The Role of Dimorphism Regulating Histidine Kinase (Drk1) in the Pathogenic Fungus Paracoccidioides brasiliensis Cell Wall.

Dimorphic fungi of the Paracoccidioides genus are the causative agents of paracoccidioidomycosis (PCM), an endemic disease in Latin America with a high incidence in Brazil. This pathogen presents as infective mycelium at 25 °C in the soil, reverting to its pathogenic form when inhaled by the mammalian host (37 °C). Among these dimorphic fungal species, dimorphism regulating histidine kinase (Drk1) plays an essential role in the morphological transition. These kinases are present in bacteria and fungi but absent in mammalian cells and are important virulence and cellular survival regulators. Hence, the purpose of this study was to investigate the role of PbDrk1 in the cell wall modulation of P. brasiliensis. We observed that PbDrk1 participates in fungal resistance to different cell wall-disturbing agents by reducing viability after treatment with iDrk1. To verify the role of PbDRK1 in cell wall morphogenesis, qPCR results showed that samples previously exposed to iDrk1 presented higher expression levels of several genes related to cell wall modulation. One of them was FKS1, a ß-glucan synthase that showed a 3.6-fold increase. Furthermore, confocal microscopy analysis and flow cytometry showed higher ß-glucan exposure on the cell surface of P. brasiliensis after incubation with iDrk1. Accordingly, through phagocytosis assays, a significantly higher phagocytic index was observed in yeasts treated with iDrk1 than the control group, demonstrating the role of PbDrk1 in cell wall modulation, which then becomes a relevant target to be investigated. In parallel, the immune response profile showed increased levels of proinflammatory cytokines. Finally, our data strongly suggest that PbDrk1 modulates cell wall component expression, among which we can identify ß-glucan. Understanding this signalling pathway may be of great value for identifying targets of antifungal molecular activity since HKs are not present in mammals.

Neutrophil-suppressive activity over T-cell proliferation and fungal clearance in a murine model of Fonsecaea pedrosoi infection.

Neutrophils are essential to control several fungal infections. These cells are commonly known for their pro-inflammatory activities. However, some studies have demonstrated the anti-inflammatory properties of neutrophils during certain infectious diseases, culminating in the inhibition of T cell proliferation. Chromoblastomycosis (CBM) is a deep and progressive mycosis that affects thousands of people worldwide. Although neutrophil infiltrates are observed in the lesion histopathology, the fungus can overtake the immune system response and destroy the host-infected tissue. The present study demonstrated that neutropenic animals had an increase in the IL-6 production in the spleen and liver, followed by a lower fungal burden in these organs up to 14 days of infection. Neutropenic animals also showed a lower F. pedrosoi-specific antibody production 14-days post infection and higher T-cell proliferation in the in vitro experiments after stimulation with F. pedrosoi-purified proteins. Taken together, our results suggest that the presence of regulatory neutrophils in the mouse model of F. pedrosoi infection could act favoring the spread of the fungus and the chronicity of the infection. These findings shed light on the CBM treatment, which might target neutrophil polarization as a new therapy approach to treat CBM lesions.

Extracellular Vesicles From Sporothrix brasiliensis Yeast Cells Increases Fungicidal Activity in Macrophages.

Sporotrichosis is a subcutaneous mycosis and is distributed throughout the world, although most cases belong to endemic regions with a warmer climate such as tropical and subtropical areas. The infection occurs mainly by traumatic inoculation of propagules. Similarly, to other organisms, Sporothrix brasiliensis display many biological features that aid in its ability to infect the host, such as extracellular vesicles, bilayered biological structures that provides communication between host cells and between fungi cells themselves. Recently, research on Sporothrix complex have been focused on finding new molecules and components with potential for therapeutic approaches. Here, we study the relationship among EVs and the host's macrophages as well as their role during infection to assess whether these vesicles are helping the fungi or inducing a protective effect on mice during the infection. We found that after cocultivation with different concentrations of purified yeasts EVs from Sb, J774 macrophages displayed an increased fungicidal activity (Phagocytic Index) resulting in lower colony-forming units the more EVs were added, without jeopardizing the viability of the macrophages. Interleukins IL-6, IL-10, and IL-12 were measured during the infection period, showing elevated levels of IL-12 and IL-6 in a dose-dependent manner, but no significant change for IL-10. We also assessed the expression of important molecules in the immune response, such as MHC class II and the immunoglobulin CD86. Both these molecules were overexpressed in Sb yeasts infected mice. Our results indicate that EVs play a protective role during Sporothrix brasiliensis infections.

