Endogenous reference genes are commonly used to normalize expression levels of other genes with the assumption that the expression of the former is constant in different tissues and in different physiopathological conditions. Whether this assumption is correct it is, however, still matter of debate. In this study, we searched for stably expressed genes in 384 cDNA array hybridization experiments encompassing different tissues and cell lines.Several genes were identified whose expression was highly stable across all samples studied. The usefulness of 8 genes among them was tested by normalizing the relative gene expression against test genes whose expression pattern was known. The range of accuracy of individual endogenous reference genes was wide whereas consistent information could be obtained when information pooled from different endogenous reference genes was used.This study suggests that even when the most stably expressed genes in array experiments are used as endogenous reference, significant variation in test gene expression estimates may occur and the best normalization is achieved when data from several endogenous reference genes are pooled together to minimize minimal but significant variation among samples. We are presently optimizing strategies for the preparation of endogenous reference gene mixtures that could yield information comparable to that of data pooled from individual endogenous reference gene normalizations. Endogenous reference alse referred to as house keeping genes defines in biology the theoretical assumption that certain genes are ubiquitously expressed in nucleated cells possibly because their stable expression is essential for cell survival and welfare in all physio-pathological circumstances. In practical terms, endogenous reference genes provide a useful constant reference to normalize the expression of test genes in different tissues and in different conditions. This is obviously important when estimates of gene expression are provided in relative terms rather than absolute units of measurement. Thus, endogenous reference genes are used as common denominator in biological fractions where the expression of a test gene is described as the relative ratio over an arbitrarily selected internal control presumed to be stably expressed in all circumstances relevant to the experiment -3. Most Powerful insights in patterns of gene expression could be attained recently through cDNA or oligonucleotide-based global transcript analysis tools that apply a constant reference system to determine ratios of gene expression across large data sets ,29. The Reproducibility assessment of the data set through determination of reference concordance. This was achieved by repeating 14 reference experiments using the melanoma cell line A375 as test sample (Cy5) co-hybridized with pooled PBMC as reference (Cy3). To test for inter-array and printing variation, slide number one and every other 25 slides in sequential order of printing (100 slides per printing set) were used for the repetitive A375 / pooled PBMC hybridizations. In addition, to assess labeling bias, reciprocal labeling was alternatively applied as previously described [Identification of putative endogenous reference genes was performed on 384 array experiments of unequivocal quality by selecting genes that had demonstrated high reference concordance (>90% of the genes in the arrays) and ranking them from the lowest to the highest variance of Log2 test / reference ratios across all array experiments. 3) Validation of the candidate endogenous reference genes as predictor of relative gene expression in large data series. For this purpose, we tested the relative estimates of expression of the melanocytic lineage-restricted melanoma differentiation antigen gp100/Pmel17 (gp100) [Validation of endogenous reference gene-based normalization of gene expression by qRT-PCR and protein expression.In the present study, we tested a set of 419 consecutive experiments performed on a 17,000 gene cDNA array platform to which RNA from neoplastic or normal tissues were consistently co-hybridized with a differentially-labeled reference RNA derived from peripheral blood mononuclear cells (PBMC) pooled from six normal donors. The following steps were pursued: 1) escribed . In this (gp100)  in melan. Thirteen arrays with missing data in more than 30% of the spots and 8 arrays with irregular distribution according to M-A plots (M and A representing respectively log-ratios and average intensities) [log2Cy5/Cy3 ratios were normalized by approximating median values to zero. Spots with ratios > 100 or < 1/100 were truncated at 100 and 1/100 respectively. Data normalization was done by median centering Log2ratio of all genes for each array. For data normalization we used certain arbitrary cutoff criteria to remove spots with weak signal. Weak signal approximating background fluorescence is not reliable and the corresponding log ratios are often distorted resulting in disproportionately high or low values that would bias the statistical results. Because the interpretation of spots with low signal is difficult to make we have adopted the policy of excluding them to focus the analysis on genes whose expression pattern is more reliably tracked by the array tool. In regard to spots with a ratio of >100 or <-1/100, most of them are due to extremely weak signal in one channel which generates a disproportionately extreme value. Although there is no published "gold standard" for the selection of such cutoffs, in practice the parameters that we used for this study are most commonly accepted by investigators and reasonable because allow retention of most of the data in the array excluding only the most extreme and least reliable information.The GenePix Pro 4.0 software was used for array image analysis and calibration. All statistical analyses were performed with the SPLUS package nsities)  were exct test as those with ratios significantly different between the 7 forward and 7 reciprocal experiments after reversal of the reciprocal values (P < 0.05) and the median ratio difference between the 7 forward and 7 reciprocal experiments was larger than 1.5 fold. The genes whose variances of the log ratio across all experiments were among the top 1 percentile of variance of all genes were identified as discordant due to hybridization bias. In addition, genes with more than 50% missing values were excluded. Overall 1,343 out of 16,738 were judged potentially discordant and were excluded from further analysis. The remaining 15,395 concordant genes were used for subsequent analyses.To select from the data base the most reliable genes, we first assessed reproducibility by testing the level of concordance in 14 reference experiments. Reference concordance relies on the expectation that results obtained by repeatedly hybridizing the same test and reference material should perfectly collimate and the degree of deviation from such prediction estimates experimental variance. Concordance can be easily measured by periodically re-hybridizing the same test sample with the constant reference sample. We analyzed a matrix of 7 forward and 7 reciprocally-labeled replicate array experiments hybridized periodically every other 25 cDNA array slides. Reciprocal labeling was applied to measure labeling bias . Data geWe studied 384 of 419 consecutive array experiments remaining after the exclusion of the 14 reference concordance experiments and 21 experiments judged of poor quality due to missing data or irregular distribution by M-A plot analysis. Selection of candidate endogenous reference genes followed these steps.log2 ratio). Consequently, genes with fold changes < 1.46 from zero across all 384 arrays were categorized as candidate endogenous reference genes and were ranked according to ascending values of SD of Log ratio  were used to calculate experimental variance Table . Median Log2 ratio had to be within 2 SD from zero and the average intensities of both channels across all samples need to be higher than 2,000. The intensity parameter was added to ensure that the selected endogenous reference genes were expressed at a relatively high level in most tissues to mitigate excessive fluctuations in Log2 ratios occurring when a low value is applied as denominator. This is important when a reference gene is applied as a denominator in the equation used to normalize the ratio of other tests genes; in such cases robust gene expression decreases the range of ratios resulting from the analysis decreasing, therefore, the experimental variance. Sixty-nine genes fit these criteria. The range of mean Log2 ratio was from 0 to 0.15 and the SD of mean Log2 ratio was from 0.27 to 0.42. Analysis of 7 replicate array based on the same group of candidate endogenous reference genes demonstrated a similar distribution of mean Log2 ratio and SD of mean Log2 ratios (with exception of TFAP2B) suggesting that this variance could be attributed to predominantly experimental noise. Among these genes we further selected for validation 11 that had high mean fluorescence intensity  were found errors in endogenous reference gene selection ."Leave-one-out cross-validations" were used to estimate the error ,34. In e. We found a good correlation between the expression patterns observed by us and that reported by the Affymetrix GeenChip array (data not shown). In addition, a cluster of endogenous reference genes suggested by Applied Biosystems for standardization during qRT-PCR was analyzed by evaluating the SD of their Log2 ratios across the 384 arrays. As data indicated in Table Log2 ratios comparable to that of the endogenous reference genes identified in this study, the corresponding percentage of the arrays in which Log2 ratios were within 2 SD from zero ranged between 0.68–0.85 which is significantly lower than the frequency in which the same parameter fell for the 69 candidate house keeping genes identified here (>95%). In addition, we looked at the variation in our arrays of genes classically used as endogenous reference genes including: ribosomal protein L32, HRPT, β-actin and tubulin-α3. In all cases the mean SD of Log2Ratio was relatively larger than for the endogenous reference genes identified in Table The expression of each candidate endogenous reference gene identified in this study was then compared with available information about the expression of the same genes in 12 normal human tissues reported by the Affymetrix HG-U95A-E probe sets To validate the usefulness of candidate endogenous reference genes in large sample populations we developed a new tool that we are planning to use in the future for validation of gene expression across extensive data sets. This tool displays cDNA libraries originated from different tissues or cell lines individually spotted on a solid surface. The principle of this technology is similar to RNA dot blot which uses RNA isolated from samples and transferred to membrane making, therefore, the transcriptome array a high-density dot blot. The labeled DNA probe of interest is hybridized to the immobilized complementary strain of mRNA. A reference gene hybridization will carried out simultaneously to estimate the relative expression of the gene of interest compared with the reference gene. The new tool we describe here, termed transcriptome array, utilizes cDNA generated from source mRNA for target immobilization to improve the stability of the immobilized targets and differentially fluorescence-labeled test and reference probes (RNA or double strained DNA) then can co-hybridize on to the same spots. Using a validated RNA amplification technology , large qTo test the usefulness of various endogenous reference gene we resorted to the well characterized expression of the melanocytic lineage-specific gene gp100/Pmel17  that is Log2 Cy5 / Cy3 ratios between melanocytic and non-melanocytic tissues. gp100 was, as expected, expressed more in melanocytic lesions with high degree of significance no matter what gp100 / endogenous reference gene combination was used. The range of significance, however, varied