RESUMO
The trans-sialidase from the trypomastigote stage of Trypanosoma cruzi was metabolically labeled with [3H]-palmitic acid and purified by immunoprecipitation with a monoclonal antibody. The action of PI-PLC on the immunoprecipitate released a lipid that was analyzed by TLC. Lyso-1-O-hexadecylglycerol and N-palmitoyl-sphinganine were obtained in a 1:3 ratio. A comparison with the GPI anchors present in the different stages of T. cruzi was made.
Assuntos
Glicosilfosfatidilinositóis/metabolismo , Lipídeos de Membrana/metabolismo , Neuraminidase/metabolismo , Trypanosoma cruzi/enzimologia , Animais , Cromatografia em Camada Fina , Camundongos , Fosfatidilinositol Diacilglicerol-Liase , Fosfoinositídeo Fosfolipase C , Especificidade por Substrato , Fosfolipases Tipo C/metabolismoRESUMO
trans-Sialidase (TS) is an enzymatic activity described only for trypanosomes that is involved in the invasion of host cells by Trypanosoma cruzi. The enzyme that is shed by the parasite is made of two domains, the C-terminal region containing immunodominant amino acid repeats that define the SAPA antigen and the N-terminal domain that contains the putative region for enzymatic activity. The SAPA antigen induces a strong humoral response detected shortly after infection, both in humans and in mice. This response is directed to the immunodominant domain but is irrelevant in terms of neutralization of TS activity. We now show that TS activity can be detected in sera from acutely infected mice. However, mice infected with a T. cruzi strain whose growth can be controlled by the host did not have detectable levels of TS activity in sera. In fact, sera from these mice were able to abolish TS activity. This inhibition was due to the presence of specific antibodies directed against the enzymatic domain of the protein since they also abolish the activity of a recombinant molecule lacking the immunodominant amino acid repeats. The neutralizing antibodies were present from day 30 after the infection, while antibodies to the immunodominant repeats were detected by day 8 postinoculation, suggesting that the in vivo role of these repeats is to defect the humoral response to the repeat domain until the infection is established.
Assuntos
Anticorpos Antiprotozoários/sangue , Doença de Chagas/imunologia , Neuraminidase/imunologia , Trypanosoma cruzi/imunologia , Animais , Masculino , Camundongos , Camundongos Endogâmicos C3H , Neuraminidase/antagonistas & inibidores , Neuraminidase/sangueAssuntos
Glicoproteínas/genética , Neuraminidase/genética , Trypanosoma cruzi/enzimologia , Trypanosoma cruzi/genética , Sequência de Aminoácidos , Animais , Antígenos de Protozoários/genética , Sequência de Bases , Primers do DNA/genética , DNA de Protozoário/genética , Genes de Protozoários , Glicoproteínas/imunologia , Glicoproteínas/metabolismo , Dados de Sequência Molecular , Neuraminidase/imunologia , Neuraminidase/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Sequências Repetitivas de Ácido Nucleico , Trypanosoma cruzi/imunologiaRESUMO
In contrast to lymphocytic choriomeningitis virus, another arenavirus, Junin virus (JV), the etiologic agent of Argentine hemorrhagic fever, when inoculated into suckling mice, induces lethal meningoencephalitis characterized by a delayed-type hypersensitivity (DTH)-like immune response. However, the adult BALB/c mouse is resistant to infection and no DTH reaction can be seen. This different viral sensitivity may be related to the development of an antigen non-specific DTH-suppressor cell pathway at work in the adult mouse. When the resistant mice are treated with cyclophosphamide (Cy) (50 mg/kg each dose) given at days -1,+1,+4 (zero: infection day), animals become susceptible and develop DTH reaction in brain that leads to death. We analyze the influence of the timing of Cy administration on the suppressor system developing after infection. It was found that Cy depletes the previously described JV-induced suppressor populations (Tsv) but a new suppressor cell (Tsv*) is disclosed bearing the Thy 1+ Ly1+2- phenotype which is unable to depress DTH in Cy-treated animals. With only two doses of Cy corresponding to days -1 and +1, the target of Tsv* cells is depleted but the third dose is still required to achieve full depletion of Tsv cells which are able to employ the Cy-resistant antigen-specific suppressor cells as targets. Since the Cy treatment is able to deplete the Tsv population together with the target of Tsv* cells, animals became unable to regulate lethal DTH reaction. Thus, a cellular explanation for an empirically established Cy schedule able to abrogate the adult mouse resistance to JV is proposed.
Assuntos
Arenavirus do Novo Mundo/imunologia , Ciclofosfamida/farmacologia , Febre Hemorrágica Americana/imunologia , Hipersensibilidade Tardia/imunologia , Linfócitos T Reguladores/imunologia , Animais , Animais Lactentes , Modelos Animais de Doenças , Suscetibilidade a Doenças , Eritrócitos/imunologia , Febre Hemorrágica Americana/complicações , Hipersensibilidade Tardia/induzido quimicamente , Hipersensibilidade Tardia/etiologia , Depleção Linfocítica , Meningoencefalite/induzido quimicamente , Meningoencefalite/etiologia , Meningoencefalite/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Baço/citologia , Baço/imunologiaRESUMO
Junin virus (JV) infection of adult (resistant) BALB/c mice induces antigen non-specific delayed-type hypersensitivity (DTH) suppressor T cells, termed Tsv, bearing the Thy-1+, Ly-1+2- phenotype. These cells may be related to survival to infection since DTH reaction is associated with lethal meningoencephalitis. Employing several xenogeneic red blood cell (RBC) sensitization schedules to induce different cell subpopulations, we have attempted to establish the target of JV-induced suppressor cells (Tsv). The target of Tsv cells was actually included in the antigen-specific suppressor cell compartment, as demonstrated for the RBC system. Tsv cells were able to trigger suppressor cells to act without loss of their specificity. The presence of two sets of sheep RBC-induced DTH suppressor cells bearing the Ly-1+2- and Ly-1-2+ phenotypes was disclosed in low (10(6))-dose sensitized mice. Both sets were simultaneously required by Tsv to achieve DTH suppression. In contrast, in high (10(8] SRBC-dose sensitized animals treated with cyclophosphamide (doses of 50 mg/kg), a single Ly-1-2+ suppressor cell was required.
