RESUMEN
The thermally-dimorphic systemic fungal group includes several important human pathogens: Blastomyces dermatitides, Coccidioides immitis and C. posadasii, Histoplasma capsulatum, Paracoccidioides brasiliensis, P. lutzii, and Talaromyces (Penicillium) marneffei. They usually are geographically restricted and have natural habitats in soil or in plants, and when fungal propagules invade mammalian host by inhalation, they initiate an inflammatory reaction that can result in self-resolution of the infection or cause an acute or chronic disease. In the setting of the AIDS pandemic and the developments in modern medicine, such as immunosuppressive therapy in cancer surgery patients and in transplantation and autoimmune diseases, the incidence of endemic mycoses has progressively increased. Another important factor of the increased incidence of systemic mycoses in certain regions is the progressive devastation of tropical and subtropical forests. In this review, we focus on two of the most important systemic mycoses: paracoccidioidomycosis and histoplasmosis, and their major characteristics in epidemiology, clinical aspects and laboratorial diagnosis.
Asunto(s)
Antifúngicos/farmacología , Histoplasma/efectos de los fármacos , Histoplasmosis/diagnóstico , Histoplasmosis/tratamiento farmacológico , Paracoccidioides/efectos de los fármacos , Paracoccidioidomicosis/diagnóstico , Paracoccidioidomicosis/tratamiento farmacológico , Antifúngicos/química , Histoplasma/aislamiento & purificación , Histoplasmosis/epidemiología , Humanos , Pruebas de Sensibilidad Microbiana , Paracoccidioides/aislamiento & purificación , Paracoccidioidomicosis/epidemiologíaRESUMEN
Silencing of SOCS1 protein with shRNAi lentivirus (shR-SOCS1) led to partial reversion of the tumorigenic phenotype of B16F10-Nex2 melanoma cells. SOCS1 silencing inhibited cell migration and invasion as well as in vitro growth by cell cycle arrest at S phase with increased cell size and nuclei. Down-regulation of SOCS1 decreased the expression of epidermal growth factor receptor, Ins-Rα, and fibroblast growth factor receptors. The present work aimed at analyzing the SOCS1 cell signaling and expression of proteins relevant to tumor development. An RNA microarray analysis of B16F10-Nex2 melanoma cells with SOCS1 silenced by shRNAi-SOCS1 was undertaken in comparison with cells transduced with the empty vector. Among 609 differentially expressed genes, c-Kit, Met and EphA3 cytokine/tyrosine-kinase (TK) receptors were down regulated. A significant decrease in the expression of TK receptors, the phosphorylation of mediators of ERK1/2 and p38 pathways and STAT3 (S727) were observed. Subcutaneous immunization with shR-SOCS1-transduced viable tumor cells rendered protection against melanoma in a syngeneic model, with decreased expression of PD-L1 and of matrix metallo-proteinases (MMPs) and CD-10 in those cells. The present work shows the role of SOCS1 in murine melanoma development and the potential of SOCS1-silenced tumor cells in raising an effective anti-melanoma immune response.
Asunto(s)
Antígeno B7-H1/metabolismo , Progresión de la Enfermedad , Transición Epitelial-Mesenquimal , Inmunidad , Melanoma Experimental/inmunología , Melanoma Experimental/patología , Proteína 1 Supresora de la Señalización de Citocinas/metabolismo , Animales , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/metabolismo , Proteínas Morfogenéticas Óseas/metabolismo , Linfocitos T CD8-positivos/inmunología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Transición Epitelial-Mesenquimal/genética , Regulación Neoplásica de la Expresión Génica , Silenciador del Gen , Melanoma Experimental/genética , Antígenos Específicos del Melanoma/metabolismo , Ratones Endogámicos C57BL , FN-kappa B/metabolismo , Sustancias Protectoras/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Transducción de Señal , Proteínas Smad/metabolismo , Factor de Transcripción AP-2/metabolismo , Transcripción Genética , Factor de Crecimiento Transformador beta/metabolismo , Microambiente Tumoral , Regulación hacia Arriba/genéticaRESUMEN
BACKGROUND: Malignant melanoma is an aggressive type of skin cancer, and despite recent advances in treatment, the survival rate of the metastatic form remains low. Nifuroxazide analogues are drugs based on the substitution of the nitrofuran group by benzofuroxan, in view of the pharmacophore similarity of the nitro group, improving bioavailability, with higher intrinsic activity and less toxicity. Benzofuroxan activity involves the intracellular production of free-radical species. In the present work, we evaluated the antitumor effects of different benzofuroxan derivatives in a murine melanoma model. METHODS: B16F10-Nex2 melanoma cells were used to investigate the antitumor effects of Benzofuroxan derivatives in vitro and in a syngeneic melanoma model in C57Bl/6 mice. Cytotoxicity, morphological