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1.  The role of adjuvants in therapeutic protection against paracoccidioidomycosis after immunization with the P10 peptide 
Paracoccidioidomycosis (PCM), a common chronic mycosis in Latin America, is a granulomatous systemic disease caused by the thermo-dimorphic fungus Paracoccidioides brasiliensis. The glycoprotein gp43 is the main antigen target of P. brasiliensis and a 15-mer internal peptide (QTLIAIHTLAIRYAN), known as P10, defines a major CD4+-specific T cell epitope. Previous results have indicated that, besides having a preventive role in conventional immunizations prior to challenge with the fungus, protective anti-fungal effects can be induced in P. brasiliensis-infected mice treated with P10 administered with complete Freund’s adjuvant (CFA). The peptide elicits an IFN-γ-dependent Th1 immune response and is the main candidate for effective immunotherapy of patients with PCM, as an adjunctive approach to conventional chemotherapy. In the present study we tested the therapeutic effects of P10 combined with different adjuvants [aluminum hydroxide, CFA, flagellin, and the cationic lipid dioctadecyl-dimethylammonium bromide (DODAB)] in BALB/c mice previously infected with the P. brasiliensis Pb18 strain. Significant reductions in the number of colony forming units of the fungus were detected in lungs of mice immunized with P10 associated with the different adjuvants 52 days after infection. Mice treated with DODAB and P10, followed by mice treated with P10 and flagellin, showed the most prominent effects as demonstrated by the lowest numbers of viable yeast cells as well as reductions in granuloma formation and fibrosis. Concomitantly, secretion of IFN-γ and TNF-α, in contrast to interleukin (IL)-4 and IL-10, was enhanced in the lungs of mice immunized with P10 in combination with the tested adjuvants, with the best results observed in mice treated with P10 and DODAB. In conclusion, the present results demonstrate that the co-administration of the synthetic P10 peptide with several adjuvants, particularly DODAB, have significant therapeutic effects in experimental PCM.
doi:10.3389/fmicb.2012.00154
PMCID: PMC3343455  PMID: 22586420
Paracoccidioides brasiliensis; paracoccidioidomycosis; P10; adjuvants; dioctadecyl-dimethylammonium bromide; FliC flagellin; aluminum hydroxide; complete Freund’s adjuvant
2.  T Helper 1–Inducing Adjuvant Protects against Experimental Paracoccidioidomycosis 
Immunostimulatory therapy is a promising approach to improving the treatment of systemic fungal infections such as paracoccidioidomycosis (PCM), whose drug therapy is usually prolonged and associated with toxic side effects and relapses. The current study was undertaken to determine if the injection of a T helper (Th) 1–stimulating adjuvant in P. brasiliensis–infected mice could have a beneficial effect on the course of experimental PCM. For this purpose, mice were infected and treated with complete Freund's adjuvant (CFA), a well-established Th1 experimental inductor, or incomplete Freund's adjuvant (IFA - control group) on day 20 postinfection. Four weeks after treatment, the CFA-treated mice presented a mild infection in the lungs characterized by absence of epithelioid cell granulomas and yeast cells, whereas the control mice presented multiple sites of focal epithelioid granulomas with lymphomonocytic halos circumscribing a high number of viable and nonviable yeast cells. In addition, CFA administration induced a 2.4 log reduction (>99%) in the fungal burden when compared to the control group, and led to an improvement of immune response, reversing the immunosuppression observed in the control group. The immunotherapy with Th1-inducing adjuvant, approved to be used in humans, might be a valuable tool in the treatment of PCM and potentially useful to improve the clinical cure rate in humans.
Author Summary
P. brasiliensis is a thermally dimorphic human pathogenic fungus that causes paracoccidioidomycosis (PCM), the most prevalent human systemic mycosis in Latin America, whose drug therapy is usually prolonged and associated with toxic side effects and relapses. Although immunostimulatory therapy is a promising approach to improving the treatment of fungal infections as PCM, few studies have been reported. In the current study, we verified that a single-dose administration of an adjuvant that induces T helper (Th) 1 immune response (complete Freund's adjuvant [CFA]) in P. brasiliensis–infected mice was sufficient to break the lack of immune response to the fungus observed in infected mice. Four weeks after treatment, the CFA-treated mice presented a mild infection in the lungs characterized by preserved lung structure and small fungal burden, whereas control mice that had been treated with incomplete Freund's adjuvant presented many granulomatous lesions and high fungal burden. The immunotherapy with Th1-inducing adjuvant might be a valuable tool in the treatment of PCM and potentially useful for faster and efficient cure of PCM in humans.
doi:10.1371/journal.pntd.0000183
PMCID: PMC2263123  PMID: 18335066
3.  Synthetic Peptides Mimic gp75 from Paracoccidioides brasiliensis in the Diagnosis of Paracoccidioidomycosis 
Mycopathologia  2012;174(1):1-10.
Paracoccidioidomycosis (PCM) is a systemic granulomatous disease, endemic in Latin America, caused by the thermal dimorphic fungus Paracoccidioides brasiliensis. Although some fungal antigens have already been characterized and used for serological diagnosis, cross-reactions have been frequently observed. Thus, the examination of fungal forms in clinical specimens or isolation of P. brasiliensis by culture is still the most frequent method for the diagnosis of this mycosis. In this study, a random peptide phage display library was used to select mimotopes of P. brasiliensis, which were employed as antigens in an indirect enzyme-linked immunosorbent assay. The protective monoclonal antibody against experimental PCM (anti-gp75) was used as molecular target to screen a phage display library. That approach led to a synthetic peptide named P2, which was synthesized and tested against PCM patients’ sera to check whether it was recognized. There was significant recognition of P2 by sera of untreated PCM patients when compared with normal human sera. Sera from treated PCM group, patients with other mycosis or co-infected with HIV had much lower recognition of P2 than untreated patient group. The test showed a sensitivity of 100 and 94.59% of specificity in relation to human sera control. These data indicate a potential use of P2 as diagnostic tool in PCM. Its application for serological diagnosis of PCM may contribute to the development and standardization of simpler, faster and highly reproducible immunodiagnostic tests at low cost.
doi:10.1007/s11046-011-9518-3
PMCID: PMC3368115  PMID: 22249604
gp75; Paracoccidioides brasiliensis; Paracoccidioidomycosis; Peptides; Phage display
4.  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 3×106 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.
