In vivo imaging of trypanosomes for a better assessment of host-parasite relationships and drug efficacy.

The advances in microscopy combined to the invaluable progress carried by the utilization of molecular, immunological or immunochemical markers and the implementation of more powerful imaging technologies have yielded great improvements to the knowledge of the interaction between microorganisms and their hosts, notably a better understanding of the establishment of infectious processes. Still today, the intricacies of the dialog between parasites, cells and tissues remain limited. Some improvements have been attained with the stable integration and expression of the green fluorescence protein or firefly luciferase and other reporter genes, which have allowed to better approach the monitoring of gene expression and protein localization in vivo, in situ and in real time. Aiming at better exploring the well-established models of murine infections with the characterized strains of Trypanosoma cruzi and Trypanosoma vivax, we revisited in the present report the state of the art about the tools for the imaging of Trypanosomatids in vitro and in vivo and show the latest transgenic parasites that we have engineered in our laboratory using conventional transfection methods. The targeting of trypanosomes presented in this study is a promising tool for approaching the biology of parasite interactions with host cells, the progression of the diseases they trigger and the screening of new drugs in vivo or in vitro.

Intravenous immunoglobulin increases survival time in the acute phase of experimental Chagas disease.

Chagas disease induced by Trypanosoma cruzi (Tc) infection is an important cause of mortality and morbidity affecting the cardiovascular system for which presently available therapies are insufficient and largely inadequate. Intravenous immunoglobulin (IVIg) is a therapeutic preparation containing normal polyspecific IgG obtained from plasma pools of several thousand healthy donors and is used in several autoimmune, inflammatory and infectious diseases. In the study of heart from mice chronically infected with Tc, we observed that IVIg restores type 1 atrioventricular block or bradycardia. In the present study, we investigated the effects of IVIg in acute Tc infection. Intravenous immunoglobulin administration after the first week of infection was associated with an increase in survival time. Taken together, results observed in the chronic and in the acute phase associate IVIg treatment with a favourable outcome in T. cruzi infection.

Immunotherapy of Trypanosoma cruzi infections.

The protozoan parasite Trypanosoma cruzi, causative agent of Chagas' disease, is transmitted to man and other mammals by triatominae insects, or 'kissing bugs'. Since its discovery in 1909, by Carlos Chagas, this parasite has been the object of several publications in the domains of immunology, cellular biology and of control gene organization, regulation and expression. Although much progress has been made concerning prophylaxis of Chagas' disease, particularly vector eradication, additional cases of infection and disease development still occur every day throughout the world. Whilst infection was largely limited in the past to vector transmission in endemic areas of Latin America, its impact has increased in terms of congenital and blood transmission, transplants and recrudescence following immunosuppressive states. Reports on new insect vectors adapted to the parasite and domestic animals infected in more developed countries, emphasize the continuing worldwide public health issue. Therapy against this parasite is limited and cure is subjected to several criteria, such as susceptibility of the parasite strain, age of the host and stage of the disease. The ability of Trypanosoma cruzi to induce important and various host immune system dysfunctions makes the development of effective vaccines a laborious and complex task. These considerations strengthen the latent significance of Chagas' disease and encourage the search for new preventive procedures and the research on rational vaccines.

Increased Trypanosoma cruzi invasion and heart fibrosis associated with high transforming growth factor beta levels in mice deficient in alpha(2)-macroglobulin.

