Murine alpha-2-macroglobulin increase during inflammatory responses and tumor growth.

OBJECTIVE AND DESIGN: To determine the alpha-2-macroglobulin (alpha2M) levels in mice during acute and chronic inflammatory responses. MATERIALS AND METHODS: Inflammation was induced by one of the following stimuli: carrageenin, zymosan, lipopolysacharide, thioglycollate, bacilli Calmette Guerin, PPD (in pre-immunized and non-immunized animals) and tumor cells. The concentration of alpha2M was determined in plasma or peritoneal liquid by electroimmunoassay. RESULTS: In all the treatments employed, the plasma levels of alpha2M were higher than in untreated animals. This increase varied from 9%, 24 h after injection up a maximum of 66% 72 h post-injection. When compared to animals injected only with saline, the increases were significant 48 h after treatment with either zymosan or LPS, and 72 h after treatment with either thioglycollate or carrageenin. Treatment with BCG triggers an increase in alpha2M levels after 24 h (18.60%) and 48 h (27.90%). Immunized mice presented higher levels of this protein than non-immunized animals after challenge with PPD. The growth of Ehrlich tumor cells in the peritoneal cavity was directly correlated with the local levels of alpha2M which increased 3.5 fold, 10 days after injection. CONCLUSIONS: These results strongly indicate that in mice, the concentration of alpha2M can increase during acute and chronic inflammatory reactions with kinetics dependent on the particular kind of inflammatory agent.

Study of pulmonary experimental paracoccidioidomycosis by analysis of bronchoalveolar lavage cells: resistant vs. susceptible mice.

Adult Swiss (susceptible) and BALB/c (non-susceptible) mice were inoculated by the intravenous route with 1 x 10(6) yeast cells of Paracoccidioides brasiliensis, strain 18. Immunologic parameters, histopathology and features of the bronchoalveolar lavage (BAL) were evaluated at week 2, 4, 8 and 16 post-infection. The pulmonary infection was progressive in Swiss mice and regressive in Balb/c mice. The numbers of total cells, lymphocytes and polymorphonuclear neutrophils increased in BAL, as well as the percentages of giant cells, and CD4 and CD8 positive cells. The ultrastructural study of BAL cells revealed a predominance of macrophages and a frequency of 13.2% of type II pneumocytes. As the infection progressed, the number of fungal cells and spreading macrophages, as well as the stimulated release of H2O2 by macrophages, increased. The animals exhibited an exacerbation of the humoral immune response and a depression of cellular immunity during the infection. There was a good correlation between the intensity and the pattern of the pulmonary histopathology and the cellular findings in the BAL. The present model reproduces some anatomoclinical patterns of the human disease and shows that BAL may be a useful tool in monitoring the pulmonary infection caused by P. brasiliensis.

Serological follow-up of patients with paracoccidioidomycosis treated with itraconazole using Dot-blot, ELISA and western-blot.

Twenty-seven mycologically proven cases of paracoccidioidomycosis (PCM) were treated with itraconazole (100-200 mg/day in month 1 and 100 mg/day until month 6-8) and evaluated clinically and serologically, up to 3.5 years post-therapy, using Dot-blot and ELISA for measuring the titers of IgG, IgA and IgM anti-P.brasiliensis antibodies and Western-blot for determining IgG, IgA and IgM antibodies against the antigen components of the fungus. Before treatment, 81.5% (Dot-blot) and 84% (ELISA) of the patients presented elevated IgG anti-P.brasiliensis antibody titers which dropped slightly with treatment. On the other hand, the percentages of pre-treatment high-titered sera for IgA and IgM anti-P.brasiliensis were lower (51.9% and 51.8%: Dot-blot; 16.5 and 36%: ELISA, respectively) but the titers tended to become negative more frequently with treatment. Prior to treatment, the percentages of positivity for IgG, IgA and IgM anti-P.brasiliensis antibodies in Western-blot were 96%, 20.8% and 41.6%, respectively. Antigens with molecular weights varying from 16-78 kDa, from 21-76 kDa and from 27-78 kDa were reactive for IgG, IgA and IgM antibodies, respectively. The most frequently reactive antigenic components had molecular weights of 27, 33 and 43 kDa for IgG, and 70 for IgA and IgM antibodies. During the period of study, the patients responded well to treatment. The present data confirm the diversity and complexity of the humoral response in PCM, and the importance of utilizing different serological tests to detect IgG, IgA and IgM anti-P. brasiliensis antibodies.

