A primary goal of acute treatment for depression is clinical remission of symptoms. Most meta-analyses of remission rates involve randomized controlled trials (RCTs) using patients from psychiatric settings, but most depressed patients are treated in primary care. The goal of this study was to determine remission rates obtained in RCTs of treatment interventions for Major Depressive Disorder (MDD) conducted in primary care settings.Potentially relevant studies were identified using computerized and manual search strategies up to May 2003. Criteria for inclusion included published RCTs with a clear definition of remission using established outcome measures.A total of 13 studies (N = 3202 patients) meeting inclusion criteria were identified. Overall remission rates for active interventions ranged between 50% and 67%, compared to 32% for pill placebo conditions and 35% for usual care conditions.Remission rates in primary care studies of depression are at least as high as for those in psychiatric settings. It is a realistic goal for family physicians to target remission of symptoms as an optimal outcome for treatment of depression. Major depressive disorder (MDD) is one of the most common and disabling of medical conditions . The CanThere are many effective treatments for MDD, including psychotherapy and antidepressants. Traditionally, efficacy in randomized controlled trials (RCTs) for depression has been determined on the basis of score changes in rating scales such as the Hamilton Depression Rating Scale (HDRS)  or the MSuch findings suggest that the goals of acute treatment (approximately the first 8–12 weeks or so of treatment) for MDD should be clinical remission, a clinical state distinguished by minimal residual symptoms, rather than just response -13. ClinIncreasing numbers of treatment studies are now explicitly reporting both clinical response and remission rates in assessment of outcome. A meta-analysis of 8 antidepressant studies of venlafaxine versus selective serotonin reuptake inhibitors [SSRIs] and placebo reported mean remission rates of 45%, 35%, and 25%, respectively . A subseAll the studies in these systematic reviews involved patients in psychiatric or mixed settings. However, most people suffering from MDD will be managed in the primary care setting . ApproxiPotentially relevant studies were identified using computerized and manual search strategies. The computerized search conducted in June, 2003 included the databases: Medline, Psych Info, Embase, Biosis, Cochrane Database of Systematic Reviews, and Cochrane Controlled Trials Register and Current Controlled Trials (1981–May 2003). The search terms used were 'depressive disorder' or 'depression' combined with 'primary care' and 'remission' and/or variants. The bibliographies of relevant articles were also manually searched. Two reviewers (MYD and RWL) collected and independently assessed abstracts for inclusion criteria. Disagreements were resolved with consensus.Studies were included if they were RCTs with original data comparing one or more interventions  and published in English. Only studies of predominantly adult populations, as opposed to exclusively child or elderly patient populations, were included. Although the focus was principally upon patients with MDD , the criteria for a diagnosis of MDD was intentionally broad in order to capture the heterogeneity of the sample and allow the results to be as generalizable as possible. Included studies also had to use a standardized outcome measure  and proTwo independent reviewers (MYD and EEM) extracted data from studies using a checklist developed for this study, with disagreements resolved by a third reviewer (RWL). A conservative measure of remission rate was calculated from each study using an intent-to-treat analysis , even ifThe type of intervention was classified as placebo, "usual care" by clinician (standard treatment by a patient's own physician), psychotherapy treatment only, antidepressant treatment only, psychotherapy plus antidepressant treatment, or program intervention .Each set of rates was pooled based on a Bayesian approach to meta-analysis using the Fastpro software program (version 1.7) by Eddy and Hasselblad. Readers interested in a more detailed discussion of this approach should refer to Eddy et al . The pooThe initial electronic and bibliographic search found 63 articles of which 47 warranted more detailed review based on the published abstract. Of these, 34 articles were excluded due to methodology , lack of remission criteria 18 studies), diagnostic criteria  and age criteria   ranged between 50% and 67%. These rates are equivalent to, or indeed greater than, those reported in meta-analyses of studies examining pharmacological or psychological interventions for depression in psychiatric populations, in which the overall remission rates ranged between 35% and 46% -24. On tsymptoms .While it was not within the scope of the current study to compare the effectiveness of different treatment interventions in improving remission rates, we can report on the trends we observed in the data. Antidepressant and psychotherapy interventions delivered in isolation showed similar remission rates (54% for both). Combination antidepressant plus psychotherapy interventions showed somewhat higher rates (67%), although this category included only 1 arm with only 35 patients. Program interventions had a mean remission rate of 50%, and all treatment interventions fared better than either placebo (32%) or usual care (35%).The studies identified in our review were quite heterogeneous in nature, ranging from those that looked solely at the effects of a particular pharmacological agent, through to complex program initiatives that incorporated a variety of interventions at different levels of care. This heterogeneity limits our ability to make broad comments about remission rates in primary care, but was not unexpected, as we wanted to capture the diversity of treatment interventions for depression currently being tested in this setting. Other potential limitations of the study include that fact that we only assessed published studies written in English and that we used a conservative measure of remission rate. Finally, we also used the definition of remission as specified by each individual study. While these definitions were similar to those widely used in RCTs conducted in psychiatric settings, and thus are useful for comparison, there is current controversy about depression scales and which cutoff scores indicate true remission of symptoms .This meta-analysis serves to answer an important clinical question about the feasibility of obtaining remission of symptoms of MDD in primary care patients. Our results indicate that this is a realistic goal in this population, although further research is still required to determine whether certain treatment modalities (or combinations of treatment interventions) are superior to others in achieving higher remission rates. Future research should also focus upon developing pragmatic strategies for general practitioners to implement evidence-based guidelines concerning the treatment of depression to clinical remission.MYD and EEM conducted the data extraction, wrote the initial draft of the manuscript, interpreted results, and revised the manuscript. PW provided statistical consultation and analysis, and revised the manuscript. JEA interpreted the results and revised the manuscript. RWL conceived the initial idea, developed the method, interpreted results, revised the manuscript, and provided financial resources for the study. All authors read and approved the final manuscript.RWL is on advisory/speaker boards or has received research funds from: AstraZeneca, Biovail, Canadian Network for Mood and Anxiety Treatments, Eli Lilly, GlaxoSmithKline, Janssen-Ortho, Litebook, Inc., Lundbeck, Merck, Organon, Roche, Shire, Servier, and Wyeth.The pre-publication history for this paper can be accessed here:Studies excluded from the reviewClick here for file


Annotation of sequences that share little similarity to sequences of known function remains a major obstacle in genome annotation. Some of the best methods of detecting remote relationships between protein sequences are based on matching sequence profiles. We analyse the superfamily specific performance of sequence profile-profile matching. Our benchmark consists of a set of 16 protein superfamilies that are highly diverse at the sequence level. We relate the performance to the number of sequences in the profiles, the profile diversity and the extent of structural conservation in the superfamily.ROC10, varying from 0.95 down to 0.01. These large differences persist even when the profiles are trimmed to approximately the same level of diversity.The performance varies greatly between superfamilies with the truncated receiver operating characteristic, Although the number of sequences in the profile (profile width) and degree of sequence variation within positions in the profile (profile diversity) contribute to accurate detection there are other superfamily specific factors. Currently some of the best methods for detecting relationships between protein sequences below the so-called twilight zone of sequence similarity are offered by iterative search algorithms such as PSI-BLAST  which, iHere, we examine how the performance of remote homolog detection by profile-profile methods varies between particular superfamilies. Since superfamilies are believed to constitute sets of remote homologs, detection of same-superfamily relationships is an important task for bioinformatics, and with the increasing number of structures becoming available, improvement in this area will help build a complete structural map of sequence space. In this paper, we use a set of superfamilies that are very sequence diverse to benchmark profile-profile methods. By sequence diverse, we mean that the superfamily has many domains that show no detectable sequence similarity to each other; this lack of detectable sequence similarity means this set is a difficult benchmark for remote homolog detection methods.profile width, we mean the number of sequences in the profile, defined in contrast to profile length and by diversity we mean the degree of sequence variation within positions in the profile. In particular, Panchenko suggested that there may be an optimum level of profile diversity [Previous work has shown that the performance of profile-profile methods is chiefly determined by the width and diversity of the profiles. By iversity , whilst iversity .We examine the performance of profile-profile matching with regard to specific superfamilies with both the full profiles generated from a PSI-BLAST search, and with profiles that are trimmed to similar width and diversity. Significant differences in recognition performance exist between superfamilies for both the full and trimmed profiles. This suggests that performance of profile-profile matching is not simply a function of profile width and diversity. We examine how the performance relates to the structural diversity of superfamilies and find that structurally conserved superfamilies are recognised more successfully than structurally diverse superfamilies.Table ROC10 (see Methods for definition) for each superfamily. The figure shows that there is a large variation in performance with respect to superfamily for both the full profiles and the trimmed profiles.Figure ROC10 values ≥ 0.7, the fibronectin, thioredoxin-like, (trans)glycosidases, immunoglobulin and FAD/NAD(P)-binding have ROC10 > 0.2 and the remaining 8 superfamilies all perform poorly, having a performance less than 0.1.For the full profiles, the