The role of dimorphism regulating histidine kinase (Drk1) in the pathogenic fungus Paracoccidioides brasiliensis cell wall

Dimorphic fungi of the Paracoccidioides genus are the causative agents of paracoccidioidomycosis (PCM), an endemic disease in Latin America with a high incidence in Brazil. This pathogen presents as infective mycelium at 25 °C in the soil, reverting to its pathogenic form when inhaled by the mammalian host (37 °C). Among these dimorphic fungal species, dimorphism regulating histidine kinase (Drk1) plays an essential role in the morphological transition. These kinases are present in bacteria and fungi but absent in mammalian cells and are important virulence and cellular survival regulators. Hence, the purpose of this study was to investigate the role of PbDrk1 in the cell wall modulation of P. brasiliensis. We observed that PbDrk1 participates in fungal resistance to different cell wall-disturbing agents by reducing viability after treatment with iDrk1. To verify the role of PbDRK1 in cell wall morphogenesis, qPCR results showed that samples previously exposed to iDrk1 presented higher expression levels of several genes related to cell wall modulation. One of them was FKS1, a β-glucan synthase that showed a 3.6-fold increase. Furthermore, confocal microscopy analysis and flow cytometry showed higher β-glucan exposure on the cell surface of P. brasiliensis after incubation with iDrk1. Accordingly, through phagocytosis assays, a significantly higher phagocytic index was observed in yeasts treated with iDrk1 than the control group, demonstrating the role of PbDrk1 in cell wall modulation, which then becomes a relevant target to be investigated. In parallel, the immune response profile showed increased levels of proinflammatory cytokines. Finally, our data strongly suggest that PbDrk1 modulates cell wall component expression, among which we can identify β-glucan. Understanding this signalling pathway may be of great value for identifying targets of antifungal molecular activity since HKs are not present in mammals.

Fonsecaeapedrosoi Conidia and Hyphae Activate Neutrophils Distinctly: Requirement of TLR-2 and TLR-4 in Neutrophil Effector Functions.

Chromoblastomycosis is a chronic and progressive subcutaneous mycosis caused mainly by the fungus Fonsecaea pedrosoi. The infection is characterized by erythematous papules and histological sections demonstrating an external layer of fibrous tissue and an internal layer of thick granulomatous inflammatory tissue containing mainly macrophages and neutrophils. Several groups are studying the roles of the innate and adaptive immune systems in F. pedrosoi infection; however, few studies have focused on the role of neutrophils in this infection. In the current study, we verify the importance of murine neutrophils in the killing of F. pedrosoi conidia and hyphae. We demonstrate that phagocytosis and reactive oxygen species during infection with conidia are TLR-2- and TLR-4-dependent and are essential for conidial killing. Meanwhile, hyphal killing occurs by NET formation in a TLR-2-, TLR-4-, and ROS-independent manner. In vivo experiments show that TLR-2 and TLR-4 are also important in chromoblastomycosis infection. TLR-2KO and TLR-4KO animals had lower levels of CCL3 and CXCL1 chemokines and impaired neutrophil migration to the infected site. These animals also had higher fungal loads during infection with F. pedrosoi conidia, confirming that TLR-2 and TLR-4 are essential receptors for F. pedrosoi recognition and immune system activation. Therefore, this study demonstrates for the first time that neutrophil activation during F. pedrosoi is conidial or hyphal-specific with TLR-2 and TLR-4 being essential during conidial infection but unnecessary for hyphal killing by neutrophils.

