Enhanced prevalence of T cells expressing TCRBV8S2 and TCRBV8S3 in hearts of chronically Trypanosoma cruzi-infected mice

We have analysed the relative T cell receptor (TCR) BV gene usage in T cells from hearts and spleens of CBA/HJ mice chronically infected with the Tulahuén strain of Trypanosoma cruzi. During chronic infection, CBA/HJ mice recruit T cells at the major site of inflammation (i.e. the heart), with over-representation of certain TCRBV gene subfamilies (TCRBV8S2 and TCRBV8S3). In contrast, no signal or a very weak message from a limited number of T cells was recorded from one heart of the control group. No alteration of TCRBV distribution was recorded in spleens of chronically infected CBA/HJ. Our findings indicate that there is a preferential TCRBV gene usage in the T cell response in the hearts of chronically infected mice. Furthermore, the pattern of CDR3 lengths in inflammatory T cells was altered.     

Protective role of nitric oxide in ocular toxoplasmosis

AIMS: To evaluate the role of nitric oxide (NO) in ocular involvement during systemic toxoplasmosis. METHODS: C57B1/6 mice were infected with Toxoplasma gondii strain ME49. The synthesis of NO was inhibited by an intraperitoneal injection of aminoguanidine every 8 hours, starting on the day of infection. Control infected mice received phosphate buffered saline vehicle alone. After 14 days, the ocular lesions were evaluated by histopathological examination. The expression of NO synthase induced in the spleen by toxoplasma infection was evaluated by immunostaining. The production of NO by the spleen cells of infected mice was measured by the colorimetric assay of Griess in the supernatant of cultures stimulated with toxoplasma antigen or concanavalin A. RESULTS: The inhibition of NO production in T gondii infected mice resulted in a marked increase in the symptoms of ocular inflammation. We observed a strong induction of NO synthase expression in the spleen of infected animals. In culture, the spleen cells from these mice produced high levels of NO in response to T gondii antigens. This elevation of NO synthesis was suppressed in the presence of aminoguanidine. CONCLUSION: This study indicates that NO plays a crucial role in the protection against T gondii infection as reflected by the severity of the ocular involvement.     

Direct and indirect exposure to air pollution

Glutathione (GSH), trypanothione (T(SH)2) and glutathionyl spermidine (GSH-SP) concentrations were determined in the Tulahuén and LQ strains and the DM 28c clone of Trypanosoma cruzi. The concentrations of GSH, T(SH)2 and GSH-SP, expressed as nmol of GSH per g of parasite fresh weight, were 60.1, 397.8 and 103.9, respectively, for the Tulahuén strain. For the DM 28c clone, the values were 113.9, 677.9 and 164.1, respectively, and for the LQ strain they were 199.1, 1100.5 and 55.3, respectively. When the parasites were treated with 10 microM nifurtimox or 50 microM benznidazole for 2 h, the concentrations of all three reduced thiols decreased strongly. The total amount of T(SH)2 decreased by more than 50%. Treatment of the parasites with 5 mM buthionine sulfoximine, an inhibitor of GSH synthesis, for 6 h diminished the concentrations of the reduced thiols by between 27% and 53% with respect to the controls. Cyclohexylamine, an inhibitor of spermidine synthesis, decreased the concentrations of T(SH)2 and GSH-SP but not that of GSH. It is possible to conclude from this study that trypanothione is the most important thiol involved in the detoxication of nifurtimox and benznidazole in T. cruzi and that electrophilic reduced metabolites of both drugs are most probably conjugated with GSH, T(SH)2 and GSH-SP, thus decreasing their concentrations. GSH biosynthesis is an important drug target.     