changes and reactive oxygen species (ROS) were assessed by a diphenyltetrasolium reagent, optical and fluorescence microscopy, respectively. Annexin-V binding and mitochondrial integrity were analyzed by flow cytometry. Western blotting and colorimetry identified cell signaling proteins. RESULTS: Benzofuroxan N-Br and N-I derivatives were active against murine and human tumor cell lines, exerting significant protection against metastatic melanoma in a syngeneic model. N-Br and N-I induce apoptosis in melanoma cells, evidenced by specific morphological changes, DNA condensation and degradation, and phosphatidylserine translocation in the plasma membrane. The intrinsic mitochondrial pathway in B16F10-Nex2 cells is suggested owing to reduced outer membrane potential in mitochondria, followed by caspase -9, -3 activation and cleavage of PARP. The cytotoxicity of N-Br and N-I in B16F10-Nex2 cells is mediated by the generation of ROS, inhibited by pre-incubation of the cells with N-acetylcysteine (NAC). The induction of ROS by N-Br and N-I resulted in the inhibition of AKT activation, an important molecule related to tumor cell survival, followed by upregulation of BIM. CONCLUSION: We conclude that N-Br and N-I are promising agents aiming at cancer treatment. They may be useful in melanoma therapy as inducers of intrinsic apoptosis and by exerting significant antitumor activity against metastatic melanoma, as presently shown in syngeneic mice.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Apoptosis/efectos de los fármacos , Benzoxazoles/química , Benzoxazoles/uso terapéutico , Melanoma Experimental/metabolismo , Proteínas de la Membrana/metabolismo , Proteína Oncogénica v-akt/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Apoptosis/fisiología , Proteínas Reguladoras de la Apoptosis/antagonistas & inhibidores , Proteína 11 Similar a Bcl2 , Benzoxazoles/farmacología , Línea Celular Tumoral , Células HeLa , Humanos , Masculino , Melanoma Experimental/tratamiento farmacológico , Proteínas de la Membrana/antagonistas & inhibidores , Ratones , Ratones Endogámicos C57BL , Proteína Oncogénica v-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
Paracoccidioidomycosis (PCM), caused by Paracoccidioides spp, is an important endemic mycosis in Latin America. There are two recognized Paracoccidioides species, P. brasiliensis and P. lutzii, based on phylogenetic differences; however, the pathogenesis and disease manifestations of both are indistinguishable at present. Approximately 1,853 (~51,2%) of 3,583 confirmed deaths in Brazil due to systemic mycoses from 1996-2006 were caused by PCM. Antifungal treatment is required for patients with PCM. The initial treatment lasts from two to six months and sulfa derivatives, amphotericin B, azoles and terbinafine are used in clinical practice; however, despite prolonged therapy, relapses are still a problem. An effective Th1-biased cellular immune response is essential to control the disease, which can be induced by exogenous antigens or modulated by prophylactic or therapeutic vaccines. Stimulation of B cells or passive transference of monoclonal antibodies are also important means that may be used to improve the efficacy of paracoccidioidomycosis treatment in the future. This review critically details major challenges facing the development of a vaccine to combat PCM.
Asunto(s)
Antígenos Fúngicos/inmunología , Vacunas Fúngicas/inmunología , Paracoccidioides/inmunología , Paracoccidioidomicosis/terapia , Vacunas de ADN/inmunología , Animales , Antígenos de Neoplasias/inmunología , Glicoproteínas/inmunología , Humanos , Ratones , Paracoccidioidomicosis/inmunología , Fragmentos de Péptidos/inmunologíaRESUMEN
SUMMARYParacoccidioidomycosis (PCM), caused by Paracoccidioides spp, is an important endemic mycosis in Latin America. There are two recognized Paracoccidioides species, P. brasiliensis and P. lutzii, based on phylogenetic differences; however, the pathogenesis and disease manifestations of both are indistinguishable at present. Approximately 1,853 (~51,2%) of 3,583 confirmed deaths in Brazil due to systemic mycoses from 1996-2006 were caused by PCM. Antifungal treatment is required for patients with PCM. The initial treatment lasts from two to six months and sulfa derivatives, amphotericin B, azoles and terbinafine are used in clinical practice; however, despite prolonged therapy, relapses are still a problem. An effective Th1-biased cellular immune response is essential to control the disease, which can be induced by exogenous antigens or modulated by prophylactic or therapeutic vaccines. Stimulation of B cells or passive transference of monoclonal antibodies are also important means that may be used to improve the efficacy of paracoccidioidomycosis treatment in the future. This review critically details major challenges facing the development of a vaccine to combat PCM.
