In vitro organ culture (IVOC) represents a gold standard model to study enteropathogenic E. coli (EPEC) infection of human intestinal mucosa. However, the optimal examination of the bacterial-host ...cell interaction requires a directional epithelial exposure, without serosal or cut surface stimulation. A polarized IVOC system (pIVOC) was developed in order to overcome such limitations: apical EPEC infection produced negligible bacterial leakage via biopsy edges, resulted in enhanced colonization compared with standard IVOC, and showed evidence of bacterial detachment, as in natural rabbit EPEC infections. Examination of mucosal innate immune responses in pIVOC showed both interleukin (IL)-8 mRNA and protein levels were significantly increased after apical EPEC infection. Increased IL-8 levels mainly depended on flagellin expression as fliC-negative EPEC did not elicit a significant IL-8 response despite increased mucosal colonization compared with wild-type EPEC. In addition, apical application of purified flagella significantly increased IL-8 protein levels over non-infected controls. Immunofluorescence staining of EPEC-infected small intestinal biopsies revealed apical and basolateral distribution of Toll-like receptor (TLR) 5 on epithelium, suggesting that EPEC can trigger mucosal IL-8 responses by apical flagellin/TLR5 interaction ex vivo and does not require access to the basolateral membrane as postulated in cell culture models.
We used bovine intestinal organ culture to study infection by enterohemorrhagic Escherichia coli serogroups O157, O26, and O111. We show colonization and attaching and effacing lesion formation on ...explants derived from the ileum, colon, and rectum. Intimin and Tir were detected at the sites of adherent bacteria; Tir was essential for colonization.
1 Centre for Paediatric Gastroenterology, Royal Free Hospital, Imperial College, London, UK
2 Division of Cell and Molecular Biology, Imperial College, London, UK
Correspondence Alan D. Phillips ...a.phillips{at}medsch.ucl.ac.uk
Enterohaemorrhagic Escherichia coli (EHEC) are an important cause of diarrhoeal and renal disease in man. Studies of a single prototypic O157 : H7 strain have shown tropism for follicle-associated epithelium (FAE) of distal ileal Peyer's patches without colonization of either small or large intestine. This study determined tropism in a range of Shiga toxin (Stx)-negative EHEC strains and looked for factors that might induce colonic colonization using human in vitro intestinal organ culture (IVOC). An FAE-restricted colonization was confirmed in two strains; four strains additionally colonized ileal villous surfaces, and adhesion to proximal small intestinal FAE was observed. All strains showed minimal adhesion to non-FAE regions of proximal small intestinal and to the transverse colon. Extensive large-bowel IVOC studies using three O157 : H7 strains, an O26 : H11 and an O103 : H2 strain, and tissue from caecum to rectum found colonization and attaching/effacing lesion formation in only 4 of 113 (3.5 %) IVOCs. Colonic adhesion was not enhanced by altering the IVOC technique or environment. Co-incubation of O157 : H7-infected ileal FAE with colonic samples enhanced colonic colonization, producing a novel, non-intimate adhesive phenotype. Thus, in the initial stages of colonization Stx-negative EHEC preferentially infect FAE and villi of the terminal ileal region ex vivo ; colonic colonization is infrequently observed as an initial event but may represent a subsequent stage of infection.
Abbreviations: A/E, attaching/effacing; D4, fourth part of duodenum; EAEC, enteroaggregative Escherichia coli ; EHEC, enterohaemorrhagic Escherichia coli ; EPEC, enteropathogenic Escherichia coli ; FAE, follicle-associated epithelium; IVOC, in vitro organ culture; LEE, locus of enterocyte effacement; Lpf, long polar fimbriae; PP, Peyer's patches; REPEC, rabbit enteropathogenic E coli ; SEM, scanning electron microscopy; Stx, Shiga toxin
These authors contributed equally to this work.
Autosomal recessive, congenital chloride diarrhea (CLD) is a form of persistent secretory diarrhea, presenting with polyhydramnios and intractable diarrhea from birth. CLD is caused by mutations in ...the SLC26A3 gene, encoding a Na+-independent Cl/HCO3- exchanger. The diagnosis is generally made on the basis of high fecal chloride concentration in patients with serum electrolyte homoeostasis corrected by salt substitution. We aimed to evaluate the role of diagnostic genetic testing in CLD.
Clinical and laboratory data were collected from 8 unrelated children diagnosed as having or suspected to have CLD. The evaluation included physical examination, routine clinical chemistry, and SLC26A3 mutation analysis by direct sequencing of DNA extracted from buccal swabs or peripheral leukocytes.
CLD was initially diagnosed on high fecal chloride concentrations in 7 patients, and by mutation analysis in 1 patient. In 3 of these patients the correct diagnosis was made more than 6 months after birth. We identified SLC26A3 mutations on both alleles in all 8 patients with CLD, including 3 novel missense and 4 novel truncating mutations. We present a compilation of reported SLC26A3 mutations and polymorphisms.
The diagnosis and therapy of CLD were considerably delayed in 3 of 8 patients from this series, highlighting the potential of misdiagnosing CLD. We add 7 novel mutations, including 3 missense changes of highly conserved residues to a total of 41 mutations in this gene. Molecular analysis is efficient and should be considered as a means of early diagnosis of CLD, especially if the clinical diagnosis remains uncertain.
