- Pistole, Thomas
- University of New Hampshire
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- End date
- To determine whether porcine mannan-binding lectin (MBL) enhances the binding of S. Typhimurium and Salmonella enterica serovar Enteritidis to human polymorphonuclear neutrophilic leukocytes (PMN) and whether the presence of MBL affects the overall cytokine expression of these host defense cells.
- To determine the effect of S. Typhimurium on the apoptosis of human neutrophils that have transmigrated across a model intestinal epithelial monolayer.
- To determine the role of porins of S. Typhimurium and S. Enteritidis on the type and time course of apoptosis in human intestinal epithelial cells
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- NON-TECHNICAL SUMMARY: Foodborne disease in humans due to Salmonella continues to be a major health concern. Natural or innate defenses are considered important in preventing infection but the basis for this protection is poorly understood.
APPROACH: OBJECTIVE 1. Overall approach. MBL will be purified by affinity chromatography, using immobilized protein A to remove residual IgG, and, if needed, gel exclusion chromatography. Salmonella serovars will be grown and maintained in Luria-Bertani medium. PMNs will be isolated from freshly obtained human blood. [The University's Institutional Review Board for the Protection of Human Subjects has approved this protocol.] Binding will be assessed by light microscopy and flow cytometry. Cytokine production will be detected by ELISA. Data will be analyzed using analysis of variance and, where appropriate, Tukey's pairwise matrix comparison. OBJECTIVE 2. Overall approach. For this study wild type S. Typhimurium will be used. Human neutrophils will be isolated from blood by density centrifugation. Some neutrophil samples will be transmigrated through a model intestinal epithelial monolayer. These and control neutrophils will then be allowed to phagocytose S. Typhimurium. Apoptosis will be measured using an ELISA system that detects DNA fragmentation, a characteristic of apoptosis; an assay to quantify caspase-3, which is the beginning protein in the apoptosis signal cascade; and a visual system to detect DNA fragmentation in single cells. Data will be collected over a 24-hour period, then plotted and compared to neutrophils not exposed to S. Typhimurium. OBJECTIVE 3. Overall approach. Bacterial induced apoptosis will be assessed by DNA fragmentation, using both quantitative (ELISA) and qualitative (TUNEL assay) systems. We will also measure levels of caspase-3 to determine if the apoptosis occurring in these systems is caspase-dependent. Porin-deficient mutants (BHKC1 [ompC::Tn10]; BHKD1 [ompD::Tn5]) of S. Typhimurium 14028 have been created in our laboratory. An ompC mutant (SA4474 [ompC396::Tn10]) of S. Enteritidis RKS53 was obtained from the Salmonella Genetic Stock Centre. These transposon-containing mutants are grown and maintained on media supplemented with tetracycline (for Tn10 mutants) or kanamycin (for Tn5 mutant). A non-invasive strain of E. coli, F18, is also included.
PROGRESS: 2005/10 TO 2008/09
OUTPUTS: Presentations to professional audiences such as, Graduate and undergraduate students in the field of immunology, and Researchers in the field of immunology.
PARTICIPANTS: Individuals on the project. Dr. Bochiwe Hara-Kaonga. Dr. Hara-Kaonga was responsible for the design of and the studies on the role of porins in adherence of Salmonella to human cells. She is currently an assistant professor in the the Biomedical Sciences Program, Rochester Institute of Technology. Dr. Imola Daniel. Dr. Daniel was responsible for the design of and the studies on the role of porcine mannan-binding lectin on the adherence of Salmonella to human cells. She is currently a medical resident in a hospital in metropolitan New York city. Mr. Christopher Wrocklage. Mr. Wrocklage was responsible for the design of and the studies on the induction of apoptosis in human neutrophils by Salmonella. He is currently in the Inflammation Department, Wyeth Research, Cambridge, MA. Collaborators on the project. Dr. Alice L. Givan, Research Associate Professor of Physiology and Director, Englert Cell Analysis Laboratory, Dartmouth Medical School, provided support for the flow cytometric studies. Dr. Beth McCormick, the Mucosal Immunology and Developmental Biology Laboratories, Mass General Hospital, and Associate Professor of Pediatrics and Microbiology, Harvard Medical School, Boston, MA, provided support for the transepithelial migration model. Training or professional development. Six undergraduate students participated in research associated with this project.
TARGET AUDIENCES: Target audiences-professional. Wrocklage, C. C. & T. G. Pistole. 2006. Salmonella enterica serovar Typhimurium promotes apoptosis in activated neutrophils. Innate Immunity: Receptors, Response & Regulation. A joint meeting of the Society for Leukocyte Biology and the International Endotoxin and Innate Immunity Society. San Antonio, TX. Nov. 9-11.
IMPACT: 2005/10 TO 2008/09
Salmonellosis is a leading cause of foodborne illness in humans. Despite extensive studies on the microorganism and the leading reservoirs of this pathogen (domestic animals and their products), the incidence of infections due to Salmonella remains high. The focus of this project was to examine key components of the early events in Salmonella infections to determine if intervention strategies could be proposed for preventing the clinical manifestations of this disease. Based on our work we now know the following information about Salmonella infections and the early host defenses associated with it. First, we have confirmed, using human intestinal cells and macrophages, what we had previously shown in animals models, namely that a major outer membrane protein, OmpD, contributes to the initial adherence of Salmonella to both of these cells. Microbial adherence to intestinal epithelial cells is a major route of entry into the body from the intestinal lumen. This information provides a focus point for intervention strategies that can block this adherence, thus aborting a potential infection. Secondly, although Salmonella can induce both humoral and cell-mediated immune responses, these are often not effective in typical cases of salmonellosis. Innate defensives, on the other hand, because they are present without the need for an induction period, may provide an early defense. Mannan-binding lectin, an evolutionarily highly conserved protein found in human serum, can enhance the adherence of Salmonella to human defense cells such as macrophages. We have shown that porcine mannan-binding lectin, like its human counterpart, can also enhance this association. Thus, exogenously administered mannan-binding lectin may be an early therapeutic regimen for abrogating a potential infection due to Salmonella. Finally, microorganisms have been shown to modulate the natural rate of apoptosis in key defense cells as a means of reducing the effectiveness of these natural responders to microbial infection. Our studies indicate that Salmonella grown under anaerobic conditions, as might be expected in the intestinal lumen, enhance the rate of apoptosis in neutrophils that have been activated by passage through an intestinal epithelial monolayer, simulating the migration of these host cells into the lumen during a nascent Salmonella infection. We suggest that this alteration of the rate of apoptosis is a virulence mechanism for this pathogen and thus another target for intervention approaches to prevent overt infection.
- Funding Source
- Nat'l. Inst. of Food and Agriculture
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- Natural Toxins
- Bacterial Pathogens
- Meat, Poultry, Game