RESUMOA paracoccidioidomicose (PCM), causada por Paracoccidioides spp, é importante micose endêmica na América Latina. Com base em diferenças filogenéticas, existem duas espécies reconhecidas de Paracoccidioides, P. brasiliensis e P. lutzii, no entanto, a patogênese e as manifestações clínicas de ambas são indistinguíveis atualmente. Aproximadamente 1853 (~51,2%) de 3583 mortes confirmadas, atribuídas a micoses sistêmicas de 1996-2006, no Brasil foram causadas por PCM. Tratamento antifúngico é necessário para pacientes com PCM. O tratamento inicial dura de dois a seis meses e derivados de sulfa, anfotericina B, azóis e terbinafina são utilizados na prática clínica; no entanto, apesar da terapêutica prolongada, as recaídas ainda são um problema. Uma resposta imune celular eficaz, tendendo a Th1, é essencial para controlar a doença que pode ser induzida por antígenos exógenos, ou moduladas por vacinas profiláticas ou terapêuticas. A estimulação de células B ou a transferência passiva de anticorpos monoclonais também são meios importantes que podem ser utilizados para melhorar a eficácia do tratamento da paracoccidioidomicose no futuro. Esta revisão detalha criticamente os principais desafios que o desenvolvimento de uma vacina para combater a PCM enfrenta.
Asunto(s)
Animales , Humanos , Ratones , Antígenos Fúngicos/inmunología , Vacunas Fúngicas/inmunología , Paracoccidioides/inmunología , Paracoccidioidomicosis/terapia , Vacunas de ADN/inmunología , Antígenos de Neoplasias/inmunología , Glicoproteínas/inmunología , Paracoccidioidomicosis/inmunología , Fragmentos de Péptidos/inmunologíaRESUMEN
Vaccination with peptide 10 (P10), derived from the Paracoccidioides brasiliensis glycoprotein 43 (gp43), induces a Th1 response that protects mice in an intratracheal P. brasiliensis infection model. Combining P10 with complete Freund's adjuvant (CFA) or other adjuvants further increases the peptide's antifungal effect. Since dendritic cells (DCs) are up to 1,000-fold more efficient at activating T cells than CFA, we examined the impact of P10-primed bone-marrow-derived DC vaccination in mice. Splenocytes from mice immunized with P10 were stimulated in vitro with P10 or P10-primed DCs. T cell proliferation was significantly increased in the presence of P10-primed DCs compared to the peptide. The protective efficacy of P10-primed DCs was studied in an intratracheal P. brasiliensis model in BALB/c mice. Administration of P10-primed DCs prior to (via subcutaneous vaccination) or weeks after (via either subcutaneous or intravenous injection) P. brasiliensis infection decreased pulmonary damage and significantly reduced fungal burdens. The protective response mediated by the injection of primed DCs was characterized mainly by an increased production of gamma interferon (IFN-γ) and interleukin 12 (IL-12) and a reduction in IL-10 and IL-4 compared to those of infected mice that received saline or unprimed DCs. Hence, our data demonstrate the potential of P10-primed DCs as a vaccine capable of both the rapid protection against the development of serious paracoccidioidomycosis or the treatment of established P. brasiliensis disease.
Asunto(s)
Células Dendríticas/inmunología , Vacunas Fúngicas/inmunología , Glicoproteínas/inmunología , Paracoccidioides/inmunología , Paracoccidioidomicosis/prevención & control , Paracoccidioidomicosis/terapia , Fragmentos de Péptidos/inmunología , Vacunación/métodos , Animales , Proliferación Celular , Citocinas/metabolismo , Vacunas Fúngicas/administración & dosificación , Pulmón/microbiología , Pulmón/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Linfocitos T/inmunología , Vacunas de Subunidad/administración & dosificación , Vacunas de Subunidad/inmunologíaRESUMEN
The presence of bats in caves, attics, ceilings, and roofs is important epidemiologically as they can increase the chance of human acquisition of pathogens, including Histoplasma capsulatum. Brazilian urban areas contain many species of bats, especially insectivorous bats, that are attracted by a wide range of readily available food and shelter. From August 2003 to December 2008, we analysed 2427 bats in the São Paulo State region. Homogenates of the livers and spleens of the bats were plated on specific medium to identify animals infected with H. capsulatum. The fungus was isolated from 87 bats (3·6%). The infected bats were identified as Molossus molossus (74), Nyctinomops macrotis (10), Tadarida brasiliensis (1), Molossus rufus (1) and Eumops glaucinus (1), all insectivorous species. The data presented are a relevant contribution to the epidemiology of H. capsulatum in densely populated urban areas such as in São Paulo State, especially since histoplasmosis is not included in the mandatory disease notification system.
