RESUMEN
Leishmania infantum causes a chronic infectious disease named visceral leishmaniasis (VL). We employed a non-human primate model to monitor immune parameters over time and gain new insights into the disease. Rhesus macaques were infected with L. infantum and the T helper and B cell immunological profiles characterized during acute and chronic phases of infection. Parasite detection in visceral compartments during the acute phase was associated with differentiation of effector memory CD4 T cells and increased levels of Th1 transcripts. At the chronic phase, parasites colonized novel lymphoid niches concomitant with increased expression of IL10. Despite the occurrence of hypergammaglobulinemia, the production of parasite-specific IgG was poor, being confined to the acute phase and positively correlated with the frequency of an activated memory splenic B cell population. We noticed the expansion of a splenic CD4 T cell population expressing CXCR5 and Bcl-6 during acute infection that was associated with the differentiation of the activated memory B cell population. Moreover, the number of splenic germinal centers peaked at one month after infection, hence paralleling the production of specific IgG. However, at chronic infection these populations contracted impacting the production of parasite-specific IgG. Our study provides new insights into the immune events taking place in a physiologically relevant host and a mechanistic basis for the inefficient humoral response during VL.
Asunto(s)
Centro Germinal/inmunología , Inmunidad Humoral , Leishmania infantum/inmunología , Leishmaniasis Visceral/inmunología , Bazo/inmunología , Células TH1/inmunología , Animales , Femenino , Regulación de la Expresión Génica/inmunología , Centro Germinal/parasitología , Centro Germinal/patología , Interleucina-10/inmunología , Leishmaniasis Visceral/patología , Macaca mulatta , Masculino , Proteínas Proto-Oncogénicas c-bcl-6/inmunología , Receptores CXCR5/inmunología , Bazo/parasitología , Bazo/patología , Células TH1/patologíaRESUMEN
Understanding the complex interactions between Leishmania and dendritic cells (DCs) is central to the modulation of the outcome of this infection, given that an effective immune response against Leishmania is dependent on the successful activation and maturation of DCs. We report here that Leishmania infantum promastigotes successfully infect mouse bone marrow-derived DCs without triggering maturation, as shown by a failure in the up-regulation of CD40 and CD86 expression, and that parasites strongly counteract the lipopolysaccharide-triggered maturation of DCs. A small increase in interleukin (IL)-12 and IL-10 transcription and secretion and a decrease in IL-6 were observed in infected cells. This arrested DC maturation state is actively promoted by parasites because heat-killed or fixed parasites increased cytokine and costimulatory molecule expression. At a molecular level, L. infantum rapidly induced activation of phosphatidylinositol 3-kinase/Akt and extracellular signal-regulated kinase 1/2, whereas no effect was observed in the c-Jun N-terminal kinase and p38 mitogen-activated protein kinase proinflammatory pathways. Moreover, parasites actively promoted cleavage of the nuclear factor-κB p65(RelA) subunit, causing its impairment. The blockade of phosphatidylinositol 3-kinase/Akt by either treatment of bone marrow-derived DCs with wortmannin or transfection with an Akt dominant-negative mutant resulted in a strong decrease in infection rates, revealing for the first time a crucial role of this pathway on Leishmania engulfment by DCs. Overall, our data indicate that activation of Akt and impairment of nuclear factor-κB are responsible for immunogenicity subversion of L. infantum-infected DCs.
