Extracellular calreticulin is present in the joints of patients with rheumatoid arthritis and inhibits FasL (CD95L)-mediated apoptosis of T cells

addresses: Peninsula College of Medicine and Dentistry, and University of Exeter, Exeter, UK.types: Journal Article; Research Support, Non-U.S. Gov'tThe definitive version is available at www3.interscience.wiley.comThe binding of FasL (CD95L) to its receptor, Fas (CD95), induces apoptosis. Studies have shown that in patients with rheumatoid arthritis (RA), T lymphocytes are resistant to FasL-induced apoptosis in vivo but are susceptible to FasL-induced apoptosis in vitro. Dysfunction in this mechanism may be an important contributor to the pathophysiology of RA. Thus, the present study was undertaken to determine which factors might inhibit FasL-Fas binding in vivo and those that would inhibit apoptosis of T lymphocytes in an in vitro model system

Transcriptomic analysis of enteropathy in Zambian children with severe acute malnutrition.

BACKGROUND: Children with severe acute malnutrition (SAM), with or without diarrhoea, often have enteropathy, but there are few molecular data to guide development of new therapies. We set out to determine whether SAM enteropathy is characterised by specific transcriptional changes which might improve understanding or help identify new treatments. METHODS: We collected intestinal biopsies from children with SAM and persistent diarrhoea. mRNA was extracted from biopsies, sequenced, and subjected to a progressive set of complementary analytical approaches: NOIseq, Gene Set Enrichment Analysis (GSEA), and correlation analysis of phenotypic data with gene expression. FINDINGS: Transcriptomic profiles were generated for biopsy sets from 27 children of both sexes, under 2 years of age, of whom one-third were HIV-infected. NOIseq analysis, constructed from phenotypic group extremes, revealed 66 differentially expressed genes (DEGs) out of 21,386 mapped to the reference genome. These DEGs include genes for mucins and mucus integrity, antimicrobial defence, nutrient absorption, C-X-C chemokines, proteases and anti-proteases. Phenotype - expression correlation analysis identified 1221 genes related to villus height, including increased cell cycling gene expression in more severe enteropathy. Amino acid transporters and ZIP zinc transporters were specifically increased in severe enteropathy, but transcripts for xenobiotic metabolising enzymes were reduced. INTERPRETATION: Transcriptomic analysis of this rare collection of intestinal biopsies identified multiple novel elements of pathology, including specific alterations in nutrient transporters. Changes in xenobiotic metabolism in the gut may alter drug disposition. Both NOIseq and GSEA identified gene clusters similar to those differentially expressed in pediatric Crohn's disease but to a much lesser degree than those identified in coeliac disease. FUND: Bill & Melinda Gates Foundation OPP1066118. The funding agency had no role in study design, data collection, data analysis, interpretation, or writing of the report

Gene expression profiles compared in environmental and malnutrition enteropathy in Zambian children and adults

Background:: Environmental enteropathy (EE) contributes to growth failure in millions of children worldwide, but its relationship to clinical malnutrition has not been elucidated. We used RNA sequencing to compare duodenal biopsies from adults and children with EE, and from children with severe acute malnutrition (SAM), to define key features of these malnutrition-related enteropathies. Methods:: RNA was extracted and sequenced from biopsies of children with SAM in hospital (n=27), children with non-responsive stunting in the community (n=30), and adults living in the same community (n=37) using an identical sequencing and analysis pipeline. Two biopsies each were profiled and differentially expressed genes (DEGs) were computed from the comparisons of the three groups. DEG lists from these comparisons were then subjected to analysis with CompBio software to assemble a holistic view of the biological landscape and IPA software to interrogate canonical pathways. Findings:: Dysregulation was identified in goblet cell/mucin production and xenobiotic metabolism/detoxification for both cohorts of children, versus adults. Within the SAM cohort, substantially greater induction of immune response and barrier function, including NADPH oxidases was noted, concordant with broadly reduced expression of genes associated with the brush border and intestinal structure/transport/absorption. Interestingly, down regulation of genes associated with the hypothalamic-pituitary-adrenal axis was selectively observed within the cohort of children with stunting. Interpretation:: Gene expression profiles in environmental enteropathy and severe acute malnutrition have similarities, but SAM has several distinct transcriptional features. The intestinal capacity to metabolise drugs and toxins in malnourished children requires further study. Funding:: Bill & Melinda Gates Foundation (OPP1066118