Asunto(s)
Quirópteros/microbiología , Histoplasma/aislamiento & purificación , Histoplasmosis/veterinaria , Animales , Brasil , Quirópteros/clasificación , Femenino , Histoplasmosis/microbiología , Hígado/microbiología , Masculino , Prevalencia , Bazo/microbiologíaRESUMEN
Antitumor immune responses are associated with proinflammatory cytokines, whereas tumor-developing animals generally have increased the production of immunosuppressive cytokines. Here, we show that splenocytes from C57Bl/6 mice resistant to low doses of B16F10-Nex2 melanoma cells produced twofold or higher interferon-γ (IFN-γ)/interleukin-10 (IL-10) ratios, whereas cells from tumor-bearing animals produced predominantly IL-10. IL-10-knockout (IL-10KO) mice were significantly more resistant to B16F10-Nex2 development, producing increased amounts of IL-12 and IFN-γ. To neutralize IL-10 in vivo, aiming at cancer therapy, recombinant eukaryotic plasmid expressing the soluble extracellular region of the murine IL-10 receptor α-chain was constructed (pcDNA3-sIL-10R). Plasmid-treated melanoma-challenged animals showed extended survival time, the protective response was IFN-γ dependent and enhanced by co-immunization with a plasmid expressing IL-12. Dendritic cells (DCs) from IL-10KO mice, primed with B16F10-Nex2 antigens (TAg), secreted increased amounts of T-helper 1-type cytokines and increased the expression of surface activation markers. Vaccination of C57Bl/6 mice with TAg-activated IL-10KO DCs, as well as with TAg-primed DCs from C57Bl/6 mice transfected with pcDNA3-sIL10R plasmid, significantly increased animal survival. In conclusion, an IFN-γ-dependent protective response was induced against B16F10-Nex2 cells by neutralization of IL-10 with pcDNA3-sIL10R plasmid. This effect was enhanced by association with IL-12 gene therapy (80% protection), and could be mediated by TAg-primed DCs.
Asunto(s)
Terapia Genética/métodos , Inmunoterapia Adoptiva/métodos , Interferón gamma/inmunología , Interleucina-12/genética , Melanoma Experimental/terapia , Receptores de Interleucina-10/genética , Animales , Línea Celular Tumoral , Células Dendríticas/inmunología , Humanos , Interleucina-12/biosíntesis , Interleucina-12/inmunología , Masculino , Melanoma Experimental/genética , Melanoma Experimental/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Plásmidos/genética , Receptores de Interleucina-10/biosíntesis , Receptores de Interleucina-10/inmunología , TransfecciónRESUMEN
The protective role of specific antibodies against Paracoccidioides brasiliensis is controversial. In the present study, we analyzed the effects of monoclonal antibodies on the major diagnostic antigen (gp43) using in vitro and in vivo P. brasiliensis infection models. The passive administration of some monoclonal antibodies (MAbs) before and after intratracheal or intravenous infections led to a reduced fungal burden and decreased pulmonary inflammation. The protection mediated by MAb 3E, the most efficient MAb in the reduction of fungal burden, was associated with the enhanced phagocytosis of P. brasiliensis yeast cells by J774.16, MH-S, or primary macrophages. The ingestion of opsonized yeast cells led to an increase in NO production by macrophages. Passive immunization with MAb 3E induced enhanced levels of gamma interferon in the lungs of infected mice. The reactivity of MAb 3E against a panel of gp43-derived peptides suggested that the sequence NHVRIPIGWAV contains the binding epitope. The present work shows that some but not all MAbs against gp43 can reduce the fungal burden and identifies a new peptide candidate for vaccine development.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Antígenos Fúngicos/inmunología , Proteínas Fúngicas/inmunología , Glicoproteínas/inmunología , Paracoccidioides/patogenicidad , Paracoccidioidomicosis/inmunología , Paracoccidioidomicosis/prevención & control , Tráquea/microbiología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Antígenos Fúngicos/química , Línea Celular , Células Cultivadas , Epítopos/química , Proteínas Fúngicas/química , Glicoproteínas/química , Inmunización Pasiva , Inyecciones Intravenosas , Macrófagos Alveolares/microbiología , Macrófagos Peritoneales/microbiología , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Paracoccidioides/inmunología , Paracoccidioidomicosis/diagnóstico , Paracoccidioidomicosis/parasitología , Fagocitosis , Resultado del TratamientoRESUMEN
Ajoene has been described as an antithrombotic, anti-tumour, antifungal, antiparasitic and antibacterial agent. This study deals with the efficacy of ajoene to treat mice intratracheally infected with Paracoccidioides brasiliensis. The results indicate that ajoene therapy is effective in association with antifungal drugs (sulfametoxazol/trimethoprim), showing a positive additive effect. Ajoene-treated mice developed Th1-type cytokine responses producing higher levels of IFN-gamma and IL-12 when compared to the infected but untreated members of the control group. Antifungal activity of ajoene involves a direct effect on fungi and a protective pro-inflammatory immune response. Reduction of fungal load is additive to chemotherapy and therefore the combined treatment is mostly effective against experimental paracoccidioidomycosis.
