RESUMO
Guanine-nucleotide exchange factors (GEFs) stimulate the intrinsic GDP/GTP exchange activity of Ras and promote the formation of active Ras-GTP, which in turn controls diverse signalling networks important for the regulation of cell proliferation, survival, differentiation, vesicular trafficking, and gene expression. RasGEF1b is a GEF, whose expression is induced in macrophages on stimulation with toll-like receptor (TLR) agonists. Here, we showed that in vitro RasGEF1b expression by macrophages is mostly induced by TLR3 (poly I:C) and TLR4 (lipopolysaccharyde) through the MyD88-independent pathway. In vivo infection with the protozoan parasites Trypanosoma cruzi and Plasmodium chabaudi induced RasGEF1b in an MyD88-, TRIF-, and IFN-gamma-dependent manner. Ectopically expressed RasGEF1b was found, mostly, in the heavy membrane fraction of HEK 293T, and by confocal microscopy, it was found to be located at early endosomes. Computational modelling of the RasGEF1b-Ras interaction revealed that RasGEF1b interacts with the binding domain site of Ras, a critical region for interacting with GEFs involved in the activation of Ras-Raf-MEK-ERK pathway. More important, RasGEF1b was found to be closely associated with Ras in live cells and to trigger Ras activity. Altogether, these results indicate that on TLR activation, RasGEF1b may trigger Ras-like proteins and regulate specific biological activities described for this subtype of GTPases.
Assuntos
Endossomos/metabolismo , Receptores Toll-Like/fisiologia , Fatores ras de Troca de Nucleotídeo Guanina/biossíntese , Animais , Células CHO , Cricetinae , Cricetulus , Endossomos/química , Feminino , Células HEK293 , Humanos , Mediadores da Inflamação/metabolismo , Mediadores da Inflamação/fisiologia , Lipopolissacarídeos/farmacologia , Macrófagos Peritoneais/química , Macrófagos Peritoneais/metabolismo , Macrófagos Peritoneais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores Toll-Like/metabolismo , Fatores ras de Troca de Nucleotídeo Guanina/metabolismo , Fatores ras de Troca de Nucleotídeo Guanina/fisiologiaRESUMO
CD8+ T lymphocytes have been reported to play a major role in the protective immune response against acute infection with Toxoplasma gondii. In order to further assess the role of CD8+ cells in resistance against this protozoan we examined the ability of beta 2m-deficient mice, which fail to express MHC class I molecules and peripheral CD8+ lymphocytes, to survive tachyzoite challenge following vaccination with an attenuated parasite mutant. Surprisingly, vaccination of beta 2m-deficient mice induced strong resistance to lethal challenge, with > 50% surviving beyond 3 months. Vaccinated beta 2m-deficient mice, but not control heterozygotes, showed a five- to six-fold expansion in spleen cell number and approximately 40% of the splenocytes were found to express the NK markers NK1.1 and asialo GM1. Spleen cells from the vaccinated beta 2m-deficient animals failed to kill either infected host cells or the NK target YAC-1. However, high levels of IFN-gamma were secreted when the cells were cultured in vitro with soluble T. gondii lysate, and this response was abolished by NK1.1+ but not CD4+ and CD8+ lymphocyte depletion, implicating the NK1.1+ population as the major source of IFN-gamma. More importantly, vaccine-induced immunity in beta 2m-deficient mice was completely abrogated by in vivo administration of antibody to NK1.1, asialo GM1, or IFN-gamma. Together, the data suggest that in class I-deficient mice vaccinated against T. gondii, the absence of CD8+ effector cells is compensated for by the emergence of a population of NK1.1+ and asialo GM1+ cells which lack cytolytic activity, and that the protective action of these cells against the parasite is attributable to IFN-gamma production. The induction of this novel NK population may provide an approach for controlling opportunistic infections in immunocompromised hosts.
Assuntos
Antígenos CD8/imunologia , Genes MHC Classe I , Células Matadoras Naturais/imunologia , Linfócitos/imunologia , Toxoplasmose Animal/imunologia , Microglobulina beta-2/deficiência , Animais , Células Cultivadas , Cruzamentos Genéticos , Citotoxicidade Imunológica , Feminino , Citometria de Fluxo , Imunização , Interferon gama/farmacologia , Depleção Linfocítica , Linfócitos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Baço/imunologia , Linfócitos T/imunologia , Vacinas AtenuadasRESUMO
Lymphoproliferation, chronic B cell activation resulting in hypergammaglobulinemia, and profound immunodeficiency are prominent features of a retrovirus-induced syndrome designated murine acquired immunodeficiency syndrome (MAIDS). In vivo treatment of infected mice with recombinant interleukin 12 (IL-12) beginning at the time of infection or up to 9 wk after virus inoculation markedly inhibited the development of splenomegaly and lymphadenopathy, as well as B cell activation and Ig secretion. Treatment with IL-12 also had major effects in preventing induction of several immune defects including impaired production of interferon gamma (IFN-gamma) and IL-2 and depressed proliferative responses to various stimuli. The therapeutic effects of IL-12 on the immune system of mice with MAIDS were also associated with reduced expression of the retrovirus that causes this disease (BM5def), with lesser effects on expression of ecotropic MuLV. IL-12 treatment was not effective in IFN-gamma knockout mice or in infected mice treated simultaneously with IL-12 and anti-IFN-gamma. These results demonstrate that induction and progression of MAIDS are antagonized by IL-12 through high-level expression of IFN-gamma and may provide an experimental basis for developing treatments of retrovirus-induced immune disorders with similar immunopathogenic mechanisms.
