Batrachochytrium dendrobatidis was identified from sick and dead frogs. Since then, several lines of evidence suggest that B. dendrobatidis may be involved in frog declines: the fungus has been found on frogs in afflicted areas; lab studies show that it's highly pathogenic to some frog species; and pathological evidence links it to host mortality. But with little information about the prevalence of this fungal infection in wild frogs, or how the disease impacts frogs in the wild, the causal role of this chytrid fungus remains unclear.Amphibian declines have reached crisis proportions in various parts of the world. In many areas, habitat loss is the likely culprit. But when mass die-offs suddenly occurred in relatively undisturbed habitats, the cause was far less obvious. Fourteen species suffered either extinctions or major declines in the pristine rainforests of Queensland, Australia, between 1979 and 1993. It was suggested in 1996 that some unknown disease had spread through the populations, but no pathogen was discovered until 1998, when the fungus B. dendrobatidis on frogs in their natural habitat, Richard Retallick et al. focused on six species living in the high-elevation rainforest streams of Eungella National Park in Queensland, Australia, where frog losses were “particularly catastrophic.” Two species vanished between 1985 and 1986: the Eungella Gastric-Brooding Frog (Rheobatrachus vitellinus), which is now thought extinct, and the Eungella Torrent Frog (Taudactylus eungellensis), which later reappeared in a few small populations. Other local frog species escaped this period relatively unscathed.To evaluate the effects of B. dendrobatidis had yet to be identified, but Retallick retained the toe tips, and the authors tested the toes for disease in 2002–2003. Fungal infections were found in two species—T. eungellensis and Litoria wilcoxii/jungguy ; the other four species were infection-free. L. wilcoxii/jungguy did not decline to any great extent during the 1985–1986 die-off.Retallick captured frogs from six sites from 1994 to 1998, clipped one or two toe tips from each frog to age and identify them, and then released the frogs back into the wild. At the time, T. eungellensis frogs was greatest at three particular sites, which showed peak infections during cooler months. Prevalence of infection was highest during winter and spring, but did not vary from year to year, suggesting that the infection is now endemic. Fungal infections were found in 27.7% of L. wilcoxii/jungguy frogs, with no evidence that prevalence differed among sites, seasons, or individuals . The probability of recapture was significantly lower for frogs that were already infected when first captured. While this might suggest a correlation between infection and death, it's impossible to distinguish death from simple failure to recapture the animal. On further analysis, McCallum and colleagues found no evidence that survival differed between infected and uninfected frogs, suggesting that this potentially devastating amphibian disease now coexists with the frogs, with little effect on their populations.The proportion of infected T. eungellensis, a rainforest frog listed as endangered, “now persist with stable infections of B. dendrobatidis.” While these findings do not exonerate the fungus as the agent of mass declines, they do rule out the possibility that the fungus caused the decline, then vanished from the area, allowing frog populations to recover. The authors allow that it's possible that B. dendrobatidis did not cause the initial T. eungellensis declines. Or alternately, the fungus could have emerged as a novel pathogen in the ecosystem, causing massive casualties before some form of evolutionary response took hold. Surviving frog populations may have evolved resistance to the pathogen, for example, or less virulent strains of the fungus may have evolved. If it turns out that frog populations can develop resistance to the chytrid fungus, the researchers point out, then a conservation program of captive breeding and selecting for resistance could potentially thwart the extinction of these, and other, critically endangered frogs. A critical next step, then, is to determine whether frogs and fungus do coevolve.These results, the authors conclude, “show unequivocally” that remaining populations of


The purpose of this study was to evaluate the role of study quality assessment of primary studies in cancer practice guidelines.Reliable and valid study quality assessment scales were sought and applied to published reports of trials included in systematic reviews of cancer guidelines. Sensitivity analyses were performed to evaluate the relationship between quality scores and pooled odds ratios (OR) for mortality and need for blood transfusion.Results found that that whether trials were classified as high or low quality depended on the scale used to assess them. Although the results of the sensitivity analyses found some variation in the ORs observed, the confidence intervals (CIs) of the pooled effects from each of the analyses of high quality trials overlapped with the CI of the pooled odds of all trials. Quality score was not predictive of pooled ORs studied here.Had sensitivity analyses based on study quality been conducted prospectively, it is highly unlikely that different conclusions would have been found or that different clinical recommendations would have emerged in the guidelines. Quality assessment of trials included in systematic reviews of evidence is a resource intensive and scientifically controversial endeavour. On the one hand, the routine use of quality assessment in the development of systematic reviews is encouraged by the Evidence-based Practice Center Program of the Agency for Healthcare Research & Quality (AHRQ) and the Cochrane Collaboration, two well respected groups that coordinate a substantial number of systematic reviews -3. IndeeThe concept of incorporating study quality assessment into systematic review methodology has also found empirical support. There is evidence that studies of lower methodological quality tend to report larger treatment effects than high quality studies -7. For eAlthough this seminal work yields compelling results, these findings are not universal and the issue is not without detractors -14. SomeTogether these results suggest the study quality issue is controversial and that the merits of this methodological step in systematic review requires thoughtful analysis. Indeed, West et al conclude with recommendations advocating for research dedicated to comparing quality rating systems and the role of quality assessment within individual clinical contexts and for studies targeted at determining specific quality factors that make a difference in final quality scores .The Practice Guidelines Initiative of the Cancer Care Ontario's Program in Evidence-based Care (PEBC) uses the Guidelines Development Cycle to create cancer practice guidelines comprised of a systematic review of the research literature, an interpretation and consensus of the evidence by members of the guideline development team, clinical recommendations informed by the evidence, and an external review process by Ontario clinicians -19. We f1. What valid and reliable quality assessment instrument would be most appropriate for our context?2. How is study quality currently being used in published systematic reviews of cancer trials and what is the relationship between effect size and study quality in this disease area?3. What impact would study quality assessment have on the clinical recommendations made in evidence-based practice guidelines developed by the PEBC?For a comprehensive review of the strengths and weaknesses of quality assessment instruments, readers are referred to the 2002 West et al. evidence report commissioned by the AHRQ . For ourTo locate systematic reviews on oncology topics, the strategy suggested by Moher et al for finding systematic reviews  was combThe validated scales were applied by two methodologists (MJ and MC) to studies reported in any our practice guidelines that included a pooled analysis based on at least ten randomized trials related to the main guideline question. Intraclass correlation coefficients with 95% confidence intervals (CI) were calculated using a random sample of the RCTs to assess inter-rater reliability, one coefficient calculated for each of the scales used. Because of budgetary limitations for staff time, the analysis was conducted on 18 randomly selected studies rather on the whole group of articles. This is a methodological limitation as fewer studies result in larger confidence intervals and less precise estimates. This may account for the difference in reliability ratings we found for the Sindu scale compared to published norms  based on total quality score. Where the scale developer suggested a cut-off point for low versus high quality, this was used. Where no cut point was specified, the observed median study quality score was used as the dividing point between low and high quality. Meta-analyses were repeated with the high quality studies. Because there would never be a situation in which guideline developers would consider low quality studies only, a meta-analysis using this sample of the studies was not conducted.Four scales meeting our criteria were found; two instruments, Jadad et al  and Cho The literature review located 32 published systematic reviews on oncology-related topics that included some measure of study quality. Five of the reviews examined changes in pooled estimates of effect size of mortality rates when meta-analysis was restricted to high-quality randomized trials -30. As sThree of the PEBC practice guidelines included at least 10 RCTs in their systematic reviews of the evidence and were eligible for inclusion in this evaluation -33: concAt the conclusion of our study, we identified a fourth practice guideline which originally did not meet our 10 RCT inclusion criteria, but later did so after it was updated. The guideline focused on the role of erythropoietin (EPO) in the management of cancer patients with non-hematologic malignancies . Unlike Intraclass correlation coefficients used to established inter-rater reliability were 0.71 for the 3-item Jadad scale (95% CI, 0.38 to 0.88), 0.80 for the 6-item Jadad scale (95% CI, 0.54 to 0.92), 0.62 for the Sindhu scale (95% CI, 0.24 to 0.84), and 0.63 for the Downs & Black Scale (95% CI, 0.25 to 0.84). Disagreements were resolved by consensus. Where consensus could not be reached, a third rater (MB) assessed the items and provided the tiebreaker score.While the total quality scores emerging from each of the different scales did all significantly correlate with one another (range r = .35 to r = .73), there was considerable variation in the classification of studies as high quality or low quality as a function of the scale