RESUMO
Complement-depleted and -non-depleted BALB/c mice were inoculated with Leishmania (Leishmania) amazonensis promastigotes into the hind footpad to study the role of the complement system in cutaneous leishmaniasis. Total serum complement activity was measured by hemolytic assay and C3 fragment deposit at the inoculation site was determined by direct immunofluorescence in the early period of infection, i.e., at 3, 24, 48 h and 7 days post-infection. The inflammatory reaction and the parasite burden were evaluated in the skin lesion at 7 and 30 days post-infection. Total serum complement activity decreased in the early phase of infection, from 3 to 24 h, in non-depleted mice compared to non-infected and non-depleted mice. C3 fragment deposit at the site of parasite inoculation was present throughout the period of infection in non-depleted mice. In contrast, no C3 fragment deposit was observed at the inoculation site in complement-depleted mice. Complement-depleted mice showed a significant decrease in the inflammatory response and a significant increase in the number of parasites (70.0 +/- 5.3 vs 5.3 +/- 1.5) at 7 days of infection (P<0.05). A higher number of parasites were also present at 30 days of infection at the inoculation site of complement-depleted mice (78.5 +/- 24.9 vs 6.3 +/- 5.7). These experiments indicate that complement has an important role at the beginning of experimental cutaneous leishmaniasis caused by L. (L.) amazonensis by controlling the number of parasites in the lesion.
Assuntos
Proteínas do Sistema Complemento/fisiologia , Leishmania , Leishmaniose Cutânea/imunologia , Animais , Ativação do Complemento , Complemento C3/fisiologia , Ensaio de Atividade Hemolítica de Complemento , Leishmaniose Cutânea/parasitologia , Depleção Linfocítica , Masculino , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Complement-depleted and -non-depleted BALB/c mice were inoculated with Leishmania (Leishmania) amazonensis promastigotes into the hind footpad to study the role of the complement system in cutaneous leishmaniasis. Total serum complement activity was measured by hemolytic assay and C3 fragment deposit at the inoculation site was determined by direct immunofluorescence in the early period of infection, i.e., at 3, 24, 48 h and 7 days post-infection. The inflammatory reaction and the parasite burden were evaluated in the skin lesion at 7 and 30 days post-infection. Total serum complement activity decreased in the early phase of infection, from 3 to 24 h, in non-depleted mice compared to non-infected and non-depleted mice. C3 fragment deposit at the site of parasite inoculation was present throughout the period of infection in non-depleted mice. In contrast, no C3 fragment deposit was observed at the inoculation site in complement-depleted mice. Complement-depleted mice showed a significant decrease in the inflammatory response and a significant increase in the number of parasites (70.0 ± 5.3 vs 5.3 ± 1.5) at 7 days of infection (P < 0.05). A higher number of parasites were also present at 30 days of infection at the inoculation site of complement-depleted mice (78.5 ± 24.9 vs 6.3 ± 5.7). These experiments indicate that complement has an important role at the beginning of experimental cutaneous leishmaniasis caused by L. (L.) amazonensis by controlling the number of parasites in the lesion.
