive control, the upregulation of the integrin was only poor. In all cases, the upregulation of the integrin was only of brief duration and returned to normal levels after 24 h (figure 6d). Only a slight and short increase in CD69 after 6 h was determined in neutrophils in a few cases. Most volunteers exhibited no CD69 increase in neutrophils. Even less CD69 expression was found on monocytes and macrophages where CD69 stimulation is associated with the production of prostaglandines, leukotriens and TNF-α. Only minor CD69 expression was observed on T-cells and on B-cells (figure 6c). No increase of CD25 and CD71 (also on monocytes) was detected (figure 6a,6b). Figure 6 Activation marker expression after LK565 application CD25, CD71, CD69 and CD11b expression on different cell types after LK565 exposure relative to starting value (n = 15, error bars show standard error). Discrete MHC class II (HLA-DR) upregulation on macrophages and lymphocytes was observed (figure 7a). We found no further evidence of an adaptive immune response via antibody production. None of the serum samples during our clinical trial exhibited any development of specific antibodies (IgM, IgG) versus the contrast agent (figure 5b). Figure 7 HLA-DR expression after LK565 application HLA-DR expression relative to starting value and differential cell count after LK565 application (n = 15, error bars show standard error). Discussion The results show that there is only a slight activation of leukocytes esp. of phagocytes after application of LK565, a particular contrast agent based on a polymer of the naturally occurring amino aspartic acid. No specific antibody development against the contrast agent was detected. Thus, the risk of a major immunological activation cascade after repeated dosage can be regarded as minor. Since LK565 does not lead to an adaptive immune activation in this study, the possibility of an overexpression of IgE, which plays a key role in allergic diseases, also becomes more unlikely. Although none of the 15 volunteers enrolled in this phase I study developed an allergic-type reaction, such reactions could, however, occur in further phases of clinical testing or application. An allergic reaction against a pharmaceutical drug is a rare event which is impossible to exclude even in large clinical studies. An increase in CD11b/CD18 is connected with phagocytosis and the diapedesis readiness of phagocytes. LK565 is eliminated from the blood stream via phagocytosis, therefore neutrophils exhibited an increase in CD11b expression and phagocytosis activity. This is also connected to a temporary higher intracellular TNF-α level in monocytes. On lymphocytes, especially T-cells, the cascade of CD69, CD25 and CD71 is closely related to an immune response. After expression of CD69, the interlinkage of the receptor leads to proliferation and secretion of IL-2, IFN-γ and TNF-α. After effective activation, the T-cells secrete IL-2 which activates the development of the high-affinity IL-2 receptor CD25 and therefore launches cell proliferation with an expression of CD71 after 3 – 4 days. During our study after LK565 application no major activation cascade was detected on either T-cells or B-cells. As mentioned above, the best contrast performance for echocardiography was obtained at a LK565 dosage of 0.4 mg/kg. Opacification of both ventricles was not enhanced by a further increase in dosage. Bearing in mind the fact that the contrast agent is phagocytosed and uptake capacity is limited, it is recommended that the daily dosage does not exceed 90 mg, which is equivalent up to three echocardiograms per day. Conclusions To minimize the risk of an undesirable adverse event such as an anaphylactoid reaction, immunological studies should be included in clinical trials for new UCAs. The use of LK565 as another new ultrasound contrast agent (UCA) with a comfortable duration of signal enhancement esp. in echocardiography should be encouraged as a means to provide additional diagnostic information without causing a major activation cascade or triggering an adaptive immune response. This can be an advantage for the "difficult-to-image" patient with adverse reactions related to other UCAs. Competing interests H.K. Maerz received salary for 6 months from Dr. F. Koehler Chemie GmbH, Alsbach-Haehnlein, Germany, the developer/manufacturer of LK565. R. Zotz is co-author of one of several patents related to LK565 (Zotz R, Erbel R, Krone V, Magerstädt M, Walch A (1992): Ultrasonic contrast agents, processes for their preparation and the use thereof as diagnostic and therapeutic agents. United States Patent 5.137.928). Authors' contributions BF: Prepared and edited the manuscript as well as the figures und performed echocardiographic evaluations; HKM: Drafted the manuscript and participated in the design of the study esp. immunological aspects; SO: Carried out TNF-α evaluations, recruited and coordinated the clinical management of the patients; SP: Carried out expression pattern analysis, recruited and coordinated the clinical management of the patient; IL: Participated in the study design; US: Reviewed the manuscript according to study design and immunological aspects PW and TG: Reviewed the manuscript and edited the figures; RZ: Participated in the development of LK565, the study design and coordination. 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Int J Card Imaging 1994 10 195 203 7876659 Schneider M Arditi M Barrau MB Brochot J Broillet A Ventrone R Yan F BR1: a new ultrasonographic contrast agent based on sulfur hexafluoridefilled microbubbles Invest Radiol 1995 30 451 457 8557510 Grayburn P Perflenapent emulsion (EchoGen): a long-acting phase shift agent for contrast echocardiography Clin Cardiol 1997 20 I12 I18 9383597 Cohen JL Cheirif J Segar DS Gillam LID Gottdiener JS Hausnerova E Bruns DE Improved left ventricular endocardial border delineation and opacification with OPTISON (FS069), a new echocardiographic contrast agent. Results of a phase-III multicenter trial J Am Coll Cardiol 1998 32 746 752 9741522 10.1016/S0735-1097(98)00311-8 Zotz R Genth S Grande J Walch A Ziehn P Krone V Schuler G Polymer microparticles for right and left heart echocardiography and imaging myocardial perfusion after peripheral vein injection Z Kardiol 1996 85 924 931 9082670 Zotz RJ Schenk S Kuhn A Schlunken S Krone V Bruns W Genth S Schuler G Safety and efficacy of LK565 – a new polymer ultrasound contrast medium Z Kardiol 2001 90 419 26 11486577 10.1007/s003920170152 Maerz HK Okorokow S Polata S Lehmann 1 Schneider P Zotz R The Fate of ECHO Beads 11 – Immunological Response or Not? Immunbiol 1999 200 361 Morrissette N Gold E Aderem A The macrophage – a cell for all seasons Trends Biol 1999 9 199 210 10.1016/S0962-8924(99)01540-8 Hofsli E Bakke 0 Nonstad U Espevik T A flow cytometric and immunofluorescence microscopic study of tumor necrosis factor production and localization in human monocytes Cell Immunol 1989 122 405 415 2548738 Testi R D'Ambrosio D De Maria R Santoni A The CD69 receptor: a multipurpose cell-surface trigger for hematopoietic cells Immunol Today 1994 15 479 483 7945773 10.1016/0167-5699(94)90193-7 Werfel T Boeker M Kapp A Rapid expression of the CD69 antigen on T cells and Natural Killer cells upon antigenic stimulation of peripheral blood mononuclear cell suspensions Allergy 1997 52 465 469 9188933 Arva E Andersson B Kinetics of cytokine release and expression of lymphocyte cell-surface activation markers after in vitro stimulation of human peripheral blood mononuclear cells with Streptococcus pneumoniae Scand J Immunol 1999 49 237 243 10102640 10.1046/j.1365-3083.1999.00470.x Prieto J Beatty PG Clark EA Patarroyo M Molecule mediating adhesion of T and B cells, monocytes and granulocytes to vascular endothelial cells Immunology 1988 63 631 637 3259203 Thompson HL Matsushima K Human polymorphonuclear leukocytes stimulated by tumour necrosis factor-alpha show increased adherence to extracellular matrix proteins which is mediated via the CD11b 18 complex Clin Exp Immunol 1992 90 280 285 1358490 Craston R Koh M McDermott A Ray N Prentice HG Lowdell MW Temporal dynamics of CD69 expression on lymphoid cells J Immunol Meth 1997 209 37 45 10.1016/S0022-1759(97)00143-9 Lundgren-Akerlund E Olofsson AM Berger E Arfors KE CD11b/CD18-dependent polymorphnuclear leukocyte interaction with matrix proteins in adhesion and migration Scand J Immunol 1993 37 569 574 8097895

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==== Front Health Res Policy SystHealth Research Policy and Systems1478-4505BioMed Central London 1478-4505-2-71536311210.1186/1478-4505-2-7CommentaryPrevention of non-communicable diseases in Pakistan: an integrated partnership-based model Nishtar Sania [email protected] Heartfile, 1-Park Road, Chak Shahzad, Islamabad, Pakistan2004 13 9 2004 2 7 7 27 8 2004 13 9 2004 Copyright © 2004 Nishtar; licensee BioMed Central Ltd.2004Nishtar; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Development and implementation of non-communicable disease (NCD) prevention polices in the developing countries is a multidimensional challenge. This article highlights the evolution of a strategic approach in Pakistan. The model is evidence-based and encompasses a concerted and integrated approach to NCDs. It has been modelled to impact a set of indicators through the combination of a range of actions capitalizing on the strengths of a public-private partnership. The paper highlights the merits and limitations of this approach. The experience outlines a number of clear imperatives for fostering an enabling environment for integrated NCD prevention public health models, which involve roles played by a range of stakeholders. It also highlights the value that such partnership arrangements bring in facilitating the mission and mandates of ministries of health, international agencies with global health mandates, and the non-profit private sector. The experience is of relevance to developing countries that have NCD programs running and those that need to develop them. It provides an empirical basis for enhancing the performance of the health system by fostering partnerships within integrated evidence-based models and permits an analysis of health systems models built on shared responsibility for the purpose of providing sustainable health outcomes. ==== Body Background Non-communicable diseases exhort a considerable toll on individuals, societies and health systems [1,2]. Located in South Asia, Pakistan has a population of 150 million and a per-capita health expenditure of US $ 18 [3]. NCDs and injuries are amongst the top ten causes of mortality and morbidity in Pakistan [4]; estimates indicate that they account for approximately 25% of the total deaths within the country [5]. NCDs contribute significantly to adult mortality and morbidity and impose a heavy economic burden on individuals, societies and health systems [6]. In most cases, it is the economically productive workforce, which bears the brunt of these diseases. Existing population-based morbidity data on NCDs in Pakistan show that one in three adults over the age of 45 years suffers from high blood pressure [7]. The prevalence of diabetes is reported at 10% whereas 40% men and 12.5% women use tobacco in one form or the other [8,9]. Karachi reports one of the highest incidences of breast cancer for any Asian population [10]. In addition, estimates indicate that there are one million severely mentally ill and over 10 million individuals with neurotic mental illnesses within the country [11]. Furthermore, 1.4 million road traffic crashes were reported in the country in the year 1999; of these, 7000 resulted in fatalities [12]. Established evidence highlights the potential to limit NCD mortality and morbidity through appropriate public health strategies aimed at disease prevention, risk factor control and health promotion [13]. Addressing NCDs in a developing country such as Pakistan is a multidimensional challenge with implications at different levels and necessitates a two fold action. Firstly, lobbying for appropriate investments and policies to facilitate their inclusion in the development and health agenda [14], and secondly, developing scientifically valid, culturally appropriate and resource-sensitive models incorporating and integrating the multidisciplinary range of actions relevant for NCD prevention. In Pakistan, the public-private tripartite partnership led by Heartfile (a non-profit NGO registered under the Societies Registration act of 1860 in Pakistan) and constituted additionally by the Ministry of Health and the WHO Pakistan office has recently released the National Action Plan for Non-Communicable Disease Prevention, Control and Health Promotion in Pakistan (NAP-NCD) to achieve national goals for the prevention and control of NCDs [15]. This paper discusses the strengths and limitations of this initiative and highlights the value that such partnership arrangements bring in facilitating the missions and mandates of various partners. Merits The present exercise is the first opportunity to mount a truly 'national plan of action' aimed at preventing and controlling NCDs with the Governments' commitment to NCD prevention as a priority and to enlist a broad range in inputs from within Pakistan for addressing a challenging issue. The NAP-NCD outlines a concerted and comprehensive approach; one that incorporates both policies and actions. It is set within a long-term and life-course perspective and calls for an institutional, community and public policy level change. It has been designed to overcome the tendency to rely on a disjointed set of small scale projects, factoring integration at six levels: grouping NCDs so that they can be targeted through a set of actions, harmonizing actions, integrating actions with existing public health systems, incorporating contemporary evidence-based concepts, combining prevention and health promotion and harnessing the potential within partnerships. Disease domain integration the term NCDs is technically reserved for a group of preventable diseases that are linked by common risk factors: cardiovascular diseases, some chronic lung conditions, cancer and diabetes fall within this category. However NAP-NCD also includes injuries and mental health within this framework as country requirements necessitate that these be addressed within a combined strategic framework through synchronized public health measures. There are many common grounds for combining public health actions to address these diseases. Action level integration the NAP-NCD delivers an Integrated Framework for Action (IFA) [16]; this is modelled to impact a set of indicators through the combination of actions across the range of NCDs in tandem with rigorous formative research. The IFA emanates from the concept highlighted in Fig 1; within this framework, it encompasses two sets of strategies; those that are common across the entire range of NCDs and others that are specific to each NCD domain. The first strategy includes a behavioural change communication strategy, reorientation of health services strategy and surveillance, while the second pertains to legislative and regulatory matters. Figure 1 Paradigm for NCD prevention, control and health promotion Systems level integration the approach adopted horizontally integrates NCD prevention with existing public health and social welfare infrastructure. It thus contributes to strengthening of the pubic health configuration and reorients health services to a more preventive orientation. Integration of concepts NAP-NCD packages several contemporary and novel approaches. The population approach includes a behavioural research and social marketing-guided communication strategy and active role of local opinion leaders and educational institutions. Reorientation of health services includes scaling up of professional capacity and basic infrastructure in health care facilities and ensuring availability of, and access to, essential drugs at all levels of health care. The IFA packages a common population surveillance mechanism for all NCD's (with the exception of cancer). The model includes population surveillance of common risk factors and combines a module on population surveillance of injuries and mental health. The model has also been adapted for program evaluation. Combining prevention and health promotion prevention is concerned with avoiding diseases whereas health promotion is about improving health and wellbeing. Both approaches are overlapping and complimentary and can be present in the same programme with similar activities and hold different meanings for two groups of targeted populations with different results. The public health approach to NCDs offers one of the best opportunities to combine prevention and health promotion to improve multiple positive outcomes; an approach NAP-NCD has capitalized on. Public-private partnership dimension this initiative created a mechanism for visible involvement and participation of relevant ministries, educational institutions, NGOs and leadership foci at a national consultation level and created avenues for their participation in the process that led to its development. In addition, all the key elements and advantages that stand to be gained from comprehensive grouping and maximizing on partnerships have been built upon: integration with the existing health system, inter-sectoral and intra-health-sector collaborations, linkage with the national health policy and partnerships with the private sector. NAP-NCD recognizes the scope of partnerships in public health activities and outlines a scope of interventions that are built on shared responsibility, allowing for agencies to participate according to their own missions, mandates, interests and resources. NAP-NCD fosters partnerships and interface arrangements between the public and private sectors so that the federal government is not solely responsible for getting these programmes out to the communities, but can rely on groups and national organizations that have complementary mandates. These partnerships are in harmony with national health priorities, complement state initiatives and are optimally integrated with national health systems. Value to participating agencies Ministry of Health Reproductive health and communicable disease prevention and control have traditionally been priority areas for the Ministry of Health. Prevention and control of NCDs did not previously feature as part of the National Health Policy of 2001. There were therefore no specific programs in the national and provincial health departments and no budgetary line for NCD prevention up until the signing of the agreement, which lay the terms of reference for developing NAP-NCD [17]. By leveraging on the technical strengths of a private sector partner, the MOH was able to acquire a scientifically sound plan incorporating broad-based consensus. By adopting this Plan, the MoH has included NCD prevention and control as part of its policy framework. The Federal Government has also shown commitment to implement the plan. Budgetary allocations have been made from the Ministry of Health's existing resources for its first phase of implementation [18]; these will support the establishment of a surveillance system and a behavioural change communication campaign through the media; in addition a training program has been introduced into the work-plan of Lady Health Workers (LHWs), Pakistan's field force of health care providers at the grass roots level in 17 districts targeting a population of approximately 10 million. Heartfile has previously pilot tested this approach in partnership with the MoH [19]. Since Heartfile has the major participatory role in implementing these activities; this approach allows the government to fulfil a policy obligation to include the private sector in national programs outside of a 'contractual' mode. It therefore serves as an empirical basis for health sector reform in the area of public-private collaboration. Overall funding for prevention and health promotion in the national health budget has been increased. The next stage of implementation of NAP-NCD includes reorientation of health services and a comprehensive school health program. A proposal is already in the funding pipeline to seek additional resources from the governments' development budget and from donor sources. World Health Organization As part of its global mandate, WHO provides technical support to 'priority national programs' through its Joint Government/WHO Program Review Mission (JPRM) program; this is a regular biannual budgetary line. In addition, 'extra-budgetary' resources are provided for 'WHO priority programs' such as the polio campaign. In the year 2003, US $ 843 million were allocated for 192 countries under the former and 1.4 billion as part of the latter [20]. For the year 2001–2003, Pakistan was allocated a budget of US $ 20 million [21]. However by convention, these resources have always been used by public sector institutes and health professionals with public sector affiliations. Within the context of the implementation of the first phase of NAP-NCD, for the first time in Pakistan, the JPRM 2004–05 has made allocations to support activities which are being implemented by an NGO albeit in an official relationship with the MoH [22]. WHO will therefore gain experience in working in a country model in which the private sector can be supported through the JPRM resources. Heartfile Heartfile has been planning and implementing national media campaigns and community-based projects for cardiovascular disease prevention incorporating social marking approaches [23,24]. Although pilot activities have previously been conducted in partnership with the MoH; the NGO activities were previously not integrated with national programs. By partnering in this program, NGOs activities will contribute to the country's National Plans within the framework of priorities set by broad-based national consensus; will be implemented through existing structures and monitored through one evaluation mechanism. Its activities will therefore contribute to achieving national goals. Currently, the NGO draws support from donor funds through 'project aid'. In future, the NGOs funding is likely to be compromised with shifting donor focus on 'programme aid', as part of which, donors provide funds through national budgets. Partnering in this program therefore contributes to sustainability of the NGO as this provides a mechanism for sustained funding. Limitations The ingredients in this public health strategy are sound; however there are several limitations of this approach. Firstly, it needs to be supported by a clear, strong and sustained political commitment; secondly, the successful implementation of this plan requires the setting up of appropriate infrastructure and public health workforce with adequate capacity for core public health functions. This has implications for the need to build capacity and related infrastructure as a parallel process. The public-private partnership dimension of this plan emanates from within the overall 'development policy framework', which encourages private-sector participation in state activities. However it does have its own challenges. This experience therefore presents a clear imperative for addressing ethical, methodological, accountability, sustainability and governance issues in public-private and other multi-stakeholder arrangements [25-27]. Implications for generalisability Useful lessons can be learnt from this experience both by developing countries and low resource settings that have NCD programs running and others that do not. Most developing countries have limited capacity for NCD prevention and control [28]. There is limited experience with building 'integrated models' and 'partnerships' for NCD prevention suited to low resource situations. The Action Plan therefore serves as an empirical basis for an integrated approach to NCDs on one hand, and an experimental basis of health sector reform in the area of public-private collaboration on the other. This example is also relevant to NGOs in other developing countries, who receive financial support from donors through project aid as it serves as an empirical basis for the integration of NGO activities with national plans and goals. Evaluation The desired impact of this intervention is positive change in population health behaviours; therefore its ultimate success can be judged by changes in population outcomes, which can only be assessed over a period of time. However the process evaluation framework of the Action Plan outlined in the IFA is modelled to access how the program achieves its effects and includes the evaluation resource inputs, description of activities and intermediate outcomes. A review of the Logical Framework Analysis of the Action Plan and the IFA has shown progress against many of the process indicators [29]. Conclusions Notwithstanding that there are several limitations of this strategy, it does provide the empirical basis for an integrated response to NCD prevention and health promotion in a developing country setting. The IFA is an innovative tool, which helps to set country targets and defines integrated actions to meet those targets. However future efforts must also seek to integrate strategies with communicable disease control, particularly in areas where a life course approach is pursued; this will enable the development of sustainable public health systems for all disease. It is hoped that the findings from this program can be a basis for policy change. ==== Refs World Health Organization The World Health Report 2002 2002 Geneva: World Health Organization World Health Organization The world health report 2001 2001 Geneva: World Health Organization World Development Indicators 2001 2001 The World Bank, Washington Hyder AA Morrow RH Lost Healthy Life Years in Pakistan in 1990 Am J Public Health 2000 90 1235 1240 10937003 Federal Bureau of Statistics, Statistics Division Pakistan Demographic Survey 2001 2003 Islamabad, Pakistan: Government of Pakistan World Health Organization World Health Report 2000 – Health Systems:Improving Performance 2000 Geneva, Switzerland: World Health Organization Pakistan Medical Research Council National Health Survey of Pakistan 1990–94 1998 Islamabad, Pakistan: Pakistan Medical Research Council, Network Publication Service Shera AS Rafique G Khuwaja IA Ara J Baqai S King H Pakistan National Diabetes Survey: prevalence of glucose intolerance and associated factors in Shikarpur, Sindh province Diabet Med 1995 12 1116 1121 8750223 Pakistan Medical Research Council Pakistan Health Education Survey (ISBN: 969-499-003-3 [pbk]) 2003 Islamabad, Pakistan: Pakistan Medical Research Council Bhurgri Y Bhurgri A Hasan SH Usman A Faridi N Malik J Khurshid M Zaidi SM Pervez S Kayani N Hashmi KZ Bashir I Isani Z Sethna F Ahsan H Zaidi ZA Naseeruddin S Zaidi SA Alam SM Cancer patterns in Karachi division (1998–1999) J Pak Med Assoc 2002 52 244 246 12481632 Planning Commission A Report of the sub-working group on mental health care in Pakistan, 1987 – National Programme of Mental Health for Pakistan 1987 Islamabad, Pakistan: Planning Commission, Government of Pakistan National Transport Research Centre Manual of road safety improvement by the use of low cost engineering countermeasures 1999 Islamabad, Pakistan: National Transport Research Centre, National Highway Authority and Finnroad OY Puska P Tuomilehto J Nissinen A Vartiainen E editors The NorthKarelia Project: 20 years of Results and Experiences 1995 Helsinki (Finland). Helsinki, National Public Health Institute Yach D Gould CL Hawkes C Hofman K The Global Burden of Chronic Diseases: dispelling Myths and Identifying Levers for Change 2004 National Action Plan for non-communicable diseases prevention, control and health promotion (accessed May 12, 04) Integrated Framework for Action: National Action Plan for non-communicable diseases prevention, control and health promotion (accessed May 12, 04) Memorandum of Understanding: National Action Plan for non-communicable diseases prevention, control and health promotion (accessed February 21, 04) National Action Plan for the Prevention, control and health promotion of NCDs in Pakistan Planning Commission (Health) Ministry of Health, Government of Pakistan F.NO.25-21/2003-(Planning) Heartfile Lodhran CVD prevention project (accessed February 21, 04) Proposed program budget for 2002–03; 54th World Health Assembly – Provisional Agenda Item 12.2 (A54/INF.Doc/8) 11 May, 2001; WHO World Budget 2002–03 (accessed May 1, 04) Pakistan Budget (accessed May 12, 04) World Health Organization: Pakistan Joint Program Review Mission, Pakistan (2004–06) Work-plan title: National Action Plan for NCD prevention and control; WHO Eastern Mediterranean Office (EMRO) classification 66 Nishtar S Mirza YA Jehan S Hadi Y Yusuf S Shahab S Badar A Newspapers as a tool for cardiovascular health promotion in the developing country: a Heartfile experience J Health Commun 2004 9 355 369 15371087 10.1080/10810730490468603 Nishtar S Cardiovascular disease prevention in low resource settings: lessons from the Heartfile experience in Pakistan Ethn Dis 2003 13 S38 148 Health Action International, 2002 Statement to 109th WHO Executive Board meeting; January 2002 (accessed June 22, 04) Nishtar S Public-private partnerships in health – a global call toaction Health Research Policy and Systems 2004 2 5 15282025 10.1186/1478-4505-2-5 Widdus R Public private partnerships for health require thoughtful evaluation Bull World Health Organ 2003 81 235 12764487 Alwan A MacLean D Mandil A Assessment of National Capacity for Non Communicable Diseases Prevention and Control 2001 Geneva, Switzerland. World Health Organization Logical Framework Analysis: National Action Plan for non-communicable diseases prevention, control and health promotion (accessed February 21, 04)

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==== Front J Transl MedJournal of Translational Medicine1479-5876BioMed Central London 1479-5876-2-321538314510.1186/1479-5876-2-32ResearchBRCA1-mediated repression of select X chromosome genes Jazaeri Amir A [email protected] Gadisetti VR [email protected] Olga [email protected] Ulrike A [email protected] Christos [email protected] Edison T [email protected] H Hilger [email protected] Cindy J [email protected] Jeff [email protected] J Carl [email protected] From the Center for Cancer Research of the National Cancer Institute. Building 31, Room 3A11, 31 Center Drive, MSC 2440, Bethesda, MD 20892-2440 USA2 Max Planck Institute for Molecular Genetics. Ihnestrasse 73, 14195 Berlin Germany3 Jules Bordet Institute. Microarray Unit, 121 Bld. de Waterloo, 1000 Brussels, Belgium4 Genome Institute of Singapore, 1 Science Park Rd., The Capricorn #05-01, Singapore Sicence Park II 117528, Singapore5 From the Departments of Surgery and Medicine of the Memorial Sloan-Kettering Cancer Center 1275 York Ave., New York, New York, 10021 USA2004 21 9 2004 2 32 32 6 7 2004 21 9 2004 Copyright © 2004 Jazaeri et al; licensee BioMed Central Ltd.2004Jazaeri et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Recently BRCA1 has been implicated in the regulation of gene expression from the X chromosome. In this study the influence of BRCA1 on expression of X chromosome genes was investigated. Complementary DNA microarrays were used to compare the expression levels of X chromosome genes in 18 BRCA1-associated ovarian cancers to those of the 13 "BRCA1-like" and 14 "BRCA2-like" sporadic tumors (as defined by previously reported expression profiling). Significance was determined using parametric statistics with P < 0.005 as a cutoff. Forty of 178 total X-chromosome transcripts were differentially expressed between the BRCA1-associated tumors and sporadic cancers with a BRCA2-like molecular profile. Thirty of these 40 genes showed higher mean expression in the BRCA1-associated samples including all 11 transcripts that mapped to Xp11. In contrast, four of 178 total X chromosome transcripts showed significant differential expression between BRCA1-associated and sporadic tumors with a BRCA1-like molecular profile. All four mapped to Xp11 and showed higher mean expression in BRCA1-associated tumors. Re-expression of BRCA1 in HCC1937 BRCA1-deficient breast cancer cell resulted in the repression of 21 transcripts. Eleven of the 21 (54.5%) transcripts mapped to Xp11. However, there was no significant overlap between these Xp11 genes and those found to be differentially expressed between BRCA1-associated and sporadic ovarian cancer samples. These results demonstrate that BRCA1 mediates the repression of several X chromosome genes, many of which map to the Xp11 locus. ==== Body Introduction The mechanisms by which mutations in BRCA1 and BRCA2 tumor suppressor genes lead to carcinogenesis are incompletely understood. It remains to be established whether pathways involved in BRCA1 and BRCA2-associated tumorigenesis are also altered in sporadic cancers. Two recent reports demonstrated that BRCA1 and BRCA2-associated tumors have distinct expression profiles in both breast [1] and ovarian [1,2] cancers. With respect to ovarian cancers, two additional novel patterns of gene expression were observed. First, the same set of genes that distinguished BRCA1 and BRCA2-associated tumors also segregated the sporadic (not BRCA1 or BRCA2-associated) ovarian cancers into 2 subgroups consisting of "BRCA1-like" and "BRCA2-like" gene expression profiles. This observation lends support to the hypothesis that the same or similar dichotomous molecular pathways are affected in major subgroups of both hereditary and sporadic ovarian tumors. Second, a disproportionate number of genes located on the Xp11 locus showed statistically significant higher expression in the BRCA1-associated tumors when compared to sporadic tumors. Related to this observation, Ganesan and colleagues recently demonstrated that BRCA1 colocalizes with XIST RNA covering the inactive X chromosome [3]. These investigators showed that repression of BRCA1 led to the increased expression of a green fluorescent protein (GFP) transgene targeted to the inactive X chromosome. However, it remains unknown whether BRCA1 mediates any changes in expression of normal X chromosome genes and whether any such changes are global (affecting the entire X chromosome) or specific to certain genes. The goal of this study was to investigate further the influence of BRCA1 on the expression of transcripts mapped to the X chromosome. For this purpose the BRCA-associated and sporadic ovarian cancer gene expression data set was analyzed with respect to the expression of 178 transcripts mapped to the X chromosome. Additional in vivo and in vitro experiments employing an X chromosome enriched cDNA microarray were also performed to further evaluate the expression patterns of X chromosome genes in a more comprehensive manner. Materials and Methods Comparison of gene expression between BRCA-linked and sporadic ovarian cancers The first part of this investigation consisted of a de novo analysis of the large publicly available data set generated by previous microarray experiments with respect to the 178 X chromosome specific genes [2]. Thus, the description of tumor samples used, BRCA1 and BRCA2 genotyping, tissue processing, and RNA extraction and amplification, and microarray technique were previously published [2]. In addition, detailed protocols describing RNA amplification and microarray hybridization methods are available at (under "Alternative Methods and Protocols"). Use of an X-chromosome enriched cDNA microarray for evaluating gene expression differences in BRCA1-associated and sporadic ovarian cancers For the second part of this investigation a recently developed cDNA microarray enriched in X chromosome transcripts was used. The developmental rationale and approach for this cDNA microarray are described in detail elsewhere [4]. The X-enriched microarray chip used in this investigation consisted of 5,296 features of which 2,879 mapped to the X chromosome. For the purposes of this investigation analysis was limited to only the X chromosome features. Since the cDNAs on this array had not been "sequence-verified" prior to spotting, the cDNA clones for all genes found to be differentially expressed between tumor samples and in cell line experiments were sequenced to ensure positive identification of the transcripts. Those transcripts for which PCR amplification did not result in a product or multiple bands were identified were eliminated from the final analysis. Adenovirus-Mediated BRCA1 expression in HCC1937 cells Tissue culture techniques and adenoviral infection of HCC1937 cells was performed as described previously [5]. Briefly, cells were plated 24 h before the infection at a density 7 x 105 cells per 100 mm plate. The cells were infected at 250 plaque-forming units per cell with adenovirus encoding full-length human BRCA1 or green fluorescent protein (GFP) cDNAs (the latter used as an irrelevant infection control). Twenty-four hours later cells were harvested and RNA was purified using Trizol Reagent (Life Technologies, Inc.) according to manufacturer's instruction. Experiments employing cDNA microarrays In studies evaluating gene expression differences between ovarian tumor samples using the X-enriched cDNA microarrays, combined RNA from 10 human cell lines (Stratagene, La Jolla, CA) was used as the reference RNA. In studies involving the HCC1937 cell line, gene expression in BRCA1 virally infected cells was directly compared to that of GFP infected controls. The logarithmic expression ratios for the spots on each array were normalized by subtracting the median logarithmic ratio for the same array. Data were filtered to exclude spots with a size of less than 25 μm, an intensity of less than two times background, or less than 300 units in both red and green channels and to exclude any poor quality or missing spots. In addition, any features found to be missing in greater than 20% of the arrays were not included in the analysis. Statistical comparison between tumor groups was performed with the "BRB Array Tools" software , consisting of a modified F test with P < 0.005 considered statistically significant. This stringent P value was selected in lieu of the Bonferroni correction for multiple comparisons, which was deemed excessively restrictive. For microarray experiments involving the HCC1937 cells infected with BRCA1 or GFP the geometric mean of BRCA1:GFP expression ratio from two separate microarray experiments was used. Genes exhibiting a mean expression ratio change of two-fold or greater were considered significant. Quantitative RT-PCR 1 μg of total RNA was reverse transcribed in 50 μl reaction and 5 μl of cDNA was then used for PCR reaction according to Applied Biosystems technical manual. Separate reaction of the same samples with β-actin was performed for normalization purposes. The difference in threshold number of cycles between the ARAF1 and β-actin was then calculated and converted into real fold difference. All measurements were done in triplicates and the results were averaged. Probes for ARAF1 and β-actin were purchased from Applied Biosystems Inc. Results It was previously shown that when compared to sporadic cancers BRCA1-associated ovarian tumors were characterized by higher mean expression levels of genes mapped to Xp11 [2]. This observation was confirmed when considering all genes mapped to the X chromosome (Fig. 1A). Eleven of the 178 X chromosome mapped transcripts were differentially expressed between the BRCA1-associated and sporadic cancers (P < 0.005). Of these 11 transcripts, six (55%) were located on Xp11. Because the sporadic tumors could be divided into subgroups with "BRCA1- and BRCA2-like" expression profiles [2], one may anticipate differences in the expression levels of genes mapped to Xp11 when comparing each of these sporadic cancer subgroups to the BRCA1-associated tumors. The expression levels of X chromosome genes in the 18 BRCA1-associated cancers was compared to those of the 13 BRCA1-like and 14 BRCA2-like sporadic tumors (Fig. 1A,1B,1C,1D). Only 4 genes showed significant differential expression between the BRCA1-associated and the sporadic tumors with a BRCA1-like molecular profile (Fig. 1B). All 4 mapped to Xp11 and showed higher mean expression in BRCA1-associated tumors. In contrast, 40 of the 178 X-chromosome transcripts were differentially expressed between the BRCA1-associated tumors and sporadic cancers with a BRCA2-like molecular profile (Fig. 1C top panel). Thirty of the 40 transcripts showed higher mean expression in the BRCA1-associated samples including all 11 genes that mapped to Xp11 (Fig. 1C top panel). These data suggest that BRCA1 may be involved in the regulation of gene expression from the X-chromosome. Furthermore, there appears to be a role for BRCA1 in suppressing the expression of several genes mapped to the Xp11 locus that were all higher expressed in BRCA1-associated tumors. This pattern of expression was observed when the BRCA1-associated samples were compared to all sporadic cancers regardless of their expression profile characterization as BRCA1- or BRCA2-like. Figure 1 X chromosome gene expression differences BRCA1-associated (B followed by a number) and sporadic (C followed by a number) ovarian cancers (P < 0.005). Genes are shown as hierarchical clusters (using centered correlation and average linkage), samples were not clustered. The red and green color intensities represent expression levels shown as standard normal deviation (Z score) values from each gene's mean expression level (represented as black) across all compared tumor samples. The symbol for each gene is followed by the I.M.A.G.E. clone number of the corresponding cDNA spotted on the array. A. Genes differentially expressed between BRCA1-associated tumors and all sporadic samples. B. Genes differentially expressed between BRCA1-associated cancers and sporadic tumors characterized as "BRCA1-like" based on gene expression profile as described in reference 2. C. Genes differentially expressed between BRCA1-associated tumors and sporadic cancers characterized as "BRCA2-like" based on gene expression profile as described in reference 2. D. An X chromosome enriched cDNA microarray was used to further investigate gene expression differences among a subset of BRCA1-associated and BRCA2-like sporadic tumors. The results of these experiments confirmed the findings observed above in (C). The significance of this differential pattern of gene expression between the BRCA1-associated and sporadic cancers is unclear at this time. However, higher expression from Xp11 may be related to the earlier age of presentation of epithelial ovarian cancers in BRCA1 mutation carriers compared to tumors in BRCA2 mutation carriers and patients with sporadic ovarian cancer [6]. This observation cannot be solely explained by an earlier occurrence of a "second hit" as modeled by Knudson's two-hit hypothesis [7] because the age of presentation of ovarian cancer in BRCA2 tumors is no different than that observed in patients with sporadic epithelial ovarian cancers [2,6], thus indicating a BRCA1-specific effect on the age of presentation. In order to confirm and expand on the above noted differences in gene expression between BRCA1-associated and sporadic tumors, an X-chromosome enriched cDNA microarray was used [4]. Due to the limited availability of resources this cDNA microarray was used to evaluate gene expression in a representative subset of 9 BRCA1-associated and 8 sporadic tumors. The sporadic tumors were selected from the subgroup with a "BRCA2-like profile" as these samples showed more robust differences in X chromosome gene expression in the above noted experiments using our conventional cDNA microarray (Fig. 1B and 1C). Twenty-one transcripts showed significant differential expression between the BRCA1-associated and sporadic tumors with a BRCA2-like profile (P < 0.005). Consistent with our earlier findings and despite the smaller number of samples used for this comparison, the majority of transcripts exhibited higher mean expression in BRCA1-associated samples including all but one of the transcripts located on Xp11 (Fig. 1D). This pattern of expression was confirmed using quantitative RT-PCR of the ARAF1 gene in a representative sample of BRCA1-associated and sporadic ovarian cancer samples (Fig. 2). Figure 2 Quantitative RT-PCR evaluation of ARAF1 expression confirms cDNA microarray data. Average of three RT-PCR repeats for each sample is shown on top left panel. ARAF1 expression levels derived by cDNA microarray relative to universal reference RNA is shown in the bottom left panel. Mean expression levels are graphed to the right; error bars represent standard error of mean. We next sought to determine if differences in X chromosome gene expression between BRCA1-associated and sporadic tumors were directly mediated by BRCA1 as oppose to other, possibly confounding, features of these tumors. The HCC1937 breast cancer cells that are either homozygous or hemizygous for the BRCA15382insC mutation were used as a model. Gene expression in HCC1937 cells following virally mediated expression of wild-type BRCA1 was compared to gene expression following viral infection of GFP which was employed as an irrelevant infection control. Prior to using the X-chromosome enriched array, the validity of this approach was tested using a 7.5 K microarray whose features included BRCA1. This preliminary experiment demonstrated that viral infection did in fact result in a 3.4 fold higher BRCA1 expression compared to the GFP control (data not shown). BRCA1 expression was also demonstrated using Western blotting which confirmed BRCA1 protein expression following viral infection (Fig. 3). Figure 3 Adenovirus-mediated BRCA1 expression in HCC1937 cells. Twenty-one X-chromosome transcripts demonstrated at least a two-fold mean expression change across two independent experiments following BRCA1 infection. It is notable that all 21 transcripts (representing 16 unigene clusters) showed decreased expression following BRCA1 transfection with a median repression of 2.4 fold. This uniform repression is particularly significant because median based normalization of logarithmic ratios was employed in microarray data analysis. Thus, the median log expression ratio for all 5,296 features on the array was adjusted to zero (corresponding to an expression ratio of 1.0). Following this median based normalization, the median log expression ratio of the transcripts mapped to the X chromosome (2,879 of the total 5,296 features on the array) was also very close to zero (median log2 ratio = 0.016 corresponding to an expression ratio of 1.01). Thus, the observed down regulation of genes does not appear to be a result of the X-enriched composition of the cDNA microarray used in these experiments nor can it be explained in terms of a global down regulation of all X chromosome transcripts. Rather, the observed repression appears to be a specific effect of BRCA1 expression on these 21 transcripts. Eleven of the 21 (52.4%) transcripts map to Xp11 and represent 8 unigene clusters (Table 1). The most highly repressed (11.6-fold) transcript was that of LOC139135 (Hs.160594) whose protein product contains a PAS domain and is weakly similar to the circadian locomoter output cycles kaput (CLOCK) protein of human and several other organisms. The PAS domain is a highly conserved motif essential for sensing changes in a variety of different environmental conditions including light, oxygen tension and redox potential [8,9]. HIF-1 alpha and EPAS-1, two other PAS containing proteins, are upregulated in a number of human tumors and play an important role in angiogenesis by stimulating VEGF expression [9-11]. LOC139135 deserves additional investigation of its role as a possible protooncogene subject to regulation by BRCA1. Table 1 X chromosome genes showing two-fold or greater change following BRCA1 expression in HCC1937 BRCA1-null cells. Name Clone ID* Locus Unigene Fold repression Comments LOC139135 C03503 Xq28 Hs.160594 11.6 Similar to CLOCK protein EST 32930 Xp11 Hs.99070 8.9 EST 34280 Xp11.2 Hs.99070 8.5 CSTF2 1705354 Xq22.1 Hs.693 5.5 EST 1535341 Xq13 Hs.444962 5.4 EST 1614299 Xq13 Hs.197801 4.3 JM11 1913391 Xp11.23 Hs.417068 3.5 ZNF6 1564783 Xq13 Hs.326801 3.0 ZNF6 5201496 Xq13 Hs.326801 2.4 LOC158572 2070337 Xp11.23 Hs.408191 2.8 LOC158572 1880263 Xp11.2 Hs.408191 2.4 LOC158572 470925 Xp11.23 Hs.408191 2.1 EST 4402168 Xq26 Hs.175894 2.6 KLF8 2148451 Xp11.21 Hs.411296 2.5 KLF8 2148451 Xp11.21 Hs.411296 2.3 EST 2516780 Xp11.23 Hs.293317 2.4 Moderately similar to PAGE-5 protein EST 2659258 Xq28 Hs.312560 2.2 ED1 2030638 Xq12-q13 Hs.105407 2.2 Weakly similar to PAGE-5 protein TIMP1 172210 Xp11.23 Hs.446641 2.1 EST 2111889 Xp11.23 Hs.163473 2.0 Weakly similar to XAGE-5 protein FLJ23614 1938584 Xq26 Hs.28780 1.9 *Integrated Molecular Analysis of Genomes and their Expression (I.M.A.G.E.) Consortium cDNA clone identification. Two transcripts representing the same unigene cluster, Hs. 99070, showed greater than 8 fold repression following BRCA1 expression in HCC1937 cells (Table 1). In addition, an EST (clone ID 32930) belonging to this same unigene cluster was found to be significantly higher expressed in BRCA1-associated ovarian cancers compared to sporadic tumors (Fig. 1D). As such this gene may be one potentially important target of BRCA1 regulation of gene expression from the X chromosome. There was no other overlap between the list of genes differentially expressed following BRCA1 expression in HCC1937 cells and the list of genes differentially expressed between BRCA1-associated and sporadic ovarian cancers. The lack of a broader overlap between the list of genes repressed following BRCA1 expression in the HCC1937 and those differentially expressed between BRCA1-associated and sporadic ovarian cancers is notable. This signifies that BRCA1's influence over transcription is unlikely to be gene specific and rather may involve more global influences over transcription such chromatin remodeling and changes in methylation states. BRCA1 expression led to the down regulation of several ESTs homologous to PAGE-5, a member of the cancer-testis antigen group of genes (MAGE, GAGE, PAGE, etc.). These ESTs are likely to represent as yet undiscovered members of this family of genes that are known for their characteristic pattern of expression, usually limited to the testes and tumors [12,13]. Intriguing parallels exist between expression characteristics of cancer testis antigens and expression changes mediated by BRCA1. The vast majority of cancer-testis antigen genes are located within discrete loci on the X chromosome [13]. Our results demonstrate that BRCA1 represses the expression of clusters of genes on Xp11, Xp21-p22, Xq13, and Xq26-q28 (Table 1), which correspond to the genomic location of several major cancer testis antigen gene clusters [12,13]. Furthermore, high expression of BRCA1 in pachytene spermatids [14,15] correlates with a significant down regulation of at least one cancer testis antigen, MAGE-B4 [16]. Finally, recent reports have documented the aberrant expression of several cancer-testis antigens in a significant portion of ovarian cancers and linked their expression to drug-resistance [17,18]. Although not completely understood, the expression of cancer testis antigens is thought to be, at least partially regulated by DNA methylation [12]. These data point to changes in DNA methylation as another possible mechanism involved in the BRCA1-mediated repression of gene clusters within the X chromosome. Further investigation of the sequence and genomic organization of genes in these loci will be useful for elucidating features responsible for the co-regulation of these genes. Discussion Whether any of these BRCA1-regulated X chromosome genes are involved in ovarian carcinogenesis and / or tumor progression remains to be determined. However, several lines of evidence support a possible connection between the X chromosome and ovarian neoplasia. First, the loss of a number of regions in the X-chromosome has been associated with ovarian agenesis or premature ovarian failure, which are commonly observed in Turner syndrome and related disorders [19,20]. Thus, the X chromosome is likely to contain genes involved in ovarian maintenance [19]. Aberrant overexpression of such potential ovarian survival/growth regulators on the X chromosome through a mechanism involving the loss of BRCA1 may be involved in ovarian carcinogenesis and/or tumor progression. Chromosome X alterations have been reported in sporadic ovarian carcinomas and borderline tumors [21,22]. Non-random X inactivation has been reported in populations of hereditary ovarian and breast cancer syndrome patients including BRCA1 mutation carriers [23,24]. Finally, in a comparison of gene expression between matched primary and recurrent chemoresistant ovarian cancer samples from the same patient XIST was the most differentially expressed gene and its expression was negatively correlated with response to paclitaxel chemotherapy [25]. Until recently, a mechanistic explanation for how BRCA1 may affect the expression of multiple genes on the X chromosome was lacking. Evidence for the existence of one such mechanism has been provided by Ganesan and colleagues who have demonstrated the co-localization and interaction between XIST and BRCA1 [3]. This interaction was shown to be sufficient and necessary to repress the expression of a green fluorescent protein transgene introduced into the inactive X chromosome. Our investigation shows that genes endogenous to the X chromosome are also repressed by BRCA1 and that genes on certain loci are preferentially affected. The exact nature of this interaction and possible differential effects on gene expression from various regions of the X chromosome remain to be determined. It is unclear whether BRCA1's effect involves changes in XIST RNA expression. Ganesan et al. did not observe such an effect, but other investigators have reported a two-fold increase in XIST RNA levels following BRCA1 expression [26]. Using an X chromosome enriched microarray that has previously been shown to be able to detect changes in XIST expression associated in X chromosome polysomies [4], no increase in XIST RNA was observed following BRCA1 expression. BRCA1 may target XIST in such a way as to bring about changes in the expression of various loci on the X chromosome. It is also possible that BRCA1 may be acting independent of XIST through a different mechanism such regulation of DNA methylation. Alterations in DNA methylation play an integral role in the normal process of X chromosome inactivation and are also involved in the characteristic expression of cancer-testis antigens most of which reside on the X chromosome as discussed above. One of the unexplained features of germ-line BRCA1 mutations is the overwhelmingly disproportionate risk of cancer in female carriers. One hypothesis put forth to explain this observation is that estrogen is the inciting event by leading to deregulated proliferation and carcinogenesis in hormonally responsive tissues [27]. An alternative, non-mutually exclusive, hypothesis is that the deregulated expression of an X-linked gene normally under BRCA1 control may play a role in predisposing women to carcinogenesis. This a plausible scenario if BRCA1 proves to be involved in the process of X-chromosome inactivation and /or gene dosage regulation for those genes on the X chromosome that do not undergo inactivation. Accordingly, the lack of a need for X chromosome inactivation and X-linked gene dose adjustment in men may explain why male BRCA1 mutation carriers do not have the same increased risk for cancers. Future studies will be aimed at testing these hypotheses. ==== Refs Hedenfalk I Duggan D Chen Y Radmacher M Bittner M Simon R Meltzer P Gusterson B Esteller M Kallioniemi OP Wilfond B Borg A Trent J Gene-expression profiles in hereditary breast cancer N Engl J Med 2001 344 539 548 11207349 10.1056/NEJM200102223440801 Jazaeri AA Yee CJ Sotiriou C Brantley KR Boyd J Liu ET Gene expression profiles of BRCA1-linked, BRCA2-linked, and sporadic ovarian cancers J Natl Cancer Inst 2002 94 990 1000 12096084 10.1093/jnci/94.13.990 Ganesan S Silver DP Greenberg RA Avni D Drapkin R Miron A Mok SC Randrianarison V Brodie S Salstrom J Rasmussen TP Klimke A Marrese C Marahrens Y Deng CX Feunteun J Livingston DM BRCA1 supports XIST RNA concentration on the inactive X chromosome Cell 2002 111 393 405 12419249 10.1016/S0092-8674(02)01052-8 Sudbrak R Wieczorek G Nuber UA Mann W Kirchner R Erdogan F Brown CJ Wohrle D Sterk P Kalscheuer VM Berger W Lehrach H Ropers HH X chromosome-specific cDNA arrays: identification of genes that escape from X-inactivation and other applications Hum Mol Genet 2001 10 77 83 11136717 10.1093/hmg/10.1.77 Aprelikova O Pace AJ Fang B Koller BH Liu ET BRCA1 is a selective co-activator of 14-3-3 sigma gene transcription in mouse embryonic stem cells J Biol Chem 2001 276 25647 25650 11384963 10.1074/jbc.C100265200 Boyd J Sonoda Y Federici MG Bogomolniy F Rhei E Maresco DL Saigo PE Almadrones LA Barakat RR Brown CL Chi DS Curtin JP Poynor EA Hoskins WJ Clinicopathologic features of BRCA-linked and sporadic ovarian cancer JAMA 2000 283 2260 2265 10807385 10.1001/jama.283.17.2260 Knudson AG Jr Mutation and cancer: statistical study of retinoblastoma Proc Natl Acad Sci USA 1971 68 820 823 5279523 Ponting CP Aravind L PAS: a multifunctional domain family comes to light Curr Biol 1997 7 R674 677 9382818 10.1016/S0960-9822(06)00352-6 Zhulin IB Taylor BL Dixon R PAS domain S-boxes in Archaea, Bacteria and sensors for oxygen and redox Trends Biochem Sci 1997 22 331 333 9301332 10.1016/S0968-0004(97)01110-9 Stassar MJ Devitt G Brosius M Rinnab L Prang J Schradin T Simon J Petersen S Kopp-Schneider A Zoller M Identification of human renal cell carcinoma associated genes by suppression subtractive hybridization Br J Cancer 2001 85 1372 1382 11720477 10.1054/bjoc.2001.2074 Sivridis E Giatromanolaki A Gatter KC Harris AL Koukourakis MI Association of hypoxia-inducible factors 1alpha and 2alpha with activated angiogenic pathways and prognosis in patients with endometrial carcinoma Cancer 2002 95 1055 1063 12209691 10.1002/cncr.10774 Kirkin AF Dzhandzhugazyan KN Zeuthen J Cancer/testis antigens: structural and immunobiological properties Cancer Invest 2002 20 222 236 11901543 10.1081/CNV-120001150 Ohman Forslund K Nordqvist K The melanoma antigen genes – any clues to their functions in normal tissues? Exp Cell Res 2001 265 185 194 11302683 10.1006/excr.2001.5173 Zabludoff SD Wright WW Harshman K Wold BJ BRCA1 mRNA is expressed highly during meiosis and spermiogenesis but not during mitosis of male germ cells Oncogene 1996 13 649 653 8760307 Blackshear PE Goldsworthy SM Foley JF McAllister KA Bennett LM Collins NK Bunch DO Brown P Wiseman RW Davis BJ Brca1 and Brca2 expression patterns in mitotic and meiotic cells of mice Oncogene 1998 16 61 68 9467943 10.1038/sj.onc.1201506 Osterlund C Tohonen V Forslund KO Nordqvist K Mage-b4, a novel melanoma antigen (MAGE) gene specifically expressed during germ cell differentiation Cancer Res 2000 60 1054 1061 10706124 Yakirevich E Sabo E Lavie O Mazareb S Spagnoli GC Resnick MB Expression of the MAGE-A4 and NY-ESO-1 cancer-testis antigens in serous ovarian neoplasms Clin Cancer Res 2003 9 6453 6460 14695148 Duan Z Duan Y Lamendola DE Yusuf RZ Naeem R Penson RT Seiden MV Overexpression of MAGE/GAGE genes in paclitaxel/doxorubicin-resistant human cancer cell lines Clin Cancer Res 2003 9 2778 2785 12855658 Simpson JL Rajkovic A Ovarian differentiation and gonadal failure Am J Med Genet 1999 89 186 200 10727994 10.1002/(SICI)1096-8628(19991229)89:4<186::AID-AJMG3>3.0.CO;2-5 Zinn AR Tonk VS Chen Z Flejter WL Gardner HA Guerra R Kushner H Schwartz S Sybert VP Van Dyke DL Ross JL Evidence for a Turner syndrome locus or loci at Xp11.2-p22.1 Am J Hum Genet 1998 63 1757 1766 9837829 10.1086/302152 Evans MF McDicken IW Herrington CS Numerical abnormalities of chromosomes 1, 11, 17, and X are associated with stromal invasion in serous and mucinous epithelial ovarian tumours J Pathol 1999 189 53 59 10451488 10.1002/(SICI)1096-9896(199909)189:1<53::AID-PATH393>3.0.CO;2-U Hu J Khanna V Jones MM Surti U Genomic imbalances in ovarian borderline serous and mucinous tumors Cancer Genet Cytogenet 2002 139 18 23 12547152 10.1016/S0165-4608(02)00603-9 Buller RE Sood AK Lallas T Buekers T Skilling JS Association between nonrandom X-chromosome inactivation and BRCA1 mutation in germline DNA of patients with ovarian cancer J Natl Cancer Inst 1999 91 339 346 10050867 10.1093/jnci/91.4.339 Kristiansen M Langerod A Knudsen GP Weber BL Borresen-Dale A-L Orstavik KH High frequency of skewed X inactivation in young breast cancer patients J Med Genet 2002 39 30 33 11826021 10.1136/jmg.39.1.30 Huang KC Rao PH Lau CC Heard E Ng SK Brown C Mok SC Berkowitz RS Ng SW Relationship of XIST expression and responses of ovarian cancer to chemotherapy Mol Cancer Ther 2002 1 769 776 12492109 Welcsh PL Lee MK Gonzalez-Hernandez RM Black DJ Mahadevappa M Swisher EM Warrington JA King MC BRCA1 transcriptionally regulates genes involved in breast tumorigenesis Proc Natl Acad Sci USA 2002 99 7560 7565 12032322 10.1073/pnas.062181799 Welcsh PL King MC BRCA1 and BRCA2 and the genetics of breast and ovarian cancer Hum Mol Genet 2001 10 705 713 11257103 10.1093/hmg/10.7.705

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J Transl Med. 2004 Sep 21; 2:32

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J Transl Med

10.1186/1479-5876-2-32

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==== Front J Circadian RhythmsJournal of Circadian Rhythms1740-3391BioMed Central London 1740-3391-2-51534742210.1186/1740-3391-2-5ResearchDaily rhythms in plasma levels of homocysteine Lavie Lena [email protected] Peretz [email protected] Unit of Anatomy and Cell Biology, Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa 31096, Israel2004 3 9 2004 2 5 5 24 4 2004 3 9 2004 Copyright © 2004 Lavie and Lavie; licensee BioMed Central Ltd.2004Lavie and Lavie; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background There is accumulated evidence that plasma concentration of the sulfur-containing amino-acid homocysteine (Hcy) is a prognostic marker for cardiovascular morbidity and mortality. Both fasting levels of Hcy and post methionine loading levels are used as prognostic markers. The aim of the present study was to investigate the existence of a daily rhythm in plasma Hcy under strictly controlled nutritional and sleep-wake conditions. We also investigated if the time during which methionine loading is performed, i.e., morning or evening, had a different effect on the resultant plasma Hcy concentration. Methods Six healthy men aged 23–26 years participated in 4 experiments. In the first and second experiments, the daily rhythm in Hcy as well as in other amino acids was investigated under a normal or an inverse sleep-wake cycle. In the third and fourth, Hcy concentrations were investigated after a morning and evening methionine loading. To standardize food consumption in the first two experiments, subjects received every 3 hours 150 ml of specially designed low-protein liquid food (Ensure® formula). Results In both the first and second experiments there was a significant daily rhythm in Hcy concentrations with a mid-day nadir and a nocturnal peak. Strikingly different 24-h patterns were observed in methionine, leucine, isoleucine and tyrosine. In all, the 24-h curves revealed a strong influence of both the sleep-wake cycle and the feeding schedule. Methionine loading resulted in increased plasma Hcy levels during both morning and evening experiments, which were not significantly different from each other. Conclusions There is a daily rhythm in plasma concentration of the amino acid Hcy, and this rhythm is independent of sleep-wake and food consumption. In view of the fact that increased Hcy concentrations may be associated with increased cardiovascular risks, these findings may have clinical implications for the health of rotating shift workers. ==== Body Background Experimental results accumulated in recent years have revealed that plasma concentration of the sulfur-containing amino-acid homocysteine (Hcy) is a prognostic marker for cardiovascular morbidity and mortality [1-5]. Plasma concentrations of Hcy in excess of 15 μmol/L under fasting conditions were associated with increased risk of cardiovascular mortality [6]. Furthermore, some patients having normal fasting levels of plasma Hcy were shown to have abnormally high levels of Hcy after methionine loading [7]. In most epidemiological studies, the differences between fasting concentrations of Hcy of cardiovascular patients and normal controls did not amount to more than 10–15%. Studies conducted during the 1960s have demonstrated that plasma levels of several amino acids vary in a daily manner. Feigin, Klainer and Beisel [8] were the first to report on daily rhythms in serum levels of total amino acids in adult men. The peak levels of the total integrated amino acids occured between 1200 and 2000 with a minimum level at 0400. Wurtman, Chou and Rose [9] reported on a daily rhythm in plasma concentration of tyrosine with a nocturnal nadir and a morning peak, which represented a two-fold increase in plasma tyrosine level. This rhythm persisted when subjects were maintained on a two-week low protein diet. Subsequently, the same group [10] extended their findings to 15 additional amino acids. Tyrosine, tryptophan, phenylalanine, methionine, cysteine, and isoleucine, underwent the greatest daily changes while alanine, glycine and glutamic acid showed the least. Hussein et al [11] reported that the daily fluctuations of plasma free amino acids were significantly affected by the dietary conditions. In none of these studies, however, were the levels of amino acids determined during the sleep period or under uniform dietary conditions. More recently, plasma Hcy levels were also shown to vary in a daily manner in humans with an evening peak and a morning nadir [12]. Significant daily rhythmicity was found in obese diabetic patients but not in normal controls. Since plasma samples were obtained every 3 hours and no attempt was made to examine how sleep affected the pattern of secretion, it is difficult to determine whether these findings bear any clinical significance. In rats, plasma Hcy demonstrated a 24-h rhythm with a nocturnal peak and a daytime nadir. Pinealectomy did not change the phase of the rhythm or its nocturnal elevation, but it did significantly increase mean plasma Hcy [13]. In the present study, we further investigated the possible existence of a daily rhythm in plasma Hcy under strictly controlled nutritional and sleep-wake conditions. We also investigated if the time during which methionine loading is performed, i.e., morning or evening, had a different effect on the resultant plasma Hcy concentration. Methods Subjects Six healthy men aged 23–26 years participated in 4 experiments. All were students who maintained a normal and regular sleep-wake cycle for at least three months prior to the studies. They were screened to ensure an adequate state of health by physical examination, detailed medical history and blood testing. All had a normal body weight (mean body mass index (BMI) = 23.5 ± 1.6 Kg/m2). They were instructed to avoid alcohol and coffee beverages during the 24 hours that preceded each of the experimental periods. The study was approved by the local Human Ethics Committee, and subjects gave written informed conset before being enrolled in the first experiment. Subjects were paid for their participation. Procedure In the first and second experiments, daily rhythms in Hcy as well as in other amino acids were investigated under a normal or an inverse sleep-wake cycle. In the third and fourth experiments, Hcy concentrations were investigated after morning and evening methionine loading. Experiment 1 Subjcts were admitted to the laboratory at 1800 for a period of 24 hours, after having a normal day. A catheter was inserted into an antecubital vein and was kept patent by a drip of saline. Electrodes were attached for polysomnographic monitoring to determine sleep stages. These included EEG, EMG, EOG, respiration by respiratpry belt and nasal thermistor, and oximetry. Starting at 1900, 5-ml blood samples were drawn every hour until 1900 on the next day. Thoughout this period subjects were either in a supine or a sitting position in individual rooms where they could read, use their personal computers or watch television. From 2300 to 0800 the room lights were turned off during the sleep period. Blood samples were collected into EDTA treated tubes, immediately centrifuged at 4°C, and plasma was stored at -70°C until assay. Hourly blood sampling during sleep continued with minimal disturbance to subjects' sleep. To standardize food consumption and to provide adequate energy intake, subjects received every 3 hours 150 ml of specially designed liquid food (Ensure® formula) with the following composition: proteins (5.49 g, 84% caseinate, 16% soy – 14.7% of the calories), fat (5.3 g, 32% of calories), carbohydrates (20 g, 77% corn syrup, 23% sucrose, 53.3% of calories), vitamins and minerals, in 77 ml water. No other food except for water was allowed. Experiment 2 Thes second experiment was identical to Experiment 1 except for the fact that the sleep period was delayed from 2300-0800 to 0720-1500. As before, subjects were admitted to the laboratory at 1800 and blood was withdrawn every hour starting at 1900 until 1900 on the next day. Sleep was monitored polygraphically as described before. Food was provided as in Experiment 1. Experiments 3 and 4 In these experiments, we conducted a methionine loading test at two times: 0900 and 2100. The selection of these times was based on the results of the first two experiments that demonstrated a daily nadir and a nocturnal peak in Hcy levels (see below). At the start of each methionine loading test, subjects were administered 100 mg/kg body weight methionine, mixed in fruit juice. Blood samples (5 ml) were taken into EDTA treated tubes before methionine loading, designated as time 0, and then at +2, +4, +6 and +8 hours after methionine administration. Light carbohydrate rich meals were provided at +1 and +6 hours after the methionine loading in each of the test periods. Measurement of amino acids and vitamins Plasma amino acids levels (Hcy, methionine, leucine, isoleucine and tyrosine) were measured in duplicates using a Biochrom 20 Amino-Acid analyzer (Pharmacia Biothech, Cambridge, UK) as described before [5]. The mean intra-assay CV was less than 3%. All samples from a single individual were analysed in a single run. In view of their involvment in Hcy metabolism, serum levels of folic acid and vitamin B12 were also measured in all samples of all subjects using commercially available kits from Abbott. The assays were performed on an Abbott IMX analyzer that utilizes ion capture technology for folate determination and microparticle enzyme immunoassay (MEIA) technology for B12. The assays were performed according to the manufacturers' instructions and used quality control sera supplied by Abbott. Statistical analysis Repeated measurements ANOVA was used to compare the means of the amino acids between the first two experiments. To obtain the average 24-h Hcy curves, each individual data point was replaced by a z-transformation based on the individual 24-h mean and standard deviation, before averaging across subjects. Then, each of the individual time series was subjected to Cosinor analysis to determine its amplitude and acrophase. Since Experiment 1 was perfomed during the summer (August) and Experiment 2 was performed during early winter (late November), approximately 2 months after the change from Summer daylight-saving time to Winter time, during which the clock in Israel was advanced by one hour, the 24-h curves of the first experiment were advanced by 1 hour before the analysis. Then repeated measurements ANOVA was used to determine differences in acrophase between the experiments. In the third and fourth experimens, the concentrations of Hcy at times 0, 2, 4, 6, and 8 hours after methionine loading were analysed by repeated measures ANOVA to determine if there were any significant morning-evening differences in Hcy levels. Results All subjects successfully completed the four experiments. In experiment 1 when they slept from 2300 to 0700, average sleep latency was 22.2 ± 7.3 min, total sleep time was 407.3 ± 51.8 min, and sleep efficiency was 77.7 ± 9.2%. In experiment 2 when they slept from 0720 to 1500, average sleep latency was 4 ± 3.1 min, total sleep time was 371.5 ± 59.4 min, and sleep efficiency was 83.6 ± 12.2%. In spite of the reversal of the sleep-wake cycle, the 24 h means and coefficients of variation of Hcy in the two experiments were very similar to each other, 8.82 μmol/L and 29.7% and 8.51 μmol/L and 27.7%, in experiments 1 and 2, respectively. None of the subjects had abnormal Hcy levels (>15 μmol/L) at any point across the 24 hours. Figure 1 presents the average z-transformed 24-h curves of Hcy in the two experiments. In spite of the reversal of the sleep-wake cycle, the 24-h pattern of Hcy was remarkeably similar. In both experiments there was a midday nadir and a nocturnal peak in Hcy levels. In absolute terms, the daily rhythm in Hcy represents a change from nadir to peak values of 6.7 to 9.83 μmol/L (46.7%) and 7.4 to 10.55 μmol/L (42.6%), in experiments 1 and 2, respectively. Analysis of variance showed no significant difference in the average amplitude of the z-transformed rhythms of the two experiments, as determined by the cosinor analysis: 0.81 ± 0.19, and 1.07 ± 0.22 μmol/L, for experiment 1 and 2, respectively. There was, however, a significant difference between the timing of the average acrophase which was earlier by approximately 2 hours in experiment 1 than in experiment 2 (22:47 ± 0:45 vs. 0:54 ± 1:14, t = 3.77; p < .01). Figure 1 Daily rhythms in plasma concentration of Homocysteine. Rhythms were measured in 6 subjects who slept from 23:00 to 07:00 (Night sleep) or from 07:20 to 15:00 (Day sleep). Blood was withdrawn every hour starting at 19:00 until 19:00 the next day. Individual data points were transformed to Z-scores before averaging across subjects. For clarity purposes standard errors of data points are not presented. Magnitude of standard errors was approximatly 10% of mean values. Strikingly different 24-h patterns were observed for the other amino acids: methionine, leucine, isoleucine and tyrosine. In all, the average z-transformed 24-h curves revealed a strong influence of both the sleep-wake cycle and the feeding schedule. Their level was notably lower during the sleep period, regardless of its timing, and increased every two hours in synchrony with the times of feeding. This pattern is exemplified in Figure 2 for methionine. Identical patterns were observed for leucine, isoleucine and tyrosine (data not shown). Figure 2 Daily rhythms in plasma concentration of methionine. Rhythms were measured in 6 subjects who slept from 23:00 to 07:00 (Night sleep) or from 0720 to 1500 (Day sleep). Blood was withdrawn every hour starting at 19:00 until 19:00 the next day. Individual data points were transformed to Z scores before averaging across subjects. For clarity purposes standard errors of data points are not presented. Magnitude of standard errors was approximatly 10% of mean values. Note the large pulses in methionine concentrations that appeared in synchrony with the times of feeding. We did not find any evidence for rhythmicity in the concentrations of B12 and folic acid. While folic acid showed a linear increase throughout the study period, the 24-h pattern of B12 was rather constant with slight elevation during the night time (data not shown). Methionine loading As expected, methionine loading resulted in increased plasma Hcy levels during both morning and evening experiments (Figure 3). Analysis of variance did not reveal overall significant differences between morning and evening post-methionine Hcy levels. However, inspection of Hcy levels at each of the time points separately revealed some interesting trends. Before methionine loading, as could be expected from the daily rhythm in Hcy found in experiments 1 and 2, morning Hcy level tended to be lower by 1.18 μmol/L than the evening level (p < .11, paired t-test, two tailed). Moreover, the increase in Hcy from time 0 to 2 hours after loading was greater by a mean of 2.8 μmol/L in the evening than in the morning (p < .09, paired t-test, two tailed). This resulted in evening and morning levels of Hcy of 26.66 and 23.86 μmol/L, respectively. These differences became much smaller at +4, +6 and +8 after the loading. Figure 3 Plasma concentration of homocysteine before and after methionine loading. Shown are the means and standard deviations of plasma concentration of homocysteine in 6 subjects before (0 hr) and 2, 4, 6 and 8 hours after methionine loading at 09:00 and 21:00. Discussion The present study demonstrated that under strictly controlled dietary conditions plasma levels of Hcy shows significant daily rhythmicity, which is independent of the 24-h cycle of sleep and wake, with a peak at around 2200 to 2400. Previously, similar rhythmicity in Hcy with an evening peak was reported in obese diabetic patients by Bremner et al [12] and with nocturnal peak in rats by Baydas et al [13]. We further extended these findings by demonstrating that daily rhythms exist also in normal young adults. In contrast to Hcy, there was no daily rhythmicity in methionine, leucine, isoleucine and tyrosine, in which the 24-h pattern followed both the timing of sleep and the feeding schedule. Homocysteine is a non-protein sulfur containing amino acid, and an intermediate in the metabolism of the essential amino acid methionine. The metabolism of Hcy is accomplished by two major pathways, remethylation into methionine and transsulfuration to cystationine [14]. In remethylation, Hcy acquires a methyl group from N-5-methyltetrahydrofolate or from betaine to form methionine. The reaction with N-5-methyltetrahydrofolate is vitamin B12 dependent while the reaction with betaine is not. In the transsulforation pathway, Hcy condenses with serine to form cystationine in an irreversible reaction catalyzed by the pyridoxal-5'-phosphate (PLP)-containing enzyme, cystationine beta synthase. Although we do not have any information as yet on the underlying mechanism responsible for the daily rhythm in plasma Hcy, it is most probably related to the balance between its rates of production and disposal. A high Hcy concentration could be due to an elevated production rate, a decreased rate of transsulforation, a decreased rate of remethylation to methionine, or any combination of these processes. The fact that the range of the daily variations in the plasma levels of Hcy is on the same order of magnitude as those seen in mild hyperhomocysteinemia, may suggest that the two phenomena share a common underlying mechanism. Mild hyperhomocystenemia seen under fasting conditions is due to mild impairement in the methylation pathway. This may be caused by folate or B12 deficiencies, or by methylenetetrahydrofolate reductase thermolability. The variations in plasma vitamin concentrations, however, could not provide an explanation for the daily rhythms in Hcy. The 24-pattern of folate levels showed a linear increase from the beginning to the end of the study. Although the plasma concentrations of vitamin B12 varied across the 24 hours – in contrast however to what was expected if B12 were involved in the daily rhythm in Hcy, ie, increasing levels of B12 associated with decreasing levels of Hcy – the 24-h pattern in B12 was parallel to that of Hcy with a daytime nadir and a night time peak. Thus, it is unlikely that a daily rhythm in plasma vitamin concentrations can explain the daily rhythm in Hcy. The methionine loading test has been used to test the individual's ability to dispose of methionine through the transsulforation pathway [14]. The fact that the differences between Hcy levels after morning and evening methionine loading were rather small and limited to the first 2 hours after the loading may indicate that the transsulforation pathway does not play a role in generating Hcy rhythmicity. A different possibility that cannot be ruled out at this point is the involvement of the Hcy cellular export mechanism. The small amount of plasma Hcy is the result of a cellular export mechanism that is essential for keeping intracellular concentrations low to avoid potentially Hcy cytotoxic effects. Thus the daily rhythm in plasma Hcy may reflect variations in the activity of the cellular export mechanism, which result in varying levels of Hcy disposed to the plasma at different phases of the 24 hours rather than in its rate of metabolism. Further studies are needed to test this possibility. Finally, what may be the clinical implications of the present findings? We would like to suggest that the existence of a daily rhythm in Hcy concentration may have possible health-related consequences to shift workers, who were shown to be at an increased cardiovascular risk [15]. Firstly, reversing the meals' schedule to a nocturnal orientation such that the time of major meal coincides with the time of the physiological peak of Hcy may have at least transient cardiovascular consequences. It was shown that an increase in Hcy concentration rapidly induces impaired elasticity of the coronary microvascular and central arterial circulation [16,17], conditions predictive of increased cardiovascular events rate [18]. Furthermore, even small physiological increments in Hcy concentration, induced by low-dose methionine or dietary animal protein meals that are more relevant to shift workers, induce a dose-related graded impairement in endothelial functioning [19]. Thus, consuming methionine or animal-protein-rich foods during the middle of the night may result in a greater risk of severe transient impairment in endothelial function than when a similar meal is consumed at the habitual lunch time during the day. Although we did not find significant differences in Hcy concentrations after methioning loading at 0900 and 2100, as expected, morning levels tended to be lower, and the initial increase in Hcy during the first 2 hours after loading was greater by a mean of 2.8 μmol/L in the evening than in the morning. This difference bordered on statistical significance. It is possible that, had we performed the methinine loading closer to the time of the nocturnal peak in Hcy, between 10 PM and midnight, this day-night difference would have been larger. Secondly, we do not know how the desynchronization between the circadian system and the enviornment which occurs in rotating shift workers may affect the rhythm in Hcy concentrations and its overall plasma concentration. Recently, Martins et al [20] reported that long-haul bus drivers working shifts had higher concentrations of Hcy than a control group of day workers. In a study just completed in our laboratory we found that rotating shift workers who complained of disturbed sleep had significantly higher concentrations of Hcy than permanent day workers, or shift workers without sleep disturbances (paper submitted to press). Furthermore, life-style related factors like smoking and heavy coffee consumption that were shown to be associated with increased Hcy concentration [21,22], are more prevalent among shift workers than among day workers [23], and may also contribute to increased Hcy concentration. Of note, decreasing levels of melatonin induced by pinealectomy in rats were reported to be associated with increased plasma concentrations of Hcy, while treatment with exogenous melatonin restored it to basal concentrations [24]. Thus, suppression of melatonin by bright light during night work may be also associated with increased Hcy concentration. In view of the fact that Hcy is a risk factor for cardiovascular morbidity, more research is needed on the possible role of hyperhomocysteinemia as a cardiovascular risk factor in shift workers. Conclusions Our results demonstrated a daily rhythm in plasma concentrations of Hcy with a nocturnal peak that was independent of sleep-wake cycle and food consumption. There were no comparable rhythms in the concentrations of methionine, leucine, isoleucine and tyrosine, nor in the concentrations of B12 and folic acid. Methionine loading at 9 AM and 9 PM produced a comparable time-dependent increase in Hcy concentrations with a tendency toward a higher increase in the evening during the first 2 hours after loading. In view of the possible involvement of Hcy in cardiovascular morbidity, and of the increased cardiovascular morbidity in shift wokers, these findings may have implications to shift workers health. List of abbreviations Hcy – homocysteine EEG – Electroencephalography EMG – electromyography EOG – electrooculography EDTA – ethylanediaminetetraacetic acid CV – coefficient of variation ANOVA – analysis of variance Competing interests None declared. Author's contribution PL and LL co-designed the study, supervised the data collection and data analysis and wrote the paper. Acknowledgements The authors are grateful to Aya Hefetz, Ziva Tzabary and Faten Barbara who help in different stages of the data collection. This study was supported by a grant to PL and LL from the Division of Labor Inspection, Ministry of Industry, Trade and Labor. ==== Refs McCully KS Vascular pathology of homocysteinemia: implications for the pathogenesis of arteriosclerosis Am J Pathol 1969 56 111 128 5792556 Mayer EL Jacobson DW Robinson K Homocysteine and coronary atherosclerosis J Am Coll Cardiol 1996 27 517 527 8606260 10.1016/0735-1097(95)00508-0 Stampfer MJ Malinow MR Willett WC Newcomer LM Upson B Ullmann D Tishler PV Hennekens CH A prospective study of plasma homocysteine and risk of myocardial infarction in US Physicians JAMA 1992 268 877 881 1640615 10.1001/jama.268.7.877 Graham IM Daly EL Refsum HM Plasma homocysteine as a risk factor for vascular disease; The European concerted action project JAMA 1997 277 1775 1781 9178790 10.1001/jama.277.22.1775 Lavie L Perelman A Lavie P Plasma homocysteine levels in obstructive sleep apnea: association with cardiovascular morbidity Chest 2001 120 900 908 11555527 10.1378/chest.120.3.900 Nygard O Nordrehaug JE Refsum H Ueland PM Farstad M Vollset SE Plasma homocysteine levels and mortality in patients with coronary artery disease N Engl J Med 1997 337 230 236 9227928 10.1056/NEJM199707243370403 Van der Griend R Haas FJ Duran M Biesma DH Meuwissen OJ Banga JD Methionine loading test is necessary for detection of hyperhomocysteinemia J Lab Clin Med 1998 132 67 72 9665374 10.1016/S0022-2143(98)90027-0 Feigin RD Klainer AS Beisel WR Circadian periodicity of blood amino-acids in adult men Nature 1967 215 512 514 6057913 Wurtman RJ Chou C Rose CM Daily rhythm in tyrosine concentration in human plasma: persistence on low-protein diets Science 1967 158 660 662 4383136 Wurtman RJ Rose CM Chou C Larin FF Daily rhythms in the concentrations of various amino acids in human plasma N Engl J Med 1968 279 171 175 5658678 Hussein MA Young VR Murray E Scrimshaw NS Daily fluctuation of plasma amino acid levels in adult men: effect of dietary tryptophan intake and distribution of meals J Nutr 1971 10 61 69 5540526 Bremner WF Holmes EW Kanabrocki EL Hermida RC Ayala D Garbincius J Third JL Ryan MD Johnson M Foley S Shirazi P Nemchausky BA Scheving LE Circadian rhythm of serum total homocysteine in men Am J Cardiol 2000 86 1153 1156 11074221 10.1016/S0002-9149(00)01181-4 Baydas G Gursu FM Cikim G Canpolat S Yasar A Canatan H Kelestimur H Effects of pinealectomy on the levels and the circadian rhythm of plasma homocytsteine in rats J Pineal Res 2002 33 151 155 12220329 10.1034/j.1600-079X.2002.02901.x Selhub J Homocysteine metabolism Annu Rev Nutr 1999 19 217 246 10448523 10.1146/annurev.nutr.19.1.217 Knutsson A Boggild H Shiftwork and cardiovascular disease: review of disease mechanisms Rev Environ Health 2000 15 359 372 11199246 Nestel PJ Chronopoulos A Cehun M Arterial stiffness is rapidly induced by raising the plasma homocysteine concentration with methionine Atherosclerosis 2003 171 83 86 14642409 10.1016/j.atherosclerosis.2003.08.003 Tawakol A Forgione MA Stuehlinger M Alpert NM Cooke JP Loscalzo J Fischman AJ Creager MA Gewirtz H Homocysteine impairs coronary microvascular dilator function in humans J Am Coll Cardiol 2002 40 1051 1058 12354427 10.1016/S0735-1097(02)02069-7 Meaume S Rodnichi A Lynch A Aortic pulse wave velocity as a marker of cardiovascular disease in subjects over 70 years old J Hypertens 2001 19 871 877 11393669 10.1097/00004872-200105000-00006 Chambers JC Obeid OA Kooner JS Physiological increments in plasma homocysteine induce vascular endothelial dysfunction in normal human subjects Arterioscler Thromb Vasc Biol 1999 19 2922 2927 10591670 Martins PJF D'Almeida V Vergani N Perez ABA Tufik S Increased plasma homocysteine levels in shift working bus drivers Occup Environ Med 2003 60 662 666 12937187 10.1136/oem.60.9.662 Panagiotakos DB Pitsavos C Zeimbekis A Chrysohoou C Stefanadis C The association between lifestyle-related factors and plasma homocysteine levels in healthy individuals from the "ATTICA" study Intern J Cardiol 2004 Urgert R van Vliet T Zack PL Katan M Heavy coffee consumption and plasma homocysteine: a randomized controlled trial in healthy volunteers Am J Clin Nutr 2000 72 1107 1110 11063436 Boggild H knutsson A Shift work, risk factors and cardiovascular disease Scand J Work Environ Health 1999 25 85 99 10360463 Baydas G Gursu MF Cikim G Canatan H Homocysteine levels are increased due to lack of melatonin in pinealectomized rats: is there a link between melatonin and Homocysteine? J Pineal Res 2002 32 63 64 11841603 10.1034/j.1600-079X.2002.01856.x

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J Circadian Rhythms. 2004 Sep 3; 2:5

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J Circadian Rhythms

10.1186/1740-3391-2-5

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==== Front Malar JMalaria Journal1475-2875BioMed Central London 1475-2875-3-321535020610.1186/1475-2875-3-32ResearchA weather-driven model of malaria transmission Hoshen Moshe B [email protected] Andrew P [email protected] Virtual Population Laboratory, Department of Physics, University of Liverpool, Liverpool L69 7ZE, UK2 Department of Geography, University of Liverpool, P.O. Box 147, Liverpool, L69 3BX, UK2004 6 9 2004 3 32 32 4 3 2004 6 9 2004 Copyright © 2004 Hoshen and Morse; licensee BioMed Central Ltd.2004Hoshen and Morse; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Climate is a major driving force behind malaria transmission and climate data are often used to account for the spatial, seasonal and interannual variation in malaria transmission. Methods This paper describes a mathematical-biological model of the parasite dynamics, comprising both the weather-dependent within-vector stages and the weather-independent within-host stages. Results Numerical evaluations of the model in both time and space show that it qualitatively reconstructs the prevalence of infection. Conclusion A process-based modelling structure has been developed that may be suitable for the simulation of malaria forecasts based on seasonal weather forecasts. ==== Body Background The importance of climate as a driving force of malaria transmission has been known since the earliest days of research on this devastating parasitic disease. However, it is only with the advent of effective weather forecasting techniques that this knowledge may be implemented numerically. Seasonal climate forecasting (with up to six months lead time) has developed rapidly in recent years with a number of atmospheric climate modelling groups showing evidence of skill and reliability in their systems. Because of the chaotic nature of the atmosphere, seasonal forecasts are necessarily probabilistic. These probabilistic predictions are derived from multiple integrations of deterministic climate models. These models successfully predicted the onset and demise of the 1997/1998 El Nino event and its impact on weather in Africa [1]. That event in East Africa was associated with devastating malaria epidemics[2] and, consequently, the health community has shown an increasing interest in the use of seasonal forecasts for predicting epidemics of climate related diseases[3]. The DEMETER project was aimed to advance the concept of seasonal climate forecasts based on multi-model ensembles. The DEMETER coupled models and the DEMETER retrospective forecast (hindcast) integrations are described elsewhere [4]. The European Centre for Medium Range Weather Forecasting (ECMWF) second-generation global weather re-analysis data set ERA-40, is being used to test the accuracy ("skill") of the hindcasts. Central to the DEMETER project is an evaluation of the potential of seasonal climate forecasts for end-user communities, such as those concerned with agricultural output and malaria epidemic control[5]. ERA is thus being used as the "gold standard" for the weather forecasts, and in the research presented here is being used as a daily weather database for all Africa. The aim of the MALSAT group at the School of Tropical Medicine along with the Department of Geography of the University of Liverpool was the assessment of the methodological issues raised by driving a dynamic malaria model with seasonal climate forecasts. In this paper, the first phase is presented, namely the formulation of the model and the development of a dynamic mathematical model of malaria transmission, which can be driven by daily meteorological variables (rainfall and temperature, as provided by ERA-40). Additional research assessed the future risk of malaria epidemics in probabilistic terms[4]. Mathematical models of malaria transmission Mathematical models of malaria span nearly a century and are well established. Macdonald[6] reformulated the pioneering model of Ross[7] and identified mosquito vector longevity as the single most important variable in the force of transmission. Further modelling work established vectorial capacity as a practical means of assessing the effectiveness of control measures aimed at the vector[8] and many refinements in modelling technique have since been applied[9,10]. However, these models have, until recently, been dependent on the unrealistic assumption of quasi-static vector numbers[8] and unvarying parasite development rates. Where variation in mosquito numbers has been introduced [11], this was achieved using pseudo-climate, a seasonal variation in mosquito numbers, but not involving variations in vector and parasite development rates, and definitely not in relation to the climate that is actually experienced, i.e. changing weather[12]. In a new report[13], varying mosquito biting frequencies were indeed simulated, but not the co-varying mosquito and parasite weather dependent dynamics. Climate (as distinct from weather) models of malaria transmission have been developed in recent years to improve our understanding of the likely impact of climate change on malaria transmission. For example, Craig et al. [14] developed a fuzzy-logic climate-based distribution model which they suggest could be used to look at the impact of climate change on malaria transmission and, combined with population, morbidity and mortality data, to estimate the burden of disease and aid strategic control of malaria. Lindsay and Birley[15] used a simple mathematical model to look at the effects of temperature on the ability of Anopheles maculipennis to transmit Plasmodium vivax malaria. Martens [16] used a rules-based modelling approach to examine how climate change might affect global malaria transmission. Lindsay and Martens[17] used a similar model to look at the implications of climate change scenarios on highland malaria in Africa and, more specifically, in Zimbabwe. Hay et al. [18] analysed the potential effects of climate change on highland malaria, using a regression approach, and Rogers and Randolph[19] used a statistical model to determine that the global impact of climate change on malaria distribution will be minimal. The relationships identified and applied in the body of research on climate change and malaria transmission highlight the possibility of explicitly relating malaria transmission both spatially and temporally to climatic variables such as temperature, rainfall and (less clearly) humidity. It is, therefore, possible to use these relationships to drive the currently available models of malaria transmission, although, to-date, none of these models are designed to indicate temporal changes in transmission dynamics based on weather[20]. In order to be able to predict within-season and between-year variation in weather-related malaria risk, the model must be driven by varying weather. This paper describes in detail the development of a weather-driven dynamic mathematical malaria model, the final output of which is new infections in the human host. Preliminary results of its numerical evaluation in time and space are presented. The choice of a causal mathematical model rather than a statistical model is based on the knowledge that the former is better suited to extrapolations to novel situations (e.g. when interventions are introduced), and for investigating the non-linear impacts of short-lived changes in driving meteorological determinants. The present malaria model is designed to be used for two distinct but related functions (a) to determine the impact of weather variables on model output (malaria cases/infection) for given interventions and (b) to determine the impact of specific control interventions on model outputs by modifying model parameters. For the former function, the model can be driven with meteorological variables, from ground-based observations, satellite or modelled weather data, seasonal climate forecasts (and potentially) climate change scenarios. In the second function, the value of a malaria early warning system (MEWS) in terms of triggering earlier or scaling up intervention efforts (e.g. residual spraying) in epidemic years may be assessed. In both cases, the biological processes are included as a series of interlinked sub-models and thus represented as coupled delay differential equations. Each new item of knowledge (such as improvements in the structure of the dynamic equations, due to novel experimental or field data) may be immediately used in the model. New developments such as, for example, the ominous spread of drug resistance may also be immediately incorporated into the model, as, in this example, a reduction in the parasite clearance rate. The implications of all such changes may then be assessed as a quantitative amendment to the prediction. This is done independently for each location and, therefore, the model can be fitted to local conditions, where relevant data are available, or to regional parameters when such data are lacking. Sensitivity analysis can also be used to establish the relative importance of obtaining more accurate data on each parameter Thus, central to the analysis is the development of a new weather-dependent mathematical dynamic model which no longer attempts to calculate a single constant epidemiological per-case multiplication rate, but follows the temporal progress of the prevalence of infection within a population through seasons and years, To simulate the stochastic elements of the model, delay differential equations based on probabilistic transition between groups rather than on Monte Carlo modelling, as has been undertaken by Gu et al. [13], have been formulated.. Methods Data-source The ERA-40 weather reanalysis data set was chosen because it is the reference data for DEMETER and can provide daily estimates of a range of potentially significant weather variables for the whole globe. This data set was prepared by ECMWF and consists of weather reanalysis data for the whole globe for 40 years (1960–2000) and builds on previous reanalysis data ERA-15 which had been used in an earlier analysis of multi-model ensembles seasonal forecasts . These three- to six-hourly data are stored on the ECMWF site and extracted by local software at the site by the user. Weather variables were extracted from the database as gridded data at one degree (~111 km.) and twelve-hour resolution for the African region. Ideally, daily averaged temperature, accumulated rainfall and humidity would have been the variables. However, reanalysis-based daily average temperature correlated poorly with station data (which is more representative of typical local conditions), and daily minimum temperature proved even worse, and humidity too was poorly modelled. Thus, the analysis used surface (2 m above ground level) daily maximum temperature, offset by -5°C (to roughly represent mean temperature) and total rainfall estimates as input variables. As accumulated rainfall (puddles etc.) is more important than daily rainfall, Rd (dekadal rainfall), the sum of the previous 10 days of rainfall., were used. There are four malaria parasites (Plasmodium spp.), which cause disease in humans. The focus here is on Plasmodium falciparum, as it is the principal life-threatening parasite species and is most common in Africa. The temporal resolution of the model was based on the nocturnal activity of the vector and the fact that empirical vector observations are usually made at no better than daily time-resolution. Therefore, the simulation time-step is a single day (24 hours). It was assumed that the dynamics at the grid-points used do not interact significantly and can be treated as independent, as the distance between grid-points is far greater than the normal mosquito flight distance of roughly 1 to 2 km [21-23]. To simulate large-range transmission by human movement (due to a migrating workforce for example), a small, constant influx of infected people is assumed for each grid point. Biological model Human malaria disease is caused in the individual by an infective mosquito biting a non or semi-immune human. After some two weeks the first gametocytes are produced, independent of ambient temperature. A second mosquito biting the infected human thereafter may ingest gametocytes, which after fertilization pass through the gut wall, develop and ultimately produce sporozoites which become infective when they migrate to the mosquito salivary glands. This process is ambient temperature-dependent. As transmission is less dependent on the number of parasites than on the infective status of the carriers, human and arthropod, only the infection and infectiousness status of the carrier populations are simulated (see Table 1 and 2). Modelling the vector population The most common vector of falciparum malaria in Africa is Anopheles gambiae (s.l.) [24,25]. As the female mosquito needs to feed on blood to enable ovum development, its entire life cycle must be modelled. The blood may come also from other mammals, such as cattle (which are not Plasmodium hosts), and the mosquito's anthropophilic tendency is an important factor in establishing the intensity of transmission. While the anthropophily varies between regions, at this stage it is assumed constant. The male does not bite and, therefore, does not transmit the disease, and as there are always sufficient males to impregnate the mature females, there is no need to simulate the males' dynamics. The female life is divided into two major parts: the immature stages (egg, larval and pupae), and the mature stage, where onset of maturity is defined as the time of the first flight, which is shortly followed by the first bite. The importance of this division is twofold. First, the immature mosquitoes do not participate in the infection cycle and are, thus, basically in a waiting period, which limits rapid vector population growth. Second, the survivorship (defined as the probability to survive 24 hours) and development rate (part of stage completed in 24 hours) have a different dependence on weather conditions for mature and immature mosquitoes. A schematic representation of the mosquito life cycle is presented in Fig. 1. Figure 1 Schematic presentation of the life cycle of Anopheles gambiae (s.l.). Immature mosquitoes progress at temperature-dependent rate m. They are liable to die at daily rate 1-s. Upon completion of immature process they form mature mosquitoes which begin a gonotrophic cycle with progress rate PR. They are liable to die at a rate of 1-a per day. New mosquitoes are being imported with rate trickle2. Each mosquito as oviposition lays gRd eggs. Immature populations Hitherto, the immature population dynamics have not been involved explicitly or clearly in malaria modelling. The immature forms are water-bound and are thus totally dependent on the existence of water bodies. High temperatures in breeding sites and evaporation (resulting in elimination of puddles, following the cessation of rain) are generally lethal (see below). Unfortunately, reports giving quantified relationships of temperature and rainfall/humidity dependence of the mosquito dynamics are in short supply, although some data are available [26]. Their further publication would assist the development of models. Eggs are posited by mosquitoes in pools. As a mosquito must find water to reproduce, the oviposition rate is roughly assumed to be proportionate to both the ovipositing mosquito number and to the dekadal (ten-daily) rainfall Rd filling the pool. Thus a mosquito's probability to oviposit, and for the larvae to survive, is proportional to the amount of water it finds. The inclusion of hydrology and soil typemay improve the understanding of the connection between rainfall and breeding-sites. This dependence on rainfall is valid for areas in which the surface water is dependent only on rainfall. However, in constant mosquito habitats such as stable pools or rice-fields, lack of rain may not limit growth of larvae, but natural predators may curb the growth of larval populations. As the movement between the human location (where a bloodmeal is taken) and the ovipostion site is dangerous and energy consuming, this distance is a major factor in determining the probability of oviposition. Future numeric data for this would be of great value. There is a shortage of detailed information on the varied survivorship and stage-dependent developmental progress in natural habitats as functions of weather conditions. There are, however, certain limited sources for derivation of information[27,28]. Jepson et al. [28] measured the length of each of the three stages (egg Le, larvae L1 and pupae Lp) of An. gambiae s.l. in 11 different breeding habitats (typically sunlit pools), in which the daytime water temperature was measured. Maturation rate, m, is defined as the fraction of the total immature stage covered in a single day, and is the inverse of the sum of the duration of the immature stages[29,30] m = 1/(Le+L1 + Lp)     (1). Thus the maturation rate is a function of temperature for these stages. Even though it is known that high temperatures are detrimental to larvae survival, there are no published numerical data (such as Ndegwa et al. [31] found for Trypanosoma Congolese) which would allow the introduction of this element. The most important cause of larval weather-attributable death is probably desiccation[32]. However, in some circumstances eggs can survive for weeks without water[33], and so an immature mosquito rainfall-dependent daily mortality rate, actually resulting in total clearance of the population, is not used in the model. Lack of rain will cause the numbers to be reduced in any case, as above. The overcrowding of immature mosquitoes may result in significant differences in both larval/pupal Survival and also in body size (and thus survival probability of emerging adults). Larval and purpal predation in well-established pools (as opposed to transitory puddles) has significant effects on population development [34]. The model does not currently account for these factors, but assumes a fixed per diem survival rate, s, of 90% [30]. This will be amended, by the aid of new data being collected now, for further model development. The immature population is, thus, simulated as a set of ν virtual boxes with populations I(n) (n = 1..ν; each box representing the inverse of ν, the length of the immature phase in days). At each simulated day the whole population of each box is multiplied by the per diem survival rate σ and moved on by mν boxes (with m, as above, the per diem fractional maturation rate). New eggs (box number 1) are laid as a fraction of the number of ovipositing adult females (as shall be discussed). Thus, at each time step, t, the immature population at stage s, I(s,t), has the dynamics: I(s+mn,t+1) = σ I(s,t)     (2). Maturing pupae (reaching age n and above) are removed and enter the mature mosquito dynamics according to: i.e. all immature mosquitoes within one day of maturation (at stages above ν(1-m)) will become mature mosquitoes the next day, if they survive(s). Mature mosquito dynamics Mature mosquitoes begin their adult life with their first, nuptial flight, during which fertilization occurs. In the model, a small constant trickle (trickle2) of young uninfected mosquitoes (representing a new imported population) is added to the population of maturing mosquitoes. Afterwards, the development of the fertilized eggs requires the intake of protein, i.e. blood meals. Although sometimes more than one initial blood meal is required before egg maturation can occur, and fed mosquitoes that are unable to develop mature eggs are best described as pre-gravid[35], this fraction is neglected at this stage of the model, and all blood meals are assumed to allow, at least potentially, egg maturation. At this level of model development, situations where blood meals are interrupted or when partially fed mosquitoes complete their meal on a second host are ignored. The rate of development of each brood of eggs in a vector is dependent on temperature and, to a lesser degree, on external humidity (probably as a result of the stress caused by a harsher dry environment on the vector). Detinova[36] detected a "degree-day" dependence of the time for the preparation of a brood in An. maculipennis (the gonotrophic cycle, Gc) and hence also of the time for biting, which may be expressed as GC = 1+Dd/(T-Tc)     (4); where Dd is the number of degree-days required, Tc the threshold beneath which development halts and T is the daily average temperature. Both Dd and Tc are dependent on humidity. In highly humid conditions, Dd = 37 from Detinova's data. In the tropics GC is typically about three days depending on temperature. The temperature dependence of An. gambiae is assumed similar until further data become available. As temperature is not assumed constant (on the contrary, the model is interested in its variation), a daily progress rate (part of gonotrophic cycle covered in one day): PR = 1/GC is calculated. This assumes that the temperature dependence of the rate is constant throughout the gonotrophic cycle, which is implicit in the degree-day concept. The completion of a cycle may be established when the sum of daily PR values reaches 1. 37 "boxes" (corresponding to the 37 degree days) are constructed, between which the mosquitoes progress in steps of PR reduced by multiplication by the survival rate (which shall be elaborated below). At the end of a gonotrophic cycle (upon arrival at box 37), each mosquito oviposits and then begins a new cycle. The success of oviposition is dependent on the existence of water-bodies, and hence on dekadal rainfall, and we assume that each ovipositing female lays γRd viable eggs, where γ is a constant. The following day these eggs begin the immature mosquito cycle (as above). It seems that the survivorship of mosquitoes is only weakly dependent on their age[6,9,37-39], in spite of some conflicting evidence[40]. The stage most dangerous to the adult mosquito in this model is the feeding stage, consisting of the approach to the mammal for the bite, the duration of the bloodmeal (with the corresponding irritation to the mammal) and the escape to a resting point afterwards. This risk is likely to be increased in unfavourable weather conditions (high temperatures and low humidity), but this has not been investigated yet. Thus, in the present model, the survival of the mosquito per gonotrophic cycle is a constant, a, independent of the duration of the cycle [15]. Estimates of the constant typically vary between 0.4–0.6, and are bound more tightly as 0.48–0.54 by some groups [41-43]. The per diem survival is thus calculated by P = α1/Gc. As GC is weather-dependent, so is the daily survival. It was assumed that survivorship is independent of the infective state[6,44], even though there are some reports that being infected is harmful to the mosquito. Combining these, it may be possible to write for δφ, the daily change in the total number of mosquitoes (Nm) is the difference between the new mosquitoes maturing (and not dying in the period) and the fraction of the mature mosquitoes dying (the daily cycle completion rate 1/GC multiplied by the death rate 1-α): As mentioned, the parasite within-vector dynamics is superimposed on the mosquito dynamics. Mosquitoes are assumed to bite human hosts randomly (independent of their infective status) and thus the proportion of infectious humans (Hi) rather than non-infectious (Hn) bitten is the human infectious ratio r = Hi/(Hi+Hn)     (6). Non-random biting by mosquito vectors is well described[45] and this could be incorporated in the model at a future stage. The preference for human biting over cattle is described by the human blood index (B, the proportion of bites on humans, of total bites), which is high (0.6+) for anthropophilic An. gambiae s.s. (even though the tendency varies between strains and regions) and generally much lower for zoophilic Anopheles arabiensis[46]. Of course, when cattle are far more abundant than humans, the effective B would be reduced. A fraction χ of mosquitoes that bite infective humans become themselves infected and thus the mosquito infection per bite probability is MIP = χ B r     (7) It is generally assumed that infected mosquitoes stay so for life. The sporogonic cycle (SC) (the process of fertilization of the macrogametocyte, formation of the oocyst, ookinete, penetration of the midgut and then the subsequent development of the sporozoites which dwell in the salivary glands) for P, falciparum lasts 111 degree-days above 18°C[36]. The daily sporogonic progress (in degree days) is thus SR = 111/SC. The infectivity of a specific mosquito over its lifetime is dependent on the number of bites it makes after the completion of a sporogonic cycle following the first bite of an infective host. To combine the gonotrophic and sporogonic processes each of the 37 box-stages of the gonotrophic cycle are sub-divided into 112 sub-sections, numbered 0 to 111, representing progress in degree-days. The 0 subsection reflects an uninfected mosquito. The mosquito population is governed by the following dynamics. An infected mosquito sub-population, M(s,Ss,t), at stage s of the gonotrophic cycle and at stage Ss of the sporogonic cycle (in sporogonic-cycle degree days) at time t (in calendar days) progresses each day by gonotrophic rate PR and by the sporogonic rate SR: M(s+PRSs+SR,t+1) = pM(s,Ss,t)     (8). A finite fraction (1-p) of the mosquito population which dies and thus does not make the transition. Upon completion of the gonotrophic cycle, the process restarts. Upon the completion of the sporogonic cycle the mosquito remains at the infectious stage. If the mosquito is not infected at biting, it remains uninfected throughout the gonotrophic cycle: M(s+PR,0,t+1) = pM(s,0,t) (cycle without infection);     (9a) but upon biting an infectious human, an uninfected mosquito has a finite probability of either becoming infected M(PR,SR,t+1) = pM(0,0,t)MIP (new infection)     (9b) or not: M(PR,0,t+1) = pM(0,0,t)(l-MIP).     (9c) Mosquitoes may arrive at the uninfected biting stage M(0,0,t+1) by two processes, either just after maturation or else by completing an uninfected gonotrophic cycle: New eggs are laid by mosquitoes completing a gonotrophic cycle: This means that all mosquitoes located less than PR from the end of the gonotrophic cycle will oviposit. Their number must be summed over all infection states 0...111. As discussed above, the average brood size is dependent on rainfall by a multiplicative constant γ. In the present report all the initial mosquitoes were non-infected. Modelling the infected host population Obviously, the focus is on the infected host population dynamics, which reflects the diseased population. The simulation of this population is based on the following assumptions: All hosts and mature mosquitoes are equivalent except for their infection status. Acquired immunity is not accounted for. Thus, the model reflected the prevalence of malaria infection in the population unless the population is largely non-immune in which case it reflects the prevalence of malaria disease. Immune individuals are assumed to be potential carriers, even though not at personal risk. This issue is contended within the modelling community, and future models will allow for both possibilities. The crude human death rate is taken as low enough to be unimportant over the time scale. Specifically, the malaria-induced death rate does not influence transmission patterns. Newly infected patients are not infectious for two weeks, during the intra-hepatic phase of the disease and the early erythrocytic stage, before gametocytaemia rises sufficiently for significant transmission: H(h+l,t+l) = δH(s,t) (13>h>l)     (12) Malaria clearance is a slow process. Patients may become uninfected at a constant rate (first order process). The rate selected (δ= 0.97 per day, based on MacDonald's work[6]) enables 90% of the population to clear their infection after 80 days, but other values of lower clearance do not show significantly different results. Super-infection is not accounted for, as for low- to medium-level transmission this effect is of secondary importance, although this may be built into subsequent models. The initial population is assumed to be non-infected, but new infections are being introduced at a constant low rate (trickle = 0.01 into a population of 100 every four days). This has the technical benefit of not changing the total population considerably over the simulation period, but limiting the influence of initial conditions found when a large infected population is assumed, or the dying out of the disease after a short initial dry period. This reflects the constant pressure of low-scale transmission by migration (infected migrant workers or troops for instance). This would be quite similar to the case of static communities with low-level external contact [47]. A human bitten by an infectious mosquito may become infected. The finite probability for a bite by an infectious mosquito causing human infection is integrated into the constant c of the reverse process. There is a probability, HIR, of a human being bitten by an infectious mosquito each day. This rate depends on the abundance of infectious mosquitoes and of human hosts [48]. There are three components of the human infectious population at time t+1, H(14,t+1): (i) Individuals remaining so from time t, (ii) those who complete the hepatic latent period and (iii) new imports: H(14,t+1) = (H(14,t)+H(13,t))(δ) + trickle     (13a). A host may be uninfected at time t+1, by either (1) remaining uninfected with probability (1-HIR), or else (2) being an infected host (S(H(s,t))) and clearing his/her infection with probability (1-δ): H(0,t+1) = (1-HIR)H(0,t)+(1-δ)S(H(s,t))     (13b) A newly infected host begins the latent phase: H(1,t+1) = HIR H(0,t).     (13c) The described modelling process was used to establish the fit of the model to a time series of clinical data from Hwange District, Matabeleland, Zimbabwe [49]. Due to the strong dependence of such a local clinical data-set on local weather conditions, the model was driven by station weather data, taken from a CD obtained from NCDC () using Victoria Falls weather station (WMO ID 678430). Weather values for days with missing data (some 10% of all days) were filled by averaging data from adjacent days. Using the steps described above, the model was run using the ERA-40 weather reanalysis for every grid point covering the African continent (a rectangle from 37°N 18 W to 35 S 52 E, with a mask for areas covering the ocean) over the time period 1987–2000. To allow a spin-up period for the model weather data for 1987 was run twice, while not storing the daily output for the first run, but allowing the first run's output of host, parasite and mosquito situation at the end of the year to be the initial conditions for the "real" run. This allows the simulation of more climatologically realistic starting conditions. Variations in the length of the spin-up period gave similar results. The average prevalence and incidence for the period is then established. Next, the variation of incidence during the period from interannual means was calculated, and hence the standard deviation of annual incidence. This value serves as an estimate of the extent of anomalous malaria, thus reflecting epidemics, beyond holoendemicity and seasonal variation. Results Development of immature Anopheles gambiae s.l Fig. 2 presents the rate of maturation of larvae using the data from Jepson et al. [28]. The x-axis is the average temperature in Celsius and the y-axis is the fraction of the whole larval stage covered in a single day at the given temperature. The straight line is the best-fit (with standard errors) Figure 2 Larvae maturation. Rate of development of larvae as a fraction of the complete development cycle as a function of the water temperature in Celsius. Data based on that of Jepson et al. Line best fit by least squares. X-axis average water temperature, Y-axis: rate of development (in 1/days) as the reciprocal of length of cycle. m = 0.011 (± 0.001) T-0.2 (± 0.26) (1/day)     (14) Note that the report is for water temperature in shallow pools, which may be significantly higher than the ambient temperature. A few points suggest themselves. To begin with, the intercept with the X-axis is around 18°C. Even though the variation for this value is large, it suggests a lower limit for larval development. Beyond this point, it seems that the assumption in the theoretical methodology of linearity of the development rate with temperature is justified. New data being collected may allow a more thorough validation. Using the proposed rain-dependent daily survivorship (S) and the length of cycle (1/m), the per-cycle rain-dependent survivorship is simply S1/m. The survivorship for the immature stage, by temperature for different values of S, is depicted in Fig. 3. Not surprisingly, larval development increases with rain and temperature. The temperature-linked increase in survival is, however, limited by rainfall. This interdependence of the influence of the two climatic factors limits the regions and times of vector abundance and, hence, also the transmission of malaria. As weather is not constant in reality, numerical integration of the process is required. Figure 3 Larvae survivorship. The probability of a new Anopheles gambiae larva surviving to maturity as function of ambient temperature for different values of per diem survivorship. X-axis temperature in °C. Y-axis the probability of completing development until maturity. Lines from top to bottom daily survivorships of 0.9, 0.8 and 0.7 respectively. Development of mature Anopheles gambiae s.l To stress the importance of the mature-stage dynamics temperature dependence, Figs. 4 and 5 depict the three processes: biting, development of sporozoites within the vector and the vector survival probability as a function of time, for two constant temperatures (assuming humid conditions), 28°C and 19°C. The main points that can be seen from the figures are discussed in turn. At time 0 a female mosquito bites an infected human and begins egg-production, concluding with oviposition. At the end of this process, the gonotrophic cycle, it will bite again and so on as long as it survives. Meanwhile, the mosquito's survival proability drops. Thus the number of mosquitoes which may survive to become infectious from the initial bite at time 0 is decreasing. Meanwhile, the parasites acquired by that initial bite are developing, a process, which may last many gonotrophic cycles. When this process is complete, any surviving mosquitoes become infectious. Thus, the transmission probability is the sum of all survival probabilities after the completion of a sporogonic cycle (when the ascending line reaches 100%). At T = 28°C the sporogonic cycle is completed within less than 12 days, and thus at the next bite over 5% of initial mosquitoes will survive, thus infecting an uninfected human. At T = 19°C the sporogonic cycle lasts for months, and the survival probability of mosquitoes by then is extremely small. This explains the strong transmission in tropical regions and the lack of transmission in temperate zones. Figure 4 Vector dynamics and probability of transmission. The biting cycle (periodic spikes, arbitrary scale), vectorial probability of survival by day (descending line, left axis) and fraction of sporogonic cycle completed (rising line, right axis) for constant temperature. (Ambient temperature 28°C). Figure 5 Vector dynamics and probability of transmission. The biting cycle (periodic spikes, arbitrary scale), vectorial probability of survival by day (descending line, left axis) and fraction of sporogonic cycle completed (rising line, right axis) for constant temperature (Ambient temperature 19°C) Fig. 6 shows the fit of the model simulation (based on station data for 1995–1998) to clinical data from Hwange, Zimbabwe[49]. The following graphs are presented: the rain time series, the incidence as calculated by the model and the number of cases of malaria disease as reported by district. In this case we see the main peaks of the 1996–7 epidemic expressed in the model results. It can de seen that the malaria is driven, both according to the model and according to the clinical reports, by the intense rainfall. The rainfall is, however, a local station set, while the malaria clinical results represent a district, in which there was a certain level of prevention and vector control, as well as treatment of cases, all of which prevent the fast exponential increase in case number and predicted by a model, well into the rainy season. Thus, one would expect the model results plot to be spikier than the clinical report. This is, in fact, a general aspect of process-based models, which predict exponential growth of prevalence. The modelling of intervention is an issue under ongoing research. To assess the location of epidemic regions, Fig. 7 presents a map of the anomalous malaria incidence according to the present model for the fourteen years 1987–2000. This was achieved by calculating the interannual epidemic incidence. The anomalies, calculated as standard deviation of annual incidence for each grid-point, are presented in the map. The intensity of spots is proportional to the value of the standard deviation in absolute terms. The regions for which malaria prevalence is usually high (averaging 20%) are blotted out, to differentiate between endemic and epidemic zones. This figure focuses on a different aspect of malaria distribution than usual. The regions in which MEWS will have the greatest benefit are those in which malaria variation is largest. The most pronounced regions are the fringes of endemic transmission. This includes especially the Sahel in West Africa, wide regions of East Africa, ranging deep into Somalia, and in Southern Africa most of Zimbabwe and part of Namibia. Figure 6 Rainfall, reported cases and modelled cases for Hwange, Zimbabwe 1995–1998. Left Y-axis: Case prevalence according to model and according to clinical reports for Hwange, Zimbabwe years 1995–199851. Model results are rescaled. Right Y-axis: rainfall in mm, using station data (NCDC). Blue line Rainfall. Dashed red line Modelled cases. Solid line Reported cases. Discussion The weather-based dynamic malaria model has been driven here using a reanalysis-climate data set, which is considered to represent the actual historic meteorology (at the appropriate temporal and spatial scale) for the range of climatic variables predicted by the hindcasts in the DEMETER multi-model system. In addition, this reanalysis-based dataset is used to derive the initial conditions of the hindcasts and verification dataset for DEMETER hindcasts. Thus an assessment of the value of the biological malaria model driven by ERA weather data represents the potential value of a "perfect" forecast of climatic conditions to the prediction of malaria epidemics. The assessment of this ability of the weather-driven model to describe interannual variability in malaria infection rates given a perfect forecast, is the basis for a realistic assessment of the benefit associated with the use of operational (and, therefore, unvalidated and imperfect) seasonal climate forecasts in the context of a malaria early warning system. The dynamic approach claims that total knowledge of the initial state and of the equations, as well as of the external driving forces, allows total knowledge of all future states. However, in real situations, this precise knowledge is often lacking. In many cases, bifurcations of the motion in phase-space due to minute perturbations of initial conditions, may result in large differences in the result, i.e. chaos. Chaotic behaviour may, however, be limited by the application of saturation limits to certain model parameters (such as a self-limiting proliferation rate). In addition, the knowledge of the equations themselves is often limited, being typically a linearization of behaviour of empirical data. Thus, there is considerable uncertainty in the result. The dynamic, deterministic method may, however, be used numerically, experimentally, by using small steps so as to validate the stability of the equations. For this reason, the improvement of the understanding of both the ability of weather forecasts to predict weather and of biological models to predict disease, presents a path to the understanding of probabilistic solutions to non-linear epidemic prediction problems. The capability of seasonal climate forecasts to predict anomalous seasonal climate conditions has improved considerably over the last decade. In particular, the El Niño/La Niña cycle has been correlated with extreme weather conditions throughout the globe[50]. This cycle has been repeatedly related to malaria epidemics. If the existence of an anomalous season can be predicted with skill, and if the relation between anomalous weather and localized malaria epidemics can be determined from time series analysis of a number of events[51], one may assess the probability of unusual increases in malaria transmission resulting from anomalous seasonal climates. This stage is, however, limited to reconstructions. The good fit in Fig. 2 suggests that the rate of maturation of larvae is linear with temperature. As the measurements were not from a controlled experiment, but from naturally occurring pools, the fit is surprisingly good. Fig. 3 shows the relation between surviving progeny and weather, using rainfall-determined oviposition and the dependence of maturation on temperature. This requires high temperatures (20+ °C) and at least moderate rainfall (10–20 mm/dekad (10 day period)). These conditions suit the regions of known habitation of An. gambiae s.l. In some regions, the existence of permanent waterbodies, such as slow-flowing rivers, lakes or swamps may provide suitable breeding sites, and thus make up for the shortage in rainfall, as far as the larvae are concerned. Even though the mature mosquito survival will be considerably reduced by the low humidity[52], malaria incidence during dry seasons will be possible[53]. These effects are not yet implemented in this model. The requirement for high temperatures for malarial transmission is further illustrated by the plots in Figs. 4 and 5. Even though mosquitoes may well survive and multiply during the summer in temperate regions, they may not become vectors for transmission of falciparum malaria, unless the temperature remains in the high 20s (°C) for considerable periods. For this reason falciparum malaria is associated heavily with tropical regions, while in pre-eradication malarious Europe P.. vivax was the dominant malaria parasite species. Further improvements should be added to the model. These may be achieved as new relevant numerical information becomes available on the biological processes, which were here handled somewhat heuristically. For example, the relationship between rainfall and larvae survivorship was simplified due to the lack of data. Laboratory and field, meteorological and entomological data may establish its true form. The relative importance of the various parameters, assessed by sensitivity analysis by variation of individual parameters is of great interest. This will allow field scientists to focus their efforts in establishing the values of the most critical parameters. In a parallel paper this has been presented. However, a full multivariate evaluation is still underway, using novel parallel computing methods. The model currently ignores both antiparasitic immunity (immunity to infection) and antitoxic immunity (immunity to disease) – the sharp distinction between which may not exist in reality[54,55]. Neither of these forms of immunity is relevant in areas where 'true' epidemics result from climate anomalies, as it is widely assumed that in these areas the population is largely non-immune and that severe morbidity and mortality due to malaria may occur in all age groups. An age-stratified model is being undertaken at present, but its verification will require considerable input from new targeted longitudinal studies. However, where the model may be used to predict the impact of an intervention amongst a semi-immune or immune population then both forms of immunity may be significant factors in determining the transmission dynamics of malaria infection[56]. Thus, in endemic areas there will be a need to separate the population between adults and under-fives, and collect suitable data. This work is underway at present. Numerical evaluations of the model in both time and space show that it has a good first order approximation to the prevalence of infection across the continent. It captures well both the seasonality and interannual variability of infection at the test site in Zimbabwe. Note that there is a large level of under-reporting of clinical cases (due to lack of access to health services) and also over-diagnosis of malaria, which often confound correlations (Barnish, personal communication) [57,58]. The model, when run with the following inputs (Table 1), is able to capture the gross spatial dynamics of malaria transmission across the African continent (Fig. 7). The model represents well both the endemic stable areas of transmission[14], as the shaded regions where average prevalence of infection is above 20%, and the epidemic zones in red, which are not detected by more standard methods. Of course, as with both these previous models, it is clear that there are inherent limitations in trying to fit one model to the entire African continent. In the two examples cited this has been overcome by splicing together two separate models. The development of strain-specific datasets of entomological time-series will provide the basis for separate dynamic models for areas of varying relative abundances of anopheline species. This is one of the reasons for the northern limit of malaria epidemics as depicted being somewhat conservative. Table 1 Values of parameters used B 0.5 Dd 37 degree days H 14 calendar days MIP 0.5 N 37 SC 111 degree days α 0.5. χ 0.5. γ 1.0 δ 0.9716. ν 14. Figure 7 Spatial epidemicity of malaria. Interannual standard deviation of incidence of infection as determined from the model run with daily ERA-40 data for the period 1987–2000. Regions with average prevalence rates of >20% (stable malaria) are shaded. Note that since the results are annual averages the values are likely to be less than those recorded from point prevalence surveys during the period of peak malaria transmission. Stronger variation, both between seasons and interannual, may be expected. Thus malaria transmission will continue further north than suggested, but will also be less stable. There are other points to consider. The most obvious discrepancy between our results and those of previous workers is that of high prevalence rates being predicted for Somalia – an area where malaria transmission in normally low. The results are created from a relatively short time series and may be disproportionately influenced by the anomalously high rainfall recorded in 1997/1998 in this area, and in East Africa in general, which corresponded with unusually high rates of transmission[3]. The malaria epidemic in southern Africa 1996–7, resulting from the heavy rains during the 1995–6 and 1996–7 rainy seasons in Zimbabwe and its region are reflected by the strong anomaly there. The epidemic character of the East African highlands is well represented too. The southward spread of actual malaria cases, as opposed to model cases, is limited by large-scale prevention schemes in South Africa. Obviously, the crude resolution of the weather data driving the model (approx 111 km2) means that the model can only represent the most marked changes in transmission potential over large geographic regions – appropriate to seasonal climate forecasts. This is expressed in the poor spatial resolution as regards Madagascar, in which the spatial variation of prevalence is not represented. Despite the limitations, which may be overcome in specific regions by using daily weather data at a finer spatial resolution formed by downscaling (a process under considerable meteorological research), the model is capable of describing in general terms the spatial, seasonal and inter-annual variability of malaria transmission in Africa. The usage of Tmax-5 as a surrogate for daily temperature was an ad hoc attempt to create a single value for a wide spectrum varying in time and place. The diurnal variation is, for example, usually far greater in dry regions than in humid conditions. Our attempt was to use a single value which bears relation to something measurable, namelyTmax. In fact, the two-metre model temperature value, was has been used may not have been the ideal, as larval development is closer to ground, but that would have required detailed soil data, which was beyond the scope of this research. The usage of reanalysis data is obviously inferior to high quality station data. Convective precipitation is highly local and is not well correlated to the averaging required by meteorological reanalysis. For example, the heavy rains in north-western Zimbabwe (end 1995) were under-expressed in ERA-40, partly due to the scale of the reanalysis data, and thus the local malaria incidence using that data is too low. The integration of the data in a reanalysis model in itself makes numerous physical assumptions. In some cases, the result is poor quality of even some large-scale processes, such as the El-Niño of 1997 in East Africa. High quality station data at the pan-African level is, however, not available. The stations are irregularly dispersed, and not all produce complete data sets, having many missing days. As the model requires daily values, interpolation over large areas with varying orographic characteristics is required with the inherent uncertainty this brings. In addition, the relation between mosquito habitat microclimate and station data too is unknown. High temporal and spatial resolution of weather data will improve the modelling attempts. Among other issues, the continuous measurement of weather data at malaria epidemiological and entomological research and surveillance sites now introduced will allow development of coupled malaria and meteorological data sets, which will be more effective for future analysis. Development of seasonal climate forecasting tools over large geographic areas, however, will remain for some time partly dependent on verification by reanalysis. Thus, malaria epidemic seasonal early warning will also be linked to this imperfect data source, though perhaps improved by novel downscaling methods. Conclusions This paper presents a first step in the preparation of a weather-driven dynamical model of malaria transmission, for use with both observed weather data and seasonal climate forecasts. The model incorporates the stages of the malaria vector and their dependence on temperature and rainfall, and part of the within-host parasite population dynamics. Some of these elements lack concrete theoretical and empirical development, requiring further input. Further work, under work at present, will enable the employment of such a model in the prediction of outbreaks based on skilful weather forecasts. Authors' contributions MBH formulated the mathematical model, prepared the code and ran the program. APM lead the applications model work package in DEMETER.. Both authors read and approved the final manuscript. Table 2 List of symbols used in text B Human blood index, the preference of a mosquito to bite humans and not other animals Dd Length of gonotrophic cycle in degree days Gc Length of gonotrophic cycle in days H Hepatic stage in days HIR Human new infection rate Hi Number of mosquitoes biting infected humans in a day Hn Number of mosquitoes biting uninfected humans in a day H(s,t) Human population at stage s of the development of infection at time t. s = 0 symbolises an uninfected host. I(s,t) Immature mosquito population at stage s of maturation cycle (in degree days) at time t Le, L1, Lp Length of egg, larval and pupal stage of mosquito maturation in days M(s,Ss,t) Mature mosquito population which is at stage s of gonotrophic cycle and stage Ss of sporogonic cycle at time t. MIP Infection probability of a single mosquito for each bite m Maturation rate of larvae in reciprocal days N Number of sections into which the gonotrophic cycle was divided. p Mosquito population daily survival rate PR Fraction of gonotrophic cycle covered in one day r Fraction of infected humans out of total human population Rd Dekadal (ten daily) rainfall in mm R0 Single case multiplication factor: number of secondary cases induced per case s Dummy variable representing stage of development in degree days SC Length of sporogonic cycle in degree days SR Daily progression of sporozoites in degree days SS Stage of sporogonic cycle in degree days. SS = 0 represents an uninfected mosquito Tc Threshold temperature for gonotrophic or sporogonic cycle α Fractional per-gonotrophic cycle survival of mosquito. χ Fraction of mosquitoes biting infective humans that become themselves infected. γ Ratio of brood of each ovipositing mosquito to rainfall. δ Fractional per diem survival rate of human infection. Hence 1-d is the daily infection clearance rate. φ Total number of mosquitoes. ν Number of sections into which the larval cycle was divided. σ Per diem larval survival rate Acknowledgements The authors would like to express their gratitude to Drs. Doblas-Reyes and Palmer for their contributions to the research. This research was funded as part of European Community grant number EVK2-CT-1999-00024 funding the project DEMETER. 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==== Front Respir ResRespiratory Research1465-99211465-993XBioMed Central 1465-9921-5-121537739510.1186/1465-9921-5-12ResearchAspergillus antigen induces robust Th2 cytokine production, inflammation, airway hyperreactivity and fibrosis in the absence of MCP-1 or CCR2 Koth Laura L [email protected] Madeleine W [email protected] Xin Liu [email protected] Salina [email protected] Xiaozhu [email protected] Israel F [email protected] Barrett J [email protected] David J [email protected] Lung Biology Center, Department of Medicine, University of California, San Francisco, California, USA2 Cardiovascular Research Institute, University of California, San Francisco, California, USA3 Program in Immunology, University of California, San Francisco, California, USA4 Gladstone Institute of Cardiovascular Disease, University of California, San Francisco, California, USA5 Department of Adult Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, USA2004 15 9 2004 5 1 12 12 29 5 2004 15 9 2004 Copyright © 2004 Koth et al; licensee BioMed Central Ltd.2004Koth et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Asthma is characterized by type 2 T-helper cell (Th2) inflammation, goblet cell hyperplasia, airway hyperreactivity, and airway fibrosis. Monocyte chemoattractant protein-1 (MCP-1 or CCL2) and its receptor, CCR2, have been shown to play important roles in the development of Th2 inflammation. CCR2-deficient mice have been found to have altered inflammatory and physiologic responses in some models of experimental allergic asthma, but the role of CCR2 in contributing to inflammation and airway hyperreactivity appears to vary considerably between models. Furthermore, MCP-1-deficient mice have not previously been studied in models of experimental allergic asthma. Methods To test whether MCP-1 and CCR2 are each required for the development of experimental allergic asthma, we applied an Aspergillus antigen-induced model of Th2 cytokine-driven allergic asthma associated with airway fibrosis to mice deficient in either MCP-1 or CCR2. Previous studies with live Aspergillus conidia instilled into the lung revealed that MCP-1 and CCR2 play a role in anti-fungal responses; in contrast, we used a non-viable Aspergillus antigen preparation known to induce a robust eosinophilic inflammatory response. Results We found that wild-type C57BL/6 mice developed eosinophilic airway inflammation, goblet cell hyperplasia, airway hyperreactivity, elevations in serum IgE, and airway fibrosis in response to airway challenge with Aspergillus antigen. Surprisingly, mice deficient in either MCP-1 or CCR2 had responses to Aspergillus antigen similar to those seen in wild-type mice, including production of Th2 cytokines. Conclusion We conclude that robust Th2-mediated lung pathology can occur even in the complete absence of MCP-1 or CCR2. ==== Body Background Monocyte chemoattractant protein-1 (MCP-1, also known as CCL2) and its receptor, CCR2, have been the focus of intense interest due to increasing awareness of their association with debilitating human diseases, including asthma [1-3] and pulmonary fibrosis [4-7]. Since MCP-1 attracts and activates a variety of cells, including monocytes, immature dendritic cells, basophils, natural killer cells, and a subset of T lymphocytes [8-17], MCP-1 may have multiple roles in the immune response. Models of Th1 or Th2 inflammation applied to mice deficient in either MCP-1 or CCR2 have clearly shown important roles for this chemokine and its receptor in the development of inflammation [18-24]. However, results obtained using allergen-induced models of asthma (ovalbumin and cockroach antigen) in CCR2-deficient mice are varied, showing either increased, decreased or unchanged Th2 inflammation and airway hyperreactivity (AHR) [25-27], possibly due to differences in the allergen models or strains of mice used. These experiments with CCR2-deficient mice do not directly address the role of MCP-1, which is just one of several MCP chemokines that can bind to CCR2. Although MCP-1-deficient mice have been reported to have defects in Th2 responses [18,19], the effects of MCP-1 deletion in allergen-induced allergic experimental asthma have not been previously reported. In addition to Th2 inflammation, airway fibrosis is another important feature of human asthma. Blease and colleagues [28,29] examined the contributions of MCP-1 and CCR2 to the development of fibrosis following intratracheal administration of Aspergillus fumigatus conidia to A. fumigatus sensitized mice. Airway fibrosis was significantly increased in mice treated with MCP-1 neutralizing antibody and in CCR2-deficient mice. However, these increases in fibrosis were seen in the setting of impaired clearance of conidia and a markedly increased neutrophilic inflammatory response, suggesting that the increased fibrosis might be attributable simply to an impaired antifungal response. Previous studies involving other models of allergic asthma applied to CCR2-deficient mice did not examine whether airway fibrosis occurred in these models or whether development of fibrosis was dependent on CCR2 expression [25-27]. Consequently, the role of MCP-1 and CCR2 in the development of allergen-induced lung fibrosis is not well established. In this study, we hypothesized that the effects of MCP-1 are mediated through CCR2 and that MCP-1 and CCR2 are independently required for the development of experimental allergic asthma. To test this hypothesis, we subjected mice deficient in either MCP-1 or CCR2 to an Aspergillus antigen model of Th2-cytokine-driven allergic asthma associated with significant airway fibrosis and measured pulmonary inflammation, cytokine production, AHR and fibrosis. Methods Mice Breeding pairs of Mcp-1+/+ and Mcp-1-/- mice [19] and Ccr2+/+ and Ccr2-/- mice [21] were generated as previously described. Mice were bred and maintained under specific pathogen-free conditions in the Laboratory Animal Resource Center at San Francisco General Hospital. All mice were backcrossed nine times with C57BL/6 mice (Jackson Laboratory, Bar Harbor, ME). Deletion of Mcp-1 or Ccr2 genes was confirmed by PCR. Similar numbers of male and female six-week-old mice were used for the study. The UCSF Institutional Animal Care and Use Committee approved all experimental protocols. Aspergillus Antigen Sensitization Protocol The Aspergillus fumigatus antigen preparation consisted of a mixture of culture filtrate (300 μg protein/mouse) and mycelial extract (80 μg protein/mouse) in PBS (Cellgro by Mediatech, Inc, Herndon, VA). Culture filtrates and mycelial extract were prepared as described previously [30]. For sensitization, anesthetized six-week old mice were given 50 μl of Aspergillus antigen intranasally five times at four-day intervals. Control mice were given 50 μl of PBS according to the same schedule as Aspergillus antigen-treated mice. All measurements and samples were obtained from mice four days after the final Aspergillus antigen administration, which was 20 days after the first challenge. Our group has previously found that airway reactivity measured four days after the final Aspergillus antigen challenge was similar to reactivity measured at earlier time points (on the same day as the final challenge or one day after the final challenge) [30]. Determination of Airway Reactivity Mice were anesthetized and paralyzed by intraperitoneal injection of etomidate (28 mg/kg) (Bedford Laboratories, Bedford, OH) and pancuronium bromide (0.1 mg/kg) (Baxter Healthcare Corporation, Irvine, CA). A tracheal cannula was inserted via a midcervical incision and the mice were ventilated using a Harvard model 683 rodent ventilator (9 μl/g tidal volume, 150 breaths per minute) (Harvard Apparatus, Holliston, MA). Using a whole body plethysmograph, airflow resistance was calculated during baseline breathing and in response to serially increasing doses of intravenous acetylcholine chloride (0.032, 0.100, 0.316, 1.00, and 3.16 μg/gm body weight) (Sigma, St. Louis, MO). The log of the concentration of acetylcholine (μg/gm) required for a 200% increase in total lung resistance, designated log PC200, was reported. Bronchoalveolar Lavage (BAL) After completion of the airway physiology measurements, the lungs were lavaged five times with 0.8-ml aliquots of sterile PBS. The lavage fluid was pooled and centrifuged, and the cell pellet was treated with red-blood-cell lysing buffer (Sigma, Saint Louis, MO). After being washed, the samples were resuspended in PBS. Total leukocytes were counted using a hemacytometer. Differential cell counts were determined by cytocentrifugation and Diff-Quik staining (Dade Behring Inc., Newark, DE) followed by microscopic examination of at least 300 cells. Thoracic Lymph Node Isolation and Lung Histology Thoracic lymph nodes were harvested from mice exposed to Aspergillus antigen. Lungs were then removed en bloc and the left mainstem bronchus was firmly sutured closed. The left lung was removed by cutting the left mainstem distal to the suture. It was then frozen in liquid nitrogen and stored at -70°C until processed for hydroxyproline content. The right lung was inflated to 20 cm water pressure with 10% neutral buffered formalin (VWR Scientific Products, West Chester, PA) and fixed in 10% formalin for more than 48 h. Fixed lungs were embedded in paraffin, sectioned at 5 μm thickness, and stained with either hematoxylin and eosin (H&E), periodic acid Schiff (PAS), or trichrome by the Pathology Department of San Francisco General Hospital using standard protocols. The proportion of peribronchial inflammatory cells that were eosinophils was determined by counting inflammatory cells surrounding airways with lumens of 100–200 μm (measured on the short axis) on H&E stained sections. We analyzed 500 total cells (100 cells from each of five airways) for each animal studied. Analysis of Cytokine Production by Cells To prepare single-cell suspensions for cytokine analyses, isolated lymph nodes were gently minced using a syringe plunger and cells were passed through 70-μm cell strainers. Red blood cells were removed by hypotonic lysis at room temperature. Lymph node cells were counted, centrifuged, and resuspended in RPMI medium 1640 (Cellgro by Mediatech, Inc, Herndon, VA) supplemented with FCS (10% vol/vol) (Hyclone, Logan, UT), penicillin (100 U/ml) (Cellgro by Mediatech, Inc, Herndon, VA), streptomycin (100 μg/ml) (Cellgro by Mediatech, Inc, Herndon, VA), phorbol 12 myristate 13-acetate (PMA) (25 ng/m) (Sigma, Saint Louis, MO), and ionomycin (1 μg/ml) (Sigma, Saint Louis, MO) to a final concentration of 5 million cells per ml. Cells were then aliquoted into 96-well plates and incubated at 37°C. After 40 h, cell supernatants were harvested and stored at -70°C until they were analyzed. ELISA for IL-4, -5, -13 and IFN-γ was performed on stimulated lymph node cell supernatant per the manufacturer's protocols (R&D Systems, Minneapolis, MN). Determination of MCP-1 For quantitation of MCP-1 levels in BAL fluid, C57BL/6 wild-type mice were treated with the previously described Aspergillus antigen protocol. Four days after the final Aspergillus antigen administration, lungs from Aspergillus antigen- and PBS-treated mice were lavaged two times with 0.6-ml aliquots of sterile PBS. The samples were centrifuged and the supernatants were collected and stored at -70°C until analysis. ELISA for MCP-1 was performed on cell-free BAL fluid per the manufacturer's protocol (R&D Systems, Minneapolis, MN). Measurement of Serum Total IgE Concentration Sera were obtained from blood collected by cardiac puncture from Aspergillus antigen- or PBS-treated mice after airway responsiveness measurement. Serum total IgE concentration was determined by a sandwich ELISA using complementary antibody pairs for mouse IgE (clone R35-72 and R35-118) obtained from Pharmingen (Pharmingen, San Diego, CA) according to the manufacturer's instructions. Color development was achieved using streptavidin-conjugated horseradish peroxidase (Pharmingen, San Diego, CA) followed by addition of HRP substrate (ABTS, Sigma, Saint Louis, MO). Determination of Lung Hydroxyproline Content Lungs were analyzed for hydroxyproline content as previously described [31] with slight modification. Lungs were homogenized in distilled water and incubated with 50% trichloroacetic acid on ice for 20 min. Samples were centrifuged and the pellet was mixed with 12 N hydrochloric acid and baked at 110°C for 14–18 h until samples were charred and dry. The samples were resuspended in 2 ml deionized water by incubating for 72 h at room temperature applying intermittent vortexing. Serial dilutions of trans-4-hydroxy-L-proline standard (Sigma, Saint Louis, MO) were prepared. 200 μl of vortexed sample (or standard) was added to 500 μl 1.4% chloramine T/0.5 M sodium acetate/10% isopropanol (Fisher Scientific, Pittsburgh, PA) and incubated for 20 min at room temperature. Next, 500 μl of Ehrlich's solution (1.0 M p-dimethylaminobenzaldehyde, 70% isopropanol/30% perchloric acid) (Fisher Scientific, Pittsburgh, PA) was added, mixed, and incubated at 65°C for 15 min. After samples returned to room temperature, the optical density of each sample and standard was measured at 550 nm and the concentration of lung hydroxyproline was calculated from the hydroxyproline standard curve. Statistical Analysis Statistical significance for treatment effect was determined by analysis of variance with post-ANOVA t tests corrected for multiple comparisons using Bonferroni adjustment. These statistical analyses were performed using statistical software STATA 5.0 (Stata Corporation, College Station, TX) and R [32] (The R Foundation for Statistical Computing, Vienna, Austria). All tests were two-tailed with a p-value of 0.05 for statistical significance. Results Aspergillus antigen airway challenge induces MCP-1 production We used a model system that involved repeated intranasal challenges with Aspergillus antigen over a 20-day period. To determine whether antigen challenge induces MCP-1 production in the airway, we measured MCP-1 protein levels in BAL fluid from wild-type C57BL/6 mice on day 20. MCP-1 levels were markedly higher in Aspergillus antigen-treated mice (46.3 ± 12.7 pg/ml, mean ± SE) than in PBS-treated mice (5.8 ± 1.3 pg/ml), (P = 0.01). MCP-1- and CCR2-deficient mice develop airway inflammation in response to Aspergillus antigen The Aspergillus antigen induction of MCP-1 was accompanied by a significant degree of lung inflammation as assessed by BAL fluid cell counts and lung histology. In wild-type mice, Aspergillus antigen induced a >20-fold increase in BAL fluid cell numbers (Fig. 1) and the development of prominent infiltrates in peribronchovascular spaces and scattered infiltrates in the lung parenchyma (Fig. 2A and 2B). The inflammatory infiltrates consisted of numerous eosinophils as well as other cell types. Figure 1 Aspergillus antigen induced similar increases in BAL fluid cell counts in wild-type, Mcp-1-/- and Ccr2-/- mice. Total cells, macrophages, eosinophils, and lymphocytes are expressed as mean BAL fluid total cell counts ± SE from wild-type, Mcp-1-/- and Ccr2-/- mice (PBS-treated, N = 5 mice/group; Aspergillus antigen-treated, N = 8 mice/group; Aspergillus antigen exposure and sample collection are described in methods). Neutrophils represented <0.5% of total cells for all groups. The data shown are from one experiment and representative of three separate experiments. Asterisks (*) indicate values that are statistically significantly different (p < 0.001) compared to PBS controls. To determine the airway inflammatory response to Aspergillus antigen in the absence of MCP-1 or its receptor, CCR2, we used mice with targeted disruptions of the genes that encode MCP-1 and CCR2. Since mouse strain differences are associated with major differences in antigen reactivity in many model systems, the mice used here were produced by extensive backcrossing into a C57BL/6 genetic background. Both MCP-1- and CCR2-deficient mice developed marked airway inflammation in response to Aspergillus antigen (Figs. 2C and 2D). The BAL fluid cell counts from Aspergillus antigen-treated MCP-1- and CCR2-deficient mice revealed significantly greater numbers of all cell types than in PBS-treated controls (p < 0.001). The numbers of macrophages, lymphocytes and neutrophils were not significantly different from those in Aspergillus antigen-treated wild-type mice (Fig. 1). The BAL fluid eosinophil response in MCP-1- and CCR2-deficient mice was slightly (~30–40%) smaller than in wild-type mice, but this difference did not reach statistical significance (Fig. 1). The fraction of peribronchial inflammatory cells that were eosinophils was not significantly different among wild-type mice (51 ± 13%, mean ± standard deviation), CCR2-deficient mice (52 ± 6%), and MCP-1-deficient mice (37 ± 13%) (N = 5 mice/group). These findings indicate that there was a robust inflammatory response to Aspergillus antigen even in the absence of MCP-1 or CCR2. Figure 2 Aspergillus antigen-induced lung inflammation appears similar in wild-type, Mcp-1-/- and Ccr2-/- mice. H&E stained lung sections from PBS- or Aspergillus antigen-treated wild-type, Mcp-1-/- and Ccr2-/- mice. Representative normal airway from wild-type control mice (A) (similar findings from Mcp-1-/- and Ccr2-/- control mice are not shown). Representative lung sections from Aspergillus antigen-treated wild-type (B), Mcp-1-/- (C) and Ccr2-/- mice (D) demonstrate intense peribronchiolar and perivascular inflammation. Aspergillus antigen exposure and sample collection are described in methods. Magnification: 20× objective. MCP-1- and CCR2-deficient mice develop AHR and produce mucus in response to Aspergillus antigen To determine airway reactivity to acetylcholine in mice exposed to Aspergillus antigen or to vehicle (PBS) alone, we compared airway reactivity of PBS- and Aspergillus-antigen-treated mice 4 days after the final challenge as described in the methods section. Measurements from this time point were previously found to be comparable to those from earlier time points [30]. In the experiment shown in Fig. 3, the PBS-treated group included a mixture of wild-type, Mcp-1-/-, and Ccr2-/- mice since preliminary experiments showed similar airway reactivity between PBS-treated wild-type, Mcp-1-/-, and Ccr2-/- mice (not shown). Aspergillus-antigen-treated wild-type, Mcp-1-/-, and Ccr2-/- mice each had significantly lower PC200 values than did PBS-treated controls (P < 0.001), indicating the development of AHR (Fig. 3). Although there appeared to be a trend toward less airway reactivity in Aspergillus-antigen-treated Mcp-1-/- and Ccr2-/- mice than in Aspergillus-antigen-treated wild-type mice, this trend was not statistically significant and was not observed in two additional Aspergillus-antigen-challenge experiments comparing wild-type mice to either Mcp-1-/- or Ccr2-/- mice separately (data not shown). Figure 3 Aspergillus antigen induced AHR in wild-type, Mcp-1-/- and Ccr2-/- mice. Airway reactivity in response to intravenous acetylcholine was measured invasively. Data are expressed as log PC200 and lower values indicate higher airway response. Aspergillus antigen exposure and the airway measurement protocol are described in methods (PBS-treated, N = 12 mice; Aspergillus antigen-treated, N = 8–10 mice/group;). The data shown are from one experiment and representative of three separate experiments. Asterisks (*) indicate values that are statistically significantly different (p < 0.001) compared to PBS controls. To determine if Aspergillus-antigen challenge results in increased mucus production, we analyzed lung histology by PAS-staining. As shown in Fig. 4A, there was minimal PAS staining in the airway epithelium of control mice. In contrast, Aspergillus-antigen-treated mice from all three groups showed accumulation of PAS-stained material in epithelial cells (Fig. 4B,4C,4D), indicating that Aspergillus antigen airway challenge resulted in mucus production by goblet cells. These findings indicate that Aspergillus antigen induces AHR and mucus production even in the absence of MCP-1 or CCR2. Figure 4 Aspergillus antigen induced goblet cell hyperplasia in wild-type, Mcp-1-/- and Ccr2-/- mice. Representative PAS-stained lung sections from PBS-treated wild-type mice (A) showed minimal PAS-positive staining (similar findings from Mcp-1-/- and Ccr2-/- control mice are not shown). Aspergillus antigen-treated wild-type (B), Mcp-1-/- (C) and Ccr2-/- mice (D) showed magenta staining in epithelial cells, which represents mucus. Aspergillus antigen exposure and sample collection are described in methods. Magnification, 40× objective. Th2 cytokine and IgE production is similar in Aspergillus antigen-treated wild-type, Mcp-1-/- and Ccr2-/- mice To determine if deletion of MCP-1 or CCR2 alters the cytokine response to Aspergillus antigen, we assayed Th1 and Th2 cytokines in stimulated cell supernatants prepared from thoracic lymph nodes isolated from Aspergillus antigen-treated mice. (PBS-treated mice had much smaller thoracic lymph nodes and it was not possible to reliably obtain sufficient numbers of cells from these mice for comparison.) MCP-1- and CCR2-deficient mice had concentrations of the cytokines IL-4, IL-5, IL-13 and IFN-γ generally similar to those in wild-type mice (Fig. 5A,5B,5C,5D). There was a trend toward lower IL-4 production in cells from Ccr2-/- mice, but this difference was not statistically significant. In addition, sera from Aspergillus-antigen-treated mice and control mice were assayed for serum total IgE levels. As shown in Fig. 5E, Aspergillus antigen induced increases in serum IgE in wild-type, Mcp-1-/-, and Ccr2-/- mice similar to those in control mice. Figure 5 Aspergillus antigen-treated wild-type, Mcp-1-/- and Ccr2-/- mice demonstrated intact Th2 cytokine production and induction of IgE. For cytokine determination, draining lymph node cells from Aspergillus antigen-treated wild-type, Mcp-1-/- and Ccr2-/- mice were isolated and stimulated with PMA/ionomycin for 40 hr and cytokine levels for IL-4 (A), IL-5 (B), IL-13 (C), and IFN-γ (D) were quantitated by ELISA. Serum IgE (E) from Aspergillus antigen-treated wild-type, Mcp-1-/- and Ccr2-/- mice and control mice were measured by ELISA. In (A-D), bars represent mean ± SE; in (E), results are expressed as the common log of IgE concentration where each circle represents a single PBS- or Aspergillus antigen-treated mouse and horizontal lines represent the mean of each group (PBS-treated, N = 5 mice/group; Aspergillus antigen-treated, N = 8–9 mice/group). Aspergillus antigen exposure and sample collection are described in methods. Asterisks (*) indicate values that are statistically significantly different (p < 0.001) compared to PBS controls. Aspergillus antigen-induced lung fibrosis develops in the absence of MCP-1 or CCR2 To determine whether Aspergillus antigen-induced airway fibrosis develops in the absence of MCP-1 or CCR2, we measured lung hydroxyproline content in PBS- and Aspergillus-antigen-challenged mice (Fig. 6). Aspergillus antigen treatment resulted in a two-fold increase in lung hydroxyproline, a measure of collagen content. This effect was very similar in wild-type, Mcp-1-/-, and Ccr2-/- mice. Histopathologically, lung sections from PBS-treated mice had normal lung architecture and minimal evidence of trichrome staining (Fig. 7A). Lung sections from mice treated with Aspergillus antigen had clear increases in trichrome staining in a peribronchiolar distribution (Fig. 7B,7C,7D). There were no apparent differences in trichrome staining in wild-type mice as compared to either MCP-1- or CCR2-deficient mice after allergen challenge. Figure 6 Aspergillus antigen induced similar lung fibrosis in wild-type, Mcp-1-/- and Ccr2-/- mice. Left lungs from Aspergillus antigen- or PBS-treated wild-type, Mcp-1-/- and Ccr2-/- mice were analyzed for total hydroxyproline content as described in methods. Results are expressed as mean ± SE (N = 10 mice/group). Aspergillus antigen exposure and sample collection are described in methods; data are representative of two separate experiments. Asterisks (*) indicate values that are statistically significantly different (p < 0.001) compared to PBS controls. Figure 7 Increased airway subepithelial collagen deposition after treatment with Aspergillus antigen. Representative lung sections from PBS-treated mice show minimal trichrome staining around small airways (A) (similar findings from Mcp-1-/- and Ccr2-/- control mice are not shown). Increased trichrome staining is noted around small airways in Aspergillus antigen-treated wild-type (B), Mcp-1-/- (C) and Ccr2-/- (D) mice. Blue staining around airways represents collagen. Aspergillus antigen exposure and sample collection are described in methods. Magnification, 20× objective. Discussion We hypothesized that MCP-1 and its receptor, CCR2, are independently required for the development of Aspergillus-antigen-induced allergic asthma. We found that wild-type C57BL/6 mice challenged with Aspergillus antigen developed robust Th2 responses associated with pulmonary inflammation, AHR, mucus production and fibrosis. Surprisingly, neither MCP-1 nor CCR2 was critical for the development of these lung pathologies, since robust responses were also seen in mice with deletions of genes encoding either protein. These results demonstrate that neither MCP-1 nor CCR2 are required for the development of experimental allergic asthma induced by exposure to Aspergillus antigen. Our results stand in contrast to some previous reports showing important roles for MCP-1 or CCR2 in other models of allergic asthma [25,27,33]. Although the precise explanation of these differences is not clear, there are several experimental factors that may contribute. For example, the choice of antigen and the route of sensitization differ between models. We used antigens prepared from Aspergillus, an important allergen in some people with asthma, and administered it exclusively to the respiratory tract, presumably a relevant route for sensitization in asthma. Previous studies have used ovalbumin [25,26,33] or cockroach antigen [27] and have used intraperitoneal antigen injections to sensitize prior to antigen challenge. CCR2-deficient mice have been shown to have defects in recruitment of antigen-presenting cells to the peritoneum [21,34,35], suggesting that CCR2 could be important for sensitization when antigen is administered to the peritoneum. Another factor that differs between studies is timing. We studied mice at 4 days after the final allergen challenge, when all aspects of the Aspergillus antigen-induced experimental asthma phenotype are present. Campbell et al. found that the administration of MCP-1 antibody could inhibit AHR in cockroach antigen sensitized and challenged mice at very early time points (1 and 8 h post challenge) but not later (24 h after challenge) [27]. The effect on AHR at 1 and 8 h was ascribed to MCP-1's ability to activate mast cells, which are important in some asthma models but not in others [36]. Genetic background may also be an important factor, since mouse strains vary widely in their response to airway antigen challenge [37]. Previous experimental asthma studies involving CCR2-deficient mice have used mice of mixed genetic backgrounds [25-27], whereas we used mice that had been backcrossed nine times to C57BL/6 and therefore have a more homogenous genetic background. Some of the specifics of our experimental system may therefore account for the lack of a requirement for MCP-1 and CCR2. However, MacLean et al. [26] used an allergic asthma model involving ovalbumin, intraperitoneal sensitization, and mice of mixed genetic backgrounds and found that CCR2-deficient mice had intact responses to allergen challenge. This indicates that the lack of a requirement for CCR2 is not unique to a single asthma model. It also highlights the difficulty in pinpointing the experimental factors that account for the diverse results reported by various investigators. Of note, neither MCP-1 nor CCR2 was critical for inflammatory cell migration to the lungs after Aspergillus antigen challenge. We found that Aspergillus antigen-induced monocyte recruitment (as measured by counting BAL fluid macrophages) was intact in both MCP-1- and CCR2-deficient mice. While intact alveolar macrophage recruitment in response to airway instillation of Saccharopolyspora rectivirgula has been reported in CCR2-deficient mice [38], other in vivo models have demonstrated requirements for MCP-1 and CCR2 in monocyte/macrophage recruitment [19,39-42]. Our finding indicates that other chemoattractants are sufficient for maximal monocyte/macrophage recruitment in this Aspergillus antigen model. In support of this observation, a recent microarray-based analysis of gene expression changes in a similar asthma model found that 14 different chemokines (including MCP-1/JE) were induced by Aspergillus antigen challenge [43]. However, we did find that MCP-1 and CCR2 may have indirect effects on eosinophil recruitment in response to Aspergillus antigen. While there was marked eosinophil recruitment to the lungs in MCP-1- and CCR2-deficient mice, there was a trend toward fewer eosinophils than in wild-type mice. Since MCP-1 is not a chemoattractant for eosinophils (which lack CCR2), this trend suggests that MCP-1 may have indirect effects on eosinophil recruitment in this model. A more dramatic decrease of eosinophil recruitment has been seen following neutralization of MCP-1 in another model, but that effect was associated with other signs of impaired Th2 immunity [33]. Although there may be some role for MCP-1 and CCR2 in eosinophil recruitment, robust inflammatory responses to Aspergillus antigen occurred even in the complete absence of either of these molecules. In contrast to our results indicating a robust Th2 response in MCP-1- and CCR2-deficient mice after Aspergillus antigen challenge, diminished Th2 cytokine production has been reported in studies of MCP-1 neutralization or deletion in different models [19,20,33,44,45]. In studies involving CCR2-deficient mice, the results have been more heterogenous, suggesting that CCR2 deletion may increase [25,28], decrease [24], or have no effect on Th2 responses [26]. As mentioned previously, the explanation for these different Th2 responses in CCR2-deficient mice is not clear, and may suggest that complex pathways involving other CCR2 ligands or MCP-1 receptors [46] are operational in different models of inflammation. However, if these pathways exist and were important in the model we used, we would have expected to find that deletion of MCP-1 and CCR2 had different effects. Instead, we observed that MCP-1- and CCR2-deficient mice were similar in all respects, including cytokine production, IgE production, and AHR. Our results support the idea that the role of MCP-1 and CCR2 in the development of allergic responses may be dependent upon the experimental model used. The role of MCP-1 and CCR2 in the development of allergen-induced airway fibrosis has not been extensively explored. Previous findings of increased pulmonary fibrosis in CCR2-deficient mice compared to wild-type mice after treatment with Aspergillus conidia were accompanied by neutrophilic inflammation and the inability of CCR2-deficient mice to clear the organism normally [28,29]. Consequently, the persistence of Aspergillus organisms in the airway may have altered the fibrotic response. Other studies involving different experimental systems have suggested that MCP-1 and CCR2 may directly or indirectly contribute to the development of fibrosis. Gharaee-Kermani et al. [47] found that MCP-1 directly induced increased production of collagen by cultured fibroblasts, although the role of CCR2 was not explored in that report. MCP-1 and CCR2 may also indirectly influence fibrosis via their effects on inflammatory cells. Previous studies showed that CCR2-deficient mice developed less pulmonary fibrosis in response to three different stimuli, including intratracheal bleomycin instillation, than did wild-type mice [48,49]; however, those studies did not test the requirement for MCP-1 in the development of fibrosis. In C57BL/6 mice, bleomycin induces a robust inflammatory response that consists of neutrophils and lymphocytes, with a smaller component of eosinophils [50], in contrast to our allergen model. Thus, it is possible that the relative abundance or types of recruited cells in response to a particular airway challenge greatly influence the character or extent of lung fibrosis mediated by MCP-1 or CCR2.. Therefore, based on these previously published results we might have expected MCP-1 and CCR2 to be critical to the development of allergen-mediated fibrosis. However, we found that MCP-1-deficient and CCR2-deficient mice each developed marked fibrosis following Aspergillus antigen challenge, similar to wild-type mice. Our result, in contrast to the reported requirement for CCR2 in the development of bleomycin-induced pulmonary fibrosis, suggests that different cell types and mediators may be operational in allergen-induced airway fibrosis than those observed in bleomycin-induced lung fibrosis. Conclusions In conclusion, this study demonstrates that pulmonary inflammation, Th2 immune responses, Th2-mediated airway pathology, and lung fibrosis are remarkably intact despite the complete absence of MCP-1 or CCR2 in an Aspergillus antigen-driven model of allergic airway disease. Previous studies have demonstrated roles for MCP-1 and CCR2 in other models of inflammation and fibrosis, including different allergic airway disease models [25,27,33]. Those findings indicate that the role of MCP-1 and CCR2 in allergic responses and in fibrosis depends on the models used, although it is difficult to identify which experimental factors determine whether MCP-1 and CCR2 are required. Both MCP-1 and CCR2 may be good therapeutic targets for some diseases. However, the variable involvement of these potential targets in animal models indicates that it may be extremely challenging to predict which human diseases are most likely to benefit from this approach. Abbreviations AHR, airways hyperreactivity; BALF, bronchoalveolar lavage fluid. Authors' contributions LLK conceived of the experiment, carried out all experiments and prepared the manuscript. MWR assisted in collection and analysis of mouse samples. XLB performed all mouse airway measurements. SC and XH performed antigen challenge and assisted in collection and analysis of mouse samples. IFC and BJR provided the targeted knock-out mice, provided expert advice and interpretation of the study's results. DJE participated in the study's design, coordination and final revisions of the manuscript. All authors read and approved the final manuscript. 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==== Front Biomed Eng OnlineBioMedical Engineering OnLine1475-925XBioMed Central London 1475-925X-3-301536125410.1186/1475-925X-3-30ResearchAnalysis of cardiac signals using spatial filling index and time-frequency domain Faust Oliver [email protected] U Rajendra [email protected] SM [email protected] Lim Choo [email protected] Dept. of ECE, Ngee Ann Polytechnic, Singapore 5994892 Dept. of Biomedical Engineering, Nanyang Technological University, Singapore2004 10 9 2004 3 30 30 14 5 2004 10 9 2004 Copyright © 2004 Faust et al; licensee BioMed Central Ltd.2004Faust et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Analysis of heart rate variation (HRV) has become a popular noninvasive tool for assessing the activities of the autonomic nervous system (ANS). HRV analysis is based on the concept that fast fluctuations may specifically reflect changes of sympathetic and vagal activity. It shows that the structure generating the signal is not simply linear, but also involves nonlinear contributions. These signals are essentially non-stationary; may contain indicators of current disease, or even warnings about impending diseases. The indicators may be present at all times or may occur at random in the time scale. However, to study and pinpoint abnormalities in voluminous data collected over several hours is strenuous and time consuming. Methods This paper presents the spatial filling index and time-frequency analysis of heart rate variability signal for disease identification. Renyi's entropy is evaluated for the signal in the Wigner-Ville and Continuous Wavelet Transformation (CWT) domain. Results This Renyi's entropy gives lower 'p' value for scalogram than Wigner-Ville distribution and also, the contours of scalogram visually show the features of the diseases. And in the time-frequency analysis, the Renyi's entropy gives better result for scalogram than the Wigner-Ville distribution. Conclusion Spatial filling index and Renyi's entropy has distinct regions for various diseases with an accuracy of more than 95%. ==== Body Background Bio-signals are essentially non-stationary signals; they display a fractal like self-similarity. They may contain indicators of current disease, or even warnings about impending diseases. The indicators may be present at all times or may occur at random – in the time scale. However, to (study and) pinpoint anomalies in voluminous data collected over several hours is strenuous and time consuming. Therefore, computer based analytical tools for in-depth study and classification of data over day long intervals can be very useful in diagnostics. Electrocardiography deals with the electrical activity of the heart. Monitored by placing sensors at defined positions on chest and limb extremities of the subject, electrocardiogram (ECG) is a record of the origin and propagation of the electric action potential through cardiac muscle. It is considered a representative signal of cardiac physiology, useful in diagnosing cardiac disorders. The state of cardiac health is generally reflected in the shape of ECG waveform and heart rate. It may contain important pointers to the nature of diseases afflicting the heart. However, bio-signals being non-stationary signals, this reflection may occur at random in the time scale. (That is, the disease symptoms may not show up all the time, but would manifest at certain irregular intervals during the day.) Therefore, for effective diagnostics, the study of ECG pattern and heart rate variability signal (instantaneous heart rate against time axis) may have to be carried out over several hours. HRV is a useful signal for understanding the status of the autonomic nervous system (ANS). The interest in the analysis of heart rate variability (HRV), that is, the fluctuations of the heart beating in time, is not new. And much progress was achieved in this field with the advent of cheap and massive computational power, which provoked many recent advances. HRV is a non-invasive measurement of cardiovascular autonomic regulation. Specifically, HRV is a measurement of the interaction between sympathetic and parasympathetic activity in autonomic functioning. There are two main approaches for analysis: time domain analysis of HRV [for standard deviation of normal to normal intervals (SDNN)]; and frequency domain analysis [for power spectrum density (PSD)]. The latter provides high frequency (parasympathetic activity) and low frequency (sympathetic and parasympathetic activity) and total power (sympathetic/parasympathetic balance) values [1-3]. Recent results on HRV signal analysis show that its dynamic behavior involves non-linear components that also contribute in the signal generation and control [4]. The autonomic nervous system (ANS) modulates the cardiac pacemaker and provides beat-to-beat regulation of the cardiovascular rhythm. Application of wavelet transformation techniques to beat-to-beat heart rate variations (HRV) provides an important non-invasive tool for monitoring the autonomic nervous system functioning. The cardiovascular system is a complex system that includes heart and vessels. ECG and HRV are two methods for study it. Hence, many attempts have been made to analyze these signals and extract information about the cardiovascular system. Most of the methods used are linear and it has been recognized that nonlinear methods may be more suitable for analyzing signals that originate from complex nonlinear living systems [5]. Recent developments in non-linear analysis have provided various methods for the study of the complex cardiovascular system [6]. It is now generally recognized that many processes generated by the biological system can be described in an effective way by using the methods of nonlinear dynamics. The nonlinear dynamical techniques are based on the concept of chaos, which was first introduced with applications to complicated dynamical systems in meteorology [7]. Since then, it has been applied to medicine and biology [8,9]. A particularly active area for the application of chaos theory has been cardiology [10,11], where many aspects have been addressed including whether chaos can be used to represent healthy or diseased state [12]. A complex system like cardiovascular system can not be linear in nature and by considering it as a nonlinear system can lead to better understanding of the system dynamics. Recent studies have also stressed the importance of nonlinear techniques to study HRV in both health and disease. The progress made in the field using measures of chaos has attracted scientific community applying these tools in studying physiological systems, and HRV is no exception. There have been several methods of estimating invariants from nonlinear dynamical systems reported in the literature. Recently, Fell et al and Radhakrishna et al have tried the nonlinear analysis of ECG and HRV signals respectively [13,14]. Also, Addison at al showed that coordinated mechanical activity in the heart during ventricular fibrillation may be made visible in the surface ECG using wavelet transform [15]. Rajendra et al, [16] have classified the HRV signals using Artificial Neural Networks (ANN) and Fuzzy equivalence relation. Recently, Renyi's entropy is used for texture analysis by Grigorescu et al [17]. Gokcay et al have applied Renyi's entropy to clustering and analyze the resulting staircase nature of the performance function that can be expected during learning [18]. In this work, different heart rate signals are analyzed using spatial filling index and time frequency techniques. Renyi's entropy is evaluated for the different cardiac abnormalities. Methods ECG data for the analysis was obtained from MIT-BIH arrhythmia database [19]. Prior to recording, the ECG signals were processed to remove noise due to power line interference, respiration, muscle tremors, spikes etc. The R peaks of ECG were detected using Tompkins's algorithm [20]. The ECG data contains eight different classes representing eight different diseases. The number of datasets chosen for each of the eight classes is given in Table 1. The Normal class contains datasets from people where no cardiac abnormality was diagnosed. The remaining classes are named according to the diagnosed cardiac abnormality, premature ventricular contraction (PVC), Complete Heart Block (CHB), Sick Sinus Syndrome (SSS), Congestive heart failure (CHF), Ishemic/Dilated cardiomyoapathy (ISCDIL), Atrial Fibrillation (AF), and ventricular fibrillation (VF). Table 1 Number of subjects in various groups Type Normal PVC CHB SSS CHF ISCDIL AF VF Number of datasets 60 60 20 20 40 20 35 45 Each dataset is taken consists of more than10,000 samples and the sampling frequency of the data is 360 Hz. The interval between two successive QRS complexes is defined as the RR interval (tr-r seconds) and the heart rate (beats per minute) is given as: HR = 60/tr-r     (1) Spatial Filling Index Let the signal be represented by the coordinates of a point X(k) in phase space. Then the dynamical behavior of the signal is reconstructed by succession of these points X(k) in the phase space. Phase space reconstructions are based on the analysis of dynamic systems by delay maps. The vectors X(k) in the multidimensional phase space are constructed by time delayed values of the time series, which determine the coordinates of the phase space plot. X(k) = {x (k), x((k + τ), ...,x (k + (E-1)τ)} for k = 1,2,...,N - (E - 1)τ     (1) where X(k) is one point of the trajectory in the phase space at time k, x(k + τ) are the coordinates in the phase space corresponding to the time delayed values of the time series, τ is the time delay between the points of the time series considered and E is the embedding dimension, which is the number of coordinates of the phase space plot. The attributes of the reconstructed phase space plot depend on the choice of value of τ. One way to choose τ is to take it as the time it takes the autocorrelation function of the data to decay to 1/e [21]. Another method is to take the first minimum in the graph of average mutual information [22], which appears to be better since it considers the nonlinear structure in the signal. It has been established using this method that the value of 7 for τ is the best choice for ECG signals and 5 for HRV signals [23]. From the given signal x (1), x(2), ..., x (N), a matrix AE is obtained as where E is the number of dimensions and M is related to N by the equation: M = N - (E -1)τ     (3) By plotting column 2 of matrix A against column 1 (for the case E = 2), the phase space plot for two dimensions is obtained. Similarly, the first three columns of matrix A3 represent a phase space plot in three dimensions. Now, a normalized matrix BE is obtained by dividing each element of AE by xmax where xmax = max |x(k)| 1 ≤ k ≤ N     (5) The matrix B2 (in two dimensions) is hence represented as In two dimensions, the phase space plot corresponding to the normalized matrix spans from -1 to +1 on either axis. The phase space area is now divided into small square areas of size {R × R |R ∈ Real, 2/R ∈ Integer}. Then the number of grids in the normalized phase space is n = 2/R. A matrix C is now obtained with its elements c(i,j) equal to the number of phase space points falling in a grid g(i,j). The matrix C is called the phase space matrix and its elements are divided by m, where This division yields P(i,j), the probability of a phase space point falling in a grid g(i,j). A matrix Q is now formed by squaring each element of P to get q(i,j) as the elements of Q. The sum of elements of matrix Q is calculated as The spatial filling index η is defined as: η = s / n2     (9) Now the value of η is used to quantify the degree of variability in the test signals. Time-Frequency analysis There are three common approaches to generating the time-frequency (TF) plots. These are the short Time Fourier Transform; the Wigner-Ville based bilinear distributions and the Continuous Wavelet Transform. In this investigation the latter two were used. Wigner-Ville analysis The Wigner-Ville distribution (WVD) is defined as: where z(t) is the analytic signal and h(τ) is a window function. The results where obtained using a Hamming window. This window attenuates the interferences oscillating perpendicularly to the frequency axis. The WVD satisfies a large number of desirable mathematical properties. In particular, the WVD is always real-valued; it preserves time and frequency shifts and satisfies the marginal properties. Moreover, the WVD conserves the Energy of the signal. We obtain the Energy (Ex) by integrating the WVD of z all over the time frequency plane: With the Energy conservation property the WVD can be interpreted in terms of probability density: expression (10) is the Fourier transform of an acceptable form of characteristic function for the distribution of energy. Therefore, the WVD can be used to obtain the information content of a signal; this thought is further extended in Section 4.3. Continuous Time Wavelet Transform (CWT) analysis A 'wavelet' implies a small wave of finite duration and finite energy, which is correlated with the signal to obtain the wavelet coefficients [24]. The reference wavelet is known as the mother wavelet, and the coefficients are evaluated for the entire range of dilation and translation factors [25]. Initially the mother wavelet is shifted (translated) continually along the time scale for evaluating the set of coefficients at all instants of time. In the next phase, the wavelet is dilated for a different width – also normalized to contain the same amount of energy as the mother wavelet – and the process is repeated for the entire signal. The wavelet coefficients are real numbers usually shown by the intensity of a chosen color, against a two dimensional plane with y-axis representing the dilation (scaling factor) of the wavelet, and the x-axis, its translation (shift along the time axis). Thus the wavelet transform plot (scalogram) can be seen as a color pattern against a two dimensional plane. In the CWT the wavelet coefficients are evaluated for infinitesimally small shifts of translation as well as scale factors. That is, the color intensity of each pixel in the scalogram is separately evaluated, and the resulting pattern contains information about the size and location of the 'event' occurring in the time domain [26,27]. Since the dilated wavelet is normalized to contain the same amount of energy as the mother wavelet; the scalogram representation of even high frequency, low energy 'events' occurring in the time scale are more conspicuous than in the Fourier Transform. Thus the color patterns in the scalogram can be useful in highlighting the abnormalities specific to different types of disease. MATLAB version 6.1 is used to plot the various scalogram plots. For a given wavelet Ψa,b(t), the coefficients are evaluated using Eq. (12): The wavelet, defined as ,...small wave of finite duration and finite energy...' has also zero mean value, is energy normalizing coefficient, and Ψa,b(t) is the mother wavelet; a → scale factor ; b → translation factor. Just like the WVD, the CWT representation preserves also the energy of the signal. The total energy (Ex) is obtained by integrating over all scale and translation factors: The scalogram patterns thus obtained also depend on the wavelet chosen for analysis. Bio-signals usually exhibit self similarity patterns in their distribution, and a wavelet which is akin to its fractal shape would yield the best results in terms of clarity and distinction of patterns. In the present work, the analysis is based on the Morlet wavelet. This wavelet gives good result compared to all the other wavelets. The Morlet wavelet function is given by: Renyi's Entropy (RE) The previous sections detailed WVD and CWT as two methods to represent a signal in the time-frequency domain. This section is concerned with the interpretation of the time-frequency representation. The signals represent measurements taken from patients being either normal or suffering from different vascular diseases. The goal is to find a measure which allows classifying the different signals according to the medical conditions. One interesting information that one may obtain from the time-frequency representation is the number of elementary signals present in the current observation. This leads to the following question: How much separation between two elementary signals must one achieve in order to be able to conclude that there are two signals present rather then one? A solution to this problem is given by applying an information measure to a time-frequency distribution of a signal. This can be done, because CWT and WVD preserve the energy of the signal. Unfortunately, the well known Shannon information can not be applied to the time-frequency representation of a signal, because it contains negative values. One information measure, which allows negative values in the distribution, is Renyi's entropy. This information measure was used to analyze the time-frequency representation of the measurement data. Renyi's entropy definition is derived from his proposed theory of means [28] where φ(.)- is a continuous and strictly monotonic function subclass of Kolmogorov-Nagumo functions. To satisfy the constraints of an information measure I(pk)- any information measure Simplifying the above relation, we have The third order Renyi's entropy (α = 3) is calculated from the WVD time-frequency representations as follows: similarly, the third order Renyi's entropy is calculated form the CWT as follows: The result produced by this measure ( and ) is expressed in bits: If one elementary signal yields zero bit of information (20), then two well separated elementary singles will yield one bit of information (21), four well speared elementary singles will yield two bits of information (22), and so on. It shows that for different cardiac signals the Renyi's entropy in the time-frequency domain is different. One-Way Analysis of Variance (ANOVA) The purpose of one-way ANOVA is to find out whether data from several groups have a common mean. That is, to determine whether the groups are actually different in the measured characteristic. One-way ANOVA is a simple special case of the linear model. The one-way ANOVA form of the model is where: yij = α.j + εij • yij is a matrix of observations in which each column represents a different group. • α.j is a matrix whose columns are the group means. (The "dot j" notation means that applies to all rows of the jth column. That is, the value αij is the same for all i.) • εij is a matrix of random disturbances. The model posits that the columns of y are a constant plus a random disturbance. You want to know if the constants are all the same. Results The result section compares the three different analyzing methods. Each of these methods results in a single parameter for each of the datasets. For Phase Space the parameter used for comparison is the spatial filling index (η) defined in Equation (9). For WVD the parameter is the third order Renyi's entropy () defined in Equation (14). For CWT the parameter is also the third order Renyi's entropy () defined in Equation (15). For each dataset these three parameters (, and η) where calculated. Table 2 shows the mean and the variance (normalized by N-1 where N is the sequence length) of these parameters for each of the data classes. The p-value, also shown in Table 2, results form the ANOVA test for each of the parameters. Table 2 Results for various cardiac abnormalities Type SSS PVC CHB NORMAL CHF AF ISCDIL VF p-value η Phase Space 1.56 ± 0.08 3.77 ± 11.82 7.07 ± 0.57 6.76 ± 1.61 7.71 ± 0.20 2.26 ± 0.05 7.44 ± 1.24 3.77 ± 8.78 0.00005 Wigner-Wille 3.38 ± 2.74 4.79 ± 1.46 5.65 ± 0.61 4.00 ± 1.52 2.10 ± 0.61 3.31 ± 2.45 4.07 ± 2.78 4.44 ± 0.96 0.021 Scalogram 2.84 ± 1.89 2.25 ± 1.06 3.01 ± 0.29 1.67 ± 0.84 1.78 ± 0.82 2.04 ± 1.37 2.15 ± 0.57 3.29 ± 0.33 0.001 The proposed technique was applied to a number of different signals, both normal and abnormal. Some of the normal and abnormal signals used in the analysis, along with their two dimensional plots are shown in the Figures 1,2,3,4,5,6,7,8. Figure 1 Heart rate in representative subject with SSS; (a) Phase space plot (b) Scalogram (c) Wigner-Ville distribution Figure 2 Heart rate in representative subject with PVC; (a) Phase space plot (b) Scalogram (c) Wigner-Ville distribution Figure 3 Heart rate in representative subject with CHB; (a) Phase space plot (b) Scalogram (c) Wigner-Ville distribution Figure 4 Heart rate in representative subject with Normal; (a) Phase space plot (b) Scalogram (c) Wigner-Ville distribution Figure 5 Heart rate in representative subject with CHF; (a) Phase space plot (b) Scalogram (c) Wigner-Ville distribution Figure 6 Heart rate in representative subject with AF; (a) Phase space plot (b) Scalogram (c) Wigner-Ville distribution Figure 7 Heart rate in representative subject with Ishemic/Dilated Cardiomyopathy; (a) Phase space plot (b) Scalogram (c) Wigner-Ville distribution Figure 8 Heart rate in representative subject with VF; (a) Phase space plot (b) Scalogram (c) Wigner-Ville distribution For the time frequency plots the normalized frequency is shown over the hart rate values. It is not useful to state an absolute frequency, because such a value is not relevant for the cardiac system under observation. Moreover, the relative frequency representation allows comparing the time frequency analysis results form varying observation intervals. As example, the observation interval for the VF data is significantly shorter as for the rest of the data, but still the results can be compared. Discussion The resulting phase space plots for various types of disease are shown in Figure 1(a),2(a),3(a),4(a),5(a),6(a),7(a),8(a). In SSS – III (Sick Sinus Syndrome – III, Bradycardia-Tachycardia) there is a continuous variation of heart rate between Bradycardia and Tachycardia. The phase space plot spreads over a larger area (Figure 1(a)). In the Ectopic beat abnormality; there would be a sudden impulsive jump in the heart rate. This may be due to a Premature-Ventricular beat in the ECG signal. This is indicated as a sudden spike in the phase space plot (Figure 2(a)). In Complete Heart Block (CHB) cases, as the atrio-ventricular node fails to send electrical signals rhythmically to the ventricles, the heart rate remains low. The phase space plot reduces almost to a point, indicating very little change with time (Figure 3(a)). For Normal cases, the phase space plot looks like a cluster of points (Figure 4(a)). In the Congestive heart failure (CHF), the heart rate variation is lower and hence the phase space plot spread in a very small area (Figure 5(a)). In the Atrial Fibrillation (AF), heart rate signal records highly erratic variability; this is depicted as scattering of points in the phase space plot (Figure 6(a)). In the case of Ischemic/Dilated cardiomyopathy, the ventricles are unable to pump out blood to the normal degree. Here the heart rate variation is very small. And hence the phase space plot will be almost a point (Figure 7(a)). And its phase space plot resembles that of Normal class. Finally, in VF, the heart rate variation is high and hence the phase space plot is randomly distributed (Figure 8(a)). The contour plots of scalogram and Wigner-Ville distribution plot for the different abnormalities are shown in figures 1(b),1(c),2(b),2(c),3(b),3(c),4(b),4(c),5(b),5(c),6(b),6(c),7(b),7(c),8(b),8(c) respectively. In the contour plot of scalogram (Figure 1(b)), for SSS, there is clear indication of variation of high frequency and low frequency in the form of irregular circles at these frequencies. In PVC (Figure 2(b)), a irregular circle is shown at high frequency indicating the spike of the signal. These irregular circles or contours are at low frequencies for CHB (Figure 3(b)), indicating smaller R-R variation. In normal case (Figure 4(b)) these contours are in the middle frequency due to variation in the R-R interval. In CHF (Figure 5(b)) and Ischemic/Dialted cardiomyopathy (Figure 7(b)), the R-R variation is extremely low. Hence the contours are aligned at the low frequency. In AF (Figure 6(b)), due to very high R-R variation are shown as irregular contours at high frequency. For VF, this R-R variation is slightly low and as result the contours are aligned at the middle of the contour plot (Figure 8(b)). The contour plots of the Wigner-Ville distribution does not indicate as clearly as contour plot of scalogram for various cardiac diseases. The spatial filling index decreases or increases from the normal class for the abnormal subjects in different ranges (Table 2) depending on the R-R variation. This value decreases for the abnormalities of high R-R variation and increases for CHB, CHF and Ishemic/Dilated cardiomyoapathy, which has low R-R variation. This parameter has excellent 'p' value for various classes (0.00005). The Renyi's entropy has high value for cardiac abnormalities like Ischemic/Dilated cardiomyopathy, CHB, VF and it decreases for Normal, PVC, AF, SSS and CHF. This RE gives lower 'p' value for scalogram than Wigner-Ville distribution and also, the contours of scalogram visually show the features of the diseases. Hence, in the time-frequency analysis, the Renyi's entropy gives better result for scalogram than the Wigner-Ville distribution. Conclusion Considering heart as a nonlinear complex system and processing various cardiovascular signals (HRV) seems to provide very useful information for detection of abnormalities in the condition of the heart that is not available by conventional means. In this paper, a phase space and the time-frequency analysis of these cardiac signals using spatial filling index and Renyi's entropy has been proposed for detecting cardiac dysfunction. The ANOVA test was used to compare the different analyzing methods. The Renyi's entropy gives better result for the scalogram than the Wigner-Ville distribution. The evaluation of the proposed technique on a larger data set will improve the efficacy of the technique. It is left as future work to compare the different methods with more sophisticated statistical methods, such as post hoc comparisons. It is hoped that the graphical representation along with its corresponding analytical index and Renyi's entropy proposed here will find potential applications in computer analysis of cardiac patients' status in intensive care units. ==== Refs Myers G Martin GJ Magid NM Barnett PS Schaad JW Weiss JS Lesch M Singer DH Power Spectral Analysis of Sudden Cardiac Death: Comparison to Other Methods IEEE Transactions on Biomedical Engineering 1986 1149 1156 3817848 Akselrod S Godo D Ubel FA Shannon DC Baeger AC Cohen RJ Power spectrum analysis of heart rate fluctuation: a quantitative probe of beat-to-beat cardiovascular control Science 1981 220 222 6166045 Saul P Yukata AL Berger RD Leonard SL Wilson CS Cohen RJ Assessment of Autonomic Regulation in Chronic Congestive Heart Failure by Heart Rate Spectral Analysis American Journal of Cardiology 1988 1292 1299 3376889 10.1016/0002-9149(88)91172-1 Iyengar N Peng CK Goldberger AL Lipsitz LA Age-related alterations in the fractal scaling of cardiac interbeat interval dynamics American journal of Physiology 1996 271 R1078 1084 8898003 Abarbanel HI Analysis of Observed Chaotic Data 1996 Springer Verlag, Berlin Karrakchou M Rheymer KV Vesin JM Pruvot E Kunt M Improving cardiovascular monitoring through modern techniques IEEE Engineering in Medicine and Biology Magazine 1996 68 78 10.1109/51.537062 May RM Simple mathematical models with very complicated dynamics Nature 1976 261 459 467 934280 Tsonis PA Tsonis A Chaos: Principles and implications in biology Computer Applications in Biosciences 1989 5 27 32 Eberhart RC Chaos theory for biomedical engineer IEEE Engineering in Medicine and Biology Magazine 1989 41 45 10.1109/51.35577 Glass L Zeng WZ Complex bifurcations and chaos in simple theoretical models of cardiac oscillations Annals of the New York Academy of Sciences 1990 316 327 2197927 Denton TA Diamond GA Fascinating rhythms: a primer on chaos theory and its applications to cardiology American Heart Journal 1990 1419 1440 2248188 10.1016/0002-8703(90)90258-Y Lipsitz LA Goldberger A Loss of complexity and aging Journal of the American Medical Association 1992 1806 1809 1482430 10.1001/jama.267.13.1806 Fell J Mann K Roschke J Gopinath MS Nonlinear analysis of continuous ECG during sleep I reconstruction Biological Cybernetics 2000 477 483 10879431 10.1007/s004220050600 Radhakrishna RA Vikram Kumar Yergani Narayana Dutt D Vedavathy TS Characterizing Chaos in heart rate variability time series of panic disorder patients In Proceedings of ICBME, Biovision Bangalore, India 2001 163 167 Addison PS Watson JN Clegg Gareth R Steen PA Robertson CE Finding Coordinated Atrial Activity During Ventricular Fibrillation Using Wavelet Decomposition IEEE Engineering In Medicine and Biology Magazine 2002 58 61 11935988 10.1109/51.993194 Rajendra Acharya U Pubbanna Bhat SS Iyengar Ashok Rao Summet Dua Classification of heart rate using artificial neural network and fuzzy equivalence relation Pattern Recognition 2003 61 68 10.1016/S0031-3203(02)00063-8 Grigorescu SE Petkov N Texture analysis using Renyi's generalized entropies In IEEE International Conference on Image Processing 2003 241 244 Gokcay E Principe JC A new clustering evaluation function using Renyi's information potential In International Conference on Acoustics, Speech, and Signal Processing (ICASSP) 2000 Ichimaru Y Moody GB Development of the polysomnographic database on CD-ROM Psychiatry and Clinical Neurosciences 1999 175 177 10459681 10.1046/j.1440-1819.1999.00527.x Jiapu P Tompkins WJ Real Time QRS Detector algorithm IEEE Transactions on Biomedical Engineering 1985 230 23 3997178 Bangham JA Marshall S Image and signal processing with mathematical morphology Electronics & Communication Engineering Journal 1998 117 128 Mallat S Characterization of signals from multiscale edges IEEE Transactions on Pattern Analysis and Machine Intelligence 1992 1019 1033 Falconer K Fractal Geometry 1990 J Wiley and Sons, New York Vetterli M Herley C Wavelets and filter banks: theory and design IEEE Transactions on Signal Processing 1992 2207 2232 10.1109/78.157221 Raghuveer MR Bopardikar AS Wavelet Transforms Introduction to theory and applications 1998 Addison Wesley Longman Inc Vetterli M Kovacevic J Wavelets and Subband Coding 1995 Englewood Cliffs, NJ: Prentice-Hall Grossmann A Kronland-Martinet R Morlet J Combes JM, Grossmann A Reading and understanding continuous wavelet transforms In Wavelets: Time-Frequency Methods and Phase Space 1990 Tchamitchian Ph 2 20 Waheed K Salam FM A Data-Derived Quadratic Independence Measure for Adaptive Blind Source Recovery in Practical Applications In 45th IEEE International Midwest Symposium on Circuits and Systems, Tulsa, Oklahoma August 4–7 2002 473 476

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==== Front J Negat Results BiomedJournal of Negative Results in Biomedicine1477-5751BioMed Central London 1477-5751-3-31538002210.1186/1477-5751-3-3ResearchThe contrast-enhanced Doppler ultrasound with perfluorocarbon exposed sonicated albumin does not improve the diagnosis of renal artery stenosis compared with angiography Teixeira Odila UN [email protected] Luiz A [email protected] Hélio Bernardes [email protected] Hypertension Unity, Heart Institute (InCor), São Paulo Medical School, Brazil2004 20 9 2004 3 3 3 26 3 2004 20 9 2004 Copyright © 2004 Teixeira et al; licensee BioMed Central Ltd.2004Teixeira et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. There are no studies investigating the effect of the contrast infusion on the sensitivity and specificity of the main Doppler criteria of renal artery stenosis (RAS). Our aim was to evaluate the accuracy of these Doppler criteria prior to and following the intravenous administration of perfluorocarbon exposed sonicated albumin (PESDA) in patients suspected of having RAS. Thirty consecutive hypertensive patients (13 males, mean age of 57 ± 10 years) suspected of having RAS by clinical clues, were submitted to ultrasonography (US) of renal arteries before and after enhancement using continuous infusion of PESDA. All patients underwent angiography, and haemodynamically significant RAS was considered when ≥50%. At angiography, it was detected RAS ≥50% in 18 patients, 5 with bilateral stenosis. After contrast, the examination time was slightly reduced by approximately 20%. In non-enhanced US the sensitivity was better when based on resistance index (82.9%) while the specificity was better when based on renal aortic ratio (89.2%). The predictive positive value was stable for all indexes (74.0%–88.0%) while negative predictive value was low (44%–51%). The specificity and positive predictive value based on renal aortic ratio increased after PESDA injection respectively, from 89 to 97.3% and from 88 to 95%. In hypertensives suspected to have RAS the sensitivity and specificity of Duplex US is dependent of the criterion evaluated. Enhancement with continuous infusion of PESDA improves only the specificity based on renal aortic ratio but do not modify the sensitivity of any index. renal artery stenosisultrasoundPESDAangiographyhypertension ==== Body Introduction Renal artery stenosis is the most frequent cause of secondary hypertension [1] which is potentially treatable with angioplasty, endovascular stent placement or surgical revascularization [2,3]. The angiography remains the gold standard, however, is invasive, expensive, and potentially harmful specially in patients with compromised renal function or diabetes [4]. Over the past few years, there has been extensive research for a reliable, noninvasive, and nonionizing imaging method to screen for renal artery stenosis (RAS) [5]. Magnetic resonance (MR) angiography, captopril renography and duplex ultrasonography have all been assessed for this purpose [6,7]. Duplex ultrasonography (US) is safe and widely available, but its use as a screening tool of renal artery stenosis does not have universal acceptance because of the lack of standardization in examination protocols and diagnostic criteria, as well as the wide differences in reported accuracy among different laboratories [8,9]. In addition, despite the use of color Doppler and other technological improvements, the localization of the main renal arteries deep within the abdomen has rendered direct visualization of these vessels difficult [10]. There is a 10 to 20 percent rate of failure due to the operator's inexperience, the presence of obesity, overlying bowel gas or respiratory motion [11]. The proposed criteria for the detection of renal artery stenosis by direct Doppler include an increased peak systolic velocity, an increase in renal aortic ratio and also an increased resistance index [12]. The mean sensitivity and specificity of the Duplex US based on these criteria varies, respectively, from 10 to 93% and from 37% to 100%, according to different reports [8-10,12-20]. There are few reports comparing these criteria for the detection of RAS [15]. Recently, the use of microbubble echo-enhancing agents in combination with harmonic Doppler imaging has been proposed to improve Doppler signal intensity in multiple vascular sites [21]. Thus, it would be expected to improve the operator's ability to visualize the renal arteries, and to significantly reduce the number of equivocal examinations [11,21-23]. In addition, contrast-enhanced harmonic Doppler US can currently provide objective functional assessment of RAS through analysis of time-intensity renal enhancement curve [22]. MISSOURIS et al [23] have reported data of microbubble Levovist® echo-enhancing ultrasonography in hypertensives with renal artery stenosis. They demonstrated a sensitivity of 85% and a specificity of 79% without contrast and a sensitivity of 94% and a specificity of 88% with contrast, besides an important reduction in the time of procedure. The echo-enhancing agent PESDA (perfluorocarbon exposed sonicated albumin) is a second-generation agent, containing high molecular weight gas, whose use results in higher stability and better reflections of Doppler signs [24]. The PESDA is broadly used in echocardiography [24], but till date there is no studies using PESDA contrast in echo-enhanced US of renal arteries. Also there are no studies that investigate the effect of the contrast infusion on the sensitivity and specificity of the different Doppler criterion mentioned above. The purpose of our study was to evaluate the accuracy of the main color Doppler criteria of the renal arteries prior to and following the intravenous administration of PESDA in patients suspected of having renal arterial stenosis. These results were compared with those from conventional angiography, which was regarded as the standard of reference. As a secondary objective, the feasibility, time of examination and safety of US with PESDA infusion was analyzed. Methods Study Population Thirty patients (13 males/ 17 females), with a mean age of 57 ± 12 years (range 16–77 years) were enrolled in the study, that was performed at Heart Institute of São Paulo University. The only inclusion criterion was a clinical suspicion of renal arterial stenosis that required conventional or digital subtraction angiography for diagnosis. The suspicion of renovascular hypertension was based on the presence of one or more of the following clinical features: resistant hypertension, progressive renal failure with no recognized cause, atherosclerotic disease in other circulatory site (coronary, peripheral or cerebrovascular disease), renal failure induced by ACE inhibitors; renal asymmetry; retinopathy grade lll or lV (Keith Wagener) with diastolic blood pressure over 125 mmHg [1]. Exclusion criteria were as follows: patients with a renal transplant; patients who had a renal arterial stent and those referred because they were suspected of having renal arterial restenosis; patients who received any iodinated agent in the previous 24 hours, and patients with acute myocardial infarction or stroke. All patients underwent conventional angiography or digital subtraction angiography. The patients were assigned randomly in a 2-steps imaging protocol: 1) acquisition of a baseline non-enhanced Doppler ultrasound study by a Sequoia Echography System (Acuson, Siemens, Mountain View, CA, USA); 2) a continuos infusion of contrast PESDA, 0,1 ml per kg of weight in a rate of infusion of 2 ml per minute; c) after infusion, we adjust the gain according to the observed gain intensity increase for optimal filling of the vessel lumen in color mode and delineation of the spectrum envelope in duplex mode. The ultrasound scans were done in different positions to evaluate all segments of the main renal arteries: a) supine position to visualize the origin and proximal portion, b) epigastric transverse scans of the aorta to identify the right artery (anterolateral) and left artery (posterolateral), c) the sagital or coronal scan from a flank approach to identify the medium and distal portions of the arteries [[20],26,27]. Peak systolic (PSV) and diastolic (PDV) velocities of the aorta and renal arteries, and calculation of resistance index (RI), pulsatility index (PI) and renal aortic ratio (RAR) were obtained in all segments of renal arteries [[20],26-27]. The following spectral Doppler diagnostic criterion for renal arterial stenosis were used: a) PSV > 150 cm/s [26-27]; b) RAR > 3.0 [18]; c) RI > 0,80 [17]. The same examiner performed all examinations and the confirmation of the presence of stenosis was done in consensus with another examiner, both of them blinded to the results of the angiography. We also collected clinical data, including number of drugs, serum creatinine and values of blood pressure in baseline conditions. The results of PESDA-enhanced and non-enhanced ultrasound examinations were compared with those from intraarterial angiography. A hemodynamically significant stenosis was defined as diameter reduction of 50% or more at angiography, because it has been widely used in the recent literature [13]. The radiologist and clinician interpreting the study were blinded to the Doppler examination results. Secondary efficacy variables included the duration of each Doppler examination, the detection of supernumerary arteries and adverse effects. The study was approved according to local legal requirements and informed consent was obtained before ultrasound examination from all patients. Results Patient Characteristics All patients underwent digital subtraction angiography and non-enhanced US. One patient did not receive the infusion of contrast, because the venous access was not possible. Renal arterial stenosis of 50% or greater was detected at angiography in 18 (60%) patients, 5 of whom had bilateral stenosis. Renal arterial stenosis was excluded in 12 patients. Thus, stenosis by angiography was detected in 23 arteries, while 37 arteries did not present. The clinical and demographic data of patients according to the presence of stenosis are presented in the Table 1. The patients with stenosis were older than patients with no stenosis (p = 0.013), while we did not observe differences in the other demographic and clinical data. Table 1 Demographic and clinical data of 30 patients according to the presence of RAS at angiography Patients with no stenosis (n = 12) Patients with stenosis (n = 18) p Age (years) 43 ± 15 57 ± 14 0,013* BMI 25,5 ± 5,2 26,5 ± 3,4 0,528 SP (mmHg) 158 ± 29 162 ± 26 0,738 DP (mmHg) 97 ± 15 96 ± 16 0,900 HR 74 ± 12 74 ± 11 0,866 Cr 1,21 ± 0,51 2,01 ± 1,50 0,088 BMI: corporeal mass index SP: systolic pressure DP: diastolic pressure; HR: heart rate Cr: serum creatinine (normal < 1,50 mg/dl); RAS = renal artery stenosis Feasibility Overall, all patients had the renal arteries assessable with non-enhanced US or after injection of PESDA, although in one patient with a high body mass index, the assessment was better after contrast. Despite an expectation of at least 20% accessory arteries, we did not find any in our population. Accuracy As stenosis is mostly in ostium and proximal portion of the arteries, we considered for diagnosis of renal artery stenosis the indexes obtained by echo-Doppler in these arterial segments. The mean values of PSV, PDV, RAR, RI and PI in these segments are showed in the tables 2 and 3. In the ostium, the values of PSV and RAR were higher in arteries with stenosis, in either enhanced or non-enhanced US. In the proximal segment, only RAR values were higher in arteries with stenosis, in both enhanced and non-enhanced US. Table 2 Mean values of Doppler indexes obtained with non-enhanced or enhanced with PESDA ultrassonography in the ostium of renal arteries Non-enhanced Enhanced p Arteries without stenosis n = 37 Arteries with stenosis n = 23 Arteries without stenosis n = 37 Arteries with stenosis n = 23 Stenosis vs no stenosis Enhanced vs no enhanced PSV (cm/s) 1,49 ± 0,76 2,26 ± 1,15 1,49 ± 0,65 2,01 ± 1,27 0,001 0,975 PDV (cm/s) 0,31 ± 0,25 0,42 ± 0,34 0,32 ± 0,19 0,37 ± 0,38 0,229 0,596 RRA 1,43 ± 0,67 2,36 ± 1,34 1,19 ± 0,54 2,19 ± 1,45 <0,001 0,145 RI 0,77 ± 0,20 0,84 ± 0,12 0,79 ± 0,11 0,76 ± 0,27 0,601 0,291 PI 1,83 ± 0,90 2,23 ± 0,97 1,97 ± 0,77 1,97 ± 0,99 0,332 0,588 PSV: peak systolic velocity; PDV: peak diastolic velocity; RAA: renal/aortic ratio RI: resistance index; PI: pulsatility index; US = ultrasonography Table 3 Mean values of Doppler indexes obtained with non-enhanced or enhanced with PESDA ultrassonography in the proximal portion of renal arteries Non-enhanced Enhanced p Arteries without stenosis n = 37 Arteries with stenosis n = 23 Arteries without stenosis n = 37 Arteries with stenosis n = 23 Stenosis vs no stenosis Enhanced vs no enhanced PSV (cm/s) 1,56 ± 0,79 2,12+/-1,22 1,68+/-0,86 2,01+/-1,27 0,059 0,973 PDV (cm/s) 0,36+/-0,22 0,38+/-0,31 0,33+/-0,21 0,38+/-0,36 0,589 0,618 RAR 1,54+/-0,84 2,16+/-1,25 1,30+/-0,60 1,98+/-1,36 0,008 0,103 RI 0,76+/-0,11 0,83+/-0,11 0,78+/-0,13 0,76+/-0,26 0,193 0,793 PI 1,74+/-0,68 2,31+/-0,91 2,06+/-1,01 2,05+/-1,07 0,526 0,375 PSV: peak systolic velocity; PDV: peak diastolic velocity; RAA: renal/aortic ratio RI: resistance index; PI: pulsatility index; US = ultrasonography The sensitivity, specificity, positive predictive value and negative predictive value of each Doppler index for the detection of RAS (Table 4) were calculated based on the values standardized in the literature. In terms of renal arteries, we observe that the sensitivity and specificity depend on the index used. Thus, in non-enhanced US the sensitivity was better when based on RI (82.9%) while the specificity was better when based on RAR (89.2%). The PPV was stable for all indexes (74.0%–88.0%) while NPV was low (44%–51%). The specificity and PPV based on RAR increased after PESDA injection respectively, to 97.3% and 95%. Table 4 Sensitivity and specificity of non-enhanced and enhanced Doppler US for the detection of RAS in 60 arteries a) Non-enhanced Sensitivity Specificity PPV NPV RRA (<3,0) 56,2% 89,2% 88% 44% PSV (<150) 69,7% 64,9% 75% 45% RI (<0,80) 82,9% 56,8% 74% 51% b) Enhanced Sensitivity Specificity PPV NPV RRA (<3,0) 33,3% 97,3% 95% 40% PSV (<150) 61,9% 64,9% 72% 42% RI (<0,80) 76,2% 43,2% 66% 42% RRA: ratio renal/aortic PSV: peak sistolyc velocity(cm/s) RI: resistive index PPV = positive predictive value, NPV = negative predictive value RAS = Renal artery stenosis; US = ultrasonography The receiver operating characteristic curves for each Dopplerdiagnostic criterion showed that the area under the receiver operating characteristic curve for resistance index was greater than the area under the curve for peak systolic velocity and renal aortic ratio. For renal aortic ratio, the cutoff point that provided the best accuracy, 2.7 gave a specificity of 96% but a low sensitivity (60%). For peak systolic velocity, no precise cutoff point could be identified between arteries with stenosis and those without stenosis. For resistance index, a threshold of 0.8 led to a sensitivity of 70% and a low specificity of 56.8%. Secondary variables and safety The median examination time was 35 minutes for enhanced Doppler US and 29 minutes for non-enhanced Doppler US, i.e., a small but significant reduction of 17% (p = 0.03). Only one patient presented adverse events to be potentially related to the injection of PESDA, including sensation of coldness, palpitation and dyspnea. There was no severe adverse event. Discussion Although the technique of renal arterial US scanning has been well established for years, a lot of difficulties in reliably identifying main and accessory renal arteries remain [8-10,25]. Most of these dificulties are related to the patient obesity, the presence of bowel gas, excessive respiratory movement, and the depth and tortuosity of the renal arteries [8,16]. The time expended in the examination can be too long as almost 60 minutes [19], and failure of technique varies from 9 to 25%. In our study, non-enhanced Doppler US showed a feasibility rate of 100%, similar to some single centers, but higher than the majority of studies using this technique (58–90%) [11]. Indeed, two recently published studies reported feasibility not exceeding 11% and 12% [22,23]. One of the reasons of our high rate of feasibility probably is related to the quality of the machine, which allowed a scan imaging with an excellent definition. To our knowledge, the present study is the first randomized study in a selected group of hypertensive patients in which renal arterial color Doppler flow US with and without a continuous infusion of PESDA was compared against the reference standard of angiography. The infusion of PESDA did not alter the feasibility that remains 100%. In a multicentric study [11] using Levovist as the US contrast, the infusion increased by 20% the number of patients in whom renal arteries could be evaluated, including difficult cases such as those involving patients who are obese and patients with impaired renal function. However, some centers participating of the study also presented a feasibility of 100% and the Levovist infusion did not interfere in the results. In our study only one obese patient had a better visualization of renal artery after contrast infusion, and in all patients with renal failure, the non-enhanced US was able to localize renal arteries. The most important conclusion from this study is that both sensitivity and specificity of Doppler US of renal arteries are strongly dependent on the criterion used, and the infusion of PESDA contrast seems not to improve it significantly, although we observe a slight increase in specificity. Thus, the best sensitivity was obtained when based on resistance index <0.8 (82.9%) but at expense of a low specificity (56.8%). On the other hand, the best specificity was obtained with renal aortic ratio >3 (89.2%), but the sensitivity was low (56.2%). In addition, the sensitivity and specificity for a peak systolic velocity of 1.5 m/sec showed intermediate values, respectively, 61.9% and 64.9%. An analysis of previously published studies [8-10,12,15,16,20] based on non enhanced Doppler evaluation of the renal artery clearly shows that the diagnostic criteria and respective threshold values fluctuate from one report to the other. Miralles et al [15] reported a sensitivity of 87.3% and a specificity of 91.5% for a higher peak systolic velocity (>1.98 m/sec) and a higher renal aortic ratio (>3.3), while Olin et al [12] reported a sensitivity of 98% and specificity of 98% for a quite similar criteria. Helenon et al [10] quoted a sensitivity of 89% and a specificity of 99% with use of a peak systolic velocity cutoff point similar to our study (1.5 m/sec) but taking into account the presence of poststenotic turbulence and not renal aortic ratio. Moreover, in the multicentric study cited above comparing non enhanced and enhanced Doppler US [11], renal aortic ratio was more accurate than peak systolic velocity in the diagnosis of a renal arterial stenosis greater than 50%, but it was difficult to determine a precise cutoff point. In the same study, the authors demonstrated, in terms of patients, a sensitivity of 80.0% and a specificity of 80.8%, but according to renal arteries the sensitivity was lower (66.7%) and the specificity was higher (90.4%). These latter results were quite similar to our results based on RAR criteria, also evaluated according to renal arteries. These facts, determination of accuracy in terms of renal arteries and not in terms of patients, can explain in part the differences encountered between our conclusions and those from the studies mentioned above. The continuous infusion of PESDA contrast increased moderately the specificity for renal aortic ratio criteria from 89.2% to 97.3% but at the expense of a significant decrease of sensitivity from 56.2% to 33.3%. For the another criteria, peak systolic velocity and resistance index the infusion of PESDA decreased mildly or did not affect the sensitivity and specificity. MISSOURIS et al [23] have reported an increase of sensitivity from 85% to 94% and of specificity from 79% to 88% after injection of microbubble Levovist® in hypertensives with renal artery stenosis. In a more recent multicentric study the contrast Levovist did not affect either sensitivity or specificity: sensitivity was 80.0%–83.7%, whereas specificity moderately increased from 80.8% to 83.6% or 86.2%, depending on the subgroups of comparable patients. In two single-center studies in which the value of Doppler US examination after intravenous injection of contrast agents for the diagnosis of renal arterial was also evaluated it was demonstrated an improvement in sensitivity, which increased from 83% to 95% in one study and from 75% to 100% in the other [22,23]. However, both of these studies were based on a limited number of patients with a very low feasibility rate of 11% and 12% at baseline examination, respectively. In addition, Melany et al [22] reported that contrast Levovist injection did not improve specificity, as we also demonstrated with PESDA infusion. There is no consensus whether contrast agent injection potentially reduces examination duration. In our study, we found a significant reduction of mean examination time after contrast infusion (17%). In other study, it was reported that the use of Levovist dramatically reduced the mean examination time from 24.5 minutes to 13.5 minutes [23]. This advantage could be of potential economic interest, but subsequent studies have to confirm more significant differences. PESDA was well tolerated and did not compromise the safety of US. This excellent patient tolerance has already been demonstrated in stress echocardiograph studies that used PESDA as contrast agent [24]. The small number of patients impose some limitations to the present study, However, the high prevalence of renal artery stenosis in this selected group of hypertensives counterbalance this limitation. In conclusion, the detection of renal artery stenosis by Doppler US depends on the criteria used and infusion of PESDA contrast seems not to improve the accuracy, despite a reduction in the examination duration and an increase in specificity based on one Doppler criterion. Also, the feasibility of US is dependent of the quality of the machine, and the infusion of contrast does not add advantages if the performance of the US machine is excellent. However, it remains unknown if the PESDA infusion can improve feasibility if the machine does not have a good imaging quality. So, there is a need for establishing a consensus opinion regarding Doppler useful criteria and thresholds for the diagnosis of renal arterial stenosis, regardless of the US equipment used or infusion of ultrasonographic contrast. ==== Refs Mann SJ Pickering TG Detection of renovascular hypertension. State of the art: 1992 Ann Intern Med 1992 117 845 853 1416561 Rankin SC Saunders AJ Cook GJ Scoble JE Renovascular disease Clin Radiol 2000 55 1 12 10.1053/crad.1999.0338 Textor SC Revascularization in atherosclerotic renal artery disease Kidney Int 1998 53 799 811 9507232 Boijsen E Baums A Renal angiography: techniques and hazards; anatomic and physiologic considerations Vascular and Interventional radiology 1997 4 Little, Brown and Company 1101 1131 Rosner MH Renovascular hypertension: can we identify a population at high risk? South Med J 2001 94 1058 1064 11780674 Safian RD Textor SC Renal-artery stenosis N Engl J Med 2001 344 431 442 11172181 10.1056/NEJM200102083440607 Bloch MJ An evidence-based approach to diagnosing renovascular hypertension Curr Cardiol Rep 2001 3 477 484 11602079 Berland LL Koslin DB Routh WD Keller FS Renal artery stenosis: prospective evaluation of diagnosis with color duplex US compared with angiography. Work in progress Radiology 1990 174 421 423 2404314 Desberg AL Paushter DM Lammert GK Hale JC Troy RB Novick AC Nally J. V., Jr. Weltevreden AM Renal artery stenosis: evaluation with color Doppler flow imaging Radiology 1990 177 749 753 2243982 Helenon O el Rody F Correas JM Melki P Chauveau D Chretien Y Moreau JF Color Doppler US of renovascular disease in native kidneys Radiographics 1995 15 833 54; discussion 854-65. 7569132 Claudon M Plouin PF Baxter GM Rohban T Devos DM Renal arteries in patients at risk of renal arterial stenosis: multicenter evaluation of the echo-enhancer SH U 508A at color and spectral Doppler US. Levovist Renal Artery Stenosis Study Group Radiology 2000 214 739 746 10715040 Olin JW Piedmonte MR Young JR DeAnna S Grubb M Childs MB The utility of duplex ultrasound scanning of the renal arteries for diagnosing significant renal artery stenosis Ann Intern Med 1995 122 833 838 7741367 Halpern EJ Deane CR Needleman L Merton DA East SA Normal renal artery spectral Doppler waveform: a closer look Radiology 1995 196 667 673 7644627 Handa N Fukunaga R Uehara A Etani H Yoneda S Kimura K Kamada T Echo-Doppler velocimeter in the diagnosis of hypertensive patients: the renal artery Doppler technique Ultrasound Med Biol 1986 12 945 952 2950645 10.1016/0301-5629(86)90063-3 Miralles M Cairols M Cotillas J Gimenez A Santiso A Value of Doppler parameters in the diagnosis of renal artery stenosis J Vasc Surg 1996 23 428 435 8601884 Postma CT Bijlstra PJ Rosenbusch G Thien T Pattern recognition of loss of early systolic peak by Doppler ultrasound has a low sensitivity for the detection of renal artery stenosis J Hum Hypertens 1996 10 181 184 8733037 Radermacher J Chavan A Bleck J Vitzthum A Stoess B Gebel MJ Galanski M Koch KM Haller H Use of Doppler ultrasonography to predict the outcome of therapy for renal-artery stenosis N Engl J Med 2001 344 410 417 11172177 10.1056/NEJM200102083440603 Simoni C Balestra G Bandini A Rusticali F [Doppler ultrasound in the diagnosis of renal artery stenosis in hypertensive patients: a prospective study] G Ital Cardiol 1991 21 249 255 1894119 Souza de Oliveira IR Widman A Molnar LJ Fukushima JT Praxedes JN Cerri GG Colour Doppler ultrasound: a new index improves the diagnosis of renal artery stenosis Ultrasound Med Biol 2000 26 41 47 10687791 10.1016/S0301-5629(99)00119-2 Strandness D. E., Jr. Duplex imaging for the detection of renal artery stenosis Am J Kidney Dis 1994 24 674 678 7942827 Nanda NC History of echocardiographic contrast agents Clin Cardiol 1997 20 I7 11 9383596 Melany ML Grant EG Duerinckx AJ Watts TM Levine BS Ability of a phase shift US contrast agent to improve imaging of the main renal arteries Radiology 1997 205 147 152 9314976 Missouris CG Allen CM Balen FG Buckenham T Lees WR MacGregor GA Non-invasive screening for renal artery stenosis with ultrasound contrast enhancement J Hypertens 1996 14 519 524 8761903 Porter TR Li S Kilzer K Smaller intravenous perfluorocarbon-containing microbubbles produce greater myocardial contrast with intermittent harmonic imaging and better delineation of risk area during acute myocardial ischemia J Am Soc Echocardiogr 1997 10 792 797 9356942 Davidson RA Wilcox CS Newer tests for the diagnosis of renovascular disease Jama 1992 268 3353 3358 1453529 10.1001/jama.268.23.3353

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==== Front Int Semin Surg OncolInternational seminars in surgical oncology : ISSO1477-7800BioMed Central London 1477-7800-1-61537738310.1186/1477-7800-1-6ReviewPlanned neck dissection following chemo-radiotherapy in advanced HNSCC Gupta Tejpal [email protected] Jai Prakash [email protected] Department of Radiation Oncology, Clinical Research Centre, Advanced Centre for Treatment Research & Education in Cancer (ACTREC), Tata Memorial Centre, Kharghar, Navi Mumbai: 410208, INDIA2 Department of Radiation Oncology, Tata Memorial Hospital, Parel, Mumbai: 400 012, INDIA2004 17 9 2004 1 6 6 30 6 2004 17 9 2004 Copyright © 2004 Gupta and Agarwal; licensee BioMed Central Ltd.2004Gupta and Agarwal; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Neck dissection has traditionally played an important role in the management of patients with regionally advanced head and neck squamous cell carcinoma (HNSCC) treated with radical radiotherapy alone. However, with the incorporation of chemotherapy in the therapeutic strategy for advanced HNSCC and resultant improvement in outcome the routine use of post chemo-radiotherapy neck dissection is being questioned. Methods Published data for this review was identified by systematically searching MEDLINE, CANCERLIT & EMBASE databases from 1995 until date with restriction to the English language. Results There is lack of high quality evidence on the role of planned neck dissection in advanced HNSCC treated with chemo-radiotherapy. A systematic literature search could identify only one small randomized controlled trial (Level I evidence) addressing this issue, albeit with major limitations. Upfront neck dissection followed by chemo-radiotherapy resulted in better disease-specific survival as compared to chemoradiation only. Several single arm prospective and retrospective reports were also identified with significant heterogeneity and often-contradictory conclusions. Conclusions Planned neck dissection after radical chemo-radiotherapy achieves a high level of regional control, but its ultimate benefit is limited to a small subset of patients only. Unless there are better non-invasive ways to identify residual viable disease, the role of such neck dissection shall remain debatable. A large randomized controlled trial addressing this issue is needed to clarify its role and provide evidence-based answers. chemo-radiotherapyHNSCCand neck dissection ==== Body Introduction The optimal management of the neck in loco-regionally advanced head & neck squamous cell carcinomas (HNSCC) following primary chemo-radiotherapy remains controversial [1,2]. Traditionally neck dissection (Fig 1) was thought to improve neck control in patients with regionally advanced disease (N2–N3 disease) treated with radical radiotherapy alone [3,4]. However, with the incorporation of chemotherapy in the therapeutic strategy for advanced HNSCC and resultant improvement in outcome [5,6], the routine use of post chemo-radiotherapy neck dissection is being questioned [7,8]. Some authors recommend neck dissection for bulky nodal disease after chemo-radiation as part of organ preservation protocol in an elective manner, regardless of the response in the neck provided the primary is controlled. Others argue that it is an ineffective procedure and should be abandoned. Nevertheless, most investigators agree that elective neck dissection be performed for patients with less than a complete response in the neck after combined modality therapy to optimize regional control. This review attempts to provide the discerning reader a bird's eye view of the available evidence on this controversial issue. Figure 1 Sketch anatomy of standard neck incision Methods Literature search strategy Published data for this review was identified by systematically searching the MEDLINE, CANCERLIT & EMBASE databases from 1995 until date with restriction to the English language. "Head & Neck cancer" OR "HNSCC" was combined with "chemo-radiotherapy" OR "chemo-radiation" as Medical Subject Heading (MeSH) terms and each of the following phrase used as text words: "adjuvant neck dissection"; "planned neck dissection"; and "neck management". Relevant cross-references were also considered. Results & Discussion The evidence There is only one small randomized control trial (American Society of Clinical Oncology [9] Level I evidence) evaluating the role of planned neck dissection in advanced HNSCC treated with primary chemo-radiotherapy. Carinci [10] et al randomly assigned patients with advanced unresectable HNSCC to either elective neck dissection followed by chemo-radiotherapy (Group I, n = 23) or chemo-radiotherapy alone (Group II, n = 31). The two groups were reasonably well balanced for known prognostic factors. The 2-and 5-year disease-specific survival rates significantly favored the surgical arm (52% and 26% for Group I versus 29% and 0% for Group II respectively). A Cox regression analysis adjusted for T-stage, N-stage, age and gender showed that only therapy (Group I versus II) reached a positive and significant odds ratio in association with the probability of death (p = 0.0366 in favor of neck dissection). This study however suffers from major limitations. Firstly, the trial methodology was not detailed adequately to assess the validity of the interpretations. The investigators neither specified the method of randomization (why was the distribution unequal in the two arms) nor about stratification on known prognostic factors. Secondly, the numbers of patients in each arm were too small to draw any definite conclusions without ruling out an element of bias. Thirdly, the radiotherapy delivery was suboptimal (only 60–65 Gy with conventional fractionation) for sterilizing advanced HNSCC, in which case the addition of neck dissection was expected to improve outcome. Finally, since neck dissection was done upfront rather than after chemo-radiotherapy, the results of this trial cannot be directly extrapolated to the issue under consideration. In absence of high quality evidence, the best available evidence tempered with clinical judgment often guides decision-making. Two of the recently published reports [11,12] somewhat at contradiction with each other are briefly discussed to illustrate the dilemma. Argiris [11] et al evaluated 131 patients with HNSCC having N2–N3 disease treated on concurrent chemo-radiotherapy protocols. Neck dissection was performed in 92 (70%) patients, either before (n = 31) or after chemo-radiotherapy (n = 61). With a median follow-up of 4.6 years, the 5-year loco-regional progression-free-survival (PFS) was significantly better in patients with planned neck dissection as compared to those without neck dissection (88% versus 74% respectively, p = 0.02) The addition of neck dissection to chemo-radiotherapy resulted in only one neck failure in 92 patients (neck PFS 99%) versus six neck failures in 39 patients (neck PFS 82%) not undergoing neck dissection (p = 0.0007). Neck dissection was however, not beneficial in patients with a complete clinical response (CCR). Of the 92 patients with a CCR, 62 underwent neck dissection, of which only 1 relapsed in the neck (neck PFS 98%). The neck PFS of 92% (2 neck failures) in the 30 patients in CCR who did not undergo neck dissection was not significantly different (p = 0.21). On subset analysis, in patients with N3 disease (n = 27), there was either a trend or a statistically significant advantage in all the survival parameters for the neck dissection arm. In contrast, in patients with N2 disease (n = 104), only the neck control improved with neck dissection. The local PFS, distant PFS, and the overall survival were similar irrespective of neck dissection. The authors concluded that in patients with N3 stage and less than CCR it was necessary to add neck dissection for optimal disease control, whereas in patients with N2 disease in CCR, neck dissection could safely be omitted without compromising outcome. Brizel [12] et al identified 108 patients with nodal disease from a cohort of 154 patients on concurrent chemo-radiation protocols. A modified neck dissection was performed in 65 (60%) of 108 patients. With a median follow up of 4 years for surviving patients, the neck control rate was 100% for N1 patients irrespective of neck dissection being performed or not. Their disease-free-survival (DFS) was 70% with no differences relative to neck dissection. In N2–N3 patients, a CCR was achieved in 43 (55%) patients. Ten patients with local progression or systemic dissemination were excluded from analysis. Of the 52 patients undergoing neck dissection in N2–N3 group, only 1 regional relapse was seen, in contrast to 3 neck failures out of 16 in those not undergoing dissection (p = 0.05). The 4-year DFS was 75% for N2–N3 patients with a CCR and neck dissection versus 53% for those with CCR but no neck dissection (p = 0.08). The 4-year overall survival was also better for the dissection arm (77% versus 50% respectively, p = 0.04). The authors concluded that the policy of neck dissection in patients with N2–N3 disease even in CCR is justified to optimize loco-regional control and survival. Apart from the afore-mentioned two reports, there are a few reasonably large studies (involving >50 patients: Table 1) and several smaller ones, both prospective and retrospective published in the last decade trying to define the benefit of such intervention with conflicting results [2,7,13-24]. However, significant heterogeneity in selection criteria as well as variable treatment schedules and response assessment methodology amongst these reports introduces a great deal of bias precluding any definitive conclusions. Table 1 Neck failure in selected series of chemo-radiotherapy for HNSCC treated with or without ND Author (year) No of pts (n) Pts in CCR Neck failures (overall) Neck failure (pts in CCR) Remark(s) ND done ND not done ND done ND not done McHam2 (2003) N2–N3: 109 65 5/76 4/33 1/32 4/33 ND needed for all N2–N3 patients Grabenbauer7 (2003) N0–N3: 142 97 Only patients with CCR offered ND 9/56 4/41 No clear benefit of ND after CCR Clayman13 (2001) N2–N3: 66 29 5/18 6/48 0/4 0/25 ND not needed for patients in CCR Stenson14 (2000) N2–N3: 69 30 1/69 NA 0/30 NA All 69 pts had ND; needed for N2–N3 Robbins15 (1999) N2–N3: 52 (56 heminecks) 33 1/34 2/20 0/16 0/17 Good control with ND for N2–N3 Lavertu16 (1999) N1–N3: 78 55 8/78 neck failures in all NK NK ND done for all pts with N2–N3 Lavertu17 (1997) N0–N1: 47 43 0/6 4/38 0/5 4/38 ND needed for all N2–N3 patients even in CCR N2–N3: 53 30 1/35 3/12 0/18 3/12 ND = neck dissection; CCR = clinical complete response; pts = patients; NA = not applicable; NK = not known The potential benefit of planned neck dissection after a course of intensive chemo-radiotherapy in terms of improved regional control with or without an impact on survival needs to be weighed against the expected morbidity associated with the surgical procedure [12,14,25]. One argument put forward in favor of planned neck dissection even for patients in CCR is the high rate of pathological positivity (30%–50%) depending upon the meticulousness of sectioning by the pathologist [14,17]. However, a significant majority of them actually may represent microscopic non-viable residual disease only, as has been demonstrated by Strasser using Ki-67 proliferating index [26], unlikely to relapse later. Proponents of neck dissection also argue that the ultimate success rate of salvage neck dissection after a relapse in the neck treated with full dose chemo-radiotherapy is small, whereas the morbidity is high [10,25]. In contrast, the morbidity of a planned neck dissection is at best modest, when scheduled between 6–12 weeks from end of chemo-radiotherapy [7,14,17], which is supposed to be the time window between acute and chronic radiation injury. Conclusions & Recommendations Planned neck dissection after radical chemo-radiotherapy achieves a high level of regional control, but its ultimate benefit is limited to a small subset of patients only. The morbidity of such dissection is small, but significant. Its impact on survival is yet to be completely realized. In the majority of patients it is either unnecessary because there is no residual disease in the neck or futile because of unsalvageable primary recurrence or distant metastases. Nevertheless, it is recommended that planned neck dissection be performed for patients with less than a complete response in the neck after combined modality therapy to optimize regional control, provided the primary is controlled and there is no evidence of distant metastases. It should also be performed as part of salvage surgery for locally persistent or residual disease at primary site. The criterion for planned neck dissection for patients with advanced nodal disease with a CCR in the neck following chemo-radiotherapy should incorporate not only the nodal staging but also the actual size of the involved lymph nodes. Unless there are better non-invasive ways to identify residual viable disease, which could include functional imaging like Positron Emission Tomography and biological assays like hypoxia markers, the role of such neck dissection shall remain debatable. A large randomized controlled trial across several institutions addressing these issues is needed to clarify the role of planned neck dissection in advanced HNSCC treated with primary chemo-radiotherapy and provide evidence-based answers. Source of funding No source of funding involved in this review Competing interest or Conflict of interest None declared. Authors' Contributions Dr JP proposed the idea of systematic review on the issue Dr TG did the literature search & prepared the manuscript Dr JP critically reviewed and revised the manuscript ==== Refs Garg M Beitler JJ Controversies in Management of the Neck in Head and Neck Cancer Curr Treat Options Oncol 2004 5 35 40 14697155 McHam SA Adelstein DJ Rybicki LA Lavertu P Esclamado RM Wood BG Strome M Carroll MA Who merits a neck dissection after definitive chemoradiotherapy for N2–N3 squamous cell head and neck cancer Head Neck 2003 25 791 798 12966502 10.1002/hed.10293 Parsons JT Mendenhall WM Cassisi NJ Stringer SP Million RR Neckdissection after twice-a-day radiotherapy: Morbidity and recurrence rates Head Neck 1989 11 400 404 2807879 Mendenhall WM Villaret DB Amdur RJ Hinerman RW Mancuso AA Planned neck dissection after definitive radiotherapy for squamous cell carcinoma ofthe head and neck Head Neck 2002 24 1012 1018 12410537 10.1002/hed.10187 Pignon JP Bourhis J Domenge C Designe L Chemotherapy added to loco-regional treatment for head and neck squamous-cell carcinoma: Three meta-analyses of updated individual data (for the Meta-Analysis of Chemotherapy on Head and Neck Cancer (MACH-NC) Collaborative group Lancet 2000 355 949 955 10768432 10.1016/S0140-6736(00)90011-0 Browman GP Hodson DI Mackenzie RJ Bestic N Zuraw L Cancer Care Ontario Practice Guideline Initiative Head and Neck Cancer Disease Site Group Choosing a concomitant chemotherapy and radiotherapy regimen for squamous cell head and neck cancer: A systematic review of the published literature with subgroup analysis Head Neck 2001 23 579 589 11400247 10.1002/hed.1081 Grabenbauer GG Rodel C Ernst-Stecken A Brunner T Hornung J Kittel K Steinhart H Iro H Sauer R Schultze-Mosgau S Neck dissection following radiochemotherapy of advanced head and neck cancer-for selected cases only? Radiother Oncol 2003 66 57 63 12559521 10.1016/S0167-8140(02)00193-7 Corry J Smith JG Peters LJ The concept of a planned neck dissection is obsolete Cancer J 2001 7 472 474 11769857 American Society of Clinical Oncology (ASCO) Clinical practice guidelines for the use of chemotherapy and radiotherapy protectants J Clin Oncol 1999 17 3333 3355 10506637 Carinci F Cassano L Farina A Pelucchi S Calearo C Modugno V Nielsen I Api P Pastore A Unresectable primary tumor of head and neck: does neck dissection combined with chemoradiotherapy improve survival J Craniofac Surg 2001 12 438 443 11572248 10.1097/00001665-200109000-00007 Argiris A Stenson KM Brockstein BE Mittal BB Pelzer H Kies MS Jayaram P Portugal L Weing BL Rosen FR Haraf DJ Vokes EE Neck dissection in the combined-modality therapy of patients with locoregionally advanced head and neck cancer Head Neck 2004 26 447 455 15122662 10.1002/hed.10394 Brizel DM Prosnitz RG Hunter S Fisher SR Clough RL Downey MA Scher RL Necessity for adjuvant neck dissection in setting of concurrent chemoradiation for advanced head-and-neck cancer Int J Radiat Oncol Biol Phys 2004 58 1418 1423 15050318 10.1016/j.ijrobp.2003.09.004 Clayman GL Johnson CJ II Morrison W Ginsberg L Lippman SM The role of neck dissection after chemoradiotherapy for oropharyngeal cancer with advanced nodal disease Arch Otolaryngol Head Neck Surg 2001 127 135 139 11177029 Stenson KM Haraf DJ Pelzer H Recant W Kies MS Weichselbaum RR Vokes EE The role of cervical lymphadenectomy after aggressive concomitant chemoradiotherapy: The feasibility of selective neck dissection Arch Otolaryngol Head Neck Surg 2000 126 950 956 10922226 Robbins KT Wong FSH Kumar P Hartsell WF Vieira F Mullins B Barry Niell H Efficacy of targeted chemoradiation and planned selective neckdissection to control bulky nodal disease in advanced head and neck cancer Arch Otolaryngol Head Neck Surg 1999 125 670 675 10367925 Lavertu P Adelstein DJ Saxton JP Sesic M Eliachar I Strome M Larto MA Wood BG Aggressive concurrent chemo-radiotherapy for squamous cell head and neck cancer: An 8-year single-institution experience Arch Otolaryngol Head Neck Surg 1999 125 142 148 10037279 Lavertu P Adelstein DJ Saxton JP Secic M Wanamaker JR Eliachar I Wood BG Strome M Management of the neck in a randomized trial comparing concurrent chemotherapy and radiotherapy with radiotherapy alone in resectable stage III and IV squamous cell head and neck cancer Head Neck 1997 19 559 566 9323143 10.1002/(SICI)1097-0347(199710)19:7<559::AID-HED1>3.3.CO;2-L Wanebo H Chougule P Ready N Safran H Ackerley W Koness RJ McRae R Nigri P Leone L Radie-Keane K Reiss P Kennedy T Surgical resection is necessary to maximize tumor control in function-preserving, aggressive chemoradiation protocols for advanced squamous cancer of the head and neck (stage III and IV) Ann Surg Oncol 2001 8 644 650 11569779 10.1245/aso.2001.8.8.644 Newkirk KA Cullen KJ William Harter K Picken CA Sessions RB Davidson BJ Planned neck dissection for advanced primary head and neckmalignancy treated with organ preservation therapy: disease control and survival outcomes Head Neck 2001 23 73 79 11303636 10.1002/1097-0347(200102)23:2<73::AID-HED1001>3.0.CO;2-6 Ahmed KA Robbins KT Wong F Salazar JE Efficacy of concomitant chemoradiation and surgical salvage for N3 nodal disease associated with upper aero-digestive tract carcinoma Laryngoscope 2000 110 1789 1793 11081585 10.1097/00005537-200011000-00002 Sanguineti G Corvo R Benasso M Margarino G Sormani MP Roncallo F Mereu P Bacigalupo A Vitale V Management of the neck after alternating chemoradiotherapy for advanced head and neck cancer Head Neck 1999 21 223 228 10208665 10.1002/(SICI)1097-0347(199905)21:3<223::AID-HED7>3.3.CO;2-R Weisman RA Christen RD Jones VE Kerber CW Seagren SL Orloff LA Glassmeyer SL Howell SB Robbins KT Observations on control of N2 and N3 neck disease in squamous cell carcinoma of the head and neck by intra-arterial chemoradiation Laryngoscope 1998 108 800 805 9628492 10.1097/00005537-199806000-00005 Dagum P Pinto HA Newman JP Higgins JP Terris DJ Goffinet DR Fee WE Jr Management of the clinically positive neck in organ preservation for advanced head and neck cancer Am J Surg 1998 176 448 452 9874431 10.1016/S0002-9610(98)00240-2 Koch WM Lee DJ Eisele DW Miller D Poole M Cummings CW Forastiere AA Chemo-radiotherapy for organ preservation in oral and pharyngeal carcinoma Arch Otolaryngol Head Neck Surg 1995 121 974 980 7646865 Narayan K Crane CH Kleid S Hughes PG Peters LJ Planned neck dissection as an adjunct to the management of patients with advanced neck disease treated with definitive radiotherapy: For some or for all? Head Neck 1999 21 606 613 10487947 10.1002/(SICI)1097-0347(199910)21:7<606::AID-HED4>3.3.CO;2-7 Strasser MD Gleich LL Miller MA Saavedra HI Gluckman JL Management implications of evaluating the N2 and N3 neck after organ preservation therapy Laryngoscope 1999 109 1776 1780 10569406 10.1097/00005537-199911000-00010

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==== Front Reprod Biol EndocrinolReproductive biology and endocrinology : RB&E1477-7827BioMed Central London 1477-7827-2-651535299810.1186/1477-7827-2-65ResearchThe C677T methylenetetrahydrofolate reductase variant and third trimester obstetrical complications in women with unexplained elevations of maternal serum alpha-fetoprotein Björklund Natalie K [email protected] Jane A [email protected] Cheryl R [email protected] Lorne E [email protected] Carol E [email protected] Bernard N [email protected] Department of Biochemistry & Medical Genetics, University of Manitoba, Winnipeg, Canada2 Department of Pediatrics & Child Health, University of Manitoba, Winnipeg, Canada3 Department of Community Health Sciences, University of Manitoba, Winnipeg, Canada4 Department of Obstetrics and Gynecology & Reproductive Sciences, University of Manitoba, Winnipeg, Canada2004 7 9 2004 2 65 65 27 5 2004 7 9 2004 Copyright © 2004 Björklund et al; licensee BioMed Central Ltd.2004Björklund et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Introduction The C677T MTHFR variant has been associated with the same third trimester pregnancy complications as seen in women who have elevations of maternal serum α-fetoprotein (MSAFP). We hypothesized that these women with third trimester pregnancy complications and MSAFP elevations would have an increased frequency of the variant compared to an abnormal study control group (women with MSAFP elevations without pregnancy complications) as well as to normal population controls. Methods Women who had unexplained elevations of MSAFP in pregnancy were ascertained retrospectively. The frequency of the C677T MTHFR variant among those women with unexplained elevations of MSAFP who had experienced later pregnancy complications was compared to that of women with unexplained elevations of MSAFP without complications as well as to that of the previously established Manitoba frequency. Results Women who had complications of pregnancy and an unexplained MSAFP elevation had a higher allele frequency for the C677T MTHFR variant (q = 0.36,) compared to women with MSAFP elevations and normal pregnancy outcomes (q = 0.25, OR 1.73 95% CI 1.25–2.37, p = 0.03). The frequency was also higher than that of the population controls (q= 0.25, OR 1.70 95% CI 1.11–2.60, p = 0.007). The frequency in women with MSAFP elevations without pregnancy complications was not significantly different from that of the population controls (p = 0.41). Conclusion Women with unexplained elevations of MSAFP and who experience complications in later pregnancy are more likely to have one or two alleles of the C677T MTHFR variant. ==== Body Background Significant elevations of amniotic fluid and maternal serum alpha-fetoprotein (MSAFP) have been shown to be associated with spina bifida and other neural tube defects (NTD). The province of Manitoba, Canada offers province-wide midtrimester MSAFP screening to all pregnant women. It has been recognized that some pregnant women with midtrimester unexplained elevations of MSAFP [1,2]. Increased total plasma homocysteine alters placental function and has been associated with the same complications that are associated with unexplained elevated MSAFP [3-7]. The C677T MTHFR variant has also been associated with complications of pregnancy in some, but not all, studies [8-10] C677T MTHFR may therefore contribute to complications of pregnancy by elevating serum homocysteine. Poor placental function could result in both an unexplained elevation of MSAFP and complications of pregnancy. We therefore hypothesized that women with third trimester pregnancy complications and MSAFP elevations (cases) would have an increased frequency of the variant compared to the Manitoba population (population controls) or women with MSAFP elevations without pregnancy complications (study controls) if they had low folate intake. Methods Background to methods In a small pilot study of 32 couples, we found that women who had an unexplained elevation of MSAFP and a normal midtrimester fetal ultrasound, and their partners, had a significantly increased C677T MTHFR frequency compared to Manitoba newborns (RR 1.42, 95% CI 1.08–1.85, p = 0.012, two tailed) [11]. The newborn study that examined 977 anonymous consecutive neonatal screening blood spots showed that 36% of Manitoba newborns were heterozygous and 7% were homozygous for C677T MTHFR [12] (q = 0.25). Subsequently, on evaluation of the pregnancy outcomes of our pilot study women, we noted that, among eight women who had gone on to experience complications of pregnancy, the odds ratio for having the C677T MTHFR allele was 2.3 times higher than in the Manitoba population. However, the result was not statistically significant (p = 0.151, two tailed) indicating the frequency was increased but, this could have been a random result. Ascertainment and recruitment of study population All pregnant women in Manitoba are eligible for routine serum screening through the voluntary MMSSP. In Manitoba, an elevation of MSAFP is defined as 2.3 multiples of the median (MOM) or greater. Candidates for inclusion in this study were women with an unexplained MSAFP elevation (i.e. not due to fetal anomalies, incorrect estimation of gestational age, previously unrecognized fetal demise, or multiple gestation) with either a complicated or uncomplicated pregnancy outcome. After appropriate approvals had been obtained from The University of Manitoba Health Research Ethics Board, review of the screening records began in 1999 and took three years. For a study using a two step consent to participate methodology administered by mail, the expected response rate (after excluding lost to follow-up) would be 20% [13]. Our goal was 1000 invitations. We anticipated this would result in approximately 120 participants. This would be double the minimum number of participants suggested by the power analysis we had conducted for the pilot study. To increase our response rate further, we added telephone follow-up for invited potential participants who were non-responders [14]. All screening records from 1995–1999 were reviewed, accounting for 783 invitations. Records for 2000–2002 were reviewed systematically as outcome information on each pregnancy became available to MMSSP. Records for 1990–1994 were then reviewed systematically in order to bring the total up to 1000. If a woman had more than one pregnancy with an elevation of MSAFP screened by the MMSSP, only the first pregnancy encountered in the retrospective review was used for the study. Previous or subsequent pregnancies were not included. Women with preexisting conditions known to influence pregnancy outcome, such as essential hypertension, and mothers of babies with major congenital anomalies were excluded. Eight women who had relinquished their babies for adoption or whose babies were placed in foster care were also excluded. Women who met the inclusion criteria were divided into two groups for analysis. Cases were defined as women with pregnancies complicated by one of the complications previously shown to be associated with an unexplained elevation of MSAFP at midtrimester [2]. These include: intrauterine growth restriction (IUGR) (<10th percentile), pregnancy induced hypertension, preeclampsia, eclampsia, postpartum hemorrhage, retained placenta requiring manual delivery, abruptio placenta, premature delivery (<36 weeks gestation or requiring specialized neonatal care for prematurity) and unexplained fetal demise. Study controls were women with normal outcomes which were defined as those with delivery at term ≥ 36 weeks gestation), no complications of pregnancy, a normal placenta and a healthy baby. Definition of complications was based on ICDC-9 codes in the MMSSP outcome charts for each patient [15] which are then confirmed later by chart review for all those with a positive MMSSP result. All women ascertained as having unexplained MSAFP elevations and who fit the inclusion criteria above, were invited by letter to participate. The previously reported newborn study provided population control group data [12]. Study questionnaires Women who agreed to participate in the study were mailed the appropriate questionnaires and blood requisitions. The questionnaire included a semi-quantitative food frequency questionnaire (FFQ) based on standard methodology but, modified to suit Manitoba residents and previously validated for this population by biochemical analysis during the pilot study [11,16]. The survey included questions on vitamin supplement intake to determine preconceptional or prenatal supplementation as well as current use of vitamins. Dietary intake of folate and folic acid from supplements, and intake of the cofactors B12 and B6, were calculated from the FFQ for intake both during pregnancy and at the time of the study. A correction of an additional 0.1 mg for folic acid fortification that began in Canada in 1998 was included for pregnancies that began after fortification [17]. FFQ analyses were performed with the researcher blinded as to the status of the mother. Laboratory analysis Total plasma homocysteine, red blood cell folate, and serum folate were determined using established methodology [18,19]. Samples were processed on site with clotting and separation by spinning. Sera was stored at 4°C during shipping to the central laboratory and until processing. DNA was extracted from whole blood and C677T MTHFR genotyping was performed using previously established methodology [11,20,21]. Genotyping and biochemical analyses were performed also blinded. Statistical analysis Chi-squared analysis (one tailed unless otherwise noted) was used for allele frequency. Comparisons of potentially confounding factors between the case group and the study control group were undertaken. Parametric data were analyzed with the Student's t test for difference between means with Bonferroni correction for multiple comparisons. Data not normally distributed were analyzed using the nonparametric Mann-Whitney Rank Sum Test. Linear regression was used to test the validity of the dietary survey. A multivariate analysis included age, smoking, maternal weight at the time of MSAFP testing, presence of C677T MTHFR, gender and weight of infant, biochemical parameters, and FFQ results for folate, B12 and B6, both at the time of the survey and for during the pregnancy was undertaken. In order to avoid convergence due to the large number of variables, the analysis was completed in subsets of six variables. Variables with the higher association scores from these analyses were then combined for further testing in various combinations using stepwise multiple linear regression. Also linear regression analysis of each continuous variable with genotype results was performed. Corrections for multiple comparisons were included. Software used was NCSS Statistical Systems for Windows [22]. Results Participation rates Nine hundred and ninety four women were identified as eligible (342 cases and 652 controls). Of the 590 women successfully contacted, 130 (22%) agreed to participate (56 cases and 74 controls). Four hundred and four women were lost to follow-up. Cases were more likely to choose to participate than controls and this difference was significant (1.5, p = 0.030). There was no difference in the proportions of cases and controls that were lost to follow-up (p = 0.157). We had anticipated a 20% response rate and we achieved 24%. Genotype results Genotypes were available for 54 cases and 73 controls for this analysis. Results are summarized in Table 1. The allele frequency for the C677T MTHFR variant in the Manitoba population has been previously established to be q = 0.25. Women who had complications of pregnancy and an unexplained MSAFP elevation had a higher allele frequency for the C677T MTHFR variant (q = 0.36) compared to women with MSAFP elevations and normal pregnancy outcomes (q = 0.25, OR 1.73 95% CI 1.25–2.37, p = 0.03). The frequency was also higher than in the population controls (q = 0.25, OR 1.70 95% CI 1.11–2.60, p = 0.007). The frequency in women without pregnancy complications and MSAFP elevations (study controls) was not significantly different than that seen in population controls (p = 0.41). Table 1 Comparison of allele frequency of C677T MTHFR between cases, study controls, and population controls. Subjects C/C (%) C/T (%) T/T (%) Comparing to study controls* OR (95%CI) Comparing to population* OR (95%CI) Cases N = 54 21 (39) 27 (50) 6 (11) 1.73 (1.25–2.37) (p = 0.033) 1.70 (1.11–2.60) (p = 0.007) Study Controls N = 73 40 (55) 30 (41) 3 (4) ~ 0.98 (0.46–1.55) (p = 0.410) Population N = 977 557 (57) 352 (36) 68 (7) 0.98 (0.46–1.55) (p = 0.410) ~ * χ2 comparison of allele frequency (total T and C) in each group, one tailed. Cases: women with unexplained elevations of MSAFP who had subsequent complications of pregnancy, (C = 69, T = 39) Controls: women who had unexplained elevations of MSAFP and no subsequent complications (C = 110, T = 36) Population controls were 977 newborns (C = 1466, T = 488) [12]. C/C = normal type, C/T = heterozygous for thermolabile variant, T/T = homozygous for thermolabile variant. The case and study control groups included women at various stages of their child bearing years. Only one woman recruited as a control subject had a previous or subsequent pregnancy with an unexplained elevation of MSAFP and complications. She was a heterozygote for C677T MTHFR. No case subjects had a previous or subsequent pregnancy with an unexplained elevation of MSAFP and a normal outcome, but four case subjects had had a previous or subsequent pregnancy with complications after an elevated MSAFP. If the case versus control classification had been based on whether or not a woman had ever had a pregnancy with an unexplained elevation of MSAFP followed by complications, the association would still be present when compared to the population control (q = 0.3636, OR 1.72, 95%CI 1.27–2.61, p = 0.0055). Biochemical results Heterozygotes and homozygotes for C677T MTHFR had lower average values (r = 0.978, p = 0.019) for serum folate than those who did not have the variant. None of the women were deficient in either serum folate (defined as <7.0 nmol/L) or red blood cell folate (defined as <430 nmol/L RBC). There was no significant difference in mean homocysteine levels (Table 2). Table 2 Comparison of the parametric characteristics of women with unexplained elevations of MSAFP according to those with and without complications of pregnancy Characteristic Mean Cases (SD) Mean Controls (SD) p value MSAFP result 2.78 (± 0.62) 3.16 (± 3.76) 0.398 weeks gestation 17.1 (± 1.61) 16.9 (± 1.40) 0.432 μg/folate/day in pregnancy2 1216 (± 915) 1010 (± 892) 0.206 μg/folate/day at time of study2 557 (± 341) 523 (± 498) 0.588 erc folate (nmol/L RBC) 1234 (± 289) 1208 (± 317) 0.632 serum folate (nmol/L) 32.3 (± 5.80) 32.3 (± 5.71) 0.956 serum homocysteine (μmol/L) 7.8 (± 2.26) 8.4 (± 2.80) 0.246 μg B12/day in pregnancy2 12.4 (± 5.37) 13.4 (8.31) 0.488 μg B12/day at time of study2 8.9 (± 12.26) 8.6 (± 10.83) 0.899 mg B6/day in pregnancy2 8.4 (± 9.69) 7.2 (± 9.01) 0.461 mg B6/day at time of study2 6.0 (± 13.35) 5.5 (± 11.10) 0.792 mother's age at delivery 31 (± 4.19) 30 (± 5.19) 0.251 mother's weight in Kg 76 (± 17.26) 69 (± 16.09) 0.0131 1 This value is not significant after Bonferroni correction for multiple comparisons. See discussion. 2Data was skewed due to a small number of women in both groups taking large dose vitamin supplements. When these women were removed from the analysis the result remained nonsignificant. Validity of surveys Seven cases and one study control declined to fill out their dietary surveys. Mean values were inserted in the multivariate analysis for these eight women. The validity of the dietary survey was demonstrated again for this study by linear regression analysis. Consistent with known homocysteine metabolism [23], a negative correlation existed between serum homocysteine and both red blood cell folate (r = 0.945 p = 0.0052) and serum folate (r = 0.932, p = 0.0001). Higher intake of dietary folate (including synthetic folic acid from supplements) as reported by the FFQ for the time of study was associated with higher serum folate (r = 0.941, p = 0.0001) and higher red blood cell folate (r = 0.949, p = 0.0166). We did several checks to determine that the women were answering their surveys accurately. Comparisons of specific data items available in the women's MMSSP charts at the time of pregnancy with the data reported in the surveys showed excellent agreement for every item examined indicating women answered questions accurately. Women who reported smoking (as a quantitative value from 0–3 based on 1/2 packs/day smoked) showed a negative correlation with serum folate (r = 0.923, p = 0.0062) consistent with accurately reporting their smoking habits [24]. Based on the results of these tests of the validity of our surveys, we are confident that the information provided by our participants was accurate. Analysis of FFQ survey and MMSSP data The ethnicity of the infants born to the case mothers (based on the ethnicity reported for the infants grandparents) was 84% Caucasian, 5% Aboriginal. Mixed ethnicity was reported for 11% of the infants with one parent Caucasian and the other parent Aboriginal, or rarely Black or Asian. The ethnic distribution was the same for controls and is typical for the Manitoba population [25,26]. There were also no significant differences between cases and controls with respect to their place of residence within the province (such as rural versus urban address). There were no significant differences in dietary and supplemental intake of folate, B12, or B6, or in the biochemical parameters of case and control mothers. There was no difference in the percentage of cases and controls who reported taking prenatal vitamin supplements during pregnancy (37/48 cases and 55/72) or taking vitamin and/or folate supplements preconceptionally (17/48 cases and 25/72 study controls). We attempted to divide our cases into smaller groups by type of pregnancy complication. We also separated isolated IUGR and IUGR associated with hypertensive disorders of pregnancy. Most of the groups lacked power for statistical analysis due to small numbers. However, normotensive women whose fetus had IUGR (N = 12) had a higher frequency of the C677T MTHFR variant compared to the population controls (q = 0.33, OR 2.58 95% CI, 1.78–3.73, p = 0.013). Homozygosity for the C677T MTHFR variant is associated with IUGR in women who do not take vitamin supplements according to one large study of Canadian women [10,27]. Our findings are in agreement with this result as only 3/12 women took supplements. We found this effect in a group of combined heterozygous and homozygous women. There was a trend towards higher mean weight for mothers who had complications at the time of MSAFP test in the individual comparisons, but this was not significant after correction for multiple comparisons (Table 2 and 3). The multivariate analysis did not reveal any unexpected associations, but it did show the importance of maternal weight as a variable (r = 0.933, p = 0.024). This was also not unexpected given that some of the complications we were examining are associated with obesity [28]. Even after controlling for women's weight in the multivariate analysis, the higher frequency of C677T MTHFR among cases remained significant (r = 0.734, p = 0.0462). There was no association between weight and MTHFR status (r = 0.431, p = 0.679). Table 3 Comparison of the nonparametric characteristics of women with unexplained elevations of MSAFP according to those with and without complications of pregnancy. Nonparametric Characteristics Cases Controls p value Location  Winnipeg 44 63 0.854  southern city 3 2  southern town 3 5  southern rural 8 8  northern city 2 1  northern town 1 4  northern rural 5 7 ethnicity 0.972  Caucasian 52 66  Aborginal 2 2  Mixed Caucasian/Aboriginal 7 6  Mixed Caucasian/Black 1 3  Asian 1 1  Mixed Caucasian/Asian 1 1  Unknown 3 1 diabetes in pregnancy 2/67 1/80 0.207 maternal smoking present (0 = nonsmoker, 1–3 = half pks/day increments) 0 = 47, 1 = 11, 2 or more = 9 0 = 60, 1 = 8, 2 or more = 12 0.721 gender of baby 27 females, 40 males 42 females, 38 males 0.352 parity = number of women 0 = 37, 1 = 20, 2 = 7, 3 or more = 3 0 = 40, 1 = 29, 2 = 7, 3 or more = 4 0.254 previous miscarriages 11/67 15/80 0.203 previous case pregnancy 1 0 0.967 Discussion Unexplained elevations in MSAFP are known to be associated with an increased risk for complications of pregnancy [2]. Others have reported that presence of the C677T MTHFR variant in pregnant women with low folate intake is associated with increased risk for pregnancy complications [2,29-31]. The unique finding of this study is an increase in the frequency of the C677T MTHFR variant among women with normal folate intake, who went on to have complications of pregnancy after an unexplained elevation of MSAFP (Table 1). The lack of folate deficiency in this population was unexpected, given previous research which showed that 23.6% of Newfoundland and Labrador women are folate deficient at their first prenatal visit [32]. As our study was retrospective, we did not have data on levels during pregnancy. It has recently been shown that the C677T MTHFR variant does not affect maternal serum homocysteine levels in pregnancy among women who take prenatal multivitamins [8]. Also a recent prospective study shows that there is no difference in homocysteine levels at midtrimester between women who later develop preeclampsia and those who do not [33]. As is the situation with NTDs, lack of folate deficiency by current definitions in a non-pregnant woman may not indicate that her folate intake is adequate for pregnancy. This would especially be true for women with the C677T MTHFR variant. Reexamination of the current definition of what constitutes a normal biochemical result for folate intake for women of child bearing age should be undertaken to clarify this. We suggest that the negative effects of the C677T MTHFR variant are more likely to occur in early pregnancy before women began taking prenatal vitamins because the majority of our study participants took prenatal vitamins, but only 35% took preconceptional supplements. We suspect that reduced methylation interfering with cell proliferation in the placenta as originally suggested by Eskes (2000) [3]. In conclusion, using a retrospective case/control study, we have found that women with unexplained MSAFP elevations who have complications in later pregnancy are more likely to have the C677T MTHFR allele. Our resultsdo not suggest that C677T MTHFR predisposes a woman to having an elevation of MSAFP level (as we did not compare the C677T MTHFR frequency in women with and without elevated MSAFP), but having one or more copies of this variant predisposes such screen positive women to having complications in later gestation. It remains to be seen if other risk factors can be identified which can more accurately define this high risk group. Authors' contributions All authors participated in original study design except CS. CG, BC and CS all acted as principal investigators for funding. NB assisted with all grant proposals. NB undertook the review of the individual MSAFP files and MMSSP database searches, designed the dietary and family history surveys, classified cases and controls, acted as study coordinator handling all aspects of participant contact, and provided data analysis. All authors also participated actively with NB for various aspects of the study. CG provided MTHFR genotyping. LS provided biochemical analysis. NB drafted the original manuscript with assistance from BC. CG and BC handled ethics approval assisted by NB. All authors read and approved the final manuscript. Acknowledgments We would like to thank R Singal, C Mesa, C Richmond, L Erdile, S Marles, A Chudley, C McLean, M Henke, K MacDonald, M Coggrave and M Baird for assistance with this study. We would also like to thank our participants, without whom such work would not be possible. This study was funded by the Children's Hospital Foundation of Manitoba, the Spina Bifida and Hydrocephalus Association of Canada, the Garrod Association of Canada, the Manitoba Medical Services Foundation, and University of Manitoba Graduate Fellowships. ==== Refs Ross HL Elias S Maternal serum screening for fetal genetic disorders Obstet Gynecol Clin North Am 1997 24 33 47 9086517 van Rijn M van der Schouw YT Hagenaars AM Visser GH Christiaens GC Adverse obstetric outcome in low-and high-risk pregnancies: predictive value of maternal serum screening Obstet Gynecol 1999 94 929 934 10576178 10.1016/S0029-7844(99)00467-6 Eskes TK Homocysteine and human reproduction Clin Exp Obstet Gynecol 2000 27 157 167 11214939 Eskes TK Clotting disorders and placental abruption: homocysteine–a new risk factor Eur J Obstet Gynecol Reprod Biol 2001 95 206 212 11301173 10.1016/S0301-2115(00)00492-9 Raijmakers MT Zusterzeel PL Roes EM Steegers EA Mulder TP Peters WH Oxidized and free whole blood thiols in preeclampsia Obstet Gynecol 2001 97 272 276 11165594 10.1016/S0029-7844(00)01127-3 Ferguson SE Smith GN Walker MC Maternal plasma homocysteine levels in women with preterm premature rupture of membranes Med Hypotheses 2001 56 85 90 11133260 10.1054/mehy.2000.1116 Vollset SE Refsum H Irgens LM Emblem BM Tverdal A Gjessing HK Monsen AL Ueland PM Plasma total homocysteine, pregnancy complications, and adverse pregnancy outcomes: the Hordaland Homocysteine study Am J Clin Nutr 2000 71 962 968 10731504 Powers RW Dunbar MS Gallaher MJ Roberts JM The 677 C-T methylenetetrahydrofolate reductase mutation does not predict increased maternal homocysteine during pregnancy Obstet Gynecol 2003 101 762 766 12681883 10.1016/S0029-7844(02)03120-4 Morrison ER Miedzybrodzka ZH Campbell DM Haites NE Wilson BJ Watson MS Greaves M Vickers MA Prothrombotic genotypes are not associated with pre-eclampsia and gestational hypertension: results from a large population-based study and systematic review Thromb Haemost 2002 87 779 785 12038776 Infante-Rivard C Rivard GE Yotov WV Genin E Guiguet M Weinberg C Gauthier R Feoli-Fonseca JC Absence of association of thrombophilia polymorphisms with intrauterine growth restriction N Engl J Med 2002 347 19 25 12097536 10.1056/NEJM200207043470105 Björklund NK Evans JA Greenberg CR Association of thermolabile methylenetetrahydrofolate reductase (C677T) with elevated maternal serum alpha fetoprotein. [Abstract] Am J Hum Genet 2000 67 417 10877980 10.1086/302999 Mogk RL Rothenmund H Evans JA Carson N Dawson AJ The frequency of the C677T substitution in the methylenetetrahydrofolate reductase gene in Manitoba Clin Genet 2000 58 406 408 11140843 10.1034/j.1399-0004.2000.580513.x Angus VC Entwistle VA Emslie MJ Walker KA Andrew JE The requirement for prior consent to participate on survey response rates: a population-based survey in Grampian BMC Health Serv Res 2003 3 21 14622444 10.1186/1472-6963-3-21 Fowler FJ JrGallagher PM Stringfellow VL Zaslavsky AM Thompson JW Cleary PD Using telephone interviews to reduce nonresponse bias to mail surveys of health plan members Med Care 2002 40 190 200 11880792 10.1097/00005650-200203000-00003 International Classification of Diseases, 9th revision, Clinical Modification 1994 4 Utah: Medicode Publications Björklund NK Evans JA Greenberg CR Folic acid supplementation: more work is needed CMAJ 2000 163 1129 1130 11079054 Health Canada Health Canada Primary Prevention of Neural Tube Defects with Folic Acid. [Electronic Citation] 2003 Government of Canada Shipchandler MT Moore EG Rapid, fully automated measurement of plasma homocyst(e)ine with the Abbott IMx analyzer Clin Chem 1995 41 991 994 7600701 Wilson DH Herrmann R Hsu S Biegalski T Sohn L Forsythe C Novotny M Beggs M Manderino G Ion capture assay for folate with the Abbott IMx analyzer Clin Chem 1995 41 1780 1781 7497624 Greenberg CR Hamerton JL Nigli M Wrogemann K DNA studies in a family with Duchenne muscular dystrophy and a deletion at Xp21 Am J Hum Genet 1987 41 128 137 3475976 Rady PL Szucs S Grady J Hudnall SD Kellner LH Nitowsky H Tyring SK Matalon RK Genetic polymorphisms of methylenetetrahydrofolate reductase (MTHFR) and methionine synthase reductase (MTRR) in ethnic populations in Texas; a report of a novel MTHFR polymorphic site, G1793A Am J Med Genet 2002 107 162 168 11807892 10.1002/ajmg.10122 NCSS Statistical Systems for Windows Kaysville, Utah: Dr Jerry L Hintze 2001 Chango A Potier DC Boisson F Guilland JC Barbe F Perrin MO Christides JP Rabhi K Pfister M Galan P Hercberg S Nicolas JP 5, 10-methylenetetrahydrofolate reductase common mutations, folate status and plasma homocysteine in healthy French adults of the Supplementation en Vitamines et Mineraux Antioxydants (SU.VI.MAX) cohort Br J Nutr 2000 84 891 896 11177206 Pagan K Hou J Goldenberg RL Cliver SP Tamura T Effect of smoking on serum concentrations of total homocysteine and B vitamins in mid-pregnancy Clin Chim Acta 2001 306 103 109 11282100 10.1016/S0009-8981(01)00402-8 Chodirker BN Erdile LB MacDonald KM Harman CR Cheang MS Evans JA MSAFP levels in aboriginal Canadian women Can J Public Health 1994 85 424 426 7534622 Population by ethnic origin, 1996 Census, provinces and territories. [Electronic Citation] Statistics Canada 2003 Infante-Rivard C Rivard GE Gauthier R Theoret Y Unexpected relationship between plasma homocysteine and intrauterine growth restriction Clin Chem 2003 49 1476 1482 12928228 10.1373/49.9.1476 Castro LC Avina RL Maternal obesity and pregnancy outcomes Curr Opin Obstet Gynecol 2002 14 601 606 12441699 10.1097/00001703-200212000-00005 Lachmeijer AM Arngrimsson R Bastiaans EJ Pals G ten Kate LP de Vries JI Kostense PJ Aarnoudse JG Dekker GA Mutations in the gene for methylenetetrahydrofolate reductase, homocysteine levels, and vitamin status in women with a history of preeclampsia Am J Obstet Gynecol 2001 184 394 402 11228493 10.1067/mob.2001.109393 Gris JC Quere I Monpeyroux F Mercier E Ripart-Neveu S Tailland ML Hoffet M Berlan J Daures JP Mares P Case-control study of the frequency of thrombophilic disorders in couples with late foetal loss and no thrombotic antecedent–the Nimes Obstetricians and Haematologists Study5 (NOHA5) Thromb Haemost 1999 81 891 899 10404763 Zusterzeel PL Visser W Blom HJ Peters WH Heil SG Steegers EA Methylenetetrahydrofolate reductase polymorphisms in preeclampsia and the HELLP syndrome Hypertens Pregnancy 2000 19 299 307 11118403 10.1081/PRG-100101991 House JD March SB Ratnam S Ives E Brosnan JT Friel JK Folate and vitamin B12 status of women in Newfoundland at their first prenatal visit CMAJ 2000 162 1557 1559 10862228 D'Anna R Baviera G Corrado F Ientile R Granese D Stella NC Plasma homocysteine in early and late pregnancies complicated with preeclampsia and isolated intrauterine growth restriction Acta Obstet Gynecol Scand 2004 83 155 158 14756732 10.1111/j.0001-6349.2004.00291.x

