Is prolonged rotavirus infection a common cause of protracted diarrhoea?
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Child↗
Biomedical subjects
A D Phillips
Publications and source records attributed to A D Phillips.
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.
Adult↗
The genetically programmed down-regulation of lactase in children.
BACKGROUND & AIMS: Intestinal lactase activity is high in all healthy human babies, but in adults a genetic polymorphism, which acts in cis to the lactase gene, determines high or low messenger RNA (mRNA) expression and activity (lactase persistence and nonpersistence, respectively). Our aim was to investigate the onset of expression of this polymorphism in children. METHODS: Activities were analyzed in relation to age in normal biopsy specimens from a 20-year collection of diagnostic specimens. In a smaller set of 32 samples, aged 2-132 months, RNA was extracted for semiquantitative reverse-transcription polymerase chain reaction. Marker polymorphisms were used to determine the allelic origin of lactase mRNA transcripts. RESULTS: Analysis of 866 children showed evidence that the lactase persistence/nonpersistence polymorphism began before 5 years of age. The 32 children tested had high lactase mRNA and activity. Six children aged 2-16 months showed equal expression of two alleles, 2 children aged 7 and 14 months showed slightly asymmetric expression, and 7 children aged 22-132 months showed very asymmetric expression, the second allele being undetectable in the 11-year-old, as previously seen in lactase-persistent heterozygote adults. CONCLUSIONS: Genetically programmed down-regulation of the lactase gene is detectable in children from the second year of life, although the onset and extent are somewhat variable.
Adolescent↗
Enteropathogenic and enterohaemorrhagic Escherichia coli: more subversive elements.
Enteropathogenic (EPEC) and enterohaemorrhagic Escherichia coli (EHEC) constitute a significant risk to human health worldwide. Both pathogens colonize the intestinal mucosa and, by subverting intestinal epithelial cell function, produce a characteristic histopathological feature known as the 'attaching and effacing' (A/E) lesion. Although EPEC was the first E. coli to be associated with human disease in the 1940s and 1950s, it was not until the late 1980s and early 1990s that the mechanisms and bacterial gene products used to induce this complex brush border membrane lesion and diarrhoeal disease started to be unravelled. During the past few months, there has been a burst of new data that have revolutionized some basic concepts of the molecular basis of bacterial pathogenesis in general and EPEC pathogenesis in particular. Major breakthroughs and developments in the genetic basis of A/E lesion formation, signal transduction, protein translocation, host cell receptors and intestinal colonization are highlighted in this review.
Animals↗
Syndrome of intractable diarrhoea with persistent villous atrophy in early childhood: a clinicopathological survey of 47 cases.
BACKGROUND: The syndrome of intractable diarrhoea of infancy is heterogeneous and includes several diseases with diverse aetiologies. This study determines whether diagnostic categories can be defined on the basis of clinicopathological analysis. METHODS: European Society of Paediatric Gastroenterology, Hepatology and Nutrition members were surveyed to identify cases of intractable diarrhoea with persisting small intestinal enteropathy. A retrospective clinicopathological analysis was performed on cases showing life-threatening diarrhoea within the first 24 mo of life and requiring total parenteral nutrition, which were characterized by persistent villous atrophy, and resistance to therapy. RESULTS: Forty-seven infants were identified with intractable diarrhoea. Villous atrophy was of varying degrees with (group I, n = 24) or without (group II, n = 18) lamina propria mononuclear cell infiltration. Group I presented later, had gut autoantibodies, and a higher prevalence of protein-losing enteropathy; a subset (group Ia, n = 12) also had extraintestinal symptoms of autoimmunity associated with a later onset of larger volume diarrhoea. Group II presented early; 8 cases (group IIa) had phenotypic abnormalities and a low birth weight; the remaining 10 (group IIb) showed mild-to-moderate villous atrophy, epithelial tufting, and abnormal crypts. Group III included five patients in whom no specific features were recognised. Twenty-one (45%) died at a median age of 24 months, 20 (43%) remained dependent on parenteral (n = 16) or enteral tube (n = 4) feeding, 4 (9%) received elimination diets plus other therapies, and 2 (4%) were lost to follow-up. CONCLUSIONS: Clinicopathological analysis allowed distinct disease groups to be identified, allowing a provisional classification to be made. This straightforward approach forms a basis for future research in this exceptionally difficult paediatric condition.
