Luz Elena Cano

Nitric Oxide Participation in the Fungicidal Mechanism of Gamma Interferon-Activated Murine Macrophages against Paracoccidioides brasiliensis Conidia

ABSTRACT Paracoccidioidomycosis, a systemic mycosis restricted to Latin America and produced by the dimorphic fungus Paracoccidioides brasiliensis, is probably acquired by inhalation of conidia produced by the mycelial form. The macrophage (Mφ) represents the major cell defense against this pathogen; when activated with gamma interferon (IFN-γ), murine Mφs kill the fungus by an oxygen-independent mechanism. Our goal was to determine the role of nitric oxide in the fungicidal effect of Mφs on P. brasiliensis conidia. The results revealed that IFN-γ-activated murine Mφs inhibited the conidium-to-yeast transformation process in a dose-dependent manner; maximal inhibition was observed in Mφs activated with 50 U/ml and incubated for 96 h at 37°C. When Mφs were activated with 150 to 200 U of cytokine per ml, the number of CFU was 70% lower than in nonactivated controls, indicating that there was a fungicidal effect. The inhibitory effect was reversed by the addition of anti-IFN-γ monoclonal antibodies. Activation by IFN-γ also enhanced Mφ nitric oxide production, as revealed by increasing NO2 values (8 ± 3 μM in nonactivated Mφs versus 43 ± 13 μM in activated Mφs). The neutralization of IFN-γ also reversed nitric oxide production at basal levels (8 ± 5 μM). Additionally, we found that there was a significant inverse correlation (r = −0.8975) between NO2− concentration and transformation ofP. brasiliensis conidia. Additionally, treatment with any of the three different nitric oxide inhibitors used (arginase,NG-monomethyl-l-arginine, and aminoguanidine), reverted the inhibition of the transformation process with 40 to 70% of intracellular yeast and significantly reduced nitric oxide production. These results show that IFN-γ-activated murine Mφs kill P. brasiliensis conidia through thel-arginine–nitric oxide pathway.

Treatment of paracoccidioidomycosis with ketoconazole: A three-year experience

The results of ketoconazole therapy in 38 patients with active paracoccidioidomycosis are described. Treatment consisted of a 200 mg tablet a day for 6 months. Evaluation was accomplished by means of a scoring system and the results were as follows: none of the patients worsened during therapy, one was found to be unchanged, five had minor improvement, 330 had major improvement, and there was complete resolution of the pretherapy conditions in 13. These findings plus the lack of toxicity of the drug and the facility for oral administration, make of ketoconazole a first line drug for the treatment of paracoccidioidomycosis.

Residual pulmonary abnormalities in adult patients with chronic paracoccidioidomycosis: prolonged follow-up after itraconazole therapy.

Itraconazole effectively controls active paracoccidioidomycosis but appears not to hinder lung fibrosis. Clinical records and chest radiographs from 47 itraconazole-treated patients with prolonged posttherapy follow-up (mean follow-up period, 5.6 years) were analyzed; the radiographs were interpreted following pneumoconiosis standards that consider the lungs as 6 fields and grade damage according to the number of fields involved. Infiltrative lesions were observed at diagnosis in 93.6% of the patients. Fibrosis was observed in 31.8% of the patients at diagnosis and had not cleared at the end of the observation period in any of these patients. Fibrosis also developed de novo in 11 patients (25%), so that by the end of the follow-up period it was seen in 53.2% of patients overall. Fibrosis correlated with severity of infiltrates at diagnosis: fibrosis was present in 83% of patients with very severe infiltration and in 12.5% of patients with minor infiltration. Among patients with severe infiltration, fibrosis was present in 30%; this increased (to 75%) when bullae were concomitantly present at diagnosis. Prompt initiation of treatment is necessary to avoid the development of fibrosis.

