Intimin and the host cell--is it bound to end in Tir(s)?

Intimate bacterial adhesion to the intestinal epithelium is a pathogenic mechanism shared by several human and animal enteric pathogens, including enteropathogenic and enterohaemorrhagic Escherichia coli. Two bacterial protein partners involved in this intimate association have been identified, intimin and Tir. Some key remaining questions include whether intimin specifically interacts with one or more host-cell-encoded molecules and whether these contacts are a prerequisite for the subsequent intimate intimin-Tir association. Recent data support the hypothesis that the formation of a stable intimin-Tir relationship is the consequence of intimin protein interactions involving both host and bacterial components.

Impact of rotavirus infection on a paediatric hospital in the east end of London.

AIMS: To study the impact of confirmed rotavirus infection at a paediatric hospital; to use the data to obtain a minimum estimate of the cost of treating reported cases of rotavirus in England and Wales. METHODS: Data were obtained on all patients with rotavirus over a two year period. Information was collected on 386 patients with rotavirus infection who were treated at the 120 bed Queen Elizabeth Hospital for Children in East London. This included the virus serotype, the patient's age, whether they required intravenous infusion, duration of hospital stay, numbers of patients treated in the casualty department, and numbers who had to be admitted. Treatment costs were obtained from the Finance Department of the Hospitals for Sick Children. RESULTS: The minimum cost of treating patients, excluding the cost of medical staff at the hospital, was estimated to be 95,400 pounds a year. One hundred and forty eight (38%) patients were admitted to the wards and a further 49 patients developed symptoms while in hospital. Intravenous infusion was required by 18 patients. The mean duration of hospital stay was 5.5 days. One hundred and eighty nine (49%) patients were treated with oral rehydration solution in casualty, given advice, and sent home. Ninety four per cent of the patients were aged under 2 years. The findings were comparable with those obtained in a study at Texas Children's Hospital, USA. The G serotype (VP7) of rotavirus did not influence the severity of infection. CONCLUSION: Rotavirus infections accounted for a significant number of patients treated in casualty, admissions to hospital, and bed occupancy in a paediatric hospital. The estimated cost of treating reported cases of rotavirus in England and Wales is in excess of 6.3 pounds million a year.

Cryptosporidiosis in immunocompetent children.

Cryptosporidial oocysts were identified by modified Ziehl-Neelsen stain in the stools of seven (3.2%) of 213 children with acute or chronic diarrhoea and one (0.9%) of 112 controls. All children with cryptosporidia were immunocompetent. Four of the index cases had a short illness (3-14 days) with watery diarrhoea, vomiting (2), and abdominal pain (2). Two index cases had chronic diarrhoea for over four months and failure to thrive. Both had a small intestinal enteropathy; one had cryptosporidial oocysts in stool specimens two months apart and the other had cryptosporidial schizonts attached to the jejunal mucosa. One index case had a colitis of indeterminate cause. Four of the index cases had recently travelled abroad. There had been an outbreak of gastroenteritis in the family of one of the index cases, and three affected sisters and an asymptomatic brother had oocysts in their stools. Cryptosporidial infestation seems to be associated with acute gastroenteritis and sometimes with chronic diarrhoea and small bowel damage in immunocompetent children.

Edwardsiella tarda induces plasma membrane ruffles on infection of HEp-2 cells.

Interaction of two clinical Edwardsiella tarda isolates with HEp-2 cells was investigated. By electron microscopy we observed at 1 h post infection that E. tarda induced formation of extensive plasma membrane projections resembling membrane ruffles. The ruffles did not coincide with adhering bacteria. Only few invading bacteria were seen. Vacuolated nuclear membrane was occasionally observed. Three hours post infection, E. tarda induced a contact-dependent cell lysis, revealing the host cell cytoskeleton and nucleus. Only one of the E. tarda strains was seen residing within the host cell remains. The results indicate that E. tarda-induced membrane ruffles may involve a distinct mechanism of bacterial pathogenesis.

Adhesion of enteroaggregative Escherichia coli to formalin-fixed intestinal and ureteric epithelia from children.

The adhesion characteristics of enteroaggregative Escherichia coli (EAggEC) to the mucosal surfaces of formalin-fixed paediatric intestinal and ureteral tissue were studied. The technique offers a means of overcoming the problem of limited tissue access in childhood and a way of examining the initial steps of bacterial adhesion. Five EAggEC strains isolated from children with diarrhoea in the UK and a well characterised, prototype EAggEC strain (221) were examined. Five of the six EAggEC strains showed preferential adhesion to jejunal mucosa with limited adhesion to ileum and colon. Five of the six also adhered to ureteric tissue. EAggEC can adhere to proximal, as well as distal, regions of the gastrointestinal tract in children, a previously unrecognised characteristic.