Atrophy↗
Pretransplant management and small bowel-liver transplantation in an infant with microvillus inclusion disease.
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Atrophy↗
Role of intimin and bundle-forming pili in enteropathogenic Escherichia coli adhesion to pediatric intestinal tissue in vitro.
Attaching and effacing (A/E) lesion formation is central to enteropathogenic Escherichia coli (EPEC) pathogenesis. In vitro experiments with human epithelial cell lines have implicated virulence plasmid-encoded bundle-forming pili (BFP) in initial binding and intimin in intimate attachment and A/E lesion formation. This study investigated the role of BFP and intimin in EPEC interactions with pediatric small intestinal biopsy tissue in in vitro organ culture. Organ culture infections (2 to 8 h) were performed with E2348/69 (a wild-type EPEC O127:H6 clinical isolate) and E2348/69 derivatives including CVD206 (eae deficient), CVD206(pCVD438) (eae-complemented CVD206), CVD206(pCVD438/01) (expressing intimin, which is nonfunctional due to a single amino acid substitution), JPN15 (spontaneous EPEC adherence factor virulence plasmid-cured E2348/69), and 31-6-1(1) (E2348/69 with a TnphoA insertion inactivation mutation in the virulence plasmid-encoded bfpA gene). Scanning and transmission electron microscopy revealed that after 8 h E2348/69 and CVD206 (pCVD438) (both Int+ BFP+) adhered to all specimens, causing A/E lesions with surrounding microvillous elongation. JPN15 and 31-6-1(1) (both Int+ BFP-) adhered and caused A/E lesions although bacteria adhered in "flat," two-dimensional groups. CVD206 and CVD206(pCVD438/01) (both Int- BFP+) did not adhere to any sample, and no pathological tissue changes were seen. Thus, in human intestinal organ culture, BFP do not appear to be involved in the initial stages of EPEC nonintimate adhesion but are implicated in the formation of complex, three-dimensional colonies via bacterium-bacterium interactions. Intimin appears to play an essential role in establishing colonization of EPEC on pediatric small intestinal tissue.
Adhesins, Bacterial↗
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.
Bacterial Adhesion↗
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.
Animals↗
Down regulation of intimin expression during attaching and effacing enteropathogenic Escherichia coli adhesion.
Enteropathogenic Escherichia coli (EPEC) produces attaching and effacing (A/E) lesions in the intestinal mucosa. The intimate bacterial adhesion associated with A/E lesion formation is promoted by intimin, a 94-kDa EPEC surface protein. Anti-intimin antisera raised in rabbits by using the purified 280-amino-acid cell binding domain of intimin as the immunogen were employed in immunofluorescence and immunoelectron microscopical studies to investigate the expression of intimin by classical EPEC strain E2348/69 (O127:H6) and defined E2348/69 derivatives during culture growth and A/E bacterium adhesion to cultured HEp-2 cells. In stationary-phase broth cultures, only a small fraction of E2348/69 bacteria expressed intimin, and of those that did, immunolabelling revealed a uniform distribution of intimin over the bacterial surface; increased numbers of bacteria expressing intimin were detected when E2348/69 was grown in tissue culture medium, an effect not seen with strain JPN15, a virulence plasmid-cured derivative of E2348/69. Strain CVD206, an eaeA mutant of E2348/69, did not stain with the anti-intimin antisera, but strain CVD206(pCVD438), containing a functional eaeA gene, stained uniformly. After a 3-h incubation of HEp-2 cells with strain E2348/69, double immunofluorescence labelling of intimin and cellular actin revealed strong intimin expression by all A/E bacteria, but after 6 h of incubation, intimin expression by most E2348/69 bacteria was greatly reduced or not detected. This effect on intimin expression was not observed with strain JPN15 but was restored for strain JPN15(pCVD450) harboring the virulence plasmid-encoded per genes. These results indicate that surface expression of intimin is regulated by environmental factors during bacterial growth and following A/E lesion formation and that virulence plasmid-encoded genes participate in these regulation processes.
Actins↗
Chronic diarrhoea in AIDS.
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Acquired Immunodeficiency Syndrome↗
Congenital enterocyte heparan sulphate deficiency with massive albumin loss, secretory diarrhoea, and malnutrition.
