ASPM, which encodes the human homologue of a fly protein essential for spindle function, are the most common known cause of MCPH. Here we have isolated large genomic clones containing the complete ASPM gene, including promoter regions and introns, from chimpanzee, gorilla, orangutan, and rhesus macaque by transformation-associated recombination cloning in yeast. We have sequenced these clones and show that whereas much of the sequence of ASPM is substantially conserved among primates, specific segments are subject to high Ka/Ks ratios (nonsynonymous/synonymous DNA changes) consistent with strong positive selection for evolutionary change. The ASPM gene sequence shows accelerated evolution in the African hominoid clade, and this precedes hominid brain expansion by several million years. Gorilla and human lineages show particularly accelerated evolution in the IQ domain of ASPM. Moreover, ASPM regions under positive selection in primates are also the most highly diverged regions between primates and nonprimate mammals. We report the first direct application of TAR cloning technology to the study of human evolution. Our data suggest that evolutionary selection of specific segments of the ASPM sequence strongly relates to differences in cerebral cortical size.Primary microcephaly (MCPH) is a neurodevelopmental disorder characterized by global reduction in cerebral cortical volume. The microcephalic brain has a volume comparable to that of early hominids, raising the possibility that some MCPH genes may have been evolutionary targets in the expansion of the cerebral cortex in mammals and especially primates. Mutations in  ASPM gene is associated with abnormally small brain size. Comparison of the ASPM gene from several primate species suggests it as a target of evolutionary selection for increased brain sizeMutation of the  ASPM gene  is an autosomal recessive neurodevelopmental disorder in which the brain fails to achieve normal growth. The affected individuals have severe reduction in brain size; however, the gyral pattern is relatively well preserved, with no major abnormality in cortical architecture . MoreoveSPM gene .ASPM gene encodes a 10,434-bp-long coding sequence (CDS) with 28 exons, and spans 65 kb of genomic DNA at 1q31. ASPM contains four distinguishable regions: a putative N-terminal microtubule-binding domain, a calponin-homology domain, an IQ repeat domain containing multiple IQ repeats , and a C-terminal region , is localized in the mitotic centrosome and is known to be essential for both the organization of the microtubules at the spindle poles and the formation of the central mitotic spindle during mitosis and meiosis. Mutations in asp cause dividing neuroblasts to arrest in metaphase, resulting in reduced central nervous system development ((Mus musculus) brain, the Aspm gene is expressed specifically in the sites of active neurogenesis. Expression in the embryonic brain was found to be greatest in the ventricular zone, which is the site of cerebral cortical neurogenesis , gorilla (Gorilla gorilla), orangutan (Pongo pygmaeus), and rhesus macaque (Macaca mulatta) were isolated by transformation-associated recombination (TAR) cloning in yeast (Saccharomyces cerevisiae), the technique allowing direct isolation of a desirable chromosomal region or gene from a complex genome without constructing its genomic library  microsatellite expansions, three Alu insertions, including retroposition of AluYi9 in the orangutan–gorilla–chimpanzee–human clade, and retroposition of a new macaque-specific AluY subfamily similar to human AluYd2. Analysis of eight different macaque individuals showed that this particular insertion is polymorphic in the macaque population (data not shown), and thus the insertion appears to be very recent. One macaque-specific 245-bp-long insertion is linked to expansion of a 49-bp-long, minisatellitelike array. The remaining macaque-specific insertion (50 bp) is nonrepetitive. A closer analysis suggests that the insert is not a processed pseudogene of known genes (data not shown).We have compared complete gene sequences from primate species with a 65-kb, full-size human rst exon . ASPM seervation H, and paervation . Multiplervation B. Seven Alu–Alu recombination . Except for orangutan and rhesus macaque, two or more ASPM CDSs were used for analysis. ASPM proteins showed the same overall length and domain structure  are more variable. Indeed, nonsynonymous substitutions in hominoid primates (From the DNA and protein conservation profiles I, ASPM sprimates C and in primates D and nonprimates E are neaprimates H and supprimates . It is nprimates .p < 0.05). Also the model that the complete gorilla–chimpanzee–human clade is evolving at one ω rate different from that in the rest of the tree is well supported (p < 0.01). Because ASPM consists of regions with different degrees of sequence conservation  revealed an elevated value in the human branch A. Accordtion see , we sepation see B the contion see C. The lition see D. As seetion see D. It is tion see . TherefoASPM polymorphism in those species (see ASPM mutant haplotypes are not representative of wild-type variation so were not used in these comparisons. For African green monkey, five synonymous and five nonsynonymous changes were found between two sequences. The gorilla and chimpanzee CDSs in particular showed an apparently high degree of replacement polymorphism. Gorilla polymorphism included 35 point mutations (15 silent mutations and 21 replacements). Chimpanzee sequences differed in five synonymous and 11 nonsynonymous sites. In order to interpret this seemingly high level of observed polymorphism, intraspecific diversity was compared to interspecific diversity using the McDonald and Kreitman test (p < 0.001) to conclude that the pattern of gorilla polymorphism is therefore different from the divergence pattern. Indeed gorilla polymorphism is less than variation resulting from divergence: within species, the ω ratio is 1.43 for gorillas compared to 2.2 for the divergence between the gorilla common ancestor and the human–chimpanzee common ancestor. Intraspecific variation, although seemingly unusual in showing so many replacement substitutions in both chimpanzee and gorilla, is less than or in line with what we have observed for ASPM divergence between species. Therefore, relaxation of selection cannot explain the high nonsynonymous/synonymous substitution ratios among African hominoids, further supporting the idea that adaptation has occurred in ASPM.Sequencing of two CDSs in African green monkey, three in gorilla, and three in chimpanzee allowed us to look for cies see E. Human man test . In the ASPM gene, which is involved in determining the size of the human brain and in which mutations lead to MCPH. The ASPM homologue in the fruit fly is essential for spindle function, suggesting a role for this gene in normal mitotic divisions of embryonic neuroblasts. Complete gene homologues from five primate species were isolated and sequenced. In agreement with the predicted critical role of ASPM in brain development, both coding and noncoding regions of ASPM homologues showed a marked degree of conservation in humans, other hominoids, and Old World monkeys. The differences found in noncoding regions were small insertions/deletions and lineage-specific insertions of evolutionarily young Alu elements into introns.In this study, we applied TAR cloning technology to investigate molecular evolution of the ASPM begins well before brain expansion suggests that the molecular evolution of ASPM in hominoids may indeed be an example of a molecular “exaptation”  selection in human and African ape lineages. Such “mosaic” selection has been previously described for other proteins . When ouptation” , in thatASPM, rapidly evolving residues are mainly concentrated in the IQ repeat domain containing multiple IQ motifs, which are calmodulin-binding consensus sequences. While there is no direct evidence yet, it is likely that the function of human ASPM is modulated through calmodulin or calmodulinlike protein(s). Previous interspecies comparisons of ASPM proteins have shown a consistent correlation of greater protein size with larger brain size mainly because of the number of IQ repeats (asp homologue of the nematode Caenorhabditis elegans contains two IQ repeats, the fruit fly—24 IQ repeats, and the mouse—61 IQ repeats, and there are 74 IQ repeats in humans (ASPM homologues in the nonhuman primates examined here contain the same number of IQ repeats as human, supporting the idea that repeat expansion occurred prior to the anthropoid divergence (which gave rise to New World monkeys, Old World monkeys, and hominoids) and possibly even earlier in primate evolution. IQ motifs are seen in a wide variety of proteins, but the ASPM proteins in primates are unique, because they have the largest known number of IQ repeats. Given the proposed role of ASPM in regulating divisions of neuronal progenitors, both the number of repeats and the particular amino acid substitutions in the IQ repeats may be strongly related to brain evolution.In the case of  repeats . For exan humans . ASPM hoASPM gene mutations which lead to MCPH provide a direct link between genotype and phenotype. ASPM is yet another example on the growing list of positively selected genes that show both accelerated evolution along the human lineage and involvement in simple Mendelian disorders  is barely different from that in gorillas  and common chimpanzees   , yet accears ago . TherefoHow might evolutionary changes in the ASPM protein affect cerebral cortical size? One potential mechanism might be that changes in ASPM induce changes in the orientation of the mitotic spindle of neuroblasts. Normally, neural precursor cells can have mitotic spindles oriented parallel to the ventricle or perpendicular to the ventricle. Mitoses in which daughter cells are oriented next to one another at the ventricular zone are typically “symmetric” in that a single progenitor cell generates two progenitor cells, causing exponential expansion of the progenitor pool. In contrast, mitoses that generate daughter cells that are vertically arranged are typically “asymmetric” so that one daughter cell becomes a postmitotic neuron, whereas the other daughter cell remains as a progenitor, causing only a linear increase in cell number. Control of this proliferative symmetry can cause dramatic alterations in cerebral cortical size , and so ASPM gene evolution in hominoids. Here, we have demonstrated the utility of TAR cloning for evolutionary sequence comparisons among humans and other primates. In addition, the ASPM TAR clones isolated in these studies could provide valuable reagents for studying ASPM gene regulation in its natural sequence context. Overall, we anticipate this technology will be extremely useful in studying the evolution of other genes that may be responsible for uniquely human traits.Functional genomics studies are clearly needed to elucidate the exact nature of the molecular mechanisms affected by Human Molecular Genetics shortly after this paper was submitted.The related paper by ASPM gene from the human (Homo sapiens), chimpanzee (Pan troglodytes), gorilla (Gorilla gorilla), orangutan (Pongo pygmaeus), and rhesus macaque (Macaca mulatta) genomes, a TAR vector containing two unique hooks was constructed. Two targeting sequences were designed, 131 bp 5′ and 151 bp 3′, from the available human genomic sequence of ASPM (positions 155,758–155,888 and 92,922–93,071 in the BAC RP11–32D17 [GI:16972838]). The targeting sequences were PCR amplified from the genomic DNA using two specific primers  in agarose plugs. Spheroplast transformation experiments were