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1.
Clin Exp Allergy ; 34(9): 1379-87, 2004 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-15347370

RESUMO

BACKGROUND: Eosinophils are major effector cells in allergic diseases. After their recruitment to sites of inflammation, they contribute to the pathophysiology of the disease by releasing granule proteins and cytokines. Suplatast tosilate (IPD-1151T), a new anti-allergic agent, has shown beneficial effect in the treatment of asthma, associated with reduced bronchoalveolar lavage eosinophil infiltration and eosinophilic cationic protein (ECP) release in serum and sputum. OBJECTIVE: We investigated whether suplatast tosilate could exert direct effects on human eosinophil activation. METHODS: Eosinophils from hypereosinophilic patients or normal donors were purified by Percoll gradient and the magnetic cell separation system. Chemotaxis was studied using the Boyden chamber technique using three chemoattractants, formyl-methionine-leucine-phenylalanine (fMLP), IL-5 and eotaxin. Oxidative metabolism was determined by a luminol-dependent chemiluminescence assay after activation with eotaxin or secretory IgA (sIgA). The release of ECP and eosinophil derived neurotoxin (EDN) was measured by radioimmunoassay and cytokine production was determined by ELISA following activation with sIgA or anti-CD28. RESULTS: The chemotactic response to fMLP, IL-5 and eotaxin was significantly inhibited by IPD-1151T. Suplatast tosilate was partially inhibiting the release of reactive oxygen species (ROS) induced by eotaxin and sIgA. Activation by sIgA and CD28 ligation resulted in the release of ECP and EDN, which was inhibited by IPD-1151T. Upon activation by anti-CD28, only IL-13 production was inhibited by IPD-1151T, whereas release of IL-2 and IFN-gamma was not affected. IL-10 release induced by sIgA was also inhibited by IPD-1151T. Additionally, the pro-inflammatory cytokine IL-6, which was secreted following anti-CD28 and sIgA stimulation, was strongly inhibited by IPD-1151T. CONCLUSION: Through inhibition of chemotaxis, IPD-1151T might limit the number of eosinophils at the inflammation site. Furthermore, it could reduce the pathological potential of eosinophils by inhibiting the release of ROS and cationic proteins, main inflammatory mediators produced by eosinophils. Moreover, the inhibition of immunoregulatory cytokines released by eosinophils could locally modify the immune response.


Assuntos
Antialérgicos/uso terapêutico , Sulfonatos de Arila/uso terapêutico , Antígenos CD28/imunologia , Eosinofilia/imunologia , Hipersensibilidade/imunologia , Imunoglobulina A Secretora/imunologia , Compostos de Sulfônio/uso terapêutico , Antialérgicos/imunologia , Sulfonatos de Arila/imunologia , Quimiocina CCL11 , Quimiocinas CC/imunologia , Quimiotaxia de Leucócito/imunologia , Citocinas/imunologia , Proteína Catiônica de Eosinófilo/metabolismo , Neurotoxina Derivada de Eosinófilo/metabolismo , Eosinófilos/metabolismo , Humanos , Interleucina-5/imunologia , N-Formilmetionina Leucil-Fenilalanina/imunologia , Oxirredução , Espécies Reativas de Oxigênio/metabolismo , Compostos de Sulfônio/imunologia
2.
Allergy ; 58(5): 397-406, 2003 May.
Artigo em Inglês | MEDLINE | ID: mdl-12752326

