Differential contribution of neutrophilic granulocytes and macrophages to nitrosative stress in a host-parasite animal model

Tyrosine nitration is a hallmark for nitrosative stress caused by the release of reactive oxygen and nitrogen species by activated macrophages and neutrophilic granulocytes at sites of inflammation and infection. In the first part of the study, we used an informative host-parasite animal model to describe the differential contribution of macrophages and neutrophilic granulocytes to in vivo tissue nitration. To this purpose common carp (Cyprinus carpio) were infected with the extracellular blood parasite Trypanoplasma borreli (Kinetoplastida). After infection, serum nitrite levels significantly increased concurrently to the upregulation of inducible nitric oxide synthase (iNOS) gene expression. Tyrosine nitration, as measured by immunohistochemistry using an anti-nitrotyrosine antibody, dramatically increased in tissues from parasite-infected fish, demonstrating that elevated NO production during T. borreli infection coincides with nitrosative stress in immunologically active tissues. The combined use of an anti-nitrotyrosine antibody with a panel of monoclonal antibodies specific for several carp leukocytes, revealed that fish neutrophilic granulocytes strongly contribute to in vivo tissue nitration most likely through both, a peroxynitrite- and an MPO-mediated mechanism. Conversely, fish macrophages, by restricting the presence of radicals and enzymes to their intraphagosomal compartment, contribute to a much lesser extent to in vivo tissue nitration. In the second part of the study, we examined the effects of nitrosative stress on the parasite itself. Peroxynitrite, but not NO donor substances, exerted strong cytotoxicity on the parasite in vitro. In vivo, however, nitration of T. borreli was limited if not absent despite the presence of parasites in highly nitrated tissue areas. Further, we investigated parasite susceptibility to the human anti-trypanosome drug Melarsoprol (Arsobal), which directly interferes with the parasite-specific trypanothione anti-oxidant system. Arsobal treatment strongly decreased T. borreli viability both, in vitro and in vivo. All together, our data suggest an evolutionary conservation in modern bony fish of the function of neutrophilic granulocytes and macrophages in the nitration process and support the common carp as a suitable animal model for investigations on nitrosative stress in host-parasite interactions. The potential of T. borreli to serve as an alternative tool for pharmacological studies on human anti-trypanosome drugs is discussed.     

Trypanoplasma borreli cysteine proteinase activities support a conservation of function with respect to digestion of host proteins in common carp

Trypanoplasma borreli is an extracellular parasite that is transmitted by a leech vector and is naturally found in the blood of cyprinid fish. High parasitemia and associated severe anemia together with splenomegaly are typical of infection of common carp, Cyprinus carpio L. Papain-like cysteine proteinases expressed by trypanosome parasites contribute to the pathogenicity of trypanosomes, and are considered an important target for the development of new trypanocidal drugs. T. borreli is a member of the Parabodonida, sharing a common ancestor with the other Kinetoplastida. We demonstrate the presence of a cysteine proteinase expressed by T. borreli. Alignment of the sequence with other kinetoplastid cysteine proteinase sequences supports the phylogenetic hypotheses based on analyses of ribosomal RNA genes. We expressed the T. borreli cysteine proteinase in Escherichia coli, refolded the purified protein into a biologically active proteinase and showed it has cathepsin L-like activity. Addition of the (non)active proteinase to in vitro-derived carp head kidney-derived macrophages did not significantly modulate macrophage activity. Immunization of carp with the recombinant proteinase did induce a very high increase in proteinase-specific antibodies but only slightly lowered parasitemia. Digestion of host hemoglobin and immunoglobulin by the cysteine proteinase likely contribute to the pathogenicity of T. borreli. The possibility that digestion by the cysteine proteinase of host transferrin could contribute to an innate activation profile of macrophages in vivo is discussed. Our findings suggest a conservation of function with respect to cysteine proteinase activity in the Parabodonida in support of the hypotheses on the phylogeny of the Kinetoplastida.     

