细菌脂多糖促进克氏锥虫抗原诱导的自身免疫性心肌炎

目的观察细菌脂多糖（LPS）对克氏锥虫抗原诱导自身免疫性心肌炎的影响。方法克氏锥虫抗原辅以完全弗氏佐剂免疫A／J小鼠，LPS经腹腔接种方式给药，其后加强免疫2次，28d后，测定心肌肌球蛋白特异的迟发型超敏反应（DTH）及自身抗体，并取鼠心脏观察心肌炎症情况。结果LPS提高锥虫抗原诱导的心肌肌球蛋白特异性DTH及自身抗体。并且25μg LPS与锥虫抗原免疫的小鼠可见明显的心肌炎。结论LPS能够促进心肌肌球蛋白特异的自身免疫反应及锥虫抗原诱导的自身免疫性心肌炎。     

Initial induction of immunity, followed by suppression of responses to parasite antigens during Trypanosoma cruzi infection of mice

Infection of a relatively resistant strain of mice (C57BL/6J) with the protozoan parasite Trypanosoma cruzi results in both the induction of parasite-specific T-helper cells and nonspecific suppressor cells. A time course study of the activation of help and suppression revealed that parasite-specific T-helper cell activity increases very early in infection (less than 12 days) at a time when suppression of non-parasite-specific responses and suppressor cell activity is increasing. Between 12 and 14 days of infection, the T-helper cell response to T. cruzi, as measured by the antibody response to hapten-T. cruzi in vitro, is suddenly and dramatically regulated. As reported previously, plastic and G-10 adherent cells appear to be responsible for the regulation of antibody responses to heterologous antigen during T. cruzi infection. These adherent suppressor cells are also responsible for the suppression of antibody responses to hapten-T. cruzi following the first 2 weeks of infection. Suppressor cells continue to regulate the parasite-specific response well into chronic infection even though the response to hapten-T. cruzi appears to return to normal levels. These results are the first to directly implicate nonspecific suppressor cells in the regulation of anti-T. cruzi humoral immune responses.     

Modulation of T-cell responsiveness during Trypanosoma cruzi infection: analysis in different lymphoid compartments

Spleen and lymph node cells of Trypanosoma cruzi-infected mice were studied for mitogen-induced responsiveness in terms of proliferation and lymphokine production (IL-2, IFN-gamma). Splenocyte (SP) as well as lymph node cell (LN) proliferation and IL-2 production were depressed during the acute phase of the infection. Proliferative capacity of LN cells recovered completely and that of SP partially during the chronic phase. In contrast to these suppressive effects, the mitogen-induced IFN-gamma response was enhanced. In vitro co-incubation of normal SP or LN cells with trypomastigotes resulted in a reduced mitogen-induced cell proliferation and IL-2 secretion, similar to those seen with cells taken from infected mice. In contrast, trypomastigotes exerted a stimulatory activity on the mitogen-induced IFN-gamma response of both SP and LN cells. Addition of lymph node cells from T. cruzi-infected mice (LN-I) to lymph node cells of control mice (LN-C) suppressed strongly the mitogen-induced responsiveness of such cocultures. A marginal level of suppression was recorded in cocultures of spleen cells from infected mice (SP-I) and control spleen cells (SP-C). The potent suppressive cells within LN-I populations were identified as macrophage-like and such cells were absent in SP-C and peritoneal exudate cells from T. cruzi infected animals.     

Trypanosoma cruzi variants with reduced virulence obtained by mutagenesis

Summary Previous reports have indicated that by mutagen treatment of mouse tumour cells in vitro it is possible to obtain at high frequency stable tumour cell variants that fail to form tumours in syngeneic mice because of increased immunogenicity. By analogy with these tumour cell variants, we examined whether variants with reduced virulence could be obtained by mutagen treatment of trypomastigotes derived from a Trypanosoma cruzi strain that was adapted to culture and produced lethal infections in DBA/2 mice at a dose of 5 × 10⁴ parasites. A very large frequency of T. cruzi clones were obtained that failed to provoke an acute lethal infection after injection of 5 × 10⁵ parasites. Most of these variants with reduced virulence (vir^-) multiplied actively in normal mice until day 8 after injection. After that time the parasitaemia decreased gradually. For most variants a low level of residual parasitaemia persisted for more than 100 days. Unlike the situation encountered with mouse tumour cell variants it was not possible to demonstrate the presence of new antigens on the T. cruzi vir^- variants. However, these variants seemed to have acquired an increased immunogenicity since they provoked the rejection of virulent parasites injected concomitantly. Mice that had been immunized with living vir^- clones were protected against a challenge with a virulent clone derived from the original parasite population.     

