To assess reinfection of BALB/c mice with different Toxoplasma gondii strains, the animals were prime infected with the non-virulent D8 strain and challenged with virulent recombinant strains. Thirty days after challenge, brain cysts were obtained from surviving BALB/c mice and inoculated in Swiss mice to obtain tachyzoites for DNA extraction and PCR-RFLP analysis to distinguish the different T. gondii strains present in possible co-infections. Anti-Toxoplasma immune responses were evaluated in D8-primed BALB/c mice by detecting IFN-³ and IL-10 produced by T cells and measuring immunoglobulin levels in serum samples. PCR-RFLP demonstrated that BALB/c mice were reinfected with the EGS strain at 45 days post prime infection (dpi) and with the EGS and CH3 strains at 180 dpi. High levels of IFN-³ were detected after D8 infection, with no significant difference between 45 and 180-day intervals. However, higher IL-10 levels and higher plasmatic IgG1 and IgA were detected from samples obtained 180 days after infection. BALB/c mice were susceptible to reinfection with different recombinant T. gondii strains and this susceptibility correlated with enhancement of IL-10 production.

In Chagas disease, understanding how the immune response controls parasite growth but also leads to heart damage may provide insight into the design of new therapeutic strategies. Tumor necrosis factor-alpha (TNF-α) is important for resistance to acute Trypanosoma cruzi infection; however, in patients suffering from chronic T. cruzi infection, plasma TNF-α levels correlate with cardiomyopathy. Recent data suggest that CD8-enriched chagasic myocarditis formation involves CCR1/CCR5-mediated cell migration. Herein, the contribution of TNF-α, especially signaling through the receptor TNFR1/p55, to the pathophysiology of T. cruzi infection was evaluated with a focus on the development of myocarditis and heart dysfunction. Colombian strain-infected C57BL/6 mice had increased frequencies of TNFR1/p55+ and TNF-α+ splenocytes. Although TNFR1-/- mice exhibited reduced myocarditis in the absence of parasite burden, they succumbed to acute infection. Similar to C57BL/6 mice, Benznidazole-treated TNFR1-/- mice survived acute infection. In TNFR1-/- mice, reduced CD8-enriched myocarditis was associated with defective activation of CD44+CD62Llow/- and CCR5+ CD8+ lymphocytes. Also, anti-TNF-α treatment reduced the frequency of CD8+CCR5+ circulating cells and myocarditis, though parasite load was unaltered in infected C3H/HeJ mice. TNFR1-/- and anti-TNF-α-treated infected mice showed regular expression of connexin-43 and reduced fibronectin deposition, respectively. Furthermore, anti-TNF-α treatment resulted in lower levels of CK-MB, a cardiomyocyte lesion marker. Our results suggest that TNF/TNFR1 signaling promotes CD8-enriched myocarditis formation and heart tissue damage, implicating the TNF/TNFR1 signaling pathway as a potential therapeutic target for control of T. cruzi-elicited cardiomyopathy.

Cepas de Toxoplasma gondii que apresentam o genótipo I/III são associadas a toxoplasmose ocular adquirida em humanos. No presente trabalho, nós utilizamos um modelo da doença em camundongos para caracterizar mecanismos imunológicos envolvidos na resistência do hospedeiro à infecção por aquelas cepas. Escolhemos as cepas D8, G2 e P-Br, que causam infecção crônica em camundongos, semelhante à toxoplasmose humana. Camundongos deficientes em MyD88, IFN-G e IL-12 foram susceptíveis a infecções com todas as três linhagens do parasita. Esses dados indicam a importância de mecanismos inatos no controle da infecção. Por outro lado, o haplótipo do MHC não influenciou na resistência/susceptibilidade, na medida em que linhagens de camundongos com um mesmo "background'' genético, mas diferentes haplótipos de MHC (H2b e H2d) apresentam o índice de mortalidade e número de cistos semelhantes após a infecção com aquelas cepas do parasita. Em contraste, o "background'' genético de C57BL/6, mas não o haplótipo de MHC, foi crítico para o desenvolvimento de inflamação intestinal causada pelas cepas estudadas. Finalmente, com relação aos mecanismos efetores, observamos que linfócitos B e T CD8+ controlam a sobrevivência após infecção. Por outro lado, a ativação da enzima óxido nítrico sintase induzida foi um fator importante para controle do número de cistos cerebrais em camundongos infectados com cepas do Tipo I/III. Esses achados são relevantes para o melhor entendimento dos mecanismos imunológicos envolvidos na proteção e patogênese durante infecção com T. gondii.