Asunto(s)
Antiinfecciosos/uso terapéutico , Disulfuros/uso terapéutico , Ajo/química , Paracoccidioides/efectos de los fármacos , Paracoccidioidomicosis/tratamiento farmacológico , Combinación Trimetoprim y Sulfametoxazol/química , Animales , Antiinfecciosos/química , Antiinfecciosos/farmacología , Productos Biológicos , Terapias Complementarias , Disulfuros/química , Ratones , Ratones Endogámicos BALB C , Pruebas de Sensibilidad Microbiana , Paracoccidioides/inmunología , Paracoccidioides/aislamiento & purificación , Paracoccidioidomicosis/inmunología , Sulfóxidos , Combinación Trimetoprim y Sulfametoxazol/uso terapéuticoRESUMEN
Propionibacterium acnes has been described as a potent adjuvant to immune responses in vitro and in vivo. Presently, we analysed the modulation of peritoneal exudate cells (PEC) by heat-killed P. acnes or its purified soluble polysaccharide (PS), both injected intraperitoneally in C57Bl/6 mice, aiming at their recruitment and cytotoxicity. Both treatments induced an increase in macrophages, immature dendritic cells, B1a lymphocytes and NK1.1(+) CD3(+) cells. The bacterium caused a remarkable increase in a NK1.1(+) CD3(+) CD4(-) CD8(-) cells subpopulation, whereas the PS component seemed responsible for the recruitment of mainly macrophage cells. To assess P. acnes and PS adjuvant effect on PEC cytotoxicity we evaluated their in vitro effect on murine B16F10 melanoma cells. The effector cells from the heat-killed bacteria and PS-treated groups lysed melanoma cells in co-cultures with PEC. Mice genetically deficient in IFN-gamma, when stimulated with P. acnes or PS, had reduced PEC cytotoxicity, and the cytotoxic effect was completely abrogated in PEC from iNOS(-/-) mice. The tumoricidal activity of PEC from P. acnes-treated mice was mediated by macrophages and NKT cells stimulated with IL-12. In PS-treated mice the cytotoxicity was mediated mainly by macrophages. Moreover, both treatments increased IL-4 and IFN-gamma production by NKT cells. In conclusion, we show that P. acnes act mainly by recruiting and activating NKT double-negative cells in PEC, which were shown to be tumoricidal in vitro when induced by IL-12. Macrophages induced by both P. acnes and PS have their antitumour effect dependent on NO production.
Asunto(s)
Líquido Ascítico/citología , Citotoxicidad Inmunológica , Células Asesinas Naturales/inmunología , Lipopolisacáridos/inmunología , Macrófagos Peritoneales/inmunología , Propionibacterium acnes/inmunología , Animales , Líquido Ascítico/inmunología , Muerte Celular/inmunología , Citocinas/metabolismo , Exudados y Transudados/citología , Exudados y Transudados/inmunología , Femenino , Factores Inmunológicos/inmunología , Inmunofenotipificación , Inyecciones Intraperitoneales , Lipopolisacáridos/aislamiento & purificación , Activación de Macrófagos/inmunología , Melanoma Experimental , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Óxido Nítrico/metabolismo , Células Tumorales CultivadasRESUMEN
Immunization with peptide P10, derived from gp43, and chemotherapy were used together in an attempt to improve treatment of paracoccidioidomycosis and prevent relapses. The combined treatment showed an additive protective effect when administered at 48 h or 30 days after intratracheal challenge. Its use is recommended to improve regular chemotherapy and reduce the duration of treatment.
Asunto(s)
Antifúngicos/uso terapéutico , Proteínas Fúngicas/inmunología , Paracoccidioides/inmunología , Paracoccidioides/patogenicidad , Paracoccidioidomicosis/tratamiento farmacológico , Paracoccidioidomicosis/terapia , Péptidos/inmunología , Adyuvantes Inmunológicos/uso terapéutico , Animales , Terapia Combinada , Proteínas Fúngicas/administración & dosificación , Vacunas Fúngicas/administración & dosificación , Vacunas Fúngicas/inmunología , Humanos , Inmunización , Intubación Intratraqueal , Masculino , Ratones , Ratones Endogámicos BALB C , Pruebas de Sensibilidad Microbiana , Paracoccidioides/aislamiento & purificación , Péptidos/administración & dosificación , VirulenciaRESUMEN
Paracoccidioides brasiliensis, a thermodimorphic fungus, is the causative agent of paracoccidioidomycosis (PCM), the most prevalent systemic mycosis in Latin America. Pathogenicity appears to be intimately related to the dimorphic transition from the hyphal to the yeast form, which is induced by a shift from environmental temperature to the temperature of the mammalian host. Little information is available on the P. brasiliensis genes that are necessary during the pathogenic phase. We have therefore undertaken Suppression Subtraction Hybridization (SSH) and macroarray analyses with the aim of identifying genes that are preferentially expressed in the yeast phase. Genes identified by both procedures as being more highly expressed in the yeast phase are involved in basic metabolism, signal transduction, growth and morphogenesis, and sulfur metabolism. In order to test whether the observed changes in gene expression reflect the differences between the growth conditions used to obtain the two morphological forms rather than differences intrinsic to the cell types, we performed real-time RT-PCR experiments using RNAs derived from both yeast cells and mycelia that had been cultured at 37 degrees C and 26 degrees C in either complete medium (YPD or Sabouraud) or minimal medium. Twenty genes, including AGS1 (alpha-1,3-glucan synthase) and TSA1 (thiol-specific antioxidant), were shown to be more highly expressed in the yeast cells than in the hyphae. Although their levels of expression could be different in rich and minimal media, there was a general tendency for these genes to be more highly expressed in the yeast cells.
