Low-level LASER therapy accelerates fungal lesions cicatrization by increasing the production of Th1 and Th2 cytokines.

Paracoccidioidomycosis (PCM) is a systemic mycosis with serious clinical consequences in which the use of antifungal drugs requires long-term treatment. Therefore, we studied the effect of low-level LASER therapy (LLLT) to evaluate its prospects as a complementary treatment for PCM and improve the clinical response to the disease. OBJECTIVES: Our study focused on the resolution of lesions caused by fungal infection using a subcutaneous air pouch model of infection. METHODS: We evaluated cell profile and cytokines, fungi viability, and the presence of fibroblasts and fibrocytes at the site of infection. Inoculation of P. brasiliensis (Pb) was performed using a subcutaneous air pouch model and the LLLT irradiation was performed on alternate days on the rear paws of mice for 10 days, after which the cells from the air pouch were collected and analyzed. RESULTS: In animals irradiated with LLLT, the influx of cells to the air pouch was reduced, but they were more activated and produced pro-inflammatory (IL-12, IL-17 and TNF-α) and neutrophil (PMN) activating cytokines (IL-8, GM-CSF and γ-IFN). A better resolution of the infection, evidenced by the reduction in the number of viable fungi with preserved morphology in the air pouch, and an increase in the number of fibrocytes, indicating a healing profile were also observed. CONCLUSION: LLLT decreased the influx of PMN, but those presents were highly activated, with increased fungicidal activity. LLLT irradiation also resulted in earlier cicatrization at the site of infection, leading to a better outcome of the infection. These data are favorable to the use of LLLT as a complementary therapy in PCM.

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.

Role of paracoccin on Paracoccidioides brasiliensis virulence and susceptibility to antifungal drugs in the Galleria mellonella larvae model.

Paracoccin (PCN), a Paracoccidioides brasiliensis glycoprotein, has been reported to play roles in fungal biology and paracoccidioidomycosis pathogenesis. Lectin and chitinase domains account for the PCN's dual roles as an immunomodulatory agent and virulence factor. Soluble PCN injected in P. brasiliensis infected mice, by interacting with TLRs' N-glycans, drives the host immune response toward a protective Th1 axis. Otherwise, mice infection with yeasts overexpressing PCN (ov-PCN) revealed that PCN acts as a fungal virulence factor, thanks to its chitinase activity on the cell wall, resulting in resistance to phagocytes' fungicidal activity and development of severe paracoccidioidomycosis. Because antifungal drug administration follows the disease diagnosis, we studied the PCN effect on yeast resistance or susceptibility to antifungal agents. Using a paracoccidioidomycosis model developed in Galleria mellonella larvae, we confirmed the observation, in the murine host, that ov-PCN yeasts display maximum virulence compared to wild-type (wt-PCN) or PCN-silenced (kd-PCN) yeasts. PCN overexpression accounted for the highest susceptibility of P. brasiliensis to antifungal and reduced relative mRNA expression of genes encoding proteins related to cell wall remodeling. The lowest virulence, detected in infection with kd-PCN yeasts, correlated with the lowest susceptibility to antifungals and impact on genes for cell wall remodeling. So, we defined that the grade of endogenous PCN production influences the P. brasiliensis virulence and susceptibility to antifungal drugs, as well as the expression of genes related to cell wall remodeling. We postulate that this variable gene expression is mechanistically associated with P. brasiliensis virulence changes.

Virulence Vs. Immunomodulation: Roles of the Paracoccin Chitinase and Carbohydrate-Binding Sites in Paracoccidioides brasiliensis Infection.

