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Hernandez O.,University of Antioquia | Hernandez O.,Cellular and Molecular Biology Unit | Almeida A.J.,University of Minho | Gonzalez A.,Medical and Experimental Mycology Group | And 8 more authors.
Infection and Immunity | Year: 2010

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. Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Naranjo T.W.,Medical and Experimental Mycology Group | Naranjo T.W.,Pontifical Bolivarian University | Lopera D.E.,Medical and Experimental Mycology Group | Diaz-Granados L.R.,Pontifical Bolivarian University | And 4 more authors.
Microbes and Infection | Year: 2010

A comparative study, based on histopathologic findings (inflammation, cellularity, and fibrosis) and immunologic parameters (pro-inflammatory and anti-inflammatory cytokines), was carried out in order to evaluate the effects of itraconazole (ITC) treatment and its starting time in a BALB/c murine model of chronic pulmonary paracoccidioidomycosis (PCM), induced by intranasal inoculation of Paracoccidioides brasiliensis (Pb) conidia. Two different groups of mice were exposed to ITC therapy beginning at the 4th or 8th week after Pb infection, respectively. ITC was administered daily, via gavage, for a period of sixty days. At weeks 0, 4, 8, 12 and 16 the animals were sacrificed and their lungs removed for histology staining with hematoxylin and eosin (H&E), Masson's trichromic and Gomori-Grocott; pulmonary levels of IL-1β, TNF-α, IFN-γ, IL-13 and TGF-β were also measured by ELISA. The development or absence of the principal pulmonary PCM sequela, lung fibrosis, was directly related to the therapy's starting time. This and other histopathologic findings were related to the behavior of cytokine levels. © 2010 Institut Pasteur.

Gonzalez A.,University of Texas at San Antonio | Gonzalez A.,Medical and Experimental Mycology Group | Gonzalez A.,University of Antioquia | Gonzalez A.,Pontifical Bolivarian University | And 2 more authors.
Microbial Pathogenesis | Year: 2011

Production of reactive oxygen species (ROS) resulting from phagocytic NADPH oxidase (NOX2) activity has been reported to contribute to host defense against numerous microbial pathogens. In this study we explored the role of NOX2 production in experimental coccidioidomycosis, a human respiratory disease caused by a soil-borne fungal pathogen. Activated and non-activated macrophages isolated from either NOX2 -/- knock-out or wild type (WT) mice showed comparable ROS production and killing efficiency in vitro when infected with parasitic cells of Coccidioides. Both mouse strains also revealed similar fungal burden in their lungs and spleen at 7 and 11 days after intranasal challenge with Coccidioides spores, although the NOX2 -/- mice died earlier than the WT strain. Immunization of the NOX2 -/- and WT mice with a live, attenuated vaccine strain of Coccidioides also resulted in comparable reduction of the fungal burden in both lungs and spleen. These combined results initially suggested that NOX2 activity and ROS production are not essential for protection against Coccidioides infection. However, the reduced survival of non-vaccinated NOX2 -/- mice correlated with high, sustained numbers of lung-infiltrated neutrophils on days 7 and 11 postchallenge, an expansion of the regulatory T cell population in infected lungs in the knock-out mice, and elevated concentrations of pro-inflammatory cytokines and chemokines in lung homogenates compared to infected WT mice. Although NOX2-derived ROS appeared to be dispensable for both innate and acquired immunity to pulmonary Coccidioides infection, evidence is presented that NOX2 production plays a role in limiting pathogenic inflammation in this murine model of coccidioidomycosis. © 2011 Elsevier Ltd.

Lopera D.,Medical and Experimental Mycology Group | Naranjo T.W.,Medical and Experimental Mycology Group | Naranjo T.W.,University of Medellín | Cruz O.G.,Programa de Computacao Cientifica | And 5 more authors.
PLoS Neglected Tropical Diseases | Year: 2011

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-1β, IL-4, IL-5, IL-6, IL-10, IL12p70, IL-13, IL-17, Eotaxin, G-CSF, MCP1, MIP1α, GM-CSF, IFN-γ, MIP1β 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,MIP1β 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. © 2011 Lopera et al.