Trypanosoma cruzi proteinases are involved in host cell invasion in human patients and in mouse models. In mice, murine alpha(2)-macroglobulin (MAM) and murinoglobulin are circulating plasma proteinase inhibitors that also have important roles in inflammation and immune modulation. To define their role in experimental Chagas disease, we investigated the susceptibility to T. cruzi infection of mice that are deficient only in alpha2-macroglobulins (AM-KO) or in both MAM and monomeric murinoglobulin-1 (MM-KO), relative to the wild type (WT). Despite the high parasite load, parasitemia was lower in AM-KO and MM-KO mice than in WT mice. Nevertheless, we observed a significantly higher parasite load in the hearts of AM-KO and MM-KO mice, i.e., more amastigote nests and inflammatory infiltrates than in WT mice. This result demonstrates a protective role for MAM in the acute phase of murine T. cruzi infection. We further demonstrated in vitro that human alpha2-macroglobulins altered the trypomastigote morphology and motility in a dose-dependent way, and that also impaired T. cruzi invasion in cardiomyocytes. Finally, we demonstrated that the levels of transforming growth factor beta in AM-KO mice increased significantly in the third week postinfection, concomitant with high amastigote burden and important fibrosis. Combined, these in vivo and in vitro findings demonstrate that the MAM contribute to the resistance of mice to acute myocarditis induced by experimental T. cruzi infection.

Parasite polyclonal activators: new targets for vaccination approaches?

Taking into consideration that the immune response following infection promotes the expansion of lymphocyte clones that are essentially non-specific, ensuring both parasite evasion and persistence inside the host, what would be the major consequences of this polyclonal response to the development of immunopathology? We favor the hypothesis that the polyclonal B cell responses triggered by the infection is responsible of the host susceptibility and is a major contributor to the maintenance of a progressive disease. In particular, the activation of B cells by parasite mitogens would contribute to the class determination of T cell responses and to the inhibition of macrophages - target cells for parasite multiplication and also responsible for parasite clearance. We also envisage that the activation of T cells by parasite 'superantigens', and the ensuing energy and deletion of these cells, processes that are frequently observed, would contribute for the immunosuppression as well as to parasite escape and persistence in the host. We had concentrated our efforts on the study of the non-specific aspects of the immune response following Trypanosoma cruzi infection. We aimed at finding new strategies to modulate and control the mechanisms leading to both the immunosuppression and the development of chronic auto-immunity leading to rational vaccine approaches against parasite infection and immunopathology.

Significant association between the skewed natural antibody repertoire of Xid mice and resistance to Trypanosoma cruzi infection.

The Xid mutation predominantly affects the development of B cells and consequently the levels and composition of natural antibodies in sera. In contrast to the congenic and susceptible BALB/c strain, immunodeficient BALB.Xid mice display a resistant phenotype both to acute Trypanosoma cruzi infection and to the development of severe cardiopathy. Because natural antibodies are known to be basically self-antigen driven, IgM and IgG natural antibody repertoires (NAR) were compared before and during infection in these two strains. The analysis revealed fundamental alterations of IgM and IgG NAR in pre- and post-infected Xid mice. In particular, relatively increased natural (pre-existing) autoreactive IgG, dominated by the unique recognition of a single band in autologous heart extracts, was typical for uninfected Xid mice. This natural autoreactive IgG directed to heart antigens disappeared early after infection not only in Xid, but also in individual BALB/c mice that survived the acute infection. Conversely, the subgroup of BALB/c mice that died early after infection presented the most pronounced instances of the rapid, relative increase of IgM reactivities to self and non-self proteins. These results suggest that self-reactive NAR may play a role in an immunoregulatory mechanism relevant for the determination of susceptibility/resistance to infections. This may act either by influencing specific responses, or by modulating the self-aggressive components responsible for pathology.

A B-cell mitogen from a pathogenic trypanosome is a eukaryotic proline racemase.

Lymphocyte polyclonal activation is a generalized mechanism of immune evasion among pathogens. In a mouse model of Trypanosoma cruzi infection (American trypanosomiasis), reduced levels of polyclonal lymphocyte responses correlate with resistance to infection and cardiopathy. We report here the characterization of a parasite protein with B-cell mitogenic properties in culture supernatants of infective forms, the cloning of the corresponding gene and the analysis of the biological properties of its product. We characterized the protein as a co-factor-independent proline racemase, and show that its expression as a cytoplasmic and/or membrane-associated protein is life-stage specific. Inhibition studies indicate that availability of the racemase active site is necessary for mitogenic activity. This is the first report to our knowledge of a eukaryotic amino acid racemase gene. Our findings have potential consequences for the development of new immune therapies and drug design against pathogens.