carried out as previously described in ASPM, yeast transformants were examined by PCR using diagnostic primers specific for exon 2 and exon 27 of ASPM  by homologous recombination in yeast using a BAC/NeoR retrofitting vector, BRV1, and then transformed into a recA DH10B E. coli strain (NotI, HindIII, EcoRI, and PstI digestions. The promoter regions of the chimpanzee, gorilla, orangutan, and rhesus macaque (approximately 3.2 kb) and exon 18 of the red-chested mustached tamarin (Saguinus labiatus) and black-handed spider monkey (Ateles geoffroyi) (approximately 4.7 kb) were PCR amplified using a pair of specific primers  using TRIzol reagent . Reverse transcription and 5′- and 3′-RACE reactions were performed using SMART RACE cDNA Amplification Kit . RNAs were extracted from primate cell lines (African green monkey ASPM genes were directly sequenced from BAC DNAs (ASPM homologues were run on a PE-Applied Biosystem 3100 Automated Capillary DNA Sequencer (Applied Biosystems, Foster City, United States). Primer pairs for cDNA sequencing were designed based on the human ASPM mRNA sequence. Primer sequences are available upon request. All sequences were named and numbered according to the clone/accession identifier.Chimpanzee, gorilla, orangutan, and rhesus macaque TAR clones containing full-size BAC DNAs . Forwardhttp://baboon.math.berkeley.edu/mavid/)  (http://pbil.univ-lyon1.fr/software/seaview.html) (http://www.hgmp.mrc.ac.uk/Software/EMBOSS/) for sequence analysis. Short nucleotide patterns associated with genome rearrangements were searched using FUZZNUC (EMBOSS). We searched for the following recombinogenic motifs: chi-like octamer (GCWGGWGG), immunoglobulin heptamer (GATAGTG), translin (ATGCAGN(0,4)GCCCWSW and GCNCWSCTN(0,2)GCCCWSSW), topoisomerase II (RNYNNCNNGYNGKNYNY), topoisomerase IId (GTNWAYATTNATNNR), topoisomerase IIi (YYCNTASYGGKYYTNNC), and V(D)J recombinase (CACAGTGN(12/23)ACAAAAACC). For short or highly ambiguous patterns (topo-isomerase II), no mismatches were allowed; for longer motifs (translin, V(D)J recombinase) up to two mismatches were permitted. Prediction of CpG islands was performed by CPGPLOT (EMBOSS) with default parameters . CENSOR (http://www.girinst.org/Censor_Server-Data_Entry_Forms.html)  were used for identification of repetitive elements. Minisatellites were detected by TANDEM REPEAT FINDER   ASPM sequence. Primate CDSs were deduced from the ASPM gene alignment with human sequences. Synonymous and nonsynonymous substitutions were detected by SNAP (http://www.hiv.lanl.gov/content/hiv-db/SNAP/WEBSNAP/SNAP.html). Codon maximum likelihood (ML) in CODEML in PAML v. 3.13 (http://abacus.gene.ucl.ac.uk/software/paml.html) (http://pbil.univ-lyon1.fr/software/phylowin.html) (http://paup.csit.fsu.edu/index.html) and corrected for multiple substitutions using the Tamura-Nei algorithm.Genomic sequences were aligned using MAVID (/mavid/) ; proteinialign/) . Alignmeew.html) . We havems.html)  and REPET FINDER . ASPM se/hgBlat) . First, ml.html)  has beenml.html) . For larin.html) . SegmentCommentaryASPM is an AT-rich gene (GC content 36.4%–37%) and, as expected . On the other hand, the 3′ breakpoint sequences are diverged from the chi consensus (second box). Both Alu elements in the alignment are shown from the first position and end at the same position, and thus positions in one element correspond to positions in the other Alu copy. As can be seen, the breakpoint position in the first AluSp repeat exactly corresponds to the breakpoint position within the second AluSz element, suggesting homologous recombination between the two repeats.Both orangutan breakpoints are located within 5′ portions of two (163 KB PDF).Click here for additional data file.Figure S2ASPM segment (percent same), and a schematic alignment of segmental duplications. The ASPM segment (black) shares similarity with 24 segmental duplications that contain additional sequences and are present on several human chromosomes. The ASPM copy and three duplications on Chromosome 7 share the same L1P4 terminal insertion, which is absent from all other duplications. The tree on the left shows evolutionary relationships among the duplications estimated by the neighbor-joining method.From left to right: phylogeny, chromosomal position, band name, identity to (169 KB PDF).Click here for additional data file.Figure S3The amino acid identity in the conserved regions is 85.44%, 49.39%, and 68.74% for exon 3, exon 4, and the IQ domain, respectively. In addition, while the alignment of conserved regions is completely gap-free, the variable domains exhibit several gaps including a large deletion in the mouse IQ domain (human positions 2655–2943).(97 KB PDF).Click here for additional data file.Table S1ASPM and lower case letters indicate cloning sites.Upper case letters indicate sequences homologous to (118 KB PDF).Click here for additional data file.Table S2p-value is in parentheses. The section over the diagonal is calculated using the Pearson (linear) correlation coefficient; under the diagonal are correlations obtained using the Spearman's rank coefficient—nonparametric). Nontrivial or interesting significant correlations are shown in bold and italics. The CAI represents the mean for all species . The ω ratio, Ka, and Ks (rows/columns 2, 3, and 4) correspond to all branches of the phylogenetic tree. They were obtained using a ML model with one fixed ω ratio for all branches.All correlations were obtained for the same 100-amino-acid-/300-nucleotide-long, nonoverlapping windows. The first value shows the correlation coefficient; Click here for additional data file.http://www.ncbi.nlm.nih.gov/Genbank/) accession number for the human ASPM mRNA sequence used in this study is NM_018136. The sequence data from chimpanzee, gorilla, orangutan, and rhesus macaque full-length ASPM have been submitted to GenBank under accession numbers AY497016, AY497015, AY497014, and AY497013. The sequence data from chimpanzee, gorilla, and African green monkey ASPM cDNA have been submitted to GenBank under accession numbers AY508452, AY508451, and AY486114. The sequence data from spider monkey and tamarin exon 18 have been submitted to GenBank under accession numbers AY497017 and AY497018.The GenBank (


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.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.In this single photon emission computed tomography (SPECT) study with [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. Decreased serotonin transporter densities (SERT) have been recorded from the brains of depressed patients ,2, whichIn 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 .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.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 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) . The sevThe 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.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.123I] nor-β-CIT  was diluted in a volume of 10 ml physiological saline. The specific activity was higher than 1.8 × 1011 Bq/μmol [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 [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 ,11. The D - 1. The striatal uptake was pooled.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  . The speFunctional 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.The reproducibility of the SPECT scan had been previously studied with eleven healthy subjects . 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).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.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 . 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 .Three studies have been published on the association between SERT availability and the serotonin transporter genotype. Two of these were performed on healthy subjects ,23, whilWe 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.None declared.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.DAT: Dopamine transporterHDRS: Hamilton Depression Rating ScaleSD: Standard deviationSERT: Serotonin transporterSPECT: Single photon emission computed tomographyThe pre-publication history for this paper can be accessed here:


The role of inflammation in Alzheimer's disease (AD) has been controversial since its first consideration. As with most instances of neuroinflammation, the possibility must be considered that activation of glia and cytokine networks in AD arises merely as a reaction to neurodegeneration. Active, healthy neurons produce signals that suppress inflammatory events, and dying neurons activate phagocytic responses in microglia at the very least. But simultaneous with the arrival of a more complex view of microglia, evidence that inflammation plays a causal or exacerbating role in AD etiology has been boosted by genetic, physiological, and epidemiological studies. In the end, it may be that the semantics of "inflammation" and glial "activation" must be regarded as too simplistic for the advancement of our understanding in this regard. It is clear that elaboration of the entire repertoire of activated microglia – a phenomenon that may be termed "malactivation" – must be prevented for healthy brain structure and function. Nevertheless, recent studies have suggested that phagocytosis of Aβ by microglia plays an important role in clearance of amyloid plaques, a process boosted by immunization paradigms. To the extent that this clearance might produce clinical improvements (still an open question), this relationship thus obligates a more nuanced consideration of the factors that indicate and control the various activities of microglia and other components of neuroinflammation. Alzheimer's disease (AD) is a progressive degeneration of neural structure and function that arises in the cerebral cortex. Behaviorally, affected individuals usually present with semantic difficulty, followed by a deficiency in episodic memory, spatial disorientation, sleep disturbances, depression, agitation, loss of longer memories, general difficulty with the activities of daily living, and eventually, death. Neuropathological findings include a relatively high number of extracellular deposits of the amyloid β-peptide (Aβ), argyrophillic cytoskeletal aggregates in neurons, accumulation of α-synuclein, loss of synapses, loss of cholinergic and adrenergic fibers, loss of pyramidal neurons, and cerebral amyloid angiopathy (CAA) – deposition of Aβ around blood vessels.Most of the AD correlates above have been connected in some way to inflammation. For instance, the plaques – comprised primarily of aggregated amyloid β-peptide (Aβ) – are inundated with microglia that show profiles of morphology and gene expression consistent with inflammation. Indeed, if one characterizes any activity by microglia as a sign of "neuroinflammation," it can be said that inflammatory responses have been evident in AD for at least 40 years . But, itDespite these indications, there are reasons to believe that the changes observed in glia and inflammatory cytokines constitute a compensatory response in AD. Indeed, some investigators have been reluctant to apply the term "inflammation" to the constellation of events related to AD pathology. Some of the cytokines and other gene products expressed in peripheral sites of inflammation are present in the AD brain, but there is no apparent vasodilation or extravasation of neutrophils. In general, there seems to be less of the molecular and cellular behavior that is responsible for bystander tissue damage in peripheral inflammation. This journal was founded partially out of recognition that "neuroinflammation" is distinct. In essence, the concept reflects a compromise befitting the difficult line that must be