Protective immunity induced by a Trypanosoma cruzi soluble extract antigen in experimental Chagas' disease. Role of interferon gamma

CBA/J mice can be protected against lethal infection with Trypanosoma cruzi by treatment using T. cruzi soluble extract antigen (TCSE). In vivo administration of TCSE (400 μg/mouse) into naive mice increased the cellular proliferative response to Con A and elevated the levels of IFN-γ. The production of IFN-γ was extremely important in controlling the replication of the parasite since the protective activity of TCSE was completely abrogated by in vivo treatment with an anti IFN-γ neutralizing antibody. These results suggest that depending on the level, cytokine production results in the control of replication of the parasite in experimental Chagas'disease.     

IN VITRO RESPONSE OF v-Ha-ras TRANSGENIC MOUSE LYMPHOCYTES AFTER IN VIVO TREATMENT WITH ALCOHOL

Oncomouse is a transgenic mouse carrying an activated v-Ha-ras oncogene under the control of the mouse mammary tumor virus promoter. The objective of this paper was to learn if the in vitro secretion of IL-2 and IFN-γ and the release of sIL-2R by Oncomice splenocytes and thymocytes depended on the presence of the oncogene product, on the in vivo pretreatment with alcohol, or on the in vitro treatment with cocaine or morphine. Oncomice thymocytes released less sIL-2R than FVB thymocytes. Alcohol did not increase sIL-2R release in Oncomice as it did in FVB mice thymocytes. Oncomice thymocytes secreted more IFN-γ than FVB thymocytes, their secretion was downregulated by in vivo treatment with alcohol, while it was upregulated in FVB thymocytes. IFN-γ secretion was lower in Oncomice splenocytes from animals receiving alcohol. Oncomice thymocytes and splenocytes responded in a nearly opposite fashion to their FVB counterparts. Therefore, the in vivo treatment with alcohol modified the in vitro response to cocaine or morphine in an oncogene-dependent and -independent manner. Hence, our results further emphasize the role of v-Ha-ras oncogene in defining the host immune response, and of alcohol in modulating such response.     

PSK and OK-432-induced immunomodulation of inducible nitric oxide (NO) synthase gene expression in mouse peritoneal polymorphonuclear leukocytes and NO-mediated cytotoxicity

We investigated whether PSK (a polysaccharide from the mycelia of Coriolus versicolor) or OK-432 (a streptococcal preparation) can up-regulate inducible nitric oxide synthase (iNOS) gene expression and nitric oxide (NO) production in mouse peritoneal polymorphonuclear leukocytes (PMNs). Six hrs after intraperitoneal injection of mice with PSK (2500 μg/mouse) or OK-432 (100 μg/mouse), mouse peritoneal PMNs were restimulated with PSK (500 μg/ml) or OK-432 (10 μg/ml) plus 100 U/ml of mouse interferon-gamma (IFN-γ) in vitro. Northern blot analysis showed strong synergism between both PSK and OK-432 and IFN-γ for the induction of iNOS gene expression. NO production by PMNs was increased up to 20 μM (2 μM/10⁶ PMNs/24 hrs) as measured by the Griess reagent method when PMNs were restimulated with PSK or OK-432 plus IFN-γ for 24 hrs, although tumor cell killing was not detected. NO concentrations of more than 80 μM were required for P815 tumor cell killing. These results suggest that PMNs produce NO after stimulation with PSK or OK-432 in combination with IFN-γ and may regulate the immune system in vivo, although the NO production induced by these agents is insufficient for tumor cell killing in vitro.     