Lymphocyte polyclonal activation: a pitfall for vaccine design against infectious agents.

In this article, Bernardo Reina-San-Martin, Alain Cosson and Paola Minoprio summarize the marked alterations in the immune system functions after infection that might account for the poor success of effective parasite vaccine development. Many of the studies on oligoclonal B- and T-cell responses to parasite antigens aiming at vaccination strategies would seem to ignore more general, and perhaps fundamental, aspects of parasite-immune system interactions. In essence, because of its consequences on immunopathology and parasite escape, the authors ascribe a central importance in the pathogenesis of parasitic diseases to the 'nonspecific' polyclonal lymphocyte activation that occurs during infection. Hence, novel targets and strategies for immune intervention should be considered.

Changes in the cytokine profile of lupus-prone mice (NZB/NZW)F1 induced by Plasmodium chabaudi and their implications in the reversal of clinical symptoms.

We have previously observed that aged lupus-prone (NZB/NZW)Fl (BWF1) mice when infected with Plasmodium chabaudi show an improvement in their clinical lupus-like symptoms. In order to study the mechanisms involved in the long-lasting protective effect of the P. chabaudi infection in lupus-prone mice we analysed specific aspects of the cellular response, namely the profiles of cytokine mRNA expression and cytokine secretion levels in old BWF1 mice, in comparison with uninfected age-matched BWF1 mice and infected or uninfected BALB/c mice. Two months after infection, cells from BWF1 mice were stimulated with concanavalin A (Con A) and demonstrated a recovery of T cell responsiveness that reached the levels obtained with BALB/c cells. Old BWF1 mice showed high levels of interferon-gamma (IFN-gamma) and IL-5 production and correspondingly low levels of IL-2 and IL-4 secretion before infection with P. chabaudi. Infection did not modify the IFN-gamma levels of BWF1 T cells, whereas it considerably increased the secretion of the Th2-related cytokines IL-4, IL-5 and IL-10. In addition, only BWF1 T cells showed increased mRNA expression of tumour necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta (TGF-beta). This counter-regulatory cytokine network of infected BWF1 mice may be involved in the improvement of their lupus symptoms. The results of our investigations using the complex model of P. chabaudi infection can be extended and, by using more restricted approaches, it may be possible to explain the multiple regulatory defects of lupus-prone mice.

Theileria annulata in CD5(+) macrophages and B1 B cells.

Theileria parasites infect and transform bovine leukocytes. We have analyzed laboratory-established Theileria sp.-infected leukocyte lines and observed that transformed macrophages express CD5. Low-level expression of CD5 by macrophages was further confirmed on three independent Theileria annulata clinical isolates from Tunisia. Interestingly, the fourth CD5(+) clinical isolate (MB2) was morphologically different, expressed surface immunoglobulin M (IgM) and BoLA class II, and had rearranged Ig light-chain genes. To demonstrate that MB2 did indeed contain CD5(+) B cells, individual clonal lines were obtained by limiting dilution, and CD5 expression and Ig gene rearrangement were confirmed. This suggests that in natural infections T. annulata can invade and transform CD5(+) B cells.

A Trypanosoma cruzi alkaline antigen induces polyclonal B-cell activation of normal murine spleen cells by T-cell-independent, BCR-directed stimulation.

We have previously reported that a cytosolic alkaline fraction (FI) obtained from epimastigotes of Trypanosoma cruzi promotes the activation, proliferation and differentiation of normal murine B cells into antibody-secreting plasmocytes. Neither the mechanism nor the cells involved in the FI-induced polyclonal B-cell activation were established. In this work we report that accessory cells are required for FI-induced polyclonal B-cell activation as no proliferative responses were obtained following treatment of normal spleen mononuclear cells (NSMC) with L-leucine methyl ester. Furthermore, FI did not induce the expression of CD25 on T cells and it promoted the proliferation of a T-cell-depleted population, indicating that it acts in a T-independent manner. We observed that NSMC were stimulated in vitro by FI-released cytokines, such as interleukin (IL)-4, IL-6 and IL-10, which are involved in B-cell proliferation and differentiation. Interestingly, while significant amounts of interferon-gamma (IFN-gamma) were found in culture supernatants we did not observe detectable levels of IL-2. Additionally, we found that B-cell receptor (BCR) and major histocompatibility complex (MHC) class II antigens were involved in the proliferative response induced by FI because antibodies directed against cell-surface immunoglobulin M (IgM), CD45 and MHC class II molecules inhibited the FI-induced B-cell proliferation. CD40 ligand (CD40L) did not participate in such a phenomenon.