alpha/beta-Hydrolases, Cytochrome c and S-adenosyl superfamilies perform well, all having with ROC10 values greater than 0.2, while the rest are still less than 0.1.After trimming, although performance is reduced, the overall pattern of performance still remains. All the well recognised superfamilies (with the exception of the (trans)glycosidases and thioredoxin-like) still show ROC10 of 0.7 when using trimmed profiles despite having, on average, less than 20 sequences in the profile.The fact that the performance varies greatly between superfamilies despite the trimming of the profiles indicates that the profile generation is not the only limiting step in the performance of profile-profile methods. One might have thought that, for instance, the bad recognition of 4-helical cytokines is due to the small number of homologs drawn from the profile-building stage. Whilst this still may be true, it is not necessarily true: the Cytochrome c superfamily still shows a Figure ROC10 for each superfamily. The figure shows a correlation between the mean RMSD of each super-family and its ROC10 value. The figure shows that superfamilies with a mean RMSD of less than 2 Å tend to be well recognised by profile-profile methods, whilst the structurally diverse superfamilies are not.Figure It may be the case that despite the absence of any discernible global sequence similarity within our dataset some local patterns of conservation do exist. These patterns may be present more strongly in some superfamilies than in others. In order to examine this possibility we constructed multiple structure based sequence alignments for each of the 16 superfamilies and then looked down the columns of the multiple sequence alignments to examine the extent of conservation at each position (see Methods section).ROC10) versus conservation. Apart from the cytochrome c superfamily (an outlier with a high ROC10 of 0.7 despite a conservation score of 0.2 because the superfamily has a conserved CxxCH motif that facilitates detection), the well performing superfamilies -binding and fibronectin with ROC10 values for the trimmed profiles of at least 0.25) have conservation measures of greater than 0.25. This suggests that some superfamilies although highly sequence diverse, may retain some patterns of conservation that facilitate recognition. Further investigation of the functional implications of this variation would be a next step.Figure ROC10). The P-loop and Viral coat superfamilies have low conservation scores and and large structural diversity reflected by high RMSD values. In contrast, the fibronectin and immunoglobulin superfamilies have higher conservation values (both around 0.28) and lower RMSDs (around 1.5Å). However the figure does not show any clear correlation between conservation and RMSD.Figure Our results suggest that profile profile methods can detect remotely related sequences for some superfamilies significantly better than for others. In our dataset the sequence identity between domains in all the superfamilies is low (not greater than 10% as defined by the ASTRAL). Although the mean width and diversity of the profiles varies across the superfamilies this does not appear to be the only factor contributing to the differences in detection.ROC10 dropping from 0.70 to 0.22. However trimming had no effect on performance for the FAD/NAD(P)-binding superfamily, and only resulted in a small reduction in performance for the immunoglobulins and the cytochrome c superfamilies. Importantly the membership of the top ranking superfamilies in terms of performance did not change after trimming.The effect of the trimming varied depending on superfamily. For the best performing profile  the trimming reduced the performance by about 50% (from 0.95 to 0.43) but the effect on the rank was small dropping from first place to second. Similarly the trimming impacted significantly on the performance of the S-adenosyl methyl transferases with Although the overall level of sequence similarity within our dataset is low (not more than 10% identity) the different superfamilies exhibit different levels of conservation at positions within the multiple structure based alignments. These conserved positions may facilitate recognition. The extent to which they constrain the structures leading to less diverse alignments is unclear. We recognise also that our measure of conservation and also the use of RMSD as a measure of structural diversity both have their shortcomings. It would be interesting to identify and extract a conserved core and represent structural profiles as combination of core profiles separated by regions of variable length.There exist large superfamily specific differences in the performance of profile profile matching for the detection of remote sequence relationships. Some superfamilies can be detected far more successfully than others. The width and diversity of the profiles are important factors in successful recognition. However these are not the only factors that contribute to these superfamily specific differences.We took release 1.63 of ASTRAL  which prFor each domain of each of the 16 superfamilies we executed a five round PSI-BLAST [To produce trimmed profiles, we take the full profile and remove the bottom sequence (corresponding to the most remote homolog) until a stopping criterion is reached. The stopping criterion is based on Neff, a statistic previously used for this task ,6,7. NefWe use the program COMPASS  to perfoi of superfamily j, , the sequence profile corresponding to  was not included in the sequence profile library. This procedure was carried out twice: firstly with the full profiles, and the again with the trimmed profiles.To assess the performance of profile-profile matching, each domain of each of the 16 superfamilies was used as a query and its sequence profile was matched against a library of sequence profiles representing the dataset. A profile database was then created using the 543 profiles. When matching the profile of domain ROC10 as a statistic that describes the performance of the profiles for a particular super-family. ROCn is defined as , where T is the total number of true hits possible and ti is the number of true positives with a score better than the ith false hit. Variance in the ROC10 statistic was calculated using the method given in [We use To evaluate the structural diversity within each superfamily, each member of a superfamily was structurally compared to every other member. For all the domains in a superfamily we perform pairwise structural alignments using the program SAP  to all oi and j at positions xi, xj in the superposition, is defined to be ((1 + RMSD)(1 + |xi - xj|))-1. A detailed explanation and analysis of this method is given in [To create a structure based multiple alignment of a superfamily, we first made all pairwise structural comparisons between all pairs within a superfamily using SAP ,12. We tgiven in .We used the Taylor Venn diagram  to assigJAC carried out the benchmarking and wrote the necessary code and helped to prepare the manuscript. MASS conceived of the study, provided input into the design and refined the manuscript. All authors read and approved the final manuscript.


Whiskers don't fossilize, so it's hard to say when they first evolved. But it's quite likely they emerged along with mammals, over 200 million years ago. To elude the eye (and feet) of ungainly dinosaurs, it's thought these shrew-like prototypes foraged at night and sought refuge underground, where the sensory advantages of whiskers would come in handy. Nocturnal animals use whiskers much like the blind use walking sticks: to navigate their surroundings, explore close objects, and avoid running into things.Whiskers, or vibrissae, connect to nerves, blood vessels, and muscles. These special connections allow rats, for example, to actively “whisk” the surface of objects and discern fine differences in texture, just as we move our fingertips along a surface to pick up details. In the wild, whisking helps rats navigate unfamiliar terrain to find food. But how does the brain know what the animal is touching?Rat whiskers scan surfaces in a rhythmic motion that excites sensory receptor cells embedded in their whisker pad. Receptors in each whisker shaft are innervated by several hundred “first-order neurons” that relay sensory signals to second-order neurons in the brain stem, then on to third-order neurons in the thalamus, and finally on to the cortex, where sensory stimuli are integrated in cell clusters called barrels.Ehsan Arabzadeh, Erik Zorzin, and Mathew Diamond work with rats to investigate how sensory receptors extract fundamental features from complex and diverse stimuli to encode texture. Not much is known about how receptor and cortical neurons respond to active whisking along irregular surfaces, though responses to simple stimuli  suggest that neurons might represent texture by encoding kinetic features of whisker vibrations, in particular, velocity. In a new study, Diamond and colleagues investigate the connection between textures, whisker vibrations, and neural codes: do distinct textures produce distinct vibrations? If so, how are these vibrations encoded and reported?The authors first collected kinetic data of whiskers moving across different textured surfaces. Stimulating cranial nerve VII of anesthetized rats (the motor nerve) generated whisking movements akin to those seen in conscious rats; the kinetics of these movements and the vibrations of the whisker shafts were measured under different conditions, including no contact with objects (“free whisk”), contact with smooth objects, and contact with various grades of sandpaper. These vibrations were then “played back” to other rats, while measuring the neuronal activity at two critical stages in the sensory pathway: the first-order neurons that innervate the whiskers and the barrel cortex neurons that integrate the incoming signal.Altogether, the authors collected a neural dataset consisting of first-order recordings, barrel cortical cluster recordings, and simultaneous paired recordings from both sites, all in response to playback of the library of texture-related vibrations. This approach afforded the opportunity to directly compare encoding of information at both levels in the sensory pathway. These recordings show, the authors argue, that temporally distinct firing patterns in the trigeminal ganglion (the cell bodies of the first-order neurons) and cortex captured the kinetic features of the texture-induced vibrations. Each texture's “kinetic signature” is encoded by a characteristic, temporally precise firing pattern associated with whisker movement. Compared to free whisking, coarse sandpaper produced irregular bursts of high and low velocity, and both first-order and cortical neurons fired far more impulses for coarse sandpaper than for free whisks. The authors then used stimuli consisting of random velocities to uncover the “tuning curves” of neurons, and simulations showed that these neuronal tuning curves could perfectly predict the real neural responses to textures.Noting the close match between the simulated and natural responses, Diamond and colleagues conclude that the texture-induced firing patterns observed in the first-order and cortical neurons suggest that neurons selectively encode elemental kinetic features—namely, high velocity—to tell rats what they're whisking. This selectivity allows even a single whisker to transmit significant bits of texture-specific information to the brain. Interesting as rat whisking may be, these findings have relevance beyond the world of whiskered beings, shedding light on the underlying neural processes that translate touch into recognition.