Effect of Crotalus durissus terrificus (Laurenti, 1768) venom on the evolution of Ehrlich ascites tumor

The effect of Crotalus durissus terrificus (LAURENTI, 1768) venom on the evolution of Erlich ascites tumor cells was evaluated. Thus, 30-day-old male mice of the Swiss strain were inoculated intraperitoneally with 1x103 tumor cells. Then, 7 groups of animals were formed: 3 control groups (physiological, venom and tumor) and 4 experimental groups that received different doses of venom. The experimental groups received 5 intraperitoneal venom injections on the 1st, 4th, 7th, 10th and 13th days after tumor implantation. On the 14th day, 5 animals from each one og the groups were sacrificed, and the varibles such as the total and differential counts of cells in the peritoneal cavity and functional state of peritoneal macrophages by macrophage spreading were evaluated. The other 5 remaining animals were kept in the laboratory for 60 days for observation of their survival percentage. The results obtained were statistically analysed by the Kruskal-Wallis test at 5 per cent significance level. It was observed that Crotalus durissis terrificus venom increases survival time of mice, but does not increase mortality percentage. This venom also increases the percentage of macrophage spreading. We suggest that snake venoms can cause inhibition of tumor growth by activating the inflammatory reaction, mainly the macrophages, stimulating the production of TNF-alpha, IL-1, IL-6 and IL-8. These cytokines may act on tumor cells by different mechanisms, inducing its complete elimination.

Isolation of fungi from nature in the region of Botucatu, State of Sao Paulo, Brazil, an endemic area of Paracoccidioidomycosis

In an attempt to isolate Paracoccidioides brasiliensis from nature 887 samples of soil from Botucatu, SP, Brazil, were collected cultured in brain heart infusion agar supplement with dextrose, in potato dextrose agar and in yeast extract starch dextrose agar, all with antibiotics, at 25º and 37ºC. Five thermo-dependent dimorphic fungi morphologically resembling P. brasiliensis were isolated; two from armadillo holes; further studies of the biology, antigenicity and genetic features of the five dimorphic fungi are necessary to clarify their taxonomy and their possible relation to P.brasiliensis. In addition, 98 dematiaceous fungi and 581 different soecies of Aspergillus spp. were also isolated. Our findings emphasize that armadillos and their environment are associated with thermo-dimorphic fungi and confirm the ubiquity of pathogenic dematiaceous fungi and Aspergillus spp.

Isolation of fungi from nature in the region of Botucatu, state of São Paulo, Brazil, an endemic area of paracoccidioidomycosis.

In an attempt to isolate Paracoccidioides brasiliensis from nature 887 samples of soil from Botucatu, SP, Brazil, were collected cultured in brain heart infusion agar supplemented with dextrose, in potato dextrose agar and in yeast extract starch dextrose agar, all with antibiotics, at 25 degrees and 37 degrees C. Five thermo-dependent dimorphic fungi morphologically resembling P. brasiliensis were isolated; two from armadillo holes; further studies of the biology, antigenicity and genetic features of the five dimorphic fungi are necessary to clarify their taxonomy and their possible relation to P. brasiliensis. In addition, 98 dematiaceous fungi and 581 different species of Aspergillus spp. were also isolated. Our findings emphasize that armadillos and their environment are associated with thermo-dimorphic fungi and confirm the ubiquity of pathogenic dematiaceous fungi and Aspergillus spp.

Paracoccidioides brasiliensis antigen batches from the same isolate show immunological and biochemical differences.

