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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. Source


Hernandez J.M.,Pontifical Bolivarian University | Munoz-Cadavid C.O.,Medical and Experimental Mycology Group | Gonzalez A.,Medical and Experimental Mycology Group | Gonzalez A.,University of Antioquia
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. Source


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. Source


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. Source


Munoz C.,Medical and Experimental Mycology Group | Zuluaga A.,Medical and Experimental Mycology Group | Restrepo A.,Medical and Experimental Mycology Group | Tobon A.,Medical and Experimental Mycology Group | And 4 more authors.
Diagnostic Microbiology and Infectious Disease | Year: 2012

A total of 98 respiratory specimens from 88 patients suspected of having Pneumocystis jirovecii pneumonia (PcP) were evaluated using a previously reported nested polymerase chain reaction (PCR) assay for mitochondrial large subunit rRNA (mtLSUrRNA). In addition, samples from patients with other pulmonary infections and a sizeable DNA collection from other fungal pathogens were studied. A panfungal PCR assay amplifying the ITS1-ITS2 regions were also used to identify all fungal DNAs. All samples positive for mtLSUrRNA-PCR were evaluated to determine mutations in dihydropteroate synthase (DHPS) and dihydrofolate reductase (DHFR) genes. All PCR-amplified products were sequenced. Of the 98 clinical specimens, 13 (13.2%) were positive by GMS stain and mtLSUrRNA-PCR, while 32 (32.6%) that were GMS stain-negative gave positive results with mtLSUrRNA-PCR. All the sequences corresponding to the 45 products amplified by mtLSUrRNA-PCR showed 99% or greater identity with P. jirovecii. The mtLSUrRNA-PCR exhibited 86% sensitivity and 98% and 96.6% specificity when results were compared to those corresponding to negative controls and other proven clinical entities, respectively. We found mutations in the DHPS gene in 3 (7.7%) patients, 2 located at codon 55 and 1 at codon 57. One patient showed a synonymous substitution at nucleotide position 312 in the DHFR gene. These results suggest that mtLSUrRNA-PCR is a useful test for diagnosing PcP. In contrast to other studies, this study found a low prevalence of mutations in the DHPS and DHFR genes in Colombian patients. © 2012 Elsevier Inc.. Source

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