Author Summary
Paracoccidioidomycosis (PCM) is a fungal infection caused by the dimorphic fungus Paracoccidioides brasiliensis. It occurs preferentially in rural workers in whom the disease is severe and may cause incapacitating pulmonary sequelae. Assessment of disease progression and treatment outcome normally includes chest x-rays or CT studies. Existing experimental PCM models have focused on several aspects, but none has done a radiologic or image follow-up evaluation of pulmonary lesions considered as the fungus primary target. In this study, the lungs of mice infected with fungal conidia were studied sequentially during the chronic stage of their experimental mycosis by noninvasive high resolution medical computed tomography, and at time of sacrifice, also by histopathology to characterize pulmonary abnormalities. Three basic lung lesion patterns were revealed by both techniques: nodular-diffuse, confluent and pseudo-tumoral which were located mainly around the hilus thus accurately reflecting the situation in human patients. The experimental design of this study 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 pulmonary diseases. The findings we are reporting open new avenues for experimental research, increase our understanding of the mycosis pathogenesis and consequently have repercussions in patients' care.
doi:10.1371/journal.pntd.0000726
PMCID: PMC2894136  PMID: 20614019
5.  Serology of Paracoccidioidomycosis Due to Paracoccidioides lutzii 
Paracoccidioides lutzii is a new agent of paracoccidioidomycosis (PCM) and has its epicenter localized to the Central-West region of Brazil. Serological diagnosis of PCM caused by P. lutzii has not been established. This study aimed to develop new antigenic preparations from P. lutzii and to apply them in serological techniques to improve the diagnosis of PCM due to P. lutzii. Paracoccidioides lutzii exoantigens, cell free antigen (CFA), and a TCA-precipitated antigen were evaluated in immunodiffusion (ID) tests using a total of 89 patient sera from the Central-West region of Brazil. Seventy-two sera were defined as reactive for P. brasiliensis using traditional antigens (AgPbB339 and gp43). Non-reactive sera for traditional antigens (n = 17) were tested with different P. lutzii preparations and P. lutzii CFA showed 100% reactivity. ELISA was found to be a very useful test to titer anti-P. lutzii antibodies using P. lutzii-CFA preparations. Sera from patients with PCM due to P. lutzii presented with higher antibody titers than PCM due to P. brasiliensis and heterologous sera. In western blot, sera from patients with PCM due to P. lutzii were able to recognize antigenic molecules from the P. lutzii-CFA antigen, but sera from patients with PCM due to P. brasiliensis could not recognize any P. lutzii molecules. Due to the facility of preparing P. lutzii CFA antigens we recommend its use in immunodiffusion tests for the diagnosis of PCM due to P. lutzii. ELISA and western blot can be used as complementary tests.
Author Summary
Tropical diseases, such as paracoccidioidomycosis, are the most common type of neglected diseases. From 1980 to 1995, 3,181 deaths from paracoccidioidomycosis occurred in Brazil, representing the eighth most common cause of death from predominantly chronic or recurrent types of infectious and parasitic diseases, showing considerable magnitude and low visibility. Paracoccidioidomycosis is traditionally assumed to be caused solely by Paracoccidioides brasiliensis, but a new species, Paracoccidioides lutzii, was discovered in the Central-Western region of Brazil. Thus, new antigenic preparations and tests for an accurate differential diagnosis between these two species appear to be needed. This study aimed to develop new antigenic preparations from P. lutzii isolates to improve the diagnosis of paracoccidioidomycosis. We used patient serum samples predominantly from the Central-Western region of Brazil. Various antigenic preparations were tested, and a cell free antigen derived from P. lutzii was an excellent antigen for serological diagnosis and able to diagnose 100% of sera from patients with PCM due to P. lutzii.
doi:10.1371/journal.pntd.0002986
PMCID: PMC4102441  PMID: 25032829
6.  Therapeutic DNA Vaccine Encoding Peptide P10 against Experimental Paracoccidioidomycosis 
Paracoccidioidomycosis (PCM), caused by Paracoccidioides brasiliensis, is the most prevalent invasive fungal disease in South America. Systemic mycoses are the 10th most common cause of death among infectious diseases in Brazil and PCM is responsible for more than 50% of deaths due to fungal infections. PCM is typically treated with sulfonamides, amphotericin B or azoles, although complete eradication of the fungus may not occur and relapsing disease is frequently reported. A 15-mer peptide from the major diagnostic antigen gp43, named P10, can induce a strong T-CD4+ helper-1 immune response in mice. The TEPITOPE algorithm and experimental data have confirmed that most HLA-DR molecules can present P10, which suggests that P10 is a candidate antigen for a PCM vaccine. In the current work, the therapeutic efficacy of plasmid immunization with P10 and/or IL-12 inserts was tested in murine models of PCM. When given prior to or after infection with P. brasiliensis virulent Pb 18 isolate, plasmid-vaccination with P10 and/or IL-12 inserts successfully reduced the fungal burden in lungs of infected mice. In fact, intramuscular administration of a combination of plasmids expressing P10 and IL-12 given weekly for one month, followed by single injections every month for 3 months restored normal lung architecture and eradicated the fungus in mice that were infected one month prior to treatment. The data indicate that immunization with these plasmids is a powerful procedure for prevention and treatment of experimental PCM, with the perspective of being also effective in human patients.
Author Summary
Paracoccidioidomycosis (PCM) is the predominant systemic mycosis in Latin America causing half of the total deaths among systemic fungal infectious diseases in Brazil. Chemotherapy is the standard treatment, but the long time required, severe cases of immunosuppression and frequent relapses indicate that additional methods should be introduced such as immunotherapy combined with antifungal drugs. Previously, the protective activity of P10, a peptide derived from the major diagnostic antigen gp43, was demonstrated, alone or combined with chemotherapy. P10 elicited a vigorous IFN-γ mediated Th-1 immune response. Presently, the reduction of fungal load, and even sterilization, was attempted using a specific DNA vaccine encoding P10. Plasmid pcDNA3 expression vector with P10 insert was tested as a vaccine in intratracheally infected BALB/c and B10.A mice. Our results showed that vaccination with pP10 induced a significant reduction of the fungal burden in the lung. Co-vaccination of pP10 with a plasmid encoding mouse IL-12 proved to be even more effective in the elimination of the fungus with virtual sterilization in a long term infection and treatment assay system. The data suggest that immunization with these plasmids, without the need of an adjuvant, could be used in the prevention and treatment of PCM in human patients.
doi:10.1371/journal.pntd.0001519
PMCID: PMC3289603  PMID: 22389734
7.  Additive Effect of rPb27 Immunization and Chemotherapy in Experimental Paracoccidioidomycosis 
PLoS ONE  2011;6(3):e17885.
Paracoccidioidomycosis, PCM, the major systemic mycosis in Latin America, is caused by the termally dimorphic fungus Paracoccidioides brasiliensis and requires extended periods of chemotherapy with a significant frequency of relapsing disease. The search for new alternatives of treatment is necessary. rPb27 is an antigenic protein from P. brasiliensis that already showed a significant protective activity as a vaccine for PCM in experimental models. The cDNA of rPb27 was subcloned into a pET-DEST 42 plasmid, expressed in E. coli with a his-tag and purified by affinity chromatography. Immunization with this recombinant protein and chemotherapy were used together in an attempt to improve treatment of PCM. For this, BALB/c mice were challenged with pathogenic P. brasiliensis strain and after immunized with rPb27, in the presence of Corynebacterium parvum and Al(OH)3, some groups were also treated with fluconazole. After 40 days of treatment, the combined drug/rPb27 administration controlled PCM in the liver and spleen, with long lasting protection, and largely preserved tissues structures of these organs. Additionally, in the lungs after 40 days of treatment there was a significant reduction in the fungal load and size of lesions. At the same time, the levels of TNF-α were higher than infected-only mice. Moreover, significant levels of anti-rPb27 specific IgG1, IgG2a and IgG2b isotypes were detected in the sera of mice immunized with rPb27 fluconazole treated or not. These results showed an additive protective effect of rPb27 immunization and chemotherapy, suggesting that an rPb27-based vaccine can be used to enhance PCM antifungal treatment.
doi:10.1371/journal.pone.0017885
PMCID: PMC3053394  PMID: 21423771
8.  Characterization of gp70 and Anti-gp70 Monoclonal Antibodies in Paracoccidioides brasiliensis Pathogenesis  
Infection and Immunity  2003;71(11):6534-6542.