extensively depending upon the gp100 / endogenous reference gene combination. This difference was considered representative of the ability of different endogenous reference genes to normalize for tissue-specific gene expression patterns . There was, however, a big range in the discriminatory capacity among endogenous reference genes with NEDD8, RAB3 and FZD6 providing the highest predictive value  and β-actin being one of the least reliable .As endogenous reference genes we chose β-Actin and new candidate genes identified by this study (see previous section). The test genes  were separately hybridized to the transcriptome array. Each gene labeled with Cy3 was co-hybridized with individual endogenous reference genes labeled with Cy5. We then compared Log2 ratios obtained with all endogenous reference genes and those obtained using the genes that provided the best five or the best three results . The basis for this test was that non-melanocytic lesions uniformly should not express gp100 and, therefore, the Log2 ratios for any gp100 / endogenous reference gene combination should be constant resulting in very low SD. SD for non-melanocytic lesions were much lower using pooled endogenous reference genes for the normalization . Similar results were obtained when results from the best 5 and best 3 endogenous reference genes were pooled together. In addition, even if pooling data together did not provide the highest level of discrimination compared to the best results obtained with selected individual endogenous reference genes, the capacity of pooled data sets to discriminate gp100 expression between melanocytic and non melanocytic samples was still very high . Interestingly, different endogenous reference genes provided not only different levels of discrimination but also provided different estimates of gp100 expression in melanocytic lesions with gp100 / endogenous reference gene Log2 ratio above  or below  0  the discriminatory capacity of endogenous reference genes in samples with known patterns of expression of gp100. We selected 6 previously characterized ,36 melanAlthough the concept of endogenous reference genes is appealing it may be unrealistic to expect that a gene could be equally expressed in all eukaryotic cells in all physiopathological conditions. Thus, the endogenous reference gene concept can only approximate such ideal biological behavior. In fact, numerous studies noted that the expression of the ostensible endogenous reference genes varies according to distinct physio-pathological situations and can be affected by experimental manipulation ,26,27,41In this study, therefore, we looked for relative stable endogenous reference gene challenging a 17,000 cDNA clone data base against approximately 400 specimens that included a broad variety of normal and neoplastic tissues as well as cell lines. Indeed, few previously unnamed genes approximated the ideal endogenous reference gene by being expressed consistently in more than 95% of the specimens analyzed within a variance that was barely and insignificantly different from what could be expected from intrinsic experimental fluctuation . However, gene expression analysis is done to determine such specificity and in most cases no previous knowledge of tissue specificity is truly available. One may argue that, in spite of the extreme variability in normalization power demonstrated by various endogenous reference genes, still significant differences could extracted between the melanocytic and non-melanocytic samples no matter what endogenous reference genes were used. This statement is correct in this experimental situation where hundreds of samples could be compared with the transcriptome array. However, in cases when only few samples are analyzed the difference in discriminatory capacity becomes critical. For instance, when randomly selected gp100 / endogenous reference gene The lowest limit of detection in the transcriptome array of specific probes in term of copy numbers is currently under investigation. Decreasing concentrations of β-actin alone printed the array spots as internal controls suggest that the detection range is from 30 ng / spot to 3 pg / spot when standard fluorescent intensity range parameters are applied. The higher limit of detection before saturation can be easily adjusted to avoid under estimates of highly expressed genes, therefore, increasing the dynamic range of detection. This is achieved by adjusting the fluorescent intensity right below the saturation for both channel and subsequently calculating the abundance of reference gene expression using serially diluted internal control spots as standard curve. With few exceptions, the expression of most genes can be estimated with relative accuracy using this semi-quantitative approach.Since in most cases endogenous reference genes are selected based on previous knowledge of their usefulness for a particular experimental situations, it is likely that the best consistency could be achieved in such circumstances when averages of several endogenous reference gene normalizations are used. This may represent a reasonable solution particularly if a cocktail of endogenous reference genes identically labeled could be used as reference within a single experiment. We are presently testing various combinations of lineage specific genes and pools of endogenous reference genes for hybridization to the transcriptome array and for qRT-PCR. Such attempts, however, have been quite disappointing so far as results obtained using mixture of endogenous reference genes do not yield information comparable to that achievable when results obtained with the same endogenous reference genes used individually are pooled together. This is possibly due to imbalanced density of various endogenous reference genes in different samples with predominant effects of some over others. Therefore, extensive work in the future will be aimed at equilibrating the interactions among endogenous reference genes in various experimental conditions to test whether mixtures of them could be used in multiplex for normalization of large expression analysis studies.Our observations, together with previous work by others indicate that much caution should be taken when using endogenous reference to normalize the expression of test genes. It is likely that previous analyses based on single endogenous reference genes might have been strongly biased by the individual selections and the interpretation of the results should looked at with caution. In particular, this study suggests that even when the most stably expressed genes in array experiments are used as endogenous reference genes, significant variation in test gene expression estimates may occur and the best normalization is achieved when data from several endogenous reference genes are pooled together to minimize minimal but significant variation among samples. We are presently optimizing strategies for the preparation of endogenous reference gene mixtures that could yield information comparable to that of data pooled from individual endogenous reference gene normalizations.The list of samples used for cDNA microarray hybridization or for preparation of the transcriptome array is available upon request. Most melanoma and renal carcinoma cell lines were generated at the Surgery Branch, National Cancer Institute (NCI), National Institutes of Health (NIH), Bethesda, MD and maintained in RPMI  supplemented with 10% fetal calf serum (Biosource). Cell lines were harvested by Trypsin /Versene (Biosource) digestion. PBMC were collected by leukapheresis from six unrelated normal donors in the Department of Transfusion Medicine, Clinical Center, NIH and purified by Ficoll gradient separation. Surgically removed tumor tissues were collected from the Tissue Network , Surgery Branch and CPSB specimen bank, NCI, . Fine needle aspiration (FNA) biopsies were obtained from patients with metastatic melanoma referred to the Surgery Branch, NCI for immunotherapy. Total RNA was isolated using Qiagen RNeasy kit and its quality and quantity was estimated using Agilent Bioanalyzer 2000 .in vitro into anti-sense RNA (aRNA) and reverse-transcribed into fluorescence labeled cDNA for hybridization to 17,000 gene cDNA-based microarrays [Total RNA extracted from test and reference samples was transcribed roarrays ,44. Pool.cDNA (UniGene cluster) microarrays were printed at the Immunogenetics Section, DTM, CC, NIH with a configuration of 32 × 24 × 23 containing 17,500 elements. Clones used for printing were selected from the Research Genetics RG_HsKG_031901 8 k clone set and the 9k RG_Hs_seq_ver_070700 40 k clone set. The 17,500 spots included 12,072 uniquely named genes, 875 duplicate genes and about 4,000 expression sequence tags. For a complete list of genes included in the Hs-CCDTM-17.5k-1px printing please visit our web site at .A collection of aRNA-based libraries was prepared from 960 frozen tissue samples or cell lines and individual aRNAs were reverse transcribed into cDNA in the presence of 1 μl of dN6 primer (8 μg/μl), 6 μl of first strand buffer, 3 μl of 10 mM dNTP, 3 ul of 50 mM dTT, 1.5 μl of Rnasin and 10 μl of aRNA (6–12 ug) with addition of 3 μl of Superscript II (BRL) in 5.5 μl volume after heating to 65°C for 5 minutes. Reactions were carried out in 96-well plates at 42°C for 90 minutes followed with addition of 1 μl Rnase H and heating for 20 min at 37°C. cDNA were further purified by utilizing CentriCept gelfitration plates to remove non incorporated primer and dNTP. Purified cDNA were transferred to 384-well plates and dried by speedvac. Samples were re-suspended in 13 μl of 3 × SSC followed by shaking at 3,000 rpm for 15 min. Reconstituted cDNA libraries from individual samples and spiked reference gene at different concentration were printed on to poly-L-lysine coated glass slides at the concentration of 0.5–1 μg/μl using the OmniGrid  printer and Telechem printing pins . Each 100 μm diameter spot was duplicated at a 250 μm distance. After complete exsiccation, slides were post-processed as described at . In order to generate single strand fluorescence labeled cDNA for hybridization, modified specific 5' primers with an extension of the T7 promoter region were used for PCR amplification. Double strand PCR fragments (300–800 bp) were used as template for in vitro transcription to generate single stranded mRNA. 3 μg of amplified RNA were then reverse transcribed into cDNA in present of 4 μl of first strand buffer, 1 μl dN6 primer , 2 μl 10 X low T-dNTP , 2 μl Cy-dUTP , 2 μl 0.1 M DTT, 1 μl RNasin, 3 μg amplified mRNA in 8 μl DEPC H2O. After heating to 65°C for 5 minutes and cooling to 42°C, 2 μl of SSII was added and the labeling reaction was carried out at 42°C for 90 min. Probe purification and hybridization were performed as previously described [Specific PCR primers for the amplification of each gene probe were designed using Primer 3 program ragments 0–800 bp escribed .max = 518 nm) and at the 3' end with the quencher dye molecule 6-carboxytetramethyl-rhodamine . Total RNA was used for gene expression validation. Measurement of gene expression was performed using the ABI 7900HT sequence detection system  as previously described [Primer and probe sets from each candidate genes were designed using the Primer Express 2.0 program  and synthesized by BioSource. Taqman probes were labeled at the 5' end with the reporter dye molecule 6-carboxy-fluorescein (FAM; emission λescribed ,45. Messescribed .PJ validated the system combining high-throughput data with gene-directed analysis by quantitative real-time PCR. YZ performed statistical analysis. YN, MCP, DN, VM and KS participated in the preparation and utilization of array experiments used for the identification of stably expressed genes. NH, HS and PRT provided a large library of samples necessary for the development of the transcriptome array. FMM supervise the project as principal investigator.EW provided leadership for the development of cDNA arrays, transcriptome array and for the overall conduct of the experiments.