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==== Front Reprod Biol EndocrinolReproductive biology and endocrinology : RB&E1477-7827BioMed Central London 1477-7827-2-661537394410.1186/1477-7827-2-66ResearchComparison in gene expression of secretory human endometrium using laser microdissection Yanaihara Atsushi [email protected] Yukiko [email protected] Shinji [email protected] Keiko [email protected] Tadateru [email protected] Takashi [email protected] Department of Obstetrics and Gynecology, Showa University School of Medicine, Hatanodai, Shinagawa-ku, Tokyo, Japan2004 17 9 2004 2 66 66 12 7 2004 17 9 2004 Copyright © 2004 Yanaihara et al; licensee BioMed Central Ltd.2004Yanaihara et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background The endometrium prepares for implantation under the control of steroid hormones. It has been suggested that there are complicated interactions between the epithelium and stroma in the endometrium during menstrual cycle. In this study, we demonstrate a difference in gene expression between the epithelial and stromal areas of the secretory human endometrium using microdissection and macroarray technique. Methods The epithelial and stromal areas were microdissected from the human endometrium during the secretory phase. RNA was extracted and amplified by PCR. Macroarray analysis of nearly 1000 human genes was carried out in this study. Some genes identified by macroarray analysis were verified using real-time PCR. Results In this study, changes in expression <2.5-fold in three samples were excluded. A total of 28 genes displayed changes in expression from array data. Fifteen genes were strongly expressed in the epithelial areas, while 13 genes were strongly expressed in the stromal areas. The strongly expressed genes in the epithelial areas with a changes >5-fold were WAP four-disulfide core domain 2 (44.1 fold), matrix metalloproteinase 7 (40.1 fold), homeo box B5 (19.8 fold), msh homeo box homolog (18.8 fold), homeo box B7 (12.7 fold) and protein kinase C, theta (6.4 fold). On the other hand, decorin (55.6 fold), discoidin domain receptor member 2 (17.3 fold), tissue inhibitor of metalloproteinase 1 (9 fold), ribosomal protein S3A (6.3 fold), and tyrosine kinase with immunoglobulin and epidermal growth factor homology domains (5.2 fold) were strongly expressed in the stromal areas. WAP four-disulfide core domain 2 (19.4 fold), matrix metalloproteinase 7 (9.7-fold), decorin (16.3-fold) and tissue inhibitor of metalloproteinase 1 (7.2-fold) were verified by real-time PCR. Conclusions Some of the genes we identified with differential expression are related to the immune system. These results are telling us the new information for understanding the secretory human endometrium. ==== Body Introduction Many studies have sought to understand the mechanism of implantation. Recently, the rate of pregnancy in the in vitro fertilization and embryo transfer (IVF-ET) cycle has declined, and this has been attributed to a decrease in the rate of implantation. The recently developed laser microdissection method has gained widespread use throughout the research field. Information about cells can be determined without contamination by using this method. Moreover, with the macroarray technique, which was already widely used for this purpose, the profiling of the gene expression of specific cells types has become possible. Torres et al. have already reported differences in gene expression between cell types or regions within the monkey endometrium using laser microdissection and differential display [1,2]. Identification of cell-specific proteins, which are expressed in the endometrium during the secretory phase, has been performed using a multi-disciplinary approach in the same trial. IGF-II mRNA is expressed in the mid-to-late secretory phase and in early pregnancy [3]. During decidualization, interstitial collagen in the mouse endometrium increases [4]. Collagen IV and laminin reactivity increased in the basal lamina and underlying subepithelial stroma as pregnancy proceeds [5]. The most abundant expression of IL-15 mRNA during the menstrual cycle is observed in the midsecretory phase [6]. Leukaemia inhibitory factor (LIF) is known as an indispensable factor for implantation and is expressed in the glandular epithelium at the time of implantation in human endometrium [7-9]. In this study, we demonstrate differential in gene expression between the epithelial and stromal areas obtained from secretory human endometrium using laser microdissection and the macroarray method. Confirmation of differential expression of candidate genes was performed by real-time PCR. Materials and Methods Materials Human endometrium was obtained from 8 patients (25–38 years old) with normal menstrual cycles (28~30 days) during the mid secretory phase. These patients had had at least one intrauterine pregnancy in the past (3 patients for Microarray analysis, 5 patients for real-time PCR). Part of the endometrial biopsy was obtained with a curetting technique. The day of the menstrual cycle was determined by the patient's history, plasma progesterone levels (9.8~17.3 ng/ml) and the histological criteria of Noyes et al [10]. These patients did not receive any hormonal therapy. Informed consent was obtained from all patients who participated in this study. The Institutional Review Boards of Showa University approved the use of human subjects and the procedures. Methods Laser Microdissection and RNA extraction The endometrium was embedded in OCT compound and frozen immediately in isopentane that had been cooled in the liquid nitrogen. This freezing block was sliced by a cryomicrotome at 8 μm thickness. Frozen sections were fixed in 100% methanol for 3 min and stained with 1% toluidine blue. The section was laser-microdissected by the PALM MicroBeam system (PALM Microlaser. Technologies A.G.) for epithelial and stromal areas and collected in a small tube (Fig 1a,1b,1c,1d). Approximately 30–50 sections were laser-microdissected. Contamination with non-target components was monitored morphologically. Total RNA was extracted from the tissue section using the acid guanidinium-phenol-chloroform method [11]. Figure 1 Human endometrial epithelial areas (a-b) and stromal areas (c-d) were laser-microdissected. (200×) Macroarray The RNAs obtained were synthesized from cDNA using a modified oligo (dT) primer and the BD SMART™ PCR cDNA Synthesis Kit (BD Biosciences Clontech, Palo Alto, CA). cDNA was PCR amplified for 24–29 cycles according to the user manual. (BD Atlas™ SMART™ Probe amplification Kit (BD Biosciences Clontech, Palo Alto, CA)). 550 ng of cDNA sample was labeled with α-32P dCTP (3000 Ci/mmol) using a randam primer. Labeled probes were hybridized to a nylon array (BD Atlas™ Nylon cDNA Expression Arrays, Human 1.2 Array (BD Biosciences Clontech, Palo Alto, CA)) in ExpressHyb solution at 68°C overnight. After hybridization, the nylon membrane was washed with 2 × standard saline citrate (SSC) + 1% sodium dodecyl sulphate (SDS) (WAKO Pure Chemical Ltd, Japan) once, twice with 1.0 × SSC + 0.5% SDS at 68°C [12,13]. The membrane was exposed to a phosphor screen (Fujifilm, Japan) for 24 hours and scanned using a STORM 830 Scanner and IMAGEQUANT 4.1-J (Molecular Dynamics). Hybridization signal intensities for individual genes were a subtracted from the background and normalized to the signals for GAPDH and the beta-actin gene, respectively, using AIS (Analytical Imaging Station) Array™ (IMAGING Research INC.). Each normalized a gene expression signal the epithelial and stromal areas was compared, and was automatically calculated as a ratio [14,15]. In this study, a change in expression <2.5-fold in all three samples was excluded. Real-time PCR RNA was reverse transcribed using oligo (dT) primers by TaKaRa RNA PCR Kit (AMV) Ver 2.1 (TAKARA BIO INC, Shiga, Japan) according to the manufacturer's instructions. PCR was performed using the ABI PRISM 7700 Sequence Detection System. TaqMan Universal PCR MasterMix and Assays-on-Demand Gene Expression probes (Applied Biosystems) were used for the PCR step (Assay ID for MMP7; Hs00159163 m1, WFDC2; Hs00707910 s1, TIMP1; Hs00171558 m1 Decorin; Hs00370385 m1). Primer sequences are not publicly available, although their validity has been established by the manufacturer. The expression values obtained were normalized against those from the control human GAPDH [16]. Statistical significance was determined by the Wilcoxon test and defined as p < 0.05. Results Microdissection and Microarray Secretory endometrium was collected from 8 patients (3 patients for Microarray, 5 patients for real-time PCR). Each sample was carefully dissected by laser microdissection for epithelial and stromal areas (Fig 1a to 1d). Total RNA was extracted and subjected to macroarray with nearly 1000 genes on the nylon membrane. Fifteen genes were strongly expressed in the epithelial areas (Table 1), while 13 genes were strongly expressed in the stromal areas (Table 2). Mean values are shown in Tables 1 and 2. Genes strongly expressed in the epithelial areas that increased >5-fold in expression included WAP four-disulfide core domain 2 (WFDC2), matrix metalloproteinase 7 (MMP7), homeo box B5, msh homeo box homolog, homeo box B7 and protein kinase C, theta (PKC theta). On the other hand, decorin, discoidin domain receptor member 2 (DDR2), tissue inhibitor of metalloproteinase 1 (TIMP1), ribosomal protein S3A, and tyrosine kinase with immunoglobulin and epidermal growth factor homology domains (Tie1) were strongly expressed in the stromal areas. Table 1 Expressed gene list in epithelial areas. Ratio GENE BANK LOCUS LINK Gene Name Classifications 44.1 X63187 10406 WAP four-disulfide core domain 2 inhibitors of proteases 40.1 X07819 4316 matrix metalloproteinase 7 metalloproteinases 19.8 M92299 3215 homeo box B5 CDK inhibitors 18.8 M97676 4487 msh homeo box homolog 1 (Drosophila) transcription activators and repressors 12.7 M16937 3217 homeo box B7 transcription activators and repressors 6.4 L07032 5588 protein kinase C, theta intracellular kinase network members 4.8 X59798 595 cyclin D1 cyclins 4.5 M97796 3398 inhibitor of DNA binding 2, dominant negative helix-loop-helix protein transcription activators and repressors 3.9 X02920 5265 serine proteinase inhibitor, clade A inhibitors of proteases 3.8 D14520 688 Kruppel-like factor 5 basic transcription factors 2.9 U24166 22919 microtubule-associated protein, RP/EB family, member 1 adaptors and receptor-associated proteins 2.9 X67055 3699 pre-alpha (globulin) inhibitor inhibitors of proteases 2.8 U26710 868 Cas-Br-M (murine) ectropic retroviral transforming sequence b adaptors and receptor-associated proteins 2.7 D45132 7799 PR domain containing 2, with ZNF domain transcription activators and repressors 2.5 AF059244 10047 cystatin 8 inhibitors of proteases Ratio shows average of 3 samples Table 2 Expressed gene list in stromal areas Ratio GENE BANK LOCUS LINK Gene Name Classifications 55.6 M14219 1634 decorin cell surface antigens 17.3 X74764 4921 discoidin domain receptor family, member 2 intracellular transducers 9 X03124 7076 tissue inhibitor of metalloproteinase 1 extracellular secreted proteins 6.3 M77234 6189 ribosomal protein S3A ribosomal proteins 5.2 X60957 7075 tyrosine kinase with immunoglobulin and epidermal growth factor homology domains intracellular transducers 4.9 M57399 5764 pleiotrophin (heparin binding growth factor 8) growth factors 4.8 M62424 2149 coagulation factor II (thrombin) receptor intracellular transducers 3.5 S40706 1649 DNA-damage-inducible transcript 3 other apoptosis-associated proteins 3.3 M81757 6223 ribosomal protein S19 other cell cycle proteins 2.8 J00123 5179 proenkephalin neuropeptides 2.8 M15395 3689 integrin, beta 2 major histocompatibility complex 2.8 U32944 8655 dynein, cytoplasmic, light polypeptide other apoptosis-associated proteins 2.6 D15057 1603 defender against cell death 1 other apoptosis-associated proteins Ratio shows average of 3 samples Real-time PCR Real-time PCR (Taqman analysis) was used to verify the changes in expression of certain candidate genes that are highly expressed in the array. Five samples were used for this study. WFDC2 and MMP7, which are both strongly expressed in the epithelial areas and decorin and TIMP1, which are both strongly expressed in the stromal areas by the cDNA array were chosen for verification. Each value was corrected for differences in loading relative to GAPDH mRNA expression. WFDC2 and MMP7 mRNA expression increased by 9.4- and 9.7-fold, respectively, compared to that of stromal cells. Decorin and TIMP1 mRNA expression increased by 16.3-and 7.2-fold, respectively, in stromal cells compared to that of epithelial cells. Statistically significant changes in expression of these genes were observed (p < 0.05) (Fig 2A,2B,2C and 2D). Figure 2 A: WFDC2 mRNA expression in the secretory phase of the endometrium was determined by real-time PCR (n = 5). Values were normalized to GAPDH mRNA expression. Epithelial areas WFDC2 mRNA expression is shown relative to that of the stromal areas. The mean change is 19.4-fold. Statistical analysis was carried out using Wilcoxon test. B: MMP7 mRNA expression in the secretory phase of the endometrium was determined by real-time PCR (n = 5). Values are normalized to GAPDH mRNA expression. Epithelial areas MMP7 mRNA expression is shown relative to that of the stromal areas. The mean change is 9.7-fold. Statistical analysis was carried out using Wilcoxon test. C: Decorin mRNA expression in the secretory phase of the endometrium was determined by real-time PCR (n = 5). Values were normalized to GAPDH mRNA expression for each sample. Stromal areas decorin mRNA expression is shown relative to that of the epithelial areas. The mean change is 16.3-fold. Statistical analysis was carried out using Wilcoxon test. D: TIMP1 mRNA expression in the secretory phase of the endometrium was determined by real-time PCR (n = 5). Values are normalized to GAPDH mRNA expression for each sample. Stromal areas TIMP1 mRNA expression is shown relative to that of the epithelial areas. The mean change is 7.2-fold. Statistical analysis was carried out using Wilcoxon test. Discussion To date, various methods have been used for understanding the function of the endometrium. It is a well-known fact that epithelial cells and stromal cells in the endometrium play specific roles and are influenced by steroid hormones. However, it is very difficult to understand the molecular composition of each cell type as a function of time during the menstrual cycle. One of the problems of a cell culture experiment is that separation cultivation makes changes the composition of the cells. This is especially true as these cells are influenced by neighboring cells in vivo. It has recently become possible to acquire the information about the cell by the microdissection method. In this study, laser microdissection was used to isolate epithelial and stromal areas from the human endometrium. RNA was amplified by PCR and global gene expression was demonstrated by cDNA macroarray. Twenty-eight genes were identified in this study. These constitute only 2.8% of the 1000 genes on the array. Although this seems to be a small number, these genes were expressed at least 2.5-fold greater in all three samples and normalized to two house keeping genes. A similar percentage (1.2–5.8%) of genes with differential expression were reported using array analysis [17-19]. However, included genes below the 2.5-fold that we established as a criterion for inclusion in this study should be considered. Recently, some papers focused on endometrial gene expression have been reported. However, lots of them were compared between phases in the menstrual cycle. While Okulicz et al. demonstrated a difference in the gene expression between cell compartments in the monkey endometrium, the genes they identified are not the same as ours [1,2]. One of the reasons for this is because they tried to find new genes using differential display RT-PCR. Fifteen of 1000 genes were strongly expressed in the epithelial areas compared to the stromal areas. Of these, WFDC2 and MMP 7 were strongly expressed in the epithelial areas as confirmed by real-time PCR. WFDC2 was originally described as an epididymis-specific protein is expressed in a number of normal human tissues. A possible role for this gene in sperm maturation is indicated by amino acid similarities to extracellular proteinase inhibitors of genital tract mucous secretions [20]. Although WFDC2 has been recently reported in the secretory endometrium of monkey, this is the first report of its localization in the epithelium of the human endometrium [21]. However, the physiological role of this gene in the endometrium is presently unknown. Baboon endometrial epithelia express MMP 7 was reported by Cox et al [22]. The highest expression of MMP 7 occurred on day 7 of pregnancy in the rat uterus [23,24] and has been reported to have close associations with tumor invasion and metastasis [25,26]. HOXB gene induction is related to the immune system, and is specifically associated with IL-2-induced NK cell proliferation [27,28]. Although Hox-7 is reported to be in human cervical tumor tissue [29], this is the first report or its localization in human endometrium. Msh genes play a role in the regulation of cell-cell adhesion [30]. Friedmann et al. reported that regulated expression of homeobox genes Msx-1 and Msx-2 in mouse mammary gland development suggests a role in hormone action and epithelial-stromal interactions [31]. PKC theta cooperates with Vav1 to induce JNK activity in T-cells [32,33]. Take Catalano et al. report that JNK pathways are altered by RU486 which is an antiprogestins. PKC therefore seems to be important factor for control secretory endometrium [17]. Of the strongly expressed genes in the stromal aeas, decorin and TIMP1 gene expression were verified by real-time PCR. San Martin et al. reported the expression of decorin which is a leucine-rich proteoglycan in the mouse uterine and suggested it localized in the undifferentiated interimplantation site stroma [34]. Some reports also demonstrated its presence in the human uterin cervix and myometrium, but not in human endometrium [35,36]. DDR2 is a new type of receptor tyrosine kinases, and is thought to be involved in the metastasis of some tumors. Its ligand is fibrillar collagen, which suggests a role in controlling celluar responses to the extracellular matrix [37]. The changes in the extracellular matrix may play an important role in implantation, in invasion of trophoblastic cells and in the maintenance of pregnancy [38]. Decidualized stromal cells stained strongly positive for TIMP-1 [39]. Matrix metalloproteinases and their endogenous inhibitors, tissue-specific inhibitors of matrix metalloproteinases, play key roles in the cyclic remodeling events that occur in the human endometrium in preparation for pregnancy [40]. Ribosomal protein S3A is through direct or indirect actions on B and T cells and cytokine secretion, could participate in the immunoregulatory processes that play a role in the balance of the Th1 and Th2 immune response [41]. It has been recently said that the ratio of Th1 to Th2 influences pregnancy. Ribosomal protein S3A may be an interesting factor for stromal cell research. In this study, these results show the fact that many of the genes, which are related to the immune system, are expressed in the endometrium during the mid secretory phase of the menstrual cycle. This time, however, differences in gene expression between cell compartments of the endometrium were considered. Interactions among cells are key factors in understanding endometrial function. Acknowledgments The authors are indebted to Dr. Takumi Yanaihara, Professor Emeritus of Showa University, Tokyo, Japan for advice. We also wish to thank Miss. Momoko Negishi Miss. Kaori Mitukawa and Mr. Hiroshi Chiba for their technical assistance.This study was supported by Health and Labour Sciences Research Grants. ==== Refs Torres MS Ace CI Okulicz WC Assessment and application of laser microdissection for analysis of gene expression in the rhesus monkey endometrium Biol Reprod 2002 67 1067 1072 12297519 Okulicz WC Ace CI Torres MS Gene expression in the rhesus monkey endometrium: differential display and laser capture microdissection Front Biosci 2003 8 d551 8 12700029 Giudice LC Dsupin BA Jin IH Vu TH Hoffman AR Differential expression of messenger ribonucleic acids encoding insulin-like growth factors and their receptors in human uterine endometrium and decidua J Clin Endocrinol Metab 1993 76 1115 1122 8496300 10.1210/jc.76.5.1115 Teodoro WR Witzel SS Velosa AP Shimokomaki M Abrahamsohn PA Zorn TM Increase of interstitial collagen in the mouse endometrium during decidualization Connect Tissue Res 2003 44 96 103 12745676 MacIntyre DM Lim HC Ryan K Kimmins 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survival of human melanoma through stroma remodeling Cancer Res 2001 61 8306 8316 11719464 Riehle MM Bennett AF Long AD Genetic architecture of thermal adaptation in Escherichia coli Proc Natl Acad Sci U S A 2001 98 525 530 11149947 10.1073/pnas.021448998 Haslett JN Sanoudou D Kho AT Bennett RR Greenberg SA Kohane IS Beggs AH Kunkel LM Gene expression comparison of biopsies from Duchenne muscular dystrophy (DMD) and normal skeletal muscle Proc Natl Acad Sci U S A 2002 99 15000 15005 12415109 10.1073/pnas.192571199 Catalano RD Yanaihara A Evans AL Rocha D Prentice A Saidi S Print CG Charnock-Jones DS Sharkey AM Smith SK The effect of RU486 on the gene expression profile in an endometrial explant model Mol Hum Reprod 2003 9 465 473 12837923 10.1093/molehr/gag060 Carson DD Lagow E Thathiah A Al-Shami R Farach-Carson MC Vernon M Yuan L Fritz MA Lessey B Changes in gene expression during the early to mid-luteal (receptive phase) transition in human endometrium detected by high-density microarray screening Mol Hum Reprod 2002 8 871 879 12200466 10.1093/molehr/8.9.871 Kao LC Tulac S Lobo S Imani B Yang JP Germeyer A Osteen K Taylor RN Lessey BA Giudice LC Global gene profiling in human endometrium during the window of implantation Endocrinology 2002 143 2119 2138 12021176 10.1210/en.143.6.2119 Bingle L Singleton V Bingle CD The putative ovarian tumour marker gene HE4 (WFDC2), is expressed in normal tissues and undergoes complex alternative splicing to yield multiple protein isoforms Oncogene 2002 21 2768 2773 11965550 10.1038/sj.onc.1205363 Ace CI Okulicz WC Microarray profiling of progesterone-regulated endometrial genes during the rhesus monkey secretory phase Reprod Biol Endocrinol 2004 2 54 15239838 10.1186/1477-7827-2-54 Cox KE Sharpe-Timms KL Kamiya N Saraf M Donnelly KM Fazleabas AT Differential regulation of stromelysin-1 (matrix metalloproteinase-3) and matrilysin (matrix metalloproteinase-7) in baboon endometrium J Soc Gynecol Investig 2000 7 242 248 10964024 10.1016/S1071-5576(00)00062-9 Rechtman MP Zhang J Salamonsen LA Effect of inhibition of matrix metalloproteinases on endometrial decidualization and implantation in mated rats J Reprod Fertil 1999 117 169 177 10645258 10.1530/jrf.0.1170169 Feng J Woessner J. F., Jr. Zhu C Matrilysin activity in the rat uterus during the oestrous cycle and implantation J Reprod Fertil 1998 114 347 350 10070364 10.1530/jrf.0.1140347 Tanioka Y Yoshida T Yagawa T Saiki Y Takeo S Harada T Okazawa T Yanai H Okita K Matrix metalloproteinase-7 and matrix metalloproteinase-9 are associated with unfavourable prognosis in superficial oesophageal cancer Br J Cancer 2003 89 2116 2121 14647147 10.1038/sj.bjc.6601372 Huachuan Z Xiaohan L Jinmin S Qian C Yan X Yinchang Z Expression of matrix metalloproteinase-7 involving in growth, invasion, metastasis and angiogenesis of gastric cancer Chin Med Sci J 2003 18 80 86 12903787 Quaranta MT Petrini M Tritarelli E Samoggia P Care A Bottero L Testa U Peschle C HOXB cluster genes in activated natural killer lymphocytes: expression from 3'-->5' cluster side and proliferative function J Immunol 1996 157 2462 2469 8805646 Care A Testa U Bassani A Tritarelli E Montesoro E Samoggia P Cianetti L Peschle C Coordinate expression and proliferative role of HOXB genes in activated adult T lymphocytes Mol Cell Biol 1994 14 4872 4877 7911974 Shim C Zhang W Rhee CH Lee JH Profiling of differentially expressed genes in human primary cervical cancer by complementary DNA expression array Clin Cancer Res 1998 4 3045 3050 9865919 Lincecum JM Fannon A Song K Wang Y Sassoon DA Msh homeobox genes regulate cadherin-mediated cell adhesion and cell-cell sorting J Cell Biochem 1998 70 22 28 9632104 10.1002/(SICI)1097-4644(19980701)70:1<22::AID-JCB3>3.0.CO;2-5 Friedmann Y Daniel CW Regulated expression of homeobox genes Msx-1 and Msx-2 in mouse mammary gland development suggests a role in hormone action and epithelial-stromal interactions Dev Biol 1996 177 347 355 8660900 10.1006/dbio.1996.0168 Moller A Dienz O Hehner SP Droge W Schmitz ML Protein kinase C theta cooperates with Vav1 to induce JNK activity in T-cells J Biol Chem 2001 276 20022 20028 11274147 10.1074/jbc.M011139200 Isakov N Altman A Protein kinase C(theta) in T cell activation Annu Rev Immunol 2002 20 761 794 11861617 10.1146/annurev.immunol.20.100301.064807 San Martin S Soto-Suazo M De Oliveira SF Aplin JD Abrahamsohn P Zorn TM Small leucine-rich proteoglycans (SLRPs) in uterine tissues during pregnancy in mice Reproduction 2003 125 585 595 12683929 10.1530/rep.0.1250585 Berto AG Sampaio LO Franco CR Cesar R. M., Jr. Michelacci YM A comparative analysis of structure and spatial distribution of decorin in human leiomyoma and normal myometrium Biochim Biophys Acta 2003 1619 98 112 12495820 10.1016/S0304-4165(02)00446-4 Ludmir J Sehdev HM Anatomy and physiology of the uterine cervix Clin Obstet Gynecol 2000 43 433 439 10949747 10.1097/00003081-200009000-00003 Vogel W Gish GD Alves F Pawson T The discoidin domain receptor tyrosine kinases are activated by collagen Mol Cell 1997 1 13 23 9659899 10.1016/S1097-2765(00)80003-9 Iwahashi M Muragaki Y Ooshima A Yamoto M Nakano R Alterations in distribution and composition of the extracellular matrix during decidualization of the human endometrium J Reprod Fertil 1996 108 147 155 8958841 10.1530/jrf.0.1080147 Zhang J Salamonsen LA Tissue inhibitor of metalloproteinases (TIMP)-1, -2 and -3 in human endometrium during the menstrual cycle Mol Hum Reprod 1997 3 735 741 9357997 10.1093/molehr/3.9.735 Chou CS Tai CJ MacCalman CD Leung PC Dose-dependent effects of gonadotropin releasing hormone on matrix metalloproteinase (MMP)-2, and MMP-9 and tissue specific inhibitor of metalloproteinases-1 messenger ribonucleic acid levels in human decidual Stromal cells in vitro J Clin Endocrinol Metab 2003 88 680 688 12574199 10.1210/jc.2002-021277 Cordeiro-Da-Silva A Borges MC Guilvard E Ouaissi A Dual role of the Leishmania major ribosomal protein S3a homologue in regulation of T- and B-cell activation Infect Immun 2001 69 6588 6596 11598026 10.1128/IAI.69.11.6588-6596.2001

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==== Front RetrovirologyRetrovirology1742-4690BioMed Central London 1742-4690-1-271537394310.1186/1742-4690-1-27ResearchProduction of infectious human immunodeficiency virus type 1 does not require depletion of APOBEC3G from virus-producing cells Kao Sandra [email protected] Eri [email protected] Mohammad A [email protected] Hiroaki [email protected] Sandrine [email protected] Ritu [email protected] Klaus [email protected] Laboratory of Molecular Microbiology, Viral Biochemistry Section; National Institute of Allergy and Infectious Diseases, NIH; Building 4, Room 310; 4 Center Drive, MSC 0460; Bethesda, MD 20892-0460, USA2004 17 9 2004 1 27 27 8 7 2004 17 9 2004 Copyright © 2004 Kao et al; licensee BioMed Central Ltd.2004Kao et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background The human immunodeficiency virus Vif protein overcomes the inhibitory activity of the APOBEC3G cytidine deaminase by prohibiting its packaging into virions. Inhibition of APOBEC3G encapsidation is paralleled by a reduction of its intracellular level presumably caused by the Vif-induced proteasome-dependent degradation of APOBEC3G. Results In this report we employed confocal microscopy to study the effects of Vif on the expression of APOBEC3G on a single cell level. HeLa cells dually transfected with Vif and APOBEC3G expression vectors revealed efficient co-expression of the two proteins. Under optimal staining conditions approximately 80% of the transfected cells scored double-positive for Vif and APOBEC3G. However, the proportion of double-positive cells observed in identical cultures varied dependent on the fixation protocol and on the choice of antibodies used ranging from as low as 40% to as high as 80% of transfected cells. Importantly, single-positive cells expressing either Vif or APOBEC3G were observed both with wild type Vif and a biologically inactive Vif variant. Thus, the lack of APOBEC3G in some Vif-expressing cells cannot be attributed to Vif-induced degradation of APOBEC3G. These findings are consistent with our results from immunoblot analyses that revealed only moderate effects of Vif on the APOBEC3G steady state levels. Of note, viruses produced under such conditions were fully infectious demonstrating that the Vif protein used in our analyses was both functional and expressed at saturating levels. Conclusions Our results suggest that Vif and APOBEC3G can be efficiently co-expressed. Thus, depletion of APOBEC3G from Vif expressing cells as suggested previously is not a universal property of Vif and thus is not imperative for the production of infectious virions. ==== Body Background Replication of human immunodeficiency virus type 1 (HIV-1) in most primary cells and some immortalized T cell lines is dependent on the expression of a functional Vif protein. In the absence of Vif, virus replication is restricted by a host factor that was recently identified as CEM15 (now referred to as APOBEC3G) [1], a host cytidine deaminase targeting DNA substrates in vitro [2] but whose role in normal cells is unknown. In the absence of Vif, APOBEC3G is efficiently incorporated into virus particles [3-9] where it causes extensive cytidine to uracil changes on the viral minus-strand cDNA during reverse transcription [5,10-12]. The conversion of cytidine to deoxyuridine on the minus-strand cDNA either results in guanine to adenine changes on the viral plus-strand cDNA to yield highly mutated viral genomes or triggers the degradation of the deaminated minus strand cDNA through the action of a DNA repair mechanism that involves removal of the uracil base by uracil DNA glycosylase and subsequent endonucleolytic cleavage at the abasic sites by apyrimidinic endonuclease (reviewed in [13,14]). While both mechanisms are detrimental to virus replication, the reported inability of vif-defective viruses grown in restrictive cells to reverse transcribe the viral genome into full-length cDNA is more consistent with the latter mechanism involving the degradation of deaminated viral cDNA [15-19]. Vif is a 23-kDa basic protein that is expressed late during infection in a Rev-dependent manner [20]. Immunocytochemical analyses revealed a largely cytoplasmic localization of Vif [21-23]. However, Vif is efficiently incorporated into HIV particles during productive infection through an interaction with viral genomic RNA and associates with viral nucleoprotein complexes [22,24-26]. In the presence of Vif, the steady-state levels of cell-associated APOBEC3G – as judged by immunoblot analysis – are reduced by 3–10 fold [3-8,27,28]. This Vif-dependent reduction in APOBEC3G levels has been attributed to proteasome-dependent degradation of the protein and requires a direct interaction of Vif with APOBEC3G [3,6-8]. Like Vif, APOBEC3G is a cytoplasmic protein. In fact, co-immunoprecipitation analyses demonstrated an interaction of Vif and APOBEC3G in transiently transfected cells [3,5,6,27,29-32]. The formation of stable Vif:APOBEC3G complexes seemed to be at odds with the reported proteasome-dependent degradation of APOBEC3G in Vif-expressing cells [3,6-9,27,28]. Indeed, the identification of Vif:APOBEC3G complexes in mixtures of cell extracts that had been individually transfected to express either Vif or APOBEC3G suggested that the stable interaction of Vif and APOBEC3G during co-immunoprecipitation may occur after cell lysis [6]. Thus, the co-immunoprecipitation of Vif and APOBEC3G from cell extracts is not necessarily an indication of the existence of stable intracellular complexes. Quite to the contrary, Marin et al reported a profound effect of Vif on the expression of APOBEC3G on a single cell level. They found that expression of Vif in transiently transfected COS7 cultures resulted in an almost complete segregation of cells expressing either APOBEC3G or Vif [6]. Interestingly, this segregation of Vif and APOBEC3G into separate cells was seen only for wild type Vif. In fact, only 10% of cells expressing wild type Vif were double-positive while 95% of cells expressing an inactive Vif variant also contained APOBEC3G [6]. The current study aims at characterizing in more detail the effects of Vif on the expression of human APOBEC3G on a single cell level. The study was initiated because of the apparent discrepancy between the drastic effects of Vif on APOBEC3G reported by Marin et al and our own finding of only moderate effects of Vif on APOBEC3G expression in transiently transfected cells. In our study, Vif was expressed from a subviral construct in a Tat- and Rev-dependent manner while APOBEC3G was expressed either in a Tat-dependent manner from an HIV-1-LTR-based vector or independently from a CMV-promoter-based expression vector. The Tat-dependent APOBEC3G expression vector was used to restrict APOBEC3G expression to cells also expressing Tat (and thus Vif). Confocal microscopic analysis of HeLa cells transiently transfected with Vif and APOBEC3G expression vectors revealed significant variations in the number of double-positive cells in identical samples ranging from as low as 40% to as high as 80% of transfected cells depending on fixation method and antibodies employed. Importantly, the appearance of cells expressing only Vif or APOBEC3G was observed both with wild type Vif and a biologically inactive variant and thus cannot be explained by Vif-induced degradation of APOBEC3G. Finally, despite the efficient co-expression of Vif and APOBEC3G, viruses produced in these cultures were fully infectious. We therefore conclude that the Vif-induced exclusion of APOBEC3G from virus-producing cells reported by Marin et al [6] does not apply to our system and because of that is not a universal property of all Vif proteins. This implies that elimination of APOBEC3G is not an obligate requirement for the production of infectious viruses from APOBEC3G-expressing cells. Results Expression of Vif in the context of a proviral vector only moderately reduces cellular APOBEC3G levels A number of previous studies reported the efficient Vif-dependent degradation of APOBEC3G by cellular proteasomes [3,6,8,28]. However, we and others noted only a moderate reduction of the cellular APOBEC3G levels in response to Vif expression [4,5]. This is exemplified in figure 1 where APOBEC3G was expressed either in the presence or absence of Vif. Specifically, HeLa cells were transfected with pcDNA-APO3G together either with wild type pNL-A1 (Fig. 1A, lane 2) or its vif-defective variant, pNL-A1vif(-) (lane 3). Mock transfected cells were included as a control (lane 1). Cells were harvested 24 hr post-transfection and whole-cell lysates were subjected to immunoblot analysis as described in Methods using an APOBEC3G-specific polyclonal antibody (Fig. 1A, top panel) or a Vif-specific monoclonal antibody (Fig. 1A, middle panel). To control for loading errors, the filters were re-probed with an antibody to α-tubulin (Fig. 1A, bottom panel). Consistent with our previous results, expression of Vif from pNL-A1 only moderately reduced the steady-state levels of APOBEC3G in HeLa cells. Quantitation of the data confirmed that expression of APOBEC3G in the presence of Vif was reduced by only about 20% (Fig. 1B). Figure 1 Vif has a moderate effect on APOBEC3G steady-state levels. (A) HeLa cells were transfected with pNL-A1 and pcDNA-APO3G vector DNA at a 4:1 ratio. As control, mock-transfected cells (lane 1) and cells transfected with the Vif-deficient pNL-A1ΔVif construct and pcDNA-APO3G vector DNA at a 4:1 ratio (lane 3) were included. Cell lysates were processed for immunoblotting as described in Methods and APOBEC3G and Vif-specific proteins were identified using an APOBEC3G-specific polyclonal antibody (α-APO3G) or a Vif-specific monoclonal antibody #319 (α-Vif). Tubulin was identified using an antibody to α-tubulin. (B) APOBEC3G-specific bands were acquired by densitometric scanning of the film and were quantified using the Fuji ImageGauge 4.0 software (Fuji Photofilm Co, LTD). Results are expressed as percent of the Vif-negative control, which was defined as 100%. Co-expression of APOBEC3G and Vif in HeLa cells An earlier study investigating the coexpression of Vif and APOBEC3G by confocal microscopy concluded that APOBEC3G was virtually excluded from Vif-expressing COS7 cells [6]. To verify this observation, we investigated the effects of Vif on APOBEC3G expression on a single cell level by performing a series of immunocytochemical analyses. For that purpose, HeLa cells were transfected with the Vif expression vector pNL-A1 together with pcDNA-APO3G for the expression of human APOBEC3G. Cells were grown on cover slips, fixed 24 hr later with cold methanol, and stained with antibodies to APOBEC3G (Fig. 2, panels A & D) and Vif (panels B & E). The results of this experiment show that APOBEC3G can be expressed in Vif-positive cells (white arrow heads) without a dramatic reduction in its expression level when compared to Vif-negative cells (yellow arrow heads). Furthermore, these data confirm that APOBEC3G is localized to the cytoplasm while Vif was observed in this experiment in some cells both in the cytoplasm and the nuclei of cells (red arrow heads). Finally, we also observed cells expressing Vif that were APOBEC3G-negative (blue arrow heads). Overlay of the Vif and APOBEC3G channels revealed a partial co-localization of Vif and APOBEC3G in the cytoplasm apparent by the yellow staining in panels C & F of figure 2. Interestingly, a significant number of cells in this experiment were single-positive expressing either APOBEC3G or Vif alone. The appearance of Vif-positive, APOBEC3G-negative cells could be explained by a Vif-dependent restriction of APOBEC3G expression as proposed by Marin et al [6]. However, cells expressing Vif only were rare when compared to cells expressing APOBEC3G alone (data not shown). The preponderance of APOBEC3G single positive cells cannot be explained by a Vif-dependent restriction but more likely represents a technical, albeit reproducible, artifact. Figure 2 Co-expression of Vif and APOBEC3G in HeLa cells. HeLa cells were transfected with pNL-A1 and pcDNA-APO3G at a 1:1 molar ratio. Transfected cells were grown on cover slips, fixed in methanol and processed for confocal microscopic analysis as described in Methods. Cells were stained with a rabbit polyclonal antibody to APOBEC3G (A & D) and a monoclonal Vif antibody (B & E). APOBEC3G was visualized using a Texas red-conjugated secondary antibody; Vif was visualized with a Cy2-conjugated secondary antibody. Panels C and F are merged images of panels A & B and D & E, respectively. Arrow heads are defined as follows: white = APOBEC3G:Vif-double-positive cells; yellow = Vif-negative cells; red = cells exhibiting nuclear and cytoplasmic staining for Vif. Tat-dependent expression of APOBEC3G reduces the fraction of single-positive cells In the experiment shown in figure 2, APOBEC3G was expressed under the control of a CMV promoter while Vif was expressed from the HIV-LTR promoter under the regulatory control of Tat and Rev. Because of the independent expression of Vif and APOBEC3G it cannot be ruled out that the large number of single-positive cells in that experiment – while statistically improbable – was caused by the selective transfection of cells with either the Vif or the APOBEC3G expression vector. To check this possibility, we expressed APOBEC3G from the HIV-1 long terminal repeat (LTR) promoter driven vector pHIV-APO3G [4]. Because of its dependence on Tat, APOBEC3G expression from pHIV-APO3G is restricted to cells also expressing Vif. Indeed, transfection of pHIV-APO3G into cells in the absence of pNL-A1 or any other Tat expression vector did not reveal any APOBEC3G expression as judged by immunofluorescence analysis or immunoblotting attesting to the strict Tat-dependence of this APOBEC3G expression vector (data not shown). In addition of measuring the context-dependent expression of APOBEC3G, we also wanted to determine the influence of the fixation procedure on the efficiency of Vif:APOBEC3G co-staining. It is well known that the choice of fixative can affect the ability of a given antibody to recognize a specific epitope on its target protein. Frequently, epitopes are masked because of the folding properties of a protein in vivo or because of pre-existing protein-protein interactions that may compete for antibody binding. To test this possibility we compared a formaldehyde fixation procedure employed previously [6] with the methanol fixation procedure employed in our own studies [22]. HeLa cells were transfected with pNL-A1 and pHIV-APO3G at a 1:1 molar ratio. Cells were fixed 24 hr later either with methanol (MeOH) as in figure 2 or with formaldehyde (FA) as described in Methods. Cells were stained with Vif- and APOBEC3G-specific antibodies as described in figure 2. The results of this experiment show that expression of APOBEC3G under the control of the HIV-1 LTR indeed increased the proportion of double-positive cells both in methanol-fixed samples (Fig. 3, panels A-C) and formaldehyde-fixed specimens (Fig. 3, panels D-F). This suggests that the high proportion of single-positive cells observed in figure 2 was not the result of a Vif-dependent restriction of APOBEC3G but was caused by the independent expression of APOBEC3G from a Tat-independent promoter. Again, in methanol-fixed samples APOBEC3G expression levels in Vif-positive cells (Fig. 3A, white arrow heads) were indistinguishable from those observed for neighboring Vif-negative cells (Fig. 3A, yellow arrow head). Interestingly, the APOBEC3G fluorescent intensity appeared to be reduced in Vif-positive formaldehyde-fixed specimens when compared to Vif-negative cells or cells expressing low levels of Vif (Fig. 3D; compare white and yellow arrow heads). Because the methanol-fixed samples did not show a Vif-dependent reduction in APOBEC3G signals in these experiments, we conclude that the reduction in APOBEC3G signals observed in formaldehyde-fixed samples is not the result of Vif-induced degradation of APOBEC3G but is a technical artifact. Figure 3 Tat-dependent expression of APOBEC3G. HeLa cells were transfected with pNL-A1 and pHIV-APO3G at a 1:1 molar ratio. Transfected cells were grown on cover slips for 24 hr and then either fixed with ice-cold methanol (panels A-C) or with formaldehyde buffer as described in Methods (panels D-F). Cells were stained with an APOBEC3G-specific antibody (A & D) and a Vif monoclonal antibody (B & E) as in figure 2 and analyzed on a confocal microscope. Panels C & F are overlays of panels A & B and D & E, respectively. Arrow heads are defined as follows: white = APOBEC3G:Vif-double-positive cells; yellow = Vif-negative cells; blue = APOBEC3G negative cells. Co-expression of Vif and APOBEC3G: Protein degradation or epitope masking? For a more quantitative analysis and to determine possible effects that arise from the use of different antibodies, we extended the experiment shown in figure 3 to include three different antibodies for the identification of APOBEC3G. As before, HeLa cells were co-transfected with a 1:1 ratio of pNL-A1 and pHIV-APO3G plasmid DNAs. Cells were grown on cover slips and fixed 24 hr later either with formaldehyde (Fig. 4, panels A-C) or methanol (panels D-F) as described in figure 3. Cells were then stained with either a monoclonal antibody to the C-terminal Myc-epitope in APOBEC3G together with a polyclonal Vif antibody (Fig. 4, panels A & D), or a polyclonal Myc antibody together with a monoclonal Vif antiserum (Fig. 4, panels B & E). A third set of cells was stained with a polyclonal APOBEC3G-specific antiserum together with the monoclonal Vif antibody (Fig. 4, panels C & F). Representative fields are shown for each combination. Figure 4 Effect of fixation method and antibody choice on co-expression of Vif and APOBEC3G. HeLa cells were transfected with pNL-A1 and pHIV-APO3G as described in figure 3. Cells were grown on cover slips for 24 hr and then either fixed with ice-cold methanol (panels A-C) or with formaldehyde buffer as in figure 3 (panels D-F) and stained with the following combinations of antibodies: (A & D) polyclonal Vif + anti-Myc MAb 9E10; (B & E) anti-Vif MAb #319 + anti-Myc polyclonal antibody; (C & F) anti-Vif MAb #319 + anti-APO3G polyclonal antibody. Vif was visualized using Cy2-conjugated secondary antibodies (green) and APOBEC3G was visualized with Texas red-conjugated antibodies (red). Areas of overlap appear as yellow. To quantify the results, multiple optical fields were analyzed (n = 5–10) with a total of at least 100 transfected cells for each parameter. As can be seen in figure 5, methanol-fixed samples showed a relatively modest variation among the three antibodies used. All three antibodies identified between 45% to 60% of the cells as double-positive for Vif and APOBEC3G. In contrast, formaldehyde-fixed samples exhibited a larger antibody-dependent variation. Staining with the 9E10 monoclonal antibody to the Myc-epitope in APOBEC3G yielded the lowest efficiency of staining and identified little more than 40% of the transfected cells as double-positive for Vif and APOBEC3G. In contrast, staining of APOBEC3G was more efficient with the polyclonal Myc antibody, which identified approximately 80% of the transfected cells as double-positive in formaldehyde-fixed samples. Finally, the polyclonal APOBEC3G-specific antibody was slightly less efficient for the staining of FA-fixed samples than methanol-fixed samples and identified about 40% of the formaldehyde-fixed samples as double-positive. Since all samples were derived from the same transfected culture, variations in the co-expression of Vif and APOBEC3G in the individual samples can only be explained by the differential sensitivity of the antibodies to the fixation procedure. Figure 5 Quantitative analysis of Vif and APOBEC3G co-expression. Samples from figure 4 were analyzed for the expression of Vif (grey bars) or APOBEC3G (white bars) or for double-positive cells (black bars). Between 5 and 10 independent optical fields were analyzed to yield at least 100 transfected cells. Error bars reflect standard deviations calculated from multiple optical fields. The results obtained with methanol-fixed samples (MeOH) are on the left; results from formaldehyde-fixed samples (FA) are on the right. Exclusion of APOBEC3G from cells expressing biologically inactive Vif protein Under optimal conditions, wild type Vif and APOBEC3G were coexpressed in about 80% of transfected cells (see figure 5). Thus, 20% of the transfected cell population either was expressing Vif but not APOBEC3G or was single-positive for APOBEC3G. To investigate whether the presence of such single-positive cells is due to an activity of Vif or is a general characteristic of transiently transfected cells, we studied the effects of a biologically inactive Vif variant. For this purpose, we employed a Vif mutant carrying a deletion of residues 23–45 in Vif. We previously showed that this mutant is unable to rescue viral infectivity in APOBEC3G-expressing cells [4]. Like wild type Vif, VifΔ23–43 was expressed in the context of the subviral expression vector pNL-A1. HeLa cells were cotransfected with pNL-A1/VifΔ23–43 and pHIV-APO3G, fixed with methanol and processed for confocal microscopy as described for figure 2. As shown in figure 6, coexpression of VifΔ23–43 and APOBEC3G yielded a significant number of double-positive cells (white arrow heads). However, as observed before with wild type Vif, we also identified cells that were Vif-positive but had significantly reduced APOBEC3G levels (Fig. 6, blue arrow heads) or cells that were APOBEC3G positive but did not express Vif (yellow arrow heads). As with wild type Vif, overlay of the Vif and APOBEC3G image channels suggested a partial colocalization of the two proteins. In cells, in which Vif had spontaneously collapsed into a perinuclear aggregate (green arrow head), APOBEC3G did not exhibit a similar change in subcellular distribution. This is in contrast to the Vif-induced reorganization of vimentin reported previously [22]. Thus, VifΔ23–43 is either unable to interact with APOBEC3G or forms complexes that are unstable. These results also imply that the partial colocalization of APOBEC3G and Vif noted in this study may not reflect a true physical interaction of the two proteins. Figure 6 Co-expression of APOBEC3G and a biologically inactive Vif variant. HeLa cells were transfected with pHIV-APO3G and pNL-A1/VifΔ23–43, encoding a biologically inactive Vif variant. Cells were fixed in methanol and stained with the monoclonal Vif antibody (MAb #319; green) and a rabbit polyclonal antibody to APOBEC3G (red) as described above. APOBEC3G is shown in panel A; panel B depicts samples stained for Vif; panel C is the merged image of panels A & B. White and yellow arrow heads depict APOBEC3G:Vif double-positive and Vif-negative cells, respectively. Blue arrow heads point to double-positive cells that show reduced levels of APOBEC3G; the green arrow head depicts a cell where Vif is concentrated around the microtubule organizing center without a similar effect on APOBEC3G. Rescue of viral infectivity and Vif-induced reduction of cellular APOBEC3G levels are not directly linked The combined results from the experiments shown in figures 2,3,4,5,6, do not support the notion that Vif expression leads to the elimination of APOBEC3G from Vif-positive cells. It can be argued, however, that under the experimental conditions employed in our experiments, the Vif expression levels were insufficient or ineffective. To control for this possibility, we compared the infectivity of viruses produced in the presence of various ratios of Vif and APOBEC3G. To allow a direct comparison with the experiments shown in figures 2 to 6, Vif and APOBEC3G were expressed in trans from pNL-A1 and pHIV-APO3G respectively in the presence of a Vif-defective NL4-3 proviral vector. The ratios of Vif to APOBEC3G expression vector were 1:1, 2:1, and 5:1, respectively. Note that the Vif to APOBEC3G ratio in the experiments shown in figures 2 to 4 was 1:1 throughout. A Vif-negative sample was analyzed as negative control. Virus-containing supernatants were harvested 24 hr after transfection, normalized for equal reverse transcriptase activity and used for the infection of LuSIV indicator cells. Relative virus infectivity was determined by comparing the Tat-dependent expression of luciferase in the target cells (Fig. 7). Interestingly, viruses produced at the lowest Vif:APOBEC3G ratio were virtually as infectious as viruses produced in the presence of higher levels of Vif. In fact, increasing the Vif:APOBEC3G ratio to 2:1 or 5:1 did not significantly increase viral infectivity. Instead, at the 5:1 ratio viral infectivity was slightly reduced, presumably due to the inhibitory effect of Vif at high concentrations as reported previously [39]. Taken together, our data suggest that the inability of Vif to prevent co-expression of APOBEC3G in transiently transfected HeLa cells is not caused by sub-optimal levels of Vif or a lack of Vif activity in our system. Figure 7 Vif efficiently rescues viral infectivity. HeLa cells were transfected with the vif-defective proviral vector pNL4-3vif(-) together with pNL-A1 and pHIV-APO3G at 1:1, 2:1, or 5:1 molar ratios. Cell lysates and purified, concentrated viral extracts were analyzed by immunoblotting using antibodies to APOBEC3G (APO3G), Vif (MAb #319), or an HIV-positive human serum for the identification of viral capsid protein (CA). Virus-containing, filtered supernatants were normalized for equal reverse transcriptase activity and used for the infection of the LuSIV indicator cell line [38]. Virus-induced luciferase activity was measured 24 hr after infection as described in Methods. Relative light units (RLU), which are directly proportional to the infectivity of the viruses, are shown. Error bars reflect standard deviations from duplicate experiments. Discussion APOBEC3G is able to deaminate cytidine residues on the HIV minus-strand cDNA and cause hypermutation of the viral genome. Nevertheless, HIV-1 is able to efficiently replicate in APOBEC3G expressing cells thanks to the activity of the accessory protein Vif. One of the prerequisites for the antiviral activity of APOBEC3G is that it is packaged into the virions where it selectively targets the viral minus-strand cDNA [5,10-12,40,41] and there is convincing evidence that HIV-1 Vif plays an important role in inhibiting the encapsidation of APOBEC3G. The question of how Vif accomplishes this remains under investigation. A number of groups have reported on the rapid Vif-induced degradation of APOBEC3G by cellular proteasomes [3,6-9,27,28]. Consistent with this, treatment of cells with proteasome inhibitors was found to increase APOBEC3G expression levels despite the presence of Vif [6,7,9,28]. This is contrasted by other reports that noted only a moderate effect of Vif on cellular APOBEC3G levels [4,5]. In fact, our own studies with proteasome inhibitors did not yield a significant increase in APOBEC3G levels in the presence of Vif (manuscript in preparation). Nevertheless, the currently prevailing opinion is that Vif inhibits the encapsidation of APOBEC3G by inducing its rapid degradation in virus-producing cells. While the results from our own study argue against a depletion of APOBEC3G in Vif-expressing cells – thus implying that Vif can rescue viral infectivity despite the presence of APOBEC3G in virus-producing cells – it is important to point out that our data do not rule out the possibility that Vif – under different experimental conditions – can indeed mediate the proteasome dependent degradation of APOBEC3G. In fact, expression of Vif from a codon-optimized vector consistently had a more pronounced effect on APOBEC3G steady-state levels than Vif expressed from pNL-A1 even though the Vif expression levels from the codon-optimized construct were consistently several-fold lower than those from pNL-A1 (manuscript in preparation). Experiments are ongoing to study the differential effects of Vif expressed from pNL-A1 and Vif expressed from a codon-optimized vector on APOBEC3G stability. However, these results could suggest that the effect of Vif on APOBEC3G steady-state levels may be influenced by the context in which Vif is expressed. At any rate, despite our inability to observe Vif-dependent cellular depletion of APOBEC3G, we were invariably able to recover fully infectious HIV under conditions were the intracellular levels of APOBEC3G were only moderately affected. We therefore conclude that (i) Vif has the ability to rescue viral infectivity even in the presence of APOBEC3G and (ii) that intracellular depletion of APOBEC3G and rescue of viral infectivity may be functionally separable activities of Vif. For now, the reason for the differences in the sensitivity of APOBEC3G to Vif noted by us versus other research groups remains unexplained. APOBEC3G can form oligomeric structures and is able to interact with Vif. It is therefore possible that such complexes undergo conformational changes that can mask epitopes thus limiting the access of antibodies used in the experiments. Thus, the discrepancy between our findings of the coexpression of Vif and APOBEC3G in the majority of cells and the virtual exclusion of APOBEC3G from Vif-expressing cells reported by Marin et al. [6] may be attributed at least in part to differences in the experimental protocols. It is unlikely that the observed discrepancies are strain-specific variations. To this end we have compared the activities of two HIV-1 Vif isolates, HXB2 and NL4-3, which differ by 18 amino acids (9.4%), and found them to be equally active against APOBEC3G (manuscript in preparation). It is unclear why cotransfection of pHIV-APO3G with pNL-A1 produces a fraction of cells that are single-positive for Vif or for APOBEC3G. Since APOBEC3G expression from the pHIV-APO3G vector is strictly Tat-dependent, the results cannot be explained by differential transfection of cells with individual plasmids. Also, this phenomenon is clearly not a consequence of Vif function, since similar results were observed in the presence of a biologically inactive Vif variant (Fig. 6) or when APOBEC3G was co-expressed with HIV-1 Gag in the absence of Vif (data not shown). The inability of Vif expressed from pNL-A1 to deplete APOBEC3G is consistent with our previous inability to observe APOBEC3G degradation in kinetic studies [4]. More recent in-depth kinetic analyses of APOBEC3G employing multiple epitope tags and various antibodies confirm these initial findings and suggest that – instead of inducing APOBEC3G degradation – Vif induces conformational changes in APOBEC3G that affect the ability of antibodies to interact with the protein (manuscript in preparation). Experiments are ongoing to study the nature of the APOBEC3G/Vif complexes and to further decipher the mechanism(s) by which Vif inhibits the encapsidation of APOBEC3G under conditions of no or low intracellular degradation. Conclusions Expression of Vif and APOBEC3G in our experimental setup does not lead to the elimination of APOBEC3G from Vif expressing cells. In fact, more than 80% of successfully transfected cells efficiently co-expressed both proteins. Similar results were observed when a biologically inactive Vif variant was co-expressed with APOBEC3G suggesting that the absence of APOBEC3G in some of the Vif-positive cells is not due to Vif-mediated APOBEC3G degradation but reflects a general characteristic of the transient expression system. Moreover, APOBEC3G expression levels were very similar for Vif-positive and Vif-negative cells as judged from the immunostaining consistent with the only modest reduction in APOBEC3G steady-state levels observed in our immunoblot analyses. Nevertheless, viruses produced under such conditions were fully infectious in the presence but not in the absence of Vif attesting to the biological activity of all the proteins involved and demonstrating that Vif was expressed at saturating levels. We conclude that production of infectious viruses from APOBEC3G expressing cells is dependent on Vif but does not necessitate APOBEC3G exclusion from virus-producing cells. Methods Plasmids The full-length molecular clone pNL4-3 [33] was used for the production of wild type infectious virus. For transient expression of Vif, the subgenomic expression vector pNL-A1 [34] was employed. This plasmid expresses all HIV-1 proteins except for gag and pol products. A vif-defective variant of pNL-A1, pNL-A1vif(-) was constructed by deletion of an NdeI/PflMI fragment [4]. Plasmid pNL-A1/VifΔ23–43 expresses a Vif variant carrying a 21 amino acid deletion (residues 23 to 43) in its vif gene as reported elsewhere [4]. This Vif variant is inactive and does not target APOBEC3G [4]. Plasmids pcDNA-APO3G and pHIV-APO3G are vectors for the expression of human APOBEC3G under the control of the CMV immediate early promoter or the HIV promoter, respectively, and were constructed as described elsewhere [4]. Antisera Serum from an HIV-positive patient (APS) was used to detect HIV-1-specific capsid (CA) proteins. A monoclonal antibody to Vif (MAb #319) was used for all immunoblot analyses and some of the immunohistochemical analyses as indicated in the text and was obtained from Michael Malim through the NIH AIDS Research and Reference Reagent Program [23,35,37] For all other immunocytochemical analyses our Vif-specific polyclonal antibody (Vif93) was employed. APOBEC3G, carrying a C-terminal Myc epitope tag was identified either with the Myc-specific 9E10 monoclonal antibody or a polyclonal antibodies to the Myc epitope tag (both antibodies were obtained from Sigma-Aldrich, St. Louis). Alternatively, APOBEC3G was identified using a polyclonal rabbit serum against recombinant APOBEC3G [4]. Tubulin was identified using a monoclonal antibody to α-tubulin (Sigma-Aldrich, St. Louis). Tissue culture and transfections HeLa cells were propagated in Dulbecco's modified Eagles medium (DMEM) containing 10% fetal bovine serum (FBS). LuSIV cells are derived from CEMx174 cells and contain a luciferase indicator gene under the control of the SIVmac239 LTR [38]. These cells were obtained through the NIH AIDS Research and Reference Reagent Program and were maintained in complete RPMI 1640 medium supplemented with 10% FBS and hygromycin B (300 μg/ml). For transfection of HeLa cells, cells were grown in 25 cm2 flasks to about 80% confluency. Cells were transfected using LipofectAMINE PLUS™ (Invitrogen Corp, Carlsbad CA) following the manufacturer's recommendations. A total of 5–6 μg of plasmid DNA per 25 cm2 flask was used. Cells were harvested 24 hr post-transfection. Transfection efficiency in our analyses was generally 30% to 40%. Preparation of virus stocks Virus stocks were prepared by transfecting HeLa cells with appropriate plasmid DNAs. Virus-containing supernatants were harvested 24 hr after transfection. Cellular debris was removed by centrifugation (3 min, 3000 × g) and clarified supernatants were filtered (0.45 μm) to remove residual cellular contaminants. For determination of viral infectivity, unconcentrated filtered viral supernatants were used for the infection of indicator cells. For immunoblot analysis of viral proteins, virus particles (7 ml) were concentrated by ultracentrifugation through 4 ml of 20% sucrose in PBS as described before [4]. Immunoblotting For immunoblot analysis of intracellular proteins, whole cell lysates were prepared as follows: Cells were washed once with PBS, suspended in PBS (400 μl/107 cells), and mixed with an equal volume of sample buffer (4% sodium dodecyl sulfate, 125 mM Tris-HCl, pH 6.8, 10% 2-mercaptoethanol, 10% glycerol, and 0.002% bromphenol blue). Proteins were solubilized by boiling for 10 to 15 min at 95°C with occasional vortexing of the samples to shear chromosomal DNA. Residual insoluble material was removed by centrifugation (2 min, 15000 rpm in Eppendorf Minifuge). Viral proteins were obtained by boiling concentrated viral pellets in a 1:1 mixture of PBS and sample buffer. Cell lysates and viral extracts were subjected to SDS-PAGE; proteins were transferred to PVDF membranes and reacted with appropriate antibodies as described in the text. Membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (Amersham Biosciences, Piscataway NJ) and visualized by enhanced chemiluminescence (ECL, Amersham Biosciences). Infectivity assay To determine viral infectivity, virus stocks were normalized for equal reverse transcriptase activity and used to infect LuSIV cells (5 × 105) in a 24-well plate total volume 1.2 to 1.4 ml. Cells were incubated for 24 hours at 37°C. Cells were then harvested and lysed in 150 μl of Promega 1x reporter lysis buffer (Promega Corp., Madison WI). To determine the luciferase activity in the lysates, 50 μl of each lysate were combined with luciferase substrate (Promega Corp., Madison WI) by automatic injection and light emission was measured for 10 seconds at room temperature in a luminometer (Optocomp II, MGM Instruments, Hamden CT). Immunocytochemistry For the analysis of transfected HeLa cells, cells were scraped off the flasks 3 hr after transfection and reseeded into 12 well plates containing 0.13 mm cover slips. Cells were grown for 15 to 24 hrs at 37°C in DMEM containing 10% FBS. Cells were then fixed at -20°C in precooled methanol (-20°C) for 10 min followed by two washes in PBS or fixed in FA buffer (5% formaldehyde + 2% sucrose in PBS) for 20 min at room temperature followed by two washes in PBS. Coverslips were stored in PBS at 4°C until use. FA-fixed samples were permeabilized for 30 min at room temperature in permeabilization buffer (1% Triton X-100, 10% sucrose in PBS) prior to incubation with antibodies. For antibody staining, cover slips were incubated in a humid chamber at 37°C for 30 min with primary antibodies at appropriate dilutions in 1% BSA in PBS. Cover slips were washed once in PBS (5 min, room temp) and incubated with Texas-Red- or Cy2-conjugated secondary antibodies (diluted in 1% BSA in PBS) for 30 min at 37°C in a humid chamber. Cover slips were then washed twice with PBS and mounted onto microscope slides with glycerol gelatin (Sigma-Aldrich, St. Louis) containing 0.1M N-propyl gallate (Sigma-Aldrich, St. Louis) to prevent photo bleaching and were stored at 4°C in the dark until analyzed by confocal microscopy. Confocal Microscopy For confocal microscopy, a Zeiss LSM410 inverted laser scanning microscope was employed. The microscope was equipped with a krypton/argon mixed-gas laser and was operated by the Microcosm Renaissance 410 (v2.3.4) software package. Images were acquired with a Plan-Apochromat 63x/1.4 oil immersion objective (Carl Zeiss, Thornwood). Additional optical magnification (up to 5-fold) was achieved using the zoom feature of the image acquisition software. For two-color analysis, objects were excited using 488/568 nm laser lines. Green and red emissions were recorded through appropriate filters (515–540 nm band pass filter for Cy2 and 590 nm long pass filter for Texas-Red) and stored in separate (red and green) image channels. At the same time, bright field images (Nomarski optics) were collected and stored in a third (blue) channel. Image quality was enhanced during data acquisition using the Renaissance 410 line average feature (8 or 16x). Competing Interests None declared. Authors' Contributions S. K. carried out immunoblot analyses, infectivity assays, and was involved in the construction of plasmids and the production of antibodies. E.M., M.A.K., H.T., S.O., and R.G. participated in immunoblot analyses, infectivity studies, sample preparations, data validation, and overall experimental design. K.S. conceived of the study, performed IFA analyses, and coordinated the study. S.K. and K.S. participated in the writing of the manuscript. All authors read and approved the final manuscript. Acknowledgements We thank Michael Malim for the Vif monoclonal antibody (MAb #319) and Jason Roos and Janice Clements for the LuSIV indicator cell line. Both reagents were obtained through the NIH Research and Reference Reagent Program. Part of this work was supported by a Grant from the NIH Intramural AIDS Targeted Antiviral Program to K.S. ==== Refs Sheehy AM Gaddis NC Choi JD Malim MH Isolation of a human gene that inhibits HIV-1 infection and is suppressed by the viral Vif protein Nature 2002 418 646 650 12167863 10.1038/nature00939 Harris RS Petersen-Mahrt SK Neuberger MS RNA Editing Enzyme APOBEC1 and Some of Its Homologs Can Act as DNA Mutators Mol Cell 2002 10 1247 1253 12453430 10.1016/S1097-2765(02)00742-6 Stopak K de Noronha C Yonemoto W Greene WC HIV-1 Vif blocks the antiviral activity of APOBEC3G by impairing both its translation and intracellular stability Mol Cell 2003 12 591 601 14527406 10.1016/S1097-2765(03)00353-8 Kao S Khan MA Miyagi E Plishka R Buckler-White A Strebel K The Human Immunodeficiency Virus Type 1 Vif Protein Reduces Intracellular Expression and Inhibits Packaging of APOBEC3G (CEM15), a Cellular Inhibitor of Virus Infectivity J Virol 2003 77 11398 11407 14557625 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J Virol 1995 69 4166 4172 7769676 Roos JW Maughan MF Liao Z Hildreth JE Clements JE LuSIV cells: a reporter cell line for the detection and quantitation of a single cycle of HIV and SIV replication Virology 2000 273 307 315 10915601 10.1006/viro.2000.0431 Akari H Fujita M Kao S Khan MA Shehu-Xhilaga M Adachi A Strebel K High Level Expression of Human Immunodeficiency Virus Type-1 Vif Inhibits Viral Infectivity by Modulating Proteolytic Processing of the Gag Precursor at the p2/Nucleocapsid Processing Site J Biol Chem 2004 279 12355 12362 14722068 10.1074/jbc.M312426200 Harris RS Sheehy AM Craig HM Malim MH Neuberger MS DNA deamination: not just a trigger for antibody diversification but also a mechanism for defense against retroviruses Nat Immunol 2003 4 641 643 12830140 10.1038/ni0703-641 Yu Q Konig R Pillai S Chiles K Kearney M Palmer S Richman D Coffin JM Landau NR Single-strand specificity of APOBEC3G accounts for minus-strand deamination of the HIV genome Nat Struct Mol Biol 2004 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==== Front BMC BioinformaticsBMC Bioinformatics1471-2105BioMed Central London 1471-2105-5-1291535787810.1186/1471-2105-5-129Research ArticleCross-species comparison significantly improves genome-wide prediction of cis-regulatory modules in Drosophila Sinha Saurabh [email protected] Mark D [email protected] Ulrich [email protected] Ulrike [email protected] Eric D [email protected] Center for Studies in Physics and Biology, The Rockefeller University, 1230 York Ave, New York, NY10021, USA2 Laboratory of Developmental Neurogenetics, The Rockefeller University, 1230 York Ave, New York, NY10021, USA2004 9 9 2004 5 129 129 8 7 2004 9 9 2004 Copyright © 2004 Sinha et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background The discovery of cis-regulatory modules in metazoan genomes is crucial for understanding the connection between genes and organism diversity. It is important to quantify how comparative genomics can improve computational detection of such modules. Results We run the Stubb software on the entire D. melanogaster genome, to obtain predictions of modules involved in segmentation of the embryo. Stubb uses a probabilistic model to score sequences for clustering of transcription factor binding sites, and can exploit multiple species data within the same probabilistic framework. The predictions are evaluated using publicly available gene expression data for thousands of genes, after careful manual annotation. We demonstrate that the use of a second genome (D. pseudoobscura) for cross-species comparison significantly improves the prediction accuracy of Stubb, and is a more sensitive approach than intersecting the results of separate runs over the two genomes. The entire list of predictions is made available online. Conclusion Evolutionary conservation of modules serves as a filter to improve their detection in silico. The future availability of additional fruitfly genomes therefore carries the prospect of highly specific genome-wide predictions using Stubb. ==== Body Background Several computational approaches to the problem of predicting cis-regulatory modules ('CRM's) have been reported recently. Berman et al. [1], Markstein et al. [2] and Halfon et al. [3] predicted CRM's involved in body patterning in the fly, and experimentally verified their predictions. The underlying principle in these algorithms was to detect dense clusters of binding sites, as determined by matches (above some threshold) to catalogued transcription factor weight matrices. The algorithm of Rajewsky et al. [4], called Ahab, avoided the use of thresholds on weight matrix matches by a probabilistic modeling of CRM's. Ahab predictions within the segmentation gene network were subjected to extensive experimental validation, with excellent overall success (Schroeder et al. [5]). Most predicted CRM's, when placed upstream of a reporter gene, faithfully reproduce one or more aspects of the endogenous gene expression pattern. Moreover, an analysis of binding site composition over the entire set of validated modules reveals that Ahab's prediction of binding sites correlates well with expression patterns produced by the modules and suggests basic rules governing module composition. The Stubb algorithm (Sinha et al. [6]) extended Ahab's approach by incorporating the use of two-species sequence information. Stubb also allows the option of scoring positional correlations between binding sites, but this option was not exercised in this study. For each sequence window analyzed, Stubb first computes the homologous sequence in the second species and aligns them using LAGAN (Brudno et al. [7]). The sequence is then partitioned into "blocks" (contiguous ungapped aligned regions of high percent identity) and non-blocks (sequence fragments between consecutive blocks, in either species). Putative binding sites in blocks are scored under an assumption of common evolutionary descent, using a probabilistic model of binding site evolution. Thus a "weak" site that is well conserved will score higher, while a "strong" site that is poorly conserved will have its score down-weighted. The score of the sequence window includes contributions from binding sites in blocks as well as in non-blocks. Stubb is implemented so that it can be run either on single species or two species data. In the single species mode, it is practically identical to the Ahab program. The Stubb software is available for download from In this paper, we present evidence that the exploitation of cross-species comparison (between D. melanogaster and D. pseudoobscura) using Stubb can lead to a significant improvement in the accuracy of genome-wide CRM prediction. To our knowledge, this is the first direct evaluation of the effect of cross-species comparison on CRM prediction on a genome-wide scale. Another important contribution of this paper is to present a benchmark for evaluating genome-wide CRM prediction tools, collected from the BDGP database and the literature, and curated by manual inspection of several hundred expression patterns. Using the same benchmark, we evaluate the effect of varying how background sequence information is incorporated in the algorithm, since this is the only tunable parameter in the Stubb program, other than the module length. We are thus able to suggest the optimal parameter settings for genome-wide CRM prediction using Stubb. Finally, we report all genome-wide predictions for cis-regulatory modules involved in anterior-posterior patterning in the early fly embryo, using both single-species and two-species Stubb, many of which make a strong case for experimental validation. Segmentation gene network The transcription control paradigm we use as our test system is the segmentation of the anterior-posterior (ap) axis during early Drosophila embryogenesis, which has long been one of the preferred arenas for studying transcription control in vivo. The segmentation genes form a hierarchical network that, in a process of stepwise refinement, translates broad, overlapping expression gradients into periodic patterns of 14 discrete stripes, which prefigure the 14 segments of the larva (for reviews see St Johnston & Nusslein-Volhard [8]; Rivera-Pomar & Jackle [9]; Furriols & Casanova [10]). The maternal factors form gradients stretching along the entire ap axis of the embryo, the zygotic "gap" factors are expressed in one or more broad slightly overlapping domains; together they generate the 7-stripe patterns of the pair-rule genes; finally, the segment-polarity genes are expressed in 14 stripes. The regulation within the segmentation gene hierarchy is almost entirely transcriptional, and most of the participating genes are transcription factors themselves, activating (in the case of the maternal factors) or repressing (most gap factors) the transcription of genes at the same level or below. In most cases, the relevant binding sites are clustered within a small interval of 0.5–1 kb; these CRM's typically contain binding sites for multiple transcription factors and multiple binding sites for each factor. The clustering and the combinatorial and redundant nature of the input facilitate the computational search for segmentation control elements. Since the expression patterns of the segmentation genes are typically complex, their control regions often contain multiple separate CRM's controlling different aspects of the pattern. The segmentation paradigm has been used as a test system for the computational detection of CRMs by us and others (Rajewsky et al. [4], Schroeder et al. [5], Berman et al. [1], Grad et al. [11]). Here, as before (Schroeder et al. [5]), we use the maternal and zygotic gap factors Bicoid, Hunchback, Caudal, Knirps, Krüppel, Giant, Tailless, Dstat, and the TorRE binding factor as input to Stubb. The binding site specificity of each factor is characterized by a position weight matrix that is based on a collection of experimentally verified binding sites. Evaluation methodology The complete genomes of two fruitflies, D. melanogaster and D. pseudobscura have been sequenced, and Stubb was used to predict CRM's in the D. melanogaster genome. This was done in two modes – (i) STUBBSS, where Stubb is run on D. melanogaster genomic sequence alone, and (ii) STUBBMS, where Stubb uses orthologous sequence data from D. pseudobscura to help predict CRM's in D. melanogaster. For each mode of execution, we obtain a separate list of predicted CRM's, sorted in order of confidence in the prediction. The ideal test for our purpose would be to compare the accuracy of these two sorted lists. However, the set of experimentally verified CRM's involved in this system is sparse compared to the size of the system – roughly 50 CRM's are known (including the 15 new modules from Schroeder et al. [5]), while the number of target genes is several hundreds, by our estimate. Hence, direct evaluation of the success-rate of predictions is not feasible, and we use an alternative source of information to evaluate predictions, as described next. A functional CRM directs the expression of a gene, by definition, and typically this gene is located in close proximity to the CRM. Hence, we may map the list of predicted CRM's to a list of predicted blastoderm-patterned genes – for each CRM predicted by Stubb, the nearest gene is identified, and if this gene is less than a threshold distance of 20 Kbp away, it is predicted to be a blastoderm-patterned gene. The resulting list of predicted "patterned genes" may now be evaluated for accuracy. (Any duplicates in the list are removed before evaluation.) The Berkeley Drosophila Genome Project (BDGP) has catalogued the expression patterns of a large number of genes in D. melanogaster, at various stages of development. We considered such a catalogue of 2167 genes, obtained from BDGP and from the literature. (See Test Genes [Additional File 1].) Visual inspection of the expression patterns of these genes revealed that 286 of them can be classified as having patterned expression along the anterior-posterior axis. (See Materials and Methods; also Patterned Genes [Additional File 2].) Hence, our benchmark is the entire set of 2167 genes, the "positive" set is the 286 ap-patterned genes, the remaining 1881 forming the "negative" set. This enables us to evaluate the accuracy of lists of patterned genes predicted by STUBBSS and STUBBMS, and compare their performance. We note that some accuracy is lost in the translation of a list of predicted CRM's to the predicted genes it is mapped to, as per the mapping defined above. For instance, it is known that CRM's may control a gene located at large distances, i.e., further than the distance threshold of 20 Kb used in the mapping procedure. Also, it is possible that a CRM is located close to two genes, and directs the expression of both genes, or only of the farther gene, being somehow insulated from the nearer one. To address these concerns, we repeat our evaluation with a slightly different mapping from the one described above. A caveat that remains is that there may be genomic sequences that are functional, in the sense that they are capable of directing a specific blastoderm pattern in reporter gene constructs, but whose activity is 'silenced' in native genomic context and does not translate to patterning of any gene. Also, the CRM may direct expression of the gene only at post-blastodermal stages, so that the gene is not included in the "positive" test set of blastoderm patterned genes. Conversely, it may also happen that a predicted CRM lies close to a patterned gene, thereby being counted as a true positive, but the predicted CRM is not the sequence responsible for the gene's regulation. We assume that such effects are not biased against either algorithm. Results STUBBMS performs significantly better than STUBBSS Figure 1a shows the results of our evaluation procedure on STUBBSS and STUBBMS. These results are for the best choice of parameters for each algorithm – local, 1st order background for STUBBSS and global, 2nd order background for STUBBMS. (The meanings of these parameter values are explained later in this section.) The x-axis is the number of unique genes that are predicted by the algorithm (by progressively decreasing its score threshold) and are in the set of 2167 genes with expression information. On the y-axis we plot how many of those predicted genes are in the "positive" set (i.e., have an ap blastoderm pattern.) Thus, the y-axis is the specificity of the algorithm. We observe that STUBBMS performs significantly better than STUBBSS. For instance, to predict 100 genes correctly, STUBBSS has to make 343 predictions, while STUBBMS only has to make 267 predictions. Figure 1b plots the difference in the number of correct predictions as a fraction of the number of correct STUBBSS predictions, i.e., the percentage change in specificity for the same number of predictions made by either algorithm. We find a typical improvement of over 20%, even when over 300 overall predictions are made by each algorithm. Figure 1c shows the progression of each algorithm's prediction specificity in a moving window of 50 predictions. We find that STUBBMS has a significantly higher hit rate for the first ~120 predictions, after which both algorithms perform comparably. Even for the lower ranked predictions (i.e., those below rank 120), we find a specificity of 20 – 35% with STUBBMS, which is roughly twice the random expectation of 13% based on 286 positives in 2167 genes. In order to further scrutinize the difference in predictions made by the two modes of Stubb, we focused on the points where their difference is most pronounced. Thus, in the top 102 unique gene predictions (for which we have information), STUBBSS reports 39 positives, while STUBBMS scores 61 hits, an improvement of over 56%. In comparison, the random expectation is ~13.5 hits. Thus the predictions of both STUBBSS and STUBBMS are significantly enriched in patterned genes (P < 10-12 and <10-37 respectively, Binomial Proportions test). Further examination of the top 102 gene predictions made by each algorithm revealed that 24 true positives are common to both lists. STUBBMS reports 37 true positives not discovered by STUBBSS, while the latter reports 15 true positives not found by the former. Similar results are seen for the top 311 predictions (another peak in Figure 1b): 70 correct predictions were common to both algorithms, 42 were predicted by STUBBMS only, and 21 by STUBBSS only. Thus there is substantial exclusivity in the sets of true positives of each algorithm. We next examined separately the following three sets of genes: (i) INTERSECTION (predicted by both algorithms in the top 311) (ii) MS-ONLY (predicted only by STUBBMS) and (iii) SS-ONLY (predicted only by STUBBSS). Table 1 shows the break-down of these sets in terms of the strength of expression of their member genes. Overall, 124 of the 286 patterned genes, i.e., about 43%, are strongly expressed. We find in Table 1 that the sets INTERSECTION and MS-ONLY have more strongly expressed genes than weak and intermediate ones, and the opposite trend is seen in the set SS-ONLY. One possible strategy that uses two-species sequence is to make predictions using STUBBSS on each of the two genomes separately and then intersect the respective lists. We found this strategy to be very restrictive – for instance, with a particular score threshold, STUBBSS predicts 205 unique genes in D. melanogaster, but intersecting these predictions with a similar number of top predictions in D. pseudoobscura gives only 68 unique genes, 33 of which are patterned. Of the top 68 predictions made in D. melanogaster alone, 29 are patterned. Thus the "intersection" strategy yields only a modest improvement over the single-species search, and does so at the price of significantly reducing the total number of predictions. Similar results were obtained when intersecting modules instead of gene predictions. We have noted above that the evaluation method is influenced by the way we map the predicted CRM's to predicted genes. To offset potential biases induced by this mapping, we repeated our analysis with a slightly different evaluation procedure, borrowing from the approach of Grad et al. [11]. We now traverse the sorted list of CRM's and count a CRM as a prediction if either of its two flanking genes has expression information. Furthermore, we designate a prediction to be "correct" if either of the two flanking genes has a blastoderm pattern. The assumption, as in Grad et al. [11], is that any predicted CRM near a blastoderm-patterned gene is a functional CRM responsible for some aspect of the pattern. Also, we are now counting modules rather than genes, i.e. we are allowing for multiple hits to the same gene. Figure 2a plots the results of STUBBSS and STUBBMS as per this new method of counting predictions and hits. We again notice a significant improvement in STUBBMS. For instance, in the top 300 CRM predictions for which a neighboring gene has expression information, STUBBMS makes 160 correct predictions while STUBBSS scores 121 hits. The gap between STUBBSS and STUBBMS increases as more predictions are considered, so that the improvement consistently stays above 20%, as seen in Figure 2b. For the remainder of this section, our evaluation method will use the more stringent mapping described earlier, wherein the nearest gene is predicted. Since our test data is in the form of lists of genes, we adhere to the evaluation strategy that counts genes. It is clear from Figures 1 and 2 that counting modules rather than genes improves the prediction accuracy, due to multiple CRM predictions for some blastoderm-patterned genes. The default mapping from CRM's to genes used in our evaluations predicts a gene to be patterned only if its proximal end is less than 20 Kb from the CRM. Schroeder et al. [5] studied the range of locations of experimentally verified CRM's relative to the gene. They found that while there is a clustering of CRM's within the proximal 5 Kb region upstream, downstream or intronic of a gene, it is not unusual to have CRM's more than 10 Kb away from the regulated gene. Nelson et al. [12] observe that for D. melanogaster, the intergenic space on either side of a gene has a mean of 2 Kb – 10 Kb, depending on the complexity of the gene's function. We repeated our evaluation with different values of the distance threshold, and found that lower thresholds (5 Kb, 10 Kb) decrease the recovery rate, while higher thresholds (50 Kb) do not affect performance. (Data not shown.) Genes in the segmentation hierarchy often have multiple aspects to their expression pattern, with more than one CRM regulating them. We therefore measured how the Stubb predictions fare if we required that each predicted gene be evidenced by at least two predicted CRM's. This heuristic improves the performance of STUBBSS more prominently than that of STUBBMS, though much fewer predictions are made by either algorithm. (See Figure 3.) While 342 unique gene predictions were made by STUBBSS (Figure 1), we now observe that only 105 predictions are made using the same score threshold and the new way of counting predictions. Thus, it appears that STUBBSS performance is open to considerable improvement in the top ~100 predictions, by using either the multiple CRM restriction or the second species' sequence data. The two-species strategy however is able to increase specificity without loss of sensitivity. We have, in all tests reported in this paper, used as input a set of 2167 genes whose expression patterns are available either from BDGP or from the literature. BDGP has a supplementary list of 2065 genes for which only textual annotation has been made public, since these genes have been found to be either (i) ubiquitously expressed at all developmental stages, (ii) not expressed at any stage, or (iii) only maternally expressed. (See Additional Genes [Additional File 3].) Inclusion of these supplementary genes in our data set would approximately halve the overall fraction of patterned genes. When we examine the performance curves of STUBBSS and STUBBMS for this pattern-diluted data set (Figure 4), we find that STUBBMS shows an improvement over STUBBSS similar in proportion to that in the default data set, even though the prediction specificity of both programs suffers a drop (as compared to that in Figure 1a), typically in the range of 10–30%. Note, however, that this is substantially lower than the 50% drop one would expect by chance, given that the total number of genes has almost doubled, while the number of patterned genes remains constant. Characteristics of genes predicted by STUBBMS Our annotations of the blastoderm patterned genes also include whether the gene expression is strong, weak or of intermediate strength; if it has a dorsal-ventral (dv) modulation in addition to the primary anterior-posterior pattern; and if the gene belongs to a "core" set of 48 genes that have been shown experimentally to be required for the segmentation of the embryo (Schroeder et al. [5]). We were therefore able to examine the characteristics of the genes correctly predicted by Stubb, along these axes of information. The top 135 correct (gene) predictions made by STUBBMS were examined progressively, 20 predictions at a time. (That is, the correct predictions ranked I to I+19 were examined, with I being incremented in steps.) In each step, we computed the fraction of the 20 genes that belonged to the following three non-exclusive categories: (i) genes with dv (in addition to ap) modulation, (ii) genes with strong expression pattern, and (iii) genes in the "core" set of 48 genes. These values are reported in Figure 5. We find that 1. Genes with dorsal-ventral aspects to their blastoderm pattern are more frequent at lower ranks of prediction; i.e., the top predictions are enriched in genes with anterior-posterior patterns only. 2. Core genes are predominantly found in the top predictions. 3. Genes found at higher ranks are somewhat more likely to be strongly expressed. The first two observations imply that the genes more directly involved in the ap axis formation are recovered at better ranks, and that the lower rank genome-wide predictions are richer in derivative patterns characteristic of genes with more complex regulatory inputs (pair-rule factors, dv factors etc.). The same trends were found for the correct predictions made by STUBBSS. (Data not shown.) Optimal parameter settings for Stubb We next evaluate the effect of varying how background sequence information is incorporated in the Stubb algorithm. This is the only configurable aspect of the program, other than the module length. (In a separate test, we ran Stubb with a module length of 700 instead of the default value of 500, and found no significant difference in the prediction specificity curve.) One important parameter is the "Markov order" of background. A value of k for this parameter means that local correlations are assumed to be present at the level of (k+1)-mers, i.e., the random probability of seeing a particular base at a position depends on the bases seen at the previous k positions. (For readers familiar with the studies of Rajewsky et al. [4] and Schroeder et al. [5], "background k" in those studies is the same as a (k-1)th order background in the terminology of this paper.) We vary this parameter to take the values k = 1 and k = 2, in different runs. The other parameter is the actual sequence used by Stubb to measure background nucleotide frequencies. Here the two options are (i) to use the current sequence window as background, or (ii) to use a pre-specified sequence (or collection of sequences) as background. We call these two the "local" and "global" background models respectively. For the "global" model, we input into Stubb 150 Kb of sequence from non-coding regions of the D. melanogaster genome, collected from the five chromosome arms 2L, 2R, 3L, 3R, and X. Figure 6 plots the specificity curves (as in Figure 1a) for each combination of parameter values tested. Figure 6a reports on different variants of STUBBSS, while Figure 6b plots the performance of STUBBMS. We find that STUBBSS performs best with a local, 1st order background, though the other parameter values produce only slightly different results. On the other hand, the effect of background parameters on two-species Stubb is more pronounced, with the best choice being a global, 2nd order background. Using a global 1st background order gives almost identical results (data not shown), hence we infer that a global background is the optimal choice for STUBBMS. As mentioned earlier, the STUBBSS program implements the same class of algorithm as the Ahab algorithm of Rajewsky et al. [4], with some technical differences, and therefore the two programs should produce similar results. We sought to verify this claim by running the Ahab program (with 1st and 2nd order Markov backgrounds), and comparing its performance to that of Stubb. (Ahab can only be run in the local background mode.) Figure 7a shows that there is not a significant difference between Stubb and Ahab CRM predictions. All the above runs were on genomic sequence with tandem repeats masked by the Tandem Repeats Finder program of Benson [13]. We have found that this heuristic improves genome-wide CRM prediction by Stubb. To substantiate this claim, we ran STUBBSS and STUBBMS on raw (unmasked) genomic sequence. Figure 7b plots the results. We find that both STUBBSS and STUBBMS perform better on masked data than on unmasked data. However, when Stubb is used to analyze shorter sequences (such as the upstream and downstream regions of a gene of interest), we have found unmasked sequence to be more useful, since false positives are less of a concern. Discussion The Stubb program is an extension of Ahab, with the important feature that it can handle two-species data within its probabilistic framework. The two programs differ in their underlying optimization method, with Stubb using an Expectation-Maximization approach in contrast to Ahab's conjugate gradient method. Performance evaluation of the two programs shows little difference between them, implying that the algorithm is robust to the actual optimization method used. Another technical difference between Ahab and Stubb is in the manner that orientation of binding sites is treated. While Stubb assumes a uniform prior on the orientation of a binding site, Ahab picks the best orientation for each site, with the caveat that probabilities are not strictly normalized. An important component of Stubb is the alignment step where the two species are aligned (using LAGAN) and blocks of high sequence similarity are extracted. (See Methods.) The parameters used in LAGAN runs were obtained from Emberly et al. [14], who derived the alignment parameters that maximize the overlap between experimentally verified binding sites and blocks of sequence conservation. They also studied the effect of changing the alignment algorithm (LAGAN from Brudno et al. [7]versus SMASH from Zavolan et al. [15]) for CRM's in the two fly species, and found no significant difference. Finally, the similarity thresholds we use for defining conserved blocks (10 bp or longer, with >70% identity) were obtained by trying a broad range of values, and choosing those that produced the best results, as per our genome-wide evaluation. Tandem repeat masking is a common pre-processing step for many sequence analysis applications involving binding sites. These repeats are short locally duplicated sequences, that may or may not be related to binding sites. It is not clear a priori how tandem repeats should affect module detection – repeats similar to binding sites of the system may improve sensitivity when they occur in CRM's; but if repeats resembling binding sites occur by chance in non-functional regions, prediction specificity may suffer. The occurrence of tandem repeats marks statistical deviation from Stubb's probabilistic model of sequence generation. In our tests, we found that repeats distract the algorithm more than they help, as manifested in better performance on repeat-masked sequence. (See Figure 7b.) This may be because two of the weight matrices in our collection (Hunchback and Caudal) resemble a poly-T stretch. Therefore, the poly-A or poly-T tandem repeats that occur promiscuously in the genome may be confused with sites of these two weight matrices. A recently published tool for genome-wide CRM prediction, called PFR-Searcher (Grad et al. [11]), first identifies "phylogenetically footprinted regions" or "PFR"s, that are sequences conserved between the two fly species, and then searches for a subset of these that are most similar in content to an input set of promoters. Their approach differs from Stubb in the nature of prior information input to the algorithm. While Stubb uses an input set of weight matrices, the training data for PFR-Searcher is a set of CRM's which, in their approach, is itself provided by a similarity search among PFR's of co-regulated genes. PFR-Searcher therefore has the advantage of not requiring knowledge of the transcription factor weight matrices relevant to the system. However, its ability to predict the binding site composition of potential CRM's is therefore more limited as compared to Stubb. (The Stubb program computes an average "parse" of the predicted module into its constituent binding sites for various transcription factors.) Grad et al. [11]report an evaluation of their algorithm on a test system very similar to ours, but with enough minor differences to make a direct comparison of performance impossible. For instance, the entire list of CRM's predicted in their evaluation corresponds, as per our CRM → gene mapping, to a set of only 46 unique genes, of which 31 are patterned. Twenty of these 31 correct predictions are also found in the top 46 gene predictions of STUBBMS, indicating a good degree of overlap between the two methods, at least in their highest ranked predictions. A fair and comprehensive comparison of the predictive power of these two algorithms is an interesting topic for future work, and it will be even more interesting to run STUBBMS only on PFR's detected by their criteria. Regarding the recovery of patterned genes by Stubb, several observations can be made. Of the 286 genes with ap patterns, we recover roughly half at a score cut-off of 10, using STUBBMS. Why is the other half not found? While it is obvious that lowering the cut-off will detect more patterned genes, there are other reasons why a patterned gene may be missed by Stubb. Some genes are likely to be lost due to the distance filter we have imposed (CRM to nearest gene <20 kb), since the regulatory regions of some genes (e.g., homeotic genes) are likely to be larger than that. More importantly, most of the patterned genes that are not part of the core transcriptional machinery have derivative patterns that reflect a more complex input (binding sites for pair rule factors, d-v factors etc.) and thus will only be recovered to the extent their input has a solid maternal/gap component. Conversely, there are at least two reasons for reporting false positives (roughly two thirds at a score cutoff of 10). The presence of an insulator could prevent the interaction between a CRM and its nearest basal promoter. More likely is a scenario where the predicted CRM's do drive expression but at post-blastoderm stages. All gap factors are active in multiple tissues in later development and therefore CRM's with dominant or exclusive gap input may well be active in these later contexts. These caveats affect all current CRM detection algorithms, and accounting for such additional axes of information as genomic context and module composition rules will be a difficult but important challenge for the future. A very interesting observation comes from the analysis in Table 1: Genes predicted by STUBBSS only, and not by STUBBMS, have weak or intermediate expression pattern more often than strong expression. This means that the CRM's that are not well-conserved between the two species (and hence not picked up by STUBBMS) typically correspond to weakly expressed genes. This ties in with previous studies (e.g., Domazet-Loso & Tautz [16]) that found fast evolving genes in Drosophila to be expressed relatively weakly. The Stubb program not only predicts cis-regulatory modules genome-wide, it additionally outputs the binding site profile of each predicted CRM, i.e., the locations and probabilities of binding sites in the CRM. Schroeder et al [5] use the corresponding feature in Ahab for a systematic analysis of the composition of all known or validated segmentation CRMs. The use of STUBBMS improves such binding site predictions. It is easy to adapt the program to take as input orthologous CRM's from the two species, and highlight the changes in terms of their binding site compositions. This leads to a powerful bioinformatic tool to predict regulatory changes between the two fly species. We can thus obtain hypotheses about changes in expression patterns, which can be verified experimentally. We have examined a representative collection of CRM's, and experimentally verified several of the changes predicted by Stubb, thereby building a catalogue of the different modes of cis-regulatory evolution. The results of this study will be reported in the near future. Conclusions We have seen that the use of a second fly genome significantly improves genome-wide module prediction. Since STUBBMS uses a natural "two-species" extension of the algorithm of STUBBSS, this finding is largely a statement about the inherent potential of cross-species comparison as a paradigm for improving functional genomics. The STUBBMS program also has a natural extension to incorporate more than two genomes, and it will be very interesting to see how much of a difference a third genome makes. The genome of D. yakuba is expected to be sequenced soon, and since this species is closer to D. melanogaster, it may help better discriminate conserved regulatory modules. Methods Alignment of D. melanogaster and D. pseudobscura D. melanogaster sequences were obtained from Flybase Release 3. The analysis was limited to the five chromosome arms 2L, 2R, 3L, 3R, and X. D. pseudobscura contigs were obtained from (February 2003 Release). Based on Blast results, we created a mapping, called "CONTIGMAP", between regions of the D. melanogaster genome and D. pseudobscura contigs, each region typically being tens of Kb long. This mapping is many to many, i.e., different regions of D. melanogaster may map to the same contig, and the same (or overlapping) region in D. melanogaster may map to two or more D. pseudobscura contigs. For each entry (M, P) in CONTIGMAP, where M is the D. melanogaster region and P is the D. pseudobscura contig, the LAGAN alignment program (Brudno et al. [7]) was run, with parameters gap start = -6, gap extension = 0, match = 1, and mismatch = -2, and all contiguous ungapped blocks of alignment, with length 10 bp or more and 70% identity or more, were extracted. In cases where the same region in D. melanogaster was mapped to multiple contigs, the density of LAGAN blocks was then used to choose exactly one mapping contig. Stubb runs Tandem repeats in the input sequences were masked with the Tandem Repeat Finder program of Benson [13], with parameter settings: (match = 2, mismatch = 5, indel = 5, match probability = 0.75, indel probability = 0.2, minimum score = 20, maximum period = 500). STUBBSS was run on the D. melanogaster genome with a sliding window of length 500 bp, in shifts of 50 bp. The input weight matrices for the maternal and gap transcription factors Bcd, Hb, Cad, Kni, Kr, Tll, Dstat and the torRE binding factor were obtained from Rajewsky et al. [4] and Schroeder et al. [5]. A weight matrix for the transcription factor Gt was constructed from known functional sites collected from the literature. STUBBMS was run on each entry (M, P) in CONTIGMAP, using a sliding window of length 500 bp on the D. melanogaster sequence M, in shifts of 50 bp. Thus, STUBBMS was not run on regions of D. melanogaster that are not aligned with some D. pseudoobscura contig. The weight matrices used were the same as in STUBBSS runs. The locations of the blocks computed in the alignment step (above) were input to STUBBMS, and the input value of the neutral mutation rate was 0.5, the value being chosen due to its better performance over alternatives tested. Each genome-wide run of Stubb produces, for each starting position of the sliding window, a score that measures the likelihood of the sequence having a cluster of binding sites. The next step is to extract the coordinates of each window that scores better than all other windows overlapping it. Such windows correspond to local "peaks" in the score profile along the genome. All such "peak" windows with scores above a certain threshold are sorted in decreasing order of their score, to produce a sorted list of predicted CRM's. Each window in this list is annotated with useful information including the identity and relative location of its neighboring genes. The list is then filtered to retain only those predicted CRM's where Stubb predicts occurrences of at least two weight matrices. This is a heuristic that incorporates the combinatorial nature of CRM's, i.e., their tendency to have sites for multiple transcription factors (activators as well as repressors.) Finally, any predicted CRM that overlaps with an exon is removed from the list before evaluation. The predictions made by STUBBMS and STUBBSS are listed in the files "Predicted CRM's – two species" (Additional File 4) and "Predicted CRM's – single species" (Additional File 5), respectively. Annotation of gene expression database The 792 genes which the BDGP expression database lists as showing expression during blastoderm (embryonic stages 4–6) were visually inspected. From this list, we removed genes with ubiquitous expression (426; this also removes the presumably very small number of genes whose ubiquitous expression is controlled by separate "regional" modules), extremely faint or irreproducible expression (31), or expression in pole cells or yolk nuclei only (64), as well as genes whose expression is modulated along the dv axis only (13). The remaining 258 genes show patterned expression in the somatic portion along the ap axis of the blastoderm embryo; 28 known segmentation genes not captured in the BDGP expression database were added to the list, for a total of 286 genes showing ap patterned blastoderm expression. These genes were further categorized by expression level (strong, intermediate, weak) and type of pattern (ap, ap+dv, dv+ap). ap includes gap, pair rule and segment polarity-like patterns (e.g., Kr, fkh, eve); ap+dv denotes ap pattern with some dv modulation (e.g., kni, so, en); dv+ap denotes dv pattern with some ap modulation (e.g., neur). Authors' contributions SS and EDS worked out the details of genome-wide Stubb runs. SS performed the Stubb runs, collected all statistics from the runs, and drafted the manuscript. EDS suggested several analyses reported. MDS, UU, and UG annotated the genes for expression pattern, suggested many of the analyses in the Results and Discussion sections, and wrote parts of the manuscript. All authors read and approved the final manuscript. Note added in proof A recently published paper (Berman et al: Genome Biol 2004, 5:R61, published 20 August 2004.) also evaluates the effect of cross-species comparison on CRM prediction in Drosophila. Supplementary Material Additional File 1 Test Genes List of 2167 genes with expression information. (Source BDGP + literature.) Click here for file Additional File 2 Patterned Genes List of 286 genes with blastoderm pattern, as determined by manual inspection of in-situ expression pattern. (Source BDGP + literature.) Click here for file Additional File 3 Additional Genes List of 2065 genes that are ubiquitous, maternal only, or not expressed. (Source BDGP.) Click here for file Additional File 4 Predicted CRM's – two species List of predicted CRM's using two-species Stubb with a global, 2nd order background, sorted in decreasing order of Stubb score. Click here for file Additional File 5 Predicted CRM's – single species List of predicted CRM's using single-species Stubb with a local, 1st order background, sorted in decreasing order of Stubb score. This list of predictions, as well as that in Additional File 4 (above), is meant to be as inclusive as possible; therefore, the specificity of the lowest ranked predictions may be poor. Click here for file Acknowledgements Support was provided by the NSF under grant DMR0129848, the NIH under grant GM066434-02, and the Keck Foundation (to SS). Figures and Tables Figure 1 (a) Effect of two-species data on performance. The x-axis is the number of unique genes predicted to be ap patterned in the blastoderm, restricted to those for which we have expression information. The y-axis is the number of predictions that are actually blastoderm patterned. STUBBSS and STUBBMS represent the single-species and two-species Stubb runs respectively. (b) The increase in number of correct predictions from STUBBSS to STUBBMS, as a percentage of the correct predictions made by STUBBSS. (c) The specificity values for a moving window of 50 predictions. The y-axis is the number of correct predictions as a fraction of the number of predictions, which is 50 for each window. Figure 2 (a) Effect of two-species data on performance, using a less stringent evaluation method than in Figure 1. The x-axis is the number of predicted CRM's, restricted to those for which at least one of the two nearest genes has expression information. A prediction is deemed "correct" if either of its two nearest genes has a blastoderm-specific pattern. (b) The increase in number of correct predictions from STUBBSS to STUBBMS, as a percentage of the correct predictions made by STUBBSS. Figure 3 Prediction accuracy under the requirement that each predicted gene be evidenced by at least two predicted CRM's. "MULT" refers to this strategy, while "DEFAULT" refers to the default mapping of CRM's, without this restriction. (a) STUBBSS (b) STUBBMS. Figure 4 Effect of two-species data on performance, using a more complete data set that includes genes with ubiquitous, maternal-only, or no expression. The axes are as in Figure 1a. (The x-axis has a greater range than in Figure 1a.) Figure 5 Annotation of blastoderm patterned genes predicted by STUBBMS. "DV+AP pattern": Genes with dorso-ventral aspect of pattern, in addition to anterior-posterior pattern. "Strong expression": Genes with strong expression. "Core gene": A set of 48 genes experimentally known to be required for segmentation. The x-axis represents correctly predicted genes. Each point represents, for a moving window of 20 correctly predicted genes, the fraction of genes in the appropriate category. Figure 6 Effect of different parameter settings in Stubb, on prediction accuracy. (a: Single species Stubb, b: two-species Stubb.) The axes have the same interpretation as Figure 1a. Terminology in legend: "SS" – STUBBSS; "MS" – STUBBMS; "BKG1" and "BKG2": 1st and 2nd order background respectively; "LOCAL" and "GLOBAL": local and global background models respectively. (The axes are not to the same scale in (a) and (b).) Figure 7 (a) Comparison between Ahab and Stubb, local background of orders 1 and 2 in different runs. All runs are on D. melanogaster genome only. (b) Effect of tandem repeat masking of input sequences. "MS_MASKED" and "MS_UNMASKED": STUBBMS, with global background model of order 2, on masked and unmasked sequence respectively. "SS_MASKED" and "SS_UNMASKED": STUBBSS, with local background model of order 1, on masked and unmasked sequence respectively. Table 1 Expression patterns of predicted genes. Top 311 genes predicted as being patterned, by STUBBSS and STUBBMS. "INTERSECTION": Genes correctly predicted by both methods. "MS-ONLY": Genes correctly predicted by STUBBMS and not by STUBBSS. "SS-ONLY": Genes correctly predicted by STUBBSS and not by STUBBMS. STRONGLY EXPRESSED WEAK + INTERMEDIATE INTERSECTION 41 29 MS-ONLY 23 19 SS-ONLY 7 14 ==== Refs Berman BP Nibu Y Pfeiffer BD Tomancak P Celniker SE Levine M Rubin GM Eisen MB Exploiting transcription factor binding site clustering to identify cis-regulatory modules involved in pattern formation in the Drosophila genome Proc Natl Acad Sci U S A 2002 99 757 62 11805330 10.1073/pnas.231608898 Markstein M Markstein P Markstein V Levine MS Genome-wide analysis of clustered Dorsal binding sites identifies putative target genes in the Drosophila embryo Proc Natl Acad Sci U S A 2002 99 763 8 11752406 10.1073/pnas.012591199 Halfon MS Grad Y Church GM Michelson AM Computation-based discovery of related transcriptional regulatory modules and motifs using an experimentally validated combinatorial model Genome Res 2002 12 1019 28 12097338 Rajewsky N Vergassola M Gaul U Siggia ED Computational detection of genomic cis-regulatory modules applied to body patterning in the early Drosophila embryo BMC Bioinformatics 2002 3 30 12398796 10.1186/1471-2105-3-30 Schroeder MD Pearce M Fak J Fan H Unnerstall U Emberly E Rajewsky N Siggia ED Gaul U Transcriptional Control in the Segmentation Gene Network of Drosophila PLoS Biology 2004 2 Sinha S van Nimwegen E Siggia ED A probabilistic method to detect regulatory modules Bioinformatics 2003 19 i292 301 12855472 10.1093/bioinformatics/btg1040 Brudno M Do CB Cooper GM Kim MF Davydov E Green ED Sidow A Batzoglou S NISC Comparative Sequencing Program LAGAN and Multi-LAGAN: efficient tools for large-scale multiple alignment of genomic DNA Genome Res 2003 13 721 31 12654723 10.1101/gr.926603 St Johnston D Nusslein-Volhard C The origin of pattern and polarity in the Drosophila embryo Cell 1992 68 201 219 1733499 10.1016/0092-8674(92)90466-P Rivera-Pomar R Jackle H From gradients to stripes in Drosophila embryogenesis: filling in the gaps Trends Genet 1996 12 478 483 8973159 10.1016/0168-9525(96)10044-5 Furriols M Casanova J In and out of Torso RTK signalling EMBO J 2003 22 1947 1952 12727862 10.1093/emboj/cdg224 Grad YH Roth FP Halfon MS Church GM Prediction of similarly-acting cis-regulatory modules by subsequence profiling and comparative genomics in D. melanogaster and D. pseudoobscura Bioinformatics Nelson CE Hersh BM Carroll SB The regulatory content of intergenic DNA shapes genome architecture Genome Biol 2004 5 R25 15059258 10.1186/gb-2004-5-4-r25 Benson G Tandem repeats finder: a program to analyze DNA sequences Nucleic Acids Res 1999 27 573 80 9862982 10.1093/nar/27.2.573 Emberly E Rajewsky N Siggia ED Conservation of regulatory elements between two species of Drosophila BMC Bioinformatics 2003 4 57 14629780 10.1186/1471-2105-4-57 Zavolan M Rajewsky N Socci ND Gaasterland T SMASHing regulatory sites in DNA by human-mouse sequence comparisons In Proceedings of the 2003 IEEE Bioinformatics Conference (CSB2003) 277 286 Domazet-Loso T Tautz D An evolutionary analysis of orphan genes in Drosophila Genome Res 2003 13 2213 9 14525923 10.1101/gr.1311003

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BMC Bioinformatics. 2004 Sep 9; 5:129

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==== Front BMC Health Serv ResBMC Health Services Research1472-6963BioMed Central London 1472-6963-4-241535300310.1186/1472-6963-4-24Research ArticleHow patient-physician encounters in critical medical situations affect trust: results of a national survey Shenolikar Rahul A [email protected] Rajesh [email protected] Mark A [email protected] Division of Management, Policy and Community Health, University of Texas School of Public Health, 1200 Herman Pressler, Houston, TX 77030, USA2 Wake Forest University Schools of Law and Medicine, 2000 West First Street, Piedmont Plaza II, 2nd Floor, Winston-Salem, NC 27104, USA, Winston-Salem, USA2004 7 9 2004 4 24 24 1 3 2004 7 9 2004 Copyright © 2004 Shenolikar et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Patients' trust in physicians and in the medical profession is vital for a successful patient-physician relationship. Trust is especially salient in critical medical situations, such as serious side-effects, hospitalizations, and diagnoses of serious medical conditions, but most trust studies have been done with the general population or in routine primary care settings. This study examines the association between patient-physician encounters in such critical medical situations and patients' trust in their physician and in the medical profession in general. Methods A random national telephone survey was conducted using validated multi-item questionnaire measuring trust and satisfaction with physicians and with the medical profession. A seven item questionnaire measured the patient-physician encounters in critical medical situations. A total of 1117 subjects aged 20 years and older with health insurance were included for analyses. Spearman rank order correlations were used to determine the association of encounter variables with trust in physicians and the medical profession. Results Prescription of medications by primary care physicians that patients believed might have side effects was negatively correlated with trust in physician (ρ = -0.12, p < 0.001, n = 1045) in multivariate analysis. A primary care physician evaluating the patient for a condition the patient believed was serious was positively correlated with trust in physician (ρ= 0.08, p < 0.01). Being hospitalized was positively correlated with trust in the medical profession (ρ = 0.12, p < 0.01, n = 475). Conclusion Hospitalization, perceived seriousness of condition, and concerns about the risks of medications were found to be associated with patient trust in physicians or the medical profession. These findings highlight the salience of trust in serious physician-patient encounters and the role that patient vulnerability plays in determining patient trust. ==== Body Background Patients' trust in their physicians is vital for a successful treatment relationship [1,2], which is important for achieving desired treatment outcomes [3-6]. Trust in physicians is a positive acceptance of a vulnerable situation in which patients believe that physicians will care for their interests [7]. Previous studies have identified three sets of factors that are associated with trust: patient characteristics, physician characteristics, and relationship factors. [2,8-26] Numerous factors, such as choice of physician, length of relationship, and managed care settings have been found to be among the stronger predictors of trust.[16] However, previous studies have not examined the association between trust and the patient-physician encounters in critical medical situations. Experience of interactions in some critical medical situations is potentially a very important factor because it affects the salience of trust, and because of the role that vulnerability is thought to play in the psychology of trust. [7] Some examples of critical medical situations are those in which physicians diagnose serious medical conditions, perform surgery, or prescribe medication that might have serious side effects. The heightened vulnerability created by these situations could have pronounced effects, either positive or negative, on trust in one's physician or in the medical profession. The objective of this exploratory study is to examine the possible associations between patients' experiences in critical medical situations and their trust in their physician and in the medical profession in general. Methods Sample selection The national sample was selected by random digit dialing, with the sampling frame generated by a random sample from a proprietary database of working residential telephone exchanges in the continental United States. The sampling frame was provided by Survey Sampling, Inc. of Westport, Connecticut. Survey Sampling, Inc. maintains a database of working residential telephone exchanges in the continental United States. In selecting the numbers to be called in this study, an exchange was randomly selected and then a random number between 0000 and 9999 was generated to complete the number. This process was repeated until a sufficient quantity of numbers had been generated. Between April-June 1999, a total of 4028 numbers dialed (minimum 15 attempts each) yielded 2637 (65 %) responses. Households were excluded with no one over the age of 20 (n = 66) or where the adult respondent with the next birthday did not have health insurance (n = 151) or had not seen a health professional at least twice during the past two years (n = 248). Respondent selection within eligible households was done using the next birthday method. Contacts with the 2172 potentially eligible individuals resulted in the following dispositions: 1117 (51.4%) were interviewed; 571 (26.3%) refused; 484 (22.2%) were unable to participate (not home, ill, non-English-speaking). Instrument All subjects were asked a core set of questions about their regular health care provider, demographic characteristics, satisfaction with care, physical and mental health, and preferences regarding seeking care and making medical decisions. Satisfaction was measured in two ways: a single item on patients' satisfaction with their physicians and a 12-item scale on patients' satisfaction with the healthcare that they have been receiving from all sources during the past few years. Two validated trust scales were used [12,22], each using a 5-point Likert scale [a 10 item physician trust scale (Cronbach's α = 0.93), and an 11 item medical profession trust scale (Cronbach's α = 0.92)]. The physician trust scale asked mainly about trust in primary care physicians. Items in both the scales represent four dimensions of trust (fidelity, competence, honesty, global). Physician trust was measured by the sum of 10 items scores, ranging from 10 to 50, with a higher score indicating more trust. Trust in the medical profession was measured by 11 item scores ranging from 11 to 55, with a higher score indicating more trust. Patient satisfaction with health care was measured using a previously validated 12-item 60 point scale. [27] Other variables thought to be related to physicians trust were measured as follows: whether one had enough choice in selecting a physician (yes/no); number of years with physician; willingness to recommend to friends (strongly agree to strongly disagree; past disagreement or dispute with the physician (yes/no); desire to switch physicians (strongly to strongly disagree). Due to interview length (25 minutes), only half of the subjects that were randomly selected were asked about trust in the medical profession. [12] A questionnaire naming seven medical situations with dichotomous responses (Yes/No) was developed to identify encounters with physicians separately for the patient's primary physicians and for other physicians if their services were utilized. The items in the questionnaire were created after expert review by a panel of physicians, behavioral scientists, and health lawyers and piloted in patient focus groups.[22]These items asked whether over the past five years, the subject had been hospitalized, had undergone minor (non-anesthesia) or major surgery, had been prescribed medications that they thought could have serious side effects, had been evaluated for possible or actual cancer or for another serious medical condition, or had been referred to a specialist. Analyses Dependent variables for the study were physician trust and medical profession trust. Independent variables included patient-physician encounter variables and other significant variables mentioned in the previous section identified from a previous study. [5] These hypothesized predictors were tested for their bivariate association with trust scores using Spearman rank-order correlations. Finally, partial Spearman correlations between significant predictors in the bivariate analyses (adjusting for other confounders, e.g. we examined correlation between subjects who had undergone major surgery and physician trust adjusting for other variables such as their satisfaction with healthcare, poor physical health, number of visits to physician, whether their physician was a foreign physician, long waiting time with physician, and disputes with physician) and the corresponding measures of trust were estimated. All statistical analyses were conducted using STATA statistical software (College Station, TX). [28] Results Table 1 presents descriptive statistics on the study population. One study population refers to the respondents who were asked about trust in physicians, and the second study population refers to respondents who responded to questions about trust in the medical profession. Complete data for analyses were obtained for 1045 subjects for the physician trust analysis, and 475 subjects for the analysis of trust in the medical profession. Mean patient characteristics, including the encounter variables, did not differ very much across the two study populations. Table 2 presents results of the bivariate Spearman rank-order correlations of the encounter variables with corresponding measures of trust in primary care physician. Prescribing medications to patients that they thought would have serious side effects, overnight hospitalization, and evaluating patients for a serious condition were found to be significantly correlated to physician trust (all significant at p < 0.05) as well as trust in the medical profession (all significant at p < 0.05) while the physician performing a surgery (p < 0.05) perceived as major by the patient was significantly correlated to only trust in physicians. Significant predictors in the bivariate analysis were included in the multivariate partial correlation analysis. Table 3 shows the partial Spearman rank order correlations between different correlates of trust, adjusting for each other. Patient-physician encounter variables significant in bivariate analyses were included. Physician trust was negatively correlated with prescription of a medication that a patient thought had serious side-effects (p < 0.01). An encounter which included being evaluated for a serious condition other than cancer was associated with higher trust in physicians (p < 0.01). Trust in the medical profession was significantly higher in patients who had been hospitalized by their primary care physician (p < 0.01). None of the other encounter variables were associated with profession trust. In other findings (detailed results not shown), we did not identify any physician-patient encounter variable that was significantly correlated with trust in the subject's primary physician among those who had an interaction with a physician other than their primary physician (33% of study population). Discussion These findings suggest that the patient-physician encounters in critical medical situations are associated with patients' trust in physicians. Of seven conditions that indicate more serious experiences, four were found to have a significant relationship with either trust in one's primary physician or trust in the medical profession. The three conditions not found to be significantly related to trust include the two that were less threatening than the others – minor surgery, and referral to a specialist. The significance of critical medical situations might arise either from the salience of trust in these situations, or from the vulnerability that is created by more serious medical conditions or procedures. It is important to note that the relationship with trust was positive for two intensity indicators, but negative for a third. The positive relationship lends support to the theory that trust arises from vulnerability and therefore is potentially higher when there is a greater need to trust. [7] However, vulnerability can also give rise to distrust. The negative correlation with the variable relating to medication might arise from the fact that the wording of this variable may have indicated that the physician made a mistake in prescribing the wrong medication. Other encounter variables were neutral regarding physician competence. Another interesting pattern that emerged in this initial, exploratory analysis is the type of trust that related to different encounter variables. Most variables were related to trust in the subject's primary physician, but overnight hospitalization was also related to increased trust in the medical profession, and when other predictors of trust were controlled for, significance remained only for trust in the medical profession. This is consistent with the fact that hospital treatment is more of a team effort that reflects on the performance of the medical system. Other encounter variables that were significant for both types of trust in the bivariate analysis remained significant only for physician trust after adjusting for other predictors of trust. This is consistent with the theory that the primary object of trust in most treatment settings is the treating physician. Due to the limitations of this study, these findings and interpretations should be regarded as preliminary. This was an exploratory cross-sectional study whose particular inclusion criteria resulted in a sample that does not exactly correspond to the socio-economic distribution of the general United States population. The cross-sectional design leaves open the possibility that current levels of trust could affect recall of past events. The study asked about the impact of previous encounters on current physician trust, but it should be noted that the current physician may not be the one involved in previous encounters. Another limitation is that it was not possible to know the degree of vulnerability associated with each type of experience. Also, most measures were based on subjects' unverified reports. Summarizing the results, this study found significant associations between patient-physician encounters in medical situations and trust in primary physicians and in the medical profession. These associations reflect the role that vulnerability plays in the psychology of trust. Future research should focus on identifying in further detail which forms of encounters affect which types of trust, and in what directions. Also, more research is needed to understand why these encounters affect trust and what factors modify these relationships. Such research could help to identify threats to trust and further maintain trust and trustworthy conditions. This is especially important in the current era in which many people fear that trust in medical care is rapidly eroding.[16] Competing interests None declared. Authors' contributions RB and RAS conceived the paper. RB and MAH were responsible for data collection. RB and RAS conducted the data analyses. RAS was primarily responsible for writing the paper. The manuscript was reviewed and critically revised by RB and MAH. Pre-publication history The pre-publication history for this paper can be accessed here: Acknowledgments This study was supported by a grant from the Robert Wood Johnson Foundation Figures and Tables Table 1 Descriptive Statistics of Study Population Variable ⇓ Physician Trust Population*(n = 1045) Medical profession Trust Population** (n = 475) Physician trust [10–50] 40.7 (6.1) 40.6 (6.1) Profession trust [11–55] 33.5 (6.9) Satisfaction with health care [12–60] 41.9 (7.6) 41.0 (7.3) Age [21–40 years] 34.1% 34.4% Age [41–64 years] 36.4% 41.2% Age [65 years and above] 29.5% 24.4% Male gender 33% 33% Latin origin 4% 5% Non white 24% 17% Low income (< $20,000/year) 17% 18% Poor physical health 13% 14% Adequate choice of physician 74% -- Changed physicians 49% 48% Physician primary health decision maker [1–10] 5.3 (1.2) 5.3 (1.2) Always better to seek medical help 44% 53% Foreign physician 9% -- Long waiting time with physician 16% -- Patients who had also accessed a non-primary care physician 33% 32% Outside interactions with physician 31% -- Number of visits to physician [1–4] 2.6 (1.2) -- Encounter variables Yes No Yes No Major Surgery 10% (105/1045) 90% 10% (48/475) 90% Minor surgery 18% (188/1045) 72% 16% (76/475) 84% Prescribed medication that you thought might have serious side effect 15% (156/1045) 85% 16% (76/475) 84% Overnight hospitalization 21% (220/1045) 79% 21% (99/475) 79% Evaluated for possible or actual cancer 41% (428/1045) 59% 42% (199/475) 58% Evaluated for another condition that you thought might be serious 40% (418/1045) 60% 43% (204/475) 57% Referred to another doctor 57% (596/1045) 43% 57% (270/475) 43% Note: Standard deviations () and ranges [] wherever applicable are indicated in parentheses and brackets. Number of lifetime visits to the physician was categorized as follows: value of 1 denotes 2–5 visits, 2 denotes 6–10 visits, 3 denotes 11–20 visits, 4 denotes more than 21 visits. * This column presents distribution of characteristics among persons who responded to items on physician trust. ** This column presents distribution of characteristics among persons who responded to items on general medical profession trust. Table 2 Bivariate Spearman Rho Correlations between types of trust and patient-physician encounter variables in critical medical situations Dependent variable ⇒ Independent variables ⇓ Physician Trust (n = 1045) Medical Profession Trust (n = 475) Major surgery 0.078* 0.012 Minor surgery 0.021 -0.00 Prescribed medication that you thought might have serious side effect -0.12** -0.1* Overnight hospitalization 0.072* 0.13** Evaluated for possible or actual cancer 0.048 -0.02 Evaluated for another condition that you thought might be serious 0.093** -0.098* Referred to another doctor 0.045 -0.013 Notes: Partial Spearman correlations are presented * Significance at the 5% level ** Significance at 1% level for the test examining if partial Spearman ρ = 0 Dependent variable: Physician trust [10–50], and medical profession trust [11–55]. [] Range Table 3 Partial correlation analysis results examining the relationship between patient-physician encounters and trust, adjusting for each other Dependent Variable ⇒ Physician Trust (n = 1045) Dependent Variable ⇒ Medical Profession Trust (n = 475) Independent Variables Independent Variables Encounter variables Encounter variables Major surgery (1 = yes, 0 = no) 0.057 Prescribed medication that might have a serious side effect (1 = yes,0 = no) -0.12** Prescribed medication that might have a serious side effect (1 = yes,0 = no) -0.03 Overnight hospitalization (1 = yes,0 = no) -0.014 Overnight hospitalization (1 = yes,0 = no) 0.12** Evaluated for another condition you thought to be serious (1 = yes,0 = no) 0.08** Evaluated for another condition you thought to be serious (1 = yes,0 = no) -0.079 Other independent variables Other independent variables Satisfaction with health care (1 = yes,0 = no) 0.53** Physician trust 0.21** Poor physical health (1 = yes,0 = no) -0.077* Satisfaction with health care -0.045** Number of visits to physician 0.099** Age -0.12** Age-squared 0.13** Foreign physician (1 = yes,0 = no) -0.09** Poor mental health (1 = yes,0 = no) -0.084 Long waiting time with physician (1 = yes,0 = no) -0.13** Low income 0.016 Disputes with physician (1 = yes,0 = no) -0.055 Changed physicians (1 = yes,0 = no) -0.27** Outside interactions with physicians (1 = yes,0 = no) 0.085** Physician primary health decision maker (1 = yes,0 = no) 0.12** Adequate choice in selecting physicians (1 = yes,0 = no) 0.12** Always better to seek medical than self-treat (1 = yes,0 = no) 0.17** Non-Physician is Primary care Provider (1 = yes,0 = no) 0.089** Notes: Partial Spearman correlations are presented * Significance at 5% level ** Significance at 1% level for the test examining if partial Spearman ρ = 0 Dependent variable: Physician trust [10–50] and medical profession trust [11–55]. [] Range ==== Refs Kao AC Green DC Davis NA Koplan JP Cleary PD Patient's trust intheir physicians: Effects of choice, continuity, and payment method J Gen Intern Med 1998 13 681 686 9798815 10.1046/j.1525-1497.1998.00204.x Pearson S Raeke L Patients' trust in physicians: Many theories, few measures, and little data J Gen Intern Med 2000 15 509 513 10940139 10.1046/j.1525-1497.2000.11002.x Stewart MA Effective physician-patient communication and health outcomes: a review CMAJ 1995 152 1423 1433 7728691 Sherbourne CD Hays RD Ordway L DiMatteo MR Kravitz RL Antecedents of adherence to medical recommendations: results from the Medical Outcomes Study J Behav Med 1992 15 447 468 1447757 Hall JA Roter DL Datz NR Meta-analysis of correlates of provider behavior in medical encounters Med Care 1988 26 657 675 3292851 Anderson RM Funnel MM Butler PM Arnold MS Fitzgerald JT Feste CC Patient empowerment. Results of a randomized controlled trial Diabetes Care 1995 18 943 949 7555554 Hall MA Dugan E Zheng B Mishra AK Trust in physicians and medical institutions: what is it, can it be measured, and does it matter? Milbank Q 79 613 639 11789119 10.1111/1468-0009.00223 Mechanic D Schlesinger M The impact of managed care on patients' trust in medical care and their physicians JAMA 1996 275 1693 1697 8637148 10.1001/jama.275.21.1693 Emanuel EJ Dubler NN Preserving the physician-patient relationship in the era of managed care JAMA 1995 273 323 329 7815662 10.1001/jama.273.4.323 Mechanic D Changing medical organization and the erosion of trust Milbank Q 1996 74 171 189 8632733 Gray BH Trust and trustworthy care in the managed care era Health Aff 1997 16 34 49 10.1377/hlthaff.16.1.34 Hall M Camacho F Dugan E Balkrishnan R Trust in the medical profession: Conceptual and measurement issues Health Serv Res 2002 37 1419 1439 12479504 10.1111/1475-6773.01070 Thom D Campbell B Patient-physician trust: an exploratory study J Fam Pract 1997 44 169 176 9040520 O'Malley AS Forrest C Primary care performance and the patient-physician relationship for low-income women J Gen Intern Med 2002 17 66 73 11903777 10.1046/j.1525-1497.2002.10338.x Montaglione C The physician-patient relationship: Cornerstone of patient trust, satisfaction, and loyalty Manag Care Q 1999 7 5 21 10620959 Balkrishnan R Dugan E Camacho FT Hall MA Trust and satisfaction with physicians, insurers, and the medical profession Med Care 2003 41 1058 1064 12972845 10.1097/01.MLR.0000083743.15238.9F DiMatteo M Hays R The significance of patients' perceptions of physician conduct: A study of patient satisfaction in a family care center J Community Health 1980 6 18 34 7430419 Krupat E Bell RA Kravitz RL Thom D Azari R When physicians andpatients think alike: patient-centered beliefs and their impact on satisfaction and trust J Fam Pract 2001 50 1057 1062 11742607 Anderson L Dedrick R Development of the trust in physician scale: A measure to assess interpersonal trust in patient-physician relationships Psychol Rep 1990 67 1091 1100 2084735 Thom DH Ribisl KM Stewart AL Luke DA Further validation and reliability testing of the trust in physician scale Med Care 1999 37 510 517 10335753 10.1097/00005650-199905000-00010 Steine S Finset A Laerum E A new brief questionnaire (PEQ) developed in primary health care for measuring patients' experience of interaction, emotion and consultation outcome Fam Pract 2001 18 410 418 11477049 10.1093/fampra/18.4.410 Hall MA Zheng B Dugan E Camacho F Kidd KE Mishra A Balkrishnan R Measuring patients' trust in their primary care providers Med Care Research & Review 2002 59 293 318 10.1177/1077558702059003004 Keating NL Green DC Kao AC Gazmararian JA Wu VY Cleary PD How are patients' specific ambulatory care experiences related to trust, satisfaction, and considering changing physicians? J Gen Intern Med 2002 17 29 38 11903773 10.1046/j.1525-1497.2002.10209.x Garrison G Bernard M Rasmussen N 21st-Century health care: The effect of computer use by physicians on patient satisfaction at a family medicine clinic Fam Med 2002 34 362 368 12038718 Grumbach K Selby J Damberg C Bindman AB Quesenberry C JrTruman A Uratsu C Resolving the gatekeeper conundrum: What patients value in primary care and referrals to specialists JAMA 1999 281 261 266 9918482 Murphy J Chang H Montgomery Rogers WH Safran DG The quality of physician-patient relationships J Fam Pract 2001 50 123 129 11219559 Hall JA Feldstein M Fretwell MD Rowe JW Epstein AM Older patients' health status and satisfaction with medical care in an HMO population Med Care 1990 28 261 270 2314135 STATA Statistical Software, release 50 College Station, TX 2000

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BMC Health Serv Res. 2004 Sep 7; 4:24

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==== Front BMC GenomicsBMC Genomics1471-2164BioMed Central London 1471-2164-5-641535787510.1186/1471-2164-5-64SoftwareA method for detecting and correcting feature misidentification on expression microarrays Tu I-Ping [email protected] Marci [email protected] Maximilian [email protected] Branimir I [email protected] Patrick O [email protected] David [email protected] Michael J [email protected] Functional Genomics Facility, Stanford University School of Medicine, Stanford, CA, USA2 Department of Biochemistry, Stanford University School of Medicine, Stanford, CA, USA3 Oncology Division, Department of Medicine, Stanford University School of Medicine, Stanford, CA, USA4 Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ, USA5 Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA, USA6 Institute of Statistical Science, Academia Sinica, Taipei, Taiwan, R.O.C2004 9 9 2004 5 64 64 6 7 2004 9 9 2004 Copyright © 2004 Tu et al; licensee BioMed Central Ltd.2004Tu et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Much of the microarray data published at Stanford is based on mouse and human arrays produced under controlled and monitored conditions at the Brown and Botstein laboratories and at the Stanford Functional Genomics Facility (SFGF). Nevertheless, as large datasets based on the Stanford Human array began to accumulate, a small but significant number of discrepancies were detected that required a serious attempt to track down the original source of error. Due to a controlled process environment, sufficient data was available to accurately track the entire process leading to up to the final expression data. In this paper, we describe our statistical methods to detect the inconsistencies in microarray data that arise from process errors, and discuss our technique to locate and fix these errors. Results To date, the Brown and Botstein laboratories and the Stanford Functional Genomics Facility have together produced 40,000 large-scale (10–50,000 feature) cDNA microarrays. By applying the heuristic described here, we have been able to check most of these arrays for misidentified features, and have been able to confidently apply fixes to the data where needed. Out of the 265 million features checked in our database, problems were detected and corrected on 1.3 million of them. Conclusion Process errors in any genome scale high throughput production regime can lead to subsequent errors in data analysis. We show the value of tracking multi-step high throughput operations by using this knowledge to detect and correct misidentified data on gene expression microarrays. ==== Body Background Expression microarrays, with the capability to measure the mRNA expression level of tens of thousands of genes simultaneously, have found broad application in both clinical and basic research [1-7]. With the generation of large data sets from microarray experiments, statistical methods are needed to extract useful information. Many methods have had specific implementations written for the analysis of gene expression data, such as various forms of clustering, self ordered maps, singular value decomposition and significance analysis [8-13]. However, these methods all rely on the assumption that there are no gross process errors in the original data. Previous analyses of systematic errors in microarray data have focused on problems at the level of sample preparation, labelling, or hybridization. This report focuses on steps in the microarray production process prior to hybridization that may ultimately result in errors in the underlying data. Much of the microarray data published at Stanford is based on mouse and human arrays produced under controlled and monitored conditions at the Brown and Botstein laboratories and at the Stanford Functional Genomics Facility (SFGF). Nevertheless, as large datasets based on the Stanford Human array began to accumulate, a small but significant number of discrepancies were detected that required a serious attempt to track down the original source of error. Due to a controlled process environment, sufficient data was available to accurately track the entire process leading to up to the final expression data. In this paper, we describe our statistical methods to detect the inconsistencies in microarray data that arise from process errors, and discuss our technique to locate and fix these errors. We are able to fix those errors that originate at the level of any microtiter plate used during a multi-step microarray production process. The major process fail points in cDNA microarray production at Stanford are shown in Table 1. It is at these points that misidentifications can occur. Other types of processes resulting in expression data will have their own possible fail points. Regardless of the particular process, for the sake of subsequent error checking it is important to track every instance where samples have moved from one microtiter plate to another, or from microtiter plate to microarray. Our process involves four such reallocations: From an archival 96-well block to a 96-well growth block, then to a 96-well PCR plate, then to four 384-well print plates and finally to 250 10–40 thousand element microarrays. Even in highly automated bar-coded and vision controlled systems the possibility exists that plates might become swapped, skipped, misordered, or rotated by 180 degrees during one of the process steps. Our own process is fairly well automated, but like most academic and commercial efforts our process involves hand loading of robots. For example in our case, during the transfer from 96 to 384-well format, it is possible to accidentally rotate a 96-well plate or misorder the 96 or 384-well plates. During printing a 384-well plate might be accidentally skipped, swapped or printed in the wrong orientation (rotated by 180 degrees). Even with the best engineering controls to prevent plate rotations, the potential exists for misidentified plates. Inconsistencies in our data were first detected both by visual inspection of microarray data as well as the appearance of anomalously large first components in singular value decomposition analyses of Ovarian Tumour data [6] traced to different production batches of arrays. Our algorithm was developed to detect and repair these types of errors, using the similarities in expression levels between sets of spots from different microarrays. The algorithm was used to check all of the microarray data in our database for which there was a sufficient process record. The general idea, illustrated in Figure 1, is as follows: Partition the microarray expression data from a single microarray into sets based on the various microtiter plates the samples have been in throughout their process history. For example, at Stanford we keep our process fairly simple, with a minimum of plate changes, so our spotting material can be said to have existed in either 96 well or 384-well format during its entire process history. Thus, we partition the data into sets corresponding to the 96 or 384-well plate history. Next, an expression vector for each element of the partition (each plate) based on absolute (not relative) expression levels is formed and compared to every other expression vector from every other plate on many other array batches. A rank matrix of correlation coefficients is formed which should be close to unity on the diagonal and far from unity off the diagonal. Non-unitary diagonal elements indicate problems with that plate comparison. A rank comparison of the best correlations can be made to find swapped plates. The algorithm can be repeated assuming a rotated plate to check to see if the discrepancy can be attributed to a plate rotation. It should be noted that in cases where no process error is indicated, the method can still indicate the presence of problem plates, print batches or PCR rounds that should be flagged for particular attention in downstream analyses. Our algorithm (named MuFu for "MixupFixup") is for arrays produced in the Brown and Botstein laboratories and the Stanford Functional Genomics Facility. However, the ideas are general and can be applied to many other types of high throughput operations. In most of the research studies using our human microarray, a common reference [14] is compared against a tumour or tissue specimen. The common reference is usually labelled with the Cy3 dye, pseudo-colour green in most visualizations of the data, while the sample specimens are labelled with Cy5 dye, pseudo-colour red. Subtle corrections due to background subtraction issues and normalization are not important for this analysis. Because a common reference is used in a large number of hybridization experiments at Stanford, all of the Cy3 intensities (or Cy5 in some cases) across various kinds of experiments are comparable. We measure the similarities of two sets of spots by taking the correlations between the common reference intensities of these two sets of spots. For those experiments that do not use common reference, the comparison is made using experiments with similar samples in either the Cy3 or Cy5 channel. Figure 1 MuFu flowchart. Flow of the MuFu algorithm. Looping and re-partitions of the data are not shown. Results Finding misidentified data This example is from the analysis of experiments from an ovarian tumor dataset[6] that first led us to develop MuFu. Here, it was noticed that similar tissues were not clustering across batch boundaries as expected. Also, an anomalously large first component in a singular value decomposition analysis[9] pointed to problems at the array batch level. After some detective work, visual inspection uncovered anomalies in certain print plates as seen in Figure 2. We were able to isolate the problem to distinct sets of 96-well plates that had been swapped during an upstream process step, probably during the transfer from 96-well plates to 384-well plates. Not wishing to go through this sort of process again and again, MuFu was developed to more succinctly recapitulate this finding and apply it to all data. Figure 2 Ovarian tumour data. Visual inspection of the anomalous spots from the Ovarian tumor data before and after applying MuFu. In each case the top row shows that the feature alignment is inconsistent with the contents of the plate. In the bottom row features group together as expected. With MuFu, a check using a 384-well plate partition of the data shows a discrepancy but no obvious plate rotation or misidentification. We then repartition and repeat our tests at the 96-well plate level. The results are shown in Table 2a where we show a 12 way pair wise comparison. The first four comparisons are for batches whose print plates are made from the first PCR round. The middle four compare the first PCR round to the second, and the last four compare batches from the second PCR round. Mismatches are evident in the middle set of comparisons. From Table 2b we see that the distance matrix identifies a match between plate h and plate i and plate n and plate o for all four comparisons of the first PCR round to the second. Table 2 Test for 96 well plate handling error. a) In this table we see that the middle bank of comparisons indicates a discrepancy between data from the first PCR run and the second, at the 96-well plate level. b) A check of the distance matrix results show that the swapped plates are h and i in one case, and n and o in the other. Bold indicates P-value = 0.3, italic indicates P-values between 1.0E-03 and 1.0E-04, while regular font indicates P-values < 1.0E-04. a) R_{m, m} PCR Round 1 vs 1 PCR Round 1 vs 2 PCR Round 2 vs 2 Plates A1 v A2 A2 v A3 A3 v A4 A4 v A5 A2 v B1 A3 v B2 A4 v B3 A5 v B4 B1 v B2 B5 v B3 B1 v B4 B5 v B4 {a, a} 1 1 1 1 1 1 1 2 1 1 2 1 {b, b} 1 1 1 1 1 1 1 1 7 1 3 1 {c, c} 1 1 1 1 1 1 1 1 1 1 1 1 {d, d} 2 1 1 1 1 1 5 9 1 1 1 1 {e, e} 1 1 1 1 1 1 1 2 1 1 2 1 {f, f} 1 1 1 1 1 1 1 1 1 1 1 1 {g, g} 3 1 1 1 1 1 1 1 1 2 1 1 {h, h} 1 1 1 1 343 322 408 294 1 1 1 1 {i, i} 2 1 1 1 421 290 402 359 1 1 1 1 {j, j} 1 1 1 1 2 1 1 1 2 1 4 1 {k, k} 1 1 1 1 2 1 1 1 1 1 1 1 {l, l} 1 1 1 1 1 2 1 1 1 1 1 2 {m, m} 1 1 1 1 1 1 1 1 1 1 1 1 {n, n} 1 1 1 1 255 141 20 167 1 1 1 1 {o, o} 1 1 1 1 330 248 357 277 3 1 5 6 {p, p} 1 1 1 1 1 3 1 1 3 2 1 1 {q, q} 1 1 1 1 4 3 3 2 1 1 1 1 {r, r} 1 1 1 1 1 1 1 5 1 1 5 1 {s, s} 3 1 1 1 1 1 1 1 1 1 1 1 {t, t} 1 1 1 1 1 1 1 1 1 1 1 1 {u, u} 1 1 1 1 1 1 1 1 1 1 1 1 {v, v} 1 1 1 1 1 1 7 1 1 1 1 1 {w, w} 2 1 1 1 1 16 1 2 1 1 1 1 b) A2 v B1 A3 v B2 A4 v B3 A5 v B4 D_{mm} D_{mn} D_{mm} D_{mn} D_{mm} D_{mn} D_{mm} D_{mn} MEAN 0.33 0.97 0.34 0.89 0.44 0.98 0.37 0.96 SD 0.2 0.11 0.2 0.13 0.21 0.11 0.21 0.12 {h, h} 0.99 1.02 1.07 1 {i, i} 1.09 1 1.07 1.06 {h, i} 0.22 0.09 0.33 0.31 {i, h} 0.28 0.07 0.11 0.14 {n, n} 0.98 0.91 0.85 0.92 {o, o} 1.01 1.02 1.07 1.02 {n, o} 0.1 0.51 0.45 0.52 {o, n} 0.04 0.17 0.11 0.05 In all four comparisons, the two distributions resolve themselves well, as can be seen in Figure 3, leading to the conclusion that h and i are swapped, and n and o are swapped, most likely in the plates from the first PCR round. The ambiguity is broken by sequencing a small sampling of wells, which confirms that the misidentifications are in the print plates from the first round of PCR, and not the second. In Figure 4 we show the effect on the Ovarian data after the correction has been applied. Also, in Figure 2 we show how, via visual inspection of the actual spots on an array, one can verify that the fix has properly reorganized the data. The figure shows spots from the four different affected 96-well plates before and after the fix is applied. Figure 3 Match, mismatch distance distributions. Good separation between match and mismatch distance distributions at the 96-well plate level lends confidence to our ability to discriminate between chance matches and actual matches. The green bars refer to the distance distributions of matched plates and the red bars for mis-matched plates. Figure 4 Ovarian tumour clusters. In Ovarian tumor data it was noticed that similar experiments were not clustering as expected (upper cluster diagram). Using MuFu we were able to isolate the problem to a distinct set of 96-well plates that had been swapped during an upstream process step, probably during the transfer from 96-well plates to 384-well plates. The samples cluster together as expected after correction (lower cluster diagram). Finding a 384-well plate rotation We compare four arrays A1, A2, A3 and A4 from a particular print production batch A to four arrays B, C, D and E from four other distinct print production batches. The total number of 384 well microtiter plates in print batch A is 113. The results of the plate rotation test are shown in Table 3a. Here we see plate p, identified in both the rank and rotated-rank matrices across print batches is a clear candidate for a plate rotation. The data from the distance matrix comparisons is shown in Table 3b. In Figure 5 we show the distributions of the diagonal elements of the distance matrix and the rotated-distance matrix. These distributions are well resolved and allow us to easily distinguish data due to a plate rotation from noisy data. Table 3 Test for rotated plates. a) Plate rotation results from the rank matrices R and R' . The flagged comparisons indicate a plate rotation for plate p. b) Plate rotation distance matrix comparison. The data meet the criteria for a plate rotation. a) A1 v B A2 v C A3 v D A4 v E Plates R_{mm} R'_{mm} R_{mm} R'_{mm} R_{mm} R'_{mm} R_{mm} R'_{mm} {i, i} 1 102 1 95 1 82 1 93 {j, j} 1 18 1 102 1 16 1 64 {k, k} 1 52 1 99 1 102 1 41 {l, l} 6 108 3 35 9 76 1 22 {m, m} 1 51 1 79 1 85 1 68 {n, n} 1 14 1 35 1 41 1 93 {o, o} 1 92 1 92 1 102 1 76 {p, p} 78 1 48 1 46 1 65 1 {q, q} 1 117 1 96 1 87 1 111 {r, r} 1 118 1 110 1 115 1 112 {s, s} 1 116 1 108 1 109 1 113 {t, t} 1 69 1 93 3 20 1 103 {u, u} 1 116 1 103 1 72 1 84 {v, v} 1 83 1 104 1 85 1 64 b) A1 v B A2 v C A3 v D A4 v E D_{mm} D'_{mm} D_{mm} D'_{mm} D_{mm} D'_{mm} D_{mm} D'_{mm} MEAN 0.26 0.96 0.23 0.88 0.19 0.81 0.21 0.88 SD 0.14 0.1 0.14 0.14 0.1 0.17 0.13 0.14 {pp} 0.95 0.84 0.85 0.97 {pp} 0.21 0.14 0.23 0.24 P-value 4.10E-07 3.20E-14 6.60E-06 6.30E-08 2.10E-11 3.20E-04 2.50E-09 2.40E-06 Figure 5 Plate rotation distance comparison. Histogram of the distance comparisons for the plate rotated and not rotated cases. The distributions are well separated, where the green bars refer to the distance distributions for non-rotated plates and the red bars for rotated plates. Finding a misidentified 384-well plate In this example we compare four arrays A1, A2, A3 and A4 from a particular print batch A to four other arrays B, C, D and E from four other distinct print batches. As seen in Table 4a no plate is found to be a candidate for plate rotation, however we do find that plate q has a poor self-match comparison. Indeed, when the distance matrix is examined, we see the plate matches instead plate r across the four array comparisons. We check the three criteria for plate misidentification and summarize these data in Table 4b. In Figure 6 we show the distributions of the match and mismatch distributions. These distributions are quite distinct and give us confidence that we can distinguish the proper match from an accidental match. Table 4 Test for swapped plates. a) Plate r is seen here to have a problem, but from the table we see that it is most certainly not a plate rotation. b) A check of the distance matrix shows the {r, q} comparison to be quite good and satisfies the criteria for a match, indicating that plates r and q have been accidentally swapped. a) A1 v B A2 v C A3 v D A4 v E Plates R_{mm} R'_{mm} R_{mm} R'_{mm} R_{mm} R'_{mm} R_{mm} R'_{mm} {i, i} 1 82 1 48 1 107 1 76 {j, j} 1 45 1 87 1 87 1 89 {k, k} 1 18 1 45 1 63 1 15 {l, l} 1 85 1 106 1 103 1 93 {m, m} 1 30 1 49 1 53 1 37 {n, n} 1 115 1 113 1 37 1 102 {o, o} 1 24 1 64 1 82 1 100 {p, p} 1 110 1 102 1 104 1 93 {q, q} 1 118 1 111 1 110 1 91 {r, r} 77 7 48 12 51 22 16 23 {s, s} 1 55 1 44 1 90 1 88 {t, t} 1 115 1 101 1 110 1 99 {u, u} 1 118 1 93 1 74 1 95 {v, v} 1 15 1 13 1 29 1 71 b) A1 v B A2 v C A3 v D A4 v E D_{mm} D_{mn} D_{mm} D_{mn} D_{mm} D_{mn} D_{mm} D_{mn} MEAN 0.19 0.93 0.25 0.94 0.28 0.95 0.2 0.81 SD 0.14 0.07 0.19 0.07 0.17 0.07 0.16 0.1 {rq} 0.3 0.26 0.25 0.34 {rr} 0.97 0.95 0.98 0.72 P-value 1.30E-08 0 1.10E-04 0 1.90E-05 0 5.80E-04 1.30E-06 Figure 6 Misidentification distance comparison. Misidentified plate distance histogram showing good separation between the match and mismatch distance distributions. The green bars refer to the distance distributions of matched plates and the red bars for mis-matched plates. Discussion Out of the 265 million features checked in our database using MuFu, problems were detected and corrected on 1.3 million of them. That we were able to find and correct both previously known and unknown problems gives us confidence in the algorithm. That so few problems existed overall (0.5%) reassures us as to the robustness of our process in general. Microarray data, by its sheer volume, presents interesting laboratory information management challenges. The data arrive at the investigators desk after a significant number of steps. We have found that the better you track production, quality control, and experimental steps, the better chance you have of uncovering the reasons behind discrepancies that may appear in the data. Often, statistical analyses look only at the data presented as final expression values or ratios, without taking into account relevant quality control data. In our effort to understand our errors and the source of large systematic discrepancies in our data we have found the MuFu algorithm a useful test of certain classes of process errors, and as a check for general problems with specific process steps or microtiter plates. We use MuFu to find, verify and fix problems that can be attributed to an error in plate processing. We also flag plates for which we can find no specific problem but we see yield inconsistent results. These may be plates that, at some point in the process, had a problem (cross contamination, a PCR problem) that was not detected while the process step was being carried out. The fact that we can test the data, using our standard quality control hybridizations, for these types of quality issues is reassuring and has helped us gain confidence in our data. Obviously, there are many other classes of error that creep into microarray data. Aside from the gross process errors that are amenable to detection, as we have described here, there is also a large class of more subtle systematic errors that can contribute to the overall systematic error on the expression ratio measurement. Isolating the source of these individual errors is sometimes quite difficult. Properties of the microarray feature such as spot size, shape and uniformity can contribute, but the majority of systematic errors are introduced at the time the experiment is performed. Slide post processing, RNA quality, labelling, hybridization and washing all lend the possibility for introducing systematic errors. Improvements in protocols and hybridization apparatus have helped reduce these errors and should continue to do so in the future. As these sources of error are identified and eliminated, expression microarrays will continue to provide progressively more sensitive measures of gene expression. Conclusions Process errors in any genome scale high throughput production regime can lead to subsequent errors in data analysis. We have shown the value of tracking multi-step high throughput operations by using this knowledge to detect and correct misidentified data on gene expression microarrays. We generalized our procedure using a simple heuristic, which found and fixed several problems with the proper assignment of gene identifier with physical microarray feature. We found thirteen print runs (9K arrays) that had four plates mistracked, six print runs with single 384 plate rotations, and one instance of a plate rotation at the 96 well plate level. One skipped plate was detected, as well as one plate printed twice. Out of the 265 million features checked in our database using MuFu, problems were detected and corrected on 1.3 million of them. That we were able to find and correct both previously known and unknown problems gives us confidence in the algorithm. That so few problems existed overall (0.5%) reassures us as to the robustness of our process in general. A list of corrected arrays can be found at . Methods We follow the simple heuristic outlined here. The flowchart for the program is shown in Figure 1. In the figure we do not include additional loops needed to repartition the data in different ways for different scenarios. Data partitioning 1. Begin by partitioning the gene expression data on an array into subsets according to plate. Other partitions can also be made but the plate level partition is the most useful for our purposes. Let Aij be the intensity measurements of array A, subset i and gene index j. The measurements are usually of the channel (Cy3 or Cy5) used as a common reference. For example, if we partition the data by the 384-well plate each feature once occupied at some point in its process history, A is the array id, i is the plate id, and j is the well index between 1–384. Let Ai be the vector (Ai1,...,Ai384). In our vernacular this is the 384-well plate expression vector for plate i. Let Bij andBi be the similar definitions for array B. We also reverse the data vectors from array A and label it Ai' . In our notation, Ai' = (Ai384,...,Ai1) is the expression vector for a plate rotated by 180°. 2. Generate a distance matrix {Dmn, 1 ≤ m, n ≤ N} in which each element Dmn = 1 - corr(Am, Bn). For the 384-well plate example, Am is the 384-well expression vector for plate m on array A, Bn is the 384-well expression vector for plate n on array B, and Dmn is the distance between the two vectors in this 384 dimensional space. We also generate the corresponding rotated-distance matrix {D'mn, 1 ≤ m, n ≤ N} for the plate rotation case, in which each element D'mn = 1 - corr(A'm, Bn). We tried several correlation functions including Euclidean and Pearson but found the Pearson to be best suited to this task. 3. Generate a rank matrix {Rmn, 1 ≤ m, n ≤ N} by converting the distances to ranks such that the row m in the rank matrix is the order statistic of the row m of the distance matrix. We also generate the corresponding rotated-rank matrix {R'mn} for {D'mn}. Ideally, for the case where we are comparing identical subsets from two different arrays we expect the diagonal elements of the rank matrix to all be equal to one, which means that each subset of genes from the first array matches its corresponding subset in the second array the best. In general, due to the statistical variation in array data, the diagonal elements are not all equal to one, even if there are no misidentification errors. The examples show that this does not hinder us from making a clear distinction between real problems in the data and statistical fluctuations. Identification of rotated plates A plate rotation may have an affect on a single microarray batch if it occurs during array printing or may persist across many print batches if it happens during a 96-well (PCR) process step. In any case, the mismatch will persist across many array comparisons. To check for plate rotations in a print batch, we compare an array sample (A1, A2, A3, A4 in the example) from the print batch in question to a control sample of arrays (B, C, D, E in the example) selected from several distinct print batches. By comparing the rank R, and rotated-rank R', matrix assignments for comparisons across array batch boundaries we can quickly flag possible rotated plates. A visualization of this test is shown in Table 3a. In the table we have flagged the top 5% of all ranks in the rotated column and the bottom 95% in the unrotated column. If the flags agree across all comparisons, we have a strong indication that a plate rotation has occurred. If we see a flag raised in this test, but we cannot attribute it to a plate rotation, this may indicate a different class of process error. In particular, if the flag is raised for all pairwise comparisons of the batch being tested (in this case batch A) against all of the control batches (in this case batches B, C, D and E) in the non-rotated case, we conclude that the corresponding flagged plate or partition from batch A may be misidentified. Note that in the limit that the partitions of the array are all uniform in expression ratios there is a 5% probability of a spurious flag. For this reason it is better to use high quality, highly variegated control arrays for such tests. Next, to better resolve ambiguous cases and to check our rank matrix determination we use the distance rather than rank matrix. If, for example, a plate x is to be considered a rotated plate, the following three criteria must be met. 1. D'xx <Dxx. The rotated-distance must be less than the non-rotated distance. 2. D'xx is close to the mean of the distribution, {Dmm, 1 ≤ m ≤ N}, and Dxx is close to the mean of the distribution {D'mm, 1 ≤ m ≤ N}. 3. D'xx is an outlier of the distribution {D'mm, 1 ≤ m ≤ N}, and Dxx is an outlier of the distribution {Dmm, 1 ≤ m ≤ N}. The second and third criteria above are valid if the distributions of {D'mm, 1 ≤ m ≤ N} and {Dmm, 1 ≤ m ≤ N} separate well. In Figure 5 we showed that our data support this model. Finding misidentified plates If a plate has consistently poor self-match rankings and a plate rotation has been excluded as a possible source of error, this next step tests for a plate misidentification. From the rank matrix we can identify the best alternative match. We test a suspect plate x for a match with plate y with the following criteria: 1. Dxy <Dxx. The mismatch distance is shorter than the match distance. 2. Dxy is close to the mean of the distribution {Dmm, 1 ≤ m ≤ N}, and Dxx is close to the mean of the distribution {Dmn, 1 ≤ m ≠ n ≤ N}. 3. Dxx is an outlier of the distribution {Dmm, 1 ≤ m ≤ N}, and Dxy is an outlier of the distribution {Dmn, 1 ≤ m ≠ n ≤ N}. Again, the second and third criteria above are valid if the distributions of {Dmm, 1 ≤ m ≤ N} and {Dmn, 1 ≤ m ≠ n ≤ N} are well resolved. Authors' contributions IT, MF developed the heuristic and carried out analysis and implementation. MS brought this problem to our attention and provided test data. MD provided advice and checked our work with an alternative technique. All authors read and approved the final manuscript. Table 1 Process steps The process fail points for cDNA based microarray production. Steps shown in italics are accessible to the testing methods outlined here. Earlier steps may require sequencing to test. The process ID is used to identify steps where the problems, if any, are found. Process ID Problem Type Note -999 Unidentified 0.0 Source.General persists across all arrays at clone, 96 level 0.1 Source.Contamination 0.2 Source.MisID 1.0 Prep.General persists across all arrays at clone, 96 level 1.1 Prep.Contamination 1.2 Prep.MisID 1.2.1 Prep.MisID.OrderError 1.2.2 Prep.MisID.Rot 1.3 Prep.Fail 2.0 PCR.General persists for 1 PCR round at 96 level 2.1 PCR.Contamination 2.2 PCR.MisID 2.3 PCR.Fail 2.4 PCR.Cleanup 3.0 Replate.General persists for 1 PCR round at 96 level 3.1 Replate.Contamination 3.2 Replate.MisID 3.2.1 Replate.MisID.OrderError 3.2.2 Replate.MisID.96Rot 3.2.3 Replate.MisID.384Rot 4.0 Print.General persists for 1 print run batch at 384 level 4.1 Print.Contamination 4.2 Print.MisID 4.2.1 Print.MisID.OrderError 4.2.2 Print.MisID.Rot 4.3 Print.Fail dried out plate, too concentrated, too weak etc. 5.0 Scan.General anything after array production Acknowledgements We thank members of the Brown and Botstein labs for encouragement and support. This work was supported in part by a grant from the National Cancer Institute. ==== Refs Alizadeh A Probing lymphocyte biology by genomic-scale gene expression analysis J Clin Immunol 1998 18 373 379 9857281 10.1023/A:1023293621057 Lossos IS Alizadeh AA Eisen MB Chan WC Brown PO Botstein D Staudt LM Levy R Ongoing immunoglobulin somatic mutation in germinal center B cell-like but not in activated B cell-like diffuse large cell lymphomas Proc Natl Acad Sci U S A 2000 97 10209 13 10954754 10.1073/pnas.180316097 DePrimo SE Diehn M Nelson JB Reiter RE Matese J Fero M Tibshirani R Brown PO Brooks JD Transcriptional programs activated by exposure of human prostate cancer cells to androgen Genome Biol 2002 3 RESEARCH0032 12184806 10.1186/gb-2002-3-7-research0032 Jeffrey SS Fero MJ Børresen-Dale A-L Botstein D Expression array technology: applications for the diagnosis and treatment of breast cancer Mol Interv 2002 2 101 109 14993355 10.1124/mi.2.2.101 Nielsen TO West RB Linn SC Alter O Knowling MA O'Connell JX Zhu S Fero M Sherlock G Pollack JR Molecular characterisation of soft tissue tumours: a gene expression study Lancet 2002 359 1301 7 11965276 10.1016/S0140-6736(02)08270-3 Schaner ME Ross DT Ciaravino G Sorlie T Troyanskaya O Diehn M Wang YC Duran GE Sikic TL Caldeira S Gene expression patterns in ovarian carcinomas Mol Biol Cell 2003 14 4376 86 12960427 10.1091/mbc.E03-05-0279 Higgins JP Shinghal R Gill H Reese JH Terris M Cohen RJ Fero M Pollack JR van de Rijn M Brooks JD Gene expression patterns in renal cell carcinoma assessed by complementary DNA microarray Am J Pathol 2003 162 925 32 12598325 Hastie T Tibshirani R Eisen MB Alizadeh A Levy R Staudt L Chan WC Botstein D Brown P 'Gene shaving' as a method for identifying distinct sets of genes with similar expression patterns Genome Biol 2000 1 RESEARCH0003 11178228 10.1186/gb-2000-1-2-research0003 Alter O Brown PO Botstein D Singular value decomposition for genome-wide expression data processing and modeling Proc Natl Acad Sci U S A 2000 97 10101 6 10963673 10.1073/pnas.97.18.10101 Troyanskaya O Cantor M Sherlock G Brown P Hastie T Tibshirani R Botstein D Altman RB Missing value estimation methods for DNA microarrays Bioinformatics 2001 17 520 5 11395428 10.1093/bioinformatics/17.6.520 Eisen MB Spellman PT Brown PO Botstein D Cluster analysis and display of genome-wide expression patterns Proc Natl Acad Sci U S A 1998 95 14863 8 9843981 10.1073/pnas.95.25.14863 Sherlock G Analysis of large-scale gene expression data Brief Bioinform 2001 2 350 62 11808747 Tusher VG Tibshirani R Chu G Significance analysis of microarrays applied to the ionizing radiation response Proc Natl Acad Sci USA 2001 98 5116 21 11309499 10.1073/pnas.091062498 Natalia Novoradovskaya MLW Lee Basehore S Alexey Novoradovsky Robert Pesich Jerry Usary Mehmet Karaca Olga Aprelikova Winston Wong K Michael Fero Charles Perou M David Botstein Jeff Braman Universal Reference RNA as a standard for microarray experiments Accepted for publication – BMC Genomics 2004

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==== Front BMC GenomicsBMC Genomics1471-2164BioMed Central London 1471-2164-5-661536960210.1186/1471-2164-5-66Research ArticleA high-resolution radiation hybrid map of chicken chromosome 5 and comparison with human chromosomes Pitel Frédérique [email protected] Behnam [email protected] Mireille [email protected] Richard PMA [email protected] Florence [email protected] Sophie [email protected] Katia [email protected] Suzanne [email protected] Denis [email protected] Sandrine [email protected] Martien AM [email protected] Madeleine [email protected] Alain [email protected] Laboratoire de Génétique Cellulaire, INRA, Castanet-Tolosan, 31326, France2 UMR Génétique Animale, INRA-ENSAR, Route de St Brieuc, Rennes, 35042, France3 Animal Breeding and Genetics group, Wageningen University, Wageningen, 6709 PG, The Netherlands2004 15 9 2004 5 66 66 19 5 2004 15 9 2004 Copyright © 2004 Pitel et al; licensee BioMed Central Ltd.2004Pitel et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background The resolution of radiation hybrid (RH) maps is intermediate between that of the genetic and BAC (Bacterial Artificial Chromosome) contig maps. Moreover, once framework RH maps of a genome have been constructed, a quick location of markers by simple PCR on the RH panel is possible. The chicken ChickRH6 panel recently produced was used here to construct a high resolution RH map of chicken GGA5. To confirm the validity of the map and to provide valuable comparative mapping information, both markers from the genetic map and a high number of ESTs (Expressed Sequence Tags) were used. Finally, this RH map was used for testing the accuracy of the chicken genome assembly for chromosome 5. Results A total of 169 markers (21 microsatellites and 148 ESTs) were typed on the ChickRH6 RH panel, of which 134 were assigned to GGA5. The final map is composed of 73 framework markers extending over a 1315.6 cR distance. The remaining 61 markers were placed alongside the framework markers within confidence intervals. Conclusion The high resolution framework map obtained in this study has markers covering the entire chicken chromosome 5 and reveals the existence of a high number of rearrangements when compared to the human genome. Only two discrepancies were observed in relation to the sequence assembly recently reported for this chromosome. ==== Body Background Chicken is the first major agricultural species for which the complete genome sequencing was undertaken. This is partly due to its position as a model species in various fields of biology including embryo development, oncology, immunology and evolution [1]. Moreover, as it is the only bird species for which the genome study is so advanced, very much is expected from its use in comparative genome analyses for annotation, including that of the human genome, by detection of conserved sequences [2,3]. Its intermediate phylogenetic position between mammals and fishes will also certainly provide valuable information on the evolution of vertebrate karyotypes. Radiation hybrid maps have a resolution power intermediate to that of the genetic and BAC contig maps and are also a powerful tool for the mapping of ESTs and genes by simple PCR. They are thus useful at two levels: first, they can be used constructively as scaffolds for a correct genome assembly or for detecting and correcting misassembled portions of the genome; second, before obtaining whole annotated genome sequences, they are very efficient tools for inter-species comparative genome analyses through the easy mapping of genes and ESTs [4-7]. The successful production of a RH panel in chicken is quite recent [8], and therefore RH maps are only available for a limited number of chromosomes [9-11]. Having identified QTL (Quantitative Trait Loci) for fatness on chicken chromosome 5 [12], our objective was to build a high-resolution and gene-rich RH map for this chromosome, as a basis for high precision comparative mapping with human and for the development of new polymorphic markers. The available human/chicken comparative mapping data indicated conservation of synteny between GGA5 and portions of HSA11, HSA14 and HSA15. In addition, two genes from HSA1 had also been shown to be located on GGA5 [13]. This information was used to develop markers from chicken EST sequence data orthologous to genes in these human regions, in addition to the existing markers from the chicken chromosome 5 genetic map. While in the process of finishing our map, the first draft sequence assembly of the chicken genome was released (March 1st, 2004). The quality of both the GGA5 RH map and of the sequence assembly was therefore checked by alignment of all the markers by BLAST searches. Results and discussion Development of EST markers In addition to the 21 microsatellite markers from the genetic map, and 9 primer pairs chosen either from available primer data in the literature or designed using the gene sequence deposited in Genbank/EMBL, 156 primer pairs were chosen from chicken EST markers selected on the basis of the known conservations of synteny between human and chicken using the ICCARE (Interactive Comparative Clustering and Annotation foR Est) software . Constraints on the design of primers were to avoid presence of long introns, whose position and length was predicted on the basis of the orthologous human gene structure, and to design primers in the most divergent regions of the human/chicken alignment, to limit cross-amplification with the hamster DNA present in the hybrids. One hundred and thirty nine primer pairs out of 156 (89.1%) enabled a successful amplification and the subsequent mapping of the corresponding genes, confirming the high success rate obtained when using the ICCARE software for designing chicken PCR primers based on EST data [10]. Construction of the GGA5 RH map Altogether, genotyping data was obtained for a total of 169 markers, comprising 148 gene fragments (of which 139 developed using ICCARE) and 21 microsatellites from the GGA5 genetic map. Two-point analysis using a LOD threshold of 6 enabled to constitute a group of 134 markers, including all the microsatellite markers from the genetic map. The remaining 35 markers correspond to the external boundaries of the regions of conserved synteny with human, from which ESTs were chosen for marker development and map either to other chromosomes for which RH maps were developed (GGA1, 10, 18 or 24) or to unknown regions (data not shown). After multipoint analysis, a 1000:1 framework map 1315.6 cR6000 long, comprising a total of 73 markers including 12 microsatellites and 61 ESTs was obtained. The remaining 61 markers are located relative to the framework map within confidence intervals, to build a comprehensive map (figure 1). Figure 1 Comparison RH / genetic maps for chicken chromosome 5. The framework RH map is 1315.6 cR6000 long. Position of markers included only in the comprehensive map is indicated with error bars to the left of the framework map. Markers for which the genetic position is known (Schmid et al, 2000) are indicated by links to the genetic map (middle). Retention frequency along the map is represented on the right. To compare the RH and the genetic maps, the best possible position of the non-framework common markers had to be estimated. That of the markers on the RH map was computed by the Carthagene program and is indicated in addition to the confidence interval. For the genetic map, the central position of the marker's confidence interval was used as their most probable position. As a result, the order of the markers on the RH map matches exactly that of the same markers on genetic map [13], with only one notable discrepancy concerning the position of BRF1 (figure 1). However, when the position of this gene was checked on the sequence assembly, the agreement was with the RH map, suggesting the position of this gene on the genetic map is erroneous. An average retention frequency of 21.4% was observed for the 134 GGA5 markers studied here, although with a high variation, with values ranging from 6.8% to 55.7%. This finding is within the range observed in other studies reported on this panel: 21.9% overall retention using 42 markers chosen genome-wise [8], 24 % for GGA4 [11], 20.1 for GGA7 [10] and 18% for GGA15 [9]. As already noticed for several species including human [14,15] or cow [16], but also for chicken chromosomes 4 and 7 [10,11], a centromeric effect is detected when observing retention frequencies of markers along the map, with a higher retention of markers in the region between 50 to 200 cR, in which the retention culminates at a value of 55.7%, whereas it is around 15% for the rest of the chromosome (400 cR downwards). Alignment of the RH map to the genomic sequence A preliminary data set based on the first draft chicken genome assembly has been deposited into public databases by a team led by R. Wilson and W. Warren, from the Washington University School of Medicine in St. Louis (1st March, 2004, ). We compared our data with the GGA5 sequence, by using BLASTN searches and sequence alignments. The agreement between the RH framework map and the sequence orders is almost perfect (figure 2), although with a few discrepancies, most of them suggesting possible improvements to be made in the sequence assembly. Figure 2 Comparison between RH map and chicken genome assembly. The RH map (left) obtained in this study is compared to the draft sequence assembly (right, ). For each marker on the framework map, a line joins both positions (cR and Mb) together. Discrepancies or missing data are indicated. Unknown: sequence of unknown location in the assembly; absent: sequence not found (no BLAST hit); 5_random: sequence attributed to GGA5, but whose position is unknown precisely in the assembly. First, a group of markers (GPR48, PAX6, SPON1 and CSTF3), that we developed on the basis of the conservation of synteny between GGA5 and HSA11, is assigned to GGA3 in the sequence assembly. Three of these markers are on the framework map and for all four, the RH genotypings obtained are very similar to those obtained with the flanking RH framework markers SLC17A6 and ARNTL (two-point LOD scores ranging from 7.1 to 15.7), both located on the GGA5 sequence assembly. Furthermore, when two-point analysis of the four markers was computed with the flanking markers LOC134957 (1.2 Mb to GPR48) and SLC22A3 (0.6 Mb to CSTF3) suggested in the GGA3 sequence assembly, LOD scores were equal to zero. This part of the genome assigned to a wrong chromosome on the sequence assembly covers a region at least 50 cR long, corresponding to a distance of 2 to 3 Mb, as estimated from the cR to Mb ratio. Indeed, the length of the sequence between the two extreme markers CSTF3 and GPR48 on the GGA3 assembly is 2.703 Mb. The retention frequency of these four markers is amongst the highest of all, suggesting that their location is close to the centromere and that the possible sequence assembly problems are related to this proximity, perhaps due to repetitive sequences. Second, we observed an inversion of the gene order for a segment in the upper part of the chromosome (first 86.2 cR or 3.08 Mb, figure 2) adjacent to the group we described as wrongly assigned to GGA3 in the sequence assembly. As the difference of likelihood between our 1000:1 framework map and the map order in this area suggested from the assembly is higher than 1015, we considered that order of the RH map is the correct one. This could also be due to assembly difficulties close to the centromere region. Third, several markers absent in the sequence assembly could be localised on the RH map (figure 2). Most of these markers belong to regions for which sequence information is available, but that couldn't be incorporated in the sequence assembly at all (Unknown) or that could be assigned to GGA5, but without a clear location (5_random). In addition, one gene (MAX) also appeared to belong to a region with no sequence available (no blast hit). This gene had previously been located on the cytogenetic map to the short arm of GGA5 [17], so we consider our data as a confirmation. Fourth, we observed a discrepancy in the local order of the two framework map markers MCW238 and GTF2A1. However, the difference of likelihood between our framework map and the same map with an inversion of these two markers is only 103.7. It is therefore difficult to conclude as to which between the sequence and the RH map presents the correct order. From these data we conclude that radiation hybrid maps can be useful to help detect errors in the draft sequence assembly and for mapping genes either absent or of unknown location in the assembly. Comparison cR6000/cM/kb The average cR/cM ratio is 6.5 when calculated over the whole map length. This relatively high value, as compared to the 4 cR/cM obtained for GGA7 [10], must be inflected by the disparity observed along the chromosome (figure 1). This heterogeneity actually reflects disparities in the recombination rate along the chromosome, with recombination events more frequent at the end of the long arm. The agreement between the gene order found on RH map and the sequence assembly is very high. Considering only the q arm of the chromosome, the cR/Mb ratio is 22.9, or 43.7 kb per cR. This ratio, similar to that obtained for GGA2 (S. Leroux, personal communication), is quite lower than the 63 kb/cR and 61 kb/cR values obtained for GGA15 [9] and GGA7 [10] respectively, suggesting a higher resolution for the larger chromosomes. This result can have two origins: first, the kb/cR ratio is not constant from one chromosome to another, regardless of their physical length [18,19,14]; second, the previous calculations were based on physical length values estimated from cytogenetic studies: 21 Mb for GGA15 [20] and 41 Mb for GGA7 [21]. If we consider the actual chromosome length based on sequence assembly, these chromosomes are shorter than previously estimated, with values of 12.4 and 37.3 Mb , the ratio is thus now closer to the value we obtain here for GGA5. Comparative mapping Figure 3 and figure 4 (see additional file 2) synthesize the comparative maps generated by us between GGA5 and its human and mouse counterparts. As indicated earlier [13,22-28], conserved synteny was observed between this chicken chromosome and portions of human chromosomes 11, 14 and 15. No correspondence was detected with HSA1, as is also supported by the GGA5 sequence assembly . The results indicate a high number of chromosomal rearrangements in the chicken and human lineages in the region corresponding to GGA5. The results presented in figures 3 and 4 make us conclude that, as previously observed [10,29], the number of synteny blocks is higher between chicken and mouse than between chicken and human. The high number of intra-chromosomal rearrangements within the regions of conserved synteny between birds and mammals is in accordance with results obtained for other chromosomes, e.g., GGA7 [10], GGA10 [25], GGA15 [20], and chicken regions homologous to HSA19 [30]. Figure 3 Comparative positions between chicken, human and mouse genomes for the framework map genes. The position of each gene on the chicken, human and mouse maps is given: chicken chromosome (GGA), cR position (this study); human chromosome (HSA), Mb position, and mouse chromosome (MMU), Mb position. The position used for human and mouse genes are from EnsMart v19.1 (human build 34, update v19.34a.1; mouse build 30, update v19.30.1 – ). Coloured blocks indicate the blocks of conserved gene order, using the human as reference. Conclusions We have built a high resolution RH map of chicken chromosome 5 using the ChickRH6 panel. In doing this, we fulfilled our objective of obtaining a detailed comparative map of GGA5, providing jointly a source of potential polymorphic markers and of candidate genes for QTL mapping on this chromosome. At the end of our work, the first draft chicken genome assembly was released and we aligned it to our GGA5 RH map. Although we detected a few errors to correct, this allowed us to demonstrate the high quality of the sequence assembly, which may have benefited from a low frequency of repeated elements. In the near future, the ChickRH6 panel will be used to assist in improving the chicken genome assembly. This is clearly needed in the regions for which the genetic map is still not complete, such as some microchromosomes, but also for parts of macrochromosomes, as shown in this study. Methods Development of markers Twenty one microsatellite markers distributed along GGA5 were chosen from the genetic map. Their primer sequences are available at . Human and mouse genes from regions for which available comparative mapping data suggested a conservation of synteny with GGA5 were selected for marker development. Except for CKB, IGF2 and RYR3 for which primers were chosen from the literature, and 6 other genes for which primers were designed from sequences deposited in Genbank/EMBL, primers pairs were designed from the available chicken EST sequence of orthologs defined using the ICCARE (Interspecific Comparative Clustering and Annotation foR ESTs) software (T. Faraut, ). The exonic structure of the genes was taken into account by extrapolating the information available from an alignment to the human genomic sequence. A link with the Primer3 software allowed us to design the primers. Primer data for markers amplifying successfully and accession numbers of the sequences used as a basis for primer design, are indicated in Table 1 (see additional file 1). Radiation hybrids – PCR amplification The generation of the RH panel has already been described [8]. The final panel is composed of 90 clones with an average retention frequency of 21.9%. PCR amplifications were carried out for each marker in 15 μl reactions containing 25 ng DNA, 0.2 μM of each primer, 0.3 U of Taq polymerase (Life Technologies-GIBCO BRL), 20 mM Tris-HCl pH 8.4, 50 mM KCl, 0.05% W-1 detergent, 2 mM MgCl2, 0.2 mM dNTP. Amplifications were carried out on a GeneAmp PCR System 9700 thermocycler (Applied Biosystem). The first 5 min denaturation was followed by 30 cycles, each of denaturation at 94°C for 30 s, annealing at Tm for 30 s and elongation at 72°C for 30 s. PCR products were analyzed on 2% agarose gels, electrophoresed in 1 X TBE buffer, and visualized by ethidium bromide staining. Each marker was genotyped twice and a third genotyping was performed in cases of discrepancies between the first two experiments. Map construction The genotyping data obtained was analyzed with the Carthagene software [31,32]. A group of GGA5 markers was defined by two-point analysis using a LOD threshold of 6. By using all the markers from this group, a 1000:1 framework map (a map whose likelihood is at least 1000 fold higher than the next possible highest likelihood using the same markers in alternate orders) was built under a haploid model. This framework was constructed using a stepwise locus adding strategy, starting from the triplet of markers whose order is the most likely ("buildfw" option). The framework map thus automatically built was further improved towards larger distance coverage by removing markers that prevented its extension. The different provisional framework maps were checked by using a simulated annealing greedy algorithm testing for possible improvements of the map by inversion of large fragments, and a flips algorithm testing all possible local permutations within a sliding window of six markers. After validation of the framework map built under the haploid model, the distances between markers of the framework were re-evaluated under a diploid model. Finally, markers not included in the framework map were mapped relative to it, to determine their most likely positions. The human and mouse reference maps were built from data available through EnsMart v19.1 (14th January 2004 – ). RH maps were drawn with MapChart 2.0 [33]. Sequence comparison Sequences for all the mapped fragments were used for a BLAST search over the entire chicken genome assembly at the Ensembl chicken site to determine their position in the sequence. The sequence assembly map of our markers was visualised with MapChart 2.0 [33]. Authors' contributions FP and BA carried out most of the molecular studies. FP drafted the manuscript. MM made the RH panel. RC and MG were involved in the GGA5 study. FV, SL, KF and SB were involved in the characterization of the panel. We use the Carthagene software thanks to DM. Construction of the maps was done after fruitful discussions with MM and SL. AV and MD conceived the study, and participated in its design and coordination. AV finalised the manuscript. All authors read and approved the final manuscript. Supplementary Material Additional File 2 Comparative maps of chicken chromosome 5 and human chromosomes 11, 14 and 15. The framework RH map (this study) is shown on the left. Conserved blocks are indicated by coloured plain boxes. Empty boxes show HSA regions for which the chicken homologous part of the genome is not positioned on GGA5. Click here for file Additional File 1 Primer pairs for the studied gene fragments Accession numbers for the chicken EST sequences from which the primers were chosen are given in this Excel file Click here for file Acknowledgements This work was financially supported by the Génopole de Toulouse Midi-Pyrénées and the GIS AGENAE. ==== Refs Dodgson JB Chicken genome sequence: a centennial gift to poultry genetics Cytogenet Genome Res 2003 102 291 296 14970719 10.1159/000075765 Thomas JW Touchman JW Blakesley RW Bouffard GG Beckstrom-Sternberg SM Margulies EH Blanchette M Siepel AC Thomas PJ McDowell JC Maskeri B Hansen NF Schwartz MS Weber RJ Kent WJ Karolchik D Bruen TC Bevan R Cutler DJ Schwartz S Elnitski L Idol JR Prasad AB Lee-Lin SQ Maduro VV Summers TJ Portnoy ME Dietrich NL Akhter N Ayele K Benjamin B Cariaga K Brinkley CP Brooks SY Granite S Guan X Gupta J Haghighi P Ho SL Huang MC Karlins E Laric PL Legaspi R Lim MJ Maduro QL Masiello CA Mastrian SD McCloskey JC Pearson R Stantripop S Tiongson EE Tran JT Tsurgeon C Vogt JL Walker MA Wetherby KD Wiggins LS Young AC Zhang LH Osoegawa K Zhu B Zhao B Shu CL De Jong PJ Lawrence CE Smit AF Chakravarti A Haussler D Green P Miller W Green ED Comparative analyses of multi-species sequences from targeted genomic regions Nature 2003 424 788 793 12917688 10.1038/nature01858 Margulies EH Blanchette M Haussler D Green ED Identification and characterization of multi-species conserved sequences Genome Res 2003 13 2507 2518 14656959 10.1101/gr.1602203 Martins-Wess F Milan D Drogemuller C Vobeta-Nemitz R Brenig B Robic A Yerle M Leeb T A high resolution physical and RH map of pig chromosome 6q1.2 and comparative analysis with human chromosome 19q13.1 BMC Genomics 2003 4 20 12744726 10.1186/1471-2164-4-20 Lahbib-Mansais Y Tosser-Klopp G Leroux S Cabau C Karsenty E Milan D Barillot E Yerle M Hatey F Gellin J Contribution to high-resolution mapping in pigs with 101 type I markers and progress in comparative map between humans and pigs Mamm Genome 2003 14 275 288 12682780 10.1007/s00335-002-2236-x Gautier M Hayes H Eggen A A comprehensive radiation hybrid map of bovine Chromosome 26 (BTA26): comparative chromosomal organization between HSA10q and BTA26 and BTA28 Mamm Genome 2003 14 711 721 14694908 10.1007/s00335-003-3014-0 Larkin DM Everts-van der Wind A Rebeiz M Schweitzer PA Bachman S Green C Wright CL Campos EJ Benson LD Edwards J Liu L Osoegawa K Womack JE de Jong PJ Lewin HA A cattle-human comparative map built with cattle BAC-ends and human genome sequence Genome Res 2003 13 1966 1972 12902387 Morisson M Lemiere A Bosc S Galan M Plisson-Petit F Pinton P Delcros C Feve K Pitel F Fillon V Yerle M Vignal A ChickRH6: a chicken whole-genome radiation hybrid panel Genet Sel Evol 2002 34 521 533 12270108 10.1051/gse:2002021 Jennen DG Crooijmans RP Morisson M Grootemaat AE Van Der Poel JJ Vignal A Groenen MA A radiation hybrid map of chicken chromosome 15 Anim Genet 2004 35 63 65 14731233 Morisson M Jiguet-Jiglaire C Leroux S Faraut T Bardes S Feve K Genet C, Pitel F Milan D Vignal A Development of a gene-based radiation hybrid map of chicken chromosome 7 and comparison to human and mouse Mammalian Genome, in press Rabie TSKM Crooijmans RPMA Morisson M Andryszkiewicz J Van der Poel JJ Vignal A Groenen MAM A radiation hybrid map of chicken chromosome 4 Mammalian Genome 2004 15 560 569 15366376 10.1007/s00335-004-2362-8 Pitel F Lagarrigue S Le Roy P Plisson-Petit F Amigues Y Neau A Cahaner A Hillel J Sourdioux M Leclercq B Vignal A Douaire M A two-step procedure for fat QTL identification in meat type chickens. 7th World Congress on Genetics Applied to Livestock Production, 19-23 Août, Montpellier, France, 04-37, texte intégral 2002 Schmid M Nanda I Guttenbach M Steinlein C Hoehn M Schartl M Haaf T Weigend S Fries R Buerstedde JM Wimmers K Burt DW Smith J A'Hara S Law A Griffin DK Bumstead N Kaufman J Thomson PA Burke T Groenen MA Crooijmans RP Vignal A Fillon V Morisson M Pitel F Tixier-Boichard M Ladjali-Mohammedi K Hillel J Maki-Tanila A Cheng HH Delany ME Burnside J Mizuno S First report on chicken genes and chromosomes 2000 Cytogenet Cell Genet 2000 90 169 218 11124517 10.1159/000056772 Gyapay G Schmitt K Fizames C Jones H Vega-Czarny N Spillett D Muselet D Prud'homme JF Dib C Auffray C Morissette J Weissenbach J Goodfellow PN A radiation hybrid map of the human genome Hum Mol Genet 1996 5 339 346 8852657 10.1093/hmg/5.3.339 Stewart EA McKusick KB Aggarwal A Bajorek E Brady S Chu A Fang N Hadley D Harris M Hussain S Lee R Maratukulam A O'Connor K Perkins S Piercy M Qin F Reif T Sanders C She X Sun WL Tabar P Voyticky S Cowles S Fan JB Cox DR et al. 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==== Front BMC PhysiolBMC Physiology1472-6793BioMed Central London 1472-6793-4-121533313410.1186/1472-6793-4-12Research ArticleSeasonal ovulatory activity exists in tropical Creole female goats and Black Belly ewes subjected to a temperate photoperiod Chemineau Philippe [email protected] Agnès [email protected]é Yves [email protected] Gilles [email protected] Didier [email protected] Equipe de Neuroendocrinologie et Maîtrise des Fonctions Saisonnières, Unité de Physiologie de la Reproduction et des Comportements, UMR INRA-CNRS-Haras Nationaux-Univ. F. Rabelais, 37380 Nouzilly, France2 C.R.A.A.G. Station de Recherches Zootechniques, BP 1232, 94195 Pointe à Pitre cedex, France3 Departement Santé Animale 37380 Nouzilly, France2004 27 8 2004 4 12 12 25 2 2004 27 8 2004 Copyright © 2004 Chemineau et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Seasonality of ovulatory activity is observed in European sheep and goat breeds, whereas tropical breeds show almost continuous ovulatory activity. It is not known if these tropical breeds are sensitive or not to temperate photoperiod. This study was therefore designed to determine whether tropical Creole goats and Black-Belly ewes are sensitive to temperate photoperiod. Two groups of adult females in each species, either progeny or directly born from imported embryos, were used and maintained in light-proof rooms under simulated temperate (8 to 16 h of light per day) or tropical (11 – 13 h) photoperiods. Ovulatory activity was determined by blood progesterone assays for more than two years. The experiment lasted 33 months in goats and 25 months in ewes. Results Marked seasonality of ovulatory activity appeared in the temperate group of Creole female goats. The percentage of female goats experiencing at least one ovulation per month dramatically decreased from May to September for the three years (0%, 27% and 0%, respectively). Tropical female goats demonstrated much less seasonality, as the percentage of goats experiencing at least one ovulation per month never went below 56%. These differences were significant. Both groups of temperate and tropical Black-Belly ewes experienced a marked seasonality in their ovulatory activity, with only a slightly significant difference between groups. The percentage of ewes experiencing at least one ovulation per month dropped dramatically in April and rose again in August (tropical ewes) or September (temperate ewes). The percentage of ewes experiencing at least one ovulation per month never went below 8% and 17% (for tropical and temperate ewes respectively) during the spring and summer months. Conclusions An important seasonality in ovulatory activity of tropical Creole goats was observed when females were exposed to a simulated temperate photoperiod. An unexpected finding was that Black-Belly ewes and, to a lesser extent, Creole goats exposed to a simulated tropical photoperiod also showed seasonality in their ovulatory activity. Such results indicate that both species are capable of showing seasonality under the photoperiodic changes of the temperate zone even though they do not originate from these regions. ==== Body Background Seasonality of reproduction is a common feature in sheep and goat breeds of temperate latitudes [1,2] and seems to have been present for millennia in the sheep and goat breeding systems [3]. The annual breeding season begins in summer in Ile-de-France ewes and in autumn in Alpine goats and ends in winter in both species, resulting in a marked seasonality in birth dates of lambs and kids. In goats and sheep, this seasonality is under photoperiodic control. In experimental conditions, long days inhibit and short days stimulate sexual activity (goats: [4-6] sheep: [1]). However, under natural conditions of temperate countries, goats, as well as sheep [7,8], probably have an endogenous rhythm that is synchronized by photoperiod such that breeding occurs during autumn/winter and anovulation (anestrus) occurs during spring/summer. When transferred to equatorial conditions (12 h of light per day, with a limited control of temperature change amplitudes), Suffolk ewes (a European breed) cycled at irregular intervals with no clear anovulatory season [9]. In contrast, when transferred to tropical photoperiodic conditions where the annual amplitude of photoperiodic changes exists but is lower than in temperate regimen, Alpine goats do not greatly modify the seasonal characteristics of their breeding season, and long periods of anestrus and anovulation are still present during spring and summer as in control females maintained under temperate photoperiod [10]. Local breeds of sheep [11-15] and goats [12,16,17] under their native tropical conditions, are either non-seasonal breeders or exhibit only a weak seasonality of reproduction. The females of these breeds ovulate and exhibit estrus almost the whole year round, even though short periods of anovulation and anestrus are detected in some females. Two main hypotheses can be raised to explain the near-absence of seasonality: either the females are insensitive to photoperiod, or the amplitude of the photoperiodic changes is too small. It is thus interesting to determine whether absence of seasonality persists when females of these breeds are subjected to major annual changes in the amplitude of day length, the prevailing conditions in temperate regions, or whether seasonality appears as it does in most temperate breeds. In the present experiment, seasonal ovulatory activities were assessed in tropical Creole goats and Black-Belly ewes. These two breeds originate from the Carribean Islands, where they have been bred for 3–4 centuries, and constitute the progeny of African tropical breeds (see Methods). The animals of the present experiment were imported into Europe and experimentally subjected for more than two years in light-proof rooms to an annual photoperiodic regimen simulating that of temperate regions (TE group), and compared to females under tropical cycle simulating that of a tropical region (TR group). Results Ovulatory activity in Creole goats Ovulatory activity demonstrated marked differences between experimental groups over the course of the experiment. Individual ovulatory activity is presented in Figure 1. Marked seasonality of ovulatory activity appeared in female goats of the TE group; the seasonal inactivity occurred from May to September for the three years of the study (Figures 1 &2). The percentage of female goats experiencing at least one ovulation per month dramatically decreased from May to September for the three years (0%, 27% and 0%, respectively). All female goats experienced an anovulatory period during the first spring/summer season (1990), only one of them continued its ovulatory activity in 1991, and all of them stopped again in May 1992. In contrast, female goats exposed to the TR photoperiodic cycle demonstrated much less seasonality as the percentage of goats experiencing at least one ovulation per month never went below 56% (minima for the three years of study: 56%, 56%, 57%). Two female goats cycled continuously during the course of the experiment, one female goat in mid-1990 and three in mid-1991 continued their ovulatory activity during spring and summer, and four females were still cycling at the end of the experiment (June 1992). These differences between the two groups led to significant differences in some, but not all, parameters. The percentage of goats experiencing at least one ovulation per month (Figure 2) was significantly lower in the TE than in the TR group in May (P < 0.05), June, July, August and September 1990 (P < 0.001), tended to be lower in May 1991 (P < 0.10), and was lower again in May (P < 0.05) and June 1992 (P < 0.001). In both groups the females which stopped their ovulatory activity did so at roughly the same date (April-May) in the 3 years of the experiment (Table 1). TR goats began their first breeding season significantly earlier than TE goats in 1990, but this difference did not appear in the second breeding season (Table 1). Variances of the dates of end of the 2nd and 3rd breeding seasons were significantly higher in TR goats (Table 1). The duration of the anovulatory period in 1990 was significantly shorter in TR than in TE goats and the duration of the 1990–1991 breeding season was significantly longer in TR than in TE goats (Table 2). The 1991 anovulatory period and the 1991–1992 breeding season did not differ between groups (Table 2). Variances of the duration of the 2nd anovulatory season and of the 3rd breeding season were significantly higher in TR goats (Table 2). Ovulatory activity in Black-Belly ewes Ovulatory activity demonstrated marked variations in both experimental groups over the course of the experiment. Individual ovulatory activity is presented in Figure 3. From October to March of the first year, all females were cycling in both groups (100% of ewes showed at least one ovulation per month; Figure 3 &4). In the TR group, ovulatory activity dropped in April, remained low (2 ewes cycling) in May and June, then rose again in July and August to reach its maximum from September to April of the next year; minimum activity was observed again from May to August, before a maximal activity in September and October. Ewes of the TE group roughly followed the same pattern, with a later onset of cyclicity in September of the first year (% of ewes cycling in August P < 0.001); and a later end in May-June of the second year (% of ewes cycling in May P < 0.01). The percentage of cycling females never went below 17% (2 ewes cycling). One ewe in the TE group never stopped cycling during the course of the experiment. Mean dates of the end of the breeding season did not differ between TR and TE ewe groups in the first and second year (April, Table 3). The onset of the breeding season occurred earlier in TR than in TE for the first year, but not for the second year (Table 3). Thus, duration of the first and second anestrous seasons and/or duration of the sexual season did not differ between TR and TE ewe groups (Table 4). Variances of the durations of anovulatory and of breeding seasons were significantly higher in TE ewes (Table 4). Discussion The two tropical breeds of Creole goats and Black-Belly ewes used in the present study demonstrated clear seasonal breeding activity with a definite cut-off when maintained under the simulated extensive photoperiodic variations of temperate areas. As many other goat and sheep breeds originating and raised under a temperate climate, these two breeds imported from the tropics displayed cessation of ovulatory activity in spring and summer, i.e. the usual months for anovulation and anestrus in a temperate climate. Even though it appears that their anovulatory season seemed shorter than European breeds of goats [e.g. Alpine, [10]] and sheep [e.g.. Ile-de-France, [18]], almost all Black-Belly ewes stopped their ovulatory activity for about 4 months and Creole goats for 2.5 months. Black-Belly ewes stopped their ovulatory activity late in the year (second half of April) as compared to the majority of breeds, for example Ile-de-France breed [mid-January; [18]) or the majority of British breeds [19]. On the other hand, they started their breeding season later that these breeds, showing a more "primitive" type of breeding season, similar to those displayed by the Moufflon [20], Romanov [21] or Icelandic [22] breeds of sheep. A similar observation could be made for the end of the breeding season of the Creole goats maintained under simulated temperate photoperiod: they stopped late in the season (April-May) compared to temperate breeds [February, [10]]. However, this was not true for the onset of the breeding season which generally started in September-October in both goat breeds. The control group of goats maintained under simulated tropical photoperiodic variations displayed significantly less seasonality. The percentage of goats showing at least one ovulation per month was significantly higher in May and June for 2 years out of 3, and did not drop to 0 as it did in the temperate group of goats. A relatively high number of female goats did not experience an anovulatory season either at all or during some years of the experiment, and those that did showed a significantly shorter anovulatory period during the first year of the experiment. Thus, the comparison between Creole female goats maintained under simulated temperate photoperiod and control females placed under simulated tropical photoperiod leads to the conclusion that their breeding season is sensitive to large photoperiodic variations. In contrast, Black-belly ewes maintained under simulated tropical photoperiodic variations did not differ greatly from those maintained under simulated temperate photoperiod. The two groups of ewes and goats used here differed in various parameters, some of which could explain the photoperiod x species interaction observed here: (a) Recipient ewes in which embryos were implanted in autumn were maintained under natural photoperiod. It is known that light changes during pregnancy may affect the progeny's photoperiod sensitivity, especially regarding the onset of puberty in sheep [23-25] and in rodents [26]. This was not the case in the goats, as these experimental animals were the 3nd or 4th progeny of females imported as embryos. (b) Experimental ewes were artificially raised in TE vs TR photoperiod from birth to 6 months old from the start of the experiment, whereas female goats were raised under simulated tropical photoperiod until the start of the experiment at one or two years old. (c) Black-Belly ewes were included in the experiment at 6 months of age, whereas Creole goats were 12 and 24 months old at the start of the experiment. These three main differences between the experiments in female goats and ewes could possibly explain this photoperiod x species interaction. However, it may also come from a real difference of sensitivity to non-photoperiodic factors (such as temperature changes or activity, see later) between the two species. The spontaneous ovulatory activity demonstrated in the present experiment by the Black-Belly ewes from the TR group were very different from those registered earlier in their natural breeding conditions on the island of Martinique in the tropics, where they cycled all year round [15]. The Creole goats from the simulated tropical photoperiod did not display here the same results as females of the same flock raised in their natural breeding conditions on the island of Guadeloupe in the tropics, where they also cycled almost continuously [17]. This unexpected difference suggests that other external cues may act in combination with photoperiod to inhibit breeding activity. The fact that all animals simultaneously stopped cycling in spring, suggests that an external physical cue could be involved. The temperature of the light-proof rooms in which the experiments were performed was not controlled and the high and low-amplitude variations of air temperature of the tropics were not applied to our experimental animals. Such a cue may interact with photoperiod and enhance the negative effects of the limited photoperiodic changes, which do not appear in normal tropical conditions. To our knowledge, very few experiments have been carried out in sheep and/or goats to determine the role played by low temperature in the appearance of seasonality. It has been demonstrated that inversion of the temperature rhythm does not entrain ovulatory activity in ewes of a European breed maintained under an equatorial photoperiodic schedule [27] and that low temperatures in the summer time may induce an 8-week advance in the onset of the annual breeding season in dark-faced ewes [28]. In Suffolk ewes, a seasonal breed, the maintenance of ewes under an equatorial regimen with a limited but efficient control of temperature change amplitude, induced cycles at irregular intervals with no clear anovulatory season [9] However, in other species it has been clearly demonstrated that the combination of photoperiod and temperature is responsible for seasonal changes in reproductive activity [29,30]. Thus, it is possible that, in the absence of a major cue (photoperiod), seasonal ovulatory activity of females of the TR groups has been entrained by temperature. In Syrian hamsters, exercise by access to a running wheel can completely inhibit the short-day induced regression of the testis [31,32]. Experimental Creole goats and Black Belly ewes of the present experiment were raised in light-proof rooms where physical exercise was limited, whereas in their original management conditions where the initial observations were done [15,17], animals were maintained at pasture. Thus, it is possible that in their original management conditions, physical exercise prevented the inhibitory effects of the 13 hours of light that was observed in our experimental light-proof rooms in the TR groups of goats and ewes. The sheep and goat breeds used here are local breeds of the Carribean islands. Even though their presence in these islands is associated with human history, they are considered as the progeny of tropical but not European ancestors, because they originate from the West coast of tropical Africa (see Methods). In these areas all sheep and goat breeds are considered as non or low seasonal breeders. Thus, the sensitivity of the Creole goats and Black-Belly ewes observed here under temperate photoperiod could be hypothetized as a true sensitivity of these breeds, not a simple inheritance of a trait coming from European ancestors. Conclusions A marked seasonality in the ovulatory activity of tropical Creole goats and Black-Belly ewes was induced when females were exposed to a simulated temperate photoperiod. Unexpectedly, and differing from the results obtained in their original breeding location, Black-Belly ewes and, to a lesser extent, Creole goats exposed to a simulated tropical photoperiod also showed significant seasonality in their ovulatory activity. Such results indicate that both species are capable of showing seasonality under the the photoperiodic changes of the temperate zone even though they do not originate from these regions. Methods Production of experimental animals from deep-frozen embryos Experimental animals from both species were produced after importation of deep-frozen embryos. The embryos were thawed and re-implanted into recipient females of Saanen goats for Creole embryos (1983) and of Ile-de-France sheep for Black-Belly embryos (1997). Embryos from donor Creole goats were collected as previously described [33] from 4 genetic families considered as representative of the native population of the Caribbean island of Guadeloupe (French West Indies). This breed has been raised in Guadeloupe for several centuries and probably originates from the West coast of Africa which it was imported from in the 17th-18th centuries [34-37]. Creole goats from Guadeloupe have many similarities with the "West African dwarf goat" regarding their size, coat color, fertility and prolificacy, growth rate and horn shapes [34,37]. Common genetic markers were found between Creole goat from Guadeloupe and West African goats [36], which reinforced the hypothesis of an African origin for the Creole goat. The first generation of animals, originating from deep-frozen imported embryos, was not used in the experiment. They were raised, with their progeny, under tropical photoperiodic conditions in light-proof buildings, as described later. The 3rd and 4th generations, constituting a sufficient number of animals, made up the two experimental groups. Embryos from donor Black-Belly ewes were collected using the technique described by Heyman et al. [38]. The donor females belonged to an INRA flock raised in Guadeloupe and were part of the 6 different families constituting this flock, bred from genitors from Martinique (F.W.I.) [39,40] and Barbados. This flock was considered to present characteristic production traits of the Black-Belly sheep population of the Caribbean [41]. As for goats, Black-Belly sheep is considered to have an African origin about 3 to 4 centuries ago [11,42]. This is confirmed by their phenotypic characteristics of hair sheep (i.e. not wool sheep), including performance traits [43]. After checking the absence of Blue-tongue virus in the collection media, embryos were re-implanted into Ile-de-France ewe lambs, 2 embryos inserted per recipient ewe. In the case of sheep, the first generation originating from deep-frozen embryos was used directly in the present experiment. After birth, all lambs were immediately placed under artificial suckling conditions, in the two experimental groups in light-proof rooms under tropical or temperate photoperiod, until the start of blood sampling for progesterone determinations. Animals and feeding conditions Both experiments were performed at the INRA Station near Tours (latitude 47°25 North). - Thirty three Creole female goats were used from October 1989 when the animals were one (n = 15 females) and two (n = 18 females) years old, for 33 months to June 1992 . They were divided into two groups (n = 17 TR and 16 TE) in visual and tactile contact, with entire and vasectomized Creole bucks, but separated by a fence. Each group was maintained in a separate light-proof room throughout the experiment. Feeding conditions were constant throughout the experiment. The animals were fed once daily with a diet of 240 g of barley, 60 g of wheat, 700 g of meadow hay and 300 g of barley straw. No flushing was performed. They had free access to water and to mineral blocks containing oligoelements and vitamins. - Twenty four Black Belly ewes were used from September 1998 when the animals were 6 months old, for 25 months, to October 2000. They were divided into two groups (n= 12 TR and 12 TE) in visual and tactile contact, with entire Black Belly rams, but separated by a fence. Each group was maintained in a separate light-proof room throughout the experiment. Feeding conditions were constant throughout the experiment. The animals were fed once daily with a diet of 150 g of corn, 110 g of barley, 45 g of dehydrated protein complement and 400 g of hay. No flushing was performed. They had free access to water and to mineral blocks containing oligoelements and vitamins. Photoperiodic treatments Within each species, one group was subjected to the large photoperiodic changes prevailing at the 45° North latitude (8 to 16 h of light per day from winter to summer solstices); this group was called the temperate group (TE). The other group was subjected to the limited photoperiodic changes prevailing at the 16° North latitude (11 to 13 h of light per day from winter to summer solstices); this group was called the tropical group (TR). In all rooms, photoperiod was regulated by an electric clock that operated bulbs providing an intensity of 300 lux, lateral to the animals' eyes. The photoperiod was adjusted by 15 min shifts (more or less frequent depending on the slope of the natural changes in daylength) to produce a complete photoperiodic cycle every 365 days. The four rooms were adjacent and of the same size (30 m2). Temperature was not controlled but variations were parallel to those monitored outside but with a lower amplitude (minimum +1°C in January, maximum +29°C in August). Measurements and samplings Liveweight of Creole goats at the beginning of the experiment was 24.6 (± 3.4, sd) kg in group TE and 25.5 (± 3.0) kg in group TR. Liveweight was measured monthly and showed a regular increase until the end of the experiment (55.7 ± 7.5 and 56.2 ± 5.2 kg for TE and TR respectively). Liveweight of Black-Belly at the beginning of the experiment was 33.2 (± 2.5 sd) kg in group TE and 31.9 (± 2.7) kg in group TR. Liveweight was measured monthly and showed a regular increase until the end of the experiment (49.5 ± 5.9 and 47.0 ± 5.8 kg for TE and TR respectively). Ovulatory activity was assessed twice weekly in goats up to end October 1991 and once weekly thereafter; and once weekly in ewes, using blood samples for the progesterone radioimmunoassay. A rapid assay was performed using the technique described by Terqui and Thimonier [44]. When progesterone concentration was lower than 1.0 ng per ml of plasma in female goats and 0.75 ng/ ml of plasma in ewes, the female was considered to be in the follicular phase of the cycle or in anovulation. Definitions and analysis of results, statistical tests The first occurrence of a positive Progesterone sample was considered as the date of the first ovulation of the season, and the last occurrence of a positive Progesterone sample was considered as the date of the last ovulation of the season. Mean duration of ovulatory activity is the number of days between first and last ovulation in the same breeding season. Mean duration of anovulation is the number of days between last ovulation in a breeding season and first ovulation of the next season. Females which did not present cessations of their ovulatory activity were not included in the calculations of duration of the breeding seasons or duration of the anovulatory periods. In September and October 2000, individual blood samplings were stopped in ewes that had resumed their ovulatory activity. Mean dates of onset and end of the breeding season, durations of the breeding season and of anovulatory activity were compared between groups using an unpaired t-test. Variances were compared with F-Tests. Percentages of females showing at least one ovulation per month were analyzed using the Chi2 method. (Statview®, Abacus Concept, Berkeley, Ca, USA). All procedures were performed in accordance with French legal requirements and with the Ministry of Agriculture authorization for animal experimentation nb A37801 . Authors' contributions The authors contributed equally to this work. PC conceived the study, and was responsible for its design and coordination. AD followed the experiment in goats and DC the experiment in sheep. YC and GA performed all the embryo transfer procedures, and were in charge of pathological analyses. AD, DC and PC analysed the data. PC drafted the manuscript. All authors read, corrected and approved the final manuscript. Acknowledgements The authors wish to thank the staff in charge of the flocks in Nouzilly, especially Pierre Pignon for goats, Jean-Claude Braguer and Francis Dupont for sheep. They also want to thank the staff in charge of Creole goats and Black-Belly ewes in Guadeloupe, especially Hugues Varo and Paul Despois, and Dominique Martinez from CIRAD-EMVT in Guadeloupe for his help in Blue-Tongue determinations of the donor and recipient ewes. They would also like to thank Sylvie Canépa, Claudette Fagu, and Christophe Gauthier of the RIA laboratory in Nouzilly for performing the progesterone assays, Jacques Thimonier for his help at the beginning of the goat experiment and Benoît Malpaux for suggestions and corrections regarding the present manuscript. Figures and Tables Figure 1 Individual ovulatory activity in Creole goats maintained under two different photoperiodic conditions. Two groups of Creole female goats were maintained in light-proof rooms and subjected to either simulated temperate photoperiodic conditions (TE goats 1-16 at the bottom of the figure, 8 to 16 hours of light per day from winter to summer solstice), or simulated tropical photoperiodic conditions (TR goats 17-33 upper part of the figure, 11 to 13 hours of light per day from winter to summer solstice) for 33 months. Ovulatory activity was assessed by twice-weekly (first 25 months) then once-weekly (last 8 months) determinations of plasma progesterone by radioimmunoassay. One line represents one female goat. A single dot represents one progesterone measurement above 1 ng/ml of plasma and is considered as indicative of the presence of at least one corpus luteum. Female goats which died or were excluded from the experiment are indicated by an "x". Figure 2 Ovulatory activity in Creole goats maintained under two different photoperiodic conditions. Two groups of Creole female goats were maintained in light-proof rooms and subjected to either simulated temperate photoperiodic conditions (TE solid line closed circles, 16 goats, 8 to 16 hours of light per day from winter to summer solstice), or simulated tropical photoperiodic conditions (TR dashed line open squares, 17 goats, 11 to 13 hours of light per day from winter to summer solstice) for 33 months. Ovulatory activity was assessed by twice-weekly (first 25 months), then once-weekly (last 8 months) determinations of plasma progesterone by radioimmunoassay. Results are expressed as the percentage of female goats experiencing at least one ovulation per month. Significant differences between groups are indicated. Figure 3 Individual ovulatory activity in Black-Belly ewes maintained under two different photoperiodic conditions. Two groups of Black-Belly ewes were maintained in light-proof rooms and subjected to either simulated temperate photoperiodic conditions (TE ewes 1-12 at the bottom of the figure, 8 to 16 hours of light per day from winter to summer solstice) or simulated tropical photoperiodic conditions (TR ewes 13-24 upper part of the figure, 11 to 13 hours of light per day from winter to summer solstice) for 25 months. Ovulatory activity was assessed by once-weekly determinations of plasma progesterone by radioimmunoassay. One line represents one ewe. A single dot represents one progesterone measurement above 0.75 ng/ml of plasma and is considered as indicative of the presence of at least one corpus luteum. All ewes were present until the end of the experiment. Figure 4 Ovulatory activity in Black-Belly ewes maintained under two different photoperiodic conditions. Two groups of Black-Belly ewes were maintained in light-proof rooms and subjected to either simulated temperate photoperiodic conditions (TE solid line closed circles, 12 ewes, 8 to 16 hours of light per day from winter to summer solstice) or simulated tropical photoperiodic conditions (TR dashed line open squares, 12 ewes, 11 to 13 hours of light per day from winter to summer solstice) for 25 months. Ovulatory activity was assessed by once-weekly determinations of plasma progesterone by radioimmunoassay. Results are expressed as the percentage of ewes experiencing at least one ovulation per month. Significant differences between groups are indicated. Table 1 Mean dates ± S.E.M. in days, for onset and end of ovulatory activity in Creole goats maintained under two different photoperiodic conditions. 1st breeding season 1989–1990 2nd breeding season 1990–1991 3rd breeding season 1991–1992 onset end onset end onset end Temperate regimen NA 20/04 ± 4 10/10 ± 11 22/04 ± 5 11/07 ± 7 08/05 ± 4 Tropical regimen NA 14/04 ± 7 18/6*** ± 8** 02/05 ± 11* 23/07 ± 15** 02/05 ± 12** Two groups of Creole female goats were maintained in light-proof rooms and subjected to either simulated temperate photoperiodic conditions (TE, 16 goats, 8 to 16 hours of light per day from winter to summer solstice), or simulated tropical photoperiodic conditions (TR, 17 goats, 11 to 13 hours of light per day from winter to summer solstice) for 33 months. Ovulatory activity was assessed by determinations of plasma progesterone by radioimmunoassay. Means and variances were compared between groups within each column. Significant differences between means and between variances are indicated by asterisks (*:p < 0.05, **: P < 0.01, ***: P < 0.001). NA: not available. Table 2 Mean durations in days ± S.E.M. of anovulatory and ovulatory seasons in Creole goats maintained under two different photoperiodic conditions. 1st anovulatory season (1990) 2nd anovulatory season (1991) 2nd breeding season (90–91) 3rd breeding season (91–92) Temperate regimen 173.0 ± 8.0 79.6 ± 9.5 194.5 ± 9.5 301.8 ± 7.8 Tropical regimen 65.0*** ± 8.4 81.6 ± 21.6* 304.3*** ± 10.8 265.5 ± 31.3*** Two groups of Creole female goats were maintained in light-proof rooms and subjected to either simulated temperate photoperiodic conditions (TE, 16 goats, 8 to 16 hours of light per day from winter to summer solstice), or simulated tropical photoperiodic conditions (TR, 17 goats, 11 to 13 hours of light per day from winter to summer solstice) for 33 months. Ovulatory activity was assessed by determinations of plasma progesterone by radioimmunoassay. Means and variances were compared between groups within each column. Significant differences between means and between variances are indicated by asterisks (*:p < 0.05, ***: P < 0.001). Table 3 Mean dates ± S.E.M. in days, for onset and end of ovulatory activity in Black-Belly ewes maintained under two different photoperiodic conditions. 1st breeding season 1998–1999 2nd breeding season 1999–2000 3rd breeding season 2000–2001 onset end onset end onset end Temperate regimen NA 22/04 ± 7 15/09 ± 3 25/04 ± 12 22/09 ± 5 NA Tropical regimen NA 09/04 ± 4 05/08*** ± 5* 22/04 ± 4* 13/09 ± 3 NA Two groups of Black-Belly ewes were maintained in light-proof rooms and subjected to either simulated temperate photoperiodic conditions (TE, 12 ewes, 8 to 16 hours of light per day from winter to summer solstice) or simulated tropical photoperiodic conditions (TR, 12 ewes, 11 to 13 hours of light per day from winter to summer solstice) for 25 months. Ovulatory activity was assessed by determinations of plasma progesterone by radioimmunoassay. Means and variances were compared between groups within each column. Significant differences between means and between variances are indicated by asterisks (*:p < 0.05, ***: P < 0.001). NA: not available. Table 4 Mean durations in days ± S.E.M. of anovulatory and ovulatory seasons in Black-Belly ewes maintained under two different photoperiodic conditions. 1st anovulatory season (1999) 2nd anovulatory season (2000) 2nd breeding season (99–2000) Temperate regimen 134.2 ± 14.0 134.3 ± 14.0 237.8 ± 13.3 Tropical regimen 118.4 ± 7.5* 144.3 ± 5.8* 260.6 ± 6.7* Two groups of Black-Belly ewes were maintained in light-proof rooms and subjected to either simulated temperate photoperiodic conditions (TE, 12 ewes, 8 to 16 hours of light per day from winter to summer solstice) or simulated tropical photoperiodic conditions (TR, 12 ewes, 11 to 13 hours of light per day from winter to summer solstice) for 25 months. Ovulatory activity was assessed by determinations of plasma progesterone by radioimmunoassay. Means and variances were compared between groups within each column. 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Existence de rythmes endogènes PhD Thesis Univ F Rabelais Tours Fr 1989 Dyrmundsson OR Studies on the breeding season of Icelandic ewes and ewe lambs J Agric Sci 1978 90 275 281 Herbosa CG Wood RI I'Anson H Foster DL Prenatal photoperiod and the timing of puberty in the female lamb Biol Reprod 1994 50 1367 1376 8080924 Sunderland SJ O'Callaghan D Boland MP Roche JF Effect of photoperiod before and after birth on puberty in ewe lambs Biol Reprod 1995 53 1178 1182 8527523 Helliwell RJ Wallace JM Aitken RP Racey PA Robinson JJ The effect of prenatal photoperiodic history on the postnatal endocrine status of female lambs Anim Reprod Sci 1997 47 303 314 9360769 10.1016/S0378-4320(97)00018-3 Goldman BD Pattern of melatonin secretion mediates transfer of photoperiod information from mother to fetus in mammals Sci STKE 2003 192 PE29 Wodzicka-Tomaszweska M Hutchinson JCD Bennett JW Control of the annual rhythm of breeding in ewes: effect of an equatorial daylength with reversed thermal seasons J Agric Sci 1967 68 61 67 Dutt RH Temperature and light as factors in reproduction among farm animals J Dairy Sci 1960 43 123 144 Larkin JE Freeman DA Zucker I Low ambient temperature accelerates short-day responses in Siberian hamsters by altering responsiveness to melatonin J Biol Rhythms 2001 16 76 86 11220782 Larkin JE Jones J Zucker I Temperature dependence of gonadal regression in Syrian hamsters exposed to short day lengths Am J Physiol Regul Integr Comp Physiol 2002 282 R744 752 11832395 Gibbs FP Petterborg LJ Exercise reduces gonadal atrophy caused by short photoperiod or blinding of hamsters Physiol Behav 1986 37 159 162 3526367 10.1016/0031-9384(86)90399-9 Menet JS Vuillez P Saboureau M Pévet P Inhibition of hibernation by exercise is not affected by intergeniculate leaflets lesion in hamsters Physiol Regul Integr Comp Physiol 285 R690 R700 Chemineau P Procureur R Cognie Y Lefevre PC Locatelli A Chupin D Production, freezing and transfer of embryos from a bluetongue-infected goat herd without bluetongue transmission Theriogenology 1986 26 279 290 10.1016/0093-691X(86)90147-0 Labat P Guillaume Cavellier père, Paris Nouveau voyage aux Isles d'Amérique 1792 8 Chemineau P Cognie Y Xande A Peroux F Alexandre G Levy F Shitalou E Beche JM Sergent D Camus E Barre N Thimonier J Le "cabrit créole" de Guadeloupe et ses caractéristiques zootechniques : monographie Elev Méd Vét Pays Trop 1984 37 225 238 Pépin L Recherche de polymorphisme génétique chez les caprins. 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Description des principaux phénotypes identifiés et étude de quelques caractères morphologiques Rev Elev Méd Vét Pays Trop (Supp) 1991 75 82 Mahieu M Aumont G Alexandre G Élevage intensif des ovins tropicaux à la Martinique INRA Prod Anim 1997 10 21 32 Thomas DL Wildeus S Hair sheep genetic resources of the Americas (review paper) In Proceedings Hair Sheep Research Symposium, Ste Croix USVI 1991 St Croix, University of the Virgin Islands 3 20 June 28, 29, 1991 Fitzhugh HA Bradford GE Hair Sheep of Western Africa and the Americas Westview Press, Boulder, Co, USA 1983 Naves M Alexandre G Leimbacher F Mandonnet N Menendez Buxadera A Le point sur les programmes de gestion des ressources génétiques chez les espèces de ruminants dans la Caraïbe INRA Prod Anim 2001 14 181 192 Terqui M Thimonier J Nouvelle méthode radioimmunologique rapide pour l'estimation du niveau de progestérone plasmatique. Application pour le diagnostic précoce de gestation chez la brebis et la chêvre C R Acad Sci D Sci Vie 1974 279 1109 1112

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==== Front BMC NeurosciBMC Neuroscience1471-2202BioMed Central London 1471-2202-5-341537739110.1186/1471-2202-5-34Research ArticleHeme oxygenase-2 gene deletion attenuates oxidative stress in neurons exposed to extracellular hemin Regan Raymond F [email protected] Jing [email protected] Luna [email protected] Department of Emergency Medicine, Thomas Jefferson University, 1020 Sansom Street, 239 Thompson Building, Philadelphia, PA 19107, USA2004 17 9 2004 5 34 34 30 6 2004 17 9 2004 Copyright © 2004 Regan et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Hemin, the oxidized form of heme, accumulates in intracranial hematomas and is a potent oxidant. Growing evidence suggests that it contributes to delayed injury to surrounding tissue, and that this process is affected by the heme oxygenase enzymes. In a prior study, heme oxygenase-2 gene deletion increased the vulnerability of cultured cortical astrocytes to hemin. The present study tested the effect of HO-2 gene deletion on protein oxidation, reactive oxygen species formation, and cell viability after mixed cortical neuron/astrocyte cultures were incubated with neurotoxic concentrations of hemin. Results Continuous exposure of wild-type cultures to 1–10 μM hemin for 14 h produced concentration-dependent neuronal death, as detected by both LDH release and fluorescence intensity after propidium iodide staining, with an EC50 of 1–2 μM; astrocytes were not injured by these low hemin concentrations. Cell death was consistently reduced by at least 60% in knockout cultures. Exposure to hemin for 4 hours, a time point that preceded cell lysis, increased protein oxidation in wild-type cultures, as detected by staining of immunoblots for protein carbonyl groups. At 10 μM hemin, carbonylation was increased 2.3-fold compared with control sister cultures subjected to medium exchanges only; this effect was reduced by about two-thirds in knockout cultures. Cellular reactive oxygen species, detected by fluorescence intensity after dihydrorhodamine 123 (DHR) staining, was markedly increased by hemin in wild-type cultures and was localized to neuronal cell bodies and processes. In contrast, DHR fluorescence intensity in knockout cultures did not differ from that of sham-washed controls. Neuronal death in wild-type cultures was almost completely prevented by the lipid-soluble iron chelator phenanthroline; deferoxamine had a weaker but significant effect. Conclusions These results suggest that HO-2 gene deletion protects neurons in mixed neuron-astrocyte cultures from heme-mediated oxidative injury. Selective inhibition of neuronal HO-2 may have a beneficial effect after CNS hemorrhage. ==== Body Background Hemin is a potent oxidant that accumulates in intracranial hematomas and may contribute to neural cell injury [1,2]. It is also the preferred substrate for heme oxygenase-2, the constitutively-expressed isoform that accounts for most CNS heme oxygenase (HO) under normal conditions [3]. In pathologic states, HO frequently has an antioxidant effect, putatively due to the protection provided by increased cellular bilirubin, decreased heme, and up-regulation of other antioxidants [4-7]. However, in models that are relevant to CNS hemorrhage, HO inhibitors have surprisingly been found to be protective [8-10]. All HO inhibitors that are currently available have numerous non-specific actions that may complicate the interpretation of experimental results, including inhibition of nitric oxide synthase and guanyl cyclase, and modification of voltage-gated calcium currents [11-14]. Some may also have a direct antioxidant effect that is unrelated to HO inhibition [15]. In order to investigate HO-2 in heme-mediated injury more specifically, we have cultured neurons and astrocytes derived from HO-2 knockout mice and genetically-similar wild type controls. In recent studies, we observed that astrocytes derived from mutant mice were more vulnerable to hemin [16]. Conversely, HO-2 gene deletion decreased the vulnerability of neurons to hemoglobin [17]. Neither wild type nor knockout astrocytes were injured by hemoglobin at the micromolar concentrations that are feasible in vitro. HO-2 gene deletion per se did not result in a compensatory increase in HO-1 in these cultures, and produced minimal or no change in other cellular antioxidants [16,17]. The disparate effects of HO-2 gene deletion on hemin toxicity to astrocytes and hemoglobin toxicity to neurons may reflect the inability of neurons to tolerate the products of heme metabolism, i.e. iron, carbon monoxide, and bilirubin. Alternatively, it may reflect the different oxidant properties of hemin and hemoglobin. Although the oxidant effect of hemoglobin may be due in part to hemin release to membrane lipids [18], other mechanisms may also contribute. Extracellular hemoglobin undergoes autoxidation, which produces superoxide [19]. In addition to being an oxidant, superoxide reacts with globin amino acids in a complex fashion to generate a variety of reactive species, including thiyl radicals, hydroxyl radicals, and hydrogen peroxide [20,21]. It is also noteworthy that hemoglobin is highly water soluble while hemin is quite lipophilic; their accumulation in separate cellular compartments may lead to a different pattern of site-specific oxidative damage [22,23]. The present study was designed to test the effect of HO-2 gene deletion on the oxidative neuronal injury produced by extracellular hemin. We specifically tested the hypothesis that targeted deletion of the HO-2 gene attenuated oxidative cell injury in a primary cell culture model of hemin toxicity. Results Effect of HO-2 gene deletion on hemin neurotoxicity In preliminary experiments, we observed that overnight (14 h) exposure to low micromolar concentrations of hemin consistently produced morphologic evidence of neuronal injury in wild-type cultures (Fig. 1). This time interval was therefore used for cytotoxicity studies. Consistent with prior observations in pure astrocyte cultures [24], no morphologic evidence of injury was observed in the astrocyte monolayer at hemin concentrations up to 10 μM. In order to specifically assess neuronal injury in this study, the concentrations used were limited to this range. In wild-type cultures, cell injury as quantified by LDH release was observed at 1 μM hemin and then increased exponentially, to release of 69.7 ± 8.6% of neuronal LDH at 3 μM (Fig 2A). The calculated EC50 was 1.85 μM. LDH release was significantly reduced in knockout cultures subjected to the same treatment. At 3 μM hemin, only 12.6 ± 4.1% of LDH had been released at this time point. Control experiments demonstrated that these low hemin concentrations do not interfere with the LDH assay. Cell death was also quantified by analysis of fluorescence intensity after staining cultures with propidium iodide. Using this method, widespread neuronal death was also observed at 3–10 μM hemin in wild type cultures, and the calculated EC50 was 1.05 μM. Propidium staining of nuclei was significantly reduced in cultures prepared from HO-2 knockout mice (Fig. 2B). The maximal fluorescence was produced by exposure to 10 μM hemin, which was 37.0 ± 3.2% of that observed in control sister cultures treated with NMDA to kill all neurons. Effect of HO-2 gene deletion on markers of cell oxidation In order to assess reactive oxygen species formation after hemin exposure, cultures were stained with 20 μM dihydrorhodamine 123 after 4 hour hemin exposure. This time interval was used because it preceded cell lysis, and therefore allowed cell retention of the reduced fluorophore. A marked increase in fluorescence was observed in cultures prepared from wild type mice (Fig. 3). This signal was concentrated in neuronal cell bodies and processes. In contrast, fluorescence in cultures prepared from HO-2 knockout mice was minimal, and did not exceed that observed in cultures subjected to medium exchanges only. In order to further investigate the effect of HO-2 gene deletion on oxidative stress produced by hemin, cells were harvested after 4 hour hemin exposure. Protein carbonyl groups (i.e. aldehydes and ketones), which are markers of oxidation, were then derivatized and detected with a dinitrophenylhydrazone antibody. Increased immunoreactivity was apparent in lysates of wild type cultures treated with hemin (Figure 4). A prominent band was present at approximately 44 kDa, along with a higher molecular weight smear. At 10 μM hemin, the carbonyl signal intensity in wild type cultures was 2.3-fold higher than in cultures exposed to culture medium only, compared with only 1.3-fold higher in knockout cultures. Hemin neurotoxicity is attenuated by iron chelators Based on our prior observations in astrocytes and neuroblastoma cells [24,25], we hypothesized that the toxic product produced by hemin breakdown in primary murine neurons was iron. In order to test this hypothesis, the effect of iron chelators on hemin neurotoxicity in wild type cultures was assessed. Most cell death, as detected by both LDH release and PI staining, was prevented by concomitant treatment with phenanthroline, a lipid-soluble iron chelator (Fig. 5). Deferoxamine, which is water soluble, was less potent; its effect when applied at a concentration tenfold greater than that of hemin reached statistical significance only when injury was assessed by PI staining. Discussion In prior experiments, we demonstrated that targeted deletion of the HO-2 gene in primary neuron/astrocyte cultures did not alter expression of HO-1, and had little or no effect on other cellular antioxidants [17]. This simplified system therefore permits investigation of HO-2 without the confounding compensatory effects that have been observed in whole animal models [26,27]. We have previously reported that HO-2 gene deletion increased the vulnerability of astrocytes to hemin, the preferred substrate of HO, in cultures containing only astrocytes. The present study targeted neurons in mixed neuron/astrocyte cultures by using hemin concentrations that did not injure astrocytes [24]. In this model, the opposite was observed. HO-2 deletion attenuated hemin-induced ROS formation and reduced levels of oxidized proteins. Consistent with the oxidative nature of hemin toxicity, neuronal death was reduced in knockout cultures. Although hemin is a highly reactive pro-oxidant, its breakdown as catalyzed by the heme oxygenases generates biologically active and potentially toxic products. Prior in vitro studies suggest that neurons are particularly vulnerable to these, i.e. iron, carbon monoxide, and bilirubin [28-30]. The present results suggest that when neurons are provided with an excess of HO substrate, the toxicity of breakdown products outweighs any benefit provided by hemin removal. The protective effect of iron chelators suggests that this phenomenon is at least partly due to iron neurotoxicity. Inorganic iron is toxic to cultured cortical neurons, with an EC50 of approximately 10 μM [17]. The lower EC50 for hemin is not surprising, given its lipophilicity and accumulation in cell membranes [31]. The latter phenomenon likely accounts for the greater efficacy of phenanthroline, which unlike deferoxamine is lipophilic. Deferoxamine is quite effective against hemoglobin neurotoxicity in this culture system [32], suggesting that hemoglobin releases its iron either in the medium or in an aqueous cellular compartment. The present results are consistent with observations that heme oxygenase inhibitors are protective in models of CNS hemorrhage [2,8,9], in contrast to the beneficial effect of HO in ischemia [33]. In a recent study, Koeppen et al. [2] observed that repeated administration of tin mesoporphyrin protected thalamic neurons from the delayed degeneration that occurred in tissue adjacent to injected autologous blood. Similarly, Huang et al. [9] observed that tin protoporphyrin attenuated edema formation after stereotactic hemoglobin injection into the rat striatum. It is noteworthy that the number of astrocytes per neuron is significantly higher in the human brain than in rodents [34]. The deleterious effect of HO inhibition on heme mediated injury to astrocytes may therefore be less prominent in animal models than in clinical intracerebral hemorrhage [35]. The disparate effect of HO on neurons and astrocytes exposed to extracellular hemin suggests that it may be somewhat difficult to target it effectively after CNS hemorrhage. All currently available HO inhibitors inhibit both HO-2, which is predominantly neuronal in vivo [36], and HO-1, which is induced mainly in glial cells [37]. The protection that these non-selective agents provide to neurons may be negated by their deleterious effect on astrocytes. Further investigation is needed for the development of strategies that would permit the selective inhibition or down-regulation of HO-2 in neurons. Conclusions Targeted deletion of the heme oxygenase-2 gene mitigates oxidative stress in cultured neurons exposed to hemin, and is cytoprotective. Selective inhibition of neuronal heme oxygenase may have a beneficial effect after CNS hemorrhage. Methods Cell cultures The HO-2 knockout mice which were used in this study are descended from mutants produced by Poss et al. [38], and have a C57BL/6 X 129/Sv genetic background. All mice were obtained from our local breeding colony, and were provided with food and water ad libitum and a 12 hour light/dark cycle. All breeding mice were the offspring of heterozygotes. Genotype was determined by polymerase chain reaction (PCR) using genomic DNA isolated from tail clippings; primers were previously published [17]. Cortical cell cultures were prepared from fetal mice at gestational age 15–17 d as previously described [39]. Under a dissecting microscope, cortices were dissected free from other brain tissue, minced with forceps, and incubated in medium containing 0.075%-acetylated trypsin at 37°C for one hour. Tissue was then collected by low speed centrifugation, and was dissociated by trituration through a flamed Pasteur pipette in plating medium containing Eagle's minimal essential medium (MEM), 5% fetal bovine serum (Hyclone, Logan, UT), 5% heat inactivated equine serum (Hyclone), glutamine (2 mM), and glucose (23 mM). The cell suspension was diluted with additional plating medium, and cells were plated on confluent astrocyte cultures in 24-well plates (Primaria, Falcon) at a density of 3 hemispheres/plate. Cultures were incubated at 37°C in a humidified atmosphere containing 5% CO2/95% air. Two-thirds of the culture medium was replaced at days 4 and 8 in vitro with MEM containing 10% equine serum, 2 mM glutamine, and 23 mM glucose. After ten days in vitro this feeding procedure was performed daily. Hemin exposure Hemin was freshly prepared as a 1 mM stock solution and was diluted to the desired concentration with minimal essential medium containing 10 mM glucose (MEM10). Experiments were conducted at 12–16 days in vitro. Serum was washed out of cultures with MEM10 (> 1000X dilution) prior to addition of hemin. Cultures were incubated at 37°C in a 5% CO2 atmosphere for the entire exposure interval. Detection of reactive oxygen species ROS formation was quantified by staining with dihydrorhodamine 123 (DHR, Molecular Probes, Eugene, OR), which is a cell-permeable, non-fluorescent compound that is oxidized by cellular peroxides to fluorescent rhodamine [40]. Fluorescence intensity is directly proportional to cellular oxidative stress. In order to prevent oxidation of DHR by hemin in the medium, cultures were washed free of hemin prior to DHR addition. After incubation with 20 μM DHR in MEM10 for 15 min, the medium was replaced, and cultures were imaged using a Nikon inverted microscope with epifluorescence attachment. Images were captured immediately after illumination (25 msec exposure). Photomicrographs of random 100X fields were analyzed with IPLab image analysis software (Scanalytics, Inc., Fairfax, VA). The low fluorescence in control cultures exposed to experimental medium only was subtracted from mean values to define the signal associated with hemin exposure. Detection of protein oxidation Protein oxidation was assessed using the Oxyblot™ kit (Chemicon, Inc., Temecula, CA). At the end of the hemin exposure interval, culture medium was aspirated, and cells were washed and then harvested in 100 μl of lysis buffer containing 210 mM mannitol, 70 mM sucrose, 5 mM HEPES, 1 mM EDTA, and 0.1% sodium dodecyl sulfate. Carbonyl groups were derivatized to 2, 4-dintrophenylhydrazone (DNP-hydrazone) by reaction with 2, 4-dinitrophenylhydrazine, following the manufacturer's instructions. Proteins were then separated on a 12% polyacrylamide gel and were transferred onto a polyvinylidene difluoride Imobilon-P transfer membrane filter (Millipore, Billerica, MA) using a semidry transfer apparatus (Bio-Rad, Hercules, CA). Carbonylated proteins were detected with rabbit anti-DNP (1:150) followed by goat anti-rabbit IgG (1:300). Immunoreactive proteins were visualized using Super Signal West Femto Reagent (Pierce Biotechnology, Rockford, IL) and Kodak ImageStation 400. Quantification of cell death After examination of cultures using phase contrast microscopy, cell death was quantified by measurement of LDH activity in the culture medium, as previously described [41]. To facilitate comparisons, values were scaled to the mean value in sister cultures exposed to NMDA 300 μM for 40 h. This treatment releases essentially all neuronal LDH in this system without injuring astrocytes [42]. Since the low micromolar concentrations of hemin that were used in this study do not injure cultured cortical astrocytes [24], the contribution of astrocyte LDH to the total signal is negligible. Cell death was also quantified by staining with propidium iodide (13 μg/ml for 15 min). When viewed with a rhodamine filter, the nuclei of cells with disrupted membranes stain red, while cells with intact membranes exclude propidium. Random 100X fields were captured with a Nikon Diaphot epifluorescence microscope and were analyzed with IPLab image analysis software. As with LDH data, fluorescence intensity was scaled to that in sister cultures treated with NMDA 300 μM for 40 h, which kills all neurons. Propidium iodide fluorescence was not observed in cells that had an astrocyte phenotype after treatment with NMDA or hemin at the concentrations used in this study. Abbreviations DHR: dihydrorhodamine; DNP: dinitrophenylhydrazone; HO: heme oxygenase; LDH: lactate dehydrogenase; MEM10: minimal essential medium containing 10 mM glucose; NMDA: N-methyl-D-aspartate; PI: propidium iodide; ROS: reactive oxygen species. Authors' contributions RFR designed the study, collected and analyzed data, and wrote the manuscript. JC also participated in data collection and analysis, and edited the manuscript. LBZ participated in genotyping and edited the manuscript. All authors reviewed and approved the final manuscript. Acknowledgements This study was supported by a grant from the National Institutes of Health (1RO1NS042273) and from the Pennsylvania/Delaware affiliate of the American Heart Association. We thank Dr. Frank Sharp for providing the HO-2 knockout mice that were used to establish our colony. Figures and Tables Figure 1 Morphologic changes in wild type and knockout cultures exposed to hemin. Phase contrast photomicrographs of cultures exposed for 14 h to: A) Experimental medium (MEM10) only; neurons rest on a monolayer of astrocytes, and aggregate in groups which send out an array of processes; B) hemin 3 μM, wild type culture; most neurons and processes have degenerated; C) hemin 3 μM, knockout culture; neurons with intact processes persist. Figure 2 Heme oxygenase-2 gene deletion attenuates the neurotoxic effect of hemin. Cultures were treated with indicated concentrations of hemin for 14 h. Injury was assessed by A) LDH activity in the medium, which is scaled to that in sister cultures treated with 300 μM NMDA for 40 h (= 100), which releases essentially all neuronal LDH; B) fluorescence intensity of cultures stained with propidium iodide, again scaled to that in sister cultures treated with NMDA. *P < 0.05, ***P < 0.001 v. knockout cultures treated with same concentration of hemin, Bonferroni multiple comparisons test. Figure 3 Heme oxygenase-2 gene deletion reduces production of cellular reactive oxygen species, assessed with dihydrorhodamine 123. Cultures were treated with 3 μM hemin for 4 h. Hemin was then washed out, and cultures were treated with 20 μM dihydrorhodamine 123 for 15 min and imaged. A) Wild type culture; fluorescence is localized to neuronal cell bodies and processes; B) Knockout culture; fluorescence is markedly diminished. Fluorescence intensity is quantified in arbitrary units. Figure 4 Heme oxygenase-2 gene deletion reduces protein oxidation in hemin-treated cultures. Top: Representative immunoblot of protein lysates from wild type (WT) or HO-2 knockout (KO) neuron/astrocyte cultures treated for 4 h with indicated hemin concentrations, stained with anti-DNP antibody to detect derivatized carbonyl groups. M: molecular weight standard with attached DNP residues. Bottom: The mean protein carbonyl signal intensity (± SEM, n = 5/condition) was normalized to that in wild-type cultures exposed to culture medium only (= 1.0) *P < 0.05; ***P < 0.001 versus signal in wild type cultures exposed to the same hemin concentration, Bonferroni multiple comparisons test. Figure 5 Effect of iron chelators on hemin toxicity. Wild-type cultures were treated with 3 μM hemin for 14 h, alone or with indicated concentrations of deferoxamine (DFO) or phenanthroline (PHE). Injury was assessed by A) LDH activity in the culture medium, which is scaled to that in sister cultures treated with 300 μM NMDA for 40 h (= 100), which releases essentially all neuronal LDH; B) fluorescence intensity of cultures stained with propidium iodide, again scaled to that in sister cultures treated with NMDA. ***P < 0.001 v. cultures treated with hemin only, Bonferroni multiple comparisons test. ==== Refs Letarte PB Lieberman K Nagatani K Haworth RA Odell GB Duff TA Hemin: levels in experimental subarachnoid hematoma and effects on dissociated vascular smooth muscle cells J Neurosurg 1993 79 252 255 8331409 Koeppen AH Dickson AC Smith J Heme oxygenase in experimental intracerebral hemorrhage: the benefit of tin-mesoporphyrin J Neuropathol Exp Neurol 2004 63 587 597 15217087 Trakshel GM Kutty RK Maines MD Resolution of rat brain heme oxygenase activity: absence of detectable amount of the inducible form (HO-1) Arch Biochem Biophys 1988 260 732 739 3124761 Stocker R Yamamoto Y McDonagh AF Glazer AN Ames BN Bilirubin is an antioxidant of possible physiologic significance Science 1987 235 1043 1046 3029864 Doré S Takahashi M Ferris CD Hester LD Guastella D Snyder SH Bilirubin, formed by activation of heme oxygenase-2, protects neurons against oxidative stress injury Proc Natl Acad Sci USA 1999 96 2445 2450 10051662 10.1073/pnas.96.5.2445 Taille C El-Benna J Lanone S Dang MC Ogier-Denis E Aubier M Boczkowski J Induction of heme oxygenase-1 inhibits NAD(P)H oxidase activity by down-regulating cytochrome b558 expression via the reduction of heme availability J Biol Chem 2004 279 28681 28688 15123630 10.1074/jbc.M310661200 Balla G Jacob HS Balla J Rosenberg M Nath K Apple F Eaton JW Vercellotti GM Ferritin: A cytoprotective strategem of endothelium J Biol Chem 1992 267 18148 18153 1517245 Wagner KR Hua Y de Courten-Myers GM Broderick JP Nishimura RN Lu SY Dwyer BE Tin-mesoporphyrin, a potent heme oxygenase inhibitor, for treatment of intracerebral hemorrhage: in vivo and in vitro studies Cell Mol Biol (Nolsy-le-grand) 2000 46 597 608 Huang FP Xi G Keep. 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Evidence consistent with a site-specific hydroxyl radical generation caused by iron ions bound to the deoxyribose molecule J Inorg Chem 1987 29 289 299 Regan RF Kumar N Gao F Guo YP Ferritin induction protects cortical astrocytes from heme-mediated oxidative injury Neuroscience 2002 113 985 994 12182902 10.1016/S0306-4522(02)00243-9 Goldstein L Teng ZP Zeserson E Patel M Regan RF Hemin induces an iron-dependent, oxidative injury on human neuron-like cells J Neurosci Res 2003 73 113 121 12815715 10.1002/jnr.10633 Dennery PA Spitz DR Yang G Tatarov A Lee CS Shegog ML Poss KD Oxygen toxicity and iron accumulation in the lungs of mice lacking heme oxygenase-2 J Clin Invest 1998 101 1001 1011 9486970 Dennery PA Visner G Weng Y-H Nguyen X Lu F Zander D Yang G Resistance to hyperoxia with heme oxygenase-1 disruption: role of iron Free Radic Biol Med 2003 34 124 133 12498987 10.1016/S0891-5849(02)01295-9 Liu R Liu W Doctrow SR M. Baudry Iron toxicity in organotypic cultures of hippocampal slices: role of reactive oxygen species J Neurochem 2003 85 492 502 12675926 Miro O Casademont J Urbanomarquez A Cardellach F Mitochondrial cytochrome C oxidase inhibition during carbon monoxide poisoning Pharmacology and Toxicology 1998 82 199 202 9584335 Silva RF Rodriguez CM Brites D Rat cultured neuronal and glial cells respond differently to toxicity of unconjugated bilirubin Pediatr Res 2002 51 535 541 11919342 Shaklai N Shviro Y Rabizadeh E Kirschner-Zilber I Accumulation and drainage of hemin in the red cell membrane Biochim Biophys Acta 1985 821 355 366 4063370 10.1016/0005-2736(85)90106-3 Regan RF Panter SS Neurotoxicity of hemoglobin in cortical cell culture Neurosci Lett 1993 153 219 222 8327197 10.1016/0304-3940(93)90326-G Doré S Sampei K Goto S Alkayed NJ Guastella D Blackshaw S Gallagher M Traystman RJ Hurn PD Koehler RC Snyder SH Heme oxygenase-2 is neuroprotective in cerebral ischemia Mol Med 1999 5 656 663 10602774 Nedergaard M Ransom B Goldman SA New roles for astrocytes: Redefining the functional architecture of the brain Trends Neurosci 2003 26 523 530 14522144 10.1016/j.tins.2003.08.008 Regan RF Guo YP Kumar N Heme oxygenase-1 induction protects murine cortical astrocytes from hemoglobin toxicity Neurosci Lett 2000 282 1 4 10713382 10.1016/S0304-3940(00)00817-X Ewing JF Maines MD In situ hybridization and immunohistochemical localization of heme oxygenase-2 mRNA and protein in normal rat brain: differential distribution of isozyme 1 and 2 Mol Cell Neurosci 1992 3 559 570 Matz P Turner C Weinstein PR Massa SM Panter SS Sharp FR Heme-oxygenase-1 induction in glia throughout rat brain following experimental subarachnoid hemorrhage Brain Res 1996 713 211 222 8724993 10.1016/0006-8993(95)01511-6 Poss KD Thomas MJ Ebralidze AK TJ OD Tonegawa S Hippocampal long-term potentiation is normal in heme oxygenase-2 mutant mice Neuron 1995 15 867 873 7576635 10.1016/0896-6273(95)90177-9 Regan RF Choi DW The effect of NMDA, AMPA/kainate, and calcium channel antagonists on traumatic cortical neuronal injury in culture Brain Res 1994 633 236 242 7907934 10.1016/0006-8993(94)91544-X Royall JA Ischiropoulos H Evaluation of 2',7'-dichlorofluorescin and dihydrorhodamine 123 as fluorescent probes for intracellular H2O2 in cultured endothelial cells Arch Biochem Biophys 1993 302 348 355 8387741 10.1006/abbi.1993.1222 Regan RF Rogers B Delayed treatment of hemoglobin neurotoxicity J Neurotrauma 2003 20 111 120 12614593 10.1089/08977150360517236 Koh JY Choi DW Vulnerability of cultured cortical neurons to damage by excitotoxins: Differential susceptibility of neurons containing NADPH-diaphorase J Neurosci 1988 8 2153 2163 3385492

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BMC Neurosci. 2004 Sep 17; 5:34

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==== Front BMC Infect DisBMC Infectious Diseases1471-2334BioMed Central London 1471-2334-4-351536733510.1186/1471-2334-4-35Research ArticleNosocomial outbreak of neonatal Salmonella enterica serotype Enteritidis meningitis in a rural hospital in northern Tanzania Vaagland Hogne [email protected] Bjørn [email protected]üger Carsten [email protected] Naftali [email protected] Roland [email protected] Nina [email protected] Department of Otolaryngology/Head & Neck Surgery, Haukeland University Hospital, N-5021 Bergen, Norway2 Centre for International Health, University of Bergen, N-5021 Bergen, Norway3 Institute of Medicine, University of Bergen, N-5021 Bergen, Norway4 Haydom Lutheran Hospital, Mbulu, Tanzania5 Deipe Stegge 71, D-48653 Coesfeld, Germany6 Department of Medicine, Haukeland University Hospital, N-5021 Bergen, Norway2004 14 9 2004 4 35 35 27 6 2004 14 9 2004 Copyright © 2004 Vaagland et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Clinicians at Haydom Lutheran Hospital, a rural hospital in northern Tanzania noted an unusually high case-fatality rate of pediatric meningitis and suspected an outbreak of an unknown agent or an organism resistant to the empirical therapy. Methods We established a provisional microbiology laboratory to investigate the suspected outbreak. Blood and spinal fluid specimens were taken from children below the age of seven years with suspected meningitis. The blood and spinal fluid specimens were inoculated in commercial blood culture bottles and locally prepared Thayer-Martin medium in slanted tubes, respectively. The bacterial isolates were sent to Norway for further investigation, including susceptibility testing and pulsed-field gel-electrophoresis (PFGE). Results Among 24 children with suspected meningitis and/or septicemia, five neonates had meningitis caused by Salmonella enterica serotype Enteritidis, all of whom died. Two children had S. Enteritidis septicemia without meningitis and both survived. Genotyping with PFGE suggested a clonal outbreak. The salmonella strain was resistant to ampicillin and sensitive to gentamicin, the two drugs commonly used to treat neonatal meningitis at the hospital. Conclusion The investigation reminds us that nontyphoidal salmonellae can cause meningitis associated with very high case-fatality rates. Resistance to multiple antimicrobial agents increases the risk of treatment failure and may have contributed to the fatal outcome in all of the five patients with salmonella meningitis. The investigation indicated that the outbreak was nosocomial and the outbreak subsided after hygienic measures were instituted. Establishing a provisional microbiological laboratory is a valuable and affordable tool to investigate and control outbreaks even in remote rural areas. ==== Body Background Nontyphoidal salmonellae are a common cause of food-borne illnesses. In Africa, nontyphoidal salmonellae are the most common cause of bloodstream infections in children younger than five years [1]. While meningitis caused by nontyphoidal salmonellae is uncommon in economically developed countries [2], it is more frequent in tropical countries, particularly in children younger than six months, and associated with higher case-fatality rates than meningitis caused by other bacteria [1,3-5]. At Haydom Lutheran Hospital, a rural hospital in northern Tanzania, clinicians noted an extraordinarily high case-fatality rate (>60%) from pediatric meningitis in the period January 1998 to April 2000. It was suspected that the high case-fatality rate of meningitis was due to an outbreak of an unusual etiological agent, or an organism resistant to the hospital's standard empirical treatment, which was ampicillin and gentamicin for infants (<2 months) and penicillin and chloramphenicol for older children. Thus, we established a provisional microbiology laboratory to identify the causative agents and to facilitate the implementation of effective preventive measures. Methods From a total of 360 children admitted from July to August 2000, blood and/or spinal fluid specimens were collected from 24 children aged one day to six years (median age 23.5 days) with suspected meningitis and/or septicaemia, after careful evaluation by the attending pediatrician. Blood and spinal fluid specimens were inoculated in BBL SeptiChek blood-culture bottles (Becton Dickinson, Sparks, MD USA) and on locally prepared non-selective Thayer-Martin medium in slanted tubes, respectively. All cultures were incubated at 35°C for 5 days and inspected daily for bacterial growth. Positive bacterial specimens were shipped to Institute of Medicine, Haukeland University Hospital, Norway, for further study. The total cost of the laboratory reagents used on site was $475. Positive specimens were identified using standard laboratory methods [6]. The susceptibilities of the isolates to antimicrobial agents were examined by disk diffusion method on PDM medium (AB Biodisk, Solna, Sweden) [7]. The isolates were genotyped with pulsed-field gel electrophoresis (PFGE). Statistics were calculated with Stata 8 for Mac OSX (Stata Corporation, College Station, TX). Results On clinical grounds, twenty-four children were considered to have possible sepsis or meningitis. Blood culture was taken from 23 children, but 13 had received prior antibiotic treatment. Spinal fluid culture was taken from 16 children, of whom ten had received prior antibiotic treatment. Both blood culture and spinal fluid culture was obtained from a total of 15 children. Salmonella enterica serotype Enteritidis was isolated from seven patients, of whom four had positive cultures from both blood and spinal fluid and three from blood only. One of the three patients, who had S. Enteritidis isolated only from blood culture, had pus in the spinal fluid, and was therefore considered a case of S. Enteritidis meningitis, resulting in a total of five cases of S. Enteritidis meningitis. One isolate each of Staphylococcus aureus and Streptococcus pyogenes were isolated from spinal fluid and three isolates of coagulase-negative staphylococci were isolated from blood. Antimicrobial susceptibility testing showed that all the S. Enteritidis isolates were resistant to chloramphenicol and ampicillin, intermediate resistant to cefuroxime, and sensitive to gentamicin, cefotaxime and ciprofloxacin. As shown in Figure 1, the PFGE patterns were identical for all the eleven S. Enteritidis isolates except one, which differed by only one band (Figure 1 shows a total of 17 strains, 6 of which are duplicate isolates from the same patients). All children with S. Enteritidis infections were neonates (median age 15 days, range: 8 to 27 days), whereas those with other infections or negative cultures, on average, were older (median age 4 months, range: 1 day to 6 years). All children with S. Enteritidis infections had been delivered at Haydom Lutheran Hospital, and five out of the seven never left the hospital before they became ill. Table 1 shows the characteristics of the patients with S. Enteritidis infection. All five children with S. Enteritidis meningitis died. The two children with S. Enteritidis sepsis survived. Five (29%) of the 17 children without verified S. Enteritidis infection also died. Among the 24 children investigated, S. Enteritidis meningitis was associated with a relative risk of 3.8 (95% confidence interval 1.8 to 8.1) for fatal outcome. Discussion The finding of an outbreak of bacteremia and meningitis caused by S. Enteritidis was not anticipated. Genotyping with PFGE suggested a clonal outbreak. This genotyping information, the susceptibility patterns and the clinical information that all children with S. Enteritidis infections were born at the hospital and that the majority never left the hospital before they became ill, strongly suggest that the outbreak was nosocomial. Nontyphoidal salmonella infections are frequently associated with animal reservoirs and infection usually originates from food products. Nosocomial spread of nontyphoidal salmonellae, particularly in neonatal wards, is known from the literature [8]. Neonates are at particular risk of infection because of relatively reduced gastric acidity and peristalsis [1]. In previous nosocomial outbreaks caused by salmonellae, the sources of infection have been related to the use of contaminated medications, diagnostics, blood products, banked human milk, the use of raw eggs or yeast in tube feeding and improper disinfection of devices such as rubber tubes for oropharyngeal suction [8,9]. The exact source of the outbreak at the hospital was not established. Direct food-borne transmission was unlikely, since the neonates at the hospital were fed on breast milk. Possible sources include contaminated instruments, clothes or bathing facilities for the newborn. Spread of infection may have occurred by baby-to-baby transmission or via family members and/or hospital staff. The medical staff at the hospital was informed about the findings, and immediate interventions in the form of hygienic measures were instituted, including the reinforcement of disinfection and hand-washing practices. Data from the hospital annual reports shows that the case-fatality rate from pediatric meningitis dropped from >60% before the intervention to 40% by 2001. The S. Enteritidis strain responsible for the outbreak was resistant to two of the first-line drugs, ampicillin and chloramphenicol, but sensitive to gentamicin. This finding is not surprising, since multi-drug-resistant S. Enteritidis has been reported from this region for decades [10]. The children at the hospital were treated with a combination of ampicillin and gentamicin. The high case-fatality rate in these patients implies that de facto monotherapy with gentamicin may be suboptimal as treatment for S. Enteritidis meningitis considering that gentamicin traverses the blood-brain-barrier poorly, is bound to proteins and inhibited by the acidity in infected cerebrospinal fluid. However, it also reflects the poor prognosis of neonatal S. Enteritidis meningitis regardless of therapy. Molyneux reported fatal outcome for 58% of children with meningitis caused by nontyphoidal salmonellae despite routine treatment with chloramphenicol, to which all bacterial isolates were sensitive in vitro [4]. Many authorities recommend third-generation cephalosporins as empirical chemotherapy for meningitis caused by gram-negative bacteria [11], not only because of high bactericidal activity due to low minimum inhibitory concentrations (MICs), but also because they penetrate the blood-brain-barrier better than both gentamicin and chloramphenicol. A third-generation cephalosporin, such as cefotaxime would have been an excellent therapeutic option in this case. However, the price of the newer cephalosporins is often prohibitive in the setting of low-income countries such as Tanzania. Ciprofloxacin is generally not recommended for use in children due to potential adverse effects, but can be resorted to for treatment of life-threatening infections with multidrug-resistant nontyphoidal salmonellae [12]. The standard empirical treatment regimen at the hospital could not be changed due to financial constraints. Infections caused by nontyphoidal salmonellae in children in Africa are more common during the rainy season and have been associated with malaria, anemia and malnutrition [1]. However, there is considerable overlap between these medical conditions, all of which may be more frequent during the rainy season. This investigation was performed during the dry season. Infections caused by nontyphoidal salmonellae have also been associated with HIV infection [1]. We do not know the HIV status of the children involved in this outbreak, however, in this part of Tanzania, the rate of HIV infection is less than 0.5% [13]. Conclusions There are a number of lessons to be learned from this investigation. We are reminded that S. Enteritidis can cause meningitis, which carries a very high case-fatality rate [1,3,4]. Our findings support former reports that S. Enteritidis can easily spread by nosocomial transmission, particularly in neonatal wards. Resistance to multiple antimicrobial agents increases the risk that empirical therapy will fail, especially in settings where modern cephalosporins are not affordable. Without adequate laboratory facilities, correct diagnosis and treatment of bacterial meningitis and septicemia in children remains a challenge. However, the report also shows that establishing a provisional microbiology laboratory can be a valuable and affordable tool to investigate and curb epidemics even in the setting of remote rural Africa, provided there is a proficient laboratory willing to assist. Competing interests None declared. Authors' contributions HV was the principal investigator, participated in the planning and execution of the work, including performing the on-site microbiological procedures, and analysis of data, and was the main responsible author. BB participated in planning, microbiological investigations in Norway, data analysis and writing. CK and NN undertook clinical investigation and sample collection and participated in the writing. RJ was responsible for the microbiological investigations in Norway and participated in the data analysis and writing. NL was the project coordinator and participated in planning, data analysis and writing. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: Acknowledgements We thank all nursing and medical staff at Haydom Lutheran Hospital who cared for the children and facilitated this work and Dr Bjørg Evjen Olsen, who provided important information, including the relevant hospital annual reports. We are particularly grateful to the director of the hospital Dr Ole Hallgrim Evjen Olsen for his willingness and interest in having this investigation undertaken. Figures and Tables Figure 1 Pulsed-field gel-electrophoresis (PFGE) of digested DNA from 17 isolates of Salmonella enterica serotype Enteritidis obtained from seven children with meningitis and/or septicemia. The isolates were obtained from blood cultures of all seven children and spinal fluid culture of four children. Six of the isolates in the gel are duplicate isolates from the same children. Table 1 Characteristics of neonates with meningitis (n = 5) and sepsis (n = 2) caused by Salmonella enterica serovar Enteritidis at Haydom Lutheran Hospital, Northern Tanzania, in July and August 2000 Patient characteristic Pat. 1 Pat. 2 Pat. 3 Pat. 4 Pat. 5 Pat. 6 Pat. 7 Sex F F M F F F M Mode of delivery SVD SVD Sectio SVD SVD SVD SVD Age at onset 8 d 7 d 8 d 15 d 24 d 14 d 13 d Age at discharge (*death) 20 d 21 d 32 d* 21 d* 29 d* 28 d* 17 d* Birth weight 2350 g 4000 g 3300 g 2620 g 3600 g ND 2800 g Fever + - + + + + - Neck stiffness - - - - - - - Fits - - - + + + + Bulging fontanel - - - + + - - S. Enteritidis in blood culture + + + + + + + S. Enteritidis in CSF culture ND - ND + + + + CSF WBC ND 10/μL Pus Pus >3000/μL ND 32/μL Clinical diagnosis  - Meningitis - - + + + + +  - Septicemia + + + - - + +  - Malaria + + - - + - - Outcome Alive Alive Death Death Death Death Death Pat. = Patient, F = Female, M = Male, SVD = Spontaneous vaginal delivery, d = Days, "+" = Present, "-" = Absent, CSF = Cerebrospinal fluid, ND = No data, WBC = White blood cells ==== Refs Graham SM Molyneux EM Walsh AL Cheesbrough JS Molyneux ME Hart CA Nontyphoidal Salmonella infections of children in tropical Africa Pediatr Infect Dis J 2000 19 1189 1196 11144383 Saphra I Winter JW Clinical manifestations of salmonellosis in man; an evaluation of 7779 human infections identified at the New York Salmonella Center N Engl J Med 1957 256 1128 1134 13452006 Barclay N High frequency of salmonella species as a cause of neonatal meningitis in Ibadan, Nigeria. A review of thirty-eight cases Acta Paediatr Scand 1971 60 540 544 5209324 Molyneux EM Walsh AL Malenga G Rogerson S Molyneux ME Salmonella meningitis in children in Blantyre, Malawi, 1996-1999 Ann Trop Paediatr 2000 20 41 44 10824212 10.1080/02724930092057 Sirinavin S Chiemchanya S Vorachit M Systemic nontyphoidal Salmonella infection in normal infants in Thailand Pediatr Infect Dis J 2001 20 581 587 11419499 10.1097/00006454-200106000-00007 Murray PR Baron EJ Jorgensen JH Pfaller MA Yolken RH Manual of Clinical Microbiology 2003 8 Washington D. C., ASM Press Bergan T Bruun JN Digranes A Lingaas E Melby KK Sander J Susceptibility testing of bacteria and fungi. Report from "the Norwegian Working Group on Antibiotics" Scand J Infect Dis Suppl 1997 103 1 36 9253777 Khan MA Abdur-Rab M Israr N Ilyas M Ahmad F Kundi Z Ghafoor A Transmission of Salmonella worthington by oropharyngeal suction in hospital neonatal unit Pediatr Infect Dis J 1991 10 668 672 1923680 Weikel CS Guerrant RL Nosocomial salmonellosis Infect Control 1985 6 218 220 3894270 Petat E Carteron B Reguer M Lemmens P Vandepitte J Ghysels G Étude des Shigella et Salmonella isolées au Burundi de 1980 a 1985 Bull Soc Pathol Exot Filiales 1987 80 171 179 3608004 Quagliarello VJ Scheld WM Treatment of bacterial meningitis N Engl J Med 1997 336 708 716 9041103 10.1056/NEJM199703063361007 Wessalowski R Thomas L Kivit J Voit T Multiple brain abscesses caused by Salmonella enteritidis in a neonate: successful treatment with ciprofloxacin Pediatr Infect Dis J 1993 12 683 688 8105442 Hinderaker SG Krüger C Olsen BE Naman N Bergsjo P Olsen OHE Low HIV-seroprevalence in pregnant women in a rural area in Tanzania Acta Obstet Gynecol Scand 2001 80 1152 1153 11846719 10.1034/j.1600-0412.2001.801218.x