Neutrophil response to mucosal infection.

Total white cell counts were reviewed in paediatric in-patients with viral gastroenteritis, bacterial gastroenteritis, delayed recovery following acute gastroenteritis, viral lower respiratory tract infections and cow's milk protein intolerance. The prevalence of neutrophilia was not different in the five groups. Neutropenia was common in association with the presence of viruses in stool or sputum, and was significantly more common in these groups than in patients with bacterial gastroenteritis and cow's milk protein intolerance. Neutropenia has not been previously reported in viral gastroenteritis. It was transient in nature and not related to age, sex, weight or antibiotic treatment; no pancreatic disorders were noted.

Royal Society of Tropical Medicine and Hygiene meeting at Manson House, London, 14 December 1995. Enteropathogenic Escherichia coli--mucosal infection models.

The formation of attaching and effacing (A/E) lesions is central to the pathogenesis of enteropathogenic Escherichia coli (EPEC)-mediated disease in humans and Citrobacter rodentium-mediated transmissible colonic hyperplasia in mice. Closely related outer membrane proteins, known as intimins, are required for formation of the A/E lesion by both EPEC and C. rodentium. In this study we found similar ultrastructural damage in small intestinal biopsies from an EPEC-infected child and large bowel specimens from C. rodentium-infected mice. The C. rodentium-infected large bowel biopsies revealed massive hyperplastic reactions and the infected human small intestinal biopsies showed an increase in total crypt cell number and mitotic index. EPEC-infected small intestinal organ cultures revealed bacteria adhering in a localized pattern and evidence of A/E lesions. Covaspheres coated with a biologically active cell-binding domain of intimin also adhered to cells in a localized fashion but did not induce the characteristic A/E lesions.

Small intestinal mucosa in childhood in health and disease.

A great increase in the knowledge of human small intestinal mucosal pathology has resulted from the use of the peroral small intestinal biopsy technique. This paper highlights some different applications of electron microscopy to such biopsy samples in order to obtain diagnostic criteria and to further the understanding of enteric processes in childhood in health and disease. The importance of control data in childhood is emphasized and differences are described between adult and childhood mucosa. The possibilities of quantitative studies at the ultrastructural level and the link between structure and function are demonstrated by studies on microvillous appearance in relation to disaccharidase enzyme levels; the potential for experimental work is shown in studies using tracer molecules to investigate antigen permeability of the small intestine; and finally descriptive observations is highlighted in a section concerned with parasitic, bacterial and viral infections of the small intestine in childhood.

A comprehensive pathological survey of duodenal biopsies from dogs with diet-responsive chronic enteropathy.

BACKGROUND: The detailed pathological phenotype of diet-responsive chronic enteropathy (CE) and its modulation with dietary therapy remain poorly characterized. HYPOTHESIS/OBJECTIVES: Key mucosal lesions of diet-responsive CE resolve with dietary therapy. METHODS: This was a prospective observational study of 20 dogs with diet-responsive CE. Endoscopic duodenal biopsies collected before and 6 weeks after the start of a dietary trial were assessed by means of qualitative and quantitative histopathological, immunohistochemical, and ultrastructural criteria. Control duodenal biopsies were obtained from 10 healthy Beagle dogs on 1 occasion. RESULTS: Compared with control dogs, the CE dogs had higher villus stunting scores and higher overall WSAVA scores, a lower villus height-to-width ratio, and higher lamina propria density of eosinophils. The CE dogs also had ultrastructural lesions of the mitochondria and brush border. In common with other studies in which the disease and control populations are not matched for breed, age, sex, and environment, these comparisons should be interpreted with caution. Comparing biopsies collected at presentation and 6 weeks after starting the dietary trial, mean lamina propria mononuclear cell score and lamina propria densities of eosinophils and mononuclear cells decreased. Dietary therapy also improved ultrastructural lesions of the mitochondria and brush border, eliciting a decrease in intermicrovillar space and an increase in microvillus height. CONCLUSIONS AND CLINICAL IMPORTANCE: In dogs with diet-responsive CE, the remission of clinical signs with dietary therapy is associated with subtle decreases in lamina propria density of eosinophils and mononuclear cells, and resolution of ultrastructural lesions of the enterocyte.

Aberrant response to commensal Bacteroides thetaiotaomicron in Crohn's disease: an ex vivo human organ culture study.