Assuntos
Linfócitos B/imunologia , Interleucina-12/uso terapêutico , Síndrome de Imunodeficiência Adquirida Murina/imunologia , Animais , Anticorpos Monoclonais/farmacologia , Linfócitos B/efeitos dos fármacos , Sequência de Bases , Primers do DNA , Feminino , Citometria de Fluxo , Hipoxantina Fosforribosiltransferase/biossíntese , Interferon gama/imunologia , Interleucina-12/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Síndrome de Imunodeficiência Adquirida Murina/fisiopatologia , Síndrome de Imunodeficiência Adquirida Murina/terapia , Tamanho do Órgão/efeitos dos fármacos , Reação em Cadeia da Polimerase , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêutico , Esplenomegalia/prevenção & controle , Fatores de TempoRESUMO
Human immunodeficiency virus (HIV) 1 transgenic mice expressing low or undetectable levels of viral mRNA in lymphoid tissue were infected with the intracellular protozoan Toxoplasma gondii. Exposure to this parasite resulted in an increase in HIV-1 transcript in lymph nodes, spleens, and lungs during the acute phase of infection and in the central nervous system during the chronic stage of disease. In vivo and ex vivo experiments identified macrophages as a major source of the induced HIV-1 transcripts. In contrast, T. gondii infection failed to stimulate HIV-1 transcription in tissues of two HIV-1 transgenic mouse strains harboring a HIV-1 proviral DNA in which the nuclear factor (NF) kappa B binding motifs from the viral long terminal repeats had been replaced with a duplicated Moloney murine leukemia virus core enhancer. A role for NF-kappaB in the activation of the HIV-1 by T. gondii was also suggested by the simultaneous induction of NF-kappaB binding activity and tumor necrosis factor alpha synthesis in transgenic mouse macrophages stimulated by exposure to parasite extracts. These results demonstrate the potential of an opportunistic pathogen to induce HIV-1 transcription in vivo and suggest a mechanism for the in vivo dissemination of HIV-1 by macrophages.
Assuntos
Infecções Oportunistas Relacionadas com a AIDS/virologia , Regulação Viral da Expressão Gênica , Infecções por HIV/virologia , HIV-1/crescimento & desenvolvimento , Macrófagos/virologia , Toxoplasmose Animal/virologia , Infecções Oportunistas Relacionadas com a AIDS/complicações , Animais , Modelos Animais de Doenças , Infecções por HIV/complicações , Repetição Terminal Longa de HIV , HIV-1/genética , Camundongos , Camundongos Transgênicos , NF-kappa B/metabolismo , Provírus/genética , Provírus/crescimento & desenvolvimento , RNA Mensageiro/biossíntese , RNA Viral/biossíntese , Toxoplasmose Animal/complicações , Transcrição Gênica , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Leishmania major promastigotes were found to avoid activation of mouse bone marrow-derived macrophages (BMM0) in vitro for production of cytokines that are typically induced during infection with other intracellular pathogens. Coexposure of BMM0 to the parasite and other microbial stimuli resulted in complete inhibition of interleukin (IL) 12 (p40) mRNA induction and IL-12 release. In contrast, mRNA and protein levels for IL-1(alpha), IL-1(beta), tumor necrosis factor (TNF) alpha, and inducible NO synthase (iNOS) were only partially reduced, and signals for IL-10 and monocyte chemoattractant protein (MCP-1/JE) were enhanced. The parasite could provide a detectable trigger for TNF-alpha and iNOS in BMM0 primed with interferon (IFN) gamma, but still failed to induce IL-12. Thus IL-12 induction is selectively impaired after infection, whereas activation pathways for other monokine responses remain relatively intact. Selective and complete inhibition of IL-12(p40) induction was observed using BMM0 from either genetically susceptible or resistant mouse strains, as well as IL-10 knockout mice, and was obtained using promastigotes from cutaneous, visceral, and lipophosphoglycan-deficient strains of Leishmania. The impaired production of the major physiological inducer of IFN-gamma is suggested to underlie the relatively prolonged interval of parasite intracellular survival and replication that is typically associate with leishmanial infections, including those producing self-limiting disease.