Paracoccin (PCN) is a bifunctional protein primarily present in the cell wall of Paracoccidioides brasiliensis, a human pathogenic dimorphic fungus. PCN has one chitinase region and four potential lectin sites and acts as both a fungal virulence factor and an immunomodulator of the host response. The PCN activity on fungal virulence, mediated by the chitinase site, was discovered by infecting mice with yeast overexpressing PCN (PCN-ov). PCN-ov are characterized by increased chitin hydrolysis, a narrow cell wall, and augmented resistance to phagocytes' fungicidal activity. Compared to wild-type (wt) yeast, infection with PCN-ov yeast causes a more severe disease, which is attributed to the increased PCN chitinase activity. In turn, immunomodulation of the host response was demonstrated by injecting, subcutaneously, recombinant PCN in mice infected with wt-P. brasiliensis. Through its carbohydrate binding site, the injected recombinant PCN interacts with Toll-like receptor 2 (TLR2) and Toll-like receptor 4 (TLR4) N-glycans on macrophages, triggers M1 polarization, and stimulates protective Th1 immunity against the fungus. The PCN-treatment of wt yeast-infected mice results in mild paracoccidioidomycosis. Therefore, PCN paradoxically influences the course of murine paracoccidioidomycosis. The disease is severe when caused by yeast that overexpress endogenous PCN, which exerts a robust local chitinase activity, followed by architectural changes of the cell wall and release of low size chito-oligomers. However, the disease is mild when exogenous PCN is injected, which recognizes N-glycans on systemic macrophages resulting in immunomodulation.

Therapeutic Vaccination with Cationic Liposomes Formulated with Dioctadecyldimethylammonium and Trehalose Dibehenate (CAF01) and Peptide P10 Is Protective in Mice Infected with Paracoccidioides brasiliensis.

The peptide P10 is a vaccine candidate for Paracoccidioidomycosis, a systemic mycosis caused by fungal species of the genus Paracoccidioides spp. We have previously shown that peptide P10 vaccination, in the presence of several different adjuvants, induced a protective cellular immune response mediated by CD4+ Th1 lymphocytes that was associated with the increased production of IFN-γ in mice challenged with a virulent isolate of Paracoccidoides brasiliensis. Cationic liposomes formulated with dioctadecyldimethylammonium and trehalose dibehenate (DDA/TDB, termed also CAF01-cationic adjuvant formulation) have been developed for safe administration in humans and CAF01 liposomes are utilized as an adjuvant for modulating a robust Th1/Th17 cellular response. We evaluated the efficacy of the adsorption of peptide P10 to CAF01 cationic liposomes and used the generated liposomes to vaccinate C57Bl/6 mice infected with P. brasiliensis. Our results showed that P10 was efficiently adsorbed onto CAF01 liposomes. The vaccination of infected mice with cationic liposomes formulated with DDA/TDB 250/50 µg/mL and 20 µg of P10 induced an effective cellular immune response with increased levels of Th17 cytokines, which correlated with significant decreases in the fungal burdens in lungs and protective granulomatous tissue responses. Hence, cationic liposomes of DDA/TDB 250/50 µg/mL with 20 µg of P10 are a promising therapeutic for safely and effectively improving the treatment of paracoccidioidomycosis.

Paracoccidioidomycosis in the northern region of Rio Grande do Sul / Paracoccidioidomicose na região norte do Rio Grande do Sul / Paracoccidioidomycosis en la región norte del Rio Grande d o Sul

Background and Objectives: Paracoccidioidomycosis (PCM) is a systemic disease caused by the dimorphic fungus Paracoccidioides brasiliensis found in the tropical and subtropical regions of Latin America. This study aimed to perform a retrospective analysis of PCM cases from the northern region of Rio Grande do Sul, Brazil. Methods: A total of 200 records of PCM cases diagnosed at the local reference pathology laboratory from 1995 to 2015 were analyzed Results: Of the patients, 185 were male and 15 female. Patients ranged in age from 31 to 80 years, the largest pro portion ( being aged between 51 and 60 years and living or working in the countryside. Clinical samples were mostly obtained from the oral cavity, followed by the oropharynx, lungs, brain, skin, and prostate. Conclusion: PCM is endemic in the south o f Brazil, as the local economy is largely based on agricultural activities, favoring the contact of the population with P. brasiliensis . Due to the great similarity between PCM symptoms and other respiratory diseases, the differential diagnosis is essentia l for the correct treatment of the disease and to avoid its progression.(AU)