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BMC Infect Dis. 2004 Sep 14; 4:35

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==== Front BMC BiotechnolBMC Biotechnology1472-6750BioMed Central London 1472-6750-4-211535787210.1186/1472-6750-4-21Methodology ArticleA high-throughput cell migration assay using scratch wound healing, a comparison of image-based readout methods Yarrow Justin C [email protected] Zachary E [email protected] Nicholas J [email protected] Timothy J [email protected] Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA2 The Institute of Chemistry and Cell Biology (ICCB), Harvard Medical School, Boston, MA 02115, USA3 School of Chemistry and Centre for Biomolecular Sciences, University of St Andrews, North Haugh, St Andrews, UK2004 9 9 2004 4 21 21 6 5 2004 9 9 2004 Copyright © 2004 Yarrow et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Cell migration is a complex phenomenon that requires the coordination of numerous cellular processes. Investigation of cell migration and its underlying biology is of interest to basic scientists and those in search of therapeutics. Current migration assays for screening small molecules, siRNAs, or other perturbations are difficult to perform in parallel at the scale required to screen large libraries. Results We have adapted the commonly used scratch wound healing assay of tissue-culture cell monolayers to a 384 well plate format. By mechanically scratching the cell substrate with a pin array, we are able to create characteristically sized wounds in all wells of a 384 well plate. Imaging of the healing wounds with an automated fluorescence microscope allows us to distinguish perturbations that affect cell migration, morphology, and division. Readout requires ~1 hr per plate but is high in information content i.e. high content. We compare readouts using different imaging technologies, automated microscopy, scanners and a fluorescence macroscope, and evaluate the trade-off between information content and data acquisition rate. Conclusions The adaptation of a wound healing assay to a 384 well format facilitates the study of aspects of cell migration, tissue reorganization, cell division, and other processes that underlie wound healing. This assay allows greater than 10,000 perturbations to be screened per day with a quantitative, high-content readout, and can also be used to characterize small numbers of perturbations in detail. ==== Body Background When wounded or scratched, cell monolayers respond to the disruption of cell-cell contacts and an increased concentration of growth factors at the wound margin by healing the wound through a combination of proliferation and migration [1-3]; these processes reflect the behavior of individual cells as well as the properties of the cell sheet as a surrogate tissue. To perform a wound healing assay, a wound is typically introduced in a cell monolayer using an object such as a pipette tip or syringe needle and the assay is performed on an individual coverslip or in a multiwell plate. The monolayers recover and heal the wound in a process that can be observed over a timecourse of 3–24 hrs. The wound heals in a stereotyped fashion – cells polarize toward the wound, initiate protrusion, migrate, and close the wound. Progression of these events can be monitored by manually imaging samples fixed at timepoints or by time-lapse microscopy. Wound healing assays are a classic and commonly used method for studying cell migration and the biology underlying it [4]. They have been used with multiple cell types and, as the monolayers heal the wound in a characteristic manner, they have been used to study cell polarization, matrix remodeling, cell migration, and numerous other processes [5-7]. Wound healing assays have been used both for detailed cell biological studies and for the discovery and validation of small molecule leads and other perturbations that affect cell migration [8-11]. The role of the Rho family GTPases, Rac, Rho, and Cdc42, in the establishment of polarity and the regulation of actin cytoskeletal structures has been studied using wound healing [12-14], as has the role of p53 in migration [15], and orientation of the microtubule organization center (MTOC) and the Golgi apparatus [16-18]. The assay has also been used as a proxy for angiogenesis, metastasis, and other physiological and pathophysiological processes [19-24]. In order to perform high throughput screening of cell migration, we developed a wound healing assay in a 384 well plate format that does not require expensive reagents, provides consistently shaped wounds, can provide detailed information on numerous processes involved in cell migration, and provides a quantitative, information-rich readout. We use multiple imaging technologies to assay the results and compare their relative merits. Results Adaptation of wound healing to a 384 well format For the development of a high-throughput wound healing assay we chose to use BS-C-1 cells, a cell type with a classic wound healing response on glass coverslips [Figure 1A]. BS-C-1 cells were seeded in clear-bottom 384 well plates at high density and allowed to form monolayers overnight. We found that wound healing was observable between 3 and 24 hrs after wounding with a pipette tip or syringe needle. Significant cell migration could be seen at 3 hrs with lamella and protrusions at the wound margin. After 7 hrs, cell migration could be observed easily with a low magnification (4×) objective, and after 24 hrs wounds were completely healed [Figure 1B, Additional file: 1]. Because of the ease of distinguishing phenotypes at 7 and 24 hrs, we have used these timepoints for the assay. To adapt this assay for parallel screening, we needed a method for introducing uniformly sized wounds in the same position of each well. We used a 96 well floating-pin transfer device – a tool primarily used for the transfer of solutions between plates. A floating pin array, with foam padding placed between the top plate and the pins, provides an adaptive stop to pin height and overcomes problems with plate planarity. We have adapted a 24-channel aspirator in the same manner for small-scale work. To wound all 384 wells in a plate, the 96 well pin array is placed in the corner of a well, pushed down to engage all pins with the surface of the plate, and then moved laterally to produce the wound. This is then repeated in three neighboring wells to cover the plate and produce uniform wounds throughout [Figure 1C]. After the cells were wounded, we introduced perturbations to individual wells (in our case, small molecules). We then allowed the cells to recover for 7 hrs or 24 hrs before fixing, staining, and imaging each plate. Comparison of imaging technologies We used four different imaging technologies to analyze the results of this assay, each with distinct advantages and drawbacks [summarized in Table 1]. We will discuss these approaches in order of image resolution, from highest to lowest: automated fluorescence microscopy after 7 hrs recovery, fluorescence and transmitted-light scanners after 7 hrs, and a fluorescence macroscope after 24 hrs. Automated microscopy For highest resolution imaging of the assay we chose an end point of 7 hrs, when migration can be clearly seen, and used an automated fluorescence microscope to image individual wells after fixing and staining for filamentous actin and DNA [Figure 1D]. The microscope is a standard inverted fluorescence instrument. Augmented with an x-y stage, it moves between plate wells and a piezoelectric z-motor on the objective gives a focused image. Capturing images with a 4× objective provided sufficient resolution to determine the extent of migration and the morphology of the cells at the wound margin. From these data, we defined four distinct phenotypes [Figure 1D]. A control well shows polarization of the cells toward the wound and concerted migration of the cell sheet, with neighboring cells connected and moving together into the wound. Wells showing decreased migration or aberrant morphology are readily apparent by visual inspection as are wells showing an increase in the number of mitotic cells, which manifest as bright spheres (in the actin channel) in an otherwise intact and adherent monolayer [Figure 1D]. Phenotypes that cause disruption of the monolayer, are considered toxic though we have not shown them to be. Using automated microscopy, the image resolution is relatively high and the time required to image an entire plate is relatively long. Imaging one 384-well plate takes ~1 hr at 4× magnification. At 10× magnification, ~1.5 hrs are required per plate because at higher magnification two images per well must be taken to ensure that the wound edge is captured. We found it more informative to observe the entirety of the wound at 4× rather than parts of it at 10×, despite the higher resolution in the latter case. Scanners A fluorescence scanner can be used to determine the extent of cell migration at the 7 hr timepoint. By staining filamentous actin and using a fluorescence scanner with a 42 μm/pixel resolution setting, we can observe consistent differences between normal and inhibited migration. Control wells show a veil of less-densely stained, migrating cells that extend into the wound with a concomitant decrease in wound width [Figure 2A]. Titration of a compound that blocks cell migration (the actin inhibitor cytochalasin D) shows complete inhibition of migration at 1 μM, as seen by a sharply delineated wound edge and a wider wound width [Figure 2A]. A simple transmitted-light scanner, normally used for scanning documents and costing less than $1,200, can also be used to monitor wound healing at 7 hrs and only requires that cells are stained with a dye. Figure 2B shows images of wells stained with Coomassie Brilliant Blue. Wells treated with cytochalasin D show inhibition of wound healing similar to that observed with the fluorescence scanner. Inhibited wells stain darkly at the wound margin while normal migration can be seen by a more diffuse wound margin, denoting migrating cells [Figure 2B]. In both cases, scanners do not provide specific information on cell morphology or other subtle effects in the 7 hr assay; however, acquisition time is greatly decreased. For the fluorescence scanner, acquisition time is 26 minutes at the resolution and image quality shown (42 μm/pixel and medium quality). For the conventional scanner, acquisition time is 8.5 minutes (at 10 μm/pixel, 2400 dpi) and cell staining with Coomassie Brilliant Blue takes only 10 minutes. Macroscope – Tundra or LeadSeeker The lowest resolution imaging technology that we tested, a fluorescence macroscope had a resolution of ~100 μm/pixel. Detecting wound healing at this resolution required an incubation time of 24 hours after wounding as differences are not readily seen with this method at the 7 hr timepoint. Cells are wounded and allowed to recover for 24 hours before fixing, staining for filamentous actin, and imaging. The macroscope captures an image of the entire plate. At this magnification and resolution, an unhealed wound is seen as a non-staining, black streak within the monolayer [Figure 3]. In contrast, wounds that have healed completely are seen as lower-intensity, grey streaks. The time required for acquisition of images using this technique, is limited only by the fluorescence signal and was typically ~5 seconds per plate. Automated image analysis of wound healing images With all of the imaging techniques discussed here, we initially scored the assay by visual inspection. Visual inspection is fast, information-rich, and can distinguish subtle effects. This method proved useful during adaptation and optimization of the assay in high-throughput format. However, screening large numbers of perturbations by visual inspection is limited by subjectivity, operator fatigue, and the lack of quantifiable metrics. Thus, we developed an automated image analysis routine to provide a rapid and quantitative measurement of the wound healing assay with images captured using the automated microscope (automated methods for the other readouts could also be developed). Using images captured by automated microscopy with a 4× objective, we are able delineate measurable characteristics of the wound. After 7 hrs recovery, actin staining defines the extent of healing as well as the morphology of the cells and, because of cell polarization, DNA staining defines the approximate starting point of the migrating cells [Figure 4A]. Applying a standard set of image processing filters to threshold the image, we defined the area of the wound not staining for nuclei and the area not staining for actin. The difference between these two areas defines an annulus containing the lamella of the cells at the wound front (hereafter, lamellar region) [Figure 4A]. With the lamellar region defined, we can extract several metrics that characterize migration. Measurements that have been useful in quantifying the phenotypes of migration inhibition and aberrant morphology include: area of lamellar region, width of lamellar region, and the smoothness of the wound margin. Automated analysis and visual inspection are complementary approaches. To illustrate this, we compare the two approaches using the results from one 384 well plate of a screen for small molecules that affect wound healing [Figure 4B]. The automated analysis values for each well are plotted and the average lamellar width from control wells is marked as a black line, with three standard deviations above and below that average marked as red dashed lines. Visual inspection of this same plate is illustrated on the graph by color-coding each well. Wells where migration is inhibited or morphology affected are shown in green; compounds that affect the well in other ways – wells with fewer cells, increased mitotic index, or disrupted monolayers – are shown in red; and compounds that show no significant deviation from control are shown in blue. As can be seen, the automated analysis picks up 4 wells that show inhibition of wound healing with lamellar widths more than 3 standard deviations from the mean and with less stringent bounds (2 standard deviations) 7 compounds are found to inhibit. Other compounds identified by visual inspection were not picked up by automated analysis. Validation of wound healing as a high-throughput assay We have used methods described in this paper (notably, automated microscopy) to screen ~1,000 bioactive and ~20,000 random drug-like compounds. We were able to reproducibly identify compounds that affected wound healing with different effects including: inhibiting migration, affecting morphology, blocking completion of mitosis, and disrupting the cell monolayer. The details of these results will be presented elsewhere. Discussion We present a cell migration assay in a 384 well plate format through the adaptation of tissue culture cell wound healing. We have also compared the readouts provided by four distinct imaging techniques technologies – automated microscopy, fluorescence and transmitted-light scanners, and a fluorescence macroscope – for their relative acquisition speed, image resolution, and information content. High-content image-based screening is often performed at high magnification, however, we have found that low magnification images are information-rich and can be sufficient for observation of detailed phenomena. New screens utilizing imaging technologies are often explicitly developed to image plates at the highest resolution possible given time constraints (10× or higher), rather than at the lowest resolution required to discern differences between wells by visual inspection or automated analysis. Increased magnification further requires acquisition of a greater number of images in order to guard against sampling error. The more sophisticated imaging technologies also carry a hefty price tag. We show here that images taken at magnifications of 4× and lower still allow us to easily discern wells in which wound healing is inhibited and morphology affected. Low magnification imaging is an underutilized method and may be particularly effective for screens that monitor gross changes in protein localization such as nuclear transport, or protein transport from Golgi to plasma membrane; processes in which changes in localization might manifest as changes in the image texture of the stained cell monolayer. While the choice of imaging technologies represents a tradeoff between resolution and information, we think that they should be used together rather than suggesting that they are mutually exclusive. One can iteratively image a given assay plate and take advantage of the best aspects of each technique. For example, a screen for inhibitors of cell migration could initially be performed using a lower resolution technique, taking advantage of the speed to identify interesting wells before imaging just those wells at higher resolution. Related to this, one can re-stain assay plates with different molecular markers. Plates initially screened with a marker that most easily defines the phenotype of interest can be subsequently re-stained with markers that provide different information. In this way, one can consider an assay plate a resource to which one can return to ask new questions. Similar to the tradeoffs seen with different imaging technologies, different image processing techniques like automated image analysis and visual inspection have their distinct advantages but also provide complementary approaches. Our scheme for automated analysis of wound healing allows us to rapidly and easily identify wells in which wound healing is significantly inhibited or morphology clearly affected. However, the automated analysis missed a number of interesting wells that were identified by visual inspection. In part this is because the analysis is based on only a few measures – lamellar width, area, and smoothness. Any automated analysis based on measurable parameters will be limited to phenotypes that manifest along those parameters and might miss more subtle or complex phenotypes that "jump off the screen" when viewed by eye. Automated analysis can also be confounded by artifacts within the plate, dust particles, precipitates, or fluorescent small molecules. Instead of being thought of as a complete answer, automated analysis can enrich for wells that differ from the norm. We don't address automated analysis with the other imaging techniques, but from what can be seen by eye, automated approaches that measure the intensity of staining at the wound edge, or the steepness to which the intensity at the wound edge drops off should provide a good measure of wound healing at very low magnification and resolution. As with any complex phenotypic assay, cell migration during wound healing can be inhibited by effects on global cellular processes – e.g. inhibition of protein translation, disruption of metabolism, disruption of ion homeostasis, etc. – and validation of the specificity of new perturbations is required. Even if the molecular target of the perturbation is unknown, this assay can be adapted or used to distinguish compounds that specifically inhibit or potentiate cell migration. For example, partial inhibition of migration with low concentrations of cytochalasin D could create a sensitized screen for cell migration and used to find suppressors or enhancers. Other complementary approaches include comparative screens across multiple cell lines that migrate differently and the use of specific trophic factors and cognate cell lines such as VEGF and endothelial cells. In our studies, we used still another approach: a series of counter screens that eliminated from our pool of hits, toxic compounds and compounds that do not inhibit processes of interest (Yarrow et. al. unpublished results). This approach has worked well and results will be reported elsewhere. Conclusions The adaptation of a wound healing assay to a 384 well format facilitates the study of aspects of cell migration, tissue reorganization, cell division, and other processes that underlie wound healing. This assay allows greater than 10,000 perturbations to be screened per day with a quantitative, information-rich readout, and can also be used to characterize small numbers of perturbations in detail. Methods Tissue culture and 384 plate preparation BS-C-1 (ATCC CCL-26) cells were grown in DMEM, 10% FCS, and antibiotics. Cells were plated in black 384 well plates with clear bottoms (Corning Costar 3712) at a density of 8500 cells/well in a volume of 50 μl using a liquid dispenser (Labsystems Multidrop). Plates were spun briefly at 500 rpm for ~30 s to in a tabletop centrifuge (Sorval RT7 plus). Cells were incubated overnight (37°C 5% CO2) and wounding was preformed 12 hours later. Wound healing assay, cell fixation, and staining Wound healing was performed using a 96 well floating-pin transfer device with a pin diameter of 1.58 mm coming to a flat point at the tip with a diameter of 0.4 mm (VP Scientific VP-408FH). Foam backing was inserted between the plates of the pin array to provide a resistive stop and the external guide pins were bent to allow greater movement in the z-axis. The pin array was placed in the top corner of a well, pushed down into the plate to engage all pins, and then pulled toward the user. This was repeated in the three neighboring wells to cover all 384. Plates were returned to the tissue culture incubator for 7 or 24 hours before fixation. Cells were fixed after removal of the media with a wand aspirator (VP scientific VP-186L) used along with the Labsystems Multidrop for all liquid handling. Fixation solution (100 mM K-Pipes pH 6.8, 10 mM EGTA, 1 mM MgCl2, 0.2% Triton X-100, 3.7% Formaldehyde) was added as 30 μl and incubated for 15 min. Wells were aspirated and washed 2× with TBS with 0.1% Triton-X 100 (TBS-Tx) and stained. For experiments involving the automated microscope and macroscope, cells were stained in TBS-Tx with TRITC-phalloidin (Sigma P1591) 0.5 μg/ml and Hoechst (Sigma B2261) 0.1 μg/ml as 15 μl per well for 15 minutes. Wells were washed 2× with TBS-Tx and imaged. For experiments using the fluorescence plate scanner, after fixation, cells were incubated in TBS-Tx with 2% BSA (AbDil) for 30 minutes, incubated with mouse anti-actin antibody (Chemicon MAB1501) at 1:10,000 in AbDil for 45 minutes, washed 2× with TBS-Tx, incubated with secondary antibodies appropriate for the plate scanner (Molecular Probes A-21057), washed 2× with TBS-Tx and imaged. For experiments using the transmitted-light scanner, after fixation, cells were incubated with SDS-Page gel staining solution (0.25% Coomassie Brilliant Blue R-250, 50% methanol, 10% acetic acid) for 10 minutes, washed 2× with TBS and imaged. Imaging Automated microscopy We used a NikonTE300 inverted fluorescence microscope with filter wheel (Sutter Lamda10-2), x-y stage (Prior H107N300), and piezoelectric-motorized objective holder (Physik Instrumente P-723.10). Images were captured on a CCD camera (Hammamatsu OrcaER). Metamorph software (Universal Imaging Corporation) running the "Screen Acquisition" drop-in allowed coordination of software-based auto-focusing, movement between wells, imaging, and image evaluation. Images were acquired using a 4× or 10× objective with 2 × 2 binning. Exposure times were ~300 ms for TRITC-phalloidin and ~10 ms for Hoechst. An individual plate took ~1 hr to image at 4× and ~1.5 hrs at 10×. Individual black and white actin images (.tif) were compiled as a .stk file and scrolled through using keystrokes to visually annotate. Visual inspection of 1 plate of images (384) took 10 minutes. Transmitted light scanner An Epson 1680 scanner was used with the positive film setting. A scanning resolution of 1200 dpi (equivalent to 20 μm/pixel) gave an acceptable image with a read time for one plate of 3.5 minute. The image shown in Figure 2 was 2400 dpi (10 μm/pixel) and read time was 8.5 minutes. We also found that using the document scanning mode (with no transmitted light attachment) worked well. In this case, a lamp with a paper diffuser (placed on top of the plate) was used for even illumination. Fluorescence scanner An Odyssey scanner (LiCor) was tested at all combinations of scanning resolution and image quality. Scanning at a resolution setting of 42 um with medium image quality was found to be the optimal balance between speed and image quality. The scanner was used as per the manual, with no modifications. Fluorescence macroscope A Tundra macroscope, (Imaging Research – now available as the Leadseeker from Amersham [25]) a 12 cm telecentric lens with a N/A of 0.45, mounted with a -50°C cooled, thinned, back illuminated CCD camera for image capture, and a motorized stage to hold the plates – all enclosed in a light-tight box. The software was used as per the manual. To image the underside of plates, they were sealed (Corning Costar 6570) while containing TBS and inverted. Exposure times were ~5 seconds. Automated image analysis Wound healing images were analyzed using software written using Visual Basic 6.0 (Microsoft) and Halcon 6.0.1 (MVTec Software) but could be implemented with most basic image analysis software. This software iterates the analysis over images specified by the Metamorph .HTS file and returns values to an Excel spreadsheet. Non-DNA staining region (Additional file: 2A) Hoechst images are convolved with a Laplacian-of-a-Gaussian (σ = 2 pixels) kernel. The resulting image is thresholded for pixels of value 0 and binarized. After a binary morphological opening (an erosion followed by a dilation) with a disc of radius 2.5 pixels, the largest contiguous region that does not touch the image edge is defined, and holes within this region are filled. Non-actin staining region (Additional file: 2B) A Kirsch edge detection filter is applied to actin images and the resulting image is thresholded at a manually set value (changed when needed to account for variation in staining intensity). The largest contiguous region that does not touch the image edge is defined and holes within this binary region are filled. Definition of measures (in pixels) lamellar area: area of annulus defined by the difference between the non-actin staining area and the non-DNA staining area; lamellar width: (lamellar area)/(perimeter length of the non-DNA staining region); lamellar smoothness (Additional file: 2C): (Perimeter of the non-actin staining region)/(perimeter of the morphological closing (dilation followed by erosion) of the non-actin staining region with a disc of radius 10 pixels). Authors' contributions JCY developed the assay and analysis. ZEP wrote the software for automated analysis. NJW initiated a project for in vivo actin cytoskeleton small molecule inhibitors. TJM provided support and enthusiasm for the project. Supplementary Material Additional file 1 Wounds generated with the 96 well floating-pin array heal in a characteristic and measurable manner. (A) Wounds generated with the 96 well floating-pin array healed for 0, 3, 7, 12, or 24 hours, were processed as above, and wound regions were measured using automated analysis. Normalized average values and standard deviations of both the area and median width of the non-actin staining region and the non-DNA staining region (see Methods for definitions) are shown. The non-DNA staining region at the 24 hr timepoint was automatically not measured (asterisks) because the non-actin staining region was 0. At least 24 wells of each condition were measured. Click here for file Additional file 2 Sample images illustrating automated analysis of wound healing images. (A) Processing of DAPI image to generate non-DNA staining region. (B) Processing of actin image to generate non-actin staining image. (C) Comparison of lamellar wound region with its morphological closing allows assessment of lamellar smoothness. Click here for file Acknowledgements We'd like to thank Yan Feng for his contribution in setting up the automated microscopy rig; Rebecca Ward for her support of the project; Jim Horne for his masterful machining skills; Steve Miller, Thomas Mayer, Maria Wigger, Scott Lokey, and Jeff Peterson for common cause; and Mimi Shirasu-Hiza for help with the manuscript and great discussions. JCY and ZEP were supported by Howard Hughes Medical Institute pre-doctoral fellowships. Funding provided to TJM through NIH GM048027-12. Figures and Tables Figure 1 Adaptation of a wound healing assay to a 384 well plate format. (A) Images from a time-lapse sequence of BS-C-1 cells migrating on a coverslip to heal a wound. Bar 20 μm. (B) Images of wounds 0, 3, 7, 12, and 24 hrs after wounding show characteristic protrusion of lamella at 3 hrs, migration by 7 hrs, and eventual healing of the wound at 24 hrs. Cells are stained for filamentous actin. Bar 100 μm. (C) Schematic of the protocol used for screening. The 96 well pin array (shown) produces wounds with consistent shape and placement within each well. Image of 25 wells stained for filamentous actin 7 hrs after wounding. Bar 500 μm. (D) The primary phenotypes observed with an automated microscope in a small molecule screen (4× objective, 7 hr healing). Wells often showed a combination of these phenotypes. Cells are stained for filamentous actin and DNA. The dotted yellow line notes the approximate edge of the initial wound (based on the high density of nuclei). Bar 100 μm. Figure 2 The use of scanners to assay wound healing after 7 hrs recovery. (A) An image of actin-stained wells obtained using a fluorescence scanner. Normal healing wounds (left two columns) can be distinguished from wells treated with a titration of the actin cytoskeleton disrupter cytochalasin D (white box) by the width of the wound and staining at the wound margin. (B) An image of Coomassie-stained wells obtained using a transmitted-light scanner. Normal healing wounds have a more diffuse wound margin, denoting cell migration into the wound, than wells treated with cytochalasin D (white box). Bar 1 mm. Figure 3 The use of a fluorescence macroscope to assay wound healing after 24 hrs recovery. Fluorescence macroscope image of wells stained for filamentous actin 24 hrs after wounding. A well showing reduced wound healing can be easily distinguished (middle well white box) from wells showing complete healing which are seen as light grey streaks. Bar 1 mm. Figure 4 Automated analysis of wound healing images. (A) An image showing normal migration and the same image after automated analysis defines regions. Extracting characteristics of the lamellar region (blue) allows us to distinguish between the phenotypes that affect wound healing (Figure 1). Stains: red – filamentous actin; green – DNA. Bar 100 μm. (B) Automated and visual analysis are complementary approaches. Automatically generated lamellar width data is plotted for a 384 well plate. Compounds found to affect wound healing by visual analysis are shown in green, and wells that differed from the control in other ways (increased mitotics, a disrupted monolayer, etc.) are shown in red. Mean lamellar width (black line) and three standard deviations (red dashed lines) are plotted for control wells. Data collected from screening one 384 well plate of small molecules. Table 1 Comparison of four imaging methods to readout the wound healing assay Device Resolution Observable phenotypes Timepoint for assaying Acquisition speed Cost of device Automated microscope 3 μm/pixel Decreased wound healing, aberrant morphology, increased mitotic index 7 and 24 hr ~1 hr/plate with 4× ~1.5 hr/plate with 10× ~$200,000 Fluorescence scanner 42 μm/pixel Decreased wound healing 7 and 24 hr 26 minutes/plate ~$50,000 Conventional scanner 10 μm/pixel Decreased wound healing 7 and 24 hr 8.5 minutes/plate ~$1,000 Fluorescence macroscope ~100 μm/pixel Decreased wound healing 24 hr 5 seconds/plate ~$500,000 ==== Refs Wong MK Gotlieb AI The reorganization of microfilaments, centrosomes, and microtubules during in vitro small wound reendothelialization J Cell Biol 1988 107 1777 1783 3182937 10.1083/jcb.107.5.1777 Coomber BL Gotlieb AI In vitro endothelial wound repair. Interaction of cell migration and proliferation Arteriosclerosis 1990 10 215 222 1969263 Zahm JM Kaplan H Herard AL Doriot F Pierrot D Somelette P Puchelle E Cell migration and proliferation during the in vitro wound repair of the respiratory epithelium Cell Motil Cytoskeleton 1997 37 33 43 9142437 10.1002/(SICI)1097-0169(1997)37:1<33::AID-CM4>3.0.CO;2-I Lampugnani MG Cell migration into a wounded area in vitro Methods Mol Biol 1999 96 177 182 10098136 10.1385/1-59259-258-9:177 Lu KV Jong KA Rajasekaran AK Cloughesy TF Mischel PS Upregulation of tissue inhibitor of metalloproteinases (TIMP)-2 promotes matrix metalloproteinase (MMP)-2 activation and cell invasion in a human glioblastoma cell line Lab Invest 2004 84 8 20 14631378 10.1038/sj.labinvest.3700003 Herren B Garton KJ Coats S Bowen-Pope DF Ross R Raines EW ADAM15 overexpression in NIH3T3 cells enhances cell-cell interactions Exp Cell Res 2001 271 152 160 11697891 10.1006/excr.2001.5353 Huang C Rajfur Z Borchers C Schaller MD Jacobson K JNK phosphorylates paxillin and regulates cell migration Nature 2003 424 219 223 12853963 10.1038/nature01745 Mc Henry KT Ankala SV Ghosh AK Fenteany G A non-antibacterial oxazolidinone derivative that inhibits epithelial cell sheet migration Chembiochem 2002 3 1105 1111 12404636 10.1002/1439-7633(20021104)3:11<1105::AID-CBIC1105>3.0.CO;2-S Vogt A Pestell KE Day BW Lazo JS Wipf P The antisignaling agent SC-alpha alpha delta 9, 4-(benzyl-(2-[(2,5-diphenyloxazole-4-carbonyl)amino]ethyl)carbamoyl)- 2-decanoylaminobutyric acid, is a structurally unique phospholipid analogue with phospholipase C inhibitory activity Mol Cancer Ther 2002 1 885 892 12481409 Klein-Soyer C Cazenave JP Herbert JM Maffrand JP SR 25989 inhibits healing of a mechanical wound of confluent human saphenous vein endothelial cells which is modulated by standard heparin and growth factors J Cell Physiol 1994 160 316 322 7518823 Fischer EG Stingl A Kirkpatrick CJ Migration assay for endothelial cells in multiwells. Application to studies on the effect of opioids J Immunol Methods 1990 128 235 239 2139083 10.1016/0022-1759(90)90215-H Nobes CD Hall A Rho GTPases control polarity, protrusion, and adhesion during cell movement J Cell Biol 1999 144 1235 1244 10087266 10.1083/jcb.144.6.1235 Etienne-Manneville S Hall A Integrin-mediated activation of Cdc42 controls cell polarity in migrating astrocytes through PKCzeta Cell 2001 106 489 498 11525734 10.1016/S0092-8674(01)00471-8 Fenteany G Janmey PA Stossel TP Signaling pathways and cell mechanics involved in wound closure by epithelial cell sheets Curr Biol 2000 10 831 838 10899000 10.1016/S0960-9822(00)00579-0 Sablina AA Chumakov PM Kopnin BP Tumor suppressor p53 and its homologue p73alpha affect cell migration J Biol Chem 2003 278 27362 27371 12750388 10.1074/jbc.M300547200 Yvon AM Walker JW Danowski B Fagerstrom C Khodjakov A Wadsworth P Centrosome reorientation in wound-edge cells is cell type specific Mol Biol Cell 2002 13 1871 1880 12058055 10.1091/mbc.01-11-0539 Magdalena J Millard TH Machesky LM Microtubule involvement in NIH 3T3 Golgi and MTOC polarity establishment J Cell Sci 2003 116 743 756 12538774 10.1242/jcs.00288 Magdalena J Millard TH Etienne-Manneville S Launay S Warwick HK Machesky LM Involvement of the Arp2/3 complex and Scar2 in Golgi polarity in scratch wound models Mol Biol Cell 2003 14 670 684 12589062 10.1091/mbc.E02-06-0345 Philp D Huff T Gho YS Hannappel E Kleinman HK The actin binding site on thymosin beta4 promotes angiogenesis Faseb J 2003 17 2103 2105 14500546 Moon EJ Lee YM Lee OH Lee MJ Lee SK Chung MH Park YI Sung CK Choi JS Kim KW A novel angiogenic factor derived from Aloe vera gel: beta-sitosterol, a plant sterol Angiogenesis 1999 3 117 123 14517429 10.1023/A:1009058232389 Ma PC Kijima T Maulik G Fox EA Sattler M Griffin JD Johnson BE Salgia R c-MET mutational analysis in small cell lung cancer: novel juxtamembrane domain mutations regulating cytoskeletal functions Cancer Res 2003 63 6272 6281 14559814 Chung RS Adlard PA Dittmann J Vickers JC Chuah MI West AK Neuron-glia communication: metallothionein expression is specifically up-regulated by astrocytes in response to neuronal injury J Neurochem 2004 88 454 461 14690533 Morgan LW Wieslander A Davies M Horiuchi T Ohta Y Beavis MJ Craig KJ Williams JD Topley N Glucose degradation products (GDP) retard remesothelialization independently of D-glucose concentration Kidney Int 2003 64 1854 1866 14531821 10.1046/j.1523-1755.2003.00265.x Saga Y Mizukami H Takei Y Ozawa K Suzuki M Suppression of cell migration in ovarian cancer cells mediated by PTEN overexpression Int J Oncol 2003 23 1109 1113 12963992 Ramm P Imaging systems in assay screening Drug Discov Today 1999 4 401 410 10461149 10.1016/S1359-6446(99)01378-1

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==== Front BMC GenomicsBMC Genomics1471-2164BioMed Central London 1471-2164-5-691538002910.1186/1471-2164-5-69Research ArticleComparative genomics of cyclin-dependent kinases suggest co-evolution of the RNAP II C-terminal domain and CTD-directed CDKs Guo Zhenhua [email protected] John W [email protected] Department of Biology, East Carolina University, Howell Science Complex N 108, Greenville, NC 27858, USA2004 20 9 2004 5 69 69 10 3 2004 20 9 2004 Copyright © 2004 Guo and Stiller; licensee BioMed Central Ltd.2004Guo and Stiller; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Cyclin-dependent kinases (CDKs) are a large family of proteins that function in a variety of key regulatory pathways in eukaryotic cells, including control over the cell cycle and gene transcription. Among the most important and broadly studied of these roles is reversible phosphorylation of the C-terminal domain (CTD) of RNA polymerase II, part of a complex array of CTD/protein interactions that coordinate the RNAP II transcription cycle. The RNAP CTD is strongly conserved in some groups of eukaryotes, but highly degenerate or absent in others; the reasons for these differences in stabilizing selection on CTD structure are not clear. Given the importance of reversible phosphorylation for CTD-based transcription, the distribution and evolutionary history of CDKs may be a key to understanding differences in constraints on CTD structure; however, the origins and evolutionary relationships of CTD kinases have not been investigated thoroughly. Moreover, although the functions of most CDKs are reasonably well studied in mammals and yeasts, very little is known from most other eukaryotes. Results Here we identify 123 CDK family members from animals, plants, yeasts, and four protists from which genome sequences have been completed, and 10 additional CDKs from incomplete genome sequences of organisms with known CTD sequences. Comparative genomic and phylogenetic analyses suggest that cell-cycle CDKs are present in all organisms sampled in this study. In contrast, no clear orthologs of transcription-related CDKs are identified in the most putatively ancestral eukaryotes, Trypanosoma or Giardia. Kinases involved in CTD phosphorylation, CDK7, CDK8 and CDK9, all are recovered as well-supported and distinct orthologous families, but their relationships to each other and other CDKs are not well-resolved. Significantly, clear orthologs of CDK7 and CDK8 are restricted to only those organisms belonging to groups in which the RNAP II CTD is strongly conserved. Conclusions The apparent origins of CDK7 and CDK8, or at least their conservation as clearly recognizable orthologous families, correlate with strong stabilizing selection on RNAP II CTD structure. This suggests co-evolution of the CTD and these CTD-directed CDKs. This observation is consistent with the hypothesis that CDK7 and CDK8 originated at about the same time that the CTD was canalized as the staging platform RNAP II transcription. Alternatively, extensive CTD phosphorylation may occur in only a subset of eukaryotes and, when present, this interaction results in greater stabilizing selection on both CTD and CDK sequences. Overall, our results suggest that transcription-related kinases originated after cell-cycle related CDKs, and became more evolutionarily and functionally diverse as transcriptional complexity increased. ==== Body Background Cyclin-dependent kinases (CDKs) belong to a large protein family with 13 members described so far in human cells including CDKs1-11, along with PCTAIRE and PFTAIRE kinases named after conserved domain sequences [1]. These kinases are essential for cell cycle progression, and also are involved in control of transcription, DNA repair and post-mitotic cellular process [2-4]. Generally, CDKs1-6, PCTAIRE and PFTAIRE have been linked to cell cycle regulation, and CDKs7, 8 and 9 to control of RNA polymerase II (RNAP II) transcription [4-8]. The functions of CDKs10 and 11 have not been defined clearly, but recent research implicates them in coordination of transcription and RNA-processing [9-13]. Among the most important and broadly studied roles of CDKs in transcription is the reversible phosphorylation of the C-terminal domain (CTD) of the largest subunit (RPB1) of RNAP II. The CTD consists of multiple repeats of an evolutionarily conserved heptapeptide with the consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7 [14]. The number of repeats varies among different organisms, ranging from 26–27 in yeast to 52 in mammals [15,16] with 8 repeats in yeast and 28 repeats in human cells required for viability [15,17,18]. Both biochemical and genetic evidence places the CTD in a central position in the 'mRNA factory,' where it functions as a platform for interactions with processing factors and other transcription-related proteins [19,20]. More than a passive scaffold, reversible phosphorylation of the CTD regulates the cycling of RNAP II between a hypophosphorylated (IIO) form, which is competent to enter the preinitiation complex, and a hyperphosphorylated (IIA) form capable of processive transcript elongation [21]. Throughout this cycle the CTD binds essential transcription-related proteins that help to regulate gene expression, promote efficient elongation, and effectively couple transcription to pre-mRNA processing [19-24]. To date at least five of the CDKs (CDK1, 2, 7, 8 and 9) have been shown to phosphorylate the CTD in vitro; they all have been referred to as 'CTD kinases' [25-28]. Both CDK7 and CDK8 are found tightly associated with the pre-initiation complex and are involved in transcriptional regulation [29]. The CDK9 subunit of P-TEFb (positive transcription elongation factor b) induces hyper-phosphorylation of the CTD and stimulates elongation. Unlike CDKs 7, 8 and 9, which have demonstrated interactions with the CTD in vivo, CDK1 and CDK2 are primarily cell-cycle related kinases [4]. CDK2 has been characterized functionally only human and Drosophila in mammals and its role in Tat-dependent HIV-1 transcription is still unclear [27,28]. Although phosphorylation of yeast RNAP II by CDK1 (CDC2) can inhibit transcription in vitro, the role of the CDK1 in mRNA synthesis in vivo is not, as yet, clearly understood. It has been proposed as a candidate for mitotic RNAP II inactivation by inhibition of CDK7 CTD-kinase activity [26]. In animals and yeasts, interactions between the CTD and CTD-specific kinases have become a focal point of biochemical and genetic investigations of RNAP II transcription and transcription-linked mRNA processing [25,26,30]. However, the ancestry and evolutionary relationships among CTD kinases have not been investigated thoroughly. Evolutionary analyses of the RNAP II CTD show that canonical CTD heptads are strongly conserved only in a subset of eukaryotic groups. In evolutionary trees based on RPB1 sequences, all eukaryotic groups in which the CTD is strongly conserved appear to be descended from a single common ancestor (descendents of this ancestor have been referred to as the "CTD-clade") [31]. The reasons for differential conservation of the CTD have not been clarified, nor have evolutionary correlations been established between strong conservation of CTD structure and the presence of essential CTD/protein interactions. In addition, although the functions of various CDKs are reasonably well characterized in mammals and yeasts, very little is known for most other eukaryotes, and the overall evolution of CDKs has been investigated only in animals and yeasts [32]. Therefore, a comparative evolutionary study also can provide clues as to which CDK orthologs, and presumably CDK functions, are present over a broad range of eukaryotic diversity. Here we present a comparative genomic analysis of CDKs, using complete genomes from members of the "CTD clade" (animals, plants, yeasts and Microsporidia), as well as from other diverse eukaryotic organisms lacking a canonical CTD (Trypanosoma, Plasmodium and Giardia), to explore the evolutionary relationships between the CTD and CTD kinases. We also provide a phylogenetic distribution of CDKs from a wide range of organisms, suggesting new hypotheses regarding the emergence and evolution of different members of the CDK family. Results We identified 133 CDK family members, 123 from animals, plants, yeasts, and four protists from which genome sequences have been completed, and 10 additional CDKs from incomplete genome sequences of organisms with known CTD sequences (Table 1). Although all of sequences are included in our supplemental phylogenetic analysis (additional file 1), only 101 of them are included in the major phylogenetic analysis (Fig. 1); a large plant-specific amplification of CDK9-like kinases (the phylogenetic weight of these sequences disrupts the CDK9 sub-clade) and sequences from incomplete genomes are excluded (see Fig. 1 and additional file 1 legends for further explanation). The nomenclature for kinases from Arabidopsis followed Joubès et al. (2000) and Vandepoele et al. (2002) [33,34] (Table 1). The catalytic core base, Gly-rich motif and T-loop, required for characterized CDK function, appear to be conserved across all defined and putative kinase sequences analyzed (additional file 2). The 50% majority rule consensus tree of 4,000 likelihood trees, sampled from the posterior probability distribution from Bayesian phylogenetic inference, is shown in Figure 1. This tree provides strong support for grouping a number of previously uncharacterized CDKs, from a variety of organisms, with defined CDKs from animals and yeast. Overall, however, very little support is found for relationships among different CDK orthologous groups. Table 1 CDK-related kinases used in this study. Species Genes gi number Abbreviations Trypanosoma brucei Cdc2-related kinase2 397162 TbCrk2 Cdc2-related kinase3 397365 TbCrk3 Cdc2-related kinase6 23392965 TbCrk6 Cdc2-like kinase 10458 TbCdc2L Leishmania major Cdc2-related kinase 1 9857049 LmCrk1 Cdc2-related kinase 3 15526337 LmCrk3 Giardia lamblia Cdc2-like1 29248279 GlCdc2L1 Cdc2-like2 29245850 GlCdc2L2 Cdc2-like3 29250990 GlCdc2L3 Cdc2-like4 29249431 GlCdc2L4 CAKlike 29249713 GlCAKlike Cryptosporidium parvum Cdc2-like kinase 3329529 CpCdc2L Plasmodium falciparum MO15-related kinase 23507945 PfMrk PK5 23619490 PfPk5 PK6 23618947 PfPk6 Crk1 23510162 PfCrk1 Crk3 23509994 PfCrk3 Crk4 23957709 PfCrk4 Dictyostelium discoideum Cdc2 kinase 167686 DdCdc2 Cdc2-related protein 167696 DdCrp Cdk7 1705721 DdCdk7 Cdk8 15778146 DdCdk8 Cdk9-like kinase 28828850 DdCdk9L Entamoeba histolytica Cdc2 kinase 543971 EhCdc2 Guillardia theta Cdc2 kinase 13812042 GtCdc2 Saccharomyces cerevisiae Cdc28 115915 ScCdc28 Pho85 295932 ScPho85 Kin28 1199540 ScKin28 Cdk8/Srb10 2131219 ScSrb10 Ctk1 486235 ScCtk1 Bur1 218486 ScBur1 Cak1 1480663 ScCak1 Schizosaccharomyces pombe Cdc2 173359 SpCdc2 PhoA 19075421 SpPhoA Mcs6 19113141 SpMcs6 Cdk8/Srb10 7493197 SpSrb10 AC2F3.15 19115305 SpAC2F3.15 Cdk9 32363142 SpCdk9 Csk1 299548 SpCsk1 BC18H10.5 3006177 SpBC18H10.5 Encephalitozoon cuniculi Cdc2-related kinaseA 19173516 EcCrkA Cdc2-related kinaseB 19069621 EcCrkB Cdc2-related kinaseC 19171093 EcCrkC Cdc2-related kinaseD 19074929 EcCrkD Cdc2-related kinaseE 19173349 EcCrkE Cdk7 like kinase 19068706 EcCdk7 Drosophila melanogaster Cdk1 115921 DmCdk1 Cdc2c 7708 DmCdk2 Cdk4 1523997 DmCdk4 Cdk5 1523999 DmCdk5 Cdk7 1336061 DmCdk7 Cdk8 1718193 DmCdk8 Cdk9 24658274 DmCdk9 Dcdrk 541654 DmDcdrk CG6800 23171908 DmCG6800 Pitslre 1524005 DmPitslre CG7597 24668136 DmCG7597 EiP63E 1524003 DmEip63E Caenorhabditis elegans K03E5.3 3158523 CeK03E5.3 Cdk1 5001728 CeCdk1 Cdk4 21902501 CeCdk4 Cdk5 5001732 CeCdk5 Zc123.4 21913082 CeZc123.4 Pctaire1 5001730 CePctaire1 Cdc2-like kinase5 7494824 CeB0385.1 Cdk7 5031478 CeCdk7 Cdk8 32563668 CeCdk8 Cdk9 17507939 CeCdk9 B0495.2 2499649 CeB0495.2 Zc504.3 897712 CeZc504.3 H01G02.2 7504821 CeH01G02.2 Homo sapiens Cdk1 115922 HsCdk1 Cdk2 29849 HsCdk2 Cdk3 4557439 HsCdk3 Cdk4 33304135 HsCdk4 Cdk5 7434324 HsCdk5 Cdk6 21885467 HsCdk6 Pctaire1 13623189 HsPctaire1 Pctaire2 21542571 HsPctaire2 Pctaire3 30583437 HsPctaire3 Pftaire1 6912584 HsPftaire1 Cdk7 13529020 HsCdk7 Cdk8 1000491 HsCdk8 Cdk9 12805029 HsCdk9 Cdk10 6226784 HsCdk10 Cdk11 16357492 HsCdk11 Cdc2-Like kinase5 10443222 HsCdc2L5 Cdc2-related kinase with RS domain 7107392 HsCrkRS Cell cycle related kinase 23344742 HsCCRK Oryza sativa CdkA.1 20343 OsCdkA.1 CdkA.2 266410 OsCdkA.2 CdkB2.1 7489567 OsCdkB2.1 CdkB1.1 34907628 OsCdkB1.1 R2 231707 OsCdk7 CdkE 12039362 OsCdkE CdkC.1 31442141 OsCdkC.1 OJ991113_30.14 38344237 OsCAD41330 B1015E06.16 34903661 OsB1015E06.16 P0560B06.11 34914693 OsP0560B06.11 P0453E05.113 28460677 OsP0453E05.113 P0450A04.129 34899281 OsP0450A04.129 P0498H04.21 42408343 OsP0498H04.21 P0435E12.11 46390990 OsP0435E12.11 P0482D04.8 34907029 OsP0482D04.8 OJ1562.H01.5 38424086 Os1562.H01.5 Arabidopsis thaliana CdkA1 30693081 AtCdkA.1 CdkB1.1 30694007 AtCdkB1.1 CdkB1.2 42569740 AtCdkB1.2 CdkB2.1 30699181 AtCdkB2.1 CdkB2.2 18394928 AtCdkB2.2 CAK1 15235518 AtCdkF CAK2 15147864 AtCdkD.3 CAK3 15147866 AtCdkD.1 CAK4 20521156 AtCdkD.2 CdkE 10177042 AtCdkE CdkC.1 30698081 AtCdkC.1 CdkC.2 11346412 AtCdkC.2 F12B7.13 17065202 AtF12B7.13 K9H21.7 17064770 AtK9H21.7 K9L2.5 15241455 AtK9L2.5 T22H22.5 25405751 AtT22H22.5 T12H1.1 15229881 AtT12H1.1 K16E14.2 26449318 AtK16E14.2 F21B7.1 7488248 AtF21B7.1 AT4g22940 15235867 At4g22940 F8L10.9 15219169 AtF8L10.9 F26A9.10 42572067 AtF26A9.10 AT4g10010 30681286 At4g10010 F14J9.26 18391043 AtF14J9.26 F6A14.22 15221833 AtF6A14.22 F1M20.1 25406336 AtF1M20.1 AAF21469.1 6649591 AtAAF21469.1 T4P13.34 42570106 AtT4P13.34 Note: The sequences in bold are the additional sequences from incomplete genomes and uncharacterized CDK9 like-kinases from Arabidopsis and Oryza included in supplemental phylogenetic tree (additional file 1). Figure 1 Unrooted 50% majority consensus tree from 4,000 ML trees sampled from the Bayesian posterior probability distribution. Support values are shown above the internode from Bayesian inference/distance bootstrap respectively. Only values above 50% are reported and values under 50% are indicated by (-). 100% values are indicated by (+). CDK names in blue are from organisms that fall into the "CTD-clade" in RPB1 phylogenetic analyses (see Fig. 2); and those in red are from groups in which the CTD is not strongly conserved. Inferred groups of CTD-directed CDKs 7, 8 and 9 are shown in bold. A large group of unidentified CDKs from Arabidopsis and Oryza, which appear to represent a plant-specific amplification of CTK9, were excluded from this analysis to determine whether identified plant CDK9s show a specific phylogenetic affinity to either the BUR1 or CTK1 subgroup. All identified plant sequences are included in an expanded analysis shown in additional file 1. In this unrooted tree the highly diversified cell-cycle kinases defined in humans, CDKs1-6, fall into a large cluster with 69% Bayesian support. This grouping includes CDKs from all organisms examined in the study. Among these putative cell-cycle CDKs, some plant and protistan kinases can be assigned with reasonable confidence to specific CDK groups. For example, apparent orthologs of human CDK1 are found in other animals (Drosophila and Caenorhabditis), yeasts, both plants (Arabidopsis and Oryza), Encephalitozoon and Giardia (Fig. 1). Likewise, putative orthologs of CDK5 were identified in all organisms examined, except for the two plants (Fig. 1). A number of other sequences, such as TbCrk2 and 3 from Trypanosoma, cluster with cell-cycle kinases but not clearly with any specific CDK family. Significantly, and consistent with the results of Liu and Kipreos (2000) [32], CDK5 and PCTAIRE-like kinases from fungi and animals form a strongly supported group, indicating their close relationship (Fig. 1). In contrast to cell-cycle kinases, our phylogenetic results failed to identify a clear ortholog of any transcription-related CDKs from two of the complete genomes examined, Trypanosoma brucei and Giardia lamblia. This includes strongly supported clades of presumed orthologs of human CDKs7-11 respectively. A well-defined CDK7 family is recovered, including sequences from yeasts, the microsporidian, plants, and animals. These are the primary groups that make up the "CTD-clade," in which the RNAP II CTD is invariably conserved (Fig. 2). CDK7 shows an interesting sister relationship to HsCCRK from human and apparent orthologs from Drosophila, Caeorhabditis and Arabidopsis. In Arabidopsis, four possible CDK7 orthologs were found, as reported previously by Shimotohno and colleagues (2003) [35]; however, AtCdkF (CAK1) is quite divergent from the core CDK7 family and related specifically to HsCCRK in our analyses. PfMRK from Plasmodium, suggested previously to be a CDK7 [36], does not fall within the well-defined CDK7 group, but clusters with another Plasmodium kinase. The a priori hypothesis that PfMRK belongs in the core CDK7 group is strongly rejected with our data set in a likelihood paired-sites test. Figure 2 Hypothesis of RNA polymerase II evolution inferred from phylogenetic analyses of RPB1 sequences conserved regions A-H. The tree displayed, after Stiller and Cook [60] had the highest likelihood of all trees sampled from the posterior probability distribution in 106 generations of Bayesian inference. Organisms with genomes included in this study are in larger/bold font, and whether each of the three primary CTD kinases (CDKs7,8,9) are present in this genome, as inferred from global phylogenetic analyses and distributions of CDK kinases (see Fig. 1), are indicated next to the name. Names in blue indicate the presence of repeated heptads at the RPB1 C-terminus, which includes several from protistan organisms that do not conform to the consensus sequence or known structural requirements of the canonical CTD [60]; names in red have no tandem-heptapeptide structure whatsoever. The node supporting a "CTD-clade," in which the consensus sequence and repetitive structure of the CTD are invariably conserved, occurred in 98% of the 8000 trees sampled from the Bayesian posterior probability distribution. See references 31 and 60 for a more complete phylogenetic treatment of the origin and conservation of the CTD. Likewise, GlCAKlike (gi: 292497120) has been proposed as a CDK7 from Giardia, based on nearest sequence similarity to Kin28 in a more limited comparison to CDK sequences from fission yeast [38]. In our expanded analyses of CDKs from 11 completed genomes, we find no evidence supporting an orthologous relationship to CDK7 for this, or any Giardia sequence. The a priori hypothesis that GlCAKlike belongs in the core CDK7 group also is strongly rejected in a likelihood paired-sites test. A robust CDK8 family is recovered with strong support values in both distance bootstrap and Bayesian inference. Like CDK7, this family includes putative orthologs only from members of the "CTD-clade," specifically yeasts, animals and plants. Although the microsporidian Encephalitozoon is a member of the RNAP II "CTD clade," TBlastN searches of the complete genome of Encephalitozoon found six CDKs but none show a phylogenetic affinity to CDK8. A CDK9 grouping also is supported as monophyletic with representative CDKs from yeasts, Encephalitozoon, animals, plants and Plasmodium. This group is divided into two well-defined sub-clades. One of them consists of BUR1 from yeast along with CDK9 orthologs from animals; the other contains CTK1 from yeast, CDC2L5 and CrkRS from human, and apparent orthologs from Drosophila and Caenorhabditis, both plants, and Plasmodium. A putative CDK9 also is found in Encephalitozoon, but falls at the base of the larger CDK9 grouping and does not associate clearly with either subgroup (Fig. 1). Plants also contain a large number of putative CDKs that show strong phylogenetic affinity to CDK9 (additional file 1). These kinases appear to represent a plant-specific amplification of CDK9, although their functions have not been determined experimentally. Human CDK10 and CDK11 group with apparent orthologs from other animals, plants, fission yeast, and PfCRK1 from Plasmodium. Once again, no kinases from either Trypanosoma or Giardia show any phylogenetic affinity to this group. Discussion A suggestion of co-evolution between the RNAP II CTD and CTD kinases At least five CDKs have the capacity to phosphorylate RNAP II CTD repeats in vitro or in vivo [25-28]. Kinases that appear to be related closely to CDK1, which can phosphorylate the CTD in vitro, are present in all organisms sampled; however, it is not clear that CDK1 interacts with the CTD in vivo or is essential for CTD function. CDK2 was found only in human and Drosophila and, based on our analyses from a diverse group of eukaryotes, appears to be derived from within a larger CDK1 family. In any case, according to its restricted phylogenetic distribution, possible CTD/CDK2 interactions cannot explain the conservation of CTD structure in diverse members of the "CTD-clade." Evolutionary investigations of RPB1 sequences show that canonical CTD heptads are conserved strongly in only a subset of eukaryotic groups, all apparently descended from a single common ancestor [31]. This "CTD-clade" is composed of animals, plants, fungi, and related protistan groups, including microsporidians, chytridiomycetes, choanoflagellates and slime molds (Fig. 2). A handful of organisms that do not fall inside the "CTD-clade" do have tandemly repeated C-terminal heptads. For example, RPB1 from Plasmodium falciparum contains a short set of seven tandem C-terminal repeats. Based on codon usage and comparative alignment with sequences from other Plasmodium species, these heptads are best explained by a recent tandem duplication of a single heptad motif in P. falciparum or its immediate ancestor [31]. No other apicomplexan RPB1 contains tandemly repeated heptads, nor does the nearest evolutionary relative of the apicomplexans (Fig. 2). Although vestigial or convergent heptad repeats are found in a few organisms scattered across the eukaryotic evolutionary tree, strong stabilizing selection on CTD structure appears to be restricted to those eukaryotic lineages found in the "CTD-clade" (Fig. 2). In our analyses of CDKs, members of this "CTD-clade" are precisely the same eukaryotes to which clear orthologs of CDK7 and CDK8 are restricted. When sequences recovered from additional but incomplete eukaryotic genomes are included in phylogenetic analyses, distribution of these two kinases remains tightly correlated with strong conservation of canonical CTD repeats (see additional file 1). Moreover, unlike CDK1, the primary characterized function of both of these kinases is to mediate RNAP IIA/IIO cycling through reversible phosphorylation of CTD residues [19-24]. Taken together, these findings suggest that the RNAP II CTD has undergone a co-evolutionary process with CDK7 and CDK8. If phylogenetic results based on CDK and RPB1 sequences reflect evolutionary history, the inference of a "CTD-clade" in both sets of analyses suggests that CDK7 and 8 originated as part of a major shift in the mechanics of RNAP II transcription in the ancestor of the "CTD-clade" [31]. It was in that ancestor that reversible phosphorylation of the CTD became a central organizing principle for regulating the transcription cycle, and laid the foundation for more complicated mechanisms of transcriptional control in these organisms. Such a profound shift in the mechanics of RNAP II transcription would explain why the CTD is conserved so strongly in members of the CTD-clade, but not in many other eukaryotic lineages [31]. In this scenario, other known or putative CTD kinases (certainly CDK1 and apparently CDK9) originated before canalization of a CTD-based RNAP II transcription cycle, and were adapted later as CTD kinases. It also is possible that the co-evolution inferred from comparisons of the phylogenetic distribution of RPB1 and CDKs7/8 does not reflect the pattern of evolutionary history but, instead, results from functional constraints driven by CTD/CDK interactions. Both GlCAKlike from Giardia and PfMrk from Plasmodium have been suggested previously to be orthologs of CDK7 [36,38]; these hypotheses are rejected strongly by our phylogenetic analyses. Assuming these kinases really are CDK7s, then their failure to cluster with other orthologs must be due to phylogenetic artifacts, frequently referred to as "long-branch attraction" [39], that can be common when rates of evolution vary dramatically among sequences. The large amounts of sequence divergence of PfMrk and GlCAKlike from other CDK7s, along with a complete degeneration of the CTD in Giardia species and apicomplexans as a group, are unlikely to coincidental. It is possible that those organisms retaining a RNAP II transcription cycle mediated by CDK7 and 8 kinase activity form distinct clades, in both RPB1 and kinase derived trees, because both sets of proteins share parallel modes of evolution driven by their physical interactions. In this case, the observation of co-evolution between the CTD and CTD-directed kinases need not have a phylogenetic basis, only a functional one. Most putative CDKs from Giardia and Trypanosoma, and several from Plasmodium, do not associate strongly with any established CDK family. It is reasonable to assume that at least some of these kinases are orthologs of defined CDK groups, but have diverged to the point that they are not recognizable using sequence-based phylogenetic methods. Although such a scenario may have disturbing implications for the use of these methods across broad evolutionary distances, particularly when functional interactions among sequences are unknown or poorly understood, it cannot be ruled out as an explanation for our observations. Analyses of additional genomes from diverse eukaryotes are required, both to verify our observations of co-evolution between the CTD and CTD-directed kinases, and to determine its bases. General evolutionary trends in the CDK family Kinases from protistan organisms In an effort to understand the broader evolutionary history of CDKs, three deep-branching protists with complete genomes, Plasmodium falciparum, Trypanosoma brucei and Giardia lamblia, were included in our study. Our Blast searches detected 15 putative kinases from these protists; six from Plasmodium falciparum, four from Trypanosoma brucei and five from Giardia lamblia (Table 1). The phylogenetic positions and orthologous relationships of these kinases generally are not well defined by phylogenetic analyses (Fig. 1). Four of them (GlCdc2L3, GlCAKlike, TbCrk6 and PfCrk4), along with two microsporidian kinases (EcCrkB and EcCrkD) branched close to ScCak1 and SpCsk1, cyclin-activating kinases from yeasts. All of these sequences are highly divergent, and it is difficult to determine, whether their branching positions are due to a phylogenetic artifact or a phylogenetic relationship. As noted above, GlCAKlike kinase has been proposed as a Giardia CDK7 ortholog based on JTT distance data [38], a relationship not supported by our broader phylogenetic analyses. Moreover, there are no experimental data reported on the functions of any of these kinases. Other putative protistan CDKs, GlCdc2L4, PfMrk and PfPk6, scatter among CDKs from other organisms, but with no statistical confidence for any implied relationship. Our most strongly supported results indicate that six of these kinases (TbCdc2L, TbCrk2 and 3, PfPk5, GlCdc2L1 and L2) belong to cell-cycle related kinase families CDK1 and CDK5. In particular, PfPk5 is well-supported as an ortholog of CDK5. In addition, two kinases from Plasmodium (PfCrk1 and PfCrk3) appear to be transcription-related kinases, PfCrk1 groups with the CDK10/11 family, and PfCrk3 with CDK9. The phylogenetic distribution of protistan kinases indicate that cell-cycle related kinases are present, or at least their functions are more strongly conserved (see discussion above regarding CTD/CDK co-evolution), in a more diverse array of eukaryotes than are transcription-related kinases. This pattern also is seen in a more widely-sampled analyses including CDKs from a number of organisms with incompletely sequenced genomes, including Dictyostelium discoideum that has a canonical RNAP II CTD, and Leishmania major, Cryptosporidium parvum and Entamoeba histolytica, which all lack a CTD (see additional file 1). Thus, the overall results suggest that cell-cycle related kinases are more ancient than transcription-related kinases, and probably ancestral to them, and that their core functions are more similar across the broad diversity of eukaryotic lineages. It will be interesting to see whether these preliminary hypotheses are supported as more genomes are sequenced completely, particularly from diverse protistan organisms. Cell-cycle related kinases Our analyses support well-defined groups for cell-cycle kinases CDK1, CDK4/6 and CDK5. An ortholog of either CDK1 or CDK5 is found in all of the organisms in our study, and these two families appear to be closely related. TbCrk3 was proposed as a functional homolog of CDK1 in Trypanosoma [40]; here it groups among cell-cycle kinases, but is not specifically related to CDK1. CDK4/6 appears to be present only in human, Drosophila and C. elegans. The CDK5 family has undergone expansion in metazoans, including PFTAIRE and PCTAIRE kinases, and putative orthologs of CDK5 are detected in Plasmodium, Trypanosoma and Giardia. Interestingly, no CDK from plants associates strongly with the CDK5 group, while the CdkB-type kinases, which are specific to plants, branch as sister to a broader CDK1/CDK5 clade. Our overall results suggest that cell-cycle kinases have undergone extensive and independent evolutionary diversification in different eukaryotic lineages, and it may be difficult to classify many of them based on orthologous relationships in phylogenetic analyses. It may be that functional homologies, once established experimentally, will prove to be more consistent criteria for designating CDK groups. The CDK7 family Clear orthologs of CDK7 from animals, plants, yeasts and Microsporidian are strongly supported as a core family, with CDK-activating kinase from Arabidopsis (AtCdkF), and its apparent orthologs from animals, branching as a sister group. In addition to their role as CTD kinases, members of the CDK7 family in plants, animals and fission yeast can function as a CDK-activating kinase (CAK) [41,42]. Unlike animals and yeast, however, four CDK7-like of CAKs were isolated from Arabidopsis [35]. AtCdkF (AtCAK1), which groups with human CCRK and apparent orthologs from Drosophila and Caenorhabiditis, exhibits only CAK activity but no CTD kinase activity. Consistent with the phylogenetic relationships recovered in our analysis, human CCRK and other animal orthologs were recently shown to have CAK activity [43]. In contrast, AtCdkD3 (AtCAK2) and AtCdkD2 (AtCAK4) display both CAK and CTD kinase activity and, along with a single CDK7 from rice, are included in a strongly supported CDK7 clade. Interestingly, and despite its high sequence similarity to AtCdkD3, no kinase activity was reported from AtCdkD1 (AtCAK3) [35]. Apparently CAKs in Arabidopsis have diversified substantially, and may be regulated in different ways from those in yeast, animals, and even rice. ScCAK1 and SpCSK1 from yeasts also have CAK activity; however, despite their functional similarity to kinases in the CCRK group, they do not group with animal or plant CAKs (Fig. 1). Interestingly, in the single most likely tree recovered in our expanded Bayesian analysis of 133 sequences, ScCAK1 and SpCsk1 group with other CAKs in the sister clade to CDK7 (additional file 1); however, there is no support for this placement in the Bayesian probability distribution. ScCAK1 and SpCSK1 sequences are highly divergent from all CDKs, and the regulation of CAK activity in yeast is very different from that of animals and plants [42,44]. Thus, alternative lines of evidence may be required to determine whether there is any specific evolutionary relationship among all CAKs. The CDK 8 family CDK 8 (SRB10 in yeast) is a component of the multi-subunit Mediator complex, which transduces signals from cis regulatory elements to RNAP II; it is proposed to inhibit transcription initiation by phosphorylation of the CTD. CDK8/SRB10 and its partner cyclin C/SRB11, together with SRB8 and SRB9, form a specific sub-module that is variably associated with the RNAP II holoenzyme, and potentially with the free mediator complex [45]. Apparent orthologs of CDK8 form a well-defined group, including sequences from plants, animals and yeasts. Interestingly, although a member of the CTD clade (Fig. 2 and note that all microsporidian RPB1 genes isolated to date encode a CTD), no ortholog of CDK8 was identified from Encephalitozoon. Our further blast results (unpublished data) failed to identify any of the units of the CDK8/SRB10 (SRBs8-11) sub-module in the Microsporidia suggesting a loss of CDK8/SRB10 unit from these highly reduced parasites. Although the CDK8/SRB10 sub-module has been implicated in negative regulation of transcription by phosphorylation of TFIIH, leading to the inhibition of the TFIIH CTD kinase and transcription [46], the exact mechanism still is unclear. Recent research shows that the Mediator containing this sub-module is isolated only in free form, not associated with RNAP II. In contrast, Mediator lacking this sub-module associates with the polymerase [47]. There also is experimental evidence that negative Mediator-RNAP II regulation by the SRB8-11 sub-module is evolutionarily conserved from yeast to humans [47]. Therefore, the absence of identifiable components of the SRB8-11 sub-module in Encephalitozoon suggests CDK8/SRB10 function is absent from the Microsporidia. The loss of CDK8 from Microsporidia, along with absolute conservation of CDKs7 and 9 in all members of the "CTD-clade" (Figs. 1 and 2) implies that interactions between the CTD and Mediator complex are less strongly entrained into essential RNAP II function, than are those regulated by TFIIH and P-TEFB kinase activity. The CDK 9 family CDK9 is a component of the P-TEFb complex, which is a positive-acting RNAP II transcription elongation factor [48,49]. Research has focused on P-TEFb from animals and budding yeast. A definitive yeast homolog of animal P-TEFb has not yet been determined from functional studies, but two candidates have emerged: the BUR1 complex and the CTDK-I complex [26]. Based on our blast and phylogenetic analyses, BUR1 and CTK1 (subunit of CTDK-I complex) are found in two distinct but related kinase groups, each with orthologs from other eukaryotes. BUR1 is identified as the specific ortholog of CDK9 from metazoans, budding yeast and probably the Microsporidia. Unexpectedly, the CDC2-like5 kinases and CrkRS from animals are highly supported as orthologs of CTK1 from yeasts. Although their functions are not yet clear [50], our results suggest that human CDC2-like5 kinases and CrkRS have CDK9 function. Recent analyses of CrkRS (CDC2-related kinase with an RS-rich domain) suggest that it has CTD kinase activity and helps to link transcription directly to intron splicing [51]. This CTK1 clade also contains putative CDK9 (CdkC) kinases from plants and as well as a CDC2-like kinase from Plasmodium (PfCRK3). The latter is the only apparent ortholog of a CTD-directed kinase (CDKs 7, 8 or 9) identified in our analyses from any organism outside the "CTD-clade." It remains to be determined whether PfCRK3 possesses the P-TEFb function of CTK1, since it is the only protistan sequence present in either CDK9 sub-group, and the RNAP II CTD has not been conserved in apicomplexans or their closest relatives (Fig. 2). In addition to the two previously identified copies of CDK9 (CdkC1 and CdkC2) from Arabidopsis, and one from Oryza (CdkC1) [33,34], our Blast searches also retrieved a large group of CDK9-like sequences (14 from Arabidopsis and 8 from Oryza) (Table 1). These kinases are annotated as "Cdc2-like" in databases and some of them also were identified in previous analyses of CDK evolution [38]. With one exception (Os1562.H01.5), all of these kinases group in a single cluster, with 100% support, and as sister to previously identified CDK9s of Arabidopsis and Oryza (additional file 1). Os1562.H01.5 (Gi: 38424086) from Oryza is extremely similar to OsCdkC1 and very likely a second copy of CdkC (CDK9) from Oryza. There is no evidence of biological functions for these kinases as yet, but our results indicate that they are part of a large CDK9 complex specific to plants. The CDK10/11 family In this group, orthologs of CDK10 are found only in human and Drosophila, while CDK11 occurs in human, Drosophila and Caenorhabditis. Three putative CDK11 orthologs were found in plants (two from Arabidopsis and one from Oryza). CDK10 has been implicated in the regulation of the G2/M phase of the cell cycle [52], but a cyclin partner has yet to be defined. Only one protein associated with CDK10, ETS2 transcription factor, has been identified so far, suggesting a link to transcription [9]. CDK11 associates with cyclin L as a partner, and is a proposed component of a signaling pathway that helps to coordinate transcription and RNA-processing events [10-13]. The close relationship between the CDK10 and CDK11 may reflect evolutionary and/or mechanistic similarities, but neither kinase family has been well characterized functionally. In addition, BC18H10 from S. pombe and PfCRK1 from Plasmodium show close relationships to the CDK10/11 family, but no function has yet been determined for these kinases either. Conclusions The apparent co-evolution between the CTD and certain CTD-specific kinases suggests an explanation for strong stabilizing selection on CTD structure in some eukaryotes, and its complete degeneration in others. Based on the genomes examined in this study, either the origins of CDK7 and CDK8 in an unknown ancestor of the "CTD-clade," or the canalization of reversible phosphorylation of the CTD in some eukaryotic groups but not others, could account for the variation seen in RPB1 C-terminal structure. In either case, once thoroughly "locked" into RNAP II function, the CTD must have recruited other transcription and processing related proteins into a growing machinery of the "transcriptosome" [53]. Our results suggest that was the case for several CDKs that clearly predate the canalization of CTD-based RNAP II transcription; further genomic analyses are underway to look for other protein-protein interactions that could be responsible for strong evolutionary conservation of the CTD in members of the "CTD-clade." This work also provides a new perspective on the overall evolution CDKs and evolutionary relationships among kinase families. Our combined genomic and phylogenetic analyses suggest that transcription-related kinases originated later than cell cycle-related CDKs. Finally, our results point to potential functions for a variety of previously uncharacterized kinases, based on their apparent orthologous relationships to defined CDKs. Additional completed genomes, particularly those from broadly diverse protists (especially non-parasitic forms), will be critical to address these questions further. Such comparative analyses will be invaluable in helping to guide experimental studies, which ultimately are required to verify the functional properties of each putative CDK. Methods Identification and alignment of protein sequences Representatives of all previously identified CDKs from budding yeast and human were obtained from Genbank, and used as probes in TBlastN and PSI-Blast [54] against the National Center for Biotechnology Information (NCBI), and additional specific complete genome databases, with an absolute cut-off of E<0.001. To confirm the identities of putative CDKs detected by the TBlastN, each identified sequence was used as a query in reciprocal Blast searches, to verify that it retrieved the original query sequences, and global sequence alignments were performed to confirm putative homologies to CDKs, according to the CDC-related kinase characterized motifs that use CDK2 as the model [55]. Initially, a number of inferred protein sequences were grouped into six subsets according to clear similarities to specific CDK family orthologs. These subgroups first were aligned in CLUSTAL X [56], and the resulting sub-alignments then were aligned with each other and adjusted through visual inspection and comparison to the kinase alignment of Liu and Kipreos (2000) [32]. Regions that could not be aligned reliably were excluded from subsequent phylogenetic analysis. The resulting alignment included 233 positions including gaps (See additional data file 2 and 3 for the original and final aligned matrices used in this study). Phylogenetic analysis Maximum-likelihood (ML) estimates of substitution parameters were made with the program TREEPUZZLE-50 [57] assuming a mixed model for variation among sites, with one category for invariable sites and a four-category discrete approximation to Γ-distribution, and the JTT weighting matrix for probability of change among amino acids. Further analyses were performed in MrBayes 3.0 b4 [58] using metropolis-coupled Markov chain Monte Carlo analysis. Four simultaneous Markov chains were run, also under an invariant + Γ rate model and a JTT substitution matrix. Four chains, one heated, were run for 500,000 generations, beginning with random a priori trees. Trees were sampled from the posterior probability distribution every 100 generations. The empirical burn-in required for likelihoods to converge was less than 100,000 generations; an additional 400,000 generations were run and the first 100,000 were excluded from analysis of Bayesian posterior probabilities. Thus, a total of 4,000 trees were examined to determine the 50% majority-rule consensus tree and Bayesian support values. In addition, 1000 distance (PROTDIST + NEIGHBOR) bootstrap replicates were performed in PHYLIP 3.573 [59], also using a JTT substitution model. Several a priori alternative hypotheses regarding CDK7 evolution were compared by KHT likelihood paired-sites tests [37]. Trees were constrained to require PfMRK from Plasmodium or GlCAKlike from Giardia, which previously have been characterized as a CDK7 orthologs [36,38], to group with the well-defined CDK7 clade. All most parsimonious trees retaining these constrained relationships were tested against the fully resolved Bayesian consensus tree to determine whether the a priori hypotheses of orthologous relationships to CDK7 were significantly worse than the Bayesian consensus tree. Authors' contributions ZG was primarily responsible for database searching and assembly of CDK genes. ZG and JWS performed phylogenetic analysis. ZG drafted the manuscript and figures and JWS contributed editorial revisions. All authors read and approved the final manuscript. Supplementary Material Additional File 1 The single most likely tree, with branch lengths, recovered from 16,000 ML trees in the posterior probability distributions of four separate iterations of Bayesian inference. Thirty-two additional sequences were added to this analysis, and are indicated in bold in Table 1. They represent CDKs identified in incomplete genomes of organisms from which CTD structure is known, as well as a large amplification of apparent plant-specific orthologs of CDK9 from Arabidopsis and Oryza. The phylogenetic weight of these latter plant sequences disrupts inferred relationships among CDK9 orthologs as shown in Fig. 1. Support values are from Bayesian inference and only values above 50% are shown. As in Fig. 1, CDK names in red are from groups in which the CTD is not strongly conserved, those in blue from members of the "CTD-clade." Inferred groups of CTD-directed CDKs 7, 8 and 9 are shown on the tree. Click here for file Additional File 2 Original protein sequence alignment. Click here for file Additional File 3 Edited protein sequence alignment. Click here for file Acknowledgements The material is based on work supported by the National Science Foundation under grant MCB#0133295. 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Biol Evol 2000 17 1061 1074 10889219 Carty SM Greenleaf AL PhosphoCTD-association proteins in the nuclear proteome link transcription to DNA/chromatin modification and RNA procession Mol Cell Proteomics 2002 1 598 610 12376575 10.1074/mcp.M200029-MCP200 Altschul SE Madden TL Schaffer AA Zhang J Zhang Z Miller W Lipman DJ Gapped BLAST and PSI-BLAST: a new generation of protein database search programs Nucleic Acids Res 1997 25 3389 3402 9254694 10.1093/nar/25.17.3389 DeBondt HL Rosenblatt J Jancarik J Jones HD Morgan DO Kim SH Crystal-structure of cyclin-dependent kinase-2 Nature 1993 363 595 602 8510751 10.1038/363595a0 Thompson JD Higgins DG Gibson TJ Clustal W improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice Nucleic Acids Res 1994 22 4673 4680 7984417 Strimmer K von Haeseler A Quartet puzzling: A quartet maximum-likelihood method for reconstructing tree topologies Mol Biol Evol 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==== Front BMC Fam PractBMC Family Practice1471-2296BioMed Central London 1471-2296-5-201538002610.1186/1471-2296-5-20Research ArticlePsychosocial risk factors for obesity among women in a family planning clinic Rohrer James E [email protected] Barbara M [email protected] Department of Family and Community Medicine Texas Tech University Health Sciences Center, USA2 Associate Professor and Regional Chair of the Department of Psychiatry (Amarillo), Texas Tech University Health Sciences Center, 1400 Coulter Blvd, Amarillo TX 79106, USA2004 20 9 2004 5 20 20 11 6 2004 20 9 2004 Copyright © 2004 Rohrer and Rohland; licensee BioMed Central Ltd.2004Rohrer and Rohland; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background The epidemiology of obesity in primary care populations has not been thoroughly explored. This study contributes to filling this gap by investigating the relationship between obesity and different sources of personal stress, mental health, exercise, and demographic characteristics. Methods A cross-sectional survey using a convenience sample. Five hundred women who attended family planning clinics were surveyed and 274 provided completed answers to all of the questions analyzed in this study. Exercise, self-rated mental health, stress, social support, and demographic variables were included in the survey. Multiple logistic regression analysis was performed. Results After adjusting for mental health, exercise, and demographic characteristics of subjects, analysis of the data indicated that that being having a large family and receiving no support from parents were related to obesity in this relatively young low-income primary care sample, but self-reported stress and most types of social support were not significant. Conclusion Obesity control programs in primary care centers directed at low-income women should target women who have large families and who are not receiving support from their parents. ==== Body Background Many variables may influence eating behavior and therefore may also influence obesity, including depression, anxiety, stress, social support, race, ethnicity, education and income [1-8]. While the national media and federal websites emphasize the importance of physical activity in controlling body weight, exercise alone is not effective for this purpose. In fact, some research shows no relationship between exercise and body weight in community samples [1]. Therefore, the important questions are: what other risk factors in addition to exercise may affect obesity and how can they be changed? Studies of stress as a risk factor for obesity are limited. In fact, few epidemiological studies have been reported in primary care journals. An exception is found in a report by Sammel et al, who included a stress index in their study of weight gain among women in their late reproductive years (ages 35–47) [1]. Three hundred and thirty-six women were followed for four years. A 14-item Perceived Stress Scale was used to assess the degree to which situations were stressful to the subject. Women who gained more than ten pounds were not different in regard to this stress measure than other subjects. Stress can be measured in different ways. The personal circumstances experienced by individuals might be expected to have a direct effect on depression, anxiety, and general health status. After all, when conflict arises within a family, the psychological consequences can be dramatic. Even though the relevance of various sources of personal stress to obesity has not previously been examined in the community health literature, their potential importance is worthy of investigation. The purpose of the study reported here was to investigate the importance of personal stressors in determining obesity. The sample was drawn from a low-income female population: women using a family planning clinic for primary care. Exercise, social support, mental health and other personal characteristics were measured and held constant in order to determine the independent effect of different sources of personal stress on obesity. Methods A cross-sectional survey of primary care patients (adult non-pregnant women) attending one of five Planned Parenthood clinics in the Panhandle of Texas was conducted. The Amarillo Institutional Review Board granted exempt status to the study because no protected health information was collected. Planned Parenthood provide a valuable and unique sampling frame because it supplies basic primary care to low-income women in this area, including birth control, but not abortions. Eligibility for the study was limited to patients who were over age 18 and not pregnant. Questionnaires were placed on a table in waiting areas, with a poster inviting participation. Clinic staff also handed out survey forms. Subjects placed the completed forms in a sealed box. Sealed boxes containing survey forms were returned to Texas Tech for data entry. Microsoft Access was used for data entry. Five hundred forms were distributed. Twenty surveys were returned by persons ineligible for study participation and were excluded from the sample. The final data set was comprised of 345 subjects. Computing the response rate as completed returns divided by eligibles (345/(500-20) produces a participation rate of .719. Complete cases were available from 274 subjects for the multivariate analysis. Measures The dependent variable was obesity. Body mass index (BMI) was computed as weight in pounds times 703 divided by height in inches squared. BMI greater than 30 was classified as obese, and made up about 20 percent of the sample. Cases with missing heights and weights were dropped. Independent variables were stress, social support, age, race/ethnicity, education, income, number of persons in the home, exercise, marital status, anxiety, and depression. Key instruments are discussed below. Health items were taken from the Duke Health Profile [9]. Mental health was measured in terms of feeling depressed or sad and nervousness. Possible responses for all of the mental health items were "Yes, describes me exactly", "Somewhat describes me," or "No, doesn't describe me at all." Stress and social support items were taken from the Duke Social Support and Stress Scale (DUSOCS). The DUSOCS contains items addressing personal support from various sources. A person who stresses the respondent is defined as one who causes problems or makes life more difficult [10]. Respondents were asked how much they were stressed by spouses, parents and children. Possible responses were "None," "Some," "A Lot," and "There is No Such Person". Categories were combined into 'none' versus 'a lot or some.' Exercise was measured in terms of times per week (none, one day, two days, three days, four days, more than four). Age, number of persons in the home, race/ethnicity (non-Hispanic white, Hispanic, other), marital status, and educational level (less than high school, high school or GED, more than high school) were used to control for demographic differences among subjects. The median age was 25. Age was categorized as 18–21, 21–30 or 31 or over. Breaks in the age distribution were made at the first and third quartile. Statistical analysis Chi-square tests were used to test for the relationship between each independent variable and obesity. Variables that were significant at p < .10 in univariate tests were included in a multiple logistic regression analysis. EpiInfo 3.2.2 was used for data analysis. Results Nearly half of respondents were classified as being obese (47.8 percent). About half of the respondents reported 'a lot or some' stress from parents or a spouse. 'A lot or some' stress from a child was experienced by about 40 percent. More than three-fourths of respondents said they receive 'a lot or some' support from a spouse or parent. About sixty percent received 'a lot or some' support from a child. Over 90 percent reported 'a lot or some' support from a friend. Table 1 shows the results of the univariate chi-square tests. Of the three stress variables, only parent stress met the selection criterion for inclusion in the logistic regression model (p = .0988). Support from parents was marginally related to obesity (p = .0542) while support from a child was significantly to obesity (p = .0390). Table 1 Psychosocial Risk Factors and Percent Obese in Family Planning Clinics (Chi-square tests) Pct Obese Pct Not Obese p Overall 47.8 52.2 Nervous .6064  None 46.7 53.3  Some 45.2 54.8  A lot 57.1 42.9 Depression .1944  None or some 45.6 54.4  A lot 59.4 40.6 Stress from Parents .0988  None 52.8 47.2  A lot or some 42.7 57.3 Stress from Spouse .8084  None 49.1 50.9  A lot or some 46.8 53.2 Stress from Child .1285  None 44.6 55.4  A lot or some 54.2 45.8 Support from Spouse .1607  None 55.4 44.6  A lot or some 45.6 54.4 Support from Child .0390  None 39.5 60.5  A lot or some 42.2 47.8 Support from Parents .0542  None 61.7 38.3  A lot or some 45.2 54.8 Support from Friend 3472  A lot or some 45.9 54.1  None 57.1 42.9 The sample was comprised of relatively young women, with most being under 30 years of age. Most respondents lived with two or more other people and most had high school degrees. More than one-fourth of respondents were married. Over one-fourth were Hispanic and over half were non-Hispanic White. Most had incomes under $30,000 per year. Over 35 percent got no exercise at all. Table 2 shows the results of univariate chi-square tests for the demographic variables and for exercise. Obesity differed significantly by the number of persons in the home (p = .0047), level of education (.0060), income level (p = .0328), and marital status (p = .0183). Over 58 percent of married respondents were obese, compared to 42.5 percent of unmarried persons. Person who lived alone were much less likely to be obese than persons who lived with four or more people (32.5 percent vs 64.8 percent). Over sixty percent of those lacking high school education were obese, whereas only about 40 percent of those who had more than a high school education were obese. The $10–20,000 income category had the lowest percent obese (36.1). Table 2 Other Risk Factors and Percent Obese in Family Planning Clinics (Chi-square tests) Pct Obese Pct Not Obese p Days of Exercise per Week .3857  None 49.6 50.4  One 57.1 42.9  Two 53.4 46.6  Three 39.6 60.4  Four 31.8 68.2  Five or more 41.7 58.3  Missing 54.5 45.5 Number of persons in home .0047  None 32.5 67.5  One to three 44.7 55.3  Four or more 64.8 35.2 Education .0060  Less than high school 63.6 36.4  High school degree or equivalent 55.3 44.7  More than high school 39.7 60.3 Race/Ethnicity .3747  White, non-hispanic 44.6 55.4  Hispanic 51.1 48.9  Other 54.8 45.2 Income .0328  Less than $10,000 54.2 45.8  $10–20,000 36.1 63.9  $20–30,000 56.4 43.6  Over $30,000 46.3 53.7 Marital Status .0183  Married 58.3 41.7  Other 42.5 57.5 Age .3857  Less than 21 49.2 50.4  21–30 44.6 55.4  over 30 53.7 46.3 'Some' or 'a lot; of nervousness was reported by about one-third of respondents, while over one-third said they were depressed 'some' or 'a lot'. Neither depression nor anxiety was retained for use in the multivariate model, since significance levels were below .10. Variables that were significant at p < .10 were included in the multiple logistic regression model (see Table 3). Women who reported no support from parents had greater odds of being obese (adjusted odds ratio (AOR) = 2.17, p = .0420). Stress from parents and support from a child had no independent relationship with obesity. Persons who lived in homes of four or more were more likely to be obese (AOR = 4.05, p = .0089). Being in the $10,000 to $20,000 income category lowered the odds of obesity in comparison to the under $10,000 category (AOR=.4864, p = .0267). Table 3 Unconditional Logistic Regression of Obesity in Family Planning Clinics (N = 274) Variable Odds Ratio (Conf. Interval) P Stress from parents  (none vs a lot or some) 1.2427 (.73–2.13) .4294 Support from child  (none vs a lot or some) .8949 (.51–1.57) .6994 Support from parent  (none vs a lot or some) 2.1710 (1.03–4.58) .0420 Number in home  One to three vs none 1.8413 (.80–4.24) .1518  Four or more vs none 4.0503 (1.42–11.55) .0089 Income  $10–20 vs less than 10 .4864 (.26–.92) .0267  $20–30 vs less than 10 1.1426 (.54–2.40) .7248  over $30 vs less than 10 .4945 (.20–1.19) .1176 Marital status  Other vs married .6095 (.32–1.16) .1340 Education  High school vs less 1.1435 (.43–3.07) .7900  More than HS vs less .7489 (.29–1.91) .5439 Comparisons of cases with missing obesity information to cases with complete obesity information revealed no significant differences in regard to age, marital status, or income. However, ethnicity and education were significantly different for persons who were missing obesity information. Missings were more likely to be Hispanic or other than non-Hispanic white. Missings also were less likely to have a high school degree or higher. Discussion According to our univariate analysis, the profile of an obese woman in this low-income population is having a large family, less than a high school education, and being married. They also were more likely to fall into income groups above or below the $10,000 to $20,000 range. Variables assessing stress from various sources were not significant at p < .05. Multivariate analysis revealed that receiving no support from parents was independently related to higher rates of obesity, while women in the $10,000 to $20,000 income category were less likely to be obese. The reasons for the income differences are not clear, though varying access to food assistance may offer a partial explanation. Additional investigation of the relationship between diet and income among low-income women is needed. The findings of this study should be treated with caution since it is based on a convenience sample and may not be representative of the population from which it was drawn. In addition, the response rate was not optimal and also a number of cases were dropped from the analysis due to missing information, which reduced statistical power and could have biased our conclusions. However, since the sample was not randomly selected we cannot be sure that dropping cases with missing data made the sample less representative of the low-income female primary care population. An additional limitation of the study was its cross-sectional in design which does not allow for proving causal relationships. Because of these limitations, our results must be considered suggestive rather than definitive. Nevertheless, the findings may be important to primary care physicians, epidemiologists and others who study the determinants of obesity in clinic populations. We could not demonstrate a significant relationship between self-reported stress and obesity in this relatively young, female population after adjustment for other variables. However, personal stress may have indirect effects on obesity, an issue not investigated in this study. Furthermore, the relationship between self-reported (perceived) stress with objective (psychological and physiological) measures of stress in this population group are unknown. Consistent with the findings of our study, Sammel et al did not find stress to be related to obesity in their study of women aged 35–47 [1]. Exercise was not significantly related to obesity in our data. Interestingly, Sammel et al also found no relationship between exercise and obesity 1]. Kaplan et al reported that physical activity was related to obesity in older Canadians, but their sample was quite large (N = 5,980) thus giving them more statistical power [3]. Obesity rates increased with age in our univarate analyses. This is consistent with what has been reported by other investigators [1,3]. We found no independent relationship with education or race, which agrees with Sammel but conflicts with other research [3,4,8]. This contradiction might be due to the fact that our sample was constrained to include primarily low-income women; obesity may be more strongly related to income than race, ethnicity or educational level. We could not show self-assessed depression to be predictive of obesity. Several other investigators have examined this issue, with some seeing obesity as a consequence of depression and others regarding depression to be a result of obesity. In a large study by Carpenter et al [11], increased BMI in women was associated with major depressive disorder as diagnosed in a structured interview using DSM-IV criteria. A study of the National Health and Nutrition Examination Survey (NHANES) also found that obesity was related to depression. Furthermore, Sammel et al reported that weight gain was related to baseline depression, providing some support that depression may be a cause rather than a consequence of obesity [2]. Goodman and Whitaker studied the development and persistence of adolescent obesity and found that depressed mood at baseline was an independent risk factor of persistent obesity [4]. Obesity may also increase the risk of depression in women due to stigma and social isolation related to obesity, particularly among women in western cultures. Since our depression variable was drawn from a single question, it may have contained too much measurement error to permit it to achieve statistical significance in our data set. The reasons why large families increase the risk of obesity are not entirely clear. One obvious mechanism is that, since women traditionally prepare meals, they may have more frequent opportunities to consume food and households with large families are more likely to have greater volumes of food available. Conclusion The research question for this paper was about the risk factors for obesity in a low-income female population in a single community. Our study differs from some other studies of obesity by its inclusion of several types of personal stress as well as social support and mental health measures. We were able to show that personal stress, as defined and measured in this study, was not an important risk factor for obesity in this population group. A limitation of this study is that it does not address occupational stress. Work-related stress has been shown to be related to the health of employees [12-14]. However, since many of the subjects in this sample were not employed, it was not feasible to test hypotheses regarding the relationship between work stress and obesity. Another limitation of the study is its cross-sectional design, which precludes making firm conclusions regarding causality. Despite these limitations, our findings have significance for public health practice related to weight control. Health promotion programs that seek to educate and encourage healthier eating behaviors in low-income female populations should focus on women who are not receiving support from their parents and have large families of their own. In addition, income and eligibility for food assistance may affect dietary practices in unexpected ways, so primary care providers should explore this issue. Competing interests None declared. Authors' contributions JR planned the study, organized the survey, and wrote the first draft of the results. BR wrote the section addressing mental health and obesity. Pre-publication history The pre-publication history for this paper can be accessed here: Acknowledgements This study was partially supported by the Women's Health and Research Institute of Amarillo. Tanya Fraley organized data collection and data management. Support provided by Planned Parenthood of Amarillo was vital to the success of the project. ==== Refs Sammel MD Grisso JA Freeman EW Hollander L Liu L Liu S Nelson DB Battistini M Weight gain among women in the late reproductive years Fam Pract 2003 20 401 409 12876110 10.1093/fampra/cmg411 Onyike CU Crum RM Lee HB Lyketsos CG Eaton W Is Obesity Associated with Major Depression? Results from the Third National Health and Nutrition Examination Survey Am J Epidemiol 2003 158 1139 1147 14652298 10.1093/aje/kwg275 Kaplan MS Huguet N Newson JT McFarland BH Lindsay J Prevalence and Correlates of Overweight and Obesity Among Older Adults: Findings From the Canadian National Population Health Survey J Gerontol A Biol Sci Med Sci 2003 58 1018 1030 14630884 Goodman E Whitaker RC A Prospective Study of the Role of Depression in the Development and Persistence of Adolescent Obesity Pediatrics 2002 110 497 504 12205250 10.1542/peds.110.3.497 Mustillo S Worthman C Erkanli A Keeler G Angold A Costello EJ Obesity and Psychiatric Disorder: Developmental Trajectories Pediatrics 2003 111 851 859 12671123 10.1542/peds.111.4.851 Goodman E Slap GB Huang B The Public Health Impact of socioeconomic Status on Adolescent Depression and Obesity Am J Public Health 2003 93 1844 1850 14600051 Allen J Markovitz J Jacobs DR Knox SS Social Support and Health Behavior in Hostile Black and White Men and Women in CARDIA Psychosom Med 2001 63 609 618 11485115 Haas JS Lee LB Kaplan CP Sonnemorn D The Association of Race, Socioeconomic Status, and Health Insurance Status With the Prevalence of Overweight Among children and Adolescents Am J Public Health 2003 93 2105 2110 14652342 Parkerson GR Broadhead WE Tse C-KJ The Duke Health Profile, A 17-Item Measure of Health and Dysfunction Med Care 1990 28 1056 1072 2250492 Parkerson GR Broadhead WE Tse CK Validation of the Duke Social Support and Stress Scale Fam Med 1991 23 357 60 1884930 Carpenter KM Hasin DS Allison DB Faith MS Relationships between Obesity and DSM-IV Major Depressive Disorder, Suicide Ideation, and Suicide Attempts: Results from a General Population Study Am J Public Health 2000 90 251 257 10667187 Pikhart H Bobak M Siegrist J Pajak A Pywik S Kyshergvi J Gostatas A Skodova Z Marmot M Psychosocial work characteristics and self rated health in four post-communist countries J Epidemiol Health 2001 55 624 630 10.1136/jech.55.9.624 Ala-Mursula L Vahtera J Kivmaki M Kevin MV Pentti J Employee control over working times: associations with subjective health and sickness absences J Epidemiol Community Health 2002 56 272 278 11896134 10.1136/jech.56.4.272 Eriksen W Bruusgaard D Knardahl S Work factors as predictors of sickness absence: a three month prospective study of nurses' aides Occup Environ Med 2003 60 272 278 10.1136/oem.60.4.271

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BMC Fam Pract. 2004 Sep 20; 5:20

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10.1186/1471-2296-5-20

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==== Front BMC Infect DisBMC Infectious Diseases1471-2334BioMed Central London 1471-2334-4-361538002510.1186/1471-2334-4-36Research ArticleSuboptimal clinical response to ciprofloxacin in patients with enteric fever due to Salmonella spp. with reduced fluoroquinolone susceptibility: a case series Slinger Robert [email protected] Marc [email protected] Anne E [email protected] Karam [email protected] Peter [email protected] Christiane [email protected] Baldwin [email protected] Division of Infectious Disease, Children's Hospital of Eastern Ontario, 401 Smyth Rd, Ottawa, ON, Canada2 Division of Microbiology, The Ottawa Hospital, 501 Smyth Rd, Ottawa, ON, Canada3 Division of Infectious Disease, The Ottawa Hospital, 501 Smyth Rd, Ottawa, ON, Canada2004 20 9 2004 4 36 36 22 4 2004 20 9 2004 Copyright © 2004 Slinger et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Salmonella spp. with reduced susceptibility to fluoroquinolones have higher than usual MICs to these agents but are still considered "susceptible" by NCCLS criteria. Delayed treatment response to fluoroquinolones has been noted, especially in cases of enteric fever due to such strains. We reviewed the ciprofloxacin susceptibility and clinical outcome of our recent enteric fever cases. Methods Salmonella enterica Serotype Typhi (S. Typhi) and Serotype Paratyphi (S. Paratyphi) blood culture isolates (1998–2002) were tested against nalidixic acid by disk diffusion (DD) and agar dilution (AD) and to ciprofloxacin by AD using NCCLS methods and interpretive criteria. Reduced fluoroquinolone susceptibility was defined as a ciprofloxacin MIC of 0.125–1.0 mg/L. The clinical records of patients treated with ciprofloxacin for isolates with reduced fluoroquinolone susceptibility were reviewed. Results Seven of 21 (33%) S. Typhi and S. Paratyphi isolates had reduced susceptibility to fluoroquinolones (MIC range 0.125–0.5 mg/L). All 7 were nalidixic acid resistant by DD (no zone) and by AD (MIC 128- >512 mg/L). The other 14 isolates were nalidixic acid susceptible and fully susceptible to ciprofloxacin (MIC range 0.015–0.03 mg/L). Five of the 7 cases were treated initially with oral ciprofloxacin. One patient remained febrile on IV ciprofloxacin until cefotaxime was added, with fever recurrence when cefotaxime was discontinued. Two continued on oral or IV ciprofloxacin alone but had prolonged fevers of 9–10 days duration, one was switched to IV beta-lactam therapy after remaining febrile for 3 days on oral/IV ciprofloxacin and one was treated successfully with oral ciprofloxacin. Four of the 5 required hospitalization. Conclusions Our cases provide further evidence that reduced fluoroquinolone susceptibility of S. Typhi and S. Paratyphi is clinically significant. Laboratories should test extra-intestinal Salmonella spp. for reduced fluoroquinolone susceptibility. ==== Body Background Salmonella enterica Serotype Typhi (S. Typhi) and Serotype Paratyphi (S. Paratyphi) with reduced susceptibility to fluoroquinolones are common in India and Southeast Asia [1]. Such isolates have elevated minimum inhibitory concentrations (MICs) to ciprofloxacin and other fluoroquinolones, although they are still considered "susceptible" using current National Committee for Clinical Laboratory Standards (NCCLS) interpretive criteria [2]. Reduced susceptibility to fluoroquinolones most often arises from point mutations in the quinolone resistance determining region (QRDR) of the gyrA gene which encodes the A subunit of DNA gyrase. These mutations lead to resistance to nalidixic acid, a quinolone, and resistance to this agent can thus be used as an indicator of reduced fluoroquinolone susceptibility [3]. Although the mechanisms of resistance have been defined, there is still little information on the clinical significance of reduced susceptibility to fluoroquinolones in Salmonella. Limited published data suggest that treatment of these infections with a fluoroquinolone may result in a delay in clinical response and possibly treatment failure [2]. As a result of this clinical data, NCCLS currently recommends testing of extra-intestinal Salmonella isolates for nalidixic acid resistance as a marker for reduced fluoroquinolone susceptibility [4]. Others have suggested a re-evaluation of the current fluoroquinolone MIC breakpoints for Salmonella [2] while some recommend differentiating between "low level resistance" (MICs of 0.125 – 1.0 mg/L) and "high level resistance" (MICs >1.0 mg/L) [5]. A poor response to ciprofloxacin therapy in a patient with typhoid fever at our hospital prompted us to review retrospectively the ciprofloxacin MICs and clinical outcomes of other enteric fever cases that were treated with a fluoroquinolone. Methods We tested 21 S. Typhi and S. Paratyphi blood culture isolates recovered from patients at The Ottawa Hospital and The Children's Hospital of Eastern Ontario between 1998–2002. Susceptibility testing was performed using NCCLS methods and interpretive criteria [4,6,7]. Disk Diffusion testing was performed using nalidixic acid (30 μg), ampicillin (10 μg), trimethoprim-sulfamethoxazole (1.25 μg/23.75 μg), and chloramphenicol (30 μg) disks (Oxoid, U.K.) on Mueller-Hinton agar plates (PML Microbiologicals, Mississauga, ON). MICs to nalidixic acid (Sigma-Aldrich, St-Louis, MO) and ciprofloxacin (Bayer Inc.) were determined by agar dilution using Mueller-Hinton agar (Becton Dickinson and Company, Sparks, MD). Reduced susceptibility to fluoroquinolones was defined as a ciprofloxacin MIC of 0.125–1.0 mg/L [3]. We sequenced the QRDR region of the gyrA gene for all isolates with reduced susceptibility to fluoroquinolones to detect nucleotide mutations. PCR amplification and DNA sequencing was performed using primers [8] and conditions previously described [9]. Sequencing was performed by the Ottawa Genome Centre DNA Sequencing Institute using the Big Dye Terminator v 3.1 method. Sequences were compared to the gyrA sequence of the fully susceptible S. Typhimurium NCTC 74 (accession number X78977, EMBL GenBank database) [8] and to 3 fully susceptible S. Typhi isolates. Pulsed field gel electrophoresis (PFGE) was performed on isolates with reduced susceptibility to fluoroquinolone using XbaI. Medical records of patients who were treated with ciprofloxacin for infections due to isolates with reduced fluoroquinolone susceptibility were reviewed to determine clinical outcomes. Results Laboratory Seven of 21 isolates or 33% (4 of 12 S. Typhi and 3 of 9 S. Paratyphi) were nalidixic acid resistant by disk diffusion, all with no zones of inhibition. These isolates were all nalidixic acid resistant with an MIC range of 128->512 mg/L, and demonstrated reduced susceptibility to fluoroquinolones (ciprofloxacin MIC range 0.125–0.5 mg/L). The 14 nalidixic acid susceptible isolates by disk diffusion (zone size range 20–25 mm) were also susceptible by agar dilution (MIC range 2–4 mg/L), and none had reduced susceptibility to fluoroquinolones (ciprofloxacin MIC range 0.015–0.03 mg/L). Multidrug resistance was identified in 5 of 21 isolates (24%) with resistance to ampicillin, trimethoprim-sulfamethoxazole, and chloramphenicol. All 7 isolates with reduced susceptibility to fluoroquinolones had nucleotide point mutations in the gyrA gene that resulted in amino acid substitutions: 5 had a Ser83 to Phe change, one had a Ser83 to Tyr change, and one had an Asp87 to Asn change. PFGE of the 7 resistant isolates showed 3 of 4 S. Typhi to have identical patterns. These 3 strains were also multidrug resistant to ampicillin, trimethoprim-sulfamethoxazole and chloramphenicol. The fourth strain, which was susceptible to ampicillin, trimethoprim-sulfamethoxazole and chloramphenicol, was a closely related strain (2 band difference). Two of 3 S. Paratyphi had identical PFGE patterns, with the third being possibly related (4 band difference) [10]. All 3 isolates of S. Paratyphi were susceptible to the other antibiotics tested. Clinical (Table 1- see Additional file 1) Infections with the 7 isolates with reduced susceptibility to fluoroquinolones were all acquired in the Indian subcontinent. Five of the 7 patients with infections due to these isolates were treated initially with ciprofloxacin. The clinical and microbiological findings for these 5 are summarized in Table 1 (see Additional file 1). S. Typhi isolates from cases 1, 3 and 4 were also resistant to ampicillin, trimethoprim-sulfamethoxazole and chloramphenicol. The Salmonella isolates from the other 2 cases were susceptible to these antibiotics. Of note, 3 of 4 patients with S. Typhi had fever durations well in excess of the expected 95% upper confidence limit of 3.9 days found in a pooled analysis of multiple studies. These studies involved patients with predominantly nalidixic acid susceptible S. Typhi who were treated with fluoroquinolones [1]. The patient infected with S. Paratyphi with reduced susceptibility to fluoroquinolone was judged to be failing ciprofloxacin, since there was no clinical improvement after 3 days of treatment. Although fever of this duration is common, patients with enteric fever given fluoroquinolones may show some clinical improvement by this time. Three days with no amelioration of symptoms has been used as indicator of a poor response to therapy in some studies [11,12]. Thus, this patient possibly may have had a poor response to therapy due to the elevated ciprofloxacin MIC. None of the 5 had a recurrence of bacteremia after antibiotics were discontinued, and none died. Discussion Fluoroquinolones have been considered the treatment of choice for enteric fever. As noted, a mean fever clearance time of only 3.9 days (for predominantly nalidixic acid-susceptible strains) has been reported, which is shorter than for other agents [1]. Calculated clinical and microbiologic failure rates of 2.1% and 0.4%, respectively, are also low in comparison to other antibiotics [1]. Relapse rates (1.2%) and fecal carriage rates (1.5%) have also been lower than for the traditional drugs, trimethoprim-sulfamethoxazole and chloramphenicol. However, reduced susceptibility to fluoroquinolones jeopardises the usefulness of these agents. It is now seen frequently in S. Typhi and S. Paratyphi isolates acquired in the Indian subcontinent and Southeast Asia [1], where common clones appear to be circulating. In fact, the common PFGE profile of 3 of our S. Typhi isolates with reduced susceptibility to fluoroquinolones appears to be the same as that described for S. Typhi strains from India, Pakistan, Bangladesh, and Tajikistan [13]. The gyrA point mutations in our isolates have also been previously described in isolates from these countries [1,3]. Several lines of evidence support the clinical significance of elevated fluoroquinolone MICs in Salmonella spp., including case reports, animal experiments, and pharmacodynamic modelling [2]. In a report of patients treated with short courses of oral ofloxacin [11], the median time to fever clearance was greater for patients infected with nalidixic acid resistant S. Typhi than for those with nalidixic acid susceptible strains. In addition, one third of nalidixic acid resistant infections required re-treatment vs. 0.8% of infections due to susceptible strains. The authors concluded that short courses (less than 5 days) of oral fluoroquinolone therapy should not be used for treating nalidixic acid resistant isolates. Our small series adds to the evidence that there is a delayed clinical response to fluoroquinolone therapy in this setting, and demonstrates that fever may be prolonged even with long courses of fluoroquinolone therapy, and even when given parenterally in some cases. Until results from randomized controlled trials of enteric fever due to strains with reduced susceptibility to fluoroquinolones are available, the best treatment regimen is uncertain. Current options include higher dose and longer duration of fluoroquinolones, 3rd generation cephalosporins and azithromycin, either alone or in combination [1]. If susceptible, agents such as ampicillin, trimethoprim-sulfamethoxazole, and chloramphenicol may also be considered, but rates of resistance to these agents are generally too high to recommend as first-line empiric therapies [1]. In our series, resistance to all these agents was high among the isolates with reduced fluoroquinolone susceptibility (3 of 7 or 43%). When fluoroquinolones are given, it is important to bear in mind that two pharmacokinetic/pharmacodynamic ratios are predictive of successful treatment with these agents [2]. These are the ratio of peak serum antimicrobial level to the MIC, and the ratio of the 24-hour area under the serum concentration-versus-time curve to the MIC (AUC/MIC). Higher dosing will maximize these ratios, and thus when oral ciprofloxacin is given for suspected enteric fever, use of 750 mg rather than 500 mg bid seems logical. It is important that microbiology laboratories have procedures to detect Salmonella strains that have reduced susceptibility to fluoroquinolones. The current NCCLS guidelines (2004) recommend that all extra-intestinal isolates of Salmonella be tested for resistance to nalidixic acid in order to detect reduced fluoroquinolone susceptibility [4]. In addition, it is recommended that physicians be informed that isolates that test fluoroquinolone "susceptible" but nalidixic acid resistant may not be eradicated with fluoroquinolone therapy. There have also been suggestions to change the NCCLS breakpoints to reflect the risk of treatment failure of Salmonella spp. with reduced susceptibility to fluoroquinolones [1]. This is clearly a difficult decision, since fluoroquinolones have previously been the most active antibiotic class for treatment of enteric fever. Some patients with these isolates may do poorly on short courses and lower doses of fluoroquinolones but potentially could respond adequately to higher doses and longer durations of treatment. Rather than reclassifying all isolates with reduced susceptibility to fluoroquinolones as being resistant to ciprofloxacin, others have recommended differentiating between low-level (MIC of 0.125 – 1.0 mg/L) and high-level (MIC >1.0 mg/L) ciprofloxacin resistance based on the MIC range [5]. It is not completely clear at present which of these is the optimal approach for laboratory detection of Salmonella spp. with reduced fluoroquinolone susceptibility. Not all laboratories routinely perform MICs and many rely on commercially-available automated susceptibility methods that cannot currently detect reduced fluoroquinolone susceptibility. The current NCCLS recommendations to test for nalidixic resistance are easy to implement for routine testing but may have reduced specificity [3]. Laboratories need to decide which approach is best for their workflow and available resources. Conclusions Given the accumulating evidence, including our own clinical experience, enteric fever due to Salmonella spp. with reduced susceptibility to fluoroquinolones is clinically significant. It is important that laboratories test S. Typhi and S. Paratyphi, as well as other extra-intestinal Salmonella isolates, for reduced susceptibility to fluoroquinolones. Competing interests None declared. Authors' contributions RS: participated in the design of study and authored first draft; MD: conceived study, coordinated susceptibility testing, and revised manuscript; AM: conceived study and reviewed clinical data; KR: participated in study design and coordinated pulsed field gel analysis; PJ: reviewed clinical data and helped coordinate susceptibilty testing; CB: carried out molecular genetic studies and participated in sequence alignment; BT: conceived study and supervised study, revised manuscript All authors read and approved of the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: Supplementary Material Additional File 1 Clinical and microbiological information for patients treated with ciprofloxacin for S. Typhi or S. Paratyphi isolates with reduced fluoroquinolone susceptibility provides information regarding clinical course for patients with isolates with reduced fluoroquinolone susceptibility as well as antibiotic susceptibility and gene mutation results for these isolates Click here for file ==== Refs Parry CM Hien TT Dougan G White NJ Farrar JJ Typhoid Fever N Engl J Med 2002 347 1770 1782 12456854 10.1056/NEJMra020201 Crump JA Barrett TJ Nelson JT Angulo FJ Reevaluating Fluoroquinolone Breakpoints for Salmonella enterica Serotype Typhi and for Non-Typhi Salmonellae Clin Infect Dis 2003 37 75 81 12830411 10.1086/375602 Hakanen A Kotilainen P Jalava J Siitonen A Huovinen P Detection of Decreased Fluoroquinolone Susceptibility in Salmonellas and Validation of Nalidixic Acid Screening Test J Clin Microbiol 1999 37 3572 7 10523554 NCCLS Performance Standards for Antimicrobial Susceptibility Testing; Fourteenth Informational Supplement NCCLS document M100-S14 Wayne, PA : NCCLS 2004 Threlfall EJ Skinner JA Ward LR Detection of Decreased In Vitro Susceptibility to Ciprofloxacin in Salmonella enterica Serotypes Typhi and Parathypi A J Antimicrob Chemother 2001 48 740 1 11679569 10.1093/jac/48.5.740 NCCLS Performance Standards for Antimicrobial Disk Susceptibility Tests; Approved Standard-Eighth Edition NCCLS document M2-A8 Wayne, PA : NCCLS 2003 NCCLS Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically: Approved Standard-Sixth Edition NCCLS document M7-A6 Wayne, PA : NCCLS 2003 Griggs DJ Gensberg K Piddock LJ Mutations in gyrA Gene of Quinolone-Resistant Salmonella Serotypes Isolated from Humans and Animals Antimicrob Agents Chemother 1996 40 1009 13 8849216 Brown JC Shanahan PMA Jesudason MV Thomson CJ Amyes SGB Mutations Responsible for Reduced Susceptibility to 4-Quinolones in Clinical Isolates of Multi-Resistant Salmonella Typhi in India J Antimicrob Chemother 1996 37 891 900 8737139 Tenover FC Arbeit RD Goering RV Mickelsen PA Murray BE Persing DH Swaminathan B Interpreting Chromosomal DNA Restriction Patterns Produced by Pulsed-Field Gel Electrophoresis: Criteria for Bacterial Strain Typing J Clin Microbiol 1995 33 2233 9 7494007 Wain J Hoa NTT Chinh NT Vinh H Everett MJ Diep S Day NPJ Solomon T White NJ Piddock LJV Parry CM Quinolone-Resistant Salmonella typhi in VietNam: Molecular Basis of Resistance and Clinical Response to Treatment Clin Infect Dis 1997 25 1404 1410 9431387 John M Decreasing Clinical Response of Quinolones in the Treatment of Enteric Fever Indian J Med Sci 2001 55 189 94 11665388 Hampton MD Ward LR Rowe B Threlfall EJ Molecular Fingerprinting of Multidrug-Resistant Salmonella enterica Serotype Typhi Emerg Infect Dis 1998 4 317 320 9621206

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BMC Infect Dis. 2004 Sep 20; 4:36

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BMC Infect Dis

10.1186/1471-2334-4-36

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==== Front BMC Infect DisBMC Infectious Diseases1471-2334BioMed Central London 1471-2334-4-381538314910.1186/1471-2334-4-38Research ArticleA comparative ultrastructural and molecular biological study on Chlamydia psittaci infection in alpha-1 antitrypsin deficiency and non-alpha-1 antitrypsin deficiency emphysema versus lung tissue of patients with hamartochondroma Theegarten Dirk [email protected] Olaf [email protected] Helmut [email protected] Mathias [email protected] Alessandro [email protected] Georgios [email protected] Grigori [email protected] Konrad [email protected] Institute of Pathology, Ruhr University Bochum, BG-Kliniken Bergmannsheil, Bürkle-de-la-Camp-Platz 1, D-44789 Bochum, Germany2 Institute of Bacterial Infections and Zoonoses, Friedrich-Loeffler-Institute (FLI), Federal Research Institute for Animal Health, Naumburger Strasse 96 a, D-07743 Jena, Germany3 Department of Thoracic Surgery, Ruhrlandklinik, Tüschener Weg 40, D-45239 Essen, Germany2004 21 9 2004 4 38 38 2 7 2004 21 9 2004 Copyright © 2004 Theegarten et al; licensee BioMed Central Ltd.2004Theegarten et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Chlamydiales are familiar causes of acute and chronic infections in humans and animals. Human pulmonary emphysema is a component of chronic obstructive pulmonary disease (COPD) and a condition in which chronic inflammation manifested as bronchiolitis and intra-alveolar accumulation of macrophages is common. It is generally presumed to be of infectious origin. Previous investigations based on serology and immunohistochemistry indicated Chlamydophila pneumoniae infection in cases of COPD. Furthermore, immunofluorescence with genus-specific antibodies and electron microscopy suggested involvement of chlamydial infection in most cases of pulmonary emphysema, but these findings could not be verified by PCR. Therefore, we examined the possibility of other chlamydial species being present in these patients. Methods Tissue samples from patients having undergone lung volume reduction surgery for advanced alpha-1 antitrypsin deficiency (AATD, n = 6) or non-alpha-1 antitrypsin deficiency emphysema (n = 34) or wedge resection for hamartochondroma (n = 14) were examined by transmission electron microscopy and PCR. Results In all cases of AATD and 79.4% of non-AATD, persistent chlamydial infection was detected by ultrastructural examination. Intra-alveolar accumulation of macrophages and acute as well as chronic bronchiolitis were seen in all positive cases. The presence of Chlamydia psittaci was demonstrated by PCR in lung tissue of 66.7% AATD vs. 29.0% non-AATD emphysema patients. Partial DNA sequencing of four positive samples confirmed the identity of the agent as Chlamydophila psittaci. In contrast, Chlamydophila pneumoniae was detected only in one AATD patient. Lung tissue of the control group of non-smokers with hamartochondroma was completely negative for chlamydial bodies by TEM or chlamydial DNA by PCR. Conclusions These data indicate a role of Chlamydophila psittaci in pulmonary emphysema by linking this chronic inflammatory process to a chronic infectious condition. This raises interesting questions on pathogenesis and source of infection. ==== Body Background Several species of the family Chlamydiaceae are well-known etiological agents of acute and chronic infections in humans and animals [1,2]. The first description of chlamydial respiratory disease in humans referred to psittacosis, also known as ornithosis, and dates back to 1879 [3]. Chlamydia (C.) psittaci, the agent responsible for this disease, has had several different names and, according to a recent proposal, should now be called Chlamydophila (Cp.) psittaci [4]. A century later, in 1986, Grayston et al. discovered another chlamydial respiratory agent, strain TWAR, which was later assigned to the species C. pneumoniae [5,6] currently reclassified as Chlamydophila pneumoniae [4]. Meanwhile, a variety of respiratory conditions in humans has been shown to be associated with this agent. Evidence of Cp. pneumoniae infection based on serology was reported in severe cases of chronic obstructive pulmonary disease (COPD), in which emphysema is dominant [7,8], as well as in exacerbations of COPD [9] and in persistent infections of the respiratory tract [10,11]. The detection rate of Cp. pneumoniae by immunohistochemical staining was elevated in lung tissue from subjects with COPD, but controls were not completely negative [12]. Initially Cp. pneumoniae was thought to be virulent for humans only, but recent descriptions of isolates from horse, koala, frog and reptiles suggest a wider host spectrum and even the possibility of zoonotic transmission [13-15]. Our previous investigations by means of immunofluorescence using a genus-specific antiserum against chlamydial LPS and scanning as well as transmission electron microscopy showed infection of the alveolar parenchyma and the bronchioles by Chlamydia spp. in patients having undergone lung volume reduction surgery for advanced pulmonary emphysema [16,17]. Accumulation of alveolar macrophages as well as different forms of bronchiolitis and focal pneumonia accompanying emphysematic changes were found regularly [18]. In preliminary examinations using an established nested PCR with DNA hybridization [19], DNA specific of Cp. pneumoniae was detected in two out of ten cases [20]. But this detection rate was far lower than that in electron microscopy or immunofluorescence using genus-specific antibodies, which showed Chlamydia spp. in over 80% [17]. Because of this fact PCR was extended to other Chlamydiaceae. Here we report the results of a more detailed study involving a larger number of cases and samples including controls. Methods Samples Lung tissue of adequate quality from patients with advanced emphysema undergoing lung volume reduction surgery was used for the present study [18,21]. Samples examined by transmission electron microscopy (TEM) included five specimens from alpha-1 antitrypsin deficiency (AATD) and 34 from non-AATD patients. PCR examinations were conducted on six AATD and 31 non-AATD specimens. History showed a status of cigarette smoking with over ten packyears in most of these patients (91.7%). There were two non-smokers among nine patients with AATD. Furthermore patients with hamartochondroma undergoing wedge resection were reviewed for clinical data (A.M.) and histology. Normal lung tissue of 14 non-smokers taken with resection of hamartochondroma was selected as a control group. Statistical analysis was done using SPSS, version 11.5 (SPSS Inc., Chicago, USA) on a PC running Windows XP Professional (Microsoft, Redmond, USA) as operating system. A test value below 0.05 was considered to be statistically significant. Light Microscopy and Transmission Electron Microscopy (TEM) Formalin-fixed lung tissue was embedded in paraffin wax (Tissuewax™; Medite GmbH, Burgdorf, Germany), slides of 3–7 μm thickness were cut using a rotatory microtome (Microm GmbH, Walldorf, Germany) and stained by hematoxylin and eosin. For TEM, tissue was fixed in 2.5% buffered glutaraldehyde or 3.5% formaldehyde and embedded in epon after postfixation with osmium tetroxide and block contrastation with uranyl acetate. In the cases of the hamartochrondroma control group, cores with diameters of 0.60 cm and 0.24 cm were obtained from paraffin blocks using a prototypical self-made manual tissue puncher (a device for tissue microarray construction developed by M.W.). These cores were used to select an area well defined by light microscopy for PCR (0.60 cm cores) and TEM (0.24 cm cores). For TEM, tissue was dewaxed with xylene and processed as described above. Semithin sections (prepared on a Reichert Om U3 ultramicrotome; Reichert, Vienna, Austria) were stained with basic fuchsin and methylene blue to define blocks of adequate quality. Ultrathin sections from two to five blocks were stained with lead citrate and examined using a Zeiss EM 900 transmission electron microscope (Zeiss, Oberkochen, Germany). Polymerase Chain Reaction (PCR) Tissue from two different resources was used. Firstly, frozen tissue was collected immediately after resection and stored at -80°C (n = 31). Secondly, paraffin-embedded tissue (PET) containing histologically discernible inflammation sites was used, and sections or 0.60 cm cores were dewaxed using xylene (n = 12 and 14 controls). In five cases, material was available as both frozen and PET. DNA was isolated from lung tissue using the High Pure PCR Template Preparation Kit (Roche Diagnostics, Mannheim, Germany) according to the instructions of the manufacturer. Five μl of the DNA extract were used as template in PCR. Samples were tested forC. psittaci and Cp. pneumoniae by a modified version of the nested PCR procedure described by Kaltenböck et al. [22], which targets the ompA gene. The first step was genus-specific amplification using primers 191CHOMP (5'-GCI YTI TGG GAR TGY GGI TGY GCI AC-3') and CHOMP371 (5'-TTA GAA ICK GAA TTG IGC RTT IAY GTG IGC IGC-3'). For the second amplification, we used 1 μl of the genus-specific product and primer combination 218PSITT (5'-GTA ATT TCI AGC CCA GCA CAA TTY GTG-3') / CHOMP336s (5'-CCR CAA GMT TTT CTR GAY TTC AWY TTG TTR AT-3') for C. psittaci, or 201CHOMP (5'-GGI GCW GMI TTC CAA TAY GCI CAR TC-3') / PNEUM268 (5'-GTA CTC CAA TGT ATG GCA CTA AAG A-3'), for Cp. pneumoniae, respectively. The sizes of specific amplicons are: 576–597 bp (genus-specific product), and 389–404 bp for C. psittaci or 244 bp for Cp. pneumoniae after nested PCR. A detailed protocol of the procedure is contained in [23]. DNA sequencing In order to discriminate the different members of the C. psittaci-group [4], five μl of the final DNA extract served as template for PCR amplification of the 16S rRNA signature region. Amplicon bands were cut out of agarose gels, extracted using the QIAquick Gel Extraction Kit (QIAGEN, Hilden, Germany), and subjected to cycle sequencing using the BigDye™ Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Darmstadt, Germany). The oligonucleotides 16SIGF (5'-CCG CGT GGA TGA GGC AT-3') and 16SIGR (5'-TCA GTC CCA GTG TTG GC-3') were used as amplification and sequencing primers [4]. Nucleotide sequences were determined on an ABI Prism 310 Genetic Analyzer (Applied Biosystems). Results Light microscopy The median value of age was 46.5 in AATD, 58.0 in non-AATD emphysema and 64.5 in hamartochondroma. The rate of females varied between 33.3% in AATD, 37.5% in non-AATD and 42.9% in hamartochondroma. Histology revealed destruction of the alveoli, intra-alveolar accumulation of macrophages, and acute alongside chronic bronchiolitis in all cases of AATD (Fig. 1A,1B) and non-AATD emphysema (Fig. 2A,2B) consistent with previous examinations. In cases of hamartochondroma only some macrophages and mucus in the bronchioli could be detected (Fig. 3). Figure 1 Histology of alpha-1 antitrypsin deficiency emphysema. In alpha-1 antitrypsin deficiency, advanced panacinar destruction of the lung parenchyma and accumulation of macrophages (A, hematoxylin eosin, original magnification ×40), as well as severe acute and chronic bronchiolitis are seen (B, periodic acid Schiff's reaction, original magnification ×100). Figure 2 Histology of non-alpha-1 antitrypsin deficiency emphysema. In non-alpha-1 antitrypsin deficiency emphysema, chronic respiratory bronchiolitis, destruction of the alveolar architecture, prominent accumulation of macrophages (A, hematoxylin eosin, original magnification ×40) and marked bronchiolitis of the terminal bronchioles is found (B, hematoxylin eosin, original magnification ×100). Figure 3 Histology of normal lung tissue in patients with hamartochondroma. In cases of hamartochondroma, only some macrophages and mucus can be detected in the bronchioli (hematoxylin eosin, original magnification ×15). No signs for emphysema or bronchiolitis could be detected. Transmission electron microscopy TEM images illustrate that the cell and tissue morphology appeared severely destroyed in emphysema samples. Chlamydial elementary and reticular bodies of 0.2 to 0.8 μm diameter were found on the surface of alveolar or bronchiolar epithelium and showed adherence to microvilli as previously described [16,17]. Reticular and some elementary bodies were seen in AATD (Fig. 4) and non-AATD emphysema (Fig. 5A,5B,5C), they were scattered within the interstitium (Fig. 5A) and also assembled in groups (Fig. 5B). Perinuclear inclusions could be detected in fibroblasts (Fig. 5C). Altogether, in 32 cases (82%) typical morphological structures indicating persistent chlamydial infection were present. In seven cases of emphysema, chlamydial bodies could not be detected or the findings were ambiguous. Detection rates were higher in AATD emphysema (5/5 = 100%) than in non-AATD emphysema (27/34 = 79.4%, Table 1). The control group of patients with hamartochondroma showed no signs of chlamydial infection in TEM. Figure 4 Transmission electron microscopy of alpha-1 antitrypsin deficiency emphysema. Chlamydial bodies (arrows) and destruction of the interstitial connective tissue are seen in alpha-1 antitrypsin deficiency. Ultrastructure is less well preserved after fixation in formaldehyde. Figure 5 Transmission electron microscopy of non-alpha-1 antitrypsin deficiency emphysema. In non-alpha-1 antitrypsin deficiency destruction of the connective tissue and chlamydial bodies are detected (A-C). Higher magnification reveals different developmental stages of chlamydial bodies within a lytic area (B) and perinuclear inclusions (arrows) (C). Table 1 Detection of Chlamydia spp. in emphysema by TEM and PCR Groups Cases C. psittaci C. pneumoniae Fisher Yates test vs. controls AATD emphysema TEM 5 5 (100%) 0.00009 PCR 6 4 (66.7%) 1 (16.7%) 0.00310* Non AATD emphysema TEM 34 27 (79.4%) 0.00000 PCR 31 9 (29.0%) 0 (0%) 0.03989 Controls TEM 14 0 (0%) PCR 14 0 (0%) 0 (0%) AATD = alpha-1 antitrypsin deficiency, * = for C. psittaci only Polymerase Chain Reaction (PCR) Examination by PCR revealed the presence of C. psittaci-specific DNA in four (66.7%) specimens with AATD and nine (29%) with non-AATD emphysema (Fig. 6, Table 1). In one case of AATD, the amplicon was identified as Cp. pneumoniae. PCR was negative in all cases with hamartochondroma. The detection rate for C. psittaci in emphysematic tissue was higher from PET than from frozen material (50% vs. 21.9%, Fisher Yates test n. s.). In five cases, where both PET and frozen tissue were examined, one patient was positive in TEM and another one in PCR. Figure 6 Detection of chlamydiae by nested omp A-PCR from frozen lung tissue of patients with advanced emphysema (samples N1 to N43). DNA was extracted from tissue samples, subjected to nested amplification, and PCR products were electrophoresed on 2% agarose gels. The amplicon of approximately 400 bp is specific for Chlamydia psittaci. Strain DC 5 of Chlamydophila psittaci was used as positive amplification control, nc1 and nc2 are negative (reagent) controls. Lane M shows the 100-bp ladder (Invitrogen, Karlsruhe, Germany). DNA sequencing To confirm the identity of the chlamydial species, DNA from four of the positive samples, i.e. N16, N25, N26, and N33, was sequenced in the 16S rRNA signature region (approximately 300 bp, Fig. 7). A BLAST search of these sequences revealed close to 100 % homology to the species Cp. psittaci. Figure 7 Sequence alignment of four tissue samples and reference strains (16S signature region). The samples N16, N25, N33, and N26 were sequenced in the 16S signature region. A BLAST search confirmed the species as Cp. psittaci. Discussion Strains of Cp. psittaci are known to cause infections in over 130 avian species and 32 other domestic and wild animals. Classical psittacosis represents a systemic disease in psittacine birds of acute, protracted, chronic or subclinical manifestation. Avian strains of the agent are known to be pathogenic to humans, the symptoms being mainly non-specific and influenza-like, but severe pneumonia, endocarditis and encephalitis are not uncommon [24,25]. The possibility of persistent infection in man was first described in the 1950s [26]. In the present study and in previous investigations, transmission electron microscopy revealed elementary bodies as well as typical and aberrant reticular bodies, thus indicating active infection alongside persistent infection [10,16,17]. Chlamydiae could not be detected in each case, but the rate of positive findings in TEM and PCR (Table 1) was comparable to that of Cp. pneumoniae in atherosclerosis [27,28]. Pear-shaped elementary bodies as typically found in Cp. pneumoniae infection [29] were not observed. While the findings of TEM are more indicative of Cp. psittaci infection [30], it must be noted that this method provides no clear-cut differentiation among Chlamydiaceae species, for even strains of the same species exhibit different morphology at the various developmental stages. Rather unexpectedly, Cp. pneumoniae was detected only in one sample by PCR, not indicating an important role of this agent in the cases examined here. The fact that, apart from psittacosis, Cp. psittaci has not been associated with human respiratory disorders in recent decades may be a question of sensitivity and specificity of detection. Particularly PCR with its capability to specifically identify all individual species of Chlamydiaceae at a detection limit of less than one inclusion-forming unit has opened up new possibilities in this respect. Higher detection rates of Cp. psittaci in tissue with histological evidence of inflammation in comparison to unselected frozen tissue (6/12 = 50% vs. 7/32 = 21.9%, Fisher Yates test n.s.) indicate an association with regional activity of infection and inflammation. Besides psittacosis, Cp. psittaci has been recently associated with chronic inflammation in patients with ocular adnexal lymphomas [31]. In COPD, activated macrophages and neutrophils produce matrix metalloproteinases which are relevant in the development of emphysema [32,33]. The release of matrix metalloproteinases was shown to be stimulated by cytokines produced in the course of chlamydial infection [34] and by chlamydial heat shock protein 60 as well [35]. These findings represent a link to the established pathogenetic concepts in pulmonary emphysema. Cp. psittaci was found at comparable and statistically significant rates in AATD and non AATD emphysema. Conclusions The fact that the chlamydial agent present in the emphysema tissue was identified by DNA sequencing as an avian serovar of Cp. psittaci provides an important indication on the source of infection. It is conceivable that the patients were infected through contact with birds, although this could not be verified for lack of relevant data on history. The detection rate of chlamydiae in cases of AATD emphysema vs. non-AATD emphysema was clearly higher, thus indicating a relevant role of Cp. psittaci infection in this disorder, or at least a higher susceptibility of AATD patients for an infection of their lungs with Cp. psittaci. Further investigations concerning smokers and non-smokers, pathogenetic relevance and zoonotic implications are required. In any circumstances, Cp. psittaci has to be considered an underestimated pathogen with considerable importance in public health, although the various facets of its specific impact have yet to be evaluated. Competing interests None declared. Authors' contributions Dirk Theegarten has designed and organized this study, done light microscopy, written most of the manuscript, participitated in its statistical analysis and reviewed results of transmission electron microscopy. Olaf Anhenn participitated in designing the study, collected the data, performed statistical analysis and reviewed the manuscript. Helmut Hotzel carried out PCR analysis and DNA sequencing. Konrad Sachse has done the sequence alignments and written the parts of the manuscript concerning molecular biology and veterinary aspects, and also participated in PCR analysis. Mathias Wagner developed and used the tissue puncher for this study. Georgios Stamatis has done lung volume reduction surgery. Alessandro Marra and Georgios Stamatis have done the clinical parts. Alessandro Marra has reviewed the history of the patients. Grigori Mogilevski performed transmission electron microscopy. All authors have discussed and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: ==== Refs Peeling RW Brunham RC Chlamydiae as pathogens. New species and new issues Emerg Infect Dis 1996 2 307 319 8969247 Schachter J Stephens RS Infection and disease epidemiology In Chlamydia Intracellular biology, pathogenesis, and immunity 1999 Washington: American Society for Microbiology Press 139 169 Ritter J Beitrag zur Frage des Pneumotyphus. Eine Hausepidemie in Uster (Schweiz) betreffend Dtsch Arch Klin Med 1879 25 53 96 Everett KDE Bush RM Andersen AA Emended description of the order Chlamydiales, proposal of Parachlamydiaceae fam. nov. and Simkaniaceae fam. nov., each containing one monotypic genus, revised taxonomy of the family Chlamydiaceae, including a new genus and five new species, and standards for the identification of organisms Int J Syst Bacteriol 1999 49 415 440 10319462 Grayston JT Kuo CC Wang SP Altman J A new Chlamydia psittaci strain, TWAR, isolated in acute respiratory tract infection New Engl J Med 1986 315 161 168 3724806 Grayston JT Wang S-P Allegra L, Blasi F History of Chlamydia pneumoniae (TWAR) In Chlamydia pneumoniae The Lung and the Heart 1999 Milan: Springer 1 8 Hertzen Lv Alakärppä H Koskinen R Liippo K Surcel H-M Leinonen M Saikku P Chlamydia pneumoniae infection in patients with chronic obstructive pulmonary disease Epidemiol Infect 1997 118 155 164 9129592 10.1017/S095026889600725X Hahn DL Chlamydia pneumoniae, asthma, and COPD: what is the evidence? Ann Allergy Asthma Immunol 1999 83 339 344 10582711 Mogulkoc N Karakurt S Isalska B Bayindir Ü Celikel T Korten V Colpan N Acute purulent exacerbation of chronic obstructive pulmonary disease and Chlamydia pneumoniae infection Am J Respir Crit Care Med 1999 160 349 353 10390424 Beatty WL Morrison RP Byrne G Persistent Chlamydiae. From cell culture to a paradigm for chlamydial pathogenesis Microbiol Rev 1994 58 686 699 7854252 Hammerschlag MR Chirgwin K Roblin PM Gelling M Dumornay W Mandel L Smith P Schachter J Persistent infection with Chlamydia pneumonia following acute respiratory illness Clin Infect Dis 1992 14 178 182 1571425 Wu L Skinner SJM Lambie N Vuletic JC Blasi F Black PN Immunohistochemical staining forChlamydia pneumoniae is increased in lung tissue from subjects with chronic obstructive pulmonary disease Am J Respir Crit Care Med 2000 162 1148 1151 10988144 Berger L Volp K Mathews S Speare R Timms P Chlamydia pneumoniae in a free-ranging giant barred frog (Mixophyes iteratus) from Australia J Clin Microbiol 1999 37 2378 2380 10364623 Hotzel H Grossmann E Mutschmann F Sachse K Genetic characterization of a Chlamydophila pneumoniae isolate from an african frog and comparison to currently accepted biovars Syst Appl Microbiol 2001 24 63 66 11403400 Bodetti TJ Jacobson E Wan C Hafner L Pospischil A Rose K Timms P Molecular evidence to support the expansion of the hostrange of Chlamydophila pneumoniae to include reptiles as well as humans, horses, koalas and amphibians Syst Appl Microbiol 2002 25 146 152 12086181 Theegarten D Stamatis G Morgenroth K The role of persisting infections in the pathogenesis of pulmonary emphysema. Electron microscopy reveals a probable bacterial colonization of the alveolar space and the bronchioles Pathol Res Pract 1999 195 89 92 10093827 Theegarten D Mogilevski G Anhenn O Stamatis G Jaeschock R Morgenroth K The role of Chlamydia in the pathogenesis of pulmonary emphysema. Electron microscopy and immunofluorescence reveal corresponding findings as in atherosclerosis Virchows Arch 2000 437 190 193 10993281 10.1007/s004280000242 Theegarten D Teschler H Stamatis G Konietzko N Morgenroth K Pathologisch-anatomische Befunde bei der chirurgischen Lungenvolumenreduktion des fortgeschrittenen Emphysems Pneumologie 1998 52 702 706 10028842 Maass M Bartels CB Engel PM Mamat U Sievers H-H Endovascular presence of viable Chlamydia pneumoniae is a common phenomenon in coronary artery disease J Am Coll Cardiol 1998 31 827 831 9525555 10.1016/S0735-1097(98)00016-3 Theegarten D Mogilevski G Anhenn O Stamatis G Maass M Morgenroth K Evidence for chronic Chlamydia pneumoniae infection in pulmonary emphysema Eur Respir J 2000 16 333s Cassina PC Teschler H Konietzko N Theegarten D Stamatis G Two-year results after lung volume reduction surgery in alpha-1-antitrypsin deficiency versus smoker's emphysema Eur Respir J 1998 12 1028 1032 9863992 10.1183/09031936.98.12051028 Kaltenböck B Schmeer N Schneider R Evidence for numerous omp 1 alleles of porcine Chlamydia trachomatis and novel chlamydial species obtained by PCR J Clin Microbiol 1997 35 1835 1841 9196204 Sachse K Hotzel H Sachse K, Frey J Detection and differentiation of chlamydiae by nested PCR In PCR detection of microbial pathogens: Methods and protocols 2002 216 Meth Mol Biol 231 246 Crosse BA Psittacosis: a clinical review J Infect 1990 21 251 259 2273272 Salisch H Malottki Kv Ryll M Hinz K-H Chlamydial infections of poultry and human health Worlds Poult Sci J 1996 52 279 308 Meyer KF Eddie B Human carrier of the psittacosis virus J Infect Dis 1951 88 109 125 14832489 Campbell LA Kuo C-C Grayston JT Chlamydia pneumoniae and cardiovascular disease Emerg Infect Dis 1998 4 571 579 9866733 Shor A A pathologist's view of organisms and human atherosclerosis J Infect Dis 2001 183 1428 1429 11294683 10.1086/319873 Chi EY Kuo CC Grayston JT Unique ultrastructure in the elementary body of Chlamydia sp. strain TWAR J Bacteriol 1987 169 3757 3763 3611029 Vanrompay D Charlier G Ducatelle R Haesebrouck F Ultrastructural changes in avian Chlamydia psittaci serovar A-, B-, and D-infected Buffalo Green Monkey cells Infect Immun 1996 64 1265 1271 8606089 Ferreri AJ Guidoboni M Ponzoni M De Conciliis C Dell'Oro S Fleischhauer K Caggiari L Lettini AA Dal Cin E Ieri R Freschi M Villa E Boiocchi M Dolcetti R Evidence for an association between Chlamydia psittaci and ocular adnexal lymphomas J Natl Cancer Inst 2004 96 586 594 15100336 10.1093/jnci/djh102 Barnes P Chronic obstructive pulmonary disease New Engl J Med 2000 343 269 280 10911010 10.1056/NEJM200007273430407 Shapiro SD The macrophage in chronic obstructive pulmonary disease Am J Respir Crit Care Med 1999 160 S29 32 10556166 Rasmussen SJ Eckmann L Quayle AJ Shen L Zhang Y-X Anderson DJ Fierer J Stephens RS Kagnoff MF Secretion of proinflammatory cytokines by epithelial cells in response to Chlamydia infection suggests a central role for epithelial cells in Chlamydial pathogenesis J Clin Invest 1997 99 77 87 9011579 Kol A Sukhova GK Lichtman AH Libby P Chlamydial heat shock protein 60 localizes in human atheroma and regulates macrophage tumor necrosis factor-α and matrix metalloproteinase expression Circulation 1998 98 300 307 9711934

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==== Front BMC Pregnancy ChildbirthBMC Pregnancy and Childbirth1471-2393BioMed Central London 1471-2393-4-191536959710.1186/1471-2393-4-19Research ArticleA failed RCT to determine if antibiotics prevent mastitis: Cracked nipples colonized with Staphylococcus aureus: A randomized treatment trial [ISRCTN65289389] Amir Lisa Helen [email protected] Judith [email protected] Suzanne M [email protected] Mother and Child Health Research, La Trobe University, Melbourne, Australia2 Department of Microbiological Research, Microbiology and Infectious Diseases, Royal Women's and Children's Hospitals, Melbourne, Australia2004 16 9 2004 4 19 19 26 2 2004 16 9 2004 Copyright © 2004 Amir et al; licensee BioMed Central Ltd.2004Amir et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background A small, non-blinded, RCT (randomised controlled trial) had reported that oral antibiotics reduced the incidence of mastitis in lactating women with Staphylococcus aureus (S. aureus)- colonized cracked nipples. We aimed to replicate the study with a more rigorous design and adequate sample size. Methods Our intention was to conduct a double-blind placebo-controlled trial to determine if an antibiotic (flucloxacillin) could prevent mastitis in lactating women with S. aureus-colonized cracked nipples. We planned to recruit two groups of 133 women with S. aureus-colonized cracked nipples. Results We spent over twelve months submitting applications to five hospital ethics committees and seven funding bodies, before commencing the trial. Recruitment to the trial was very slow and only ten women were randomized to the trial after twelve months, and therefore the trial was stopped early. Conclusions In retrospect we should have conducted a feasibility study, which would have revealed the low number of women in these Melbourne hospitals (maternity wards and breastfeeding clinics) with damaged nipples. The appropriate use of antibiotics for breastfeeding women with cracked nipples still needs to be tested. ==== Body Background Mastitis is a common problem for breastfeeding women [1,2]. Before planning a trial to reduce the number of lactating women who develop mastitis, we reviewed the literature to identify factors that may be associated with mastitis and to examine previous trials. A relatively small number of trials was identified which included mastitis as one of the outcome measures (see Table) [3-13]. Using historical controls, prophylactic topical penicillin ointment was found to be ineffective [3], while hand disinfectant at the mother's bedside appeared to reduce mastitis [7]. A Finnish study examined "breast massage" (which appears to be a variation of "nipple toughening") and found no impact of this practice on mastitis [10]. Table 1 Trials to prevent mastitis Author, date, country Subjects Aim Control(presence/absence/type) Intervention Sample size Outcome: mastitis Hesseltine et al 1948, USA[3] Patients at the Chicago Lying-In Hospital: July – Sept 1946 Does topical penicillin ointment on mother's nipples prevent mastitis? Historical controls: July 1933 to Dec 1946 Penicillin ointment (2,000 units per treatment) on nipples after feeds (6–8 weeks) Intervention 865; Controls 40,629 Intervention: 53 women with mastitis, 6.1%, and 18 with abscess, 2%; Control: 210 women with abscess, 0.51% Sasse 1973, Germany (in German)[4] Postnatal women in the Frauenclinik der Freien Universitat Berlin-Charlottenburg, 1967 Does an antibiotic spray to mother's nipples prevent mastitis? Historical controls Nabectin Puder Spray(neomycin and bacitracin) applied to nipples, plus hand disinfection for nurses and mothers before handling breasts. Intervention130; Controls100 Intervention: 7% mastitis by 2 months; Control: 23% Berger & Pusteria 1981 Switzerland[5] Postnatal women in the Women's Hospital, University of Berne(reported in 1962 [22]) Does nipple ointment prevent mastitis? One group used a nipple ointment without the active ingredient. Not a RCT. Six nipple ointments: (a) boric-acid Vaseline with Peruvian balsam, (a) 1,000 (a) 1.5% (b) chlortetracycline, (b) 1,000 (b) 0.7% (c) chlorquinadol ointment, (c) 1,000 (c) 0.4% (d) base of chlorquinadol ointment (without active ingredient), (d) 1,000 (d) 0.4% (e) calcium pantothenicum, (e) 2,000 (e) 0.8% (f) dihydrofolliculin benzoate and tyrothrycin (f) 1,500 (f) 0.5% Kovalev 1990, Russia (in Russian)[6] Does treating cracked nipples with laser therapy prevent mastitis? Unclear from abstract Laser treatment to damaged nipples 329 women with damaged nipples Intervention reduced mastitis from 18.6% to 3.7% Sytnik 1990, Russia [8] (in Russian)[8] Does bifidobacterium prevent mastitis? Unclear from abstract Bifidobacterium 160 women Mastitis reduced from 6.88% to 1.25% Peters and Flick-Fillies1991, Germany[7,23] Postnatal women in St Hildegardis Hospital, Mainz, 1989–1991 Does the use of bedside hand disinfectant prevent mastitis? Historical controls: 12 months (Sep 1989-Jun 1990, May-Jun 1991) Bed-side disinfectant dispensers: 12 months (Jul 1990-April 1991, Jul-Aug 1991) Intervention: 1095; Control 1230 Intervention 8 women, 0.65%; Control 32 women 2.9%; p <0.001 Waldenstrom and Nilsson 1994, Sweden[9] Women giving birth at South Hospital, Stockhom Is birth centre care beneficial for breastfeeding? Does it increase duration and reduce complications (including mastitis)? RCT Birth centre care compared to standard care Intervention 617; Control 613. Postal questionnaire 2 months postpartum. "Milk stasis" (fever and swelling, redness and tenderness in one of the breasts): Intervention 26%; Control 19% (p = 0.002). "Mastitis" (infective breast treated with antibiotics): Intervention 1%, Control < 1% (p = 0.07) Jonsson & Pulkkinen 1994, Finland[10] Women in South-West Finland Does antenatal / postnatal breast massage prevent mastitis? Concurrent controls. "Breast massage with the hands, a brush, a coarse towel or a sponge before and / or after delivery" Intervention 255, Control 400. Questionnaire 5–12 weeks postpartum at outpatient visit. Overall incidence of mastitis was 24%. No difference in incidence of mastitis (no details given). "This physical training of the nipples neither decreases or increases the frequency of mastitis" (p86) Evans et al 1995, Australia[11] Postnatal women at Flinders Medical Centre, Adelaide Does prolonged feeding on one breast per feed reduce breastfeeding complications, including mastitis? Historical controls: 5 months Advice to feed from one breast per feed and only offer the second breast if the baby still showed signs of hunger rather than standard care of both breasts at each feed: 5 months Intervention 150; Control 152 Telephone interview at 6 months postpartum: Intervention 15%; Control 18% Gunn et al 1998, Australia[12] Women giving birth in one metropolitan hospital and one rural hospital in Victoria, 1995 Does an early visit to a general practitioner reduce problems (including mastitis) compared to the standard six-week postnatal visit? RCT General practitioner visit at one week compared to standard six week visit Intervention 232; Control 243 Postal questionnaire at 3 months. Intervention 11.6%; Control 15.6% (Odds Ratio 0.71, 95%CI: 0.42, 1.20) Livingstone & Stringer, Canada[13] Women attending the Vancouver Breastfeeding Center with a cracked nipple and S. aureus positive culture. Are oral or topical antibiotics more effective in the treatment of S. aureus-colonized cracked nipples than standard care? RCT (not blind to treatment group or outcome) 4 groups: Assessment at 7 days. Oral antibiotics: 1/19, 5%; Other groups: 16/65 (25%) (Fisher exact 0.1) (a) oral antibiotics (a) 19 (b) topical mupirocin (b) 25 (c) topical fusidic acid (c) 17 (d) standard care (d) 23 The authors of one trial were convinced that their intervention was effective, despite methodological difficulties [13,14]. Livingstone and Stringer conducted a randomised trial for women with cracked nipples with positive cultures for Staphylococcus aureus (S. aureus), in Canada[13]. They compared topical antibiotics, oral antibiotics and "optimal breastfeeding advice" and found better improvement in nipple healing in the women given oral antibiotics. In addition, they found 16 women out of 65 (25%) given non-systemic treatment developed mastitis within 7 days, compared to 1 of 19 women (5%) given systemic antibiotics (chi-square, p = 0.065) [not 0.005 as stated in their abstract]. The authors have concluded that cracked nipples colonized with S. aureus should be "treated aggressively with systemic antibiotics". However, the chi-square test used by the authors is inappropriate because one cell contains an expected value less than 5. Using Fisher's exact test, the p value is 0.10 [15]. As the Livingstone and Stringer trial had been published in a major lactation journal and was likely to be very influential in practice [13], it needs to be replicated in a more rigorous manner in order to assess the usefulness and safety of the intervention. Our intention was to replicate that study with an adequate sample size, rigorous definitions of nipple damage and mastitis, and double blinding of the intervention. Methods The aim of our study was to prevent mastitis in breastfeeding women with cracked nipples colonized with S. aureus. A randomised controlled trial was conducted: participating women were randomized to receive a seven day course of either an oral antibiotic (flucloxacillin) or identical placebo capsules. A follow-up visit was arranged one week after recruitment for women with positive nipple culture for S. aureus. Women with negative nipple culture were followed up by telephone at one week. All women received a final telephone interview at six weeks. The primary outcome was the incidence of mastitis in each group in the week following recruitment. In the study by Livingstone and Stringer [13] 30% of women with S. aureus-colonized cracked nipples who received only breastfeeding advice developed mastitis within one week. In order to detect a 50% decrease in incidence, ie mastitis occurring in 15% of women receiving oral antibiotics, a sample size of 133 women in each group is required, with 95% confidence and 80% power. Sample size was calculated using Epi-Info 6. A previous study in Australia found that 62% (13/21) of cultures from breastfeeding women with cracked nipples were positive for S. aureus [16]. An earlier study by Livingstone and colleagues found that 54% of cracked nipples of mothers with infants younger than one month were positive for S. aureus (27/50) [17]. Assuming that 50% of cracked nipples are positive for S. aureus, we would need 133 × 2 × 2 = 532 women with cracked nipples, to recruit two groups of 133 women with S. aureus-colonized cracked nipples. To allow for loss to follow-up, it was planned to recruit 570 women. A review of the literature on the topic of nipple damage found a lack of consistency in assessment of nipple damage [18]. Many reports have not provided a clear description of the assessment process. Some of the more recent studies have provided a more detailed description, such as Brent et al's Nipple Attribute Score and Duffy et al's Nipple Trauma Index [19,20]. The Nipple Trauma Index used by Duffy and colleagues in Western Australia appeared to be useful, however a request for more information about this instrument was not successful (E. Duffy, email 28 February 2001) [20]. Our definitions of nipple damage are as follows: mild 1 or 2 mm wide; moderate 3–9 mm wide; severe: greater than 10 mm wide and / or yellow colour visible in crack. In addition to a clinical assessment, a more permanent record of nipple damage was created using digital photography. It was planned for the photographs to be reviewed independently by three lactation consultants, in order to allow a thorough assessment of nipple damage and changes over time, rather than relying on the clinical assessment alone. (As the trial ended prematurely, this did not take place). Furthermore, although the WHO defines mastitis as an inflammation of the breast [21], there is no generally agreed definition of mastitis for research purposes. The definition of mastitis used for this study was that a woman reported: • at least two breast symptoms (pain, redness, lump) and • at least one of fever or 'flu-like symptoms. Foreseen problems Multi-centred trial As we intended to recruit over 500 women we planned a multi-centred trial, involving a number of public and private maternity hospitals in inner Melbourne. All hospitals provide a breastfeeding clinic staffed with International Board Certified Lactation Consultants for women having breastfeeding difficulties following hospital discharge. The public hospitals, where women tend to have shorter hospital stays, also provide home visits by domiciliary midwives post discharge. It was foreseen that there would be replication in the requirements of the hospital ethics committees and logistical difficulties for one researcher (LA) to conduct the study on multiple sites. Each hospital had its own research ethics committee (or committees) and different forms to submit (at the time of this study). Approval was obtained from the Ethics Committees at La Trobe University (20/11/2000), Royal Women's Hospital (6/9/2000), Mercy Hospital for Women (12/2/2001), Frances Perry House (23/8/2001), Freemasons Maternity Hospital (15/3/2001) and Cabrini Private Hospital (24/04/02). One private hospital did not appear to have a procedure in place to deal with a research proposal. Negotiations continued with this hospital from late 2000 until mid-2002 when the hospital insisted that we sign a Sponsor Indemnity Form, which the university advised us against. The researcher visited the postnatal wards and breastfeeding clinics of these hospitals each day or second day and asked a senior member of the nursing staff if there were any breastfeeding women with damaged nipples in the ward. The staff member introduced the researcher to the woman in order to inform the woman about the study and invite her to participate in the trial. Also, the researcher asked the domiciliary midwives to inform women at home with a cracked nipple about the trial. If the woman were interested in the study, the midwife gave the researcher the woman's name and phone number. After a telephone discussion, the researcher would visit her at home to assess her eligibility. Thus, the researcher was visiting a number of hospitals on a daily basis and making home visits to potential participants and follow-up visits to participants one week after recruitment. Therefore, if the researcher was going to be unavailable one week, she could not recruit women the week prior (as she would not be able to follow them up). Funding All potential participants had a specimen collected from their nipple crack for culture and sensitivity. As this was collected for the purpose of research rather than clinical practice, it was necessary to seek funding for the cost of the microbiological assessment. We intended to recruit 570 women, therefore substantial funds were required. A number of applications (seven) were submitted to local, national and international funding bodies in 2001. A funding application to the Medical Research Foundation for Women and Babies for 2002 was successful (A$15,000). Delay between recruitment and randomization We recognized that there would be a delay between recruitment (when the initial data and nipple specimen were collected) and randomization (when the result was available). The Microbiology laboratory faxed the result to the researcher (or the researcher contacted the laboratory on weekends). However, the minimum time was 2 days for the laboratory to identify S. aureus and up to 6 days in one instance (mean 3.6). The delay meant that women would be at home when the results were available and the researcher was required to visit the participant at her home to deliver the capsules. In addition to the inconvenience, a small number of women had already developed mastitis by the time the researcher contacted her with the result. Unforeseen problems Production of placebo capsules It was expected that a local company specializing in the preparation of placebos for drug trials would prepare the identical capsules. A common practice is to cover the active capsule with a larger capsule; participants are unaware if their capsule contains the active capsule or an inert substance. However, when the company realized that the active capsule contained a penicillin-like drug they were unable to participate, as they do not have a license for penicillin. Finally, the pharmaceutical company, CSL Ltd, provided us with identical empty capsules as well as active flucloxacillin capsules. A pharmacy technician at the pharmacy department at the Royal Women's Hospital opened each capsule manually and inserted glucose powder. Randomisation was conducted in blocks of ten, stratified according to hospital. Ten bottles were prepared for each hospital prior to the trial commencing (further capsules were not needed). Participation Not all the women who were eligible for the trial were interested in taking part (see Figure 1, ROBIn Trial Profile). Some women expressed a reluctance to take antibiotics, others were overwhelmed with the difficulties they were experiencing and preferred not to participate in a trial. The researchers had previously conducted studies involving breastfeeding women which had high rates of participation and had expected women to be more interested in taking part in a trial that aimed to prevent mastitis. We should have expected a lower participation rate as this study involved the possibility of taking a medication, in particular an antibiotic. Figure 1 ROBIn Trial Profile Less than anticipated incidence of cracked nipples A total of approximately 17,000 women give birth in these hospitals each year. We estimated that 80% of women start breastfeeding, 5% develop cracked nipple(s), 80% would be eligible and 95% would agree to participate, thus there would be 537 eligible women per year. We anticipated that we would recruit approximately ten women with cracked nipples per week. It would therefore take 57 weeks (57 × 10) to recruit the total sample. However, recruitment was slow, as very few women were identified with damaged nipples. Hospital staff made unsolicited remarks that nipple damage was seen much less frequently than in the past. Midwives have been trained to help women position the baby and attach the baby at the breast; women are reporting the presence of nipple pain and any nipple damage is usually identified at an early stage. In the past, women may have continued to breastfeed with poor attachment of the baby to the breast, resulting in more severe damage, whereas at the time of the study maternity staff were likely to suggest "resting" the damaged nipple and expressing the milk by hand or electric pump until the damage had healed. Results Recruitment began at two hospitals in November 2001, two others in February 2002 and a fifth hospital in June 2002. Recruitment was slow as few women had damaged nipples. During the months of the trial, the rate of recruitment decreased rather than increased. Additionally, the flucloxacillin supplied by CSL were labeled to use before the end of November 2002. Therefore it was decided to stop recruiting, once a twelve-month recruiting period had elapsed. The trial stopped recruiting on the 14th November 2002. Of the 158 women referred to the study as possible participants, 48 women were eligible (ie they had a cracked nipple, were not allergic to penicillin, did not have concurrent "nipple thrush" and had adequate English). Twenty-six of these women refused (10 not interested, 9 didn't want to take antibiotics, 7 other reason given). Therefore, 22 were potentially eligible in that they had at least one cracked nipple and consented to take part in the trial if the results of the nipple swab confirmed S. aureus. Thirteen of the nipple cultures were positive and ten women were randomized to receive flucloxacillin (n = 5) or placebo capsules (n = 5). Two women had already developed mastitis prior to receiving the results and the third woman had developed a rash and did not want to take the capsules. All women were followed-up at one week and six weeks. Of the ten women in the RCT, one woman in the placebo group developed mastitis (not in the first week of the trial, baby was 32 days old, 28 days after randomization). Three women reported that they had not taken the capsules. When the study was unblinded it showed that all three were in the placebo group. Discussion This trial experienced a number of problems, both foreseen and unforeseen. In the trial conducted by Livingstone and Stringer, there is no mention of women refusing to participate in the study or not taking the treatment they were allocated [13]. It is not reported if any woman developed mastitis in the period between collection of the swab, the clinician receiving the result and the woman being given her allocated treatment regime – indeed the paper does not state that women had to return to the breastfeeding clinic for this. Possibly, women attending a breastfeeding clinic are more likely to comply with treatment regimes than women who are invited to participate in a trial. We thought the estimate of 5% of breastfeeding women developing a cracked nipple was a conservative estimate. For example, in Western Australia, Duffy et al had found that 6% of women in their intervention group had cracked nipples, compared to 69% in their control group [20]. However, on visiting the postnatal wards and breastfeeding clinics in inner Melbourne, it was not unusual to find that the staff were unable to identify any women with damaged nipples. And of the women who were assessed, more than half did not have a cracked nipple. Therefore, nipple damage appears to be uncommon in breastfeeding women in Melbourne. Conclusions In retrospect, we should have conducted a pilot or feasibility study before commencing the trial. The appropriate use of antibiotics for breastfeeding women with cracked nipples still needs to be tested. We hope our experience will be useful for others planning trials of mastitis or nipple damage. Competing interests None declared. Authors' contributions All authors contributed to the design of the trial, LA reviewed the literature, conducted the trial, and wrote the first draft of the paper. All authors approved the final draft of the paper. Pre-publication history The pre-publication history for this paper can be accessed here: Acknowledgements Thank you to the women who participated and the hospital staff who were involved at the Royal Women's Hospital, Frances Perry House, Mercy Hospital for Women, Freemasons Maternity Hospital and Cabrini Private Hospital. Lisa Amir has received a National Health and Medical Research Council Medical Public Health PhD Scholarship, a grant from the Medical Research Foundation for Women and Babies and postgraduate support grants from the Faculty of Health Sciences, La Trobe University. CSL Ltd donated the flucloxacillin capsules and empty placebo capsules. ==== Refs Kinlay JR O'Connell DL Kinlay S Incidence of mastitis in breastfeeding women during the six months after delivery: a prospective cohort study Med J Aust 1998 169 310 12 9785526 Foxman B D'Arcy H Gillespie B Bobo JK Schwartz K Lactation mastitis: occurrence and medical management among 946 breastfeeding women in the United States Am J Epidemiol 2002 155 103 14 11790672 10.1093/aje/155.2.103 Hesseltine HC Freundlich CG Hite KE Acute puerperal mastitis: Clinical and bacteriologic studies in relation to penicillin therapy Am J Obstet Gynecol 1948 55 778 88 Sasse H Gillert K-E Hoffbauer H [Prevention of mastitis by measures against bacterial invasion of the nipple] [Article in German] Med Welt 1973 24 549 51 4704285 Berger M Pusteria E Inflammation of the female breast Sandorama 1981 II 25 27 Kovalev MI [Prevention of lactation mastitis by the use of low-intensity laser irradiation] [Article in Russian] Akush Ginekol (Mosk) 1990 57 61 2339766 Peters F Flick-Fillies D Hand disinfection to prevent puerperal mastitis Lancet 1991 338 831 1681208 10.1016/0140-6736(91)90728-8 Sytnik SI Panichev VA Dautov AG [The use of bifidumbacterin in the prophylaxis of mastitis] [Article in Russian] Vrach Delo 1990 98 100 2368375 Waldenstrom U Nilsson CA No effect of birth centre care on either duration or experience of breastfeeding, but more complications: findings from a randomised controlled trial Midwifery 1994 10 8 17 8159123 10.1016/0266-6138(94)90004-3 Jonsson S Pulkkinen MO Mastitis today: incidence, prevention and treatment Ann Chir Gynaecol Suppl 1994 208 84 87 8092782 Evans K Evans R Simmer K Effect of the method of breast feeding on breast engorgement, mastitis and infant colic Acta Paediatr 1995 84 849 52 7488804 Gunn J Lumley J Chondros P Young D Does an early postnatal check-up improve maternal health: results from a randomised trial in Australian general practice Br J Obstet Gynaecol 1998 105 991 97 9763051 Livingstone VH Stringer LJ The treatment of Staphylococcus aureus infected sore nipples: A randomized comparative study J Hum Lact 1999 15 241 46 10578803 Livingstone V Stringer LJ The treatment of Staphylococcus aureus infected sore nipples: a randomized comparative study (Letter) J Hum Lact 2001 17 116 117 10578803 Amir LH Lumley J The treatment of Staphylococcus aureus infected sore nipples: A randomized comparative study (Letter) J Hum Lact 2001 17 115 16 11847821 Amir LH Garland SM Dennerstein L Farish SJ Candida albicans : Is it associated with nipple pain in lactating women? Gynecol Obstet Invest 1996 41 30 34 8821881 Livingstone VH Willis CE Berkowitz J Staphylococcus aureus and sore nipples Can Fam Physician 1996 42 654 59 8653033 Braund D Amir L Review of the management of nipple pain and damage Topics in Breastfeeding Set XIII 2001 Melbourne: Lactation Resource Centre Brent N Rudy SJ Redd B Rudy TE Roth LA Sore nipples in breast-feeding women Arch Pediatr Adolesc Med 1998 152 1077 82 9811284 Duffy E Percival P Kershaw E Positive effects of an antenatal group teaching session on postnatal nipple pain, nipple trauma and breast feeding rates Midwifery 1997 13 189 96 9511686 World Health Organization Mastitis: Causes and management WHO/FCH/ CAH/0013 2000 Berger M [On 534 cases of puerperal mastitis] [Article in German] Gynaecologia 1962 153 111 15 13867673 Peters F Flick-Fillies D Ebel S [Hand disinfection as the central factor in prevention of puerperal mastitis. Clinical study and results of a survey] [Article in German] Geburtshilfe Frauenheilkd 1992 52 117 120 1568632

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==== Front BMC MedBMC Medicine1741-7015BioMed Central London 1741-7015-2-341536959010.1186/1741-7015-2-34Research ArticleDelta-9 tetrahydrocannabinol (THC) inhibits lytic replication of gamma oncogenic herpesviruses in vitro Medveczky Maria M [email protected] Tracy A [email protected] Thomas W [email protected] Herman [email protected] Peter G [email protected] Department of Medical Microbiology and Immunology, MDC Box 10, University of South Florida, and the H. Lee Moffitt Cancer Center, 12901 Bruce B. Downs Blvd, Tampa, FL 33612-4799, USA2004 15 9 2004 2 34 34 20 2 2004 15 9 2004 Copyright © 2004 Medveczky et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background The major psychoactive cannabinoid compound of marijuana, delta-9 tetrahydrocannabinol (THC), has been shown to modulate immune responses and lymphocyte function. After primary infection the viral DNA genome of gamma herpesviruses persists in lymphoid cell nuclei in a latent episomal circular form. In response to extracellular signals, the latent virus can be activated, which leads to production of infectious virus progeny. Therefore, we evaluated the potential effects of THC on gamma herpesvirus replication. Methods Tissue cultures infected with various gamma herpesviruses were cultured in the presence of increasing concentrations of THC and the amount of viral DNA or infectious virus yield was compared to those of control cultures. The effect of THC on Kaposi's Sarcoma Associated Herpesvirus (KSHV) and Epstein-Barr virus (EBV) replication was measured by the Gardella method and replication of herpesvirus saimiri (HVS) of monkeys, murine gamma herpesvirus 68 (MHV 68), and herpes simplex type 1 (HSV-1) was measured by yield reduction assays. Inhibition of the immediate early ORF 50 gene promoter activity was measured by the dual luciferase method. Results Micromolar concentrations of THC inhibit KSHV and EBV reactivation in virus infected/immortalized B cells. THC also strongly inhibits lytic replication of MHV 68 and HVS in vitro. Importantly, concentrations of THC that inhibit virus replication of gamma herpesviruses have no effect on cell growth or HSV-1 replication, indicating selectivity. THC was shown to selectively inhibit the immediate early ORF 50 gene promoter of KSHV and MHV 68. Conclusions THC specifically targets viral and/or cellular mechanisms required for replication and possibly shared by these gamma herpesviruses, and the endocannabinoid system is possibly involved in regulating gamma herpesvirus latency and lytic replication. The immediate early gene ORF 50 promoter activity was specifically inhibited by THC. These studies may also provide the foundation for the development of antiviral strategies utilizing non-psychoactive derivatives of THC. ==== Body Background The Kaposi's Sarcoma-Associated Herpesvirus (KSHV/HHV-8) is the likely cause of Kaposi's sarcoma in AIDS patients, aging individuals and organ transplant patients [for review, see [1]]. KSHV is also implicated in AIDS-associated primary effusion lymphoma (PEL) and a subset of cases of the lymphoproliferative disorder multicentric Castleman's disease. Epstein-Barr virus (EBV) belongs to the same group of herpesviruses and is also involved in human malignancies such as Burkitt's lymphoma, Hodgkin's disease, nasopharingeal carcinoma, and AIDS-associated lymphoma [for review, see [2]]. Related viruses such as herpesvirus saimiri (HVS) of monkeys and the murine gamma herpesvirus 68 (MHV-68) have been developed as animal models [3-7]. The DNA genomes of these oncogenic viruses persist in nuclei of lymphoid cells in a latent episomal circular form and a few of these cells can produce small amounts of infectious virus. In response to extracellular signals, the latent virus can be reactivated leading to production of more infectious virus progeny. This switch from latent to lytic infection is thought to be important in the pathogenesis of herpesviruses and the spread of infection within the organism and among individuals. Reactivation of latent virus is often initiated by extracellular signals activating through receptors and various transcription factors. Transcription factors activate the promoter of the critical viral gene open reading frame 50 (ORF 50) of KSHV, HVS, MHV 68 or its homologue Rta of EBV [8-16]. The ORF 50 protein is also a transcription factor and further boosts production of its own mRNA [10,13,16]. An important function of ORF50/Rta is to activate early lytic genes [1,2] involved in DNA synthesis. After synthesis of early proteins, the process culminates with expression of late genes leading to production of virion components, virus assembly, release of progeny virus and cell death. The major psychoactive cannabinoid compound of marijuana, Δ9 tetrahydrocannabinol (THC), has been shown to modulate and primarily suppress immune responses against various pathogens [for review see [17,18]]. THC binds to and activates either one of the two cannabinoid receptors (CB1 and CB2) located on the surface of both brain and lymphoid cells [17-26]. CB1 and CB2 belong to the family of G protein-coupled receptors characterized by seven transmembrane loops interacting with the ligand on the outer surface of the cell. The receptors also contain an intracellular signaling domain. Several endogenous natural ligands of the CB receptor family termed endocannabinoids have been described. One example is anandamide, a lipid eicosanoid compound generated by the arachidonic acid pathway. The CB receptors are conserved through various vertebrate species including mammals and even fish. They have been shown in various tissues to modulate signaling and gene activation in response to short-lived endocannabinoid ligands or THC. However, THC has been also proposed to influence cellular function by other mechanisms due to its hydrophobic nature and likely association with lipid structures such as cell membranes [for review, see [17,18]]. This membrane-mediated effect is clearly less specific as all types of cells may be subject to it. Since THC is an immune modulator we hypothesized that it may have an effect on gamma herpesvirus replication and/or latency. The data presented show that THC inhibits reactivation and lytic replication of these herpesviruses, possibly through inhibition of the ORF 50 promoter. Methods Tissue culture cell lines, virus, and THC The KSHV positive primary effusion lymphoma cell line BCBL-1 isolated by Don Ganem and co-workers [27], and BC-3 isolated by Ethel Cesarman and co-workers [28], were obtained through the US National Institutes of Health AIDS Research and Reference Reagent Program (Rockville, MD, USA) and from the American Type Culture Collection (Manassas, VA, USA). The EBV positive P3HR1 cell line was from George Miller [29]. MHV 68 was obtained from Jeffrey Sample (St. Jude Children's Research Hospital, Memphis TN USA). NIH3T12 cells were from Samuel Speck (Yerkes Primate Research Center, Emory University, Atlanta, GA, USA). HVS strain 484 was isolated as described [30]. Owl monkey kidney (OMK) cells were from Danny Daniel (New England Research Primate Center, Southborough, MA, USA). THC was obtained from the National Institute on Drug Abuse, NIH (Bethesda, MD, USA). Three independent batches of synthetic THC were tested that gave very similar results. The purity of these THC preparations exceeded 99%. Antiviral assay based on the Gardella gel method Cell suspensions were loaded in wells of a vertical agarose gel as described [31,32]. The cell layer was then overlaid with a lysis solution containing SDS and pronase resulting in gentle cell lysis and liberation of cellular and viral DNA. The gel was subjected to electrophoresis. Latent episomal DNA migrates much more slowly in these gels than linear replicating DNA [31,32]. After electrophoresis, Southern blotting was performed; DNA was transferred to nitrocellulose followed by hybridization with radiolabeled cloned viral DNA as described [32]. Radioactive images were analyzed and quantitated by a phosphoimager. The antiviral effect (IC50) was quantitated by comparing the values obtained from the episomal bands with those from linear bands as described [32]. Virus yield reduction and cytotoxicity assays NIH3T12 cells were infected with MHV 68, OMK cells with HVS, and both cell types with HSV-1 at a multiplicity of infection of 2 in the presence or absence of various concentrations of THC dissolved in DMSO. Control cultures were treated with DMSO. IC50 was determined by measuring virus titers in THC treated and control samples after one cycle of freeze-thawing of the cultures. Additional controls were uninfected cells grown in the presence of THC or DMSO. THC or DMSO was present throughout the experiment. Dual luciferase promoter assays To evaluate the effect of THC on the KSHV ORF 50 promoter, a DNA fragment corresponding to the promoter region upstream of the mapped mRNAs [reference [9], nucleotides 70513–71513] was cloned into the basic firefly luciferase vector (Promega, Inc., Madison Wisconsin). To assay the effect of THC on the MHV 68 ORF 50 gene, a DNA fragment containing the 0.4 kb full length ORF 50 promoter (nucleotides 66242–66652) cloned upstream of the firefly luciferase was provided by Dr. Samuel Speck [46,47]. About 5 μg KSHV ORF 50 firefly plasmid DNA was transfected into BCBL-1 cells. About 5 μg MHV 68 ORF 50 firefly plasmid DNA was transfected into NIH312 cells. Control renilla luciferase expression vector (under the control of the CMV immediate early promoter) was included and co-transfected along with the ORF 50 reporter constructs. Cells were incubated for 48 h with either 5 μg/ml THC solution or an equal amount of DMSO. Luciferase activities were determined using a dual luminometer. Results THC inhibits KSHV and EBV DNA replication To evaluate the effect of THC on KSHV replication, we selected BCBL-1 and BC-3 lymphoblastoid cells because these cultures spontaneously produce small amounts of virus, and we used a previously published antiviral drug testing protocol [32] to determine whether THC induces or inhibits virus replication. Dead cells from BCBL-1 or BC-3 lymphoblastoid cell cultures were removed by Ficoll gradients. Cells were cultured in RPMI 1640 growth medium containing 10% serum for 3 days in the presence or absence of various concentrations of THC (dissolved in DMSO). Control cells were cultured in the presence of 0.1% DMSO (this concentration of DMSO was also used in THC treated cultures). Cells were suspended and loaded in wells of a vertical agarose gel, then overlaid with a lysis buffer to remove proteins from DNA. After lysis, slowly migrating episomal DNA representing the resident latent viral genome, and fast migrating linear DNA representing lytic virus replication, were separated by electrophoresis. After electrophoresis, viral DNA was visualized by Southern blotting. Figure 1, left panel shows that control DMSO-treated BCBL-1 cells spontaneously produce a small amount of linear DNA, indicating lytic replication/reactivation in a subpopulation of the cells. Various concentrations of THC showed concentration-dependent inhibition of linear but not episomal KSHV DNA. These data were reproducible and similar observations were made in five independent experiments. Based on these experiments we calculated the 50% inhibitory concentration (IC50) of THC at 1 μg/ml or about 3.3 μM. To evaluate whether another KSHV strain is also susceptible to THC, the BC-3 cell line was also tested. In these cells, THC had a similar inhibitory effect on KSHV reactivation as observed with BCBL-1 cells (not shown). Two experiments analogous to the one described for KSHV were performed with the EBV transformed B cell line P3HR1. P3HR1 cell cultures were grown in the presence of the phorbol ester TPA, because EBV did not reactivate spontaneously and TPA was essential to induce lytic EBV replication. The cultures were also supplemented with either various concentrations of THC dissolved in DMSO or with equivalent volumes of DMSO. Fig. 1, right panel, shows a representative experiment. The autoradiogram indicates that THC inhibited EBV linear DNA synthesis. Based on quantitative analysis of the radioactive bands and extrapolation of data, the 50% inhibitory concentration (IC50) for THC was estimated at around 0.9 μg/ml or about 3 μM. THC inhibits MHV 68 and HVS lytic replication in monolayer cells We examined the effects of THC on the replication of MHV68 and HVS, two rhadinoviruses related to KSHV and to lesser extent to EBV. These viruses can infect monolayer cells and are suitable for testing the effects of compounds on lytic virus replication by virus yield reduction assays, a more widely used test for evaluating antiviral drugs. NIH3T12 cells were infected with MHV68 at a multiplicity of infection of 2 in the presence or absence of various concentrations of THC dissolved in DMSO. Control cultures were treated with DMSO. THC or DMSO was present throughout the experiment. Cell cultures were incubated for 48 h. As shown in Figure 2, a typical full cytopathic effect of MHV 68 was observed in the control infected cell culture treated with DMSO; most adherent cells become detached from the plate, and remaining loosely adhered cells were round and denser than uninfected controls. However, when infected cells were cultured in the presence of 1.25 μg/ml or higher concentrations of THC, they remained indistinguishable from uninfected control cells and remained adhered to the plate. The effect of 0.6 μg/ml of THC was intermediate between uninfected cells and the full cytopathic effect. These results indicated a protective effect of THC against destruction of host cells by the virus and suggested that THC may inhibit MHV 68 replication. Similar results were obtained with HVS in owl monkey kidney cells (not shown). To quantitatively determine antiviral effects of THC, yield reduction assays were performed. NIH3T12 cell cultures were infected with MHV 68 and incubated with THC or DMSO dilutions for 48 h. Cell-associated virus was liberated by freeze-thawing. After low speed centrifugation, virus titer was determined in the culture supernatants. Figure 3 shows that virus yield (expressed as infectious units of virus per ml) was highly significantly inhibited by THC. Inhibition of virus yield was over 300-fold at 10 μg/ml THC. The 50% inhibitory concentration (IC50) was estimated at around 0.6 μg/ml, equivalent to about 1.9 μM. Similar results were obtained from two independent experiments. Similar and reproducible results were obtained with HVS in monkey kidney cells (not shown). THC is not cytotoxic to murine NIH3T12 or owl monkey kidney (OMK) cells Because the observed effects might be due to a non-specific toxic effect of THC, we tested whether THC alters cell division or morphology in NIH3T12 and owl monkey kidney (OMK) cells. Monolayers of these cells were prepared at about 25% confluency and cultured for 2 days in the presence of THC concentrations ranging 0.6–10 μg/ml. A series of photographs (Figure 4) show that THC treated cultures were indistinguishable from control cultures, formed confluent monolayers, and showed no altered morphology. Owl monkey kidney (OMK) cells also showed no toxicity in an analogous experiment (not shown). The effect of higher THC concentrations on cell division was also determined. Exponentially growing BCBL-1 or NIH12 cells were cultured for two days in the presence of various concentrations of THC or DMSO and cell counts were determined. The 50% cell division inhibitory concentration of THC for BCBL-1 cells was around 33 μM. NIH3T12 cells were less sensitive and the 50% inhibitory concentration was around 99 μM (not shown). THC has no comparable effect on HSV-1 lytic replication We next examined whether or not the THC inhibitory effect can be observed against other herpesviruses and tested whether drug treatment suppresses the replication of the alpha herpesvirus, HSV-1. Monolayers of NIH3T12 were prepared, infected with about 100 infectious units of HSV-1, and cultured for 3 days in the presence of THC concentrations ranging 0.6–10 μg/ml. A series of photographs shows (Figure 5) that typical plaques developed in THC treated cultures, indistinguishable from those in control cultures. HSV-1 replication in OMK was also unaffected by THC in an analogous experiment (not shown). We also examined the effects of THC on production of HSV-1 virus yield in NIH3T12 cells. Cells were infected with HSV-1 at a multiplicity of infection of 2 in the presence or absence of various concentrations of THC dissolved in DMSO. Control cultures were treated with DMSO. THC or DMSO was present throughout the experiment. Cell cultures were incubated for 24 h and cell-associated virus was liberated by freeze-thawing. After low speed centrifugation the HSV-1 titer was determined in the culture supernatants. Figure 6 shows that THC had no significant inhibitory effect on replication of HSV-1 in NIH3T12 cells. THC inhibits the ORF 50 promoter To evaluate the effect of THC on the MHV 68 and KSHV ORF 50 promoters, luciferase reporter assays were performed. To assay the effect of THC on the MHV 68 gene, a DNA construct published by the Speck laboratory [46,47] containing the 0.5 kb full length ORF 50 promoter cloned upstream of the firefly luciferase was transfected into NIH312 cells. Control renilla luciferase expression vector (under the control of the CMV immediate early promoter) was included and co-transfected with the ORF 50 reporter. Cells were incubated for 48 h with either 5 μg/ml THC or equal amount of DMSO. Luciferase activities were determined using a dual luminometer. The results show (Table 1) that 5 μg/ml THC suppressed the ORF 50 promoter about 7.4 fold. In contrast, the control CMV promoter activity was only reduced by 35%. Similar results were obtained with the KSHV ORF 50 promoter construct containing 1 kb of the promoter sequence. In the transfected BCBL-1 cells, 5 μg/ml THC inhibited the ORF 50-driven luciferase activity almost 4-fold. Interestingly, the co-transfected CMV promoter was slightly stimulated by THC in these cells. These results were obtained from three independent transfection experiments for both the MHV 68 and KSHV promoter assays. Cannabinoid receptor antagonists can reverse the inhibitory effect of THC on KSHV replication To investigate whether the observed antiviral effects of THC are mediated through the cannabinoid receptors, two antagonists of THC, SR141716A (which acts on CB1) and SR144528 (which inhibits CB2; a gift from Dr. Pierre Casallas, Sanofi Recherche), were tested in the standard BCBL-1 KSHV reactivation assay (Figure 7). BCBL-1 cells were incubated with or without THC and in the presence of these compounds. The THC inhibition of KSHV DNA synthesis was not reversed by treatment with a single receptor antagonist (not shown). The antagonists had no effect on spontaneous KSHV reactivation. However, Figure 7 shows that they reversed the inhibition of linear DNA synthesis by 1.25 μg/ml THC. Discussion To summarize the antiviral effects of THC and to compare THC with well-characterized antiviral drugs, we compiled data from the literature as well as from our own experimental results. Table 2 shows the 50% inhibitory concentrations (IC50) of four known antiviral drugs and of THC. Data on the antiviral effects of acyclovir, PFA and ganciclovir on KSHV were obtained from our Gardella gel assays [32]. Usherwood et al. [33] also used a Gardella gel-based assay to determine the effect of acyclovir against MHV 68 in transformed B cell line S11. The data regarding the effects of four antiviral drugs against MHV 68 and on cell division are from the work of Neyts and De Clercq [34] using standard lytic virus replication inhibition assays on monolayers. The THC antiviral results are from the data described in this paper (Gardella gels for KSHV using BCBL-1 cells and NIH12 antiviral assays for MHV 68). These data suggest that THC is a potent and selective antiviral agent against KSHV comparable with some well-characterized anti-herpesvirus compounds. THC is even more potent and selective against MHV 68 than acyclovir, ganciclovir and foscarnet. Cidofovir appears to be most potent in these in vitro experiments; however, this drug is known to cause serious side effects and is toxic to the kidney in humans. As outlined earlier, THC can modulate and inhibit the activation of immune cells, so it is not entirely surprising that it can down-regulate the reactivation of viruses residing in lymphocytes, as shown by the data. However, the antiviral effect of THC is not cell specific, since MHV 68 and HVS replication was also strongly inhibited by THC in NIH3T12 or owl monkey kidney monolayer cell cultures. This observation suggests that THC either directly or indirectly targets a viral gene shared by these herpesviruses. The data presented in Figure 7 suggest that THC may inhibit KSHV replication through the cannabinoid receptors. When BCBL-1 cells were treated with THC the receptor antagonists partially reversed this effect, suggesting a role for the CB receptors expressed by BCBL-1 cells. However, more studies are required to evaluate whether CB1, CB2 or both receptors are involved. As discussed in the Introduction, ORF 50/Rta is a critical gene for both reactivation of latent virus and lytic replication in monolayers. Interestingly, ORF 50/Rta activation involves cAMP signaling [12,35]. In contrast, cannabinoid receptor binding has been shown to down-regulate the level of activated CREB through a decrease in cyclic AMP synthesis [17,18,26,36]. Therefore, one possible explanation is that THC inhibits cAMP signaling, leading to decrease of ORF 50/Rta-mediated transcription and block of virus replication. This antiviral mechanism of THC is supported by our data. Luciferase reporter assays showed that in the presence of THC, initiation of transcription of ORF 50 mRNA in both KSHV and MHV 68 is markedly reduced (4-fold and 7.4-fold, respectively) as compared with the CMV immediate early promoter. These data suggest selective inhibition of the ORF 50 promoter of MHV 68 and KSHV by THC. However, it is also anticipated that THC may also block other cellular and viral genes,, as this drug has been shown to cause a wide range of changes in lymphocyte gene expression [17,18]. Conclusions Early studies have attempted to evaluate whether THC has effects on HSV-1 and HSV-2 replication [37-44]. Most of these studies concluded that THC directly or indirectly enhances replication/reactivation of these viruses, although Lancz et al. [42] showed that a very high concentration (330 μM) decreases the infectivity of virions. Our data presented in this paper show no effect of THC on HSV replication at lower concentrations. To resolve these conflicting observations, investigation of this issue should continue in the light of new advances in herpesvirus molecular biology and cannabinoid research. Interestingly, statistical analysis indicates a lower incidence of Kaposi's sarcoma in HIV positive women using non-intravenous drugs [45]. About 5.4% of HIV positive women with no drug use developed KS, whereas none of the 47 women in this study who only used marijuana suffered from KS [ref. [45], and James Goedert, personal communication]. This report, however, involved relatively few individuals so further analysis of a larger cohort is warranted. We believe that studies on cannabinoids and herpesviruses are important to continue because there are obvious potential benefits. Better understanding may lead to the development of specific non-psychoactive drugs that may inhibit reactivation of oncogenic herpesviruses. Competing interests None declared. Authors' contributions MMM carried out most of these studies. TAS carried out some of the luciferase assays. TWK and HF participated in the design of the study. PGM conceived the study and participated in its design and overall coordination. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: Acknowledgements We thank Dr. Samuel Speck for providing the MHV 68 ORF 50 luciferase reporter construct. This work was partly supported by a grant from the National Institute of Health RO1 DA10683. Figures and Tables Figure 1 THC Inhibits of KSHV and EBV linear DNA replication. BCBL-1 or P3HR1 cell cultures were grown in standard RPMI/fetal calf serum medium. Cultures were supplemented with either various concentrations of THC dissolved in DMSO (as indicated) or with equivalent volumes of DMSO. After three days, cells were analyzed for latent episomal and lytic linear viral genomes as described [31,32]. Briefly, 106 cells were loaded in wells of a vertical agarose gel then overlaid with a lysis solution containing SDS and pronase, resulting in gentle cell lysis and liberation of cellular and viral DNA. The gel was subjected to electrophoresis. After electrophoresis, Southern blotting was performed and DNA was transferred to nitrocellulose followed by hybridization with radiolabeled overlapping cosmid clone probes of KSHV, representing the entire genome, as described [32] (left panel) or with radiolabeled cloned Bam W fragment of EBV (right panel). Latent episomal DNA migrates much more slowly in these gels than linear replicating DNA [31,32]. Arrows on the autoradiograms indicate the position of both species of DNA. Figure 2 THC inhibits MHV 68 cytopathic effect in NIH3T12 cells. Photograph of NIH3T12 infected with MHV 68 at a multiplicity of 2. Plates were cultured for 48 h in the presence or absence of various concentrations of THC (0.625–10 μg/ml) or equivalent volumes of DMSO. Figure 3 THC inhibits MHV 68 virus yield in NIH3T12 cells. NIH3T12 monolayer cultures propagated in 24-well plates were infected and cultured in the presence of various concentrations of THC or equivalent DMSO. Forty-eight hours after infection, control cultures showed complete cytopathic effect and destruction of cells. To liberate cell-associated virus, cultures were subjected to a cycle of freeze-thawing; cells were homogenized and virus was titrated by end point dilution in 96-well plates. Figure 4 THC is not toxic to NIH3T12 cells. NIH3T12 cells seeded at about 1/4th of confluency were cultured for 48 h in the presence of various concentrations of THC (0.625–10 μg/ml) and photographed. Figure 5 THC has no significant effect on plaque formation of HSV-1 in NIH3T12 cells. NIH3T12 monolayer cultures propagated in 6-well plates were infected with about 100 infectious units of HSV-1 per plate and cultured in the presence of various concentrations of THC or equivalent DMSO solvent. Photographs were taken four days after infection. Figure 6 THC has no significant effect on virus yield of HSV-1 in NIH3T12 cells. NIH3T12 monolayer cultures propagated in 24-well plates were infected with HSV-1 at a multiplicity of 2 and cultured in the presence of various concentrations of THC or equivalent DMSO. Twenty-four hours after infection, all cultures showed complete cytopathic effect and destruction of cells. To liberate cell-associated virus, cultures were subjected to a cycle of freeze-thawing; cells were homogenized and virus was titrated. Figure 7 Effect of receptor antagonists on THC mediated suppression of KSHV reactivation. BCBL-1 cells were cultured in the presence of 1.25 μg/ml THC (lane THC), 1.25 μg/ml THC plus 5× molar excess of SR141716A and SR144528 for 72 h (lane Anti1+2), or in medium containing equivalent amount of solvent (DMSO). Cells were analyzed for episomal control and linear replicating DNA as described in Figure 1. Table 1 Relative luciferase activity of extracts from control and THC treated cells. NIH 3T3 cells co-transfected with MHV 68 ORF 50 plus CMV control (top rows of the table). BCBL-1 cells were co-transfected with the KSHV ORF 50 plus CMV control (bottom rows of the table). Data represent averages from three independent co-transfection experiments. Treatment of NIH 3T3 cells MHV 68 ORF 50 promoter (firefly luciferase) CMV renilla luciferase DMSO 100% +/- 14% 100% +/- 11% 5 μg/ml THC 13.5% +/- 1.5% 65.8% +/- 9% Treatment of BCBL-1 cells KSHV ORF 50 (firefly luciferase) CMV renilla luciferase DMSO 100% +/- 14% 100% +/- 3% 5 μg/ml THC 28% +/- 13% 128% +/- 3% Table 2 Comparison of 50% antiviral and cell division inhibitory concentrations (IC50) and selectivity (viral versus cellular IC50) of selected antiviral drugs and THC Inhibitor compound KSHV IC50 (μM) Cellular IC50 (μM) Selectivity index (viral versus cellular IC50) Acyclovir 75 [ref. 32] Not done Ganciclovir 5.1 [ref. 32] Not done Foscarnet 97 [ref. 32] Not done Cidofovir 0.05 [ref. 32] Not done THC 3.3 (this work) 33 (this work) 10 (this work) MHV 68 IC50 (μM) Acyclovir 6 [ref. 34] 100 [ref. 33] 182 [ref. 34] 30 [ref. 34] Ganciclovir 28 [ref. 34] 108 [ref. 34] 3.7 [ref. 34] Foscarnet 120 [ref. 34] 1413 [ref. 34] 11 [ref. 34] Cidofovir 0.08 [ref. 34] 78 [ref. 34] 10,000 [ref. 34] THC 1.9 (this work) 99 (this work) 52 (this work) ==== Refs Olsen S Moore P Medveczky P, Friedman H, Bendinelli M Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8) and the etiology of KS In Herpesviruses and Immunity 1998 New York, NY: Plenum Publishing Co 115 147 Kieff E Fields BN, Knipe DM, Howley PM Epstein-Barr virus and its replication In Fields Virology 1996 Philadelphia, PA: Lippinscott Raven 2343 2396 Melendez LV Daniel MD Hunt RD Garcia FG An apparently new herpesvirus from primary kidney cultures of the squirrel monkey (Saimiri sciureus) Lab Anim Care 1968 l8 374 38l Melendez LV Hunt RD Daniel MD Garcia F Fraser CEO Herpesvirus saimiri II. 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==== Front BMC PsychiatryBMC Psychiatry1471-244XBioMed Central London 1471-244X-4-271536310510.1186/1471-244X-4-27Research ArticleElevated midbrain serotonin transporter availability in mixed mania: a case report Tolmunen Tommi [email protected] Mikko [email protected] Pirjo Irmeli [email protected] Hanna [email protected] Pasi [email protected] Ritva [email protected] Jyrki [email protected] Jari [email protected] Johannes [email protected] Department of Psychiatry, Kuopio University Hospital, 70210 Kuopio, Finland2 Department of Clinical Physiology and Nuclear Medicine, Kuopio University Hospital, 70210 Kuopio, Finland3 Department of Clinical Radiology, Kuopio University Hospital, 70210 Kuopio, Finland4 Department of Forensic Psychiatry, University of Kuopio, 70240 Kuopio, Finland2004 13 9 2004 4 27 27 16 4 2004 13 9 2004 Copyright © 2004 Tolmunen et al; licensee BioMed Central Ltd.2004Tolmunen et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Results obtained from brain imaging studies indicate that serotonin transporter (SERT) and dopamine transporter (DAT) densities are altered in major depression. However, no such studies have been published on current mania or hypomania. Case presentation In this single photon emission computed tomography (SPECT) study with [123I]nor-β-CIT we present a case with simultaneous symptoms of major depression and hypomania. She had an elevated serotonin transporter availability (SERT) in the midbrain and elevated dopamine transporter availability (DAT) in the striatum, which normalised in a one-year follow-up period during which she received eight months of psychodynamic psychotherapy. Conclusions To our knowledge, this is the first report on SERT and DAT associated with mania. In our case the availability of both SERT in the midbrain and DAT in the striatum were elevated at baseline and declined during psychotherapy, while the SERT and DAT of the depressed controls increased during psychotherapy. Symptoms of hypomania in the case were alleviated during psychotherapy. Clinical recovery was also reflected in the Hamilton Depression Rating Scale (HDRS) scores. ==== Body Background Decreased serotonin transporter densities (SERT) have been recorded from the brains of depressed patients [1,2], which have also been reported to normalise during psychotherapy [3,4]. A few studies have also revealed altered levels of dopamine transmission in major depression [5,6]. However, in a search of the PubMed database we found no serotonin-specific brain imaging studies on mania or hypomania. In this paper we present a case with simultaneous symptoms of major depression and hypomania. The structural diagnostic classifications DSM-IV and ICD-10 are unable to diagnose this kind of mixed state, even though similar cases have been presented earlier in the literature [7]. Materials and methods Case The index subject was a 25-year-old female with a history of two periods of major depression according to the DSM-IV-R criteria. She had also had hypomanic periods that did not completely fulfil the criteria of mania, and at baseline she fulfilled the criteria of hypomania, moderate depression and dysthymia (296.32, 300.40). She was overactive, restless and irritated and coped with a reduced amount of sleep. She had difficulty in concentrating on one thing at a time, and she appeared very lively and talkative. She was therefore diagnosed with bipolar mood disorder type II. In other words, our index patient had a mixed mania without fulfilling the criteria of full-blown mania during the baseline scan. Controls The 6 female depressed controls were diagnosed to have severe or moderate depression (296.22, 296.23, 296.32 or 296.33). Three of them also fulfilled the criteria for dysthymia (300.40) and three had previously suffered periods of major depression. They were participants of our larger study on the effects of psychotherapy on SERT and DAT densities to be reported in full elsewhere. The group of 10 healthy controls consisted mainly of employees of Kuopio University Hospital and medical students. Depression was an exclusion criterion for the control group. Controls had previously received no psychotropic medication or other psychiatric treatment and were physically healthy. Psychiatric evaluation Psychiatric diagnosis was based on clinical assessment and verified for all study subjects by a trained independent psychiatrist using the Structured Clinical Interview for DSM-IV-R (SCID-I) [8]. The severity of depression was assessed with the 17-item Hamilton Depression Rating Scale (HDRS) [9]. Setting, therapist and therapy The depressed controls (see Table for sociodemographic characteristics) received psychotherapy for 12 months in an outpatient clinic of the Department of Psychiatry of Kuopio University Hospital. The case discontinued the therapy after eight months because she felt it was no longer useful to her. The case and the depressed controls had received no psychiatric treatment or medication prior the SPECT imaging and they were treated without medication. Psychotherapy sessions occurred twice a week, 80 sessions per year. The psychotherapists had formal postgraduate professional training in psychodynamic psychotherapy. The study design was approved by the ethics committee of Kuopio University Hospital. Table 1 Characteristics of the participants. Values of the controls are means ± SD. Case Depressed controls n = 6 Healthy controls n = 10 Age, y 25 27.2 (5.9) 26.3 (5.9) Duration of depressive episode, months 9 10.6 (6.9) - SERT availability in midbrain at baseline 1.51 1.08 (0.12) 1.28 (0.12) SERT availability in midbrain at 12 months 1.36 1.23 (0.20) - DAT availability in striatum at baseline 2.75 2.48 (0.35) 2.45 (0.25) DAT availability in striatum at 12 months 2.61 2.70 (0.45) - HDRS score at baseline 15 17.67 (3.44) - HDRS score at 12 months 5 12.66 (5.09) - Abbreviations: DAT: Dopamine transporter, HDRS: Hamilton Depression Rating Scale SD: Standard deviation, SERT: Serotonin transporter. Participants of the study were right-handed and all were female. The depressed and the healthy controls were age-matched with the case (Table). The healthy controls were not followed up. Imaging procedure SPECT imaging was performed on the Wednesday after the menstrual bleeding that preceded the psychotherapy and on 12-month follow-up. A dose of 185 MBq of [123I] nor-β-CIT (supplied by MAP Medical Technologies OY, Tikkakoski Finland) was diluted in a volume of 10 ml physiological saline. The specific activity was higher than 1.8 × 1011 Bq/μmol [10]. The dose was slowly injected into the right antecubital vein in a dimly lit and quiet room. Serial SPECT scans (5 min, 6 h and 24 h) were performed on a dedicated Siemens MultiSPECT 3 gamma camera with fan-beam collimators (Siemens Medical Systems; Hoffman Estates I11., USA) [11]. Head positioning was monitored during acquisition by using two position lasers. Data analysis The SPECT scans were decay-corrected and reconstructed with Butterworth-filtered back-projection in a 128 × 128 matrix with a pixel size of 3 × 3 mm, and were attenuation-corrected with Chang's algorithm [10,11]. The imaging resolution was 8–9 mm. The SPECT slices were consecutively summarised to the slice thickness of 6 mm and re-aligned using a Siemens semi-automatic brain quantification program and the Talairach coordinates [12]. The slices were rotated and re-aligned so that transaxial (x-direction), sagittal (y-direction) and coronal (z-direction) slices were at right angles to each other. Region of interest placement was based on a Siemens semi-automatic brain quantification program. The lower threshold of 60% of the maximum count was used to reduce the volume averaging and partial volume errors. Regions of interest were the cerebellum, striatum and the midbrain. It was assumed that the cerebellum (reference region) corresponds to a two-compartment model (unbound tracer in arterial blood and free plus non-specifically bound tracer in the tissue) [13]. The specific binding in ml/ml for SERT and DAT was calculated using a graphical plot [13]. The slope of this plot is equal to the distribution volume ratio: (Region - Cerebellum)/Cerebellum = VD - 1. The striatal uptake was pooled. Functional neuroanatomy by means of SPECT was confirmed using magnetic resonance imaging (MRI) within two weeks of the SPECT imaging. If any cerebral focal abnormalities or organic brain diseases were detected by MRI scan, the patient was excluded from our study. The participants of our study thus had normal findings. Reproducibility of SPECT The reproducibility of the SPECT scan had been previously studied with eleven healthy subjects (5 males and 6 females; age range 20–39 y). SPECT studies were performed twice with a 12-month interval. The correspondence between the studies was good, with the mean difference being 0.00 ± 0.08 (SD = standard deviation) for SERT (mean and SD: 1.27 ± 0.11 and 1.27 ± 0.14, respectively) and 0.04 ± 0.18 for DAT (mean and SD: 2.49 ± 0.28 and 2.45 ± 0.27, respectively). The intraclass correlation coefficient was 0.82 (p < 0.01) and 0.79 (p < 0.01), respectively (unpublished data). Statistics The Student's t-test was used to compare the depressed controls and the healthy controls and paired samples t-test to compare SERT densities of the depressed controls at baseline and on follow-up. A p-value of less than 0.05 was considered as the criterion for statistical significance. Results The background characteristics of the case and the controls are presented in the table. The case and the depressed controls had HDRS scores of 14 or more. The midbrain SERT availability did not correlate with HDRS scores in depressive controls either at baseline or on follow-up. Neither did the change in the HDRS score correlate with changes in SERT or DAT capacities under therapy. The case had an elevated SERT availability in the midbrain at baseline, while the depressed controls had decreased levels compared to the healthy controls (t = 3.17, p < 0.01; Table and Figure 1). The SERT availability of the index case was two standard deviations (SD) higher than the mean SERT availability of the depressed controls and almost two SD higher than the mean SERT availability of the healthy controls. The MRI scans of the case and the controls were normal. At the twelve-month follow-up, the HDRS scores of both the case and the depressed controls had decreased. The mean decrease in HDRS scores in depressed patients during the follow-up period was 5 (SD 3.3; t = 3.7, p = 0.02). The SERT availability in the midbrain had decreased in the case by 9.9% (Fig. 2) and increased in the depressed controls by 12.5% (t = 3.00, p = 0.03) during the one year of psychotherapy. Figure 1 SERT densities of the healthy controls, depressed controls and of the index subject at baseline. Figure 2 Transaxial slices of [123I]nor-β-CIT scans in the index subject at baseline (A and B) and after 12 months (C and D). A and C indicate midbrain SERT binding and B and D striatal DAT binding. A 10% step colour scale is shown on the right and the cerebellum was used in its normalization. DAT densities in the striatum were elevated in the case (Table, Fig. 2.) and slightly elevated in the depressed controls at baseline compared to the healthy controls. On 12-month follow-up the DAT availability in the striatum of the case had decreased by 5.1%, while DAT densities in the depressed controls had increased by 8.8%. The increase in DAT binding was not significant (data not shown). Discussion To our knowledge, this is the first report on SERT and DAT associated with a current mania. In our case the densities of both SERT in the midbrain and DAT in the striatum were elevated at baseline and they decreased during psychotherapy, while the SERT and DAT of the depressed controls increased during psychotherapy. Clinical recovery was also reflected in the change in the HDRS score. Furthermore, the index case had no symptoms of hypomania after the psychotherapy. Ichimiya et al. [14] found increased SERT binding of the radioligand in the thalamus in a sample of patients with either major depression (n = 7) or bipolar disorder (n = 6) compared to healthy controls (n = 21). In their study the bipolar patients were either depressed or euthymic prior to brain imaging. There were no significant differences in binding potentials in the midbrain compared to the mood-disorder patients and the healthy controls. Bipolar patients had slightly higher binding potentials in the midbrain than the healthy controls, while patients with major depression had slightly lower binding potentials than the healthy controls. However, these differences were not significant. The patients of Ichimiya and co-workers were euthymic or depressed, so we cannot compare our results with theirs. It is clinically well known that anti-depressive medication, including serotonin-selective drugs, may aggravate mania [15]. If the serotonergic functions had already been elevated in mania and had then been further enhanced by anti-depressive medication, it is reasonable that this might further increase the manic symptoms. In animal models, the administration of an amino-acid mixture lacking the cathecolamine precursors tyrosine and phenylalanine decreases the availability of plasma tyrosine to the brain, which further diminishes cathecolamine synthesis [16]. In a few clinical human studies, decreasing tyrosine availability has had a positive effect on the symptoms of acute mania [17,18]. Tyrosine depletion has also led to decreased dopamine functions in healthy volunteers [19]. In positron emission tomography, a tyrosine-free amino-acid mixture increased striatal binding of the dopamine receptor ligand in healthy volunteers, which may reflect lowered presynaptic dopamine release [20]. SPECT studies revealing increased amphetamine-induced dopamine release in bipolar disorder patients have also been published [21]. The above-mentioned results indicate that acute mania may be associated with elevated dopaminergic functions in the central nervous system. A decrease in the DAT availability of the striatum was associated with clinical recovery in our case, which may support this hypothesis. The DAT densities of the depressed controls were only slightly above the level of the healthy controls. Laasonen-Balk et al. [5] have previously observed a greater increase in DAT densities in depressed patients. Because of the small sample size of this study there was insufficient statistical power to draw any conclusions in this issue. Our results support previous findings that the SERT densities of depressed patients are lower than in healthy controls [1,2,4]. Previous studies on neurotransmitters in association with mania have mostly dealt with dopamine. We cannot completely rule out the bias of nor-β-cit binding to the noradrenergic transporters (NORT). The noradrenergic cell-body rich nucleus ceruleus is close to our target region. This is assumed to contain the nucleus raphe, which is mostly comprised of serotonergic cell-bodies. The dopamine cell-body rich substantia nigra is also located close to our region of interest. However, nor-β-cit is considered to be more serotonin-specific than previous radioligands [10]. Three studies have been published on the association between SERT availability and the serotonin transporter genotype. Two of these were performed on healthy subjects [22,23], while the third concerned abstinent alcoholics and healthy controls [24]. In the study of Van Dyck and co-workers the short homozygotes had a significantly greater SERT availability than the long-short heterozygotes, which indicates a complex relationship between the genotype and SERT availability. Furlong et al. [25] found an association between promoter allele 2 of the SERT gene and bipolar disorder. Studies combining brain imaging and data of genotype are also recommended in the evaluation of bipolar disorder. PET would be a more valid method if adequate radioligands were available [14], as PET provides absolute rather than relative values for transporter availability. Conclusion We have found no previous imaging studies showing that serotonin plays a role in mania. Further research is needed to determine whether our findings could be generalised to all manic states. Competing interests None declared. Authors' contributions TT planned the study and analysed and interpreted the data. MJ also interpreted the data. PIS initiated and planned the study and organised the psychotherapy setting. HM took care of the imaging procedures. PA organised the psychotherapy setting. RV was responsible for the MRI scans and the interpretation of the scans. JK performed SPECT analyses. JT participated in the design of the study. JL initiated and planned the study project. All authors read, critically revised and approved the final manuscript. Abbreviations DAT: Dopamine transporter HDRS: Hamilton Depression Rating Scale SD: Standard deviation SERT: Serotonin transporter SPECT: Single photon emission computed tomography Pre-publication history The pre-publication history for this paper can be accessed here: Acknowledgements Financial support: JL and PIS have got a grant from Signe and Ane Gyllenberg Foundation, Helsinki, Finland. Written consent was obtained from the patient for publication of this study. ==== Refs Malison RT Price LH Berman R van Dyck CH Pelton GH Carpenter L Sanacora G Owens MJ Nemeroff CB Rajeevan N Baldwin RM Seibyl JP Innis RB Charney DS Reduced Brain Serotonin Transporter Availability in Major Depression as Measured by (4-iodophenyl) tropane and Single Photon Emission Computed Tomography Biol Psychiatry 1998 44 1090 1098 9836013 10.1016/S0006-3223(98)00272-8 Eggers B Hermann W Barthel H Sabri O Wagner A Hesse S The degree of depression in Hamilton rating scale is correlated with the availability of presynaptic serotonin transporters in 23 patients with Wilson's disease J Neurol 2003 250 576 580 12736737 10.1007/s00415-003-1039-7 Viinamäki H Kuikka J Tiihonen J Lehtonen J Change in monoamine transporter density related to clinical recovery: A case-control study Nordic J Psychiatry 1998 52 39 44 10.1080/080394898422553 Laasonen-Balk T Viinamäki H Kuikka JT Husso-Saastamoinen M Lehtonen J Tiihonen J 123 I-beta-CIT binding and recovery from depression: a six-month follow up study Eur Arch Psychiaty Clin Neurosci 2004 254 152 155 Laasonen-Balk T Kuikka JT Viinamäki H Husso-Saastamoinen M Lehtonen J Tiihonen J Striatal dopamine transporter density in major depression Psychopharmacology 1999 144 282 285 10435396 10.1007/s002130051005 Brunswick DJ Amsterdam JD Mozley PD Newberg A Greater availability brain dopamine transporters in major depression shown by [99mTc]TRODAT-1 SPECT imaging Am J Psychiatry 2003 160 1836 1841 14514499 10.1176/appi.ajp.160.10.1836 Benazzi F Bipolar II depressive mixed state: finding a useful definition Compr Psychiatry 2003 44 21 7 12524632 10.1053/comp.2003.50000 American Psychiatric Association Diagnostic and Statistical Manual of Mental Disorders (4th edn) (DSM-IV) 1994 Washington, DC:APA Hamilton M A rating scale for depression J Neurol Neurosurg Psychiat 1960 23 56 62 14399272 Hiltunen J Åkerman KK Kuikka JT Bergström KA Halldin C Nikula T Räsänen P Tiihonen J Vauhkonen M Karhu J Kupila J Länsimies E Farde L Iodine-123 labeled nor-β as a potential tracer for serotonin transporter imaging in the human brain with single-photon emission tomography Eur J Nucl Med 1998 25 19 23 9396870 10.1007/s002590050189 Kuikka JT Tenhunen-Eskelinen M Jurvelin J Kiiliäinen H Physical performance of the Siemens MultiSPECT3 gamma camera Nucl Med Commun 1993 14 490 497 8321489 Talairach J Tournoux P Referentially oriented cerebral MRI anatomy Thieme, New York 1993 Acton PD Kushner SA Kung MP Mozley PC Plossl K Kung HF Simplified reference region model for the kinetic analysis of [99mTc]TRODAT-1 binding to dopamine transporters in nonhuman primates using single-photon emission tomography Eur J Nucl Med 1990 26 518 26 10382097 10.1007/s002590050420 Ichimiya T Suhara T Sudo Y Okubo Y Nakayama K Masahiro N Inoue M Yasuno F Takano A Maeda J Shibuya H Serotonin Transporter Binding in Patients with mood Disorders: A PET Study with [11C](+)McN5652 Biol Psychiatry 2002 51 715 722 11983185 10.1016/S0006-3223(01)01351-8 American Psychiatric Association Practice guideline for treatment of patients with bipolar mood disorder (revision) Am J Psychiatry 2002 159 S4 McTavish SFB Cowen PJ Effect of a tyrosine-free amino acid mixture on regional brain catecholamine synthesis and release Psychopharmacology 1999 141 182 188 9952043 10.1007/s002130050823 McTavish SFB McPherson MH Harmer CJ Clark L Sharp T Goodwin GM Cowen PJ Antidopaminergic effects of dietary tyrosin depletion in healthy subjects and patients with manic illness Br J of Psychiat 2001 179 356 360 10.1192/bjp.179.4.356 Scarnà A Gijsman HJ McTavish SFB Harmer CJ Cowen PJ Goodwin GM Effects of a branched-chain amino acid drink in mania Br J of Psychiatr 2003 283 210 213 10.1192/bjp.182.3.210 Gijsman HJ Scarnà A Harmer CJ McTavish SB Odontiadis J Cowen PJ Goodwin GM A dose-finding study on the effects of branch chain amino acids on surrogate markers of brain dopamine function Psychopharmacology 2002 160 192 197 11875637 10.1007/s00213-001-0970-5 Montgomery JA McTavish SFB Cowen PJ PET measurement of the effect of dietary tyrosine depletion on [11C]raclopride binding Journal of Psychopharmacology 2002 16 A42 Anand A Verhoeff P Seneca N Zoghbi SS Seibyl JP Charney DS Innis RB Brain SPECT Imaging of Amphetamine-Induced Dopamine Release in Euthymic Bipolar Disorder Patients Am J Psychiatry 2000 157 1108 1114 10873919 10.1176/appi.ajp.157.7.1108 Jacobsen LK Staley JK Zoghbi SS Seibyl JP Kosten TR Innis RB Gelernter J Prediction of dopamime transporter binding availability by genotype: A preliminary report. Am J Psychiatry 2000 157 1700 1703 11007732 10.1176/appi.ajp.157.10.1700 van Dych CH Malison RT Staley JK Jacobsen LK Seibyl JP Laruelle M Baldwin RM Innis RB Gelernter J Central Serotonin Transporter Availability measured With [123I]β-CIT SPECT in Relation to Serotonin Transporter Genotype Am J Psychiatry 2004 161 525 531 14992979 10.1176/appi.ajp.161.3.525 Heinz A Jones D Mazzanti C Goldman D Ragan P Hommer D Linnoila M Weinberger DR A relationship between serotonin transporter genotype and in vivo protein expression and alcohol neurotoxicity Biol Psychiatry 2000 47 643 649 10745057 10.1016/S0006-3223(99)00171-7 Furlong RA Ho L Walsh C Rubinsztein JS Jain S Paykel ES Easton DF Rubinsztein DC Analysis and Meta-Analysis of Two Serotonin Transporter Gene Polymorphisms in Bipolar and Unipolar Affective Disorders Am J Med Genet (Neuropsychiatr Genet) 1998 81 58 63 10.1002/(SICI)1096-8628(19980207)81:1<58::AID-AJMG11>3.3.CO;2-W

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BMC Psychiatry. 2004 Sep 13; 4:27

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BMC Psychiatry

10.1186/1471-244X-4-27

oa_comm

==== Front BMC PsychiatryBMC Psychiatry1471-244XBioMed Central London 1471-244X-4-271536310510.1186/1471-244X-4-27Research ArticleElevated midbrain serotonin transporter availability in mixed mania: a case report Tolmunen Tommi [email protected] Mikko [email protected] Pirjo Irmeli [email protected] Hanna [email protected] Pasi [email protected] Ritva [email protected] Jyrki [email protected] Jari [email protected] Johannes [email protected] Department of Psychiatry, Kuopio University Hospital, 70210 Kuopio, Finland2 Department of Clinical Physiology and Nuclear Medicine, Kuopio University Hospital, 70210 Kuopio, Finland3 Department of Clinical Radiology, Kuopio University Hospital, 70210 Kuopio, Finland4 Department of Forensic Psychiatry, University of Kuopio, 70240 Kuopio, Finland2004 13 9 2004 4 27 27 16 4 2004 13 9 2004 Copyright © 2004 Tolmunen et al; licensee BioMed Central Ltd.2004Tolmunen et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Results obtained from brain imaging studies indicate that serotonin transporter (SERT) and dopamine transporter (DAT) densities are altered in major depression. However, no such studies have been published on current mania or hypomania. Case presentation In this single photon emission computed tomography (SPECT) study with [123I]nor-β-CIT we present a case with simultaneous symptoms of major depression and hypomania. She had an elevated serotonin transporter availability (SERT) in the midbrain and elevated dopamine transporter availability (DAT) in the striatum, which normalised in a one-year follow-up period during which she received eight months of psychodynamic psychotherapy. Conclusions To our knowledge, this is the first report on SERT and DAT associated with mania. In our case the availability of both SERT in the midbrain and DAT in the striatum were elevated at baseline and declined during psychotherapy, while the SERT and DAT of the depressed controls increased during psychotherapy. Symptoms of hypomania in the case were alleviated during psychotherapy. Clinical recovery was also reflected in the Hamilton Depression Rating Scale (HDRS) scores. ==== Body Background Decreased serotonin transporter densities (SERT) have been recorded from the brains of depressed patients [1,2], which have also been reported to normalise during psychotherapy [3,4]. A few studies have also revealed altered levels of dopamine transmission in major depression [5,6]. However, in a search of the PubMed database we found no serotonin-specific brain imaging studies on mania or hypomania. In this paper we present a case with simultaneous symptoms of major depression and hypomania. The structural diagnostic classifications DSM-IV and ICD-10 are unable to diagnose this kind of mixed state, even though similar cases have been presented earlier in the literature [7]. Materials and methods Case The index subject was a 25-year-old female with a history of two periods of major depression according to the DSM-IV-R criteria. She had also had hypomanic periods that did not completely fulfil the criteria of mania, and at baseline she fulfilled the criteria of hypomania, moderate depression and dysthymia (296.32, 300.40). She was overactive, restless and irritated and coped with a reduced amount of sleep. She had difficulty in concentrating on one thing at a time, and she appeared very lively and talkative. She was therefore diagnosed with bipolar mood disorder type II. In other words, our index patient had a mixed mania without fulfilling the criteria of full-blown mania during the baseline scan. Controls The 6 female depressed controls were diagnosed to have severe or moderate depression (296.22, 296.23, 296.32 or 296.33). Three of them also fulfilled the criteria for dysthymia (300.40) and three had previously suffered periods of major depression. They were participants of our larger study on the effects of psychotherapy on SERT and DAT densities to be reported in full elsewhere. The group of 10 healthy controls consisted mainly of employees of Kuopio University Hospital and medical students. Depression was an exclusion criterion for the control group. Controls had previously received no psychotropic medication or other psychiatric treatment and were physically healthy. Psychiatric evaluation Psychiatric diagnosis was based on clinical assessment and verified for all study subjects by a trained independent psychiatrist using the Structured Clinical Interview for DSM-IV-R (SCID-I) [8]. The severity of depression was assessed with the 17-item Hamilton Depression Rating Scale (HDRS) [9]. Setting, therapist and therapy The depressed controls (see Table for sociodemographic characteristics) received psychotherapy for 12 months in an outpatient clinic of the Department of Psychiatry of Kuopio University Hospital. The case discontinued the therapy after eight months because she felt it was no longer useful to her. The case and the depressed controls had received no psychiatric treatment or medication prior the SPECT imaging and they were treated without medication. Psychotherapy sessions occurred twice a week, 80 sessions per year. The psychotherapists had formal postgraduate professional training in psychodynamic psychotherapy. The study design was approved by the ethics committee of Kuopio University Hospital. Table 1 Characteristics of the participants. Values of the controls are means ± SD. Case Depressed controls n = 6 Healthy controls n = 10 Age, y 25 27.2 (5.9) 26.3 (5.9) Duration of depressive episode, months 9 10.6 (6.9) - SERT availability in midbrain at baseline 1.51 1.08 (0.12) 1.28 (0.12) SERT availability in midbrain at 12 months 1.36 1.23 (0.20) - DAT availability in striatum at baseline 2.75 2.48 (0.35) 2.45 (0.25) DAT availability in striatum at 12 months 2.61 2.70 (0.45) - HDRS score at baseline 15 17.67 (3.44) - HDRS score at 12 months 5 12.66 (5.09) - Abbreviations: DAT: Dopamine transporter, HDRS: Hamilton Depression Rating Scale SD: Standard deviation, SERT: Serotonin transporter. Participants of the study were right-handed and all were female. The depressed and the healthy controls were age-matched with the case (Table). The healthy controls were not followed up. Imaging procedure SPECT imaging was performed on the Wednesday after the menstrual bleeding that preceded the psychotherapy and on 12-month follow-up. A dose of 185 MBq of [123I] nor-β-CIT (supplied by MAP Medical Technologies OY, Tikkakoski Finland) was diluted in a volume of 10 ml physiological saline. The specific activity was higher than 1.8 × 1011 Bq/μmol [10]. The dose was slowly injected into the right antecubital vein in a dimly lit and quiet room. Serial SPECT scans (5 min, 6 h and 24 h) were performed on a dedicated Siemens MultiSPECT 3 gamma camera with fan-beam collimators (Siemens Medical Systems; Hoffman Estates I11., USA) [11]. Head positioning was monitored during acquisition by using two position lasers. Data analysis The SPECT scans were decay-corrected and reconstructed with Butterworth-filtered back-projection in a 128 × 128 matrix with a pixel size of 3 × 3 mm, and were attenuation-corrected with Chang's algorithm [10,11]. The imaging resolution was 8–9 mm. The SPECT slices were consecutively summarised to the slice thickness of 6 mm and re-aligned using a Siemens semi-automatic brain quantification program and the Talairach coordinates [12]. The slices were rotated and re-aligned so that transaxial (x-direction), sagittal (y-direction) and coronal (z-direction) slices were at right angles to each other. Region of interest placement was based on a Siemens semi-automatic brain quantification program. The lower threshold of 60% of the maximum count was used to reduce the volume averaging and partial volume errors. Regions of interest were the cerebellum, striatum and the midbrain. It was assumed that the cerebellum (reference region) corresponds to a two-compartment model (unbound tracer in arterial blood and free plus non-specifically bound tracer in the tissue) [13]. The specific binding in ml/ml for SERT and DAT was calculated using a graphical plot [13]. The slope of this plot is equal to the distribution volume ratio: (Region - Cerebellum)/Cerebellum = VD - 1. The striatal uptake was pooled. Functional neuroanatomy by means of SPECT was confirmed using magnetic resonance imaging (MRI) within two weeks of the SPECT imaging. If any cerebral focal abnormalities or organic brain diseases were detected by MRI scan, the patient was excluded from our study. The participants of our study thus had normal findings. Reproducibility of SPECT The reproducibility of the SPECT scan had been previously studied with eleven healthy subjects (5 males and 6 females; age range 20–39 y). SPECT studies were performed twice with a 12-month interval. The correspondence between the studies was good, with the mean difference being 0.00 ± 0.08 (SD = standard deviation) for SERT (mean and SD: 1.27 ± 0.11 and 1.27 ± 0.14, respectively) and 0.04 ± 0.18 for DAT (mean and SD: 2.49 ± 0.28 and 2.45 ± 0.27, respectively). The intraclass correlation coefficient was 0.82 (p < 0.01) and 0.79 (p < 0.01), respectively (unpublished data). Statistics The Student's t-test was used to compare the depressed controls and the healthy controls and paired samples t-test to compare SERT densities of the depressed controls at baseline and on follow-up. A p-value of less than 0.05 was considered as the criterion for statistical significance. Results The background characteristics of the case and the controls are presented in the table. The case and the depressed controls had HDRS scores of 14 or more. The midbrain SERT availability did not correlate with HDRS scores in depressive controls either at baseline or on follow-up. Neither did the change in the HDRS score correlate with changes in SERT or DAT capacities under therapy. The case had an elevated SERT availability in the midbrain at baseline, while the depressed controls had decreased levels compared to the healthy controls (t = 3.17, p < 0.01; Table and Figure 1). The SERT availability of the index case was two standard deviations (SD) higher than the mean SERT availability of the depressed controls and almost two SD higher than the mean SERT availability of the healthy controls. The MRI scans of the case and the controls were normal. At the twelve-month follow-up, the HDRS scores of both the case and the depressed controls had decreased. The mean decrease in HDRS scores in depressed patients during the follow-up period was 5 (SD 3.3; t = 3.7, p = 0.02). The SERT availability in the midbrain had decreased in the case by 9.9% (Fig. 2) and increased in the depressed controls by 12.5% (t = 3.00, p = 0.03) during the one year of psychotherapy. Figure 1 SERT densities of the healthy controls, depressed controls and of the index subject at baseline. Figure 2 Transaxial slices of [123I]nor-β-CIT scans in the index subject at baseline (A and B) and after 12 months (C and D). A and C indicate midbrain SERT binding and B and D striatal DAT binding. A 10% step colour scale is shown on the right and the cerebellum was used in its normalization. DAT densities in the striatum were elevated in the case (Table, Fig. 2.) and slightly elevated in the depressed controls at baseline compared to the healthy controls. On 12-month follow-up the DAT availability in the striatum of the case had decreased by 5.1%, while DAT densities in the depressed controls had increased by 8.8%. The increase in DAT binding was not significant (data not shown). Discussion To our knowledge, this is the first report on SERT and DAT associated with a current mania. In our case the densities of both SERT in the midbrain and DAT in the striatum were elevated at baseline and they decreased during psychotherapy, while the SERT and DAT of the depressed controls increased during psychotherapy. Clinical recovery was also reflected in the change in the HDRS score. Furthermore, the index case had no symptoms of hypomania after the psychotherapy. Ichimiya et al. [14] found increased SERT binding of the radioligand in the thalamus in a sample of patients with either major depression (n = 7) or bipolar disorder (n = 6) compared to healthy controls (n = 21). In their study the bipolar patients were either depressed or euthymic prior to brain imaging. There were no significant differences in binding potentials in the midbrain compared to the mood-disorder patients and the healthy controls. Bipolar patients had slightly higher binding potentials in the midbrain than the healthy controls, while patients with major depression had slightly lower binding potentials than the healthy controls. However, these differences were not significant. The patients of Ichimiya and co-workers were euthymic or depressed, so we cannot compare our results with theirs. It is clinically well known that anti-depressive medication, including serotonin-selective drugs, may aggravate mania [15]. If the serotonergic functions had already been elevated in mania and had then been further enhanced by anti-depressive medication, it is reasonable that this might further increase the manic symptoms. In animal models, the administration of an amino-acid mixture lacking the cathecolamine precursors tyrosine and phenylalanine decreases the availability of plasma tyrosine to the brain, which further diminishes cathecolamine synthesis [16]. In a few clinical human studies, decreasing tyrosine availability has had a positive effect on the symptoms of acute mania [17,18]. Tyrosine depletion has also led to decreased dopamine functions in healthy volunteers [19]. In positron emission tomography, a tyrosine-free amino-acid mixture increased striatal binding of the dopamine receptor ligand in healthy volunteers, which may reflect lowered presynaptic dopamine release [20]. SPECT studies revealing increased amphetamine-induced dopamine release in bipolar disorder patients have also been published [21]. The above-mentioned results indicate that acute mania may be associated with elevated dopaminergic functions in the central nervous system. A decrease in the DAT availability of the striatum was associated with clinical recovery in our case, which may support this hypothesis. The DAT densities of the depressed controls were only slightly above the level of the healthy controls. Laasonen-Balk et al. [5] have previously observed a greater increase in DAT densities in depressed patients. Because of the small sample size of this study there was insufficient statistical power to draw any conclusions in this issue. Our results support previous findings that the SERT densities of depressed patients are lower than in healthy controls [1,2,4]. Previous studies on neurotransmitters in association with mania have mostly dealt with dopamine. We cannot completely rule out the bias of nor-β-cit binding to the noradrenergic transporters (NORT). The noradrenergic cell-body rich nucleus ceruleus is close to our target region. This is assumed to contain the nucleus raphe, which is mostly comprised of serotonergic cell-bodies. The dopamine cell-body rich substantia nigra is also located close to our region of interest. However, nor-β-cit is considered to be more serotonin-specific than previous radioligands [10]. Three studies have been published on the association between SERT availability and the serotonin transporter genotype. Two of these were performed on healthy subjects [22,23], while the third concerned abstinent alcoholics and healthy controls [24]. In the study of Van Dyck and co-workers the short homozygotes had a significantly greater SERT availability than the long-short heterozygotes, which indicates a complex relationship between the genotype and SERT availability. Furlong et al. [25] found an association between promoter allele 2 of the SERT gene and bipolar disorder. Studies combining brain imaging and data of genotype are also recommended in the evaluation of bipolar disorder. PET would be a more valid method if adequate radioligands were available [14], as PET provides absolute rather than relative values for transporter availability. Conclusion We have found no previous imaging studies showing that serotonin plays a role in mania. Further research is needed to determine whether our findings could be generalised to all manic states. Competing interests None declared. Authors' contributions TT planned the study and analysed and interpreted the data. MJ also interpreted the data. PIS initiated and planned the study and organised the psychotherapy setting. HM took care of the imaging procedures. PA organised the psychotherapy setting. RV was responsible for the MRI scans and the interpretation of the scans. JK performed SPECT analyses. JT participated in the design of the study. JL initiated and planned the study project. All authors read, critically revised and approved the final manuscript. Abbreviations DAT: Dopamine transporter HDRS: Hamilton Depression Rating Scale SD: Standard deviation SERT: Serotonin transporter SPECT: Single photon emission computed tomography Pre-publication history The pre-publication history for this paper can be accessed here: Acknowledgements Financial support: JL and PIS have got a grant from Signe and Ane Gyllenberg Foundation, Helsinki, Finland. Written consent was obtained from the patient for publication of this study. ==== Refs Malison RT Price LH Berman R van Dyck CH Pelton GH Carpenter L Sanacora G Owens MJ Nemeroff CB Rajeevan N Baldwin RM Seibyl JP Innis RB Charney DS Reduced Brain Serotonin Transporter Availability in Major Depression as Measured by (4-iodophenyl) tropane and Single Photon Emission Computed Tomography Biol Psychiatry 1998 44 1090 1098 9836013 10.1016/S0006-3223(98)00272-8 Eggers B Hermann W Barthel H Sabri O Wagner A Hesse S The degree of depression in Hamilton rating scale is correlated with the availability of presynaptic serotonin transporters in 23 patients with Wilson's disease J Neurol 2003 250 576 580 12736737 10.1007/s00415-003-1039-7 Viinamäki H Kuikka J Tiihonen J Lehtonen J Change in monoamine transporter density related to clinical recovery: A case-control study Nordic J Psychiatry 1998 52 39 44 10.1080/080394898422553 Laasonen-Balk T Viinamäki H Kuikka JT Husso-Saastamoinen M Lehtonen J Tiihonen J 123 I-beta-CIT binding and recovery from depression: a six-month follow up study Eur Arch Psychiaty Clin Neurosci 2004 254 152 155 Laasonen-Balk T Kuikka JT Viinamäki H Husso-Saastamoinen M Lehtonen J Tiihonen J Striatal dopamine transporter density in major depression Psychopharmacology 1999 144 282 285 10435396 10.1007/s002130051005 Brunswick DJ Amsterdam JD Mozley PD Newberg A Greater availability brain dopamine transporters in major depression shown by [99mTc]TRODAT-1 SPECT imaging Am J Psychiatry 2003 160 1836 1841 14514499 10.1176/appi.ajp.160.10.1836 Benazzi F Bipolar II depressive mixed state: finding a useful definition Compr Psychiatry 2003 44 21 7 12524632 10.1053/comp.2003.50000 American Psychiatric Association Diagnostic and Statistical Manual of Mental Disorders (4th edn) (DSM-IV) 1994 Washington, DC:APA Hamilton M A rating scale for depression J Neurol Neurosurg Psychiat 1960 23 56 62 14399272 Hiltunen J Åkerman KK Kuikka JT Bergström KA Halldin C Nikula T Räsänen P Tiihonen J Vauhkonen M Karhu J Kupila J Länsimies E Farde L Iodine-123 labeled nor-β as a potential tracer for serotonin transporter imaging in the human brain with single-photon emission tomography Eur J Nucl Med 1998 25 19 23 9396870 10.1007/s002590050189 Kuikka JT Tenhunen-Eskelinen M Jurvelin J Kiiliäinen H Physical performance of the Siemens MultiSPECT3 gamma camera Nucl Med Commun 1993 14 490 497 8321489 Talairach J Tournoux P Referentially oriented cerebral MRI anatomy Thieme, New York 1993 Acton PD Kushner SA Kung MP Mozley PC Plossl K Kung HF Simplified reference region model for the kinetic analysis of [99mTc]TRODAT-1 binding to dopamine transporters in nonhuman primates using single-photon emission tomography Eur J Nucl Med 1990 26 518 26 10382097 10.1007/s002590050420 Ichimiya T Suhara T Sudo Y Okubo Y Nakayama K Masahiro N Inoue M Yasuno F Takano A Maeda J Shibuya H Serotonin Transporter Binding in Patients with mood Disorders: A PET Study with [11C](+)McN5652 Biol Psychiatry 2002 51 715 722 11983185 10.1016/S0006-3223(01)01351-8 American Psychiatric Association Practice guideline for treatment of patients with bipolar mood disorder (revision) Am J Psychiatry 2002 159 S4 McTavish SFB Cowen PJ Effect of a tyrosine-free amino acid mixture on regional brain catecholamine synthesis and release Psychopharmacology 1999 141 182 188 9952043 10.1007/s002130050823 McTavish SFB McPherson MH Harmer CJ Clark L Sharp T Goodwin GM Cowen PJ Antidopaminergic effects of dietary tyrosin depletion in healthy subjects and patients with manic illness Br J of Psychiat 2001 179 356 360 10.1192/bjp.179.4.356 Scarnà A Gijsman HJ McTavish SFB Harmer CJ Cowen PJ Goodwin GM Effects of a branched-chain amino acid drink in mania Br J of Psychiatr 2003 283 210 213 10.1192/bjp.182.3.210 Gijsman HJ Scarnà A Harmer CJ McTavish SB Odontiadis J Cowen PJ Goodwin GM A dose-finding study on the effects of branch chain amino acids on surrogate markers of brain dopamine function Psychopharmacology 2002 160 192 197 11875637 10.1007/s00213-001-0970-5 Montgomery JA McTavish SFB Cowen PJ PET measurement of the effect of dietary tyrosine depletion on [11C]raclopride binding Journal of Psychopharmacology 2002 16 A42 Anand A Verhoeff P Seneca N Zoghbi SS Seibyl JP Charney DS Innis RB Brain SPECT Imaging of Amphetamine-Induced Dopamine Release in Euthymic Bipolar Disorder Patients Am J Psychiatry 2000 157 1108 1114 10873919 10.1176/appi.ajp.157.7.1108 Jacobsen LK Staley JK Zoghbi SS Seibyl JP Kosten TR Innis RB Gelernter J Prediction of dopamime transporter binding availability by genotype: A preliminary report. Am J Psychiatry 2000 157 1700 1703 11007732 10.1176/appi.ajp.157.10.1700 van Dych CH Malison RT Staley JK Jacobsen LK Seibyl JP Laruelle M Baldwin RM Innis RB Gelernter J Central Serotonin Transporter Availability measured With [123I]β-CIT SPECT in Relation to Serotonin Transporter Genotype Am J Psychiatry 2004 161 525 531 14992979 10.1176/appi.ajp.161.3.525 Heinz A Jones D Mazzanti C Goldman D Ragan P Hommer D Linnoila M Weinberger DR A relationship between serotonin transporter genotype and in vivo protein expression and alcohol neurotoxicity Biol Psychiatry 2000 47 643 649 10745057 10.1016/S0006-3223(99)00171-7 Furlong RA Ho L Walsh C Rubinsztein JS Jain S Paykel ES Easton DF Rubinsztein DC Analysis and Meta-Analysis of Two Serotonin Transporter Gene Polymorphisms in Bipolar and Unipolar Affective Disorders Am J Med Genet (Neuropsychiatr Genet) 1998 81 58 63 10.1002/(SICI)1096-8628(19980207)81:1<58::AID-AJMG11>3.3.CO;2-W

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2021-01-04 16:03:41

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BMC Med Inform Decis Mak. 2004 Sep 15; 4:16

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BMC Med Inform Decis Mak

10.1186/1472-6947-4-16

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==== Front World J Surg OncolWorld Journal of Surgical Oncology1477-7819BioMed Central London 1477-7819-2-301535020110.1186/1477-7819-2-30Technical InnovationsSingle group study to evaluate the feasibility and complications of radiofrequency ablation and usefulness of post treatment position emission tomography in lung tumours Kang Shijun [email protected] Rongcheng [email protected] Wangjun [email protected] Hubing [email protected] Xuelin [email protected] Yuru [email protected] Department of Oncology, Nanfang Hospital, Guangzhou, P.R.China, 5105152 PET Center, Nanfang Hospital, Guangzhou, P.R.China, 5105153 Medical Image Center, Nanfang Hospital, Guangzhou, P.R.China, 5105152004 6 9 2004 2 30 30 23 5 2004 6 9 2004 Copyright © 2004 Kang et al; licensee BioMed Central Ltd.2004Kang et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background There is genuine need to develop interventional treatment options for management of lung tumors. Radiofrequency ablation (RFA) is one such alternative being promoted to treat lung tumors recently. Larger studies should help define RFA's further development. Furthermore fluorodeoxyglucose positron emission tomography (PET) has been reported to be an accurate indicator of treatment response in variety of tumors. This study focuses on the evaluating the feasibility of RFA and usefulness of PET scan in lung tumors after RFA procedure. Patients and methods Between November 1999 and May 2002, 50 patients with primary or metastasis pulmonary tumors underwent RFA procedure. The electrode was guided to the target areas using computerized tomography (CT). Tumors smaller than 3.5 cm were given single RFA, while tumors larger than 3.5 cm received RFA to multiple sites. Maximum 4 lesions or 6 target areas were treated during one operating procedure. Whole body and/or lung PET images were acquired; identical site CT images and chest X-ray were taken 1 week before and after RFA. Results Of the 50 patients, 17 had single lesions while rest had multiple lesions. Tumors smaller than 3.5 cm were completely dissipated after RFA. In tumors larger than 3.5 cm, the part within 3.5 cm diameter dissipated. While CT showed that tumor image became larger 1 to 2 weeks after RFA procedure. PET demonstrated tumor destruction in 70% cases, compared to 38% in CT. Conclusion The present study shows RFA to be safe and effective treatment option for lung tumors. PET is superior to CT in evaluation the effectiveness of RFA treatment shortly after the procedure. ==== Body Background Lung cancer continues to be the leading cause of cancer deaths in United States [1]. The overall prognosis of lung cancer is still dismal despite all current early detection and treatment efforts. Only about 20–25% of lung cancers can potentially be cured by surgery. The majority of patients presents with locally advanced or metastatic disease, and treatments essentially rely on external beam irradiation, chemotherapy or a combination of both [2]. Thus other interventional palliative treatment options have been developed for these lesions. Radiofrequency ablation (RFA), is an imaging-guided percutaneous ablative procedure, that has been suggested to be an effective treatment option for patients with non-small cell lung cancer (NSCLC) and metastatic disease who are not suitable candidates for surgery [3,4]. Guided by computed tomography (CT), physicians are able to localize the tumor and determine the optimal approach. During RFA, current passing through tissue from the active electrode leads to ion agitation and frictional heat generation. This leads to irreparable cellular damage and coagulation necrosis [5]. Recently a number of studies reported its application in malignant lung tumours. Accurate assessment of treatment response remains one of the major problems. PET has been reported to be an accurate indicator of treatment response in variety of tumors [13-17]. However, its use has been limited to evaluating disease stage in lung tumors [18-24]. PET imaging, provides proliferation and metabolism information, is sensitive and specific to diagnose malignant lesions from benign. Coleman and colleagues has provided substantial information in evaluating the role of PET in management of lung cancers [21-23]. In this report we focus on evaluating the feasibility of RFA, its complication and on evaluating the role of PET on RFA response in lung tumors. Patients and methods Between November 1999 and May 2002, 50 patients with either primary or metastatic lung tumors were enrolled in to a prospective single group trial. Patient characteristics are detailed in Table 1. Patients with bleeding potentials or serious heart, liver and renal failures were excluded. Antibiotics and medicines for prevention of bleeding were given regularly. Every patient underwent a chest Flurodeoxyhlucose postron emission (PET) and CT scan before procedure. Table 1 Patient characteristics Patients (n = 50) Patient characteristic No % Age, years  Median 51  Range 35–74 Sex  Male 32 64  Female 18 36 Origins  Primary lung tumors 23 46  Metastases from breast 13 26  Metastases from colon 9 18  Metastases from other places 5 10 No. of patient with lesions  Single lesion 17 34  Multiple lesions 33 66 Total lesions received RFA 120 Patients received a chest X-ray and CT for preoperative evaluation and a repeat scan after RFA procedure. A PET scan was performed one week after the treatment. The Radiofrequency ablation was carried out using RF-2000 generator and related software purchased from Radio Therapeutics Corporation, USA; PET imaging was done using an Advance 2 Scanner (General Electric Medical Systems, WI, USA). Patients received general anesthesia along with local infiltration of Lidocaine. The electrodes were directed to target areas during RFA procedure using CT scan. The initial power applied was 50 W, which was subsequently increased to a maximum 90 W over several minutes. RFA continued for 5 to 15 min until roll off was achieved, which continued for 2 min to stop. Tumors smaller than 3.5 cm were given full heating energy only once, while tumors larger than 3.5 cm received multiple RFA to different areas. Maximum 4 lesions or 6 target areas were treated during one procedure. One to two weeks after the procedure and a 4 hour fast, patients were taken for PET scan. They were made to rest for 15 min, and then received 18F-FDG 296 MBq – 440 MBq (8 mCi -12 mCi) intravenously. After another period of rest lasting for 45–60 min, the whole body and/or lung images was acquired by PET scanner. PET was also acquired at 5–8 bed positions, typically from the base of skull to the mid thigh, which was identical to the CT protocol used in the present study. The complications of the treatment are detailed in table 2 and results are summarized in table 3. Table 2 Complications of Radiofrequency ablation Patients (n = 50) Complication No % Fever 10 20 Congested pneumonia 6 12 Pneumothorax 9 18 Hemothorax 1 2 Table 3 Early effectiveness of RFA by various techniques Tumor destruction demonstrated Technique No % All 50 patients received  PET 35 70  CT 19 38  X-ray 13 26 Results After RFA procedure a number of complications were seen. Fever and/or congested pneumonia were commonest complications seen in 32% of patients; however, they were cured in a week with antibiotics treatment. Pneumothorax occurred during procedures in 18% and the patients were treated with aspiration. Five of these had small pneumothorax that did not require and treatment. One patient had hemothorax which required intercostals drainage (ICD) which was removed 2 days later. These were no life threatening events or deaths. Post procedural PET demonstrated the effectiveness of RFA on lung tumors. Tumors smaller than 3.5 cm showed complete response after RFA (Figure 1). In tumors larger than 3.5 cm, the part within 3.5 cm diameter dissipated, while the part outside this 3.5 cm area remained (Figure 2). Damage to the normal tissue outside the tumor was not extensive in any cause. Figure 1 PET images taken before and after RFA treatments: coronal (A, B), and Sagittal (C, D) views of PET scans of lung cancer. (A, C) were taken before RFA treatment; (B, D) were taken two weeks after RFA treatment. Figure 2 PET images taken before and after RFA treatments. This patient had a tumor size larger than 3.5 cm. PET scans were taken 1 week before (A) and 2 weeks after (B) RFA treatment. The Chest X-ray and CT showed that tumor image became larger 1 to 2 weeks after RFA procedure (Figure 3). These may result from partial tissue damages, bleeding, acute inflammation or pneumonia, and support the routing use of antibiotics and haemostatic drugs after RFA. The tumor destruction was picked up by PET much effectively when compared to CT scan or chest X-ray. Figure 3 CT images taken before and after RFA treatments. The same patient PET images were shown as Figure 1. (A) was taken before RFA treatment, (B) was taken 2 week after RFA treatment. Discussion Since RFA ablates lung tumors directly and locally, marginal tissues surrounding the tumor are frequently partially damaged leading to occasional pneumonia. It is difficult for regular CT and/or chest X-rays to discriminate pathological-physiological tissue damage and fibrillation from the treatment effect of RFA. PET on the other hand provides information on functional and metabolic activity anatomically, and is the only available technique which can specifically diagnose tumors or necrosis after surgery and radiotherapy effectively [28]. Our experience too proves that PET is particularly superior to CT in its ability to evaluate the effectiveness of RFA treatment early after therapy. RFA is a relatively noninvasive, well-tolerated approach. It could destruct tumor completely within the effective diameter while avoiding the surgery, side effects of radiotherapy and toxicity of high dose chemotherapy. Our observations suggest that RFA can kill lung tumors smaller than 3.5 cm after a single RFA procedure. The effect of RFA appears to be limited within 3.5 cm diameter area with the current instruments. However, this also suggests that RFA may not damage the normal tissues surrounding the small tumors. The malignant lesions dissipated in 1 to 2 weeks, while the surrounding tissue stayed intact. While at this period regular chest CT and chest X-ray may show enlarged lesion images. This is in agreement with other reports. With improvements in technology, RFA in combination with other options may further reduce the morbidity and mortality of cancer deaths [11]. Though complications do occur, they are usually curable. RFA results in a higher rate of complete necrosis and requires fewer treatment cycles compared to traditional chemotherapy or radiotherapy. Besides CT guidance help to localize the tumor and determine the optimal approach further optimizes specific of targeting the tumor. For patients with non-small cell lung malignancy who are not candidates for surgery owing to poor cardio respiratory reserve, RFA alone or followed by conventional radiation therapy or chemotherapy may prove to be a treatment option [11]. For patients with metastatic disease, RFA may be suitable for treatment of a small tumor or reduce symptoms caused by large tumor burden. This technique can be used as a primary technique or in conjunction with other interventional procedures [11]. Further randomized controlled trials comparing RFA with conventional palliative treatment are needed before RFA can be accepted as a routine treatment modality. Survival of patient and quality of life issues too need be addressed. Conclusions Despite inherent deficiency of trial design our single group study clearly demonstrates that RFA can be an effective treatment option for lung tumors. Unlike other interventional techniques, RFA provide controlled regions of coagulation necrosis with a single application to an area with 3.5 cm diameter. RFA may cure small lung tumor, reduce tumor burden in larger lesions and may be combined with external beam radiation and/or systemic chemotherapy for further improvements. PET provides functional and metabolic activity anatomically and is particularly superior to CT in evaluation the effectiveness shortly after RFA procedure. Absence of follow-up information and randomization in the current study are two major fallacies which need to be addressed in subsequent studies. Authors' contributions SJK is the leading physician and drafted the manuscript. RL, WL, HW, XZ, YM all participated in the study, patient management, literature search and preparation of manuscript. All authors have read and approved the final version of the manuscript. Competing interests None declared. ==== Refs Williams MD Sandler AB The epidemiology of lung cancer Cancer Treat Res 2001 105 31 52 11224993 Noppen N Interventional palliative treatment options for lung cancer Ann Oncol 2002 13 Suppl 4 247 250 12401697 Dupuy DE Mayo-Smith WW Abbott GF DiPetrillo T Clinical applications of radio-frequency tumor ablation in the thorax Radiographics 2002 22 Spec No S259 69 12376615 Chhajed PN Tamm M Radiofrequency heat ablation for lung tumors: potential applications Med Sci Monit 2003 9 ED5 7 14586281 Gazelle GS Goldberg SN Solbiati L Livraghi T Tumor ablation with radio-frequency energy Radiology 2000 217 633 646 11110923 Wong CY Salem R Raman S Gates VL Dworkin HJ Evaluating 90Y-glass microsphere treatment response of unresectable colorectal liver metastases by [18F]FDG PET: a comparison with CT or MRI Eur J Nucl Med Mol Imaging 2002 29 815 820 12029557 10.1007/s00259-002-0787-4 Wang W Larson SM Fazzari M Tickoo SK Kolbert K Sgouros G Yeung H Macapinlac H Rosai J Robbins RJ Prognostic value of [18F]fluorodeoxyglucose positron emission tomographic scanning in patients with thyroid cancer J Clin Endocrinol Metab 2000 85 1107 1113 10720047 10.1210/jc.85.3.1107 Price DT Coleman RE Liao RP Robertson CN Polascik TJ DeGrado TR Comparison of [18 F]fluorocholine and [18 F]fluorodeoxyglucose for positron emission tomography of androgen dependent and androgen independent prostate cancer J Urol 2002 168 273 280 12050555 10.1097/00005392-200207000-00090 Voth M Opfermann T Gottschild D [The value of fluorodeoxyglucose positron emission tomography (FDG-PET) in differentiation of pancreatic lesions] Zentralbl Chir 2003 128 375 378 12813634 10.1055/s-2003-40030 Lowe VJ Dunphy FR Varvares M Kim H Wittry M Dunphy CH Dunleavy T McDonough E Minster J Fletcher JW Boyd JH Evaluation of chemotherapy response in patients with advanced head and neck cancer using [F-18]fluorodeoxyglucose positron emission tomography Head Neck 1997 19 666 674 9406745 10.1002/(SICI)1097-0347(199712)19:8<666::AID-HED4>3.3.CO;2-C Peschina W [F-18 fluorodeoxyglucose positron emission tomography in lung lesions] Wien Med Wochenschr 2002 152 265 268 12138653 10.1046/j.1563-258X.2002.02019.x Berlangieri SU Scott AM Knight SR Fitt GJ Hennessy OF Tochon-Danguy HJ Clarke CP McKay WJ F-18 fluorodeoxyglucose positron emission tomography in the non-invasive staging of non-small cell lung cancer Eur J Cardiothorac Surg 1999 16 Suppl 1 S25 30 10536941 Hunter GJ Hamberg LM Choi N Jain RK McCloud T Fischman AJ Dynamic T1-weighted magnetic resonance imaging and positron emission tomography in patients with lung cancer: correlating vascular physiology with glucose metabolism Clin Cancer Res 1998 4 949 955 9563889 Coleman RE PET in lung cancer staging Q J Nucl Med 2001 45 231 234 11788815 Coleman RE Value of FDG-PET scanning in management of lung cancer Lancet 2002 359 1361 1362 11978328 10.1016/S0140-6736(02)08388-5 Gonzalez-Stawinski GV Lemaire A Merchant F O'Halloran E Coleman RE Harpole DH D'Amico TA A comparative analysis of positron emission tomography and mediastinoscopy in staging non-small cell lung cancer J Thorac Cardiovasc Surg 2003 126 1900 1905 14688703 10.1016/S0022-5223(03)01036-5 Marom EM McAdams HP Erasmus JJ Goodman PC Culhane DK Coleman RE Herndon JE Patz E. F., Jr. Staging non-small cell lung cancer with whole-body PET Radiology 1999 212 803 809 10478250 Burt BM Humm JL Kooby DA Squire OD Mastorides S Larson SM Fong Y Using positron emission tomography with [(18)F]FDG to predict tumor behavior in experimental colorectal cancer Neoplasia 2001 3 189 195 11494112 10.1038/sj.neo.7900147 Coleman RE FDG imaging Nucl Med Biol 2000 27 689 690 11091113 10.1016/S0969-8051(00)00139-6 Liao WJ Luo RC Kang SJ Wu HB Wang CB Luo YL Zhang MJ Investigation of short-term therapy results for radiofrequency ablation by positron emission tomography Di Yi Jun Yi Da Xue Xue Bao 2002 22 376 377 12390754 Kostakoglu L Goldsmith SJ 18F-FDG PET evaluation of the response to therapy for lymphoma and for breast, lung, and colorectal carcinoma J Nucl Med 2003 44 224 239 12571214 Akhurst T Downey RJ Ginsberg MS Gonen M Bains M Korst R Ginsberg RJ Rusch VW Larson SM An initial experience with FDG-PET in the imaging of residual disease after induction therapy for lung cancer Ann Thorac Surg 2002 73 259 64; discussion 264-6 11834020 10.1016/S0003-4975(01)03257-X Herrera LJ Fernando HC Perry Y Gooding WE Buenaventura PO Christie NA Luketich JD Radiofrequency ablation of pulmonary malignant tumors in nonsurgical candidates J Thorac Cardiovasc Surg 2003 125 929 937 12698158 10.1067/mtc.2003.18 Rose DM Allegra DP Bostick PJ Foshag LJ Bilchik AJ Radiofrequency ablation: a novel primary and adjunctive ablative technique for hepatic malignancies Am Surg 1999 65 1009 1014 10551746

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World J Surg Oncol. 2004 Sep 6; 2:30