Asunto(s)
Células Dendríticas/inmunología , Leishmania infantum/inmunología , Leishmaniasis Visceral/inmunología , FN-kappa B/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Animales , Ciclo Celular/inmunología , Ciclo Celular/fisiología , Diferenciación Celular/inmunología , Células Cultivadas , Células Dendríticas/metabolismo , Células Dendríticas/patología , Células Dendríticas/fisiología , Regulación hacia Abajo/genética , Regulación hacia Abajo/fisiología , Activación Enzimática/fisiología , Femenino , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Evasión Inmune/inmunología , Evasión Inmune/fisiología , Leishmania infantum/genética , Leishmania infantum/metabolismo , Leishmaniasis Visceral/metabolismo , Leishmaniasis Visceral/patología , Ratones , Ratones Endogámicos BALB C , FN-kappa B/antagonistas & inhibidores , FN-kappa B/metabolismo , Organismos Modificados Genéticamente , Fosfatidilinositol 3-Quinasas/fisiología , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/fisiología , Transducción de Señal/fisiologíaRESUMEN
There is ongoing interest to identify signaling pathways and genes that play a key role in carcinogenesis and the development of resistance to antitumoral drugs. Given that histone deacetylases (HDACs) interact with various partners through complex molecular mechanims leading to the control of gene expression, they have captured the attention of a large number of researchers. As a family of transcriptional corepressors, they have emerged as important regulators of cell differentiation, cell cycle progression, and apoptosis. Several HDAC inhibitors (HDACis) have been shown to efficiently protect against the growth of tumor cells in vitro as well as in vivo. The pancreatic cancer which represents one of the most aggressive cancer still suffers from inefficient therapy. Recent data, although using in vitro tumor cell cultures and in vivo chimeric mouse model, have shown that some of the HDACi do express antipancreatic tumor activity. This provides hope that some of the HDACi could be potential efficient anti-pancreatic cancer drugs. The purpose of this review is to analyze some of the current data of HDACi as possible targets of drug development and to provide some insight into the current problems with pancreatic cancer and points of interest for further study of HDACi as potential molecules for pancreatic cancer adjuvant therapy.
Asunto(s)
Inhibidores de Histona Desacetilasas/uso terapéutico , Histona Desacetilasas/metabolismo , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/enzimología , Transducción de Señal , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Diferenciación Celular/efectos de los fármacos , Inhibidores de Histona Desacetilasas/farmacología , Humanos , Neoplasias Pancreáticas/irrigación sanguínea , Neoplasias Pancreáticas/inmunología , Transducción de Señal/efectos de los fármacosRESUMEN
PURPOSE: To evaluate the in vitro and in vivo pancreatic anticancer activity of a nano-sized formulation based on novel polyallylamine grafted with 5% mole cholesteryl pendant groups (CH(5)-PAA). METHODS: Insoluble novel anticancer drug, Bisnaphthalimidopropyldiaaminooctane (BNIPDaoct), was loaded into CH(5)-PAA polymeric self-assemblies by probe sonication. Hydrodynamic diameters and polydispersity index measurements were determined by photon correlation spectroscopy. The in vitro cytotoxicity evaluation of the formulation was carried out by the sulforhodamine B dye assay with human pancreatic adenocarcinoma BxPC-3 cells, while for the in vivo study, Xenograff mice were used. In vitro apoptotic cell death from the drug formulation was confirmed by flow cytometric analysis. RESULTS: The aqueous polymer-drug formulation had a mean hydrodynamic size of 183 nm. The drug aqueous solubility was increased from negligible concentration to 0.3 mg mL(-1). CH(5)-PAA polymer alone did not exhibit cytotoxicity, but the new polymer-drug formulation showed potent in vitro and in vivo anticancer activity. The mode of cell death in the in vitro study was confirmed to be apoptotic. The in vivo results revealed that the CH(5)-PAA alone did not have any anti-proliferative effect, but the CH(5)-PAA-drug formulation exhibited similar tumour reduction efficacy as the commercial drug, gemcitabine. CONCLUSIONS: The proposed formulation shows potential as pancreatic cancer therapeutics.
Asunto(s)
Adenocarcinoma/patología , Antineoplásicos/farmacología , Carcinoma Ductal Pancreático/patología , Nanotecnología , Naftalimidas/farmacología , Neoplasias Pancreáticas/patología , Polímeros , Animales , Apoptosis , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Citometría de Flujo , Humanos , Técnicas In Vitro , Concentración 50 Inhibidora , Ratones , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
We have characterized a Leishmania protein belonging to the silent information regulator 2 (SIR2) family [SIR2 related protein 1 (SIR2RP1)] that might play an immunoregulatory role during infection through its capacity to trigger B-cell effector functions. We report here that SIR2RP1 leads to the proliferation of activated B cells, causing increased expression of major histocompatibility complex (MHC) II and the costimulatory molecules CD40 and CD86, which are critical ligands for T-cell cross-talk during the development of adaptive immune responses. In contrast, B cells isolated from Toll-like receptor 2 (TLR2) knockout mice were unable to respond to the SIR2RP1 stimulus. Similarly, SIR2RP1 induced the maturation of dendritic cells (DCs) in a TLR2-dependent manner with the secretion of pro-inflammatory cytokines [interleukin (IL)-12 and tumour necrosis factor (TNF)-alpha] and enhanced the costimulatory properties of DCs. Nevertheless, immunization assays demonstrated that TLR2-deficient mice were able to mount a specific humoral response to SIR2RP1. Interestingly, further investigations showed that macrophages were activated by SIR2RP1 even in the absence of TLR2. Therefore, a different type of interplay between SIR2RP1 and the major antigen-presenting cells in vivo could explain the immune response observed in TLR2-deficient mice. Together, these results demonstrate that TLR2 signalling contributes to SIR2RP1 recognition by innate immune host cells.