Virulence profiling and quantification of verocytotoxin-producing Escherichia coli O145:H28 and O26:H11 isolated during an ice cream-related hemolytic uremic syndrome outbreak

In September-October 2007, a mixed-serotype outbreak of verocytotoxin-producing Escherichia coli (VTEC) O145:H28 and O26:H11 occurred in the province of Antwerp, Belgium. Five girls aged between 2 and 11 years developed hemolytic uremic syndrome, and seven other coexposed persons with bloody diarrhea were identified. Laboratory confirmation of O145:H28 infection was obtained for three hemolytic uremic syndrome patients, one of whom was coinfected with O26:H11. The epidemiological and laboratory investigations revealed ice cream as the most likely source of the outbreak. The ice cream was produced at a local dairy farm using pasteurized milk. VTEC of both serotypes with indistinguishable pulsed-field gel electrophoresis patterns were isolated from patients, ice cream, and environmental samples. Quantitative analysis of the ice cream indicated concentrations of 2.4 and 0.03 CFU/g for VTEC O145 and O26, respectively. Virulence typing revealed that the repertoire of virulence genes carried by the O145:H28 outbreak strain was comparable to that of O157 VTEC and more exhaustive as compared to the O26:H11 outbreak strain and nonrelated clinical strains belonging to these serotypes. Taken together, these data suggest that O145:H28 played the most important role in this outbreak

Shiga Toxin-Mediated Hemolytic Uremic Syndrome: Time to Change the Diagnostic Paradigm?

Hemolytic uremic syndrome (HUS) is caused by enterohemorrhagic Escherichia coli (EHEC) which possess genes encoding Shiga toxin (stx), the major virulence factor, and adhesin intimin (eae). However, the frequency of stx-negative/eae-positive E. coli in stools of HUS patients and the clinical significance of such strains are unknown.Between 1996 and 2006, we sought stx-negative/eae-positive E. coli in stools of HUS patients using colony blot hybridization with the eae probe and compared the isolates to EHEC causing HUS. stx-negative/eae-positive E. coli were isolated as the only pathogens from stools of 43 (5.5%) of 787 HUS patients; additional 440 (55.9%) patients excreted EHEC. The majority (90.7%) of the stx-negative/eae-positive isolates belonged to serotypes O26:H11/NM (nonmotile), O103:H2/NM, O145:H28/NM, and O157:H7/NM, which were also the most frequent serotypes identified among EHEC. The stx-negative isolates shared non-stx virulence and fitness genes with EHEC of the corresponding serotypes and clustered with them into the same clonal complexes in multilocus sequence typing, demonstrating their close relatedness to EHEC.At the time of microbiological analysis, approximately 5% of HUS patients shed no longer the causative EHEC, but do excrete stx-negative derivatives of EHEC that lost stx during infection. In such patients, the EHEC etiology of HUS is missed using current methods detecting solely stx or Shiga toxin; this can hamper epidemiological investigations and lead to inappropriate clinical management. While maintaining the paradigm that HUS is triggered by Shiga toxin, our data demonstrate the necessity of considering genetic changes of the pathogen during infection to adapt appropriately diagnostic strategies

The host metabolite D-serine contributes to bacterial niche specificity through gene selection

Escherichia coli comprise a diverse array of both commensals and niche-specific pathotypes. The ability to cause disease results from both carriage of specific virulence factors and regulatory control of these via environmental stimuli. Moreover, host metabolites further refine the response of bacteria to their environment and can dramatically affect the outcome of the host–pathogen interaction. Here, we demonstrate that the host metabolite, D-serine, selectively affects gene expression in E. coli O157:H7. Transcriptomic profiling showed exposure to D-serine results in activation of the SOS response and suppresses expression of the Type 3 Secretion System (T3SS) used to attach to host cells. We also show that concurrent carriage of both the D-serine tolerance locus (dsdCXA) and the locus of enterocyte effacement pathogenicity island encoding a T3SS is extremely rare, a genotype that we attribute to an ‘evolutionary incompatibility’ between the two loci. This study demonstrates the importance of co-operation between both core and pathogenic genetic elements in defining niche specificity