RNA interference (RNAi) can potently reduce target gene expression in mammalian cells and is in wide use for loss-of-function studies. Several recent reports have demonstrated that short double-stranded RNAs (dsRNAs), used to mediate RNAi, can also induce an interferon-based response resulting in changes in the expression of many interferon-responsive genes. Off-target gene silencing has also been described, bringing into question the validity of certain RNAi-based approaches for studying gene function. We have targeted the plasminogen activator inhibitor-2 (PAI-2 or SERPINB2) mRNA using lentiviral vectors for delivery of U6 promoter-driven PAI-2-targeted short hairpin RNA (shRNA) expression. PAI-2 is reported to have anti-apoptotic activity, thus reduction of endogenous expression may be expected to make cells more sensitive to programmed cell death.As expected, we encountered a cytotoxic phenotype when targeting the PAI-2 mRNA with vector-derived shRNA. However, this predicted phenotype was a potent non-specific effect of shRNA expression, as functional overexpression of the target protein failed to rescue the phenotype. By decreasing the shRNA length or modifying its sequence we maintained PAI-2 silencing and reduced, but did not eliminate, cytotoxicity. ShRNA of 21 complementary nucleotides (21 mers) or more increased expression of the oligoadenylate synthase-1 (OAS1) interferon-responsive gene. 19 mer shRNA had no effect on OAS1 expression but long-term selective pressure on cell growth was observed. By lowering lentiviral vector titre we were able to reduce both expression of shRNA and induction of OAS1, without a major impact on the efficacy of gene silencing.Our data demonstrate a rapid cytotoxic effect of shRNAs expressed in human tumor cell lines. There appears to be a cut-off of 21 complementary nucleotides below which there is no interferon response while target gene silencing is maintained. Cytotoxicity or OAS1 induction could be reduced by changing shRNA sequence or vector titre, but stable gene silencing could not be maintained in extended cell culture despite persistent marker gene expression from the RNAi-inducing transgene cassette. These results underscore the necessity of careful controls for immediate and long-term RNAi use in mammalian cell systems. Gene silencing is a powerful tool with which to study protein function. Gene inactivations in mice have revolutionised the way we study both basic biology and a plethora of disease types ,2. Gene Maintaining RNAi-inducing dsRNA below 30 nucleotides in length was thought to avoid activation of the interferon-induced anti-viral response. PKR is a key anti-viral regulator and its expression can be induced by the interferon response . PKR is In this work we used a simple approach for gene silencing in human tumor cell lines, using lentiviral vectors for stable delivery of shRNAs. We aimed to study the effects of targeting the plasminogen activator inhibitor-2 (PAI-2 or SERPINB2) mRNA on cell survival in the presence of pro-apoptotic stimuli. In addition to its inhibitory activity on the urokinase plasminogen activator, PAI-2 is thought to have anti-apoptotic properties ,25. Its Using lentiviral vectors for delivery of RNAi-inducing expression cassettes we achieved potent PAI-2 gene silencing, accompanied by a rapid cytotoxic effect. The degree of cytotoxicity was proportional to shRNA length and induction of an interferon response gene could be detected when shRNA of 21 complementary base pairs or more was expressed. The phenotype was not target gene specific, as PAI-2 overexpression failed to rescue cytotoxicity and control hairpins were also cytotoxic. Using lower vector titre, reduced shRNA expression and interferon response induction was measured without compromising gene silencing. Using a 19 complementary base pair shRNA expression vector, which reduced PAI-2 expression and induced no initial cytotoxicity or interferon response, transduced cell marker gene expression was maintained but gene silencing lost in long-term cell culture. Our results highlight the need for careful controls to monitor specificity and maintenance of gene silencing when using RNAi for stable loss-of-function studies in mammalian cells.Lentiviral vectors were generated which deliver an expression cassette for human U6 promoter-driven expression of short hairpin RNA (shRNA), with exact homology to the human PAI-2 mRNA. The vector expression cassette also bears the enhanced green fluorescent protein (GFP) gene under the control of the EF-1α promoter, and an internal ribosome entry site (IRES) sequence  also reduced PAI-2 mRNA and protein levels. Equal sample loading for immunoblots was confirmed by Ponceau S staining of nitrocellulose membranes (data not shown) and the presence of equal amounts of a PAI-2 monoclonal antibody-reactive non-specific band in each blot . In parallel, we transduced IS-1 cells with the sh119 vector or a GFP control vector. We achieved high percentage transduction rates which were monitored for over two weeks Figure . GFP posHere we report the use of lentiviral vectors for the delivery of expression cassettes designed for RNAi-induced stable knock-down of gene expression. We undertook this approach because of the promise of RNAi to easily create cell lines that are specifically deficient in one protein component. By using lentiviral vectors for shRNA expression, high transduction efficiencies can be achieved, avoiding effects due to clonal selection of phenotypically different cells.We successfully targeted the PAI-2 mRNA with the aim of studying the effects of reducing PAI-2 activity on cell sensitivity to apoptosis-inducing stimuli. PAI-2 activity has previously been implicated in protection from apoptosis . The cytWe were able to detect increased expression of an interferon response gene, OAS1, in cells transduced with all hairpins of 21 or more base-paired nucleotides. A 21 mer hairpin induced the most potent OAS1 induction. These results, and those of others , suggestOverexpression of functional PAI-2 did not rescue the cytotoxic effects or OAS1 induction observed in cells transduced with a series of vectors for expression of different length shRNAs. This result, the cytotoxicity associated with scrambled sequence hairpin-encoding constructs, and the same selective pressure seen on the growth of transduced HeLa cells, which do not express detectable PAI-2 mRNA or protein , initially appeared stable, compared to control-transduced cells. PAI-2 protein levels were clearly reduced 10 days after transduction and selective pressure on cell growth appeared to be minimal, as the percentage of sh119 GFP positive cells was apparently stable at about 80 %, 17 days after transduction. However, after cell sorting of GFP positive cells and prolonged cell culture of over one month after transduction, the PAI-2 antigen measured in sh119s (s for selected) cells was restored to control-transduced cell levels. Despite maintenance of transduction marker expression, gene silencing was absent. The GFP expression levels in selected sh119s cells was reduced after one month of growth, compared to cells monitored three days after transduction. It is possible that we selected cells with a greatly reduced, non-cytotoxic shRNA expression level, as we have detected a reduced number of integrated vector copies. The negative selective effect on cells transduced with the sh119 vector was not due to the suppression of PAI-2 expression, as PAI-2 overexpressing cells showed the same negative selection.In all experiments in which a selective pressure on growth was apparent on shRNA-expressing cells, reduced percentage GFP positive cells and reduced GFP expression in transduced cells was measured over time. We hypothesised that very high expression levels of the various shRNAs is cytotoxic. This could occur via high numbers of transcriptionally active vector integration events or integration at transcriptionally active chromosomal regions. Both might be controlled using a tightly regulatable expression system, which has been described , but mayOur data demonstrate the importance of appropriate controls for using RNAi, as proposed in a recent editorial . These iIn the light of our own data and other recent reports -22, soluOur study demonstrates vector-derived RNAi in tumor cell lines and points towards the necessity of careful, but clearly feasible, controls when using RNAi for stable gene suppression in short- and long-term experiments.Isreco-1 (IS-1) cells were a gift from Dr. B. Sordat . 293T cells were a gift from Dr. D. Trono . HeLa cells were purchased from the European collection of cell cultures, ECACC number: 93021013. All cells were maintained in DMEM supplemented with 10 % Fetal Bovine Serum and 10 mM HEPES pH 7.4 (IS-1 medium) (purchased from Invitrogen).PEF-1α-iresGFP region of the plasmid. A schematic of the gene transfer cassette is given in Figure Gene transfer plasmids, for RNA interference using lentiviral vectors, were constructed using the backbone of ploxEWiresGFP, a gift from Dr. P. Salmon, Geneva University Medical Centre. The human U6 gene was amplified by PCR using HeLa cell genomic DNA as template and the oligonucleotides U6-ClaI-F 5' GATC ATCGATAAGGTCGGGCAGGAAGAGGGCCTATTTCCC 3' and U6-ClaI-R 5' GATCATCGATTGGTAAACCGTGCACCGGCGATAAACG 3'. The 483 base pair PCR product was digested with ClaI, inserted into the ClaI site of pTRE2 hyg (Clontech), and its sequence verified by DNA sequencing. The U6 promoter and gene sequence corresponded to nucleotides 65 to 527 of Genbank accession number M14486. This plasmid was used as template for PCR reactions to amplify U6 promoter-driven expression cassettes. Each PCR product included the U6 promoter with shRNA-encoding sequences beginning at the U6 +1 site, and a run of 6 or 7 thymidine bases for an RNA polymerase III transcription termination signal. Each PCR introduced flanking ClaI restriction sites. PCR products were directly cloned into the pGEM Teasy (Promega) plasmid and sequenced. ClaI fragments of positive clones were excised and ligated into the unique ClaI site of ploxEWiresGFP. The U6 promoter cassettes in the resulting lentiviral vector plasmids were verified by DNA sequencing. Each construct used for vector production had the same orientation of the U6 expression cassette with respect to the 4 or 1 × 105 cells/well. Upon addition of vectors, plates were centrifuged for one hour at 1000 g in the presence of 8 μg/ml polybrene . After 24 hours, target cells were washed twice with PBS and cultured in IS-1 medium until analysis. Where indicated, viral titre was determined by transducing cells with 10 μl of lentiviral vector conditioned medium from 293T cell producer cells and measuring the % of GFP positive cells by flow cytometry. We routinely achieve 106 effective transducing units per ml of producer cell conditioned medium, which results in a typical multiplicity of transduction of 10. Comparisons between cell populations transduced with different vectors is made by flow cytometry analysis of GFP positive cells.Lentiviral vectors were produced by three plasmid co-transfection of 293T cells, essentially as described previously . VectorsAnti-human PAI-2 monoclonal antibody 3750 was purchased from American Diagnostica. PE-labelled goat anti-mouse antibodies were purchased from Pharmingen. HRP-conjugated goat anti-mouse antibodies were purchased from Bio-Rad.Flow cytometry was