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OBJECTIVE: To investigate the effect of pre and postnatal exposure to a glyphosate-based herbicide on glucose metabolism and liver histology in adult F1 mice offspring. METHODS: Female mice (C57Bl/6) received 0.5% of glyphosate (Roundup Original DI®) in drinking water or purified water (Glyphosate Group and Control Group respectively) during pregnancy and lactation. Offspring (F1) were submitted to glucose and insulin tolerance tests and euthanized on postnatal day 150. Body and plasma parameters, and liver histology were analyzed. RESULTS: Exposure to glyphosate reduced maternal body weight gain during pregnancy and lactation, with no impacts on litter size. Pre and postnatal exposure to glyphosate did not affect body parameters but increased glucose tolerance on postnatal day 60. In spite of glucose tolerance normalization by postnatal day 143, this effect was associated with higher insulin sensitivity relative to mice in the Control-F1 Group. Mice in the Glyphosate-F1 Group had mild and moderate lobular inflammation in the liver. CONCLUSION: Maternal exposure to glyphosate affected insulin sensitivity and caused hepatic inflammation in adult F1 mice offspring.
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Herbicidas , Resistencia a la Insulina , Efectos Tardíos de la Exposición Prenatal , Animales , Femenino , Glucosa/metabolismo , Glicina/análogos & derivados , Herbicidas/metabolismo , Herbicidas/toxicidad , Humanos , Inflamación/inducido químicamente , Insulina , Hígado/metabolismo , Ratones , Embarazo , Efectos Tardíos de la Exposición Prenatal/inducido químicamente , Efectos Tardíos de la Exposición Prenatal/metabolismo , Ratas , Ratas Wistar , GlifosatoRESUMEN
Maternal obesity increases the risk of nonalcoholic fatty liver disease (NAFLD) in offspring. The Roux-en-Y gastric bypass (RYBG) is effective for achieving weight loss and ameliorates NAFLD. To determine whether these benefits are maintained after pregnancy and/or lactation, and whether they modulate hepatic morphofunction in the next generation, we evaluated hepatic lipid metabolism in Western diet (WD)-obese female rats that underwent RYGB and in their F1 offspring at adulthood. Female Wistar rats consumed a WD from 21 to 130 days of age, before being submitted to RYGB (WD-RYGB-F0) or SHAM (WD-SHAM-F0) operations. After 5 weeks, these females were mated with control male breeders, and the male and female F1 offspring were identified as WD-RYGB-F1 and WD-SHAM-F1. WD-RYGB-F0 dams exhibited lower serum lipids levels, but severe hepatic steatosis and pathological features of advanced liver injury. The hepatic proteins involved in lipogenesis were reduced in WD-RYGB-F0, as were the genes related to ß-oxidation and bile acids (BAs). Although the female and male WD-RYGB-F1 groups did not exhibit hepatic steatosis, the livers of female WD-RYGB-F1 demonstrated higher amounts of lipogenic genes and proteins, while male WD-RYGB-F1 presented a similar downregulation of lipogenic factors to that seen in WD-RYGB-F0 dams. In contrast, maternal and offspring groups of both sexes displayed reductions in the expressions of genes involved in BAs physiology and gluconeogenesis. As such, RYGB aggravates NAFLD after pregnancy and lactation and induces a gender-dependent differential expression of the hepatic lipogenesis pathway in offspring, indicating that female WD-RYGB-F1 may be an increased risk of developing NAFLD.
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Derivación Gástrica , Enfermedad del Hígado Graso no Alcohólico , Adulto , Animales , Dieta Alta en Grasa/efectos adversos , Femenino , Derivación Gástrica/efectos adversos , Humanos , Lactancia , Lipogénesis , Hígado/metabolismo , Masculino , Enfermedad del Hígado Graso no Alcohólico/etiología , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Obesidad/complicaciones , Obesidad/cirugía , Embarazo , Ratas , Ratas WistarRESUMEN
ABSTRACT Objective: To investigate the effect of pre and postnatal exposure to a glyphosate-based herbicide on glucose metabolism and liver histology in adult F1 mice offspring. Methods: Female mice (C57Bl/6) received 0.5% of glyphosate (Roundup Original DI®) in drinking water or purified water (Glyphosate Group and Control Group respectively) during pregnancy and lactation. Offspring (F1) were submitted to glucose and insulin tolerance tests and euthanized on postnatal day 150. Body and plasma parameters, and liver histology were analyzed. Results: Exposure to glyphosate reduced maternal body weight gain during pregnancy and lactation, with no impacts on litter size. Pre and postnatal exposure to glyphosate did not affect body parameters but increased glucose tolerance on postnatal day 60. In spite of glucose tolerance normalization by postnatal day 143, this effect was associated with higher insulin sensitivity relative to mice in the Control-F1 Group. Mice in the Glyphosate-F1 Group had mild and moderate lobular inflammation in the liver. Conclusion: Maternal exposure to glyphosate affected insulin sensitivity and caused hepatic inflammation in adult F1 mice offspring.