RESUMO

BACKGROUND: The aim of this study was to investigate the effects of ketotifen on different parameters of human eosinophil functions, namely chemotaxis, oxidative metabolism and mediator release, induced after activation. METHODS: Eosinophils from hypereosinophilic patients or normal donors were purified by Percoll gradient and the magnetic cell separation system. Chemotaxis was studied using the Boyden chamber technique using three potent chemoattractants: formyl-methionine-leucine-phenylalanine (fMLP), interleukin (IL)-5 and eotaxin. Oxidative metabolism was determined by a luminol-dependent chemiluminescence assay after activation with eotaxin or secretory immunoglobulin A (sIgA). The release of eosinophil cationic protein (ECP) and eosinophil derived neurotoxin (EDN) was measured by radioimmunoassay after activation with sIgA. RESULTS: At pharmacologically active concentrations and in a dose-dependent manner, ketotifen significantly inhibited the chemotaxis of eosinophils to fMLP, IL-5 and eotaxin. The production of reactive oxygen species induced by eotaxin and sIgA was decreased by ketotifen, showing a more pronounced effect when cells were activated by eotaxin. Activation by sIgA resulted in ECP and EDN release, which was partially inhibited by ketotifen. CONCLUSIONS: Through inhibition of chemotaxis, ketotifen might limit the number of eosinophils at the inflammation site during allergic reaction. Furthermore, inhibition by ketotifen of main inflammatory mediators release suggests a potential role of the drug in limiting the pathological potential of eosinophils.


Assuntos
Antialérgicos/farmacologia , Quimiotaxia de Leucócito/efeitos dos fármacos , Eosinófilos/efeitos dos fármacos , Cetotifeno/farmacologia , Proteínas Sanguíneas/efeitos dos fármacos , Estudos de Casos e Controles , Sobrevivência Celular/efeitos dos fármacos , Quimiocina CCL11 , Quimiocinas CC/antagonistas & inibidores , Fatores Quimiotáticos de Eosinófilos , Relação Dose-Resposta a Droga , Neurotoxina Derivada de Eosinófilo , Eosinófilos/metabolismo , Oftalmopatias/tratamento farmacológico , Humanos , Interleucina-5/antagonistas & inibidores , N-Formilmetionina Leucil-Fenilalanina/antagonistas & inibidores , Oxirredução/efeitos dos fármacos , Espécies Reativas de Oxigênio/antagonistas & inibidores , Ribonucleases/efeitos dos fármacos
3.
Eur J Appl Physiol ; 88(3): 288-93, 2002 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-12458373

RESUMO

Recommendations for the requirements for protein intake amount usually to 0.8-1.0 g x kg(-1) body mass x day(-1) in adolescents without any reference to the undertaking of acute exercise or to the training status. The present investigation intended to determine the nitrogen balance and protein intake in 8 healthy male non-active adolescents and 11 adolescent soccer players, both groups aged about 15 years. An assessment of nutrient intake was obtained by analysing 7 day food records collected by a questionnaire. Nitrogen excretion rate was determined and nitrogen balance was calculated from the mean daily protein intake and the urinary excretion. The results showed that the nutritional status of the two groups was similar. Nevertheless, we found that their diets were quite inappropriate in terms of the intakes of carbohydrate, some minerals (zinc, calcium, magnesium), vitamins (A, B6, D) and fibre. A positive nitrogen balance was observed from a mean protein intake of 1.57 g x kg(-1) body mass x day(-1) in these adolescents, whether they were non-active or athletes. Thus, the present investigation indicated that the growth and development in non-active adolescents and in adolescent soccer-players give rise to a need for a higher protein intake than is usually recommended. However, the higher protein requirements did not seem to be related only to the increased energy expenditure imposed by the exercise training in the soccer-player group.


Assuntos
Fenômenos Fisiológicos da Nutrição do Adolescente , Proteínas Alimentares/administração & dosagem , Nitrogênio/metabolismo , Educação Física e Treinamento , Futebol , Adolescente , Metabolismo Energético , Crescimento/fisiologia , Humanos , Masculino , Valores de Referência
4.
Vaccine ; 19(20-22): 2803-11, 2001 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-11282190

RESUMO

In this study, the impact of Th1-inducing cytokine gene co-delivery and antigen boosting on humoral and cellular responses induced by multiepitopic DNA immunization in mice have been investigated. Intramuscular injection of mixed DNA constructs encoding for HIV-1 Gag, Tat and Nef proteins, co-administered with the DNA encoding for interleukin-18 (IL-18) have been used. The effect of boosting with the recombinant proteins was also evaluated on the outcome of the responses in DNA-primed mice. It was demonstrated that at least two DNA immunizations were necessary to generate virus specific Th-1 responses detected by the presence of cytotoxic T lymphocyte (CTL) and by the secretion of IL-2 and IFN-gamma, but not IL-4 and IL-10, in antigen-stimulated splenocyte cultures. Interestingly, co-delivery of Th-1-inducing IL-18 gene was able to shorten by 2 weeks, the CTL induction time, and to increase the antigen-induced secretion of IL-2 and IFN-gamma. Furthermore, IL-18 co-delivery enhanced antigen-specific lymphoproliferative responses, and this was most evident in mice that were primed and boosted with plasmid DNA. However, the induction of detectable antibodies in mice required two DNA vaccinations and a protein boost. In contrast to the effects on cell-mediated immunity, co-administration of IL-18-plasmid resulted in decreased antibody titers against viral proteins.