Partial protection of mice against Trypanosoma cruzi after immunizing with the TcY 72 antigenic preparation.

A 72 kDa Trypanosoma cruzi glycoprotein recognized by the 164C11 monoclonal antibody (IgM isotype) was purified by preparative electrophoresis. The antigenic preparation obtained, named TcY 72, was used to immunize C57Bl/10 mice. The following results were observed after immunization: (1) induction of higher titres of IgG than IgM antibodies, as evaluated by indirect immunofluorescence; (2) significant DTH after injection of epimastigotes in mice footpads; (3) peak parasitemia in immunized mice was significantly reduced and animals were negative by 13 days post-infection, although the mice still succumb to infection; (4) the phenotypic analysis of spleen cell populations showed a decrease in the CD4/CD8 ratio in immunized mice. Taken as a whole, these findings indicate that TcY 72 is immunogenic and potentially important for protective immunity.

X-linked immunodeficiency affects the outcome of Schistosoma mansoni infection in the murine model.

The incidence of the X-linked immunodeficiency (Xid) on the outcome of Schistosoma mansoni infection has been evaluated through a comparative analysis of parasitological and immune parameters in two different mouse strains: control BALB/c and BALB. Xid mice which carry the Xid mutation and lack B1 (CD5+ B) cells. This study clearly demonstrates that infected B1 cell-deficient animals display a higher susceptibility to S. mansoni infection as revealed by an increase in the tissue egg loads and a significantly elevated mortality, as well as an increase in the granuloma densities. The analysis of the humoral and the cellular responses, conducted in the same experimental conditions, indicates differences in terms of cytokine production after specific antigenic stimulation of splenocytes. Larger amounts of IFN-gamma and IL-4 are observed in BALB. Xid mice while IL-10 production is reduced. In parallel, the study of the specific antibody isotype profiles shows higher amounts of specific IgE and IgG1 antibodies and lower amounts of IgM and IgA in BALB. Xid mice. Taken together, these observations support the idea that B cells are playing a role in the ability of mice to tolerate infection with Schistosoma mansoni.

A 24,000 MW Trypanosoma cruzi antigen is a B-cell activator.

Trypanosoma cruzi, the causative agent of Chagas' disease, is a protozoan parasite that infects humans and other mammals in Central and Latin America. Several alterations of the immune response after infection have been described, such as severe immunosuppression of both cellular and humoral responses and massive polyclonal B- and T-cell activation, including the expansion of self-reactive clones. We have investigated the effects of the intraperitoneal injection of a recombinant 24,000 MW T. cruzi-specific antigen (rTc24) on the immune response of normal and deficient strains of mice. We analysed the in vivo and ex vivo levels of lymphocyte activation and the proliferative responses to rTc24 by determining the expression of CD69 activation marker and the levels of thymidine incorporation by spleen cells. The numbers of antibody-producing cells were determined by ELISPOT and the levels of immunoglobulin in the sera by isotype-specific enzyme-linked immunosorbent assay. We observed an increased [3H]thymidine ([3H]TdR) incorporation by spleen cells after rTc24 stimulation in vivo and in vitro. This proliferative activity induced by rTc24 was independent of the mouse strain used in the experiments (including C3H/HeJ mice) and ruled out the possibility that rTc24 preparations were contaminated by lipopolysaccharide. The injection of rTc24 protein induced preferentially the activation of B cells, as determined by the increased expression of CD69 molecules on IgM+ spleen cells. Considerable increases of IgM-secreting B cells were determined in both athymic and euthymic BALB/c mice. Mice that are deficient in B cells (BALB.Xid) responded to rTc24 but to a lesser extent. These increases in IgM B-cell numbers were accompanied by elevated levels of IgM immunoglobulins in the sera of injected animals. Our results suggest a role for rTc24 in B-cell activation.