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ADN Complementario/análisis , ADN de Hongos/análisis , Perfilación de la Expresión Génica , Genes Fúngicos/fisiología , Paracoccidioides/genética , ADN Complementario/aislamiento & purificación , Regulación Fúngica de la Expresión Génica , Hibridación de Ácido Nucleico , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Saccharomyces cerevisiae/genética , Técnica de SustracciónRESUMEN
The 43 kDa glycoprotein (gp43) of Paracoccidioides brasiliensis is the major diagnostic antigen of paracoccidioidomycosis (PCM), a prevalent fungal infection in South America. A 15-mer sequence from gp43, denominated P10, induced T-CD4+ T helper 1 cellular immune responses in mice of three different haplotypes and protected against intratracheal challenge by a virulent isolate of P. brasiliensis. In an attempt to improve delivery of P10, a promiscuous antigen also presented by human leucocyte antigen-DR alleles, aiming at immunotherapy, we synthesized a multiple antigen peptide with the protective T-cell epitope expressed in a tetravalent 13-mer analog of P10 (M10). M10 induced specific lymph node cell proliferation in mice preimmunized with peptides in complete Freund's adjuvant (CFA). In addition, M10 immunization without CFA significantly protected intratracheally infected mice. We conclude that M10 is a candidate for an anti-PCM vaccine. In this report we describe: (1) the synthesis of M10; (2) the induction of M10-elicited T-cell response and (3) in vivo protection of mice immunized with M10 and challenged by a virulent strain of P. brasiliensis.
Asunto(s)
Antígenos Fúngicos/inmunología , Epítopos de Linfocito T/inmunología , Proteínas Fúngicas/inmunología , Glicoproteínas/inmunología , Paracoccidioidomicosis/prevención & control , Péptidos/síntesis química , Péptidos/inmunología , Secuencia de Aminoácidos , Animales , Femenino , Vacunas Fúngicas/inmunología , Inmunización , Activación de Linfocitos/inmunología , Masculino , Ratones , Datos de Secuencia Molecular , Paracoccidioides/inmunologíaRESUMEN
Experiments were performed to determine whether sialic acids are expressed in two dermatophytes: Trichophyton rubrum and T. mentagrophytes, similarly to other fungal pathogens. Chemical extraction of mycelia and microconidia followed by high-performance thin-layer chromatography and colorimetric assays were all negative for sialic acid. Incubation of dermatophytes in the presence of Limax flavus agglutinin, specific for sialic acid, was negative in a fluorescence staining test. We have also used other lectins that bind to sialic acid and/or other sugar residues, and these ligands coupled to fluorescein strongly stained these fungi. Such fluorescence staining was not abolished or reduced when fungi were pretreated with sialidase. Different strains of influenza virus which recognize sialic acid residues were also used, but no agglutination of the dermatophytes was observed. Based on these methods, which successfully revealed the presence of sialic acids in other fungal pathogens, we show that these monosaccharides do not occur in both dermatophyte species. Thus, sialic acids do not seem to play a role in the pathogenicity of these fungi.