Antibody-dependent cytotoxicity of Trypanosoma cruzi antigen-coated mouse cell lines by eosinophils and neutrophils

Eosinophils and neutrophils are shown to be cytotoxic against two syngeneic mouse cell lines cells when these are coated with T. cruzi antigen and anti-T. cruzi antibody. Activity is detected within 5 h of incubation. Highest levels of cytotoxicity are obtained at antibody dilutions of 1:100 and 1:1000, while antiserum at 1:10 is shown to be inhibitory. Eosinophils show significant activity at an effector to target ratio of 5:1. No cytotoxicity occurs in the absence of either antigen, antibody or effector cells. This phenomenon may be a model for the tissue destruction in acute T. cruzi infection, where the lysis of trypanosomes may lead to antigen coating of host cells, followed by antibody-dependent granulocyte-mediated cytotoxicity of the host cells.     

Melatonin treatment reduces the severity of experimental Trypanosoma cruzi infection

Prior studies show that melatonin enhances the immune response. This study investigated the possible therapeutic effects of melatonin during the course of Trypanosoma cruzi infection. T. cruzi-infected male Wistar rats were orally treated with 5 mg/kg body weight/day of melatonin. Animals treated with melatonin showed a significant reduction in the number of blood trypomastigotes during the acute phase of infection compared with untreated animals (P<0.05). A significant increase in leucocytes numbers during the peak of parasitaemia was also observed (P<0.05). Moreover, both prior and concomitant treatment with melatonin increased interleukin-2 levels, especially 9 days postinfection (P<0.05). Histopathological observations of heart tissue revealed that melatonin administration also resulted in fewer and smaller amastigote burdens, and less inflammatory infiltrate and tissue disorganization, indicating a reduced parasitism of this tissue. These results show that melatonin is effective in controlling parasite replication and suggest that melatonin might serve as an effective therapeutic agent in the treatment of American trypanosomiasis.     

Trypanosoma cruz/-induced decrease in the level of interferon-7 receptor expression by resting and activated human blood lymphocytes

A substantial proportion of human peripheral blood mononuclear cells (PBMC) manifested a decreased capacity to express membrane interferon-γ receptors (IFN-γR) when co-cultured with Trypanosoma cruzi. Among the lymphocytes, B cells accounted for the bulk of this effect, evidenced by a marked drop in the proportion of CD19+ or CD20+ cells expressing IFN-γR. Decreased IFN-γR expression by B lymphocytes was seen as early as 3 h after co-culture with T. cruzi and persisted for at least 24 h. The parasite had no detectable effect on CD19, CD20 or DR antigen expression by B lymphocytes. Neither the proportion ofB cells expressing these markers nor the membrane density of these molecules varied significantly in the presence of T. cruzi. In PBMC cultures stimulated with Staphlyococcus aureus Cowan I (SACI), T. cruzi decreased the percentages of both IFN-γR+ and IFN-R +^bnght (cells expressing above-normal levels of surface IFN-γR) B lymphocytes. Cell-free filtrates of T. cruzi suspensions reproduced the suppressive effects of living parasites on IFN-γR expression by B cells. When T. cruzi was present, the intracellular levels of IFN-γR molecules in resting or SACI-activated B lymphocytes, represented by fluorescence intensity, were well below control values, suggesting that decreased surface expression resulted from suppressed IFN-γR synthesis. Among T (CD3+) cells, 10–8% to 39–6% (7 donors) expressed surface IFN-γR and did so at a very low level. These percentages were also reduced by T. cruzi. If occurring in the host, downregulated expression of IFN-γR could curtail the utilization of IFN-γ, known to play a critical role in host defence against T. cruzi infection.     

