Brazilian Red Propolis shows antifungal and immunomodulatory activities against Paracoccidioides brasiliensis.

ETHNOPHARMACOLOGICAL RELEVANCE: Paracoccidioidomycosis (PCM) is a systemic mycosis with high prevalence in South America and especially in Brazil with severe clinical consequences that need broadened therapeutic options. Propolis is a natural resin from bees used in folk medicine for centuries with the first report in the ancient history of Egypt by Eberly papyrus, in Middle-Ages used to wash the newborn's umbilical cord and World War II as antiseptic or antibiotics. Nowadays it is a natural product worldwide consumed as food and traditionally used for oral and systemic diseases as an anti-inflammatory, antimicrobial, antifungal, and other diseases. Brazilian red propolis (BRP) is a new type of propolis with a distinguished chemical profile and biological activities from propolis (green) with pharmacological properties such as antimicrobial, anti-inflammatory, antioxidant, and others. AIM OF STUDY: Thus, the main purpose of this study was to investigate the direct in vitro and ex vivo effect of BRP on Paracoccidioides brasiliensis. MATERIAL AND METHODS: Antifungal activity of different concentrations of BRP on a virulent P. brasiliensis isolate (Pb18) was evaluated using the microdilution technique. Also, mice splenic cells co-cultured with Pb18 were treated with BRP at different times and concentrations (only Pb18 = negative control). Mice were inoculated with Pb18 and treated with different concentrations of BRP (50-500 mg/mL) in a subcutaneous air pouch. In this later experimental model, macroscopic characteristics of the air pouch were evaluated, and cellular exudate was collected and analyzed for cellular composition, mitochondrial activity, total protein reactive oxygen specimens (ROS), and nitric oxide production, as well as the number of viable fungal cells. RESULTS: The in vitro experiments showed remarkable direct antifungal activity of BRP, mainly with the highest concentration employed (500 mg/mL), reducing the number of viable cells to 10% of the original inoculum after 72 h incubation. The splenocytes co-cultivation assays showed that BRP had no cytotoxic effect on these cells, on the contrary, exerted a stimulatory effect. This stimulation was also observed on the PMNs at the air pouch, as verified by production of ROS and total proteins and mitochondrial activity. This activation resulted in enhanced fungicidal activity, mainly with the 500 mg/mL concentration of BRP. An anti-inflammatory effect was also detected, as verified by the smaller volume of the BRP-treated air pouch as well as by an earlier shift from neutrophils to mononuclear cells present in the infection site. CONCLUSION: Our results strongly suggest, for the first time in the literature, that Brazilian Red propolis has four protective mechanisms in experimental paracoccidioidomycosis: activating neutrophils, exerting a direct antifungal effect, preventing fungal dissemination, and controlling excessive inflammation process.

Identification of Potentially Therapeutic Immunogenic Peptides From Paracoccidioides lutzii Species.

Paracoccidioidomycosis (PCM) is an endemic mycosis in Latin America caused by the thermodimorphic fungi of the genus Paracoccidioides spp. Paracoccidioides lutzii (PL) is one of the 5 species that constitute the Paracoccidioides genus. PL expresses low amounts of glycoprotein (Gp) 43 (PLGp43) and PLGp43 displays few epitopes in common with the P. brasiliensis (PB) immunodominant antigen PBGp43, which is commonly used for serological diagnosis of PCM. This difference in structure between the glycoproteins markedly reduces the efficiency of serological diagnosis in patients infected with PL. We previously demonstrated that peptide 10 (P10) from the PBGp43 induces protective immune responses in in vitro and in vivo models of PB PCM. Since, P10 has proven to be a promising therapeutic to combat PB, we sought to identify peptides in PL that could similarly be applied for the treatment of PCM. PL yeast cell proteins were isolated from PL: dendritic cell co-cultures and subjected to immunoproteomics. This approach identified 18 PL peptides that demonstrated in silico predictions for immunogenicity. Eight of the most promising peptides were synthesized and applied to lymphocytes obtained from peptide-immunized or PL-infected mice as well as to in vitro cultures with peptides or dendritic cells pulsed the peptides. The peptides LBR5, LBR6 and LBR8 efficiently promoted CD4+ and CD8+ T cell proliferation and dendritic cells pulsed with LBR1, LBR3, LBR7 or LBR8 stimulated CD4+ T cell proliferation. We observed increases of IFN-γ in the supernatants from primed T cells for the conditions with peptides without or with dendritic cells, although IL-2 levels only increased in response to LBR8. These novel immunogenic peptides derived from PL will be employed to develop new peptide vaccine approaches and the proteins from which they are derived can be used to develop new diagnostic assays for PL and possibly other Paracoccidioides spp. These findings identify and characterize new peptides with a promising therapeutic profile for future against this important neglected systemic mycosis.