Intracellular PRRs Activation in Targeting the Immune Response Against Fungal Infections.

The immune response against fungal infections is complex and exhibits several factors involving innate elements that participate in the interaction with the fungus. The innate immune system developed pattern recognition receptors that recognize different pathogen-associated molecular patterns present both on the surface of the fungi cell wall and on their genetic material. These receptors have the function of activating the innate immune response and regulating a subsequent adaptive immune response. Among pattern recognition receptors, the family of Toll-like receptors and C-type lectin receptors are the best described and characterized, they act directly in the recognition of pathogen-associated molecular patterns expressed on the wall of the fungus and consequently in directing the immune response. In recent years, the role of intracellular pattern recognition receptors (TLR3, TLR7, TLR8, and TLR9) has become increasingly important in the pathophysiology of some mycoses, as paracoccidioidomycosis, cryptococcosis, aspergillosis, and candidiasis. The recognition of nucleic acids performed by these receptors can be essential for the control of some fungal infections, as they can be harmful to others. Therefore, this review focuses on highlighting the role played by intracellular pattern recognition receptors both in controlling the infection and in the host's susceptibility against the main fungi of medical relevance.

High Glucose Environments Interfere with Bone Marrow-Derived Macrophage Inflammatory Mediator Release, the TLR4 Pathway and Glucose Metabolism.

Macrophages may be a crucial aspect of diabetic complications associated with the inflammatory response. In this study, we examined how hyperglycaemia, a common aspect of diabetes, modulates bone marrow-derived macrophages (BMDMs) under an inflammatory stimulus. To perform this study, BMDMs from non-diabetic and diabetic (60 mg/kg alloxan, i.v.) male C57BL/6 mice (CEUA/FCF/USP-488) were cultured under normal (5.5 mM) and high glucose (HG, 25 or 40 mM) conditions and stimulated or not stimulated with lipopolysaccharide (LPS, 100 ng/mL). Compared to the BMDMs from the normoglycaemic mice, the LPS-stimulated BMDMs from the diabetic mice presented reduced TLR4 expression on the cell surface, lower phagocytic capacity, and reduced secretion of NO and lactate but greater oxygen consumption and greater phosphorylation of p46 SAPK/JNK, p42 ERK MAPK, pAKT and pPKC-δ. When the BMDMs from the non-diabetic mice were cultured under high-glucose conditions and stimulated with LPS, TLR4 expression was reduced on the cell surface and NO and H2O2 levels were reduced. In contrast, the diabetic BMDMs cultured under high glucose conditions presented increased levels of lactate and reduced phosphorylation of AKT, PKC-δ and p46 SAPK/JNK but enhanced phosphorylation of the p46 subunit of SAPK/JNK after LPS stimulation. High glucose levels appear to modify macrophage behaviour, affecting different aspects of diabetic and healthy BMDMs under the same LPS stimulus. Thus, hyperglycaemia leaves a glucose legacy, altering the basal steady state of macrophages.

Silencing of OCH1 unveils the role of Sporothrix schenckii N-linked glycans during the host-fungus interaction.

BACKGROUND: Sporothrix schenckii is a neglected fungal pathogen for the human being and other mammals. In several fungal systems, Och1 is a Golgi α1,6-mannosyltransferase with a key function in the synthesis of N-linked glycans; which are important elements during the host-fungus interplay. The role of OCH1 in fungal virulence seems to be species-specific, being an essential component for Candida albicans virulence and dispensable during the interaction of Aspergillus fumigatus with the host. METHODS: Here, we silenced S. schenckii OCH1 and characterized the phenotype of the mutant strains. RESULTS: The mutant strains did not show defects in the cell or colony morphology, the growth rate or the ability to undergo dimorphism; but the cell wall changed in both composition and exposure of inner components at the surface. When interacting with human monocytes, the silenced strains had a reduced ability to stimulate TNFα and IL-6 but stimulated higher levels of IL-10. The interaction with human macrophages was also altered, with reduced numbers of silenced cells phagocytosed. These strains showed virulence attenuation in both Galleria mellonella and in the mouse model of sporotrichosis. Nonetheless, the cytokine levels in infected organs did not vary significantly when compared with the wild-type strain. CONCLUSION: Our data demonstrate that OCH1 silencing affects different aspects of the S. schenckii-host interaction.

