Decreased apoptosis in the ileum and ileal Peyer's patches: a feature after infection with rabbit enteropathogenic Escherichia coli O103.

Significant changes occur in intestinal epithelial cells after infection with enteropathogenic Escherichia coli (EPEC). However, it is unclear whether this pathogen alters rates of apoptosis. By using a naturally occurring weaned rabbit infection model, we determined physiological levels of apoptosis in rabbit ileum and ileal Peyer's patches (PP) and compared them to those found after infection with adherent rabbit EPEC (REPEC O103). Various REPEC O103 strains were first tested in vitro for characteristic virulence features. Rabbits were then inoculated with the REPEC O103 strains that infected cultured cells the most efficiently. After experimental infection, intestinal samples were examined by light and electron microscopy. Simultaneously, ileal apoptosis was assessed by using terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) and caspase 3 assays and by apoptotic cell counts based on morphology (hematoxylin-and-eosin staining). The highest physiological apoptotic indices were measured in PP germinal centers (median = 14.7%), followed by PP domed villi (8.1%), tips of absorptive villi (3.8%), and ileal crypt regions (0.5%). Severe infection with REPEC O103 resulted in a significant decrease in apoptosis in PP germinal centers (determined by TUNEL assay; P = 0.01), in the tips of ileal absorptive villi (determined by H&E staining; P = 0.04), and in whole ileal cell lysates (determined by caspase 3 assay; P = 0.001). We concluded that REPEC O103 does not promote apoptosis. Furthermore, we cannot rule out the possibility that REPEC O103, in fact, decreases apoptotic levels in the rabbit ileum.

Segmented filamentous bacteria prevent colonization of enteropathogenic Escherichia coli O103 in rabbits.

Despite their distribution in the intestines of many mammals, including man, segmented filamentous bacteria (SFB) have not been found in rabbits, nor has any function been identified for these uncultivable microbes. New Zealand White rabbits were infected with rabbit enteropathogenic Escherichia coli O103 (REPEC O103) derivatives, followed up clinically, and randomly killed 1-4 days after inoculation. Intestinal tissue samples were examined by electron and light microscopy to search for SFB and to evaluate REPEC O103 colonization. Twelve of 21 rabbits showed SFB colonization on ileal absorptive villi. The presence of SFB was correlated with lack of REPEC 0103 ileal colonization (P<.01) and disease. Rabbits without SFB were always colonized by this pathogen. SFB appear to inhibit intestinal colonization by REPEC O103 and thus protect against REPEC 0103 disease. SFB colonization in rabbits is also described for the first time.

In vivo interactions of rabbit enteropathogenic Escherichia coli O103 with its host: an electron microscopic and histopathologic study.

A family of human and animal pathogens, including enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC), trigger formation of 'attaching and effacing' lesions on cultured and intestinal epithelial surfaces. However, our understanding of these events in vivo is incomplete. To further study these interactions in a natural infection model, weaned rabbits were infected with rabbit enteropathogenic E. coli O103 (REPEC O103), followed clinically, and infected tissues were evaluated by electron and light microscopy. Of the 36 rabbits challenged, morbidity and mortality were 65 and 23%, respectively. Twenty-four hours after infection, expression of fimbriae-like organelles was observed on the bacterial surface. Microvilli of ileal Peyer's patches (PP) became disorganized, and intestinal mucus secretion increased which coincided with intraluminal binding of the pathogen in the proximal colon. Forty-eight hours after infection, there was conspicuous lack of fimbriae-like organelle expression, while bacterial adherence preferentially occurred at the domed villi of PP. Seventy-two hours after infection, broad morphological heterogeneity was noted within pedestals beneath attached bacteria, including extended pseudopods. We conclude that REPEC O103 express surface organelles during initial exposure to the host, that the initial target sites of adherence are the domed villi of ileal PP, and that increased mucus secretion occurs during REPEC O103 infection. As well, extended pseudopod formation was demonstrated in vivo.