Assuntos
Vacinas contra a AIDS/imunologia , HIV-1/imunologia , Interleucina-18/genética , Vacinas de DNA/imunologia , Animais , Citocinas/biossíntese , Epitopos , Feminino , Produtos do Gene gag/genética , Produtos do Gene gag/imunologia , Produtos do Gene nef/genética , Produtos do Gene nef/imunologia , Produtos do Gene tat/genética , Produtos do Gene tat/imunologia , Anticorpos Anti-HIV/sangue , Imunização , Imunização Secundária , Camundongos , Camundongos Endogâmicos BALB C , Linfócitos T Citotóxicos/imunologia , Produtos do Gene nef do Vírus da Imunodeficiência Humana , Produtos do Gene tat do Vírus da Imunodeficiência Humana
5.
Vaccine ; 19(1): 95-102, 2000 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-10924791

RESUMO

Many different HIV-1 vaccine strategies have been developed, but as yet none has been completely successful. Promising results from combined DNA prime/protein boost vaccines have been reported. Specific immune responses generated by DNA vaccines can be modulated by the co-delivery of genes coding for cytokines. In this study, we have used the intradermal route by needle injection of a plasmid coding for the HIV-1 Nef accessory protein. We show that DNA prime/protein boost vaccine combinations increase the humoral and cellular immune responses against HIV-1 Nef and that the co-injection of DNA encoding Interleukin-18 (IL-18) modulates the specific immune response towards a Th1 type.


Assuntos
Vacinas contra a AIDS/administração & dosagem , DNA Viral/administração & dosagem , Produtos do Gene nef/imunologia , HIV-1/imunologia , Interleucina-18/imunologia , Vacinas de DNA/administração & dosagem , Vacinas contra a AIDS/imunologia , Animais , Formação de Anticorpos/efeitos dos fármacos , DNA Viral/imunologia , Feminino , Produtos do Gene nef/administração & dosagem , Produtos do Gene nef/biossíntese , Produtos do Gene nef/genética , HIV-1/genética , Imunidade Celular/efeitos dos fármacos , Imunização Secundária , Interleucina-18/genética , Camundongos , Camundongos Endogâmicos BALB C , Plasmídeos/genética , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/imunologia , Vacinas de DNA/imunologia , Proteínas Virais/administração & dosagem , Proteínas Virais/imunologia , Produtos do Gene nef do Vírus da Imunodeficiência Humana
6.
J Infect Dis ; 179(1): 83-91, 1999 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-9841826

RESUMO

The role of recombinant interleukin-16 (rIL-16) in regulating human immunodeficiency virus type 1 (HIV-1) replication in endogenously infected cells has been investigated. Cultures of CD8 cell-depleted mitogen-activated lymphocytes from 22 of 26 HIV-1-infected subjects presented variable levels of secreted p24 antigen. The presence of rIL-16 throughout the 14-day culture period dramatically inhibited p24 release into the culture supernatants. This effect was found to be mediated through inhibition of viral transcription but to be independent of the induced levels of other cytokines or chemokines known to regulate viral replication. Analysis of serum samples from HIV-1-infected subjects over a period of 8 years showed maintained or even increased IL-16 levels during the whole asymptomatic phase and a significant drop on progression to disease. These results strongly support a potential therapeutic value of rIL-16 in HIV-1 infection and the use of serum IL-16 levels to monitor disease progression.