maintained between effective cell-mediated immune responses and damage to the precious components of the CNS. Microglia elevate their expression of neurotrophic factors under many of the same conditions in which they show inflammation-related responses such as phagocytosis, retraction of processes, release of excitotoxins, and production of IL-1β and IL-6 and tumor necrosis factor ; in factRecent experiments with anti-Aβ immunization have highlighted another beneficial effect of "activated" microglia: removal of Aβ. It has long been recognized that microglia can efficiently phagocytose and at least partially degrade Aβ both in vitro and in vivo. But the persistence of amyloid plaques suggests that microglia are stymied in this regard during the development of AD or in the deposition of Aβ in mice transgenically engineered to produce large amounts of the peptide. Introduction of antibodies recognizing Aβ, either by active vaccination or by passive immunization , results in removal of some Aβ deposits and/or prevention of their formation. Although the phenomenon has been studied most rigorously in the transgenic mouse models, similar clearance of parenchymal plaques seems to have occurred in two human subjects that participated in an Aβ-vaccine trial ,15. And et al. [+ cells presumed to be microglia. The experimental paradigm was one of passive immunization of transgenic mice at nearly two years of age, old enough to have accumulated substantial Aβ deposits. Consistent with expectations, injection of anti-Aβ antibody diminished deposits in the parenchyma, even those that were mature enough to stain with Congo red. However, vascular deposition of Congo-red staining was elevated by approximately four-fold in the anti-Aβ-treated animals. Pfeiffer et al. found similar results in another transgenic line [et al. now show that the regional accumulation of vascular amyloid was accompanied by an elevated index of hemorrhages and a congregation of CD45+ cells, presumed to be microglia [+ cells observed by Wilcock and coworkers are microglia or another cell type. It is also unclear whether the accumulation of amyloid and inflammatory cells at the blood vessels represents an arrested state in Aβ clearance or simply a bottleneck in the transport, one that would eventually yield to complete removal of the peptide. However, the appearance of CAA in the human subjects that suffered from acute encephalitis suggests that the vascular accumulation is an untoward event, created or facilitated by inflammation. Another vascular irregularity caused by Aβ has been linked to inflammatory events in both transgenic mice and isolated human blood vessels [Wilcock et al.  have nowet al.  – and a nic line . Furthericroglia . Given ticroglia , one inticroglia . In thos vessels .malactivation" will be applied here to microglial activation which produces neurodegeneration. One obvious question is whether there might be a mode of "activation" that permits phagocytosis while limiting malactivation. In fact, stimulation of Fc receptors – the antibody receptors that initiate a good deal of antibody-triggered phagocytosis – can inhibit cytotoxicity in macrophages [The apparent contributions of inflammatory mechanisms to both Aβ clearance and vascular pathology illustrate a somewhat unique example of microglial ambivalence. While many had argued that microglial "activation" by Aβ was at least partially responsible for AD-associated degeneration, others had pointed to microglial phagocytosis as a desirable consequence of activation. For the purposes of discussion, the term "rophages . Similarrophages . Evidencrophages . Althougrophages . Interesrophages .While some have argued that CAA is of little consequence in AD , the elaAD: Alzheimer's diseaseAβ: amyloid β-peptideCAA: cerebral amyloid angiopathyIL-1, -6, -8: interleukin-1, -6, -8NSAID: nonsteroidal antiinflammatory drugTGFβ: transforming growth factor βThe author(s) declare that they have no competing interests.


Our recent study has shown that HIV-1 nef-derived miRNAs are produced in HIV-1 persistently infected cells. Furthermore, nef short hairpin RNA (shRNA) that corresponded to a predicted nef miRNA (~25 nt, miR-N367) can block HIV-1 Nef expression in vitro and the suppression by shRNA/miR-N367 would be related with low viremia in an LTNP (15-2-2). In the 15-2-2 model mice, the weight loss, which may be rendered by nef was also inhibited by shRNA/miR-N367 corresponding to suppression of nef expression in vivo.Here, we show the possibility that nef/U3 miRNAs produced in HIV-1-infected cells may suppress both Nef function and HIV-1 virulence through the RNAi pathway.These data suggest that  The human immunodeficiency virus (HIV), which infect humans cause acquired immunodeficiency syndrome (AIDS), which has reached pandemic levels in some societies, especially those in Southern Africa and Southeast Asia . Given tC. elegans germline [Drosophila germline, and the response against invading viruses in plants [et al., 2002), fragile X mental retardation [RNAi is a defense mechanism against aberrant transcripts that may be produced during viral infection and mobilization of transposons ,4. The Rgermline ,6, silenn plants . Post-trn plants . In contn plants . The matn plants ,11. At tardation ,14 and cardation  have beenef, is located at the 3' end of the viral genome and partially overlaps the 3'-long terminal repeat (LTR). The nef gene is uniquely conserved in HIV-1, HIV-2 and simian immunodeficiency virus (SIV) and is not essential but important for viral replication in vivo [nef gene is expressed during HIV infection and often accounts for up to 80% of HIV-1 specific RNA transcripts during the early stages of viral replication [nef dsRNA containing the 3'-LTR regions, obtained from long-term non-progressor (LTNP) AIDS patients, actually inhibited the transcription of HIV-1 [cis-expression of mutated F12-HIV-1 nef inhibits replication of highly productive NL43-HIV-1 strain, which is not related to down-regulation of CD4 [nef gene cloned from the provirus of naturally occurring HUT-78 T cells infected with the supernatant of the peripheral blood mononuclear cells (PBMCs) of an HIV-seropositive non-producer patient, induces a block of viral replication [nef RNAs may be a cis-regulatory factor for HIV-1 replication [One of the human immunodeficiency virus type 1 (HIV-1) coding accessory genes,  in vivo . The nefof HIV-1 . Furthern of CD4 ,19. It hlication .nef-derived miRNAs are produced in HIV-1-infected cells. The results presented here show that nef short hairpin RNAs (shRNAs) corresponding to the nef miRNAs efficiently block RNA stability or mRNA translation, perhaps an indication that HIV-1 regulates its own replication by using nef miRNAs.In the current study, we have established the link between miRNAs and HIV infections by demonstrating that nef miRNAs and HIV-1-infected cells. To achieve this purpose, we extracted total RNA from HIV-1 IIIB strain persistently infected MT-4 T cells and northern blot analysis was performed using eight probes against the nef coding region, as shown in Figure nef region. However HIV-1 major transcripts were not detected (data not shown), indicating that the structure of the small RNA molecules could be double-stranded RNAs (miRNAs). Some variability was observed when the quantity of the miRNAs was compared with the total of the major transcripts. A maximum of 3.2% of miRNAs was detected by using #367 probe when compared with total HIV-1 transcripts, and the minimum of 0.3% was detected by using probe# 90 -encoded miRNAs were identified. Thus, during the preliminary stages of this study, our curiosity was fixed on the need to find out if indeed there was any relationship between  90 Fig. .nef miRNA, ~25-nt RNAs were gel purified, cloned and sequenced. The sequences from the nef miRNA clones were 5'-acugaccuuuggauggugcuucaa-3' or similar ones, corresponded to the nucleotides approximately 420 to 443 conserved region of nef (miR-N367). The most notable feature of this analysis is that it has proven beyond reasonable doubts that nef-derived miRNAs are produced in HIV-1 infected cells.To randomly clone the nef expression by the nef miRNA, we constructed eight shRNAs homologous to the native miRNA or probes used in Figure nef gene expression or not  as a positive or negative control. All the shRNA-expressing plasmids including the controls were co-transfected into Jurkat T cells with pYM2.2 and cell fluorescence resulting from the expression of EGFP reporter gene was quantified by flow cytometry. The sinef176, 190, 367/miR-N367 and control siegfp all showed efficient reduction, but the sinef 007, 084, 299, 468 and 580 constructs gave only modest reductions, and no suppression was observed with si(-) and luc -based live vector. The PFV vector expressed HIV-1 SF2 nef gene as a reporter and the STYLE vector expressed shRNA as effectors. The full-length nef gene was inserted into the bel-2 portion of a PFV clone (22) in frame to obtain pPFV/nef , was collected at 72 h after transfection. For preparation of the STYLE vectors to deliver the shRNAs, the env gene portion of pSKY3.0 was replaced with the shRNA expression cassette under the control of the H1 promoter. The pSTYLE was produced -PCR. The integration of the DNAs into the genome of Jurkat T cells was also confirmed by PCR of the LTR, gag and/or nef regions . The most efficient sinef176, 190 and 367/miR-N367 vectors for reduction in nef expression  suppressed Luc activity when compared to controls .Since different host gene products are required for siRNA-mediated RNAi and miRNA-mediated translational repression with  in vivo . To testBecause extracellular Nef is internalized into human and mouse lymphocytes and macrophages -27, we ein vitro [PPARγ mRNA and body weights of mice. Significant PPARγ mRNA expression in intestinal adipose tissue of group 3, but not group 1 and 2, was detected on day 2 , our results show that nef-derived miRNAs are produced in HIV-1 infected cells, and support the possibility that miRNA and siRNA may be functionally identical, at least in a retrotransposon such as HIV. Recent studies have revealed that miRNAs and siRNAs could block mRNA expression by similar mechanisms [nef miR-N367 could regulate nef expression even in vivo. In our unpublished data, HIV-1 LTR promoter activity was inhibited by miR-N367 (nt number 379 to 449 of SF2 nef, 71-nt) expression, of which activity was dependent on negative responsive element (NRE) of U3 region (our unpublished data). Although no mismatch shRNA against region #367 was active, the miR-N367 from HIV-1 genome may have some mismatches and effectively inhibit HIV-1 transcription. Further the effects of siRNAs of Tc1, in particular those to the terminal inverted repeats derived from read-through transcription of entire transposable elements, were presented for silencing transposase gene expression by RNAi machinery in germ lines of C. elegans, [in vivo, however the inserted nef gene in the foamy retrotransposon may represent miRNAs which could inhibit nef mRNA expression by presumably an identical mechanism to that observed of siRNAs. Thus, RNAi might serve as a new sequence-specific therapeutic arsenal in AIDS prevention and possibly treatment.In this study, whereas siRNA has been reported to inhibit hepatitis B virus replication chanisms  and thatchanisms  and EBV-chanisms . Our resin vivo.Overall, our results indicate that nef shRNA transduced into T cell line inhibited HIV-1 transcription. Further, nef miRNAs could be produced from infected T cells and can block the trans-activity of Nef as well as HIV-1 replication on its own via the cis-action of nef. These functions of nef via RNAi pathways