GSTT1 and GSTM1 null variants in Mestizo and Amerindian populations from northwestern Mexico and a literature review

Abstract The GSTT1 and GSTM1 genes are key molecules in cellular detoxification. Null variants in these genes are associated with increase susceptibility to developing different types of cancers. The aim of this study was to determine the prevalence of GSTT1 and GSTM1 null genotypes in Mestizo and Amerindian individuals from the Northwestern region of Mexico, and to compare them with those reported worldwide. GSTT1 and GSTM1 null variants were genotyped by multiplex PCR in 211 Mestizos and 211 Amerindian individuals. Studies reporting on frequency of GSTT1 and GSTM1 null variants worldwide were identified by a PubMed search and their geographic distribution were analyzed. We found no significant differences in the frequency of the null genotype for GSTT1 and GSM1 genes between Mestizo and Amerindian individuals. Worldwide frequencies of the GSTT1 and GSTM1 null genotypes ranges from 0.10 to 0.51, and from 0.11 to 0.67, respectively. Interestingly, in most countries the frequency of the GSTT1 null genotype is common or frequent (76%), whereas the frequency of the GSMT1 null genotype is very frequent or extremely frequent (86%). Thus, ethnic-dependent differences in the prevalence of GSTT1 and GSTM1 null variants may influence the effect of environmental carcinogens in cancer risk.

GSTT1 and GSTM1 null variants in Mestizo and Amerindian populations from northwestern Mexico and a literature review.

The GSTT1 and GSTM1 genes are key molecules in cellular detoxification. Null variants in these genes are associated with increase susceptibility to developing different types of cancers. The aim of this study was to determine the prevalence of GSTT1 and GSTM1 null genotypes in Mestizo and Amerindian individuals from the Northwestern region of Mexico, and to compare them with those reported worldwide. GSTT1 and GSTM1 null variants were genotyped by multiplex PCR in 211 Mestizos and 211 Amerindian individuals. Studies reporting on frequency of GSTT1 and GSTM1 null variants worldwide were identified by a PubMed search and their geographic distribution were analyzed. We found no significant differences in the frequency of the null genotype for GSTT1 and GSM1 genes between Mestizo and Amerindian individuals. Worldwide frequencies of the GSTT1 and GSTM1 null genotypes ranges from 0.10 to 0.51, and from 0.11 to 0.67, respectively. Interestingly, in most countries the frequency of the GSTT1 null genotype is common or frequent (76%), whereas the frequency of the GSMT1 null genotype is very frequent or extremely frequent (86%). Thus, ethnic-dependent differences in the prevalence of GSTT1 and GSTM1 null variants may influence the effect of environmental carcinogens in cancer risk.

THE POWER OF THE SMALL: THE EXAMPLE OF Paracoccidioides brasiliensis CONIDIA.

Research on Paracoccidioides brasiliensis has centered in the yeast cell probably because of the lack of distinctive features in the mycelium. In 1942 and for the first time, lateral conidia were noticed in the fungus' hyphae. Later on, Brazilian, Venezuelan and Argentinean researchers described "aleurias" when the fungus was grown in natural substrates. In 1970 authors became interested in the conidia and were able to obtain them in large numbers and treat them as individual units. Their shape and size were defined and the presence of all the elements of a competent eukaryotic cell were demonstrated. Conidia exhibited thermal dimorphism and, additionally, when given intranasally to BALB/c male mice, they converted into yeasts in the lungs and produce progressive pulmonary lesions with further dissemination to other organs. Studies on the phagocyte-conidia interaction were revealing and showed that these versatile structures allow a better understanding of the host- P. brasiliensis interactions.

THE POWER OF THE SMALL: THE EXAMPLE OF Paracoccidioides brasiliensis CONIDIA / O poder do pequeno: o exemplo dos conídios do Paracoccidioides brasiliensis

SUMMARYResearch on Paracoccidioides brasiliensis has centered in the yeast cell probably because of the lack of distinctive features in the mycelium. In 1942 and for the first time, lateral conidia were noticed in the fungus' hyphae. Later on, Brazilian, Venezuelan and Argentinean researchers described "aleurias" when the fungus was grown in natural substrates. In 1970 authors became interested in the conidia and were able to obtain them in large numbers and treat them as individual units. Their shape and size were defined and the presence of all the elements of a competent eukaryotic cell were demonstrated. Conidia exhibited thermal dimorphism and, additionally, when given intranasally to BALB/c male mice, they converted into yeasts in the lungs and produce progressive pulmonary lesions with further dissemination to other organs. Studies on the phagocyte-conidia interaction were revealing and showed that these versatile structures allow a better understanding of the host- P. brasiliensisinteractions.