Toxoplasma gondii strains displaying the Type I/III genotype are associated with acquired ocular toxoplasmosis in humans. Here, we used a mice model to characterize some immunological mechanisms involved in host resistance to infection with such strains. We have chosen the Type I/III strains D8, G2 and P-Br, which cause a chronic infection in mice that resembles human toxoplamosis. Mice deficient of molecules MyD88, IFN-gamma, and IL-12 were susceptible to all three parasite strains. This finding indicates the importance of innate mechanisms in controlling infection. On the other hand, MHC haplotype did not influenced resistance/susceptibility; since mice lineages displaying a same genetic background but different MHC haplotypes (H2b or H2d) developed similar mortality and cyst numbers after infection with those strains. In contrast, the C57BL/6 genetic background, and not MHC haplotype, was critical for development of intestinal inflammation caused by any of the studied strains. Finally, regarding effector mechanisms, weobserved that B and CD8+ T lymphocytes controlled survival,whereas the inducible nitric oxide synthase influenced cyst numbers in brains of mice infected with Type I/III strains. These findings are relevant to further understanding of the immunologic mechanisms involved in host protection and pathogenesis during infection with T. gondii.

A resposta imune induzida por uma proteína recombinante de Leishmania (Leishmania) amazonensis de 33 kD (Larp33) foi avaliada em linfócitos de indivíduos vacinados com a Leishvacin® e em camundongos através de vacinação. Larp33 foi expressa em Escherichia coli após clonagem de um fragmento genômico de L. (L.) amazonensis de 2,2 kb no vetor pDS56-6His. Larp33 foi reconhecida por anticorpos IgG presentes no soro de indivíduos vacinados com Leishvacin® e induziu proliferação em linfócitos desses indivíduos em níveis comparáveis ao antígeno total de Leishmania. A análise por imunoblot indicou que Larp33 corresponde a uma proteína de aproximadamente 40 kD expressa em promastigotas de L. (L.) amazonensis e L. (Viannia) braziliensis. Hibridização com sonda de DNA correspondente a parte do fragmento clonado, também demonstrou que o gene codificador desta proteína é expresso em promastigotas destas duas espécies. Larp33 em associação com BCG (Bacille Calmette-Guerin) foi capaz de induzir 75% de proteção em camundongos C75BL/10 e BALB/c, suceptíveis à infecção por L. (L.) amazonensis. Linfócitos dos camundongos protegidos produziram IL-2 e IFN-<FONT FACE="Symbol">g</font> em resposta a Larp33. Nossos resultados indicam que Larp33 é imunogênica para linfócitos de indivíduos vacinados com Leishvacin ® e protetora para camundongos contra a infecção por L. (L.) amazonensis

In the search for Leishmania recombinant antigens that can be used as a vaccine against American Cutaneous Leishmaniasis, we identified a Leishmania (Leishmania) amazonensis recombinant protein of 33 kD (Larp33) which is recognized by antibodies and peripheral blood leukocytes (PBL) from subjects vaccinated with Leishvacin ®, Larp33 was expressed in Escherichia coli after cloning of a 2,2 kb Sau3A digested genomic fragment of L. (L.) amazonensis into the pDS56-6 His vector. Immunoblotting analysis indicated that Larp33 corresponds to an approximately 40-kD native protein expressed in promastigotes of L.(L.) amazonensis and L. (Viannia) braziliensis. Northern blots of total RNA also demonstrated that the gene coding for this protein is expressed in promastigotes of the major lineages of Leishmania causing American Cutaneous Leishmaniasis. Larp33 induced partial protection in susceptible mouse strains (BALB/c and C57BL/10) against L. (L.) amazonensis after vaccination using Bacille Calmette-Guerin (BCG) as adjuvant. In vitro stimulation of splenocytes from BALB/c protected mice with Larp33 elicited the secretion of IL-2 and IFN-<FONT FACE="Symbol">g</font>, suggesting that a Th1 cell-mediated protective response is associated with the resistance observed in these mice. As revealed by its immunogenic and antigenic properties, this novel recombinant antigen is a suitable candidate to compose a vaccine against cutaneous leishmaniasis