To stain filamentous actin, cells were incubated with Alexa 488-conjugated phalloidin (Molecular Probes). crypt regions (0.5%). Severe infection with REPEC O103 resulted in a significant decrease in apoptosis in PP germinal centers (determined by TUNEL assay; = 0.01), in the tips of ileal absorptive villi (determined by H&E staining; = 0.04), and in whole ileal cell lysates (determined by caspase 3 assay; = 0.001). We concluded that REPEC O103 does not promote apoptosis. Furthermore, we cannot rule out the possibility that REPEC Esmolol O103, in fact, decreases apoptotic levels in the rabbit ileum. Enteropathogenic (EPEC) is the leading cause of bacterium-mediated infantile diarrhea, killing several hundred thousand children every year (19, 30). Natural animal disease models such as rabbits infected with rabbit EPEC serogroup O103 (REPEC O103) have been used to study pathogenesis. Apart from its chromosome-encoded adhesive factor (AF/R2) that triggers initial diffuse adherence to intestinal epithelial cells, REPEC O103 possesses the same virulence factors and mechanisms as human EPEC. It secretes several effector proteins via a type III secretion system (EspA, EspB, EspD, and Tir), it produces the outer membrane protein intimin, and it is a Shiga toxin-negative strain (1, 28, 32). Binding between intimin and its translocated receptor, Tir, results in the formation of attaching-and-effacing lesions characterized by intimate attachment between the bacterium and the host epithelial cell with effacement of microvilli resulting with the pathogen residing upon actin-rich pedestals (9, 21). Despite significant research, the pathogenic mechanisms by which EPEC causes diarrheal disease remain undefined. Specific host responses could include apoptotic changes in macrophages and/or intestinal epithelial cells, as have been reported for a variety of pathogens in vitro. However, to date, neither histopathologic reports nor in vivo investigations have KRT17 described changes in apoptotic activities due to EPEC infection (10, 38). Invasive and/or toxin-producing enteropathogens such as species, enterohemorrhagic (EHEC), and increase apoptosis in macrophages and/or epithelial cells in vivo (18, 20, 23, 27, 29, 34, 42, 45, 46). Similarly, and enteroinvasive increase apoptosis in vitro (22, 45). Cell lines such as T84 and HeLa cells infected with EPEC also show features of apoptosis (6). However, the cell permeability to vital dyes such as trypan blue and propidium iodide that was observed is not a definitive feature of apoptosis (late apoptotic cells are propidium iodide positive), and dye-positive cells were not consistently seen beneath adherent bacteria. In addition, infected cells rarely showed distinct morphologic Esmolol characteristics of apoptosis, including DNA breakdown, and the increase in apoptotic signals was, in general, much weaker than that caused by invasive or toxin-producing pathogens. Other investigators found that EPEC, REPEC serogroup O15 (RDEC-1), and directly inhibit the production of interleukin-2 (IL-2), IL-4, and gamma interferon, suggesting that these pathogens do not increase apoptosis Esmolol (26). In addition, researchers have speculated that EPEC adherence potentially stimulates some antiapoptotic pathways within the host cell due to activation of protein kinase C, tyrosine kinases, and the nuclear transcription factor NF-B, leading to the expression of IL-8 (7, 36, 39). Based on these conflicting reports, we set out to determine the influence of EPEC infection on apoptotic intestinal activities in vivo by using the naturally infected, weaned rabbit model. Several REPEC O103 strains isolated in different countries were first tested in vitro for Esp and Tir protein secretion, plasmid profile, and adherence. Animals were then inoculated with strains expressing characteristic representative virulence traits. We evaluated the incidence of Esmolol apoptosis in sections from the ileum and ileal Peyer’s patches (PP), sites that REPEC colonizes (14). The methods used included terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) and caspase 3 assays and counting of apoptotic cells based on their characteristic morphology in sections stained with hematoxylin and eosin (H&E). We found that apoptotic activities in the rabbit ileum and ileal PP were significantly decreased when REPEC O103 disease was fully established. Esmolol MATERIALS AND METHODS Bacterial strains and culture conditions. Various REPEC O103 strains isolated from weaned rabbits with heavy diarrhea were kindly provided by Jorge Blanco (Laboratorio de Referencia de standard, serotype O1? ECRC O103REPEC O103United States (1986C1987)? ECRC 88-0990/103-2REPEC O103Hungary (1988)? 85/150REPEC O103:K-:H2Belgium31C, St, Te, Tr?.