that was applied Table . For exaThe 20 comparisons from the 18 trials included in the head and neck concomitant therapy systematic review yielded 2, 14, 12 and 14 high quality studies, respectively, when the Jadad 3-item, the Jadad 6-item, the Sindhu and the Downs & Black scales was used. Although both Jadad 6-item and Downs & Black scales both assessed 14 comparisons to be from high quality studies, only 11 of these 14 studies were the same. For the 12 comparisons from studies categorized as high quality with the Sindhu scale, 10 of these were also rated high quality by both the Jadad 6-item and the Downs & Black scales, the other 2 were rated as high quality by the Jadad 6-item scale only. There was 1 comparison from a study rated as high quality by the Jadad 6-item scale only and two from studies rated as high quality by the Downs & Black scale only.Mortality data  used for the meta-analysis included in the guideline reports were available for two guidelines and need for blood transfusion data were available for the third ,32,34. FOnly the 6-item Jadad scale was applied to the studies of the EPO guideline and the data were pooled to calculate an overall risk ratio for blood transfusion . The risSeveral conclusions can be drawn from this study and review of the literature. First, there are established methods for assessing the quality of randomized controlled trials in which data on adequate reliability and validity were available. West et al uncovered 32 scales, check lists and component systems concerned with evaluating RCTs ; more thAlthough all of the scales we used have established reliability and validity estimates, we found that the number of trials categorized as high quality or low quality depended specifically on the scale that was applied. For the head and neck cancer systematic review, the number comparisons from high quality studies ranged from 2  to 14 . The range for the colon cancer review was 0 to 9. There was also considerable variability regarding the specific quality category in which each trial was placed. These finding are consistent with those of Juni et al [The lack of consistency of study classification from one scale to the next and the lack of clear cut-off criteria for users to employ when measuring quality of studies, presents a challenge to guideline developers when they need to make choice about which instrument they ought to adopt if the choose to adopt an instrument at all. Rather than clear evidence driving our decisions, we considered other features of the instrument in our decision making. Of the rating scales we examined, our preferred choice would be the Jadad 6-item instrument. In contrast to the others considered in this report, this instrument is relatively easy to implement and interpret and good inter-rater reliability was established. Further, although the 3-item version of the Jadad scale is most commonly used, we found the original 6-item version to be more relevant in our clinical context as it provides greater variation in scores. In the cancer discipline, few trials are placebo-controlled and treatment allocation tends to be poorly reported. In contrast to the pain trials which were profiled in the development phase of the Jadad instrument, the majority of the items in the 3-item version (randomization and blinding items) yield no variation in scores in our context and are, therefore, not useful to discriminate among cancer trials. The 6-item version of the scale more aptly differentiates quality across studies and includes more quality domains for which an empirical basis has been established .Another conclusion that can be drawn from this study is that effect size can be related to study quality but that the nature of the relationship in one clinical area may not generalize to another clinical area. Some of the original work examining the role of study quality reinforces the need to be mindful of the variation among studies included in systematic review -7. HowevWe conducted sensitivity analysis on the systematic reviews comprising the guidelines developed by the PEBC. Only four systematic reviews among 36 eligible practice guidelines included more than 10 trials with data appropriate for pooling; three from which we could extract data. Although there was some variation in the odds ratios observed, the confidence intervals of the pooled effects from each of the analyses of high quality trials overlapped with the confidence intervals of the pooled odds with all of the trials. In no case would the conclusions based on these results be affected by restricting the meta-analysis to only high quality studies; the recommendations remained the same. Had sensitivity analysis based on study quality been conducted prospectively, it is highly unlikely that different conclusions would have been drawn from the systematic review or that different clinical practice guidelines would have been formulated.Together, these findings lead to our final conclusion that measuring study quality did not translate into altered conclusions from a systematic review in the oncology domain for the outcomes we used here. Thus, at this time we have decided that measuring study quality using a numerical assessment scale for the purposes of sensitivity analysis will not be a routine part of our guideline development program. We will, however, encourage guideline developers to describe the variation among studies and to point out methodologic flaws. In addition, it will be important for us to repeat this study looking at other outcome measures, such as quality of life and adverse effects, as they become more routinely reported in primary cancer research and incorporated into our practice guidelines. Outcomes other than those studied here may be more sensitive to the issues of study quality.This study highlights a strategy that may be useful for guideline programs to utilize in making decisions regarding the methods employed in their guideline development process. It is important that scientific inquiry be maintained in studying the value and role of study quality assessment rather than accepting its role as convention. By exploring it within a specific clinical context one can identify it's most appropriate application.The author(s) declare that they have no competing interests.MB, MJ and GB conceived of the project idea and developed the protocol. MB, MJ, and MC conducted by study. SH provided statistical advice and AJ provided conceptual advice. The manuscript was drafted initially by MB and MJ. All authors contributed to the final version submitted for publication.The pre-publication history for this paper can be accessed here:


Mast cell (MC)-derived serine proteases have been implicated in a variety of inflammatory processes. We have previously shown that rat peritoneal MC (PMC) express mRNA for protease activated receptor 2 (PAR-2), a G-coupled receptor activated by trypsin-like proteases. Recent evidence also suggests that MC-induced inflammation can be mediated through PAR. Therefore, we hypothesized that specific PAR-2 agonist peptides (PAR-2ap) induce protease release from PMC.Western blot analysis of PMC supernatants revealed that a PAR-2ap, tc-LIGRLO (10 μM), stimulated the release of rat MC protease (RMCP)-1, RMCP-5 and carboxypeptidase-A. The release was evident by 20 min but further increased up to 8 h. To study the biological effects of protease release we tested supernatants from tc-LIGRLO, tc-OLRGIL (inactive control peptide) and antigen-activated PMC for proteolytic activity by seeding with TNF (150 pg/ml), incubating for 8 h at 37°C, and measuring TNF remaining in the supernatants. Supernatants from tc-LIGRLO-stimulated PMC degraded 44 % of seeded TNF (n = 5). Moreover, this TNF proteolysis was dependent on the concentration of tc-LIGRLO used to stimulate PMC, and was significantly inhibited (94 %) by soybean trypsin inhibitor. Antigen and tc-OLRGIL induced no significant release of such proteolytic activity.These data indicate that a PAR-2ap induces the release of proteases from mast cells, which may degrade extracellular cytokines and other substrates thus modulating the inflammatory response. Peptides that are similar in sequence to the tethered ligand domains of PAR-2, such as SLIGRL-NH2 (SLI) or tc-LIGRLO-NH2 (tc-LIG) are able to interact directly with the activation site and act as potent agonists [Protease activated receptor-2 (PAR-2) has been identified on a variety of cell types including eosinophils , neutropagonists .A growing number of studies have identified a role for PAR-2 in inflammation. There is a delayed onset of inflammation in PAR-2 knock out mice , and PARin vivo could be MC-mediated. The administration of PAR-2ap or trypsin into the rat hind paw enhanced vascular permeability and caused edema formation, which can be abolished by repeated pre-treatment with compound 48/80, known to deplete the MC of its granular content [et al., 1999 [The ability of serine proteases to activate MC and the observation that MC express PAR-2 ,13, sugg content . By contl., 1999  showed tActivation of PAR-2 on the surface of mast cells could act as part of an autocrine and paracrine positive feedback loop through the release of serine proteases that could activate further PAR-2 on mast cells or other neighboring cells. Therefore, we investigated the direct effects of PAR-2ap on the release of serine proteases from purified PMC and the effects of these released proteases on extracellular protein degradation. In particular we studied the release of rat mast cell protease-1 (RMCP-1), RMCP-5 and carboxypeptidase A (CPA).To identify proteases released by mast cells following PAR-2ap stimulation we activated PMC with tc-LIG (10 μM), and analyzed the supernatants for various mast cell proteases by western blotting, using antisera against the amino-terminal sequences of RMCP-5 and MC-CPA and an antiserum against RMCP-1 protein. In supernatants from tc-LIG-treated PMC one band for RMCP-1 (30 kDa), two bands for RMCP-5 (34 and 35 kDa), and three bands for CPA (40, 41 and 42 kDa) were detected Fig. . The PARNippostrongylus brasiliensis antigen (Nippo Ag) (10 We/ml) induced no detectable release of any of the three proteases studied , which however, was associated with protease release , tc-LIG (10 μM), tc-OLR (10 μM) or compound 48/80 (0.5 μg/ml) for 20 min or 8 hr at 37°C and the supernatants were collected. These supernatants or media were then seeded with 150 pg/ml of rat recombinant TNF and incubated for an additional 8 hr. At the end of the incubation TNF was measured by ELISA and the proteolytic activity was calculated as % degraded TNF (as discussed in the methods section). Proteolytic activity in the supernatants of sham-treated cells was subtracted from that in the supernatants of activated PMC. Supernatants from sham-treated PMC showed significant loss of seeded TNF (17 ± 7 % at 