Asunto(s)
Ácidos Siálicos/biosíntesis , Trichophyton/metabolismo , Aglutinación , Cromatografía Líquida de Alta Presión , Colorimetría , Técnica del Anticuerpo Fluorescente Directa , Lectinas/metabolismo , Mitocondrias/metabolismo , Micelio/química , Micelio/metabolismo , Neuraminidasa/farmacología , Orthomyxoviridae/metabolismo , Ácidos Siálicos/análisis , Especificidad de la Especie , Trichophyton/efectos de los fármacosRESUMEN
The activity of a phosphatase was characterized in intact mycelial forms of Fonsecaea pedrosoi, a pathogenic fungus that causes chromoblastomycosis. At pH 5.5, this fungus hydrolyzed p-nitrophenylphosphate (p-NPP) to p-nitrophenol (p-NP) at a rate of 12.78 +/- 0.53 nmol p-NP per h per mg hyphal dry weight. The values of Vmax and apparent Km for p-NPP hydrolyses were measured as 17.89 +/- 0.92 nmol p-NP per h per mg hyphal dry weight and 1.57 +/- 0.26 mmol/l, respectively. This activity was inhibited at increased pH, a finding compatible with an acid phosphatase. The enzymatic activity was strongly inhibited by classical inhibitors of acid phosphatases such as sodium orthovanadate (Ki = 4.23 micromol/l), sodium molybdate (Ki = 7.53 micromol/l) and sodium fluoride (Ki = 126.78 micromol/l) in a dose-dependent manner. Levamizole (1 mmol/l) and sodium tartrate (10 mmol/l), had no effect on the enzyme activity. Cytochemical localization of the acid phosphatase showed electrondense cerium phosphate deposits on the cell wall, as visualized by transmission electron microscopy. Phosphatase activity in F. pedrosoi seems to be associated with parasitism, as sclerotic cells, which are the fungal forms mainly detected in chromoblastomycosis lesions, showed much higher activities than conidia and mycelia did. A strain of F. pedrosoi recently isolated from a human case of chromoblastomycosis also showed increased enzyme activity, suggesting that the expression of surface phosphatases may be stimulated by interaction with the host.
Asunto(s)
Ascomicetos/enzimología , Pared Celular/enzimología , Cromoblastomicosis/microbiología , Monoéster Fosfórico Hidrolasas/metabolismo , Ascomicetos/crecimiento & desarrollo , Ascomicetos/metabolismo , Pared Celular/metabolismoRESUMEN
Polyclonal and monoclonal antibodies (MAbs) have been raised against B16F10 cells collected from growing tumors in vivo or grown in culture media supplemented with normal mouse serum to avoid xenogeneic reactivity. Antibody binding to glutaraldehyde-fixed melanoma cells and Melan A melanocytes was assayed using chemiluminescent-enzyme-linked immunosorbent assay (CL-ELISA) for increased sensitivity. Most of the reactivity of antitumor polyclonal IgG (92%) was inhibitable by a carbohydrate pool consisting of melibiose, mannose, lactose, and sialic acid. Two monoclonal IgG(2a) antibodies, A4 and B11, had their reactivity to melanoma cells completely and specifically inhibited by melibiose. MAb A4 did not bind to alpha-galactosyl residues abundantly expressed in a protozoan mucin used as substrate, and its binding to the tumor cells was not affected by alpha-galactosidase treatment or addition of alpha-methyl-galactopyranoside or raffinose. Recognition of a mimotope similar to melibiose is suggested. MAb is cytotoxic in vitro in a complement-mediated reaction and effectively neutralizes melanoma cells protecting syngeneic mice against tumor development in vivo. This MAb is thus an important tool for further studies on antitumor adjuvant therapy combined with other agents associated with immuno- and chemotherapy of invasive melanoma.
Asunto(s)
Melanoma/metabolismo , Melibiosa/química , Animales , Anticuerpos Monoclonales/metabolismo , Anticuerpos Antineoplásicos/administración & dosificación , Antígenos de Neoplasias , Vacunas contra el Cáncer/química , Metabolismo de los Hidratos de Carbono , Carbohidratos/farmacología , Separación Celular , Proteínas del Sistema Complemento , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática , Epítopos , Citometría de Flujo , Galactosa/química , Glutaral/farmacología , Humanos , Hibridomas , Inmunoglobulina G/metabolismo , Inmunoterapia , Lactosa/metabolismo , Antígeno MART-1 , Manosa/metabolismo , Melanoma Experimental , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Microscopía Fluorescente , Mucinas/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Proteínas de Neoplasias/farmacología , Conformación Proteica , Trypanosoma cruziRESUMEN
CD43, the major transmembrane sialoglycoprotein of neutrophils, monocytes, T lymphocytes and platelets, is highly glycosylated and its high sialic acid content contributes to the strongly negative charge of cells. In this study the role of CD43 in melanoma development was addressed using CD43 -/- mice (null mutated for the corresponding gene or knockout [KO]). Growth of B16F10 melanoma was retarded in the KO mice compared with the wild-type CD43+/+ control (WT). A marked difference in lung colonization and other metastatic foci was observed in the KO and WT mice up to 15 days after intravenous injection of tumour cells. The initial resistance of KO mice was reversed with time, and in the long term there was no difference in the survival rate of the two animal groups. Transient resistance was attributed to increased adhesion of thrombin-activated platelets and leukocytes to melanoma and endothelial cells in KO mice. In the KO mice tumour emboli were found in the central portion of the lung more than at the lung periphery immediately after intravenous injection, in contrast to the WT mice. Activation of melanoma adhesion receptors by thrombin or TRAP stimulated lung colonization in WT but not KO mice. Therefore, the correlation of tumour embolism and metastasis in short-term experiments depends on the nature and stability of interactions between the tumour and the blood/endothelial cells of the host.