Abstract Background A rapid assessment (RA) tool was developed to rapidly and inexpensively identify key areas of weakness and to yield an overall performance score for vital registration (VR) ...systems. This standardised tool has now been applied in more than 70 countries, but there has been no formal evaluation of its validity or relationship with measures of socioeconomic development. Similarly, there is very little empirical research on whether better VR systems are associated with better health outcomes. We aimed to compare the RA scores to independent assessments of VR completeness and data quality; explore the relationship between the RA results and selected national socioeconomic indicators; and investigate how the RA scores correlate with key mortality indicators, controlling for levels of national income and other variables. Methods We regressed the overall scores (%) of the RA from 70 countries on a series of development indicators, including national income and education. We measured the correlation between overall and sub-component RA scores against alternative estimates of national VR completeness and quality. We regressed RA scores against child and adult mortality, controlling for national income. Findings The RA scores were consistent with external measures of completeness and quality and were robustly correlated with a number of indicators of socioeconomic development, confirming their predictive value for assessing VR systems. Completeness of civil registration was associated with better health outcomes, independent of level of national income. Interpretation Our research demonstrates the utility of RA as an overall system indicator, as well as its predictive validity in relation to development indicators. VR is not only a by-product of development that will come about organically along with economic, governance, and political maturity, but also contributes directly and indirectly to desirable development outcomes, including better health. The close correlation between assessment scores with widely available development indices enhances the utility of the tool for guiding civil registration and vital statistics development strategies. The correlation between VR systems functionality and key health and development outcomes supports our hypothesis that VR is not merely a source of data but is a driver of development and improved health status in its own right. Funding Australian Overseas Aid.
Intestinal colonization by enteropathogenic and enterohemorrhagic Escherichia coli requires the locus of enterocyte effacement-encoded type III secretion system. We report that NleC and NleD are ...translocated into host cells via this system. Deletion mutants induced attaching and effacing lesions in vitro, while infection of calves or lambs showed that neither gene was required for colonization.
The human pathogen enterohemorrhagic Escherichia coli (EHEC) O157:H7 colonizes human and animal gut via formation of attaching and effacing lesions. EHEC strains use a type III secretion system to ...translocate a battery of effector proteins into the mammalian host cell, which subvert diverse signal transduction pathways implicated in actin dynamics, phagocytosis, and innate immunity. The genomes of sequenced EHEC O157:H7 strains contain two copies of the effector protein gene nleH, which share 49% sequence similarity with the gene for the Shigella effector OspG, recently implicated in inhibition of migration of the transcriptional regulator NF-κB to the nucleus. In this study we investigated the role of NleH during EHEC O157:H7 infection of calves and lambs. We found that while EHEC ΔnleH colonized the bovine gut more efficiently than the wild-type strain, in lambs the wild-type strain exhibited a competitive advantage over the mutant during mixed infection. Using the mouse pathogen Citrobacter rodentium, which shares many virulence factors with EHEC O157:H7, including NleH, we observed that the wild-type strain exhibited a competitive advantage over the mutant during mixed infection. We found no measurable differences in T-cell infiltration or hyperplasia in colons of mice inoculated with the wild-type or the nleH mutant strain. Using NF-κB reporter mice carrying a transgene containing a luciferase reporter driven by three NF-κB response elements, we found that NleH causes an increase in NF-κB activity in the colonic mucosa. Consistent with this, we found that the nleH mutant triggered a significantly lower tumor necrosis factor alpha response than the wild-type strain.
Enterotoxin-producing Staphylococcus aureus may cause severe inflammatory intestinal disease, particularly in infants or immunodeficient or elderly patients. They are also recognized to be associated ...with sudden infant death syndrome. Little is known, however, about mucosal responses to staphylococci.
The mucosal lesion in three infants with staphylococcal enterocolitis was assessed by immunohistochemistry and electron microscopy. The organisms underwent extensive molecular analysis. Their toxins were assessed for capacity to induce T-cell activation and host mucosal responses examined by in vitro organ culture. Epithelial responses were studied by coculture with HEp-2 and Caco-2 cells.
Intestinal biopsies from the patients showed marked epithelial damage with mucosal inflammation. The three staphylococci, representing two distinct clones, were methicillin-sensitive, producing SEG/I enterotoxins and Rho-inactivating EDIN toxins. Their enterotoxins potently activated T cells, but only whole organisms could induce in vitro enteropathy, characterized by remarkable epithelial desquamation uninhibited by tacrolimus. EDIN-producing staphylococci, but not their supernatants, induced striking cytopathy in HEp-2 epithelial cells but not in Caco-2 cells. Although HEp-2 and Caco-2 cells produced similar IL-8, CCL20, and cathelicidin LL37 responses upon bacterial exposure, only Caco-2 cells expressed mRNA for the β-defensins HBD2 and HBD3, while HEp-2 cells were unable to do so.
Staphylococci induce enterocolitis by a combination of direct enterocyte cytopathy mediated by EDIN toxins, disrupting the epithelial barrier, and enterotoxin superantigen-induced mucosal T-cell activation. Gut epithelial production of β-defensins may contribute to host defense against invasive staphylococcal disease.
Shiga toxins are associated with haemolytic uraemic syndrome but human intestinal epithelium does not express the Gb3 receptor. We describe Gb3 expression and Shiga toxin binding in histologically ...normal intestine and demonstrate that the pattern is unaltered in inflammatory disease states. Gb3 expression and Shiga toxin binding were identified in Paneth cells in both normal and inflamed mucosae.
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