RESUMOA investigação sobre Paracoccidioides brasiliensis tem-se centrado na célula de levedura, provavelmente devido à falta de características distintas no micélio. Em 1942 e, pela primeira vez, conídios laterais foram notados nos hifas dos fungos. Mais tarde, pesquisadores brasileiros, venezuelanos e argentinos descreveram "aleurias" quando o fungo foi cultivado em substratos naturais. Em 1970, os autores se interessaram pelos conídios e foram capazes de obtê-los em grande número e tratá-los como unidades individuais. A sua forma e tamanho foram definidos, e a presença de todos os elementos de uma célula eucariótica competente foram demonstrados. Conídios apresentam dimorfismo térmico e, além disso, quando administrados por via intranasal a camundongos BALB/c machos, são convertidos em leveduras nos pulmões e produzem lesões pulmonares progressivas com posterior disseminação para outros órgãos. Estudos sobre a interação de fagócitos-conídios foram reveladores e mostraram que estas estruturas versáteis permitem melhor compreensão das interacções entre hospedeiro e P. brasiliensis.

Validation and clinical application of a nested PCR for paracoccidioidomycosis diagnosis in clinical samples from Colombian patients

Paracoccidioidomycosis is a systemic and endemic mycosis, restricted to tropical and subtropical areas of Latin America. The infection is caused by the thermal dimorphic fungus Paracoccidioides brasiliensisand Paracoccidioides lutzii. The diagnosis of paracoccidioidomycosis is usually performed by microscopic examination, culture and immunodiagnostic tests to respiratory specimens, body fluids and/or biopsies; however these methods require laboratory personnel with experience and several days to produce a result. In the present study, we have validated and evaluated a nested PCR assay targeting the gene encoding the Paracoccidioides gp43membrane protein in 191 clinical samples: 115 samples from patients with proven infections other than paracoccidioidomycosis, 51 samples as negative controls, and 25 samples from patients diagnosed with paracoccidioidomycosis. Additionally, the specificity of the nested PCR assay was also evaluated using purified DNA isolated from cultures of different microorganisms (n= 35) previously identified by culture and/or sequencing. The results showed that in our hands, this nested PCR assay for gp43 protein showed specificity and sensitivity rates of 100%. The optimized nested PCR conditions in our laboratory allowed detection down to 1 fg of P. brasiliensisDNA.

Pentoxifylline immunomodulation in the treatment of experimental chronic pulmonary paracoccidioidomycosis.

BACKGROUND: Pentoxifylline (PTX) is a methylxanthine compound with immunomodulatory and antifibrotic properties. The simultaneous use of PTX and antifungal therapy (itraconazole) has previously been evaluated in an experimental model of pulmonary paracoccidioidomycosis (PCM), a systemic fungal disease caused by the fungus Paracoccidioides brasiliensis (Pb) and characterized by chronic inflammation and lung fibrosis that appears even after a successful course of antifungal therapy. The results revealed prompt and statistically significant reductions in inflammation and fibrosis when compared to itraconazole alone. However, the effect of monotherapy with PTX on the host response to PCM has not been well-documented. Our aim was to determine the effect of PTX on the course of pulmonary lesions and on the local immune response. RESULTS: At the middle and end of treatment, the Pb-infected-PTX-treated mice exhibited significant reductions in lung density compared to the Pb-infected-non-treated mice as assessed by the quantification of Hounsfield units on high-resolution computed tomography (HRCT) (p <0.05 by Kruskal-Wallis test); additionally, at the end of therapy, the lung areas involved in the inflammatory reactions were only 3 vs. 22 %, respectively, by histomorphometry (p <0.05 by Mann-Whitney test), and this reduction was associated with a lower fungal burden and limited collagen increment in the pulmonary lesions. PTX treatment restored the levels of IFN-γ, MIP-1ß, and IL-3 that had been down-regulated by Pb infection. Additionally, IL-12p70, IL-10, IL-13, and eotaxin were significantly increased, whereas Regulated upon Activation, Normal T cell Expressed and Secreted (RANTES) levels were decreased in the lungs of the Pb-infected-PTX-treated mice compared to the non-treated group. CONCLUSIONS/SIGNIFICANCE: This study showed that PTX therapy administered at an "early" stage of granulomatous inflammation controlled the progress of the PCM by diminishing the pulmonary inflammation and the fungal burden and avoiding the appearance of collagen deposits in the pulmonary lesions.