Drosophila immune response involves three types of hemocytes (‘blood cells’). One cell type, the lamellocyte, is induced to differentiate only under particular conditions, such as parasitization by wasps. Here, we have investigated the mechanisms underlying the specification of lamellocytes. We first show that collier (col), the Drosophila orthologue of the vertebrate gene encoding early B-cell factor (EBF), is expressed very early during ontogeny of the lymph gland, the larval hematopoietic organ. In this organ, Col expression prefigures a specific posterior region recently proposed to act as a signalling centre, the posterior signalling centre (PSC). The complete lack of lamellocytes in parasitized col mutant larvae revealed the critical requirement for Col activity in specification of this cell type. In wild-type larvae, Col expression remains restricted to the PSC following parasitization, despite the massive production of lamellocytes. We therefore propose that Col endows PSC cells with the capacity to relay an instructive signal that orients hematopoietic precursors towards the lamellocyte fate in response to parasitization. Considered together with the role of EBF in lymphopoiesis, these findings suggest new parallels in cellular immunity between Drosophila and vertebrates. Further investigations on Col/EBF expression and function in other phyla should provide fresh insight into the evolutionary origin of lymphoid cells. The lamellocyte is induced to differentiate under conditions such as parasitization by wasps and is shown in this study to require collier, the orthologue of the vertebrate early B-cell factor Drosophila shares several features with the analogous process in vertebrates. A first population of embryonic hemocyte precursors (prohemocytes) is specified from the head mesoderm very early during embryogenesis. At the end of larval stages and the onset of metamorphosis, a second population of hemocytes is released from a specialised hematopoietic organ, the larval lymph gland  is expressed in the lymph glands at the end of embryogenesis   , a key for (EBF) . We showDrosophila lymph glands at the end of embryogenesis  are lethal at the late embryonic stage  parasitization by producing lamellocytes. This dedicated cellular response is maximal in wild-type (wt) larvae 48 h after wasp egg-laying 06949 AN293 06949 . Whereas1 larvae D and 2E.2R)AN293 . In no cestation , col1 ise33C-Gal4 line, which drives expression in lymph glands   , with a pen-lacZ . Around pen-lacZ G, which pen-lacZ . This phpen-lacZ . Thus, wal cells , consistal cells . No prod lz-Gal4  and hml- drivers , respectAt the end of larval stages, the lymph gland is composed of four to six paired lobes. The two anterior (primary) lobes that formed in the embryo  contain Ser mutant cells in the larval lymph glands further indicated that Ser-expressing cells are responsible for activation of Lz expression in surrounding cells and their commitment to a crystal cell fate (Col expression in a posterior cluster of cells of the primary lobes is reminiscent of that of Serrate (Ser), a Notch ligand . The Serell fate . Togetheell fate F–3H. Howell fate   Col expression defines a specific group of cells within the lymph glands; (ii) lamellocyte differentiation, which is an exclusive feature of lymph gland hematopoiesis, depends upon Col activity; and (iii) the massive production of lamellocytes that follows parasitization does not involve changes in Col expression. We thus propose a two-step signalling model for induction of lamellocytes in response to wasp egg-laying A. AccordDrosophila cellular immune response, and EBF function in B-cell development in vertebrates, suggests that Col/EBF function was co-opted early during the evolution of cellular immunity. A puzzling question remains, however, of how the cell-autonomous function of EBF in B-cell development, and the non–cell-autonomous function of Col in lamellocyte development, could relate to an ancestral Col/EBF function. We would like to propose that the ancestral expression of Col/EBF in a subset of hematopoietic cells conferred on these cells the ability to respond to signals from circulating immune supervisors (generically designated as macrophages in Drosophila PSC on the other. Although admittedly highly speculative, this proposal takes into account the following considerations. B-cell development represents the default fate of lymphoid progenitors (Pax5. The −/−Pax5 pro–B-cells retain the ability to generate a whole range of both ‘innate’ myeloid and lymphoid cells (Pax5, acting downstream of EBF, for which there is no known counterpart in Drosophila hematopoiesis. Second, the co-opting of Col activity for lamellocyte differentiation in larval hematopoiesis most likely came on top of a preexisting hematopoietic system, such as that operating in Drosophila embryos (B- and T-lymphocytes mediate adaptive immunity, a phylogenetically recent component of the immune system as it is found only in gnathostomes . How adagenitors . Althougid cells . Thus, t embryos . FurtherL. boulardi for 2–4 h, then allowed to develop at the appropriate temperature and analysed 24 or 48 h later. Hemocyte observation and counting, and lacZ staining of lamellocytes, were as previously described (Unless otherwise stated, all fly stocks were maintained at 25 °C on standard medium, and genotypes were verified with marked balancer chromosomes. For wasp infection, second instar larvae were submitted to egg-laying by escribed .srpD-Gal4 transgene: A distal promoter fragment, between 8.8 and 6 kb upstream of the srp transcription start site and a 340-bp fragment overlapping this site were amplified by PCR using 5′-GCTAGCGACGCGTGATGCAACTTAATCAA-3′ and 5′-CTGCAGTTTATGAATGGAAGACGCGGACG-3′ primers, and 5′-CTGCAGACGGCCAAGTCCAACAACAACAA-3′ and 5′-GGATCCCTGTTGCTGCTGTAACTGTTGAT-3′ primers, respectively, then fused before subcloning upstream of the Gal4 coding sequence in a pCaSpeR vector. Transgenic lines were obtained by standard procedures. Because they are embryonic-lethal at 25 °C, the srpD-Gal4/UAS-col animals were kept at 18 °C before shifting to 25 °C at the second larval instar.The Immunostaining and in situ hybridisation of larval lymph glands and embryos were performed as in


Combined treatment modalities using TRAIL and cytotoxic drugs revealed highly additive effects in different tumour cell lines. Little is known about the efficacy and underlying mechanistic effects of a combined therapy using TRAIL and ionising radiation in solid tumour cell systems. Additionally, little is known about the effect of TRAIL combined with radiation on normal tissues.TRAIL (Tumour cell systems derived from breast- (MDA MB231), lung- (NCI H460) colorectal-  and head and neck cancer  were treated with a combination of TRAIL and irradiation using two different time schedules. Normal tissue cultures from breast, prostate, renal and bronchial epithelia, small muscle cells, endothelial cells, hepatocytes and fibroblasts were tested accordingly. Apoptosis was determined by fluorescence microscopy and western blot determination of PARP processing. Upregulation of death receptors was quantified by flow cytometry.The combined treatment of TRAIL with irradiation strongly increased apoptosis induction in all treated tumour cell lines compared to treatment with TRAIL or irradiation alone. The synergistic effect was most prominent after sequential application of TRAIL after irradiation. Upregulation of TRAIL receptor DR5 after irradiation was observed in four of six tumour cell lines but did not correlate to tumour cell sensitisation to TRAIL. TRAIL did not show toxicity in normal tissue cell systems. In addition, pre-irradiation did not sensitise all nine tested human normal tissue cell cultures to TRAIL.Based on the in vitro data, TRAIL represents a very promising candidate for combination with radiotherapy. Sequential application of ionising radiation followed by TRAIL is associated with an synergistic induction of cell death in a large panel of solid tumour cell lines. However, TRAIL receptor upregulation may not be the sole mechanism by which sensitation to TRAIL after irradiation is induced. Tumour necrosis factor related apoptosis inducing ligand) is one of the most promising anti-cancer agent being currently under investigation (for review see [TRAIL (view see -5). Initview see ,7. In thview see ,9. Howevview see . This isview see .in vivo. In this regard it has been shown that mice lacking TRAIL display a significantly reduced capacity to eliminate syngenic tumour cells in the liver [One possible explanation for the tumour specifity of TRAIL could lie in its potential role as a mediator of tumour immune surveillance he liver ,13.Tumour necrosis factor) or CD95. Most important in this regard is the fact that at least five, instead of only two resp. one, different TRAIL receptors have been identified: The proapoptotic DR4/Trail-R1 [Although TRAIL is a member of the death receptor ligand family certain differences exist to the well characterised ligands TNF . KeyWith only very few exceptions , apoptosInterestingly, both  pathways are interconnected on several levels. In case of death receptor activation, the propagation of the apoptotic signal is enhanced by caspase-8 mediated activation of Bid . Bid likAlthough TRAIL induces apoptosis when given alone, it has been shown that combination of TRAIL with cytotoxic drugs as 5-fluorouracil, etoposide, paclitaxel, actinomycin C and cisplatin has an even higher apoptotic efficacy -59,6.Whereas abundant data therefore support the combination of TRAIL with cytotoxic drugs, only limited studies of TRAIL combined with ionising radiation have been performed ,33,60-62All biochemicals were obtained from Sigma-Aldrich chemicals  unless otherwise specified. Hoechst 33342 was purchased from Calbiochem and dissolved in distilled water as 1.5 mM stock solution.2. Normal human epithelial cells  and hepatocytes were obtained from Clonetics/Cambrex . Cell culture was performed according to the manufacturer's protocols.The tumour cell lines MDA-MB 231, HCT-15, Colo 205, NCI H460, FaDu DD and SCC-4 cells were purchased from ATCC . HSF6/ HSF7 fibroblasts, HUVEC and SMC were kindly provided from H-P. Rodemann and R. Kehlbach . Respectively. All cells were grown in RPMI 1640 medium  and maintained in a humidified incubator at 37°C and 5% COTRAIL induced apoptosis was induced with recombinant human TRAIL/TNFSF10  in concomitant or sequential application with irradiation.Cells were irradiated with 6 MV Photons using a Siemens Mevatron linear accelerator with a dose rate of 4 Gy per min at room temperature.Apoptosis induction was quantified by counting of cells with a characteristic apoptotic morphology after DNA staining with Hoechst 33342. Cells were stained by incubation with Hoechst 33342 at a final concentration of 1.5 μM for 15 min. Microscopy was performed