Enhanced protection by melatonin and meloxicam combination in experimental infection by Trypanosoma cruzi

The aim of this study was to evaluate a possible synergism between melatonin and meloxicam in up‐regulating the immune response in male Wistar rats infected with Trypanosoma cruzi during immunosuppression phenomenon, which characterizes the acute phase of the Chagas’ disease. Male Wistar rats were infected with the Y strain of T. cruzi. Experiments were performed on 7, 14 and 21 days post‐infection. Several immunological parameters were evaluated including γ‐interferon (IFN‐γ), interleukin‐2 (IL‐2), nitric oxide (NO) and prostaglandin E₂ (PGE₂). The combined treatment with melatonin and meloxicam significantly enhanced the release of IL‐2 and INF‐γ into animals’ serum, when compared with the infected control groups during the course of infection. Furthermore, the blockade of PGE₂ synthesis and the increased release of NO by macrophage cells from T. cruzi‐infected animals contributed to regulate the production of Th1 subset cytokines significantly reducing the parasitaemia in animals treated with the combination of both substances. Therefore, our results suggest that the association of melatonin and meloxicam was more effective in protecting animals against the harmful actions of T. cruzi infection as compared with the treatments of meloxicam or melatonin alone.     

Effect of protective and non-protective antibodies in the phagocytosis rate of Trypanosoma cruzi blood forms by mouse peritoneal macrophages

The phagocytosis of Trypanosoma cruzi blood forms by mouse peritoneal macrophages is significantly enhanced by sera from chronic chagasic patients, rabbits and mice presenting 'lytic antibodies' (LA) which are associated with resistance and active infections as well as 'conventional serology antibodies' (CSA) which are immunoglobulins involved in the positivity of serological diagnostic tests. The phagocytosis rate, however, is not influenced by sera from mice immunized with T. cruzi antigen or chagasic patients submitted to specific treatment, both displaying only CSA but not LA. The efficacy of LA in increasing phagocytosis is related to their ability to bind to epitopes of living trypomastigotes, a property lacking in CSA that bind only to fixed parasites. This phenomenon is apparently the reason for the low effectiveness of antigens used for vaccination in Chagas' disease which only induce CSA, immunoglobulins apparently unable to mediate a number of regular effector immune mechanisms such as complement-mediated lysis, antibody-dependent cell cytotoxicity and phagocytosis.     

Specific suppression of delayed hypersensitivity expression to Trypanosoma cruzi in mice

The absence of cutaneous delayed type hypersensitivity (DTH) expression was investigated in Trypanosoma cruzi infected mice. Neither spleen cells nor peritoneal exudate cells from infected mice transferred DTH to normal recipients in local or systemic adoptive transfer experiments. Expression of DTH in T. cruzi immunized mice was suppressed specifically by Thy-1 negative spleen cells from acutely and subacutely infected animals. Suppression was observed only upon systemic transfer, but not when infected mice spleen cells were added to DTH effector cells and transferred to normal recipients. These results suggest that cutaneous DTH expression in acutely infected mice, might be blocked by mechanisms other than those described for suppression of lymphocyte proliferation and of DTH induction to T. cruzi.     

Changes in the mouse sciatic nerve action potential after epineural injection of sera from Trypanosoma cruzi infected mice

Pathology of chronic Chagas' disease involves peripheral nervous system (PNS) compromise. A high prevalence of antibodies reacting with nervous system antigens has been found in the sera of patients and infected animals, although their physiological role in mediating PNS tissue damage is unknown. Here, we demonstrate that epineural injection of sera from Trypanosoma cruzi infected mice affects the characteristics of the sciatic nerve action potential (SNAP) depending on the parasite strain. Sera from mice infected with the reticulotropic/neurotropic RA strain with reactivity against sciatic nerve (RA/Ne+ sera) induced delays on latency and diminished amplitudes 4 days after injection. Sera from mice infected with the myotropic CA-I strain failed to affect SNAP. Purified immunoglobulin (Ig)G from RA/Ne+ also diminished the amplitude of SNAP. Deposits of IgG labelling axonal fibres and/or myelin sheaths were detected in nerves injected with RA/Ne+ sera. No major histological damage or parasite DNA was found in those nerves. The SNAP changes after sera injection were similar to those observed in mice injected with trypomastigotes in the epineurum 17 days before and in chronically infected animals. This investigation suggests that autoantibodies triggered as a consequence of T. cruzi infection are able to mediate, at least in part, the electrophysiological abnormalities observed in PNS during the course of Chagas' disease.     

