RESUMO
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.
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Citocinas , Terapia com Luz de Baixa Intensidade , Paracoccidioidomicose , Células Th1 , Células Th2 , Animais , Camundongos , Citocinas/metabolismo , Células Th1/imunologia , Células Th1/metabolismo , Paracoccidioidomicose/radioterapia , Paracoccidioidomicose/imunologia , Paracoccidioidomicose/patologia , Células Th2/imunologia , Células Th2/metabolismo , Paracoccidioides/imunologia , Camundongos Endogâmicos BALB C , MasculinoRESUMO
Paracoccidioidomycosis (PCM), initially reported in 1908 in the city of São Paulo, Brazil, by Adolpho Lutz, is primarily a systemic and neglected tropical mycosis that may affect individuals with certain risk factors around Latin America, especially Brazil. Paracoccidioides brasiliensis sensu stricto, a classical thermodimorphic fungus associated with PCM, was long considered to represent a monotypic taxon. However, advances in molecular taxonomy revealed several cryptic species, including Paracoccidioides americana, P. restrepiensis, P. venezuelensis, and P. lutzii, that show a preference for skin and mucous membranes, lymph nodes, and respiratory organs but can also affect many other organs. The classical diagnosis of PCM benefits from direct microscopy culture-based, biochemical, and immunological assays in a general microbiology laboratory practice providing a generic identification of the agents. However, molecular assays should be employed to identify Paracoccidioides isolates to the species level, data that would be complemented by epidemiological investigations. From a clinical perspective, all probable and confirmed cases should be treated. The choice of treatment and its duration must be considered, along with the affected organs, process severity, history of previous treatment failure, possibility of administering oral medication, associated diseases, pregnancy, and patient compliance with the proposed treatment regimen. Nevertheless, even after appropriate treatment, there may be relapses, which generally occur 5 years after the apparent cure following treatment, and also, the mycosis may be confused with other diseases. This review provides a comprehensive and critical overview of the immunopathology, laboratory diagnosis, clinical aspects, and current treatment of PCM, highlighting current issues in the identification, treatment, and patient follow-up in light of recent Paracoccidioides species taxonomic developments.
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Paracoccidioides , Paracoccidioidomicose , Humanos , Paracoccidioidomicose/diagnóstico , Paracoccidioidomicose/tratamento farmacológico , Paracoccidioidomicose/epidemiologia , Brasil , PeleRESUMO
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.
Assuntos
Paracoccidioides , Paracoccidioidomicose , Fatores de Virulência/metabolismo , Animais , Antifúngicos/uso terapêutico , Parede Celular/metabolismo , América Central/epidemiologia , Proteínas Fúngicas/metabolismo , Glucanos/metabolismo , Glicoproteínas/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Melaninas/metabolismo , Paracoccidioides/efeitos dos fármacos , Paracoccidioides/isolamento & purificação , Paracoccidioides/metabolismo , Paracoccidioides/patogenicidade , Paracoccidioidomicose/epidemiologia , Paracoccidioidomicose/metabolismo , Paracoccidioidomicose/patologia , Paracoccidioidomicose/terapia , Prevalência , América do Sul/epidemiologiaRESUMO
The antifungal drug therapy often employed to treat paracoccidiodomycosis (PCM), an important neglected fungal systemic infection, leads to offensive adverse effects, besides being very long-lasting. In addition, PCM compromises the oral health of patients by leading to oral lesions that are very painful and disabling. In that way, photodynamic therapy (PDT) arises as a new promising adjuvant treatment for inactivating Paracoccidioides brasiliensis (Pb), the responsible fungus for PCM, and also for helping the patients to deal with such debilitating oral lesions. PDT has been linked to an improved microbial killing, also presenting the advantage of not inducing immediate microbial resistance such as drugs. For the present study, we investigated the generation of reactive oxygen species (ROS) by using the fluorescent probes hydroxyphenyl fluorescein (HPF) and aminophenyl fluorescein (APF) after toluidine blue (TBO-37.5 mg/L)-mediated PDT (660 nm, 40 mW, and 0.04 cm2 spot area) and the action of TBO-PDT upon Pb cultures grown for 7 or 15 days in semisolid Fava Netto's culture medium; we also targeted oral PCM manifestations by reporting the first clinical cases (three patients) to receive topic PDT for such purpose. We were able to show a significant generation of hydroxyl radicals and hypochlorite after TBO-PDT with doses around 90 J/cm2; such ROS generation was particularly useful to attack and inactivate Pb colonies at 7 and 15 days. All three patients reported herein related an immediate relief when it came to pain, mouth opening, and also the ability to chew and swallow. As extracted from our clinical results, which are in fact based on in vitro outcomes, TBO-PDT is a very safe, inexpensive, and promising therapy for the oral manifestations of PCM.