Molecular and functional characterization of carp TNF: a link between TNF polymorphism and trypanotolerance?

Two carp tumor necrosis factor alpha (TNFalpha) genes have been cloned and sequenced. Both TNF1 and TNF2 sequences have several polymorphisms in the 3' UTR and TNF2 has a polymorphism in the coding sequence. Lipopolysaccharide and the protozoan blood flagellate Trypanoplasma borreli induced expression of TNFalpha in carp head kidney phagocytes when added in vitro. Differential expression was observed, with TNF2 being higher expressed than TNF1. We used the TNFalpha-specific inhibitor pentoxifylline to demonstrate the involvement of carp TNFalpha in the induction of nitric oxide and in the stimulation of cell proliferation. In addition, two carp lines differing in their resistance to T. borreli were typed for the TNF2 polymorphism and association between one isoform and resistance was found.     

The immunological response of CBA mice to P. yoelii. I. General characteristics, the effects of T-cell deprivation and reconstitution with thymus grafts.

Experimental infection of normal CBA mice with the parasite Plasmodium berghei yoelii (P. yoelii) resulted in a mild, non-fatal and self-limiting infection which lasted for 15-17 days. Animals which recovered from the primary infection were immune to reinfection though parasites could be detected in the kidneys of such mice 4 weeks after recovery from infection. (No plasmodia were demonstrated in the peripheral blood and other tissues examined.) In T cell-deprived mice, P. yoelii infections resulted in a progressive parasitaemia and proved fatal in 35-40 days. Studies of fluorescent antibody levels and morphological changes in the spleens of infected normal and T cell-deprived mice showed that while normal mice produced high levels of IgG1, IgG2 and IgM antiplasmodial antibodies and developed a strong and sustained germinal centre response, in T cell-deprived animals the production of IgG1 antibodies was almost completely abolished and the germinal centre response severely impaired. Reconstitution of T cell-deprived mice with syngeneic thymus grafts resulted in partial restoration of immunological responsiveness. P. yoelii infections in these reconstituted animals ran a self-limiting course akin to that seen in normal CBA mice; the level of protective immunity and the germinal centre response correlated with the degree of reconstitution achieved.     

The role of CD4+ T cells in the protective immune response to Plasmodium chabaudi in vivo

CD4+ T cells are an essential component of the protective immune response to Plasmodium chabaudi. In order to determine whether the presence of CD4+ T cells is necessary throughout a primary infection for a protective immune response to develop mice were depleted of their CD4+ T cells in vivo by treatment with specific antibodies. Removal of CD4+ T cells during the acute phase of infection renders mice incapable of clearing their infection. In contrast, removal of CD4+ T cells after this time did not affect their ability to control their parasitaemia. The ability to control parasitaemia correlated with appearance of malaria-specific IgG antibodies. Our data, therefore, suggest a mechanism requiring the presence of CD4+ T cells during the acute pre-IgG period. Later, after IgG has been produced, this mechanism is no longer required.     

Cultivation of bloodstream forms of Trypanosoma carassii, a common parasite of freshwater fish

Trypanosoma carassii (syn. T. danilewskyi) is a widespread parasite of carp and other cyprinid as well as some noncyprinid freshwater fish. It lives extracellularly in the blood and tissues of its hosts, causing chronic infections. In this paper the isolation of T. carassii from fish blood and the propagation and cloning of bloodstream forms in vitro are described. By several criteria, cultured and fish-derived trypomastigotes are indistinguishable. The culture system should be useful for the biochemical characterization of this trypanosome and its interaction with the fish immune system. Received: 10 September 1997 / Accepted: 26 November 1997     

Circulating concentrations of soluble granzyme A and B increase during natural and experimental Plasmodium falciparum infections