Justificativa e Objetivos: A paracoccidioidomicose (PCM) é uma doença sistêmica causada pelo fungo dimórfico Paracoccidioides brasiliensis , o qual é encontrado nas regiões tropicais e subtropicais da América Latina. Este estudo objetivou realizar uma análise retrospectiva dos casos de PCM na região nor te do Rio Grande do Sul, Brasil . Métodos: Foram anal isados 200 prontuários relativos aos casos de PCM de 1995 até 2015 diagnosticados pelo laboratório de patologia referência na região . Resultados: Destes pacientes, 185 eram homens e 15 mulheres. Os pacientes tinham idade variando de 31 a 80 anos, sendo que a maior proporção (35,5%) tinha entre 51 e 60 anos e viviam ou trabalhavam na zona rural Os materiais clínicos eram provenientes em sua maioria da cavidade oral, seguido da região orofaríngea, pulmão, cérebro, provenientes em sua maioria da cavidade oral, seguido da região orofaríngea, pulmão, cérebro, pele e próstata. pele e próstata. Conclusão: O sul do Brasil O sul do Brasil é região endêmica de PCM, devido a sua é região endêmica de PCM, devido a sua economia estar centrada na agricultura, o que propicia o contato do homem com o fungo. economia estar centrada na agricultura, o que propicia o contato do homem com o fungo. Devido à grande semelhança dos sintomas da PCM com outras doenças respiratórias, o Devido à grande semelhança dos sintomas da PCM com outras doenças respiratórias, o diagnóstico diferencial é relevante para que se diagnóstico diferencial é relevante para que seja realizado o tratamento correto da doença e ja realizado o tratamento correto da doença e para que seu avanço seja evitado. para que seu avanço seja evitado.(AU)

Justificación y objetivos: L a paracoccidioidomicosis (PCM) es una enfermedad sistémica c ausada por el hongo dimorfo Paracoccidioides brasiliensis , que se encuentra en las regiones tropicales y subtropicales de América Latina. Este estudio tuvo como objetivo realizar un análisis retrospectivo de casos de PCM en la región nor te de Rio Grande do Sul, Brasil . Métodos: se analizaron 200 registros médicos relacionados con casos de PCM de 1995 a 2015 diagnosticado s por el laboratorio de patología de referencia en la región . Resultados: De estos pacientes, 185 eran hombres y 15 mujeres. Los pacientes tenían edades comprendidas entre 31 y 80 años, con la mayor proporción (35.5%) entre 51 y 60 años y viviendo o trabajando en el campo. Los materiales clínicos provenían principalmente de la cavidad oral, seguidos de la región orofaríngea, pulmón, cerebro, piel y próstata. Conclusión: El sur de Brasil es una región endémica de PCM, debido a que su economía se centra en la agricultura, que proporciona el contacto del hombre con el hongo. Debido a la gran similitud de los síntomas de PCM con otras enfermedade s respiratorias, el diagnóstico diferencial es relevante para el tratamiento correcto de la enferm edad y para evitar su progreso.(AU)

Paracoccin: Purification and Validation of Its Lectin and Enzymatic Properties.

Studies on the effects of components derived from the human pathogenic fungi Paracoccidioides brasiliensis have identified paracoccin (PCN), as a bifunctional protein with lectin (GlcNAc-binding) and enzymatic (chitinase) activities, able to induce modulation of host immune response. Endogenous PCN acts as a fungal virulence factor, whereas exogenous purified PCN, administered to the host, confers protective immunity in a murine model of paracoccidioidomycosis. The immunomodulation induced by purified-PCN injection has characterized it as an agent applicable in the therapy and vaccine against paracoccidioidomycosis. This section describes methods for PCN purification and validation of its lectin and enzymatic activities. It includes detailed protocols to obtain homogeneous PCN from P. brasiliensis yeasts, as well as to purify recombinant PCN from transformed heterologous microorganisms.

