To study tissue-mediated control of immunity, we are using 4 different systems. 1. DENDRITIC CELLS FROM VARIOUS LOCATIONS AS ANTIGEN PRESENTING CELLS: We are studying the effect of different environments on the activation of naive and previously activated T cells. we had previously shown that dendritic cells isolated from mesenteric lymph nodes (which drain the gut) tend to induce naive T cells to produce Il4, IL10 and TGFbeta, while dendritic cells grown from bone marrow or isolated from spleen tend to induce interferon gamma (IFNg) and tumor necrosis factor (TNF). We are now studying the effect of offering antigen in different locations in vivo, and of stimulating T cells in various organs in organospecific culture systems. 2. ANTIGENS GIVEN BY DIFFERENT ROUTES GIVE DIFFERENT RESPONSES: we used the protective antigen (PA) of Anthrax as antigen, and gave it either subcutaneously (SQ) or intraperitoneally (IP). when given SQ, the PA immunizes and leads to protection from anthrax toxin challenge. When given IP, it does not. in contrast, the same antigen given with complete adjuvant immunizes by both routes. 3. THE GUT: a. EFFECT OF COMMENSAL BACTERIA ON THE HEALTH OF INTESTINAL TISSUE: we are studying, by microarray analysis of laser microdissected sections of small intestine, the response of various tissues (eg lamina propria, epithelium, crypts) to the presence or absence of commensal bacteria. Preliminary data suggest that 1) many more cells produce anti-microbial peptides than previously thought. 2) antimicrobial peptides are made by epithelial cells, not just Paneth cells b. THE EFFECT OF ANTIBIOTICS ON THE HEALTH OF INTESTINAL TISSUE. Mice were given a cocktail of antibiotics for several weeks and then jejunum tissue was assessed by microarray analysis. We found that antibiotics cause expression changes in a number of genes expressed by normal gut epithelium. Some of these are indirect effects due to the loss of commensal bacteria. Others, however, are direct effects on jejunal tissue, as the changes were also found in antibiotic-treated germ free mice. These direct effects were mostly concentrated in mitochondrial functions. c. THE EFFECT OF B CELLS ON THE HOMEOSTASIS OF THE GUT. Preliminary results suggest that in the absence of B cells, fat metabolism and leptin levels are affected. Thus B cells have a strong effect on normal gut function. We are now analyzing the gut flora in B cell containing and mutant mice to see if the effect of B cells is direct or indirect via a change in the flora. 4. THE MOUTH: a. THE EFFECT OF COMMENSAL ORGANISMS ON NORMAL MOUTH DEVELOPMENT: In order to evaluate the role of oral bacteria in the homeostasis of oral tissues, oral bacterial samples and oral tissues (tongue, mandible, cervical lymph nodes) from germfree vs. conventional mice have been under collection. The RNA samples of oral epithelia obtained from 6 month old mice are under RNA microarray analysis. b. FUSOBACTERIUM NUCLEATUM AS A POTENTIAL VACCINE VECTOR: In order to determine the potential of Fusobacterium nucleatum (a normal human commensal bacterium) as a mucosal vaccine carrier, we are characterizing the adaptive immune responses to F. nucleatum after sublingual administration. Early results show that both IgA and IgG2a antibody responses and Th1/Th17 T cell cytokine profiles are elicited. We are currently working on optimizing the immunization protocol. c. P gingivalis (the bacterium that causes gingivitis) stimulates dendritic cells to produce IL-5 and not IL12p70. e coli, in contrast, stimulate TNF and IL-6 production. We are studying the interactions of these two bacteria and their effects on the stimulation of dendritic cells. Early results suggest that they inhibit each other.
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