BACKGROUND: The molecular basis of protein-losing enteropathy is unknown. However it has been shown that sulphated glycosaminoglycans may be important in regulating vascular and renal albumin loss. METHODS: We describe three baby boys who presented within the first weeks of life with massive enteric protein loss, secretory diarrhoea, and intolerance of enteral feeds. All required total parenteral nutrition and repeated albumin infusions. No cause could be found in any case despite extensive investigations, including small intestinal biopsy sampling, which were repeatedly normal. FINDINGS: By specific histochemistry, we detected gross abnormality in the distribution of small intestinal glycosaminoglycans in all three infants, with complete absence of enterocyte heparan sulphate. The distribution of vascular and lamina propria glycosaminoglycans was, however, normal. INTERPRETATION: The presentation of these infants suggests that enterocyte heparan sulphate is important in normal small intestinal function.
Albumins↗
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.
Bacterial Adhesion↗
Adhesion of enteroaggregative Escherichia coli to pediatric intestinal mucosa in vitro.
Organ cultures of small- and large-intestinal mucosa from children were used to examine the interactions of enteroaggregative Escherichia coli (EAEC) with human intestine. Mucosae from patients aged between 3 and 190 months were cultured with five EAEC strains isolated from infants with diarrhea in the United Kingdom and with two well-described prototype EAEC strains, 17-2 and 221. The prototype strains adhered to jejunal, ileal, and colonic mucosae. The wild-type strains also adhered to this tissue but showed a variable pattern of adhesion: two adhered to all intestinal levels, one adhered to jejunum and ileum, one adhered to ileum only, and one adhered to ileum and colon. Adherence was in an aggregative or stacked-brick pattern, resembling that seen on HEp-2 cells. Electron microscopy of infected small intestinal mucosa revealed bacteria in association with a thick mucus layer above an intact enterocyte brush border, which contained extruded cell fragments. This mucus layer was not present on controls. EAEC adherence to colonic mucosa was associated with cytotoxic effects including microvillous vesiculation (but without evidence of an attaching/effacing lesion), enlarged crypt openings, the presence of intercrypt crevices, and increased epithelial cell extrusion. These results demonstrate that in vitro organ culture of intestinal mucosa from children can be used to investigate EAEC pathogenesis in childhood directly. EAEC strains appear able to colonize many regions of the gastrointestinal tract, without overt changes to small intestinal mucosa but with cytotoxic effects on colonic mucosa.
Adult↗
T84 cells in culture as a model for enteroaggregative Escherichia coli pathogenesis.
Enteroaggregative Escherichia coli (EAEC) is an important cause of persistent diarrhea in many developing parts of the world, yet the pathogenetic mechanisms of EAEC diarrhea are unknown. Experiments with animal models suggest that EAEC strains damage the intestinal mucosa, and a putative cytotoxin has been described. To characterize the mucosal effects of EAEC, we studied strain 042, which we have shown to cause diarrhea in adult volunteers. Strain 042 was incubated in an in vitro organ culture model with biopsy-derived normal intestinal mucosa from pediatric patients. Strain 042 adhered strongly to samples of jejunal, ileal, and colonic mucosa. In addition, scanning electron microscopic examination of in vitro-infected intestinal biopsies revealed cytotoxic effects marked by exfoliation of mucosal epithelial cells. To develop an in vitro model to study these effects, we incubated 042 with polarized monolayers of the human intestinal epithelial cell lines Caco-2 and T84. Strain 042 adhered strongly to T84 cells but not to Caco-2 cells. T84 cells infected with 042 displayed marked toxic effects, most prominently in areas where bacteria were adhering. The apical membrane of damaged cells exhibited vesiculation and shedding of microvilli. The cytoplasm of affected cells displayed subnuclear vacuolization, and in some cases, nuclei of affected cells became separated from the surrounding cytoplasm. Severely affected cells ruptured, releasing their nuclei. Vacuolated remnant cells were seen throughout the monolayer. Strain 042 was not internalized by T84 cells. We concluded that EAEC strain 042 alters intestinal cell morphology, ultimately leading to cell death. Although the factor(s) required for this effect remains to be elucidated, T84 cells may serve as a valuable model in EAEC pathogenesis studies.
Bacterial Adhesion↗
Intimin from enteropathogenic Escherichia coli restores murine virulence to a Citrobacter rodentium eaeA mutant: induction of an immunoglobulin A response to intimin and EspB.
