RESUMEN

The interactions between cell and cellular matrix confers plasticity to each body tissue, influencing the cellular migratory capacity. Macrophages rely on motility to promote their physiological function. These phagocytes are determinant for the control of invasive infections, and their immunological role largely depends on their ability to migrate and adhere to tissue. Therefore, they interact with the components of the extracellular matrix through their adhesion receptors, conferring morphological modifications that change their shape during migration. Nevertheless, the need to use in vitro cell growth models with the conditioning of three-dimensional synthetic matrices to mimic the dynamics of cell-matrix interaction has been increasingly studied. This becomes more important to effectively understand the changes occurring in phagocyte morphology in the context of infection progression, such as in Chagas disease. This disease is caused by the intracellular pathogen Trypanosoma cruzi, capable of infecting macrophages, determinant cells in the anti-trypanosomatid immunity. In the present study, we sought to understand how an in vitro extracellular matrix model interferes with T. cruzi infection in macrophages. Using different time intervals and parasite ratios, we evaluated the cell morphology and parasite replication rate in the presence of 3D collagen I matrix. Nevertheless, microscopy techniques such as scanning electron microscopy were crucial to trace macrophage-matrix interactions. In the present work, we demonstrated for the first time that the macrophage-matrix interaction favors T. cruzi in vitro replication and the release of anti-inflammatory cytokines during macrophage infection, in addition to drastically altering the morphology of the macrophages and promoting the formation of migratory macrophages.

RESUMEN

PURPOSE: Trypanosoma caninum exhibits atypical epimastigote forms under axenic conditions. This study aimed to analyze this evolutionary form under different cultivation conditions and provide more information about this evolutionary form. METHODS: We selected a T. caninum isolate with a high percentage of aflagellar epimastigote forms in axenic cultures. Two separate growth curves were generated for T. caninum cultured in Schneider axenic medium and co-cultured with the DH82 cell line, followed by analysis and quantification of evolutionary forms using bright field microscopy. In addition, ultrastructural analysis of T. caninum was performed under both cultivation conditions. RESULTS: The growth curves of T. caninum under axenic and co-cultivation conditions exhibited similar profiles. However, in the axenic culture, the number of parasites was three times higher at the peak of the exponential phase than in the co-culture. In contrast to that in the axenic culture, in which only the epimastigote forms were observed along the entire curve, during co-cultivation with the DH82 cell line, differentiation was observed for the trypomastigote and spheromastigote forms in low proportions. These results demonstrated that when cultured alone, the T. caninum isolate preserved the aflagellar epimastigote form, but in the presence of DH82 canine macrophages, they differentiated into evolutionary forms, particularly trypomastigote forms. Moreover, this study is the first to describe the presence of lipid bodies, structure described as the parasite's nutritional reserve, throughout the body of T. caninum. CONCLUSIONS: These findings describe biological and ultrastructural aspects of epimastigote aflagellar and suggest that this evolutionary form may be involved in the biological cycle of T. caninum, still unknown.

Asunto(s)

Trypanosoma cruzi , Animales , Cultivo Axénico , Línea Celular , Medios de Cultivo , Perros , Macrófagos/parasitología

RESUMEN

Chagas disease is a neglected illness caused by Trypanosoma cruzi and its treatment is done only with two drugs, nifurtimox and benznidazole. However, both drugs are ineffective in the chronic phase, in addition to causing serious side effects. This context of therapeutic limitation justifies the continuous research for alternative drugs. Here, we study the in vitro trypanocidal effects of the non-steroidal anti-inflammatory drug nimesulide, a molecule that has in its chemical structure a toxicophoric nitroaromatic group (NO2). The set of results obtained in this work highlights the potential for repurposing nimesulide in the treatment of this disease that affects millions of people around the world.