Assuntos
Animais , Masculino , Camundongos , Proteínas do Sistema Complemento , Leishmania , Leishmaniose Cutânea , Complemento C3 , Ensaio de Atividade Hemolítica de Complemento , Técnica Direta de Fluorescência para Anticorpo , Leishmaniose Cutânea , Depleção Linfocítica , Camundongos Endogâmicos BALB CRESUMO
Infection of Balb/c mice with Echinococcus granulosus protoscoleces constitutes the model for secondary hydatid infection. The immune response of Balb/c mice infected with E. granulosus is characterized by secretion of antibodies specific for carbohydrate epitopes and production of type-2 cytokines. A role for glycoconjugates in the induction of type-2 responses has been suggested in other host--parasite systems. Although glycoconjugates are immunogenic in E. granulosus infection, the role of these molecules in the establishment of the type-2 response has never been analysed. In this study, a carbohydrate rich fraction (E4+) from E. granulosus protoscoleces was obtained using the monoclonal antibody E492/G1 specific for the moiety Galalpha(1,4)Gal which is widely represented in protoscoleces and other E. granulosus antigenic preparations. The results showed that E4+ was immunogenic in Balb/c mice evoking an antibody response mainly directed against carbohydrate epitopes. In addition, splenocytes from E4+-immunized mice showed suppressed proliferative responses to Con A and E4+ induced IL-10 secretion by E4+-primed and naive splenocytes. The fraction E4+ also was immunogenic in infected mice during early infection. In this case also, splenocytes from infected mice as well as peritoneal cells from infected or naive mice, when stimulated in vitro with E4+, secreted IL-10. Collectively, these results suggest that E4+ may be involved in immunosuppression phenomena and, by stimulating IL-10 secretion, may contribute to the induction and sustaining of the type-2 cytokine response established in early experimental infection.
Assuntos
Antígenos de Helmintos/imunologia , Equinococose/imunologia , Echinococcus/imunologia , Glicoconjugados/imunologia , Células Th2/imunologia , Animais , Anticorpos Anti-Helmínticos/sangue , Citocinas/biossíntese , Equinococose/parasitologia , Echinococcus/crescimento & desenvolvimento , Echinococcus/metabolismo , Imunização , Terapia de Imunossupressão , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Using A.SW, A.CA, B10.S and B10.M congenic mouse strains, we measured the IgG specific humoral immune responses against sonicated and live Trypanosoma cruzi epimastigotes. Genes located in the A background (A.SW and A.CA strains) mediate higher IgG responses against the parasite antigenic complexes than those located in the B background (strains B10.S and B10.M), regardless of the H2 haplotypes. Thus, non H2 genetic elements seem to be more important in determining differences in the total IgG immune response against T. cruzi. Whether a detectable H2 effect, in favor of the H2(s) haplotype, occurred in the A or B background, was contingent on the immunisation protocol used. Thus, the H2(s) haplotype mediates a higher IgG response in the A background, if immunised with live epimastigotes, and in the B background against sonicated epimastigotes. Most likely this represents a complex sequence of events, controlled by non-MHC genes, involving antigen handling and processing and depending on the physical form of antigen delivery.
Assuntos
Anticorpos Antiprotozoários/biossíntese , Doença de Chagas/imunologia , Imunoglobulina G/biossíntese , Trypanosoma cruzi/imunologia , Animais , Feminino , Haplótipos , Imunização , Ensaio Imunorradiométrico , Camundongos , Camundongos Congênicos , Trypanosoma cruzi/genéticaRESUMO
The aims of this study were to investigate whether a Th1- or a Th2-type response is stimulated in the first stages of experimental infection with Echinococcus granulosus, and to determine whether live or dead protoscoleces equally contribute to such Th1/Th2-type polarization. Live parasites stimulated the production of IL-10, IL-4 and IL-5 as early as week 1 postinoculation. The levels of IL-10 and IL-4 decreased towards week 4 p.i. and that of IFN gamma increased. The production of specific antibodies was characterized by high levels of systemic IgG1 and local IgM and IgG3 (measured in peritoneal lavages). In contrast, dead parasites induced elevated levels of IL-4, IFN gamma, IL-10 and IL-5 on week 1 postinoculation followed by a decrease of IFN gamma and an increase of IL-4. Low levels of specific antibodies were stimulated by dead parasites both systemically and in the peritoneal cavity. These results show that E. granulosus infection induced an early Th2-type response and that live parasites stimulated stronger antibody responses than dead parasites. In addition, they strongly suggest that both phenomena were modulated by live protoscoleces.