IN VITRO RESPONSE OF v-Ha-ras TRANSGENIC MOUSE LYMPHOCYTES AFTER IN VIVO TREATMENT WITH COCAINE

Oncomouse is a transgenic mouse carrying an activated v-Ha-ras oncogene under the control of the mouse mammary tumor virus promoter. The objective of this paper was to learn if the in vitro secretion of IL-2 and IFN-γ and the release of sIL-2R by Oncomice spleen and thymus cells depended on the presence of the oncogene product, on the in vivo pretreatment with cocaine, or on the in vitro treatment with cocaine or morphine. Oncomice thymocytes from different experimental groups released less sIL-2R than FVB thymocytes. Oncomice thymocytes secreted more IFN-γ than FVB thymocytes. Oncomice thymocytes cultured in the presence of Con A and cocaine showed a diminished release of sIL-2R and a lower secretion of IFN-γ, a phenomenon not observed in FVB thymocytes. IFN-γ secretion was lower in Oncomice splenocytes. In general, Oncomice thymocytes and splenocytes responded in a nearly opposite fashion to their FVB counterparts. In this study, the in vitro response to mitogens, cocaine or morphine depended on genetic background and not on the in vivo pretreatment with cocaine. Our results emphasize the role of the v-Ha-ras oncogene in defining the host immune response.     

Oral administration of aqueous extract of Carthami Flos induces macrophage activation and preferentially potentiates type 1 helper T-cell response in vivo

In vivo immunomodulatory activity of aqueous extract of Carthami Flos (AECF) was investigated using a mouse model immunized with keyhole limpet hemocyanin. Serum level of Ag-specific IgG2a was significantly elevated by oral administration of AECF but not IgG1. However, no selective B-cell proliferation by AECF was observed in vivo. Ag-specific proliferation and IFN-γ and IL-5 production of draining lymph node T cells also was higher in AECF-treated mice when compared with water-treated control mice. However, AECF failed to enhance nonspecific T-cell response under CD3 stimulation. These results led us to hypothesize that AECF potentiates Ag-specific T-cell response, possibly through activation of antigen presenting cells (APC) other than B cells. Functional assessment of splenic macrophages showed that AECF administration significantly enhances IL-12 production as well as APC activity for IFN-γ production and STAT-4 activation by T cells. Collectively, these data strongly support that AECF preferentially potentiates immune response polarized toward TH1 and for which increased activation of macrophages is most likely to be responsible. The present data implicate a possible application of AECF to potentiate cellular immunity and, we hope, prevent intracellular infections.     

Activation of cytotoxic T lymphocyte responses and attenuation of tumor growth in vivo by Andrographis paniculata extract and andrographolide

The stimulatory effect of Andrographis paniculata extract and andrographolide on cytotoxic T lymphocyte (CTL) production was determined in BALB/c mice by Winn's neutralization assay using CTL sensitive EL4 thymoma cells as target cell. Extract and andrographolide showed a significant increase in CTL production in both the in vivo and in vitro models. The survival time of EL4 cells alone in animals was only 27.1 days and it was increased to 51.1 and 44.5 days in extract- and andrographolide treated animals with percentage increase in life span (%ILS) of 88.5 and 64.2, respectively. The survival rate of animals administered EL4 cells incubated with alloimmunized spleen cells (effector cells) from normal BALB/c mice was 35.8 (%ILS 32.1). When this group was treated with 10 doses of extract and andrographolide the life span was further increased to 52.1 days (%ILS 92.2 ) and 48.1 days (%ILS 77.4). Survival days of animal carrying EL4 cells incubated with alloimmunized spleen cells (effector cells) from extract and andrographolide treated animals were 55.5 and 50.3 days respectively. When these animals continued with extract and andrographolide treatment for 10 days their life spans were significantly increased to 62 and 53.8 days, respectively. The level of cytokines such as Interlevkin (IL)-2 and Interferon (IFN)-γ also was enhanced in these animals when they were treated with extract and andrographolide. This study demonstrated that A. paniculata extract and andrographolide stimulate the CTL production through enhanced secretion of IL-2 and IFN-γ by T cells and thereby inhibit the tumor growth.     