using a Zeiss Axiovert 200 microscope  using an excitation wavelength filter of 380 nm. All apoptotic rates were means of at least three independent experiments. The given error bars represent the standard error of the mean from independent measurements of the same cell batch.6) were lysed for 30 min in a lysis buffer containing 25 mM HEPES, 0.1% SDS, 0.5% deoxycholate, 1% Triton X-100, 10 mM EDTA, 10 mM NaF and 125 mM NaCl on ice. After removing insoluble material by centrifugation for 10 min at 12.000 g, 20 μg lysate was separated by SDS-PAGE. Blotting was performed employing a tank blotting apparatus  onto Hybond C membranes . Equal protein loading was confirmed by Ponceau S staining (Sigma). Blots were blocked in PBS buffer containing 0.05 % Tween 20 and 5% bovine serum albumin at 4°C over night. Primary antibodies were detected after repeated washings with PBS/Tween 20 (0.05%) of the membrane, using a secondary antibody  diluted in PBS/Tween and incubated for 3 hours at room temperature and washed three times with PBS/Tween. Detection of antibody binding was performed employing enhanced chemoluminescence . PARP cleavage was tested using a polyclonal antibodies for cleaved and uncleaved PARP from Boehringer  in a 1: 1000 dilution. Monoclonal antibodies for caspase 8 were a gently gift from Prof. K. Schultze-Osthoff and used in a 1:45 dilution. β-Actin  antibody was used in a 1: 5000 dilution.Cells (1 × 106) were washed twice with PBS and incubated for 30 min with PE-labeled anti-R1/DR4 or -R2/DR5-antibody  at a dilution of 1: 400 in 0,5% FCS/PBS. FACS analyses of superficial receptor expression was performed according to manufacturer's protocol with the Quantibrite™ kit from BD .Cells . No upregulation of DR4/R1 was found in all tested tumour cell lines. Instead, a subtle downregulation of DR4/R1 after irradiation was observed fig. . Fig. 4BAs stated above, hardly any data on normal tissue toxicity after combined treatment are available. We therefore analysed the effect of irradiation plus TRAIL on human hepatocytes, fibroblasts (HSF6 and 7), epithelial cells from prostata (PrEC), kidney (RPTEC), breast (HMEC) and small airways (SAEC), endothelial cells from umbilical cord (HUVEC) and small muscle cells (SMC). Normal tissue cells were treated with 10 Gy and a tenfold higher dosage of TRAIL (1 ng/ml) as used for tumour cells. For evaluation of apoptosis strict morphologic criteria as condensation of chromatin and nuclear fragmentation were used. 48 h after combined treatment no relevant sensitisation of normal tissue cells to TRAIL induced apoptosis could be detected in all cultures. Moreover, even preirradiation did not sensitise normal cells to TRAIL induced apoptosis as observed in tumour cells table .Based on the rationale that radiation and TRAIL induce cell death via distinct but overlapping cell death pathways, tumour cell lines and normal tissue cultures were subjected to either radiation or TRAIL alone or combined with varying application schedules. Our data show that combining radiation with TRAIL induces apoptosis in a significantly higher percentage than either treatment alone. It is important to note that for TRAIL stimulation in our experiments on tumour cells very low concentrations of TRAIL were used (0.1 ng/ml). The pharmacodynamic properties of TRAIL, especially the peak plasma levels in humans, are not known. Since it is likely that toxicity rises with higher doses of TRAIL, the observation of pronounced effects on tumour cell kill at such low doses is particularly intriguing.Any combination of radiation with TRAIL proved to be more effective than either one alone; however depending on dose level and schedule of stimulation less than additive, additive and synergistic effects were detectable. When TRAIL was applied simultaneously with irradiation three of the six cell systems reacted synergistically. In contrast, five of the six cell systems reacted with synergistic effects when TRAIL was given sequentially 12 hours after irradiation. One of the cell lines displayed only less than additive effects.Statistical analysis confirmed, that synergistic effects are more pronounced and occur with greater likelihood after sequential application of TRAIL after irradiation when compared to concomitant treatment schedules.-/- HCT-116 cells and mouse embryonic fibroblasts showing that NF-κB may also be important for a irradiation induced upregulation of death receptors [Possible explanations for the positive interaction of TRAIL and DNA damaging agents including ionising radiation are being disputed. Since the sensitisation was associated with upregulation of the DR5 receptor in some experimental settings ,64,68-70eceptors .Recently, it was shown in overexpression experiments of NF-κB that its subunits c-Rel and RelA regulate expression of cell death molecules in a differential manner. This suggests that RelA, in contrast to c-Rel, acts as a survival factor by inhibiting expression of DR4/DR5 and caspase-8 and up-regulating cIAP1 and cIAP2 . This prRecently, it has been shown that the interaction of 5-FU with TRAIL is strictly Bax but not Bak dependent in HCT 116 cells . Thus, tHowever, the general mechanism of the positive interaction of TRAIL with irradiation remains unclear. It may ultimately turn out, that manifold mechanisms exist and only some mechanisms will be operative in a single cell system .The second part of our experiments the toxicity of TRAIL combined with radiation on normal tissues. In order to be able to draw relevant conclusions for normal tissue cells , 10 foldOur data suggest that TRAIL has great potential in cancer treatment, especially in sequential combination with radiotherapy. We did not observe any sensitising effect of the sequential treatment on TRAIL sensitivity in normal tissue cells. Xenograft experiments designed to answer the questions regarding the short and long term efficacy of a combination of radiation with either TRAIL or TRAIL specific antibodies are underway in our laboratory.APAF, apoptosis protease activating factor; FADD, Fas-associated death domain protein; NFκB, nuclear factor κB; PARP, poly-ADP-ribosyl polymerase; TNF, tumor necrosis factor; TRAIL, TNF-related apoptosis inducing ligand. HMEC: human mammary epithelial cells, PrEC: human prostate epithelial cells, RPTEC: epithelial cells of renal proximal tubule, SAEC: small airways epithelial cells, HUVEC: human epithelial cells of umbilical vein, SMC: smooth muscle cells, HSF 6,7: human fibroblastsThe author(s) declare that they have no competing interests.PM performed FACS-analysis and microscopic evaluation of Hoechst stained cells, analyzed the data, participated in the conception of the trial and participated in the preparation of the manuscript. AS participated in microscopic evaluation of tumour cell apoptosis and accomplished analysis of the resulting data. VJ participated in receptor analysis and microscopic evaluation of Hoechst stained cells. HF carried out Western blotting. PTD participated on the preparation of the manuscript. WB performed isobologramm analysis. CB participated in the conception, design of the study, coordination of the study as well as preparation of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Cross-species gene-expression comparison is a powerful tool for the discovery of evolutionarily conserved mechanisms and pathways of expression control. The usefulness of cDNA microarrays in this context is that broad areas of homology are compared and hybridization probes are sufficiently large that small inter-species differences in nucleotide sequence would not affect the analytical results. This comparative genomics approach would allow a common set of genes within a specific developmental, metabolic, or disease-related gene pathway to be evaluated in experimental models of human diseases. The objective of this study was to investigate the feasibility and reproducibility of cross-species analysis employing a human cDNA microarray as probe.As a proof of principle, total RNA derived from human and bovine fetal brains was used as a source of labelled targets for hybridisation onto a human cDNA microarray composed of 349 characterised genes. Each gene was spotted 20 times representing 6,980 data points thus enabling highly reproducible spot quantification. Employing high stringency hybridisation and washing conditions, followed by data analysis, revealed slight differences in the expression levels and reproducibility of the signals between the two species. We also assigned each of the genes into three expression level categories- i.e. high, medium and low. The correlation co-efficient of cross hybridisation between the orthologous genes was 0.94. Verification of the array data by semi-quantitative RT-PCR using common primer sequences enabled co-amplification of both human and bovine transcripts. Finally, we were able to assign gene names to previously uncharacterised bovine ESTs.Results of our study demonstrate the harnessing and utilisation power of comparative genomics and prove the feasibility of using human microarrays to facilitate the identification of co-expressed orthologous genes in common tissues derived from different species. Microarrays are routinely used for large scale transcriptome analyses and have been widely and successfully employed for simultaneously monitoring the expression of a potentially unlimited number of genes in parallel, thus providing the basis for identifying genes differentially expressed in distinct cell-types, developmental stages, disease states and cells subjected to exogenous reagents . The rapThe advantages of cross-species hybridisation are two-fold. First, cross-species gene-expression comparison is a powerful tool for the discovery of evolutionarily conserved mechanisms and pathways of expression control. The advantage of cDNA microarrays in this context is that broad areas of homology are compared and hybridization probes are sufficiently large so that small inter-species differences in nucleotide sequence would not affect the analytical results. This comparative genomics approach would allow a common set of genes within a specific developmental, metabolic, or disease-related gene pathway to be evaluated in experimental models of human diseases. Second, the use of microarrays in studies in mammalian species other than human and rodents, for example nonhuman primates, bovine, sheep and porcine may advance our understanding of human health and disease, for example the use of animal models in drug target validation. However, the inavailability of adequate sequence data and commercial cDNA and oligonucleotide microarrays keeps this technology beyond the reach of investigators working on economically and scientifically important large domestic species such as cattle, pigs and sheep. A potential solution to this problem is the use of cross-species hybridisations, i.e, human