Assuntos
Anticorpos Anti-Helmínticos/imunologia , Echinococcus/imunologia , Células Th1/imunologia , Células Th2/imunologia , Animais , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Cavidade PeritonealRESUMO
Resistance to the Leishmaniae is associated with interferon (IFN)-gamma mediated activation of macrophages. In this study, Balb/c mice were infected with three Leishmania strains that cause progressively growing cutaneous lesions without obvious dissemination: L. mexicana mexicana giving rise to rapidly growing lesions, and L. (Viannia) panamensis and L. mexicana-like, which both cause slowly developing lesions. The rate of lesion growth was compared to induction of early local and systemic IFN-gamma responses. All the three parasite strains induced increased levels of IFN-gamma transcripts 24 h after infection. Infection with the more aggressive strain resulted in a notably lower IFN-gamma response when compared to infection with the two low pathogenic strains. Interleukin-4 (IL-4) mRNA appeared 7 days after infection with L. (Viannia) panamensis and L. mexicana-like but not with L. mexicana mexicana. Thus, virulence of these Leishmania strains could not be associated with induction of IL-4 during the first week after infection. In addition, none of the Leishmania strains induced detectable mRNA for IL-12 or inducible nitric oxide synthase (iNOS). These data present a picture somewhat different from that which has been described in L. major experimental infection.
Assuntos
Interferon gama/biossíntese , Interleucina-4/biossíntese , Leishmania mexicana/imunologia , Leishmaniose Cutânea/imunologia , Animais , Expressão Gênica , Interferon gama/genética , Interleucina-4/genética , Leishmaniose Cutânea/patologia , Linfonodos/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , RNA Mensageiro/genética , Baço/parasitologia , Fatores de TempoRESUMO
Trypanosoma rangeli experimental murine infections were performed in order to study parasitemias and anti-parasite antibody levels. Three groups of mice were used: a) mice infected with metatrypomastigotes derived from infected bugs; b) mice which received four reinoculations of metatrypomastigotes and c) mice immunosuppressed with cyclophosphamide. The results showed that bloodstream parasites can be found from the first day post inoculation reaching a peak at day 5 or 7 and then start to decline. Parasites disappeared completely from the circulation after 20-25 days. However in the immunosuppressed group, parasites were found in blood up to 45 days post infection. The humoral immune response was monitored using an ELISA test and low levels of specific IgG and IgM immunoglobulins were found. However the IgG titers were lower than the IgM. One could conclude that IgM was the predominant immunoglobulin isotype induced in a T. rangeli experimental infection because the highest titers were observed in the reinoculated group. IgM antibodies also showed the most prominent crossreactivities with T. cruzi antigens.
Assuntos
Anticorpos Antiprotozoários/imunologia , Trypanosoma cruzi/imunologia , Tripanossomíase/imunologia , Animais , Reações Cruzadas/imunologia , Ciclofosfamida/administração & dosagem , Camundongos , Parasitemia/patologiaRESUMO
If the H-2 congenic mouse strains A.SW (H-2n) and A.CA (H-2f), are infected with Trypanosoma cruzi, a 45 kDa protein (Tc45), present in cultured epimastigotes and blood trypomastigotes, is recognized only by the A.SW strain sera. In order to explore the possibility that among seropositive humans the response to Tc45 is also highly variable, 81 chagasic human sera (as defined by the HemAve agglutination test, Polychaco S.A.I.C., Buenos Aires, Argentina) were tested in a direct (epimastigote antigenic complex directly bound to the solid phase) and indirect immunoradiometric assay (IRMA) (Tc45, from a partially purified preparation, bound to the solid phase, by means of a monoclonal antibody). Sixty nine of these sera reacted in both the direct and indirect assays, 11 were negative in both assays (these samples may correspond to false positives detected by the commercial agglutination test) and only one reacted with the antigenic complex but not with Tc45. Reactivity of the human sera with the epimastigote antigenic extract was relatively homogenous, while reactivity with Tc45 was extremely variable. No statistical correlation was determined between the two variables. Given the high variability of the human response to Tc45, ranging from negative to highly positive, together with the immunogenetic restriction previously described in the murine model, we speculate that human MHC may also modulate the response to this molecule.
