Interleukin-1ß (IL-1ß) promotes susceptibility of Toll-like receptor 5 (TLR5) deficient mice to colitis.

BACKGROUND: The extent to which numerous strains of genetically engineered mice, including mice lacking Toll-like receptor 5 (T5KO), display colitis is environment dependent. Gut microbiota underlie much of the variation in phenotype. Accordingly, embryonic rederivation of T5KO mice ameliorated their spontaneous colitis despite only partially correcting elevated proinflammatory gene expression. It was postulated that endogenous anti-inflammatory pathways mediated the absence of overt inflammation in these mice when their gut microbiota were reset. Consequently, it was hypothesised that neutralisation of the anti-inflammatory cytokine interleukin 10 (IL-10) might induce uniform colitis in T5KO mice, and thus provide a practical means to study mechanisms underlying their inflammation. METHODS: Two distinct strains of non-colitic T5KO mice, as well as mice lacking MyD88, Toll-like receptor 4 (TLR4), IL-1 receptor (IL-1R) and various double knockouts (DKOs) were treated weekly for 4 weeks with 1 mg/mouse of IL-10 receptor neutralising antibody (IL-10R mAb) and colitis assayed 1 week later. The composition of the caecal microbiota was determined by 454 pyrosequencing of 16S rRNA genes. RESULTS: Anti-IL-10R mAb treatment led to severe uniform intestinal inflammation in both strains of T5KO mice. Such neutralisation of IL-10 signalling did not cause colitis in wild-type littermates nor mice lacking TLR4, MyD88 or IL-1R. The susceptibility of T5KO mice to this colitis model was not rescued by absence of TLR4 in that T4/T5 DKO mice displayed severe colitis in response to anti-IL-10R mAb treatment. IL-1ß signalling was crucial for this colitis model in that IL-1R/T5 DKOs were completely protected from colitis in response to IL-10R mAb treatment. Lastly, it was observed that blockade of IL-10R function was associated with changes in the composition of gut microbiota, which were observed in mice that were susceptible and resistant to IL-10R mAb-induced colitis. CONCLUSION: Regardless of whether they harbour a colitogenic microbiota, loss of TLR5 predisposes mice to colitis triggered by immune dysregulation via an IL-1ß-dependent pathway.

Comparing outcomes of biopsy-proven anti-neutrophil cytoplasmic autoantibody-associated glomerulonephritis patients treated with cyclophosphamide in the 20th and 21st centuries: a 23-year study.

BACKGROUND: Anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis (AAV) is a multisystem autoimmune disorder associated with significant morbidity and mortality. Approximately 80-90% of patients have circulating ANCAs. Long-term outcomes appear to be improving. This retrospective study analyses the incidence and patient outcomes over a period of 23 years at a single tertiary centre. METHODS: Outcomes of patients diagnosed with AAV between 1 January 1988 and 31 December 2010 were collected retrospectively. Data including patient demographics, age of diagnosis, dates of starting renal replacement therapy, death and biochemistry results were collected. Patients were divided into two cohorts (1988-99 and 2000-10) and analysed using Stata software (StataCorp, College Station, TX, USA). RESULTS: A complete dataset was obtained for 273 patients. Of these patients, 101 were diagnosed between 1988 and 1999 while 172 were diagnosed between 2000 and 2010. The number of patients diagnosed with AAV increased from 2.2/million in 1988 to 10.3/million in 2010. A higher proportion of patients (56.4%) in the earlier cohort presented with creatinine >500 µmol/L compared with the later cohort (30.2%; P < 0.001). Overall patient survival improved significantly between the two cohorts. Cohort 1 had a median survival of 59 months compared with 125 months for Cohort 2 (P = 0.003). CONCLUSIONS: This study shows that AAV is being diagnosed at an earlier stage, resulting in improved outcomes. This may be because of improvements in the management of AAV and chronic kidney disease.

Factors predisposing nursing home resident to inappropriate transfer to emergency department. The FINE study protocol.

BACKGROUND: Each year, around one out of two nursing home (NH) residents are hospitalized in France, and about half to the emergency department (ED). These transfers are frequently inappropriate. This paper describes the protocol of the FINE study. The first aim of this study is to identify the factors associated with inappropriate transfers to ED. METHODS/DESIGN: FINE is a case-control observational study. Sixteen hospitals participate. Inclusion period lasts 7 days per season in each center for a total period of inclusion of one year. All the NH residents admitted in ED during these periods are included. Data are collected in 4 times: before transfer in the NH, at the ED, in hospital wards in case of patient's hospitalization and at the patient's return to NH. The appropriateness of ED transfers (i.e. case versus control NH residents) is determined by a multidisciplinary team of experts. RESULTS: Our primary objective is to determine the factors predisposing NH residents to inappropriate transfer to ED. Our secondary objectives are to assess the cost of the transfers to ED; study the evolution of NH residents' functional status and the psychotropic and inappropriate drugs prescription between before and after the transfer; calculate the prevalence of potentially avoidable transfers to ED; and identify the factors predisposing NH residents to potentially avoidable transfer to ED. DISCUSSION: A better understanding of the determinant factors of inappropriate transfers to ED of NH residents may lead to proposals of recommendations of better practice in NH and would allow implementing quality improvement programs in the health organization.