We investigated the occurrence of antigenic and biochemical variability among Paracoccidioides brasiliensis antigen batches prepared according to the same protocol. Initially (experiment #1), we analyzed two antigen lots of two human isolates (Bt1 & Bt2), cultured in two media (PYG: bactopeptone, yeast extract, glucose; MMM: McVeigh & Morton medium) in SDS-PAGE and in two immunological tests (immunodiffusion-ID and footpad swelling test-FPT). Afterwards (experiment #2), we compared the antigenic profile of three antigen batches from three human isolates (Bt1, Bt2 & Bt3) by two-dimensional immunoelectrophoresis (2 D-IEP) against a reference system for P. brasiliensis antigens. In experiment #1, there were important intra- and inter-strain antigenic differences between batches of the fungal isolates cultured on both media. The block titration of the antigen batches for the immunological tests revealed correlation between protein concentration and biological activity in ID and no correlation in FPT. In experiment #2, the reference system for P. brasiliensis showed 26 antigen peaks. There were important differences between batches prepared from the same isolate and between batches from different isolates. Our data suggested the occurrence of instability in the synthesis of antigenic components by a same P. brasiliensis isolate, under controlled incubation conditions.

Antitumor effect of snake venoms

The search for biological antitumor agents has been pursued for over half a century. Snake venom has been shown to possess a wide spectrum of biological activities. The objectives of the present review are to evaluate the existing controversies on this subject published in a number of papers and to propose probable explanations for the phenomena observed. We reported our results obtained in a study, in which we evaluated the action of the venoms of Crotalus durissus terrificus and Bothrops jararaca on Ehrlich ascites tumor cells. We noticed an important antitumor effect, mainly with Bothrops jararaca venom, as well as an increase in the functional activity of macrophages. We also observed an increase in the number of mononuclear and polymorphonuclear cells with Bothrops jararaca venom. Considering these findings, we postulate that both Bothrops jararaca and Crotalus durissus terrificus venoms can act directly on tumor cells. In addition, we propose an indirect mechanism, based on the stimulation of the inflammatory response, to inhibit tumor growth and to promote its rejection.

Optimization of a mouse immunization protocol with Paracoccidioides brasiliensis antigens.

The objectives of the present study were to optimize the protocol of mouse immunization with Paracoccidioides brasiliensis antigens (Rifkind's protocol) and to test the modulation effect of cyclophosphamide (Cy) on the delayed hypersensitivity response (DHR) of immunized animals. Experiments were carried out using one to four immunizing doses of either crude particulate P. brasiliensis antigen or yeast-cell antigen, followed by DHR test four or seven days after the last immunizing dose. The data demonstrated that an immunizing dose already elicited response; higher DHR indices were obtained with two or three immunizing doses; there were no differences between DHR indices of animals challenged four or seven days after the last dose. Overall the inoculation of two or three doses of the yeast-cell antigen, which is easier to prepare, and DHR test at day 4 simplify the original Rifkind's immunization protocol and shorten the duration of the experiments. The modulation effect of Cy on DHR was assayed with administration of 2.5, 20 and 100 mg/kg weight at seven day intervals starting from day 4 prior to the first immunizing dose. Only the treatment with 2.5 mg Cy increased the DHR indices. Treatment with 100 mg Cy inhibited the DHR, whereas 20 mg Cy did not affect the DHR indices. Results suggest an immunostimulating effect of low dose of Cy on the DHR of mice immunized with P. brasiliensis antigens.

The use of a mixture of somatic and culture filtrate antigens in the evaluation of the immune response to Paracoccidioides brasiliensis.

Three Paracoccidioides brasiliensis antigens, namely a culture filtrate preparation, a somatic antigen and a mixture of equal parts of the two, were tested by two serological techniques against sera from patients with paracoccidioidomycosis, and in an in vivo delayed hypersensitivity model in mice. The antigen mixture was more sensitive than the two individual antigens for the evaluation of the humoral and cellular immune response to P. brasiliensis, both in man and in experimental animals.