Validation and clinical application of a nested PCR for paracoccidioidomycosis diagnosis in clinical samples from Colombian patients.

Paracoccidioidomycosis is a systemic and endemic mycosis, restricted to tropical and subtropical areas of Latin America. The infection is caused by the thermal dimorphic fungus Paracoccidioides brasiliensis and Paracoccidioides lutzii. The diagnosis of paracoccidioidomycosis is usually performed by microscopic examination, culture and immunodiagnostic tests to respiratory specimens, body fluids and/or biopsies; however these methods require laboratory personnel with experience and several days to produce a result. In the present study, we have validated and evaluated a nested PCR assay targeting the gene encoding the Paracoccidioides gp43 membrane protein in 191 clinical samples: 115 samples from patients with proven infections other than paracoccidioidomycosis, 51 samples as negative controls, and 25 samples from patients diagnosed with paracoccidioidomycosis. Additionally, the specificity of the nested PCR assay was also evaluated using purified DNA isolated from cultures of different microorganisms (n=35) previously identified by culture and/or sequencing. The results showed that in our hands, this nested PCR assay for gp43 protein showed specificity and sensitivity rates of 100%. The optimized nested PCR conditions in our laboratory allowed detection down to 1fg of P. brasiliensis DNA.

Response to therapy in patients with cryptococcosis and AIDS: Association with in vitro susceptibility to fluconazole.

BACKGROUND: The implications of the Cryptococcus neoformans resistance to fluconazole on patient therapy have not been fully elucidated due to the discordant results found in published studies. AIMS: To establish the influence of C. neoformans resistance to fluconazole in the therapy of individuals with cryptococcosis and AIDS. METHODS: This study retrospectively compared the clinical course of patients with cryptococcosis according to the level of fluconazole resistance of their C. neoformans isolates. RESULTS: This study included 71 episodes of cryptococcosis, defined as those isolates of C. neoformans obtained from patients with mycosis, of which 36 isolates were sensitive to fluconazole, 20 susceptible dose-dependent (SDD), and 15 were resistant. There were 5 treatment failures in the consolidation phase; two occurred in patients who had a susceptible strain, 2 in patients who had SDD strains, and one in a patient who had a resistant strain. During the maintenance treatment, relapses occurred in 4 of 33 patients (12%), seen during the follow-up period, none of which occurred in the group with resistant isolates. There were no significant differences in survival time free of treatment failure (p=0.65) or survival time free of failure or relapse (p=0.38). These results were not affected when tested in a Cox model that included age, CD4T lymphocyte counts, and use of antiretroviral therapy. CONCLUSIONS: In HIV patients with cryptococcosis, the resistance of C. neoformans appeared not to increase the risk of failure or relapse during treatment.

Diagnostic value of culture and serological tests in the diagnosis of histoplasmosis in HIV and non-HIV Colombian patients.

We determined the value of culture and serological tests used to diagnose histoplasmosis. The medical records of 391 histoplasmosis patients were analyzed. Diagnosis of the mycosis was assessed by culture, complement fixation, and immunodiffusion tests; 310 patients (79.5%) were male, and 184 patients (47.1%) were infected with human immunodeficiency virus (HIV). Positivity value for cultures was 35.7% (74/207), reactivity of serological tests was 95.2% (160/168), and a combination of both methodologies was 16.9% (35/207) for non-HIV patients. Positivity value for cultures was 75.0% (138/184), reactivity of serological tests was 92.4% (85/92), and a combination of both methodologies was 26.0% (48/184) for HIV/acquired immunodeficiency syndrome (AIDS) patients; 48.1% (102/212) of extrapulmonary samples from HIV/AIDS patients yielded positive cultures compared with 23.1% (49/212) in non-HIV patients. Lymphocyte counts made for 33.1% (61/184) of HIV/AIDS patients showed a trend to low CD4+ numbers and higher proportion of positive cultures. These results indicate that culture is the most reliable fungal diagnostic method for HIV/AIDS patients, and contrary to what is generally believed, serological assays are useful for diagnosing histoplasmosis in these patients.