Assuntos
Arenavirus do Novo Mundo/imunologia , Linfócitos T Reguladores/imunologia , Animais , Ciclofosfamida/farmacologia , Relação Dose-Resposta Imunológica , Epitopos/imunologia , Eritrócitos/imunologia , Hipersensibilidade Tardia/imunologia , Terapia de Imunossupressão , Camundongos , Camundongos Endogâmicos , Linfócitos T Reguladores/efeitos dos fármacosRESUMO
Several recombinant Trypanosoma cruzi proteins previously isolated were used as antigens to analyse antibody specificities present in sera from human infections. Some parasite proteins such as SAPA (Shed Acute Phase Antigen) are antigenic early after infection. Others, like antigens 1 and 30, are antigenic mainly during the chronic phase of the infection. To understand why different proteins are antigenic at different periods of infection, specificities of antibodies present in the sera of infected mice were compared with the antigens expressed by parasites collected directly from blood. Parasites collected during the acute parasitaemia peak expressed not only antigen SAPA, but also antigens 1 and 30. However, only antibodies against SAPA were frequently observed during the early period and also in the chronic phase of murine infection. Long-lasting antibodies against SAPA were detected regardless of the mouse and parasite strains used. Furthermore, all 8 recombinant clones detected in a T. cruzi expression library with pooled sera from acutely infected mice were homologous to the SAPA gene. These results show that even though parasites from the acute parasitaemia peak in mice may express simultaneously several proteins known to be antigenic, only antibodies against SAPA were consistently detected.
Assuntos
Anticorpos Antiprotozoários/biossíntese , Antígenos de Protozoários/imunologia , Doença de Chagas/parasitologia , Trypanosoma cruzi/imunologia , Doença Aguda , Animais , Anticorpos Antiprotozoários/imunologia , Especificidade de Anticorpos , Doença de Chagas/sangue , Doença de Chagas/imunologia , Doença Crônica , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos NusRESUMO
A monospecific polyclonal antibody obtained against a cysteine proteinase isolated from epimastigotes of Trypanosoma cruzi was used for the immunocytochemical localization of the protein by electron microscopy and to analyse the role played by cysteine proteinase in the process of T. cruzi-host cell interaction. Cytoplasmic structures that correspond to elements of the endosomal-lysosomal (reservosome) system found in epimastigote, amastigote and trypomastigote forms reacted intensely with colloidal gold-labelled antibodies using on-section indirect labelling. The surface of most of the tissue culture-derived trypomastigotes was not labelled. However, the flagellar pocket of this form was labelled. All epimastigotes obtained from axenic cultures and amastigote-like forms found in the supernatant of vertebrate cells heavily infected with T. cruzi had their surface intensely labelled, indicating also the surface localization of the protein. Incubation of the parasites in the presence of a sub-agglutinating concentration of the anti-cysteine proteinase antibody led to a marked increase in their uptake by macrophages. In contrast, addition of the F(ab')2 portion of the same antibody significantly reduced the uptake of the parasites by the macrophages. The results obtained strongly suggest an important participation of cysteine proteinase in the process of T. cruzi-macrophage interaction.
Assuntos
Cisteína Endopeptidases/metabolismo , Interações Hospedeiro-Parasita/fisiologia , Trypanosoma cruzi/enzimologia , Animais , Anticorpos Antiprotozoários , Cisteína Endopeptidases/imunologia , Glutamato Desidrogenase/metabolismo , Imuno-Histoquímica , Técnicas In Vitro , Macrófagos/parasitologia , Camundongos , Microscopia Imunoeletrônica , Trypanosoma cruzi/imunologia , Trypanosoma cruzi/ultraestruturaRESUMO
Intracerebral inoculation of Junin virus (JV) in all susceptible mouse models available induces a lethal meningoencephalitis compatible with a delayed-type hypersensitivity (DTH) immune response. In contrast, adult BALB/c mice prove resistant to infection and no DTH response is seen. JV inoculation in adult BALB/c mice induces DTH suppression to unrelated antigens such as sheep red blood cells. (SRBC). This suppression is mediated by JV-induced T cells (Tsv), which are operative from 1 to 24 days post-infection (p.i.), and seems to be related to adult mouse survival. The presence of JV-induced contrasuppressor cells (CS) bearing the Thy-1+, Ly 1+2- phenotype, able to abrogate Tsv cells-mediated suppression, is described here. Thus, CS cells may be involved in the mechanism by which mice avoid over-exposure to Tsv-mediated DTH suppression. Such CS cells were found in the spleen of inoculated animals and may also be induced by transferring JV-free Tsv cells to 'naive' recipients, in which JV inoculation then induces morbidity.