Activation of CD4+ and CD8+ parasite -specific T-cells by macrophages infected with live T. cruzi amastigotes.

T. cruzi-infected macrophages are potential candidates for the presentation of parasite antigens to T. cruzi-specific T lymphocytes. To assess this question, we examine the ability of peritoneal exudate macrophages to process exogenous live or dead parasites and to activate defined populations of T. cruzi-specific immune T-cells. Macrophages infected with live amastigotes activated both lymph node CD4+ and spleen CD8 + T-primed cells that proliferated and secreted cytokines. Lymph node CD4+ T-cells produced IFN-gamma and IL-10 while CD8 + T-cells produced IFN-gamma. In contrast, macrophages pulsed with dead parasites activated only lymph node CD4+ T-cells, which proliferated and secreted IFN-gamma. Interestingly, the immunization with heat-killed parasites primed mice for CD8+ T-cells which were expanded in vitro by recognition of infected macrophages. Taken together, these results demonstrated that amastigote infected macrophages present parasite peptides associated with MHC I and II molecules, activating both CD4 + and CD8+ T-cells. Furthermore, the development of T. cruzi-specific CD8+ T-cells in vivo using the immunization protocol with non-living parasites as described in this report could be explored for further studies on the role of CTL in the outcome of infection.

Purification of F(ab')2 anti-snake venom by caprylic acid: a fast method for obtaining IgG fragments with high neutralization activity, purity and yield.

Pooled horse plasma containing antibodies against Crotalus durissus terrificus whole venom were digested with pepsin at an enzyme-substrate ratio of 8:1, pH 3.1, for 40 min and the F(ab')2M fragments purified by adding 8.7% caprylic acid (pH 5.0). For comparison, F(ab')2B purified by precipitation with ammonium sulphate and uncleaved IgG purified with caprylic acid were also prepared. Fab' fragments were obtained by reduction and alkylation of F(ab')2B. The anti-whole C.d. terrificus venom titers, determined by Dot-Blot were 12,800 (IgG), 6400 [F(ab')2B], 4800 [F(ab')2M] and 3200 (Fab'B). Immunochemical analysis of these fragments by SDS gel electrophoresis, Western blot and by double immunodiffusion revealed that the solution containing F(ab')2M was free of IgG and of other plasma proteins, whereas that containing F(ab')2B was not. One milligram of either F(ab')2B, F(ab')2M or Fab'B was able to neutralize respectively 20.7 micrograms, 20.2 micrograms and 13.8 micrograms of C.d. terrificus venom.

Experimental paracoccidioidomycosis: early immunosuppression occurs in susceptible mice after infection with pathogenic fungi.

The course of Paracoccidioides brasiliensis infection depends on the natural resistance of the host and on the virulence of the fungus isolate. In the present study the immune response to sheep red blood cells (SRBC) was investigated in susceptible (B10.A) and resistant (A/SN) mice after intraperitoneal (ip) infection with pathogenic (Pb18) and apathogenic (IVIC Pb267) P. brasiliensis isolates. Infection with Pb18 yeast cells quickly induces a state of immunodepression only in the susceptible mice. This immunosuppression was not observed when mice were inoculated with IVIC Pb267 yeast.

Characterization of the spleen B-cell compartment at the early and late blood-stage Plasmodium chabaudi malaria.

Polyclonal B-cell activation is a feature of the early spleen cell response to blood-stage Plasmodium chabaudi malaria. Immunity to blood-stage malaria is guaranteed by the generation of B cells able to produce parasite-specific antibodies mainly from the immunoglobulin (Ig)G2a isotype. In the present study, we characterized the spleen B-cell compartment during blood-stage P. chabaudi infection. The numbers of B220(+) and B220(LOW) CD138(+) (plasma) cells increased sharply between days 4 and 7 post-infection (p.i.). At this time B220(+) cells expressed surface (s)IgM, but nearly all B220(LOW) CD138(+) cells showed concomitantly intracellular (i)IgM and IgG2a. Both follicular and marginal zone B cells were activated expressing high amounts of CD69. At day 40 p.i., B220(LOW) CD138(+) cell population was still increased but, differently from acute infection, 61.1% of these cells were positive for iIgG2a while only 14.2% expressed iIgM. Moreover, at days 20 and 40 p.i., 29.2% and 13.0% of B220(+) cells expressed sIgG2a, respectively. According to cell size and expression of CD80, CD86, CD11b, CD44 and CD38, B220(+) sIgG2a(+) cells had a phenotype characteristic of activated/memory B cells. Furthermore, 14.1% of B220(+) sIgG2a(+) cells at day 30 p.i. expressed a marginal zone B-cell phenotype. Importantly, B cells from 40-day-infected mice were very efficient in presenting parasite antigens leading to proliferation of both CD4(+) and CD8(+) cells. Our results contribute for understanding the dynamics of B cells during P. chabaudi infection, underlying the mechanisms of antigen presentation and antibody production, which are essential for the acquisition of protective immunity against malaria.