20 min and 22 ± 5 % at 8 hr) as compared to media. At both 20 min and 8 hr of treatment supernatants from tc-LIG-treated MC (10-0.1 μM) showed a greater loss of seeded TNF compared to supernatants of sham-treated MC (p < 0.05), suggesting tc-LIG-mediated activation induced the release of proteolytic activity. Proteolytic activity, following subtraction of spontaneous proteolytic activity released, was 44 ± 5 % at 8 hr and 30 ± 4 % at 20 min following PMC activation with 10 μM of tc-LIG  before seeding with TNF. SBTI inhibited TNF loss from the supernatants of tc-LIG (10 μM) stimulated PMC by 82% Fig. , confirmWe have previously shown that PMC express PAR-2 mRNA, that can be regulated by cytokines and PAR-2ap . We haveNippo Ag.In this study, we provided the first direct evidence for serine protease release from PMC measured by Western blot analysis of the supernatants, in addition to proteolytic activity assays. The sizes of released RMCP-1 (~30 kDa), RMCP-5 (2 close bands, ~34 kDa) and CPA (3 close bands, ~41 kDa) are similar to the sizes of the stored forms of these proteases that we published previously . The difin vivo and in vitro. The release of RMCP-2 by rat mucosal mast cells has been reported to be induced by antigen challenge in parasitic infections, and during anaphylaxis [2+ ionophore and antigen activation of PMC [Previous studies have shown that activation of MC to release protease activity may be induced by a variety of agents both phylaxis -21. The phylaxis . Furthern of PMC .In our experiments FcεRI-mediated PMC stimulation did not release detectable levels of RMCP-1, RMCP-5 or CPA, or any proteolytic activity with the ability to degrade TNF. It is possible that FcεRI-mediated activation induces low levels of protease release which is undetectable by Western blotting. Furthermore, the lack of demonstrable proteolytic activity in the supernatants does not necessarily indicate that proteases are not released since it may be due to an FcεRI-mediated simultaneous release of protease inhibitors stored in the MC. Indeed, MC produce and release secretory leukocyte protease inhibitor (a chymase inhibitor) and latexin (a CPA inhibitor) ,24. FinaGiven that RMCP-1 and RMCP-5 are present in the supernatants of tc-LIG-stimulated MC, it is likely that these proteases are involved in the TNF degradation. However, antibodies to RMCP-1 inhibited TNF degradation by supernatants of sham treated cells but did not affect the additional degradation of supernatants from tc-LIG activated MC (unpublished observation). We cannot rule out that PAR-2ap activated PMC release other proteases, including the tryptases RMCP-6 and RMCP-7 , which min vivo. It may be that such proteolytic activity directed against TNF, and possibly other cytokines, is an important anti-inflammatory function for mast cell serine proteases.Our data further suggest that MC may regulate TNF function by releasing proteases that can directly degrade this cytokine. Given that both TNF and serine proteases are stored and released from MC, our present findings suggest an important mechanism by which MC may regulate TNF function In vitro, MC tryptase can stimulate histamine release by human tonsillar [The expression of PAR-2 by mast cells and the involvement of MC in PAR-2-mediated inflammation has been controversial. onsillar  and guinonsillar  MC, but onsillar . Tryptasonsillar , indicatonsillar  and rat onsillar  mast celonsillar , but othonsillar . Taken ttrans-cinnamoyl group, which acts to stabilize the peptide and prevent its degradation by aminopeptidases. It is unlikely that the different sensitivity to proteases can explain fully the difference between the effects of the two PAR-2ap peptides. It is also unlikely that the trans-cinnamoyl modification on tc-LIG is solely responsible for tc-LIG-mediated activation of MC, because it is also present on the reverse sequence peptide tc-OLR, but does not have the same effects with tc-LIG on protease release, as shown in this study, or in the release of β-hex, as we showed before.In a previous study we have shown that only one of two PAR-2ap (tc-LIG) activates β-hex release from PMC . The othCompound 48/80 along with other cationic compounds can activate MC by directly interacting with a pertussis toxin sensitive component . Our pre2 has been identified pharmacologically in murine vascular smooth muscle [Recently, a new receptor activated by the PAR-2 activating peptide tc-LIGRLO-NHh muscle . In thatOur study provides evidence that a PAR-2ap, tc-LIG, activates MC to release proteases and proteolytic activity that could potentially have both pro- and anti-inflammatory functions. We further showed that these proteases may degrade extracellular proteins and affect the inflammatory environment in areas of mast cell activation. Although the presence and function of PAR-2 on MC is still controversial, our findings indicate that PAR-2 may be part of an autocrine loop. PAR-2 activation leads to the release of serine proteases which in turn may further activate more PAR-2 receptors on mast cells and also on other cells.2-terminal sequences were synthesized at Zymogenetics Inc, Seattle, WA, and used to immunize rabbits to develop specific polyclonal anti-protease antibodies. Professor H. Miller, Edinburgh, Scotland, kindly provided rabbit antibody to RMCP-1.Compound 48/80, 4-methylumbelliferyl-N-acetyl-β-D-glucosaminide (β-hexosaminidase (β-hex) substrate) and soybean trypsin inhibitor (SBTI) were purchased from Sigma Chemical Co. (St. Louis, MO). PAR-2ap and PAR-2 control peptides (PAR-2cp) were synthesized by the Peptide Synthesis Facility, Faculty of Medicine, University of Calgary. These peptides were determined to be ≥ 95 % pure by mass spectrometry and HPLC. Polyclonal RMCP-5 and CPA antibodies were produced and characterized as described previously . BrieflyNippostrongylus brasiliensis, by a single subcutaneous injection of 3000 third-stage larvae in 0.5 mL of saline as described previously [Outbred male Sprague-Dawley rats (weight 250–500 g) were purchased from Charles River Canada Inc., (St. Constant, Quebec). Rats were maintained in an isolation room with filter-topped cages to minimize unwanted infections. For the experiments where MC were activated through their IgE receptor, rats were sensitized to eviously . The expFifteen mL of ice-cold Hepes-buffered (10 mM, pH 7.3) Tyrodes buffer supplemented with 0.1% BSA was injected into the peritoneal cavity of each rat for the isolation of PMC. MC in peritoneal lavage fluids were enriched by centrifugation through a discontinuous density gradient of Percoll, as described previously . Recover6 cells/mL. Cells were placed in 1.5 mL Eppendorf tubes or in 48 well plates, incubated at 37°C for 10 min, and then the same volume of pre-warmed (37°C) PAR-2ap or controls in complete RPMI were added, to give a final cell concentration of 0.5 × 106 cells/mL. The cells were incubated for different times (20 min to 8 hr) depending on the experiment.After isolation and enrichment, PMC were rested in RPMI (Invitrogen, Burlington, Ontario) supplemented with 5% FBS for 2 hr at 37°C. After incubation, the cells were washed twice by centrifugation (150 g) and resuspended in RPMI at 1 × 10Nippostrongylus brasiliensis Antigen /mL [To measure spontaneous release of mediators by PMC, cells were mixed with media alone. As positive controls, either compound 48/80 (0.5 μg/mL) or  (WE)/mL ) were miSupernatants were concentrated (10×) using Centricon (YM-10) centrifugal filter devices (Millipore, Bedford, MA). For Western blot analysis, proteins were transferred electrophoretically (25 V, 35 min) to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad Laboratories, Mississauga, ON) using the Semi-Dry Trans Blot System. The membranes were blocked in Tris-buffered saline containing 0.02% Tween, 5% w/vmilk (Bio-Rad Laboratories) and 5 % v/vgoat serum (Invitrogen) for 1 hr. The membranes were probed with 1/1000 dilution of anti-RMCP-1, 1/600 anti-CPA, 1/5000 anti-RMCP-5 and then incubated with donkey anti-rabbit IgG HRP-conjugated antibody (1:5000). Protein bands were detected by enhanced chemiluminescence using ECL Western blotting detection system (Amersham Pharmacia Biotech, Quebec, Canada).β-hex was measured in the supernatants and cell pellets, as described . ResultsTo measure release of proteolytic activity, supernatants from stimulated PMC (containing secreted TNF) were transferred to a 96-well plate. After 2 min incubation, exogenous TNF or medium was added to the supernatants and mixed to give a final concentration of 150 pg/ml. Plates were incubated at 37°C for 8 hr and then TNF content was measured by ELISA. Percent TNF proteolysis was calculated by the following formula:% TNF degraded = 1 - (TNF recovered / (rat recombinant TNF seeded + measured TNF release) × 100)For protease inhibition experiments SBTI (1 mg/ml) was added to the supernatants before seeding with TNF. The supernatants were then processed as above and used to measure TNF degradation.Supernatants from activated PMC were analysed for TNF using a rat TNF ELISA kit (Endogen, Woburn, MA), according to manufacturer's instruction. The sensitivity of the TNF assay was < 10 pg/ml. To exclude the possibility that proteases contained in PMC supernatants interfere with ELISA determination of TNF we incubated the TNF antibody coated wells with PMC supernatants washed them and then added specified amounts of TNF for determination. Pre-incubation with PMC supernatants did not affect the ability to measure TNF, indicating that the proteases in PMC supernatants do not degrade the antibodies of the assay.t-test and ANOVA.All values are given as mean ± standard error of mean (SEM) for the numbers of experiments noted and statistical analyses were performed using the Student's β-hex β-hexosaminidaseCPA carboxypeptidase-A2 (PAR2-cp)LSI LSIGRL-NHMC mast cellPAR protease-activated receptorPAR-ap protease-activated receptor-agonist peptidePAR-cp protease-activated receptor-control peptidePMC peritoneal mast cellsRMCP-1,5 rat mast cell protease-1, 5SBTI soybean trypsin inhibitor2 (PAR2-ap)SLI SLIGRL-NHtrans-cinnamoyl-LIGRLO-NH2tc-LIG trans-cinnamoyl-OLRGIL-NH2tc-OLR Nippo Ag Nippostrongylus brasiliensis Antigen.WE worm equivalentHNA carried out the majority of the experiments presented and drafted the manuscript. This work was part of his MSc thesis. GRS assisted with mast cell isolation and some of the activation experiments and participated in the experimental design. JLW participated in the design of the study. MDH participated in the design of the study. ADB participated in the design of the study, supervised the work shown and made substantial contributions in manuscript preparation. HV participated in the design of the study and contributed in the preparation of the final manuscript. All authors read and approved the final manuscript.