sequence-based arrays as tools for undertaking comparative genome expression studies. Such analyses have been performed using ape brain RNA as target on a human oligonucleotide array  and pig,in vitro production of embryos for stem cell derivation and somatic nuclear transfer cloning. Employing the bovine model and sensitive RT-PCR assays, it has been shown that the majority of embryos derived from such sources display distinct mRNA expression patterns in a variety of developmentally important genes compared to their in vivo derived counterparts [in vitro production and/or nuclear transfer of which significant oversize is a predominant feature [A crucial step employing domestic animals in biomedicine is genetic modification which requires extensive embryo and embryo-related technologies, such as terparts . Some of feature . Analysi feature ,11 and aImportant criteria for evaluating any microarray system include the reproducibility of the data generated, the specificity of detection of the targeted gene, and the validity of the results that identify and establish differential gene expression. The experiments described here show the systematic validation of cross-species microarray analysis, with emphasis on the reproducibility and statistical analysis of generated data using standard microarray data analysis tools.Specifically, we investigated the feasibility and reproducibility of cross-species hybridisation of orthologous genes within a defined developmental and metabolic pathway using as a test case and the first description of its kind, human and bovine fetal brain RNA as Cy-dye labelled targets on a human cDNA microarray. The microarray is composed of 349 genes each spotted 20 times to ensure reproducible validation by independent technologies such as semi-quantitative RT-PCR as carried out in this study, or alternatively, Real-Time PCR and Northern blot analysis.HPRT and β-ACTIN  were employed as endogenous guide dots to enable accurate grid placement prior to image analysis. These spots can be seen as intense yellow signals at the periphery of each block as portrayed in Figure OCT-4 and Arabidopsis cDNAs  were included as negative controls to monitor hybridisation-specificity. The transcription factor OCT-4 is expressed in human embryonic stem cells and primordial germ cells and is down-regulated upon differentiation [The human cDNA microarray used in this study consisted of 349 fully sequenced and annotated cDNAs as described in the Supplemental Table ntiation .Classification of the genes according to Gene Ontology annotations (molecular function) and chromosomal location Figures  and 1C dOur experimental design incorporated dye-swaps and four replicated hybridisations. Three hundred and forty nine cDNAs, each representing a unique gene were spotted 20 times on each microarray. Four independent experiments were performed with human and bovine brain, respectively. Within each experiment the 20 replicates were averaged to yield a reliable signal for the respective probe. In the next step, the replicated signals from the different experiments were averaged to compute overall characteristics. The overall correlation of expression of the human and bovine genes is shown in Figure Additionally, we computed MA-plots  in orderth cycle of a standard PCR reaction. cDNAs were considered as "detected" when their average BG-tag was above 0.9.In order to measure whether a given gene was significantly expressed, we compared its cDNA's signal to a signal distribution derived from negative controls represented by approx. 2,500 empty spot positions on the array. After quantification of each array a low non-zero intensity is assigned to each of these empty spots reflecting the amount of background signal on the array. Since these positions are spread uniformly over the array, the distribution of these signals reflects the distribution for signal noise and is an indicator whether signals are at the background level or reflect reliable expression levels. For each cDNA we counted the relative proportion of empty positions on the array that are smaller than the actual observed intensity (BG-tag). BG-tags from replicated experiments for the same cDNA were averaged. Thus, high values (close to one) indicate that the cDNA is expressed in the respective tissue whereas low values reflect noise. The limit of visual detection of a spot corresponds to a BG-tag level of 0.9, however there is a grey zone around this value. Comparison with RT-PCR analyses showed  that thiWe grouped the 349 genes into three classes of expression levels with respect to their intensity values above background levels  with class 1 as low (not-detected), class 2 as medium (possibly detected) and class 3 as high level of expression (detected), respectively. Within class 1, are 19 bovine and 17 human genes. Class 2, comprises 26 bovine and 17 human genes and finally class 3 has 304 bovine and 315 human genes, respectively. The genes within expression class 1 have signal intensities below the detection level of our microarray analysis platform and as such these genes can be designated as "Absent"  across the replicated experiments. Histograms are shown in Figure Figure ZNF278, APOARGC, KIAA1609 and MGC12904 – highlighted as diamond shapes in Figure Three statistical tests that judge the significance of differences in the levels of gene expression in human and bovine fetal brains were employed  as described previously . Tests wAPOARGC). Thus, Holm's setp-wise correction would start with an adjusted experiment level P-value of p = 0.05/349 = 1.43e-04. Similarly, measuring the false discovery rate by qvalues [Furthermore, taking into account corrections for multiple testing, no gene is differentially expressed at the global experiment significance level of 0.05. For example, the lowest P-value of the Welch test is 1.98e-03, , solute transport- (SLC11A3), protein assembly- (CALNEXIN), anion channel- (VDAC2) and as an endogenous reference, the housekeeping gene, β-ACTIN. Primer sequences were designed to co-amplify the orthologous genes  are expressed at a low level  in human and twenty genes (5.7%) in bovine with variability in expression levels in the four hybridisation experiments. In addition, approximately 305 genes have a CV of less than 0.5 with human brain (87%) compared to 280 with bovine brain (80%). However, this level of variability is low and can be expected due to the fact that the probe set used are of human origin. Moreover, the slight decrease in reproducibility due to species and nucleotide sequence differences can be compensated for by increasing the number of independent repetitions  of the experiments. The unexpectedly low level of variability in gene expression levels between human and bovine fetal brain can also be attributed to the 80 data points examined per gene and the four replicate hybridisations  carried out for each species. Our finding further emphasises the need for sufficient technical and biological replicates in all microarray experiments.CDC27: 0.95, CDC6: 1.36, PCNA: 1.07, respectively, whereas the deduced ratios derived from the semi-quantitative RT-PCR assay were 0.59 for CDC27, 0.35 for CDC6 and 0.87 for PCNA, respectively.In assessing differential gene expression in bovine and human fetal brain, we observed that the expression levels of the individual orthologous genes were roughly in the same broad range. For example, if one considers genes involved in pathways of cell cycle control, the microarray deduced ratios (bovine:human) were for SLC11A3 and CALNEXIN are 0.65 vs. 0.98 and 0.88 vs. 0.97. An illustration of co-amplification with common primers and also the confirmation of the array data for a selection of genes can be seen in Figures Similarly the comparative ratios for The similar expression ratio of the orthologous genes implicated in cell cycle control and initiation of eukaryotic genome replication which is conserved in all eukaryotes highlights the feasibility and importance of our study in examining conserved pathways operative within the fetal brains of human and bovine and of course across other species using human cDNA microarrays. A similar finding has also been confirmed when comparing yeast co-regulated genes against the archaeal and bacterial operons. This implies that the components of the protein translation process are conserved across organisms at the expression level with minor specific differences .We also identified four significantly differentially expressed genes as judged by the Wilcoxon test. The Wilcoxon test is known to be more conservative than the Student's t-test and the Welch-test, however, we have more confidence in this test since it is distribution-free, in particular it does not depend on the partly unrealistic assumption of an underlying Gaussian distribution.ZNF278 and APOARGC were 1.45 – and 1.66 -fold over-expressed in human whereas KIAA1609 and MGC12904 were 1.60 – and 1.37-fold over-expressed in bovine. ZNF278 encodes a zinc finger-containing transcription factor that acts as a transcriptional repressor and also implicated in small round cell tumours [APOARGC encodes a protein with hydrolase activity. MGC12904 and KIAA1609 are uncharacterised ESTs so comments cannot be made with regard to their function.The genes  tumours . The genOrthologous genes between human and mouse and between human and rat both have a mean of approximately 85% sequence identity ,23. In tFurthermore, using this protocol of cross-species hybridisation, we have compared gene expression in bovine unfertilised oocytes and blastocyst using a larger microarray (The Human Ensembl Chip) comprising 15,500 fully sequenced and annotated genes and ESTs. The correlation co-efficient between the RNA samples is 0.26, thus reflecting the diversity between oocyte derived maternal transcripts and embryonic transcripts derived from the blastocyst .In summary, this study highlights the significance and utilisation power of comparative genomics and also demonstrates the feasibility of using human cDNA microarrays to facilitate the identification of differentially expressed genes in human and bovine fetal brain. Our results indicate that cross-species hybridisation is not only a useful short-term solution for studying species for which gene maps, cDNA or oligo microarrays are not yet available, but also possesses tremendous power in enabling the unravelling of common evolutionary evolved mechanisms in different species.For the generation of probes for spotting, cDNA inserts from a single 384-well plate of non-redundant, fully sequenced, annotated human cDNA collection (Human Ensembl set RZPD1.1) were employed-  and a second partial plate consisting of control genes and empty wells to be used for data normalisation were amplified by PCR in a 384-well format. Bacterial clones were transferred to the PCR plates using 384-well replicators . PCR amplifications were carried out in a