Parengyodontium album Isolated from Cutaneous Lesions of a Pacific White-Sided Dolphin (Lagenorhynchus obliquidens) During Treatment for Paracoccidioidomycosis Ceti.

The prominence of seafood in Japan motivates close monitoring of its seas and marine lives for potentially pathogenic fungi. During the treatments of the male Pacific white-sided dolphin (Lagenorhynchus obliquidens) for paracoccidioidomycosis ceti (PCM-C), 5 white and floccose colonies showing identical genotype and morphological characteristics were isolated from two skin biopsy samples of cutaneous granulomatous lesions in 2018. The isolates were identified as Parengyodontium album known as one of fungal species having abilities to produce industrially important proteases, and to become a causative agent for emerging mycosis based on morphological and molecular biological characteristics. These lesions consisted of non-malignant pearl-like structures of hyperplastic keratinocytes. Interestingly, although the isolates could grow at 35 °C, their DNA sequences were phylogenetically located in a cluster consisting of environmental and clinical isolates lacking the ability to grow at 35 °C, based on previous reports. The opportunistic infection we observed in the dolphin might be caused by immune disorder due to PCM-C. Notably, although P. album is recognized as non-harmful, and has significant industrial importance and antitumor activity, it has potential to cause not only superficial but also systemic infection, and presents difficulties in treatment because of its high resistance to antifungal compounds.

Virulence factors of Paracoccidioides brasiliensis as therapeutic targets: a review.

Paracoccidiodomycosis (PCM) is a systemic mycosis caused by the fungus Paracoccidioides brasiliensis and Paracoccidioides lutzii. The disease requires long and complicated treatment. The aim of this review is to address the fungal virulence factors that could be the target of the development of new drugs for PCM treatment. Virulence factors favoring the process of fungal infection and pathogenicity are considered as a microbial attribute associated with host susceptibility. P. brasiliensis has some known virulence factors which are 43 kDa glycoprotein (gp 43) which is an important fungal antigen, 70 kDa glycoprotein (gp 70), the carbohydrates constituting the fungal cell wall α-1,3, glucan and ß-1,3-glucan, cell adhesion molecules and the presence of melanin pigments. The discovery and development of drugs that interact with these factors, such as inhibitors of ß-1,3-glucan, reduced synthesis of gp 43, inhibitors of melanin production, is of great importance for the treatment of PCM. The study of virulence factors favors the understanding of pathogen-host relationships, aiming to evaluate the possibility of developing new therapeutic targets and mechanisms that these molecules play in the infectious process, favoring the design of a more specific treatment for this disease.

Antifungal activity of two oxadiazole compounds for the paracoccidioidomycosis treatment.

Paracoccidioidomycosis (PCM) is a neglected disease present in Latin America with difficulty in treatment and occurrence of serious sequelae. Thus, the development of alternative therapies is imperative. In the current work, two oxadiazole compounds (LMM5 and LMM11) presented fungicidal activity against Paracoccidioides spp. The minimum inhibitory and fungicidal concentration values ranged from 1 to 32 µg/mL, and a synergic effect was observed for both compounds when combined with Amphotericin B. LMM5 and LMM11 were able to reduce CFU counts (≥2 log10) on the 5th and 7th days of time-kill curve, respectively. The fungicide effect was confirmed by fluorescence microscopy (FUN-1/FUN-2). The hippocratic screening and biochemical analysis were performed in Balb/c male mice that received a high dose of each compound, and the compounds showed no in vivo toxicity. The treatment of experimental PCM with the new oxadiazoles led to significant reduction in CFU (≥1 log10). Histopathological analysis of the groups treated exhibited control of inflammation, as well as preserved lung areas. These findings suggest that LMM5 and LMM11 are promising hits structures, opening the door for implementing new PCM therapies.