performed using Becton Dickinson FACScan, FACStrack or FACScalibur instruments at the Geneva University Medical Centre flow cytometry facility. GFP was detected in cells detached and resuspended in FACS buffer comprising 1 % BSA in PBS supplemented with 0.05 % sodium azide. For the detection of intracellular PAI-2, a method adopted from Dr. M. Ranson  was used. Cells were detached and fixed in 0.25 % paraformaldehyde (PAF)/PBS for one hour on ice. Cells were permeabilised in 0.1 % saponin/PBS for 30 minutes at room temperature. Fixed, permeabilised cells were incubated for 30 minutes in PBS/0.5 % BSA/0.1 % saponin containing 2 μg/ml 3750 anti-PAI-2 monoclonal antibody, washed twice in PBS/0.1 % saponin and incubated for 30 minutes in 0.1 % saponin/0.5 % BSA/PBS/goat anti-mouse-PE antibodies. Finally, cells were washed twice in 0.1% saponin/PBS, twice in PBS and resuspended in 2.5 % PAF for flow cytometric analysis.Sorting of live GFP positive cells was performed using a FACStar+ instrument (Becton Dickinson).2, 5 mM DTT and 1 mM PMSF for one hour on ice. Lysates were centrifuged at 16000 g for 5–10 minutes to remove nuclei and precipitates. Supernatant protein concentrations were measured using the Bio-Rad protein assay with BSA in lysis buffer as a standard. Cell lysates were separated by reducing SDS-PAGE and transferred to nitrocellulose membranes. Equal total protein lysate was used for each blot, between 2.5 and 10 μg depending on the assay. Membranes were blocked for 1 hour at room temperature in TBS-0.1 % Tween 20/5 % non-fat milk, and probed using antibodies in TBS-0.1 % Tween 20/5 % non-fat milk. The 3750 anti-PAI-2 antibody was used at a concentration of 1 μg/ml.Cells in suspension were lysed in 10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 0.5 % NP-40, 3 mM MgClPhase contrast microscopy was performed using a Zeiss Axiovert 100 M instrument on live cells. Images were collected using a Hamamatsu CCD camera (ORCA-100).To measure PAI-2 functional activity, IS-1 and IS-1 PAI-2 cells were lysed in 1 % NP-40, 150 mM NaCl and 50 mM Tris pH 8.0. Thereafter, 6 μg of cleared total cell lysate was incubated with and without 10 U of low molecular weight urokinase (u-PA). 3 μg total cell lysate samples were then subjected to reducing SDS-PAGE and immunoblotting as for other PAI-2 immunoblots.® reagent (Invitrogen). cDNA was generated using ImpromII® reverse transcriptase (Promega) and random hexamers, according to the manufacturers instructions, typically using 1 μg of total RNA per reaction. Quantitative PCR was performed using an Applied Biosystems Prism 7000 instrument using Applied Biosystems SYBR® green master mix reagent and oligonucleotide pairs to detect hypoxanthine phosphoribosyl transferase (HPRT), PAI-2 and oligoadenylate synthase-1 (OAS1) cDNA. 5' to 3' primer sequences were as follows: HPRT forward TATTGTAATGACCAGTCAACAG, HPRT reverse GGTCCTTTTCACCA GCAAG, PAI-2 forward GGGTCAAGACTCAAACCAAAG, PAI-2 reverse CCTTTGAAGTAGACAGCATTC, OAS1 forward AGGTGGTAAAGGGT GGCTCC and OAS1 reverse ACAACCAGGTCAGCGTCAGAT. Data were analysed using Applied Biosystems Prism software and the ΔCT method. Briefly, target gene expression was normalised to the HPRT endogenous reference gene for each sample. The difference between mean threshold PCR cycle values for target and control genes gave the ΔCT value. This was then calibrated to the control sample in each experiment to give the ΔΔCT value, where the control had a ΔΔCT value of 0. The fold target gene expression, compared to the calibrator value, is given by the formula 2-ΔΔCT. Error bars represent the standard deviation of each target gene value, after evaluating the expression 2-ΔΔCT+s and 2-ΔΔCT-s, where s is the standard deviation of the ΔΔCT value. All reactions were performed in duplicates or triplicates.Total RNA was isolated from cells using RNA preparation kits from Qiagen or TRIZOLmirVana™ Probe Construction and miRNA Detection kits from Ambion (Austin), according to the manufacturers instructions. These kits employ in vitro transcription for radiolabelled probe generation and an RNase protection protocol for detection of small RNA expression, respectively. Briefly, radiolabelled RNA probes incorporating α-32P-UTP (Amersham) were constructed using T7 polymerase-driven transcription templates. Templates were designed to generate RNAs which hybridize to the endogenous Mir-16 miRNA, as a control, or the first 19 nucleotides of the sh325 shRNA (see table Expression of shRNAs was detected using the RF carried out all of the experiments in this study and contributed to its conception, design and description. EKOK conceived the study and participated in its design and description. Both authors approved the final manuscript.


Conservative treatment remains the standard of care for treating mild to moderate carpal tunnel syndrome despite a small number of well-controlled studies and limited objective evidence to support current treatment options. There is an increasing interest in the usefulness of wrist magnetic resonance imaging could play in predicting who will benefit for various treatments.Two hundred patients with mild to moderate symptoms will be recruited over 3 1/2 years from neurological surgery, primary care, electrodiagnostic clinics. We will exclude patients with clinical or electrodiagnostic evidence of denervation or thenar muscle atrophy.We will randomly assign patients to either a well-defined conservative care protocol or surgery. The conservative care treatment will include visits with a hand therapist, exercises, a self-care booklet, work modification/ activity restriction, B6 therapy, ultrasound and possible steroid injections. The surgical care would be left up to the surgeon (endoscopic vs. open) with usual and customary follow-up. All patients will receive a wrist MRI at baseline.Patients will be contacted at 3, 6, 9 and 12 months after randomization to complete the Carpal Tunnel Syndrome Assessment Questionnaire (CTSAQ). In addition, we will compare disability (activity and work days lost) and general well being as measured by the SF-36 version II. We will control for demographics and use psychological measures  as well as EDS and MRI predictors of outcomes.We have designed a randomized controlled trial which will assess the effectiveness of surgery for patients with mild to moderate carpal tunnel syndrome. An important secondary goal is to study the ability of MRI to predict patient outcomes. Carpal tunnel syndrome (CTS) is the most common peripheral nerve entrapment syndrome, with an annual incidence of 50–150 cases/100,000. It is an important cause of workplace morbidity ). TypicaA Cochrane literature review  for randAlthough there is generally a lack of rigorous scientific support for non-surgical treatments for CTS , there iAlthough not commonly used, Ebenbichler  found thDiagnostic criteria for grading CTS severity as "mild" and "moderate" are not well established. Electrodiagnostic studies (EDS) do not correlate well with clinical severity and have not been shown to accurately predict outcomes for patients with mild to moderate CTS -42.We designed a randomized clinical trial to compare surgical release to non-surgical treatment for patients with mild to moderate CTS. We will examine the association between outcome, as measured by symptoms and functional status, and baseline variables such as symptoms, function, occupational risk factors, EDS measures, demographics, signs and symptoms, and prior treatments. Our primary endpoint is at 12 months.In addition, our study will evaluate the ability of high resolution magnetic resonance imaging (MRI) of the wrist to predict who will benefit from surgical release. MRI has the potential to offer new insight into the diagnosis and management of patients with hand and wrist neurological symptoms. Unlike electrodiagnostic studies, MRI directly visualizes the median nerve and can detect abnormalities of both configuration (nerve compression) as well as signal  -46. EithFinally, we will gather utilization data for each arm of the study to test the hypothesis that the incremental cost-effectiveness ratio of surgery falls below conventional cost-effectiveness thresholds.We have designed a multi-center, randomized trial comparing surgical release to a multi-component, non-surgical therapy. The study protocol was approved by the University of Washington Human Subjects Division and all participants provide written informed consent.The two major goals of this study are to determine: 1) if surgery compared with conservative therapy benefits patients with mild or moderate carpal tunnel syndrome, and, 2) if high resolution magnetic resonance imaging (MRI) of the median nerve can identify patients for whom early surgery might be more efficacious than conservative therapy.Patients with mild to moderate CTS are recruited from six participating sites in the Puget Sound region of Washington State: the University of Washington (UW) affiliated practice sites , Virginia Mason Medical Center (Seattle), the Seattle Hand Surgery , Proliance Surgeons , and Management Services Organization of Washington in Tacoma. We recruit patients within the primary care clinics as well as the specialty referral clinics that treat patients with carpal tunnel syndrome. These clinics include neurological surgery, neurology, orthopedic surgery, and physical medicine and rehabilitation. Additionally, we identify potential subjects in the electrophysiology laboratories at each of our participating sites.Prior to study participation, patients are required to have a physician confirmation of suspected CTS and to obtain an EDS with or without an electromyeogram (EMG). We define mild to moderate carpal tunnel syndrome based on electrodiagnostic studies (EDS) and clinical findings. Specifically, patients are eligible for the study if EDS demonstrates any one of the following: (1) wrist median motor nerve conduction latency greater than or equal to > = 4.4 milliseconds (ms), (2) a 10 cm (thumb to wrist) median to radial sensory nerve ratio difference of > 0.5 ms, (3) an 8 cm mid-palm median to ulnar sensory nerve difference > 0.3 ms, (4) a 14 cm (digit four to wrist) median to ulnar difference of > 0.4 ms, or (5) a combined sensory index ,48 ≥ 1.0Other study inclusion criteria are: age 18 years or older, no previous hand or wrist surgery on the study hand, no previous carpal tunnel release on the contralateral hand in the previous 6 months, symptoms in at least two digits in a median motor nerve pattern, able and willing to answer research questionnaires in English, and classification using the hand diagrams as at least "possible CTS",50. ExclPatients with evidence of severe CTS on EDS, EMG, or clinical findings are excluded from the study. Severe CTS is defined as a median motor amplitude of ≤ 3.8 mV, EMG evidence of denervation, or thenar atrophy.Study participants are required to have not improved after a minimum of two weeks of standard non-surgical treatment  and be willing to schedule surgery within one week if randomized to the surgical arm of the trial. Lack of improvement with conservative care is defined by any of the following: (1) < 0.75 point improvement  on the CFor patients with bilateral CTS, we designate a "study hand" based on the following priorities: 1) most severe according to patient reporting, 2) most severe based on electrodiagnostic reports, and 3) the dominant hand.We use a 50/50 