Defective nitric oxide effector functions lead to extreme susceptibility of Trypanosoma cruzi-infected mice deficient in gamma interferon receptor or inducible nitric oxide synthase

Trypanosoma cruzi, the causative agent of Chagas' disease, induces an innate and adaptive host immune response during the acute phase of infection. These responses were analyzed by comparing mouse lines deficient for the gamma interferon (IFN-gamma) receptor (IFN-gammaR(-/-)) or deficient for inducible nitric oxide synthase (iNOS(-/-)). Both lines were highly susceptible, with similar and dramatically increased parasite burdens and severe histopathology and were incapable of surviving even very low doses, exhibiting similar mortality kinetics. This pathophysiological correlation has a common cause, since both mutant mouse strains were unable to respond to infection by producing nitric oxide (NO) with the consequence that mutant macrophages had impaired trypanocidal activities. These in vivo and subsequent in vitro studies further demonstrated that an IFN-gamma-dependent pathway of iNOS induction is crucial for efficient NO production and mandatory for resisting acute infection with T. cruzi. Despite this defect, both mutant mouse strains had a rather normal proinflammatory cytokine response (interleukin-12 [IL-12], IFN-gamma, IL-6), with the exception of an impaired tumor necrosis factor alpha and IL-1alpha response in IFN-gammaR(-/-) mice, demonstrating that only the latter two cytokines are dependent on IFN-gamma activation. Moreover, polarization of T cells in type 1 and type 2 T-helper (Th1/Th2) and cytotoxic T (Tc1/Tc2) cells as well as T. cruzi-specific antibody responses were normal in IFN-gammaR(-/-) mice, demonstrating that IFN-gamma is not necessary for the promotion of T-cell differentiation and T. cruzi-specific antibody responses.     

Erectile insufficiency as first symptom of HTLV I/II associated myelopathy. Case report

People infected with Trypanosoma cruzi remain so for life, yet only 30-40% of these individuals develop characteristic chagasic cardiomyopathies. Similarly, when infected with the Brazilian strain of T. cruzi, DBA/2 mice develop severe cardiac damage while B10.D2 mice do not. To better understand the immunological parameters that may be involved in the disease process, we have used this murine model (DBA/2 vs B10.D2) and compared the changes in cytokine production during the course of infection with T cruzi. Concanavalin A (Con A) stimulation of spleen cells harvested during the acute phase (day 30) resulted in similarly high levels of IFN-gamma in both mouse strains. However, the amount of IFN-gamma in supernatants from cultures of B10.D2 spleen cells initiated during the chronic phase (day 72) was at subacute levels, whereas secretion by chronic DBA/2 spleen cells remained high. In addition, Con A-stimulated spleen cells from acute DBA/2 mice produced approximately twice as much IL-10 and significantly more IL-4 than cells from B10.D2 mice. IL-4 secretion remained low by cells from chronic B10.D2 mice, but when using cells from chronic DBA/2 mice, levels continued to increase beyond the already high levels secreted by cells harvested during the acute phase. Proliferative responses to Con A stimulation by spleen cells from DBA/2 mice were significantly higher than those from B10.D2 mice in both the acute and chronic phases. These data suggest that enhanced responses in DBA/2 mice, which may be related to a higher parasite burden, a lack of down-regulation, and/or the onset of autoimmune phenomena, correlate with the more severe cardiomyopathy seen in pathopermissive mice.     

Is epsilon-aminocaproic acid as effective as aprotinin in reducing bleeding with cardiac surgery?: a meta-analysis

Infection of BALB/c mice with Trypanosoma cruzi resulted in up-regulated expression of Fas and Fas ligand (FasL) mRNA by splenic CD4+ T cells, activation-induced CD4+ T cell death (AICD), and in Fas: FasL-mediated cytotoxicity. When CD4+ T cells from infected mice were co-cultured with T. cruzi-infected macrophages, onset of AICD exacerbated parasite replication. CD4+ T cells from T. cruzi-infected FasL-deficient BALB gld/gld mice had no detectable AICD in vitro and their activation with anti-TCR did not exacerbate T. cruzi replication in macrophages. However, infection of BALB gld/gld mice with T. cruzi resulted in higher and more prolonged parasitemia, compared to wild-type mice. Secretion of Th2 cytokines IL-10 and IL-4 by CD4+ T cells from infected gld mice was markedly increased, compared to controls. In addition, in vivo injection of anti-IL-4 mAb, but not of an isotype control mAb, reduced parasitemia in both gld and wild-type mice. These results indicate that, besides controlling CD4+ T cell AICD and parasite replication in vitro, an intact Fas: FasL pathway also controls the host cytokine response to T. cruzi infection in vivo, being required to prevent an exacerbated Th2-type immune response to the parasite.     

