Immune responses to Campylobacter (C. jejuni or C. coli) infections: a two-year study of US forces deployed to Thailand.

Campylobacter spp. is a leading cause of diarrheal disease among US troops deployed to Thailand for exercise. We investigated the importance of immunological analysis and immune responses against Campylobacter infection in US troops deployed to Thailand. Blood and fecal samples were collected from volunteered soldiers with diarrhea and from healthy controls. Stool culture was performed to identify the pathogens. Campylobacter-specific antibodies, antibody secreting cells and cytokines were measured. Several bacterial protein fragments in the outer membrane extract of Campylobacter spp., were identified by an immunoblot analysis with plasma and fecal antibodies. Among all of the diarrheal cases, 35% were Campylobacter-positive. Based on antibody titers in plasma and in fecal extract and antibody secreting cells: 6% of healthy controls, 32% of the Campylobacter culture-negative diarrheal cases, and 85% of the Campylobacter culture-positive diarrheal cases were positive for Campylobacter. Our results indicate that the measurement of Campylobacter-specific antibodies in plasma and fecal extract samples is a good marker of exposure to Campylobacter, and this test may be a useful diagnostic tool for seroepidemiological studies. Elicited antibodies against several bacterial outer membrane protein fragments suggest that these protein fragments are vital in providing protective immunity against Campylobacter.

Evaluation of an intragastric challenge model for Shigella dysenteriae 1 in rhesus monkeys (Macaca mulatta) for the pre-clinical assessment of Shigella vaccine formulations.

Shigellosis is a worldwide disease, characterized by abdominal pain, fever, vomiting, and the passage of blood- and mucus-streaked stools. Rhesus monkeys and other primates are the only animals that are naturally susceptible to shigellosis. A suitable animal model is required for the pre-clinical evaluation of vaccines candidates. In this study, the minimal dose of Shigella dysenteriae1 1617 strain required to produce dysentery in four of five (80% attack rate) monkeys using an escalating dose range for three groups [2 × 10(8) , 2 × 10(9) and 2 × 10(10) colony forming unit (CFU)] was determined. In addition, the monkeys were re-infected. The identified optimal challenge dose was 2 × 10(9) CFU; this dose elicited 60% protection in monkeys when they were re-challenged with a one log higher dose (2 × 10(10) CFU). The challenge dose, 2 × 10(10) CFU, produced severe dysentery in all monkeys, with one monkey dying within 24 h, elicited 100% protection when re-challenged with the same dose. All monkeys exhibited immune responses. This study concludes that the rhesus monkey model closely mimics the disease and immune response seen in humans and is a suitable animal model for the pre-clinical evaluation of Shigella vaccine candidates. Prior infection with the 1617 strain can protect monkeys against subsequent re-challenges with homologous strains.

Establishment of a non-human primate Campylobacter disease model for the pre-clinical evaluation of Campylobacter vaccine formulations.

Campylobacter jejuni is a common cause of enteritis worldwide. The mechanisms by which C. jejuni causes disease are unclear. Challenge studies in humans are currently considered unethical due to the possibility of severe complications, such as Guillain-Barré syndrome. Campylobacter infection in non-human primates closely mimics the disease and immune response, seen in humans. In this study, we attempted to determine the minimal dose of a pathogenic C. jejuni 81-176 strain required for clinical signs and symptoms of disease (> or = 80% attack rate) in Macaca mulatta monkeys using an escalating dosage (three doses for three monkey groups: 10(7), 10(9) and 10(11) cfu). Eighty percent of the monkeys challenged with highest dose (10(11) cfu) had mild disease, but the 80% attack rate (moderate diarrhea in 80% of the monkeys) was not achieved. However, 100% of monkeys showed IgA seroconversions (three-fold over pre-challenge titers). The elicited immune response was challenge dose-dependent. Campylobacter antigen specific fecal s-IgA responses were observed in all challenged groups but the response was not dose-dependent. Only IgM antibody secreting cells response was observed against Campylobacter antigens. The elicited immune response in three groups of rhesus monkeys was dose-dependent, indicating this monkey model can be used for pre-clinical evaluation of Campylobacter candidate vaccines, however these adult rhesus monkeys are less prone to Campylobacter infection.

Cyclins D1, E, A expression and synergistic cytotoxicity to a cholangiocarcinoma cell line from recombinant TNF-alpha and PHA supernate.

We studied the cytotoxic effects of recombinant TNF-alpha and supernate of phytohemagglutinin stimulated peripheral blood mononuclear cells individually and in combination against a cholangiocarcinoma cell line. Levels of cyclins D1, E and A in the cell line were detected by immunoblotting, and the cell cycle stage was assayed by propidium iodide staining followed by flow cytometry analysis. Viable and apoptotic cells were assessed by trypan blue dye exclusion, DAPI staining, agarose DNA laddering and propidium iodide staining. At the beginning of each experiment, the majority of cholangiocarcinoma cells expressed cyclin A and were in S phase as determined by propidium iodide staining. Treatment of such cells with recombinant TNF-alpha resulted in cytotoxic effects clearly evident at 36 hours post exposure. There was a synergistic killing effect when recombinant TNF-alpha was combined with PHA supernate and this effect could be partly neutralized by monoclonal anti TNF-alpha, interleukin (IL)-2, IL-12 and IFN-gamma.