computer-generated block randomization, stratified by enrollment site. The block size is randomly varied between 4 and 12 to reduce the potential for clinicians or research staff to predict treatment allocation. The treatment assignment is centrally administered and concealed in consecutively numbered opaque envelopes.Patients are randomized to receive either a surgical release of the median nerve or a package of multiple, non-surgical treatments tailored for individual patients. For those randomized to surgery, we attempt to schedule the surgery within two weeks of allocation or as soon as possible. Surgery is performed by a board-certified neurological surgeon or orthopedic surgeon. Either open or endoscopic surgery can be performed, depending on the surgeon's preference. Surgical patients receive clinical follow-up just as they would if not in the study. Typically, this includes a follow-up visit within one week for wound and suture management and several post-operative follow-up visits with a hand therapist to perform median nerve and tendon gliding exercises.We created expert focus groups and reviewed the relevant body of literature to develop the non-surgical treatment arm of study. Our focus group included experts in orthopedics, neurosurgery, physical medicine and rehabilitation, hand therapy, biostatistics, health services, behavioral science, and health economics.The non-surgical treatment is directed by a hand, occupational, or physical therapist and includes a minimum of three visits, separated by 6 weeks each. At the first visit, each patient receives an educational booklet, a prescription for NSAIDs (if they have not previously tried them), and specific exercises for their hand. The educational booklet details the hand exercises, describes the causes of carpal tunnel syndrome, and lists resources for obtaining additional information. Work and activity modifications are prescribed at the discretion of the hand therapist and additional hand therapy visits are prescribed as needed. Patients allocated to the non-surgical arm who have already undergone extensive physical therapy can opt to get ultrasound treatment immediately (see below).2, in pulsed mode 1:4, and 15-minutes each. Three months after randomization, patients return for the final non-surgical evaluation. If improved, they are instructed in a self-care maintenance program and advised to return to their provider if their symptoms worsen. If symptoms are not improved, patients are referred to a study surgeon for evaluation for crossover to receive surgery or corticosteroid injection.Patients in the non-surgical arm return six weeks after the randomization for a study visit. If a patient reports improvement, no changes to their therapy are made. For patients who do not improve, we offer oral vitamin B-6 supplements (100 mg per day) and ultrasound in addition to the existing therapy regimen. The ultrasound regimen used in this study consists of up to 12 sessions per week (for 6 weeks) of focused ultrasound at 1 Mhz, 1.0 W/sqr cmOutcomes measures are collected for patients in person at baseline and via telephone interviews at 3, 6, 9, and 12 months after randomization. Although it is impossible to blind providers and patients to the treatment the assignment, the telephone interviewer is blinded to study participants' treatment assignment.Carpal Tunnel Syndrome Assessment Questionnaire (CTSAQ) is the primary outcome measure. The CTSAQ is a self-report CTS functional status and symptom severity questionnaire with established validity, reliability and responsiveness [The functional status scale of the siveness ,53. The SF-36 Health Survey version 2  has been used to assess general health status in samples of patients with a variety of diseases, including CTS [The ding CTS . It consSymptom Check List SCL-90 12-item Somatization and 13-item Depression scales [Study participants also complete the n scales -57. PartPain Catastrophizing Scale is used as both a predictor and a secondary outcome. A substantial volume of research had consistently found substantial associations between pain-related catastrophizing and pain-related disability [The 13-item sability -62. We aIn addition to the outcome measures, we obtain information on several other variables at baseline. This includes information on demographics, occupational risk factors, medical co-morbidity, hand pain history, work status, and litigation/compensation issues. The first three items of the Alcohol Use Disorders Identification Test (AUDIT) are administered to assess the frequency of alcohol use, amount of alcohol consumption, and binge drinking -65.resource use questionnaire (RUQ) at 3, 6, 9, and 12 months post-enrollment.We are also collecting information on the costs of care following enrollment. We aim to estimate the cost to health purchasers and society based on billing and medical records, and a detailed We attempted to either measure or abstract from the medical records information that is generally included in the hand physical examination at the time of enrollment. The hand physical included measurements of patient height, weight, dominant hand, 2-point discrimination (measured at digits #2 and #5), wrist width and thickness (for MRI correlation), 2-point pinch strength (average of three efforts), Semmes-Weinstein monofilament test (from 1.65 to 6.65), Tinel sign, Phalen sign (held greater than 1 minute), and flick test. Not every site routinely completed all sensibility and/or strength tests.Mackinnon-Dellon disk-criminators™ are used to test the static two-point discrimination at the second and fifth digits as a measure of sensibility to correlate to the MRI findings. We tested each digit a minimum of three times and until the tester was confident that a clear endpoint was reached. The test was performed on both hands even in patients with unilateral disease. The disk prongs are held perpendicular to the long axis of the finger. The prongs are placed upon the skin only with sufficient pressure for a patient to determine that he is being stimulated. The three-point pinch strength is tested bilaterally, with the non-study hand being first. We recorded the mean of three serial efforts of the key pinch measurement  using a B&L Engineered  30 lbs. pinch gauge calibrated to +/- 1% accuracy.Tinel sign is positive if tapping over the median nerve at the distal wrist crease for approximately 10 seconds reproduces the pain, numbness and or tingling in the patients hand or wrist. Phalen sign is positive if while holding both wrists flexed at 90 degrees with the dorsum of the hands in opposition to each other for a minimum of 1 minute produces dysesthesias and pain. A flick test is positive if in their response to being asked "What do you actually do with your hand(s) when your symptoms are at their worst?" a patients gestures by flicking the wrist(s) -74.All subjects who enter the study undergo wrist MRI except for subjects who have MRI contraindications , are claustrophobic, who exceed the weight limit of the MRI table, who have scheduling difficulties, or refuse the MRI. We use phased-array surface coils to obtain high resolution images of the carpal tunnel and median nerve. The imaging protocol consists of a fast T1-weighted gradient echo coronal localizer, an axial T1-weighted series and a short-tau inversion recovery (STIR) T2-weighted series. This protocol has established reliability and, because of the short imaging times, patients usually are in the MR scanner for only 15–20 minutes. Imaging parameters for the axial series are listed in table The MRI is interpreted by a neuroradiologist without knowledge of the demographic data, clinical findings, or electrodiagnostic study findings. The key imaging variables are the degree and length of signal abnormality within the median nerve, flattening or swelling of the median nerve, although other qualitative and quantitative measurements will be made as well table .Patients receive a follow-up interview via telephone at 3, 6, 9 and 12 months. Hand specific symptom and functional disability scores, along with general health and psychological instruments are collected. For each interview, we attempt to contact participants a minimum of three times, varying the day and time of the call. In instances where we are unable to reach a person by phone, the survey instruments are mailed along with a postage paid return envelope. Some patients also consent to allow us to contact them for follow-up using e-mail.All data are collected onto hardcopies and entered into an MS Access database using a web-based data entry system that requires double entry of data to reduce errors in transcribing. Error reporting to identify out of range answers, inconsistent replies and compliance monitoring is routinely performed.We developed stopping rules using CTSAQ symptom scores, disability reporting, adverse event rates and rates of "red flag" answers to a question asking about thoughts of suicide on the SCL-90. A safety officer monitors these variables for group differences, and also monitors response variables, missing data, and protocol compliance.A formal evaluation of efficacy will be conducted after 100 patients have been randomized to test for group differences in the CTSAQ symptom severity score. We adopted O'Brien-Fleming boundaries for discontinuation, and therefore maintain an overall type I error rate of 5%. Additional stopping criteria include a suicidal ideation rate difference of 10%, a difference in the change from baseline rate of functional disability  of 20%, or a difference in the number of days lost from work of 20%. The nature and severity of the adverse events will also be considered individually by the safety officer. Finally, individual item response rates on all answered questionnaires with less than 20% being incomplete, failure to collect greater than 75% at three-month follow-up, or enrollment rates below the expected rate by more than 25% provided grounds for the safety officer to recommend changes or stop the study.In the primary analysis the CTSAQ functional score at 12 months will be compared between the surgical and non-surgical treatment arms, using conventional t-tests and ANCOVA techniques to adjust for baseline values. The analysis will be conducted on an intention-to-treat basis.Secondary analyses will include a comparison of secondary outcome measures  for the two treatment arms. Adjusted analyses of CTSAQ functional status and secondary outcomes at 1 year will be conducted using linear regression methods. To characterize the time evolution in the primary and secondary outcomes we will use linear mixed models  to analyze the repeated measures obtained at all follow-up visits. Finally, we will use exploratory regression methods to determine if baseline disability, psychological factors, MRI variables, and electrodiagnostic measures correlate with clinical outcomes.We will use aggregate results of patient outcomes on the CTSAQ and SF-36 scores to perform a descriptive analysis, uncontrolled for other baseline factors. We will explore factors that predict improvement in CTSAQ  and general health scores as measured by the SF-36. Specific analysis and tests used will depend on the distribution of the tested values. We will also test the independent associations of the various mental health domains, the work-related risk factors, and physical findings to changes in the CTSAQ. Finally, we will use linear regression