Quantitation of 35S promoter in maize DNA extracts from genetically modified organisms using real-time polymerase chain reaction, part 2: interlaboratory study.

The European Committee for Standardization (CEN) and the European Network of GMO Working Laboratories have proposed development of a modular strategy for stepwise validation of complex analytical techniques. When applied to the quantitation of genetically modified organisms (GMOs) in food products, the instrumental quantitation step of the technique is separately validated from the DNA extraction step to better control the sources of uncertainty and facilitate the validation of GMO-specific polymerase chain reaction (PCR) tests. This paper presents the results of an interlaboratory study on the quantitation step of the method standardized by CEN for the detection of a regulatory element commonly inserted in GMO maize-based foods. This is focused on the quantitation of P35S promoter through using the quantitative real-time PCR (QRT-PCR). Fifteen French laboratories participated in the interlaboratory study of the P35S quantitation operating procedure on DNA extract samples using either the thermal cycler ABI Prism 7700 (Applied Biosystems, Foster City, CA) or Light Cycler (Roche Diagnostics, Indianapolis, IN). Attention was focused on DNA extract samples used to calibrate the method and unknown extract samples. Data were processed according to the recommendations of ISO 5725 standard. Performance criteria, obtained using the robust algorithm, were compared to the classic data processing after rejection of outliers by the Cochran and Grubbs tests. Two laboratories were detected as outliers by the Grubbs test. The robust precision criteria gave values between the classical values estimated before and after rejection of the outliers. Using the robust method, the relative expanded uncertainty by the quantitation method is about 20% for a 1% Bt176 content, whereas it can reach 40% for a 0.1% Bt176. The performances of the quantitation assay are relevant to the application of the European regulation, which has an accepted tolerance interval of about +/-50%. These data were fitted to a power model (r2 = 0.96). Thanks to this model, it is possible to propose an estimation of uncertainty of the QRT-PCR quantitation step and an uncertainty budget depending on the analytical conditions.

Characterization and event specific-detection by quantitative real-time PCR of T25 maize insert.

T25 is one of the 4 maize transformation events from which commercial lines have so far been authorized in Europe. It was created by polyethylene glycol-mediated transformation using a construct bearing one copy of the synthetic pat gene associated with both promoter and terminator of the 35S ribosomal gene from cauliflower mosaic virus. In this article, we report the sequencing of the whole T25 insert and the characterization of its integration site by using a genome walking strategy. Our results confirmed that one intact copy of the initial construct had been integrated in the plant genome. They also revealed, at the 5' junction of the insert, the presence of a second truncated 35S promoter, probably resulting from rearrangements which may have occurred before or during integration of the plasmid DNA. The analysis of the junction fragments showed that the integration site of the insert presented high homologies with the Huck retrotransposon family. By using one primer annealing in the maize genome and the other in the 5' end of the integrated DNA, we developed a reliable event-specific detection system for T25 maize. To provide means to comply with the European regulation, a real-time PCR test was designed for specific quantitation of T25 event by using Taqman chemistry.

Quantification of the 35S promoter in DNA extracts from genetically modified organisms using real-time polymerase chain reaction and specificity assessment on various genetically modified organisms, part I: operating procedure.

A highly sensitive quantitative real-time assay targeted on the 35S promoter of a commercial genetically modified organism (GMO) was characterized (sF/sR primers) and developed for an ABI Prism 7700 Sequence Detection System and TaqMan chemistry. The specificity assessment and performance criteria of sF/sR assay were compared to other P35S-targeted published assays. sF/sR primers amplified a 79 base pair DNA sequence located in a part of P35S that is highly conserved among many caulimovirus strains, i.e., this consensus part of CaMV P35S is likely to be present in many GM events. According to the experimental conditions, the absolute limit of detection for Bt176 corn was estimated between 0.2 and 2 copies of equivalent genome (CEG). The limit of quantification was reached below 0.1% Bt176 content. A Cauliflower Mosaic Virus control (CaMV) qualitative assay targeted on the ORF III of the viral genome was also used as a control (primers 3F/3R) to assess the presence of CaMV in plant-derived products. The specificity of this test was assessed on various CaMV strains, including the Figwort Mosaic Virus (FMV) and solanaceous CaMV strains. Considering the performance of sF/sR quantification test, the highly conserved sequence, and the small size of the amplicon, this assay was tested in a collaborative study in order to be proposed as an international standard.

A microarray-based detection system for genetically modified (GM) food ingredients.

A multiplex DNA microarray chip was developed for simultaneous identification of nine genetically modified organisms (GMOs), five plant species and three GMO screening elements, i.e. the 35S promoter, the nos terminator and the nptII gene. The chips also include several controls, such as that for the possible presence of CaMV. The on-chip detection was performed directly with PCR amplified products. Particular emphasis was placed on the reduction of the number of PCR reactions required and on the number of primers present per amplification tube. The targets were biotin labelled and the arrays were detected using a colorimetric methodology. Specificity was provided by specific capture probes designed for each GMO and for the common screening elements. The sensitivity of the assay was tested by experiments carried out in five different laboratories. The limit of detection was lower than 0.3% GMO for all tests and in general around 0.1% for most GMOs. The chip detection system complies with the requirements of current EU regulations and other countries where thresholds are established for the labelling of GMO.