Asunto(s)

Enfermedad de Chagas/tratamiento farmacológico , Enfermedad de Chagas/parasitología , Reposicionamiento de Medicamentos , Sulfonamidas/uso terapéutico , Trypanosoma cruzi/fisiología , Animales , Muerte Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Estadios del Ciclo de Vida/efectos de los fármacos , Ratones Endogámicos BALB C , Parásitos/efectos de los fármacos , Sulfonamidas/química , Sulfonamidas/farmacología , Trypanosoma cruzi/efectos de los fármacos , Trypanosoma cruzi/crecimiento & desarrollo , Trypanosoma cruzi/ultraestructura

RESUMEN

Leprosy reactional episodes are acute inflammatory events that may occur during the clinical course of the disease. Type 1 reaction (T1R) is associated with an increase in neural damage, and the understanding of the molecular pathways related to T1R onset is pivotal for the development of strategies that may effectively control the reaction. Interferon-gamma (IFN-γ) is a key cytokine associated with T1R onset and is also associated with autophagy induction. Here, we evaluated the modulation of the autophagy pathway in Mycobacterium leprae-stimulated cells in the presence or absence of IFN-γ. We observed that IFN-γ treatment promoted autophagy activation and increased the expression of genes related to the formation of phagosomes, autophagy regulation and function, or lysosomal pathways in M. leprae-stimulated cells. IFN-γ increased interleukin (IL)-15 secretion in M. leprae-stimulated THP-1 cells in a process associated with autophagy activation. We also observed higher IL15 gene expression in multibacillary (MB) patients who later developed T1R during clinical follow-up when compared to MB patients who did not develop the episode. By overlapping gene expression patterns, we observed 13 common elements shared between T1R skin lesion cells and THP-1 cells stimulated with both M. leprae and IFN-γ. Among these genes, the autophagy regulator Translocated Promoter Region, Nuclear Basket Protein (TPR) was significantly increased in T1R cells when compared with non-reactional MB cells. Overall, our results indicate that IFN-γ may induce a TPR-mediated autophagy transcriptional program in M. leprae-stimulated cells similar to that observed in skin cells during T1R by a pathway that involves IL-15 production, suggesting the involvement of this cytokine in the pathogenesis of T1R.

Asunto(s)

Autofagia/genética , Interleucina-15/genética , Lepra/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Células Cultivadas , Niño , Citocinas/genética , Femenino , Expresión Génica/genética , Humanos , Interferón gamma/genética , Lepra/microbiología , Masculino , Persona de Mediana Edad , Mycobacterium leprae/patogenicidad , Piel/metabolismo , Piel/microbiología , Células THP-1/metabolismo , Adulto Joven

RESUMEN

BACKGROUND: Lutzomyia longipalpis-derived cell line (Lulo) has been suggested as a model for studies of interaction between sandflies and Leishmania. OBJECTIVES: Here, we present data of proteomic and gene expression analyses of Lulo cell related to interactions with Leishmania (Viannia) braziliensis. METHODS: Lulo cell protein extracts were analysed through a combination of two-dimensional gel electrophoresis and mass spectrometry and resulting spots were further investigated in silico to identify proteins using Mascot search and, afterwards, resulting sequences were applied for analysis with VectorBase. RESULTS: Sixty-four spots were identified showing similarities to other proteins registered in the databases and could be classified according to their biological function, such as ion-binding proteins (23%), proteases (14%), cytoskeletal proteins (11%) and interactive membrane proteins (9.5%). Effects of interaction with L. (V.) braziliensis with the expression of three genes (enolase, tubulin and vacuolar transport protein) were observed after an eight-hour timeframe and compared to culture without parasites, and demonstrated the impact of parasite interaction with the expression of the following genes: LLOJ000219 (1.69-fold), LLOJ000326 (1.43-fold) and LLOJ006663 (2.41-fold). CONCLUSIONS: This set of results adds relevant information regarding the usefulness of the Lulo cell line for studies with Leishmania parasites that indicate variations of protein expression.

Asunto(s)

Leishmania braziliensis , Leishmania , Proteómica , Psychodidae , Animales , Línea Celular , Leishmania/genética , Leishmania braziliensis/genética , Psychodidae/parasitología , Transcriptoma

RESUMEN

INTRODUCTION: Musca domestica is resistant to many insecticides; hence, biological control is a suitable alternative. METHODS: We evaluated the lethality of strain Btk176 towards the larval and adult M. domestica and the histopathological effects in the larvae midgut. RESULTS: We observed 99% larval and 78.9% adult mortality within 48 hours of spore ingestion (dosage, 2.4×108 CFU/ml). The histopathological effects were consistent with cytotoxicity. PCR analysis showed the presence of the cry1Ba gene. Transmission electron microscopy revealed a bipyramidal parasporal body. Thurigiensin activity was not detected. CONCLUSIONS: The serovar, Btk176 might be a potential biocontrol agent for houseflies.