Evidence for a protective role of tumor necrosis factor in the acute phase of Trypanosoma cruzi infection in mice.

A possible role for tumor necrosis factor (TNF) alpha during Trypanosoma cruzi infection was explored by using transgenic mice expressing in blood high levels of a soluble TNFR1-FcIgG3 fusion protein, which neutralizes the effects of TNF in vivo. Nontransgenic littermates were used as controls. The transgenic mice showed high susceptibility to T. cruzi infection. Inocula sublethal for control mice resulted in over 80% mortality associated with higher levels of parasites in the blood. In histological sections of the hearts of transgenic mice, large parasitic clusters without inflammatory cell infiltrates around the parasites were seen, while smaller parasitic clusters associated with leukocytes were seen in control mice. No difference in specific antibody response or lymphocyte composition of the spleen was found between transgenic and control mice, although the unresponsiveness of spleen cells to concanavalin A stimulation in vitro, typical of the acute phase of T. cruzi infection, was less pronounced in transgenic mice. Infected transgenic mice produced higher levels of gamma interferon than did control mice. These results confirm that TNF is involved in mechanisms leading to parasite clearance and protection from death in the acute phase of T. cruzi infection. More importantly, the data reveal that TNF is necessary for the establishment of effective tissue inflammation and parasite load control in acute experimental Chagas' disease myocarditis.

V beta 6-bearing T cells are involved in resistance to Trypanosoma cruzi infection in XID mice.

BALB.xid mice, carrying an X-linked mutation leading to the absence of CD5+ B cells, are highly resistant to Trypanosoma cruzi infection. These mice clear blood parasites in the acute phase of infection and do not develop the inflammatory infiltration characteristically observed in the chronic phase of susceptible strains of mice. We have shown that the resistance of BALB.xid is dependent on the production of high levels of IFN-gamma. Natural (adoptive foster) or artificial (in vivo injection of blocking antibodies) treatments of BALB.xid induced deletion of CD4+ and CD8+ cells bearing V beta 6 TCR. The absence of V beta 6 lymphocytes considerably reduced resistance to infection. Furthermore, in BALB.xid lacking this minor fraction of the T cell repertoire, almost 50% of the IFN-gamma production is lost. This indicates that V beta 6-bearing T cells are either directly or indirectly involved in the production of IFN-gamma and, thus, important for an effective immune response during the acute phase of experimental Chagas' disease.

Murine acariasis. II. Immunological dysfunction and evidence for chronic activation of Th-2 lymphocytes.

The authors describe the immunological profile of BALB/c mice with Mite-Associated Ulcerative Dermatitis (MAUD)-like disease, due to Myocoptes musculinus (Koch 1844) infestation. The disease probably involves allergic mechanisms and is characterized by erythematous and pruritic skin lesions, widespread hair loss, lymphadenopathy, lymphocytopenia, granulocytosis and wasting. Affected individuals had much reduced numbers of pre-B and B cells in bone marrow and B cells in blood; decreased T-cell numbers in peripheral lymphoid organs and blood; hypergammaglobulinaemia with selective increases of IgG1, IgE and IgA, and depletion on IgM and IgG3, the same isotype distribution being detected in splenic plasmocytes; qualitative modifications of the serum antibody reactivity pattern; and increased production of IL-4 with decreased IL-2 production after in vitro polyclonal stimulation of T cells. Taken together, these results suggest that infestation by M. musculinus in BALB/c mice leads to a significant immunological disorder resulting in a T-helper-2 (Th-2) type response, with marked systemic consequences. This pathological condition may thus provide a useful model system for the immunobiological perturbation associated with chronic allergic disease.