Assuntos
Infecções por HIV/terapia , HIV-1/efeitos dos fármacos , HIV-1/fisiologia , Interleucina-16/uso terapêutico , Replicação Viral/efeitos dos fármacos , Síndrome da Imunodeficiência Adquirida/imunologia , Síndrome da Imunodeficiência Adquirida/terapia , Síndrome da Imunodeficiência Adquirida/virologia , Adulto , Estudos de Casos e Controles , Citocinas/biossíntese , Proteína do Núcleo p24 do HIV/metabolismo , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/genética , Humanos , Técnicas In Vitro , Interleucina-16/sangue , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/virologia , Proteínas Recombinantes/uso terapêutico , Transcrição Gênica/efeitos dos fármacos
9.
Gene ; 174(1): 19-26, 1996 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-8863724

RESUMO

The cloning and sequencing of the gene coding for Trypanosoma cruzi elongation factor 1 alpha (TcEF-1 alpha) was performed by screening a T. cruzi genomic library with a probe obtained through the polymerase chain reaction (PCR) amplification of T. cruzi DNA using two oligonucleotides deduced from the sequence of T. brucei EF-1 alpha. Southern blot analysis of T. cruzi digested genomic DNA and Northern blot hybridized with the labeled probe revealed that one copy of TcEF-1 alpha exist in the genome of the parasite. Indirect immunofluorescence technique using anti-EF-1 alpha antibodies and epimastigotes harvested after different days of in vitro culture showed that EF-1 alpha is localised in the cytoplasm of the parasites from the exponential growth phase. Surprisingly, during the stationary phase (ageing parasites), EF-1 alpha was found in the nucleus. Furthermore, treatment of parasites with the antibiotic drug geneticin (G418) which induces the death of epimastigotes by apoptosis showed selective localization of EF-1 alpha in the nucleus of dying parasites. This observation supports the notion already reported in the case of mammalian cells that EF-1 alpha could participate in the transcription processes and possibly in the case of T. cruzi, in the expression regulation of genes involved in the control of cell death. The possible transfection and genomic manipulation of T. cruzi may provide a model to study the role of TcEF-1 alpha in this phenomenon.


Assuntos
Apoptose/genética , Núcleo Celular/genética , Fatores de Alongamento de Peptídeos/genética , Trypanosoma cruzi/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Regulação da Expressão Gênica , Biblioteca Genômica , Dados de Sequência Molecular , Fator 1 de Elongação de Peptídeos , Fatores de Alongamento de Peptídeos/ultraestrutura , Trypanosoma cruzi/ultraestrutura
10.
Exp Parasitol ; 81(4): 453-61, 1995 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-8542986

RESUMO

We have previously isolated and characterized a Trypanosoma cruzi cDNA encoding a polypeptide with a molecular mass of 52 kDa (Tc52) sharing significant homology to glutathione S-transferase. In the present study, by molecular and immunological approaches, we showed that Tc52 is preferentially expressed by dividing forms of the parasite: (e.g., epimatigotes and amastigotes). Moreover, we could identify the reactive antigen in different T. cruzi strains. A different pattern of reactivity on immunoblots was observed in the case of Trypanosoma rangeli. Furthermore, immunofluorescence assays using T. cruzi epimastigote culture forms revealed that the reactive antigen is localized within cytoplasmic organelles morphologically ressembling the structures previously designated as the reservosome found mostly at the posterior end of the parasite. Furthermore, the antibodies did not react against trypomastigotes which emerged from infected fibroblasts, whereas amastigotes showed polar fluorescence. Immunogold labeling and electron micrographs further revealed that the Tc52 protein is mainly associated with organelles composed of a large network of multivesicular structures, the latter being more abundant in epimastigotes. Taken together, these results demonstrated that Tc52 is associated with organelles composed of a multivesicular network and appears to be developmentally regulated, being fully expressed by parasite dividing forms.


