Specific depletion of myeloid-derived suppressor cells by the chemotherapeutic agent 5-Fluorouracil enhances protective immune response in Paracoccidioidomycosis.

BACKGROUND: Paracoccidioidomycosis (PCM), a systemic mycosis in Latin America, is regulated by suppressive mechanisms mediated by tolerogenic plasmacytoid-dendritic-cells and regulatory T-cells. Our recent studies revealed that myeloid-derived suppressor cells (MDSCs), are important mediators in PCM. Their suppressive activity on Th1/Th17 immunity was shown to be mediated by inhibitory effect of IL-10, IDO-1 and PD-L1. Studies revealed the chemotherapeutic drug 5-Fluorouracil (5-FU) as a selective MDSC apoptosis-inducing agent, but its in vivo effect on infectious processes remains poorly investigated. METHODS: MDSCs and other leukocytes were evaluated in the lungs of 5-FU-treated mice after four, six, and eight weeks of P. brasiliensis infection. Disease severity and immunological response were evaluated in MDSCs-depleted. RESULTS: 5-FU treatment caused a significant reduction of pulmonary MDSCs and fungal loads. The specific depletion of MDSCs by 5-FU reduced all pulmonary CD4+ T-cell populations (Th1, Th2, Th17, and Treg) resulting in improved tissue pathology and increased survival rates. Importantly, this reduction was concomitant with increased frequencies of Th1/Th17 cells and the increased levels of Th1/Th2/Th17 cytokines in the lungs and liver of treated mice suggesting an early and efficient protective effect of these cells. Furthermore, the immuneprotection conferred by the specific depletion of MDSCs by 5FU treatment could be reversed by the adoptive transfer of MDSCs. CONCLUSIONS: 5-FU treatment depletes lung-MDSCs of P. brasiliensis-infected mice resulting in enhanced immunity. The protective effect of 5-FU treatment in pulmonary PCM suggests that the specific depletion of MDSCs can be viewed as a potential immunotherapeutic tool for PCM.

MDSCs use a complex molecular network to suppress T-cell immunity in a pulmonary model of fungal infection.

Background: Paracoccidioidomycosis (PCM) is a systemic endemic fungal disease prevalent in Latin America. Previous studies revealed that host immunity against PCM is tightly regulated by several suppressive mechanisms mediated by tolerogenic plasmacytoid dendritic cells, the enzyme 2,3 indoleamine dioxygenase (IDO-1), regulatory T-cells (Tregs), and through the recruitment and activation of myeloid-derived suppressor cells (MDSCs). We have recently shown that Dectin-1, TLR2, and TLR4 signaling influence the IDO-1-mediated suppression caused by MDSCs. However, the contribution of these receptors in the production of important immunosuppressive molecules used by MDSCs has not yet been explored in pulmonary PCM. Methods: We evaluated the expression of PD-L1, IL-10, as well as nitrotyrosine by MDSCs after anti-Dectin-1, anti-TLR2, and anti-TLR4 antibody treatment followed by P. brasiliensis yeasts challenge in vitro. We also investigated the influence of PD-L1, IL-10, and nitrotyrosine in the suppressive activity of lung-infiltrating MDSCs of C57BL/6-WT, Dectin-1KO, TLR2KO, and TLR4KO mice after in vivo fungal infection. The suppressive activity of MDSCs was evaluated in cocultures of isolated MDSCs with activated T-cells. Results: A reduced expression of IL-10 and nitrotyrosine was observed after in vitro anti-Dectin-1 treatment of MDSCs challenged with fungal cells. This finding was further confirmed in vitro and in vivo by using Dectin-1KO mice. Furthermore, MDSCs derived from Dectin-1KO mice showed a significantly reduced immunosuppressive activity on the proliferation of CD4+ and CD8+ T lymphocytes. Blocking of TLR2 and TLR4 by mAbs and using MDSCs from TLR2KO and TLR4KO mice also reduced the production of suppressive molecules induced by fungal challenge. In vitro, MDSCs from TLR4KO mice presented a reduced suppressive capacity over the proliferation of CD4+ T-cells. Conclusion: We showed that the pathogen recognition receptors (PRRs) Dectin-1, TLR2, and TLR4 contribute to the suppressive activity of MDSCs by inducing the expression of several immunosuppressive molecules such as PD-L1, IL-10, and nitrotyrosine. This is the first demonstration of a complex network of PRRs signaling in the induction of several suppressive molecules by MDSCs and its contribution to the immunosuppressive mechanisms that control immunity and severity of pulmonary PCM.

