Additive effect of P10 immunization and chemotherapy in anergic mice challenged intratracheally with virulent yeasts of Paracoccidioides brasiliensis

Paracoccidioidomycosis is a systemic granulomatous disease manifested in the acute/subacute or chronic forms. The anergic cases of the acute/subacute form are most severe, leading to death threatening conditions. Drug treatment is required to control the disease but the response in anergic patients is generally poor. A 15-mer peptide from the major diagnostic antigen gp43, named P10, induces a T-CD4+ helper-1 immune response in mice of different haplotypes and protects against intratracheal challenge with virulent P. brasiliensis. Presently, P10 immunization and chemotherapy were associated in an attempt to improve antifungal treatment in Balb/c mice made anergic by adding dexamethasone to the drinking water. The combined drug/peptide treatment significantly reduced the lung CFUs in infected anergic mice, largely preserved lung alveolar structure and prevented fungal dissemination to liver and spleen. Results recommend that a P10-based vaccine should be associated to chemotherapy for improved treatment of paracoccidioidomycosis aiming especially at anergic cases.     

Immunization with P10 Peptide Increases Specific Immunity and Protects Immunosuppressed BALB/c Mice Infected with Virulent Yeasts of Paracoccidioides brasiliensis

Paracoccidioidomycosis is a systemic granulomatous disease caused by Paracoccidioides spp. A peptide from the major diagnostic antigen gp43, named P10, induces a T-CD4⁺ helper-1 immune response in mice and protects against intratracheal challenge with virulent P. brasiliensis. Previously, we evaluated the efficacy of the P10 peptide alone or combined with antifungal drugs in mice immunosuppressed and infected with virulent isolate of P. brasiliensis. In the present work, our data suggest that P10 immunization leads to an effective cellular immune response associated with an enhanced T cell proliferative response. P10-stimulated splenocytes increased nitric oxide (NO) production and induced high levels of IFN-γ, IL-1β and IL-12. Furthermore, significantly increased concentrations of pro-inflammatory cytokines were also observed in lung homogenates of immunized mice. P10 immunization was followed by minimal fibrosis in response to infection. Combined with antifungal drugs, P10 immunization most significantly improved survival of anergic infected mice. Administration of either itraconazole or sulfamethoxazole/trimethoprim together with P10 immunization resulted in 100 % survival up to 200 days post-infection, whereas untreated mice died within 80 days. Hence, our data show that P10 immunization promotes a strong specific immune response even in immunocompromised hosts and thus P10 treatment represents a powerful adjuvant therapy to chemotherapy.     

Serological detection of antibodies against Paracoccidioides brasiliensis in dogs with leishmaniasis

The aim of this study was to detect antibodies against Paracoccidioides brasiliensis in dogs seropositive and seronegative for leishmaniasis. Sera from 836 dogs (449 positive and 387 negative to leishmaniasis) were analysed by ELISA and the immunodiffusion test using gp43 and exoantigen, respectively. The analysis of the 836 serum samples by ELISA and the immunodiffusion test showed a positivity of 67.8 % and 7.3%, respectively, for P. brasiliensis infection. The dogs positive to leishmaniasis showed a higher reactivity to gp43 (79.9%) and exoantigen (12.7%) than the negative ones (54.0% and 1.0%, respectively). The higher reactivity to P. brasiliensis antigens may be due to cross-reactivity or a co-infection of dogs by Leishmania and P. brasiliensis. The lower correlation (0.187) observed between reactivity to gp43 and Leishmania antigen reinforces the latter hypothesis.     