Extracellular Vesicles From Sporothrix brasiliensis Are an Important Virulence Factor That Induce an Increase in Fungal Burden in Experimental Sporotrichosis.

Sporotrichosis is a mycosis that affects the skin, lymphatic system and other organs in humans and animals. The disease has a worldwide distribution, with endemic areas in Brazil, and is caused by a complex of species, including Sporothrix brasiliensis. Some fungi release extracellular vesicles (EVs) that can interact with the host cell and modulate the host immune response. The aim of this study was to analyze the participation of S. brasiliensis EVs in the modulation of dendritic cells (DCs) and in the control of infection in vivo. Our results showed that in vitro, the EVs isolated from S. brasiliensis induced an increase in the phagocytic index and fungal burden in DCs. In addition, we observed a significant increase in IL-12p40 and TNF-α cytokine production. Then, the EVs were inoculated into BALB/c mice before subcutaneous infection with yeast, and the lesion was analyzed after 21, 35, and 42 days. An increase in fungal burden and lesion diameter were observed after 21 days in mice inoculated with a high concentration of EVs. However, after 35 days, we observed a regression of the lesion, which persisted until 42 days after infection. Interestingly, we observed an increase in fungal burden in these mice. In addition, we observed the presence of immunogenic components and proteins that could be related with virulence in EVs. These results suggest that EVs can play an important role in virulence and modulation of the host immune system during experimental S. brasiliensis infection.

Notch Signaling is Required for Dendritic Cell Maturation and T Cell Expansion in Paracoccidioidomycosis.

The Notch signaling pathway participates in several cellular functional aspects. This signaling has an important role in targeting both DC maturation and DC-mediated T cell responses. Thus, it is essential to investigate the influence of this signaling pathway in the role played by DCs in the pathogenesis of experimental paracoccidioidomycosis. This disease is a granulomatous and systemic mycosis that mainly affects lung tissue and can spread to any other organ and system. In this study, we demonstrated that bone marrow-derived DCs infected with yeasts from Paracoccidioides brasiliensis strain 18 performed efficiently their maturation after the activation of Notch signaling, with an increase in CD80, CD86, CCR7, and CD40 expression and the release of cytokines such as IL-6 and TNF-α. We observed that the inhibition of the γ-secretase DAPT impaired the proliferation of T cells induced by DC stimulation. In conclusion, our data suggest that Notch signaling contributes effectively to the maturation of DCs and the DC-mediated activation of the T cell response in P. brasiliensis infections.

An immunoproteomic approach revealing peptides from Sporothrix brasiliensis that induce a cellular immune response in subcutaneous sporotrichosis.

Sporothrix brasiliensis is the most virulent fungus of the Sporothrix complex and is the main species recovered in the sporotrichosis zoonotic hyperendemic area in Rio de Janeiro. A vaccine against S. brasiliensis could improve the current sporotrichosis situation. Here, we show 3 peptides from S. brasiliensis immunogenic proteins that have a higher likelihood for engaging MHC-class II molecules. We investigated the efficiency of the peptides as vaccines for preventing subcutaneous sporotrichosis. In this study, we observed a decrease in lesion diameters in peptide-immunized mice, showing that the peptides could induce a protective immune response against subcutaneous sporotrichosis. ZR8 peptide is from the GP70 protein, the main antigen of the Sporothrix complex, and was the best potential vaccine candidate by increasing CD4+ T cells and higher levels of IFN-γ, IL-17A and IL-1ß characterizing a strong cellular immune response. This immune environment induced a higher number of neutrophils in lesions that are associated with fungus clearance. These results indicated that the ZR8 peptide induces a protective immune response against subcutaneous sporotrichosis and is a vaccine candidate against S. brasiliensis infection.

