Imunoglobulinas e C3 no granuloma paracoccidióidico

Utilizou-se o modelo experimental de paracoccidioidomicose, em camundongos, induzida pela inoculação endovenosa de suspensão de formas cerebriformes do P. brasiliensis (cepa Bt2; 1x10(6) formas viáveis/animal), para avaliar, após 2, 4, 8, 16 e 20 semanas: 1. A presença de imunoglobulinas e C3 nos granulomas pulmonares, por imunofluorescência direta; 2. A resposta imune humoral (imunodifusão) e celular (teste do coxim plantar), e 3. A histopatologia das lesões. Os camundongos apresentaram resposta imunocelular positiva desde a 2a. semana, com depressão transitória na 16a. semana, e anticorpos desde a 4a. semana, com pico na 16a. semana. Os granulomas pulmonares foram epitelióides, com numerosos fungos e microabscesses; a extensão das lesões foi progressiva até a 16a. semana, com regressão discreta na 20a. semana. Desde a 2a. semana, houve deposição de IgG e C3 na parede dos fungos no interior dos granulomas e a presença de células IgG positivas no halo linfomononuclear periférico; estes achados foram máximos entre a 4a. e 16a. semanas. Não se detectou depósito de IgG e C3 no interstício dos granulomas. IgG e C3 parecem exercer papel precoce e importante na defesa do hospedeiro contra o P. brasiliensis, contribuindo possivelmente para a destruição dos fungos e bloqueando a difusão de antígenos para fora dos granulomas.

Reação de microimunodifusão em gel de ágar no diagnóstico sorológico da paracoccidioidomicose

Utilizou-se técnica de microimunodifusão dupla em gel de ágar para a medida quali e quantitativa de anticorpos circulantes anti - P. brasiliensis, comparando-se os resultados com o macrométodo. Todos os 103 soros de pacientes portadores de paracoccidioidomicose foram positivos no micrométodo contra 87% de positividade no macrométodo. Os 83 soros de pacientes sem paracoccidioidomicose foram negativos em ambas as reações. Os títulos dos soros positivos tenderam a ser mais elevados no micrométodo, que forneceu bandas de precipitação mais nítidas e fáceis de serem lidas. O micrométodo é de realização simples, utiliza pequena quantidade de material e possibilita o teste simultâneo de 102 soros. Acreditamos que ele poderá substituir o macrométodo, especialmente em laboratórios de grande rotina sorológica.

Transcriptome profiling of Paracoccidioides brasiliensis yeast-phase cells recovered from infected mice brings new insights into fungal response upon host interaction

Paracoccidioides brasiliensis is a fungal human pathogen with a wide distribution in Latin America. It causes paracoccidioidomycosis, the most widespread systemic mycosis in Latin America. Although gene expression in P. brasiliensis had been studied, little is known about the genome sequences expressed by this species during the infection process. To better understand the infection process, 4934 expressed sequence tags (ESTs) derived from a non-normalized cDNA library from P. brasiliensis (isolate Pb01) yeast-phase cells recovered from the livers of infected mice were annotated and clustered to a UniGene (clusters containing sequences that represent a unique gene) set with 1602 members. A large-scale comparative analysis was performed between the UniGene sequences of P. brasiliensis yeast-phase cells recovered from infected mice and a database constructed with sequences of the yeast-phase and mycelium transcriptome (isolate Pb01) (https://dna.biomol.unb.br/Pb/), as well as with all public ESTs available at GenBank, including sequences of the P. brasiliensis yeast-phase transcriptome (isolate Pb18) (http:// www.ncbi.nlm.nih.gov/). The focus was on the overexpressed and novel genes. From the total, 3184 ESTs (64.53%) were also present in the previously described transcriptome of yeast-form and mycelium cells obtained from in vitro cultures (https://dna.biomol.unb.br/Pb/) and of those, 1172 ESTs (23.75% of the described sequences) represented transcripts overexpressed during the infection process. Comparative analysis identified 1750 ESTs (35.47% of the total), comprising 649 UniGene sequences representing novel transcripts of P. brasiliensis, not previously described for this isolate or for other isolates in public databases. KEGG pathway mapping showed that the novel and overexpressed transcripts represented standard metabolic pathways, including glycolysis, amino acid biosynthesis, lipid and sterol metabolism. The unique and divergent representation of transcripts in the cDNA library of yeast cells recovered from infected mice suggests differential gene expression in response to the host milieu.