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INTRODUÇÃO: À medida que a população envelhece e a expectativa de vida aumenta, a incidência global e a prevalência de AVC isquêmico tendem a aumentar significativamente. Nesse contexto, surge a necessidade de avaliar novos marcadores preditores de mortalidade, como a contagem absoluta de monócitos, relação linfócitos sobre monócitos, relação neutrófilos sobre linfócitos e níveis de proteína C reativa ultrassensível, que além de serem de fácil acesso e baixo custo, sugerem indicar desfecho no paciente com AVC agudo. OBJETIVOS: o objetivo deste estudo foi avaliar a associação dos marcadores inflamatórios com a mortalidade de pacientes com AVC isquêmico. MÉTODOS: trata-se de um estudo retrospectivo observacional a partir de prontuários eletrônicos e exames laboratoriais de pacientes com AVC isquêmico em uma unidade hospitalar de Cascavel/PR. Uma análise estatística descritiva foi conduzida para determinar o perfil dos pacientes segundo o desfecho e aplicado um modelo de regressão logística para verificar as variáveis associadas a mortalidade. Foram considerados significativos apenas os dados com p-valor <0,05. RESULTADOS: Dos 65 pacientes que foram admitidos no estudo, 50 receberam alta hospitalar e 15 foram a óbito no hospital. Entre os marcadores inflamatórios, a relação de neutrófilos sobre linfócitos (OR 1,55; p-valor <0,01) mostrou-se significativamente associada a maior chance de óbito. Os pacientes que faleceram apresentaram níveis superiores de PCR ultrassensível, maior contagem absoluta de monócitos, relação linfócitos sobre monócitos diminuída, e relação neutrófilos sobre linfócitos elevada. CONCLUSÃO: a relação de neutrófilos sobre linfócitos elevada pode estar significativamente associada ao desfecho desfavorável após um AVC isquêmico
IINTRODUCTION: As the population ages and life expectancy increases, the global incidence and prevalence of ischemic stroke tends to rise significantly. In this context, the need arises to evaluate new predictive markers of mortality, such as absolute monocyte count, lymphocyte-to-monocyte ratio, neutrophil-to-lymphocyte ratio and C-reactive protein (CRP) levels which, besides being easily accessible and affordable, manage to predict the outcome in patients with acute stroke. OBJECTIVES: the aim of this study was to evaluate the association between inflammatory markers and the mortality in ischemic stroke patients. METHODS: this is a retrospective observational study based on the analysis of electronic medical records and laboratory tests of in-patients who suffered an ischemic stroke in Cascavel/PR. A descriptive statistical analysis was conducted to determine patients´ profile according to the outcome and a logistic regression model was applied in order to verify the variables associated with mortality. Only data with a p-value <0,05 was considered. RESULTS: Out of the 65 patients who suffered an ischemic stroke included in the study, 50 were discharged and 15 died in hospital. Among the inflammatory markers, the neutrophil-tolymphocyte ratio (OR 1.55; p-value <0,01) was associated with a greater chance of death. Patients who died presented with higher levels of ultra-sensitive CRP, higher absolute monocyte count, lower lymphocyte-to-monocyte ratio and higher neutrophil-to- lymphocyte ratio. CONCLUSION: the elevated neutrophil-to-lymphocyte ratio may be significantly associated with negative outcomes following an ischemic stroke
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Humanos , Masculino , Femenino , Adulto , Persona de Mediana Edad , Anciano , Anciano de 80 o más Años , Adulto Joven , Accidente Cerebrovascular Isquémico/mortalidad , Accidente Cerebrovascular Isquémico/epidemiología , Inflamación/sangre , Recuento de Células Sanguíneas , Comorbilidad , Prevalencia , Estudios Retrospectivos , Factores de RiesgoRESUMEN