total volume of 25 μl consisting of 1x PCR buffer, 1.5 M Betaine , 1U Taq polymerase, 200 mM of each dNTP , 25 pmoles of M13-forward (5'GTAAAACGACGGCCA3') and M13-reverse (5'CAGGAAACAGCTATGAC3') primers respectively. Cycling parameters typically consisted of an initial denaturation step at 95°C for 5 min followed by 35 cycles of denaturation at 95°C for 1 min, annealing at 55°C for 1 min, elongation at 72°C for 1 min, then a final elongation step at 72°C for 10 min. All amplified products were analysed by agarose gel electrophoresis and in all cases single bands were obtained per gene.Purification was carried out using isopropanol precipitations. In brief, 24 μl of the PCR product was transferred into 384-well Genetix plates  previously filled with 18 μl of isopropanol per well. The samples were mixed by gentle vortexing after sealing the plates with transparent tape. After precipitation overnight at -20°C, the plates were spun at 3,500 rpm for 2 hrs at 21°C, the isopropanol decanted off and the plates dried briefly in a SpeedVac without heating. The pellets were resuspended in 6 μl of spotting solution ). Random sampling of cDNAs revealed concentrations ranging between 200 to 300 ng/μl. Finally the samples in each well were spotted twenty times  with four slit pins arranged in a 2x 2y printhead . Prior to every spotting run the performance of all components of the robot is carefully tested – in particular the performance of the washing station in test runs designed to detect possible sample carry over. A modified Genetix Q-array , controlled by a novel in-house software was used for the arraying of the samples on SuperAmine™ aminosilane-coated microscope slides . Post-processing of the arrays was performed using 1,2-dichloroethane and the acylating catalyst N-methylimidazole following protocols described previously .Human brain RNA was isolated from a medically terminated fetus at 10 weeks gestation. This was provided by the MRC-funded Human Embryonic Tissue Bank maintained at the Institute of Child Health and University College London, England. Bovine fetal brain was obtained from a 3–4 months old bovine fetus at a local abattoir near Mariensee, Germany. Immediately after slaughter of the pregnant female, the tissue was plunged into liquid nitrogen and transported to the laboratory. Brain tissues were homogenised using a Dounce homogeniser, total RNA isolated using TRIzol reagent (Invitrogen) and further purified using phenol/chloroform extractions and precipitation with ethanol after DNase 1 (Promega) treatment. All procedures were as described by the manufacturers. RNA Purity, integrity and concentrations were evaluated on the Agilent 2100 Bioanalyzer. High quality RNAs with A260/A280 ratio over 1.8 with intact ribosomal 28S and 18S RNA bands were utilised for subsequent labelling reactions.°C for 5 min., left at room temperature for 10 min and then cooled on ice for 2 min. The following reagents were then added: 4 μl first-strand buffer, 2 μl 0.1 M DTT, 0.5 μl dNTP mix , 1 μl of 1.0 mM Cy5- or Cy3-dUTP (Amersham Pharmacia) and 1 μl (200 U/μl) Superscript II (Invitrogen). The labelling reaction was carried out at 42°C for 1.5 hrs. The reaction was stopped with 4 μl of 0.5 M EDTA. The input RNA was hydrolysed by the addition of 2 μl of 2.5 M NaOH and incubated at 37°C for 15 mins. followed by neutralization with 10 μl HEPES free acid . Labelled cDNAs (four replicates per species which included dye-swaps) were purified from unincorporated Cy-dyes using Microcon YM-30 purification columns (Millipore). All labelled cDNAs were routinely analysed on a Fuji scanner (FL8-8000) and by ethidium bromide staining to ascertain dye incorporation and size-range of synthesized cDNAs. After concentrating cDNAs by evaporation in a SpeedVac, labelled targets were resuspended in 20 μl of hybridisation buffer . After thorough resuspension, the cDNA was denatured by heating at 95°C for 5 mins followed by 20 mins. at 42°C to enable annealing of the blocking reagents to repetitive sequences within the target cDNAs. The hybridisation mixture consisting of Cy3-labelled bovine and Cy5-labelled human RNA and vice versa was placed on the blocked array under a 24 × 40 mm coverslip . To maintain humidity inside the chamber, 20 μl of 3x SSC was added to the reservoir wells. The chamber was then tightly sealed and slides incubated at 42°C for 18 hrs in a waterbath. Slides were washed twice in 0.2x SSC / 0.1% SDS and then twice in 0.2x SSC. Washes were carried out at room temperature with 10 min durations per wash. Finally, the slides were dried by centrifugation at 1100 rpm for 10 min.Four independent labelling (including dye-swaps) reactions per species were carried out using 25 μg total RNA from both human and bovine per labelling reaction. Direct incorporation of Cy3 and Cy5 during reverse transcription was carried out in a 20 μl reaction volume using 1 μg of anchored oligo-dT primer. The RNA/primer mix was incubated at 70Fluorescence images were captured using an Affymetrix 428 scanner  with appropriate gains on the photomultiplier tube (PMT) to obtain the highest intensity without saturation. A 16 bit TIFF image was generated for each channel for subsequent image analyses. Image analysis was carried out by placing the centre of each spot manually (grid-finding step) using the software AIDA (Raytest-Germany) and then by quantifying in a pre-defined neighbourhood around this spot centre using a two-dimensional Gaussian distribution (quantification step).j the median signal, medj, was computed and a multiplicative factor was calculated according to medref/medj, where medref is the global derived from the average intensity values of the cDNAs across the full batch of experiments. The multiplicative factor was used to adjust the signal of the ith cDNA in the jth experiment, xij, by xij * medref/medj.Data analysis comprised two distinct parts. In the first part, data were normalised to eliminate extrinsic influencing factors and artefacts not attributable to the probe-target interaction. For each tissue (human and bovine brain) under analysis the whole batch of experimental replicates was normalized simultaneously. Normalisation should eliminate multiplicative technical bias between the different experiments and result in the same median signal level for each experiment. In a first step, the local background of each spot was subtracted from the spot's signal intensity. Then, for each experiment In the second part, a number of numerical characteristics were calculated in order to quantify the cross-species comparison. These characteristics were signal detection value, signal reproducibility and statistical significance of differential expression. Signal detection was judged by a number of empty positions that were spread across the array. For each experiment, the proportion of signals of empty positions lower than the actual spot signal was calculated. Then across all repetitions, the average proportion was kept as the signal detection value. Employing this procedure, a signal strength was quantified for each cDNA. Signal reproducibility was judged by calculating the co-efficient of variation (CV) for each cDNA across all experiments.In order to judge differential expression of a gene in human and bovine brain we calculated three statistical tests: Student's t-test, Welch-test and Wilcoxon's rank sum test for each cDNA based on the signal series derived from human and bovine experimental repetitions. The three tests make different assumptions on the signal series. Whereas the first two assume that the series are Gaussian distributed, the Wilcoxon test is distribution-free. Low P-values calculated by these tests indicate significant differences in signal intensity within the human and bovine samples .The list of primers, annealing temperatures and genes under investigation is shown in Table °C for 5 mins and cooled on ice. Thereafter, the following components were added sequentially, 4.0 μl of 5x RT. Buffer (Invitrogen), 2.0 μl of 0.1 M DTT (Invitrogen) 1.0 μl of (10 mM) dNTP (Amersham) and 1.0 μl (200 U/μl) Superscript II (Invitrogen). After pulse spinning, incubation was carried out at 42°C for 1.5 hrs.For primer annealing and reverse transcription, 1.0 μl (2.5μg/μl) of DNase1 treated human and bovine total RNA was added to 2.0 μl (50 μM) Oligo-dT primer plus 9.0 μl of RNAse free water. The mixture was spun briefly and heated to 702, 2.0 μl of the first strand cDNA  and distilled water to 50 μl. All reagents were purchased from Invitrogen. Cycling parameters consisted of an initial denaturation step at 95°C for 5 min followed by 30 cycles of denaturation at 95°C for 30 sec, annealing for 30 sec, elongation at 72°C for 30 sec, then a final elongation step at 72°C for 5 min. Primer sequences, annealing temperatures and predicted size of cDNA products are shown in Table ACTIN was used as endogenous control.Gene-specific PCR amplifications were carried out in a total volume of 50 μl consisting of 5 μl of 10x PCR buffer, 0.2 μl (5 U/μl) Taq Polymerase, 1.0 μl (10 mM) dNTP, 2.5 μl of each (20 μM) primer, 1.5 μl (50 mM) MgClJ.A. conceived the study's idea, designed and optimised the protocols, carried out the hybridisations and was pivotal in developing the analysis plan and writing the manuscript.R.H. developed the analysis plan, analysed the data and was pivotal in writing the manuscript.D.H and M.N designed the cross-species primers and carried out the semi-quantitative RT-PCR.W.W. performed the initial image analysis.A.BK. selected the clones used as probes for creating the Chip and carried out the assignment of orthologous genes.T.B. assisted in the semi-quantitative RT-PCR analysisJ.W.C. was involved in the conceptualisation and writingC.H. was instrumental in the Chip design and production.H.N. is the Head of the Department of Biotechnology at the Institute for Animal Science and was involved in the conceptualisation and writing.H.L. is the Head of the Department of Vertebrate Genomics at the Max Planck Institute for Molecular Genetics.Summary of the expression data and the corresponding gene annotations. The terms used under each column are explained viz;IMAGE ID: Gene specific identifier assigned by the IMAGE consortiumGene: HUGO gene nameChromosome: Chromosomal location of geneDescription: Gene descriptionMolecular Function: Assigned function by the Gene Ontology ConsortiumBG-tag-bovine: Signal detection probability in the bovine samplesBG-tag-human: Signal detection probability in the human samplesCV-bovine: Co-efficient of variation between replicate signal intensitiesCV-human: Co-efficient of variation between replicate signal intensitiesMean-bovine: Mean signal across all hybridisation experiments using bovine RNAlog-Mean-bovine: Logarithm (base 2) of the mean valueMean-human: Mean signal across all hybridisation experiments using human RNAlog-Mean-human: Logarithm (base 2) of the mean valueRatio(bovine vs human): Ratio of the mean valueslog2(Ratio): Log-ratio (base 2) of the mean valuesP-value-Student's-t-test: P-value of Student's t-test using the replicate experimentsP-value-Welch-test: P-value of the Welch test using the replicate experimentsP-value-Wilcoxon-test: P-value of Wilcoxon's test using the replicate experimentsMatch: A BLAST hit with E-value < 1.0e-15 was found (1) or not (0)Homology: Classification of sequence homology between human gene sequence and bovine ESTs. "ortholog" = best match in both directions, "paralog" = bovine EST has its best match with another human sequence.TIGR-BTGI0-51503- Matches: Best results of the BLAST matches using the Ensembl- annotated gene sequence with the TIGR- databaseIdentity: %-identity of best matchOverlap: Overlap in base pairsClick here for file