may allow persistently low pathogenic or latent infection as observed in HIV-infected non-progressors. Cumulatively, these data suggest that Nef may be involved in both viral replication and the disease progression, the findings, which may facilitate new strategies for HIV control nef sequence, because of the inclusion of viruses, which were also subtype B. The SF2 contained full-length nef reading frame as indicated in Table Patient selection is showed in Table 6 cells per ml for 3 days.HeLa and BHK cells were grown in Dulbecco's modified Eagle Medium (DMEM) (GIBCO, Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and antibiotics. CRFK cells were grown in Iscove's Modified Dulbecco's Medium (IMDM) (GIBCO) with 10% FBS and antibiotics. Jurkat T cells and MT-4 T cells persistently infected with HIV-1 IIIB strain were cultured in RPMI-1640 medium (GIBCO) supplemented with 10% FBS and antibiotics. The packaging cells (CRFKsugi) were made by transfecting CRFK cells with 10 μg of the pFFenv with Lipofectin Reagent (Invitrogen) and selecting transformants after culture for 14 days with 25 μg/ml of hygromycine B (Invitrogen). After 14 days, FFV Env protein expression was measured by flow cytometry and immunoblot analyses with FFV-infected cat B serum . The pPFThe details of plasmid constructs and the primer sequences used in cloning strategies are shown in supplementary file see .Flow cytometry was performed with a FACS Calibur (Becton Dickson, San Jose, CA) as described previously .Firefly Luc assay was performed using the Luciferase Assay System (Promega) as described previously . ImmunobThe concentration of p24 supernatant was determined by an antigen capture assay (Beckman Coulter, Fullerton, CA) according to the manufacturer's instructions.Confocal laser microscopy analysis was performed as described previously .SalI and cloned into SalI site of pBluescript SK(-), followed by sequence analysis. The secondary structures of RNAs were predicted by GENETYX-MAC program (Software Development Co. Ltd, Tokyo, Japan).Total RNAs were extracted from HIV-1 IIIB or SF2 persistently infected or uninfected MT-4 T cells using TRIzol reagent (Invitrogen). Approximately 40 μg of total RNA was treated with RNase A and T1 (Sigma, St. Louis, MO) as described previously , subjectgag, VIII (5'-tgtggtggaatgccactag-3') and IX (5'-attgtcatggaattttgta-3') for PFV LTR, XI (5'-tcttacagaccagtaacaa-3') and XII (5'-gtcaatcattacatctgca-3') for PFV gag, XIII (5'-aactactagtacccttcagg-3') and XIV (5'-aaaactcttgctttatggcc-3') for HIV-1 gag, XV (5'-atgggtggcaagtcaaaacg-3') and XVI (5'-tcagcagtctttgtagtactccg-3') for HIV-1 nef, XVII (5'-gttatgggtgaaactctgggagat-3') and XVIII (5'-atgttcctgaacataatcgtc-3') for PPARγ, XIX (5'-gacaacggctccggcatgtgcaaag-3') and XX (5'-ttcacggttggccttagggttcag-3') for β-actin, respectively. The nested PCR followed RT reaction was performed as described previously [nef or gag bands/integrating number of β-actin X 100.Semi-quantitative RT-PCR analysis was performed using the ThermoScript RT-PCR System (Invitrogen, CA., USA) according to the manufacturer's protocol with the following primers: III (5'-atcatgggccaaagagaattc-3') and IV (5'-aaatttcactcaatcgagcc-3') for FFV LTR, VI (5'-aggacctgaaaggcatg-3') and VII (5'-ttgttgagatcgtcccg-3') for FFV eviously . PCR pro5 IFU of PFV/nef and STYLE-367 by intravenous (i.v.) injection. RT-PCR analyses were performed 2 days after infection. For histological analysis, cryostat sections were prepared from both human and mouse tissues. The fixed sections were rinsed with PBS and incubated with 5% BSA for at least 1 hr to inhibit nonspecific binding of antibodies. Sections were incubated overnight at 4°C with anti-Nef rabbit serum, F3 or 305 mAb, and incubated with peroxidase or FITC conjugated secondary antibodies. The washed sections were incubated in 0.03% 3,3'diaminobenzidine (Sigma) solution in 0.05 M Tris buffer with 0.01% H2O2 for development of peroxidase activity. After counterstaining with hematoxylin or methylgreen, the sections were dehydrated and mounted.Balb/c and C3H/Hej mice were raised under specific pathogen-free (SPF) conditions. Mice were infected with 1 ml of 10Data were analysed using a one-way ANOVA analysis with a post-hoc Fisher's test. P values of 0.05 or more were determined for that of cut off.HIV-1 human immunodeficiency virus type 1miRNA microRNAnt, nucleotidesLTNP long-term non-progressorsshRNA short hairpin RNAAIDS acquired immunodeficiency syndromeRNAi RNA interferencesiRNA small interfering RNALTR long terminal repeatSIV simian immunodeficiency virusPBMCs peripheral blood mononuclear cells;EBV Epstein-Barr virus;EGFP enhanced green fluorescence proteinPFV prototype foamy virusTA trichostatinA IFU infectious unitsFFV feline foamy virusRT reverse transcriptionm.o.i., multiplicity of infectionmAb monoclonal antibody;Ner Nef receptor molecule;NRE negative responsive element.The authors declare that they have no competing interests.in vivo studies and tissue analyses. Y.I. and H.O. participated in data validation and overall experimental design. E.A.B. and N.K.S. carried out the clinical, sequencing, and virological studies and the writing of the manuscript. Y.R.F. participated in the design of the study and coordinated it. All authors read and approved the final manuscript.S.O. carried out northern analyses, immunoblot analyses, RNAi assays and was involved in the construction of plasmids. M.I. and Y.T. participated in Click here for file


Traditional non-steroidal anti-inflammatory drugs (NSAIDs) are a widely used class of therapy in the treatment of chronic pain and inflammation. The drugs are effective and can be relatively inexpensive thanks to available generic versions. Unfortunately the traditional NSAIDs are associated with gastrointestinal complications in a small proportion of patients, requiring costly co-therapy with gastro-protective agents. Recently, a new class of non-steroidal anti-inflammatory agents known as coxibs has become available, fashioned to be safer than the traditional NSAIDs but priced considerably higher than the traditional generics. To help physicians choose appropriately and cost-effectively from the expanded number of anti-inflammatory therapies, scientific bodies have issued clinical practice guidelines and third party payers have published restricted reimbursement policies. The objective of this study is to determine whether an educational intervention can prompt physicians to adjust their prescribing in accordance with these expert recommendations.This is an ongoing, randomized controlled trial. All primary care physicians in Manitoba, Canada have been randomly assigned to a control group or an intervention study group. The educational intervention being evaluated consists of an audit and feedback mechanism combined with optional participation in a Continuing Medical Education interactive workshop. The primary outcome of the study is the change, from pre-to post-intervention, in physicians' appropriate prescribing of non-steroidal anti-inflammatory therapies for patients requiring chronic treatment. Three classes of non-steroidal anti-inflammatory therapies have been identified: coxib therapy, traditional NSAID monotherapy, and traditional NSAID therapy combined with gastro-protective agents. Appropriate prescribing is defined based on international clinical practice guidelines and the provincial drug reimbursement policy in Manitoba. Traditional non-steroidal anti-inflammatory drugs (NSAIDs) are a widely prescribed class of therapy used to relieve pain and inflammation. The drugs have been shown to be effective for a variety of common disorders (hence their widespread use), most notably chronic osteoarthritis and rheumatoid arthritis. They are relatively inexpensive due to the available generic versions, but unfortunately have clinically important drawbacks related to their gastrointestinal (GI) toxicity . Each yeIn the last five years, a new class of non-steroidal anti-inflammatory agents has become available to physicians, fashioned specifically to be safer than the traditional NSAIDs but priced at least two to three times higher than the generic versions of traditional NSAIDs . The newTo assist physicians in selecting from the different classes of anti-inflammatory agents, various scientific bodies have published clinical guidelines and third party payers have issued restricted reimbursement criteria -24. ThesWith these guidelines and policies on prescribing in place, it is now important for physicians to adjust their prescribing practices accordingly. This is true both for rheumatology specialists and for physicians in the primary care setting where osteoarthritis patients are frequently managed. Unfortunately, experience and educational research show that simply making guidelines available does not elicit behavioural change from physicians -33. In tThe current use of anti-inflammatory drugs suggests a need for strategies that will prompt physicians to change their prescribing practice in accordance with the expert recommendations. Preferably, strategies should be investigated that have proved successful at altering physician behaviour in other settings.In this paper, we describe a randomized controlled study, which we are presently conducting, to evaluate the impact of an educational intervention on primary care physicians' prescribing of non-steroidal anti-inflammatory therapies. The study, being conducted in the province of Manitoba, Canada, tests the hypothesis that this intervention will significantly improve physician appropriate prescribing of anti-inflammatory drugs in compliance with international clinical practice guidelines and Manitoba's restricted drug reimbursement policy -22. The MAAUI is a province-wide population-based study. All primary care physicians  registered with the College of Physicians and Surgeons of Manitoba and practicing in Manitoba since July 1995 were eligible to enter MAAUI. The study excluded residents, new physician graduates, physicians registered as specialists, and physicians who started practicing in the province of Manitoba after July 1995. The eligible study population thus totalled approximately 884 physicians.All eligible physicians were automatically entered into the MAAUI protocol. Physicians who were randomized to intervention group of the research were given the option to withdraw from the study. Six family physicians who agreed to act as facilitators for the MAAUI CME workshop were not included in the study population.The MAAUI study design is depicted in Figure This study design was chosen for several reasons. First, it offered the scientific rigour that is associated with randomization and the use of a control group. It also ensured that both rural and urban Manitoban physician groups had an equal opportunity to take part in and learn from the study intervention. The study design was also particularly amenable to testing an intervention that required physicians to congregate in common locations (i.e. for the CME workshop). The 1:2 ratio of study groups helped to ensure that there were a sufficient number of study participants who would consider attending the CME workshops.Physicians allocated to the intervention arm of MAAUI were mailed a package that included an introduction to the study, audit and feedback material, and an invitation to participate in a CME workshop. Physicians in the control arm received no package.The audit and feedback material consisted of a "Personalised Prescribing Profile"  at the University of Manitoba, using administrative data from the Population Health Research Data Repository. This repository contains anonymized encounter-based records of individuals' interactions with