PURPOSE: Obesity is predominant in women of reproductive age. Roux-en-Y gastric bypass (RYGB) is the most common bariatric procedure that is performed in obese women for weight loss and metabolic improvement. However, some studies suggest that this procedure negatively affects offspring. Herein, using Western diet (WD)-obese female rats, we investigated the effects of maternal RYGB on postnatal body development, glucose tolerance, insulin secretion and action in their adult male F1 offspring. METHODS: Female Wistar rats consumed a Western diet (WD) for 18 weeks, before being submitted to RYGB (WD-RYGB) or SHAM (WD-SHAM) operations. After 5 weeks, WD-RYGB and WD-SHAM females were mated with control male breeders, and the F1 offspring were identified as: WD-RYGB-F1 and WD-SHAM-F1. RESULTS: The male F1 offspring of WD-RYGB dams exhibited decreased BW, but enhanced total nasoanal length gain. At 120 days of age, WD-RYGB-F1 rats displayed normal fasting glycemia and glucose tolerance but demonstrated reduced insulinemia and higher glucose disappearance after insulin stimulus. In addition, these rodents presented insulin resistance in the gastrocnemius muscle and retroperitoneal fat, as judged by lower Akt phosphorylation after insulin administration, but an increase in this protein in the liver. Finally, the islets from WD-RYGB-F1 rats secreted less insulin in response to glucose and displayed increased ß-cell area and mass. CONCLUSIONS: RYGB in WD dams negatively affected their F1 offspring, leading to catch-up growth, insulin resistance in skeletal muscle and white fat, and ß-cell dysfunction. Therefore, our data are the first to demonstrate that the RYGB in female rats may aggravate the metabolic imprinting induced by maternal WD consumption, in their male F1 descendants. However, since we only used male F1 rats, further studies are necessary to demonstrate if such effect may also occur in female F1 offspring from dams that underwent RYGB operation.
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Glucemia , Peso Corporal , Derivación Gástrica/efectos adversos , Insulina/sangre , Páncreas/metabolismo , Páncreas/fisiopatología , Animales , Femenino , Masculino , Madres , Obesidad/cirugía , Ratas , Ratas WistarRESUMEN
OBJECTIVE: To investigate the effects of sericin extracted from silkworm Bombyx mori cocoon on morphophysiological parameters in mice with obesity induced by high-fat diet. METHODS: Male C57Bl6 mice aged 9 weeks were allocated to one of two groups - Control and Obese, and fed a standard or high-fat diet for 10 weeks, respectively. Mice were then further subdivided into four groups with seven mice each, as follows: Control, Control-Sericin, Obese, and Obese-Sericin. The standard or high fat diet was given for 4 more weeks; sericin (1,000mg/kg body weight) was given orally to mice in the Control-Sericin and Obese-Sericin Groups during this period. Weight gain, food intake, fecal weight, fecal lipid content, gut motility and glucose tolerance were monitored. At the end of experimental period, plasma was collected for biochemical analysis. Samples of white adipose tissue, liver and jejunum were collected and processed for light microscopy analysis; liver fragments were used for lipid content determination. RESULTS: Obese mice experienced significantly greater weight gain and fat accumulation and had higher total cholesterol and glucose levels compared to controls. Retroperitoneal and periepididymal adipocyte hypertrophy, development of hepatic steatosis, increased cholesterol and triglyceride levels and morphometric changes in the jejunal wall were observed. CONCLUSION: Physiological changes induced by obesity were not fully reverted by sericin; however, sericin treatment restored jejunal morphometry and increased lipid excretion in feces in obese mice, suggesting potential anti-obesity effects.