Studies on inflammatory response induced by Ehrlich tumor in mice peritoneal cavity.

In the present study we investigated the inflammatory response induced by the inoculation of Ehrlich tumor cells (EAT) into the peritoneal cavity of mice. It was found that after inoculation of 10(3) EAT cells, the number of peritoneal leukocytes remained unchanged till the sixth day. Subsequently, the number of cells increased as a consequence of tumor growth. EAT cells did not induce influx of PMN leukocytes till six days after tumor implantation, but a significant influx was observed on the tenth day. Inoculation of the tumor cells did not induce production of H2O2 by peritoneal cells at any time examined and induced low levels of macrophage spreading only until the third day after tumor implantation but not later on. The levels of thromboxane in the peritoneal cavity were not affected by the presence of the tumor, whereas prostaglandin E2 levels were significantly increased at all times examined. The biological significance of these results on the evolution and escape of the tumor from host defense mechanisms is under investigation.

Inhibition of Ehrlich ascites tumor in vivo by PAF-antagonists.

Several lines of evidence support that PAF modulates the inflammatory and immune responses, and that tumors may inhibit both these processes. In the present study we analysed the effect of PAF antagonists on the growth of Ehrlich Ascites Tumor (EAT) in vivo. Mice were inoculated intraperitoneally with 1 x 10(3) EAT cells and the tumor growth evaluated by counting the number of peritoneal cells, 1,6 and 10 days after tumor implantation. BN 52021 was administered intraperitoneally, intravenously or subcutaneously once or twice a day, at 1.0, 2.5, 5.0 and 20.0 mg/kg. Control animals received 0.1 ml of the vehicle in the same schedule. It was found that i.p. and i.v. administration of BN 52021 (5 mg/kg, twice a day) significantly inhibited EAT growth (80.8% and 56.0% respectively). Other routes and doses were less effective. Another PAF antagonist, SRI 63441 (5 mg/kg, i.p., twice a day) also inhibited EAT growth (80.4%). The BN 52021 added to EAT cells in culture, at concentration of 10(-3) and 10(-4) M, did not affect the viability and proliferation of tumors cells. In an attempt to understand the mechanism of this inhibition, we analyzed the peritoneal macrophages for spreading ability and H2O2 release. It was found that 24 h after tumor implantation there was an increase in the spreading ability of peritoneal macrophages (75%) and that, as the tumor grew, the spreading index fell to control levels ( less than 10%). (5 mg/kg/twice a day) the spreading remained elevated (50-60%) at all the times examined. Release of H2O2, measured by horseradish peroxidase-phenol red oxidation, was below detectable levels throughout tumor growth.(ABSTRACT TRUNCATED AT 250 WORDS)

[Neurocryptococosis and immunosuppression: experimental model in mice]. / Neurocriptococose e imunossupressão: modelo experimental em camundongos.

In order to accomplish a model for immunosuppression in experimental conditions two lots of 50 young mice were divided into groups of 10, and submitted to dexamethasone ingestion. Two experiments were considered in the study. In the first experiment conditions for immunosuppression were established in 50 mice, by per os supply of several concentrations of dexamethasone in the water given for normal daily intake ad libitum. Criteria of immunosuppression considered include: hair standing on end; leukopenia by decrease of lymphocyte population; spleen atrophy in relation to controls, with severe hypoplasy of all lymphoid structures. In the second experiment another lot of 50 mice in similar conditions established for the first experiment and concerning the immunosuppression were submitted to intraperitoneal inoculation of near 10(6) Cryptococcus neoformans. Culture suspensions of cryptococci isolated from cerebrospinal fluid samples of human neurocryptococcosis cases were used for this purpose. Although fungi could be recognized in lungs, liver and spleen from every mice inoculated, only those with central nervous system involvement (near by an half of all) died. The discussion included considerations about the role of the blood-brain barrier function in the findings registered.