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World J Surg Oncol

10.1186/1477-7819-2-30

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==== Front Environ HealthEnvironmental Health1476-069XBioMed Central London 1476-069X-3-81536309810.1186/1476-069X-3-8ResearchAnogenital distance in human male and female newborns: a descriptive, cross-sectional study Salazar-Martinez Eduardo [email protected] Patricia [email protected] Edith [email protected] Matthew P [email protected] Mauricio [email protected] National Institute of Public Health, Av. Universidad 655, Col. Santa Ma. Ahuacatitlan, 62508 Cuernavaca, Morelos, Mexico2 Mexican Institute of Social Security, Boulevard Benito Juarez #18 Tercer piso Col. Centro, C.P. 62000, Cuernavaca, Morelos, Mexico3 National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, MD A3-05, PO Box 12233, Research Triangle Park, North Carolina 27709, USA2004 13 9 2004 3 8 8 18 3 2004 13 9 2004 Copyright © 2004 Salazar-Martinez et al; licensee BioMed Central Ltd.2004Salazar-Martinez et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background In animal studies of the effects of hormonally active agents, measurement of anogenital distance (AGD) is now routine, and serves as a bioassay of fetal androgen action. Although measurement of AGD in humans has been discussed in the literature, to our knowledge it has been measured formally in only two descriptive studies of females. Because AGD has been an easy-to-measure, sensitive outcome in animals studies, we developed and implemented an anthropometric protocol for measurement of AGD in human males as well as females. Methods We first evaluated the reliability of the AGD measures in 20 subjects. Then measurements were taken on an additional 87 newborns (42 females, 45 males). All subjects were from Morelos, Mexico. Results The reliability (Pearson r) of the AGD measure was, for females 0.50, and for males, 0.64. The between-subject variation in AGD, however, was much greater than the variation due to measurement error. The AGD measure was about two-fold greater in males (mean, 22 mm) than in females (mean, 11 mm), and there was little overlap in the distributions for males and females. Conclusion The sexual dimorphism of AGD in humans comprises prima facie evidence that this outcome may respond to in utero exposure to hormonally active agents. ==== Body Background In animal studies of the effects of hormonally active agents, measurement of anogenital distance (AGD) is now routine [1-16], and serves as a bioassay of fetal androgen action. In rodents, perineal growth is dihydrotestosterone-dependent [17], males have a greater AGD than females, and use of AGD to sex newborns is standard [18]. In animals AGD is correlated at only modest levels with body weight [19], because these measures reflect the effects of endocrine axes that are largely independent. AGD usually tracks through life, varies by dose of antiandrogen, and can be predictive of other androgen-responsive outcomes [20]. Although measurement of AGD in humans has been discussed in the literature [19,21-23], to our knowledge it has been measured formally in only two descriptive studies of females [24,25]. Because AGD has been an easy-to-measure, sensitive outcome in animal studies, we developed and implemented an anthropometric protocol for measurement of AGD in human males as well as females. This work constitutes a modest step towards evaluation of AGD in human males as a potentially useful anthropometric measure and indicator of in utero androgen status. Methods Subjects A cross-sectional study was conducted among the newborn children of women admitted for delivery to the Dr. Ernesto Meana San Román General Hospital in Jojutla, Morelos, Mexico, in 1999. This hospital provides medical care to low socioeconomic status and uninsured populations. The study included 87 newborn infants, none of whom had congenital defects or had been admitted to the neonatal intensive care unit. All infants were born at term (≥38 weeks gestation), except for one (32 weeks). The infants were of both sexes and were born after spontaneous cephalic delivery or caesarean section. Within 6 hours of birth, a structured questionnaire about family background and obstetric history was administered to the mothers, and anthropometric measurements were taken on the newborns. Anthropometry Anthropometric measurements were taken of weight, length, head circumference, and AGD. AGD was measured as follows: the newborn infant was in the dorsal decubitus position; both hips were flexed and light pressure was exerted on the infant's thighs until the examiner's hand touched the subject's abdomen. Measurements were made with Vernier calipers. Distance was measured from the center of the anus to the posterior convergence of the fourchette (where the vestibule begins) in female infants [24]; and from the center of the anus to the junction of the smooth perineal skin with the rugated skin of the scrotum in male infants (Figure 1). Gestational age was estimated according to the Dubowitz scoring system [26]. Figure 1 Schematic Diagram of Measurements Done, by Sex Reliability Before any contact with the 87 subjects in the main study, the personnel performing the anthropometry examined 20 other neonates; all of whom were born after ≥38 weeks gestation. In this standardization training, 7 female infants and 13 male infants were measured twice by each observer. A sufficient time interval (30 minutes) was allotted between each measurement so that the second would not be influenced by the observer's memory of the first. These data were used to examine the reliability of measures and sources of variance. Statistical analysis The reliability of the anthropometric measures was calculated as the Pearson correlation coefficient between the paired measures. The observations taken by the two observers were not statistically different when compared using a paired t-test (results not shown). Analysis of variance (ANOVA) with a random effect term for subject was used to estimate between-subject, between-observer, and within-observer components of variance, by sex. For the main study, a linear regression analysis was used to evaluate birth weight, birth length, and gestational age as predictors of AGD. Age of the mother, number of pregnancies, and time elapsed between birth and measurement were not important predictors (or confounders) of AGD in univariate or multivariate models and were not considered further in the analysis. To examine influential values and the overall fit of the model, we conducted an analysis of residuals, but found nothing of note. The protocol was approved by human subjects committees at the National Institute of Public Health in Mexico and the National Institute of Environmental Health Sciences in the U.S. Results Among the 20 subjects in the standardization exercise, the between-subject coefficient of variation was greater for measures of AGD in females than for the other measures (Table 1). The reliability of the AGD measures were lower than for the traditional measures of anthropometry, with the female value being slightly lower than that for males. The variances estimated from the ANOVA were, for females: between-subjects, 7.9; between-observers, 0.6; and within-observer, 0.0. For males, the values were: between-subjects, 3.5; between-observers, 0.0; and within-observer, 0.1. The relative size of the variance components was unchanged when birth weight was included in the models. Thus, the between-subject variation in AGD was much greater than the variation due to measurement error. Table 1 Mean, coefficient of variation (CV), and reliability of anthropometric measurements in 20 newbornsa Measurement Mean CV Reliability Weight (kg) 3.01 0.13 1.00 Length (cm) 48.9 0.03 0.97 Head Circumference (cm) 34.2 0.03 0.98 Anogenital distance 18 0.31 0.91  Female 11 0.27 0.50  Male 21 0.09 0.64 a 7 females and 13 males. Among the 87 subjects in the main study, the birth weight, length, and head circumference were as expected in a population from southern Mexico (Table 2) [27]. The AGD measure was about two-fold greater in males than in females, and there was little overlap in the distributions for males and females (Figure 2). The correlation of AGD with body weight was 0.64 in females and 0.48 in males. Table 2 Distribution of selected characteristics in 87 newborns, Mexico, 1999a Variable Female n = 42 Male n = 45 Anogenital distance (mm) Mean 11 21 SD 2 3 Median 11 22 25th percentile 10 20 75th percentile 11 23 Weight (g) Mean 3070 3060 SD 408 440 Median 3060 3110 25th percentile 2870 2800 75th percentile 3310 3290 Length (cm) Mean 48.6 48.7 SD 1.4 2.2 Median 48.6 48.7 25th percentile 47.5 48.0 75th percentile 49.6 49.9 Head circumference (cm) Mean 337 341 SD 10.9 16.7 Median 337 341 25th percentile 330 334 75th percentile 345 350 aSD, standard deviation Figure 2 Distribution of Anogenital Distance (AGD), by Sex In the crude models of AGD in females, weight, length, and gestational age all appeared to be predictive (Table 3). The adjusted results, however, suggested that weight of the newborn was the most important correlate, based on the p value being lower than for length or gestational age. For males, weight and length were more important than gestational age as determinants, and this pattern was seen also in the adjusted results (Table 4). Length had a slightly larger R2 and slightly lower p value, suggesting it may be a marginally better predictor than weight in males. In a model of data for males that included weight, length, and gestational age, the p values for both length and gestation were less than 0.05, although the coefficient for gestation was negative. In a model of AGD based on data for males and females combined (results not shown), after adjustment for weight, the term for sex was clearly important (β for males = 10.9 mm, standard error = 0.4, p < 0.0001; change in R2 due to addition of sex to model = 0.86). Table 3 Regression coefficients for anogenital distance as a function of characteristics at birth, femalesa Variable Crude Adjustedb Coefficient 95% CI p value R2 Coefficient 95% CI p value R2 Birth weight 0.002  0.002 0.003 0.000 0.41 0.002c  0.001 0.003 0.000 0.43 Birth length 0.319 -0.005 0.642 0.061 0.09 0.141c -0.189 0.471 0.407 0.22 Gestational age 1.296  0.516 2.076 0.002 0.21 0.501d -0.282 1.283 0.217 0.43 aUnits for regression coefficients are mm of AGD per unit characteristic (g, cm, or weeks). Results based on 42 females. CI, confidence interval. b Multivariate adjusted regression coefficients (adjustment factors listed below). c Adjusted for gestational age. d Adjusted for weight of newborn infant. Table 4 Regression coefficients for anogenital distance as a function of characteristics at birth, malesa Variable Crude Adjustedb Coefficient 95% CI p value R2 Coefficient 95% CI p value R2 Birth weight 0.003  0.001 0.005 0.001 0.23  0.004c  0.002 0.006 0.001 0.27 Birth length 0.671  0.348 0.995 0.000 0.28  0.914c  0.499 1.329 0.000 0.33 Gestational age 0.356 -0.258 0.971 0.262 0.03 -0.560d -1.284 0.165 0.137 0.27 aUnits for regression coefficients are mm of AGD per unit characteristic (g, cm, or weeks). Results based on 45 males. CI, confidence interval. b Multivariate adjusted regression coefficients (adjustment factors listed below). c Adjusted for gestational age. d Adjusted for weight of newborn infant. Discussion The AGD measures employed in the present study reflect the location of the caudal border of the genital swelling, an embryologic structure that differentiates into the labia majora in females and the scrotum in males. After the indifferent stage of the external genitalia, the critical events determining the sexual dimorphism of AGD in humans begin when, relative to the anus, the genital swelling, urethral folds, and possibly the genital tubercle, move ventrally under the influence of androgens [28]. Elongation of the genital tubercle, which becomes the phallus, also occurs at this time. The difference between males and females in our data demonstrates sexual dimorphism of this particular measure of AGD. The two-fold difference in the aspect of AGD that we measured is not reflected in the schematic diagrams of human sexual differentiation we have seen [29,30], which is likely due to the previous lack of formal measures. Direct comparison of our results with those in the two other studies with measures of anus-to-fourchette (AF) distance in female newborns [24,25] is hampered by different eligibility criteria, and possibly different ethnicities, in the three studies. For example, Callegari et al.'s subjects had a mean weight of 2,530 g; Phillips et al. did not present mean birth weight but subjects were required to have a birth weight above 2,750 g; and in our study the mean birth weight among females was 3,060 g. The mean AF distance in the Callegari et al. study was 10.9 mm; in the Phillips et al. study was 16.1 mm in Jews and 16.5 in Bedouins, and in the present study was 11 mm. Callegari reported no ethnic differences in their population (62.6% Hispanic, 28.7% black, and 8.7% white). Despite the ethnic-specific mean values noted above, Phillips et al. reported that Jewish females had a greater AF distance than did Bedouins. The similarity of the mean AF distance measures in the present study and the Callegari et al. study is surprising given the difference in mean birth weights, and suggests ethnic differences, or a systematic difference in how the measurements were done. Compared with established anthropometric measures on newborns, the reliability of the AGD measures were lower. The lower reliability of the AGD measures is likely due to several factors. The AGD measures depend on indistinct landmarks on soft tissues. Structures such as "the center of the anus" or the posterior fourchette are not clearly demarcated. Any slight traction or pressure applied to the perineum or surrounding structures could alter measures. Finally, compared with established anthropometric measures on newborns, the AGD dimensions are smaller, thus measures done with the naked eye on a subject unlikely to hold still are inherently at a disadvantage. Use of two observers, one to restrain the subject and one to do the measurements could result in improved reliability compared to our approach, which employed one observer. Compared with adult humans, the size of the genitals at birth is large relative to the body overall [28]. Yet the genital size is, of course, still determined in part by overall body dimensions and age. The need to adjust AGD for overall body dimension is well known in animal experiments [19]. In humans, the best approach to such adjustment remains unclear. Our data suggest that for the aspect of AGD we measured, adjustment for body weight is reasonable. A complete assessment of AGD in humans would include more measurements than were done in our study. In neonatal rodents, measurement of AGD is relatively straightforward and is the distance from the genital tubercle to the anus. In older animals or humans of any age, however, questions arise as to which measure is most informative. For example, in human males, rather than a genital tubercle, the presence of the phallus and testicles at birth means that a number of measurements are possible. The measurement in the present study, from the posterior scrotal-perineal junction, represents only one such measurement. Ideally we would have done genital tubercle measurements in males and females, but we did not. Whether sexual dimorphism exists in the distance from the anus to the genital tubule (penile base in males) would be useful to know. While one might expect that penile length may be a good measure of androgenization among males, difficulties obtaining a reliable measure mean that alternative measures, such as AGD, are worth investigating. Effects of endocrinopathies on AGD in humans have been described, but only to a limited degree. A rare form of congenital adrenal hyperplasia that causes incomplete masculine development has been reported to cause decreased AGD in boys [21]. Details on how the measurement was done (and the measured values), however, were not presented [22,23]. Callegari et al. [24] measured the distance from the anus to the fourchette (same as what we did) and in addition measured the distance from the anus to the clitoris; the ratio of these two measures in three newborn females with congenital adrenal hyperplasia was increased relative to normal newborn females. Earlier case reports on females with adrenogenital syndrome noted labiosacral fusion, but again, no formal measures were published [23]. The utility of AGD measures in humans is further supported by experimental data in primates showing that in utero exposure of females to androgenic agents increased AGD [1]. The purported mechanism by which androgens increase AGD in females is by inducing "labioscrotal fusion" (in normal males fusion begins caudally and proceeds ventrally, presumably androgens in females act the same way) [24]. This mechanism, however, does not account for why males who are not fully androgenized would have a decreased AGD, unless AGD in males is defined as being from tip of penis to the center of the anus. A set of formal AGD measures on subjects with selected congenital endocrinopathies or birth defects could be useful in evaluating whether this outcome is uniformly responsive to gross stimuli, and may help discern details of normal embryology and the consequences of disrupting it. Conclusions In summary, we have shown that an aspect of genital dimension that reflects migration of the genital swelling is sexually dimorphic in humans. Whether this particular measure, or other measures of AGD in humans, has any utility as markers of exposure in utero to hormonally active agents remains to be seen. Abbreviations AF: anus-fourchette AGD: anogential distance ANOVA: analysis of variance CI: confidence interval Competing interests None declared. Authors' contributions ES participated in the design of the study, carried out the measurements, and wrote the first draft of the manuscript. PR participated in the study coordination and data management. EY carried out and coordinated the measurements. ML originated the idea that AGD measurements in human males may be useful, revised the manuscript, and analyzed the data. MH conceived of the study and participated in its design and coordination. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: Acknowledgements We are grateful to Alma Gonzalez-Rodriquez, MD, from the Instituto Nacional de Salud Publica, Cuernavaca, Mexico, for taking the anthropometric measurements of the infants, to the administrators of Dr. Ernesto Meana San Román General Hospital in Jojutla, Morelos, Mexico, for their cooperation and support, and to Grace Kissling, PhD, for statistical support. ==== Refs Hendrickx AG Korte R Leuschner F Neumann BW Prahalada S Poggel A Binkerd PE Gunzel P Embryotoxicity of sex steroidal hormone combinations in nonhuman primates: I. Norethisterone acetate + ethinylestradiol and progesterone + estradiol benzoate (Macaca mulatta, Macaca fascicularis, and Papio cynocephalus) Teratology 1987 35 119 127 3563930 Gray LE JrOstby JS Kelce WR Developmental effects of an environmental antiandrogen: the fungicide vinclozolin alters sex differentiation of the male rat Toxicol Appl Pharmaco 1994 129 46 52 10.1006/taap.1994.1227 You L Casanova M Archibeque-Engle S Sar M Fan LQ Heck HA Impaired male sexual development in perinatal Sprague-Dawley and Long-Evans hooded rats exposed in utero and lactationally to p,p'-DDE Toxicol Sci 1998 45 62 73 10.1006/toxs.1998.2515 Mylchreest E Sar M Cattley RC Foster PM Disruption of androgen-regulated male reproductive development by di(n-butyl) phthalate during late gestation in rats is different from flutamide Toxicol Appl Pharmacol 1999 156 81 95 10198273 10.1006/taap.1999.8643 Gray LE JrWolf C Lambright C Mann P Price M Cooper RL Ostby J Administration of potentially antiandrogenic pesticides (procymidone, linuron, iprodione, chlozolinate, p,p'-DDE, and ketoconazole) and toxic substances (dibutyl- and diethylhexyl phthalate, PCB 169, and ethane dimethane sulphonate) during sexual differentiation produces diverse profiles of reproductive malformations in the male rat Toxicol Ind Health 1999 15 94 118 10188194 10.1191/074823399678846664 Ostby J Kelce WR Lambright C Wolf CJ Mann P Gray LE Jr The fungicide procymidone alters sexual differentiation in the male rat by acting as an androgen-receptor antagonist in vivo and in vitro Toxicol Ind Health 1999 15 80 93 10188193 10.1191/074823399678846718 McIntyre BS Barlow NJ Wallace DG Maness SC Gaido KW Foster PM Effects of in utero exposure to linuron on androgen-dependent reproductive development in the male Crl:CD(SD)BR rat Toxicol Appl Pharmacol 2000 167 87 99 10964759 10.1006/taap.2000.8998 Nagao T Ohta R Marumo H Shindo T Yoshimura S Ono H Effect of butyl benzyl phthalate in Sprague-Dawley rats after gavage administration: a two-generation reproductive study Reprod Toxicol 2000 14 513 532 11099877 10.1016/S0890-6238(00)00105-2 Fielden MR Halgren RG Tashiro CH Yeo BR Chittim B Chou K Zacharewski TR Effects of gestational and lactational exposure to Aroclor 1242 on sperm quality and in vitro fertility in early adult and middle-aged mice Reprod Toxicol 2001 15 281 292 11390173 10.1016/S0890-6238(01)00129-0 McIntyre BS Barlow NJ Foster PM Androgen-mediated development in male rat offspring exposed to flutamide in utero: permanence and correlation of early postnatal changes in anogenital distance and nipple retention with malformations in androgen-dependent tissues Toxicol Sci 2001 62 236 249 11452136 10.1093/toxsci/62.2.236 Gray LE Ostby J Furr J Wolf CJ Lambright C Parks L Veeramachaneni DN Wilson V Price M Hotchkiss A Orlando E Guillette L Effects of environmental antiandrogens on reproductive development in experimental animals Hum Reprod Update 2001 7 248 264 11392371 10.1093/humupd/7.3.248 McIntyre BS Barlow NJ Foster PM Male rats exposed to linuron in utero exhibit permanent changes in anogenital distance, nipple retention, and epididymal malformations that result in subsequent testicular atrophy Toxicol Sci 2002 65 62 70 11752686 10.1093/toxsci/65.1.62 Shimamura M Kodaira K Kenichi H Ishimoto Y Tamura H Iguchi T Comparison of antiandrogenic activities of vinclozolin and D,L-camphorquinone in androgen receptor gene transcription assay in vitro and mouse in utero exposure assay in vivo Toxicology 2002 174 97 107 11985887 10.1016/S0300-483X(02)00044-6 Miyata K Yabushita S Sukata T Sano M Yoshino H Nakanishi T Okuno Y Matsuo M Effects of perinatal exposure to flutamide on sex hormones and androgen-dependent organs in F1 male rats J Toxicol Sci 2002 27 19 33 11915366 Wang XQ Fang J Nunez AA Clemens LG Developmental exposure to polychlorinated biphenyls affects sexual behavior of rats Physiol Behav 2002 75 689 696 12020734 10.1016/S0031-9384(02)00673-X Turner KJ Barlow NJ Struve MF Wallace DG Gaido KW Dorman DC Foster PM Effects of in utero exposure to the organophosphate insecticide fenitrothion on androgen-dependent reproductive development in the Crl:CD(SD)BR rat Toxicol Sci 2002 68 174 183 12075120 10.1093/toxsci/68.1.174 Bowman CJ Barlow NJ Turner KJ Wallace DG Foster PM Effects of in utero exposure to finasteride on androgen-dependent reproductive development in the male rat Toxicol Sci 2003 74 393 406 12773767 10.1093/toxsci/kfg128 Marty MS Chapin RE Parks LG Thorsrud BA Development and maturation of the male reproductive system Birth Defects Res Part B Dev Reprod Toxicol 2003 68 125 136 12866704 Gallavan RH JrHolson JF Stump DG Knapp JF Reynolds VL Interpreting the toxicologic significance of alterations in anogenital distance: potential for confounding effects of progeny body weights Reprod Toxicol 1999 13 383 390 10560587 10.1016/S0890-6238(99)00036-2 Gray LE JrOstby J Monosson E Kelce WR Environmental antiandrogens: low doses of the fungicide vinclozolin alter sexual differentiation of the male rat Toxicol Ind Health 1999 15 48 64 10188191 10.1191/074823399678846646 Goldman AS Bongiovanni AM Induced genital anomalies Ann N Y Acad Sci 1967 142 755 767 5340449 Bongiovanni AM Root AW The adrenogenital syndrome N Engl J Med 1963 268 1283 9 1342–1351 13968788 Bongiovanni AM Kellenbenz G The adrenogenital syndrome with deficiency of 3β-hydroxysteroid dehydrogenase J Clin Invest 1962 41 2086 2092 13968789 Callegari C Everett S Ross M Brasel JA Anogenital ratio: measure of fetal virilization in premature and full-term newborn infants J Pediatr 1987 111 240 243 3612396 Phillip M De Boer C Pilpel D Karplus M Sofer S Clitoral and penile sizes of full term newborns in two different ethnic groups J Pediatr Endocrinol Metab 1996 9 175 179 8887140 Dubowitz LM Dubowitz V Goldberg C Clinical assessment of gestational age in the newborn infant J Pediatr 1970 77 1 10 5430794 Flegal KM Launer LJ Graubard BI Kestler E Villar J Modeling maternal weight and height in studies of pregnancy outcome among Hispanic women Am J Clin Nutr 1993 58 145 151 8338040 Langman J Medical Embryology: Human development – Normal and Abnormal 1975 3 Baltimore: The Williams & Wilkins Company 160 200 Hannon TS Fuqua JS Eugster EA, Pescovitz OH Sexual differentiation Developmental Endocrinology 2002 Totowa, NJ: Humana Press 261 291 Quigley CA De Bellis A Marschke KB el-Awady MK Wilson EM French FS Androgen receptor defects: historical, clinical, and molecular perspectives Endocr Rev 1995 16 271 321 7671849 10.1210/er.16.3.271

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Environ Health. 2004 Sep 13; 3:8

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Environ Health

10.1186/1476-069X-3-8

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==== Front Mol CancerMolecular Cancer1476-4598BioMed Central London 1476-4598-3-241535300110.1186/1476-4598-3-24ResearchRNA-binding proteins to assess gene expression states of co-cultivated cells in response to tumor cells Penalva Luiz OF [email protected] Michael D [email protected] Simon M [email protected] Hedwig [email protected] Jack D [email protected] Department of Molecular Genetics and Microbiology, Center for RNA biology, 414 Jones Building, Research Drive, Duke University Medical Center, Durham, North Carolina 27710 USA2 Duke Bioinformatics Shared Resource, Duke University Medical Center, Durham, North Carolina 27710 USA3 Children's Cancer Research Institute. UTHSCSA. Mail code 7784 7703 Floyd Curl Dr. San Antonio, TX 78229-3900 USA4 Bayer Corporation 85 T.W. Alexander Drive Research Triangle Park NC 27709 USA5 Institut fur Krebsforschung Abt. Fur Angewandte und Experimentelle Onkologie Borschkegasse 8a A-1090 Wien Austria2004 7 9 2004 3 24 24 25 6 2004 7 9 2004 Copyright © 2004 Penalva et al; licensee BioMed Central Ltd.2004Penalva et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Tumors and complex tissues consist of mixtures of communicating cells that differ significantly in their gene expression status. In order to understand how different cell types influence one another's gene expression, it will be necessary to monitor the mRNA profiles of each cell type independently and to dissect the mechanisms that regulate their gene expression outcomes. Results In order to approach these questions, we have used RNA-binding proteins such as ELAV/Hu, poly (A) binding protein (PABP) and cap-binding protein (eIF-4E) as reporters of gene expression. Here we demonstrate that the epitope-tagged RNA binding protein, PABP, expressed separately in tumor cells and endothelial cells can be used to discriminate their respective mRNA targets from mixtures of these cells without significant mRNA reassortment or exchange. Moreover, using this approach we identify a set of endothelial genes that respond to the presence of co-cultured breast tumor cells. Conclusion RNA-binding proteins can be used as reporters to elucidate components of operational mRNA networks and operons involved in regulating cell-type specific gene expression in tissues and tumors. ==== Body Background Many recent studies have described the use of microarrays to identify genes expressed in different types of cancers (reviewed in [1,2]. Most of these transcriptomic studies monitor the steady state levels of expressed mRNAs in order to derive the "molecular signatures" of tumors [2]. However, the gene expression profile of a whole tumor corresponds to the combined profiles of the different cell types contained within it (e.g. endothelial cells, T-cells, cancer cells, stromal cells, etc.). Moreover, the multiple cell types present in a tumor or organ are interdependent and exchange biochemical signals as a means of cell-cell communication [3]. An important example of cell-cell communication is evident in angiogenesis, the mechanism by which new blood vessels vascularize tumors and other organs (reviewed in [4]). Monitoring the dynamics of gene expression in each cell type of a tumor during angiogenesis will advance understanding of tumorigenesis as well as organogenesis, in general. Methods have been devised to generate mRNA samples from specific types of tumor cells. These include microdissection, laser capture (reviewed in [4-6], and cell sorting based on specific membrane markers [7]. Here we demonstrate that RNA-binding proteins can be used to isolate mRNA populations representing total cell mRNA from specific types of cells, as well as discrete mRNA subpopulations that represent post-transcriptionally regulated subsets of mRNAs that encode functionally related proteins. We propose that these represent genes whose regulation is important for tumor growth and maintenance. RNA binding proteins play a key role in post-transcriptional regulation, participating in splicing, mRNA transport and localization, mRNA stability and translation (for overview see ref. [8]). Our lab has devised biochemical and immunological approaches to gene expression profiling by using RNA-binding proteins as reporters of discrete mRNA subsets in metazoan cells [8-10]. For example, we identified subpopulations of mRNAs that are associated with ELAV/Hu RNA-binding proteins that are expressed in specific cell types [10]. While we and other labs have demonstrated the isolation of mRNA subsets that are potentially co-regulated using RNA binding proteins as reporters of gene expression, methods have not been described that provide information about coordinated posttranscriptional regulation within specific types of cells during tumorigenesis and development. Moreover, because many different mRNA-binding proteins in specific cell types are known to interact with unique subpopulations of mRNAs encoding functionally related proteins [9-15] they can be informative of the dynamic effects of cells on one another. Therefore, it will be necessary to assess changes in gene expression that occur when cells such as tumor cells and endothelial cells interact in order to understand growth control and critical processes such as angiogenesis. In this study, we define a model system for using poly (A) binding protein (PABP) to recover mRNAs from specific cell-types in mixed cell cultures. Using this approach, we were able to determine how the gene expression profiles of endothelial cells change in response to the presence of breast cancer cells. Among the advantages of this approach are: a) no manipulations or treatments are required prior to the preparation of cell extracts, b) the recovered mRNA population can be identified directly using genomic methods, and c) RNA binding proteins can be engineered for expression in different cell types using various molecular tags in order to discriminate cell-specific mRNA populations. These studies provide a methodological basis for creating mouse models in which different types of cells within a tumor express RNA binding proteins to reveal unique populations of posttranscriptionally regulated mRNAs. Results and Discussion The goals of these experiments are to validate procedures for the isolation and characterization of discrete mRNA sub-populations associated with RNA binding proteins expressed in specific cell types within a tumor or organ in order to assess the responses of cells to their surroundings. Earlier studies have shown that mRNA subpopulations in single cell types reflect the functions of the RNA binding proteins with which they associate and can provide key information about post-transcriptional regulatory mechanisms of gene expression [8-11,13,15-18]. In model organisms, such information can be obtained by expressing epitope-tagged RNA binding proteins using tissue-specific promoters [19] or by using virus-specific receptors (M.D.B., L.O.F.P. and J.D.K. unpublished). In this study we demonstrate the feasibility of this approach by using two different cell types in culture that each express specific RNA binding proteins as reporters of gene expression profiles. Comparison of total mRNA of PY4.1 endothelial cells with their PABP-associated mRNA patterns Microarray analysis was used to compare the gene expression profiles obtained using total RNA and PABP-associated RNA of PY 4.1 endothelial cells (Figure 1 and supplementary data). These patterns were highly reproducible and consistent. Very few if any qualitative differences were observed when comparing these mRNA patterns indicating that the same set of expressed genes was detected in both preparations. However, quantitative differences were observed between some of the PABP-associated mRNA levels and those of the total mRNA population (Figure 1 and supplementary data). Approximately 19% of the expressed mRNAs varied more than two fold. In the case of the total mRNA profile (transcriptome), the signal intensity reflects the steady-state level of each mRNA, while the signal intensity of the PABP associated messages likely correlates with their translational activity [20]. PABP is an essential RNA binding protein that is highly conserved among eukaryotic organisms. It mediates interactions between polyadenylated mRNA sequences at the 3' ends of mRNAs and the eIF-4G protein [21]. Interactions of eIF-4G with the cap-binding protein, eIF-4E, are believed to circularize the mRNA and to prepare it for association with ribosomes. Many studies have shown that PABP is involved in activating the stability and translation of mRNAs to which it is bound (reviewed in [22,23], by protecting the poly (A) tail from exonuclease attack [24], preventing mRNA decapping [25], by promoting mRNA maturation [26] and by stimulating the initiation of translation [20]. Most studies are in general agreement that PABP functions as a translational activator by facilitating the assembly of mRNAs and ribosomes. On the average, a single PABP is expected to recognize approximately fifteen adenylate residues, suggesting that approximately ten molecules of PABP are bound to the average mRNA that has a poly (A) stretch of 150–200 in length [27]. Figure 1 Comparison of the gene expression profiles of total RNA and PABP-associated mRNA populations. Total RNA from PY4.1 murine endothelial cells and mRNA immunoprecipitated from cell extracts using anti-PABP serum were radiolabeled and hybridized on 1.2 mouse Atlas arrays (CLONTECH). The image overlay comparing total RNA with PABP-associated gene expression profiles was derived using the Atlas software with a global normalization showing quantitative, but not qualitative differences. Several reports indicate that substantial differences can be found when comparing the steady state levels of mRNAs (transcriptome) with proteins (proteome) in the same cell population [28,29]. The accumulated levels of some proteins and their corresponding mRNAs can vary by as much as 30-fold [28-30]. The differential between steady state levels of mRNA and protein are expected to be more dramatic under conditions in which post-transcriptional regulation plays a major role. For example, following T cell-activation or during neuronal differentiation, translational control is thought to affect a significant proportion of the proteomic outcome [31,32]. It is possible that gene expression profiles obtained by immunoprecipitating mRNA-PABP complexes may reflect the functional state of protein production from these mRNAs [10]. For the purposes of this study, PABP is used as a functionally relevant RNA-binding protein with which to compare changes in bound mRNAs across gene expression profiles. Expression of tagged PABP does not interfere with cell growth In order to compare mRNA profiles from mixed cell populations, we prepared two different cell lines stably expressing different epitope tagged PABPs. Figure 2 outlines the experimental approach. T98G human glioma cells and PY4.1 mouse endothelial cells were co-cultured, cell extracts were prepared and antibodies against PABP, Flag-PABP and G10-PABP were used to immunoprecipitate the mRNP complexes in order to generate mRNA populations for gene expression analysis. Concerns that the epitopes represented in the tags might affect the results were addressed using PY4.1 cells stably expressing either Flag-tagged PABP or G10-tagged PABP. Cell extracts from both of the stable cell lines grown separately were prepared and immunoprecipitated with the respective antibodies. The two mRNA populations generated by this procedure were compared using an RNAse protection assay (RPA) and a microarray analysis. The results showed no significant qualitative or quantitative differences between these profiles in that 96% of the genes detected by microarray were within 1.5-fold of one another (data not shown). Figure 2 A) Experimental design for sorting cell type specific mRNA populations using RNA binding proteins in mixed cell cultures. Two cell lines from two different species (murine endothelial PY4.1 and human glioma tumor T98G) are engineered to express G10-tagged and FLAG-tagged PABP, respectively. Cell-type specific gene expression profiles are obtained from co-cultured cells or mixed cell extracts after immunoprecipitation with specific antibodies against the different tags. The RNA samples are phenol extracted, precipitated and subsequently analyzed by RPAs or microarrays. B) Western blots of cell lines expressing tagged-PABP. Immunoblots of extracts from T98G cells expressing Flag-PABP were probed with anti-Flag antibody, while extracts from PY4.1 and PY4.1 cells expressing G10-PABP were probed with anti-G10 antibody. Control blots of extracts of T98G and PY4.1 cells with anti-sera against PABP. C) Comparison between the overall levels of PABP of T98G cells and T98G cells expressing Flag-PABP. Immunoblots of extracts from T98G cells and T98G cells expressing Flag-PABP were probed with anti-sera against PABP and anti-α tubulin antibody, as a loading control. D) Comparison of the cell cycle status of T98G and T98G expressing Flag-PABP cells – Cells were arrested at G0/G1 by serum deprivation and stimulated to re-enter the cell cycle by addition of serum. At the indicated times, aliquots of cells were processed for FACS analysis to determine the population distribution in G1, S, and G2 stages of the cell cycle. No differences between of the cell cycle of T98G cells and T98G cells expressing Flag-PABP were observed. A potential complication for this type of analysis is that expression of a tagged-RNA binding protein, in this case PABP, could affect cell growth. While these cell lines appeared unaffected morphologically, the levels of PABP in cell lines expressing tagged-PABP and respective control cell lines were evaluated and compared by Western blotting. No substantial change in overall PABP expression was observed when expressing exogenous tagged PABP (Figure 2C). This result was expected, since PABP has been shown to inhibit the translation of its own mRNA by binding to poly (A) sequences found in the 5' UTR. This fortuitous auto-regulatory mechanism is believed to keep the level of PABP constant in the cell, thereby avoiding excessive overexpression [33]. No changes in cell growth or mortality of the cell lines used in this study or in other cells lines expressing tagged-PABP were observed. Moreover, the cell cycle kinetics of T98G cells expressing Flag-PABP and cells that were subsequently stimulated by serum addition were compared to those of T98G cells containing the empty vector, pCMVneo and no substantial differences were observed using fluorescent cell sorting (Figure 2D). We conclude that expression of neither the authentic PABP, nor the tagged-PABP has untoward effects on the growth and homeostasis of these cells. PABP does not exchange between mRNAs in cell extracts Early studies of PABP binding to mRNA indicated that a dynamic exchange or hopping of PABP from mRNA to mRNA might be an important aspect of its function [27,34]. For the expression profiling methods described above to be precise, it is critical to avoid post-lysis exchange (or adventitious reassortment) of PABP with mRNAs. In other words, does free mRNA in a cell extract displace the mRNA originally bound to PABP during the incubation period; or instead, does free PABP in an extract exchange by binding to available mRNAs? PABP is a good test model in this case because it has been suggested to "hop" based on in vitro studies, and it is a highly abundant RNA-binding protein. To examine these possibilities, we added increasing amounts of competitor poly (A) RNA with an average length of 550 nucleotides to lysates of mouse endothelial PY4.1 and human glioma T98G cells. After immunoprecipitation with anti-PABP serum, mRNAs were isolated from the pellets and analyzed using a highly sensitive multi-probe RNase Protection Assay (RPA) as described previously [10]. Figure 3 shows that the mRNAs originally bound to PABP were not displaced by the competing poly (A) RNA even at concentrations as high as 1000 fold excess. The inability of free poly (A) RNA to compete bound PABP off of endogenous mRNA reflects a stable interaction between PABP and endogenous mRNA. These data suggest that exchange of mRNA into PABP RNPs is not likely to distort gene expression profiles obtained by immunoprecipitating PABP, and in addition, this observation is not compatible with a previous "hopping model" for PABP [27]. Figure 3 Reassortment of mRNA and PABP was not detected in cell lysates. The potential for displacement of PABP was tested by adding increasing amounts (0–1500 μg) of pure competitor poly (A) to 400 μl of cell lysates of PY4.1 and T98G cells prior to incubation with antibody-coated (anti-PABP) beads. Following immunoprecipitation, mRNAs were isolated from the pellets and analyzed using the RNase Protection Assay (RPA) mouse angiogenesis (mAngio) and human tumor suppressor (hTS1) multi-probe sets. Unprotected RNA probes were used to identify the nature of the different sized protected fragments. The experiment shows that the interaction between PABP and the endogenous mRNA targets cannot be disrupted by competing poly (A) RNA. We have also addressed the potential problem of having a pool of free PABP in an extract that could be available to bind mRNA during incubation. Sucrose gradient analysis indicated that this is very unlikely since the majority of PABP was found in heavy polysomes and associated with mRNA, while only a small percentage was found in the upper portion of the sucrose gradients (H.S. and J.D.K., unpublished data). In total, these results demonstrate that reassortment of PABP in these cell extracts was not a significant limitation to using PABP RNPs for gene expression profiling of bound mRNAs. As noted above, it has been suggested that yeast PABP uses a "hopping" mechanism in vivo by moving from RNA to RNA [27,34]. While this experiment is not a direct test of that hypothesis, these data are not consistent with a hopping or exchange of PABP among the mRNAs in our cell extracts, but suggest instead that PABP forms a stable RNP complex with polyadenylated transcripts. Detection of cell-specific mRNAs using epitope-tagged PABP The question of whether reassortment of PABP occurs among mRNAs in cell extracts was also examined using lysates from mixed mouse and human cells. We used two different cell lines, murine endothelial PY4.1 and human glioblastoma T98G, that express G10-PABP and Flag-PABP, respectively (Figure 2B). T98G cells have a volume approximately 3 to 4 times larger than PY4.1 cells. These cell lines were co-cultured in an approximate 1:1 cell ratio and subsequently lysed, or in separate experiments, lysates of each were mixed at equivalent amounts of total protein prior to immunoprecipitation. Both approaches gave the same results. Using the G10 antibody to precipitate only PY4.1 mRNAs and Flag antibody to precipitate T98G mRNAs we were able to examine the separate populations using a multiprobe RPA (Figure 4). Thus, by mixing a human (T98G) and a murine (PY4.1) cell line we could take advantage of the species-specific RPA probe sets. Given our interest in tumor angiogenesis, we used mouse angiogenesis (mAngio) and human tumor suppressor (hTS1) RPA probe sets after they were tested for cross species hybridization. Both probe sets showed good specificity of discrimination with the exception of the L32 and GAPDH control genes as expected (Figure 4A and 4B). In Figure 4C and 4D, it is apparent that the same expressed genes were detected whether using total RNA, or mRNA obtained by immunoprecipitation with anti-PABP, anti-Flag or anti-G10 antibodies. Extracts from mixed cell lines were immunoprecipitated and analyzed using both human and mouse probe sets (Figure 4E,4F and 4G). Both mouse and human mRNAs were detected when anti-PABP rabbit serum was used to precipitate both endogenous and exogenous PABP, while immunoprecipitations with anti-G10 and anti-Flag antibodies enriched the mRNA population for each species with only minor background from the other species. While the discrimination obtained in these experiments was excellent, a low degree of background due to non-specific binding of mRNA to the agarose beads was consistently observed even in the absence of antibody. Figure 4 RNase Protection Assay (RPA) of mRNAs from mixed mouse and human cell lines. Species specificity of multiprobe RPAs using: A) the human tumor suppressor probe set (hTS1), and B) the mouse angiogenesis RPA probe set (mAngio), was verified using total RNA extracted from murine PY4.1 cells and human T98G cells. Unprotected RNA probes were used to identify the nature of the different sized protected fragments. C) RPA gene expression profile of PY4.1 cells expressing G10-PABP obtained with total RNA and with mRNA derived from immunoprecipitations with anti-PABP serum or anti-G10 antibody D) RPA gene expression profile of T98G cells expressing Flag-PABP obtained from total RNA and from mRNA derived from immunoprecipitations with anti-PABP serum or anti-Flag antibody. E, F and G) Mixed extracts from cells expressing T98G Flag-PABP and PY4.1 G10-PABP were immunoprecipitated with anti-PABP serum or anti-Flag or anti-G10 antibodies. The mRNA populations generated by immunoprecipitations were analyzed with RPA of both the mouse angiogenesis (mAngio) and the human tumor suppressor probe (hTS1) sets. GAPDH and L32 are controls in both probe sets and show cross species hybridization. The asterisk in B and G also indicate a band resulting from cross species hybridization. The experiments indicate that species-specific mRNA populations can be isolated and quantified by the use of distinct tagged-PABPs. Analysis of PABP-associated mRNA populations using microarrays In order to evaluate the degree of mRNA enrichment over background using a genome–wide methodology, we analyzed immunoprecipitated mRNAs from mixed cell populations on CLONTECH Atlas arrays. We first tested these arrays for cross species hybridization using total RNA and it was minor (data not shown). The mouse RNA on human Atlas arrays did not show any detectable cross hybridization signal, while the human RNA on mouse Atlas arrays showed a small percent (2–3%) of cross hybridizing mRNAs (Figure 5B, blue squares). Figure 5 Discrimination of the gene expression profiles of mixed human and mouse cell lines using microarrays. Cell extracts from T98G Flag-PABP and PY4.1 G10-PABP cells were prepared, mixed and immunoprecipitated with both anti-Flag and anti-G10 antibodies. The mRNA populations generated by both immunoprecipitations were analyzed on human and mouse 1.2 CLONTECH arrays. When anti-Flag antibodies were used, the T98G mRNA population was enriched in relation to the PY4.1 mRNA population. When anti-G10 antibodies were used, the PY4.1 mRNA population was enriched in relation to the T98G mRNA population. To identify the PABP-associated mRNAs in the T98G Flag-PABP and PY4.1 G10-PABP cells, extracts were prepared as described above, followed by immunoprecipitation with either anti-Flag or anti-G10 antibodies. The mRNA populations generated by both immunoprecipitations were analyzed on human and mouse 1.2 Atlas arrays. Cross-species hybridization was monitored and genes showing cross-species reactivity were eliminated from consideration. A comparison of Flag versus G10 PABP-associated mRNAs was performed to assess the degree of enrichment. In an average experiment for the mouse genes, 91 % (184 out of 202 detected genes) were enriched at least 4 fold in the G10 PABP population when compared to the Flag PABP population. For the human genes, 82.4 % (122 out if 148) were enriched at least 4 fold in the Flag PABP population in relation to the G10 PABP population (Figure 5 and supplementary data). Changes in gene expression induced by co-cultivation of PY4.1 endothelial cells with 4T1 breast cancer cells Having demonstrated that the approach we described using PABP can be used to efficiently recover cell type specific mRNAs from mixed cell types, we addressed the consequences of cell-cell communication and changes in gene expression that were induced in the endothelial cells by co-cultivation with the tumor cell. The goal of these experiments is to gain insight into how endothelial cells respond to the presence of cancer cells in cell culture as a first approximation of changes in gene expression that may be involved in early stages of angiogenesis. We used two murine cell lines, the PY4.1 line described above and a 4T1 breast tumor cell line that can produce tumors and spread by metastasis in nude mice. Figure 6 represents a schematic view of two branches of our experimental strategy. In the first line of inquiry, PY4.1 cells stably expressing Flag-PABP and 4T1 cells were co-cultured. The number of plated cells of each type was calculated based upon measurements of their different growth rates. After 48 hours the co-cultured cells reached confluence. Approximately 50% of the petri dish surface was covered with PY4.1 cells and 50% was covered with 4T1 cells. Cells were harvested and extracts were prepared as described above. In the second line of inquiry, the two cell types were plated separately. Cells were harvested after 48 hours of incubation, the extracts were prepared, and mixed proportionally to those used in the first experimental line of investigation. Subsequently, extracts from the co-cultured and the mixed cells were immunoprecipitated with anti-Flag antibodies and mRNAs analyzed on microarrays. The comparison between the two mRNA populations (co-culture versus mix) was used to detect changes in the gene expression profile of PY4.1 cells as a response to the presence of 4T1 cells. In a separate experiment, a comparison between PY4.1 total RNA labeled with Cy3 and Cy5 was performed to rule out dye bias (not shown). Figure 6 Effects of co-cultivated mouse tumor cells on gene expression profile of mouse endothelial cells. PY4.1 cells expressing Flag-PABP and 4T1 cells were grown either separately or together in a co-culture. Cell extracts from the co-culture and from a mixture of the monocultures were prepared. Immunoprecipitation of extracts with anti-Flag antibodies generated two distinct PY4.1 cells mRNA populations that were compared by microarray. The comparison revealed PY4.1 cellular genes that changed their gene expression profile in response to presence of 4T1 breast cancer cells. As expected, the great majority of the genes expressed in PY4.1 cells were not altered in their levels of expression while in co-culture. Of interest, a small number of genes were consistently upregulated in four independent experiments. To assess the consistency of the fold change, we plotted the p-value (from modified t-test) against the average fold change (Figure 7). A 'volcano plot' summarizes both the magnitude of change and the corresponding statistical significance for all genes. We sorted the candidate genes according to their p-values (Table 1), and the top-20 genes were identified and are listed along with comments concerning their biological function(s). [See Supplementary Data for the complete microarray analysis – ]. Several of the genes present in this population are gene expression regulators that fall into two major categories: RNA binding proteins and DNA binding proteins/transcription factors. We expected to find gene expression regulators as part of an early response to cell surface interactions or secreted factors from the other cell line. As with any biochemical cascade event, changes in the expression of global regulators as well as structural genes (such as those encoding membrane or cytoskeletal proteins) often precede downstream alterations in the expression of other important genes. Among the RNA binding proteins identified in our screen were stem-loop binding protein, a highly conserved RNA binding protein that binds a stem loop structure in the 3'UTR of histone mRNAs and is required for both processing and translation of histone messages [35], Brul4, the mouse homologue of Drosophila bruno, a translation repressor which functions at the early steps of embryogenesis [36], and quaking, described as a key gene involved in the myelination of the central nervous system and other regulatory functions [37]. It should be noted that we did not identify PY4.1 genes whose expression decreased as a response to the presence of 4T1 cells. Figure 7 "Volcano" plot of p-value versus fold change in expression level. Dashed line indicates the cutoff of the top 20 enriched genes shown in Table 1. Table 1 List of the top 20 PY4.1 genes that were upregulated in response to the presence of 4T1 tumor cells. Genes are classified according to their biological function. Gene expression regulators (GR). Genes involved in metabolism (M). Genes related to cell cycle or cell division (C). Genes encoding structural proteins (S). Other genes (O). Name, classification gbID fold P value biological function 1-Heterogeneous nuclear ribonucleoproteinH1, (G R) NM_021510 2.6 6.40E-06 RNA binding, RNA processing and modification 2-High mobility group box 1, (G R) NM_010439 2.7 9.78E-06 DNA binding, nitric oxide biosynthesis, inflammation mediator, cell differentiation 3-Prothymosinalpha, (C) NM_008972 3.7 1.38E-05 cell proliferation, cell division 4-RIKEN cDNA2610016F04 gene, (G R) AK009120 2.6 4.59E-05 putative DNA binding, transcritionfactor 5-ATPase, H+ transporting, (M) NM_024173 1.8 4.92E-05 hydrogen-exporting, ATPaseactivity, phosphorylativemechanism 6-RIKEN cDNA2510010F10 gene, (O) AF215660 1.8 7.45E-05 described as a carnitinedeficiency-associated gene 7-Stem-loop binding protein, (G R) NM_009193 2.1 7.88E-05 RNA binding, histonemRNA processing 8-Quaking, (G R) NM_021881 1.9 7.79E-05 RNA binding, participates in myelination 9-RIKEN cDNA2410004I17 gene, (O) AK010391 2 8.26E-05 unknown 10-Purinerich element binding protein A, (G R) NM_008989 2.8 8.95E-05 DNA and RNA binding, association with rough endoplasmic reticulum, postnatal brain development 11-Similar to isopentenyl-diphosphatedelta isomerase, (M) BC004801 3 9.80E-05 cholesterol biosynthesis, steroid biosynthesis 12-P53 apoptosis effectorrelated to Pmp22, (O) NM_022032 2.4 1.11E-04 induction of apoptosis 13-Tumor differentially expressed 1, like, (S) NM_019760 2 1.17E-04 plasma membrane 14-RIKEN cDNA5830409B12 gene, (S) AK017914 7.6 9.73E-05 putative cytoskeleton associated protein 15-G7e protein, (S) NM_033075 2 1.42E-04 resembles viral envelope genes 16-Procollagenlysine, 2-oxoglutarate 5-dioxygenase 2, (M) NM_011961 2.8 1.43E-04 protein metabolism 17-Receptor-like tyrosine kinase, (M) L21707 1.7 2.08E-04 ATP binding, kinaseactivity 18-RIKEN cDNA4930506D01 gene, (G R) BC006745 2.8 1.77E-04 putative transcription factor 19-MusmusculusBRUL4 (Brul4) mRNA, (G R) AF314173 5.8 1.91E-04 RNA binding, translation regulator 20-CyclinI, (C) NM_017367 2.3 1.67E-04 cell cycle, cyclin-dependent protein kinaseregulator activity relevant references 1-none 2-Gastroenterology. 2002. 123:790–802; J LeukocBiol. 2002. 72:1084–1091 3-Peptides. 2000. 21:1433–1446; IntJ Biochem Cell Biol. 1999. 31:1243–1248. 4-none 5-J BiolChem. 2002. 277:36296–36303; Gene. 2003. 302: 147–153. 6-Biochim BiophysActa. 2002. 1577:437–444. 7-J. Cell Sci. 2002. 115: 4577–4586. 8-Neuron. 2002. 36:815–829; Nucleic Acids Res. 2003.31:4616–4624 9-none 10-J BiolChem. 2002.277:37804–37810; Mol Cell Biol. 2003. 23:6857–6875 11-J Mol Evol. 2003. 57: 282–291. 12-Genes Dev. 2000. 14: 704–718. 13-J Exp Biol. 2000. 203:447–457. 14-none 15-Genomics. 1996. 15:5–12 16-J BiolChem. 2003. 278:40967–40972. 17-none 18-J Immunol. 1995. 154:1157–1166; Proc NatlAcadSciU S A. 1992. 89:11818–11822. 19-CytogenetGenome Res. 2002.97:254–260. 20-Gene. 2000. 256: 59–67. The presence of several RNA binding proteins among the top-20 genes affected by co-cultivation may result in downstream effects on gene expression, and we plan to examine the target mRNAs of these RNA binding proteins in endothelial cells. This should help elucidate additional post-transcriptional pathways and networks regulating cell growth mechanisms and tumorigenesis [9]. Conclusion This study describes changes in the gene expression profile of an endothelial cell when co-cultivated with a tumor cell by isolating ribonucleoprotein complexes and identifying their associated mRNAs using genomic arrays. Moreover, it presents a model system that can be used to elucidate post-transcriptional operons in specific types of cells by using various RNA binding proteins from mixed cell cultures as a novel approach to understanding how cell-cell communication affects gene expression during tumorigenesis and organogenesis. Methods Cell lines and media Murine endothelial PY4.1 cells were kindly provided by Dr. Christopher Kontos, Duke University Medical Center. Human gliobastoma T98G and murine breast cancer 4T1 cells were obtained from American Type Culture Collection. All cell lines were maintained in DMEM Medium (Gibco) supplemented with 10% Fetal Bovine serum. Constructs and stable cell lines The ORF of human PABP I containing the Flag tag (GACTACAAGGACGACGATGACAAG) or the G10 tag (CCACCATGGCT AGCATGACTGGTGGACAGCAAATGGGT) at the 5' end was cloned into the pCMV-Neo retroviral vector [38]. Stable lines expressing the Flag-PABP (T98G and PY 4.1 cells) and the G10-PABP (PY 4.1 cells) were obtained according the protocol described in the Pantropic Retroviral Expression System (Clontech). Antibodies Monoclonal anti-G10 antibodies were obtained as previously described [39]. Antibodies against Flag and α-tubulin were obtained from SIGMA. A PABP carboxy-terminal (last 172 amino acids) was prepared by cloning a PCR product into the pGEXCS expression vector. The protein was purified by their affinity to glutathione beads (Amersham Biosciences). The purified proteins were dialyzed against 1 × PBS, 20% glycerol and sent to COVANCE Inc., where a rabbit was immunized. Protein preparation and Western analysis Protein extracts were prepared from T98G and PY4.1 cell lines by homogenization in polysomal lysis buffer [10]. 50 μg of extract were fractionated by electrophoresis in 10% polyacrylamide-SDS Laemmli gels. Proteins were transferred to nitrocellulose membranes using a transfer cell (Bio-Rad). After blocking with 5% nonfat milk in PBS-Tween 20 buffer, the membranes were incubated with anti-PABP rabbit serum (1:10,000 dilution), anti-Flag antibody (1:1,500 dilution) or anti-G10 antibody (1:10,000 dilution). Anti-rabbit or anti-mouse HPC IgGs (Amersham Biosciences) were used as secondary antibodies at a 1:3000 dilution. Blots were developed using an ECL detection kit (Amersham Biosciences) and exposed to film. Cell cycle experiments Analysis of the cell cycle of T98G cells was performed as described [40]. Immunoprecipitation of mRNP complexes from cell lysates Cell lysates and immunoprecipitation of mRNP complexes were essentially performed as described [41]. Polyadenylated RNA (free poly-A) used in competition experiments was obtained from Amersham Biosciences. RNase Protection Assay Total or immunoprecipitated RNAs were assayed by RNase protection by using the PharMingen Riboquant assay according to the manufacturer's recommendations (45014K). mAngio-1 (mouse angiogenesis) and hTS1 (human tumor suppressor) template sets were used (551418 and 556161, respectively). Protected riboprobe fragments were visualized on a phosphorimaging screen (Molecular Dynamics). Phosphorimages were scanned by using the Molecular Dynamics STORM 860SYSTEM at 100 μm resolution and analyzed by using Molecular Dynamics IMAGE QUANT software (version 5.0). Clontech microarrays, probing and analysis cDNA array analysis was performed by using Atlas Mouse and Human 1.2 Arrays (CLONTECH). Probing of cDNA arrays was performed as described in the CLONTECH Atlas cDNA Expression Arrays User Manual (PT3140-1). Reverse-transcribed probes were radiolabeled with 32P α-dATP (Amersham Biosciences). After hybridization, the array membrane was washed and the results were visualized on a phosphorimaging screen (Molecular Dynamics). Phosphorimages were scanned by using the Molecular Dynamics STORM 860SYSTEM at 100 μm resolution and stored as .gel files. Images were analyzed by using ATLASIMAGE 2.01 software (CLONTECH). Global normalization was used when arrays being compared had approximately the same number of positive hits. Printed oligo arrays, probing and analysis Printed oligo arrays using the Operon Mouse Oligo set version 2.0 (16,423 genes) were produced by the Duke Microarray Core Facility. Protocols used for preparation of slides, labeling, amplification, hybridization and scanning are described in . GenePix data were normalized with pin-tip specific lowess normalization [42]. Differentially expressed genes were identified with a moderated t-test, which shrinks the estimated sample variances towards a pooled estimate [43]. This moderated t-test is more robust when the number of arrays is small. The candidate gene list is sorted by the p-value. All calculations were conducted using the bioconductor package [44]. List of abreviations ELAV – embryonic lethal abnormal vision RPA – RNase Protection Assay PABP – Poly A binding protein Author's contribution LOFP was responsible for the experimental design and performed Western blots, immunoprecipitations, RPAs and microarray experiments. MDB helped perform RPAs and microarray experiments. SML performed statistic analysis of microarray data and prepared the webpage with supplementary data. HS generated the stable cell lines used in this study and performed the cell cycle experiment. JDK conceived the project and assisted in experimental design. Supplementary material Acknowledgments We thank Amy Sims for technical help, and Juan Valcarcel and Karl Simin for comments on the manuscript, as well as S.G. Gao and his group for technical help. 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into the gag region J Virol 1987 61 1639 1646 3502707 Lutz-Freyermuth C Query CC Keene JD Quantitative determination that one of two potential RNA-binding domains of the A protein component of the U1 small nuclear ribonucleoprotein complex binds with high affinity to stem-loop II of U1 RNA Proc Natl Acad Sci U S A 1990 87 6393 6397 1696729 Lisztwan J Marti A Sutterluty H Gstaiger M Wirbelauer C Krek W Association of human CUL-1 and ubiquitin-conjugating enzyme CDC34 with the F-box protein p45(SKP2): evidence for evolutionary conservation in the subunit composition of the CDC34-SCF pathway Embo J 1998 17 368 383 9430629 10.1093/emboj/17.2.368 Penalva LOF Tenenbaum SA Keene JD Shonenberg D Gene Expression Analysis of mRNP Complexes Methods in Molecular Biology – mRNA Processing and Metabolism 2004 257 Totowa, NJ: Human Press 125 134 10.1385/1-59259-750-5:125 Yang YH Dudoit S Luu P Lin DM Peng V Ngai J Speed TP Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation Nucleic Acids Res 2002 30 e15 11842121 10.1093/nar/30.4.e15 Smyth GK Linear Models and Empirical Bayes Methods for Assessing Differential Expression in Microarray Experiments Statistical Applications in Genetics and Molecular Biology 2004 3 Article 3 Dudoit S Gentleman RC Quackenbush J Open source software for the analysis of microarray data Biotechniques 2003 Suppl 45 51 12664684

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==== Front RetrovirologyRetrovirology1742-4690BioMed Central London 1742-4690-1-261536310910.1186/1742-4690-1-26Short ReportIdentification of the 15FRFG domain in HIV-1 Gag p6 essential for Vpr packaging into the virion Zhu Henghu [email protected] Heng [email protected] Ling-Jun [email protected] Institute for Molecular Virology, St. Louis University School of Medicine, St. Louis, USA2 Department of Plant Pathology, China Agricultural University, Beijing, China2004 13 9 2004 1 26 26 9 9 2004 13 9 2004 Copyright © 2004 Zhu et al; licensee BioMed Central Ltd.2004Zhu et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The auxiliary regulatory protein Vpr of HIV-1 is packaged in the virion through interaction with the Gag C-terminal p6 domain. Virion packaging of Vpr is critical for Vpr to exert functions in the HIV-1 life cycle. Previous studies suggest that Vpr interacts with a (Lxx)4 domain in p6 for virion packaging. In the present study, mutational analysis of HIV-1 Gag p6 domain was performed in the context of the HIV-1 genome to examine the effect on virion packaging of Vpr. Surprisingly, Ala substitutions for Leu44 and Phe45 in the (Lxx)4 domain or deletion of the whole (Lxx)4 domain (amino acid #35–52 of the Gag p6 domain) did not affect Vpr virion packaging. Vpr virion packaging was normal when amino acid #1–23 of the Gag p6 domain was preserved. Most importantly, Ala substitutions for Phe15, Arg16 and Phe17 in the context of amino acid #1–23 of the Gag p6 domain abolished Vpr virion packaging. Single Ala substitutions for Phe15 and Phe17 also abolished Vpr virion packaging, whereas Ala substitution for Arg16 had no effect. Our studies have revealed a novel signal sequence for Vpr packaging into the HIV-1 virion. The 15FRFG domain in p6 resembles the FxFG repeat sequences commonly found in proteins of the nuclear pore complex. These results have provided novel insights into the process of virion packaging of Vpr and suggest for the first time that Vpr may recognize the FxFG domain for both virion packaging and association with nuclear pores. ==== Body Findings Vpr is a 15 kDa auxiliary regulatory protein of HIV-1 produced in the late phase of the viral life cycle and packaged in the virion [1-3]. Thus, Vpr has the capacity to function both in the early phase and the late phase of the viral life cycle. A number of biological activities have been assigned to Vpr, including nuclear localization [4-6], transcriptional effects [7,8], cell cycle arrest at the G2/M check point [9-13], and pro- and anti-apoptotic activities [14-18]. In most cases the direct cellular target for Vpr remains to be identified. It is possible that Vpr has multiple unrelated functions to facilitate HIV-1 interaction with the host cells. Alternatively, some of the biological activities of Vpr may be explained by a common mechanism. Transiently expressed Vpr localizes in the nucleus, and specific nuclear localization signals have been identified in Vpr [6]. Vpr nuclear transport has been correlated with interaction with importin a [19]. However, the nuclear localization of Vpr appears to be more complicated since Vpr is also found to interact with residents of the nuclear pore complex [20]. Notably, Vpr is found to interact with the FG repeat domain of rat Poml21, which is a nuclear pore protein [20]. However, in similar assays Vpr fails to interact with the FG repeat domain of other nuclear pore proteins [20]. Thus, the exact specificity of this interaction remains uncharacterized. Virion packaging of Vpr is through interaction with the Gag C-terminal p6 domain [1]. With vaccinia expression of HIV-1 Gag and Vpr, a (Lxx)4 domain (amino acid #35–46) in HIV-1 p6 was determined to be essential for virion packaging of Vpr [21]. Fusion of MLV Gag with the HIV-1 p6 domain allows the fusion protein to package Vpr [22]. Under this condition, single point mutations of L45A or F46A within the (Lxx)4 domain abolish Vpr virion packaging [22]. The direct interaction of HIV-1 p6 with Vpr appears to be rather weak, detectible only by using a sensitive in vitro assay [23]. The dissociation constant for the p6-Vpr complex is between 18–75 μM [23]. It is hypothesized that this weak interaction may be enhanced during the process of virion packaging when Gag forms oligomers [23]. Secondary interactions between Vpr and other regions of Gag may also aid virion packaging of Vpr [24]. Interestingly, the HIV-1 p6 also has well-characterized domains for binding cellular sorting factors Tsg101 and AIP1 [25,26]. Whether these interactions influence Vpr virion packaging remains unclear. In this study, sequences in HIV-1 Gag p6 domain required for Vpr virion packaging was dissected in the context of the HIV-1 genome. Surprisingly, the previously identified (Lxx)4 domain in p6 is shown non-essential for Vpr virion packaging. Instead, a 15FRFG domain in HIV-1 Gag p6, 4 amino acid residues downstream of the Tsg101-binding domain, is found critical for Vpr virion packaging. Since FxFG domains also occurs in nuclear pore proteins, the current finding also suggests for the first time that Vpr may recognize the same FxFG domain for both virion packaging and association with nuclear pores. Thus, the FxFG domain appears to be a favorite signal for in vivo recognition by Vpr. We discuss the impact of this finding in the context of the HIV-1 life cycle. To examine the biochemical process of Vpr virion packaging, we introduced various Gag p6 mutations into an HIV-1 genome containing partial deletion of the Pol gene and HA-tagged ubiquitin in place of the Nef gene. This modified HIV-1 genome was used to facilitate construction of p6 mutants and to examine ubiquitination of HIV-1 proteins. All HIV-1 genomic constructs were based on the p89.6 plasmid [27] and their sequences were confirmed by automatic sequence analysis. p89.6/Po1-/R+ and p89.6/Pol-/R- constructs were described before [28]. A BamHI site was inserted at the beginning of the Nef ORF in a subclone of p89.6 carrying the 3' half of the HIV-1 genome, p89.6/3'[27], to generate p89.6/3'-BamHI. This modification also resulted in deletion of the 5' region of Nef ORF up to the KpnI site. Subsequently, the HA-Ub coding sequence was PCR-amplified from the pCMV-HA-Ub plasmid [29] with primer 1 AGTTACGGATCCATGGCATAGCTACCCTTATGACGTC and primer 2 CATTCAGGATCCTACCCACCTCTGAGACGGAGGACCAG, digested with BamHI and inserted into the p89.6/3'-BamHI plasmid to generate p89.6/3'-HA-Ub. The EcoRI/PstI-blunt fragment of p89.6/3'-HA-Ub was ligated to the EcoRI/SmaI sites of p89.6/Pol-/R+ and p89.6/Pol-/R- to generate p89.6/HA-Ub/R+ and p89.6/HA-Ub/R-constructs, respectively (labeled as HA-Ub/R+ and HA-Ub/R- in Fig. 1). Figure 1 HIV-1 genomic constructs and requirements for Vpr virion packaging. A) All viral constructs were based on the p89.6/HA-Ub/R+. Pr-/R+: genomic construct carrying the wild type p6 and a premature stop codon for the protease ORF immediately after the p6 stop codon. All other clones were derived from the Pr-/R+ construct. Bold-typed regions represent binding sites for Tsg101, Vpr (this study), and AIP1. B) Effects of p6 mutations on virion packaging of Vpr. Experimental conditions are described in "Findings". Left panels: Gag and Vpr Western blots with virion samples. Right panels: top two panels are Western blots of virion samples, whereas the bottom panel is Western blot of Vpr immunoprecipitated from cell lysates. C) Comparision of the 15FxFG domain in HIV-1 Gag p6 with the FxFG domains in human Pom121. HIV-1 p6 sequence is derived from isolate 89.6 [27], and the human Poml21 sequence is derived from GenBank accession number BC008794. Numbers indicate the amino acid positions in the proteins. The p89.6/Pr-/R+ and p89.6/Pr-(LF)a/R+ constructs were prepared by inserting a PstI/StuI digested PCR DNA fragment into the PstI/BalI sites of p89.6/HA-Ub/R+. For p89.6/Pr-/R+, PCR was performed with the p89.6/5' clone as the template [27], and primer 3 GGTACATCAGGCCATCTCACC and primer 4 CTGACCAGGCCTCCCGGGTTATTTTATTGTGACGAGGGGTCGTTGC. For p89.6/Pr-(LF)a/R+, PCR was performed with the same template and primer 3 and primer 5 CTGACCAGGCCTCCCGGGTTATTTTATTGTGACGAGGGGTCGTTGCCTGCGGC TGATCTGAGGGAAGC. For constructs p89.6/Pr (Lxx)-/R+, p89.6/Pr-(1–23)/R+ and p89.6/Pr (FRF)a/R+, the PCR DNA was digested with PstI/SmaI and ligated into the PstI/SmaI sites of p89.6/Pr- (LF)a/R+. For p89.6/Pr- (Lxx)-/R+, PCR was performed with the p89.6/5' template and primer 3 and primer 6 GTACTACCCGGGAGGCCTTTATTCCTTGTCTATCGGCTCCTGC. For p89.6/Pr-(l-23)/R+, PCR was performed with primer 3 and primer 7 GTACTACCCGGGAGGCCTTTATTGAGTTGTTGTCTCCTCCCCAAACC. For p89.6/Pr- (FRF)a/R+, PCR was performed with primer 3 and primer 8 GTACTACCCGGGAGGCCTTTATTGAGTTGTTGTCTCCTCCCCGGCCGCGGCGC TCTCTGCTGG. The construct p89.6/Pr-F15A/R+, p89.6/Pr-R15A/R+, and p89.6/Pr-F17A/R+ were prepared in the same way as p89.6/Pr-(1–23)/R+, except that the PCR was performed with primer 3 and a new primer instead of primer 7: primer 9 (for p89.6/Pr-F15A/R+) ACTCGACCCGGGAGGCCTTTATTGAGTTGTTGTCTCCTCCCCAAACCTGGCGC TCTCTGCTGG, primer 10 (for p89.6/Pr- R16A/R+) ACTCGACCCGGGAGGCCTTTATTGAGTTGTTGTCTCCTCCCCAAACGCGAAGC TCTCTGC, and primer 11 (for p89.6/Pr- F17A/R+) ACTCGACCCGGGAGGCCTTTATTGAGTTGTTGTCTCCTCCCCGGCCCTGAAGC TCTC. The construct p89.6/Pr- (l-23)/R+/Δ Ub was prepared by removing the BamHI-BamHI fragment, encoding the HA-tagged Ub gene, from the p89.6/Pr-(1–23)/R+ construct. Cell culture and transfection were performed under conditions described previously [18]. To obtain HIV-1 virions, three days after transfection, culture supernatant was clarified by a low speed centrifugation followed by filtration through a 0.45 nm filter. The clarified culture supernatant was subjected to centrifugation through a 20% sucrose cushion in the SW50.1 rotor at 33,000 rpm for 1 hour. Virions from transfected 293 cells were examined for the presence of Gag and Vpr by Western blot analysis. As shown, Gag p55, p24, p17 as well as Vpr were all detected in the virions with the R+ genome (Fig. 1B, lane 1). With the HIV-1 genome containing a premature stop codon in Vpr (R- genome), no Vpr was detected in the virion (lane 2). We subsequently prepared a protease-truncated construct based on the R+ genome, named Pr-/R+, and observed normal Vpr virion packaging (Fig. 1B, lane 3). As expected, Gag p55 was not processed with the Pr-/R+ construct due to the loss of protease. Surprisingly, normal Vpr virion packaging was still observed with the Pr- (LF)a/R+ construct (lane 4), which contains L44A/F45A double mutations in the Gag p6 domain (Fig. 1A) that are reported to abolish Vpr packaging in the context of the MLV Gag/HIV-1 p6 fusion construct [22]. The whole (Lxx)4 domain was then deleted from p6 to generate the Pr-(Lxx)-/R+ construct, and again normal Vpr packaging was detected (Fig. 1B, lane 5). The Pr-(Lxx)-/R+ construct still maintains a 15FRFG domain in p6 which resembles the FxFG domain frequently observed in resident proteins of the nuclear pore [30]. To examine the potential involvement of this domain in Vpr packaging, another p6 deletion construct was prepared, with only aa #1–23 of p6 preserved (Fig. 1A). As shown, normal Vpr virion packaging was also observed for this construct, Pr-(1–23)/R+ (Fig. 1B, lane 6). Subsequently, 15FRF residues were all substituted by Ala residues to generate the Pr-(FRF)a/R+ construct (Fig. 1A). Importantly, this mutant failed to package Vpr into the virion (Fig. 1B, lane 7). To examine the roles of individual amino acid residues in the 15FRFG domain during Vpr packaging, Phe15, Arg16 and Phe17 were individually substituted by Ala (Fig. 1A). As shown, while single F15A and F17A mutations abolished Vpr packaging (Fig. 1B, lanes 8 and 10), R16A mutation had no effect (lane 9). Since all of the HIV-1 constructs expressed HA-tagged ubiquitin (HA-Ub), the HA-Ub coding sequence was removed from the Pr-(1–23)/R+ construct. As shown, removal of HA-Ub had no effect on Vpr virion packaging (Fig. 1B, lane 11). Analysis of cell lysates showed that all HIV-1 genomic constructs expressed the same amount of Vpr in the cell (Fig. 1B, lanes 5–11, bottom panel). These results strongly suggest that the 15FRFG domain is critical for Vpr virion packaging. In this report we provide evidence that HIV-1 Vpr is packaged into the virion through the previously unrecognized 15FRFG domain in the Gag p6 domain. The Vpr packaging function of the 15FRFG domain is preserved when amino acid #1–23 of p6 is retained. This function is abolished when 15FRF are substituted by Ala residues. Our conclusion is further supported by the finding that Ala substitutions for Phe15 and Phe17abolish Vpr packaging whereas Ala substitution for Arg16 has no effect. Previous studies have shown that a (Lxx)4 repeat domain in Gag p6 is essential for Vpr virion packaging [21,22]. The exact reason for the discrepancy is unclear. However, the previous studies were based on vaccinia expression of Gag and Vpr [21] or on the MLV Gag/HIV-1 p6 fusion constructs [22]. It is possible that different experimental conditions affect the virion packaging of Vpr. Alternatively, different HIV-1 strains may prefer the 15FRFG domain or the (Lxx)4 domain for Vpr packaging. It is noticeable that although the 15FRFG domain is highly conserved among different HIV-1 strains, it is replaced with 15FRSG in the HIV-1 Hxb2 strain (GenBank accession number K03455) and 15VRFG in the Yu-2 strain (GenBank accession number AF287352). Future studies may reveal if an engineered FRFG domain in these HIV-1 strains can allow Vpr packaging in the absence of the (Lxx)4 domain. Significantly, the 15FRFG domain of p6 resembles the FxFG domains of certain nucleoporins with respect to both the FxFG core and the following hydrophilic residues rich in Ser/Thr residues (Fig. 1C). Thus, Vpr appears to recognize the same sequence for both virion packaging and association with the nuclear envelope for transport into the nucleus. We hypothesize that the FxFG domain is one of the most important signals for Vpr recognition in vivo. It may govern Vpr function during both the late phase and the early phase of the HIV-1 life cycle. Vpr interaction with nucleoporins has been reported before [20]. In particular, Vpr is found to interact with the FG repeat domain of Pom121 and more weakly with that of Nsp1p [20]. It has been suggested that the FG residues in these FG repeats constitute the hydrophobic core that is critical for recognition by other proteins [30]. However, the property of this hydrophobic core and the specificity of protein-protein recognition are critically dependent on the neighboring residues preceding the FG residues, so that the FxFG, GLFG, and other types of FG repeats may be involved in different protein-protein interactions [30]. Comparison of the Gag FxFG domain with the seven of the FxFG repeats of the human Pom121 reveals that these FxFG domains are followed by a sequence rich in Ser/Thr residues (Fig. 1C) which may be critical for the function of the FxFG domain. The roles of these Thr residues in Vpr virion packaging remain to be dissected. It is likely that Vpr recognizes the FxFG domain and not other types of FG repeats. Single Ala substitution for Phe15 in the 15FRFG domain of p6 abolishes Vpr virion packaging (Fig. 1). The nucleoporin Nup159p does not interact with Vpr [20], and its FG repeat domain contains eight PxFG repeats and no FxFG repeat. In contrast, the FG repeat domain of the Vpr-interacting nucleoporin Pom121 contains seven copies of the FxFG repeats and six copies of the PxFG repeat. Another nucleoporin that interacts with Vpr weakly, Nsp1p, has a large number of FxFG repeats. However, it is expected that nucleoporins function in the context of a large protein complex and their conformations and interaction with Vpr may be influenced by the presence of other interaction partners. List of abbreviations MLV: murine leukemia virus; Ub: ubiquitin. Competing interests None declared. Authors' contributions HZ and HJ participated in the construction of mutant HIV-1 genomes, cell culture, transfection, and Western blot analyses. LZ conceived of the study and participated in its design, coordination and execution. All authors read and approved the final manuscript. Acknowledgments This work has been supported by a NIH/NIHBL grant (HL61952). The authors are grateful to Drs. G. Chinnadurai and D. Grandgenett (St. Louis University) for valuable discussions during the progress of the project, and A. Vora (St. Louis University) for assistance with preparation of HIV-1 virions. ==== Refs Paxton W Connor RI Landau NR Incorporation of Vpr into human immunodeficiency virus type 1 virions: requirement for the p6 region of gag and mutational analysis J Virol 1993 67 7229 7237 8230445 Cohen EA Dehni G Sodroski JG Haseltine WA Human immunodeficiency virus vpr product is a virion-associated regulatory protein J Virol 1990 64 3097 3099 2139896 Yuan X Matsuda Z Matsuda M Essex M Lee TH Human immunodeficiency virus vpr gene encodes a virion-associated protein AIDS Res Hum Retroviruses 1990 6 1265 1271 2150318 Zhao LJ Mukherjee S Narayan O Biochemical mechanism of HIV-I Vpr function. 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and mediates G2 accumulation by a mechanism which differs from DNA damage checkpoint control J Virol 1996 70 2324 2331 8642659 He J Choe S Walker R Marzio DiP Morgan DO Landau NR Human immunodeficiency virus type 1 viral protein R (Vpr) arrests cells in the G2 phase of the cell cycle by inhibiting p34cdc2 activity J Virol 1995 69 6705 6711 7474080 Re F Braaten D Franke EK Luban J Human immunodeficiency virus type 1 Vpr arrests the cell cycle in G2 by inhibiting the activation of p34cdc2-cyclin B J Virol 1995 69 6859 6864 7474100 Yao XJ Rougeau N Ghislaine D Lemay J Cohen EA Analysis of HIV-1 Vpr determinants responsible for cell growth arrest in Saccharomyces cerevisiae Retrovirology 2004 1 21 15312229 10.1186/1742-4690-1-21 Stewart SA Poon B Jowett JB Xie Y Chen IS Lentiviral delivery of HIV-1 Vpr protein induces apoptosis in transformed cells Proc Natl Acad Sci USA 1999 96 12039 12043 10518572 10.1073/pnas.96.21.12039 Yasuda J Miyao T Kamata M Aida Y Iwakura Y T cell apoptosis causes peripheral T cell depletion in mice transgenic for the HIV-1 vpr gene Virology 2001 285 181 192 11437653 10.1006/viro.2001.0964 Fukumori T Akari H Iida S Hata S Kagawa S Aida Y Koyama AH Adachi A The HIV-1 Vpr displays strong anti-apoptotic activity FEBS Lett 1998 432 17 20 9710242 10.1016/S0014-5793(98)00824-2 Somasundaran M Sharkey M Brichacek B Luzuriaga K Emerman M Sullivan JL Stevenson M Evidence for a cytopathogenicity determinant in HIV-1 Vpr Proc Natl Acad Sci USA 2002 99 9503 9508 12093916 10.1073/pnas.142313699 Jian H Zhao LJ Pro-apoptotic Activity of HIV-1 Auxiliary Regulatory Protein Vpr Is Subtype-dependent and Potently Enhanced by Nonconservative Changes of the Leucine Residue at Position 64 J Biol Chem 2003 278 44326 44330 14506268 10.1074/jbc.C300378200 Vodicka MA Koepp DM Silver PA Emerman M HIV-1 Vpr interacts with the nuclear transport pathway to promote macrophage infection Genes Dev 1998 12 175 185 9436978 Fouchier RA Meyer BE Simon JH Fischer U Albright AV Gonzalez-Scarano F Malim MH Interaction of the human immunodeficiency virus type 1 Vpr protein with the nuclear pore complex J Virol 1998 72 6004 6013 9621063 Lu YL Bennett RP Wills JW Gorelick R Ratner L A leucine triplet repeat sequence (LXX)4 in p6gag is important for Vpr incorporation into human immunodeficiency virus type 1 particles J Virol 1995 69 6873 6879 7474102 Kondo E Gottlinger HG A conserved LXXLF sequence is the major determinant in p6gag required for the incorporation of human immunodeficiency virus type 1 Vpr J Virol 1996 70 159 164 8523520 Jenkins Y Pornillos O Rich RL Myszka DG Sundquist WI Malim MH Biochemical analyses of the interactions between human immunodeficiency virus type 1 Vpr and p6(Gag) J Virol 2001 75 10537 10542 11581428 10.1128/JVI.75.21.10537-10542.2001 de Rocquigny H Petitjean P Tanchou V Decimo D Drouot L Delaunay T Darlix JL Roques BP The zinc fingers of HIV nucleocapsid protein NCp7 direct interactions with the viral regulatory protein Vpr J Biol Chem 1997 272 30753 30759 9388214 10.1074/jbc.272.49.30753 Strack B Calistri A Craig S Popova E Gottlinger HG AIP1/ALIX is a binding partner for HIV-1 p6 and EIAV p9 functioning in virus budding Cell 2003 114 689 699 14505569 10.1016/S0092-8674(03)00653-6 Garrus IE von Schwedler UK Pornillos OW Morham SG Zavitz KH Wang HE Wettstein DA Stray KM Cote M Rich RL Myszka DG Sundquist WI Tsg101 and the vacuolar protein sorting pathway are essential for HIV-1 budding Cell 2001 107 55 65 11595185 10.1016/S0092-8674(01)00506-2 Collman R Balliet JW Gregory SA Friedman H Kolson DL Nathanson N Srinivasan A An infectious molecular clone of an unusual macrophage-tropic and highly cytopathic strain of human immunodeficiency virus type 1 J Virol 1992 66 7517 7521 1433527 Zhang S Feng Y Narayan O Zhao LJ Cytoplasmic retention of HIV-1 regulatory protein Vpr by protein-protein interaction with a novel human cytoplasmic protein VprBP Gene 2001 263 131 140 11223251 10.1016/S0378-1119(00)00583-7 Treier M Staszewski LM Bohmann D Ubiquitin-dependent c-Jun degradation in vivo is mediated by the delta domain Cell 1994 78 787 798 8087846 Ryan KJ Wente SR The nuclear pore complex: a protein machine bridging the nucleus and cytoplasm Curr Opin Cell Biol 2000 12 361 371 10801463 10.1016/S0955-0674(00)00101-0

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==== Front PLoS BiolPLoS BiolpbioplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 1550286810.1371/journal.pbio.0020327Research ArticleDevelopmentNeuroscienceIn VitroSensitivity to Oxidative Stress in DJ-1-Deficient Dopamine Neurons: An ES- Derived Cell Model of Primary Parkinsonism DJ-1 Protects from Oxidative StressMartinat Cecile 1 Shendelman Shoshana 1 Jonason Alan 1 Leete Thomas 1 Beal M. Flint 2 Yang Lichuan 2 Floss Thomas 3 Abeliovich Asa [email protected] 1 1Departments of Pathology and Neurology, Center for Neurobiology and Behavior, and Taub Institute, Columbia UniversityNew York, New YorkUnited States of America2Department of Neurology and Neuroscience, Weill Medical College of Cornell UniversityNew York, New YorkUnited States of America3Institute of Developmental Genetics, GSF-National Research Center for Environment and HealthNeuherbergGermany11 2004 5 10 2004 5 10 2004 2 11 e32728 4 2004 29 7 2004 Copyright: © 2004 Martinat et al.2004This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. DJ-1 Is a Redox-Dependent Molecular Chaperone that Inhibits α-Synuclein Aggregate Formation A New Cell Model for Parkinson's Disease Molecules That Cause or Prevent Parkinson's Disease The hallmark of Parkinson's disease (PD) is the selective loss of dopamine neurons in the ventral midbrain. Although the cause of neurodegeneration in PD is unknown, a Mendelian inheritance pattern is observed in rare cases, indicating a genetic factor. Furthermore, pathological analyses of PD substantia nigra have correlated cellular oxidative stress and altered proteasomal function with PD. Homozygous mutations in DJ-1 were recently described in two families with autosomal recessive Parkinsonism, one of which is a large deletion that is likely to lead to loss of function. Here we show that embryonic stem cells deficient in DJ-1 display increased sensitivity to oxidative stress and proteasomal inhibition. The accumulation of reactive oxygen species in toxin-treated DJ-1-deficient cells initially appears normal, but these cells are unable to cope with the consequent damage that ultimately leads to apoptotic death. Furthermore, we find that dopamine neurons derived from in vitro–differentiated DJ-1-deficient embryonic stem cells display decreased survival and increased sensitivity to oxidative stress. These data are consistent with a protective role for DJ-1, and demonstrate the utility of genetically modified embryonic stem cell–derived neurons as cellular models of neuronal disorders. Dopaminergic neurons, derived from DJ-1-deficient embryonic stem cells, display decreased survival and increased sensitivity to oxidative stress ==== Body Introduction Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by rigidity, slowed movement, gait difficulty, and tremor at rest (Dauer and Przedborski 2003). The pathological hallmark of PD is the relatively selective loss of dopamine neurons (DNs) in the substantia nigra pars compacta in the ventral midbrain. Although the cause of neurodegeneration in PD is unknown, a Mendelian inheritance pattern is observed in approximately 5% of patients, suggesting a genetic factor. Extremely rare cases of PD have been associated with the toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, which is taken up specifically by DNs through the dopamine transporter and is thought to induce cellular oxidative stress. Population-based epidemiological studies have further supported roles for genetic and environmental mechanisms in the etiology of PD (Dauer and Przedborski 2003; Jenner 2003). The identification of several genes that underlie familial forms of PD has allowed for the molecular dissection of mechanisms of DN survival. Autosomal dominant mutations in α-synuclein lead to a rare familial form of PD (Polymeropoulos et al. 1997), and there is evidence that these mutations generate toxic, abnormal protein aggregates (Goldberg and Lansbury 2000) and cause proteasomal dysfunction (Rideout et al. 2001). A majority of patients with sporadic PD harbor prominent intracytoplasmic inclusions, termed Lewy bodies, enriched for α-synuclein (Spillantini et al. 1998), as well as neurofilament protein (Trojanowski and Lee 1998). Mutations in a second gene, Parkin, lead to autosomal recessive PD (Hattori et al. 2000). Parkin is a ubiquitin ligase that appears to participate in the proteasome-mediated degradation of several substrates (Staropoli et al. 2003). Homozygous mutations in a third gene, DJ-1, were recently associated with autosomal recessive primary parkinsonism (Bonifati et al. 2003). DJ-1 encodes a ThiJ domain protein of 189 amino acids that is broadly expressed in mammalian tissues (Nagakubo et al. 1997). Interestingly, DJ-1 was independently identified in a screen for human endothelial cell proteins that are modified with respect to isoelectric point in response to sublethal doses of paraquat (Mitsumoto and Nakagawa 2001; Mitsumoto et al. 2001), a toxin that generates reactive oxygen species (ROS) within cells and has been associated with DN toxicity (McCormack et al. 2002). Gene expression of a yeast homolog of DJ-1, YDR533C, is upregulated in response to sorbic acid (de Nobel et al. 2001), an inducer of cellular oxidative stress. These results suggest a causal role for DJ-1 in the cellular oxidative stress response. Surprisingly, animal models that harbor genetic lesions that mimic inherited forms of human PD, such as homozygous deletions in Parkin (Goldberg et al. 2003; Itier et al. 2003) or overexpression of α-synuclein (Masliah et al. 2000; Giasson et al. 2002; Lee et al. 2002), have failed to recapitulate the loss of dopamine cells. An alternative approach, the genetic modification of midbrain DNs in vitro (Staropoli et al. 2003), is potentially useful but limited by the difficulty and variability in culturing primary postmitotic midbrain neurons. Other studies have focused on immortalized tumor cell lines, such as neuroblastoma cells, but these may not accurately model the survival of postmitotic midbrain neurons. Here we show that DJ-1-deficient cells display increased sensitivity to oxidative stress. DNs appear to be particularly sensitive to the loss of DJ-1. The initial accumulation of ROS is normal in DJ-1-deficient cells, but subsequent cellular defenses to ROS are impaired, leading to increased apoptosis. Results Generation of DJ-1-Deficient ES Cells To investigate the normal cellular function of DJ-1 and the pathogenic mechanism of the PD mutations, we generated cells deficient in DJ-1. A murine embryonic stem (ES) cell clone, F063A04, that harbors a retroviral integration at the DJ-1 locus was obtained through the German Gene Trap Consortium (http://tikus.gsf.de) (Figure 1A; Floss and Wurst 2002). This integration is predicted to disrupt the normal splicing of DJ-1, leading to the generation of a truncated protein that lacks the carboxy-terminal domain required for dimerization and stability (unpublished data). Of note, a mutation that encodes a similarly truncated protein (at the human DJ-1 exon 7 splice acceptor) has been described in a patient with early-onset PD (Hague et al. 2003). Figure 1 DJ-1-Deficient ES Cells Are Sensitized to Oxidative Stress (A) Schematic map of the murine DJ-1 gene in clone F063A04. The retroviral insertion places the engrailed-2 (En2) intron, the splice acceptor (SA), and the β-galactosidase/neomycin resistance gene fusion (β-geo) between exons 6 and 7. (B) Southern blot analysis of KpnI-digested genomic DNA from DJ-1 homozygous mutant (–/–), WT (+/+), and heterozygous (+/–)cells, probed with murine DJ-1 cDNA. WT DNA shows a predicted 14-kb band (WT), whereas the mutant allele migrates as a 9-kb band (insertion). (C) Western blot (WB) of ES cell lysates from WT (+/+), DJ-1 heterozygous (+/–), and mutant homozygous (–/–) clones with antibodies to murine DJ-1 (α-DJ-1) or β-actin (α-β-actin). DJ-1 migrates at 20 kDa, β-actin at 45 kDa. (D) ES cells were exposed to 0, 5, 10, and 20 μM H2O2 for 15 h and viability was assayed by MTT. Responses of DJ-1 heterozygous cells (diamonds) and DJ-1 knockout clones 9 (open circles), 16 (solid circles), 23 (squares), and 32 (triangles) are shown. ** p ≤ 0.01; *** p ≤ 0.0001. (E and F) Cell death of DJ-1 heterozygous and DJ-1-deficient cells (clone 32) after exposure to H2O2 (10 μM) was quantified by staining with PI and an antibody to AV with subsequent FACS analysis. AV staining marks cells undergoing apoptosis, whereas PI staining indicates dead cells. * p ≤ 0.05. (G) DJ-1 heterozygous (+/–) and knockout (clone 32; –/–) cells were assayed at 1, 6, and 24 h after treatment with 10 μM H2O2 by Western blotting for cleaved PARP (89 kDa), which indicates apoptosis. No band is seen for cleaved PARP or β-actin for the DJ-1-deficient cells at 24 h due to cell death. Data represent means ± standard error of the mean (SEM) and were analyzed by ANOVA with Fisher's post-hoc test. To generate ES cell subclones homozygous for the trapped DJ-1 allele, clone F063A04 was exposed to a high dose of the antibiotic G418, which selects cells that are homozygous for the neomycin resistance gene insertion (Mizushima et al. 2001). Several homozygous mutant ES cell subclones (that had undergone gene conversion at the DJ-1 locus) were identified by Southern blotting (Figure 1B). To confirm that the trapped allele leads to the loss of wild-type (WT) DJ-1 protein, cell lysates from homozygous DJ-1-deficient (also termed “knockout” here) ES cell clones as well as the parental heterozygous clone were analyzed by Western blotting using polyclonal antibodies to the amino-terminal region of DJ-1 (amino acids 64–82) or full-length DJ-1 protein (unpublished data). Neither full-length nor truncated DJ-1 protein products were detected in knockout clones (Figure 1C), consistent with instability of the predicted truncated DJ-1 product. In addition, no full-length DJ-1 RNA was detected in cultures of knockout cells (Figure S1). In contrast, heterozygous and WT ES cells express high levels of DJ-1. Initial phenotypic analysis of knockout subclones indicated that DJ-1 is not essential to the growth rate of ES cells in culture, consistent with the viability of humans homozygous for DJ-1 mutations. DJ-1 Protects Cells from Oxidative Stress and Proteasomal Inhibition DJ-1 has been hypothesized to function in the cellular response to oxidative stress. To investigate the role of DJ-1 in the oxidative stress response in vivo, DJ-1-deficient knockout and heterozygous ES cell clones were analyzed for cell viability in the context of increasing concentrations of H2O2. Heterozygous cells were used as controls because the knockout subclones were derived from these. Cell viability was initially determined by MTT assay (which detects reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide [MTT] by metabolic enzymes) in triplicate (Fezoui et al. 2000). Exposure to H2O2 led to significantly greater toxicity in the DJ-1-deficient cells; similar results were obtained with multiple knockout subclones in independent experiments (Figures 1D and 2A). In contrast, in the absence of toxin, heterozygous and knockout cells displayed comparable viability in the MTT assay (Figure S2). Consistent with the MTT assay, fluorescence-activated cell sorting (FACS) analysis of cells stained with annexin V (AV) and propidium iodide (PI) revealed increased death of knockout cells compared to heterozygous cells in the context of H2O2 exposure (Figure 1E). The increase in AV-positive cells implicated an apoptotic mechanism of cell death (Figure 1F). Furthermore, when exposed to H2O2, knockout cells displayed potentiated cleavage of poly(ADP-ribose)polymerase-1 (PARP) in a pattern indicative of an apoptotic death program (Gobeil et al. 2001) (Figure 1G). Figure 2 Specificity and Mechanism of Altered Toxin Sensitivity in DJ-1-Deficient Cells (A–C) Cell viability of DJ-1 heterozygous cells (solid bar) and DJ-1-deficient knockout clone 32 cells (open bar) after 15 h exposure to H2O2 (A), lactacystin (B), or tunicamycin (C) as assayed by MTT reduction. * p ≤ 0.05. (D) DJ-1-deficient knockout cells (clone 32) were transiently transfected with plasmids containing WT human DJ-1 vector (solid bar) and PD-associated L166P mutant DJ-1 vector (gray bar); as a control, knockout cells were also transfected with vector alone (open bar). 48 h after transfection, cells were exposed to 10 μM H2O2 for 15 h and then assayed by MTT reduction. WT human DJ-1 significantly enhanced survival of the knockout cells, whereas the L166P mutant did not. Similar results were obtained at 20 μM H2O2 and with a second DJ-1-deficient clone (unpublished data). Transfection efficiency exceeded 90% in all cases and protein expression level was comparable for human WT and L166P mutant DJ-1 as determined by Western blotting (Figure S1). * p ≤ 0.05. (E) DJ-1-deficient cells (clone 32; open bar) and control heterozygous cells (solid bar) were assayed for intracellular formation of ROS in response to H2O2 treatment (15 min, 1 or 10 μM) using DHR and FACS analysis. (F) Protein carbonyl levels were measured by spectrophotometric analysis of DNP-conjugated lysates from DJ-1-deficient (clone 32; solid red line) and control heterozygous cells (dashed blue line). Data are shown as the mean ± SEM and were analyzed by ANOVA with Fisher's post-hoc test. Additional toxin exposure studies demonstrated that DJ-1-deficient cells were sensitized to the proteasomal inhibitor lactacystin (Figure 2B), as well as to copper (Figure S2), which catalyzes the production of ROS. We did not observe altered sensitivity to several other toxins, including tunicamycin (an inducer of the unfolded protein response in the endoplasmic reticulum; Figure 2C), staurosporine (a general kinase inhibitor that induces apoptosis) (Figure S2), or cycloheximide (an inhibitor of protein translation) (unpublished data). WT but Not PD-Associated L166P Mutant DJ-1 Protects Cells from Oxidative Stress To confirm that altered sensitivity to oxidative stress is a consequence of the loss of DJ-1, we performed rescue experiments by overexpressing WT or mutant human DJ-1 in knockout ES cells. Plasmids encoding human Flag-tagged WT DJ-1, Flag-tagged PD-associated L166P mutant DJ-1, or vector alone, were transiently transfected into DJ-1-deficient clones, and these were subsequently assayed for sensitivity to H2O2 using the MTT viability assay. DJ-1-deficient cells transfected with a vector encoding Flag-WT human DJ-1 were effectively rescued in terms of viability in the presence of H2O2 (Figure 2D); Thus, viability in rescued knockout cells mimicked the viability of untransfected heterozygous cells in the context of H2O2 treatment (Figure 2A and 2D). In contrast, transfection of knockout cells with a vector encoding the PD-associated L166P mutant DJ-1 did not significantly increase the viability of H2O2-treated knockout cells (Figure 2D). Baseline cell viability in the absence of toxin exposure was not altered by DJ-1 overexpression, and Western blotting of lysates from transfected cells with an antibody specific to human DJ-1 demonstrated that transfected Flag-WT DJ-1 and Flag-L166P mutant DJ-1 accumulated comparably (Figure S2). DJ-1 Deficiency Does Not Alter the H2O2-Induced Intracellular ROS Burst We hypothesized that DJ-1 either alters the initial accumulation of intracellular ROS in response to H2O2 exposure, or that it functions downstream of the ROS burst and protects cells from consequent damage. Therefore, we quantified the accumulation of ROS in response to H2O2 treatment in knockout and heterozygous cells using the ROS-sensitive fluorescent indicator dye dihydrorhodamine-123 (DHR) and FACS analysis. Initial ROS accumulation (at 15 min after stimulation) appeared unaltered in the DJ-1-deficient cells in comparison to control heterozygous cells (Figure 2E). Consistent with this, accumulation of protein carbonyls, an index of oxidative damage to proteins (Sherer et al. 2002), appeared normal initially (at 1 h after toxin exposure; Figure 2F). However, at 6 h after toxin exposure, a point at which knockout cells already display increased apoptosis (as indicated by PARP cleavage; see Figure 1G), protein carbonyl accumulation was robustly increased in the DJ-1-deficient cells. These data suggest that initial ROS accumulation is not altered by DJ-1 deficiency, but that the mutant cells are unable to appropriately cope with the consequent damage. Consistent with this result, no antioxidant or peroxiredoxin activity with purified DJ-1 protein in vitro was detected (S.S. and A.A., personal communication). DJ-1 Is Required for Survival of ES Cell–Derived DNs Several methods have been established for the differentiation of ES cells into DNs in vitro (Morizane et al. 2002). To extend our analysis of DJ-1 function to DNs, we differentiated DJ-1-deficient ES cells or control heterozygous cells into DNs in vitro by coculture with stromal cell–derived inducing activity (SDIA; Figure 3A) (Morizane et al. 2002; Barberi et al. 2003). DNs were quantified by immunohistochemistry for tyrosine hydroxylase (TH; a marker for DNs and other catecholaminergic cells), or by analysis of dopamine transporter uptake activity (a quantitative DN marker) (Han et al. 2003). Production of DNs appeared to be significantly reduced in knockout ES cell cultures compared to parental heterozygous cultures at 18 days in vitro (DIV) as determined by both dopamine uptake and TH immunoreactivity (Figures 3B and 3C; 4A–4L). In contrast, general neuronal production did not appear altered in this assay in terms of the postmitotic neuronal marker TuJ1 (a monoclonal antibody specific to neuronal, not glial, class III β-tubulin) (Figures 3E and 4A–4L′); other neuronal subtypes also appeared normal, including GABAergic (Figures 3D and 4A′–4L′) and motor neurons (HB9-positive; Figure S3). To investigate whether the reduction in DNs in DJ-1-deficient cultures is due to defective generation or survival, a time course analysis was performed. At early time points (8 and 12 DIV), dopamine uptake activity was comparable in WT and DJ-1-deficient cultures, whereas subsequently the DJ-1-deficient cultures appeared defective (Figure 3F). Consistent with this, intracellular dopamine accumulation (as quantified using high-performance liquid chromatography) was significantly reduced in DJ-1-deficient cultures (6.4 ± 1.5 ng dopamine/mg protein) relative to control heterozygous cultures (66.0 ± 17.4 ng/mg) at 35 DIV. These data strongly suggest that DJ-1 deficiency leads to loss of DNs, rather than simply to downregulation of cell marker expression. Figure 3 DJ-1-Deficient ES Cell Cultures Display Reduced DN Production (A) The SDIA coculture method. DJ-1 knockout or control heterozygous ES cells are cocultured with mouse stromal cells (MS5) in the absence of serum and leukemia inhibitory factor for 18 DIV. (B) DN production was quantified at 18 DIV by 3H-dopamine uptake assay. DJ-1-deficient ES cell cultures were defective relative to heterozygous control cultures. (C–D) Neuron production was quantified by immunohistochemical analysis as a percent of TuJ1-positive colonies that express TH (C) or GABA (D). Quantification of TH and GABA immunostaining was performed on all colonies in each of three independent wells. Colonies were scored as positive if any immunostained cells were present. * p ≤ 0.05. (E) The absolute number of TuJ1-positive colonies was not significantly different between the two genotypes. (F) Kinetic analysis of DN differentiation in DJ-1-deficient cultures (clone 32, solid square) and heterozygous controls (open circle) as quantified by 3H-dopamine uptake assay. * p ≤ 0.05. (G) DJ-1-deficient (open bar) and heterozygous control (closed bar) cultures differentiated for 9 DIV and then exposed to 6-OHDA at the indicated dose for 72 h. DNs were quantified by 3H-dopamine uptake assay. Data represent the means ± SEM and were analyzed by ANOVA followed by Fisher's post-hoc test. * p ≤ 0.05. Figure 4 Neuronal Differentiation of DJ-1-Deficient and Control Heterozygous ES Cell Cultures (A–L) DJ-1 heterozygous (+/–; A–F) and knockout (–/– [clone 32]; G–L) cultures were differentiated by SDIA for 18 DIV and immunostained with antibodies to TH (green) and TuJ1 (red). Images of both (Merge) are also shown. (A′–L′) Immunostaining of DJ-1 heterozygous (+/–, A′–F′) and deficient (–/–, G′–L′) cultures with antibodies for GABA (green) and TuJ1 (red). Scale bar, 50 μm. Images of both (Merge) are also shown. We hypothesized that DJ-1-deficient DNs may be sensitized to oxidative stress, akin to DJ-1-deficient undifferentiated ES cells. To test this, DN cultures from DJ-1-deficient or heterozygous control ES cell cultures at 9 DIV were exposed to oxidative stress in the form of 6-hydroxydopamine (6-OHDA), a DN-specific toxin that enters DNs through the dopamine transporter and leads to oxidative stress and apoptotic death (Dauer and Przedborski 2003). DJ-1-deficient DNs displayed an increased sensitivity to oxidative stress in this assay (Figure 3G). Post-hoc analysis of the data indicates that the difference among genotypes is maximal at an intermediate dose of toxin (50 μM); at the highest dose of 6-OHDA employed (100 μM), the difference is lessened (because the heterozygote is increasingly affected as well), indicating that DJ-1-mediated protection is limited. Although we cannot exclude a role for DJ-1 in the late-stage differentiation of DNs, these data suggest that DJ-1 deficiency leads to reduced DN survival and predisposes these cells to endogenous and exogenous toxic insults. RNAi “Knockdown” of DJ-1 in Midbrain Embryonic DNs Leads to Increased Sensitivity to Oxidative Stress To confirm the role of DJ-1 in primary midbrain DNs, DJ-1 expression was inhibited by RNA interference (RNAi) in embryonic day 13.5 (E13.5) murine primary midbrain cultures by lentiviral transduction of short hairpin RNAs (shRNAs) (Figure 5) (Rubinson et al. 2003). E13.5 midbrain cultures (Staropoli et al. 2003) were transduced with a lentiviral vector that includes a gene encoding the green fluorescent protein marker eGFP, along with shRNAs homologous to murine DJ-1. DJ1-shRNA virus-infected cells displayed efficient silencing of DJ-1 gene expression to 10%–20% of control vector-infected cultures (as determined by Western blotting [Figure 5Q]). Transduction efficiency, as assessed by visualization of the fluorescent eGFP marker, exceeded 95% in all cases (Figure 5I and unpublished data). After 7 DIV, cultures were exposed to H2O2 for 24 h and then evaluated for DN survival as quantified by immunostaining for TH and dopamine transporter (DAT). Figure 5 RNAi “Knockdown” of DJ-1 in Primary Embryonic Midbrain DNs Display Increased Sensitivity to Oxidative Stress (A–P) Primary midbrain cultures from E13.5 embryos were infected with lentiviral vectors encoding DJ-1 shRNA (or vector alone) under the regulation of the control vector (A–H) or the U6 promoter (I–P). Cells were cultured for 1 wk after infection and then exposed to H2O2 (5 μM; E–H and M–P) for 24 h. Cultures were immunostained for TH (B, F, J, and N) or DAT (C, G, K, or O) and visualized by confocal microscopy. Images containing all stains are included (Merge; D, H, L, and P). Scale bar, 100 μm. (Q) Cell lysates prepared from midbrain primary cultures infected with DJ-1 shRNA lentivirus (or control vector) were analyzed by Western blotting for murine DJ-1 or β-actin. (R–T) Quantification of TH, DAT, and GFP signal was performed on ten randomly selected fields in each of three wells for each condition. Red triangles, DJ-1 shRNA treated; black circles, control vector. Data represent the means ± SEM and were analyzed by ANOVA followed by Fisher's post-hoc test. * p ≤ 0.05. Midbrain cultures transduced with DJ-1 shRNA virus and with control vector displayed similar numbers of TH-positive neurons in the absence of exposure to H2O2 (Figure 5A–D, 5I–L, and 5R–S). In contrast, in the presence of H2O2, DJ-1-deficient cultures displayed significantly reduced DN survival as quantified by immunohistochemistry for TH or DAT (Figure 5E–H, 5M–P, 5R–S). These studies were repeated three times with similar results. The reduction in DAT immunoreactivity appears to be more robust than the reduction in TH-positive cell number in the context of H2O2; this may reflect the differential localization of DAT to DN processes, whereas TH is primarily in the cell body. As we described in a previous manuscript, nondopaminergic cells in the E13.5 primary midbrain cultures are predominantly GABAergic neurons (90%–95%) (Staropoli et al. 2003). Total embryonic midbrain neurons transduced with either DJ-1 shRNA or vector displayed comparable survival in the context of toxin exposure, suggesting that DJ-1 deficiency leads to a relatively specific alteration in DN survival (Figure 5T). These data are consistent with the analyses of ES cell–derived DNs above and indicate that DJ-1 is required for the normal survival of midbrain DNs in the context of toxin exposure. Discussion In this study we present evidence that DJ-1 is an essential component of the oxidative stress response of DNs. DJ-1-deficient cells display an apparently normal initial burst of ROS in response to H2O2, but they are unable to cope with the consequent toxicity, culminating in apoptosis. Additionally, we find that DJ-1 deficiency sensitizes cells to the proteasomal inhibitor lactacystin but not other toxic stimuli such as tunicamycin. Proteasomal inhibition induces the accumulation of short-lived and misfolded cytoplasmic proteins, leading to oxidative stress and apoptosis (Demasi and Davies 2003). ROS and proteasomal inhibition have previously been correlated with PD pathology (Dauer and Przedborski 2003), and it is therefore tempting to hypothesize that DJ-1 mutations lead to PD because of an increased sensitivity to such stressors. The apparent cell-type specificity of DN impairment in patients with the Parkinsonism-associated DJ-1 mutation is not predicted by the ubiquitous expression of DJ-1 (Nagakubo et al. 1997). In this study, we find that DJ-1 protects both dopaminergic and nondopaminergic cells from oxidative insult. However, DJ-1-deficient DNs appear to be especially sensitive to oxidative insult, suggesting relative cell-type specificity to the consequences of DJ-1 deficiency. Similar results are observed in DJ-1-knockout ES cell–derived DNs (which are devoid of any detectable DJ-1) and in primary DNs with DJ-1 levels reduced by RNAi “knockdown.” However, we find that even in the absence of exogenous toxin exposure, the knockout ES cell–derived DNs display reduced survival, whereas survival of the primary embryonic midbrain RNAi knockdown DNs appears to be similar to WT cells. We hypothesize that this discrepancy reflects the activity of residual DJ-1 (approximately 10%–20%) in the RNAi knockdown cultures. Alternatively, the knockout ES cell–derived DNs may be exposed to a greater degree of oxidative stress in vitro than are the knockdown-derived DNs even in the absence of added toxin. The mechanism by which DNs are preferentially targeted for destruction in the absence of DJ-1 is unclear. It has been proposed that DNs are subject to high levels of endogenous oxidative stress that may relate to dopamine metabolism (Jenner and Olanow 1998). DJ-1 is structurally modified in the context of cellular oxidative stress (Mitsumoto and Nakagawa 2001), suggesting a possible function. Two recent studies (Yokota et al. 2003; Taira et al. 2004) investigated the role of DJ-1 in the oxidative stress response of neuroblastoma tumor cells. Both studies used RNAi to perturb the expression of DJ-1 in neuroblastoma tumor cell lines, and suggested that DJ-1 deficiency sensitizes cells to oxidative stress; these results are consistent with our data. Taira et al. (2004) further reported that overexpression of DJ-1 in neuroblastoma cells leads to a reduction in ROS accumulation and hypothesized that DJ-1 may harbor antioxidant activity in vivo. In contrast, we find that ES cells that are deficient in DJ-1 display a normal initial burst of ROS in the context of H2O2. Consistent with this, we fail to detect DJ-1 antioxidant activity in vitro (Shendelman et al. 2004). Finally, this study presents a novel, ES cell-based genetic approach to the study of neurodegenerative disorders. Mouse genetic models of disease are often limited by the inherent variability of animal experiments, the limited mouse life span, and the difficulties in manipulating whole animals. For instance, genetic rescue experiments and toxicological dose-response studies are impractical in whole animals. Furthermore, genetic cell models are more readily amenable to molecular dissection of disease mechanisms than are whole animals. Thus, genetically altered, ES cell–derived neurons are likely to be generally useful as cellular models of neurodegenerative disorders. Future studies may also utilize available human ES cells to investigate species differences. Materials and Methods Cell culture. Undifferentiated ES cells were cultured using standard techniques (Abeliovich et al. 2000). SDIA differentiation of ES cell cultures to DNs was performed as described in Kawasaki et al. (2000), except that ES cells were plated at a density of 500 cells/cm2 rather than approximatively 1,000 cells/cm2, and were cocultured with the MS5 mouse stromal cell line (Barberi et al. 2003). For rescue experiments, cells were plated at a cell density of 1.4 × 106 cells/well. Transfections with plasmids encoding human Flag-WT DJ-1, PD-associated L166P mutant DJ-1, or vector alone, were performed using Lipofectamine 2000 (Life Technologies) for 18–36 h according to the manufacturer's instructions (Staropoli et al. 2003). 24 hours post-transfection, cells were split into 96-well plates and treated as described below. Primary cultures and lentiviral transductions were performed as described in Staropoli et al. (2003). Generation of knockout ES cell clones The pT1ATGβgeo gene trap vector, which includes βgeo, a fusion of the genes for β-galactosidase and neomycin resistance, is present between exons 6 and 7 of the murine DJ-1 gene, as determined by cDNA sequencing of trapped transcripts and genomic analysis (Figure 1A). To select for ES cell subclones homozygous for the trapped DJ-1 allele, we treated clone F063A04 with 4 mg/ml G418. Several subclones that were homozygous for the mutant DJ-1 allele were identified by Southern blotting (see Figure 1B), and three were chosen for further experimentation: clones 9, 23, and 32. To confirm that the trapped allele leads to the loss of wild-type (WT) DJ-1 protein, cell lysates from these clones, as well as from the parental heterozygous clone, were analyzed by Western blotting using polyclonal antibodies to the amino-terminal region of DJ-1 or the full-length DJ-1 protein. For Western blotting, cells were resuspended in 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 0.2% Triton X-100, and incubated at 4 °C, rotating for 20 min. Cleared lysate was prepared by centrifuging the lysate at 13,000 rpm for 10 min at 4 °C. Antibodies. A rabbit polyclonal antibody to DJ-1 was generated against the synthetic polypeptide QNLSESPMVKEILKEQESR, which corresponds to amino acids 64–82 of the mouse DJ-1 protein. Antiserum was produced by the Polyquick polyclonal antibody production service of Zymed Laboratories (South San Francisco, California, United States). The antiserum was affinity-purified on a peptide-coupled Sulfolink column (Pierce Biotechnology, Rockford, Illinois, United States) according to the manufacturer's instructions. Antibody was used at a dilution of 1:200 for immunohistochemistry and Western blotting as described (Staropoli et al. 2003). Immunohistochemistry was performed with a rabbit polyclonal antibody to TH (PelFreez, Rogers, Arizona, United States; dilution 1:1000), the mouse monoclonal antibody to neuronal class III β-tubulin TuJ1 (Covance, Princeton, New Jersey, United States; dilution 1:500), and a rabbit polyclonal antibody to GABA (Sigma, St. Louis, Missouri, United States; dilution 1:1000). Western blotting was performed using a polyclonal antibody to cleaved PARP (Cell Signaling Technology, Beverly, Massachusetts, United States; dilution 1:500), a monoclonal antibody to DJ-1 (Stressgen Biotechnologies, San Diego, California, United States; dilution 1:1000), and a mouse monoclonal antibody to β-actin (Sigma, 1:500). In vivo assays. ES cells plated in 96-well format (2.3 × 104 cells/well) were treated for 15 h with H2O2 in ES cell medium deficient in β-mercaptoethanol (Abeliovich et al. 2000). Cell viability (as a percent of untreated control) was determined by MTT assay in triplicate (Fezoui et al. 2000). AV/PI (Molecular Probes, Eugene, Oregon, United States) staining was performed according to the manufacturer's instructions. For DHR staining (Molecular Probes) (Walrand et al. 2003), cells were preincubated for 30 min with DHR (5 μM), washed with PBS, then treated with H2O2 in ES cell medium deficient in β-mercaptoethanol for 15 min at 37 °C. The FACS analysis was performed using a FACSTAR sorter (Becton-Dickinson, Palo Alto, California, United States). Dopamine uptake assays were performed as described (Farrer et al. 1998). Reported values represent specific uptake from which nonspecific uptake, determined in the presence of mazindol, was subtracted. Uptake values were normalized for protein content with the BCA kit (Pierce). For 6- hydroxydopamine (6-OHDA, Sigma) treatment, the drug was diluted in the differentiation medium (Kawasaki et al. 2000) and medium was changed every day for 72 h. Primary midbrain embryonic cultures were prepared and transduced with lentiviral vectors as described in Staropoli et al. (2003). The DJ-1 shRNA vector was generated by insertion of annealed oligonucleotides 5′-TGTCACTGTTGCAGGCTTGGTTCAAGAGACCAAGCCTGCAACAGTGACTTTTTTC-3′ and 5′-ACAGTGACAACGTCCGAACCAAGTTCTCTGGTTCGGACGTTGTCACTGAAAAAAGAGCT-3′ into the LentiLox vector (Rubinson et al. 2003). For cellular dopamine quantification, cultures were incubated in standard differentiation medium supplemented with L-DOPA (50 μM) for 1 h to amplify dopamine production, as described in Pothos et al. (1996). Subsequently, cells were washed in PBS and then lysed in 0.2 M perchloric acid. Dopamine levels were quantified by HPLC (Yang et al. 1998) and normalized for protein content as above. Expression vectors. The cDNA for human DJ-1 was PCR-amplified from a human liver cDNA library (Clontech, Palo Alto, California, United States). For expression of DJ-1 in ES cell rescue experiments, DJ-1 was cloned into the expression vector pcDNA3.1 (Invitrogen, Carlsbad, California, United States) containing a Flag peptide sequence at the N-terminus using standard cloning techniques. Flag-L166P DJ-1 (pcDNA3) was generated by PCR-mediated mutagenesis. Protein carbonyl analysis. For protein carbonyl quantitation (Bian et al. 2003), cells were plated (1.4 × 105 cells per well), grown for 24 h, and then treated with 10 μM H2O2 as indicated. Cells were lysed in 200 μl lysis buffer and cleared lysate was prepared as described above. An aliquot of 40 μl from each time point was added to 2 M HCl (120 μl) with or without 10 μM 2,4-dinitrophenyl-hydrazine and incubated for 1 h at 24 °C with shaking. Proteins were then TCA-precipitated and resuspended in 200 μl of 6 M guanidinium chloride. Absorbance was measured at 360 nm, and DNP-conjugated samples were normalized for protein concentration with the underivitized control samples. Supporting Information Figure S1 Quantitative Real-Time PCR for DJ-1 Gene Expression (A) Real-time PCR analyses of DJ-1 cDNA in WT (+/+), heterozygous (+/–), and knockout (–/–) cultures. Each expression value was normalized to that of β-actin and expressed relative to the respective value of the WT (+/+) control. These gene expression patterns were replicated in at least three independent PCR experiments. Total RNA from ES cells differentiated with the SDIA method for 18 days was isolated using the Absolutely RNA Miniprep kit (Stratagene, La Jolla, California, United States). Synthesis of cDNA was performed using the SuperScript first strand synthesis system for RT-PCR (Invitrogen). Real-time PCR reactions were optimized to determine the linear amplification range. Quantitative real-time RT-PCRs were performed (Stratagene MX3000P) using the QuantiTect SYBR Green PCR Master Mix (Qiagen, Valencia, California, United States) according to the manufacturer's instructions. DJ-1 primer sequences were 5′-CGAAGAAATTCGATGGCTTCCAAAAGAGCTCTGGT-3′ and 5′-CAGACTCGAGCTGCTTCACATACTACTGCTGAGGT-3′; primers used for β-actin were 5′-TTTTGGATGCAAGGTCACAA-3′ and 5′-CTCCACAATGGCTAGTGCAA-3′. For quantitative analyses, PCR product levels were measured in real time during the annealing step, and values were normalized to those of β-actin. (B) Ethidium bromide staining of the PCR products obtained after 29 cycles for DJ-1 (625 bp) and β-actin (350 bp). (704 KB EPS). Click here for additional data file. Figure S2 Analysis of DJ-1-Deficient ES Cells (A and B) Cell viability of DJ-1 heterozygous cells (solid bar) and DJ-1-deficient knockout clone 32 (open bar) after exposure to CuCl2 or staurosporine at the doses indicated. (C) MTT values of untreated DJ-1-deficient ES cell clones and the control heterozygous cells. Assays were performed exactly as in Figure 2, but in the absence of toxin. (D) MTT values of untreated DJ-1-deficient ES cells transfected with vector alone or various DJ-1-encoding plasmids. Transfection and expression of WT DJ-1 or mutant forms of DJ-1 does not alter the basal metabolic activity or viability of the cells. (E) Western blotting of extracts from ES cells transfected with vectors harboring WT human DJ-1 or the L166P mutant. (545 KB EPS). Click here for additional data file. Figure S3 Immunocytochemistry for HB9 and GABA Neurons in DJ-1-Deficient and Control Heterozygous ES Cells Both cell cultures were differentiated by SDIA for 18 DIV. Cells were fixed with 4% paraformaldehyde and stained with mouse monoclonal antibodies against HB9 (gift from T. Jessell, dilution 1:50) and rabbit polyclonal antibodies against GABA (Sigma, dilution 1:1000) as in Figure 5. Scale bar, 50 μM. (5.5 MB TIF). Click here for additional data file. Accession Numbers The GenBank (http://www.ncbi.nlm.nih.gov/) accession numbers of the genes discussed in this paper are α-synuclein (NM_000345), Parkin (AB009973), and DJ-1 (AB073864). We thank O. Hobert and L. Clark for critical reading of the manuscript. SS is funded by the Integrated Graduate Program Training Grant (National Institutes of Health); CM is funded by the American Parkinson's Disease Association; AA receives funding from the Spitzer, Rockefeller Brothers, Taub and M. J. Fox Foundations, National Institute of Neurological Disorder and Stroke (NINDS) and National Institute of Aging (NIA), and is a Culpeper and Beeson scholar. Conflicts of interest. The authors have declared that no conflicts of interest exist. Author contributions. CM, SS, and AA conceived and designed the experiments. CM, SS, AJ, TL, and AA performed the experiments. CM, SS, AJ, and AA analyzed the data. CM, SS, AJ, MFB, LY, TF, and AA contributed reagents/materials/analysis tools. CM, SS, and AA wrote the paper. Academic Editor: Huda Y. Zoghbi, Baylor College of Medicine Citation: Martinat C, Shendelman S, Jonason A, Leete T, Beal MF, et al. (2004) Sensitivity to oxidative stress in DJ-1-deficient dopamine neurons: An ES-derived cell model of primary parkinsonism. PLoS Biol 2(11): e327. Abbreviations 6-OHDA6-hydroxydopamine DATdopamine transporter DHRdihydrorhodamine-123 DIVd in vitro DNdopamine neuron E[number]embryonic day [number] ESembryonic stem FACSfluorescence-activated cell sorter MTT3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide PARPpoly(ADP-ribose)polymerase-1 PIpropidium iodide PDParkinson's disease RNAiRNA interference ROSreactive oxygen species SDIAstromal cell–derived inducing activity SEMstandard error of the mean shRNAshort hairpin RNA THtyrosine hydroxylase WTwild-type ==== Refs References Abeliovich A Schmitz Y Farinas I Choi-Lundberg D Ho WH Mice lacking α-synuclein display functional deficits in the nigrostriatal dopamine system Neuron 2000 25 239 252 10707987 Barberi T Klivenyi P Calingasan NY Lee H Kawamata H Neural subtype specification of fertilization and nuclear transfer embryonic stem cells and application in parkinsonian mice Nat Biotechnol 2003 21 1200 1207 14502203 Bian K Gao Z Weisbrodt N Murad F The nature of heme/iron-induced protein tyrosine nitration Proc Natl Acad Sci U S A 2003 100 5712 5717 12709594 Bonifati V Rizzu P van Baren MJ Schaap O Breedveld GJ Mutations in the DJ-1 gene associated with autosomal recessive early-onset parkinsonism Science 2003 299 256 259 12446870 Dauer W Przedborski S Parkinson's disease: Mechanisms and models Neuron 2003 39 889 909 12971891 Demasi M Davies KJ Proteasome inhibitors induce intracellular protein aggregation and cell death by an oxygen-dependent mechanism FEBS Lett 2003 542 89 94 12729904 de Nobel H Lawrie L Brul S Klis F Davis M Parallel and comparative analysis of the proteome and transcriptome of sorbic acid-stressed Saccharomyces cerevisiae Yeast 2001 18 1413 1428 11746603 Farrer M Wavrant-De Vrieze F Crook R Boles L Perez-Tur J Low frequency of α-synuclein mutations in familial Parkinson's disease Ann Neurol 1998 43 394 397 9506559 Fezoui Y Hartley DM Walsh DM Selkoe DJ Osterhout JJ A de novo designed helix-turn-helix peptide forms nontoxic amyloid fibrils Nat Struct Biol 2000 7 1095 1099 11101888 Floss T Wurst W Functional genomics by gene-trapping in embryonic stem cells Methods Mol Biol 2002 185 347 379 11769000 Giasson BI Duda JE Quinn SM Zhang B Trojanowski JQ Neuronal α-synucleinopathy with severe movement disorder in mice expressing A53T human α-synuclein Neuron 2002 34 521 533 12062037 Gobeil S Boucher CC Nadeau D Poirier GG Characterization of the necrotic cleavage of poly(ADP-ribose) polymerase (PARP-1): Implication of lysosomal proteases Cell Death Differ 2001 8 588 594 11536009 Goldberg MS Lansbury PT Is there a cause-and-effect relationship between α-synuclein fibrillization and Parkinson's disease? 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PLoS Biol. 2004 Nov 5; 2(11):e327

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==== Front PLoS BiolPLoS BiolpbioplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 1550286910.1371/journal.pbio.0020333Research ArticleMicrobiologyEubacteriaProtein Thiol Modifications Visualized In Vivo Thiol Modifications Visualized In VivoLeichert Lars I 1 Jakob Ursula [email protected] 1 1Department of Molecular, Cellular, and Developmental Biology, University of MichiganAnn Arbor, MichiganUnited States of America11 2004 5 10 2004 5 10 2004 2 11 e33316 4 2004 3 8 2004 Copyright: © 2004 Leichert and Jakob.2004This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. Oxidative Stress Inactivates Cobalamin-Independent Methionine Synthase (MetE) in Escherichia coli A New Way to Look at Oxidative Stress Thiol-disulfide interconversions play a crucial role in the chemistry of biological systems. They participate in the major systems that control the cellular redox potential and prevent oxidative damage. In addition, thiol-disulfide exchange reactions serve as molecular switches in a growing number of redox-regulated proteins. We developed a differential thiol-trapping technique combined with two-dimensional gel analysis, which in combination with genetic studies, allowed us to obtain a snapshot of the in vivo thiol status of cellular proteins. We determined the redox potential of protein thiols in vivo, identified and dissected the in vivo substrate proteins of the major cellular thiol-disulfide oxidoreductases, and discovered proteins that undergo thiol modifications during oxidative stress. Under normal growth conditions most cytosolic proteins had reduced cysteines, confirming existing dogmas. Among the few partly oxidized cytosolic proteins that we detected were proteins that are known to form disulfide bond intermediates transiently during their catalytic cycle (e.g., dihydrolipoyl transacetylase and lipoamide dehydrogenase). Most proteins with highly oxidized thiols were periplasmic proteins and were found to be in vivo substrates of the disulfide-bond-forming protein DsbA. We discovered a substantial number of redox-sensitive cytoplasmic proteins, whose thiol groups were significantly oxidized in strains lacking thioredoxin A. These included detoxifying enzymes as well as many metabolic enzymes with active-site cysteines that were not known to be substrates for thioredoxin. H2O2-induced oxidative stress resulted in the specific oxidation of thiols of proteins involved in detoxification of H2O2 and of enzymes of cofactor and amino acid biosynthesis pathways such as thiolperoxidase, GTP-cyclohydrolase I, and the cobalamin-independent methionine synthase MetE. Remarkably, a number of these proteins were previously or are now shown to be redox regulated. A differential thiol-trapping technique combined with two- dimensional gel analysis has been developed and used to visualize thiol-disulfide exchange reactions, which act as switches in redox-regulated proteins ==== Body Introduction Cysteine is one of the most rarely used amino acids in the proteins of most organisms studied so far (Pe'er et al. 2004). Therefore, when highly conserved in proteins, it usually plays crucial roles in the structure, function, or regulation of the protein. This is due to the ability of thiol groups to stabilize protein structures by forming covalent disulfide bonds and to coordinate metal ions, as well as due to their high reactivity and redox properties. Proteins in the extracellular space and oxidizing cell compartments (e.g., endoplasmic reticulum and periplasm) often rely on disulfide bonds to support their correct folding and maintain their structural stability (Bardwell 1994). Cytosolic proteins, on the other hand, are present within the reducing environment of the cytosol. Here, cysteine residues are reduced and often found in binding pockets of substrates, coenzymes, or metal cofactors (e.g., in zinc binding dehydrogenases), or are present in the active site of enzymes, where they directly participate in the catalytic reaction (e.g., in cysteine proteases). Moreover, cysteine residues are also often involved in redox reactions, where transfer of electrons proceeds via thiol-disulfide exchange reactions. Importantly, the activity of all these cytosolic enzymes usually depends on the preservation of the reduced state of the cysteine residue(s) involved. The very same properties that make cysteine the perfect amino acid for redox reactions, metal coordination and thiol-disulfide interchanges, also make cysteines extremely vulnerable to oxidation by reactive oxygen species (ROS). ROS arise transiently during normal metabolism as toxic byproducts of respiration and have been shown to accumulate under conditions of oxidative stress. Over the past few years, an increasing number of thiol-containing proteins has been identified that use ROS as a mediator to quickly regulate their protein activity (Linke and Jakob 2003). This new class of redox-regulated proteins includes the molecular chaperone Hsp33, which we discovered in 1999 (Jakob et al. 1999), metabolic enzymes (e.g., glyceraldehyde-3-phosphate dehydrogenase [GapDH]) (Cotgreave et al. 2002), prokaryotic and eukaryotic transcription factors (OxyR and Yap1) (Rainwater et al. 1995; Zheng et al. 1998; Kang et al. 1999; Kuge et al. 2001; Kim et al. 2002), kinases (protein kinase C and Raf) (Gopalakrishna and Jaken 2000; Hoyos et al. 2002), and phosphatases (PTP1B and PTEN) (Barrett et al. 1999; Leslie et al. 2003). What all these proteins have in common are highly reactive cysteine residues that are quickly and reversibly modified upon exposure to oxidative stress. These modifications include disulfide bond formation (e.g., in Hsp33, RsrA, and OxyR), nitrosylation (e.g., in Ras and OxyR), glutathionylation (e.g., in PTP1B, GapDH, and OxyR), or sulfenic acid formation (e.g., in PTP1B and OxyR). These modifications cause significant conformational changes and either lead to the activation (e.g., in Hsp33, OxyR, PKC, and Raf-kinase) or inactivation (e.g., in p53 and PTEN) of the respective protein's function. Upon return to non–oxidative stress conditions, cellular reductants such as the small molecule glutathione as well as cellular reductases like thioredoxin and glutaredoxin rapidly reduce the cysteine modifications and restore the original protein activity. These findings suggested that basically any protein with reactive cysteine residue(s) has the potential of being redox regulated. Many important regulatory proteins such as zinc finger proteins contain clusters of conserved cysteines and are, therefore, attractive targets for redox regulation. Over the past few years, several proteomic strategies have been developed to identify proteins that undergo thiol modifications in vivo. These methods addressed very specific questions and were used to either identify disulfide-bonded or glutathionylated proteins under oxidative stress conditions in vivo (Fratelli et al. 2002; Cumming et al. 2004), thioredoxin-targeted proteins in chloroplasts and Escherichia coli (Motohashi et al. 2001; Yano et al. 2001; Kumar et al. 2004), or target proteins of periplasmic thiol-disulfide oxidoreductases (Hiniker and Bardwell 2004; Kadokura et al. 2004). None of these methods, however, generated a general and global overview of thiol-modified proteins in vivo. We have now invented a differential thiol-trapping technique combined with two-dimensional (2D) gel analysis to monitor the in vivo thiol status of cellular proteins upon variations in the redox homeostasis of the cells. To test our method, we analyzed the thiol-disulfide status of proteins in aerobically growing E. coli cells and confirmed that the majority of proteins with thiol modifications are localized to the oxidizing environment of the periplasm. We found that the periplasmic thiol-disulfide oxidoreductase DsbA is responsible for these protein thiol modifications and identified novel DsbA substrate proteins. We then used our method to visualize directly the extent of ROS-induced thiol oxidation during aerobic growth and identified a number of cytosolic proteins with reactive cysteine residues that require functional thioredoxin to maintain their reduced thiol state. Finally, we analyzed the thiol status of cellular proteins in cells that were exposed to H2O2-induced oxidative stress and discovered a select group of new potentially redox-regulated proteins in vivo. Results A Differential Trapping Technique to Detect Oxidatively Modified Proteins In Vivo We have developed an innovative technique that allows us to monitor globally the in vivo thiol status of cellular proteins upon variations in the redox homeostasis of the cells. This method is based on the sequential reaction of two variants of the thiol-modifying reagent iodoacetamide (IAM) with accessible cysteine residues in proteins (Figure 1). Wild-type or mutant cells that were grown exponentially in glucose-minimal medium at 37 oC were exposed to the desired oxidative stress treatment. Then, cells were treated with trichloracetic acid (TCA) to rapidly quench thiol-disulfide exchange reactions. All accessible thiol groups were then alkylated with cold, unlabeled IAM under denaturating conditions. In a next step, all reversible thiol modifications that developed during normal growth or oxidative stress treatment (e.g., disulfide bonds and sulfenic acids) were reduced with DTT, and the newly accessible thiol groups were modified with 14C-labeled IAM. Therefore, radioactivity was specifically incorporated into proteins that originally contained thiol modifications. High ratios of 14C activity/protein are predicted for proteins with thiol modifications while low ratios of 14C activity/protein are predicted for proteins whose thiol groups are not significantly modified in vivo (Figure 1). Figure 1 Schematic Overview of Our Differential Thiol-Trapping Technique Under normal growth conditions (top), a hypothetical cytoplasmic protein present within the complex mixture of the crude whole-cell extract is fully reduced. Upon incubation in TCA, all thiol-disulfide exchange reactions are quenched and the cells lyse. In the first thiol-trapping step, the protein is denatured and incubated with IAM. Accessible thiol groups are quickly carbamidomethylated (CAM) and blocked for the subsequent reduction/alkylation steps. After TCA precipitation and washing, DTT is added to reduce oxidized cysteines, and 14C-labeled IAM is used to modify potentially newly released, accessible cysteines. Under oxidative stress conditions (bottom) the cysteine residues become modified (e.g., sulfenic acid and disulfide bonds). In the first trapping step, IAM cannot attack the oxidized disulfide bond. Only after reduction with DTT are the cysteines accessible to the 14C-labeled IAM. Therefore, the 14C radioactivity correlates with the degree of thiol modification in the protein. The differentially trapped protein species are chemically identical regardless of their original thiol-disulfide status. This ensures their identical migration behavior on 2D gel electrophoresis. The differentially trapped protein extract was then separated by 2D gel electrophoresis. Importantly, due to this specific trapping technique, all accessible thiol groups of each protein were carbamidomethylated to an extent that is independent of the original thiol-disulfide status of the protein. This ensures their identical migration behavior on 2D gels. The 2D gels were stained with colloidal Coomassie blue to get a measure of the total protein content. The 14C radioactivity, which correlates to the degree of thiol modification in the individual spots, was determined by exposing the dried gels to phosphor screens. Then, the 14C activity/protein ratio was visualized and quantified. The Majority of Oxidized Proteins Are Present in the Periplasm of E. coli In order to test our method, we analyzed the steady-state thiol-disulfide status of cellular proteins in wild-type E. coli. E. coli strains were grown in minimal medium to mid-logarithmic phase, and the cells were harvested. The cysteines were thiol trapped using our differential thiol-trapping technique and separated on 2D gels. To analyze the extent of thiol modification and the distribution of thiol-modified proteins in an unbiased way, we focused first on the 100 most abundant proteins on our colloidal Coomassie blue–stained 2D gels. We set the total spot intensity of all 100 protein spots on the colloidal blue gel to 100% and determined the relative spot intensity for each protein. We then quantified the 100 corresponding 14C activity spots on the phosphor image, set their combined total spot intensities to 100%, and determined again the relative spot intensity for each protein. Finally, we determined the 14C activity/protein ratios for each of the 100 proteins. Low 14C activity/protein ratios were predicted for non-thiol-modified proteins presumably present in the reducing environment of the cytoplasm, while high ratios of 14C activity/protein were predicted for proteins with cellular thiol modifications such as those present in the oxidizing milieu of the E. coli periplasm. As shown in Figure 2, the majority of proteins (91 proteins) had a 14C activity/protein ratio below 2.0, while nine proteins showed a higher than 2.0-fold ratio. Figure 2 Overall Thiol-Disulfide State of Cellular Proteins in Exponentially Growing E. coli Wild-Type Cells (A) Colored overlay of the Coomassie blue–stained 2D gel (shown in green) and the phosphor image (shown in red) of a differentially trapped protein extract from exponentially growing E. coli wild-type cells. Proteins with a high ratio of 14C activity/protein appear red; proteins with a low ratio appear green. Protein spots with a ratio of 14C activity/protein greater than 2.0 are indicated by an arrow, while circles label abundant proteins without cysteines. (B) Distribution of the 14C activity/protein ratio in the 100 most abundant protein spots found on a Coomassie blue–stained gel. Bars representing spots with a ratio higher than 2.0 are colored red and are labeled with the name of the protein(s) they represent. (C) Distribution of the 14C activity/protein ratio in the 100 most intense protein spots found on the phosphor images. Bars representing spots with a ratio higher than 2.0 are colored red and are labeled with the name of the protein(s) they represent. (D) Regular and reverse trapping of exponentially growing E. coli wild-type cells. Details of colored overlays of stained protein gels (shown in green) and phosphor images (shown in red) of cell extracts upon regular trapping (top) and reverse trapping (bottom). Mass spectrometric identification of a large number of these proteins suggested that our differential thiol trapping is indeed very selective for proteins with thiol modifications. From the nine protein spots with a 14C activity/protein ratio greater than 2.0, six are known periplasmic proteins such as the periplasmic oligopeptide permease (OppA) and glycerol-uptake protein (UgpB ), as well as proteins associated with the outer membrane like the outer membrane porin protein A (OmpA) (Figure 2A and 2B). Importantly, all of these proteins harbor at least two cysteines, suggesting that they may form structural disulfide bonds in the oxidizing environment of the periplasm. Three potentially cytoplasmic proteins were found to have high 14C activity/protein ratios: dihydrolipoyl transacetylase (AceF), lipoamide dehydrogenase (Lpd), and the stringent starvation protein (SspA) (Figure 2A and 2B). AceF and Lpd correspond to the enzymes E2 and E3 of the pyruvate dehydrogenase complex. Lpd contains a reactive cysteine pair in the active site that undergoes disulfide bond formation during the regeneration of the disulfide bond of the covalently bound cofactor lipoamide of AceF (Massey and Veeger 1960). Detection of these proteins in our analysis is an excellent indication that we obtained an in vivo snapshot of proteins that use disulfide bond formation in their catalytic cycle and shows that the method can detect the redox state of covalently bound thiol-containing cofactors as well. SspA contains only one cysteine residue. This cysteine residue might be glutathionylated in vivo under steady-state conditions. Alternatively, however, SspA might co-migrate with the low-abundance periplasmic protein arginine binding protein ArtI that harbors two highly conserved cysteines and migrates at a very similar position on periplasmic extract gels (A. Hiniker, personal communication). The majority of cytoplasmic proteins that contain numerous cysteines, on the other hand, revealed a 14C activity/protein ratio below 2.0, including the very abundant elongation factor EF-Tu (Tu elongation factor [TufB]-IF1) with three cysteine residues (14C activity/protein ratio = 1.3 ± 0.5) and isocitrate dehydrogenase with seven cysteine residues (14C activity/protein = 1.2 ± 0.5). Protein spots that showed extremely low 14C activity/protein ratios (less than 0.2) included the very abundant outer membrane porin protein E as well as the cytoplasmic proteins P-specific transport protein and trigger factor , all of which do not contain any cysteine residues (Figure 2A). These results indicated that under our labeling conditions, IAM was quite specific for cysteine residues, and labeling of non-thiol-containing amino acids could be neglected. Because many proteins with thiol modifications are low-abundance periplasmic proteins, we performed a similar analysis but focused now on the 100 most heavily 14C-modified proteins rather than on the 100 most abundant proteins (Figure 2C). A number of proteins that showed a high 14C activity/protein ratio were periplasmic proteins that we had previously identified. In addition, however, the low-abundance periplasmic proteins periplasmic histidine binding protein (HisJ), ArtJ, DsbA, and the periplasmic dipeptide binding protein (DppA) were identified as proteins with a very high degree of thiol modification (14C activity/protein ratio > 2.0) (Figure 2A and 2C). All four proteins are known to be localized to the periplasm of E. coli and again contain at least one pair of cysteine residues. Both DppA and HisJ have recently been identified as substrates of the disulfide oxidase DsbA (Hiniker and Bardwell 2004), confirming that they do contain disulfide bonds in vivo. To obtain an idea about the sensitivity of our method, we closely analyzed DsbA, a protein that contains two cysteine residues and that has been found to be fully oxidized in wild-type E. coli cells using our technique as well as conventional thiol-trapping methods (Kishigami et al. 1995). Although DsbA was only a faint spot on protein gels (318 spots showed a stronger signal), it was among the most abundant spots on the phosphor image (the 27th most intense spot). This showed that even in a low-abundant protein such as DsbA, the presence of only two thiol-modified cysteines is fully sufficient to create a clearly detectable 14C signal. Regular and Reverse Thiol Trapping—Determining Redox States In Situ Quantitative analysis of the ratio of oxidized and reduced protein species in vivo can be used to determine the redox state of the protein in the cell providing that the oxidation mechanism of the protein is known (Watson and Jones 2003). We therefore considered that if our regular thiol trapping was completely alkylating all reactive cysteine residues, the 14C activity/protein ratio of a defined protein spot should correspond to the amount of oxidized protein. To then visualize and quantify the amount of reduced protein species in the same protein spot, we decided to perform a reverse trapping in parallel. In the reverse trapping, all free, accessible cysteines were immediately alkylated with radioactive IAM, while oxidatively modified cysteines were alkylated with cold IAM after their reduction. Therefore, the 14C activity/protein ratio should now correspond to the amount of reduced protein in the respective protein spot. We confirmed that proteins that had very high ratios of 14C activity/protein such as oxidized OmpA IF1 and IF2 in our regular trapping had very low ratios of 14C activity/protein (0.2 ± 0.08) in our reverse-trapped samples (Figure 2D). A mostly reduced protein such as succinyl-CoA synthetase, which had a low ratio of 0.7 ± 0.2 under regular trapping conditions, on the other hand showed a very high ratio of 2.8 ± 0.7 under reverse-trapping conditions. Based on these results, we considered that the comparison of the 14C activity/protein ratio of defined protein spots in regular and reverse-trapped samples could give us the ratio of oxidized and reduced protein under steady-state conditions, if the reaction mechanism of the protein oxidation was known. This should then allow us to determine the half-cell potential or redox state of the cellular proteins in vivo. To test our approach, we decided to calculate the redox state of Lpd, a cytosolic enzyme that we identified in our screen to be partly oxidized under steady-state conditions. Oxidative decarboxylation of pyruvate goes along with the reduction of lipoamide, the prosthetic group of AceF. To regenerate this complex, the disulfide bond in dihydrolipoamide is re-oxidized by the active-site disulfide bond of Lpd, which itself donates its electrons to the prosthetic flavin adenine dinucleotide and ultimately to nicotinamide adenine dinucleotide. Because the standard redox potentials of Lpd and the dihydrolipoic acid/lipoic acid redox pair of AceF are known (Table 1) (Maeda-Yorita et al. 1991; Nelson et al. 2000), we determined the 14C activity/protein ratio of Lpd under regular and reverse-trapping conditions and determined its redox state in vivo. Table 1 Standard Redox Potentials, Redox Potentials, and 14C Activity/Protein Ratios in the Reverse and Regular Trapping Experiments of Selected Redox Pairs a  Maeda-Yorita et al. (1991) b Determined from the ratios obtained in the trapping experiment and the standard redox potential c  Nelson et al. (2000) d Determined from the ratios obtained in the trapping experiment, assuming that AhpC is in equilibrium with the cellular redox potential as represented by the GSH/GSSG redox pair e Calculated from the concentrations of GSH and GSSG in E. coli DHB4 (Aslund et al. 1999), assuming that AhpC is in equilibrium with the cellular redox potential as represented by the GSH/GSSG redox pair f Based on the formation of intermolecular dimers, a ratio of [AhpCred]2/[AhpCox] was used (Ellis and Poole 1997) The in vivo redox state of Lpd was found to be –0.261 ± 0.009 V (pH 7.0). This was in excellent agreement with the proposed flow of electrons through this multienzyme system, showing that Lpd is able to oxidize the dihydrolipoic acid in AceF (E 0 = −0.290). A reliable direct measurement of the redox potential of the dihydrolipoic acid/lipoic acid redox couple in AceF was not possible because of the very low signal that we found for AceF in the reverse-trapping experiment. This indicated that AceF is mostly in its thiol-oxidized state. This probably reflects the fact that the prosthetic group is only accessible to Lpd in its reduced state (Nelson et al. 2000), which presumably allows AceF to keep its prosthetic group oxidized even within the very reducing environment of the cytosol. The redox potential that we determined for Lpd also suggested that the components of this multienzyme complex are neither in equilibrium with one another nor with the overall GSH/GSSG redox potential in the cell (−0.24 V at pH 7.0, an intracellular concentration of 5 mM GSH, and a ratio of GSH/GSSG of 223:1 in E. coli DHB4 [Aslund et al. 1999]). These results showed that the calculation of (standard) redox potentials for proteins in vivo using our differential trapping technique was possible when sufficient amounts of reduced and oxidized species could be detected and when their ratios could be reliably quantified. Then, this technique proved to be very useful to estimate the direction of electron flow in metabolic pathways in vivo. We also calculated the standard redox potential of alkylhydroperoxide reductase small subunit (AhpC), a protein that uses disulfide bond formation to detoxify alkylhydroperoxides, assuming that under steady-state conditions it is in equilibrium with the overall cellular redox potential. We calculated a standard redox potential for AhpC of −0.257 ± 0.009 V. This agrees well with studies in Helicobacter pylori (Baker et al. 2001) and our findings (see below) that suggested that thioredoxin (E 0 = −0.270 V) (Krause et al. 1991) might play a direct role in the catalytic cycle of AhpC. Identification of the In Vivo Substrate Proteins of DsbA We found that our method specifically and reliably detected proteins with thiol modifications in vivo. This suggested to us that our method should also be an excellent tool to determine the in vivo substrate specificity of cellular thiol-disulfide oxidoreductases. We therefore decided to first compare the thiol-disulfide status of proteins in wild-type E. coli and strains that lack DsbA, the enzyme that is responsible for disulfide bond formation in the periplasm of E. coli. Previously, only a few DsbA substrates have been identified. The studies that addressed this question in the past relied either on the formation of covalent intermediates between an active-site cysteine mutant of DsbA and substrate proteins (Kadokura et al. 2004), or on the instability and premature degradation of periplasmic proteins that are no longer stabilized by disulfide bonds because of the absence of DsbA (Hiniker and Bardwell 2004). We grew wild-type E. coli cells and cells lacking the chromosomal copy of DsbA (dsbA::kan) to mid-logarithmic phase, harvested the cells, and differentially thiol-trapped the cysteines. As shown in Figure 3 and Table 2, using our new thiol-trapping technique, we identified a number of proteins that showed significantly less or no thiol modification in dsbA deletion strains than in wild-type strains. Among the proteins that we selected for mass spectrometric analysis were known DsbA substrate proteins (e.g., OmpA, DppA, organic solvent tolerance protein [Imp], and HisJ) as well as a number of proteins that have not yet been associated with DsbA (e.g., ArtJ and UgpB). Because all of these proteins are periplasmic and have at least one conserved pair of cysteines, it appears very likely that these proteins are also substrate proteins of DsbA. Figure 3 Identification of In Vivo Substrate Proteins of the Periplasmic Disulfide Bond Oxidase DsbA (A) Colored overlay of the stained 2D gel (shown in green) and the phosphor image (shown in red) of differentially trapped protein extract from exponentially growing E. coli wild-type cells. Proteins with a high 14C activity/protein ratio appear red, while proteins with a low ratio appear green. Proteins that were found to have significantly lower ratio of 14C activity/protein in the dsbA::kan strain (B and C) are labeled. (B) Overlay of the stained 2D gel (shown in green) and the phosphor image (shown in red) of a differentially trapped protein extract from exponentially growing dsbA::kan cells. Proteins that were found to have a significantly lower 14C activity/protein ratio in dsbA::kan cells than in wild-type strain (A) are marked with an arrowhead. A circle marks the position of DsbA on the wild-type gel. (C) Overlay of the stained 2D gel (shown in green) and the phosphor image (shown in red) of a differentially trapped protein extract from E. coli dsbA::kan cells growing under oxygen limitation. Arrowheads label proteins that were found to have a significantly lower 14C activity/protein ratio than the wild-type strain grown under oxygen limitation. A circle marks the position of DsbA on the wild-type gel. Table 2 Identification of the In Vivo Substrates of DsbA a The activity/protein ratio of the given protein spot on gels from differentially trapped extracts from E. coli DHB4 ( wild-type) was divided by the corresponding activity/protein ratio on gels from differentially trapped extracts from E. coli LL029 (dsbA::kan) b The p-value according to the TTEST function of Excel 2000 (Microsoft, Redmond, Washington, United States) c The activity/protein ratio of the given protein spot on gels from differentially trapped extracts from E. coli DHB4 ( wild-type) grown under oxygen limitation was divided by the corresponding ratio on gels from differentially trapped extracts from E. coli LL029 (dsbA::kan) grown under oxygen limitation d Localization according to Swiss-Prot (http://us.expasy.org/sprot/) (Boeckmann et al. 2003) or PSORTB (http://www.psort.org/psortb/index.html) (Gardy et al. 2003). CP, cytoplasmic; IM, inner membrane; OM, outer membrane; PP, periplasmic e Imp and the outer membrane protein YaeT migrate to the same position in the 2D gels Interestingly, the oxidation state of the known DsbA substrate protein OmpA, as well as of some other periplasmic proteins, appeared to be only moderately affected by the lack of DsbA (Figure 3A and 3B). Because the process of folding and disulfide bond formation in OmpA has been shown to be reasonably fast (half time, t 1/2 = 5 min) even in the absence of DsbA (Bardwell et al. 1991), we considered that air oxidation was probably responsible for the disulfide bond formation in proteins such as OmpA under steady-state conditions. To investigate the role that air oxidation might play in the disulfide bond formation of periplasmic proteins in vivo, we performed the same thiol-trapping experiments using wild-type and dsbA::kan cells that were grown under very limiting oxygen conditions. Under those oxygen-limited conditions the 14C activity/protein ratio of OmpA and other periplasmic proteins was dramatically decreased compared to wild-type cells and also significantly lower than in dsbA::kan cells grown under normal oxygen conditions (Figure 3C; Table 2). These results not only confirmed that the thiol modifications that we detected with our differential thiol-trapping technique are indeed in vivo modifications and are not introduced during the aerobic lysis and trapping of the sample, but also clearly showed that DsbA is not absolutely necessary for the thiol oxidation of certain periplasmic proteins under aerobic conditions. Under low-oxygen conditions, however, which occur in stationary phase or when cells are grown micro-aerobically, functional DsbA is absolutely required for the successful disulfide bond formation in the E. coli periplasm. TrxA Protects a Large Number of Intracellular Proteins from Oxidation While DsbA promotes disulfide bond formation in the E. coli periplasm, the thioredoxin and glutaredoxin systems reduce disulfide bonds in the E. coli cytoplasm. These systems not only prevent the formation of unwanted disulfide bonds in cytoplasmic proteins, which often lead to the inactivation of the respective proteins, but also play important regulatory roles in the cell. For instance, the oxidative stress response in both prokaryotes and eukaryotes is rapidly attenuated by glutaredoxins and thioredoxins, which reduce and inactivate the oxidative stress transcription factors OxyR or Yap1p (Carmel-Harel and Storz 2000). Therefore, analysis of the proteins that use these systems for their specific reduction will help us to identify cytosolic proteins that use thiol modifications in their functional life cycle. To analyze the substrate specificity of cytoplasmic thiol-disulfide oxidoreductases, we decided to compare the thiol-disulfide status of proteins in a thioredoxin null mutant and the isogenic wild-type E. coli strain. Importantly, strains that lack the trxA gene do not exhibit a general disulfide stress phenotype (Prinz et al. 1997), which minimizes potential secondary thiol modifications in proteins that could be otherwise attributed to those stresses (Derman et al. 1993). The dramatic alteration in the oxidation state of a large number of cellular proteins in a ΔtrxA strain as compared to a wild-type E. coli strain is clearly visible (Figure 4). Of the 100 proteins that were selected based on their high level in 14C activity, 37 protein spots showed a more than 2-fold further increase in thiol modification in ΔtrxA strains compared to wild-type strains, where functional thioredoxin is apparently working successfully to keep them reduced. Proteins whose 14C activity/protein ratio did not change in the absence of thioredoxin included the majority of periplasmic proteins that have been identified before, as well as some highly abundant cytoplasmic proteins that harbor presumably inaccessible or unreactive cysteines. Figure 4 Identification of the In Vivo Substrate Proteins of Thioredoxin A (A) Colored overlay of the stained 2D gel (shown in green) and the phosphor image (shown in red) of differentially trapped protein extract from exponentially growing E. coli wild-type cells. Proteins with a high ratio of activity per protein appear red, proteins with a low ratio appear green. Proteins that were found to have a significantly higher ratio of activity per protein in the trxA− strain (B) are labeled. (B) Overlay of the stained 2D gel (shown in green) and the phosphor image (shown in red) of differentially trapped protein extract from exponentially growing E. coli ΔtrxA cells. Proteins that were found to have a significantly higher ratio of 14C activity/protein in the ΔtrxA strain are labeled with an arrow. All 37 spots were selected for mass spectrometric analysis, and resulted in the identification of 27 individual proteins, 23 of which have either been shown or are predicted to be localized to the reducing environment of the cytoplasm and to contain at least one and up to ten cysteine residues (Table 3). Of those, at least six proteins (e.g., thioredoxin-linked thiol peroxidase [Tpx], AhpC, GapDH, and aconitase B [AcnB]) have been previously shown to be targets of thioredoxin in plants (Yamazaki et al. 2004) or, very recently, in E. coli (Kumar et al. 2004) (Table 3). Thirteen proteins have cysteine residues in the active site or the cofactor-binding site (e.g., aspartate semialdehyde dehydrogenase, citrate synthase, and γ-glutamyl phosphate reductase). Five proteins are known metal-binding proteins either coordinating zinc via cysteines (e.g., cobalamin-independent methionine synthase [MetE] and carbonic anhydrase [YadF]), binding iron (e.g., alcohol/acetaldehyde dehydrogenase[AdhE]), or harboring iron-sulfur clusters (e.g., AcnB and succinate dehydrogenase [SdhB]) (Table 3). Reactive and/or accessible cysteine residues appear to make these proteins particularly vulnerable to small amounts of ROS, such as hydrogen peroxide (H2O2), which are known to be produced as toxic byproducts of cellular respiration in aerobically growing cells (Costa Seaver and Imlay 2001). That H2O2 must be produced during aerobic growth became also obvious when we found the detoxifying enzymes AhpC and the thioredoxin-linked Tpx to be largely oxidized. Both proteins use disulfide bond formation to detoxify peroxides and appear to require functional thioredoxin to regenerate their reduced thiol status. Table 3 In Vivo Substrate Proteins of Thioredoxin A a The 14C activity/protein ratio of the given protein spot on gels from differentially trapped extracts from E. coli DHB4 ( wild-type) was divided by the corresponding 14C activity/protein ratio on gels from differentially trapped extracts from E. coli WP570 (ΔtrxA) b The p-value according to the TTEST function of Excel 2000 c Localization according to Swiss-Prot (http://us.expasy.org/sprot/) (Boeckmann et al. 2003) or PSORTB (http://www.psort.org/psortb/index.html) (Gardy et al. 2003). CP, cytoplasmic; IM, inner membrane; OM, outer membrane; PP, periplasmic Table 3 Continued The in vivo snapshot of the thiol status of proteins in thioredoxin-defective strains shows for the first time, to our knowledge, the detrimental effects of small amounts of ROS generated during aerobic growth on cytosolic proteins, and the important role that thioredoxin A plays in regenerating these proteins. A surprisingly large number of cytosolic proteins appear to harbor such oxidation-sensitive cysteine residues that require the constant presence of the reducing thioredoxin system under aerobic growth. We cannot exclude at this point the possibility that the other cellular redox system glutaredoxin is overwhelmed by the accumulation of thiol-modified proteins in thioredoxin-deficient strains as well. This might be the reason why we detect thiol-modified MetE, one of the very few known glutathionylated E. coli proteins (Hondorp and Matthews 2004), which presumably requires functional glutaredoxin for its regeneration. It is also important to note that we focused only on the 100 most intense protein spots on the phosphor image. A significant number of additional proteins showed an at least 2-fold further increase in their 14C activity/protein ratio in trxA− cells as compared to wild-type cells. These are likely also substrates of thioredoxin and remain to be identified (Figure 4). Only three periplasmic proteins were found to be increasingly thiol-modified in the absence of cytoplasmic thioredoxin; Tpx, OppA , and the methionine-binding protein MetQ (YaeC). In the case of Tpx, thioredoxin A has been suggested to be an essential component in its functional regulation. This makes Tpx a potential substrate protein of the periplasmic thiol-disulfide oxidoreductase systems DsbC or DsbG, which are connected to cytoplasmic thioredoxin via the membrane protein DsbD. Absence of thioredoxin in the cytoplasm would lead to the accumulation of oxidized DsbC and DsbG in the periplasm, which would then no longer be able to reduce and/or isomerize disulfide bonds in periplasmic proteins such as Tpx; this would explain the accumulation of oxidized proteins in the periplasm. The same could apply for the other two periplasmic proteins that we identified in this study. Alternatively, however, thiol modifications that occur and are not reduced in the cytoplasm might prevent their efficient transport, or might impair potential attachments of lipid anchors. The latter might be the case for the lipid protein MetQ (YaeC), whose single cysteine is predicted to be linked to a lipid anchor. Identification of Proteins Sensitive to Oxidative Stress Over the past few years, an increasing number of redox-sensitive proteins have been identified that use the oxidation state of reactive cysteine residues as a regulatory switch. Oxidative stress–induced thiol modifications lead to conformational changes and to the transient activation or inactivation of the respective protein. Upon return to non–oxidative stress conditions, cellular reductants such as the thioredoxin system rapidly reduce the cysteine modifications and restore the original protein activity. The observation that the function of so many different cysteine-containing proteins is regulated by the redox conditions of the environment, suggests that basically any protein with one or more reactive and exposed cysteines has the potential of being redox-regulated. Because many important regulatory and biosynthetic proteins contain clusters of cysteines in the active site or cofactor-binding site, they can therefore be considered attractive and potential targets for this novel form of functional regulation. To investigate thiol modification under conditions of exogenous oxidative stress, wild-type E. coli cells were grown to mid-logarithmic phase and exposed to oxidative stress treatment by addition of H2O2. Here, we focused on the 100 most heavily 14C-labeled proteins after 10 min of H2O2 treatment, and compared their 14C activity/protein ratio to the ratio immediately before and 2, 5, and 30 min after the addition of H2O2 (Figure 5; Table 4). From the 100 most thiol-modified proteins in oxidatively stressed E. coli cells, seven proteins showed increasing thiol modification upon exposure to oxidative stress. These included H2O2-detoxifying enzymes such as Tpx, as well as a number of biosynthetic enzymes such as MetE, GTP cyclohydrolase (FolE), and phosphoglycerate dehydrogenase (SerA). Figure 5 Identification of Oxidative Stress–Sensitive Proteins In Vivo (A) Overlay of the stained 2D gel (shown in green) and the phosphor image (shown in red) of differentially trapped protein extracts from H2O2-stressed E. coli wild-type cells. Proteins that were found to have a significantly higher ratio of 14C activity/protein after 10 min of H2O2 treatment than in untreated cells are labeled. (B) Time course of the oxidation of proteins in E. coli wild-type cells upon treatment with H2O2. Details of the colored overlays of stained protein gels (shown in green) and phosphor images (shown in red) of cell extracts taken before (Co) and 2, 5, 10, and 30 min after addition of H2O2 to the cells. The selected proteins are FolE, Tpx, MetE, and TufB. Bar charts on the right show the oxidation-dependent change in ratio of 14C activity/protein of the protein spot labeled by an arrowhead. (C) Time course of the oxidation of MetE in E. coli wild-type cells upon treatment with 1 mM diamide. Details of the colored overlays of stained protein gels (shown in green) and autoradiographs taken on X-ray films (shown in red) of cell extracts taken before (Co) and 2, 10, and 30 min after addition of diamide to the cells. Table 4 Oxidation Stress Sensitive Proteins in E. coli a The activity/protein ratio of the given protein spot on gels from differentially trapped extracts from E. coli DHB4 ( wild-type) treated with 4 mM H2O2 was divided by the corresponding ratio on gels from differentially trapped extracts from untreated E. coli DHB4 b The p-value according to the TTEST function of Excel 2000 c Localization according to Swiss-Prot (http://us.expasy.org/sprot/) (Boeckmann et al. 2003) or PSORTB (http://www.psort.org/psortb/index.html) (Gardy et al. 2003). CP, cytoplasmic; PP, periplasmic Importantly, in the companion paper by Hondorp and Matthews (2004), MetE has been shown to be redox-regulated in vivo and in vitro. The surface-exposed cysteine 645 in MetE was found to be particularly sensitive to oxidative stress–induced thiol modifications. The authors showed that this thiol modification transiently inactivated the enzyme, which provided an excellent explanation for the observed methionine auxotrophy that accompanies oxidative stress in E. coli. To compare the kinetics and extent of our H2O2-induced thiol modification of MetE with the diamide-induced glutathionylation observed by Hondorp and Matthews, we analyzed the time course of MetE modification upon exposure of E. coli cells to 1 mM diamide. As shown in Figure 5C, MetE was maximally thiol-modified within 2 min of diamide treatment and maintained its high level of thiol modification over at least 30 min of incubation. This was in excellent agreement with the in vivo thiol trapping conducted by Hondorp and Matthews, who showed that at their first time point of 15 min, all of MetE was in the oxidized state. The second metabolic enzyme that we identified as being particularly sensitive to oxidative stress was FolE, which catalyzes the committed step in the synthesis of the one-carbon donor tetrahydrofolate. Analysis of its crystal structure revealed that FolE contains a cysteine-coordinating zinc center, which escaped prior detection because of its high oxidation sensitivity in vitro (Rebelo et al. 2003). Air-oxidation of FolE leads to the inactivation of the enzyme. We have now observed that FolE is one of the major targets of H2O2 treatment in E. coli, with a more than 5-fold increase in 14C activity/protein ratio upon H2O2 treatment. This suggested that FolE is also transiently oxidized and inactivated upon oxidative stress in vivo, a finding that would clearly make physiological sense. Tetrahydrofolate is a highly oxidation-sensitive compound, and synthesizing it under oxidative stress conditions would be extremely wasteful for the cell. Analysis of the time course of thiol modification in FolE during H2O2-induced oxidative stress treatment showed a steady increase in thiol modification. This was in contrast to the time course of thioredoxin substrate proteins such as Tpx, whose thiol modification peaked around 2–5 min after the start of the oxidative stress treatment (Figure 5B). This suggests that FolE is indeed a protein whose redox state is not controlled by thioredoxin and that is especially sensitive to oxidative stress treatment. A large number of cysteine-containing cytoplasmic proteins (e.g., isocitrate dehydrogenase), as well as all of the identified periplasmic proteins, did not show any significant increase in oxidation-induced thiol modification. This indicated that under oxidative stress conditions, the majority of cytosolic proteins remain reduced and confirmed that the periplasmic proteins were already fully oxidized under aerobic growth conditions. The probably best-known redox-regulated proteins in E. coli, the oxidative stress transcription factor OxyR and the molecular chaperone Hsp33, were not among the 100 most thiol-modified proteins after 10 min of oxidative stress. This was not very surprising, given that OxyR is a low-abundance protein and Hsp33 has an extremely low pI (pH 4.35) and cannot be detected by our 2D gel system. The fact, however, that we identified a number of proteins that either have been shown (Tpx and MetE) or were predicted (FolE) to undergo thiol modification upon oxidative stress in vivo, made us very confident that thiol modifications play regulatory or functional roles in the other proteins that we discovered as well (e.g., GlyA, PheT, and SerA). All of these proteins have numerous cysteine residues, which are either surface exposed or might play other functional roles that have not yet been identified (Table 4). Detailed biochemical analysis is now required to investigate the exact role that thiol modifications play in these potentially redox-regulated proteins. Conclusion: A Widely Applicable New Method to Visualize Thiol Modifications In Vivo Over the past few years, a variety of reversible oxidative cysteine modifications have been discovered that regulate the activity of eu- and prokaryotic proteins. The most prominent modification is disulfide bond formation, but also transient glutathionylation of cysteines or oxidation to sulfenic acid have been found to play an important regulatory role in many proteins (Barrett et al. 1999; Kim et al. 2002). These modifications are usually transiently introduced by specific stress conditions such as peroxide or disulfide stress and are resolved by cellular thiol-disulfide oxidoreductases such as thioredoxin and glutaredoxin, which are usually upregulated during these stress conditions (Potamitou et al. 2002). Several new proteomic strategies have now been developed to probe for potential substrate proteins of cellular thiol-disulfide oxidoreductases and proteins that undergo thiol modifications during stress conditions. These strategies involve the use of radioactive glutathione to detect glutathionylated proteins under oxidative stress conditions (Fratelli et al. 2002), fluorescent thiol-reactive dyes to identify thioredoxin-targeted proteins (Yano et al. 2001), or active-site mutants of thiol-disulfide oxidoreductases, whose failure to complete the enzymatic reaction leads to an irreversible disulfide crosslink between thiol-disulfide oxidoreductase and substrate, which can then be identified by SDS-PAGE or diagonal PAGE in combination with mass spectrometry (Motohashi et al. 2001; Balmer et al. 2003; Kadokura et al. 2004). Very recently a study also examined proteins that interact very tightly with tap-tagged thioredoxin in E. coli (Kumar et al. 2004). This technique, however, was unable to distinguish between proteins that require thiol-disulfide exchange reactions with thioredoxins (i.e., enzymatic substrates) and proteins that simply associate with thioredoxin without the involvement of thiol chemistry. This was especially emphasized by the finding that at least 25% of the thioredoxin-associated proteins identified by Kumar et al. did not contain any cysteines. With the help of these various methods, a number of proteins have been identified that serve as substrate proteins of different thiol-disulfide oxidoreductases in E. coli and other organisms. We have now developed a technique that allows us to globally monitor and compare the thiol-disulfide status of all cellular proteins that can be resolved in 2D gels. With this technique, the substrate proteins of all major oxidoreductase systems can be identified and dissected simply by comparing the thiol status of proteins in the appropriate mutant strains. In prokaryotes, for instance, substrate overlap and differences between the thioredoxin A and C can be analyzed by simply comparing the thiol disulfide status in trxA− and trxC− cells. The substrate specificity of the complete thioredoxin system can then be distinguished from the substrate specificity of the glutaredoxin systems by comparing the thiol-disulfide status of cellular proteins in strains lacking one of the systems altogether. Most importantly, this technique should be widely applicable to many different cell types and organisms. Preliminary experiments in yeast, for instance, showed that AdhE, GapDH, and SdhB are major targets for oxidative thiol modifications in yeast (data not shown). We found the respective E. coli homologs to be among the most redox-sensitive proteins in strains lacking thioredoxin A. The reason why this method is applicable to both pro- and eukaryotic cells is the rapid thiol-quenching step that involves the incubation and lysis of cells in the presence of TCA. This immediately stops all thiol-disulfide exchange reactions. All subsequent trapping steps are then conducted with the soluble proteins under denaturating conditions. These unique features should allow us and others to monitor and visualize the in vivo thiol status of cellular proteins upon exposure of various cells and organisms to virtually every physiological or pathological condition that is accompanied by oxidative stress. Materials and Methods Bacterial strains. E. coli DHB4 (F′ lac-pro lacI Q/Δ(ara-leu)7697 araD139 ΔlacX74 galE galK rpsL phoR Δ(phoA)PvuII ΔmalF3 thi) (herein referred to as wild-type), WP570 (DHB4 ΔtrxA) (Prinz et al. 1997), and LL029 (DHB4 dsbA::kan) were grown aerobically in glucose MOPS minimal medium (Neidhardt et al. 1974) containing 40 μg/ml L-leucine and 10 μM thiamine at 37 °C. Oxygen-limited cultures were grown in completely full 15-ml screw cap tubes. E. coli LL029 was obtained by P1 transduction of a dsbA::kan insertion mutation into DHB4. The dsbA-null strain AH55 was used as the source of the P1 transduction (Hiniker and Bardwell 2004). Harvest of cell samples. Wild-type E. coli and the respective mutant cells were grown to an OD600 of 0.4 at 37 oC. To expose wild-type E. coli cells to oxidative stress treatment, the cells were then treated with 4 mM H2O2 or 1 mM diamide for the duration indicated. Then 1.8 ml of the cell culture was harvested directly into 200 μl of ice-cold 100% (w/v) TCA and stored on ice for at least 20 min. Differential thiol trapping of cellular proteins. The TCA-treated cells were centrifuged (13,000g, 4 °C, 30 min), and the resulting pellet was washed with 500 μl of ice-cold 10% (w/v) TCA followed by a wash with 200 μl of ice-cold 5% (w/v) TCA. The supernatant was removed completely, and the pellet was resuspended in 40 μl of denaturing buffer (6 M Urea, 200 mM Tris-HCl (pH 8.5), 10 mM EDTA, and 0.5 % [w/v] SDS) supplemented with 100 mM IAM. This first alkylation procedure irreversibly modified all free thiol groups that were made accessible by the urea and SDS-denaturation of the proteins. After 10 min of incubation at 25 °C, the reaction was stopped by adding 40 μl of ice-cold 20% (w/v) TCA. After 20 min of incubation on ice, the alkylated proteins were centrifuged again, and the pellet was washed with TCA as described before. The protein pellet was then dissolved in 20 μl of 10 mM DTT in denaturing buffer to reduce all reversible thiol modifications such as disulfide bonds and sulfenic acids. After a 1-h incubation at 25 °C, 20 μl of a solution of 100 mM radioactively labeled [14C-1]-IAM in denaturing buffer was added to titrate out the DTT and to irreversibly alkylate all newly reduced cysteines. The reaction mixture was incubated for 10 min at 25 °C. The reaction was stopped by adding 40 μl of 20% (w/v) TCA. After precipitation on ice and subsequent centrifugation, the pellet was washed first with TCA and then three times with 500 μl of ice-cold ethanol (for schematic overview see Figure 1). Reverse-trapping experiments were conducted as described except that the first alkylation procedure was performed with [14C-1]-IAM while the second alkylation step was performed with unlabeled IAM. For protein identification purposes, thiol-trapping experiments using nonradioactive IAM in both alkylation steps were performed in parallel. 2D gel electrophoresis. The pellet of the thiol-trapped proteins was dissolved in 500 μl of rehydration buffer (7 M urea, 2 M thiourea, 1% [w/v] Serdolit MB-1, 1% [w/v] dithiothreitol, 4% [w/v] Chaps, and 0.5% [v/v] Pharmalyte 3–10), and the 2D gel electrophoresis was performed as previously described (Hiniker and Bardwell 2004). Staining of the gels, storage phosphor autoradiography, and image analysis. Gels were stained using colloidal Coomassie blue stain (Neuhoff et al. 1985) and scanned using an Expression 1680 scanner with transparency unit (Epson America, Long Beach, California, United States) at 200-dpi resolution/16-bit grayscale. Phosphor images were obtained by exposing LE Storage Phosphor Screens (Amersham Biosciences, Piscataway, New Jersey, United States) to dried gels for 7 d. The phosphor image screens were read out with the Personal Molecular Imager FX (Biorad, Hercules, California, United States) at a resolution of 100 μm. The original image size of the phosphor image was changed to a resolution of 200 dpi with PhotoShop 7.0 (Adobe Systems, San Jose, California, United States). The phosphor images and images of the stained proteins were analyzed using Delta 2D Software (Decodon, Greifswald, Germany). Data analysis. For each of the described experiments, at least four individually trapped samples of cultures were obtained from at least two independent cell cultures. The only exceptions were the time course of H2O2 treatment at the time points 2, 5, and 30 min, the DsbA experiments under oxygen limitation, and the time course of diamide treatment. For each of the experiments, the phosphor image with the highest overall 14C activity was chosen for spot detection. The 100 most abundant spots were chosen from the detected set of spots and the boundaries transferred to all other phosphor images and protein gel images using the Delta 2D “transfer spots” function. The absolute intensity for each of these 100 spots on the protein gels and the phosphor image was determined to quantitatively describe the amount of protein and 14C activity for each protein spot. These absolute spot intensities were then normalized over all 100 spots (for trapping and reverse-trapping of wild-type cells, and for all H2O2 experiments). This normalization scheme was changed when the thiol-disulfide status of the dsbA mutant strain was analyzed. This was based on the consideration that a large number of the most intense spots on the phosphor images are heavily thiol-modified periplasmic proteins, which are putative DsbA substrate proteins. Normalizing over those protein spots would largely affect our data analysis. We therefore decided to normalize over four of the most abundant intracellular spots, TufB isoform (IF) 1, GapA IF 1, AhpC, and GroEL, whose thiol-disulfide status was not affected by the absence or presence of DsbA. In the case of the trxA mutant strain, similar considerations led us to normalize over four of the most abundant periplasmic protein spots, OmpA IF 1, OmpA IF 2, HisJ, and ArtJ, whose thiol-disulfide status was not influenced by the lack of TrxA activity. Finally, the ratio of 14C activity/protein was calculated by dividing the normalized intensity of the protein spot on the phosphor image by the corresponding normalized intensity of the Coomassie blue–stained protein spot. For a protein to be considered significantly thiol-modified, the average of the 14C activity/protein ratio for a given protein spot had to be at least 1.5-fold above the average of the 14C activity/protein ratio of this protein under control conditions. Identification of proteins from 2D gels. Thiol-trapped samples using nonradioactive IAM in both alkylation steps were separated on 2D gels and used to excise proteins of interest. These proteins were identified by Peptide Mass Fingerprinting at the Michigan Proteome Consortium (http://www.proteomeconsortium.org). Supporting Information Accession Numbers The Swiss-Prot (http://www.ebi.ac.uk/swissprot/) accession numbers for the gene products discussed in this paper are 30S ribosomal subunit protein S2 (P02351), 50S ribosomal subunit protein L5 (P02389), AceF (P06959), AcnB (P36683), AdhE (P17547), AhpC (P26427), and γ-glutamyl phosphate reductase (P07004), ArtI (P30859), ArtJ (P30860), aspartate semialdehyde dehydrogenase (P00353), carbonic anhydrase (P36857), carbonic anhydrase (P36857), citrate synthase (P00891), DAHP synthetase (P00886), DppA (P23847), DsbA (P24991), GapA (P06977), glutamyl-tRNA synthetase (P04805), GroEL (P06139), GTP cyclohydrolase I (P27511), HisJ (P39182), Hsp33 (P45803), Imp (P31554), isocitrate dehydrogenase (P08200), Lpd (P00391), MetE (P25665), MetQ/YaeC (P28635), NusA (P03003), OmpA (P02934), OppA (P23843), OxyR (P11721), phenylalanyl-tRNA synthetase beta-subunit (P07395), phosphate import ATP-binding protein (P07655), phosphoribosylaminoimidazole synthetase (P08178), phosphoribosylaminoimidazole-succinocarboxamide synthetase (P21155), phosphotransferase system enzyme I (P08839), PhoU (P07656), porin protein E (P02932), P-specific transport protein (P06128), pyruvate kinase I (P14178), SdhB (P07014), SerA (P08328), serine hydroxymethyltransferase (P00477), SspA (P05838), succinyl-CoA synthetase (P07459), Tpx (P37901), trigger factor (P22257), TufB (P02990), and UgpB (P10904). We are very grateful to Neeraj Korde for automating our database searches. Peptide Mass Fingerprinting data were provided by the Michigan Proteome Consortium (http://www.proteomeconsortium.org, which is supported in part by funds from The Michigan Life Sciences Corridor. We thank Prof. Dr. Fritz Scholz for helpful discussion on the topic. We would also like to thank Dr. James Bardwell for many helpful suggestions and for critically reading this manuscript. The National Institutes of Health Grant GM065318 and a Burroughs Wellcome Fund Career Award to UJ supported this work. Conflicts of interest. The authors have declared that no conflicts of interest exist. Author contributions. UJ conceived and designed the experiments. LIL performed the experiments. LIL and UJ analyzed the data and wrote the paper. Academic Editor: Joan Valentine, University of California, Los Angeles Citation: Leichert LI, Jakob U (2004) Protein thiol modifications visualized in vivo. PLoS Biol 2(11): e333. 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Reaction mechanism of GTP cyclohydrolase I J Mol Biol 2003 326 503 516 12559918 Schirch V Hopkins S Villar E Angelaccio S Serine hydroxymethyltransferase from Escherichia coli Purification and properties J Bacteriol 1985 163 1 7 3891721 Tame JR Dodson EJ Murshudov G Higgins CF Wilkinson AJ The crystal structures of the oligopeptide-binding protein OppA complexed with tripeptide and tetrapeptide ligands Structure 1995 3 1395 1406 8747465 Varghese S Tang Y Imlay JA Contrasting sensitivities of Escherichia coli aconitases A and B to oxidation and iron depletion J Bacteriol 2003 185 221 230 12486059 Wagner T Shumilin IA Bauerle R Kretsinger RH Structure of 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase from Escherichia coli Comparison of the Mn(2+)*2-phosphoglycolate and the Pb(2+)*2-phosphoenolpyruvate complexes and implications for catalysis J Mol Biol 2000 301 389 399 10926516 Watson WH Jones DP Oxidation of nuclear thioredoxin during oxidative stress FEBS Lett 2003 543 144 147 12753922 Yamazaki D Motohashi K Kasama T Hara Y Hisabori T Target proteins of the cytosolic thioredoxins in Arabidopsis thaliana Plant Cell Physiol 2004 45 18 27 14749482 Yano H Wong JH Lee YM Cho MJ Buchanan BB A strategy for the identification of proteins targeted by thioredoxin Proc Natl Acad Sci U S A 2001 98 4794 4799 11274350 Zheng M Aslund F Storz G Activation of the OxyR transcription factor by reversible disulfide bond formation Science 1998 279 1718 1721 9497290 Zhou ZS Peariso K Penner-Hahn JE Matthews RG Identification of the zinc ligands in cobalamin-independent methionine synthase (MetE) from Escherichia coli Biochemistry 1999 38 15915 15926 10625458

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==== Front PLoS BiolPLoS BiolpbioplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 1550287010.1371/journal.pbio.0020336Research ArticleCell BiologyMicrobiologyMolecular Biology/Structural BiologyEubacteriaOxidative Stress Inactivates Cobalamin-Independent Methionine Synthase (MetE) in Escherichia coli Oxidative Stress Inactivates MetEHondorp Elise R 1 Matthews Rowena G [email protected] 1 2 1Department of Biological Chemistry, University of MichiganAnn Arbor, MichiganUnited States of America2Biophysics Research Division and Life Sciences Institute, University of MichiganAnn Arbor, MichiganUnited States of America11 2004 5 10 2004 5 10 2004 2 11 e33614 4 2004 2 8 2004 Copyright: © 2004 Hondorp and Matthews.2004This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. Protein Thiol Modifications Visualized In Vivo Shut Down, Don't Stress Out In nature, Escherichia coli are exposed to harsh and non-ideal growth environments—nutrients may be limiting, and cells are often challenged by oxidative stress. For E. coli cells confronting these realities, there appears to be a link between oxidative stress, methionine availability, and the enzyme that catalyzes the final step of methionine biosynthesis, cobalamin-independent methionine synthase (MetE). We found that E. coli cells subjected to transient oxidative stress during growth in minimal medium develop a methionine auxotrophy, which can be traced to an effect on MetE. Further experiments demonstrated that the purified enzyme is inactivated by oxidized glutathione (GSSG) at a rate that correlates with protein oxidation. The unique site of oxidation was identified by selectively cleaving N-terminally to each reduced cysteine and analyzing the results by liquid chromatography mass spectrometry. Stoichiometric glutathionylation of MetE by GSSG occurs at cysteine 645, which is strategically located at the entrance to the active site. Direct evidence of MetE oxidation in vivo was obtained from thiol-trapping experiments in two different E. coli strains that contain highly oxidizing cytoplasmic environments. Moreover, MetE is completely oxidized in wild-type E. coli treated with the thiol-oxidizing agent diamide; reduced enzyme reappears just prior to the cells resuming normal growth. We argue that for E. coli experiencing oxidizing conditions in minimal medium, MetE is readily inactivated, resulting in cellular methionine limitation. Glutathionylation of the protein provides a strategy to modulate in vivo activity of the enzyme while protecting the active site from further damage, in an easily reversible manner. While glutathionylation of proteins is a fairly common mode of redox regulation in eukaryotes, very few proteins in E. coli are known to be modified in this manner. Our results are complementary to the independent findings of Leichert and Jakob presented in the accompanying paper (Leichert and Jakob 2004), which provide evidence that MetE is one of the proteins in E. coli most susceptible to oxidation. In eukaryotes, glutathionylation of key proteins involved in protein synthesis leads to inhibition of translation. Our studies suggest a simpler mechanism is employed by E. coli to achieve the same effect. Under conditions of oxidative stress, methionine auxotrophy in E. coli is a result of glutathionylation of the enzyme that catalyzes the final step of methionine biosynthesis (MetE) ==== Body Introduction As a consequence of living in an aerobic world, organisms face the challenge of maintaining a favorable cellular redox status. Oxidative stress is caused by imbalances between the production and disposal of reactive oxygen species, which can damage proteins, lipids, and nucleic acids. Bacteria encounter reactive oxygen intermediates that are generated as byproducts of aerobic metabolism or during challenge by the immune systems of hosts. Thus, understanding the effects of oxidative stress on the cell, as well as elucidating cellular defense mechanisms, is of considerable interest. Over the past several decades, multiple observations in Escherichia coli have been reported that collectively suggest a link between oxidative stress, methionine limitation, and the enzyme that catalyzes the final step in methionine biosynthesis, cobalamin-independent methionine synthase (MetE). In E. coli, the sulfur of methionine comes from cysteine, which in turn obtains the sulfur group from sulfate via the sulfate assimilation pathway (Figure 1). In catalyzing the formation of methionine, MetE lies at the intersection between the methyl cycle and the one-carbon pathway. Several studies involving cellular adaptations to stress conditions have reported unusual expression of MetE under stress conditions. Following the transition from anaerobic to aerobic growth in minimal medium, wild-type E. coli cells were found to rapidly induce MetE to a level comprising almost 5% of the total cellular protein (Smith and Neidhardt 1983). Similarly, MetE was massively overexpressed in a temperature-sensitive mutant of groEL. GroEL is a heat-shock protein that is instrumental in helping to correctly fold cellular proteins. When the groEL strain growing in rich medium was shifted to the nonpermissive temperature, MetE was the major soluble protein synthesized (Horwich et al. 1993). The additional inability of this strain to grow on minimal medium suggests that MetE may not be correctly functioning in these cells. Furthermore, Candida albicans MetE has been found to be heat and estrogen inducible, and is believed to play an important role in the yeast stress response and virulence (Burt et al. 1999). The basis for the overproduction of MetE in all of these experiments has been unclear. Figure 1 Pathways of Methionine Biosynthesis and One-Carbon Metabolism in E. coli In addition, several independent researchers have reported an association between stress conditions and methionine limitation in E. coli. Early studies found that strains lacking the genes coding for the manganese and iron superoxide dismutases (sodA and sodB, respectively) were sensitive to oxidative challenge and had an oxygen-dependent growth requirement for the sulfur-containing amino acids cysteine and methionine (Carlioz and Touati 1986; Benov et al. 1996), which was attributed to leakage of sulfite from the cell. In trxA grxA (thioredoxin 1 and glutaredoxin 1) and grxA grxB grxC gor (glutaredoxins 1, 2, and 3 and glutathione reductase) strains of E. coli, cells required cysteine or methionine for growth, presumably because of oxidative inactivation of 3′-phosphoadenylylsulfate (PAPS) reductase (Russel et al. 1990; Vlamis-Gardikas et al. 2002; Lillig et al. 2003). Similarly, the growth of E. coli was dramatically slowed following a shift to higher temperature, unless the medium was supplemented with methionine (Ron and Davis 1971). This heat-induced methionine auxotrophy was traced to temperature sensitivity of homoserine transsuccinylase (MetA), the enzyme that catalyzes the first step in methionine biosynthesis (Ron and Davis 1971; Ron and Shani 1971; Gur et al. 2002); however, MetE has also been subsequently identified as a thermolabile protein in heat-treated E. coli (Mogk et al. 1999). It is intriguing that MetE was a major aggregation-prone protein at a temperature (45 °C) well below the melting temperature of the purified protein (55 °C) (E. Hondorp and R. Matthews, unpublished data). Aerobic heat shock appears to be associated with oxidative stress (Benov and Fridovich 1995), suggesting that the aggregation of MetE might relate to oxidative stress, rather than heat per se. Though oxidative stress undoubtedly has multiple effects, taken together, these observations suggest that MetE may be inactivated by biological oxidants, thereby decreasing the production of methionine within the cell. Methionine biosynthesis involves several enzymes (Figure 1); the final step is catalyzed by either cobalamin (B12)-independent methionine synthase (the metE gene product) or B12-dependent methionine synthase (the metH gene product). Both enzymes employ a catalytic zinc to transfer a methyl group from methyltetrahydrofolate to L-homocysteine to form methionine (equation 1). While mammals have only the MetH homolog, organisms that do not synthesize or transport vitamin B12 (e.g., yeast, fungi, and plants) use only the B12-independent isoform (Matthews 1984). The E. coli genome contains both metE and metH but they are differentially expressed: functional MetH is only expressed in the presence of B12, which also serves to repress MetE expression. In the absence of exogenously supplied B12, MetE is the sole source of de novo methionine synthesis (Greene 1996). In this study, we investigated the effects of oxidative stress on MetE and methionine availability within the cell. Transient oxidative stress conditions induced by diamide were found to elicit a methionine auxotrophy in E. coli growing in minimal medium, which was traced to an effect on MetE. In vitro experiments demonstrated that oxidized glutathione (GSSG) reversibly oxidizes MetE at a rate that correlates with enzyme inactivation. GSSG was found to specifically glutathionylate MetE and induce a conformational change. In vivo thiol-trapping experiments provide direct evidence that MetE is readily oxidized in cells experiencing a variety of oxidative stress conditions. Moreover, oxidation of MetE is associated with a cellular dependence upon methionine for growth, suggesting that oxidant-mediated inactivation of MetE may have a profound impact on the stressed cell. Results Methionine Becomes Limiting for Growth in Cells Subjected to Oxidative Stress Previous observations have suggested a link between oxidative stress and a growth requirement for methionine in E. coli. We investigated the duration of growth lags induced by oxidative stress in cells growing in glucose-minimal medium containing or lacking methionine. In the experiment shown in Figure 2A and 2B, oxidative stress was induced by addition of the thiol oxidant diamide, which rapidly penetrates cells and oxidizes intracellular thiols. Diamide treatment does not kill cells, but results in an abrupt arrest of growth for a length of time that is dependent on the initial concentration of diamide. Cells resume normal growth once the thiol status is restored (Kosower and Kosower 1995). E. coli cultures logarithmically growing in glucose-minimal medium with and without methionine experienced a lag in growth upon challenge with diamide (Figure 2A). However, cells grown in medium containing methionine resumed growth significantly faster than those needing to synthesize methionine. Higher diamide concentrations increased the duration of the growth lag and augmented the effect of methionine on resumption of growth. Figure 2 Methionine Limitation during Oxidative Stress Induced by Diamide and H2O2 (A) Cells of wild-type E. coli strain W3110 growing exponentially in glucose-minimal medium were diluted into the same medium with (filled circles) or without (open circles) 0.2 mM L-methionine. Cultures were allowed to grow to an OD600 of approximately 0.5, and then diamide was added to a final concentration of 2 mM (red) or 3 mM (blue), and growth was monitored. (B) Cultures were grown as in (A), except that they were diluted into medium with (filled circles) or without (open circles) 0.2 mM L-homocysteine. At an OD600 of approximately 0.5, diamide (final concentration, 2 mM) was added to one set of cultures (red). (C) Cells of wild-type E. coli strain W3110 were grown in the presence or absence of L-methionine as described in (A). At an OD600 of approximately 0.2, H2O2 was added to one set of cultures (green) to a final concentration of 3 mM (a concentration found to be high enough to induce a significant slowing of growth). (D) Cultures were grown as in (C) except that they were diluted into medium with (filled circles) or without (open circles) 0.2 mM L-homocysteine. Note the different time scales between (A,B) and (C,D). The biosynthesis of methionine involves several reactions (see Figure 1). If a step upstream of the reaction catalyzed by MetE were responsible for methionine limitation during diamide treatment, addition of homocysteine to the medium should decrease the growth lag imposed by diamide. No effect of homocysteine addition was seen on the duration of the growth lag in diamide-treated cells (Figure 2B), even though homocysteine can support the growth of cells that lack enzymes upstream of MetE in the biosynthetic pathway (Urbanowski and Stauffer 1989). Similar results were obtained when hydrogen peroxide (H2O2) was used to induce oxidative stress. Addition of methionine to the medium significantly decreased the growth lag produced by H2O2 treatment (Figure 2C), while homocysteine was ineffective and even amplified the lag (Figure 2D). The mechanism by which homocysteine appears to have potentiated the H2O2 stress is not known; however, it was clearly not protective. Taken together, the growth experiments suggest that methionine becomes limiting under oxidative stress, and that the bottleneck to de novo biosynthesis of methionine lies in the terminal step catalyzed by MetE. Inactivation of MetE In Vitro Is Correlated with Cysteine Oxidation The methionine auxotrophy observed under oxidative stress conditions could reflect an increased demand for methionine or a decreased rate of de novo synthesis due to inactivation of MetE. To distinguish between these possibilities, we assayed the activity of the purified enzyme upon challenge with GSSG (Figure 3). Nearly complete loss of methionine synthase activity was observed following addition of GSSG. This process was readily reversible upon incubation with a reductant such as dithiothreitol (DTT) or reduced glutathione (GSH) (data not shown). Activity loss was correlated with thiol oxidation, as assessed by dithio-1,4-nitrobenzoic acid (DTNB) titration of the trichloroacetic acid (TCA)–precipitated protein. GSSG treatment led to thiol oxidation, which proceeded with approximately the same rate constant (0.06 min−1) as loss of activity (0.07 min−1). Figure 3 In Vitro Treatment of MetE with GSSG Leads to Loss of Activity and Cysteine Oxidation At time zero, GSSG (final concentration, 5 mM) was added to 50 μM MetE in 100 mM Tris chloride (pH 7.2). MetE activity was measured in samples removed at the indicated time points as described in the Materials and Methods. These assays are made over a 30-s time period and initiated by addition of MetE, so that oxidation of MetE by GSSG is minimal during the time required for measurement. Also, at the indicated times, an aliquot of the protein was precipitated with TCA, and the extent of cysteine oxidation was determined by DTNB titration. MetE activity loss (red) occurred with a rate constant of 0.07 min−1, while cysteine oxidation (blue) occurred with a rate constant of 0.06 min−1. While the rate of thiol oxidation could be measured with DTNB, the absolute number of cysteines oxidized was ambiguous. MetE contains seven cysteines, yet DTNB titration of the fully reduced enzyme gave values between 7.2 and 9.0 cysteines for different protein preparations, and oxidation of MetE led to an average decrease of 1.8 cysteines per mol. Thus it was important to use independent means to determine the number of cysteines oxidized by GSSG. Oxidation of MetE by GSSG would be expected to proceed by initial formation of a mixed disulfide between MetE and glutathione (equation 2). An inter- or intramolecular disulfide bond could then be generated by attack on the mixed disulfide by a second cysteine residue. These two possibilities can be distinguished by using mass spectrometry to determine the molecular mass of the oxidized protein. The reduced protein was found to be 84,530 ± 6 Da (expected mass, 84,542, −0.01% deviation), whereas the mass of the protein oxidized by GSSG was 84,835 ± 8 Da. The mass difference of 305 ± 10 Da between oxidized and reduced MetE is consistent with the addition of a single glutathione adduct (expected mass, 306 Da). Formation of an intramolecular disulfide bond would only decrease the mass by 2 Da, which would not be detected by mass spectrometry in such a large protein. To obtain evidence for stoichiometric glutathionylation of MetE, the oxidized protein was reduced with DTT to release the glutathione adduct. The DTT and GSH were separated from the protein using a Microcon concentrator. The filtrate was assayed for GSH using fluorescamine, which reacts with primary amines to form a fluorescent product. The reduced protein did not release GSH (0.0 mol GSH per mol protein) while oxidized protein released 0.9 mol GSH per mol of protein. Thus both mass spectrometry and the fluorescamine assay were consistent with glutathionylation of MetE by GSSG. MetE Is Glutathionylated at Cysteine 645 In order to determine the site of oxidation and verify that an intramolecular disulfide was not being formed concomitant with oxidation, we performed disulfide mapping experiments based on the method of Wu and Watson (1997). MetE was cyanylated with 1-cyano-4-dimethylamino-pyridinium tetrafluoroborate (CDAP) under denaturing conditions at pH 3, and then cleaved immediately N-terminally to the cyanylated cysteines in 1 N ammonia, resulting in the formation of 2-iminothiazolidine-4-carboxyl (itz) peptides (Figure 4). Cleavage will not occur at cysteines that have been protected from cyanylation by oxidation. Following cleavage, any disulfide bonds were reduced with DTT and the resulting peptide fingerprint analyzed by liquid chromatography mass spectrometry (LC-MS). Figure 4 Strategy for Mapping of Disulfide Bonds Formed during Oxidative Inactivation of MetE In Vitro Protein sulfhydryls are cyanylated with CDAP under denaturing, acidic conditions, and then cleaved N-terminally to each cyanylated cysteine with 1 N ammonia. This treatment results in an N-terminal fragment and a series of itz peptides. The mixture is then completely reduced with DTT and analyzed by LC-MS. Cleavage does not occur adjacent to cysteines that are oxidized. Thus sites of oxidation can be deduced from the mass fingerprint. This approach allows for relatively straightforward mapping of disulfide bonds. The acidic pH employed minimizes the possibility of disulfide bond scrambling. Furthermore, the ability to cleave N-terminally to each cyanylated cysteine allows cleavage between closely spaced, even adjacent, cysteines. These factors provide a significant advantage over traditional methods of peptide mapping, particularly for MetE, which contains cysteines spaced only two residues apart at the active site (cysteines 643 and 645). Moreover, for large proteins, most proteolytic and chemical cleavage methods have the potential to generate numerous peptides, whereas this approach produces a more limited number. LC-MS analysis of the cyanylated and cleaved reduced MetE allowed for identification of all but one of the expected peptides (Figure 5A; Table 1). High performance liquid chromatography (HPLC) peaks with masses corresponding to each of the expected peptides were observed, except for the dipeptide itz643–644, which is too small to be detected. Cyanylated cysteines can also undergo a β-elimination side reaction instead of cleavage. Susceptibility of cyanylated cysteines to β-elimination depends on the neighboring residues (Wu and Watson 1998). In MetE, cysteine 726 readily formed the β-elimination product I, and β-elimination at cysteine 560 produced a small amount of product H. Identification of these side products provides additional evidence that cysteines 560 and 726 were reduced and available for cyanylation. Disulfide mapping of a mutant MetE, MetEC>A1–4, in which cysteines 323, 353, 516, and 560 were changed to alanines, further confirmed the mass assignments. Only peaks corresponding to cleavage next to the three remaining cysteines (643, 645, and 726) were observed (Figure 5B; Table 1). Figure 5 Disulfide Mapping of Reduced MetE Reduced MetE should be cleaved N-terminally to each of its seven cysteines to generate eight protein fragments as shown in the diagram, where peptide A comprises residues 2–322, B is itz323–352, C is itz353–515, D is itz516–559, E is itz560–642, F is itz645–725, and G is itz726–753. It is assumed that itz643–644 is too small to be resolved. β-elimination of cyanylated cysteines can also occur; H is itz516–642 with β-elimination at cysteine 560, and I is itz645–753 with β-elimination at cysteine 726. (A) The HPLC trace at 280 nm for disulfide mapping of fragments derived from cyanylation and cleavage of reduced MetE. Following derivitization, the samples were chromatographed on a C4 reversed-phase column as described in the Materials and Methods. Peaks were assigned from comparison of masses determined by mass spectrometry and predicted masses (see Table 1). (B) The HPLC trace for disulfide mapping of MetEC>A1–4 (green), which only contains cysteines 643, 645, and 726, overlaid with that of the wild-type reduced protein (blue). Peaks corresponding to ABCDE, G, and I were identified from the mass data (see Table 1). The mass for one peak could not be determined, but was assigned as F by comparison to the wild-type trace. Table 1 Mass Spectrometry Data for Disulfide Mapping Experiments The average mass obtained for each peak described in Figures 5 and 6 is listed. The β-elimination products were found to react with the low levels of acetic acid present in the LC-MS solvent to generate an acetate (Ac) adduct. Masses for both the β-elimination product and the Ac adduct were observed as indicated. Peak G consistently contains two unidentified fragments in addition to itz726–752 Disulfide mapping data for the oxidized protein following cyanylation, cleavage, and reduction with DTT (Figure 6A; Table 1), were similar to results for the reduced protein, suggesting that most of the cysteines in MetE remained reduced. Peptides A, B, C, D, E, and H provide evidence that cysteines 323, 353, 516, 560, and 643 were reduced. Peptides G and J indicate that cysteine 726 was reduced. The pattern is consistent with the initial oxidation occurring at cysteine 645 alone. Peptide J is the expected product obtained when MetE is initially oxidized at cysteine 645, with β-elimination at cysteine 726, and is then reduced. Figure 6 Disulfide Mapping of Oxidized MetE Reveals That Cysteine 645 Is Glutathionylated (A) LC-MS analysis of fragments generated by cyanylation, cleavage, and total reduction (see Figure 4) of GSSG-oxidized (red) and reduced (blue) MetE. For oxidized MetE, peaks corresponding to F and I were not found. Instead, a peak was observed with a mass corresponding to J, itz643–753, with one reduced uncyanylated cysteine and β-elimination at the other cysteine. As discussed in the text, β-elimination occurs preferentially at cysteine 726 in the reduced enzyme, and we infer that the reduced uncyanylated cysteine is cysteine 645. (B) LC-MS analysis of the cleavage products in (A) prior to DTT reduction (black) or following total reduction (red). Prior to DTT reduction, a peak is observed that has a mass corresponding to that expected for K, itz643–753, with a glutathione adduct at one cysteine and β-elimination at the other cysteine. As discussed in the text, β-elimination occurs preferentially at cysteine 726 in the reduced enzyme, and we infer that the glutathionylated cysteine is cysteine 645. The peak containing fragment J, the product of DTT reduction, is no longer apparent. The broad peak, indicated by the asterisk, appeared to contain a mixture of fragments with masses that could not be deconvoluted. These were attributed to disulfide-linked byproducts formed under the basic cleavage conditions prior to reduction with DTT. LC-MS analysis of the oxidized protein fragments prior to DTT reduction yielded an almost identical pattern to that obtained after reduction (Figure 6B; Table 1). However, a peptide, K, corresponding to itz643–753 with a glutathione adduct and β-elimination, was observed instead of J. It is not possible to distinguish which cysteine, 645 or 726, is glutathionylated and which has undergone β-elimination based simply on the masses of peptides J and K. However, the presence of peptide G in all traces at a level similar to that of reduced MetE indicates that cysteine 726 must be reduced. Moreover, the high susceptibility of cysteine 726 to undergo β-elimination (as evidenced by peak I in Figure 5A)—contrasted with the complete lack of a β-elimination product at cysteine 645—argues that β-elimination occurs at cysteine 726 in the oxidized enzyme and glutathionylation occurs at cysteine 645. In order to confirm that cysteine 645 is the only site of oxidation, the inactivation of various MetE mutants was analyzed. MetEC>A1–4 contains only cysteine 645 and the zinc-binding ligands, cysteines 643 and 726; the latter ligands are required for catalytic activity. This mutant has approximately 90% of the activity of the native protein, and the kinetics of inactivation by GSSG are nearly identical to that of the wild-type protein (Figure 7A). However, mutation of cysteine 645 to alanine (MetEC>A5) dramatically altered the course of inactivation by GSSG. The slow partial loss of activity seen with MetEC>A5 may reflect weakening of zinc binding for the mutant enzyme, allowing glutathione to compete with MetE for zinc. Figure 7 Cysteine 645 Is Critical for Sensitivity of MetE to Oxidation (A) The activity of 50 μM wild-type MetE (black), MetEC>A1–4 (blue), and MetEC>A5 (red) was assayed following addition of GSSG to a final concentration of 5 mM. 100% activity refers to the specific activity of the fully reduced enzyme prior to GSSG addition (27 min−1 for wild-type, 24 min−1 for mutant). (B) The change in absorbance at 325 nm was used to monitor diamide reduction after addition of 500 μM diamide to buffer (light blue), 50 μM wild-type MetE (black), and 50 μM MetEC>A5 (red). (C) Scheme for the reaction of MetE with diamide. In the first step, attack of cysteine 645 on diamide forms an inactive but stable complex. In vivo, where GSH is present in high concentration, attack of GSH leads to the formation of MetE-S-SG and reduced diamide. Further evidence implicating cysteine 645 as the site of sensitivity to oxidation was obtained by monitoring the oxidation of MetE by diamide. The reaction of diamide with thiols can be followed at 325 nm, where diamide absorbs strongly, but the reduced product does not (Kosower and Kosower 1995). Addition of MetE to diamide results in a rapid decrease in absorbance at 325 nm (Figure 7B). However, MetEC>A5 does not appear to be oxidized by diamide. This observation suggests that diamide oxidation of MetE in vitro involves an initial attack of cysteine 645 on diamide to form an inactive covalent complex (Figure 7C). In the presence of accessible thiols, such as the millimolar concentrations of GSH present in vivo, this complex can then readily react to form glutathionylated MetE and reduced diamide. Thus, although diamide oxidation does not result in glutathionylation of MetE in the absence of glutathione, this experiment demonstrates the extreme reactivity of cysteine 645 to oxidizing agents. Glutathionylation of MetE Induces a Conformational Change In order to further characterize the effects of GSSG oxidation on MetE, the reduced and oxidized forms of the protein were subjected to gel filtration. The elution of glutathionylated MetE was markedly shifted in comparison to that for the reduced protein (Figure 8A). The change in hydrodynamic radius seen upon oxidation suggested that GSSG oxidation had altered the conformation of MetE. Therefore, to confirm a conformational difference between reduced and oxidized MetE, we analyzed the two forms of the protein by circular dichroism (CD) spectroscopy and limited tryptic digestion. The similarity between the CD spectra of oxidized and reduced MetE suggests that the oxidized protein has not undergone a gross structural rearrangement and is still largely intact (Figure 8B). However, the deviation in the two spectra between 210 and 230 nm is consistent with a conformational change upon glutathionylation. Tryptic digestion of oxidized MetE appeared to occur with different kinetics compared to that of the reduced protein (Figure 8C). The oxidized protein was more stable against tryptic digestion than the reduced form, again consistent with a conformational change upon MetE oxidation. Figure 8 Glutathionylation of MetE Induces a Conformational Change (A) Gel filtration analysis of reduced (blue), GSSG-oxidized (red), or a mixture of reduced and GSSG-oxidized (black) MetE. (B) Far UV CD spectra of reduced (blue) and GSSG-oxidized (red) MetE. (C) SDS-PAGE analysis of a digestion of native MetE with 0.08% trypsin. The band corresponding to the molecular weight of the holo-protein is marked with an asterisk. (D) GroEL (2 μM) was incubated with 1 μM reduced (blue) or GSSG-oxidized (red) MetE and the proteins were then subjected to gel filtration. MetE has previously been shown to be an in vivo substrate of the chaperone GroEL (Houry et al. 1999). Since oxidation of MetE was found to induce a conformational change, we tested whether GroEL bound preferentially to either form of MetE. GroEL and reduced MetE were incubated together and then injected onto a gel filtration column (Figure 8D). Separate peaks corresponding to GroEL and reduced MetE were observed, indicating that reduced MetE did not associate with GroEL. However, when the glutathionylated protein was incubated with GroEL, only the GroEL peak was observed. The GroEL complex is large enough (approximately 800 kDa) that binding of MetE would not be expected to shift the elution time significantly. Thus, the disappearance of the peak corresponding to oxidized MetE strongly suggests that GroEL specifically binds glutathionylated MetE, leading to coelution. The Equilibrium Constant for Glutathionylation of MetE Is Consistent with MetE Oxidation In Vivo To gain a better understanding of the thermodynamic sensitivity of MetE to oxidation, we measured the equilibrium constant for formation of the inactive mixed disulfide. MetE was incubated with varied ratios of GSH and GSSG until equilibrium was reached, and then activity was assayed (Figure 9A). The equilibrium constant Kmix (equation 2) was determined to be 1.4 and found to be independent of GSH concentration. If formation of an intramolecular disulfide bond were responsible for MetE inactivation, the equilibrium constant should be dependent on the GSH concentration (Gilbert 1995). The independence of the equilibrium constant from GSH concentration provides further support that glutathionylation is the mechanism of inactivation. Figure 9 The Equilibrium Constant for MetE Oxidation Is Consistent with MetE Inactivation In Vivo (A) MetE was incubated with the indicated [GSH]/[GSSG] ratios at 1.1 mM (yellow), 2.3 mM (red), 4.6 mM (green), or 9.1 mM (blue) GSH until equilibrium was reached, as judged by a constant level of activity with time. MetE activity was then assayed to determine the relative amount of active reduced enzyme. The equilibrium constant, Kmix (see equation 2) was determined to be 1.4 from the plot of relative MetE activity versus [GSH]/[GSSG]. This value was independent of the GSH concentration and dependent only on the [GSH]/[GSSG] ratio, consistent with glutathionylation as the mechanism of inactivation. (B) Cellular redox potentials are often referenced in terms of the GSH–GSSG couple, which depends upon [GSH]2/[GSSG]. Since inactivation of MetE occurs via glutathionylation (which is dependent on [GSH]/[GSSG]), a redox potential (which is dependent on [GSH]2/[GSSG]) for inactivation of MetE (by equilibrium titration with GSH/GSSG) cannot be determined. In order to provide a physiological context for the Kmix determined in vitro, the equilibrium fraction of MetE expected to be active at different glutathione concentrations, 1 mM (red), 2 mM (green), and 5 mM (blue), was determined as a function of the cellular potential as described in the text. The regions designated “normal growth” and “oxidative stress” were inferred from published estimates of typical redox potentials in cells experiencing these conditions (Gilbert 1990). In order to estimate the extent that MetE inactivation may be expected in vivo, both the redox potential, which is determined by the [GSH]2/[GSSG] ratio, and the total glutathione concentration in the cell ([GSH] + 2[GSSG]) need to be taken into account. Based on the equilibrium constant for GSSG oxidation, MetE activity at equilibrium was calculated for typical cellular glutathione concentrations (1–5 mM [Kosower and Kosower 1978; Åslund et al. 1999]) at various potentials as determined by the GSH–GSSG redox couple (Figure 9B). (At each potential, Eh, the ratio of [GSH]2/[GSSG] was calculated from the Nernst equation: where Eo is –0.252 V at pH 7.2 (using a value of –0.24 V at pH 7.0 with an adjustment of –6.2 mV per 0.1 increase in pH [Schafer and Buettner 2001]), R is 8.314 V·C/mol·K, T is 310 K, n is 2, and F is 96,485 C/mol. Based on a total glutathione concentration ([GSH] + 2[GSSG]) of 1, 2, or 5 mM, and the calculated [GSH]2/[GSSG], [GSH] and [GSSG] can be determined at each cellular potential. The ratio of [GSH]/[GSSG] may then be used to calculate the relative amount of MetE activity expected at equilibrium by where Kmix is 1.4.) For E. coli growing under normal conditions (–250 mV to –280 mV [Hwang et al. 1992]), more than 97% of the cellular MetE would be expected to be reduced and active. However, in a severely oxidizing environment (greater than –150 mV), inactive MetE may accumulate to an appreciable level. In comparison, Hsp33, which is a redox-regulated chaperone, has a midpoint potential of –170 mV (Jakob et al. 1999). Therefore, thermodynamically, under conditions where Hsp33 is 50% active, MetE would be expected to be 29%–54% oxidized for glutathione concentrations of 1–5 mM. Although GSSG activates Hsp33 via formation of an intramolecular disulfide bond and inactivates MetE by glutathionylation, this suggests that both proteins may have similar sensitivities to oxidation. However, these calculations assume that the oxidation state of MetE is in equilibrium with GSH and GSSG. Deviations from theoretical values could occur if MetE were oxidized or reduced in vivo by a mechanism other than simple thiol-disulfide exchange. Comparison of the actual amount of oxidized MetE to measurements of the physiological concentrations of GSH and GSSG could therefore provide an indication of the in vivo mechanism of oxidation. Nevertheless, while MetE inactivation by GSSG may be energetically favorable under oxidizing conditions, the apparently slow kinetics make it difficult to conclude whether or not oxidation actually occurs in vivo. MetE Is Oxidized In Vivo during Oxidative Stress Thus, to determine whether our observations are physiologically relevant, we needed to develop a method whereby the oxidation of MetE could be directly observed in vivo. To this end, thiol-trapping experiments were performed as outlined in Figure 10A. Cells growing under normal or oxidative conditions were briefly incubated with iodoacetamide, which is able to quickly penetrate the cell membrane and efficiently alkylate all available reactive thiols. The proteins were precipitated with TCA, and oxidized cysteines were reduced with DTT. The sulfhydryls exposed under these conditions were trapped with iodoacetic acid, which adds a negative charge to the protein for each cysteine trapped. Differently charged forms of the protein were separated by vertical slab isoelectric focusing, and MetE was visualized by immunoblotting. This method of analysis should readily distinguish between glutathionylated MetE (one additional negative charge as compared to reduced MetE) and MetE with an intramolecular disulfide bond (two additional negative charges as compared to reduced MetE). Glutathionylated MetE prepared in vitro is more acidic than fully reduced MetE, as indicated by comparing lanes 4 and 3 in Figure 10B. Figure 10 In Vivo Thiol-Trapping Experiments (A) The strategy for in vivo thiol trapping involves brief incubation with iodoacetamide, which is able to efficiently alkylate all available thiols. Oxidized cysteines in the TCA-precipitated proteins are reduced, and the sulfhydryls exposed are then trapped with iodoacetic acid, which adds a negative charge. The two forms of the protein are separated by isoelectric focusing, and MetE is visualized by immunoblotting. Oxidized MetE results in a more acidic band on the gel than reduced MetE. (B) Thiol-trapping experiments were performed on a trxA gor strain (WP843) of E. coli (lane 2) as well as the isogenic wild-type strain (DHB4) (lane 1). The position of MetE on the gel may be compared with that of the reduced (lane 3) and GSSG-oxidized (lane 4) purified protein. In vivo thiol-trapping experiments were performed with logarithmically growing wild-type and trxA gor strains of E. coli. Strains mutant in genes for one of the thioredoxins (trxA) and glutathione reductase (gor) are impaired in their ability to reduce disulfide bonds, resulting in a highly oxidizing cytoplasmic environment (Prinz et al. 1997). Comparison of the thiol status of MetE in a trxA gor mutant strain (WP843) with that in the isogenic wild-type strain (DHB4) revealed that approximately 50%–60% of the MetE in the trxA gor strain was oxidized in vivo (Figure 10B). Moreover, the shift in the isoelectric point for MetE that was oxidized in vivo is identical to the mobility of glutathionylated MetE prepared in vitro. E. coli strains containing mutations in trxB (which specifies thioredoxin reductase) and gor experience more severe disulfide stress than trxA gor strains (Prinz et al. 1997). Cells require the presence of DTT in the medium to grow at rates comparable to wild-type strains; growth rapidly halts when DTT is removed. This strain cannot be grown in minimal medium, where wild-type MetE is expressed, so a trxB gor strain containing a plasmid specifying MetE (which expresses MetE independently of the cellular methionine concentration) was grown in Luria-Bertani (LB) medium containing DTT. Oxidized MetE was observed within 90 min of DTT removal, while MetE was fully reduced in the presence of DTT (Figure 11A and 11B). Again, the shift in the isoelectric point of MetE oxidized in vivo was indistinguishable from that of glutathionylated MetE prepared in vitro. Together, these experiments provide evidence that MetE is glutathionylated during steady-state growth in two different strains of E. coli that are intrinsically oxidatively stressed. Figure 11 MetE Is Oxidized When E. coli Experience Oxidative Stress (A) Growth of a trxB gor strain (WP778) containing a plasmid expressing MetE was monitored by measurement of the OD600 during growth in LB medium supplemented with 4 mM DTT (filled symbols). At an OD600 of approximately 0.4, cells were filtered to remove the DTT, resuspended in fresh LB medium, and growth was monitored (open circles). (B) In vivo thiol-trapping experiments were performed before and after removal of DTT from the medium. Lanes 1 and 7 contain thiol-trapped samples of purified reduced MetE, and lanes 2 and 8 contain thiol-trapped samples of glutathionylated MetE prepared in vitro by treatment of MetE with GSSG. (C) Growth of wild-type E. coli (strain W3110) in glucose-minimal medium was monitored by the OD600 with (filled circles) and without (open circles) the addition of diamide (at OD600 approximately 0.5) to a final concentration of 0.9 mM. (D) In vivo thiol-trapping experiments were carried out to assess the oxidation state of MetE at various times following diamide addition. Lanes 1, 2, and 8 contain thiol-trapped samples of purified reduced and/or diamide-oxidized MetE. In order to establish a direct link between oxidative inactivation of MetE and the methionine auxotrophy observed during transient oxidative stresses, we performed thiol-trapping experiments on cells challenged with diamide. When wild-type E. coli cells growing exponentially in glucose-minimal medium were treated with 0.9 mM diamide, MetE was found to be completely oxidized during the lag in growth (Figure 11C and 11D). Identical levels of reduced MetE reappeared within 60 min of the challenge, just prior to the resumption of normal cellular growth. Thus oxidation of MetE is associated with the cellular methionine limitation (described earlier) that is imposed by addition of diamide. Furthermore, these results are consistent with those of Leichert and Jakob (2004), which independently show that MetE is rapidly oxidized in E. coli (wild-type strain DHB4) within 2 min of diamide treatment. Discussion Protein glutathionylation has been increasingly recognized as an important mode of regulation in eukaryotes; however, in E. coli, only the activities of OxyR and PAPS reductase have been reported to be modulated by formation of mixed disulfides with glutathione (Kim et al. 2002; Lillig et al. 2003; Potamitou Fernandes and Holmgren 2004). In this study, we have expanded this list by clearly demonstrating that E. coli MetE is also inactivated by glutathionylation in a manner that is stoichiometric and specific for cysteine 645. Moreover, oxidation of MetE was directly observed in vivo concomitant with a methionine auxotrophy induced by oxidative stress. In general, protein glutathionylation may occur by two different mechanisms. Proteins can undergo thiol-disulfide exchange with GSSG to generate the glutathionylated product (Kmix equilibrium in equation 2). Alternatively, either cysteine residues on the protein or the GSH sulfhydryl may be oxidized to a more reactive intermediate, such as a sulfenic acid, followed by formation of the mixed disulfide. In vivo glutathionylation of proteins via thiol-disulfide exchange is dependent on changes in the [GSH]/[GSSG] ratio, while formation of a reactive intermediate would result in glutathionylation independent of the [GSH]/[GSSG] ratio. Alterations in [GSH]/[GSSG] ratios have been associated with increased protein-glutathione mixed disulfides (Chai et al. 1994a); however, there are also reports of extensive glutathionylation occurring in vivo without a measurable change in GSSG concentration or total glutathione levels (Chai et al. 1994a, 1994b). This has been taken as evidence to support a mechanism involving a reactive sulfhydryl intermediate (Thomas et al. 1995). The ratios of [GSH] to [GSSG] in wild-type (DHB4) and trxA gor strains (FÅ378) growing in LB medium were previously reported to be 223 ± 35 and 18 ± 7, respectively (Åslund et al. 1999). Though our experiments were performed with E. coli growing in minimal medium, assuming that the [GSH]/[GSSG] ratios are comparable, more than 99% of the MetE in the wild-type strain (DHB4) should be reduced. Similarly, based on the equilibrium constant for thiol-disulfide exchange with MetE, only approximately 7% of the MetE is expected to be oxidized in the trxA gor strain (WP843). Yet 50%–60% of the MetE was found to be oxidized in this strain by thiol-trapping experiments. A [GSH]/[GSSG] ratio of less than 1.4 would be required to accumulate that much oxidized MetE (which corresponds to a redox potential above –174 for 5 mM glutathione and above –152 mV for 1 mM glutathione). Moreover, in vitro oxidation of MetE by GSSG is rather slow, further suggesting that glutathionylation of MetE via thiol-disulfide exchange is not the primary mechanism functioning in vivo. Rather, the in vitro and in vivo data for MetE are consistent with initial formation of a reactive intermediate followed by glutathionylation of cysteine 645, resulting in enzyme inactivation. Our results are complementary to the observations of Leichert and Jakob (2004) that MetE is one of the proteins most susceptible to oxidation in a trxA strain. Since E. coli lacking trxA do not experience a substantial amount of cytosolic disulfide bond formation (Prinz et al. 1997), the appearance of oxidized MetE within this environment delineates the extreme sensitivity of the protein to oxidation. The concurrent presence of oxidized peroxide-detoxifying enzymes (alkylhydroperoxide reductase [AhpC] and thioredoxin-linked thiolperoxidase [Tpx]) suggests that endogenously produced reactive oxygen species (e.g., H2O2) accumulate in the trxA strain, since thioredoxin appears to be required to regenerate the reduced thiol status of these proteins. Our preliminary in vitro experiments indicate that glutathionylated MetE is inefficiently reduced by thioredoxin (data not shown), which is not unexpected since glutaredoxins are generally more specific for glutathione-protein adducts (Potamitou Fernandes and Holmgren 2004). Thus, taken together, our study and that of Leichert and Jakob (2004) suggest that the oxidation of MetE observed in trxA strains is mediated by elevated reactive oxygen species, perhaps due to an overwhelmed glutaredoxin system. Leichert and Jakob also observed significant oxidation of MetE in wild-type cells treated with H2O2, further supporting the premise that MetE is acutely vulnerable to reactive oxygen species. Protein glutathionylation has been suggested to play a protective role in oxidative stress. Studies with purified eukaryotic proteins have shown that incubation with oxidants (e.g., H2O2) results in irreversible modification, whereas oxidation in the presence of GSH (as would be expected in vivo) yielded reversible glutathionylation (Klatt and Lamas 2000; Hamann et al. 2002; Mallis et al. 2002). Similarly, we found that addition of an excess of H2O2 to purified MetE appeared to cause irreversible oxidation, presumably because of the formation of sulfinic (–SO2) or sulfonic (–SO3) acid adducts (data not shown). Thus, glutathionylation of MetE following the formation of a reactive intermediate may serve the dual purpose of modulating enzyme activity while protecting the active site from more extensive damage. Reduction by cellular thioreductases could then readily reactivate the enzyme. Our data strongly suggest that glutathionylation is reversible in vivo as well as in vitro. Upon the addition of diamide to cells growing in medium lacking methionine, growth is abruptly halted and cells become limited for methionine. In the experiments shown in Figure 11C and 11D, all of the MetE in the cell is oxidized at the 15- and 30-min time points, whereas it is completely reduced after 60 min, just prior to the resumption of growth. Within this time period, the concentration of MetE within the cell does not appear to change, based on the intensity of the band on the immunoblot. In particular, there is no decrease in intensity of the band corresponding to oxidized enzyme between 15 and 30 min, suggesting that MetE is not being degraded. While a lack of change could indicate a balance between new protein synthesis and degradation, we argue that cells that are not growing and are limited for methionine should have a decreased rate of protein synthesis. If new synthesis of MetE were responsible for the reappearance of reduced MetE, all of the oxidized MetE (estimated at 1% of the total protein or 60 μM MetE) must be degraded and new protein synthesized to the same level between 30 and 60 min. Thus, although we cannot definitively rule out the possibility that the reappearance of reduced MetE is due to new protein synthesis, we feel that it is far more likely that MetE is re-reduced once the diamide in the cell is dissipated. Proteins containing mixed disulfides with glutathione are typically reduced by one of the three glutaredoxin isoforms. The glutaredoxins Grx1 (grxA) and Grx3 (grxC) are structurally distinct from Grx2 (grxB), yet all three proteins are capable of reducing glutathionylated PAPS reductase (Lillig et al. 2003). The levels of Grx2 and Grx3 in E. coli are considerably higher than that of Grx1; however, the three proteins employ different modes of regulation. Grx3 concentrations are relatively constant, whereas OxyR induces Grx1, and levels of Grx2 are growth phase dependent. Grx2 has the highest catalytic activity of the isoforms and has been estimated to contribute up to 80% of the total glutaredoxin activity (Potamitou Fernandes and Holmgren 2004). Since glutaredoxins catalyze the reduction of proteins with high efficiency and specificity, it will be of interest to determine the precise protein or proteins that are responsible for reduction and reactivation of oxidized MetE in vivo. Based on a recent crystal structure of the Thermatoga maritima MetE homolog, cysteine 645 is located at the entrance to the active site (R. Pejchal and M. Ludwig, personal communication). This places the sulfhydryl in direct contact with the solvent, where it is accessible to oxidation and reduction. Glutathionylation of cysteine 645 at the entrance to the active site may sterically prevent substrate binding, leading to the observed inactivation of the enzyme. Addition of the glutathione adduct also appears to correlate with a weakened binding of the catalytic zinc to the enzyme (data not shown). The active site of MetE is located in a cleft formed between two domains (R. Pejchal and M. Ludwig, personal communication). Addition of the glutathione tripeptide to cysteine 645 may provide enough bulk to push the two domains apart, giving rise to the observed conformational change and increased hydrodynamic radius. CD spectroscopy indicates that oxidation does not result in gross unfolding of MetE (see Figure 8B), yet GroEL was found to bind only to the glutathionylated protein, and not to reduced MetE. Separation of the domains upon addition of glutathione could expose hydrophobic surfaces that allow GroEL to recognize and bind to one of the domains, since the holoenzyme (84.5 kDa) is too large to be accommodated within the cavity formed by the GroEL–GroES complex (Xu et al. 1997). Earlier observations identified MetE as an in vivo substrate for GroEL (Houry et al. 1999); here, we showed that GroEL specifically binds to the glutathionylated protein. GroEL binding may be required to present the enzyme for reduction, as well as to prevent oxidized enzyme from aggregating. Alternatively, binding to GroEL could target the inactive enzyme for eventual degradation. Oxidant-mediated inactivation of MetE may have broad implications for the cell. Initiation of translation in E. coli requires formylated methionine, and it has been suggested that depletion of methionine and one-carbon pools could block protein translation (Gold 1988). We postulate that the development of methionine auxotrophy via inactivation of MetE may therefore protect stressed cells by slowing the initiation of protein translation. This could safeguard cells from rapid synthesis of peptides under adverse conditions, allowing cellular processes to attend to managing and detoxifying the stress. Interestingly, the conditions that induce a heat shock response in E. coli also affect the translational capacity of the cell, implicating the rate of protein synthesis in triggering of this stress response (VanBogelen and Neidhardt 1990). Studies with rat hepatocytes provide support for a role of protein glutathionylation in the control of protein synthesis under oxidative stress conditions. Upon the addition of t-butyl hydroperoxide, rapid inhibition of protein translation was observed, accompanied by an increase in the levels of protein mixed disulfides. It was hypothesized that glutathionylation of key proteins involved in protein synthesis was responsible for the inhibition (Latour et al. 1999). Furthermore, peroxide stress has been found to result in reversible inhibition of protein synthesis in Saccharomyces cerevisiae (Shenton and Grant 2003). This effect was irreversible in cells that lack glutathione, suggesting that protein glutathionylation may protect the cellular translational capacity from irreversible damage. Thus, mechanisms for downregulating the rate of protein translation may be employed by a wide spectrum of organisms experiencing oxidative stress. The methionine limitation imposed by oxidative stresses could also play a significant role in bacterial quorum sensing. S-adenosylmethionine, which is directly formed from methionine, is the precursor to autoinducer AI-2 (see Figure 1) (Chen et al. 2002). A growing body of evidence suggests that AI-2 communicates the metabolic state and growth potential of the cell rather than simply providing a density-dependent signal (DeLisa and Bentley 2002; Xavier and Bassler 2003). Moreover, a shift in the metabolic activity of the cell is postulated to be responsible for modulation of AI-2 signaling in response to a host of environmental stresses (DeLisa et al. 2001a, 2001b). Aerobiosis, H2O2 treatment, and heat shock were all found to result in decreased AI-2 levels, while addition of DTT or glucose served to increase the accumulation of AI-2 (DeLisa et al. 2001b). Oxidant-mediated inactivation of MetE imposes a methionine limitation, which would then lower the availability of substrate for AI-2 production. Decreased levels of AI-2 in response to methionine-limiting conditions could provide a simple means of communicating the cellular metabolic potential under stress conditions, thereby helping cells to adapt to a non-ideal or even hostile environment. In conclusion, we have established that MetE, a highly expressed protein in E. coli, is inactivated by glutathionylation of cysteine 645 in vitro. Our results, coupled with the findings of Leichert and Jakob (2004), demonstrate oxidation of MetE in E. coli undergoing oxidative stress, as evidenced by high levels of the oxidized protein in three intrinsically stressed strains (trxA, trxA gor, and trxB gor) as well as wild-type cells challenged with diamide or H2O2. Our results further show that oxidation of MetE is associated with a methionine limitation imposed by oxidative stress. Hence this study provides insight into a previously unknown, but important aspect of the E. coli cellular response to oxidative stress. Materials and Methods Materials. Electrophoresis-grade urea, Chaps, ampholytes (pH 4–8), ammonium persulfate, TEMED, 10 N sodium hydroxide, and 85% phosophoric acid were obtained from Genomic Solutions (Ann Arbor, Michigan, United States). Iodoacetate and iodoacetamide were purchased from Fluka (Sigma-Aldrich, St. Louis, Missouri, United States). (6S)-5-methyltetrahydropteroyltriglutamate was synthesized from pteroyltriglutamate (Schircks Laboratories, Jona, Switzerland), as described previously (Matthews 1986). L-Homocysteine was prepared by hydrolysis of L-homocysteine thiolactone (Drummond et al. 1995). Polyclonal rabbit antibodies to purified wild-type MetE were generated by Lampire Biological Laboratories (Pipersville, Pennsylvania, United States). All other chemicals were obtained from Sigma (St. Louis, Missouri, United States). GroEL was generously provided by Z. Xu (University of Michigan, Ann Arbor, Michigan, United States). Bacterial strains and plasmids. Wild-type K-12 strain W3110 was obtained from F. C. Neidhardt (University of Michigan, Ann Arbor, Michigan, United States). Wild-type E. coli K-12 strain DHB4 and isogenic E. coli K-12 strains WP843 (trxA gor) and WP778 (trxB gor) (Prinz et al. 1997) were obtained from U. Jakob (University of Michigan). Overexpression of mutant MetE proteins was performed in E. coli K-12 strain GW2531 (metE) (Mulligan et al. 1982). The MetE mutants MetEC>A5 and MetEC>A1–4 were constructed by overlap extension PCR (Ge and Rudolph 1997) using Pfu Turbo (Strategene, La Jolla, California, United States). Plasmid pJG816 (González et al. 1992), specifying wild-type MetE, was used as the template, and the primers used for overlap extension are shown in Table 2. The products were purified by gel electrophoresis and ligated into alkaline phosphatase–treated pJG816. Plasmids containing the mutant genes were isolated after transformation into competent XL1-blue cells by electroporation. Single Cys→Ala mutations of Cys323 (pEM1), Cys353 (pEM2), Cys560 (pEM4), and Cys645 (pEM5) were constructed by overlap extension using pJG816 as the template. For the construction of pEM3 (Cys353Ala, Cys516Ala), pEM2 was used as the template instead of pJG816. For construction of pEM1, pEM2, pEM3, and pEM4, the purified products and pJG816 were digested with BssHII and StuI, while for pEM5, AatII and BssHII were used. MetEC>A1–4 was constructed by subcloning of fragments containing the individual mutations from the appropriate plasmids. Plasmids pEM1 and pEM3 were digested with MluI; the fragment containing the Cys323Ala mutation from pEM1 was ligated into the alkaline phosphatase–treated pEM3 vector to generate pEM6. Similarly, pEM6 was digested with AgeI and BssHII and then ligated into pEM4 that had been digested with the same enzymes to generate pEM7. Plasmids containing pEM5 (MetEC>A5) and pEM7 (MetEC>A1–4) were transformed into E. coli strain GW2531. Mutations were confirmed by DNA sequencing of the entire metE gene. Table 2 Sequences of Primers Used in the Construction of MetE Mutants a A pair of complementary primers was used for each mutant. Only the sequences of the A primers are shown. Primers nB are the reverse complement of primers nA for each mutation, n = 1 to 5. Changes in the sequence are underlined b Nucleotide number is assigned according to the GenBank sequence M87625 of metE. Translation begins at base pair 1 in the sequence Expression and purification of wild-type and mutant MetE proteins. Wild-type MetE was expressed using strain GW2531/pJG816, MetEC>A5 was expressed using strain GW2531/pEM5, and MetEC>A1–4 was expressed from strain GW2531/pEM7. Six 1-l portions of LB medium supplemented with 100 μg/ml ampicillin and 0.5 mM zinc sulfate were inoculated with 3 ml of an exponentially growing culture in the same medium (OD600 ∼1). Cultures were incubated at 37 °C with shaking at 250 rpm for approximately 30 h before harvesting. The proteins were purified as described previously (González et al. 1992), except that an 800-ml gradient from 100 to 400 mM potassium phosphate (pH 7.2) was used. Purified proteins were dialyzed into 50 mM potassium phosphate buffer (pH 7.2) containing 500 μM DTT, concentrated, and stored at –80 °C. Culture conditions For examining the effect of oxidative stress in minimal medium, cultures were grown aerobically at 37 °C in a rotary water bath shaker in glucose-minimal morpholinopropane sulfonate (MOPS) medium (Neidhardt et al. 1974) supplemented with 10 μM thiamine. Exponentially growing cultures in the same medium were diluted to an OD600 of approximately 0.02–0.04. Where diamide was added to cultures, the concentration of the diamide stock was determined spectrophotometrically (ɛ296 = 3,000 M−1cm−1) (Kosower and Kosower 1995). H2O2 concentrations were determined similarly (ɛ240 = 43.6 M−1cm−1) (Hildebrandt and Roots 1975; Poosch and Yamazaki 1986). Protein concentration determination and activity assay. The molar absorption coefficient for MetE at 280 nm was determined to be 157,000 ± 1000 M−1cm−1 using the Endelhoch method (Pace et al. 1995). The specific activity of MetE was measured using the modified enzyme assay previously described (González et al. 1996), but omitting DTT from the assay mix. Exclusion of DTT did not affect the measured activity. For experiments monitoring enzyme inactivation, aliquots containing 650 pmol of MetE were withdrawn from the oxidation reaction and mixed with activity assay reagents which had been pre-equilibrated to 37 °C. The final assay mixture contained 10 mM potassium phosphate buffer (pH 7.2), 50 mM Tris chloride (pH 7.2), 100 μM magnesium sulfate, 2 mM L-homocysteine, 66 μM (6S)-5-methyltetrahydropteroyltriglutamate, and enzyme in a total volume of 400 μl. After a 30-s incubation at 37 °C, the reaction was quenched by addition of 100 μl of 5 N hydrochloric acid/60% formic acid, followed by heating at 88 °C for 10 min. After cooling on ice, the reaction was centrifuged at 14,000g for 5 min at 4 °C to remove precipitated protein. The amount of tetrahydrofolate product (converted to methenyltetrahydrofolate by formic acid) was quantitated by its absorbance at 350 nm. Excess GSSG used to oxidize the enzyme did not significantly alter activity during the 30-s assay. Oxidant effects on cysteine oxidation and enzyme activity. MetE (50 μM) in 100 mM Tris chloride (pH 7.2) was equilibrated to 37 °C, and GSSG was added (final concentration, 5 mM) to initiate the oxidation reaction. The GSSG concentration was established by assay with glutathione reductase (Zander et al. 1998). At time points, aliquots were removed for measurement of cysteine oxidation or enzyme activity (described above). To determine the extent of oxidation, aliquots (100 μl) of the reaction mixture were removed and quenched with 100 μl of ice-cold 20% TCA (w/v). Samples were incubated on ice at least 30 min and then pelleted by centrifugation at 14,000g for 30 min at 4 °C. The supernatant was removed, and the pellet was dissolved in 100 mM potassium phosphate buffer (pH 7.3) containing 0.25 mM DTNB, 6 M guanidinium chloride, and 1 mM EDTA. Complete dissolution of the pellet required vortexing for 15–20 min. Reduced cysteines were quantitated by measuring the absorbance at 412 nm after subtraction of a blank containing DTNB and buffer but no protein. The extent of oxidation was determined using the fraction of reduced cysteines at the indicated time compared to the value at time zero (taken as 100%). Preparation of oxidized MetE. Purified MetE was desalted into 100 mM Tris chloride (pH 7.2) to remove DTT from the storage buffer. The reduced protein (50 μM) was then incubated with 10 mM GSSG for 90 min at 37 °C. Samples were concentrated using Microcon concentrators (Millipore, Billerica, Massachusetts, United States), and GSSG was removed using Bio-Gel P-6 columns (BioRad, Hercules, California, United States) equilibrated with 100 mM Tris chloride (pH 7.2). Reduction and reactivation of GSSG-oxidized MetE was accomplished by incubation of the oxidized enzyme with 2 mM reductant at 37 °C. For preparation of samples of diamide-oxidized MetE to be used as standards for in vivo thiol-trapping assays, diamide (1 mM) was incubated with MetE (10 μM) in 100 mM Tris chloride (pH 7.2) at 37 °C. After 45 min, diamide was removed by gel filtration using Bio-Gel P-6 columns. Fluorescamine assay for glutathionylation of MetE. Reduced and GSSG-oxidized MetE preparations were exchanged into 50 mM potassium phosphate (pH 7.2) using Bio-Gel P-6 columns. Proteins were then washed three times with the same buffer using Centricon concentrators. MetE (50 μM) was incubated with 2 mM DTT in 50 mM potassium phosphate (pH 7.2) in a volume of 200 μl for 1 h at 37 °C. Each solution was then loaded into a Microcon concentrator and centrifuged at 12,000g for 5 min at room temperature. Aliquots (100 μl) of the filtrate were added to 1.7 ml of 200 mM borate buffer (pH 9.0) containing 1 mM p-(hydroxymercuri)benzoic acid. While vortexing, 0.6 ml of 1 mg/ml fluorescamine (in acetone) was added. The fluorescence emission of the samples was measured at 485 nm with excitation at 390 nm. A range of GSH concentrations (0–125 mM) was assayed to generate a standard curve. GSH concentrations were determined by DTNB titration (Riddles et al. 1983). The stoichiometry of glutathionylation was calculated by subtracting the results obtained in the absence of DTT from the amount of glutathione released by incubation with DTT and dividing by the enzyme concentration (50 μM). Each form of MetE was assayed in duplicate from two separate preparations. Preparation of disulfide mapping samples. CDAP dissolved in 200 μl of 100 mM citrate buffer (pH 3) containing 6 M guanidine hydrochloride was added to 100 μl of MetE in 100 mM Tris chloride (pH 7.2) and incubated for 15–20 min at room temperature. The stock concentrations were adjusted so that CDAP was approximately 25-fold in excess over the total thiol concentration. CDAP was removed from the solution by gel filtration, using Bio-Gel P-6 columns equilibrated with 6 M guanidine hydrochloride solution (brought to pH ∼7–8 with potassium hydroxide). Concentrated ammonium hydroxide was added (final concentration 1 M [pH ∼12]), and the reaction was incubated at room temperature for approximately 75 min. Samples were then lyophilized to remove the ammonia and stored at –80 °C. Prior to LC-MS analysis, DTT was added (final concentration was approximately 200 times the concentration of MetE) and allowed to incubate at room temperature for 30 min. Solutions were then diluted into 6 M guanidine hydrochloride solution (adjusted to pH 3 with acetic acid) and characterized by LC-MS. LC-MS analysis. Samples (250–500 pmol) were injected onto a Vydac (Hesperia, California, United States) 250 mm × 2.1 mm C4 column (214MS52) using an analytical HPLC Surveyor from ThermoFinnigan (San Jose, California, United States). The column was equilibrated with 97% solvent A (water with 0.1% v/v acetic acid and 0.02% v/v TFA) and 3% solvent B (acetonitrile with 0.1% v/v acetic acid and 0.02% v/v TFA), and samples were eluted at a flow rate of 0.25 ml/min with a three-step linear gradient: 3% B for 15 min, 3% to 30% B in 9 min, and 30% to 65% B in 35 min. For LC-MS of the holo-protein, samples were desalted using a two-step linear gradient: 20% B for 10 min, and 20% to 70% B in 25 min. Fractionation was monitored by the UV chromatogram recorded at 280 nm. The eluent was diverted to waste for the first 15 min, and then directly infused into the electrospray ionization source of a ThermoFinnigan LCQ mass spectrometer. Mass spectral data were obtained in the positive mode, and ESI spectra were deconvoluted using the BioMass software provided by ThermoFinnigan. Diamide oxidation of purified MetE. Oxidation of MetE by diamide was monitored at 37 °C using a Hi-Tech Scientific (Salisbury, United Kingdom) SF-61DX2 stopped-flow spectrophotometer equipped with a xenon light source for single wavelength detection. Diamide (1 mM) and MetE (100 μM) in 100 mM Tris chloride (pH 7.2) were mixed, and reduction of diamide was monitored at 325 nm. Size exclusion chromatography of oxidized and reduced MetE and interaction with GroEL. MetE (20 pmol) was injected onto an Alltech (Lexington, Kentucky, United States) macrosphere size exclusion HPLC column (250 mm × 4.6 mm) with a 300-Å pore size (7-μm particle size) and run at 0.5 ml/min with 20 mM MOPS (pH 7.5) containing 100 mM potassium chloride and 5 mM magnesium chloride. For GroEL interaction experiments, GroEL (2 μM) was incubated with MetE (1 μM) in 20 mM MOPS (pH 7.5) containing 100 mM potassium chloride and 5 mM magnesium chloride for 30 min at 37 °C before analysis by size exclusion chromatography. CD measurements. Reduced and oxidized MetE were buffer exchanged into 10 mM potassium phosphate buffer (plus 500 μM DTT for the reduced protein) (pH 7.2). The final filtrates were collected and used as a blank. Samples were centrifuged for 1 h at 14,000g at 4 °C and then diluted to 0.4 mg/ml. Far UV CD spectra were recorded using a JASCO (Easton, Maryland, United States) J-810 spectropolarimeter and 0.1 cm cuvettes. Absorbance spectra of the same sample were recorded on a JASCO V-550 UV/Vis spectrophotometer to determine the precise protein concentration. The data were then normalized to a protein concentration of 4.5 μM MetE. Limited tryptic digestion. Aliquots containing 2.5 mg/ml reduced or oxidized MetE in 50 mM potassium phosphate (pH 7.2) were digested with 0.02% (w/v) trypsin at room temperature. At indicated times, 5-μl samples were quenched with 10 μg of 1-tosylamido-1-lysyl chloromethylketone, and then analyzed by SDS-PAGE (10% gel). Determination of the equilibrium constant for oxidation of MetE by GSSG. MetE (10 μM) was incubated with varying concentrations of GSH and GSSG in 100 mM Tris chloride (pH 7.2) at 37 °C. GSH (Riddles et al. 1983) and GSSG (Zander et al. 1998) stock concentrations were measured as described. After 5.5 h, aliquots were removed and MetE activity was assayed. Since A350 directly correlates to the amount of product in the activity assay, relative MetE activity was calculated by: where A350(x) is the absorbance at 350 nm for sample x, A350(oxid) is for completely oxidized MetE (2 mM GSSG, no GSH), and A350(red) is for completely reduced MetE (2 mM GSH, 4 μM [trace] GSSG). The equilibrium constant, Kmix, was then determined by plotting relative MetE activity versus the [GSH]/[GSSG] ratio. In vivo thiol-trapping experiments. All E. coli cultures used for in vivo thiol-trapping experiments were grown aerobically at 37 °C. E. coli wild-type (DHB4) and trxA gor (WP843) strains were grown in glucose-minimal MOPS medium supplemented with 10 μM thiamine and all amino acids except methionine (Neidhardt et al. 1974), with 0.4 mM arginine used instead of 5.2 mM. Tetracyclin (20 μg/ml) was included in the medium for the trxA gor strain. Overnight cultures of exponentially growing cells were diluted to an OD600 of approximately 0.02 into the same medium. Cultures were allowed to grow to an OD600 of approximately 0.4, and samples were removed for thiol trapping as described below. For in vivo thiol-trapping experiments performed on the trxB gor strain, a plasmid expressing MetE (pJG816) was transformed into electrocompetent trxB gor (strain WP778) cells by electroporation. An overnight culture of WP778/pJG816 in LB medium containing 20 μg/ml kanamycin, 50 μg/ml ampicillin, and 4 mM DTT was diluted to an OD600 of approximately 0.02 into the same medium (without kanamycin). Care was taken to ensure that an overnight culture from the same inoculum failed to grow when DTT was not included in the medium. The 50-ml culture was grown to an OD600 of approximately 0.4, and then filtered into a prewarmed sterile filter flask. The cells were washed twice with prewarmed sterile LB medium to remove residual DTT, and resuspended in 50 ml of prewarmed LB medium. At the indicated time points following DTT removal, samples were taken for alkylation as described. To assay the oxidation status of MetE in diamide-treated cells, wild-type E. coli were grown in glucose-minimal MOPS medium without methionine as in the growth experiments described above, except that diamide (0.9 mM, final concentration) was added at an OD600 of approximately 0.4. Aliquots were removed at intervals following diamide addition for treatment with alkylating agents. In order to trap oxidized thiols in vivo, 1 ml of culture was added to 250 μl of 0.5 M iodoacetamide in 100 mM Tris chloride (pH 7.2) that had been pre-equilibrated at 37 °C. Samples were incubated 2 min at 37 °C, quenched by the addition of 140 μl of 100% (w/v) ice-cold TCA, and incubated on ice for at least 30 min. Precipitated proteins were pelleted by centrifugation (14,000g) for 30 min at 4 °C, washed with cold 10% TCA (once) followed by ethanol (twice), and then dried by incubating approximately 20 min in a 37-°C incubator. The pellet could then be stored at –80 °C prior to secondary alkylation with iodoacetic acid. Pellets were dissolved in 50 μl of alkylation buffer containing DTT (0.1 M Tris chloride [pH 8.7] containing 0.2 M potassium chloride, 1 mM EDTA, 8 M urea, and 10 mM DTT), and incubated 15 min at room temperature. Newly exposed cysteines were trapped by adding 12.5 μl of 0.5 M iodoacetate (104 mg dissolved in 0.5 ml of 1 M potassium hydroxide, followed by addition of 0.5 ml of 1 M Tris chloride [pH 7.3]) and incubating 2 min at room temperature. For some samples, excess DTT was added to quench the reaction; however, this was found to not be necessary. Alkylated proteins were then frozen in liquid nitrogen and stored at –80 °C prior to analysis by isoelectric focusing. For the reduced and oxidized MetE standards, 25 μl of 0.5 M iodoacetamide was added to 100 μl of 10 μM MetE in 100 mM Tris chloride (pH 7.2) and incubated for 1 min at 37 °C. The reaction was quenched by addition of 125 μl of 20% (w/v) ice-cold TCA and incubation on ice for at least 30 min. The proteins were pelleted by centrifugation (14,000g) at 4 °C for 30 min and then washed with cold 10% TCA (once) and either ether (3 times) or 1 M Tris chloride (pH 7.2) followed by water. Pellets were then alkylated with iodoacetate as described for the in vivo culture samples. Vertical slab isoelectric focusing. The vertical slab isoelectric focusing protocol developed was based on that of Savinova and Jagus (1997) and Robertson et al. (1987). Gels contained 8 M urea, 9.6% glycerol, 3.75% acrylamide, 5% ampholytes, and 1.25% Chaps. Briefly, 6.0 g of urea was dissolved in 2.4 ml of 18 MΩ water, 2.4 ml of 50% glycerol, and 1.56 ml of acrylamide solution (28.36% acrylamide/1.62% bis-acrylamide). 600 μl of ampholytes (pH 4–8) and 780 μl of 20% Chaps were added, and the resulting solution was degassed for approximately 10 min. 25 μl of 10% APS and 20 μl of TEMED were added, and 8 cm × 8 cm × 1.0 cm gels were poured. Gels were allowed to polymerize approximately 30 min and then used immediately. Lanes were carefully washed with cathode buffer. The sample buffer solution contained 9.5 M urea, 5% chaps, 4.5% glycerol, 0.5 mM lysine–hydrochloride, 2% ampholytes (pH 4–8), 50 mM DTT, and 0.15% SDS. Care was taken not to increase the solution temperature above 30 °C to avoid breaking down urea, which could then carbamylate proteins. Aliquots of the sample buffer were snap-frozen in liquid nitrogen and stored at –20 °C. An Xcell Surelock Mini-cell gel apparatus from Invitrogen was assembled with the freshly prepared gel, and degassed cathode (upper, 0.1 M sodium hydroxide) and anode (lower, 10 mM phosphoric acid) solutions were added. Samples were diluted at least 10-fold into sample buffer and loaded. Power was applied for 1 h at 100 V followed by 1 h at 200 V and finally 30 min at 500 V. Gels were then stained as described previously (Garfin 1990) or blotted. Immunoblotting. Gels were electroblotted onto PVDF membrane (Amersham Pharmacia, Piscataway, New Jersey, United States) according to the manufacturer's (Invitrogen) protocol using 1/2X Towbin buffer. Proteins were immunoblotted with a polyclonal rabbit antibody to MetE (1:3,000) and immune complexes were revealed using a fluorescein-linked anti-rabbit antibody (ECF western blotting kit, Amersham Pharmacia) followed by direct detection using a Molecular Dynamics (Sunnyvale, California, United States) Storm phosphorimager. Supporting Information Accession Numbers The Swiss-Prot (http://www.ebi.ac.uk/swissprot/) accession numbers for the gene products discussed in this paper are AhpC (P26427), glutaredoxin 1 (P00277), glutaredoxin 2 (P39811), glutaredoxin 3 (P37687), glutathione reductase (P06715), GroEL (P06139), iron superoxide dismutases (P09157), manganese superoxide dismutase (P00448), MetA (P07623), MetE (C. albicans) (P82610), MetE (E. coli) (P25665), MetE (T. maritima) (Q9X112), MetH (P13009), OxyR (P11721), PAPS reductase (P17854), thioredoxin 1 (P00274), thioredoxin reductase (P09625), and Tpx (P37901). We are very grateful to M. Prydzial and J. Omnaas for helpful instruction concerning the LC-MS analysis, as well as use of the instrument, which is shared within the medicinal chemistry department (University of Michigan). We thank W. Wu and J. T. Watson (Michigan State University) for some preliminary mass spectrometry results. In addition, we are grateful to U. Jakob and J. Winter for assistance with the CD experiments. We thank R. Pejchal and M. Ludwig for sharing unpublished data. This work was supported in part by National Institutes of Health Research Grant GM24908 (RGM) and a National Science Foundation Graduate Research Fellowship (ERH). Conflicts of interest. The authors have declared that no conflicts of interest exist. Author contributions. ERH and RGM conceived and designed the experiments. ERH performed the experiments. ERH and RGM analyzed the data and wrote the paper. Academic Editor: Joan Valentine, University of California, Los Angeles Citation: Hondorp ER, Matthews RG (2004) Oxidative stress inactivates cobalamin-independent methionine synthase (MetE) in Escherichia coli. PLoS Biol 2(11): e336. Abbreviations AhpCalkylhydroperoxide reductase B12cobalamin CDcircular dichroism CDAP1-cyano-4-dimethylamino-pyridinium tetrafluoroborate DTNBdithio-1,4-nitrobenzoic acid DTTdithiothreitol GSHreduced glutathione GSSGoxidized glutathione H2O2hydrogen peroxide HPLChigh performance liquid chromatography itz2-iminothiazolidine-4-carboxyl LBLuria-Bertani LC-MSliquid chromatography mass spectrometry MetAhomoserine transsuccinylase MetEcobalamin-independent methionine synthase MetHcobalamin-dependent methionine synthase MOPSmorpholinopropane sulfonate PAPS3′-phosphoadenylylsulfate TCAtrichloroacetic acid Tpxthioredoxin-linked thiolperoxidase ==== Refs References Åslund F Zheng M Beckwith J Storz G Regulation of the OxyR transcription factor by hydrogen peroxide and the cellular thiol-disulfide status Proc Natl Acad Sci U S A 1999 96 6161 6165 10339558 Benov L Fridovich I Superoxide dismutase protects against aerobic heat shock in Escherichia coli J Bacteriol 1995 177 3344 3346 7768839 Benov L Kredich NM Fridovich I The mechanism of the auxotrophy for sulfur-containing amino acids imposed upon Escherichia coli by superoxide J Biol Chem 1996 271 21037 21040 8702868 Burt ET O'Connor C Larsen B Isolation and identification of a 92-kDa stress induced protein from Candida albicans Mycopathologia 1999 147 13 20 10872511 Carlioz A Touati D Isolation of superoxide dismutase mutants in Escherichia coli : Is superoxide dismutase necessary for aerobic life? 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Salmonella typhimurium and Escherichia coli J Bacteriol 1989 171 3277 3281 2656646 VanBogelen RA Neidhardt FC Ribosomes as sensors of heat and cold shock in Escherichia coli Proc Natl Acad Sci U S A 1990 87 5589 5593 2198567 Vlamis-Gardikas A Potamitou A Zarivach R Hochman A Holmgren A Characterization of Escherichia coli null mutants for glutaredoxin 2 J Biol Chem 2002 277 10861 10868 11741965 Wu J Watson JT A novel methodology for assignment of disulfide bond pairings in proteins Protein Sci 1997 6 391 398 9041641 Wu J Watson JT Optimization of the cleavage reaction for cyanylated cysteinyl proteins for efficient and simplified mass mapping Anal Biochem 1998 258 268 276 9570840 Xavier KB Bassler BL LuxS quorum sensing: More than just a numbers game Curr Opin Microbiol 2003 6 191 197 12732311 Xu Z Horwich AL Sigler PB The crystal structure of the asymmetric GroEL-GroES-(ADP)7 chaperonin complex Nature 1997 388 741 750 9285585 Zander T Phadke ND Bardwell JCA Disulfide bond catalysts in Escherichia coli Methods Enzymol 1998 290 59 74 9534151

