Adjuvant effect of LPS and killed Propionibacterium acnes on the development of experimental gastrointestinal nematode infestation in sheep.

Gastrointestinal helminthic infection is an important worldwide sheep disease. The emergence of anthelminthic resistance has led to drives to seek new means of therapeutic control of helminthiasis in sheep. Several data demonstrated the adjuvant effect of Propionibacterium acnes on resistance to infection. Herein, we evaluate the adjuvant effect of the commercial suspension containing LPS and P. acnes on experimental helminthiasis. Sheep received three doses of LPS and P. acnes commercial suspension or saline 0.9% (control group). Both groups received orally Haemonchus contortus infective larvae on day 0. Parasitological, haematological, lymphoproliferation analysis, IL-5 and IgE determination were made once a week until 35th day after infection. Our results revealed increase on packed cell volumes on day 14, in LPS + P. acnes treated group. On 21st and 35th days after infection in the same group occurred increase on circulating eosinophils and lymphocytes, and also in the lymphoproliferative response to mitogen. On 35th day, the faecal eggs peak in LPS + P. acnes treated group was significantly lower than control. A negative correlation between faecal eggs counts and circulating eosinophils in the immunostimulant treated group was also observed. Our findings suggest that LPS + P. acnes suspension can be used as a strategy to control helminthiasis in sheep.

Chronic Chagas' disease cardiomyopathy patients display an increased IFN-gamma response to Trypanosoma cruzi infection.

One-third of all Trypanosoma cruzi -infected patients eventually develop chronic Chagas' disease cardiomyopathy (CCC), a particularly lethal inflammatory dilated cardiomyopathy, where parasites are scarce and heart-infiltrating mononuclear cells seem to be the effectors of tissue damage. Since T. cruzi is a major inducer of interleukin-12 production, the role of inflammatory cytokines in the pathogenesis of CCC was investigated. We assayed cytokine production by peripheral blood mononuclear cells (PBMC) from CCC and asymptomatic T. cruzi -infected (ASY) individuals, as well as by T cell lines from endomyocardial biopsies from CCC patients. PBMC from CCC and ASY patients produced higher IFN-gamma levels than normal (N) individuals in response to B13 protein and phytohaemagglutinin PHA; IFN-gamma high responders (> or =1 ng/ml) were 2-3 fold more frequent among CCC patients than ASY individuals. Conversely, IL-4 production in response to the same stimuli was suppressed among T. cruzi -infected patients. The frequency of PHA-induced IFN gammaproducing cells on PBMC was significantly higher among CCC than ASY and N individuals. IFN-gamma and TNF-alpha were produced by ten out of ten PHAstimulated T cell lines from CCC patients; IL-2 and IL-10 were produced by four out of ten and one out of ten lines, respectively; IL-4, IL-1alpha, IL-1beta, IL-6 and IL-12 were undetectable. Our results suggest that CCC and ASY patients may respond differentially to the IFN-gamma-inducing stimulus provided by T. cruzi infection. Given the T(1)-type cytokine profile of heart-infiltrating T cell lines from CCC patients, the ability to mount a vigorous IFN-gamma response may play a role on the differential susceptibility to CCC development.

Retro-inverso peptide analogues of Trypanosoma cruzi B13 protein epitopes fail to be recognized by human sera and peripheral blood mononuclear cells.

Retro inverso (RI) analogues of antigenic synthetic peptides, which are made of D-amino acids with a reversed sequence, may mimic the side chain conformation of natural all-L peptides. RI analogues were cross-reactively recognized by antibodies and CD4+ T cells reactive against natural all-L synthetic peptides or native proteins in animal models. Since peptides containing D-amino acids are highly resistant to proteolytic digestion, cross-reactive RI analogues may be ideal for in vivo administration to humans as synthetic peptide vaccines or immunomodulators. B13 is an immunodominant tandemly repetitive protein from Trypanosoma cruzi, a protozoan parasite that is the causative antigen of Chagas' disease. In order to test whether RI peptides can be recognized by human antibody and T cells, we synthesized two all-L peptides containing the immunodominant B (S12) and T (S15.7) cell epitopes of B13 protein from T. cruzi and their retro (R, made of all-L amino acids with reversed sequence), inverso (I, made of all-D amino acids) and RI analogues. Recognition of peptides S12, S12-R, S12-I and S12-RI by anti-B13 antibodies in sera from T. cruzi-infected patients was tested in competitive ELISA assay with recombinant B13 protein as the solid phase antigen. Peptides S15.7 and its topological analogues were tested at the 10-50 microM range in proliferation assays on peripheral blood mononuclear cells (PBMC) from S15.7-responder individuals. The median percentage inhibition of B13 ELISA for peptide S12 was 94%, while those of the RI analogue or the other topological analogues were below 12%. While peptide S15.7 was recognized by PBMC from all subjects tested, none recognized the RI analogue of the S15.7 T cell epitope. Our results indicate that cross-reactivity with natural epitopes is not an universal property of RI analogues. This may limit the general applicability of the use of cross-reactive RI analogues as human vaccines and immunotherapeutic agents.

Molecular mimicry between cardiac myosin and Trypanosoma cruzi antigen B13: identification of a B13-driven human T cell clone that recognizes cardiac myosin

Previous reports from our group have demonstrated the association of molecular mimicry between cardiac myosin and the immunodominant Trypanosoma cruzi protein B13 with chronic Chagas´disease cardiomyopathy at both the antibody and heart-infiltrating T cell level. At the peripheral blood level, we observed no difference in primary proliferative responses to T. cruzi B13 protein between chronic Chagas´cardiopathy patients, asymptomatic chagasics and normal individuals. In the present study, we investigated whether T cells sensitized by T. cruzi B13 protein respond to cardiac myosin. T cell clones generated from a B13-stimulated T cell line obtained from peripheral blood of a B13-responsive normal donor were tested for proliferation against B13 protein and human cardiac myosin. The results showed that one clone responded to B13 protein alone and the clone FA46, displaying the highest stimulation index to B13 protein (SI=25.7), also recognized cardiac myosin. These data show that B13 and cardiac myosin share epitopes at the T cell level and that sensitization of a T cell with B13 protein results in response to cardiac myosin. It can be hypothesized that this also occurs in vivo during T. cruzi infection which results in heart tissue damage in chronic Chagas´disease cardiomyopathy.

Molecular mimicry between cardiac myosin and Trypanosoma cruzi antigen B13: identification of a B13-driven human T cell clone that recognizes cardiac myosin.

Previous reports from our group have demonstrated the association of molecular mimicry between cardiac myosin and the immunodominant Trypanosoma cruzi protein B13 with chronic Chagas' disease cardiomyopathy at both the antibody and heart-infiltrating T cell level. At the peripheral blood level, we observed no difference in primary proliferative responses to T. cruzi B13 protein between chronic Chagas' cardiopathy patients, asymptomatic chagasics and normal individuals. In the present study, we investigated whether T cells sensitized by T. cruzi B13 protein respond to cardiac myosin. T cell clones generated from a B13-stimulated T cell line obtained from peripheral blood of a B13-responsive normal donor were tested for proliferation against B13 protein and human cardiac myosin. The results showed that one clone responded to B13 protein alone and the clone FA46, displaying the highest stimulation index to B13 protein (SI = 25.7), also recognized cardiac myosin. These data show that B13 and cardiac myosin share epitopes at the T cell level and that sensitization of a T cell with B13 protein results in response to cardiac myosin. It can be hypothesized that this also occurs in vivo during T. cruzi infection which results in heart tissue damage in chronic Chagas' disease cardiomyopathy.