Interaction between Paracoccidioides brasiliensis conidia and the coagulation system: involvement of fibrinogen

The infectious process starts with an initial contact between pathogen and host. We have previously demonstrated that Paracoccidioides brasiliensis conidia interact with plasma proteins including fibrinogen, which is considered the major component of the coagulation system. In this study, we evaluated the in vitro capacity of P. brasiliensis conidia to aggregate with plasma proteins and compounds involved in the coagulation system. We assessed the aggregation of P. brasiliensis conidia after incubation with human serum or plasma in the presence or absence of anticoagulants, extracellular matrix (ECM) proteins, metabolic and protein inhibitors, monosaccharides and other compounds. Additionally, prothrombin and partial thromboplastin times were determined after the interaction of P. brasiliensis conidia with human plasma. ECM proteins, monosaccharides and human plasma significantly induced P. brasiliensis conidial aggregation; however, anticoagulants and metabolic and protein inhibitors diminished the aggregation process. The extrinsic coagulation pathway was not affected by the interaction between P. brasiliensis conidia and plasma proteins, while the intrinsic pathway was markedly altered. These results indicate that P. brasiliensis conidia interact with proteins involved in the coagulation system. This interaction may play an important role in the initial inflammatory response, as well as fungal disease progression caused by P. brasiliensis dissemination.

Utilidad de la muestra de la lámina ungular en el diagnóstico de onicomicosis / Usefulness of nail plate sample in the diagnosis of onychomycosis

Introducción. La sensibilidad de las pruebas convencionales (examen directo, cultivo) para el diagnóstico de la onicomicosis (25 a 80%), representa un problema para la decisión terapéutica del dermatólogo. Objetivo. Determinar la exactitud diagnóstica de la muestra de la lámina ungular en pacientes con diagnóstico clínico de onicomicosis. Metodología. Es un estudio prospectivo de pruebas diagnósticas en 50 pacientes con sospecha de onicomicosis. Se tomó muestra de la lámina ungular con cortaúñas estéril en el área de onicólisis para pruebas micológicas (KOHcultivo) y de histopatología (hematoxilina y eosina y ácido peryódico de Schiff), y muestra de detritos mediante raspado del lecho para prueba micológica. La toma de muestras y el procesamiento de las pruebas se realizaron en laboratorios de referencia y se interpretaron de manera ciega e independiente. La muestra de detritos se consideró la prueba estándar. Resultados. Se observó compromiso de los pies en 90% de los pacientes, 86,6% con afectación del primer dedo. La prueba micológica de detritos fue positiva en 80% de los casos, encontrándose estructuras micóticas en el examen directo en 72% y aislamiento al cultivo en 64%. En la lámina ungular, la sensibilidad fue de 87,5% y la especificidad de 80%; el cociente de probabilidades positivo fue 4,4. Cinco muestras positivas con la tinción PAS fueron negativas en la prueba estándar. La sensibilidad neta aumentó a 95% mediante el análisis de las pruebas en paralelo de la lámina ungular. La mayoría de los aislamientos fueron especies de Candida (77,3% en detritos y 75,9% en la lámina ungular), y C. parapsilosis fue el aislamiento más frecuente. Conclusión. Se propone la muestra de la lámina ungular para pruebas micológicas y tinción de PAS, como complemento a la muestra de detritos para el diagnóstico de onicomicosis.

Structural and topographic dynamics of pulmonary histopathology and local cytokine profiles in Paracoccidioides brasiliensis conidia-infected mice.

BACKGROUND: Paracoccidioidomycosis (PCM), an endemic systemic mycosis caused by the fungus Paracoccidioides brasiliensis (Pb), usually results in severe lung damage in patients. METHODS AND FINDINGS: Considering the difficulties to sequentially study the infection in humans, this work was done in mice inoculated intranasally with infective Pb-conidia. Lungs of control and Pb-infected mice were studied after 2-hours, 4, 8, 12 and 16-weeks post-infection (p.i) in order to define histopathologic patterns of pulmonary lesions, multiplex-cytokine profiles and their dynamics during the course of this mycosis. Besides the nodular/granulomatous lesions previously informed, results revealed additional non-formerly described lung abnormalities, such as periarterial sheath inflammation and pseudotumoral masses. The following chronologic stages occurring during the course of the experimental infection were defined: Stage one (2-hours p.i): mild septal infiltration composed by neutrophils and macrophages accompanied by an intense "cytokine burst" represented by significant increases in IL-1α, IL-1ß, IL-4, IL-5, IL-6, IL-10, IL12p70, IL-13, IL-17, Eotaxin, G-CSF, MCP1, MIP1α, GM-CSF, IFN-γ, MIP1ß and TNFα levels. Stage two (4-weeks p.i): presence of nodules, evidence of incipient periarterial- and intense but disperse parenchymal- inflammation, abnormalities that continued to be accompanied by hyper-secretion of those cytokines and chemokines mentioned in the first stage of infection. Stages three and four (8 and 12-weeks p.i.): fungal proliferation, inflammation and collagenesis reached their highest intensity with particular involvement of the periarterial space. Paradoxically, lung cytokines and chemokines were down-regulated with significant decreases in IL-2,IL-3,IL-5,IL-9,IL-13,IL-15,GM-CSF,IFN-γ,MIP1ß and TNFα. Stage five (16-weeks p.i.): inflammation decreased becoming limited to the pseudotumoral masses and was accompanied by a "silent" cytokine response, except for PDGF, MIG, RANTES and IL12p40 which remained up-regulated for the duration of the experiment. CONCLUSIONS: Results of this study identified both classic and novel patterns corresponding to histopathologic and immunologic responses occurring during the course of experimental PCM.