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==== Front PLoS BiolPLoS BiolpbioplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 1550287110.1371/journal.pbio.0020340Research ArticleEcologyEvolutionZoologyArthropodsHomo (Human)Genetic Analysis of Lice Supports Direct Contact between Modern and Archaic Humans Modern and Archaic Humans in ContactReed David L [email protected] 1 Smith Vincent S 2 Hammond Shaless L 3 Rogers Alan R 4 Clayton Dale H 3 1Florida Museum of Natural History, Dickinson Hall, University of FloridaGainesville, FloridaUnited States of America2Graham Kerr Building, DEEB, IBLS, University of GlasgowGlasgowScotland3Department of Biology, University of UtahSalt Lake City, UtahUnited States of America4Department of Anthropology, University of UtahSalt Lake City, UtahUnited States of America11 2004 5 10 2004 5 10 2004 2 11 e34011 5 2004 3 8 2004 Copyright: © 2004 Reed et al.2004This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. Did We or Didn't We? Louse Genetic Analysis Says Yes Parasites can be used as unique markers to investigate host evolutionary history, independent of host data. Here we show that modern human head lice, Pediculus humanus, are composed of two ancient lineages, whose origin predates modern Homo sapiens by an order of magnitude (ca. 1.18 million years). One of the two louse lineages has a worldwide distribution and appears to have undergone a population bottleneck ca. 100,000 years ago along with its modern H. sapiens host. Phylogenetic and population genetic data suggest that the other lineage, found only in the New World, has remained isolated from the worldwide lineage for the last 1.18 million years. The ancient divergence between these two lice is contemporaneous with splits among early species of Homo, and cospeciation analyses suggest that the two louse lineages codiverged with a now extinct species of Homo and the lineage leading to modern H. sapiens. If these lice indeed codiverged with their hosts ca. 1.18 million years ago, then a recent host switch from an archaic species of Homo to modern H. sapiens is required to explain the occurrence of both lineages on modern H. sapiens. Such a host switch would require direct physical contact between modern and archaic forms of Homo. A phylogenetic analysis reveals that humans have two types of head lice and that one must have switched from an ancient to a modern human host, suggesting these humans had contact ==== Body Introduction One of the most intensely debated topics in evolutionary biology pertains to the origin of modern Homo sapiens. The debate concerns the precise manner in which anatomically modern humans arose from archaic ancestors. Empirical studies tend to support one of two prominent models of human origins, the Recent African Replacement model (Stringer and Andrews 1988) or the Multiregional Evolution model (Wolpoff et al. 1994). The Recent African Replacement model, as originally proposed, suggests that modern humans arose from an archaic ancestor in Africa ca. 130,000 years ago, and then replaced archaic humans in Asia, Africa, and Europe without introgression between archaic and modern humans. The Multiregional Evolution model (as proposed by Wolpoff et al. [1994] and revisited by Wolpoff et al. [2000]) suggests that gene flow existed not only among populations of modern Homo sapiens, but also between modern H. sapiens and archaic forms of Homo (e.g., Homo neanderthalensis and Homo erectus), which led to some degree of regional continuity. Both models can be subdivided into many variants. There are two common variants of the Multiregional Evolution model. In one variant, the transition from archaic to modern humans occurs incrementally across a large geographic region (i.e., both within and outside Africa); in the other variant, the transition from archaic to modern humans arises first in Africa then spreads through gene flow outside of Africa. This latter variant is very similar to a Diffusion Wave model recently put forth by Eswaran (2002). Both types of models of human origins (the Recent African Replacement and Multiregional Evolution models) have been examined with both human fossil and genetic data, but no single model or variant has been supported by all the data. Fossils provide the only source of data available for most species of archaic humans and are therefore crucial to understanding the origin of modern humans. Unfortunately, missing taxa and fragmentary fossils limit our ability to reconstruct human evolutionary history based solely on fossil data. Molecular (DNA sequence) data have provided additional insight into the recent evolutionary history of humans, but these data are limited mainly to extant human populations. Ancient DNA was recently sequenced from H. neanderthalensis (Krings et al. 1997, 1999, 2000) and a 24,000-year-old specimen of modern H. sapiens (Caramelli et al. 2003), but even these ancient DNA studies do not agree on hypotheses of modern human origins (Templeton 2002; Serre et al. 2004). Only a few ancient specimens have been examined molecularly, and additional sequences are slow to emerge. Furthermore, DNA may never be retrieved from some specimens because it is difficult, if not impossible, to liberate sequenceable DNA from poorly preserved (Krings et al. 1997) or very old (Paabo and Wilson 1991) fossil material. Therefore, the degree to which we can reconstruct human evolutionary history depends, in part, upon additional types of data. Several recent studies have inferred portions of human evolutionary history from the evolutionary history of their parasites (Chan et al. 1992; Ho et al. 1993; Ong et al. 1993; Escalante et al. 1998; Ashford 2000; Leal and Zanotto 2000; Hoberg et al. 2001). Parasites can be a powerful tool for reconstructing host evolutionary history because they provide data that are independent of host data. For example, human papillomaviruses (Chan et al. 1992; Ho et al. 1993; Ong et al. 1993), tapeworms (Hoberg et al. 2001), and malarial parasites (Escalante et al. 1998) each have evolutionary origins in Africa, consistent with most human fossil and molecular data. Human T-cell leukaemia/lymphoma virus (HTLV) sequences show that most human viral strains are closely related to those of Old World apes and monkeys (Leal and Zanotto 2000). In contrast, some Native American strains of HTLV have closer affinities to viral strains from Asian primates, suggesting a dual origin for this virus in humans (Leal and Zanotto 2000 and references therein). Ashford (2000) recently reviewed the use of parasites as markers of human evolutionary history, pointing out that five parasites of humans (lice, tapeworms, follicle mites, a protozoan, and bedbugs) have closely related taxonomic pairs that suggest periods of host geographic isolation. Unfortunately, none of these five pairs has been studied rigorously with the primary goal of inferring host evolutionary history. Of these parasites, the ones most likely to provide the greatest insight into human evolutionary history are those that are known to have had a long-term coevolutionary association with their hosts, such as lice (Insecta: Phthiraptera) (Page 2003). Lice are obligate parasites of mammals or birds that complete their entire life cycle on the body of the host; they cannot survive more than a few hours or days off the host (Buxton 1946). Mammal lice are closely tied to their hosts in both ecological (Reed and Hafner 1997) and evolutionary (Hafner et al. 1994) time. The lice found on primates are quite host specific, with most species occurring only on a single species of host (Durden and Musser 1994). Host specificity is reinforced by the fact that primate lice require direct physical contact between hosts for transmission (Buxton 1946; Durden 2001; Canyon et al. 2002; Burgess 2004). Host specificity often goes hand in hand with long-term coevolutionary patterns between hosts and parasites (Page 2003), making primate lice excellent candidates for inferring host evolutionary history. Humans are parasitized by two species of lice: head/body lice (Pediculus humanus), the focus of this paper, and pubic lice(Pthirus pubis), which serve as a phylogenetic outgroup in this study. P. humanus is found in two forms (head and body lice) that are morphologically similar, but ecologically distinct. Body lice live primarily in clothing and move onto the skin to feed once or twice a day. Head lice are confined to the scalp and feed more frequently. Body lice vector the bacteria responsible for epidemic typhus, trench fever, and relapsing fever; head lice are not known to vector any agent of human disease under natural conditions (Buxton 1946). Recent molecular work by Leo et al. (2002) showed that, despite the ecological differences between head and body lice, the two forms are not genetically distinct. Kittler et al. (2003) confirmed this finding but also discovered two deeply divergent clades within P. humanus that are uncorrelated with the head and body louse forms. The divergent clades of lice stand in contrast to mitochondrial sequence data from extant human populations, which coalesce to a single lineage very rapidly. The shallow coalescence in human mitochondrial sequence data is likely the result of a recent population bottleneck and subsequent population expansion (Rogers and Harpending 1992), which obscures much of the evolutionary history of humans prior to the bottleneck. The deep divergences within P. humanus have the potential to reveal aspects of human evolutionary history that cannot be recovered from human DNA markers. We reconstructed the evolutionary history of P. humanus and several outgroup taxa using both morphological and molecular data. First, we used louse morphological data to test for patterns of cospeciation between primate lice and their hosts. Then we collected molecular data from a subset of the same taxa and calculated divergence dates for nodes in the louse phylogeny. This broad phylogenetic approach allowed us to date the origin of the human louse, P. humanus, and to date the two divergent lineages within the species. Finally, we collected population genetic data for P. humanus to compare with population-level characteristics of extant humans. Taken together, our phylogenetic and population-level data provide a well-resolved picture of the evolutionary history of P. humanus, which can be used to indirectly infer human evolutionary history. Specifically, we compared three distinct models of modern human origins (Recent African Replacement without Introgression, Multiregional Evolution, and Diffusion Wave) to see which model best fits the data from human lice. Results Phylogenetic Analyses and Divergence Estimates Both the morphological and molecular data sets produced a single phylogenetic relationship for the louse species in Figure 1. The phylogeny shows that Pediculus species on chimpanzees and humans are sister taxa, which together with Pthirus form a clade that is sister to Pedicinus, the most basal member of the ingroup (Figure 1). Bootstrap support for these relationships is high. Reconciliation analysis using Treemap v. 2.0 (M. A. Charleston and R. D. M. Page, software distributed by authors) revealed significant congruence (p < 0.01) between the louse and primate phylogenies, thus validating the assumption of cospeciation (Kittler et al. 2003). Reconciliation analysis using Treemap showed four cospeciation events and one host switch. One particular node of cospeciation determined that as cercopithecoid and hominoid primates diverged 20–25 million years ago (MYA) (Benefit 1993; Leakey et al. 1995), Pedicinus diverged from the lineage leading to Pediculus and Pthirus. Since the nodes of cospeciation in congruent host and parasite trees are contemporaneous, the louse tree can be calibrated using the host tree. Figure 1 Phylogeny of Primate Lice from Morphological and Molecular Data The phylogeny is a strict consensus of morphology and a 1,525-bp fragment of COI and Cytb. Branch lengths were determined from the molecular data. Numbers in parentheses are bootstrap values from molecular and morphological data, respectively. Divergence dates are direct estimates from mtDNA data (see text). Louse images from light microscopy were taken by VSS. We used the date of 22.5 ± 2.5 MYA to calibrate the split between Pedicinus and Pthirus + Pediculus in the louse tree. This, in turn, yielded a divergence time of 11.5 MYA for the Pthirus/Pediculus split and 5.6 MYA for the split between Pediculus schaeffi and P. humanus (Table 1). Our estimated divergence between chimp and human lice (5.6 MYA) is strikingly similar to the 5.5 MYA estimates for the chimp/human divergence based on both mitochondrial and nuclear sequence data (Stauffer et al. 2001). To test the original calibration date of 22.5 MYA, we used the molecular estimate of the chimp/human split (5.5 MYA; Stauffer et al. 2001) to reverse calibrate the louse tree. This younger calibration point resulted in divergence estimates that were nearly identical to those from the previous calibration. For example, the 5.5 MYA calibration resulted in an estimated divergence of 22.65 MYA for the split between Pedicinus and Pthirus + Pediculus. Estimates of divergence time error were calculated from bootstrapped data sets (Table 1). Other studies have shown that louse mitochondrial DNA (mtDNA) sequences evolve at a rate two to three times faster than that of host sequence rates (Page 1996; Page et al. 1998). The lice in this study are evolving at ca. 2.3 times the rate of their primate hosts, when nucleotide substitutions are estimated under a best-fit model of sequence evolution. Table 1 Mean (± Standard Deviation), Minimum, and Maximum Estimates of Divergence Times (in Millions of Years) of Louse Lineages from 100 Bootstrapped Data Matrices Divergence dates were calculated without rate-smoothing methods because our data do not depart from the assumptions of a molecular clock (see text). Direct estimates of divergence times were calculated using the original data set (1,525-bp mtDNA) rather than the bootstrapped matrices. Trees were calibrated with a cercopithecoid/hominoid primate divergence of 22.5 MYA (from fossil evidence) except for the Pedicinus dates (asterisks), which were calibrated from the 5.5 MYA estimate of the human (P. humanus)-chimp (P. schaeffi) divergence date from molecular data (see text) Phylogenetic analysis revealed two divergent clades within P. humanus (6% uncorrected sequence divergence for cytochrome oxidase subunit I [COI] and cytochrome b [Cytb]). One of the two lineages in our data set is worldwide (WW) in distribution (Figure 2, Worldwide clade), contains both head and body louse forms, as determined by discriminant function analysis (Figure 3), and has a most recent common ancestor (MRCA) 0.54 MYA (Table 1). Even within this WW clade head and body lice are not reciprocally monophyletic, and a constraint to enforce such monophyly can be rejected using a Shimodaira-Hasegawa test (p < 0.01) (Shimodaira and Hasegawa 1999). The other lineage (Figure 2, New World clade) is restricted to the New World (NW), contains only the head louse form, and has a MRCA only 0.15 MYA. The MRCA of all P. humanus was 1.18 MYA, which predates by a considerable margin the origin of modern H. sapiens based on mtDNA (≤0.20 MYA; Cann et al. 1987; Vigilant et al. 1991; Ingman et al. 2000) as well as fossil evidence (0.15–0.16 MYA; White et al. 2003). Figure 2 Molecular Phylogeny of P. humanus from Geographically Diverse Human Populations This species exhibits distinct “head” and “body” forms, which differ in ecology, and slightly in size. Head lice (black lettering) are smaller than body lice (red lettering) and are confined to the scalp, whereas body lice live primarily in clothing. Haplotypes shown in green were found in both head and body lice. There are no fixed genetic differences between the head and body forms, suggesting a lack of reproductive isolation, despite the fact that the two forms can be distinguished using discriminant function analysis of morphological data. These results are consistent with experimental data showing that head lice can transform morphologically into body lice within a few generations (Levene and Dobzhansky 1959). The Worldwide clade (red branches) shares a MRCA ca. 0.54 MYA and the geographically restricted New World clade (blue branches) has a much younger MRCA, ca. 0.15 MYA. Asterisks denote samples from Leo et al. (2002) Figure 3 Plot of the First and Second Canonical Discriminant Functions for Specimens of Adult Head/Body Lice (P. humanus) and Pubic Lice (Pthirus pubis) Solid points denote reference specimens of known identity acquired from museum collections. Unfilled points denote newly collected specimens used in the molecular analyses. In all cases the discriminant function analysis successfully classified each unknown case with a probability of >0.95. Our estimate of the age of the MRCA for P. humanus (1.18 MYA) is much older than that reported by Kittler et al. (2003), which was only 0.53 MYA based on mtDNA. Their estimate of 0.53 MYA was determined using a mtDNA sequence from a specimen of the chimp louse, P. schaeffi, that is quite aberrant when compared to other primate lice. Phylogenetic analysis of the Kittler et al. Cytb data (downloaded from GenBank), combined with our own data, shows that the Kittler et al. sequence for P. schaeffi is 40% divergent from P. humanus and 40% divergent from our own sequence of P. schaeffi. Phylogenetic analysis places their specimen of P. schaeffi outside all other primate lice and even outside the rodent louse (Figure 4), whereas our specimen of P. schaeffi is sister to P. humanus, based on both morphology and molecular data. We think that the Kittler et al. specimen has been attributed to the species P. schaeffi in error. In contrast to the mitochondrial data reported by Kittler et al. (2003), our analysis of their nuclear elongation factor (EF1-alpha) sequences produces a MRCA for P. humanus that is ca. 2 MYA. Similarly, 18S rRNA sequences for P. humanus from Yong et al. (2003), combined with an 18S rRNA sequence from P. schaeffi, provide a MRCA for P. humanus that is ca. 2 MYA (for GenBank accession numbers, see Supporting Information). Together, these mitochondrial and nuclear markers support a MRCA for P. humanus greater than 1.18 MYA, which is an order of magnitude older than the MRCA for its human host. Figure 4 Neighbor-Joining Tree Using a Best-Fit Model of Nucleotide Substitution (Tamura-Nei + Γ) for a Combined Data Set of Cytb Sequences from Our Study and from Kittler et al. (2003) The clades of P. humanus identified by Kittler et al. (2003) are nearly identical to those from our data, with the exception of their basal African clade, which was not represented in our data set. One clade contains both head lice and body lice and is WW in distribution. Another clade is comprised solely of head lice from the NW (our data) and Europe (samples from Kittler et al. 2003), and the most basal clade contains isolates 4, 18, and 33 from Kittler et al. (2003), which are head lice from Africa. The size of the triangles representing the three clades are proportional in size to the number of taxa within the clade. This phylogeny is rooted with a divergent louse, Dennyus hirundinus, which is a bird louse in the suborder Amblycera. Note the placement of the Kittler et al. (2003) specimen of P. schaeffi, which falls outside all other primate lice and the rodent louse Fahrenholzia. Population Genetic Analysis of P. humanus We can calculate an expected date of mitochondrial coalescence for P. humanus if we assume for the moment that the entire population of lice mated at random (i.e., panmixia). The estimate of expected coalescence is based on the effective female population size (Nef), which was estimated from the sample of all P. humanus specimens to be 1.1 million female lice from the equation Θ = 2Nefμ. The estimate of Nef provides an expected coalescence time for the two divergent mitochondrial lineages of P. humanus of 1.10 million generations or ca. 0.11 MYA, which is an order of magnitude younger than the observed divergence time of 1.18 MYA. In a large randomly mating population consisting of 1.1 million female lice, one would expect to maintain two distinct haplotypes for only ca. 0.11 million years (MY). This suggests that we can reject panmixia if we assume that Nef prior to the bottleneck was roughly similar to what we see today. If estimates of Nef were drastically higher (ca. 60 million female lice) prior to the bottleneck, then expected time to coalescence could be much longer. The Fst value, a measure of genetic population differentiation, calculated for the WW and NW clades was 0.96, indicating substantial population structure, which also supports the rejection of panmixia. The WW clade of P. humanus shows evidence of a recent population expansion (Fu and Li's D* = −2.80 [Fu and Li 1993]; p < 0.02). We estimated the date of this population expansion from the mismatch distribution. The estimate was calculated by comparing the average pairwise difference within the WW clade of P. humanus (4.21 mutations) to the pairwise difference between P. humanus and P. schaeffi (220 mutations), which diverged 5.6 MYA. The population expansion of the WW clade is estimated to be 0.11 MYA, similar to the estimated date of population expansion of modern humans out of Africa ca. 0.10 MYA (di Rienzo and Wilson 1991; Rogers and Harpending 1992; Harpending et al. 1993). In contrast, the NW clade of P. humanus does not exhibit the signature of a recent population expansion (Fu and Li's D* = 0.17), but instead shows a more stable population size. Contemporaneous Divergences in Pediculus and Archaic Homo spp. The age of the MRCA of P. humanus dates to 1.18 MYA (for mtDNA), which is roughly midway between the estimated ages of H. neanderthalensis (0.60 MY) and H. erectus (1.8 MY). We used a maximum likelihood (ML) analysis to test whether our two divergent lineages of lice could have diverged in tandem with H. sapiens and H. neanderthalensis (Neandertals). H. neanderthalensis is the only other species of Homo for which DNA sequence data are available (Krings et al. 1999). The test evaluated whether relative branch lengths (scaled according to mutation rate) in the host tree, specifically for the branch between H. sapiens and H. neanderthalensis, are consistent with the parasite DNA sequence data (Huelsenbeck et al. 1997). In cospeciating assemblages, host and parasite branch lengths are highly correlated due to a shared evolutionary history (Page 1996). A likelihood ratio test (LRT) rejected (p < 0.0001) the H. sapiens/H. neanderthalensis split as a node of cospeciation with the two clades of P. humanus because the branch length between H. sapiens and H. neanderthalensis is far too short to explain the louse DNA sequence data. In other words, the split between H. sapiens and H. neanderthalensis is too recent to have been contemporaneous with the divergence of the two lineages of lice. If one artificially lengthens the branch between H. sapiens and H. neanderthalensis to approximate the split between H. sapiens and H. erectus (anywhere from 1.2 to 1.8 MYA), the LRT fails to reject this hypothesis of cospeciation. Discussion Morphological and molecular data agree that primates and their lice have been cospeciating for over 20 MY. Indeed, it is this cospeciation that permits us to use host fossil evidence to calibrate portions of the louse phylogenetic tree. This has resulted in the discovery of two extant lineages of human lice that diverged 1.18 MYA. This ancient divergence is surprising because humans, and presumably their lice, are thought to have passed through a population bottleneck ca. 0.05–0.10 MYA (Rogers and Harpending 1992). Such bottlenecks reduce genetic diversity by eliminating uncommon haplotypes, thereby making it less likely that multiple haplotypes survive bottleneck events. For example, mtDNA sequences from human populations coalesce to a single lineage very quickly (≤0.20 MYA), presumably the result of the population bottleneck. The deep divergences found in P. humanus could conceivably be the result of sequencing a nuclear copy of a mitochondrial gene. However, several lines of evidence strongly suggest otherwise. Because we amplified two different mitochondrial genes (COI and Cytb) that show the same divergent lineages and similar percent sequence divergences, copies of both mitochondrial genes would have had to enter the nucleus simultaneously, which is unlikely. In addition, we amplified each gene with a nested set of overlapping primers, and we never amplified more than one gene copy, even during bouts of cloning. Nucleotide base composition for our COI and Cytb data do not deviate from the mean values for all louse COI and Cytb sequences in GenBank (unpublished data), which would not be the case for a nuclear copy of a mitochondrial gene. Finally, the deep divergences seen in our mitochondrial genes are confirmed by preliminary analyses of nuclear data (EF1-alpha and 18S rRNA, unpublished data). Therefore, we are confident that the DNA sequences used in this study are mitochondrial in origin, and we must attempt to explain the occurrence of such ancient mitochondrial haplotypes in human lice. Gene Trees and Ancient Polymorphisms Gene trees (e.g., mitochondrial lineages) can be considerably older than species trees, and therefore our louse mitochondrial lineages could predate the actual origin of the species P. humanus (i.e., its speciation time). It is useful to determine an expected time to coalescence from the estimated Nef of 1.1 million female lice, even though this estimate seems high for a parasite of humans, who themselves have had very small effective population sizes (as few as 10,000 individuals) and recently went through a population bottleneck (Rogers and Harpending 1992). Although we do not necessarily expect human and louse effective population sizes to be directly correlated, it is difficult to imagine that humans could have maintained such a large effective population of lice during a bottleneck event. Regardless, the expected time to coalescence was estimated to be 0.10 MYA, an order of magnitude younger than the observed divergence time of 1.18 MYA. The deeper gene tree that our data provide also could have been produced either by balancing selection or by subdivision of the louse population into several distinct groups with very limited gene flow. A Fu and Li test does not detect balancing selection when both lineages of P. humanus are evaluated together (p = 0.11); therefore, we must consider the alternative explanation of extensive population subdivision. Population Substructure and Host Geographic Isolation Substantial isolation among populations of lice on modern H. sapiens could disrupt gene flow and allow the retention of very old lineages, making the age of P. humanus seem much older than it actually is. However, there is no evidence of such pervasive geographic isolation in the modern human hosts of these lice. Other species of lice have been shown to have substantial geographic substructure (i.e., isolation) even when hosts show no geographic isolation (Johnson et al. 2002). If populations of P. humanus are more highly subdivided than those of their hosts, then we might expect P. humanus to have retained ancient mitochondrial polymorphisms, even through host bottleneck events. One prediction of this hypothesis would be that both clades of P. humanus (the WW and NW clades) would show signs of the recent population expansion of humans during the last ca. 0.10 MY. However, only the WW clade shows evidence of this event, which very closely matches the timing of human population expansion. Because the WW clade is commonly found worldwide, and shows a population expansion concurrent with that of modern H. sapiens, we conclude that this lineage has a common evolutionary history with modern H. sapiens. In contrast, the NW clade appears to have diverged from the WW lineage 1.18 MYA, and has had a distinctly different evolutionary history. We are left unable to explain the retention of two ancient louse lineages, each with a different evolutionary history, within the confines of a single host, modern H. sapiens. Given the history of cospeciation between primate lice and their hosts, it is necessary to look beyond modern H. sapiens to determine whether the two divergent lineages of P. humanus are legacies of a more ancient divergence. Contemporaneous Divergences in Pediculus and Archaic Homo spp. ML analyses rejected H. neanderthalensis as having diverged from H. sapiens contemporaneously with the two divergent lineages of lice. The mitochondrial MRCA of Neandertals and humans is 0.60 MYA (Krings et al. 1997), which is only about half as old as the MRCA of the two ancient lineages of P. humanus, 1.18 MYA. The same ML test failed to reject the codivergence of these lice with H. erectus and H. sapiens when their divergence was set anywhere between 1.2 and 1.8 MYA. Therefore, the deep divergence within P. humanus is entirely consistent with a cospeciation event within the genus Homo ca. 1.2–1.8 MYA, but not 0.60 MYA. Unfortunately, no DNA sequence data exist for H. erectus or any other archaic species of Homo to enable a more direct test of cospeciation. There is much debate regarding the past 2 MY of hominid evolution. However, one area of broad agreement is that, prior to 2 MYA, our ancestors were confined to Africa, then left the continent ca. 1.8 MYA. This first migration out of Africa resulted in archaic species of Homo that were widespread in distribution, and at times both contemporaneous with, and geographically isolated from, the lineage leading to modern H. sapiens (Figure 5). The 1.18 MY of isolation required to preserve the two ancient louse lineages must have occurred, in part, among these archaic species of Homo. It should be noted here that some interpretations of the Multiregional Evolution model do not necessarily consider modern H. sapiens to be a distinctly different species from archaic humans (e.g., H. erectus and H. neanderthalensis). We refer to them as “species” mostly for convenience of writing. Whereas the WW lineage has population genetic characteristics that are similar to those of modern H. sapiens, the geographically restricted NW lineage does not. It likely evolved on a now extinct species of Homo only to switch to modern H. sapiens very recently. For example, Figure 5 depicts one possible scenario where the NW lineage evolved on H. erectus and switched to modern H. sapiens. Interestingly, Hoberg et al. (2001) reported that two species of tapeworms of humans diverged ca. 0.78–1.71 MYA, and one of the two species, Taenia asiatica, is entirely restricted to Asia. This is consistent with the depiction in Figure 5, if one assumes that T. asiatica evolved on H. erectus. Although divergence dates are not available, it is intriguing that some Native American strains of HTLV have closer affinities to Asian primate strains than to other human strains of HTLV, suggesting an independent Asian origin of this virus in humans. One must still explain how these parasites came to be on modern H. sapiens, but taken together, the parasitological evidence (especially the deep divergences in tapeworms and lice) suggests that they might have evolved on H. erectus and switched recently to H. sapiens. If true, this implies that H. erectus was contemporaneous with modern H. sapiens in eastern Asia, as suggested by Swisher et al. (1996), and it begs a discussion of recent human origins. Figure 5 Temporal and Geographical Distribution of Hominid Populations Redrawn from Stringer (2003) This figure depicts one view of human evolutionary history based on fossil data. Other interpretations differ primarily in the taxonomy and geographical distribution of hominid species. The temporal distribution of the two divergent lineages of P. humanus is superimposed on the hominid tree to show host evolutionary events that were contemporaneous with the origin of P. humanus. Whereas the NW lineage is depicted on H. erectus in this figure, several alternative hypotheses are consistent with our data when other evolutionary histories of hominids are considered (unpublished data). The WW clade is shown in red and the NW clade in blue (see text for descriptions of clades). Recent Human Origins Explanations of modern human origins are dominated by two competing models, Recent African Replacement and Multiregional Evolution. The Recent African Replacement model assumes that anatomically modern H. sapiens arose as the result of a speciation event ca. 0.13 MYA, and then replaced without introgression (i.e., admixture) non-African archaic humans (e.g., H. neanderthalensis and H. erectus). In contrast, the sensu stricto Multiregional Evolution model assumes that modern H. sapiens evolved from early African descendants (up to 2 MYA). Characteristics of modernity were spread geographically through intercontinental gene flow, but local regional characteristics were maintained through admixture between modern and archaic forms (Wolpoff et al. 1994). Most debates on modern human origins in the recent literature focus on one central question: “Was there admixture (i.e., introgression) between modern and archaic humans?” Both the Recent African Replacement and Multiregional Evolution models have been proposed with numerous variations that include introgression, one of which was recently put forth by Eswaran (2002). Eswaran's Diffusion Wave model proposes that a diffusion wave of modern H. sapiens left Africa (ca. 0.13 MYA) and replaced archaic humans through a process of introgression, natural selection, and gradual demic expansion. The evolutionary history of P. humanus is somewhat consistent with all three models of modern human origins mentioned above; however, the number of ad hoc assumptions required to reconcile host and parasite evolutionary histories varies among the three views of human origins. Each model can account for the deep divergence between the two clades of P. humanus because each recognizes divergences between archaic species of Homo ca. 2 MYA. However, the model that best fits the louse data must account not only for the 1 MY of isolation between archaic and modern forms of lice, but also for a recent population expansion in just one louse lineage, the WW clade. The model must also explain how archaic louse DNA might have been incorporated into the lice of modern H. sapiens. The sensu stricto model of Multiregional Evolution (Wolpoff et al. 1994) predicts continual gene flow between the geographically separated populations of humans following their early migration out of Africa (ca. 2 MYA), which is inconsistent with the louse data. This intercontinental gene flow among humans is required in the Multiregional Evolution model to maintain the continuity of the species H. sapiens. This scenario does not provide the louse populations with the degree of isolation necessary (ca. 1.18 MY) to maintain the two divergent louse lineages, unless we assume that gene flow between human populations was considerably greater than gene flow among their populations of lice. There is no reason to assume such a disparity in gene flow between hosts and parasites. The Multiregional Evolution model also predicts that we should detect the same genetic fingerprint of recent population expansion in both clades of P. humanus, which we do not. The Recent African Replacement model provides the isolation necessary between archaic and modern forms, because it assumes that modern H. sapiens left Africa ca. 0.10 MYA, more than a million years after archaic species of Homo left Africa, and that the modern and archaic humans remained distinct (i.e., no introgression). Furthermore, it explicitly assumes a recent population expansion in modern H. sapiens, which would account for the population expansion seen in our WW clade. However, in the strict sense, this model also predicts that modern H. sapiens replaced archaic forms of humans without introgression (i.e., hybridization), which leaves no obvious mechanism for archaic louse DNA to reach the lice of modern H. sapiens. This lack of host introgression implies, but does not require, a lack of direct physical contact between modern and archaic humans. It is conceivable that direct contact between modern and archaic humans was sufficient to allow the lice to switch hosts without making the assertion that the hosts were interbreeding. Therefore, the Recent African Replacement model is fairly consistent with the louse data, so long as one assumes some level of direct contact (e.g., fighting, sharing/stealing of clothing, etc.) between modern and archaic humans. Eswaran's Diffusion Wave model (2002) is similar to the Multiregional Evolution model in that it permits some level of introgression between modern and archaic humans. However, it is also similar to the Recent African Replacement model in that it assumes the same recent population expansion of modern humans out of Africa (ca. 0.10 MYA), thus providing both the isolation and population expansion necessary to accommodate our louse data. The additional assumption of introgression between modern and archaic forms of humans, which is proposed to have occurred only in the last 0.10 MY, provides a ready vehicle that would have transported archaic louse DNA into the modern louse population. Eswaran's model applied to lice suggests that at the beginning of the diffusion wave of modern H. sapiens leaving Africa (ca. 0.13 MYA), modern and archaic humans had distinct types of lice owing to ≥1 MY of isolation. As modern humans began to replace archaic forms, direct contact between hosts during introgression allowed archaic lice to switch to modern H. sapiens hosts. As previously stated, the recent literature addressing human origins boils down to models that do not permit introgression (strict-sense replacement models) and those of many types that do (admixture models, including variants allied with both the Multiregional Evolution and Recent African Replacement models). All things being equal, our parasite data are most consistent with a limited amount of admixture between modern and archaic humans, because this process presents the opportunity for host switching. However, introgression between modern and archaic humans over a protracted period of time would erode the isolation required to maintain the two louse lineages that we have observed. For example, some variants of the Multiregional Evolution model reject a single origin of modernity in Africa ca. 0.13 MYA in favor of a piecemeal acquisition of modern traits over a long period of time. This long-term admixture is precisely what would disrupt the isolation required to maintain the two louse lineages. Eswaran's Diffusion Wave model, on the other hand, confines admixture to the last ca. 0.10 MY. Our data cannot directly address whether host introgression occurred, because nonsexual, direct contact between hosts is sufficient for parasite transmission. We are confident that “direct contact” would be required for a host switch because these obligate parasitic lice cannot move between individuals without direct physical contact (Buxton 1946; Durden 2001; Canyon et al. 2002; Burgess 2004) and furthermore, they die within 24 h of being removed from their host. However, an examination of Pthirus pubis, the human pubic louse, might shed light on the subject of human admixture because unlike head and body lice, pubic lice are primarily transmitted during intercourse. If our scenario involving lice switching from H. erectus to H. sapiens were true, then the host switch would have brought together two long-separated taxa of lice. It is impossible to know whether this long separation affected the reproductive compatibility of the two louse taxa once reunited. Discriminant function analysis shows no morphological differences between members of the two divergent molecular haplotypes of head lice (see Figure 3). There are other well-defined species of lice (e.g., see Johnson et al. 2002) whose populations show even greater sequence divergence (19% uncorrected sequence divergence) and yet have no discernible morphological differences between populations. It is likely that the two long-separated types of lice have experienced some level of introgression since their secondary contact on modern H. sapiens. The recency of this introgression of archaic louse DNA into modern lice also accounts for the younger coalescence time for the NW clade (0.15 MYA) compared to the WW clade (0.54 MYA). Presumably, the archaic form (i.e., morphotype) of louse either was extirpated along with its host or was assimilated into modern P. humanus. Regardless of the mechanism, ancient louse lineages can be found among the lice of modern H. sapiens. A recent review by Ashford (2000) reported five parasites that occur on humans as closely related pairs of taxa (lice, tapeworms, follicle mites, a protozoan, and bedbugs). The fact that there are five such pairs caused Ashford to ask, were humans once two distinct populations that rejoined after a long separation? The ancient divergences seen in mitochondrial data from P. humanus are clearly consistent with some level of long-term host isolation, and preliminary evidence from nuclear markers (EF1-alpha and 18S rRNA) reveals similarly ancient divergences (unpublished data). Furthermore, the two tapeworm species from humans showed amazingly concordant divergences (Hoberg et al. 2001) and distributional patterns. Our data suggest that the isolation Ashford refers to may be between species of Homo rather than within modern H. sapiens itself. We conclude that the parasites may be very useful in the study of human evolutionary history, because they represent an independent marker of human evolution that has yet to be studied in detail. Materials and Methods Specimen collection and preparation. We collected human head and body lice (P. humanus) from many localities, ranging from remote areas such as the Papua New Guinea highlands to metropolitan areas like Boston (Table 2). We also obtained P. schaeffi (from chimpanzees), Pthirus pubis (from humans), Pedicinus hamadryas (from baboons), and Fahrenholzia pinnata (from a rodent) to use as outgroup taxa in phylogenetic analyses. All lice were preserved in 95% EtOH and stored at −80 °C. DNA was extracted from lice by separating the thorax and abdomen and placing both in digestion buffer (Qiagen DNeasy tissue kit; Qiagen, Valencia, California, United States). Digestion proceeded for 48 h at 55 °C, then followed the manufacturer's protocol. After digestion, each louse was reassembled on a microscope slide as a voucher specimen corresponding to each DNA sequence. Voucher specimens were deposited in the Price Institute of Phthirapteran Research (PIPeR) collection at the University of Utah. Table 2 Specimens of P. humanus and Outgroup Taxa Examined in This Study, Their Collection Locality, and Number of Specimens Examined a From this study b From Leo et al. (2002) c Downloaded from GenBank (see Supporting Information for accession numbers) Phylogenetic analyses: morphological data. Considerable morphological variation exists among different species of primate lice. We examined 155 unordered morphological characters for 113 specimens of P. humanus (from humans), P. schaeffi (from chimpanzees), Pthirus pubis (from humans), Pthirus gorillae (from gorillas), Pedicinus hamadryas (from baboons), and F. pinnata (from a rodent). Morphological data were scored in the software package MacClade v. 4.05 (W. P. Maddison and D. R. Maddison; Sinauer, Sunderland, Massachusetts, United States), and heuristic searches consisting of random stepwise addition (1,000 replicates) and tree bisection/reconnection branch swapping were performed in PAUP* v. 4.0b10 (D. L. Swofford; Sinauer). Branch support was estimated with bootstrapping (tree bisection/reconnection swapping, 1,000 replicates). The complete data matrix is available from TreeBASE (http://www.treebase.org/) as study accession number SN1969. Primate-louse cospeciation. The morphological data set (six species, 155 characters) was compared to the host phylogeny ((((human, chimp), gorilla), baboon), rodent) using reconciliation analysis in Treemap v. 2.0 with default parameters. Treemap determines whether the two phylogenies are more congruent than expected by chance based on randomizations of both the host and parasite phylogeny. Significant congruence between host and parasite phylogenies is interpreted as being the result of a shared evolutionary history (i.e., repeated bouts of cospeciation). Discriminant function analysis. We examined the morphology of P. humanus lice in detail to test for morphological correlates of the differences detected at the molecular level. Busvine (1978) examined a large series of head and body louse specimens and found no discrete morphological differences between the two forms. However, he noted that several morphological characters related to size and shape might be useful in this regard. To test this hypothesis we measured head width, thoracic width, total body length, and second-leg tibia length from a series of 50 slide-mounted adult museum specimens collected by earlier workers prior to our study. The microhabitat (head or body, i.e., clothing) from which these museum specimens were collected was well documented. Canonical discriminant analysis was used to build a predictive model to attempt to distinguish between the head and body forms of P. humanus. The predictor variables were used to build a set of discriminant functions that maximized variation among groups while minimizing within-group variation. The first two canonical discriminant functions explained 100% of the variation within the data. These discriminant functions, which were built using existing museum specimens, were then applied to our newly collected specimens in a blind test to determine whether the specimens could be identified as head or body lice from morphology alone. We were able to classify our samples as head or body lice with a probability of ≥0.95. Indeed, the assignment of adult specimens proved to be 100% accurate when checked against microhabitat data for the new specimens. Phylogenetic analyses: molecular data. Fresh specimens suitable for the collection of molecular data were obtained for five of the six species of lice. We sequenced 1,525 combined base pairs (bp) of the mitochondrial (mtDNA) genes COI (854 bp) and Cytb (671 bp) from 69 individuals of P. humanus, P. schaeffi, Pthirus pubis, Pedicinus hamadryas, and F. pinnata. PCR primers were as follows: (5′−3′) COI, C1-J-1718 GGAGGTTTTGCTAATTGATTAG and H7005 CCGGATCCACNACRTARTANGTRTCRTG; Cytb, L11122 GAAATTTTGGGTCWTTRCTNGG and H11823 GGCATATGCGAATARGAARTATCA. PCR parameters included 94 °C for 30 s, 48 °C for 30 s, and 72 °C for 1.5 min (five cycles), then 30 cycles of 94 °C for 30 s, 52 °C for 30 s, and 72 °C for 1.5 min. Amplified fragments were sequenced in both directions, assembled using Sequencher v. 4.1 (GeneCodes, Ann Arbor, Michigan, United States), and deposited in the NCBI database (see Supporting Information). To ensure that we were not amplifying nuclear copies of mitochondrial genes, we performed additional PCR amplifications using nested sets of overlapping primers. The computer program modelTest (Posada and Crandall 1998) was used as a guide to determine a best-fit (Cunningham et al. 1998) ML model for the molecular data. This model (GTR+I+G) was incorporated into ML branch and bound and heuristic searches in PAUP* with 100 bootstrap replicates. An LRT was used to compare ML estimates from a clock-enforced and an unconstrained analysis. Our data did not depart significantly from the assumption of a molecular clock. Dating nodes in the louse phylogeny. We used the significant cospeciation shown between the primate and louse phylogenies (see Results) as a basis for dating nodes in the louse phylogeny. We used a calibration point of 22.5 ± 2.5 MYA for the split between Pedicinus and Pediculus + Pthirus. The date is based on fossil evidence (Benefit 1993; Leakey et al. 1995) of the split between cercopithecoid primates that host only lice in the genus Pedicinus and hominoid primates that host only lice in the genera Pthirus and Pediculus (Durden and Musser 1994). All reconciliations of the host and parasite trees in our cospeciation analysis determined that this particular host/parasite node represents a cospeciation event (see Results). Using the computer software r8s (M. J. Sanderson, software distributed by the author), we constrained the divergence of Pedicinus + Pthirus/Pediculus to 22.5 ± 2.5 MYA and allowed all other nodes in the louse tree to be determined from our DNA sequence data. Error estimates on divergence dates were calculated by generating 100 bootstrapped data matrices in Phylip (J. Felsenstein, software distributed by the author). Each of these bootstrapped datasets was calibrated with the same 22.5 ± 2.5 MYA divergence. Cospeciation within Homo An ML-based analysis was used to determine whether deep divergences in the louse tree were contemporaneous with divergences of now extinct species of Homo. We calculated the branch length between H. sapiens and H. neanderthalensis (the only species of Homo for which DNA sequence data are available) based on the human mitochondrial hypervariable region II of the D loop (see Supporting Information). This value was scaled according to the average distance between these two taxa and their sister taxon (chimpanzee), providing a relative branch length within the primate tree (e.g., the branch between human and Neandertal is one-fifth the length of the branch that unites them with the chimpanzee). This relative branch length was incorporated into a louse constraint tree, in effect forcing the two clades of P. humanus to conform to a prescribed relative branch length. The resulting likelihood score was compared with the unconstrained tree score using an LRT (d.f. = taxa − 2). If the constrained and unconstrained tree scores are not significantly different, then the host tree topology and branch lengths describe the parasite DNA sequence data as well as the parasite tree itself (Huelsenbeck and Crandall 1997). However, if a significant difference is detected, then the host tree does not fit the parasite data well enough to be explained by cospeciation, and the hypothesis of cospeciation is rejected. Population genetic analyses. Population genetic analyses were performed on a pruned dataset, which contained only specimens of P. humanus (i.e., no outgroup taxa). The computer software package DnaSP (Rozas and Rozas 1999) was used to generate mismatch distributions, to calculate Fu and Li's D* statistic for the P. humanus clades, and to calculate additional population parameters (e.g., Fst and Θ). Ten additional haplotypes from Leo et al. (2002) were used in these population-level analyses (see Supporting Information). To estimate an expected time to coalescence for P. humanus, we used estimates of theta (Θ, from the software package DnaSP [Rozas and Rozas 1999]) and mutation rate (μ) for P. humanus (see below) to calculate louse Nef from the equation Θ = 2Nefμ. The mutation rate (μ = 9.0 × 10−9 substitutions per site per generation) was calculated by determining the expected number of substitutions per site between P. humanus and P. schaeffi under the Tamura-Nei + Γ model of nucleotide substitution. This mutation rate for P. humanus is roughly five to six times faster than that of human mtDNA, excluding the D-loop (Ingman et al. 2000), when both mutation rates are scaled to absolute time (i.e., number of substitutions per site per year). Nef was then used to determine the expected time to coalescence (in generations) given the formula 2Nef (1 − 1/n), where n is the number of haplotypes detected in the population. One can also ask the similar question, what is the probability that two lineages, which are expected to differ by ke substitutions, actually differ by k* or more substitutions? The expression used, (ke /(1+ ke) ^ k*), is derived from the geometric distribution (see, for example, Golding and Strobeck 1982) and for our data suggests that the large number of substitutions found between the WW and NW clades is far greater than that which is expected (p < 0.0006). Supporting Information Accession Numbers GenBank (http://www.ncbi.nlm.nih.gov/Genbank/) accession numbers for items discussed in the text are as follows: the EF1-alpha sequences from Kittler et al. (2003), AY316794–AY316834; the 18S rRNA sequences for P. humanus from Yong et al. (2003), AY236410–AY236418, AF139478–AF139482, AF139484, AF139486, and AF139488; the 18S rRNA sequence from P. schaeffi, AY695939; the Cytb sequence from P. schaeffi, AY316793; the human mitochondrial hypervariable region II of the D loop, M76311, AY195756, AY217615, AF282972, AF142095, X97709, X98472, X93336, X93337, X93347, and X93348; the ten haplotypes from Leo et al. (2002) used in population-level analyses, AF320286; the divergent louse Dennyus hirundinus shown in Figure 4, AF545694 and U96434. The amplified PCR fragments discussed in Materials and Methods have been deposited in the NCBI database under accession numbers AY695939–AY696069. We thank F. Adler, C. Bell, S. Bush, J. Coyne, M. Gitzendanner, M. Hafner, H. Harpending, K. Johnson, W. Potts, J. Seger, and three reviewers for helpful comments. For providing specimens we thank D. Colwell (Agriculture and Agri-Food Canada), K. Rice (Southwest Foundation for Biomedical Research), R. Todd (Insect Control and Research, Inc.), D. Altschuler (National Pediculosis Association), J. O'Connell (Boston Health Care for the Homeless Program), D. Dean (Salt Lake City Public Health Center), Debby Cox (Chimpanzee Sanctuary, Wildlife Conservation Trust, and The Jane Goodall Institute), and Nigel Hill (London School of Hygiene and Tropical Medicine). We also thank C. Lyal and P. Brown at the Natural History Museum, London, and D. Arnold, K.C. Emerson Entomology Museum, Stillwater, Oklahoma, for access to museum collections of Phthiraptera. We thank J. Light for sequences of the rodent outgroup taxon, Fahrenholzia. This research was supported by a University of Utah Funding Incentive Seed Grant and National Science Foundation (NSF) grants to DHC (DEB-0107947 and DEB-0118794), an NSF Postdoctoral Fellowship to DLR (DBI-0102112), and a Wellcome Trust Junior Biodiversity Fellowship to VSS. Conflicts of interests. The authors have declared that no conflicts of interest exist. Author contributions. DLR, VSS, and DHC designed the study. DLR, VSS, and SLH collected and analyzed the data. DLR, VSS, ARR, and DHC wrote the paper. Academic Editor: Nick Barton, University of Edinburgh Citation: Reed DL, Smith VS, Hammond SL, Rogers AR, Clayton DH (2004) Genetic analysis of lice supports direct contact between modern and archaic humans. PLoS Biol 2(11): e340. Abbreviations COIcytochrome oxidase subunit I Cytbcytochrome b HTLVhuman T-cell leukaemia/lymphoma virus LRTlikelihood ratio test MLmaximum likelihood MRCAmost recent common ancestor mtDNAmitochondrial DNA MYmillion years MYAmillion years ago Nefeffective female population size NWNew World WWworldwide ==== Refs References Ashford RW Parasites as indicators of human biology and evolution J Med Microbiol 2000 49 771 772 10966223 Benefit BR The permanent dentition and phylogenetic position of Victoriapithicus from Maboko Island, Kenya J Hum Evol 1993 25 83 172 Burgess IF Human lice and their control Annu Rev Entomol 2004 49 457 481 14651472 Busvine JR Evidence from double infestations for the specific status of human head lice and body lice (Anoplura) Syst Entomol 1978 3 1 8 Buxton PA The louse 1946 Baltimore Williams and Wilkins Co 164 Cann RL Stoneking M Wilson AC Mitochondrial DNA and human evolution Nature 1987 325 31 36 3025745 Canyon DV Speare R Muller R Spatial and kinetic factors for the transfer of head lice (Pediculus capitis) between hairs J Invest Dermatol 2002 119 629 631 12230505 Caramelli D Lalueze-Fox C Vernesi C Lari M Casoli A Evidence for a genetic discontinuity between Neandertals and 24,000-year-old anatomically modern Europeans Proc Natl Acad Sci U S A 2003 100 6593 6597 12743370 Chan SY Bernard HU Ong CK Chan SP Hofmann B Phylogenetic analysis of 48 papilomavirus types and 28 subtypes and variants: A showcase for the molecular evolution of DNA viruses J Virol 1992 66 5714 5725 1326639 Cunningham CW Zhu H Hillis DM Best-fit maximum likelihood models for phylogenetic inference: Empirical tests with known phylogenies Evolution 1998 52 978 987 di Rienzo A Wilson AC Branching pattern in the evolutionary tree for human mitochondrial DNA Proc Natl Acad Sci U S A 1991 88 1597 1601 2000368 Durden LA Samuel WM Pybus MJ Kocan AA Lice (Phthiraptera) Parasitic diseases of wild mammals 2001 Ames Iowa State University Press 3 17 Durden LA Musser GG The sucking 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Fujinaga K The genetic drift of human papillomavirus type 16 is a means of reconstructing prehistoric viral spread and the movement of ancient human populations J Virol 1993 67 6413 6423 8411343 Hoberg EP Alkire NL de Queiroz A Jones A Out of Africa: Origins of the Taenia tapeworms in humans Proc R Soc Lond B Biol Sci 2001 268 781 787 Huelsenbeck JP Crandall KA Phylogeny estimation and hypothesis testing using maximum likelihood Annu Rev Ecol Systematics 1997 28 437 466 Huelsenbeck JP Ronquist F Yang Z Statistical tests of host-parasite cospeciation Evolution 1997 51 410 419 Ingman M Kaessmann H Paabo S Gyllensten U Mitochondrial genome variation and the origin of modern humans Nature 2000 408 708 713 11130070 Johnson KP Williams BL Drown DM Adams RJ Clayton DH The population genetics of host specificity: Genetic differentiation in dove lice (Insecta: Phthiraptera) Mol Ecol 2002 11 25 38 11903902 Kittler R Kayser M Stoneking M Molecular evolution of Pediculus humanus and the origin of clothing Curr Biol 2003 13 1414 1417 12932325 Krings M Stone A Schmitz RW Krainitzki H Stoneking M Neandertal DNA sequences and the origin of modern humans Cell 1997 90 19 30 9230299 Krings M Geisert H Schmitz RW Krainitzki H Paabo S DNA sequence of the mitochondrial hypervariable region II from the Neandertal type specimen Proc Natl Acad Sci U S A 1999 96 5581 5585 10318927 Krings M Capelli C Tschentscher F Geisert H Meyer S A view of Neandertal genetic diversity Nat Genet 2000 26 144 146 11017066 Leakey MG Ungar PS Walker A A new genus of large primate from the late Oligocene of Lothidok, Turkana District, Kenya J Hum Evol 1995 28 519 531 Leal ES Zanotto PMA Viral diseases and human evolution Mem Inst Oswaldo Cruz 2000 95 193 200 Leo NP Campbell NJH Yang X Mumcuolglu K Barker SC Evidence from mitochondrial DNA that head and body lice of humans (Phthiraptera: Pediculidae) are conspecific J Med Entomol 2002 39 662 666 12144300 Levene H Dobzhansky T Possible genetic difference between the head louse and the body louse Am Nat 1959 873 347 353 Ong C Chan S Campo M Fujinaga K Mavromara-Nazos P Evolution of human papillomavirus type 18: An ancient phylogenetic root in Africa and intratype diversity reflect coevolution with human ethnic groups J Virol 1993 67 6424 6431 8411344 Paabo S Wilson AC Miocene DNA sequences—a dream come true? Curr Biol 1991 1 45 46 15336206 Page RD Lee PL Becher SA Griffiths R Clayton DH A different tempo of mitochondrial DNA evolution in birds and their parasitic lice Mol Phylogenet Evol 1998 9 276 293 9562986 Page RDM Temporal congruence revisited: Comparison of mitochondrial DNA sequence divergence in cospeciating pocket gophers and their chewing lice Syst Biol 1996 45 151 167 Page RDM Tangled trees: Phylogeny, cospeciation, and coevolution 2003 Chicago University of Chicago Press 378 Posada D Crandall KA MODELTEST: Testing the model of DNA substitution Bioinformatics 1998 14 817 818 9918953 Reed DL Hafner MS Host specificity of chewing lice on pocket gophers: A potential mechanism for cospeciation J Mammal 1997 78 655 660 Rogers AR Harpending H Population growth makes waves in the distribution of pairwise genetic differences Mol Biol Evol 1992 9 552 569 1316531 Rozas J Rozas R DnaSP version 3: An integrated program for molecular population genetics and molecular evolution analysis Bioinformatics 1999 15 174 175 10089204 Serre D Langaney A Chech M Teschler-Nicola M Paunovic M No evidence of Neandertal mtDNA contribution to early modern humans PLoS Biol 2004 2 e57 10.1371/journal.pbio.0020057 15024415 Shimodaira H Hasegawa M Multiple comparisons of log-likelihoods with applications to phylogenetic inference Mol Biol Evol 1999 16 1114 1116 Stauffer RL Walker A Ryder OA Lyons-Weiler M Hedges SB Human and ape molecular clocks and constraints on paleontological hypotheses J Hered 2001 92 469 474 11948213 Stringer CB Out of Ethiopia Nature 2003 423 692 695 12802315 Stringer CB Andrews P Genetic and fossil evidence for the origin of modern humans Science 1988 239 1263 1268 3125610 Swisher CC Rink WJ Anton SC Schwarcz HP Curtis GH Latest Homo erectus of Java: Potential contemporaneity with Homo sapiens in Southeast Asia Science 1996 274 1870 1874 8943192 Templeton AR Out of Africa again and again Nature 2002 416 45 51 11882887 Vigilant L Stoneking M Harpending H Hawkes K Wilson AC African populations and the evolution of human mitochondrial DNA Science 1991 253 1503 1507 1840702 White TD Asfaw B DeGusta D Gilbert H Richards GD Pleistocene Homo sapiens from Middle Awash, Ethiopia Nature 2003 423 742 747 12802332 Wolpoff MH Thorne AG Smith FH Frayer DW Pope GG Nitecki M Nitecki D Multiregional evolution: A world-wide source for modern human populations Origins of anatomically modern humans 1994 New York Plenum Press 174 199 Wolpoff MH Hawkes J Caspari R Multiregional, not multiple origins Am J Phys Anthropol 2000 112 129 136 10766948 Yong Z Fournier PE Rydkina E Raoult D The geographical segregation of human lice preceded that of Pediculus humanus capitis and Pediculus humanus humanus C R Biol 2003 326 565 574 14558476

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PLoS Biol. 2004 Nov 5; 2(11):e340

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==== Front PLoS BiolPLoS BiolpbioplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 1550287210.1371/journal.pbio.0020341Research ArticleGenetics/Genomics/Gene TherapyDrosophilaX Chromosome Sites Autonomously Recruit the Dosage Compensation Complex in Drosophila Males Dosage Compensation in DrosophilaFagegaltier Delphine 1 Baker Bruce S [email protected] 1 1Department of Biological Sciences, Stanford UniversityStanford, CaliforniaUnited States of America11 2004 5 10 2004 5 10 2004 2 11 e34120 5 2004 9 8 2004 Copyright: © 2004 Fagegaltier and Baker.2004This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. High Affinity: Making Up for Being Male It has been proposed that dosage compensation in Drosophila males occurs by binding of two core proteins, MSL-1 and MSL-2, to a set of 35–40 X chromosome “entry sites” that serve to nucleate mature complexes, termed compensasomes, which then spread to neighboring sequences to double expression of most X-linked genes. Here we show that any piece of the X chromosome with which compensasomes are associated in wild-type displays a normal pattern of compensasome binding when inserted into an autosome, independently of the presence of an entry site. Furthermore, in chromosomal rearrangements in which a piece of X chromosome is inserted into an autosome, or a piece of autosome is translocated to the X chromosome, we do not observe spreading of compensasomes to regions of autosomes that have been juxtaposed to X chromosomal material. Taken together these results suggest that spreading is not involved in dosage compensation and that nothing distinguishes an entry site from the other X chromosome sites occupied by compensasomes beyond their relative affinities for compensasomes. We propose a new model in which the distribution of compensasomes along the X chromosome is achieved according to the hierarchical affinities of individual binding sites. Evidence is presented that the existing model for dosage compensation in Drosophila is incorrect, and a new model is proposed ==== Body Introduction Most X chromosomal genes are essential or relevant to both sexes. To cope with the difference in the number of copies of these genes in females (XX) and males (XY), organisms have evolved a variety of mechanisms, collectively termed dosage compensation, to equalize the levels of X-linked gene products in the two sexes. In Drosophila males the expression of most of the genes on the single X chromosome is doubled. At least six protein-coding genes, collectively referred to as male specific lethals (msls), are required for dosage compensation (Baker et al. 1994; Marin et al. 2000; Meller 2000): msl-1, msl-2, and msl-3, whose functions remain unknown; maleless (mle), encoding an RNA helicase; males absent on the first (mof), encoding a histone acetyltransferase; and jil-1, encoding a histone kinase. The products of these genes, together with noncoding RNAs encoded by the RNA on the X genes(roX1 and roX2) (Amrein and Axel 1997; Meller et al. 1997; Franke and Baker 1999), are all reproducibly associated with hundreds of locations along the length of the polytenized salivary gland X chromosome in males. MOF has been shown both in vivo and in vitro to acetylate H4Lys16, a specific histone modification also found at sites where compensasomes are associated with the male X (Hilfiker et al. 1997; Smith et al. 2000; Akhtar and Becker 2001). Recently, JIL-1, which phosphorylates H3Ser10, was shown to be enriched at the MSL binding sites in males (Wang et al. 2001). Thus, MSL proteins and roX RNAs are thought to function in a ribonucleoprotein complex (compensasome) to mediate dosage compensation by altering chromatin structure of the male X chromosome (Stuckenholz et al. 1999; Franke and Baker 2000). In females translational repression of msl-2 mRNA by the Sex-lethal protein (SXL) prevents formation of compensasomes and hence dosage compensation (Bashaw and Baker 1997; Kelley et al. 1997). The processes and constraints that generate the observed distribution of compensasomes along the male X chromosome are unknown. Although the hundreds of places where compensasomes are found along the X chromosome are referred to as “sites,” they are in fact not points, but rather bands (small segments of chromosome) that roughly span the size range of salivary chromosome bands seen with DNA stains (i.e., a few tens to several hundreds of kilobases in length). Thus, both the locations and the extents of these sites are somehow specified. Furthermore, the compensasome bands do not correspond to the bands where DNA is condensed (Baker et al. 1994; Kelley et al. 1999; Demakova et al. 2003). In addition, non-dosage-compensated X-linked genes (e.g., LSP1-α) are scattered throughout the X chromosome and can reside next to dosage-compensated genes (Baker et al. 1994). Since there is no known DNA-binding component in the compensasome, and consensus DNA sequences required for binding have not yet been identified, an understanding of the distribution of compensasomes along the X chromosome needs to encompass not only how complexes are targeted to these several hundred sites, but also how the ends of each band are delimited. A proposal for how the distribution of compensasome bands along the X chromosome is generated (Kelley et al. 1999) has come from the following findings. MSL-1 and MSL-2 represent core components of the complex: The presence of both is required for either to bind, and none of the other MSL proteins binds to the X chromosome in an msl-1 or msl-2 mutant male (Lyman et al. 1997). Furthermore, in males mutant for mle, msl-3, or mof, binding of MSL-1 and MSL-2 is only maintained at a limited number of sites (35–40) on the X chromosome, which include the roX1 and roX2 genes (Lyman et al. 1997; Kelley et al. 1999). Finally, roX transgenes inserted into an autosome retain binding of compensasomes, and in addition show compensasome binding in the autosomal region flanking the insertion site, a phenomenon termed spreading (Kelley et al. 1999). Based on these observations, a reasonable model (Kelley et al. 1999) emerged suggesting that the 35–40 sites of MSL-1 and MSL-2 binding on the X seen in mle, msl-3, or mof mutants represent nucleation sites or entry sites for the complex. From these sites, newly assembled compensasomes would spread in cis along the X to form the hundreds of final sites observed in a wild-type male. In this spreading model, roX RNAs would also be required for compensasome assembly (Park et al. 2003). However, there is to date no direct evidence that entry sites and spreading play any role in the processes that generate the normal pattern of compensasome binding along the X chromosome. We thus directly tested this model by analyzing various pieces of the X chromosome transposed or translocated to autosomal locations for their ability to bind compensasomes and initiate spreading. Results The spreading model implies that a piece of the X chromosome translocated to an autosome must contain at least one of the 35–40 “entry” sites if that piece of the X is to recruit compensasomes and become dosage compensated. We looked at MSL binding in various chromosome rearrangements that inserted small pieces of X chromosome into autosomal locations. Table 1 summarizes the translocations, transpositions, and duplications examined. The insertions in the first set (lines I to XI) range in size from about 1% to 15% of the length of the X, and the corresponding stretch of X chromosome for each contains 1–19 distinguishable MSL bands. These insertions were examined in heterozygous condition so we could readily identify the junctions between X chromosomal and autosomal material. When large enough, they appear as a loop of unpaired chromosome protruding from the paired autosomes. We found that transpositions containing one (lines VI to VIII) or several (lines I to V) previously described entry sites (Lyman et al. 1997) showed consistent MSL binding along the inserted piece (Table 1; Figure 1A, 1B, and 1D). Surprisingly, transposed pieces of X chromosome lacking any entry site also showed MSL binding when inserted into an autosome (Table 1, lines IX to XI; Figure 1C, 1E, and 1F). For all of these 11 transpositions the binding pattern observed and the intensity of MSL bands reproducibly matched the expected pattern of that piece of the X chromosome in a wild-type male. Even the smallest piece we looked at (line X, approximately 200 kb) showed one to two MSL bands (Figure 1C). Thus, we found that any piece of the X chromosome moved to an autosomal location is able to bind compensasomes, whether or not the transposed piece of X chromosome contains an entry site. This finding suggests that each of the hundreds of MSL bands observed on the X in males carries the information necessary and sufficient to attract compensasomes, and does not require adjacent entry sites. Figure 1 MSL Binding to Pieces of X Chromosome Inserted into Autosomes Salivary glands from males heterozygous for each transposition were fixed (47% acetic acid in phosphate-buffered saline, then lactic acid/water/acetic acid [1:2:3]), squashed on slides, treated with anti-MSL-1 antibodies and a secondary Cy3 anti-rabbit immunoglobulin G antibody, then counterstained with DAPI and viewed using a Zeiss Axiophot microscope. Both duplications and transpositions were able to attract compensasomes, whether or not they contained predicted entry sites. (A) Line II. (B) Line I, which contains the roX1 gene. (C) Line X shows one to two bands on the smallest transposition we studied; the intensity of the second band was variable even on the X chromosome. (D) Line IV. (E) Line IX. (F) Line XI. Breakpoints (described in Table 1) were verified by cytology when possible and/or with specific probes by in situ hybridization. Gray value images were pseudo-colored and merged. Table 1 Summary of the Transpositions Studied: Transpositions, Duplications, and Reciprocal Translocations Variations in both the number of bands observed in the transpositions and their intensity are due to variable accessibility of the piece examined on the squash and the orientation of the chromosomes when flattened for observation. Similar variations were observed on the intact X. No additional MSL binding was observed into autosomal regions flanking translocated X material or onto autosomal material inserted onto the X chromosome. We found a breakpoint in line VI to be at 3F1 instead of 3E7–8, and 5A instead of 4A in line XIII. Line III contains a piece of the X inserted into a pericentric inversion of the second chromosome, while line IV carries an inversion of 77D5–81 See Materials and Methods for precise genotypes aES, number of entry/high-affinity sites present in each transposition according to our observations and previous studies (Lyman et al. 1997) bNumber of MSL bands observed in a wild-type background on each piece of the X inserted onto an autosome (lines I to XI), or number of MSL bands observed in autosomal regions inserted into the X (lines XII to XV) cN, percentage of nuclei showing additional bands in autosomal regions flanking the site of insertion of a piece of the X chromosome; the number of nuclei scored is presented in parentheses Tp, transposition; Dp, duplication; T, reciprocal translocation Interestingly, duplications showed binding both along the autosomal insertion and on the X chromosome (lines II and XI), indicating that the supply of compensasomes is not limiting in these circumstances. We also tested homozygous transpositions and duplications for MSL binding in males and found that we could recover MSL binding on each homozygous transposed piece (unpublished data) as well as on the X. Thus, even three copies of the same segment of the X chromosome (two of the duplication plus the original piece on the X) were able to maintain MSL binding. This result extends previous data showing that, by using specific msl-2 transgenes escaping SXL repression, ectopic expression of MSL-2 in females induced binding to both X chromosomes, in a pattern identical to the single X of a wild-type male (Bashaw and Baker 1997). Therefore, binding occurs regardless of the location and number of copies of the X-linked targeted sequences. The determinations listed in Table 1 of how many entry sites each of the transpositions contains were made by comparing the reported breakpoints of each rearrangement to the described locations of entry sites (Lyman et al. 1997). As cytological determinations can vary, we directly confirmed the presence or absence of entry sites by examining MSL binding in an msl-3 or mle mutant background for a subset of these transpositions (Figure 2). Each line used in these experiments contained the transposed region from the X inserted into an autosome and a wild-type X chromosome. For line XI we found that, in mle mutant individuals, MSL binding was undetectable in either the transposed region (3A5–E8) inserted at 87E17 (Figure 2A–2E) or in this region in the wild-type X. As expected, the same is true when only a subset of this region is duplicated: Line X did not show binding in mle mutants to region 3C2–3C6 on the X or to the transposition of that region inserted at 61D (Figure 2F–2K). These findings confirm that lines X and XI do not contain entry sites. Similarly, we confirmed that transpositions inferred to contain entry sites in two lines (IV and VI) did in fact contain such sites. Thus, for line IV in an mle mutant background we observed MSL binding to one to three sites on both the transposition and the corresponding region of the X (Figure 2N and 2P), while for line VI in an msl-3 mutant background we observed one site of MSL binding on both the transposition and the corresponding region of the X (Figure 2S). These findings are consistent with those of Lyman et al. (1997), who reported two entry sites in the region encompassed by the transposition in line VI, and one entry site in the region encompassed by the transposition in line IV. Our findings firmly establish that isolated subregions of the X chromosome display normal patterns of compensasome binding irrespective of whether they contain entry sites, and thus suggest that entry sites do not play a distinct role in the establishment of compensasome binding along the X as postulated by the spreading hypothesis. Hereafter we will refer to entry sites as high-affinity sites, their original name (Lyman et al. 1997). During the course of this study, Oh et al. (2004) have reported similar results for binding of compensasomes to transpositions from lines I, VIII, and IX. However, the scale of the analysis and the limited number of rearrangements did not yield the same conclusions. Figure 2 MSL Binding to Autosomal Duplications of X Chromosome Pieces in mle or msl-3 Mutant Larvae Salivary glands from w; pr mle 12.17/cn bw mle; Dp (1;3)/msl2Δ10 or w; Dp (1;2)/msl2Δ21; msl3p/msl3p females were squashed and stained as described in Figure 1, followed by in situ hybridization with a biotinylated probe specific for regions carried by each duplication (Lavrov et al. 2004) and incubation with Oregon green-coupled streptavidin. Conditions throughout the procedure were adjusted to maximize MSL staining. Specific biotinylated probes (green bars) appear in green in merges (A, F, I, L, O, Q, and R) and as bright bands in (B, D, G, J, and M). MSL bands are shown in red in merges and in (P) and as bright bands in (C, E, H, K, N, and S). DAPI stain is blue. MSL binding is absent from duplications or the matching region on the X in line XI (3A5–3E8) (A–E) and line X (3C2–3C6) (F–K) in mle mutants, confirming that they lack any entry sites. Probe maps region 3D–E in (A–E) and 3C in (F–K). (L–P) Illustrated are the one to three bands detected in mle mutant nuclei on the duplicated region from line IV (2C1–3C5) (O and P) and on the same segment on the X (M and N). (O) and (P) are from another nucleus. (Q–S) A single band is detected at the 3F1 breakpoint of the duplication (3C2–3F1, line VI) in msl-3 mutant nuclei (S), corresponding to the weakest band of the doublet at 3F on the X. Note the weak signal on duplications compared to the same region on the X chromosome. Probe maps region 2D5–3A2 in (L–P) and 3D–E in (Q–S). The two high-affinity sites identified to date correspond to the roX1 and roX2 genes (Kageyama et al. 2001; Park et al. 2003), and it was the fact that roX transgenes inserted into autosomal locations are able to induce spreading—binding of the MSLs to some autosomal sequences surrounding a roX transgene insertion site—that led to the hypothesis that spreading gives rise to the wild-type distributions of compensasome bands along the male X chromosome. We therefore examined whether autosomal transpositions of a piece of the X were able to induce spreading. In cells heterozygous for each of the transpositions listed above we never observed additional MSL binding to the autosomal regions either cis or trans to the insertion site (Table 1; see Figure 1). We also did not observe additional MSL binding in males homozygous for the transpositions described above. This was true irrespective of the number of high-affinity sites contained in the transpositions. Interestingly, lines I and V, which each contain several high-affinity sites, including the roX1 or roX2 gene, respectively, showed no spreading in males wild-type for the MSLs (see Figure 1B). The dichotomy between our results and those obtained with roX transgenes suggests that spreading may be a phenomenon restricted to some roX transgenes (see below) and not an aspect of dosage compensation. To further assess if spreading in cis occurs on the X chromosome, we next asked if the complex could spread from the X onto an autosomal piece attached to the X by a reciprocal translocation. We tested two reciprocal translocations that interchanged large portions of the X and 3R or 2L (see Table 1, lines XII and XIII, respectively). Both translocations separate roX1 (3F) and roX2 (10C) genes from one another and thus both pieces of each translocation contain a roX locus. Anti-MSL-1 staining revealed the absence of any bands on either of the 3R or 2L pieces of these translocations (Figure 3), while the pattern observed on the two transposed pieces of the X was normal. These results strengthen the idea that spreading may be a phenomenon restricted to roX transgenes, since the breakpoints in line XII (10A) and line XIII (5A) are relatively close to the roX2 (10C) and roX1 (3F) loci, respectively. Figure 3 Compensasomes Do Not Spread from the X Chromosome onto Autosomal Regions Inserted on the X (A) Females expressing MSL-2 from an msl2Δ3–21 transgene and bearing a reciprocal translocation between the X and second chromosome (line XIII) do not show additional bands in the regions of the 2L arm juxtaposed to X chromosome material. (B) MSL binding pattern on the X chromosome of a wild-type male. (C and D) The autosomal region 81F–82F10–11 does not show MSL binding when inserted at 3D in the single X of a male (line XV) (C) or in MSL-2-expressing females heterozygous for the same transposition (D). Note that the MSL binding pattern on the X chromosome is not altered by the insertion. The light band (arrow) maintained on the wild-type unpaired region of the X of a female heterozygous for the transposition is also present next to the same insertion at 3D on the unique X chromosome of a male (compare C and D). We also tested two small transpositions of autosomal regions into the X (Table 1, lines XIV and XV; Figure 3C): Neither of them showed MSL binding, even weak, to any part of the inserted autosomal sequences. Furthermore, females either heterozygous or homozygous for these transpositions and expressing ectopic MSL-2 did not show any MSL bands in either of these insertions of autosomal material into the X, although they displayed normal MSL binding both to the unpaired X region (in heterozygotes) and along the paired portions of the two X chromosomes (Figure 3D). Thus, insertion of a piece of an autosome into the X does not disrupt MSL binding to either the unpaired X homologue at the insertion site or the regions of the X immediately flanking the site of insertion of autosomal material. Moreover, these results are inconsistent with the model derived from the roX transgene studies where MSL binding is observed both in the autosomal regions adjacent to the insertion site and on the wild-type autosomal homologue. Discussion In summary, we have used chromosome rearrangements to test two central aspects of the proposed spreading model of dosage compensation in Drosophila. It is worth noting that our experiments were a priori neutral: They could have provided compelling evidence for or against the spreading model. In both cases our results are inconsistent with the clear predictions of that model. First, we show that pieces of the X chromosome inserted into an autosome bind compensasomes in precisely the pattern characteristic of that piece of the X at its endogenous location on the X, and this property is independent of the presence of sites previously described as entry sites. Second, compensasomes do not spread from the X into autosomal pieces inserted into, or translocated onto, the X. Moreover, there is not spreading of compensasomes from autosomal insertions of pieces of the X chromosome into the autosomal regions flanking the insertion, even when such pieces contain a roX gene close to the breakpoint. These results suggest that spreading in cis is not part of the process of dosage compensation in flies. We thus propose that all of the hundreds of sites along the X chromosome where compensasomes are found in wild-type males are competent to independently recruit compensasomes. Our findings raise several questions regarding previous data. Are the 35–40 sites that attract partial complexes in mle or msl-3 mutants qualitatively different from the other sites at which MSL bands are found in wild-type, and if so, how? Why do roX transgenes induce additional binding to adjacent autosomal sequences? With respect to the potential heterogeneity of compensasome binding sites, while most of the relevant data are indirect (only the roX1 and roX2 genes are identified binding sites), the data are consistent with the simple view that the binding sites are homogeneous in terms of their function, but have varying affinities for compensasomes. Our finding that pieces of X chromosome transposed to autosomal locations display normal patterns of compensasome binding, irrespective of whether or not they contain high-affinity sites, removes the one functional distinction between binding sites that had been proposed. That there are not two classes of binding sites in terms of affinity for compensasomes, but rather a continuum of affinities, is strongly suggested by the recent report of Demakova et al. (2003), who carefully characterized the number and locations of compensasome bands in mutant females expressing various limiting amounts of MSL-2. They found only four bands in the most limiting case, and progressively higher numbers of bands as more MSL-2 protein was expressed. Interestingly, the intermediate 40 sites at which complete complexes are assembled in these conditions exactly matched with the 35–40 high-affinity sites bound by partial complexes in mle or msl-3 mutants. Their data are consistent with a model in which compensasomes continue to bind site specifically to additional sites after all high-affinity sites are occupied, as opposed to spreading from high-affinity sites as previously proposed. Given these findings, a reasonable scenario as to how dosage compensation is achieved would be the following. As MSL expression begins, the high-affinity sites progressively sequester nascent partial or full complexes in the early stages of dosage compensation. When the amount of available complexes or its components increases, sites of higher affinity would accumulate more complexes, while low-affinity sites would remain undetectable, until the former have preferentially assembled sufficient amounts of complexes to make components available for sites with lower affinities. Thus, the compensasomes would progressively bind to different sites along the X according to the different affinities of these sites. Consistent with our model, we found that in mle or msl-3 mutants, duplications maintain binding of partial complexes at the high-affinity sites (Figure 2N, 2P, and 2S), though with a lower affinity than the same site on the X. The latter observation suggests that, in conditions where components of the complex are limiting, binding might also be dependent on the location of these sequences in the cell (see discussion on spreading below). That compensasome binding sites would have a range of affinities is also consistent with what is known about DNA-binding proteins, which recognize with varying affinities a range of binding sites whose sequences are related to a common consensus. Variations from the consensus can allow temporal and quantitative modulation of individual genes, or subsets of genes. That compensasome binding sites are also likely to vary in sequence, and hence affinities, comes from what is known about sex chromosome evolution in Drosophila species (Marin et al. 1996, 2000). During the course of sex chromosome evolution in this genus there are a number of cases in which new X chromosomes have evolved, and in all cases examined to date, this has been accompanied by the new X chromosome gradually acquiring compensasome binding sites as the new Y chromosome, its former homologue, degenerates. The selective advantage of dosage compensation for each gene is determined both by the state of degeneration of the allele on the new Y chromosome and by the degree to which a gene in males requires its function, and thus its expression, to match the output of both wild-type female X chromosomes (Marin et al. 2000). Hence, one would expect individually evolved binding sites to exhibit a range of affinities for compensasomes. Finally, we note that each of the final compensasome bands on the X chromosome displays a reproducible but specific intensity, likely to reflect not only different affinities for compensasomes, but also the length of X chromosome encompassed in each band. The last issue we wish to address is spreading. The fact that, in chromosome rearrangements that juxtapose pieces of X and autosome, we never observed spreading, even when entry sites or roX genes were near the breakpoints, suggests that spreading does not exist naturally on the X chromosome, and is not required to establish the final pattern of binding in Drosophila males. Yet spreading from roX transgenes is very well documented in a variety of situations. We therefore suggest that spreading is a phenomenon specific to the roX transgenes, and a consequence of the key function of roX RNAs in dosage compensation. In particular, we propose that the roX genes are the sites of assembly of compensasomes using newly synthesized roX RNAs, just as the ribosomal RNA genes are the sites where ribosomes are assembled. Thus, roX transgenes would generate a high local concentration of compensasomes in their vicinity, competing with other chromatin-binding factors that normally bind to nearby autosomal sequences. In some cases, compensasomes would displace these other factors, resulting in a new compensasome band in the autosomal region flanking the transgene (spreading). Several features of spreading are consistent with this proposal. First, additional bands corresponding to spreading from roX transgenes contain roX RNA and the H4Lys16 modification, suggesting that they correspond to mature complexes (Kelley et al. 1999). Second, transcription from a roX transgene is required to observe spreading of the complex onto neighboring regions (Park et al. 2002, 2003). Third, roX transgenes show variable and often no additional bands in a wild-type background, suggesting that spreading is largely dependent on the insertion site and its environment on the autosomes. One possibility would be that these roX transgenes lacking spreading are inserted next to sites bound by factors normally counteracting the effect of compensasomes on the autosomes. Such a view is supported by recent data showing that association of compensasomes at some roX1 transgenes can overcome the effect of methylation-mediated silencers (Kelley and Kuroda 2003). Finally, MSL-1 and MSL-2 co-overexpression leads to mislocalization of partial MSL complexes to the autosomes and the centromere, as well as a dramatic decompaction of the X (Oh et al. 2003), a male-specific phenotype also observed in both iswi or nurf mutants, two chromatin regulators (Deuring et al. 2000; Badenhorst et al. 2002; Corona et al. 2002). Thus, increasing locally the amount of available complexes can induce new binding of MSL complexes to usually non-dosage-compensated regions. Molecular studies of dosage compensation in flies, worms, and mammals have revealed some striking similarities between these systems. In all three systems dosage compensation is achieved by a widespread modification of the structure of X chromosome chromatin, and in mammals and flies this involves specific modifications of histones. Dosage compensation in mammals and flies is also similar in that noncoding RNAs are essential components of the dosage compensation machinery. With respect to the other components of the dosage compensation machinery the situation is less clear. While compensasome-related complexes might be present in mammals (orthologs of msl-1, -2, -3, mle, and mof genes exist in mammalian genomes), some of them have identified functions not related to dosage compensation, and orthologs of msl-1, -2, and -3 were not found in Caenorhabditis elegans (Marin 2003). Up until now it had also been thought that spreading was involved in dosage compensation in all three systems (Park et al. 2002; Oh et al. 2003; Csankoversuszki et al. 2004; Okamoto et al. 2004). However, our findings indicate that in flies each of the bands on the X chromosome at which compensasomes are found in males is able to independently attract those complexes. Thus, at the interband level spreading does not appear to be part of the dosage compensation process in flies. However, it should be noted that our results do not address either how compensasomes are distributed across the tens of kilobases of DNA that likely comprise individual compensasome bands in salivary gland chromosomes, or how that distribution is achieved; it is possible that, at the level of single bands, spreading may be part of the process of dosage compensation. Materials and Methods Fly strains and genetic crosses Flies were raised on standard cornmeal-yeast-agar medium. Fly stocks containing transpositions were obtained from the Bloomington Drosophila Stock Center. Their genotypes are: Tp(1;2)rb+71 g, ct6 v1/C(1)DX, y1 w1 f1 (line I); Df(1)ct-J4, In(1)dl-49, f1/C(1)DX, y1 w1 f1; Dp(1;3)sn13a1/+ (line II); Tp(1;2)sn+72d, f1 car1/C(1)DX, y1 f1; Dp(?;2)bwD, bwD (line III); Tp(1;3)wvco, v1 f1: in wvco/ClB, B36d (line IV); Tp(1;3)v+74c/FM7a (line V); Tp(1;2)w-ec, ec64d cm1 ct6 sn3/C(1)DX, y1 w1 f1 (line VI); Tp(1;3)f+71b/FM6 (line VII); Tp(1;3)JC153, v1/FM7a (line VIII); Tp(1;3)sta, sta1: sssta/FM3 (line IX); Tp(1;3)wzh, sc1 z1 wzh (line X); Df(1)w258–45, y2 sn3/C(1)DX, y1 w1 f1; Dp(1;3)w+67k/+ (line XI); T(1;3)v, vA/FM6 (line XII); Tp(2;1)odd1.10, b1 pr1 cn1 sca1/CyO (line XIII); Df(2 l)sc19–7/In(2 l)CyLtR In(2R)Cy, Cy1 amosRoi-1 cn2 sp2 or Dp(2;1)B19, y1 ed1 dpo2 cl1 (line XIV); Dp(3;1)2–2, w1118; Df(3R)2–2/TM3, Sb1 (line XV). Breakpoints and insertion site are referred in Table 1. Some lines contain additional rearrangements referenced in Lindsey and Zimm (1992). Depending on their genotype, each line was crossed to Canton-S males or females for studies of MSL binding in their male progeny. For homozygous transpositions studies, stocks were balanced to give w; Tp(1;2)/Cyo-GFP or w; Tp(1;3)/TM3-GFP stocks. Non-GFP third instar male larvae were dissected for analysis. For autosome-to-X transpositions, females from lines XIV and XV were mated with w; msl2Δ3–21/CyoGFP or Dp(A;1)/Y; msl2Δ3–21/CyoGFP males. Non-GFP female larvae were dissected. For mle and msl-3 mutant analysis, stocks were balanced to give w; Tp(1;2)/CyoGFP; msl3p/TM3-GFP or w; prmle12.17/CyoGFP; Tp(1;3)/TM3-GFP stocks. Females were crossed to w; msl3p/CyoGFP; msl2Δ3–10/TM3-GFP or mle1cnbw/CyoGFP; msl2Δ3–21/TM3-GFP males, respectively. Non-GFP third instar female larvae were dissected for salivary glands polytene chromosomes analysis. Lines expressing MSL-2 from transgenes msl2Δ3–21 and msl2Δ3–10 are described in Bashaw and Baker (1995). Mle and msl-3 mutants are described in Fukunaga et al. (1975), Kuroda et al. (1991), and Gorman et al. (1995). All crosses to generate larvae for immunostaining were carried out at 18 °C. Polytene chromosome immunostaining Glands from male third instar larvae were dissected in PBS/0.7% NaCl, prefixed in 45% acetic acid for 10 s, and then fixed for 2–3 min in lactic acid/water/acetic acid (1:2:3) solution on siliconized coverslips. Glands were squashed and coverslips flipped off after freezing the slides in liquid nitrogen. Slides were then incubated in PBS for 15 min followed by incubation with affinity-purified anti-MSL-1 antibodies (dilution 1:100) as described previously (Gorman et al. 1995). Chromosomes were viewed under epifluorescence optics on a Zeiss Axiophot microscope or a confocal microscope; pictures were taken using Spot software and colored. Immunofluorescent in situ hybridization of polytene chromosomes Clones RP-98 17.E.2, RP-98 03.D.13, and RP-98 48.O.22 from the Drosophila melanogaster BAC library (BACPAC Resources, Oakland, California, United States) were used to map regions 3D–E, 3C, and 2D5–3A2, respectively. Specific probes were obtained from BAC clone DNA preparations using the Bionick Labelling System (Invitrogen, Carlsbad, California, United States) according to the manufacturer's instructions. Squashes were prepared as described above. Immunostaining with affinity-purified anti-MSL-1 antibodies was followed by incubation with the appropriate biotinylated probe according to the method of Lavrov et al. (2004). Supporting Information Accession Numbers The LocusLink (http://www.ncbi.nlm.nih.gov/LocusLink/) accession numbers for the genes and gene products discussed in this paper are jil-1 (LocusLink 39241), mle (LocusLink 35523), mof (LocusLink 31518), msl-1 (LocusLink 35121), msl-2 (LocusLink 33565), msl-3 (LocusLink 38779), roX1 (LocusLink 43963), roX2 (LocusLink 44673), and SXL (LocusLink 44872). We thank Greg Bashaw for the msl2Δ10 and msl2Δ21 lines, and would like to express our gratitude to Victoria Meller, Joseph Lipsick, Liqun Luo, and the Bloomington Stock Center for providing stocks, and to Marc Tessier-Lavigne for the use of the microscope. Zeina Ferzli gave inestimable help with squashes, and Guennet Bohm with skillful preparation of fly media. We thank members of the Bruce Baker and Mike Simon labs for helpful discussions and encouragement, and Mark Siegal and Dev Manoli for comments on the manuscript. This work is supported by grants from the National Institute of General Medical Sciences. Conflicts of interest. The authors have declared that no conflicts of interest exist. Author contributions. DF and BSB conceived and designed the experiments. DF performed the experiments. DF and BSB analyzed the data. DF and BSB wrote the paper. Academic Editor: R. Scott Hawley, Stowers Institute for Medical Research Citation: Fagegaltier D, Baker BS (2004) X chromosome sites autonomously recruit the dosage compensation complex in Drosophila males. PLoS Biol 2(11): e341. Abbreviations Dpduplication mlemaleless mofmales on the first mslmale specific lethal roXRNA on the X Sxlsex lethal Ttranslocation Tptransposition ==== Refs References Akhtar A Becker PB The histone H4 acetyltransferase MOF uses a C2HC zinc finger for substrate recognition EMBO Rep 2001 2 113 118 11258702 Amrein H Axel R Genes expressed in neurons of adult male Drosophila Cell 1997 88 459 469 9038337 Badenhorst P Voas M Rebay I Wu C Biological functions of the ISWI chromatin remodeling complex NURF Genes Dev 2002 16 3186 3198 12502740 Baker BS Gorman M Marin I Dosage compensation in Drosophila Annu Rev Genet 1994 28 491 521 7893138 Bashaw GJ Baker BS The msl-2 dosage compensation gene of Drosophila encodes a putative DNA-binding protein whose expression is sex specifically regulated by Sex-lethal Development 1995 121 3245 3258 7588059 Bashaw GJ Baker BS The regulation of the Drosophila msl-2 gene reveals a function for Sex-lethal in translational control Cell 1997 89 789 798 9182767 Corona DF Clapier CR Becker PB Tamkun JW Modulation of ISWI function by site-specific histone acetylation EMBO Rep 2002 3 242 247 11882543 Csankoversuszki G McDonel P Meyer BJ Recruitment and spreading of the C. elegans dosage compensation complex along X chromosomes Science 2004 303 1182 1185 14976312 Demakova OV Kotlikova IV Gordadze PR Alekseyenko AA Kuroda MI The MSL complex levels are critical for its correct targeting to the chromosomes in Drosophila melanogaster Chromosoma 2003 112 103 115 14579126 Deuring R Fanti L Armstrong JA Sarte M Papoulas O The ISWI chromatin-remodeling protein is required for gene expression and the maintenance of higher order chromatin structure in vivo Mol Cell 2000 5 355 365 10882076 Franke A Baker BS The rox1 and rox2 RNAs are essential components of the compensasome, which mediates dosage compensation in Drosophila Mol Cell 1999 4 117 122 10445033 Franke A Baker BS Dosage compensation rox! Curr Opin Cell Biol 2000 12 351 354 10801462 Fukunaga A Tanaka A Oishi K Maleless, a recessive autosomal mutant of Drosophila melanogaster that specifically kills male zygotes Genetics 1975 81 135 141 812765 Gorman M Franke A Baker BS Molecular characterization of the male-specific lethal-3 gene and investigations of the regulation of dosage compensation in Drosophila Development 1995 121 463 475 7768187 Hilfiker A Hilfiker-Kleiner D Pannuti A Lucchesi JC mof, a putative acetyl transferase gene related to the Tip60 and MOZ human genes and to the SAS genes of yeast, is required for dosage compensation in Drosophila EMBO J 1997 16 2054 2060 9155031 Kageyama Y Mengus G Gilfillan G Kennedy HG Stuckenholz C Association and spreading of the Drosophila dosage compensation complex from a discrete roX1 chromatin entry site EMBO J 2001 20 2236 2245 11331589 Kelley RL Kuroda MI The Drosophila roX1 RNA gene can overcome silent chromatin by recruiting the male-specific lethal dosage compensation complex Genetics 2003 164 565 574 12807777 Kelley RL Wang J Bell L Kuroda MI Sex lethal controls dosage compensation in Drosophila by a non-splicing mechanism Nature 1997 387 195 199 9144292 Kelley RL Meller VH Gordadze PR Roman G Davis RL Epigenetic spreading of the Drosophila dosage compensation complex from roX RNA genes into flanking chromatin Cell 1999 98 513 522 10481915 Kuroda MI Kernan MJ Kreber R Ganetzky B Baker BS The maleless protein associates with the X chromosome to regulate dosage compensation in Drosophila Cell 1991 66 935 947 1653648 Lavrov S Dejardin J Cavalli G Combined immunostaining and FISH analysis of polytene chromosomes Methods Mol Biol 2004 247 289 303 14707354 Lindsey DL Zimm GG The genome of Drosophila melanogaster 1992 San Diego Academic Press 804 1066 Lyman LM Copps K Rastelli L Kelley RL Kuroda MI Drosophila male-specific lethal-2 protein: Structure/function analysis and dependence on MSL-1 for chromosome association Genetics 1997 147 1743 1753 9409833 Marin I Evolution of chromatin-remodeling complexes: Comparative genomics reveals the ancient origin of “novel” compensasome genes J Mol Evol 2003 56 527 539 12698291 Marin I Franke A Bashaw GJ Baker BS The dosage compensation system of Drosophila is co-opted by newly evolved X chromosomes Nature 1996 383 160 163 8774878 Marin I Siegal ML Baker BS The evolution of dosage-compensation mechanisms Bioessays 2000 22 1106 1114 11084626 Meller VH Dosage compensation: Making 1X equal 2X Trends Cell Biol 2000 10 54 59 10652515 Meller VH Wu KH Roman G Kuroda MI Davis RL roX1 RNA paints the X chromosome of male Drosophila and is regulated by the dosage compensation system Cell 1997 88 445 457 9038336 Oh H Park Y Kuroda MI Local spreading of MSL complexes from roX genes on the Drosophila X chromosome Genes Dev 2003 17 1334 1339 12782651 Oh H Bone JR Kuroda MI Multiple classes of MSL binding sites target dosage compensation to the X chromosome of Drosophila Curr Biol 2004 14 481 487 15043812 Okamoto I Otte AP Allis CD Reinberg D Heard E Epigenetic dynamics of imprinted X inactivation during early mouse development Science 2004 303 644 649 14671313 Park Y Kelley RL Oh H Kuroda MI Meller VH Extent of chromatin spreading determined by roX RNA recruitment of MSL proteins Science 2002 298 1620 1623 12446910 Park Y Mengus G Bai X Kageyama Y Meller VH Sequence-specific targeting of Drosophila roX genes by the MSL dosage compensation complex Mol Cell 2003 11 977 986 12718883 Smith ER Pannuti A Gu W Steurnagel A Cook RG The Drosophila MSL complex acetylates histone H4 at lysine 16, a chromatin modification linked to dosage compensation Mol Cell Biol 2000 20 312 318 10594033 Stuckenholz C Kageyama Y Kuroda MI Guilt by association: Non-coding RNAs, chromosome-specific proteins and dosage compensation in Drosophila Trends Genet 1999 15 454 458 10529808 Wang Y Zhang W Jin Y Johansen J Johansen KM The JIL-1 tandem kinase mediates histone H3 phosphorylation and is required for maintenance of chromatin structure in Drosophila Cell 2001 105 433 443 11371341

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==== Front PLoS BiolPLoS BiolpbioplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 1550287310.1371/journal.pbio.0020351Research ArticleEcologyEvolutionInfectious DiseasesMicrobiologyZoologyEpidemiology/Public HealthFrogsYeast and FungiEndemic Infection of the Amphibian Chytrid Fungus in a Frog Community Post-Decline Endemic Infection of Chytrid FungusRetallick Richard W. R 1 2 3 McCallum Hamish [email protected] 2 Speare Rick 4 1School of Tropical Biology, James Cook UniversityTownsville, QueenslandAustralia2Department of Zoology and Entomology, University of QueenslandSt Lucia, QueenslandAustralia3School of Life Sciences—Biology, Arizona State UniversityTempe, ArizonaUnited States of America4Amphibian Diseases Group, School of Public Health and Tropical Medicine, James Cook UniversityTownsville, QueenslandAustralia11 2004 5 10 2004 5 10 2004 2 11 e35126 3 2004 12 8 2004 Copyright: © 2004 Retallick et al.2004This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. Endangered Frogs Coexist with Fungus Once Thought Fatal The chytrid fungus Batrachochytrium dendrobatidis has been implicated in the decline and extinction of numerous frog species worldwide. In Queensland, Australia, it has been proposed as the cause of the decline or apparent extinction of at least 14 high-elevation rainforest frog species. One of these, Taudactylus eungellensis, disappeared from rainforest streams in Eungella National Park in 1985–1986, but a few remnant populations were subsequently discovered. Here, we report the analysis of B. dendrobatidis infections in toe tips of T. eungellensis and sympatric species collected in a mark-recapture study between 1994 and 1998. This longitudinal study of the fungus in individually marked frogs sheds new light on the effect of this threatening infectious process in field, as distinct from laboratory, conditions. We found a seasonal peak of infection in the cooler months, with no evidence of interannual variation. The overall prevalence of infection was 18% in T. eungellensis and 28% in Litoria wilcoxii/jungguy, a sympatric frog that appeared not to decline in 1985–1986. No infection was found in any of the other sympatric species. Most importantly, we found no consistent evidence of lower survival in T. eungellensis that were infected at the time of first capture, compared with uninfected individuals. These results refute the hypothesis that remnant populations of T. eungellensis recovered after a B. dendrobatidis epidemic because the pathogen had disappeared. They show that populations of T. eungellensis now persist with stable, endemic infections of B. dendrobatidis. Recapture experiments provide evidence that some amphibian species can now persist with infections of the pathogenic chytrid fungus and suggests, for example, that frogs and fungus might be coevolving ==== Body Introduction Increasingly, the amphibian chytrid fungus (Batrachochytrium dendrobatidis) has been implicated as a major contributor to global catastrophic declines in frog populations (Berger et al. 1998; Daszak et al. 1999, 2003). It has been found on frogs in areas where catastrophic declines were reported, it has been shown in the laboratory to be highly pathogenic to some species, and there is pathological evidence to link this fungal parasite to host mortality (Berger et al. 1998). The pathogen may therefore be capable of producing the extremely high mortality observed during declines. However, little information is available on the impact of the fungus on individuals in the field, rather than the laboratory. Furthermore, little has been published on the prevalence of infection among frog populations as a whole, as distinct from the prevalence among morbid frogs only. In addition to data on the prevalence of the pathogen among morbid animals, information on the prevalence of a putative pathogen in the population in general is important to determine the potential effect of the pathogen on the host population (McCallum and Dobson 1995). In Queensland, Australia, there have been extinctions or major declines of at least 14 frog species in undisturbed, high-elevation rainforest streams, commencing in 1979–1981 in the Conondale and Blackall Ranges (26°50′ S, 152°41′ E), followed in 1985–1986 in the Eungella region of the Clarke Range (21°07′ S, 148°29′ E), and, in 1990–1995, in the Wet Tropics bioregion (17°22′ S, 145°49′ E) (Laurance et al. 1996; McDonald and Alford 1999). Laurance et al. (1996, 1997) suggested that an epidemic disease was responsible for all these declines, without proposing an agent. Despite the presence of B. dendrobatidis in ill and dead frogs collected from the Big Tableland in the Wet Tropics in 1993, it was not recognised as a pathogenic organism until 1998 (Berger et al. 1998) and was described as a new species of fungus in 1999 (Longcore et al., 1999). B. dendrobatidis has been suggested as the causative agent of many of the east coast Australian declines (Berger et al. 1999a), although only the decline at Big Tableland (McDonald and Alford 1999) had direct evidence of an association with the presence of B. dendrobatidis. In this paper, we report the retrospective analysis of B. dendrobatidis infection on toe tips collected between 1994 and 1998 from six species of frogs at Eungella National Park in east-central Queensland, Australia (Figure 1). Declines at this location were particularly catastrophic. Between 1985 and 1986, the Eungella Gastric-Brooding Frog (Rheobatrachus vitellinus) disappeared suddenly from relatively undisturbed rainforest streams, and it is now considered to be extinct (McDonald 1990; Campbell 1999; Department of Environment and Heritage 2003). During the same period, the Eungella Torrent Frog (Taudactylus eungellensis) also disappeared, but it was later found to have persisted in a few small populations (McDonald 1990; Couper 1992; McNellie and Hero 1994; Retallick et al. 1997), which are the subject of this paper. A suite of other species that coexisted with R. vitellinus and T. eungellensis showed no evidence of decline. Unfortunately, the extent of infection with B. dendrobatidis in the frog community at Eungella during the period of decline is unknown. The first record of B. dendrobatidis at Eungella was from a moribund frog collected in 1995 (Berger et al. 1999a). Figure 1 Location of Study Sites 1, Mount David Creek; 2, Mount William Creek; 3, Dooloomai Falls; 4, Rawson Creek; 5, Picnic Ground Creek; and 6, Tree Fern Creek. Results We detected B. dendrobatidis on 71 (15.0%) of the 474 toes assessed. Four species showed no sign of infection, while we detected infections on 58 (18.4%) of 316 toes of T. eungellensis and 13 (34%) of 47 toes of frogs identified at the time as Litoria lesueuri (Table 1). Recently, the taxonomy of L. lesueuri has been revised (Donellan and Mahoney, 2004), with North Queensland members of the complex being either L. jungguy or L. wilcoxii. From the reported distributions of these species, our “L. lesueuri” may have been either one of the species or even hybrids (Donnellan and Mahoney, 2004; Michael Mahoney, personal communication) These species can be distinguished only with genetic information. We therefore describe them as L. wilcoxii/jungguy for the remainder of this paper. Table 1 Prevalence of Infection of B. dendrobatidis in All Frog Toes Examined Sample sizes are in parentheses. A small number of cases with uncertain diagnoses have been omitted Using a logistic model with season (summer, autumn, winter, and spring), species, and site as predictor variables, we found significant effects of each variable on infection, corrected for the effects of the other variables (for season, the change in deviance [Δdev] = 15.25, df = 3, p = 0.0061; for site, Δdev = 13.56, df = 5, p = 0.02; for species, Δdev = 17.32, df = 5, p = 0.004). The most parsimonious model (i.e., one that minimizes the Akaike Information Criterion [AIC]) included each of these predictors but no interaction terms. Further analysis was concentrated on the two species on which B. dendrobatidis was detected, namely T. eungellensis and L. wilcoxii/jungguy. Infection in T. eungellensis The proportions of frogs that were infected in the three largest populations (at Rawson Creek, Dooloomai Falls, and Tree Fern Creek [Figure 1]) differed significantly among those sites (Δdev = 12.84, df = 2, p = 0.001). A significantly smaller proportion was infected at Rawson Creek (10.5%) than at Dooloomai Falls (26.7%) or Tree Fern Creek (25.0%). There was a marginally significant difference overall in the infection levels among males, females, and subadults (Δdev = 5.9, df = 2, p = 0.052). However, when site was included in the model, any suggestion of a difference in infection level between males, females, or subadults disappeared. When we separated the main T. eungellensis populations, it was apparent that the difference in prevalence between those categories was influenced by the population at Tree Fern Creek, where 46% of males (n = 13) and no females or subadults were infected (Table 2). When we grouped all sites and times, the estimated overall prevalence of infection was 18.1%. Table 2 Prevalence of Infection of B. dendrobatidis in T. eungellensis by Age/Sex Class and Site, Pooled over Sampling Times Sample sizes are shown in parentheses. A small number of metamorphlings and adult frogs, the sex of which was not recorded, have been excluded At these three sites, levels of infection among T. eungellensis varied significantly among seasons (Δdev = 14.605, df = 3, p = 0.002), but not among years (Δdev = 3.433, df = 3, p = 0.26). Further, there was no evidence that the pattern of seasonal variation in infection changed among years (Δdev = 5.561, df = 6, p = 0.49). Infection was most prevalent (37.8%) during the winter months (1 June to 31 August) and least prevalent (11.3%) during the summer months (1 December to 28/29 February). Comparing the two sites with the largest sample sizes, Dooloomai Falls and Rawson Creek, there was no evidence that they had differing seasonal patterns of infection (Δdev = 5.32, df = 3, p = 0.150), although the level of infection overall was much higher at Dooloomai Falls (log odds ratio = 1.1612, standard error [se] = 0.3586, p = 0.0012). Infection levels were much higher in winter and spring combined than in summer and autumn (log odds ratio = 1.360, se = 0.3589, p = 0.00015), and there was no evidence of infection levels differing between winter and spring or between summer and autumn (Δdev = 1.107, df = 2, p = 0.5). The seasonal changes in infection at the two sites are shown in Figure 2. Figure 2 Seasonal Patterns of Prevalence of B. dendrobatidis in T. eungellensis Solid circles show the observed prevalence, with binomial 95% confidence limits, in frogs pooled over years and age/sex class. The dashed line shows the prevalence predicted from the best-fitting logistic model. Numbers in brackets above each error bar are the sample sizes. Infection in L. wilcoxii/jungguy B. dendrobatidis was detected in 13 of the 47 L. wilcoxii/jungguy examined, giving an estimated prevalence of 27.7%, with a 95% confidence interval ranging from 15.6% to 42.6%. There was no evidence that prevalence of infection differed among males, females, and subadults (Δdev = 2.32, df = 2, p = 0.31); among sites (Δdev = 0.175, df = 2, p = 0.91); or among seasons (Δdev = 1.44, df = 1, p = 0.7) (each of the above Δdev terms is corrected for the other terms in the model). Comparing the prevalence of infection between L. wilcoxii/jungguy and T. eungellensis was hampered by the fact that prevalence of infection on T. eungellensis differed between sites, and that the sampled populations of the two frog species had largely disjunct distributions. When the data were pooled over all sites, there was insufficient evidence to reject a null hypothesis of equal prevalence in the two species, whether the effects of season were allowed for (Δdev = 0.15, df = 1, p = 0.7) or not (Δdev = 2.09, df = 1, p = 0.15). Influence of Infection on Recapture and Survival Table 3 shows the numbers of L. wilcoxii/jungguy and T. eungellensis recaptured at any stage later in the study, grouped by their infection status at first capture. A logistic model with recapture as the response and species and infection status as predictors produced no evidence that the effect of B. dendrobatidis infection on recapture probability differed between the species (Δdev = 0.002, df = 1, p = 0.96). In both species, the probability of recapture was significantly lower for infected frogs than for uninfected frogs (Δdev = 5.34, df = 1, p = 0.02; log odds ratio = –0.6464, se = 0.2856), correcting for the substantially higher overall recapture rate of T. eungellensis relative to L. wilcoxii/jungguy. Table 3 Recapture and Survival of Infected and Uninfected Frogs Shown are numbers of marked L. wilcoxii/jungguy and T. eungellensis frogs that were infected (+ve) and uninfected (-ve) and recaptured during the 4-y monitoring study, and maximum periods of time over which those frogs were known to survive after being toe-clipped This simple analysis suggests that B. dendrobatidis may affect survival, but confounds the probability of recapturing a frog that is present at a site with the probability that a frog is still present at the site. The more sophisticated analysis that follows separates these two components, although it cannot distinguish between death and permanent emigration from the site. For brevity, we refer to continued presence at the site as “survival.” Investigating mark–recapture data for both Rawson Creek and Dooloomai Falls, we found that the best model (on the basis of minimizing the AIC) for which there were sufficient data to estimate almost all parameters had the probability of recapture varying with time but not group (i.e., infected and uninfected males, females, and subadults), and the survival probability differing between the groups but constant through time. However, further analysis in both cases showed that there was no evidence that survival differed between infected and uninfected frogs: At both sites, the group effect was due to survival being highest in females, intermediate in males, and lowest in subadults (Figure 3; Table 4). In the case of Tree Fern Creek, which had a smaller total sample size than the other two sites, and in which only males were infected, the best model had recapture probabilities constant with both time and group, and survival varying with group. At this site, there was some evidence that infected males had lower survival than uninfected males (Figure 3; Table 4). Figure 3 Estimated Quarterly Survival of Age/Sex Classes of Taudactylus eungellensis at Three Sites Survival for uninfected frogs is shown with circles, and survival for infected frogs is shown with squares; 95% confidence limits around each point are also shown. Where there is no survival estimate for infected frogs, there were insufficient data to estimate the parameter. Sample sizes are given in Table 2. Table 4 Summary of Results of Mark-Recapture Modelling on T. eungellensis “AICc” is the corrected Akaike Information Criterion; “ΔAICc” is the change in AICc from the “best” model; and “Model Likelihood” is the likelihood of the model relative to the best model. A parametric bootstrap analysis using the most complex model that could be parameterised from the capture histories (a model with additive effects of group and time on both recapture and survival) indicated that in each case the goodness of fit was adequate Discussion Our results show unequivocally that remnant populations of T. eungellensis, a rainforest frog that almost disappeared as a result of major die-offs in the Eungella area in the mid 1980s, now persist with stable infections of B. dendrobatidis. This does not imply that this pathogen cannot have been responsible for the decline. One hypothesis that can be discarded, however, is that B. dendrobatidis drove frog populations to low levels, consequently became locally extinct itself, and that frog populations subsequently recovered or stabilised in the absence of the pathogen. Two other hypotheses are consistent with our observed results. It may be that B. dendrobatidis was not responsible for the initial decline of T. eungellensis populations. These populations declined more than a decade before this chytrid was formally identified, and the declines occurred over a very short period (McDonald 1990). Because of the rapidity of the declines, no samples were collected during the period of declines, so it is not possible to examine for the presence of B. dendrobatidis. Although it is not possible to eliminate all environmental factors as being responsible for the decline of T. eungellensis and the disappearance of R. vitellinus from Eungella in 1985, the failure to detect substantial climatic anomalies in the area at that time (McDonald 1990) means that this explanation is unlikely to account for the sudden declines. Another possibility consistent with our results is that B. dendrobatidis was a pathogen novel to the ecosystem in 1985, and that it was indeed responsible for the declines. Available molecular evidence (Morehouse et al. 2003), although not conclusive, is consistent with the hypothesis that B. dendrobatidis is a recently emerged disease agent. Populations of T. eungellensis may have recovered or stabilised following evolution of resistance to the pathogen in the frogs, or evolution of less-pathogenic strains of the fungus. Rapid coevolutionary changes in both host and pathogen following the introduction of a novel pathogen are to be expected, with the best-known example being the coevolution of myxomatosis and its rabbit hosts within a few years of the introduction of the virus to Australia in 1953 (May and Anderson 1983; Fenner and Fantini 1999). Our study provides no direct evidence for such coevolution in frogs and B. dendrobatidis, but it identifies this as an important area for future research. If it could be shown that susceptible frog populations were able to develop resistance to the amphibian chytrid, then captive breeding and artificial selection for resistance would provide an avenue for management of this threat to critically endangered species. It is intriguing that we found similar seasonal fluctuations in infection levels within each year, but no evidence of variation among the 4 y of the study. This suggests strongly that B. dendrobatidis has become endemic and relatively stable in prevalence in these populations. Given that frog numbers and diversity remained broadly similar over this period, it suggests that some form of host–pathogen equilibrium has become established, in contrast to the epizootic that may have occurred 10 y previously. There are some obvious methodological limitations in our study that we must acknowledge. Infection status was determined by histological examination of toe tips at the time of first capture. Toe tips are often examined for B. dendrobatidis because the feet of frogs are a particularly favoured body location for infection by the fungus (Berger 2001). However, in light infections, B. dendrobatidis can occur in microscopic clusters, which can potentially be missed in histological sections. Hence, a proportion of frogs found to be negative for B. dendrobatidis using histology at first capture may actually have been infected. This possibility of false negatives (Berger 2001; Berger et al. 2002) suggests that the infection prevalences found in this study may underestimate the true prevalence of B. dendrobatidis within these frog populations. For individuals classed as infected at the time of first capture, and that subsequently survived for extended periods, it is not known whether their infection persisted or was cleared after first capture. There is evidence that some frog species can clear B. dendrobatidis infection: 50% of experimentally infected Mixophyes fasciolatus held at 27 °C with confirmed infection subsequently cleared the infection (Berger 2001; Berger et al. 2004). The prevalences we report are therefore estimated from the prevalence of infection in frogs captured for the first time in the period under consideration. In this study, infection levels of B. dendrobatidis were significantly higher during winter and spring than during summer and autumn. A survey of ill and dead frogs from eastern Australia from 1995 to 1999 showed a similar seasonal prevalence of chytridiomycosis (Berger et al. 2004). In the laboratory, the growth of B. dendrobatidis has been shown to peak at about 23 °C, with death or arrested growth occurring in vitro at temperatures above 30 °C (Longcore et al. 1999; Johnson et al. 2003; Piotrowski et al. 2004). Further, infection in some frog species can be cleared in the laboratory by exposing them to temperatures in excess of 37 °C (Woodhams et al. 2003). At Eungella, 23 °C is a typical daytime maximum temperature for winter, with water and air temperatures along streams generally remaining at or below 23 °C for the entire 24-h daylength period. In summer, however, daytime air temperatures along Eungella's streams regularly exceed 23 °C, and at sites such as Rawson Creek can reach as high as 37 °C (R. Retallick, unpublished data). The different infection levels among sites for T. eungellensis reported in Table 2 may be correlated with the degree of sunlight and warmth that reaches the streams. The streams at Dooloomai Falls and Tree Fern Creek are well shaded and remain relatively cool and damp for much of the year. At those sites, the average canopy gap above the centre of the streams (a coarse measure of how much sunlight reaches the stream) are 0.35 m and 1.90 m respectively (R. Retallick, unpublished data). Rawson Creek is considerably wider (average canopy gap = 5.10 m) and thus receives more sunlight. With access to sunny microhabitats, where surface temperatures can exceed ambient air temperatures, frogs at “warmer” streams such as Rawson Creek may be less subject to infection at any time of the year, or may be able to reduce infection through thermoregulation. The idea that frogs may avoid, control, or eliminate infection by differential use of their environment warrants considerable and immediate attention. Such a process may prove to be critical to the relationship between B. dendrobatidis and frog populations in the wild. Our mark–recapture analysis did not find consistent evidence that T. eungellensis infected with B. dendrobatidis at the time of first capture had a lower survival rate than uninfected T. eungellensis. Failure to reject the null hypothesis of no effect of infection on survival cannot, of course, be used as evidence in favor of the null hypothesis, and it is worth noting that the point estimate of survival for infected males was lower than that for uninfected males at each site. Only at Tree Fern Creek, however, did the best-supported model include an infection term. There was also evidence that infection influenced the proportion of frogs that were recaptured (Table 3). Together with the observation that some infected T. eungellensis survived for extended periods (a maximum of 1,089 d), our results show that infection with B. dendrobatidis did not inevitably lead to rapid death in T. eungellensis. Both epidemiological theory and observations suggest that where a pathogen drives a host species to extinction, there is likely to be a reservoir host within which the pathogen has a reduced effect and is therefore maintained at a higher prevalence (McCallum and Dobson 1995, 2002). L. wilcoxii/jungguy appears not to have declined at the same time as T. eungellensis and R. vitellinus and, in our data, it has a high prevalence of infection. It therefore is a candidate reservoir host. Whether the prevalence of B. dendrobatidis differs between the sibling species L. wilcoxii and L. jungguy at sites where they are sympatric would be an interesting question, but cannot be answered from our study. The L. lesueuri complex has a widespread distribution throughout streams on the east coast of Australia (Barker et al. 1995; Donellan and Mahony 2004) and could therefore play a substantial role in the maintenance and spread of chytrid infection throughout that region. If a species is acting as a reservoir, the prevalence of infection in that species should be higher than in species that are declining as a result of infection (McCallum and Dobson 1995). The prevalence of infection we observed in L. wilcoxii/jungguy exceeded that observed in T. eungellensis, but we do not have clear evidence that this represents a higher prevalence in the populations as a whole. It is also possible that B. dendrobatidis exists as a saprophyte in the environment independent of amphibians (Longcore et al. 1999; Johnson and Speare 2003), or may use tadpoles (which seem relatively little affected by the pathogen) as a reservoir (Daszak et al. 1999). We did not detect infection in any of the 50 individuals of Litoria revelata or the 42 individuals of L. chloris we examined, which demonstrates that those species had a lower prevalence of chytridiomycosis than T. eungellensis and L. wilcoxii/jungguy. We did not record any infected frogs of any species from the two sites at which L. revelata was collected. Litoria chloris, however, has been shown to carry infection in other areas (Speare and Berger 2004) and in laboratory experiments (Woodhams et al. 2003). Sample sizes in our study for these other frog species may be too small to draw any reliable conclusions about whether they become infected with B. dendrobatidis in the wild. Materials and Methods Between 1994 and 1998, 36 excursions to Eungella National Park were made as part of a mark–recapture study of populations of frogs associated with streams. Frogs were captured from six study sites (see Figure 1), which were visited monthly from March 1994 to June 1996, in February and September 1997, and in February and May 1998. When caught for the first time, frogs were toe-clipped using the numbering system devised by Hero (1989) and then released alive. Amputated toe tips were preserved and individually stored in vials filled with 70% ethanol or 10% formalin. To minimize effects on the animals, no further toes were taken from subsequent recaptures of marked frogs; recaptured frogs were identified and released alive. All frog species encountered were monitored, with a special focus on T. eungellensis because of its precarious conservation status and history of decline. In 1997, toes from six species (278 individual frogs) were histologically prepared in transverse sections stained with Ehrlich's haematoxylin for skeletochronological assessment (see Castanet et al. [1996] for a description of the technique). We subsequently reexamined these sections for B. dendrobatidis (Berger et al. 1999b; Pessier et al. 1999). On average, 160 sections per individual were examined. Despite the sections not being stained with eosin, which highlights the keratinised layer of the epidermis where B. dendrobatidis occurs (Berger et al. 1999b; Pessier et al. 1999), most diagnoses were unambiguous. A small number, however, were not, and those samples were excluded from the analysis. For this assessment, samples were labelled “positive” only when the examiners were convinced that the sample was infected with B. dendrobatidis. Samples in which no infection was found were labelled “negative.” For this study, a further 196 archived toes of T. eungellensis were analysed by the Australian Animal Health Laboratory in Geelong, Australia, for B. dendrobatidis infection using histopathology with immunoperoxidase staining (Berger et al. 2002). The immunoperoxidase stain improves the sensitivity of diagnosis by highlighting structures that are equivocal with haematoxylin and eosin, and that would otherwise be diagnosed as negative. With the software MARK (Cooch and White 2002), we used mark–recapture modelling to investigate the survival of T. eungellensis at different sites. Frogs were grouped into six categories on the basis of their status at first capture (infected and uninfected males, females, and subadults). For both mark–recapture and analysis of prevalence, we grouped the data into 3-mo austral seasons (in Australia, summer runs from 1 December to the end of February, etc.). Mark–recapture modelling of survival requires that several assumptions be satisfied. Most critically, every marked individual present in the population at a given time has the same probability of recapture as all other members of its group, and the same probability of surviving to the next time interval. We used a parametric bootstrap (Cooch and White 2002) to test the goodness of fit of our capture history sets to these assumptions. We analysed prevalence data using logistic models implemented in R (Version 1.6.2) (R Development Core Team 2003). Since the mark–recapture study was performed with no knowledge of the infection status of individual animals, and the examination of histology was carried out with no knowledge of details of the fate of individual animals in the field, the study was double-blinded in design. We are grateful to Diana Mendez, Alex Hyatt, and the diagnostic staff at the Australian Animal Health Laboratory for diagnosis of chytrid infection, and the School of Public Health and Tropical Medicine, James Cook University, for work space and technical support. Funding for the original fieldwork was supplied by a grant from Australian Nature Conservation Agency (now Environment Australia) to J-M Hero, RA Alford, and KR McDonald, and an Australian Post-Graduate Research Award to R Retallick. This study was completed under multiple Queensland Parks and Wildlife Service permits, and ethics approval was obtained from the Experimentation Ethics Review Committee (Biological Sciences, James Cook University). Financial support for the retrospective analysis was provided by the Australian Research Council and the US National Science Foundation Integrated Research Challenges in Environmental Biology scheme. Some of the analysis was carried out by H McCallum while he was a visiting scientist at the Centre for Population Biology, Silwood Park. Conflicts of interest. The authors have declared that no conflicts of interest exist. Author contributions. RWRR and RS conceived and designed the experiments. RWRR performed the experiments. HIM analyzed the data. HIM wrote the paper. Academic Editor: Georgina M. Mace, Institute of Zoology, Zoological Society of London Citation: Retallick RWR, McCallum H, Speare R (2004) Endemic infection of the amphibian chytrid fungus in a frog community post-decline. PLoS Biol 2(11): e351. Abbreviations AICAkaike Information Criterion Δdevchange in deviance sestandard error ==== Refs References Barker J Grigg G Tyler M A field guide to Australian frogs 1995 Chipping Norton Surrey Beatty and Sons 407 Berger L Diseases in Australian frogs [dissertation] 2001 Townsville, Queensland James Cook University 331 Available from: James Cook University library (http://www.jcu.edu ) Berger L Speare R Hyatt A Campbell A Chytrid fungus and amphibian declines: Overview, implications and future directions Declines and disappearances of Australian frogs 1999a Canberra Environment Australia 23 33 Berger L Speare R Kent A Diagnosis of chytridiomycosis in amphibians by histologic examination Zoos' Print J 1999b 15 184 190 Berger L Speare R Daszak P Green DE Cunningham AA Chytridiomycosis causes amphibian mortality associated with population declines in the rain forests of Australia and Central America Proc Natl Acad Sci U S A 1998 95 9031 9036 9671799 Berger L Hyatt AD Olsen V Hengstberger SG Boyle D Production of polyclonal antibodies to Batrachochytrium dendrobatidis and their use in an immunoperoxidase test for chytridiomycosis in amphibians Dis Aquat Organ 2002 48 213 220 12033708 Berger L Speare R Hines HB Marantelli G Hyatt AD Effect of season and temperature on mortality in amphibians due to chytridiomycosis Aust Vet J 2004 82 434 439 15354853 Campbell A Declines and disapppearances of Australian frogs 1999 Canberra Environment Australia 234 Castanet J Francillon-Vieillot H Bruce RC Age estimation in desmognathine salamanders assessed by skeletochronology Herpetologica 1996 52 160 171 Cooch E White G Using MARK—A gentle introduction 2002 http://www.phidot.org/software/mark/docs/book/ . Accessed 25 August 2004 Couper PJ Hope for our missing frogs Wildl Aust 1992 29 11 12 Daszak P Berger L Cunningham AA Hyatt AD Green DE Emerging infectious diseases and amphibian population declines Emerg Infect Dis 1999 5 735 748 10603206 Daszak P Cunningham AA Hyatt AD Infectious disease and amphibian population declines Divers Distrib 2003 9 141 150 Department of the Environment and Heritage National list of threatened fauna Available: <http://www.deh.gov.au/cgi-bin/sprat/public/publicthreatenedlist.pl?wanted=fauna#ENDANGERED%20FROGS > 2003 Accessed 21 August 2004 Donnellan SC Mahony MJ Allozyme, chromosomal and morphological variability in the Litoria lesueuri species group (Anura: Hylidae) including a description of a new species Aust J Zool 2004 52 1 28 Fenner F Fantini B Biological control of vertebrate pests: The history of myxomatosis—An experiment in evolution 1999 Wallingford CABI Publishing 352 Hero JM A simple code for toe clipping anurans Herpetol Rev 1989 20 66 67 Johnson ML Speare R Survival of Batrachochytrium dendrobatidis in water: Quarantine and disease control implications Emerg Infect Dis 2003 9 922 925 12967488 Johnson M Berger L Philips L Speare R Fungicidal effects of chemical disinfectants, UV light, desiccation and heat on the amphibian chytrid, Batrachochytrium dendrobatidis Dis Aquat Organ 2003 57 255 260 14960039 Laurance WF McDonald KR Speare R Epidemic disease and the catastrophic decline of Australian rain forest frogs Conserv Biol 1996 10 1 9 Laurance WF McDonald KR Speare R In defense of the epidemic disease hypothesis Conserv Biol 1997 11 1030 1034 Longcore JE Pessier AP Nichols DK Batrachochytrium dendrobatidis gen. et sp. nov., a chytrid pathogenic to amphibians Mycologia 1999 91 219 227 May RM Anderson RM Epidemiology and genetics in the coevolution of parasites and hosts Proc R Soc Lond B Biol Sci 1983 219 281 313 6139816 McCallum HI Dobson AP Detecting disease and parasite threats to endangered species and ecosystems Trends Ecol Evol 1995 10 190 194 21237000 McCallum H Dobson A Disease, habitat fragmentation and conservation Proc R Soc Lond B Biol Sci 2002 269 2041 2049 McDonald K Alford R Campbell A A review of declining frogs in Northern Queensland Declines and disappearances of Australian frogs 1999 Canberra Environment Australia 14 22 McDonald KR Rheobatrachus Liem and Taudactylus Straughan and Lee (Anura: Leptodactylidae) in Eungella National Park, Queensland: Distribution and decline Trans R Soc South Aust 1990 114 187 194 McNellie M Hero J-M Mission Amphibian: The search for the missing rainforest frogs of Eungella Wildl Aust 1994 31 22 23 Morehouse EA James TY Ganley ARD Vilgalys R Berger L Multilocus sequence typing suggests that the chytrid pathogen of amphibians is a recently emerged clone Mol Ecol 2003 12 395 403 12535090 Pessier AP Nichols DK Longcore JE Fuller MS Cutaneous chytridiomycosis in poison dart frogs (Dendrobates spp.) and White's tree frogs (Litoria caerulea) J Vet Diagn Invest 1999 11 194 199 10098698 Piotrowski JS Annis SL Longcore JE Physiology of Batrachochytrium dendrobatidis a chytrid pathogen of amphibians Mycologia 2004 96 9 15 21148822 R Development Core Team R: A language and environment for statistical computing. Vienna: R Foundation for Statistical Computing. Available: http://www.R-project.org 2004 Accessed 22 August 2004 Retallick R Hero JM Alford R Adult population monitoring and larval ecology of the stream-dwelling frogs at Eungella National Park: ANCA/QDOE 1997 63 Speare R Berger L Chytridiomycosis in amphibians in Australia. Available: http://www.jcu.edu.au/school/phtm/PHTM/frogs/chyspec.htm 2004 Accessed 26 Feb 2004 Woodhams DC Alford RA Marantelli G Emerging disease of amphibians cured by elevated body temperature Dis Aquat Organ 2003 55 65 67 12887256

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==== Front PLoS BiolPLoS BiolpbioplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 1550287410.1371/journal.pbio.0020362Research ArticleMolecular Biology/Structural BiologyNeuroscienceIn VitroDJ-1 Is a Redox-Dependent Molecular Chaperone That Inhibits α-Synuclein Aggregate Formation DJ-1 Is a Redox-Dependent ChaperoneShendelman Shoshana 1 Jonason Alan 1 Martinat Cecile 1 Leete Thomas 1 Abeliovich Asa [email protected] 1 1Departments of Pathology and Neurology, Center for Neurobiology and Behavior, and Taub Institute, Columbia University, College of Physicians and SurgeonsNew York, New YorkUnited States of America11 2004 5 10 2004 5 10 2004 2 11 e36228 4 2004 19 8 2004 Copyright: © 2004 Shendelman et al.2004This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. Sensitivity to Oxidative Stress in DJ-1-Deficient Dopamine Neurons: An ES-Derived Cell Model of Primary Parkinsonism A New Cell Model for Parkinson's Disease Molecules That Cause or Prevent Parkinson's Disease Parkinson's disease (PD) pathology is characterized by the degeneration of midbrain dopamine neurons (DNs) ultimately leading to a progressive movement disorder in patients. The etiology of DN loss in sporadic PD is unknown, although it is hypothesized that aberrant protein aggregation and cellular oxidative stress may promote DN degeneration. Homozygous mutations in DJ-1 were recently described in two families with autosomal recessive inherited PD (Bonifati et al. 2003). In a companion article (Martinat et al. 2004), we show that mutations in DJ-1 alter the cellular response to oxidative stress and proteasomal inhibition. Here we show that DJ-1 functions as a redox-sensitive molecular chaperone that is activated in an oxidative cytoplasmic environment. We further demonstrate that DJ-1 chaperone activity in vivo extends to α-synuclein, a protein implicated in PD pathogenesis. The interaction of the proteins DJ-1 and α- synuclein described here may be important for understanding the molecular basis of Parkinson's disease ==== Body Introduction Parkinson's disease (PD) is a progressive movement disorder that is characterized pathologically by the relatively selective degeneration of midbrain DNs and the presence of prominent intracytoplasmic neuronal inclusions, termed Lewy bodies (Dauer and Przedborski 2003). The identification of several genes that underlie familial forms of primary parkinsonism has allowed for the molecular dissection of mechanisms of dopamine neuron (DN) survival. Autosomal dominant mutations in α-synuclein (αSyn) lead to a rare familial form of primary Parkinsonism (Polymeropoulos et al. 1997), and there is evidence that these mutations generate toxic, abnormal protein aggregates (Goldberg and Lansbury 2000) and proteasomal dysfunction (Rideout et al. 2001). Of note, Lewy body inclusions are particularly enriched for αSyn (Spillantini et al. 1998) and neurofilament protein subunits (Trojanowski and Lee 1998). Mutations in a second gene, Parkin, lead to autosomal recessive primary Parkinsonism (Hattori et al. 2000). Parkin is a ubiquitin ligase that appears to participate in the proteasome-mediated degradation of several substrates (Staropoli et al. 2003). Homozygous mutations in a third gene, DJ-1, were recently associated with autosomal recessive primary Parkinsonism (Bonifati et al. 2003). DJ-1 encodes a ThiJ domain protein of 189 amino acids that is broadly expressed in mammalian tissues (Nagakubo et al. 1997). Interestingly, DJ-1 was independently identified in a screen for human endothelial cell proteins that are modified with respect to isoelectric point in response to sublethal doses of paraquat (Mitsumoto and Nakagawa 2001; Mitsumoto et al. 2001), a toxin which generates reactive oxygen species (ROS) within cells and has been associated with DN toxicity (McCormack et al. 2002). Gene expression of a yeast homolog of DJ-1, YDR533C, is upregulated in response to sorbic acid (de Nobel et al. 2001), an inducer of cellular oxidative stress. These data suggest a causal role for DJ-1 in the cellular oxidative stress response. ThiJ domain proteins are highly conserved and have been associated with several functions including protease and chaperone activities (Halio et al. 1996; Du et al. 2000). The crystal structure of DJ-1 demonstrates the presence of a highly conserved nucleophile elbow-like domain at cysteine 106, but the relative position of this residue differs from that of a structurally related ThiJ protease, PH1704, and does not appear to be permissible for proton transfer and protease catalysis (Wilson et al. 2003). Furthermore, DJ-1 forms an asymmetric homodimer with a prominent carboxy-terminal helical region present at the dimerization interface, which appears to limit access to the nucleophile elbow-like domain (Huai et al. 2003; Lee et al. 2003; Wilson et al. 2003). DJ-1 displays significant homology to the carboxy-terminal domain of the Escherichia coli HPII catalase, as both proteins are divergent members of the type I glutamine amidotransferase family. Interestingly, the carboxy-terminal DJ-1 homology domain of HPII catalase lacks catalase activity, but rather appears to function as a chaperone in the correct folding of the catalytic core of the protein, and in thermal enzyme stability (Chelikani et al. 2003). Taira et al. (2004) recently reported that purified DJ-1 harbors catalase activity, and that overexpression of DJ-1 by transfection of neuroblastoma tumor cells inhibits the accumulation of ROS. In contrast, analysis of DJ-1-deficient cells (Martinat et al. 2004) revealed that such cells display an apparently normal initial accumulation of ROS, indicating that DJ-1 likely functions in a protective role downstream of ROS insult. Consistent with this, DJ-1-deficient cells are predisposed to apoptotic death in the context of oxidative stress (Martinat et al. 2004). Here we demonstrate that DJ-1 functions as a redox-regulated molecular chaperone that is activated in an oxidizing environment. DJ-1 chaperone activity extends in vivo to αSyn, a protein that has been implicated in PD pathogenesis. DJ-1 activity is abrogated by the L166P mutation, associated with primary Parkinsonism, as a consequence of defective dimerization and reduced stability. Results DJ-1 Lacks Apparent Protease and Antioxidant Activities In Vitro DJ-1 homologs have been reported to harbor protease (Halio et al. 1996; Du et al. 2000; Lee et al. 2003) and amidotransferase activities (Horvath and Grishin 2001). However, crystal structure analyses of DJ-1 suggest that this protein may not retain such catalytic activities (Honbou et al. 2003a; Huai et al. 2003; Lee et al. 2003; Tao and Tong 2003; Wilson et al. 2003). Consistent with this, purified DJ-1 preparations failed to display in vitro protease activity toward a variety of synthetic or natural substrates, and, similarly, DJ-1 lacked antioxidant (Table S1) or catalase activities (Figure S1) in vitro. Furthermore, cells deficient in DJ-1 appear unaltered in the initial accumulation of ROS in the context of acute oxidative stress (Martinat et al. 2004). DJ-1 Is a Redox-Dependent Molecular Chaperone Every organism responds to ROS and other toxic environmental stresses by overexpressing a highly conserved set of heat shock proteins (Hsps), many of which function as molecular chaperones to assist other proteins in folding. Hsp31, an E. coli ThiJ domain protein, has been shown to function as a molecular chaperone in vitro (Sastry et al. 2002; Malki et al. 2003). We hypothesized that DJ-1 may similarly function as a protein chaperone to protect cells from ROS. DJ-1 chaperone activity was quantified in the suppression of heat-induced aggregation of citrate synthase (CS) and glutathione S-transferase (GST), two well-characterized protein chaperone assays. These proteins lose their native conformation and undergo aggregation during incubation at 43 °C and 60 °C, respectively. Addition of 0.5–4.0 μM polyhistidine (His)-tagged DJ-1 was found to effectively suppress the heat-induced aggregation of 0.8 μM CS (Figure 1A). The chaperone activity was independent of the His tag used for purification, as cleavage and removal of the His tag did not alter DJ-1 chaperone function (unpublished data). DJ-1 chaperone activity is comparable to that of a well-described small cytoplasmic chaperone, human Hsp27. In contrast, RNase A failed to demonstrate chaperone activity and served as a negative control. Interestingly, the Parkinsonism-associated L166P DJ-1 mutation abrogated chaperone activity relative to the wild-type (WT) protein (Figure 1B). Figure 1 DJ-1 Is a Redox-Dependent Molecular Chaperone (A) Aggregation of CS was monitored at 43 °C after addition of either 0.8 μM CS alone (black), or along with 8.0 μM RNase A (purple), 0.5 μM DJ-1 (aqua), 2.0 μM DJ-1 (blue), 4.0 μM DJ-1 (red), or 2.0 μM Hsp27 (green). (B) Aggregation of 0.8 μM CS after 30 min at 4 °C (unfilled bar) is inhibited by 4.0 μM WT DJ-1 (black bar) but not 4.0 μM L166P mutant DJ-1 (gray bar). Data are shown as the mean ± SEM and were analyzed by ANOVA with Fisher's post-hoc test. * p < 0.05. (C) Aggregation of insulin (26 μM) B chains induced by 20 mM DTT at 25 °C. Insulin alone (black) or in the presence of 4.0 μM RNase A (purple), 0.5 μM DJ-1 (aqua), 2.0 μM DJ-1 (blue), 4.0 μM DJ-1 (red), or 2.0 μM Hsp27 (green). (D) CS thermal aggregation (unfilled bar) is suppressed by 4 μM DJ-1 (black bar), but chaperone activity is abrogated upon incubation of DJ-1 with 0.5 mM DTT for 10 min at 4 °C (gray bar). Further treatment of DTT-reduced DJ-1 with 10 mM H2O2 for 10 min at 4 °C leads to reactivation of CS suppression (hatched bar). Data are shown as the mean ± SEM and were analyzed by ANOVA with Fisher's post-hoc test. * p < 0.05. DJ-1 similarly functioned as a molecular chaperone in the context of the heat-induced aggregation of GST (see Figure S1). In contrast, DJ-1 failed to display activity in a third chaperone assay, aggregation suppression of reduced insulin (Figure 1C). Reduction of the disulfide bonds between the A and B chains of insulin with dithiothreitol (DTT) leads to aggregation of the B chains. Hsp27 effectively inhibited the aggregation of insulin in the presence of 20 mM DTT, whereas neither DJ-1 nor the negative control protein RNase A displayed chaperone activity in this assay. As the insulin aggregation assay is performed in a reduced environment, we hypothesized that DJ-1 chaperone activity may be redox regulated. Interestingly, such a redox switch in a molecular chaperone has been described in Hsp33 (Jakob et al. 1999), a dimeric bacterial Hsp unrelated to DJ-1. To test the redox regulation of DJ-1, we assayed chaperone activity in the CS aggregation assay in the presence or absence of the reducing agent DTT. DJ-1 chaperone activity in the CS aggregation assay was completely abrogated by preincubation of DJ-1 with 0.5 mM DTT in aggregation buffer for 10 min at 4 °C (Figure 1D). DTT did not significantly alter CS aggregation in the absence of DJ-1 and did not modify suppression of CS aggregation by Hsp27 (unpublished data). To further test whether redox regulation might govern DJ-1 chaperone activity, reactivation studies using reduced DJ-1 were performed. DTT-reduced DJ-1 was incubated with H2O2 (10 mM in aggregation buffer for 10 min at 4 °C followed by dialysis against aggregation buffer for 2 h), and subsequently chaperone activity was measured in the CS thermal aggregation assay. H2O2 effectively reactivated the chaperone activity of DTT-treated DJ-1 (Figure 1D). This was not an indirect effect of residual H2O2 on CS aggregation, as H2O2 treatment of CS increased aggregation (unpublished data). These results suggest that redox regulation of DJ-1 is reversible and is regulated by the redox environment. Molecular chaperones typically display marked stability to thermal stress (Sastry et al. 2002). Consistent with this, the ultraviolet-circular dichroism (CD) spectrum of WT DJ-1 is consistent with a well-folded protein, and thermal denaturation of WT DJ-1 revealed a cooperative thermal unfolding transition at approximately 75 °C (see Figure S1). In contrast, the CD spectrum of the DJ-1 L166P mutant protein is typical of a partially unfolded polypeptide, suggesting that the L166P mutation causes a significant loss of helical structure. The mutant protein does not exhibit a thermal unfolding transition in the range studied (0–90 °C). DJ-1 Inhibits the Generation of αSyn Aggregates We extended the analysis of DJ-1 chaperone function to a candidate DJ-1 substrate, αSyn (Figure 2). The aggregation of αSyn has been implicated in familial and sporadic forms of PD, as mutations associated with autosomal dominant familial primary Parkinsonism alter the propensity of αSyn to aggregate (Conway et al. 2000a), and as αSyn fibrils are a major constituent of the Lewy body intracytoplasmic inclusions that typify PD pathology (Spillantini et al. 1997). In vitro, monomeric αSyn is disordered or “natively unfolded” in dilute solution (Weinreb et al. 1996). Incubation of purified WT human αSyn for 2 h at 55 °C results in the generation of high molecular weight multimers that likely represent protofibrils (Figure 2A and 2B) (Volles et al. 2001; Gosavi et al. 2002). This treatment does not result in formation of mature amyloid fibrils, as determined by Congo red staining (see Figure S1). WT DJ-1 effectively inhibits the formation of soluble αSyn protofibrils at a molar ratio of 1:2 (DJ-1: αSyn). In contrast, L166P mutant DJ-1, GST, and Hsp27 (Figure 2A and 2B) failed to inhibit the generation of αSyn protofibrils. Figure 2 DJ-1 Inhibits Formation of αSyn Protofibrils and Fibrils In Vitro (A) Purified αSyn (200 μM) was incubated for 2 h at 55 °C in the presence of WT DJ-1, L166P mutant DJ-1, GST, or Hsp27 (all at 100 μM). WT DJ-1 inhibits accumulation of αSyn protofibrils in vitro, while L166P mutant DJ-1, GST, and Hsp27 do not. (B) Suppression of αSyn protofibril formation by WT DJ-1 (in triplicate) was quantified as compared to GST (as a negative control) and mutant L166P DJ-1. Data are shown as the mean ± SEM and were analyzed by ANOVA with Fisher's post-hoc test. * p < 0.05. (C) Purified αSyn (200 μM) was incubated for 1 wk at 37 °C in the presence of WT DJ-1, L166P mutant DJ-1, or GST (all at 100 μM). WT DJ-1 inhibits formation of mature Congo red–positive αSyn fibrils. Data are shown as the mean ± SEM and were analyzed by ANOVA with Fisher's post-hoc test. * p < 0.05. αSyn protofibrils have been shown to be an intermediate in the formation of mature amyloid fibrils. Because DJ-1 chaperone activity is effective at inhibiting the accumulation of αSyn protofibrils, we sought to investigate the role of this activity in the generation of Congo red–positive mature fibrils. Congruently, WT DJ-1 inhibited formation of Congo red-positive αSyn fibrils, while L166P DJ-1 and GST did not (Figure 2C). Thus, DJ-1 seems to inhibit formation of αSyn fibrils by preventing formation of αSyn high molecular weight oligomers, or protofibrils. Interestingly, PD-associated clinical mutations in αSyn appear to accelerate oligomerization and protofibril formation (Volles et al. 2001). DJ-1 Chaperone Activity In Vivo We sought to investigate the chaperone activity of DJ-1 toward αSyn in vivo. αSyn has been shown to form aggregates that consist of both protofibrils and mature amyloid fibrils in the context of oxidative stress (such as FeCl2 treatment [Lee and Lee 2002; Lee et al. 2002]) in neuroblastoma cells. We evaluated the activity of DJ-1 overexpression on αSyn aggregation in this tissue culture model system. Briefly, CAD murine neuroblastoma cells (Staropoli et al. 2003) were transfected with Flag epitope-tagged αSyn (Flag-αSyn), differentiated via serum withdrawal, and exposed to FeCl2 (2 mM) for 18 h. Treatment with FeCl2 induced accumulation of αSyn in the Triton X-100-insoluble fraction, which has been shown to correlate with αSyn protofibrils (Lee and Lee 2002). Overexpression of WT DJ-1, but not L166P clinical mutant DJ-1, significantly inhibited the accumulation of Triton X-100-insoluble αSyn (Figure 3A and 3B). DJ-1 overexpression did not alter the accumulation (Figure 3A) or half-life of soluble αSyn, as determined by pulse-chase kinetic analysis (Figure S2). Thus, DJ-1 overexpression is sufficient to inhibit the formation of αSyn aggregates in vivo, consistent with the in vitro analysis. Figure 3 Overexpression of WT DJ-1 Inhibits Aggregation of αSyn In Vivo (A) CAD murine neuroblastoma cells were transfected with Flag-αSyn along with WT DJ-1, L166P clinical mutant, or vector alone, and were differentiated in vitro via serum withdrawal. Cells were subsequently treated with 2 mM FeCl2 (Fe), 5 μM lactacystin (LC), or media alone (0). Triton X-100-soluble (Tx-100 sol) and Triton X-100-insoluble (Tx-100 insol) fractions were analyzed by Western blotting. Upon FeCl2 treatment, αSyn accumulates in the Triton X-100-insoluble fraction, and accumulation of insoluble αSyn is inhibited by overexpression of WT DJ-1 (left) but not the L166P clinical mutant (right). (B) Triton X-100-insoluble αSyn as quantified by NIH Image J of a Western blot (from [A]). (C) Heterozygous (+/–) and DJ-1 deficient (–/–) ES cells were differentiated using the embryoid body protocol. Cells were transfected with Flag-αSyn (F-αSyn), and, after 48 h, treated with 2 mM FeCl2 or with media alone for 18 h. Cell lysates were analyzed by Western blotting for αSyn or β-actin. In the Triton X-100-soluble fraction (Tx-100 sol), DJ-1 accumulated to a similar extent in the knockout and control cells. In contrast, αSyn accumulation in the insoluble pool (Tx-100 insol) was detectable only in the knockout cells, and this was further promoted by FeCl2 treatment. (D) CAD cells transfected with Flag-αSyn (F-αSyn) along with WT DJ-1 (or vector alone) were treated with 2 mM FeCl2 or media alone for 18 h. Triton X-100-soluble cell lysates were immunoprecipitated with a mouse monoclonal antibody for the Flag epitope and Western blotted for DJ-1. FeCl2 treatment induces association of Flag-αSyn with WT DJ-1. Lysates represent 20% input of the immunoprecipitation (IP α-Flag). The Triton X-100 soluble pool of DJ-1 is reduced by αSyn overexpression (but not vector control), particularly in the context of FeCl2 treatment (bottom). (E) DJ-1 colocalizes with αSyn in the Triton X-100-insoluble fraction upon FeCl2 treatment. The Western blot from (A) was stripped and reprobed for DJ-1. To investigate whether DJ-1 is necessary to inhibit αSyn aggregation in vivo, we utilized DJ-1 “knockout” embryonic stem (ES) cells, which display increased sensitivity to oxidative stress. DJ-1 homozygous knockout or control heterozygous ES cells (heterozygous cells were used as controls because they were the source of the knockout subclones) were differentiated in vitro using the embryoid body protocol (Martinat et al. 2004) and transfected with Flag-αSyn or control vector. Upon differentiation, both endogenous αSyn and transfected Flag-αSyn are accumulated to a similar extent in the soluble fraction of knockout and control cell lysates, as determined by Western blotting with an antibody for αSyn. In contrast, DJ-1-deficient cells (but not control cells) additionally accumulate Triton X-100-insoluble αSyn (both endogenous αSyn and transfected Flag-αSyn), which likely corresponds to protofibril aggregates (Lee and Lee 2002). As predicted, FeCl2 treatment further promoted the accumulation of insoluble αSyn in DJ-1-deficient cells but not in control heterozygous cells (Figure 3C). Interestingly, transfection of Flag-αSyn into undifferentiated knockout or control ES cells in the presence or absence of FeCl2 treatment did not lead to the accumulation of insoluble Flag-αSyn (see Figure S2), consistent with a prior study suggesting a role for neuronal differentiation in the generation of insoluble αSyn aggregates (Lee et al. 2002). To investigate the mechanism of DJ-1 activity toward αSyn, we performed coimmunoprecipitation experiments on untreated and FeCl2-treated CAD cells transfected with DJ-1 and Flag-αSyn (or control vector) as above. Triton X-100-soluble cell lysates were immunoprecipitated with a mouse monoclonal antibody for the Flag epitope, and Western blots were probed with a rabbit polyclonal antibody for DJ-1. DJ-1 failed to interact with Flag-αSyn in the absence of pretreatment with FeCl2, but an association was evident in FeCl2-treated cell lysates (Figure 3D). Furthermore, overexpression of αSyn (but not vector control) leads to a reduction in the soluble pool of DJ-1, particularly in the context of FeCl2 treatment, indicating that DJ-1 additionally associates with an insoluble fraction of αSyn (Figure 3D, bottom panel). Consistent with this, we found that a significant fraction of DJ-1 protein localizes to the insoluble fraction upon FeCl2 treatment (Figure 3E) in cells that have been cotransfected with Flag-αSyn. To further evaluate αSyn aggregation, we performed immunohistochemical analyses of CAD cells transfected with αSyn along with DJ-1 or control vector (Figure 4). Overexpression of αSyn in neuroblastoma cells induces the formation of visible cytoplasmic aggregates (Lee and Lee 2002) (Figure 4J–4L). Additional overexpression of WT DJ-1 significantly decreased the number of cells containing αSyn aggregates (Figure 4D–4F and 4M), whereas the L166P DJ-1 mutant fails to do so (Figure 4G–4I and 4M). However, DJ-1 does not appear to colocalize with αSyn aggregates, suggesting that DJ-1 functions at an early step in the formation of mature aggregates (Figure 4N–4S). Figure 4 DJ-1 Inhibits Formation of αSyn Intracytoplasmic Inclusions (A–L) CAD murine neuroblastoma cells were transfected with WT DJ-1 (A–F), L166P DJ-1 (G–I) or vector control (J–L), along with Flag-αSyn (D–L) or vector control (A–C) and differentiated in vitro by serum withdrawal for 72 h. Cells were fixed and stained with a mouse monoclonal antibody for αSyn and ToPro3, a nuclear dye, and images were obtained by confocal microscopy. Transfection of Flag-αSyn induced formation of intracytoplasmic inclusions (arrows). Scale bar, 20 μm. (M) Quantification of cells with inclusions was performed on ten random images from each of three wells per condition. Images were quantified by an observer blinded to the experiment. A significantly lower percentage of cells harbor inclusions in the context of WT DJ-1 overexpression. Aggregation is expressed as the percentage of cells containing αSyn aggregates per frame. Total cell number per frame, as determined by ToPro3 staining, did not differ significantly (Figure S3). Data are shown as the mean ± SEM, and were analyzed by ANOVA with Fisher's post-hoc test. * p < 0.05. (N–S) Cells were fixed and stained with a monoclonal antibody for αSyn and a polyclonal antibody that recognizes both transfected human DJ-1 and endogenous murine DJ-1. DJ-1 does not appear to colocalize with the αSyn aggregates. Scale bar, 20 μm. In a separate set of experiments, we assayed the ability of DJ-1 to inhibit aggregation of a second substrate, neurofilament light subunit (NFL). Overexpression of a mutant form of human NFL, Q333P, by transient transfection of CAD murine neuroblastoma cells, leads to the accumulation of intracytoplasmic inclusions (Perez-Olle et al. 2002). Co-overexpression of WT DJ-1 along with mutant NFL significantly inhibited the accumulation of NFL inclusions (Figure 5), whereas overexpression of the L166P Parkinsonism-associated mutant form of DJ-1 with NFL failed to inhibit the accumulation of aggregates. Coimmunostaining for DJ-1 and NFL indicated that DJ-1 does not colocalize with the NFL inclusions (Figure 5M–5R). DJ-1 did not appear to alter the expression of NFL (Figure S3). These data are consistent with our analysis of DJ-1 chaperone activity toward αSyn and indicate that DJ-1 harbors chaperone activity toward a range of substrates in vivo. Figure 5 DJ-1 Inhibits Formation of NFL Intracytoplasmic Inclusions (A–L) CAD cells were transfected with an aggregation-prone mutant NFL (Q333P) plasmid, as well as WT human DJ-1 plasmid (that also harbors GFP; E–H), L166P mutant DJ-1 (that also harbors GFP; I–L), or control GFP vector (A–D). After 72 h in culture, cells were fixed and stained with a mouse monoclonal antibody for NFL and ToPro3, a nuclear dye. Scale bar, 100 μm. (M–R) CAD cell transfectants, as above, were fixed and stained with a polyclonal antibody for NFL (Perez-Olle et al. 2002) along with a mouse monoclonal antibody specific for the transfected human DJ-1. Scale bar, 20 μm. (S) Quantification of CAD cell NFL aggregates was performed using confocal microscopy. Images from tenrandomly selected fields in each of three wells were quantified for the presence of aggregates for each condition and presented as a percentage of total cells per field. Total cell number was determined by ToPro3 nuclear staining and did not differ significantly (Figure S3). Data are shown as the mean ± SEM and were analyzed by ANOVA with Fisher's post-hoc test. * p < 0.05. DJ-1 Function Requires Cysteine 53 The DJ-1 crystal structure suggests the presence of two highly reactive cysteines, cysteine 106 (Lee et al. 2003; Wilson et al. 2003) and cysteine 53 (Honbou et al. 2003b). To test whether reactive cysteines play a critical role in the function or regulation of DJ-1 activity, we mutagenized each cysteine in DJ-1 to alanine (Figure 6). Surprisingly, mutation of cysteine 106, at the predicted nucleophile elbow of DJ-1, does not alter the basal activity (Figure 6A) or the DTT sensitivity (See Figure S1) of DJ-1 chaperone function. In contrast, mutation of cysteine 53, which is present at the dimeric interface of DJ-1, completely abrogates chaperone activity. Similarly, mutation of all three cysteines in DJ-1 (cysteine 106, cysteine 53, and cysteine 47) leads to the loss of chaperone function. The cysteine mutations do not alter DJ-1 dimerization (Figure 6D) or the apparent stability of DJ-1 in vivo (unpublished data), unlike the L166P Parkinsonism-associated mutation. Figure 6 DJ-1 In Vitro Chaperone Activity and In Vivo Oxidative Stress Protection Activity Require Cysteine 53 but Not Cysteine 106 (A) DJ-1 cysteine-to-alanine mutants C106A, C53A, and a triple mutant that harbors mutations at all three cysteines in DJ-1 (C106A/C53A/C46A), as well as L166P, were tested for in vitro chaperone activity by CS aggregation suppression assay. (B) Self-association of DJ-1 cysteine mutants. Murine neuroblastoma CAD cells were transiently cotransfected with Flag-tagged human DJ-1 vectors (either WT or mutant) along with WT YFP-tagged human DJ-1. Lysates were immunoprecipitated with anti-Flag antibodies and probed by Western blotting with an antibody specific for human DJ-1. WT Flag-DJ-1, C106A DJ-1, C53A DJ-1, and C106A/C53A/C46A DJ-1 effectively coprecipitated WT GFP-DJ-1, whereas the L166P mutant Flag-DJ-1 failed to do so. Lysates represent 20% of the input for the immunoprecipitate; Flag-DJ-1 migrates at 22 kDa, and YFP-DJ-1 migrates at 50 kDa. (C) DJ-1-deficient ES cells were transiently transfected with vector alone, WT DJ-1, or DJ-1 cysteine mutants, and exposed to 10 μM H2O2 for 15 h followed by MTT assay. The viability of the cells in the absence of drug treatment was not altered by the expression of WT or mutant DJ-1). Data are shown as the mean ± SEM and were analyzed by ANOVA with Fisher's post-hoc test. * p < 0.05 (D) Expression levels of WT and mutant forms of DJ-1 were comparable as determined by Western blotting for human DJ-1 and β-actin. DJ-1-deficient ES cells display increased sensitivity to oxidative stress, and this phenotype can be “rescued” by overexpression of WT DJ-1 but not PD-associated L166P mutant DJ-1 (Martinat et al. 2004). We further investigated the activity of the cysteine-mutant forms of human DJ-1 in vivo in the complementation of DJ-1-deficient ES cells. Cysteine 106–mutant DJ-1 robustly rescued DJ-1 knockout cells from H2O2 toxicity, consistent with the in vitro chaperone activity assay (Figure 6C). In contrast, cysteine 53 and the triple-cysteine mutant forms of DJ-1 failed to protect from H2O2 toxicity. These data support a role for cysteine 53–dependent chaperone activity in DJ-1-mediated ROS protection, and demonstrate a direct correlation between DJ-1 in vitro chaperone activity and cellular protection from oxidative stress. Our data are consistent with the prior observation that mutation of cysteine 53 to alanine abrogates the low–isoelectric point variant that is induced by oxidative stress (Honbou et al. 2003a). Discussion We provide evidence that DJ-1 functions as a cytoplasmic redox-sensitive molecular chaperone in vitro and in vivo. This activity extends to αSyn and the neurofilament subunit NFL, proteins implicated in PD pathology. In a companion article (Martinat et al. 2004), we show that DJ-1 deficiency sensitizes cells to oxidative stress, leading to increased apoptosis in the context of an ROS burst. Taken together, our data strongly support the notion that DJ-1 functions as a redox-dependent protein chaperone to mitigate molecular insults downstream of an ROS burst. Oxidation-modified proteins have been shown to accumulate in the context of normal aging and PD, and may participate in the generation of protein aggregates in neurodegenerative disorders (Jenner 2003). It is of interest to identify relevant in vivo substrates for DJ-1 activity in the context of DNs in PD. Our data suggest that DJ-1 activity extends to multiple targets, reminiscent of other small protein chaperones (Gusev et al. 2002), and consistent with this, DJ-1 activity is not ATP-dependent (unpublished data). Candidate substrates for DJ-1 chaperone activity in the context of PD include αSyn and neurofilament proteins, based on their presence in PD protein inclusions. Our data suggest that DJ-1 functions to suppress protein aggregates in the cytoplasm. It is possible that DJ-1 plays additional roles in the mitochondria or nucleus, as has been suggested (Bonifati 2003; Canet-Aviles 2004), although DJ-1 appears to remain localized diffusely in the cytoplasm with or without toxin treatment in our studies (see Figure S2). Our data indicate that DJ-1 can suppress an early step in the formation of αSyn aggregates, the generation of high molecular weight oligomers (protofibrils). Interestingly, it has been suggested that such protofibrils, rather than the large fibrillar aggregates, may underlie αSyn toxicity in vivo (Volles et al. 2001). DJ-1 inhibits the aggregation of αSyn in differentiated cells in vivo, and loss of DJ-1 leads to increased accumulation of insoluble αSyn. DJ-1 appears to associate with αSyn in the Triton X-100-soluble fraction of FeCl2-treated lysates, and DJ-1 colocalizes with αSyn in the Triton X-100-insoluble fraction in the context of FeCl2 treatment. However, DJ-1 does not colocalize with the punctate protein aggregates visible by immunostaining in the case of either αSyn or NFL. This supports the notion that DJ-1 functions at an early step in the aggregation process, when the substrate protein may be misfolded, but has not yet formed a mature aggregate. We hypothesize that DJ-1 may promote the degradation of such misfolded proteins, either through the proteasome or through other cellular pathways such as chaperone-mediated autophagy. A recent study investigated the chaperone activity of WT DJ-1 in vitro toward CS and concluded that redox regulation was not a significant factor (Lee et al. 2003). This is most likely a consequence of the use of only oxidizing conditions (0.5 mM H2O2) but not reducing conditions in the described chaperone assays (Lee et al. 2003). A second report failed to detect DJ-1 chaperone activity in vitro (Olzmann et al. 2003), but importantly, this study employed only reducing conditions in which DJ-1 chaperone activity is abrogated. In the present study we demonstrate that DJ-1 chaperone activity is inhibited by reducing conditions, and can be stimulated by oxidation. Thus, in the normal reducing environment of the cell, DJ-1 may be inactive. Production of ROS and alteration of the redox state of the cytoplasm may activate DJ-1 chaperone activity as a mechanism of coping with protein aggregation and misfolding. We find that that the PD-associated L166P mutant DJ-1 fails to function as a molecular chaperone in vivo or in vitro. Consistent with this, in a companion article (Martinat et al. 2004), we show that this mutant fails to complement DJ-1 knockout cells in vivo, even when overexpressed at artificially high levels (Martinat et al. 2004). Furthermore, the L166P mutant form fails to dimerize even when expressed at WT levels. Thus, although prior studies (Miller et al. 2003) and our analyses (unpublished data) have found that the L166P PD-associated DJ-1 mutation leads to decreased protein stability, it is apparent that even overexpression of the L166P mutant protein does not restore function. The L166P clinical phenotype is not due simply to reduced levels of DJ-1 protein, and, furthermore, we do not observe evidence of altered subcellular localization of the L166P mutant protein (Figure 4M-4R). Rather, our studies favor a model by which the pathological mechanism of this mutation is a consequence of altered structure and resultant loss of function. Mutation of cysteine 53 in DJ-1 abrogates both chaperone and protective functions of this protein. Interestingly, cysteine 53 has previously been implicated as a reactive cysteine required for the in vivo modification of DJ-1 to a lower isoelectric point in response to oxidative stress (Honbou et al. 2003a), consistent with a role for such redox regulation in vivo. In contrast, cysteine 106, which has been reported to be sensitive to oxidative modification in vitro (Wilson et al. 2003), does not appear to be required for the in vitro and in vivo DJ-1 activities. Materials and Methods Cell culture and in vivo assays. Undifferentiated ES cells, CAD neuroblastoma cells, and HeLa cells were cultured using standard techniques (Abeliovich et al. 2000; Staropoli et al. 2003). Transfections were performed using Lipofectamine 2000 (Life Technologies, Carlsbad, California, United States) for 18–36 h according to the manufacturer's instructions. For in vivo αSyn aggregation assays, CAD cells were transfected with Flag-αSyn (pcDNA3) or DJ-1 (pCMS), and medium was replaced with medium without serum. Cells were cultured without serum to induce differentiation for 48 h post-transfection, at which time the medium was exchanged for medium alone or containing 2 mM FeCl2 and 5 μM lactacystin. Cells were treated with toxin for 18 h, then lysed or fixed with 4% PFA. Cell lysis was performed by resuspending cells in 50 mM Tris (pH 7.6), 150 mM sodium chloride, 0.2% Triton X-100, and protease inhibitor cocktail (Sigma, St. Louis, Missouri, United States). Cells were incubated on ice for 20 min and Triton X-100-soluble and -insoluble fractions were separated via centrifugation at 13,000 rpm for 15 min. Quantification of CAD cell aggregates was performed using a Zeiss LSM Pascal confocal microscope (Zeiss, Oberkochen, Germany) with a 20× long working distance lens. Images were imported to NIH Image J for analysis. Images from tenrandomly selected fields in each of three wells were quantified for each condition. Cells containing at least one intracytoplasmic aggregate, independent of size or number per cell, were scored as positive for aggregates. This number was divided by the number of transfected cells per field, determined by GFP fluorescence. ES cell culture and in vitro differentiation Mouse ES cells were propagated and differentiated as described (Martinat et al. 2004). ES cells were differentiated via the embryoid body protocol. Cells were transfected with Flag-αSyn (pCMS) using Lipofectamine 2000 as per the manufacturer's instructions. 48 h post-transfection, cells were treated with 2 mM FeCl2 (or media alone) for 18 h. Antibodies. An anti-DJ-1 rabbit polyclonal antibody was generated against the synthetic polypeptide QNLSESPMVKEILKEQESR, which corresponds to amino acids 64–82 of the mouse protein. Antiserum was produced using the Polyquick polyclonal antibody production service of Zymed Laboratories (South San Francisco, California, United States). The antiserum was affinity purified on a peptide-coupled Sulfolink column (Pierce Biotechnology, Rockford, Illinois, United States) according to the manufacturer's instructions. Antibody was used at a dilution of 1:200 for immunohistochemistry and Western blotting as described (Staropoli et al. 2003). Immunohistochemistry was performed with a rabbit polyclonal antibody to DJ-1 (Martinat et al. 2004), TH (PelFreez, Rogers, Arizona, United States; dilution 1:1000), and a rabbit polyclonal antibody to GABA (Sigma; dilution 1:1000). Western blotting was performed using monoclonal antibody to DJ-1 (Stressgen Biotechnologies, San Diego, California, United States; dilution 1:1000), a monoclonal antibody to αSyn LB509 antibody (Zymed), and a monoclonal antibody to β-actin (Sigma; dilution 1:500). Mouse monoclonal antibody to NFL (Sigma; dilution 1:200) and rabbit polyclonal antibody to NFL (Perez-Olle et al. 2002). ToPro3 (Molecular Probes, Eugene, Oregon, United States; dilution 1:1000) was used as a nuclear dye. Expression vectors. DJ-1 cDNA was PCR amplified from human liver cDNA (Clontech, Palo Alto, California, United States) and cloned into the expression vectors pET-28a (Novagen, Madison, Wisconsin, United States) or pcDNA3.1 (Invitrogen, Carlsbad, California, United States). Flag-DJ-1 and all described mutants were generated by PCR-mediated mutagenesis using standard techniques. In vitro preparation of WT and mutant DJ-1. His-tagged recombinant human WT or L166P DJ-1 was produced in E. coli BL21 cells induced with 1 mM IPTG for 4 h at 37 °C. Bacterial pellets were resuspended in 50 mM sodium phosphate (pH 6.8) and 300 mM sodium chloride, and lysed by sonication. Lysates were cleared by centrifugation at 20,000 × g for 20 min, and the supernatant was incubated with NTA-Ni-conjugated agarose resin for 1 h at 4 °C. The resin was subsequently washed five times with 20 resin volumes of lysis buffer containing 20 mM imidazole, and protein was eluted in five fractions of two resin volumes of lysis buffer containing 250 mM imidazole. Recombinant protein elutions were confirmed to be of > 99% purity by SDS-PAGE and colloidal Coomassie staining. Aggregation assays. CS aggregation was performed in 40 mM HEPES (pH 7.8), 20 mM potassium hydroxinde, 50 mM potassium chloride, and 10 mM ammonium sulfate, and monitored in a thermostat-controlled fluorescence spectrophotometer with excitation and emission wavelengths at 500 nm and slit widths at 2.5 nm. Insulin aggregation was performed as described (Giasson et al. 2000). CS, insulin, RNase A, and GST were obtained from Sigma; human Hsp27 was obtained from Stressgen. αSyn protofibril and fibril formation assays were performed essentially as described (Uversky et al.). Briefly, protofibrils were formed by incubation of 200 μM WT synuclein with 100 μM DJ-1 or control chaperone protein in PBS for 2 h at 55 °C. Samples were mixed with SDS loading buffer and analyzed by SDS-PAGE and Western blotting using αSyn LB509 antibody (Zymed). Quantitation of high molecular weight αSyn was performed using NIH Image J. Integrated pixel intensity of high molecular weight synuclein for each sample was normalized to monomeric synuclein intensity. For fibril formation, αSyn and chaperone proteins (as described above) were incubated with shaking for 1 wk at 37 °C. Fibril formation was assessed by Congo red (Conway et al. 2000b). Supporting Information Figure S1 Additional Structural and Functional Analyses of DJ-1 In Vitro (A) DJ-1 catalase activity was quantified as compared to catalase I (5 μg/ml). DJ-1 does not display catalase activity even at concentrations as high as 5 mg/ml. (B) Addition of DJ-1 at 5 mg/ml does not alter catalase activity of the catalase I-positive control, indicating that there are no inhibitory elements present in the DJ-1 preparation. (C) Purity of bacterially produced DJ-1 utilized in the in vitro assays was assessed to be > 99% by SDS-PAGE and colloidal Coomassie staining. (D) GST thermal aggregation (0.4 μM, black circles) is suppressed by WT DJ-1 (2 μM, red squares) and by positive control Hsp27 (2 μM, green stars), but not by L166P mutant DJ-1 (2 μM, blue triangles) or by RNase A (2 μM, purple diamonds). (E) Far-ultraviolet CD spectra of WT DJ-1 (blue triangles) and the L166P mutant (red squares); mean residue ellipticity (Θ) equals °C · cm2 · dmol−1. The mutant protein displays significantly reduced secondary structure. CD spectra of DJ-1 (40 μM in 10 mM PBS [pH 7.4]) were recorded on an Aviv 62A sCD spectrometer at 4 °C in a 0.02-cm path length cuvette, and α-helix and β-sheet content were estimated as described (Sreerama and Woody 2003). Based on an initial evaluation of the spectra, the WT spectrum was analyzed using a basis set appropriate for folded proteins, whereas the mutant spectrum was analyzed using a basis set suited for unstructured proteins. Thermal stability was determined by monitoring the change in mean residue ellipticity ([Θ], equal to °C · cm2 · dmol−1) at 222 nm as a function of temperature. Thermal melts were performed in 4 °C increments with an equilibration time of 1 min and an integration time of 30 sec, using a 0.1-cm path length cuvette. (F) Thermal denaturation curves for WT and mutant L166P DJ-1; mean residue ellipticity (Θ)222 is equal to °C · cm2 · dmol−1 at 222 nm. (G) Redox regulation is unaffected by the C106A mutation. Redox regulation of C106A DJ-1 was assayed via DTT inactivation (0.5 mM) in the CS aggregation suppression assay. (H) Protofibril preparations (as in Figure 2A and 2B, incubated for 2 h at 55 °C) do not contain Congo red–positive mature fibrils. Untreated αSyn preparations (open bars) and protofibril preparations (filled bars) were subjected to Congo red analysis as in Figure 2C. (1.2 MB PDF). Click here for additional data file. Figure S2 Additional Studies of DJ-1 Chaperone Activity In Vivo (A) Undifferentiated ES cells were transfected with Flag-αSyn and treated with 2 mM FeCl2 (Fe) or media alone (0) as described in Figure 3. As expected, undifferentiated ES cultures do not express endogenous αSyn. Furthermore, the transfected Flag-αSyn does not accumulate in the Triton X-100-insoluble fraction of undifferentiated cells, in contrast to differentiated cultures. (B) Overexpression of WT DJ-1 does not significantly alter the half-life of soluble Flag-αSyn. CAD murine neuroblastoma cells were stably transfected with Flag-tagged human α-synuclein using standard techniques. 2 × 105 cells in a 24-well format were transiently transfected with eukaryotic expression constructs encoding WT human DJ-1 or empty vector. After 36 h, cells were starved for 1 h with DMEM lacking cysteine and methionine and supplemented with 8% dialyzed FBS. Cells were pulsed for 2 h with 10 μCi[35S]-L-Met/L-Cys (EasyTides; Perkin Elmer, Wellesley, California, United States) per well, washed twice, and chased at the indicated intervals with complete medium. Flag-αSyn was immunoprecipitated with Flag antibody-conjugated agarose beads (Sigma), subjected to SDS-PAGE, and visualized by autoradiography. (C) Flag-αSyn from (B) was quantitated using NIH Image J. (815 KB PDF). Click here for additional data file. Figure S3 Additional Studies of DJ-1 Mutations (A) Overexpression of WT DJ-1 or L166P DJ-1 in the context of αSyn aggregation does not alter cell number. Cells from Figure 4M were quantified via ToPro3 nuclear staining and are expressed as number of cells per field from ten independent fields in each of three wells. Data are shown as the mean ± SEM and were analyzed by ANOVA with Fisher's post-hoc test. * p < 0. (B) Overexpression of WT DJ-1 or L166P mutant DJ-1 in the context of Q333P mutant NFL aggregation does not alter cell number. GFP positive transfected cells from Figure 5A–5L were quantified and are expressed as number of transfected cells per field from ten independent fields in each of three wells. Data are shown as the mean ± SEM and were analyzed by ANOVA with Fisher's post-hoc test. * p < 0. (C) Overexpression of WT DJ-1, but not L166P mutant DJ-1, rescues cells from Q333P mutant NFL toxicity. HeLa cells were transfected with Q333P mutant NFL along with WT human DJ-1, L166P mutant DJ-1, or vector control. After 72 h, cells were assayed by MTT reduction assay (which detects reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide by metabolic enzymes) (Martinat et al. 2004). Data are shown as the mean ± SEM and were analyzed by ANOVA with Fisher's post-hoc test. * p < 0. (D) C53A mutant DJ-1 is unable to rescue cells from Q333P mutant NFL toxicity. Undifferentiated ES cells were transfected with Q333P mutant NFL along with WT human DJ-1, C53A mutant DJ-1, or vector control. After 72 h, cells were assayed by MTT reduction assay (Martinat et al. 2004). Data are shown as the mean ± SEM and were analyzed by ANOVA with Fisher's post-hoc test. * p < 0. (E) Coexpression of DJ-1 with NFL does not alter NFL expression levels. CAD cells were transfected with Q333P mutant NFL and vector, WT DJ-1, C53A mutant DJ-1, or L166P mutant DJ-1. Cells were differentiated for 72 h and lysed to produce Triton X-100-soluble and -insoluble fractions. Lysates were exposed to Western blotting with an antibody against transfected human NFL. NFL is present only in the insoluble fraction, and expression of WT or mutant DJ-1 does not alter NFL expression levels. (685 KB PDF). Click here for additional data file. Figure S4 DJ-1 Localization Does Not Appear Altered by FeCl2 Treatment CAD cells were transfected with WT DJ-1 and differentiated by serum withdrawal for 72 h. Cells were treated with medium alone (A–F) or medium with 2 mM FeCl2 (G–L) for 18 h prior to fixation with PFA. Cells were immunostained with rabbit anti-DJ-1 as described, followed by donkey anti-rabbit Cy5 (A, D, G, and J). Nuclei (B, E, H, and K) were visualized by incubation with the nuclear stain ToPro3 prior to imaging. (1.6 MB PDF). Click here for additional data file. Table S1 DJ-1 Lacks Protease and Antioxidant Activities (45 KB DOC). Click here for additional data file. We thank Yaacov Hod and David Eliezer for assistance at the early stages of this project. SS is funded by the Integrated Graduate Program Training Grant (National Institutes of Health [NIH]); and CM is funded by the American Parkinson's Disease Association. AA receives funding from the Spitzer, Taub, Rockefeller Brothers, and MJ Fox Foundations, National Institute of Neurological Diseases and Stroke, and National Institute on Aging, and is a Culpeper and Beeson scholar. Conflicts of interest. The authors have declared that no conflicts of interest exist. Author contributions. SS, ASJ, CM, and AA conceived and designed the experiments. SS, ASJ, CM, TL, and AA performed the experiments. SS, ASJ, CM, and AA analyzed the data. SS, ASJ, CM, and AA contributed reagents/materials/analysis tools. SS, ASJ, CM, and AA wrote the paper. Academic Editor: Huda Y. Zoghbi, Baylor College of Medicine Citation: Shendelman S, Jonason A, Martinat C, Leete T, Abeliovich A (2004) DJ-1 is a redox-dependent molecular chaperone that inhibits α-synuclein aggregation formation. PLoS Biol 2(11): e362. Abbreviations αSynα-synuclein CDcircular dichroism CScitrate synthase DNdopamine neuron DTTdithiothreitol ESembryonic stem Flag-αSynFlag epitope-tagged αSyn GSTglutathione S-transferase Hispolyhistidine Hspheat shock protein NFLneurofilament light subunit PDParkinson's disease ROSreactive oxygen species WTwild-type ==== Refs References Abeliovich A Schmitz Y Farinas I Choi-Lundberg D Ho WH Mice lacking α-synuclein display functional deficits in the nigrostriatal dopamine system Neuron 2000 25 239 252 10707987 Beal MF Oxidatively modified proteins in aging and disease Free Radic Biol Med 2002 32 797 803 11978481 Bonifati V Rizzu P van Baren MJ Schaap O Breedveld GJ Mutations in the DJ-1 gene associated with autosomal recessive early-onset parkinsonism Science 2003 299 256 259 12446870 Canet-Aviles RM Wilson MA Miller DW Ahmad R McLendon C The Parkinson's disease protein DJ-1 is neuroprotective due to cysteine-sulfinic acid-driven mitochondrial localization Proc Natl Acad Sci U S A 2004 101 24 9103 9108 15181200 Chelikani P Donald LJ Duckworth HW Loewen PC Hydroperoxidase II of Escherichia coli exhibits enhanced resistance to proteolytic cleavage compared to other catalases Biochemistry 2003 42 5729 5735 12741830 Conway KA Lee SJ Rochet JC Ding TT Harper JD Accelerated oligomerization by Parkinson's disease linked α-synuclein mutants Ann N Y Acad Sci 2000a 920 42 45 11193175 Conway KA Harper JD Lansbury PT Fibrils formed in vitro from α-synuclein and two mutant forms linked to Parkinson's disease are typical amyloid Biochemistry 2000b 39 2552 2563 10704204 Dauer W Przedborski S Parkinson's disease: Mechanisms and models Neuron 2003 39 889 909 12971891 de Nobel H Lawrie L Brul S Klis F Davis M Parallel and comparative analysis of the proteome and transcriptome of sorbic acid-stressed Saccharomyces cerevisiae Yeast 2001 18 1413 1428 11746603 Du X Choi IG Kim R Wang W Jancarik J Crystal structure of an intracellular protease from Pyrococcus horikoshii at 2-Å resolution Proc Natl Acad Sci U S A 2000 97 14079 14084 11114201 Giasson BI Duda JE Murray IV Chen Q Souza JM Oxidative damage linked to neurodegeneration by selective α- synuclein nitration in synucleinopathy lesions Science 2000 290 985 989 11062131 Goldberg MS Lansbury PT Is there a cause-and-effect relationship between α-synuclein fibrillization and Parkinson's disease? 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W Structural composition of β(I)- and β(II)-proteins Protein Sci 2003 12 384 388 12538903 Staropoli JF McDermott C Martinat C Schulman B Demireva E Parkin is a component of an SCF-like ubiquitin ligase complex and protects postmitotic neurons from kainate excitotoxicity Neuron 2003 37 735 749 12628165 Taira T Saito Y Niki T Iguchi-Ariga SM Takahashi K DJ-1 has a role in antioxidative stress to prevent cell death EMBO Rep 2004 5 213 218 14749723 Tao X Tong L Crystal structure of human DJ-1, a protein associated with early onset Parkinson's disease J Biol Chem 2003 278 31372 31379 12761214 Trojanowski JQ Lee VM Aggregation of neurofilament and α-synuclein proteins in Lewy bodies: Implications for the pathogenesis of Parkinson disease and Lewy body dementia Arch Neurol 1998 55 151 152 9482355 Volles MJ Lee SJ Rochet JC Shtilerman MD Ding TT Vesicle permeabilization by protofibrillar α-synuclein: Implications for the pathogenesis and treatment of Parkinson's disease Biochemistry 2001 40 7812 7819 11425308 Weinreb PH Zhen W Poon AW Conway KA Lansbury PT NACP, a protein implicated in Alzheimer's disease and learning, is natively unfolded Biochemistry 1996 35 13709 13715 8901511 Wilson MA Collins JL Hod Y Ringe D Petsko GA The 1.1-Å resolution crystal structure of DJ-1, the protein mutated in autosomal recessive early onset Parkinson's disease Proc Natl Acad Sci U S A 2003 100 9256 9261 12855764

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PLoS Biol. 2004 Nov 5; 2(11):e362

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==== Front PLoS BiolPLoS BiolpbioplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 1550287510.1371/journal.pbio.0020363Research ArticleBioinformatics/Computational BiologyCell BiologyGenetics/Genomics/Gene TherapyMolecular Biology/Structural BiologyHomo (Human)Human MicroRNA Targets Human MicroRNA TargetsJohn Bino 1 Enright Anton J 1 2 Aravin Alexei 3 Tuschl Thomas 3 Sander Chris 1 Marks Debora S [email protected] 4 1Computational Biology Center, Memorial Sloan-Kettering Cancer CenterNew York, New YorkUnited States of America2Wellcome Trust Sanger InstituteCambridgeUnited Kingdom3Laboratory of RNA Molecular Biology, The Rockefeller UniversityNew York, New YorkUnited States of America4Department of Systems Biology, Harvard Medical SchoolBoston, MassachusettsUnited States of America11 2004 5 10 2004 5 10 2004 2 11 e36318 5 2004 20 8 2004 Copyright: © 2004 John et al.2004This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. MicroRNA is a Major Regulator MicroRNAs (miRNAs) interact with target mRNAs at specific sites to induce cleavage of the message or inhibit translation. The specific function of most mammalian miRNAs is unknown. We have predicted target sites on the 3′ untranslated regions of human gene transcripts for all currently known 218 mammalian miRNAs to facilitate focused experiments. We report about 2,000 human genes with miRNA target sites conserved in mammals and about 250 human genes conserved as targets between mammals and fish. The prediction algorithm optimizes sequence complementarity using position-specific rules and relies on strict requirements of interspecies conservation. Experimental support for the validity of the method comes from known targets and from strong enrichment of predicted targets in mRNAs associated with the fragile X mental retardation protein in mammals. This is consistent with the hypothesis that miRNAs act as sequence-specific adaptors in the interaction of ribonuclear particles with translationally regulated messages. Overrepresented groups of targets include mRNAs coding for transcription factors, components of the miRNA machinery, and other proteins involved in translational regulation, as well as components of the ubiquitin machinery, representing novel feedback loops in gene regulation. Detailed information about target genes, target processes, and open-source software for target prediction (miRanda) is available at http://www.microrna.org. Our analysis suggests that miRNA genes, which are about 1% of all human genes, regulate protein production for 10% or more of all human genes. This computational analysis provides evidence that as many as 10% of human genes are targets for regulation by small RNA molecules called microRNAs ==== Body Introduction The Functions of MicroRNAs In the past three years, several hundred novel genes encoding transcripts containing short double-stranded RNA hairpins, named microRNAs (miRNAs), were identified in plants and animals (Lee et al. 1993; Reinhart et al. 2000, 2002; Lagos-Quintana et al. 2001, 2002, 2003; Lau et al. 2001; Lee and Ambros 2001; Llave et al. 2002a; Mette et al. 2002; Mourelatos et al. 2002; Park et al. 2002; Ambros et al. 2003b; Aravin et al. 2003; Brennecke et al. 2003; Dostie et al. 2003; Grad et al. 2003; Houbaviy et al. 2003; Lai et al. 2003; Lim et al. 2003a, 2003b; Palatnik et al. 2003). More recently, miRNAs have also been identified in a large DNA virus, the Epstein Barr virus, and are likely to be found in other viruses (Pfeffer et al. 2004). The cellular functions of most animal miRNAs are unknown. More than ten years after the discovery of the first miRNA gene, lin-4 (Chalfie et al. 1981; Lee et al. 1993), we know that miRNA genes constitute about 1%–2% of the known genes in eukaryotes. Investigation of miRNA expression combined with genetic and molecular studies in Caenorhabditis elegans, Drosophila melanogaster, and Arabidopsis thaliana have identified the biological functions of several miRNAs (recent review, Bartel 2004). In C. elegans, lin-4 and let-7 were first discovered as key regulators of developmental timing in early larval developmental transitions (Ambros 2000; Abrahante et al. 2003; Lin et al. 2003; Vella et al. 2004). More recently lsy-6 was shown to determine the left–right asymmetry of chemoreceptor expression (Johnston and Hobert 2003). In D. melanogaster, miR-14 has a role in apoptosis and fat metabolism (Xu et al. 2003) and the bantam miRNA targets the gene hid involved in apoptosis and growth control (Brennecke et al. 2003). In mouse, miR-181a modulates hematopoietic differentiation (Chen et al. 2004), and miR-196 directs the cleavage of Hox-B8 transcripts (Yekta et al. 2004). miRNAs have specificity. In a range of organisms, miRNAs are differentially expressed in developmental stages, cell types, and tissues (Lee and Ambros 2001; Lagos-Quintana et al. 2002; Sempere et al. 2004). In particular, differential expression has been observed in mammalian organs (Lagos-Quintana et al. 2002; Krichevsky et al. 2003; Sempere et al. 2004) and embryonic stem cells (Houbaviy et al. 2003). Estimates in worm show that there are approximately 1,000 molecules of miRNA per cell, with some cells exceeding 50,000 molecules (Lim et al. 2003b). This dynamic range of regulation of miRNA expression underscores the regulatory functional importance of miRNAs. The Mechanism of miRNA Action How do miRNAs pair with their target messages? miRNAs cause the translational repression or cleavage of target messages (Doench and Sharp 2004). Some miRNAs may behave like small interfering RNAs (siRNAs) that direct mRNA cleavage between the nucleotide positions 10 and 11 in the siRNA:mRNA target duplex (Tuschl et al. 1999; Zamore et al. 2000; Elbashir et al. 2001; Hutvágner and Zamore 2002a; Llave et al. 2002b; Martinez et al. 2002; Bartel 2004; Yekta et al. 2004). It appears that the extent of base pairing between the small RNA and the mRNA determines the balance between cleavage and degradation (Hutvágner and Zamore 2002a). Recent examples of cleavage of target messages are, in mouse, mir-196 guiding cleavage of Hox-B8 transcripts (Yekta et al. 2004) and, in Epstein Barr virus, miR-BART2, a virus-encoded miRNA, guiding the cleavage of transcripts for virus DNA polymerase (gene BALF5) (Pfeffer et al. 2004). While cleavage of mRNA is a straightforward process, the details of the mechanism of translational repression are unknown. The following rules for matches between miRNA and target messages have been deduced from a range of experiments. (1) Asymmetry: experimentally verified miRNA target sites indicate that the 5′ end of the miRNA tends to have more bases complementary to the target than its 3′ end. Loopouts in either the mRNA or the miRNA between positions 9 and 14 of the miRNA have been observed or deduced (Brennecke et al. 2003; Johnston and Hobert 2003; Lin et al. 2003; Vella et al. 2004). Recent experiments show some correlation between the level of translational repression and the free energy of binding of the first eight nucleotides in the 5′ region of the miRNA (Doench and Sharp 2004). However, confirmed miRNA:mRNA target pairs can have mismatches in this region (Moss et al. 1997; Johnston and Hobert 2003). (2) G:U wobbles: wobble base pairs are less common in the 5′ end of a miRNA:mRNA duplex, and recent work shows a disproportionate penalty of G:U pairing relative to standard thermodynamic considerations (Doench and Sharp 2004). (3) Cooperativity of binding: many miRNAs can bind to one gene (Reinhart et al. 2000; Ambros 2003; Vella et al. 2004), and the target sites may overlap to some degree (Doench and Sharp 2004). Given the overlap between the siRNA and miRNA pathways, it is reasonable to assume that rules of regulation in the siRNA pathway will partly apply to miRNA target recognition (Hutvágner and Zamore 2002b; Boutet et al. 2003; Doench et al. 2003). Lately, detailed characteristics associated with siRNA functionality were identified: low G/C content, a bias towards low internal stability at the3′ terminus, lack of inverted repeats, and strand base preferences (positions 3, 10, 13, and 19) (Jackson et al. 2003; Reynolds et al. 2004). These observations may provide clues for better quantitative description of miRNA:mRNA interaction. Regions adjacent or near to the target site can be important for miRNA specificity. In lin-41, a 27-nucleotide (nt) intervening sequence between two consecutive let-7 sites is necessary for its regulation (Vella et al. 2004). Because of lack of conservation of this 27-nt intervening sequence in C. briggsae, incorporation of a corresponding rule is premature. Maturation of miRNAs and Assembly in RNA-Induced Silencing Complex miRNAs are transcribed as longer precursors, termed pre-miRNAs (Lee et al. 2002), sometimes in clusters and frequently in introns (25% of human miRNAs; Table S1). Upon transcription, miRNAs undergo nuclear cleavage by the RNase III endonuclease Drosha, producing the 60–70-nt stem-loop precursor miRNA (pre-miRNA) with a 5′ phosphate and a 2-nt 3′ overhang (Lee et al. 2003). The pre-miRNA is subsequently transported across the nuclear membrane, dependent on the protein exportin 5 (Lund et al. 2003; Yi et al. 2003). Dicer cleaves the pre-miRNA in the cytoplasm about two helical turns away from the ends of the pre-miRNA stem loop, producing double-stranded RNA. A helicase unwinds the cleaved double-stranded RNA in a strand-specific direction (Khvorova et al. 2003; Schwarz et al. 2003). One of the unwound strands is subsequently incorporated into a ribonuclear particle (RNP) complex, RNA-induced silencing complex (RISC) (Hutvágner and Zamore 2002a; Martinez et al. 2002). Every RISC contains a member of the Argonaute protein family, which tightly binds the RNA in the complex (Hammond et al. 2001; Hutvágner and Zamore 2002a; Martinez et al. 2002; Mourelatos et al. 2002). There are at least eight members of the Argonaute family in mammals (Sasaki et al. 2003), and only a small subset has been functionally characterized. The Argonautes and Dicer bind single-stranded RNA via their PAZ domains (Lingel et al. 2003; Sasaki et al. 2003; Song et al. 2003; Yan et al. 2003), and the known structures of the PAZ domains may have implications for prediction of miRNA targets (Lingel et al. 2003; Song et al. 2003; Yan et al. 2003). Association of mRNAs and miRNAs with Fragile X Mental Retardation Protein Among the prime candidates for miRNA control are the genes that are posttranscriptionally regulated. The mRNA-binding protein fragile X mental retardation protein (FMRP) is involved in the regulation of local protein synthesis (Antar and Bassell 2003) and binds 4% of mRNAs expressed in the rat brain, as tested in vitro (Brown et al. 2001). The loss of function of FMRP causes fragile X syndrome, the most prevalent form of mental retardation (one in every 2,000 children). Over the past three years a number of different groups have identified in vivo mRNA cargoes of FMRP. The Warren and Darnell laboratories have identified ligands by co-immunoprecipitation followed by microarray analysis, complemented by extraction of polyribosomal fractions (Brown et al. 2001). They discovered that FMRP and one of its three RNA-binding domains specifically binds to G-rich quartet motifs (Brown et al. 2001; Darnell et al. 2001; Denman 2003; Miyashiro et al. 2003). Three more studies found that mRNAs containing U-rich motifs bind recombinant FMRP in vitro and associate with FMRP-containing mRNPs in vivo (Chen et al. 2003; Denman 2003). Lastly, antibody-positioned RNA amplification as a primary screen followed by traditional methods identified over 80 FMRP-regulated mRNAs, with a combination of G-quartet and U-rich motifs in their mRNA sequences (Miyashiro et al. 2003). Independently, FMRP has been shown to be associated with RISC components and miRNAs (Jin et al. 2004). The Drosophila homolog of FMRP (FXR) and the Vasa intronic gene were identified as components of RISC (Caudy et al. 2002). More recent studies have proved that mammalian FMRP interacts with miRNAs and with the components of the miRNA pathways including Dicer and the mammalian orthologs of Argonaute (AGO) 1 (Ishizuka et al. 2002; Jin et al. 2004). Given the association of FMRP with Argonaute-containing complexes, we propose and investigate the hypothesis that the cargoes carried by FMRP are also miRNA targets, and we derive hypotheses of specific pairing interactions. Here, we predict miRNA targets in five vertebrate genomes as a way of facilitating experiments and exploring a number of open questions. What proportion of all genes is regulated by miRNAs? How many genes are regulated by each miRNA? Are specific cellular processes targeted by specific miRNAs or by miRNAs in general? What is the extent of cooperativity in miRNA:mRNA binding? Results Prediction of miRNA Targets Using currently known mammalian miRNA sequences, we scanned 3′ untranslated regions (UTRs) from the human (Homo sapiens), mouse (Mus musculus), and rat (Rattus norvegicus) genomes for potential target sites. The scanning algorithm was based on sequence complementarity between the mature miRNA and the target site, binding energy of the miRNA–target duplex, and evolutionary conservation of the target site sequence and target position in aligned UTRs of homologous genes. We identified as conserved across mammals a total of 2,273 target genes with more than one target site at 90% conservation of target site sequence (Tables S2 and S3) and 660 target genes at 100% conservation. We also scanned the zebrafish (Danio rerio) and fugu (Fugu rubripes) fish genomes for potential targets using known and predicted miRNAs (Figure 1; Tables S4 and S5) and identified 1,578 target genes with two or more conserved miRNA sites between the two fish species. Figure 1 Target Prediction Pipeline for miRNA Targets in Vertebrates The mammalian (human, mouse, and rat) and fish (zebra and fugu) 3′ UTRs were first scanned for miRNA target sites using position-specific rules of sequence complementarity. Next, aligned UTRs of orthologous genes were used to check for conservation of miRNA–target relationships (“target conservation”) between mammalian genomes and, separately, between fish genomes. The main results (bottom) are the conserved mammalian and conserved fish targets, for each miRNA, as well as a smaller set of super-conserved vertebrate targets. In addition to the analysis of 3′ UTRs, we also scanned all protein-coding regions for high-scoring miRNA target sites. For convenience, these results are reported both as hits in cDNAs (coding plus noncoding; Table S6) and as hits in coding regions (Table S7), with cross-references in the UTR target tables (number of hits in the coding region for each UTR in Tables S2 and S3). The algorithm and cutoff parameters were chosen to provide a flexible mechanism for position-specific constraints and to capture what is currently known about experimentally verified miRNA target sites: (1) nonuniform distribution of the number of sequence-complementary target sites for different miRNAs; (2) 5′–3′ asymmetry (the complementary pairing of about ten nucleotides at the 5′ end is more important than that of the ten nucleotides at the 3′ end [Doench and Sharp 2004], and the matches near the 3′ end can to a limited extent compensate for weaker 5′ binding); and (3) influence of G:U wobbles on binding (Doench and Sharp 2004). In choosing these parameters, we drew on experience from careful analysis of target predictions in Drosophila (Enright et al. 2003) as well as proposed human targets of virus-encoded miRNAs (Pfeffer et al. 2004). To facilitate evaluation of predicted targets and design of new experiments, we provide methods and results in a convenient and transparent form. We make the miRanda software freely available under an open-source license, so that researchers can adjust the algorithm, numerical parameters, and position-specific rules. We also provide web resources, including a viewer for browsing potential target sites, conserved with or without positional constraints, on aligned UTRs, with periodic updates (http://www.microrna.org, as well as links to these targets from the miRNA registry site RFAM (http://www.sanger.ac.uk; Griffiths-Jones 2004). We provide both high-scoring targets, as strong candidates for validation experiments, and lower-scoring targets, which may have a role in broader background regulation of protein dose. Expression information (see Table S3) for miRNAs and mRNAs provides an additional filter for validation experiments, in addition ranking target sites by complementarity and evolutionary conservation. Validation of Target Predictions Only a small number of target sites of target genes regulated by miRNAs have been experimentally verified, so we sought direct and indirect evidence to help validate or invalidate the proposed set of mammalian targets. (1) We compared predicted targets with experimentally verified targets in mammals, C. elegans, and D. melanogaster, as well as their mammalian homologs. (2) We compared predicted target numbers from real and shuffled miRNA sequences and estimated the rate of false-positive predictions. (3) We assessed the enrichment of miRNA targets in mRNAs that are known cargoes of FMRP, an RNA-binding protein known to be involved in translational regulation. Agreement with known targets We previously used known miRNA sites for the let-7 and lin-4 miRNAs in Drosophila to develop the target prediction method and check for consistency (Enright et al. 2003). More recent experimental target identification provides independent control data. Recent work in C. elegans (Vella et al. 2004) has narrowed the originally reported list of six target sites for let-7 in the UTR of lin-41 down to three elements, two target sites, and a 27-nt intervening sequence (a possible binding site for another factor). The surviving two target sites have high alignment scores, S = 115 and S = 110, while the other four sites are below threshold (Enright et al. 2003), fully consistent with the experimental results. As one of the confirmed sites has a single-residue bulge, target prediction methods that require a perfect run of base pairs near the 5′ end of the miRNA would not detect it, while our method does. lsy-6, a recently experimentally identified miRNA in C. elegans, controls left–right neuronal asymmetry via cog-1, an Nkx-type homeobox gene; the cog-1 gene has a target site in its 3′ UTR, which also has a high score (S = 125) and passes the conservation filter. Experiments in D. melanogaster have identified six new miRNA–target gene pairs: miR-7 targets the notch signaling genes HLHm3, HLHm4, and hairy, and miR-2b targets the genes reaper, grim, and sickle (Stark et al. 2003). Consistent with these experiments, our target predictions in D. melanogaster (Enright et al. 2003) ranked HLHm3, hairy, and HLHm4 at positions 1, 3, and 7, respectively, in the list of 143 target genes for miR-7 (Enright et al. 2003). Similarly, our predictions ranked reaper, grim, and sickle at positions 3, 11, and 19, respectively, among the other 120 predicted target genes for miR-2c. We also predicted miR-6 to target this group of pro-apoptotic genes, with sites that have lower scores than the miR-2 family but are conserved in D. pseudoobscura. Unfortunately, one cannot in general use these predicted and then validated target sites (Stark et al. 2003) for the derivation of new prediction rules, as the set of targets tested is limited to the type predicted and is not exhaustive. Indirect validation comes from the prediction that mammalian orthologs of some of the known miRNA targets in C. elegans and D. melanogaster are miRNA targets. An example is the proposed conservation of the miRNA–target relationship lin-4:lin-28 (we use the notation miRNA:mRNA for a miRNA–target pair), first discovered in worm (Moss and Tang 2003): we detect target sites in human lin-28 for the lin-4 miRNA homolog miR-125. We also confirm the human analog of a let-7:lin-28 relation predicted in C. elegans (Reinhart et al. 2000). In summary, the predicted target sites on human lin-28 are miR-125 (1 site), let-7b (2 sites; Moss and Tang 2003), miR-98 (2 sites), and miR-351 (1 site). Another known lin-4 and let-7 target in C. elegans is lin-41. The human homolog of lin-41 (sequence provided by F. J. Slack, personal communication) and another closely related gene (encoding Tripartite motif protein 2) are predicted as high-ranking targets of let-7 and miR-125 (the human homolog of lin-4) (see Tables S2 and S3). Another known instance of miRNA target regulation in worms is the regulation of cog-1 by the lsy-6 miRNA (Johnston and Hobert 2003). Although there is no obvious homolog of lsy-6 in mammals, the vertebrate homolog of the target gene cog-1, nkx-6.1, is a conserved target for five different miRNAs in our predictions (see Table S2). The comparison of our results with known targets shows that our method can detect most (but not all) known target sites and target genes at reasonably high rank. However, given the small number of experimentally verified miRNA–target pairs, additional validation tests are desirable, such as statistical tests using randomization of miRNA sequences to estimate false positives. Estimate of false positives As a computational control of the validity of the prediction method, one can perform a statistical test that attempts to estimate the probability that a predicted site is incorrect. Here, a “false positive” is a predicted target site of a real miRNA on a real mRNA that has passed all relevant thresholds but is incorrect in that it is not biologically meaningful. The statement “not biologically meaningful” is rarely clearly defined, but can reasonably be taken to mean that no functionally effective miRNA:mRNA interaction occurs under conditions of co-expression at physiological concentration, where “functionally effective” is defined in terms of detectable changes of phenotypic attributes. Technically, an estimate of the false-positive rate can be obtained by computing (directly or via randomization) the background distribution of scores for biologically non-meaningful miRNA target sites and then deriving the probability that a non-meaningful target site passes all score thresholds, i.e., for a single aggregate score, that the incorrect site has a score T > Tc, where Tc is a fixed threshold that may be, in general, different for each miRNA. We chose to estimate the background distribution using shuffled miRNAs obtained by swapping randomly selected pairs of bases of each given miRNA 1,000 times, keeping the nucleotide composition constant. The shuffled miRNA sequences were scanned against human, mouse, and rat 3′ UTR sequences exactly as for the prediction procedure for real miRNA sequences. In the procedure, a miRNA:mRNA match site is predicted to be a target site if it passes three thresholds, S > Sc for match score, |ΔG| > |ΔGc| for free energy of duplex formation, and C > Cc for conservation, where C reflects a binary evaluation of orthology of mRNAs, similarity of position of the site on the mRNA, and a threshold percentage of conserved residues in the two mRNA target sites. Finally, the predicted target sites for a set of shuffled miRNAs are counted and then averaged over a total of ten randomized runs. The percentage of false positives for target transcripts with more than two, three, and four sites is 39%, 30%, and 24%, respectively, using a non-permissive conservation threshold of 100% for target site sequences (Figure 2). In addition, the false-positive rate for single sites with a score of more than 110 is approximately 35%. Figure 2 Distribution of Transcripts with Cooperativity of Target Sites and Estimated Number of False Positives Each bar reflects the number of human transcripts with a given number of target sites on their UTR. Estimated rate of false positives (e.g., 39% for ≥2 targets) is given by the number of target sites predicted using shuffled miRNAs processed in a way identical to real miRNAs, including the use of interspecies conservation filter. To provide a realistic estimate of false positives using randomization, the distribution of scores from random trials (“random-false”) should be similar to the distribution of incorrect (non-meaningful) hits from real trials (“real-false”). The difference between these two distributions is difficult to compute in principle, as very few validated correct predictions are known at present. For human sequences, without any conservation filter, we obtained a total of 2,538,431 predicted target sites for real miRNAs, and, for shuffled miRNAs, on average, 2,033,701 (± 82,172) target sites—a difference of 20%. This difference may be indicative of a biological signal in the raw score (S) and energy (ΔG) calculated by the miRanda algorithm or may be due to different polynucleotide compositions of shuffled miRNAs compared to real miRNAs. Even if this difference represents a real effect, by far the most predictive criterion for accurate target detection is conservation of target sites across species, and not alignment scores or energies (20% compared to a factor of three, see Figure 2; Table S8). As a consequence, the current set of predicted targets rests heavily on the criterion of conservation of miRNA:mRNA match between different species. We believe this to be essentially true for all currently published target prediction methods. Indirect experimental support: FMRP-associated mRNAs An excellent opportunity to test our target predictions comes from experiments showing the association of mRNAs and miRNAs with proteins involved in translational control, even if these experiments do not provide information on specific miRNA:mRNA pairings. In particular, FMRP, which may regulate translation in neurons, not only associates with hundreds of mRNAs (Brown et al. 2001; Chen et al. 2003; Denman 2003; Miyashiro et al. 2003; Waggoner and Liebhaber 2003) and with miRNAs (Jin et al. 2004), but also associates with components of the miRNA processing machinery, Dicer, and the mammalian homologs of AGO1 and AGO2 (Jin et al. 2004). If all FMRP-bound mRNAs are regulated by miRNAs, one should see a large enrichment of predicted targets among such mRNAs. We tested this hypothesis with 397 FMRP-associated mRNAs taken from a number of recent experiments (Brown et al. 2001; Chen et al. 2003; Denman 2003; Miyashiro et al. 2003; Waggoner and Liebhaber 2003). Are FMRP-bound messages enriched in predicted targets? Using five different datasets (Table S9), we predicted that 74% of FMRP-associated messages are miRNA target genes (294 of 397 mRNAs). This corresponds to an enrichment factor of about five compared to the 59 targets one would expect from our analysis in a randomly chosen set of 397 mRNAs, where 59/397 equals 4,462/29,785 (4,462 predicted mammalian target mRNAs pass the 90% conservation filter for one or more sites per transcript out of a total of 29,785 transcripts). This suggests that in the 397 FMRP target genes, 59 should pass the filters. The enrichment factor does not vary much with the cutoff parameters used in target prediction (data not shown), but is subject to some uncertainty because of potential false-positive predictions. The enrichment of miRNA:FMRP interaction is consistent with the hypothesis that translational control involving FMRP protein is executed in a complex that involves one or more miRNAs interacting with transcripts at specific sites. Note that this analysis supports the validity of target gene prediction, not the identity of the controlling miRNA or the accuracy of specific sites. An additional validation test involved FMRP cargoes that had been identified in more than one study, using independent experimental methods. For example, the mRNAs of 14 genes (Brown et al. 2001) were overrepresented in both the polyribosome fraction of mouse fragile X cells and in co-immunoprecipitation with mouse brain mRNPs that contain FMRP. Almost all of the 14 genes are predicted targets with more than one conserved site (11 of 12 annotated UTRs; Table S9). In some cases, expression data provide additional support: postsynaptic density protein 95 (PSD95)–associated (SAPAP4), a neuron-specific protein, is regulated by many miRNAs highly expressed in rat brain primary cortical neurons (Kim et al. 2004). In summary, the three validation approaches (retrospective, statistical, and indirect experimental) suggest that the current version of the miRanda algorithm, in spite of clear limitations, can predict true miRNA targets at reasonable accuracy, provided that (1) the targets are detected as conserved and (2) the gene contains more than one miRNA target site or a single high-scoring site (S > 110, approximately, including sites with almost perfect complementarity suggestive of mRNA cleavage). Overview of Mammalian miRNA Target Genes More than 2,000 mammalian targets. We predicted 2,273 genes as targets with two or more miRNA target sites in their 3′ UTRs conserved in mammals at 90% target site conservation (see Tables S2 and S3). This means we predicted approximately 9% of protein-coding genes to be under miRNA regulation. In addition, we predicted another 2,128 genes with only one target site, but the false-positive rate for these is significantly higher (Figure 2). Of these, the top-scoring 480 genes (S > 110) have an estimated false-positive rate comparable to that of genes with multiple sites and thus also are good candidates for experimental verification. Some of the genes with single sites may contain additional sites that we cannot detect for a number of reasons, including truncated UTRs. A significant subset of the total number of single-site target genes (7%) has near complementary single sites. These near complementary sites may indicate cleavage, for which additional sites may not be necessary. The targets listed in Table 1 were selected for variety of function, variation in number of sites, and varied extent of conservation (some are also conserved in fish). Somewhat surprisingly, the number of predicted targets per miRNA varies greatly, from zero (for seven miRNAs) to 268 (for let-7b), but the distribution is nonuniform (mean = 7.1, standard deviation = 4.7; Figure 3). This indicates a range of specificity for most miRNAs and suggests that regulation of one message by one miRNA is rare. Figure 3 Multiplicity and Cooperativity in miRNA–Target Interactions One miRNA can target more than one gene (multiplicity) (A), and one gene can be controlled by more than one miRNA (cooperativity) (B). The distributions are based on ordered (ranked) lists and decay approximately exponentially (approximate straight line in log-linear plot). (A) Some miRNAs appear to be very promiscuous (top left), with hundreds of predicted targets, but most miRNAs control only a few genes (bottom right). (B) Some target genes appear to be subject to highly cooperative control (top left), but most genes do not have more than four targets sites (bottom right). Although specific values are likely to change with refinement of target prediction rules, the overall character of the distribution may well be a biologically relevant feature reflecting system properties of regulation by miRNAs. Table 1 Selection of Predicted miRNA Targets Add “ENSG00000” to the beginning of the identifiers to derive Ensembl identifiers. All miRNA–target relationships shown here are conserved in mammals, i.e., homologous miRNAs target transcripts of homologous genes at similar UTR positions with similar local sequence. Genes that are predicted to be targets in both mammals and fish are in bold. Where the miRNA–target relationship is also conserved in non-mammalian vertebrates, the miRNA is in bold a Contains conserved CPE motif N.A., not available Functional analysis We analyzed the distribution of functional annotation for all targets of all miRNAs using Gene Ontology (GO) terms (see Materials and Methods; Table S10) and domain annotations from InterPro (Mulder et al. 2003). The target genes reflected a broad range of biological functions (Figure S1). The most enriched GO term was “ubiquitin-protein ligase activity,” with 3.3-fold enrichment (Table S10). Since ubiquitination is a process controlling the quantity of specific proteins in a cell at specific times, miRNA regulation of components of the ubiquitin pathway could increase protein levels. Other overrepresented functional terms were “neurogenesis” (3.2-fold), “protein serine/threonine kinase” (2.5-fold), and “protein-tyrosine kinase activity” (2.5-fold). The four domains most overrepresented in predicted targets relative to all genes were Homeobox domain, 5.3-fold; KH domain, 4.0-fold; and Guanine-nucleotide dissociation stimulator CDC25 domain, 3.4-fold (Figure S1; Table S10). Interestingly, KH domains are RNA-binding domains found in a wide range of proteins such as hnRNPk, FMR1, and NOVA-1. In addition to the Homeobox domain, other DNA-binding domains and domains associated with chromatin regulation were also enriched, suggesting that miRNAs in animals target the transcription machinery disproportionately, as they do in plants. Another overrepresented domain was semaphorins (3.0-fold). The semaphorins and plexins (semaphorin receptors) are involved in axon guidance, angiogenesis, cell migration, the immune system, and the adult nervous system (Pasterkamp and Verhaagen 2001). Many semaphorins and their receptors are predicted targets of brain-expressed miRNAs (e.g., let-7c, miR-125b, miR-153, miR-103, miR-323, miR-326, and miR-337). The plexins dimerize with Neuropilin (NP1) to signal the Semaphorin ligand attachment; neuropilin is a predicted high-ranking target of let-7g and miR-130, both brain-expressed miRNAs. A significant proportion of ephrin receptors (seven out of ten genes) and ephrin ligands (five out of seven genes) are predicted targets. The family of ephrins is involved in boundary formation, cell migration, axon guidance, synapse formation, and angiogenesis, and the ephrin ligand, EphA2, which contains a conserved cytoplasmic polyadenylation element (CPE) motif, is considered to be under translational regulation in axon growth cones (Steward and Schuman 2003). Although many members of the ephrin family are predicted targets of brain-expressed miRNAs, they appear to be targeted by different miRNAs, consistent with differential regulation. In Drosophila, both ephrin and EphR, closest to the mammalian B class of the ephrin family, also are predicted miRNA targets. Do specific miRNAs target particular functional groups? We analyzed each miRNA individually for GO term and domain enrichment (Table S10). The targets of some miRNAs were strongly enriched in certain categories, e.g., miR-105 in “small GTPase mediated signal transduction” (5-fold), miR-208 in “transcription factor” (6-fold), and miR-7, which lies in the intron of the hnRNPk (an RNA-binding protein) gene, in “RNA binding proteins.” Neuronal differentiation of embryonic carcinoma cells by retinoic acid in both mice and humans is coupled to induction of let-7b, miR-30, miR-98, miR-103, and miR-135 (Sempere et al. 2004), and their targets are enriched in “neurogenesis” (3.5-fold). miR-124a and miR-125, both highly and specifically expressed in brain, preferentially target RNA-binding proteins. Thirty-one new miRNAs (miR-322–miR-352) cloned from rat neuronal polyribosomes have a large number of neuronal target genes and share many targets, e.g., miR-352 and miR-327 target 5HT-2c, and miR-340, -328, -326, -331, and -333 potentially target beta-catenin, which is implicated in various stages of neural differentiation. Two highly expressed miRNAs in the thymus, miR-181a and miR-142–3p are key components of a molecular circuitry that modulates hematopoietic lineage (Chen et al. 2004). Ectopic expression of miR-181a causes a 2-fold increase in the cells of the B cell lymphoid lineage. Some of our high-ranking targets for miR-181a may provide clues for the mechanism of this effect. Germ cell nuclear factor GCNF (NR6A1) (the second-highest-ranked target for miR-181a) is expressed in the thymus and bone marrow. miR-181a itself is encoded on the antisense strand of an intron of GCNF. We also predict that the gene Bcl11b, known to affect B cell growth, is a target of miR-181a, ranking third, as well as Lim/homeobox protein LHX9, recently found expressed in developing thymus (Woodside et al. 2004). FMRP cargo mRNAs regulated by miRNAs FMRP is composed of several RNA-binding domains (two KH and one RRG) that bind messages. The specific binding motifs for FMRP on messages are incompletely known, but are thought to include G-quartet patterns and/or U-rich sequences (Dolzhanskaya et al. 2003; Ramos et al. 2003). We predicted 294 mRNAs known to be FMRP cargoes as miRNA targets (see Table S9). The most reliable of these (Table 2) reflect high confidence in experimental identification of FMRP association or conservation of target site between mammals and fish. Table 2 Selected FMRP Cargoes Predicted as miRNA Targets Transcripts for genes (Gene and ID) are described as FMRP cargoes in several studies (DR) and predicted here as targets of specific miRNAs (miRNA). Selected from a total of 294 such targets a Reference from which data was extracted b Homologous miRNA–mRNA pair conserved in fish c Additional miRNAs are predicted to target the gene (number in parentheses): APP (9), BASP1 (4), Capicua (2), DLG3 (7), and DLG4 (5) d The miRNA has multiple target sites on the gene e The 3′ UTR of the gene contains a CPE motif (Table S11) Alzheimer's disease amyloid protein Amyloid precursor protein (APP) is an FMRP-bound protein that is translationally regulated. The APP transcript contains a 29-nt motif at position 200 in the 3′ UTR that is known to aid destabilization of the APP mRNA in certain nutrient conditions and that binds nucleolin, a protein associated with RNPs containing FMRP (Rajagopalan and Malter 2000). In addition, there is an 81-nt sequence at position 630 in the APP 3′ UTR that is required for the TGFbeta-induced stabilization of the APP mRNA (Amara et al. 1999). We predicted APP as a target, with a total score of S = 708 with a minimum of eight miRNA sites, including two let-7 top-ranking sites that are conserved in human, mouse, and rat. One of the predicted miRNA target sites in the APP UTR lies in the 81-nt region (Figure 4), and another is within 30 nt of the motif at position 200. Figure 4 Potential miRNA Target Sites in the 3′ UTRs of Selected Genes Nucleotide sequence conservation between the 3′ UTRs of human and the closest mouse or rat orthologous genes is averaged for each block of 40 base pairs (long rectangles; white indicates 0% identical nucleotides, black indicates 100% identical nucleotides, and grey indicates intermediate values). The positions of target sites for specific miRNAs (triangles above rectangles, with numbers indicating miR miRNAs, e.g. “130” is “mir-130”) are, in general, distributed nonuniformly. Sequence motifs other than target sites (triangles below rectangles) are mRNA stability elements (APP), a G-quartet (DLG4), and an AU-rich element (ELAVL1), representing possible protein-binding sites. Detailed alignments between the miRNA and the predicted target sites (arbitrary selection) illustrate, in general, stronger match density at the 5′ end of miRNAs than at the 3′ end, as required by the algorithm and as observed in experimentally validated targets. The nonconserved nucleotides in the target sites are highlighted in red. Gene names map to the following Ensembl identifiers (142192 is ENSG00000142192, etc.): APP, 142192; CPEB2, 137449; DLG4, 132535; EFNB1, 090776; EIF2c1, 092847; ELAVL1, 066044; EPHB1, 154928; EPHB3, 182580; FMR1, 102081; FMR2, 155966; FXR1, 114416; FXR2, 129245; and PTEN, 171862. Other APP-interacting proteins, APP-binding family B member 1 (mir-9, miR-340, and miR-135b), APP-binding family member 2 (let-7 and miR-218), and APP-binding family 2 (miR-188 and miR-206) were also predicted targets, some of which had near exact target site matches. In summary, the APP gene appears to be subject to translational regulation by the combinatorial control of a number of different miRNAs. PSD95 and synaptic processes PSD95 and similar scaffolding molecules, link the NMDA receptor with intracellular enzymes that mediate signaling; this process is involved in the development and maintenance of synaptic function and synaptic plasticity, and interference in this process is implicated in schizophrenia and bipolar disorder (Beneyto and Meador-Woodruff 2003). FMRP binds PSD95 and is required for mGluR-dependent translation of PSD95 (Todd et al. 2003). PSD95 is a high-ranking target of miR-125, miR-135, miR-320, and miR-327, all of which are either exclusively expressed in brain or enriched in brain tissue (Lagos-Quintana et al. 2002; Krichevsky et al. 2003; Sempere et al. 2004). In particular, large transcript numbers of miR-125b are found copurified with polyribosomes in rat neurons in (Kim et al. 2004). PSD95 has one reported G-quartet in its 3′ UTR at position 648 (Todd et al. 2003), further suggesting it as an in vivo FMRP target. We predicted an additional G-quartet site at position 205–235 in the 3′ UTR of PSD95. One of the miRNA (miR-125) target sites overlaps with the G-quartets, raising the possibility that miRNAs directly compete with FMRP to bind the message in this location. Likewise, NAP-22, which has three miRNA target sites (see Table S9), has a miR-207 target site that overlaps with a G-quartet (Darnell et al. 2001). Other PSD95 family members are also involved in synaptic processes, in particular, in the integration of NMDA signaling in the synaptic membrane. All PSD95 family members in mammals (also known as discs large 1–5), SAP90, and CamKII are predicted miRNA targets (see Table S9), as well as mGluR, the protein product of which is an agonist that induces the rapid translation of PSD95 (Todd et al. 2003) and three NMDA receptor subunits (see Table S9). These results suggest that miRNAs may be involved in NMDA and glutamate receptor signaling to coordinate and integrate information, with specificity achieved through the combinatorial action of different miRNAs. Components of RNPs Regulated by miRNAs FMRP-associated proteins FMRP binds its own mRNA, implying negative feedback if the binding inhibits FMRP production (Ceman et al. 1999). The fact that miRNAs target transcripts for FMRP and FMRP-binding proteins suggests another negative feedback loop in which high levels of these proteins inhibit their own production (depending, of course, on the concentration of miRNAs and mRNAs) (Figure 4). The genes for six FMRP-associated (not associated at the same time) proteins, hnRNP A1, Pur-alpha, Pur-beta, Staufen, AGO-2, and PABP, are predicted miRNA targets. This indicates that FMRP-containing RNPs are under miRNA regulation. FXR2, a gene similar to FMR1 is also a miRNA target in human, mouse, rat, and fish. Details of the implied feedback regulation and differential control of RNP action remain to be determined. RISC. Our data suggest that the RNAi–miRNA machinery itself is under miRNA regulation; for example Dicer appears to be controlled by let-7 and miR-15b; Ago-1 by let-7 and miR-29b/c; Ago-2 by miR-138; Ago-3 by miR-138, miR-25, and miR-103; and Ago-4 by miR-27a/b. Dicer and two of the Argonautes also are predicted to be targets in both zebrafish and fugu. The let-7 sites on the 3′ UTR of Dicer and Ago-1 (Figure 4) will accommodate most of the let-7 variants with similar scores. The variants of let-7 are expressed in a wide range of tissues and developmental stages, suggesting broad regulation of Dicer and Ago-1 by miRNAs. In contrast, the only miRNA that targets Ago-2 is miR-138, which has so far been cloned only once in the cerebellum (Lagos-Quintana et al. 2002). The target site for miR-138 has only one mismatch at position 8; this may induce a siRNA-like cleavage of the message (Hutvágner and Zamore 2002a; Doench et al. 2003). Ago-3 is also a top target for miR-138, with only two mismatches in its site. We suggest that some miRNAs targeting this machinery (e.g., let-7, miR-27, miR-29, and miR-103) are expressed fairly widely, while others (e.g., miR-138 and miR-25) have lower and more restricted expression. Other RNPs. The highly conserved RNA-binding proteins, ELAV-like proteins (HuR, HuB, HuC, and HuD), contain three RNA-recognition motifs, which bind AU-rich elements in 3′ UTRs of a subset of target mRNAs (Good 1995). These AU-rich elements increase the proteins' cytoplasmic stability and increase translatability (Perrone-Bizzozero and Bolognani 2002). Experiments have identified 18 mRNAs bound to HuB in retinoic-acid-induced cells; of the 14 we were able to map unambiguously, 12 are predicted miRNA target genes: Elavl1 (known to regulate its own mRNA), Gap-43, c-fos, PN-1, Krox-24, CD51, CF2R, CTCF, NF-M, GLUT-1, c-myc, and N-cadherin (Tenenbaum et al. 2000). Three of the ELAV-like genes themselves are also targets of a large number of miRNAs (see Tables S2 and S3; Figure 4). This is yet another example of miRNAs predicted to target the bound messages of RNA-binding proteins and of the regulation of RNA-binding genes by miRNAs. Cytoplasmic Polyadenylation Binding Proteins Regulated by miRNAs We predicted all four human cytoplasmic polyadenylation binding proteins (CPEBs) known in mammals as miRNA targets ranked within the top 170 target genes with 6–20 sites in their UTRs (Figure 4; Table S11). Indeed, CPEB2 is the highest-ranking gene of all transcripts. The orthologs to CPEB1 in fish and fly (known as orb in D. melanogaster) are also predicted as targets. CPEB is an RNA-binding protein first shown to activate translationally dormant mRNAs by regulating cytoplasmic polyadenylation in Xenopus oocytes (Hake and Richter 1994). It also regulates dendritic synaptic plasticity (Mendez and Richter 2001; Richter 2001) and dendritic mRNA transport (Mendez and Richter 2001; Huang et al. 2003) and facilitates transport of mRNAs in dendrites together with kinesin and dynein in RNPs (Huang et al. 2003). CPEB binds to its target message through the CPE motif (UUUUAU), which must be within a certain distance of the hexanucleotide AAUAAA. CPEB keeps messages in their dormant state until phosphorylated, after which it activates polyadenylation (Mendez et al. 2000), thereby activating translation or degradation (Mendez et al. 2002). In addition, CPEB co-fractionates with the postsynaptic density fraction in mouse synaptosomes, consistent with translation of stored mRNAs in dendrites being part of the mechanism of synaptic plasticity. We have three more lines of evidence suggesting the notion that translational regulation by CPEB is linked to miRNA regulation. First, our target list and the list of genes regulated by CPEB significantly overlap. There are nine genes known to be CPEB-regulated, seven of which are predicted targets: alpha-CAMIIK, Map 2, Inositol 1, 4–5-Triphosphate Receptor type 1, Ephrin A receptor class A type 2, SCP-1, and CPEB3 (Mendez and Richter 2001). Second, CPEB is known to self-regulate in D. melanogaster (Tan et al. 2001). The CPEB1 homolog in fly, orb, and CPEBs in vertebrates are predicted miRNA targets. Third, the gene most correlated in expression to the CPEB homolog in D. melanogaster is a Piwi protein (Sting), a member of the Argonaute family (Pal-Bhadra et al. 2002; Stuart et al. 2003) that is involved in translational regulation and in the RISC. Among the predicted miRNA targets, 115 genes also contained CPE motifs, which were conserved in at least two mammals in the same positions in the UTRs and are therefore candidates for CPEB regulation (Table S11; see Materials and Methods). Our predictions include HuB, HuR, Eif-4 gamma, DAZ associated protein 2, VAMP-2 (known to be posttranscriptionally regulated), Presynaptic protein SAP102, and brain-derived neurotrophic factor precursor. Taken together these data suggest that the CPEB genes, the known CPEB-regulated genes, and the predicted CPEB-regulated genes are strong miRNA target candidates and provide rich ground for experimentation. Targets of Cancer-Related miRNAs Deregulated expression of certain miRNAs has been linked to human proliferative diseases such as B cell chronic lymphocytic leukemia (Calin et al. 2002; Lagos-Quintana et al. 2003) and colorectal neoplasia (Michael et al. 2003). Recent analysis of the genomic location of known miRNA genes suggested that 50% of miRNA genes are in cancer-associated genomic regions or in fragile sites (Calin et al. 2004). The miRNAs miR-15 and miR-16 are located within a 30-kb region at Chromosome 13q14, a region deleted in 50% of B cell chronic lymphocytic leukemias, 50% of mantle cell lymphomas, 16%–40% of multiple myelomas, and 60% of prostate cancers (Calin et al. 2002). Furthermore, miR-15 and miR-16 are down-regulated, or their loci lost, in 68% of B cell chronic lymphocytic leukemias (Calin et al. 2002). Similarly, miR-143 and miR-145 are down-regulated at the adenomatous and cancer stages of colorectal neoplasia (Michael et al. 2003), and miR-155 is up-regulated in children with Burkitt lymphoma (Metzler et al. 2004). Our method predicted cancer-specific (by annotation) gene targets of miR-15a, miR-15b, miR-16, miR-143, miR-145, and miR-155. The target genes and their miRNA regulators are as follows: (1) CNOT7, a gene expressed in colorectal cell lines and primary tumors (Flanagan et al. 2003) (miR-15a); (2) LASS2, a tumor metastasis suppressor (Pan et al. 2001) (miR-15b); (3) ING4, a homolog of the tumor suppressor p33 ING1b, which stimulates cell cycle arrest, repair, and apoptosis (Shiseki et al. 2003) (miR-143); (4) Gab1, encoding multivalent Grb2-associated docking protein, which is involved in cell proliferation and survival (Yart et al. 2003) (miR-155); and (5) COL3A1, a gene up-regulated in advanced carcinoma (Tapper et al. 2001) (miR-145). miR-16 has a tantalizing number of high-ranking targets that are cancer associated and specifically involved in the Sumo pathway There is increasing evidence that Sumo controls pathways important for the surveillance of genome integrity (Muller et al. 2004). The first- and fifth-highest-ranked targets of miR-16 are Sumo-1 activating and conjugating enzymes, respectively. The top two single-site targets for miR-16 are an Activin type II receptor gene (TGFbeta signaling) and Hox-A5, both known to be dysregulated at the level of protein expression in colon cancers (Wang et al. 2001). Both of these sites show near perfect complementary matching between miR-16 and the target genes (indicating possible cleavage). Both of these target genes are also targets for another cancer related miRNA, miR-15. Targets Conserved between Mammals and Fish Roughly 55 miRNAs have identical mature sequences in fugu and mammals, and 80 have very similar sequences in the two species; additional fish miRNA sequences can be predicted with confidence based on sequence similarity. It is therefore reasonable to expect that the targets of these probably functionally homologous miRNAs are orthologous genes in the different species. To follow up on this hypothesis, we assessed conservation of mammalian miRNA–target pairs between the 2,273 mammalian and 1,578 fish miRNA targets (with more than one target site per UTR). The analysis yielded 240 target genes conserved between mammals and fish. The number 240 is probably an underestimate because of several factors, including: (1) unfinished annotation of genomes, particularly rat and fugu; (2) ambiguity in assigning orthologs; and (3) lack of UTR information. The full set of conserved target genes between fish and mammals indicates a wide functional range of conserved targets (Table S12). Many Hox genes are conserved as targets, including the miR-196 targets, Hox-A4:miR-34a, Hox-C9:let-7b (near prefect complementary match), and Hox-B5:miR-27b. Examples from the notch signaling pathway include miR-30:hairy enhancer of split 1 (Hes1) and miR-152:noggin. Targets Conserved between Vertebrates and Flies Twenty-eight of the 78 identified miRNAs in flies have apparent mammalian homologs. Based on this remarkable conservation across hundreds of millions of years, it is reasonable to expect that there is some conservation of target sites, target genes, and target pathways between flies and humans. Most strikingly we can identify hox genes and axon guidance genes as common targets between vertebrates and flies, e.g., capicua and sex combs reduced (one of the vertebrate homologs of Hox-A5). The hox gene cluster in Drosophila contains high-ranking predicted targets (Enright et al. 2003) of miR-10 and miR-iab-4, and the hox gene cluster in mammals contains high-ranking targets of miR-196. These miRNAs are themselves located in the hox gene region. We predicted miR-iab-4–3p to target abd-B in Drosophila, a gene related to the ancestral hox-7 cluster, the ancestral parent of many of the predicted targets of miR-196. Axon guidance receptors and ligands conserved as targets include Lar, ephrins, and slits. Human slit1 is a top target of miR-218, which itself is transcribed from the intron of slit2, suggesting down-regulation of slit1 by transcription of slit2. We expect that there are many more conserved targets but we are hindered by the difficulty of mapping orthologous genes between human and fish. Future work will elucidate to what extent there are common pathways regulated by common miRNAs between vertebrates and invertebrates. Target Sites in Protein-Coding Sequences Experiments suggest that miRNA target sites in metazoans are preferentially in UTRs, not in coding regions. If this is true, a correct target site prediction method should predict a larger number of targets in UTRs than in coding regions. Alternatively, target sites in coding regions may so far have escaped experimental verification, especially in plants, in which targets of miRNAs in coding regions are the rule, not the exception. To investigate this issue we computed the average density of target sites for high-scoring targets (S > 130) and before application of conservation filters. The statistical assessment of the influence of conservation filters in coding regions would have raised complicated issues, as nearly two-thirds of nucleotides in coding regions are conserved between mammalian genomes to preserve amino acid sequences. Interestingly, we found, on average, 11 pre-conservation target sites per 1 million nucleotides in coding regions, versus 15 such target sites per 1 million nucleotides in UTRs. This is consistent with a stronger “raw” prediction signal in UTRs and may indicate a lower number of biologically relevant target sites in coding regions in mammals, consistent with early experimental findings. As a guide to experimentation, we report all sites in coding regions with an alignment score above 110 for miRNAs of length up to 20 nt and an alignment score above 130 for miRNAs longer than 20 nt (scores depend on the length). These cutoff scores approximately correspond to a 75% complementary match between miRNA and target, leaving open the question of how many match pairs are needed to lead to translational inhibition in coding regions, by any mechanism. We identified 942 genes that contained such sites in their coding regions. Strikingly, there was only one site with a perfect match, and this was for the imprinted miR-127, known to be antisense to the reciprocally imprinted retrotransposon-like gene on the opposite strand (Seitz et al. 2003). Of the 942 genes, 25% have been otherwise identified as targets based on conserved target sites in their UTRs. However, only five genes have targets sites in their UTRs complementary to the same miRNA that targets the coding region (see Table S3, columns H and I). For example, miR-211 has a near perfect complementary site in the coding region of a gene of unknown function (Ensembl ID ENSG00000134030, containing an Eif-4 gamma domain) and also has two conserved “normal” sites in the UTR. Similarly, miR-198 has a site in the coding region, as well as conserved sites in the UTR region, of a sodium and chloride GABA transporter (Ensembl ID ENSG00000157103). However, we see no trend for miRNAs that have conserved sites in UTRs to have additional sites in the coding region; rather, stronger target sites for a given miRNA tend to be confined either to the UTR or the coding region and are rarely in both. Target Sites with Near Perfect Matches in cDNAs We scanned all cDNAs for high-scoring matches without using conservation to check for high-scoring targets, which we may have missed through strict conservation rules (see Table S6). Over 40 genes contain sites that have near perfect complementarity to a miRNA (S >120), and these target genes may be cleaved rather than translationally repressed as in the case of miR-196 and Hox-B8. For example miR-298, an embryonic-stem-cell-specific miRNA (Houbaviy et al. 2003), has a near match with MCL-1, and miR-328 (neuronally expressed) has a near match with LIMK-1, which is known to be involved in synapse formation and function. miR-129, expressed in mouse cerebellum, has a near perfect complementary match with Musashi-1, which is an RNA-binding gene essential for neural development, regulated in the cerebellum, and up-regulated in medulloblastoma (Yokota et al. 2004). Comparison of miRNA Target Prediction Methods Recently, several computational methods for the prediction of miRNA targets have been developed (Enright et al. 2003; Lewis et al. 2003; Rajewsky and Socci 2003; Stark et al. 2003; Kiriakidou et al. 2004; Rehmsmeier et al. 2004). Two of these have been applied to mammalian miRNAs, as described in Lewis et al. (2003) and Kiriakidou et al. (2004) . We now compare and contrast these two methods with each other and with the current version of our method, as further developed from miRanda 1.0 and as applied to mammalian and vertebrate genomes (Enright et al. 2003). We compare algorithms and target lists, as an aid to the design of experiments. The three prediction methods share the goal of identifying mRNAs targeted by miRNAs. All three use sequence complementarity, free energy calculations of duplex formation, and evolutionary arguments in developing a scoring scheme for evaluation of potential targets. Results are reported as lists of target sites and lists of target genes containing such sites. The three methods differ, however, in important technical details, such as the datasets of miRNA and UTR sequences and the algorithm and scoring scheme, as well as the report format. We now summarize these technical differences and compare the lists of resulting target genes for a common subset of miRNAs. The interpretation of such comparisons is hampered by the fact that selection criteria and the use of numerical cutoffs differ conceptually, and genomic coverage is nonuniform. In the first method, Lewis et al. used 79 miRNAs in human, mouse, and rat, seeking targets in a UTR dataset extracted from the June 2003 version of the Ensembl database. The UTR dataset had 14,300 ortholog triplets conserved between human, mouse, and rat and 17,000 ortholog pairs between human and mouse. All annotated UTRs were extended by 2 kb of 3′ flanking sequence. The algorithm required exact complementarity of a 7-nt miRNA “seed” sequence, defined as positions 2–8 from the 5′ end of the miRNA, to a potential target site on the mRNA, followed by optimization of mRNA–miRNA duplex free energies between an extended window of 35 additional bases of the mRNA and the rest of the miRNA. Target genes were ranked using a composite scoring function, which took into account all sites for a particular miRNA on a given mRNA. Conserved miRNA:mRNA pairs were required to involve orthologs of miRNA and mRNA in human, mouse, and rat, but there was no requirement for conservation of target site sequence (beyond the seed match) or position on the mRNA. Using shuffled miRNA sequences, with the constraint that shuffled controls match real miRNAs in relevant sequence properties, the false-positive rate of predictions was estimated to be 50% for target genes conserved between mouse and human, 31% for target genes conserved in human, mouse, and rat, and 22% for target genes identified in fugu as well as mammals. As a final result, Lewis et al. reported 400 conserved target genes for the 79 miRNAs. Among these targets, 107 genes were reported as conserved in the fish fugu. In the second method, Kiriakidou et al. used 94 miRNAs in human and mouse, seeking targets in a dataset of 13,000 UTRs conserved in mouse and human (from Ensembl, date not given). The algorithm used a 38-nt sliding mRNA window and calculation of miRNA–mRNA duplex free energies, keeping duplexes with energies below −20 kcal/mol. The duplexes were further filtered using a set of requirements regarding matches and loop lengths in certain positions, as derived and extrapolated from experimental tests involving a predicted target site for let-7b miRNA on the UTR of the human homolog of worm lin-28. The target site sequence was engineered into a Luciferase reporter, followed by sequence variation of the target site and test of an initial set of 15 predictions in the same reporter assay. Using shuffled miRNA sequences, and applying the same rules and parameters, the false-positive rate of predictions was estimated to be 50% for targets conserved between human and mouse. As a final result, Kiriakidou et al. reported 5,031 human targets, with 222 reported as conserved in the mouse. In the third method (this work), we used 218 mammalian miRNAs and 29,785 transcripts derived from Ensembl (Table 3) and, as a final result, report 4,467 target genes. What are the main differences between these three prediction methods? Comparison of the total number of predicted target genes is not very informative, as different datasets and cutoffs were used. We attempted to remove one of the technical differences, by explicitly comparing reported targets for the same set of 79 miRNAs used by Lewis et al. (although significant differences remained in the sets of UTR sequences used): the overlap of target genes between Kiriakidou et al. (out of 189) and Lewis et al. (out of 400) was 10.6%; the overlap between Lewis et al. (out of 400) and this work (out of 2,673) was 46%; and the overlap between Kiriakidou et al. (out of 189) and this work (out of 2,673) was 49%. In each case the totals (“out of”) are the number of target genes for the common set of 79 miRNAs and the percentage is relative to the smaller set of two compared. The obvious reason for the larger overlap with our results, 46% and 49% respectively, is the larger number of targets in our predictions, which in turn is primarily the result of choice of cutoff. Table 3 Number of Genes and 3′ UTR Sequences Used for Target Prediction 3′ Direct comparison of the three prediction methods is complicated by the fact that the noticeable differences between the target lists of the three methods are due to the aggregate effects of datasets, algorithm, including selection rules, use of conservation, and cutoffs. The following characteristics of the three methods underlie these differences and should be taken into consideration when choosing targets for experimentation. (1) As to UTR datasets, Lewis et al., with the earliest published report, used a smaller set of UTRs, with some likelihood of false positives as a result of UTR extension. The UTR sets used in this work, the third in terms of publication date, are the most comprehensive and plausibly the most reliable (as of February 2004). (2) As to miRNA datasets, there was an increase from 79 for Lewis et al. to 94 for Kiriakidou et al. to 218 miRNAs used in this work. (3) As to the cooperativity of binding, the scoring system of Lewis et al. evaluated cooperativity of multiple target sites by the same miRNA on a target gene, but disregarded multiple target sites from different miRNAs on one gene; that of Kiriakidou et al. focused on single sites; and that of this work gave high scores to multiple hits on a target gene, no matter whether these hits involved the same miRNA or different miRNAs. These tendencies are not exclusive where scores involve functions of several real numbers, with cutoffs applied to the aggregate score; e.g., our method also allows strong single target sites. (4) As to assessment of false positives using statistical methods based on shuffling, the comparison of percentages is inconclusive, as the statistics of the background distribution of true negatives is not well known. It appears certain, however, from both Lewis et al. and this work, that statistical confidence increases with the extent of conservation among increasingly distant species. (5) As to validation experiments, each of the methods used a different type and set, with mixed overall conclusions. On the reassuring side, there was direct validation of some of the predicted target sites of Lewis et al. and of Kiriakidou et al. using reporter constructs in cell lines. We found some agreement between the sites validated in this way and our predicted targets (details in Table S13), but in some cases we predicted different details of target sites for a given experimentally tested miRNA:mRNA pair. Also, Kiriakidou et al. used a series of such experiments to extrapolate from a set of specific sequence variants to general rules for identification of target sites. However, serious doubts about the validity of any set of rules persist as there is very little in vivo validation in which native levels of specific miRNAs are shown to interact with identified native mRNA targets with observable phenotypic consequences under normal physiological conditions. (6) As to differences in algorithm, one can state opinions about the strengths or weaknesses of each particular algorithm, but the relationship between each prediction method and the actual in vivo process by which miRNAs have functional interactions with their target mRNAs remains unclear or, at best, unproven. In summary, in our view, each of the three methods, including the one in this work, falls substantially short of capturing the full detail of physical, temporal, and spatial requirements of biologically significant miRNA–mRNA interaction. As such, the target lists remain largely unproven, but useful hypotheses. The predicted targets are useful in practice for the design of experiments as they increase the efficiency of validation experiments by focusing on target lists significantly enhanced in likely targets, compared to random. It is plausible that targets near the top of lists are the most likely to lead to successful experiments. Task-specific filtering of target lists for particular planned experiments is recommended, especially with respect to cooperativity of binding (more than one site for one or more miRNAs on one gene transcript) and coincidence of expression, as new data on expression patterns of miRNAs and mRNAs in different tissues become available. For example, a recommended conservative approach to the design of experiments would use all available expression information and restrict the predicted target genes to those with two or more target sites at normal threshold (S > 90) or one target site with a higher threshold (S > 110), counting only sites with up to one G:U pair in residues 2–8 counting from the 5′ end of the miRNA. To take into account the rapid development of this field and the likely close interaction of theory and experiment, we plan to periodically update our prediction method and parameters and make revised target lists available on http://www.microrna.org. Next, we discuss some conceptual consequences of the composition of our target list. Discussion How Widespread Is the Regulation of Translation by miRNA? With plausible parameters, we have predicted that close to 9% (2,273 out of 23,531) of all mammalian genes have more than one miRNA target site in their 3′ UTRs, with 1,314 being stronger candidates with more than two target sites. This could well be an underestimate of the total number of genes subject to miRNA regulation, as we have used a conservative conservation filter. On the other hand, not all predicted miRNA–mRNA pairs would have a biological consequence unless both miRNA and mRNA are expressed at the same time in the same cell and at sufficient concentration. The human genome has about 250 miRNA genes, compared to about 35,000 protein genes. Thus, the the determination that about 1% of genes (miRNAs) control the expression of more than 10% of genes is a reasonable first order estimate. It is currently not known if any miRNAs control the expression of miRNA genes, i.e., the progression from miRNA transcript to mature miRNA. How Conserved in Evolution Are miRNA Targets? As many miRNA sequences are detectably conserved across large evolutionary distances, they must be subject to strong functional constraints. These constraints are unlikely to come from single-site interactions with the target, as experimentally validated animal miRNAs rarely have perfectly matched target sites. Plausibly, the evolution of miRNAs is constrained by functional interactions with multiple targets. As a consequence, any compensatory mutation in the miRNA in response to mutations in a target site would be disruptive to the miRNA's interaction with other target sites. Co-evolution of the miRNA sequence and all of its target sequences is therefore a rare event. With these assumptions, the constraints on the local mRNA sequence of individual target sites are weaker than those on the miRNA sequence. We were therefore surprised to observe a substantial number of cases (28.6% of the 2,273 targets) with 100% conservation of target site sequence and with the target sites being within ten nucleotides of each other on the globally aligned UTRs of orthologous genes between mammals. Lacking more detailed knowledge of miRNA evolution, we draw two operational conclusions. (1) Conservation of target site sequence and position is a practical information filter for predicted target sites, reducing the rate of false positives. (2) It is very likely that new miRNAs have continuously appeared in evolution (Lai 2003) at some non-negligible rate and that the set of targets for any given miRNA has lost or gained members, even between species as close as human and mouse. It is therefore important to develop prediction tools that do not rely on conservation filters or at least allow us to make them weaker. Work on this is in progress. Multiplicity and Cooperativity Regulation by miRNAs is obviously not as simple as one miRNA–one target gene, as perhaps the early examples (lin-4 and let-7) seemed to indicate. The distribution of predicted targets reflects more complicated combinatorics, both in terms of target multiplicity (more than one target per miRNA) and signal integration (more than one miRNA per target gene). The distribution of the number of target genes (and target sites) per miRNA is highly nonuniform, ranging from zero for seven miRNAs to 268 for let-7b, with an average of 7.1 targets per miRNA. It is difficult to describe in detail, beyond the examples discussed in this text and beyond the annotation of target genes in Figure 2 and Table S3, which specific processes appear to be regulated by each miRNA or each set of co-expressed miRNAs. Groups of targets may reflect a reaction, a pathway, or a functional class (see Results). Although all miRNA–target pairs are subject to the condition of synchrony of expression, it is likely that typically one miRNA regulates the translation of a number of target messages and that, in some cases, the target genes as a group are involved in a particular cellular process. This was already known for the case of lin-4 (Ambros 2003). The number of miRNA target sites per gene is also nonuniform, with a mean of 2.4. Although we do list target genes with single miRNA sites, there is increasing evidence that, in general, two or more sites are needed in the context of repression of translation. Although the details of these distributions (see Figure 2 and Table S3) depend on technical details, such as uniform cutoff for all miRNAs and evaluation in terms of a particular, imperfect scoring system, the general features of the distributions (see Figure 3) may be generally valid. We conclude that multiplicity of targets and cooperative signal integration on target genes are key features of the control of translation by miRNAs. Neither multiplicity nor cooperativity is a novel feature in the regulation of gene expression. Indeed, regulation by transcription factors appears to be characterized, at least in eukaryotes, by analogous one-to-many and many-to-one relations between regulating factor and regulated genes (Kadonaga 2004). We are, of course, aware that the control cycles and feedback loops involving miRNAs cannot be adequately described without more detailed knowledge of the control of transcription of miRNA genes, about which little is known at present. Mechanisms of miRNA Action The role of a few animal miRNAs as posttranscriptional regulators of gene expression and, in particular, as inhibitors of translation is well established. However, the molecular mechanism of action is not well understood. Posttranscriptional control of protein levels can be achieved, for example, by cleaving the mRNA, by preventing RNP transport to ribosomes, by stalling or otherwise inhibiting translation on ribosomes, or by facilitating the formation of protein complexes near ribosomes that degrade nascent polypeptide chains. What do our results imply regarding the mechanism of action? In analogy to plant miRNAs that have near perfect sequence complementarity and facilitate mRNA degradation, our predicted targets with near perfect complementarity between miRNA and mRNA plausibly are involved in mRNA cleavage (e.g., miR-196 and miR-138; see Results). Most of these would involve single target sites. In the case of Hox-B8, cleavage has been experimentally shown in mammalian cells (Yekta et al. 2004). We estimate that fewer than 5% of miRNA targets are cleaved as a result of miRNA binding. Multiple target sites of lesser complementarity are consistent with RNP formation leading to translational inhibition, not mRNA degradation. Although we did predict single miRNA target sites for some genes, most target genes have multiple sites, indicating that cooperative binding (Doench and Sharp 2004) may be essential for formation of inhibitory RNP complexes. An interesting and somewhat paradoxical feature is seen with mRNAs bound by FMRP, some of which increased and some of which are decreased in polysome fractions in FMRP knock-out mice (Brown et al. 2001). We see no bias in which of these two sets is most enhanced as predicted miRNA targets. This ambiguity not only raises questions about details of FMRP regulation but also raises the possibility that miRNA targets may not always be translationally repressed and may instead be translationally enhanced. Improvement of Prediction Rules Current methods for predicting miRNA targets rely on conservation filters to reduce noise. Although the miRNA–mRNA pairings of experimentally validated targets were carefully used to define prediction rules (Enright et al. 2003; Lewis et al. 2003; Stark et al. 2003), the information content in sequence match scores and free energy estimates of RNA duplex formation appears to be low. What is missing? Perhaps the fine details of experimentally proven target site matches are incorrect, although in some experiments mismatches and insertions have been tested. More plausibly, the rules do not yet capture additional functionally relevant interactions of miRNAs, such as in maturation and transport. Such additional interactions remain to be described in molecular detail, such as interactions with the small RNA processing machinery (Drosha and Dicer) and with the components of RNPs (AGO and FMRP). A first step in this direction is the very recent analysis of the crystal structure of a PAZ domain of a human Argonaute protein, eIF2c1, complexed with a 9-mer RNA oligonucleotide in dimer configuration, which may represent three-dimensional interactions for the 3′ end of a miRNA (and siRNA) complexed, e.g., with Dicer or AGO (Ma et al. 2004). In this structure, each PAZ domain makes close binding contact with nine nucleotides of a single-stranded RNA. The two 3′ terminal nucleotides bind in a pocket through RNA backbone and other contacts. The remaining seven nucleotides bind PAZ through a series of backbone contacts such that nucleotides 3 to 9 are in an RNA helical conformation with bases exposed for base pairing to the second single-stranded RNA. If a 20–21-nt single-stranded RNA is bound to a PAZ domain in the same fashion, the 5′ end would be free for other interactions, such as binding to another protein domain in the RISC or base-pairing to mRNA. The conformational entropy that results when the 3′ end binds to PAZ, because the RNA helix is pre-formed, is consistent with weaker base pairing between miRNA and mRNA at the 3′ end of the miRNA, and stronger base pairing at the 5′ end. The dimeric structure of the PAZ domain (Ma et al. 2004) also raises the tantalizing possibility of cooperative binding of a dimer of two miRNA–PAZ combinations to two target sites on one or more mRNAs. In such an arrangement, seven residues at the 3′ ends of the two miRNAs (residues 3–9, but not the terminal two nucleotides) are paired in antiparallel fashion, with near perfect complementary pairing. As more details of molecular contacts become available, prediction rules will evolve and improve in accuracy. The following elements are worth considering in the next generation of target prediction rules: (1) details of strand bias as deduced from siRNA experiments (Khvorova et al. 2003), (2) contribution of sequences outside of the mRNA target sites, (3) refinement of position-dependent rules, including different gap penalties for the mRNA and the miRNA, (4) energetics of miRNA–protein binding, starting with PAZ domain interaction, and (5) translation of systematic mutational profiling experiments into scoring rules (Doench and Sharp 2004). Principles of Regulation by miRNAs Although the predicted targets are subject to error (see estimate of false positives) and the prediction rules in need of improvement, several general principles of gene regulation by miRNAs are emerging. (1) Except in cases where a highly complementary match causes cleavage of the target message, miRNAs appear to act cooperatively, requiring two or more target sites per message, for either one or several different miRNAs. (2) Most miRNAs are involved in the translational regulation of several target genes, which in some cases are grouped into functional categories. (3) miRNAs carried in the context of RNPs appear to be sequence-specific adaptors guiding RNPs to particular target sequences. miRNA regulation of cellular messages may therefore range from a switch-like behavior (e.g., cleavage of mRNA message) to a subtle modulation of protein dosage in a cell through low-level translational repression (Bartel and Chen 2004). These aspects of miRNA regulation complicate the design of experiments aiming at testing target predictions, or, more generally, at discovering biologically meaningful targets. Straightforward experiments that test one target site for one miRNA on one UTR will not be able to disentangle the effects of multiplicity or cooperativity. Tests for multiple sites on one UTR for one miRNA capture aspects of cooperativity (Doench and Sharp 2004), but still do not capture signal integration by diverse miRNAs. The most complicated situation is one in which multiple miRNAs affect multiple genes in combinatorial fashion, with fine-tuning depending on the state of the cell. We look forward to the results of ingenious experiments designed to deal with the complexity of miRNA regulation. The results of this genome-wide prediction for mammals and fish are meant to be a guide to experiments that will in time elucidate the genetic control network of regulators of transcription, translation/maturation, and degradation of gene products, including miRNAs. Materials and Methods miRNA sequences Mature human and mouse miRNA sequences were obtained from the RFAM miRNA registry (Griffiths-Jones 2004). To cover cases of incomplete data, any mouse miRNA sequence not (yet) described in humans was assumed to be present in human, with the same sequence, and vice versa. Similarly, all mouse miRNAs were assumed to be identical and present in the rat genome. These assumptions are reasonable as sequence identity for known orthologous pairs in human and mouse is, on average, 98% (with 110 out of 146 orthologous sequences being identical). In total, 218 mammalian miRNAs were used. For human target searches, 162 native miRNA sequences were available plus 17 mouse and 39 rat miRNA sequences; for mouse, 191 native, 14 human, and 13 rat sequences; and for rat, 45 native, 159 mouse, and 14 human miRNA sequences. Mature miRNA sequences for zebrafish and fugu were predicted starting from known human and mouse miRNA precursor sequences (Ambros et al. 2003a). Each precursor sequence was used, in a scan against the zebrafish supercontigs (release 18.2.1) using NCBI BLASTN (version 2.2.6; E-value cutoff, 2.0) (Altschul et al. 1990), to identify a sequence segment containing the potential zebrafish miRNA. The mammalian and fish segments were then realigned using a global alignment protocol (ALIGN in the FASTA package, version 2u65; Pearson and Lipman 1988). After testing the potential fish miRNA precursors for foldback structures (Zuker 2003), the final set of 225 predicted zebrafish miRNAs was selected. The same set of sequences was used for fugu. 3′ UTR sequences The Ensembl database (Birney et al. 2004) served as the source of genomic data. The Ensembl BioPerl application user interface was used to generate 3′ UTR sequences for all transcripts of all genes from each genome. Some transcripts are alternatively spliced from the same gene, so the total number of genes is smaller than the number of transcripts (Table 3). When no Ensembl annotated 3′ UTR sequences were available, we predicted 3′ UTRs by taking 4,000 bp of genomic sequence downstream of the end of the last exon of a transcript (Table 3). If this predicted region overlapped coding sequence on either strand, we halted 3′ UTR extension at that point. UTR orthology and alignment Orthology mappings between genes from different genomes were obtained using “orthologue tables” from the EnsMart (Kasprzyk et al. 2004) feature of the Ensembl database. Pairs of orthologous UTRs were aligned with each other using the AVID (Bray et al. 2003) alignment algorithm to facilitate analysis of conservation of position and sequence of target sites. In total, 26,205 human transcripts, representing 15,869 genes, were mapped to both mouse and rat transcripts. For zebrafish, 11,442 transcripts, representing 10,909 genes, were mapped to fugu transcripts and 11,306 transcripts mapped to human transcripts (10,063 genes). miRNA target prediction The miRanda algorithm (version 1.0; Enright et al. 2003) was used to scan all available miRNA sequences for a given genome against 3′ UTR sequences of that genome derived from the Ensembl database and—tabulated separately—against all cDNA sequences and coding regions. The algorithm uses dynamic programming to search for maximal local complementarity alignments, corresponding to a double-stranded antiparallel duplex. A score of +5 was assigned for G:C and A:T pairs, +2 for G:U wobble pairs, and −3 for mismatch pairs, and the gap-open and gap-elongation parameters were set to −8.0 and −2.0, respectively. To significantly increase the speed of miRanda runs, in calculating the optimal alignment score at positions i, j in the alignment scoring matrix, the gap-elongation parameter was used only if the extension to i, j of a given stretch of gaps ending at positions i–1, j or j–1, i (but not of stretches of gaps ending at i–k, j or j, i–k for k > 1) resulted in a higher score than the addition of a nucleotide–nucleotide match at positions i, j. Removal of this restriction with the availability of more computing power would result in a moderate increase in average loop length, but the advantages of this would probably be superceded by overall refinement of target prediction rules. Importantly, complementarity scores at the first eleven positions, counting from the miRNA 5′ end, were multiplied by a scaling factor of 2.0, so as to approximately reflect the experimentally observed 5′–3′ asymmetry; for example, G:C and A:T base pairs contributed +10 to the match score in these positions. The value of the scaling factor at each position is an adjustable parameter subject to optimization as more experimental information becomes available. Because of the ongoing discussion about the rules for target prediction, target genes (a total of 490) that contained target sites with more than one G:U wobble in the 5′ end are flagged in the Table S2. The thresholds for candidate target sites were S > 90 and ΔG < −17 kcal/mol, where S is the sum of single-residue-pair match scores over the alignment trace and ΔG is the free energy of duplex formation from a completely dissociated state, calculated using the Vienna package as in Enright et al. (2003). After finding optimal local matches above these thresholds between a particular miRNA and the set of 3′ UTRs in each genome, we asked whether target site position and sequence for this miRNA were conserved in the 3′ UTRs of orthologous genes, i.e., between human and mouse or rat, or between fugu and zebrafish. The alignments of target sites were generated transitively (UTR→miRNA→UTR) via a shared (or homologous) miRNA. We required that the positions of pairs of target sites in two species fall within ±10 residues in the aligned 3′ UTRs. Conserved target sites with sequence identity of 90% or more (human versus mouse or rat) and 70% or more (zebrafish versus fugu) were selected as candidate miRNA target sites and stored in a MySQL database. Using human as the reference species, we predicted 10,572 conserved target sites (conserved in either mouse or rat) in 4,463 human transcripts, of which 2,307 transcripts of 2,273 genes contained more than one target site. Similarly, using zebrafish as a reference species, we predicted 7,057 conserved target sites (conserved in fugu) in 4,820 zebrafish transcripts. To focus on the strongest predictions, conserved target sites for each miRNA were sorted according to alignment score, with free energy as the secondary sort criterion. In cases where multiple miRNAs targeted the same site on a transcript (or within 25 nt of a site), only the highest scoring, lowest energy miRNA was reported for that site. Functional analysis of targets. To facilitate surveys of target function and analysis of functional enrichment, InterPro domain assignments (Mulder et al. 2003) and GO (molecular function hierarchy) mappings (Ashburner and Lewis 2002) for all human genes were obtained using EnsMart. For each functional class derived from either source, we calculated its degree of under- or overrepresentation, Fclass, using the log-odds ratio of the fraction of annotated target genes with the same class (F1) and the fraction of all annotated Ensembl human genes with that class (F2): Here, N represents the number of genes of a given functional class for either target genes (Ntar) or all genes (Nall), and C represents the total number of functional classes. To eliminate bias from small counts we did not report assignments that were present in less than 1% of all annotated target genes (F1 ≤ 0.01 or F2 ≤ 0.01). Randomized trials For each random experiment all miRNAs were shuffled by randomly swapping two bases of a miRNA 1,000 times. These shuffled sequences were then searched against human, mouse, and rat 3′ UTR sequences in the same way described for the main analysis, including analysis of conservation of target site sequence and position in orthologous 3′ UTRs. A total of ten randomized experiments were performed. Counts were averaged across all experiments, and the standard deviation and other statistical measures were calculated. Analysis of FMRP-associated mRNAs We compiled a list of 464 gene identifiers of FMRP-associated mRNAs from five different publications (Brown et al. 2001; Chen et al. 2003; Denman 2003; Miyashiro et al. 2003; Waggoner and Liebhaber 2003). Among the 464 gene identifiers, 397 identifiers were mapped to the corresponding genes in our 3′ UTR dataset. The remaining 67 genes were not mapped because their published identifiers were obsolete, primarily because of their Affymetrix probeset identification numbers. To identify miRNA regulation of the 397 FMRP-associated mRNAs, these genes were then compared with the set of predicted miRNA targets. CPE motif prediction. We predicted CPE motifs in human, mouse, and rat UTRs. We used a search pattern using four criteria: (1) presence of the CPE motif UUUUAU, (2) presence of the hexanucleotide AAUAAA, (3) the CPE and the hexanucleotide motif being within 100 nucleotides of each other, and (4) the conservation of these motifs and the positions of the motifs in the mouse ortholog (Mendez and Richter 2001). Supporting Information Figure S1 Overrepresentation of the GO and Interpro Domains (347 KB PDF). Click here for additional data file. Table S1 Human miRNAs in Introns (25 KB XLS). Click here for additional data file. Table S2 Predicted Mammalian miRNA Targets by Gene (8.0 MB XLS). Click here for additional data file. Table S3 Predicted Mammalian miRNA Targets by miRNA (17.0 MB XLS). Click here for additional data file. Table S4 Predicted Fish Targets by Gene (5.6 MB XLS). Click here for additional data file. Table S5 Predicted Fish Targets by miRNA (9.8 MB XLS). Click here for additional data file. Table S6 High-Scoring miRNA Matches in Human cDNAs (601 KB XLS). Click here for additional data file. Table S7 High-Scoring miRNA Matches in Human Coding Regions (512 KB XLS). Click here for additional data file. Table S8 Estimate of False Positives (23 KB XLS). Click here for additional data file. Table S9 Predicted Targets That Are Associated with FMRP (678 KB XLS). Click here for additional data file. Table S10 Function of Targets by Interpro and GO Mapping (357 KB XLS). Click here for additional data file. Table S11 Target Genes That Contain Predicted CPE Motifs (529 KB XLS). Click here for additional data file. Table S12 Conserved Vertebrate Target Genes (621 KB XLS). Click here for additional data file. Table S13 Overlap of the Predicted Targets with Validated Gene Targets from Lewis et al. (2003) and Kiriakidou et al. (2004) (68 KB XLS). Click here for additional data file. We thank Rebecca Ward and Ulrike Gaul for intellectual input and support, Nathan Marks for help with FMRP datasets, Russell Oliver Kosik for help with compiling expression data, and Joanne Edington for computer systems support. Conflicts of interest. The authors have declared that no conflicts of interest exist. Author contributions. CS and DMS conceived and directed the project. BJ, AJE, CS, and DSM worked on the algorithm. BJ, AJE, and DSM prepared the input data. BJ and AJE wrote the computer programs. BJ, AA, TT, CS, and DSM analyzed the ouput data. TT contributed microRNA expression data. BJ, CS, and DSM wrote the paper with assistance from AJE. Academic Editor: James C. Carrington, Oregon State University Citation: John B, Enright AJ, Aravin A, Tuschl T, Sander C, et al. (2004) Human microRNA targets. PLoS Biol 2(11): e363. Abbreviations AGOArgonaute APPamyloid precursor protein CPEcytoplasmic polyadenylation element CPEBcytoplasmic polyadenylation binding protein FMRPfragile X mental retardation protein GOGene Ontology miRNAmicroRNA ntnucleotide PSD95postsynaptic density protein 95 RISCRNA-induced silencing complex RNPribonuclear particle siRNAsmall interfering RNA UTRuntranslated region ==== Refs References Abrahante JE Daul AL Li M Volk ML Tennessen JM The Caenorhabditis elegans hunchback-like gene lin-57/hbl-1 controls developmental time and is regulated by microRNAs Dev Cell 2003 4 625 637 12737799 Altschul SF Gish W Miller W Myers EW Lipman DJ Basic local alignment search tool J Mol Biol 1990 215 403 410 2231712 Amara FM Junaid A Clough RR Liang B TGF-beta(1), regulation of alzheimer amyloid precursor protein mRNA expression in a normal human astrocyte cell line: mRNA stabilization Brain Res Mol Brain Res 1999 71 42 49 10407185 Ambros V Control of developmental timing in Caenorhabditis elegans Curr 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altered mRNA translational profiles in fragile X syndrome Cell 2001 107 477 487 11719188 Calin GA Dumitru CD Shimizu M Bichi R Zupo S Frequent deletions and down-regulation of micro-RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia Proc Natl Acad Sci U S A 2002 99 15524 15529 12434020 Calin GA Sevignani C Dumitru CD Hyslop T Noch E Human microRNA genes are frequently located at fragile sites and genomic regions involved in cancers Proc Natl Acad Sci U S A 2004 101 2999 3004 14973191 Caudy AA Myers M Hannon GJ Hammond SM Fragile X-related protein and VIG associate with the RNA interference machinery Genes Dev 2002 16 2491 2496 12368260 Ceman S Brown V Warren ST Isolation of an FMRP-associated messenger ribonucleoprotein particle and identification of nucleolin and the fragile X-related proteins as components of the complex Mol Cell Biol 1999 19 7925 7932 10567518 Chalfie M Horvitz HR Sulston JE Mutations that lead to reiterations in the cell lineages of C. elegans Cell 1981 24 59 69 7237544 Chen CZ Li L Lodish HF Bartel DP MicroRNAs modulate hematopoietic lineage differentiation Science 2004 303 83 86 14657504 Chen L Yun SW Seto J Liu W Toth M The fragile X mental retardation protein binds and regulates a novel class of mRNAs containing U rich target sequences Neuroscience 2003 120 1005 1017 12927206 Darnell JC Jensen KB Jin P Brown V Warren ST Fragile X mental retardation protein targets G quartet mRNAs important for neuronal function Cell 2001 107 489 499 11719189 Denman RB Deja vu all over again: FMRP binds U-rich target mRNAs Biochem Biophys Res Commun 2003 310 1 7 14511639 Doench JG Sharp PA Specificity of microRNA target selection in translational repression Genes Dev 2004 18 504 511 15014042 Doench JG Petersen CP Sharp PA siRNAs can function as miRNAs Genes Dev 2003 17 438 442 12600936 Dolzhanskaya N Sung YJ Conti J Currie JR Denman RB The fragile X mental retardation protein interacts with U-rich RNAs in a yeast three-hybrid system Biochem Biophys Res Commun 2003 305 434 441 12745094 Dostie J Mourelatos Z Yang M Sharma A Dreyfuss G Numerous microRNPs in neuronal cells containing novel microRNAs RNA 2003 9 180 186 12554860 Elbashir SM Lendeckel W Tuschl T RNA interference is mediated by 21 and 22 nt RNAs Genes Dev 2001 15 188 200 11157775 Enright AJ John B Gaul U Tuschl T Sander C MicroRNA targets in Drosophila Genome Biol 2003 5 R1 14709173 Flanagan J Healey S Young J Whitehall V Chenevix-Trench G Analysis of the transcription regulator, CNOT7, as a candidate chromosome 8 tumor suppressor gene in colorectal cancer Int J Cancer 2003 106 505 509 12845644 Good PJ A conserved family of elav-like genes in vertebrates Proc Natl Acad Sci U S A 1995 92 4557 4561 7753842 Grad Y Aach J Hayes GD Reinhart BJ Church GM Computational and experimental identification of C. elegans microRNAs Mol Cell 2003 11 1253 1263 12769849 Griffiths-Jones S The microRNA Registry Nucleic Acids Res 2004 32 D109 D111 14681370 Hake LE Richter JD CPEB is a specificity factor that mediates cytoplasmic polyadenylation during Xenopus oocyte maturation Cell 1994 79 617 627 7954828 Hammond SM Boettcher S Caudy AA Kobayashi R Hannon GJ Argonaute2, a link between genetic and biochemical analyses of RNAi Science 2001 293 1146 1150 11498593 Houbaviy HB Murray MF Sharp PA Embryonic stem cell-specific MicroRNAs Dev Cell 2003 5 351 358 12919684 Huang YS Carson JH Barbarese E Richter JD Facilitation of dendritic mRNA transport by CPEB Genes Dev 2003 17 638 653 12629046 Hutvágner G Zamore PD A microRNA in a multiple-turnover RNAi enzyme complex Science 2002a 297 2056 2060 12154197 Hutvágner G Zamore PD RNAi: Nature abhors a double-strand Curr Opin Genet Dev 2002b 12 225 232 11893497 Ishizuka A Siomi MC Siomi H A Drosophila fragile X protein interacts with components of RNAi and ribosomal proteins Genes Dev 2002 16 2497 2508 12368261 Jackson AL Bartz SR Schelter J Kobayashi SV Burchard J Expression profiling reveals off-target gene regulation by RNAi Nat Biotechnol 2003 21 635 637 12754523 Jin P Zarnescu DC Ceman S Nakamoto M Mowrey J Biochemical and genetic interaction between the fragile X mental retardation protein and the microRNA pathway Nat Neurosci 2004 7 113 117 14703574 Johnston RJ Hobert O A microRNA controlling left/right neuronal asymmetry in Caenorhabditis elegans Nature 2003 426 845 849 14685240 Kadonaga JT Regulation of RNA polymerase II transcription by sequence-specific DNA binding factors Cell 2004 116 247 257 14744435 Kasprzyk A Keefe D Smedley D London D Spooner W EnsMart: A generic system for fast and flexible access to biological data Genome Res 2004 14 160 169 14707178 Khvorova A Reynolds A Jayasena SD Functional siRNAs and miRNAs exhibit strand bias Cell 2003 115 209 216 14567918 Kim J Krichevsky A Grad Y Hayes GD Kosik KS Identification of many microRNAs that copurify with polyribosomes in mammalian neurons Proc Natl Acad Sci U S A 2004 101 360 365 14691248 Kiriakidou M Nelson PT Kouranov A Fitziev P Bouyioukos C A combined computational-experimental approach predicts human microRNA targets Genes Dev 2004 18 1165 1178 15131085 Krichevsky AM King KS Donahue CP Khrapko K Kosik KS A microRNA array reveals extensive regulation of microRNAs during brain development RNA 2003 9 1274 1281 13130141 Lagos-Quintana M Rauhut R Lendeckel W Tuschl T Identification of novel genes coding for small expressed RNAs Science 2001 294 853 858 11679670 Lagos-Quintana M Rauhut R Yalcin A Meyer J Lendeckel W Identification of tissue-specific microRNAs from mouse Curr Biol 2002 12 735 739 12007417 Lagos-Quintana M Rauhut R Meyer J Borkhardt A Tuschl T New microRNAs from mouse and human RNA 2003 9 175 179 12554859 Lai EC microRNAs: Runts of the genome assert themselves Curr Biol 2003 13 R925 R936 14654021 Lai EC Tomancak P Williams RW Rubin GM Computational identification of Drosophila microRNA genes Genome Biol 2003 4 R42 12844358 Lau NC Lim LP Weinstein EG Bartel DP An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans Science 2001 294 858 862 11679671 Lee RC Ambros V An extensive class of small RNAs in Caenorhabditis elegans Science 2001 294 862 864 11679672 Lee RC Feinbaum RL Ambros V The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14 Cell 1993 75 843 854 8252621 Lee Y Jeon K Lee JT Kim S Kim VN MicroRNA maturation: Stepwise processing and subcellular localization EMBO J 2002 21 4663 4670 12198168 Lee Y Ahn C Han J Choi H Kim J The nuclear RNase III Drosha initiates microRNA processing Nature 2003 425 415 419 14508493 Lewis BP Shih IH Jones-Rhoades MW Bartel DP Burge CB Prediction of mammalian microRNA targets Cell 2003 115 787 798 14697198 Lim LP Glasner ME Yekta S Burge CB Bartel DP Vertebrate microRNA genes Science 2003a 299 1540 12624257 Lim LP Lau NC Weinstein EG Abdelhakim A Yekta S The microRNAs of Caenorhabditis elegans Genes Dev 2003b 17 991 1008 12672692 Lin SY Johnson SM Abraham M Vella MC Pasquinelli A The C. elegans hunchback homolog, hbl-1, controls temporal patterning and is a probable microRNA target Dev Cell 2003 4 639 650 12737800 Lingel A Simon B Izaurralde E Sattler M Structure and nucleic-acid binding of the Drosophila Argonaute 2 PAZ domain Nature 2003 426 465 469 14615801 Llave C Kasschau KD Rector MA Carrington JC Endogenous and silencing-associated small RNAs in plants Plant Cell 2002a 14 1605 1619 12119378 Llave C Xie Z Kasschau KD Carrington JC Cleavage of Scarecrow-like mRNA targets directed by a class of Arabidopsis miRNA Science 2002b 297 2053 2056 12242443 Lund E Guttinger S Calado A Dahlberg JE Kutay U Nuclear export of microRNA precursors Science 2003 303 95 98 14631048 Ma JB Ye K Patel DJ Structural basis for overhang specific small interfering RNA recognition by the PAZ domain Nature 2004 429 318 322 15152257 Martinez J Patkaniowska A Urlaub H Luhrmann R Tuschl T Single-stranded antisense siRNAs guide target RNA cleavage in RNAi Cell 2002 110 563 574 12230974 Mendez R Richter JD Translational control by CPEB: A means to the end Nat Rev Mol Cell Biol 2001 2 521 529 11433366 Mendez R Hake LE Andresson T Littlepage LE Ruderman JV Phosphorylation of CPE binding factor by Eg2 regulates translation of c-mos mRNA Nature 2000 404 302 307 10749216 Mendez R Barnard D Richter JD Differential mRNA translation and meiotic progression require Cdc2-mediated CPEB destruction EMBO J 2002 21 1833 1844 11927567 Mette MF van der Winden J Matzke M Matzke AJ Short RNAs can identify new candidate transposable element families in Arabidopsis Plant Physiol 2002 130 6 9 12226481 Metzler M Wilda M Busch K Viehmann S Borkhardt A High expression of precursor microRNA-155/BIC RNA in children with Burkitt lymphoma Genes Chromosomes Cancer 2004 39 167 169 14695998 Michael MZ O'Connor SM van Holst Pellekaan NG Young GP James RJ Reduced accumulation of specific microRNAs in colorectal neoplasia Mol Cancer Res 2003 1 882 891 14573789 Miyashiro KY Beckel-Mitchener A Purk TP Becker KG Barret T RNA cargoes associating with FMRP reveal deficits in cellular functioning in Fmr1 null mice Neuron 2003 37 417 431 12575950 Moss EG Tang L Conservation of the heterochronic regulator Lin-28, its developmental expression and microRNA complementary sites Dev Biol 2003 258 432 442 12798299 Moss EG Lee RC Ambros V The cold shock domain protein LIN-28 controls developmental timing in C. elegans and is regulated by the lin-4 RNA Cell 1997 88 637 646 9054503 Mourelatos Z Dostie J Paushkin S Sharma A Charroux B miRNPs: A novel class of ribonucleoproteins containing numerous microRNAs Genes Dev 2002 16 720 728 11914277 Mulder NJ Apweiler R Attwood TK Bairoch A Barrell D The InterPro Database, 2003 brings increased coverage and new features Nucleic Acids Res 2003 31 315 318 12520011 Muller S Ledl A Schmidt D SUMO: A regulator of gene expression and genome integrity Oncogene 2004 23 1998 2008 15021887 Pal-Bhadra M Bhadra U Birchler JA RNAi related mechanisms affect both transcriptional and posttranscriptional transgene silencing in Drosophila Mol Cell 2002 9 315 327 11864605 Palatnik JF Allen E Wu X Schommer C Schwab R Control of leaf morphogenesis by microRNAs Nature 2003 425 257 263 12931144 Pan H Qin WX Huo KK Wan DF Yu Y Cloning, mapping, and characterization of a human homologue of the yeast longevity assurance gene LAG1 Genomics 2001 77 58 64 11543633 Park W Li J Song R Messing J Chen X CARPEL FACTORY, a Dicer homolog, and HEN1, a novel protein, act in microRNA metabolism in Arabidopsis thaliana Curr Biol 2002 12 1484 1495 12225663 Pasterkamp RJ Verhaagen J Emerging roles for semaphorins in neural regeneration Brain Res Brain Res Rev 2001 35 36 54 11245885 Pearson WR Lipman DJ Improved tools for biological sequence comparison Proc Natl Acad Sci U S A 1988 85 2444 2448 3162770 Perrone-Bizzozero N Bolognani F Role of HuD and other RNA-binding proteins in neural development and plasticity J Neurosci Res 2002 68 121 126 11948657 Pfeffer S Zavolan M Grasser FA Chien M Russo JJ Identification of virus-encoded microRNAs Science 2004 304 734 736 15118162 Rajagopalan LE Malter JS Growth factor-mediated stabilization of amyloid precursor protein mRNA is mediated by a conserved 29-nucleotide sequence in the 3′-untranslated region J Neurochem 2000 74 52 59 10617105 Rajewsky N Socci ND Computational identification of microRNA targets Dev Biol 2003 267 529 535 Ramos A Hollingworth D Pastore A G-quartet-dependent recognition between the FMRP RGG box and RNA RNA 2003 9 1198 1207 13130134 Rehmsmeier M Steffen P Hochsmann M Giegerich R Fast and effective prediction of microRNA/target duplexes 2004 RNA. In press Reinhart BJ Slack FJ Basson M Pasquinelli AE Bettinger JC The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans Nature 2000 403 901 906 10706289 Reinhart BJ Weinstein EG Rhoades MW Bartel B Bartel DP MicroRNAs in plants Genes Dev 2002 16 1616 1626 12101121 Reynolds A Leake D Boese Q Scaringe S Marshall WS Rational siRNA design for RNA interference Nat Biotechnol 2004 22 326 330 14758366 Richter JD Think globally, translate locally: What mitotic spindles and neuronal synapses have in common Proc Natl Acad Sci U S A 2001 98 7069 7071 11416189 Sasaki T Shiohama A Minoshima S Shimizu N Identification of eight members of the Argonaute family in the human genome Genomics 2003 82 323 330 12906857 Schwarz DS Hutvagner G Du T Xu Z Aronin N Asymmetry in the assembly of the RNAi enzyme complex Cell 2003 115 199 208 14567917 Seitz H Youngson N Lin SP Dalbert S Paulsen M Imprinted microRNA genes transcribed antisense to a reciprocally imprinted retrotransposon-like gene Nat Genet 2003 34 261 262 12796779 Sempere LF Freemantle S Pitha-Rowe I Moss E Dmitrovsky E Expression profiling of mammalian microRNAs uncovers a subset of brain-expressed microRNAs with possible roles in murine and human neuronal differentiation Genome Biol 2004 5 R13 15003116 Shiseki M Nagashima M Pedeux RM Kitahama-Shiseki M Miura K p29ING4 and p28ING5 bind to p53 and p300, and enhance p53 activity Cancer Res 2003 63 2373 2378 12750254 Song JJ Liu J Tolia NH Schneiderman J Smith SK The crystal structure of the Argonaute2 PAZ domain reveals an RNA binding motif in RNAi effector complexes Nat Struct Biol 2003 10 1026 1032 14625589 Stark A Brennecke J Russell RB Cohen SM Identification of Drosophila microRNA targets PLoS Biol 2003 1 e60 14691535 Steward O Schuman EM Compartmentalized synthesis and degradation of proteins in neurons Neuron 2003 40 347 359 14556713 Stuart JM Segal E Koller D Kim SK A gene-coexpression network for global discovery of conserved genetic modules Science 2003 302 249 255 12934013 Tan L Chang JS Costa A Schedl P An autoregulatory feedback loop directs the localized expression of the Drosophila CPEB protein Orb in the developing oocyte Development 2001 128 1159 1169 11245581 Tapper J Kettunen E El-Rifai W Seppala M Andersson LC Changes in gene expression during progression of ovarian carcinoma Cancer Genet Cytogenet 2001 128 1 6 11454421 Tenenbaum SA Carson CC Lager PJ Keene JD Identifying mRNA subsets in messenger ribonucleoprotein complexes by using cDNA arrays Proc Natl Acad Sci U S A 2000 97 14085 14090 11121017 Todd PK Mack KJ Malter JS The fragile X mental retardation protein is required for type-I metabotropic glutamate receptor-dependent translation of PSD-95 Proc Natl Acad Sci U S A 2003 100 14374 14378 14614133 Tuschl T Zamore PD Lehmann R Bartel DP Sharp PA Targeted mRNA degradation by double-stranded RNA in vitro Genes Dev 1999 13 3191 3197 10617568 Vella MC Choi EY Lin SY Reinert K Slack FJ The C. elegans microRNA let-7 binds to imperfect let-7 complementary sites from the lin-41 3′UTR Genes Dev 2004 18 132 137 14729570 Waggoner SA Liebhaber SA Identification of mRNAs associated with alphaCP2-containing RNP complexes Mol Cell Biol 2003 23 7055 7067 12972621 Wang Y Hung C Koh D Cheong D Hooi SC Differential expression of Hox A5 in human colon cancer cell differentiation: A quantitative study using real-time RT-PCR Int J Oncol 2001 18 617 622 11179495 Woodside KJ Shen H Muntzel C Daller JA Sommers CL Expression of Dlx and Lhx family homeobox genes in fetal thymus and thymocytes Gene Expr Patterns 2004 4 315 320 15053981 Xu P Vernooy SY Guo M Hay BA The Drosophila microRNA mir-14 suppresses cell death and is required for normal fat metabolism Curr Biol 2003 13 790 795 12725740 Yan KS Yan S Farooq A Han A Zeng L Structure and conserved RNA binding of the PAZ domain Nature 2003 426 468 474 14615802 Yart A Mayeux P Raynal P Gab1, SHP-2 and other novel regulators of Ras: Targets for anticancer drug discovery? Curr Cancer Drug Targets 2003 3 177 192 12769687 Yekta S Shih IH Bartel DP MicroRNA-directed cleavage of HOXB8 mRNA Science 2004 304 594 596 15105502 Yi R Qin Y Macara IG Cullen BR Exportin-5 mediates the nuclear export of pre-microRNAs and short hairpin RNAs Genes Dev 2003 17 3011 3016 14681208 Yokota N Mainprize TG Taylor MD Kohata T Loreto M Identification of differentially expressed and developmentally regulated genes in medulloblastoma using suppression subtraction hybridization Oncogene 2004 23 3444 3453 15064731 Zamore PD Tuschl T Sharp PA Bartel DP RNAi: Double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals Cell 2000 101 25 33 10778853 Zuker M Mfold web server for nucleic acid folding and hybridization prediction Nucleic Acids Res 2003 31 3406 3415 12824337

15502875

PMC521178

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2021-01-05 08:21:20

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PLoS Biol. 2004 Nov 5; 2(11):e363

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PLoS Biol

10.1371/journal.pbio.0020363

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==== Front PLoS BiolPLoS BiolpbioplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 10.1371/journal.pbio.0020373SynopsisCell BiologyMicrobiologyMolecular Biology/Structural BiologyEubacteriaShut Down, Don't Stress Out Synopsis11 2004 5 10 2004 5 10 2004 2 11 e373Copyright: © 2004 Public Library of Science.2004This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. Oxidative Stress Inactivates Cobalamin-Independent Methionine Synthase (MetE) in Escherichia coli Exploiting Thiol Modifications ==== Body Among the many stresses faced by a cell, one of the most serious is exposure to oxidizing agents. An invading organism, for example, must defend itself against the oxidative assault mounted by a host's immune system. Since oxidation can rapidly destroy many types of molecules, cells have developed multiple means of protecting against it. Rapid mobilization of these defenses requires diversion of resources and temporary suspension of many normal cellular functions, including protein synthesis. In a new study, Elise Hondorp and Rowena Matthews show that when the Escherichia coli bacterium confronts oxidative stress, an enzyme that stands at a central point in the amino acid supply line for protein synthesis is rapidly and reversibly inactivated. Of the twenty amino acids that make up proteins, methionine plays a special role. It is the first amino acid added to every polypeptide chain, and without it, protein synthesis grinds quickly to a halt. Methionine is formed in E. coli through the action of the enzyme cobalamin-independent methionine synthase (MetE), which makes up between one and five percent of all protein in the cell. Thus, by turning off MetE in the face of oxidative stress, protein synthesis can be slowed or stopped, freeing cellular resources to be used elsewhere. Hondorp and Matthews show that in E. coli, MetE is acutely vulnerable to oxidation under a variety of conditions. These results are in accord with a companion study by Leichert and Jakob, also in PLoS Biology, showing that MetE is one of the proteins most sensitive to oxidative damage. When the active site of MetE is stressed by an oxidant, Hondorp and Matthews show, it is temporarily blocked by the attachment of a glutathione subunit. Glutathione is a small molecule that includes a reactive sulfur atom. During “glutathionylation” of MetE, a sulfur on an amino acid of the enzyme is oxidized and links up with a sulfur on glutathione. This study shows that glutathionylation occurs only on a specific amino acid (cysteine 645), which recent structural work indicates sits at the entrance to the active site. Attachment of the bulky glutathione subunit to this cysteine would be expected to block the active site, thus shutting down enzymatic activity. The results indicate that glutathionylation does indeed prevent activity of the enzyme, and furthermore, causes the enzyme to change its three-dimensional form. As the oxidative challenge abates, glutathionylation may be reversed, and the normal activity of the enzyme restored. Thus, glutathionylation of MetE may also serve to protect the active site from permanent oxidative damage. While glutathionylation is a common strategy in eukaryotes, MetE is so far one of the few proteins in bacteria known to be affected in this way. Shutting down MetE and limiting methionine production may play another important role, namely, communicating the bacterium's metabolic state to other nearby E. coli. Methionine is a precursor for the signaling molecule AI-2, which is released extracellularly and appears to serve as a key indicator of colony health and density. This information enables neighboring cells to better respond to changing and potentially hostile environments. Thus, the glutathionylation and inactivation of MetE may provide a simple mechanism by which a bacterium and its neighbors attempt to deal with oxidative stress.