BACKGROUND: Human ex vivo evidence indicating that an inappropriate immune response(s) to nonpathogenic bacteria contributes to disease pathogenesis in pediatric Crohn's disease (CD) is limited. The aim of the present study was to compare and contrast the early innate immune response of pediatric "healthy" versus CD mucosa to pathogenic, probiotic, and commensal bacteria. METHODS: "Healthy control" and CD pediatric mucosal biopsies (terminal ileum and transverse colon) were cocultured for 8 hours with E. coli O42, Lactobacillus GG (LGG), Bacteroidesthetaiotaomicron (B. theta), or stimulated with interleukin (IL)-1ß (positive control). Matched nonstimulated biopsies served as experimental controls. IL-8 was the immune marker of choice. IL-8 mRNA and protein levels were quantified by quantitative polymerase chain reaction and sandwich enzyme-linked immunosorbent assay, respectively. RESULTS: IL-8 secretion was observed when control, ileal biopsies were exposed to pathogenic O42 and probiotic LGG, with no response noted to commensal B. theta. In comparison, Crohn's ileal biopsies showed impaired ability to induce IL-8 in response to O42 and LGG. Control colonic tissue showed a limited response to O42 or B. theta and LGG significantly reduced IL-8 secretion. Unlike control tissue, however, Crohn's ileal and colonic tissue did respond to B. theta, with more enhanced expression in the colon. CONCLUSIONS: We provide the first ex vivo data to support the notion that aberrant mucosal recognition of commensal bacteria may contribute to pediatric CD. While IL-8 responses to O42 and LGG varied with disease status and anatomical location, B. theta consistently induced significant IL-8 both in ileal and colonic CD tissue, which was not seen in control, healthy tissue.

Intestinal alpha-defensin expression in pediatric inflammatory bowel disease.

BACKGROUND: Reduced alpha-defensin expression has been reported in the terminal ileum (TI) of adult patients with ileal Crohn's disease (CD). However, little is known about alpha-defensin expression in children with chronic inflammatory bowel disease (IBD). METHODS: In all, 283 intestinal biopsies were obtained from children with CD, ulcerative colitis (UC), and healthy controls. Absolute mRNA copy numbers for HD5, HD6, IL-8, Villin 1, and Tcf-4 were analyzed by reverse-transcription polymerase chain reaction (RT-PCR). HD5 immunostaining was performed on biopsy sections and patients genotyped for NOD2 mutations. RESULTS: Equal expression levels of alpha-defensins (HD5 and HD6) were found in TI biopsies of children with ileal CD (L1+L3) compared to patients with colonic disease (L2) and healthy controls. In contrast, we found significantly higher levels of alpha-defensins in the TI of children with UC compared to CD and controls. Reduced expression of Tcf-4 was observed exclusively in the duodenum and TI of CD patients with L1+L3 phenotype. We demonstrate significantly increased expression of HD5 and HD6 in the inflamed colon of IBD children (UC and CD) attributable to the presence of metaplastic Paneth cells. CONCLUSIONS: In this study no difference in alpha-defensin expression was found in the TI of CD children and controls. However, significant reduction of Tcf-4 in L1+L3 phenotype suggests that a possibly impaired PC differentiation may lead to altered HD5 and HD6 expression at some stage of disease. Additionally, substantially increased expression of alpha-defensins in the inflamed colonic mucosa of children with IBD raises the question for their potential involvement in modulating inflammation in these patients.

The medium is the messenger.

Enteropathogenic Escherichia coli (EPEC), like many bacterial pathogens, employ a type III secretion system to deliver effector proteins across the bacterial cell. In EPEC, four proteins are known to be exported by a type III secretion system--EspA, EspB and EspD required for subversion of host cell signal transduction pathways and a translocated intimin receptor (Tir) protein (formerly Hp90) which is tyrosine-phosphorylated following transfer to the host cell to become a receptor for intimin-mediated intimate attachment and "attaching and effacing" (A/E) lesion formation. The structural basis for protein translocation has yet to be fully elucidated for any type III secretion system. Here, we describe a novel EspA-containing filamentous organelle that is present on the bacterial surface during the early stage of A/E lesion formation, forms a physical bridge between the bacterium and the infected eukaryotic cell surface and is required for the translocation of EspB into infected epithelial cells.

Familial microvillous atrophy: a clinicopathological survey of 23 cases.

Twenty-three cases of microvillous atrophy were reviewed to determine clinical and morphological characteristics of the disease. Congenital and late-onset forms of presentation were clearly identified in which the late-onset cases appeared to have a better prognosis. Three different, and distinctive, appearances of the proximal small intestinal mucosa were found. Careful orientation of mucosal samples allowed a temporal sequence of events to be delineated in which the first morphological abnormality to be detected in the epithelium was the accumulation of "secretory granules"; microvillous inclusions were seen in older cells in the upper villous region. It is suggested that, in familial microvillous atrophy, diarrhoea and disorganisation of the brush border assembly occur as a consequence of a more fundamental defect that affects the intracellular traffic of certain cell components, as indicated by the accumulation of "secretory granules."