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Viabilidade Microbiana/efeitos dos fármacos , Doenças da Boca/tratamento farmacológico , Doenças da Boca/microbiologia , Paracoccidioides/efeitos da radiação , Paracoccidioidomicose/tratamento farmacológico , Paracoccidioidomicose/microbiologia , Fotoquimioterapia , Cloreto de Tolônio/uso terapêutico , Adulto , Antifúngicos/farmacologia , Corantes Fluorescentes/química , Humanos , Cinética , Masculino , Pessoa de Meia-Idade , Doenças da Boca/patologia , Paracoccidioides/crescimento & desenvolvimento , Espécies Reativas de Oxigênio/metabolismo , Cloreto de Tolônio/farmacologiaRESUMO
The NADH:ubiquinone oxidoreductase, respiratory complex I, couples electron transfer from NADH to ubiquinone with the translocation of protons across the membrane. The complex consists of a peripheral arm catalyzing the redox reaction and a membrane arm catalyzing proton translocation. The membrane arm is almost completely aligned by a 110 Å unique horizontal helix that is discussed to transmit conformational changes induced by the redox reaction in a piston-like movement to the membrane arm driving proton translocation. Here, we analyzed such a proposed movement by cysteine-scanning of the helix of the Escherichia coli complex I. The accessibility of engineered cysteine residues and the flexibility of individual positions were determined by labeling the preparations with a fluorescent marker and a spin-probe, respectively, in the oxidized and reduced states. The differences in fluorescence labeling and the rotational flexibility of the spin probe between both redox states indicate only slight conformational changes at distinct positions of the helix but not a large movement.
Assuntos
Complexo I de Transporte de Elétrons/química , Complexo I de Transporte de Elétrons/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Cisteína , Espectroscopia de Ressonância de Spin Eletrônica , Transporte de Elétrons , Escherichia coli/genética , Escherichia coli/metabolismo , Modelos Moleculares , Mutação , NAD/metabolismo , NADH Desidrogenase/química , NADH Desidrogenase/metabolismo , Oxirredução , Prótons , Ubiquinona/metabolismoRESUMO
BACKGROUND: Paracoccidioidomycosis, a chronic granulomatous fungal disease caused by Paracoccidioides brasiliensis yeast cells affects mainly rural workers, albeit recently cases in immunosuppressed individuals has been reported. Protective immune response against P. brasiliensis is dependent on the activity of helper T cells especially IFN-γ-producing Th1 cells. It has been proposed that Paracoccidioides brasiliensis is able to modulate the immune response towards a permissive state and that the thymus plays a major role in it. METHODS: In this paper, we show that acute infection of BALB/c mice with P. brasiliensis virulent isolate (Pb18) might cause alterations in the thymic environment as well as the prohibitive TCR-expressing T cells in the spleens. RESULTS: After seven days of infection, we found yeast cells on the thymic stroma, the thymic epithelial cells (TEC) were altered regarding their spatial-orientation and inflammatory mediators gene expression was increased. Likewise, thymocytes (differentiating T cells) presented higher migratory ability in ex vivo experiments. Notwithstanding, P. brasiliensis-infected mice showed an increased frequency of prohibitive TCR-expressing T cells in the spleens, suggesting that the selection processes that occur in the thymus may be compromised during the acute infection. CONCLUSION: In this paper, for the first time, we show that acute infection with Paracoccidioides brasiliensis yeast cells promotes thymic alterations leading to a defective repertoire of peripheral T cells. The data presented here may represent new mechanisms by which P. brasiliensis subverts the immune response towards the chronic infection observed in humans.