Immunohistochemical characterization of mononuclear cells in delayed hypersensitivity reactions to Paracoccidiodes brasiliensis (paracoccidioidin test)

The density and distribution of T cells, T helper cells, macrophages and B cells at the site of skin tests with a cytoplasmic Paracoccidioides brasiliensis antigen (paracoccidioidin) was studied at 24 and 48 h post-challenge in 10 patients with the chronic form of paracoccidioidomycosis and in 5 noninfected individuals. The in situ study was carried out using immunoperoxidase techniques and monoclonal antibodies. The controls showed negative skin test. In the patients, the great majority of the cells in the perivascular foci were T cells (CD43-positive cells) making up 47% and 48.6% of the total number of cells at 24 and 48 h respectively. Most of the T cells showed a T helper phenotype (CD45RO-positive cells). Approximately 25% of the cells were macrophages (CD68-positive cells) and there were very few B lymphocytes (CD20-positive cells). The present data on the microanatomy of paracoccidioidin skin test sites were consistent with a delayed type hypersensitivity pattern. Our results were comparable to those reported on skin tests for other granulomatous chronic diseases.

PATHOGENICITY AND IMMUNOGENICITY OF PARACOCCIDIOIDES-BRASILIENSIS ISOLATES IN THE HUMAN-DISEASE AND IN AN EXPERIMENTAL MURINE MODEL

The pathogenicity and immunogenicity of six recently isolated Paracoccidioides brasiliensis samples derived from patients presenting distinct and well defined clinical forms of paracoccidioidomycosis (PCM) were compared as to their virulence, tropism to different organs and ability to induce specific cellular and humoral immune response in susceptible (B10.A) inbred mice. Isolates Pb44 and Pb47 were obtained from acute cases, Pb50 from a chronic severe form, Pb45 from a chronic moderate case and both Pb56 and Pb57 from chronic mild forms of PCM. Pathogenicity and tropism of each fungal sample were evaluated by LD50% estimation, examination of gross lesions on various organs at 2, 4, 12 and 16 weeks post-infection, and by colony-forming unit (CFU) counts in the lungs at week 16 post-infection of mice. Fungal tropism in human PCM and in B10.A mice was always dissociated. A well defined relationship between virulence of the fungal sample and the clinical findings of the correspondent patient was not evident, although a tendency to higher LD50% and less intense paracoccidioidic lesions was observed in mice infected with Pb56 and Pb57. The specific DTH response patterns varied according to the infectant sample, but positive DTH reactions at the beginning of the infection and a tendency to anergy or low DTH responses at week 12 and/or week 16 post-infection were always observed. A correspondence between the DTH response in humans and in mice was noticeable only when the isolates from the most benign cases (Pb56 and Pb57) were considered. The specific antibody patterns in mice and in the correspondent patients were also not analogous. Collectively, these results indicate that an association between the fungal pathogenicity and immunogenicity in the human disease and in susceptible mice was discernible only when isolates obtained from very mild cases (Pb56 and Pb57) were considered.

Antigenic similarities to Paracoccidioides brasiliensis in thermo-dependent dimorphic fungi isolated from soil in Botucatu, SP, Brazil

We compared the antigenic characteristics of two thermo-dependent dimorphic fungi isolated from soil in Botucatu, an endemic area of paracoccidioidomycosis (PCM) and Paracoccidioides brasiliensis. The soil isolates grew as cerebriform colonies at 37 degrees C (yeast form) and as cottonous colonies at 25 degrees C (mycelial form). No pathogenicity for ddY mice or hamsters were observed. In immunodiffusion test, there were precipitation bands between the 2 soil isolates and pooled PCM patient sera. There were also common precipitation bands at 21, 50 and 58 kDa between the soil isolates antigens and PCM patient sera by Western-blotting, but no gp43 kDa band. No gene for gp43 kDa protein was detected in the soil isolates by PCR. The fact that these isolates were obtained from an endemic area of PCM and there were some antigenic similarities between the soil isolates and P. brasiliensis in immunodiffusion test and Western-blotting may have some importance in epidemiological surveys done with paracoccidioidin as well interfering with the immune response of the exposed population.

Analysis of the Paracoccidioides brasiliensis triosephosphate isomerase suggests the potential for adhesin function

Paracoccidioides brasiliensis is an important fungal pathogen. The disease it causes, paracoccidioidomycosis (PCM), ranges from localized pulmonary infection to systemic processes that endanger the life of the patient. Paracoccidioides brasiliensis adhesion to host tissues contributes to its virulence, but we know relatively little about molecules and the molecular mechanisms governing fungal adhesion to mammalian cells. Triosephosphate isomerase (TPI: EC 5.3.1.1) of P. brasiliensis (PbTPI) is a fungal antigen characterized by microsequencing of peptides. The protein, which is predominantly expressed in the yeast parasitic phase, localizes at the cell wall and in the cytoplasmic compartment. TPI and the respective polyclonal antibody produced against this protein inhibited the interaction of P. brasiliensis to in vitro cultured epithelial cells. TPI binds preferentially to laminin, as determined by peptide inhibition assays. Collectively, these results suggest that TPI is required for interactions between P. brasiliensis and extracellular matrix molecules such as laminin and that this interaction may play an important role in the fungal adherence and invasion of host cells.

