Proteção induzida pela fração de alta massa molecular de Paracoccidioides brasiliensis em camundongos BALB/c
Paracoccidioidomycosis (PCM) is an infectious, granulomatosa disease, caused for a thermally dimorphic fungus Paracoccidioides brasiliensis (Pb), that grows in the host or culture 37ºC, under the form of leavedurifoms or mycelium (filamentous) 25ºC. It is a restricted disease to Latin America and with great endemic areas in Brazil, Colombia, Venezuela and Argentina. Parts fungicus aerial constituted of hifas initiates the infection with focus in the lungs, followed of lymphatic or hematogenics dissemination for the lymph nods, liver, spleens, skin and mucosa. In the lungs or nodules granulomatosis can remain quiescent per years or to progress to unchain a disease, depending on the immune response of the host. In virtue of the increase in the ratio of disease as the PCM, over all caused for the reactivation of the quiescent focus, they use of therapeutically immunization as attempt to eliminate the quiescent organisms or properly to extinguish the infection, especially in disease whose treatment is long, as the case of the PCM, being able in some cases to provoke kidneys and liver lesions. The major challenge in experiments with vaccination against fungus, of all the times, has been to identify to substances or fungus?s inert particles as proteins or carboidrats, which can be used as vehicle in the immunotherapy process. One goals and also one of difficulties for generation of a vaccine, is the identification of a particle that can promote adequate a immune response and accomplishes. In treating to paracoccidioidomycosis, also a protection has been longed for criteriously that during some studies are correlated with the presence of exuberant immune response mediated by cells. Preliminary data indicate that immunizations stimulating the release INF-a and injections of gp43 can reduce the concentration of the P. brasiliensis in the lungs of inoculated mice and to diminish the gravity of the inflammation in the lungs . An even so diverse study has been made in the intention to get a substance that can activate the immune response mainly cellular protector, few advances have been observed. In the present work high Mass Molecular-hMM was introduced a new fraction of P. brasiliensis with the following specific objectives: a) to determine soluble level of gp43 in serum of mice experimental infected and immunized or not immunized with the fraction of hMM. b) Quantified the presence of P. brasiliensis cells in organs of mice immunized and not immunized with the fraction of hMM, using the technique of CFU, c) To analyses the reaction of hypersensitivity-DTH, using as stimulation the fraction of hMM. d) To analyze the histopathology?s characteristics of organs (Lung and Liver) with relation to the granulomatous injuries in the continuation of the experimental infection in BALB/c mice, previously immunized or not with the fraction of hMM, e) to determine in serum the levels of IgG anti-hMM, IgG anti-gp43 and of IgG anti-ExoAg, in the process of immunization with the fraction of hMM, in BALB/c mice. P. brasiliensis (Pb 18) the leveduriforme phase and gotten CFA in accordance with Camargo  was used modified for the inhibitor addition of proteases (PMSF). This sample was fractioned in column of Sephadex G200, having gotten itself it fraction of hMM, in accordance with (Marquez et al, 2003). Female mice BALB/c in 4 groups had been used divided: A) Control, b)Infected, c) Immunized with 50µg of the fraction of hMM (F-50) and infected, d) Immunized with 100µg of the fraction of hMMc (F-100) and infected. The analysis of the levels of gp43 soluble, of IgG anti-hMMc, IgG anti-gp43, IgG anti-ExoAg was carried through by ELISA, Counting of Units formatting of Colonies-CFU, with samples of lung and liver, reaction of hypersensitivity-DTH with the fraction of hMM and Histopathology Analysis had been carried after through 28 and 56 days of the infection. The results had demonstrated lesser soluble antigen levels (p<0,001) in the period of 28 days, in relation to the infected group, already for the period of 56 days, the animals immunized with F-100 of the fraction of hMM, only are that they present significant differences (p<0,001). How much to the levels of IgG antibodies anti-ExoAg and anti-gp43, we can observe that both the groups F-50 and F-100 had presented high levels of antibodies (p<0,001) in relation to excessively, this for 28 days, and 56 days (p<0,001, p<0,01), respectively. In longer period of 56 days, only the F-100 group, showed significant values (p<0,05). For both (lung and liver) the organs analyzed for the CFU, both the groups F-50 and F-100 of the fraction hMM, had presented significant P values (p<0,001) (in lung 28 days) and p<0,05 (lung-56 days), being that for liver the F-50 group presented (p<0,05) and F-100 (p<0,01), respectively. The immune response intensity in vivo determined by DTH, was developed efficiently for the animals that previously had been immunized, in both the periods of 28 and 56 days and groups F-50 and F-100. The analysis of the injuries evidenced, in immunized animals, a degree of compacting and bigger organization, without the dissemination of fungus for other organs, when compared with the animals not immunized.
Advisor:Eiko Nagakawa Itano; Eiko Nagakawa Itano [Orientador].; José Daniel Lopes; Mario Augusto Ono
Source Type:Master's Thesis
Date of Publication: