Comparative efficacy and toxicity of two vaccine candidates against Sporothrix schenckii using either Montanide™ Pet Gel A or aluminum hydroxide adjuvants in mice.

Sporotrichosis is an important zoonosis in Brazil and the most frequent subcutaneous mycosis in Latin America, caused by different Sporothrix species. Currently, there is no effective vaccine available to prevent this disease. In this study, the efficacy and toxicity of the adjuvant Montanide™ Pet Gel A (PGA) formulated with S. schenckii cell wall proteins (ssCWP) was evaluated and compared with that of aluminum hydroxide (AH). Balb/c mice received two subcutaneous doses (1st and 14th days) of either the unadjuvanted or adjuvanted vaccine candidates. On the 21st day, anti-ssCWP antibody levels (ELISA), the phagocytic index, as well as the ex vivo release of IFN-γ, IL-4, and IL-17 by splenocytes and IL-12 by peritoneal macrophages were assessed. Cytotoxicity of the vaccine formulations was evaluated in vitro and by histopathological analysis of the inoculation site. Both adjuvanted vaccine formulations increased anti-ssCWP IgG, IgG1, IgG2a, and IgG3 levels, although IgG2a levels were higher in response to PGA+CWP100, probably contributing to the increase in S. schenckii yeast phagocytosis by macrophages in the opsonophagocytosis assay when using serum from PGA+CWP100-immunized mice. Immunization with AH+CWP100 led to a mixed Th1/Th2/Th17 ex vivo cytokine release profile, while PGA+CWP100 stimulated a preferential Th1/Th2 profile. Moreover, PGA+CWP100 was less cytotoxic in vitro, caused less local toxicity and led to a similar reduction in fungal load in the liver and spleen of S. schenckii- or S. brasiliensis-challenged mice as compared with AH+CWP100. These results suggest that PGA may be an effective and safe adjuvant for a future sporotrichosis vaccine.

Anti-Saccharomyces cerevisiae (ASCA) and anti-endomysial antibodies in spondyloarthritis.

Spondyloarthritis (SpA) are diseases with increased gut inflammation. To search for (anti-Saccharomyces cerevisiae) ASCA IgA, ASCA IgG, and anti-endomysial antibodies (EmA-IgA) in a cohort of 70 patients with SpA, we found 18.6% (13/70) positive for IgA-ASCA in the SpA group and 3/57 (5.2%) in the control group (P = 0.031). ASCA IgG and EmA-IgA were found at the same frequency in SpA and controls. No relationship of ASCA IgA positivity could be established with disease activity (measured by ESR, C-reactive protein, and BASDAI), presence of uveitis, or peripheral arthritis neither with functional status measured by BASFI. SpA patients present an increase in the IgA-ASCA positivity without any relationship to disease activity, functional index, clinical profile or the presence of HLA-B27. There is no evidence of higher prevalence of EmA-IgA in SpA patients in the studied sample.

Seropositive antiaquaporin-4 antibody associated with multisegmental myelitis in a patient with paracoccidioidomycosis.

Since the description of the association between neuromyelitis optica (Devic's disease) and aquaporin 4 IgG antibody (NMO-IgG), the search for this antibody has been considered a highly recommended laboratory test when centromedullary multisegmental lesions are observed by magnetic resonance imaging (MRI). Such MRI lesions have not been confined to acute NMO myelitis because other infectious and post-infectious disorders may display a similar lesional pattern. However, NMO-IgG has not been currently searched and associated with these myelitides. The objective of this study is to report an infectious myelitis that tested positive for NMO-IgG and comment on the implications of this finding. We report the presence of NMO-IgG in one patient exhibiting centromedullary multisegmental lesions who presented Paracoccidioides brasiliensis myelitis.

The relative importance of CD4+ and CD8+T cells in immunity to pulmonary paracoccidioidomycosis.

Protective immunity in paracoccidioidomycosis (PCM) is believed to be mediated by cellular immunity, but the role of T cell subsets has never been investigated. The aim of this study was to characterize the function of CD4+ and CD8+ T cells in the immunity developed by susceptible, intermediate and resistant mice after P. brasiliensis infection. In susceptible mice, depletion of CD4+ T cells did not alter disease severity and anergy of cellular immunity but diminished antibody production. Anti-CD8 treatment led to increased fungal loads, but restored DTH reactivity. In resistant mice, both CD4+ and CD8+ T cells control fungal burdens and cytokines although only the former regulate DTH reactions and antibody production. In the intermediate strain, deficiency of whole T and CD8+ T cells but not of CD4+ T or B cells led to increased mortality rates. Thus, in pulmonary PCM: (a) irrespective of the host susceptibility pattern, fungal loads are mainly controlled by CD8+ T cells, whereas antibody production and DTH reactions are regulated by CD4+ T cells; (c) CD4+ T cells play a protective role in the resistant and intermediate mouse strains, whereas in susceptible mice they are deleted or anergic; (d) genetic resistance to PCM is associated with concomitant CD4+ and CD8+ T cell immunity secreting type 1 and type 2 cytokines.