N,N′-Thiophene-substituted polyamine analogs inhibit mammalian host cell invasion and intracellular multiplication of Trypahosoma cruzi

We studied the effects of two, N,N′-thiophene-substituted polyamine analogs (MDL 28302 and MDL 29431) on the capacities of Trypanosoma cruzi, the etiologic agent of Chagas' disease, to invade and multiply within a mammalian host cell. Both compounds inhibited infectivity significantly in a time- and concentration-dependent manner. This inhibition resulted from a selective effect on the parasite, because pretreatment of T. cruzi but not host cell cultures with either MDL 28302 or MDL 29431 reduced infectivity. The parasite gradually recovered its infective capacity after removal of unincorporated polyamine analog, denoting the reversible nature of the inhibitory effect. Some biochemical modification of MDL 28302 and MDL 29431 appeared to be required for their inhibitory activities to be exerted, since the effects of these drugs on T. cruzi infectivity were abrogated by MDL 72527, a drug known to inhibit polyamine oxidase (PAO) activity specifically. Supporting the notion of that products of MDL 28302 and MDL 29431 oxidation by PAO were involved in the activity of these compounds was the finding that PAO competitive substrates (N¹Lacetylspermine and N¹-acetylspermidine) also abolished the inhibition of T. cruzi infectivity mediated by MDL 28302 or MDL 29431. However, we can not rule out that MDL 72527 and the PAO competitive substrates might have altered an alternative mechanism because no significant polyamine oxidase activity could be demonstrated in preparations of lysed or intact T. cruzi in assays monitoring conversion of [¹⁴C]spermine to [¹⁴C]spermidine. When either MDL 28302 or MDL 29431 was added to infected cell cultures, a marked reduction in the rate of intracellular parasite growth ensued. The significance of the finding that N,N′-thiophene-substituted polyamine analogs inhibit cell invasion and cytoplasmic replication by T. cruzi resides in the fact that this pathogenic parasite requires a cytoplasmic localization to replicate in mammalian hosts.     

Mycobacterium vaccae immunization to OVA sensitized pregnant BALB/c mice suppressed placental and postnatal IL-5 and inducing IFN-gamma secretion

Although the development of atopy in the newborn is determined by a multitude of factors, an intense Th1 stimulus early in life could be protective by facilitating a switch away from Th2. Aimed to determine the effect of single Mycobacterium vaccae (M. vaccae) immunization to OVA-sensitized pregnant mice on IL-5 and IFN-γ secretion from placental lymphocytes and splenocytes of offspring.

Pregnant BALB/c mice were divided into 4 groups, OVA-sensitized + M. vaccae immunized, OVA-sensitized, M. vaccae immunized and controls. Sensitization with OVA was initiated before mating, and aerosol OVA challenge were performed during pregnancy. M. vaccae immunization was performed on the 12^th day of pregnancy. IL-5 and IFN-γ levels of placental lymphocytes were analyzed on the 18^th day of pregnancy and splenocytes of offspring on the 2^nd and 28^th days during postnatal period. A single administration of M. vaccae to OVA-sensitized pregnant mice downregulated IL-5 secretion and induced IFN-γ secretion from placental lymphocytes. On the other hand, after M. vaccae immunization downregulation of IL-5 levels and upregulation of IFN-γ secretion persisted in offspring when determined on 2^nd and 28^th days of life. Vaccination with M. Vaccae to OVA-sensitized pregnant BALB/c mice prevented Th2 immune responses by enhancing secretion of IFN-γ and lowering IL-5 levels during pregnancy and the effect persisted during the postnatal period in offspring.     