Release of soluble Granzymes (sGranzymes) is considered to reflect activation of cytotoxic T lymphocytes and NK cells. sGranzymes and a number of pro-inflammatory cytokines were measured in plasma of malaria patients with natural or experimentally induced Plasmodium falciparum infections. Concentrations of sGranzyme A and B, IL-10, IL-12p70 and CRP were significantly increased in African children presenting with clinical malaria; IL-10 and CRP concentrations were significantly correlated with disease severity. In nonimmune Dutch volunteers which were experimentally infected by P. falciparum-infected mosquitoes, sGranzyme A increment started 1-2 days prior to clinical symptoms and microscopically detectable parasitaemia. This coincided with increases in IFNgamma, IL-12p40 and IL-8, while sGranzyme B and IL-10 levels increased 24-48 h later. The elevation of sGranzyme A and IFNgamma in nonimmune volunteers suggests that NK cells are activated upon release of parasites by infected liver cells and subsequently during blood stage infection; thus, NK cells are likely involved innate immune human host resistance in the early phase of a malaria infection.     

Induction of the trypanosome lymphocyte-triggering factor (TLTF) and neutralizing antibodies to the TLTF in experimental african trypanosomiasis

We have demonstrated that African trypanosomes secrete a novel trypanokine, the trypanosome-derived lymphocyte-triggering factor (TLTF), which activates CD8+ cells to produce interferon-gamma (IFN-gamma) that in turn stimulates parasite growth. The gene for TLTF was recently cloned, and recombinant TLTF (rTLTF) showed bioactivity that was similar to native TLTF. In this work, we employed two anti-TLTF monoclonal antibodies (mAbs) to detect levels of TLTF during Trypanosoma brucei brucei (T. b. brucei ) infections in mice. Furthermore, rTLTF was utilized to assess levels of anti-TLTF antibodies. Mice with intact genes (wild type), and knockout mice with disrupted IFN-gamma (IFN-gamma-/-) or IFN-gammaR (IFN-gammaR-/-) genes were studied. The knockout mice were used in order to illustrate the role of IFN-gamma in the production of antibodies to TLTF. While wild-type mice showed high parasitaemia accompanied by high TLTF levels and low anti-TLTF antibodies at day 3 postinfection (p.i.), low TLTF was measured together with increased anti-TLTF antibodies at day 21 p.i. IFN-gamma-/- mice exhibited very low parasitaemia, TLTF and anti-TLTF antibody levels. In contrast, IFN-gammaR-/- mice revealed very high parasitaemia, increased TLTF levels, but decreased anti-TLTF antibodies. In a biological assay for TLTF, Fab' fragments of anti-TLTF antibodies dose dependently inhibited the TLTF-induced IFN-gamma production by splenocytes, suggesting a regulatory importance of these antibodies. Our data demonstrate a role of IFN-gamma in the generation of neutralizing antibodies to TLTF. Furthermore, the induction of TLTF and its antibodies may constitute a new approach for future diagnosis of African trypanosomiasis.     

Protective 'immunity' by pre-existent neutralizing antibody titers and preactivated T cells but not by so-called 'immunological memory'

Summary: The idea of immunological memory originally arose from the observation that survivors of infections were subsequently resistant to disease caused by the same infection. While most immunologists accept a special ‘remembering’ memory quality, we have argued previously and document here that increased resistance against re‐infection, i.e. immunity, reflects low‐level antigen‐driven T‐ and B‐cell responses, resulting in elevated serum or mucosal titers of protective antibodies or of activated T cells, respectively. Periodic antigen re‐exposure is from within, by persisting infection (long‐term) or by immune complexes (short‐term), or from without, by low‐level re‐infections. This simple concept is supported by clinical evidence and model experiments but is often ignored, although this concept, but not so‐called ‘immunological memory’, as defined in textbooks (i.e. earlier and better responses of a primed host), is compatible with evolutionary maternal antibody transfer of protection as well as immunity against existing infections. The concept of ‘immunity without immunological remembering memory’ explains why it is easy to generate vaccines against acute cytopathic infections, particularly those of early childhood, where neutralizing antibodies are the key to protection, because it has been validated by adoptive transfer of maternal antibodies. It also explains why we have not succeeded (yet?) to generate truly protective vaccines against persisting infections, because we cannot imitate ‘infection immunity’ that is long‐lasting, generating protective T‐ and B‐cell stimulation against variable infections without causing disease by either immunopathology or tolerance.     