Assuntos
Paracoccidioides/fisiologia , Paracoccidioidomicose/imunologia , Receptores de Antígenos de Linfócitos T/genética , Timo/microbiologia , Animais , Humanos , Ativação Linfocitária , Linfócitos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Paracoccidioidomicose/genética , Paracoccidioidomicose/microbiologia , Receptores de Antígenos de Linfócitos T/imunologia , Baço/imunologia , Células Th1/imunologia , Timo/imunologiaRESUMO
Paracoccidioidomycosis is a systemic infection prevalent in Latin American countries. Disease develops after inhalation of Paracoccidioides brasiliensis conidia followed by an improper immune activation by the host leucocytes. Dendritic cells (DCs) are antigen-presenting cells with the unique ability to direct the adaptive immune response by the time of activation of naive T cells. This study was conducted to test whether extracts of P. brasiliensis would induce maturation of DCs. We found that DCs treated with extracts acquired an inflammatory phenotype and upon adoptive transfer conferred protection to infection. Interestingly, interleukin-10 production by CD8(+) T cells was ablated following DC transfer. Further analyses showed that lymphocytes from infected mice were high producers of interleukin-10, with CD8(+) T cells being the main source. Blockage of cross-presentation to CD8(+) T cells by modulated DCs abolished the protective effect of adoptive transfer. Collectively, our data show that adoptive transfer of P. brasiliensis-modulated DCs is an interesting approach for the control of infection in paracoccidioidomycosis.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Interleucina-10/biossíntese , Paracoccidioides/imunologia , Paracoccidioidomicose/imunologia , Paracoccidioidomicose/prevenção & controle , Transferência Adotiva , Animais , Antígenos de Fungos/farmacologia , Diferenciação Celular/imunologia , Apresentação Cruzada , Citocinas/biossíntese , Células Dendríticas/citologia , Células Dendríticas/microbiologia , Feminino , Vacinas Fúngicas/imunologia , Vacinas Fúngicas/farmacologia , Mediadores da Inflamação/metabolismo , Interleucina-10/antagonistas & inibidores , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Paracoccidioidomycosis (PCM) is a systemic fungal infection, endemic in Brazil, that leads to severe morbidity and even mortality if not correctly treated. Patients may respond differently to PCM depending on the pattern of the acquired immune response developed. The onset of protective immune response is notably mediated by neutrophils (PMN) that play an important role through directly killing the fungi and also by interacting with other cell types to modulate the acquired protective immune response that may follow. In that way, this study aimed to present and compare different experimental models of PCM (intraperitoneal and subcutaneous) regarding PMN production and maturation inside femoral bone marrow and also PMN infiltration in peritoneal and subcutaneous exudates of resistant and susceptible mice. We also assessed the fungal colony forming units and the levels of soluble inflammatory mediators (LTB4, KC, IFN-γ, GM-CSF, and IL-10) inside subcutaneous air-pouches to compare the efficiency of the PMN present at this site in relation to the two main neutrophil functions: initial lysis of the invading pathogen and modulation of the acquired immune response. P. brasiliensis inoculated intraperitoneally was able to disseminate to the bone marrow of susceptible mice, causing a more marked alteration of PMN production and maturation than that observed after resistant mice infection by the same route. Subcutaneous air-pouch inoculation of P. brasiliensis elicited a controlled and limited infection that produced a PMN-rich exudate, thus favoring the study of the interaction between the fungus and the neutrophils. Susceptible mice produced higher numbers of PMN; however, these cells were less effective in killing the fungi. Inflammatory cytokines were more pronounced in resistant mice, which supports their PCM raised resistance.