Hernandez J.M.,Pontifical Bolivarian University | Munoz-Cadavid C.O.,Medical and Experimental Mycology Group | Hernndez D.L.,Ophthalmology Clinic San Diego | Montoya C.,Ophthalmology Clinic San Diego | And 2 more authors.
Medical Mycology | Year: 2012

Ocular histoplasmosis syndrome (OHS) is a significant cause of vision loss in young and middle-aged adults. We report here a case of an immunocompetent 37-year-old man who presented fever, malaise, headache, and anterior cervical lymphadenopathy for one week, after which he started to experience a sudden loss in visual acuity of his right eye. Fluorescent angiography and an optical coherent tomography demonstrated the presence of a type II choroidal neo-vascular membrane in the right eye, suggesting a diagnosis of OHS. A peripheral blood sample was tested by nested PCR to detect Histoplasma capsulatum using a set of primers known to amplify a DNA sequence coding for a specific 100-kDa protein of this fungus (Hc100-PCR). The blood sample was Hc100-PCR-positive and sequence analysis showed an identity of 97% with the reference sequence. The patient received intravitreal bevacizumab injection and itraconazol therapy, leading to an improvement in media vision acuity. In this case, the molecular test provided evidence linking the ocular lesions with an earlier infection by H. capsulatum and demonstrated that the Hc100-nested PCR assay is a valuable tool in the diagnosis of histoplasmosis. © 2012 ISHAM.

Gonzalez A.,University of Texas at San Antonio | Gonzalez A.,Medical and Experimental Mycology Group | Gonzalez A.,University of Antioquia | Gonzalez A.,Pontifical Bolivarian University | And 2 more authors.
Microbial Pathogenesis | Year: 2011

The functions of inducible nitric oxide synthase (iNOS) activity in protection against microbial insults are still controversial. In this study, we explored the role of iNOS in protection against Coccidioides infection in mice. We observed that wild type (WT) and iNOS-/- mice showed similar percent survival and fungal burden in their lungs at days 7 and 11 after intranasal challenge with Coccidioides. Vaccinated WT and iNOS-/- mice revealed comparable fungal burden in their lungs and spleen at 7 and 11 days postchallenge. However, at 11 days the non-vaccinated, iNOS-deficient mice had significantly higher fungal burden in their spleen compared to WT mice. Additionally, higher numbers of lung-infiltrated neutrophils, macrophages and dendritic cells were observed in WT mice at day 11 postchallenge compared to iNOS-/- mice. Moreover, no difference in numbers of T, B, NK or regulatory T cells, or concentrations of selected cytokines and chemokines were detected in lungs of both mouse strains at 7 and 11 days postchallenge. Although iNOS-derived NO production appears to influence the inflammatory response and dissemination of the fungal pathogen, our results suggest that iNOS activity does not play a significant role in the control of coccidioidal infection in mice and that other, still undefined mechanisms of host protection are involved. © 2011 Elsevier Ltd.

Gonzalez A.,University of Texas at San Antonio | Gonzalez A.,Medical and Experimental Mycology Group | Gonzalez A.,University of Antioquia | Gonzalez A.,Pontifical Bolivarian University | And 2 more authors.
Microbial Pathogenesis | Year: 2011

We studied the effect of the presence of Coccidioides on the production of nitric oxide (NO) by primary macrophages previously activated by IFN-γ and LPS. The fungal cells were isolated from cultures of arthroconidia that had been incubated for 24 h in a medium that supported parasitic phase growth and were co-cultured with the macrophages. These live, first-generation parasitic cells of Coccidioides, referred to as spherule initials, suppressed NO production as well as iNOS mRNA expression by activated macrophages. Phagocytosis was not required for suppression of NO. We also showed that the culture supernatant of the spherule initials was capable of suppressing NO production, and that this activity was mediated by an as yet unidentified, secreted fungal factor(s). Heat-, paraformaldehyde- or X-ray-treated spherule initials did not show this inhibitory effect. To our surprise, macrophages obtained from iNOS-deficient mice revealed phagocytic activity and killing efficiency which were comparable to that of macrophages isolated from wild type C57BL/6 mice. Although the cultured fungal pathogen can suppress NO production, this oxidative product is apparently not essential for in vitro killing of Coccidioides by activated macrophages. Our results suggest that other unidentified fungicidal mechanisms exist against Coccidioides which are apparently independent of NO production. © 2010 Elsevier Ltd.