Assuntos
Medula Óssea/imunologia , Regulação da Expressão Gênica , Interleucina-12/biossíntese , Leishmania/imunologia , Macrófagos/imunologia , Animais , Sequência de Bases , Células da Medula Óssea , Citocinas/biossíntese , Predisposição Genética para Doença , Imunidade Inata/genética , Interleucina-12/genética , Leishmania donovani/imunologia , Leishmania major/imunologia , Lipopolissacarídeos/imunologia , Ativação de Macrófagos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Mycobacterium tuberculosis/imunologia , RNA Mensageiro/análiseRESUMO
Chagas disease (CD) is caused by the infection with the protozoan haemoflagellate Trypanosoma cruzi. This disease is still a great menace to public health, and is largely neglected as it affects mostly the poorest populations of Latin America. Nonetheless, there are neither effective diagnostic markers nor therapeutic options to accurately detect and efficiently cure this chronic infection. In spite of the great advances in the knowledge of the biology of natural transmission, as well as the immunobiology of the host-parasite interaction, the understanding of the pathogenesis of CD remains largely elusive. In the recent decades, a controversy in the research community has developed about the relevance of parasite persistence or autoimmune phenomena in the development of chronic cardiac pathology. One of the most notable aspects of chronic CD is the progressive deterioration of cardiac function, derived mostly from structural derangement, as a consequence of the intense inflammatory process. Here we review the evidence supporting the multifactorial nature of Chagas heart disease comprising pathogen persistence and altered host immunoregulatory mechanisms.
Assuntos
Cardiomiopatia Chagásica/parasitologia , Trypanosoma cruzi/patogenicidade , Animais , Autoimunidade , Cardiomiopatia Chagásica/imunologia , Interações Hospedeiro-Parasita , Humanos , América Latina , Trypanosoma cruzi/fisiologia , VirulênciaRESUMO
This study was undertaken to determine if prolonged daily subcutaneous administration of ultra low dose IL-2 could influence the constitutive endogenous production of a type 1 (IFN-gamma) cytokine in patients with AIDS or AIDS-associated malignancies. Using a quantitative reverse transcription PCR assay, we demonstrate that daily administration of one type 1 cytokine, IL-2, for 3 mo increases significantly the constitutive endogenous gene expression of another type 1 cytokine, IFN-gamma, in vivo. The predominant source of IFN-gamma appears to be IL-2-expanded natural killer cells and CD8(+) T cells. Moreover, PBMC obtained from these patients during IL-2 therapy showed normalization of a profound deficit in IFN-gamma protein production after stimulation with extracts from infectious agents in vitro. Our data suggest that prolonged exogenous administration of a type 1 cytokine in a nontoxic fashion to patients with AIDS and AIDS-associated malignancies can enhance significantly the endogenous type 1 cytokine profile in vivo. Consequently, ultra low dose IL-2 therapy has the potential to improve the immunodeficient hosts' immune response to infectious pathogens that require IFN-gamma for clearance.
Assuntos
Síndrome da Imunodeficiência Adquirida/terapia , Interferon gama/metabolismo , Interleucina-2/administração & dosagem , Interleucina-2/uso terapêutico , Linfoma Relacionado a AIDS/terapia , Sarcoma de Kaposi/terapia , Síndrome da Imunodeficiência Adquirida/imunologia , Síndrome da Imunodeficiência Adquirida/metabolismo , Contagem de Linfócito CD4 , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Citometria de Fluxo , Expressão Gênica , Humanos , Imunidade Inata , Hospedeiro Imunocomprometido/efeitos dos fármacos , Hospedeiro Imunocomprometido/imunologia , Imunoterapia/métodos , Interferon gama/genética , Interleucina-10/genética , Interleucina-10/metabolismo , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Leucócitos Mononucleares/metabolismo , Lipopolissacarídeos/imunologia , Contagem de Linfócitos , Linfoma Relacionado a AIDS/imunologia , Linfoma Relacionado a AIDS/metabolismo , Reação em Cadeia da Polimerase , Sarcoma de Kaposi/imunologia , Sarcoma de Kaposi/metabolismo , Subpopulações de Linfócitos T/imunologiaRESUMO
Recent studies have documented that the immunoregulatory functions of IL-12 may play a role in promoting endogenous protective responses during infections and/or contribute to pathology resulting from unregulated cytokine expression. Pathogen induction of IL-12 elicits interferon-gamma production by natural killer cells, which contributes to early defense during certain bacterial, parasitic, and viral infections. IL-12 also facilitates the development of T helper type 1 (Th1) lymphocytes required for late protection against bacteria, parasites, and fungi. During viral infections, however, there appear to be mechanisms independent of IL-12 for inducing protective T-cell responses. In contrast, negative regulation of IL-12 during acute infections can be a key event in the establishment of chronic infection and protection against harmful excessive cellular immune response. Under appropriate conditions, IL-12 has therapeutic efficacy for promoting defense against a variety of pathogens, and for use as a vaccine adjuvant to enhance beneficial Th1 over detrimental Th2 lymphocyte responses. This information extends knowledge about the regulation of immune responses to infectious agents, and provides new insights for the development of treatment and adjuvant strategies to potentiate beneficial or inhibit detrimental endogenous immune responses.