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PMC521179

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2021-01-05 08:28:06

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PLoS Biol. 2004 Nov 5; 2(11):e373

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PLoS Biol

10.1371/journal.pbio.0020373

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==== Front PLoS BiolPLoS BiolpbioplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 10.1371/journal.pbio.0020374SynopsisMicrobiologyEubacteriaA New Way to Look at Oxidative Stress Synopsis11 2004 5 10 2004 5 10 2004 2 11 e374Copyright: © 2004 Public Library of Science.2004This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. Protein Thiol Modifications Visualized In Vivo Exploiting Thiol Modifications ==== Body Chemical reactions lie at the heart of many biological processes, from photosynthesis and respiration to cell signaling and drug metabolism. Thanks to an atmosphere rich in oxygen, many organisms use oxygen to carry out these life processes. But oxygen metabolism produces highly toxic by-products called reactive oxygen species. When oxidation outpaces detoxifying reactions, oxidative stress occurs, and accumulating reactive oxygen species are free to wreak havoc on cellular machinery. Cysteine, one of the 20 different amino acids that make up proteins, contains a thiol group, which can be modified upon oxidation. A thiol group can stabilize protein structures by forming covalent disulfide bonds and can mediate cysteine-regulated redox reactions. At the same time, however, the high reactivity of thiol groups makes them also particularly vulnerable to nonspecific reactions during conditions of oxidative stress. Over the past few years, an increasing number of proteins have been discovered that use oxidative thiol chemistry to regulate their protein activity. In PLoS Biology, Lars Leichert and Ursula Jakob describe a novel method to monitor thiol modifications in proteins subjected to varying redox conditions in a living organism, the bacteria Escherichia coli. This technique is capable of providing a global snapshot of the redox state of protein cysteines during normal and oxidative stress conditions in the cell. To detect proteins that have the ability to undergo stress-induced thiol modifications, Leichert and Jakob differentially labeled the thiol groups of thiol-modified and non-thiol-modified proteins. The proteins were then separated on two-dimensional gels based on their charge and molecular weight. If the technique worked, most thiol-modified proteins should be detected in the oxidizing environment of the E. coli periplasm (the region between the cell's membrane layers), and they were. After proving the method's ability to detect proteins whose thiol groups were oxidized, the next logical step was to determine what proteins DsbA—the enzyme that catalyzes disulfide bond formation in the E. coli periplasm—was targeting. In E. coli mutant strains that lack DsbA, Leichert and Jakob identified a number of proteins with either substantially less or no thiol modification as compared to wild-type (non-mutant) strains, suggesting that these proteins are indeed DsbA substrates. A differential thiol-trapping technique provides a snapshot of the in vivo thiol status of proteins upon variations in the redox homeostasis of cells In contrast to the periplasm, the E. coli cytoplasm contains several reducing systems. When the researchers tested a mutant strain that lacked the gene for the reducing enzyme thioredoxin, they found that a large number of proteins accumulated in an oxidized state. Many of these proteins have cysteines and require a reduced thiol status for their activity. These results demonstrated that under normal growing conditions, many proteins contain cysteine residues that are vulnerable to even small amounts of reactive oxygen species and so require the constant attention of detoxifying enzymes. In a final set of experiments, Leichert and Jakob discovered a number of proteins whose thiol groups get specifically modified in the presence of reactive oxygen species. These results start to explain some of the many metabolic changes that occur in oxidatively stressed cells. Leichert and Jakob's technique should be applicable to many different cell types and organisms and can be used to investigate the in vivo thiol status of cellular proteins exposed to virtually any physiological or pathological condition that is accompanied by oxidative stress. The next step will be to investigate just how thiol modifications mediate the various functions of redox-regulated proteins.

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PMC521180

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2021-01-05 08:21:15

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PLoS Biol. 2004 Nov 5; 2(11):e374

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PLoS Biol

10.1371/journal.pbio.0020374

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==== Front PLoS BiolPLoS BiolpbioplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 10.1371/journal.pbio.0020378SynopsisEcologyEvolutionZoologyArthropodsHomo (Human)Did We or Didn't We? Louse Genetic Analysis Says Yes Synopsis11 2004 5 10 2004 5 10 2004 2 11 e378Copyright: © 2004 Public Library of Science.2004This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. Genetic Analysis of Lice Supports Direct Contact between Modern and Archaic Humans ==== Body If you're an evolutionary biologist, the tired old saw, “You can tell a lot about a person by the company they keep,” represents a fresh new approach to a longstanding problem. Especially if the company in question is a parasite—say, for example, lice—and the problem is tracing the path of human evolution. One of the hottest debates in the study of human origins centers around whether modern Homo sapiens interbred with archaic humans. While genetic data have provided insight into recent human evolution, fossils remain the only available evidence for many archaic human species—and the fossil record is notoriously spotty, leaving the data open to multiple interpretations. Two prominent models and a subset of variants have emerged, differing mainly on the question of gene flow: one asserts that modern humans emerged from an archaic ancestor in Africa about 130,000 years ago and then replaced archaic forms in Africa, Asia, and Europe with no gene flow between them; the other proposes gene flow between modern human populations as well as interbreeding between modern and archaic forms in different parts of the world. Both models find support in the available data, but neither can claim a perfect fit with all the data, leaving the possibility of interbreeding an open question. Faced with a relative paucity of human fossil and genetic data, scientists have been forced to rely on other data sources. Mounting evidence suggests that parasites with an established coevolutionary history with their hosts can serve as a proxy for host evolutionary history, an especially handy tool in the event of insufficient host data. Following this approach in a new study, David Reed and colleagues circumvent the lack of human data by analyzing the next best thing: head lice. As host-specific, obligate parasites—that is, occurring on a single species and not able to survive off that host—lice require direct physical contact between hosts for transmission. As human parasites, lice harbor in their genetic sequence hints of the slings and arrows of evolutionary fortune (and touches of grace, for that matter) that strike their host. Recent studies of the evolutionary history of other human parasites (tapeworms, malarial parasites, and human papillomaviruses), for example, fall in line with fossil and genetic data that place our origins in Africa. Ancient nit combs (above) resemble modern ones (below). (Egyptian wooden comb courtesy of Te Papa, Wellington, New Zealand, negative number F.003884/5. Modern louse comb and head louse images by Vincent S. Smith) Two louse species parasitize humans, head/body (Pediculus humanus) and pubic (Pthirus pubis). Head and body lice obviously occupy different habitats, but are not genetically distinct. Interestingly, P. humanus contains two ancient lineages, offering the opportunity to shed light on this murky period in human evolution. To do this, Reed and colleagues had to reconstruct the evolutionary history of P. humanus, which they did using morphological and genetic analyses of this and other species of lice. They confirmed that P. humanus comprises two lineages—one contains both head and body forms and has worldwide distribution; the other contains only the head louse and is restricted to the New World—but discovered that P. humanus originated long before its H. sapiens host. Humans went through a population bottleneck around 100,000 years ago, followed by an expansion; one would expect to see the same thing in lice. Population genetics studies revealed, however, that only the worldwide lineage went through a bottleneck and subsequent expansion. The New World lineage not only maintained a relatively stable population size but followed an evolutionary path distinct from the worldwide lineage for the past 1.18 million years. It is unlikely, the authors argue, that two ancient louse lineages could embark on such different evolutionary histories on the back (or head) of a single host. More likely, the New World louse evolved on an archaic form of humans and then cast its lot with a modern version. While the split between H. sapiens and H. neanderthalensis was too recent (about 700,000 years ago) to support a concurrent split between the worldwide and New World lice lineages, the split between H. sapiens and H. erectus (about 1.8 million years ago) could. Reed and colleagues propose a scenario in which H. sapiens and H. erectus carried distinct types of lice owing to a million years or so of isolation. As the first waves of modern humans left Africa about 100,000 years ago and modern humans replaced archaic forms, the two forms engaged in enough contact—whether in the form of fighting, swapping clothes, or interbreeding—for archaic lice to make the switch to modern human hosts. Tackling the question of interbreeding, the authors suggest, might best be pursued by studying P. pubis, which requires sexual contact for transmission.

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PMC521181

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2021-01-05 08:21:15

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PLoS Biol. 2004 Nov 5; 2(11):e378

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PLoS Biol

10.1371/journal.pbio.0020378

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==== Front PLoS BiolPLoS BiolpbioplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 10.1371/journal.pbio.0020385SynopsisDevelopmentMolecular Biology/Structural BiologyNeuroscienceIn VitroA New Cell Model for Parkinson's Disease Synopsis11 2004 5 10 2004 5 10 2004 2 11 e385Copyright: © 2004 Public Library of Science.2004This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. Sensitivity to Oxidative Stress in DJ-1-Deficient Dopamine Neurons: An ES-Derived Cell Model of Primary Parkinsonism DJ-1 Is a Redox-Dependent Molecular Chaperone that Inhibits α-Synuclein Aggregate Formation Molecules That Cause or Prevent Parkinson's Disease ==== Body Clinical descriptions of Parkinson's disease remain remarkably similar to those first described by James Parkinson nearly 200 years ago. Patients with “shaking palsy” experience a progressive loss of muscle control, increased muscle rigidity, inhibited movement, and tremors. These symptoms, it was later discovered, result from the loss of dopamine-producing neurons specifically in an area of the ventral midbrain called the substantia nigra. Midbrain dopamine neurons relay chemical signals that regulate motor control and less quantifiable attributes like mood and motivation, and therefore the loss of these cells is predicted to lead to the symptoms of Parkinson's. Despite the well-characterized cellular basis of Parkinson's disease, the molecular mechanisms responsible for dopamine neurodegeneration remain unknown. There is evidence that both genetic and environmental components are involved. That a person with Parkinson's disease is three to four times more likely than an unaffected individual to have a close family member with “parkinsonian” symptoms suggests a genetic factor; furthermore, several genes have been associated with relatively rare, familial forms of the disease. For example, mutations of the protein alpha-synuclein (α-synuclein), which is found to aggregate in the brains of patients with Parkinson's, lead to a familiar parkinsonism syndrome. Mutations in a second gene called DJ-1 were recently found in two families with an inherited form of Parkinson's. Importantly, mutations in DJ-1 have previously been linked to the pesticide paraquat in unrelated research on cell stress and reactive oxygen species, and have been linked to dopamine neuron toxicity. Reactive oxygen species are molecular byproducts of oxygen metabolism that react with and damage cellular components like proteins and DNA, and there is evidence from postmortem studies that reactive oxygen species may play a role in Parkinson's disease. Part of the challenge of untangling the relative contributions of all these components stems from the difficulty in finding a model that can adequately mimic the loss of dopamine cells. In two papers published in PLoS Biology, Asa Abeliovich and colleagues make the case that a model based on mouse embryonic stem cells offers a promising platform for dissecting the disease mechanism of Parkinson's. Working with these cells, the researchers report that DJ-1-deficient cells—and especially DJ-1-deficient dopamine neurons—display heightened sensitivity to oxidative stress. In a second paper, they link DJ-1 dysfunction to alpha-synuclein aggregation. Oxidative stress has long been associated with neuronal cell death and neurodegenerative diseases like Parkinson's. Proving a causal relationship between oxidative stress and neurodegeneration, however, requires establishing a molecular mechanism. In the first paper, to explore the hypothesis that DJ-1 contributes to the cellular response to oxidative stress, Abeliovich and colleagues created mouse embryonic stem cells lacking functional copies of DJ-1 and exposed them to hydrogen peroxide, a powerful oxidizer. Compared to normal cells, DJ-1 mutants showed signs of greater toxicity and higher levels of cell death. These defects were corrected when the researchers reintroduced the protein in the mutants, confirming DJ-1's responsibility for the defects. DJ-1 protects against oxidative damage, the results show, not by inhibiting the accumulation of the reactive oxygen species associated with hydrogen peroxide, but by mitigating the damage created by them. Abeliovich and colleagues then explored DJ-1's function in dopamine neurons by inducing mutant and control embryonic stem cells to differentiate in cell cultures. Production of dopamine neurons was significantly reduced in the DJ-1-deficient cultures relative to the control cultures. And like DJ-1-deficient embryonic stem cells, DJ-1 dopamine mutants were vulnerable to oxidative stress. “DJ-1 deficiency,” the authors conclude, “leads to reduced dopamine neuron survival and predisposes these cells to endogenous and exogenous insults.” Inhibiting DJ-1 activity in neurons from the embryonic mouse midbrain produced the same results. In the second paper, Abeliovich and colleagues go on to probe the molecular basis of DJ-1's activity. There have been several leads regarding how DJ-1 functions, based on homology to related genes, including a potential role as a molecular protein chaperone; protein chaperones assist in the folding and refolding of damaged proteins, and thus play a central role in the cellular response to oxidative stress. Abeliovich and colleagues found that DJ-1 acts as an unusual molecular chaperone that is specifically induced under oxidative conditions, and acts to prevent the aggregation of cellular proteins. Interestingly, the researchers go on to show that one substrate of DJ-1 activity is alpha-synuclein, thus providing a possible mechanism linking these two molecules implicated in Parkinson's disease. Altogether, these results support a link between toxin-induced oxidative damage and disease, and provide a tractable model for studying the molecular mechanisms of neurodegenerative disease.

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PMC521182

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2021-01-05 08:21:16

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PLoS Biol. 2004 Nov 5; 2(11):e385

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PLoS Biol

10.1371/journal.pbio.0020385

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==== Front PLoS BiolPLoS BiolpbioplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 10.1371/journal.pbio.0020387SynopsisGenetics/Genomics/Gene TherapyDrosophilaHigh Affinity: Making Up for Being Male Synopsis11 2004 5 10 2004 5 10 2004 2 11 e387Copyright: © 2004 Public Library of Science.2004This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. X Chromosome Sites Autonomously Recruit the Dosage Compensation Complex in Drosophila Males ==== Body Because males and females possess different numbers of the two sex chromosomes (for instance, in mammals, XX in females versus XY in males), the potential “dose” of each gene differs. Without some compensating mechanism, female mammals would express twice the quantity of an X-linked gene as males. The same holds true in the fruitfly Drosophila, in which the female carries two X chromosomes, while the male carries only one. In mammals, dosage compensation is achieved by silencing one of the X's in the female. Drosophila takes the opposite tack, doubling the output from the single male X chromosome. It does so through the creation of “compensasomes,” protein–RNA complexes that bind to the X chromosome and boost gene transcription. One model of compensasome activity has posited a two-step mechanism, in which the complexes form only at 35–40 specific “entry sites” along the X, and then spread out to the surrounding regions. In this issue, Delphine Fagegaltier and Bruce Baker test this model and show that its predictions do not match experimental results. The compensasome complex includes half a dozen proteins collectively known as MSLs (for “male-specific lethal”), along with two pieces of RNA, roX1 and roX2. Fagegaltier and Baker reasoned that, according to the entry-site model, if a piece of the X not containing one of the entry sites was transposed to an autosome (non-sex chromosome), it should be unable to recruit MSLs and therefore be unable to form compensasomes. To test this prediction, they used autosomes into which various pieces of the X had been transposed. Contrary to prediction, they found that even the smallest pieces could recruit MSLs, whether or not they contained entry sites. Furthermore, the pattern of MSL binding was exactly the same as if the fragment of the X was still on its native chromosome, suggesting that each of the hundreds of sites at which compensasomes are found function autonomously to recruit them. Compensasomes do not spread from the X chromosome onto autosomal material translocated onto the X Another prediction of the entry-site model is that compensasomes should spread out from the entry site, along the chromosome. And here again, the model does not hold up—Fagegaltier and Baker found that even when entry sites from the X chromosome are put close to an autosomal region, compensasomes never spread from the X onto these regions. These results suggest that spreading is not an innate function of the compensasome, and further strengthens the case for autonomous recruitment all along the X. In place of the two-step “entry site plus spreading” model, the authors propose a model based on differential affinity for compensasome components. They suggest that the 35–40 “entry sites” are simply high-affinity sites that recruit MSLs first, based on intrinsic differences that allow them to bind and hold MSLs more strongly than other sites. Once these sites are occupied, additional compensasome components can bind to lower-affinity sites. This mechanism can account for observed compensasome activity without the restriction to a limited number of entry sites and the requirement for spreading. Fagegaltier and Baker note that while compensasome spreading does not normally occur during dosage compensation on the X chromosome, it has nonetheless been documented for some roX transgenes. They propose that the additional binding observed specifically around roX transgenes results from a mass action of compensasomes, as roX transgenes would act as assembly sites for compensasomes, just as ribosomal RNA genes do for ribosomes. Once formed, compensasomes may bind locally to other neighboring sites. While the details of dosage compensation and the dosage compensation complexes now clearly differ between mammals and flies, there are broad similarities, including the widespread modification of chromatin structure and the use of RNA components in the compensation machinery. A deeper understanding of the process in flies may help shed light on the details of compensation in other organisms as well.

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PMC521183

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2021-01-05 08:28:06

no

PLoS Biol. 2004 Nov 5; 2(11):e387

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PLoS Biol

10.1371/journal.pbio.0020387

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==== Front PLoS BiolPLoS BiolpbioplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 10.1371/journal.pbio.0020389SynopsisEcologyEvolutionInfectious DiseasesMicrobiologyZoologyEpidemiology/Public HealthFrogsYeast and FungiEndangered Frogs Coexist with Fungus Once Thought Fatal Synopsis11 2004 5 10 2004 5 10 2004 2 11 e389Copyright: © 2004 Public Library of Science.2004This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. Endemic Infection of the Amphibian Chytrid Fungus in a Frog Community Post-Decline ==== Body Amphibian declines have reached crisis proportions in various parts of the world. In many areas, habitat loss is the likely culprit. But when mass die-offs suddenly occurred in relatively undisturbed habitats, the cause was far less obvious. Fourteen species suffered either extinctions or major declines in the pristine rainforests of Queensland, Australia, between 1979 and 1993. It was suggested in 1996 that some unknown disease had spread through the populations, but no pathogen was discovered until 1998, when the fungus Batrachochytrium dendrobatidis was identified from sick and dead frogs. Since then, several lines of evidence suggest that B. dendrobatidis may be involved in frog declines: the fungus has been found on frogs in afflicted areas; lab studies show that it's highly pathogenic to some frog species; and pathological evidence links it to host mortality. But with little information about the prevalence of this fungal infection in wild frogs, or how the disease impacts frogs in the wild, the causal role of this chytrid fungus remains unclear. To evaluate the effects of B. dendrobatidis on frogs in their natural habitat, Richard Retallick et al. focused on six species living in the high-elevation rainforest streams of Eungella National Park in Queensland, Australia, where frog losses were “particularly catastrophic.” Two species vanished between 1985 and 1986: the Eungella Gastric-Brooding Frog (Rheobatrachus vitellinus), which is now thought extinct, and the Eungella Torrent Frog (Taudactylus eungellensis), which later reappeared in a few small populations. Other local frog species escaped this period relatively unscathed. Taudactylus eungellensis (Photo: Richard Retallick) Retallick captured frogs from six sites from 1994 to 1998, clipped one or two toe tips from each frog to age and identify them, and then released the frogs back into the wild. At the time, B. dendrobatidis had yet to be identified, but Retallick retained the toe tips, and the authors tested the toes for disease in 2002–2003. Fungal infections were found in two species—T. eungellensis and Litoria wilcoxii/jungguy (the latter consists of two species that are indistinguishable without genetic analysis); the other four species were infection-free. L. wilcoxii/jungguy did not decline to any great extent during the 1985–1986 die-off. The proportion of infected T. eungellensis frogs was greatest at three particular sites, which showed peak infections during cooler months. Prevalence of infection was highest during winter and spring, but did not vary from year to year, suggesting that the infection is now endemic. Fungal infections were found in 27.7% of L. wilcoxii/jungguy frogs, with no evidence that prevalence differed among sites, seasons, or individuals (males, females, or subadults). The probability of recapture was significantly lower for frogs that were already infected when first captured. While this might suggest a correlation between infection and death, it's impossible to distinguish death from simple failure to recapture the animal. On further analysis, McCallum and colleagues found no evidence that survival differed between infected and uninfected frogs, suggesting that this potentially devastating amphibian disease now coexists with the frogs, with little effect on their populations. These results, the authors conclude, “show unequivocally” that remaining populations of T. eungellensis, a rainforest frog listed as endangered, “now persist with stable infections of B. dendrobatidis.” While these findings do not exonerate the fungus as the agent of mass declines, they do rule out the possibility that the fungus caused the decline, then vanished from the area, allowing frog populations to recover. The authors allow that it's possible that B. dendrobatidis did not cause the initial T. eungellensis declines. Or alternately, the fungus could have emerged as a novel pathogen in the ecosystem, causing massive casualties before some form of evolutionary response took hold. Surviving frog populations may have evolved resistance to the pathogen, for example, or less virulent strains of the fungus may have evolved. If it turns out that frog populations can develop resistance to the chytrid fungus, the researchers point out, then a conservation program of captive breeding and selecting for resistance could potentially thwart the extinction of these, and other, critically endangered frogs. A critical next step, then, is to determine whether frogs and fungus do coevolve.

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PMC521184

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2021-01-05 08:21:15

no

PLoS Biol. 2004 Nov 5; 2(11):e389

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PLoS Biol

10.1371/journal.pbio.0020389

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==== Front PLoS BiolPLoS BiolpbioplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 10.1371/journal.pbio.0020396SynopsisBioinformatics/Computational BiologyCell BiologyGenetics/Genomics/Gene TherapyMolecular Biology/Structural BiologyHomo (Human)MicroRNA Is a Major Regulator Synopsis11 2004 5 10 2004 5 10 2004 2 11 e396Copyright: © 2004 Public Library of Science.2004This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. Human MicroRNA Targets ==== Body Since their discovery a decade ago, microRNAs (miRNAs) have emerged as major regulators of gene expression in eukaryotes of all kinds. Only 20 to 40 nucleotides long, a miRNA binds to a specific target sequence within a much longer messenger RNA (mRNA), inhibiting its translation and thus controlling expression of the corresponding gene even after the DNA itself has been read. Within the human genome, there are about 250 genes that code for miRNAs. Each miRNA has the potential to bind to many different transcripts. Variations in miRNA sequence dictate the gene transcripts to which each will bind most strongly. It has become clear that miRNAs play a critical role in controlling gene expression, for example, in larval developmental transitions and neuronal development in the worm Caenorhabditis elegans, growth control and apoptosis in the fruitfly Drosophila melanogaster, hematopoietic differentiation in mammals, and leaf development, flower development, and embryogenesis in the plant Arabidopsis thaliana. Despite their significance, the full range of genes miRNAs target is unknown, as is the best method for discovering them. In a new study, Debora Marks, Chris Sander, and colleagues describe an algorithm for determining the targets of miRNAs, and show they include more than 10% of all human genes. The algorithm uses three factors to evaluate whether a potential target site is likely to actually be regulated by miRNA. First, the target site must have some degree of sequence complementarity to one or more of the known miRNAs. Second, the strength with which the predicted target and its miRNA bind together, which can be calculated from the sequence and other structural factors, must be higher than some threshold. Finally, evolutionary conservation—the presence of the target–miRNA pair in different organisms—is factored in, because the likelihood that the target and miRNA actually pair in vivo is greater if the pair is found in multiple types of organisms. Using these principles, and the specific weighting they assigned to each factor, Marks and colleagues identified 2,273 genes in humans, rats, and mice that are likely targets for miRNA regulation. This is probably an underestimate of the total, since the researchers required each candidate gene to have at least two miRNA target sites. The authors identified another 2,128 genes with only one target site, but note that the false-positive rate here is likely to be high. Whatever the final number, the implication is that several thousand of our approximately 30,000 genes are under the control of miRNAs. Of special interest is that these putative targets include many genes known to be associated with the fragile X mental retardation protein, a crucial but still poorly understood player in mRNA regulation, whose absence leads to a type of mental retardation called fragile X syndrome. microRNA gene networks The researchers' findings also reinforce several emerging principles of miRNA-based regulation. First, it is widespread among multicellular eukaryotes, and sequences are surprisingly conserved. Of the 78 known miRNAs in Drosophila, 28 have close relations in mammals. Second, an individual miRNA may regulate multiple genes—Marks and colleagues found that the average miRNA interacts with seven distinct mRNAs, with a range from 0 to 268. Third, the genes regulated by a single miRNA may be functionally related, such as components of the protein degradation system or specific signal transduction pathways. Fourth, single genes may be regulated by multiple miRNAs—the gene that encodes amyloid precursor protein, for example, has at least eight miRNA sites—suggesting that expression may be combinatorially controlled by numerous cellular influences. These results provide resources for a host of experiments to elucidate the mechanism of miRNA action, which is not well understood. Several of the identified mammalian miRNA–target pairs have near-perfect matching sequences. In both plants (where miRNAs were first discovered) and animals, such matches are associated with degradation of the mRNA. The authors fully recognize that their algorithm, called miRanda, is not the last word in miRNA target identification. In order to improve both the search for targets and the algorithm itself, they are making the algorithm and full sets of results in vertebrates available free to other researchers (www.microrna.org), who can modify its parameters as experimental results and new models dictate.

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PMC521185

CC BY

2021-01-05 08:27:51

no

PLoS Biol. 2004 Nov 5; 2(11):e396

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PLoS Biol

10.1371/journal.pbio.0020396

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==== Front Respir ResRespiratory Research1465-99211465-993XBioMed Central 1465-9921-5-141538003110.1186/1465-9921-5-14ResearchCheyne-Stokes respiration in patients hospitalised for heart failure Mared Lena [email protected] Charles [email protected] Leif [email protected] Søren [email protected] Bengt [email protected] Dept of Respiratory Medicine, University Hospital, Lund, Sweden2 Dept of Cardiology, University Hospital, Malmö, Sweden3 Dept of ENT diseases, University Hospital, Lund, Sweden4 Lund Sleep Study Group, University Hospital, Lund, Sweden2004 20 9 2004 5 1 14 14 20 2 2004 20 9 2004 Copyright © 2004 Mared et al; licensee BioMed Central Ltd.2004Mared et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Previous studies showing a strong relationship between Cheyne-Stokes respiration and the severity of left ventricular systolic dysfunction have usually been done in selected patient populations with lower age and a higher proportion of males than the "typical" in-hospital patient with heart failure. The purpose of the present study was test the strength of this relationship in unselected patients admitted to hospital due to decompensated chronic heart failure. Methods We evaluated 191 patients (32% women), mean age 73 years, ready for discharge from the heart failure unit in the University Hospital of Malmö, Sweden. The patients underwent echocardiography for determination of left ventricular ejection fraction and left ventricular inner diastolic diameter. A respiratory investigation during sleep was performed the last night before discharge. Results We found that 66% of the patients had Cheyne-Stokes respiration more than 10% of the total recording time. Only 7 (3.6%) of the patients had predominantly obstructive apnoeas. There was a significant but very weak relationship between left ventricular ejection fraction and left ventricular inner diastolic diameter on one hand and Cheyne-Stokes respiration on the other. Age was a stronger determinant of Cheyne-Stokes respiration than any of the cardiac or other clinical variables. Conclusion Although presence of Cheyne-Stokes respiration indicates left ventricular dysfunction, its severity seems only weakly related to the severity of heart failure. Age was found to be a stronger determinant, which may reflect the underlying age-dependency found also in healthy subjects. Due to age restrictions or other selection criteria, the importance of age may have been underestimated in many previous studies on factors associated with Cheyne-Stokes respiration. ==== Body Background Cheyne-Stokes respiration (CSR) during sleep, is common in patients with heart failure [1,2]. Cheyne-Stokes respiration during sleep has been claimed to be an independent risk factor for death [3,4], speculatively through increased neurohumoral stress on the heart [5]. Results from other studies have, however, been contradictory [6]. It has also been claimed that sleep disturbance from Cheyne-Stokes respiration may cause daytime sleepiness [7]. Most previous studies have included selected patients with impaired left ventricular systolic function. A large proportion of heart failure patients are elderly and have relatively preserved left ventricular systolic function. However, elderly heart failure patients have often been excluded from sleep studies. Such studies are not representative of the everyday clinical spectrum of heart failure patients, including the increasing number of elderly patients usually seen in general medical wards. The proportion of women, with predominantly diastolic heart failure, may also be greater in this group. The aims of the present study were to test the strength of the relationship between left ventricular dysfunction and nocturnal Cheyne-Stokes respiration in unselected patients admitted to hospital due to decompensated chronic heart failure and to determine the correlation between Cheyne-Stokes respiration and other clinical variables to provide source material for subsequent analyses of quality of life and survival. Methods Patients All patients admitted to the Heart Failure Unit at the Department of Cardiology, Malmö University Hospital from January 1996 to november 1999 primarily due to decompensated chronic heart failure were eligible for inclusion. Malmö University Hospital serves as the main hospital for the whole population of Malmö (population 250,000) and is the only hospital in the city. Patients were excluded only if unable to comply to the study protocol due to some other condition, to complete the study questionnaire or to provide informed consent. Heart failure was diagnosed according to European Society of Cardiology guidelines for the diagnosis of heart failure [8]. All patients had been stabilised following treatment for heart failure and were studied on the day prior to planned discharge. For practical reasons not all patients discharged could be included into the study. Patients were included if they were to be discharged on a weekday and only when a nurse trained in the use of the registration apparatus was available. Furthermore, we could not register more than one patient per night. Unfortunately, the precise number of patients discharged alive from this unit during this particular period was not possible to obtain, but a crude estimate is that we have investigated between 25 and 50% of the available patients. All patients provided informed consent to participate in the study, which was performed in accordance with the principles of the Declaration of Helsinki and approved by the Medical Ethics Committee at Lund University. Clinical evaluation Ischaemic heart disease was diagnosed based on findings from previous coronary angiography, documented myocardial infarction or typical signs of ischaemia at exercise testing. Hypertension (according to the local guidelines at the time of the study) was diagnosed if blood pressure was >150/95 mmHg or the patient was receiving drug therapy for hypertension. Diabetes was diagnosed if fasting blood glucose levels were >7 mmol/l or the patient was treated with oral anti-glycaemic medication or insulin. The diagnosis of other concomitant diseases was based on patient history and/or patient records. Echocardiography Echocardiographic examinations were performed using a Hewlett-Packard Sonos 2000 (Andover, Mass, USA). Parasternal and apical views were obtained with the patient in a left lateral recumbent position. Measurements were acquired during silent respiration or end-expiratory apnoea. Left ventricular systolic function was assessed by determination of the mean left atrioventricular plane displacement (AVPD), global qualitative assessment and/or single plane ellipse (modified Simpson's rule) [9-11]. Respiration during sleep We recorded oronasal airflow by thermocouples, electrocardiogram, chest wall movement by electrical impedance, and finger pulse oximetry using the EdenTrace II Plus Multirecording System (EdenTech Corp, Eden Prairie, MN, USA) [12,13]. The recordings started when the patients went to bed and were discontinued the following morning when the patients woke up. No attempt was made to define the amount of sleep. The recordings were printed out and scored manually. Hypnotics were allowed, and were taken by 48% of the patients at the time of the study. Patients were considered to be habitual snorers if they answered "often" or "always" to the question "Do you snore loudly and disturbingly?" Scoring and analysis of breathing patterns The records were scored manually for Cheyne-Stokes respiration (gradual waxing and waning of respiration followed by a central apnoea or hypopnoea) [14,15] and for obstructive sleep apnoea. Patients with purely or predominantly obstructive sleep apnoea (n = 7) were excluded from the analyses of CSR. Patients with occasional obstructive apnoeas occuring during extended periods of CSR (n = 28) were included in the analyses of CSR. The total time spent in Cheyne-Stokes respiration was divided by the total recording time to compute the percentage of time in bed spent in Cheyne-Stokes respiration (CSR%). Since CSR% was not normally distributed, we used Spearmans rank correlation test to relate CSR% to the continuous variables age, BMI, LVEF and LVIDD. For analysis of CSR% with respect to the categorical variables gender, cerebrovascular disease, ischaemic heart disease, NYHA class, atrial fibrillation and habitual snoring, we used the Mann-Whitney U-test. There are no data in the literature to allow categorisation of patients as normal and abnormal according to any specific level of CSR%. In table 2, the data concerning CSR% are, however, categorised; this is for demonstrational purpose only. Table 2 Cheyne-Stokes respiration related to physiologic variables All CSR incl mixed apnoeas CSR% CSR% CSR% Significance <10 10–50 >50 n = 58 n = 66 n = 60 Age (years) 68.8 ± 11.7 72.1 ± 8.5 75,9 ± 8.6 p < 0.01 R=0.24 Body Mass Index (kg/m2) 26.5 ± 5.5 26.1 ± 4.3 24.6 ± 4.1 NS LVEF (%) 36.1 ± 12.1 38.8 ± 12.6 32.4 ± 11.5 P < 0.05 R=-0.17 LVIDD (mm) 55.1 ± 10.0 57.1 ± 9.4 58.0 ± 8.2 P < 0.01 R = 0.20 Ischaemic heart disease (percentage of patients) 51 59 72 p < 0.05 NYHA class 3–4 (percentage of patients) 60 39 55 NS Atrial fibrillation (percentage of patients) 29 33 40 NS Cerebrovascular disease (percentage of patients) 16 11 27 NS Habitual snorers (percentage of patients) 12 12 12 NS Males (percentage of patients) 57 77 70 NS Values are given as mean ± SD. Patients are divided according to the severity of Cheyne-Stokes respiration (quantified as percentage of total recording time); the limits are arbitrarily chosen. Significance testing is made with Spearman's rank correlation test for continuous variables and with Mann-Whitney U-test for categorical data. Results Clinical data Two hundred and three patients were included, however final analysis only included 191 patients (32% women). Three patients were excluded because of total recording time less than two hours. Eight patients were excluded due to the poor quality of their recordings and one patient was excluded because of an abnormal irregular breathing pattern that could not be categorized as Cheyne-Stokes or obstructive sleep apnoea. The aetiology of the heart failure was ischaemic heart disease in 60%, dilated cardiomyopathy in 3%, hypertension in 14%, valvular disease in 8% and other or unknown reason in 15%. Half of the patients had had heart failure diagnosis for more than a year. All but eight patients were prescribed diuretics, 43% digitalis, 70% ACE inhibitors and 28% beta-blockers. Almost half (48%) of the patients used hypnotics (usually bensodiazepines). The clinical characteristics of the study patients are presented in Table 1. Five patients were in NYHA class 1, 83 in NYHA 2, 89 in NYHA 3 and five in NYHA 4. Only 10 patients were free from concomitant disease. Twenty five percent had diabetes, 13% chronic obstructive pulmonary disease and 18% cerebrovascular disease (reversible or permanent cerebral ischaemia or haemorrhage) at any time prior to the investigation. Five percent had cancer diagnosed and treated within the last year. Table 1 Clinical characteristics of the study patients All patients Females (n = 61) Males (n = 130) Significance Age (years) 72. 6 ± 10.0 75.1 ± 8.9 71.4 ± 10.3 P < 0.05 BMI (kg/m2) 25.7 ± 4.6 25.4 ± 5.4 25.9 ± 4.3 NS LVIDD (mm) 56.5 ± 9.4 51.2 ± 9.5 59.0 ± 8.3 P < 0.001 LVEF (%) 36.2 ± 12.1 39.0 ± 13.0 34.9 ± 11.6 P < 0.05 LVEF ≥45% (percentage of patients) 26 36 22 P < 0.05 Ischaemic heart disease (percentage of patients) 60 57 62 NS NYHA 3–4 (percentage of patients) 52 57 49 NS Atrial fibrillation (percentage of patients) 35 33 35 NS Cerebrovascular disease (percentage of patients) 18 15 19 NS Habitual snorers (percentage of patients) 12 7 15 P < 0.05 Values for continuous data (age, BMI, LVIDD and LVEF) are given as mean ± SD. Significance testing is made with T-test for continuous data and chi-square for the other data. Overall 26% had heart failure due to left ventricular diastolic dysfunction (ejection fraction >45%). Female patients were older (Table 1) and more likely to have heart failure with preserved left ventricular systolic function; 36% had ejection fraction ≥45% as compared to 22% of the males (p < 0.05, chi square). There was no significant gender difference in the prevalence of ischaemic heart disease as the cause of heart failure. Respiration during sleep The average recording time was 424 (SD 75, median 444, interquartile 400–472) minutes and average CSR% 35 (SD 30, median 28, interquartile 6–59). Predominantly obstructive apnoeas were found in seven patients (3.7%) and CSR (arbitrarily defined as CSR% >10%) in 126 patients (66%). Sixty (31%) of the patients had CSR more than 50% of the recording time (Table 2). In figure 1, four examples of different breathing patterns are demonstrated. Figure 1 Examples of nocturnal respiratory recordings. The upper part of each panel is the flow signal from the thermocouples and the lower is the impedance signal from the ECG electrodes. The duration of each example is 6 minutes. Panel A depicts an unequivocal period of Cheyne-Stokes respiration. Panel B was interpreted as normal by the software of the recording device, but was interpreted by us as Cheyne-Stokes respiration. Panel C is an example of obstructive sleep apnoeas and panel D is a period of Cheyne-Stokes respiration with a small obstructive component, classified by us as Cheyne-Stokes respiration rather than obstructive sleep apnoeas. Univariate rank correlation analysis showed that CSR% was most strongly correlated to age (R = 0.24, p < 0.01, figure 2), but also to left ventricular ejection fraction (LVEF) and to left ventricular diastolic diameter (table 2). Stepwise multiple regression analysis with gender, age, BMI, LVEF and LVIDD as independent variabels confirmed the relative importance of age vs that of LVEF and LVIDD (r2 0.06 vs 0.03 and 0.03). The severity of CSR was also greater in patients with ischaemic heart disease. There were no relationships between medication (e.g. betablockers or benzodiazepines) and CSR%. Figure 2 Cheyne-Stokes respiration as a function of age. Cheyne-Stokes respiration (% of total recording time) as a function of age, linear regression line and confidence bands are drawn. Filled circles denote patients with LVEF 45 and above, i.e. patients with diastolic heart failure. Seven patients (3.6%) were found to have predominantly obstructive sleep apnoea (average AHI 16.0, SD 7.0, median 16.2, interquartile range 8.7 – 21.8). Excessive snoring, obesity or male gender were not overrepresented in this group. Discussion We have shown that CSR is common in elderly patients hospitalised due to decompensated chronic heart failure and that age was a stronger determinant of CSR than any of the cardiac or other clinical variables Selection of patients and timing of the study Malmö University Hospital serves a population of 250,000 inhabitants. In general all patients primarily admitted to hospital due to decompensated chronic heart failure are treated at the Heart Failure Unit until discharge from hospital. Due to this and to the liberal inclusion criteria we believe that the study patients are representative of the general population of patients with heart failure. Many other studies suffer from the disadvantages of a selection bias by excluding older patients, including only men or including only patients specifically referred for sleep studies [16] or for evaluation for heart transplantation [17]. One major difference between our study design and that of the majority of other studies is that we included patients immediately after an episode of decompensation, whereas most other authors have studied patients 1–3 months after discharge. We do not, however, know to what extent this approach affects nocturnal breathing patterns. The results reported by Tremel et al [2] suggest that sleep respiratory disturbances are stable during the second month after an episode of worsening heart failure, whereas there are no data, as far as we know, examining patients prior to that phase. Nonetheless, many observations of apnoeas (by nurses and relatives, posing questions to the clinician) are made during the hospitalisation. Our results are therefore relevant with respect to the factors associated with apnoeas in this situation. Respiratory disturbances during sleep At the time of the study, the most convenient and easy manageable system available at our department was the EdenTrace system. The semiquantitative nature of chest wall impedance measurement may have reduced the sensitivity of our measurements. However, by combining impedance measurement of respiratory movements, recording of oronasal airflow with thermocouples and finger pulse oxymetry with a careful visual analysis af the traces by an experienced physician (SB), we postulate that the distinction between central and obstructive events is sufficiently accurate. Furthermore that the bias introduced, would tend to underestimate, rather than overestimate the prevalence of sleep disordered breathing in the studied population. Whereas there are widely accepted standards for analysis of sleep apnoea the definition of CSR is less precise, and there is no accepted standard method for its quantification. We quantified the severity of CSR as percentage of total recording time. This approach is suggested by Ancoli-Israel et al [14] and is accepted also by the American Academy of Sleep Medicine Task Force [15] since it is simple and well suited for routine clinical use, irrespective of the technical methods available for respiratory recordings. The overall prevalence of CSR in this material was 66%, using an arbitrary limit of CSR% >10%. This figure is in the same magnitude as the 50–60% previously reported in stable heart failure outpatients [18,19] and close to that found by Ancoli-Israel (70%) in a small group of elderly, hospitalised patients [14]. We suggest that the main cause of the prevalence differences between studies is the age of the population studied, rather than the timing with regard to worsening heart failure [2]. We found 28 patients with mixed apnoeas (see figure 1 panel D for example). We believe that these patients should be considered to have a variant of CSR rather than to have an obstructive sleep apnoea syndrome (OSAS). There was no excess of snoring, obesity or male gender in this group, factors that are otherwise considered to be associated with OSAS. Furthermore, exclusion of these patients from the analyses did not change our results. It has been suggested that obstructive sleep apnoeas and CSR in heart failure patients both are part of a spectrum of periodic breathing [20,21], our data are compatible with this hypothesis. Age The most consistent result of our study is that age was more strongly related to Cheyne Stokes respiration than any other variable recorded. This corroborates data from other large studies without an upper age limit [14,16,22]. Many other studies that failed to demonstrate a similar relationship are constrained to patients below an arbitrary age limit or to patients referred for cardiac transplantation [17], which strongly affects validity for the general in-hospital patients. The strength of the association with age remained also when we excluded the youngest outliers (see figure 2) from the statistical analysis. We therefore consider our finding of age-dependency to be valid for unselected in-hospital patients with heart failure. It should be emphasized, however, that the predictive value of age was very weak, only explaining 6% of the total variability. Increasing prevalence of central apnoeas with age in normal subjects has been demonstrated by e.g. Bixler and coworkers [23], although the prevalence is much lower than that found in our patients with heart failure. Bixler et al suggest that a conservative approach should be applied when interpreting sleep studies in elderly. Our data suggest that this may also be a valid strategy for patients with heart failure. Although the presence of CSR seems to be associated with the presence of heart failure, its severity gives little information about the severity of the heart disease. Gender We found no gender effect (table 2), in contrast to the findings of e.g. Sin and coworkers [16]. One important reason may be that their 450 patients (only 15% women vs 32% in our study) were not a random sample of heart failure patients, but represent a much younger population (mean age 60 years vs 72 in our study) specifically referred for a sleep study. Indices of heart failure Heart dilatation (in our study measured as LVIDD) has been claimed to be an important factor for the development of CSR [24]. Impaired systolic function, as demonstrated by a low LVEF, is another factor that is usually considered to be associated with CSR. In our data, we found that these factors explained only 3% each of the variability of CSR. This contrasts to the results of many other studies, but is well in accordance with the findings of Sin and coworkers in their large study [16]. The idea of CSR mainly being a function of low cardiac output (as estimated by e.g. LVEF) may therefore be an oversimplification. Atrial fibrillation is a third factor that has been claimed to be associated with CSR [16,18,22], but this could not be confirmed in our study. One reason for the discrepancy may be that the patients in the studies of Sin [16] and Javaheri [18] were considerably younger than our patients, with a lower prevalence of atrial fibrillation. Thus their findings may have been confounded by an age effect. However the study of Blackshear [22] with a non-selected sample of elderly heart failure patients with a high prevalence of atrial fibrillation demonstrates a strong relationship between atrial fibrillation and CSR. The reason for the discrepancy between their results and our present study is not obvious. Concomitant diseases Patients with a history of stroke are usually excluded from studies on CSR and heart failure. This may be a limitation of the external validity of such studies, since many patients with heart failure (18% in our material) also have a history of minor or major cerebrovascular disease. We found however, quite unexpectedly, that this was not associated with a higher occurrence of CSR, thereby corroborating the findings of Blackshear et al [22]. Neither was any other concomitant disease associated with CSR. There was no association between intake of hypnotics and CSR. Conclusions Although presence of Cheyne-Stokes respiration indicates presence of left ventricular dysfunction, its severity seems only weakly related to severity of heart failure. Age was a stronger determinant, which may reflect the underlying age-dependency found also in healthy subjects. Follow-up of the current patient cohort will be performed, but from the present data, we cannot conclude if Cheyne-Stokes respiration is of clinical importance or not. Authors' contributions LM coordinated and performed the study which was designed by CC and BM. LE and CC were responsible for the cardiac investigations and SB and BM for the interpretation of the nocturnal respiratory recordings. Acknowledgements Financial support has in part been provided by the Swedish Heart and Lung Foundation and by a research grant from Aga Gas AB, Sweden. ==== Refs Javaheri S Parker TJ Wexler L Michaels SE Stanberry E Nishyama H Roselle GA Occult sleep-disordered breathing in stable congestive heart failure Ann Intern Med 1995 122 487 492 7872582 Tremel F Pépin J-L Veale D Wuyam B Siché J-P Mallion JM Lévy P High prevalence and persistence of sleep apnoea in patients referred for acute left ventricular failure and medically treated over 2 months Eur Heart J 1999 20 1201 1209 10448029 10.1053/euhj.1999.1546 Lanfranchi PA Braghiroli A Bosimini E Mazzuero G Colombo R Donner CF Giannuzzi P Prognostic value of nocturnal Cheyne-Stokes respiration in chronic heart failure Circulation 1999 99 1435 1440 10086966 Hanly PJ Zuberi-Khokhar NS Increased mortality associated with Cheyne-Stokes respiration in patients with congestive heart failure Am J Respir Crit Care Med 1996 153 272 276 8542128 Köhnlein T Welte T Tan LB Elliott MW Central sleep apnoea syndrome in patients with chronic heart disease: a critical review of the current literature Thorax 2002 57 547 554 12037232 10.1136/thorax.57.6.547 Andreas S Hagenah G Möller C Werner GS Kreuzer H Cheyne-Stokes respiration and prognosis in congestive heart failure Am J Cardiol 1996 78 1260 1264 8960586 10.1016/S0002-9149(96)00608-X Hanly P Zuberi-Khokhar N Daytime sleepiness in patients with congestive heart failure and Cheyne-Stokes respiration Chest 1995 107 952 958 7705160 The Task Force on Heart Failure of the European Society of Cardiology Guidelines for the diagnosis of heart failure Eur Heart J 1995 16 741 751 7588917 Alam M Höglund C Thorstrand C Hellekant C Hemodynamic significance of the atrioventricular plane displacement in patients with coronary disease Eur Heart J 1992 13 194 200 1555616 Alam M Höglund C Thorstrand C Longitudinal systolic shortening of the left ventricle: An echocardiographic study in subjects with and without preserved global function Clin Physiol 1992 12 443 452 1505166 Willenheimer R Cline C Erhardt L Israelsson B Left ventricular atrioventricular plane displacement: An echocardiographic technique for rapid assessment of prognosis in heart failure Heart 1997 78 230 236 9391283 Emsellem HA Corson WA Rappaport BA Hackett S Smith LG Hausfeld JN Verification of sleep apnea using a portable sleep apnea screening device South Med J 1990 83 748 752 2371595 Redline S Tosteson T Boucher MA Millman RP Measurement of sleep-related breathing disturbances in epidemiologic studies. Assessment of the validity and reproducibility of a portable monitoring device Chest 1991 100 1281 1286 1935282 Ancoli-Israel S Engler RL Friedman PJ Klauber MR Ross PA Kripke DF Comparison of patients with central sleep apnea. With and without Cheyne-Stokes respiration Chest 1994 106 780 786 8082359 American Academy of Sleep Medicine Task Force Sleep-related breathing disorders in adults: recommendations for syndrome definition and measurement techniques in clinical research Sleep 1999 22 667 689 10450601 Sin DD Fitzgerald F Parker JD Newton G Floras JS Bradley TD Risk factors for central and obstructive sleep apnea in 450 men and women with congestive heart failure Am J Respir Crit Care Med 1999 160 1101 1106 10508793 Mortara A Sleight P Pinna GD Maestri R Capomolla S Febo O La Rovere MT Cobelli F Association between hemodynamic impairment and Cheyne-Stokes respiration and periodic breathing in chronic stable congestive heart failure secondary to ischemic or idiopathic dilated cardiomyopathy Am J Cardiol 1999 84 8 900 904 10532507 10.1016/S0002-9149(99)00462-2 Javaheri S Parker TJ Liminy JD Sleep apnea in 81 ambulatory male patients with stable heart failure: types and their prevalences, consequences, and presentations Circulation 1998 97 2154 2159 9626176 Staniforth AD Kinnear WJM Starling R Cowley AJ Nocturnal desaturation in patients with stable heart failure Heart 1998 79 394 399 9616350 Alex CG Önal E Lopata M Upper airway occlusion during sleep in patients with Cheyne-Stokes respiration Am Rev Respir Dis 1986 133 42 45 3079976 Tkacova R Niroumand M Lorenzo-Filho G Bradley TD Overnight shift from obstructive to central apneas in patients with heart failure. Role of PCO2 and circulatory delay Circulation 2001 103 238 243 11208683 Blackshear JL Kaplan J Thompson RC Safford RE Atkinson EJ Nocturnal dyspnea and atrial fibrillation predict Cheyne-Stokes respirations in patients with congestive heart failure Arch Intern Med 1995 155 1297 1302 7778961 10.1001/archinte.155.12.1297 Bixler EO Vgonzas AN Ten Have T Tyson K Kales A Effects of age on sleep apnea in men. 1. Prevalence and severity Am J Respir Crit Care Med 1998 157 144 148 9445292 Tkacova R Hall MJ Liu PP Fitzgerald FS Bradley TD Left ventricular volume in patients with heart failure and Cheyne-Stokes respiration during sleep Am J Respir Crit Care Med 1997 156 1549 1555 9372674

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==== Front Health Qual Life OutcomesHealth and Quality of Life Outcomes1477-7525BioMed Central London 1477-7525-2-481536125210.1186/1477-7525-2-48ResearchHealth-related quality of life as a predictor of pediatric healthcare costs: A two-year prospective cohort analysis Seid Michael [email protected] James W [email protected] Darron [email protected] Paul S [email protected] RAND Health, 1700 Main Street, M-28, Santa Monica, California, 90407, USA2 Department of Landscape Architecture and Urban Planning, College of Architecture Texas A&M University, 3137 TAMU, College Station, Texas 77843, USA3 Department of Pediatrics, College of Medicine, Texas A&M University, 3137 TAMU, College Station, Texas 77843, USA4 MHS., San Diego, California, USA5 Center for Child Health Outcomes, 3020 Children's Way, San Diego, CA, 92123, USA2004 10 9 2004 2 48 48 30 6 2004 10 9 2004 Copyright © 2004 Seid et al; licensee BioMed Central Ltd.2004Seid et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background The objective of this study was to test the primary hypothesis that parent proxy-report of pediatric health-related quality of life (HRQL) would prospectively predict pediatric healthcare costs over a two-year period. The exploratory hypothesis tested anticipated that a relatively small group of children would account for a disproportionately large percent of healthcare costs. Methods 317 children (157 girls) ages 2 to 18 years, members of a managed care health plan with prospective payment participated in a two-year prospective longitudinal study. At Time 1, parents reported child HRQL using the Pediatric Quality of Life Inventory™ (PedsQL™ 4.0) Generic Core Scales, and chronic health condition status. Costs, based on health plan utilization claims and encounters, were derived for 6, 12, and 24 months. Results In multiple linear regression equations, Time 1 parent proxy-reported HRQL prospectively accounted for significant variance in healthcare costs at 6, 12, and 24 months. Adjusted regression models that included both HRQL scores and chronic health condition status accounted for 10.1%, 14.4%, and 21.2% of the variance in healthcare costs at 6, 12, and 24 months. Parent proxy-reported HRQL and chronic health condition status together defined a 'high risk' group, constituting 8.7% of the sample and accounting for 37.4%, 59.2%, and 62% of healthcare costs at 6, 12, and 24 months. The high risk group's per member per month healthcare costs were, on average, 12 times that of other enrollees' at 24 months. Conclusions While these findings should be further tested in a larger sample, our data suggest that parent proxy-reported HRQL can be used to prospectively predict healthcare costs. When combined with chronic health condition status, parent proxy-reported HRQL can identify an at risk group of children as candidates for proactive care coordination. Health-related quality of lifePedsQL™predictionhealthcare costsmanaged care. ==== Body Background Predicting healthcare costs for pediatric populations has been challenging [1]. Although population-based risk prediction and case-mix adjustment can be used to inform policy, set rates, and compare outcomes across providers [2], a more immediate concern for healthcare providers is to clinically manage their enrolled population. In a prospective payment system with predetermined funding limits, providers must be able to proactively case-manage those enrollees at greatest risk of poor health while remaining within designated budget constraints. If healthcare providers knew in advance – for example at the time of health plan enrollment – which children were at the greatest risk for future health problems, then healthcare resources could be proactively targeted to those children in order to minimize or prevent morbidity and associated healthcare costs. Researchers working with adult populations have linked health status with several important outcomes. In general populations, self-reported health status has been shown to be a predictor of future health services charges [3], the use of physician services and mortality in working-age adults [4], and of frailty in the elderly [5]. For chronically ill adults, self-rated health status is an independent predictor of physiologic health in diabetes and hypertension[6], and self-reported quality of life is an independent predictor of survival in cancer patients [7]. For the hospitalized elderly, functional status [8-10] and depressive symptoms [11] have been shown to be predictive of resource utilization and mortality. Several researchers have demonstrated that both diagnostic information and self-rated health status are associated with costs for general adult populations [12,13]. In pediatric populations, diagnosis-based classification systems have achieved some degree of association with healthcare costs [14,15]. However, there remain limitations with current pediatric healthcare cost prediction methods, including the underestimation of healthcare costs for chronically ill children [14]. The ideal pediatric cost prediction model for clinical management would predict healthcare costs proactively in those patients at greatest risk. Increasingly, health-related quality of life (HRQL) has become recognized as an important health outcome, some contend the most important outcome in child health services research [16-18]. Researchers have made great strides in conceptualizing and measuring HRQL for children [19-27]. HRQL has been shown to be responsive to treatment in children with rheumatic disease [28], and to be related to treatment status in children with cancer [20,29], to chronic health condition status in a general sample [19], to severity of illness within children with cardiac diagnoses [30], and to parent reports of primary care quality [31] and barriers to care [32]. Measuring HRQL in large populations has several distinct benefits. It can aid in identifying subgroups of children who are at-risk for health problems [33], in determining the burden of a particular disease or disability [34], and, at least in general populations, in informing efforts aimed at prevention and intervention [13]. While self-report is considered the standard for measuring perceived HRQL as an outcome, it is typically parents' perceptions of their children's HRQL that influence healthcare utilization [35,36]. Consequently, the objective of this study was to test the primary hypothesis that parent proxy-report of pediatric HRQL would prospectively predict pediatric healthcare costs over a two-year period. The exploratory hypothesis tested anticipated that a relatively small group of children would account for a disproportionately large percent of healthcare costs. Method Participants and Settings The study took place in San Diego, California between January 1998 and December 2000. We recruited members of a 50,000-member federally supported (Medicaid) managed care health plan. Additional inclusion criteria were that children be between 2 and 18 years of age and that the parent be able to speak either English or Spanish. We exclude children under 2 years of age because the PedsQL™ does not assess parent proxy-report HRQOL below age 2 years. In order to maximize the heterogeneity of the sample, subjects were recruited from three types of healthcare settings: children presenting at pediatricians' offices for scheduled well-child checks (n = 18, 5.7%), children at one of two hospital specialty clinics – orthopedics (n = 6, 1.9%) and cardiology (n = 7, 2.1%) – or children who had been seen at the hospital or its outpatient clinics at least three months previously (n = 286, 90.3%). The data reported here were collected as part of the initial field test to assess the reliability and validity of the PedsQL™ 4.0 Generic Core Scales [19]. Only pediatric patients reported being members of the federally supported managed care health plan are included in the current data analysis. Measures The PedsQL™ 4.0 (Pediatric Quality of Life Inventory™ 4.0) Generic Core Scales The PedsQL™ 4.0 Generic Core Scales [19] were designed to measure the core physical, mental and social health dimensions as delineated by the World Health Organization [37], and to additionally include role (school) functioning. The 23-item PedsQL™ 4.0 encompasses both physical functioning (8 items) and psychosocial (emotional, social, role) functioning (15 items) and is comprised of parallel child self-report and parent proxy-report formats. The parent proxy-report form is designed to assess the parent's perceptions of their child's HRQL. Parent proxy-report includes ages 2–4 (toddler), 5–7 (young child), 8–12 (child), and 13–18 (adolescent). Higher PedsQL™ 4.0 scores indicate better HRQL. To create Scale Scores, the mean is computed as the sum of the items divided by the number of items answered (this accounts for missing data). If more than 50% of the items in the scale are missing, the Scale Score is not computed. Imputing the mean of the completed items in a scale when 50% or more are completed is generally the most unbiased and precise method [38]. Because parent proxy-report of HRQL has been shown to be related to utilization [35,36], we used only the parent proxy-report Physical Functioning and Psychosocial Functioning Summary Scales of the PedsQL™ 4.0 in the current investigation. Chronic health condition status Parents were asked to report on the presence of a chronic health condition for their child. They read the following statement: "A chronic health condition is: (1) a physical or mental health condition (2) that has lasted or is expected to last at least 6 months and (3) interferes with your child's activities." They then responded with yes or no to the question "In the past 6 months, has your child had a chronic health condition?" If yes, the parents were asked to identify the name of the chronic health condition. Parents who answered yes or who gave the name of a chronic health condition were coded as having a child with a chronic health condition. This method has been used in previous work [19,31], and the PedsQL™ 4.0 scores for the two groups defined using this method (with and without chronic health condition) are very similar to those observed in other studies [33]. Healthcare Costs Healthcare costs were calculated as the dollar amount paid by the health plan per patient. We first determined patients' eligibility from the health plan's eligibility data files for three consecutive cumulative periods: 0–6 months, 0–12 months, and 0–24 months after the date they completed the PedsQL™ 4.0. A pediatric patient was considered eligible for health plan benefits for those periods if they were eligible for at least 5 months out of the 6-month period. We then electronically captured healthcare costs (the dollar amount paid by the health plan) for each pediatric patient for those periods in which they were eligible. We did this by matching each eligible pediatric patient with the health plan's existing databse of claims and encounter data. These data include the dollar amount spent by the health plan. Healthcare costs included hospital and emergency room costs, professional fees, durable medical equipment, home health, specialty clinic, and primary care costs. We did not have access to pharmacy or mental health costs. In California, the site of the study, treatment for 22 specific diagnoses is "carved out," or paid through a separate program (called California Children's Services; CCS) regardless of a child's health plan membership. Thus, for health plans in California, treatment of CCS-covered diagnoses might not be measured in calculating utilization. However, because California's carve out may differ from other states' methods of financing treatment for these diagnoses, and to more completely describe healthcare costs, we included the costs for procedures covered by CCS in our healthcare costs calculations. To derive these costs, we linked the procedure codes on the health plan's CCS referral with the federally supported health plan's fee schedule. These data thus represent the dollar amount the health plan would have spent had the services not been carved out. Procedure A convenience sample – subects were recruited nonsystematically when research assistants were available – was recruited at pediatrician offices and specialty clinics. These pediatric patients were identified through examination of the clinic appointment schedules. At these sites, parents of children identified as possible study participants were informed of the study by one of the research assistants after checking in for their appointment, but before being seen by their healthcare provider. Written informed consent included permission for the researchers to examine the medical record to assess utilization. After written informed consent was obtained, the parent completed the proxy-report version of the PedsQL™ 4.0. The research assistant was available at all times to answer any questions. A random sample was recruited from children and adolescents ages 2–18 years who had been seen as inpatients or outpatients at Children's Hospital and Health Center between April 1 and June 30, 1998, and who were members of the health plan. This sample excluded children with a discharge status of expired, children whose payer was from the victim/witness fund, and children whose parents had requested their phone number and address to be kept private. Research assistants called parents of children on this list and obtained verbal informed consent. The research assistant verbally administered the PedsQL™ 4.0 to parents. This research protocol was approved by the institutional review board at Children's Hospital and Health Center, San Diego (#98-020). Statistical analysis We pooled the data from the two samples. Previous reasearch on the PedsQL™ has documented the lack of mode of administration effects [19,20]. In order to test the primary hypothesis that HRQL would prospectively predict healthcare costs, multiple linear regression analyses were conducted. We examined the association between age, gender, chronic health condition status (variables typically used by health plans to predict risk), and PedsQL™ 4.0 scores with healthcare costs at each of the three cumulative follow-up periods. We did not use socioeconomic status, as eligibility criteria for membership in the health plan requires families to have incomes below a certain level, and this restricts the range of this variable. Four models were constructed for each follow-up period. Model 1 included age and gender only, Model 2 included age, gender, and chronic health condition status, Model 3 included age, gender, and PedsQL™ 4.0 scores, and Model 4 included age, gender, chronic health condition status, and PedsQL™ 4.0 scores. We report the adjusted R2, a measure of the percent of variance in the dependent variable accounted for by the predictor variables while adjusting for the complexity of the model, and the standardized regression coefficient, or beta, for each predictor. PedsQL™ 4.0 scores were skewed toward the high end of the scale and were transformed by taking the square root of the reverse of the score (sqrt(100-score)) in order to create a more normal distribution. The distribution of cost data was skewed to the lower end, with many children having little cost and a relatively smaller number of children having high costs. These data were normalized by taking the log of the costs. In order to explore whether HRQL and chronic health condition status together would define a relatively small subset of enrollees who accounted for a disproportionately large percent of healthcare costs, we divided the sample into quintiles based on the PedsQL™ 4.0 Physical Functioning Scale score and into two groups based on chronic health condition status. Those children who fell in the lowest PedsQL™ 4.0 quintile and who reported the presence of a chronic health condition were assigned to the high-risk group. We describe the percent of costs, per member costs, and per member per month costs per child accounted for by this high risk group. Results Descriptive Statistics Data was collected from the parents of 317 children (157 girls, 160 boys) ages 2 to 18 years. The average age of the children was 8.3 years (SD = 4.14) with a range of 2.03 to 17.13 years. The sample was heterogeneous with respect to race/ethnicity, with 76 (25.4%) White non-Hispanic, 155 (51.8%) Hispanic, 39 (13.0%) Black non-Hispanic, 6 (2.0%) Asian/Pacific Islander, 3 (1.0%) American Indian or Alaskan Native, 20 (6.7%) Other, and 19 (6.0%) missing. With respect to mother's education, 36.4% had less than a high school education, 46.6% had a high school diploma or some college, and 7.0% were college graduates or beyond (18.8% missing). The measures were administered in two languages – English (n = 233, 73.6%) and Spanish (n = 84, 26.4%). The sample represented both chronically ill (n = 102, 32.1%) and healthy children (n = 215, 67.9%), based on parent report of the presence of a chronic health condition. Table 1 presents the chronic health conditions reported by parents for the high risk group and the non-high risk group. Table 1 Parent-reported chronic health conditions, by high risk group status. High risk group? Total no yes name of condition None 223 0 223 ADHD 7 1 8 allergies 1 0 1 arthritis 2 0 2 asthma 25 8 33 autism 2 1 3 back and leg pain 0 1 1 Battan disease 0 1 1 bi-polar 1 0 1 breathing problems 1 0 1 bronchitis 2 0 2 cancer 1 0 1 cerebral palsy 2 2 4 chronic ear infections 2 0 2 cough 2 0 2 CSF leak 0 1 1 depression 1 0 1 developmental delay 1 0 1 dislocated shoulder 0 1 1 doesn't produce salt 1 0 1 Down Syndrome 1 0 1 ear problems 0 1 1 epilepsy 1 0 1 gallbladder problems 1 0 1 gastrointestinal problem 1 0 1 hearing 1 0 1 heart murmur 2 0 2 heart problem 1 1 2 kidney reflux 1 0 1 leukemia 1 0 1 May Hegglin syndorme 1 0 1 migraine headaches 0 1 1 muscle condition 0 1 1 muscular dystrophy 0 1 1 osteomyelitis 1 0 1 pierre robyn syndrome 1 0 1 reactive airway disease 1 0 1 Rett syndrome 1 0 1 scleroderma 0 1 1 scoliosis 0 1 1 seizures 3 1 4 speech 1 0 1 spina bifida 0 2 2 stomach aches 1 0 1 stomach problems 1 0 1 unknown genetic syndrome 0 1 1 urinary tract infections 1 0 1 There were no differences found in PedsQL™ scores between the group sampled at well-child checks or specialty clinics and that sampled by phone. All 317 children were enrolled in the health plan after 6 months, with 314 (99.0%) enrolled after 12 months, and 244 (76.9%) after 24 months. There were no differences between those enrolled versus not enrolled at 24 months in percent with a chronic health condition, race/ethnicity, mother's education, or PedsQL™ scores. The cost per member per month (pmpm) for this sample, which represents the total cost divided by the number of members divided by the number of months enrolled, was $149 at 6 months, $137 at 12 months, and $115 at 24 months. The sample included 4,954 claims (there are multiple claims in a single clinical encounter) over the 24 months. The largest category of visits was for upper respiratory infections (URIs) and related infections (10.96%). Asthma, other infections, otitis media, and pain each account for 5 to 6% of visits, with acute orthopedic conditions accounting for 2.6% of visits. These most common diagnoses account for more than a third (38.7%) of the visits, the rest is comprised of a large number of relatively low-frequency diagnoses. This distribution of diagnoses is similar to the epidemiology of childhood illness, in that much of pediatric morbidity is accounted for by a large number of relatively low frequency diagnoses [39,40]. Table 2 displays the descriptive statistics for the PedsQL™ 4.0 parent proxy-report at Time 1. Consistent with previous PedsQL™ 4.0 findings, [19] chronically ill children had lower HRQL scores than healthy children (Table 2). Table 2 Descriptive Statistics for PedsQL™ 4.0 scores N Mean SD Minimum Maximum t^ df p Total Sample  Total Scale Score 316 84.38 13.67 25.00 100.00  Physical Functioning 316 85.39 19.87 0.00 100.00  Psychosocial Functioning 317 83.78 13.73 32.14 100.00 Children with Chronic Health Condition  Total Scale Score 101 79.26 16.66 25.00 100.00 -4.73 313 0.001  Physical Functioning 100 79.69 25.00 0.00 100.00 -3.59 313 0.001  Psychosocial Functioning 102 79.27 16.59 32.14 100.00 -4.12 314 0.001 Children without Chronic Health Condition  Total Scale Score 214 86.82 11.29 44.44 100.00  Physical Functioning 215 88.17 16.30 10.00 100.00  Psychosocial Functioning 214 85.91 11.61 48.33 100.00 ^comparing chronically ill to healthy children Multiple regression analysis Table 3 displays the results of the multiple regression analyses predicting healthcare costs for 6, 12, and 24 month follow-up. As can be seen, Model 1, with age and gender as the only predictors variables, did not account for significant variance in costs. Model 2 shows that age and gender, with chronic health condition status accounted for an increasing percentage of costs as the follow-up time lengthened. This pattern holds true as well for Model 3, which included age, gender, and the PedsQL™ 4.0 scores. Model 4, comprised of age, gender, chronic health condition status, and PedsQL™ 4.0 scores, accounted for the most variance, explaining 10.1% 14.4% and 21.2% of the variance in healthcare costs at 6, 12, and 24 month follow-up intervals. Inspection of the standardized regression coefficients for each predictor in Model 4 shows that, of the four predictors used, chronic health condition status and the PedsQL™ 4.0 Physical Functioning Scale scores consistently accounted for the greatest amount of variance. Table 3 Adjusted R-square (in bold) and standardized regression coefficients (betas) for models predicting costs at 6, 12, and 24 months Follow up 6 Months (N = 318) 12 Months (N = 315) 24 Months (N = 245) Model 1 Adjusted R-square 0.010 0.001 0.004  Beta   Age -0.067 -0.037 -0.030   Gender .11 0.063 0.108 Model 2 Adjusted R-square 0.049** 0.066*** 0.130***  Beta   Age -0.063 -0.031 0.008   Gender 0.118* 0.076 0.115   Chronic Health Condition 0.180** 0.265*** 0.360*** Model 3 Adjusted R-square 0.089*** 0.103*** 0.122***  Beta   Age -0.103 -0.091 -0.079   Gender 0.080 0.030 0.076   PedsQL Physical Functioning 0.291*** 0.353*** 0.379***   PedsQL Psychosocial Functioning 0.004 0.057 0.081 Model 4 Adjusted R-square 0.101*** 0.144*** 0.212***  Beta   Age 0.098 -0.083 -0.043   Gender 0.086 0.040 0.080   Chronic Health Condition 0.126+ 0.214*** 0.312***   PedsQL Physical Functioning 0.275*** 0.326*** 0.340***   PedsQL Psychosocial Functioning 0.025 0.093 0.123+ + = p < 0.05; * = p < 0.01; *** = p < 0.001; *** = p < 0.0001; R2 = percent variance accounted for. Defining the high risk group We used the two variables accounting for most of the variance in the regression analysis – the PedsQL™ 4.0 Physical Functioning scores and chronic health condition status – to describe the percentage of costs accounted for by different groups of children. In order to create a single denominator for the percentages, we used the 241 children continuously enrolled in the health plan with complete data for this set of analyses. To create quintiles, we determined the values that divided the sample into five equal-sized groups based on PedsQL™ 4.0 Physical Functioning Scale scores. Enrollees with a score of less than 75 on the PedsQL™ 4.0's 0–100 scale were in the first quintile (N = 51; 21.0%). The second quintile (N = 45; 18.5%) was bounded by the scores 75.0 to 90.624, the third quintile (N = 48; 19.6%) by the scores 90.625 to 96.874, the fourth quintile by the scores 96.875 to 100 (N = 18; 7.3%), and the fifth quintile consisted of enrollees scoring 100 (N = 81; 33.4%). Because the distribution of these PedsQL™ 4.0 scores was skewed, we combined the fourth and fifth quintiles (N = 99; 40.7%; 2 missing). Table 4 shows the percentage of total costs accounted for by children across PedsQL™ 4.0 Physical Functioning Scale quintiles and chronic health condition status, for the three cumulative follow up periods. As can be seen, children in the high risk group (the subset of chronically ill children in the lowest quintile), account for a disproportionately large share of healthcare costs. This group, comprising just 8.7% of the sample, accounted for 37.42% of the healthcare costs over six months, 59.16 % of costs over 12 months, and 61.74% of costs over 24 months. Table 4 Percent of cost at each follow up period, by PedsQL quintile and chronic health condition status (n = 241) PedsQL Physical Functioning Quintile Lowest quintile (N = 51) 2nd quintile (N = 45) 3rd quintile (N = 48) 4th and 5th quintiles (N = 99) Chronic Health Condition Chronic Health Condition Chronic Health Condition Chronic Health Condition Yes (N = 21) No (N = 30) Yes (N = 18) No (N = 27) Yes (N = 12) No (N = 36) Yes (n = 26) No ( = 73) % of cost, 6 months 37.43 9.35 26.76 9.30 0.33 6.95 7.59 2.30 % of cost, 12 months 59.16 4.70 20.56 4.54 0.30 3.63 5.25 1.85 % of cost, 24 months 61.74 3.38 22.06 2.86 0.41 2.64 4.87 2.03 Bold = High-risk group Table 5 shows the total costs, the per member costs, and the per member per month (pmpm) costs for the high risk group and the not high risk group over the three follow-up periods. As can be seen, the high risk group was an extremely costly subset of enrollees for each of the cumulative 6 month periods, as measured by total, per member, or pmpm costs. Pmpm costs were quite disparate between the high risk group and other enrollees. For the high risk group at 6 months, pmpm was $432 (vs. $66 for the other patients), at 12 months pmpm was $809 (vs. $61), and at 24 months, pmpm was $722 (vs. $60). Table 5 Total costs, per member costs, and per member per month costs for high-risk* (N = 21) and not high-risk (N = 231) enrollees. Total Per member Per member per month Time Period High-risk Not high-risk High-risk Not high-risk High-risk Not high-risk 6 months $54,493 $91,484 $2,595 $396 $432 $66 12 months $203,875 $168,022 $9,708 $727 $809 $61 24 months $363,822 $330,846 $17,325 $1,432 $722 $60 *High risk is defined as parent reported chronic health condition and scoring in lowest quintile on PedsQL™ 4.0 Discussion This study tested the primary hypothesis that HRQL could prospectively predict healthcare cost in pediatric patients in a managed care environment. We measured age, chronic health condition status, and PedsQL™ 4.0 scores at Time 1, and prospectively measured utilization, via costs based on claims and encounter data, for three cumulative periods. These data demonstrate that parent-reported HRQL, as measured by the PedsQL™ 4.0, and chronic health condition status each accounted for significant variance in healthcare costs over 6, 12, and 24 months. The data further show how these two predictor variables, chronic health condition status, and PedsQL™ 4.0 Physical Functioning scores, define a relatively small group of enrollees that accounted for a large percentage of total healthcare costs. This high risk group displays disproportionately high costs as early as 6 months, and their pmpm costs peak at one year. This suggests the importance of managing high risk enrollees as soon as they are identified, perhaps as early as their initial enrollment. It also implies the potential for significant return on investment for better case management, even in the first six months of enrollment. The high risk group's costs remain disproportionately high throughout the 24 months of the study. This fact suggests that the method used here for identifying the high risk group succeeded in identifying children with high ongoing care needs and costs, as opposed to children with one-time health care needs. An anomalous finding was that children in the third quintile on PedsQL™ scores who had chronic health conditions were, for an unexplained reason, much less costly than their peers. It is worth comparing the mean PedsQL™ 4.0 scores for the high risk group to other published data. The high risk group had scores of 44.5 for the Physical Functioning Scale, 70.7 for the Psychosocial Summary Scale, and 61 for the Total Scale. This is placed in clinical perspective by other data showing that scores for children with cancer, in active treatment, are 65, 68, and 67 for the Physical, Psychosocial and Total scales, respectively. [31]. A hypothetical example is presented to illustrate the potential impact of these findings. In a typical health plan, the rate of chronic health conditions will most likely be between 5% [41] and 18% [42], rather than the 31.4% rate we found by selecting our sample, in part, from hospital specialty clinics. We will further conservatively assume that one-fifth (20%) of children with chronic health conditions would fall in the lowest quintile on the PedsQL™ 4.0. If this were so, then between 1% (5% chronic health condition × 20% in the lowest quintile) and 3.6% (18% chronic health condition × 20% in the lowest quintile) of enrollees in a health plan would fall into the high risk group. Thus, in a hypothetical medium to large health plan with 50,000 pediatric enrollees, the high risk group would be comprised of anywhere from 500 (1% of 50,000) to 1800 (3.6% of 50,000) children. Using the costs figures from this sample ($722 pmpm), this hypothetical high risk group represents between $8.6 and $31.2 million in costs over the course of 24 months. This example relies on speculation and is intended as a hypothetical case, for illustrative purposes only. Taken together, these findings represent an alternative method toward the prospective prediction of healthcare costs in pediatric federally supported managed care populations. While a percentage of these identified costs are inevitable, due to the costs of appropriate care for these chronically ill, poorly functioning children, the possibility exist that a proportion of these healthcare costs are avoidable. Evidence-based disease management has been shown to reduce healthcare costs and increase HRQL in certain chronic conditions such as asthma [43,44]. By identifying at-risk children with low PedsQL™ 4.0 scores, targeted interventions may avert certain future healthcare costs by ameliorating impaired HRQL when first identified. Certain limitations exist in this study. The first has to do with data not accounted for in this study. We did not have access to pharmacy and mental health costs, nor did we have access to out-of-plan expenditures. For mental health costs, however, recent data has shown that children referred for psychiatric services demonstrate child self-report and parent proxy-report PedsQL™ 4.0 Total Scale Scores comparable to children with chronic physical health conditions [45]. Those data suggest that this methodology may be useful in predicting mental health costs as well. We did not include children under the age of two. Neonatal intensive care, for example, is a large percent of the costs for Medicaid managed care plans [46]. The portion of health plan costs devoted to caring for children under two is not explored here. However, many of these costs cannot be avoided, and preventive efforts for these costs are most appropriately targeted at the prenatal and perinatal periods. Finally, we did not compare the performance of these variables to that of existing administrative risk adjustment methods currently available. The second limitation has to do with sampling issues and with generalizing these data beyond this study. Our data was not collected at enrollment, nor did we sample from the entire pool of enrollees. Further research is necessary to determine whether these findings hold true for children assessed at health plan enrollment, and to determine the extent to which the results may be influenced by the convenience sample used here. The sample was too small to use cross-validation techniques. Prediction models tend to overfit the development sample, and the predictive validity of these variables should be tested in other, larger samples. Although generalization of these findings to broader populations should be made with caution, the sample here is very diverse with respect to race/ethnicity, and thus likely to be similar to other federally support health plans. We also combined data from two different samples – specialty clinic patients, and health plan members who had been seen in the hospital or outpatient clinics at least three months after the clinical encounter. These two groups could have had unmeasured systematic differences, which could have biased the results. The third has to do with using a survey to gather these data. The costs of fielding a survey can be quite high, and, if tied to payment, survey responses are subject to "gaming". However, we submit that the potential gains from optimal management of an enrolled population will almost certainly be greater than the costs of survey administration. Moreover, while gaming might occur if health plans were to be compensated based on the HRQL of their enrolled population, the methods used here are suggested as strategies for clinical management, not for rate setting, thus reducing the incentives for gaming. We did not track refusal rates and so do not know what percent of potential participants consented to be in the study. Finally, using a survey means that parents reported on their children's chronic health condition information. Objective measures of chronic health condition would strengthen the validation process. However, in previous PedsQL™ 4.0 clinical research in pediatric patients with cancer, cardiac and rheumatic chronic health conditions, objective medical diagnosis of these chronic diseases demonstrated similar differences between healthy children and children with chronic health conditions as shown in the present findings [28-30]. Further research is necessary. First, a much larger, and randomly selected sample is necessary to confirm these results. Second, split-half validation should be performed so that the coefficients from one group are used to predict the costs in a different group. This could be done with split halves of one large group, or with two similar groups enrolled at different points in time. Given that our regression equation explains 21% of costs, further studies could be done to determine whether other variables might account for additional variance in costs. Further studies could also allow comparison and validation with the results here. Conclusion This is the first study we are aware of to use parent reports of pediatric HRQL and chronic health condition status to prospectively predict healthcare costs in a pediatric sample. These data have implications for healthcare decision makers such as pediatricians, health plan administrators, and policymakers. In a prospective payment system, providers are incentivized to actively manage high-risk patients and to provide care at the appropriate level. The idea behind such a system is that prevention and appropriate care accrues benefits to patients in the form of better health and to providers in the form of lower costs. If, as these data suggest, parent reports of HRQL can be used to predict healthcare costs, one could identify at-risk children proactively and intervene to avoid both illness and costs. In this way, these data can serve simultaneously to improve the health of children and the system that serves them. List of Abbreviations HRQL Health-related Quality of Life PedsQL™ 4.0 Pediatric Quality of Life Inventory™, Version 4.0 SD Standard Deviation CCS California Children's Services URI Upper respiratory infection PMPM per member per month Authors' Contributions MS, PSK, and JWV conceived of the research. MS and JWV supervised the data collection. MS and DS performed the data analysis. MS drafted the manuscript. JWV, PSK, and DS provided substantive input into the manuscript. All authors read and approved the final manuscript. Acknowledgements This research was supported by intramural grants from the Children's Hospital and Health Center, San Diego. The authors would like to thank Robert Kaplan, Ph.D., Richard Kronick, Ph.D., and Theodore Ganiats, MD for their valuable input into previous versions of this manuscript. ==== Refs Newhouse JP Sloss EM W.G. Manning Keeler EB Risk adjustment for a children's capitation rate. Health Care Financing Review 1993 15 39 54 10133708 Iezzoni LI Risk Adjustment for Measuring Health Care Outcomes 2003 3rd Chicago, Health Administration Press Parkerson G. R., Jr. Harrell F. 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Quality of life and pharmacoeconomics in clinical trials 1996 New York, Raven Press 427 435 Stein REK Jessop DJ A noncategorical approach to chronic childhood illness Public Health Reports 1982 97 354 362 7111657 Stein REK Bauman LJ Westbrook LE et al. Framework for identifying children who have chronic conditions: The case for a new definition Journal of Pediatrics 1993 122 342 347 8441085 Newacheck PW Taylor WR Childhood chronic illness: prevalence, severity, and impact Am J Public Health 1992 82 364 371 1536351 Newacheck PW, Strickland B Shonkoff JP Perrin JM McPherson M McManus M Lauver C Fox H Arango P An epidemiologic profile of children with special health care needs Pediatrics 1998 102 117 123 9651423 10.1542/peds.102.1.117 Greineder DK Loane KC Parks P Reduction in resource utilization by an asthma outreach program Arch Pediatr Adolesc Med 1995 149 415 420 7704170 Home-taught pediatric asthma program improves outcomes, cuts hospital, physician visits Health Care Cost Reengineering Rep 1997 2 40 43 10175060 Bastiaansen D Koot HM Bongers IL Varni JW Verhulst FC Measuring quality of life in children referred for psychiatric problems: Psychometric properties of the PedsQL(tm) 4.0 Generic Core Scales Quality of Life Research 2004 13 489 495 15085921 10.1023/B:QURE.0000018483.01526.ab Zupancic JA Richardson DK Lee K McCormick MC Economics of prematurity in the era of managed care Clin Perinatol 2000 27 483 497 10863661

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==== Front Health Qual Life OutcomesHealth and Quality of Life Outcomes1477-7525BioMed Central London 1477-7525-2-481536125210.1186/1477-7525-2-48ResearchHealth-related quality of life as a predictor of pediatric healthcare costs: A two-year prospective cohort analysis Seid Michael [email protected] James W [email protected] Darron [email protected] Paul S [email protected] RAND Health, 1700 Main Street, M-28, Santa Monica, California, 90407, USA2 Department of Landscape Architecture and Urban Planning, College of Architecture Texas A&M University, 3137 TAMU, College Station, Texas 77843, USA3 Department of Pediatrics, College of Medicine, Texas A&M University, 3137 TAMU, College Station, Texas 77843, USA4 MHS., San Diego, California, USA5 Center for Child Health Outcomes, 3020 Children's Way, San Diego, CA, 92123, USA2004 10 9 2004 2 48 48 30 6 2004 10 9 2004 Copyright © 2004 Seid et al; licensee BioMed Central Ltd.2004Seid et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background The objective of this study was to test the primary hypothesis that parent proxy-report of pediatric health-related quality of life (HRQL) would prospectively predict pediatric healthcare costs over a two-year period. The exploratory hypothesis tested anticipated that a relatively small group of children would account for a disproportionately large percent of healthcare costs. Methods 317 children (157 girls) ages 2 to 18 years, members of a managed care health plan with prospective payment participated in a two-year prospective longitudinal study. At Time 1, parents reported child HRQL using the Pediatric Quality of Life Inventory™ (PedsQL™ 4.0) Generic Core Scales, and chronic health condition status. Costs, based on health plan utilization claims and encounters, were derived for 6, 12, and 24 months. Results In multiple linear regression equations, Time 1 parent proxy-reported HRQL prospectively accounted for significant variance in healthcare costs at 6, 12, and 24 months. Adjusted regression models that included both HRQL scores and chronic health condition status accounted for 10.1%, 14.4%, and 21.2% of the variance in healthcare costs at 6, 12, and 24 months. Parent proxy-reported HRQL and chronic health condition status together defined a 'high risk' group, constituting 8.7% of the sample and accounting for 37.4%, 59.2%, and 62% of healthcare costs at 6, 12, and 24 months. The high risk group's per member per month healthcare costs were, on average, 12 times that of other enrollees' at 24 months. Conclusions While these findings should be further tested in a larger sample, our data suggest that parent proxy-reported HRQL can be used to prospectively predict healthcare costs. When combined with chronic health condition status, parent proxy-reported HRQL can identify an at risk group of children as candidates for proactive care coordination. Health-related quality of lifePedsQL™predictionhealthcare costsmanaged care. ==== Body Background Predicting healthcare costs for pediatric populations has been challenging [1]. Although population-based risk prediction and case-mix adjustment can be used to inform policy, set rates, and compare outcomes across providers [2], a more immediate concern for healthcare providers is to clinically manage their enrolled population. In a prospective payment system with predetermined funding limits, providers must be able to proactively case-manage those enrollees at greatest risk of poor health while remaining within designated budget constraints. If healthcare providers knew in advance – for example at the time of health plan enrollment – which children were at the greatest risk for future health problems, then healthcare resources could be proactively targeted to those children in order to minimize or prevent morbidity and associated healthcare costs. Researchers working with adult populations have linked health status with several important outcomes. In general populations, self-reported health status has been shown to be a predictor of future health services charges [3], the use of physician services and mortality in working-age adults [4], and of frailty in the elderly [5]. For chronically ill adults, self-rated health status is an independent predictor of physiologic health in diabetes and hypertension[6], and self-reported quality of life is an independent predictor of survival in cancer patients [7]. For the hospitalized elderly, functional status [8-10] and depressive symptoms [11] have been shown to be predictive of resource utilization and mortality. Several researchers have demonstrated that both diagnostic information and self-rated health status are associated with costs for general adult populations [12,13]. In pediatric populations, diagnosis-based classification systems have achieved some degree of association with healthcare costs [14,15]. However, there remain limitations with current pediatric healthcare cost prediction methods, including the underestimation of healthcare costs for chronically ill children [14]. The ideal pediatric cost prediction model for clinical management would predict healthcare costs proactively in those patients at greatest risk. Increasingly, health-related quality of life (HRQL) has become recognized as an important health outcome, some contend the most important outcome in child health services research [16-18]. Researchers have made great strides in conceptualizing and measuring HRQL for children [19-27]. HRQL has been shown to be responsive to treatment in children with rheumatic disease [28], and to be related to treatment status in children with cancer [20,29], to chronic health condition status in a general sample [19], to severity of illness within children with cardiac diagnoses [30], and to parent reports of primary care quality [31] and barriers to care [32]. Measuring HRQL in large populations has several distinct benefits. It can aid in identifying subgroups of children who are at-risk for health problems [33], in determining the burden of a particular disease or disability [34], and, at least in general populations, in informing efforts aimed at prevention and intervention [13]. While self-report is considered the standard for measuring perceived HRQL as an outcome, it is typically parents' perceptions of their children's HRQL that influence healthcare utilization [35,36]. Consequently, the objective of this study was to test the primary hypothesis that parent proxy-report of pediatric HRQL would prospectively predict pediatric healthcare costs over a two-year period. The exploratory hypothesis tested anticipated that a relatively small group of children would account for a disproportionately large percent of healthcare costs. Method Participants and Settings The study took place in San Diego, California between January 1998 and December 2000. We recruited members of a 50,000-member federally supported (Medicaid) managed care health plan. Additional inclusion criteria were that children be between 2 and 18 years of age and that the parent be able to speak either English or Spanish. We exclude children under 2 years of age because the PedsQL™ does not assess parent proxy-report HRQOL below age 2 years. In order to maximize the heterogeneity of the sample, subjects were recruited from three types of healthcare settings: children presenting at pediatricians' offices for scheduled well-child checks (n = 18, 5.7%), children at one of two hospital specialty clinics – orthopedics (n = 6, 1.9%) and cardiology (n = 7, 2.1%) – or children who had been seen at the hospital or its outpatient clinics at least three months previously (n = 286, 90.3%). The data reported here were collected as part of the initial field test to assess the reliability and validity of the PedsQL™ 4.0 Generic Core Scales [19]. Only pediatric patients reported being members of the federally supported managed care health plan are included in the current data analysis. Measures The PedsQL™ 4.0 (Pediatric Quality of Life Inventory™ 4.0) Generic Core Scales The PedsQL™ 4.0 Generic Core Scales [19] were designed to measure the core physical, mental and social health dimensions as delineated by the World Health Organization [37], and to additionally include role (school) functioning. The 23-item PedsQL™ 4.0 encompasses both physical functioning (8 items) and psychosocial (emotional, social, role) functioning (15 items) and is comprised of parallel child self-report and parent proxy-report formats. The parent proxy-report form is designed to assess the parent's perceptions of their child's HRQL. Parent proxy-report includes ages 2–4 (toddler), 5–7 (young child), 8–12 (child), and 13–18 (adolescent). Higher PedsQL™ 4.0 scores indicate better HRQL. To create Scale Scores, the mean is computed as the sum of the items divided by the number of items answered (this accounts for missing data). If more than 50% of the items in the scale are missing, the Scale Score is not computed. Imputing the mean of the completed items in a scale when 50% or more are completed is generally the most unbiased and precise method [38]. Because parent proxy-report of HRQL has been shown to be related to utilization [35,36], we used only the parent proxy-report Physical Functioning and Psychosocial Functioning Summary Scales of the PedsQL™ 4.0 in the current investigation. Chronic health condition status Parents were asked to report on the presence of a chronic health condition for their child. They read the following statement: "A chronic health condition is: (1) a physical or mental health condition (2) that has lasted or is expected to last at least 6 months and (3) interferes with your child's activities." They then responded with yes or no to the question "In the past 6 months, has your child had a chronic health condition?" If yes, the parents were asked to identify the name of the chronic health condition. Parents who answered yes or who gave the name of a chronic health condition were coded as having a child with a chronic health condition. This method has been used in previous work [19,31], and the PedsQL™ 4.0 scores for the two groups defined using this method (with and without chronic health condition) are very similar to those observed in other studies [33]. Healthcare Costs Healthcare costs were calculated as the dollar amount paid by the health plan per patient. We first determined patients' eligibility from the health plan's eligibility data files for three consecutive cumulative periods: 0–6 months, 0–12 months, and 0–24 months after the date they completed the PedsQL™ 4.0. A pediatric patient was considered eligible for health plan benefits for those periods if they were eligible for at least 5 months out of the 6-month period. We then electronically captured healthcare costs (the dollar amount paid by the health plan) for each pediatric patient for those periods in which they were eligible. We did this by matching each eligible pediatric patient with the health plan's existing databse of claims and encounter data. These data include the dollar amount spent by the health plan. Healthcare costs included hospital and emergency room costs, professional fees, durable medical equipment, home health, specialty clinic, and primary care costs. We did not have access to pharmacy or mental health costs. In California, the site of the study, treatment for 22 specific diagnoses is "carved out," or paid through a separate program (called California Children's Services; CCS) regardless of a child's health plan membership. Thus, for health plans in California, treatment of CCS-covered diagnoses might not be measured in calculating utilization. However, because California's carve out may differ from other states' methods of financing treatment for these diagnoses, and to more completely describe healthcare costs, we included the costs for procedures covered by CCS in our healthcare costs calculations. To derive these costs, we linked the procedure codes on the health plan's CCS referral with the federally supported health plan's fee schedule. These data thus represent the dollar amount the health plan would have spent had the services not been carved out. Procedure A convenience sample – subects were recruited nonsystematically when research assistants were available – was recruited at pediatrician offices and specialty clinics. These pediatric patients were identified through examination of the clinic appointment schedules. At these sites, parents of children identified as possible study participants were informed of the study by one of the research assistants after checking in for their appointment, but before being seen by their healthcare provider. Written informed consent included permission for the researchers to examine the medical record to assess utilization. After written informed consent was obtained, the parent completed the proxy-report version of the PedsQL™ 4.0. The research assistant was available at all times to answer any questions. A random sample was recruited from children and adolescents ages 2–18 years who had been seen as inpatients or outpatients at Children's Hospital and Health Center between April 1 and June 30, 1998, and who were members of the health plan. This sample excluded children with a discharge status of expired, children whose payer was from the victim/witness fund, and children whose parents had requested their phone number and address to be kept private. Research assistants called parents of children on this list and obtained verbal informed consent. The research assistant verbally administered the PedsQL™ 4.0 to parents. This research protocol was approved by the institutional review board at Children's Hospital and Health Center, San Diego (#98-020). Statistical analysis We pooled the data from the two samples. Previous reasearch on the PedsQL™ has documented the lack of mode of administration effects [19,20]. In order to test the primary hypothesis that HRQL would prospectively predict healthcare costs, multiple linear regression analyses were conducted. We examined the association between age, gender, chronic health condition status (variables typically used by health plans to predict risk), and PedsQL™ 4.0 scores with healthcare costs at each of the three cumulative follow-up periods. We did not use socioeconomic status, as eligibility criteria for membership in the health plan requires families to have incomes below a certain level, and this restricts the range of this variable. Four models were constructed for each follow-up period. Model 1 included age and gender only, Model 2 included age, gender, and chronic health condition status, Model 3 included age, gender, and PedsQL™ 4.0 scores, and Model 4 included age, gender, chronic health condition status, and PedsQL™ 4.0 scores. We report the adjusted R2, a measure of the percent of variance in the dependent variable accounted for by the predictor variables while adjusting for the complexity of the model, and the standardized regression coefficient, or beta, for each predictor. PedsQL™ 4.0 scores were skewed toward the high end of the scale and were transformed by taking the square root of the reverse of the score (sqrt(100-score)) in order to create a more normal distribution. The distribution of cost data was skewed to the lower end, with many children having little cost and a relatively smaller number of children having high costs. These data were normalized by taking the log of the costs. In order to explore whether HRQL and chronic health condition status together would define a relatively small subset of enrollees who accounted for a disproportionately large percent of healthcare costs, we divided the sample into quintiles based on the PedsQL™ 4.0 Physical Functioning Scale score and into two groups based on chronic health condition status. Those children who fell in the lowest PedsQL™ 4.0 quintile and who reported the presence of a chronic health condition were assigned to the high-risk group. We describe the percent of costs, per member costs, and per member per month costs per child accounted for by this high risk group. Results Descriptive Statistics Data was collected from the parents of 317 children (157 girls, 160 boys) ages 2 to 18 years. The average age of the children was 8.3 years (SD = 4.14) with a range of 2.03 to 17.13 years. The sample was heterogeneous with respect to race/ethnicity, with 76 (25.4%) White non-Hispanic, 155 (51.8%) Hispanic, 39 (13.0%) Black non-Hispanic, 6 (2.0%) Asian/Pacific Islander, 3 (1.0%) American Indian or Alaskan Native, 20 (6.7%) Other, and 19 (6.0%) missing. With respect to mother's education, 36.4% had less than a high school education, 46.6% had a high school diploma or some college, and 7.0% were college graduates or beyond (18.8% missing). The measures were administered in two languages – English (n = 233, 73.6%) and Spanish (n = 84, 26.4%). The sample represented both chronically ill (n = 102, 32.1%) and healthy children (n = 215, 67.9%), based on parent report of the presence of a chronic health condition. Table 1 presents the chronic health conditions reported by parents for the high risk group and the non-high risk group. Table 1 Parent-reported chronic health conditions, by high risk group status. High risk group? Total no yes name of condition None 223 0 223 ADHD 7 1 8 allergies 1 0 1 arthritis 2 0 2 asthma 25 8 33 autism 2 1 3 back and leg pain 0 1 1 Battan disease 0 1 1 bi-polar 1 0 1 breathing problems 1 0 1 bronchitis 2 0 2 cancer 1 0 1 cerebral palsy 2 2 4 chronic ear infections 2 0 2 cough 2 0 2 CSF leak 0 1 1 depression 1 0 1 developmental delay 1 0 1 dislocated shoulder 0 1 1 doesn't produce salt 1 0 1 Down Syndrome 1 0 1 ear problems 0 1 1 epilepsy 1 0 1 gallbladder problems 1 0 1 gastrointestinal problem 1 0 1 hearing 1 0 1 heart murmur 2 0 2 heart problem 1 1 2 kidney reflux 1 0 1 leukemia 1 0 1 May Hegglin syndorme 1 0 1 migraine headaches 0 1 1 muscle condition 0 1 1 muscular dystrophy 0 1 1 osteomyelitis 1 0 1 pierre robyn syndrome 1 0 1 reactive airway disease 1 0 1 Rett syndrome 1 0 1 scleroderma 0 1 1 scoliosis 0 1 1 seizures 3 1 4 speech 1 0 1 spina bifida 0 2 2 stomach aches 1 0 1 stomach problems 1 0 1 unknown genetic syndrome 0 1 1 urinary tract infections 1 0 1 There were no differences found in PedsQL™ scores between the group sampled at well-child checks or specialty clinics and that sampled by phone. All 317 children were enrolled in the health plan after 6 months, with 314 (99.0%) enrolled after 12 months, and 244 (76.9%) after 24 months. There were no differences between those enrolled versus not enrolled at 24 months in percent with a chronic health condition, race/ethnicity, mother's education, or PedsQL™ scores. The cost per member per month (pmpm) for this sample, which represents the total cost divided by the number of members divided by the number of months enrolled, was $149 at 6 months, $137 at 12 months, and $115 at 24 months. The sample included 4,954 claims (there are multiple claims in a single clinical encounter) over the 24 months. The largest category of visits was for upper respiratory infections (URIs) and related infections (10.96%). Asthma, other infections, otitis media, and pain each account for 5 to 6% of visits, with acute orthopedic conditions accounting for 2.6% of visits. These most common diagnoses account for more than a third (38.7%) of the visits, the rest is comprised of a large number of relatively low-frequency diagnoses. This distribution of diagnoses is similar to the epidemiology of childhood illness, in that much of pediatric morbidity is accounted for by a large number of relatively low frequency diagnoses [39,40]. Table 2 displays the descriptive statistics for the PedsQL™ 4.0 parent proxy-report at Time 1. Consistent with previous PedsQL™ 4.0 findings, [19] chronically ill children had lower HRQL scores than healthy children (Table 2). Table 2 Descriptive Statistics for PedsQL™ 4.0 scores N Mean SD Minimum Maximum t^ df p Total Sample  Total Scale Score 316 84.38 13.67 25.00 100.00  Physical Functioning 316 85.39 19.87 0.00 100.00  Psychosocial Functioning 317 83.78 13.73 32.14 100.00 Children with Chronic Health Condition  Total Scale Score 101 79.26 16.66 25.00 100.00 -4.73 313 0.001  Physical Functioning 100 79.69 25.00 0.00 100.00 -3.59 313 0.001  Psychosocial Functioning 102 79.27 16.59 32.14 100.00 -4.12 314 0.001 Children without Chronic Health Condition  Total Scale Score 214 86.82 11.29 44.44 100.00  Physical Functioning 215 88.17 16.30 10.00 100.00  Psychosocial Functioning 214 85.91 11.61 48.33 100.00 ^comparing chronically ill to healthy children Multiple regression analysis Table 3 displays the results of the multiple regression analyses predicting healthcare costs for 6, 12, and 24 month follow-up. As can be seen, Model 1, with age and gender as the only predictors variables, did not account for significant variance in costs. Model 2 shows that age and gender, with chronic health condition status accounted for an increasing percentage of costs as the follow-up time lengthened. This pattern holds true as well for Model 3, which included age, gender, and the PedsQL™ 4.0 scores. Model 4, comprised of age, gender, chronic health condition status, and PedsQL™ 4.0 scores, accounted for the most variance, explaining 10.1% 14.4% and 21.2% of the variance in healthcare costs at 6, 12, and 24 month follow-up intervals. Inspection of the standardized regression coefficients for each predictor in Model 4 shows that, of the four predictors used, chronic health condition status and the PedsQL™ 4.0 Physical Functioning Scale scores consistently accounted for the greatest amount of variance. Table 3 Adjusted R-square (in bold) and standardized regression coefficients (betas) for models predicting costs at 6, 12, and 24 months Follow up 6 Months (N = 318) 12 Months (N = 315) 24 Months (N = 245) Model 1 Adjusted R-square 0.010 0.001 0.004  Beta   Age -0.067 -0.037 -0.030   Gender .11 0.063 0.108 Model 2 Adjusted R-square 0.049** 0.066*** 0.130***  Beta   Age -0.063 -0.031 0.008   Gender 0.118* 0.076 0.115   Chronic Health Condition 0.180** 0.265*** 0.360*** Model 3 Adjusted R-square 0.089*** 0.103*** 0.122***  Beta   Age -0.103 -0.091 -0.079   Gender 0.080 0.030 0.076   PedsQL Physical Functioning 0.291*** 0.353*** 0.379***   PedsQL Psychosocial Functioning 0.004 0.057 0.081 Model 4 Adjusted R-square 0.101*** 0.144*** 0.212***  Beta   Age 0.098 -0.083 -0.043   Gender 0.086 0.040 0.080   Chronic Health Condition 0.126+ 0.214*** 0.312***   PedsQL Physical Functioning 0.275*** 0.326*** 0.340***   PedsQL Psychosocial Functioning 0.025 0.093 0.123+ + = p < 0.05; * = p < 0.01; *** = p < 0.001; *** = p < 0.0001; R2 = percent variance accounted for. Defining the high risk group We used the two variables accounting for most of the variance in the regression analysis – the PedsQL™ 4.0 Physical Functioning scores and chronic health condition status – to describe the percentage of costs accounted for by different groups of children. In order to create a single denominator for the percentages, we used the 241 children continuously enrolled in the health plan with complete data for this set of analyses. To create quintiles, we determined the values that divided the sample into five equal-sized groups based on PedsQL™ 4.0 Physical Functioning Scale scores. Enrollees with a score of less than 75 on the PedsQL™ 4.0's 0–100 scale were in the first quintile (N = 51; 21.0%). The second quintile (N = 45; 18.5%) was bounded by the scores 75.0 to 90.624, the third quintile (N = 48; 19.6%) by the scores 90.625 to 96.874, the fourth quintile by the scores 96.875 to 100 (N = 18; 7.3%), and the fifth quintile consisted of enrollees scoring 100 (N = 81; 33.4%). Because the distribution of these PedsQL™ 4.0 scores was skewed, we combined the fourth and fifth quintiles (N = 99; 40.7%; 2 missing). Table 4 shows the percentage of total costs accounted for by children across PedsQL™ 4.0 Physical Functioning Scale quintiles and chronic health condition status, for the three cumulative follow up periods. As can be seen, children in the high risk group (the subset of chronically ill children in the lowest quintile), account for a disproportionately large share of healthcare costs. This group, comprising just 8.7% of the sample, accounted for 37.42% of the healthcare costs over six months, 59.16 % of costs over 12 months, and 61.74% of costs over 24 months. Table 4 Percent of cost at each follow up period, by PedsQL quintile and chronic health condition status (n = 241) PedsQL Physical Functioning Quintile Lowest quintile (N = 51) 2nd quintile (N = 45) 3rd quintile (N = 48) 4th and 5th quintiles (N = 99) Chronic Health Condition Chronic Health Condition Chronic Health Condition Chronic Health Condition Yes (N = 21) No (N = 30) Yes (N = 18) No (N = 27) Yes (N = 12) No (N = 36) Yes (n = 26) No ( = 73) % of cost, 6 months 37.43 9.35 26.76 9.30 0.33 6.95 7.59 2.30 % of cost, 12 months 59.16 4.70 20.56 4.54 0.30 3.63 5.25 1.85 % of cost, 24 months 61.74 3.38 22.06 2.86 0.41 2.64 4.87 2.03 Bold = High-risk group Table 5 shows the total costs, the per member costs, and the per member per month (pmpm) costs for the high risk group and the not high risk group over the three follow-up periods. As can be seen, the high risk group was an extremely costly subset of enrollees for each of the cumulative 6 month periods, as measured by total, per member, or pmpm costs. Pmpm costs were quite disparate between the high risk group and other enrollees. For the high risk group at 6 months, pmpm was $432 (vs. $66 for the other patients), at 12 months pmpm was $809 (vs. $61), and at 24 months, pmpm was $722 (vs. $60). Table 5 Total costs, per member costs, and per member per month costs for high-risk* (N = 21) and not high-risk (N = 231) enrollees. Total Per member Per member per month Time Period High-risk Not high-risk High-risk Not high-risk High-risk Not high-risk 6 months $54,493 $91,484 $2,595 $396 $432 $66 12 months $203,875 $168,022 $9,708 $727 $809 $61 24 months $363,822 $330,846 $17,325 $1,432 $722 $60 *High risk is defined as parent reported chronic health condition and scoring in lowest quintile on PedsQL™ 4.0 Discussion This study tested the primary hypothesis that HRQL could prospectively predict healthcare cost in pediatric patients in a managed care environment. We measured age, chronic health condition status, and PedsQL™ 4.0 scores at Time 1, and prospectively measured utilization, via costs based on claims and encounter data, for three cumulative periods. These data demonstrate that parent-reported HRQL, as measured by the PedsQL™ 4.0, and chronic health condition status each accounted for significant variance in healthcare costs over 6, 12, and 24 months. The data further show how these two predictor variables, chronic health condition status, and PedsQL™ 4.0 Physical Functioning scores, define a relatively small group of enrollees that accounted for a large percentage of total healthcare costs. This high risk group displays disproportionately high costs as early as 6 months, and their pmpm costs peak at one year. This suggests the importance of managing high risk enrollees as soon as they are identified, perhaps as early as their initial enrollment. It also implies the potential for significant return on investment for better case management, even in the first six months of enrollment. The high risk group's costs remain disproportionately high throughout the 24 months of the study. This fact suggests that the method used here for identifying the high risk group succeeded in identifying children with high ongoing care needs and costs, as opposed to children with one-time health care needs. An anomalous finding was that children in the third quintile on PedsQL™ scores who had chronic health conditions were, for an unexplained reason, much less costly than their peers. It is worth comparing the mean PedsQL™ 4.0 scores for the high risk group to other published data. The high risk group had scores of 44.5 for the Physical Functioning Scale, 70.7 for the Psychosocial Summary Scale, and 61 for the Total Scale. This is placed in clinical perspective by other data showing that scores for children with cancer, in active treatment, are 65, 68, and 67 for the Physical, Psychosocial and Total scales, respectively. [31]. A hypothetical example is presented to illustrate the potential impact of these findings. In a typical health plan, the rate of chronic health conditions will most likely be between 5% [41] and 18% [42], rather than the 31.4% rate we found by selecting our sample, in part, from hospital specialty clinics. We will further conservatively assume that one-fifth (20%) of children with chronic health conditions would fall in the lowest quintile on the PedsQL™ 4.0. If this were so, then between 1% (5% chronic health condition × 20% in the lowest quintile) and 3.6% (18% chronic health condition × 20% in the lowest quintile) of enrollees in a health plan would fall into the high risk group. Thus, in a hypothetical medium to large health plan with 50,000 pediatric enrollees, the high risk group would be comprised of anywhere from 500 (1% of 50,000) to 1800 (3.6% of 50,000) children. Using the costs figures from this sample ($722 pmpm), this hypothetical high risk group represents between $8.6 and $31.2 million in costs over the course of 24 months. This example relies on speculation and is intended as a hypothetical case, for illustrative purposes only. Taken together, these findings represent an alternative method toward the prospective prediction of healthcare costs in pediatric federally supported managed care populations. While a percentage of these identified costs are inevitable, due to the costs of appropriate care for these chronically ill, poorly functioning children, the possibility exist that a proportion of these healthcare costs are avoidable. Evidence-based disease management has been shown to reduce healthcare costs and increase HRQL in certain chronic conditions such as asthma [43,44]. By identifying at-risk children with low PedsQL™ 4.0 scores, targeted interventions may avert certain future healthcare costs by ameliorating impaired HRQL when first identified. Certain limitations exist in this study. The first has to do with data not accounted for in this study. We did not have access to pharmacy and mental health costs, nor did we have access to out-of-plan expenditures. For mental health costs, however, recent data has shown that children referred for psychiatric services demonstrate child self-report and parent proxy-report PedsQL™ 4.0 Total Scale Scores comparable to children with chronic physical health conditions [45]. Those data suggest that this methodology may be useful in predicting mental health costs as well. We did not include children under the age of two. Neonatal intensive care, for example, is a large percent of the costs for Medicaid managed care plans [46]. The portion of health plan costs devoted to caring for children under two is not explored here. However, many of these costs cannot be avoided, and preventive efforts for these costs are most appropriately targeted at the prenatal and perinatal periods. Finally, we did not compare the performance of these variables to that of existing administrative risk adjustment methods currently available. The second limitation has to do with sampling issues and with generalizing these data beyond this study. Our data was not collected at enrollment, nor did we sample from the entire pool of enrollees. Further research is necessary to determine whether these findings hold true for children assessed at health plan enrollment, and to determine the extent to which the results may be influenced by the convenience sample used here. The sample was too small to use cross-validation techniques. Prediction models tend to overfit the development sample, and the predictive validity of these variables should be tested in other, larger samples. Although generalization of these findings to broader populations should be made with caution, the sample here is very diverse with respect to race/ethnicity, and thus likely to be similar to other federally support health plans. We also combined data from two different samples – specialty clinic patients, and health plan members who had been seen in the hospital or outpatient clinics at least three months after the clinical encounter. These two groups could have had unmeasured systematic differences, which could have biased the results. The third has to do with using a survey to gather these data. The costs of fielding a survey can be quite high, and, if tied to payment, survey responses are subject to "gaming". However, we submit that the potential gains from optimal management of an enrolled population will almost certainly be greater than the costs of survey administration. Moreover, while gaming might occur if health plans were to be compensated based on the HRQL of their enrolled population, the methods used here are suggested as strategies for clinical management, not for rate setting, thus reducing the incentives for gaming. We did not track refusal rates and so do not know what percent of potential participants consented to be in the study. Finally, using a survey means that parents reported on their children's chronic health condition information. Objective measures of chronic health condition would strengthen the validation process. However, in previous PedsQL™ 4.0 clinical research in pediatric patients with cancer, cardiac and rheumatic chronic health conditions, objective medical diagnosis of these chronic diseases demonstrated similar differences between healthy children and children with chronic health conditions as shown in the present findings [28-30]. Further research is necessary. First, a much larger, and randomly selected sample is necessary to confirm these results. Second, split-half validation should be performed so that the coefficients from one group are used to predict the costs in a different group. This could be done with split halves of one large group, or with two similar groups enrolled at different points in time. Given that our regression equation explains 21% of costs, further studies could be done to determine whether other variables might account for additional variance in costs. Further studies could also allow comparison and validation with the results here. Conclusion This is the first study we are aware of to use parent reports of pediatric HRQL and chronic health condition status to prospectively predict healthcare costs in a pediatric sample. These data have implications for healthcare decision makers such as pediatricians, health plan administrators, and policymakers. In a prospective payment system, providers are incentivized to actively manage high-risk patients and to provide care at the appropriate level. The idea behind such a system is that prevention and appropriate care accrues benefits to patients in the form of better health and to providers in the form of lower costs. If, as these data suggest, parent reports of HRQL can be used to predict healthcare costs, one could identify at-risk children proactively and intervene to avoid both illness and costs. In this way, these data can serve simultaneously to improve the health of children and the system that serves them. List of Abbreviations HRQL Health-related Quality of Life PedsQL™ 4.0 Pediatric Quality of Life Inventory™, Version 4.0 SD Standard Deviation CCS California Children's Services URI Upper respiratory infection PMPM per member per month Authors' Contributions MS, PSK, and JWV conceived of the research. MS and JWV supervised the data collection. MS and DS performed the data analysis. MS drafted the manuscript. JWV, PSK, and DS provided substantive input into the manuscript. All authors read and approved the final manuscript. Acknowledgements This research was supported by intramural grants from the Children's Hospital and Health Center, San Diego. The authors would like to thank Robert Kaplan, Ph.D., Richard Kronick, Ph.D., and Theodore Ganiats, MD for their valuable input into previous versions of this manuscript. ==== Refs Newhouse JP Sloss EM W.G. Manning Keeler EB Risk adjustment for a children's capitation rate. Health Care Financing Review 1993 15 39 54 10133708 Iezzoni LI Risk Adjustment for Measuring Health Care Outcomes 2003 3rd Chicago, Health Administration Press Parkerson G. R., Jr. Harrell F. 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2021-01-04 16:36:37

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Retrovirology. 2004 Sep 21; 1:28

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Retrovirology

10.1186/1742-4690-1-28

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==== Front BMC Public HealthBMC Public Health1471-2458BioMed Central London 1471-2458-4-391534166610.1186/1471-2458-4-39Study ProtocolThe Internet and HIV study: design and methods Elford Jonathan [email protected] Graham [email protected] Mark [email protected] Lorraine [email protected] Graham [email protected] institute of Health Sciences & St Bartholomew School of Nursing and Midwifery, City University London, 24 Chiswell Street, London EC1Y 4TY2 Department of Primary Care and Population Sciences, Royal Free and University College Medical School, Rowland Hill Street, London NW3 2PF3 MRC Social and Public Health Sciences Unit, 4 Lilybank Gardens, Glasgow G12 8RZ2004 1 9 2004 4 39 39 7 7 2004 1 9 2004 Copyright © 2004 Elford et al; licensee BioMed Central Ltd.2004Elford et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background The Internet provides a new meeting ground, especially for gay men, that did not exist in the early 1990s. Several studies have found increased levels of high risk sexual behaviour and sexually transmissible infections (STI) among gay men who seek sex on the Internet, although the underlying processes are not fully understood. Research funded by the UK Medical Research Council (2002–2004) provided the opportunity to consider whether the Internet represents a new sexual risk environment for gay and bisexual men living in London. Methods The objectives of the Internet and HIV study are to: (i) measure the extent to which gay men living in London seek sexual partners on the Internet; (ii) compare the characteristics of London gay men who do and do not seek sex on the Internet; (iii) examine whether sex with Internet-partners is less safe than with other sexual partners; (iv) compare use of the Internet with other venues where men meet sexual partners; (v) establish whether gay men use the Internet to actively seek partners for unprotected anal intercourse; (vi) determine the potential for using the Internet for HIV prevention. These objectives have been explored using quantitative and qualitative research methods in four samples of London gay men recruited and interviewed both online and offline. The four samples were: (i) gay men recruited through Internet chat rooms and profiles; (ii) HIV positive gay men attending an NHS hospital outpatients clinic; (iii) gay men seeking an HIV test in an NHS HIV testing or sexual health clinic; (iv) gay men recruited in the community. Results Quantitative data were collected by means of confidential, anonymous self-administered questionnaires (n>4000) completed on-line by the Internet sample. Qualitative data were collected by means of one-to-one interviews (n = 128) conducted either face-to-face or on-line. Conclusion The strength of the Internet and HIV study is its methodological plurality, drawing on both qualitative and quantitative research among online and offline samples, as well as taking advantage of recent advances in web survey design. The study's findings will help us better understand the role of the Internet in relation to gay men's sexual practice ==== Body Background Several studies have found elevated levels of high risk sexual behaviour among people who seek – and meet – sexual partners through the Internet [1-7] In a study conducted in a public HIV testing clinic in Denver, Colorado, for example, people who sought sex on the Internet were more likely to have had a sexually transmitted infection (STI) or report sexual exposure to a person with HIV than those who did not seek sex on the Internet [2]. They were also more likely to be male, gay and to report anal sex. While the study concluded that gay men were more likely than other participants to use the Internet to seek – and meet – sexual partners, it could not establish whether the excess sexual risk actually occurred with partners whom the men had met through the Internet. A San Francisco study also found that gay men were more likely than heterosexual men and women to use the Internet to meet sexual partners [5]. Around one-third of the gay men interviewed at a public STI clinic had used the Internet to meet a sexual partner compared with one-in-ten heterosexual men and women. The gay men in the study said that their online partners – men they met through the Internet – were more likely to be casual (ie a one night stand) than their offline partners – men they met elsewhere. Similar reports have also emerged from European studies. An investigation of gay men in London gyms found that in the year 2000 over one-third of those with Internet access had used it to look for sex [1]. When surveyed three years later, this figure had increased to nearly half [8]. HIV positive men were more likely to use the Internet to look for sex than HIV negative or never-tested men. Seeking sex on the Internet was associated with a recent STI diagnosis and high-risk sexual behaviour, ie unprotected anal intercourse (UAI) with a person of unknown or discordant HIV status [1]. This presents a risk for HIV transmission. In addition, HIV-positive Internet-sex seekers were more likely to report UAI with another HIV-positive man than those who did not seek sex in this way. This raises the possibility that HIV positive men use the Internet to meet other positive men for unprotected anal intercourse. While this does not present a risk of HIV transmission to an uninfected person, it may lead to co-infection with an STI or with another, potentially drug-resistant strain of HIV [9]. As in the U.S. studies, the London study could not establish whether the excess risk for HIV and STD occurred with sexual partners whom the men had actually met through the Internet. The association between seeking sex on the Internet and high risk sexual behaviour raises a number of important, as yet unanswered questions. Does the excess risk for HIV and STI occur with sexual partners whom men actually meet through the Internet? Does the association reflect the fact that high risk men are selectively using the Internet to look for sex? Or does the Internet in some way facilitate high risk behaviour? Is the Internet attracting a constituency of men who have little contact with the established gay scene or health promotion agencies [10]? For example, men who do not use bars and clubs or bisexual men? In other words, does the Internet represent an emerging sexual risk environment for gay men? If so, what are the underlying processes? Answers to these questions are essential if we are to use the Internet effectively for HIV prevention and sexual health promotion. To address these questions we have undertaken research funded for two years (2002–2004) by the UK Medical Research Council and the Department of Health. This has been conducted by researchers at City University London in collaboration with colleagues at the MRC Social and Public Health Sciences Unit Glasgow and University College London (see appendix 1). Research question The core research question is: Does the Internet represent a new sexual risk environment for gay/bisexual men living in London?" (referred to as "gay men" throughout the rest of this paper). And if so, what are the underlying processes? The research focuses on gay men living in London since the incidence and prevalence of HIV infection among gay men in London is higher than elsewhere in the UK [11-13]. The objectives of the research are to • measure the extent to which London gay men seek sexual partners on the Internet • compare the characteristics of gay men who do and do not seek sex on the Internet • examine whether sex with Internet-partners is less safe than with other sexual partners • compare use of the Internet with other venues such as saunas and backrooms • establish whether gay men use the Internet to actively seek partners for unprotected anal intercourse • determine the potential for using the Internet for HIV prevention Methods These objectives have been explored using both quantitative and qualitative research methods. While quantitative research methods can provide data on a range of outcomes they can rarely offer insight into underlying processes. Qualitative research, on the other hand, illuminates our understanding of human behaviour but is limited in the extent to which findings can be generalized. By adopting methodological plurality and employing both quantitative and qualitative methods, we will be able to build on the strengths of both approaches within an integrated research programme In desribing the design and methods of the Internet and HIV study we focus on sampling strategy, recruitment into the study, data collection and data analysis. Sampling Research among hard-to-reach groups such as gay men is usually based on convenience rather than probability samples [14]. For example, behavioural research among gay men in the UK has primarily been conducted among men recruited in bars, clubs, GUM clinics [15], gay pride events [16] and gyms [17]. While probability sampling would undoubtedly provide a more robust foundation for statistical analysis [18] such an approach is extremely, if not prohibitively expensive. Convenience samples have the advantage of being affordable and also provide the opportunity to focus on men with characteristics which may be of particular interest, eg men who report high risk behaviour [14]. The disadvantage of course is that such samples may introduce selection bias. This bias can be partially overcome by including samples from more than one source allowing for triangulation of data. Consequently, we recruited gay men from four different sources – one online, three offline. Each sample has specific features which are pertinent to the research question. HIV positive gay men HIV positive men are central to any research concerning HIV risk, transmission and prevention [19-21]. Furthermore, HIV positive men are more likely to use the Internet to seek sexual partners than other men [1,8]. Consequently it was decided to over-sample HIV positive men for the study to permit an in-depth examination of sexual risk behaviour and use of the Internet in this group of men. In the UK everyone who is diagnosed HIV positive is offered free treatment and care within the National Health Service (NHS) through a hospital outpatients clinic. Consequently an NHS clinic sample will be broadly representative of all those living with diagnosed HIV. Gay men seeking an HIV test Most people seeking an HIV test have been at risk of HIV infection. Furthermore, HIV negative gay men with a history of multiple repeat testing report elevated levels of high risk sexual behaviour [22]. This group therefore merits inclusion in an investigation of the Internet as an emerging sexual risk environment. In the UK, the NHS offers free voluntary counselling and testing for HIV either in dedicated HIV testing clinics or in general sexual health clinics. Since there is no charge for this service, these clinics attract a broad cross section of people. Gay men in the community Surveying gay men in the community allows us to examine the extent to which they use the Internet for seeking sexual partners and the associated risks. Previous research has shown that gay men surveyed in central London gyms are broadly representative of men "on the scene" in London ie men who go to gay bars, clubs and other venues [23]. However, whereas questionnaires distributed in London bars and clubs have to be short because of the limited time available for completion, we have found that in gym-based surveys respondents are willing to complete questionnaires that take up to 15 minutes to answer. This allows for a detailed investigation of sexual behaviour. The Internet Men who use Internet chat rooms and profiles to seek sex with other men are clearly of central importance to this research project. The Internet may attract a constituency of men who would not otherwise be included in behavioural surveys among gay men ie men who do not go to gay bars, clubs or other venues and men who have not been tested for HIV [10]. Comparing the characteristics of men recruited online with the community and clinic samples will throw these differences, where they exist, into sharp focus. Recruitment Based on previous research conducted among gay men in both clinic and community settings [1,8,22,23] we estimated that, for the quantitative arm of the study, we would needed to recruit 400–500 men in each of the four samples. This would provide sufficient power at a 5% level of significance to compare the characteristics of men who do and do not seek sex on the Internet, to compare use of the Internet with other venues such as saunas and backrooms, and to examine whether sex with Internet-partners is less safe than with other partners. For the qualitative arm of the study, we recruited at least 20 men from each of the four samples to allow us to derive accounts of Internet dating and sexual practice from a diverse range of men, selected purposively according to age, education, employment, HIV status and use of the Internet for seeking sex. HIV positive gay men Men diagnosed with HIV infection attending an outpatient treatments clinic at the Royal Free Hampstead NHS Trust hospital, London over an 8 month period (October 2002–May 2003) were invited to participate in the research. Patients with a limited command of English were ineligible for the study as were those who were too ill to complete a questionnaire. Eligible patients were approached in the clinic's waiting area by a trained member of the research team who discussed the project with them. Patients were provided with written information about the research, contact details of the research team as well as helpline numbers. Once they had provided written consent, respondents were asked to complete a pen-and-paper questionnaire in the clinic and return it in a sealed envelope to the team member (further information about the questionnaire in Research Methods below). Some patients were only in the waiting area for a short time so there wasn't an opportunity to invite them to take part in the study. Over the eight month survey period (October 2002–May 2003), 1001 individual male patients attended the clinic of whom 939 were deemed eligible for the study. Of those who were eligible, 864 were asked to complete a questionnaire and 620 did so. The response rate was 72% of men who were offered a questionnaire and 66% of all eligible men who attended the clinic. Of the 620 men who completed a questionnaire, 542 described their sexual orientation as gay or bisexual or had had sex with another man in the previous year. Of these, 523 men provided sufficient information to be included in the quantitative sample (table 1). Table 1 Number of London gay/bisexual men who participated in the Internet and HIV study 2002–2003 Quantitative arm Qualitative arm Recruitment site HIV positive HIV negative Never-tested Total HIV positive HIV negative Never-tested Total HIV treatment clinic 523 - - 523 20 - - 20 HIV testing & sexual health clinics* 15** 389 - 404 - 20 - 20 Community 2002 138 592 184 914 } 1 18 4 23 Community 2003 88 361 94 543 } Internet*** 2002 142 680 396 1218 } 17 35 13 65 Internet*** 2003 67 315 197 579 } * An additional 435 heterosexual men and 450 heterosexual women completed a questionnaire in the HIV testing clinic for the quantitative arm * * Fifteen men received an HIV positive diagnosis when they returned for their test result * * * An additional 3279 gay/bisexual men living in the UK but outside London completed the questionnaire online in 2002, 1944 in 2003 for the quantitative arm HIV positive gay men who completed a questionnaire were asked on the last page if they would be willing to have an in-depth face-to-face interview, one-to-one, with a qualitative researcher working on the project (MD) (further information about the one-to-one interviews in Research Methods below). If they agreed, the researcher contacted them to arrange a time for the interview. This could be in the researcher's office at City University London, in the hospital or at the respondent's home. In this way 20 HIV positive gay men were recruited for one-to-one interviews as part of the qualitative arm of the study (table 1). Gay men seeking an HIV test People seeking an HIV test at the same-day HIV testing clinic, Royal Free Hampstead NHS Trust Hospital over a 13 month period (October 2002–November 2003) were invited to take part in the study. Based on previous research in the HIV testing clinic, we estimated that this would generate a sample of approximately 500 gay men, 500 heterosexual men and 500 heterosexual women [22]. Although the focus of the study was gay men, data were also collected from heterosexual men and women to provide a valuable comparison. Using a similar strategy to the outpatient HIV treatments clinic, everyone seeking an HIV test was asked to participate in the research. Those who agreed completed a detailed, self-administered pen-and-paper questionnaire, after providing written consent, while they were waiting for their pre-test counselling. People with limited command of English were deemed ineligible for the study as were those who were too ill, too young (under 18 years) or too anxious to complete a questionnaire. Those attending the testing clinic on more than one occasion during the survey period were asked to only complete the questionnaire once. Over the 13 month survey period (October 2002–November 2003), 1889 individuals came to the Royal Free clinic for an HIV test of whom 1753 were eligible for the study. Of those who were eligible, 1640 were asked to complete a questionnaire and 1230 did so. The response rate was 75% of people who were offered a questionnaire and 70% of all eligible persons who attended the clinic. Of the 1230 people who completed a questionnaire, 345 described their sexual orientation as gay or bisexual, 435 as heterosexual male and 450 as heterosexual female; 334 gay/bisexual men provided sufficient information to be included in the quantitative sample. Gay men who completed a questionnaire were asked if they would be willing to have an in-depth, face-to-face interview with the qualitative researcher (MD). If they agreed, the researcher contacted them to arrange a time for the interview as described above. In this way, 16 gay men seeking an HIV test at the Royal Free were recruited for one-to-one interviews for the qualitative arm. Heterosexual men and women who completed the questionnaire were not asked to have a one-to-one interview. Previous research suggested that the majority of clinic attenders would test HIV negative while approximately 6% of gay men were expected to test HIV positive. It was considered inappropriate to ask men who had just been diagnosed HIV positive if they would be willing to have a face-to-face interview. Consequently, the qualitative sample from the HIV testing clinic solely comprised men who had tested negative. Anyone who volunteered for an interview who subsequently received a positive test result was not included in the qualitative sample. After 6 months it became apparent that while the response rate at the Royal Free HIV testing clinic was high the number of gay men seeking a test was not as large as had been expected. We decided therefore to extend recruitment to men seeking an HIV test at a sexual health clinic specifically for gay men at Barts and the London NHS Trust hospital, London. All men attending this clinic are gay. Using a similar strategy to the one developed at the Royal Free, everyone attending the gay men's sexual health clinic at Barts and the London hospital between June and November 2003 was invited to participate in the research. Those who agreed completed a self administered questionnaire while they were waiting in the clinic for their appointment. Although all men were asked to complete a questionnaire, only men seeking an HIV test were eligible for inclusion in the Internet and HIV quantitative sample. Over the 6 month survey period at the Barts and the London sexual health clinic (June – November 2003), 211 gay men came for an appointment of whom 209 were eligible for the study. Of those who were eligible, 198 were asked to complete a questionnaire and 156 did so. The response rate was 79% of men who were offered a questionnaire and 75% of all eligible men attending the clinic. Of the 156 gay men who completed a questionnaire at Barts and the London, 70 were seeking an HIV test and provided sufficient information to included in the quantitative sample for the study. These men were also asked if they were willing to have a one-to-one interview with the qualitative researcher. Four men were recruited in this way for the qualitative arm of the study. Combining the data from the two clinics, 404 gay men seeking an HIV test were recruited for the quantitative arm of the study between October 2002–November 2003, of whom 20 agreed to have a one-to-one interview for the qualitative arm (table 1). In addition 450 heterosexual women and 435 heterosexual men also completed a questionnaire for the quantitative arm. Gay men in the community Previous research has shown that gyms in central London provide a suitable environment for undertaking detailed behavioural research among gay men at risk of HIV infection [1,8,17,24,25]. In both 2002 and 2003, all men using any one of 7 central London gyms during a one-week period between January-March were invited to take part in the study. All these gyms have a substantial gay male membership. One gym was exclusively gay whereas the others estimated that gay men comprised 40–90% of their male membership [26]. All men using the gyms during the survey period were asked to complete a self-administered pen-and-paper questionnaire after providing written consent. A filter question on sexual orientation distinguished gay or bisexual men from straight men. Only gay/bisexual men were requested to answer questions on the Internet and sex. Men could complete the questionnaire in the gym or at home. Respondents returned completed questionnaires to collection boxes in the gym or by post to the research team. In the mixed gyms the number of questionnaires handed out to gay men was estimated by multiplying the total number of questionnaires distributed in the gym by the proportion of male members who were gay (according to the managers' estimates). To calculate the response rates we divided the number of questionnaires returned by gay men (as indicated on the questionnaire) by the estimated number of questionnaires handed out to gay men, as described above. In 2002, 921 gay men completed the questionnaire while in 2003, 550 men did so. Those men who provided information on their HIV status (2002, n = 914; 2003, n = 543) were included in the quantitative sample (table 1). The estimated response rate each year was 50%–60%. In both years, gay men who completed a questionnaire were asked if they would be willing to have an in-depth, face-to-face interview with the qualitative researcher (MD). If they agreed, the researcher contacted them to arrange a time for the interview as described above. Some interviewees were recruited through snowballing. In this way, 23 men were recruited from the gyms for the qualitative arm of the study (table 1). The Internet In both 2002 and 2003, men using UK chat rooms or personal profiles on gaydar ()or gay.com () were invited to take part in the study. Gaydar and gay.com are two of the UK's most popular websites for gay men (personal communication H Badenhorst, M Watson). Over a four week period in May-June each year, a series of pop-ups and banners advertised the research project in UK chatrooms and on profiles pages. Clicking on a popup or banner took men to the homepage of the online questionnaire. Men who agreed to complete the questionnaire then did so after providing informed consent – all online. For technical reasons, it was not possible to restrict banner advertising or pop-ups to London chatrooms or personal profiles alone. Instead, the advertising was restricted to UK chatrooms or profiles. Consequently, anyone entering a UK chatroom or profile during the survey period had the opportunity of completing the online questionnaire even though the target group was London gay men. In 2002, 1250 London men completed the online questionnaire while in 2003, 595 did so. Those men who provided information on their HIV status were included in the quantitative sample (2002, n = 1218; 2003, n = 579) (table 1). A further 3279 men living in the UK but outside London completed the questionnaire in 2002; 1944 in 2003. The decline in the number of respondents in 2003 compared with 2002 reflects a general pattern seen by gaydar and gay.com in other online surveys in the UK (personal communication H Badenhurst, M Watson). Estimating a response rate for the online quantitative sample is problematic [27,28]. It is impossible to gauge what proportion of chatroom and profile users saw the banners and pop-ups advertising the online survey. Nor do we know what percentage of those seeing the pop-ups and banners went on to complete the questionnaire. Based on estimates provided by and on the number of people using their Internet chatrooms and profiles during the survey periods, it is likely that less than one percent of all users completed the questionnaire. This level of response is standard for online surveys. This highlights the importance of not relying solely on respondents recruited through the Internet for research of this kind. Gay men living in London who completed the online questionnaire were asked if they would be willing to have a one-to-one interview with the qualitative researcher (MD). Those who agreed were asked to send an email to the researcher who then contacted them, also by email, to provide further information about the study and to arrange a time for the interview as described above. Interviews were either conducted online or face-to-face (see Research methods below). Of the London men who completed a questionnaire online in 2002 or 2003, 65 went on to have a one-to-one interview as part of the qualitative arm (table 1); 30 men were interviewed face-to-face while 35 were interviewed online Data collection Quantitative data The questionnaires sought detailed information on the men's socio-demographic characteristics (age, ethnicity, employment, education), sexual orientation, HIV test history (date and result of last test), history of STIs, access to and use of the Internet, seeking sex on the Internet, use of other venues (eg back rooms, saunas), as well as sexual risk behaviour in the previous 3 months, differentiating between Internet- and other sex partners. Unprotected anal intercourse (UAI) in the previous 3 months was categorized according to type (regular or casual) and HIV status of partner. Detailed information was collected about sexual behaviour with partners met through the Internet and with partners met elsewhere in order to compare the level and nature of risk with Internet and other sexual partners. Data were also collected on potential confounding factors such as recreational drug use, alcohol consumption, relationships, attitudes towards new treatments for HIV and mental health. Standard and validated questionnaire items were used extensively (copies of the questionnaires are available from JE). Core questions, included in the questionnaires for all four samples, were worded identically to ensure direct comparability between the different groups. In addition, some questions specific to each group were included eg, detailed questions on HIV test history and reasons for testing for people seeking an HIV test; questions on HIV medication, CD4 and viral load for HV positive men. The questionnaires were piloted both online and offline among gay men at the developmental stage of the study and revised in the light of any feedback and comments. People recruited offline, in clinics or gyms, completed a pen-and-paper questionnaire. All questionnaires were confidential and anonymous. They contained no information that would allow an individual respondent to be identified. For the HIV positive men, information on their most recent viral load and CD4 count was abstracted from hospital records and linked to their questionnaire without breaching confidentiality. The HIV test result of those seeking an HIV test was linked to their questionnaire, again without breaching confidentiality. Once the questionnaire and clinic data had been linked in the database individual identifiers (eg hospital numbers) were removed and destroyed to ensure anonymity. Men recruited through Internet chatrooms and profiles completed the questionnaire online. The online questionnaire was constructed by a computer programmer at gaydar () working in close collaboration with the research team. Because of their technical expertise and capacity, gaydar hosted the questionnaire on their server. All questionnaires were confidential and anonymous. Identifiers such as IP addresses were removed from the questionnaires completed online before the data were downloaded to a database. Within each sample men were asked (a) to complete only one questionnaire and (b) whether they had been in any of the other samples. For example, men who completed the questionnaire online were asked whether they had also completed a questionnaire in the gyms or clinics, etc. Qualitative data Qualitative data were collected by means of one-to-one, in-depth interviews conducted face-to-face (n = 93) or on-line (n = 35) Face-to-face interviews The interviewees were volunteers from the quantitative samples recruited in the HIV treatments clinic, HIV testing clinic, gyms or online as described above. They provided written consent for the one-to-one interview which lasted between 50 and 90 minutes and was audio-taped for transcription. The interviews were generally conducted in the research office at City University London but occasionally at the interviewee's home or in the clinic. Confidentiality was provided in two ways; first, the interviewee's contact details were not linked to the interview transcript and secondly personal identifiers such as the person's name, where he was born, lived or worked were removed from the transcripts. Once all the data had been transcribed and entered into the database, individual identifiers (eg interviewees' contact details) were destroyed. Online interviews The interviewees were volunteers from the online quantitative samples. They provided informed consent by email. Interviews were prearranged and conducted in a private room on or which only the interviewer and interviewee could enter. The interviews were synchronous and conducted entirely through text generated as online chat. Each interview lasted between 50 and 90 minutes at the end of which the interview text was copied and pasted into a Word document. Confidentiality was provided in the same manner as the face-to-face interviews.. Interviews were conducted in two phases. Phase one (n = 24 interviews) focused on how gay men used the Internet for sexual partnering while phase two (n = 104) focused on risk behaviour related to the Internet. The interviews for phase one were based on a topic guide comprising questions about age, residence, schooling, employment, relationship status and HIV testing. Social and sexual lifestyles were explored in depth paying particular attention to the role of the Internet. The topic guide included seeking sexual partners, preferences for different ways of meeting partners (including the Internet), Internet experience and skills, learning how to use the Internet for sexual partnering, online communication skills, other uses of the Internet and related media, a recent sexual episode and its relationship with the Internet. The topic guide for phase two comprised social background; sexual lifestyle and role of the Internet; risk episodes including a description of anal sex with a condom and without a condom with Internet and non-Internet partners; HIV testing (where relevant); general discussion of Internet experience and web profiles; safer sex (rules, negotiated safety, serostatus of sexual partner, disclosure, slip-ups, problems, pleasure and rationality); and sources of knowledge and skills about risk reduction (health carers, media, school, common sense). For HIV positive men the topic guide also covered their experience of HIV treatment and care (eg current diagnosis, treatments, treatment effectiveness, clinical markers) and the relationship between treatments and risk (eg prospects for new drugs, role of viral load in risk taking, reinfection); Ethics The research protocol was approved by the following ethics committees: Royal Free Hospital and Medical School Local Research Ethics Committee, the East London and The City Research Ethics Committee and City University London Research Ethics Committee. Sample characteristics and data analysis Over 4000 London gay men were recruited for the quantitative arm of the study in 2002 and 2003 from the HIV treatments and testing clinics, gyms and through the Internet. Of these, a subset of 128 men were interviewed one-to-one for the qualitative arm (table 1) Quantitative data Data from the pen-and-paper questionnaires were coded, entered into a database and verified. Data collected online were downloaded directly into a database. The background characteristics of the different samples in the quantitative arm are presented in table 2. Table 2 Background characteristics of the men in the quantitative sample, by recruitment site HIV treatment clinic HIV testing clinics Community Internet 2002–2003 (n = 523) 2002–2003 (n = 404) 2002 (n = 914) 2003 (n = 543) 2002 (n = 1218) 2003 (n = 579) n % n % n % n % n % n % Age (median; range) 38 23–70 32 17–73 35 17–79 36 18–75 33 18–70 32 18–75 Ethnicity (white) 467 89.6 342 84.7 821 90.4 484 89.6 1117 91.7 526 89.3 Employed 324 62.8 317 91.4 771 85.3 450 83.2 1007 82.7 477 80.2 Higher education 328 65.7 261 72.9 761 83.8 425 79.7 810 66.5 377 63.9 Sexual orientation "gay" 489 93.5 354 87.6 869 95.1 523 96.3 1084 89.0 524 88.1 In a relationship with a man 285 55.3 223 55.9 469 51.6 300 55.4 531 43.6 254 42.7 HIV positive 523 100.0 15 3.7 138 15.1 88 16.2 142 11.7 67 11.3 Treatments optimism 1 201 40.9 92 26.1 175 20.3 120 23.3 164 14.5 79 14.2 Treatments optimism 2 141 28.7 85 24.6 176 20.7 117 22.9 158 15.5 94 17.0 Uses recreational drugs 277 60.6 200 60.4 467 53.2 334 64.4 497 40.8 212 38.4 Felt depressed 274 55.2 85 49.6 389 44.5 253 48.6 552 45.3 274 48.7 Had suicidal thoughts 96 21.9 46 14.3 104 12.8 65 12.8 210 17.3 105 18.7 Has access to the Internet 443 86.3 367 90.8 841 92.6 499 93.1 1193 97.9 588 98.8 Uses the Internet to seek sex 223 43.6 186 46.0 400 44.4 280 52.0 1040 85.4 544 91.4 Has sex with men only 453 *96.2 352 87.6 853 93.5 509 93.7 1089 89.4 537 90.3 STI in previous 12 months 13 27.5 79 19.8 203 22.5 127 23.5 295 24.2 128 21.7 Non-concordant UAI 116 22.2 141 34.9 199 22.1 119 22.1 391 32.1 196 32.9 Concordant UAI only 58 11.1 36 8.9 135 15.0 80 14.8 175 14.4 87 14.6 Completed clinic survey 1 - - - - - - 16 3.0 - - 26 4.7 Completed gym survey 2 25 5.0 14 3.9 - - - - 24 2.0 16 2.9 Completed 2002 online survey 3 27 5.4 26 7.3 - - 54 10.2 - - - - Treatments optimism 1: Men who agreed with the statement "I am less worried about HIV now that treatments have improved" Treatments optimism 2: Men who agreed with the statement "I believe new treatments make people with HIV less infectious" Non-concordant UAI: unprotected anal intercourse in the previous 3 months with someone of unknown or discordant status Concordant UAI only: unprotected anal intercourse in the previous 3 months only with someone of the same HIV status. * as a percentage of sexually active men (some men had not had sex in the previous year) 1 Number (%) of men completing a 2003 questionnaire in the gyms or online who said they had also completed a questionnaire in the HIV testing clinic or treatments clinic 2 Number (%) of men completing a questionnaire online or in the clinics who said they'd also completed a questionnaire in a gym 3 Number (%) of men completing a questionnaire in the gyms (2003) or in the clinics who said they'd also completed the 2002 online questionnaire In all samples, the majority of men were relatively young, white and identified as gay. There were however, differences between the samples in these and other characteristics. For example, men who completed a questionnaire online were less likely to describe themselves as gay than men who completed a questionnaire in a gym (88% v 96% in 2003). On the other hand, men surveyed online were more likely to have used the Internet to look for sex (approximately 90%) than men who surveyed in the clinics or gyms (40–50%). The differences between the samples will be explored further in future analyses and subject to formal statistical testing. For the most part the four samples were independent; only a minority of men said they had completed a questionnaire in more than one recruitment site (eg in a clinic and in a gym); range 2–10%. Data analysis, using standard statistical packages, will allow us to examine; whether sex with Internet partners is of higher risk than with other men (within-person analysis) and the extent to which Internet sex seekers were specifically looking for unprotected anal intercourse either online or offline. The characteristics of those who have and have not used the Internet to find a sexual partner will be compared to explore whether selected groups of men (eg bisexual or those at high risk) seek sex on the Internet. The importance of the Internet in relation to other venues will be examined. The samples, ranging in size from 404 to 1218, are sufficiently large to allow us to detect statistically significant differences between and within the different groups at the 5% level of significance. All analyses will be conducted for HIV positive, negative and never-tested men separately and comparisons will be made within and between the four samples. Comparison of those recruited through the Internet with the community and clinic samples will indicate whether the Internet attracts men who would otherwise be hard to reach for health promotion and HIV prevention. The data collected online from men living in the UK but outside London will provide opportunities for further analysis. For example their characteristics and behaviours can be compared with those of the London men surveyed online as well as with other men living outside London surveyed in the community (eg in Scotland). The data collected online will also allow us to examine patterns of Internet use in a sample.that covers the whole of the UK. A PhD studentship, funded separately by the UK Economic and Social Research Council (ESRC), will allow us to examine methodological issues around using the Internet for data collection and research [29], such as mode effect, motivation for participating in online surveys, number of fields completed online and offline, drop outs and non-probability sampling (see appendix 2). The ESRC-funded PhD will utilize all the data collected from men living in the UK who completed an online questionnaire in 2002 or 2003. In addition, data collected in the HIV testing clinic will also allow us to explore Internet sex-seeking and risk behaviours among heterosexual men and women. Qualitative data The interview transcripts from both the face-to-face and online interviews were coded and analysed using Nvivo. The background characteristics of the men interviewed for the qualitative arm of the study are summarized in table 3. Table 3 Background characteristics of the men in the qualitative sample, by recruitment site HIV treatment clinic HIV testing clinics Community Internet Interviewed....... Face-to-face (n = 20) Face-to-face (n = 20) Face-to-face (n = 23) Online (n = 35) Face-to-face (n = 30) n n n n n Age (median; range) 38 31–59 40 25–66 35 24–51 32 20–63 39 21–60 Employed 9 18 18 30 23 Higher education 15 15 17 20 20 Has regular sexual partner 8 8 12 14 12 HIV status HIV positive 20 - 1 6 11 HIV negative - 20 18 20 15 Never-tested - - 4 9 4 Seeks sex through the Internet Rarely, never or used to 13 14 11 6 0 Seeks sex both on and offline 3 4 5 10 14 Seeks sex mostly through the Internet 4 2 7 19 16 Interviewees were recruited for qualitative interviews once they had completed a behavioural questionnaire for the quantitative arm of the study. Additional purposive criteria were adopted to ensure the sample included men from a range of age groups; of different HIV status (HIV positive, HIV negative and never tested); of varying educational attainment; both employed and unemployed; and who reported differential use of the Internet for sexual purposes. Online, as well as face-to-face (FTF) interviews were conducted to encourage the participation of gay men who used the Internet to look for sex. This combination of matching the quantitative arm in terms of recruitment sites, together with additional purposive criteria and two interviewing methods resulted in 128 qualitative interviews with a diverse range of gay/bisexual men. All interviewees lived in the greater London area. For the qualitative arm of the study, we addressed analytic bias using guidance provided by Barbour [30], Kvale [31] and Popay & Williams [32] for whom methodological rigour is best addressed through the process of qualitative research. For example, the internal logic of the study; emphasis on the quality of the craft of research together with communicative and pragmatic forms of validation [31]; "privileging subjective meaning" [32]. In our study we addressed quality in three main ways: • Iteration • Team analysis • Transparency Iteration We conducted a mapping phase to provide an empirical context for our thinking about the Internet-related sexuality of gay men and to orient further research in terms of the sampling and topic guides. We also adopted the practice of constant comparison derived from grounded theory as another form of iteration. Team analysis The research team was involved in a cycle of reflection on the data as they were generated and analysed. In the first meeting the team reviewed a transcript to identify possible themes for analysis. Notes and the transcription of the meeting contributed to the formulation of themes. At an intermediate stage the team was asked to apply the thematic framework to other transcripts. This helped to assess the utility of the framework and elaborate possible new themes. A second meeting was then held where the team reviewed the coding scheme for the qualitative research and its application to the entire data set. This process was conducted twice – first for data generated during phase 1, then for data collected in phase 2. Transparency We documented how the data were generated, catalogued and analysed to make it open to observation. The one-to-one interviews will be analysed for recurring and contradictory themes relating to the core research questions. Phase one will help orientate the qualitative research around the Internet and risk. Phase two will provide information about the range of sexual experiences linked to the Internet as well as how people communicate about sex and risk through the Internet. The qualitative interviews will allow for greater interrogation than is permitted by self-completed questionnaire alone. A number of issues will be examined including personal experiences of using the Internet for sex, partner selection, the context in which unsafe sex occurs, the emergence of social and sexual networks via the Internet and the value or meaning placed on unsafe sex with casual partners met in different settings including the Internet. Discussion Does the Internet represent a new sexual risk environment for gay men? To answer this question, we have employed a range of quantitative and qualitative research methods in online and offline samples of London gay men. The data we collect will allow us to explore in depth the association between seeking sex on the Internet and high risk behaviour and also consider the underlying processes. One of the strengths of the Internet and HIV study is its methodological plurality. Detailed behavioural data have been collected in the quantitative arm of the study from a large number of men recruited in community and clinic settings as well as through the Internet. Questionnaires were completed online by the Internet sample. On the other hand, qualitative interviews will allow for a greater understanding of the context in which unsafe sex occurs and the processes that underlie the behavioural patterns seen in the quantitative analysis. By using both quantitative and qualitative methods, we hope to garner the best that both approaches can offer within an integrated research programme Sampling men online has allowed us to develop innovative research methods for both the quantitative and qualitative arms of the study by taking advantage of recent advances in web-based data collection. The online questionnaire provided opportunities for innovation with respect to its design, format of the questions and data entry. Conducting one-to-one interviews online has opened up new ways of undertaking qualitative research. Ours is one of a small number of studies which have explored this approach to qualitative interviewing [33]. In addition, the ESRC-funded PhD examining the Internet and research methodology will provide an opportunity to consider the advantages and disadvantages of conducting research online [29]. The Internet undoubtedly offers enormous potential for HIV prevention and sexual health promotion [34,35]. Our research will reveal whether the Internet reaches a group of men who have little contact with the established gay scene or health promotion agencies. If this is the case, the Internet could provide access to an otherwise hard-to-reach group of men. Exciting as these new opportunities are, however, we still know relatively little about the efficacy of online sexual health promotion and HIV prevention [36]. Data generated by both the quantitative and qualitative arms of our study will provide a better understanding of the social and sexual networks created through the Internet. Who is using the Internet to look for sex and how are they using it? In this way the potential for using the Internet for sexual health promotion and HIV prevention can be established Competing interests None declared. Authors' contribution JE, GH and LS conceived the study; all authors participated in its design; JE was responsible for overall project management; GB was responsible for conducting the quantitative arm of study; MD was responsible for the qualitative arm; JE drafted the manuscript with input from GB and MD. All authors read, revised and approved the final manuscript. Appendix 1 The Internet and HIV study: MRC-funded research City University London, Institute of Health Sciences, St Bartholomew School of Nursing and Midwifery Professor Jonathan Elford, principal investigator Graham Bolding, research fellow (quantitative arm) Mark Davis, research fellow (qualitative arm) MRC Social and Public Health Sciences Research Unit, Glasgow Professor Graham Hart, co-investigator Royal Free and University College Medical School, London Professor Lorraine Sherr, co-investigator Collaborators , , Royal Free Hampstead NHS Trust Hospital London, Barts and the London NHS Trust Hospital, Camden Primary Care Trust, London, Central London gyms. The research was funded from 2002–2004 by the UK Medical Research Council and the Department of Health as part of its AIDS Epidemiological Research Programme (G0100159. The Internet and HIV: an examination of high risk sexual behaviour among London gay men who seek sex on the Internet) Further information about the project can be found on our website at Appendix 2 The Internet and research methodology: ESRC-funded PhD studentship City University London, School of Social Sciences and Institute of Health Sciences, St Bartholomew School of Nursing and Midwifery Alison Evans PhD candidate School of Social Sciences and Institute of Health Sciences, St Bartholomew School of Nursing and Midwifery Professor Dick Wiggins first supervisor, School of Social Sciences, Department of Sociology Professor Jonathan Elford second supervisor, Institute of Health Sciences, St Bartholomew School of Nursing and Midwifery Advisory Board City University London Graham Bolding, Mark Davis, Professor Jonathan Elford, Professor Roger Jowell, Professor Dick Wiggins MRC Social and Public Health Sciences Research Unit, Glasgow Professor Graham Hart Royal Free and University College Medical School, London Professor Lorraine Sherr Further information can be found at Pre-publication history The pre-publication history for this paper can be accessed here: Acknowledgements This research was funded by the Medical Research Council (grant number GO 100159). The authors would like to thank: , , Royal Free Hampstead NHS Trust hospital, Barts and The London NHS Trust hospital, central London gyms as well as all the men who completed a questionnaire or were interviewed one-to-one, for their participation and support; Tony Fragar whose home in Bundeena, New South Wales provided the perfect environment for writing the first draft of this paper; Edith Stokes and the staff at Mount Pleasant, Surrey, where later drafts were written ==== Refs Elford J Bolding G Sherr L Seeking sex on the Internet and sexual risk behaviour among gay men using London gyms AIDS 2001 15 1409 15 11504962 10.1097/00002030-200107270-00010 McFarlane M Bull SS Rietmeijer CA The Internet as a newly emerging risk environment for sexually transmitted diseases JAMA 2000 284 443 6 10904506 10.1001/jama.284.4.443 Rietmeijer CA Bull SS McFarlane M Patnaik J Douglas J Risks and benefits of the Internet for populations at risk for sexually transmitted infections (STIs): results of an STI clinic survey Sexually Transmitted Diseases 2003 30 15 9 12514436 10.1097/00007435-200301000-00004 Hospers H Harterink P van den Hoek K Veenstra J Chatters on the Internet: a special target group for HIV prevention AIDS Care 2002 14 539 44 12204155 10.1080/09540120220133053 Kim A Kent C McFarland W Klausner J Cruising on the Internet highway J Acquir Immune Defic Syndr 2001 28 89 93 11579282 Benotsch E Kalichman S Cage M Men who have met sex partners via the Internet: prevalence, predictors and implications for HIV prevention Archives of Sexual Behavior 2002 31 177 84 11974643 10.1023/A:1014739203657 Elford J Surfing for sex Focus: a guide to AIDS research and counselling 2002 17 1 4 Bolding G Elford J Sherr L Hart G Behavioural surveillance among gay men using London gyms 2003 preliminary report London, City University 2003 Little S Holte S Routy J Daar E Markowitz M Collier A Antiretroviral-drug resistance among patients recently infected with HIV N Engl J Med 2002 347 385s 94 10.1056/NEJMoa013552 Ross M Tikkanen R Mansson S-A Differences between Internet samples and conventional samples of men who have sex with men: implications for research and HIV interventions Social Science and Medicine 2000 51 749 58 10975234 10.1016/S0277-9536(99)00493-1 Health Protection Agency & Scottish Centre for Infection and Environmental Health AIDS/HIV quarterly surveillance tables, cumulative UK data to end September 2003 London, HPA 2003 60 03/3 Health Protection Agency Survey of prevalent HIV infections diagnosed (SOPHID) data for 2002. HPA HPA Communicable Disease Surveillance Centre UK 2003 Health Protection Agency, SCIEH, ISD, National Public Health Service for Wales, CDSC Northern Ireland, and UASSG. Renewing the focus. HIV and other sexually transmitted infections in the United Kingdom in 2002 London, Health Protection Agency 2003 McGarrigle C Fenton K Gill O Hughes G Morgan D Evans B Behavioural surveillance: the value of national coordination Sexually Transmitted Infections 2002 78 398 405 12473798 10.1136/sti.78.6.398 Dodds J Mercey D Sexual health survey of gay men London 2002 London, Royal Free and University College Medical School 2003 Reid D Weatherburn P Hickson F Stephens M Know the score: findings from the national gay men's sex survey 2001 London, Sigma Research 2002 Elford J Bolding G Sherr L High risk sexual behaviour increases among London gay men: what is the role of HIV optimism? AIDS 2002 16 1537 44 12131192 10.1097/00002030-200207260-00011 Johnson A Mercer C Erens B Copas A McManus S Wellings K Sexual behaviour in Britain: partnerships, practices and HIV risk behaviours Lancet 2001 358 1835 42 11741621 10.1016/S0140-6736(01)06883-0 Kalichman S Kelly J Rompa D Continued high-risk among HIV seropositive gay and bisexual men seeking HIV prevention services Health Psychology 1997 16 369 73 9237089 10.1037//0278-6133.16.4.369 Kalichman S HIV transmission risk behaviors of men and women living with HIV-AIDS: prevalence, predictors and emerging clinical interventions Clinical Psychology Scientific Practice 2000 7 32 47 10.1093/clipsy/7.1.32 Crepaz N Marks G Towards an understanding of sexual risk behaviour in people living with HIV: a review of social, psychological and medical findings AIDS 2002 16 135 49 11807297 10.1097/00002030-200201250-00002 Leaity S Sherr L Wells H Evans A Miller R Johnson M Repeat HIV testing: high risk behaviour or risk reduction strategy AIDS 2000 14 547 52 10780717 10.1097/00002030-200003310-00010 Elford J Behavioral surveillance in London gyms: Trends, comparisons and methodological strengths Paper poresented at the Behavioral Surveillance Collaborator's Meeting, PHLS Communicable Disease Sruveillance Centre, London 2002 Elford J Sherr L Bolding G Maguire M Serle F Watson J, Platt S Peer-led HIV prevention among gay men in London (the 4 gym project). Intervention and evaluation Researching health promotion 2000 London: Routledge Barak A Fisher W Toward an Internet-driven, theoretically-based, innovative approach to sex education The Journal of Sex Research 2001 38 324 32 Elford J Bolding G Maguire M Sherr L Sexual risk behavior among gay men in a relationship AIDS 1999 13 1407 11 10449295 10.1097/00002030-199907300-00019 Rhodes S Bowie D Hergenrather K Collecting behavioural data using the world wide web: considerations for researchers Journal of Epidemiology and Community Health 2003 57 68 73 12490652 10.1136/jech.57.1.68 Couper MP The promises and perils of web surveys London, Association for Survey Computing 2001 Fricker R Schonlau M Advantages and disadvantages of Internet research surveys: evidence from the literature Field Methods 2002 14 347 67 10.1177/152582202237725 Barbour R Checklists for improving rigour in qualitative research: a case of the tail wagging the dog? British Medical Journal 2001 322 1115 7 11337448 10.1136/bmj.322.7294.1115 Kvale S InterViews: an introduction to qualitative research interviewing Thousand Oaks: Sage 1996 Popay J Williams I Qualitative research and evidence-based health care J R Soc Med 1998 91 32 7 9797748 Davis M Bolding G Hart G Sherr L Elford J Reflecting on the experience of interviewing online: perspectives from the Internet and HIV study in London AIDS Care 2004 Ross M The Internet as a medium for HIV prevention and counselling Focus: a guide to AIDS research and counselling 2002 17 4 6 Elford J Treatments optimism, the Internet and risk: an update on HIV prevention research among gay men AIDS and Hepatitis Digest 2003 97 2 5 Bull S Lloyd L Rietmeijer C McFarlane M Recruitment and retention of an online sample for an HIV prevention intervention targeting men who have sex with men: the Smart Sex Quest project AIDS Care 2004

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==== Front BMC GenomicsBMC Genomics1471-2164BioMed Central London 1471-2164-5-671536960110.1186/1471-2164-5-67Research ArticleComplete mitochondrial genome sequence of Urechis caupo, a representative of the phylum Echiura Boore Jeffrey L [email protected] Evolutionary Genomics Department, DOE Joint Genome Institute and Lawrence Berkeley National Laboratory, 2800 Mitchell Drive, Walnut Creek, CA, USA2 Department of Integrative Biology, University of California, Berkeley, CA, USA2004 15 9 2004 5 67 67 10 2 2004 15 9 2004 Copyright © 2004 Boore; licensee BioMed Central Ltd.2004Boore; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Mitochondria contain small genomes that are physically separate from those of nuclei. Their comparison serves as a model system for understanding the processes of genome evolution. Although hundreds of these genome sequences have been reported, the taxonomic sampling is highly biased toward vertebrates and arthropods, with many whole phyla remaining unstudied. This is the first description of a complete mitochondrial genome sequence of a representative of the phylum Echiura, that of the fat innkeeper worm, Urechis caupo. Results This mtDNA is 15,113 nts in length and 62% A+T. It contains the 37 genes that are typical for animal mtDNAs in an arrangement somewhat similar to that of annelid worms. All genes are encoded by the same DNA strand which is rich in A and C relative to the opposite strand. Codons ending with the dinucleotide GG are more frequent than would be expected from apparent mutational biases. The largest non-coding region is only 282 nts long, is 71% A+T, and has potential for secondary structures. Conclusions Urechis caupo mtDNA shares many features with those of the few studied annelids, including the common usage of ATG start codons, unusual among animal mtDNAs, as well as gene arrangements, tRNA structures, and codon usage biases. mtDNAevolutiongene rearrangementannelidstrand skew ==== Body Background Mitochondrial genomes are physically separate from the nuclear genome. For animals, they are typically circular with a compact arrangement of an identical set of 37 genes [1]. For some animals, all genes are on the same strand, whereas for others they are divided between the two. The arrangement of these genes can remain stable for long periods of time; for example, human [2] and shark [3] mtDNAs have the same gene arrangement, and do those of fruit fly [4] and shrimp [5]. However, in other lineages, rearrangements have occurred much more rapidly. Many of the same processes that occur in large and complex nuclear genomes also take place in these diminutive genomes, so comparisons among mtDNAs can address general questions of genome evolution, but in a model system that is much more tractable for a large number of taxa. Toward this end, this article describes the complete mtDNA sequence of the fat innkeeper worm, Urechis caupo, the first example from the phylum Echiura. Echiurans comprise about 150 species and are commonly called spoon worms because of the shape of their extensible proboscis. Unlike annelids, they have no overt segmentation, but they develop via trochophore larvae, very similar to those of annelids. U. caupo is a pink, sausage shaped worm that lives in U-shaped burrows in the mud or sand of the intertidal or subtidal zones. Unlike other echiurans, it feeds on plankton by filtering using an elaborate mucus net. Results and discussion Gene content and organization The mtDNA of Urechis caupo is 15,113 nts in length (GenBank accession number AY619711) and contains the same 37 genes found for nearly all animal mtDNAs [see ref. [1]]. All genes are transcribed from the same strand (Fig. 1), as is the case for the two studied annelid mtDNAs, the polychaete Platynereis dumerilii [6] and the oligochaete Lumbricus terrestris [7] and for several other animal mtDNAs. The arrangement of the genes is substantially similar to those of the two annelids, and shares short regions of similarity with several other mtDNAs, as can be seen in Table 1. Figure 1 Mitochondrial gene map of the echiuran Urechis caupo. All genes are transcribed from the same DNA strand. Scaling is only approximate. Genes are designated by standard nomenclature except for tRNAs, which are identified only by the one-letter code for the corresponding amino acid, with the two serine and two leucine tRNAs differentiated by numeral as identified in Fig. 3. "nc" indicates the largest non-coding regions; it may be that transcription initiates here, but this is not known. Table 1 Mitochondrial gene arrangement identities found in pairwise comparisons between Urechis caupo and various animals. Full taxon names are given here for the annelids Lumbricus terrestris and Platynereis dumerilii, the mollusks Katharina tunicata, Loligo bleekeri, Cepaea nemoralis, and Mytilus edulis, the brachiopods Terebratulina retusa and Terebratalia transversa, the platyhelminths Fasciola hepatica, Taenia crassiceps, Echinococcus multilocularis, and Hymenolepis diminuta, the arthropods Drosophila yakuba, Anopheles gambiae, Artemia franciscana, Daphnia pulex, Apis mellifera, Locusta migratoria, Ixodes hexagonus, Rhiphicephalus sanguineus, Limulus polyphemus and Lithobius forficatus, the nematodes Trichinella spirallis, Onchocerca volvulus, Meloidogyne javanica, Ascaris suum, and Caenorhabditis elegans, the echinoderms Arabacia lixula, Asterina pectinifera, Paracentrotus lividus, Strongylocentrotus purpuratus, and Florometra serratissima, the hemichordate Balanoglossus carnosus, and the chordate Branchiostoma floridae along with the gene order most typical for vertebrates. Complete citations can be found in Boore (1999) or updated by following the "Evolutionary Genomics" link at . Contiguous gene arrangements are separated by a comma; a slash indicates a gap containing one or more unrelated genes. L. terrestris and P. dumerilii cox3, trnQ, nad6, cob, trnW, atp6, trnR, trnH, nad5, trnF/trnL2, nad1, trnI, trnK, nad3/trnT, nad4L, nad4 L. terrestris but not P. dumerilii trnL1, trnA, trnS2, trnL2/trnD, atp8 K. tunicata trnL2, nad1/nad4L, nad4/trnH, nad5, trnF L. bleekeri nad6, cob/nad4L, nad4/nad5, trnF/trnD, atp8 C. nemoralis trnL1, trnA M. edulis trnL2, nad1/trnT, nad4L T. retusa trnL2, nad1/nad4L, nad4/trnH, nad5, trnF/trnL1, trnA/trnD, atp8/cox1, cox2 T. transversa trnP, trnD F. hepatica, T. crassiceps, E. multilocularis trnI, trnK, nad3/nad4L, nad4/trnY, trnL1/trnS2, trnL2 H. diminuta trnI, trnK, nad3/nad4L, nad4/trnY, trnL1 D. yakuba, A. gambiae, A. franciscana, D. pulex nad6, cob/nad4L, nad4/trnH, nad5, trnF/trnD, atp8 A. mellifera, L. migratoria nad6, cob/nad4L, nad4/trnH, nad5, trnF I. hexagonus, R. sanguineus, L. polyphemus, L. forficatus nad6, cob/trnL2, nad1/nad4L, nad4/trnH, nad5, trnF/trnD, atp8/cox1, cox2 T. spirallis nad6, cob/nad4L, nad4/trnH, nad5, trnF/trnR, trnH/trnL1, trnA/trnD, atp8/cox1, cox2 O. volvulus trnP, trnD M. javanica trnN, trnG A. suum NONE C. elegans NONE A. lixula, A. pectinifera, P. lividus and S. purpuratus trnL2, nad1, trnI F. serratissima nad1, trnI/nad2, trnY B. carnosus trnL2, nad1/nad4L, nad4 B. floridae and the typical vertebrate arrangement trnL2, nad1, trnI/nad4L, nad4 Base composition and codon usage The U. caupo mtDNA is 62% A+T, about the same as for annelid mtDNAs (64% and 62% for P. dumerilii and L. terrestris, respectively). As is typical, all homodinucleotides and homotrinucleotides are greatly over represented relative to a random distribution and CG is the least frequent dinucleotide, both in absolute number and in the ratio of observed to expected. The gene-coding strand has a strong skew against G vs. C but about equal amounts of A vs. T; G-skew ([G-C]/[G+C]) is – 0.24 and T-skew ([T-A]/[T+A]) is – 0.016 [8]. These values show no striking variation across the length of the mtDNA. Codon usage (Table 2) reflects these values, with those ending in A or T being most frequent. In all cases except for two, where they are synonymous, NNC codons are in greater abundance than NNG codons, as is consistent with the coding strand being rich in C relative to G. The two exceptions are CGG and GGG codons, which are each in greater abundance than their respective synonyms, CGC and GGC. This invites the speculation that there is something favored about the GG dinucleotide created when G appears in the second codon position. However, this is not consistently seen, since in the remaining case, AGC codons outnumber AGG codons two-to-one. This effect has been shown to be very strong for codon usage pattern of the mtDNA of the brachiopod Terebratalia transversa [9]. Table 2 Codon usage in the 13 protein-encoding genes of the Urechis caupo mitochondrial genome. The total number of codons is 3722. The anticodon of the corresponding tRNA gene is shown in parentheses below each amino acid designation. Stop codons are not included in this analysis. Amino acid Codon N % Amino acid Codon N % Phe (F) TTT 161 4.3% Ser (S2) TCT 108 2.9% (GAA) TTC 115 3.1% (TGA) TCC 65 1.7% Leu (L2) TTA 146 3.9% TCA 74 2.0% (TAA) TTG 10 0.3% TCG 3 0.1% Tyr (Y) TAT 42 1.1% Cys (C) TGT 18 0.5% (GTA) TAC 65 1.7% (GCA) TGC 11 0.3% TER TAA --- --- Trp (W) TGA 77 2.1% TAG --- --- (TCA) TGG 21 0.6% Leu (L1) CTT 105 2.8% Pro (P) CCT 72 1.9% (TAG) CTC 62 1.7% (TGG) CCC 44 1.2% CTA 224 6.0% CCA 80 2.1% CTG 38 1.0% CCG 6 0.2% His (H) CAT 33 0.9% Arg (R) CGT 6 0.2% (GTG) CAC 55 1.5% (TCG) CGC 5 0.1% Gln (Q) CAA 84 2.3% CGA 46 1.2% (TTG) CAG 12 0.3% CGG 9 0.2% Ile (I) ATT 200 5.4% Thr (T) ACT 76 2.0% (GAT) ATC 100 2.7% (TGT) ACC 91 2.4% Met (M) ATA 171 4.6% ACA 93 2.5% (CAT) ATG 52 1.4% ACG 5 0.1% Asn (N) AAT 61 1.6% Ser (S1) AGT 7 0.2% (GTT) AAC 65 1.7% (TCT) AGC 16 0.4% Lys (K) AAA 78 2.1% AGA 62 1.7% (TTT) AAG 14 0.4% AGG 8 0.2% Val (V) GTT 49 1.3% Ala (A) GCT 75 2.0% (TAC) GTC 28 0.8% (TGC) GCC 75 2.0% GTA 99 2.7% GCA 122 3.3% GTG 18 0.5% GCG 14 0.4% Asp (D) GAT 23 0.6% Gly (G) GGT 14 0.4% (GTC) GAC 37 1.0% (TCC) GGC 27 0.7% Glu (E) GAA 73 2.0% GGA 127 3.4% (TTC) GAG 10 0.3% GGG 36 1.0% Gene initiation and termination Mitochondrial genes commonly use several alternatives to ATG as start codons. However, 11 of the 13 proteins coding genes of U. caupo mtDNA use ATG. The only exceptions are cox1, which uses GTG and nad3 which uses ATC. In the case of cox1, there is an in frame stop only three codons upstream and neither of the intervening codons is ATG. Also, this inference of starting on GTG specifies a set of amino acids well matched to those at the beginning of other Cox1 proteins. The situation for nad3 is nearly identical, with an in frame stop only four codons upstream and no intervening ATG codons. However, downstream of the inferred start are several ATA codons that can not be ruled out as alternatives. The commonality of using ATG as a start codon has also been noted for mitochondrial genes of four annelids, Platynereis dumerilii [6], Lumbricus terrestris [7], Helobdella robusta and Galathealinum brachiosum (previously considered to be of the phylum Pogonophora) [10] and a sipunculid, Phascolopsis gouldii [11]. A complete stop codon without overlap of the downstream gene is found for all except cox2, nad1, nad2, cob, and nad5 (Fig. 2). In each of these cases, it appears that an abbreviated stop codon is generated by cleavage of a downstream tRNA from the polycistronic transcript, which is then completed to a TAA stop codon by polyadenylation. However, in two of these cases (nad2 and cob), a complete stop codon could be formed by including only the next two nucleotides, and two other cases (nad1 and cox2), there is an in frame stop codon just one or two codons downstream, respectively. It is not clear how gene overlaps could be resolved from a polycistronic transcript (assuming that the genes of this mtDNA are expressed in this way), but the presence of these stop codons seems beyond coincidence. It could be that they serve as a "back up" in case translation should begin in the absence of transcript cleavage. Figure 2 A greatly abbreviated schematic of the sequence of Urechis caupo mtDNA. In the interest of brevity, the middle portion of each large gene is omitted and replaced by a numeral indicating the number of nucleotides removed. Since all mitochondrial proteins are thought to initiate with formyl-methionine, an M is placed in parentheses at the first codon position of cox1 (GTG) and nad3 (ATC) to indicate nonconformity to the genetic code. Asterisks indicate inferred stop codons whether complete or abbreviated and plus symbols mark nucleotides that would form the first in frame, complete stop codon if genes instead overlap. Transfer RNAs Twenty-two regions can be folded into the typical cloverleaf structures of the expected set of tRNAs (Fig. 3). There are several mismatched nucleotide pairs within stems; nearly all of these are flanked by multiple G-C pairs, suggesting that they may provide compensatory stability for these arms. T precedes the anticodon and a purine follows it for all tRNAs. The two serine tRNAs lack potential for folding a DHU arm, as has been found for a number of other animal mtDNAs. There is an alternative folding possible for tRNA(S2) with a six-member anticodon stem and only one nucleotide separating the acceptor and DHU stems; this unusual folding has been found for the homologous genes of some mammals. tRNA(R) also does not have potential for a normally paired DHU arm, although there are three potential nucleotide pairs if two (rather than one) nucleotides were between the DHU and anticodon stems. However, this potential pairing could, alternatively, be a coincidence, with the DHU arm having no paired stem for this tRNA. Those with paired DHU arms have stems of three to five nucleotide pairs and loops of three to eight nts. All tRNAs have potential for stems of three to six nucleotide pairs for their TΨC arms with loops of three to seven nts. One of the tRNAs for serine has the anticodon TCT; although this is often found, the alternative of GCT is otherwise common. Figure 3 The 22 inferred tRNA genes folded into the typical cloverleafstructures. Nomenclature for tRNA substructures is indicated on tRNA(V). Ribosomal RNAs As has been the case for all studied animal mtDNAs to date, two rRNA genes are identified, one for each of the small and large mitochondrial ribosomal subunits. Determining the precise ends of the rRNA transcript requires experimentation, but if it's assumed that they extend to the boundaries of the adjacent genes, then rrnS is 903 nucleotides and rrnL is 1266 nucleotides in length. These genes are arranged sequentially, but without an intervening tRNA gene as is otherwise commonly found. Non-coding regions The largest non-coding region is only 282 nts long. The region is 71% A+T and contains one palindrome of an 11 nt sequence (TCAAAAGGGGT/ACCCCTTTTGA, with a slash indicating the center), but otherwise no large repeat elements. Obviously, this has potential for forming a stem-loop structure, and it may be significant that a short sequence a few nucleotides upstream, TCAAAA, has the potential for competing with this to form a short hairpin with the TTTTGA at the end of the palindrome. There has been previous speculation that regions with potential for competing, mutually exclusive hairpins may play a role in regulating transcription and/or replication [e.g. ref. [7]]. There are four other potential hairpins in this region with stems of 5–6 bp and loops of 5–17 nt. All four nucleotides occur in homopolymers with much greater frequency than expected by chance, often in runs of four or five. The second largest non-coding region is 43 nt between trnS1 and cox3. This has no repeat elements and the base composition is unremarkable. What role, if any, these sequences have in the regulation of transcription and/or replication awaits further study. Aside from these 282 and 43 nt regions, there are only 36 total intergenic nucleotides scattered among 14 regions. In seven cases these are 2–6 nts long (CCAAA, AT, TCCC, TAAA, CATAAA, AT, and ACACCT). For the other seven cases, genes are separated by a single nucleotide, and in six of these, that nucleotide is a C. (The remaining case is a T.) The prevalence of C is consistent with the measured G-skew between the strands, although it is possible that this otherwise indicates some function of these nucleotides. Conclusions This is the first description of a complete mitochondrial genome sequence of a representative of the phylum Echiura. The genome contains the same 37 genes most commonly found in animal mtDNAs. Many features are most similar to those found for annelid mtDNAs, including A+T content, use of protein initiation codons, size and potential secondary structures of the largest non-coding region, and the relative arrangement of many genes. As in annelids examined to date, all genes are found on the same DNA strand. As noted for brachiopod mtDNA, there is a preference for G nucleotides to appear in tandem, without obvious explanation. Further description and comparison of complete mtDNA sequences will continue to produce a picture of genome evolution, particularly once sampling includes representatives of each animal phylum. Methods Molecular techniques A preparation of Urechis caupo total DNA was the kind gift of Eric Rosenthal. The entire mtDNA sequence was obtained using techniques detailed in [9]. Briefly, small fragments (450–710 nt) were amplified from cox1, cob, and rrnS using primer pairs HCO 2198/LCO 1490 [12], CytbF/CytbR [10], and 16SARL/16SBRH [13], respectively. The sequences of these fragments were determined using dye-terminator chemistry (PE Biosystems) on an ABI 377 automated DNA sequencer. Primers were then designed facing "out" from these fragments to amplify the intervening regions (~2.9 to ~8 Kb) using long-PCR protocols with rTth-XL polymerase (PE Biosystems) as in [9]. Sequences were determined from the ends of these long-PCR fragments, then internally by "primer walking". To ensure quality, all sequences were determined on both strands and base calls for all chromatograms were verified by eye. Gene annotation Genes encoding rRNAs and proteins were identified by matching nucleotide or inferred amino acid sequences to those of Lumbricus terrestris mtDNA [7]. Since it is not possible to precisely determine the ends of rRNA genes by sequence data alone, they were assumed to extend to the boundaries of flanking genes. Each protein gene start was inferred as the eligible initiation codon nearest to the beginning of its alignment with homologous genes that does not cause overlap with the preceding gene. In five cases, an abbreviated stop codon was inferred where cleavage of a downstream tRNA from the transcript would leave a partial codon of T or TA, such that subsequent mRNA polyadenylation could generate a TAA stop codon. In each case an extension of this gene to the first in frame stop codon would cause overlap with the downstream tRNA. Genes for tRNAs were identified generically by their ability to fold into a cloverleaf structure and specifically by anticodon sequence. Abbreviations cox1, cox2, cox3, cytochrome oxidase subunit I, II, and III protein genes; cob, cytochrome b gene; atp6, atp8, ATP synthase subunit 6 and 8 genes; nad1, nad2, nad3, nad4, nad4L, nad5, nad6, NADH dehydrogenase subunit 1–6, 4L genes; trnA, trnC, trnD, trnE, trnF, trnG, trnH, trnI, trnK, trnL1, trnL2, trnM, trnN, trnP, trnQ, trnR, trnS1, trnS2, trnT, trnV, trnW, trnY, transfer RNA genes designated by the one-letter code for the specified amino acid, with numerals differentiating cases where there are two tRNAs for the same amino acid. Acknowledgments I am grateful to Eric Rosenthal for Urechis caupo DNA. This work was supported by DEB-9807100 from the National Science Foundation and by contract No. DE-AC03-76SF00098 between the U.S. Department of Energy Office of Biological and Environmental Science, and the University of California, Lawrence Berkeley National Laboratory. ==== Refs Boore JL Animal mitochondrial genomes Nucleic Acids Res 1999 27 1767 1780 10101183 10.1093/nar/27.8.1767 Anderson S Bankier AT Barrell BG DeBruijn MHL Coulson AR Drouin J Eperon IC Nieflich DP Roe BA Sanger F Schreier PH Smith AJH Staden R Young IG Sequence and organization of the human mitochondrial genome Nature 1981 290 457 465 7219534 Cao Y Waddell PJ Okada N Hasegawa M The complete mitochondrial DNA sequence of the shark (Mustelus manazo): Evaluating rooting contradictions to living bony vertebrates Mol Biol Evol 1998 15 1637 1646 9866199 Clary DO Wolstenholme DR The mitochondrial DNA molecule of Drosophila yakuba: Nucleotide sequence, gene organization, and genetic code J Mol Evol 1985 22 252 271 3001325 Wilson K Cahill V Ballment E Benzie J The complete sequence of the mitochondrial genome of the crustacean Penaeus monodon: Are malacostracan crustaceans more closely related to insects than to branchiopods? Mol Biol Evol 2000 17 863 874 10833192 Boore JL Complete mitochondrial genome sequence of the polychaete annelid Platynereis dumerilii Mol Biol Evol 2001 18 1413 1416 11420379 Boore JL Brown WM Complete DNA sequence of the mitochondrial genome of the annelid worm, Lumbricus terrestris Genetics 1995 141 305 319 8536978 Perna NT Kocher TD Patterns of nucleotide composition at fourfold degenerate sites of animal mitochondrial genomes J Mol Evol 1995 41 353 358 7563121 10.1007/BF00186547 Helfenbein KG Brown WM Boore JL The complete mitochondrial genome of a lophophorate, the brachiopod Terebratalia transversa Mol Biol Evol 2001 18 1734 1744 11504853 Boore JL Brown WM Mitochondrial genomes of Galathealinum, Helobdella, and Platynereis: Sequence and gene arrangement comparisons indicate that Pogonophora is not a phylum and Annelida and Arthropoda are not sister taxa Mol Biol Evol 2000 17 87 106 10666709 Boore JL Staton J The mitochondrial genome of the sipunculid Phascolopsis gouldii supports its association with Annelida rather than Mollusca Mol Biol Evol 2002 19 127 137 11801741 Folmer O Black M Hoeh R Lutz R Vrijenhoek R DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates Mol Mar Biol Biotechnol 1994 3 294 299 7881515 Palumbi SR Hillis DM, Moritz C, Mable BK Nucleic acids II: The polymerase chain reaction Molecular Systematics 1996 Sinauer Associates, Sunderland, Massachusetts, USA 205 247

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==== Front BMC BioinformaticsBMC Bioinformatics1471-2105BioMed Central London 1471-2105-5-1321537394710.1186/1471-2105-5-132Research ArticleSimple statistical models predict C-to-U edited sites in plant mitochondrial RNA Cummings Michael P [email protected] Daniel S [email protected] Center for Bioinformatics and Computational Biology, University of Maryland, College Park, MD 20742-3360, USA2004 16 9 2004 5 132 132 7 4 2004 16 9 2004 Copyright © 2004 Cummings and Myers; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background RNA editing is the process whereby an RNA sequence is modified from the sequence of the corresponding DNA template. In the mitochondria of land plants, some cytidines are converted to uridines before translation. Despite substantial study, the molecular biological mechanism by which C-to-U RNA editing proceeds remains relatively obscure, although several experimental studies have implicated a role for cis-recognition. A highly non-random distribution of nucleotides is observed in the immediate vicinity of edited sites (within 20 nucleotides 5' and 3'), but no precise consensus motif has been identified. Results Data for analysis were derived from the the complete mitochondrial genomes of Arabidopsis thaliana, Brassica napus, and Oryza sativa; additionally, a combined data set of observations across all three genomes was generated. We selected datasets based on the 20 nucleotides 5' and the 20 nucleotides 3' of edited sites and an equivalently sized and appropriately constructed null-set of non-edited sites. We used tree-based statistical methods and random forests to generate models of C-to-U RNA editing based on the nucleotides surrounding the edited/non-edited sites and on the estimated folding energies of those regions. Tree-based statistical methods based on primary sequence data surrounding edited/non-edited sites and estimates of free energy of folding yield models with optimistic re-substitution-based estimates of ~0.71 accuracy, ~0.64 sensitivity, and ~0.88 specificity. Random forest analysis yielded better models and more exact performance estimates with ~0.74 accuracy, ~0.72 sensitivity, and ~0.81 specificity for the combined observations. Conclusions Simple models do moderately well in predicting which cytidines will be edited to uridines, and provide the first quantitative predictive models for RNA edited sites in plant mitochondria. Our analysis shows that the identity of the nucleotide -1 to the edited C and the estimated free energy of folding for a 41 nt region surrounding the edited C are the most important variables that distinguish most edited from non-edited sites. However, the results suggest that primary sequence data and simple free energy of folding calculations alone are insufficient to make highly accurate predictions. ==== Body Background RNA editing is the process whereby an RNA sequence is modified from the sequence corresponding to the DNA template. A particular form of RNA editing in plant mitochondria, by which some cytidines are converted to uridines before translation, occurs in many land plant lineages. Although cytidine to uridine conversion is most common, the reverse conversion is sometimes observed [1-4]. In plants, the phenomenon is best studied, albeit still poorly understood, in the mitochondria and plastids of angiosperms [5-8]. The majority of plant mitochondrial RNA editing occurs in coding sequences, and editing frequently changes codons, resulting in changes of amino acids, or, in some cases, creation of entirely new open reading frames [1,9,10]. These changes often result in an increase in similarity with respect to homologous protein sequences among different organisms (such as in wheat [11]), and Gray has postulated that the RNA editing process functions as a repair mechanism to correct otherwise-deleterious genomic mutations [12]. RNA editing has also been detected in introns, where it is conjectured to improve splicing efficiency [13]. The precise biochemical basis for C-to-U editing in plant mitochondria is unknown, although experimental evidence suggests a deamination reaction [14-18]. Despite substantial study, the molecular biological mechanism by which C-to-U RNA editing proceeds remains relatively obscure, although several experimental studies have implicated a role for cis-recognition [19-21]. The mechanism by which edited sites are recognized is also still poorly understood, but the importance of surrounding nucleotides has been noted [22]. A highly non-random distribution of nucleotides in the immediate vicinity of edited sites (within 10–20 nucleotides 5' and 3') is observed, but no precise consensus motif has been identified [9,16]. Additionally, previous studies suggest that inferred secondary structure is not important in site recognition for C-to-U conversion [16,19]. Identifying edited sites thus remains an open problem, one to which we have applied tree-based statistical models and an extension of such models. When applied to a similar problem (predicting peptide binding to major histocompatibility complex (MHC) class I molecules [23]), tree-based statistical methods generated very accurate models, identifying specific important residues when no precise sequence motif had previously been identified. Therefore, we were motivated to apply tree-based statistical models and an extension, random forests, to the problem of C-to-U RNA editing in angiosperm mitochondria using complete mitochondrial genome data for three species: Arabidopsis thaliana, Brassica napus and Oryza sativa. The objective for the current research was to identify sequence features that may provide insights into C-to-U editing of plant mitochondrial RNA. We address the following specific questions. Is there evidence that sufficient information exists within sequence regions flanking edited sites to accurately predict editing? Is there an association between estimated free energy of folding for short sequence regions containing edited sites and C-to-U editing? We report tree-based statistical analysis of three complete mitochondrial genomes and show that relatively simple models provide moderately accurate prediction of C-to-U edited sites. Results Tree-based statistical models Analysis of each of the three species-specific mitochondrial genome data sets yielded substantially similar results (Table 1). Using flanking nucleotides and estimates of folding energy as predictor variables, the optimistic re-substitution-based estimates for cross-validated pruned models had a mean correct classification rate of 0.705 (sensitivity [the proportion of observations correctly identified as edited] = 0.640, and specificity [the proportion of observations correctly identified as non-edited] = 0.883) across the three species. As an additional classification tree analysis, we examined a dataset generated by combining the data from the three species. These results were generally similar to those described above for the mean of the individual genome datasets. The classification tree model is shown in Figure 1; the partition is defined based on the nucleotide immediately 5' (-1 position) of the edited/non-edited site. Of the 1972 observations with pyrimidine at the -1 position, 1262 (0.64) are edited and 710 (0.36) are non-edited sites. Of the 722 observations with purine at the -1 position, 85 (0.12) are edited and 637 (0.88) are non-edited sites. Random forests Results from random forests (Table 2) were very similar to those obtained with classification trees and were somewhat more accurate. In single-species analyses, the mean accuracy rate was 0.744 (sensitivity = 0.717, specificity = 0.809). Analysis of the larger, combined data set yielded a model better than any of the single genome models with an accuracy of 0.848 (Table 2). Analysis of variable importance showed that the -1 position is overwhelmingly the most important factor in determining editing status. Other variables of lesser predictive value include estimated free energy of folding, and the -2 and +1 positions relative to the edited/non-edited site (Figure 2). Discussion Despite their simplicity, the tree-based statistical models derived here performed moderately well, with mean accuracies across species generally ~0.71. Single trees were improved upon by constructing models based on ensembles of tree-based models (random forests) each of which was built using random subsamples of the data. This sub-sampling has the effect of reducing the variance through averaging and also reducing the correlation among models. One of the advantages that random forests have over single classification trees is that they provide quantitative measures of variable importance, whereas with a simple classification tree, one is primarily limited to inferring variable importance from the frequency and location of the occurrence of variables in the model. One measure of variable importance is the decrease in the Gini index (a measure of impurity of observations at a particular node) induced by splitting on the variable, averaged over all trees [24]. In order to infer the relative importance of the predictor variables, we considered the measure of variable importance produced during the random forest run on the combined dataset, which is the most broadly representative dataset considered here. A plot of the variable importance measure for this dataset is shown in Figure 2; more important variables are shown as higher bars. The measure strongly indicates that the residue immediately 5' of the edited site (-1 position) is very important. These variable importance results are in agreement with previous work on C-to-U editing in mitochondria of Arabidopsis thaliana, which noted the -1, and -2 positions had highly non-random nucleotide distributions [9]. However, the results here differ from the past study of Arabidopsis in that we find no indication that the -17 position has much importance in edited site recognition. Also previously noted was that for 93.1% of the time [9], the -1 position contained a pyrimidine, which is the data partition found by the classification trees. The free energy results contrast with previous studies indicating that secondary structure was not important in edited site recognition [16,19]. Our results show free energy is a relatively important variable in the random forest analyses. These results therefore indicate that secondary structure, as measured by free energy of folding for the 41 nt region centered on an edited/non-edited site, does help in distinguishing edited from non-edited sites. Previous studies determined putative secondary structures for mRNA regions containing edited sites and looked for conserved structural motifs. In contrast, we used estimates of free energy of folding, which are much easier to compare quantitatively. It may be that secondary or tertiary structure is even more important in determining edited sites than shown here; however, secondary structure may not be effectively represented by the calculated estimates of free energy of folding analyzed. Conclusions Simple models based on nucleotides surrounding edited/non-edited sites and on estimated folding energies of those regions provide moderately accurate prediction of C-to-U RNA edited sites. More nuanced representation of secondary or higher-order structure in combination with variables based on the nucleotide positions found important here might improve models. Overall, the results strongly suggest that the C-to-U editing mechanism in plant mitochondria does not depend exclusively on the primary sequence immediately in the vicinity of the edited site. Methods Data sources We obtained complete mitochondrial genome sequences and information regarding edited sites from GenBank [25] for three species: Arabidopsis thaliana (L.) Heynh. (mouse-ear cress), 455 edited sites, GenBank accession number NC_001284 [9]; Brassica napus L. (rapeseed), 425 edited sites, GenBank accession number AP006444 [26]; and Oryza sativa L (rice), 486 edited sites, GenBank accession numbers AB076665 and AB076666 [27]. None of the GenBank entries noted U-to-C RNA edited sites. Variable selection Incomplete annotations in the GenBank sequences required us to algorithmically determine on which strand an edited site fell (the GenBank files sometimes supplied only a position number, with no strand information). The algorithm, implemented in a Perl script, scanned the entire GenBank file and built an in-memory representation of the layout of all genes and coding sequence regions in the genome. The strand with which an edited site was associated could then be determined by consulting the resultant genome map and checking which strand at the edited site contained a gene region. In no case were genes on both strands at an edited site, so strand localization was always unambiguous. In a few cases, however, a gene containing an edited site could not be located, or a site marked as a C-to-U edit did not contain a C in either strand. In these cases, the supposed edited site was eliminated from further consideration. Final numbers of included sites were as follows: Arabidopsis, 444; Brassica, 422; Oryza, 481. In total, 19 edited sites in the GenBank files were not included across all three species. We also constructed a set of null observations of cytidines that are not edited to uridines. In constructing a null-set, it is important to ensure that the observations are as alike as possible to the edited observations (differing only in the trait to be measured), or the resulting model may be fictive. Here, our null-set observations were non-edited cytidines chosen at random from within gene regions of the genome. Additionally, we chose cytidines such that the null set had exactly the same distribution of codon positions as did the edited set, because the distribution of edited sites within the three possible positions of a codon is highly non-random with a bias to the first two positions [9] (Table 3). For each observation, we recorded 40 nucleotide state variables: one variable for each of the 20 nucleotides sites 5' and 3' of the edited C (on the same strand). We chose a value of 20 for the number of nucleotides 5' and 3' so as to encompass the entire range of semi-conserved positions previously suggested, the most extreme of which occurs 17 bases 5' of the edited site [9]. In some cases other edited sites occurred within the 20 nucleotides 5' and 3' of the edited site used as a response variable. In these cases the edited sites as predictor variables were recorded as C. The low frequency of these sites at a particular position with respect to other edited sites results in non-significant effects, independent of how these sites are handled. In those cases where a full 20 nucleotides were not included within an annotated mRNA, the missing nucleotides were treated as unknown. Additionally, we included two variables based on free energy expressed in units of kcal/mole at 20°C: the estimated free energy of folding for each 41-nucleotide sequence (20 bases 5', the edited/non-edited base, 20 bases 3') and the change in free energy of folding between the non-edited and edited versions of the 41-nucleotide sequence. Free energies of folding were calculated using mfold [28,29] version 3.1 with program parameters except temperature at default values. Finally, we included codon position as a variable, even though the null set had been chosen so non-edited sites had the same distribution of codon position as the edited sites, as shown in Table 3. Including codon position as a predictor variable allows for possible interactions with other variables. Finally, we created a combined data set to use alongside the species-specific datasets. The combined dataset is the result of combining all edited sites from all three species (there were no observations identical in all predictor variables), and then randomly selecting negative examples from the set of those already chosen for the three individual datasets. Negative examples were chosen to exactly match the positive examples in distribution over both species and codon position. The combined dataset comprises 2,694 observations. Data analysis Tree-based statistical models We used the R language for statistical computing [30], version 1.7.1 to conduct our analyses. Analyses included tree-based statistical models using rpart [31] and random forests using the FORTRAN implementation of random forest version 3.1 [24,32]. Tree-based statistical models [33], also known as classification and regression trees (CART) [34], are generated by recursively creating binary partitions of a dataset. Each partition is based on the value of a single predictor variable chosen to best produce homogeneous collections of a nominal or ordinal response variable (classification) or to best separate low and high values of a continuous response variable (regression). More precisely, the partitions may be considered as questions of the following form: Is the observation xi ∈ A? Where A is a region of the variable space defined by some criterion of a single predictor variable. Answering such a question for all observations produces two groups: those observations for which the answer is yes (those in region A) and those for which the answer is no (xi ∉ A, those in ). Subsequent binary partitioning continues until stopping criteria (variously defined) are met [34]. The result is a classification or a regression tree: a hierarchical series of data bifurcations that depicts the partition definitions and describes the resulting data subsets defined by each partition. To address concerns about possible over-fitting models to the data we used 10-fold cross-validation and pruned trees to the shortest within 1-SE of the best tree. We assessed the significance of our tree-based statistical models through permutation where the predictor variables are randomized with respect to the response variable [35]. The frequency of observing a result value equal to or better than the observed value in 1 × 104 permutations is the estimate of the probability associated with the observed result. Random forests If one tree-based statistical model is good, then an ensemble (forest) of appropriately constructed tree models should be even better, which is the principal idea of random forests. A random forest attempts to improve upon a simple tree-based statistical model by generating a collection of such models and using them in aggregate [24,32]. Each model in a random forest is generated from a bootstrap sample of the original dataset, and at each node in each model a search for the best possible split is through a subset of variables selected at random from the bootstrap sample of predictor variables. These randomization steps decrease prediction error through variance reduction resulting from averaging and by decreasing the correlation between individual models in the ensemble [36,37]. Each of our random forest analyses comprised 1 × 104 individual models constructed by sub-sampling seven predictor variables at each node. Several model summary statistics were calculated, including sensitivity, which is the proportion of observations correctly identified as edited, specificity, which is the proportion of observations correctly identified as non-edited, and accuracy, which is the total proportion of observations correctly identified. More formally, these definitions are: sensitivity = true positives/(true positives + false negatives); specificity = true negatives/(true negatives + false positives); and accuracy = (true positives + true negatives)/total. Authors' contributions MPC conceived, designed and coordinated the study. DSM carried out the programming and statistical analyses. Both authors wrote and approved the final manuscript. Supplementary Material Additional File 1 Arabidopsis thaliana data file File is plain text, space delimited. First row is column headings with variable names: edit; + site is edited, - site is not edited; -20 through 20, nucleotide position relative to edited site; cp, codon position; fe, estimated folding energy; dfe, difference in estimated folding energy between pre-edited and edited sequences; and loc, location of focus site (position 0) in GenBank file. Each subsequent line represents a observation. Click here for file Additional File 2 Brassica napus data file File is plain text, space delimited. First row is column headings with variable names: edit; + site is edited, - site is not edited; -20 through 20, nucleotide position relative to edited site; cp, codon position; fe, estimated folding energy; dfe, difference in estimated folding energy between pre-edited and edited sequences; and loc, location of focus site (position 0) in GenBank file. Each subsequent line represents a observation. Click here for file Additional File 3 Oryza sativa data file File is plain text, space delimited. First row is column headings with variable names: edit; + site is edited, - site is not edited; -20 through 20, nucleotide position relative to edited site; cp, codon position; fe, estimated folding energy; dfe, difference in estimated folding energy between pre-edited and edited sequences; and loc, location of focus site (position 0) in GenBank file. Each subsequent line represents a observation. Click here for file Additional File 4 Combined data file File is plain text, space delimited. First row is column headings with variable names: edit; + site is edited, - site is not edited; -20 through 20, nucleotide position relative to edited site; cp, codon position; fe, estimated folding energy; dfe, difference in estimated folding energy between pre-edited and edited sequences; and loc, location of focus site (position 0) in GenBank file. Each subsequent line represents a observation. Click here for file Acknowledgements We thank AL Bazinet and MC Neel for comments on the manuscript. Figures and Tables Figure 1 Cross-validated pruned classification tree for the combined dataset. The number of edited and non-edited sites are given at each node. The single split is based on the nucleotides at position -1 relative to the edited site. Figure 2 Variable importance measures for the combined dataset. Numbered positions represent nucleotide state variables (with position zero representing the edited/non-edited site). The importance of each position is the decrease in the Gini index (a measure of impurity) induced by splitting the data on that position averaged over all trees (higher values are more important). The three variables based on estimates of free energy of folding are the codon position of the edited site (cp), estimated free energy of folding for the entire 41-nucleotide sequence centered on the edited/non-edited site (fe), and the difference in estimated free energy of folding between the edited and non-edited versions of the 41-nucleotide sequence (dfe). Table 1 Summary statistics for tree-based statistical models. Accuracy Sensitivity Specificity Arabidopsis thaliana 0.711 0.645 0.888 Brassica napus 0.693 0.630 0.887 Oryza sativa 0.709 0.645 0.874 combined 0.705 0.640 0.882 Table 2 Summary statistics for random forest models. Accuracy Sensitivity Specificity Arabidopsis thaliana 0.744 0.701 0.811 Brassica napus 0.765 0.733 0.808 Oryza sativa 0.722 0.716 0.808 combined 0.848 0.823 0.877 Table 3 Counts of C-to-U edited sites for each codon position. Codon Position Species 1 2 3 Not in Codon Arabidopsis thaliana 149 231 51 13 Brassica napus 142 243 33 4 Oryza sativa 174 230 77 0 ==== Refs Gray M RNA editing in plant organelles: A fertile field Proc Natl Acad Sci USA 1996 93 8157 8159 8710840 10.1073/pnas.93.16.8157 Maier R Zeltz P Kossel H Bonnard G Gualberto J Grienenberger J RNA editing in plant mitochondria and chloroplasts Plant Mol Biol 1996 32 343 365 8980487 Smith H Gott J Hanson M A guide to RNA editing RNA 1997 3 1105 1123 9326486 Gray M Diversity and evolution of mitochondrial RNA editing systems IUBMB Life 2003 55 227 233 12880203 Hiesel R Wissinger B Wolfgang S Brennicke A RNA Editing in plant mitochondria Science 1989 246 1632 1634 2480644 Hiesel R Combettes B Brennicke A Evidence for RNA editing in mitochondria of all major groups of land plants except the Bryophyta Proc Natl Acad Sci USA 1994 91 629 633 8290575 Malek O Lattig K Hiesel R Brennicke A Knoop V RNA editing in bryophytes and a molecular phylogeny of land plants EMBO J 1996 15 1403 1411 8635473 Freyer R Kiefer-Meyer MC Kössel H Occurance of plastid RNA editing in all major lineages of land plants Proc Natl Acad Sci USA 1997 94 6285 6290 9177209 10.1073/pnas.94.12.6285 Giegé P Brennicke A RNA editing in Arabidopsis effects 441 C to U changes in ORFs Proc Natl Acad Sci USA 1999 96 15324 15329 10611383 10.1073/pnas.96.26.15324 Wakasugi T Hirose T Tsudzuki T Kossel H Sugiura M Creation of a novel protein-coding region at the RNA level in black pine chloroplasts: The pattern of RNA editing in the gymnosperm chloroplast is different from that in angiosperms Proc Natl Acad Sci USA 1996 93 8766 8770 8710946 10.1073/pnas.93.16.8766 Gualberto JM Lamattina L Bonnard G Weil J Grienenberger J RNA editing in wheat mitochondria results in the conservation of protein sequences Nature 1989 341 660 666 2552325 10.1038/341660a0 Gray MW Covello PS RNA editing in plant mitochondria and chloroplasts FASEBJ 1993 7 64 71 Carrillo C Bonen L RNA editing status 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site recognition in higher plant mitochondria J Heredity 1999 90 338 344 10.1093/jhered/90.3.338 Chateigner-Boutin A Hanson M Cross-competition in transgenic chloroplasts expressing single editing sites reveals shared cis elements Mol Cell Biol 2002 22 8448 8456 12446765 10.1128/MCB.22.24.8448-8456.2002 Farré J Leon G Jordana X Araya A cis recognition elements in plant mitochondrion RNA editing Mol Cell Biol 2001 21 6731 6737 11564858 10.1128/MCB.21.20.6731-6737.2001 Williams M Kutcher B Mulligan R Editing site recognition in plant mitochondria: the importance of 5'-flanking sequences Plant Mol Biol 1998 36 229 37 9484435 10.1023/A:1005961718612 Segal MR Cummings MP Hubbard AE Relating genotype to phenotype: analysis of peptide binding data Biometrics 2001 57 632 643 11414594 10.1111/j.0006-341X.2001.00632.x Breiman L Random forests – random features Tech Rep 567, Department of Statistics, University of California 2001 Benson DA Karsch-Mizrachi I Lipman DJ Ostell J Wheeler DL GenBank 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thermodynamic parameters provides robust prediction of RNA secondary structure J Mol Biol 1999 288 910 940 10.1006/jmbi.1999.2700 Ihaka R Gentleman R R: a language for data analysis and graphics Comput Graph Stat 1996 5 299 314 Therneau TM Atkinson EJ An introduction to recursive partitioning using the RPART routines Tech Rep Mayo Foundation 1997 Breiman L Random Forests Machine Learning 2001 45 5 32 10.1023/A:1010933404324 Clark LA Pergibon D Statistical Models in S 1993 London: Chapman and Hall Breiman L Friedman JH Olshen RA Stone CJ Classification and Regression Trees 1984 Pacific Grove, CA: Wadsworth and Brooks Cummings MP Myers DS Mangelson M Applying permutation tests to tree-based statistical models: extending the R package rpart Tech Rep CS-TR-4581, UMIACS-TR-2004-24, Center for Bioinformatics and Computational Biology, Institute for Advanced Computer Studies, University of Maryland 2004 Breiman L Bagging predictors Mach Learn 1996 24 123 140 10.1023/A:1018054314350 Hastie TJ Tibshirani R Friedman JH The Elements of Statistical Learning 2001 New York: Springer

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==== Front BMC GenomicsBMC Genomics1471-2164BioMed Central London 1471-2164-5-681537739210.1186/1471-2164-5-68DatabasePEDRo: A database for storing, searching and disseminating experimental proteomics data Garwood Kevin [email protected] Thomas [email protected] Chris [email protected] Scott [email protected] Norman [email protected] Christopher F [email protected] Kathleen [email protected] Caroline [email protected] Anthony D [email protected] Sarah [email protected] David [email protected] Zhikang [email protected] Alistair JP [email protected] Andrew [email protected] Keith [email protected] Lena [email protected] Muriel [email protected] Peter [email protected] Julie [email protected] Kathryn S [email protected] Simon J [email protected] Andy [email protected] Simon J [email protected] Stephen G [email protected] Norman W [email protected] Department of Computer Science, University of Manchester, Oxford Road, Manchester M13 9PL, UK2 School of Biological Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, UK3 European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridgeshire CB10 1SD, UK4 School of Biomolecular Sciences, UMIST, PO Box 88, Manchester M60 1QD, UK5 School of Medical Sciences, Institute of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, UK6 Department of Molecular Microbiology, John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, UK7 Scottish Centre for Genomic Technology & Informatics, University of Edinburgh Medical School, The Chancellor's Building, Little France Crescent, Edinburgh, EH16 4S, UK8 University of Cambridge, Department of Biochemistry, Downing Site, Cambridge, CB2 1QW, UK9 Department of Chemistry, UMIST, PO Box 88, Manchester M60 1QD, UK2004 17 9 2004 5 68 68 5 8 2004 17 9 2004 Copyright © 2004 Garwood et al; licensee BioMed Central Ltd.2004Garwood et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Proteomics is rapidly evolving into a high-throughput technology, in which substantial and systematic studies are conducted on samples from a wide range of physiological, developmental, or pathological conditions. Reference maps from 2D gels are widely circulated. However, there is, as yet, no formally accepted standard representation to support the sharing of proteomics data, and little systematic dissemination of comprehensive proteomic data sets. Results This paper describes the design, implementation and use of a Proteome Experimental Data Repository (PEDRo), which makes comprehensive proteomics data sets available for browsing, searching and downloading. It is also serves to extend the debate on the level of detail at which proteomics data should be captured, the sorts of facilities that should be provided by proteome data management systems, and the techniques by which such facilities can be made available. Conclusions The PEDRo database provides access to a collection of comprehensive descriptions of experimental data sets in proteomics. Not only are these data sets interesting in and of themselves, they also provide a useful early validation of the PEDRo data model, which has served as a starting point for the ongoing standardisation activity through the Proteome Standards Initiative of the Human Proteome Organisation. ==== Body Background Bioinformatics tools and techniques depend directly or indirectly upon experimental data. However, interpreting experimental data often requires access to significant amounts of additional information about the sample used in the experiment, the conditions in which measurements were taken, the equipment used to take the measurements, etc. Recent proposals for models that capture such experimental descriptions alongside experimental results include MIAME for transcriptome data [1] and PEDRo for proteome data [2]. However, if full use is to be made of such rich data models for genomic data, these models must also be associated with comprehensive software tools for data capture, dissemination and analysis. In proteomics, which is rapidly evolving into a high-throughput experimental approach, there is (as yet) no standard representation for experimental data. As a result, limited tool support is available for disseminating, searching, comparing or analysing the results of experiments conducted using different techniques and equipment in different laboratories. Thus, while experimental results can be analysed, often in a labour-intensive manner in-house, the development of bioinformatics techniques for archiving, sharing and wider exploitation of proteomics results is still in its infancy. This paper seeks to contribute to the development of effective and systematic support for proteome data management by: 1. Describing a database for storing, searching and disseminating experimental proteomics data. This material should be relevant to the developers of future proteome data management systems in that it discusses and illustrates various design and implementation decisions that have an impact on the role and maintenance of the resulting database. 2. Making available data sets from several labs whose data have been included in the initial release of the database. These data sets themselves result from substantial experimental activities, and are representative of the sorts of information that in-house and public proteome data repositories must capture. As the database stores data in an XML format that conforms to the PEDRo (Proteomics Experimental Data Repository) data model [2], this material provides concrete examples for other users of data that conform to this schema, and should be useful for validation of specific parts of the model as input to the Human Proteome Organisation Proteome Standards Initiative (HUPO-PSI) activity on models for proteome data [3]. The database described in this paper has similar objectives and functionality to various other databases for functional genomic data. In particular, like the Gene Expression Omnibus (GEO) [4], the Stanford Microarray Database [5] and ArrayExpress [6], it contains a single category of experimental data, while accommodating the production of that data using several different experimental techniques. Like ArrayExpress, and unlike GEO, for example, the data stored in PEDRo must conform to a rich, but nevertheless deliberately constraining, data model. This model is richer than that supported by the well established SWISS-2DPAGE database [7] in that it not only contains information on protein separation and identification, but also includes detailed descriptions of experimental samples, the mass spectrometric analyses conducted, and the software used to perform protein identifications. Establishing the most appropriate kinds of data to include in a database such as PEDRo is not straightforward, as this depends on the use that is to be made of the data. In a large data repository, users may want to search for results based on widely varying criteria – for example, the proteins identified, the change in the level of a protein over time, the mechanism by which a sample was studied, etc. Furthermore, the users of a proteome data repository may themselves be diverse, and include: experimentalists with minimal direct experience of proteomics, but who are interested in proteins or organisms for which proteome studies have been conducted; proteome scientists who want to identify how successful specific techniques have been in different contexts; or mass-spectrometric analysts who want to compare their results with those of others. This wide range of potential users encourages the creation of a rich repository for proteomics data that provides detailed descriptions of many different aspects of an experiment. However, populating a database such as PEDRo is not a trivial task, as several of the different kinds of data included in PEDRo currently have to be entered manually, which is time-consuming for data providers. Even though a data entry tool has been developed to ease data entry (available from [8]), experience populating the database suggests that the creation of a data set from scratch (e.g., for a sample analysed using a single gel, for which multiple identifications have taken place) can take around a week, but that creating subsequent data sets that share some aspects of the experimental set-up is significantly less time-consuming. In addition, widespread deployment of a standard model should lead to laboratory equipment, or associated software, producing data that conforms to the standard, so the longer-term position for high-throughput laboratories should involve much lower data capture costs. It is hoped that the early provision of a collection of data sets conforming to the widely discussed PEDRo model will be useful in informing ongoing activities on the HUPO-PSI proteome data standard [3]. Construction and content Many bioinformatics databases, such as UniProt [9] and PDB [10], are associated with file formats that can be parsed by software that analyses or displays the data from the database. The Extensible Markup Language (XML [11]) has been developed in part to make the description, parsing and display of such files more systematic; thus there is a trend in bioinformatics towards the use of XML for storing or transmitting biological data [1,2]. The PEDRo database makes extensive use of XML for capturing, transmitting, storing and searching proteomics data. In particular: 1. The data-capture process uses a software tool, illustrated in Figure 1, which prompts users for values for different fields, and includes facilities for importing substantial data files, such as those representing peak lists. The tool constructs data-entry forms from the XML Schema definition of the PEDRo model. An XML Schema describes the structure of an XML document, and thus makes explicit the hierarchical structure of the document, the elements that are contained within the document, the types of those elements, and the number of times different elements may occur. The result of the data capture process is thus an XML file that corresponds to the PEDRo schema. A fragment of the XML format for a PEDRo entry is provided in Figure 2. Figure 1 The Pedro data capture tool. The Pedro data capture tool in use, editing a proteome data file. The left hand panel provides a tree-based browser for the complete document, while the right hand panel supports data entry for a specific component of the model, in this case a sample. The data capture tool is available for download from [8]. Figure 2 Sample PEDRo XML. A fragment of XML for an S. cerevisiae sample. The XML Schema from the model, plus complete data sets for the experiments described in Table 1, are available from [8]. 2. The database stores the XML captured using the data entry tool directly, using Xindice [12], an open-source XML storage system. Several different storage options exist for XML data, including: (i) storing the XML directly in a native XML repository such as Xindice; (ii) storing the XML directly using the XML storage extensions provided by commercial relational database vendors; and (iii) mapping from the XML documents onto tables for storage in a relational database. We have chosen option (i) for PEDRo. Option (ii) was not adopted because there is not yet a standard for integrating XML storage with relational databases, although this is being developed [13]. Option (iii) was not adopted because we envisage that the data model used in PEDRo will evolve to reflect the HUPO-PSI standard [3], and we wanted to avoid the need to evolve both relational and XML versions of the database in parallel. Furthermore, the emphasis for the PEDRo database is on enabling users to identify relevant experimental data sets, rather than on conducting complex searches or analyses over such data sets. For the required tasks, the query facilities provided with XML databases such as Xindice, which tend for the meantime to be based upon XPath [14] are sufficient. 3. The data are presented to users by generating web pages from the stored XML using XSLT [15], which was designed to support exactly this sort of task. This means that it has been straightforward to develop reports from the stored form of the data. Furthermore, the download format for the data is as XML documents, in the hope that this will ease the development of tools for parsing and analysing data obtained from the database. The software components used within PEDRo (and the role they play in data capture, storage and dissemination) are illustrated in Figure 3. Figure 3 PEDRo software components. The software components used in PEDRo. In essence, data flows clockwise from the top left, with three categories of user. The first category of user is the scientist who carries out data entry – this user must be intimately familiar with the experiment that has been conducted and the equipment that has been used. The result of the data capture process is a PEDRo XML file. This XML file is then checked by the database curator, who can then add the data into the database. Once in the database, the PEDRo Database Access software can be used to search the database and view its contents. The PEDRo software has been implemented over the Xindice XML database [12] using Java Server Pages [28]. The data stored in the database for each experiment is as described in the PEDRo model [2], and thus involves sample generation (e.g. organism, growth conditions, tagging), sample processing (e.g. gel properties, spot details), mass spectrometry (e.g. machine settings, peak lists) and in silico analyses (e.g. database search program used and results obtained). Table 1 provides a high-level overview of the initial data sets in the database. The data in the initial release of the database illustrates several different proteomics techniques in use, including sample processing based on classical 2D Gels and DIGE, the use of different gel imaging software, mass spectrometry using MALDI-TOF and MS/MS, and in silico data analyses using more than one program. Furthermore, the data captured covers a range of different organisms, including Saccharomyces cerevisiae, Candida albicans, Candida glabrata, Mus musculus, Arabidopsis thaliana and Streptomyces coelicolor. Table 1 Summary of database contents. A summary of the data sets included in the initial release of PEDRo. The database provides more detailed descriptions of sample generation, sample processing, mass spectrometry and in silico analyses, populating the model described in [2]. 1 Organism Saccharomyces cerevisiae Sample Generation Whole cell extracts (bead beating) Experimenter Al Brown et al. Sample Processing 2D gel Description GCN4-dependent proteins that respond to amino acid starvation Mass Spectrometry MALDI-ToF In SilicoAnalysis Peptide mass fingerprint searches (MASCOT, MS-Fit) 2 Organism Candida albicans Sample Generation Whole cell extracts (bead beating) Experimenter Al Brown et al. Sample Processing 2D gel Description GCN4-dependent proteins that respond to amino acid starvation Mass Spectrometry MALDI-ToF In SilicoAnalysis Peptide mass fingerprint searches (MASCOT, MS-Fit) 3 Organism Candida glabrata Sample Generation Whole cell extracts (bead beating) Experimenter Ken Haynes et al. Sample Processing 2D gel Description Proteins that respond to the inactivation of ACE2 Mass Spectrometry MALDI-ToF In SilicoAnalysis Peptide mass fingerprint searches (MASCOT, MS-Fit) 4 Organism Streptomyces coelicolor Sample Generation Whole cell extracts (sonication) Experimenter Andrew Hesketh Sample Processing 2D gel Description Changes in the proteome of strain M600 during growth and antibiotic production in liquid medium Mass Spectrometry MALDI-ToF In SilicoAnalysis Peptide mass fingerprint searches (MASCOT) 5 Organism Mus Musculus BALB/C Sample Generation Exfoliated jejunal epithelium prepared as per Bjerknes & Cheng, Anat Rec 1981, 199, 565. Experimenter Alan Pemberton, Pamela Knight Sample Processing 2D gel Description Trichinella spiralis infection in mice induces alterations in the proteome of the small mucosal epithelium Mass Spectrometry MALDI-ToF In SilicoAnalysis Peptide mass fingerprint searches (MASCOT, MS-FIT) 6 Organism Homo sapiens Sample Generation Gel image analysis data from 12 patients, used to assign spot picks from a preparative 2D gel Experimenter Tony Whetton, Caroline Evans Sample Processing 2D gel Description Proteomics can identify prognostic markers in CLL disease progression Mass Spectrometry MALDI-ToF In SilicoAnalysis Peptide mass fingerprint searches (MASCOT) 7 Organism Saccharomyces cerevisiae Sample Generation Saccharomyces cerevisiae, strain YMK36, butanol – and butanol + Experimenter Kathleen Carroll Sample Processing 2D gel Description Effect of butanol stress on yeast Mass Spectrometry MALDI-ToF In SilicoAnalysis Peptide mass fingerprint searches (MASCOT) 8 Organism Saccharomyces cerevisiae Sample Generation Experimenter Kathleen Carroll Sample Processing 2D gel Description Mapping Heat shock proteins in Saccharomyces cerevisiae Mass Spectrometry MALDI-ToF In SilicoAnalysis Peptide mass fingerprint searches (MASCOT) 9 Organism T. bruceii Sample Generation Gel pieces Experimenter Sarah Hart Sample Processing Bands from 1D gel Description Trypanosome Flagella Mass Spectrometry MALDI-ToF, LC MS/MS In SilicoAnalysis Mascot MS/MS These PEDRo data are significant in biological terms. For example, they include the first direct comparison of proteomic responses in two fungal species, namely responses to amino-acid starvation in the baker's yeast S. cerevisiae and the pathogenic fungus, C. albicans [16]. In addition, they include the first proteomic analysis of the medically important pathogen C. glabrata (Stead et al., Proteomic changes associated with inactivation of the Candida glabrata ACE2 virulence-moderating gene, manuscript submitted), whose genome sequence has only just been completed. The Streptomyces coelicolor M600 data set is the largest proteomics time course analysis of this strain in terms of numbers of proteins identified. It adds significantly to our knowledge of expression of some of the 20 gene sets annotated as being determinants of the biosynthesis of secondary metabolites, including antibiotics. Somewhat similar experiments, but differing in many aspects of their metadata, are reported on the SWICZ database [17]. This provides an opportunity to evaluate PEDRo in the context of related data presented in different databases. Also included are data from an experiment investigating the proteomic analysis of the mouse jejunal epithelium and its response to infection with the intestinal nematode, Trichinella spiralis [18]. Utility Web-based interfaces to biological databases tend to support one or more of the following tasks: browsing – interactively listing or navigating through database entries; searching – identifying database entries on the basis of simple restrictions on the values of one or more fields; visualising – presenting a visual representation of the data as a starting point for browsing; or querying – specifying a search that is to be conducted over the database using a query building interface or by providing inputs to pre-written (or "canned") queries. Functional genomics databases tend to emphasise browsing and searching. For example, the Stanford Microarray Database [5] supports browsing based around organisms and experiments, and more complex Boolean searches based on criteria such as experimenter, organism and category of experiment. ArrayExpress [6] supports browsing through experiments, arrays and protocols, and searching based on criteria such as species, experiment type and author. SWISS-2DPAGE [7] supports browsing by clicking on spots on gels, and searching based on criteria such as description, accession number or author. PEDRo also emphasises browsing and searching. Figures 4 and 5 illustrate the web-based interface to PEDRo, which can be accessed at [8]. In essence, the records in the database can be accessed by browsing summaries of the entries in the database, or by searching using one or more criteria. These criteria were obtained through a systematic requirements analysis with potential users from several different research groups, who were asked to comment on early versions of the interface. Overall, PEDRo provides core data access facilities that are principally intended to allow users to identify data sets that are of interest to them. As such, the PEDRo database as described should not be seen as a comprehensive query or analysis environment for proteomics data, but rather as a repository through which experimental results can be made available to a wider community. Therefore, S. cerevisiae data from PEDRo will also be made available through GIMS [19], for example, to enable the integration of these data with other sequence and functional information. Figure 4 Search page. Searching the PEDRo database. The search facility is intended to support rapid identification of PEDRo entries of interest. Searching can be conducted on one or more of the species name, experimenter, hypothesis, gene name and ORF number. Where more than one value is provided, entries are retrieved that match all the values given. In all cases, matches can be partial. Thus, for example, typing "Sacc" into the Species Name field will retrieve entries for Saccharomyces cerevisiae. Figure 5 Results page. Viewing results of the PEDRo search from Figure 4. Discussion A significant motivating factor behind the development of the PEDRo repository has been to allow informed discussion, assisted by concrete examples, into the level of detail and forms of model that are most appropriate for a proteome data repository. As the PEDRo model is being used as the starting point for the HUPO-PSI activity on models for proteome data, early validation of this model is important. The following observations have been made about the PEDRo model during the data capture process: 1. Sample description is neither very precise nor systematic. The effective description of samples is an open issue that spans different kinds of functional genomic data. For example, work is underway on the development of an ontology for characterising microarray experiments, focusing, in particular, on samples [20]. However, as the variety of organisms, genetic manipulations, extraction techniques, environmental conditions and experimental manipulations that may characterise a sample are extremely large, a mature solution to this problem may be some way off. 2. There is only limited support for relative protein abundance data (e.g. DIGE and stable isotope labelling strategies). Thus, for example, there is no place in the model to describe an expression ratio for a protein species derived from quantitative experimental strategies, only the ability to capture the 'raw' numbers. In fact, the PEDRo model was not designed to capture expression ratios, partly because such numbers are easily derived from the captured primary data, and partly because the particular method of their derivation may be contentious. It is hoped that the HUPO-PSI model will provide generic constructs for representing relationships between certain kinds of measurement (e.g. relative protein expression readings), to which can be attached the specific detail for individual techniques. However, it also seems important to avoid the pitfalls associated with overly permissive models, as these provide a less stable foundation for the developers of analytical tools than their more proscriptive counterparts. 3. The gel model is not particularly detailed. Thus, for example, there is no detailed description of the image analysis software used, the descriptions of individual spots are fairly minimal, and no details are captured on spot excision. An earlier critique of the PEDRo model for gels, and some possible extensions, is provided by [21]. It seems that, in order to provide insights for the developers of gel-based experiments, it would be appropriate for the model to be revised to provide additional details on gels. Overall, the appropriate level of detail for a proteomics repository is somewhat subjective, but can usefully be based on guiding principles; agreement as to the principles should then avoid scope-based discussions at a very fine-grained level. The current PEDRo model essentially supports the principle that enough detail should be captured about an experiment to: i. Allow results of different experiments to be analysed/compared. ii. Allow suitability of experiment design and implementation decisions to be assessed. iii. Allow protein identifications to be re-run in the future with new databases or software. There is also an additional negative principle, to the effect that the model itself should not be designed to include dependencies on characteristics relating to the configuration or properties of an individual piece of equipment. Accordingly, we have attempted to allow experimental methods and results to be described in significant detail, but without including parameters and properties that are likely to be superseded rapidly when new models of equipment are introduced, and without including parameters that can only be understood with reference to the documentation of a particular product. The data stored in PEDRo is more comprehensive for each experiment than is the case for most existing proteome databases. For example, in the longest established experimental proteomics resource, SWISS-2DPAGE [7], the emphasis is on annotated gels, and there is much less information collected on how the annotations were arrived at. Furthermore, there is an architectural distinction – SWISS-2DPAGE follows a more federated approach, with individual sites continuing to hold their own data. These other proteome data sources can be accessed through WORLD-2DPAGE, a web resource listing sites making available experimental proteomics data [22]. An example of a database that participates in WORLD-2DPAGE is the University of Alabama (UAB) Proteomics Database [23] which provides search and browsing facilities over data from its host university. As such, the emphasis is on annotated gels, and relatively few details are captured on sample processing, mass spectrometry or in silico analysis. Such design decisions are appropriate for certain categories of user of a proteomics database, but not for others. The UAB database has been designed to provide access to processed experimental results for biomedical researchers, but does not provide enough information to allow detailed comparisons of the ways in which the results were obtained. The ProteomeWeb [24] provides a wider range of tools than PEDRo (for example, for computing theoretical maps), and supports browsing of annotated gels from several bacteria and archaea. Once again, though, the data provided for each experiment are less comprehensive than in PEDRo. ProDB [25] has a certain amount in common with UAB, in that it too provides search and browsing over a database of locally produced data. In addition, ProDB features an architecture that supports the plugging-in of data-loading and analysis tools. However, the level of detail supported by the model is not obvious from the paper, which gives only part of the model, and the database was not publicly accessible at the time of writing. In consisting of a collection of tools associated with a database, ProDB thus also has a certain amount in common with SBEAMS [26] which includes a relational database of proteomic data. The SBEAMS model emphasises the description and analysis of mass spectrometry data, but seems not to support open access to experimental data at the time of writing. In terms of quantities of data, there are fewer data sets in PEDRo than in SWISS-2DPAGE, reflecting the fact that PEDRo is a newly created resource (Release 16 of SWISS-2DPAGE contains 34 reference maps), but somewhat more than in the UAB Proteomics Database. The Open Proteomics Database (OPD) supports the browsing and downloading of comparable amounts of data to those in PEDRo, and also includes mass spectrometry data, although quite a lot of the data are in flat-file format [27]. However, it is fair to say that none of the current databases is operating in the context of high-throughput experimentation, which will certainly be prevalent in the near future. Conclusions The need for wider and more systematic dissemination of experimental proteomics data is widely recognised, as argued in [27], and attested to by the ongoing work of the Proteome Standards Initiative [3]. As such, issues that need to be addressed include: i. The nature and variety of information that should be recorded about proteomics experiments. ii. The functionality that should be provided by repositories that make large-scale proteomic data available. iii. The computational architecture that should be used to provide the functionality at (ii). iv. The nature of the tools that should be developed for use with such a repository. This paper has sought to address issues (i), (ii) and (iii), with a particular emphasis on (i). Following on from [2] we believe that the provision of a collection of representative proteomic data sets conforming to a consistent model is important to the ongoing process of developing a stable and effective de jure standard for proteome data representation and sharing. This paper describes a database that includes a rich collection of representative data sets. Furthermore, the paper describes the functionality (issue ii) and architecture (issue iii) of an exploratory system for disseminating such data. In the same way as we see models for representing proteomic data evolving in the light of practical experience, we anticipate that the PEDRo repository, and the overall understanding of the data access and dissemination requirements for proteomic data, will evolve as the opportunities presented by high-throughput experimental techniques and comprehensive data sets become more fully understood. Availability and requirements The database can be accessed using a web browser at , by following the Database link. Authors' contributions KG and CG implemented the software. SJ, NM and CFT contributed to the development of earlier prototypes. TM coordinated the data capture activity. CK, CE, AW, SH, DS, ZY, AJPB, AH, KC, LH, MM, PG, JH, KSL, SJG conducted or led experimental activities that generated the data in the database, and contributed to feedback on the model. AB, SJH, SJO and NWP oversaw the database design and development activity, and the latter led the write-up. Acknowledgements Software development has been jointly funded by the BBSRC Investigating Gene Function (IGF) programme CoGeME grant (34/IGF13036) and by the UK e-Science Programme through the North-West Regional e-Science Centre. The BBSRC IGF Programme has also supported the work at Aberdeen, Cambridge, JIC and UMIST; Norman Morrison is supported by the NERC environmental genomics programme. Work in Aberdeen was also supported by the Wellcome Trust (055015, 063204) and the BBSRC (1G18883). Work at JIC was also supported by the BBSRC Exploiting Genomics Initiative. The authors would like to acknowledge the support of various colleagues in conducting the work described in this paper, including: Phil Cash, Laura Selway and Jan Walker (Aberdeen); Mike Naldrett (JIC); Alan Pemberton (Edinburgh); Richard Cawley (Manchester) and Isabelle Riba-Garcia (UMIST). ==== Refs Spellman PT Miller M Stewart J Troup C Sarkans U Chervitz S Bernhart D Sherlock G Ball C Lepage M wiatek M Marks WL Goncalves J Markel S Iordan D Shojatalab M Pizarro A White J Hubley R Deutsch E Senger M Aronow BJ Robinson A Bassett D Stoeckert CJ JrBrazma A Design and implementation of microarray gene expression markup language (MAGE-ML) Genome Biol 2002 3 research0046.1 0046.9 12225585 10.1186/gb-2002-3-9-research0046 Taylor CF Paton NW Garwood KL Kirby PD Stead DA Yin ZK Deutsch EW Selway L Walker J Riba-Garcia I Mohammed S Deery MJ Howard JA Dunkley T Aebersold R Kell DB Lilley KS Roepstorff P Yates JR Brass A Brown AJP Cash P Gaskell SJ Hubbard SJ Oliver SG A systematic approach to modeling, capturing, and disseminating proteomics experimental data Nature Biotech 2003 21 247 254 10.1038/nbt0303-247 Orchard S Zu W Julian RK Hermjakob H Apweiler R Further advances in the development of a data interchange standard for proteomics data Proteomics 2003 3 2065 2066 14625869 10.1002/pmic.200300588 Edgar R Domrachev M Lash AE Gene Expression Omnibus: NCBI gene expression and hybridisation array data repository, Nucleic Acids Research 2002 30 207 201 11752295 10.1093/nar/30.1.207 Gollub J Ball CA Binkley G Demeter J Finkelstein DB Hebert JM Hernandez-Boussard T Jin H Kaloper M Matese JC Schroeder M Brown PO Botstein D The Stanford Microarray Database: data access and quality assessment tools, Nucleic Acids Research 2003 31 94 96 12519956 10.1093/nar/gkg078 Brazma A Parkinson H Sarkans U Shojatalab M Vilo J Abeygunawardena N Holloway E Kapushesky M Kemmeren P Lara GG Oezcimen A Rocca-Serra P Sansone S-A ArrayExpress – a public repository for microarray gene expression data at the EBI, Nucleic Acids Research 2003 31 68 71 12519949 10.1093/nar/gkg091 Hoogland C Sanchez J-C Tonella L Binz P-A Bairoch A Hochstrasser DF Appel RD The 1999 SWISS-2DPAGE database Update, Nucleic Acids Research 2000 28 286 288 10592248 10.1093/nar/28.1.286 PEDRo Web Site Apweiler R Bairoch A Wu CH Barker WC Boeckmann B Ferro S Gasteiger E Huang H Lopez R Magrane M Martin MJ Natale DA O'Donovan C Redaschi N Yeh LS UniProt: the Universal Protein Knowledgebase, Nucleic Acids Res 2004 32 D115 D119 14681372 10.1093/nar/gkh131 Berman HM Westbrook J Feng Z Gilliland G Bhat TN Weissig H Shindyalov IN Bourne PE The Protein Databank, Nucleic Acids Research 28 235 242 10592235 10.1093/nar/28.1.235 Extensible Markup Language Xindice Eisenberg A Melton J SQL/XML is making good progress, ACM SIGMOD Record 2002 32 101 108 XPath XSLT Yin Z Stead D Selway L Walker J Riba-Garcia I Mclnerney T Gaskell S Oliver SG Cash P Brown AJP Divergence between Candida albicans and Saccharomyces cerevisiae in their globalresponses to amino acid starvation Proteomics 2004 2425 36 15274137 10.1002/pmic.200300760 SWICZ Pemberton AD Knight PA Wright SH Miller HRP Proteomic analysis of mouse jejunal epithelium and its response to infection with the intestinal nematode, Proteomics 2004 4 1101 1108 15048991 10.1002/pmic.200300658 Cornell M Paton NW Hedeler C Kirby P Delneri D Hayes A Oliver SG GIMS: An integrated data storage and analysis environment for genomic and functional data Yeast 2003 20 1291 1306 14618567 10.1002/yea.1047 Stoeckert C JrParkinson H The MGED ontology: a framework for describing functional genomics experiments Comparative and Functional Genomics 2003 4 127 132 10.1002/cfg.234 Jones A Wastling J Hunt E Proposal for a standard representation of two-dimensional gel electrophoresis data, Comparative and Functional Genomics 2003 4 492 501 10.1002/cfg.323 WORLD-2D-PAGE Hill A Kim H The UAB Proteomics Database Bioinformatics 2003 19 2149 51 14594723 10.1093/bioinformatics/btg281 Babnigg G Giometti CS ProteomeWeb: A web-based interface for the display and interrogation of proteomes Proteomics 2003 3 584 600 12748939 10.1002/pmic.200300396 Wilke A Bioinformatics support for high-throughput proteomics, Journal of Biotechnology 2003 106 147 156 14651857 10.1016/j.jbiotec.2003.08.009 SBEAMS Prince JT Carlson MW Wang R Lu P Marcotte EM The need for a public proteomics repository, Nature Biotec 2004 22 471 472 10.1038/nbt0404-471 Java Server Pages

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BMC Genomics. 2004 Sep 17; 5:68