Serotypes, virulence genes and intimin types of Shiga toxin (verocytotoxin)-producing Escherichia coli isolates from minced beef in Lugo (Spain) from 1995 through 2003

BACKGROUND: Shiga toxin-producing Escherichia coli (STEC) have emerged as pathogens that can cause food-borne infections and severe and potentially fatal illnesses in humans, such as haemorrhagic colitis (HC) and haemolytic uraemic syndrome (HUS). In Spain, like in many other countries, STEC strains have been frequently isolated from ruminants, and represent a significant cause of sporadic cases of human infection. In view of the lack of data on STEC isolated from food in Spain, the objectives of this study were to determine the level of microbiological contamination and the prevalence of STEC O157:H7 and non-O157 in a large sampling of minced beef collected from 30 local stores in Lugo city between 1995 and 2003. Also to establish if those STEC isolated from food possessed the same virulence profiles as STEC strains causing human infections. RESULTS: STEC were detected in 95 (12%) of the 785 minced beef samples tested. STEC O157:H7 was isolated from eight (1.0%) samples and non-O157 STEC from 90 (11%) samples. Ninety-six STEC isolates were further characterized by PCR and serotyping. PCR showed that 28 (29%) isolates carried stx(1 )genes, 49 (51%) possessed stx(2 )genes, and 19 (20%) both stx(1 )and stx(2). Enterohemolysin (ehxA) and intimin (eae) virulence genes were detected in 43 (45%) and in 25 (26%) of the isolates, respectively. Typing of the eae variants detected four types: γ1 (nine isolates), β1 (eight isolates), ε1 (three isolates), and θ (two isolates). The majority (68%) of STEC isolates belonged to serotypes previously detected in human STEC and 38% to serotypes associated with STEC isolated from patients with HUS. Ten new serotypes not previously described in raw beef products were also detected. The highly virulent seropathotypes O26:H11 stx(1 )eae-β1, O157:H7 stx(1)stx(2 )eae-γ1 and O157:H7 stx(2)eae-γ1, which are the most frequently observed among STEC causing human infections in Spain, were detected in 10 of the 96 STEC isolates. Furthermore, phage typing of STEC O157:H7 isolates showed that the majority (seven of eight isolates) belonged to the main phage types previously detected in STEC O157:H7 strains associated with severe human illnesses. CONCLUSION: The results of this study do not differ greatly from those reported in other countries with regard to prevalence of O157 and non-O157 STEC in minced beef. As we suspected, serotypes different from O157:H7 also play an important role in food contamination in Spain, including the highly virulent seropathotype O26:H11 stx(1 )eae-β1. Thus, our data confirm minced beef in the city of Lugo as vehicles of highly pathogenic STEC. This requires that control measures to be introduced and implemented to increase the safety of minced beef

High frequency of Human Cytomegalovirus DNA in the Liver of Infants with Extrahepatic Neonatal Cholestasis

BACKGROUND: Biliary atresia (BA) is the most severe hepatic disorder in newborns and its etiopathogenesis remains unknown. Viral involvement has been proposed, including the human cytomegalovirus (HCMV). The aims of the study were to use the polymerase chain reaction (PCR) to screen the liver tissue of infants with extrahepatic cholestasis for HCMV and to correlate the results with serological antibodies against HCMV and histological findings. METHODS: A retrospective study in a tertiary care setting included 35 patients (31 BA, 1 BA associated with a choledochal cyst, 2 congenital stenosis of the distal common bile duct and 1 hepatic cyst). HCMV serology was determined by ELISA. Liver and porta hepatis were examined histologically. Liver samples from infants and a control group were screened for HCMV DNA. RESULTS: Twelve patients had HCMV negative serology, 9 were positive for IgG antibodies and 14 were positive for IgG and IgM. Nine liver and seven porta hepatis samples were positive for HCMV DNA but none of the control group were positive (general frequency of positivity was 34.3% – 12/35). There was no correlation between HCMV positivity by PCR and the histological findings. The accuracy of serology for detecting HCMV antibodies was low. CONCLUSION: These results indicate an elevated frequency of HCMV in pediatric patients with extrahepatic neonatal cholestasis. They also show the low accuracy of serological tests for detecting active HCMV infection and the lack of correlation between HCMV positivity by PCR and the histopathological changes