Purification and Partial Characterization of a Paracoccidioides brasiliensis Protein with Capacity To Bind to Extracellular Matrix Proteins

ABSTRACT Microorganisms adhere to extracellular matrix proteins by means of their own surface molecules. Paracoccidioides brasiliensis conidia have been shown to be capable of interacting with extracellular matrix proteins. We aimed at determining the presence of fungal proteins that could interact with extracellular matrix protein and, if found, attempt their purification and characterization. Various extracts were prepared from P. brasiliensis mycelial and yeast cultures (total homogenates, β-mercaptoethanol, and sodium dodecyl sulfate [SDS] extracts) and analyzed by ligand affinity assays with fibronectin, fibrinogen and laminin. Two polypeptides were detected in both fungal forms. SDS extracts that interacted with all the extracellular matrix protein were tested; their molecular masses were 19 and 32 kDa. Analysis of the N-terminal amino acid sequence of the purified 32-kDa mycelial protein showed substantial homology with P. brasiliensis, Histoplasma capsulatum, and Neurospora crassa hypothetical proteins. Additionally, a monoclonal antibody (MAb) produced against this protein recognized the 32-kDa protein in the SDS extracts of both fungal forms for immunoblot. Immunofluorescence analysis revealed that this MAb reacted not only with mycelia and yeast cells, but also with conidia, indicating that this protein was shared by the three fungal propagules. By immunoelectron microscopy, this protein was detected in the cell walls and in the cytoplasm. Both the 32-kDa purified protein and MAb inhibited the adherence of conidia to the three extracellular matrix proteins in a dose-dependent manner. These findings demonstrate the presence of two polypeptides capable of interacting with extracellular matrix proteins on the surface of P. brasiliensis propagules, indicating that there may be common receptors for laminin, fibronectin, and fibrinogen. These proteins would be crucial for initial conidial adherence and perhaps also in dissemination of paracoccidioidomycosis.

A technique to collect and dislodge conidia produced by Paracoccidioides brasiliensis mycelial form

Collection and separation of mycelial propagules produced by P. brasiliensis was accomplished by agitation with glass beads, centrifugation and filtration through cotton wool. The mean number of conidia liberated per plate (approximately 1 000 000) and their viability (79%), lead us to think that it is now possible to undertake experimental studies with these propagules.

Depletion of CD8+ T Cells In Vivo Impairs Host Defense of Mice Resistant and Susceptible to Pulmonary Paracoccidioidomycosis

ABSTRACT Using a pulmonary model of infection, we demonstrated previously that A/Sn and B10.A mice are, respectively, resistant and susceptible to Paracoccidioides brasiliensis infection. Employing the same experimental model, we examined herein the role of CD8+ T cells in the course of paracoccidioidomycosis. Treatment with anti-CD8 monoclonal antibodies caused a selective depletion of pulmonary and splenic CD8+ T cells in both mouse strains. The number of pulmonary CD4+ T cells and immunoglobulin-positive cells was independent of the number of CD8+ T cells. In susceptible mice, the loss of CD8+ T cells by in vivo treatment with anti-CD8 monoclonal antibodies impaired the clearance of yeasts from the lungs and increased the fungal dissemination to the liver and spleen. The same treatment in resistant mice increased fungal dissemination to extrapulmonary tissues but did not alter the pulmonary fungal load. Furthermore, CD8+ T-cell depletion did not modify delayed-type hypersensitivity reactions of A/Sn mice but increased these reactions in B10.A mice. The production of P. brasiliensis-specific antibodies by resistant and susceptible mice depleted of CD8+ T cells was similar to that of mice given control antibody. Histopathologically, depletion of CD8+ T cells did not disorganize the focal granulomatous lesions developed by both mouse strains. These results indicate that CD8+ T cells are necessary for optimal clearance of the fungus from tissues of mice infected with P. brasiliensisand demonstrate more prominent protective activity by those cells in the immune responses mounted by susceptible animals.

Predictive value of serologic tests in the diagnosis and follow-up of patients with paracoccidioidomycosis