Assuntos
Anticorpos Antiprotozoários/análise , Antígenos de Protozoários/imunologia , Doença de Chagas/imunologia , Trypanosoma cruzi/imunologia , Testes de Aglutinação , Animais , Anticorpos Monoclonais/imunologia , Reações Falso-Positivas , Humanos , Immunoblotting , Camundongos , Camundongos Endogâmicos BALB C , Radioimunoensaio , Sensibilidade e EspecificidadeRESUMO
The present work describes the purification and characterization of antigen B (AgB), the thermostable lipoprotein from E. granulosus. Native AgB was purified to homogeneity by a new strategy involving adsorption on DEAE-Sepharose, followed by immunopurification. The purified antigen was analysed using mapped monoclonal antibodies (MoAbs) and peptide isolation by in situ digestion in gels after SDS-PAGE. Epitope mapping of 7 MoAbs using PEPSCAN, synthetic peptides and competition studies, revealed that six of them defined epitopes which clustered the N-terminal extension of a 8 kDa subunit of AgB, whilst the remaining one reacted against the stretch RGLIAEGE, corresponding to the C-terminus. The epitopes defined by the seven MoAbs were found to be present in all the subunits. Furthermore, the similarities of the peptide finger prints obtained by HPLC analysis and amino acid sequencing of tryptic peptides isolated from the 8, 16 and 24 kDa subunits, indicated that they have most if not all the amino acid sequence in common. We also found evidence that the band representing a component of an apparent molecular weight of 8 kDa in SDS-PAGE, believed to be the smallest subunit of AgB, contained at least two components, which may constitute the building blocks of the higher molecular weight subunits.
Assuntos
Antígenos de Helmintos/química , Echinococcus/imunologia , Lipoproteínas/química , Lipoproteínas/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais , Antígenos de Helmintos/genética , Antígenos de Helmintos/isolamento & purificação , Bovinos , Cromatografia de Afinidade , Echinococcus/genética , Mapeamento de Epitopos , Proteínas de Helminto/química , Proteínas de Helminto/imunologia , Proteínas de Helminto/isolamento & purificação , Lipoproteínas/isolamento & purificação , Camundongos , Dados de Sequência Molecular , Peso Molecular , Conformação ProteicaRESUMO
Formation of inflammatory lesions, one of the pathologic consequences of infection with Trypanosoma cruzi, involves intricate cell-cell interactions in which cell adhesion molecules (CAMs) are involved. Sera from 56 Chagas' disease patients grouped according to disease severity were studied for the presence of soluble intercellular adhesion molecule-1 (s-ICAM-1), soluble endothelial selectin (s-E-selectin), soluble vascular cell adhesion molecule-1 (s-VCAM-1), soluble platelet selectin (s-P-selectin), and s-CD44 were studied to determine if they could be used alone or in different combinations as markers for specific diagnostic procedures. Comparisons were made between congenitally, acutely, and chronically infected patients and aged-matched, noninfected individuals, as well as between patients with chronic Chagas' disease grouped according to the severity of their heart-related pathology. No differences in levels of s-CAMs were detected between sera from children with congenital T. cruzi infection and sera from noninfected infants born from chagasic mothers. In contrast, titers of s-ICAM-1, s-VCAM-1, s-selectin, and s-CD44 but not s-P-selectin were significantly increased in sera from patients during the acute phase of infection with T. cruzi. Titers of s-VCAM-1 and s-P-selectin were increased in chronically infected patients. A positive association with disease severity in sera from patients with chronic disease was observed for the levels of s-P-selectin. In contrast, we found no association between clinical symptoms and levels of s-VCAM-1. Patients with chronic disease with severe cardiopathy also showed diminished levels of s-CD44 in comparison with healthy controls or patients with mild disease. The results are discussed in the context of pathology of Chagas' disease.
