Sharon Ruschkowski

The objectives of the present study were to compare the effects of two regimens of serial blood sampling on the concentrations of hormones and ions during the ovulatory cycle of the domestic hen, and to examine the effectiveness of an indwelling vascular access device for repeated collection of blood samples. Single Comb White Leghorn hens were bled every 2 h over a period of 24 to 26 h, either from one oviposition to the next oviposition (OVIP-OVIP), or from 10 h prior to ovulation until the same time 24 h later (AFTN-AFTN). Whole blood was analyzed for ionized calcium concentration. Plasma was analyzed for total calcium, inorganic phosphorus, 1,25-dihydroxycholecalciferol, estradiol-17beta, and progesterone concentrations. The OVIP-OVIP regimen, using oviposition as a reference point, provided more accurate measurements of ionized calcium, inorganic phosphorus, and estradiol-17beta than did the AFTN-AFTN regimen. Either bleeding regimen was suitable for observing the patterns of 1,25-dihydroxycholecalciferol and progesterone concentrations. The decrease in bound calcium concentration observed with both regimens appeared to be an artifact of repeated blood sampling. The chance of a bird laying an egg following her second oviposition was lower following the OVIP-OVIP regimen than the AFTN-AFTN regimen. The vascular access device was a helpful tool in procuring multiple blood samples for measurement of ions and hormones during the ovulatory cycle of the domestic fowl.

Intimate attachment to the host cell leading to the formation of attaching and effacing (A/E) lesions is an essential feature of enterohemorrhagic Escherichia coli (EHEC) O157:H7 pathogenesis. In a related pathogen, enteropathogenic E. coli (EPEC), this activity is dependent upon translocation of the intimin receptor, Tir, which becomes tyrosine phosphorylated within the host cell membrane. In contrast, the accumulation of tyrosine-phosphorylated proteins beneath adherent EHEC bacteria does not occur, leading to questions about whether EHEC uses a Tir-based mechanism for adherence and A/E lesion formation. In this report, we demonstrate that EHEC produces a functional Tir that is inserted into host cell membranes, where it serves as an intimin receptor. However, unlike in EPEC, in EHEC Tir is not tyrosine phosphorylated yet plays a key role in both bacterial adherence to epithelial cells and pedestal formation. EHEC, but not EPEC, was unable to synthesize Tir in Luria-Bertani medium...

A clonal variant of serotype M1 group A streptococcus (designated M1inv+) has been linked to severe and invasive infections, including sepsis, necrotizing fasciitis and toxic shock. High frequency internalization of cultured epithelial cells by the M1inv+ strain 90-226 is dependent upon the M1 protein. Invasion of HeLa cells was blocked by an anti-M1 antibody, invasion by an M1- strain (90-226 emm1::km) was greatly reduced, and latex beads bound to M1 protein were readily internalized by HeLa cells. Beads coated with a truncated M1 protein were internalized far less frequently. Scanning electron microscopy indicated that streptococci invade by a zipper-like mechanism, that may be mediated by interactions with host cell microvilli. Initially, internalized streptococci and streptococci undergoing endocytosis are associated with polymerized actin. Later in the internalization process, streptococcal-containing vacuoles are associated with the lysosomal membrane glycoprotein, LAMP-1.

Enteropathogenic E. coli (EPEC) belongs to a group of bacterial pathogens that induce actin accumulation beneath adherent bacteria. We found that EPEC adherence to epithelial cells mediates the formation of fingerlike pseudopods (up to 10 microm) beneath bacteria. These actin-rich structures also contain tyrosine phosphorylated host proteins concentrated at the pseudopod tip beneath adherent EPEC. Intimate bacterial adherence (and pseudopod formation) occurred only after prior bacterial induction of tyrosine phosphorylation of an epithelial membrane protein, Hp90, which then associates directly with an EPEC adhesin, intimin. These interactions lead to cytoskeletal nucleation and pseudopod formation. This is the first example of a bacterial pathogen that triggers signals in epithelial cells which activates receptor binding activity to a specific bacterial ligand and subsequent cytoskeletal rearrangement.

Enteropathogenic Escherichia coli (EPEC) induces tyrosine phosphorylation of a 90-kDa protein (Hp90) in infected epithelial cells. This in turn facilitates intimate binding of EPEC via the outer membrane protein intimin, effacement of host cell microvilli, cytoskeletal rearrangement, and bacterial uptake. This phenotype has been commonly referred to as attaching/effacing (A/E). The ability of EPEC to induce A/E lesions was dependent on bacterial growth phase and temperature. Early-logarithmic-phase EPEC grown at 37 degrees C elicits strong A/E activity within minutes after infection of HeLa epithelial cells. EPEC de novo protein syntheses during the first minutes of interaction with the host cell was required to elicit A/E lesions. However, once formed, bacterial viability was not needed to maintain A/E lesions. The type of growth media and partial O2 pressure level do not seem to affect the ability of EPEC to cause A/E lesions. These results indicates that the A/E activity of EPEC ...

Helicobacter pylori is a bacterial pathogen of humans that infects the gastric mucosa. This infection has been associated with gastritis, peptic ulcers, and gastric carcinomas. Diverse in vitro studies have described efficient adherence of H. pylori to different types of epithelial cells. Because of its varied effects on host cells, we have analysed signal transduction events in H. pylori-infected epithelial cells. Our results show that H. pylori induces an increase in inositol phosphates in all cultured epithelial cells used, including HeLa, Henle 407, Hep-2, and the human gastric adenocarcinoma cell line AGS. Bacterial growth medium supernatants induce a similar response in the host cell. The increase in inositol phosphates is not related to redistribution of cytoskeletal proteins such as actin or alpha-actinin nor tyrosine-phosphorylation of host cell proteins. The inositol phosphate increase is also observed in cells infected with low or non-adherent H. pylori mutants or mutants defective in the vacuolating toxin or urease holoenzyme. These results indicate that inositol phosphate release in H. pylori-infected cells is not dependent on bacterial adherence, and that a soluble bacterial factor, but not the vacuolating toxin or urease holoenzyme, mediates such an effect.

Salmonella typhimurium, like many other intracellular pathogens, is capable of inducing its own uptake into non-phagocytic cells by a process termed invasion, and residing within a membrane-bound inclusion. During invasion it causes significant rearrangement of the host cytoskeleton, indicating that signals are transduced between the bacterium and the host cell cytoplasm, across the eukaryotic cell membrane. We found that intracellular inositol phosphate concentrations in HeLa cells increased during S. typhimurium entry and returned to normal levels after bacterial internalization. A chelator of intracellular calcium (BAPTA/AM) blocked S. typhimurium uptake into HeLa epithelial cells, but extracellular calcium chelators (BAPTA, EGTA, EDTA) had no effect on bacterial invasion. These results indicate that S. typhimurium may activate host cell phospholipase C activity to form inositol phosphates which in turn stimulate release of intracellular calcium stores to facilitate bacterial uptake.