to test relevant associations while controlling for baseline demographics, co-morbidity and other important variables to identify factors that may predict improved outcomes at 1 year.As important sub-analyses we will also determine the reliability of quantitative MR median nerve measurements, determine the correlation of symptoms and function in patients with these quantitative MR measurements as well as with electrodiagnostic studies (EDS), and construct a cost-effectiveness model to test the hypothesis that MRI is an efficient method for selecting patients with mild or moderate CTS who are likely to benefit from surgery.The prospective cohort study by Katz et al. provided data on outcome differences between surgical and non-surgical patients . They alIf we consider the difference in means observed for the pilot data (m1-m0 = 0.554) then 48+48 = 96 subjects are adequate for 80% power and a total of 64+64 = 128 are required for 90% power (refer to following table). However, since we are recruiting patients with less severe disease, we would expect smaller improvements in the scores from baseline to follow-up and hence, smaller effect sizes. Thus, it is reasonable to power the study to detect a difference in means of 0.4 rather than 0.5. Since our primary analysis will use the more efficient ANCOVA, these sample size estimates based on observed change scores (post-pre) are slightly conservative. This should allow for a small percentage of subjects lost to follow-up. Similar power calculations were conducted to assess the power to detect an impact on CTSAQ symptom scores. The pilot data suggest a 0.67 point greater reduction in symptoms for the surgical patients, and variance estimates lead to sample sizes of 30+30 = 60 total patients, and 40+40 = 80 total patients to detect 0.67 point difference  that patients with mild or moderate CTS benefit from surgery more than conservative therapy, and; 2) that high-resolution wrist MRI accurately identifies which patients with mild to moderate symptoms are more likely to benefit from surgery. We expect our findings to be more generalizable to the primary care clinicians and include milder cases of CTS than Gerritsens' study. No previous study has sufficiently considered conservative treatment options of vitamin B6, ultrasound, anti-inflammatory drugs, and hand exercises in combination versus surgical treatment. Furthermore, our study offers the unique use of wrist MRI data to establish the diagnostic and predictive understanding of carpal tunnel syndrome.The pre-publication history for this paper can be accessed here:


Few studies report on the effect of organizational factors facilitating transfer between primary and tertiary care hospitals either within an integrated health care system or outside it. In this paper, we report on the relationship between degree of clinical integration of cardiology services and transfer rates of acute coronary syndrome (ACS) patients from primary to tertiary hospitals within and outside the Veterans Health Administration (VHA) system.Prospective cohort study. Transfer rates were obtained for all patients with ACS diagnoses admitted to 12 primary VHA hospitals between 1998 and 1999. Binary variables measuring clinical integration were constructed for each primary VHA hospital reflecting: presence of on-site VHA cardiologist; referral coordinator at the associated tertiary VHA hospital; and/or referral coordinator at the primary VHA hospital. We assessed the association between the integration variables and overall transfer from primary to tertiary hospitals, using random effects logistic regression, controlling for clustering at two levels and adjusting for patient characteristics.Three of twelve hospitals had a VHA cardiologist on site, six had a referral coordinator at the tertiary VHA hospital, and four had a referral coordinator at the primary hospital. Presence of a VHA staff cardiologist on site and a referral coordinator at the tertiary VHA hospital decreased the likelihood of any transfer . Conversely, having a referral coordinator at the primary VHA hospital increased the likelihood of transfer .Elements of clinical integration are associated with transfer, an important process in the care of ACS patients. In promoting optimal patient care, clinical integration factors should be considered in addition to patient characteristics. Coronary artery disease is the leading cause of death among Americans . HospitaThe coordination and integration between primary and tertiary hospitals has important implications for integrated health care delivery systems. The Veterans Health Administration (VHA) is one of the largest vertically integrated health care delivery systems in the United States . The VHATreatment guidelines for acute coronary syndromes -10 suggeThe objective of this study was to evaluate the association between structural components of clinical integration and patient transfer rates from VHA primary hospitals to tertiary hospitals, both within and outside the VHA system for patients with ACS. We hypothesized that primary VHA hospitals with structural components of clinical integration present would have a higher rate of within-system transfer of ACS patients than primary VHA hospitals lacking these components.The VHA Access to Cardiology study was a prospective cohort study of 2,733 patients with a primary discharge diagnosis of either acute myocardial infarction (ICD9-CM 410.xx) or unstable angina (ICD9-CM 411.xx) discharged over a one year period  from 24 VHA hospitals in five regions, including Minnesota and the Dakotas, the Southwest, the Rocky Mountains, the Pacific Northwest, and Southern California. Patient demographics, clinical characteristics, and specific processes of care including hospital transfer were obtained as part of the Access to Cardiology Study.All patients admitted to one of the 12 primary VHA hospitals in the study were eligible for this analysis . The remaining 12 VHA Medical Centers were tertiary hospitals with cardiology services and cardiac catheterization laboratories on site. These were not the focus of the analysis reported in this paper. We excluded 107 patients because they were initially admitted to a private hospital and transferred into a primary VHA hospital. In addition, we excluded 3 patients who were transferred from one primary VHA hospital to another. Finally, 27 patients had missing data in the variable indicating prior history of congestive heart failure, which was included in the final analysis. As a result, a total of 725 patients from 12 primary VHA hospitals were included in these analyses. The study protocol was approved by the Human Subjects Committee at the University of Washington, and by Institutional Review Boards and Research and Development Committees at each participating VHA hospital.Patient transfer from a primary VHA hospital to a tertiary hospital (either VHA or private) was the primary outcome for this study. Secondary outcomes included both transfer from a primary VHA hospital to a tertiary VHA hospital, and transfer from a primary VHA hospital to a private (non-VHA) tertiary hospital.Transfers to a tertiary VHA hospital were considered transfers within the system, while transfers to a private hospital were considered transfers outside the system. Transfer data were available for all 725 patients in the study cohort. We constructed two binary variables for the analyses: transfer to any tertiary care hospital (yes/no), and transfer to a tertiary VHA hospital versus transfer to a private (non-VHA) hospital.The key independent variable for this study was clinical integration of cardiac services. We defined clinical integration ,13 as thThe data used to construct these measures came from on-site interviews conducted with Chiefs of Cardiology at each of the tertiary VHA hospitals associated with the primary VHA hospitals included in this study. During on-site interviews, Chiefs of Cardiology were asked to describe all of the primary VHA hospitals that refer ACS patients to them on a regular basis, and to identify the presence or absence of each of the structural elements of clinical integration. Interviews followed a structured protocol, ensuring uniform data collection. In all cases, the Chiefs of Cardiology were able to provide detailed information about the services available at both the tertiary and primary VHA hospitals. We also asked the Chief of Cardiology about the degree of competitiveness for cardiac services in the local markets for each of the primary VHA hospitals. This was an ordinal variable, with three levels: non-competitive; moderately competitive; or highly competitive market. In all cases, the Chief of Cardiology was able to answer the questions about market competition in the primary hospital market without difficulty, indicating considerable awareness of market conditions and the impact these had on their referral base.In addition, we constructed two separate variables to control for patient distance from the primary VHA hospital to which they were initially admitted, and to control for the distance between primary and tertiary VHA hospitals. The patient distance variable was measured as the distance from the patient's home zip code centroid to the primary VHA hospital. The distance between the primary and tertiary referral VHA hospitals was measured in miles using VHA national databases. We tested different specifications of the distance variables, concluding that it was best to enter the distance between primary and tertiary VHA hospital as a continuous variable, whereas it made no difference in the results of the estimation what form we used for patient distance to primary VHA hospital. In the final analyses, it was dichotomized at greater than or equal to 100 miles – approximately two hours driving time. The patient distance variable is measured at the patient level, while the hospital distance variable is measured at the hospital level.We included several measures of patient clinical characteristics, including age 65 or over; prior history of chronic obstructive pulmonary disease, bleeding disorder (such as hemophilia or anticoagulation therapy), smoking, prior percutaneous coronary intervention (PCI), or chronic heart failure; having a "Do Not Resuscitate" order, and several measures of seriousness or urgency of condition during the index admission in the primary VHA hospital: ST segment elevation on electrocardiogram or elevated cardiac enzymes at presentation; and a composite variable indicating the presence of a serious event during admission. Presence of a serious event during admission was a binary variable taking the value "1" if at least one of the following conditions was present: angina persisting more than 24 hours after admission; hypotensive episode; heart failure during admission; cardiac arrest; or positive stress test during admission. All of these variables were abstracted from the medical record.We explored the bivariate association between clinical integration variables, distance variables, patient characteristic variables, and patient transfer using one-way analysis of variance with Scheffe correction for multiple comparisons.To construct the most parsimonious models using the full set of candidate independent variables , we used backward stepwise logistic regression, beginning with all available patient clinical characteristics that have been shown to be significant in predicting mortality