Assuntos
Moléculas de Adesão Celular/sangue , Doença de Chagas/patologia , Doença Aguda , Adolescente , Adulto , Idoso , Moléculas de Adesão Celular/química , Doença de Chagas/sangue , Doença de Chagas/congênito , Doença Crônica , Selectina E/sangue , Selectina E/química , Humanos , Receptores de Hialuronatos/sangue , Receptores de Hialuronatos/química , Lactente , Molécula 1 de Adesão Intercelular/sangue , Molécula 1 de Adesão Intercelular/química , Pessoa de Meia-Idade , Selectina-P/sangue , Selectina-P/química , Índice de Gravidade de Doença , Solubilidade , Molécula 1 de Adesão de Célula Vascular/sangue , Molécula 1 de Adesão de Célula Vascular/químicaRESUMO
Monoclonal antibodies (MoAbs) specific for unique epitopes of the catalytic domain of cruzipain (Crz) were used to develop a two-site sandwich ELISA specific for native Crz. In addition, the authors developed a sandwich ELISA that allowed the detection of the protease C-terminal domain (CT) using a combination of a MoAb specific for the CT and rabbit anti-Crz IgGs. Both assays were sensitive with detection limits of 2 ng/ml and 0.7 ng/ml, respectively. The assays were assessed for applicability in detection of antigens in serum and urine from experimentally infected BALB/c mice. The antigens were already detectable in serum by the third week after infection, reached their peak by week four, and decreased during the chronic phase of the infection. Throughout the infection the relative amount of CT detected was several-fold higher than that of native Crz, and the data demonstrate that the cT exposes highly immunogenic epitopes that are absent in native Crz. Since these observations have a potential application in diagnosis, the authors analysed the degree of cross-reactivity with antigens from T. rangeli, T. brucei, Leishmania mexicana and L. panamensis, and determined that the assays were highly specific. Measurable amounts of the CT were also recorded in urine samples.
Assuntos
Doença de Chagas/enzimologia , Cisteína Endopeptidases/análise , Fragmentos de Peptídeos/análise , Trypanosoma cruzi/enzimologia , Animais , Anticorpos Monoclonais/imunologia , Cisteína Endopeptidases/imunologia , Ensaio de Imunoadsorção Enzimática , Camundongos , Camundongos Endogâmicos BALB C , Fragmentos de Peptídeos/imunologia , Proteínas de Protozoários , CoelhosRESUMO
The T cell receptor (TCR) V beta repertoire was studied in BALB/c, CBA/HJ, and CBA/J mice experimentally infected with Trypanosoma cruzi. The percentage of expression of 14 V beta chains of the variable domain of the TCR in the thymus and spleen was evaluated. In the thymus of acutely infected with BALB/c and CBA/HJ mice there was an increase in the expression of positively selected V beta families. These changes in the V beta chains usage in the thymus paralleled the enrichment of CD4+ and CD8+ single-positive T cells. During the acute infection, several changes were observed in the peripheral expression of V beta families, such as of V beta 6 in BALB/c (a 36% increase in CD8+ T cells of the corresponding levels of V beta), of V beta 8 in CBA/HJ (a 37% decrease in CD8+ cells), and of V beta chains 8 and 14 in CBA/J mice (V beta 14+CD4+ cells increased 19%, and V beta 8 expression decreased 19 and 33% in CD4+ and CD8+ cells, respectively). In chronically infected BALB/c and CBA/HJ mice, no change in the V beta families was observed, neither in the thymus nor in the spleen. In acutely infected mice, the alterations of the peripheral expression of positively selected V beta families could be due to the stimulation by T. cruzi antigens and/or cytokines; the homeostatic mechanism/s that maintains the selection of the TCR V beta repertoire did not seem to be severely affected during the infections.