Role of T Helper/Inducer Cells as well as Natural Killer Cells in Resistance to Trypanosoma cruzi Infection

T lymphocytes provide a major line of defence against many protozoan parasites. The aim of this work was to determine the role of T-cell helper/inducer subset (T h/i) in the resistance to Trypanosoma cruzi in a murine model. The importance of natural killer (NK) cells in the resistance to the parasite was also evaluated. BALB/c and C57BL/6 mice were injected with either monoclonal antibodies against L3T4, Thy 1.2, NK1.1, or with a polyclonal rabbit antiserum against NK cells (anti-asialo GM-1). The effect of in vivo administration of these antibodies was tested in separate functional assays. After antibody treatment, mice were infected with a low dose of T. cruzi in the bloodstream form. Mice depleted of, or reduced in T, T h/i, or NK cell activity all developed higher parasitaemia and had higher mortality than their control counterparts. Mice injected with anti-L3T4 monoclonal antibodies were unable to generate a specific antibody response to the parasite. Treatment of mice with alpha/beta interferon, which is known to boost NK cell activity, resulted in an enhanced resistance to the parasite. Our data indicate that T h/i cells as well as NK cells are of vital importance in controlling parasitaemia and reducing mortality in T. cruzi-infected mice. Finally, we also demonstrate that the production of antibodies specific for T. cruzi is strictly T helper cell-dependent.     

Mixed Mycobacterium tuberculosis Complex Infections and False-Negative Results for Rifampin Resistance by GeneXpert MTB/RIF Are Associated with Poor Clinical Outcomes

The Xpert MTB/RIF (Xpert) assay is becoming a principal screening tool for diagnosing rifampin-resistant Mycobacterium tuberculosis complex (MTBC) infection. However, little is known about the performance of the Xpert assay in infections with both drug-sensitive and drug-resistant strains (mixed MTBC infections). We assessed the performance of the Xpert assay for detecting rifampin resistance using phenotypic drug sensitivity testing (DST) as the reference standard in 370 patients with microbiologically proven pulmonary tuberculosis. Mixed MTBC infections were identified genetically through 24-locus mycobacterial interspersed repetitive-unit–variable-number tandem-repeat (MIRU-VNTR) analysis. Logistic regression was used to identify the factors associated with poor (defined as treatment failure, default, and death from any cause) or good (defined as cure or successful treatment completion) clinical outcomes. The analytic sensitivity of the Xpert assay for detecting rifampin resistance was assessed in vitro by testing cultures containing different ratios of drug-sensitive and drug-resistant organisms. Rifampin resistance was detected by the Xpert assay in 52 (14.1%) and by phenotypic DST in 55 (14.9%) patients. Mixed MTBC infections were identified in 37 (10.0%) patients. The Xpert assay was 92.7% (95% confidence interval [CI], 82.4% to 97.9%) sensitive for detecting rifampin resistance and 99.7% (95% CI, 98.3% to 99.9%) specific. When restricted to patients with mixed MTBC infections, Xpert sensitivity was 80.0% (95% CI, 56.3 to 94.3%). False-negative Xpert results (adjusted odds ratio [aOR], 6.6; 95% CI,1.2 to 48.2) and mixed MTBC infections (aOR, 6.5; 95% CI, 2.1 to 20.5) were strongly associated with poor clinical outcome. The Xpert assay failed to detect rifampin resistance in vitro when <90% of the organisms in the sample were rifampin resistant. Our study indicates that the Xpert assay has an increased false-negative rate for detecting rifampin resistance with mixed MTBC infections. In hyperendemic settings where mixed infections are common, the Xpert results might need further confirmation.     