Paracoccidioidomycosis (PCM) is a systemic granulomatous mycosis whose agent is Paracoccidioides brasiliensis. In the culture supernatant, the fungus expresses glycoproteins of from 13 to 148 kDa. A cell surface glycoprotein of 43 kDa is the major antigenic component of P. brasiliensis. Another expressed glycoprotein, gp70, is recognized by 96% of sera from PCM patients and is able to induce lymphoproliferation. Since, little is known about this glycoprotein, we produced monoclonal antibodies (MAbs) against gp70 to isolate the molecule from total fungus extracts and to investigate its possible role in the pathogenesis of PCM. Using these MAbs, it was observed by confocal microscopy that gp70 is located mainly in the intracellular compartment of the fungus, although it was also detected in the culture supernatant. Based on observations showing that gp43 has a down-regulatory effect on mouse peritoneal macrophages, we tested the effects of gp70 on their phagocytic ability. Purified gp70 was able to inhibit the activity of macrophages through the mannose receptors and also through the Fc receptors; the latter effect was not observed with gp43. gp70 inhibits NO and H2O2 liberation by peritoneal macrophages in vitro, as does gp43. Results obtained with gp43 led us to hypothesize that gp70 could act as an escape mechanism for fungal establishment in primary infections. To corroborate this hypothesis, we analyzed the effect of passive immunization of mice during infection with P. brasiliensis using anti-gp70 MAbs. This treatment almost completely abolished granuloma formation in the lungs, suggesting that the protein facilitates fungal establishment and progression of lesions in primary infection.
doi:10.1128/IAI.71.11.6534-6542.2003
PMCID: PMC219562  PMID: 14573675
9.  Therapeutic Administration of Recombinant Paracoccin Confers Protection against Paracoccidioides brasiliensis Infection: Involvement of TLRs 
Background
Paracoccin (PCN) is an N-acetylglucosamine-binding lectin from the human pathogenic fungus Paracoccidioides brasiliensis. Recombinant PCN (rPCN) induces a T helper (Th) 1 immune response when prophylactically administered to BALB/c mice, protecting them against subsequent challenge with P. brasiliensis. In this study, we investigated the therapeutic effect of rPCN in experimental paracoccidioidomycosis (PCM) and the mechanism accounting for its beneficial action.
Methodology/Principal Findings
Four distinct regimens of rPCN administration were assayed to identify which was the most protective, relative to vehicle administration. In all rPCN-treated mice, pulmonary granulomas were less numerous and more compact. Moreover, fewer colony-forming units were recovered from the lungs of rPCN-treated mice. Although all therapeutic regimens of rPCN were protective, maximal efficacy was obtained with two subcutaneous injections of 0.5 µg rPCN at 3 and 10 days after infection. The rPCN treatment was also associated with higher pulmonary levels of IL-12, IFN-γ, TNF-α, nitric oxide (NO), and IL-10, without IL-4 augmentation. Encouraged by the pulmonary cytokine profile of treated mice and by the fact that in vitro rPCN-stimulated macrophages released high levels of IL-12, we investigated the interaction of rPCN with Toll-like receptors (TLRs). Using a reporter assay in transfected HEK293T cells, we verified that rPCN activated TLR2 and TLR4. The activation occurred independently of TLR2 heterodimerization with TLR1 or TLR6 and did not require the presence of the CD14 or CD36 co-receptors. The interaction between rPCN and TLR2 depended on carbohydrate recognition because it was affected by mutation of the receptor's N-glycosylation sites. The fourth TLR2 N-glycan was especially critical for the rPCN-TLR2 interaction.
Conclusions/Significance
Based on our results, we propose that PCN acts as a TLR agonist. PCN binds to N-glycans on TLRs, triggers regulated Th1 immunity, and exerts a therapeutic effect against P. brasiliensis infection.
Author Summary
Paracoccidioides brasiliensis is a pathogenic fungus that causes paracoccidioidomycosis (PCM) in humans, a debilitating fungal infection that mainly affects the lungs and is widespread in Latin America. Paracoccin (PCN) is a sugar-binding protein produced by this fungus. Previous studies have shown that PCN contributes to the colonization of host tissues by the fungus and induces the production of inflammatory factors (i.e., cytokines and nitric oxide) by immune cells such as macrophages. Here we investigated the therapeutic efficacy of recombinant PCN (rPCN) on the course of P. brasiliensis infection in mice. Histopathological analysis of lungs of animals treated with rPCN showed much lower inflammation in comparison to untreated, control mice. In addition, fewer infective P. brasiliensis yeast forms were recovered from the lung of rPCN-treated animals than from that of control animals. Administration of rPCN was associated with a profile of pro- and anti-inflammatory factors in the lung that was conducive to host protection. These effects were associated with PCN binding to sugar chains linked to innate immunity receptors, namely Toll-like receptors 2 and 4. These findings reveal a mechanism by which rPCN confers protection against PCM.
doi:10.1371/journal.pntd.0003317
PMCID: PMC4256291  PMID: 25474158
10.  In Pulmonary Paracoccidioidomycosis IL-10 Deficiency Leads to Increased Immunity and Regressive Infection without Enhancing Tissue Pathology 
Background
Cellular immunity is the main defense mechanism in paracoccidioidomycosis (PCM), the most important systemic mycosis in Latin America. Th1 immunity and IFN-γ activated macrophages are fundamental to immunoprotection that is antagonized by IL-10, an anti-inflammatory cytokine. Both in human and experimental PCM, several evidences indicate that the suppressive effect of IL-10 causes detrimental effects to infected hosts. Because direct studies have not been performed, this study was aimed to characterize the function of IL-10 in pulmonary PCM.
Methodology/Principal Findings
Wild type (WT) and IL-10−/− C57BL/6 mice were used to characterize the role of IL-10 in the innate and adaptive immunity against Paracoccidioides brasiliensis (Pb) infection. We verified that Pb-infected peritoneal macrophages from IL-10−/− mice presented higher phagocytic and fungicidal activities than WT macrophages, and these activities were associated with elevated production of IFN-γ, TNF-α, nitric oxide (NO) and MCP-1. For in vivo studies, IL-10−/− and WT mice were i.t. infected with 1×106 Pb yeasts and studied at several post-infection periods. Compared to WT mice, IL-10−/− mice showed increased resistance to P. brasiliensis infection as determined by the progressive control of pulmonary fungal loads and total clearance of fungal cells from dissemination organs. This behavior was accompanied by enhanced delayed-type hypersensitivity reactions, precocious humoral immunity and controlled tissue pathology resulting in increased survival times. In addition, IL-10−/− mice developed precocious T cell immunity mediated by increased numbers of lung infiltrating effector/memory CD4+ and CD8+ T cells. The inflammatory reactions and the production of Th1/Th2/Th17 cytokines were reduced at late phases of infection, paralleling the regressive infection of IL-10−/− mice.