Asunto(s)
Leishmania infantum/inmunología , Proteínas Protozoarias/inmunología , Sirtuina 1/inmunología , Receptor Toll-Like 2/inmunología , Animales , Linfocitos B/inmunología , Antígeno B7-2/biosíntesis , Antígenos CD40/biosíntesis , Proliferación Celular , Células Cultivadas , Células Dendríticas/inmunología , Antígenos de Histocompatibilidad Clase II , Inmunidad Innata , Activación de Linfocitos/inmunología , Prueba de Cultivo Mixto de Linfocitos/métodos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Factor 88 de Diferenciación Mieloide/biosíntesis , FN-kappa B/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Receptor Toll-Like 6/biosíntesis , Regulación hacia Arriba/inmunologíaRESUMEN
Proteins of the SIR2 (Silent Information Regulator 2) family are characterized by a conserved catalytic domain that exerts unique NAD(+)-dependent deacetylase activity on histones and various other cellular substrates. Previous reports from us have identified a Leishmania infantum gene encoding a cytosolic protein termed LiSIR2RP1 (Leishmania infantum SIR2-related protein 1) that belongs to the SIR2 family. Targeted disruption of one LiSIR2RP1 gene allele led to decreased amastigote virulence, in vitro as well as in vivo. In the present study, attempts were made for the first time to explore and characterize the enzymatic functions of LiSIR2RP1. The LiSIR2RP1 exhibited robust NAD(+)-dependent deacetylase and ADP-ribosyltransferase activities. Moreover, LiSIR2RP1 is capable of deacetylating tubulin, either in dimers or, when present, in taxol-stabilized microtubules or in promastigote and amastigote extracts. Furthermore, the immunostaining of parasites revealed a partial co-localization of alpha-tubulin and LiSIR2RP1 with punctate labelling, seen on the periphery of both promastigote and amastigote stages. Isolated parasite cytoskeleton reacted with antibodies showed that part of LiSIR2RP1 is associated to the cytoskeleton network of both promastigote and amastigote forms. Moreover, the Western blot analysis of the soluble and insoluble fractions of the detergent of promastigote and amastigote forms revealed the presence of alpha-tubulin in the insoluble fraction, and the LiSIR2RP1 distributed in both soluble and insoluble fractions of promastigotes as well as amastigotes. Collectively, the results of the present study demonstrate that LiSIR2RP1 is an NAD(+)-dependent deacetylase that also exerts an ADP-ribosyltransferase activity. The fact that tubulin could be among the targets of LiSIR2RP1 may have significant implications during the remodelling of the morphology of the parasite and its interaction with the host cell.