An improved high-performance liquid chromatography (HPLC) method for detection of variations in the hydroxyproline content of tissue homogenates from Paracoccidioides brasiliensis-infected mice.

Background: Paracoccidioidomycosis (PCM) is a severe granulomatous disease. The hallmark of this mycosis is fibrin degradation and granuloma formation as a result of a wound-healing process in the context of excessive inflammation. Therefore, as the content of collagen can be assessed by the methodology described in this manuscript, we propose that the content of hydroxyproline (HYP) be employed as a new and efficient measurement of granulomatous lesions developed. To estimate the level of HYP the major byproduct of the degradation process, we hypothesized that this simple and efficient technique could serve as a marker of disease severity. Methods: Five B10.A female mice were infected with P. brasiliensis and, after 15 days, the omentum was removed, subjected to histopathological analysis or processed (i.e. deproteinized and derivatized), and further analyzed on a reverse phase HPLC using a C-18 column. The omentum of five uninfected controls was also collected and similarly analyzed. Results: Infected mice showed numerous, disseminated paracoccidioidomycotic lesions, as well as marked collagen deposits, as observed in histopathologic analysis, and high levels of HYP. Normal uninfected mice showed no granulomas, little or no deposits of collagen fibers, and very low levels of HYP, as evaluated by HPLC. Our results show that the disease intensity as evaluated number and the morphology of the granulomatous lesions were correlated to the HYP levels using small tissue samples from the omentum, the main target organ of P. brasiliensis. Conclusions: Here we propose an alternative methodology to follow disease evolution and, to some extent, fungal load in experimental P. brasiliensis infection and suggest its usefulness to other diseases with pronounced fibrin degradation.

Celecoxib exhibits antifungal effect against Paracoccidioides brasiliensis both directly and indirectly by activating neutrophil responses.

BACKGROUND: Celecoxib, an anti-inflammatory drug, combined therapies using antimicrobials and immune modulator drugs are being studied. OBJECTIVE: To assess whether Celecoxib has direct in vitro antifungal effect against the Paracoccidioides brasiliensis, the causative agent of Paracoccidioidomycosis-(PCM) and also if it improves the in vivo activity of neutrophils-(PMN) in an experimental murine subcutaneous-(air pouch) model of the disease. METHODS: The antifungal activity of Celecoxib(6 mg/mL) on P. brasiliensis-(Pb18) was evaluated using the microdilution technique. Splenocytes co-cultured with Pb18 and treated with Celecoxib(6 mg/mL) were co-cultured for 24, 48 and 72-hours. Swiss mice were inoculated with Pb18 and treated with Celecoxib(6 mg/kg) in the subcutaneous air pouch. Neutrophils were collected from the air pouch. Mitochondrial activity, reactive oxygen production, catalase, peroxidase, cytokines and chemokines, nitrogen species, total protein, microbicidal activity of PMNs and viable Pb18 cells numbers were analyzed. RESULTS: Celecoxib had no cytotoxic effect on splenocytes co-cultured with Pb18, but had a marked direct antifungal effect, inhibiting fungal growth both in vitro and in vivo. Celecoxib interaction with immune system cells in the air pouch, it leads to activation of PMNs, as confirmed by several parameters (mitochondrial activity, reactive oxygen species, peroxidase, KC and IL-6 increase, killing constant and phagocytosis). Celecoxib was able to reduce IL-4, IL-10 and IL-12 cytokine production. The number of recovered viable Pb18 decreased dramatically. CONCLUSIONS: This is the first report of the direct antifungal activity of Celecoxib against P. brasiliensis. The use of Celecoxib opens a new possibility for future treatment of PCM.

Impact of COVID-19 on paracoccidioidomycosis. Which was the most influential: The pandemic or the virus?