Interferon-gamma suppresses the growth of Toxoplasma gondii in human fibroblasts through starvation for tryptophan

The effect of human recombinant interferon-γ (IFN-γ) on Toxoplasma gondii in cultured human fibroblasts is predominantly parasitostatic. This effect is dependent upon the induction in the host cell of a potent indoleamine 2,3-dioxygenase that converts tryptophan to N-formylkynurenine. This product is, in turn, degraded to kynurenine by a formamidase. Within 24 h of treatment with IFN-γ most of the tryptophan originally present in the medium is converted to these products together with some minor metabolites. When added to the medium of infected cultures at concentrations equimolar to the tryptophan content, neither N-formylkynurenine nor kynurenine suppresses the growth of T. gondii, although at higher concentrations they are effective. The medium of uninfected cultures treated with IFN-γ for 24 h has no effect on the growth of T. gondii, when transferred to fresh cultures provided that the residual IFN-γ is first removed by ultrafiltration or neutralized with a specific monoclonal antibody. Thus minor metabolites produced from tryptophan in response to IFN-γ and excreted into the medium are not parasitostatic. When cultures treated with IFN-γ for 24 h are incubated with medium that contains [³H]tryptophan, the radioactive amino acid is converted to N-formylkynurenine and kynurenine as rapidly as it enters the cell. This degradation not only results in a very low intracellular concentration of tryptophan but also produces intracellular concentrations of tryptophan metabolites that are significantly higher than the tryptophan concentration in control cells. However, it is unlikely that either metabolite reaches intracellular concentrations that are sufficient to suppress the growth of the parasite. The parasitostatic effect of IFN-γ is most likely to result from the starvation of T. gondii for tryptophan.     

Human Lymphocyte Stimulation by Legume Lectins from the Diocleae Tribe

Lectins from eight leguminous seeds from the Diocleae tribe were compared to Concanavalin A (Con A), a well known T cell mitogen, on the stimulation of lymphocyte proliferation and Interferon γ (IFN-γ) production by peripheral blood mononuclear cells (PBMC) from normal volunteers. Lectins from Canavalia brasiliensis and Dioclea virgata induced the highest lymphocyte proliferation, both much higher than levels obtained with Con A, whereas lectins from Dioclea guianensis var. lasiophylla and from Canavalia bonariensis induced the lowest stimulation. Lectins from Dioclea rostrata, D. grandiflora, D. violacea and Cratylia floribunda induced intermediate levels of proliferation. The highest stimulation for IFN-γ production was obtained with the lectin from D. rostrata, followed by those of C. floribunda and C. brasiliensis: only the lectins from D. virgata and C. bonariensis induced an IFN-γ production lower than the one obtained by Con A-stimulation. Since all these legumes belong to the same tribe of C. ensiformis (Con A), and all are supposed to exhibit very similar lectins, it is interesting the high variation in the stimulation of lymphocyte proliferation. It is also noteworthy the dissociation between this parameter and IFN-γ production in the case of D. virgata. A detailed analysis on the structure of such lectins, and their ligand sugars on lymphocyte surface is necessary to further explore such differences.     

Natural Killer Cell Activity, Lymphocyte Proliferation, and Cytokine Profile in Tumor-Bearing Mice Treated with MAPA, a Magnesium Aggregated Polymer from Aspergillus oryzae

The present study examined the effects of MAPA, an antitumor aggregated polymer of protein magnesium ammonium phospholinoleate-palmitoleate anhydride, isolated from Aspergillus oryzae, on concanavalin A (Con A)-induced spleen cell proliferation, cytokine production and on natural killer (NK) cell activity in Ehrlich ascites tumor-bearing mice. The Ehrlich ascites tumor (EAT) growth led to diminished mitogen-induced expansion of spleen cell populations and total NK activity. This was accompanied by striking spleen enlargement, with a marked increase in total cell counts. Moreover, a substantial enhancement in IL-10 levels, paralleled by a significant decrease in IL-2 was observed, while production of IL-4 and interferon-γ (IFN-γ) was not altered. Treatment of mice with 5 mg/kg MAPA for 7 days promoted spleen cell proliferation, IL-2 production and NK cell activity regardless of tumor outgrowth. In addition, MAPA treatment markedly enhanced IFN-γ levels and reduced IL-10 production relative to EAT mice. A 35% reduction in splenomegaly with normal number of nucleated cells was also found. Altogether, our results suggest that MAPA directly and/or indirectly modulates immune cell activity, and probably disengages tumor-induced suppression of these responses. Clearly, MAPA has an impact and may delay tumor outgrowth through immunotherapeutic mechanisms.     