The carcinogenesis of colorectal cancer has been accepted by a model for a cascade of genetic alterations, named the adenoma-carcinoma sequence. In order to elucidate the carcinogenesis of the colorectal cancer more clearly, the genetic abnormalies of the non-neoplastic mucosal epithelium of the colon and rectum should be investigated. It has been speculated that colonic Paneth cell metaplasia (PaM) is one of the pre-neoplastic mucosa of colonic cancer. Therefore, we studied the propria mucosa of the right colon with PaM from the standpoints of the frequency of the K-ras codon 12 mutations (K-ras), which is initial genetic abnormality in colorectal cancer, and the loss of heterozygosity of microsatellite markers (LOH-MS), which has a relationship to development of colorectal cancer.Fifty-two regions with PaM histopathologically from 12 surgically resected right colon specimens were studied. DNA extraction of the colonic mucosa with PaM was obtained using a microdissection method, and the frequency of the K-ras of PaM was investigated by enriched polymerase chain reaction-enzyme linked mini-sequence assay, and the frequency of the LOH-MS (D2S123, D17S250 and D5S346) of PaM was examined by high resolution fluorescenced labeled PCR primers.K-ras mutation was detected in fifteen regions among 52 PaM (28.9%). All mutations were a single mutation and GGT changed to AGT in eleven and GAT in four. LOH-MS were detected in twenty-one regions among 52 PaM (40.4%) (D2S123: 35.4%, 17/48 regions, D17S250: 13.7%, 7/51 regions, and D5S346: 0%, 0/52 regions). No K-ras mutations and LOH-MS were detected in the controls .Colonic mucosa with Paneth cell metaplasia may be one of the pre-neoplastic mucosa in the development of the colonic epithelial neoplasia. Vogelstein et al.  have repHowever, the genetic abnormalities of the non-neoplastic mucosal epithelium of the colon and rectum has not been investigated, except the aberrant crypt foci  and hypeWe have speculated previously that the colorectal Paneth cell metaplasia (PaM) is one of pre-neoplastic mucosa on the development of the colorectal epithelial neoplasias , becauseThe main purpose of the present study was to investigate the frequencies of the K-ras codon 12 mutations (K-ras) and the loss of heterozygosity of dinucleotide microsatellite markers (LOH-MS) in the propria mucosa with PaM of the right colon.The materials were 12 surgically resected specimens of right colon, which had the colonic carcinomas were present, and histological diagnosis was assessed at the Department of Pathology, Juntendo Izunagaoka Hospital. Although we have intended to investigate the PaM in the normal colon of the individuals without cancer, using the biopsies specimens, it was difficult to detect the PaM in these specimens. No inflammatory bowel diseases were included in the materials. Informed consent was obtained from all the patients to investigate the genetic alterations in the current study.The specimens were fixed in 10 % buffered formalin solution and prepared by cutting the non-neoplasitic area into 3 – 5 mm sections. Each section was embedded in paraffin and stained with hematoxylin and eosin (HE), followed by immunohistochemical staining for anti-lysozyme (DAKO, Japan). Immunohistochemical stainings were performed by the avidin-biotin-peroxidase-complex method, at dilution of 1 : 100.Fifty-two colonic mucosal regions with PaM, distant from neoplastic lesion, aberrant crypt foci and hyperplastic polyp, were detected by HE staining and anti-lysozyme antibody staining, and these mucosa were used as the target regions in the current study.All paraffin blocks of the target regions and 12 paraffin blocks with no PaM as controls of each materials were used for the DNA extraction.Paraffin blocks with the target foci mentioned above were prepared for DNA extraction. The target foci were microdissected using a 20-gauge needle, comparing the slide with HE staining in the same position. The extracted DNA was diluted with 5 ml of TaKaRa DEXPAT .2, 1 μM each primer, 0.625 U Taq DNA polymerase and 1 × PCR buffer [containing 10 mM Tris-HCl(pH 8.3 at 25 degrees of temperature), 50 mM KCl and 0.001%(w/v) gelatin] in a thermal cycler. Then, 10 μL of the denatured second PCR product was hybridized with probes to detect the K-ras codon 12 wild-type (GGT) and six mutant (GAT, GCT, GTT, AGT, CGT and TGT) DNAs were immobilized, at 55 degrees of temperature for 30 minutes, and 100 μL of biotinylated A and 0.01 U of TdqDNA polypmerase were added and incubation was continued at 55 degrees of temperature for 30 minutes.Mutation of K-ras was analyzed and compared by enriched polymerase chain reaction-enzyme linked mini-sequence assay (PCR-ELMA). In PCR-ELMA ,9, upstrFor development, 100 μL of avidin-horseradish peroxidase conjugate was added and the reaction was performed at room temperature for 30 minutes. Then, 100 μL of tetramethyl-benzidine (TMB) substrate was added and the plates were left to develop in the dark at room temperature for 20 minutes. Finally, 100 μL of stop solution was contained and the light absorbance of each sample was measured by spectrophotometry (Multiskan Multisoft, Labsystems, Tokyo) with a 450 nm filter wavelength  were selected for LOH among the microsatellite markers, recommended by National Cancer Institute, because commonly it has been thought to be difficult to study the LOH using mononucleotide microsatellite markers.2, 1 μM each primer marked with fluorescent dye of three colors as blue, green and yellow, 0.625 U Taq DNA polymerase and 1 × PCR buffer [containing 10 mM Tris-HCl(pH 8.3 at 25°C), 50 mM KCl and 0.001%(w/v) gelatin] in a thermal cycler. 2. The electrophoresis was conducted 2 hours by means of an ABI-377 DNA auto-sequencer (PE Biosystems, Inc., Foster City, CA, USA). 3. A comparison was made of peaks of same marker arising from normal tissue and the propria mucosa with PaM, using the Gene Scan TM waveform analyzed softwave, and LOH were assessed. That is to say, LOH (+) was assessed when the ratio of the peak area of  was less than 70 % or was more than 143 %.These LOH-MS were investigated using high resolution fluorescenced labeled PCR primers in proportion to the method of Tsuchida et al. . The outThe data were analyzed statistically with Student's t-test (t-test) and chi-square test ; a p-value of less than 0.05 was considered to be significant.K-ras mutation was detected in fifteen regions among 52 PaM (28.9 %). All mutations were a single mutation. For K-ras mutation patterns, 11 showed GGT to AGT, and four showed to GAT.LOH-MS was detected in twenty-one regions among 52 PaM (40.4 %) (D2S123: 35.4 %, 17/48 regions, D17S250: 13.7 %, 7/51 regions, and D5S346: 0 %, 0/52 regions). No K-ras mutations and LOH-MS were detected in the controls .Thus, the frequency of both K-ras mutation and LOH-MS in the colonic mucosa with PaM were significantly higher than those of the controls (p < 0.01, chi-square test).K-ras mutations have been detected in several human neoplasias -14, and In order to conclude the carcinogenesis of the colorectal cancer more clearly, the genetic abnormality of the non-neoplastic mucosal epithelium of the colon and rectum should be investigated, however, because the colorectal cancers are derived from the colorectal mucosa. It is also important for the preventive medicine of the colorectal cancer to know the carcinogenesis of it.However, until now, the genetic abnormalities of the colorectal non-neoplastic mucosa is unclear, except the aberrant crypt foci  and hypeWe have already reported that the colorectal Paneth cell metaplasia (PaM) is one of pre-neoplastic mucosa on the development of the colorectal epithelial neoplasias , becauseTherefore, K-ras mutation and the loss of heterozygosity of microsatellite markers of PaM were investigated in this study, and the current study is thought to be the first report focusing on this.Our results showed that K-ras mutation was detected in fifteen regions among 52 PaM (28.9%), and LOH-MS was detected in twenty-one regions among 52 PaM (40.4%).Namely, K-ras mutation and LOH-MS of PaM were not rare and the frequency of those of PaM were higher than those of the normal colonic mucosa, and it came to light that some PaM had the genetic abnormalities which had a relationship to the development of colorectal cancer.Paneth cells, which are usually situated at the base of the glands of the small intestine, were first identified by Scwalbe  in 1872,Now, these cells have been one of the most famous cells in the gastro-intestinal tract, however, the detail function of these is not clear. Paneth cells are sometimes seen in the colorectal tubules, for example in the proximal colonic mucosa of elderly subjects ,17, in pDescribed above, many riddles about Paneth cell are still remained. And the gene abnormalities of colonic mucosa with PaM in the current study may be not equal to those of single Paneth cell in the colonic mucosa, because it is difficult to obtain only single Paneth cell in the colonic mucosa, even if microdissection method is used.However, we have been able to investigate the colonic mucosa with PaM, and we think it very interesting that some PaM have K-ras mutation and the loss of heterozygosity of microsatellite markers, and these PaM may be thought to be the pre-neoplastic mucosa in development of the colonic epithelial neoplasia. Further molecular studies concerning Paneth cell metaplasia in the large bowel should be warranted.Colonic mucosa with Paneth cell metaplasia may be one of the pre-neoplastic mucosa in the development of the colonic epithelial neoplasia.PaM, colonic Paneth cell metaplasia; K-ras, K-ras codon 12 mutations; LOH-MS, loss of heterozygosity of microsatellite markers; HE, hematoxylin and eosin; PCR, polymerase chain reaction; ELMA, enzyme linked mini-sequence assay


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