Assuntos
Movimento Celular , Mediadores da Inflamação/fisiologia , Ativação de Neutrófilo , Neutrófilos/imunologia , Paracoccidioidomicose/imunologia , Animais , Medula Óssea/patologia , Interleucina-10/fisiologia , Masculino , Camundongos , Neutrófilos/citologia , Paracoccidioides , Paracoccidioidomicose/patologiaRESUMO
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.
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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.
Assuntos
Antifúngicos , Celecoxib , Neutrófilos , Paracoccidioides , Paracoccidioidomicose , Animais , Paracoccidioides/efeitos dos fármacos , Paracoccidioides/imunologia , Camundongos , Celecoxib/farmacologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Paracoccidioidomicose/tratamento farmacológico , Paracoccidioidomicose/imunologia , Antifúngicos/farmacologia , Antifúngicos/uso terapêutico , Citocinas/metabolismo , Células Cultivadas , Masculino , Baço/imunologia , Baço/citologia , Baço/efeitos dos fármacos , Modelos Animais de Doenças , Espécies Reativas de Oxigênio/metabolismoRESUMO
Paracoccidioidomycosis (PCM) caused by Paracoccidioides brasiliensis (Pb), is a severe mycosis, prevalent in tropical countries. The presence of polymorphonuclear neutrophils (PMN) in lesions is conspicuous, indicating their central role in innate immunity through the direct killing of Pb and the production of cytokines that activate acquired immunity in the presence of itraconazole (Itra). The toxicity and direct antifungal activity of Itra on Pb in splenocyte co-cultures were evaluated in vitro. Itra showed no toxic effect but marked antifungal activity against Pb. Purified PMN were obtained by the subcutaneous (SC) injection of Pb into mice. Results showed the effect of Itra on the size of the air pouch produced, the cellular population that migrated to the infection site, protein, and mitochondrial metabolism patterns, production of ROS an NO, and the number of cytokines synthesized. Lower doses (3 and 10 mg/kg) of Itra did not affect the cellular profile but led to a lower influx of viable more active PMN, and increased production of ROS and proteins. At a dose of 50 mg/kg the PMN profile remained unchanged along with all other parameters analyzed remained unaltered. Decreases in most cytokine levels were inversely proportional to the Itra concentration. Lower Itra concentrations may elicit activation of the immune response because the combined effects of therapy and immune response are needed, while the higher dose does not require it. Itra also promotes the activation of the cytokines which elicit PMN activation and consequently the resolution of Pb18 infection in the air pouch.
Assuntos
Neutrófilos , Paracoccidioidomicose , Animais , Camundongos , Neutrófilos/metabolismo , Paracoccidioidomicose/tratamento farmacológico , Paracoccidioidomicose/microbiologia , Itraconazol/farmacologia , Antifúngicos/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Chumbo/metabolismo , Citocinas/metabolismo , Imunidade InataRESUMO
Live-cell RNA imaging with high spatial and temporal resolution remains a major challenge. Here we report the development of RhoBAST:SpyRho, a fluorescent light-up aptamer (FLAP) system ideally suited for visualizing RNAs in live or fixed cells with various advanced fluorescence microscopy modalities. Overcoming problems associated with low cell permeability, brightness, fluorogenicity, and signal-to-background ratio of previous fluorophores, we design a novel probe, SpyRho (Spirocyclic Rhodamine), which tightly binds to the RhoBAST aptamer. High brightness and fluorogenicity is achieved by shifting the equilibrium between spirolactam and quinoid. With its high affinity and fast ligand exchange, RhoBAST:SpyRho is a superb system for both super-resolution SMLM and STED imaging. Its excellent performance in SMLM and the first reported super-resolved STED images of specifically labeled RNA in live mammalian cells represent significant advances over other FLAPs. The versatility of RhoBAST:SpyRho is further demonstrated by imaging endogenous chromosomal loci and proteins.