Arango-Bustamante K.,Medical and Experimental Mycology Group | Restrepo A.,Medical and Experimental Mycology Group | Cano L.E.,Medical and Experimental Mycology Group | Cano L.E.,University of Antioquia | And 3 more authors.
American Journal of Tropical Medicine and Hygiene | Year: 2013

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. Copyright © 2013 by The American Society of Tropical Medicine and Hygiene.

Ruiz H.O.,University of Antioquia | Ruiz H.O.,Cellular and Molecular Biology Unit | Ruiz H.O.,Major College of Antioquia | Gonzalez A.,Medical and Experimental Mycology Group | And 7 more authors.
PLoS Neglected Tropical Diseases | Year: 2011

Background: Paracoccidioides brasiliensis is a human thermal dimorphic pathogenic fungus. Survival of P. brasiliensis inside the host depends on the adaptation of this fungal pathogen to different conditions, namely oxidative stress imposed by immune cells. Aims and Methodology: In this study, we evaluated the role of alternative oxidase (AOX), an enzyme involved in the intracellular redox balancing, during host-P. brasiliensis interaction. We generated a mitotically stable P. brasiliensis AOX (PbAOX) antisense RNA (aRNA) strain with a 70% reduction in gene expression. We evaluated the relevance of PbAOX during interaction of conidia and yeast cells with IFN-γ activated alveolar macrophages and in a mouse model of infection. Additionally, we determined the fungal cell's viability and PbAOX in the presence of H2O2. Results: Interaction with IFN-γ activated alveolar macrophages induced higher levels of PbAOX gene expression in PbWt conidia than PbWt yeast cells. PbAOX-aRNA conidia and yeast cells had decreased viability after interaction with macrophages. Moreover, in a mouse model of infection, we showed that absence of wild-type levels of PbAOX in P. brasiliensis results in a reduced fungal burden in lungs at weeks 8 and 24 post-challenge and an increased survival rate. In the presence of H2O2, we observed that PbWt yeast cells increased PbAOX expression and presented a higher viability in comparison with PbAOX-aRNA yeast cells. Conclusions: These data further support the hypothesis that PbAOX is important in the fungal defense against oxidative stress imposed by immune cells and is relevant in the virulence of P. brasiliensis. © 2011 Hernandez Ruiz et al.

PubMed | Colombia Hospital La Maria, National University of Colombia, Medical and Experimental Mycology Group and University of Antioquia
Type: Journal Article | Journal: Medical mycology | Year: 2016

Histoplasmosis is an important mycosis in the Americas; and in children with no immune system abnormalities, histoplasmosis is typically a self-limited process. In contrast, in children with immune problems, disease manifestations are frequently more severe and include dissemination. From 1984 to 2010, a retrospective study of paediatric patients who had been diagnosed with histoplasmosis was performed. A total of 45 pediatric cases of histoplasmosis were identified. The most important risk factor was malnutrition (37%), followed by environmental exposure (33%). The patients exhibited pulmonary infiltrates (83%), fever (76%), cough, constitutional symptoms (38%), headache (35%), and lymph node hypertrophy (33%). Concerning the clinical forms, 64% of the patients presented with the progressive disseminated form that frequently affected the central nervous system (48%). Diagnostic laboratory tests indicated that the cultures were positive for 80% of the patients, the agar gel immunodiffusion was reactive in 95%, the M band of the precipitate was more commonly observed (81%), and the complement fixation tests were reactive in 88% of the patients. The timely diagnosis of histoplasmosis is important, and for this reason, it is hoped that the results of this study will lead pediatricians toward a better understanding of this mycosis in children.

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