PLoS Medicine addressed the issue of ensuring equity in distributing AIDS medications. Reis and Capron discuss the study's implicationsA study by Wilson and Blower in the February issue of  Of the roughly 40 million people living with HIV , an estiGiven this gap between what can be done and what needs to be done, the people who set policies and administer programs to provide ART in high-burden countries are faced with difficult questions of distributive justice. Decisions regarding the pricing of ARTs and other care for patients with HIV/AIDS, the distribution of treatment centers, and potential measures to overcome barriers for vulnerable populations will determine who will get access to treatment and who will die. In order to deal with these crucial issues, decision-makers need guidance on how to design policies on equitable access to ART that respect human rights norms and ethical standards.PLoS Medicine [A new study by Wilson and Blower, published in the February issue of Medicine , addressMedicine . Wilson While the authors suggest that their method could be adapted to take other objectives into account, here they have taken an exclusively egalitarian approach to equity. Although this notion of equity is broadly accepted, other important approaches could have been taken into consideration. Simple equality in access can actually produce inequities (because a fair approach would differentiate among groups in the population according to their different needs); further, under some theories, those who are least advantaged generally should receive a disproportionate share of newly distributed benefits (the maximin principle) . In geogIn determining equitable spatial accessibility for the application of their model to KwaZulu–Natal, the authors used a rather rough estimation of HIV prevalence . As prevalence greatly varies between specific communities, future studies would certainly benefit from using more disaggregated data where available (see, for example, ). SimilaPlace matters, but spatial accessibility is only one factor to be overcome in ensuring equitable access to health services. Studies show that even when services are available at a near distance, factors such as temporal accessibility, disease perception, stigmatization, and outright discrimination heavily influence “effective demand” . MoreoveWilson and Blower have developed a mathematical model to determine the fair geographical distribution of ART treatment sites and have applied it to the specific setting of KwaZulu–Natal. Despite some methodological and data limitations, such studies can inform policy-makers' decisions regarding the location of HIV services. Since distance to a treatment center is strongly determinant of patients' ability to access care, WHO is developing a service availability mapping tool to monitor relative equity between districts and identify major gaps in service availability, for example, availability of ART and prevention of mother-to-child transmission programs.Not only is further research needed to refine the spatial accessibility model presented by the authors but careful attention must be paid to other factors that affect access to HIV services and to the underlying assumptions as to what would constitute fair distribution. In a recent guidance document, WHO and UNAIDS recommended that ART programs include special measures to ensure access of vulnerable and marginalized populations and women to ART . The dec


Leukemia is a clonal disorder characterized by uncontrolled proliferation of haematopoietic cells, and represents the most common form of cancer in children. Advances in therapy for childhood leukemia have relied increasingly on the use of high-dose chemotherapy often combined with stem-cell transplantation. Despite a high success rate and intensification of therapy, children still suffer from relapse and progressive disease resistant to further therapy. Thus, novel forms of therapy are required.This study focuses on dendritic cell (DC) vaccination of childhood leukemia and evaluates the in vitro efficacy of different strategies for antigen loading of professional antigen-presenting cells. We have compared DCs either loaded with apoptotic leukemia cells or transfected with mRNA from the same leukemia cell line, Jurkat E6, for their capacity to induce specific CD4+ and CD8+ T-cell responses. Monocyte-derived DCs from healthy donors were loaded with tumor antigen, matured and co-cultured with autologous T cells. After one week, T-cell responses against antigen-loaded DCs were measured by enzyme-linked immunosorbent spot (ELISPOT) assay.DCs loaded with apoptotic Jurkat E6 cells or transfected with Jurkat E6-cell mRNA were both able to elicit specific T-cell responses in vitro. IFNγ-secreting T cells were observed in both the CD4+ and CD8+ subsets.The results indicate that loading of DCs with apoptotic leukemia cells or transfection with tumour mRNA represent promising strategies for development of cancer vaccines for treatment of childhood leukemia. Leukemia represents the most common form of cancer in children. There are two main types of childhood leukemia, acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). The success rate in treatment of childhood leukemia has improved continuously over the past decades , and todImmunotherapy based on vaccination with dendritic cells (DCs) has emerged as an attractive new form of therapy for cancer in general, and DC-based vaccines have already shown promise in follicular non-Hodgkin's lymphoma, and in other hematological malignancies -7. DCs aVaccination with tumor antigen-loaded DCs has been shown to induce both Th and CTL responses, and tumor regression in some patients ,17. An i2), IL-7, IL-2 and IL-12 from R&D Systems (Minneapolis, USA). Staurosporin was obtained from (Sigma Aldrich, Saint Louise, Missouri).GM-CSF was purchased from Novartis (Basel, Switzerland), IL-4, TNFα, IL1β and IL-6 from CellGenix (Freiburg, Germany), and Prostaglandin E2 (PgE6 cells/ml for 1.5 h at 37°C in 3 ml CellGro DC medium (CellGenix, Freiburg, Germany). Non-adherent cells were collected and frozen for later use as responder cells. Adherent cells were cultured in 3 ml CellGro DC medium, supplemented with 800 U/ml GM-CSF and 10 ng/ml IL-4 every second day, until day 5 when maturation of DCs was induced by addition of maturation cocktail (10 ng/ml TNF-, 10 ng/ml IL-11000 U/ml IL-6 and 1 μg/ml PgE2) for 24 h. Characterization of DC phenotype was done by staining 0,5 × 106 cells with fluorochrome-labelled antibodies against the Lin1 panel (CD3, CD14, CD19, CD16, CD20, CD56), HLA-DR, CD1a, CD80, CD83, and CD86 (Becton Dickinson, San Jose, CA), and analyzing by FACSCalibur flow cytometry (Becton Dickinson). The mAb isotypes used were IgG1 FITC, IgG2a PE, IgG1 APC (Becton Dickinson, San Jose, CA).PBMC from healthy donors  were obtained by density gradient centrifugation (Lymphoprep, Nycomed, Norway). Monocyte-derived DCs were generated under serum-free conditions from the adherent fraction of PBMCs cultured in six-well plates at a density of 4 × 10Jurkat E6 cells obtained from American Type Culture Collection (ATCC) were exposed to 1 μM staurosporin for 3 h to induce apoptosis. Apoptotic cell death was assessed using Annexin V-FLUOS as described by the manufacturer (Boehringer Manheim, Manheim, Germany). For assessments of phagocytosis, Jurkat E6 cells were stained with the green fluorescent dye PKH-67 (Sigma Aldrich) as described in the kit manual, exposed to 1 μM staurosporin for 3 h and incubated with immature DCs at a ratio of 3:1. After 6 h, immature DCs were labelled with a red fluorescent antibody (mAb CD1a-PE). Phagocytosis of apoptotic cells was measured quantitatively by flow cytometry. Similarly, phagocytosis was visualized by confocal laser microscopy (Leica TCS SP, equipped with HeNe and Ar lasers) using apoptotic Jurkat E6 cells pre-stained with PKH-26 red fluorescent dye (Sigma Aldrich) and DCs stained with the green fluorescent dye PKH-67.6 cells using Trizol Reagent as described by the manufacturer (Invitrogen, Basel, Switzerland). Poly (A)+ mRNA was isolated from total RNA using the GenoPrep Direct mRNA kit (GenoVision, Oslo, Norway). Purified mRNA was used fresh or stored at -80°C until use. Transfection of DCs with mRNA was performed as described previously [6 cells) were mixed with mRNA, transferred to a 4-mm-gap cuvette and pulsed with a BTX ECM-830 square-wave electroporator (Genetronics Inc., San Diego, CA) using instrument settings 500 V and 1 ms. Transfected cells were incubated on ice for 30 s followed by addition of 2.0 ml cold CellGro DC medium supplemented with 10 ng/ml IL-4, 800 U/ml GM-CSF and maturation cocktail (see above), and transferred to standard culturing conditions. Transfection with EGFP-pCIpA102 mRNA (10 μg/400 μl) encoding the green fluorescence protein [Jurkat E6 cells were used as a source of tumor material. Total RNA was isolated from 20–25 × 10eviously , with mi protein  was usedThe Negative Isolation Kit  was used for isolation of CD4 and CD8 T cells according to the manufacturer's protocol. Isolation was performed on day 7 after in vitro priming, before setting up the ELISPOT assay.6) expressing Jurkat E6-cell mRNA or loaded with apoptotic Jurkat E6 cells, were co-cultured with 3 × 106 autologous non-adherent PBMC for 7 days in 1.0 ml CellGro DC medium without serum, before setting up the ELISPOT assay. The cultures were tested for INF- production in an ELISPOT assay [5, 1.0 × 105, 2.0 × 105 and 4.0 × 105 responding cells and 0.5 × 104 DCs per well. Mock transfected DCs were used as control. The assay was done in duplicate. Spots were counted manually, and the frequency of reactive T cells was calculated according to the formula: (spots with transfected DC - spots with non transfected DC)/number of T cells added.Mature DCs  and early apoptotic cells were accordingly used in all experiments. To verify apoptosis after 3 h exposure to staurosporin, cells were stained with annexin V-FLUOS and examined by flow cytometry. The assessments showed that more than 90% of the cells were annexin-V positive Fig. .To study uptake of apoptotic leukemia cells by immature DCs, Jurkat E6 cells were stained with the red fluorescent dye PKH-26 before induction of apoptosis. Immature DCs were stained with the green fluorescent dye PKH-67. Apoptotic Jurkat E6 cells were then co-incubated with immature DCs for various periods of time to determine the optimal conditions for internalization of apoptotic Jurkat E6 cells. We observed that immature DCs ingested apoptotic Jurkat E6 cells within 6 h of co-incubation. Phagocytosed apoptotic Jurkat E6 cells (red stained) were observed inside or in the process of being phagocytozed by immature DCs (green stained) by confocal microscopy Fig. .Flow cytometry was used to further determine the efficiency of DC loading with apoptotic leukemic cells. In these experiments, apoptotic Jurkat E6 cells had been pre-stained with the green fluorescent dye PKH-67 and DCs were identified by staining with PE-conjugated anti-CD1a. Highly efficient uptake of apoptotic Jurkat E6 cells was confirmed, since virtually all CD1a positive cells showed green PKH-67 staining Fig. .Following antigen loading, DCs were matured in the presence of pro-inflammatory cytokines for 24 h. Assessments by flow cytometry confirmed that this treatment led to up-regulation of CD83, and the co-stimulatory molecules CD80 and CD86, in compliance with a mature DC phenotype Fig. .Transfection of cells with tumor-derived mRNA is an alternative method for loading of DCs with tumor antigens. mRNA from the Jurkat E6 cells was isolated and electroporated into