Silvio Luis de Oliveira

Cell wall fractions from Paracoccidioides brasiliensis induce hypergammaglobulinemia.

The antibody response against the antigen sheep red blood cells (SRBC) was investigated in mice pre-treated with formalin-killedParacoccidioides brasiliensis or with cell wall fractions of the fungus. Pre-treatment withP. brasiliensis, as well as with the F1 fraction and beta-glucan significantly increased the anti-SRBC antibody response in the experimental groups as compared to the control group that received only SRBC. This immunomodulatory effect varied with the different doses employed and with pre-treatment time. We conclude that the cell wall fractions ofP. brasiliensis might play an important role in the hypergammaglobulinemia associated with Paracoccidioidomycosis.

Role of cytokines, NO, and H2O2 on the immunopathology of leptospirosis in genetically selected mice

Immune response to leptospirosis is mainly humorally mediated, and involves opsonization of leptospires for phagocytosis by macrophages and neutrophils. However, some aspects are still unknown. For a more detailed analysis of the cellular immune response to leptospirosis infection, trials were carried out in order to determine the hydrogen peroxide and nitric oxide (H2O2 and NO) production stimulated or not by Interferon-gamma. The participation of some specific cytokines, such as Tumor Necrosis Factor-a (TNF-a); Interferon-gamma (IFN-g); Interleukin-6 (IL-6); and Interleukin-4 (IL-4), in the immunopathology of this infection was also investigated. For this purpose, we analyzed the supernatant from peritoneal macrophage cell culture and the splenic cells of mice genetically selected as High (H) and Low (L) antibody producers, and inbred Balb/c mice infected with Leptospira interrogans serovar icterohaemorrhagiae. The IL-6 production varied from release peaks to inhibition in H, L, and Balb/c mice. Similar behavior was observed for IL-4, produced only by H and Balb/c mice. The three strains presented constant and elevated production of TNF-a until day 14, suggesting its effective participation in the initial phase of the infection. Meanwhile, all the three strains presented a constant and irregular IFN-g production, with release peaks between the 7th and 14th days in L mice. The H and Balb/c mice strains presented a higher tendency to Th2 response pattern, whereas L mice tended towards Th1 response.

Resposta humoral, recuperação bacteriana e lesões histológicas em camundongos geneticamente selecionados para bons e maus produtores de anticorpos e Balb/c, frente à infecção por Leptospira interrogans sorovar icterohaemorrhagiae

O presente trabalho teve por finalidade associar a cinetica da resposta humoral a recuperacao de leptospiras viaveis e a intensidade das lesoes teciduais em camundongos geneticamente selecionados para bons (High) e maus (Low) produtores de anticorpos (selecao IV-A), alem de camundongos outbreed, Balb/c, inoculados com amostra patogenica de Leptospira interrogans sorovar icterohaemorrhagiae. As linhagens High e Low (selecao IV-A) apresentam modificacoes em alguns compartimentos da resposta imune, principalmente em relacao a atividade macrofagica, representando fenotipos extremos encontrados em populacoes naturais heterogeneas. Os camundongos foram sacrificados em oito momentos apos a infeccao. A analise dos resultados revelou que a partir do 7o dia apos a infeccao, os camundongos da linhagem High apresentaram elevacao nos titulos de anticorpos estatisticamente significantes quando comparados aos camundongos da linhagem Low, mantendo assim o efeito multiespecifico. Os camundongos Balb/c apresentaram resultados intermediarios entre as duas linhagens. A producao de anticorpos colaborou como fator limitante a infeccao, pois quando obteve-se maior recuperacao de leptospiras, na fase inicial da infeccao, os titulos de anticorpos encontravam-se em elevacao. As lesoes observadas nos orgaos de camundongos infectados consistiram basicamente nos mesmos processos inflamatorios e degenerativos, que nao se alteraram, variando apenas o grau de comprometimento tecidual, de acordo com a linhagem. A linhagem high apresentou lesoes mais extensas que as apresentadas pelas linhagens low e Balb/c, sendo que nesta ultima as lesoes foram moderadas. De forma geral a linhagem High e Balb/c apresentaram um perfil de resposta Th2, com o maior indice de producao de anticorpos e gravidade das lesoes, enquanto a linhagem Low apresentou um perfil de resposta Th1.