Characteristics of Helicobacter pylori attachment to human primary antral epithelial cells.

Conventional cell lines are commonly used to study infection characteristics of the human gastric pathogen Helicobacter pylori. We sought to investigate bacterial attachment to human antral primary epithelial cells, a cell model that more closely resembles the human stomach than transformed cell lines. Primary cells were infected for 24 and 48 h with H. pylori. Morphological appearance of both the pathogen and the cells as well as features of colonization, attachment and internalization were evaluated by electron microscopy and compared to features observed with cultured AGS cells. H. pylori exhibited various shapes during colonization including the spiral, U-shaped, donut, and coccoid forms. The prevalence of each form seemed to be dependent on the infected donor tissue but, in general, changed with time to the coccoid form. Bacterial cell membranes progressively enlarged and appeared at times to be connected with microvilli. Bacterial attachment occurred to cells that were either unchanged, or had formed cup-like structures. Simultaneously, outer membrane vesicles were increasingly secreted from the bacteria, coinciding with increased cellular damage. We conclude that bacterial shape conversion, adherence and secretion of outer membrane vesicles are features of H. pylori infection. Primary gastric cell cultures closely imitate the antral environment and present an appropriate and useful model to study H. pylori pathogenesis.

Two enteropathogenic Escherichia coli type III secreted proteins, EspA and EspB, are virulence factors.

Enteropathogenic Escherichia coli (EPEC) belongs to a family of related bacterial pathogens, including enterohemorrhagic Escherichia coli (EHEC) O157:H7 and other human and animal diarrheagenic pathogens that form attaching and effacing (A/E) lesions on host epithelial surfaces. Bacterial secreted Esp proteins and a type III secretion system are conserved among these pathogens and trigger host cell signal transduction pathways and cytoskeletal rearrangements, and mediate intimate bacterial adherence to epithelial cell surfaces in vitro. However, their role in pathogenesis is still unclear. To investigate the role of Esp proteins in disease, mutations in espA and espB were constructed in rabbit EPEC serotype O103 and infection characteristics were compared to that of the wild-type strain using histology, scanning and transmission electron microscopy, and confocal laser scanning microscopy in a weaned rabbit infection model. The virulence of EspA and EspB mutant strains was severely attenuated. Additionally, neither mutant strain formed A/E lesions, nor did either one cause cytoskeletal actin rearrangements beneath the attached bacteria in the rabbit intestine. Collectively, this study shows for the first time that the type III secreted proteins EspA and EspB are needed to form A/E lesions in vivo and are indeed virulence factors. It also confirms the role of A/E lesions in disease processes.

Helicobacter pylori-infected human antral primary cell cultures: effect on gastrin cell function.

Although Helicobacter pylori infection increases gastrin secretion, it is unknown whether this is a direct effect or requires activation of the immune system. We developed an H. pylori-infected human primary antral epithelial cell culture model to address this question. This culture protocol favors growth of H. pylori, and infected cultures could be maintained for up to 48 h. These cultures were enriched for gastrin (10-40%), somatostatin (2-5%), and gastric mucin (60-80%) cells but did not contain immunocytes. Bacterial attachment occurred in a random manner within 2 h of infection, although bacterial density was lower than in sections from infected patients. After 24 or 48 h, the bacterial microcolonies were similar in size to those seen in vivo, and at 24 h ultrastructural studies demonstrated well-developed pedestal formation underlying the bacteria. Coculture with H. pylori increased basal but not stimulated gastrin secretion at all time points >2 h. In conclusion, a newly developed cell culture model has been used to characterize the interactions between H. pylori and normal human antral epithelial cells.

Sequence of an exon of the feline p53 gene--mutation in a lymphosarcoma.

Exon 8 of tumour suppressor gene p53 was sequenced in domestic cats and showed remarkable similarity to the human sequence. Only four of the 13 nucleotide differences gave rise to interspecific amino acid differences. In an investigated lymphosarcoma we detected a mutation cgg --> tgg (arginine --> tryptophan) in codon no. 282.