DCs according to previously optimized methods . FollowiELISPOT assay of INF-γ producing cells is the method of choice for assessments of T-cell responses against cancer vaccines representing a heterogeneous mixture of antigens. This assay measures in a quantitative way the number of reactive T cells in pre and post-vaccination samples and thus directly relates the effect of vaccination to in-vivo expansion of reactive T cells. Autologous T cells were stimulated with tumour-mRNA transfected DCs and with DCs loaded with apoptotic Jurkat E6 cells. Fig.  secreting T cells. However, it appeared that DCs loaded with tumour-mRNA in general were most potent in inducing T-cell responses.Immunotherapy for childhood leukemia has the potential to contribute to long-term control or cure of the disease. Until now immunotherapeutic approaches for leukemia have been limited to trials of cytokine therapy . FurtherWe observed that the frequencies of induced INF-γ producing T cells depended on the individual donor, the method of antigen-loading and the subset of T cells studied. Such variations are not surprising, since the model system used employs allogeneic cells and no effort was done to HLA match the blood donors with the Jurkat cell line in these experiments. Since the experimental system is based on the use of an allogeneic cell line, we expect multiple antigens, encoded by a broad array of polymorphisms, including other HLA alleles to be involved. We have therefore taken advantage of the genetic differences between responding cells and the leukaemia cell line by using the combined repertoires of membrane expressed and cross presented allo-antigens as a sensitive readout for immunological response in our experiments.We expected that the two different procedures would provide some differences in loading of HLA molecules with tumour-derived antigens and subsequently in the responding T-cell subsets. According to the current dogma, processed peptides from phagocytosed apoptotic cells would be directed to HLA class I molecules by a process known as cross-presentation and to HLA class II molecules by the classical pathway. Cross-priming of CTL with antitumor activity has been demonstrated with DCs loaded with apoptotic tumour cells ,21,35. SThe aim of the present study was to determine if loading of DCs with antigens derived from a tumour cell line, either as apoptotic cells or as mRNA would provide a basis for an efficient vaccine, using ELISPOT as a read-out of immune responses. It has been shown that DCs transfected with antigens encoded in tumor mRNA is capable of inducing potent T-cell responses against tumour-specific epitopes . While p secreting T cells. However, it appeared that DCs loaded with tumour mRNA in general were most potent in inducing T-cell responses.In our study we demonstrate that both DCs loaded with apoptotic Jurkat E6 cells or transfected with mRNA isolated from Jurkat E6 cells, can induce T-helper and CTL responses against antigens derived from allogeneic leukemic T-cells. We also show that the two different methods of antigen-loading did not result in any apparent differences in the phenotype of the mature DCs. In terms of immune responses both methods of antigen loading produced DCs capable of inducing INF-The author(s) declare that they have no competing interests.SJJ performed preparation of DCs and T cells, assessments of apoptosis and phagocytosis of apoptotic cells, preparation of Jurkat E6-cell mRNA and transfection of DCs, isolation of T-cell subsets CD4 and CD8 and induction of primary T cell responses. SSL did the transfection of DCs with EGFP mRNA and fluorescence microscopy of DCs. RP, FW and GG planned the project.The pre-publication history for this paper can be accessed here:


Late referral to specialist nephrological care is associated with increased morbidity, mortality, and cost. Consequently, nephrologists' associations recommend early referral. The recommendations' effectiveness remains questionable: 22–51% of referrals need renal replacement therapy (RRT) within 3–4 months. This may be due to these recommendations addressing the specialist, rather than the primary care providers (PCP).The potential of specialist intervention aiming at slowing progression of chronic renal failure was introduced individually to some 250 local PCPs, and referral strategies were discussed. To overcome the PCPs' most often expressed fears, every referred patient was asked to report back to his PCP immediately after the initial specialist examination, and new medications were prescribed directly, and thus allotted to the nephrologist's budget.In retrospective analysis, the stage of renal disease in patients referred within three months before the introductory round (group A, n = 18), was compared to referrals two years later (group B, n = 50).Relative number of patients remained stable (28%) for mild/ moderate chronic kidney disease (MMCKD), while there was a noticeable shift from patients referred severe chronic kidney disease (SCKD) (group A: 44%, group B: 20%) to patients referred in moderate chronic kidney disease (MCKD) (group A: 28%, group B: 52%).Individually addressing PCPs' ignorance and concerns noticeably decreased late referral. This stresses the importance of enhancing the PCPs' problem awareness and knowledge of available resources in order to ensure timely specialist referral. Late referral to specialist care for renal failure is associated with increased morbidity, mortality, and cost (review in,2). ConsChronic renal disease may be asymptomatic for a very long time. This stresses the importance of primary care providers in ensuring timely referral to a renal care center. Levin reviewedThe problem of late referral seems to be ubiquitous, if discussions with colleagues world- wide may be believed, and has led to numerous local, regional and national initiatives aiming at ensuring timely referral into specialist care. Some initiatives,8 succesThis article summarizes findings from a local initiative started by one of the authors (K.H.) in 1997 in the city of Dortmund, Germany.In Germany, about 90% of the population are covered by the mandatory health insurance system ("Gesetzliche Krankenversicherung"). All physicians who wish to treat these patients are organized in associations, which negotiate a budget with the insurance companies. Depending on the specialty, a typical per capita budget is then assigned per patient for a period of three months. This budget covers consultation fees, and the cost of medication prescribed. If a physicians exceeds the total budget thus calculated for his practice, the insurance companies can demand restoration of funds. Typically, a GP's per patient budget would be much lower than a nephrologist's. During the time period described here, the nephrologist's budget per patient was about ten times as much than a general practitioner's. While this budget strategy is intended as a safeguard against excess prescriptions, it has been criticized by many physicians for inducing sub- optimal treatment as practice owners comply rather with budget demands than with best practice guidelines.Dortmund, a town of 589000, has a physician to patient ratio of 141/ 100000 (German average: 156/ 100000). There are five nephrological centers, including two hospitals. Currently, 172 general practioners ("Allgemeinärzte und Praktische Ärzte"), 123 internists ("Ärzte für Innere Medizin") and 21 urologists are listed in Dortmund. One of the internist practices supervises a dialysis center, but does not provide specialized nephrological consultancy. GPs, internists and urologists were considered primary care providers, as all referrals for treatment of chronic renal failures came from one of these specialties. There were no major fluctuations of physician numbers between 1997 and 1999, the period for which data was analyzed here.Joint initiatives by all five nephrology centres in Dortmund to give PCPs a series of lectures on treatment of chronic renal disease met with little response, as the presenters regularly outnumbered the audience. In the spirit of the Declaration of Helsinki, part II.1.: , K.H. deIn order to do so, education was brought to the primary care providers, as opposed to bringing the PCPs to education. Over a period of 18 months, K.H. introduced himself and the potential of timely nephrological care in delaying or even halting the progression of chronic renal disease to PCPs- General Practitioners, and specialists in Internal Medicine and Urology. This introductory round included participation in local PCPs' round tables and PCP organized continuous medical education events, but also individually meeting some 250 PCPs for discussion. During these sessions, the topic of renal disease was subtly introduced during discussions on the more common diseases and risk factors, such as cardiovascular disease, hypertension and diabetes mellitus.During these teaching sessions and discussions, three arguments were repeatedly brought forward by the PCPs:1. Mild- moderate renal failure should be treated by the primary care providers, while the nephrologists' task was seen in providing renal replacement therapy.2. Referral to specialist care meant risking the loss of the patient to the specialist's practice .3. If patients did return from specialist consultation, they were usually carrying recommendations for costly permanent medication, such as ACE inhibitors, that put a heavy burden on the PCP's budget.The first of these arguments was addressed by the "teaching module" of the visits. PCPs were given an executive summary of the complexities of diagnostic and therapeutic procedures used in state- of- the- art nephrology, and the potential of intensified treatment by specialists aiming at slowing the progression of renal failure was explained in detail.Secondly, all patients referred were asked to report back to the referring PCP within one week of the consultation, by which time a summary of findings and appropriate counsel had been sent ahead.recommendations, drug prescriptions were handed out directly and so allotted to the much higher nephrologist's budget, thus easing the financial pressure on the primary care providers. More importantly, also the follow- up prescriptions were done in the renal care center, so that at no time the PCPs budget became endangered.Thirdly, instead of drug The present study investigates the question whether improving the PCP's knowledge about the potential of timely treatment of chronic renal failure, in combination with addressing their economic concerns, succeeded in encouraging timely referral to specialist care.2, moderate chronic kidney disease (MCKD), 60 ml/min/1.73 m2 > ECC > 20 ml/min/1.73 m2, and severe chronic renal disease (SCKD), ECC < 20 ml/min/1.73 m2. Due