Role of placental alkaline phosphatase in the internalization of trypomatigotes of Trypanosoma cruzi into HEp2 cells

BACKGROUND: In vitro, Trypanosoma cruzi invades a wide variety of mammalian cells by an unique process that is still poorly understood. Trypomastigotes adhere to specific receptors on the outer membrane of host cells before intracellular invasion, causing calcium ion mobilization and rearrangement of host cell microfilaments. OBJECTIVE: To test if placental alkaline phosphatase (PLAP), a trophoblast plasma membrane protein anchored by a glycosylphosphatidylinositol molecule, is involved in the transplacental transmission of this parasite. METHOD: We cultured HEp2 cells with the parasite and studied PLAP and actin microfilaments. The results were correlated with invasion rate. RESULTS: Human HEp2 tumour cells express PLAP. HEp2 cells infected with trypomastigotes showed alteration in their alkaline phosphatase activity and a different pattern of actin organization, compared to control cells. Perturbation of PLAP from HEp2 cells before infection with T. cruzi trypomastigotes decreased the invasion rate. CONCLUSION: Placental alkaline phosphatase could be involved in the internalization of T. cruzi into HEp2 cells, via activation of tyrosine kinase and rearrangement of actin microfilaments.     

Influence of melatonin therapy and orchiectomy on T cell subsets in male Wistar rats infected with Trypanosoma cruzi

Abstract:  Gonadal steroids exert an important influence on the host immune response during infection. Changes resulting from the absence or replacement of gonadal hormones may represent a distinct evolution of a particular parasite. Taking into account the greater susceptibility of males to parasites, the magnitude of the immune response seems to depend on the interaction of many hormones that will act synergistically with other immune cells. The aims of this research were to evaluate the effects of the luck of male sex hormones due to orchiectomy, and the influence of oral administration of melatonin on the immune response of male Wistar rats infected with the Y strain of Trypanosoma cruzi . The percentage of CD3⁺ CD4⁺ and CD3⁺ CD8⁺ lymphocyte T cell subsets were evaluated using flow cytometry and the measurement of IL-2 and IL-12. For all parameters examined, a synergistic action of melatonin and orchiectomy on the host's immune response was observed, promoting an effective response against the parasite during the acute phase of infection. These results offer insight into other possibilities for possibly controlling T. cruzi proliferation through melatonin therapy and also the stimulatory effects on host's immune response triggered by the absence of male gonadal steroids during the acute phase of infection.     

Susceptible mice present higher macrophage activation than resistant mice during infections with myotropic strains of Trypanosoma cruzi

The kinetics of macrophage activation were compared among inbred strains of mice (C3H, BALB, B6 and B10.A) that are known to differ in their relative resistance to infections with the myotropic strains (Colombian and CL) of Trypanosoma cruzi. The parameters utilized to measure macrophage activation were rapid spreading on glass surfaces, hydrogen peroxide release and tumour necrosis factor/cachectin production. Macrophages obtained from C3H (susceptible), BALB (intermediate) and B6 or B10.A (resistant) mice infected with both strains of T. cruzi began to spread rapidly at the onset of parasitaemia. Surprisingly, the amount of hydrogen peroxide released by peritoneal cells obtained from the more susceptible mouse strain (C3H) was significantly higher than in the other mouse strains. Also, only in the serum of C3H mice was tumour necrosis factor/cachectin detected. These results suggest that resistance against infections with myotropic strains of T. cruzi does not correlate with enhanced macrophage activation. It is also shown that the acquired macrophage activation is largely dependent on T-lymphocytes bearing the phenotypic marker CD4 (helper/inducer), since all parameters of macrophage activation were significantly inhibited in athymic mice or in C3H mice treated in vivo with monoclonal antibody anti-CD4+ T-cells.     

Seroprevalence of Trypanosoma cruzi in kidney transplant donors and recipients in Mexico City

Infectious diseases are common causes of morbidity and mortality among kidney transplant recipients. Chagas disease (CD) has been recognized as an emerging infectious complication of transplantation caused by the parasite Trypanosoma cruzi. CD is prevalent in Mexico, particularly in the southern coastal region. The impact on Mexican kidney transplant programs has not been previously studied prospectively. From 2009 through 2010, serum samples from 59 kidney transplant donors and 405 renal transplant recipients were screened for antibodies against T. cruzi. Serum was initially screened using a locally developed ELISA test; positive results were confirmed by an indirect immunofluorescense test, in accordance with Panamerican Health Organization/World Health Organization guidelines. None of the donors were seropositive for T. cruzi, while 8 (1.97%) kidney transplant recipients were confirmed to be seropositive for T. cruzi. None of them have developed clinical manifestations of CD, although specific screening of recipients was not performed. A prospective study is planned to define the epidemiology and outcome of CD among kidney transplant donors and recipients in Mexico more thoroughly.     