DNA vaccine encoding peptide P10 against experimental paracoccidioidomycosis induces long-term protection in presence of regulatory T cells

Paracoccidioidomycosis is a granulomatous systemic mycosis endemic in Brazil and other Latin America countries. A DNA vaccine encoding the immunoprotective peptide 10 (P10) significantly reduced the fungal burden in mice when given prior to or after intratracheal challenge with Paracoccidioides brasiliensis. Presently, the generation/expansion of CD4⁺ CD44^hi memory T cells as well as Foxp3⁺ Treg cells in mice immunized with the DNA vaccine (pcDNA3-P10) before and after infection with P. brasiliensis was investigated. Memory CD4⁺ CD44^hi T cells simultaneously with Foxp3⁺ Treg cells increased in the spleens and lungs of pcDNA3-P10 immunized mice on day 0, 30, 60 and 120 postinfection. Histopathology of the lung tissue showed minimal inflammation in immunized mice compared with the unimmunized group, suggesting a role for regulatory T cells in controlling the immunopathology. The DNA vaccine shows that the repeated immunization generates memory cells and regulatory T cells that replace the initially protective pro-inflammatory T cells conferring a long term protection while preserving the integrity of the infected tissue.     

Protection induced in BALB/c mice by the high-molecular-mass (hMM) fraction of <Emphasis Type="Italic">Paracoccidioides brasiliensis</Emphasis>

Paracoccidioidomycosis (PCM) is a granulomatous disease caused by a dimorphic fungus, Paracoccidioides brasiliensis. The present study investigated the protective activity of the P. brasiliensis high-molecular-mass (hMM) fraction (~380 kDa) in experimental murine PCM. In the first step, lymphocyte proliferation and production of IFNγ (but not IL-4) were observed in “in vitro” spleen cells (from female BALB/c mice infected (i.v.) with P. brasiliensis) that were stimulated with hMM fractions. In the second step, female BALB/c mice were previously immunized (s.c.) with hMM fraction (25 μg/protein = F-25 and 50 μg/protein = F-50), and the colony-forming units (CFU) of the lung and spleen, the histopathological characteristics of the granulomatous lesions, and plasmatic gp43 soluble antigens and anti-hMM IgG levels were analyzed at 28 and 56 days after infection. The lung and liver CFU were lower in mice previously immunized with the hMM fraction (P < 0.05). The granulomatous lesions revealed a greater degree of compaction and organization, with no dissemination of the fungus to other organs. Lower soluble antigen levels (P < 0.05) and higher IgG anti-hMM fraction (P < 0.05) were observed in immunized groups. The results for CFU, histopathology and antigenemia suggest that the hMM fraction has a protective effect in experimental paracoccidioidomycosis in BALB/c mice.     

Serological Survey of Paracoccidioidomycosis in Sheep

The objective of this study was to evaluate the prevalence of antibodies against Paracoccidioides brasiliensis in sheep from Guarapuava, Paraná State, Brazil. The seroepidemiological study was carried out in 262 sheep. The samples were analyzed by ELISA and immunodiffusion test using P. brasiliensis gp43 and exoantigen as antigens, respectively. Initially, two sheep were immunized with P. brasiliensis to evaluate whether contact with the fungal cells could induce a humoral immune response against gp43 and exoantigen from P. brasiliensis. Both animals produced antibodies against gp43 and exoantigen, the main antigens used for diagnosis and seroepidemiology of paracoccidioidomycosis. A reactivity of 37% was observed to the P. brasiliensis gp43 antigen by ELISA although no reactivity had been observed by the immunodiffusion test. Sheep under extensive grazing system showed higher frequency of positivity to P. brasiliensis (P ≤ 0.05) than those under intensive and semi-intensive systems. These data suggest that sheep may be a useful epidemiological marker of P. brasiliensis presence in the environment and reinforce that contact with soil is an important risk factor for infection.     