The impact of COVID-19 on paracoccidioidomycosis (PCM) in Argentina and the consequences generated by the pandemic are discussed. From 2018 to 3 years after the pandemic declaration, 285 proven PCM patients were registered. No association between both diseases was documented. PCM frequency decreased to extremely low levels in 2020. Mandatory social isolation and the emotional and psychological effects generated under pandemic circumstances led to delays in diagnosis, severe disseminated cases, and other challenges for diagnosis in subsequent years. Probable underdiagnosis should be considered due to the overlap of clinical manifestations, the low index of suspicion and the lack of sensitive diagnostic tools.

Clinical response to prednisone in a severe case of infant-juvenile paracoccidioidomycosis.

BACKGROUND: Paracoccidioidomycosis is a neglected tropical disease caused by fungi of the genus Paracoccidioides. A wide range of symptoms is related to the disease; however, lungs and skin are the sites predominantly affected. The disease is mostly seen in people living in rural areas in Latin America. CASE REPORT: We present a pediatric case of severe disseminated paracoccidioidomycosis that slowly responded to the antifungal treatment. Within three months, symptoms evolved into hepatosplenomegaly, necrotic cervical and abdominal lymph nodes, and splenic abscess. Clinical response to amphotericin B deoxycholate and itraconazole was slow, resulting in pleural and peritoneal cavity effusions, heart failure and shock. Amphotericin B deoxycholate was replaced by the liposomal formulation, with no response. Subsequently, prednisone was added to the treatment, which led to improvement in the clinical response. Serological Paracoccidioides antibody titers were atypical, with very low titers in the critical phase and significant increase during the convalescence phase. The infection was finally cleared up with amphotericin B deoxycholate, liposomal amphotericin B and the use of corticosteroids. Paracoccidioidomycosis serology was non-reactive two years post-discharge. CONCLUSIONS: Due to the intense inflammatory response triggered by Paracoccidioides cells, giving low-dose prednisone for a short period of time modulated the inflammatory response and supported antifungal treatment.

Autochtonal case of chronic, unifocal, pulmonary paracoccidioidomycosis with methotrexate use, in Salvador ‒ Brazil.

We report an autochthonous case of mild unifocal chronic pulmonary paracoccidioidomycosis in a 48-year-old previously healthy woman with no history of possible environmental exposures in endemic rural areas, supposedly resulting from reactivation of a latent pulmonary focus secondary to the use of methotrexate for the control of Chikungunya arthropathy. Laboratory investigation ruled out other immunosuppression. Her only symptoms were a dry cough and chest pain. Diagnosis confirmed by needle lung biopsy. There were no abnormalities on physical examination nor evidence of central nervous system involvement. MRI of the total abdomen showed no involvement of other organs. Computed chest tomography showed a favorable evolution under the use of itraconazole (200 mg/day). Different tomographic presentations findings are highlighted when performed before and after treatment. CONCLUSIONS: PCM should be considered even in a woman without a history of consistent environmental exposure and in a non-endemic geographic area.

Analysis and Interpretation of Automated Blood Count in the Treatment of Chronic Paracoccidioidomycosis.

Blood count is crucial for assessing bone marrow's cell production and differentiation during infections, gaging disease severity, and monitoring therapeutic responses. The profile of blood count in chronic forms of paracoccidioidomycosis (PCM) has been insufficiently explored. To better understand the changes in hematological cells in different stages of the PCM chronic form, we evaluated the blood count, including immature blood cells in automated equipment, before and during the treatment follow-up of 62 chronic PCM patients. Predominantly male (96.8%) with an average age of 54.3 (standard deviation SD 6.9) years, participants exhibited pre-treatment conditions such as anemia (45.2%), monocytosis (38.7%), and leukocytosis (17.7%), which became less frequent after clinical cure. Anemia was more prevalent in severe cases. Notably, hemoglobin and reticulocyte hemoglobin content increased, while leukocytes, monocytes, neutrophils, immature granulocytes, and platelets decreased. Chronic PCM induced manageable hematological abnormalities, mainly in the red blood series. Monocytosis, indicating monocytes' role in PCM's immune response, was frequent. Post-treatment, especially after achieving clinical cure, significant improvements were observed in various hematological indices, including immature granulocytes and reticulocyte hemoglobin content, underscoring the impact of infection on these parameters.

Proceedings of the second international meeting on endemic mycoses of the Americas (IMEMA) and first international symposium on implantation mycoses (ISIM).