Conclusions/Significance
Our work demonstrates for the first time that IL-10 plays a detrimental effect to pulmonary PCM due to its suppressive effect on the innate and adaptive immunity resulting in progressive infection and precocious mortality of infected hosts.
Author Summary
Paracoccidioidomycosis, the most important deep mycosis from Latin America, is acquired by inhalation of fungal spores. The pulmonary infection can remain as a quiescent infection or evolve to overt, life-threatening disease. Immunoprotection is mainly mediated by Th1 lymphocytes secreting IFN- γ, the most important macrophage activating cytokine. It is well established that the severe forms of infection are associated with elevated production of anti-inflammatory or suppressive cytokines such as IL-10. However, direct approaches investigating the role of this cytokine in pulmonary paracoccidioidomycosis were never employed. This led us to investigate the innate and adaptive aspects of immunity in pulmonary paracoccidioidomycosis using IL-10-deficient mice in comparison with their IL-10-normal counterparts. We verified that IL-10 absence leads to a regressive disease, resulting in reduced mortality rates of infected mice. This better disease outcome was associated with precocious and enhanced mechanisms of innate and adaptive immunity that allow the control of fungal growth without excessive inflammatory reactions and harmful tissue pathology. These evidences on the detrimental effects of IL-10 to pulmonary paracoccidioidomycosis suggest that therapeutic measures aimed to control IL-10 production or activity could exert a protective effect to this severe fungal pathology.
doi:10.1371/journal.pntd.0002512
PMCID: PMC3812093  PMID: 24205424
11.  Paracoccidioides brasiliensis Vaccine Formulations Based on the gp43-Derived P10 Sequence and the Salmonella enterica FliC Flagellin▿  
Infection and Immunity  2009;77(4):1700-1707.
Paracoccidioidomycosis (PCM) is a systemic granulomatous disease caused by the dimorphic fungus Paracoccidioides brasiliensis. Anti-PCM vaccine formulations based on the secreted fungal cell wall protein (gp43) or the derived P10 sequence containing a CD4+ T-cell-specific epitope have shown promising results. In the present study, we evaluated new anti-PCM vaccine formulations based on the intranasal administration of P. brasiliensis gp43 or the P10 peptide in combination with the Salmonella enterica FliC flagellin, an innate immunity agonist binding specifically to the Toll-like receptor 5, in a murine model. BALB/c mice immunized with gp43 developed high-specific-serum immunoglobulin G1 responses and enhanced interleukin-4 (IL-4) and IL-10 levels. On the other hand, mice immunized with recombinant purified flagellins genetically fused with P10 at the central hypervariable domain, either flanked or not by two lysine residues, or the synthetic P10 peptide admixed with purified FliC elicited a prevailing Th1-type immune response based on lung cell-secreted type 1 cytokines. Mice immunized with gp43 and FliC and intratracheally challenged with P. brasiliensis yeast cells had increased fungal proliferation and lung tissue damage. In contrast, mice immunized with the chimeric flagellins and particularly those immunized with P10 admixed with FliC reduced P. brasiliensis growth and lung damage. Altogether, these results indicate that S. enterica FliC flagellin modulates the immune response to P. brasiliensis P10 antigen and represents a promising alternative for the generation of anti-PCM vaccines.
doi:10.1128/IAI.01470-08
PMCID: PMC2663153  PMID: 19204092
12.  The Pathogenic Fungus Paracoccidioides brasiliensis Exports Extracellular Vesicles Containing Highly Immunogenic α-Galactosyl Epitopes▿ 
Eukaryotic Cell  2011;10(3):343-351.
Exosome-like vesicles containing virulence factors, enzymes, and antigens have recently been characterized in fungal pathogens, such as Cryptococcus neoformans and Histoplasma capsulatum. Here, we describe extracellular vesicles carrying highly immunogenic α-linked galactopyranosyl (α-Gal) epitopes in Paracoccidioides brasiliensis. P. brasiliensis is a dimorphic fungus that causes human paracoccidioidomycosis (PCM). For vesicle preparations, cell-free supernatant fluids from yeast cells cultivated in Ham's defined medium-glucose were concentrated in an Amicon ultrafiltration system and ultracentrifuged at 100,000 × g. P. brasiliensis antigens were present in preparations from phylogenetically distinct isolates Pb18 and Pb3, as observed in immunoblots revealed with sera from PCM patients. In an enzyme-linked immunosorbent assay (ELISA), vesicle components containing α-Gal epitopes reacted strongly with anti-α-Gal antibodies isolated from both Chagas' disease and PCM patients, with Marasmius oreades agglutinin (MOA) (a lectin that recognizes terminal α-Gal), but only faintly with natural anti-α-Gal. Reactivity was inhibited after treatment with α-galactosidase. Vesicle preparations analyzed by electron microscopy showed vesicular structures of 20 to 200 nm that were labeled both on the surface and in the lumen with MOA. In P. brasiliensis cells, components carrying α-Gal epitopes were found distributed on the cell wall, following a punctuated confocal pattern, and inside large intracellular vacuoles. Lipid-free vesicle fractions reacted with anti-α-Gal in ELISA only when not digested with α-galactosidase, while reactivity with glycoproteins was reduced after β-elimination, which is indicative of partial O-linked chain localization. Our findings open new areas to explore in terms of host-parasite relationships in PCM and the role played in vivo by vesicle components and α-galactosyl epitopes.
doi:10.1128/EC.00227-10
PMCID: PMC3067469  PMID: 21216942
13.  Expression of Antibodies Directed to Paracoccidioides brasiliensis Glycosphingolipids during the Course of Paracoccidioidomycosis Treatment▿  
Clinical and Vaccine Immunology  2006;14(2):150-156.