Diversity in Paracoccidioides brasiliensis . The PbGP43 gene as a genetic marker

Paracoccidioides brasiliensis is a temperature-dependent dimorphic fungus and the agent of paracoccidioidomycosis (PCM), which is prevalent in rural workers of Latin American countries. Until a decade ago, most of the studies involving P. brasiliensis used clinical isolates, since environmental samples from soil are difficult to obtain. More recently, P. brasiliensis has been isolated from infected wild and domestic animals, especially from the nine-banded armadillo Dasypus novemcinctus in Brazil. Over the years, diversity within the species has been observed at several phenotypic levels. The present review will discuss the reports focusing on genetic polymorphism, which culminated with the detection of P. brasiliensis phylogenetic species as a result of a multilocus study. Polymorphism in the PbGP43 gene is detailed. This gene encodes fungal glycoprotein gp43, a dominant P. brasiliensis antigen largely studied in the last two decades for its importance in diagnosis, immune protection, and adhesive properties to extracellular matrix-associated proteins. Fungal traits associated with genetic groups are discussed.     

Molecular phylogeny of animal pathogen Lacazia loboi inferred from rDNA and DNA coding sequences

Lacazia loboi is a geographically restricted, uncultivated fungal pathogen of humans and dolphins. Previous investigations using 18S small unit rDNA, chitin synthase 2 and gp43 DNA sequences positioned L. loboi as a close relative of Paracoccidioides brasiliensis. However, given the few individuals of L. loboi studied and the high degree of genetic variation observed in P. brasiliensis, the existence of L. loboi as an independent species has been questioned. To investigate the phylogenetic position of this species, we conducted a phylogenetic analysis using 20 L. loboi collections (L. loboi was obtained from proven cases of lacaziosis and 14 collections were maintained in mice, the others were analyzed from DNA taken directly from infected human tissue.). L. loboi DNA sequence was compared to that from 17 P. brasiliensis strains that represented the known variation in this species, and outgroup taxa in the Onygenales (Ajellomyces and Coccidioides species). Our analyses used DNA sequence from ITS rRNA, and partial coding sequences of chitin synthase 4, ADP-ribosylation factor, and gp43. Nucleotide variation among strains of L. loboi was minor but numerous nucleotide mismatches and multiple gaps were found for these gene regions among members in the Ajellomycetaceae, including P. brasiliensis. Phylogenies inferred using neighbor-joining, maximum parsimony and Bayesian analyses showed no significant conflict and depicted L. loboi as a well-supported, monophyletic group that was sister to the Paracoccidioides clade. These results argue for maintaining L. loboi as a taxon independent from Paracoccidioides within the Ajellomycetaceae.     

Inhibition of PbGP43 Expression May Suggest that gp43 is a Virulence Factor in Paracoccidioides brasiliensis

Glycoprotein gp43 is an immunodominant diagnostic antigen for paracoccidioidomycosis caused by Paracoccidioides brasiliensis. It is abundantly secreted in isolates such as Pb339. It is structurally related to beta-1,3-exoglucanases, however inactive. Its function in fungal biology is unknown, but it elicits humoral, innate and protective cellular immune responses; it binds to extracellular matrix-associated proteins. In this study we applied an antisense RNA (aRNA) technology and Agrobacterium tumefaciens-mediated transformation to generate mitotically stable PbGP43 mutants (PbGP43 aRNA) derived from wild type Pb339 to study its role in P. brasiliensis biology and during infection. Control PbEV was transformed with empty vector. Growth curve, cell vitality and morphology of PbGP43 aRNA mutants were indistinguishable from those of controls. PbGP43 expression was reduced 80–85% in mutants 1 and 2, as determined by real time PCR, correlating with a massive decrease in gp43 expression. This was shown by immunoblotting of culture supernatants revealed with anti-gp43 mouse monoclonal and rabbit polyclonal antibodies, and also by affinity-ligand assays of extracellular molecules with laminin and fibronectin. In vitro, there was significantly increased TNF-α production and reduced yeast recovery when PbGP43 aRNA1 was exposed to IFN-γ-stimulated macrophages, suggesting reduced binding/uptake and/or increased killing. In vivo, fungal burden in lungs of BALB/c mice infected with silenced mutant was negligible and associated with decreased lung ΙΛ−10 and IL-6. Therefore, our results correlated low gp43 expression with lower pathogenicity in mice, but that will be definitely proven when PbGP43 knockouts become available. This is the first study of gp43 using genetically modified P. brasiliensis.     