the provincial health care system and is derived from information received by the Department of Health, Province of Manitoba, as part of the routine provision of health care in the province. The repository includes Physician Registry files, Medical Claims and Hospital discharge files, as well as Drug Programs Information Network (DPIN) files. The DPIN contains records of all drugs dispensed by Manitoba pharmacies, regardless of who is responsible for payment.Because of the sensitive nature of the Personalised Prescribing Profiles, the individualized profiles were not seen by the MAAUI research team. Instead the packages containing the profiles were assembled and mailed out by Manitoba Health with the assistance of an independent researcher, hired by the MAAUI research team, who signed a confidentiality agreement with Manitoba Health.The CME workshop for MAAUI was entitled "The Utilization and Prescribing of Anti-inflammatory Drugs in Osteoarthritis". The workshop focused specifically on the prescribing for osteoarthritis, as it was felt that people with this disease account for a large proportion of the chronic non-steroidal anti-inflammatory use by primary care practices .Eleven CME workshops were held throughout Manitoba (four within Winnipeg and seven outside of Winnipeg) and physicians in the intervention group were invited to voluntarily attend one of the 11. The workshops were free of charge and were approved for 3.0 hours of MAINPRO-M1 credits (continuing education credits awarded by the College of Family Physicians of Canada for group learning activities). Each workshop was facilitated by a trained family physician.The workshops included three components: an introductory video, a decision tree, and a case study portion. The introductory video provided the history and rationale of MAAUI, explained aspects of the Personalised Prescribing Profile, and introduced the decision tree. The decision tree depicted a stepwise approach to the diagnosis and treatment of osteoarthritis. This aid was designed by a rheumatologist on the MAAUI research team specifically for use in the study and was based on international clinical practice guidelines and Manitoba's drug reimbursement policy -22. A taThe overall MAAUI educational intervention, including both the audit and feedback material and the optional CME interactive workshop, was chosen by the MAAUI research team based on the success of similar interventions in the past. Audit and feedback mechanisms have shown moderate success in changing physician behaviour in past studies . EquallyThe primary goal of MAAUI is to evaluate the impact of the educational intervention on physician appropriate prescribing of non-steroidal anti-inflammatory therapies for patients requiring long-term treatment. To this end, four principal outcome measures have been developed:• the change in appropriate prescribing of all non-steroidal anti-inflammatory therapy  from pre-to post-intervention;• the change in appropriate prescribing of coxibs from pre-to post-intervention;• the change in appropriate prescribing of traditional NSAID/GPA combination therapy from pre-to post-intervention; and• the change in appropriate prescribing of traditional NSAID monotherapy from pre-to post-intervention.st 2001 to March 31st 2002) minus this rate during a 10-month period immediately pre-intervention (October 1st, 2000 to July 31st, 2001). The rate of appropriate prescribing will be defined as the number of patients prescribed long-term treatment in whom the treatment was appropriate, divided by the total number of patients prescribed long-term treatment, multiplied by 100%. Otherwise expressed:These primary outcomes will be evaluated at the level of the individual physician and will be calculated using administrative data similar to that used to create the Personalised Prescribing Profiles. The change in appropriate prescribing (AP) for a given physician will be calculated by the physician's rate of appropriate prescribing during a 6-month period post-intervention . A separate focus group will be held for the workshop facilitators.st, 2000 to July 31st, 2001) as control measurements: i) physician demographics including sex, urban or rural practice location, and the number of years in practice; ii) physician volume of non-steroidal anti-inflammatory therapies prescribed annually for patients requiring chronic therapy; and iii) physician rate of appropriate prescribing of NSAIDs. These latter two measures were assessed for each of coxibs, traditional NSAID/GPA combination therapy, and traditional NSAID monotherapy.The following data were collected at baseline  on the steering committee, which is the governing body responsible for approving all aspects of study development and implementation. Merck Frosst will not participate in data analysis and will not have access to confidential individual patient or physician data.AK, CM, and GT have received speaking fees from Merck Frosst Canada. KR is the scientific writer for this manuscript, employed by the PHCRU.All of these competing interests were not specifically disclosed to study participants. Merck Frosst's sponsorship of the study, however, was disclosed in the consent form signed by participants at the MAAUI CME workshops.MD, AK, BK, and CM conceived of the study. MD, AK, L-JM, CM, and GT contributed to the development of the protocol. GT created the decision tree and list of criteria for appropriate prescribing. GT also chaired the MAAUI project steering committee. LM is project managing the study. KR performed the literature review for this manuscript and wrote the first draft and coordinated all subsequent revisions. All authors approved the final manuscript.The pre-publication history for this paper can be accessed here:Decision Tree: take-home copyThe decision tree was a component of the MAAUI CME workshop. Physicians were introduced to the decision tree during the workshop and were provided with a two-sided take-home copy. The decision tree depicts a stepwise approach to the diagnosis and treatment of osteoarthritis. a/ Side 1. b/ Side 2. Of note, on side 2 of the decision tree the term "NSAID" denotes traditional NSAIDs.click here for file


Computational protein annotation methods occasionally introduce errors. False-positive (FP) errors are annotations that are mistakenly associated with a protein. Such false annotations introduce errors that may spread into databases through similarity with other proteins. Generally, methods used to minimize the chance for FPs result in decreased sensitivity or low throughput. We present a novel protein-clustering method that enables automatic separation of FP from true hits. The method quantifies the biological similarity between pairs of proteins by examining each protein's annotations, and then proceeds by clustering sets of proteins that received similar annotation into biological groups.Using a test set of all PROSITE signatures that are marked as FPs, we show that the method successfully separates FPs in 69% of the 327 test cases supplied by PROSITE. Furthermore, we constructed an extensive random FP simulation test and show a high degree of success in detecting FP, indicating that the method is not specifically tuned for PROSITE and performs well on larger scales. We also suggest some means of predicting in which cases this approach would be successful.Automatic detection of FPs may greatly facilitate the manual validation process and increase annotation sensitivity. With the increasing number of automatic annotations, the tendency of biological properties to be clustered, once a biological similarity measure is introduced, may become exceedingly helpful in the development of such automatic methods. Computational protein annotation is a major goal of bioinformatics and annotation methods are widely used. A wide variety of annotation methods exist, many of which rely on some kind of scoring. Typically, when testing whether a protein should be given a certain annotation, a score threshold is set, and proteins that score higher than the threshold are given the annotation. Obviously, some annotation mistakes may occur. Such mistakes can be divided into false positives (FPs) and false negatives (FNs). FPs  are annotations that were mistakenly assigned to a protein (type I error). FNs (or misses) are annotations that should have been assigned to a protein but were not (type II error). Adjustment of score thresholds allows tradeoff between these two types of mistakes. FPs annotations are considered to be of graver consequence than FNs. This is partially due to the fact that introduction of a false positive annotation into a protein database may cause other proteins to become incorrectly annotated on the basis of sequence similarity ,2. A sysDue to the importance of minimizing the amount of false annotations and maintaining highly reliable protein databases, three methods are generally used to avoid false annotations. The first method is manual validation of the annotation of each protein, which creates a serious bottleneck in the addition of new proteins and annotations to the database. The second method is using high score thresholds, thus lowering the rate of FPs but also increasing the rate of FNs (resulting in a loss of sensitivity). The third method is requirement for hits from different detection methods, eliminating advantages that are unique to some methods. Thus it would be beneficial to develop means by which FP annotations could be detected automatically, allowing both high throughput and high sensitivity.Here we present such a method that uses clustering of protein functional groups to separate true positives (TPs) from FPs automatically. Our method is based on the following notions: (a) protein annotations represent biological properties; (b) protein functional groups share specific combinations of biological properties, essentially constituting "property clusters"; (c) if two proteins have very different combinations of annotations, they are unlikely to share a single functional annotation and therefore there is a high chance that one of them was given that annotation incorrectly. These notions are not obvious, but were shown to correctly indicate false annotations in some individual cases tested manually using the graphical annotation-analysis tool of PANDORA . We aim Using these ideas, the method attempts to separate a group of proteins into "property clusters", by introducing a measure that quantifies the similarity between the annotation combinations of two proteins. According to our basic notions, these clusters are likely to be in accordance with false and true hits.all the TPs were clustered together without any FPs. We applied a stringent scoring, where a partial success is considered failure.We tested our method on the PROSITE protein signature database . The datFurthermore, we constructed a random FP simulation test in order to provide a more extensive test. In this test, all 5,551 InterPro  annotatiWe begin by describing the method of property-based clustering. Given a set P of all proteins that were given a certain annotation, and that there are both FPs and TPs in P, we would like to separate the set into disjoint subsets, so that one of the subsets will include all TPs and no FPs (leaving one or more subsets containing the FPs).Annotation-based clustering is used to detect these subsets. We define an annotation as a binary property assigned to a protein (each protein may or may not have a "hit"). At the first stage, annotations from GO (Gene Ontology) , InterPrp1 and p2 is defined as:The clustering works in the following way: between each two proteins we define a similarity score that tries to quantify how much do the