Assuntos
Corantes Fluorescentes , Oligonucleotídeos , Animais , Rodaminas , Ionóforos , Microscopia de Fluorescência , RNA , MamíferosRESUMO
Protective immunity against Paracoccidioides consists of a stepwise activation of numerous effector mechanisms that comprise many cellular and soluble components. At the initial phase of non-specific innate immunity, resistance against Paracoccidioides comes from phagocytic polymorphonuclear neutrophils, natural killer (NK) cells and monocytes, supplemented by soluble factors such as cytokines and complement system components. Invariant receptors (Toll-like receptors (TLRs), Dectins) which are present in cells of the immune system, detect patterns present in Paracoccidioides (but not in the host) informing the hosts cells that there is an infection in progress, and that the acquired immunity must be activated. The role of components involved in the innate immunity of paracoccidioidomycosis is herein presented. Humoral immunity, represented by specific antibodies which control the fungi in the blood and body fluids, and its role in paracoccidioidomycosis (which was previously considered controversial) is also discussed. The protective mechanisms (involving various components) of cellular immunity are also discussed, covering topics such as: lysis by activated macrophages and cytotoxic T lymphocytes, the participation of lytic products, and the role of cytokines secreted by T helper lymphocytes in increasing the efficiency of Paracoccidioides, lysis.
RESUMO
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.
Assuntos
Antifúngicos/farmacologia , Paracoccidioides/efeitos dos fármacos , Paracoccidioidomicose/tratamento farmacológico , Própole/farmacologia , Animais , Antifúngicos/administração & dosagem , Antifúngicos/isolamento & purificação , Brasil , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Feminino , Inflamação/tratamento farmacológico , Inflamação/microbiologia , Camundongos , Neutrófilos/metabolismo , Paracoccidioidomicose/microbiologia , Própole/administração & dosagem , Própole/isolamento & purificação , Espécies Reativas de Oxigênio/metabolismoRESUMO
Fungal infections have been increasing in recent decades, mainly affecting immunocompromised individuals, although certain mycoses, such as paracoccidioidomycosis (PCM), infect immunologically competent individuals. The major problems observed regarding fungal diseases are inadequate diagnosis, prolonged treatment time, the reduced number of drugs available for treatment, in addition to the fact that there are no vaccines for clinical use. Drug combination in order to immunomodulate the immune response is a new strategy used for the treatment of mycoses, since it is difficult to develop new antifungal drugs. The aim of this study is to present and analyze strategies recently suggested for the treatment of fungi of medical interest, in particular for PCM, such as the utilization of combinations of protein fractions or dead microorganisms, as vaccinal antigens, and cellular immunotherapy. We will also propose new therapeutic alternatives, such as lipids, vitamins, synthetic or natural products as well as the use of low intensity LASER therapy (LLLT) to modulate the immune response of the host, enhancing the efficiency of the existing treatments of mycoses of medical interest and in particular of PCM.
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Antifúngicos/uso terapêutico , Imunomodulação , Paracoccidioidomicose/tratamento farmacológico , Humanos , Imunoterapia , Paracoccidioidomicose/imunologiaRESUMO
Matrix metalloproteinases (MMPs) modulate extracellular matrix turnover, inflammation and immunity. We studied MMP-9 and MMP-2 in experimental paracoccidioidomycosis. At 15 and 120 days after infection (DAI) with virulent Paracoccidioides brasiliensis, MMP-9 was positive by immunohistochemistry in multinucleated giant cells, in mononuclear cells with macrophage and lymphocyte morphologies and also in fungal cells in the lesions of susceptible and resistant mice. Using gelatin zymography, pro- and active MMP-9 and active MMP-2 were detected in all infected mice, but not in controls. Gelatinolytic activity was not observed in P. brasiliensis extracts. Semiquantitative analysis of gelatinolytic activities revealed weak or absent MMP-2 and strong MMP-9 activity in both mouse strains at 15 DAI, declining at 120 DAI. Avirulent P. brasiliensis-infected mice had residual lesions with MMP-9-positive pseudoxantomatous macrophages, but no gelatinase activity at 120 DAI. Our findings demonstrate the induction of MMPs, particularly MMP-9, in experimental paracoccidioidomycosis, suggesting a possible influence in the pattern of granulomas and in fungal dissemination.