Saccharomyces cerevisiae Expressing Gp43 Protects Mice against Paracoccidioides brasiliensis Infection

The dimorphic fungus Paracoccidioides brasiliensis is the etiological agent of paracoccidioidomycosis (PCM). It is believed that approximately 10 million people are infected with the fungus and approximately 2% will eventually develop the disease. Unlike viral and bacterial diseases, fungal diseases are the ones against which there is no commercially available vaccine. Saccharomyces cerevisiae may be a suitable vehicle for immunization against fungal infections, as they require the stimulation of different arms of the immune response. Here we evaluated the efficacy of immunizing mice against PCM by using S. cerevisiae yeast expressing gp43. When challenged by inoculation of P. brasiliensis yeasts, immunized animals showed a protective profile in three different assays. Their lung parenchyma was significantly preserved, exhibiting fewer granulomas with fewer fungal cells than found in non-immunized mice. Fungal burden was reduced in the lung and spleen of immunized mice, and both organs contained higher levels of IL-12 and IFN-γ compared to those of non-vaccinated mice, a finding that suggests the occurrence of Th1 immunity. Taken together, our results indicate that the recombinant yeast vaccine represents a new strategy to confer protection against PCM.     

Paracoccidioides lutzii Plp43 Is an Active Glucanase with Partial Antigenic Identity with P. brasiliensis gp43

Background

Paracoccidioides brasiliensis and P. lutzii cause paracoccidioidomycosis (PCM). P. brasiliensis main diagnostic antigen is glycoprotein gp43, and its peptide sequence is 81% identical with a P. lutzii ortholog here called Plp43. P. lutzii (“Pb01-like”) apparently predominates in Midwestern/Northern Brazil, where high percentages of false-negative reactions using P. brasiliensis antigens have recently been reported. The aim of this work was to produce recombinant Plp43 to study its antigenic identity with gp43.

Methodology

We expressed rPlp43 as a secreted major component in Pichia pastoris and studied its reactivity in immunoblot with PCM patients'' sera from Southwestern and Midwestern Brazil.

Principal Findings

We showed that rPlp43 is not glycosylated and bears glucanase activity. The protein did not react with anti-gp43 monoclonal antibodies in immunoblot, suggesting absence of the corresponding gp43 epitopes. Nevertheless, common epitope(s) might exist, considering that gp43-positive PCM sera recognized rPlp43 in immunoblot, while gp43-negative sera (33 out of 51) from patients resident in Midwestern Brazil were also rPlp43-negative. Two genotyped P. lutzii were from patients with gp43-negative sera, suggesting that non-reactive sera are from patients infected with this species.

Conclusion

Our data suggest that gp43 and Plp43 bear one or only a few common epitopes and that gp43 cannot be used in diagnosis of PCM patients infected with P. lutzii probably because Plp43 is poorly expressed during infection.     

Morphological, Biochemical and Molecular Approaches for Comparing Typical and Atypical Paracoccidioides brasiliensis Strains

We evaluated the morphology of typical and atypical Paracoccidioides brasiliensis strains and the expression of its 43 kDa glycoprotein (GP43). Strains of P. brasiliensis preserved under mineral oil for long periods of time presented different morphological patterns on peptone, yeast-extract and glucose (PYG) agar. The intravenous inoculation in BALB/c mice confirmed that a strain bearing morphological alterations was non-virulent. In contrast, another strain also maintained under mineral oil but which did not exhibit such morphological dysfunction was as virulent as the well characterized Pb 339 and Pb 18 strains. The expression of the main antigen expressed by P. brasiliensis, GP43, was assessed in culture filtrates by western immunoblots. Typical and atypical strains were capable of secreting the glycoprotein, except strain Pb IOC 1059. The identity of the atypical strains was confirmed by PCR using specific primers for gp43, though the single PCR-fragment varied in size for the atypical strains. The PCR fragments from an atypical strain, Pb IOC 1210, and the typical Pb 339 and Pb IOC 3698 strains were sequenced and blasted to the gp43 gene from the Pb 18 strain (GenBank AY005429). These results ensured the identity of the atypical strains as P. brasiliensis, and suggested a relationship between the alteration of morphological differentiation and the virulence factor following storage under mineral oil.     