Persistence of polyclonal B cell activation with undetectable parasitemia in late stages of experimental Chagas' disease.

The polyclonal B cell responses induced by Trypanosoma cruzi infection last for at least 6 mo after the inoculation of the parasites. In the acute phase of the disease, B cells from spleen and lymph nodes are largely stimulated, whereas a decrease in bone marrow PFC is observed. As the disease progresses, the numbers of Ig-secreting cells in the spleen, lymph nodes, and bone marrow are all enhanced. The isotype distribution of PFC, however, remains unvariable along the course of the infection, and it is characterized by the predominance of IgG2a- and IgG2b-secreting cells. No striking difference in the isotype pattern of resistant and susceptible strains of mice was observed. The continuous and long-lasting B cell stimulation generated during the infection may have important consequences in the pathology of Chagas' disease.

Polyclonal lymphocyte responses to murine Trypanosoma cruzi infection. II. Cytotoxic T lymphocytes.

Intraperitoneal infection of young adult C57BL/6 males with 10(5) blood or cloned culture forms of Trypanosoma cruzi (CL strain) induced the appearance in spleen, blood, and lymph nodes of cytotoxic effector cells detectable in a lectin-dependent 51Cr-release assay. The effector cells were conventional cytotoxic T lymphocytes (CTL), since they were Thy 1+ and Lyt 2+, and the lysis of tumour target cells was strictly dependent on the presence of lectin. CTL activity is already detectable in spleen 2 days after infection, reaches a peak at 2 weeks, and returns to normal levels during the chronic phase (1 month onwards). Increased levels of CTL activity were also detected in lymph nodes with similar kinetics, even in animals that were splenectomized prior to infection. In contrast to spleen, significant levels of CTL activity persisted in lymph nodes in the chronic phases. This functional variable correlates with the appearance of high numbers of large Lyt 2+ lymphocytes in the same organs (50 to 100-fold higher than in control, uninfected mice). Very similar responses are detected in a T. cruzi sensitive mouse strain (C3H/HeJ). It appears, therefore, that T. cruzi infection results in a large polyclonal activation of Lyt 2+ lymphocytes, some of which differentiate to effector, cytolytic functions.

Influence of acute-phase parasite load on pathology, parasitism, and activation of the immune system at the late chronic phase of Chagas' disease.

To obtain low and high parasite loads in the acute phase of Chagas' disease, A/J mice were infected with 10(3) or 10(5) Trypanosoma cruzi trypomastigotes of the Y strain and treated on day 6 with benznidazol. One year later, chronically infected mice were screened for subpatent parasitemias, tissue pathology, and immune response. Mice infected with the high parasite inoculum showed higher levels of chronic parasitemias, heart and striated muscle inflammation, and activation of the immune system than did mice infected with the low inoculum. Concerning the activation of the immune system, the main findings for high-dose-infected mice were (i) increased numbers of splenocytes, with preferential expansion of CD8(+) and B220(-) CD5(-) cells, many of them bearing a macrophage phenotype; (ii) higher frequencies of B (B220(+)), CD4(+), and CD8(+) large lymphocytes; (iii) a shift of CD4(+) cells towards a CD45RBLow phenotype; (iv) increased frequencies of both CD45RBLow and CD45RBHigh large CD4(+) cells; (v) augmented numbers of total immunoglobulin (Ig)-secreting cells, with predominance of IgG2a-producing cells; and (vi) increased production of gamma interferon and interleukin 4. In addition, these mice presented lower IgM and higher IgG2a and IgG1 parasite-specific serum antibody levels. Our results indicate that the parasite load at the acute phase of T. cruzi infection influences the activation of the immune system and development of Chagas' disease pathology at the late chronic phase of the disease.