The second international meeting on endemic mycoses of the Americas (IMEMA) and the first international symposium on implantation mycoses (ISIM) took place in Santiago del Estero, Argentina, on September 25-27, 2023. The conference provided a platform for researchers, clinicians, and experts to discuss the latest developments in the field of endemic and implantation mycoses. Topics included epidemiology, diagnostic advances, treatment strategies, and the impact of environmental factors on the spread of these fungal diseases. IMEMA and ISIM contributed to the regional discourse on the mycoses, emphasizing the importance of international cooperation in addressing these public health challenges.

IMEMA/ISIM, held in Santiago del Estero, Argentina, convened experts to discuss endemic and implantation mycoses, covering topics such as epidemiology, diagnostics, treatment, and advocacy. The event highlighted ongoing efforts in combating these diseases.

Rising rates of paracoccidioidomycosis-related hospitalizations and in-hospital deaths, Rio de Janeiro, Brazil (2010-2019).

The epidemiological dynamics of paracoccidioidomycosis (PCM) has been changing over the years. We analyzed secondary public data from the Hospital Information System of the Brazilian Unified Health System (SIH/SUS), focusing on PCM-related hospitalizations and in-hospital deaths. In the period between 2010 and 2019, 396 hospitalizations and 30 deaths were related to PCM among 7 073 334 hospitalizations registered in Rio de Janeiro. We highlight the rising rates, reflecting the increase in the number of acute forms previously reported. Urgent public health policies are essential to prevent poor outcomes related to this neglected mycosis.

Epidemiology of paracoccidioidomycosis has been changing in endemic areas. We analyzed secondary data on hospitalizations in Rio de Janeiro, an important endemic area. There is a trend on increasing rates of hospitalizations and in-hospital deaths mainly in the Metropolitan belt.

Extracellular vesicles from virulent P. brasiliensis induce TLR4 and dectin-1 expression in innate cells and promote enhanced Th1/Th17 response.

Extracellular vesicles (EVs) are membrane-enclosed nanoparticles that transport several biomolecules and are involved in important mechanisms and functions related to the pathophysiology of fungal diseases. EVs from Paracoccidioides brasiliensis, the main causative agent of Paracoccidioidomycosis (PCM), modulate the immune response of macrophages. In this study, we assessed the EVs proteome from a virulent P. brasiliensis isolated from granulomatous lesions and compared their immunomodulatory ability with EVs isolated from the fungus before the animal passage (control EVs) when challenging macrophages and dendritic cells (DCs). Proteome showed that virulent EVs have a higher abundance of virulence factors such as GP43, protein 14-3-3, GAPDH, as well as virulence factors never described in PCM, such as aspartyl aminopeptidase and a SidJ analogue compared with control EVs. Virulent extracellular vesicles induced higher expression of TLR4 and Dectin-1 than control EVs in macrophages and dendritic cells (DCs). In opposition, a lower TLR2 expression was induced by virulent EVs. Additionally, virulent EVs induced lower expression of CD80, CD86 and TNF-α, but promoted a higher expression of IL-6 and IL-10, suggesting that EVs isolated from virulent P. brasiliensis-yeast promote a milder DCs and macrophage maturation. Herein, we showed that EVs from virulent fungi stimulated a higher frequency of Th1/Tc1, Th17, and Treg cells, which gives new insights into fungal extracellular vesicles. Taken together, our results suggest that P. brasiliensis utilizes its EVs as virulence bags that manipulate the immune system in its favour, creating a milder immune response and helping with fungal evasion from the immune system.

Blocking the CTLA-4 and PD-1 pathways during pulmonary paracoccidioidomycosis improves immunity, reduces disease severity, and increases the survival of infected mice.