Paracoccidioidomycosis (PCM) is a granulomatous disease caused by the dimorphic fungus Paracoccidioides brasiliensis. The immunoglobulin classes and isotypes of antibodies directed to acidic glycosphingolipids (GSLs) and glucosylceramide of P. brasiliensis were determined by enzyme-linked immunosorbent assay of sera from 31 PCM patients. The reactivities of 38 serum samples were analyzed by considering the stage of treatment: before antifungal treatment (n = 10), during 1 to 4 months of treatment (T1-4; n = 9), during 5 to 12 months of treatment (T5-12; n = 9), and posttreatment (PT; n = 10). Sera from healthy subjects (n = 12) were used as controls. Only the GSL Pb-1 antigen, which presents the carbohydrate structure Galfβ1-6(Manα1-3)Manβ1, was reactive with the PCM patient sera. The PCM patient sera did not react with Pb-2, which lacks the Galf residue and which is considered the biosynthetic precursor of Pb-1, indicating that the Galf residue is essential for antibody reactivity. The Pb-1 glycolipid from nontreated patients elicited a primary immune response with immunoglobulin M (IgM) production and subsequent switching to IgG1 production. The IgG1 titer increased after the start of antifungal treatment (T1-4 group), and general decreases in the anti-Pb-1 antibody titers were observed after 5 months of treatment (T5-12 and PT groups). The Pb-1 antigen, an acidic GSL with terminal Galf residue, has potential application as an elicitor of the host immune response in patients with PCM.
doi:10.1128/CVI.00285-06
PMCID: PMC1797792  PMID: 17135452
14.  Genome Update of the Dimorphic Human Pathogenic Fungi Causing Paracoccidioidomycosis 
Paracoccidiodomycosis (PCM) is a clinically important fungal disease that can acquire serious systemic forms and is caused by the thermodimorphic fungal Paracoccidioides spp. PCM is a tropical disease that is endemic in Latin America, where up to ten million people are infected; 80% of reported cases occur in Brazil, followed by Colombia and Venezuela. To enable genomic studies and to better characterize the pathogenesis of this dimorphic fungus, two reference strains of P. brasiliensis (Pb03, Pb18) and one strain of P. lutzii (Pb01) were sequenced [1]. While the initial draft assemblies were accurate in large scale structure and had high overall base quality, the sequences had frequent small scale defects such as poor quality stretches, unknown bases (N's), and artifactual deletions or nucleotide duplications, all of which caused larger scale errors in predicted gene structures. Since assembly consensus errors can now be addressed using next generation sequencing (NGS) in combination with recent methods allowing systematic assembly improvement, we re-sequenced the three reference strains of Paracoccidioides spp. using Illumina technology. We utilized the high sequencing depth to re-evaluate and improve the original assemblies generated from Sanger sequence reads, and obtained more complete and accurate reference assemblies. The new assemblies led to improved transcript predictions for the vast majority of genes of these reference strains, and often substantially corrected gene structures. These include several genes that are central to virulence or expressed during the pathogenic yeast stage in Paracoccidioides and other fungi, such as HSP90, RYP1-3, BAD1, catalase B, alpha-1,3-glucan synthase and the beta glucan synthase target gene FKS1. The improvement and validation of these reference sequences will now allow more accurate genome-based analyses. To our knowledge, this is one of the first reports of a fully automated and quality-assessed upgrade of a genome assembly and annotation for a non-model fungus.
Author Summary
The fungal genus Paracoccidioides is the causal agent of paracoccidioidomycosis (PCM), a neglected tropical disease that is endemic in several countries of South America. Paracoccidioides is a pathogenic dimorphic fungus that is capable of converting to a virulent yeast form after inhalation by the host. Therefore the molecular biology of the switch to the yeast phase is of particular interest for understanding the virulence of this and other human pathogenic fungi, and ultimately for reducing the morbidity and mortality caused by such fungal infections. We here present the strategy and methods we used to update and improve accuracy of three reference genome sequences of Paracoccidioides spp. utilizing state-of-the-art Illumina re-sequencing, assembly improvement, re-annotation, and quality assessment. The resulting improved genome resource should be of wide use not solely for advancing research on the genetics and molecular biology of Paracoccidioides and the closely related pathogenic species Histoplasma and Blastomyces, but also for fungal diagnostics based on sequencing or molecular assays, characterizing rapidly changing proteins that may be involved in virulence, SNP-based population analyses and other tasks that require high sequence accuracy. The genome update and underlying strategy and methods also serve as a proof of principle that could encourage similar improvements of other draft genomes.
doi:10.1371/journal.pntd.0003348
PMCID: PMC4256289  PMID: 25474325
15.  Transcriptional and Proteomic Responses to Carbon Starvation in Paracoccidioides 
Background
The genus Paracoccidioides comprises human thermal dimorphic fungi, which cause paracoccidioidomycosis (PCM), an important mycosis in Latin America. Adaptation to environmental conditions is key to fungal survival during human host infection. The adaptability of carbon metabolism is a vital fitness attribute during pathogenesis.
Methodology/Principal Findings
The fungal pathogen Paracoccidioides spp. is exposed to numerous adverse conditions, such as nutrient deprivation, in the human host. In this study, a comprehensive response of Paracoccidioides, Pb01, under carbon starvation was investigated using high-resolution transcriptomic (RNAseq) and proteomic (NanoUPLC-MSE) approaches. A total of 1,063 transcripts and 421 proteins were differentially regulated, providing a global view of metabolic reprogramming during carbon starvation. The main changes were those related to cells shifting to gluconeogenesis and ethanol production, supported by the degradation of amino acids and fatty acids and by the modulation of the glyoxylate and tricarboxylic cycles. This proposed carbon flow hypothesis was supported by gene and protein expression profiles assessed using qRT-PCR and western blot analysis, respectively, as well as using enzymatic, cell dry weight and fungus-macrophage interaction assays. The carbon source provides a survival advantage to Paracoccidioides inside macrophages.
Conclusions/Significance
For a complete understanding of the physiological processes in an organism, the integration of approaches addressing different levels of regulation is important. To the best of our knowledge, this report presents the first description of the responses of Paracoccidioides spp. to host-like conditions using large-scale expression approaches. The alternative metabolic pathways that could be adopted by the organism during carbon starvation can be important for a better understanding of the fungal adaptation to the host, because systems for detecting and responding to carbon sources play a major role in adaptation and persistence in the host niche.
Author Summary
The species of the Paracoccidioides genus, a neglected human pathogen, represent the causative agents of paracoccidioidomycosis (PCM), one of the most frequent systemic mycoses in Latin America. Despite being phagocytosed, the fungus conidia differentiate into the parasitic yeast form that subverts the normally harsh intraphagosomal environment and survives and replicates into murine and human macrophages. It has been suggested that alternative carbon metabolism plays a role in the survival and virulence of Paracoccidioides spp. within host cells. We used large-scale transcriptome and proteome approaches to better characterize the responses of Paracoccidioides, Pb01, yeast parasitic cells, to carbon starvation. We aimed to identify important molecules used by the fungus to adapt to these hostile conditions. The shift to a starvation mode, including gluconeogenesis and ethanol increases, activation of fatty acids, and amino acid degradation are the strategies used by the pathogen to persist under this stress. Our study provides a detailed map of Paracoccidioides spp. responses under carbon starvation conditions and contributes to further investigations of the importance of alternative carbon adaptation during fungus pathogenesis.
doi:10.1371/journal.pntd.0002855
PMCID: PMC4014450  PMID: 24811072
16.  Characterization of PbPga1, an Antigenic GPI-Protein in the Pathogenic Fungus Paracoccidioides brasiliensis 
PLoS ONE  2012;7(9):e44792.