outcomes for ACS patients in prior studies. We eliminated variables from the model if the p-value for the variable was greater than 0.1. A number of the candidate variables, including many of the history and co-morbidity variables, were found to be insignificant, and we created a summary variable described above which included many of the highly significant variables from the index hospital admission (details available from authors). C-statistics for each of the final models ranged from 0.77 to 0.85. We used Stata SE version 8.2 for all analyses.We then investigated the relationship between clinical integration of cardiac services and transfer rates using random effects logistic regression , correctAmong the 12 primary VHA hospitals included in the sample, the mean rate of transfer was 42% (319 of 725). Mean rate of transfer to a tertiary VHA hospital was 31% (237 of 725), and to a private hospital was 11% (82 of 725). Most patients were transferred in order to receive cardiac catheterization or coronary revascularization. In addition, 37% of patients were treated in primary VHA hospitals that were over 250 miles from their tertiary referral VHA hospital, and 18% of patients lived over 100 miles from the primary VHA hospital to which they were admitted.Three of the 12 primary care VHA hospitals had a VHA cardiologist available at least episodically on site; six had a referral coordinator at the associated tertiary center; and four had a referral coordinator at the primary VHA hospital. The distribution of these components is shown in Figure The bivariate associations between the patient characteristic variables, clinical integration variables, and type of transfer are shown in Table Results of the random effects logistic regressions for transfer to any tertiary care hospital are shown in Table Patient factors that decreased the likelihood of transfer to any tertiary hospital included history of chronic obstructive pulmonary disease, or bleeding disorder; and having a do not resuscitate (DNR) order during the hospital admission. In addition, the further the distance between primary and tertiary VHA, the less likely patients were to be transferred at all, and the more competitive the market for cardiac care, the less likely that the patient was transferred to a tertiary care hospital.All three components of integration were significantly associated with transfer to tertiary care, although in different directions. After adjustment for patient and other characteristics, the presence of a VHA staff cardiologist and having a referral coordinator at the tertiary VHA hospital decreased the likelihood of transfer to any tertiary care hospital. In contrast, the presence of a referral coordinator at the primary VHA hospital increased the probability of transfer to a tertiary hospital.The results of this analysis are shown in Table The degree of market competition was not significantly associated with transfer to VHA versus private tertiary hospital. Neither of the distance variables were associated with transfer either to VHA or non-VHA tertiary hospitals. Furthermore, only one of the individual integration variables entered separately were significantly associated with likelihood of transfer to tertiary VHA versus private hospital, and although the parameter estimate for the variable indicating presence of a referral coordinator at the tertiary hospital was large and significant, it was very imprecise (i.e. large standard error). This is probably due to the relatively small number of patients included in the estimation (N = 319) and uneven splits among hospitals, clustered by VISN.The goal of this study was to investigate the association between measures of clinical integration of care and transfer of patients with acute coronary syndromes in the VHA. In particular, we evaluated whether structural components of clinical integration, such as the presence of referral coordinators and on-site cardiologists, were associated with patient transfer within and/or outside of the VHA healthcare system. In multivariate analysis, the presence of referral coordinators located at primary care VHA hospitals increased the overall likelihood of transfer of ACS patients. In contrast, having a VHA staff cardiologist available or a referral coordinator at a tertiary VHA hospital significantly decreased the likelihood of any transfer to a tertiary care hospital. Finally, we found that only one of the three integration components, presence of a referral coordinator at the tertiary VHA hospital, was significantly associated with transfer to a tertiary VHA hospital compared to a non-VHA tertiary hospital.Our finding that referral coordinators at primary care hospitals increase the likelihood of transfer to tertiary care hospitals is consistent with prior studies demonstrating that referral coordinators increase the ease of referral and frequency of transfer ,15-19. PHowever, our finding that the presence of a referral coordinator at a tertiary VHA hospital was negatively associated with transfer appears contradictory. It is possible that referral coordinators at the tertiary centers may facilitate consultation, which may, at least for lower risk patients, appropriately reduce the need for transfer. However, it is of some concern that these referral coordinators may be serving in a gatekeeper role with regard to transfer decisions. Future research should focus on the role and decision-making associated with these referral coordinators. Of note, when transfer did occur, the presence of a referral coordinator at the tertiary VHA hospital was positively associated with transfer to VHA facilities rather than non-VHA facilities. This suggests that referral coordinators may function differently with different kinds of patients, decreasing overall transfer rates but facilitating within-system transfer when transfer occurred.In general, we found that transfers to tertiary care were largely associated with patient characteristics appropriate to transfer: sicker and more urgent patients, except for those for whom more intensive care may not be indicated (e.g. DNR status), were significantly more likely to be transferred. In particular, patients with ST-segment elevation on their presenting electrocardiogram and abnormal cardiac enzymes were significantly more likely to be transferred, most likely for coronary revascularization. These patients are most likely to benefit from revascularization ,9, and tDistance between the patient's home and primary VHA hospital was significantly associated with increased likelihood of subsequent transfer to a tertiary care hospital. This may indicate that patients who live further from the hospital take longer to present and are therefore sicker on arrival, leading to the requirement for higher levels of care. Also of interest, distance between primary and tertiary VHA hospitals was significantly associated with a decreased likelihood of transfer, indicating that in situations where primary and tertiary VHA hospitals are further apart, primary VHA hospitals may elect to keep more ACS patients rather than transfer them at all. Future research is needed on the appropriateness of transfer of ACS patients, as it is not clear that variation in transfer based on distance between hospitals represents appropriate variation in care.The finding that cardiologist availability at the primary VHA hospitals was associated with less transfer to tertiary care hospitals may reflect that local or distant cardiology consultation was sufficient in some cases (e.g. lower risk patients) to avoid transfer. Similarly, the availability of a transfer coordinator at the tertiary VHA hospital may have provided an avenue for consultation and avoidance of transfer in some cases. Future studies are needed to define the mechanisms of association between reduced transfers and both on-site cardiology availability and tertiary hospital transfer coordinators.The findings of this study, that referral coordination is associated with transfer from primary to tertiary hospitals, but may operate differently for different types of patients, and may have one mechanism of operation within a health care system and another outside that system, have potential application outside VHA. Previous studies  have fouFirst, we were not able to conduct full-scale validation and reliability testing of the clinical integration measures, which would have required a larger sample of hospitals participating in the study to conduct split-sample validation. Second, we used structural, rather than process, elements of integration in this analysis. We focus on structural elements both because they are relatively easier to measure (present or not), and because in Donabedian's widely accepted model of quality in health care, structure precedes process and outcome ,23. ThirFourth, because transfer is closely related to patient outcomes, especially for ACS patients , carefulWe found that referral coordinators located at primary care VHA hospitals increase the overall likelihood of transfer of ACS patients. Referral coordinators at tertiary VHA hospitals and the presence of on-site cardiologists appeared to decrease the likelihood of transfer. Only one component of integration, presence of a referral coordinator at the tertiary hospital, was associated with within-system compared to out-of-system transfer. These findings have significant potential implications for the VHA. One of the goals of an integrated health care system is to maintain optimal coordination between its component parts . This stThe author(s) declare that they have no competing interests.AES participated in the design and conduct of the study, conducted the analyses and wrote the manuscript. SLP participated in conducting the project, and assisted in writing the manuscript. DJM participated in writing the manuscript. NRE participated in the design and conduct of the study. NDS participated in writing the manuscript. JSR participated in the statistical analyses and co-wrote the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