Expression of antibodies directed to Paracoccidioides brasiliensis glycosphingolipids during the course of paracoccidioidomycosis treatment.

Paracoccidioidomycosis (PCM) is a granulomatous disease caused by the dimorphic fungus Paracoccidioides brasiliensis. The immunoglobulin classes and isotypes of antibodies directed to acidic glycosphingolipids (GSLs) and glucosylceramide of P. brasiliensis were determined by enzyme-linked immunosorbent assay of sera from 31 PCM patients. The reactivities of 38 serum samples were analyzed by considering the stage of treatment: before antifungal treatment (n = 10), during 1 to 4 months of treatment (T1-4; n = 9), during 5 to 12 months of treatment (T5-12; n = 9), and posttreatment (PT; n = 10). Sera from healthy subjects (n = 12) were used as controls. Only the GSL Pb-1 antigen, which presents the carbohydrate structure Galfbeta1-6(Manalpha1-3)Manbeta1, was reactive with the PCM patient sera. The PCM patient sera did not react with Pb-2, which lacks the Galf residue and which is considered the biosynthetic precursor of Pb-1, indicating that the Galf residue is essential for antibody reactivity. The Pb-1 glycolipid from nontreated patients elicited a primary immune response with immunoglobulin M (IgM) production and subsequent switching to IgG1 production. The IgG1 titer increased after the start of antifungal treatment (T1-4 group), and general decreases in the anti-Pb-1 antibody titers were observed after 5 months of treatment (T5-12 and PT groups). The Pb-1 antigen, an acidic GSL with terminal Galf residue, has potential application as an elicitor of the host immune response in patients with PCM.

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 Addison's Disease.

Neutrophil role in pulmonary paracoccidioidomycosis depends on the resistance pattern of hosts.

The immunoprotective and immunomodulatory role of neutrophils during pulmonary infection of resistant (A/J) and susceptible (B10.A) mice to Paracoccidioides brasiliensis was investigated. First, comparative studies about early cellular influx to the lungs demonstrated higher numbers of neutrophils in susceptible rather than in resistant mice. Neutrophil depletion resulted in decreased survival times of susceptible but not resistant mice. In both mouse strains, depletion led to increased fungal burdens at Week 1 of infection; however, only susceptible mice remained with increased pulmonary fungal loads and presented a dramatic fungal dissemination to liver and spleen. At Week 1 of infection, treated and untreated B10.A and A/J mice were negative for delayed-type hypersensitivity (DTH) reactions, which remained negative for the susceptible strain. In contrast, from the second week onward, control and neutrophil-depleted, resistant mice became positive for DTH reactions. In B10.A mice, neutrophil depletion resulted in increased levels of interleukin (IL)-12 and IL-4 in the lungs, high levels of hepatic cytokines, and increased synthesis of T helper cell type 1 (Th1)- and Th2-regulated antibodies [immunoglobulin G1 (IgG1), IgA, and IgG3]. In neutrophil-depleted A/J mice, high levels of pulmonary IL-12 and granulocyte macrophage-colony stimulating factor were concomitant to diminished levels of hepatic cytokines and increased amounts of Th1-regulated isotypes (IgG2a, IgG2b, and IgG3). Differently from primary infection, neutrophil depletion did not alter immunoprotection in secondary paracoccidioidomycosis. As a whole, our data showed that the genetic patterns of hosts exert an important influence on the immunoprotective and immunoregulatory functions of neutrophils, which appear to be essential in situations devoid of cell-mediated immunity.

Glyceraldehyde-3-phosphate dehydrogenase of Paracoccidioides brasiliensis is a cell surface protein involved in fungal adhesion to extracellular matrix proteins and interaction with cells.