Paracoccidioides brasiliensis is the etiologic agent of paracoccidioidomycosis (PCM), one of the most prevalent mycosis in Latin America. P. brasiliensis cell wall components interact with host cells and influence the pathogenesis of PCM. Cell wall components, such as glycosylphosphatidylinositol (GPI)-proteins play a critical role in cell adhesion and host tissue invasion. Although the importance of GPI-proteins in the pathogenesis of other medically important fungi is recognized, little is known about their function in P. brasiliensis cells and PCM pathogenesis. We cloned the PbPga1 gene that codifies for a predicted GPI-anchored glycoprotein from the dimorphic pathogenic fungus P. brasiliensis. PbPga1 is conserved in Eurotiomycetes fungi and encodes for a protein with potential glycosylation sites in a serine/threonine-rich region, a signal peptide and a putative glycosylphosphatidylinositol attachment signal sequence. Specific chicken anti-rPbPga1 antibody localized PbPga1 on the yeast cell surface at the septum between the mother cell and the bud with stronger staining of the bud. The exposure of murine peritoneal macrophages to rPbPga1 induces TNF-α release and nitric oxide (NO) production by macrophages. Furthermore, the presence of O-glycosylation sites was demonstrated by β-elimination under ammonium hydroxide treatment of rPbPga1. Finally, sera from PCM patients recognized rPbPga1 by Western blotting indicating the presence of specific antibodies against rPbPga1. In conclusion, our findings suggest that the PbPga1gene codifies for a cell surface glycoprotein, probably attached to a GPI-anchor, which may play a role in P. brasiliensis cell wall morphogenesis and infection. The induction of inflammatory mediators released by rPbPga1 and the reactivity of PCM patient sera toward rPbPga1 imply that the protein favors the innate mechanisms of defense and induces humoral immunity during P. brasiliensis infection.
doi:10.1371/journal.pone.0044792
PMCID: PMC3443090  PMID: 23024763
17.  Immunological response to cell-free antigens of Paracoccidioides brasiliensis: relationship with clinical forms of paracoccidioidomycosis. 
Journal of Clinical Microbiology  1993;31(3):671-676.
Sera from patients with the acute (AF) and chronic (CF) forms of paracoccidioidomycosis (PCM) were tested against Paracoccidioides brasiliensis cell-free antigens by Western blot (immunoblot). The CFA preparation contained components ranging in molecular mass from 18 to 102 kDa. The immunoglobulin G (IgG) reactivity profiles were similar for patients with both forms of the disease, and the 43-kDa component was recognized by 100% of the sera. IgM antibodies from the AF- and the CF-PCM sera recognized 21 and 20 components, respectively, the AF-PCM sera reacting preferentially with components with molecular masses above 50 kDa. None of the AF-PCM sera (IgM) reacted with the 43-kDa component, and only 10% of the CF-PCM sera recognized this molecule. The IgA response was more significant in the CF-PCM group than in the AF-PCM group, and the 43- and 74-kDa components were the most reactive ones (about 40% each). Our results showed that the cell-free antigen preparation is very appropriate for the immunoblotting analysis of PCM sera, and they also showed that the detection of IgG anti-gp43 is the best marker for the diagnosis and the following up of patients with the acute or the chronic form of the disease.
Images
PMCID: PMC262839  PMID: 8458961
18.  NLRP3 Inflammasome Activation by Paracoccidioides brasiliensis 
Paracoccidioides brasiliensis is the etiologic agent of paracoccidioidomycosis (PCM), the most prevalent systemic mycosis that is geographically confined to Latin America. The pro-inflammatory cytokine IL-1β that is mainly derived from the activation of the cytoplasmic multiprotein complex inflammasome is an essential host factor against opportunistic fungal infections; however, its role in infection with a primary fungal pathogen, such as P. brasiliensis, is not well understood. In this study, we found that murine bone marrow-derived dendritic cells responded to P. brasiliensis yeast cells infection by releasing IL-1β in a spleen tyrosine kinase (Syk), caspase-1 and NOD-like receptor (NLR) family member NLRP3 dependent manner. In addition, P. brasiliensis-induced NLRP3 inflammasome activation was dependent on potassium (K+) efflux, reactive oxygen species production, phagolysosomal acidification and cathepsin B release. Finally, using mice lacking the IL-1 receptor, we demonstrated that IL-1β signaling has an important role in killing P. brasiliensis by murine macrophages. Altogether, our results demonstrate that the NLRP3 inflammasome senses and responds to P. brasiliensis yeast cells infection and plays an important role in host defense against this fungus.
Author Summary
Paracoccidioidomycosis is a systemic disease that has an important mortality and morbidity impact in Latin America. It mainly affects rural workers of Argentina, Colombia, Venezuela and Brazil. Upon host infection, one of the most important aspects that contribute to the disease outcome is the initial interaction of the Paracoccidioides brasiliensis fungus with the phagocytic cells and the induction of the inflammatory process. Among several inflammatory mediators, the cytokine interleukin-1β is of pivotal importance in this complex process. Here, we demonstrate that P. brasiliensis is sensed by the NLRP3 inflammasome, a cytoplasmatic multiprotein complex that lead to the processing and secretion of IL-1β. In addition, we described the intracellular perturbations that may be associated with NLRP3 activation such as potassium efflux, production of reactive oxygen species, and lysosomal damage. Finally, our work provides evidence for the protective role of IL-1β during fungal infection of murine macrophages.
doi:10.1371/journal.pntd.0002595
PMCID: PMC3855149  PMID: 24340123
19.  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-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.
Author Summary
Paracoccidioidomycosis (PCM), an endemic fungal infection of pulmonary origin resulting in severe disseminated disease, occurs in rural areas of most South American countries and presents several clinical forms. The infection is acquired by inhalation of specific fungal propagules, called conidia. Considering the difficulties encountered when studying the infection in humans, this work was done in mice infected by inhalation of infective fungal conidia thus mimicking the human natural infection. The lungs of mice were sequentially studied by histopathological and multiplex cytokine methods from 2 h to 16 weeks after infection to verify the course of the disease. The mycosis presented different morphologic aspects during the course of time, affecting several pulmonary compartments. Otherwise and based on the analysis of 30 cytokines, the immune response also showed heterogeneous responses, which were up or down regulated depending on the time of infection. By recognizing the different stages that correspond to the evolution of pulmonary lesions, the severity (benign, chronic or fibrotic) of the disease could be predicted and the probable prognosis of the illness be inferred.
doi:10.1371/journal.pntd.0001232
PMCID: PMC3134433  PMID: 21765962
20.  Comparative Genomic Analysis of Human Fungal Pathogens Causing Paracoccidioidomycosis 
PLoS Genetics  2011;7(10):e1002345.