Nitric oxide synthase expression in macrophages of Histoplasma capsulatum-infected mice is associated with splenocyte apoptosis and unresponsiveness

Splenic macrophages from Histoplasma capsulatum-infected mice express inducible nitric oxide synthase (iNOS), and the iNOS expression correlates with severity of the infection. We examined whether production of NO is responsible for apoptosis and the anti-lymphoproliferative response of splenocytes from mice infected with H. capsulatum. In situ terminal deoxynucleotidyl transferase nick end labeling revealed apoptotic nuclei in cryosections of spleen from infected but not normal mice. Splenocytes of infected mice were unresponsive to stimulation by either concanavalin A or heat-killed H. capsulatum yeast cells. Splenocyte responsiveness was restored by addition to the medium of NG-monomethyl-L-arginine, a known inhibitor of NO production. The proliferative response of splenocytes from infected mice was also restored by depletion of macrophages or by replacement with macrophages from normal mice. In addition, expression of iNOS returned to its basal level when the animals had recovered from infection. These results suggest that suppressor cell activity of macrophages is associated with production of NO, which also appears to be an effector molecule for apoptosis of cultured splenocytes from infected mice.     

A dichotomous role for nitric oxide during acute Toxoplasma gondii infection in mice

Production of nitric oxide by macrophages is believed to be an important microbicidal mechanism for a variety of intracellular pathogens, including Toxoplasma gondii. Mice with a targeted disruption of the inducible nitric oxide synthase gene (iNOS) were infected orally with T. gondii tissue cysts. Time to death was prolonged compared with parental controls. Histologic analysis of tissue from infected mice showed scattered small foci of inflammation with parasites in various tissues of iNOS-/- mice, whereas tissue from the parental C57BL/6 mice had more extensive tissue inflammation with few visible parasites. In particular, extensive ulceration and necrosis of distal small intestine and fatty degeneration of the liver was seen in the parental mice at day 7 postinfection, as compared with the iNOS-/- mice where these tissues appeared normal. Serum interferon gamma and tumor necrosis factor alpha levels postinfection were equally elevated in both mouse strains. Treatment of the parental mice with a NO synthase inhibitor, aminoguanidine, prevented early death in these mice as well as the hepatic degeneration and small bowel necrosis seen in acutely infected control parentals. These findings indicate that NO production during acute infection with T. gondii can kill intracellular parasites but can be detrimental, even lethal, to the host.     

Macrophages in mice acutely infected with lymphocytic choriomeningitis virus are primed for nitric oxide synthesis

Following infection of adult mice with lymphocytic choriomeningitis virus (LCMV) there is a well documented suppression of T-cell and B-cell functions concurrent with the strong anti-LCMV immune response. Macrophages have been shown to be infected and activated during acute LCMV infection and there is some evidence to indicate that there is altered antigen presentation in acutely infected mice. We have examined nitric oxide (NO) production by splenic macrophages during acute infection of adult mice. Our results show that these macrophages are primed for production of NO, that the inducible production of NO parallels the immune suppression, and that NO production is dependent on the presence of IFN gamma. However, neither in vivo nor in vitro treatment with N-monomethyl-L-arginine (NMA), a specific inhibitor of nitric oxide synthase, altered the induction or maintenance of virus-induced immune suppression in mice acutely infected with LCMV.     