Asunto(s)
ADP Ribosa Transferasas/metabolismo , Leishmania infantum/enzimología , Proteínas Protozoarias/metabolismo , Sirtuinas/metabolismo , Animales , Proliferación Celular , Supervivencia Celular , Citosol/enzimología , Leishmania infantum/citología , Proteínas Protozoarias/aislamiento & purificación , Sirtuinas/aislamiento & purificación , Tubulina (Proteína)/metabolismoRESUMEN
BACKGROUND: Alterations in HDACs gene expression have been reported in a number of human cancers. No information is available concerning the status of HDACs in pancreatic cancer tumors. The aim of the present study was to evaluate the expression levels of members of class I (HDAC1, 2,, 3), class II (HDAC4, 5, 6, and 7), and class III (SIRT1, 2, 3, 4, 5, and 6) in a set of surgically resected pancreatic tissues. METHODS: Total RNA was isolated from 11 pancreatic adenocarcinomas (PA): stage 0 (n = 1), IB (n = 1), IIB (n = 6), III (n = 1), IV (n = 2), one serous cystadenoma (SC), one intraductal papillary mucinous tumor of the pancreas (IMPN), one complicating chronic pancreatitis (CP), and normal pancreas (NP) obtained during donor liver transplantation. Moreover, six other control pancreatic were included. HDACs gene expression was conducted using quantitative real-time polymerase chain reaction (qPCR). Protein expression levels were analyzed by Western blot and their localization by immunohistochemistry analyses of cancer tissues sections. RESULTS: Remarkably, 9 of the 11 PA (approximately 81%) showed significant increase of HDAC7 mRNA levels. In contrast to PA samples, message for HDAC7 was reduced in CP, SC, and IMPN specimens. The Western blot analysis showed increased expression of HDAC7 protein in 9 out of 11 PA samples, in agreement with the qPCR data. Most of the PA tissue sections examined showed intense labeling in the cytoplasm when reacted against antibodies to HDAC7. CONCLUSION: The data showed alteration of HDACs gene expression in pancreatic cancer. Increased expression of HDAC7 discriminates PA from other pancreatic tumors.
Asunto(s)
Adenocarcinoma/enzimología , Regulación Enzimológica de la Expresión Génica , Histona Desacetilasas/metabolismo , Neoplasias Pancreáticas/enzimología , Femenino , Expresión Génica , Histonas/metabolismo , Humanos , MasculinoRESUMEN
The complement regulatory protein (CRP) of Trypanosoma cruzi is a surface glycoprotein which confers to the infectious trypomastigote forms a protection against the lytic activity of the host complement. CRP belongs to the large family of the trans-sialidase-like proteins and its sequence is highly similar to those of the flagellar FL-160 and chronic exoantigen proteins, encoded by a multigene family. To further define the gene family encoding the CRP, we investigated the protein diversity among several strains of T. cruzi through the sequencing of trypomastigote transcripts, and used a phylogenetic analysis based on the multiple alignment of these proteins with the top scoring sequences detected by a database sequence homology search. Intrastrain variations in CRP sequences revealed the existence of several copies per strain. The interstrain variability of CRP was consistent with the genetic subdivisions of T. cruzi into lineages and discrete typing units. The phylogenetic analysis based on a 227 amino acid alignment of CRP sequences with the 200 putative proteins retrieved from the protein databases (including the sequences from the T. cruzi genome project) revealed that the CRP sequences clustered with the FL-160 proteins into a monophyletic group characterized by the presence of the 12 amino acid mimicry epitope that mimics nervous tissues. The phylogeny did not differentiate between the CRP and the FL-160 proteins. The identification of this group of CRP-like proteins and the high sequence similarity observed within it open up new prospects for the exploration of the localization, structure and function of these proteins and a better understanding of their involvement in key aspects of host-parasite interactions, such as the resistance to the complement. This work provides also information for the T. cruzi genome annotation of the trans-sialidase-like putative proteins.
Asunto(s)
Variación Genética , Estadios del Ciclo de Vida/genética , Glicoproteínas de Membrana/genética , Proteínas Protozoarias/genética , Trypanosoma cruzi/genética , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Análisis por Conglomerados , Genes Protozoarios , Glicoproteínas/química , Datos de Secuencia Molecular , Familia de Multigenes , Neuraminidasa/química , Filogenia , Trypanosoma cruzi/crecimiento & desarrolloRESUMEN
Leishmaniasis causes significant morbidity and mortality worldwide, constituting an important public health problem. Leishmania infections cause a wide spectrum of diseases, ranging in severity from spontaneously healing skin lesions to fatal visceral disease. Attempts to develop an effective vaccine to control leishmaniasis have been shown to be feasible, but no vaccine is in active clinical use. The ability to create genetically modified parasites by eliminating virulence or essential genes is considered a powerful alternative in the development of an effective protective vaccine. Here, recent findings related to genetically defined live attenuated Leishmania parasites as promising vaccine candidates are reviewed.