Asunto(s)

Bacillus thuringiensis , Toxinas Bacterianas/farmacología , Moscas Domésticas/efectos de los fármacos , Insecticidas/farmacología , Larva/efectos de los fármacos , Análisis de Varianza , Animales , Recuento de Colonia Microbiana , Exotoxinas , Microscopía Electrónica de Transmisión , Control Biológico de Vectores/métodos , Reproducibilidad de los Resultados

RESUMEN

Abstract INTRODUCTION: Musca domestica is resistant to many insecticides; hence, biological control is a suitable alternative. METHODS: We evaluated the lethality of strain Btk176 towards the larval and adult M. domestica and the histopathological effects in the larvae midgut. RESULTS: We observed 99% larval and 78.9% adult mortality within 48 hours of spore ingestion (dosage, 2.4×108 CFU/ml). The histopathological effects were consistent with cytotoxicity. PCR analysis showed the presence of the cry1Ba gene. Transmission electron microscopy revealed a bipyramidal parasporal body. Thurigiensin activity was not detected. CONCLUSIONS: The serovar, Btk176 might be a potential biocontrol agent for houseflies.

Asunto(s)

Animales , Bacillus thuringiensis , Toxinas Bacterianas/farmacología , Moscas Domésticas/efectos de los fármacos , Insecticidas/farmacología , Larva/efectos de los fármacos , Recuento de Colonia Microbiana , Control Biológico de Vectores/métodos , Reproducibilidad de los Resultados , Análisis de Varianza , Microscopía Electrónica de Transmisión , Exotoxinas

RESUMEN

Epoxymethoxylawsone is a naphthoquinone derivative promising as drug candidate for the treatment of leishmaniases. In the present work the effectiveness of epoxymethoxylawsone, and meglumine antimoniate on Leishmania (Leishmania) amazonensis parasites and on mice paw lesions of infected BALB/c mice was assessed. In an intracellular amastigotes assay, the half-maximal inhibitory concentration (IC50) value for epoxymethoxylawsone was slightly higher (1.7-fold) than that found for meglumine antimoniate. The efficacy of both drugs became more evident after 48 h of exposure when either the oxirane compound and reference drug reached 18-fold and 7.4-fold lower IC50 values (0.40 ± 0.001 µM and 0.60 ± 0.02 µM), respectively. Promastigotes were also affected by epoxymethoxylawsone after 24 h of incubation (IC50 = 45.45 ± 5.0 µM), but with IC50 6-fold higher than those found for intracellular amastigotes. Cytotoxicity analysis revealed that epoxymethoxylawsone (CC50 = 40.05 ± µM) has 1.7-fold higher effects than meglumine antimoniate (CC50 = 24.14 ± 2.6 µM). Treatment of the paw lesion in infected BALB/c mice with epoxymethoxy-lawsone led to a significant 27% reduction (p < 0.05) of the lesion size, for all administrated doses, compared to the control group. Lesion reduction was also detected after mice treatment with meglumine antimoniate, reaching 31.0% (0.23 mg of Sb(V)/Kg/day and 2.27 mg of Sb(V)/Kg/day) and 64.0% (22.7 mg of Sb(V)/Kg/day). In addition, mice lesion ultrastructural changes were evidenced in amastigotes. The set of data gathered here indicate that epoxymethoxylawsone has pronounced effects on parasites and merits furthering to the preclinical stage.

Asunto(s)

Antiprotozoarios/administración & dosificación , Leishmaniasis/tratamiento farmacológico , Naftoquinonas/administración & dosificación , Animales , Antiprotozoarios/química , Antiprotozoarios/farmacología , Modelos Animales de Enfermedad , Femenino , Leishmania/efectos de los fármacos , Macrófagos/citología , Macrófagos/efectos de los fármacos , Meglumina/administración & dosificación , Meglumina/farmacología , Antimoniato de Meglumina , Ratones , Ratones Endogámicos BALB C , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Naftoquinonas/química , Naftoquinonas/farmacología , Compuestos Organometálicos/administración & dosificación , Compuestos Organometálicos/farmacología