Mice Immunization with Radioattenuated Yeast Cells of <Emphasis Type="Italic">Paracoccidiodes brasiliensis</Emphasis>: Influence of the Number of Immunizations

Paracoccidioides brasiliensis is the agent of paracoccidioidomycosis, the most prevalent mycosis in Latin America. Up to the moment no vaccine has been reported. The aim of this study was to evaluate the influence of the number of immunizations on the protection elicited by radioattenuated yeast cells of P. brasiliensis. BALB/c mice were divided into two groups that were immunized once (Group 1) or twice (Group 2), respectively. In each group, mice were divided into sub-groups that were challenged 30, 45, or 60 days after the second immunization. Organ colony-forming units (CFUs) was determined 90 days post-challenge. A significant reduction in CFUs recovery was verified in both groups, but it was higher in Group 2. Histologic alterations were observed only in Group 1. The cytokines IL-4, IL-10, and IFN-γ were produced in mice of Group 1. In Group 2, only IFN-γ was significantly detected. IgG2a predominance relative to IgG1 was also observed in Group 2. Altogether, our results indicated that mice immunized once developed a mixed Th1/Th2 response, which was less efficient in the infection control, while a trend to a Th1 pattern was obtained with two immunizations, promoting optimal elimination of P. brasiliensis yeast cells from mice tissues.     

Delayed-type hypersensitivity response in an isogenic murine model of paracoccidioidomycosis

The specific delayed-type hypersensitivity (DTH) response was evaluated in resistant (A/SN) and susceptible (B10.A) mice intraperitoneally infected with yeasts from a virulent (Pb18) or from a non-virulent (Pb265)Paracoccidioides brasiliensis isolates. Both strains of mice were footpad challenged with homologous antigens. Pb18 infected A/SN mice developed an evident and persistent DTH response late in the course of the disease (90th day on) whereas B10.A animals mounted a discrete and ephemeral DTH response at the 14th day post-infection. A/SN mice infected with Pb265 developed cellular immune responses whereas B10.A mice were almost always anergic. Histological analysis of the footpads of infected mice at 48 hours after challenge showed a mixed infiltrate consisting of predominantly mononuclear cells. Previous infection of resistant and susceptible mice with Pb18 did not alter their DTH responses against heterologous unrelated antigens (sheep red blood cells and dinitrofluorobenzene) indicating that the observed cellular anergy was antigen-specific. When fungal related antigens (candidin and histoplasmin) were tested in resistant mice, absence of cross-reactivity was noted. Thus, specific DTH responses againstP. brasiliensis depend on both the host's genetically determined resistance and the virulence of the fungal isolate.Abbreviations DTH delayed-type hypersensitivity - DNFB dinitrofluorobenzene - FN18 Fava Netto's antigen obtained from isolate Pb18 - FN265 Fava Netto's antigen obtained from isolate Pb265 - SRBC sheep red blood cells     

Immunogenicity of DNA and recombinant Sendai virus vaccines expressing the HIV-1 gag gene