Assuntos
Antígenos de Protozoários/isolamento & purificação , Organelas/química , Proteínas de Protozoários/isolamento & purificação , Trypanosoma cruzi/química , Animais , Antígenos de Protozoários/genética , Antígenos de Protozoários/imunologia , Sequência de Bases , Biomarcadores , Northern Blotting , Imunofluorescência , Glutationa Transferase/genética , Immunoblotting , Microscopia Imunoeletrônica , Dados de Sequência Molecular , Organelas/genética , Organelas/imunologia , Organelas/ultraestrutura , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Trypanosoma cruzi/genética , Trypanosoma cruzi/imunologia , Trypanosoma cruzi/ultraestrutura
11.
Cell Death Differ ; 2(4): 285-300, 1995 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17180034

RESUMO

The origin of programmed cell death (PCD) has been linked to the emergence of multicellular organisms. Trypanosoma cruzi, a member of one of the earliest diverging eukaryotes, is a protozoan unicellular parasite that undergoes three major differentiation changes and requires two different hosts. We report that the in vitro differentiation of the proliferating epimastigote stage into the G0/G1 arrested trypomastigote stage is associated with massive epimastigote death that shows the cytoplasmic and nuclear morphological features and DNA fragmentation pattern of apoptosis, the most frequent phenotype of PCD in multicellular organisms. Apoptosis could be accelerated or prevented by modifying culture conditions or cell density, indicating that extracellular signals influenced the epimastigote decision between life and death. Epimastigotes responded to complement-mediated immunological agression by undergoing apoptosis, while undergoing necrosis in response to nonphysiological saponin-mediated damage. PCD may participate into the optimal adaptation of T. cruzi to its different hosts, and the avoidance of a local competition between a G0/G1 arrested stage and its proliferating progenitor. The existence of a regulated cell death programme inducing an apoptotic phenotype in a unicellular eukaryote provides a paradigm for a widespread role for PCD in the control of cell survival, which extends beyond the evolutionary constraints that may be specific to multicellular organisms and raises the question of the origin and nature of the genes involved. Another implication is that PCD induction could represent a target for therapeutic strategies against unicellular pathogens.

12.
J Belge Radiol ; 78(3): 175-6, 1995 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-7592279

RESUMO

Florid reactive periostitis (FRP) is a benign entity, mostly involving the tubular bones of the hands and feet. It is important to distinguish FRP from malignant processes such as periosteal osteosarcoma and parosteal osteosarcoma, to give an adequate treatment and to avoid amputation of the total digit. The final diagnosis of FRP is usually based on the combination of clinical, radiological, and pathological findings. The pattern seen on the isotope bone scan can be very helpful in orienting the differential diagnosis.


Assuntos
Dedos , Periostite/diagnóstico por imagem , Neoplasias Ósseas/diagnóstico , Criança , Diagnóstico Diferencial , Feminino , Dedos/diagnóstico por imagem , Humanos , Radiografia , Cintilografia
13.
Toxicon ; 32(8): 875-82, 1994 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-7985193

RESUMO

Venom from three different snake species was tested in vitro against the protozoan parasites Trypanosoma cruzi and Leishmania donovani infantum. Two of them, Cerastes cerastes and Naja haje, exerted a significant growth inhibition of T. cruzi and L. d. infantum parasites. Heating of the venoms abolished their activity, suggesting that the active factors are thermolabile. Incubation of parasites with 125I-labelled C. cerastes venom proteins allowed preliminary identification of components which interact preferentially with the pathogens.


Assuntos
Venenos Elapídicos/farmacologia , Leishmania donovani/efeitos dos fármacos , Proteínas/análise , Trypanosoma cruzi/efeitos dos fármacos , Venenos de Víboras/farmacologia , Animais , Autorradiografia , Meios de Cultura , Venenos Elapídicos/química , Elapidae , Eletroforese em Gel de Poliacrilamida , Leishmania donovani/crescimento & desenvolvimento , Temperatura , Trypanosoma cruzi/crescimento & desenvolvimento , Venenos de Víboras/química , Viperidae
14.
Biol Cell ; 82(1): 39-44, 1994.
Artigo em Inglês | MEDLINE | ID: mdl-7735118