Ig-isotype patterns of primary and secondary B cell responses to Plasmodium chabaudi chabaudi correlate with IFN-gamma and IL-4 cytokine production with CD45RB expression by CD4+ spleen cells.

In this work, the authors analysed T and B lymphocyte subsets and cytokine production in the spleen of BALB/c mice during polyclonal lymphocyte activation (primary infection) and parasite-specific response to Plasmodium chabaudi chabaudi (secondary infection). The secondary response was evaluated in fully immunoprotected animals, 60 days after a chloroquine-cured infection. The authors observed that in polyclonal lymphocyte activation antibody-secreting cells of all isotypes increased, with predominance of IgG2a and IgG3 classes. At that time, IFN-gamma was largely produced, but IL-4/IL-5 were just slightly enhanced. In mice re-infected after 60 days, the Ig-isotype pattern was restricted to IgG1 and only IL-4/IL-5 were produced. In both responses, however, the levels of IL-2 were greatly reduced, while those of IL-10 were enhanced to similar levels. The different involvement of Th1 and Th2 cells in both responses was confirmed through analysis of CD45RB expression by CD4+ cells. The authors observed that CD45RBhigh cells were the major CD4+ subpopulation in primary infected mice, while CD45RBlow cells predominated in 60 days re-infected animals. Moreover, the great majority of activated (large) CD4+ cells in the primary infection belonged to the CD45RBhigh subset, while after reinfection most of the CD4+ large had a CD45RBlow phenotype.

The influence of T cell subsets on Trypanosoma cruzi multiplication in different organs.

Several studies in mice have strengthened the active role played either by CD4+, CD8+ or both T cell subsets in conferring resistance to Trypanosoma cruzi infection. To date, no studies reported the role played by T cell subsets on parasite multiplication in different organs. In the present work, mice were infected with CL strain of T. cruzi and T cell subset activities were blocked by i.p. injection of monoclonal antibody (mAb) directed against CD4, or IAk, or CD8 molecules. The effect of these treatments was determined by counting the number of parasite nests in heart and liver sections 16 days after infection. Our results showed that mice treated with anti-CD4 or anti-IAk mAbs presented a significant increase in the parasite load in the hearts and in the livers. Conversely, the number of parasites in hearts of anti-CD8 treated mice did not increase significantly. This treatment, however, resulted in a 20-fold increase in the number of parasites found in the liver. Simultaneous depletion of both T cell subsets by treatment of mice with anti-CD4/CD8 mAbs had, in the heart, the same effect as the CD4 depletion. Interestingly, this treatment caused a dramatic increase (200-fold) in the T. cruzi parasitism of the liver. These results indicate that the activity of T cell subsets against T. cruzi varies according to the infected organ.

Intranasal inoculation of Bordetella bronchiseptica in mice induces long-lasting antibody and T-cell mediated immune responses.

Humoral and cellular immune responses were analysed in mice inoculated intranasally with Bordetella bronchiseptica. After infection, the number of bacteria that colonized the respiratory tract of the mice increased during the first day and decreased thereafter. Total IgG levels increased as early as 14 days after infection and decreased with time after infection, whereas total IgA and IgM levels were lower but remained stable. Specific antibodies to the bacteria were mainly IgG2a and IgA and persisted up to 10 months after infection. Some of these specific antibodies were directed against adenylate cyclase-haemolysin, the bacterial factor that had been shown to be necessary for initiation of infection. The proliferation of Bordetella bronchiseptica-reactive spleen cells occurred during the acute phase of infection. T cells from infected mice produced increasing amounts of IFN gamma and IL-2 after infection. Although very low levels of IL-10 were produced, no IL-4 was detected after bacterial stimulation in vitro. These results suggest that Bordetella bronchiseptica infection induces primarily a Th1-type T-cell response. Importantly, the authors demonstrated that antibody and T-cell responses directed against bacterial determinants of the virulent strain and to purified adenylate cyclase-haemolysin were long-lasting. This observation could be due to the fact that Bordetella bronchiseptica may persist intracellularly in the host as it was demonstrated in vitro.