Immune checkpoint pathways, i.e., coinhibitory pathways expressed as feedback following immune activation, are crucial for controlling an excessive immune response. Cytotoxic T lymphocyte antigen-4 (CTLA-4) and programmed cell death protein-1 (PD-1) are the central classical checkpoint inhibitory (CPI) molecules used for the control of neoplasms and some infectious diseases, including some fungal infections. As the immunosuppression of severe paracoccidioidomycosis (PCM), a chronic granulomatous fungal disease, was shown to be associated with the expression of coinhibitory molecules, we hypothesized that the inhibition of CTLA-4 and PD-1 could have a beneficial effect on pulmonary PCM. To this end, C57BL/6 mice were infected with Paracoccidioides brasiliensis yeasts and treated with monoclonal antibodies (mAbs) α-CTLA-4, α-PD-1, control IgG, or PBS. We verified that blockade of CTLA-4 and PD-1 reduced the fungal load in the lungs and fungal dissemination to the liver and spleen and decreased the size of pulmonary lesions, resulting in increased survival of mice. Compared with PBS-treated infected mice, significantly increased levels of many pro- and anti-inflammatory cytokines were observed in the lungs of α-CTLA-4-treated mice, but a drastic reduction in the liver was observed following PD-1 blockade. In the lungs of α-CPI and IgG-treated mice, there were no changes in the frequency of inflammatory leukocytes, but a significant reduction in the total number of these cells was observed. Compared with PBS-treated controls, α-CPI- and IgG-treated mice exhibited reduced pulmonary infiltration of several myeloid cell subpopulations and decreased expression of costimulatory molecules. In addition, a decreased number of CD4+ and CD8+ T cells but sustained numbers of Th1, Th2, and Th17 T cells were detected. An expressive reduction in several Treg subpopulations and their maturation and suppressive molecules, in addition to reduced numbers of Treg, TCD4+, and TCD8+ cells expressing costimulatory and coinhibitory molecules of immunity, were also detected. The novel cellular and humoral profiles established in the lungs of α-CTLA-4 and α-PD-1-treated mice but not in control IgG-treated mice were more efficient at controlling fungal growth and dissemination without causing increased tissue pathology due to excessive inflammation. This is the first study demonstrating the efficacy of CPI blockade in the treatment of pulmonary PCM, and further studies combining the use of immunotherapy with antifungal drugs are encouraged.

The Effect of Geoclimatic Factors on the Distribution of Paracoccidioidomycosis in Mato Grosso do Sul, Brazil.

The incidence of paracoccidioidomycosis (PCM) varies in Latin America, and it is influenced by environmental factors. This study evaluated the distribution of PCM acute/subacute form (AF) cases and their correlation with geoclimatic factors in the Mato Grosso do Sul (MS) state. The study included 81 patients diagnosed with the PCM/AF at the University Hospital of the Federal University of Mato Grosso do Sul between January 1980 and February 2022. Geographic coordinates, health microregion of patient's residence, compensated average temperature, relative air humidity (RH), El Niño Southern Oscillation (ENSO), and average global temperature were analyzed. The highest incidence was observed in the Aquidauana (7/100,000 inhabitants), while Campo Grande, the state's capital, had the highest number (n = 34; 42.4%) and density (4.4 cases/km2) of cases. The number of cases increased during extended periods of the El Niño phenomenon. A positive correlation was found between higher RH and PCM/AF cases. Most PCM/AF cases were found in areas with loamy soils and RH ranging from 60.8 to 73.6%. In MS, the health microregions of PCM/AF patients are characterized by deforestation for agricultural and pasture use, coupled with loamy soils and specific climatic phenomena leading to higher soil humidity.

Paracoccidioidomycosis after eyebrow micropigmentation: Unusual reactivation or a new form of transmission by cutaneous inoculation?

We report a case of unusual paracoccidioidomycosis reactivation after eyebrow micropigmentation in a Brazilian patient. The cutaneous lesion was the only clinical manifestation. Direct cutaneous inoculation in dermal tissues with Paracoccidioides sp. is extremely rare, explaining why paracoccidioidomycosis is not classically considered a cutaneous implantation mycosis.

Estimated burden of fungal infections in Panama.