An electron microscopical investigation of faecal small round viruses.

A retrospective study of small round featureless viruses (SRVs) initially identified by negative-staining electron microscopy of stool samples was performed. A variety of technique, including immunoelectron microscopy and caesium chloride gradient centrifugation, was applied in an attempt to classify further these viruses. Over a four-year period, 64 SRV-positive samples were reported (1.8% of the stool samples sent for electron microscopy and 6.2% of the total number of positive samples), of which 53 were available for further study. A significant degree of misclassification was found. Viruses previously identified as SRVs were shown to be astrovirus (n = 14), calicivirus (n = 2), and "Norwalk-like" virus (n = 1). The majority of the 36 remaining samples were identified as parvovirus-like (n = 27) (75%), 14 of which were associated with the presence of adenovirus particles. Enteroviruses (n = 3) and hepatitis A virus (n = 1) were infrequently detected. The remaining viruses (n = 5) could not be adequately classified. Parvovirus may be the predominant SRV associated with acute diarrhoeal disease in childhood.

Intimin-mediated tissue specificity in enteropathogenic Escherichia coli interaction with human intestinal organ cultures.

The hallmark of enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) adhesion to cultured human host cells is intimate attachment and the formation of attaching and effacing (A/E) lesions. Recently, EHEC O157:H7 was shown to induce A/E lesions on human intestinal explants. Unlike EPEC, which colonized the small intestine, EHEC adhesion was restricted to follicle-associated epithelium (FAE) of ileal Peyer's patches. This study tested the hypothesis that the bacterial adhesin intimin contributes to tissue specificity. Complementing the eae gene mutation in CVD206 (derived from EPEC strain E2348/69) with EPEC eaealpha (encoding intimin-alpha) restored the ability to colonize small intestinal mucosa like the parent strain. In contrast, complementing with EHEC eaegamma (encoding intimin-gamma) resulted in the strain adhering and inducing A/E lesion on Peyer's patches, similar to EHEC. An intimin-gamma-positive O55:H7 EPEC also targeted FAE. Thus, intimin contributes to the tissue specificity of A/E lesion-forming microbial pathogens.

Abnormal expression of dipeptidylpeptidase IV activity in enterocyte brush-border membranes of children suffering from coeliac disease.

Dipeptidylpeptidase IV (DPP IV) activity has been shown cytochemically to decrease significantly in enterocytes of children suffering from coeliac disease. This decrease is due to a halving of the time available for enterocytes to express DPP IV in their brush-border membranes during development. This effect is compared with previous results showing coeliac disease to inhibit disaccharidase activities selectively.

Mechanisms of gut damage by Escherichia coli.

This chapter primarily concerns three main categories of diarrhoeagenic Escherichia coli, enteropathogenic (EPEC), enterohaemorrhagic (EHEC) and enteroaggregative (EAEC) E. coli. They have distinctive virulence factors and vary in the enteropathies they produce. The molecular biological approach has opened up the complex way in which they interact with the intestine. EPEC and EHEC show a subversive approach to colonization in that they adapt the host cell to their requirements in the formation of the attaching effacing lesion. EAEC appear to co-opt the host defence system to produce a biofilm-like colony and currently go unrecognized in routine laboratories.

Investigation of hospital-acquired rotavirus gastroenteritis using RNA electrophoresis.

Rotavirus is a common cause of diarrhoea both in the community and in the hospital. Because of this, it may be difficult to determine whether crossinfection has occurred in the hospital, an important finding as review of hygienic techniques and ward closure may be indicated. We therefore investigated the use of Polyacrylamide gel electrophoresis (PAGE) of the rotavirus RNA genome as a means of distinguishing between rotavirus strains in order to assess its role in the evaluation of apparent hospital-acquired rotavirus diarrhoea. Suspected examples of hospital-acquired rotavirus gastroenteritis were studied on an infectious diseases ward and a general infant ward. PAGE analysis demonstrated that crossinfection had not occurred on the infectious diseases ward, even though this was indicated clinically; a single source outbreak involving 11 patients was confirmed on the general infant ward, as all cases showed an identical rotavirus electropherotype. Following ward closure an endemic rotavirus electropherotype was detected, which affected 17 patients over a 3-month period. Electrophoresis of rotavirus RNA is a useful and practical technique in the analysis of hospital-acquired gastroenteritis and can indicate appropriate clinical action.