Assuntos
Doença de Chagas/imunologia , Doença de Chagas/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/análise , Baço/imunologia , Subpopulações de Linfócitos T/imunologia , Timo/imunologia , Animais , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos CBA , Especificidade da Espécie , Baço/metabolismo , Baço/parasitologia , Timo/metabolismo , Timo/parasitologiaRESUMO
An 80-kDa Trypanosoma cruzi urinary antigen (UAg) was affinity-purified from the urine of infected dogs. We demonstrated that UAg is structurally and functionally related to proteins belonging to the transferrin family, as shown by amino acid sequence and iron binding experiments. Nevertheless, monoclonal antibodies raised against UAg specifically and selectively recognized this parasite's circulating antigen. The existence of an 80-kDa T. cruzi antigen co-migrating with UAg could be confirmed when epimastigotes were metabolically labelled with [35S] methionine and then immunoprecipitated with the above mentioned antibodies. We conclude that UAg is an iron-binding T. cruzi component eliminated in the urine of the infected host.
Assuntos
Antígenos de Protozoários/metabolismo , Doença de Chagas/imunologia , Ferro/metabolismo , Trypanosoma cruzi/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Antígenos de Protozoários/imunologia , Antígenos de Protozoários/isolamento & purificação , Doença de Chagas/urina , Cães , Eletroforese em Gel de Poliacrilamida , Epitopos , Dados de Sequência MolecularRESUMO
The kinetics of humoral immune responses were investigated in mice experimentally infected with five clones of Trypanosoma cruzi isolated from different sources in Panama. Sera were collected at different timepoints post-infection. ELISA and IHA tests were used to detect antibodies against T. cruzi epimastigote antigens. The levels of T. cruzi specific antibodies increased during the course of infection; at day 90 post-infection the range was between 1:5120 and 1:10240. A high correlation was evident between ELISA and IHA results. Western blots revealed that these antibodies recognized polypeptides of 81, 76 and 71 KDa during the first weeks and 81, 76, 71, 50, 40, 28 and 12 KDa after 30-50 days. Only minor differences in antigen recognition patterns were demonstrated, suggesting that the major antigens may be represented in all clones. T. rangeli antigens were also recognized by T. cruzi seropositive sera. However, an ELISA test using antigens isolated from a genomic expression library of T. cruzi revealed that a hyperimmune rabbit serum against T. rangeli was unable to recognize the repeat sequence of SAPA (Shed Acute Phase Antigen) peptides but did recognize a number of other T. cruzi synthetic peptide antigens. The importance of these findings, in the context of Chagas' disease, is discussed.
Assuntos
Anticorpos Antiprotozoários/biossíntese , Doença de Chagas/imunologia , Trypanosoma cruzi/imunologia , Trypanosoma/imunologia , Sequência de Aminoácidos , Animais , Antígenos de Protozoários/imunologia , Doença de Chagas/patologia , Reações Cruzadas , Ensaio de Imunoadsorção Enzimática , Testes de Hemaglutinação , Camundongos , Dados de Sequência Molecular , Panamá , Parasitemia/imunologia , Peptídeos/síntese química , Peptídeos/imunologia , Coelhos , Especificidade da Espécie , Trypanosoma cruzi/patogenicidade , VirulênciaRESUMO
Estudamos a susceptibilidade a Leishmania (Viannia) panamensis em linhagens de camundongos BALB/c, DBA/2J, CBA/HJ e C57BL/6. Os camundongos da linhagem C57BL/6 eram resistentes, apresentando lesoes autolimitantes na pata. Os das linhagens BALB/c e DBA/2J eram suscetiveis, apresentando edema na pata, evidente aos 20 dias pos-infeccao que progrediu para uma lesao tipo tumoral nas fases tardias. Os animais da linhagem CBA/HJ apresentaram resistencia intermediaria. Em contraste a outros modelos de leishmaniose cutanea murina, a lesao nos camundongos infectados pela L. (V.) panamensis mostrou ser restrita ao local de inoculacao na pele. Estudamos tambem o desenvolvimento de resposta celular e anticorpos anti-Leishmania nas linhagens BALB/c e C57BL/6. A resposta proliferativa de celulas do linfonodo a antigenos de L. (V.) panamensis foi bifasica em ambas as linhagens...