New 4-methoxy-naphthalene derivatives as promisor antifungal agents for paracoccidioidomycosis treatment.

AIM: Novel 4-methoxy-naphthalene derivatives were synthesized based on hits structures in order to evaluate the antifungal activity against Paracoccidioides spp. METHODS: Antifungal activity of compounds was evaluated against P. brasiliensis and most promising compounds 2 and 3 were tested against eight clinically important fungal species. RESULTS: Compound 3 was the more active compound with MIC 8 to 32 µg.ml-1 for Paracoccidioides spp without toxicity monkey kidney and murine macrophagecells. Carbohydrazide 3 showed good synergistic antifungal activity with amphotericin B against P. brasiliensis specie. Titration assay of carbohydrazide 3 with PbHSD enzyme demonstrates the binding ligand-protein. Molecular dynamics simulations show that ligand 3 let the PbHSD protein more stable. CONCLUSION: New carbohydrazide 3 is an attractive lead for drug development to treat paracoccidioidomycoses.

Promising New Antifungal Treatment Targeting Chorismate Synthase from Paracoccidioides brasiliensis.

Paracoccidioidomycosis (PCM), caused by Paracoccidioides, is a systemic mycosis with granulomatous character and a restricted therapeutic arsenal. The aim of this work was to search for new alternatives to treat largely neglected tropical mycosis, such as PCM. In this context, the enzymes of the shikimate pathway constitute excellent drug targets for conferring selective toxicity because this pathway is absent in humans but essential for the fungus. In this work, we have used a homology model of the chorismate synthase (EC 4.2.3.5) from Paracoccidioides brasiliensis (PbCS) and performed a combination of virtual screening and molecular dynamics testing to identify new potential inhibitors. The best hit, CP1, successfully adhered to pharmacological criteria (adsorption, distribution, metabolism, excretion, and toxicity) and was therefore used in in vitro experiments. Here we demonstrate that CP1 binds with a dissociation constant of 64 ± 1 µM to recombinant chorismate synthase from P. brasiliensis and inhibits enzymatic activity, with a 50% inhibitory concentration (IC50) of 47 ± 5 µM. As expected, CP1 showed no toxicity in three cell lines. On the other hand, CP1 reduced the fungal burden in lungs from treated mice, similar to itraconazole. In addition, histopathological analysis showed that animals treated with CP1 displayed less lung tissue infiltration, fewer yeast cells, and large areas with preserved architecture. Therefore, CP1 was able to control PCM in mice with a lower inflammatory response and is thus a promising candidate and lead structure for the development of drugs useful in PCM treatment.

Paracoccidioides spp. and Histoplasma capsulatum: Current and New Perspectives for Diagnosis and Treatment.

The thermally-dimorphic systemic fungal group includes several important human pathogens: Blastomyces dermatitides, Coccidioides immitis and C. posadasii, Histoplasma capsulatum, Paracoccidioides brasiliensis, P. lutzii, and Talaromyces (Penicillium) marneffei. They usually are geographically restricted and have natural habitats in soil or in plants, and when fungal propagules invade mammalian host by inhalation, they initiate an inflammatory reaction that can result in self-resolution of the infection or cause an acute or chronic disease. In the setting of the AIDS pandemic and the developments in modern medicine, such as immunosuppressive therapy in cancer surgery patients and in transplantation and autoimmune diseases, the incidence of endemic mycoses has progressively increased. Another important factor of the increased incidence of systemic mycoses in certain regions is the progressive devastation of tropical and subtropical forests. In this review, we focus on two of the most important systemic mycoses: paracoccidioidomycosis and histoplasmosis, and their major characteristics in epidemiology, clinical aspects and laboratorial diagnosis.

Therapeutic treatment with scFv-PLGA nanoparticles decreases pulmonary fungal load in a murine model of paracoccidioidomycosis.