Paracoccidioides is a fungal pathogen and the cause of paracoccidioidomycosis, a health-threatening human systemic mycosis endemic to Latin America. Infection by Paracoccidioides, a dimorphic fungus in the order Onygenales, is coupled with a thermally regulated transition from a soil-dwelling filamentous form to a yeast-like pathogenic form. To better understand the genetic basis of growth and pathogenicity in Paracoccidioides, we sequenced the genomes of two strains of Paracoccidioides brasiliensis (Pb03 and Pb18) and one strain of Paracoccidioides lutzii (Pb01). These genomes range in size from 29.1 Mb to 32.9 Mb and encode 7,610 to 8,130 genes. To enable genetic studies, we mapped 94% of the P. brasiliensis Pb18 assembly onto five chromosomes. We characterized gene family content across Onygenales and related fungi, and within Paracoccidioides we found expansions of the fungal-specific kinase family FunK1. Additionally, the Onygenales have lost many genes involved in carbohydrate metabolism and fewer genes involved in protein metabolism, resulting in a higher ratio of proteases to carbohydrate active enzymes in the Onygenales than their relatives. To determine if gene content correlated with growth on different substrates, we screened the non-pathogenic onygenale Uncinocarpus reesii, which has orthologs for 91% of Paracoccidioides metabolic genes, for growth on 190 carbon sources. U. reesii showed growth on a limited range of carbohydrates, primarily basic plant sugars and cell wall components; this suggests that Onygenales, including dimorphic fungi, can degrade cellulosic plant material in the soil. In addition, U. reesii grew on gelatin and a wide range of dipeptides and amino acids, indicating a preference for proteinaceous growth substrates over carbohydrates, which may enable these fungi to also degrade animal biomass. These capabilities for degrading plant and animal substrates suggest a duality in lifestyle that could enable pathogenic species of Onygenales to transfer from soil to animal hosts.
Author Summary
Paracoccidioides sp. are fungal pathogens that cause paracoccidioidomycosis in humans. They are part of a larger group of dimorphic fungi causing pulmonary infections in immunocompetent people, whereas many other fungi cause opportunistic infections. We sequenced the genomes of two strains of Paracoccidioides brasiliensis and one strain of the closely related species Paracoccidioides lutzii, and compared them to other fungal genomes. We found gene family expansions specific to Paracoccidioides, including the fungal-specific kinase family. By contrast we found that dimorphic fungi as a group have lost many genes involved in carbohydrate metabolism but retained most proteases. As the growth substrates for dimorphic fungi have not been well characterized, we tested a non-pathogenic relative, Uncinocarpus reesii, for growth on 190 carbon sources. We found that U. reesii is capable of growth on a limited set of carbohydrates, but grows more rapidly on a wide range of dipeptides and amino acids. Our analysis suggests that this genetic and phenotypic preference may underlie the ability of the dimorphic fungi to infect and grow on animal hosts.
doi:10.1371/journal.pgen.1002345
PMCID: PMC3203195  PMID: 22046142
21.  TNF-α and CD8+ T Cells Mediate the Beneficial Effects of Nitric Oxide Synthase-2 Deficiency in Pulmonary Paracoccidioidomycosis 
Background
Nitric oxide (NO), a key antimicrobial molecule, was previously shown to exert a dual role in paracoccidioidomycosis, an endemic fungal infection in Latin America. In the intravenous and peritoneal models of infection, NO production was associated with efficient fungal clearance but also with non-organized granulomatous lesions. Because paracoccidioidomycosis is a pulmonary infection, we aimed to characterize the role of NO in a pulmonary model of infection.
Methodology/Principal Findings
C57Bl/6 wild type (WT) and iNOS−/− mice were i.t. infected with 1×106 Paracoccidioides brasiliensis yeasts and studied at several post-infection periods. Unexpectedly, at week 2 of infection, iNOS−/− mice showed decreased pulmonary fungal burdens associated with an M2-like macrophage profile, which expressed high levels of TGF-β impaired ability of ingesting fungal cells. This early decreased fungal loads were concomitant with increased DTH reactions, enhanced TNF-α synthesis and intense migration of activated macrophages, CD4+ and CD8+ T cells into the lungs. By week 10, iNOS−/− mice showed increased fungal burdens circumscribed, however, by compact granulomas containing elevated numbers of activated CD4+ T cells. Importantly, the enhanced immunological reactivity of iNOS−/− mice resulted in decreased mortality rates. In both mouse strains, depletion of TNF-α led to non-organized lesions and excessive influx of inflammatory cells into the lungs, but only the iNOS−/− mice showed increased mortality rates. In addition, depletion of CD8+ cells abolished the increased migration of inflammatory cells and decreased the number of TNF-α and IFN-γ CD4+ and CD8+ T cells into the lungs of iNOS−/− mice.
Conclusions/Significance
Our study demonstrated that NO plays a deleterious role in pulmonary paracoccidioidomycosis due to its suppressive action on TNF-α production, T cell immunity and organization of lesions resulting in precocious mortality of mice. It was also revealed that uncontrolled fungal growth can be overcome by an efficient immune response.
Author Summary
Paracoccidiodomycosis is a human systemic mycosis endemic in Latin America that has a wide spectrum of manifestations ranging from localized to fatal disseminated forms. Both in humans and experimental models, immunoprotection is mediated by T cell immunity whereas immunosuppression is associated with the severe forms of the disease. The literature shows that nitric oxide (NO) produced by the enzyme nitric oxide synthase-2 (NOS2 or iNOS) is the major fungicidal component of phagocytic cells. The role of NO production was previously investigated in the intra-peritoneal and intravenous murine models of Paracoccidioides brasiliensis infection. The human paracoccidioidomycosis is believed to be acquired by the respiratory route, thus our study aimed to characterize the role of NO production in a pulmonary model of infection. We verified that, paradoxically, absence of NO production by iNOS- deficient mice resulted in less severe disease and increased survival times. This was associated with increased development of cellular immunity and enhanced synthesis of TNF-α which enhances cell migration to the site of infection and contributes to the better organization of lesions. Our work highlighted the deleterious effect of excessive NO production in pulmonary paracoccidioidomycosis, and demonstrated that uncontrolled fungal growth can be overridden by an efficient immune response.
doi:10.1371/journal.pntd.0002325
PMCID: PMC3731220  PMID: 23936574
22.  Indoleamine 2,3-Dioxygenase Controls Fungal Loads and Immunity in Paracoccidioidomicosis but is More Important to Susceptible than Resistant Hosts 
Background
Paracoccidioidomycosis, a primary fungal infection restricted to Latin America, is acquired by inhalation of fungal particles. The immunoregulatory mechanisms that control the severe and mild forms of paracoccidioidomycosis are still unclear. Indoleamine 2,3-dioxygenase (IDO), an IFN-γ induced enzyme that catalyzes tryptophan metabolism, can control host-pathogen interaction by inhibiting pathogen growth, T cell immunity and tissue inflammation.
Methodology/Principal Findings
In this study, we investigated the role of IDO in pulmonary paracoccidioidomycosis of susceptible and resistant mice. IDO was blocked by 1-methyl-dl-tryptophan (1MT), and fungal infection studied in vitro and in vivo. Paracoccidioides brasiliensis infection was more severe in 1MT treated than untreated macrophages of resistant and susceptible mice, concurrently with decreased production of kynurenines and IDO mRNA. Similar results were observed in the pulmonary infection. Independent of the host genetic pattern, IDO inhibition reduced fungal clearance but enhanced T cell immunity. The early IDO inhibition resulted in increased differentiation of dendritic and Th17 cells, accompanied by reduced responses of Th1 and Treg cells. Despite these equivalent biological effects, only in susceptible mice the temporary IDO blockade caused sustained fungal growth, increased tissue pathology and mortality rates. In contrast, resistant mice were able to recover the transitory IDO blockade by the late control of fungal burdens without enhanced tissue pathology.