A Dectin-1-Caspase-8 Pathway Licenses Canonical Caspase-1 Inflammasome Activation and Interleukin-1ß Release in Response to a Pathogenic Fungus.

Background: Paracoccidioides brasiliensis is equipped with an arsenal of virulence factors that are crucial for causing infection. Our group previously defined the NLRP3 inflammasome as a mediator of P brasiliensis-induced cell damage recognition and induction of effective Th1 immune responses. However, deficiency of caspase-1 only partially reduced interleukin (IL)-1ß levels. Methods: In this study, using chemical inhibitors as well as genetically modified mice, we identify an additional pathway for IL-1ß production in response to P brasiliensis infection. Results: Paracoccidioides brasiliensis initiated caspase-8-mediated IL-1ß production, an event that was necessary for transcriptional priming and posttranslational processing of pro-IL-1ß. Caspase-8 synergizes with the canonical NLRP3 inflammasome pathway to control caspase-1 processing and IL-1ß maturation, providing a regulatory role for caspase-8 in host resistance to in vivo P brasiliensis infection. Conclusions: Taken together, these findings revealed an important role for caspase-8 in the innate immune response of host cells to P brasiliensis infection, demonstrating a connected network between noncanonical and canonical inflammasomes to coordinate IL-1ß production during fungal challenge.

Th1-Inducing Agents in Prophylaxis and Therapy for Paracoccidioidomycosis.

Adjuvants and immunomodulatory molecules could be included in the treatment of P. brasiliensis infection. In this context, we reported that the therapeutic and/or prophylactic administration of Th1-inducing agents, such as immunomodulatory lectins and adjuvants, was able to provide protection against experimental paracoccidioidomycosis. Then, we described the protocols to investigate the effect of immunomodulatory agents on the course of P. brasiliensis infection. In this sense, we detailed the measurement of fungal burden and cytokine production, and the histopathological analysis used to evaluate the most effective administration regime.

Epidemiology of paracoccidioidomycosis / Epidemiologia da paracoccidioidomicose

SUMMARYThe epidemiological characteristics of paracoccidioidomycosis were reviewed and updated. The new endemic areas in Brazil were discussed in the section regarding the geographic distribution of the mycosis. Subclinical infection with Paracoccidioides brasiliensis was discussed on the basis of skin test surveys with antigens of the fungus, seroepidemiological studies, and disease cases outside Latin America. Large case series permitted a comparison of the prevalence of the mycosis in different regions, its estimated incidence and risk factors for the development of the disease. Aspects modulating the expression of the clinical forms of paracoccidioidomycosis are also presented. This review also deals with diseases associated with the mycosis, opportunistic paracoccidioidomycosis, lethality, mortality and infection and disease in animals.

RESUMOAs características epidemiológicas da paracoccidioidomicose foram revistas e atualizadas. Novas áreas endêmicas brasileiras foram discutidas na seção de distribuição geográfica da micose. A infecção subclínica por Paracoccidioides brasiliensis foi discutida com base em pesquisas realizadas com testes cutâneos com antígenos do fungo, estudos soroepidemiológicos e em casos de doença, fora da América Latina. Grandes séries de casos permitiram a comparação da prevalência da micose em diferentes regiões, sua incidência estimada e fatores de risco para o desenvolvimento da doença. Aspectos modulando a expressão de formas subclínicas da paracoccidioidomicose foram igualmente apresentados. Esta revisão também trata de doenças associadas à micose, paracoccidioidomicose oportunista, letalidade, mortalidade e infecção e doença em animais.

Alternative oxidase plays an important role in Paracoccidioides brasiliensis cellular homeostasis and morphological transition.