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Metaloproteinases da Matriz/metabolismo , Paracoccidioides , Paracoccidioidomicose/enzimologia , Animais , Feminino , Gelatina/metabolismo , Granuloma/enzimologia , Granuloma/microbiologia , Técnicas Imunoenzimáticas , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos , Omento/enzimologia , Doenças Peritoneais/enzimologia , Doenças Peritoneais/microbiologiaRESUMO
The role of nitric oxide (NO) in granulomas of Paracoccidioides brasiliensis-infected inducible NO synthase-deficient C57BL/6 mice (iNOS KO) and their wild-type counterparts and its association with osteopontin (OPN) and matrix metalloproteinases (MMPs) was studied. At 15 days after infection (DAI), iNOS KO mice showed compact and necrotic granulomas with OPN+ macrophages and multinucleated giant cells, whereas wild-type mice developed loose granulomas with many fungi and OPN+ cells distributed throughout the tissue. In addition, high OPN levels and fungal load were observed in iNOS KO mice. Both experimental groups had MMP-9 activity. At 120 DAI, iNOS KO had smaller granulomas with OPN+ cells, lower OPN levels, lower fungal load and decreased MMP-9 activity compared with wild-type mice. These findings suggest that NO has an important role in granuloma modulation, by controlling OPN and MMP production, as well as by inducing loose granulomas formation and fungal dissemination, resulting, at later phases, in progression of paracoccidioidomycosis.
Assuntos
Granuloma/imunologia , Óxido Nítrico/imunologia , Paracoccidioides/imunologia , Paracoccidioidomicose/imunologia , Animais , Feminino , Granuloma/microbiologia , Macrófagos/imunologia , Macrófagos/microbiologia , Metaloproteinase 2 da Matriz/imunologia , Metaloproteinase 9 da Matriz/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico Sintase Tipo II/imunologia , Omento/imunologia , Omento/microbiologia , Omento/patologia , Osteopontina/imunologia , Paracoccidioidomicose/microbiologiaRESUMO
The participation of osteopontin (OPN) in Paracoccidioides brasiliensis infected mice, its association to granulomatogenesis, severity of infection, pattern of lesions, nitric oxide (NO) levels and fungal load were evaluated in this investigation. Immunohistochemistry analysis showed marked OPN staining in extracellular matrix and in macrophages and multinucleated giant cells at the center of lesions, suggesting a possible role of OPN in the distribution of these cells within the granulomas. At 15 days post-infection with a virulent P. brasiliensis isolate, OPN+ cells were more numerous and intensely immunostained in the loose granulomas of susceptible mice than in those of resistant mice. In addition, high fungal loads and low NO levels were observed in susceptible mice. At 120 days after infection, resistant mice had increased total OPN levels (ELISA) and OPN positivity in compact granulomas, higher NO levels and lower fungal loads than susceptible mice. Residual lesions associated with low OPN levels, high NO and control of fungal dissemination were observed in both mouse strains at 120 days post-infection with the slightly virulent fungal isolate. Therefore, OPN could be associated with higher severity of the disease in an early phase of infection and with a degree of control of the progressive infection.