to small proband numbers in the subgroups, the null hypothesis ("no inter- group differences") was tested by the non- parametric, two sided Chi- square test. Survival was estimated by Kaplan- Meier analysis. Statistical analysis was carried out using the SPSS v11.5 package.Retrospective analysis of patients' records. Analysis of patient records was carried out by a final year medical student (TDV). All patients had agreed to have their data used for quality control measures at the time of referral. Specifically informed consent was obtained from all patients still alive at the time of data collection. Two groups were selected by date of first contact with the nephrologist: Group A, third quarter 1997, immediately before the start of the initiative, and Group B, third quarter 1999, six months after the last visit to a primary care provider had taken place. All new patients whose records indicated referral for nephrological specialist treatment were included in the study. Criteria were subnormal creatinine clearance (ECC) or elevated serum creatinine, elevated blood pressure, proteinuria, or erythrocyturia. The patients in each of the two groups were divided into three subgroups according to their renal function: mild/moderate chronic kidney disease (MMCKD), ECC > 60 ml/min/1.73 mTable The present study is a retrospective analysis of patient data from a single nephrological referral centre, and statistics are carried out on a data set of limited size (n = 18 only in the pre- intervention group A). As such, the statistical significance of the findings is doubtful, and the results might be interpreted as akin to a case report, or a medical anecdote.As such, however, it may have a value quite different from, but equal to that of a randomized, controlled study.Aronson recently discussed the value of medical anecdotes; of the More often than not, observational studies give similar results to controlled randomized trials,13, and The positive finding of the present study is that active recruiting strategies may improve the referral pattern. Following the discussions with individual PCPs, more patients were referred to specialist care at a stage where appropriate treatment may slow or even halt the progression of chronic renal failure. This should benefit first the patient, and then in the long run society as a whole. Recent studies,15 have Late referral to specialist care in chronic renal disease remains a problem world- wide. As early as 1984, focusing on the question under which circumstances renal replacement therapy should be initiated or not, questionnaires showed significant differences in the attitudes of nephrologists and non- nephrologists towards referral. SimilarIt has been speculated that this may be because these recommendations address the specialist, rather than the primary care providers (PCP),2.At present, a medline search using the keywords "referral" and "chronic renal failure" listed a total of 81 references since 1974, with only 48 in specialist, not necessarily nephrological journals, while adding "guideline" reduced the reference list to a disappointing three references from the years 1998 and 1999, which included, however, a fully accessible online guideline in the Canadian Medical Association's journal. Neither the American, nor the European Renal Associations' guidelines,4 were fBetter results were obtained using the same search terms on a general search engine (Google), leading at first hit to the DOQI webpage, which includes the free access to full text guidelines. An extensive search using the same terms in German, however, showed no relevant results but an abundance of lay articles of dubious quality. This scarcity of qualified information might exclude those physicians not fluent in English from gathering relevant and up- to- date information. National renal care associations should therefore consider extending their educational efforts beyond their specialist members and selectively target PCPs and their respective professional associations.In the present study, two arguments brought forward by PCPs to explain their reluctance to refer early focused on the economic burden this presented to their own practice, rather than society as a whole. These two arguments concerned budget penalties if too much money was spent on the (costly) pre- dialytic patients with chronic kidney disease, and loss of patients after referral. This is an unusually frank statement.macro- economic level. Micro- economic considerations in the distribution of health care are only hinted at[The majority of 22 medline hits using the search terms "economy" and "renal disease" address the question of best cost- efficiency of treatment strategies on a Due to the retrospective design of this study, one cannot analyze to what extent the PCPs' economic considerations rather than their educational state were responsible for their referral pattern. The fact that financial concerns were the tenor of the individual discussions, however, indicates that health care planners may profit from taking this into account.The authors hope that this single- centre study will entice multi- centre RRT providers to conduct a prospective, large scale study to further investigate the relative contribution of the factors discussed here.The initiative presented here shows that timely specialist referral in renal care can be achieved. National and regional differences in the organization of health care provision may lead to variant strategies to implement optimal health care, but close collaboration with the primary care providers is essential.ECC: endogenous creatinine clearanceMCKD: moderate chronic renal diseaseMMCKD: mild/moderate chronic renal diseasePCP: primary care providerRRT: renal replacement therapySCKD: severe chronic renal diseaseThe authors declare that they have no competing interests.K.H. started the initiative, carried out all the teaching modules and gave access to the data.T.D.V. retrieved the data from archived filesZ.A.L. did statistical analysisM.L. did statistical analysisA.M. did statistical analysis and wrote the manuscript.The pre-publication history for this paper can be accessed here:


Caenorhabditis elegans. We show tight conservation of the HIF-1/VHL-1/EGL-9 hydroxylase pathway. However, persisting differential gene expression in hif-1 versus hif-1; vhl-1 double mutant worms clearly distinguished HIF-1–independent effects of VHL-1 inactivation. Genomic clustering, predicted functional similarities, and a common pattern of dysregulation in both vhl-1 worms and a set of mutants (dpy-18, let-268, gon-1, mig-17, and unc-6), with different defects in extracellular matrix formation, suggest that dysregulation of these genes reflects a discrete HIF-1–independent function of VHL-1 that is connected with extracellular matrix function.The von Hippel-Lindau (VHL) tumor suppressor functions as a ubiquitin ligase that mediates proteolytic inactivation of hydroxylated α subunits of hypoxia-inducible factor (HIF). Although studies of VHL-defective renal carcinoma cells suggest the existence of other VHL tumor suppressor pathways, dysregulation of the HIF transcriptional cascade has extensive effects that make it difficult to distinguish whether, and to what extent, observed abnormalities in these cells represent effects on pathways that are distinct from HIF. Here, we report on a genetic analysis of HIF-dependent and -independent effects of VHL inactivation by studying gene expression patterns in  Besides its known function of inactivating hypoxia-inducible factor (HIF), genetically engineered worms clearly demonstrate that there exist HIF-independent effects of the von Hippel- Lindau tumor suppressor In herercinomas , the natrcinomas .To date, the best-understood function of VHL is as a ubiquitin ligase that affects oxygen-dependent proteolytic targeting of the α subunits of hypoxia-inducible factor (HIF) . Oxygen-Caenorhabditis elegans. Whereas mammalian cells possess three HIF-α isoforms that are targeted by VHL, C. elegans has a single HIF-α homolog (HIF-1) and a single VHL homolog (VHL-1), simplifying the genetic approach  bearing defects in genes involved in extracellular matrix function, supporting the existence of a conserved non-HIF pathway connecting VHL with an as yet unknown extracellular matrix function.To address this we have used a genetic approach in approach . Since hC. elegans, a whole-genome microarray was probed to compare transcript patterns in vhl-1 versus wild-type worms (n = 1). From this array a set of genes  was assayed quantitatively by ribonuclease (RNase) protection  were strikingly downregulated by VHL-1  and the egg-laying defective phenotype of egl-9, none of the worm strains showed obvious phenotypic abnormalities. Interestingly, hif-1 corrected the phenotype of egl-9.To determine the extent to which disruption of the conserved EGL-9/HIF-1 pathway mediates these effects we studied wild-type, egl-9 worms (hif-1 inactivation abrogated the upregulation by the egl-9 mutation (nhr-57, fmo-12, egl-9, and cah-4) of the six genes contained a potential HIF-1 binding core motif (RCGTG) within an arbitrarily defined region (−1,000 to +250 nucleotides) that was conserved in Caenorhabditis briggsae  expression in normoxia was entirely dependent on HIF-1, whereas other genes retained substantial normoxic expression in hif-1 worms ((nhr-57, egl-9, and cah-4) showed modest upregulation, and one gene (phy-2) showed modest downregulation, by hypoxia that was independent of HIF-1, VHL-1, and EGL-9 , suggesting that the VHL-1–independent repressive effects on nhr-57 may be nonenzymatic.Though the six genes all conformed to the above patterns to demonstrate regulation by the EGL-9/HIF-1 pathway , there w-1 worms A. Second pathway C. Interehif-1; vhl-1 and hif-1 worms (n = 3). Fewer genes showed differential expression than in the vhl-1 versus wild-type array; however, persisting differential expression did suggest the existence of VHL-1 pathways that are independent of HIF-1  contained a potential HIF-1 binding site (HBS) within an arbitrarily defined region that was conserved in C. briggsae (see To address directly whether HIF-1–independent, VHL-1–mediated pathways exist, we performed further microarray comparisons of RNA from of HIF-1 . To teston assay , six (C0on assay B. Computgsae see . This cogsae see A, for whgsae see .p ≪ 10−5 (C. elegans genomic assemblies (for example, WS120) have shown that the entire 110-kb region containing the coregulated gene cluster is arranged in tandem with a second nearly identical segmental duplication of the locus . At this level of identity, our microarray and RNase protection analyses cannot discriminate between the two copies of each gene, so for all of our analyses we have only used the