The pathogenic fungus Paracoccidioides brasiliensis causes paracoccidioidomycosis, a pulmonary mycosis acquired by inhalation of fungal airborne propagules, which may disseminate to several organs and tissues, leading to a severe form of the disease. Adhesion to and invasion of host cells are essential steps involved in the infection and dissemination of pathogens. Furthermore, pathogens use their surface molecules to bind to host extracellular matrix components to establish infection. Here, we report the characterization of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of P. brasiliensis as an adhesin, which can be related to fungus adhesion and invasion. The P. brasiliensis GAPDH was overexpressed in Escherichia coli, and polyclonal antibody against this protein was obtained. By immunoelectron microscopy and Western blot analysis, GAPDH was detected in the cytoplasm and the cell wall of the yeast phase of P. brasiliensis. The recombinant GAPDH was found to bind to fibronectin, laminin, and type I collagen in ligand far-Western blot assays. Of special note, the treatment of P. brasiliensis yeast cells with anti-GAPDH polyclonal antibody and the incubation of pneumocytes with the recombinant protein promoted inhibition of adherence and internalization of P. brasiliensis to those in vitro-cultured cells. These observations indicate that the cell wall-associated form of the GAPDH in P. brasiliensis could be involved in mediating binding of fungal cells to fibronectin, type I collagen, and laminin, thus contributing to the adhesion of the microorganism to host tissues and to the dissemination of infection.

Adjuvant effect of synthetic oligodeoxyribonucleotides (CpG-ODN) from the Paracoccidioides brasiliensis gp43 gene on the Th2-Th1 immunomodulation of experimental paracoccidioidomycosis.

Paracoccidioidomycosis (PCM) is caused by the dimorphic fungus Paracoccidioides brasiliensis. Immunostimulatory effects of P. brasiliensis DNA and CpG-oligodeoxyribonucleotides (CpG-ODN) have shown a Th2-Th1 immunomodulation of the isogenic murine model of susceptibility, which develops a progressive and disseminating disease. In this study, we investigated the optimum time interval and doses of CpG-ODN which are able to induce Th2-Th1 immunomodulation. The optimum concentrations for the induction of a decrease in antibody production were 0.5 and 1 microg. Mice immunized twice with CpG-ODN and gp43 (5 and 7 days before the challenge) showed a 60% higher chance of survival compared with the control group (nonimmunized), and an increase in Th1 isotype (IgG2a) was also observed. In vitro assays of naive and preimmunized mice showed discrete cellular proliferation when stimulated by suitable concentrations of CpG-ODN. Type 1 cytokines interleukin-12 (IL-12) and interferon-gamma were increased in cell culture supernatants, but no significant difference was found in Th2 IL-4 cytokines in stimulated or nonstimulated cell cultures. Concerning the Th2-Th1 kinetics in experimental PCM models by adjuvant effect of CpG-ODN, there are still many questions to be answered and clarified. However, the gathering of data obtained in this investigation has led us to suggest that the modulation of Th2-Th1 in experimental PCM depends on time and CpG-ODN concentration.

Identification of immunogenic proteins from Paracoccidioides brasiliensis antigenic fractions F0, FII and FIII.

Paracoccidioides brasiliensis causes a chronic granulomatous mycosis prevalent in South America, and cell-mediated immunity is the principal mode of protection against this fungal infection. In this context, one of the strategies to discover proteins that are target of an effective immune response against P. brasiliensis is the partial sequencing of cDNA from an expression library previously screened with immunoglobulins (Ig) to generate antigen sequence tags (AST). In the present work, a P. brasiliensis yeast cDNA expression library was screened with affinity chromatography-purified IgG from rabbit sera immunized with P. brasiliensis antigenic fractions (F0, FII or FIII) or from paracoccidioidomycosis (PCM) patient sera by indirect ELISA. From 119 clones selected by the immunoscreening procedure, 40% were recognized by IgG from PCM patients, 25% were recognized by anti-F0, 8% were selected by anti-FII and 11% recognized by FIII specific antibodies. The remaining clones presented cross-reaction to all anti-sera tested. The AST homologies with previously reported sequences in the nonredundant GenBank at NCBI revealed high significant homology to fungal proteins of known function. One of them matched calcineurin B of Neurospora crassa with 35% identity and 55% similarity in amino acid sequence. We also identified an AST homologous to a Kinesin like protein from Ustilagus maydis and other fungi with 86% identity and 91% similarity. On the other hand, the vast majority of selected cDNA clones are new genes and represent 60% of the total. Prediction of transmembrane regions with the prediction transmembrane protein topology with a hidden markov model (TMHMM) revealed consensus sequences representing structural membrane segments in 28 encoded proteins.