A 32-kilodalton hydrolase plays an important role in Paracoccidioides brasiliensis adherence to host cells and influences pathogenicity.

One of the most crucial events during infection with the dimorphic fungus Paracoccidioides brasiliensis is adhesion to pulmonary epithelial cells, a pivotal step in the establishment of disease. In this study, we have evaluated the relevance of a 32-kDa protein, a putative adhesion member of the haloacid dehalogenase (HAD) superfamily of hydrolases, in the virulence of this fungus. Protein sequence analyses have supported the inclusion of PbHad32p as a hydrolase and have revealed a conserved protein only among fungal dimorphic and filamentous pathogens that are closely phylogenetically related. To evaluate its role during the host-pathogen interaction, we have generated mitotically stable P. brasiliensis HAD32 (PbHAD32) antisense RNA (aRNA) strains with consistently reduced gene expression. Knockdown of PbHAD32 did not alter cell vitality or viability but induced morphological alterations in yeast cells. Moreover, yeast cells with reduced PbHAD32 expression were significantly affected in their capacity to adhere to human epithelial cells and presented decreased virulence in a mouse model of infection. These data support the hypothesis that PbHad32p binds to extracellular matrix (ECM) proteins and modulates the initial immune response for evasion of host defenses. Our findings point to PbHAD32 as a novel virulence factor active during the initial interaction with host cells in P. brasiliensis.

Pulmonary abnormalities in mice with paracoccidioidomycosis: a sequential study comparing high resolution computed tomography and pathologic findings.

BACKGROUND: Human paracoccidioidomycosis (PCM) is an endemic fungal disease of pulmonary origin. Follow-up of pulmonary lesions by image studies in an experimental model of PCM has not been previously attempted. This study focuses on defining patterns, topography and intensity of lung lesions in experimentally infected PCM mice by means of a comparative analysis between High Resolution Computed Tomography (HRCT) and histopathologic parameters. METHODOLOGY: Male BALB/c mice were intranasally inoculated with 3 x 10(6) Paracoccidioides brasiliensis (Pb) conidia (n = 50) or PBS (n = 50). HRCT was done every four weeks to determine pulmonary lesions, quantify lung density, reconstruct and quantify lung air structure. Lungs were also analyzed by histopathology and histomorphometry. RESULTS: Three different patterns of lesions were evidenced by hrct and histopathology, as follows: nodular-diffuse, confluent and pseudo-tumoral. The lesions were mainly located around the hilus and affected more frequently the left lung. At the 4th week post-challenge HRCT showed that 80% of the Pb-infected mice had peri-bronchial consolidations associated with a significant increase in upper lung density when compared with controls, (-263+/-25 vs. -422+/-10 HU, p<0.001). After the 8th and 12th weeks, consolidation had progressed involving also the middle regions. Histopathology revealed that consolidation as assessed by HRCT was equivalent histologically to a confluent granulomatous reaction, while nodules corresponded to individual compact granulomas. At the 16th week of infection, confluent granulomas formed pseudotumoral masses that obstructed large bronchi. Discrete focal fibrosis was visible gradually around granulomas, but this finding was only evident by histopathology. CONCLUSIONS/SIGNIFICANCE: This study demonstrated that conventional HRCT is a useful tool for evaluation and quantification of pulmonary damage occurring in experimental mouse PCM. The experimental design used decreases the need to sacrifice a large number of animals, and serves to monitor treatment efficacy by means of a more rational approach to the study of human lung disease.