RESUMEN

CONTEXT: Leishmania amazonensis is the main agent of diffuse cutaneous leishmaniasis, a disease characterized by lesional polymorphism and the commitment of skin surface. Previous reports demonstrated that the Citrus genus possess antimicrobial activity. OBJECTIVE: This study evaluated the anti-L. amazonensis activity of Citrus sinensis (L.) Osbeck (Rutaceae) extracts. MATERIALS AND METHODS: Citrus sinensis dried leaves were subjected to maceration with hexane (CH), ethyl acetate (CEA), dichloromethane/ethanol (CD/Et - 1:1) or ethanol/water (CEt/W - 7:3). Leishmania amazonensis promastigotes were treated with C. sinensis extracts (1-525 µg/mL) for 120 h at 27 °C. Ultrastructure alterations of treated parasites were evaluated by transmission electron microscopy. Cytotoxicity of the extracts was assessed on RAW 264.7 and J774.G8 macrophages after 48-h treatment at 37 °C using the tetrazolium assay. In addition, Leishmania-infected macrophages were treated with CH and CD/Et (10-80 µg/mL). RESULTS: CH, CD/Et and CEA displayed antileishmanial activity with 50% inhibitory activity (IC50) of 25.91 ± 4.87, 54.23 ± 3.78 and 62.74 ± 5.04 µg/mL, respectively. Parasites treated with CD/Et (131.2 µg/mL) presented severe alterations including mitochondrial swelling, lipid body formation and intense cytoplasmic vacuolization. CH and CD/Et demonstrated cytotoxic effects similar to that of amphotericin B in the anti-amastigote assays (SI of 2.16, 1.98 and 1.35, respectively). Triterpene amyrins were the main substances in CH and CD/Et extracts. In addition, 80 µg/mL of CD/Et reduced the number of intracellular amastigotes and the percentage of infected macrophages in 63% and 36%, respectively. CONCLUSION: The results presented here highlight C. sinensis as a promising source of antileishmanial agents.

Asunto(s)

Antiprotozoarios/farmacología , Citrus sinensis/química , Leishmania/efectos de los fármacos , Macrófagos/parasitología , Extractos Vegetales/farmacología , Hojas de la Planta/química , Animales , Antiprotozoarios/aislamiento & purificación , Antiprotozoarios/toxicidad , Supervivencia Celular/efectos de los fármacos , Citrus sinensis/toxicidad , Relación Dosis-Respuesta a Droga , Concentración 50 Inhibidora , Leishmania/crecimiento & desarrollo , Leishmania/ultraestructura , Ratones , Pruebas de Sensibilidad Parasitaria , Fitoterapia , Extractos Vegetales/aislamiento & purificación , Extractos Vegetales/toxicidad , Hojas de la Planta/toxicidad , Plantas Medicinales , Células RAW 264.7 , Solventes/química

RESUMEN

Leprosy is a chronic infectious disease that may present different clinical forms according to the immune response of the host. Levels of IFN-γ are significantly raised in paucibacillary tuberculoid (T-lep) when compared with multibacillary lepromatous (L-lep) patients. IFN-γ primes macrophages for inflammatory activation and induces the autophagy antimicrobial mechanism. The involvement of autophagy in the immune response against Mycobacterium leprae remains unexplored. Here, we demonstrated by different autophagic assays that LC3-positive autophagosomes were predominantly observed in T-lep when compared with L-lep lesions and skin-derived macrophages. Accumulation of the autophagic receptors SQSTM1/p62 and NBR1, expression of lysosomal antimicrobial peptides and colocalization analysis of autolysosomes revealed an impairment of the autophagic flux in L-lep cells, which was restored by IFN-γ or rapamycin treatment. Autophagy PCR array gene-expression analysis revealed a significantly upregulation of autophagy genes (BECN1, GPSM3, ATG14, APOL1, and TPR) in T-lep cells. Furthermore, an upregulation of autophagy genes (TPR, GFI1B and GNAI3) as well as LC3 levels was observed in cells of L-lep patients that developed type 1 reaction (T1R) episodes, an acute inflammatory condition associated with increased IFN-γ levels. Finally, we observed increased BCL2 expression in L-lep cells that could be responsible for the blockage of BECN1-mediated autophagy. In addition, in vitro studies demonstrated that dead, but not live M. leprae can induce autophagy in primary and lineage human monocytes, and that live mycobacteria can reduce the autophagy activation triggered by dead mycobacteria, suggesting that M. leprae may hamper the autophagic machinery as an immune escape mechanism. Together, these results indicate that autophagy is an important innate mechanism associated with the M. leprae control in skin macrophages.