two proteins have in common from a biological perspective. The score between two proteins A1 and A2 are the set of annotations of proteins p1 and p2 respectively, i is the current annotation, and f(i) is the frequency of i in the database. This score uses the following logic: if two proteins share an annotation, they are biologically similar in some manner. The more annotations these proteins share, the more cause we have to believe that they are similar biologically. However, two proteins sharing an annotation like "Enzyme" (that appears 45,991 times in our database) should receive a worse similarity score than two proteins that share a much uncommon annotation like "Heat Shock Protein" (that appears only 832 times). This is taken into account by using log(f(i)). Obviously, one could think of different scoring schemes that would quantify this differently. For a specific example of how the score is calculated see Table where The similarity score is calculated between every two proteins in P. Next, we define the similarity score between two clusters as the arithmetic average of scores of all inter-cluster protein pairs:C1 and C2 are clusters of proteins. Starting with clusters of 1 protein each, the method begins by an initial one-step clustering which merges all clusters that have the exact same combination of annotations. Following this the primary clustering commences: At each clustering step the two clusters that have the highest similarity score are merged. At each step the contents of the clusters are evaluated, and if all TP proteins appear in one cluster without any FPs, we say that the clustering process successfully separated the TPs from the FPs. Note that we do not require all the FPs to be grouped into one cluster, due to the fact that they cannot be expected to share biological similarity amongst themselves.where Out of 327 sets of proteins that share a PROSITE signature, the method showed successful separation (as defined previously) in 227 sets, i.e. 69% of the cases. The average size of the protein sets was 156.1 and the median 76. Altogether 58,254 proteins were used for this test. The average and median FP rates (FP rate is defined as: FP/(TP+FP)) of the sets were 0.12 and 0.07 respectively. These general statistics about the test set indicate that the sets were large enough and had a high enough amount of TPs and FPs so that the chance of random success would be minimal.In order to demonstrate the method's performance in this test, we provide the following example of testing a single protein set. The set presented here is the set of all 37 proteins that matched the PROSITE "Serum albumin family" signature. Each protein in the set contains an average of 18.2 annotations . First, the score between every pair of proteins is calculated, based on their mutual annotations. Next, the proteins undergo a preliminary clustering step in which all proteins that have the exact same combination of annotations are merged into clusters. Following this, the proteins are clustered together based on their mutual similarity score. Finally, once the clustering has finished we examine the tree to see if the true positives were separated from the false positives. In the given example, there are 5 proteins that were incorrectly assigned the PROSITE annotation (FPs), and in Figure 5,551 sets of proteins were tested 15 times each and showed successful separation in 74% of the cases. Altogether 99,076 proteins were used for this test. This can be subdivided into 78% success for the sets that had 1 random protein added, 74% success for the sets that had 5 random proteins added and 68% for the sets that had 10 random proteins added. The average set size was 78 proteins. The drop in the performance by increasing the level of FPs is due to the fact that there is a higher chance that one of the randomly selected proteins will be biologically similar to the TPs. Since we consider only cases in which all FPs are detected, then there would be a higher chance of failure as the number of randomly-generated FPs increases.While the simulation of FP errors randomly provides endless amounts of test sets, which is a clear advantage over the limited test sets provided by a real database such as PROSITE, the simulation has its own limitations. The hidden assumption made by this approach is that the FP hits are independent of each other. This assumption is not necessarily true: for example, if annotation is done by means of sequence similarity, false hits may be more likely to be biologically similar to each other (e.g. belong to the same family). In fact, in many cases in the PROSITE test we find that the correct separation separates the TP proteins into one cluster and the FP into one or two clusters, suggesting that the FPs share some degree of biological similarity . This difference in the way that FP annotations are generated may also account for the difference in success rates between the PROSITE test set and the simulated test set. The way FP annotations are introduced into databases is impossible to model, but the combined success of the method on both a real database test set and on an extensive simulated test set seems promising.A further issue which concerns the simulation method is determining the amount of FPs to add to each set. Here we chose to add 1, 5 or 10 proteins to each set. This does not necessarily reflect the amount of FPs in real databases. Understandably, each database's average FP rate depends on its specific characteristics. However, the PROSITE database's average FP rate of 0.12 (median of 0.08) might give an indication as to what a typical rate is. In comparison, the average FP rate for our random simulation set was 0.11 (median of 0.07), which suggests that our choice was reasonable.We call a clustering process successful if it managed at any step to separate the false annotations. However, this step must be somehow determined automatically. There are two approaches to this: one is to use an intrinsic parameter of the clustering process that would indicate where the correct halting step is located; the other is selecting a predetermined step of the process. We chose the similarity score at each merging step as an intrinsic process parameter. When plotting the score against the progression of the clustering Figure , a knee Interestingly, we found that with certain sets the method tended to be more successful than with other sets, probably indicating that these sets are more coherent biologically. This might suggest exploring an approach in which for each annotation one could predict the level of success provided by this method. Furthermore, we used the InterPro categorization of annotations into types in order to check success in specific annotation types. InterPro divides its annotations into different categories, such as "domain", "repeat" and "family". Understandably, "family" type annotations had a ~30% higher success rate than the other annotation types, primarily due to the fact that the "family" annotations often represent protein sets that are biologically coherent whereas other types such as "repeat" or "domain" annotations are biologically diverse. This result would be expected by a method that performs a clustering based on biological similarity. This indicates that this approach should be aimed primarily at functional family annotations.However, functional families can be defined at different resolutions: an alcohol dehydrogenase belongs to the enzyme family, the dehydrogenase family and the alcohol dehydrogenase family. The test sets of the PROSITE and InterPro databases mainly represent mid-level and low-level annotations, with a typical size of tens or a few hundreds of proteins (see the statistics given previously). In order to further our understanding of the resolution in which this method is successful, we divided the protein groups into size categories and studied the relative amount of success in every size category. Figure Because multiple annotation sources were used, concerns arose regarding interdependencies amongst them. For example, InterPro is highly dependent on PROSITE, so proteins that have a PROSITE annotation will very likely be assigned an InterPro annotation as well automatically. In order to minimize this effect, we did not allow the algorithm to use the InterPro annotations that matched the PROSITE annotation which was being tested. Furthermore, in order to increase reliability of the random FP simulation test, all known PROSITE FPs were removed from InterPro prior to the test. Still, there is some concern that the results are partially biased due to annotation source interdependencies. Furthermore, it is difficult to determine whether these dependencies represent true biologically dependent properties, or simply a duplication of the same property in different sources. Keeping this difficulty in mind, our results which show different levels of success for different types of annotations (see "Prediction of success") indicate that the success of the method is more likely due to biological dependency rather than artificial duplication.It should be stressed that the clustering process is based on sufficient annotation. Therefore, it may be difficult to apply this method to proteins that are poorly annotated. Still, these cases should be relatively rare: Nearly 77% of the ~1,600,000 proteins in TrEMBL  have at It is worthwhile noting that amongst the 58,254 proteins used in these sets there were 3,587 (6%) proteins annotated by SwissProt as "hypothetical proteins". 18% of the sets that were successfully separated contained such hypothetical proteins, with an average of 8% hypothetical proteins for each such set. These results suggest that the method is capable of handling to some extent hypothetical proteins of unknown function.Another helpful approach to the problem of insufficient annotation could be the introduction of quantitative protein properties that are easily determined and show some correlation with function (i.e. the protein length, its Isoelectric point, etc.) into this method. Preliminary testing showed some positive correlation between protein length and Isoelectric point with function in certain cases (not shown).Introduction of FP annotations into protein databases can be harmful. It has been shown that once a mistaken annotation is introduced into a database, it often transfers to other proteins that are sequentially similar causing a propagation of false annotation . Due to Based on the notion that protein functional groups share specific combinations of annotations, we have introduced a method that by separating a set of proteins into biological "property clusters" shows successful separation of incorrectly annotated proteins from correctly annotated proteins. We test the method both with a manually validated test set and with a randomly constructed test set, and in both cases show a high degree of success. These results suggest that this tendency of certain annotations to appear in groups may be used as a basis of automatic methods that detect FPs. Naturally, different computer learning methods can be used to take advantage of these interdependencies of biological properties (for example see ).We created a database that includes all proteins from SwissProt 40.28 (114,033 proteins) . The datAnnotation source and the number of annotation for each (in parenthesis) are: SwissProt version 40.28 (865 keywords), InterPro version 5.2 (5,551 entries), GO as of July 2002 (5,229 terms), PROSITE version 17.5 (1,189 signatures).NK and ML conceived of the study. NK designed the method. NK implemented and developed the method. NK designed the tests and analyzed the results. NK and ML wrote the manuscript.