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.

TLR3 Is a Negative Regulator of Immune Responses Against Paracoccidioides brasiliensis.

Toll-like receptors (TLRs) comprise the best-characterized pattern-recognition receptor (PRR) family able to activate distinct immune responses depending on the receptor/adaptor set assembled. TLRs, such as TLR2, TLR4 and TLR9, and their signaling were shown to be important in Paracoccidioides brasiliensis infections. However, the role of the endosomal TLR3 in experimental paracoccidioidomycosys remains obscure. In vitro assays, macrophages of the bone marrow of WT or TLR3-/- mice were differentiated for evaluation of their microbicidal activity. In vivo assays, WT or TLR3-/- mice were infected intratracheally with Paracoccidioides brasiliensis yeasts for investigation of the lung response type induced. The cytotoxic activity of CD8+ T cells was assessed by cytotoxicity assay. To confirm the importance of CD8+ T cells in the control of infection in the absence of tlr3, a depletion assay of these cells was performed. Here, we show for the first time that TLR3 modulate the infection against Paracoccidioides brasiliensis by dampening pro-inflammatory response, NO production, IFN+CD8+T, and IL-17+CD8+T cell activation and cytotoxic function, associated with granzyme B and perforin down regulation. As conclusion, we suggest that TLR3 could be used as an escape mechanism of the fungus in an experimental paracoccidioidomycosis.

Influência de TLR3 em modelo de Paracoccidioidomicose experimental / TLR3 influence in experimental PCM model

Os receptores do tipo Toll compreendem a família de receptores de reconhecimento de padrões melhor caracterizados, que podem ativar diferentes respostas imunes, dependendo de quais receptores e conjuntos de adaptadores são utilizados. Os TLRs, como TLR2, TLR4 e TLR9, e sua sinalização foram implicados no reconhecimento de P. brasiliensis e na regulação da resposta imune, no entanto, o papel do TLR3 ainda não está claro. Assim, a compreensão da função endossomal do TLR3 na PCM experimental é crucial. Utilizamos modelos in vitro e in vivo de infecção por P. brasiliensis, camundongos C57Bl/6 e TLR3-/-, para avaliar a contribuição da TLR3 no desenvolvimento da infecção. Mostramos que ausência de TLR3 leva o aumento de óxido nítrico e a capacidade fagocítica por macrófagos nas primeiras 4 horas de interação com leveduras P. brasiliensis. Mostramos ainda que os camundongos TLR3-/- desempenham papel protetor após 30 dias de infecção intratraqueal com P. brasiliensis, mostrando diminuição do aumento de CFU, perfil de resposta Th1 e Th17, bem como aumento de células citotóxicas T CD8+ produtoras de IFN-γ e IL-17. As células citotóxicas T CD8+ mostraram ser essenciais para o controle da infecção nos camundongos TLR3-/-, uma vez que a depleção dessas células levou a progressão da doença. Em estágios iniciais, 3 e 5 dias de infecção, observamos aumento do recrutamento de neutrófilos para o pulmão. Estudos recentes indicam que o TLR3 é um receptor importante para a resposta imune na micose e sua ausência favorece a infecção por fungos. Em contraste, nossos resultados mostram que, no caso do PCM, o TLR3 é prejudicial ao hospedeiro, sugerindo que a ativação do TLR3 pode ser um possível mecanismo de escape de P. brasiliensis