Combinations of DNA and recombinant-viral-vector based vaccines are promising AIDS vaccine methods because of their potential for inducing cellular immune responses. It was found that Gag-specific cytotoxic lymphocyte (CTL) responses were associated with lowering viremia in an untreated HIV-1 infected cohort. The main objectives of our studies were the construction of DNA and recombinant Sendai virus vector (rSeV) vaccines containing a gag gene from the prevalent Thailand subtype B strain in China and trying to use these vaccines for therapeutic and prophylactic vaccines. The candidate plasmid DNA vaccine pcDNA3.1(+)-gag and recombinant Sendai virus vaccine (rSeV-gag) were constructed separately. It was verified by Western blotting analysis that both DNA and rSeV-gag vaccines expressed the HIV-1 Gag protein correctly and efficiently. Balb/c mice were immunized with these two vaccines in different administration schemes. HIV-1 Gag-specific CTL responses and antibody levels were detected by intracellular cytokine staining assay and enzyme-linked immunosorbant assay (ELISA) respectively. Combined vaccines in a DNA prime/rSeV-gag boost vaccination regimen induced the strongest and most long-lasting Gag-specific CTL and antibody responses. It maintained relatively high levels even 9 weeks post immunization. This data indicated that the prime-boost regimen with DNA and rSeV-gag vaccines may offer promising HIV vaccine regimens. Foundation item: National 863 project (2003AA219070)     

Paracoccidioidomycosis in wild monkeys from Paraná State, Brazil

The aim of this study was to evaluate the seroprevalence of Paracoccidioides brasiliensis infection in wild New World monkeys (Cebus sp. and Alouatta caraya). A total of 93 animals (Cebus sp., n = 68 and Alouatta caraya, n = 25) were captured in the Paraná River basin, Paraná State, Brazil and the serum samples were analyzed by ELISA and immunodiffusion using P. brasiliensis gp43 and exoantigen as antigens, respectively. The seropositivity observed by ELISA was 44.1% and 60% for Cebus sp. and A. caraya, respectively, while by immunodiffusion test Cebus sp. showed positivity of 2.9% only. No significant difference was observed in relation to age and sex. This is the first report of paracoccidioidomycosis in wild capuchin monkeys and in wild-black and golden-howler monkeys. The high positivity to P. brasiliensis infection in both species evaluated in our study and the positivity by immunodiffusion test in Cebus sp. suggest that natural disease may be occurring in wild monkeys living in paracoccidioidomycosis endemic areas.     

Evaluation of Paracoccidioides brasiliensis Infection in Dairy Goats

Paracoccidioides brasiliensis is the etiological agent of paracoccidioidomycosis, a systemic mycosis that affects mainly rural workers in Brazil and other Latin American countries. The participation of domestic and wild animal species in the ecoepidemiology of paracoccidioidomycosis is not well understood. The objective of this study was to evaluate P. brasiliensis infection in dairy goats. The humoral immune response to the gp43 antigen, the main antigen used for paracoccidioidomycosis serodiagnosis and seroepidemiology, was evaluated in two goats immunized with inactivated P. brasiliensis yeast cells. Both animals produced antibodies against the P. brasiliensis gp43 antigen, detected by ELISA, 2 weeks after immunization. A total of 202 goat serum samples were analyzed by ELISA and the immunodiffusion test using P. brasiliensis gp43 and exoantigen as antigens. The seropositivity observed by ELISA was 26.2 % although no reactivity was detected by immunodiffusion. The animals over 18 months of age showed significantly higher positivity (40 %) than animals aged 6–18 months (14.8 %) and 0–6 months (2.6 %). Taking into account that cross-reactivity may occur with other pathogens, the serum samples were also analyzed by ELISA using Histoplasma capsulatum exoantigen as antigen and the positivity observed was 14.3 %. The low correlation (0.267) observed between reactivity to P. brasiliensis gp43 and H. capsulatum exoantigen suggests co-infection rather than cross-reactivity. This is the first report showing serological evidence of P. brasiliensis infection in goats and reinforces that domestic animals are useful epidemiological markers of paracoccidioidomycosis.     