Ecology of Paracoccidioides brasiliensis, P. lutzii and related species: infection in armadillos, soil occurrence and mycological aspects

Abstract Paracoccidioides brasiliensis and the related species P. americana, P. restrepiensis, P. venezuelensis, and P. lutzii (Ascomycota, Ajellomycetaceae) are the etiological agents of paracoccidoidoimycosis (PCM), one of the most important systemic mycoses in Latin America. They are dimorphic fungi, with a mycelial life cycle in soil and a yeast phase associated with tissues of mammalian hosts. This study aimed to detect Paracoccidioides spp. in armadillo tissues and associated soil samples in three well‐defined geographic areas, including the Alta Floresta, an area not only endemic for PCM in the central region of Brazil but also of probable P. lutzii occurrence, whose ecology and geographic distribution are poorly elucidated. The isolates were genotyped by sequencing ITS‐rDNA and the gp43‐exon‐2 region, and by PCR‐RFLP of alpha tubulin (tub1) gene; mycological aspects such as yeast‐to‐mycelial transition, growth and conidial production in soil extract agar were also evaluated. We confirmed that while armadillos are highly infected by P. brasiliensis, including multiple infections by distinct genotypes or species (P. brasiliensis and P. americana) in the same animal, the same does not hold true for P. lutzii, which in turn seems to present less capacity for mycelial growth and conidial production, when developing in a soil‐related condition.

Immune Response to the Rhodococcus equi Infection in High and Low Antibody‐Producing Mice (Selection IV‐A)

Rhodococcus equi is a Gram‐positive, facultative intracellular bacterium which infects macrophages and causes rhodococcal pneumonia and enteritis in foals. Recently, this agent has been recognized as an opportunistic pathogen for immunocompromised humans. Several murine experimental models have been used to study R. equi infection. High (HIV‐A) and Low (LIV‐A) antibody (Ab)‐producers mice were obtained by bi‐directional genetic selections for their ability to produce antibodies against sheep and human erythrocytes (Selection IV‐A). These lines maintain their phenotypes of high and low responders also for other antigens than those of selection (multispecific effect). A higher macrophage activity in LIV‐A mice has been described for several intracellular infectious agents, which could be responsible for their intense macrophage antigens (Ag)‐handling and low Ab production. Due to these differences, LIV‐A mice were found to exhibit a better performance to trigger an effective immune response towards intracellular pathogens. The objective of this work was to characterize the immune response of Selection IV‐A against R. equi. HIV‐A and LIV‐A mice were infected with 2.0 ×106 CFU of ATCC 33701+R. equi by intravenous route. With regards to bacterial clearance and survival assays, LIV‐A mice were more resistant than HIV‐A mice to virulent R. equi. LIV‐A mice presented a higher hydrogen peroxide (H2O2) and nitric oxide (NO) endogenous production by splenic macrophages than HIV‐A mice. LIV‐A expressed the most intense cellular response, available by the Delayed‐Type Hypersensitivity (DTH) reaction, which activated macrophages and produced more H2O2 and NO. The three times higher specific antibodies titres in HIV‐A indicated that Selection IV‐A maintained the multispecific effect and the polygenic control of humoral and cellular responses also to R. equi.

Experimental visceral leishmaniasis in high and low antibody - producer mice (selection IV-A)

Leishmaniasis is a typical parasite infection whose protective immunity depends on macrophage activation. Susceptibility to Leishmania donovani infection was compared in H (high antibody responder) and L (low antibody responder) mice from selection IV-A. H mice infected intravenously with 10(7) amastigotes of L. donovani were more susceptible to infection than their L counterparts. This higher susceptibility was characterized by a higher splenic and hepatic parasite burden. An increased splenic index was observed in both lines after sixty days of infection. This splenomegaly was caused, at least partially, by an increase in the number of splenic cells as determined by direct counts of cells from spleen. The results show that selection IV-A is susceptible to visceral leishmaniasis, with the H line being more susceptible than the L line.

The role of leukotriene B4 in early stages of experimental paracoccidioidomycosis induced in phenotypically selected mouse strains

Paracoccidioidomycosis is a human systemic mycosis caused by the fungus Paracoccidioides brasiliensis. The mechanisms involved in innate immune response to this fungus are not fully elucidated. Leukotrienes are known to be critical for the clearance of various microorganisms, mainly by mediating the microbicidal function of phagocytes. We investigated the involvement of leukotriene B4 in the early stages of experimental paracoccidioidomycosis, which was induced by intratracheal inoculation of the fungus in selected mouse lines. The mouse lines utilized were produced through bi-directional phenotypic selection, endowed with maximal or minimal acute inflammatory reactivity, and designated AIRmax and AIRmin, respectively. AIRmax mice were more resistant to the infection, which was demonstrated by reduced lung fungal loads. However, the two lines produced similar amounts of leukotriene B4, and pharmacological inhibition of this mediator provoked similar fungal load increases in the two lines. The lower fungal load in the AIRmax mice was associated with a more effective inflammatory response, which was characterized by enhanced recruitment and activation of phagocytic cells and an increased production of activator cytokines. This process resulted in an increased release of fungicidal molecules and a diminution of fungal load. In both lines, leukotriene production was associated with a protective response in the lung that was consequent to the effect of this eicosanoid on the influx and activation of phagocytes.