Toll-like receptors comprise the best-characterized pattern-recognition receptor family that can activate different immune responses, depending on which receptor and adaptor set are utilized. TLRs, such as TLR2, TLR4 and TLR9, and their signaling have been implicated in the recognition of P. brasiliensis and regulation of the immune response, however, the role of TLR3 remains unclear. Thus, understanding the endosomal function of TLR3 in experimental PCM is crucial. We used in vitro and in vivo models of infection by P. brasiliensis, C57Bl/6 and TLR3-/- mice, to assess the contribution of TLR3 on development of infection. We show that absence of TLR3 leads to increased nitric oxide and phagocytic capacity by macrophages in the first 4 hours of interaction with yeasts P. brasiliensis. We also showed that TLR3-/- mice play a protective role after 30 days of intratracheal infection with P. brasiliensis, showing a decrease in the CFU increase, Th1 and Th17 response profile, as well as an increase in cytotoxic CD8+ cells producing IFN-γ and IL-17. The cytotoxic T CD8+ cells were shown to be essential for the control of infection in TLR3-/- mice, since the depletion of these cells led to the progression of the disease. In the initial stages, 3 and 5 days of infection, we observed increased recruitment of neutrophils to the lung. Recent studies indicate that TLR3 is an important receptor for the immune response in mycosis and its absence favors fungal infection. In contrast, our results show that in the case of PCM, TLR3 is detrimental to the host, suggesting that TLR3 activation may be a possible escape mechanism of P. brasiliensis

Extracellular vesicles from Sporothrix brasiliensis are an important virulence factor that induce an increase in fungal burden in experimental sporotrichosis

Sporotrichosis is a mycosis that affects the skin, lymphatic system and other organs in humans and animals. The disease has a worldwide distribution, with endemic areas in Brazil, and is caused by a complex of species, including Sporothrix brasiliensis. Some fungi release extracellular vesicles (EVs) that can interact with the host cell and modulate the host immune response. The aim of this study was to analyze the participation of S. brasiliensis EVs in the modulation of dendritic cells (DCs) and in the control of infection in vivo. Our results showed that in vitro, the EVs isolated from S. brasiliensis induced an increase in the phagocytic index and fungal burden in DCs. In addition, we observed a significant increase in IL-12p40 and TNF-alpha cytokine production. Then, the EVs were inoculated into BALB/c mice before subcutaneous infection with yeast, and the lesion was analyzed after 21, 35, and 42 days. An increase in fungal burden and lesion diameter were observed after 21 days in mice inoculated with a high concentration of EVs. However, after 35 days, we observed a regression of the lesion, which persisted until 42 days after infection. Interestingly, we observed an increase in fungal burden in these mice. In addition, we observed the presence of immunogenic components and proteins that could be related with virulence in EVs. These results suggest that EVs can play an important role in virulence and modulation of the host immune system during experimental S. brasiliensis infection.

Extracellular vesicles from Sporothrix brasiliensis are an important virulence factor that induce an increase in fungal burden in experimental sporotrichosis

Sporotrichosis is a mycosis that affects the skin, lymphatic system and other organs in humans and animals. The disease has a worldwide distribution, with endemic areas in Brazil, and is caused by a complex of species, including Sporothrix brasiliensis. Some fungi release extracellular vesicles (EVs) that can interact with the host cell and modulate the host immune response. The aim of this study was to analyze the participation of S. brasiliensis EVs in the modulation of dendritic cells (DCs) and in the control of infection in vivo. Our results showed that in vitro, the EVs isolated from S. brasiliensis induced an increase in the phagocytic index and fungal burden in DCs. In addition, we observed a significant increase in IL-12p40 and TNF-alpha cytokine production. Then, the EVs were inoculated into BALB/c mice before subcutaneous infection with yeast, and the lesion was analyzed after 21, 35, and 42 days. An increase in fungal burden and lesion diameter were observed after 21 days in mice inoculated with a high concentration of EVs. However, after 35 days, we observed a regression of the lesion, which persisted until 42 days after infection. Interestingly, we observed an increase in fungal burden in these mice. In addition, we observed the presence of immunogenic components and proteins that could be related with virulence in EVs. These results suggest that EVs can play an important role in virulence and modulation of the host immune system during experimental S. brasiliensis infection.

