Pulmonary paracoccidioidomycosis: a case report of reactivation in a patient receiving biological therapy

Abstract Paracoccidioidomycosis is an endemic disease in Latin America that is rarely associated with immunosuppression and biological therapy. Herein, we report for the first time a case of pulmonary paracoccidioidomycosis reactivation after infliximab treatment. A 47-year-old man from Brazil received infliximab to treat psoriatic spondyloarthropathy and presented with cough, dyspnea, weight loss, and fever. Chest computed tomography revealed a pulmonary nodule and biopsy confirmed paracoccidioidomycosis. Treatment with sulfamethoxazole and trimethoprim was initiated for fungal infection and infliximab was reintroduced two months later. Considering his clinical improvement and favorable radiologic evolution, antifungal therapy was discontinued after 29 months.

High interleukin-4 expression and interleukin-4 gene polymorphisms are associated with susceptibility to human paracoccidioidomycosis

Paracoccidioidomycosis (PCM) is caused by dimorphic fungi from theParacoccidioides brasiliensis complex. Previous studies have demonstrated that the severity of disease is associated with a T-helper 2 immune response characterised by high interleukin (IL)-4 production. In the present study we analysed two polymorphisms in the IL-4gene (-590 C/T and intron-3 microsatellite) in 76 patients with PCM and 73 control subjects from an endemic area. The production of IL-4 by peripheral blood mononuclear cells after antigen or phytohaemagglutinin stimulation was determined by ELISA. A significant correlation was observed between the RP2/RP2 intron-3 genotype and infection with Paracoccidioides sp.(p = 0.011), whereas the RP1/RP1 genotype was correlated with resistance. No significant correlation was observed for the IL-4promoter polymorphism. Furthermore, the low IL-4 expression observed in the control group compared with patients was associated with the RP1/RP1 genotype. These results suggest that IL-4polymorphisms might be associated with the ability of the host to control Paracoccidioides sp.infection. The relevance of this polymorphism is supported by the observation that patients with disease produce high levels of IL-4 following mitogen or antigen stimulation. The IL-4gene is located in the cytokine cluster region of chromosome 5 where other polymorphisms have also been described.

Paracoccidioidoyycosis: challenges in the develpment of a vaccine against an eendemic mycosis in the americas / Paracoccidioidomicose: desafios no desenvolvimento de uma vacina contra micose endêmica nas Américas

SUMMARYParacoccidioidomycosis (PCM), caused by Paracoccidioides spp, is an important endemic mycosis in Latin America. There are two recognized Paracoccidioides species, P. brasiliensis and P. lutzii, based on phylogenetic differences; however, the pathogenesis and disease manifestations of both are indistinguishable at present. Approximately 1,853 (~51,2%) of 3,583 confirmed deaths in Brazil due to systemic mycoses from 1996-2006 were caused by PCM. Antifungal treatment is required for patients with PCM. The initial treatment lasts from two to six months and sulfa derivatives, amphotericin B, azoles and terbinafine are used in clinical practice; however, despite prolonged therapy, relapses are still a problem. An effective Th1-biased cellular immune response is essential to control the disease, which can be induced by exogenous antigens or modulated by prophylactic or therapeutic vaccines. Stimulation of B cells or passive transference of monoclonal antibodies are also important means that may be used to improve the efficacy of paracoccidioidomycosis treatment in the future. This review critically details major challenges facing the development of a vaccine to combat PCM.

RESUMOA paracoccidioidomicose (PCM), causada por Paracoccidioides spp, é importante micose endêmica na América Latina. Com base em diferenças filogenéticas, existem duas espécies reconhecidas de Paracoccidioides, P. brasiliensis e P. lutzii, no entanto, a patogênese e as manifestações clínicas de ambas são indistinguíveis atualmente. Aproximadamente 1853 (~51,2%) de 3583 mortes confirmadas, atribuídas a micoses sistêmicas de 1996-2006, no Brasil foram causadas por PCM. Tratamento antifúngico é necessário para pacientes com PCM. O tratamento inicial dura de dois a seis meses e derivados de sulfa, anfotericina B, azóis e terbinafina são utilizados na prática clínica; no entanto, apesar da terapêutica prolongada, as recaídas ainda são um problema. Uma resposta imune celular eficaz, tendendo a Th1, é essencial para controlar a doença que pode ser induzida por antígenos exógenos, ou moduladas por vacinas profiláticas ou terapêuticas. A estimulação de células B ou a transferência passiva de anticorpos monoclonais também são meios importantes que podem ser utilizados para melhorar a eficácia do tratamento da paracoccidioidomicose no futuro. Esta revisão detalha criticamente os principais desafios que o desenvolvimento de uma vacina para combater a PCM enfrenta.