Comparative studies on immune responses to infection in susceptible B10.BR mice infected with different strains of the murine nematode parasite Trichuris muris

A comparison of immune responses to infection between groups of B10.BR mice infected with different strains of T. muris, S strain (isolated in Sobreda, Portugal), E strain (isolated in Edinburgh), and E-J strain (originally E strain, which has been maintained in our laboratory, Japan), was performed. In mice infected with E and E-J strains, most of the worms were expelled by day 32 after infection, though the expulsion was faster in E-J strain-infected mice. In contrast, no expulsion was observed in S strain-infected mice by day 32 and egg production occurred on day 32. IL-4 production occurred in concanavalin A (Con-A)-stimulated mesenteric lymph node cells (MLNC) from B10.BR mice infected with E and E-J strains, whereas no IL-4 production was observed in S strain-infected mice. IL-4 production did not occur in normal mice. In comparison with normal mice, high levels of IFN-gamma production by Con A-stimulated MLNC were detected in mice infected with every strain of T. muris. IFN-gamma production in S strain-infected mice was greater, occurred earlier and was more persistent than in mice infected with E and E-J strains. IgG1 and IgG2a antibodies to T. muris excretory/ secretory antigens were observed in B10.BR mice infected with every strain of T. muris. Antibody production showed similar kinetics. These differences in the expulsion kinetics and IL-4 production in B10.BR mice infected with S, E, and E-J strains suggest the involvement of IL-4 in protection against T. muris infection, and confirm the previous conclusion by Else et al.     

Trypanosoma cruzi-induced immunosuppression: blockade of costimulatory T-cell responses in infected hosts due to defective T-cell receptor-CD3 functioning

A model of experimental Trypanosoma cruzi murine infection with chemically induced metacyclic forms (opossum clone Dm28c) showed a marked state of T-cell unresponsiveness during acute phase, but lacked evidence of suppressor cell activity. Spleen cells from infected mice were suppressed in vitro in responses to T-cell activators concanavalin A, anti-Thy1 monoclonal antibody (MAb), and anti-CD3 MAb compared with spleen cells from control littermates. Activation with accessory cell-independent stimulus provided by immobilized anti-CD3 was defective in splenic CD4-positive T cells from infected mice, but not in such cells from control mice. No evidence of splenic suppressor cell activity was found in cell-mixing experiments using nylon-passed T cells from control and infected donors. Kinetic experiments showed that there was a discrete stage in infection when T cells were already suppressed in response to anti-CD3 but still responded to anti-CD69 MAb. In these T cells, immobilized anti-CD3 failed to enhance simultaneous CD69 responses, although anti-CD3 enhanced CD69 responses in control T cells from uninfected donors. These results demonstrate an intrinsic defect in T-cell receptor-mediated T-cell activation, which could be a mechanism generating T-cell suppression during infection by T. cruzi.     

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.     

Nitric oxide production by peritoneal macrophages of Mycobacterium bovis BCG-infected or non-infected mice: regulatory role of T lymphocytes and cytokines