The Critical Role of Notch1-TLR 4 Signaling in the Inflammatory and Fungicidal Activity of Macrophages Against Paracoccidioides brasiliensis Strain Pb18.

Paracoccidioidomycosis is a systemic mycosis of deep nature that primarily affects the lung and can spread via lymphatic and hematogenous to other organs and tissues. It is mainly caused by Paracoccidioides brasiliensis fungus which exhibits thermal dimorphism. The innate immune system mediated by macrophages is extremely important for the control of infection and is involved in the induction and regulation of immune/inflammatory response. These cells are able to recognize pathogens through pattern recognition receptors (PRRs) such as Toll-like receptors (TLR). Beyond these PRRs, the importance of Notch signaling has recently been demonstrated in the innate immune system and the regulation of macrophage activity. Our data demonstrate that the Pb18 strain of P. brasiliensis is able to activate the transcription of Notch1 receptor in J774 macrophages. Activation of this receptor with also activation of TLR 4 (via LPS) induces IL-6 production, which favors the pathogenesis. By using a γ-secretase pharmacological inhibitor (DAPT) for inhibiting the activation of Notch1 receptor on macrophages, it is possible to observe the decreased fungal burden, less production of IL-6, and increased TNF-α and phagocytosis. Taken together, these results showed that Pb18 is able to induce the transcription of Notch1 receptor on macrophages and may provide a new immunity study approach in experimental paracoccidioidomycosis.

scFv from Antibody That Mimics gp43 Modulates the Cellular and Humoral Immune Responses during Experimental Paracoccidioidomycosis.

Paracoccidioidomycosis (PCM), caused by Paracoccidioides species is a prevalent systemic and progressive mycosis that occurs in Latin America. It is caused by Paracoccidioides species. Immunization with dendritic cells transfected with a plasmid encoding the scFv (pMAC/PS-scFv) that mimics the main antigen of P. brasiliensis (gp43) confers protection in experimental PCM. DCs link innate and adaptive immunity by recognizing invading pathogens and selecting the type of effector T cell to mediate the immune response. Here, we showed that DC-pMAC/PS-scFv induces the activation of CD4+ and CD8+ T cells. Moreover, our results demonstrated that BALB/c mice infected with P. brasiliensis and treated with DC-pMAC/PS-scFv showed the induction of specific IgG production against gp43 and IFN-γ, IL-12 and IL-4 cytokines. Analysis of regional lymph nodes revealed increases in the expression of clec7a, myd88, tlr2, gata3 and tbx21, which are involved in the immune response. Taken together, our results indicate that the scFv modulates the humoral and cellular immune responses and presents epitopes to CD4+ and CD8+ T cells.

Therapeutic vaccine using a monoclonal antibody against a 70-kDa glycoprotein in mice infected with highly virulent Sporothrix schenckii and Sporothrix brasiliensis.

Sporotrichosis is a chronic granulomatous mycosis caused by the dimorphic fungi that comprise the Sporothrix complex. The latter are widely distributed in nature, developing a saprophytic mycelial form on plant debris and soil. Formerly, the S. schenckii species was thought to be the only species capable of causing sporotrichosis. However, in recent years, the existence of a group of highly genotypically and phenotypically variable species has been reported as etiologic agents of this mycosis. Recently, it has become important to study aspects such as virulence and the immune response against key members of the Sporothrix complex and to observe the presence of glycoprotein (gp) 70 and efficacy of the P6E7 monoclonal antibody against more virulent strains. The data presented here demonstrate that the strain isolated from a case of feline sporotrichosis, that is, strain 5110 (American Type Culture Collection MYA-4823) is the most virulent and the only one able to secrete gp70. This glycoprotein is apparently an important factor in the virulence of Sporothrix spp. because treatment with MAb P6E7 resulted in the reduction of fungal burden in the analyzed organs. Additional studies of the role of gp70 in modulating the immune response of the host are needed to understand the pathology of sporotrichosis.