Paracoccidioides brasiliensis is the etiologic agent of one of the most common systemic mycoses in Latin America. As a dimorphic fungus, it must adapt to different environments during its life cycle, either in nature or within the host, enduring external stresses such as temperature or host-induced oxidative stress. In this study we addressed the role of alternative oxidase (PbAOX) in cellular homeostasis during batch culture growth and the morphological transition of P. brasiliensis. Using a PbAOX-antisense-RNA (PbAOX-aRNA) strain with a 70% reduction in gene expression, we show that PbAOX is crucial for maintaining cell viability and vitality during batch culture growth of yeast cells, in what appears to be a pH-dependent manner. We also show that silencing of PbAOX drastically reduced expression levels of other detoxifying enzymes (PbY20 and PbMSOD). In addition, our data indicate that PbAOX plays a role during the morphological transition, namely, during the yeast-to-mycelia germination and mycelia/conidia-to-yeast transition, essential events during the establishment of infection by dimorphic fungal pathogens. Altogether, our findings support the hypothesis that PbAOX is important for the maintenance of cellular homeostasis, possibly by assisting redox balancing during cell growth and the morphological switch of P. brasiliensis.

Radiochemical pharmacokinetic profile of P10 peptide with antifungal properties.

Paracoccidioidomycosis (PCM) is a chronic granulomatous disease that is caused by the thermally dimorphic fungus Paracoccidioides brasiliensis. It is endemic in some countries of Latin America and can cause a high-burden fungal infection with significant morbidity and mortality. The peptide P10, which demonstrates immune protection against experimental PCM, was radiolabeled with a radioisotope and evaluated in vivo. The radiolabeling was conducted to trace the pharmacokinetics of the molecule in principal organs and tissues. This was achieved with high radiochemical purity. Biodistribution and scintigraphic imaging showed fast blood clearance that was mainly renal; however, hepatobiliar excretion was also, with marked uptake in cervical lymph nodes. This profile may be useful for the development of a prophylactic drug or vaccine for patients exposed to PCM.

Ribosomal DNA nanoprobes studied by Fourier transform infrared spectroscopy.

Paracoccidioides brasiliensis (P. brasiliensis) is a thermo-dimorphic fungus that causes paracoccidioidomycosis. Brazil epidemiological data shows that endemic areas are the subtropical regions, especially where agricultural activities predominate such as the Southeast, South, and Midwest. There are several tests to diagnose paracoccidioidomycosis, but they have many limitations such as low sensitivity, high cost, and a cross-reacting problem. In this work, gold nanoprobes were used to identify P. brasiliensis as an alternative diagnostic technique, which is easier to apply, costs less, and has great potential for application. The specific Ribosomal sequence of P. brasiliensis DNA was amplified and used to design the nanoprobes using a thiol-modified oligonucleotide. The results of positive and negative tests were done by UV-visible and Fourier Transform Infrared (FT-IR) measurements. The deconvolution of FT-IR sample spectra showed differences in the vibrational modes from the hydrogen bridge NHN and NHO bands that form the double helix DNA for samples matching the DNA sequence of nanoprobes that could be used to classify the samples.

New advances in the development of a vaccine against paracoccidioidomycosis.

Paracoccidioidomycosis (PCM) is an endemic Latin American mycosis caused by Paracoccidioides brasiliensis and also by the recently described P. lutzii. The systemic mycosis is the 10th leading cause of death due to infectious diseases in Brazil. As published, 1,853 patients died of PCM in the 1996-2006 decade in this country. The main diagnostic antigen of P.brasiliensis is the 43 kDa glycoprotein gp43, and its 15-mer peptide QTLIAIHTLAIRYAN, known as P10, contains the T-CD4(+) epitope that elicits an IFN-γ-mediated Th1 immune response, which effectively treats mice intratracheally infected with PCM. The association of peptide P10 with antifungal drugs rendered an additive protective effect, even in immunosuppressed animals, being the basis of a recommended treatment protocol. Other immunotherapeutic tools include a peptide carrying a B cell epitope as well as protective anti-gp43 monoclonal antibodies. New delivery systems and gene therapy have been studied in prophylactic and therapeutic protocols to improve the efficacy of the recognized antigens aiming at a future vaccine as co-adjuvant therapy in patients with PCM.