Assuntos
Animais , Masculino , Ratos , Leishmaniose/imunologia , Meios de Cultura , Leishmaniose/parasitologia , Leishmania/isolamento & purificação , Sensibilidade e EspecificidadeRESUMO
We studied the susceptibility to Leishmania (Viannia) panamensis in strains of mice. The C57BL/6 strain was resistant and showed self-controlled lesion at the injected foot pad. The BALB/c and DBA/2J strains were susceptible and showed a foot swelling that started day 20 post-infection and progressed to a tumour-like lesion in later period of observation. The CBA/HJ strain was found to be of intermediary resistance. In contrast to other known cutaneous leishmaniasis in mice, the lesion in L. (V.) panamensis-infected mice was restricted to the inoculation site in the skin. In addition, we studied the development of cellular response and antibodies against Leishmania antigen in BALB/c and C57BL/6 strains. The proliferative response of lymph node cells against L. (V.) panamensis antigen was biphasic in both strains. An initial response was seen on day 20, followed by a refractory period between 40 and 80 days and a second response around fourth month post-infection. The response in the latter period was higher in C57BL/6 strain than in BALB/c strain. BALB/c strain presented much higher anti-Leishmania antibody level than C57BL/6 strain. The model and the correlation of immunological variables and the course of the infection are discussed.
Assuntos
Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/biossíntese , Leishmania guyanensis/imunologia , Leishmaniose Mucocutânea/imunologia , Animais , Anticorpos Antiprotozoários/análise , Modelos Animais de Doenças , Suscetibilidade a Doenças , Feminino , Imunidade Celular , Imunização , Leishmania guyanensis/patogenicidade , Linfonodos/citologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Endogâmicos DBARESUMO
We analyzed the biological role of nitric oxide (NO) during murine Trypanosoma cruzi infection. Infection of mice with T. cruzi markedly increased NO synthesis. Administration of N-nitro-L-arginine methyl-esther (L-NAME) intraperitoneally or intragastrically diminished endogenous NO synthesis and resistance of mice to acute infection with three biologically different strains of T. cruzi. Mice protected against challenge with T. cruzi by transfer of T-cell-enriched populations from chronically infected animals, showed higher serum nitrate levels than controls non-transferred, or transferred, with T cells from non-immune mice. Administration of L-NAME abrogated transfer of resistance, suggesting NO participation in this process. Depletion of T cells from the transferred population abolished both protection and NO3- increase. On the contrary, mice chronically infected with T. cruzi showed no increased parasitemia or death upon treatment with L-NAME. The NO donor drug S-nitroso-acetyl-penicillamine was able to kill tissue culture or bloodstream trypomastigotes in vitro at biologically relevant concentrations. Conversely, NO appeared not to play a role in formation of inflammatory foci during T. cruzi infection, since infected mice treated with L-NAME showed no reduced inflammation.
Assuntos
Doença de Chagas/imunologia , Doença de Chagas/patologia , Óxido Nítrico/fisiologia , Doença Aguda , Animais , Doença de Chagas/prevenção & controle , Doença Crônica , Imunidade Inata , Camundongos , Camundongos Endogâmicos BALB C , Óxido Nítrico/imunologia , Óxido Nítrico/metabolismo , Linfócitos T/imunologia , Linfócitos T/patologia , Trypanosoma cruzi/imunologiaRESUMO
In the present study we describe the production and characterization of a panel of monoclonal antibodies (MoAbs) directed against cruzipain (Crz), the major cysteine proteinase from Trypanosoma cruzi. The five MoAbs, BD6, BF2, CG2, CH8, and DC10 were analysed with respect to affinity and specificity. None of the MoAbs cross-reacted with papain, which has regions of high homology with Crz. Treatment of the antigen with periodate did not affect the binding of the MoAbs, suggesting that they bind to the polypeptide moiety of Crz. CH8 recognized a continuous epitope located at the C-terminal extension of the proteinase that appeared to be highly immunogenic. Although the rest of the MoAbs recognized epitopes located in the catalytic domain, the enzymatic activity of Crz was not impaired by the binding of the MoAbs. Characterization of the antibody-binding sites revealed the presence of at least four separate epitopes.