Paracoccidioidomycosis (PCM) is a systemic mycosis with lymphatic dissemination that is caused by Paracoccidioides species. Treatment of PCM consists of chemotherapeutics such as itraconazole, trimethoprim, sulfamethoxazole or amphotericin B. However, several studies are aiming to develop therapeutic alternatives for the treatment of fungal infection using new molecules as adjuvants. The single-chain variable fragments (scFv) from an antibody that mimics the main fungal component incorporated within poly(lactide-co-glycolic) acid (PLGA) nanoparticles helped treat the fungal disease. After expressing the scFv in Picchia pastoris (P. pastoris), the recombinant molecules were coupled with PLGA, and the BALB/c mice were immunized before or after infection with yeast Paracoccidioides brasiliensis (P. brasiliensis). Our results showed decreased disease progression and decreased fungal burden. Taken together, our results showed an increased of IFN-γ and IL-12 cytokine production and an increased number of macrophages and dendritic cells in the pulmonary tissue of BALB/c mice treated with a high concentration of our molecule. Our data further confirm that the scFv plays an important role in the treatment of experimental PCM.

Targeting the Homoserine Dehydrogenase of Paracoccidioides Species for Treatment of Systemic Fungal Infections.

This work evaluated new potential inhibitors of the enzyme homoserine dehydrogenase (HSD) of Paracoccidioides brasiliensis, one of the etiological agents of paracoccidioidomycosis. The tertiary structure of the protein bonded to the analogue NAD, and l-homoserine was modeled by homology. The model with the best output was subjected to gradient minimization, redocking, and molecular dynamics simulation. Virtual screening simulations with 187,841 molecules purchasable from the Zinc database were performed. After the screenings, 14 molecules were selected and analyzed by the use of absorption, distribution, metabolism, excretion, and toxicity criteria, resulting in four compounds for in vitro assays. The molecules HS1 and HS2 were promising, exhibiting MICs of 64 and 32 µg · ml-1, respectively, for the Pb18 isolate of P. brasilensis, 64 µg · ml-1 for two isolates of P. lutzii, and also synergy with itraconazole. The application of these molecules to human-pathogenic fungi confirmed that the HSD enzyme may be used as a target for the development of drugs with specific action against paracoccidioidomycosis; moreover, these compounds may serve as leads in the design of new antifungals.

Animal Models and Antifungal Agents in Paracoccidioidomycosis: An Overview.

Paracoccidioides brasiliensis is the etiologic agent of paracoccidioidomycosis, the most prevalent systemic mycosis in Latin America. The morbidity and mortality associated with paracoccidioidomycosis necessitate our understanding of fungal pathogenesis and discovering of new agents to treat this infection. Animal models have contributed much to the knowledge of fungal infections and their corresponding therapeutic treatments. This is true for animal models of the primary fungal pathogens such as P. brasiliensis. This review describes the development, details and utility of animal models of paracoccidioidomycosis for studying and developing the current antifungal agents used for therapy of this fungal disease and novel agents with antifungal properties against P. brasiliensis.

Evaluation of the efficacy of antifungal drugs against Paracoccidioides brasiliensis and Paracoccidioides lutzii in a Galleria mellonella model.

Paracoccidioides brasiliensis and P. lutzii belong to a group of thermodimorphic fungi and cause paracoccidioidomycosis (PCM), which is a human systemic mycosis endemic in South and Central America. Patients with this mycosis are commonly treated with amphotericin B (AmB) and azoles. The study of fungal virulence and the efficacy and toxicity of antifungal drugs has been successfully performed in a Galleria mellonella infection model. In this work, G. mellonella larvae were infected with two Paracoccidioides spp. and the efficacy and toxicity of AmB and itraconazole were evaluated in this model for the first time. AmB and itraconazole treatments were effective in increasing larval survival and reducing the fungal burden. The fungicidal and fungistatic effects of AmB and itraconazole, respectively, were observed in the model. Furthermore, these effects were independent of changes in haemocyte number. G. mellonella can serve as a rapid model for the screening of new antifungal compounds against Paracoccidioides and can contribute to a reduction in experimental animal numbers in the study of PCM.

CHYLOTHORAX IN PARACOCCIDIOIDOMYCOSIS.

