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|Title:||The role of pertussis toxin in intestinal hypersensitivity|
|Advisor:||Perdue, Mary H.|
|Keywords:||Medical Sciences;Medical Sciences|
|Abstract:||<p>Immediate hypersensitivity (allergy) is a very common disorder, which may<br />develop after exposure of an individual to an antigen. Intestinal hypersensitivity<br />to luminal antigens has been postulated as a possible cause or triggering<br />mechanism in the pathogenesis of ulcerative colitis, Crohn's disease, eosinophilic<br />gastroenteritis, celiac disease and peptic ulcer. The mechanism by which<br />individuals became sensitized is not known but naturally occurring adjuvants<br />(bacteria and their products) may be crucial for the development of<br />hypersensitivity. In my studies, I investigated in the role of pertussis toxin, a<br />product of Bordetella pertussis, in intestinal hypersensitivity, since pertussis<br />vaccine containing attenuated bacteria was used previously for the induction of<br />anaphylaxis in experimental animals. Pertussis toxin has the enzymatic activity<br />of ADP-ribosyltransferase. which can block the function of some G proteins,<br />important elements in the transduction of signals into the cell. I sensitized rats<br />or mast cell-deficient mice with ovalbumin (OVA) plus recombinant wild type<br />pertussis toxin (wPT) as adjuvant. The reaction to secondary antigen challenge<br />was evaluated 14 days later by determining changes in short-circuit current (Isc, indication of net ion transport) in small intestinal segments mounted in Ussing chambers. Other parameters measured included evaluation of antibody levels in the circulation and histological enumeration of mast cells in the intestinal mucosa.</p> <p>Sensitization of rats with OVA and wPT resulted in enhancement of the<br />jejunal ion secretory response to secondary antigen challenge as compared to<br />rats sensitized with OVA. Injection of doses of wPT as low as 10 ng with OVA<br />caused significantly elevated secondary responses to OVA, while 50 ng wPT<br />resulted in the maximal response. The responses to OVA challenge were<br />enhanced 18.7 fold when OVA was added to the luminal side of the tissues<br />while on serosal side this effect was enhanced 2.2 fold in sensitized rats. The<br />induction of hypersensitivity by wPT adjuvant was dependent on the enzymatic<br />activity of wPT. since the enzymatically inactive mutant (mPT) did not influence<br />responses to secondary antigen challenge. This suggests that ADP-ribosylation<br />of G proteins is involved in the elevation of hypersensitivity by wPT.</p> <p>Two classes of antigen-specific antibodies, IgE and IgG₂s., were measured<br />in the circulation of sensitized rats. The levels of both antibody isotypes were<br />increased at day 14 in wPT but not mPT plus OVA injected rats, which indicates<br />that enzymatically active wPT influences antibody production. The role of<br />particular classes of antibodies was examined by passive sensitization of naive<br />animals with serum from actively sensitized rats. Ion transport responses to<br />secondary antigen challenge of intestine were present in those animals.<br />Blockage of IgE receptors (with myeloma IgEI prior to passive sensitization<br />showed that the response to OVA was totally abolished. This finding shows that<br />in this model of hypersensitivity. IgE antibodies are crucial for the response to antigen. Experiments on mast cell-deficient mutant mice revealed that mast cells<br />are a necessary element for the responses to antigen. Additionally, the number<br />of stainable mast cells in rat intestinal mucosa was significantiy increased by<br />40% on day 14 in wPT but not mPT plus OVA injected rats. This indicates that<br />wPT-induced increases in mast cell numbers are partially responsible for elevated<br />hypersensitivity responses to antigen. Experiments with the neural toxin,<br />tetrodotoxin (TTX), showed that responses to luminal (but not serosal) OVA<br />were neurally regulated in that these responses to OVA were diminished by 66%<br />after treatment of the tissues with TTX.</p> <p>Evaluation of tne responses to OVA at different days after primary<br />sensitization with OVA plus wPT revealed that the responsiveness to antigen<br />appeared between day 3 and 7 after sensitization, and it was still present on day<br />228. In rats sensitized with OVA, the responsiveness to OVA was highest on<br />day 7 and than it decreased very rapidly, showing no responses on day 56.<br />Evaluation of IgE levels in rats demonstrated a similar pattern: the IgE levels were<br />detectable for a long time in wPT plus OVA treated rats but only on day 7 in<br />OVA treated rats. This suggest that elevated IgE levels caused by wPT may<br />account for the long lasting hypersensitivity responses.</p> <p>My data showed that wPT is a very potent adjuvant for hypersensitivity<br />reactions and that the mechanism involves elevation of antigen-specific<br />antibodies, increases in the number of mucosal mast cells and enhanced neurally-mediated<br />antigen uptake.</p>|
|Appears in Collections:||Open Access Dissertations and Theses|
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