Identificación de algunos genes asociados al proceso de germinación de la conidia al micelio en Paracoccidioides brasiliensis / Identification of genes associated with germination of conidia to form mycelia in the fungus Paracoccidioides brasiliensis

Introducción. Paracoccidioides brasiliensis es un hongo dimórfico térmico, que a temperatura ambiente se presenta como un moho productor de conidias, mientras que en el huésped se comporta como una levadura de gemación múltiple. Los mecanismos moleculares que rigen la germinación de conidia a micelio aún se desconocen. Objetivo. Estudiar en P. brasiliensis la cinética del proceso de germinación de conidia a micelio y determinar los genes expresados durante este proceso mediante la construcción y el análisis de una librería EST (Expressed Sequence Tag). Materiales y métodos. Para el estudio de la cinética de germinación, se produjeron y aislaron conidias de P. brasiliensis. Estas fueron incubadas en cultivos líquidos a 18°C por 24, 48, 72 y 96 horas, y se examinaron por microscopía de luz. A partir de conidias cultivadas por 96 horas, se construyó y caracterizó una librería EST, la cual representaría los genes expresados durante el proceso de germinación. Resultados. Durante el proceso de germinación de conidia a micelio, se observó 11,7±1,2%, 30±0,6%, 43±1,3% y 66±2,4% de germinación a las 24, 48, 72 y 96 horas de incubación, respectivamente. Además, se obtuvo una librería del proceso de germinación consistente en 129 secuencias agrupadas en cuatro secuencias contiguas y siete secuencias únicas, para un total de 11 posibles genes. Ocho secuencias (72,7%) no habían sido descritas anteriormente en otras librerías informadas para este hongo y podrían representar genes específicos de la germinación de conidia a micelio. Conclusiones. Éste es el primer reporte en el que se identifican genes no descritos anteriormente, que son expresados durante la germinación de conidia a micelio, proceso de gran importancia en la biología de P. brasiliensis.

Introduction. Paracoccidioides brasiliensis is a thermo-dimorphic fungus. At room temperature it grows as a mold that produces conidia, whereas in the vertebrate host it grows as a multiple-budding yeast. The molecular mechanisms involved in the germination from the conidia to the mycelia process remain unknown. Objective. The kinetics of conidia to mycelia germination process were studied in the dimorphic fungus P. brasiliensis. Gene expression during this process was evaluated by construction and analysis of an EST library. Materials and methods. For the germination kinetics study, P. brasiliensis conidia were isolated as single cell units. Then, they were cultured at 18° C in BHI (brain-heart infusion) broth for 24, 48, 72 and 96 hr. After each perion, they were examined by light microscopy. From conidia harvested at 96 hr, an EST library was constructed; at this stage the gene expression was presumed to be maximal for the germination process. Results. During the conidia to the mycelia developmental process, the following germination rates were observed: at 24 hr, 11.7±1.2%; at 48 hr, 30±0.6%; at 72 hr, 43±1.3%; and at 96 hr, 66±2.4%. At the 96 hour stage, an EST library was constructed. It consisted of 129 sequences grouped in 4 contigs and 7 singlets for a total of 11 possible genes. Eight of the sequences had not been described previously in other EST libraries of this fungus. Conclusions. New genes were identified that were expressed during the conidia to the mycelia germination process and may represent genes specific to the germination process.

Pulmonary immune responses induced in BALB/c mice by Paracoccidioides brasiliensis conidia.

Knowledge concerning the host-Paracoccidioides brasiliensis interactions is abundant. Yet, most of the experimental studies have used yeast cells to prepare the corresponding inoculum. As these cells do not represent the naturally infecting propagules, the corresponding experiments by-pass the earlier stages of such interactions. Studies done in patients, who also harbour yeast cells, suffer from the same bias. The review presented below focuses on the immune responses of BALB/c mice infected with conidia obtained from P. brasiliensis mycelial form cultures, the fungal stage most probably existing in nature. As such, the corresponding experiments would copy the onset and course of the human infection. A large number of experimental studies done by the CIB Medical and Experimental Mycology Unit in a period of almost 25 years have been revised and extracted so as to present a comprehensive record on the immune responses induced when mice are infected intranasally with the conidia. The establishment of this mouse model has permitted the analysis of the immune responses taking place during the early and late stages post-challenge. This unique model has made possible to characterize the course of the experimental disease including the inflammatory reaction, the expression of cytokines and of the various molecules associated to these responses, all of which lead to granuloma formation. The latter structure serves as a nest for the development of fibrosis. Thus, we have also obtained a glimpse on the complexity that accompanies the fibrosis, the most common sequelae of paracoccidioidomycosis. Additionally, a concerted effort has been made to appraise the whole gamut of immune factors and related molecules that directly or indirectly, contribute to shape the pathogenesis of this Latin American mycosis.