Influence of the polyclonal activation induced by Plasmodium chabaudi on ongoing OVA-specific B- and T-cell responses.

Infection by Plasmodium chabaudi results in polyclonal activation, massive proliferation and differentiation of lymphocytes with parasite-unrelated specificities. To verify if polyclonal activation includes experienced B and T lymphocytes and if it modifies pre-established cytokine and Ig-isotype patterns, mice were immunized with ovalbumin (OVA) in alum, a condition that favours T helper 2/immunoglobulin G1 (Th2/IgG1) responses, and infected with P. chabaudi 7 or 80 days later. Polyclonal activation markedly increased the number of anti-OVA Ig-secreting cells in the spleen, an effect more patent in mice infected 7 days after OVA immunization, but also evident in mice infected after 80 days. The Ig-isotype profile predefined by immunization was not qualitatively modified by polyclonal activation. Thus, although P. chabaudi infection preferentially induces IgG2a, the expanded anti-OVA response is dominated by IgG1. Polyclonal expansion of the anti-OVA response did not yield an enlarged memory B-cell pool that could be recalled months later by OVA boosting. Moreover, polyclonal activation of anti-OVA IgG1-secreting cells did not increase this antibody in serum, a probable consequence of the high Ig turnover observed during infection. When OVA-specific T-cell cytokines were evaluated, we observed an increase of both interleukin-4 (IL-4) and interferon-gamma (IFN-gamma) in mice infected 7 days after immunization, whereas in those infected after 80 days, only IL-4 was augmented. These results suggest that polyclonal activation expands experienced B- and T-cell compartments, preserving their antibody and cytokine patterns.

Very large and isotypically atypical polyclonal plaque-forming cell responses in mice infected with Trypanosoma cruzi.

Normal C3H/HeJ mice, acutely infected with T. cruzi, develop large numbers of splenic Ig-secreting plaque-forming cells (PFC). IgG2a, IgG2b and IgG1 PFC account for over 90% of all PFC, while the numbers of IgG3- and IgA-secreting PFC are lower than in normal animals. These effects appear to be due to both T helper-dependent regulation and to a mitogenic activity associated with the parasites themselves.

Isotypic pattern of the polyclonal B cell response during primary infection by Plasmodium chabaudi and in immune-protected mice.

The primary infection by P. chabaudi induces an increase of the numbers of splenic immunoglobulin (Ig)-secreting B cells in both athymic and euthymic BALB/c mice. The isotypic pattern of the polyclonal response is restricted only in euthymic mice where IgG2a, IgG2b and IgM plaque-forming cells (PFC) predominate. In immunized animals, protected against a parasite challenge, the isotypic pattern of splenic PFC is completely different, the IgG1 and IgM isotypes constituting the main part of the response. Reinoculation of immune-protected animals induces a PFC response which is dose dependent and accentuates the characteristic isotypic profile of the immune-protected mice.

Polyclonal lymphocyte responses to murine Trypanosoma cruzi infection. I. Quantitation of both T- and B-cell responses.

Lymphoid activity was studied in spleen and lymph node cells from Trypanosoma cruzi-infected mice. Blast transformation in each lymphocyte class was assessed by dual parameter analysis for size and surface markers by both FACS and conventional immunofluorescence, while proliferative activity was measured by tritiated thymidine uptake, autoradiography, and analysis of DNA content in single cells. Acute infection results in rapid blast transformation and proliferative activity of all three lymphocyte classes (Ig+, L3T4+, and Lyt 2+). At 2 weeks of infection most cells in these organs are enlarged and more than half are dividing. By 2 and 6 months after infection (chronic phase of resistant strains), large numbers of activated B lymphocytes and, to a lesser extent, of Lyt 2+ T cells are still detected. Similar results were obtained in C57BL/6 (resistant) and C3H/HeJ (susceptible) mouse strains. The implications of this massive polyclonal lymphocyte response to the parasite for the physiopathology of acute and chronic infection are discussed.

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.