A previously healthy, 52-year-old woman presented with a nine months history of low fever and weight loss (> 30 kg). Physical examination disclosed generalized lymphadenopathy, skin lesions, abdominal distension, mild tachypnea and a left breast mass. Laboratory tests showed anemia; (prerenal) kidney injury, low serum albumin level; and negative serology for HIV and viral hepatitis. Computed tomography (neck/chest/abdomen) showed generalized lymph node enlargement, splenomegaly, pleural effusion and ascites. We performed thoracocentesis and paracentesis, and the findings were consistent with chylothorax and chylous ascites (with no neoplastic cells). Biopsies of the breast mass, skin and lymph nodes were performed and all of them showed large round yeast cells with multiple narrow-based budding daughter cells, characteristic of Paracoccidioides brasiliensis. Consequently, paracoccidioidomycosis was diagnosed, and liposomal amphotericin B was prescribed, as well as a high protein and low fat diet (supplemented with medium chain triglycerides). Even so, her clinical status worsened, requiring renal replacement therapy. She evolved with pneumonia, septic shock and respiratory failure and subsequently died. To our knowledge, this is the first description of a case with chylothorax and breast mass due to paracoccidioidomycosis. Additionally, we discuss: 1- the importance of the inclusion of this mycosis in the differential diagnosis of chylothorax and breast mass (breast cancer), especially in endemic areas; and 2- the possible mechanism involved in the development of chylous effusions.

Cyclopalladated Compound 7a Induces Apoptosis- and Autophagy-Like Mechanisms in Paracoccidioides and Is a Candidate for Paracoccidioidomycosis Treatment.

Paracoccidioidomycosis (PCM), caused by Paracoccidioides species, is the main cause of death due to systemic mycoses in Brazil and other Latin American countries. Therapeutic options for PCM and other systemic mycoses are limited and time-consuming, and there are high rates of noncompliance, relapses, toxic side effects, and sequelae. Previous work has shown that the cyclopalladated 7a compound is effective in treating several kinds of cancer and parasitic Chagas disease without significant toxicity in animals. Here we show that cyclopalladated 7a inhibited the in vitro growth of Paracoccidioides lutzii Pb01 and P. brasiliensis isolates Pb18 (highly virulent), Pb2, Pb3, and Pb4 (less virulent) in a dose-response manner. Pb18 was the most resistant. Opportunistic Candida albicans and Cryptococcus neoformans were also sensitive. BALB/c mice showed significantly lighter lung fungal burdens when treated twice a day for 20 days with a low cyclopalladated 7a dose of 30 µg/ml/day for 30 days after intratracheal infection with Pb18. Electron microscopy images suggested that apoptosis- and autophagy-like mechanisms are involved in the fungal killing mechanism of cyclopalladated 7a. Pb18 yeast cells incubated with the 7a compound showed remarkable chromatin condensation, DNA degradation, superoxide anion production, and increased metacaspase activity suggestive of apoptosis. Autophagy-related killing mechanisms were suggested by increased autophagic vacuole numbers and acidification, as indicated by an increase in LysoTracker and monodansylcadaverine (MDC) staining in cyclopalladated 7a-treated Pb18 yeast cells. Considering that cyclopalladated 7a is highly tolerated in vivo and affects yeast fungal growth through general apoptosis- and autophagy-like mechanisms, it is a novel promising drug for the treatment of PCM and other mycoses.

Validation and clinical application of a nested PCR for paracoccidioidomycosis diagnosis in clinical samples from Colombian patients

Paracoccidioidomycosis is a systemic and endemic mycosis, restricted to tropical and subtropical areas of Latin America. The infection is caused by the thermal dimorphic fungus Paracoccidioides brasiliensisand Paracoccidioides lutzii. The diagnosis of paracoccidioidomycosis is usually performed by microscopic examination, culture and immunodiagnostic tests to respiratory specimens, body fluids and/or biopsies; however these methods require laboratory personnel with experience and several days to produce a result. In the present study, we have validated and evaluated a nested PCR assay targeting the gene encoding the Paracoccidioides gp43membrane protein in 191 clinical samples: 115 samples from patients with proven infections other than paracoccidioidomycosis, 51 samples as negative controls, and 25 samples from patients diagnosed with paracoccidioidomycosis. Additionally, the specificity of the nested PCR assay was also evaluated using purified DNA isolated from cultures of different microorganisms (n= 35) previously identified by culture and/or sequencing. The results showed that in our hands, this nested PCR assay for gp43 protein showed specificity and sensitivity rates of 100%. The optimized nested PCR conditions in our laboratory allowed detection down to 1 fg of P. brasiliensisDNA.