Detection of Antibodies Against Paracoccidioides brasiliensis in Free-Range Domestic Pigs (Sus scrofa)

Paracoccidioidomycosis, caused by the thermodimorphic fungus Paracoccidioides brasiliensis, is a human systemic mycosis prevalent in Latin America. Paracoccidioidomycosis affects mainly male rural workers, causing granulomatous lesions in several organs such as the lungs, liver and spleen. The participation of other animal species in the fungus epidemiology is not well understood. The objective of this study was to evaluate the infection of free-range domestic pigs by P. brasiliensis. Serum samples from 106 pigs were analyzed by ELISA and the immunodiffusion test, using P. brasiliensis gp43 and exoantigen as antigens, respectively. The overall positivity to gp43 in ELISA was 37.7 %, although no reactivity was observed in the immunodiffusion test and nor was P. brasiliensis detected in tissue samples (spleen, lung, liver and lymph nodes) from slaughtered animals submitted to culture, histopathological examination and PCR analysis. Five pigs seronegative to gp43 were exposed to natural infection by P. brasiliensis, and all animals seroconverted 3 months after exposure. The results suggest that free-range pigs are frequently infected with P. brasiliensis but are resistant to disease development. This is the first report of paracoccidioidomycosis in pigs.     

Effect of macrophage blockade on the resistance of inbred mice toParacoccidioides brasiliensis infection

The effect of macrophage blockade on the natural resistance and on the adaptative immune response of susceptible (B10.D2/oSn) and resistant (A/Sn) mice toParacoccidioides brasiliensis infection was investigated. B10.D2/oSn and A/Sn mice previously injected with colloidal carbon were infected ip with yeast cells to determine the 50% lethal dose, and to evaluate the anatomy and histopathology, macrophage activation, antibody production and DTH reactions. Macrophage blockade rendered both resistant and susceptible mice considerably more susceptible to infection, as evidenced by increased mortality and many disseminated lesions.P. brasiliensis infection and/or carbon treatment increased the ability of macrophages from resistant mice to spread up to 25 days after treatment. In susceptible mice the enhanced spreading capacity induced by carbon treatment was impaired at all assayed periods except at 1 week after infection. Macrophage blockade enhanced DTH reactions in resistant mice, but did not alter these reactions in susceptible mice, which remained anergic. To the contrary, macrophage blockade enhanced specific antibody production by susceptible mice, but did not affect the low levels produced by resistant mice. The effect of macrophage blockade confirms the natural tendency of resistant animals to mount DTH reactions in the course of the disease and the preferential antibody response developed by susceptible mice afterP. brasiliensis infection. On the whole, macrophage functions appear to play a fundamental role in the natural and acquired resistance mechanisms toP. brasiliensis infection.     

Subcutaneous infection of mice with Paracoccidioides brasiliensis induces a peculiar pattern of inflammatory and immune responses

It is well established that resistance or susceptibility to Paracoccidioidis brasiliensis infection in mice is under strict host's genetic control. Mice from A/Sn strain inoculated by the ip route are resistant to fungal infection while infection induced in mice from B10.A strain results in a fatal disease. The early cellular events of infection in both strains are characterized by a marked neutrophilic infiltration that is more prominent in B10.A mice. A peculiar characteristic of the Paracoccidioides brasiliensis-mouse model is that the subcutaneous (sc) inoculations of the fungus either in resistant (A/Sn) or susceptible (B10.A) mice is self-curing and turns mice from the B10.A strain able to express typical DTH reaction to fungal antigens, as observed in A/Sn mice. Here we report the investigation on the early events of the inflammatory response induced by the inoculation of live fungus into the hind footpad of A/Sn (resistant) and B10.A (susceptible) mice. The influence of neutrophils on the inflammatory response and antibody titers or DTH response to gp43, the major fungal antigen, was also evaluated. Results showed a different course of the inflammatory response induced by fungal inoculation in A/Sn and B10.A mice. Neutrophil depletion before infection differently influenced the kinetics of the inflammatory process in both mice strains but did not modifythe fungal load in the lesions. In neutrophil depleted mice from both strains, a decrease in DTH response and an increase in total antibody titers to gp43 were observed. The significant increase in the fungal load in lesions seen in nude mice indicates that the self-limited infection evoked by fungal inoculation into the subcutaneous tissue is a T-cell dependent phenomenon. The implications of these observations in the pathogenesis of paracoccidioidomycosis are discussed.This revised version was published online in October 2005 with corrections to the Cover Date.