Asunto(s)
Antígenos CD , Melanoma Experimental/patología , Sialoglicoproteínas/biosíntesis , Animales , Plaquetas/metabolismo , Adhesión Celular , Endotelio Vascular/citología , Leucosialina , Pulmón/patología , Masculino , Melanoma Experimental/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Metástasis de la Neoplasia , Trasplante de Neoplasias , Activación Plaquetaria , Ácidos Siálicos/metabolismo , Trombina/metabolismo , Factores de Tiempo , Células Tumorales CultivadasRESUMEN
Glycosylphosphatidylinositol (GPI) anchors and glycoinositolphospholipids (GIPLs) from parasitic protozoa have been shown to exert a wide variety of effects on cells of the host innate immune system. However, the receptor(s) that are triggered by these protozoan glycolipids has not been identified. Here we present evidence that Trypanosoma cruzi-derived GPI anchors and GIPLs trigger CD25 expression on Chinese hamster ovary-K1 cells transfected with CD14 and Toll-like receptor-2 (TLR-2), but not wild-type (TLR-2-deficient) Chinese hamster ovary cells. The protozoan-derived GPI anchors and GIPLs containing alkylacylglycerol and saturated fatty acid chains or ceramide were found to be active in a concentration range of 100 nM to 1 microM. More importantly, the GPI anchors purified from T. cruzi trypomastigotes, which contain a longer glycan core and unsaturated fatty acids in the sn-2 position of the alkylacylglycerolipid component, triggered TLR-2 at subnanomolar concentrations. We performed experiments with macrophages from TLR-2 knockout and TLR-4 knockout mice, and found that TLR-2 expression appears to be essential for induction of IL-12, TNF-alpha, and NO by GPI anchors derived from T. cruzi trypomastigotes. Thus, highly purified GPI anchors from T. cruzi parasites are potent activators of TLR-2 from both mouse and human origin. The activation of TLR-2 may initiate host innate defense mechanisms and inflammatory response during protozoan infection, and may provide new strategies for immune intervention during protozoan infections.
Asunto(s)
Proteínas de Drosophila , Glicosilfosfatidilinositoles/fisiología , Glicoproteínas de Membrana/metabolismo , Receptores de Superficie Celular/metabolismo , Trypanosoma cruzi/inmunología , Animales , Células CHO , Línea Celular , Cricetinae , Relación Dosis-Respuesta Inmunológica , Glucolípidos/fisiología , Glicosilfosfatidilinositoles/aislamiento & purificación , Inflamación/inmunología , Inflamación/parasitología , Macrófagos/inmunología , Macrófagos/metabolismo , Glicoproteínas de Membrana/deficiencia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/fisiología , Ratones , Ratones Noqueados , FN-kappa B/fisiología , Fosfolípidos/fisiología , Receptores de Superficie Celular/deficiencia , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/fisiología , Receptores de Interleucina-2/biosíntesis , Transducción de Señal/genética , Transducción de Señal/inmunología , Receptor Toll-Like 2 , Receptor Toll-Like 4 , Receptores Toll-Like , Transfección , Trypanosoma cruzi/química , Trypanosoma cruzi/crecimiento & desarrolloRESUMEN
In the present study, we evaluated the ability of GPI-anchored mucin-like glycoproteins purified from Trypanosoma cruzi trypomastigotes (tGPI-mucin) to trigger phosphorylation of different mitogen-activated protein kinases (MAPKs) and related transcription factors in inflammatory macrophages. Kinetic experiments show that the peak of extracellular signal-related kinase (ERK)-1/ERK-2, stress-activated protein kinase (SAPK) kinase-1/mitogen-activated protein kinase (MAPK) kinase-4, and p38/SAPK-2, phosphorylation occurs between 15 and 30 min after macrophage stimulation with tGPI-mucin or GPI anchors highly purified from tGPI-mucins (tGPI). The use of the specific inhibitors of ERK-1/ERK-2 (PD 98059) and p38/SAPK-2 (SB 203580) phosphorylation also indicates the role of MAPKs, with possible involvement of cAMP response element binding protein, in triggering TNF-alpha and IL-12 synthesis by IFN-gamma-primed-macrophages exposed to tGPI or tGPI-mucin. In addition, tGPI-mucin and tGPI were able to induce phosphorylation of I kappa B, and the use of SN50 peptide, an inhibitor of NF-kappa B translocation, resulted in 70% of TNF-alpha synthesis by macrophages exposed to tGPI-mucin. Finally, the similarity of patterns of MAPK and I kappa B phosphorylation, the concentration of drugs required to inhibit cytokine synthesis, as well as cross-tolerization exhibited by macrophages exposed to tGPI, tGPI-mucin, or bacterial LPS, suggest that receptors with the same functional properties are triggered by these different microbial glycoconjugates.