Involvement of accessory cells in the Trypanosoma cruzi-induced inhibition of the polyclonal response of T lymphocytes

Infection with Trypanosoma cruzi is characterized by hyporesponsiveness of the immune system during the acute phase of infection. To better understand the immunological mechanisms affected by T. cruzi , we studied if a reduced T cell proliferative response could originate from an inability of T cells to proliferate or a functional deficiency at the level of accessory cells (AC). The inhibitory effect exerted by T. cruzi was during the induction phase of the lymphoproliferative response, suggesting the participation of AC in the hyporesponse. Then we further investigated the potential of the parasite to interfere with accessory cell-dependent and -independent pathways of human T cell proliferation. Peripheral blood mononuclear cells and peripheral blood lymphocytes from healthy individuals, enriched for T cells, were analysed with regard to their proliferative capacity using: phytohaemagglutinin, immobilized anti-CD3 monoclonal antibody (MoAb) and MoAb to the CD28 antigen, anti-CD3 MoAb and recombinant IL-2 and anti-CD3 MoAb plus phorbol myristate acetate in the presence of parasites. Significant suppression of the proliferative response was caused by the parasite only when AC were present. The parasite markedly reduced the surface expression of HLA-DR and CD11b antigens, key molecules in PHA-induced proliferation. Addition of indomethacin to the culture failed to reverse the inhibitory effect of the parasites, suggesting that prostaglandin E₂ was not involved. These data suggest that AC in contact with T. cruzi become incompetent as antigen presenting cell because they are unable to induce a normal proliferative response in T lymphocytes.     

Activation by synergism between endotoxin and lymphokines of the mouse macrophage cell line J774 against infection by Trypanosoma cruzi

The mouse macrophage cell line J774 was easily infected by T. cruzi epimastigotes which were transformed to amastigotes that multiplied inside the cells. Spleen-T-cells from T. cruzi immune mice stimulated with Concanavalin A or T. cruzi, but not with unrelated antigens, released lymphokines into the supernatants that when added to J774 cells were unable to induce complete trypanocidal activity, although they were able to delay the rate of infection by protecting the cells from being infected. Addition of bacterial lipopolysaccharide (LPS), although inactive by itself, acted synergistically with the supernatants in inducing complete trypanocidal activity without affecting the susceptibility of J774 cells to infection. Gamma-interferon (gamma-IFN) activity was detected in the supernatants, however, but was not solely responsible for the trypanocidal inducing activities, since: there was no correlation between the levels of gamma-IFN and macrophage activation; gamma-IFN alone was less effective than the supernatants alone; and two active fractions of 100,000-150,000 mol. wt and 30,000 mol. wt were separated by gel filtration chromatography of the lymphokine preparations. The latter, which showed the characteristics of gamma-IFN with respect to size, pH 2 sensitivity and antiviral activity, had some trypanocidal activity alone. However, the 100,000-150,000 mol. wt fraction was active only in the presence of LPS. Finally, this trypanocidal inducing activity of the supernatants was not due to the induction of synthesis of gamma-IFN by the J774 cells.     

Evaluation of IFN-gamma production by CD8 T lymphocytes in response to the K1 peptide from KMP-11 protein in patients infected with Trypanosoma cruzi

The cellular response mediated by MHC class I restricted CD8+ T cells has been shown to be crucial in the control of Chagas disease. The K1 peptide derived from T. cruzi KMP-11 protein has a high binding affinity to the HLA-A*0201 molecule. Nevertheless, it is not known whether this peptide is processed and displayed as an MHC class I epitope during natural infection by T. cruzi. The aim of this study was to evaluate, by ELISPOT assay, the ability of K1 peptide to activate CD8+ T lymphocytes to produce IFN-gamma. Therefore, CD8+ T lymphocytes from 22 HLA-A*0201+ individuals, 12 chronic chagasic patients and 10 uninfected controls, were analysed. The results revealed that two of the chagasic patients had IFN-gamma-secreting CD8+ T cells that were able to respond to K1 peptide with a relative frequency of 110 and 230 per million CD8+ T cells. In contrast, none of HLA-A*0201+ uninfected controls responded to K1 peptide. Responses to HLA-A*0201 restricted peptide from the influenza matrix protein were found in six chagasic patients and four uninfected controls with an average frequency of 175 and 111 cells per million CD8+ T cells, respectively. Moreover, a flow cytometric assay for degranulation showed that chagasic responders had K1-specific cytotoxic CD8+ T cells. It is shown here for the first time that the K1 peptide is efficiently processed, presented and recognized by CD8+ T lymphocytes during the natural course of Chagas disease.