IFN-gamma, but not nitric oxide or specific IgG, is essential for the in vivo control of low-virulence Sylvio X10/4 Trypanosoma cruzi parasites

Highly virulent strains of Trypanosoma cruzi are frequently used as murine models of Chagas' disease. However, these strains do not fully represent the spectrum of parasites involved in the human infection. In this paper, we analysed parasitaemia, mortality, tissue pathology and parasite-specific IgG serum levels in immune-deficient mice infected with Sylvio X10/4 parasites, a T. cruzi derived from a chagasic patient that yields very low parasitaemias and in C3H/HePAS mice induces a chronic cardiopathy resembling the human disease. IFN-gamma was identified as a crucial element for parasite control as its absence determined a drastic increase in parasitaemia, tissue parasitism, leukocyte infiltrates at the heart and striated muscles and mortality. The lack of IFN-gamma or IL-12p40, a molecule shared by IL-12 and IL-23, also resulted in spinal cord lesions and a progressive paralysis syndrome. Whereas IgG2a was the main Ig isotype in infected C57BL/6 mice, IL-12p40-KO mice produced IgG2a and IgG1 and IFN-gamma-KO mice produced only IgG1. The IFN-gamma-protective effect was not essentially mediated by nitric oxide (NO), inasmuch as infected iNOS-KO mice showed no parasitaemia and low tissue damage. Mice deficient in CD4(+) or CD8(+) T cells showed an intermediate phenotype with increased mortality and tissue pathology but no parasitaemia. Interestingly, CD28-KO mice were unable to produce anti-T. cruzi IgG antibodies but presented moderate tissue pathology and managed to control the infection. Thus, differently from infections with high virulence parasites, neither IgG, NO nor CD28-mediated signalling are essential for the non-sterile control of Sylvio X10/4 parasites.     

Enteric fever in a 6-year-old traveler caused by Salmonella enterica serotypes Typhi and Paratyphi A: laboratory detection strategies and treatment options

We report the first pediatric case of enteric fever caused by Salmonella enterica serotypes Typhi and Paratyphi A. Mixed infections are infrequently reported, potentially because detection of two different Salmonella serotypes in blood cultures is technically challenging. We suggest laboratory strategies to aid in the recognition of mixed infections.     

Modulation of experimental blood stage malaria through blockade of the B7/CD28 T-cell costimulatory pathway

Recent studies have implicated cytokines associated with CD4+ T lymphocytes of both T helper (Th)1 and Th2 subsets in resistance to experimental blood stage malaria. As the B7/CD28 costimulatory pathway has been shown to influence the differentiation of Th cell subsets, we investigated the contribution of the B7 molecules CD80 and CD86 to Th1/Th2 cytokine and immunoglobulin isotype profiles and to the development of a protective immune response to malaria in NIH mice infected with Plasmodium chabaudi. Effective blockade of CD86/CD28 interaction was demonstrated by elimination of interleukin (IL)-4 and up-regulation of interferon (IFN)-gamma responses by P. chabaudi-specific T cells and by reduction of P. chabaudi-specific immunoglobulin G1 (IgG1). The shift towards a Th1 cytokine pattern corresponded with efficient control of acute parasitaemia but an inability to resolve chronic infection. Moreover, combined CD80/CD86 blockade by using anti-CD80 and anti-CD86 monoclonal antibodies raised IFN-gamma production over that seen with CD86 blockade alone, with augmentation of this Th1-associated cytokine reducing levels of peak primary parasitaemia. These results demonstrate that IL-4 production by T cells in P. chabaudi-infected NIH mice is dependent upon CD86/CD28 interaction and that IL-4 and IFN-gamma contribute significantly, at different times of infection, to host resistance to blood stage malaria. In addition, combined CD80/CD86 blockade resulted in preferential expansion of IFN-gamma-producing T cells during P. chabaudi infection, suggesting that costimulatory pathways other than B7/CD28 may contribute to T-cell activation during continuous antigen stimulation. This study indicates a role for B7/CD28 costimulation in modulating the CD4+ T-cell response during malaria, and further suggests involvement of this pathway in other infectious and autoimmune diseases in which the Th cell immune response is also skewed.     