Conclusions/Significance
Our studies demonstrate for the first time that in pulmonary paracoccidioidomycosis, IDO is an important immunoregulatory enzyme that promotes fungal clearance and inhibits T cell immunity and inflammation, with prominent importance to susceptible hosts. In fact, only in the susceptible background IDO inhibition resulted in uncontrolled tissue pathology and mortality rates. Our findings open new perspectives to understand the immunopathology of paracoccidioidomycosis, and suggest that an insufficient IDO activity could be associated with the severe cases of human PCM characterized by inefficient fungal clearance and excessive inflammation.
Author Summary
Immunoprotection to paracoccidiodomycosis, a systemic mycosis endemic in Latin America, is mediated by T cell immunity whereas immunosuppression characterizes the severe forms of the disease. Indoleamine 2,3-dioxygenase (IDO), an enzyme mainly induced by IFN-γ, catabolizes tryptophan along the kynurenines pathway. Tryptophan deficiency has been associated with reduced pathogen growth, while elevated levels of kynurenines with suppressed immune responses. In this study, the role of IDO in pulmonary paracoccidioidomycosis was investigated using resistant and susceptible mice. In both mouse strains, IDO blockade by 1-methyl tryptophan resulted in inefficient fungal clearance accompanied by enhanced T cell immunity. Despite these equivalent biological effects, only in susceptible mice IDO inhibition caused progressive fungal growth and tissue pathology resulting in increased mortality. Our findings demonstrate for the first time that IDO exert a yet unexplored immunoregulatory role in pulmonary paracoccidioidomycosis that can be particularly important in the severe cases of the disease.
doi:10.1371/journal.pntd.0003330
PMCID: PMC4238999  PMID: 25411790
23.  Detection of Paracoccidioides brasiliensis gp70 Circulating Antigen and Follow-Up of Patients Undergoing Antimycotic Therapy 
Journal of Clinical Microbiology  2004;42(10):4480-4486.
Paracoccidioidomycosis (PCM), one of the most important systemic mycoses in Central and South America, is caused by the dimorphic fungus Paracoccidioides brasiliensis and has a high prevalence in Brazil. Glycoproteins of 43 and 70 kDa are the main antigenic compounds of P. brasiliensis and are recognized by Western blotting by 100 and 96% of PCM patient sera, respectively. In the present study, an inhibition enzyme-linked immunosorbent assay (ELISA) was used to detect gp70 in different biological samples from patients with PCM. gp70 was detected in 98.76% of 81 serum samples, with an average concentration of 8.19 μg/ml. The test was positive for 100% of the patients with the acute and chronic unifocal forms of PCM and 98.43% of the patients with the multifocal chronic form, with average concentrations of 11.86, 4.83, and 7.87 μg/ml, respectively. Bronchoalveolar lavage fluid from 23 patients with pulmonary unifocal PCM and 14 samples of cerebrospinal fluid from patients with neurological PCM were also tested for gp70 detection, with the test showing 100% sensitivity and 100% specificity, with mean gp70 concentrations of 7.5 and 6.78 μg/ml, respectively. To investigate the potential of gp70 detection by inhibition ELISA for the follow-up of PCM patients during antimycotic therapy with itraconazole (ITZ), the sera of 23 patients presenting with the chronic multifocal form of PCM were monitored at regular intervals of 1 month for 12 months. The results showed a decrease in circulating gp70 levels during treatment which paralleled the reduction in anti-P. brasiliensis antibody levels. The detection of P. brasiliensis gp70 from the biological fluids of patients suspected of having PCM proved to be a promising method for diagnosing infection and evaluating the efficacy of ITZ treatment.
doi:10.1128/JCM.42.10.4480-4486.2004
PMCID: PMC522319  PMID: 15472297
24.  5-Lipoxygenase Activity Increases Susceptibility to Experimental Paracoccidioides brasiliensis Infection 
Infection and Immunity  2013;81(4):1256-1266.
Paracoccidioidomycosis (PCM) is a systemic mycosis caused by the thermodimorphic fungus Paracoccidioides brasiliensis. Leukotrienes and lipoxins are lipid mediators produced after 5-lipoxygenase (5-LO) activation that exhibit pro- and anti-inflammatory roles, respectively. Here, we have investigated the contribution of 5-LO enzymatic activity in PCM using an experimental model of P. brasiliensis infection. B6.129 wild-type (B6.129) and 5-LO-deficient (5-LO−/−) mice were intravenously inoculated with a virulent strain of P. brasiliensis (Pb18), and the survival rate of the infected mice was investigated on different days after yeast infection. 5-LO−/− mice exhibited an increased survival rate associated with a decreased number of CFU. The resistance of 5-LO−/− during PCM was associated with augmented nitric oxide (NO) production and the formation of compact granulomas. In addition, the absence of 5-LO was associated with a diminished number of CD4+ CD25+ regulatory T cells, higher levels of gamma interferon and interleukin-12, and increased T-bet (a T-box transcription factor that directs Th1 lineage commitment) mRNA levels in the lungs. Taken together, our results show for the first time that 5-LO enzymatic activity increases susceptibility to P. brasiliensis, suggesting that this pathway may be a potential target for therapeutic intervention during PCM.
doi:10.1128/IAI.01209-12
PMCID: PMC3639621  PMID: 23381993
25.  ArtinM offers new perspectives in the development of antifungal therapy 
The thermally dimorphic fungus Paracoccidioides brasiliensis is the causative agent of paracoccidioidomycosis (PCM), the most frequent systemic mycosis that affects the rural populations in Latin America. Despite significant developments in antifungal chemotherapy, its efficacy remains limited since drug therapy is prolonged and associated with toxic side effects and relapses. In response to these challenges, it is now recognized that several aspects of antifungal immunity can be modulated to better deal with fungal infections. A common idea for halting fungal infections has been the need to activate a cell-based, pro-inflammatory Th1 immune response to improve the fungal elimination. ArtinM, a D-mannose binding lectin from Artocarpus heterophyllus, has the property of modulating immunity against several intracellular pathogens. Here, we review the immunomodulatory activity of ArtinM during experimental PCM in mice. Both prophylactic and therapeutic protocols of ArtinM administration promotes a Th1 immune response balanced by IL-10, which outstandingly reduces the fungal load in organs of the treated mice while maintaining a controlled inflammation at the site of infection. A carbohydrate recognition-based interaction of ArtinM with Toll-like receptor 2 (TLR2) accounts for initiating the immunomodulatory effect of the lectin. The precise identification of the TLR2 N-glycan(s) targeted by ArtinM may support novel basis for the development of antifungal therapy.
doi:10.3389/fmicb.2012.00218
PMCID: PMC3375580  PMID: 22715337
Paracoccidioides brasiliensis; ArtinM; immunomodulation

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