Padronização da metodologia de Dot-Blot para o diagnóstico rápido da paracoccidioidomicose / Metodology standartization of Dot-Blot for screening of paracoccidioidomycosis

A paracoccidioidomicose (PCM), micose profunda de natureza crônicagranulomatosa, apresenta maior incidência no Brasil. O diagnóstico de certezaderiva da visualização do agente etiológico, o fungo Paracoccidioides spp emamostras biológicas. No entanto, em algumas situações o acesso ao local dalesão impede a coleta do material biológico. Assim, as técnicas imunosorológicaspermitem inferir diagnóstico com certo grau de certeza, otimizandoo tempo utilizado para se obter resultados. A técnica sorológica amplamenteutilizada para o imunodiagnóstico da PCM é a imunodifusão dupla em gel deagarose (ID), com especificidade e sensibilidade variando de 65 a 100%; sendode fácil execução e não necessitando de automação. A técnica de Dot-Blot (DB) tem sido utilizada com sucesso no diagnóstico de inúmeras doençasparasitárias e infecciosas, como a toxoplasmose e a leishmaniose visceral. Nodiagnóstico da PCM, a metodologia mostrou-se promissora noacompanhamento de pacientes durante o tratamento anti-fúngico e eminquéritos soroepidemiológicos. Diante do exposto, o objetivo deste trabalho foipadronizar o ensaio de Dot-Blot visando o diagnóstico rápido da PCM,propondo que o mesmo seja uma ferramenta de triagem dos soros comsuspeita clínica para a doença. A padronização da técnica de Dot-Blot apresentou melhores resultados quando se utilizou antígeno obtido de filtradode cultura de P. brasiliensis do isolado B-339 para a sensibilização demembranas de nitrocelulose. As diluições de soro e conjugado foram de 1:40 e1:2000, respectivamente, incubando ambos em solução PBS-L 3%. Para apadronização do ensaio de Dot-Blot, 143 amostras de soro foram utilizadas echamadas de grupo controle. Destas, 23 amostras de soro foram de pacientesaparentemente sadios, 77 amostras de soro de pacientes com PCM confirmadae 43 amostras de soros com outras doenças (tuberculose, aspergilose ehistoplasmose)...

The paracoccidioidomycosis (PCM) is the greatest mycosis of chronicgranulomatous nature in Brazil. The definitive diagnosis derives from the view ofthe etiologic agent, Paracoccidioides spp in biological samples. However, insome situations, access to the site of injury prevents the collection of biologicalmaterial. Thus, immuno- serological techniques allow inferring diagnosis with adegree of certainty and optimizing time taken to get results. The serologicaltechnique widely used for immunodiagnosis of PCM is double immunodiffusionin agarose gel (DI), with a sensitivity and specificity ranging from 65 to 100%,being easy to perform and not requiring automation. The Dot-blot (DB)technique has been used successfully in the diagnosis of many infectious andparasitic diseases such as visceral leishmaniasis and toxoplasmosis. On thediagnosis of PCM, this methodology showed promise results in monitoringpatients during treatment with anti-fungal and seroepidemiological surveys.Given the above, the objective of this study was to standardize the Dot-blotassay targeting the rapid diagnosis of PCM, suggesting that it is a screeningtool for sera with clinical suspicion for the disease. The standardization of Dot-Blot showed better results when using antigen obtained from culture filtrate of P.brasiliensis isolated from B-339 to sensitize nitrocellulose membranes. Theserum and conjugated dilutions were...

Production of TNF-α, nitric oxide and hydrogen peroxide by macrophages from mice with paracoccidioidomycosis that were fed a linseed oil-enriched diet

Omega-3 polyunsaturated fatty acids (n-3 PUFA) can modulate the immune system and their primary effect is on macrophage function. Paracoccidioidomycosis (PCM) is an endemic systemic mycosis in Latin America that is caused by the dimorphic fungus Paracoccidioides brasiliensis (Pb). Macrophages are the main defence against this pathogen and have microbicidal activity that is dependent on interferon-Γ and tumour necrosis factor (TNF)-α. These cytokines stimulate the synthesis of nitric oxide (NO) and hydrogen peroxide (H2O2), leading to the death of the fungus. To study the effect of n-3 PUFA on the host immune response during experimental PCM, macrophages that were obtained from animals infected with Pb18 and fed a diet enriched by linseed (LIN) oil were cultured and challenged with the fungus in vitro. The macrophage function was analysed based on the concentrations of TNF-α, NO and H2O2. LIN oil seems to influence the production of TNF-α during the development of disease. A diet enriched with LIN oil influences the microbicidal activity of the macrophages by inducing the production of cytokines and metabolites such as NO and H2O2, predominantly in the chronic phase of infection.