Protective immunity induced in mice by F0 and FII antigens purified from Paracoccidioides brasiliensis.

Paracoccidioides brasiliensis causes a chronic granulomatous mycosis prevalent in South America, and cell-mediated immunity represents the principal mode of protection against this fungal infection. We investigated whether immunization with P. brasiliensis antigens fractionated by anionic chromatography on fast protein liquid chromatography (FPLC) could elicit protective immunity. BALB/c mice were immunized by subcutaneous injection of either 10 microg fractions 0 (F0), II (FII) or III (FIII) in the presence of 100 microg of Corynebacterium parvum and 1 mg of Al(OH)(3) and challenged with pathogenic P. brasiliensis strain. Mice immunized with F0 presented cellular and humoral immune responses with significant production of IFN-gamma, and high levels of IgG2a and IgG3 isotypes. Immunization with FII induced significant production of IFN-gamma and IL-10 associated with high levels of IgG1 and IgG2a. It was demonstrated that immunization with F0 or FII promoted significant decrease of organ colony-forming units (CFUs) in the lung after challenge infection without fungi dissemination to the spleen or liver. In contrast, FIII immunized mice develop a progressive disseminated disease to spleen and liver presented significant levels of INF-gamma, IL-10 or TGF-beta associated with high production of IgG1 and IgG2a with low production of IgG2b and IgG3 after challenge infection. Taken together, these findings suggest that antigens of F0 and FII are reliable vaccine candidates against the paracoccidioidomycosis.

Interleukin-12 protects mice against disseminated infection caused by Paracoccidioides brasiliensis but enhances pulmonary inflammation.

Paracoccidioides brasiliensis is a facultative, intracellular pathogen causing the most important deep mycosis in Latin America. As the production of IFN-gamma and induction of cell-mediated immunity to P. brasiliensis is of critical importance in host defense, the immunotherapeutic effect of exogenous IL-12 administration was studied in a murine model of susceptibility to pulmonary infection. rIL-12 treatment led to a less disseminated disease, as confirmed by decreased fungal loads in liver and spleen. Administration of rIL-12 did not affect fungal growth in the lungs, although it did induce an augmented pulmonary mononuclear cell inflammation. IL-12 treatment induced an early (week 1) increase in pulmonary IFN-gamma, but decreased cytokine and specific antibody (IgG1 and IgG3) production at week 8 after infection. These results show that IL-12 administration induces a less severe infection, but the high inflammatory response detected in the lungs precludes its possible use as a new therapeutic tool for severe paracoccidioidomycosis.

Resistance to Paracoccidioides brasiliensis infection is linked to a preferential Th1 immune response, whereas susceptibility is associated with absence of IFN-gamma production.

The secretion of interferon-gamma (IFN-gamma), interleukin-2 (IL-2), IL-4, IL-5, and IL-10 by antigen-stimulated lymph node cells, eosinophil maturation, and the antibody isotypes produced were examined during intraperitoneal infection of susceptible (B10.A) and resistant (A/Sn) mice with Paracoccidioides brasiliensis. Lymph node cells from resistant mice produced early and sustained levels of IFN-gamma and IL-2, whereas susceptible animals secreted low to undetectable amounts of these type 1 cytokines. Both mouse strains presented late and transient production of IL-4, whereas IL-10 was produced constantly throughout the course of disease. Resistant animals produced increasing levels of IL-5 in the chronic phase of the infection (from the eighth week on), whereas susceptible mice showed two peaks of IL-5 production, at the first and twelfth weeks after infection. Only the susceptible strain presented medullary and splenic eosinophilia concomitant with the raised IL-5 production. In resistant mice, the levels of IgG2a antibodies were significantly higher than those observed in susceptible mice, which preferentially secreted IgG2b and IgA isotypes. Taken together, these results demonstrate that a sustained production of IFN-gamma and IL-2 and a predominant secretion of IgG2a antibodies are associated with resistance to P. brasiliensis. In contrast, the production of low levels of IFN-gamma, early secretion of high levels of IL-5 and IL-10, eosinophilia, and a preferential secretion of IgG2b and IgA isotypes characterize the progressive disease in susceptible animals.

Depletion of CD8(+) T cells in vivo impairs host defense of mice resistant and susceptible to pulmonary paracoccidioidomycosis.