Con el objeto de determinar el valor predictivo de las pruebas serologicas (Fijacion de Complemento, FC, e Inmunodifusion en Gel de Agar, IDGA), utilizadas regularmente en nuestro laboratorio para el diagnostico y el seguimiento de la paracoccidioidomicosis, se analizaron los datos provenientes de 43 pacientes con la enfermedad. Tales pacientes fueron tratados en forma identica (Ketoconazol, 200 mg/dia por 6 meses); ademas ambas pruebas serologicas utilizaron un mismo lote de antigeno, eliminando asi posibles variables. Fuera de los sueros provenientes de los 43 pacientes con paracoccidioidomicosis (A), se estudiaron 50 sueros de pacientes con otras micosis profundas (B) y 92 sueros de personas sanas (C). Empleamos las formulas dadas por GALEN y GAMBINO6 y hallamos que la sensibilidad de ambas pruebas serologicas era comparable, sin diferencia estadisticamente significativa entre ellas (FC = 93%, IDGA - 88.4%). Dicha sesibilidad tendia a disminuir con el tratamiento, siendo mas marcada su baja durante las observaciones post-terapia. Al analizar la especificidad se encontro que la IDGA era totalmente especifica, no encontrandose falsos positivos en las poblaciones controles estudiadâs (B y C). La FC mostro un 96.7% de especificidad frente a la poblacion C y un 82% frente a la B, siendo estadistisarnente significativa la diferencia entre ambas. Basados en los datos ariteriores, se busco et valor predictivo, hallaridose que la IDGA da seguridad completa para el diagnostiso en cualquiera de los periodos evaluados no asi la FC, la que mostro disminucion de tales valores durante la terapia y la post-terapia. La curva respectiva mostro como a medida que pasaba el tiempo, disminuia la posibiildad de diferenciar la paracoccidioidomicosis (A) de otras micosis (B), manteniendose, sinembargo, la diferencia con los controles normales (C).

Fibrotic sequelae in pulmonary paracoccidioidomycosis: histopathological aspects in BALB/c mice infected with viable and non-viable Paracoccidioides brasiliensis propagules

Patients with paracoccidioidomycosis often present pulmonary fibrosis and exhibit important respiratory limitations. Based on an already established animal model, the contribution of viable and non-viable P. brasiliensis propagules to the development of fibrosis was investigated. BALB/c male mice, 4-6 weeks old were inoculated intranasally either with 4x10(6) viable conidia (Group I), or 6. 5x10(6) fragmented yeast cells (Group II). Control animals received PBS. Six mice per period were sacrificed at 24, 48, 72h (initial) and 1, 2, 4, 8, 12 and 16 weeks post-challenge (late). Paraffin embedded lungs were sectioned and stained with H&E, trichromic (Masson), reticulin and Grocot&tacute;s. During the initial period PMNs influx was important in both groups and acute inflammation involving 34% to 45% of the lungs was noticed. Later on, mononuclear cells predominated. In group I, the inflammation progressed and granulomas were formed and by the 12th week they fussed and became loose. Thick collagen I fibers were observed in 66.6% and 83.3% of the animals at 8 and 12 weeks, respectively. Collagen III, thick fibers became apparent in some animals at 4 weeks and by 12 weeks, 83% of them exhibited alterations in the organization and thickness of these elements. In group II mice, this pattern was different with stepwise decrease in the number of inflammatory foci and lack of granulomas. Although initially most animals in this group had minor alterations in thin collagen I fibers, they disappeared by the 4th week. Results indicate that tissue response to fragmented yeast cells was transitory while viable conidia evoked a progressive inflammatory reaction leading to granuloma formation and to excess production and/or disarrangement of collagens I and III; the latter led to fibrosis.

A 32-Kilodalton Hydrolase Plays an Important Role in Paracoccidioides brasiliensis Adherence to Host Cells and Influences Pathogenicity

ABSTRACT 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.

Production of pro-inflammatory cytokines during the early stages of experimental Paracoccidioides brasiliensis infection

Pro-inflammatory cytokines play an important role in both recruitment and activation of leukocytes migrating into tissues in response to invading pathogens. In this study the production of pro-inflammatory cytokines, determined by ELISA assays, and the recruitment of leukocytes into the lungs of BALB/c mice infected with Paracoccidioides brasiliensis conidia were evaluated during the early stages of infection. The results showed that infected mice had a significant increase in leukocytes in the lung during the first 4 days with a peak at day 2 post-challenge; infiltrates were composed mainly of polymorphonuclear neutrophils (PMN). Pro-inflammatory cytokines such as tumour necrosis factor alpha (TNF-alpha), interleukin (IL) 6, IL-1beta and macrophage inflammatory protein (MIP) 2 were produced at elevated levels during the first 4 days post-challenge, but only in pulmonary samples and not in sera. Additionally, during the early stages of infection, overall weight loss was recorded in infected mice. These results suggest that pro-inflammatory cytokines could be responsible for the recruitment of leukocytes into the lung during the early stages of P. brasiliensis infection. In addition, both pro-inflammatory cytokine production and leukocyte recruitment may participate in the control of infection by influencing the organization of the immune response in the host exposed to P. brasiliensis conidia.