Asunto(s)
Leishmania/inmunología , Vacunas Antiprotozoos/inmunología , Animales , Animales Modificados Genéticamente , Humanos , Leishmania/genética , Vacunas Atenuadas/inmunologíaRESUMEN
Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, is a major public health problem in most of Latin America. A key priority is the development of new treatments, due to the poor efficacy of current ones. We report here the comparative evaluation of therapeutic DNA vaccines encoding various T. cruzi antigens. ICR mice infected with 500 parasites intraperitoneally were treated at 5 and 12 days postinfection with 20 microg of plasmid DNA encoding T. cruzi antigens TSA-1, TS, ASP-2-like, Tc52 or Tc24. Treatment with plasmid encoding TS and/or ASP-2-like antigens had no significant effect on parasitemia or survival. Treatment with Tc52 DNA significantly reduced parasitemia, as well as cardiac parasite burden, and improved survival, although myocarditis was not significantly affected. Finally, treatment with plasmids encoding Tc24 and TSA-1 induced the most complete control of disease as evidenced by significant reductions in parasitemia, mortality, myocarditis and heart parasite burden. These data demonstrate that therapeutic vaccine efficacy is dependent on the antigen and suggest that DNA vaccines encoding Tc24, TSA-1, and Tc52 represent the best candidates for further studies of a therapeutic vaccine against Chagas disease.
Asunto(s)
Cardiomiopatía Chagásica/tratamiento farmacológico , Enfermedad de Chagas/tratamiento farmacológico , Vacunas Antiprotozoos/uso terapéutico , Trypanosoma cruzi , Vacunas de ADN/uso terapéutico , Animales , Antígenos de Protozoos/genética , Antígenos de Protozoos/inmunología , Cardiomiopatía Chagásica/patología , Femenino , Ratones , Ratones Endogámicos ICR , Parasitemia/tratamiento farmacológico , Plásmidos/genética , Vacunas Antiprotozoos/genética , Vacunas de ADN/genéticaRESUMEN
Direct drug screening against the mammalian stage of Leishmania has been hampered by cost and the time consuming effort required to accomplish it. The ability to derive transgenic Leishmania expressing reporter genes opened up new possibilities for the development of drug screening tests. Further developments to standardize and gather multiple informations could now be envisionned. We will discuss on such available methodologies that could improve sensitivity, reliability, versatility and the rapidity, of the screen based on intracellular model.
RESUMEN
Proteins of the SIR2 family are characterized by a conserved catalytic domain that exerts unique NAD-dependent deacetylase activity on histone and various other cellular substrates. Functional analyses of such proteins have been carried out in a number of prokaryotes and eukaryotes organisms but until now, none have described an essential function for any SIR2 genes. Here using genetic approach, we report that a cytosolic SIR2 homolog in Leishmania is determinant to parasite survival. L. infantum promastigote tolerates deletion of one wild-type LiSIR2 allele (LiSIR2+/-) but achievement of null chromosomal mutants (LiSIR2-/-) requires episomal rescue. Accordingly, plasmid cure shows that these parasites maintain episome even in absence of drug pressure. Though single LiSIR2 gene disruption (LiSIR2+/-) does not affect the growth of parasite in the promastigote form, axenic amastigotes display a marked reduction in their capacity to multiply in vitro inside macrophages and in vivo in Balb/c mice. Taken together these data support a stage specific requirement and/or activity of the Leishmania cytosolic SIR2 protein and reveal an unrelated essential function for the life cycle of this unicellular pathogenic organism. The lack of an effective vaccine against leishmaniasis, and the need for alternative drug treatments, makes LiSIR2 protein a new attractive therapeutic target.
Asunto(s)
Proliferación Celular , Supervivencia Celular , Citosol/enzimología , Leishmania infantum/citología , Sirtuinas/metabolismo , Animales , Secuencia de Bases , Cartilla de ADN , Leishmania infantum/enzimología , Ratones , Ratones Endogámicos BALB CRESUMEN
Diagnosis of leishmaniasis is frequently based on serological methods, such as direct agglutination, immunofluorescence tests and ELISA assays with Leishmania total extracts, as antigen, however due to highly inconclusive results, more reliable tests are needed. In the present study, the prevalence of antibodies to a number of recombinant proteins (LmSIR2, LmS3a, LimTXNPx, LicTXNPx and LiTXN1) highly conserved among Leishmania species, were evaluated by ELISA in Leishmania infantum infected children from an endemic area of Portugal. We found that sera from children patients had antibodies against the different recombinant proteins, LicTXNPx presented the highest immuno-reactivity compared to the other and the most often recognized in the case of acute visceral leishmaniasis (VL). Moreover, in children treated with meglumine antimoniate or amphotericin B, antibodies against some of the recombinant proteins declined, whereas conventional serology using crude extracts showed little or no difference between the pre- and post-treatment values. The highest reduction was observed in the case of antibodies against the LicTXNPx protein. These results suggest that the antibodies against LicTXNPx might be a useful constituent of a defined serological test for the diagnosis and the monitoring of the therapeutic response in VL. The monitoring and follow-up in a large-scale field trials of such marker in areas where leishmaniasis is endemic will lend support to this.