The role of T cells in pathogenesis and protective immunity to murine malaria.

T-cell-mediated immunity to a virulent strain of Plasmodium berghei NK65 (Pb NK65) and to an attenuated derivative (Pb XAT) of the strain were examined in CBA mice by the administration of monoclonal antibodies against T-cell subsets or interferon-gamma (IFN-gamma). The injection of anti-CD8+ or anti-IFN-gamma delayed the mortality of mice infected with Pb NK65, although it did not affect the parasitaemia. In the late stage of PB NK65 infection, T cells, especially CD8+ T cells, were increased in number in the liver at the expense of splenic CD8+ T cells. These CD8+ T cells released IFN-gamma in culture without antigen stimulation and were thought to induce tumour necrosis factor-alpha (TNF-alpha) production by the cells in the liver. In mice infected with Pb XAT, or mice primed with Pb XAT and then challenged with Pb NK65, CD4+ T cells had a crucial role in preventing parasite growth and in protective immunity. IFN-gamma was again the key molecule in protective immunity. These results suggest that T cells stimulated with malaria antigen play important roles both in protective immunity and pathogenesis depending upon their subsets; CD8+ T cells in pathogenesis, and CD4+ T cells in protective immunity. These apparently contradictory responses may be mediated by the same cytokine, IFN-gamma.     

Resolution of acute malarial infections by T cell-dependent non-antibody-mediated mechanisms of immunity.

While it is generally accepted that acute blood stage malarial infections are resolved through the actions of protective antibodies, we observed that resistance to acute infection with Plasmodium chabaudi adami was mediated by T cell-dependent cellular immune mechanisms independent of antibody. We now report that acute blood stage infections caused by three additional murine hemoprotozoan parasites, Plasmodium vinckei petteri, Plasmodium chabaudi chabaudi, and Babesia microti, appear to be controlled by similar T cell-dependent mechanisms of immunity. Mice rendered B cell deficient by lifelong treatment with goat anti-mouse immunoglobulin M (IgM) had IgM levels in serum of less than 0.6 micrograms/ml and contained precipitating amounts of goat anti-mouse IgM. When these B cell-deficient mice were infected with blood stage P. vinckei petteri, P. chabaudi chabaudi, or B. microti, they resolved their infections with kinetics similar to those seen in immunologically intact mice. Infected B cell-deficient mice did not produce antiparasite antibodies. As assayed by immunofluorescence, significant titers of parasite-specific antibody were present only in the sera of infected immunocompetent mice. In addition, only sera from infected immunocompetent mice immunoprecipitated metabolically labeled parasite antigens. In contrast to B cell-deficient mice, athymic nude mice failed to resolve acute P. vinckei petteri or B. microti infections. These data suggest that antibody-independent, T cell-mediated immune mechanisms play a more significant role in resisting acute blood stage infections caused by hemoprotozoa than was recognized previously.     

Trypanocidal drugs and their effect on parasitaemia,specific IgG production and protective immunity in rats infected withTrypanosoma lewisi