Assuntos
Infecções Bacterianas/imunologia , Interleucina-12/imunologia , Interleucina-12/uso terapêutico , Animais , Infecções Bacterianas/terapia , Imunidade Inata/efeitos dos fármacosRESUMO
Glycosylphosphatidylinositol (GPI) anchor and glycoinositolphospholipid (GIPL) are abundant molecules present in the membrane of parasitic protozoa that are common etiologic agents of medical and veterinary diseases. Recent studies have documented the immunostimulatory/regulatory activity of protozoan-derived GPI-anchors and related structures. Among the bioactivity displayed by the protozoan-derived GPI-anchor is the ability to elicit the synthesis of pro-inflammatory cytokines as well as nitric oxide by host macrophages. In contrast, at high concentrations GIPL and lipophosphoglycan (LPG) derived from protozoan parasites suppress several functions of the host immune system. Additionally, the protozoan-derived GPI-anchor and GIPL have been shown to serve as targets for both specific B and NK-T lymphocyte responses. This information extends our knowledge about parasite molecules that stimulate/regulate the host immune system during protozoan infection. The identification of receptor(s) and signaling pathways triggered by these GPI-related glycolipids may provide new insights for the development of therapies that inhibit detrimental immune responses or potentiate beneficial immune responses observed during infection with protozoan parasites.
Assuntos
Eucariotos/imunologia , Glicolipídeos/metabolismo , Glicosilfosfatidilinositóis/metabolismo , Fosfolipídeos/metabolismo , Infecções por Protozoários/imunologia , Transdução de Sinais , Animais , Eucariotos/química , Eucariotos/metabolismo , Eucariotos/patogenicidade , Glicolipídeos/química , Glicosilfosfatidilinositóis/química , Humanos , Ativação de Macrófagos , Macrófagos/imunologia , Macrófagos/metabolismo , Fosfolipídeos/química , Infecções por Protozoários/parasitologia , VirulênciaRESUMO
A strong activation of macrophages is observed during acute infection with Trypanosoma cruzi. Little is known, however, about the parasite molecules that are responsible for this early activation of innate immunity. Recent studies have shown the stimulatory activity of protozoan-derived glycosylphosphatidylinositol (GPI) anchors on cultured macrophages. In this review, we provide a detailed analysis of the correlation between structure and proinflammatory activity by T. cruzi-derived GPI anchors. We also cover the studies that have identified the Toll-like receptor 2 as a functional GPI receptor and have partially characterized signaling pathways triggered by T. cruzi-derived GPI anchors, which lead to the synthesis of proinflammatory cytokines in macrophages. Finally, we discuss the implications of these findings in resistance and pathogenesis during the infection with T. cruzi.
Assuntos
Doença de Chagas/imunologia , Proteínas de Drosophila , Glicosilfosfatidilinositóis/química , Glicosilfosfatidilinositóis/fisiologia , Trypanosoma cruzi/patogenicidade , Animais , Sequência de Carboidratos , Citocinas/biossíntese , Inflamação/imunologia , Lipopolissacarídeos/química , Macrófagos/imunologia , Glicoproteínas de Membrana/metabolismo , Dados de Sequência Molecular , Receptores de Superfície Celular/metabolismo , Transdução de Sinais , Relação Estrutura-Atividade , Receptor 2 Toll-Like , Receptores Toll-LikeRESUMO
Differential display reverse transcriptase-polymerase chain reaction (PCR) was used to identify genes expressed by murine macrophages exposed to glycosylphosphatidylinositol-anchored mucin-like glycoproteins isolated from Trypanosoma cruzi trypomastigotes. Among the different PCR product bands identified in the differential display gel, one showed high homology with the serum amyloid A3 protein (SAA3). Northern blot assays showed augmentation of SAA3 mRNA expression by inflammatory macrophages exposed to live trypomastigotes or parasite glycolipids, as compared to unstimulated macrophages. Our results also showed the expression of SAA3 mRNA, in liver and heart from animals in the acute phase of Chagas disease. It is important that expression of SAA3 mRNA was closely associated with tissue parasitism and presence of inflammatory cells. Together, our findings indicate the possible involvement of SAA3 protein on immunopathology of Chagas disease and establish a new infectious disease model to study the pathophysiological role of this acute-phase protein.