Using a pulmonary model of infection, we demonstrated previously that A/Sn and B10.A mice are, respectively, resistant and susceptible to Paracoccidioides brasiliensis infection. Employing the same experimental model, we examined herein the role of CD8(+) T cells in the course of paracoccidioidomycosis. Treatment with anti-CD8 monoclonal antibodies caused a selective depletion of pulmonary and splenic CD8(+) T cells in both mouse strains. The number of pulmonary CD4(+) T cells and immunoglobulin-positive cells was independent of the number of CD8(+) T cells. In susceptible mice, the loss of CD8(+) T cells by in vivo treatment with anti-CD8 monoclonal antibodies impaired the clearance of yeasts from the lungs and increased the fungal dissemination to the liver and spleen. The same treatment in resistant mice increased fungal dissemination to extrapulmonary tissues but did not alter the pulmonary fungal load. Furthermore, CD8(+) T-cell depletion did not modify delayed-type hypersensitivity reactions of A/Sn mice but increased these reactions in B10.A mice. The production of P. brasiliensis-specific antibodies by resistant and susceptible mice depleted of CD8(+) T cells was similar to that of mice given control antibody. Histopathologically, depletion of CD8(+) T cells did not disorganize the focal granulomatous lesions developed by both mouse strains. These results indicate that CD8(+) T cells are necessary for optimal clearance of the fungus from tissues of mice infected with P. brasiliensis and demonstrate more prominent protective activity by those cells in the immune responses mounted by susceptible animals.

In vitro susceptibility of chromoblastomicosis and phaehyphomycosis agents to antigungal drugs

The in vitro susceptibility of chromoblastomycosis and phaeohyphomycosis agents to antifungal drugs was appraised using the reference macrodilution method proposed by the National Committee for Clinical Laboratory Standards (NCCLS) for yeasts modified for filamentos fungi. The antifungal drugs amphotericin B, 5-fluorocytosine, intraconazole and fluconazole were tested agsints one environmental and 18 clinical isolates. This work amented the macrodilution methods proposed by NCCLS and suggests that a conidial suspension free of hyphae leads to a more reliable assay and provides for better reproducibility. The macrodilution method was performed with 10 (elevado ao 4) conidia ml-1. The MIC values ranged from 1.0 to 16.0 ug ml-1 for amphotericin B and 3.12 to 25.0 ug ml-1 for 5-fluorocytosine. A MIC range of 0.06 to 1.95 ug ml-1 was determined for itraconazole while 2.0 to 64.0 ug ml-1 was detected for fluconazole

[Specific fungal antisera production in rabbits]. / Producción de antisueros fúngicos específicos en conejo.

A method for fungic antisera production against Aspergillus fumigatus, Histoplasma capsulatum, Paracoccidioides brasiliensis and Coccidioides immitis in rabbits was evaluated. Intradermic via and antigen in the dilution used for routine tests (UD) were employed to produce positive control serum for immunodiffusion test in agar gel. A. fumigatus, H. capsulatum and C. immitis antigens were prepared as described in CDC's Procedure Manual, P. brasiliensis antigen was prepared as previously described by Pires de Camargo. All rabbits produced antibodies against the different specific antigens in the primary response peak and after each booster. The titer obtained in secondary response was similar or smaller than the primary response in all cases. However, bands of similar quality and intensity were obtained by immunoprecipitation in agar gel tests. Although the antibody titers proved to be similar, higher or lower concentration of antigen used in the primary immunization produced fewer and smeared bands, respectively. This effect was evaluated in A. fumigatus only. Specific antisera production with this method proved to be easy and yielded high quality antisera. The major advantages of this method are: a) reduced number of inoculations, b) fast and simple standardisation of the antigen needed, c) equally useful for all the fungal species used so far. Therefore we strongly recommend this method.

[The evaluation of the specific type of capsular polysaccharide of Cryptococcus neoformans as an immunogen and positive-control antigen]. / Evaluación del polisacárido capsular tipo específico de Cryptococcus neoformans como inmunógeno y antígeno control positivo.

The specific type of capsular polysaccharide of an autochthonous strain of Cryptococcus neoformans var. neoformans was obtained by using the method of selective precipitation with hexadimethrine bromide. The capsular polysaccharide was matched to lamb's erythrocytes and it was used as an immunogen in rabbits. Antibody titres of up to 1:32 were attained. Doble serial dilutions of the capsular polysaccharide were evaluated as positive control antigen by contraimmunoelectrophoresis, latex and ELISA techniques, showing biological activity.

Cellular requirements for immunomodulatory effects caused by cell wall components of Paracoccidioides brasiliensis on antibody production.