In rats infected withTrypanosoma lewisi, parasitaemia normally resolves by day 32; thereafter, the rodents become solidly immune to re-infection. Rats treated on day 5 of infection with a single i.m. dose of 35 mg/kg of the trypanocidal drug ethidium bromide (EB) had recovered from parasitaemia by day 12, where-as berenil (BE) given at 100 mg/kg more than twice the recommended dose, had no effect on parasitaemia. However, rats treated for 4 consecutive, days beginning on day 5 of infection with Lampit (LA) and Radanil (RA) at 35- mg/kg showed no parasitaemia on days 16 and 20, respectively. EB was the most effective drug in lowering the total IgG antibody as compared with the control animals, whose specific IgG titres remained elevated for over 200 days after the parasitaemia had been cleared. LA also significantly reduced the antibody levels through day 240, whereas RA only transitorily depressed the antibody levels on days 20 and 30. BE, which had no effect on parasitaemia, correspondingly failed to depress the total IgG levels. Re-challenge infection of the drug-treated, recovered animals on day 240 (208 days after the normal resolution of the infection) revealed that except for the EB group, which displayed transitory parasitaemia for 4 days, other treated and control rats completely resisted the challenge; pre-challenge antibody titres were lower than 1:160 in EB-treated animals in contrast to the levels of 1:320 or higher measured in the other drug-treated and control animals, which resisted the infection. This study indicates that protective immunity in rats infected withT. lewisi is established very early during the epimastigote phase of infection and appears to be related to the levels of specitic circulating antibodies that persist long after infection has been cleared.This study was supported by an operating grant from the Natural Sciences and Engineering Council of Canada, Research at the Institute of Parasitology is supported by the Natural, Sciences and Engineering Research Council of Canada and by the Fonds FCAR pour at la sortien a la recherche. Support for the author was provided by the Kenya General Training Fund and by the Canadian International Development Agency     

Interleukin-12-mediated resistance to Trypanosoma cruzi is dependent on tumor necrosis factor alpha and gamma interferon.

The aim of this study was to determine if interleukin-12 (IL-12) has a role in the immune response to Trypanosoma cruzi. Infection of BALB/c mice with the virulent Tulahuen strain of T. cruzi is characterized by a high-level parasitemia, pathology in the heart associated with the presence of amastigotes, and death during the acute phase of the disease. Administration of IL-12 to BALB/c mice infected with T. cruzi resulted in a reduced parasitemia and a significant delay in the time to death compared with those for infected controls. This protective effect was correlated with increased levels of gamma interferon (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) in serum. To determine if these cytokines were involved in the protective effects of IL-12, we treated infected mice with IL-12 alone or in combination with monoclonal antibodies specific for IFN-gamma or TNF-alpha. These antibodies antagonized the protective effect of exogenous IL-12. Treatment of infected mice with a polygonal antibody specific for IL-12 resulted in a significant increase in parasitemia but did not affect the time to death. These latter studies demonstrate a role for endogenous IL-12 in resistance to T. cruzi. Together, our data identify an IL-12-mediated mechanism of resistance to T. cruzi, which is dependent on IFN-gamma and TNF-alpha.     

Intranasal immunization with multivalent group A streptococcal vaccines protects mice against intranasal challenge infections

We have previously shown that a hexavalent group A streptococcal M protein-based vaccine evoked bactericidal antibodies after intramuscular injection. In the present study, we show that the hexavalent vaccine formulated with several different mucosal adjuvants and delivered intranasally induced serum and salivary antibodies that protected mice from intranasal challenge infections with virulent group A streptococci. The hexavalent vaccine was formulated with liposomes with or without monophosphorylated lipid A (MPL), cholera toxin B subunit with or without holotoxin, or proteosomes from Neisseria meningitidis outer membrane proteins complexed with lipopolysaccharide from Shigella flexneri. Intranasal immunization with the hexavalent vaccine mixed with these adjuvants resulted in significant levels of antibodies in serum 2 weeks after the final dose. Mean serum antibody titers were equivalent in all groups of mice except those that were immunized with hexavalent protein plus liposomes without MPL, which were significantly lower. Salivary antibodies were also detected in mice that received the vaccine formulated with the four strongest adjuvants. T-cell proliferative assays and cytokine assays using lymphocytes from cervical lymph nodes and spleens from mice immunized with the hexavalent vaccine formulated with proteosomes indicated the presence of hexavalent protein-specific T cells and a Th1-weighted mixed Th1-Th2 cytokine profile. Intranasal immunization with adjuvanted formulations of the hexavalent vaccine resulted in significant levels of protection (80 to 100%) following intranasal challenge infections with type 24 group A streptococci. Our results indicate that intranasal delivery of adjuvanted multivalent M protein vaccines induces protective antibody responses and may provide an alternative to parenteral vaccine formulations.