Assuntos
Glicoconjugados/imunologia , Glicolipídeos/imunologia , Macrófagos Peritoneais/imunologia , Fosfolipídeos/imunologia , Proteína Amiloide A Sérica/genética , Trypanosoma cruzi/imunologia , Animais , Linhagem Celular , Células Cultivadas , Doença de Chagas/imunologia , Doença de Chagas/parasitologia , Regulação da Expressão Gênica , Glicosilfosfatidilinositóis , Interferon gama/imunologia , Interferon gama/farmacologia , Ativação de Macrófagos/imunologia , Macrófagos Peritoneais/citologia , Macrófagos Peritoneais/efeitos dos fármacos , Masculino , Glicoproteínas de Membrana/imunologia , Lipídeos de Membrana/imunologia , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Mucinas/imunologia , RNA Mensageiro/biossínteseRESUMO
We investigated the kinetics of parasite replication, leukocyte migration, and cytokine/chemokine mRNA expression in the heart tissue from animals infected with the Colombiana strain of Trypanosoma cruzi. Cardiac tissue parasitism was noticeable at 15 days, peaked around 30 days and was dramatically reduced at 120 days postinfection (p.i.). Kinetic studies showed that the inflammatory infiltrate was dominated by the presence of alphabetaT CD3(+ )CD4(+ )CD8(-), alphabetaT CD3(+ )CD4(-)CD8(+ )lymphocytes and macrophages. The mRNA expression of the monokines IL-1beta and IL-12(p40) was elevated at 15 days p.i. and controlled at later time points. In contrast, TNF-alpha mRNA was expressed throughout the infection. Interestingly, we found that at 15 and 30 days p.i. cytokine expression was dominated by the presence of IFN-gamma mRNA, whereas at 60 days or later time points the balance of type 1 and type 2 cytokines was switched in favor of IL-4 and IL-10 mRNAs. The chemokine mRNAs encoding JE, MIP-1alpha, MIP-1beta, KC, and MIP-2 were all mainly expressed at 15 and/or 30 days p.i. and diminished thereafter. In contrast, the expression of RANTES, MIG and IP-10 mRNAs was augmented at 15 days p.i. and persisted at high levels up to 120 days p.i. Taken together, our results indicate that regulation of IFN-gamma and chemokine expression, associated with decreased tissue parasitism, may be largely responsible for the control of inflammation and immunopathology observed in the cardiac tissue of animals infected with T. cruzi.
Assuntos
Cardiomiopatia Chagásica/imunologia , Quimiocinas/genética , Citocinas/genética , Interferon gama/genética , Trypanosoma cruzi/imunologia , Animais , Células Cultivadas , Cardiomiopatia Chagásica/parasitologia , Cardiomiopatia Chagásica/patologia , Quimiocinas CC/genética , Quimiocinas CXC/genética , Modelos Animais de Doenças , Feminino , Expressão Gênica , Coração/parasitologia , Cinética , Macrófagos Peritoneais/citologia , Macrófagos Peritoneais/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Miocárdio/patologia , Parasitemia , RNA MensageiroRESUMO
The determinants of the prevalence of CD8(+) T cells in the inflamed myocardium of Trypanosoma cruzi-infected patients and experimental animals are undefined. Using C3H/He mice infected with the Colombiana strain of T. cruzi, we found that the distribution of CD4(+)/CD8(-) and CD4(-)/CD8(+) T cells in the myocardium mirrors the frequency of cells expressing the CD62L(Low)LFA-1(High)VLA-4(High) activation phenotype among CD4(+)/CD8(-) and CD4(-)/CD8(+ )peripheral blood T cells. Consistently, vascular cell adhesion molecule-1-positive endothelial cells and a fine fibronectin network surrounding VLA-4(+) mononuclear cells were found in the inflamed myocardium. Further, interferon gamma (IFN-gamma) and IFN-gamma-induced chemokines (RANTES, MIG and CRG-2/IP-10), as well as JE/MCP-1 and MIP1-alpha, were found to be the dominant cytokines expressed in situ during acute and chronic myocarditis elicited by T. cruzi. In contrast, interleukin 4 mRNA was only detected during the chronic phase. Altogether, the results indicate that the distribution of T-cell subsets in the myocardium of T. cruzi-infected mice reflects the particular profile of adhesion molecules acquired by most peripheral CD8(+) T lymphocytes and point to the possibility that multiple IFN-gamma-inducible molecules present in the inflamed tissue contribute to the establishment and maintenance of T. cruzi-induced myocarditis.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Cardiomiopatia Chagásica/imunologia , Integrinas/análise , Interferon gama/farmacologia , Selectina L/análise , Antígeno-1 Associado à Função Linfocitária/análise , Receptores de Antígenos de Linfócitos T alfa-beta/análise , Receptores de Retorno de Linfócitos/análise , Animais , Moléculas de Adesão Celular/biossíntese , Cardiomiopatia Chagásica/parasitologia , Cardiomiopatia Chagásica/patologia , Quimiocinas/biossíntese , Citocinas/biossíntese , Feminino , Imunofenotipagem , Integrina alfa4beta1 , Camundongos , Camundongos Endogâmicos C3H , Miocárdio/patologia , Parasitemia/mortalidadeRESUMO