Infection with Mycobacterium bovis bacillus Calmette-Guerin (BCG) confers mice with strong abilities to produce nitric oxide (NO) and cytokines. Because the peritoneal macrophages taken from the mice immunized with live or heat-killed BCG can produce NO without any accessory cells and stimulants, it is difficult to clarify the immune regulation on NO production by manipulating the macrophages. Therefore, we investigated the participation of immune T cells and cytokines in NO production by using in vitro co-cultures of macrophages from non-immune mice with T cells prepared from BCG-infected mice in the presence or absence of a mycobacterial antigen, purified protein derivative (PPD). Although the non-immune thioglycollate (TGB)-elicited macrophages could not produce any detectable NO in the presence of PPD, supplementation of the macrophage cultures with CD4+ T cells prepared from BCG-infected mice enabled the macrophages to produce NO. Immunocytostaining showed that the macrophages, hut not the immune T cells, expressed inducible NO synthase (iNOS), indicating that they were NO producers. PPD could only induce NO production if there was cell-cell contact of the CD4+ T cells in the immune cells and antigen-presenting macrophages were required for the NO production in response to PPD; this interaction led to the production of soluble mediators that induced NO production by the TGB macrophages. NO production by the co-cultured cells was abrogated by adding either anti-interferon-gamma(IFN-gamma) or anti-tumor necrosis factor alpha (TNF-alpha) antibody. Furthermore, the roles of immune T cells and PPD could be replaced by adding recombinant IFN-gamma together with TNF-alpha to the macrophage cultures, but neither alone was sufficient to induce NO production by the macrophages. Our present data indicate that TNF-alpha produced by PPD-stimulated macrophages and IFN-gamma produced by cell-cell interaction of BCG-immune T cells and antigen-engulfed macrophages together activate the macrophages to produce NO.     

Interleukin-18 protects mice against acute herpes simplex virus type 1 infection

We examined the effects of interleukin-18 (IL-18) in a mouse model of acute intraperitoneal infection with herpes simplex virus type 1 (HSV-1). Four days of treatment with IL-18 (from 2 days before infection to 1 day after infection) improved the survival rate of BALB/c, BALB/c nude, and BALB/c SCID mice, suggesting innate immunity. One day after infection, HSV-1 titers were higher in the peritoneal washing fluid of control BALB/c mice than in that of IL-18-treated mice. A genetic deficiency of gamma interferon (IFN-gamma), however, diminished the survival rate and the inhibition of HSV-1 growth at the injection site in the mice. Anti-asialo GM1 treatment had no influence on the protective effect of IL-18 in infected mice. IL-18 augmented IFN-gamma release in vitro by peritoneal cells from uninfected mice, while no appreciable IFN-gamma production was found in uninfected mice administered IL-18. Although IFN-gamma has the ability to induce nitric oxide (NO) production by various types of cells, administration of the NO synthase inhibitor NG-monomethyl-L-arginine resulted in superficial loss of the improved survival, but there was no influence on the inhibition of HSV-1 replication at the injection site in IL-18-treated mice. Based on these results, we propose that IFN-gamma produced before HSV-1 infection plays a key role as one of the IL-18-promoted protection mechanisms and that neither NK cells nor NO plays this role.     

Induction of alpha/beta interferon and dependent nitric oxide synthesis during Chlamydia trachomatis infection of McCoy cells in the absence of exogenous cytokine

The propensity of two Chlamydia trachomatis strains (L2/434/Bu [biovar LGV] and E/DK20/ON [biovar trachoma]) to induce putative host defense responses upon infection of McCoy (mouse) cell cultures was examined. Both strains induced production of alpha/beta interferon and nitric oxide (NO) by McCoy cells. NO synthesis was mediated by the inducible isoform of NO synthase as indicated by the ability of cycloheximide or the arginine analog NG-monomethyl-L-arginine to abolish NO production; the extent of the response was dependent upon the dose of chlamydiae applied. Incubation of McCoy cells with chloramphenicol prior to infection reduced NO production by strain 434 but not by DK20, suggesting that initial chlamydial metabolism was essential to induction by the LGV strain. Antibody inhibition studies indicated that NO synthesis was dependent upon production of alpha/beta interferon and induction via lipopolysaccharide. Overall, our findings show that chlamydiae are capable of the induction of interferon and NO in murine fibroblasts in the absence of exogenous cytokines. However, the role of NO as an antichlamydial effector could not be clearly demonstrated since treatment with an arginine analog, while suppressing NO production, gave no consistent enhancement of infected cell numbers.     