Asunto(s)

Autofagia/fisiología , Lepra/inmunología , Piel/microbiología , Adulto , Anciano , Western Blotting , Ensayo de Inmunoadsorción Enzimática , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Inmunohistoquímica , Interferón gamma/inmunología , Lepra/patología , Macrófagos/inmunología , Masculino , Microscopía Electrónica de Transmisión , Persona de Mediana Edad , Mycobacterium leprae/inmunología , Reacción en Cadena de la Polimerasa , Piel/inmunología , Piel/patología , Transcriptoma , Adulto Joven

RESUMEN

Leprosy is a chronic infectious disease that may present different clinical forms according to the immune response of the host. Levels of IFN-γ are significantly raised in paucibacillary tuberculoid (T-lep) when compared with multibacillary lepromatous (L-lep) patients. IFN-γ primes macrophages for inflammatory activation and induces the autophagy antimicrobial mechanism. The involvement of autophagy in the immune response against Mycobacterium leprae remains unexplored. Here, we demonstrated by different autophagic assays that LC3-positive autophagosomes were predominantly observed in T-lep when compared with L-lep lesions and skin-derived macrophages. Accumulation of the autophagic receptors SQSTM1/p62 and NBR1, expression of lysosomal antimicrobial peptides and colocalization analysis of autolysosomes revealed an impairment of the autophagic flux in L-lep cells, which was restored by IFN-γ or rapamycin treatment. Autophagy PCR array gene-expression analysis revealed a significantly upregulation of autophagy genes (BECN1, GPSM3, ATG14, APOL1, and TPR) in T-lep cells. Furthermore, an upregulation of autophagy genes (TPR, GFI1B and GNAI3) as well as LC3 levels was observed in cells of L-lep patients that developed type 1 reaction (T1R) episodes, an acute inflammatory condition associated with increased IFN-γ levels. Finally, we observed increased BCL2 expression in L-lep cells that could be responsible for the blockage of BECN1-mediated autophagy. In addition, in vitro studies demonstrated that dead, but not live M. leprae can induce autophagy in primary and lineage human monocytes, and that live mycobacteria can reduce the autophagy activation triggered by dead mycobacteria, suggesting that M. leprae may hamper the autophagic machinery as an immune escape mechanism. Together, these results indicate that autophagy is an important innate mechanism associated with the M. leprae control in skin macrophages.

Asunto(s)

Humanos , Masculino , Femenino , Adulto , Persona de Mediana Edad , Anciano , Adulto Joven , Piel/inmunología , Piel/microbiología , Piel/patología , Autofagia/fisiología , Ensayo de Inmunoadsorción Enzimática , Inmunohistoquímica , Western Blotting , Reacción en Cadena de la Polimerasa , Técnica del Anticuerpo Fluorescente , Interferón gamma/inmunología , Microscopía Electrónica de Transmisión , Transcriptoma , Lepra/inmunología , Lepra/patología , Macrófagos/inmunología , Mycobacterium leprae/inmunología

RESUMEN

Mycobacterium leprae, the intracellular etiological agent of leprosy, infects Schwann promoting irreversible physical disabilities and deformities. These cells are responsible for myelination and maintenance of axonal energy metabolism through export of metabolites, such as lactate and pyruvate. In the present work, we observed that infected Schwann cells increase glucose uptake with a concomitant increase in glucose-6-phosphate dehydrogenase (G6PDH) activity, the key enzyme of the oxidative pentose pathway. We also observed a mitochondria shutdown in infected cells and mitochondrial swelling in pure neural leprosy nerves. The classic Warburg effect described in macrophages infected by Mycobacterium avium was not observed in our model, which presented a drastic reduction in lactate generation and release by infected Schwann cells. This effect was followed by a decrease in lactate dehydrogenase isoform M (LDH-M) activity and an increase in cellular protection against hydrogen peroxide insult in a pentose phosphate pathway and GSH-dependent manner. M. leprae infection success was also dependent of the glutathione antioxidant system and its main reducing power source, the pentose pathway, as demonstrated by a 50 and 70% drop in intracellular viability after treatment with the GSH synthesis inhibitor buthionine sulfoximine, and aminonicotinamide (6-ANAM), an inhibitor of G6PDH 6-ANAM, respectively. We concluded that M. leprae could modulate host cell glucose metabolism to increase the cellular reducing power generation, facilitating glutathione regeneration and consequently free-radical control. The impact of this regulation in leprosy neuropathy is discussed.