RESUMO

In previous studies, we reported the characterization of three Trypanosoma cruzi proteins with molecular masses of 45, 30 and 25 kDa eluted from a glutathione agarose column (these proteins were named TcGBP). Using antibodies against TcGBP native proteins we could isolate from a lambda ZAPII epimastigote cDNA library cDNA clones encoding the 30 and 25 kDa proteins. Comparison of the two sequences with amino acid sequences in several data banks revealed that both protein sequences were highly homologous to human and Artemia salina elongation factor 1 beta. Thus, the proteins were named TcEF-1 beta 25 and TcEF-1 beta 30. In the present study we used a double immunoscreening strategy that allowed us to isolate a cDNA clone corresponding to the 45 kDa protein. The protein sequence revealed 31% identity and 61% homology with human and Artemia salina EF1 gamma and therefore was named TcEF-1 gamma. Moreover, three putative phosphorylation sites at position 51 (CSPC), at position 90 (RTPL) and at position 265 (PSPF) were found in the TcEF-1 gamma sequence. These sites are compatible with the notion that TcEF-1 gamma could be the target of phosphorylation by protein kinase(s). Random primed cDNA hybridized with a single 1.4 kb mRNA found in epimastigote, trypomastigote and amastigote forms. In addition, Southern blot analysis of genomic DNA suggested that the protein is encoded by a single gene. The TcEF-1 gamma cDNA was subcloned into the pGEX-4T-3 vector for expression in Escherichia coli.(ABSTRACT TRUNCATED AT 250 WORDS)


Assuntos
Fatores de Alongamento de Peptídeos/genética , Proteínas de Protozoários/genética , Trypanosoma cruzi/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Evolução Biológica , Western Blotting , DNA Complementar/genética , Proteínas Fúngicas , Dados de Sequência Molecular , Fator 1 de Elongação de Peptídeos , Fatores de Alongamento de Peptídeos/ultraestrutura , Proteínas Recombinantes de Fusão/genética , Homologia de Sequência do Ácido Nucleico , Trypanosoma cruzi/química
15.
Biol Cell ; 80(1): 1-10, 1994.
Artigo em Inglês | MEDLINE | ID: mdl-8054880

RESUMO

The isolation, characterization, and expression of a novel cDNA encoding a Trypanosoma cruzi polypeptide (TcAc2), homologous to various small stress proteins and glutathione S-transferases, are described. The deduced amino-acid sequence revealed two domains sharing 27% identity and an additional 27% similarity to each other suggesting that the molecule may have evolved from a single domain by a process of gene duplication and fusion. The TcAc2 cDNA was subcloned into the pGEX-2T vector for expression in E coli. In vitro translation products of epimastigote mRNA, immunoprecipitated with anti-TXepi serum, showed a major radioactive band of 52 kDa. Immunoprecipitation of [35S]methionine labelled epimastigote and trypomastigote antigens after pulse chase experiments, using anti-TcAc2 fusion protein antibodies, showed that the protein is released into the culture medium. Moreover, Western blot analysis revealed a single band of 52 kDa with epimastigote, trypomastigote and amastigote antigens. Primary structure homology searches revealed that each TcAc2 domain contained within its N-terminus significant homology to Solanum tuberosum pathogenesis-related protein PR1, soybean heat shock protein 26-A, auxin regulated clone pCNT103 from Nicotiana tabacum and Drosophila melanogaster glutathione S-transferase 27 (GST27). This finding was supported by a comparison of hydrophobicity profiles of TcAc2 and these proteins. Most of them play a central role in protection mechanisms against stress. Based on the homology between TcAc2, glutathione S-transferases (GST) and small stress proteins, it is likely that the TcAc2 gene product may play a crucial role in parasite's adaptation to its microenvironment. These molecules could be considered as members of the GST superfamily, where the T cruzi protein may take a particular place because of its internal gene duplication.