Alterations induced by Trypanosoma cruzi in activated mouse lymphocytes

Although a number of immunological anomalies have been shown to occur during the acute period of Trypanosoma cruzi infection, the contribution of the parasite has not been clarified. In this work, we co-cultured activated splenic mononuclear cells (SMC) from normal outbred (CD1) or inbred (CBA/J) mice with purified T. cruzi trypomastigotes and studied ensuing T- and B-lymphocyte alterations. In the presence of parasites, phytohaemagglutinin-stimulated SMC from either mouse background manifested a marked reduction in both lymphoproliferative capacity (i.e., 3H-thymidine incorporation) and cell membrane level of interleukin-2 receptors (IL-2R; determined by flow cytometry) relative to SMC from parasite-free cultures. Thus, substantial proportions of activated SMC either became unable to express detectable levels of IL-2R or expressed this receptor in significantly lower numbers than control SMC. Supernatants from T. cruzi suspensions reproduced these suppressive effects on phytohaemagglutinin-stimulated SMC from normal or chronically infected CD1 or CBA/J mice. Similar results were obtained with SMC activated with a bacterial lipopolysaccharide. Since IL-2R expression is required for activated lymphocytes to progress through the cell cycle and multiply to mount effective immune responses, impaired IL-2R expression by T. cruzi provides a plausible hypothesis for the wide-ranged immunosuppression that occurs in the infected host.     

Protection of mice against Trypanosoma cruzi by immunization with paraflagellar rod proteins requires T cell, but not B cell, function

Previous studies have shown that immunization of mice with the paraflagellar rod proteins (PAR) of Trypanosoma cruzi induces an immune response capable of protecting mice against an otherwise lethal challenge with this parasite. Herein, we define immunologic responses that do or do not play a critical role in PAR-mediated protection. Firstly, PAR-immunized Ab-deficient (muMT) strain mice survived an otherwise lethal T. cruzi challenge, indicating that a B cell response is not required for PAR-induced immunity. However, beta2m -/- mice, which are severely deficient in MHC class I and TCR alphabeta+ CD8+ CD4- T cells, did not survive challenge infection following PAR immunization, indicating that MHC class I/CD8+ T cell function is necessary for protection induced by PAR immunization. Surprisingly, PAR-immunized mice depleted of CD4+ T cells survived a T. cruzi challenge for >84 days postinfection while maintaining a parasitemia that is generally thought to be lethal (i.e., >10(6) trypomastigotes/ml), thus associating CD4+ T cell function with the process of parasite clearance. Consistent with this association, CD4+ T cells from PAR-immunized mice released INF-gamma and stimulated T. cruzi-infected macrophages to release nitric oxide. The importance of IFN-gamma in PAR-induced protective immunity is further indicated by the observation that PAR-immunized INF-gamma knockout mice developed an extremely high parasitemia and did not survive a challenge infection. Thus, while Ab-mediated immune mechanisms are not required for protection induced by PAR immunization, T cell responses are necessary for both elimination of bloodstream parasites and survival.     

Induction of differential T-helper-cell responses in mice infected with variants of the parasitic nematode Trichuris muris

The resistance or susceptibility of mice to infection with the intestinal nematode parasite Trichuris muris is closely correlated with polarization of T helper (Th) cell responses to the type 2 (Th2) or type 1 (Th1) subset. Comparison of infections with three isolates of T. muris (E/K, E/N, and S) in three inbred strains of mice (CBA, C57BL/10, and B10.BR) has shown that host Th response phenotype can be parasite determined. Although the mouse strains used show genetically determined variation in ability to respond to T. muris (CBA > C57BL/10 > B10.BR), the speed of worm expulsion in a given strain depended upon the isolate used for infection (E/K > E/N > S). The two isolates that induced the most effective resistance (E/K and E/N) elicited parasite-specific host antibody responses that were dominated by immunoglobulin G1 (IgG1), and antigen-stimulated T cells from infected mice released interleukin-5 in vitro. With the isolate that induced the least host resistance (S), the dominant antibody response was IgG2a, and T cells released gamma interferon in vitro. These data show clearly that parasite variant-specific factors play a major role in Th subset polarization during infection.