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==== Front BMC SurgBMC Surgery1471-2482BioMed Central London 1471-2482-4-111537738710.1186/1471-2482-4-11Research ArticleDevelopment of a perioperative medicine research agenda: a cross sectional survey Khan Nadia A [email protected] Taha [email protected] Finlay A [email protected] Andre [email protected] Norman R [email protected] William A [email protected] the Canadian Perioperative Research Group [email protected] Department of Medicine, University of Calgary, 3330 Hospital Drive NW, Calgary, AB, T2N 4N1, Canada2 Department of Medicine, University of Alberta, Walter Mackenzie Center, 8440-112 St., Edmonton, AB, T6G 2B7, Canada2004 20 9 2004 4 11 11 10 2 2004 20 9 2004 Copyright © 2004 Khan et al; licensee BioMed Central Ltd.2004Khan et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Post-operative complications are a significant source of morbidity and mortality for patients undergoing surgery. However, there is little research in the emerging field of perioperative medicine beyond cardiac risk stratification. We sought to determine the research priorities for perioperative medicine using a cross sectional survey of Canadian and American general internists. Methods Surveys were electronically sent to 312 general internists from the Canadian Society of Internal Medicine and 130 internists from the perioperative medicine research interest group within the US based Society of General Internal Medicine. The questionnaire contained thirty research questions and respondents were asked to rate the priority of these questions for future study. Results The research topics with the highest ratings included: the need for tight control of diabetes mellitus postoperatively and the value of starting aspirin on patients at increased risk for postoperative cardiac events. Research questions evaluating the efficacy and safety of perioperative interventions had higher ratings than questions relating to the prediction of postoperative risk. Questions relating to the yield of preoperative diagnostic tests had the lowest ratings (p < 0.001 for differences across these categories). Conclusion The results of this survey suggest that practicing general internists believe that interventions studies are a priority within perioperative medicine. These findings should help prioritize research in this emerging field. ==== Body Background Over 40 million people undergo non-cardiac surgery in the United States each year [1]. Postoperative cardiac complications affect 2–18% of patients alone, costing over 20 billion dollars annually in the United States [2]. Many will suffer other potentially avoidable perioperative complications such as pneumonia, hemorrhage or infection. Efforts to minimize these complications have resulted in the development of the field of perioperative medicine. Until now, research in this emerging field has focused primarily on cardiac risk stratification [3]. Despite the significant medical and economic burden of perioperative complications, few studies evaluate diagnostic testing, risk stratification for non-cardiac complications, or interventions to prevent cardiac or non-cardiac complications. Thus, there is a growing need to expand the research base in this field. Given the wide spectrum of comorbidities in the surgical patient and the potential for postoperative complications, numerous research questions still need to be answered. The purpose of this study is to identify top perceived research priorities in this field using a survey of general internists practicing perioperative medicine. Methods Participants To obtain the opinions of general internists who practice perioperative medicine, from the Canada and the United States, all general internists within the Canadian Society of Internal Medicine (n = 312) and all members of the perioperative medicine interest group of the American based Society of General Internal Medicine (n = 130), were surveyed. Physicians were excluded if they practiced in a subspecialty rather than general medicine (>90% of perioperative medicine consults are performed by general internists [4]) or did not perform preoperative consultation. Survey development and administration Research questions for the survey were generated from a Medline search of perioperative medicine studies and from a focus group of five general internists active in perioperative medicine research. The questionnaire was pre-tested by four independent general internists for clarity and to confirm face validity. Modifications were made based on this pre-testing. The questionnaire was developed in English and then translated into French for French speaking Canadian physicians by a medical translator. Subsequently, a bilingual general internist validated the French translation. The questionnaire contained 30 randomly ordered research questions conceptually divided into three themes: 1. evaluating the yield of preoperative diagnostic tests (5 questions), 2. predicting postoperative risk (6 questions) and 3. determining the efficacy of perioperative interventions (19 questions). A subset of the sample (130) was asked to specifically rank the importance of these three categories as an internal check of the reliability of the survey instrument. This subset was also asked to list any other research questions that ought to be considered for future research apart from those included in the questionnaire. Respondents were asked to rate the priority for each research question to be studied in the future on a 10 point Likert scale where 1 indicates a low priority study question and 10 is a high priority research question. For questions that have been partially answered by existing studies, respondents were specifically asked to rate the priority of "further research" in these areas. The self- administered questionnaire was electronically mailed in October 2000 (faxes were sent to those without e-mail addresses). For non-responders, second and third mail-outs were sent in November and December 2000. Statistical analyses Descriptive statistics were used to analyze the demographic data and ratings for individual questions. T-tests (two- sided) were used to compare mean response scores across different subgroups. Because of the multiple planned comparisons, the alpha was set at 0.005 to determine statistical significance. Repeated measures analysis of variance was used to analyze differences across the 3 major themes of questions using SPSS statistical software. Results After 3 mailings, we obtained 152 completed surveys (overall response rate 34%). Thirty-three respondents were then excluded because of subspecialty status or because they did not perform perioperative consults. Respondents belonging to the Society of General Internal Medicine special interest group and Canadian Society of Internal Medicine were identical in most respects except fewer Canadian Society of Internal Medicine members had academic appointments (54% vs. 100%). Table 1 shows the demographic and professional characteristics for all respondents. Table 1 Physician characteristics (n = 119) Characteristics Age (SD) 44 (9.2) Female (%) 24 Average year of graduation 1982 Number of years in practice, mean (range) 13 (0.5–35) Practice Location (%) Rural 8 Urban <50 000 8 Urban 50–250 000 25 Urban >250 000 59 Academic appointment (%) 77 Number of preoperative consults performed per month, median (range) 10 (1–100) Research questions evaluating the efficacy of perioperative interventions had higher ratings followed by questions relating to prediction of postoperative risk. Questions evaluating the yield of preoperative diagnostic tests had the lowest ratings. The differences in ratings across these general categories were statistically significant (p < 0.001) and this pattern persisted regardless of academic status or volume of consults seen. Mean scores for individual research questions, based on responses where 1 indicates low priority for future research and 10 indicates high priority, ranged from a low of 3.6 (± 2.3 standard deviation) to a high of 7.2 (± 2.1 standard deviation). Mean scores for the ten highest rated individual questions are given in Table 2. Only one respondent suggested additional research topics that were not included in the questionnaire. The full list of 30 research topics presented to respondents in the questionnaire is presented in the appendix [see Additional file 1]. Table 2 The ten highest rated perioperative research issues: mean scores* (rank) Research issues Total 95% (CI) High** volume Low volume Academic Non-academic The value of tight control of diabetes mellitus postoperatively. 7.2 (1) 6.8–7.6 7.2 (1) 7.1 (2) 7.1 (3) 7.2 (1) Starting aspirin on patients at increased risk for postoperative cardiac complications. 7.1 (2) 6.7–7.5 7.2 (2) 7.0 (4) 7.1 (4) 7.1 (2) Safety and efficacy of continuing aspirin preoperatively for those already taking aspirin. 7.0 (3) 6.6–7.4 7.0 (5) 7.1 (1) 7.1 (5) 7.0 (3) Optimal management of perioperative anticoagulation for patients with prosthetic valves. 7.0 (4) 6.5–7.4 6.9 (6) 7.1 (3) 7.0 (6) 7.0 (4) The value of starting angiotensin converting enzyme inhibitors for those at increased risk of postoperative cardiac complications. 6.9 (5) 6.5–7.3 7.2 (3) 6.7 (9) 7.0 (7) 6.9 (7) Determining the diagnostic yield of routine postoperative cardiac surveillance. 6.8 (6) 6.4–7.2 7.1 (4) 6.6 (13) 6.5 (12) 6.9 (6) Developing interventions to minimize postoperative delirium. 6.8 (7) 6.4–7.2 6.5 (9) 7.0 (6) 6.4 (14) 6.9 (5) The value of starting beta-blockers for patients at increased risk of postoperative cardiac complications. 6.7 (8) 6.2–7.2 6.4 (11) 7.0 (5) 7.2 (1) 6.6 (10) Optimal management of perioperative anticoagulation for patients with atrial fibrillation. 6.6 (9) 6.2–7.1 6.3 (12) 6.9 (7) 6.6 (11) 6.7 (9) Developing a risk stratification index for predicting postoperative pulmonary complications 6.6 (10) 6.2–7.1 6.6 (8) 6.7 (10) 6.5 (13) 6.7 (8) * Mean scores were derived from responses based on a 10-point Likert scale. A score of 10 indicates high priority and 1 indicates low priority for future research. ** Ratings from consultants who see a high volume of preoperative consults (>10/month). Mean scores for most questions were similar among physicians independent of their academic status or whether they performed a high volume of preoperative consults (defined as greater than 10 consults per month) or not. For several questions, however, differences in ranking according to academic status and volume of consultations were found, although none achieved statistical significance (Table 2). Discussion There are few areas within perioperative medicine that are well studied beyond the area of predicting cardiac risk. The American Heart Association perioperative guidelines highlighted the paucity of studies on interventions to prevent postoperative cardiac events [3]. Reflecting this statement, respondents globally rated studies that determine the efficacy and safety of interventions as higher priority for future research compared to studies predicting postoperative risk or determining diagnostic yield of tests. Within the category of intervention studies, questions on medical therapy to prevent postoperative cardiac complications were among the highest rated questions. This result is congruent to the significant prevalence, morbidity and mortality associated with postoperative cardiac complications. A study by Devereaux et al also identified this area as an important target for future research after finding considerable practice variation in the management of cardiac medications [4]. Innovative interventions for cardiac protection with antiplatelet agents, angiotensin converting enzyme inhibitors or tight glycemic control were highly rated. Despite the publication of small trials of beta blocker therapy, responding internists felt that there was a need to definitively determine the efficacy of perioperative beta blockade. Respondents may be more skeptical of adopting results from the small trials of beta blocker where methodological controversies have arisen [5]. Other intervention questions that were rated highly focused on understudied areas of perioperative anticoagulation. Determining optimal perioperative anticoagulation strategies for patients with prosthetic valves or atrial fibrillation was rated highly. Although perioperative risk stratification is well studied for identifying those at risk of cardiac events, pulmonary complications occur more frequent than cardiac complications and are associated with a longer hospital stay [6]. Reflecting this significant morbidity and cost of respiratory complications, development of a prediction rule for postoperative pulmonary complications was among the highest rated topics. Since the completion of the survey, the ratings also reflect ongoing research activity within perioperative medicine. Tight glycemic control was a high rated topic and a study examining the effect of tight glycemic control in postoperative patients in a critical care setting has been published since the completion of this survey. Also since the completion of this survey, a large multi-center trial examining the efficacy of perioperative beta blockade has been launched. Another highly rated research topic was developing a prediction rule for pulmonary complications. Recently, a study was published to identify those at increased risk of postoperative pneumonia. Two of the highly rated research priority topics have been recently published in major medical journals [7,8]. There are several potential limitations with this study. The low response rate may be felt to limit the generalizability to other general internists. However, views of those who practice perioperative medicine and who have a particular interest in the area, the research consumers, are important in developing a research agenda. We presume that physicians who practice within an area and who have a particular interest are more likely to respond to a survey than those who do not. Thus, the physicians who responded to the questionnaire and reported practicing perioperative medicine define the group of research consumers we were targeting. This study also only examined the beliefs of general internists on research priorities since greater than 90% of the perioperative medicine consultations are conducted by general internists rather than subspecialists in internal medicine in tertiary care centers [4]. Also, lower response rates are seen in physician surveys but surveys with response rates of 10–45% are published in major medical journals [9-15]. Another limitation is that anesthesiologists, cardiologists or primary care physicians perform perioperative medicine consultations and their opinions were not elicited in this survey. Another potential limitation is that not all important research questions could be examined. However, only one respondent indicated additional research topics suggesting that there were no major omissions in the research topics listed. Additionally, it is difficult to interpret meaningful differences in mean Likert response scores among individual questions. Individual scores and their confidence intervals should be interpreted to give a general idea of high- priority questions, rather than a strict ranking of research pursuits. Conclusion Perioperative medicine research is growing in response to the significant medical and economic consequences of perioperative complications. Identifying the research priorities of those who provide perioperative medical care – the consumers of research, is important. The results suggest that intervention studies are a higher priority for future research compared to studies that predict postoperative complications or determine the yield of diagnostic tests. Researchers and funding boards may use the findings of this survey to identify perceived high priority research topics within perioperative medicine. Competing interests None declared. Authors' contribution NK, TT, AF, NC, FAM, and WG contributed to the design of the project. NK and TT contributed to data collection and data analysis. NK, TT, WG contributed to writing the manuscript. AF, NC, FAM made substantive, intellectual editing contributions. Pre-publication history The pre-publication history for this paper can be accessed here: Supplementary Material Additional File 1 Perioperative medicine research questions. This appendix lists all 30 perioperative research issues from the survey, along with the mean scores rated by all respondents. Click here for file Acknowledgements The authors thank Dr. Sophia Khan for editing. Dr. Khan, Dr. McAlister and Dr. Ghali were supported by the Alberta Heritage Foundation for Medical Research during this project. Dr. Ghali is also supported by a Government of Canada Research Chair. ==== Refs Owings MF Kozak LJ Ambulatory and inpatient procedures in the United States, 1996. National Center for Health Statistics Vital Health Stat 1998 13 1 119 Mangano DT Perioperative cardiac morbidity–epidemiology, costs, problems, and solutions Western Journal of Medicine 1994 161 87 9 7941525 Guidelines for perioperative cardiovascular evaluation for noncardiac surgery. Report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines. Committee on Perioperative Cardiovascular Evaluation for Noncardiac Surgery J Am Coll Cardiol 1996 27 910 48 8613622 10.1016/0735-1097(95)99999-X Devereaux PJ Ghali WA Gibson NE Skjodt NM Ford DC Quan H Physicians' recommendations for patients undergoing noncardiac surgery Arch Intern Med 2002 162 63 11784221 10.1001/archinte.162.1.63 Devereaux PJ Ghali WA Effect of atenolol on mortality and cardiovascular morbidity after noncardiac surgery [letter;comment] New England Journal of Medicine 1997 336 1452 3 9148147 10.1056/NEJM199705153362011 Lawrence VA Hilsenbeck SG Mulrow CD Dhanda R Sapp J Page CP Incidence and hospital stay for cardiac and pulmonary complications after abdominal surgery J Gen Intern Med 1995 10 671 8 8770719 Arozullah AM Khuri SF Henderson WG Daley J Participants in the National Veterans Affairs Surgical Quality Improvement Program. Development and validation of a multifactorial risk index for predicting postoperative pneumonia after major noncardiac surgery Annals of Internal Medicine 2001 135 847 57 11712875 Van den Berghe G Wouters P Weekers F Verwalst C Intensive insulin therapy in critically ill patients N Engl J Med 2001 345 1359 1367 11794168 10.1056/NEJMoa011300 Asch DA Jedrziewski MK Christakis NA Response rates to mail surveys published in medical journals J Clin Epidemiol 1997 50 1129 36 9368521 10.1016/S0895-4356(97)00126-1 Hyman DJ Pavlik VN Self-reported hypertension treatment practices among primary care physicians: blood pressure thresholds, drug choices, and the role of guidelines and evidence-based medicine Arch Intern Med 2000 14–28 2281 6 10.1001/archinte.160.15.2281 Brodsky MA Chun JG Podrid PJ Douban S Allen BJ Cygan R Regional attitudes of generalists, specialists and subspecialists about management of atrial fibrillation Arch Intern Med 1996 156 2553 2562 8951298 10.1001/archinte.156.22.2553 Hoffman RM Papenfuss MR Buller DB Moon TE Attitudes and practices of primary care physicians for prostate cancer screening Am J Prev Med 1996 12 277 281 8874692 Friedmann PD Brett AS Mayo-Smith MF Differences in generalists' and cardiologists' estimates of baseline cardiovascular risk and outcomes of preventive therapy in cardiovascular disease Ann Intern Med 1996 124 414 421 8554250 Mendelssohn DC Kua BTK Singer PA Referral for dialysis in Ontario Arch Intern Med 1995 155 2473 2478 7503607 10.1001/archinte.155.22.2473 Robinson AR Hohmann KB Rifkin JI Topp D Gilroy CM Pickard JA Anderson RJ Physician and public opinions on quality of health care and the problem of medical errors Arch Intern Med 2002 162 2186 90 12390060 10.1001/archinte.162.19.2186

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BMC Surg. 2004 Sep 20; 4:11

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==== Front BMC DermatolBMC Dermatology1471-5945BioMed Central London 1471-5945-4-111538002410.1186/1471-5945-4-11Research ArticleUVA/UVA1 phototherapy and PUVA photochemotherapy in connective tissue diseases and related disorders: a research based review Breuckmann Frank [email protected] Thilo [email protected] Peter [email protected] Alexander [email protected] Department of Dermatology, Ruhr-University Bochum, 44791 Bochum, Germany2 Dermatology Out-Patient Clinic, Oldchurch Hospital, Romford RM7 OBE, Greater London, UK2004 20 9 2004 4 11 11 12 5 2004 20 9 2004 Copyright © 2004 Breuckmann et al; licensee BioMed Central Ltd.2004Breuckmann et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Broad-band UVA, long-wave UVA1 and PUVA treatment have been described as an alternative/adjunct therapeutic option in a number of inflammatory and malignant skin diseases. Nevertheless, controlled studies investigating the efficacy of UVA irradiation in connective tissue diseases and related disorders are rare. Methods Searching the PubMed database the current article systematically reviews established and innovative therapeutic approaches of broad-band UVA irradiation, UVA1 phototherapy and PUVA photochemotherapy in a variety of different connective tissue disorders. Results Potential pathways include immunomodulation of inflammation, induction of collagenases and initiation of apoptosis. Even though holding the risk of carcinogenesis, photoaging or UV-induced exacerbation, UVA phototherapy seems to exhibit a tolerable risk/benefit ratio at least in systemic sclerosis, localized scleroderma, extragenital lichen sclerosus et atrophicus, sclerodermoid graft-versus-host disease, lupus erythematosus and a number of sclerotic rarities. Conclusions Based on the data retrieved from the literature, therapeutic UVA exposure seems to be effective in connective tissue diseases and related disorders. However, more controlled investigations are needed in order to establish a clear-cut catalogue of indications. ==== Body Background Unlike UVB radiation that can penetrate at the most into the papillary dermis, longer wavelengths in the UVA region have the capacity to reach the subcutis as well. Accordingly, as well as due to its lesser antiproliferative activity, UVB irradiation has not been established in the treatment of sclerotic disorders except for occasional cases of graft-versus-host disease (GvHD) [1,2]. Hence, this review examines different modalities of UVA phototherapy in the treatment of connective tissue diseases and related disorders. The term irradiance (e.g., in mW/cm2), which is the most commonly used term in photobiology, relates to the subject (e.g., patient) struck by the irradiation. In photobiology, the time integral of the irradiance is commonly expressed as fluence (e.g., in J/cm2), or even more loosely as dose [3,4]. Even though mostly combined with 8-methoxypsoralene or other photochemotherapeutic agents, broad-band UVA irradiation (315–400 nm), containing both UVA1 (340–400 nm) and UVA2 (315–340 nm), was used as monotherapy e.g. in the treatment of atopic dermatitis [5]. However, this phototherapeutic option was being replaced more frequently by the usage of irradiation devices which allow a more effective treatment by the administration of selected spectra. By eliminating shorter wavelengths in the UVA2 region adverse effects such as erythema are minimized and therapeutically effective higher UV doses can be administered. Thus, today broad-band UVA may play a subordinated role in modern phototherapy anymore, except for the combined application with psoralenes, even though it is still used for a large number of patients due to its wide availability and although it has not yet been directly compared with UVA1 for many sclerosing disorders. Conventional UVA1 treatment emitts wavelengths mainly between 340 and 400 nm, but may also produce scattered radiation >530 nm including infrared irradiation (780–3000 nm). Three different pattern of UVA1 dosage have been described: high-dose UVA1 phototherapy applying UVA1 doses ranging from 90–130 J/cm2 single (975–1840 J/cm2 cumulative) irradiation, medium-dose UVA1 phototherapy admitting doses between 20–90 J/cm2 single (300–975 J/cm2 cumulative) UVA1 and last but not least low-dose UVA1 phototherapy administering single UVA1 doses of ≤ 20 J/cm2 or a cumulative doage ≤ 300 J/cm2, respectively [6-9]. Conventional UVA1 phototherapy may be accompanied by extensive heat load predominantly generated by infrared irradiation (780–3000 nm) and/or insufficient cooling systems of the phototherapy devices. For this reason, within the last years, lavish new UVA1 equipment was developed containing a special filtering and cooling system, in which a considerable amount of wavelengths >530 nm are eliminated and consequently the heat load due to heat-producing infrared radiation is strongly diminished [10]. Since more than two decades, the combination of oral 5- or 8-methoxypsoralen followed by broad-band UVA exposure is an effective treatment option in a widespread number of indications such as psoriasis and cutaneous T cell lymphoma [11-13]. Psoralens specifically belong to the best characterized agents of photosensitizing chemicals. Following its administration low-dose UVA irradiation is applied including an individual progression depending on the formation of the so-called PUVA erythema. In general, the overall mean cumulative dose has been found to comprise ≤ 400 J/cm2 UVA [14]. In contrast to systemic PUVA therapy topical PUVA, for example applied as PUVA bath, PUVA shower or PUVA cream, is mainly characterized by absent/reduced systemic side effects and the restriction to a selective limited lesional area [15]. As a consequence, systemic PUVA has mostly been replaced by topical PUVA treatment representing an efficient well-tolerable alternative to oral methoxypsoralen administration. Generally, overall mean cumulative dosage has been found to comprise ≤ 200 J/cm2 UVA [14]. Different types of UVA phototherapy were introduced as an innovative and promising therapeutic option in the treatment of inflammatory diseases such as atopic dermatitis and more recently in therapy of lymphoproliferative disorders such as cutaneous T cell lymphoma and related skin affections. No more than seven years ago, subsequent to promising clinical results of extracorporeal photochemotherapy and psoralene plus UVA (PUVA) in systemic sclerosis (SSc) and morphea [16-19], first investigations have verified the therapeutic value of UVA1 irradiation for the treatment of localized scleroderma (LS) [20-22]. However, encouraged by the clinical success and the diversity of immunomodulatory effects achieved by the use of UVA phototherapeutic regimens in a great number of different indications, additional studies focused on investigating the efficacy of UVA phototherapy in the treatment of a widespread range of sclerotic skin diseases [23]. Furthermore, series of UVA exposure may be used in the treatment of lupus erythematosus (LE), even if known as a photosensitive condition. Today, different forms of UVA phototherapy are widely used and have subsequently developed into a treatment modality of importance within the field of dermatology and rheumatology at least as an adjunctive treatment and, beside the 'initial indications', may also or even especially be indicated as a successful alternative in the treatment of skin manifestations of connective tissue diseases and related disorders. Methods For this systematic review we concentrated on the therapeutic use of UVA application in humans. The computerized bibliographic database PubMed (includes all citations from Medline and additional life science journals) without time limits (January 1966 to April 2004) was screened for original papers, case reports, letters, reviews and book articles on UVA/UVA1/PUVA. As main key words we used "UVA", "UV-A", "UVA phototherapy", "UVA1", "UV-A1" "UVA1 phototherapy", "PUVA", "PUVA phototherapy", "PUVA photochemotherapy", "ultraviolet A", "ultraviolet A1", "sclerosis", "scleroderma", "sclerosus", "sclerotic", "sclerodermoid", "morphea", and "lupus erythematosus". Other sources included monographs, textbooks, and the reference lists from all the articles retrieved. All abstracts were read and selected by two authors (F.B., T.G.) Inclusion or exclusion of articles were based on consensus. Relevant data including study design, number of patients, duration of treatment, clinical outcome, cumulative UVA doses, and adverse effects were retrieved from the articles, summarized and briefly discussed as follows. Results Systemic sclerosis SSc, affecting the connective tissue of various organs including the skin, is histologically recognized by an alteration of the microvasculature with a rarefaction of the vessels within the papillary layer, perivascular skin infiltrating T lymphocytes (mainly due to an alteration of the Th2 immune response), rapid proliferation of dermal fibroblasts exhibiting an elevated status of protein synthesis and by a resulting massive deposition of collagen in both the skin and internal organs [24-27]. UVA1 So far UVA1 phototherapy has only been reported to be effective in the clearance of acral sclerotic skin lesions of SSc patients by administering local UVA1 irradiation of the hands or forearms, respectively. First, Kobyletzki et al. reported on preliminary results about the efficacy of low-dose UVA1 phototherapy within the treatment of acrosclerosis in eight patients suffering from progressive SSc [28]. Exposure of 30 J/cm2 UVA1 was administered four times per week for eight weeks and subsequent three times per week for six weeks resulting in a total of 50 sessions and a cumulative dose of 1500 J/cm2. In 2000, Morita et al. could confirm the UVA1-induced softening of sclerosis following partial body 60 J/cm2 medium-dose UVA1 phototherapy ranging from 510 to 1740 J/cm2 cumulative dose in three patients with diffuse and one with limited SSc, later further underlined by decreased dermal decorin levels as published in 2003 [29,30]. A recent open non-randomized study including 18 patients with SSc derived acrosclerosis revealed softening of former stiffness, an increase of total skin distension, the reduction of skin thickness and an elevation of dermal collagenase activity in 16 patients following the corresponding irradiation protocol as described by Kobyletzki et al. [31]. However, whole-body UVA1 phototherapy has not yet been described, although a possible systemic impact due to the deep penetration depth seems to be imminent. PUVA Based on first studies reporting the efficacy of topical PUVA in one patient with SSc [7], Kanekura et al. described the positive outcome of former sclerotic lesions in three patients exhibiting cutaneous manifestation of progressive SSc [32]. PUVA was administered for three to eight weeks with daily doses of 0.25 J/cm2 to 0.4 J/cm2 (cumulative dosage: 3.5 J/cm2 to 9.6 J/cm2) resulting in remarkable clinical improvement of skin sclerosis index as well as stiffed fingers, hands and knees. Another small uncontrolled study investigated oral PUVA therapy for SSc. The study included four women suffering from SSc receiving a PUVA protocol consisting of 0.5 to 4 J/cm2 single dose UVA given three times a week for ten weeks and a mean cumulative dose of 70.5 J/cm2 UVA. Even though posttherapeutic skin severity scores did not alter significantly, at least microscopic analysis of the histological skin scores of all patients revealed visible improvements [33]. The last case report appeared in 2003 discussing the use of PUVA bath in a young girl, proposing that PUVA bath could also be used in childhood [34]. Localized scleroderma LS is characterized by circumscribed fibrotic plaques generally affecting the whole dermis. Thus LS, particularly when occurring in childhood, may contribute to progressive and long-lasting induration of the skin and subcutaneous tissue, growth retardation, muscle atrophy and, in severe cases, even to flexion deformities and poorly healing ulcerations [35]. UVA As to our knowledge, there has been conducted only two large study investigating low-dose broad-band UVA exposure in morphea [36,37]. Twelve patients were irradiated by doses of 20 J/cm2 broad-band UVA three times a week for a total of 20 sessions (400 J/cm2 cumulative dose). Following treatment, all patients experienced marked softening of former skin lesions accompanied by significant reduction of the mean concentration of collagen. Additionally, nine patients received 10 J/cm2 UVA single and 200 J/cm2 UVA cumulative dose. Even though different study parameters varied between both groups, no statistically significant differences could be detected in the clinical response to those doses. UVA1 High-dose UVA1 phototherapy of LS has been introduced by Stege et al. in 1997 [38]. Ten patients receiving 130 J/cm2 high-dose UVA1 therapy (30 sessions, 3900 J/cm2 UVA1 cumulative dose) were compared with seven patients treated by low-dose UVA1 phototherapy (30 sessions, 600 J/cm2 cumulative dosage) and internal controls. The authors state that high-dose UVA1 significantly reduced skin thickness and stiffness and increased elasticity of plaques. High-dose UVA1 was superior to low-dose UVA1. By contrast, a number of different studies and case series could also confirm the effectiveness of low-dose UVA1 phototherapy. In 1995, Kerscher et al. were able to discuss the first successful phototherapeutic approach of low-dose UVA1 phototherapy of LS [39]. Subsequently, the authors conducted a larger study including 20 patients suffering from LS. Patients were irradiated with low-dose UVA1 for twelve weeks (total of 30 treatment sessions, 20 J/cm2 single dose, 600 J/cm2 cumulative dose) resulting in significant clinical improvement in about 80% of the patients [20]. Two patients exhibiting subcutaneous LS did not improve. Finally, Gruss et al. analyzed and compared the effect of their low-dose UVA1 irradiation protocol on late-stage lesions, inflammatory lesions and late-stage lesions with overlying lichen sclerosus et atrophicus (LSA) [40]. All three patients responded well to therapy. In addition to low- and high-dose UVA1, medium-dose UVA1 phototherapy stands for a further phototherapeutic option. In 2001, seven patients with morphea were treated by 30 J/cm2 medium-dose UVA1 phototherapy three times weekly during a ten week period [41]. All patients reported improvement as judged by softening of the skin lesions. Recently, controlled medium-dose UVA1 treatment was performed in a total of eight patients using 48 J/cm2 UVA1 [42]. Irradiation was administered four times per week for twelve weeks resulting in an improvement of skin sclerosis by a cumulative dose of 2304 J/cm2 UVA1. Furthermore, combined therapy with calcipotriol ointment and low-dose UVA1 phototherapy seems to be highly effective at least in childhood morphea. Following a first case report, Kreuter et al. conducted a large open prospective study including 19 children suffering from LS [43,44]. UVA1 exposure was given four times a week for ten weeks (20 J/cm2 single dose UVA1, cumulative dose: 800 J/cm2) as an adjunct to twice daily topical calcipotriol application. Combined therapy resulted in a relative reduction of clinical scores of about 67%. Its successful use has also been reported regarding the variant of LS en coup de sabre (30 J/cm2 UVA1, 30 sessions) [45]. PUVA First application of PUVA bath photochemotherapy in two cases of LS was published in 1994 by Kerscher et al. UVA irradiation was administered once daily four times a week for five consecutive weeks followed by twice per week for additional five weeks (30 treatments, maximum single dose of 20 J/cm2), leading to an almost clearance of lesional skin [19]. Evaluation of 17 consecutive patients receiving PUVA bath photochemotherapy (0.2 J/cm2 to 0.5 J/cm2 initial dosage, 1.2 J/cm2 to 3.5 J/cm2 UVA maximum dose) revealed marked clinical improvement in 13 of 17 persons even after 15 treatment sessions [46]. As already mentioned above, Kanekura et al. could also verify the effectiveness of PUVA therapy, beside three patients with SSc, in one patient exhibiting generalized morphea [32]. Within the following years, a number of case reports and serial cases were able to reproduce the positive results of PUVA therapy in LS, employing higher initial and cumulative doses and more treatment sessions to achieve improvement/clearance [47-49]. Recently, Pasic et al. demonstrated that local PUVA bath may also be of certain benefit for LS in childhood [34]. Moreover, PUVA cream therapy has been successfully introduced by Grundmann-Kollmann et al. in four LS patients (cumulative dose ranging from 67.5 J/cm2 to 121 J/cm2, maximum single dose: 3.5 J/cm2) and, analogous to UVA1 phototherapy, improvement of linear scleroderma en coup de sabre treated with topical calcipotriol ointment and PUVA cream could be observed by Gambichler et al. [50,51]. Extragenital lichen sclerosus et atrophicus Extragenital LSA is an uncommon skin disease characterized by white porcelain-like sclerotic skin lesions predominantly affecting the flexor surface of the wrists, the upper part of the trunk, and the axillae. In the common form of LSA, genital involvement with atrophy of the vulval, penile, and perianal skin is usually observed [52]. Although LSA has sometimes been considered as a subspecies of LS, LSA is generally regarded as a separate entity as to its distinct clinical and histomorphological peculiarities [53-55]. UVA1 The efficiency of UVA1 phototherapy in extragenital LSA was first established by Kreuter et al. in 2001 [56]. The authors here report on the improvement of skin status following 40 sessions of long-wave UVA1 irradiation (four sessions per week for ten weeks, total of 40 treatments, 20 J/cm2 low-dose UVA1 per session, 800 J/cm2 cumulative dose). In the same year, a subsequent double casuistic was presented, both receiving the same low-dose UVA1 phototherapy and both responding to therapy with an almost complete clearance of formerly sclerosing lesions [57]. Only one year later, Kreuter et al. were able to present the improvement of extragenital LSA in ten patients, all being treated by the established standard irradiation protocol [58]. In contrast, low-dose UVA1 phototherapy of morphea with overlying LSA could not completely reverse the corresponding histopathological changes in a clinical trial in one patient [40]. PUVA As far as PUVA therapy in extragenital LSA is concerned, one case report could demonstrate a promising therapeutic attempt [59]. Interestingly, single UVA1 progressed from 0.3 to 2.3 J/cm2 resulting in a cumulative dose of 31.7 J/cm2 during a six week period. In addition to the extragenital manifestation, PUVA cream photochemotherapy has also been proven to be even effective in genitoanal lesions of LSA [60]. Nevertheless, despite the absence of any short-term side effect, UVA irradiation of genital affections should be performed extremely carefully in order to prevent long-term negative adverse consequences. Sclerodermoid graft-versus-host disease Chronic graft-versus-host disease (GvHD) is an immunological condition frequently occurring as a late consequence of allogenic bone marrow transplantation. Two subtypes, cutaneous lichenoid and sclerodermoid, have been described, based on clinical and histopathological examinations. Sclerodermoid GvHD is a severe adverse immunologic reaction with deposition of collagen in the skin and possibly other soft tissues, resulting in loss of range of motion and functional capabilities [61]. UVA1 In 2000, Grundmann-Kollmann et al. presented a patient with chronic sclerodermic GvHD, who did not respond to conventional chemotherapeutic agents [62]. Low-dose UVA1 phototherapy was successfully administered four times a week over six weeks (20 J/cm2 single dose, 480 J/cm2 cumulative dose) combined with mycophenolate mofetil therapy. Based on the potentially beneficial effect of UVA1 phototherapy in scleroderma, Staender et al. investigated the efficacy of low- or medium-dose UVA1 phototherapy, respectively [63]. Five patients (two of them after insufficient PUVA treatment) received 50 J/cm2 single-dose UVA1 irradiation five times per week for two months followed by a subsequent reduction towards three times weekly. One patient was treated by a stable dosage of 20 J/cm2 in combination with immunosuppressives and extracorporeal phototherapy. In all cases, therapy led to softening of formerly stiffed sclerotic lesions. Most recently, the positive effect of UVA1 irradiation was underlined by Calzavara Pinton et al. [64]. Five patients exhibiting sclerodermoid GvHD (localized: 4; generalized: 1) were treated with medium-dose UVA1 phototherapy three times weekly (50 J/cm2 single dose, 750 J/cm2 to 1650 J/cm2 UVA1 cumulative dose) resulting in a complete remission in three and a partial improvement in two patients. PUVA Already years ago it has been shown that systemic PUVA therapy might be of certain benefit to chronic lichenoid and recalcitrant stages of GvHD, but remains insufficient in sclerotic forms [65-67]. In 1991, another report of PUVA therapy for chronic GvHD could only demonstrate clinical improvement in lichenoid lesions, sclerodermoid skin involvement did not respond to therapy [68]. Oncoming studies including up to 40 patients were able to verify the efficiency of therapeutic PUVA administration, even though only single cases of slightly improved sclerodermatous lesions could be observed [69,70]. However, most recently, Leiter et al. performed a successful PUVA treatment in two patients suffering from sclerodermoid GvHD [71]. Inasmuch as improvement of skin involvement is concerned, after a median of 25 treatment sessions, PUVA treatment resulted in a sharp reduction of skin thickness reflected by a relative decrease of 72%. Bath PUVA was administered three to four times per week at the beginning followed by a subsequent reduction via twice to finally once weekly until improvement occurred (34 or 25 sessions, respectively; standard protocol not described; 64.0 or 14.2 J/cm2 cumulative UVA1, respectively). One of both received additional 667 J/cm2 UVA1 irradiation. Nevertheless, most authors state that UVA irradiation should only be performed as an adjunct treatment in addition to conventional chemotherapeutic regimens. Lupus erythematosus LE is an autoimmune disease including a wide spectrum of manifestations in various organ systems. LE specific skin lesions can be found in over 80% of the patients. Currently, three epidemiological forms are distinguished: discoid LE, subacute cutaneous LE and systemic LE beside the presence of a variety of specific subtypes [72]. UVA1 The first study on UVA1 phototherapy in subacute cutaneous LE appeared in 1993 [73]. A nine week series of UVA1 phototherapy leading to a cumulative dose of 186 J/cm2 had been administered. Thereafter, an impressive improvement of LE lesions was noted. In another uncontrolled study conducted in 1994, ten patients with systemic LE were treated with 6 J/cm2 for 15 sessions during a period of three weeks [74]. Four of them continued treatment for eight months. The authors could verify a marked clinical improvement combined with a decrease of autoantibody concentration. Since then, two randomized double-blind placebo-controlled cross-over studies were performed. First, McGrath et al. reported in a two-phase study two groups of patients, one receiving 6 J/cm2 UVA1 five times a week for three weeks followed by a three week exposure of placebo visible light, the other vice versa [75]. Twenty-five patients completed this phase of the study. Both procedures were followed by an unblinded exposure of progressively decreasing UVA1 levels. Taking clinical as well as serological data in account, the authors proposed that low-dose UVA1 phototherapy might be superior to visible light irradiation. Second, Poldermann et al. tried to compare exactly the two different groups in a total of eleven patients [76]. Although no statistically significant difference between the two groups could be evaluated after an exposure of three weeks including 6 J/cm2 cold-light UVA1 five times weekly, significant clinical improvement was restricted to the UVA1 group. Apart from the short term benefit following UVA1 phototherapy, Molina et al. were also able to describe a long term benefit following low-dose UVA1 treatment (once/twice per week, 6–15 J/cm2) for a mean impressive period of 3.4 years in six patients of their former study [77]. Additionally, recent data of a case report suggest that UVA1 might contribute to a reversal of brain dysfunction and may also improve covered discoid lupus lesions via unknown systemic pathways [78]. As to our knowledge, no positive effects of PUVA treatment have been reported so far. Sclerotic rarities Eosinophilic fasciitis is a rare disorder disabling joint motility closely related to profound morphea with a variable response to treatment [79]. UVA1 A case report could verify a significant clinical improvement of eosinophilic fasciitis with low dose UVA1 therapy four times a week for ten weeks, resulting in a total of 40 treatment sessions, additional to azathioprine treatment. At each treatment session 20 J/cm2 UVA1 were applied, resulting in a cumulative dose of 800 J/cm2. Afterwards the induration had softened markedly and the patient was able to close the fist again [80]. PUVA Eosinophilic fasciitis successfully treated with PUVA bath photochemotherapy was described by Schiener et al. in 2000 [81]. In their study the authors present a case report of single dose 0.3 J/cm2 PUVA four times a week for period of 35 treatments including a progression of 0.3 J/cm2 every third session. Subsequently, irradiation frequency was reduced to three times a week for three weeks or two times a week for another two weeks resulting in a total of 50 treatment sessions and a cumulative dose of 102.1 J/cm2. Pansclerotic morphea of childhood represents a severe variant of LS, often lethal even in young patients [82]. UVA1 In 1997, Gruss et al. reported the successful administration of low-dose UVA1 phototherapy in disabling pansclerotic morphea of childhood by the usage of 20 J/cm2 four times a week for eight weeks resulting in a total of 32 treatment sessions, a cumulative dose of 640 J/cm2 UVA1 and a remarkable softening of the skin [83]. Another recent study could underline the efficacy of UVA exposure [84]. PUVA In 1995, Scharffetter-Kochanek et al. presented a successful approach of PUVA therapy in disabling pansclerotic morphea of a young girl [85]. UVA was administered by a maximum singe dose of 1.8 J/cm2 four times weekly for the first two months followed by maintenance on two treatments per week for another six months. In contrast, an additional case report demonstrated one patient failing to respond to PUVA therapy as an adjunct to penicillamine treatment [86]. Scleromyxedema is a variant of lichen myxedematosus exhibiting erythematous, sclerotic and stiffed lesions beside lichenoid papules caused by an extensive dermal deposition of glycosaminoglycans with only little tendency of spontaneous remission [87-89]. PUVA Following a first promising attempt of PUVA treatment as early as 1984 [90], Adachi et al. tried systemic PUVA photochemotherapy in lichen myxedematosus administering 35 treatment session at a cumulative dose of 202 J/cm2 [91]. In this respect, the authors speculate on the inhibition of dermal fibroblasts and synthesis of mucopolysaccharides as a possible mechanism of action. Nevertheless, Schirren et al. achieved only limited beneficial effect after combined chlorambucil and PUVA therapy [92]. Scleredema adultorum Buschke, occurring secondarily to diabetes or independently, is an uncommon party sclerodermoid disease characterized by erythematous indurated skin and a mucinous dermal infiltration exhibiting increased collagen deposition [93,94]. PUVA Both PUVA bath and PUVA cream have been reported to be of benefit in patients suffering from Buschke's disease. First, bath PUVA therapy was tested in 1998 by Hager et al. in case of three patients exhibiting resistant scleredema adultorum [95]. A median of 59 treatments and a cumulative dose of 245.7 J/cm2 UVA was applied resulting in a substantial clinical improvement in all three patients. Later, Grundmann-Kollmann and co-workers introduced cream PUVA in a patient responding excellently to UVA irradiation (35 sessions, 114.5 J/cm2 total cumulative dose) [96]. As to our knowledge, UVA/UVA1 phototherapy have so far not been taken into account neither in scleredema adultorum nor in scleromyxedema. POEMS syndrome, characterized by polyneuropathy, organomegaly, endocrinopathy and elevated levels of a monoclonal protein, often exhibit scleroderma-like skin changes [97]. UVA1 Severe therapy-resistant cutaneous sclerodermatous lesions of one patient suffering from POEMS syndrome showed a remarkable improvement following low-dose UVA1 phototherapy given for 35 treatment sessions [98]. Bleomycin-induced SSc-like scleroderma may occur following application of the antitumor agent bleomycin [99]. UVA1 The authors report a case of drug induced scleroderma after bleomycin administration given due to a malignant testicular seminoma. Low-dose UVA1 phototherapy (20 J/cm2 UVA1, three to four times a week) caused an initial improvement of skin condition, but could not stop the overall progress [99]. Pansclerotic porphyria cutanea tarda is an uncommon subtype of cutaneous porphyria [100]. Simultaneously, massive exposure of organic solvents may also result in sclerotic modifications. UVA1 In 2003, Karamfilov et al. could stop progression of skin affection by the use of medium-dose UVA1 phototherapy combined with intensive physiotherapy and oral glucocorticoids in a patient with pansclerotic porphyria cutanea tarda after chronic exposure to organic solvents [101]. UVA1 irradiation was applied for a total of 30 sessions, a single-dose of 40 J/cm2 and a corresponding cumulative dose of 1200 J/cm2. Discussion Beside a high number of different side indications, systemic and especially topical PUVA treatment have been shown to provoke a remarkable clearance of psoriatic plaques and infiltrated lesions of cutaneous T cell lymphoma. On the other hand, UVA1 phototherapy achieved practical value in the treatment of inflammatory and malignant T cell related skin diseases. Typical indications regularly include exacerbated atopic dermatitis, cutaneous T cell lymphoma, parapsoriasis or mucinosis follicularis due to the induction of T cell apoptosis and dermal immunoregulation. By reason of notable collateral induction of dermal collagenase activity, UVA irradiation was subsequently introduced as a treatment alternative in LS and other sclerotic collagenoses in both dermatology and rheumatology. As far as the extensive accumulation of collagen is concerned several investigations demonstrated the long-wave UVA-induced stimulation of the synthesis of specific mRNA-levels of various matrix-metalloproteinases in cultured human fibroblasts, probably due to their lower antioxidant capacity and involvement of the protein kinase C pathway [21,102-110]. Simultaneously, an elevation of interstitial collagenase m-RNA and protein expression can be determined immunohistochemically and by the use of nucleic-acid in-situ hybridization in dermal fibroblasts [21,111]. Furthermore, several studies provided evidence that at least UVA1 irradiation induces the formation of several cytokines and soluble factors e.g. interleukin-1 and/or interleukin-6 stimulating the synthesis of collagenase, while some immunomodulatory cytokines remain unaltered [112,113]. On the other hand, UVA1 irradiation has been shown to initiate apoptotic cell death in dermal T lymphocytes [114,115]. A shift of the balance between protooncogenes (e.g., bcl-2) and tumor suppressor genes (e.g., p53) towards the induction of apoptosis seems to be one of the major effects of UVA1 irradiation [10]. Beside the involvement of singlet oxygen as an early intermediate in collagenase induction, oxidative stress has also been proven to induce lipid peroxidation in cytoplasmatic membranes and to be responsible for DNA damage [116,117]. Therefore, one causal factor of membrane alterations might be the (P)UVA-induced generation of reactive oxygen species, such as singlet oxygens or superoxide anions and hydroxyl radicals leading to lipid peroxidation, structural and functional modifications of membranes characterized by altered fluidity, increased permeability and inactivation of cellular enzymes and transport proteins [116-119]. Furthermore, singlet oxygen is able to open mitochondrial megachannels, releasing apoptosis initiating factor (AIF) and cytochrom c heading towards indirect DNA damage in T cells [116,120,121]. Besides, UVA1-induced apoptosis is triggered by receptor mechanisms, e.g. by the alternative activation of the FAS/FAS-ligand (APO-1, CD95) system in peripheral T cells [114,116,122]. If and to what extent additional modulations of impaired endothelial cells might also contribute to the posttherapeutic clinical and histological improvement still has to be investigated [123]. Nevertheless, the efficacy of different regimens of UVA phototherapy might probably, at least in parts, be due to the mechanisms of action as mentioned above. Inasmuch as LE and other autoimmune disorders are concerned, a transparent mechanism of action remains obscure. Immunohistologically, LE is at least characterized by an inflammatory T cell derived infiltration mainly of the T helper subtype combined with an impairment of T and B lymphocyte regulation, dysregulated dendritic cell abnormalities and defective clearance of immune complexes and autoantigens [124-126]. Analogous to the sclerosing disorders as mentioned above, induction of apoptosis in T lymphocytes may also represent the committed step of UVA exposure in LE. Simultaneously, UVA has been shown to directly affect presence, function and morphology of dermal and epidermal Langerhans cells, which may result in a suppression of cell-mediated immunity and a disruption of autoreactive T lymphocyte, B cell and Langerhans cell stimulating processes [127-131]. Controversially, UV radiation is often associated with exacerbating skin eruptions and photosensitivity is actually a diagnostic criterion of LE. Nevertheless, in how far improvement of skin condition after repeated irradiation might be due to adaptive decreased oxidative stress upon subsequent UVA exposures has to be evaluated [131]. Despite an extensive therapeutic administration of UVA irradiation, relatively little data are available concerning possible acute and long-term side effects. Usually, patients exhibit a dose-dependent tanning of the skin, which has been described to appear following a single minimal pigmenting dose of 50 J/cm2 UVA1 [132,133], whereas UVA1-induced erythema can usually only be observed after ≥ 90 J/cm2 single dose UVA1 [134]. After UVA1 exposure, individuals of all skin types appear to develop more or less immediate pigment darkening that is due to a reversible photochemical reaction (oxidation of melanin and its precursors and metabolites). In contrast, even after a comparably lower dosage, broad-band UVA irradiation is able to provoke erythema and delayed tanning which is due to an enzymatically controlled production of melanin polymers. After topical or systemic PUVA treatment patients exhibit the characteristic methoxsalen dose-dependent PUVA erythema ranging from three to six days following irradiation [135,136]. Additionally, systemic PUVA is often associated with nausea or vomitus. However, by considering exclusion criteria such as an autoimmune disease associated abnormally increased photosensibility, solar and heat induced urticaria or a history of polymorphous light eruption and, especially in the case of PUVA therapy, by the consequent posttherapeutic use of potent sun protection, additional clinically relevant acute side effects may usually not be expected. As far as long-term side effects are concerned no definite prediction has yet been taken. Controlled studies dealing with the carcinogenesis induced by broad-band UVA sources are still rare. Nevertheless, the induction of dermal hyperplastic elastic fibers resulting in early skin ageing following a cumulative dose of 4000–8000 J/cm2 UVA1 seems to be imminent [6]. Additionally, as already reported above, the induction of collagenases released by dermal fibroblasts is known to be an important cofactor within this process [9,137]. On the other hand, carcinogenesis of UVA1 irradiation is still poorly understood. Taking the elimination of the potentially procarcinogenetic wavelength ranging from 315–320 nm into account, one might speculate on the possibly lower risk of UVA irradiation [138]. Indeed, possible melanogenetic long-term effects of long-wavelength UV irradiation (induction of malignant melanomas) have previously been discussed [139,140]. Simultaneously, animal studies suggested the induction of squamous cell carcinomas even though provoked by 220 kJ/m2 for a period of 265 days [6]. Until today, no assignment to humans could be achieved. However, at least concentrated PUVA photochemotherapy has been found to be related to potential mutagenesis and the increased occurrence of squamous cell carcinomas as well as malignant melanomas in psoriatic patients [9,141-143]. Moreover, recent investigations concerning the effects of UVA1 irradiation on human dermal endothelium revealed the initiation of apoptotic cascades even after a comparably low dose of single 80 J/cm2 UVA1 radiation. Due to the penetration of up to 20% of UVA to the level of dermal vasculature, the induction of the programmed cell death cascades may develop to one of the main side effects of UVA phototherapeutic strategies [144]. Even though especially in case of LE sunlight exposure has been postulated to induce exacerbation in as much as half of the patients, courses of UVA irradiation can also be used in such disease. In this respect, UVA2 and UVB seem to be responsible for the induction of LE eruptions [145]. Nevertheless, different studies provide strong evidence that apoptosis associated with a shift of the balance between p53 and bcl-2, simultaneously one of the main mechanisms of action concerning UVA phototherapy, may play a role in the pathogenesis and activity of LE and might to correlate with the sequential progress of LE skin lesions [146,147]. As the peak of dermatological therapeutic usage of UVA irradiation still seems to rise, scientific research engagement is needed in order to rate its potential long-lasting negative impact. Therefore, until the evaluation of firm data UVA phototherapy should most likely be restricted in the number of cycles per year, treatment should be supervised by an experienced dermatologist and a UV pass book should be issued. Conclusion Today, by considering this widespread range of clinical and experimental studies, one might clearly conclude that different regimens of UVA phototherapy have simultaneously been developed to effective, often well-tolerated and beneficial therapeutic strategies in the treatment of a variety of sclerotic skin diseases such as SSc, LS, chronic GvHD, extragenital LSA or sclerodermoid rarities and other disorders affecting the connective tissue. In this respect, the controlled application of UVA irradiation seems to exhibit a comparably tolerable risk/benefit ratio as a minimum in case of these precise indications. Furthermore, UVA phototherapy might also be considered as an optional treatment in both the cutaneous and systemic forms of LE, although the mechanism of action remains difficult to understand. Nevertheless, therapeutic application of UVA phototherapy, especially of long-wave UVA1 phototherapy, is still 'under construction' as to its limited availability apart from selective centers of excellence and mostly uncontrolled pilot investigations or case reports especially as far as PUVA or conventional UVA1 phototherapy in sclerodermic skin affections or autoimmune disorders are concerned. From our point of view there is no doubt that UVA phototherapy could by far be much more frequently used in the treatment of connective tissue lesions of the skin and, due to its ability to affect dermal vascular structure, even of extracutaneous manifestations. However, additional research efforts are required to determine an exemplary clear-cut catalogue of indications responding to UVA irradiation. Therefore, oncoming controlled randomized studies evaluating the efficacy of UVA phototherapy in connective tissue diseases should not only focus on the assessment of further innovative indications, but also on the comparison between phototherapeutic agents and conventional immunosupressive/-modulating regimens as for example systemic glucocorticoids, azathioprine, methotrexate or cyclophosphamide as well as on the confirmation of former uncontrolled reports, not only because of the fact that the natural history of fibrotic disorders includes a period of inflammation/rapid induration followed by a prolonged period of regression even in untreated patients, but also in order to expand its usage to a widely available treatment option. Competing interests None declared. Authors' contributions FB conceived of this investigation including its methods and manuscript structure, performed the comprehensive literature search including data extraction and interpretation, and finished the paper. TG participated in the literature search. A.K. conceived of the study. P.A. participated in its design and coordination. All authors read and approved the final manuscript. Figure 1 UVA1 phototherapy in systemic sclerosis. Clinical appearence of acrosclerotic piece-meal necrosis of the first digit in SSc before (Fig. 1) and almost complete clearance following low-dose UVA1 phototherapy (Fig. 2). Figure 2 UVA1 phototherapy in systemic sclerosis. Clinical appearence of acrosclerotic piece-meal necrosis of the first digit in SSc before (Fig. 1) and almost complete clearance following low-dose UVA1 phototherapy (Fig. 2). Figure 3 UVA1 phototherapy in localized scleroderma. Macroscopic aspects of LS displaying extensive sclerosis on the chest before (Fig. 3) and after low-dose UVA1 irradiation resulting in a remarkable softening (Fig. 4). Figure 4 UVA1 phototherapy in localized scleroderma. Macroscopic aspects of LS displaying extensive sclerosis on the chest before (Fig. 3) and after low-dose UVA1 irradiation resulting in a remarkable softening (Fig. 4). Figure 5 UVA1 phototherapy in extragenital lichen sclerosus et atrophicus. Confetti-like lesions of extragenital LSA (Fig. 5) and marked improvement of following low-dose UVA1 phototherapy (Fig. 6). Figure 6 UVA1 phototherapy in extragenital lichen sclerosus et atrophicus. Confetti-like lesions of extragenital LSA (Fig. 5) and marked improvement of following low-dose UVA1 phototherapy (Fig. 6). Table 1 Overview of the different phototherapeutic strategies within the main groups of sclerotic connective tissue diseases. [Categories: A – double-blind, randomized, placebo-controlled; B – open, randomised; C – open, non-randomized; D – case series; E – case report] Disease Therapy Dosage Experience Comments Systemic sclerosis UVA - - requires evaluation UVA128–31 low-/medium-dose E, C benefit, especially suited for acrosclerosis and partial body exposure PUVA18,32–34 medium-dose E, D bath application in childhood discussed Localized scleroderma UVA36,37 low-dose C benefit, no further evaluation UVA138–45 low-/medium-/high-dose D, C no exact recommendation in favor to best dosage, benefit, combination with calcipotriol where appropriate, successful in childhood/adolescence PUVA19,32,34,46–51 high-dose E, D questionable efficacy, extreme variance in dosage, combination (cream) with calcipotriol in childhood Extragenital lichen sclerosus et atrophicus UVA - - requires evaluation UVA140,56–58 low-dose E, D, C effectiveness, disputable in combined morphea/lichen sclerosis et atrophicus PUVA59,60 low-dose E benefit, careful cream therapy for genitoanal lesions where appropriate Sclerodermoid graft- vs-host disease UVA - - requires evaluation UVA162–64 low-/medium-dose E, D partial efficacy, medium-dose possibly more effective than low-dose, combined UV/immunosuppressive therapy PUVA65–71 medium-dose E, D, C skeptical effectiveness, potentially adjunct therapy in addition to conventional chemotherapy, more effective in lichenoid than sclerodermoid lesions Lupus erythematosus UVA - - requires evaluation UVA173–78 low-dose D, C, A benefit in occasional cases, long-term application PUVA - - requires evaluation Table 2 Synopsis of recent case reports decribing various phototherapeutic alternatives in a number of sclerotic rarities. [Categories: A – double-blind, randomized, placebo-controlled; B – open, randomised; C – open, non-randomized; D – case series; E – case report] Disease Therapy Dosage Experience Comments Eosinophilic fasciitis UVA180 low-dose E benefit, no valid data available PUVA81 medium-dose E Pansclerotic morphea UVA84 low-dose E possible efficacy as an adjunct therapy, UVA183 low-dose E no valid data available PUVA85,86 medium-dose E Scleromyxedema PUVA90–92 high-dose E controversial, possible efficacy as an adjunct therapy, no valid data available Scleredema adultorum Buschke PUVA95,96 high-dose E, D possible therapeutic alternative, cream therapy, no valid data available POEMS UVA198 low-dose E benefit, no valid data available Bleomycin-induced scleroderma UVA199 low-dose E limited success, no valid data available Pansclerotic porphyria cutanea tarda UVA1101 medium-dose E benefit, no valid data available Pre-publication history The pre-publication history for this paper can be accessed here: ==== Refs van Dooren-Greebe RJ Schattenberg A Koopman RJ Chronic cutaneous graft-versus-host disease: successful treatment with UVB Br J Dermatol 1991 125 498 499 1751360 Hoffmann K Kaspar K Altmeyer P Gambichler T UV transmission measurements of small skin specimens with special quartz cuvettes Dermatology 2000 201 307 311 11146339 10.1159/000051543 Repacholi MH Mathes R Fundamentals. 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radiation: quantitative and morphological study Br J Dermatol 2003 148 291 299 12588382 10.1046/j.1365-2133.2003.05112.x Breuckmann F von Kobyletzki G Avermaete A Pieck C Kreuter A Brockmeyer NH Altmeyer P Gambichler T Mononuclear cells in atopic dermatitis in vivo: immunomodulation of the cutaneous infiltrate by medium-dose UVA1 phototherapy Eur J Med Res 2002 7 315 322 12176681 Gruner S Hofmann T Meffert H Sonnichsen N Studies on the effects of a high dose UVA-1 radiation therapy on surface markers and function of epidermal Langerhans cells Arch Dermatol Res 1993 285 283 286 8379688 Grabbe J Welker P Humke S Grewe M Schopf E Henz BM Krutmann J High-dose ultraviolet A1 (UVA1), but not UVA/UVB therapy, decreases IgE-binding cells in lesional skin of patients with atopic eczema J Invest Dermatol 1996 107 419 422 8751980 10.1111/1523-1747.ep12363402 Cohen MR Isenberg DA Ultraviolet irradiation in systemic lupus erythematosus: friend or foe? Br J Rheumatol 1996 35 1002 1007 8883441 10.1093/rheumatology/35.10.1002 Kowalzick L UV-A1-Therapie des atopischen Ekzems – derzeitiger Stand Dt Derm 1997 45 930 933 Piazena H Meffert H Schmollack KP Zum Einfluss von UVA-1-Bestrahlungen auf die Erythemempfindlichkeit des Menschen Dermatol Monatsschr 1993 179 22 24 Kowalzick L Büttner G Neuber K Kleinheinz A Weichenthal M Ring J UVA1-Kaltlicht-Bestrahlungsbehandlung des atopischen Ekzems: Positive Dosiswirkungskorrelation Hautnah Derm 1995 11 374 378 Man I Kwok YK Dawe RS Ferguson J Ibbotson SH The time course of topical PUVA erythema following 15- and 5-minute methoxsalen immersion Arch Dermatol 2003 139 331 334 12622625 10.1001/archderm.139.3.331 Ibbotson SH Dawe RS Farr PM The effect of methoxsalen dose on ultraviolet-A-induced erythema J Invest Dermatol 2001 116 813 815 11348476 10.1046/j.1523-1747.2001.01348.x von Kobyletzki G Pieck C Hoffmann K Freitag M Altmeyer P Medium-dose UVA1 cold-light phototherapy in the treatment of severe atopic dermatitis J Am Acad Dermatol 1999 41 931 937 10570376 von Bohlen F Kallusky J Woll R Die UVA-1-Kaltlichtbehandlung der atopischen Dermatitis Allergologie 1994 17 382 384 Setlow RB Grist E Thompson K Woodhead AD Wavelengths effective in induction of malignant melanoma Proc Natl Acad Sci 1993 90 6666 6670 8341684 Staberg B Wulf HC Klemp P Poulsen T Brodthagen H The carcinogenic effect of UVA irradiation J Invest Dermatol 1983 81 517 519 6644093 10.1111/1523-1747.ep12522855 Shyong EQ Lu Y Lazinsky A Saladi RN Phelps RG Austin LM Lebwohl M Wei H Effects of the isoflavone 4',5,7-trihydroxyisoflavone (genistein) on psoralen plus ultraviolet A radiation (PUVA)-induced photodamage Carcinogenesis 2002 23 317 321 11872639 10.1093/carcin/23.2.317 Stern RS PUVA Follow up Study The risk of melanoma in association with long-term exposure to PUVA J Am Acad Dermatol 2001 44 755 761 11312420 10.1067/mjd.2001.114576 Morison WL Baughman RD Day RM Forbes PD Hoenigsmann H Krueger GG Lebwohl M Lew R Naldi L Parrish JA Piepkorn M Stern RS Weinstein GD Whitmore SE Consensus workshop on the toxic effects of long-term PUVA therapy Arch Dermatol 1998 134 595 598 9606329 10.1001/archderm.134.5.595 Breuckmann F von Kobyletzki G Avermaete A Kreuter A Mannherz HG Altmeyer P Apoptosis of human dermal endothelial cells as a potential side effect following therapeutic administration of UVA1 irradiation: preliminary results Arch Dermatol Res 2002 294 303 309 12373335 Lehmann P Holzle E Kind P Goerz G Plewig G Experimental reproduction of skin lesions in lupus erythematosus by UVA and UVB radiation J Am Acad Dermatol 1990 22 181 187 2179293 Miret C Molina R Filella X Garcia-Carrasco M Claver G Ingelmo M Ballesta A Font J Relationship of p53 with other oncogenes, cytokines and systemic lupus erythematosus activity Tumour Biol 2003 24 185 188 14654712 10.1159/000074428 Baima B Sticherling M Apoptosis in different cutaneous manifestations of lupus erythematosus Br J Dermatol 2001 144 958 966 11359381 10.1046/j.1365-2133.2001.04182.x

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==== Front BMC CancerBMC Cancer1471-2407BioMed Central London 1471-2407-4-641536310810.1186/1471-2407-4-64Research ArticleTransformation and scattering activities of the receptor tyrosine kinase RON/Stk in rodent fibroblasts and lack of regulation by the jaagsiekte sheep retrovirus receptor, Hyal2 Miller A Dusty [email protected] Hoeven Neal S [email protected] Shan-Lu [email protected] Division of Human Biology, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, Seattle, WA 98109-1024, USA2 Molecular and Cellular Biology Program, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, Seattle, WA 98109-1024, USA2004 13 9 2004 4 64 64 9 7 2004 13 9 2004 Copyright © 2004 Miller et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background The envelope (Env) protein of jaagsiekte sheep retrovirus (JSRV) can transform cells in culture and is likely to be the main factor responsible for lung cancer induction by JSRV in animals. A recent report indicates that the epithelial-cell transforming activity of JSRV Env depends on activation of the cell-surface receptor tyrosine kinase Mst1r (called RON for the human and Stk for the rodent orthologs). In the immortalized line of human epithelial cells used (BEAS-2B cells), the virus receptor Hyal2 was found to bind to and suppress the activity of RON. When Env was expressed it bound to Hyal2 causing its degradation, release of RON activity from Hyal2 suppression, and activation of pathways resulting in cell transformation. Methods Due to difficulty with reproducibility of the transformation assay in BEAS-2B cells, we have used more tractable rodent fibroblast models to further study Hyal2 modulation of RON/Stk transforming activity and potential effects of Hyal2 on RON/Stk activation by its natural ligand, macrophage stimulating protein (MSP). Results We did not detect transformation of NIH 3T3 cells by plasmids expressing RON or Stk, but did detect transformation of 208F rat fibroblasts by these plasmids at a very low rate. We were able to isolate 208F cell clones that expressed RON or Stk and that showed changes in morphology indicative of transformation. The parental 208F cells did not respond to MSP but 208F cells expressing RON or Stk showed obvious increases in scattering/transformation in response to MSP. Human Hyal2 had no effect on the basal or MSP-induced phenotypes of RON-expressing 208F cells, and human, mouse or rat Hyal2 had no effect on the basal or MSP-induced phenotypes of Stk-expressing 208F cells. Conclusions We have shown that RON or Stk expression in 208F rat fibroblasts results in a transformed phenotype that is enhanced by addition of the natural ligand for these proteins, MSP. Hyal2 does not directly modulate the basal or MSP-induced RON/Stk activity, although it is possible that adaptor proteins might mediate such signaling in other cell types. ==== Body Background JSRV is an acutely oncogenic retrovirus that can induce lung tumors in newborn sheep in as little as 10 days [1]. Unlike most acutely oncogenic retroviruses, JSRV does not carry a host cell-derived oncogene. Instead, expression of the native viral Env protein is sufficient to transform cultured cells and is likely to be the active oncogene in animals [2-5]. The only other examples of such retroviruses are enzootic nasal tumor virus, a close relative of JSRV that encodes a similar transforming Env protein [6,7]; avian hemangioma virus [8], which expresses an Env protein that can induce cell proliferation in cultured cells [9]; and spleen focus-forming virus, a replication-defective virus that expresses a recombinant nonfunctional Env protein that has been shown to induce proliferation in cultured cells and in animals by activating the erythropoietin receptor and a short form of the transmembrane receptor tyrosine kinase Stk [10,11]. At least two mechanisms of transformation by JSRV Env have been identified in cultured cells. JSRV Env can transform immortalized mouse, rat, and chicken fibroblasts [2-4], and the cytoplasmic tail of Env is absolutely required for transformation [4,12-14]. Interaction of the extracellular domain of Env with the virus receptor Hyal2 appears to play no role in transformation of the rodent fibroblasts since JSRV Env does not bind to mouse Hyal2 [15], and deletion of the Hyal2-binding domain of Env did not abrogate transformation of rat fibroblasts [16]. Although there is some controversy regarding the mechanism of transformation, most studies indicate a role for the phosphatidylinositol 3-kinase (PI3K)/Akt pathway in transformation of the rodent fibroblasts [7,12,13,17]. In contrast, this pathway appears to play a smaller role in the transformation of chicken fibroblasts, but other more important pathways have yet to be identified [4,14]. A second pathway for JSRV Env transformation involves activation of the transmembrane receptor tyrosine kinase Mst1r (herein called RON for the human and Stk for the rodent forms [18,19]). RON/Stk activation is associated with cell scattering, cell transformation, and oncogenesis in animals [20]. RON/Stk activity is induced by binding of its only known ligand, macrophage stimulating protein (MSP) [21,22]. Recent evidence indicates that the JSRV receptor Hyal2 is also involved in regulating RON activity in an immortal human epithelial cell line (BEAS-2B) that naturally expresses Hyal2 and RON [5]. In its normal inactive state, RON is bound to Hyal2, and when Hyal2 binding is prevented by JSRV Env, RON becomes constitutively active in the absence of MSP stimulation. Introduction of a dominant negative form of RON into these cells blocked transformation, indicating that main transformation pathway is through RON and that JSRV might cause cancer through a novel mechanism involving RON activation [5]. Thus it appears that JSRV Env can transform cells by at least two distinct mechanisms. Furthermore, the dependence of epithelial cell transformation on activation of the RON pathway, in contrast to fibroblasts, which do not express RON and are transformed through a different pathway, indicated that the RON pathway might be more important in oncogenesis in animals because epithelial cells are the natural target for JSRV-induced cancer in sheep. We have had difficulty in further exploring transformation through the RON pathway in BEAS-2B cells due to difficulty with reproducibility of the transformation assay. Others have found that Stk can transform immortalized rodent fibroblasts [23,24], and we hypothesized that if Hyal2 does downregulate RON/Stk activity, we might be able to suppress transformation by RON/Stk by overexpressing Hyal2. In addition, we wanted to explore whether Hyal2 might modulate MSP induction of RON/Stk activity. Here, we have been able to generate 208F rat fibroblasts that express either RON or Stk and find that these cells exhibit a transformed phenotype in comparison to the parental cells. Treatment of the cells with MSP induced a dose-dependent increase in scattering/transformation. Using a variety of approaches we have been unable to detect an effect of Hyal2 on the constitutive or MSP-induced phenotypes of 208F cells expressing RON or Stk. These results argue against a direct role for Hyal2 in the regulation of RON/Stk activity. Methods Cell culture Cell lines used here include 208F Fischer rat embryo fibroblasts [25], a morphologically flat subclone of NIH 3T3 Swiss mouse embryo fibroblasts suitable for transformation studies (gift from Maxine Linial, Fred Hutchinson Cancer Research Center, Seattle; originally from Doug Lowy, National Cancer Institute, Bethesda), PJ4/LAPSN packaging cells [26] that produce the LAPSN retroviral vector [27] with a JSRV pseudotype, and PT67 10A1-MLV-pseudotype retrovirus packaging cells [28]. Unless otherwise stated, cells were grown in Dulbecco's modified Eagle medium (DMEM) with high glucose (4.5 g/L) and 10% fetal bovine serum at 37°C in a 10% CO2-air atmosphere at 100% relative humidity. Expression plasmids Mouse Stk was expressed by using a plasmid (gift from Sandra Ruscetti, National Cancer Institute, Frederick, Maryland) that contains the mouse Stk cDNA cloned into the NotI site of the pAlter-Max expression plasmid (Promega, Madison, Wisconsin). Human RON was expressed by using a plasmid (gift from Michael Lerman, National Cancer Institute, Frederick, Maryland) that contains the human RON cDNA inserted into the pCI-neo expression plasmid (Promega). The expression cassette in both of these plasmids is identical and consists of a human cytomegalovirus immediate early promoter followed by splicing signals, the cDNA, and the simian virus 40 late polyadenylation signal. Mouse Stk was also expressed using the previously described pcDNA3-based expression vector [23] (gift from Susan Waltz, University of Cincinnati, Ohio), which contains the human cytomegalovirus early promoter, no splice signals, and the bovine growth hormone polyadenylation signal. Human RON was also expressed using the previously described retroviral vector expression plasmid pMSCVpuroRON [5], in which the RON cDNA is expressed from the Moloney murine leukemia virus promoter and the puromycin resistance gene is expressed using a phosphoglycerate kinase promoter. In this vector both spliced and unspliced mRNAs are made that encode RON. Retroviral vectors The LAPSN retroviral vector encodes human placental alkaline phosphatase (AP) and neomycin phosphotransferase (Neo). LAPSN virus was made using PT67 (10A1-MLV pseudotype) or PJ4 (JSRV pseudotype) packaging cells. Retroviral vectors encoding human Hyal1, human Hyal2, mouse Hyal2 cloned from NIH 3T3 cells, and rat Hyal2 cloned from 208F rat cells (GenBank accession numbers U03056, U09577, AF535140, and AF535141, respectively) were made by insertion of the cDNAs into the LXSN vector [29] and by generation of stable PT67 retrovirus packaging cell lines that produce the vectors as described [3,15,29]. The human Hyal1 and human Hyal2 vectors present in the packaging cell clones that were used are known to be functional based on phenotypic assays of the proteins made in cells transduced with the vectors [3]. Two independent packaging cell clones that produce the rat Hyal2 vector and two independent clones that produce the mouse Hyal2 vector were used, and the correct sequence of the cDNAs in the integrated vectors in these packaging cells was confirmed by PCR amplification of the sequences followed by complete sequencing of the cDNAs. Cell transformation assay NIH 3T3 and 208F cells were transfected using calcium phosphate as previously described [15] and were monitored for appearance of transformed foci of cells for up to a month after transfection. Cellular response to MSP Recombinant human MSP activated by treatment with kallikrein (R&D Systems, Minneapolis, MN) was dissolved at a concentration of 10 μg/ml in phosphate buffered saline with calcium and magnesium containing 0.2% bovine serum albumin and was stored at -70°C. Cells were seeded at 10,000 per well (d = 16 mm) of 24-well plates. Two days after seeding, the cells were treated with 0.06 to 20 μl of MSP stock in 0.5 ml of fresh medium (MSP concentration of 1.2 to 400 ng/ml) and cellular morphology was evaluated the day after MSP addition. Treatment of cells with 20 μl buffer containing 0.2% bovine serum albumin in 0.5 ml of fresh medium had no effect on cell morphology. Results RON and Stk exhibit low transforming activity in rodent fibroblasts We tested plasmids encoding RON or Stk (pCIneoRON and pAlter-Max-Stk, respectively) for transforming activity in NIH 3T3 mouse and 208F rat fibroblasts. The cDNAs in both plasmids were driven by a strong human cytomegalovirus immediate early promoter and each contained an intron to promote high-level expression. In these assays, strong oncogenes induce transformed foci of cells in less than a week while less active oncogenes take longer to induce foci. In both cases the numbers of foci generally increase with time after transfection. Neither the RON nor the Stk expression plasmid induced transformed foci in NIH 3T3 cells (<0.2 foci per μg plasmid DNA at one month after transfection), while a plasmid expressing JSRV Env (pSX2.Jenv) induced 33 well-developed foci per μg plasmid DNA under the same conditions. This result is consistent with previous reports showing a lack of transformation by human RON [30-32], but is inconsistent with reports indicating efficient transformation of NIH 3T3 cells by mouse Stk (called "Ron" in these reports) [23,24]. We obtained the Stk expression plasmid used by the latter group (full-length mouse Ron cDNA in pcDNA3), and again could not detect transformation of NIH 3T3 cells by this plasmid (<0.2 foci per μg plasmid DNA at one month after transfection). There was some background focal growth in all plates of transfected NIH 3T3 cells one month after transfection, but this growth was identical in plates transfected with RON/Stk or control expression plasmids. In contrast, foci induced by JSRV Env were distinct and readily detected at 2 weeks after transfection. Next we tested for transformation of 208F rat fibroblasts by the RON and Stk plasmids. The Stk plasmid (pAlter-Max-Stk) induced 3 clear foci in a total of 7 dishes (0.2 foci per μg plasmid DNA), and the RON plasmid (pCIneoRON) induced 1 clear focus in a total of 5 dishes (0.1 foci per μg plasmid DNA) (foci counted 1 month after transfection, 2 μg plasmid DNA per dish, 2–3 separate experiments for each plasmid). The Stk expression plasmid pcDNA3Stk used in the earlier studies [23,24] did not induce transformed foci in the 208F cells (<0.1 foci per μg plasmid DNA). For comparison, the JSRV Env expression plasmid induced 300 to over 500 foci per dish when counted 2 weeks after transfection under the same conditions. Together these results document a very low transforming activity for the RON and Stk genes. Generation of cells expressing RON and Stk that respond to MSP Foci of transformed 208F cells were isolated from plates of cells transfected with the RON or Stk expression plasmids and were tested for their response to MSP. Cells transformed by the RON plasmid were not responsive to MSP and were discarded. Cells transformed by the Stk plasmid were responsive to MSP, and two clones (208F/Stk c12 and c13) were used for further study. Both of these clones made Stk protein of the appropriate molecular weight as judged by western analysis using RON/Stk-specific antibodies (data not shown). The response of 208F/Stk c13 cells to overnight treatment with MSP is shown in Fig. 1. Without MSP the cells were elongated and formed chains of connected cells (Fig. 1, top right panel), in contrast to the parental 208F cells which grow as flat uniformly-expanding colonies (Fig. 1, top left panel). Treatment of the 208F/Stk c13 cells with MSP resulted in dramatic scattering (Fig. 1, bottom right panel) while treatment of the parental 208F cells with MSP had no effect on cell morphology (Fig. 1, bottom left panel). The MSP response was fully reversible; incubation of cells overnight in regular growth medium reversed the scattering induced by MSP and chains of cells reformed (not shown). 208F/Stk c12 cells exhibited an even more transformed appearance than did 208F/Stk c13 cells, and many of these cells would grow in suspension, often as doublets of what appeared to be recently divided cells. These floating cells could be repeatedly regrown in new plates, showing that the cells were alive and were not simply undergoing apoptosis. Passage of the floating cells provided a simple method for maintaining the transformed phenotype of these cells, which tended to decrease with time of culture. Since we were unsuccessful in generating 208F cells expressing functional RON by isolation of transformed cells following transfection, we isolated RON-expressing cells by transfection with a RON expression vector that also expressed a selectable marker (pMSCVpuroRON), grew the cells in puromycin to select for cells expressing the marker, and isolated multiple cell clones that showed a weakly-transformed phenotype. These clonal lines were screened for scattering response to MSP, and two independent clones that had the most transformed appearance and that responded to MSP (208F/RON c9 and c10) were identified and used in further analyses. Both clones produced RON protein of the appropriate size band by western analysis using a RON/Stk-specific antibodies, and both exhibited clear surface expression of RON by FACS analysis using a RON-specific antibody while control 208F cells showed no expression (data not shown). The 208F/RON clones had a less obvious transformed phenotype than the 208F/Stk clones but still showed convincing scattering in response to MSP (see below). Hyal2 expression does not affect the phenotype of cells expressing RON or Stk We tested whether overexpression of Hyal2 would reverse the altered phenotype of 208F cells expressing RON or Stk. Since the original work in BEAS-2B human cells showed inhibition of RON activity by human Hyal2 [5], we tested human Hyal2 in the 208F/RON cells. In 208F/Stk cells we tested mouse and rat Hyal2, as well as human Hyal2, in case the rodent proteins might show more effective interaction with mouse Stk in the rat cells. To quantitate the effect of Hyal2 expression on cell phenotype, cells were seeded at low density and colonies that grew out were scored for morphology, either round and flat, round and refractile, chain-like, or scattered. Fig. 2 shows examples of the phenotypes exhibited by 208F/Stk c13 cells. The top panel shows examples of chain-like (left) and flat (right) colonies, the middle panel shows an example of a chain-like colony, and the bottom panel shows an example of a scattered colony. In initial experiments we found that transduction of the cells with control vectors encoding either AP or Hyal1 had no effect on cell morphology (data not shown), and we used these vectors as negative controls for the effects of vector transduction in subsequent experiments. We exposed 208F cells that expressed RON to retroviral vectors encoding human Hyal2, AP, or human Hyal1, grew these cells in G418 to select for cells expressing the vector, and quantitated the morphologies of the cells that grew out. The experiment was repeated by replating the G418-resistant cells at low density and repeating the morphologic quantitation. The results were similar for both cell lines in both experiments so the results for the two experiments with each of the two cell lines were averaged (Fig. 3). There was no significant change in morphology of the cells expressing human Hyal2 in comparison to those expressing human Hyal1 or AP, arguing against the hypothesis that Hyal2 can inhibit the activity of RON. Indeed, cells expressing human Hyal2 exhibited fewer flat colonies and more chain-like colonies than the other cell types, the opposite of what the hypothesis predicts, although these differences were not statistically significant (Fig. 3). To confirm that Hyal2 was expressed in these cells, we measured transduction of the cells by a JSRV-pseudotype retroviral vector, which cannot infect rat cells unless they express a functional Hyal2 receptor [15]. We confirmed that only the 208F/RON cells transduced with the human Hyal2 vector, and not those transduced with the human Hyal1 vector, were susceptible to JSRV-pseudotype LAPSN vector transduction, showing that functional human Hyal2 was indeed expressed in these cells (data not shown). Next we exposed the 208F/Stk c12 and c13 clonal cell lines to retroviral vectors encoding mouse, rat or human Hyal2, or human Hyal1, grew these cells in G418 to select for cells expressing the vector, and quantitated the morphologies of colonies that grew after plating the cells at low density (Fig. 4). There were no consistent changes in morphology in response to any of the Hyal2 proteins by comparison to cells expressing Hyal1 or no additional protein. To test for expression of the Hyal2 proteins in these cells, we exposed the cells to JSRV-pseudotype LAPSN vector and measured the transduction rate (Table 1). As expected based on previous results [15], 208F/Stk cells expressing no additional protein or expressing human Hyal1 were not transduced, cells expressing human Hyal2 were transduced efficiently, cells expressing rat Hyal2 were transduced at about 1/3 the rate of cells expressing human Hyal2, and cells expressing mouse Hyal2 were not transduced. Acquisition of JSRV vector susceptibility by the normally resistant rat 208F cells following transfer of the human and rat Hyal2 vectors indicates that the Hyal2 genes are expressed in functional forms. We cannot conclude from this assay that mouse Hyal2 is expressed in cells transduced with the mouse Hyal2 vector, but the integrated vectors in both packaging lines that were used to make the mouse Hyal2 vectors were sequenced and found to have the correct mouse Hyal2 sequence, so it is likely that normal mouse Hyal2 protein was expressed in the transduced 208F/Stk cells. Together these results indicate that the Hyal2 proteins were expressed in the transduced 208F/Stk cells and argue against the hypothesis that Hyal2 can inhibit the activity of Stk. Hyal2 overexpression in RON- or Stk-expressing 208F cells does not affect their response to MSP To quantitate cellular responses to MSP, cells seeded two days earlier were exposed to MSP at concentrations from 1.2 to 400 ng/ml (in half-log intervals) and the next day the cell morphology was examined. We measured the minimum MSP concentration at which an alteration in morphology was observed and the MSP concentration above which no further morphologic changes were observed. The right panels of Fig. 1 show an example of such extremes of morphology. The original studies showing an interaction between RON/Stk and Hyal2 studied the human proteins [5], so we first examined the effects of human Hyal2 on the MSP response of 208F/RON cells. Identical responses to MSP were observed for 208F/RON c9 and 208F/RON c10 cells that expressed human Hyal2, human Hyal1, or AP. The responses were first noticeable at 4 ng/ml MSP and were maximal at 120 ng/ml MSP. These results indicate that Hyal2 does not modulate the activation of RON in response to MSP. We next explored the possible effects of Hyal2 on the MSP response of 208F/Stk cells. We tested human, mouse, and rat Hyal2 proteins to address the possibility that only the rodent Hyal2 proteins would interact with Stk. Identical responses to MSP were observed for 208F/Stk c12 cells that expressed mouse, rat, or human Hyal2, human Hyal1, or no protein. In two independent experiments, the first effects were observed at 1.2 ng/ml MSP and the maximum effect was observed at 12 ng/ml. Identical responses were also observed for 208F/Stk c13 cells that expressed mouse, rat, or human Hyal2, human Hyal1, or no protein. In this case, the responses were first noticeable at 4 ng/ml MSP and were maximal at 40 ng/ml MSP. Untreated 208F/Stk c12 cells are more refractile and express more Stk than the 208F/Stk c13 cells (data not shown), and the higher level and basal activity of Stk in these cells likely explains their higher sensitivity to MSP. Discussion The ability of JSRV Env to transform cells in culture, the identification of Hyal2 as the cell-surface receptor for the Env protein of JSRV, and the localization of Hyal2 to the chromosome 3p21.3 lung cancer tumor suppressor locus suggested the hypothesis that JSRV might cause cancer by inhibiting a tumor suppressor activity of Hyal2 [3]. Support for this hypothesis was provided by transformation studies in the human bronchial epithelial cell line BEAS-2B, which indicated that Hyal2 could suppress the transforming activity of the RON tyrosine kinase, and that JSRV Env expression caused Hyal2 degradation and RON activation [5]. The fact that a dominant-negative kinase-dead version of RON could block transformation by JSRV Env in BEAS-2B cells indicated that the RON pathway was required for transformation. Unfortunately and despite numerous attempts we have been unable to reliably measure transformation in BEAS-2B cells using the originally described expression plasmids and transfection techniques [5]. Occasionally we observe transformed foci following transfection of the JSRV Env expression plasmid, but often cells transfected with a control non-transforming plasmid exhibit similar foci. We are able to obtain reproducible and obvious transformation of BEAS-2B cells by transducing the cells with a retroviral vector encoding JSRV Env (LJeSN vector, ref. [6]), so presumably the lack of reproducibility is due to poor Env expression following transfection. To avoid these difficulties we pursued further studies of the RON pathway in rodent fibroblast cell lines that have traditionally been used in transformation assays. Based on earlier findings that Stk could transform NIH 3T3 mouse fibroblasts [23,24], we attempted to isolate cells expressing RON or Stk by isolating transformed foci of cells following transfection of NIH 3T3 cells. However, we were unable to detect transformation of NIH 3T3 cells by RON or Stk expression plasmids, consistent with results from other groups for RON [30-32] and for Stk (Kazuo Nishigaki and Sandra Ruscetti, personal communication). Furthermore, we were unable to repeat the studies showing transformation of NIH 3T3 cells by Stk when we used the same Stk expression plasmid used in the earlier reports [23,24]. These reports showed a very high background transformation rate in the NIH 3T3 cells (50 foci per μg control DNA, ref. [24]; see photographs of cell culture dishes in Fig. 2 of ref. [23]) casting doubt on the reliability of the transformation assay. Unless a very flat subclone of NIH 3T3 cells is used in these assays, as we used here, a high and variable background transformation rate can be observed. Even here we observed a level of background morphologic changes in the NIH 3T3 cells such that low levels of transformation would be difficult to detect. In contrast, the 208F cells showed no background transformation and allowed us to detect a low frequency of transformation by RON and Stk. This low rate of transformation could be due to a requirement for a very high level of RON/Stk expression that occurs only in a small number of cells, or a requirement for mutations that activate the RON/Stk proteins and occur at low frequency. We isolated several foci of Stk-transformed 208F cells and found that these cells expressed Stk protein of the expected size by western blotting and that the cells responded to MSP, indicating that the Stk protein was not constitutively activated by mutations or deletions, and suggesting that the protein is simply overexpressed in these cells. In contrast, cells from the one focus of cells transformed by transfection of the RON gene were unresponsive to MSP and thus might have been the result of activating mutations or deletions in RON. To generate 208F cells expressing RON we transfected RON with a selectable marker and isolated cells showing a weakly transformed phenotype. We presume that the low level phenotypic alteration observed in 208F/RON and 208F/Stk cells in the absence of MSP addition is due either to low-level constitutive activity of RON/Stk or to RON/Stk activation by low levels of MSP made by the cells or present in the culture medium. Given that the parental 208F cells show no response to MSP, we conclude that the cell scattering phenotype observed after addition of MSP is a direct measure of RON/Stk activation. The transformed phenotype of these cells in the absence of added MSP and the scattering phenotype observed after treatment of the cells with MSP seem to represent variations of the same transformation phenotype. Indeed, 208F cells transformed by other oncogenes, including JSRV Env, exhibit a scattered phenotype similar to MSP treated 208F cells expressing RON or Stk. Furthermore, 208F cells expressing RON or Stk can exhibit a scattered phenotype in the absence of added MSP, especially if the cells are selected for a highly transformed phenotype by passaging substrate-independent cells that float in the culture medium. Given this interpretation, the response of the RON/Stk-expressing cells to MSP can be considered a form of ligand-dependent transformation, similar to what has been observed previously for cells expressing the receptors for human colony-stimulating factor 1 [33], insulin-like growth factor I [34], granulocyte/macrophage colony-stimulating factor [35], and a hybrid EGF receptor/Xmrk tyrosine kinase [36]. Alternatively, it may be that the basal and MSP-induced phenotypes of the RON/Stk-expressing cells are mediated by separate signaling pathways, as has been found for the related receptor tyrosine kinase MET [37]. These investigators found that a MET oncogene could induce transformed foci and an invasion phenotype in cultured rat fibroblasts, which when injected into nude mice would form tumors and would metastasize to the lungs. Two point mutations in the MET oncogene completely abrogated the invasion and metastasis phenotypes but did not affect the transformation and tumorigenesis phenotypes of the MET oncogene, indicating that separable signaling pathways were involved. We did not find any effect of human Hyal2 on the basal phenotype or the response to MSP of the 208F/RON cells, nor did we find an effect of human, rat, or mouse Hyal2 on the basal phenotype or the MSP response of 208F/Stk cells. These results argue against direct regulation of RON activity by Hyal2 as was indicated by prior experiments using BEAS-2B cells [5]. It is possible that additional factors present in BEAS-2B but not in 208F cells are required for regulation of RON activity by Hyal2. Recently we have found that Env proteins from JSRV and from the related retrovirus enzootic nasal tumor virus (ENTV) can transform Madin-Darby canine kidney (MDCK) epithelial cells, but by a mechanism different from that observed in BEAS-2B cells and similar to that observed in rodent fibroblast cell lines [38]. In particular, the cytoplasmic tail of Env is required for transformation, the PI3K/Akt pathway is activated and inhibition of PI3K activity reverses the transformed phenotype, expression of RON (which is not normally expressed in MDCK cells) does not affect transformation, and canine Hyal2 expressed by these cells appears uninvolved. These results show that the JSRV Env protein can transform epithelial cells besides BEAS-2B cells, and argue against a model for Env transformation involving different pathways that are uniquely active in epithelial cells as compared to fibroblasts. Future work will focus on the transformation pathways that are active in lung tumors induced in animals by JSRV Env. While it is possible that the RON pathway plays a role in oncogenesis, most of the evidence points to a main role for the cytoplasmic domain of Env and activation of the PI3K/Akt pathway. Conclusions We have shown that expression of the mouse or human orthologs of the receptor tyrosine kinase Mst1r (called RON in humans and Stk in rodents) can induce phenotypic changes indicative of transformation in a rat fibroblast cell line. These changes are enhanced by treatment of the cells with MSP, the natural ligand for RON/Stk, which induces a pronounced scattering/transformation response. Transformation of the BEAS-2B immortalized human epithelial cell line by JSRV appears to require RON activation, as a result of JSRV Env binding to and degradation of the virus receptor Hyal2, which appeared to negatively regulate RON activity. However, we find no evidence for direct regulation of the basal or MSP-induced RON/Stk activity by Hyal2 in rat fibroblasts. Competing interests None declared. Authors' contributions ADM designed the study, performed most of the experiments, and drafted the manuscript. NSVH and SLL performed the western blot and FACS experiments and provided intellectual input, and SLL performed the preliminary studies on BEAS-2B cells. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: Acknowledgements We thank Sandra Ruscetti, Michael Lerman, and Susan Waltz for gifts of expression plasmids and for helpful discussions, and thank Maxine Linial and Doug Lowy for the NIH 3T3 cells. This work was supported by NIH grants DK47754 and HL66947. Figures and Tables Figure 1 Scattering of 208F/Stk cells in response to MSP. 208F (left panels) and 208F/Stk c13 cells (right panels) were seeded at 2 × 104 cells per well in 24-well plates. Two days later the cells were treated overnight with 400 ng/ml MSP in growth medium (DMEM plus 10% FBS) (bottom panels) or with medium alone (top panels) and were photographed. Figure 2 Morphology of 208F/Stk colonies. Cells were seeded 5 days before photographs were taken. See text for descriptions. Figure 3 Colony morphology of 208F/RON cells is unaffected by Hyal2 expression. 208F/RON c9 and c10 cells were transduced with retrovirus vectors expressing the indicated proteins, the cells were seeded at low density, and colony morphologies were evaluated 1 week after seeding. Results are means of four experiments (two for each clone) and standard errors are shown. Figure 4 Colony morphology of 208F/Stk cells is unaffected by Hyal2 expression. 208F/Stk c12 and c13 cells were transduced with retrovirus vectors expressing the indicated proteins or were not transduced (None), the cells were seeded at low density, and colony morphologies were evaluated 1 week after seeding. In the right panel, the numbers of flat (bottom black bar) and refractile (top black bar) colonies were counted separately. In the left panel these categories were combined. The experiment was repeated with similar results. Table 1 Assay for functional expression of Hyal2 proteins in 208F/Stk cells transduced with Hyal2-expressing retroviral vectors Source of vector used to transduce cells LAPSN(PJ4) vector titer (AP+ FFU/ml) on cells: 208F/Stk c12 208F/Stk c13 No vector <10 <10 PT67/LhHyal1SN c16 <10 <10 PT67/LhHyal2SN c16 2,000 2,000 PT67/LrHyal2SN c2 800 600 PT67/LrHyal2SN c12 600 600 PT67/LmHyal2SN c7 <10 <10 PT67/LmHyal2SN c8 <10 <10 208F/Stk c12 and 208F/Stk c13 cells were exposed to vectors from the indicated vector-producing packaging cell lines and were exposed to G418 to select for expression of the vector. The cells were then exposed to JSRV-pseudotype LAPSN vector from PJ4/LAPSN cells and the apparent titers were determined. ==== Refs Sharp JM Angus KW Gray EW Scott FM Rapid transmission of sheep pulmonary adenomatosis (jaagsiekte) in young lambs. Brief report Arch Virol 1983 78 89 95 6197047 Maeda N Palmarini M Murgia C Fan H Direct transformation of rodent fibroblasts by jaagsiekte sheep retrovirus DNA Proc Natl Acad Sci U S A 2001 98 4449 4454 11296288 10.1073/pnas.071547598 Rai SK Duh F-M Vigdorovich V Danilkovitch-Miagkova A Lerman MI Miller AD Candidate tumor suppressor HYAL2 is a glycosylphosphatidylinositol (GPI)-anchored cell-surface receptor for jaagsiekte sheep retrovirus, the envelope protein of which mediates oncogenic transformation Proc Natl Acad Sci U S A 2001 98 4443 4448 11296287 10.1073/pnas.071572898 Allen TE Sherrill KJ Crispell SM Perrott MR Carlson JO DeMartini JC The jaagsiekte sheep retrovirus envelope gene induces transformation of the avian fibroblast cell line DF-1 but does not require a conserved SH2 binding domain J Gen Virol 2002 83 2733 2742 12388809 Danilkovitch-Miagkova A Duh F-M Kuzmin I Angeloni D Liu S-L Miller AD Lerman MI Hyaluronidase 2 negatively regulates RON receptor tyrosine kinase and mediates transformation of epithelial cells by jaagsiekte sheep retrovirus Proc Natl Acad Sci U S A 2003 100 4580 4585 12676986 10.1073/pnas.0837136100 Dirks C Duh F-M Rai SK Lerman MI Miller AD Mechanism of cell entry and transformation by enzootic nasal tumor virus J Virol 2002 76 2141 2149 11836391 10.1128/jvi.76.5.2141-2149.2002 Alberti A Murgia C Liu S-L Mura M Cousens C Sharp M Miller AD Palmarini M Envelope-induced cell transformation by ovine betaretroviruses J Virol 2002 76 5387 5394 11991967 10.1128/JVI.76.11.5387-5394.2002 Soffer D Resnick-Roguel N Eldor A Kotler M Multifocal vascular tumors in fowl induced by a newly isolated retrovirus Cancer Res 1990 50 4787 4793 2164448 Alian A Sela-Donenfeld D Panet A Eldor A Avian hemangioma retrovirus induces cell proliferation via the envelope (env) gene Virology 2000 276 161 168 11022004 10.1006/viro.2000.0550 Persons DA Paulson RF Loyd MR Herley MT Bodner SM Bernstein A Correll PH Ney PA Fv2 encodes a truncated form of the Stk receptor tyrosine kinase Nat Genet 1999 23 159 165 10508511 10.1038/13787 Nishigaki K Thompson D Hanson C Yugawa T Ruscetti S The envelope glycoprotein of friend spleen focus-forming virus covalently interacts with and constitutively activates a truncated form of the receptor tyrosine kinase Stk J Virol 2001 75 7893 7903 11483734 10.1128/JVI.75.17.7893-7903.2001 Palmarini M Maeda N Murgia C De-Fraja C Hofacre A Fan H A phosphatidylinositol 3-kinase docking site in the cytoplasmic tail of the Jaagsiekte sheep retrovirus transmembrane protein is essential for envelope-induced transformation of NIH 3T3 cells J Virol 2001 75 11002 11009 11602740 10.1128/JVI.75.22.11002-11009.2001 Liu S-L Lerman MI Miller AD Putative phosphatidylinositol 3-kinase (PI3K) binding motifs in ovine betaretrovirus Env proteins are not essential for rodent fibroblast transformation and PI3K/Akt activation J Virol 2003 77 7924 7935 12829832 10.1128/JVI.77.14.7924-7935.2003 Zavala G Pretto C Chow YH Jones L Alberti A Grego E De las Heras M Palmarini M Relevance of Akt phosphorylation in cell transformation induced by Jaagsiekte sheep retrovirus Virology 2003 312 95 105 12890624 10.1016/S0042-6822(03)00205-8 Liu S-L Duh F-M Lerman MI Miller AD Role of virus receptor Hyal2 in oncogenic transformation of rodent fibroblasts by sheep betaretrovirus env proteins J Virol 2003 77 2850 2858 12584308 10.1128/JVI.77.5.2850-2858.2003 Chow YH Alberti A Mura M Pretto C Murcia P Albritton LM Palmarini M Transformation of rodent fibroblasts by the jaagsiekte sheep retrovirus envelope is receptor independent and does not require the surface domain J Virol 2003 77 6341 6350 12743291 10.1128/JVI.77.11.6341-6350.2003 Maeda N Inoshima Y Fruman DA Brachmann SM Fan H Transformation of mouse fibroblasts by Jaagsiekte sheep retrovirus envelope does not require phosphatidylinositol 3-kinase J Virol 2003 77 9951 9959 12941905 10.1128/JVI.77.18.9951-9959.2003 Ronsin C Muscatelli F Mattei MG Breathnach R A novel putative receptor protein tyrosine kinase of the met family Oncogene 1993 8 1195 1202 8386824 Iwama A Okano K Sudo T Matsuda Y Suda T Molecular cloning of a novel receptor tyrosine kinase gene, STK, derived from enriched hematopoietic stem cells Blood 1994 83 3160 3169 8193352 Wang MH Wang D Chen YQ Oncogenic and invasive potentials of human macrophage-stimulating protein receptor, the RON receptor tyrosine kinase Carcinogenesis 2003 24 1291 1300 12807733 10.1093/carcin/bgg089 Wang MH Ronsin C Gesnel MC Coupey L Skeel A Leonard EJ Breathnach R Identification of the ron gene product as the receptor for the human macrophage stimulating protein Science 1994 266 117 119 7939629 Wang MH Iwama A Skeel A Suda T Leonard EJ The murine stk gene product, a transmembrane protein tyrosine kinase, is a receptor for macrophage-stimulating protein Proc Natl Acad Sci U S A 1995 92 3933 3937 7732008 Peace BE Hughes MJ Degen SJ Waltz SE Point mutations and overexpression of Ron induce transformation, tumor formation, and metastasis Oncogene 2001 20 6142 6151 11593422 10.1038/sj.onc.1204836 Peace BE Hill KJ Degen SJ Waltz SE Cross-talk between the receptor tyrosine kinases Ron and epidermal growth factor receptor Exp Cell Res 2003 289 317 325 14499632 10.1016/S0014-4827(03)00280-5 Quade K Transformation of mammalian cells by avian myelocytomatosis virus and avian erythroblastosis virus Virology 1979 98 461 465 228482 10.1016/0042-6822(79)90569-5 Rai SK DeMartini JC Miller AD Retrovirus vectors bearing jaagsiekte sheep retrovirus Env transduce human cells by using a new receptor localized to chromosome 3p21.3 J Virol 2000 74 4698 4704 10775607 10.1128/JVI.74.10.4698-4704.2000 Miller DG Edwards RH Miller AD Cloning of the cellular receptor for amphotropic murine retroviruses reveals homology to that for gibbon ape leukemia virus Proc Natl Acad Sci U S A 1994 91 78 82 8278411 Miller AD Chen F Retrovirus packaging cells based on 10A1 murine leukemia virus for production of vectors that use multiple receptors for cell entry J Virol 1996 70 5564 5571 8764070 Miller AD Rosman GJ Improved retroviral vectors for gene transfer and expression Biotechniques 1989 7 980 990 2631796 Santoro MM Penengo L Minetto M Orecchia S Cilli M Gaudino G Point mutations in the tyrosine kinase domain release the oncogenic and metastatic potential of the Ron receptor Oncogene 1998 17 741 749 9715276 10.1038/sj.onc.1201994 Williams TA Longati P Pugliese L Gual P Bardelli A Michieli P MET(PRC) mutations in the Ron receptor result in upregulation of tyrosine kinase activity and acquisition of oncogenic potential J Cell Physiol 1999 181 507 514 10528237 10.1002/(SICI)1097-4652(199912)181:3<507::AID-JCP15>3.0.CO;2-Q Zhou YQ He C Chen YQ Wang D Wang MH Altered expression of the RON receptor tyrosine kinase in primary human colorectal adenocarcinomas: generation of different splicing RON variants and their oncogenic potential Oncogene 2003 22 186 197 12527888 10.1038/sj.onc.1206075 Roussel MF Sherr CJ Mouse NIH 3T3 cells expressing human colony-stimulating factor 1 (CSF-1) receptors overgrow in serum-free medium containing human CSF-1 as their only growth factor Proc Natl Acad Sci U S A 1989 86 7924 7927 2554296 Kaleko M Rutter WJ Miller AD Overexpression of the human insulinlike growth factor I receptor promotes ligand-dependent neoplastic transformation Mol Cell Biol 1990 10 464 473 2153917 Areces LB Jucker M San Miguel JA Mui A Miyajima A Feldman RA Ligand-dependent transformation by the receptor for human granulocyte/macrophage colony-stimulating factor and tyrosine phosphorylation of the receptor beta subunit Proc Natl Acad Sci U S A 1993 90 3963 3967 7683416 Winkler C Wittbrodt J Lammers R Ullrich A Schartl M Ligand-dependent tumor induction in medakafish embryos by a Xmrk receptor tyrosine kinase transgene Oncogene 1994 9 1517 1525 8183545 Giordano S Bardelli A Zhen Z Menard S Ponzetto C Comoglio PM A point mutation in the MET oncogene abrogates metastasis without affecting transformation Proc Natl Acad Sci U S A 1997 94 13868 13872 9391119 10.1073/pnas.94.25.13868 Liu S-L Miller AD Transformation of Madin-Darby canine kidney (MDCK) epithelial cells by sheep retrovirus envelope proteins J Virol 2004

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==== Front BMC OphthalmolBMC Ophthalmology1471-2415BioMed Central London 1471-2415-4-131538788910.1186/1471-2415-4-13Research ArticleMethods of assessment of patients for Nd:YAG laser capsulotomy that correlate with final visual improvement Aslam Tariq M [email protected] Niall [email protected] Manchester Eye Hospital, Manchester, UK2 Edinburgh Eye Pavilion, Edinburgh, UK2004 23 9 2004 4 13 13 15 6 2004 23 9 2004 Copyright © 2004 Aslam and Patton; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background This paper attempts to clarify the usefulness of various simple pre-operative measures in estimating the potential for a visually successful capsulotomy. Methods 24 patients attending for capsulotomy had pre-operative measures of glare with BAT tester, visibility of posterior pole and grading of posterior capsular pearls and fibrosis seen at slit lamp. Visual function was measured before and after standardised capsulotomy. Correlations of the various preoperative measures with eventual visual function improvements were calculated. Results Pearls at slit lamp and poor posterior pole visualisation were all correlated with improvements in visual acuity and contrast sensitivity after capsulotomy. Amount of fibrosis visible at slit lamp and glare assessment were not correlated with vision improvements after laser. Conclusion Of the various measures that are taken prior to Nd : YAG capsulotomy, some correlate with eventual visual improvement but for others no clinical utility was found. Practitioners should note these findings as they are especially of use in more questionable or high-risk cases to help determine whether referral for PCO treatment by Nd: YAG capsulotomy is likely to benefit the patient. ==== Body Background Posterior capsular opacification (PCO) remains one of the most common post operative morbidities in modern day cataract surgery [1,2] and Nd:YAG posterior capsulotomy is one of the most commonly performed surgical procedures. However, the Nd: YAG capsulotomy procedure has been associated with complications such as damage to intraocular lenses [3], post operative intraocular pressure increases [4], cystoid macular oedema [4], disruption of the anterior vitreous face [5] and increased incidence of retinal detachment [6]. Until recently Nd:YAG laser treatments have cost the U.S healthcare system up to $250 million annually [7]. Apart from exposing a patient to unnecessary risk, unqualified capsulotomies worsen this burden to the developed and developing world [8]. PCO is an extremely common development in patients after cataract extraction and in many mild cases it may not be immediately obvious whether it is visually significant. Patients may have reduced vision from other undetermined causes or have some measures of visual function that are not reduced at all. Information is needed that will help the practitioners to decide on the visual significance of a patient's PCO using tools that are easily available. The specific aim of this study was to assess the correlation between preoperative measures that are easily performed (fundus visibility, capsule opacity grading and initial glare testing) with eventual visual function improvements after Nd: YAG capsulotomy. This information would be of use to practitioners when deciding whether a patient had PCO that was clinically significant. In other words, we aimed to test the clinical utility of these preoperative tests for Nd:YAG laser capsulotomy. Methods Local research ethical committee approval for the study was obtained and the study was performed in accordance with the treaty of Helsinki. 26 consecutive patients who had been referred for Nd: YAG capsulotomies for posterior capsular opacification were recruited and full informed consent was obtained. Patients excluded were those that had media opacities other than PCO or were not suitable for capsulotomy treatment. Patients in whom macular or disc pathologies were present were excluded from posterior pole assessment arm of the study as grading of visualisation would not be valid. Patients were roughly equally divided between silicone and acrylic IOLs. Prior to Nd:YAG capsulotomy, the patients visual function was assessed with their normal physiological pupil state in terms of best corrected distance and near visual acuity using Bailey Lovie logMAR charts and contrast sensitivity with and without glare, using a Pelli-Robson chart under standard illumination levels at 1 meter. Glare was tested using a Mentor brightness acuity tester (BAT) instrument (set at medium illumination levels) and recorded as the level of contrast sensitivity chart read when exposed to the BAT. Pupils were dilated with topical 1% tropicamide. After a minimum of 20 minutes, the size of the pupil was recorded using a millimetre ruler and the PCO was graded according to the slit-lamp appearance (grade 0 to 4). Use of such slit lamp grading has been established in scientific literature for PCO assessment [9-16] There are of course many other systems available for assessment of PCO [17] We chose a system based on slit lamp grading as it is the most commonly performed method of PCO assessment in practice. Many such slit lamp grading systems exist, but none have been proven to be gold standard in terms of clinical utility. After our own studies on the effect of PCO on visual function [18], we decided to use a protocol based on that described by Sellman and Lindstrom [19], recording pearls and fibrosis separately, which we feel is likely to be as clinically valid as any system that is commonly used in pre-assessment for Nd;YAG capsulotomy. All these tests were performed by one practitioner (T.A.), who was masked to fundus gradings, using the following scale, for pearls and fibrosis separately; 0 None visible at all 1 Visible but none reaching to IOL edge 2 At IOL edge 3 Well Inside IOL edge but visual axis clear 4 Across visual axis Visualisation of the posterior pole was then assessed by examining the optic disc and macula using a Volk 90D lens. Visualisation of the optic disc was subjectively graded according to the following scale (adapted from the Madurai Intraocular lens study IV [20]): 0 Clear view of optic disc margin, blood vessels at the optic disc and nerve fibre layer (NFL examined using the red-free filter) 1 Clear view of optic disc margin, but disc blood vessels and/or nerve fibre layer are not clearly seen 2 Optic disc margin, as well as disc blood vessels and nerve fibre layer are not clearly seen Visualisation of the macula was subjectively graded according to the following scale: 0 Clear view of foveal reflex, peri-foveal blood vessels and nerve fibre layer 1 Diminished foveal reflex, but clear view of peri-foveal blood vessels and nerve fibre layer 2 Blurred foveal reflex, peri-foveal blood vessels and/or nerve fibre layer The totals for the visualisations of the optic disc and the macula were combined to produce a total posterior pole visualisation score (PolVS), ranging from 0 to 4 in order of decreasing visualisation. All examinations of the posterior pole were carried out by the same examiner (NP). In order to be masked as to whether the patient was pre- or post- Nd:YAG capsulotomy, NP examined the fundus with the lenses already placed in front of the eye, thus obscuring any anterior segment view. The patients then had Nd:YAG capsulotomy by the same surgeon (TA). This involved initial setting of 1 mJ and subsequent rises of 0.5 mJ as necessary to pierce the posterior capsule. The laser treatment was initiated off axis in a horizontal line across centre, followed by a line in the vertical axis to form a cross. Any obvious lines of capsule tear were treated with laser if deemed beneficial and overall energy used was kept to a minimum. Treatments in this manner produced small capsulotomies of size 2–3 mm diameter. Size of capsulotomy was dictated by ease of making openings, and concerns over energy used. In general the aim was to create an opening using minimum energy, which might be small, but which could be enlarged at follow up visits if deemed necessary. Four weeks post Nd: YAG capsulotomy, the patients were reassessed again in terms of visual function as described earlier, by the same practitioner (TA). The pupils were dilated using 1% tropicamide and after a minimum 20-minute interval, the size of the pupil was recorded. The PolVS was again graded according to the same scales as before, by the same examiner (NP), again masked as to the state of the posterior capsule. Four weeks post-capsulotomy was chosen for re-examination, on the basis of evidence suggesting that capsulotomies enlarged progressively up to one month after Nd: YAG laser and then stabilised thereafter [21] On inspection, the data was found to be of skewed distribution. All correlation calculations were therefore performed using the Spearman's rank correlation coefficients. Means were compared using Wilcoxon signed ranks test for non-normally distributed data. Significance was at the p < 0.05 level. Because the visualisation scale was composed of a combination of sub-scales (2 items for PolVS), internal validity was determined by the Cronbach test of reliability19,20. This is a commonly used test statistic to determine the degree with which constituent items within a scale correlate with each other. An alpha coefficient of = 0.7 is considered necessary for a composite of measurements to be considered a scale. Statistical analysis was performed using SPSS for Windows (version 8.0) for all calculations. Results A total of 26 eyes of 26 patients were recruited into the study. Mean age was 75.2 years (range 52 to 90). There were 14 females and 12 males. 2 patients only had one examination pre-Nd:YAG capsulotomy, and declined any further examinations. Therefore, 24 patients were seen pre- and post- Nd:YAG capsulotomy. The PolVS scale was found to be internally reliable and consistent (α coefficient = 0.7824). Mean "pearl" grading score was 3.3 (± 1.3) and mean "fibrosis" grading score was 2.3 (± 1.4). 5 patients were omitted from PolVS score as macular or disc pathology prevented objective grading. Mean improvement of PolVS (n = 19) was 1.95 (S.D. ± 1.31) (95% C.I. 1.32 to 2.58) (p < 0.0001, Wilcoxon signed ranks test). Improvements in the PolVS score after Nd: YAG capsulotomies are shown in fig. 1. Mean improvement of distance logMAR visual acuity was 0.32 (± 0.29) (95% CI 0.20 to 0.44) and near logMAR visual acuity was 0.32 (± 0.29) (95% CI 0.20 to 0.44). Mean improvement of log contrast sensitivity was -0.41 (± 0.39) (95% CI 0.25 to 0.57) and glare testing vision was -0.35 (S.D. ± 0.41) (95% CI 0.18 to 0.52). (Improved contrast sensitivity and glare testing visual function are associated with lower grading scores.) For all the above analyses, p < 0.0001, except for mean improvement in glare testing (p = 0.001) (Wilcoxon signed ranks test). The main aim of the study was to determine which pre-operative assessments on patients attending for Nd: YAG capsulotomy would correlate with eventual visual function outcome of the patient, and thus be useful clinical measures. The results of the various correlation coefficient analyses are shown in table 1. The table demonstrates that a patient's improvement in visual function after laser capsulotomy is correlated to measurements made before treatment of pearls at slit lamp and posterior pole visibility score. Measurements of fibrosis at slit lamp and of glare do not correlate with eventual improvements in vision after capsulotomy. Discussion If it is decided to offer a patient Nd; YAG capsulotomy treatment they need to be told of the likely benefits of the treatment as well as risks. Information on potential visual function improvements for each specific patient would be especially welcome in decisions in uncertain cases and higher risk patients such as high myopes. Practitioners should have some evidence for the potential benefit to visual function before suggesting treatment. Retroilluminated photograph analysis by computer have been correlated with improvements in vision [22] but these tools are not available to most ophthalmologists in the examination room. Practitioners are able to subjectively estimate the amount of PCO on slit lamp examination of posterior capsule but often with more subtle PCO it is difficult to assess clinical relevance. It is thought by some [23] that contrast sensitivity with glare testing are likely to be particularly sensitive to visual loss from PCO, but of course these visual parameters are also open to influence from many other ocular states. Visibility of discs has been used as an assessment of the amount of PCO [20] but without convincing evidence of validity. Although all of the above tests have been used to try to assess how much benefit a particular patient may gain from capsulotomy, there has previously been little empirical evidence supporting their use. This study provides some evidence for the use and avoidance of various preoperative tests in assessing potential benefit from Nd; YAG laser capsulotomy. Preoperative glare measures There has been some controversy as to the relative usefulness of the different measures of visual function for assessing PCO severity [23-27], with some suggestion that glare assessment might be particularly useful for such anterior segment disorders [23,27] This study suggests that initial glare measures with the BAT did not correlate with eventual improvements in any visual function. Our results do, however, show a definite improvement in glare following Nd:YAG capsulotomy, all of whom underwent a small capsulotomy (2–3 mm). It appears that glare assessment with BAT is of no clinical utility when assessing the potential improvement a patient may gain from Nd:YAG capsulotomy. This may reflect the difficulties of BAT glare measurement which has been found to be inconsistent in some studies [28]. Indeed, glare assessment by any means is not simple to perform reliably and with clinical utility. Slit lamp assessment of PCO by practitioner There were two types of PCO assessed, pearls and fibrosis, both graded 0–4 with identical criteria for severity. The study shows very clearly that the fibrosis score was not significantly correlated to any improvements in visual function. The pearl score however was significant in correlating with the eventual improvement of all visual functions. This agrees with previous work using computerised analysis of retro-illumination photographs, which showed that only very central fibrosis affected vision whereas pearls could be detrimental even in para-axial locations [18] Other studies have also shown that pearls have a greater effect on vision than fibrosis [29]. The assessment of fibrosis at slit lamp is commonly performed before Nd:YAG capsulotomy, but according to this study does not correlate with eventual visual outcomes. This finding was perhaps the most poignant due to the common clinical use of amount of fibrosis at slit lamp when deciding whether a patient would benefit from capsulotomy. The finding deserves further investigation, but it may be that antero-posterior thickness of the fibrosis as well as density of opacification are important contributory variables to any visual loss. This thickness of PCO would not be easily assessed accurately and routinely at slit lamp, and was not incorporated into the grading scale used for this experiment. In contrast, clinical utility of pearl assessment is demonstrated, by its correlation with eventual improvement of visual function. Visualisation of posterior pole Visualisation of posterior pole according to the scale above correlated with improvements in visual acuity and contrast sensitivity. Using the specific criteria as described may have increased the objectivity of the test. This confirms expected agreement between the difficulty of visualization of the posterior pole and the patient's own vision of the outside world. An assessment of the posterior pole along the suggested guidelines is shown to have significant clinical utility. It may be that some of the correlations found were due to some criteria measured being closely related, for example, pearls at sit lamp and visualisation of fundus. The numbers in this analysis were unfortunately insufficient for stepwise multiple linear regression analysis, which could be used in future studies to determine the relative importance of the different measures. However the principle aims of the current study were realised with the presented experimental protocol and statistical analysis. Conclusions This study confirms the clinical value of pre-YAG measurements of pearls graded at slit lamp and posterior pole visualisation. However, other measures were not significantly correlated with improvements in visual function, namely pre-YAG testing of glare with BAT and pre-YAG grading of fibrosis seen at slit lamp. If a practitioner is uncertain as to the potential visual benefit of an Nd:YAG capsulotomy, an assessment of posterior pole visualisation or pearls at slit lamp should be useful. There is little clinical utility shown from this study in attempting glare assessment or assessment of fibrosis at slit lamp and these assessments may lead to erroneous expectations from treatment. This information is of practical value to practitioners faced with the common problem of assessing suitability of patients for Nd: YAG capsulotomy. It should especially be of use in more uncertain or high-risk cases to determine whether referral to PCO treatment by Nd: YAG capsulotomy is clinically appropriate. Competing interests The author(s) declare that they have no competing interests. Authors' contributions Both authors were involved in planning, design, execution and writing of this paper. Both read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: Figures and Tables Figure 1 Boxplots of PolVS pre- and post-Nd:YAG capsulotomy. Table 1 Correlation cross-tabulation table between improvements in visual function and pre-treatment measures of glare, slit-lamp pearls, slit-lamp fibrosis and posterior pole visibility score (Polvs). Improvement in Distance Visual Acuity Improvement in Contrast Sensitivity Pre-Nd:YAG R = -0.30 R = -0.35 Glare Testing p = 0.159* p = 0.093* Slit-lamp R = 0.67 R = 0.64 Pearls p < 0.0001 p = 0.001 Slit-lamp R = -0.01 R = 0.06 Fibrosis p = 0.950* p = 0.775* Pre-Nd:YAG R = 0.56 R = 0.50 PolVS p = 0.012 p = 0.029 R = Spearman's Rank Correlation Coefficient * = Not statistically significant ==== Refs Spalton DJ Posterior capsular opacification after cataract surgery Eye 1999 13 489 492 10627830 Bertelmann E Kojetinsky C Posterior capsule opacification and anterior capsule opacification Curr Opin Ophthalmol 2001 12 35 40 11150079 Newland TJ McDermott ML Eliott D Hazlett LD Apple DJ Lambert RJ Barrett RP Experimental neodymium: YAG laser damage to acrylic, poly(methyl methacrylate), and silicone intraocular lens materials Journal of Cataract & Refractive Surgery 1999 25 72 76 9888080 Bath PE Fankhauser F Long-term results of Nd:YAG laser posterior capsulotomy with the Swiss laser Journal of Cataract & Refractive Surgery 1986 12 150 153 3754577 Framme C Hoerauf H Roider J Laqua H Delayed intraocular lens dislocation after neodymium:YAG capsulotomy Journal of Cataract & Refractive Surgery 1998 24 1541 1543 9818350 Ranta P Tommila P Immonen I Summanen P Kivela T Retinal breaks before and after neodymium:YAG posterior capsulotomy Journal of Cataract & Refractive Surgery 2000 26 1190 1197 11008047 10.1016/S0886-3350(00)00404-1 Posterior capsule opacification ( secondary cataract) Survey of Ophthalmology 2000 45 Gillies M Brian G La Nauze J Le Mesurier R Moran D Taylor H Ruit S Modern surgery for global cataract blindness: preliminary considerations Arch Ophthalmol 1998 116 90 92 9445213 Kucuksumer Y Bayraktar S Sahin S Yilmaz OF Posterior capsule opacification 3 years after implantation of an AcrySof and a MemoryLens in fellow eyes J Cataract Refract Surg 2000 26 1176 1182 11008045 10.1016/S0886-3350(00)00583-6 Ober MD Lemon LC Shin DH Nootheti P Cha SC Kim PH Posterior capsular opacification in phacotrabeculectomy : a long-term comparative study of silicone versus acrylic intraocular lens Ophthalmology 2000 107 1868 73; discussion 1874. 11013189 10.1016/S0161-6420(00)00270-0 Westling AK Calissendorff BM Factors influencing the formation of posterior capsular opacities after extracapsular cataract extraction with posterior chamber lens implant Acta Ophthalmologica 1991 69 315 320 1927314 Legler UF Apple DJ Assia EI Bluestein EC Castaneda VE Mowbray SL Inhibition of posterior capsule opacification: the effect of colchicine in a sustained drug delivery system J Cataract Refract Surg 1993 19 462 470 8355152 Malecaze F Couderc B de Neuville S Serres B Mallet J Douin-Echinard V Manenti S Revah F Darbon JM Adenovirus-mediated suicide gene transduction: feasibility in lens epithelium and in prevention of posterior capsule opacification in rabbits Hum Gene Ther 1999 10 2365 2372 10515456 10.1089/10430349950017013 Hepsen IF Bayramlar H Gultek A Ozen S Tilgen F Evereklioglu C Caffeic acid phenethyl ester to inhibit posterior capsule opacification in rabbits J Cataract Refract Surg 1997 23 1572 1576 9456418 Odrich MG Hall SJ Worgul BV Trokel SL Rini FJ Posterior capsule opacification: experimental analyses Ophthalmic Res 1985 17 75 84 3885127 Kruger AJ Schauersberger J Abela C Schild G Amon M Two year results: sharp versus rounded optic edges on silicone lenses J Cataract Refract Surg 2000 26 566 570 10771231 10.1016/S0886-3350(00)00323-0 Aslam TM Dhillon B Werghi N Taguri A Wadood A Systems of analysis of posterior capsule opacification British Journal of Ophthalmology 2002 86 1181 1186 12234903 10.1136/bjo.86.10.1181 Aslam T. M., Aspinall P., Dhillon B, Posterior capsular morphology determinants of visual funtion Graefes Arch Clin Exp Ophthalmol 2003 208 212 12644945 Lindstrom RL Harris WS Management of the posterior capsule following posterior chamber lens implantation Journal - American Intra-Ocular Implant Society 1980 6 255 258 7410176 Prajna NV Ellwein LB Selvaraj S Manjula K Kupfer C The madurai intraocular lens study IV: posterior capsule opacification American Journal of Ophthalmology 2000 130 304 309 11020409 10.1016/S0002-9394(00)00481-5 Hu CY Woung LC Wang MC Change in the area of laser posterior capsulotomy: 3 month follow-up Journal of Cataract & Refractive Surgery 2001 27 537 542 11311620 10.1016/S0886-3350(00)00645-3 Aslam T. M., Dhillon B Neodymium:YAG laser capsulotomy: a clinical morphological analysis. Graefes Arch Clin Exp Ophthalmol 2002 240 972 976 12483318 Wilkins M McPherson R Fergusson V Visual recovery under glare conditions following laser capsulotomy Eye 1996 10 117 120 8763316 Sunderraj P Villada JR Joyce PW Watson A Glare testing in pseudophakes with posterior capsule opacification Eye 1992 6 411 413 1478316 Magno BV Datiles MB Lasa MS Fajardo MR Caruso RC Kaiser-Kupfer MI Evaluation of visual function following neodymium:YAG laser posterior capsulotomy Ophthalmology 1997 104 1287 1293 9261315 santiago MD Personal communication / Presentations ASCRS 2001 Aslam T. M., Dhillon B Use of visual function tests in posterior capsular opacification CME journal Ophthalmology 2002 6 44 50 Tan JC Spalton DJ Arden GB Comparison of methods to assess visual impairment from glare and light scattering with posterior capsule opacification J Cataract Refract Surg 1998 24 1626 1631 9850902 Cheng CY Yen MY Chen SJ Kao SC Hsu WM Liu JH Visual acuity and contrast sensitivity in different types of posterior capsule opacification J Cataract Refract Surg 2001 27 1055 1060 11489575 10.1016/S0886-3350(00)00867-1

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BMC Ophthalmol. 2004 Sep 23; 4:13

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==== Front BMC Med EducBMC Medical Education1472-6920BioMed Central London 1472-6920-4-171545391110.1186/1472-6920-4-17Research ArticleUsing email reminders to engage physicians in an Internet-based CME intervention Abdolrasulnia Maziar [email protected] Blanche C [email protected] Linda [email protected] Terry [email protected] Claire [email protected] Midge N [email protected] Norman W [email protected] Jeroan J [email protected] Division of Continuing Medical Education, University of Alabama at Birmingham, JNWB 406, 1530 3rd Avenue South, Birmingham, AL 35294-0500, USA2 Center for Outcomes and Effectiveness Research Education, University of Alabama at Birmingham, MT 401, 1530 3rd Avenue South, Birmingham, AL 35294-1170, USA3 Department of Pediatrics, University of Alabama at Birmingham, MTC 201 1600 7th Avenue South 35294-0011, USA4 Aetna Integrated Informatics, Inc. 151 Farmington Avenue Hartford, CT 06156, USA5 School of Health Related Professions, University of Alabama at Birmingham, Webb Building 564, 1530 3rd Avenue South, Birmingham, AL 35294-3361, USA6 Division of General Medicine, University of Alabama at Birmingham, FOT 720D 20th Street South, Birmingham, AL 35294-3407, USA2004 29 9 2004 4 17 17 2 6 2004 29 9 2004 Copyright © 2004 Abdolrasulnia et al; licensee BioMed Central Ltd.2004Abdolrasulnia et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Engaging practicing physicians in educational strategies that reinforce guideline adoption and improve the quality of healthcare may be difficult. Push technologies such as email offer new opportunities to engage physicians in online educational reinforcing strategies. The objectives are to investigate 1) the effectiveness of email announcements in engaging recruited community-based primary care physicians in an online guideline reinforcement strategy designed to promote Chlamydia screening, 2) the characteristics of physicians who respond to email announcements, as well as 3) how quickly and when they respond to email announcements. Methods Over a 45-week period, 445 recruited physicians received up to 33 email contacts announcing and reminding them of an online women's health guideline reinforcing CME activity. Participation was defined as physician log-on at least once to the website. Data were analyzed to determine participation, to compare characteristics of participants with recruited physicians who did not participate, and to determine at what point and when participants logged on. Results Of 445 recruited physicians with accurate email addresses, 47.2% logged on and completed at least one module. There were no significant differences by age, race, or specialty between participants and non-participants. Female physicians, US medical graduates and MDs had higher participation rates than male physicians, international medical graduates and DOs. Physicians with higher baseline screening rates were significantly more likely to log on to the course. The first 10 emails were the most effective in engaging community-based physicians to complete the intervention. Physicians were more likely to log on in the afternoon and evening and on Monday or Thursday. Conclusions Email course reminders may enhance recruitment of physicians to interventions designed to reinforce guideline adoption; physicians' response to email reminders may vary by gender, degree, and country of medical training. Repetition of email communications contributes to physician online participation. ==== Body Background Many clinical practice guidelines have been disseminated through print publications and the Internet to improve the quality of healthcare. Complex physician behavior-change interventions have been created and applied to clinical practice guidelines to determine if the guidelines are being practiced. Research indicates that the adoption of clinical practice guidelines continues to lag behind their production and dissemination [1]. Adoption of such guidelines has been improved by the use of secondary strategies, which involve education, audit and feedback, benchmarking, office system interventions, and multifaceted interventions [1]. Continuing medical education (CME) providers and others interested in improving the quality of health care have had difficulty actively engaging practicing physicians in secondary strategies that may lead to guideline adoption [2], since CME courses and other educational interventions must compete with the physician learner's multiple priorities [3]. Over the past ten years, accessibility to educational opportunities has increased with advancements in technology, particularly the Internet. The Internet offers a delivery system for educational interventions and research activities that are more convenient to the physician learner than traditional live large group lectures and seminars [4]. The Internet generally functions in a passive manner, or as a "pull" technology, allowing the user to determine when, where and how to seek information. Engaging physicians in online educational activities may require the use of "push" technologies rather than pull technologies. "Push" technologies allow information to be delivered to the user rather than requiring the user to actively search for the desired information; they require minimal effort on the part of the recipient, which greatly supports their adoption [5]. Email was the first type of online push technology [5]. Other forms of push technologies are actively used such as pop-ups, list-serves, and screen savers. Based on preliminary research presented at the Proceedings of the American Medical Informatics Association's 2002 Annual Symposium, screen savers have been shown to be effective as a reminder system in engaging physicians in bioterrorism CME activity, although the use of email has exceeded all other Internet applications [5]. Flanagan and colleagues demonstrated promise for email as a means of engaging physicians in online activities designed to reinforce guideline use. They found physician response rate to email solicitation to be 50% over a 14-month study period [6]. Beyond this, little is known about the effectiveness of email in engaging physicians in online educational interventions designed to improve the quality of healthcare [7]. The purposes of this study were to investigate 1) the effectiveness of email reminders in engaging recruited community-based primary care physicians in an online guideline reinforcement strategy that was designed to promote women's health, 2) the characteristics of physicians who respond to email announcements, as well as 3) how quickly and when they respond to email announcements. Methods This study is a subanalysis of data from a randomized controlled trial (RCT), described in detail elsewhere [8]. The goal of the RCT was to improve Chlamydia screening rates of at-risk young women by community-based primary care physicians by using a multi-faceted online guideline reinforcement intervention. Primary care physicians were recruited from a national sample of 923 eligible offices identified by a large managed care organization's administrative data. Eligible offices had at least 20 young women (ages 16–26) at risk for chlamydial infection and at least one primary care physician (Internal Medicine, Family Medicine/General Practice, Pediatrics) with Internet access. In Phase I of recruitment occurred at the office level and Phase II at the physician level. In Phase I, all eligible offices were invited to participate via facsimile; an office was designated as "recruited" when one of its physicians declared intent to participate. In Phase II, an active link to the Intervention module was e-mailed to physicians recruited in Phase I. Recruited physicians were assigned at the time of logon to either a control or Internet CME intervention arm designed to improve Chlamydia screening rates; physicians within the same office were assigned to the same arm of the study. We designated physicians as "participating" when they first engaged the Internet intervention. The study was conducted between February 1, 2002 and December 31, 2002. The intervention included a series of four modules, feedback of performance data and a quality improvement toolbox. The education modules were based upon adult learning and behavior change theory [8]. Our driving principles included case-based learning [9,10] making programs interactive by adapting to the program learners' readiness-to-change stage [11] and performance feedback for behavioral motivation and reinforcement [12] The control condition was a series of four text-based modules on topics unrelated to Chlamydia. Physicians receive 1 category 1 CME credit per module for their participation. The main outcome measure was Chlamydia screening rates. The subanalysis of this trial focuses on the use of email reminders to engage physicians in the online intervention. Physician recruitment occurred from November of 2001 to January of 2002. Following recruitment, the intervention was initiated in February of 2002 via email broadcast to recruited physicians. During 2002, four separate educational modules were offered. Each module was introduced with a series of email announcements followed by email reminders that contained tailored subject line (i.e. Dear Dr. John Smith) and text message containing the URL that would connect the physician directly to the educational module. The first three modules, emphasized: (1) young sexually active women are at high risk for asymptomatic infection that may lead to future serious health consequences; (2) newer urine-based screening allows diagnosis without a pelvic examination; and (3) infection may be effectively treated with a one-dose antibiotic. The fourth module reviewed previously introduced concepts. A total of 33 announcements and reminders were sent to recruited physicians between February 1, 2002 and December 31, 2002, which represents one reminder overall for each 1.5 weeks of the study duration. Initially, we were concerned that frequent email reminders would be considered intrusive. After a trial period, we found that we could increase the frequency of reminders without difficulty, thus fewer email reminders were sent during the initial phases of the study. For the subanalysis of this trial, the principal outcome measure is participation, defined as physician log-on at least once to the website. Data was collected electronically when physicians logged on to the intervention. In addition, several evaluation questions were used at the conclusion of the online educational activities to explore CME preferences. Descriptive analyses of patient and physician demographics are used to compare baseline characteristics of patients and physicians in the email participants versus the recruited non-participants. Statistical significance is determined with tests appropriate to the distribution of the data (chi-square for categorical variables and student-t test for continuous variable). Two-tailed tests are used for all analyses. Results and discussion Four hundred eighty physicians were recruited to participate, representing 380 physician offices. Of the 463 recruited physicians, 445 were successfully contacted by email using the addresses they had furnished at the time of recruitment. Of these 445 recruited physicians, 210 (47.2%) physicians from 190 offices logged on to at least one of the educational modules. Of the 210 physicians who logged on at least once during the 45-week study period, one hundred twenty-four (59%) returned again to log-on for Module 2, eighty-seven (41%) logged on for Module 3, and forty-four (21%) logged on to Module 4. Two hundred and ten physicians of 445 logged on at least once to the website, leaving 235 physicians as non-participants. Figure 1 represents total physician log-on by week. Analysis of log-on days indicated that participants were most likely to log-on on Monday or Thursday (see Figure 2). Log-on times were also examined and findings indicate that physicians were most likely to log-on to a module between the hours of 3 P.M. and 7 P.M (15:00–19:00). Other common times for participants to log-on included times earlier in the day, or between the hours of 8 P.M. and midnight (20:00–24:00) (see Figure 3). Figure 1 Cumulative internet engagement by physician over time Figure 2 Number of logins by day of the week. Figure 3 Number of logins by time of day. The participant characteristics (n = 210) were compared with those of recruited but non-participating physicians (n = 235) (Table 1). Age was not significant, however there were significant differences by gender, degree, and country of medical training. By race, 81.5% of the participants were Caucasian, 8.8% were Asian, 3.0% were African American, 3.5% were Hispanic and 3.0% were listed as other with no significant differences. The largest percentage of recruited physicians was family practitioners (41.3%), followed by general internists (29.4%), pediatricians (9.6%), and general practitioners (1.5%), with no significant differences. Female physicians were significantly more likely to participate than males (p = . 0001), Medical Doctors (MDs) were significantly more likely than Doctors of Osteopathy (DOs) to participate (p = . 01), and graduates of U.S. medical schools significantly more likely than graduates of international medical schools to participate (p = . 01). In addition to demographic characteristics, chlamydia screening rates of participant and non-participant physicians were compared. Baseline screening rates of nonparticipants were significantly lower than those of participants. Non-participant chlamydia screening rates were 14.6% at baseline, compared to 17.4% for participants (p < .006). Table 1 Participant* versus recruited** but non-participant characteristics Recruited Non-participants Participants p-value N 235 210 Mean Age 44.3 45.2 .143 Gender Male 60.0% 40.0% .0001 Female 40.0% 60.0% Degree DO 65.3% 34.7% .01 MD 51.9% 48.0% Medical Training International 67.5% 32.5% .01 USA 52.7% 47.3% *Participated physician engaged in at least one module **Eligible offices had at least 1 eligible physicians with at least 20 female patients who were candidates for Chlamydia screening according to the Health Employers Data and Information Set Technical Specifications, 2000. From follow-up evaluation question data, 100% of responding DOs felt that the course email reminders were effective in reminding them about educational modules compared to 92.6% of MDs; 95.9% of US medical graduates rated the reminders as useful, while only 77.8% of graduates from non-US medical schools found them useful. Concerning preferences for delivery of CME, no DOs reported web-based activities as their preferred method for lifelong learning; they preferred local (50.0%) and national meetings (50.0%). MDs were more likely to prefer web-based CME (37.0%) to local (35.1%) or national (16.6%) meetings. Female physicians reported a preference for web-based CME (50.0%), whereas male physicians preferred local (35.0%) and national (27.5%) meetings over web-based CME (25.0%). The results of this study indicate that email reminders may be useful in engaging nearly half of a group of practicing primary care physicians recruited to participate in an online women's health educational series. This data is consistent with McMahon et al.'s findings in comparing the use of email, fax and mail, finding that email reminders were more useful to increase response rates [12]. The study participation rate, (47.2%) is also consistent with the work of Flanagan et. al.'s study of participation in web decision support tools for the management of pneumonia [6]. However, the gap of up to 3 months between recruitment and the initiation of the online educational activities may have contributed to a lower participation rate. It is possible that by decreasing the gap in time between recruitment and announcement of the availability of the online educational activity, participation rates could be increased. Future study designs using email reminders should consider beginning the intervention immediately following agreement to participate or shortly thereafter. Two current studies using email reminders to promote educational courses in the prevention of glucocorticoid-induced osteoporosis and in the secondary prevention of cardiovascular disease in patients following a myocardial infarction have been designed to deliver the intervention immediately following agreement to participate [13]. Findings from follow-up evaluation question data indicate that CME providers interested in targeting specific groups of physicians may benefit from using alternative methods of CME recruitment and delivery. CME providers targeting DOs may want to explore ways to engage DOs in web-based learning activities or consider focusing activities that target DOs to local or national meetings. Providers of CME who are interested in engaging male physicians and graduates of U.S. medical schools may find email reminders useful, but they may also want to explore additional methods of recruitment. Persistent email reminders did increase physicians' response rates to online education, but response rate decreased with the number of reminders. The first three reminders produced the largest responses, with decline after the 10th reminder. Based on our experience, it would seem reasonable that providers and researchers with limited resources consider focusing their announcements/reminders on the first 3–10 encounters. Data from time of log-on underscores the advantages of asynchronous online interventions for busy clinicians. Traditional "live" online symposia scheduled for the middle of the day might appeal to the physicians who logged on between 10 AM and 2 PM (10:00–14:00), but data from this intervention suggest that many physicians have more available time later in the day for online educational activity. The investigators findings of Monday being a frequent day for log-on was unexpected, but may offer an opportunity for future study designs to include weekend email broadcasting rather than a Thursday broadcast. The topic of the online educational activity may influence response to email announcements and reminders. While baseline Chlamydia screening rates were relatively low in both groups, the significant difference between the groups may indicate that those who are likely to perform better according to clinical practice guidelines are more likely to participate in online educational strategies that reinforce their use. Or the higher screening rates may be associated with a higher degree of interest in the overall topic area of women's health. Also related to the topic addressed in this online study, previous studies of preventive practices of female physicians have indicated they are more likely than males to promote preventive practices [14] and screening [15] including Chlamydia screening among their female patients than are male physicians. The advertised educational topic for this study was an online women's health course. More female physicians than male physicians responded to the email course reminders, but the topic may have had more appeal to female physicians than to male physicians, leaving the issue of whether there are gender differences among physicians in response to email reminders unresolved. Comparisons of characteristics of participant physicians and non-participant recruited physicians may be useful to those designing online recruitment and engagement strategies for future studies. Those using email reminders to communicate with physician populations including large numbers of DOs, however, may benefit from considering blended methods of CME recruitment and delivery. Using various methods of reaching providers, may also enhance DO participation. CME providers targeting DOs may want to explore additional ways to engage DOs in web-based learning activities or consider focusing activities that target DOs at local or national meetings. Conclusions Physicians' online clinical information seeking and engagement in online education continues to grow [12]. Researchers of online interventions who are attempting to improve the quality of healthcare and physician performance should continue to study and evaluate physician online behavior. Knowing when, where, and how physicians seek information on the Internet, and how they respond to receiving specific information pushed toward them, will prove to be very useful for targeting future quality improvement interventions. Reminding physicians often via email about online educational opportunities appears to increase engagement in a community-based primary care physician audience. The early and consistent implementation of this push technology may increase physicians' utilization of interventions designed to improve practice. Authors' contributions MA participated in the statistical analysis and drafted the manuscript. BCC participated in the statistical analysis and drafting of the manuscript. LC participated in all phases of the project. TW participated in the draft of the manuscript. CS participated in the design and coordination of the study and the drafting of the manuscript. MNR participated in the coordination of the study. NWW participated in the design and implementation of the study. JJA conceived of the study and participated in all phases of the study. All authors read and approved the final manuscript. Competing interests The authors declare that they have no competing interests. Pre-publication history The pre-publication history for this paper can be accessed here: Acknowledgement This project was supported by HS11124 from the Agency for Healthcare Research and Quality. We gratefully acknowledge the biostatistical analysis and assistance of Tony Horn, Department of Preventive Medicine, University of Alabama School of Medicine, Birmingham, Alabama. ==== Refs Davis DA Taylor-Vaisey A Translating guidelines into practice. A systematic review of theoretic concepts, practical experience and research evidence in the adoption of clinical practice guidelines CMAJ 1997 157 408 16 9275952 Davis D Fox R Barnes BE Davis D, Barnes BE, Fox R The horizon of continuing professional development: five questions in knowledge translation In The Professional Development of Physicians: From Research to Practice 2003 Chicago: American Medical Association 9 24 Bennett NL Casebeer LL Kristofco RE Strasser SM Physicians' information seeking behavior in Internet use J Contin Educ Health Prof Casebeer LL Anderson JG Kristofco RE Carillo A Facilitating adoption of clinical practice guidelines at the point of use: Bridging knowledge to the point of use In Proceedings of the American Medical Informatics Association's 1998 Spring Congress: 27–30 May 1998; Philadelphia 1998 56 Key email features – email is a push technology Flanagan JR Peterson M Dayton C Strommer Pace L Plank A Walker K Carlson W Email recruitment to use web decision support tools for pneumonia In Proceedings of the American Medical Informatics Association's 2002 Annual Symposium: 9–13 November; San Antonio 2002 255 9 Casebeer LL Spettell C Allison J Designing tailored web-based instruction to improve practicing physicians' chlamydial screening rates Acad Med 2002 77 929 12228099 10.1097/00001888-200209000-00032 Casebeer LL Bennett N Kristofco R Carillo A Centor R Physician Internet medical information seeking and on-line continuing education use patterns J Contin Educ Health Prof 2002 22 33 42 12004639 Norman GR Schmidt HG Effectiveness of problem-based learning curricula: theory, practice and paper darts Medical Education 2000 34 721 8 10972750 10.1046/j.1365-2923.2000.00749.x Knowles MS Self-directed learning: a guide for learners and teachers 1988 Cambridge, UK: Cambridge Book Company Parker K Parikh SV Applying Prochaska's model of change to needs assessment, programme planning and outcome measurement Journal of Evaluation in Clinical Practice 2001 7 365 71 11737528 10.1046/j.1365-2753.2001.00299.x McMahon RS Iwamoto M Massoudi SM Yusuf HR Stevenson JM David F Chu SY Pickering LK Comparison of e-mail, fax, and postal surveys of pediatricians Pediatrics 2003 111 e299 303 12671142 10.1542/peds.111.4.e299 Casebeer LL Strasser SM Spettel CM Wall TC Weissman N Ray MN Allison JJ Designing tailored Web-based instruction to improve practicing physicians' preventive practices J Med Internet Res 2003 5 e20 14517111 Halpern-Felsher BL Ozer EM Millstein SG Wibbelsman CJ Fuster CD Elster AB Irwin CE Jr Preventive services in a health maintenance organization: How well do pediatricians screen and educate adolescent patients? Arch Pediatr Adolesc Med 2000 154 173 9 10665605 Franks P Bertakis KD Physician gender, patient gender, and primary care J Womens Health (Larchmt) 2003 12 73 80 12639371

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BMC Med Educ. 2004 Sep 29; 4:17

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==== Front BMC Med Inform Decis MakBMC Medical Informatics and Decision Making1472-6947BioMed Central London 1472-6947-4-151536733210.1186/1472-6947-4-15Technical AdvanceSeal of transparency heritage in the CISMeF quality-controlled health gateway Darmoni SJ [email protected] B [email protected] Thomas R [email protected] CISMeF, Rouen University Hospital, France & L@STICS, PSI Lab FRE CNRS 2645, France CISMeF & L@STICS, 1 rue de Germont 76031 Rouen Cedex, France2 University of Kaiserslautern Knowledge-Based Systems Group, Postfach 3049, 67653 Kaiserslautern, Germany2004 14 9 2004 4 15 15 26 4 2004 14 9 2004 Copyright © 2004 Darmoni et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background It is an absolute necessity to continually assess the quality of health information on the Internet. Quality-controlled subject gateways are Internet services which apply a selected set of targeted measures to support systematic resource discovery. Methods The CISMeF health gateway became a contributor to the MedCIRCLE project to evaluate 270 health information providers. The transparency heritage consists of using the evaluation performed on providers that are referenced in the CISMeF catalogue for evaluating the documents they publish, thus passing on the transparency label from the publishers to their documents. Results Each site rated in CISMeF has a record in the CISMeF database that generates an RDF into HTML file. The search tool Doc'CISMeF displays information originating from every publisher evaluated with a specific MedCIRCLE button, which is linked to the MedCIRCLE central repository. Starting with 270 websites, this trust heritage has led to 6,480 evaluated resources in CISMeF (49.8% of the 13,012 resources included in CISMeF). Conclusion With the MedCIRCLE project and transparency heritage, CISMeF became an explicit third party. ==== Body Background The availability of Internet health tools and services has been increasing at a phenomenal rate in recent years making the Internet a major source of knowledge for healthcare professionals, medical students and also patients and the general public. This increase has made it an absolute necessity to continually assess the quality of health information on the Internet. Indeed, creating a Web site is relatively easy, therefore uncontrolled health information can be launched by virtually anyone with access to the Internet. Peer review is often absent throughout this media as opposed to scientific journals. There have been numerous debates about the variable quality of health information on the Internet and its impact on public health [1]. There is no other field in which inaccurate, incomplete, or biased information can be potentially more damaging [2]. In the past five years, a lot of authors have scrutinized the quality of the health content available on the Internet. These studies, assessing the quality of health information, have been extensively reviewed by Eysenbach et al. [1]. In the meantime, several worldwide initiatives have been undertaken to define criteria for assessing the quality of health information on the Internet. These initiatives have been reviewed by Risk and Dzenowagis [3]. However, no consensus has been reached by healthcare professionals or consumers on how to assess the quality of health information on the Internet. As of today, access to accurate and trustworthy health information on the Internet is not an easy task; there are a great number of directories and search engines available in this new media [4]. But, there is also a need to develop reliable and quality-controlled health subject gateways to disseminate relevant trustworthy health information. Koch [5] defined quality-controlled subject gateways as Internet services which apply a rich set of quality measures to support systematic resource discovery. Considerable manual effort is used to process a selection of resources, which meet quality criteria, and to display a rich description and indexing of these resources with standards-based metadata. Regular checking and updating ensure optimal collection management. The main goal is to provide a high-quality of subject access through resource indexing using controlled vocabularies and via a practical classification structure for advanced searching and browsing. The objective of CISMeF (French acronym for Catalogue and Index of health resources in French) [6,7] is to describe and index the main health resources in French in order to assist health professionals, students and consumers in their search for electronic information available on the Internet. CISMeF is a quality-controlled subject gateway initiated by the Rouen University Hospital (RUH). Each of the following phases proposed by Koch [5], which characterise a typical quality-controlled subject gateway, are implemented in CISMeF: (a) selection and collection development, based on the Net Scoring, a list of 49 criteria to assess quality of health information (URL: ) [4], (b) collection management, (c) creation of metadata (performed by experts), (d) resource description (an extensive and documented metadata set), and (e) resource indexing (using a controlled vocabulary system). CISMeF is manually maintained. In CISMeF, a resource is defined as 1) a Web site or 2) high-quality documents from this Web site. CISMeF describes and indexes the most important sources of institutional health information in French, in order to allow them to be searched quickly and precisely. A great variety of resources are indexed, in terms of resource type (clinical guidelines, teaching material, patients information, etc.), and resource format (html, pdf, etc.). Its Universal Resource Locator (URL) is The CISMeF Web site opened in February 1995. In December 2003, 13,012 resources had been indexed, with an average of 55 new resources indexed each week. CISMeF is considered by most professionals as the reference health institutional Web site in France with as many as 25,000 unique machines visiting this Web site by working day. Doc'CISMeF is the search tool of the CISMeF gateway [7]. In 1997, because the quality of health information became an important issue for the building and maintenance of a trustworthy health gateway, the CISMeF team participated in the development of a user guide named Net Scoring [8]. The goal of the Net Scoring project was to provide a set of criteria that can be consistently used to assess the quality of health information on the Internet. Between 1997 – 2002 the CISMeF gateway selected health resources using the main criteria established by the Net Scoring initiative (source of information, disclosure, editorial review process, date of last update, and feedback mechanism) in view of the fact that the selection process is mandatory to create a trustworthy health Internet directory. Resources that are not compliant with basic ethical criteria are not included in the CISMeF database. In 2002, CISMeF became a contributor to the MedCIRCLE project (URL: ) [9]. The aim of this project is to establish a global Web of transparency for networked health information and to increase the accessibility and findability of trustworthy health websites using "Semantic Web" approaches, which essentially means to make "narrative" information on the Web accessible in a machine processable format by using RDF (Resource Description Framework) expressed in XML (eXtended Markup Language) [10]. MedCIRCLE is a collaboration of trustworthy European health subject gateways, medical associations, accreditation, certification, or rating services, which share the common goal of evaluating, describing, or indexing health information. MedCIRCLE began in March 2002 and lasted till December 2003. Whereas CISMeF initially addressed quality on a more finely grained level, i.e., the quality of documents or single web pages, MedCIRCLE focused on whole websites including information about the respective publisher. The main deliverable of this project for the CISMeF team was the evaluation and rating of 270 health information providers (or publishers) which who release health resources in French on a regular basis. Because CISMeF catalogues and indexes not only Web sites but also and mainly quality-controlled documents from those health publishers, we introduced the concept of "transparency transitivity" or " transparency heritage". It consists in applying to these documents the evaluation performed for their publishers, thus passing on the transparency label from the publishers to their documents. Methods Providing transparency related metadata Health professionals have begun to realize that it is their responsibility to guide consumers and patients to the best available medical information on the web. Many national governments and medical societies have acknowledged that it is their responsibility to help users to identify "good quality" information sources and have begun to develop national health gateways (such as HealthinSite in Australia, NHS Direct in the UK, or Healthfinder in the USA), portal sites and other forms of "infomediaries" such as seals of approval [2] or certification mechanisms in an effort to help consumers to locate trustworthy information resources. However, current approaches do not harness any of the advantages of the Web as a decentralized, distributed information system. There is a need for "next generation" tools, including intelligent knowledge-based tools, allowing consumers to positively and actively identify reliable health information that suits their needs. The three application partners of MedCIRCLE, besides CISMeF in France, were the Agency for Quality in Medicine (AQuMed) in Germany and the Official Medical College of Barcelona (COMB). AQuMed was founded in March 1995 as a joint institution of the German Medical Association and the National Association of Statutory Health Insurance Physicians. AQuMed established a health gateway (URL: ) for laypersons, listing consumer health information sites. Before MedCIRCLE, documents had been evaluated using the DISCERN instrument [11]. COMB (URL: ) represents the medical profession of Barcelona. To this date, in the project "Web Medica Acreditada", COMB has accredited more than 300 Spanish health websites from Spain and Latin America [12]. The Knowledge Management Department of the German Research Center for Artificial Intelligence DFKI GmbH provided consultancy services especially in the area of ontology modeling. DFKI also provided the technical infrastructure and development resources for the project. CISMeF terminology The CISMeF team is composed of five medical librarians, two medical informaticians, one engineer, three Ph.D. and two Master students in Computer Science. CISMeF uses two standard tools for organizing information: the MeSH (Medical Subject Headings) thesaurus from the US National Library of Medicine, and several metadata element sets [13]: (a) 11 of 15 items of the Dublin Core metadata format to describe and index all the health resources included in CISMeF (author or creator, date, description, format, identifier, language, publisher, resource type, rights, subject and keywords, and title), (b) the 11 elements of the Educational category from Learning Object Metadata (LOM) for teaching resources, (c) specific metadata for evidence-based medicine resources (indication of the level of evidence and the method to calculate it) which also describe the health content [14], and (d) the HIDDEL metadata set (Health Information Disclosure, Description and Evaluation Language) [15]. Description of the HIDDEL language HIDDEL is a metadata language and an ontology, which enables the expression of descriptive and evaluative annotations in XML/RDF. The first version of HIDDEL was initially developed during the MedCERTAIN project (MedPICS Certification and Rating of Trustworthy Health Information on the Net, ) [16]. HIDDEL evolved from MedPICS [17], a basic rating vocabulary for medical information conforming to the Platform for Internet Content Selection (PICS) [18]. HIDDEL is used to enhance transparency of health information on the Internet. HIDDEL is based on existing quality criteria such as the Health On the Net (HON) Code of Conduct [2]. It was developed together with a quality management process model. HIDDEL can be used by information providers for self-disclosure, but also by third parties such as quality-controlled health gateways, to evaluate health information providers. It presents three levels of evaluation: (a) self-disclosure (b) evaluation by non-medical experts, and (c) evaluation by medical experts. As a quality-controlled subject gateway, CISMeF uses HIDDEL only as a third-party. The HIDDEL vocabulary can be downloaded freely from the MedCIRCLE Web site, as long as the sources are acknowledged and requests for changes or expansions are fed back to the community. At present HIDDEL is available in four languages: English, German, French and Spanish. The use of this controlled vocabulary enables automatic translation (except for free text). The heritage process was made possible because of HIDDEL's dual structure: on the one hand, Infoprovider metadata, describing to the health information provider (e.g., the name of the person responsible for the quality of the web site), and on the other hand, Sitespecific metadata devoted to one Web site evaluation (e.g., language). In CISMeF, we have applied Sitespecific metadata to each resource (mainly quality-controlled documents) from a publisher already included in the CISMeF database. The name of the person responsible for the quality of the Web site, which is one of the Infoprovider metadata, is the same for every document of the Web site. On the contrary, the language of the document, which is one of the Sitespecific metadata, may vary from one document to another. The CISMeF team implemented the whole HIDDEL structure in the CISMeF database, which involved the creation of triggers, thus ensuring automated transfer from CISMeF to HIDDEL metadata, and the creation of new forms (interface recasting) to deal with non-CISMeF metadata. Because the HIDDEL elements are optional and repeatable, CISMeF has selected a number of 70 metadata among the 305. Most of the metadata previously used in CISMeF and in particular the Dublin Core are also included in the HIDDEL language. These metadata were automatically triggered in the HIDDEL language. Interoperability The interoperability process consists of an exchange of RDF files, containing experts' annotations "written" in HIDDEL. The semantic-based Archer Annotation System deals with RDF annotations reception. Archer is a Web application that allows annotating health information Web sites using the HIDDEL vocabulary. It is a technical platform and an organizational infrastructure that can be used by consumers, health information providers, and third party rating services. The first version of Archer was implemented as a part of MedCERTAIN, and further enhanced in the course of the successor project MedCIRCLE to allow the exchange of metadata between third party rating organizations. On another ground, through its search engine Doc'CISMeF, CISMeF provides external links to Archer backend servlets, and internal links to rated sites disclosure (see Figure 1). Since August 2002 the CISMeF team has embedded RDF metadata (URL: ) into the generated HTML pages, making them not only machine-readable (as every HTML page is) but also machine-processable. Therefore, one of the main goals of this metadata element set was fulfilled easily: it became interoperable with other Internet services. Moreover, an RDF Scheme describing CISMeF specific metadata was created (URL: ). In a more pragmatic way, interoperability relies on a 3 steps process (see Figure 2): (1) RDF files generation: a Java program (RDFWriter.class) formats evaluation data according to a MedCIRCLE RDF Schema of annotations; (2) RDF files export: a Java program (RDFSender) sends RDF files to the MedCIRCLE web server using HTTP Post; (3) Reception and ID allocation: for each transmitted file, the MedCIRCLE Web server sends back an ID number that will be used to access the exported metadata. Results The CISMeF team in the MedCIRCLE consortium has evaluated and annotated the main health information providers (or publishers) included in the CISMeF database: national agencies, medical societies, universities and hospitals. CISMeF first checks the publishers' information without asking the health information provider to self-declare any metadata as described in the MedCERTAIN quality management process. CISMeF used HIDDEL to select and evaluate the 270 health publishers most represented in CISMeF and made the results of their evaluations explicit and accessible using RDF metadata. These were exported into the searchable MedCIRCLE Open Directory. Each site rated in CISMeF has a record in the CISMeF database that generates an RDF into HTML file. The search tool Doc'CISMeF (URL: ) displays the information originating from each of the publishers that were evaluated with a specific MedCIRCLE button, which is linked to the MedCIRCLE central repository where HIDDEL metadata elements are displayed (see Figure 3). Seal of trust, such as the one developed by HON, is a "quality seal" or a "seal of approval": i.e. the HON logo provides an accreditation, whereas the MedCIRCLE seal is not a "quality seal" but a "transparency seal": it is a button allowing health professionals and consumers to access metainformation. The presence of a MedCIRCLE button on a health Web site does not imply, in any way, that the site meets minimum required standards. This decision is left up to the user. In contrast, a seal of trust is a quality seal: i.e. every Web site with a seal of approval (such as the HON seal) has been previously accredited a third party. Nonetheless, every resource included in CISMeF and those evaluated by the MedCIRCLE process are quality-controlled. The MedCIRCLE consortium takes a very neutral approach and does not impose but strongly recommends certain procedures or minimum metadata, taking into account that collaborating gateways, accreditors, certifiers, raters may approach from very different angles. CISMeF has applied full heritage from the evaluated publishers: each document from a MedCIRCLE rated publisher, indexed in CISMeF, will also receive the MedCIRCLE button of the publisher with the same link to MedCIRCLE central repository. The idea is to keep the common Infoprovider elements for every document, and to use CISMeF metadata to disclose Sitespecific elements specific to each document, in particular the indexing with the MeSH thesaurus. At the end of the project in December 2003, starting from 270 websites, the translation from CISMeF metadata to HIDDEL, led to 6,480 evaluated resources in CISMeF in September 2003 (49.8% of the 13,012 resources included in CISMeF). All CISMeF selected HIDDEL metadata (70 out of 305) are displayed in the Doc'CISMeF record in RDF into HTML. The top five publishers indexed in CISMeF, which produced trustworthy documents in French are: Grenoble Medical School (N = 435), Health Canada (n = 275), Strasbourg Medical School (N = 263), and French Ministry of Health (N = 248). Every new document (e.g. clinical guideline or teaching material) included in CISMeF from one of the 270 main publishers evaluated through the MedCIRCLE process inherits automatically: (a) a MedCIRCLE button linking to the repository and (b) HIDDEL metadata included in CISMeF database and displayed in the Doc'CISMeF record in RDF into HTML. On the other hand, a document that comes from one of the 270 publishers evaluated within the MedCIRCLE project but not included in the CISMeF database will not receive a MedCIRCLE button in the CISMeF gateway. However, this more global transitivity could be applied in a more generic search tool such as Google. Since February 2003, when the MedCIRCLE button became operational in the CISMeF gateway, the CISMeF team has decided to go on (keep) applying the MedCIRCLE transparency process after the end of the project with the following rules: (a) check every month if there is a new publisher with five documents already included in CISMeF and (b) if so, begin the MedCIRCLE process for these publishers and apply transparency heritage for their respective documents. We applied these rules after the end of the EU-funding project. In March 2004, the CISMeF database contained a complete evaluation through the MedCIRCLE process for 346 publishers (+76, as compared to the EU-grant proposal) and 7,053 documents from these publishers (53.3% of the 13,227 resources included in CISMeF). In the MedCIRCLE repository, end-users can access an aggregate view of what people say about a certain Web site by clicking: The CISMeF gateway is one of many possible producers of trustworthy metadata regarding a health information provider. Metadata from the Open Directory can also be fed into search engines and other gateways. Discussion As the number of health related Web sites worldwide has been estimated as being around 100,000, complete coverage by a single third party evaluation body is impossible. Instead, a collaborative approach as shown in this paper has to be promoted, whereby different rating services or organizations use comparable standards and a common metadata language. More recently, the Health on the Net Foundation has developed a HON tool bar in the course of the EU-funded Active Health (Active Environment for Health Promotion and Disease Prevention, URL: ) consortium (URL: ): this HON tool bar is indicating if the site is accredited directly by HON or indirectly by one specific accredited Web sites as health gateways such as CISMeF or MedlinePlus. In this context, HON is creating a seal of quality trust heritage where MedCIRCLE is creating a seal of transparency heritage. A formal evaluation of these two examples of heritage (quality seal vs. transparency seal) is mandatory to check their respective hypothetical added value. One of the main findings of the MedCIRCLE consortium in the course of the project has been that there is no absolute objective quality of a Web site. Quality is to a certain degree subjective, may vary in time and also according to the eye of the beholder. A Web site that a consumer looking for health information finds acceptable one day may be unacceptable another day. For example, a consumer may search general information for one drug (e.g. after a TV show) and finds advice on a patient information Web site. Later, the same consumer searches for the same drug for his/her child. But this time, he/she checks if the respective Web site is sponsored by a pharmaceutical company. He/she comes to the same Web site as before, but this time this Web site will be unacceptable. The context of the search changed, making a general advice acceptable and a specific advice unacceptable. By providing metadata about health related websites, MedCIRCLE allows the health information consumers to decide themselves if a website is of good quality. Trust is improved by enhancing transparency. More globally, the MedCIRCLE consortium will lead to a safer Internet by providing a seal of transparency to over 7,000 resources on the Internet via the CISMeF search engine. The precaution principle is now widely accepted in the European Union, which develops Action Plans, such as the Action Plan for Safer Use of the Internet (URL: ), which partially granted the MedCIRCLE consortium. The MedCIRCLE button does not directly fulfill the objective to only identify reliable health information. This objective is in fact fulfilled by th HON initiative. In contrast, the MedCIRCLE consortium indirectly fulfill this objective (i.e. to identify only reliable health information) by providing a seal of transparency. The Netizen (citizen on the Internet) has the active role to evaluate the reliability when reading the HIDDEL metadata after clicking the MedCIRCLE button. With a seal of trust, the role of the Netizen is more passive but leads to a faster trust information access. Nevertheless, a drawback of the MedCIRCLE button is a possible misunderstanding by end-users, who might confuse it with a quality seal and therefore may forget to actively press the MedCIRCLE button, which may hinder their valid judgement. Here again, a formal evaluation of theses two approaches (transparency vs. trust) is necessary. While using Net Scoring, CISMeF was acting as an implicit third party. One of the main results of the MedCIRCLE project, from the CISMeF team's point of view, is that CISMeF proceeded from being an implicit to being an explicit third party thanks to the creation of the MedCIRCLE button now used for 346 publishers (2.6% of the resources included in CISMeF) and 7,053 resources (53.3% of the resources included in CISMeF), which multiplies by 20 the number of resources with a seal of trust. The HIDDEL language is totally embedded in the CISMeF metadata element sets. This allows a very easy interoperability with MedCIRCLE tools, and more specifically Archer. Semantic Web approaches already used in the MedCIRCLE project could open up new ways for educating health professionals and consumers and reaching less savvy health professionals and consumers, because part of the intelligence and knowledge currently required to critically appraise information on the health professional or consumer Web site could be built into the search tools. The feasibility of this approach has been already demonstrated by CISMeF but also by the German (AQuMed) and Spanish partners (COMB). The impact on health professionals and consumers is subject to ongoing investigation within the MedCIRCLE project. The Semantic Web may provide the health professional or the consumer with a greater capacity to determine the reliability of a given health information provider or service than the Web in its current form. Conclusion With the MedCIRCLE project and transparency heritage, CISMeF became an explicit third party. Competing interests None declared. Authors' contributions SJD had the original idea of seal transparency and drafted the manuscript. DH and TRRB developed respectively in France and Germany the programs to implement the seal transparency in CISMeF & MedCIRCLE Web sites. Pre-publication history The pre-publication history for this paper can be accessed here: Acknowledgments The MedCIRCLE project was partially funded by the European Union under the Action Plan for Safer Use of the Internet (URL: ). Figures and Tables Figure 1 Interoperability between CISMeF and Archer Figure 2 Interacting java classes to export rating results into the central repository Figure 3 Links between Doc'CISMeF and MedCIRCLE repository ==== Refs Eysenbach G Powell J Kuss O Sa ER Empirical studies assessing the quality of health information for consumers on the world wide web: a systematic review JAMA 2002 287 2691 700 12020305 10.1001/jama.287.20.2691 Boyer C Selby M Scherrer JR Appel RD The health on the net code of conduct for medical and health Websites Comput Biol Med 1998 28 603 610 9861515 10.1016/S0010-4825(98)00037-7 Risk A Dzenowagis J Review of internet information quality initiatives J Med Internet Res 2001 3 e28 11772543 Flannery MR Cataloging Internet resources Bull Med Libr Assoc 1995 83 211 215 7599587 Koch T Quality-controlled subject gateways: definitions, typologies, empirical overview Online Information Review 2000 24 24 34 10.1108/14684520010320040 Darmoni SJ Leroy JP Thirion B Baudic F Douyère M Piot J CISMeF: a structured Health resource guide Methods Inf Med 2000 39 30 35 10786067 Darmoni SJ Thirion B Leroy JP Douyere M Lacoste B Godard C Rigolle I Brisou M Videau S Goupy E Piott J Quéré M Ouazir S Abdulrab H A search tool based on 'encapsulated' MeSH thesaurus to retrieve quality health resources on the Internet Med Inform Internet Med 2001 26 165 178 11706927 10.1080/14639230110064488 Darmoni SJ Leroux V Daigne M Thirion B Santamaria P Duvaux C Albert A, Roger-France FH, Degoulet D, Fieschi M Critères de qualité de l'information de santé sur l'Internet In Santé et Réseaux Informatiques Informatique et Santé 1998 Paris: Springer Verlag France 162 174 Mayer MA Darmoni SJ Fiene M Köhler C Roth-Berghofer T Eysenbach G MedCIRCLE – Modeling a Collaboration for Internet Rating, Certification, Labeling and Evaluation of Health Information on the Semantic World-Wide-Web Stud Health Technol Inform 2003 95 667 672 14664064 Berners-Lee T Hendler J Publishing on the semantic web Nature 2001 410 1023 1024 11323639 10.1038/35074206 Charnock D Shepperd S Needham G Gann R DISCERN: an instrument for judging the quality of written consumer health information on treatment choices J Epidemiol Community Health 1999 53 105 11 10396471 Sarrias R Mayer MA Latorre M Accredited medical web: an experience in Spain J Med Internet Res 2000 2 e5 11720924 Thirion B Loosli G Douyère M Darmoni SJ Metadata element set in a Quality-Controlled Subject Gateway: a step to an health semantic Web Stud Health Technol Inform 2003 95 707 712 14664071 Darmoni SJ Haugh MC Lukacs B Boissel JP Quality of health information about depression on internet. Level of evidence should be gold standard BMJ 2001 322 1367 11409403 10.1136/bmj.322.7298.1367 Eysenbach G Yihune G Lampe K Cross P Brickley D A metadata vocabulary for self- and third-party labeling of health web-sites: Health Information Disclosure, Description and Evaluation Language (HIDDEL) Proc AMIA Symp 2001 169 73 11825174 Eysenbach G Yihune G Lampe K Cross P Brickley D MedCERTAIN: Quality Management, Certification and Rating of Health Information on the Net Proc AMIA Symp 2000 230 234 11079879 Eysenbach G Diepgen TL Labeling and filtering of medical information on the internet Methods Inf Med 1999 38 80 88 10431511 Resnick P Miller J PICS: Internet access control without censorship Communications of the ACM 1996 39 87 93 10.1145/236156.236175 Gaudinat A Joubert M Aymard S Falco L Boyer C Fieschi M WRAPIN: new generation health search engine using UMLS knowledge sources for MeSH term extraction from health documentation Medinfo 2004 356 360 15360834

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==== Front Nutr JNutrition Journal1475-2891BioMed Central London 1475-2891-3-151536959410.1186/1475-2891-3-15ResearchLycopene from two food sources does not affect antioxidant or cholesterol status of middle-aged adults Collins JK [email protected] BH [email protected] PL [email protected] P [email protected] RA [email protected] BA [email protected] USDA, ARS, South Central Agricultural Research Laboratory, Lane, OK, USA2 Dept. Nutritional Sciences, Oklahoma State University, Stillwater, OK, USA3 Dept. Statistics, Oklahoma State University, Stillwater, OK, USA4 Retired, USDA, ARS, Citrus and Subtropical Products Laboratory, Winter Haven, FL, USA5 USDA, ARS, Diet and Human Performance Laboratory, Beltsville, MD, USA2004 15 9 2004 3 15 15 13 8 2004 15 9 2004 Copyright © 2004 Collins et al; licensee BioMed Central Ltd.2004Collins et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Epidemiological studies have reported associations between reduced cardiovascular disease and diets rich in tomato and/or lycopene. Intervention studies have shown that lycopene-containing foods may reduce cholesterol levels and lipid peroxidation, factors implicated in the initiation of cardiovascular disease. The objective of this study was to determine whether consumption of lycopene rich foods conferred cardiovascular protection to middle-aged adults as indicated by plasma lipid concentrations and measures of ex vivo antioxidants. Methods Ten healthy men and women consumed a low lycopene diet with no added lycopene (control treatment) or supplemented with watermelon or tomato juice each containing 20 mg lycopene. Subjects consumed each treatment for three weeks in a crossover design. Plasma, collected weekly was analyzed for total cholesterol, high density lipoprotein cholesterol (HDL-C) and triglyceride concentrations and for the antioxidant biomarkers of malondialdehyde formation products (MDA), plasma glutathione peroxidase (GPX) and ferric reducing ability of plasma (FRAP). Data were analyzed using Proc Mixed Procedure and associations between antioxidant and lipid measures were identified by Pearson's product moment correlation analysis. Results Compared to the control diet, the lycopene-containing foods did not affect plasma lipid concentrations or antioxidant biomarkers. Women had higher total cholesterol, HDL-C and triglyceride concentrations than did the men. Total cholesterol was positively correlated to MDA and FRAP while HDL-C was positively correlated to MDA and GPX. GPX was negatively correlated to triglyceride concentration. Conclusions The inclusion of watermelon or tomato juice containing 20 mg lycopene did not affect plasma lipid concentrations or antioxidant status of healthy subjects. However, plasma cholesterol levels impacted the results of MDA and FRAP antioxidant tests. ==== Body Background Watermelons and tomatoes are good sources of the carotenoid lycopene [1,2]. However, bioavailability of lycopene is not directly related to plant content, and depends in a large part upon plant matrix effects. In tomatoes, heat processing and homogenization breaks protein-carotenoid complexes, releases lycopene from cell wall linkages and improves human uptake of this compound [3-6], while heat processing is not necessary for adequate uptake of lycopene from watermelon juice [7]. Extracts of both foods exhibit antioxidant activity in vitro and function is attributed to lycopene since isolated lycopene demonstrates strong oxygen and peroxy radical scavenging properties [8-10]. Recent epidemiological studies have linked reductions in risks of cardiovascular disease with diets rich in lycopene containing foods. These reductions in risk have been primarily attributed to the antioxidant properties of lycopene [11,12]. Improved antioxidant parameters of lymphocytes have been reported in clinical trials that supplemented diets with 16.5 mg and 40 mg /day of lycopene from tomato puree and tomato juice, respectively [13,14]. Other clinical trials have shown reductions in low-density lipoprotein (LDL) oxidation resulting from lycopene supplementation [15-17]. LDL contains unsaturated fatty acids and can be oxidized by free radicals and peroxidizing agents. Since lycopene is primarily attached to LDL in plasma, it may protect against atherosclerosis through inhibition of lipid peroxidation and foam cell production [12,18]. Other studies have assessed response of plasma lipids to lycopene-rich diets. In one study, six healthy men were supplemented with 60 mg/day for three months with tomato lycopene (LycoRed) with a 14% reduction in LDL-C and no change in HDL-C [19]. Researchers concluded that lycopene was involved in controlling cholesterol synthesis and found the same results in a macrophage cell study [19]. It is not known if other lycopene containing foods can act ex vivo as antioxidants or alter cholesterol levels. The objectives of this study were to compare the ability of two lycopene containing foods, tomato and watermelon to provide cardiovascular protection to middle-aged adults by measuring changes in cholesterol levels and antioxidant ex vivo biomarkers. Methods Experimental Design Samples for this study came from a larger study, which has been reported in detail [7]. This study was a diet-controlled, repeated measures crossover design with ten healthy non-smoking subjects, five men (average age 49 years) and five women (average age 51 years) recruited from the Beltsville, MD area (Table 1). In addition to a base diet, which provided 34% of energy from fat and minimal amounts of lycopene, subjects were randomly assigned to receive three dietary treatments for 3 weeks each: 1) control (no added lycopene); 2) 20.1 mg lycopene per day from watermelon juice; and 3) 18.4 mg lycopene per day from tomato juice. All subjects followed a low-lycopene diet for two weeks before the first treatment and during the four-week washout periods between treatments. Total study duration was 19 weeks. During treatment periods, all meals were prepared and consumed Monday through Friday at the Beltsville Human Nutrition Research Center's Human Studies Facility, and weekend meals were packed for off-site consumption. Blood was drawn from fasted subjects before treatment (the day before the start of study and on the first day of the study), prior to treatment and weekly during treatment. Plasma was separated from whole blood by centrifugation and stored at -80°C until analyzed for cholesterol and antioxidant activity. Table 1 Description of human clinical study participants. Gender N Age (Range) yr BMI (Range) Kg/m2 Men 5 49 (43–68) 26.3 (23.0–29.5) Women 5 51 (35–63) 29.1 (23.5–34.5) Juice Treatments Watermelon juice for the study was prepared at a pilot plant at the USDA Citrus and Subtropical Products Laboratory, Winter Haven, FL without heat treatment as previously described [7]. Canned commercial tomato juice (Campbell's, Camden, NJ) was used for the tomato intervention. Juices were analyzed for carotenoid content using established extraction procedures with reversed phase HPLC with photo diode array detector (Waters Corp, Franklin, MA) [7]. For watermelon treatment, subjects were given one bottle of juice (260 g each) at breakfast, lunch and dinner, which provided daily totals of 20.1 mg lycopene, 0.90 mg phytoene, 0.45 mg phytofluene and 2.5 mg beta carotene. The juice contained 94% trans lycopene and 6% cis isomers, primarily 5-cis and 13-cis with minimal amounts of other cis isomers [7]. For tomato juice treatment, subjects were given one serving (122 g each) at breakfast and dinner, which provided daily totals of 18.4 mg lycopene, 2.1 mg phytoene, 1.1 mg phytofluene and 0.6 mg beta carotene with 89% of the lycopene as trans lycopene and 10.8% cis isomers, primarily identified as 5-cis, 9-cis, 13-cis, and 15-cis, and minimal amounts of other cis isomers [7]. Cholesterol Analysis Plasma samples were thawed on ice for four hours then mixed by vortexing, prior to preparing for assays. Serum total cholesterol and triglyceride concentrations were determined enzymatically using kits from Roche Diagnostics (Sommerville, NJ). Serum HDL-cholesterol was determined by a direct method (Unimate HDL Direct ; Roche Diagnostics, Indianapolis, IN) that utilizes the combined action of polymers, polyanions, and detergent to solubilize cholesterol from HDL but not from VLDL, LDL, and chylomicrons as previously described [20]. Analysis was performed on a Cobas-Fara II Clinical Analyzer (Montclair, NJ) using commercially available calibrators and quality control standards (Roche Diagnostics, Indianapolis, IN). Plasma Glutathione Peroxidase Assay Plasma from subjects was analyzed for plasma glutathione peroxidase using an ELISA kit (OXIS Internatl., Portland, OR). Two replicates per sample of 20 μl of plasma were diluted 1:25 with TRIS-HCl buffer then pippetted into pre-coated polyclonal antibodies microplate wells specific for human plasma glutathione peroxidase (GPX). The amount of enzyme present was determined by reaction with para-nitrophenyl-phosphate and was read using a microplate reader at 405 nm (Elx 808 Ultra Microplate Reader, Bio-Tek Instruments Inc., Winooski, VT). The concentration of plasma GPX was determined from a standard curve for each plate using five dilutions of GPX standard. Plasma lipid peroxidation Malondialdehyde compounds were determined colorimetrically using a commercial kit specific for measuring free and total malondialdehyde compounds (OXIS Internatl., Portland, OR). Two replicates per sample of 210 μl of plasma were added to each test tube with 11 μl of 500 mM butylated hydroxytoluene and 5.3 μl of concentrated hydrochloric acid. Tubes were capped, mixed then incubated at 60°C for 80 minutes, cooled to room temperature and 680 μl of N-methyl-2-phenylindole in acetonitrile was added. Then tubes were mixed, and centrifuged at 13,000 g for 5 minutes. New tubes were prepared and 660 μl of clear supernatant was added with 115 μl of concentrated HCl. Tubes were capped, mixed and incubated at 45°C for 60 minutes. Samples were centrifuged at 13,000 g for 5 min and the supernatant was read on a spectrophometer at 575 nm. Concentration of samples was determined using a five point standard curve. Ferric reducing ability of plasma assay This assay was conducted according to previously published methods [1]. In brief, three reagents were used: 1) sodium acetate, acetic acid buffer (pH 3.6); 2)10 mmol/L solution of 2, 4,6-tripyridyl-s-triazine in a 40 mmol/L solution of hydrochloric acid (Sigma, St. Louis, MO); and 3) 20 mmol/L solution of ferric chloride hexahydrate prepared in double deionized water. The FRAP reagent was prepared daily with 25 ml of reagent one, 2.5 ml reagent two and three that were heated to 37°C before using [21]. The assay was conducted with 10 uL of plasma that was diluted with 30 μl of ddi water. Sample was added to reagent in cuvettes with an autosampler and then read on a COBAS FARA II spectrofluorometric centrifugal analyzer (Roche, Montclair, NJ) at 593 nm at four minutes. FRAP values were determined from a five point curve using a trolox (vitamin E analog) standard. Standard curves were run after every 90 samples. Experimental procedures for the clinical trial were approved by the Institutional Review Board at the Johns Hopkins University Bloomberg School of Hygiene and Public Health; subjects gave their written informed consent to participate. The plasma cholesterol and antioxidant studies were approved by the Institutional Review Board at Oklahoma State University, Stillwater, OK. Data were analyzed using Proc Mixed Procedure and mean separation was performed using LSMEANS, correlation analysis was performed using Spearman's Correlation Coefficient Analysis (SAS Statistical Analysis Software, version 8.2, SAS Institute, Cary, NC). Results Because there were significant four way interactions with gender × intervention period × treatment × weeks with MDA, FRAP, GPX and cholesterol analysis, trends by treatment, intervention period or week of treatment were not seen. Supplementing the diet with 20 mg/day of lycopene of either food did not change the plasma antioxidant status of the subjects and values ranged from 0.66–2.20, 540–1094, and 1296–2596 :mol/L for MDA, FRAP and GPX respectively. These levels are similar to levels reported for healthy subjects in other studie [22,23]. Intervention with 20 mg of lycopene to the diet of subjects did not alter their total cholesterol, HDL-C or triglyceride status. However, there were gender differences and the women had higher average levels of plasma triglycerides, total cholesterol and HDL-C than men (Figure 1). The higher cholesterol levels for women compared to men in this study were not unusual since women in this age range often have higher cholesterol levels than men, a phenomenon related to decreased estrogen production [24,25]. In this study, menopausal information was not recorded. Figure 1 Mean plasma total cholesterol, triglycerides, and high density lipoprotein (HDL) cholesterol (mg/dl) of 5 men and 5 women supplemented for 3 week with no lycopene (control), 20 mg lycopene from watermelon juice and 20 mg lycopene from tomato juice. *Represents significance p < 0.05. There was a significant positive correlation between each pair of total cholesterol and MDA and MDA and FRAP and between HDL-C and MDA and HDL-C and GPX. A significant negative correlation was found between triglycerides and GPX (Table 2). Table 2 Spearman's Correlation Coefficients between antioxidant tests of malondialdehyde (MDA) ferric reducing ability of plasma (FRAP), and plasma glutathione peroxidase (GPX) and cholesterol measurements. Variable MDA FRAP GPX Total cholesterol 0.547** 0.325** 0.003 HDL-C 0.563** 0.059 0.294** Triglycerides 0.219 0.037 -0.229* MDA . 0.474** 0.180 FRAP 0.474** . 0.077 Correlations significant at the 0.05* and 0.01** level. Because of the correlation between cholesterol concentrations and antioxidant analysis, a preliminary analysis of data was conducted to determine if cholesterol levels impacted antioxidant results. Subjects were separated into two groups based upon baseline concentrations of plasma triglycerides, total cholesterol and LDL-C above or below 200, 180 and 160, respectively. Five subjects, two men and three women, fit the criteria of moderately hypercholesterolemic (Table 3). Table 3 Separation of subjects by baseline cholesterol levels from a watermelon and tomato juice lycopene intervention study, n = 5 for each group. Cholesterol Group Total Cholesterol mg/dl Triglycerides mg/dl HDL-Cmg/dl 1 229.3 ± 4.9 190.9 ± 12.8 59.1 ± 2.9 2 176.6 ± 2.5 129.7 ± 5.2 46.9 ± 2.1 Data represents mean ± SE Analyses showed an interaction of cholesterol level × treatment period × treatment factor for MDA and FRAP analysis. Higher MDA and FRAP levels were found in the group having higher cholesterol levels compared to the other group (Table 4). No trend with cholesterol level and glutathione peroxidase was found. Table 4 Total, triglyceride, high density lipoprotein (HDL-C cholesterol and antioxidant analysis of malondialdehyde (MDA), glutathione peroxidase (GPX) and ferric reducing ability of of plasma (FRAP) after 4 week lycopene depletion and three weeks of intervention with watermelon and tomato juice (20 mg lycopene/day). Subjects were separated into 2 groups based upon cholesterol levels (see Table 3). Cholesterol Group Analysis Depletion SE Control SE Watermelon SE Tomato SE Total Cholesterol (mg/dl) 220.9 ± 9.5 223.4 ± 7.9 224.6 ± 8.2 233.6 ± 6.2 Triglycerides (mg/dl) 185.9 ± 16.9 181.7 ± 16.9 198.9 ± 18.3 174.7 ± 15.6 1 HDL-C (mg/dl) 56.9 ± 5.15 58.65 ± 4.31 58.00 ± 5.96 59.38 ± 4.55 MDA (umol/L) 1.21 ± 0.11 1.12 ± 0.11 1.15 ± 0.12 1.37 ± 0.11 GPX (umol/L) 2728 ± 219 2728 ± 222 2263 ± 169 2574 ± 187 FRAP (umol/L) 831.6 ± 24.9 871.7 ± 26.7 900.9 ± 25.2 861.6 ± 23.4 Total Cholesterol (mg/dl) 182.6 ± 4.2 173.3 ± 3.3 186.1 ± 6.4 173.1 ± 2.8 Triglycerides (mg/dl) 129.4 ± 8.3 128.7 ± 7.6 189.4 ± 4.9 135.4 ± 10.7 2 HDL-C (mg/dl) 42.5 ± 3.3 43.6 ± 2.8 44.7 ± 4.0 38.3 ± 2.8 MDA (umol/L) 0.53 ± 0.03 0.56 ± 0.04 0.48 ± 0.04 0.54 ± 0.03 GPX (umol/L) 2129 ± 151 2111 ± 154 2292 ± 168 2229 ± 160 FRAP (umol/L) 743.9 ± 33.6 762.9 ± 30.7 780.9 ± 32.4 756.7 ± 36.0 Discussion We found no improvement in the antioxidant status of healthy middle-aged adults supplemented with two lycopene-containing foods. In previous antioxidant studies, reduced lipid peroxidation was reported in subjects supplemented from one to four weeks with 5 to 45 mg lycopene containing tomato products [15,16,23,26]. However, in each of these studies, the diet was not controlled. When healthy elderly subjects in a diet controlled study were supplemented with 13.3 mg of tomato lycopene (LycoRed) for 12 weeks, lycopene intervention did not significantly change LDL oxidation, as measured by the rate of conjugated diene production [27]. The reports from lycopene intervention studies that measured FRAP activity are not in agreement. One study reported improvement in FRAP levels of plasma in subjects supplemented with tomato juice and olive oil [28], while two other tomato juice intervention studies reported no improvement in plasma antioxidant levels after lycopene supplementation as measured by Trolox equivalent antioxidant capacity (TEAC), radical trapping antioxidant parameter assay (TRAP), and FRAP [3,23]. Researchers in one study found that the FRAP assay was more accurate when measuring the antioxidant activity of water-soluble antioxidants [23]. They thought full expression of the antioxidant activity was not identified from lycopene in this assay since it is a lipophyllic compound. Curiously, both watermelon and tomato contain other water-soluble compounds that are reported to have antioxidant activity that reacts in vitro in the FRAP assay [9,29]. In this study, contribution of these water-soluble compounds in changes in plasma FRAP activity with either food intervention compared to the control was not found. Unlike a previous report by Fuhrman et al., neither lycopene intervention with watermelon nor tomato affected cholesterol levels [19]. Differences in results may have been due to lycopene dosage level. In that study [19] the subjects were supplemented with 60 mg/day for three months, however diet was not controlled. Fruits and vegetables are excellent sources of antioxidant compounds and the average American consumes only 1.5 and 3.1 servings per day [45]. In many of the studies where antioxidant protection with lycopene containing foods was reported, subjects consumed their normal diet that may or may not have met the recommended servings of fruits and vegetables [13,23,26,31,32]. Increasing fruit and vegetable consumption to 12 servings per day compared to 5.8 servings, without the addition of other diet interventions, reduced a biomarker of DNA oxidative damage (8-hydroxydeoxyguanosine) by 32% [33]. In a controlled trial where subjects were supplemented with tomato juice but restricted in total fruit and vegetable consumption and exposed to low levels of ozone, researchers found reduced DNA strand breaks compared to placebo controls [34]. Because this study controlled for other phytochemical containing fruits and vegetables, the DNA protection was attributed to tomato juice phytochemicals [34]. The positive correlation between total cholesterol and MDA antioxidant analysis has been reported in studies with hypercholesterolemic subjects compared to normocholesterolemic subjects [35,36]. The MDA assay measures lipid peroxidation products, and a higher level of lipids available to react with peroxidizing agents results in higher MDA values [36,37]. The trend correlating higher FRAP with higher cholesterol levels has not been previously reported. The significance of this trend is speculative, since the FRAP assay measures the oxidation and reduction potential of compounds based on the reduction of the ferric to ferrous iron [38], lipid peroxidation products may have contributed to the oxidation/reduction potential of the reaction. Conclusions Long-term supplementation studies where diet is controlled will probably be necessary to identify the benefits provided by lycopene. There may be real health benefits associated with lycopene especially since it is stored in various tissues and exhibits strong antioxidant activity in vitro [8,10,39,40]. Also the body of epidemiological evidence points to the protection provided against cardiovascular disease and some cancers with lycopene containing foods [11,12,41,42]. Recent cancer intervention studies have reported beneficial effects on prostate cancer from lycopene food supplementation [43,44]. The health benefits associated with diets providing lycopene are most likely long-term. Therefore, the findings of the present study should not be interpreted as a lack of health benefits from regular consumption of lycopene-rich foods. The interaction between cholesterol levels and antioxidant values needs more research. Contradictory findings of this study with other ex vivo antioxidant studies may be due to the cholesterol levels of subjects thus warranting further research. Competing interests None declared. Note Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. All programs and services of the U.S. Department of Agriculture are offered on a nondiscriminatory basis without regard to race, color, national origin, religion, sex, age, marital status, or handicap. The article cited was prepared by a USDA employee as part of his/her official duties. Copyright protection under U.S. copyright law is not available for such works. Accordingly, there is no copyright to transfer. The fact that the private publication in which the article appears is itself copyrighted does not affect the material of the U.S. Government, which can be freely reproduced by the public. Authors' contributions JKC: conception and design of the study, drafted the manuscript, BHA: design of study, editing, PLC:design of the study, statistical analysis, PPV: conception of study, editing, RAB: editing, technical assistance, BAC:conception of study, editing. All authors read and approved the final manuscript. Acknowledgements Research supported in part by a grant from the National Watermelon Promotion Board, Orlando, FL. Researchers would like to acknowledge the following individuals for their technical assistance: Kari Callicoat, Edralin Lucas and Jarrod King. ==== Refs Holden AL Eldridge AL Beecher GR Buzzard IM Bhagwat SA Davis CS Douglas LW Gebhardt SE Haytowitz DB Schakel S Carotenoid Content of U.S. Foods: An Update of the Database J Food Comp Anal 1999 12 169 196 10.1006/jfca.1999.0827 Perkins-Veazie P Collins JK Pair SD Roberts W Lycopene content differs among red-fleshed watermelon cultivars J Science of Food and Agric 2001 81 1 5 10.1002/1097-0010(20010101)81:1<1::AID-JSFA819>3.3.CO;2-1 Bohm V Bitsch R Intestinal absorption of lycopene from different matrices and interactions to other carotenoids, the lipid status, and the antioxidant capacity of human plasma Eur J Nutr 1999 38 118 125 10443333 10.1007/s003940050052 Shi J Le Maguer M Lycopene in tomatoes: chemical and physical properties affected by food processing Crit Rev Biotechnol 2000 20 293 334 11192026 Gartner C Stahl W Sies H Lycopene is more bioavailable from tomato paste than from fresh tomatoes Am J Clin Nutr 1997 66 116 122 9209178 het Hof KH de Boer BC Tijburg LB Lucius BR Zijp I West CE Hautvast JG Weststrate JA Carotenoid bioavailability in humans from tomatoes processed in different ways determined from the carotenoid response in the triglyceride-rich lipoprotein fraction of plasma after a single consumption and in plasma after four days of consumption J Nutr 2000 130 1189 1196 10801917 Edwards AJ Vinyard BT Wiley ER Brown ED Collins JK Perkins-Veazie P Baker RA Clevidence BA Consumption of watermelon juice increases plasma concentrations of lycopene and beta-carotene in humans J Nutr 2003 133 1043 1050 12672916 Di Mascio P Kaiser S Sies H Lycopene as the most efficient biological carotenoid singlet oxygen quencher Arch Biochem Biophys 1989 274 532 538 2802626 Djuric Z Powell LC Antioxidant capacity of lycopene-containing foods Int J Food Sci Nutr 2001 52 143 149 11303462 Stahl W Nicolai S Briviba K Hanusch M Broszeit G Peters M Martin HD Sies H Biological activities of natural and synthetic carotenoids: induction of gap junctional communication and singlet oxygen quenching Carcinogenesis 1997 18 89 92 9054593 10.1093/carcin/18.1.89 Rissanen TH Voutilainen S Nyyssonen K Salonen R Kaplan GA Salonen JT Serum lycopene concentrations and carotid atherosclerosis: the Kuopio Ischaemic Heart Disease Risk Factor Study Am J Clin Nutr 2003 77 133 138 12499332 Gianetti J Pedrinelli R Petrucci R Lazzerini G De Caterina M Bellomo G De Caterina R Inverse association between carotid intima-media thickness and the antioxidant lycopene in atherosclerosis Am Heart J 2002 143 467 474 11868053 10.1067/mhj.2002.120776 Pool-Zobel BL Bub A Muller H Wollowski I Rechkemmer G Consumption of vegetables reduces genetic damage in humans: first results of a human intervention trial with carotenoid-rich foods Carcinogenesis 1997 18 1847 1850 9328185 10.1093/carcin/18.9.1847 Riso P Pinder A Santangelo A Porrini M Does tomato consumption effectively increase the resistance of lymphocyte DNA to oxidative damage? Am J Clin Nutr 1999 69 712 718 10197573 Rao AV,Shen,H. Effect of low dose lycopene intake on lycopene bioavailability and oxidative stress Nutrition Research 2002 22 1125 1131 10.1016/S0271-5317(02)00430-X Agarwal S Rao AV Tomato lycopene and low density lipoprotein oxidation: a human dietary intervention study Lipids 1998 33 981 984 9832077 Kiokias S Gordon MH Dietary supplementation with a natural carotenoid mixture decreases oxidative stress Eur J Clin Nutr 2003 57 1135 1140 12947433 10.1038/sj.ejcn.1601655 Arab L Steck S Lycopene and cardiovascular disease Am J Clin Nutr 2000 71 1691S 1695S 10837319 Fuhrman B Elis A Aviram M Hypocholesterolemic effect of lycopene and beta-carotene is related to suppression of cholesterol synthesis and augmentation of LDL receptor activity in macrophages Biochem Biophys Res Commun 1997 233 658 662 9168909 10.1006/bbrc.1997.6520 Arjmandi BH Sohn E Juma S Murthy SR Daggy BP Native and partially hydrolyzed psyllium have comparable effects on cholesterol metabolism in rats J Nutr 1997 127 463 469 9082031 Benzie IF Strain JJ The ferric reducing ability of plasma (FRAP) as a measure of "antioxidant power": the FRAP assay Anal Biochem 1996 239 70 76 8660627 10.1006/abio.1996.0292 Drai J Bannier E Chazot C Hurot JM Goedert G Jean G Charra B Laurent G Baltassat P Revol A Oxidants and antioxidants in long-term haemodialysis patients Farmaco 2001 56 463 465 11482779 10.1016/S0014-827X(01)01063-1 Bub A Watzl B Abrahamse L Delincee H Adam S Wever J Muller H Rechkemmer G Moderate intervention with carotenoid-rich vegetable products reduces lipid peroxidation in men J Nutr 2000 130 2200 2206 10958813 Schwab US Vogel S Lammi-Keefe CJ Ordovas JM Schaefer EJ Li Z Ausman LM Gualtieri L Goldin BR Furr HC Lichtenstein AH Varying dietary fat type of reduced-fat diets has little effect on the susceptibility of LDL to oxidative modification in moderately hypercholesterolemic subjects J Nutr 1998 128 1703 1709 9772139 Castelo-Branco C Blumel JE Roncagliolo ME Haya J Bolf D Binfa L Tacla X Colodron M Age, menopause and hormone replacement therapy influences on cardiovascular risk factors in a cohort of middle-aged Chilean women Maturitas 2003 45 205 212 12818466 10.1016/S0378-5122(03)00140-3 Maruyama C Imamura K Oshima S Suzukawa M Egami S Tonomoto M Baba N Harada M Ayaori M Inakuma T Ishikawa T Effects of tomato juice consumption on plasma and lipoprotein carotenoid concentrations and the susceptibility of low density lipoprotein to oxidative modification J Nutr Sci Vitaminol (Tokyo) 2001 47 213 221 11575576 Carroll YL Corridan BM Morrissey PA Lipoprotein carotenoid profiles and the susceptibility of low density lipoprotein to oxidative modification in healthy elderly volunteers Eur J Clin Nutr 2000 54 500 507 10878653 10.1038/sj.ejcn.1601046 Lee A Thurnham DI Chopra M Consumption of tomato products with olive oil but not sunflower oil increases the antioxidant activity of plasma Free Radic Biol Med 2000 29 1051 1055 11084294 10.1016/S0891-5849(00)00440-8 Takeoka GR Dao L Flessa S Gillespie DM Jewell WT Huebner B Bertow D Ebeler SE Processing effects on lycopene content and antioxidant activity of tomatoes J Agric Food Chem 2001 49 3713 3717 11513653 10.1021/jf0102721 Patterson RE Haines PS Popkin BM Diet quality index: capturing a multidimensional behavior J Am Diet Assoc 1994 94 57 64 8270756 10.1016/0002-8223(94)92042-7 Torbergsen AC Collins AR Recovery of human lymphocytes from oxidative DNA damage; the apparent enhancement of DNA repair by carotenoids is probably simply an antioxidant effect Eur J Nutr 2000 39 80 85 10918989 10.1007/s003940050006 Pool-Zobel BL Bub A Liegibel UM Treptow-van Lishaut S Rechkemmer G Mechanisms by which vegetable consumption reduces genetic damage in humans Cancer Epidemiol Biomarkers Prev 1998 7 891 899 9796634 Thompson HJ Heimendinger J Haegele A Sedlacek SM Gillette C O'Neill C Wolfe P Conry C Effect of increased vegetable and fruit consumption on markers of oxidative cellular damage Carcinogenesis 1999 20 2261 2266 10590217 10.1093/carcin/20.12.2261 Samet JM Hatch GE Horstman D Steck-Scott S Arab L Bromberg PA Levine M McDonnell WF Devlin RB Effect of antioxidant supplementation on ozone-induced lung injury in human subjects Am J Respir Crit Care Med 2001 164 819 825 11549539 Hussein O Frydman G Frim H Aviram M Reduced susceptibility of low density lipoprotein to lipid peroxidation after cholestyramine treatment in heterozygous familial hypercholesterolemic children Pathophysiology 2001 8 21 28 11476969 10.1016/S0928-4680(01)00061-X Sakuma N Hibino T Sato T Ohte N Akita S Tamai N Sasai K Yoshimata T Fujinami T Levels of thiobarbituric acid-reactive substance in plasma from coronary artery disease patients Clin Biochem 1997 30 505 507 9316747 10.1016/S0009-9120(97)00050-7 Quasim T McMillan DC Talwar D Sattar N O'Reilly DS Kinsella J Lower concentrations of carotenoids in the critically ill patient are related to a systemic inflammatory response and increased lipid peroxidation Clin Nutr 2003 22 459 462 14512033 10.1016/S0261-5614(03)00044-X Svilaas A Sakhi AK Andersen LF Svilaas T Strom EC Jacobs D.R.,Jr. Ose L Blomhoff R Intakes of Antioxidants in Coffee, Wine, and Vegetables Are Correlated with Plasma Carotenoids in Humans J Nutr 2004 134 562 567 14988447 Erdman J.W.,Jr. Bierer TL Gugger ET Absorption and transport of carotenoids Ann N Y Acad Sci 1993 691 76 85 8129321 Parker RS Absorption, metabolism, and transport of carotenoids FASEB J 1996 10 542 551 8621054 Giovannucci E Rimm EB Liu Y Stampfer MJ Willett WC A prospective study of tomato products, lycopene, and prostate cancer risk J Natl Cancer Inst 2002 94 391 398 11880478 10.1093/jnci/94.5.391 De Stefani E Boffetta P Brennan P Deneo-Pellegrini H Carzoglio JC Ronco A Mendilaharsu M Dietary carotenoids and risk of gastric cancer: a case-control study in Uruguay Eur J Cancer Prev 2000 9 329 334 11075886 10.1097/00008469-200010000-00007 Kucuk O Sarkar FH Sakr W Djuric Z Pollak MN Khachik F Li YW Banerjee M Grignon D Bertram JS Crissman JD Pontes EJ Wood D.P.Jr. Phase II randomized clinical trial of lycopene supplementation before radical prostatectomy Cancer Epidemiol Biomarkers Prev 2001 10 861 868 11489752 Bowen P Chen L Stacewicz-Sapuntzakis M Duncan C Sharifi R Ghosh L Kim HS Christov-Tzelkov K van Breemen R Tomato sauce supplementation and prostate cancer: lycopene accumulation and modulation of biomarkers of carcinogenesis Exp Biol Med (Maywood ) 2002 227 886 893 12424330 U.S. Dept. of Agriculture, Agriculture Research Service 2000 Servings Intakes by U.S. Children and Adults: 1994-1996, 1998. Online. ARS Community Nutrition Research Group web site available at , accessed Sept. 23, 2004.

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==== Front Mol CancerMolecular Cancer1476-4598BioMed Central London 1476-4598-3-231535020410.1186/1476-4598-3-23EditorialOpen Access gains attention in scholar communication Chiao Paul J [email protected] Christian [email protected] Molecular Cancer, BioMed Central Ltd, Middlesex House, 34-42 Cleveland Street, London W1T 4LB, UK2004 6 9 2004 3 23 23 22 4 2004 6 9 2004 Copyright © 2004 Chiao and Schmidt; licensee BioMed Central Ltd.2004Chiao and Schmidt; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ==== Body Open Access is one of the attempts to maximize the exchange of information, and therefore benefits the scholar communication [1]. Molecular Cancer offers Open Access to all of its content, thereby providing a platform to present information to specialists and the public in order to further promote free exchange of ideas, concepts and findings in all fields of cancer-related biomedical science. All the published articles in the journal are determined by the peer review process. Open Access has following broad benefits for science and the general public: • All articles become freely and universally accessible online; so an author's work can be read by anyone at no cost. • The authors hold copyright for their work and grant anyone the right to reproduce and disseminate the article, provided that it is correctly cited. • A copy of the full text of each Open Access article is permanently archived in an online repository separate from the journal, such as PubMed Central, the US National Library of Medicine's full-text repository of life science literature, the repositories at the University of Potsdam in Germany, at INIST in France and in e-Depot, the National Library of the Netherlands' digital archive of all electronic publications. • Authors are assured that their work is disseminated to the widest possible audience. This is accentuated by the authors being free to reproduce and distribute their work, for example by placing it on their institution's website. It has been suggested that free online articles are more highly cited because of their easier availability [2]. • The information available to researchers will not be limited by their library's budget, and the widespread availability of articles will enhance literature searching. • The results of publicly funded research will be accessible to all interested readers and not just those with access to a library with a subscription. As such, Open Access could help to increase public interest in, and support of, research. Please note that this public accessibility may become a legal requirement in the USA if the proposed Public Access to Science Act is made law [3]. • A country's economy will not influence its scientists' ability to access articles because resource-poor countries (and institutions) will be able to read the same material as wealthier ones, although creating access to the internet is another matter. Molecular Cancer published a number of interesting papers, and the list of the top ten most accessed articles is available at . All papers accepted by Molecular Cancer appear as 'accepted manuscript' on the web pages and are subsequently included in PubMed. A fully formatted portable document file is available approximately two to three weeks after acceptance along with a web-version of the article. The on-line publication, to the exclusion of print, has many advantages: Coloured pictures can be presented along with large sets of supporting data (movies, tables, pictures, et cetera) without additional charges. In addition, the on-line submission process allows a fast and effective handling of papers and allows authors to check the status of their submitted manuscript(s). There is no limitation in space, but concise papers are more likely to be read. The peer review policy, described in [4], ensures a fair evaluation of the work. We wish to thank our authors for sending their work to Molecular Cancer, all members of the editorial board and the reviewers for their ongoing support for Open Access publishing and for aiming higher standards for Molecular Cancer. The acceptance rate of Molecular Cancer did not change significantly, compared to the last report [4]. One out of three incoming articles are accepted for publication at Molecular Cancer after revisions. In addition to indexing in PubMed, PubMed Central and other search engines, Molecular Cancer is working closely with the Institute for Scientific Information to ensure that citation analysis of our articles will be available. Competing interests PJC is Editor-in-Chief and CS is Deputy Editor of this journal. Both do not receive any remuneration for their efforts but they are exempted from the article processing fee for this journal. ==== Refs Salva U Open access already exists Science 2004 303 1467 Lawrence S Free online availability substantially increases a paper's impact Nature 2001 411 521 11385534 10.1038/35079151 Open Access law introduced Deora AB Schmidt C Sclabas GM First anniversary of Molecular Cancer: achievements and future goals Mol Cancer 2003 2 26 10.1186/1476-4598-2-26

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==== Front Health Qual Life OutcomesHealth and Quality of Life Outcomes1477-7525BioMed Central London 1477-7525-2-521538314810.1186/1477-7525-2-52ResearchAbility to perform activities of daily living is the main factor affecting quality of life in patients with dementia Andersen Christian K [email protected] Kim U [email protected] Anette [email protected] Kjeld [email protected]ørensen Per [email protected] MUUSMANN Research & Consulting, Haderslevvej 36, 6000 Kolding, Denmark2 Global Health Economics & Outcomes Research, Novo Nordisk A/S 2880 Bagsværd, Denmark3 Center for Dementia Research, Odense University Hospital, Sdr. Boulevard 29 5000 Odense, Denmark2004 21 9 2004 2 52 52 27 4 2004 21 9 2004 Copyright © 2004 Andersen et al; licensee BioMed Central Ltd.2004Andersen et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Dementia is a chronic illness associated with a progressive loss of cognitive and intellectual abilities, such as memory, judgment and abstract thinking. The objective of this study was to assess the health utilities of patients with dementia in Europe and identify the key factors influencing their Health-Related Quality of Life (HRQol). Methods This study used cross-sectional data from the Odense study; a Danish cohort of patients aged 65–84 living in Odense, Denmark. A total of 244 patients with mild to severe dementia were interviewed together with a caregiver about their health status and activities of daily living (ADL). Alzheimer's disease was diagnosed according to the NINCDS-ADRDA criteria for probable dementia. Vascular dementia and other types of dementia were diagnosed according to the DSM-IIIR criteria. Severity of dementia was defined by score intervals on the Mini Mental State Examination score: mild (MMSE 20–30), moderate (MMSE 10–19), and severe (MMSE 0–9). Based on the ADL information, the patients' dependency level was defined as either dependent or independent. Questions from the Odense Study were mapped into each of the five dimensions of the EQ-5D in order to assess patients' HRQol. Danish EQ-5D social tariffs were used to value patients' HRQol. A regression analysis of EQ-5D values was conducted with backward selection on gender, age, severity, ADL level and setting in order to determine the main factor influencing HRQoL. Results The EQ-5D weight in patients independent upon others in ADL was 0.641 (95% CI: [0.612–0.669]), and in those dependent upon others was 0.343 (95% CI: [0.251–0.436]). Conclusion Dependency upon others to perform ADL was the main factor affecting HRQoL. ==== Body Background Dementia is a chronic illness associated with a progressive loss of cognitive and intellectual abilities, such as memory, judgment and abstract thinking. Cognitive disabilities are those that impact an individual's ability to access, process, or remember information. People with profound cognitive disability will need assistance with nearly every aspect of daily living. The most visible manifestation of dementia is the progressive inability – proportional to the severity of the disease – to perform activities of daily living (ADL) and the subsequent loss of independence [1]. Progressive deterioration in the cognitive, functional and behavioural domains eventually brings patients to the later stages of dependency and, in most cases, to institutionalisation, which is linked to an increased need in caregiver assistance [2]. A patient's level of dependency is a global measurement reflecting a certain level of severity, resource consumption and Quality-of-Life (QoL) [3]. Measuring the QoL of patients suffering from dementia can take several forms. Firstly, QoL can be measured using generic health indices like the other disease specific measures. Recently several scales have been developed and validated specifically for dementia patients such as the Quality of Life-Alzheimer's Disease (QOL-AD). Another alternative to assess QoL is to use utility measurements, which are preference-based [4]. Preference-based measures evaluate the patient's preference for a health state instead of measuring the frequency and the severity of symptoms or disabilities. In order to use quality-adjusted life years (QALYs) as outcome measures for cost-effectiveness analyses, utility-weighted measures of Health-Related Quality-of-Life (HRQoL) are required. These attribute a single number to a health state using a common unit of measure allowing comparison between different strategies [5]. In general, however, HRQoL is not as broad a concept as QoL. One of the more reliable and newer tools used to measure HRQoL in a wide range of health conditions and treatments including dementia is the EQ-5D [6]. It is a generic measure designed to complement disease specific outcome measures and health characterises on five dimensions: mobility, self-care, ability to perform usual activities, pain, and anxiety/depression. It provides a descriptive health profile and a single index value for health status and, as such, it can be used to estimate utility in pharmacoeconomic evaluations of new pharmacological treatments. Health utilities have already been measured in AD in the US and Canada using the Health Utility Index (HUI) [7]. In the UK, the EuroQol instrument has been used to investigate whether HRQol data could be obtained from proxies, such as family caregivers [8]. In France, the EuroQol instrument has been administered to patients with dementia in order to determine the feasibility, reliability, and validity of the French version of the EuroQol instrument [9]. However, health utilities are not reported from the two latter studies. Furthermore, HRQol data are needed in order to carry out cost-effectiveness analysis for a Danish setting. Based on data collected alongside an epidemiological study conducted in Odense Denmark, we attempted to assess the health utilities of patients with dementia and identify the key factors influencing their HRQoL. Methods Population Data were derived from the Odense study, an epidemiological survey in which the objective was to estimate the prevalence and incidence of dementia in Denmark [10,11] In this study, a total of 244 patients with dementia agreed to participate in an interview accompanied by a relative or caregiver. The study was approved by the Scientific-Ethical Committee of the Counties of Funen and Vejle, Denmark, and by the Danish Data Protection Agency. Demented patients were classified by type of dementia and by severity of dementia. Alzheimer's disease (AD) was diagnosed according to the NINCDS-ADRDA criteria for probable dementia [12]. Vascular dementia and other types of dementia were diagnosed according to the DSM-IIIR criteria [13]. Severity of dementia was diagnosed according to the Clinical Dementia Rating (CDR) scale [14] and the Mini Mental State Examination (MMSE) [15]. The complete examination programme is described in Andersen, Lolk et al, 1997 [10]. Assessments All interviews were conducted by a certified nurse in the patient's home., Patient's and caregiver's socio-economic and socio-demographic status as well patients' health status and ADL were recorded. In the event that a relative was not present during the interview, a professional caregiver verified information provided by the patient. Each interview included the following information: - sociodemographic questions (age, gender, setting). - activity of daily living (ADL) questionnaire using 7 items describing patients' ability to perform physical activities (personal care, dressing, mobility and personal toiletry) and psychosocial activities (activities in the home and hobbies inside and outside of the home). Each activity was scored using a four-point Likert scale anchored at the ends with 1 = "Unable to perform the activity" and 4 = "Perform the activity without help from others". The physical ADL ranged between 4 (worst state) and 16 (best state), while the psychosocial ADL scored between 3 (worst state) and 12 (best state) [16]. - Questions mapped into each of the five dimensions of the EQ-5D: mobility, self-care, usual activities, pain/discomfort, and anxiety/depression as presented in the Table 1[17]. Table 1 Mapping questions from the Odense Study into the EQ-5D EQ-5D The Odense Study Mobility Mobility  1. I have no problems in walking around.  • Without help from others.  2. I have some problems in walking around.  • Needs some help from others.  • Needs help from others.  3. I am confined to bed.  • Unable to walk around without help. Personal care Personal care/dressing  1. I have no problems with self-care.  • Without help from others.  2. I have some problems with self-care.  • Needs some help from others.  • Needs help from others.  3. I am unable to wash or dress myself.  • Unable to wash or dress without help. Usual activities Hobbies in the home  1. I have no problems with performing my usual activities.  • Without help from others.  2. I have some problems with performing my usual activities.  • Needs some help from others.  • Needs help from others.  3. I an unable to perform my usual activities.  • Unable to perform hobbies in the home. Pain/discomfort Patient's assessment of own health status  1. I have no pain or discomfort.  • Very good.  2. I have moderate pain or discomfort.  • Good/fair.  3. I have extreme pain or discomfort.  • Poor. Anxiety/depression Patient's experience of emotional problems  1. I am not anxious or depressed.  • Never.  2. I am moderately anxious or depressed.  • Sometimes.  3. I am extremely anxious or depressed.  • Often. For the mobility dimension of the EuroQol instrument, we assumed that patients that were able to walk without assistance from others had no problems in performing this activity, whereas patients who were unable to walk unassisted were classified as confined to bed. Patients that needed help from others were classified as having some mobility problems. Two questions from the ADL instrument in the Odense Study were used to classify patients on the EuroQol personal care dimension. Patients that performed both activities without help from others were classified as having no problems on this dimension. Patients in need of help with either personal care or dressing or both were classified as having some problems. Only patients unable to wash and dress without help from others were classified as such on the EuroQol instrument. The patient's ability to carry out hobbies in the home was used as a proxy for their ability to perform usual activities. For the pain/discomfort dimension of the EuroQol instrument, it was assumed that patient assessment of their own health status covered this dimension. Therefore, if they found their health status to be very good they were classified as having no pain or discomfort. A good or fair assessment was categorised as having moderate pain or discomfort, whereas a poor assessment was assumed to correspond to extreme pain or discomfort. In the Odense Study, patients stated how often they experienced emotional problems, whereas the health state being described by the EuroQol instrument refers to the patient's health at the time of filling in the instrument. Thus, the questions in the Odense Study included an aspect of time that the EuroQol does not cover. To overcome this, it was assumed that the occurrence of emotional problems converts to the degree of anxiety or depressions. That is, patients that never experience emotional problems were assumed not to be anxious or depressed, whereas patients that sometimes or often experience problems converted to moderate or extreme anxiety or depression, respectively. The procedure of mapping returned a five-digit code, where the first digit referred to the patient's mobility level; the second to the patient's level on personal care; and so forth. This five-digit code described a health state for which we looked up the HRQoL utility weight in a table of EQ-5D tariffs. The EQ-5D tariffs take values between zero and one, where zero is the worst imaginable health status, and one is the best imaginable health status. We used Danish EQ-5D tariffs from a survey based on the time trade-off technique [18]. Patients classification Based on their cognitive and functional scores patients were classified by severity and dependency level. Severity Status The severity of a patient's dementia was defined by score intervals on the MMSE [15]. Those scoring ≥20 were considered as having mild dementia, while patients scoring between 10 and 19 were classified as having moderate dementia. Patients scoring ≤9 were classified as suffering from severe dementia. MMSE scores were not available for 30 patients (21 AD patients and 9 patients suffering from vascular dementia). In order to determine these patients degree of dementia we used the CDR score to classify them into the above three severity groups. Patients with a CDR score of 0.5 were classified as mild, patients with a CDR of 1 were classified as moderate and patients with a CDR of 2 to 3 were classified as severe [19]. Dependency Status Patients were classified by their ability to perform physical and psychosocial activities of daily living (ADL) This resulted in a classification of either dependent or independent [3]. A binary variable was based on a non-hierarchical cluster analysis [20]. Firstly, we identified possible initial seeds for the analysis. The seeds were identified from a cross table of the physical ADL score and the psychosocial ADL score. Combinations of the physical and psychosocial ADL scores with five or more observations were included in the cluster analysis as possible seeds. Secondly, we carried out the cluster analysis using the PROC FASTCLUS procedure in SAS 8.2 (SAS Institute Inc., Cary, NC, USA) in order to identify two clusters. One cluster included patients with low scores on both the physical and psychosocial ADL scales. As low scores on both instruments meant that patients required help from others in performing the activities in question, they were classified as "Dependent". The other cluster included patients with high scores on both ADL scales and they were classified as "Independent". As these were composite criteria, the characteristics of the two groups of dependency were analysed. Statistical Analysis After analysing the descriptive results of the EQ-5D scores, we performed a regression analysis of EQ-5D scores on sociodemographic and clinical characteristics. Sociodemographic characteristics included gender, age and setting (living in the community or institutionalised) and clinical characteristics took into account the level of severity (Mild, Moderate, and Severe), the type of dementia (AD, vascular or other) and ADL status (independent or dependent). The regression analysis was performed with backward selection (level 5%) in order to determine the main factors influencing QoL. Because of heteroscedasticity, we estimated the heteroscedasticity consistent covariance matrix, which was used to calculate test statistics for the coefficients. Observations with missing data were automatically excluded from the analyses. That is, observations with insufficient information to establish a EQ-5D weight, e.g. that information was lacking to determine a patient's mobility level on the EQ-5D instrument. Results Table 2 illustrates the characteristics of patients included in the Odense Study by type of dementia. Of the 244 patients, 164 (67%) suffered from Alzheimer's disease (AD) and 80 suffered from vascular or other types of dementia. On average, AD patients were 3.9 years younger (95% confidence interval: [2.5 – 5.3]) than patients suffering from vascular or other types of dementia and more AD patients lived in a nursing home (p = 0.03). The remainder of patient characteristics in Table 2 did not significantly differ between the two groups. Table 2 Background Characteristics of Patients in the Odense Study Patients suffering from Alzheimer's disease Patients suffering from vascular or other types of dementia All patients suffering from dementia Number of patients 164 (67.2%) 80 (32.8%) 244 Number of females 89 (54.3%) 33 (41.3%) 122 (50.0%) Mean (SD) age in years 79.4 (5.14) 75.5 (5.21) 78.1 (5.47) Mean (SD) MMSE score 20.6 (4.62) 21.6 (4.96) 21.0 (4.75) Severity of dementia  Mild (MMSE 20–30) 91 (55.5%) 49 (61.3%) 140 (57.4%)  Moderate (MMSE 10–19) 51 (31.1%) 23 (28.8%) 74 (30.4%)  Severe (MMSE 0–9) 22 (13.4%) 8 (10.0%) 30 (12.3%) Mean (SD) physical ADL score 14.1 (3.08) 13.7 (2.96) 14.0 (3.04) Mean (SD) psychosocial ADL score 9.3 (2.81) 9.0 (2.39) 9.2 (2.68) Living in the community 132 (80.5%) 73 (91.3%) 205 (84.0%) Tables 3 and 4 show the results from the cluster analysis, which was used to classify patients, according to their ADL status, as either independent or dependent in performing activities of daily living. Table 3 presents cluster characteristics. Of the 244 patients with dementia, 38 (16%) were classified as dependent, and 206 (84%) were classified as independent in the performance of ADL. Dependent patients were, on average, more severely stricken and institutionalised than independent patients. Table 3 Cluster Description Dependent Independent p-value Number of patients 38 (16%) 206 (85%) Number of females 17 (45%) 105 (51%) 0.4801 Mean (SD) age in years 79.3 (5) 77.9 (5) 0.1493 Number of AD patients 24 (63%) 140 (68%) 0.5622 Mean (SD) MMSE score 16.8 (6) 21.4 (4) <0.0001 Mean (SD) physical ADL score 7.9 (2) 15.1 (1) <0.0001 Mean (SD) psychosocial ADL score 4.7 (2) 10.1 (2) <0.0001 Number of patients living in the community 16 (42%) 189 (92%) <0.0001 Table 4 Number of Patients by Dependency Status and Type of Dementia ADL status Patients suffering from Alzheimer's disease Patients suffering from vascular or other types of dementia All Dependent 24 (15%) 14 (18%) 38 (16%) Independent 140 (85%) 66 (83%) 206 (84%) All 164 (100%) 80 (100%) 244 (100%) Table 4 shows the number of patients by dependency status and type of dementia. Due to of missing data, EQ-5D values were estimated for only 211 patients upon 244 demented patients. Results of comparison of EQ-5D value in different subgroups of patients were shown in Table 5. Patient QoL seems to decrease when severity and dependency increase as well as when patients are institutionalised. Table 5 Mean EQ-5D weights by patient subgroups N Mean SD Severity Subgroups Mild MMSE > 20 135 0.636 (0.2109) Moderate 9 < MMSE < 20 64 0.596 (0.2152) Severe MMSE < 10 12 0.486 (0.2191) Dependency Subgroups Independent 193 0.641 (0.1952) Dependent 18 0.343 0.2324) Setting subgroups Community 191 0.621 (0.2173) Institution 20 0.564 (0.1861) Table 6 shows the results of a regression analyses. The results from the full model that included all predictors, as well as results from the reduced model that included only significant predictors are presented. Table 6 Results of the Regression Analysis of EQ-5D TTO Tariff on Severity of Dementia, ADL Status and Setting (n = 211) Full model Reduced model Variable Coefficient Asymptotic standard error p-value Coefficient Asymptotic standard error p-value Constant 0.579 0.0371 <0.0001 0.641 0.0140 <0.0001 Gender (0 = male, 1 = female) 0.045 0.0266 0.0948 - - - Age 0.003 0.0029 0.3553 - - - Type of dementia (0 = Other, 1 = AD) 0.006 0.0327 0.8522 - - - Severity of dementia Moderate 0.043 0.0280 0.1258 - - - Severe -0.079 0.0534 0.1405 - - - Dependency status (0 = independent, 1 = dependent) -0.289 0.0534 <0.0001 -0.297 0.0551 <0.0001 Setting (0 = community, 1 = nursing home) 0.027 0.0368 0.4672 - - - R2 0.1776 0.1502 Patients dependent upon others to perform activities of daily living clearly had a lower QoL than independent patients. The QoL of independent patients suffering from dementia as assessed by their capacity to perform activities of daily living was 0.641 (95% confidence interval: [0.612 – 0.669]) whereas the QoL of dependent patients for the same assessment was 0.343 (95% CI: [0.251 – 0.436]). Severity of dementia and setting has no statistically significant impact on QoL. Discussion For the first time, this study provides health utilities for patients with dementia in Denmark. This study has shown that the factor that most affects the HRQoL of a patient with dementia is their dependency status as defined based by their ability to perform activities of daily living. The type of dementia doesn't seem to have a great an influence on patient's HRQoL, and severity does not appear to discriminate significantly between health utilities. However, due to missing data – particularly among patients with severe dementia – caution must be exercised when interpreting the results. In the utility results previously measured by Neumann et al. [7] using the Health Utility Index (HUI), AD patients' utilities decreased significantly with their severity levels. However, this could be explained by the fact that the EQ-5D does not consider cognition as a separate attribute, unlike the HUI scales. Despite this difference, results obtained with the HUI and the EQ-5D instruments were within the same range. As the EQ-5D values were estimated based on mapped questions, it raises the possibility of quotation bias and goodness of fit. Also, both patients and caregivers answered questions. Yet, with AD – and especially when patients are severely demented – it is impossible to collect non-proxy measurements in the later stages. The same methodology was performed in previous evaluations without knowing the impact of the difference between caregivers' and patients' perceptions. A particular strength of this study was that all data have been collected in conjunction with an epidemiological study wherein patients with dementia had been examined carefully and dementia criteria were explicitly stated. Conclusion Measuring HRQoL is as important as measuring disease severity, progression, symptom response, cognition and behavioural disturbance when assessing the impact of disease and determining proper intervention in the treatment and management of dementia. However, HRQoL is difficult to assess in a disease such as dementia for which patients suffer from cognitive disabilities. Based on study results and as previously shown by Kurz et al. [3], dependency level greatly influences patients' HRQoL and, when viewed as a global measure, reflects a certain level of HRQoL. Determining dependency levels could be considered as an indirect evaluation of HRQol. Other studies with disease specific questionnaires such as QoL-AD are needed to confirm these findings. Abbreviations ADL Activities of Daily Living EQ-5D EuroQol – Five Dimension Scale EuroQol EuroQol Scale DSM-IIIR Diagnostic and Statistical Manual of Mental Disorders 3rd edition HRQol Health-Related Quality of Life MMSE Mini Mental State Examination QoL Quality of Life SAS Statistical Analytical Software Authors Contributions CKA is principal author and responsible for quality control. K W-J provided mapping of the questions from the Odense Study into the five dimensions of the EQ-5D. AL and KA provided data analysis and data analyses of the Odense database. P K-S provided access to the Odense database. ==== Refs Potkin SG The ABC of Alzheimer's disease: ADL and improving day-to-day functioning of patients Int Psychogeriatr 2002 14 7 26 12636178 10.1017/S1041610203008640 Bullock R Hammond G Realistic expectations: the management of severe Alzheimer disease Alzheimer Dis Assoc Disord 2003 17 S80 85 14512821 10.1097/00002093-200307003-00004 Kurz X Scuvee-Moreau J Rive B Dresse A A new approach to the qualitative evaluation of functional disability in dementia Int J Geriatr Psychiatry 2003 18 1050 1055 14618558 10.1002/gps.1009 Bennett KJ Torrance GW Spilker B Measuring heralth state preferences and utilities: rating scale, time trade-off, and standard gamble techniques Quality of Life and Pharmacoeconomics in Clinical Trials 1996 2 Philadelphia: Lippincott-Raven 253 265 Dolan P Drummond M, McGuire A Output measures and valuation in health In Economic evaluation in health Care Merging theory with Practice 2001 Oxford University Press Brooks R EuroQol: the current state of play Health Policy 1996 37 53 72 10158943 10.1016/0168-8510(96)00822-6 Neumann PJ Sandberg EA Araki SS Kuntz KM Feeny D Weinstein MC A comparison of HUI2 and HUI3 utility scores in Alzheimer's disease Med Decis Making 2000 20 413 422 11059474 Coucill W Bryan S Bentham P Buckley A Laight A EQ-5D in patients with dementia: an investigation of inter-rater agreement Med Care 2001 39 760 771 11468496 10.1097/00005650-200108000-00003 Ankri J Beaufils B Novella JL Morrone I Guillemin F Jolly D Ploton L Blanchard F Use of the EQ-5D among patients suffering from dementia J Clin Epidemiol 2003 56 1055 1063 14614996 10.1016/S0895-4356(03)00175-6 Andersen K Lolk A Nielsen H Andersen J Olsen C Kragh-Sorensen P Prevalence of very mild to severe dementia in Denmark Acta Neurol Scand 1997 96 82 87 9272182 Andersen K Nielsen H Lolk A Andersen J Becker I Kragh-Sørensen P Incidence of very mild to severe dementia and Alzheimer's disease in Denmark Neurology 1999 52 85 90 9921853 McKhann G Drachman D Folstein M Katzman R Price D Stadlan EM Clinical diagnosis of Alzheimer's disease: report of the NINCDS-ADRDA Work Group under the auspices of Department of Health and Human Services Task Force on Alzheimer's Disease Neurology 1984 34 939 944 6610841 American Psychiatric Association Diagnostic and statistical manual of mental disorders 1987 3 Washington DC. American Psychiatric Association Berg L Clinical Dementia Rating (CDR) Psychopharmacol Bull 1988 24 637 639 3249765 Folstein MF Folstein SE McHugh PR "Mini-mental state". A practical method for grading the cognitive state of patients for the clinician J Psychiatr Res 1975 12 189 198 1202204 10.1016/0022-3956(75)90026-6 Salling AL Stimulation af patients aktivitie og udvikling (Stimulation of patient's activity and development. In Danish) Doctoral thesis 1990 Copenhagen. Danish Nurses Organisation Ullerup K An economic evaluation of donepezil (Aricept) for the treatment of Alzheimer dementia using a Markov cohort simulations model (In Danish) 1999 Institute of Public Health Working Paper, Odense University 1 Pedersen KM Wittrup-Jensen KU Brooks R Gudex C Valuation of health. The theory of quality adjusted life years and a Danish application (In Danish) 2003 University of SOuthern Denmark Press Reisberg B Franssen E Souren L Kenowski S Auer S Wimo A, Jönsson B, Karlsson G, Winblad B Severity scales Health Economics of Dementia 1998 Wiley: Chichester 327 357 Sharma A Applied Multivariate Techniques 1996 John Wiley & Sons

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Health Qual Life Outcomes. 2004 Sep 21; 2:52

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Health Qual Life Outcomes

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==== Front Health Qual Life OutcomesHealth and Quality of Life Outcomes1477-7525BioMed Central London 1477-7525-2-501537738410.1186/1477-7525-2-50ReviewCaring for family members with chronic physical illness: A critical review of caregiver literature Lim Jung-won [email protected] Brad [email protected] School of Social Work, University of Southern California, 669 MRF Building, West 34th Street, & 102W, Los Angeles, CA 90089-0411, USA2004 17 9 2004 2 50 50 29 1 2004 17 9 2004 Copyright © 2004 Lim and Zebrack; licensee BioMed Central Ltd.2004Lim and Zebrack; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. This article reviews 19 studies (1987–2004) on quality of life for family caregivers helping those with chronic physical illness. Here we explore the concepts of and instruments used to measure caregivers' quality of life. We were particularly interested in understanding stress-related variables and documenting factors influencing quality of life based on family stress theory. Findings show that various positive and negative terms equated with quality of life were used to measure them. Results indicate that stress-related variables as possible predictors influencing caregivers' quality of life include: patient and caregiver characteristics, stressors, stress appraisal, stress coping methods, and social support. Our recommendations touch upon applying theory for intervention, developing measurement, making operable the concepts for measuring, and the need for longitudinal and comprehensive study. Family caregiversFamily stress theoryQuality of life ==== Body Background Recent reforms in U.S. health care systems mean that individuals with long-term, complex health problems are being cared for at home by family members [1]. Specifically, changes in medical practice resulting in shorter impatient hospital stays and the search for outpatient substitutes such as home-based care have brought cost savings to both hospitals and consumers. A study reported that home-based care reduced the cost per patient treated by 44% overall compared with hospital-based treatment [2]. Despite such cost-effectiveness, this trend means that an increased financial, physical, and emotional responsibility falls upon family members who care for a person with chronic physical illness [3]. Now, more than 25 million Americans serve as family caregivers for that population. Their work, if it were part of the market economy, would have an economic value of nearly $257 billion in 2000, which is equal to 20 % of the total for all health-care expenditures [4]. For example, family caregivers are more frequently called upon to use daunting and complex equipment at home. They also deal with extensive coordination of care, including symptom management, disability, mobility, and dressings. In the face of these increasing challenges and responsibilities, caregivers often feel tired, isolated, and overwhelmed, because they lack support, training, information and a sympathetic ear. Furthermore, some family caregivers who are employed report missing work, taking personal days, and quitting or retiring early to provide care [5]. Thus, chronic illness affects not only the lives of those suffering from disease but also those of family members who care for them. Attending to the impacts of chronic illness on family members is important because the physical and emotional health of family caregivers has the potential to influence the health, welfare and successful rehabilitation of persons with such chronic illness [6]. Existing studies document how caring for chronically ill family members or significant others at home influences multiple aspects of caregivers' lives. These effects are physical, psychological and social and may include worsened physical health, impaired social and family life, and increased stress, anxiety and depression ([7-9]). Placing these conditions experienced by caregivers in the context of family stress theory and quality of life advances our understanding of caregivers' experiences by examining how multiple aspects of caregivers' lives – their quality of life – may be partly influenced by other existing environmental stressors, stress appraisal, coping methods and social support. The importance of family stress theory in studying normative family transitions and adaptation to major life changes and illness is based on the central role that family strengths and capabilities play in understanding and explaining psychological and behavioral outcomes [10]. In family stress theory, the family is "viewed as encountering hardships and changes as an inevitable part of family life over the life cycle" [11]. Given that caring for a seriously ill family member arguably is (or is quickly becoming) an inevitable part of family life, current research has begun examining family stressors, stress appraisal, coping methods, and social support as they influence QOL outcomes, or as they attenuate the effects of other patient or caregiver characteristics or health-related variables on caregiver QOL ([12-15]). Quality of life is a construct that encompasses health and functioning, socioeconomic status, psychological, emotional and spiritual aspects, and family [16]. Ferrell [17] confirmed the influence of such multidimensional aspects on the QOL for breast cancer survivors. Wyatt & Friedman [18] also identified concerns related to QOL in those with chronic physical illness, and suggested that considering multidimensional aspects of QOL for them is essential. Although some scholars have different points of view regarding the dimensions of QOL, most researchers generally agree that QOL is multidimensional, subjective, and relating to a state of physical, psychological, social, spiritual well-being [19]. However, QOL for caregivers includes more aspects such as burden and family functioning [20]. Further, some researchers use QOL together with life satisfaction, adaptation, health, and distress ([12,13,21-23]). Such comprehensive consideration has led to an awareness of QOL as a broader and more appropriate concept for determining how caregiving affects family members [1]. Therefore, it is meaningful to address QOL comprehensively in this paper. This work will also consider 1) the lack of consensus on concepts like stressors, stress appraisal, coping methods, and social support, and 2) inconsistent results regarding factors influencing caregivers' QOL. To address the lack of review papers on this topic as well as inconsistent results among empirical studies, we undertook a thorough review of the literature. We were particularly interested in research that reported psychosocial or QOL outcomes and accounted for family stress variables as correlates or explanatory variables. The purpose of this paper is twofold: to review caregiver QOL impacts (loosely-defined) organized around family stress theory, and to discuss the implications of findings for future research (i.e., measurement, hypothesis testing, refinement of concepts and constructs). Methods Only published peer reviewed research articles were included in this review. Several methods were applied in searching the literature. First, articles were limited to those published in English between January 1, 1987, and January 31, 2004. Second, participants were caregivers of patients over 21 years of age with chronic physical illnesses. Third, a study was excluded if patients were receiving hospice care. Fourth, a computer search was conducted in February 2004 to review the databases of MedLine and PubMed by using the following key words: caregivers, caregiving, chronic illness, quality of life, adjustment, life satisfaction, burden, distress, and family stress theory. The terms "health," "stress," and "coping" were also used for study retrieval. Databases also were searched for review articles published during the same time period. Additional sources for empirical reports included reference lists from published studies. Of the more than 220 articles identified, there were only 16 empirical studies based on family stress theory that focused on the caregivers of patients with chronic physical illness and measured the QOL of caregivers. Two additional articles were identified from searching review articles. Finally, one more article was found in the references of these 18 articles. Thus, a total of 19 articles are included in this review (Appendix 1 [See additional file]). Results Concepts and Instruments Used to Measure Caregiver Quality of Life Reviewed studies used diverse outcomes and models to indicate caregivers' QOL. Psychosocial outcomes falling with varied dimensions of QOL and measured included adaptation, mental health, life satisfaction, stress, emotional distress, health, caregiver burden, and depression. Given the variety of terms equated with QOL, both positive and negative terms were used to measure the QOL. Three studies ([12,13,21]) used positive terms such as 'adaptation' and 'life satisfaction.' Seven studies ([15,22-27]) measured QOL using negative terms such as 'caregiving burden,' 'depression,' 'stress outcomes,' and 'emotional distress.' Other research used neutral terms including 'health,' 'mental health outcomes,' and 'quality of life.' Of 19 reviewed articles, only one used a single measure of QOL per se: the Caregiver Quality of Life Index containing items related to physical, emotional, social, and financial wellbeing [28]. Eight papers reported use of a single instrument based either on a modified patient questionnaire or on other concepts (burden, general health, and mood status) indicating various QOL dimensions. Ten reported studies administered a composite measurement combining some concepts. Table 1 shows diverse outcomes and instruments used to measure caregivers' QOL. Table 1 Concepts and measurements of quality of life Outcome (QOL) Single Measurement Composite Measure Quality of life 1. Caregiver quality of life index – cancer [28] 1.1) Stress, 2) Anxiety, 3) Depression, 4) Health [7] 2. Multidimensional quality of life scale – cancer [33] 2.1) Physical health, 2) Emotional health, 3. Quality of life index [32] 3) Use of psychotropic drugs, 4) Caregivers' social life, 5) Financial status [30] 1.1) Depression, 2) Life satisfaction, 3) Health [12] Adaptational outcome (Adaptation) 2.1) Caregiver's level of life satisfaction, 2) Depression 3) Subjective symptoms of stress [13] Mental health outcome 1.1) Depression, 2) Quality of life [29] Life satisfaction 1. 8-item, open-ended questionnaire [21] Stress outcome (stress response, distress) 1. Brief symptom inventory [34] 1.1) Yielding of role, 2) Physical health, 3) Anxiety [24] 2.1) Anxiety, 2) Depression, 3) Stress [25] 3.1) Burden, 2) Depression, 3) Anxiety [26] Emotional (Psychological) distress 1. Profile of mood states – short form [22] 2. General health questionnaire [23] Health 1. the Medical Outcome Study 36-item 1.1) Caregiver mental health, Short Form Health Survey [31] 2) Caregiver physical health [14] Caregiving Burden 1. Zarit burden scale 27 Depression 1.1) depression, 2) strain [15] Factors Influencing Caregiver Quality of Life Studies of variables influencing caregivers' QOL are summarized in Table 2. Variables examined are categorized here into patient characteristics, caregiver characteristics, stressors, stress appraisal, stress coping methods, and social support. Table 2 Factors influencing caregiver quality of life Predictors Significant Mediating variables Quality of life Significant Not found Patient characteristics Performance status [7], [24], [26], [28], [34] Age [7] [15] Gender [15] Depression [7], [29] The kind of illness [30] Pain / symptoms (severity of illness) [29, 32] Caregiver characteristics Age [7], [24] [15] Gender [7], [15] [24] Physical disability [7] Income [29], [32] Initial quality of life [29] Educational level [22], [28] Health problem [26] Depression [29] Anger [26] Anxiety [26] STRESSOR Primary Objective context [13] [14] Caregiving demands [13], [22] Patient impairment [12], [15] Duration of care [29], [25] Intensity of care [29], [25] ADL Dependency [7], [24], [26] [29] Stress types [25] Caregiver overload [24] Recurrence [15] Problem behavior [24] Secondary Subjective context [13] Caregiving demands [13] Role change [21] [29] Responsibility [21] Caregiver experiences [29] Life style interference [22] Stress Appraisal Appraisal [12], [14] [12], [14], [23], [31] Perceived control [13] Differences in the perception (pt & caregiver) [33] Stress Coping Methods Coping responses [12], [13], [25], [14], [15] [12], [14], [26], [31] [24], [32] Social support Perceived adequacy of social support [14], [25], [34] [14], [21], [26], [31] Social life and social network [12], [15], [12], [7] Family life (Quality of relationship and Marital adjustment) [7], [29], [32] Loneliness [7] Resources [26] Formal support [24] Patient characteristics Nine articles examined the association between patient characteristics (including performance status, age, gender, depression, type of illness, pain, and symptoms) and caregiver QOL. Generally, there were significant correlations between caregiver QOL and the patient's physical and emotional characteristics as related to the illness. Seven studies ([7,29,24,26,28,31]) found that the patient's performance status, type of illness, and depression were related to the caregiver's QOL. However, two ([29,32]) showed that pain and physical symptoms were not related to the caregiver QOL. Two other articles investigated the relationship between patient age and caregiver QOL, but there were no consistent results. Schumacher, Dodd, & Paul [15] reported a relationship between patient gender and caregiver QOL, with caregivers of male patients reporting higher levels of strain. Caregiver characteristics Eight of 19 articles examined the relationship between caregiver characteristics and QOL. A caregiver's age, gender, physical disability, income, initial QOL, educational level, health problem, depression, anger, and anxiety were addressed as characteristics. Three studies examined the association between a caregiver's age and QOL, with two ([7,24]) reporting that older age of the caregiver is associated with increased stress. Two of three articles investigating the relationship between a caregiver's gender and QOL reported that females were more likely to be depressed ([7,15]). In addition, the caregiver's physical disability, income, initial quality of life, educational level, health problem, depression, anger, and anxiety were consistently found significant in reducing their QOL. Stressors Eleven articles investigated the relationship between stressors and caregiver QOL, but they did not show consistent results. To varying degrees, caregiving demands, patient impairment, the duration and intensity of care, ADL (activities of daily living) dependency, stressor types, caregiver overload, how much assistance is given the caregiver, recurrence of illness and problem behavior in the patient were identified as primary stressors. Secondary stressors were caregiving demands, role change, responsibility, caregiver experience, and life-style interference. Seven studies ([7,12,13,15,24-26]) found that primary stressors were related to reductions in caregiver QOL. However, five articles ([13,14,22,29,25]) did not find any association between primary stressors and QOL. Two ([13,21]) found a significant relationship between secondary stressors and lower QOL. In contrast, another [29] found that secondary stressors were unrelated. Vedhara, Shanks, Anderson, & Lightman [25] investigated the relative importance of stressor types on stress outcomes. Their study demonstrated that stressor types (e.g. daily hassles, caregiving-specific stressors, and life events) determined the stress outcomes, with the proportion of variance accounted for by the stressor indices (which ranged from 20% to 53%). Winslow [24] found that caregiver overload was positively related to caregiver anxiety. The hypothesized direct effect of a care receiver's problem behavior on the caregiver's yielding up his/her role was also supported by the findings. That is, higher levels of a care receiver's problem behavior as the primary stressor were more likely to lead to patient institutionalization. Examining the stress process in family caregivers of persons receiving chemotherapy, Schumacher et al. [15] corroborated that modest but significant negative relationships were found between caregiver strain and patient functional status as well as disease recurrence. Haley, Levine, Brown, & Bartolucci [12] also found a significant positive correlation between patient impairment on the IADL (instrumental activities of daily living) and caregiver depression scores. Wallhagen [13] found that the subjective context and subjective demands of caregiving as well as the objective context were associated with the caregiver's adaptation, including level of life satisfaction, depression, and subjective symptoms of stress. Aspects of the caregiving situation assessed by both the objective and subjective context indices included caregiver competence, social resources, the physical environment, and socioeconomic status or perceived financial adequacy. The objective and subjective demands of caregiving included caregiving responsibilities, instrumental activities of daily living, and personal demands. Examining determinants of caregiver outcomes through a longitudinal study, Nijboer, Trienmstar, Tempelaar, Sanderman, & Van den [29] considered various stressors as both mediating and predicting variables. Some stressors, including duration of care, intensity of care, ADL dependency of patients, and role change, were not related to caregiver QOL. On the other hand, caregiver experience (i.e. disrupted schedule, financial problems, lack of family support, loss of physical strength, and self-esteem) was a significant mediating variable affecting the relationship between caregiver, patient, care characteristics and a caregiver's mental health. They found that all caregiver experiences were related in the expected direction to the caregiver's level of depression. With regard to a caregiver's QOL, only the loss of physical strength and its impact on the caregiver's self-esteem appeared to be related significantly, also in the expected direction. Negative caregiver experiences were associated with low income, living with the patient exclusively, distressed relationship, high level of patient dependency, and high involvement in caregiving tasks. Cameron, Franche, Cheung, & Stewart [22] specifically examined the mediation of lifestyle interference as the secondary stressor between the amount of care provided and emotional distress. The results supported that lifestyle interference mediated the relationship between caregiving assistance and overall mood disturbance. However, they did not find the relationship between caregiver QOL and caregiving demands to be the primary stressor. Coping methods Eight reports examined the relationship between coping methods and caregiver QOL. Five studies ([12-15,25]) found that stress coping methods operated as a mediating variable affecting the relationship between predictor variables and caregiver QOL. Four studies ([9,12,14,26]) concluded that coping methods significantly predicted the caregiver's QOL. Two studies ([12,14]) showed that stress coping methods operated as both predictor and mediating variables, where two others ([32,24]) found no such significant relationship. Haley et al. [12] found that coping responses were significant mediators and predictors of all three outcome variables: depression, life satisfaction, and self-related health. They showed that when caregivers used logical analysis and problem-solving coping strategies, they enjoyed a higher QOL. Information seeking was related to a better health outcome, and affective regulation was related to better outcomes in health and life satisfaction. Emotional discharge was actually related to higher levels of caregiver depression. Goode, Haley, Roth, & Ford [14] found that initially higher proportions of approach versus avoidance coping predicted better health over time. This suggests that relatively greater use of approach coping may help optimize caregivers' health over time. They also examined mediated effects on physical and mental health outcomes. Changes in approach coping percentage were directly related to changes in depression for self-care stressors and memory and behavior problems, indicating that as relative levels of approach coping increase, depression decreases. Of the four coping strategies, Wallhagen [13] discovered that, only wishful thinking mediated the perceived control and outcome variables. That is, wishful thinking coping behavior had a negative relationship with all adaptation variables. The higher levels of perceived control also reported using wishful thinking behavior. Schumacher et al. [24] studied how coping mediated the relationship between strain and depression. A modest but significant negative relationship was found between caregiver strain and coping efficacy. Predictably, caregiver depression was also significantly related to coping efficacy. That is, caregivers who experienced less coping efficacy were more depressed. Stress appraisal Six articles examined the association between stress appraisal and caregiver QOL. Two ([12,14]) demonstrated that stress appraisal operated as both a predictor and mediating variable. Four articles ([33,13,23,9]) investigated how stress appraisal was associated with caregiver QOL as a predictor variable only. Thus, most research investigating the relation between stress appraisal and caregiver QOL showed their significant relationship. Haley et al. [12] found that measures of caregiver appraisal were consistently related to caregiver outcome and operated as mediator and predictor variables. When caregivers appraised patients' behavioral problems and disability as highly stressful and appraised themselves as lacking in self-efficacy, they experienced higher levels of caregiver depression. Goode et al. [14] found that changes in one domain of caregiving stress, memory and behavior problems produced changes in stressfulness appraisals as a mediating process. Changes in stressfulness appraisals were then positively associated with changes in depression and health symptoms. These results may suggest that the appraised stressfulness of memory and behavior problems mediates the relation between these problems and caregiver health outcomes. In contrast, Wallhagen [13] did not validate the hypothesis that perceived control mediates the objective and subjective aspects of caregiving and caregiver adaptation. However, he found that perceived control had a direct relationship with life satisfaction and depression. Thus, perceived control was associated with a higher level of life satisfaction and lower levels of depression and subjective symptoms of stress. In addition to its direct relationships with the outcome variables, perceived control also had an indirect relationship with both life satisfaction and depression through its direct connection to wishful thinking. Miaskowski, et al. [33] investigated whether differences in patients' and family caregivers' perceptions of pain experience influence patient and caregiver outcomes. In terms of the QOL measures, significant differences were found for psychological well-being, interpersonal well-being, nutrition and the total QOL score, with reporting lower scores for patients whose pain intensity scores were non-congruent with their family caregivers. These data suggest that non-congruence in the patient's and the caregiver's perceptions of the patient's pain may result in a poorer QOL score for the patient. Social support A majority of the articles addressed social support as a mediator and predictor variable. Six articles ([12,14,15,24,25,34]) reported that social support mediated both predictor and outcome variables. Seven ([7,12,14,21,29,26,32]) showed a direct relationship between social support and caregiver QOL. Specific items of social support addressed here were perceived adequacy of social support, social life, social network, family life (quality of relationship and marital adjustment), loneliness, resources, and formal support. Examining predictors of adaptational outcome among dementia caregivers, Haley et al. [12] found that social support and activity were significant predictor variables of caregiver outcome. They discovered that higher levels of social network size, activity, and satisfaction with network were related to better outcomes, particularly life satisfaction and health. Social support and activity also mediated the stressors and caregiver outcomes, including depression, life satisfaction, and health. In the study conducted by Schumacher et al. [15], caregiver depression was significantly related to perceived adequacy of social support. Predictably, caregivers with less social support were more depressed. Social support was found to mediate the relationship between functional status and depression. Goode et al. [14] found that initial levels of social support also protected physical health changes over time. Those caregivers who reported higher initial levels of social support resources actually showed improved health over time. Initial satisfaction with level of social support provided the same beneficial effect in preventing physical health problems or promoting health improvements. Vedhara et al. [25] assessed the predictive stability of psychosocial mediators over a six month period. The results of the regression analyses revealed stable predictive relationships between the mediator factors and the stress response indices. Anxiety was predicted by seeking social support at six months. Ergh, Rapport, Coleman, & Hanks [34] also examined predictors of caregiver distress among 60 caregivers of patients with a traumatic brain injury. In this study, social support showed a direct relationship to family functioning. As well, social support powerfully moderated the caregiver's psychological distress. That is, in the absence of adequate social support, caregiver distress increased with performance status in care recipients. On the other hand, in studying how formal supports affect stress outcomes in family caregivers of Alzheimer's patients, Winslow [24] considered formal supports as mediators. He found that no formal support mediated primary stressors and caregiver characteristics in the directions hypothesized. Others ([21,26,7,29,32]) showed that social support was a predictor variable affecting caregiver QOL. Discussion Nineteen studies have been reviewed to understand stress-related variables and to examine how each factor influences a caregiver's QOL. Factors were identified from the literature based on family stress theory and included patient characteristics, caregiver characteristics, stressors, stress appraisal, coping methods, and social support. In this section, we will discuss method as well as family stress theory based on the literature reviewed. Theoretical and Clinical Considerations Family stress theory provides a way of viewing the family's efforts over time to adapt to multiple stressors through using family resources and perceptual factors as a coping process aimed at achieving family balance [35]. A family situation addresses multiple changes and demands simultaneously, not single stressors. Secondary stressors, such as role change, responsibility, and caregiving demands, emerge from the primary stressors and these strains often may be difficult to resolve. They become instead a source of chronic strain. Chronic strain causes a build-up of unresolved stressors and contributes to undesirable characteristics in the family environment [36]. Although most of the reviewed papers addressed stressors as factors influencing caregiver QOL, the focus was on primary rather than secondary stressors. As a result, researchers may be overlooking stressors without considering chronic illness as a source of chronic strain changing the family system. Therefore, it is imperative to consider secondary as well as primary factors for understanding stress on caregivers of patients with chronic physical illnesses. Resources for the family are the psychological, social, interpersonal, and material characteristics of individual family members, of the family unit, and of the community. That meets family demands and needs. When families have insufficient resources, their needs and demands are not adequately met. As a result, this contributes to increased conflict in the family environment [35]. Although this theory emphasizes resources, the focus on resources is remarkably broad [26]. For instance, some works ([13,14,23,26]) have addressed social support and coping as components of resources. A lack of clarity is likely to bring inconsistent research results and thus may cause problems of generalization. Instead we need to clearly prescribe each concept. Family stress theory suggests that stress may be perceived or experienced both as a crisis and a challenge to be overcome [10]. The family may perceive the stressor as having caused a crisis. Or the family may accept it and see it as a challenge. Perception of the stressor as a challenge suggests that, over time, families engaged in a constructive effort to manage the stressor will redefine their total situation [35]. This explanation regarding stress perception may artificially dichotomize the stressor as a crisis or a challenge. However, perception is so subjective that each family member may differently interpret an event occurring within a family system. The subjective characteristics of perception may impede measuring the cognitive patterns of each person. Even if caregivers perceive stressors as a challenge and have volition to cope with stressors, we must consider the possibility that recovery from crisis may not occur among caregivers of patients with chronic illness. In other words, many caregivers may have financial burdens caused by continuous treatment and tests. Additionally, although personal characteristics change, environmental characteristics of patients and caregivers, such as social prejudice against the chronically ill, may not change. As shown by inconsistent results from previous research findings, the assumption that caregivers recover from the crisis and achieve some level of adaptation needs to be re-considered. Family stress theory also emphasizes the need for intervention following clear assessment. In the Resiliency Model [37], practitioner interventions were directed at restoring the balance between family stressors and resources. This can be the first intervention to assess whether family behavior is adaptive or maladaptive. Robinson [38] maintains that in collaboration with the family it is necessary to develop a plan, for managing stress. The plan includes the following points: (a) commitment of all family members to work on the problems; (b) inclusion of all past successful coping strategies; (c) brainstorming of all possible strategies; (d) use of strategies that are flexible, reality-oriented, and open to expression of emotions; and (e) discussion of possible outcomes of all strategies. Figley & McCubbin [36] suggested that a family crisis should be an opportunity for family interventionists to promote family well-being. Social workers can not only make use of the community and its programs and services in support of families under stress, but also more importantly use the situation to improve the family's problem-solving skills, coping repertoire, and overall interpersonal relationships. Besides those interventions mentioned above, the reviewed papers suggested other interventions for caregivers and health care professionals on the basis of family stress theory. These interventions emphasized the need for health professionals' involvement in the ongoing care of cancer patients and their families to monitor increasing demands [29], the importance of educating family members in effective ways to communicate [33], and the need for long-term counseling and early involvement of caregivers [6]. Based on strategies mentioned above, social workers can use individual therapy, family therapy, education, and problem-solving programs as interventions for caregivers of patients with chronic illness. When social workers so apply family stress theory, they must maximize resources because resources can positively influence coping, perception, and adaptation. After finding resources, it may be helpful to educate caregivers, focusing on their perceptions, problems-solving, and coping skills. In family stress theory, resources, perception, and coping mutually interact, and thus may influence QOL among family members. As a result, synergistic interventions that integrate all factors may be more effective. Finally, family interventions must consider longer-term effects or the sustained effect, because coping methods and perception may change over time, depending on circumstances. Methodological Considerations Some studies of caregivers and family stress theory address various outcome variables, including QOL, adaptation, life satisfaction, emotional distress, and caregiving burden ([12,13,28]). As well, most research uses concepts such as stress, perception, coping, and social support as predictor or mediating variables. These concepts are so subjective that they are hard to define and measure using concrete methods. For example, Schumacher et al [15]'s study measured both perceived efficacy of coping strategies and perceived adequacy of social support with only one-item indicators. Thus, at first, operationalization of concepts is required to reduce the gap between theory and research and clear the way for measure. As well, specific instruments to measure the complex and multidimensional phenomena need to be developed. For a number of reasons, we need longitudinal studies in research applying family stress theory to caregivers of the chronically ill. Otherwise we cannot understand longer-term or sustained effects. First, although a patient with chronic illness may have completed treatment, recurrence of the illness is possible. Second, coping methods and appraisal may change depending on the circumstances, because family stress theory is dynamic and influenced by both internal and external environments. A longitudinal study may see the attrition of participants over time. Due to the nature of chronic illness, many patients may have died or experienced recurrence at the follow-up evaluation. Therefore, caregivers of these patients need to be followed through the end to assess predictors and consequences of patient death and recurrence of illness. Family stress theory is very complex and comprehensive. As a result, it is difficult to conduct research using the full model. Examples of this complexity include interactions among various factors, interactions and transactions over time, individual dynamics within the family system, and the balance between family demands and the family. Considering these multidimensional aspects, it would take much hard work to test the full model of family stress theory. However, we need to try. Otherwise, we may never understand the complex relation between individual, family, and environment. Supplementary Material Additional File 1 Appendix 1: Review of empirical findings on quality of life of family caregivers. Author, topic, significant predictor variables, mediating variables, outcome variables, measurement, and intervention of 19 reviewed articles are shown in detail. Click here for file ==== Refs Canam C Acorn S Quality of life for family caregivers of people with chronic health problems. Rehabilitation Nursing 1999 24 192 196 10754909 Moalosi G Floyd K, Phatshwane J, Moeti T, Binkin N, Kenyon T. Cost-effectiveness of home-based care versus hospital care for chronically ill tuberculosis patients, Francistown, Botswana Int J Tuberc Lung Dis 2003 7 S80 85 12971658 Rees J O'Boyle C, MacDonagh R Quality of life: Impact of chronic illness on the partner Journal of the Royal Society of Medicine 2001 94 563 566 11691892 Services U.S. Department of Health and Human Family Caregivers: Our Heros on the Frontlines of Long-Term Care 2003 E Rorer United hospital fund study finds changes in nation's health care system place daunting new burdens on 25 million unpaid family caregivers who provide nearly $ 200 billion in essential services New York 1998 New York Han B Haley WE Family caregiving for patients with stroke: Review and analysis Stroke 1999 30 1478 1485 10390326 Jones DAS Peters TJ Caring for elderly dependents: Effects on the carer's quality of life Age Ageing 1992 21 421 428 1471580 Northhouse L Swain C Adjustment of patients and husbands to the initial impact of breast cancer Nursing Research 1987 36 221 225 3648695 Spiegel K Leproult R, Van Cauter E Impact of sleep debt on metabolic and endocrine function Lancet 1999 354 1435 1439 10543671 10.1016/S0140-6736(99)01376-8 Figley CR Treating stress in families 1989 New York, Brunner/Mazel McCubbin M McCubbin H Families coping with illness: The resiliency model of family stress, adjustment, and adaptation 1993 Madison, Wisconsin, University of Wisconsin-Madison Haley WE Levine EG, Brown SL, Bartolucci AA Stress, appraisal, coping, and social support as predictors of adaptational outcome among dementia caregivers Psychology and Aging 1987 2 323 330 3268224 10.1037//0882-7974.2.4.323 Wallhagen MI Perceived control and adaptation in elder caregivers: development of an explanatory model Int J Aging Hum Dev 1992 36 219 237 1302245 Goode KT Haley WE, Roth DL, Ford GR Predicting longitudinal changes in caregiver physical and mental health: A stress process model Health Psychology 1998 17 190 198 9548710 10.1037//0278-6133.17.2.190 Schumacher KL Dodd MJ, Paul SM The stress process in family caregivers of persons receiving chemotherapy Research in Nursing & Health 1993 16 395 404 8248566 Ferrans C Quality of life: Conceptual issues Seminars in Oncology Nursing 1990 6 248 254 2274721 Ferrell B The quality of lives Oncology Nursing Forum 1996 23 909 916 8829161 Wyatt G Friedman LL Long-term female cancer survivors: Quality of life issues and clinical implications Cancer Nursing 1996 19 1 7 8904381 10.1097/00002820-199602000-00001 Zebrack B Quality of life long-term survivors of Leukemia and Lymphoma Journal of Psychosocial Oncology 2000 18 39 59 Edwards B Ung L Quality of life instruments for caregives of patients with cancer: A review of their psychometric properties Cancer Nursing 2002 25 342 349 12394561 10.1097/00002820-200210000-00002 Boyle D et al. Caregiver quality of life after autologous bone marrow transplantation Cancer Nursing 2000 23 193 203 10851770 10.1097/00002820-200006000-00006 Cameron JI Franche R, Cheung AM, Stewart DE Lifestyle interference and emotional distress in family caregivers of advanced cancer patients Cancer 2002 94 521 527 11900237 10.1002/cncr.10212 Pot AM Deeg DJH, Dyck RV Psychological distress of caregivers: Moderator effects of caregiver resources? Patient Education and Counseling 2000 41 235 240 12024547 10.1016/S0738-3991(99)00081-6 Winslow BW Effects of formal supports on stress outcomes in family caregivers of Alzheimer's patients Research in Nursing & Health 1997 20 27 37 9024475 10.1002/(SICI)1098-240X(199702)20:1<27::AID-NUR4>3.3.CO;2-S Vedhara K Shanks N, Anderson S, Lightman S The role of stressors and psychosocial variables in the stress process: A study of chronic caregiver stress Psychosomatic Medicine 2000 62 374 385 10845351 Vitalino PP Russo J, Young HM, Teri L, Maiuro RD Predictors of burden in spouse caregivers of individuals with Alzheimer's disease Psychology and Aging 1991 6 392 402 1930756 10.1037//0882-7974.6.3.392 Lowenstein A The perception of caregiving burden on the part of elderly cancer patients, spouses and adult children The Journal of Collaborative Family Healthcare 2000 18 337 346 Weitzner MA McMillan SC, Jacobsen PB Families caregiver quality of life: Differences between curative and palliative cancer treatment settings. J Pain Symptom Manage 1999 17 418 429 10388247 10.1016/S0885-3924(99)00014-7 Nijboer C Triemstar M, Tempelaar R, Sanderman R, Van den bos GAM Determinants of caregiving experiences and mental health of partners of cancer patients Cancer 1999 86 577 588 10440685 10.1002/(SICI)1097-0142(19990815)86:4<577::AID-CNCR6>3.0.CO;2-S Clipp EC George LK Dementia and cancer: a comparison of spouse caregivers The Gerontologist 1993 33 534 541 8375684 Helder DI Kaptein AA, Van Kempen GM, Weinman J, Van Houwelingen JC, Roos RA Living with huntington's disease: Illness perceptions, coping mechanisms, and spouses' quality of life International Journal of Behavioral Medicine 2002 9 37 52 12112995 10.1207/S15327558IJBM0901_03 Dunn SA Bonner PN, Lewis SL, Meize-Grochowski R Quality of life for spouses of CAPD patients ANNA Journal 1994 21 237 247 8080335 Miaskowski C Differences in patients' and family caregivers' perceptions of the pain experience influence patient and caregiver outcomes Pain 1997 72 217 226 9272806 10.1016/S0304-3959(97)00037-7 Ergh TC Rapport LJ, Coleman RD, Hanks RA Predictors of caregiver and family functioning following traumatic brain injury: Social support moderates caregiver distress Journal of Head Trauma Rehabilitation 2002 17 155 174 11909512 McCubbin HI Patterson JM Systemic assessment of family stress, resources and coping: Tools for research, education and clinical intervention 1981 St. Paul, Minnesota, Department of Family Social Science Figley CR McCubbin M Stress and the family 1983 New York, Brunner/Mazel McMillan SC Mahon M The impact of hospice services on the quality of life of primary caregivers Oncology Nursing Forum 1994 21 1189 1195 7971429