Asunto(s)

Humanos , Células de Schwann/metabolismo , Células de Schwann/microbiología , Lepra Tuberculoide/metabolismo , Línea Celular , Ácido Láctico/metabolismo , Metabolismo Energético , Glucosa/metabolismo , Glucosafosfato Deshidrogenasa/metabolismo , Metionina/análogos & derivados , Metionina/farmacología , Mitocondrias/metabolismo , Mycobacterium leprae/metabolismo

RESUMEN

Leprosy is a chronic granulomatous disease caused by Mycobacterium leprae affecting the skin and peripheral nerves. Despite M. leprae invasion of the skin and keratinocytes importance in innate immunity, the interaction of these cells in vitro during M. leprae infection is poorly understood. Conventional and fluorescence optical microscopy, transmission electronic microscopy, flow cytometry and ELISA were used to study the in vitro interaction of M. leprae with the HaCaT human keratinocyte cell line. Keratinocytes uptake of M. leprae is described, and modulation of the surface expression of CD80 and CD209, cathelicidin expression and TNF-α and IL-1ß production of human keratinocytes are compared with dendritic cells and macrophages during M. leprae interaction. This study demonstrated that M. leprae interaction with human keratinocytes enhanced expression of cathelicidin and greatly increased TNF-α production. The highest spontaneous expression of cathelicidin was by dendritic cells which are less susceptible to M. leprae infection. In contrast, keratinocytes displayed low spontaneous cathelicidin expression and were more susceptible to M. leprae infection than dendritic cells. The results show, for the first time, an active role for keratinocytes during infection by irradiated whole cells of M. leprae and the effect of vitamin D on this process. They also suggest that therapies which target cathelicidin modulation may provide novel approaches for treatment of leprosy.

Asunto(s)

Queratinocitos/inmunología , Queratinocitos/microbiología , Lepra/inmunología , Lepra/microbiología , Mycobacterium leprae/inmunología , Mycobacterium leprae/patogenicidad , Péptidos Catiónicos Antimicrobianos/metabolismo , Antígeno B7-1/metabolismo , Moléculas de Adhesión Celular/metabolismo , Línea Celular , Células Dendríticas/inmunología , Células Dendríticas/microbiología , Células Dendríticas/patología , Humanos , Inmunidad Celular , Interleucina-1beta/biosíntesis , Queratinocitos/patología , Lectinas Tipo C/metabolismo , Lepra/patología , Macrófagos/inmunología , Macrófagos/microbiología , Macrófagos/patología , Fagocitosis , Receptores de Superficie Celular/metabolismo , Factor de Necrosis Tumoral alfa/biosíntesis , Catelicidinas

RESUMEN

Transmission electron microscopy can yield useful information in a range of scientific fields; it is capable of imaging at a significantly higher resolution than light microscopes and has been a very useful tool in the identification of morphological changes of the dermis as well as assessment of changes in the extracellular matrix. Our aim is to characterize by electron microscopy the cellular profile of lesions caused by Sporothrix schenckii from the sporotrichosis epidemic in its zoonotic form that occurs in Rio de Janeiro, Brazil.

Asunto(s)

Enfermedades de los Gatos/patología , Piel/diagnóstico por imagen , Esporotricosis/patología , Animales , Brasil/epidemiología , Enfermedades de los Gatos/epidemiología , Gatos/microbiología , Epidemias , Humanos , Microscopía Electrónica de Transmisión , Piel/microbiología , Piel/patología , Sporothrix , Esporotricosis/epidemiología , Esporotricosis/veterinaria , Ultrasonografía , Zoonosis/epidemiología , Zoonosis/patología