The epidemiology of travel-associated campylobacteriosis is still largely unclear, and various known risk factors could only explain limited proportions of the recorded cases.Using data from 28,704 notifications of travel-associated campylobacteriosis in Sweden 1997 to 2003 and travel patterns of 16,255 Swedish residents with overnight travel abroad in the same years, we analysed risks for travel-associated campylobacteriosis in 19 regions of the world, and looked into the seasonality of the disease in each of these regions.The highest risk was seen in returning travellers from the Indian subcontinent , and the lowest in travellers from the other Nordic countries . In Africa, large differences in risk between regions were noted, with 502 /100,000 in travellers from East Africa, compared to 76/100,00 from West Africa and 50/100,000 from Central Africa. A distinct seasonal pattern was seen in all temperate regions with peaks in the summer, while no or less distinct seasonality was seen in tropical regions. In travellers to the tropics, the highest risk was seen in children below the age of six.Data on infections in returning travellers together with good denominator data could provide comparable data on travel risks in various regions of the world. Campylobacter infection is a zoonotic disease, observed in most parts of the world. The disease is caused by Campylobacter jejuni, or less commonly Campylobacter coli. It is estimated to cause 5–14% of diarrhoea, worldwide [Campylobacter infection has emerged to be most important bacterial cause of gastrointestinal infection. Animals  are the major reservoir for the bacteria. Campylobacter does not easily grow in food, but the critical infective dose is low [orldwide . Also ine is low .Unlike salmonellosis with well-known routes of transmission, the epidemiology of campylobacteriosis is still largely unclear, and various known risk factors could only explain limited proportions of the recorded cases . Known rCampylobacter infection is a leading cause together with infections due to enterotoxigenic Escherichia coli (ETEC), salmonellosis, and shigellosis [Approximately 80 million persons from industrialized countries travel every year to places in Africa, Asia, Pacific Islands, Latin America and remote areas of Eastern Europe, and between, 25 and 50 % of travellers to these areas experience travellers' diarrhoea -12. Abougellosis ,13.In this study we have used returning travellers to Sweden as sentinels to estimate the comparative risks for travel-associated campylobacteriosis in 19 regions of the world, and looked into the seasonality of the disease in each of these regions.Campylobacteriosis is a notifiable disease according to the Swedish communicable disease act. Cases are notified in parallel to the Swedish Institute for Infectious Disease Control (SMI) by the clinician having seen the patient  and the laboratory having diagnosed the pathogen (laboratory notification). At the SMI the notifications from the two sources are merged into case records, using a unique personal identification number issued to all Swedes, and used in all health care contacts. The clinical notifications contain epidemiological information of relevance, including country of infection. For this study we retrieved notification information  from the national surveillance database  on all cData on travel patterns were obtained from a commercial database, the Swedish Travel and Tourist Database (TDB) . This daCampylobacter infection (cases) as numerator and extrapolated total numbers of travellers from the TDB as denominator. The actual numbers of interviewed persons (controls) were used for the calculations of 95% confidence intervals (95% CI) for the estimates, using the formula:The age, sex and geographical distribution of the interviewees in the TDB, were standardised against the total population of Sweden to give an extrapolation of the actual number of travellers to each country during the seven years. We then estimated risks per 100,000 travellers  using notifications on In risk ± 1.96*√ (1/cases+1/controls)eTo adjust for possible confounding and test for interaction, we also calculated odds ratios (OR) with corresponding 95% CI for the same exposures with a logistic regression model.In an initial crude analysis, odds ratios (ORs) for all exposures  on the outcome campylobacteriosis were analysed, with the lowest incidence in each category used as reference. Confounding was then assessed using Mantel-Haenszel stratification. ORs for exposures with significant association with the outcome were included in a logistic regression analysis if they were shown to contribute significantly to the model in a Wald test. The presence of significant interaction was tested with tests for homogeneity. For each region we analysed seasonality separately . All analyses were done using the Stata 6.0 software .Notification data is regulated by the Swedish Communicable disease act, and contain full personal identification. The TDB contains aggregated data only. The Medical Ethics Committee of the Karolinska Institute, Stockholm, Sweden, approved the study.Of 53,223 persons notified with campylobacteriosis in the period 1997 to 2003, 28,704 (54%) were travel-associated, corresponding to 42.3 cases per 100,000 travellers , East Africa , East Asia , North Africa  and Arab countries/Iran . Adjusting for age, sex, and month in the logistic regression model did not change the rank between the regions  versus 40.8 cases per 100,000 95 % CI 39.7–41.9). After adjusting for destination, age, and month in the multivariate logistic regression model, the risks were reversed with a significantly higher OR in males . However, travel destination was an effect modifier on the association between sex and campylobacteriosis, and this higher risk for males were only significant for travellers returning from a European country   and in small children 0–6 years old . Also the association between age and campylobacteriosis was modified by travel destination, and in travellers from tropical destinations, especially from Africa and Asia/Oceania the highest risk was seen in the youngest children , Western Europe , Eastern Europe , North America , Southern Europe , Northern Africa , Arab countries and Iran , and Australia/New Zealand . In the Eastern Mediterranean the peak risk was seen in the spring , and in Russia and former USSR in late fall . In the tropical regions the seasonality was considerably less distinct. In East Asia the risk peak was in December with nadir in May  and in the Caribbean in February with nadir in September . In the Indian Subcontinent, Sub-Saharan Africa, and Central/South America no distinct, significant seasonal peaks could be identified.In this study we report the risks for travel-associated campylobacteriosis and seasonality of the risks in various parts of the world, based on more than 28,000 notified cases. The large number of cases gives more precise risk estimates for this disease than in previous studies, although the estimates are given for quite large regions in parts of the world with few Swedish travellers. The denominator data from the TDB has previously been used in studies on dengue fever  and rickCampylobacter infections. One should therefore be cautious in drawing conclusions from the magnitude of the figures, and rather focus on the relative risks between the various regions, as estimated by the odds ratios. Since the data are all from the same source, the risk figures from the various regions are directly comparable. However, there may be a tendency of investigating travellers from the tropics more vigorous than travellers from e.g. the other Nordic countries, thus underestimating the risks in nearby countries. However, such selection bias could likely not explain the huge differences between say West Africa and East Africa.Notification data only reflects a small (but unknown) proportion of all travel-related Since we have no comparable data on the length of stay among the cases in our study, we were not able to include length of stay in our logistic regression model. However, the TDB clearly shows a longer median stay among travellers in far-away destinations. For instance the median stay in Spain was 6 nights, while in Thailand it was 14 nights. On the other hand, only cases detected after the return to Sweden are included in the analysis. The disease data therefore mainly reflect infections contracted during the last week of stay at the travel destination. Differences in length of travel are therefore to some extent evened out.It has previously been suggested that the risk of travellers' diarrhoea is higher during the first two weeks in highly endemic areas ,18. The Campylobacter jejuni infection was 10 per 100,000 travellers returning from Spain and Portugal, and 50, 60, and 80 per 100,000 returning travellers from China, Thailand and India, respectively [The differences in risk between various regions were considerable, not only between industrialized and developing countries, but also between different developing countries. The Indian Subcontinent, East Africa, East Asia, and North Africa stood out as special high-risk areas. In a recent Finnish study, the risk of travel associated ectively . The lowectively ,21, but Age, sex and season (month of travel/infection) were identified as possible confounders for the association between travel destination and risk for campylobacteriosis and were thus included in the logistic regression model. All three variables contributed significantly to the model, but the overall effect of these confounders did not alter the rank order between the regions in the logistic regression model compared to the crude analysis.The highest risks were seen in young adults and small children, and especially in the tropics the highest risks were seen in the youngest. This is consistent with previous findings that in developed countries the disease most of hits children below the age of 5 years and young adults, while in developing countries it is most often seen in children below the age