Assuntos
Anticorpos Monoclonais/biossíntese , Anticorpos Antiprotozoários/biossíntese , Cisteína Endopeptidases/imunologia , Trypanosoma cruzi/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Antiprotozoários/imunologia , Afinidade de Anticorpos , Especificidade de Anticorpos , Ensaio de Imunoadsorção Enzimática , Mapeamento de Epitopos , Camundongos , Camundongos Endogâmicos BALB C , Testes de Precipitina , Proteínas de Protozoários , Trypanosoma cruzi/enzimologiaRESUMO
We analysed the production of nitric oxide (NO) intermediates by cells from BALB/c mice infected with either virulent (Tulahuén or RA) or avirulent (CA-1) strains of Trypanosoma cruzi. Peritoneal or spleen cells from mice infected with T. cruzi released NO when incubated without further stimuli. Cells from mice during the acute stage of infection accumulated higher levels of inducible NO synthase mRNA and produced both, before and after lypopolysaccharide stimulation, higher amounts of NO than cells from mice chronically infected with T. cruzi. NO synthesis showed similar kinetics in connection with all three strains of T. cruzi, but cells from mice inbred with the Tulahuén or RA strains released higher levels of IFN-gamma, an activator of the NO pathways, than cells from mice infected with the CA-1 strain. In vivo administration of L-Ng-monomethyl-L-arginine (L-NMMA), a competitive inhibitor of NO synthase, increased the susceptibility of mice to T. cruzi. We conclude that infection with T. cruzi induces NO production, and suggest that NO plays a role in the resistance against the parasite.
Assuntos
Doença de Chagas/metabolismo , Óxido Nítrico/biossíntese , Aminoácido Oxirredutases/metabolismo , Animais , Arginina/análogos & derivados , Arginina/farmacologia , Sequência de Bases , Primers do DNA , Modelos Animais de Doenças , Feminino , Interferon gama/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Óxido Nítrico/antagonistas & inibidores , Óxido Nítrico Sintase , Cavidade Peritoneal/citologia , RNA Mensageiro/metabolismo , Baço/citologia , Trypanosoma cruzi/patogenicidade , Virulência/efeitos dos fármacos , ômega-N-MetilargininaRESUMO
Leishmania (Viannia) panamensis infected Balb/c mice developed a progressive swelling in the injected footpad that grew to a tumour-like lesion from day 80 onwards. We did not observe any typical ulcera, necrosis or metastasis to other parts of the skin. Neither did we observe any histopathological changes in liver or spleen during the experiment. At the site of injection, we observed progressive changes ranging from a moderate, mixed inflammatory infiltrate with few leishmania amastigotes in the macrophages to an extensive inflammation composed of monomorphic vacuolated macrophages containing large numbers of parasites. A granulomatous pattern with presence of epithelioid cells and a few multinucleated giant cells was observed at the initial phase of the infection. During later stages, focal necrosis with polymorphonuclear neutrophils was seen. Lymph nodes presented granulomatous lesions in the subcapsular area, numerous plasma cells in the medullary cords and macrophages with leishmania organisms in dilated cortical sinuses at the 4th and the 6th months of infection. This Leishmania (Viannia) panamensis infected Balb/c mice seems to be a good model for continued studies of the pathogenesis of cutaneous leishmaniasis and also for drug trials in the development of new therapeutic tools.