In a previous study, we reported an increase in the number of immunoglobulin-secreting cells and the augmentation of antibody production (IgM and IgG3) against unrelated antigens (sheep erythrocytes or bovine serum albumin (BSA)) in mice infected with the fungus Paracoccidioides brasiliensis as well as in mice inoculated with its cell wall preparation (CW). The immunomodulatory effect of the live fungus and CW preparation was dose-dependent and mainly restricted to the i.p. inoculation simultaneously to the BSA challenge by the i.v. route. In the present study, we investigated the active component of CW preparation upon the phenotype and also the degree of activation of possible target peritoneal cells involved in those phenomena. An insoluble polysaccharide fraction (F1 fraction) mainly composed of beta-glucan and chitin, and the purified beta-glucan (BGPb) behaved as CW in the augmentation of early antibody production. The peritoneal mononuclear inflammatory cells induced by CW, F1 fraction and BGPb were highly positive to alpha-naphthyl esterase staining; released low H2O2; expressed high levels of MHC-Ia(d) molecules and produced inflammatory cytokines such as tumour necrosis factor-alpha (TNF-alpha) and IL-6. Phenotypic analysis by flow cytometry and immunohistochemical techniques of the inflammatory cells responding to F1 fraction showed a prevalence of (CD11b/CD18, Mac-1)+ peritoneal macrophages. In addition, s.c. inoculation of F1 fraction resulted in the formation of nodular, localized and not progressive granulomatous lesions with an accumulation of (CD11b/C18)+ macrophages. Adoptive transferred Mac-1 macrophages to immunized syngeneic recipient mice were able to cause an increase in anti-BSA antibody production. These results suggest that inflammatory (CD11b/CD18)+ macrophages may be related to immunological disturbances, caused by cell wall components of P. brasiliensis.

Biochemical, antigenic and allergenic characterization of crude extracts of Drechslera (Helminthosporium) monoceras.

In a previous study with airborne mould extracts we verified that Drechslera (Helminthosporium) monoceras presented stronger reactions than those presented by 42 other moulds isolated in São Paulo city. In the present study, we evaluated the biochemical composition and the antigenicity of crude extracts obtained from vegetative and conidial stage of D. monoceras using Czapeck broth (CB) modified and tris-HCl for extraction. The maximum values of total proteins and lipids were verified in the crude extract obtained in the 28th day of growth, and maximum values of carbohydrates were observed in the extracts of the 16th, 22nd and 26th days. The fractionated proteins by SDS-PAGE presented bands with molecular weights between 14.4 to 67 Kd; the 28th day extract showed a larger number of bands. The carbohydrates and amino acids were characterized by thin-layer chromatography. The antigenicity of the crude extracts was verified by immunodiffusion reaction in agar against rabbit hyperimmune sera. Precipitation lines were observed in all studied extracts and common antigenic molecular populations. Based on the above results, the 28th day extract was selected to verify the induction of IgE antibody responses in immunizations of Balb/c and cAF-1 mice, and titer by passive cutaneous anaphylaxis test using Wistar rats. The maximum titers obtained were 160 in cAF-1 mice and 1.280 in Balb/c mice. The results suggest that the 28th day extract contains allergenic fractions and should be chosen for future studies related to fractionation, characterization and standardization in diagnostic methods and immunotherapy.

The role of somatic structure of the fungus Paracoccidioides brasiliensis upon B cell activation in experimental paracoccidioidomycosis.

In this study, we report an increase of the number of antibody-secreting cells and the augmentation of antibody production against unrelated antigens in mice infected with the fungus P. brasiliensis, as well as in mice inoculated with cell wall preparation isolated from P. brasiliensis (CW). The immunomodulatory effect of the live fungus and the CW preparation was dose-dependent, and their actions were mainly restricted to the i.v. or i.p. inoculation simultaneously with the sheep erythrocyte challenge by the i.v. route or restricted to i.p. inoculation of CW when bovine serum albumin (BSA) antigen was used. The dependence of antibody production on different routes of CW inoculation was correlated with the number of antigen-specific B cells in the spleen as determined by direct and reverse plaque-forming cell assays. The immunization schedules using CW preparation caused a preferential production of IgM and IgG3 antibodies. The results also showed that the hyperactive humoral immune response of mice induced by i.p. inoculation of CW was devoid of polyclonal B cell activation compared with the effects observed for the lipopolysaccharide (LPS)-treated groups. Paracoccidioides brasiliensis CW components may have potent immunological properties related to the non-specific B cell activation found in paracoccidioidomycosis.