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.

EFFECT OF MACROPHAGE BLOCKADE ON THE RESISTANCE OF INBRED MICE TO PARACOCCIDIOIDES-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 to Paracoccidioides 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 ail 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 nor 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 after P. brasiliensis infection. on the whole, macrophage functions appear to play a fundamental role in the natural and acquired resistance mechanisms to P. brasiliensis infection.

GRANULOMATOUS REACTION IN THE HAMSTER-CHEEK POUCH INDUCED BY KILLED PARACOCCIDIOIDES-BRASILIENSIS

We have studied the role of Paracoccidioides brasiliensis viability in the morphology of paracoccidioidomycotic granulomas in the hamster cheek pouch, an immunologically privileged site. Naive (N = 75) and previously sensitized (N = 50) two-month old male hamsters (Mesocricetus auratus) were inoculated into the pouch with 5 x 10(5) live or heat- or formalin-killed fungi. Previously sensitized animals presented a positive footpad test and immunodiffusion demonstrable antibodies (titer 1/32), at the time of sacrifice; naive animals were always negative for those immunological tests. The histological results showed that, like viable P. brasiliensis, killed fungi evoke typical epithelioid granulomas in 100% of animals, even in the absence of immunodiffusion or footpad test detectable immune response. The granulomas elicited by killed fungi were devoid of giant cells or a mononuclear cell halo, suggesting that live proliferating fungi or their products may be involved in these events.

Monoclonal antibodies against the 43,000 da glycoprotein from Paracoccidioides brasiliensis modulate laminin-mediated fungal adhesion to epithelial cells and pathogenesis

The surface glycoprotein gp43, a highly immunogenic component of Paracoccidioides brasiliensis, is used in the serodiagnosis of paracoccidioidomycosis (PCM) and has recently been shown to specifically bind the extracellular matrix protein laminin, Binding to laminin induces the increased adhesion of the fungus to epithelial cells; a hamster testicle infection model has shown that the gp43-dependent binding of fungal cells to laminin enhances their pathogenicity in vivo. We report on the production and characterization of 12 monoclonal antibodies against the gp43 that recognize peptide sequences in the molecule detecting at least three different epitopes as well as different isoforms of this antigen. MAbs interfered in the fungal pathogenicity in vivo either by inhibiting or enhancing granuloma formation and tissue destruction, Results suggest that P. brasiliensis propagules may start infection in man by strongly adhering to human lung cells, Thus, laminin-mediated fungal adhesion to human lung carcinoma (A549) cells was much more intense than to Madin-Darby canine kidney cells (MDCK), indicating differences in binding affinity, Subsequent growth of fungi bound to the lung cells could induce the granulomatous inflammatory reaction characteristic of PCM. Both steps are greatly stimulated by laminin binding in infective cells expressing gp43.

Fungicidal activity of human monocyte-derived multinucleated giant cells induced in vitro by Paracoccidioides brasiliensis antigen

Multinucleated giant cells (MGC) are characteristic cells in granulomatous disorders such as paracoccidioidomycosis (PCM) and also are formed in vitro from peripheral blood mononuclear cells by several stimuli. In this study, the authors investigated in vitro formation of MGC derived from monocytes of healthy individuals, stimulated with Paracoccidioides brasiliensis antigen (PbAg), compared with other stimuli such as IFN-gamma and supernatant of Con-A-stimulated peripheral blood mononuclear cells (CM-ConA). Besides, the fungicidal activity of monocytes and monocyte-derived MGC challenged with P. brasiliensis were compared, at a ratio of one fungus per 50 monocytes. Results demonstrated that PbAg, IFN-gamma, and CM-ConA stimuli were able to induce MGC generation, with fusion indices significantly higher than control cultures. Striking results were observed when MGC induced by PbAg and IFN-gamma presented higher fungicidal activity than monocytes, submitted to the same stimuli, showing a better capacity of these cells to kill P. brasiliensis. In summary, the results suggest that PbAg is able to induce MGC generation, and these cells presented higher fungicidal activity against P. brasiliensis than monocytes.

Interactions between TLR2, TLR4, and mannose receptors with gp43 from Paracoccidioides brasiliensis induce cytokine production by human monocytes

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Inhibitory effect of PGE(2) on the killing of Paracoccidioides brasiliensis by human monocytes can be reversed by cellular activation with cytokines

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)