Asunto(s)
Anticuerpos Antiprotozoarios/sangre , Inmunoglobulina G/sangre , Leishmania infantum/inmunología , Leishmaniasis Visceral/diagnóstico , Leishmaniasis Visceral/inmunología , Peroxirredoxinas/inmunología , Animales , Anticuerpos Antiprotozoarios/biosíntesis , Biomarcadores , Niño , Humanos , Inmunoglobulina G/biosíntesis , Leishmaniasis Visceral/sangre , Leishmaniasis Visceral/terapia , Monitorización Inmunológica/métodosRESUMEN
BACKGROUND: In yeast and Caenorhabditis elegans, Silent Information Regulator (SIR2) proteins have been shown to be involved in ageing regulation. In Leishmania, the LmSIR2rp was originally isolated from the excreted/secreted material of the Leishmania parasites. Among the function(s) of this protein in Leishmania biology, we have documented its implication in parasite survival, and in particular in Leishmania amastigotes. In this paper we question the role of the excreted/secreted form of the protein. In particular we wonder if the Leishmania Sir2 homologue is involved in some aspect of its biological function(s), in various components and pathways, which could promote the host cell survival. To test this hypothesis we have mimicked an intracellular release of the protein through constitutive expression in mouse L929 fibrosarcoma cells. RESULTS: Our results demonstrate that the LmSIR2 protein was properly expressed by fibroblasts and that LmSIR2 is localized both in the cytoplasm and the nucleus of all the transformed cell clones. Unexpectedly, we found that cells expressing LmSIR2 presents reduced saturation cell density ranging from 40% to 60% and expressed an acidic (pH6.0) beta-galactosidase activity, which is known to be a senescence biomarker. As a consequence, we observed that LmSIR2 positive fibroblasts were more permissive towards Leihmania infection. CONCLUSIONS: LmSIR2 is able to substantially interfere with the host cell physiology. Thus, it is tempting to speculate that these modifications could help Leishmania to survive for a long period in a cell with reduced capacity to multiply or respond to immunologic stimuli. The potential implications of our finding during the in vivo infection process are discussed.
RESUMEN
Silent information regulator 2 (SIR2) proteins are NAD-dependant deacetylases found in organisms ranging from bacteria to human. In eukaryotes, these proteins are involved in many biological processes including transcriptional repression, metabolism, ageing, or apoptosis. Here, we have shown that Sirtinol, a commercially available inhibitor of SIR2 deacetylases, significantly inhibits the in vitro proliferation of Leishmania infantum axenic amastigotes in a dose-dependent manner. This activity is stage specific since sirtinol did not affect the in vitro growth of parasite promastigotes. Growth arrest in amastigotes is associated with genomic DNA fragmentation, a process reminiscent of apoptosis. Interestingly parasites carrying extra copies of the LmSIR2 gene were less susceptible to the sirtinol mediated cell death. Altogether, these results constitute novel evidences that Leishmania SIR2 proteins play a role in the control of the parasite apoptotic phenomenon.