Lysozyme plays a dual role against the dimorphic fungus Paracoccidioides brasiliensis / A lisozima desempenha um papel duplo contra o fungo dimórfico Paracoccidioides brasiliensis

In order to determine the role of lysozyme, an antimicrobial peptide belonging to the innate immune system, against the dimorphic fungus Paracoccidioides brasiliensis, co-cultures of the MH-S murine alveolar macrophages cell line with P. brasiliensis conidia were done; assays to evaluate the effect of physiological and inflammatory concentrations of lysozyme directly on the fungus life cycle were also undertaken. We observed that TNF-α-activated macrophages significantly inhibited the conidia to yeast transition (p = 0.0043) and exerted an important fungicidal effect (p = 0.0044), killing 27 percent more fungal propagules in comparison with controls. Nonetheless, after adding a selective inhibitor of lysozyme, the fungicidal effect was reverted. When P. brasiliensis propagules were exposed directly to different concentrations of lysozyme, a dual effect was observed. Physiologic concentrations of the enzyme facilitated the conidia-to-yeast transition process (p < 0.05). On the contrary, inflammatory concentrations impaired the normal temperature-dependant fungal transition (p < 0.0001). When yeast cells were exposed to lysozyme, irrespective of concentration, the multiple-budding ability was badly impaired (p < 0.0001). In addition, ultra-structural changes such as subcellular degradation, fusion of lipid vacuoles, lamellar structures and interruption of the fibrilar layer were observed in lysozyme exposed conidia. These results suggest that lysozyme appears to exert a dual role as part of the anti-P. brasiliensis defense mechanisms.

Com a finalidade de determinar o papel da lisozima, um peptídeo antimicrobiano que pertence ao sistema imune inato, contra o fungo dimórfico Paracoccidioides brasiliensis, foram feitas co-culturas de uma linha de macrófagos alveolares murinos (MH-S) com as conídias do fungo na presença ou não do TNF-α e/ou um inibidor da lisozima; também foram feitos ensaios que avaliaram o efeito das concentrações fisiológicas e inflamatórias de lisozima diretamente sobre o ciclo de vida do fungo. Observamos que os macrófagos ativados com a citoquina tiveram um efeito significativo na inibição da transição conídia/levedura (p = 0,0043) e exerceram um efeito fungicida importante (p = 0,0044), matando mais de 27 por cento das propágulas do fungo em comparação com os macrófagos não ativados. No entanto, após ser o inibidor seletivo da lisozima adicionado, o efeito fungicida foi revertido. Quando os propágulos do fungo foram expostos diretamente a diferentes concentrações da lisozima, um duplo efeito foi observado. Assim, as concentrações fisiológicas da enzima facilitaram o processo de transição conídia-levedura (p < 0,05). Contrariamente, as concentrações inflamatórias prejudicaram a transição fúngica (p < 0,0001). Quando as leveduras foram expostas a qualquer concentração de lisozima, sua capacidade de multi-brotação foi gravemente prejudicada (p < 0,0001). Além disso, mudanças ultra-estruturais, como a sub degradação, a fusão dos vacúolos dos lípidos, estruturas lamelares e interrupção da camada fibrilar foram observadas em conídios expostos à lisozima. Estes resultados sugerem que a lisozima poderia exercer um duplo papel no mecanismo antifúngico contra P. brasiliensis.

Melanina: implicaciones en la patogénesis de algunas enfermedades y su capacidad de evadir la respuesta inmune del hospedero / Melanin: implications in some disease pathogenesis and its capacity to evade the host immune response

La melanina es uno de los pigmentos más comunes y de mayor distribución en la naturaleza. Es responsable de la coloración de plantas y animales; se encuentra en los ojos, el cabello, la piel, el plumaje, la cáscara de los huevos, la cutícula de los insectos, la tinta de los cefalópodos y en la pared y el citoplasma de muchos microorganismos. En los humanos este pigmento se ha encontrado también fuera de la piel, en las neuronas de la sustancia nigra y en los hepatocitos. Entre los microorganismos que se han reportado como productores de melanina tenemos Vibrio cholerae, Mycobacterium leprae, Bacillus thurigiensis, Pseudomonas aeruginosa, Schistosoma mansoni, Fasciola gigantita Trichuris suis, Alternaria alternata, Aspergillus niger, Blastomyces dermatitidis, Candida albicans, Cladosporium carionii, Coccidioides immitis, Cryptococcus neoformans, Exophiala (Wangiella) dermatitidis, Fonsecaea pedrosoi, Histoplasma capsulatum, Paracoccidioides brasiliensis, Penicillium marneffei, Pneumocystis carinii (jirovecii), Scedosporium prolificans, Scytalidium dimidiatum y Sporothrix schenckii; esto sin tener en cuenta los hongos dematiáceos, entre muchos otros. Esta revisión pretende hacer un compendio de las más recientes publicaciones sobre melanina relacionadas principalmente con su función, su importante contribución a la supervivencia en el ambiente y durante la infección, como factor de virulencia en diversos microorganismos, principalmente en hongos patógenos, y su papel como agente inmunomodulador, así como la reducida susceptibilidad que confiere contra muchos de los antimicóticos usados en la actualidad.