Assuntos
DNA de Protozoário/genética , Glutationa Transferase/biossíntese , Proteínas de Choque Térmico/biossíntese , Sequências Repetitivas de Ácido Nucleico , Trypanosoma cruzi/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Clonagem Molecular , Primers do DNA , Sondas de DNA , DNA Complementar , Drosophila melanogaster/genética , Escherichia coli , Biblioteca Gênica , Glutationa Transferase/genética , Glutationa Transferase/isolamento & purificação , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/isolamento & purificação , Metionina/metabolismo , Dados de Sequência Molecular , Plantas/genética , Reação em Cadeia da Polimerase , RNA Mensageiro/biossíntese , Mapeamento por Restrição , Homologia de Sequência de Aminoácidos , Radioisótopos de Enxofre
16.
Parasitology ; 107 ( Pt 5): 497-507, 1993 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-8295789

RESUMO

In the present study we have used antibodies to Leishmania major promastigote antigens which were eluted from a glutathione-agarose column (LmGbp) and could identify several parasite components among different Leishmania species by using immunoprecipitation and Western blot techniques. The results also showed that some of LmGbp are present among the molecules released into the culture medium. Moreover, immunofluorescence assays clearly demonstrated that LmGbp are expressed by intracellular amastigotes. The electron micrographs of thawed cryosections of L. major-infected cells revealed that the antigens were associated with the membrane of the phagocytic vacuole. Moreover, the Western blot technique allowed us to identify, using other Leishmania species extracts and anti-LmGbp antibodies, a major polypeptide of an apparent molecular mass of 66 kDa. Immunofluorescence studies suggested that the 66 kDa polypeptide is associated with intracytoplasmic vesicles. Cryosections of Leishmania promastigotes improved the fine structure preservation of the organelles and enabled a number of features to be seen, particularly the structures considered as vesicles, which appeared as a complex tubulo-vesicular structure resembling mammalian cell endosomes and Leishmania organelles previously named 'megasomes'. Further studies using antibodies against the native 66 kDa protein will be needed to investigate the localization of the protein at the ultrastructural level and to follow its intracellular vesicular traffic.


Assuntos
Antígenos de Protozoários/análise , Grânulos Citoplasmáticos/ultraestrutura , Leishmania/ultraestrutura , Organelas/ultraestrutura , Animais , Antígenos de Protozoários/biossíntese , Antígenos de Protozoários/isolamento & purificação , Western Blotting , Cromatografia de Afinidade , Grânulos Citoplasmáticos/metabolismo , Imunofluorescência , Leishmania/classificação , Leishmania/metabolismo , Leishmania major/ultraestrutura , Metionina/metabolismo , Microscopia Imunoeletrônica , Peso Molecular , Organelas/metabolismo , Especificidade da Espécie , Radioisótopos de Enxofre
17.
Res Immunol ; 144(8): 553-63, 1993 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-8303073

RESUMO

Three Trypanosoma cruzi glutathione-binding proteins (TcGBP) of 45, 30 and 25 kDa presenting glutathione S transferase activity were characterized from T. cruzi epimastigotes. We show here that immunization of mice using TcGBP and complete Freund's adjuvant (CFA) did not protect the animals against a challenge with bloodstream trypomastigotes. In contrast, immunization of mice using TcGBP in association with Bordetella pertussis plus alum (BpAI) resulted in greatly diminished parasitaemia and significantly protected the animals from lethal infection. Using TcGBP mixed with BpAI and a lower challenge dose, we obtained strongly diminished parasitaemia and 100% protection in terms of survival. Only sera from mice immunized with TcGBP plus BpAI were able to kill trypomastigotes by complement-mediated lysis, whereas sera from mice immunized with TcGBP plus CFA did not. Interestingly, sera from mice immunized with TcGBP plus BpAI showed significant levels of specific IgE, IgG2a and IgG2b antibodies, whereas these isotypes were not detected in sera from mice immunized with TcGBP in CFA. All these levels were increased in sera of protected animals. These results demonstrate that TcGBP antigens can confer resistance to T. cruzi acute infection in mice, and suggest a possible functional role for both IgE and IgG2 isotypes in the induction of protective immunity.