RESUMEN

In the present study, we characterized the in vitro modulation of NETs (neutrophil extracellular traps) induced in human neutrophils by the opportunistic fungus Cryptococcus neoformans, evaluating the participation of capsular polysaccharides glucuronoxylomanan (GXM) and glucuronoxylomannogalactan (GXMGal) in this phenomenon. The mutant acapsular strain CAP67 and the capsular polysaccharide GXMGal induced NET production. In contrast, the wild-type strain and the major polysaccharide GXM did not induce NET release. In addition, C. neoformans and the capsular polysaccharide GXM inhibited PMA-induced NET release. Additionally, we observed that the NET-enriched supernatants induced through CAP67 yeasts showed fungicidal activity on the capsular strain, and neutrophil elastase, myeloperoxidase, collagenase and histones were the key components for the induction of NET fungicidal activity. The signaling pathways associated with NET induction through the CAP67 strain were dependent on reactive oxygen species (ROS) and peptidylarginine deiminase-4 (PAD-4). Neither polysaccharide induced ROS production however both molecules blocked the production of ROS through PMA-activated neutrophils. Taken together, the results demonstrate that C. neoformans and the capsular component GXM inhibit the production of NETs in human neutrophils. This mechanism indicates a potentially new and important modulation factor for this fungal pathogen.

Asunto(s)

Cryptococcus neoformans/química , Polisacáridos Fúngicos/administración & dosificación , Galactanos/administración & dosificación , Polisacáridos/administración & dosificación , Cryptococcus neoformans/patogenicidad , Trampas Extracelulares , Polisacáridos Fúngicos/química , Galactanos/química , Humanos , Neutrófilos/efectos de los fármacos , Polisacáridos/química , Especies Reactivas de Oxígeno/metabolismo

RESUMEN

Representatives of the genus Trypanosoma have been traditionally found in epimastigote, espheromastigote and trypomastigote flagellated forms in axenic cultures. Trypanosoma caninum is a trypanosomatid that has recently been reported infecting dogs in endemic areas of canine leishmaniasis in Brazil. It presents specific biological characteristics and it is found exclusively on healthy skin. Here, we describe the evolutive forms of this parasite showing not only the forms commonly found in culture, but also epimastigote forms with no free flagellum. The study was conducted using scanning and transmission electron microscopy and, we demonstrate that typical flagellated epimastigotes originate from forms without flagellum, although the latter may remain without differentiation in the culture. Two hypotheses are considered and discussed in this paper: (i) the aflagellated epimastigotes are a typical developmental forms of T. caninum and (ii) the emergence of these aflagellated forms could be resultant from a disturbed process during cell division caused by interfering specific proteins, which leads to inability to form and regulate the flagellum length. In any case, considering that T. caninum is a parasite that is still little studied, the information brought by our study adds data which may be useful to clarify aspects on the cell cycle of this intriguing parasite that has been found in different regions of Brazil.

Asunto(s)

Cultivo Axénico , Flagelos/ultraestructura , Trypanosoma/crecimiento & desarrollo , Trypanosoma/ultraestructura , Animales , Brasil , Perros , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , Trypanosoma/aislamiento & purificación

RESUMEN

Fibrocytes are important for understanding the progression of many diseases because they are present in areas where pathogenic lesions are generated. However, the morphology of fibrocytes and their interactions with parasites are poorly understood. In this study, we examined the morphology of peripheral blood fibrocytes and their interactions with Leishmania (L.) amazonensis . Through ultrastructural analysis, we describe the details of fibrocyte morphology and how fibrocytes rapidly internalise Leishmania promastigotes. The parasites differentiated into amastigotes after 2 h in phagolysosomes and the infection was completely resolved after 72 h. Early in the infection, we found increased nitric oxide production and large lysosomes with electron-dense material. These factors may regulate the proliferation and death of the parasites. Because fibrocytes are present at the infection site and are directly involved in developing cutaneous leishmaniasis, they are targets for effective, non-toxic cell-based therapies that control and treat leishmaniasis.