To determine the frequency, risk factors and mortality of nosocomial pneumonia a prospective study was conducted in the intensive care units. In the study period, 2402 patients were included. The nosocomial pneumonia was defined according to the Centers for Disease Control Criteria. Overall, 163 (6.8%) of the patients developed nosocomial pneumonia and 75.5% (n = 123) of all patients with nosocomial pneumonia were ventilator-associated pneumonia. 163 patients who were admitted to the intensive care unit during the same period but had no bacteriologic or histologic evidence of pneumonia were used as a control group. The APACHE II score, coma, hypoalbuminemia, mechanical ventilation, tracheotomy, presence of nasogastric tube were found as independent risk factors. Crude and attributable mortality were 65% and 52.6%, respectively. The mortality rate was five times greater in the cases . The mean length of stay in the intensive care unit and hospital in the cases were longer than controls (p < 0.0001). Patients requiring mechanical ventilation have a high frequency of nosocomial pneumonia. Pseudomonas aeruginosa, Acinetobacter spp., Stenotrophomonas maltophilia) are associated with higher mortality [Nosocomial pneumonia (NP) is the most frequent nosocomial infection in the intensive care units (ICU). The reported frequency varies with the definition, the type of hospital or ICU, the population of patients, and the type of rate calculated. In the recent studies, the incidence was reported as 6.8–27% -4. In anortality ,11,12. Portality .Studies on NP are mainly reported from the United States and European countries, whereas studies from around the world are missing. The aims of this study were to assess incidence, risk factors and mortality of NP in Eurasian intensive care units.2) blocker therapy, clinical outcome, length of stay in ICU and in the hospital.Between February 2001 and February 2002 a prospective study was conducted among intensive care units (ICU) patients of the Erciyes University Hospital. This university hospital is a teaching hospital and full time intensivists care the patients in ICUs. Patients from the surgical ICU (SICU) (24 beds), medical ICU (MICU) (9 beds) and burn unit (7 beds) were included. The SICU consist of 8 neurosurgical (NICU), 8 general surgery (GICU) and 8 cardiac surgery (CICU) beds. Patients older than 16 yr of age were included. The same infection control doctor collected data and intensivist reviewed the diagnosis of pneumonia. Data collection included physical examination findings, APACHE II scores on admission, consciousness, risk factors , prior surgery, immunosuppression, prior antimicrobial and antacid or histamine type 2  purulent endotracheal secretions with a Gram stain showing one or more types of bacteria [NP was considered when new and persistent (more than 48 h) pulmonary infiltrates not otherwise explained appeared on chest radiographs. Moreover, at least two of the following criteria were also required: 1) fever >38°C; 2) peripheral leukocyte count >10 000/mmbacteria . VAP wasbacteria . AdmissiExtra length of stay was calculated comparing the extra stay after onset of pneumonia in the cases and after a reference date  in the control group.Giemsa stains of sputum samples were performed for all patients. Sputum samples, containing more than 25 polimorphonuclear leukocyte (pnl) and less than 10 (×100) epithel were classified as purulent. If necessary, samples were obtained by nasotracheal aspiration. In that case, samples containing more than 10 pnl (×1000) were defined as purulent. Quantitative cultures of all purulent samples were performed using standard methods. Susceptibility testing was performed by disc diffusion method. In the absence of an alternative diagnosis a bronchoalveolar lavage was performed. In some case, pleural fluid was obtained by thoracentesis and examined for cell count, smear, Gram- and Giemsa-staining and microbiological culture.All data were evaluated using SPSS. Parameters were compared using univariate and multivariate logistic regression and chi-square tests. Student t test was used to compare the extra length of stay. Data were given as mean ± SD and a p-value of <0.05 was accepted as significant.2 = 19.7, p < 0.0001). Length of stay in the MICU was significantly higher than in the SICU, 21.3 ± 21.4 versus 16.2 ± 8.8 days, respectively (p < 0.05). Characteristics of patients who developed NP are shown in table During the study period, 2402 patients were admitted to the ICUs. Distribution of patients by ICU and length of stay in the ICU are shown in table 2 = 6.6, p < 0.01). Consequently, the incidence of VAP was higher for MICU- than SICU-patients . Furthermore, the length of ventilation was higher for patients admitted to MICU (6.3 ± 4.0) than SICU (5.1 ± 3.7), but the difference was not statistically significant.Overall, 17% of all patients requiring MV (n-724) developed VAP. Thereby, VAP accounts for 75.5% (n = 123) of all patients with NP (n = 163) during the study period  Table . MechaniDuring the study period patients received 3128 ventilation days, with an average duration of 11.3 ± 10.0 days per ventilated patient. The device-related incidence rate for VAP was 39.3/1000 ventilation days. The incidence per 1000 ventilation days was 41.9 in SICU, 36.6 in MICU, 66.0 in NICU, 38.0 in GICU, and 9.1 in CICU patients. The mean onset day of NP after MV was 4.2 ± 3.9 days.Univariate analysis suggested the following risk factors for the development of NP: the APACHE II score, coma, COPD, uremia, hypoalbuminemia, MV, tracheotomy, enteral feeding, presence of nasogastric tube and previous treatment with broad-spectrum antibiotic (Table A. baumannii (29.6%), P. aeruginosa (20.6%), Klebsiella pneumoniae (14.4%) were the most common.187 pneumonia episodes were observed during the study period, resulting in the isolation of 257 microorganisms. The most commonly isolated pathogens were Gram-negative bacteria (85.6%). Among these pathogens, Empiric antibiotic therapy was based on previous surveillance cultures and the Gram stain results. Therapy was adjusted according to the reports of susceptibility testing.Crude and attributable mortality were 65% and 52.6%, respectively. The mortality in patients without NP was 26.4% Table . The risThe mean length of stay in the ICU and hospital for the patients with NP were 18.04 ± 14.74 days and 23.49 ± 16.44 days, respectively. The mean length of stay in the ICU and hospital for the control group 3.10 ± 3.03 and 9.64 ± 5.08 days, respectively. This difference was statistically significant (p < 0.0001). The extra stay in the control group was 4.36 ± 3.87 and 17.04 ± 14.17 in the patients (p < 0.001).The incidence of NP was reported different in different studies, which may be justified by the presence of different populations with variable ages, underlying diseases, and other associated risk factors. Incidence ranges from 6.8 to 27% -4 and alMV increases the risk of NP by 3- to 10-fold -23, resuComa was described as another important risk factor for NP. In these patients, local defense mechanisms of the respiratory airway are altered, allowing microorganisms to better attach to and colonize the mucosal surface. Furthermore, depression of the level of consciousness significantly increases the chance of aspiration, and as a result development of NP ,28. In o2 antagonists did not increase the risk of NP.The causative agents of NP differ by the study population and diagnostic techniques but generally Gram-negative bacteria are the most common ones ,4,28-33.In the literature, tracheotomy is described as a significant risk factor for NP. Bronchial colonization during the procedure and (prolonged) continuation of sedation after the procedure will furthermore increase the occurrence of NP , a fact The role of advanced age and high APACHE II scores as risk factors of NP are still under discussion. While Kollef et al.  report tPatients with NP have a significantly higher morbidity and mortality ,35,38. HAs a result of the increased morbidity, patients with VAP remain hospitalized for 4–17 days longer than controls -38. ThisIn conclusion, NP is a major cause of morbidity and mortality in ICU patients. Especially patients on mechanical ventilation are at high risk. Studies determining the impact of "old" and "new" risk factors of NP should repeatedly be performed in order to effectively guide the implementation of preventive measures methods.


Defending against attack is one of the most important challenges facing any organism. But while sticks and stones may break the bones of a lion, microscopic threats such as bacteria require different weapons. And it's not just we humans who have this problem—insects are prey to bacterial infections too. Their immune systems, however, rely on a far simpler set of defenses than those found in mammals. Exactly how one insect immune system recognizes bacteria, and how it fights off the invader, is the subject of a new study in this issue by Johann Deisenhofer and colleagues.Drosophila, has long been known to use a set of molecular sentries called “peptidoglycan recognition proteins,” or PGRPs, that circulate in the fly's bloodstream. When a PGRP recognizes a bacterial invader, it triggers a cascade of events whose ultimate product is a group of antimicrobial compounds that attack and kill the bacteria.The fruitfly, While the family of PGRPs has been extensively studied, exactly how they recognize their target bacteria has been less clear. At the cellular level, recognition requires contact, and the part of the bacterium the PGRP recognizes is, as its name implies, the peptidoglycan. A peptidoglycan is a special sort of molecular polymer found primarily on bacterial cell walls. Peptidoglycan forms when chains of sugar molecules (the glycans) are cross-linked by amino acids (the peptides) to form a meshwork that helps keep the bacterium from bursting under the osmotic strain of its contents.Drosophila defensive reaction. Deisenhofer and colleagues set out to determine whether this difference in triggering ability of particular peptidoglycans was linked to differences in the PGRPs that recognize them. To do this, they determined the three-dimensional structure of one PGRP, called PGRP-SA. They worked out not only the overall shape of PGRP-SA, but also which amino acids sat where on the convoluted surface of the protein.There are several types of peptidoglycans that differ in their precise sugar and amino acid constituents and in their ability to trigger the What they found on that surface was an extended groove down one entire side of the protein. To test whether this groove was indeed the recognition site for peptidoglycan, the group introduced a series of mutations to critical amino acids along the groove, testing each new form for its ability to bind peptidoglycan. Indeed, the binding and defense-triggering ability was worse for almost every mutant, demonstrating conclusively that the normal protein uses the groove to bind and recognize peptidoglycan.Then the team made a surprising discovery. They found that when PGRP-SA comes in contact with bacterial peptidoglycan, it begins to cleave the links between amino acids in the peptide portion of the peptidoglycan. This in itself is not so amazing—animals make plenty of peptide-cleaving proteins. But this protein has a difference, one which makes it unique in the animal kingdom.To understand this difference, consider your two hands. They are mirror images of each other, alike yet not the same. No amount of twisting and turning will allow you to superimpose one exactly on the other—if you align the fingers, the knuckles will point in opposite directions, and if the knuckles point the same way, the fingers are all mismatched. This type of relationship between mirror images, called chirality (from the Greek for “hand”), is found in amino acids as well, a result of the three-dimensional geometry that radiates from their central atom.Drosophila PGRP-SA was able to break apart this “L,D” (levo-dextro) linkage, making it, in their words, “the first eukaryotic protein exhibiting such an activity specific for peptide bonds existing only in prokaryotes.”All the amino acids used by all known animal species are exclusively of the “left-handed” form, and the protein-digesting enzymes we make are designed specifically for these L-amino acids. Bacteria, however, link left-handed and right-handed amino acids together to form peptidoglycan. What Deisenhofer's team discovered was that unlike any other known animal enzyme, the What does it all mean? Deisenhofer and colleages' results are yet another demonstration that at the molecular level, understanding structure is the key to understanding function. They also show that when it comes to defense, it helps to be able to fight with both hands.