Validation and clinical application of a nested PCR for paracoccidioidomycosis diagnosis in clinical samples from Colombian patients.

Paracoccidioidomycosis is a systemic and endemic mycosis, restricted to tropical and subtropical areas of Latin America. The infection is caused by the thermal dimorphic fungus Paracoccidioides brasiliensis and Paracoccidioides lutzii. The diagnosis of paracoccidioidomycosis is usually performed by microscopic examination, culture and immunodiagnostic tests to respiratory specimens, body fluids and/or biopsies; however these methods require laboratory personnel with experience and several days to produce a result. In the present study, we have validated and evaluated a nested PCR assay targeting the gene encoding the Paracoccidioides gp43 membrane protein in 191 clinical samples: 115 samples from patients with proven infections other than paracoccidioidomycosis, 51 samples as negative controls, and 25 samples from patients diagnosed with paracoccidioidomycosis. Additionally, the specificity of the nested PCR assay was also evaluated using purified DNA isolated from cultures of different microorganisms (n=35) previously identified by culture and/or sequencing. The results showed that in our hands, this nested PCR assay for gp43 protein showed specificity and sensitivity rates of 100%. The optimized nested PCR conditions in our laboratory allowed detection down to 1fg of P. brasiliensis DNA.

Low-level laser therapy to the mouse femur enhances the fungicidal response of neutrophils against Paracoccidioides brasiliensis.

Neutrophils (PMN) play a central role in host defense against the neglected fungal infection paracoccidioidomycosis (PCM), which is caused by the dimorphic fungus Paracoccidioides brasiliensis (Pb). PCM is of major importance, especially in Latin America, and its treatment relies on the use of antifungal drugs. However, the course of treatment is lengthy, leading to side effects and even development of fungal resistance. The goal of the study was to use low-level laser therapy (LLLT) to stimulate PMN to fight Pb in vivo. Swiss mice with subcutaneous air pouches were inoculated with a virulent strain of Pb or fungal cell wall components (Zymosan), and then received LLLT (780 nm; 50 mW; 12.5 J/cm2; 30 seconds per point, giving a total energy of 0.5 J per point) on alternate days at two points on each hind leg. The aim was to reach the bone marrow in the femur with light. Non-irradiated animals were used as controls. The number and viability of the PMN that migrated to the inoculation site was assessed, as well as their ability to synthesize proteins, produce reactive oxygen species (ROS) and their fungicidal activity. The highly pure PMN populations obtained after 10 days of infection were also subsequently cultured in the presence of Pb for trials of protein production, evaluation of mitochondrial activity, ROS production and quantification of viable fungi growth. PMN from mice that received LLLT were more active metabolically, had higher fungicidal activity against Pb in vivo and also in vitro. The kinetics of neutrophil protein production also correlated with a more activated state. LLLT may be a safe and non-invasive approach to deal with PCM infection.

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.

Role of omega-3 polyunsaturated fatty acids in the production of prostaglandin E2 and nitric oxide during experimental murine paracoccidioidomycosis.

There has recently been increased interest in the potential health effects of omega-3 polyunsaturated fatty acids on the immune system. Paracoccidioidomycosis is the most important endemic mycosis in Latin America. Macrophages have a fundamental role and act as first line of organism defense. The purpose of this study was to analyze the effect of n-3 fatty acids on the production of PGE2 and NO by mice infected with Pb18 and fed a diet enriched with LNA for 8 weeks. To study the effect of omega-3 fatty acids on macrophage activity during experimental paracoccidioidomycosis, mice were infected with Pb18 and fed a diet supplemented with LNA. PGE2 in the serum of animals was analyzed and NO in the supernatants of macrophages cultured and challenged in vitro with Pb18 was measured. Omega-3 fatty acids seemed to decrease the production of PGE2 in vivo in the infected group fed an LNA-supplemented diet during the 4th and 8th weeks of the experiment. At the same time, we observed an increase in synthesis of NO by peritoneal macrophages in this group. Omega-3 fatty acids thus appear to have an immunomodulatory effect in paracoccidioidomycosis.