Cytokine production in lungs and adrenal glands of high and low antibody producing mice infected with Paracoccidioides brasiliensis

Mice genetically selected for high (H) and low (L) antibody production (HIV-A and LIV-A) were used in an experimental model of paracoccidioidomycosis. In a previous work, it was observed that male HIV-A animals were more susceptible to the infection due to adrenal gland damage. Male HIV-A and LIV-A animals were intravenously inoculated with Paracoccidioides brasiliensis (strain 18) and sacrificed 2, 4, 6, 8 and 10 weeks after inoculation. At each time interval, lungs and adrenals were removed to estimate recoverability of the fungus, as well as to determine Th1 (IFN-gamma, TNF-alpha) and Th2 (IL-4 and IL-10) cytokine profiles. While viable fungi recoverability from the lungs of HIV-A mice was higher after 4 and 8 weeks, there was less fungal recovery from the adrenals of LIV-A animals after the 2nd week, with total fungal elimination after the 8th week. With regard to Th2 cytokines, there was an inhibition in IL-4 production in the organs from infected animals, the extent of which varied according to the organ and the time period after initiation of infection. IL-10 production was found to be lower in both organs. Determination of Th1 cytokines revealed that IFN-gamma production increased in both organs, mainly in the adrenal of LIV-A after 8 and 10 weeks, when these animals showed a total fungal elimination. A significant difference was observed between HIV-A and LIV-A concerning TNF-alpha production in both organs and at all recovery times, in that LIV-A produced a higher level of this cytokine, mainly in the adrenal. These results may explain the high susceptibility of HIV-A to P. brasiliensis infection, is due, at least in part, to adrenal involvement. The higher production of Th1 cytokines by LIV-A in comparison to HIV-A mice may account for LIV-A resistance to P. brasiliensis infection. Our data reveal the importance of this experimental model in the study of the adrenal involvement in paracoccidioidomycosis, since this gland may be highly compromised in the patients, leading to the development of Addisons Disease.

Immune response to Mycobacterium bovis-AN5 infection in genetically selected mice (selection IV-A)

Mice genetically selected for high (H) and low (L) antibody production (Selection IV-A) were used as murine experimental model. The aim of the present work was to evaluate the macrophagic activity and to characterize the immune response in Mycobacterium bovis-AN5 infected mice (3X107 bacteria). The response profile previously observed in such strains was not similar to that obtained during M. bovis infection; however, it corroborated works carried out using Selection I, which is very similar to Selection IV-A regarding infection by M. tuberculosis and Bacillus Calmette-Guerin (BCG). Considering bacterial recovery, LIV-A mice showed higher control of the infectious process in the lungs than in the spleen, whereas HIV-A mice presented more resistance in the spleen. With respect to macrophagic activity, hydrogen peroxide (H2O2) was probably not involved in the infection control since there was an inhibition in the production of this metabolite. Nitric oxide (NO) and TNF-a production seemed to be important in the control of bacterial replication and varied according to the strain, period and organ. Evaluation of the antibody production indicated that the multi-specific effect commonly observed in these strains was not the same in the response to M. bovis. Antibody concentrations were higher in LIV-A than in HIV-A mice at the beginning of the infection, being similar afterwards. Such data were compared with delayed-type hypersensitivity (DTH), which was more intense in HIV-A than in LIV-A mice, indicating that antibody production is independent of the capability to trigger DTH reactions and that cellular and humoral responses to M. bovis antigens show a polygenic control and an independent quantitative genetic regulation. Differences were observed among organs and metabolites, suggesting that different mechanisms play an important role in this infection in natural heterogeneous populations, indicating that NO, TNF-a and Th1 cytokines are involved in the infection control.

Identificação da Leptospira interrogans sorovar pomona em camundongos geneticamente selecionados, para a alta e baixa produção de anticorpos, através da técnica de imunoperoxidase em tecido renal e isolamento bacteriano em meio de Fletcher

The present work had the objective of identifying the presence of Leptospira interrogans serovar pomona in mice that had been genetically selected for high and low response to antibodies. All the animals were subjected to bacterial isolation, immunohistochemical analysis (immunoperoxidase) in renal tissue sections and hematoxylin-eosin staining. The immunoperoxidase technique was little more sensitive than culturing, but both were good parameters for agent identification. More severe renal lesions were present at times when there were greater numbers of bacteria isolated in culture medium. Mice of the lineage HIV-A were able to eliminate the Leptospira more efficiently and faster than the lineage LIV-A could. However, the study demonstrated that both lineages of the IV-A selection were efficient in controlling the infectious process.