Asunto(s)

Fibroblastos/parasitología , Leishmania/fisiología , Leishmaniasis/fisiopatología , Leucocitos Mononucleares/parasitología , Análisis de Varianza , Animales , Fibroblastos/ultraestructura , Citometría de Flujo , Interacciones Huésped-Parásitos/fisiología , Mesodermo/citología , Ratones Endogámicos BALB C/parasitología , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , Óxido Nítrico/análisis , Óxido Nítrico/biosíntesis , Cultivo Primario de Células , Estadísticas no Paramétricas , Factores de Tiempo

RESUMEN

Fibrocytes are important for understanding the progression of many diseases because they are present in areas where pathogenic lesions are generated. However, the morphology of fibrocytes and their interactions with parasites are poorly understood. In this study, we examined the morphology of peripheral blood fibrocytes and their interactions with Leishmania (L.) amazonensis . Through ultrastructural analysis, we describe the details of fibrocyte morphology and how fibrocytes rapidly internalise Leishmania promastigotes. The parasites differentiated into amastigotes after 2 h in phagolysosomes and the infection was completely resolved after 72 h. Early in the infection, we found increased nitric oxide production and large lysosomes with electron-dense material. These factors may regulate the proliferation and death of the parasites. Because fibrocytes are present at the infection site and are directly involved in developing cutaneous leishmaniasis, they are targets for effective, non-toxic cell-based therapies that control and treat leishmaniasis.

Asunto(s)

Animales , Fibroblastos/parasitología , Leishmania/fisiología , Leishmaniasis/fisiopatología , Leucocitos Mononucleares/parasitología , Análisis de Varianza , Citometría de Flujo , Fibroblastos/ultraestructura , Interacciones Huésped-Parásitos/fisiología , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , Mesodermo/citología , Ratones Endogámicos BALB C/parasitología , Óxido Nítrico/análisis , Óxido Nítrico/biosíntesis , Cultivo Primario de Células , Estadísticas no Paramétricas , Factores de Tiempo

RESUMEN

The Parinaud oculoglandular syndrome is a rare eye disease caused by different etiologic agents, including bacteria, viruses and fungi. It is characterized by a granulomatous conjunctivitis, accompanied by adjacent preauricular lymphadenopathy and can bring consequences if not treated promptly. We present a case of Parinaud oculoglandular syndrome caused by Sporothrix schenckii from the sporotrichosis epidemic in its zoonotic form occurring in Rio de Janeiro, Brazil.

RESUMEN

BACKGROUND: Visceral leishmaniasis is the most serious form of leishmaniasis and can be lethal if left untreated. Currently available treatments for these parasitic diseases are frequently associated to severe side effects. The leaves of Croton cajucara are used as an infusion in popular medicine to combat several diseases. Previous studies have demonstrated that the linalool-rich essential oil from C. cajucara (white sacaca) is extremely efficient against the tegumentary specie Leishmania amazonensis. In this study, we investigated the effects of the 7-hydroxycalamenene-rich essential oil from the leaves of C. cajucara (red sacaca) against Leishmania chagasi, as well as on the interaction of these parasites with host cells. METHODS: Promastigotes were treated with different concentrations of the essential oil for determination of its minimum inhibitory concentration (MIC). In addition, the effects of the essential oil on parasite ultrastructure were analyzed by transmission electron microscopy. To evaluate its efficacy against infected cells, mouse peritoneal macrophages infected with L. chagasi promastigotes were treated with the inhibitory and sub-inhibitory concentrations of the essential oil. RESULTS: The minimum inhibitory concentrations of the essential oil and its purified component 7-hydroxycalamenene against L. chagasi were 250 and 15.6 µg/mL, respectively. Transmission electron microscopy analysis revealed important nuclear and kinetoplastic alterations in L. chagasi promastigotes. Pre-treatment of macrophages and parasites with the essential oil reduced parasite/macrophage interaction by 52.8%, while it increased the production of nitric oxide by L. chagasi-infected macrophages by 80%. CONCLUSION: These results indicate that the 7-hydroxycalamenene-rich essential oil from C. cajucara is a promising source of leishmanicidal compounds.

Asunto(s)

Antiprotozoarios/farmacología , Croton/química , Leishmania/efectos de los fármacos , Aceites Volátiles/farmacología , Extractos Vegetales/farmacología , Sesquiterpenos/farmacología , Animales , Antiprotozoarios/química , Células Cultivadas , Femenino , Estadios del Ciclo de Vida/efectos de los fármacos , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/metabolismo , Ratones , Pruebas de Sensibilidad Microbiana , Óxido Nítrico/metabolismo , Aceites Volátiles/química , Péptido Hidrolasas/análisis , Péptido Hidrolasas/metabolismo , Extractos Vegetales/química , Sesquiterpenos/química