Assuntos
Granuloma/metabolismo , Osteopontina/metabolismo , Paracoccidioides , Paracoccidioidomicose/metabolismo , Análise de Variância , Animais , Contagem de Colônia Microbiana , Modelos Animais de Doenças , Feminino , Granuloma/microbiologia , Imuno-Histoquímica , Camundongos , Óxido Nítrico/metabolismo , Omento/química , Osteopontina/análise , Índice de Gravidade de DoençaRESUMO
BACKGROUND: Paracoccidioidomycosis (PCM) is a systemic mycosis commonly found in Latin America that is caused by distinct species of Paracoccidioides genus: Paracoccidioides brasiliensis complex (S1, PS2, PS3 and PS4) and Paracoccidioides lutzii. Its pathobiology has been recently explored by different approaches to clarify the mechanisms of host-pathogen interactions underpinning PCM. The diversity of clinical forms of this disease has been attributed to both host- and fungus-related factors. METHODOLOGY/PRINCIPAL FINDINGS: For better understanding of the molecular underpinnings of host-fungus interactions, we evaluated in vivo virulence of nine Paracoccidioides brasiliensis complex isolates and correlated it to protein expression profiles obtained by two-dimensional gel electrophoresis. Based on the recovery of viable fungi from mouse organs, the isolates were classified as those having low, moderate, or high virulence. Highly virulent isolates overexpressed proteins related to adhesion process and stress response, probably indicating important roles of those fungal proteins in regulating the colonization capacity, survival, and ability to escape host immune system reaction. Moreover, highly virulent isolates exhibited enhanced expression of glycolytic pathway enzymes concomitantly with repressed expression of succinyl-CoA ligase beta chain, a protein related to the tricarboxylic acid cycle. CONCLUSIONS/SIGNIFICANCE: Our findings may point to the mechanisms used by highly virulent P. brasiliensis isolates to withstand host immune reactions and to adapt to transient iron availability as strategies to survive and overcome stress conditions inside the host.
Assuntos
Proteínas Fúngicas/imunologia , Regulação Fúngica da Expressão Gênica/imunologia , Paracoccidioides , Paracoccidioidomicose/imunologia , Animais , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Paracoccidioides/imunologia , Paracoccidioides/isolamento & purificação , Paracoccidioides/patogenicidade , Paracoccidioidomicose/patologiaRESUMO
Cancer-associated fibroblasts constitute a vital subpopulation of the tumor stroma and are present in more than 90% of epithelial carcinomas. The overexpression of the serine protease fibroblast activation protein (FAP) allows a selective targeting of a variety of tumors by inhibitor-based radiopharmaceuticals (FAPIs). Of these compounds, FAPI-04 has been recently introduced as a theranostic radiotracer and demonstrated high uptake into different FAP-positive tumors in cancer patients. To enable the delivery of higher doses, thereby improving the outcome of a therapeutic application, several FAPI variants were designed to further increase tumor uptake and retention of these tracers. Methods: Novel quinoline-based radiotracers were synthesized by organic chemistry and evaluated in radioligand binding assays using FAP-expressing HT-1080 cells. Depending on their in vitro performance, small-animal PET imaging and biodistribution studies were performed on HT-1080-FAP tumor-bearing mice. The most promising compounds were used for clinical PET imaging in 8 cancer patients. Results: Compared with FAPI-04, 11 of 15 FAPI derivatives showed improved FAP binding in vitro. Of these, 7 compounds demonstrated increased tumor uptake in tumor-bearing mice. Moreover, tumor-to-normal-organ ratios were improved for most of the compounds, resulting in images with higher contrast. Notably two of the radiotracers, FAPI-21 and -46, displayed substantially improved ratios of tumor to blood, liver, muscle, and intestinal uptake. A first diagnostic application in cancer patients revealed high intratumoral uptake of both radiotracers already 10 min after administration but a higher uptake in oral mucosa, salivary glands, and thyroid for FAPI-21. Conclusion: Chemical modification of the FAPI framework enabled enhanced FAP binding and improved pharmacokinetics in most of the derivatives, resulting in high-contrast images. Moreover, higher doses of radioactivity can be delivered while minimizing damage to healthy tissue, which may improve therapeutic outcome.