Data published on Panamanian fungal disease are scarce, mostly case reports. To date, there is no paper that compiles the burden of fungal disease Here we estimate for the first time the incidence and prevalence of fungal diseases in Panama. Data on fungal disease were obtained from different search engines: PubMed, Google Scholar, Scielo and Lilacs. For population and at risk diseases, we used statistics from worldometer, UNAIDS, and WHO. Incidence, prevalence, and absolute numbers were calculated based on the population at risk. Panamanian population in 2022 was 4,429,739. We estimated that 85,530 (1.93 %) people suffer from fungal diseases. The most frequent fungal infection was recurrent Candida vaginitis (3285/100,000). There are 31,000 HIV-infected people in Panama and based on the number of cases not receiving anti-retroviral therapy (14,570), and previous reports of prevalence of opportunistic infections, we estimated annual incidences of 4.0/100,000 for cryptococcal meningitis, 29.5/100,000 for oral candidiasis, 23.1/100,000 for esophageal candidiasis, 29.5/100,000 for Pneumocystis pneumonia, 15.1/100,000, and for histoplasmosis. For chronic pulmonary aspergillosis (CPA) and fungal asthma we used data from Guatemala and Colombia to estimate COPD and asthma prevalence and WHO report for tuberculosis. We estimated annual incidences of 6.1/100,000 for invasive aspergillosis and prevalence of 31.5/100,000 for CPA, 60.2/100,000 for allergic bronchopulmonary aspergillosis, and 79.5/100,000 for severe asthma with fungal sensitisation. Other incidence estimates were 5.0/100,000 for candidaemia, 0.20/100,000 for mucormycosis, and 4.97/100,000 for fungal keratitis. Even though this report on burden of fungal disease is a forward step, more epidemiological studies to validate these estimates are needed.

Standardization of Semi-Quantitative Dot Blotting Assay-Application in the Diagnosis, Follow-Up, and Relapse of Paracoccidioidomycosis.

INTRODUCTION: This study standardized a semi-quantitative dot blotting assay (DB) and a quantitative real-time polymerase chain reaction (qPCR) to detect specific antibodies for Paracoccidioides brasiliensis and its DNA in PCM patients. METHODOLOGY: We evaluated 42 confirmed PCM patients upon admission using a serological double agar gel immunodiffusion test (DID), DB, and molecular tests (qPCR in total blood). The control groups included 42 healthy individuals and 37 patients with other infectious diseases. The serological progress during treatment was evaluated in eight patients, and there was a relapse diagnosis in ten patients using the Pb B.339 strain antigen. The cut-off points for the serological tests were determined by a receiver operator characteristic curve. RESULTS: The DB and DID tests showed similar accuracy, but the DB identified lower antibody concentrations. Cross-reactions were absent in the DB assay. In the relapse diagnoses, DB exhibited much higher sensitivity (90%) than DID (30%). CONCLUSIONS: A DB assay is easier and faster than a DID test to be performed; DB and DID tests show the same accuracy, while blood qPCR is not recommended in the diagnosis at the time of admission; cross-reactions were not observed with other systemic diseases; DB and DID tests are useful for treatment monitoring PCM patients; and a DB assay is the choice for diagnosing relapse. These findings support the introduction of semi-quantitative DB assays in clinical laboratories.

Rapid Classification of Serum from Patients with Paracoccidioidomycosis Using Infrared Spectroscopy, Univariate Statistics, and Linear Discriminant Analysis (LDA).

Paracoccidioidomycosis (PCM) is a systemic mycosis that is diagnosed by visualizing the fungus in clinical samples or by other methods, like serological techniques. However, all PCM diagnostic methods have limitations. The aim of this study was to develop a diagnostic tool for PCM based on Fourier transform infrared (FTIR) spectroscopy. A total of 224 serum samples were included: 132 from PCM patients and 92 constituting the control group (50 from healthy blood donors and 42 from patients with other systemic mycoses). Samples were analyzed by attenuated total reflection (ATR) and a t-test was performed to find differences in the spectra of the two groups. The wavenumbers that had p < 0.05 had their diagnostic potential evaluated using receiver operating characteristic (ROC) curves. The spectral region with the lowest p value was used for variable selection through principal component analysis (PCA). The selected variables were used in a linear discriminant analysis (LDA). In univariate analysis, the ROC curves with the best performance were obtained in the region 1551-1095 cm-1. The wavenumber that had the highest AUC value was 1264 cm-1, achieving a sensitivity of 97.73%, specificity of 76.01%, and accuracy of 94.22%. The total separation of groups was obtained in the PCA performed with a spectral range of 1551-1095 cm-1. LDA performed with the eight wavenumbers with the greatest weight from the group discrimination in the PCA obtained 100% accuracy. The methodology proposed here is simple, fast, and highly accurate, proving its potential to be applied in the diagnosis of PCM. The proposed method is more accurate than the currently known diagnostic methods, which is particularly relevant for a neglected tropical mycosis such as paracoccidioidomycosis.

Molecular detection of Paracoccidioides spp. in environmental samples from the Southwestern Amazon.