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Fármacos Antiobesidad/uso terapéutico , Dieta Alta en Grasa , Obesidad/tratamiento farmacológico , Sericinas/uso terapéutico , Tejido Adiposo/patología , Animales , Fármacos Antiobesidad/farmacología , Peso Corporal/efectos de los fármacos , Colesterol/análisis , Dieta Alta en Grasa/efectos adversos , Ingestión de Alimentos/efectos de los fármacos , Hígado Graso/patología , Tránsito Gastrointestinal/efectos de los fármacos , Prueba de Tolerancia a la Glucosa , Hígado/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Obesos , Obesidad/etiología , Obesidad/fisiopatología , Reproducibilidad de los Resultados , Sericinas/farmacología , Factores de Tiempo , Resultado del Tratamiento , Triglicéridos/análisis , Aumento de Peso/efectos de los fármacosRESUMEN
ABSTRACT Objective To investigate the effects of sericin extracted from silkworm Bombyx mori cocoon on morphophysiological parameters in mice with obesity induced by high-fat diet. Methods Male C57Bl6 mice aged 9 weeks were allocated to one of two groups - Control and Obese, and fed a standard or high-fat diet for 10 weeks, respectively. Mice were then further subdivided into four groups with seven mice each, as follows: Control, Control-Sericin, Obese, and Obese-Sericin. The standard or high fat diet was given for 4 more weeks; sericin (1,000mg/kg body weight) was given orally to mice in the Control-Sericin and Obese-Sericin Groups during this period. Weight gain, food intake, fecal weight, fecal lipid content, gut motility and glucose tolerance were monitored. At the end of experimental period, plasma was collected for biochemical analysis. Samples of white adipose tissue, liver and jejunum were collected and processed for light microscopy analysis; liver fragments were used for lipid content determination. Results Obese mice experienced significantly greater weight gain and fat accumulation and had higher total cholesterol and glucose levels compared to controls. Retroperitoneal and periepididymal adipocyte hypertrophy, development of hepatic steatosis, increased cholesterol and triglyceride levels and morphometric changes in the jejunal wall were observed. Conclusion Physiological changes induced by obesity were not fully reverted by sericin; however, sericin treatment restored jejunal morphometry and increased lipid excretion in feces in obese mice, suggesting potential anti-obesity effects.
RESUMO Objetivo Investigar os efeitos da sericina extraída de casulos de Bombyx mori na morfofisiologia de camundongos com obesidade induzida por dieta hiperlipídica. Métodos Camundongos machos C57Bl6, com 9 semanas de idade, foram distribuídos em Grupos Controle e Obeso, que receberam ração padrão para roedores ou dieta hiperlipídica por 10 semanas, respectivamente. Posteriormente, os animais foram redistribuídos em quatro grupos, com sete animais cada: Controle, Controle-Sericina, Obeso e Obeso-Sericina. Os animais permaneceram recebendo ração padrão ou hiperlipídica por 4 semanas, período no qual a sericina foi administrada oralmente na dose de 1.000mg/kg de massa corporal aos Grupos Controle-Sericina e Obeso-Sericina. Parâmetros fisiológicos, como ganho de peso, consumo alimentar, peso das fezes em análise de lipídios fecais, motilidade intestinal e tolerância à glicose foram monitorados. Ao término do experimento, o plasma foi coletado para dosagens bioquímicas e fragmentos de tecido adiposo branco; fígado e jejuno foram processados para análises histológicas, e amostras hepáticas foram usadas para determinação lipídica. Resultados Camundongos obesos apresentaram ganho de peso e acúmulo de gordura significativamente maior que os controles, aumento do colesterol total e glicemia. Houve hipertrofia dos adipócitos retroperitoneais e periepididimais, instalação de esteatose e aumento do colesterol e triglicerídeos hepáticos, bem como alteração morfométrica da parede jejunal. Conclusão O tratamento com sericina não reverteu todas as alterações fisiológicas promovidas pela obesidade, mas restaurou a morfometria jejunal e aumentou a quantidade de lipídios eliminados nas fezes dos camundongos obesos, apresentando-se como potencial tratamento para a obesidade.