Profile of total IgG, IgG1, IgG2, IgG3 and IgG4 levels in sera of patients with paracoccidioidomycosis: treatment follow-up using Mexo and rPb27 as antigens in an ELISA

The levels of total of IgG, IgG1, IgG2, IgG3 and IgG4 were evaluated in 54 patients with chronic paracoccidioidomycosis (PCM) before, during and after treatment using an enzyme-linked immunosorbent assay with Mexo and recombinant Pb27 (rPb27) as the antigens. Mexo was effective in distinguishing PCM patients from individuals in the negative control group (NC) based on total IgG and rPb27 performed worse than Mexo when these two groups were compared. IgG1, IgG2, IgG3 and IgG4 could not be used to clearly distinguish PCM patients from those in the NC group using either antigen. There was no clear relationship between antibody levels and the period of treatment. The majority of patients presented with decreased antibody levels during treatment, with no statistically significant differences among the different periods of treatment. Only IgG4 presented a negative correlation between its levels and clinical improvement during treatment. In total, 65 percent of untreated PCM patients showed reactivity against IgG4 when the Mexo antigen was used and this reactivity decreased over the course of treatment. There was a tendency towards decreasing antibody levels during treatment, but these antibody levels did not necessarily clear after the treatment was stopped. Mexo was useful for PCM diagnosis using total IgG; however, more studies are necessary before this antigen can be used in measuring the levels of total IgG and its subclasses for monitoring patients during treatment.

AIDS-associated paracoccidioidomycosis in a patient with a CD4+ T-cell count of 4 cells/mm³ / Paracoccidioidomicose associada a Aids em paciente com a contagem total de quatro células T-CD4+

We describe a case of a patient presenting with HIV and paracoccidioidomycosis co-infection. At the time of diagnosis total CD4+ T-cell count was 4 cells/mm3. Histopathology revealed tuberculoid granulomas, scarce CD4+ T cells, a moderate number of CD8+ cells and the absence of Foxp3+ cells. Most of the cutaneous lesions healed after two weeks of treatment with amphotericin B. After 14 months the patient is still under antiretroviral therapy and no clinical evidence of recurrence of the mycosis has been observed.

Neste trabalho apresenta-se paciente com coinfecção paracoccidioidomicose/Aids. No momento do diagnóstico, a contagem de células T CD4 + era 4 células. No exame histopatológico, observou-se a presença de granulomas tuberculóides bem formados e na imunohistoquímica, ausência de células Foxp3, raros linfócitos T CD4+ e presença de células T CD8+ em moderada quantidade. Com duas semanas de uso da anfotericina B, verificou-se a regressão de grande parte das lesões cutâneas. Após 14 meses, o paciente encontra-se em uso de terapia antiretroviral e sem evidências de atividade da micose.

Imunologia da paracoccidioidomicose / Immunology of paracoccidioidomycosis

Paracoccidioidomicose é a mais prevalente micose sistêmica na América Latina, em pacientes imunocompetentes, sendo causada pelo fungo dimórfico Paracoccidioiddes brasiliensis. O estudo da sua imunopatogênese é importante na compreensão de aspectos relacionados à história natural, como a imunidade protetora, e à relação entre hospedeiro e parasita, favorecendo o entendimento clínico e a elaboração de estratégias terapêuticas. O polimorfismo clínico da doença depende, em última análise, do perfil de resposta imune que prevalece expresso pelo padrão de citocinas teciduais e circulantes, além da qualidade da resposta imune desencadeada, que levam ao dano tecidual.

Paracoccidioidomycosis is the most prevalent systemic mycosis in Latin America, among immunecompetent patients. It's caused by the dimorphic fungus Paracoccidioiddes brasiliensis. Investigations regarding its immunopathogenesis are very important in the understanding of aspects related to natural history, as the protective immunity, and the relationship between host and parasite; also favoring the knowledge about clinical patterns and the elaboration of therapeutic strategies. The disease clinical polymorphism depends, at least, of the immune response profile according to the tissue and blood released citokynes, resulting in tissue damage.

Humoral response of paracoccidioidomycosis sera in hamsters with different Venezuelan isolates

Humoral response of paracoccidioidomycosis sera in hamsters with different Venezuelan isolates. Paracoccidioidomycosis (PCM) is a progressive systemic mycosis caused by the fungus Paraccocidioides brasiliensis (Pb), endemic to Venezuela and Latin America. In this study, eight different Venezuelan isolates obtained from patients with PCM, were inoculated intraperitoneally in Syrian hamsters (Cricetus auratus) and studied by immune-serum. Each strain was collected by gently scraping the surface of the culture medium (Sabouraud Dextrose Agar) and suspended in 3ml of 0.15 M phosphate-buffered saline. The antigen obtained was called Paraccocidioides brasiliensis Crude Antigen (CAP). Immunoblotting results showed that the immune-sera from hamsters recognized at least 3 bands: one over 200 kDa, and two of 80 and 15-20 kDa. This study suggests that IgG anti-CAP can reveal a significant variability in the eight Venezuelan isolates. Sera from 88 infected hamsters were evaluated by ELISA with eight different CAPs and Western blot with CAP 37383. ELISA results showed that, the antigen of the virulent isolate 37383 had the highest percentage (38%) of positivity, while the non-virulent isolate 1458 had the lowest one (13.6%). Furthermore, scanning densitometry revealed that the isolate 37383 had less bands than the non-virulent isolates. These results suggest that the ELISA test with CAP 37383 can detect circulating antibodies, and that this virulent isolate may be useful for the diagnosis of PCM, and to monitor disease responses to treatments. Rev. Biol. Trop. 57 (3): 505-513. Epub 2009 September 30.

La Paracoccidioidomicosis (PCM), es una micosis sistémica causada por el hongo Paraccocidioides brasiliensis (Pb), endémica en Venezuela y Latino América. En este estudio ocho diferentes aislados venezolanos, obtenidos de pacientes con PCM, fueron inoculados intraperitonealmente en hámsteres y fueron estudiados por ELISA e inmunoblotting. Los antígenos obtenidos de P. brasiliensis fueron llamados, Antígeno Crudo (CAP). Los resultados del immunoblotting mostraron que los sueros inmunes de hámsteres reconocieron al menos tres bandas: una sobre 200, y otras de 80, y 15-20 kDa. Este estudio sugiere que la IgG anti-CAP muestra una variabilidad en los ocho aislados Venezolanos. Sueros de 88 hámsteres infectados fueron evaluados usando ELISA, el antígeno del aislado virulento 37383 mostró el más alto porcentaje de positividad (38%) en los sueros de los hámsteres estudiados. El aislado novirulento 1458 mostró un porcentaje bajo de positividad (13.6%). Además, un escaneo densitométrico reveló que el aislado 37383 tiene menos bandas que el otro aislado no-virulento. Por lo tanto, estos resultados sugieren que el ensayo de ELISA con CAP 37383 puede detectar anticuerpos circulantes y este aislado virulento puede ser útil para el diagnostico de PCM, y para el monitoreo de la respuesta al tratamiento de la enfermedad.

Serum proteins and fractions, HDL-cholesterol and total IgG and IgE levels in cases of acute and chronic paracoccidioidomycosis / Proteínas séricas e frações, HDL-colesterol e níveis de IgG e IgE totais na paracoccidioidomicose aguda e crônica

This study evaluated serum protein fractions, HDL-cholesterol, total immunoglobulin G and total immunoglobulin E levels in patients with acute and chronic paracoccidioidomycosis, by means of electrophoresis, enzymatic reaction and immunoenzymatic assay. The results demonstrated elevated levels of total immunoglobulin G, total immunoglobulin E, alpha-2 and gamma-globulins, which were more evident in acute than in chronic PCM, but no increase in HDL-cholesterol levels. There was a correlation between the levels of total immunoglobulin E and gamma-globulins and the alpha-2 and beta-globulin fractions in the acute form and between beta and gamma-globulins in both the acute and the chronic form. In conclusion, changes in total immunoglobulin G and immunoglobulin E levels and in the electrophoretic profile may be important markers for the prognosis and therapeutic follow-up of PCM cases, especially because protein electrophoresis is a simple laboratory test that can be applied when specific PCM serological tests are not available. In addition, levels of the gamma-globulin fraction greater than 2.0g/dl may suggest that the patient is developing a more severe form of PCM.

Este trabalho avaliou as frações de proteínas séricas, HDL-colesterol e imunoglobulina G e imunoglobulina E totais em pacientes com paracoccidioidomicose aguda e crônica por eletroforese, reação enzimática e ensaio imunoenzimático. Os resultados demonstraram aumento dos níveis de imunoglobulina G e imunoglobulina E totais, alfa-2 e gama-globulinas, mais evidente na forma aguda que na forma crônica, e não nos níveis de HDL-colesterol. Houve correlação entre níveis de imunoglobulina E total e gama-globulinas e fração alfa-2 e beta-globulinas na forma aguda e entre beta e gama-globulinas nas formas aguda e crônica. Concluindo, alterações nos níveis de imunoglobulina G e imunoglobulina E totais e no perfil eletroforético podem ser importantes marcadores para prognóstico e acompanhamento terapêutico da PCM, especialmente por ser a eletroforese de proteínas um exame laboratorial simples que pode ser empregado em situações onde a sorologia específica para PCM não está disponível. Adicionalmente, níveis da fração gama-globulinas acima de 2,0g/dL podem sugerir que o paciente esteja desenvolvendo uma forma mais grave de PCM.

Human serum antibody reactivity towards Paracoccidioides brasiliensis antigens treated with sodium metaperiodate / Reatividade de anticorpos de soros humanos a antígenos de Paracoccidioides brasiliensis tratados com metaperiodato sódico

In this study, we evaluated the profile of anti-Paracoccidioides brasiliensis immunoglobulin isotypes in serum from patients with the acute and chronic forms of paracoccidioidomycosis, using the whole Paracoccidioides brasiliensis antigen and the antigen treated with sodium metaperiodate. All the immunoglobulin isotypes present in the serum from patients with the acute and chronic forms of paracoccidioidomycosis presented higher reactivity towards the whole antigen than to the antigen treated with metaperiodate (P < 0.05). The reactivity of IgG and IgM to the antigen treated with metaperiodate was greater in serum from patients with the acute form of the disease (P < 0.05), while IgA was more reactive in serum from patients with the chronic form (P < 0.05). There was greater reactivity of IgG1 and IgG2 to the whole antigen and the antigen treated with metaperiodate in the serum from patients with paracoccidioidomycosis than there was in serum from patients with other parasitic infections (P < 0.05). Furthermore, IgG1 from patients with the acute form recognized the 19kDa, 27kDa and 31kDa antigens in the western blot test. Thus, the results suggest that modifications to the epitopes of Paracoccidioides brasiliensis antigens may help to improve the immunodiagnosis of paracoccidioidomycosis.

Neste trabalho, foi avaliado o perfil de isotipos de imunoglobulinas anti-Paracoccidioides brasiliensis em soros de pacientes com formas crônica e aguda de paracoccidiodomicoses usando antígeno total e tratado com meta-periodato. Todos os tipos de imunoglobulinas presentes nos soros de pacientes com formas aguda e crônica apresentaram alta reatividade ao antígeno total quando comparado ao tratado com meta-periodato (P < 0,05). Houve maior reatividade de IgG e IgM anti-antígeno tratado com meta-periodato em soros de pacientes com forma aguda da doença (P < 0,05), enquanto IgA foi mais reativa em soros da forma crônica (P < 0,05). Houve maior reatividade de IgG1 e IgG2 com antígeno total e tratado com meta-periodato em soros de pacientes comparados aos com outras parasitoses (P < 0,05). Além disso, IgG1 de pacientes com a forma aguda reconhecem antígenos de 19kDa, 27kDa e 31kDa por western blot. Assim, os resultados sugerem que alterações nos epitopos de antígenos de Paracoccidioides brasiliensis podem auxiliar no aprimoramento do imunodiagnóstico da paracoccidioidomicose.

Detecção molecular de Paracoccidioides brasiliensis em amostras de tecido e sangue periférico de pacientes portadores de Pacoccidioidomicose: correlação com perfis histopatológico, sorológicos e produção de TNF-alfa / Molecular detection of Paracoccidioides brasiliensis in tissue samples and peripheral blood of patients with Paracoccidioidomycosis: correlation with histopathological profiles, and production of serum TNF-alfa

A paracoccidioidomicose (PCM) é uma doença granulomatosa sistêmica, causada pelo Paracoccidioides brasiliensis, um fungo imperfeito e termodimórfico. Embora o contágio ocorra principalmente pela via aérea, as vias transcutânea e mucosa não devem ser desconsideradas, devido à possibilidade de invasão através de pele ou mucosas. No presente estudo, a detecção molecular de P. brasiliensis por Nested-PCR foi realizada em fragmentos de biópsias de lesões cutâneo-mucosas de pacientes com PCM ativa e comparada a outras técnicas utilizadas como rotina no diagnóstico dessa micose. Para tanto, amostras de 29 pacientes atendidos no Serviço de Dermatologia, da Faculdade de Medicina de Botucatu/UNESP, foram coletadas no período de abril/2004 a abril/2007. Na análise histopatológica observou-se granulomas compactos bem organizados, algumas vezes ao lado de áreas contendo granulomas frouxos mal definidos. Tal técnica foi positiva em todas as amostras avaliadas, com presença de grande quantidade de células fúngicas em 65,5% dos casos, algumas com multi-brotamentos. Para as análises sorológicas, na técnica de ELISA, 74,1% dos casos apresentaram títulos de 1/12800, 11,1% apresentaram 1/3200, 3,7% apresentaram 1/800 e 11,1% foram negativos. Já para imunodifusão (ID), 3,5% apresentaram título de 1/512, 55,1% apresentaram títulos de 1/32, 3,5% apresentaram 1/16, 3,5% apresentaram 1/8, 10,3% apresentaram 1/4, 6,9% apresentaram 1/2 e 17,2% foram negativos. Foi observado um aumento nos níveis de TNF-alfa , nos pacientes na forma grave, produzido por monócitos não estimulados, em comparação à forma leve e moderada, e ao controle. A produção desta citocina por monócitos estimulados com LPS também se mostrou gradativamente maior quanto maior a gravidade dos pacientes. Além disso, na análise do seguimento dos pacientes nota-se uma diminuição significativa nos níveis da citocina no período 60 dias pós-início do tratamento em comparação com o período pré-tratamento...

Use of immunoblotting assay improves the sensitivity of paracoccidioidomycosis diagnosis

The purpose of this work was to evaluate two serological assays: double immunodiffusion (DI) and immunoblotting (IB) in immunodiagnosis of paracoccidioidomycosis (PCM). We evaluated by IB assay 23 sera samples from patients with clinical confirmation of PCM, all of them with negative DI results against culture filtrate from Paracoccidioides brasiliensis isolate 113. For IB, as well as for comparative DI assay, we employed soluble components of the cell wall outer surface (SCCWOS) from P. brasiliensis isolate 113 cultivated at 36°C in Fava-Neto's agar medium for 5 and 10 days. Among the 20 sera samples analyzed by DI, 13 (65 percent) were negative and 7 (35 percent) were positive against SCCWOS obtained on the 5th and 10th days. By IB assay, 95.4 percent and 100 percent of sera reacted against gp43 and gp70 present in SCCWOS from the 5th day and 95.6 percent recognized these fractions when evaluated against SCCWOS from the 10th day. Our results demonstrated that the use of an immunoenzymatic assay significantly improves the sensitivity of PCM immunodiagnosis and also suggests that at least two serological tests for antibody detection should be adopted in cases of questionable diagnosis.

Kinetics of IFN-gamma, TNF-alpha, IL-10 and IL-4 production by mononuclear cells stimulated with gp43 peptides, in patients cured of paracoccidioidomycosis

We analyzed the kinetics of cytokine production by mononuclear cells from 17 patients who had been treated for paracoccidioidomycosis, using the stimulus of gp43 peptide groups (43kDa glycoprotein of Paracoccidioides brasiliensis) at 0.1 and 1µM, gp43 (1µg/ml) and crude Paracoccidioides brasiliensis antigen (PbAg; 75µg/ml). IFN-gamma production was a maximum at 144 hours in relation to the G2 and G8 peptide groups at 1µM and was greatest at 144 hours when stimulated by gp43 and by PbAg. The maximum TNF-alpha production was at 144 hours for the G2 group (0.1µM) and for gp43. IL-10 production was highest after 48 and 72 hours for G7 and G6 at 1µM, respectively. We also suggest the best time for analysis of IL4 production. These results may contribute towards future studies with gp43 peptides and encourage further investigations with the aim of understanding the influence of these peptides on the production of inflammatory and regulatory cytokines.

Analisamos a cinética da produção de citocinas de células mononucleares de 17 pacientes com paracoccidioidomicose tratada, usando como estímulo: grupos de peptídeos da gp43 (glicoproteina de 43kDa de Paracoccidioides brasiliensis) a 0,1 e 1µM, gp43 (1µg/mL) e antígeno bruto de Paracoccidioides brasiliensis - AgPb (75µg/mL). A produção de IFN-gama foi máxima em 144 horas frente aos grupos de peptídeos G2 e G8 a 1µM e maior em 144 horas quando estimuladas por gp43 e por AgPb. A produção de TNF-alfa foi máxima em 144 horas para G2 (0,1µM) e para gp43. A produção de IL-10 foi maior após 48 e 72 horas para G7 e G6 a 1µM, respectivamente. Sugerimos também o melhor período para a análise da produção de IL4. Tais resultados podem contribuir para estudos com peptídeos da gp43, estimulando investigações posteriores visando entender a influência de tais peptídeos na produção de citocinas inflamatórias e regulatórias.

Failure of nitric oxide production by macrophages and decrease in CD4+ T cells in oral paracoccidioidomycosis: possible mechanisms that permit local fungal multiplication

A paracoccidioidomicose é uma doença granulomatosa crônica que induz resposta inflamatória e imune específica. A participação do óxido nítrico (NO), produto da enzima óxido nítrico sintase induzível (iNOS), como uma importante molécula fungicida contra o fungo Paracoccidioides brasiliensis tem sido demonstrada. Com o objetivo de melhor caracterizar as lesões orais da paracoccidioidomicose (OP), propusemos estudo imunohistoquímico das células iNOS+ e das células CD45RO+, CD3+, CD8+, CD20+, CD68+ e mastócitos. As amostras foram distribuídas em grupos de acordo com o número de fungos viáveis por mm2. Nossos resultados demonstraram leve imunomarcação para iNOS nas células gigantes multinucleadas (MNGC) e na maioria das células mononucleares (MN), e a proporção de células MN/MNGC iNOS+ na OP foi comparável a do grupo Controle (tecido bucal clinicamente saudável). Adicionalmente, nossa análise revelou similaridade no número de células CD4+ entre o Controle e o grupo de OP com elevado número de fungos. Estes achados sugerem que a baixa expressão de iNOS e a diminuição de células CD4+ na OP podem representar possíveis mecanismos que permitiram a multiplicação local do fungo e a manutenção das lesões bucais ativas.

Ação da cloroquina sobre a infecção experimental murina por Paracoccidioides brasiliensis: atividade macrofágica, produção de citocinas e expressão de receptores para transferrina / Action of chloroquine on the murine experimental infection by Paracoccidioides brasiliensis: macrophage activity, production of cytokines and expression of receptors for transferrin

O íon ferro é um fator de crescimento essencial para todos os organismos. Dessa forma, trabalhos têm demonstrado que microorganismos, incluindo fungos como o Paracoccidioides brasiliensis, utilizam o estoque intracelular de ferro nos macrófagos para crescimento e multiplicação. No entanto, drogas utilizadas na clínica médica conseguem interferir no metabolismo férrico celular, diminuindo a concentração desse íon, desfavorecendo o crescimento de microorganismos intracelulares. Estudos realizados em nosso laboratório têm demonstrado que o tratamento com uma dessas drogas, a Cloroquina (Clor), uma base fraca, que bloqueia o metabolismo férrico, diminui significativamente a recuperação de fungos viáveis no pulmão de camundongos infectados por Paracoccidioides brasiliensis. Assim, o objetivo do presente trabalho foi avaliar a ação da Clor sobre a evolução da infecção experimental murina, através da recuperação de fungos viáveis a partir de pulmão, fígado e baço de animais infectados, e avaliação da atividade de macrófagos através da produção de citocinas (TNF-alfa, IL-6 e IL-10), da determinação da produção de H202 e NO e da expressão de receptores para transferrina. Células de camundongos tratados apresentaram produção de altos níveis de H202, baixos níveis de NO que foram associados a um aumento na expressão de receptores para transferrina, e supressão da produção de citocinas TNF-alfa, IL-6 e IL-10. No entanto, apesar dessas ações da Clor sobre a atividade macrofágica, concluímos que a diminuição da recuperação de fungos viáveis de pulmão, fígado e baço após o tratamento com Clor, foi associada à ação direta dessa droga sobre o metabolismo férrico, diminuindo a disponibilidade de ferro intracelular para o crescimento do P. brasiliensis.

The low efficiency of dendritic cells and macrophages from mice susceptible to Paracoccidioides brasiliensis in inducing a Th1 response

In the present study we evaluated T cell proliferation and Th lymphokine patterns in response to gp43 from Paracoccidioides brasiliensis presented by isolated dendritic cells from susceptible and resistant mice. T cell proliferation assays showed that dendritic cells from susceptible mice were less efficient than those from resistant mice. The pattern of T cell lymphokines stimulated by dendritic cells was always Th1, although the levels of IL-2 and IFN-gamma were lower in T cell cultures from susceptible mice. To determie whether different antigen-presenting cells such as macrophages and dendritic cells stimulated different concentrations of Th1 lymphokines, the production of IFN-gamma and IL-2 was measured. It was observed that dendritic cells were more efficient than macrophages in stimulating lymphoproliferation in resistant mice. However, no significant difference was observed for IFN-gamma or IL-2 production. When cells from susceptible mice were used, macrophages were more efficient in stimulating lymphoproliferation than dendritic cells, but no difference was observed in the production of Th1 cytokine. Taken together, these results suggest the lower efficiency of dendritic cells and macrophages from B10.A mice in stimulating T cells that secrete Th1 lymphokines in vitro, an effect that may be involved in the progression of the disease in vivo

Paracoccidioides brasiliensis: a mycologic and immunochemical study of two strains

Os autores estudaram do ponto de vista micologico, imunoquimico e de sua biologia molecular, duas amostras de Paracoccidioides brasiliensis, uma isolada do solo, no municipio de IBIA (MG) por Silva-Vergara et al (1996,1998) denominada IBIA e outra, BAT, cultivada de um caso humano de paracoccidioidomicose em Ribeirao Preto (SP) por Freitas da Silva (1996). Tais amostras apresentam colonias cotonosa (M) e leveduriforme (L ou Y), sendo patogenicas para cobaios inoculados por via testicular, produzindo orquite granulomatosa e/ou supurativa. Do ponto de vista imunoquimico, atraves de provas de Imunodifusao dupla, Imunoeletroforese e Western Blotting, foi demostrada a presenca da gp43

Cytokines produced by suscesptible and resistant mice in the course of Paracoccidioides brasiliensis infection

Paracoccidioidomycosis (PCM) is the most prevalent deep mycosis in Latin America and presents a wide spectrum of clinical manifestations. We established a genetically controlled murine model of PCM, where A/Sn mice develop an infection which mimics the benign disease (immune responses which favor cellular immunity) and B10.A animals present the progressive disseminated form of PCM (preferential activation of B cells and impairment of cellular immune responses). To understand the immunoregulatory phenomena associated with resistance and susceptibility in experimental PCM, A/Sn and B10.A mice were studied regarding antigen-elicited secretion of monokines (TNF-alpha and TGF-beta) and type-1 (IL-2 and IFN-gamma) and type-2 (IL-4,5,10) cytokines. Total lymph node cells from resistant mice infected ip with P. brasiliensis produced early and sustained levels of IFN-gamma and IL-2; type-2 cytokines (IL-4 and IL-05) started to appear 8 weeks after infection. In contrast, susceptible mice produced low levels of IFN-gamma concomitant with significant levels of IL-5 and IL-10 early in the infection. In the chronic phase of the disease, susceptible animals presented a transitory secretion of IL-2, and IL-4. In the pulmonary infection IL-4, IL-5 and IL-10 were preferentially detected in the lung cells washings of susceptible animals. After in vitro challenge with fungal antigens, normal peritoneal macrophages from B10.A mice secreted high levels of TGF-beta and low levels of TNF-alpha. In contrast, macrophages from A/Sn animals released high levels of TNF-alpha associated with a small production of TGF-beta. The in vivo depletion of IFN-gamma not only abrogated the resistance of A/Sn mice but also diminished the relative resistance of B10.B animals. The in vivo depletion of IL-4 did not alter the disease outcome, whereas administration of rIL-12 significantly enhanced resistance in susceptible animals. Taken together, these results suggest that an early secretion of high levels of TNF-alpha and IFN-gamma followed by a sustained secretion of IL-2 and IFN-gamma plays a dominant role in the resistance mechanisms to P. brasiliensis infection. In contrast, an early and ephemeral secretion of low levels of TNF-alpha and IFN-gamma associated with production of IL-5 IL-10 and TGF-beta characterizes the progressive disease of susceptible animals.

Isolation of Paracoccidoides brasiliensis from armadillos (Dasypus novemcinctus) captured in an endemic area of paracoccidioidomycosis

Paracoccidioides brasiliensis, the causative agent of paracoccidioidomycosis (PCM), was first isolated from the Amazonian gerion where the mycosis is uncommon. In the present study, we report on the high incidence of PCM infection in armadillos from a hyperendemic region of the disease. Four nine-banded armadillos (Dasypus novemcinctus) were captured in the endemic area of Botucatu, Sao Paulo, Brazil, killed by manual cervical dislocation and autopsied under sterile conditions. Fragments of lung, spleen, liver and mesenteric lymph nodes were precessed for histology, cultured on Mycosel agar at 37ºC, and homogenized for inoculation into the testis and peritoneum of hamster. The animals were killed from week 6 to week 20 postinoculation and fragments of liver, lung, spleen, testis, and lymph nodes were cultured on brain heart infusion agar at 37ºC. Paracoccidioides brasiliensis was isolated from three armadillos both by direct organ culture and from the liver, spleen, lung, and mesenteric lymph node hamster. In addition, one positive armadillo presented histologically proven PCM disease in a mesenteric lymph node. The three aramdillos isolates (Pb-A1, Pb-A2, and Pb-A4) presented thermodependent dimorphism, urease activity, and casein assimilation, showed amplification of the gp43 gene, and were highly virulent in intratesticulary inoculation hamster. The isolates expressed the gp43 glycoprotein, the immunodominant antigen of the fungus, and reacted with a pool of sera from PCM patients. Taken together, the present data confirm that armadillos are a natural reservoir of P. brasiliensis and demonstrate that the animal is a sylvan host to the fungus