Susceptibility of the different developmental stages of the asexual (schizogonic) erythrocyte cycle of Plasmodium chabaudi chabaudi to hyperimmune serum, immunoglobulin (Ig)G1, IgG2a and F(ab')2 fragments.

The mechanisms by which antibodies interfere with Plasmodium growth are still under debate. Characterizing the asexual erythrocyte stages susceptible to antibodies from hyperimmune individuals is therefore a relevant contribution to vaccine research. In this study, using a virulent and synchronous murine malaria parasite, Plasmodium chabaudi chabaudi AJ, we have shown that trophozoites and circulating schizonts are not the main targets for antibodies from hyperimmune serum. In drug-cured mice challenged with a high inoculum of ring-infected erythrocytes, parasitemias do not decline until the moment of erythrocyte rupture, suggesting that effector mechanisms operate immediately prior to reinvasion. Confirming these findings, treatment of primary-infected mice with hyperimmune serum inhibited the generation of new ring forms, but did not alter the numbers of schizont-infected erythrocytes, despite the fact that these cells were recognized by immunoglobulin (Ig)G antibodies. When these mice were treated with IgG1 or IgG2a purified from hyperimmune serum, both subclasses limited reinvasion, but IgG2a showed a stronger protective activity. The fact that Fc digestion decreases but does not abrogate protection suggests that both Fc-dependent and independent mechanisms participate in this process. Treatment with cobra venom factor did not interfere with the antibody-mediated protection, ruling out the participation of the complement system in both lysis and phagocytosis of merozoites or infected erythrocytes. Therefore, in mice suffering from P. c. chabaudi AJ malaria, merozoite neutralization seems to be a major mechanism of protection conferred by hyperimmune serum antibodies. However, FcgammaR-mediated interactions, or other mechanisms not yet defined, may also contribute to inhibit erythrocyte reinvasion.

Most parasite-specific CD8+ cells in Trypanosoma cruzi-infected chronic mice are down-regulated for T-cell receptor-alphabeta and CD8 molecules.

The present study shows that CD8+ T lymphocytes expressing low levels of T-cell receptor (TCR)alphabeta, CD8 and CD3 accumulate in the spleen, blood, peritoneum and liver, but not in the lymph nodes of mice chronically infected with Trypanosoma cruzi. Analysis of spleen lymphocytes reveals that most CD8LOW TCRLOW T cells have an experienced phenotype (CD44HIGH CD62LLOW and CD45RA,B,CLOW). These cells have small size, lack activation markers such as CD69, CD25 and CD11b (Mac-1), and do not spontaneously secrete cytokines, suggesting they are at the resting state. When stimulated in vitro with T. cruzi-infected macrophages, TCRLOW CD8LOW T cells behave as parasite-specific memory cells, readily responding with interferon-gamma (IFN-gamma) production. Indeed, among parasite-activated CD8+ lymphocytes, IFN-gamma production was mostly due to TCRLOW CD8LOW cells. Upon in vitro stimulation with anti-CD3/CD28 monoclonal antibodies, down-regulated cells produce IFN-gamma and tumour necrosis factor-alpha, but not interleukin IL-10 or IL-4. Our results indicate that despite parasite persistence, most T. cruzi-specific experienced CD8+ cells are resting. Nevertheless, when encountering infected macrophages these cells differentiate to Tc1 effectors.

A new mutant hairless mouse with limph node hyperplasia and late onset of autoimmune pathology

Adult BALB/c male mice were injected with a single dose of ethyl nitroso urea (ENU; 250 mg/Kg, ip) and mated to C57BL/6, DBA/2 AND A/J adult females 13 weeks later. F1 males were mated with BALB/c females and F2 females were then backcrossed to the F1 parents. One BALB/c male mouse thus teated gave origin to a mutant presenting hair and skin alterations similar to those of natural hairless mutants. The new mutation is located on chromosome 14 near the Es10 locus, and probably at the same locus for the hairless mutation. Similar to the hairless mouse, this new mutant has a normal phenotype at birth and after three weeks starts to loose hair which is never replaced. Additionally, the skin becomes thickened and wrinkled. One feature that distinguishes this mutant from other hairless mice is the peculiar enlargement of its axillary and cervical lymph nodes. The new mutant develops membranoproliferative glomerulonephritis similar to the rhino mouse, one of the hariless allele mutants already described in the literature, but with a much later onset