Paracoccidioidomycosis is an infection with the potential for environmental dissemination, especially in regions of hot and humid climate, where human cases have been recorded in the Southwestern Amazon of Brazil, specifically in the state of Acre. Despite studies providing information about the presence of these fungi in soil and animal samples, such as armadillos, further investigations are still needed to determine the epidemiological distribution of the genus Paracoccidioides. The aim of this study was to detect the occurrence of Paracoccidioides fungi in the Southwestern Amazon. To achieve this, 60 soil samples were collected from armadillo burrows on rural properties in the in the municipalities of Acrelândia, Bujari, Plácido de Castro, Rio Branco, Sena Madureira, and Senador Guiomard, located in the state of Acre, Brazil. Fungal DNA was extracted from these samples using the DNEASY® PowerSoil kit-Quiagen, followed by Nested PCR technique with ITS4 and ITS5 as external primers, and PBITS-E and PBITS-R as internal primers. DNA amplification products of about 380 bp compatible with Paracoccidioides spp. were detected in six samples (10%), being sequenced and identified as P. brasiliensis. These findings indicate that the soils of the Acre state could be considered a potential source for Paracoccidioides spp., suggesting that local infections are likely.

Contribution to the study of inflammasome and programmed cell death in paracoccidioidomycosis oral lesions.

BACKGROUND: Paracoccidioidomycosis is the most prevalent systemic mycosis in Latin America, with a high incidence in Brazil, Colombia and Venezuela, and constitutes a serious public health problem, a frequent cause of morbidity and disability for work. Some mechanisms of cell death are described as important tools in infectious processes. When apoptosis is blocked, RIPK (Receptor-interacting protein kinase) 3 dependent, a caspase-independent form of cell death, can limit the replication and spread of pathogens. Some molecules that mediate necroptosis include RIPK3 and have been extensively studied due to their signalling mechanism and pathological function. RIPK3 activates NLRP1 and NLRP3-mediated inflammasome formation. Caspase-1 has an important role in processing the cytokines ILß and IL18 to their active form. Such molecules are part of the inflammasome characterization, whose caspase-1-dependent activation promotes the death of pyroptotic cells and the secretion of proinflammatory cytokines. Knowledge about the mechanisms of pathogen-mediated cell death can be useful for understanding of the pathogenesis of infections and inflammatory conditions. OBJECTIVE: The objective of this work was to identify the mechanisms of programmed cell death and inflammasome components in human oral mucosal lesions of paracoccidioidomycosis through immunohistochemical methods and identification of RIPK-3, IL1ß, IL18, NLRP-1 and caspase-1. Thirty specimens were included, and a histopathological analysis of the lesions was performed using haematoxylin-eosin staining. RESULTS: Our results on in situ expression of inflammasome elements and programmed cell death showed increased expression of IL-1ß, NLRP-1, caspase-1 and RIPK-3. We suggest that inflammasome complex participate in the immunopathogenesis in paracoccidioidomycosis oral lesions in an interplay with RIPK3.

Structure of glyceraldehyde-3-phosphate dehydrogenase from Paracoccidioides lutzii in complex with an aldonic sugar acid.

The pathogen Paracoccidioides lutzii (Pb01) is found in South America countries Colombia, Ecuador, Venezuela and Brazil, especially in the central, west, and north regions of the latter. It belongs to the Ajellomycetaceae family, Onygenales order, and is typically thermodimorphic, presenting yeast cells when it grows in animal tissues, but mycelia when in the environment, where it produces the infectious propagule. This fungus is one of the etiologic agents of Paracoccidioidomycosis (PCM), the most important endemic fungal infection in Latin America. Investigations on its genome have contributed to a better understanding about its metabolism and revealed the complexity of several metabolic glycolytic pathways. Glyceraldehyde-3-Phosphate Dehydrogenase from Paracoccidioides lutzii (PlGAPDH) is considered a moonlighting protein and participates in several biological processes of this pathogen. The enzyme was expressed and purified, as seen in SDS-PAGE gel, crystallized and had its three dimensional structure (3D) determined in complex with NAD+, a sulphate ion and d-galactonic acid, therefore, a type of 'GAA site'. It is the first GAPDH structure to show this chemical type in this site and how this protein can bind an acid derived from oxidation of a linear hexose.