Asunto(s)
Benzamidas/farmacología , Inhibidores Enzimáticos/farmacología , Inhibidores de Histona Desacetilasas , Leishmania infantum/efectos de los fármacos , Naftoles/farmacología , Sirtuinas/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Apoptosis/efectos de los fármacos , Fragmentación del ADN/efectos de los fármacos , ADN Protozoario/efectos de los fármacos , ADN Protozoario/metabolismo , Citometría de Flujo , Histona Desacetilasas/biosíntesis , Histona Desacetilasas/metabolismo , Humanos , Leishmania infantum/enzimología , Leishmania infantum/genética , Leishmania infantum/crecimiento & desarrollo , Leishmaniasis Visceral/parasitología , Datos de Secuencia Molecular , Alineación de Secuencia , Sirtuinas/biosíntesis , Sirtuinas/metabolismoRESUMEN
A number of features occurring during host-parasite interactions in Chagas disease caused by the protozoan parasite, Trypanosoma cruzi, and Leishmaniasis, caused by a group of kinetoplastid protozoan parasites are reminiscent of those observed in cancer diseases. In fact,although the cancer is not a single disease, and that T.cruzi and Leishmania are sophisticated eukaryotic parasites presenting a high level of genotypic variability the growth of the parasites in their host and that of cancer cells share at least one common feature, that is their mutual capacity for rapid cell division. Surprisingly, the parasitic diseases and cancers share some immune evasion strategies. Consideration of these immunological alterations must be added to the evaluation of the pathogenic processes. The molecular and functional characterization of virulence factors and the study of their effect on the arms of the immune system have greatly improved understanding of the regulation of immune effectors functions. The purpose of this review is to analyze some of the current data related to the regulatory components or processes originating from the parasite that control or interfere with host cell physiology. Attempts are also made to delineate some similarities between the immune evasion strategies that parasites and tumors employ. The elucidation of the mode of action of parasite virulence factors toward the host cell allow not only provide us with a more comprehensive view of the host-parasite relationships but may also represent a step forward in efforts aimed to identify new target molecules for therapeutic intervention.
Asunto(s)
Interacciones Huésped-Parásitos/inmunología , Leishmania/inmunología , Leishmania/patogenicidad , Neoplasias/inmunología , Trypanosoma cruzi/inmunología , Trypanosoma cruzi/patogenicidad , Animales , Antígenos de Protozoos , Apoptosis , Enfermedad de Chagas/inmunología , Enfermedad de Chagas/parasitología , Proteínas del Sistema Complemento/metabolismo , Citocinas/biosíntesis , Humanos , Leishmaniasis/inmunología , Leishmaniasis/parasitología , Activación de Linfocitos , Modelos Inmunológicos , Óxido Nítrico/biosíntesisRESUMEN
The Silent Information Regulator (SIR2) family of genes have been cloned from a variety of species ranging from bacteria to man. In previous studies, we reported the characterization of a Leishmania major gene encoding a protein with extensive homology to yeast SIR2p and expressed by different Leishmania species and parasite developmental stages and thus termed LmSIR2. Unlike the yeast SIR2p, LmSIR2p is mainly localized within the cytoplasm. In the present study, sequencing of a homologue encoding gene in another Leishmania species, Leishmania infantum, revealed 93% overall amino acid identity with L. major SIR2 gene. Further, using L. infantum as a recipient for a plasmid vector (pTEX) which allows overexpression of LmSIR2p led to the accumulation of the protein in the parasite cytoplasm of both promastigote and amastigote forms and a striking increase in the survival of amastigotes, the vertebrate stage of the parasite, when maintained under normal axenic culture conditions. This phenotype was also observed when L. infantum parasites were transfected with a cosmid vector (CLHyg), isolated from a L. infantum cosmid library, carrying the L. infantum SIR2 gene (CLHyg-LiSIR2). In contrast, no effect was observed on survival of the promastigote forms (insect stage) under similar culture conditions. However, when the glucose was used as a unique source of energy under starvation conditions, the viability of promastigotes was significantly enhanced. Moreover, we showed that amastigote forms in the stationary phase of culture died with a feature of apoptosis as revealed by the appearance of YOPRO-1 positive cells and that expression of LmSIR2 protein substantially delays this phenomenon. Taken together, these results demonstrate the existence of SIR2-related proteins encoding genes in different Leishmania species and suggest that LmSIR2p could participate among other factors in the control of cell death.