Hepatitis C virus (HCV) infection is a significant problem among patients undergoing maintenance hemodialysis (HD). We conducted a prospective multi-center study to evaluate the effect of dialysis machine separation on the spread of HCV infection.Twelve randomly selected dialysis centers in Tehran, Iran were randomly divided into two groups; those using dedicated machines (D) for HCV infected individuals and those using non-dedicated HD machines (ND). 593 HD cases including 51 HCV positive (RT-PCR) cases and 542 HCV negative patients were enrolled in this study. The prevalence of HCV infection in the D group was 10.1% (range: 4.6%– 13.2%) and it was 7.1% (range: 4.2%–16.8%) in the ND group. During the study conduction 5 new HCV positive cases and 169 new HCV negative cases were added. In the D group, PCR positive patients were dialyzed on dedicated machines. In the ND group all patients shared the same machines.In the first follow-up period, the incidence of HCV infection was 1.6% and 4.7% in the D and ND group respectively (p = 0.05). In the second follow-up period, the incidence of HCV infection was 1.3% in the D group and 5.7% in the ND group (p < 0.05).In this study the incidence of HCV in HD patients decreased by the use of dedicated HD machines for HCV infected patients. Additional studies may help to clarify the role of machine dedication in conjunction with application of universal precautions in reducing HCV transmission. Hepatitis C virus (HCV) transmission occurs mainly through large or repeated direct percutaneous punctures to blood vessels; for example repeated injections for drug abuse . Less frMolecular virological studies have clearly shown the nosocomial transmission of HCV to hemodialysis patients,,17 but tAmong 40 HD centers in Tehran, we randomly selected centers one by one to reach a total number of 593 patients (12 centers) to enroll in this study. Selected centers were randomly divided in to dedicated (D) and non-dedicated (ND) HD machine groups, including 297 patients in D (4 centers) and 296 patients in ND group (8 centers). ELISA III checked all patients for HCV antibody detection before enrolling in the study. Positive cases were confirmed by RT-PCR. Only patients who were HCV positive by RT-PCR were considered to be HCV infected. Out of 593 HD cases 51 were RT-PCR positive (30 in the D groups and 21 in the ND group), and 542 were HCV negative (267 in the D group and 275 in the ND group). The prevalence of HCV infection in the D group was 10.1% (range: 4.6%– 13.2%) and was 7.1% (range: 4.2%–16.8%) in ND group. During the study conduction, 5 new HCV positive cases (1 in D group and 4 in ND group) and 169 new HCV negative cases entered the study. Information regarding age, sex, occupation , HCV infected relatives, previous peritoneal dialysis, surgery during last 2 months, duration of hemodialysis, number of blood product transfusions, history of organ transplantation, and the causes of ESRD was collected. The obtained history of IV drug abuse, tattooing, and multiple sex partners was not reliable.442 patients (254 cases in the D and 192 in the ND group) were followed for 9 months (first follow up population). 281 patients (160 cases in the D and 121 cases in the ND group) who remained within our study were followed for an additional 9 months (Second follow up population). Histories of surgeries or blood product transfusion were obtained at each follow-up; with no significant difference found between the D and the ND groups. There were no significant differences between the D and the ND groups in the number of patients lost to follow-up due to death, renal transplantation or transfer to a different hospital change (data not shown).Patients were dialyzed for 4 or 4.5 hours, 2 or 3 times weekly, using standard HD techniques by Cuprophane and Polysulfone dialyzers. All included HD patients were HIV and HBs-Ag negative. Dialysis membranes were low-pressure and used only once and HD machines were bleached and rinsed between dialysis sessions according to the manufacturers' instruction. Socioeconomic level was essentially similar between D and ND groups. The only difference between two groups of HD centers was that in-group D, HCV positive patients were assigned to a dedicated HD machine, but in-group ND, HD HCV positive and negative patients were not assigned to dedicated machines. All machines were located in dialysis wards and not in separate rooms in both groups.Patient to staff ratio in the D and the ND groups was not statistically different (3.1 and 3.4 respectively) and all staff members were negative for anti-HCV. To prevent HCV transmission, educational courses were held for the staff to reemphasize the CDC hygienic guidelines; however, an interview of all nurses directly involved in patient care disclosed some deviation from CDC hygienic guidelines. The minority of nurses remembered situations when they had failed to change their gloves due to an urgent adjustment of a hemodialysis machine. A checklist was used respecting hemodialysis-specific infection control practices and new gloves were applied for each individual patient. Nevertheless, masks, aprons and protective glasses were not universally used. In all centers, all patients had specific dialysis stations assigned to them, and chairs and beds were cleaned after each use. Handling and storage of medications and hand washing were not done in the same or adjacent areas to those where used equipment or blood samples were handled. One of the ND centers was excluded from the study due to non-adherence to CDC hygienic guidelines in the first months of the study.Statistical analysis was performed using SPSS 10.5 software. Comparisons between groups were made by the chi-square test method for categorical variables and by the t-test for quantitative variables.The mean age was 49.5 years (range from 12–84), 58.7% were male, and the mean HD duration was 21.6 months. The etiology of end-stage renal disease was hypertension in 36% followed by diabetes in 28% and glomerulonephritis in 10.5%. 15.5% of patients in the dialysis centers had a one-time history of kidney transplantation, and 2.2% had undergone transplantation twice. The demographic data for two groups is illustrated in table In the first follow-up period, the incidence of HCV infection was 1.6% and 4.7% in the dedicated and the non-dedicated groups (p = 0.05). In the second follow-up period, the incidence was 1.3% in the dedicated and 5.7% in the non-dedicated groups (p < 0.05) table .The possibility of an intradialytic spread of HCV appeared to be very low and the treatment of HCV infected patients with dedicated machines was not strictly required -26. AlthSome authors recommended that it is sufficient to treat every dialysis patient as potentially infectious, strictly adhering to the "universal precautions for prevention of HCV transmission", to prevent the spread of HCV in dialysis units ,33 and iIn our study population, the prevalence of HCV infection was approximately the same in both groups at the beginning of the study, but significantly lower incidence of HCV infection in D group may show that machine dedication strategies can be effective to reduce HCV transmission at least in our HD centers.Considering the prevalence of HCV infection and adherence to adequate infection control measures, HD machine dedication may help to decrease transmission of HCV infection in our dialysis units.However rigorous implementation of precaution measures remains a cornerstone for prevention of HCV transmission among patients undergoing maintenance hemodialysis, but as unpredictable accidents can always take place in hemodialysis units; machine dedication may play a more important role in prevention of HCV transmission. Further studies are needed to evaluate the possible roles of machine dedication in the presence of strict adherence to hygienic precautions.Dr AA Shamshirsaz designed the draft questionnaires and study protocol, managed the coordination of the surveys and drafted the manuscript. . Dr. Kamgar participated in drafting the manuscript and data collection, coordinated the study and designed the protocol. Dr Bekheirnia conceived of and designed the study, performed the statistical analysis, drafted the manuscript and helped design the protocol. Dr Bouzari helped draft the manuscript, participated in statistical analysis and managed paraclinical surveys. Dr Habibzadeh drafts the manuscript, participated in statistical analysis and helped in data collection and physical examination. Dr Pourzahedgilani participated in physical exam, filled out questionnaires and searched scientific sources. Dr V Broumand participated in drafting the manuscript. Dr Moradi participated in statistical analysis. Dr. Ayazi, Dr. Hashemi, Dr. A.H Shamshirsaz, Dr. Borghei and Dr. Haghighi took part in physical exams and filling questionnaires. Dr. B Broumand facilitated the study design and progress.The pre-publication history for this paper can be accessed here:


Many Canadian cities are experiencing ongoing infectious disease and overdose epidemics among injection drug users (IDUs). In particular, Human Immunodeficiency Virus (HIV) and hepatitis C Virus (HCV) have become endemic in many settings and bacterial and viral infections, such as endocarditis and cellulitis, have become extremely common among this population. In an effort to reduce these public health concerns and the public order problems associated with public injection drug use, in September 2003, Vancouver, Canada opened a pilot medically supervised safer injecting facility (SIF), where IDUs can inject pre-obtained illicit drugs under the supervision of medical staff. The SIF was granted a legal exemption to operate on the condition that its impacts be rigorously evaluated. In order to ensure that the evaluation is appropriately open to scrutiny among the public health community, the present article was prepared to outline the methodology for evaluating the SIF and report on some preliminary observations. The evaluation is primarily structured around a prospective cohort of SIF users, that will examine risk behavior, blood-borne infection transmission, overdose, and health service use. These analyses will be augmented with process data from within the SIF, as well as survey's of local residents and qualitative interviews with users, staff, and key stakeholders, and standardised evaluations of public order changes. Preliminary observations suggest that the site has been successful in attracting IDUs into its programs and in turn helped to reduce public drug use. However, each of the indicators described above is the subject of a rigorous scientific evaluation that is attempting to quantify the overall impacts of the site and identify both benefits and potentially harmful consequences and it will take several years before the SIF's impacts can be appropriately examined. Many Canadian cities are currently experiencing Human Immunodeficiency Virus (HIV) and hepatitis C virus (HCV) epidemics as a result of illicit injection drug use ,2. OtherIn over two dozen European cities and more recently in Sydney, Australia, safer injection facilities (SIFs), where injection drug users can inject pre-obtained illicit drugs, have been implemented in an effort to reduce the community and public health impacts of illicit drug use . SIF typWhile it must be stressed that limited quantitative data are presently available, various reports have credited SIFs with a number of public health and community benefits including: improving the health and social functioning of their clients , while rOn September 22, 2003 Vancouver, Canada opened North America's first government sanctioned SIF pilot study . FederalPrior to the opening of the SIF, a major concern with the evaluation related to willingness of the target community to use the injection facility . In ordeIn brief, the aims of Insite are to reduce public injection drug use and the unsafe disposal of syringes in public spaces, the reduction of overdoses and infectious disease risk, and improve access to healthcare services among IDUs. The methodology for evaluating these aims is described below and involves both a prospective cohort design and additional data sources including evaluation of community impacts.The framework for the Vancouver SIF evaluation was designed prior to the SIF's opening and involved a number of methodological approaches. In light of the lack of existing quantitative efficacy data , the exiThe Vancouver SIF evaluation is somewhat unique because of the availability of a number of pre-existing data sources. These data sources include the community health and safety evaluation (CHASE) cohort, which is a community recruited virtual cohort of Downtown Eastside residents that prospectively and retrospectively examines health service use in the community by linking to administrative health record databases. In addition, the Vancouver Injection Drug Users Study (VIDUS) is an ongoing prospective cohort study of injection drug users that involves semi-annual serology of HIV and HCV as well as a semi-annual questionnaire . VIDUS ap > 0.05).In addition, in order to augment these data sources and to allow for close examination of the characteristics of Insite clients over time, a prospective cohort of Insite users has also been established. The Scientific Evaluation of Supervised Injecting (SEOSI) cohort is based on a representative sample of Insite users. The sample is derived through random recruitment of Insite users who are offered an informed consent to enroll into the study. Random recruitment involves attending the SIF at times of the day that are randomly selected using a random number generation program in SPSS, and inviting all users who use the SIF at this time to enroll in the study. As with VIDUS, participants provide a blood sample and conduct an interviewer-administered questionnaire. The SEOSI questionnaire deals with items that are particularly relevant to Insite, such as risk behaviours, public drug use, satisfaction with Insite, and access to medical care and addiction treatment services. All SEOSI participants provide informed consent to link to the Insite database so that SIF use can be tracked, as well as informed consent to access administrative health record databases in the community. As of September 1, 2004 over 900 Insite users have been enrolled into SEOSI and comparisons of socio-demographic variables (age, gender, etc) has shown that the SEOSI cohort is statistically similar to the overall cohort of insight users ; Reliability (e.g., the ability of staff to perform the service dependably); Responsiveness (e.g., the willingness of staff to help clients and provide prompt service); Assurance (e.g., security, credibility and courtesy); and Empathy (e.g., ease of access, approachability and effort taken to understand clients' requirements). Similarly, reasons for avoiding the service are measured among IDUs in VIDUS who have not used Insite.These above prospective cohort data will be augmented by a number of other data sources including: process indicators, measures of community satisfaction and perceived impact, standardized measures of public order, and qualitative and quantitative measures of the health of the target population. The collection of each of these data sources is described below.In order to track service use in the database at an individual level, while allowing for participant anonymity, each client must select a unique client 'handle' or nickname. The SIF database has a search function that allows for rapid searches based on demographic information, such as birth date, if an individual forgets their handle. Similar anonymous tracking of individual clients is commonly used at needle exchanges and other services for illicit injection drug users .A primary purpose of the evaluation is to measure process indicators related to service uptake within the SIF, and this is enabled through the Insite database. The database tracks what drugs participants are consuming (heroin, cocaine, etc) and what services, such as nursing care and counseling services, are accessed by each client. For instance, in the month of May 2004, over 1300 unique visits were logged into the database.Community satisfaction and the perceived impact of the SIF on business persons are measured through a community survey that is performed in person among street recruited residents and at street-level businesses. The survey is similar to surveys being used in the Sydney SIF trial, and examines perceived changes in the neighborhood after the SIF's opening. In addition, staff satisfaction with the operation of the facility is measured through focus groups and qualitative interviews with staff persons. These interviews focus on how service delivery can be improved and on what measures can be taken to ensure staff safety and satisfaction.Standardized measures of public order were undertaken to examine the impact of the SIF on several indicators of public injection drug use. In brief, the survey protocol involves measuring specified public order indicators within an a priori defined geographical area in the neighborhood and at a priori defined times of the week. Data collection times are spread evenly throughout the week and involved walking through the study zone in the same pattern. Measures of discarded syringes, injection-related litter, and public injection drug use are all measured prospectively. An evaluation of these indicators has recently been described in detail .Following the opening of the SIF in September 2003, there was widespread support among the target population with a steady increase in uptake during the first few weeks. The site reached virtual capacity within two months and currently an approximate average of 500 injections take place each day in the site. The busiest times of the day are mid-afternoon and early evening at which times demand often exceeds capacity and waiting times to get into the 12 seat injection room can result in participants obtaining syringes and injecting elsewhere. Whether the wait times are disproportionately affecting specific populations is presently being investigated. Utilization also fluctuates daily, peaking on the days leading up to, and following welfare day. Exit surveys of IDU clients have been widely supportive of the service and high levels of satisfaction with the service among Insite staff have been reported. Contrary to the suggestion that cocaine users would be unwilling to use the SIF , approxiDespite the chaotic behaviours often associated with injection drug use, overall staff safety has been high and the instances of verbal or physical abuse by clients are managed efficiently as per the service's protocols. In outstanding circumstances, Vancouver Police Department has been called to remove disruptive clients, and support and assistance from the police in this regard has been very positive. Overall the staff remains very committed to the activities at Insite and staff satisfaction has been high.Overdoses, from a range of illicit drugs, are commonly observed in the SIF. The severity of these overdoses range from lowered respiration rate to severe emergency situations that have required the administration of naloxone and ambulance responses. Given the high levels of illness (for instance HIV and hepatitis C co-infection) and drug using behaviours (unknown substances of unknown purity) of the target population, it is not inconceivable that a fatality could occur in the SIF despite staff supervision and emergency response.There have been no instances where used syringe borrowing has been seen within Insite. These behaviours are common among street based injectors and it is well recognized that these activities promote the spread of blood-borne infections. It is also noteworthy that alcohol swabs to clean the injection site, and clean water and cookers are all provided to optimize hygenic injection procedures. Research of street-based IDU in Vancouver has shown that alcohol swabs are rarely used, and that non-hygenic water sources, such as puddle water, are commonly used. It is also noteworthy that within the SIF, safer hygenic injection practices are taught by the nursing staff to IDUs who have never been shown how to inject safely.In addition to supervising injections, teaching safer injecting practices, and responding to overdoses, there has been substantial health intervention within Insite. In particular, referrals to medical care at St Paul's Hospital are common as well as referrals to community health centres. Early intervention for primary medical care concerns, such as abscesses, is commonly provided by the Insite nursing team, and coverage with public health interventions, such as flu shots, has been provided to Insite users. In addition, addictions counseling occurs on site and there have been many referrals to detoxification programs and methadone maintenance therapy.Overall, Insite has attracted the target population and preliminary evidence suggests that the experiences within Insite as well as the community impact have been consistent with the experience of over two dozen European settings where SIF exist, and more recently Sydney, Australia. The examination of early changes in public order has been completed and there is strong evidence of improvement in several indicators including public drug use .However, each of the indicators described above is the subject of a rigorous scientific evaluation that is attempting to quantify the overall impacts of the site and identify both benefits and potentially harmful consequences over a multi-year period. This evaluation is primarily structured around a prospective cohort design that will involve the longitudinal measurement of health and community indicators over the next several years. As such, it will be some time before the overall impact of Insite on a number of outcomes, such as blood-borne infections and IDUs behavior, can be adequately quantified.