of 2 years, with an annual incidence of 40–60 % . The datCampylobacter infection. This pattern is also seen in domestically acquired Swedish campylobacteriosis cases, where 56% of the notified cases in 2003 were males, and in the US where campylobacteriosis is more common in males of all age groups [In travellers returning from Europe, male gender was an independent risk factor for e groups ,9. The he groups .In each of the 19 regions in the study, we looked closely at the seasonality of the disease. As has previously been shown ,6-9, theCampylobacter infection was seen between November and April [In the tropical regions, seasonal peaks of campylobacteriosis have not previously been recognized . Also innd April . With onData on infections in returning travellers together with good denominator data could provide comparable data on travel risks in various regions of the world. This study has revealed large and unexplained regional incidence differences, e.g. between East and Central Africa. The very distinct seasonal pattern seen in all temperate regions could not be discerned in the tropics.The author(s) declare that they have no competing interests.KE raised the original idea of the study, did the statistical analyses, and prepared the first draft of the manuscript. YA contributed with in depth knowledge of campylobacteriosis and revised the draft manuscript. Both authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Slc26a4+/+) or lack a complete Slc26a4 gene (Slc26a4-/-).Pendred syndrome, a common autosomal-recessive disorder characterized by congenital deafness and goiter, is caused by mutations of SLC26A4, which codes for pendrin. We investigated the relationship between pendrin and deafness using mice that have (+ concentration were measured with double-barreled microelectrodes. Currents generated by the stria marginal cells were recorded with a vibrating probe. Tissue masses were evaluated by morphometric distance measurements and pigmentation was quantified by densitometry.Expression of pendrin and other proteins was determined by confocal immunocytochemistry. Expression of mRNA was determined by quantitative RT-PCR. The endocochlear potential and the endolymphatic KSlc26a4-/- mice was increased and tissue masses in areas normally occupied by type I and II fibrocytes were reduced. Slc26a4-/- mice lacked the endocochlear potential, which is generated across the basal cell barrier by the K+ channel KCNJ10 localized in intermediate cells. Stria vascularis was hyperpigmented, suggesting unalleviated free radical damage. The basal cell barrier appeared intact; intermediate cells and KCNJ10 mRNA were present but KCNJ10 protein was absent. Endolymphatic K+ concentrations were normal and membrane proteins necessary for K+ secretion were present, including the K+ channel KCNQ1 and KCNE1, Na+/2Cl-/K+ cotransporter SLC12A2 and the gap junction GJB2.Pendrin was found in the cochlea in apical membranes of spiral prominence cells and spindle-shaped cells of stria vascularis, in outer sulcus and root cells. Endolymph volume in These observations demonstrate that pendrin dysfunction leads to a loss of KCNJ10 protein expression and a loss of the endocochlear potential, which may be the direct cause of deafness in Pendred syndrome. PDS gene SLC26A4, which codes for the protein pendrin . Slides were washed in PBS-TX and incubated for 1 h at 25°C with appropriate secondary antibodies at a 1:1000 dilution in PBS-TX with 1–3% BSA . Actin filaments were visualized by staining with Alexa 488 conjugated phalloidin (Molecular Probes). After incubation, slides were washed with PBS-TX, mounted with FluorSave , and viewed by confocal microscopy . Laser scanning brightfield images were collected to document structural preservation, for morphometric analysis and for analysis of pigmentation.Animals were deeply anesthetized with sodium pentobarbital (100 mg/kg i.p.) and transcardially perfused with a Cl2. Temporal bones were removed and two preparations of inner ear tissues were obtained by microdissection: (1) stria vascularis without spiral ligament, and (2) spiral ganglia including the surrounding bone and the organ of Corti. The dissection medium was changed twice during the microdissection and isolated tissues were washed three times to minimize cross-contamination. The Slc26a4-/-genotype was verified by the observation of large otoconia in the utricular macula  were measured with double-barreled microelectrodes  + A × [Na+]), where Vi is an offset term, S is slope, A is selectivity, and [Na+] is Na+ concentration. Calibrations were made immediately after withdrawal of the electrodes from the cochlea. Plasma K+ concentrations were obtained using a blood analyzer .Animals were anesthetized with inactin . The endocochlear potential and the endolymphatic [Kectrodes . MeasureData are presented as mean ± sem; n denotes the number of experiments. Differences were considered significant when p < 0.05.In situ hybridization in the cochlea has suggested that pendrin mRNA is expressed in cells that reside immediately beneath the spiral prominence on the lateral wall of the external sulcus [Slc26a4+/+) mice using an established antibody [data not shown). Strong expression of pendrin was observed not only in outer sulcus epithelial cells, as predicted from in situ hybridization data, but also in root cells, in apical membranes of spiral prominence surface epithelial cells and in apical membranes of spindle-shaped cells that are part of stria vascularis  support a role for pendrin in HCO3 - secretion into endolymph.l sulcus . To deteantibody . Staininris Fig. . The pree kidney . HCO3 - te cells ,19. CO2 + channel that is expressed in strial marginal cells, and ZO-1, a tight junction protein that labels basal cells and thereby delineates the boundaries of stria vascularis. Dual labeling experiments demonstrated that pendrin is expressed in spindle-shaped cells, which are surface epithelial cells in stria vascularis adjacent to strial marginal cells near the borders of both the spiral prominence and Reissner's membrane . These observations suggest that a primary event leading to deafness in Slc26a4-/- mice, and potentially in patients suffering from Pendred syndrome, is the loss of the endocochlear potential. Degeneration of hair cells is probably a response to the loss of the endocochlear potential.The onset of pendrin expression during development of the mouse inner ear has been determined to be embryonic day (ED) 13 . Morpholdulthood . Interesdulthood . These osurvival , is estasurvival  and may  hearing . We hypo-/- Fig. . No signtic Fig.  or plasm+ channel KCNJ10 located in intermediate cells [+ from the intrastrial space into endolymph and keep the K+ concentration low adjacent to the KCNJ10 K+ channel [Slc26a4-/- mice, we first determined whether intermediate cells are present in stria vascularis, since a loss of intermediate cells is known to lead to a loss of the endocochlear potential [Slc26a4-/- mice  Slc26a4+/+ and both young and old (~12 month) Slc26a4-/- mice, assessed amounts of isolated tissues by quantification of 18S rRNA, and quantified the expression of KCNJ10 mRNA. Quantities of stria vascularis isolated from these different mice were similar, since no significant differences were found in the numbers of 18S rRNA molecules (log(rRNA) = 9.46 ± 0.08, n = 17, pooled data). In contrast, quantities of spiral ganglia isolated from young and old Slc26a4-/- mice (log(rRNA) = 9.04 ± 0.18, n = 4 and 9.29 ± 0.15, n = 6) were significantly smaller than in Slc26a4+/+ mice (log(rRNA) = 9.48 ± 0.19, n = 7), consistent with morphometric data (see below). Expression of KCNJ10 mRNA was normal in stria vascularis and spiral ganglia of young Slc26a4-/- mice but significantly reduced in old Slc26a4-/- mice . The absence of a change in tissue height is consistent with the presence of intermediate cells [Slc26a4-/- mice was less prominent, spiral ligament thinner and spiral limbus flatter . In addition, we observed an apparent degeneration of stria vascularis, including an increase in pigmentation and an irregular pattern of the tight junctions of marginal cells . KCNQ1 and KCNE1, subunits of the secretory K+ channel, were co-localized in the apical membrane of strial marginal cells; the Na+/2Cl-/K+ cotransporter SLC12A2, which is located in the basolateral membrane of strial marginal cells, was found in stria vascularis; and the gap junction protein GJB2 was found in spiral ligament of Slc26a4+/+ and Slc26a4-/- mice (Fig. Slc26a4-/- mice is consistent with the finding of KCNQ1 mRNA expression (Fig. + secretion by strial marginal cells or a compromise of gap junction mediated K+ cycling.The observation that endolymphatic and perilymphatic Kndolymph ,31. In oecretion ,32,33, a cycling . K+ secrice Fig. . Co-locaice Fig. . Express+ concentrations in spite of an enlargement of this fluid compartment. We found that Slc26a4-/- mice lack the endocochlear potential because they do not express KCNJ10 protein. Intermediate cells protect stria vascularis by converting CO2 to HCO3 - and by detoxifying free radicals (Fig. 2 and free radicals are generated by the large numbers of mitochondria in the metabolically highly active strial marginal cells. Intermediate cells employ carbonic anhydrase to convert CO2 to HCO3 - [3 - into endolymph, results in an accumulation of HCO3 - and an alkalinization of the intrastrial spaces. This extracellular alkalinization may enhance free radical stress, since it may inhibit the uptake of cysteine and thereby limit production of the protective glutathione [+ channels has been shown to be controlled by the cytosolic pH and free radicals, which encode the metabolic state of the cell [+ channel in strial intermediate cells, which is essential for the generation of the endocochlear potential, is probably the direct cause of deafness in Slc26a4-/- mice and patients suffering from Pendred syndrome.We described locations of pendrin expression in the cochlea and vestibular labyrinth and detected normal endolymphatic Kals Fig. . CO2 ando HCO3 - ,19 and co HCO3 - -37. It itathione . Supportthe cell . SuppresNone declared.+ concentration. JHL carried out current measurements on strial marginal cells. SMW, LAE, IER and EDG provided the mice and the pendrin-specific antibody. PW and DCM conceived the study. PW wrote the manuscript. All authors read and approved the final manuscript.PW designed and coordinated the immunocytochemical, morphometrical and molecular biological experiments. EMI and BA carried out confocal immunocytochemistry on cryosections. SJ and SVJ carried out confocal microscopy on whole-mounts of stria vascularis. SVJ and RJM carried out quantitative RT-PCR. DCM designed and coordinated electrophysiological experiments. TW carried out measurements of the endocochlear potential and the endolymphatic KThe pre-publication history for this paper can be accessed here: