In vitro Arthrographis kalrae biofilm formation: Scanning electron microscopy and cytotoxic analysis.

To our knowledge, this study represents the first demonstration of Arthrographis kalrae biofilm formation in vitro by scanning electron microscopy and the distinct cytotoxic activity between planktonic and biofilm extracts on RAW 264.7 cell line. Higher activity was observed with biofilm. It could impact host immune response, that require furthers study.

Immunohistochemical Cross-Reactivity Between Arthrographis kalrae and Highly Pathogenic Coccidioides posadasii, Histoplasma capsulatum, and Paracoccidioides Fungal Species.

Recently, we have reported serological cross-reactivity between paracoccidioidomycosis ceti and paracoccidioidomycosis, histoplasmosis, and coccidioidomycosis. However, data on the interaction of Arthrographis kalrae with the above pathogenic fungal infections are lacking. A. kalrae is a widely occurring ascomycetous fungus; causes superficial and deep mycoses; shows thermally dependent dimorphism; and has a genomic profile related to the above-mentioned fungal species. Our study aims to investigate cross-reactivity using eight murine sera, obtained from experimental infection with two A. kalrae isolates. The murine sera were incubated with fungal cells of A. kalrae, Coccidioides posadasii, Histoplasma capsulatum, Paracoccidioides sp., and P. brasiliensis. Thirty murine sera, obtained from experimental infection with six isolates of H. capsulatum, sera from three cases of dolphin paracoccidioidomycosis ceti, two human sera from patients with paracoccidioidomycosis, and a serum sample from a healthy person with a history of coccidioidomycosis, were also incubated with A. kalrae fungal cells and the respective fungal cells that caused the infection as positive controls. Sera derived from the mice infected with A. kalrae reacted strongly when incubated with the Paracoccidioides sp., P. brasiliensis, and C. posadasii, but no positive reaction was observed against the fungal cells of H. capsulatum. The murine sera infected with three out of six isolates of H. capsulatum, and all cetacean and human serum samples reacted positively with the fungal cells of A. kalrae. The present study demonstrated serological cross-reactions among A. kalrae infection, coccidioidomycosis, paracoccidioidomycosis, paracoccidioidomycosis ceti, and histoplasmosis.

Immunomodulation over the course of experimental Arthrographis kalrae infection in mice.

Arthrographis kalrae is occasionally described as an opportunistic human pathogen. This study investigated the immune response to A. kalrae during murine experimental infection (7, 14, 28 and 56 days post infection). The fungal load was higher in the early phase and mice presented with neurological syndrome over the course of the infection. There was a gradual increase in the level of anti-A. kalrae IgG and increased levels of DTH at 14 days. There was decreased IFN-γ (14-56 days) and an increase in IL-4 (7 and 56 days). Decreased levels of cytokines (IFN-γ, IL-4, IL-10 and IL-17) were observed in the brain at 56 days p.i. The results suggest that the immune response during murine A. kalrae infection modulates to the pattern of Th2 response. This study shows for the first time the cytokines and cellular immunomodulation that occur in response to an experimental infection with A. kalrae in mice.

Arthrographis kalrae soluble antigens present hemolytic and cytotoxic activities.

Arthrographis kalrae is a dimorphic, cosmopolitan and neurotropic fungus that has been described as a rare human pathogen. This study investigated the hemolytic and cytotoxic activities of A. kalrae cell-free antigens (CFAs). Total CFAs and their Sephadex chromatography fractions were tested on mouse erythrocytes for hemolysis and on the P3U1 cell line for cytotoxicity. Hemolytic and cytotoxic activities were detected in distinct molecular mass (MM) fractions. Additionally, antibodies against isogenic erythrocytes sensitized with CFAs (anti-E-CFAs) inhibited hemolysis but not cytotoxicity. Hemolysis was not affected by heating, and a higher reactivity was detected in the carbohydrate-rich fractions, which decreased after reduction by periodate treatment. The pioneering nature of this work is due to the demonstration of the cytotoxic activity in A. kalrae and the suggestion that this activity may be due to molecules distinct from the hemolytic factor, with the latter potentially being a component with a high MM.

Carbohydrate-rich high-molecular-mass antigens are strongly recognized during experimental Histoplasma capsulatum infection.

INTRODUCTION: During histoplasmosis, Histoplasma capsulatum soluble antigens (CFAg) can be naturally released by yeast cells. Because CFAg can be specifically targeted during infection, in the present study we investigated CFAg release in experimental murine histoplasmosis, and evaluated the host humoral immune response against high-molecular-mass antigens (hMMAg. >150 kDa), the more immunogenic CFAg fraction. METHODS: Mice were infected with 2.2 x 104 H. capsulatum IMT/HC128 yeast cells. The soluble CFAg, IgG anti-CFAg, IgG anti-hMMAg, and IgG-hMMAg circulating immune complexes (CIC) levels were determined by enzymelinked immunosorbent assay, at days 0, 7, 14, and 28 post-infection. RESULTS: We observed a progressive increase in circulating levels of CFAg, IgG anti-CFAg, IgG anti-hMMAg, and IgG-hMMAg CIC after H. capsulatum infection. The hMMAg showed a high percentage of carbohydrates and at least two main immunogenic components. CONCLUSIONS: We verified for the first time that hMMAg from H. capsulatum IMT/HC128 strain induce humoral immune response and lead to CIC formation during experimental histoplasmosis.

Carbohydrate-rich high-molecular-mass antigens are strongly recognized during experimental Histoplasma capsulatum infection / Antígenos de alta massa molecular ricos em carboidratos são fortemente reconhecidos durante infecção experimental por Histoplasma capsulatum

INTRODUCTION: During histoplasmosis, Histoplasma capsulatum soluble antigens (CFAg) can be naturally released by yeast cells. Because CFAg can be specifically targeted during infection, in the present study we investigated CFAg release in experimental murine histoplasmosis, and evaluated the host humoral immune response against high-molecular-mass antigens (hMMAg. >150 kDa), the more immunogenic CFAg fraction. METHODS: Mice were infected with 2.2x10(4) H. capsulatum IMT/HC128 yeast cells. The soluble CFAg, IgG anti-CFAg, IgG anti-hMMAg, and IgG-hMMAg circulating immune complexes (CIC) levels were determined by enzymelinked immunosorbent assay, at days 0, 7, 14, and 28 post-infection. RESULTS: We observed a progressive increase in circulating levels of CFAg, IgG anti-CFAg, IgG anti-hMMAg, and IgG-hMMAg CIC after H. capsulatum infection. The hMMAg showed a high percentage of carbohydrates and at least two main immunogenic components. CONCLUSIONS: We verified for the first time that hMMAg from H. capsulatum IMT/HC128 strain induce humoral immune response and lead to CIC formation during experimental histoplasmosis.

INTRODUÇÃO: Durante a histoplasmose, os antígenos solúveis de Histoplasma capsulatum (CFAg) podem ser liberados naturalmente pelas células leveduriformes. Considerando que os CFAg constituem um alvo específico durante a infecção, no presente estudo nós investigamos a liberação de CFAg durante a histoplasmose murina experimental, e avaliamos a resposta imune humoral do hospedeiro contra antígenos de alta MM (hMMAg; >150 kDa), altamente imunogênicos. MÉTODOS: Camundongos foram infectados com 2.2x10(4) leveduras de H. capsulatum, cepa IMT/HC128. Os níveis de CFAg solúveis, IgG anti-CFAg, IgG anti-hMMAg, e também de imunocomplexos circulantes (CIC) IgG-hMMAgs foram determinados por ELISA nos dias 0, 7, 14 e 28 após a infecção. RESULTADOS: Após a infecção por H. capsulatum, observamos um aumento progessivo de CFAg circulantes, IgG anti-CFAg, IgG anti-hMMAg, e também de CIC IgG-hMMAgs. Os hMMAg apresentaram alta porcentagem de carboidratos e, pelo menos, dois componentes imunogênicos. CONCLUSÕES: Mostramos pela primeira vez que os hMMAg de H. capsulatum cepa IMT/HC128 induzem resposta imune humoral e levam à formação de CIC durante a histoplasmose experimental.

High molecular mass fraction in clinical isolates of Paracoccidioides brasiliensis.

INTRODUCTION: Different serum levels of the IgG/IgE for Paracoccidioides brasiliensis high mass molecular (hMM) fraction (~366 kDa) in the acute and chronic forms of the disease have been reported. Considering the nonexistence of hMM fraction investigation involving clinical isolates of P. brasiliensis, the present study aimed to investigate the presence of the hMM fraction (~366 kDa) in cell free antigens (CFA) from P. brasiliensis clinical isolates. METHODS: CFA from 10 clinical isolates and a reference strain (Pb18) were submitted to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by gel image capturing and densitometer analysis. Additionally, CFA from 20 isolates and Pb18 were analyzed by capture ELISA (cELISA) using polyclonal (polAb) or monoclonal (mAb) antibodies to the hMM fraction. RESULTS: The presence of the hMM component was observed in CFA of all samples analyzed by SDS-PAGE/densitometry and by cELISA. In addition, Pearson's correlation test demonstrated stronger coefficients between hMM fraction levels using pAb and mAb (R = 0.853) in cELISA. CONCLUSIONS: The soluble hMM fraction was present in all the P. brasiliensis clinical isolates analyzed and the reference strain Pb18, which could be used as a source of this antigen. The work also introduces for first time, the cELISA method for P. brasiliensis hMM fraction detection. Analysis also suggests that detection is viable using polAb or mAb and this methodology may be useful for future investigation of the soluble hMM fraction (~366 kDa) in sera from PCM patients.

High molecular mass fraction in clinical isolates of Paracoccidioides brasiliensis / Fração de alta massa molecular em isolados clínicos de Paracoccidioides brasiliensis

INTRODUCTION: Different serum levels of the IgG/IgE for Paracoccidioides brasiliensis high mass molecular (hMM) fraction (~366kDa) in the acute and chronic forms of the disease have been reported. Considering the nonexistence of hMM fraction investigation involving clinical isolates of P. brasiliensis, the present study aimed to investigate the presence of the hMM fraction (~366kDa) in cell free antigens (CFA) from P. brasiliensis clinical isolates. METHODS: CFA from 10 clinical isolates and a reference strain (Pb18) were submitted to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by gel image capturing and densitometer analysis. Additionally, CFA from 20 isolates and Pb18 were analyzed by capture ELISA (cELISA) using polyclonal (polAb) or monoclonal (mAb) antibodies to the hMM fraction. RESULTS: The presence of the hMM component was observed in CFA of all samples analyzed by SDS-PAGE/densitometry and by cELISA. In addition, Pearson's correlation test demonstrated stronger coefficients between hMM fraction levels using pAb and mAb (R = 0.853) in cELISA. CONCLUSIONS: The soluble hMM fraction was present in all the P. brasiliensis clinical isolates analyzed and the reference strain Pb18, which could be used as a source of this antigen. The work also introduces for first time, the cELISA method for P. brasiliensis hMM fraction detection. Analysis also suggests that detection is viable using polAb or mAb and this methodology may be useful for future investigation of the soluble hMM fraction (~366kDa) in sera from PCM patients.

INTRODUÇÃO: Diferentes níveis sorológicos de IgG/IgE contra a fração de alta massa molecular (hMM) (~366kDa) de Paracoccidioides brasiliensis têm sido encontrados na PCM aguda e crônica. Considerando a inexistência de investigação sobre esta fração em isolados clínicos de P. brasiliensis, o objetivo deste estudo foi investigar a presença da fração hMM (~366kDa) no preparado livre de células (CFA) de P. brasiliensis obtidos de isolados clínicos. MÉTODOS: CFA de 10 isolados e de cepa de referência (Pb18) foram submetidas à eletroforese em gel de SDS-poliacrilamida (SDS-PAGE) seguida de captura de imagem e análise por densitometria. Adicionalmente, CFA de 20 isolados e de Pb18 foram analisados por ELISA captura (cELISA) utilizando anticorpos policlonal (polAb) ou monoclonal (mAb) para fração hMM. RESULTADOS: A presença do componente de hMM foi observada em todas as amostras analisadas por SDS-PAGE/densitometria e por cELISA. Adicionalmente, o teste de correlação de Pearson demonstrou forte relação entre os níveis de fração hMM usando pAb e mAb (R = 0.853) no cELISA. CONCLUSÕES: Conclui-se que a fração hMM está presente em todos os isolados clínicos de P. brasiliensis analisados e no isolado referencial, sugerindo a possibilidade dos mesmos serem utilizados como fonte desta fração antigênica. Este trabalho também introduz pela primeira vez o método de cELISA para detecção da fração hMM de P. brasiliensis, sugerindo que detecção utilizando anticorpos polAb ou mAb é viável e essa metodologia poderá ser útil para investigação futura desta fração solúvel (~366kDa) em soros de pacientes com PCM.