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==== Front Genet Vaccines TherGenetic Vaccines and Therapy1479-0556BioMed Central London 1479-0556-2-131536311110.1186/1479-0556-2-13CommentarySkipping the co-expression problem: the new 2A "CHYSEL" technology de Felipe Pablo [email protected] Centre for Biomolecular Sciences, School of Biology, Biomolecular Sciences Building, University of St. Andrews, North Haugh, St. Andrews KY16 9ST, Scotland, UK2004 13 9 2004 2 13 13 30 6 2004 13 9 2004 Copyright © 2004 de Felipe; licensee BioMed Central Ltd.2004de Felipe; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The rapid progress in the field of genomics is increasing our knowledge of multi-gene diseases. However, any realistic hope of gene therapy treatment for those diseases needs first to address the problem of co-ordinately co-expressing several transgenes. Currently, the use of internal ribosomal entry sites (IRESs) is the strategy chosen by many researchers to ensure co-expression. The large sizes of the IRESs (~0.5 kb), and the difficulties of ensuring a well-balanced co-expression, have prompted several researchers to imitate a co-expression strategy used by many viruses: to express several proteins as a polyprotein. A small peptide of 18 amino acids (2A) from the foot-and-mouth disease virus (FMDV) is being used to avoid the need of proteinases to process the polyprotein. FMDV 2A is introduced as a linker between two proteins to allow autonomous intra-ribosomal self-processing of polyproteins. Recent reports have shown that this sequence is compatible with different sub-cellular targeting signals and can be used to co-express up to four proteins from a single retroviral vector. This short peptide provides a tool to allow the co-expression of multiple proteins from a single vector, a useful technology for those working with heteromultimeric proteins, biochemical pathways or combined/synergistic phenomena. ==== Body Introduction For the last 20 years, the gene therapy field has centred many of its efforts on finding ways to deliver a therapeutic gene to certain target cells in order to produce a therapeutic result. It was soon clear that it was necessary to deliver at least two genes, because a reporter/marker gene was needed in order to track the expression of the therapeutic gene (normally not easy to detect). There has been a large increase in vector development during these years, with the appearance of many new viral and non-viral vectors. However, since the late 1980s, few improvements have been made 'inside' those vectors. The linkage of the two genes of interest (therapeutic and reporter) has remained the same. The different strategies known for co-expression were reported during the 1980s -splicing, multiple promoters, fusions, reinitiation and IRESs-, at the same time that the first gene therapy experiments were being performed (for a review [1]). During the 1990s, nearly all those strategies were abandoned in favour of the IRESs. In bicistronic mRNAs bearing an IRES sequence, the first cistron is translated by scanning ribosomes that enter via the 5' end. The cloning of an IRES sequence downstream of the first cistron, allows the internal entry of ribosomes that translate the second cistron. As each cistron is translated from a different translational initiation event, both translations are uncoupled, and the proteins are not obtained in an equimolecular proportion ("imbalance") leading to a large excess of the first protein. The drive to co-express more than two genes, opening the door to therapies for muti-gene deficiencies, was halted by the inability of vector technology to guarantee a reliable co-expression. Nevertheless, IRESs were the first strategy that met with some success, and several polycistronic vectors able to co-express up to 4 genes were developed during the 1990's [2]. However, two main problems blocked the successful use of large and complex polycistronic vectors: the large size and imbalance of most IRESs which makes it very difficult to predict the level of expression of the downstream cistron [3]. This commentary discusses several recent publications that use self-processing polyproteins as a novel strategy for co-ordinated co-expression of several genes. Discussion Although gene therapy has employed the viruses as vectors, the co-expression strategies previously described have not taken advantage of the dominant ways in which viruses achieve co-expression in cells. It is the polyprotein strategy that many viruses use to co-express most of their proteins, or even all of them (as in picornaviruses). Not surprisingly, this strategy is indeed used by cells, although not very often, in particular for the co-ordinated secretion of different proteins and peptides. Recently, several groups have been trying to utilize this co-expression strategy. One of the possibilities is to introduce the target site for a cellular proteinase between two cistrons cloned in frame forming a single open reading frame (ORF; [4]). In this way the polyprotein is synthesized as a fusion protein that post-translationally is proteolytically cleaved to yield the discrete proteins of interest. Unfortunately, this strategy has several practical difficulties: (1) the polyprotein to be cleaved must reside, or at least pass through, the same compartment as the proteinase, (2) the cleavage is not always independent of the context, (3) the cleavage may be incomplete and unpredictable, (4) efficient cleavage will only be produced in cells actively expressing the proteinase, and (5) the post-translational cleavage is not compatible with all possible sub-cellular targetings. In many ways, a co-translational strategy such as reinitiation, which would be independent of cellular or viral factors, would be desirable. In reinitiation, ribosomes first translate an upstream cistron, although highly inefficiently, then resume translation of the second, downstream, cistron. Reinitiation was indeed tried in the 1980s, but the large imbalance makes it unsuitable for co-expression of even two genes (reviewed in [1]). The foot-and-mouth disease virus (FMDV) 2A sequence as a co-expression tool Picornaviruses, the same family of viruses to first provide the IRESs, encode all their proteins in a long single ORF that is cleaved post-translationally by viral proteinases. However, it was described in the 1980's that at one position, the polyprotein of some picornaviruses (such as FMDV) underwent a rapid co-translational self-processing. It was soon realised that the key was a small 18aa peptide (2A) that directed its own separation from the growing polyprotein. During the last decade, this mechanism has been studied in detail, resulting in a simple model: the small 2A peptide, during its translation, interacts with the exit tunnel of the ribosome to induce the "skipping" of the last peptide bond at the C-terminus of 2A. The crucial point is that the ribosome is able to continue translating the downstream gene, after releasing the first protein fused in its C-terminus to 2A (reviewed in [5]). This type of sequence has been termed CHYSEL (cis-acting hydrolase element). From a biotechnological standpoint, all that is needed is to clone the coding sequence of 2A, followed by the codon for the first amino acid of the next FMDV protein (2B), in frame between the two genes one wishes to co-express. The synthesis of the peptide bond between the last amino acid (Gly) of 2A and the first (Pro) of 2B is skipped, producing an upstream protein with a C-terminal tail of 18aa (2A) and a downstream protein with a Pro at the N-terminus. The extra sequences have minimal effect on the activity of most proteins and none on their stability. In fact, the 2A peptide has been used as an efficient tag for immunoprecipitation and Western blotting, although commercial antibodies are not yet available. Interestingly, additional CHYSEL sequences have been found in viruses other than FMDV (for a review of these "2A-like" sequences, see [5]). Broad applicability of 2A The initial publications using this strategy have shown that 2A skipping can be used in the typical viral vectors used for gene therapy (retrovirus and adeno-associated virus) to reliably co-express many reporter proteins (neomycin phosphotransferase, NEO; puromycin N-acetyl transferase, PAC; green fluoresecent protein, GFP, etc) and therapeutic proteins (Herpes simplex virus-1 thymidine kinase, HSV1TK; interleukin-12, IL-12; viral antigens, etc.) in transient transduced or stable cells lines and in animals. A full list of publications using 2A is available on the web [6]. Several publications in the past few months have shown the potential of this new co-expression strategy [7-9]. Co-ordinating the co-expression of all your genes Up to four genes have been successfully co-expressed from plasmids and retrovirus using several copies of the FMDV 2A or other 2A-like sequences (to avoid direct repeats in retroviruses) [8,9]. Not only was co-expression effective, its co-ordination was also apparent [7,9] (Fig. 1), and the imbalance in the level of the proteins expressed was low (determined to 1.2 [8]). These properties allowed polycistronic vectors bearing pac in the last position to easily generate stable cell lines co-expressing two upstream genes [7,9]. Figure 1 Co-ordinated co-expression to different compartments in HeLa cells. A single ORF was designed with the fluorescent genes eyfp and ecfp plus the puromycin resistant gene pac [9]. These genes were cloned flanking FMDV 2A sequences. An internal signal-anchor from the human β-1,4 galactosyltransferase (GT) was fused to the 5' end of the ecfp for Golgi targeting. During its translation, the self-processing of this polyprotein produced EYFP-2A that diffused to the cytoplasm and nucleus (due to its small size), while GT-EYFP-2A was co-translationally targeted to the Golgi apparatus by the GT signal (some protein also stays in the endoplasmic reticulum, due to the continuous cycling between these compartments). Two fields are shown, in both cases the cell on the left shows a high level of expression of both proteins that were expressed at lower levels in the cell on the right, illustrating the co-ordination obtained with the 2A co-expression strategy. PAC was able to confer resistance to puromycin. Images were taken 48 hours post-transfection. Bar represents 10 μm. Putting your proteins where they should be The CHYSEL strategy of co-expression is also compatible with the most disparate sub-cellular localisations [7-10]. Proteins processed by 2A from polyproteins were targeted to the cytosol, nucleus, mitochondria, endoplasmic reticulum, Golgi apparatus, plasma membrane (both, by transmembrane proteins and by cytosolic attachment due to myristoylation) and the extra-cellular compartment. Post-translationally targeted cytosolic proteins as well as co-translationally secreted and transmembrane proteins type I, II and III, have been successfully co-translated. Only one combination of co-translational signals was not correctly targeted [9]. Designing complex polyproteins for multi-gene deficiency The results reported in reference [8] should be particularly interesting for researchers in the gene therapy field. They provide a good example of the potential of the 2A co-expression strategy, introducing up to four genes in a single vector. Furthermore, they show the utility of this strategy to reconstruct a very delicate heteromultimeric protein complex on the cell surface (T-cell receptor:CD3 complex, TCR:CD3; Fig. 2A). It is known that all six subunits are necessary for the efficient formation of the TCR:CD3 complex and just two retroviral vectors were sufficient to reconstruct it in transfected 293T or infected 3T3 cells: one encoding both subunits of the T-cell receptor and the other the four subunits of the CD3 complex (Fig. 2B). Figure 2 Self-processing polyproteins to reconstruct the TCR:CD3 complex. (A) Schematic diagram of the TCR:CD3 complex spanning the cytoplasmic membrane. The T-cell receptor (TCR) is formed by two subunits and the other four proteins assemble in three dimers to form the CD3 complex. The square boxes in the cytoplasmic sequences of the CD3 subunits represent the immunoreceptor tyrosine-based activation motifs (ITAMs). (B) To express the TCR:CD3 complex in cells, two retroviral vectors were designed to carry the two ORFs drawn here [8]. In the retrovirus encoding the four CD3 subunits, three different 2A sequences were used to avoid deletions due to direct repetitions. Lethally irradiated CD3εΔP/ΔP × CD3ζ -/- mice (lacking all four CD3 subunits) were transplanted with bone marrow from wt C57BL/6 mice or CD3εΔP/ΔP × CD3ζ -/- mice transduced with a retrovirus encoding the four CD3 subunits, and in both cases TCR surface expression was detected and the T cells proliferated normally after immune stimulation. Bone marrow from CD3εΔP/ΔP × CD3ζ -/- mice without CD3 transduction did not restore T-cell development. T cells were also reconstituted in sub-lethally irradiated RAG-1-/- mice (lacking mature T and B lymphocytes) in which bone marrow from CD3εΔP/ΔP mice (lacking CDε and with a severe inhibition of CD3γ and CD3δ), transduced with a retrovirus encoding these three subunits (via two 2A sequences), was used for a transplant into the RAG-1-/- mice. The same experiment using three vectors encoding the CD3 subunits separately was unsuccessful. Conclusions The development of FMDV 2A as a cloning tool is an example of how dangerous pathogenic viruses can be harnessed by biotechnology for human benefit. Their molecular "tricks" (as IRES or CHYSEL sequences) are gradually becoming part of the biotechnologists' toolbox. The development of the polycistronic vectors here discussed is a big step forward, a decade and a half after the launching of the very first gene therapy trial with the aim of introducing in blood cells just a single therapeutic gene, adenosine deaminase (ADA), and the NEO marker [11]. These results represent a considerable advance in the correction of diseases that involve heteromultimeric proteins, several enzymes involved in a biochemical pathway or various proteins for combined/synergistic effects. 2A is not a magic tool that is going to solve all our problems, but it will help to pave the way for gene therapy. Competing interests None declared. Acknowledgements I would like to thank Drs. M. D. Ryan, M. C. Thomas and M. C. López for critical reading of the manuscript, and Drs. G. Luke and L. E. Hughes for helpful discussions on the topics of this paper. The author is supported by the Biotechnology and Biological Sciences Research Council (BCB). ==== Refs de Felipe P Polycistronic viral vectors Curr Gene Ther 2002 2 355 378 12189721 Fussenegger M The impact of mammalian gene regulation concepts on functional genomic research, metabolic engineering, and advanced gene therapies Biotechnol Prog 2001 17 1 51 11170478 10.1021/bp000129c Mizuguchi H Xu Z Ishii-Watabe A Uchida E Hayakawa T IRES-dependent second gene expression is significantly lower than cap-dependent first gene expression in a bicistronic vector Mol Ther 2000 1 376 382 10933956 10.1006/mthe.2000.0050 Gäken J Jiang J Daniel K van Berkel E Hughes C Kuiper M Darling D Tavassoli M Galea-Lauri J Ford K Kemeny M Russell S Farzaneh F Fusagene vectors: a novel strategy for the expression of multiple genes from a single cistron Gene Ther 2000 7 1979 1985 11175308 10.1038/sj.gt.3301341 Ryan MD Luke G Hughes LE Cowton VM ten Dam E Li X Donnelly MLL Mehrotra A Gani D Semler BL, Wimmer E The aphto- and cardiovirus "primary" 2A/2B polyprotein "cleavage" Molecular Biology of Picornaviruses 2002 Washington, ASM Press 213 223 Dr. Martin Ryan's laboratory web page Lorens JB Pearsall DM Swift SE Peelle B Armstrong R Demo SD Ferrick DA Hitoshi Y Payan DG Anderson D Stable, stoichiometric delivery of diverse protein functions J Biochem Biophys Methods 2004 58 101 110 14980783 10.1016/S0165-022X(03)00147-7 Szymczak AL Workman CJ Wang Y Vignali KM Dilioglou S Vanin EF Vignali DAA Correction of multi-gene deficiency in vivo using a single 'self-cleaving' 2A peptide-based retroviral vector Nat Biotechnol 2004 22 589 594 15064769 10.1038/nbt957 de Felipe P Ryan MD Targeting of Proteins Derived from Self-Processing Polyproteins Containing Multiple Signal Sequences Traffic 2004 5 616 626 15260831 10.1111/j.1398-9219.2004.00205.x El Amrani A Barakate A Askari BM Li X Roberts AG Ryan MD Halpin C Coordinate expression and independent subcellular targeting of multiple proteins from a single transgene Plant Physiology 2004 135 16 24 15141063 10.1104/pp.103.032649 Muul LM Tuschong LM Soenen SL Jagadeesh GJ RamseY WJ Long Z Carter CS Garabedian EK Alleyne M Brown M Bernstein W Schurman SH Fleisher TA Leitman SF Dunbar CE Blaese RM Candotti F Persistence and expression of the adenosine deaminase gene for 12 years and immune reaction to gene transfer components: long-term results of the first clinical gene therapy trial Blood 2003 101 2563 2569 12456496 10.1182/blood-2002-09-2800

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==== Front Kinetoplastid Biol DisKinetoplastid Biology and Disease1475-9292BioMed Central London 1475-9292-3-31537738510.1186/1475-9292-3-3Original ResearchVariable Surface Glycoprotein RoTat 1.2 PCR as a specific diagnostic tool for the detection of Trypanosoma evansi infections Claes Filip [email protected] Magda [email protected] Toyo [email protected] Phelix AO [email protected] Bruno [email protected]üscher Philip [email protected] Faculty of Agriculture and Applied Biological Sciences, K. U. Leuven, Department of Animal Science, Kasteelpark Arenberg 30, 3000 Leuven, Belgium2 Prince Leopold Institute of Tropical Medicine, Department of Parasitology, Nationalestraat 155, Antwerpen, Belgium3 International Livestock Research Institute (ILRI), Nairobi, Kenya2004 17 9 2004 3 3 3 1 6 2004 17 9 2004 Copyright © 2004 Claes et al; licensee BioMed Central Ltd.2004Claes et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Based on the recently sequenced gene coding for the Trypanosoma evansi (T. evansi) RoTat 1.2 Variable Surface Glycoprotein (VSG), a primer pair was designed targeting the DNA region lacking homology to other known VSG genes. A total of 39 different trypanosome stocks were tested using the RoTat 1.2 based Polymerase Chain Reaction (PCR). Results This PCR yielded a 205 bp product in all T. evansi and in seven out of nine T. equiperdum strains tested. This product was not detected in the DNA from T. b. brucei, T. b. gambiense, T. b. rhodesiense, T. congolense, T. vivax and T. theileri parasites. The Rotat 1.2 PCR detects as few as 10 trypanosomes per reaction with purified DNA from blood samples, i.e. 50 trypanosomes/ml. Conclusion PCR amplification of the RoTat 1.2 VSG gene is a specific marker for T. evansi strains, except T. evansi type B, and is especially useful in dyskinetoplastic strains where kDNA based markers may fail to amplify. Furthermore, our data support previous suggestions that some T. evansi stocks have been previously misclassified as T. equiperdum. ==== Body Background Surra is an animal disease occurring in Africa, Asia and Latin America, caused by Trypanosoma evansi. T. evansi belongs to the subgenus Trypanozoon, together with T. equiperdum and T. brucei. The parasite can infect different host species and is mechanically transmitted by different biting flies such as Tabanidae and Stomoxys as well as by vampire bats such as Desmodus rotondus [1]. Camels and horses are very susceptible to the infection and death can occur within weeks or months. Moreover, T. evansi infections of cattle and buffaloes usually lead to a pronounced immunosuppression resulting in an increased susceptibility to other opportunistic diseases such as Pasteurella and anthrax [2]. Diagnosis of a T. evansi infection usually starts with clinical symptoms or the detection of antibodies to T. evansi. Conclusive evidence of T. evansi infection, however, relies on detection of the parasite in the blood or tissue fluids of infected animals. Unfortunately, parasitological techniques cannot always detect ongoing infections as the level of parasitaemia is often low and fluctuating, particularly during the chronic stage of the disease [3]. As an alternative to parasitological tests, DNA detection based on PCR has been proposed. Trypanozoon specific primers have been designed previously: TBR primers which target a 177 bp repeat [4], pMUTEC primers targeting a retrotransposon [5] and ORPHON primers which target the spliced leader sequence [6]. Most of them have been tested on cattle [7,8], water buffaloes [9] or goats [10]. PCR tests for diagnosis of T. congolense and T. vivax infections exist as well [11]. The development of a PCR test that would be able to differentiate between the different members of the Trypanozoon subgenus still remains a challenging issue. For T. evansi infections, the only specific test available so far is based on the detection of a kinetoplast DNA sequence [12,13]. However, the existence of dyskinetoplastic trypanosomes such as T. evansi RoTat 5.1 [14] and E152 [12] casts doubt about the diagnostic potential of such tests to detect all infections caused by T. evansi parasites. Recently, Ventura et al. [15] developed a PCR (PCR-Te664) for the detection of T. evansi based on a Random Amplified Polymorphic DNA (RAPD) fragment. The taxon specificity of this PCR remains uncertain since it was only tested on nine T. evansi strains, one T. equiperdum, two T. b. gambiense and one T. b. rhodesiense. Following evidence that the variable epitope of RoTat 1.2 VSG is expressed by all T. evansi strains tested so far [16], and that the gene encoding RoTat 1.2 VSG is present in all T. evansi but not in T. brucei isolates [17], we designed primers derived from the sequence of this VSG cDNA. In this article we will present and discuss the results obtained with these primers and compare them to the results we obtained using the PCR-Te664. Results PCR RoTat 1.2 : taxon specificity The 39 different trypanosome stocks used in this study are listed in Table 1 [see Additional file 1]. They were derived from a wide range of hosts and from distinct geographical locations. In all PCR runs, RoTat 1.2 DNA was used as a positive control. As shown in Figure 1, the RoTat 1.2 PCR yielded a 205 bp amplicon in the positive control (lane 1) as well as in all other T. evansi populations (lanes 3–8). Moreover, the same fragment was found in seven out of the nine T. equiperdum populations tested. The T. equiperdum BoTat 1.1 was PCR negative (lane 10), while the T. equiperdum OVI strain yielded a PCR product shorter than 205 bp (lane 11) probably due to mispriming. All other tested trypanosome populations, including six T. b. brucei, eight T. b. gambiense, five T. b. rhodesiense, two T. congolense, one T. vivax and one T. theileri, were negative. (lanes 18–40). As a negative control, a PCR-mix without template DNA was included (lane 2). Sequencing of the positive samples revealed that all amplicon were identical (data not shown). The weak band in OVI did not yield sufficient material to enable sequencing. Figure 1 PCR specificity results for the different Trypanosoma (T.) species and subspecies in this study. Lane 1 pos. control RoTat 1.2, Lane 2 neg. control, Lanes 3–8 (T. evansi) are, respectively, AnTat 3.1, STIB 816, Zagora I.17, Colombia, Merzouga 56, CAN 86 K; Lanes 9–17 (T. equiperdum) are, respectively, AnTat 4.1, BoTat 1.1, OVI, STIB 818, Alfort, Hamburg, SVP, Am. Strain, Can. Strain ; Lanes 18–23 (T.b.brucei) are, AnTat 1.8, AnTat 2.2, AnTat 5.5, KETRI 2494, TSW 196, STIB 348; Lanes 24–31 (T.b.gambiense) are, respectively, AnTat 9.1, AnTat 11.6, AnTat 22.1, NABE, SEKA, ABBA, LIGO, LiTat 1.6; Lanes 32–36 (T.b. rhodesiense) are STIB 884, STIB 850, AnTat 25.1/S, Etat 1.2/S, AnTat 12.1/S ; Lanes 37–38 (T. congolense) are IL1180, TRT 17; Lane 39 (T. vivax) is ILRAD 700 and Lane 40 (T. theileri) is MELSELE ; Lanes M 100 bp molecular marker (MBI Fermentas, Germany). PCR RoTat 1.2 : analytical sensitivity A tenfold dilution series (105 trypanosomes down to 1 trypanosome per 200 μl sample) of RoTat 1.2 trypanosomes in mouse blood was prepared to determine the analytical sensitivity of the PCR. As shown in figure 2, the PCR was able to detect as few as 10 trypanosomes per PCR reaction, which corresponds with a lower detection limit of 50 trypanosomes per ml. In principle, this limit can still be lowered if a blood sample of 200 μl extracted with the QIAamp DNA mini kit is eluted in less than 200 μl. Figure 2 Analytical sensitivity of the RoTat 1.2 PCR. Lanes M 100 bp molecular marker (MBI Fermentas, Germany); lane 1: 105 trypanosomes, lane 2: 104 trypanosomes, lane 3: 103 trpyanosomes, lane 4: 102 trypanosomes, lane 5: 10 trypanosomes, lane 6: 1 trypanosome, lane 7: 0.1 trypanosome, lane 8: negative control. PCR-Te664 : taxon specificity To evaluate the RoTat 1.2 diagnostic system alongside other published methods, we compared our method to the PCR-Te664 method as published by Ventura et al. [15] using the same trypanosome stocks. The PCR-Te664 method yielded the expected amplicon in all seven T. evansi strains and in seven out of nine T. equiperdum. As with the RoTat 1.2 PCR only T. equiperdum strains OVI and BoTat 1.1 remained negative. Unexpectedly, four out of six T. b. brucei (AnTat 2.2, AnTat 5.2, TSW 196 and KETRI 2494) and two T. b. gambiense type II strains (ABBA and LIGO) tested positive in this PCR (data not shown). Discussion This study was initiated to develop a specific PCR test that would be able to distinguish T. evansi from the other members of the Trypanozoon subgenus. The study is an extension of the initial observation that the RoTat 1.2 VSG gene only is found in T. evansi and not in T. brucei strains [17]. This study mainly focused on the presence and expression of the RoTat 1.2 VSG gene in T. evansi rather than the use of this VSG in diagnosis of Salivarian trypanosomes. Previously, other research groups have used VSG genes as target sequences for PCR detection of T.b. gambiense infections (sleeping sickness). In these studies, five different primers derived from VSG genes, AnTat 11.17, LiTat 1.3, 117, 2 K and U2 were used in PCR screening of different trypanosome populations, originating from distinct geographical locations [18-20]. AnTat 11.17 based PCR tests were capable of distinguishing T.b. gambiense from T.b. brucei parasites from most foci of sleeping sickness in countries such as Nigeria, Cameroon, Côte d'Ivoire, R. P. Congo/Brazza. and Sudan. However, populations originating from the Moyo focus in North-west Uganda and from Cameroon were shown to be negative in AnTat 11.17 and in LiTat 1.3 (2 K) PCRs respectively. According to Bromidge et al. [18], this might be due to antigenic variation and genetic evolution of the VSG genes. On the other hand, the presence of 117 and U2 genes was shown to be a common feature among all T. brucei populations tested. In T. evansi, a similar phenomenon may occur in certain Kenyan isolates. A recent study by Ngaira et al. [21] pointed out that some T. evansi stocks in the Isiolo district in Kenya seem to lack the Rotat 1.2 VSG gene. It is believed that these stabilates belong to the T. evansi type B group. So far, this type of T. evansi has only been observed in this specific region in Kenya [22,23]. To our knowledge, all other T. evansi isolated elsewhere, are from the classical T. evansi type A group. Thus, we assume that, except for these few Kenyan strains belonging to the type B group, our PCR is specific for T. evansi. Compared to the PCR-Te664 presented by Ventura et al. [15], the PCR RoTat 1.2 seems to have a higher taxon specificity, since no reaction with T. b. brucei, nor with T. b. gambiense type II was observed. However, regarding T. equiperdum, both PCR test positive for the same seven T. equiperdum strains and are both negative for the BoTat 1.1 and OVI strains. Since the RAPD fragment (AF397194) shares no homology with the Rotat 1.2 VSG gene (AF317914) and is not found within the expression site of trypanosomes, both sequences can be considered as independent molecular markers. Based on the observations with both markers, it appears that on the genomic level the Botat 1.1 and the OVI strains are different from the other T. equiperdum and T. evansi strains. The observed analytical sensitivity with the RoTat 1.2 PCR is comparable to what was reported for the Te664 PCR (25 cells per reaction) [15]. The presence of a RoTat 1.2 specific DNA sequence in some T. equiperdum strains corresponds with the serological evidence that rabbits experimentally infected with these strains develop RoTat 1.2 specific lytic antibodies within 30 days post infection [24]. In contrast, rabbits infected with the BoTat 1.1 clone and the OVI strain, which are negative in the present PCR, did not produce specific antibodies to the RoTat 1.2 clone when tested in immune trypanolysis. This might be explained by the loss of the RoTat 1.2 gene in the OVI and the BoTat 1.1 strain. It is also possible that there has been a sequence drift at the sites where these primers could bind. However, we hypothesize that RoTat 1.2 VSG truly is T. evansi specific and that RoTat 1.2 PCR positive T. equiperdum strains are actually T. evansi and not T. equiperdum. Indeed, in a previous molecular characterization study using Random Amplified Polymorphic DNA (RAPD) and the Multiplex-endonuclease Genotyping Approach (MEGA) it appeared that the T. equiperdum collection is not as homogenous as previously believed and that the generally followed concept that T. equiperdum is very closely related to T. evansi and more distant from T. b. brucei, seems incorrect. From the cluster analysis on the available strains, it appeared that only two clusters can be identified: a homogeneous T. evansi/T. equiperdum cluster and a more heterogeneous T. b. brucei/T. equiperdum cluster [25]. Interestingly, all strains of that homogeneous T. evansi/T. equiperdum cluster are all PCR RoTat 1.2 VSG positive while the strains found in the more heterogeneous T. b. brucei/T. equiperdum cluster, in casu BoTat 1.1 and OVI are PCR RoTat 1.2 VSG negative. Conclusions PCR amplification of the RoTat 1.2 VSG gene is a specific marker for T. evansi strains, except T. evansi type B, and is especially useful in dyskinetoplastic strains where kDNA based markers may fail to amplify. Furthermore, our data support previous suggestions that some T. evansi stocks have been previously misclassified as T. equiperdum. Methods Trypanosome populations A total of 39 different trypanosome populations were used in this study. They belong to 39 stocks and six species, isolated from a variety of host species at distinct geographical locations (Table 1 [see Additional file 1]). Only three T. equiperdum strains, BoTat 1.1, OVI and STIB 818 are well documented, i.e. known origin and host. The other six are putative T. equiperdum, based on publications or on their use as reference strains in different national dourine reference laboratories [26-30]. Preparation of trypanosome DNA Procyclic trypanosome populations were grown in vitro in Cunningham's medium [31] and in the Kit for In Vitro Isolation (KIVI) [32]. Pure procyclic trypanosomes were obtained by repeated centrifugation (20 min., 2000 g) and sediment washes with Phosphate Glucose Sacharose buffer (PGS) (38 mM Na2HPO4.2H20, 2 mM NaHPO4, 80 mM glucose, 100 mM sacharose, pH 8.0). Bloodstream form trypanosomes were expanded in mice and rats and were purified from the blood by di-ethyl-amino-ethyl (DEAE) chromatography [33], followed by repeated centrifugation (20 min., 2000 g) and sediment washes with Phosphate Buffered Saline Glucose (PSG) (38 mM Na2HPO4.2H20, 2 mM NaHPO4, 80 mM glucose, 29 mM NaCl, pH 8.0). Trypanosome sediments were subsequently stored at -80°C. Twenty μl of trypanosome sediment (approximately 2.107 cells) were resuspended in 200 μl of Phosphate Buffered Saline (PBS) (8.1 mM Na2HPO4.2H20, 1.4 mM NaHPO4, 140 mM NaCl, pH 7.4) and the trypanosome DNA was extracted using the commercially available QIAamp DNA mini kit (Westburg, Leusden, The Netherlands), resulting in pure DNA in 200 μl of TE buffer. The typical yield of DNA extracted from a 20 μl pellet was 150 ng/μl or 30 μg total DNA. The extracts obtained were diluted 200 times in water and divided into aliquots of 2 ml in microcentrifuge tubes for storage at -20°C. For trypanosome dilution series, 180 μl of each heparinized blood sample were mixed with an equal volume of the Qiagen AS-1 storage buffer and subsequently extracted using the QIAamp DNA blood mini kit (Westburg, Leusden, The Netherlands) resulting in 200 μl of extracted DNA in Millipore water. Manipulation was performed according to the manufacturer's instructions. PCR RoTat 1.2 Primers were derived from the RoTat 1.2 VSG sequence (AF317914), recently cloned and sequenced by Urakawa et al. [17]. Using DNA sequence homology search programs to interrogate databases at TIGR (The Institute for Genomic Research) and GenBank, primer sequences were identified within the region (608–812 bp) lacking homology with any other known VSG sequence present in the databases. Primer sequences (in 5'-3' direction) and annealing temperatures were as follows: RoTat 1.2 Forward GCG GGG TGT TTA AAG CAA TA, Tann. 59°C and RoTat 1.2 Reverse ATT AGT GCT GCG TGT GTT CG, Tann. 59°C. Twenty μl of extracted DNA were mixed with 30 μl of a PCR-mix containing: 1 U Taq DNA recombinant polymerase (Promega, UK), PCR buffer (Promega, UK), 2.5 mM MgCl2 (Promega, UK), 200 μM of each of the four dNTPs (Roche, Mannheim, Germany) and 0.8 μM of each primer (Gibco BRL, UK). All amplifications were carried out in a Biometra® Trio-block thermocycler. Cycling conditions were as follows: denaturation for 4 min. at 94°C, followed by 40 amplification cycles of 1 min. denaturation at 94°C, 1 min. primer-template annealing at 59°C and 1 min. polymerization at 72°C. A final elongation step was carried out for 5 min. at 72°C. Twenty μl of the PCR product and ten μl of a 100 bp size marker (MBI Fermentas, Germany) were subjected to electrophoresis in a 2 % agarose gel (25 min. at 100 V). Gels were stained with ethidium bromide (0.5 μg/ml) (Sigma, USA) and analyzed on an Imagemaster Video Detection System (Pharmacia, UK). PCR Te-664 PCR on purified DNA samples was performed using primers and PCR conditions according to Ventura et al. [15]. Only the Taq DNA polymerase was purchased from another distributor, i.e. Promega (UK) instead of Gibco BRL (UK). Competing interests None declared. Authors' contributions FC carried out the molecular work and drafted the manuscript. MR and TU participated in the molecular analysis. PM, BG and PB participated in the design and co-ordination of the study. All authors read and approved the final manuscript. Supplementary Material Additional File 1 Table 1. Data on the different Trypanosoma (T.) populations used in this study Click here for file Acknowledgements This study received financial support from the International Livestock Research Institute in Nairobi, Kenya and the Institute for the Promotion of Innovation by Science and Technology in Flanders, Belgium (IWT). Trypanosoma spp. stabilates were kindly provided by: T Baltz, University of Bordeaux II, France; T De Waal, Onderstepoort Veterinary Institute, South Africa; R Brun, Swiss Tropical Institute Basel, Switzerland; P-H Clausen, Free University Berlin, Germany; J Hagebock†, National Veterinary Services Laboratories, United States Department of Agriculture, USA; Z Lun, Zhongshan University, P.R. China; and V Zablotsky, All-Russian Research Institute for Experimental Veterinary Medicine (VIEV), Russia. ==== Refs Hoare CA The trypanosomes of mammals 1972 Oxford, Blackwell Scientific Publications 1 749 Stephen LE StephenLE Trypanosomiasis A veterinary perspective 1986 Oxford, Pergamon Press Nantulya VM Trypanosomiasis in domestic animals: the problems of diagnosis Rev Sci Tech 1990 9 357 367 2132685 Moser DR Cook GA Ochs DE Bailey CP McKane MR Donelson JE Detection of Trypanosoma congolense and Trypanosoma brucei subspecies by DNA amplification using the polymerase chain reaction Parasitology 1989 99 57 66 2797872 Wuyts N Chokesajjawatee N Panyim S A simplified and highly sensitivity detection of Trypanosoma evansi by DNA amplification Southeast Asian J Trop Med Public Health 1994 25 266 271 7855639 Pereira de Almeida PJL Ndao M Van Meirvenne N Geerts S IAEA Diagnostic evaluation of PCR in goats infected with Trypanosoma brucei brucei 1997 Vienna, International Symposium on Diagnosis and Control of Livestock Diseases using Nuclear and Related Techniques 68 Clausen P-H Wiemann A Patzelt R Kakaire D Poetzsch C Peregrine A Mehlitz D Use of a PCR assay for the specific and sensitive detection of Trypanosoma spp. in naturally infected dairy cattle in peri-urban Kampala, Uganda Ann N Y Acad Sci 1998 849 21 31 9668446 Omanwar S Rao JR Basagoudanavar SH Singh RK Butchaiah G Direct and sensitive detection of Trypanosoma evansi by polymerase chain reaction Acta Vet Hung 1999 47 351 359 10497829 Holland WG Claes F My LN Thanh NG Tam PT Verloo D Büscher P Goddeeris B Vercruysse J A comparative evaluation of parasitological tests and a PCR for Trypanosoma evansi diagnosis in experimentally infected water buffaloes Vet Parasitol 2001 97 23 33 11337124 10.1016/S0304-4017(01)00381-8 Pereira de Almeida PJL Ndao M Goossens B Osaer S PCR primer evaluation for the detection of trypanosome DNA in naturally infected goats Vet Parasitol 1998 80 111 116 9870363 10.1016/S0304-4017(98)00205-2 Majiwa PAO SonesKR DNA probe- and PCR-based methods for the detection of trypanosomes Twenty-second Meeting of the International Scientific Councel for Trypanosomiasis Research and Control (ISCTRC) Kampala, Uganda,25-29 October 1993 1995 Nairobi, OAU/STRC 53 58 Masiga DK Gibson WC Specific probes for Trypanosoma (Trypanozoon) evansi based on kinetoplast DNA minicircles Mol Biochem Parasitol 1990 40 279 284 2163493 10.1016/0166-6851(90)90049-R Artama WT Agay MW Donelson JE DNA comparisons of Trypanosoma evansi (Indonesia) and Trypanosoma brucei spp. Parasitology 1992 104 67 74 1319564 Diall O Camel trypanosomosis in Mali: contribution to the diagnosis and the epidemiology 1993 Vrije Universiteit Brussel 1 91 Ventura RM Takeda GF Silva RAMS Nunes VLB Buck GA Teixeira MMG Genetic relatedness among Trypanosoma evansi stocks by random amplification of polymorphic DNA and evaluation of a synapomorphic DNA fragment for species-specific diagnosis Int J Parasitol 2001 1 11 11286188 Verloo D Magnus E Büscher P General expression of RoTat 1.2 variable antigen type in Trypanosoma evansi isolates from different origin Vet Parasitol 2001 97 183 189 11390070 Urakawa T Verloo D Moens L Büscher P Majiwa PAO Trypanosoma evansi: cloning and expression in Spodoptera fugiperda insect cells of the diagnostic antigen RoTat 1.2 Exp Parasitol 2001 99 181 189 11888244 10.1006/expr.2001.4670 Bromidge T Gibson W Hudson K Dukes P Identification of Trypanosoma brucei gambiense by PCR amplification of variant surface glycoprotein genes Acta Trop 1993 53 107 119 8098897 10.1016/0001-706X(93)90023-5 Dukes P Gibson WC Gashumba JK Hudson KM Bromidge TJ Kaukus A Asonganyi T Magnus E Absence of the LiTat 1.3 (CATT antigen) gene in Trypanosoma brucei gambiense stocks from Cameroon Acta Trop 1992 51 123 134 1354930 10.1016/0001-706X(92)90054-2 Cross M Taylor MC Borst P Frequent loss of the active site during variant surface glycoprotein expression site switching in vitro in Trypanosoma brucei Mol Cell Biol 1998 18 198 205 9418867 Ngaira JM Njagi ENM Ngeranwa JJN Olembo NK PCR amplification of RoTat 1.2 VSG gene in Trypanosoma evansi isolates in Kenya Vet Parasitol 2004 120 23 33 15019140 10.1016/j.vetpar.2003.12.007 Gibson WC Wilson AJ Moloo SK Characterisation of Trypanosoma (Trypanozoon) evansi from camels in Kenya using isoenzyme electrophoresis Res Vet Sci 1983 34 114 118 6836172 Borst P Fase-Fowler F Gibson WC Kinetoplast DNA of Trypanosoma evansi Mol Biochem Parasitol 1987 23 31 38 3033499 10.1016/0166-6851(87)90184-8 Claes F Verloo D De Waal DT Urakawa T Majiwa P Goddeeris BM Büscher P Expression of RoTat 1.2 cross-reactive variable antigen type in Trypanosoma evansi and T. equiperdum Ann N Y Acad Sci 2002 969 174 179 12381586 Claes F Agbo EC Radwanska M te Pas MFW Baltz T De Waal DT Goddeeris BM Claassen E Büscher P How does Trypanosoma equiperdum fit into the Trypanozoon group? A cluster analysis by RAPD and Multiplex-endonuclease genotyping approach Parasitology 2003 126 425 431 12793646 10.1017/S0031182003002968 Barrowman PR Experimental intraspinal Trypanosoma equiperdum infection in a horse Onderstepoort J Vet Res 1976 43 201 202 1023092 Van Meirvenne N Immunologische aspekten van de relatie tussen salivaria trypanosomen en de zoogdiergastheer 1977 Universitaire Instelling Antwerpen, Antwerpen 1 240 Baltz T Etude de la variation antigénique chez Trypanosoma equiperdum 1982 L'Université de Bordeaux II, Bordeaux 1 84 Hagebock JM Chieves L Frerichs WM Miller CD Evaluation of agar gel immunodiffusion and indirect fluorescent antibody assays as supplemental tests for dourine in equids Am J Vet Res 1993 54 1201 1208 8214884 Lun Z-R Allingham R Brun R Lanham SM The isoenzyme characteristics of Trypanosoma evansi and Trypanosoma equiperdum isolated from domestic stocks in China Ann Trop Med Parasitol 1992 86 333 340 1463353 Cunningham I New culture medium for maintenance of tsetse tissues and growth of trypanosomatids J Protozool 1977 24 325 329 881656 Aerts D Truc P Penchenier L Claes Y Le Ray D A kit for in vitro isolation of trypanosomes in the field: first trial with sleeping sickness patients in the Congo Republic Trans R Soc Trop Med Hyg 1992 86 394 395 1440814 10.1016/0035-9203(92)90234-4 Lanham SM Godfrey DG Isolation of salivarian trypanosomes from man and other mammals using DEAE-cellulose Exp Parasitol 1970 28 521 534 4993889 10.1016/0014-4894(70)90120-7

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Kinetoplastid Biol Dis. 2004 Sep 17; 3:3

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Kinetoplastid Biol Dis

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==== Front Int J Health GeogrInternational Journal of Health Geographics1476-072XBioMed Central London 1476-072X-3-201545012110.1186/1476-072X-3-20MethodologyUse of satellite imagery in constructing a household GIS database for health studies in Karachi, Pakistan Ali Mohammad [email protected] Shahid [email protected] Jin-Kyung [email protected] Shamoon [email protected] Rion Leon [email protected] Qamaruddin [email protected] Camilo J [email protected] Zulfiqar [email protected] International Vaccine Institute, SNU Research Park, San 4–8 Bongcheon-7 dong, Kwanak-gu, Seoul, Korea2 Aga Khan University, Pediatric Department, Karachi, Pakistan3 Techno-Consult International, Karachi, Pakistan2004 28 9 2004 3 20 20 16 8 2004 28 9 2004 Copyright © 2004 Ali et al; licensee BioMed Central Ltd.2004Ali et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Household-level geographic information systems (GIS) database are usually constructed using the geographic positioning system (GPS). In some research settings, GPS receivers may fail to capture accurate readings due to structural barriers such as tall buildings. We faced this problem when constructing a household GIS database for research sites in Karachi, Pakistan because the sites are comprised of congested groups of multi-storied building and narrow lanes. In order to overcome this problem, we used high resolution satellite imagery (IKONOS) to extract relevant geographic information. Results The use of IKONOS satellite imagery allowed us to construct an accurate household GIS database, which included the size and orientation of the houses. The GIS database was then merged with health data, and spatial analysis of health was possible. Conclusions The methodological issues introduced in this paper provide solutions to the technical barriers in constructing household GIS database in a heavily populated urban setting. ==== Body Introduction Geographic data are increasingly being employed in health studies [1]. By studying disease patterns in space, we can understand the relationships between socioecological exposure and illness [2,3]. Such understanding may help the formulation of need based healthcare systems and health intervention programs. Geographic methods provide a wide spectrum of geographic scales from local to global for analyzing health and health-related data. Regional variation in disease incidence be attributed to regional or global differences in ecological or socio-environmental phenomena [4]. Local-level geographic variation of disease obtained from fine resolution geographic data can provide clues about the spatial variability [5], and may pinpoint areas where health interventions are needed. One way to facilitate the measurement of local variation in health outcomes is to create household-level geographic information systems (GIS) database. Household locations can be captured by using GPS (global positioning system) receivers [6,7]. However precise geographic data on households are an absolute requirement for critical examination of local variation of the disease and its association with the environment [8]. A large variety of GPS receivers are available in the market and different GPS receivers provide different levels of accuracy. A low cost receiver can capture data with an accuracy of 5 to 10 meters provided that they are configured properly and the satellites have good alignment at the time the data are collected [9]. The alignment of the GPS satellite constellation at a particular time can be measured using GPS trip planning software. It is essential that the GPS receiver has a clear "view" of at least four GPS satellites which can be obstructed by large structures such as buildings or mountains. In congested urban settings, collecting household locations in narrow lanes using the GPS can be challenging. Faced by such challenges we explored satellite imagery in order to acquire household GIS data in urban slums in Karachi, Pakistan. This paper describes the methods used to construct the household GIS database and the technical barriers one might encounter during the construction of a database. The household geographic information systems project Geographic studies have been considered as one of the research disciplines of large Vi (antigen) typhoid vaccine effectiveness trials as well as typhoid disease burden studies [10]. The Vi typhoid vaccine provides a comparable degree of protection to the whole-cell type but with less severe side effects. Only one dose is required for a course of vaccination. The studies are part of the Diseases of the Most Impoverished (DOMI) program, a multi-country, multi-disciplinary health research program aimed to accelerate the development and introduction of a new generation vaccines against cholera, typhoid fever, and shigellosis in several Asian countries. The program involves a number of parallel activities including epidemiological studies, social science studies, and vaccine technology transfer. The local collaborator of the household GIS project in Karachi is the Pediatric Department of the Aga Khan University Hospital, Karachi, Pakistan. Technical support for the project was provided by Techno-Consult International, Karachi, Pakistan. The aim of the project was to construct a spatial database that includes household locations, study area boundary with administrative units, and other geographic features such as hospitals/clinics, schools, mosques, roads, lanes, and water bodies. The project area included four Karachi slums including Sultanabad, Hijrat Colony, Rehri Goth, and Sherpao Colony (Figure 1). Sultanabad and Hijrat Colony are adjacent areas near the port of Karachi, and Rehri Goth and Sherpao Colony are about two kilometers apart located 20 kilometers South East of Karachi. In 2002, a census was conducted in the four slums to enumerate the study population. The list of households and their addresses were obtained from the population database. Figure 1 The study sites in Karachi, Pakistan. The geographic position of the four study sites along with other geographic characteristics of Karachi are shown in the map. The base map A commercially available map of Karachi was used as the base map for this GIS project. The base map was georeferenced with four identifiable landmarks using handheld GPS receivers with accuracy of approximately five meters. This accuracy was considered sufficient to identify the study areas, to order satellite imagery, and to conduct subsequent ground surveys. After georeferencing the map, the main geographic features such as roads, hospitals/healthcare centres, and other city landmarks were digitized and incorporated into the baseline GIS database (Figure 1). The satellite imagery Satellite imagery is available at different spatial, temporal, and spectral resolutions [11]. Different sensors capture images of the earth surface in different spectral resolutions, which allow different surface features to be differentiated. At the time of the project, the highest resolution commercially available satellite imagery was the one-meter panchromatic from the IKONOS (Space Imaging, Inc) satellite. We acquired a panchromatic IKONOS image (Figure 2) for two study slums, Sultanabad and Hijrat. We found that the one-meter panchromatic imagery was not helpful for separating lanes from building and mud roofs from open ground. We therefore acquired the four-meter multispectral IKONOS imagery for the two other study slums Rehri Goth and Sherpao Colony where many houses are made of mud. The result was more appropriate for our purposes even with the loss of spatial resolution. Figure 2 IKONOS image of Sultanabad, Karachi, Pakistan. The landscape of the Sultanabad study area obtained from IKONOS satellite imagery. Image processing and georeferencing The satellite images were enhanced using an image processing software package (ERDAS Imagine, Atlanta, USA) to facilitate the digitization of house parcel boundaries. High precision, dual frequency GPS units (Trimble 4000 ssi) were used to capture data at several identifiable points on the images to be used as ground control points (GCPs). To transfer images into a GIS database, it must be geometrically rectified to a known coordinate system on the basis of a number of GCPs [12]. Most of the GCPs were selected from the periphery of the study area so that possible errors would converge towards middle of the area. After locating GCPs on the satellite image and identifying them on the ground, GPS readings were obtained at centimeter level accuracy. The GPS data were collected in the WGS-84 (World Geodatic Systems-84) datum in the latitude/longitude system and were subsequently transformed into the Universal Transverse Mercator (UTM) Zone 42-North system. The GCP coordinates within the UTM projection were then integrated with the satellite images using the ERDAS Imagine software for georeferencing. The resultant root mean square (RMS) errors were approximately two meters, which was considered sufficiently accurate for the purpose of constructing the GIS database. Digitization of house parcels After georeferencing the images were resampled, converted into JPEG files, and were imported in AutoCAD Version 14 (Cadopolis.com Inc., Canada). These processes allowed the parcel boundaries to be delineated through heads-up digitizing. The resampled satellite imagery was inserted as backdrop in AutoCAD. The image was aligned to correct its scale, translation and rotation by using two GCPs located at the corners of the image. After the alignment of the raster images in AutoCAD the house parcels were digitized. One image was used on multiple workstations to digitize different portions of the image, which were subsequently merged to form a complete area map of house parcels (Figure 3). Figure 3 House parcels added to the satellite image, Sultanabad, Karachi, Pakistan. The house parcels drawn using AutoCAD superimposed on to the IKONOS satellite imagery. Ground survey The georeferenced image and the household parcel maps were used during a ground survey. The survey team consisted of civil engineers skilled in drawing household parcels and other field staff who conducted the census survey. The ground survey included verification of the size and orientation of household structures, resketching of the structures where needed, and locating specific household parcels so that they could be given their unique address identification number (ID) which was created during census survey (Figure 4). The address ID of each household was marked on the walls or doors at the time of census survey. Each time, the ground survey was started from a known location on the image, the household address ID was verified, and the IDs were marked on the hard copy map. Incorrect sizes or orientations of the digitized house parcels were also corrected during the ground survey. Figure 4 The address ID is used to link house parcels to population database, Sultanabad, Karachi. The unique census ID (address ID) of the household assigned to each parcel is shown inside the house parcel (the map in right side). The ID is used to link household census and disease surveillance data. After completing the ground surveys, the maps were updated using AutoCAD, and the address IDs were added to household parcels in the database. Finally, the household parcel AutoCAD files were imported as polygons into the ArcGIS software package (ESRI Inc., USA). The process included several checks for missing households, duplicate address IDs, and misplacement of address IDs and data were corrected when an error was found. The corrected data were validated by randomly selecting several household parcels (about 2%) from different zones of the study area and verifying their position on ground. At this stage, we observed no discrepancies in the data between ground verification and the satellite based maps suggesting that the household level GIS database is highly accurate. Implementation of the health GIS (HGIS) study The HGIS database is composed of spatial and non-spatial components (Figure 5). The spatial component consists of geographic features of households, roads, rivers, prominent places (e.g., hospitals), schools, and administrative boundaries. Each type of geographic feature was drawn in a single map layer. For instance, although both hospitals and schools were spatially referenced by points, we created two map layers for these two types of geographic features. The non-spatial component of the database consists of household-level data that include household socioeconomic status and individual-level data such as vaccination and disease history. The database relationships for patients with target diseases are shown in Figure 5. Figure 5 Entity relationship of the health study GIS database, Karachi, Pakistan. Inter-relationship between spatial and non-spatial database and intra-relationship of the database tables are shown here for the GIS-based health study research project. The descriptions of the entity relationship are descried in the texts. Entity relationships between data tables are shown as lines, and logical relationships between entities have parentheses around them (Figure 5). In the logical relationship (1,N), "1" indicates each entity should be linked to an entity on the other end, and "N" indicates multiple entities can be linked to an entity at the other end. Similarly, the "0" in (0,1) indicates not all entities will be linked to an entity at the other end. The "1" of the relationship indicates not more than one entity will link to an entity at the other end. For example, the relationship of "member" towards "patient" is shown as (0,1). Here "0" indicates not all records in "member" are to be linked in "patient," and "1" indicates not more than one record of the "member" can be linked to a record in "patient". Similarly, "1" in the relationship (1,N) of "patient" towards "member" indicates all records in "patient" should be linked to "member', and "N" indicates multiple records in "patient" can be linked to a record in the "member". Conclusion In this paper, we have outlined methodological issues involved in the construction of a household GIS database using satellite-based technology in a situation where the GPS was not appropriate. To our knowledge, this approach has never been reported, but may offer greater value in constructing household GIS databases compared to that based on GPS. Our household GIS offers size and orientation of individual houses in dense urban environment. Such database can be instrumental in health and disease studies because they facilitate the integration of socioecological and environmental factors that may influence health. Future health studies may benefit by using satellite-based technology to construct household GIS databases. Acknowledgement This work was supported by the Diseases of the Most Impoverished Program, funded by the Bill and Melinda Gates Foundation and coordinated by the International Vaccine Institute. The authors wish to thank Drs. Michael Emch and Lorenz von Seidlein for their valuable comments and suggestions in improving this paper. ==== Refs McGlashan ND McGlashan ND Medical geography: an introduction In Medical geography: techniques and field studies 1972 London: Methuen 3 15 Hunter JM Chapel Hill The geography of health and disease In The challenge of medical geography 1974 NC: University of North Carolina 1 3 Jones K Duncan C Individuals and their ecologies: analysing the geography of chronic illness within multilevel modeling framework Health & Place 1995 1 27 40 10.1016/1353-8292(95)00004-6 Kennedy S A geographic regression model for medical statistics Soc Sci Med 1988 26 119 129 3353743 10.1016/0277-9536(88)90051-2 Mayer JD The role of spatial analysis and geographic data in the detection of disease cauation Soc Sci Med 1983 17 1213 1221 6623127 10.1016/0277-9536(83)90014-X Ali M Emch M Ashley C Streatfield PK Implementation of a Medical Geographic Information System: Concepts and Uses Journal of Health, Population, and Nutrition 2001 19 100 110 11503345 Wilkinson D Tanser F GIS/GPS to document increased access to comminity-based treatment for tuberculosis in Africa The Lancet 1999 354 394 395 10437874 10.1016/S0140-6736(99)01893-0 Wilkinson P Grundy C Landon M Stevenson S Gatrell AC, Löytönen M GIS in Public Health In GIS and Health (GISDATA Series 6) 1998 Taylor & Francis 179 189 Kennedy M The global positioning system and GIS 2002 Second Taylor & Francis Camilo JA Claudia MG Ochiai RL Danovaro-Holliday MC Page AL Thiem VD Park JK Koo HW Park E Wang XY Elyazeed RA Ali M Ivanoff B Pang T Xu ZY Clemens JD The role of epidemiology in the introduction of typhoid fever Vi polysaccharide vaccines in Asia Journal of Health, Population and Nutrition Jensen JR Introductory Digital Image Processing: A Remote Sensing Perspective 2004 3 Prentice Hall Gao J Non-differential GPS as an alternative source of plainmetric control for rectifying satellite imagery Photogrammetric Engineering & Remote Sensing 2001 67 49 55

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Int J Health Geogr. 2004 Sep 28; 3:20

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Int J Health Geogr

10.1186/1476-072X-3-20

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==== Front Ann Clin Microbiol AntimicrobAnnals of Clinical Microbiology and Antimicrobials1476-0711BioMed Central London 1476-0711-3-171536959310.1186/1476-0711-3-17ResearchIncidence, risk factors and mortality of nosocomial pneumonia in Intensive Care Units: A prospective study Alp Emine [email protected]üven Muhammet [email protected]ıldız Orhan [email protected] Bilgehan [email protected] Andreas [email protected] Mehmet [email protected] Clinical Microbiology and Infectious Disease, Faculty of Medicine, Erciyes University, Kayseri, Turkey2 Intensive Care Unit, Faculty of Medicine, Erciyes University, Kayseri, Turkey3 Medical Microbiology, University Medical Centre St Radboud, Nijmegen, The Netherlands2004 15 9 2004 3 17 17 7 7 2004 15 9 2004 Copyright © 2004 Alp et al; licensee BioMed Central Ltd.2004Alp et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. To determine the frequency, risk factors and mortality of nosocomial pneumonia a prospective study was conducted in the intensive care units. In the study period, 2402 patients were included. The nosocomial pneumonia was defined according to the Centers for Disease Control Criteria. Overall, 163 (6.8%) of the patients developed nosocomial pneumonia and 75.5% (n = 123) of all patients with nosocomial pneumonia were ventilator-associated pneumonia. 163 patients who were admitted to the intensive care unit during the same period but had no bacteriologic or histologic evidence of pneumonia were used as a control group. The APACHE II score, coma, hypoalbuminemia, mechanical ventilation, tracheotomy, presence of nasogastric tube were found as independent risk factors. Crude and attributable mortality were 65% and 52.6%, respectively. The mortality rate was five times greater in the cases (OR: 5.2; CI 95%: 3.2–8.3). The mean length of stay in the intensive care unit and hospital in the cases were longer than controls (p < 0.0001). Patients requiring mechanical ventilation have a high frequency of nosocomial pneumonia. Hospital infectionsventilator-associated pneumoniadeath rates ==== Body Background Nosocomial pneumonia (NP) is the most frequent nosocomial infection in the intensive care units (ICU). The reported frequency varies with the definition, the type of hospital or ICU, the population of patients, and the type of rate calculated. In the recent studies, the incidence was reported as 6.8–27% [1-4]. In an one day point prevalence study in European ICUs, ICU-acquired pneumonia accounted for 46.9% of nosocomial infections [5]. The National Nosocomial Infections Surveillance (NNIS) system reported that NP accounts for 31% of all nosocomial infections in intensive care units [6]. The risk of pneumonia is increased in the intubated patients receiving mechanical ventilation (MV) and the ventilator associated pneumonia (VAP) frequencies varied between 7–70% in different studies [7-9]. NP developed at a rate of 0.9 cases per 1000 patient-days in non-ventilated patients versus rates of 20.6 cases per 1000 patient-ventilator-days and 14.8 cases per 1000 patient-days in patients who received any MV [10]. NP is also associated with high morbidity and mortality in ICUs. The increasing incidence of infections caused by antibiotic-resistant pathogens contributes to the seriousness of these infections. The mortality rate reaches to 20–50%, and also NP caused by high-risk pathogens (Pseudomonas aeruginosa, Acinetobacter spp., Stenotrophomonas maltophilia) are associated with higher mortality [1,11,12]. Patients with NP, stay 1 to 2 weeks longer than those without NP and result in higher costs [13]. Studies on NP are mainly reported from the United States and European countries, whereas studies from around the world are missing. The aims of this study were to assess incidence, risk factors and mortality of NP in Eurasian intensive care units. Methods Between February 2001 and February 2002 a prospective study was conducted among intensive care units (ICU) patients of the Erciyes University Hospital. This university hospital is a teaching hospital and full time intensivists care the patients in ICUs. Patients from the surgical ICU (SICU) (24 beds), medical ICU (MICU) (9 beds) and burn unit (7 beds) were included. The SICU consist of 8 neurosurgical (NICU), 8 general surgery (GICU) and 8 cardiac surgery (CICU) beds. Patients older than 16 yr of age were included. The same infection control doctor collected data and intensivist reviewed the diagnosis of pneumonia. Data collection included physical examination findings, APACHE II scores on admission, consciousness, risk factors (intubation, MV, presence of nasogastric tube, enteral nutrition, tracheotomy), prior surgery, immunosuppression, prior antimicrobial and antacid or histamine type 2 (H2) blocker therapy, clinical outcome, length of stay in ICU and in the hospital. 163 patients who were admitted to the ICU during the same period but had no bacteriologic or histologic evidence of pneumonia were used as a control group. In the ICUs infection control doctor collects active surveillance data routinely and empiric antibiotic therapy is directed at the most prevalent and virulent pathogens reported in these data. Appropriate antibiotic therapy included the administration of at least one antibiotic with in vitro activity against the bacterial pathogens isolated from the patient's respiratory secretions, as well as from blood and pleural fluid when applicable [14]. NP was considered when new and persistent (more than 48 h) pulmonary infiltrates not otherwise explained appeared on chest radiographs. Moreover, at least two of the following criteria were also required: 1) fever >38°C; 2) peripheral leukocyte count >10 000/mm3; 3) purulent endotracheal secretions with a Gram stain showing one or more types of bacteria [15]. VAP was considered when its onset occurred after 48 h of MV and was judged not to have been incubated before starting MV [16]. Admission APACHE II score was used to determine the severity of the illness, and attributable mortality was registered, as were laboratory values, electrocardiogram, x-ray, and arterial blood gas values. Extra length of stay was calculated comparing the extra stay after onset of pneumonia in the cases and after a reference date (the mean value of the extra stay after onset of pneumonia in the cases) in the control group. Microbiology Giemsa stains of sputum samples were performed for all patients. Sputum samples, containing more than 25 polimorphonuclear leukocyte (pnl) and less than 10 (×100) epithel were classified as purulent. If necessary, samples were obtained by nasotracheal aspiration. In that case, samples containing more than 10 pnl (×1000) were defined as purulent. Quantitative cultures of all purulent samples were performed using standard methods. Susceptibility testing was performed by disc diffusion method. In the absence of an alternative diagnosis a bronchoalveolar lavage was performed. In some case, pleural fluid was obtained by thoracentesis and examined for cell count, smear, Gram- and Giemsa-staining and microbiological culture. Statistical Analysis All data were evaluated using SPSS. Parameters were compared using univariate and multivariate logistic regression and chi-square tests. Student t test was used to compare the extra length of stay. Data were given as mean ± SD and a p-value of <0.05 was accepted as significant. Results During the study period, 2402 patients were admitted to the ICUs. Distribution of patients by ICU and length of stay in the ICU are shown in table I. Overall, 163 (6.8%) of the 2402 patients developed NP; 105 (5.8%) SICU- and 58 (11.7%) MICU-patients. The demographics of the NP patients and control group are shown in table II. The percentage of NP in NICU, GICU and CICU were 7.8%, 6.3% and 1.2%, respectively. During the study period no burn unit patient developed NP. The incidence of NP in MICU-patients was much higher than in SICU-patients (X2 = 19.7, p < 0.0001). Length of stay in the MICU was significantly higher than in the SICU, 21.3 ± 21.4 versus 16.2 ± 8.8 days, respectively (p < 0.05). Characteristics of patients who developed NP are shown in table III. Table I Numbers of patients and length of stay in ICU ICU No. of beds Total patient (n) Total length of stay (d) Mean length of stay (d) SICU 24 1806 5594 3.1  NICU 8 767 2213 2.9  GICU 8 636 2132 3.4  CICU 8 403 1249 3.1 MICU 9 495 2086 4.2 Burn Unit 7 111 1765 15.9 Total 40 2402 9445 3.9 Table II Demographic Factors of Study Patients* NP patients (n = 163) Control (n = 163) t p Age 53.30 ± 16.05 51.50 ± 16.87 0,985 >0,05 Admission APACHE II 10.86 ± 3.42 10.18 ± 4.56 1,526 >0,05 Gender 0,454 >0,05  Male 98 (60) 102 (63)  Female 65 (40) 61 (37) Diabetes mellitus 22 (14) 32 (20) 1,49 >0,05 COPD 36 (22) 11 (7) 4,027 <0.001 Cardiovascular disease 31 (19) 19 (12) 1,848 >0,05 Uremia 36 (22) 12 (7) 3,823 <0.001 Neoplasia 29 (18) 17 (10) 1,914 >0,05 Immunosuppressive therapy 6 (4) 3 (2) 1,013 >0,05 Coma 143 (88) 23 (14) 19,58 <0,001 Trauma 35 (22) 85 (52) 6,038 <0,001 * Data presented as mean ± SD or No. (%) Table III Characteristics of patients Characteristics MICU NICU GICU CICU Total Age (mean ± SD) 52.9 ± 15.1 50.25 ± 17.57 58.28 ± 14.38 55.00 ± 14.68 53.3 ± 16.1 Admission APACHE II, (mean ± SD) 11.2 ± 3.3 10.43 ± 3.12 11.03 ± 3.67 11.20 ± 6.38 10.9 ± 3.4 NP APACHEII, (mean ± SD) 16.2 ± 5.1 14.73 ± 3.94 16.30 ± 4.79 14.80 ± 6.37 15.6 ± 4.7 Length of stay in ICU (d, mean ± SD) 21.3 ± 21.4 14.42 ± 8.87 19.47 ± 8.20 12.20 ± 3.96 18.0 ± 14.7 Length of hosp. stay (d, mean ± SD) 25.0 ± 22.5 21.60 ± 11.93 24.78 ± 12.13 18.40 ± 8.26 23.5 ± 16.4 Overall, 17% of all patients requiring MV (n-724) developed VAP. Thereby, VAP accounts for 75.5% (n = 123) of all patients with NP (n = 163) during the study period (OR: 5,4; 95% CI: 3,36–8,75; p < 0,001) (Table IV). Mechanical ventilation was more frequently used in MICU patients than SICU patients (X2 = 6.6, p < 0.01). Consequently, the incidence of VAP was higher for MICU- than SICU-patients (X2 = 29.2, p < 0.0001). Furthermore, the length of ventilation was higher for patients admitted to MICU (6.3 ± 4.0) than SICU (5.1 ± 3.7), but the difference was not statistically significant. Table IV Rate of VAP in ICUs ICU No. of patients required MV No. of patients with VAP (%) SICU 550 72 (13.1)  NICU  141  38 (27.0)  GICU  157  31 (19.7)  CICU  252  3 (1.2) MICU 163  51 (31.3) Burn unit 11 0 (0) Total 724 123 (17.0) During the study period patients received 3128 ventilation days, with an average duration of 11.3 ± 10.0 days per ventilated patient. The device-related incidence rate for VAP was 39.3/1000 ventilation days. The incidence per 1000 ventilation days was 41.9 in SICU, 36.6 in MICU, 66.0 in NICU, 38.0 in GICU, and 9.1 in CICU patients. The mean onset day of NP after MV was 4.2 ± 3.9 days. Univariate analysis suggested the following risk factors for the development of NP: the APACHE II score, coma, COPD, uremia, hypoalbuminemia, MV, tracheotomy, enteral feeding, presence of nasogastric tube and previous treatment with broad-spectrum antibiotic (Table V). However, multivariate logistic regression showed that the APACHE II score (OR: 1.23; 95% CI: 1.13–1.33), coma (OR: 2.83; 95% CI: 1.24–6.47), hypoalbuminemia (OR: 2.23; 95% CI: 1.01–4.93), MV (OR: 3.35; 95% CI: 1.71–6.56), tracheotomy (OR: 6.03; 95% CI: 1.36–26.76) and presence of nasogastric tube (OR: 2.68; 95% CI: 1.33–5.41) were significant independent predictive factors for the development of NP. Table V Results of univariate analysis of potential risk factors for NP Risk Factors OR 95% Confidence Interval p Age 1.0 0.99 – 1.02 ns APACHE II 1.3 1.22 – 1.38 <0.001 Coma 6.6 3.75 – 11.48 <0.001 Trauma 1.7 0.93 – 2.97 ns COPD 3.9 1.91 – 8.01 <0.001 Diabetes mellitus 0.6 0.35 – 1.16 ns Central nervous system disorder 1.1 0.71 – 1.77 ns Uremia 3.6 1.78–7.14 <0.001 Hypoalbuminemia 3.3 1.99–5.61 <0.001 Mechanical ventilation 5.4 3.36–8.75 <0.001 Tracheotomy 12.5 3.75–41.89 <0.001 Enteral feeding 13.9 6.38–30.13 <0.001 Presence of nasogastric 6.3 3.89–10.18 <0.001 Previous antibiotic treatment 3.3 1.94–5.62 <0.001 Immunosupressive therapy 2.0 0.50–8.29 ns Antacids or H2 antagonist therapy 0.6 0.25–1.36 ns Thoracoabdominal surgery 1.0 0.63–1.69 ns ns: non-significant 187 pneumonia episodes were observed during the study period, resulting in the isolation of 257 microorganisms. The most commonly isolated pathogens were Gram-negative bacteria (85.6%). Among these pathogens, A. baumannii (29.6%), P. aeruginosa (20.6%), Klebsiella pneumoniae (14.4%) were the most common. Empiric antibiotic therapy was based on previous surveillance cultures and the Gram stain results. Therapy was adjusted according to the reports of susceptibility testing. Crude and attributable mortality were 65% and 52.6%, respectively. The mortality in patients without NP was 26.4% (Table VI). The risk of death was five times higher for patients with NP (OR: 5.2; 95% CI: 3.2–8.3; p < 0,001). The mortality rates were high in high risk pathogens (Table VII). The appropriateness of the empiric therapy did not contribute to increased mortality (Table VIII). Table VI Comparisons of outcomes between NP and control group NP group n (%) Control group n (%) X2 p Mortality 106 (65.0) 43 (26.4) 47.5 <0.0001 Improve 57 (35.0) 120 (73.6) Attributable mortality 52.6% Table VII Mortality rates in high risk pathogens Microorganism Mortality/Total (%) Gram negative 61/97 (62.9)  A. baumannii 31/42 (73.8)  P. aeruginosa 19/28 (67.9) Gram positive 10/15 (66.7)  MRSA 10/14 (71.4) Table VIII Appropriateness of empiric therapy and mortality Appropriate n (%) Inappropriate n (%) X2 p Survive 43/121 (35.5) 14/42 (33.3) 0.005 >0.05 Death 78/121 (64.5) 28/42 (66.7) The mean length of stay in the ICU and hospital for the patients with NP were 18.04 ± 14.74 days and 23.49 ± 16.44 days, respectively. The mean length of stay in the ICU and hospital for the control group 3.10 ± 3.03 and 9.64 ± 5.08 days, respectively. This difference was statistically significant (p < 0.0001). The extra stay in the control group was 4.36 ± 3.87 and 17.04 ± 14.17 in the patients (p < 0.001). Discussion The incidence of NP was reported different in different studies, which may be justified by the presence of different populations with variable ages, underlying diseases, and other associated risk factors. Incidence ranges from 6.8 to 27% [1-4] and also in this study it was 6.8%. Development of NP varies according to the different type of ICUs. Craven et al. [17] reported that the rate of pneumonia was higher in MICU but the difference was not significant. In the present study, the rate of NP and VAP was significantly higher in MICU than SICU, possible due to the differences in the proportion of patients that needed MV and the duration of MV. MV increases the risk of NP by 3- to 10-fold [1,18-23], resulting in an VAP incidence of 7 to 70% [7-9]. Generally, the duration of mechanical ventilation increases the risk of pneumonia. Cook et al. [24] reported that the rate of VAP increased 3% per day in the first week of ventilation, 2% per day in the second week, and 1% per day in the third week. In this study, 75.5% of the cases with NP occurred in ventilated patients. From 724 patients who required MV 123 (17%) developed VAP. Accordingly, patients on MV had a 3-fold higher risk to develop NP than the non-ventilated patients. Consequently, the use of non-invasive MV should be preferred whenever possible, since it has lower rates of nosocomial infections [25-27]. Coma was described as another important risk factor for NP. In these patients, local defense mechanisms of the respiratory airway are altered, allowing microorganisms to better attach to and colonize the mucosal surface. Furthermore, depression of the level of consciousness significantly increases the chance of aspiration, and as a result development of NP [3,28]. In our study, comatose patients had a 2-fold increased risk of NP. The causative agents of NP differ by the study population and diagnostic techniques but generally Gram-negative bacteria are the most common ones [3,4,28-33]. Colonization of the oropharynx, trachea or stomach with Gram-negative pathogens has been identified as a risk factor for NP [15,31]. Also in our study, the most common pathogens were Gram-negative bacteria. Furthermore, prior antibiotic therapy and COPD, leading to colonization with Gram-negative aerobic pathogens, were reported to be risk factors for the development of NP [11,28,30,34]. In our patient population, univariate analysis suggested that previous antibiotic treatment and COPD increased the risk of pneumonia, but interestingly they were not independent risk factors in multivariate analysis. Furthermore, the presence of a naso-gastric tube was found to be a risk factor in our study population. Naso-gastric tubes impair the function of the gastroesophageal sphincter and increase the risk of maxillary sinusitis, oropharyngeal colonization and reflux, all of which may lead to migration of bacteria [35]. Accurate evaluation of nutritional status and early initiation of enteral feeding is important in ICUs patients and can aid to preserve the gastrointestinal epithelium and prevent bacterial colonization. However, it may also increase the risk of gastric distention, colonization, aspiration, and pneumonia. Though, to reduce the risk of NP, it is important to avoid unnecessary enteral nutrition [30]. In univariate analysis, we found enteral feeding as a risk factor, but in multivariate analysis it was not an independent risk factor. For a long time it was assumed that increased gastric pH levels e.g. after the use of antacids, would allow Gram-negative microorganisms to multiply in the stomach, and consequently lead to an increased rate of NP. Our study results confirm what was reported by George et al. [36], namely that the use antacids or H2 antagonists did not increase the risk of NP. In the literature, tracheotomy is described as a significant risk factor for NP. Bronchial colonization during the procedure and (prolonged) continuation of sedation after the procedure will furthermore increase the occurrence of NP [28], a fact that was also seen in our patients. Patients with tracheotomy had a 7-fold increased risk of NP. The role of advanced age and high APACHE II scores as risk factors of NP are still under discussion. While Kollef et al. [37] report them as significant risk factors, earlier investigations do not support this [1,30]. In our present study, the APACHE II score was a significant risk factor for the development of NP; suggested that the severity of the general condition of the patient was important. Besides, uremia was found as a risk factor in univariate analysis. Patients with NP have a significantly higher morbidity and mortality [12,35,38]. Heyland et al. [38] reported the crude mortality rate of VAP 23.7% and an attributable mortality rate 32.3%. However, numerous studies have demonstrated that severe underlying illness predisposes patients in the ICU to the development of pneumonia, and their mortality rates are, as a result, high. Survival in patients with NP primarily by the degree of severity of illness at the time of diagnosis [23,39,40]. On the other hand, this does not exclude the possibility that certain subgroups of patients, such as patients with VAP caused by antibiotic resistant bacteria may have had extra attributable mortality rate [1]. In our study, the crude mortality rates for cases and controls were 65.0% and 26.4%, respectively and the mortality rates were highest in high risk pathogens. The mortality rate was five times greater in cases and attributable mortality of NP was 52.6%. Recent clinical investigations suggest that patients receiving inappropriate initial therapy have a greater mortality rate compared to patients receiving antibiotics to which the isolated bacteria were sensitive. However, in this study there was no statistically difference between the mortality rates of the patients who received appropriate and inappropriate initial therapy. As a result of the increased morbidity, patients with VAP remain hospitalized for 4–17 days longer than controls [35-38]. This observation was confirmed in the present study. The incidence of NP and VAP in MICU were significantly higher than in SICU patients and consequently the length of stay in the MICU was significantly higher than in the SICU. In conclusion, NP is a major cause of morbidity and mortality in ICU patients. Especially patients on mechanical ventilation are at high risk. Studies determining the impact of "old" and "new" risk factors of NP should repeatedly be performed in order to effectively guide the implementation of preventive measures methods. ==== Refs Fagon JY Chastre J Hance AJ Montravers P Novara A Gibert C Nosocomial pneumonia in ventilated patients: A cohort study evaluating attributable mortality and hospital stay Am J Med 1993 94 281 288 8452152 10.1016/0002-9343(93)90060-3 Vanhems P Lepape A Savey A Jambou P Fabry J Nosocomial pulmonary infection by antimicrobial-resistant bacteria of patients hospitalized in intensive care units: risk factors and survival J Hosp Infect 2000 45 98 106 10860686 10.1053/jhin.2000.0752 Rello J Ausina V Castella J Net A Prats G Nosocomial respiratory tract infections in multiple trauma patients. Influence of level of consciousness with implications for therapy Chest 1992 102 525 529 1643942 Richards MJ Edwards JR Culver DH Gaynes RP Nosocomial infections in medical intensive care units in the United States. National Nosocomial Infections Surveillance System Crit Care Med 1999 27 887 893 10362409 10.1097/00003246-199905000-00020 Vincent JL Bihari DJ Suter PM The prevalence of nosocomial infection in intensive care units in Europe. Results of the European Prevalence of Infection in Intensive Care (EPIC) Study. EPIC International Advisory Committee JAMA 1995 274 639 644 7637145 10.1001/jama.274.8.639 Richards MJ Edwards JR Culver DH Gaynes RP Nosocomial infections in combined medical-surgical intensive care units in the United States Infect Control Hosp Epidemiol 2000 21 510 515 10968716 Fagon JY Chastre J Domart Y Nosocomial pneumonia in patients receiving continous mechanical ventilation: prospective analysis of 52 episodes with use of a protected specimen brush and quatitative culture techniques Am Rev Respir Dis 1989 139 877 884 2930067 Craven DE Steger KA Barber TW Preventing nosocomial pneumonia: state of the art and perspectives for the 1990s Am J Med 1991 91 44S 53S 1928191 10.1016/0002-9343(91)90343-V Andrews CP Coalson JJ Smith JD Johanson WG Jr Diagnosis of nosocomial bacterial pneumonia in acute, diffuse lung injury Chest 1981 80 254 258 7273874 George DL Mayhall CG Nosocomial pneumonia Hospital Epidemiology and Infection Control 1996 Baltimore: Williams & Wilkins 175 195 Kollef MH Ventilator-associated pneumonia JAMA 1993 270 1965 1970 8411554 10.1001/jama.270.16.1965 Kollef MH Silver P Murphy DM Trovillion E The effect of late-onset ventilator-associated pneumonia in determining patient mortality Chest 1995 108 1655 1662 7497777 Strausbaugh LJ Mandell GL, Bennett JE, Dolin R Nosocomial Respiratory Infections Principles and Practice of Infectious Diseases 2000 Churchill Livingstone, Philadelphia 3020 3028 Blot S Vandewoude K Colardyn F Nosocomial bacteremia involving Acinetobacter baumannii in critically ill patients: a matched cohort study Intensive Care Med 2003 29 471 475 12577148 Centers for Disease Control and Prevention Guidelines for prevention of nosocomial pneumonia MMWR Recomm Rep 1997 46 1 79 Bonten MJM Controversies on diagnosis and prevention of ventilator-associated pneumonia Diagn Microbiol Infect Dis 1999 34 199 204 10403099 10.1016/S0732-8893(99)00040-1 Craven DE Kunches LM Lichtenberg DA Nosocomial infection and fatality in medical and surgical intensive care units patients Arch Intern Med 1988 148 1161 1168 3365084 10.1001/archinte.148.5.1161 Bell RC Coalson JJ Smith JD Johanson WG Multiple organ system failure and infection in adult respiratory distress syndrome Ann Intern Med 1983 99 293 298 6614678 Chevret S Hemmer M Carlet J Langer M Incidence and risk factors of pneumonia acquired in intensive care units. Results from a multicenter prospective study on 996 patients. European Cooperative Group on Nosocomial Pneumonia Intensive Care Med 1993 19 256 264 8408934 Langer M Mosconi P Cigada M Mandelli M Long-term respiratory support and risk of pneumonia in critically ill patients. Intensive Care Unit Group of Infection Control Am Rev Respir Dis 1989 140 302 305 2764365 Celis R Torres A Gatell JM Almela M Rodriquez-Roisin R Agusti-Vidal A Nosocomial pneumonia. A multivariate analysis of risk and prognosis Chest 1988 93 318 324 3338299 Cross AS Roup B Role of respiratory assistance devices in endemicnosocomial pneumonia Am J Med 1981 70 681 685 6938128 10.1016/0002-9343(81)90596-9 Chastre J Fagon JY Ventilator-associated pneumonia Am J Respir Crit Care Med 2002 165 867 903 11934711 Cook DJ Walter SD Cook RJ Incidence of and risk factors for ventilator associated pneumonia in critically ill patients Ann Intern Med 1998 129 433 440 9735080 Antonelli M Conti G Rocco M A comparison of noninvasive positive-pressure ventilation and conventional mechanical ventilation in patients with acute respiratory failure N Engl J Med 1998 339 429 435 9700176 10.1056/NEJM199808133390703 Nourdine K Combes P Carton MJ Beuret P Cannamela A Ducreux JC Does noninvasive ventilation reduce the ICU nosocomial infection risk? A prospective clinical survey Intensive Care Med 1999 25 567 573 10416907 10.1007/s001340050904 Girou E Schortgen F Delclaux C Association of noninvasive ventilation with nosocomial infections and survival in critically ill patients JAMA 2000 284 2361 2367 11066187 10.1001/jama.284.18.2361 Ewig S Torres A El-Ebiary M Bacterial colonization patterns in mechanically ventilated patients with traumatic and medical head injury Am J Respir Crit Care Med 1999 159 188 198 9872838 Georges H Leroy O Guery B Alfandari S Beaucaire G Predisposingfactors for nosocomial pneumonia in patients receiving mechanical ventilation and requiring tracheotomy Chest 2000 118 767 774 10988201 10.1378/chest.118.3.767 Memish ZA Cunningham G Oni GA Djazmati W The incidence and riskfactors of ventilator-associated pneumonia in a Riyadh Hospital Infect Control Hosp Epidemiol 2000 21 271 273 10782591 Garrouste-Orgeas M Chevret S Arlet G Oropharyngeal or gasric colonization and nosocomial pneumonia in adult intensive care unit patients Am J Respir Crit Care Med 1997 156 1647 1655 9372689 Rello J Torres A Microbial causes of ventilator-associated pneumonia Semin Respir Infect 1996 11 24 31 8885060 Rello J Quintana E Ausina V Incidence, etiology, and outcome of nosocomial pneumonia in mechanically ventilated patients Chest 1991 100 439 444 1864118 Soler N Torres A Ewig S Bronchial microbial patients in severe exacerbations of chronic obstructive pulmonary disease (COPD) requiring mechanical ventilation Am J Respir Crit Care Med 1998 157 1498 1505 9603129 Leal-Noval SR Marquez-Vacaro JA Garcia-Curiel A Nosocomial pneumonia in patients undergoing heart surgery Crit Care Med 2000 28 935 940 10809262 10.1097/00003246-200004000-00004 George DL Falk PS Wunderink RG Epidemiology of ventilator-acquired pneumonia based on protected bronchoscopic sampling Am J Respir Crit Care Med 1998 158 1839 1847 9847276 Kollef MH Prentice D Shapiro SD Mechanical ventilation with or without daily changes of in-line suction catheters Am J Respir Crit Care Med 1997 156 466 472 9279225 Heyland DK Cook DJ Griffith L Keenan SP Brun-Buisson C The attributable morbidity and mortality of ventilator-associated pneumonia in the critically ill patient Am J Respir Crit Care Med 1999 159 1249 1256 10194173 Rello J Rue M Jubert P Survival in patients with nosocomial pneumonia: Impact of the severity of illness and the etiologic agent Crit Care Med 1997 25 1862 1867 9366771 10.1097/00003246-199711000-00026 Rello J Diaz E Pneumonia in the intensive care unit Crit care Med 2003 31 2544 2551 14530765 10.1097/01.CCM.0000089928.84326.D2

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==== Front BMC BiolBMC Biology1741-7007BioMed Central London 1741-7007-2-211537738110.1186/1741-7007-2-21Research ArticleNormal histone modifications on the inactive X chromosome in ICF and Rett syndrome cells: implications for methyl-CpG binding proteins Gartler Stanley M [email protected] Kartik R [email protected] Ping [email protected] Theresa K [email protected] Jeff [email protected] Uta [email protected] R Scott [email protected] Department of Medicine, University of Washington, Seattle, WA, USA2 Department of Genome Sciences, University of Washington, Seattle, WA, USA3 Department of Genetics, Stanford University, Stanford, CA, USA2004 20 9 2004 2 21 21 23 6 2004 20 9 2004 Copyright © 2004 Gartler et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background In mammals, there is evidence suggesting that methyl-CpG binding proteins may play a significant role in histone modification through their association with modification complexes that can deacetylate and/or methylate nucleosomes in the proximity of methylated DNA. We examined this idea for the X chromosome by studying histone modifications on the X chromosome in normal cells and in cells from patients with ICF syndrome (Immune deficiency, Centromeric region instability, and Facial anomalies syndrome). In normal cells the inactive X has characteristic silencing type histone modification patterns and the CpG islands of genes subject to X inactivation are hypermethylated. In ICF cells, however, genes subject to X inactivation are hypomethylated on the inactive X due to mutations in the DNA methyltransferase (DNMT3B) genes. Therefore, if DNA methylation is upstream of histone modification, the histones on the inactive X in ICF cells should not be modified to a silent form. In addition, we determined whether a specific methyl-CpG binding protein, MeCP2, is necessary for the inactive X histone modification pattern by studying Rett syndrome cells which are deficient in MeCP2 function. Results We show here that the inactive X in ICF cells, which appears to be hypomethylated at all CpG islands, exhibits normal histone modification patterns. In addition, in Rett cells with no functional MeCP2 methyl-CpG binding protein, the inactive X also exhibits normal histone modification patterns. Conclusions These data suggest that DNA methylation and the associated methyl-DNA binding proteins may not play a critical role in determining histone modification patterns on the mammalian inactive X chromosome at the sites analyzed. ==== Body Background Although it has been known for some time that histone modifications play a role in gene expression [1], it is only in the last several years that the details of these modifications have been more fully described. Acetylation and methylation of histone tails, for example, exhibit characteristic patterns for expressed and repressed genes in all eukaryotes studied [2]. This generality of histone modification and gene expression holds for eukaryotes with and without DNA methylation, indicating that DNA methylation is not required for histone modification. In organisms with DNA methylation, however, interactions between histone modification and DNA methylation do appear to exist. In Neurospora, histone methylation appears to determine DNA methylation patterns [3,4]. In Arabidopsis, non-CpG DNA methylation also appears to be determined by histone methyltransferases, whereas CpG methylation does not [5,6]. In mammals, there is considerable evidence suggesting that methyl-CpG binding proteins may play a significant role in histone modification through their association with histone deacetylases [7-11]. Mutations in the MeCP2 methyl-DNA binding protein, which are the cause of most Rett syndrome cases [12], support this model, because human male and female cells with MECP2 mutations exhibit histone hyperacetylation [10]. Histone hyperacetylation was also observed in mice with Mecp2 mutations [13]. Thus, DNA methylation is upstream of histone modification in this model of methyl-DNA binding proteins and histone modification. Another possibility is that DNA methyltransferases themselves may target histone deacetylases through a noncatalytic domain, leading to histone modifications that are independent of other methyl-DNA binding proteins [14]. We are especially interested in the X chromosome with respect to the question of the relationship between DNA methylation and histone modification. The mammalian X chromosome is unusual in that about a thousand gene-associated CpG islands are hypermethylated on the inactive X and hypomethylated on the active X. Except for imprinted loci, methylation patterns at most other regions of the genome are similar between homologs. Histone modification differences known to be associated with either silent or expressed chromatin also distinguish the active and inactive X chromosomes [15-19]. Thus, the mammalian X chromosome inactivation system would appear ideal for testing whether or not a methyl DNA binding protein – histone modification pathway exists for the inactive X chromosome. To examine more fully the possible relationships between DNA methylation and histone modification, we have utilized cell cultures from individuals with a human hypomethylation disease called the ICF syndrome. This disease is clinically characterized by "Immune deficiency, Centromeric region instability, and Facial anomalies". In most cases, the molecular defects result from mutations in the DNMT3B methyltransferase gene [20-22]. Certain heterochromatic regions are markedly hypomethylated as a result of these mutations, including the CpG islands on the inactive X chromosome that are associated with genes [23] and LINE-1 elements [24]. If DNA methylation is upstream of histone modification, the histones on the inactive X should not be modified to a silent form in ICF cells. Our results indicate, however, that these histones do have modifications typical of silenced genes, suggesting that methyl-DNA binding proteins may not be critical with respect to histone modification on the inactive X chromosome. In addition, we examined clonal primary fibroblast cultures from two individuals with Rett syndrome and found that the histone modification pattern of the inactive X is not affected by mutations in MECP2. This suggests that this specific methyl-DNA binding protein does not have a major role in silencing the inactive X through histone modification. Results Cytological analysis of histone modification DNMT3B mutant cells (ICF syndrome) We examined histone modifications known to be associated with the inactive X chromosome in two ICF cell lines and normal control cells. Specific histone modifications including histone H3 and H4 acetylation, and histone H3 methylation at K4 and K9, were examined using antibodies to stain nuclei and metaphases [15-17]. We also examined histone macroH2A1 staining, which is known to be concentrated on the inactive X at interphase [25]. One hundred or more interphase nuclei that had an obvious sex chromatin body by DAPI staining were scored for histone modification. For acetylated histone (H3 and H4) and K4-methylated histone H3, the majority (>60%) of nuclei showed a characteristic hole at the sex chromatin body in both normal and ICF cells (Fig. 1A). For histone H3 methylation at K9, the frequency of positive cells (Fig. 1B) was lower (about 50%). We often noted a positive signal for methylated K4 histone H3 in an otherwise negative-staining sex chromatin region in both normal and ICF cells (Fig. 1A). This signal appears to derive from the DXZ4 locus that was previously reported by Chadwick and Willard [19] as having active-type histone modifications. DXZ4 is a megabase-sized region known to be hypermethylated on the active X and hypomethylated on the inactive X in normal cells [26]; this locus appears to be modified normally in ICF cells. Surprisingly, we did not observe this signal on metaphase spreads, suggesting that our resolution on these preparations may be much lower than on interphase chromatin. For metaphase spreads (acetylated histone H3 and H4 and histone H3 methylated at K4) we analyzed 50 cells per line, and in the great majority of analyzable metaphases (>80%) a single lightly-labeled chromosome was detected (Fig. 2). In some of these cells, the tip of the short arm was labeled, as would be expected for the pseudoautosomal region (Fig. 2). The expected patterns of macroH2A1 histone concentration and histone modification on the inactive X were found in cells derived from ICF individuals and in control normal cells (Figs. 1 and 2 and Table 1). At the cytological level, therefore, no difference could be found between normal and ICF cells with respect to the histone modifications on the inactive X. MECP2 mutant cells (Rett syndrome) We examined histone modifications and macrohistone association in clones from two individuals heterozygous for a mutation in MECP2, an X-linked gene that is subject to X inactivation [12]. MECP2 mutations lead to Rett syndrome, and the protein product codes for a methyl-CpG binding protein known to recruit a transcriptional silencing complex that deacetylates histones. In Rett syndrome and in mice with mutant Mecp2, histones exhibit hyperacetylation [10,13], as would be expected if this methyl-DNA binding protein functions upstream of histone modification. In one case, we had complementary clones with either the mutant (1786YY) or wild type MECP2 allele (1786QQ) on the inactive X, and in the second case, we had one clone with the wild type MECP2 on the inactive X (1789V) [27]. For all the histone modifications we examined (H3 and H4 acetylation and H3 K4 and K9 methylation), the cytological patterns on the active and inactive X chromosomes in mutant MeCP2-expressing cells were indistinguishable from those in clones expressing the wild type allele or in other control cultures (Fig. 1). These results suggest that MeCP2 does not function in determining these histone modification patterns on the inactive X chromosome. ChIP analysis To verify our cytological histone modification results at the gene level, we searched for promoter polymorphisms at seven X-linked loci (G6PD, NEMO, MECP2, SYBL1, AR, FMR1, and PGK1) in ICF cells so that we could employ allele-specific chromatin immunoprecipitation (ChIP) analysis. We restricted our search for polymorphisms to the promoter region, as several reports have indicated that marked differences in histone modifications between active and inactive alleles are seldom detected at other regions [28-30]. We found useful promoter polymorphisms at two loci, SYBL1 (synaptobrevin-like gene in the pseudoautosomal portion of Xq28) and AR (androgen receptor in Xq12). Previously, one of us (RSH) has reported on a ChIP study at the SYBL1 locus in male ICF cells where the inactive Y allele had reactivated and the histone modification pattern was that of an active gene [31]. Here we report on ChIP studies at the SYBL1 and AR loci in ICF female cultures using antibodies to histone H3 dimethylated at K4 and to acetylated histone H3. Both loci are subject to X inactivation, and the inactive X alleles remain inactive in ICF cells even though the 5' CpG islands are hypomethylated [23]. In the case of the SYBL1 inactive X allele, the methylation level is reduced by over 90% with most chromosomes exhibiting no methylation. An XhoI restriction site polymorphism in the untranslated exon 1 of SYBL1 permitted separation of the active and inactive alleles in cloned cells. A CAG repeat number polymorphism in the 5' coding region of the AR gene (1.3 kb downstream of the transcription start site according to reference sequence NM000044) was informative in one ICF sample (PT 4) and in several controls, thus permitting separation of the active and inactive alleles in cloned cells, and in cultures with highly skewed X inactivation. The antibodies were highly specific under the amplification conditions chosen, so that a strong signal was seen for the pull-down experiment with antibody and little or no signal for the "no antibody" control (Fig. 3A). The fluorescent amplification products from the AR gene were then separated on an automated sequencer according to CAG repeat number. Two major peaks are detected in the input control DNA, corresponding to the active (A) and inactive (I) X alleles, differing in CAG repeat number (Fig. 3B). An allele was determined to be from the active X by RT-PCR analysis (data not shown). The lesser "shadow band" peaks, labeled S, probably derive from PCR errors. In the methylated K4 H3 and acetylated H3 antibody ChIPs, a single peak predominates in both normal and ICF cells (Figs. 3B and 3C) that corresponds to the active X allele. Our ChIP analysis of the inactive X at the SYBL1 locus in an ICF female (PT3) also showed normal histone H3 hypoacetylation and K4 H3 hypomethylation in spite of the very low levels of DNA methylation in this CpG island region (Fig. 4). These data, therefore, agree with our cytological observations in that only the active X alleles are positive for the histone modifications known to be associated with active genes, though a minor portion of the inactive X allele was found to precipitate with the acetylated H3 antibody in both normal and ICF cells (Fig. 3C and data not shown). Discussion The major observation reported here is that ICF cells, despite being hypomethylated at gene- and L1-associated CpG islands on the inactive X chromosome, exhibit the same histone modification patterns as inactive Xs in normal cells. In addition, we show that cells mutant for MeCP2, a methyl DNA binding protein, also exhibit normal histone modification patterns on the inactive X. These results imply that DNA methylation and/or this methyl DNA binding protein are not critical for determining histone modification patterns on the inactive X chromosomes. Two major questions can be raised about our results: (1) is the sensitivity of the cytological histone modification assay too low to detect active-type histone modifications on the ICF inactive X? and (2) is the extent of methylation on the ICF inactive X greater than is suggested by CpG island and LINE-1 methylation patterns? The cytological results imply that most of the genes on the inactive X in ICF cells are subject to inactivation, a conclusion supported by our allele-specific expression analyses of individual genes, such as AR, in ICF cells ([23] and data not shown). For genes subject to X inactivation in ICF cells, we expect histone modifications at the gene level to be similar to those detected cytologically at the chromosome level, and this is what we have shown here for the AR gene. For genes that escape X inactivation in ICF cells, we expect their histone modification patterns to be those of expressed genes, and one of us (RSH) has previously reported this to be the case for the SYBL1 gene ([31] and data not shown). We did not detect these active patterns cytologically, suggesting that there are no large blocks of genes escaping inactivation in ICF cells except at the Xp pseudoautosomal region, which normally contains escaping genes (Fig. 2). Methylation levels at inactive X-linked CpG islands in ICF cells are decreased by an average of 89% from normal as determined by bisulfite analyses at the G6PD, FMR1, and SYBL1 loci, and many of the cloned alleles analyzed were completely unmethylated like active X alleles [23]. It is possible that DNA methylation at other CpG-rich regions (e.g., Alu and LINE-1 elements) on the X chromosome might be differentially methylated and play a role in the X chromosome histone modification pathway. One of us (RSH) has recently shown that LINE-1 elements are hypermethylated on both active and inactive X chromosomes in normal cells but, surprisingly, they are hypomethylated on the inactive X and hypermethylated on the active X in ICF cells [24]. These results argue against a role for LINE-1 methylation in histone modification on the inactive X chromosome. A more complete DNA methylation analysis of the ICF and normal inactive Xs needs to be done, however, because other widespread sequences may be hypermethylated on the ICF inactive X that could direct histone modifications. Because we know that promoter methylation is important in gene expression, it seems reasonable that if DNA methylation were directly involved in the histone modification pathway, CpG island methylation would play a critical role. Further support for this idea comes from the fact that histone modifications distinguishing active and inactive X-linked genes are concentrated in promoter regions [28,32]. In fact, Rougeulle et al. [32] propose that the promoter-restricted histone modification seen at X-linked loci may be unique to monoallelically-expressed genes and provide them with an epigenetic mark. That DNA methylation is not critical to the developmental appearance of histone modifications is further supported by recent murine studies showing that differential histone modification of the Xs in early development precede differential developmental appearance of DNA methylation [33,34]. The fact that DNA methylation does not appear to be critical to the development of histone modifications in X-linked gene expression should not be confused with a more important role for DNA methylation in maintaining repression of X-linked genes. Some years ago we showed that the earliest events in reactivating inactive X-linked genes were hemidemethylation followed by chromatin hypersensitivity, and then transcription factor binding and transcription [35,36]. More extensive studies have recently pointed to a similar conclusion [11,37,38]. Thus, DNA methylation appears to play a dominant role in maintaining repression, even though it is a late event in establishing silent chromatin. We can also consider the implication of this work for the proposed role of methyl-CpG binding proteins in the histone modification pathway. Our ICF cell data and the results from murine developmental studies, showing that histone modification of X-linked genes precedes DNA methylation, argue against such a role for the X chromosome. A role for methyl-DNA binding proteins in the histone modification pathway is supported by studies with Rett syndrome cells where a methyl-DNA binding protein, MeCP2, is mutated. In both humans and mice with Rett syndrome mutations, general hyperacetylation of histones was reported, albeit at different sites. In human cell lines, H4 was hyperacetylated preferentially at K16 [10], while in mouse mutant tissues hyperacetylation was reported specifically at H3K9 [10,13]. In our work, however, we saw no major effect of two different MECP2 mutations on inactive X histone modification. The recent discovery that LINE-1 elements on the inactive X are methylated by a methyltransferase distinct from the one that carries out the same modification on the active X raises the possibility that the inactive X could have its own modification rules [24]. We must consider, therefore, the possibility that the inactive X chromosome does not utilize methyl-DNA binding proteins in the histone modification pathway. Such a possibility would fit with the failure to detect protein footprints at promoters on the inactive X chromosome, whereas they are readily detectable on the active X [36,39-42]. It should be noted that only a small fraction of possible histone modifications have been elucidated at this time, and it is possible that histone modification on the inactive X that depends on methyl-DNA binding protein(s) will be found in the future. Finally, we would like to comment on the implication of this study regarding the inactive X silencing complex. Systems controlling gene expression tend to be multilayered and the X inactivation system is no exception. We know that silencing on the inactive X involves XIST RNA, DNA methylation, histone modification patterns, chromatin sensitivity, and delayed replication. It is our opinion that these factors tend to act in a more or less independent manner, as we have suggested several times in the past [23,43-46]. For example, promoter demethylation of inactive X-linked genes, as occurs in ICF cases, does not necessarily lead to reactivation; markedly advanced replication time must also be present for reactivation to take place [23]. The present study would appear to add further support to this idea. Conclusions The inactive X chromosome in mammalian cells is characterized by a particular set of histone modifications. It has been suggested that methyl-DNA binding proteins may be involved in these modifications through their interactions with histone deacetylases. We have investigated this idea by studying histone modification patterns on the inactive X in ICF and Rett syndrome cells. ICF cells are hypomethylated on the inactive X, in contrast to normal cells, and the Rett cells we studied were deficient in MeCP2, a specific X-linked methyl-DNA binding protein. We found that the histone modification patterns on the inactive X in these mutant cells were indistinguishable from those in normal cells. We conclude that DNA methylation and the associated methyl-DNA binding proteins do not appear to play a critical role in determining histone modification patterns on the mammalian inactive X chromosome, either globally or at the level of the promoter. Methods Cells and cell culture Mutant fibroblast cell cultures included two from female ICF individuals, whose DNMT3B mutations have been previously described [20], and complementary clonal cultures from an individual (Rett 1) heterozygous for a mutation (1155del132) in the MECP2 gene [27]. In one clone (1786YY), the mutant gene is on the active X, leading to a culture with nonfunctional MeCP2 protein; the complementary control clone (1786QQ) has functional MeCP2 because the mutant gene is on the inactive X. In another clone (1789V), derived from a Rett individual with the mutation R106W (Rett 2), the active X carried the mutant allele. Normal fibroblast cultures were obtained from commercial sources. For chromatin precipitation studies, the ICF fibroblast clones were immortalized with hTERT, as previously described [47]. Cells were grown in AmnioMax-C100 (Gibco Invitrogen Corp.; Carlsbad, CA) and harvested in trypsin:EDTA (Gibco Invitrogen Corp.) under standard conditions [46,48]. Cytology For analysis of interphase stages, cells were plated on alcohol-washed 22 mm square cover slips in 35 × 10 mm Petri dishes. On the following day the medium was removed and the cells were washed once with PBS followed by fixation in 95% ethanol:5% acetic acid for 1 min at room temperature. The rest of the procedure followed the "Immunocytochemistry Protocol" of Upstate (Lake Placid, NY). DAPI-stained slides mounted in antifade were examined with a Nikon Microphot FXA microscope and images were captured with a Nikon Coolpix 995 digital camera. The inactive X was recognized under DAPI staining as sex chromatin. Absence of a particular histone modification on the inactive X was seen as a hole or gap at the sex chromatin location. For analysis of metaphase cells, we plated cells in 150 × 25 mm tissue culture dishes and added colcemid (Gibco Invitrogen Corp.) 48 h later (0.1 μg/ml for 2 h). The medium was removed and the cells were washed once with Hanks' balanced salt solution, followed by trypsinization (Gibco Invitrogen Corp.) with slight agitation to collect metaphase cells. Serum was added to stop tryptic action and the cells were recovered by centrifugation, then placed in hypotonic solution (3 mg/ml KCl and 1 mg/ml sodium citrate) at 37°C for 15 min. Cells were collected on to slides using a Cytospin centrifuge and then fixed in 95% ethanol:5% acetic acid for 1 min. The rest of the procedure followed the Upstate protocol mentioned above, followed by DAPI staining, mounting in antifade, and examination with a Nikon Microphot FXA microscope. Antibodies used to detect histone modifications were obtained from Upstate and included: "Anti-acetyl-Histone H4," recognizing acetylated lysines 5, 8, 12, and 16, "Anti-acetyl-Histone H3 (Lys 9)," "Anti-dimethyl-Histone H3 (Lys 9)," "Anti-dimethyl-Histone H3 (Lys 4)," and "Anti-Histone macroH2A1." ChIP studies Chromatin immunoprecipitation was performed using the protocol of Upstate with slight modifications. For each experiment, a near-confluent 75 cm2 tissue culture flask (about 3 × 106 cells) was washed with PBS and treated with 4% formaldehyde (pH > 7) for 10 min at 37°C. Protease inhibitor cocktail (Complete) from Roche Diagnostics (Indianapolis, IN) was used in place of individual inhibitors, and Protein A-Separose 4B (Zymed Laboratories Inc.; South San Francisco, CA) was used to collect immune complexes. After elution of immune complexes, they were heated at 65°C for 4 h to reverse crosslinks, and the DNA was recovered with a "QIAquick" PCR purification kit from Qiagen Inc. (Valencia, CA). Primary amplification of AR DNA was across the polymorphic 5' CAG repeat region as previously described [23], using 27–35 cycles of PCR amplification with 10% of the immunoprecipitated material or 50 ng of input DNA in a 50 μl reaction volume. Allele-specific analysis was performed by amplifying the primary product with a 5'-FAM-labeled nested primer as previously described [23], using 6–15 cycles of PCR amplification with 2–10 μl primary product in a 50 μl reaction volume. All amplification conditions were chosen so as to produce visible products by ethidium staining only for antibody-precipitated material, and not for "no antibody" controls. The fluorescent products were then run on an ABI PRISM 310 capillary sequencer (Applied Biosystems; Foster City, CA) to separate alleles differing in CAG repeat number and analyzed using GeneScan software (Applied Biosystems). Allele-specific expression analysis by RT-PCR was performed on DNaseI-treated RNA using a similar procedure [23]. Analysis of the 5' region of the SYBL1 gene was performed similarly to that of AR, except the allele-specific reaction entailed XhoI digestion of the primary amplification product followed by these products being separated by agarose gel electrophoresis. Conditions for PCR amplification and XhoI digestion were as previously described [31]. List of abbreviations ac = acetylated ChIP = chromatin immune precipitation DAPI = 4,6-diamidino-2-phenylidole FB = fibroblast FAM = 5-carboxyfluorescein FITC = fluorescein-isothiocyanate ICF = immune deficiency, centromeric region instability, facial anomalies H3 = histone 3 H4 = histone 4 K4 = lysine 4 K9 = lysine 9 LINE-1 = long interspersed nuclear element 1 MeCP2 = methyl-CpG binding protein WT = wild type Xa = active X chromosome Xi = inactive X chromosome Authors' contributions SMG and RSH conceived the study design, supervised and coordinated its progress, and drafted and prepared the final manuscript. KRV and PL carried out the cell culture and cytological studies. TKC carried out the ChIP analyses. JT and UF developed the cloned Rett cell cultures. All authors read and approved the manuscript. Acknowledgments We thank the Geron Corporation for the hTERT cDNA and the laboratory of the late James K. McDougall for transfection reagents and protocols. Christine Disteche and Charles Laird provided helpful comments on the manuscript. This study was funded by grants from the NIH [HD16659, NS40163 (UF), T32 GM08748 (JT), and the March of Dimes Birth Defects Foundation (UF)]. Figures and Tables Figure 1 The interphase inactive X in normal and mutant cells: histone modification and macroH2A1 association. Photomicrograph examples of normal, ICF, and Rett fibroblasts that were FITC-labeled using antisera to various modified histones. Arrows point to sex chromatin on DAPI-stained cells, and to the corresponding sex chromatin site in the FITC-labeled photo. A. Normal, ICF, and Rett fibroblasts FITC-labeled using antisera to acetylated histone H4 (acH4), acetylated histone H3 (acH3), and dimethylated K4 histone H3 (meK4H3). Note that the sex chromatin body is not stained by these antibodies and appears as a hole or a gap that occasionally contains a prominent dot (see insets). This FITC-stained dot appears to correspond to the DXZ4 domain, as described in the text. B. Normal and ICF fibroblasts labeled with antibody to dimethylated K9 histone H3 (meK9H3) and macrohistone H2A1 (macroH2A). Figure 2 Histone modification on the inactive X in metaphases from normal and ICF fibroblasts (FB). Examples are shown of normal and ICF (PT3 and PT4) metaphase cells labeled with antisera to acetylated histone H4 (anti-acH4) and dimethylated K4 histone H3 (anti-meK4H3). Arrows point to X chromosomes stained with DAPI that correspond to Xs stained lightly with FITC-antibodies. Note the densely-stained acH4 region on the tip of the inactive X short arm in PT4. Such staining is also seen in normal cells and corresponds to the actively transcribed pseudoautosomal region. These observations indicate that on the inactive X, H4 is hypoacetylated and K4 H3 is hypomethylated in both normal and ICF cells. Figure 3 ChIP analysis of histone modification at the promoter region of the AR gene. Allele-specific ChIP analysis for H3 acetylation and H3 K4 methylation was performed using the highly polymorphic CAG repeat region in the 5' coding region of the androgen receptor in Xq12. A. "Normal" isoX fibroblasts with highly skewed inactivation (GM00088) and cloned fibroblasts from normal (1871) and ICF (PT4) cultures were subjected to ChIP and the immunoprecipitated material was amplified for AR analysis in two stages; nested FAM-labeled primers were used in the second stage for size analysis on an automated sequencer. The ethidium bromide-stained agarose gel of the secondary PCR products show strong signals in the input (Inp), anti-H3 dimethyl K4 antibody (mK4), and anti-H3 acetyl K9, K14 (acH3), but little or no signal in the "no antibody" controls (-). The weak bands with slower migration correspond to heteroduplexes with the primary amplimer, but these do not interfere with the allele-specific analysis of the denatured product. B. and C. Allele-specific analyses of the products shown in A, using an automated sequencer, revealed that the active X alleles (Xa) in normal cells (1871 and GM00088) are hypermethylated at H3 K4 (H3meK4) and hyperacetylated at H3 K9, K14 (AcetylH3), whereas the corresponding inactive X (Xi) alleles are hypomethylated and hypoacetylated. ICF cells (PT4) showed the same active X versus inactive X modifications as normal cells. "Shadow bands" (s) probably correspond to PCR errors. Allele assignment was ascertained by a similar method using allele-specific RT-PCR with DNased RNA. Figure 4 ChIP analysis of histone modification at the SYBL1 promoter region. Allele-specific ChIP analysis for H3 acetylation and H3 K4 methylation was performed using an XhoI polymorphism in the 5' UTR of the SYBL1 gene in the Xq28 pseudoautosomal region that is also present on the Y chromosome. In normal male cells, the Y-linked locus is inactivated, hypermethylated, and late-replicating as is the inactive X allele in female cells [23,31,49]. To determine if this region has abnormal histone modifications on the X-inactivated but DNA hypomethylated ICF female X, ChIP assays were performed for acetylated histone H3 (acH3) and K4-methylated histone H3 (mK4) in normal male lymphoblasts and in PT3 ICF female fibroblasts; the ethidium bromide-stained gels of each sample are shown before and after digestion with XhoI. The undigested alleles (XhoI+) are 268 bp; the digested alleles (XhoI-) result in fragments of 108 and 260 bp. The ChIP assay for acetylated histone H3 (acH3) shows that only the XhoI-digested allele (XhoI+) is hyperacetylated in a normal male lymphoblast (NMLB1), and this corresponds to the active X allele (Xa) by RT-PCR (data not shown). An hTERT-transformed clone of PT3 ICF fibroblasts was also analyzed by ChIP. This clone has normal monoallelic expression of SYBL1 even though the promoter region is extremely hypomethylated as determined by bisulfite methylation analysis of DNA. The inactive X allele in the PT3 clone is hypoacetylated at histone H3 and hypomethylated at H3K4 because only the active X allele (XhoI-) is immunoprecipitated with either the acetylated or K4-methylated histone H3 antibodies (although a small portion of the inactive X also appears to have been precipitated by the acetylated H3 antibody). 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==== Front BMC Dev BiolBMC Developmental Biology1471-213XBioMed Central London 1471-213X-4-121538315510.1186/1471-213X-4-12Research ArticleDifferential dynamics of histone H3 methylation at positions K4 and K9 in the mouse zygote Lepikhov Konstantin [email protected] Jörn [email protected] Department of Natural Sciences – Technical Faculty III FR 8.3, Biological Sciences, Institute of Genetics/Epigenetics, University of Saarland, Saarbrücken, Germany2004 21 9 2004 4 12 12 25 5 2004 21 9 2004 Copyright © 2004 Lepikhov and Walter; licensee BioMed Central Ltd.2004Lepikhov and Walter; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background In the mouse zygote the paternal genome undergoes dramatic structural and epigenetic changes. Chromosomes are decondensed, protamines replaced by histones and DNA is rapidly and actively demethylated. The epigenetic asymmetry between parental genomes remains at least until the 2-cell stage suggesting functional differences between paternal and maternal genomes during early cleavage stages. Results Here we analyzed the timing of histone deposition on the paternal pronucleus and the dynamics of histone H3 methylation (H3/K4mono-, H3/K4tri- and H3/K9di-methylation) immediately after fertilization. Whereas maternal chromatin maintains all types of histone H3 methylation throughout the zygotic development, paternal chromosomes acquire new and unmodified histones shortly after fertilization. In the following hours we observe a gradual increase in H3/K4mono-methylation whereas H3/K4tri-methylation is not present before latest pronuclear stages. Histone H3/K9di-methylation is completely absent from the paternal pronucleus, including metaphase chromosomes of the first mitotic stage. Conclusion Parallel to the epigenetic asymmetry in DNA methylation, chromatin modifications are also different between both parental genomes in the very first hours post fertilization. Whereas methylation at H3/K4 gradually becomes similar between both genomes, H3/K9 methylation remains asymmetric. ==== Body Background It is now generally accepted that the properties of a particular DNA sequence in cells are not solely defined by the nucleotide sequence itself, but by "epigenetic" modifications as well. Epigenetic modifications imply the methylation of cytosine residues in CpG dinucleotides and covalent modifications of core histones. These modifications allow for flexible, but heritable at the same time, reprogramming of the genome. In histone H3 five lysine residues can be methylated (K4, K9, K27, K36 and K79) [1]. Methylation at K4 and K9 play opposite roles in structuring repressive or accessible chromatin domains, with K4 methylation associated with transcriptionally active chromatin and K9 methylation with inactive chromatin in higher eukaryotes [2]. In addition, these lysine residues can be mono-, di- or tri-methylated, which contributes to the distinct qualities of H3/K4 and H3/K9 methylation. Similar to H3/K9 methylation, DNA methylation is associated with silenced chromatin and there appeared to be an interplay between the two epigenetic modifications. It is still an open question whether DNA methylation directs H3/K9 methylation or other way around, for both scenarios the experimental evidences do exist [3,4]. It recently has been shown that in mammalian cells the maintenance DNA methyltransferase DNMT1 is associated with proteins involved in chromatin reprogramming, including histones deacetylases, and is required for the establishment of H3/K9 methylation [5]. Various experimental data suggest that the DNA methylation causes multiple changes in local nucleosomes, such as deacetylation of histones H3 and H4, prevents H3/K4 methylation and induces H3/K9 methylation [6]. The fertilization of mouse egg causes dramatic changes in organization of both paternal and maternal genomes. Initially arrested in metaphase II oocyte completes the meiosis, forming haploid maternal pronucleus and extruding the second polar body. The densely packed with protamines sperm DNA decondences, protamines get exchanged by histones and DNA undergoes active demethylation. The demethylation in the early mouse zygote occurs asymmetrically on paternal DNA and affects different classes of repetitive and single copy sequences, but not the control regions of imprinted genes [7,8]. Previous studies have shown the exclusive localization of methylated H3/K9 in maternal pronucleus of the mouse zygote, which additionally marks the epigenetic asymmetry between maternal and paternal pronuclei [9-11]. Here we examine time dependent changes of chromatin structure in the mouse zygote, focusing on the dynamics of the acquisition of histones in the paternal pronucleus and methylation status of histone H3 at positions K4 and K9. Results and discussion In order to obtain mouse zygotes at different stages of development and to provide the precise timing for fertilization we used in vitro fertilization of mature mouse oocytes. Histones and methylated H3/K4 and H3/K9 were detected by using indirect immunofluorescence. In our experiments we used antibodies, which specifically recognize mono- or tri-methylated forms of H3/K4, and di-methylated H3/K9. The zygotes were analyzed after 3, 5, 8, 10, 12 and 18 hours incubation of mature oocytes with capacitated sperm from donor males. After 18 hours most of embryos were found to be at metaphase and some already at telophase stage of the first mitotic division. Even using in vitro fertilization, the obtained zygotes are not completely synchronous in their development and it is more appropriate to use PN stages classification, which is based on the morphological changes of both pronuclei [8,12]. Appearance of histones on paternal chromosomes We performed the immunostaining against core histones (anti-PanHistone antibodies) in all the stages tested in combination with antibodies, recognizing the specific methylated forms of histone H3. This served as a positive control for the immunostaining procedure and allowed us to follow the dynamics of histone acquisition in the paternal pronucleus. Histones were first detected shortly after the penetration of sperm into the oocyte and the beginning of the decondensation of sperm chromatin. According to PN stages classification we could clearly detect histones on paternal pronucleus at late PN0/early PN1 stages (approx. 3–5 hours p.f.), exactly when the global demethylation starts [8] (Fig. 1). Figure 1 Dynamic changes in chromatin of zygotes at different pronuclear stages. DNA is visualized by DAPI (blue colour) staining. Mouse monoclonalanti PanHistones antibodies were detected by fluorescein conjugated anti-mouse secondary antibodies (green colour). Specific rabbit polyclonal antibodies, recognizing H3/K4monoMe (a), H3/K4triMe (b) or H3/K9diMe (c) were detected by Rhodamine Red-X conjugated anti-rabbit secondary antibodies (red colour). Dynamic changes in H3/K4 methylation in paternal genome Probing the mouse zygotes at different stages with antibodies specifically recognizing either mono- or tri-methylated H3/K4 revealed that these types of modifications are associated with maternal genome through all zygotic stages, including mature oocyte and seem to be rather ubiquitous (Fig. 1a,1b). As for the paternal pronucleus – we detect the appearance of H3/K4mono-methylation in the beginning of PN1 (approx. 5 hours p.f.) stage (Fig. 1a), only slightly delayed compared to the appearance of core histones (Fig. 2). By PN3 – PN4 stages both paternal and maternal pronuclei show equal staining intensity. This indicates that H3/K4 specific histone methyltransferase, possibly Set9 [13], is quite active in the early zygote and methylates histone H3 after it is incorporated into the nucleosomes. In contrast to that, it has been shown recently that H3/K9 specific histone methyltransferase is inactivated immediately after the fertilization by yet unknown active mechanism, which involves de novo synthesis of some specific factors [11]. H3/K4tri-methylation becomes detectable later, starting from PN4 stage (approx. 8–10 hours p.f.) and the difference in antibodies staining intensity between paternal and maternal pronuclei becomes indistinguishable in the last pronuclear stage PN5 (approx 12 hours p.f.) (Fig. 1b) and in metaphase stage of first mitosis approximately 16 hours p.f. (Fig. 3a). The fact that H3/K4 first becomes mono-methylated and several hours later tri-methylated suggests progressive methylation of histone H3 at lysine 4. We also suggest that histone H3 gets incorporated into the nucleosomes being unmethylated and then undergoes methylation because we observe first the appearance of histones and then H3/K4mono-methylation. In contrast to that – acetylation of histones H3 and H4 happens before they are incorporated into the nucleosomes, and after the nucleosome assembly they can get deacetylated by histone deacetylases (HDACs) whenever required [14]. But no histone demethylase has been found so far. Figure 2 Distribution of histones and H3/K4monoMe in the zygotes at late PN0 stage. At this stage histones (green signal) are detectable in both male (♂) and female (♀) pronuclei, whereas H3/K4monoMe (red signal) is only detectable in female pronucleus and polar body (pb). Figure 3 Distribution of H3/K4triMe and H3/K9diMe in metaphase chromosomes during the latter portion of the first cell cycle. (a) Distribution of H3/K4triMe. Paternally and maternally derived chromosomes show equal staining pattern along the whole length of chromosomes. (b) Distribution of H3/K9diMe. This type of modification is not detectable on paternal chromosomes and in maternal chromosomes is mostly associated with centromeres. H3/K9 methylation but not H3/K4 defines the genomes asymmetry in the mouse zygote In order to compare the patterns of H3/K4 and H3/K9 methylation we performed the immunostaining of mouse zygotes using antibodies, which recognize di-methylated H3/K9. Our results are in the agreement with earlier observations that H3/K9 methylation is only attributed to the maternal genome and is completely absent from the paternal [9-11] (Fig. 1c, Fig. 3b). In normal somatic cells the absence or disruption of H3/K9 methylation leads to the chromosome instability and affects chromosomes segregation during mitosis [15]. Therefore the absence of H3/K9 methylation on paternal chromosomes is rather surprising and compromises its role in chromosomes segregation. The epigenetic asymmetry between paternal and maternal genomes is observed till 2-cell stage and is characterized by low levels of DNA methylation and H3/K9 methylation in paternal genome [8,10,11,16]. In case with H3/K4 methylation – the asymmetry is observed only in the beginning of the zygotic development and is indistinguishable in the metaphase stage of the first mitotic division (Fig. 3a). Recent data from Liu et al. suggest that H3/K9 methylation does not depend on DNA methylation [11], but it is only paternal DNA which gets demethylated in the mouse zygote and at the same time it does not have detectable H3/K9 methylation. According to data published by Santos et al. [17,18] DNA demethylation starts at PN1 stage, i.e. at a time when we first observe the appearance of H3/K4mono-methylation (PN1 stage, Fig. 1a), and is completed at PN3 stage when H3/K4mono-methylation in paternal pronucleus reaches approximately the same level as in the maternal (Fig. 1a). This fact is raising the question if such a coincidence might indicate that DNA demethylation and the establishment of H3/K4 methylation are interdependent. Demethylation of paternal DNA upon the fertilization is not a universal phenomenon for mammalian species. In bovine zygote paternal DNA becomes only partially demethylated, while in sheep and rabbit zygotes the demethylation is hardly detectable [17,18]. The analysis of chromatin modification in early zygotes of these species might help to get an answer if DNA demethylation depends on, or is directed by the specific chromatin modifications. Conclusions Unlike H3/K9 methylation, methylation of H3/K4 is not attributed only to the maternal genome but appears shortly after the acquisition of histones by paternal pronucleus. The methylation of H3/K4 is progressive and by first mitotic division reaches approximately same level as in maternal genome. Methods In vitro fertilization of mouse oocytes As sperm and oocytes donors we used (C57BL/6 X CBA)F1 mice. Mature oocytes were collected 14 hours post human chorionic gonadotropin injection according to standard procedures [19]. Sperm isolation and in vitro fertilization (IVF) procedures were performed as described in [20]. Briefly: the sperm was isolated from cauda epididimus of donor males and capacitated in pre-gassed HTF medium for 1,5 hours. Isolated oocytes in cumulus cell mass were placed into 100 μl drop of HTF medium with capacitated sperm and incubated in CO2 incubator for 3, 5, or 8 hours. For longer incubation time the oocytes were incubated with sperm in HTF medium for 8 hours and then transferred into the drop of pre-gasses and pre-warmed M16 medium and incubated further for 2, 4 or 10 hours. Immunofluorescence staining After the removal of zona pellucida by treatment with Acidic Tyrode's solution fertilized oocytes were fixed for 20 min in 3.7% paraformaldehyde in PBS, and permeabilized with 0.2% Triton X-100 in PBS for 10 min at room temperature. The fixed zygotes were blocked overnight at 4°C in 1% BSA, 0.1% Triton X-100 in PBS. After blocking the embryos were incubated in the same solution with either anti PanHistones (mouse polyclonal, Roche), anti mono-methyl H3/K4 (rabbit polyclonal, Abcam), anti tri-methyl H3/K4 (rabbit polyclonal, Abcam) or anti di-methyl H3/K9 (rabbit polyclonal, a gift from T. Jenuwein [21] antibodies at room temperature for 1 hour, followed by several washes and incubation for 1 hour with anti-mouse secondary antibodies coupled with fluorescein (Sigma-Aldrich), and anti-rabbit secondary antibodies coupled with Rhodamine Red-X (Jackson ImmunoResearch Laboratories Inc.). After final washes the zygotes were placed on slides and mounted with a small drop of Vectashield (VectorLab) mounting medium containing 0.5 μg 4,6-diamino-2-phenylindole (DAPI). At least 20 zygotes have been analyzed for each stage of zygotic development. Immunofluorescence microscopy The slides were analyzed on Zeiss Axiovert 200 M inverted microscope equipped with the fluorescence module and B/W digital camera for imaging. The images were captured, pseudocoloured and merged using AxioVision software (Zeiss). Authors' contributions KL conducted the experimental part of the work and co-wrote the manuscript. JW coordinated the study and co-wrote the manuscript. All authors read and approved the final manuscript. Acknowledgements This work was supported by the grant from Deutsche Forschungsgesellschaft (DFG) WA 1029/2-1. We thank Sven Oliger for his help with breeding the mice used in this study, Sabine Reither and Tarang Khare for participation in discussions and for their help in writing the manuscript, Auke Boersma and Susan Marschall for their invaluable help with the establishment of in vitro fertilization technique in our laboratory. We are grateful to Thomas Jenuwein for providing the antibodies against H3/K9diMe. ==== Refs Lachner M O'Sullivan RJ Jenuwein T An epigenetic road map for histone lysine methylation J Cell Sci 2003 116 2117 2124 12730288 10.1242/jcs.00493 Lachner M Jenuwein T The many faces of histone lysine methylation Curr Opin Cell Biol 2002 14 286 298 12067650 10.1016/S0955-0674(02)00335-6 Soppe WJ Jasencakova Z Houben A Kakutani T Meister A Huang MS Jacobsen SE Schubert I Fransz PF DNA methylation controls histone H3 lysine 9 methylation and heterochromatin assembly in Arabidopsis Embo J 2002 21 6549 6559 12456661 10.1093/emboj/cdf657 Tamaru H Zhang X McMillen D Singh PB Nakayama J Grewal SI Allis CD Cheng X Selker EU Trimethylated lysine 9 of histone H3 is a mark for DNA methylation in Neurospora crassa Nat Genet 2003 34 75 79 12679815 10.1038/ng1143 Xin H Yoon HG Singh PB Wong J Qin J Components of a pathway maintaining histone modification and heterochromatin protein 1 binding at the pericentric heterochromatin in Mammalian cells J Biol Chem 2004 279 9539 9546 14665632 10.1074/jbc.M311587200 Hashimshony T Zhang J Keshet I Bustin M Cedar H The role of DNA methylation in setting up chromatin structure during development Nat Genet 2003 34 187 192 12740577 10.1038/ng1158 Oswald J Engemann S Lane N Mayer W Olek A Fundele R Dean W Reik W Walter J Active demethylation of the paternal genome in the mouse zygote Curr Biol 2000 10 475 478 10801417 10.1016/S0960-9822(00)00448-6 Santos F Hendrich B Reik W Dean W Dynamic reprogramming of DNA methylation in the early mouse embryo Dev Biol 2002 241 172 182 11784103 10.1006/dbio.2001.0501 Cowell IG Aucott R Mahadevaiah SK Burgoyne PS Huskisson N Bongiorni S Prantera G Fanti L Pimpinelli S Wu R Gilbert DM Shi W Fundele R Morrison H Jeppesen P Singh PB Heterochromatin, HP1 and methylation at lysine 9 of histone H3 in animals Chromosoma 2002 111 22 36 12068920 10.1007/s00412-002-0182-8 Arney KL Bao S Bannister AJ Kouzarides T Surani MA Histone methylation defines epigenetic asymmetry in the mouse zygote Int J Dev Biol 2002 46 317 320 12068953 Liu H Kim JM Aoki F Regulation of histone H3 lysine 9 methylation in oocytes and early pre-implantation embryos Development 2004 131 2269 2280 15102709 10.1242/dev.01116 Adenot PG Mercier Y Renard JP Thompson EM Differential H4 acetylation of paternal and maternal chromatin precedes DNA replication and differential transcriptional activity in pronuclei of 1-cell mouse embryos Development 1997 124 4615 4625 9409678 Nishioka K Chuikov S Sarma K Erdjument-Bromage H Allis CD Tempst P Reinberg D Set9, a novel histone H3 methyltransferase that facilitates transcription by precluding histone tail modifications required for heterochromatin formation Genes Dev 2002 16 479 489 11850410 10.1101/gad.967202 Verreault A De novo nucleosome assembly: new pieces in an old puzzle Genes Dev 2000 14 1430 1438 10859162 Peters AH O'Carroll D Scherthan H Mechtler K Sauer S Schofer C Weipoltshammer K Pagani M Lachner M Kohlmaier A Opravil S Doyle M Sibilia M Jenuwein T Loss of the Suv39h histone methyltransferases impairs mammalian heterochromatin and genome stability Cell 2001 107 323 337 11701123 10.1016/S0092-8674(01)00542-6 Mayer W Smith A Fundele R Haaf T Spatial separation of parental genomes in preimplantation mouse embryos J Cell Biol 2000 148 629 634 10684246 10.1083/jcb.148.4.629 Shi W Dirim F Wolf E Zakhartchenko V Haaf T Methylation Reprogramming and Chromosomal Aneuploidy in In Vivo Fertilized and Cloned Rabbit Preimplantation Embryos Biol Reprod 2004 71 340 347 15028628 Beaujean N Taylor J Gardner J Wilmut I Meehan R Young L Effect of Limited DNA Methylation Reprogramming in the Normal Sheep Embryo on Somatic Cell Nuclear Transfer Biol Reprod 2004 71 185 193 14998909 Hogan Brigid Manipulating the mouse embryo : a laboratory manual 1994 2nd Plainview, N.Y., Cold Spring Harbor Laboratory Press xvii, 497 p. Peters AH Kubicek S Mechtler K O'Sullivan RJ Derijck AA Perez-Burgos L Kohlmaier A Opravil S Tachibana M Shinkai Y Martens JH Jenuwein T Partitioning and plasticity of repressive histone methylation states in mammalian chromatin Mol Cell 2003 12 1577 1589 14690609 10.1016/S1097-2765(03)00477-5

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==== Front BMC GenetBMC Genetics1471-2156BioMed Central London 1471-2156-5-281538505610.1186/1471-2156-5-28Research ArticleNo major association between TGFBR1*6A and prostate cancer Kaklamani Virginia [email protected] Lisa [email protected] Diana [email protected] Junjian [email protected] Nathan [email protected] Carole [email protected] Harry [email protected] Yu [email protected] Habibul [email protected] Kenneth [email protected] Boris [email protected] Cancer Genetics Program, Division of Hematology/Oncology, Department of Medicine and Robert H. Lurie Comprehensive Cancer Center, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA2 Clinical Genetics Service, Memorial Sloan-Kettering Cancer Center, New York, New York, USA3 Human Genetics Program, Department of Pediatrics, New York University Medical Center, New York, N.Y., 10016, USA4 Department of Epidemiology of Mailman School of Public Health and Herbert Irving Comprehensive Cancer Center, Columbia University, New York, New York, USA2004 22 9 2004 5 28 28 26 5 2004 22 9 2004 Copyright © 2004 Kaklamani et al; licensee BioMed Central Ltd.2004Kaklamani et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Prostate cancer is the most commonly diagnosed cancer in men and one of the leading causes of cancer deaths. There is strong genetic evidence indicating that a large proportion of prostate cancers are caused by heritable factors but the search for prostate cancer susceptibility genes has thus far remained elusive. TGFBR1*6A, a common hypomorphic variant of the type I Transforming Growth Factor Beta receptor, is emerging as a tumor susceptibility allele that predisposes to the development of breast, colon and ovarian cancer. The association with prostate cancer has not yet been explored. A total of 907 cases and controls from New York City were genotyped to test the hypothesis that TGFBR1*6A may contribute to the development of prostate cancer. TGFBR1*6A allelic frequency among cases (0.086) was slightly higher than among controls (0.080) but the differences in TGFBR1*6A genotype distribution between cases and controls did not reach statistical significance (p = 0.67). Our data suggest that TGFBR1*6A does not contribute to the development of prostate cancer. ==== Body Background Transforming Growth Factor Beta (TGF-ß) is one of the most potent inhibitor of cell growth [1]. Almost all cancer cells loose the ability to be growth inhibited by TGF-ß, which makes loss of TGF-ß growth inhibition a hallmark of cancer development [2]. TGFBR1*6A is a common variant of the type I TGF-ß receptor, TGFBR1 [3]. TGFBR1*6A (*6A) has a deletion of three GCG triplets coding for alanine within a nine alanine (9A) repeat sequence of TGFBR1 (*9A) exon 1, resulting in a six alanine (6A) repeat sequence. The 9-bp deletion that differentiates *6A from *9A is located within the predicted signal sequence cleavage region. In vitro studies have demonstrated that TGFBR1*6A responds less effectively than TGFBR1 to TGF-ß growth inhibitory signals [4,5]. The additional findings of an overrepresentation of TGFBR1*6A heterozygotes and homozygotes among patients with a diagnosis of cancer as compared with the general population led us to postulate that TGFBR1*6A might act as a tumor susceptibility allele [5]. Two recent meta-analyses show that TGFBR1*6A carriers may have an increased risk of breast, colon and ovarian cancer [6,7]. To test the hypothesis that TGFBR1*6A may contribute to the development of prostate cancer, we conducted a case control study of patients with biopsy verified prostate cancer cases and geographically and ethnic-status matched controls. Results A total of 907 cases and controls were genotyped for TGFBR1*6A. The mean age of cases was significantly higher than controls (p < 0.01) but there were no differences in ethnic status between the two groups. There were 59 TGFBR1*6A heterozygotes and three TGFBR1*6A homozygotes among cases, 62 TGFBR1*6A heterozygotes and 1 TGFBR1*6A homozygote among controls. TGFBR1*6A allelic frequency among cases (0.086) was slightly higher than among controls (0.080) but the differences in TGFBR1*6A genotype distribution between cases and controls did not reach statistical significance (p = 0.67) (Table 1). Effect estimates from conditional logistic regression were similar (OR 1.01, 95% CI 0.29–3.52) to those from unconditional logistic regression (OR 0.96, 95% CI 0.56–1.64). Analyses restricted to subjects aged 40 years and above controlling age on a continuous scale produced essentially the same effect estimates (OR 0.94, 95% CI 0.56–1.60). To examine the possibility that TGFBR1*6A is associated with early onset prostate cancer, we determined the prostate cancer risk for individuals above and below the age of 55. Among younger patients with prostate cancer we found that 13 of 59 were TGFBR1*6A carriers yielding an allelic frequency of 0.119, one of the highest TGFBR1*6A allelic frequency ever reported. Only 45 of 367 controls in the same age range were TGFBR1*6A carriers yielding an allelic frequency of 0.063, which is similar to the TGFBR1*6A allelic frequency found among 3,451 healthy controls from Europe and the U.S. [7]. The association between carrier status of TGFBR1*6A and prostate cancer in younger age group was significant after adjustment for race (OR 2.13, 95% CI 1.06–4.27) but was not significant after adjustment for race and age strata within groups (OR 2.11, 95% CI 0.98–4.57) (Table 2). While in the older age group we did not observe a significant association either (OR 0.57, 95% CI 0.30–1.10), the test for multiplicative interaction between age and carrier status of TGFBR1*6A was significant (p = 0.01). Table 1 Distribution of Age, Ethnicity, and TGFBR1 Genotypes and Adjusted Odds Ratios of Prostate Cancer by TGFBR1 Genotype Status Cases (N = 442) Controls (N = 465) P-value 1 Adjusted OR (95% CI)2 N % N % TGFBR1 genotype  9A/9A 380 86.0 402 86.5 0.67 1.00 (ref)  9A/6A 59 13.4 62 13.3 0.96 (0.56–1.64)  6A/6A 3 0.6 1 0.2 Age  20–40 1 0.2 205 44.1 <0.01  41–60 126 28.5 204 43.9  61–80 308 69.7 55 11.8  80+ 7 1.6 1 0.2 Race  White 396 89.6 415 89.3 1.00  Black 26 5.9 29 6.3  Hispanic 8 1.8 8 1.7  Asian 2 0.5 2 0.4  Other 1 0.2 2 0.4  Unknown 9 2.0 9 1.9 1p-value for Chi-Square or Fisher's Exact Test (comparing proportions) 2 OR was adjusted for age strata and race, based on dominant model. Table 2 Adjusted Odds Ratios of prostate cancer by age groups (> 55, <= 55 years old) Age group/ Genotypes Cases N (% within age strata) Controls N (% within age strata) OR (95% CI)1 OR (95% CI)2 P for testing multiplicative interaction Age = 55  9A/9A 46 (78.0) 322 (87.7) 1.00 1.00  9A/6A or 6A/6A 13 (22.0) 45 (12.3) 2.13 (1.06–4.27) 2.11 (0.98–4.57) Age > 55 0.01  9A/9A 334 (87.2) 80 (81.6) 1.00 1.00  9A/6A or 6A/6A 49 (12.8) 18 (18.4) 0.64 (0.36–1.17) 0.57 (0.30–1.10) 1 OR was adjusted for race. 2 OR was adjusted for race and age strata within age groups Discussion Prostate cancer is the most common cancer and the second most common cause of cancer death among U.S. men [8]. A similar pattern is observed throughout the western world. There is strong epidemiologic evidence indicating that a large proportion of prostate cancers are caused by heritable factors. The most convincing data is a study of 44,788 Scandinavian twins showing that 42% of prostate cancer cancers may be caused by shared genes [9]. Despite intense efforts led by several research teams, the search for prostate cancer susceptibility genes has thus far remained elusive. Recent studies suggest that carriers of deleterious mutations of the BRCA2 gene have an increased prostate cancer risk [10]. However, given the low prevalence of deleterious BRCA2 mutations in the general population, it is unlikely to account for a significant proportion of prostate cancer cases. Approximately 14% of the general population carries at least one copy of TGFBR1*6A, which makes it the most common candidate tumor susceptibility allele reported to date. While there is growing evidence that TGFBR1*6A predisposes to the development of breast, colon and ovarian cancer, our data do not suggest that it predisposes to the development of prostate cancer. We have previously shown that TGFBR1*6A homozygotes have an O.R of 2.69 and 2.02 for ovarian and colon cancer, respectively. The present study has the power to detect an O.R. for prostate cancer of 1.70 or higher and therefore rules out a major association between TGFBR1*6A and prostate cancer. However, it does not exclude a smaller O.R., which might have clinical relevance given the high TGFBR1*6A allelic frequency in the general population. It is possible that age differences in cases and controls affected the allele frequencies observed. If the TGFBR1*6A allele predisposes to a lethal malignancy such as prostate cancer, however, its frequency could be higher, not lower, in a younger cohort. Thus, the younger mean age of controls could result in a bias toward the null hypothesis, resulting in a stronger association than that observed. The intriguing findings of a high TGFBR1*6A allelic frequency among prostate cancer cases diagnosed before the age of 55 have to be cautiously interpreted given the fact that this group only included 46 patients. We have previously shown that TGFBR1*6A is not associated with an increased risk of bladder cancer [6]. Our results suggest that the association between TGFBR1*6A and prostate cancer is at best very weak but further studies are needed to formally exclude an association with early onset prostate cancer. Methods DNA was extracted from lymphocytes of blood specimens from 465 consecutive individuals diagnosed with adenocarcinoma of the prostate who received care at the outpatient urology clinic at Memorial Sloan-Kettering Cancer Center from April 2000 to September 2002. The blood samples were collected following completion of diagnostic studies. They were unselected for age or family history. Clinical and pathological records were reviewed to confirm the diagnosis of prostate cancer in all subjects. Once pathological diagnosis of prostate cancer was confirmed, the age of diagnosis was recorded, and all other identifying links were destroyed. The study design and anonymization method were approved by the Memorial Sloan-Kettering Cancer Center Institutional Review Board. A population of 465 healthy male controls aged 20 to 87 years with well-defined ethnic background who had donated blood for various reasons (predominantly pre-natal screening for non-cancer disease) constituted the control group. Controls were matched to the cases on ethnicity and were from the same geographic locations as the prostate cancer cases. None of the controls had any personal history of cancer at the time of blood donation. This was ascertained by a questionnaire completed by each control. Exact age information was not available for 205 controls since it was not collected prospectively but the age range (20 to 40) was known. All personal identifiers were permanently removed from both cases and controls. DNA was extracted by standard technique using the Qiagen DNA extraction kit. The PCR primers used were 5'-CCA CAG GCG GTG GCG GCG CGA TG-3' in the forward direction and 5'-CGT CGC CCC CGG GAF CAG CGC CGC-3' in the reverse direction. A standard solution was prepared using the Clontech Advantage® GC rich kit (BD-Biosciences Clontech, Palo Alto, CA). The PCR reaction mixture included 20 ng of genomic DNA in a 10-µL reaction volume and the following concentration of other reagents: primers (0.25 µM each), 1X GC genomic PCR reaction buffer, 1.625 mM Mg2+, 0.2 mM dNTPs and 0.16 µL of Advantage-GC genomic polymerase mix. Polymerase chain reaction cycling conditions consisted of an initial denaturation period of 3 minutes at 94°C, then 35 cycles of denaturation for 30 seconds at 94°C and annealing/extension for 2 minutes at 72°C, followed by a final extension step of 5 minutes at 72°C. Quality controls were run on a 2% agarose gel. The ABI Prism 310 Genetic Analyzer (Applied Biosystems, Foster City, CA) was used for data acquisition. A peak at 115 base pairs corresponded to TGFBR1 allele, whereas a peak at 107 base pairs corresponded to the TGFBR1*6A variant. The rare equivocal results were confirmed by cloning of the PCR product followed by automated sequencing. Samples were read by two independent investigators unaware of the case /control status. Ten percent of samples were randomly selected and run for quality assurance. Concordance rate was 100%. Statistical analysis Distributions of TGFBR1 genotypes, age, and ethnicity were compared between cases and controls using Fisher's exact tests. To test the hypothesis that the hypomorphic TGFBR1*6A gene is related to an increased prostate cancer risk, adjusted odds ratios of prostate cancer were estimated using both conditional and unconditional logistic regression models. Both models were run since the matched controls of cases with missing genotypes had to be excluded in the conditional models but could be included in the unconditional models. Adjusted odds ratios of prostate cancer were estimated comparing carriers of TGFBR1*6A versus non-carriers under dominant models. Potential confounders such as age (in four strata) and ethnicity were controlled in the analysis. Whether the effects of TGFBR1*6A on prostate cancer differ by age was evaluated by stratified analysis and tests for multiplicative interaction. A small p value indicates that interaction of age and gene is statistically significant on the multiplicative level. For the unconditional models, sensitivity analysis was conducted to evaluate the impact of the fact that the exact age of some controls with age 20–40 years is unknown (N = 126). With 442 cases and 465 controls, the power to detect an OR of 1.7 and 2 in the present study was 0.86 and 0.98, respectively, based on a two-tailed test at the 0.05 significance level. Authors' contributions All authors made substantial contributions to this paper, including conceiving of the ideas, discussion and writing. All authors read and approved the final manuscript. Acknowledgements This work was supported in part by grants CA89018 and CA90386 (B.P.) from the National Cancer Institute (Bethesda, MD) and a gift from the Mander Foundation (Chicago, IL). Dr Pasche is the recipient of a Career Development Award from the Avon Foundation, New York, NY. ==== Refs Siegel PM Massague J Cytostatic and apoptotic actions of TGF-beta in homeostasis and cancer Nat Rev Cancer 2003 3 807 820 14557817 10.1038/nrc1208 Bian Y Kaklamani V Reich J Pasche B TGF-beta signaling alterations in cancer Cancer Treat Res 2003 115 73 94 12613193 Pasche B Luo Y Rao PH Nimer SD Dmitrovsky E Caron P Luzzatto L Offit K Cordon-Cardo C Renault B Satagopan JM Murty VV Massague J Type I transforming growth factor beta receptor maps to 9q22 and exhibits a polymorphism and a rare variant within a polyalanine tract Cancer Res 1998 58 2727 2732 9661882 Chen T de Vries EG Hollema H Yegen HA Vellucci VF Strickler HD Hildesheim A Reiss M Structural alterations of transforming growth factor-beta receptor genes in human cervical carcinoma Int J Cancer 1999 82 43 51 10360819 10.1002/(SICI)1097-0215(19990702)82:1<43::AID-IJC9>3.3.CO;2-S Pasche B Kolachana P Nafa K Satagopan J Chen YG Lo RS Brener D Yang D Kirstein L Oddoux C Ostrer H Vineis P Varesco L Jhanwar S Luzzatto L Massague J Offit K T beta R-I(6A) is a candidate tumor susceptibility allele Cancer Res 1999 59 5678 5682 10582683 Kaklamani Virginia G. Hou Nanjiang Bian Yiansong Reich Jennifer Offit Kenneth Michel Loren S. Rubinstein WS Rademaker Alfred Pasche Boris TGFBR1*6A and Cancer Risk: A Meta-Analysis of Seven Case-Control Studies J Clin Oncol 2003 21 3236 3243 12947057 10.1200/JCO.2003.11.524 Pasche Boris Kaklamani Virginia G. Hou Nanjiang Young Taya Rademaker Alfred Peterlongo Paolo Ellis Nathan Offit Kenneth Caldes Trinidad Reiss Michael Zheng Tongzhang TGFBR1*6A and Cancer: A Meta-Analysis of 12 Case-Control Studies J Clin Oncol 2004 22 756 758 14966109 10.1200/JCO.2004.99.271 Jemal Ahmedin Tiwari Ram C. Murray Taylor Ghafoor Asma Samuels Alicia Ward Elizabeth Feuer Eric J. Thun Michael J. Cancer Statistics, 2004 Ca: a Cancer Journal for Clinicians 2004 54 8 29 14974761 Lichtenstein P Holm NV Verkasalo PK Iliadou A Kaprio J Koskenvuo M Pukkala E Skytthe A Hemminki K Environmental and heritable factors in the causation of cancer - Analyses of cohorts of twins from Sweden, Denmark, and Finland N E J Med 2000 343 78 85 10.1056/NEJM200007133430201 Edwards SM Kote-Jarai Z Meitz J Hamoudi R Hope Q Osin P Jackson R Southgate C Singh R Falconer A Dearnaley DP Ardern-Jones A Murkin A Dowe A Kelly J Williams S Oram R Stevens M Teare DM Ponder BA Gayther SA Easton DF Eeles RA Two Percent of Men with Early-Onset Prostate Cancer Harbor Germline Mutations in the BRCA2 Gene Am J Hum Genet 2003 72 1 12 12474142 10.1086/345310

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BMC Genet. 2004 Sep 22; 5:28

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==== Front BMC GenomicsBMC Genomics1471-2164BioMed Central London 1471-2164-5-721545391510.1186/1471-2164-5-72Methodology ArticleStrengths and weaknesses of EST-based prediction of tissue-specific alternative splicing Gupta Shobhit [email protected] Dorothea [email protected] Bernhard [email protected] Martin [email protected] Stefan A [email protected] Computational Molecular Biology, Max Planck Institute for Molecular Genetics, Ihnestr. 73, D-14195 Berlin – Germany2 German Resource Center for Genome Research, INF 580, 69120 Heidelberg – Germany2004 28 9 2004 5 72 72 26 6 2004 28 9 2004 Copyright © 2004 Gupta et al; licensee BioMed Central Ltd.2004Gupta et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Alternative splicing contributes significantly to the complexity of the human transcriptome and proteome. Computational prediction of alternative splice isoforms are usually based on EST sequences that also allow to approximate the expression pattern of the related transcripts. However, the limited number of tissues represented in the EST data as well as the different cDNA construction protocols may influence the predictive capacity of ESTs to unravel tissue-specifically expressed transcripts. Methods We predict tissue and tumor specific splice isoforms based on the genomic mapping (SpliceNest) of the EST consensus sequences and library annotation provided in the GeneNest database. We further ascertain the potentially rare tissue specific transcripts as the ones represented only by ESTs derived from normalized libraries. A subset of the predicted tissue and tumor specific isoforms are then validated via RT-PCR experiments over a spectrum of 40 tissue types. Results Our strategy revealed 427 genes with at least one tissue specific transcript as well as 1120 genes showing tumor specific isoforms. While our experimental evaluation of computationally predicted tissue-specific isoforms revealed a high success rate in confirming the expression of these isoforms in the respective tissue, the strategy frequently failed to detect the expected restricted expression pattern. The analysis of putative lowly expressed transcripts using normalized cDNA libraries suggests that our ability to detect tissue-specific isoforms strongly depends on the expression level of the respective transcript as well as on the sensitivity of the experimental methods. Especially splice isoforms predicted to be disease-specific tend to represent transcripts that are expressed in a set of healthy tissues rather than novel isoforms. Conclusions We propose to combine the computational prediction of alternative splice isoforms with experimental validation for efficient delineation of an accurate set of tissue-specific transcripts. ==== Body Background The large difference between cells from different tissues is the consequence of a complex regulatory machinery guiding the tissue specific expression of genes and their transcripts. Several genes have been described to exhibit differential splicing patterns for different tissues (E.g. PDE1C [1]; IRF-3 [2]) that result either in alternative proteins or affect the regulation of the respective gene product [3]. Due to the large number of genes generating alternative transcripts as well as by the complicated splicing machinery involving a large variety of different proteins, mis-splicing events are also frequently observed. Some of these artificial splice isoforms are already linked to specific diseases like Hemophilia A, Marfan syndrome etc. [4,5]. The resource mainly used to predict tissue-specific expression is the rapidly expanding repertoire of expressed sequence tags (ESTs) in the public databases representing a wide spectrum of tissue types. Unlike serial analysis of gene expression (SAGE) which mainly yields the tissue specific expression of genes [6], the EST data additionally allow the identification of alternatively spliced transcripts [7-11]. Besides the detection of the existence of alternative splice isoforms the tissue annotation of ESTs can be used for the computational prediction of the expression pattern of these transcripts where the tissue-wise count of transcript-specific ESTs with respect to a random background distribution defines an expression level [12-14]. Transcripts that are significantly over-represented by ESTs derived from a single tissue are usually defined as being tissue-specifically expressed. However, different cDNA construction protocols like normalization [15] include subtractive hybridization and PCR amplification steps introducing an artificial enrichment of ESTs from lowly abundant transcripts. The level of enrichment depends on the number of normalization/amplification steps performed, measured as Cot or Rot [16]. This inconsistency in the correlation of the number of ESTs observed for a transcript and its real expression level may affect the reliability of computational predictions of tissue-specifically expressed transcript. Since the EST-based prediction of expression patterns might already be error-prone because of the lack of sufficient numbers of EST sequences for each tissue this might be further complicated by different cDNA library protocols. Consequently, EST data related to normalized cDNA libraries are excluded from analysis in several computational approaches that aim at predicting tissue-specific expression [13,17]. Because of these uncertainties we combined our computational prediction of alternative splice variants and their expression pattern with experimental validation of these iso-forms via RT-PCR on 40 different tissue samples in order to evaluate the predictive potential of ESTs. Results The EST-based prediction of alternative splice iso-forms revealed 427 genes each contributing at least one potential tissue-specifically expressed variant. These variants show specificity for 28 different tissue types, where brain, testis and placenta account for approximately half of these transcripts (see additional file 1). Many of these genes (n = 210) exhibit isoforms that were exclusively detected due to ESTs derived from normalized libraries. These form a significant fraction (p-value: 8e-19) of the total genes that show tissue specific transcripts, since the number of ESTs derived from normalized libraries (896,645) is only 30% the total EST count (3,084,576) in tissues for which tissue specific isoforms exist. Out of the 20 isoforms tested experimentally (see additional file 3 for details of experiments), 15 isoforms could be successfully verified in some tissue (Table 1). The remaining five variants are either likely to resemble rare transcripts according to the respective library construction protocol, or as in case of a disease-specific isoform (Hs.272688), the appropriate tissue sample was not available for experimental testing. Only four of the isoforms predicted based on the basis of normalized libraries could be validated using the standard RT-PCR conditions. For five additional isoforms a more refined protocol had to be applied in order to detect bands of significant strength. More sensitive PCR conditions frequently revealed expression in more tissues indicating low expression of the isoforms in these tissues. These results show the tendency of normalized libraries to be enriched for low-abundant transcripts. Table 1 RT-PCR validation results for tissue and disease-specific splice isoforms. The experiments are categorized into three groups viz. tissue specific isoforms predicted via ESTs related to non-normalized libraries (1 to 4), tissue specific isoforms predicted only via ESTs derived from normalized libraries (5 to 16) and disease-specific isoforms (17 to 20). For some of the variants represented by normalized libraries, standard PCR did not reveal the isoforms. However, five of these isoforms were detected using refined PCR conditions. The experiments frequently validated the isoforms and the tissue type, but the predicted specificity was rarely verified. Isoform Gene Chr. Unigene EST Evidence ESTs Cycles Isoform Specificity Comment (Most sensitive PCR) Norm. Level 1 Unknown 11 Hs.112250 testis 3 39 + + 2 ISGF3G 14 Hs.1706 stomach 10 39 + - Ubiquitous 3 MRPL42 12 Hs.112110 stomach-lymph 5 39 + - Ubiquitous 4 SGN3 17 Hs.6076 testis 3 39 - ? 5 PC326 13 Hs.279882 testis 9 39 + + testis [36] Rot-5 6 LMO7 13 Hs.5978 brain 5 39 + - brain, testis, eye(?) [18] 7 HRD1 8 Hs.274122 brain 3 39 + - brain, eye, thymus, salivary gland, kidney 8 Unknown 1 Hs.24119 pancreas 4 39 + - approx. 10 tissues Cot-20 9 BCLG 12 Hs.11962 testis 4 39,78 ?,+ +,+ Cot-5 10 RBPMS 8 Hs.80248 placenta 4 39,78 ?,+ -,- Ubiquitous 11 SCML1 X Hs.109655 testis 12 39,78 ?,+ +,- approx. 6 tissues Rot-5 12 WNK1 12 Hs.432900 kidney 3 39,78 ?,+ +,- Digestive system [28] Cot-25 13 NY-CO-10 5 Hs.23557 testis 3 39,78 -,+ ?,+ Cot-5 14 Unknown 11 Hs.169100 testis 3 39,78 -,- ?,? Rot-5 15 Unknown 16 Hs.48396 breast 4 39,78 -,- ?,? Cot-230 16 CIDE-A 18 Hs.249129 breast 4 39,78 -,- ?,? Cot-230 17 KCNAB2 1 Hs.298184 tumor 29 39 + - Ubiquitous 18 SNRP70 19 Hs.174051 stomach ascites 25/26 39 + - Ubiquitous 19 RAB1 14 Hs.227327 tumor 39/95 39 + - fetal(kidney, thymus, liver, spleen), ovary [19] 20 Unknown 7 Hs.272688 tumor 12 39,78 -,- ?,? relevant tumor sample not in set The predicted expression of the isoforms in a single tissue could not be confirmed for half of the variants analyzed (standard conditions). However, the isoforms were always detected to be expressed in the tissue that was originally predicted by our software. The observed expression pattern of the 'unspecific' isoforms ranges from expression in only a few, sometimes related tissues (LMO7 [18]: brain, eye, testis, Fig. 2; HRD1: brain, eye, thymus, salivary gland, kidney) to ubiquitous expression (MRPL42, ISGF3G). Those variants that were validated to be specifically expressed frequently originate from testis. Increasing the sensitivity of the RT-PCR revealed another testis-specific variant. At the same time the variants of the genes WNK1 and SCML1 were no longer defined as being tissue-specifically expressed since they were now also detected in a few additional tissues (Table 1: isoform 11 & 12). The number of genes with transcripts exclusively expressed in tumors is relatively large (1120) as compared to the number of genes revealing tissue specific isoforms. Interestingly, 2 out of 4 such disease-related transcripts (Table 1: isoform 17–20) were ubiquitously expressed although the large number of ESTs covering these variants was suggesting a high significance of the prediction. The tumor associated isoform described by Wang et al. [19] was observed to be expressed in several fetal tissues along with ovary. Figure 2 RT-PCR validation experiment of a putative brain-specific isoform (LMO7). (A) The additional exon is detected in all tissues (primers F1, R1). (B) The primer pair F1-R2 located on exons flanking the extra exon results in two products where the shorter one is observed in brain, testis and eye (weak band). The predicted brain-specific expression pattern is, in fact, not specific. The entire dataset for human as well as the gel images from the RT-PCR experiments is available at . Discussion Consistent with previous work [11] our approach of combining computational and experimental validation yields a high success rate in predicting the existence of splice variants. In line with the expected general enrichment of clones derived from lowly expressed transcripts in normalized cDNA libraries our experimental results confirm the expression of the predicted low abundance transcripts. Consequently, those isoforms that could not be validated experimentally may also reflect real biological signatures of extremely rare transcripts since they are often represented just by heavily normalized libraries (Cot 230, CIDE-A + Hs.48396). While the methods used in the construction of normalized libraries (PCR amplification, subtraction, size selection) increase the sensitivity of the detection of transcripts they unfortunately disturb the rough correlation between the expression level of a transcript and the observed number of related clones that is usually maintained in non-normalized libraries. Therefore, in these cases, the larger number of ESTs found for a specific transcript will profess to deal with a higher expressed transcript, also implying a higher confidence in the prediction although the sequences may be derived from the same although amplified clone. From the computational point of view the artificially increased number of ESTs affects the likelihood to predict tissue-specifically expressed transcripts since the prediction mainly relies on the count of ESTs [12,13]. Nevertheless, our experimental results show that especially isoforms predicted to be expressed exclusively in testis could be successfully validated, while other isoforms frequently appear to be expressed in a set of additional tissues that were not suggested by the ESTs. Surprisingly, the absence of supporting EST evidence for the variants LMO7 and ISGF3G is not caused by the lack of the respective cDNA libraries but may rather reflect differences in the tissue samples (e.g. enrichment of different cell types from the same organ, developmental differences) used for library construction. In the context of tumors, our data shows that the predicted tumor-specific expression of isoforms derived from ESTs usually tends not to reflect the experimentally validated expression pattern. Rather it suggests expression in a collection of different tissues although the large number of related ESTs derived from tumor would imply a high confidence in the EST based prediction. Since tumor cells often show an up-regulation of a larger number of transcripts involved in various pathways [20,21] the tumor-specific transcripts predicted based on the EST data may just reflect this general deregulation of gene expression. The large number of predicted tumor-related isoforms further supports this hypothesis. Nevertheless, some transcripts detected via EST data may still serve as potential tumor markers like in case of the gene PRAME [22] where the EST data as well as the experimental data suggests specific expression in testis and in a variety of different tumors (see additional file 2). Overall, ESTs are an extremely powerful tool to reliably unravel alternative transcripts independent of the level of expression. The functional relevance of the low abundant transcripts is not yet clear, especially if the isoforms do not affect the coding sequence. These isoforms may either be related to processes like nonsense-mediated decay (NMD: [23,24]) or they might be some kind of non-functional leakage of the splicing machinery. Nevertheless, since many lowly expressed genes are already known to have important regulatory functions [25-27] this may also hold true for a not yet defined fraction of the alternative isoforms we detected via normalized libraries. In contrast to the prediction of the existence of isoforms, the task of predicting their expression pattern is much more error-prone since EST data always covers only a subset of potential tissues with variable sensitivity. The fuzzy terminology of tissue-specific expression that is frequently used to describe significant expression in a discrete tissue or a set of tissues, is therefore strongly biased by the sensitivity of computational and experimental methods (SCML1; WNK1 [28]). Beside these technical difficulties, the definition of specificity may also depend on the regulatory network that mediates tissue-specificity. While isoforms expressed in testis are specifically expressed in a more strict sense, other isoforms are expressed in a small set of (not necessarily related) tissues eventually pointing to alternative regulatory mechanisms acting with different stringency, e.g. involving transcription factors [29], [30] and/or DNA methylation [31,32]. Conclusions The separate evaluation of EST data from non-normalized as well as from normalized cDNA libraries will help to categorize transcripts into highly and lowly abundant ones thus facilitating the integration of EST-based predictions with expression data from microarray experiments. We suggest that large-scale analysis of tissue-specific transcripts should be ideally based on a computational prediction of isoforms that ranks candidate transcripts, tightly coupled with experimental validation via RT-PCR or DNA microarray experiments [33]. Such an approach will lead to a comprehensive set of verified isoforms suitable for a wide range of applications in the functional analysis of the regulation of tissue-specific expression. This will also improve the detection of tumor-related isoforms that do not just reflect a general up-regulation of gene expression. Methods The basis of our work is the tissue/tumor annotation of ESTs is GeneNest database [34] and the quality prediction of alternative splicing [11], visualized in SpliceNest database [10]. Library classification The cDNA libraries of the GeneNest database were semi-automatically categorized into non-normalized, normalized/subtracted and PCR-based libraries by screening for the appropriate keywords in the original annotation of the respective EMBL database entries. All libraries for which none of the keywords were found were defined as being non-normalized. PCR-based libraries like those derived by ORESTES PCR were not used for the current analysis. Additionally, to avoid miscounting caused by PCR amplification, ESTs of the same library and with identical start/end positions in the alignment were treated as a single sequence. Since the level of normalization of different libraries may differ depending on the number of rounds of subtractive hybridizations performed, we also extracted the normalization level (measured as Cot or Rot: [16]) as far as it was noted in the respective entries. Increasing Cot-values hereby reflect the enrichment of clones derived from low abundant transcripts in the respective cDNA library. Besides the categorization of cDNA libraries according to the construction methods used we further split these groups into libraries derived from healthy or disease tissue. Finally, ESTs of the four groups of cDNA libraries (healthy/non-normalized, healthy/normalized, disease/non-normalized, disease/normalized) were either analyzed separately or data of normalized and non-normalied libraries were combined. Prediction of tissue specific alternative splicing Alternative splice isoforms in the SpliceNest database are revealed by aligning EST consensus sequences (putative transcripts) related to one gene to the appropriate genomic sequence. Significant differences in the boundaries of the putative exons are interpreted as alternative splicing events. For all exon-exon-boundaries that define a certain splice iso-form the annotation of ESTs covering the respective boundary is evaluated. Isoforms overrepresented by ESTs from particular tissue are tagged as putative tissue/tumor specific splice isoforms. Several parameters (e.g. number of ESTs from a particular tissue, number of ESTs from other tissues, number of associated mRNA sequences etc.) are computed for these isoforms and finally stored in a relational database system. The refined set of tissue and tumor specific variants is then generated by setting the requirement of at least 3 ESTs in both alternative forms. Fig. 1 describes such a prediction using GeneNest and SpliceNest visualizations. Since the counts of ESTs per tissue-specific splice event were frequently below 5, we considered it inappropriate to apply statistical methods as were used by Xu et. al. ([12]). Figure 1 Detection of brain specific splicing in gene LMO7. The top part of the figure is a visualization of gene LMO7 in SpliceNest, showing parts of three transcripts with exons displayed as red blocks, connected by lines representing introns. The middle exon of the top transcript (Hs5978.1) is missing in the second transcript (Hs5978.2) and is therefore highlighted as an alternative splice event (green bar). The boundaries corresponding to this exon as well as the corresponding intron are visualized as vertical lines in the GeneNest database (left and right box respectively). Both regions are covered by several ESTs depicted by horizontal arrows with corresponding tissues encoded in coloured rectangles towards the left of each EST. Upon comparing the tissue distribution of these alternative regions it is evident that the middle exon of transcript Hs5978.1 is covered by ESTs derived from several tissues, while the corresponding exon junction that lacks this middle exon, in transcript Hs5978.2, is represented by ESTs derived from brain only, thereby revealing this as a brain specific splice event. Experimental verification A set of putative tissue specific (n = 16) and disease-related (n = 4) alternative splice events was arbitrarily selected for RT-PCR experiments. PCR primers were generated on the alternatively spliced exon as well as on either side of the event (Fig. 2) using the primer design software GenomePRIDE ([35]). For the subsequent RT-PCR experiment, total RNA was prepared using the single-step guanidinum method according to the manufacturer's instructions (TRIZOL, Gibco BRL). First strand cDNA synthesis was carried out in 20 μl reaction using the Omniscript Reverse transcriptase (Qiagen) and the oligo(dT) primers with 2 μg of total RNA. RT-PCR was carried out in a 20 μl reaction in 1 × buffer [1.5 mM Mg2+, 0.2 mM dNTPs, 0,4 μM primers each, 1 Unit of Taq polymerase (Roche)] and 1 μl of cDNA. Amplification steps were as follows: 95°C for 90 sec; 9 cycles of 94°C for 20 sec, 64°C for 10 sec decreasing the annealing temp for 1°C with each cycle (touchdown), 72°C for 20 sec; followed by 30 cycles of 94°C for 20 sec, 55°C for 10 sec, 72°C for 20 sec, followed by an extension at 72°C for 10 min. For the refined PCR, the amplification step was repeated with identical PCR conditions but with 2 μl of PCR product instead of 1 μl of cDNA. All PCR products were resolved on 2% agarose gels run at 90 V/20 cm for 1.5 h in TAE buffer. Gels were then manually examined for exact size, genomic contamination and the tissues in which the bands are observed. As a control, a fraction of variants were sequenced using the ABI Prism BigDye Terminators and the ABI Prism 3100 sequencer (Applied Biosystems). Authors' contributions SG wrote the prediction software as well as designed PCR primers for experimental analysis. SH and MV provided guidance for the computational work. DZ performed the RT-PCR experiments with the guidance of BK. Supplementary Material Additional File 1 List of tissues for which tissue specific transcripts are predicted. This is a text file with a listing of all tissues for which specific trascripts exist along with the number of ESTs related to individual tissues. Furthermore, the ESTs derived from normalized libraries and the specific variants predicted via such ESTs are also listed. Click here for file Additional File 3 Detailed description of RT-PCR experiments This is an excel file containing the primer sequences used for RT-PCR experiments along with detailed comments on the gel pictures subsequently obtained. Click here for file Additional File 2 RT-PCR picture (jpeg file) showing the expression pattern of gene PRAME This gene shows specific expression for several tumor types, along with testis as the only normal tissue. Click here for file Acknowledgements We thank Ms. Bianca Horeis for performing RT-PCR experiments. This work is supported by a grant from the German Human Genome Project (DHGP Grant 01KW0302). ==== Refs Yan C Zhao AZ Bently JK Beavo JA The calmodulin-dependent phosphodiesterase gene PDElc encodes several functionally different splice variants in a tissue specific manner J Biol Chem 1996 271 25699 25706 8810348 10.1074/jbc.271.41.25699 Karpova AY Howley PM Ronco LV Dual utilization of an acceptor/donor splice site governs the alter native splicing of the IRF-3 gene Genes Dev 2000 14 2813 2818 11090129 10.1101/gad.813800 Jin X Turcott E Englehardt S Mize GJ Morris DR The two upstream open reading frames of oncogene mdm2 have different translational regulatory properties J Biol Chem 2003 278 25716 25721 12730202 10.1074/jbc.M300316200 Caceras JF Kornblihtt AR Alternative splicing: multiple control mechanisms and involvement in human disease Trends Genet 2002 18 186 193 11932019 10.1016/S0168-9525(01)02626-9 Cooper TA Mattox W The regulation of splice-site selection and its role in human disease Am J Hum Genet 1997 61 259 266 9311728 Yamamoto M Wakatsuki T Hada A Ryo A Use of serial analysis of gene expression (SAGE) technology J Immunol Methods 2001 250 45 46 11251221 10.1016/S0022-1759(01)00305-2 Modrek B Resch A Grasso C Lee C Genome-wide detection of alternative splicing in expressed sequences of human genes Nucleic Acids Res 2001 29 2850 2859 11433032 10.1093/nar/29.13.2850 Kan Z States D Gish W Selecting for functional alternative splices in ESTs Genome Res 2002 12 1837 1845 12466287 10.1101/gr.764102 Thanaraj TA Stamm S Clark F Riethoven JJ Le Texier V Muilu J ASD: the Alternative Splicing Database Nucleic Acids Res 2004 32 D64 D69 14681360 10.1093/nar/gkh030 Coward E Haas SA Vingron M SpliceNest: visualization of gene structure and alternative splicing based on EST clusters Trends Genet 2002 18 53 55 10.1016/S0168-9525(01)02525-2 Gupta S Zink D Korn B Vingron M Haas SA Genome-wide identification and classification of alternative splicing based on EST data Bioinformatics 2004 Xu Q Modrek B Lee C Genome-wide detection of tissue-specific alternative splicing in the human transcriptome Nucleic Acids Res 2002 30 3754 3766 12202761 10.1093/nar/gkf492 Megy K Audic S Claverie J Heart-specific genes revealed by expressed sequence tag (EST) sampling Genome Biol 2002 3 RESEARCH0074 12537563 Xu Q Lee C Discovery of novel splice forms and functional analysis of cancer-specific alternative splicing in human expressed sequences Nucleic Acids Res 2003 31 5635 5643 14500827 10.1093/nar/gkg786 Bonaldo MF Lennon G Soares MB Normalization and subtraction: two approaches to facilitate gene discovery Genome Res 1996 6 791 806 8889548 Sagerstrom CG Sun BI Sive HL Subtractive cloning: past, present, and future Annu Rev Biochem 1997 66 751 783 9242923 10.1146/annurev.biochem.66.1.751 Schmitt AO Specht T Beckmann G Dahl E Pilarsky CP Hinzmann B Rosenthal A Exhaustive mining of EST libraries for genes differentially expressed in normal and tumour tissues Nucleic Acids Res 1999 27 4251 4260 10518618 10.1093/nar/27.21.4251 Putilina T Jaworski C Gentleman S McDonald B Kadiri M Wong P Analysis of a human cDNA containing a tissue-specific alternatively spliced LIM domain Biochem Biophys Res Commun 1998 252 433 439 9826547 10.1006/bbrc.1998.9656 Wang Z S LH Yang H Gere S Hu Y Buetow KH Lee MP Computational analysis and experimental validation of tumor-associated alternative RNA splicing in human cancer Cancer Res 2003 63 655 657 12566310 Corn PG El-Deiry WS Derangement of growth and differentiation control in oncogenesis Bioassays 2002 24 83 90 10.1002/bies.10036 Malumbres M Carnero A Cell cycle deregulation: a common motif in cancer Prog Cell Cycle Res 2003 5 5 18 14593696 Matsushita M Yamazaki R Ikeda H Kawakami Y Preferentially expressed antigen of melanoma (PRAME) in the development of diagnostic and therapeutic methods for hematological malignancies Leuk Lymphoma 2003 44 439 444 12688312 10.1080/1042819021000035725 Hillman RT Green RE Brenner SE An unappreciated role for RNA surveillance Genome Biol 2004 5 R8 14759258 10.1186/gb-2004-5-2-r8 Lewis BP Green RE Brenner SE Evidence for the widespread coupling of alternative splicing and nonsense-mediated mRNA decay in humans Proc Natl Acad Sci USA 2003 100 189 192 12502788 10.1073/pnas.0136770100 Hao H Tyshenko MG Walker VK Dihydrofolate reductase of Drosophila. 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BMC Genomics. 2004 Sep 28; 5:72

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10.1186/1471-2164-5-72

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==== Front BMC ImmunolBMC Immunology1471-2172BioMed Central London 1471-2172-5-221538315210.1186/1471-2172-5-22Research ArticleMAPK-dependent regulation of IL-1- and β-adrenoreceptor-induced inflammatory cytokine production from mast cells: Implications for the stress response Chi David S [email protected] S Matthew [email protected] Shannon [email protected] Karen [email protected] Ellis [email protected] Steven A [email protected] Shau-Ku [email protected] Guha [email protected] Department of Internal Medicine, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, Tennessee 37614, USA2 The Johns Hopkins Asthma and Allergy Center, Baltimore, MD 21224, USA2004 21 9 2004 5 22 22 24 3 2004 21 9 2004 Copyright © 2004 Chi et al; licensee BioMed Central Ltd.2004Chi et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Catecholamines, such as epinephrine, are elaborated in stress responses, and mediate vasoconstriction to cause elevation in systemic vascular resistance and blood pressure. Our previous study has shown that IL-1 can induce mast cells to produce proinflammatory cytokines which are involved in atherogenesis. The aim of this study was to determine the effects of epinephrine on IL-1-induced proatherogenic cytokine production from mast cells. Results Two ml of HMC-1 (0.75 × 106 cells/ml) were cultured with epinephrine (1 × 10-5 M) in the presence or absence of IL-1β (10 ng/ml) for 24 hrs. HMC-1 cultured alone produced none to trace amounts of IL-6, IL-8, and IL-13. IL-1β significantly induced production of these cytokines in HMC-1, while epinephrine alone did not. However, IL-6, IL-8, and IL-13 production induced by IL-1β were significantly enhanced by addition of epinephrine. The enhancing effect appears to involve NF-κB and p38 MAPK pathways. Flow cytometry showed the presence of β1 and β2 adrenoreceptors on resting mast cells. The enhancing effect of proatherogenic cytokine production by epinephrine was down regulated by the β1 and β2 adrenoceptor antagonist, propranolol, but not by the β1 adrenoceptor antagonist, atenolol, suggesting the effect involved β2 adrenoceptors. The enhancing effect of epinephrine on proatherogenic cytokine production was also down regulated by the immunosuppressive drug, dexamethasone. Conclusions These results not only confirm that an acute phase cytokine, IL-1β, regulates mast cell function, but also show that epinephrine up regulates the IL-1β induction of proatherogenic cytokines in mast cells. These data provide a novel role for epinephrine, a stress hormone, in inflammation and atherogenesis. ==== Body Background Atherogenesis involves the cellular infiltration of several cell types, including monocytes, T lymphocytes, and mast cells. Cytokine secretion by these cells and endothelial cells are contributing factors in the growth and propagation of atherosclerotic plaques as well as the stability and degradation of fibrous caps. Cytokines implicated in atherogenesis include Interleukin (IL)-1β, IL-6, IL-8, IL-13, and Tumor Necrosis Factor (TNF) [1,2]. IL-1β is secreted mainly by macrophages and virtually by every cell type in the body. IL-1β is produced in response to various stimulants, such as cytokines, bacteria, and viruses, but most interestingly to epinephrine [3]. IL-1β has a broad range of functions which includes activation of neutrophils, endothelial cells, monocytes, T-cells, and mast cells. It may also induce procoagulant changes in endothelial tissue. IL-6 induces an acute phase response consisting of increased fibrinogen synthesis and thrombocytosis with increased vascular permeability. The detection of IL-6 in the blood of patients suffering from unstable angina suggests that nuclear factor-kappa B (NF-κB) activation may be occurring at the vascular level in patients with heart disease [4-7]. IL-8 is in the CXC family of chemokines and functions to recruit neutrophils to the site of inflammation. IL-13 exerts multiple effects on cell differentiation and function of monocytes/macrophages. It can also suppress the cytotoxic function of monocytes/macrophages and the production of proinflammatory cytokines by these cells [8,9]. Mast cells are found preferentially around blood vessels and beneath the epithelium of the skin and mucus membranes [1,10-12]. Traditionally, mast cells are responsible for allergy and asthma pathogenesis. Typically, mast cell activation occurs in response to cross-linkage of the high affinity IgE receptor (FcεRI) by antigen and IgE [12]. Activation may also occur in response to a range of agents, such as pathogens, cytokines, and even oxidized low density lipoprotein (ox-LDL). After activation, key mediators secreted by mast cells include preformed mediators like histamine, proteoglycans, proteases, and several cytokines and growth factors [1]. Mast cells have been observed in both aortic atherosclerotic lesions and in coronary arteries. The large numbers of mast cells found in the adventitia of arteries and in the intima are in proportion to the severity of heart disease [13]. The study of the distribution, activation, and phenotype of mast cells in lesions of 250 specimens of human carotid arteries by Jeziorski, et al. further supports the role of mast cells in atherogenesis [14]. They demonstrated significant numbers and focal accumulations of mast cells in association with macrophages and extensive activation/degranulation at all developmental stages of atherosclerotic lesion development. It now appears likely that inflammatory events and mast cells play an important role in atherogenesis as recently reviewed by us [1,2]. Stress is known to influence immune function [15-17]. An immunoregulatory effect of the sympathetic nervous system in stress has been indicated for some time [18]. Catecholamines, such as epinephrine, norepinephrine, and dopamine, are elevated in stress responses, and mediate vasoconstriction and an increase in blood pressure as a result of increased peripheral vascular resistance. In disorders such as sepsis, cardiovascular disease, or cocaine abuse, catecholamines are elaborated in excess. Sustained increases in circulating catecholamines by infusion of epinephrine or norepinephrine have been shown to cause moderate cardiovascular and metabolic effects [19]. Catecholamines induce aggravation of aortic and coronary atherosclerosis in monkeys [20] and play a direct role in atherogenesis and cardiovascular disease [21]. Epinephrine and norepinephrine increase the uptake of low density lipoprotein in atheroscelotic plaques in rabbits and rats [22] as well as enhance proliferation of rat endothelial and smooth muscle cells [23]. It has been reported that norepinephrine increases adherence and chemotaxis of macrophages [24]. Epinephrine also upregulates the surface expression of L-selectin on monocytes in vitro [25]. Most recently, we have reported that nitric oxide production from macrophages induced by LPS is enhanced by catecholamines [26]. Both epinephrine and IL-1 are involved in acute phase responses seen in stress and in coronary artery disease. Studies have shown that norepinephrine can induce IL-1β mRNA in mycocardial tissue [27] and that infusion of IL-1β in animal models can induce expression of catecholamines [28,29]. These data suggest that, in some conditions, both IL-1β and catecholamines can be delivered to tissues that can then mediate additive or modulatory effects. Moreover, as reviewed by Gidron Y et al., [30] stress in conjunction with the release of catecholamines and proinflammatory cytokines, can potentiate atherogenesis. Hence, studies of the interactions between catecholamines, monokines and inflammatory cell activation are especially relevant. The aim of the study was to determine whether epinephrine affects IL-1β induced proatherogenic cytokine production in mast cells, a phenomenon previously not described. Our results indicated that epinephrine synergized with IL-1β in the production of proatherogenic cytokines, suggesting a potential role for this interaction in inflammatory and atherogenic states. Results Epinephrine enhances IL-1β-induced IL-6, IL-8, and IL-13 production in mast cells Human mast cell line, HMC-1, was incubated with IL-1β at various concentrations for 24 hours. The cell free supernatants of the cultures were harvested and subjected to IL-6 assay. HMC-1 cultured in medium alone produced trace amounts of IL-6. The IL-6 production from HMC-1 cultures treated with IL-1β was significantly increased in a dose-dependent manner (p < 0.0001) (Fig. 1). Since there was no significant difference in the IL-6 production induced by IL-1β at concentrations of 10 and 50 ng/ml, 10 ng/ml of IL-1β has been used to induce cytokine production in HMC-1 for the rest of the experiments. Epinephrine (Epi) alone at a concentration of 10-3 M did not induce production of IL-6 in HMC-1 (Fig. 2). When epinephrine at 10-3 to 10-7 M concentration was added simultaneously with IL-1β into the cultures, the production of IL-6 was enhanced significantly (p < 0.05) compared with that induced by IL-1β alone (Fig. 2). Since the physiological concentration of epinephrine in plasma is 0.11 – 0.27 × 10-6 M [31], we decided to use epinephrine at a supramaximal concentration of 1 × 10-5 M for the rest of the experiments. In addition to IL-6, the enhancing effect of epinephrine was also observed in the production of IL-8 and IL-13 from IL-1β-induced HMC-1 cells (Fig. 3). Figure 1 IL-1β induces IL-6 production from HMC-1 cells. To each well of a 6 well culture plate, two ml of HMC-1 mast cells (0.75 × 106cells/ml) were cultured with IL-1β (1, 10, and 50 ng/ml) for 24 hours. The cultures were carried out in triplicate. Supernatants were harvested for measuring IL-6 by ELISA. By Student's t-test analysis, * indicates p < 0.0001, when compared with the medium alone. + indicates p < 0.0005, when compared with the IL-1 (1 ng/ml) group. Figure 2 Enhancing effect of epinephrine on IL-6 production from IL-1β-induced HMC-1 cells. To each well of a 6 well culture plate, two ml of HMC-1 mast cells (0.75 × 106 cells/ml) were cultured with epinephrine (1 × 10-3 to 1 × 10-7 M) in the presence and absence of IL-1β (10 ng/ml) for 24 hrs in triplicate. Supernatants were harvested for measuring IL-6 by ELISA. By Student's t-test analysis, * indicates p < 0.05, when compared with the IL-1β-treated group. Figure 3 Effect of propranolol (Pro) and atenolol (Ate) on the enhancing effect of epinephrine (Epi) on production of IL-6 (A), IL-8 (B), and IL-13 (C) from IL-1β-induced HMC-1 cells. To each well of a 6 well culture plate, two ml of HMC-1 mast cells (0.75 × 106 cells/ml) were cultured alone (Medium), or in the presence of IL-1β (10 ng/ml), Epi (1 × 10-5 M), Pro (1 × 10-4 to 1 × 10-6 M), Ate (1 × 10-4 to 1 × 10-6 M), and the combinations of these reagents for 24 hrs in triplicate. Supernatants were harvested for measuring IL-6, IL-8, and IL-13 by ELISA. IL-8 and IL-13 production were not detected in the Medium, Epi, Pro, and Ate alone groups. In A and B, by Student's t-test analysis, * and + indicate p < 0.05, when compared with the IL-1β-treated group, and the IL-1β plus Epi group, respectively. In C, * indicates p < 0.01, when compared with the IL-1β-treated group; p values for ++, +, ##, and # were <0.00005, <0.0005, <0.01, and <0.05, when compared with the IL-1β plus Epi group. To measure proatherogenic cytokine gene expression, HMC-1 were treated with IL-1β, epinephrine, and IL-1β plus epinephrine for 6 hours and harvested for transcriptional analysis via RT-PCR. IL-1β-treated HMC-1 showed increased IL-6 mRNA transcription as seen with densitometry, while epinephrine alone appeared to have no effect. When IL-1β and epinephrine were added together to HMC-1, IL-6 mRNA expression increased over IL-1β treatment alone (Fig. 4). The intensities of the cytokine and house keeping gene (HPRT) bands were measured by densitometry, and the ratio of the cytokine to the house keeping gene was calculated and assigned as the intensity index. The intensity indices for IL-6 were 0.36 for the control, 0.39 for IL-1β alone, 0.33 for epinephrine alone, and 0.54 for IL-1β plus epinephrine. IL-1β activated IL-8 mRNA production but epinephrine had no effect on IL-8 transcripts. IL-1β and epinephrine treatment together further increased IL-8 mRNA production (Fig. 4). The intensity indices for IL-8 were 0.17 for the control, 0.52 for IL-1β alone, 0.20 for epinephrine alone, and 0.64 for IL-1β plus epinephrine. The results with IL-13 expression showed the same pattern. IL-1β was a good inducer of IL-13 transcription while epinephrine alone only minimally induced IL-13 mRNA. The combined stimulus of IL-1β and epinephrine significantly increased IL-13 mRNA production over that seen with each stimulus alone (Fig 4). Intensity indices for IL-13 were 0.22 for the control, 0.57 for IL-1β alone, 0.20 for epinephrine alone, and 0.64 for IL-1β plus epinephrine. To evaluate further the ability of epinephrine to induce IL-13 transcription at a molecular level, we transiently transfected HMC-1 cells with minimal promoter sequences as described in the materials and methods. IL-1β at 10 ng/ml significantly increased IL-13 promotor activity as detected by luciferase expression (data not shown). Epinephrine did not enhance IL-13 promoter activity suggesting that post-transcriptional mechanisms may be involved in the IL-13 induction. It is likely that epinephrine either prolongs IL-13 mRNA half life and/or enhances IL-13 secretory processes from the mast cell in response to IL-1-stimulation. Figure 4 RT-PCR analysis for IL-6, IL-8, and IL-13 in HMC-1 treated with IL-1β and epinephrine. HMC-1 were treated for 6 hours with IL-1β with and without epinephrine and harvested for RNA preparation. RNA was subjected to RT-PCR with specific primers for target genes. HPRT was used as a house keeping gene to ensure equal loading. IL-6 gene expression was increased with IL-1β treatment and further increased with IL-1β plus epinephrine. Epinephrine alone had no effect on IL-6 gene expression in HMC-1. IL-8 and IL-13 showed similar results with a more robust expression of gene transcripts at this time point. Enhancing effect of epinephrine on proatherogenic cytokine production from IL-1β-induced HMC-1 is down regulated by adrenoceptor antagonists Since our previous study has shown that the effect of epinephrine on nitric oxide synthesis is mediated by β-adrenoceptors [26], β-adrenergic receptor antagonists (propranolol and atenolol) were used to block the enhancing effect of epinephrine on proatherogenic cytokine production in HMC-1. Propranolol (Pro) and atenolol (Ate) at a concentration of 1 × 10-4 M did not affect the cell viability in the cultures (88 and 90%, respectively, while that of the medium control was 85%), nor induced production of IL-6, IL-8 or IL-13 (Fig. 3). When propranolol at 1 × 10-4 and 1 × 10-5 M was used in the culture, it significantly reduced the enhancing effect of epinephrine on IL-6 production (p < 0.05, Fig. 3A). Propranolol at 1 × 10-4 M also significantly reduced the enhancing effect of epinephrine on IL-8 production (p < 0.05, Fig. 3B), and at 1 × 10-4 and 1 × 10-5 M significantly reduced the enhancing effect of epinephrine on IL-13 production (p < 0.00005 and 0.0005, respectively, Fig. 3C). However, atenolol only significantly reduced the enhancing effect of epinephrine on IL-13 production (p < 0.05, Fig. 3C), but not on IL-6 or IL-8 production (Fig. 3A and 3B). Expression of β1 and β2 adrenergic receptors on mast cells In order to further identify whether the enhancing effect of epinephrine on proatherogenic cytokine production is through the β-adrenoceptor, HMC-1 cells were incubated with rabbit polyclonal antibodies against β1 and β2 adrenergic receptors followed by a FITC-labeled second antibody. By flow cytometry analysis, β1 and β2 adrenergic receptors were found in small amounts on HMC-1 (18.6 and 11.7% respectively) (Fig 5). Figure 5 Detection of β1 and β2 adrenergic receptors on HMC-1 cell by flow cytometry analysis. Resting HMC-1 were harvested and stained with a purified rabbit polyclonal antibody to either β1 or β2 adrenergic receptor and counter stained with a secondary goat anti-rabbit FITC conjugated antibody. Normal rabbit serum and the FITC conjugated goat anti-rabbit Ig G antibody was used as a staining control. Enhancing effect of epinephrine on proatherogenic cytokine production from IL-1β-induced HMC-1 is down regulated by immunosuppressants Since glucocorticoids are very effective treatment strategies for inflammatory disease, dexamethasone was used to determine its effect on atherogenic cytokine production in HMC-1. Dexamethasone (Dex, 1 × 10-7 M) alone did not induce proatherogenic cytokine production (Fig. 6). However, Dex significantly inhibited the enhancing effect of epinephrine on IL-6 production (p < 0.05, Fig. 6A). The cell viability of the cultures was not different between the medium control (70%) and Dex (64%) groups. Dex also significantly inhibited the enhancing effect of epinephrine on IL-8 and IL-13 production (p < 0.05, Fig. 6B and 6C). When Dex was included in the IL-1β-treatment, it slightly decreased the cytokine production when compared to the IL-1β alone, but the decrease was not significant (Fig. 6). Figure 6 Effect of dexamethasone (Dex) on the enhancing effect of epinephrine (Epi) on production of IL-6 (A), IL-8 (B), and IL-13 (C) from IL-1β-induced HMC-1 cells. To each well of a 6 well culture plate, two ml of HMC-1 mast cells (0.75 × 106 cells/ml) were cultured alone (Medium), or in the presence of IL-1β (10 ng/ml), Epi (1 × 10-5 M), Dex (1 × 10-7 M), and the combinations of these reagents for 24 hrs in triplicate. Supernatants were harvested for measuring IL-6, IL-8, and IL-13 by ELISA. * p < 0.005, when compared with the medium control, + p < 0.05 compared to the IL-1β-treated group, and ++ p < 0.05 compared to the IL-1β plus Epi group. Role of NF-κB activation in the enhancing effect of epinephrine on proatherogenic cytokine production from IL-1β-induced HMC-1 NF-κB is an important transcription factor that mediates the transcription of many proinflammatory cytokine genes. To study the role NF-κB plays in the enhancing effect of epinephrine on proatherogenic cytokine production from IL-1β-induced HMC-1, NF-κB activation was analyzed in HMC-1 cultures. NF-κB translocation, as seen by a shift in oligonucleotide binding in EMSA gels, was not seen in control or epinephrine treated cells (Fig. 7). A marked increase of NF-κB nuclear binding activity was observed in samples stimulated with IL-1β and IL-1β plus epinephrine for one hour but started to diminish after two hours (Fig. 7). Not only did IL-1β plus epinephrine have no further effects on NF-κB translocation over IL-1β treatment alone, it seemed to decrease after one and two hours of stimulation. Figure 7 Effects of IL-1β and epinephrine on NF-κB translocation in HMC-1. HMC-1 were treated for 1 and 2 hours with IL-1β and epinephrine. NF-κB translocation was analyzed by a shift in oligonucleotide binding in EMSA gels. After one hour of treatment, NF-κB translocation is increased in the IL-1β treated cells but not in the untreated or epinephrine treated cells. Addition of IL-1β plus epinephrine does not further enhance NF-κB translocation. After two hours of treatment, NF-κB translocation in HMC-1 starts to decrease. Role of p38 MAPK activation in the enhancing effect of epinephrine on proatherogenic cytokine production from IL-1β-induced HMC-1 Because of its importance in cytokine signaling, phosphorylated p38 MAPK was also assayed. After 30 minutes of activation, the HMC-1 were lysed to be analyzed for p38 activation by Western blot. The presence of phosphorylated p38 was greatly increased in the epinephrine and IL-1β plus epinephrine samples (Fig. 8). IL-1β alone had small effects on p38 activation at this time point while control levels were virtually nonexistent. Figure 8 Phosphorylated and total p38 MAPK in HMC-1 cells treated with IL-1β, epinephrine, and IL-1β plus epinephrine. HMC-1 were treated for 30 minutes with the indicated reagents and harvested for phosphorylated p38 expression by Western blot. Unphosphorylated p38 was used as loading control to show total MAPK expression. IL-1β treated cells showed a small amount of p38 activation while the bulk of p38 was activated with epinephrine. IL-1β plus epinephrine had no additional effects over epinephrine alone. Enhancing effect of epinephrine on proatherogenic cytokine production from IL-1β-induced HMC-1 is down regulated by NF-κB and p38 MAPK inhibitors To confirm the role of NF-κB and p38 MAPK in the enhancing effect of epinephrine on proatherogenic cytokine production from IL-1β-induced HMC-1, Bay 11, an NF-κB inhibitor [32], and SB203580, a specific inhibitor of p38 MAPK [33], were added to the cultures. By themselves, neither Bay 11 (1 × 10-5 M) nor SB203580 (1 × 10-5 M) affected the cell viability of the cultures (92 and 87%, respectively, while that of the medium control was 92%), nor did they induce proatherogenic cytokine production (Fig. 9). However, Bay 11 and SB203580 significantly inhibited the enhancing effect of epinephrine on IL-6 production (p < 0.0005 and p < 0.00005, respectively, Fig. 9A). Bay 11 decreased the IL-1β-epinephrine induced IL-8 production but not significantly, however SB203580 did significantly inhibit the enhancing effect of epinephrine on IL-8 production (p < 0.05, Fig. 9B). Bay 11 and SB203580 significantly inhibited the enhancing effect of epinephrine on IL-13 production (p < 0.00005 and p < 0.0001, respectively, Fig. 9C). Figure 9 Effect of Bay 11 and SB203580 on the enhancing effect of epinephrine (Epi) on production of IL-6 (A), IL-8 (B), and IL-13 (C) from IL-1β-induced HMC-1 cells. To each well of a 6 well culture plate, two ml of HMC-1 mast cells (0.75 × 106 cells/ml) were cultured alone (Medium), or in the presence of IL-1β (10 ng/ml), Epi (1 × 10-5 M), Bay 11 (1 × 10-5 M), SB 203580 (1 × 10-5 M), and the combinations of these reagents for 24 hrs in triplicate. Supernatants were harvested for measuring IL-6, IL-8, and IL-13 by ELISA. IL-8 production was not detected in the Medium, Epi, Bay 11 alone groups, while IL-13 production was not detected in the Bay 11 and SB 203580 alone groups. In A, by Student's t-test analysis, * indicates p < 0.005, when compared with the IL-1β-treated group, and + and ++ indicate p < 0.0005 and <0.00005, when compared with the IL-1β plus Epi group. In B, * indicates p < 0.05, when compared with both the IL-1β-treated group, and the IL-1β plus Epi group. In C, * indicates p < 0.005, when compared with the IL-1β-treated group, and + and ++ indicate p < 0.00005 and <0.0001, when compared with the IL-1β plus Epi group. Discussion Inflammatory cytokines play an important role in atherogenesis. Acute phase response (APR) proteins have been demonstrated as risk factors for atherosclerotic heart disease [34]. Recent studies also suggest a prominent role for the APR in cerebrovascular disease and brain ischemia [35]. The APR culminates in the secretion of inflammatory cytokines such as IL-6, TNF-α", and IL-1 resulting in the synthesis of several proteins including C-reactive protein, fibrinogen, serum amyloid A protein, and ceruloplasmin [36,37]. These cytokines are intimately involved with the stress response [38]. These cytokines can also induce transcriptional regulation of complement genes that have been shown to play a role in cardiovascular disease [39]. Catecholamines are elaborated in stress responses which mediate vasoconstriction and elevate systemic vascular resistance and blood pressure. Catecholamines induce aggravation of aortic and coronary atherosclerosis in monkeys [20] and play a direct role in atherogenesis and cardiovascular disease [21]. Thus, it is important to understand the interaction between epinephrine and IL-1β with respect to atherogenic cytokine production. In this study, IL-1β, an acute phase cytokine, activated mast cells to produce proatherogenic cytokines, IL-6, IL-8, and IL-13, in a dose-dependent manner (Fig 1, 2, and 3). These results confirm our previous report that IL-1β regulates mast cell function [40]. These results also show that epinephrine significantly up regulated the IL-1β induction of proatherogenic cytokines in mast cells giving new insight into neuronal regulation of the immune system. The gene expression of these proatherogenic cytokines was also increased in IL-1β-induced HMC-1 cells by addition of epinephrine, suggesting that the enhancing effect of proatherogenic cytokine production is a result of increased cytokine gene transcription (Fig. 4). These data provide a novel role for epinephrine in inflammation and atherogenesis. IL-1β signaling probably synergizes with β2-adrenoreceptor-mediated signaling pathways in inducing proatherogenic cytokine production. Several reports have shown that the effect of catecholamines on immune function is due to β-adrenoceptors [41-45]. Flow cytometry data indicated that HMC-1 cells express both β1 and β2 adrenoceptors in small amounts (Fig. 5). The result showed that the enhancing effect of proatherogenic cytokine production by epinephrine is down regulated by β1 and β2 adrenoceptor antagonist, propranolol, but not by β1 specific adrenoceptor antagonist, atenolol, further suggesting the enhancing effect involves β2 adrenoceptors (Fig. 3). The down regulation by propranolol does not appear to be due to cytotoxicity of the antagonist since there is no difference in viabilities between the propranolol-treated and untreated cell cultures. It was interesting to see that propranolol caused a reduction of production of IL-13 to an amount that was much lower than that treated with IL-1β only (Fig. 3). It may be that epinephrine-induced enhancement of IL-13 production is more sensitive to the propranolol blocking. Activated NF-κB has been demonstrated in atheromatous plaques and has been shown to play a role in atherogenesis [46]. To study the mechanism of the enhancing effect of epinephrine on proatherogenic cytokine production from IL-1β-induced mast cells, NF-κB and p38 MAPK activations were investigated. Control samples and epinephrine alone samples did not induce NF-κB activation. However, a marked increase in NF-κB activation was observed in samples stimulated with IL-1β and IL-1β plus epinephrine (Fig. 7). NF-κB activation was seen early at one hour and began to fade by two hours. NF-κB also was not increased by the addition of epinephrine to IL-1β and even seemed to decrease it at both time points suggesting that NF-κB is needed for cytokine induction but not for the enhancing effect. The presence of phosphorylated p38 MAPK was greatly increased in the epinephrine and IL-1β plus epinephrine samples but only minimally activated with IL-1β alone at a 30 minute incubation time point (Fig. 8). SB203580 blocked the IL-1β and IL-1β plus epinephrine effect on IL-6, IL-8, and IL-13 expression suggesting that p38 plays an important role in signaling from both IL-1β and epinephrine. The double stimulation of p38, early by IL-1β and later by epinephrine, may explain the enhancing effect on the production of IL-6, IL-8, and IL-13 in mast cells. The enhancing effect of epinephrine on proatherogenic cytokine production was also down regulated by immunosupressants, such as Dex. Dex at the concentration used in this study did not affect the cell viability of the culture, suggesting the down regulation effect of the drugs is not due to toxic effect. Dex also slightly, but not significantly, decreased IL-1β-induced cytokine production in mast cells (Fig. 6). Taken all together, these results indicate that β2-adrenoceptor antagonists and glucocorticoids may have clinical potential in stress-mediated disease and atherogenesis. All the signaling pathways induced by IL-1β and epinephrine in mast cells are complex and beyond the scope of this manuscript. However, two important inflammatory pathways, NF-κB and p38 MAPK, have been shown. IL-1β release from immune challenge and epinephrine elevated from stress response can jointly stimulate mast cells to increase IL-6, -8, and -13 production above that which is seen with either stimulus alone. The exact mechanisms are unclear, but we have shown that IL-1β is a strong inducer of NF-κB while epinephrine is a strong inducer of p38 MAPK. Neither NF-κB nor p38 MAPK was activated further by IL-1β plus epinephrine compared to either stimulus alone nor was the promotor activity of IL-13 increased by the double stimulus as seen by luciferase activity of a IL-13 reporter gene construct. These data would suggest that IL-1β is activating IL-6, IL-8, and IL-13 by NF-κB while p38 MAPK activation is enhancing protein production by inducing other transcription factors, stabilizing the gene mRNA, or other forms of post-translational modification. These mechanisms are summarized in Fig. 10. Figure 10 Schematic presentation showing the possible route of IL-6, IL-8, and IL-13 signaling. Endogenous IL-1β production may occur with immune challenge by cytokines, bacteria, and viruses, and any microtrauma in the body while epinephrine is released in states of stress or sympathetic nervous system activation. The pathways activated by these signals converge on IL-6, IL-8, and IL-13 genes to induce cytokine production that is greater than either signal alone. IL-1β activates the NF-κB pathway which leads to significant amounts of IL-6, IL-8, and IL-13 production. Epinephrine activates the p38 MAPK pathway which may activate other transcription factors or stabilize the IL-6, 8, and 13 mRNA. From our data it is evident that IL-1β and epinephrine do not combine to further activate NF-κB or the promotor activity of the IL-13 gene. The importance of IL-6, IL-8, and IL-13 are listed in the figure. Conclusions In conclusion, stress related catecholamines, such as epinephrine, synergized with IL-1β in gene expression and production of proatherogenic cytokines, IL-6, IL-8, and IL-13 in mast cells. The enhancing effect of proatherogenic cytokine production by epinephrine on IL-1β-induced mast cells was down regulated by β-adrenoceptor antagonist, propranolol, and the immunosuppressant Dex. These data support a novel role for catecholamines in disorders such as inflammation and atherogenesis. These data also indicate that β-adrenoceptor antagonists and immunosuppressants may be used preventively and therapeutically for modulation of the catecholamine – proatherogenic cytokine axis in disease states. Methods Mast cell culture and the induction of cytokine production in HMC-1 cells HMC-1 cell line, established from a patient with mast cell leukemia, were graciously provided by Dr. Butterfield (Mayo Clinic, Rochester, MN). These cells were maintained in RPMI 1640 media (GibcoBRL, Rockville, MD), supplemented with 5 × 10-5 M 2-mercaptoethanol (Sigma Chemical Company, St. Louis, MO), 10 mM HEPES (GibcoBRL), Gentamycin 50 μg/ml, 5 μg/ml insulin transferrin, 2 mM L-glutamine, and 5% heat inactivated fetal bovine serum (Atlanta Biologicals, Atlanta, GA), at 37°C and in 5% CO2 mixture [33]. HMC-1 cells were cultured and maintained in 25 cm2 flasks. To each well of a 6 well culture plate, two ml of HMC-1 mast cells at 0.75 × 106 cells/ml concentration were cultured with epinephrine at 1 × 10-5 M concentration in the presence and absence of IL-1β (10 ng/ml) for 24 hrs. The cultures were carried out in triplicate. Supernatants were harvested for measuring IL-6, IL-8, and IL-13 by ELISA and cell viability and numbers of the culture were analyzed. ELISA for cytokine proteins Cytokine ELISA was performed for the following cytokines: IL-6, IL-8, and IL-13. ELISA was carried out on cell-free culture supernatants using commercially available ELISA kits, according to manufacturers instructions as earlier described (R&D Systems, Minneapolis, MN; Immunotech, Westbrook, ME; Genzyme, Cambridge, MA). Results were analyzed on an ELISA plate reader (Dynatech MR 5000 with supporting software) [47,48]. Measurement of cell viability of the cultures At the end of incubation, the cells were subjected to the viability count by trypan blue (TB) dye exclusion technique. Two tenths ml of cell cultures were mixed with 0.05 ml of TB, applied to hemocytometer, and counted under a microscope. The cell viability is calculated by dividing the number of live cells (unstained cells) by the total number of all cells (TB-stained and unstained cells) and expressed as a percent. Analysis of cytokine gene expression by RT-PCR HMC-1 were treated with the appropriate reagents and allowed to incubate at 37°C before being harvested for RNA. RNA was extracted from HMC-1 (3 × 106 cells) by the addition of 1 ml of RNAzol B (Tel-Test, Inc., Friendswood, Texas) [49]. After shaking for 1 minute the samples were centrifuged at 12,000 × g for 15 minutes at 4°C. The aqueous layer was washed twice with 0.8 ml phenol : chloroform (1:1, v/v), centrifuged at 12,000 × g for 15 minutes at 4°C, washed once with 0.8 ml of chloroform and centrifuged at 12,000 × g for 15 minutes at 4°C again. Isopropanol was added to the aqueous phase, and the preparation was frozen at -20°C overnight. The following day, the samples were centrifuged at 12,000 × g for 30 minutes at 4°C. The RNA pellet was washed with 1 ml 75% ethanol and allowed to air dry until all moisture was gone. The pellet was resuspended in DEPC water and quantitated by optical density readings at 260 nm. cDNA was synthesized with murine leukemia virus reverse transcriptase (2.5 U/μl), 10 × PCR buffer (500 mM KCl, 100 mM Tris-HCl, pH 8.3), 1 mM each of the nucleotides dATP, dCTP, dGTP and dTTP; RNase inhibitor (1 U/μl), MgCl2 (5 mM), and oligo(dT)16 (2.5 μM) as a primer. The samples were incubated at 42°C for 20 minutes, 99°C for 20 minutes, and 5°C for 5 minutes in a DNA thermocycler (Perkin-Elmer Corp., Norwalk, CT) for reverse transcription. PCR of cDNA was done with MgCl2 (1.8 mM), each of the dNTPs (0.2 mM), AmpliTaq polymerase (1 U/50 μl), and paired cytokine-specific primers (0.2 nM of each primer) to a total volume of 50 μl. Cycles consisted of 1 cycle of 95°C for 2 min, 35 cycles of 95°C for 45 sec, 60°C for 45 sec, and 72°C for 1 min 30 sec, and lastly, 1 cycle of 72°C for 10 min. Ten microliters of the sample were electrophoresed on a 2% agarose gel and stained with ethidium bromide for viewing. Primer sequences used are as follows: HPRT: 5' CGA GAT GTG ATG AAG GAG ATG G 3' and 5' GGA TTA TAC TGC CTG ACC AAG G 3'; IL-6: 5' ATG AAC TCC TTC TCC ACA AGC GC 3' and 5' GAA GAG CCC TCA GGC TGG ACT G 3'; IL-8: 5' ATG ACT TCC AAG CTG GCC GTG GCT 3' and 5' TCT CAG CCC TCT TCA AAA ACT TCT C 3'; and IL-13: 5' GGA AGC TTC TCC TCA ATC CTC TCC TGT T 3' and 5' GCG GAT TCG TTG AAC CGT CCC TCG CGA AA 3'. Densitometry was done by normalizing target genes to house keepers using Un-Scan-It Version 5.1 software (Orem, UT). The PCR experiment was repeated twice. NF-κB assay in HMC-1 HMC-1 were stimulated with PMA, IL-1β and/or epinephrine and then harvested for EMSA analysis [49,50]. Cells were washed with PBS and mixed with one hundred microliters of hypotonic buffer which contains: 10 mM HEPES pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol (DTT), 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 1 μM aprotinin, 1 μM pepstatin, 14 μM leupeptin, 50 mM NaF, 30 mM β-glycerophosphate, 1 mM Na3VO4, and 20 mM p-nitrophenyl phosphate. Cells were incubated over ice for 30 minutes and then vortexed after the addition of 6.25 μl of 10% of Nonidet P-40. After 2 minutes of centrifugation at 30,000 × g, supernatants were kept at -80°C while the pellets were collected and vortexed every 20 minutes for 3 hours in 60 ml of a hypertonic salt solution: 20 mM HEPES pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 12 mM DTT, 1 mM PMSF, 1 μM aprotinin, 1 μM pepstatin, 14 μM leupeptin, 50 mM NaF, 30 mM β-glycerophosphate, 1 mM Na3VO4, and 20 mM p-nitrophenyl phosphate. Nuclear translocation of NF-κB was analyzed by the Electrophoretic Mobility Shift Assay (EMSA) using the nuclear fraction. Seven micrograms of nuclear protein were added to 2 ml of binding buffer (Promega, Madison, WI), and 35 fmol of double stranded NF-κB consensus oligonucleotide (5' AGT TGA GGG GAC TTT CCC AGG C 3') (Promega, Madison, WI) end labeled with γ-P32 ATP (Amersham Biosciences, Piscataway, NJ). The samples were incubated at room temperature for 20 minutes and run on a 5% nondenaturing polyacrylamide gel for 2 hours. The gel was then dried on a Gel-Drier (Bio-Rad Laboratories, Hercules CA) and exposed to Kodak X-ray film at -80°C. Detection of p38 MAPK by Western blot Cells were treated and lysed in lysis buffer (50 mM Tris HCL, 150 mM NaCl, 1 mM EDTA, 1% Triton × 100, and 0.1% SDS) to be analyzed for p38 MAPK activation by Western blot [29]. Briefly, 50 μg of sample protein was diluted 1:2 with Laemmli buffer (Bio-Rad laboratories, Hercules, CA) and boiled for 10 minutes in a sand bath. The resultant sample was then run in a Bio-Rad Modular Mini Electrophoresis System (Hercules, CA) on a 10% polyacrylamide gel for 1 hour and then transferred to a 0.2 μm nitrocellulose membrane (Bio-Rad laboratories, Hercules, CA) for 1 hour. The blot was then incubated in blocking buffer (1% BSA and 0.1% Tween in PBS) for 1 hour at room temperature with gentle agitation. Rabbit anti-human Phospho-p38 MAPK (Thr180/Tyr182) polyclonal antibody (Calbiochem, San Diego, CA) was diluted 1:1000 in blocking buffer and incubated on the blot overnight at 4°C with gentle agitation. After the primary antibody was removed the blot was washed three times for 10 minutes each with agitation in the wash buffer (0.1% Tween in PBS). The blot was then incubated in horse radish peroxidase conjugated mouse anti-rabbit Ig's antibody (human adsorbed, Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1:5000 in blocking buffer. The blot remained in the secondary antibody for 1 hour at room temperature. The blot was then washed again and covered with Super Signal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL) for 5 minutes. The blot was then exposed to acetate transparency film (Kodak, Rochester, NY) and developed. The same protocol was repeated for total p38 MAPK analysis. Analysis of β-adrenoceptor by flow cytometry Resting HMC-1 were centrifuged, washed in PBS at room temperature, and resuspended in 100 μl of PBS. The cells were incubated for 20 minutes with rabbit polyclonal anti β1 or β2 adrenergic receptor antibodies (Santa Cruz, Santa Cruz, CA) using normal rabbit serum as a control. The samples were washed with PBS with 0.01% sodium azide and resuspended in 100 ml PBS. FITC labeled goat anti-rabbit Ig's antibody was added to the samples and allowed to bind for 20 minutes. The samples were once again washed with PBS with 0.01% sodium azide and resuspended in 100 μl of PBS. In addition, HMC-1 were pretreated with normal rabbit serum and incubated with FITC labeled goat anti-rabbit Ig's antibody as a control for nonspecific binding [51]. Cell suspensions were then gated and analyzed based on fluorescence using a Becton Dickinson FACSCalibur 4-color flow cytometer (San Diego, CA) and histograms generated on WinMDI 2.8 software (kindly provided by Joseph Trotter over the internet). IL-13 promotor analysis HMC-1 were treated with IL-1β (10 ng/ml), epinephrine (10-5 M), and IL-1β plus epinephrine to investigate IL-13 promotor activity. Untreated cells were used as a control. Transient transfection assays were performed using a reporter gene construct containing the minimal promoter sequence of IL-13. The promoter sequence (-233 to + 50, relative to the transcription initiation site) of the IL-13 gene was fused to the luciferase coding sequence. Plasmid DNA was obtained with double-cesium chloride purification (BioServe Biotechnologies, Laurel, MD), while SuperFect reagent (Qiagen) was used for transient transfections of HMC-1 cells. Two micrograms of plasmid DNA and 8 μl SuperFect reagent were used for transfection of 1 × 106 HMC-1 cells. Luciferase expression was monitored by chemiluminescence of cell lysates 24 hrs after transfections using the Enhanced Luciferase Assay Kit (Analytical Luminescence Laboratory, Ann Arbor, MI). Statistical analysis of the data All experiments were done in triplicate. The data were analyzed by Student's two-tailed t-test using Statistica software (StatSoft, Inc., Tulsa, OK). All data were reported as means ± SE. A p-value of less than 0.05 was considered significant. List of abbreviations used HMC-1, human mast cell – 1 Epi, epinephrine Pro, propranolol Ate, atenolol Dex, dexamethasone MAPK, mitogen-activated protein kinase Author's contributions DSC designed experiments, oversaw research, and wrote paper. SMF designed and conducted experiments and wrote paper. SP helped with experiments. KC conducted experiments. EK conducted experiments. SAL conducted experiments. SKH conducted experiments. GK oversaw research. Acknowledgments This work was supported in part by Public Health Service Grant HL-63070 from the National Institutes of Health, the Chair of Excellence in Medicine (State of Tennessee Grant 20233), The Ruth R. Harris Endowment, and RDC of ETSU. ==== Refs Kelley JL Chi DS Abou-Auda W Smith JK Krishnaswamy G The molecular role of mast cells in atherosclerotic cardiovascular disease Mol Med Today 2000 6 304 308 10904247 10.1016/S1357-4310(00)01747-0 Krishnaswamy G Kelley J Johnson D Youngberg G Stone W Huang SK Bieber J Chi DS The human mast cell: functions in physiology and disease Front Biosci 2001 6 D1109 D1127 11532608 Cannon JG Evans WJ Hughes VA Meredith CN Dinarello CA Physiological mechanisms contributing to increased interleukin-1 secretion J Appl Physiol 1986 61 1869 1874 3491062 Biasucci LM Vitelli A Liuzzo G Altamura S Caligiuri G Monaco C Rebuzzi AG Ciliberto G Maseri A Elevated levels of interleukin-6 in unstable angina Circulation 1996 94 874 877 8790019 Paul H Whitaker JH Downs CJ Alturjuman AM Martin E Krishnaswamy G Khan AA Hiremagular S Cantor K Chi DS Plasma interleukin-6, fibrinogen levels, and fibrinogen promoter polymorphism in patients with coronary artery disease Chest 1997 112 S67S Rus HG Vlaicu R Niculescu F Interleukin-6 and interleukin-8 protein and gene expression in human arterial atherosclerotic wall Atherosclerosis 1996 127 263 271 9125317 10.1016/S0021-9150(96)05968-0 Blum A Miller H Role of cytokines in heart failure Am Heart J 1998 135 181 186 9489963 Borish LC Steinke JW Cytokines and chemokines J Allergy Clin Immunol 2003 111 S460 S475 12592293 10.1067/mai.2003.108 Stassen M Muller C Arnold M Hultner L Klein-Hessling S Neudorfl C Reineke T Serfling E Schmitt E IL-9 and IL-13 production by activated mast cells is strongly enhanced in the presence of lipopolysaccharide: NF-kappa B is decisively involved in the expression of IL-9 J Immunol 2001 166 4391 4398 11254693 Bradding P Holgate ST The mast cell as a source of cytokines in asthma Ann N Y Acad Sci 1996 796 272 281 8906234 Bradding P Human mast cell cytokines Clin Exp Allergy 1996 26 13 19 8789538 Church MK Levi-Schaffer F The human mast cell J Allergy Clin Immunol 1997 99 155 160 9042038 Wissler RW New insights into the pathogenesis of atherosclerosis as revealed by PDAY. Pathobiological Determinants of Atherosclerosis in Youth Atherosclerosis 1994 108 S3 20 7802726 Jeziorska M McCollum C Woolley DE Mast cell distribution, activation, and phenotype in atherosclerotic lesions of human carotid arteries J Pathol 1997 182 115 122 9227350 10.1002/(SICI)1096-9896(199705)182:1<115::AID-PATH806>3.0.CO;2-9 Riley V Psychoneuroendocrine influences on immunocompetence and neoplasia Science 1981 212 1100 1109 7233204 Eskola J Ruuskanen O Soppi E Viljanen MK Jarvinen M Toivonen H Kouvalainen K Effect of sport stress on lymphocyte transformation and antibody formation Clin Exp Immunol 1978 32 339 345 668205 Hedfors E Holm G Ohnell B Variations of blood lymphocytes during work studied by cell surface markers, DNA synthesis and cytotoxicity Clin Exp Immunol 1976 24 328 335 1277581 Landmann RM Muller FB Perini C Wesp M Erne P Buhler FR Changes of immunoregulatory cells induced by psychological and physical stress: relationship to plasma catecholamines Clin Exp Immunol 1984 58 127 135 6478647 Tulen JH Moleman P Blankestijn PJ Mani V van Steenis HG Boomsma F Psychological, cardiovascular, and endocrine changes during 6 hours of continuous infusion of epinephrine or norepinephrine in healthy volunteers Psychosom Med 1993 55 61 69 8446743 Kukreja RS Datta BN Chakravarti RN Catecholamine-induced aggravation of aortic and coronary atherosclerosis in monkeys Atherosclerosis 1981 40 291 298 7332608 Bauch HJ Grunwald J Vischer P Gerlach U Hauss WH A possible role of catecholamines in atherogenesis and subsequent complications of atherosclerosis Exp Pathol 1987 31 193 204 3622722 Born GV Recent evidence for the involement of catecholamines and of macrophages in atherosclerotic processes Ann Med 1991 23 569 72 1756026 Hauss WH Bauch HJ Schulte H Adrenaline and noradrenaline as possible chemical mediators in the pathogenesis of arteriosclerosis Ann NY Acad Sci 1990 598 91 101 2248466 Ortega E Garcia JJ De la FM Modulation of adherence and chemotaxis of macrophages by norepinephrine. Influence of ageing Mol Cell Biochem 2000 203 113 117 10724339 10.1023/A:1007094614047 Rainer TH Lam N Cocks RA Adrenaline upregulates monocyte L-selectin in vitro Resuscitation 1999 43 47 55 10636317 10.1016/S0300-9572(99)00121-5 Chi DS Qui M Krishnaswamy G Li C Stone W Regulation of nitric oxide production from macrophages by lipopolysaccharide and catecholamines Nitric Oxide 2003 8 127 132 12620376 10.1016/S1089-8603(02)00148-9 Briest W Elsner C hemker J Muller-Strahl G Zimmer HG Norepinephrine-induced expression of cytokines in isolated biventricular working rat hearts Mol Cell Biochem 2003 245 69 76 12708746 10.1023/A:1022861609896 MohanKumar SM Smith CL MohanKumar PS Central adaptation to chronic administration of interleukin-1beta (IL-1beta) in rats Brain Res Bull 2003 62 71 76 14596894 10.1016/j.brainresbull.2003.09.001 Kaur D Cruess DF Potter WZ Effect of IL-1alpha on the release of norepinephrine in rat hypothalamus J Neuroimmunol 1998 90 122 127 9817439 10.1016/S0165-5728(98)00062-9 Gidron Y Gilutz H Berger R Huleihel M Molecular and cellular interface between behavior and acute coronary syndromes Cardiovasc Res 2002 56 15 21 12237162 10.1016/S0008-6363(02)00537-0 Kopin IJ Catecholamine metabolism (and the biochemical assessment of sympathetic activity) Clin Endocrinol Metab 1977 6 525 546 22412 Krishnaswamy G Martin R Walker E Li C Hossler F Hall K Chi DS Moraxella catarrhalis induces mast cell activation and nuclear factor kappaB-dependent cytokine synthesis Front Biosci 2003 8 a40 a47 12456364 Fitzgerald SM Lee SA Hall HK Chi DS Krishnaswamy G Human Lung Fibroblasts Express IL-6 in Response to Signaling Following Mast Cell Contact Am J Respir Cell Mol Biol Haverkate F Thompson SG Pyke SD Gallimore JR Pepys MB Production of C-reactive protein and risk of coronary events in stable and unstable angina. European Concerted Action on Thrombosis and Disabilities Angina Pectoris Study Group Lancet 1997 349 462 466 9040576 10.1016/S0140-6736(96)07591-5 Hallenbeck JM Significance of the inflammatory response in brain ischemia Acta Neurochir Suppl (Wien) 1996 66 27 31 8780793 Cermak J Key NS Bach RR Balla J Jacob HS Vercellotti GM C-reactive protein induces human peripheral blood monocytes to synthesize tissue factor Blood 1993 82 513 520 8329706 Baumann H Gauldie J The acute phase response Immunol Today 1994 15 74 80 7512342 10.1016/0167-5699(94)90137-6 Elenkov IJ Chrousos GP Stress hormones, proinflammatory and antiinflammatory cytokines, and autoimmunity Ann N Y Acad Sci 2002 966 290 303 12114286 Volanakis JE Transcriptional regulation of complement genes Annu Rev Immunol 1995 13 277 305 7612224 10.1146/annurev.iy.13.040195.001425 Krishnaswamy G Hall K Youngberg G Hossler F Johnson D Block WA Huang SK Kelley J Chi DS Regulation of eosinophil-active cytokine production from human cord blood-derived mast cells J Interferon Cytokine Res 2002 22 379 388 12034046 10.1089/107999002753675811 Sigola LB Zinyama RB Adrenaline inhibits macrophage nitric oxide production through beta1 and beta2 adrenergic receptors Immunology 2000 100 359 363 10929058 10.1046/j.1365-2567.2000.00029.x Christensen JD Hansen EW Frederiksen C Molris M Moesby L Adrenaline influences the release of interleukin-6 from murine pituicytes: role of beta2-adrenoceptors Eur J Pharmacol 1999 378 143 148 10478575 10.1016/S0014-2999(99)00448-3 Ben Eliyahu S Shakhar G Page GG Stefanski V Shakhar K Suppression of NK cell activity and of resistance to metastasis by stress: a role for adrenal catecholamines and beta-adrenoceptors Neuroimmunomodulation 2000 8 154 164 11124582 10.1159/000054276 Boomershine CS Lafuse WP Zwilling BS Beta2-adrenergic receptor stimulation inhibits nitric oxide generation by Mycobacterium avium infected macrophages J Neuroimmunol 1999 101 68 75 10580815 10.1016/S0165-5728(99)00134-4 Hasko G Shanley TP Egnaczyk G Nemeth ZH Salzman AL Vizi ES Szabo C Exogenous and endogenous catecholamines inhibit the production of macrophage inflammatory protein (MIP) 1 alpha via a beta adrenoceptor mediated mechanism Br J Pharmacol 1998 125 1297 1303 9863660 Brand K Page S Rogler G Bartsch A Brandl R Knuechel R Page M Kaltschmidt C Baeuerle PA Neumeier D Activated transcription factor nuclear factor-kappa B is present in the atherosclerotic lesion J Clin Invest 1996 97 1715 1722 8601637 Krishnaswamy G Smith JK Mukkamala R Hall K Joyner W Yerra L Chi DS Multifunctional cytokine expression by human coronary endothelium and regulation by monokines and glucocorticoids Microvasc Res 1998 55 189 200 9657919 10.1006/mvre.1998.2079 Krishnaswamy G Smith JK Srikanth S Chi DS Kalbfleisch JH Huang SK Lymphoblastoid interferon-alpha inhibits T cell proliferation and expression of eosinophil-activating cytokines J Interferon Cytokine Res 1996 16 819 827 8910767 Fitzgerald SM Chi DS Hall HK Reynolds SA Aramide O Lee SA Krishnaswamy G GM-CSF induction in human lung fibroblasts by IL-1beta, TNF-alpha, and macrophage contact J Interferon Cytokine Res 2003 23 57 65 12744771 10.1089/107999003321455453 Li C Browder W Kao RL Early activation of transcription factor NF-kappaB during ischemia in perfused rat heart Am J Physiol 1999 276 H543 H552 9950856 Smith JK Chi DS Krishnaswamy G Srikanth S Reynolds S Berk SL Effect of interferon alpha on HLA-DR expression by human buccal epithelial cells Arch Immunol Ther Exp (Warsz) 1996 44 83 88 8915510

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==== Front BMC Plant BiolBMC Plant Biology1471-2229BioMed Central London 1471-2229-4-161537738910.1186/1471-2229-4-16Research ArticleLong-distance transport of L-ascorbic acid in potato Tedone Luigi [email protected] Robert D [email protected] Salvatore [email protected] Sophie [email protected] Roberto [email protected] Unit of Plant Biochemistry, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK2 Dipartimento di Scienze delle Produzioni Vegetali, Universita degli Studi di Bari, Italy3 University of Naples "Federico II", Department of Soil, Plant and Environmental Sciences, Via Universita' 100 – 80055 Portici, Italy2004 17 9 2004 4 16 16 18 8 2004 17 9 2004 Copyright © 2004 Tedone et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Following on from recent advances in plant AsA biosynthesis there is increasing interest in elucidating the factors contributing to the L-ascorbic acid (AsA) content of edible crops. One main objective is to establish whether in sink organs such as fruits and tubers, AsA is synthesised in situ from imported photoassimilates or synthesised in source tissues and translocated via the phloem. In the current work we test the hypothesis that long-distance transport is involved in AsA accumulation within the potato tuber, the most significant source of AsA in the European diet. Results Using the EDTA exudation technique we confirm the presence of AsA in the phloem of potato plants and demonstrate a correlation between changes in the AsA content of source leaves and that of phloem exudates. Comparison of carboxyflourescein and AgNO3 staining is suggestive of symplastic unloading of AsA in developing tubers. This hypothesis was further supported by the changes in AsA distribution during tuber development which closely resembled those of imported photoassimilates. Manipulation of leaf AsA content by supply of precursors to source leaves resulted in increased AsA content of developing tubers. Conclusion Our data provide strong support to the hypothesis that long-distance transport of AsA occurs in potato. We also show that phloem AsA content and AsA accumulation in sink organs can be directly increased via manipulation of AsA content in the foliage. We are now attempting to establish the quantitative contribution of imported AsA to overall AsA accumulation in developing potato tubers via transgenic approaches. ==== Body Background L-Ascorbic acid (AsA), the reduced form of vitamin C, is an essential antioxidant for many biological systems and must be obtained via the diet by humans, primates and a few other animals which are unable to synthesise AsA endogenously [1]. The main dietary source of AsA for all these organisms are plants and insufficient intake of this micronutrient results in the onset of a debilitating disease (scurvy) and eventually death. In spite of its obvious relevance for humankind, our understanding of how plants synthesise AsA is still rudimentary. It was only in 1998 that an evidence-backed AsA biosynthetic pathway in plants was put forward [2]. The original proposal was supported by the characterisation of Arabidopsis thaliana AsA-deficient mutants (vtc1) which were defective in the activity of a pathway enzyme [3]. Since then, other AsA-deficient A. thaliana mutants have been identified which do not seem to be affected in any of the known pathway genes [4,5]. Additionally, up-regulation of AsA accumulation in plants has been achieved via ectopic over-expression of biosynthetic genes unrelated to the proposed biosynthetic pathway [6-9]. This has led to proposals of additional steps, branches or alternative routes to the original pathway [7,9,10]. Many of the recent advances in plant AsA biosynthesis have been obtained through investigations of model systems such as tobacco or Arabidopsis thaliana [2,3,9,11]. On the other hand, our understanding of the mechanisms controlling AsA accumulation in the edible parts of crop plants (e.g. fruits and vegetables), which represent the main dietary source of vitamin C [12] remains limited. One problem is that AsA functions outside the chloroplasts are much less understood compared with those associated with photosynthetic metabolism [13]. We have also no biological or taxonomic explanation for the massive variability in AsA contents in sink organs such as fruits which can contain over 3000 mg/100 g FW in the fruits of camu camu (Mirciaria dubia) [14] or less than 10 mg/100 gFW as is the case for grapes, apples or plums [15]. Large variability is also found in non-green vegetables [16] whilst dry seeds are completely devoid of AsA [e.g. [17]]. These examples highlight the high degree of tolerance for AsA content in storage organs and suggest the biological feasibility for the development of AsA-rich crop products. To this end, one key question that needs to be answered is whether AsA accumulation in sink organs occurs as a result of biosynthesis in situ or import from the foliage. Recently Franceschi and Tarlyn [18] observed long distance movement of 14C-AsA from leaves to flowers and root tips in model systems such as A. thaliana and Medicago sativa. Research in our laboratory demonstrated the occurrence of AsA in the phloem of a number of crop plants and we also observed that the plant phloem was capable of supporting active AsA biosynthesis from a number of precursors [19]. We have previously shown that rates of AsA accumulation are highest soon after organ formation in sink organs such as blackcurrant berries [20] and potato tubers [21] when sink activity is strongly induced [22]. Taken together, these findings indicate that sink-source relationships may play an important role in defining AsA accumulation in sink organs. In this work we have extended our investigation on AsA accumulation in potato tubers, the main source of vitamin C in the European diet [23]. We present here evidence for the implication of long-distance transport in AsA accumulation in potato tubers. Additionally, we show that artificial increase of AsA content in the foliage results in phloem AsA enrichment and AsA accumulation in tuberising stolons. Our findings have implications for the development of strategies for increasing the nutritional value of crop plants. Results Detection of AsA in the phloem Fig. 1 shows HPLC chromatograms of potato source leaf phloem exudates collected from severed petioles in buffer containing either EDTA or CaCl2. Total AsA (AsAt; L-ascorbic acid + dehydroascorbic acid) appeared as the largest peak of absorbance at 245 nm retained by the column in both cases. When exudates were collected in the presence of CaCl2 instead of EDTA, all exudate derived peaks were strongly reduced due to the reduction in exudation caused by callose gelation [24]. In experiments to test the stability of authentic AsA (final concentration 0.1 μM) in the presence of EDTA or CaCl2 exudation buffers less than 5% oxidation was observed in either case over 90 min (data not shown). AsA localisation to the vascular tissue was confirmed histochemically in sections of potato stems and tubers incubated with ethanolic AgNO3 at 3°C (Fig. 2). Intense deposition of metallic silver was observed in the vasculature of stems. In tubers metallic silver deposits appeared as short strands or well defined spots, sometimes in the perimedullary zone and also in the cortex. Although AgNO3 staining and CFDA treatments could not be carried out on the same section due to interference between the treatments, a clear similarity was observed in the pattern of metallic silver deposits and the distribution of fluorescence in stem and tuber sections. No AgNO3 staining was observed in control sections pre-incubated with 1% CuSO4 for 18 h in order to oxidise AsA (data not shown) [25]. AsA distribution during tuber development Fig. 3 shows AsAt distribution along the axis of stolons and developing tubers. In non-swelling stolons the AsAt content was maximal in the apical section with a sharp basipetal decline such that the more basal sections contained approximately 10% of the AsAt content found in the apex. Tuberising stolons showed a 50% reduction in the AsAt content of the apical section. This was accompanied by increases in the subapical 3–10 mm sections. In developing tubers there was a very substantial increase in the AsAt content in the sub-apical region, corresponding to the swelling area. AsA transport Figure 4 shows the changes in AsAt content of source leaves of potato plants acclimatised for 14 days in cabinets with artificial diurnal dark/light cycles. The cabinets were off-phased so that plants at different stages in their lighting regime were available at any given time. The AsAt content of source leaves was monitored for 24 h in plants sampled simultaneously from the two cabinets. At the end of the dark phase, leaf AsAt content was approximately 12 mg/100 gFW and it progressively increased following artificial sunrise. After 10 h of light, the leaf AsAt content increased to approximately 30 mg/100 gFW after which it leveled off. Following artificial sunset the AsAt level gradually fell back to approximately 12 mg/100 gFW within 6–10 h. Table 1 shows the AsAt content of source leaves and tuberising stolons collected at 12.00 h from the light-phase or dark-phase plants (after collection of phloem exudate for 90 min in a prehumidified atmosphere in leaves). In the table are also reported the chromatographic AsAt peak areas from phloem exudates collected from source leaves and tuberising stolons of the same plants. The exudate data are reported as peak areas as the exact volume of exudate collected could not be established. Whilst the leaves from light-phase plants contained over twice the AsAt level of leaves from dark-phase plants, no significant difference was found in the AsAt content of tuberising stolons from the two sets of plants. The AsAt peak area in chromatograms of light-phase leaf exudates was 1.8-fold larger than that obtained from dark-phase leaves. In tuberising stolons the increase in exudate peak area was more pronounced (4.6-fold). The correlation between leaf AsAt content and the AsAt levels of phloem exudates was also investigated in glasshouse-grown plants following the supply of a range of AsA biosynthetic precursors using the flap technique [18]. Figure 5 shows the chromatographic AsAt peak area of exudates from leaves pre-treated for 24 h with 20 mM MES pH 5.5, 2 mM CaCl2 alone (control) or containing precursors at a final concentration of 25 mM. Incubation with D-glucose (D-Glc) resulted in a slight (10%) reduction in leaf AsAt content compared with the control but no significant change in AsAt was detected in the exudates. By contrast, supply of L-galactose (L-Gal) or L-galactono-1,4-lactone (L-GalL) increased the AsAt content of leaves (4.9 and 6.2-fold respectively) and, more substantially, of exudates (10.8 and 11.2-fold respectively). With all treatments, the replacement of EDTA with CaCl2 in collection wells resulted in a significant reduction of AsAt peak area in the exudate chromatograms. An artificial increase of AsAt content in the totality of source foliage of whole plants was obtained through the supply of the direct AsA precursor L-GalL via the flap technique for 24 h to all terminal leaflets of the four lowermost nodes. At the end of the incubation period the AsAt content of source leaves was 2.2-fold higher than in control plants (Fig. 6). AsAt content also significantly increased in sink organs such as flowers (33%) and, more substantially in developing tubers (80%) compared with the control. No changes in AsAt levels were observed in petioles, stems or non-tuberising stolons. Discussion AsA in the potato phloem We have previously shown that AsA represented the largest peak of absorption at 245 nm retained by the column when phloem sap of potato source leaves obtained by aphid stylectomy was analysed by HPLC [19]. In the present study we obtained very similar results from phloem samples collected from different organs of the potato plant by a different approach i.e. the EDTA exudation technique [24]. Localisation of AsA to the vascular tissue of stems and developing tubers was further demonstrated histochemically exploiting the specific interaction between AsA and AgNO3 at low temperature with ensuing formation of metallic silver deposits [25]. In particular, the distribution of metallic silver in developing tubers generated a pattern reflecting the intense phloem anastomosis typical of these storage organs [26] and resembling the phloem network involved in the unloading of labelled assimilates in plants supplied with 14CO2 [22]. This was further evidenced by the close similarity between the silver nitrate staining and distribution of fluorescence in tubers following supply of CFDA to source leaves. We have previously demonstrated that the distribution of CF in developing potato tubers can be used to identify symplastic phloem unloading in these organs [22], thus the similar distribution of CF and metallic silver deposits in developing tubers implies that transfer of phloem AsA to storage parenchyma cells occurs with the mass flow of assimilates. This hypothesis is further supported by the changes in AsAt distribution along the stolon axis following tuber induction which closely resemble the changes in sucrose content and radiolabelled assimilate distribution observed in plants labelled with 14CO2 [22]. The decline of AsAt in the apices of tuberising stolons accompanied by its accumulation in the subapical region may thus reflect the induction of symplastic phloem unloading in this region resulting in a distal migration of sink activity from the apex. However, this pattern also correlates with changes in the mitotic index along the axis as a result of tuber induction with cessation of cell division in the apical region of the stolon [27] and its activation in secondary meristems within the swelling region [28]. This may be relevant in view of the purported role of AsA in cell division [29]. Long-distance AsA transport in potato In preliminary experiments, we observed diurnal changes in the AsAt content of mature source leaves of potato. Circadian or diurnal oscillations of AsA content in photosynthetic tissues are known [e.g. [30]] as well as light-induction of AsA accumulation in leaves [e.g. [31]] and sink organs. Light-induced expression of specific AsA biosynthetic enzymes has been observed [e.g. [30]] as well as a general increase in the AsA biosynthetic flux [11]. By growing plants in cabinets with out-of-phase light/dark regimes we were able to simultaneously sample plants in the dark or light phase when maximal differences in foliage AsAt content occurred. Leaf AsAt content showed diurnal changes with the maximal levels (observed during the light-phase) 2-fold higher than the lowest levels observed in the dark. Phloem exudates obtained from source and sink organs of light phase plants always showed significantly higher AsAt levels than exudates obtained from dark phase plants. Indeed, the AsAt content of exudates from tuberising stolons of light phase plants was over 4-fold higher that of dark-phase plants. Our findings indicate that changes in source leaf AsA biosynthesis rapidly impact on the phloem AsAt content resulting in transport of de novo synthesised AsA directly to developing sinks. This was also confirmed in experiments where exogenous AsA precursors such as L-GalL or L-Gal were supplied to source leaves via the flap technique, a treatment which resulted in substantial AsAt enrichment in phloem exudates. We were also able to more than double the AsAt content of the source foliage of whole plants by "bulk" supply of exogenous L-GalL to the majority of source leaves for 24 h. This resulted in significant AsAt increases in sink organs such as flowers and developing tubers. To our knowledge this is the first demonstration that AsA accumulation in the foliage results in AsA increases in storage organs and it may explain the positive effect of light irradiation on AsA content of fruits and vegetables [32]. The rapid changes in phloem AsA concentration which reflected changes in mesophyll AsA content indicate that AsA storage in the foliage does not occur, unlike the case of assimilated carbohydrates. It is tempting to speculate that AsA produced within mesophyll cells is directly taken up by SE/CC complex for translocation, as also deduced from the results reported by Franceschi and Tarlyn [18]. The kinetics of AsA transport have been studied across a number of plant membranes such as the plasmlemma, the chloroplast membrane, the thylakoid membrane and the tonoplast [33]. However, to date no transporter has been identified although at least 12 genes encoding putative nucleobase/ascorbate transporters (NATs) have been identified in the Arabidopsis genome. NATs belong to a superfamily of integral membrane transporters, which move purines, pyrimidines or ascorbic acid across biological membranes and which also comprises mammalian transporters specific for ascorbate [34]. Functional characterisation of these transporters is in progress [35]. As the plant phloem contains the complete enzymic complement for AsA biosynthesis [19], we have already speculated that the indirect transfer of AsA from the mesophyll to the phloem could involve the transport of the non-charged intermediate L-Gal across membranes. We show here that exogenous supply of L-Gal or L-GalL to source leaves leads to AsA enrichment of phloem exudates and AsA accumulation in tuberising stolons and developing tubers. Furthermore, we have observed uptake of L-[1-14C]Gal by source phloem of potato (data not shown), as already demonstrated in N. benthamiana [19]. Conclusions The evidence we provide here for long-distance transport of AsA in potato plants, corroborates earlier findings in A. thaliana and M. sativa [18] and suggests that source-sink AsA translocation may be a general occurrence in plants. What remains to be established is the relative contribution of phloem-derived AsA (whether synthesised in the mesophyll or in the phloem itself) to overall AsA accumulation in potato tubers. Slices excised from developing potato tubers can synthesise AsA from a variety of substrates (Hancock and Viola, unpublished). Moreover, microtubers obtained in vitro from nodal cuttings cultured in the dark with sucrose as the sole carbon source contain similar amounts of AsA as field-grown tubers [21]. These latter findings in particular are difficult to reconcile with the hypothesis of a major role played by long-distance AsA transport in tuber AsA accumulation. The development of transgenic potato plants with tissue and organ-specific down-regulation of AsA biosynthesis will be required to address this issue. Methods Plant material and growth conditions Potato plants cv. Desiree were grown in 40 cm pots in unheated glasshouses under natural light in compost. In order to subject plants to artificial light/dark cycles, plants were transferred after 35 days to Sanyo Fitotron 1700 controlled environment cabinets and maintained for a further 14 days on 14 h–10 h light-dark cycles with day and night temperatures of 22°C and 15°C respectively. Light was provided by 60 W incandescent lamps to provide a photon flux of 900 μmol m2 s-1 at the top of the canopy. Relative humidity was maintained at a constant 70% and plants were watered daily. In all cases experiments were performed on tuberising plants after 40–60 days from planting. Throughout the text stolons are defined as non-swelling (uniform diameter along terminal 15 mm) or tuberising (swelling 2–5 mm diameter). Swellings between 5–10 mm diameter are defined as developing tubers. Quantification of AsA in plant tissues Tissue was extracted in a mortar and pestle with ice-cold 5% metaphosphoric acid (MPA) containing 5 mM tris(2-carboxyethyl)phosphine hydrochloride TCEP (9:1 v/w). Samples were then held on ice for 60 min to allow reduction of dehydroascorbic acid to AsA therefore, all data are reported as total AsA pool (AsAt) i.e. reduced L-ascorbic acid + dehydroascorbic acid. Samples were then centrifuged at 16000 g for 5 min at 1°C and AsAt in the supernatant quantified by HPLC according to the method of Hancock et al [36]. Briefly, 20 μl of sample supernatant were injected onto a 300 × 7.8 mm ID Coregel 64H ion exclusion column (Interaction Chromatography, San Jose, CA, USA) with a 4 × 3 mm ID carbo-H+ guard cartridge (Phenomenex, Macclesfield, UK) maintained at 50°C. Mobile phase was 8 mM H2SO4 at 0.6 ml min-1 and AsAt was detected at 245 nm using a Gynkotech UVD 340S diode array detector (Dionex, Camberley, UK). Detection of AsA in the phloem Phloem exudates were collected from the petiole of source leaves or tuberising stolons using an adaptation of the method developed by King and Zeevart [24]. Following excision of the organs, a portion of the petiole (5 mm) or stolon (10 mm) was removed under water, the sample was rinsed and the cut end transferred to a 0.6 ml reaction tube containing 200 μl 15 mM EDTA pH 7.5. In the case of petioles, samples were transferred to a pre-humidified atmosphere at 20°C and exudate collected for 90 min in the dark. In the case of stolons, exudates were collected from the cut end which remained attached to the plant and moist paper was wrapped around the top of the reaction tube to minimise evaporation. Control samples were run in parallel in which petioles or stolons were incubated in 5 mM CaCl2 pH 7.5 to induce callose gellation and reduce exudation [24]. At the end of the incubation, MPA and TCEP were added to the samples to a final concentration of 5% and 5 mM respectively. Following centrifugation (16000 g, 1°C, 5 min), AsAt concentration was determined by HPLC as described above. Histochemical localization of AsA in tubers using the AgNO3 method was carried out as previously described [19]. Briefly, tubers were hand sliced to form approximately 2 mm sections, washed in distilled water and fixed and stained in 5% (w/v) AgNO3 dissolved in 66% (v/v) aqueous ethanol containing 5% (v/v) glacial acetic acid at 3°C in the dark for up to 24 h. The reaction was stopped by washing the tissue twice for 15 min in ethanolic ammonium hydroxide (95% (v/v) 70% ethanol, 5% (v/v) NH4OH ACS reagent, Sigma-Aldrich, Dorset, UK) [25]. Finally the tissue was transferred to 70% (v/v) ethanol and stored at 3°C prior to photography. Transport of carboxyfluorescein in potato plants Phloem transport through potato stems into developing tubers was visualised using the fluorescent transportable molecule carboxyflourescein (CF) as previously described [22]. Plants were labelled with 20 ml of an aqueous carboxyflourescein diacetate (CFDA) solution (1 mg ml-1) introduced via open stomata on the abaxial leaf surface using a plastic syringe. The acetylated compound is able to diffuse across cell membranes unlike its deacetylated derivative CF which is produced in vivo by endogenous esterases and is used as a marker for phloem strands and symplastic unloading from the phloem [37]. Plants were left to translocate CF for 5 h prior to hand sectioning (2 mm) and examination of stem and sink tissues for fluorescence using a MRC2000 confocal microscope (Bio-Rad, Hemel Hemstead, UK). Supply of precursors to leaves Leaf AsAt levels were manipulated using an adaptation of the 'flap' technique [18]. An incision (15 mm) was made either side of the midrib of terminal leaflets and the 'flap' formed was placed into a 0.6 ml eppendorf tube containing 500 μl of 20 mM MES pH 5.5, 2 mM CaCl2 alone or with the addition of various intermediates at a final concentration of 25 mM for 24 h. At the end of the incubation period, leaflets were ground in liquid nitrogen and extracted in 5% MPA containing 5 mM TCEP (9:1 v/w) and the AsAt content measured by HPLC. For measurement of phloem exudates, treated leaflets were excised under water and placed in eppendorf tubes for collection of phloem exudates as described previously. In some experiments the terminal leaflets of the four lower nodes on all stems (between 8 and 10 per plant) were simultaneously supplied with 500 μl 20 mM MES pH 5.5, 2 mM CaCl2 alone or containing 25 mM L-GalL for 24 h. Four independent plants were used for each treatment. At the end of the incubation individual plants were separated into flowers, source leaves, leaf petioles, plant stems, non-tuberising stolons (terminal 15 mm), or tuberising stolons (swelling portion). Tissues were immediately frozen in liquid nitrogen and lyophilised. Lyophilised tissue was ground to a powder and 3 × 1 g fractions of each tissue were extracted in 5% MPA, 5 mM TCEP (19:1 v/w) and the AsAt content of each extract analysed by HPLC to give an average value for each tissue. No developing tubers larger than 5 mm diameter were present in the plants. Authors' contributions LT undertook most of the physiological and biochemical experiments. RDH participated in the design and coordination of the study, the biochemical experimentation and the writing of the manuscript. SA participated in the physiological, biochemical and histochemical experiments. SH undertook the CFDA labeling and silver staining of potato plants and participated in production of the figures. RV conceived the study, participated in its design and coordination and drafted the manuscript. Acknowledgements This work was supported by the Scottish Executive Environment and Rural Affairs Department. Figures and Tables Figure 1 HPLC traces of potato leaf exudates. The terminal leaflets of potato leaves were excised from glasshouse plants, petioles were re-cut under water and the freshly cut end transferred to 200 μl 5 mM CaCl2 (trace A) or 200 μl 15 mM EDTA (trace B). Samples were transferred to a saturated atmosphere to exude in the dark for 90 min. The exudation medium was brought to 5% MPA, 5 mM TCEP, centrifuged and injected onto a Coregel 64H column. Traces were recorded at 245 nm using a diode array detector and insets show the absorbance spectra of the peaks at 12.2 min. Trace C was authentic AsA (2.27 nmol) which gave a peak height of 42 mAU. The unbound peak at 6.3 min contained MPA. Figure 2 Comparison of CF and AgNO3 staining in potato tissues. Plants were labelled with aqueous CFDA and left to translocate for 5 h prior to harvesting. Tubers or stems were hand sectioned and sections were either incubated in mineral oil and examined under a confocal microscope for CF fluorescence or stained for AsA using ethanolic AgNO3. Confocal images are shown on the left and silver stained sections on the right of each panel. A, stem (bar = 2 mm); B, developing tuber (bar = 3 mm); C, large developing tuber (bar = 5 mm). Figure 3 Distribution of AsAt along the axes of stolons and developing tubers. Non-swelling stolons, tuberising stolons or developing tubers were excised from glasshouse grown plants and sliced into 1 mm transverse sections which were quickly weighed and immediately frozen in liquid nitrogen. Samples were ground to a powder, extracted in 5% MPA, 5 mM TCEP (9:1 v/w) and AsAt in the extract supernatant estimated by HPLC. Values are represented as mean ± SE, n = 3. Figure 4 Changes in AsAt content of potato leaves as a function of light. Glasshouse grown plants were transferred to off-phased controlled environment chambers (panels A and B) 14 days prior to the start of the experiment. Environment chambers were on a 10 h dark – 14 h light cycle as indicated by the brown and yellow panels respectively. At the times shown, source leaves (leaves on the lower four nodes of each stem) were removed from each of three plants, ground in liquid nitrogen and extracted in 5% MPA, 5 mM TCEP (9:1 v/w) prior to quantification by HPLC. Values are represented as mean ± SE, n = 3. Figure 5 AsAt content of petiole exudates after treatment of source leaves with AsA precursors. Terminal leaflets of glasshouse grown potato plants were supplied with 500 μl of the substrate indicated (25 mM) via a 'flap' whilst still attached to the plant. After 24 h incubation, leaflets were removed for AsAt determination in tissues and collection of exudates (90 min). Bars represent the mean exudate AsAt peak area ± SE (n = 4) and values in boxes represent the mean tissue AsAt content (mg/100 gFW) ± SE (n = 4). mAUt = milli-absorbance units (λ245 nm) × time Figure 6 Effect of precursor supply to source leaves on AsAt content in sink tissues. Terminal leaflets of all leaves of the lower four nodes of each stem of four potato plants were incubated with 500 μl 20 mM MES pH 5.5, 2 mM CaCl2 alone (control) or containing 25 mM L-GalL for 24 h. At the end of incubation the indicated tissues were removed from plants, snap frozen in liquid nitrogen and lyophilised. After lyophilisation, tissues were powdered and extracted in 5% MPA, 5 mM TCEP (19:1 v/w) prior to estimation of the AsA content in each tissue by HPLC. Values are presented as means ± SE, n = 4. Table 1 Effect of light on AsAt content in source leaves, tuberising stolons and exudates Tissue Cycle AsAt in tissue (mg/100 gFW) AsAt in exudate (peak area mAUt) Leaf Light 42.5 ± 3.1 1.1 ± 0.1 Dark 19.1 ± 3.0 0.6 ± 0.05 Tuberising stolon Light 4.1 ± 0.3 7.9 ± 0.8 Dark 4.5 ± 0.4 1.7 ± 0.9 Glasshouse grown plants were transferred to off-phased controlled environment chambers 14 days prior to the start of the experiment. Environment chambers were on a 10 h dark – 14 h light cycle (see Fig. 4). At 12:00 h source leaves and tuberising stolons were removed from 3 plants and a sub-sample used for tissue AsAt quantification. For the determination of AsAt in leaf phloem exudates, petioles were re-cut under water and placed into EDTA or CaCl2 exudation buffer for 90 min in a prehumidified chamber in the dark. For determination of stolon phloem exudates the cut end of the stolon attached to the plant was re-cut under water and placed in the appropriate exudation solution. 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