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 (formerly C. freundii biotype 4280)-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 (Int(EPEC)) and C. rodentium (Int(CR)). A secreted protein, EspB (formally EaeB), is also necessary for A/E-lesion formation. Here we report that expression of a cloned Int(EPEC), encoded by plasmid pCVD438, restores murine virulence to an intimin-deficient mutant of C. rodentium DBS255. Replacement of Cys937 with Ala abolished the ability of the cloned EPEC intimin to complement the deletion mutation in DBS255. Ultrastructural examination of tissues from wild-type C. rodentium and DBS255(pCVD438)-infected mice revealed multiple A/E lesion on infected cells and loss of contact between enterocytes and basement membrane. Histological investigation showed that although both wild-type C. rodentium and DBS255(pCVD438) colonized the descending colon and induced colonic hyperplasia in orally infected 21-day-old mice, the latter strain adhered to epithelial cells located deeper within crypts. Nonetheless, infection with the wild-type strain was consistently more virulent, as indicated by a higher mortality rate. All the surviving mice, challenged with either wild-type C. rodentium or DBS255(pCVD438), developed a mucosal immunoglobulin A response to intimin and EspB. These results show that C. rodentium infection provides a relevant, simple, and economic model to investigate the role of EPEC proteins in the formation of A/E lesions in vivo and in intestinal disease.
Adhesins, Bacterial↗
Epithelial cell proliferation in childhood enteropathies.
BACKGROUND/AIM: The aim of this study was to investigate epithelial cell turnover in childhood enteropathy to establish whether common disease related mechanisms operate. Levels of epithelial cell proliferation were measured in children with food intolerance (cows' milk protein intolerance and coeliac disease), and after infection with Giardia lamblia, Cryptosporidium, and enteropathogenic Escherichia coli. METHODS: Comparative measures of epithelial cell proliferation were performed by recording mitotic activity and MIB-1 immunoreactivity in proximal small intestinal biopsy specimens. RESULTS/CONCLUSIONS: A hyperplastic crypt response was evident in all of the disease states examined and was particularly pronounced in coeliac disease and in infection with enteropathogenic E coli, where mitotic and MIB-1 labelling indices were significantly raised above control values. In contrast with coeliac disease, increased crypt cell production rates in enteropathogenic E coli infection were also due to an expansion of the crypt proliferation compartment, without a comparable increase in crypt cell numbers. Crypt hyperplasia is therefore a common tissue response to mucosal damage in food allergy and infection, although disease specific mechanisms are evident.
Animals↗
Human intestinal development in a severe-combined immunodeficient xenograft model.
The present work describes a severe-combined immunodeficient murine xenograft model used to investigate human gastrointestinal ontogenesis. Specifically, the study has tested whether carefully selected regions of human fetal gut are able to undergo region-specific morphogenesis and epithelial cytodifferentiation when transplanted subcutaneously into immunodeficient mice. In addition, double-label in situ hybridisation techniques, utilising specific human and mouse DNA probes, have been adopted to characterise host and donor cell types and to investigate the potential developmental roles for non-epithelial cells in the regulation of epithelial differentiation pathways in vivo. Human fetal small and large bowel developed to form a characteristic mucosa 10 weeks after transplantation, which displayed clear region-specific structural and functional gradients. The initial phase of xenograft epithelialisation closely resembled the stratified type of epithelium which is present during early fetal gastrointestinal development. Idiosyncratic epithelial differentiation pathways were recorded during xenograft regeneration, with an absence of Paneth cells and an abundance of enteroendocrine cells when compared with developed xenograft and paediatric intestine. Such differences may, therefore, be important in ensuring rapid and region-specific development in the absence of conventional luminal stimuli and hormonal changes that occur normally during pregnancy. In situ hybridisation demonstrated an exclusively human origin for the intestinal xenograft epithelium and muscularis mucosa and externa. Although the submucosa and lamina propria were comprised of a chimeric mixture, murine cells were rarely seen to contact with the epithelium, which interacted primarily with human myofibroblasts and human intraepithelial lymphocytes. It is proposed that a 'selection' process operates to maintain species-specific cellular interactions, and this mechanism may subsequently play an important role in regulating epithelial cell differentiation, orchestrated in part by juxtaposed non-epithelial cell types.
Absorption↗