Twenty-seven Trypanosoma cruzi strains, susceptible or naturally resistant to the nitroderivatives benznidazole and nifurtimox, were analyzed using the following molecular markers: (i) isoenzyme patterns of six enzymes; (ii) genetic variability assayed by randomly amplified polymorphic DNA (RAPD) with two different primers; and (iii) gene probes for P-glycoprotein (TcPGP), hypoxanthine-guanine phosphoribosyltransferase (HGPRT), the ribosomal RNA gene (rDNA) and the mini-exon gene (MEX), RAPD and isoenzyme profiles divided the T. cruzi strains into three groups, whereas the gene probes divided the T. cruzi strains in two groups. Strains classified as group I or II by RAPD or zymodemes Z1 or Z2 by isoenzyme analysis were either susceptible or naturally resistant to the nitroderivatives. In contrast, strains classified as group III by RAPD and zymodeme ZB by isoenzyme analysis were only drug susceptible and showed polymorphisms for HGPRT and TcPGP. No correlation was observed between drug susceptibility and polymorphisms of rDNA and MEX. Eighteen T. cruzi strains isolated from different geographic regions were included in this study. Thus, from a total of 45 T. cruzi strains analyzed, all 19 of zymodeme B were susceptible to the experimental treatment independent of their geographic origin.
Assuntos
Nifurtimox/farmacologia , Nitroimidazóis/farmacologia , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Animais , Brasil , Doença de Chagas/parasitologia , DNA de Protozoário/genética , DNA Ribossômico/genética , Resistência a Medicamentos , Éxons/genética , Genes de Protozoários , Variação Genética , Glucose-6-Fosfato Isomerase/análise , Humanos , Hipoxantina Fosforribosiltransferase/genética , Isoenzimas/análise , Polimorfismo Genético , Técnica de Amplificação ao Acaso de DNA Polimórfico , Trypanosoma cruzi/classificação , Trypanosoma cruzi/enzimologia , Trypanosoma cruzi/genéticaRESUMO
Microbial stimuli such as bacterial lipopolysaccharide (LPS) or glycosylphosphatidylinositol-mucins derived from Trypanosoma cruzi trypomastigotes (tGPI-mucins) are effective stimulators of the synthesis of cytokines by macrophages. Here, we evaluated the ability of cyclic AMP mimetic or elevating agents to modulate TNF-alpha and IL-12 synthesis by murine inflammatory macrophages. Cholera Toxin (ChTx) inhibited tGPI-mucins (2.5 nM) or LPS (100 ng ml(-1)) induced TNF-alpha and IL-12(p40) synthesis in a concentration-dependent manner. Similarly, the cyclic AMP mimetics, 8-bromo cyclic AMP or dibutyryl cyclic AMP, or prostaglandin (PG) E2 inhibited the synthesis of both cytokines by macrophages exposed to microbial stimuli. The protein kinase A inhibitor H-89 partially reversed the inhibitory effects of dibutyryl cyclic AMP and PGE2 on both IL-12(p40) and TNF-alpha synthesis. Pretreatment of macrophages with dibutyryl cyclic AMP or ChTx augmented the synthesis of IL-10 triggered by microbial products. Elevation of cyclic AMP inhibited the synthesis of TNF-alpha, but not IL-12(p40), by inflammatory macrophages from IL-10 knockout mice. Kinetic studies showed that synthesis of both TNF-alpha and IL-10 peaked at 8 h and IL-12 at 24 h after stimulation with microbial stimuli. Together, our findings favour the hypothesis that the cyclic AMP inhibitory activity on IL-12(p40) but not on TNF-alpha synthesis is dependent on de novo protein synthesis, most likely involving IL-10, by macrophages stimulated with microbial products. Accordingly, dibutyryl cyclic AMP inhibited IL-12(p40) synthesis only when added before or at the same time of the stimuli. In contrast, the effect of this cyclic AMP analogue on TNF-alpha synthesis was protracted and observed even 2 h after the addition of the stimuli.