names of the distal copy and genes from the proximal copy were excluded from computational analyses.Interestingly, all six genes validated by RNase protection assay to be negatively regulated by VHL-1 in a HIF-1–independent manner localize within 45 kb on Chromosome V . We applied single-linkage clustering (nearest-neighbor method)  to identp ≪ 10−5 B. Recentegl-9; hif-1 versus hif-1 worms  . Further experiments on dpy-18; hif-1 double mutant worms clearly indicated that the effects of DPY-18 on this group of genes were (like the effects of VHL-1) HIF-1 independent   and the e (DMOG)  were tesgon-1 (heterozygote), mig-17, and unc-6 mutant worms but not in the cuticle-associated dpy-11, bli-4, or sqt-3 mutant worms  that are distinct from protein hydroxylation. These experiments indicated that the six genes were, to varying extents, upregulated in the basement membrane-associated nt worms . Conversnt worms  and SQT-vhl-1 inactivation in different genetic backgrounds, these data clearly distinguish HIF-1–dependent and –independent effects of VHL-1 on gene expression. Somewhat surprisingly, all of the VHL-regulated genes analyzed fell into one of two patterns: independent of HIF-1 and EGL-9 and dependent on DPY-18, LET-268, GON-1, MIG-17, and UNC-6, or the reverse, suggesting that they reflect perturbation of two discrete aspects of VHL-1 function.By comparing the effects of vhl-1 versus wild-type array underlines the importance of the HIF-1 pathway in VHL-1 function. Computational analysis revealed that five of these genes (F22B5.4, nhr-57, fmo-12, egl-9, and cah-4) have at least one HIF-1 binding core motif (RCGTG) that is conserved in C. briggsae within an arbitrarily defined (−1,000 to +250 nucleotides) promoter region, suggesting that they are direct HIF-1 transcriptional targets. Several genes  have mammalian homologs that are HIF targets  regulation by VHL-1 were located within 45 kb on Chromosome V. Analysis of the microarray data revealed that there was indeed a single, highly significant (p < 10−5) chromosomal cluster of genes negatively regulated by VHL-1 in a HIF-1–independent manner and that in total this cluster extended over 110 kb to include F56A4.9, Y45G12C.9, Y45G12C.12, and Y45G12C.2 in addition to the six genes validated by RNase protection assay. The chromosomal localization of genes in C. elegans is not random, with functionally related genes located close to one another   (unc-6 (encoding the extracellular guidance protein, netrin)  , gon-1 aoteases) , and unc netrin) —suggest  netrin) . These aronectin . Most turonectin . Howeverronectin . Though Escherichia coli strain HT115(DE3) expressing double-stranded (ds) RNA on Nematode Growth Medium containing 1 mM isopropyl-β-D-thiogalactopyranoside (ITPG) and 50 μg/ml ampicillin for 72 h. Plasmids for ds RNA production were derivatives of the L4440 vector and were obtained from J. Ahringer (Cambridge, United Kingdom); ds RNA sequences are available on WormBase (http://www.wormbase.org). Wild-type worms were Bristol strain (N2); mutant strains were obtained from the Caenorhabditis Genetics Center (gon-1 worms (maintained at 18 °C). The double mutants hif-1; vhl-1, egl-9; hif-1, and dpy-18; hif-1 were constructed using either fog-2 or unc-51 to mark hif-1(+); the double mutant egl-9; vhl-1 was constructed using unc-42 to mark egl-9(+); PCR was used to confirm homozygosity.Worms were studied as mixed-stage populations or as synchronized populations following brief exposure to sodium hypochlorite. Exposure to hypoxia (2% or 0.1% oxygen), DIP (200 μM), and DMOG (1 mM) was for 18 h . RNA ints Center . Strainsvhl-1 worms and hif-1 versus hif-1; vhl-1 worms were performed on independent samples of RNA (n = 1 and 3, respectively), using near full-genome C. elegans DNA microarrays ; primary array data are available on the SMD (http://genome-www.stanford.edu/microarray) and are also shown in hif-1 versus hif-1; vhl-1 microarray comparisons (n = 3) the log2 fold change was calculated as the mean of the three log2 transformed fold changes. To test for significant upregulation, the mean log2 fold change was compared with zero using a Student's t test. The genes were ranked by amplitude of fold upregulation and a subset of genes was selected for potential validation by RNase protection assays ; (b) mean Cy3-dUTP and Cy5-dUTP background-corrected signal intensities exceeding 300 and 100 U, respectively (lower intensities than these were difficult to detect by RNase protection assay); and (c) high spot quality as judged by manual inspection. For the hif-1 versus hif-1; vhl-1 microarray comparisons (n = 3), genes (which had been filtered as described above) were considered to be differentially expressed if the mean fold change was greater than 2.0  as reciprocal best matches by BLASTN, initiated with the C. elegans gene coding sequence (a single ortholog of F56A4.2 could not be defined). Translation initiation sites  were inferred in both C. briggsae and C. elegans through alignment with the C. elegans coding sequence (WormBase, WS117). Sequences encompassing the 1,000 nucleotides upstream to 250 nucleotides downstream of the translation start sites for orthologous genes were aligned using DNA Block Aligner (C. elegans and C. briggsae.(1) Identification of potential HBSs see . Ortholo Aligner  with the Aligner , identifhttp://genome.ucsc.edu/). The software used for simulations and clustering was implemented in Perl and is available on request.(2) Single-linkage analysis to determine spatial clusters of VHL-1–dependent, HIF-1–independent genes see . A maximhttp://genome-www.stanford.edu/microarray.Primary microarray data can be viewed at Table S1vhl-1 (green, channel 1) versus wild-type (red, channel 2) comparison.Primary microarray data for the (6.5 MB XLS).Click here for additional data file.Table S2hif-1; vhl-1 (green, channel 1) versus hif-1 (red, channel 2) microarray comparisons. Continued in Tables Primary microarray data for the three independent (6.6 MB XLS).Click here for additional data file.Table S3Continuation of (6.6 MB XLS).Click here for additional data file.Table S4Continuation of Tables (6.6 MB XLS).Click here for additional data file.http://www.ebi.ac.uk/arrayexpress/) under accession number E-SMDB-23.Primary array data have been deposited in ArrayExpress (H. sapiens VHL gene discussed in this paper can be found in Online Mendelian Inheritance in Man (OMIM) under accession number 608537 (http://www.ncbi.nlm.nih.gov:80/entrez/dispomim.cgi?id=608537).The C. elegans genes discussed in this paper (bli-4, C01B4.6, C01B4.7, C01B4.8, C01B4.9, cah-4, dpy-10, dpy-11, dpy-18, egl-9, F21C3.5, F22B5.4, F56A4.2, F56A4.9, F56A4.10, fmo-12, fog-2, gon-1, hif-1, let-268, mig-17, nhr-57, phy-2, phy-3, sqt-3, unc-4, unc-6, unc-42, unc-51, unc-52, vhl-1, Y45G12C.2, Y45G12C.9, and Y45G12C.12) can be found in the WormBase database by including the name of the gene at the end of the URL (e.g., for bli-4,http://wormbase.org/db/gene/gene?name=bli-4).The


When pathogens enter your body, most wind up engulfed (by phagocytes), poisoned (by stomach acids), or flushed out of your system. These defenses kick in when a bacterium's toxic secretions bind to cellular receptors that trigger a chain of events ending in an innate response. A wide variety of cells and chemicals, including phagocytes and cytokines, initiate the inflammatory reaction that redirects innate defenses in the bloodstream to the infected site.If all goes well, the innate system successfully contains and eliminates a pathogen at the site of infection. But if infection persists, so will the inflammatory response, and these first responders turn traitor. It's thought that the immune response rather than the bacteria, for example, causes diarrhea in food poisoning. And when infection leads to death—as happens in septic shock—the immune response may be just as culpable as the infectious microbe.Drosophila and the food-borne pathogen Salmonella. Working with mutant strains of both fly and bacteria, the authors identified genes important to the development of infection and disease and showed that the host's reaction can indeed be lethal.To better understand the interplay between pathogen and host in the onset of infection and disease, David Schneider and colleagues turned to the genetically compliant fruitfly Drosophila phagocytes find, engulf, and kill invading microbes and then alert the rest of the immune system—and how Salmonella circumvents these defenses to initiate disease.Pathogens possess various means to infect their host, unleashing toxins and secreting molecules that enhance virulence by breaching cell membranes and altering the intracellular environment. Fruitflies combat these intrusions with various innate responses, including phagocytosis. In this study, the authors investigated how Salmonella strain (S. tyhphimurium) that produces two pathogenicity complexes, called type III secretion apparatuses, which shuttle virulence molecules through the host's cell membrane and into its cytoplasm. One complex, SPI1, facilitates cell entry while the other, SPI2, retools the intracellular environment to suit bacterial growth. The authors created a series of less virulent Salmonella strains and examined their effects on wild-type (nonmutant) flies. In addition, they looked at infections of wild-type Salmonella in flies carrying mutations in two critical immune response pathways .Schneider and colleagues used a Salmonella died much faster than the Toll mutants and had far more bacteria in their blood. When Schneider and colleagues correlated Salmonella population numbers with fly fate, they discovered something surprising. In other bacteria models, flies die when bacterial pathogen numbers reach a critical mass. Here, Salmonella populations hit a ceiling and the flies died with comparatively few bacteria. In flies infected with Salmonella strains lacking either one or the other virulence complex, the flies survived despite increased bacterial growth.Imd mutants infected with nonmutant eiger gene—a homolog of the human TNF gene—live longer with Salmonella infections. This is the same phenomenon seen in TNF-induced septic shock, when patients die as much from their immune system's response to infection as from the bacteria itself. Since many of the proteins involved in the Drosophila imd pathway have counterparts in the mammalian antibacterial immune response, the model described here can help identify the genetic agents of metabolic collapse associated with bacterial infections in humans.The authors explain this surprising finding with a model in which the fly's immune response produces substances that ultimately engineer its own destruction. Their model is supported by experiments showing that flies carrying a mutation in the