Assuntos
Proteínas de Transporte/imunologia , Glutationa , Proteínas de Protozoários/imunologia , Trypanosoma cruzi/imunologia , Adjuvantes Imunológicos , Hidróxido de Alumínio , Animais , Anticorpos Antiprotozoários/sangue , Bordetella pertussis , Células Cultivadas , Doença de Chagas/prevenção & controle , Ensaio de Imunoadsorção Enzimática , Adjuvante de Freund , Imunoglobulina E/sangue , Imunoglobulina G/sangue , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Radioimunoensaio
18.
J Immunol ; 151(5): 2676-89, 1993 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-7689612

RESUMO

In the present study, we investigate the immunoprotective properties of trypomastigote excretory-secretory Ag (ESA) in experimental models. In the case of BALB/c mice, the immunization with ESA resulted in the reduction of parasitemia during acute infection and a significant level of protection in terms of mortality with more than 60% survival, whereas none of the mice in the control groups survived after 39 days postinfection. The same experiments performed in Fischer rats showed a high degree of protection against acute lethal infection with 100% survival, whereas 20 to 40% of rats in the control groups survived the acute phase of T. cruzi infection. Mouse and rat immune sera presented trypanolytic activity against Trypanosoma cruzi infective forms, and recognized two major parasite components of 85 and 24 kDa. The analysis of specific isotype profiles showed a predominance of IgG1, IgG2a, and IgG2b antibody responses. Rat antisera to ESA were then used to screen a trypomastigote cDNA library. Several clones were identified, all of which encoded for the 24-kDa protein. Using a mAb (Tcr7) produced against the native protein, the 31-kDa recombinant fusion protein was purified by affinity chromatography. The antisera to the recombinant protein used in IFA and immunoelectron microscopy showed that the localization of the 24-kDa protein differs among T. cruzi developmental stages. Protection experiments were performed in BALB/c mice using two synthetic peptides (20-40 and 109-124) derived from the primary sequence of the 24-kDa polypeptide. The results obtained clearly indicated that the peptide 109 to 124 containing a putative T cell epitope represents the most protective epitope, which induced 30 to 50% of protection against mortality during acute infection, whereas the percent survival in the control groups (OVA and 20-40 OVA peptide-immunized mice) was around 16%. Moreover, analysis of T cell proliferation in response to OVA-coupled peptides clearly indicated that only the 109 to 124 peptide had the capacity to induce the proliferation of T cells from peptide-immunized mice. Interestingly, only the 109 to 124-coupled peptide induced the proliferation of T cells from T. cruzi-infected mice.


Assuntos
Antígenos de Protozoários/imunologia , Epitopos/análise , Fragmentos de Peptídeos/imunologia , Linfócitos T/imunologia , Trypanosoma cruzi/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Antiprotozoários/sangue , Soros Imunes/imunologia , Imunização , Isotipos de Imunoglobulinas/sangue , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Coelhos , Ratos , Proteínas Recombinantes/imunologia
20.
Eur J Cell Biol ; 59(1): 68-79, 1992 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-1281773

RESUMO

The morphological and biochemical events following Trypanosoma cruzi trypomastigote-fibronectin (Fn) interactions have been studied. Adhesion of trypomastigotes to Fn-coated surfaces is followed by Fn degradation. The proteolytic cleavage of Fn was demonstrated by qualitative and quantitative measurement of Fn degradation after its exposure to trypomastigotes as well as polyacrylamide gel analysis of Fn proteolysis by a parasite protease (s). The released Fn peptide fragments stimulated the transformation of trypomastigotes to amastigotes. The gelatin (45 kDa) and heparin (40 kDa) binding fragments were shown to be able to promote trypomastigote differentiation. In contrast, native Fn and the 120 kDa fragment (cell attachment domain) were inactive. Complementary investigations showed that the gelatin and heparin binding fragments stimulated parasite RNA synthesis and protein synthesis and phosphorylation but not DNA replication and increased parasite intracellular cAMP concentrations. These findings suggest that the proteolysis of Fn by parasite proteases, which occurs under physiological conditions, might facilitate invasion of target cells by trypomastigotes. The Fn peptides released during this process may act as "growth factor-like" substances.


Assuntos
Fibronectinas/farmacologia , Peptídeos/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Animais , Sítios de Ligação , Ligação Competitiva , Diferenciação Celular/efeitos dos fármacos , Fibronectinas/metabolismo , Peptídeos/metabolismo , Fosforilação/efeitos dos fármacos , Biossíntese de Proteínas , RNA/biossíntese , Transdução de Sinais , Trypanosoma cruzi/crescimento & desenvolvimento
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