Assuntos
AMP Cíclico/fisiologia , Interleucina-12/biossíntese , Lipopolissacarídeos/farmacologia , Macrófagos/metabolismo , Trypanosoma cruzi/fisiologia , Fator de Necrose Tumoral alfa/biossíntese , Animais , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Glicosilfosfatidilinositóis/farmacologia , Interleucina-10/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Mucinas/farmacologiaRESUMO
Two proviral HIV transgenic mouse models, one bearing wild-type HIV proviral DNA and the other a modified provirus in which the viral LTRs contained the core enhancer of the Moloney murine leukemia virus (MLV), were compared. The MLV/HIV chimeric LTR, in which the MLV enhancer replaced the NF-kappa B-binding motifs, was transcriptionally active in human and murine cells in vitro and virus containing the chimeric LTR was replication competent in human cell cultures. Transgenic mice derived from microinjections of chimeric MLV/HIV proviral DNA transcribed HIV genes at a greater frequency and at higher levels than wild-type HIV proviral transgenic mice. MLV/HIV mice were also more apt to develop disease; wasting, periocular infections, and a degenerative myopathy characterized the most predominant phenotype. The tissue specificities of the wild-type and chimeric LTRs in transgenic mice were remarkably similar, but a significant difference was apparent in lymphoid cells. Basal level and LPS-inducible HIV gene expression occurred in peritoneal and bone marrow-derived macrophages from wild-type HIV transgenic mice. In contrast, HIV gene expression in macrophages from MLV/HIV mice was undetectable, even following LPS induction. However, cultured splenocytes from MLV/HIV mice supported HIV proviral gene transcription better than splenocytes from HIV mice, particularly after induction with LPS or anti-IgD antibody but not with concanavalin A. These data suggest that in transgenic mice, the HIV and MLV/HIV LTRs display a differential tropism for macrophages and B cells, respectively. HIV and MLV/HIV transgenic mice represent alternative models amenable to in vivo studies of HIV gene regulation in lymphoid cells, the induction of HIV-related disease and the evaluation of anti-HIV therapies.
Assuntos
Regulação Viral da Expressão Gênica , HIV-1/genética , Vírus da Leucemia Murina/genética , Provírus/genética , Animais , Técnicas de Transferência de Genes , Células HeLa , Humanos , Camundongos , Camundongos TransgênicosRESUMO
Transparent objects (phase objects) are not visible in a standard brightfield optical microscope. In order to see such objects the most used technique is phase-contrast microscopy. In phase-contrast microscopy the contrast observed is proportional to the optical path difference introduced by the object. If the index of refraction is uniform, phase-contrast microscopy then yields a measure of the thickness profile of phase objects. We show that by slightly defocusing an optical microscope operating in brightfield, phase objects become visible. We modeled such an effect and show that the image contrast of a phase object is proportional to the amount of defocusing and proportional to the two-dimensional Laplacian of the optical path difference introduced by the object. For uniform index of refraction, defocusing microscopy then yields a measure of the curvature profile of phase objects. We extended our previous model for thin objects to thick objects. To check our theoretical model, we use as phase objects polystyrene spherical caps and compare their curvature radii obtained by defocusing microscopy (DM) to those obtained with atomic force microscopy (AFM). We also show that for thick curved phase objects one can reconstruct their thickness profiles from DM images. We illustrate the utility of defocusing microscopy in biological systems to study cell motility. In particular, we visualize and quantitatively measure real-time cytoskeleton curvature fluctuations of macrophages (a cell of the innate immune system). The study of such fluctuations might be important for a better understanding of the engulfment process of pathogens during phagocytosis.
Assuntos
Processamento de Imagem Assistida por Computador , Microscopia/métodos , Animais , Calibragem , Movimento Celular , Citoesqueleto/fisiologia , Citoesqueleto/ultraestrutura , Macrófagos/fisiologia , Macrófagos/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Força Atômica , Modelos Teóricos , Óptica e Fotônica , RefratometriaRESUMO
Immunoaffinity-purified antibodies against soluble Schistosoma mansoni egg antigens (SEA) were isolated from the sera of patients with schistosomiasis mansoni. Similarly, antibodies against Trypanosoma cruzi epimastigote antigens were obtained from sera of patients with Chagas' disease. These antibody preparations were used in culture to demonstrate the presence of anti-idiotypic T lymphocytes in peripheral blood mononuclear cell preparations from patients with either schistosomiasis mansoni or Chagas' disease, or with both of these infections. Only cells from patients with schistosomiasis or both infections proliferated upon exposure to the anti-SEA antibodies. Conversely, only cells from patients with Chagas' disease or both infections responded to anti-epimastigote antibodies. Western blot analysis of SEA and epimastigote antigens, developed by patients' sera or by immunoaffinity-purified antibody preparations, substantiated that anti-SEA immunoaffinity-purified antibodies only reacted with components of SEA, and anti-epimastigote immunoaffinity-purified antibodies only reacted with components of epimastigote antigenic preparation. These studies demonstrate the presence of anti-idiotypic T lymphocytes in the peripheral blood of patients with schistosomiasis or Chagas' disease which are specific for idiotypes generated during these infections.