Asunto(s)
Apoptosis/genética , Leishmania infantum/genética , Sirtuinas/genética , Secuencia de Aminoácidos , Animales , División Celular/genética , Clonación Molecular , Citoplasma/enzimología , ADN Protozoario/química , ADN Protozoario/genética , Regulación Enzimológica de la Expresión Génica , Leishmania infantum/enzimología , Leishmania infantum/crecimiento & desarrollo , Datos de Secuencia Molecular , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Factores de Tiempo , TransfecciónRESUMEN
There is a rapidly expanding interest into the glutathione S-transferases (GSTs) and the structurally related molecules. Many of the latter have been identified as members of conserved protein families sharing structural and some times functional properties being particularly involved in heat-shock response, drug resistance and carcinogenesis. Also, evidence is emerging that members of the GST super family from some pathogens could exert immunomodulatory functions toward the cell of the immune system, involving separate profiles of cytokine gene transcription and different patterns of cell growth, illustrating therefore the 'one gene-dual function' phenomenon. The implication of these biological properties for pathogenesis is discussed.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Glutatión Transferasa/farmacología , Parásitos/enzimología , Secuencia de Aminoácidos , Animales , Citocinas/biosíntesis , Citocinas/genética , Evolución Molecular , Regulación de la Expresión Génica , Humanos , Datos de Secuencia Molecular , Oxidación-Reducción , Parásitos/patogenicidad , Alineación de Secuencia , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunologíaRESUMEN
The intracellular protozoan parasite Trypanosoma cruzi is the etiological agent of Chagas' disease. We have previously characterized a T. cruzi virulence factor named Tc52 sharing structural and functional properties with the thioredoxin and glutaredoxin protein family. Single mutant parasite clones (Tc52(+/-)) exhibiting low virulence in vitro and in vivo were obtained by targeted Tc52 gene replacement. In this report, we have extended our study to analyze the immune response and the disease phenotype in Tc52(+/-)-infected BALB/c mice, during the acute and chronic phases of the disease. Significantly lower parasitemia were found in Tc52(+/-)-infected mice, as compared to wild-type parasite (WT)-infected ones. However, the expansion of all classes of lymphocytes and macrophages was similar for both clones. Furthermore, except for IgG2b levels which were higher in the case of WT-infected mice, all classes of Ig presented no significant difference for WT and Tc52(+/-)-infected animals. Interestingly, a lack of suppression of IL-2 production and of T-cell proliferation inhibition was observed in the case of spleen cells from Tc52(+/-)-infected mice. Finally, the pattern of inflammation process was different and characterized as diffused in the case of Tc52(+/-)-infected mice, or presenting numerous foci in the case of WT-infected mice. Localization of the Tc52 protein in tissue sections and infected heart cell primary cultures by immunofluorescence and immunogold labeling, respectively, revealed the presence of Tc52 at the amastigote surface and associated to aggregates within host cell vesicles. Taken together, these results reinforce the notion of Tc52 being a virulence factor playing a role in the phenotype of the immune response associated to the infection and on the course of the disease.
Asunto(s)
Enfermedad de Chagas/inmunología , Proteínas Protozoarias/genética , Proteínas Protozoarias/fisiología , Trypanosoma cruzi/genética , Trypanosoma cruzi/patogenicidad , Animales , Anticuerpos Antiprotozoarios/sangre , Apoptosis , Células Cultivadas , Enfermedad de Chagas/parasitología , Enfermedad de Chagas/patología , Citocinas/biosíntesis , Citocinas/genética , Ensayo de Inmunoadsorción Enzimática , Marcación de Gen , Corazón/parasitología , Inmunoglobulinas/sangre , Interferón gamma/biosíntesis , Ratones , Ratones Endogámicos BALB C , Músculo Esquelético/química , Músculo Esquelético/parasitología , Músculo Esquelético/patología , Miocardio/química , Miocardio/patología , Proteínas Protozoarias/análisis , Proteínas Protozoarias/inmunología , Bazo/citología , Bazo/inmunología , Trypanosoma cruzi/inmunología , Trypanosoma cruzi/fisiologíaRESUMEN
The parasitic protozoa Trypanosoma cruzi and Leishmania sp release a variety of molecules into their mammalian hosts (ESA: excretory-secretory products). The effects of these ESA on the host cell function may participate in the establishment of a successful infection, in which the parasite persists for a sufficient period of time to complete its life cycle. A number of regulatory components or processes originating from the parasite that control or regulate the metabolism and the growth of host cell have been identified. The purpose of the present review is to analyze some of the current data related to the parasite ESA that interfere with the host cell physiology. Special attention is given to members of conserved protein families demonstrating remarkable diversity and plasticity of function (ie, glutathione S-transferases and related molecules; members of the trans-sialidase and mucin family; and members of the ribosomal protein family). The identification of parasite target molecules and the elucidation of their mode of action toward the host cell represents a step forward in efforts aimed at an immunotherapeutic or pharmacological control of parasitic infection.