Morfologia, virulência e perfil genético de Paracoccidioides brasiliensis: avaliação pré e pós-passagem em modelo murino de isolados provenientes de forma clínica crônica humana da paracoccidioidomico / Morphology, virulence and genetic profile of P. brasiliensis pre evaluation and post-passage in a murine model of chronic isolates from human clinical form of paracoccidioidomico

Estudos sobre os aspectos morfológicos, genéticos e de virulência, inerentes ao Paracoccidioides brasiliensis antes e depois da interação com hospedeiro experimental são de grande importância para. Oito isolados avaliados não apresentaram diferenças macro e micromorfológicas póspassagem em animal quando comparadas a morfologia pré-passagem. Entretanto eles diferiram entre si em relação à micromorfologia, in vitro, com diferenças na gemulação e presença de filamentos à temperatura de 36oC. Em relação à virulência, segundo os critérios adotados, índice esplênico, reisolamento de células fúngicas, lesões em diferentes órgãos e taxa de sobrevivência, o isolado Pb235CRS foi considerado o mais virulento; os isolados Pb261, Pb31MAS, Pb285, Pb246, Pb281 apresentaram virulência intermediária e os isolados Pb29EE e Pb639 foram os menos virulentos. A genotipagem por RAPD dos isolados demonstrou modificações no perfil de bandas do DNA após interação com o hospedeiro animal, aos 100 dias da inoculação, porém não foi possível correlacionar esses dados com os demais obtidos. Os resultados obtidos em nosso estudo apontam que diferenças entre isolados existem e podem interferir na evolução da doença...

Resistance of melanized yeast cells of Paracoccidioides brasiliensis to antimicrobial oxidants and inhibition of phagocytosis using carbohydrates and monoclonal antibody to CD18

Paracoccidioides brasiliensis, a thermal dimorphic fungal pathogen, produces a melanin-like pigment in vitro and in vivo. We investigated the involvement of carbohydrates and monoclonal antibody to CD18, on phagocytosis inhibition, involving macrophage receptors and the resistance of melanized fungal cells to chemically generated nitric oxide (NO), reactive oxygen species (ROS), hypochlorite and H2O2. Our results demonstrate that melanized yeast cells were more resistant than nonmelanized yeast cells to chemically generated NO, ROS, hypochlorite and H2O2, in vitro. Phagocytosis of melanized yeast cells was virtually abolished when mannan, N-acetyl glucosamine and anti-CD18 antibody were added together in this system. Intratracheal infection of BALB/c mice, with melanized yeast cells, resulted in higher lung colony forming units, when compared to nonmelanized yeast cells. Therefore, melanin is a virulence factor of P. brasiliensis.

Microdilution procedure for antifungal susceptibility testing of Paracoccidioides brasiliensis to amphotericin b and itraconazole

In vitro tests employing microdilution to evaluate fungal susceptibility to antifungal drugs are already standardized for fermentative yeasts. However, studies on the susceptibility of dimorphic fungi such as Paracoccidioides brasiliensis employing this method are scarce. The present work introduced some modifications into antifungal susceptibility testing from the European Committee on Antimicrobial Susceptibility Testing (EUCAST), concerning broth medium and reading time, to determine minimal inhibitory concentration (MIC) of amphotericin B and itraconazole against Paracoccidioides brasiliensis. Yeast-like cells of P. brasiliensis (Pb18 strain) were tested for susceptibility to amphotericin B and itraconazole in RPMI 1640 medium, supplemented with 2 percent glucose and nitrogen source and incubated at 35ºC. The MIC of amphotericin B and itraconazole against Pb18 were respectively 0.25 µg/mL and 0.002 µg/mL. The results of minimal fungicidal concentration (MFC) showed that amphotericin B at 0.25 µg/mL or higher concentrations displayed fungicidal activity against Pb18 while itraconazole at least 0.002 µg/mL has a fungistatic effect on P. brasiliensis. In conclusion, our results showed that the method employed in the present study is reproducible and reliable for testing the susceptibility of P. brasiliensis to antifungal drugs.