Assuntos
Reações Antígeno-Anticorpo , Doença de Chagas/imunologia , Leucócitos Mononucleares/imunologia , Esquistossomose mansoni/imunologia , Animais , Anticorpos Anti-Helmínticos/imunologia , Anticorpos Antiprotozoários/imunologia , Antígenos de Helmintos/imunologia , Antígenos de Protozoários/imunologia , Western Blotting , Doença de Chagas/complicações , Cromatografia de Afinidade , Humanos , Idiótipos de Imunoglobulinas/imunologia , Ativação Linfocitária , Schistosoma mansoni/imunologia , Esquistossomose mansoni/complicações , Linfócitos T/imunologia , Trypanosoma cruzi/imunologiaRESUMO
With the exception of assays for the detection of antibodies promoting complement-mediated lysis of Trypanosoma cruzi, serologic tests have generally failed to assess the effectiveness of chemotherapy for Chagas' disease. Conventional serology, although useful for the diagnosis of infection, is not capable of determining which patients have been cured. Here we demonstrate that a high proportion of antibodies detected by conventional serology (using fixed epimastigotes or trypomastigotes or crude extracts obtained therefrom) are directed against the carbohydrate residue galactosyl alpha 1- > 3 galactose (Gal alpha 1- > 3 Gal), a determinant also recognized by antibodies from noninfected healthy volunteers. In a study of 14 cured patients with long-term followup, we found that the persistently positive reactions detected using conventional serology were largely eliminated following immunoadsorption with melibiose. Because of their wide distribution among microorganisms of intestinal and pulmonary microflora, these carbohydrate determinants may keep stimulating lymphocytes previously primed by T. cruzi Gal alpha 1- > 3 Gal epitopes, thereby accounting for false-positive results in cured patients. Consistent with this proposition, enzyme-linked immunosorbent assays performed with two distinct T. cruzi antigen preparations that lack the Gal alpha 1- > 3 Gal epitope, namely purified GP57/51 and trypomastigote-shed antigens, were indeed capable of determining a cure after chemotherapy, albeit to a different degree. Collectively, the data indicate that conventional immunoassays prepared with highly specific T. cruzi antigens can be useful in the assessment of a cure after chemotherapy.
Assuntos
Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/imunologia , Doença de Chagas/diagnóstico , Glicoproteínas/imunologia , Trypanosoma cruzi/imunologia , Doença Aguda , Adsorção , Adulto , Animais , Especificidade de Anticorpos , Doença de Chagas/tratamento farmacológico , Criança , Doença Crônica , Reações Cruzadas , Cisteína Endopeptidases , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Seguimentos , Humanos , Pessoa de Meia-Idade , Nifurtimox/uso terapêutico , Nitroimidazóis/uso terapêutico , Resultado do Tratamento , Tripanossomicidas/uso terapêutico , Trypanosoma cruzi/químicaRESUMO
The tachyzoite of the human pathogen Toxoplasma gondii is morphologically indistinguishable from the proliferative stages of some other zoonotic coccidia, including Sarcocystis. To determine the identity of such coccidia obtained from human tissues and other sources, we compared riboprints (through restriction enzyme analysis of the polymerase chain reaction [PCR]-amplified small subunit rRNA gene) of the following protozoa: the RH and ts-4 strains of T. gondii, lines OH3 and S11, which are two recently isolated T. gondii-like parasites from Brazil, Neospora caninum, Sarcocystis species, and the malarial parasite Plasmodium berghei. In addition, the protozoan genomes were examined by PCR for homologs of surface antigen genes of T. gondii, and by Southern hybridization to the heterologous rRNA gene probe pSM 389. Strains OH3, S11, ts-4, and RH shared identical riboprints, and OH3, S11, and ts-4 have p22 and p30 surface antigen gene structures similar to RH. In contrast, riboprints for N. caninum and T. gondii differ with respect to Dde 1 sites, and moreover, their genomes vary significantly from one another at both the p22 and p30 gene loci. The riboprints of Sarcocystis and P. berghei differ markedly from T. gondii and N. caninum and from each other. Bam HI pSM 389 restriction fragment length polymorphisms differentiate ts-4 from RH, OH3, and S11. Our results confirm that OH3 and S11 are indeed T. gondii, but that N. caninum and T. gondii are likely to be separate species, thereby resolving previous uncertainties concerning the identity of these parasites. Together, the variation in riboprints and surface antigen gene structure reflects the phylogenetic diversity among these coccidia, and in addition, confirms the value of riboprinting in the identification of apicomplexan parasites such as T. gondii.