Melanin is one of the common pigments in nature; it is responsible for pigmentation in plants and animals. It is found in skin, eyes, feathers, egg shell, hair, insect cuticle, cuttlefish ink and wall and/or cytoplasm from many microorganisms. Melanin in humans is also present in substantia nigra and hepatocytes. Some microorganisms that have been reported producing melanin are: Vibrio cholerae, Mycobacterium leprae, Bacillus thurigiensis, Pseudomonas aeruginosa, Schistosoma mansoni, Fasciola gigantita, Trichuris suis, Alternaria alternata, Aspergillus niger, Blastomyces dermatitidis, Candida albicans, Cladosporium carionii, Coccidioides immitis, Cryptococcus neoformans, Exophiala (Wangiella) dermatitidis, Fonsecaea pedrosoi, Histoplasma capsulatum, Paracoccidioides brasiliensis, Penicillium marneffei, Pneumocystis carinii (jirovecii), Scedosporium prolificans, Scytalidium dimidiatum, Sporothrix schenckii, and most of the dematiaceous fungi. This review is focused on recent international publications concerning melanin analysing its capacity to survive in nature and during infection inside the host and its evasion of the immune response. Melanin acts as an inmunomodulador particle and it is known that its presence in many of microorganisms could protect them from microbicidal agents presently used.

Expression and arrangement of extracellular matrix proteins in the lungs of mice infected with Paracoccidioides brasiliensis conidia.

Extracellular matrix (ECM) proteins are important modulators of migration, differentiation and proliferation for the various cell types present in the lungs; they influence the immune response as well as participate in the adherence of several fungi including Paracoccidioides brasiliensis. The expression, deposition and arrangement of ECM proteins such as laminin, fibronectin, fibrinogen, collagen and proteoglycans in the lungs of mice infected with P. brasiliensis conidia has been evaluated in this study, together with the elastic fibre system. Lungs of BALB/c mice infected with P. brasiliensis conidia were analysed for the different ECM proteins by histological and immunohistochemical procedures at different times of infection. In addition, laser scanning confocal microscopy and scanning electron microscopy were used. During the early periods, the lungs of infected animals showed an inflammatory infiltrate composed mainly of polymorphonuclear neutrophils (PMNs) and macrophages, while during the later periods, mice presented a chronic inflammatory response with granuloma formation. Re-arrangement and increased expression of all ECM proteins tested were observed throughout all studied periods, especially during the occurrence of inflammatory infiltration and formation of the granuloma. The elastic fibre system showed an elastolysis process in all experiments. In conclusion, this study provides new details of pulmonary ECM distribution during the course of paracoccidioidomycosis.

Paracoccidioides brasiliensis conidia recognize fibronectin and fibrinogen which subsequently participate in adherence to human type II alveolar cells: involvement of a specific adhesin.

We examined the ability of Paracoccidioides brasiliensis conidia to interact with fibronectin, fibrinogen and with A549 cells, in order to establish the nature of the molecules involved. Conidia bound to immobilized proteins in a concentration-dependent manner. Antibodies against fibronectin and fibrinogen inhibited the fungal adherence to the corresponding proteins; as did laminin and fibronectin, but not fibrinogen when added in soluble form; however, the fibrinogen fragment D interfered with adhesion in a significant manner. Various monosaccharides and RGD/RGDS peptides had no effect on adherence to fibronectin or fibrinogen, while N-acetylneuraminic acid (NANA) abolished adherence to both proteins. Additionally, these proteins were detected on the surface of A549 cells. Inhibition assays showed a significant decrease in fungal adherence when A549 cells were treated with anti-fibrinogen, anti-fibronectin antibodies and a purified adhesin of P. brasiliensis (32-kDa protein); or when conidia were treated with these soluble proteins, mAb anti-32-kDa protein, RGD peptides and NANA. These results suggest that fibrinogen and fibronectin facilitate the adherence of conidia to A549 cells probably through the interaction with adhesin-type molecules or a sialic acid based recognition system. These interactions appear to play a role in the initial fungal attachment to the lung, and consequently, also in the pathogenesis of paracoccidioidomycosis.