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Animales , Masculino , Fármacos Antiobesidad/uso terapéutico , Sericinas/uso terapéutico , Obesidad/tratamiento farmacológico , Factores de Tiempo , Triglicéridos/análisis , Peso Corporal/efectos de los fármacos , Tránsito Gastrointestinal/efectos de los fármacos , Aumento de Peso/efectos de los fármacos , Tejido Adiposo/patología , Colesterol/análisis , Reproducibilidad de los Resultados , Resultado del Tratamiento , Fármacos Antiobesidad/farmacología , Sericinas/farmacología , Ingestión de Alimentos/efectos de los fármacos , Hígado Graso/patología , Dieta Alta en Grasa/efectos adversos , Prueba de Tolerancia a la Glucosa , Hígado/metabolismo , Ratones Endogámicos C57BL , Ratones Obesos , Obesidad/etiología , Obesidad/fisiopatologíaRESUMEN
Until the moment, no study explored conjunctively the physical activity relation, using the inflammatory biomarkers, with the periodontitis. This way, the objective of this experiment was to evaluate the muscular tissue behavior of rats submitted to physical exercise in aquatic environment with experimental disease. Twenty-four male Wistar rats were divided in 4 groups: 1) control and sedentary (CS); 2) control and active (CA); 3) with the periodontal disease and sedentary (PDS); with the periodontal disease and active (PDA). On the group that the periodontitis was induced, it was for ligature and the groups with active swimming activity have practiced it for 4 weeks. At the end of 30 days the animals were euthanized and a portion of the gingival tissue and the soleus muscle were removed and underwent analysis by ELISA and morphological and morphometrical analysis of the muscle. Data drawn from the analysis was analyzed through ANOVA and Tukey. Results have shown that there is a higher expression of TNF-α in the gingival tissue and on the muscular tissue of the rats that underwent the induced periodontitis independently of the physical activity (PDS and PDA), as a meaningful decrease on the conjunctive tissue on the groups with induced periodontitis, that have or have not undergone active swimming activity which could suggest a predisposition to muscular injury or difficulty of muscular recovering on these groups. Therefore, it was possible to highlight a correlation between the periodontal disease and the muscle morphological changes, and, moreover, the physical swimming activity promoting an acceleration of the regeneration of the muscle tissue
Hasta este momento, ningún estudio ha explorado conjuntamente la relación de la actividad física con la periodontitis utilizando biomarcadores de inflamación. Siendo así, el objetivo de este estudio fue evaluar el comportamiento del tejido muscular de ratas con periodontitis experimental al realizar ejercicio físico en medio acuático. Veinticuatro ratas Wistar machos fueron divididas en cuatro grupos: 1) control y sedentario (CS); 2) control y ejercicio (CA); 3) con enfermedad periodontal y sedentario (PDS); 4) con enfermedad periodontal y ejercicio (PDA). En los grupos con periodontitis, la enfermedad periodontal fue inducida por ligadura y los grupos con ejercicio realizaron natación durante cuatro semanas. A los treinta días, los animales fueron sacrificados y una parte del tejido de las encías y del músculo soleo se resecaron y utilizaron para análisis con ELISA y para análisis morfológicos y morfométricos. Los datos obtenidos fueron analizados y evaluados a través de los tests ANOVA y TUKEY. Los datos mostraron una mayor expresión de TNF-α tanto en el tejido de las encías como en el tejido muscular de los ratones sometidos a periodontitis inducida independiente del ejercicio físico (PDS y PDA). Se percibió también una disminución significativa en el tejido conjuntivo en los grupos con periodontitis inducida sometidos o no al ejercicio de natación, lo que podría sugerir una predisposición a lesión muscular o una dificultad en la reparación de las lesiones musculares de esos grupos. Por lo tanto, fue posible destacar una correlación entre la enfermedad periodontal y los cambios morfológicos musculares y, además, que la actividad física de natación favoreció una aceleración de la regeneración del tejido muscular
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Ratas , Periodontitis/fisiopatología , Músculo Esquelético/fisiología , Ejercicio Físico/fisiología , Modelos Animales de Enfermedad , Natación/fisiología , Factor de Necrosis Tumoral alfa/análisis , Regeneración/fisiologíaRESUMEN
AIMS: Hypothalamic obesity is a severe condition without any effective therapy. Bariatric operations appear as an alternative treatment, but the effects of this procedure are controversial. We, herein, investigated the effects of duodeno-jejunal bypass (DJB) surgery upon the lipid profile and expression of genes and proteins, involved in the regulation of hepatic lipid metabolism, in hypothalamic obese (HyO) rats. METHODS: During the first 5days of life, male newborn Wistar rats received subcutaneous injections of monosodium glutamate [4g/kg body weight, HyO group] or saline (control, CTL group). At 90days of life, HyO rats were randomly submitted to DJB (HyO DJB) or Sham-operations (HyO Sham group). Six months after DJB, adiposity, hepatic steatosis and lipid metabolism were verified. KEY FINDINGS: HyO Sham rats were obese, hyperinsulinemic, insulin resistant and dyslipidemic. These rats had higher liver contents of trygliceride (TG) and presented disorganization of the hepatocyte structures, in association with higher hepatic contents of acetyl-CoA carboxylase (ACC), fatty acid synthase (FASN), and stearoyl-CoA desaturase-1 mRNAs and protein. DJB surgery normalized insulinemia, insulin resistance, and dyslipidemia in HyO rats. TG content in the liver and the hepatic microscopic structures were also normalized in HyO DJB rats, while the expressions of ACC and FASN proteins were decreased in the liver of these rodents. SIGNIFICANCE: The DJB-induced amelioration in hepatic steatosis manifested as a late effect in HyO rats, and was partly associated with a downregulation in hepatic de novo lipogenesis processes, indicating that DJB protects against liver steatosis in hypothalamic obesity.
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Hígado Graso/metabolismo , Hígado Graso/cirugía , Derivación Gástrica , Metabolismo de los Lípidos , Obesidad/metabolismo , Obesidad/cirugía , Acetil-CoA Carboxilasa/metabolismo , Animales , Ácido Graso Sintasas/metabolismo , Hígado Graso/patología , Masculino , Obesidad/inducido químicamente , Obesidad/patología , Ratas , Glutamato de Sodio , Estearoil-CoA Desaturasa/metabolismo , Triglicéridos/metabolismoRESUMEN
Paracoccidioidomycosis is the most prevalent systemic mycosis in Latin America. It is caused by the temperature-dependent dimorphic fungus Paracoccidioides brasiliensis. The P. brasiliensis cell wall is a dynamic outer structure, composed of a network of glycoproteins and polysaccharides, such as chitin, glucan and N-glycosylated proteins. These glycoproteins can interact with the host to affect infection rates, and are known to perform other functions. We inhibited N-linked glycosylation using tunicamycin (TM), and then evaluated the expression of P. brasiliensis genes related to cell wall remodeling. Our results suggest that cell wall synthesis related genes, such as ß-1,3-glucanosyltransferase (PbGEL3), 1,3-ß-D-glucan synthase (PbFKS1), and α-1,4-amylase (PbAMY), as well as cell wall degrading related genes, such as N-acetyl-ß-D-glucosaminidase (PbNAG1), α-1,3-glucanase (PbAGN), and ß-1,3-glucanase (PbBGN1 and PbBGN2), have their expression increased by the N-glycosylation inhibition, as detected by qRT-PCR. The observed increases in gene expression levels reveal possible compensatory mechanisms for diminished enzyme activity due to the lack of glycosylation caused by TM.
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Toxoplasma gondii is an obligate intracellular protozoan parasite found worldwide that is able to chronically infect almost all vertebrate species, especially birds and mammalians. Chitinases are essential to various biological processes, and some pathogens rely on chitinases for successful parasitization. Here, we purified and characterized a chitinase from T. gondii. The enzyme, provisionally named Tg_chitinase, has a molecular mass of 13.7 kDa and exhibits a Km of 0.34 mM and a Vmax of 2.64. The optimal environmental conditions for enzymatic function were at pH 4.0 and 50 °C. Tg_chitinase was immunolocalized in the cytoplasm of highly virulent T. gondii RH strain tachyzoites, mainly at the apical extremity. Tg_chitinase induced macrophage activation as manifested by the production of high levels of pro-inflammatory cytokines, a pathogenic hallmark of T. gondii infection. In conclusion, to our knowledge, we describe for the first time a chitinase of T. gondii tachyzoites and provide evidence that this enzyme might influence the pathogenesis of T. gondii infection.
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Quitinasas/inmunología , Activación de Macrófagos/inmunología , Macrófagos Peritoneales/inmunología , Proteínas Protozoarias/inmunología , Toxoplasma/inmunología , Secuencia de Aminoácidos , Animales , Quitinasas/genética , Quitinasas/metabolismo , Cromatografía Liquida , Citocinas/inmunología , Citocinas/metabolismo , Citoplasma/enzimología , Interacciones Huésped-Parásitos/inmunología , Concentración de Iones de Hidrógeno , Mediadores de Inflamación/inmunología , Mediadores de Inflamación/metabolismo , Cinética , Macrófagos Peritoneales/metabolismo , Macrófagos Peritoneales/parasitología , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Microscopía Confocal , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Proteínas Protozoarias/química , Espectrometría de Masas en Tándem , Temperatura , Toxoplasma/enzimología , Toxoplasma/fisiologíaRESUMEN
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.