T-cell Ig and mucin domain-containing protein (Tim)-2 regulates murine allergic conjunctivitis during the effector phase

T-cell Ig and mucin domain-containing protein (Tim)-2 is associated with Th2-dependent immune responses. Here, we investigated whether administration of anti-Tim-2 Abs affects the development of murine experimental allergic conjunctivitis (EC), a Th2-mediated disease. While treatment with anti-Tim-2 Abs in vivo during the induction phase did not affect the severity of EC, treatment during the effector phase augmented EC. The in vitro stimulation of ragweed (RW)-primed splenocytes with RW in the presence of anti-Tim-2 Abs induced significantly more IL-5 and IL-13 production but significantly less IFN-γ production compared with the control splenocytes treated with normal rat IgG. In addition, the transfer of the anti-Tim-2 Ab-treated splenocytes induced significantly more severe EC than the control splenocytes. Thus, Tim-2 negatively regulates the Th2 differentiation of Ag-primed T-cells and the development of EC during the effector phase.     

Purification and characterization of a soluble nucleoside diphosphate kinase in Trypanosoma cruzi

A soluble nucleoside diphosphate kinase (NDP kinase) was purified and characterized in epimastigote forms of Trypanosoma cruzi. The enzyme was purified by affinity chromatography on Blue-agarose and Q-Sepharose columns and by FPLC on a Superose 12 column. A membrane-associated NDP kinase was identified which accounts for 30% of total enzymatic activity. Western blot analysis of the soluble NDP kinase revealed a 16.5-kDa monomer recognized by polyclonal antibodies to NDP kinase from Dictyostelium discoideum, Candida albicans or human. Most of the T. cruzi NDP kinase is found in the cell as a hexamer composed of 16.5-kDa monomers. The K_m values of the enzyme for ATP, GDP and dTDP were 0.2 ± 0.008 mM, 0.125 ± 0.012 mM and 0.4 ± 0.009 mM, respectively. The parasite enzyme was stable, remained active at 65°C and was found to tolerate up to 2.5 M urea. The 16.5-kDa subunit was phosphorylated with [γ-³²P]ATP or thiophosphorylated with [³⁵S]GTPγS. The incubation of the ³²P-labelled phosphoenzyme with unlabelled nucleoside 5′-diphosphates resulted in the formation of ³²P-labelled nucleoside 5′-triphosphates without strict base specificity, indicating that the reaction mechanism of the T. cruzi enzyme is the same as reported for other NDP kinases. When the phosphoenzyme was incubated with a mixture of nucleoside 5′-diphosphates, GTP was preferentially formed.     

Treatment of H. pylori infected mice with antioxidant astaxanthin reduces gastric inflammation, bacterial load and modulates cytokine release by splenocytes

Helicobacter pylori is a Gram-negative bacterium affecting about half of the world population, causing chronic gastritis type B dominated by activated phagocytes. In some patients the disease evolves into gastric ulcer, duodenal ulcer, gastric cancer or MALT lymphoma. The pathogenesis is in part caused by the immunological response. In mouse models and in human disease, the mucosal immune response is characterized by activated phagocytes. Mucosal T-lymphocytes are producing IFN-γ thus increasing mucosal inflammation and mucosal damage. A low dietary intake of antioxidants such as carotenoids and vitamin C may be an important factor for acquisition of H. pylori by humans. Dietary antioxidants may also affect both acquisition of the infection and the bacterial load of H. pylori infected mice. Antioxidants, including carotenoids, have anti-inflammatory effects. The aim of the present study was to investigate whether dietary antoxidant induced modulation of H. pylori in mice affected the cytokines produced by H. pylori specific T-cells. We found that treatment of H. pylori infected mice with an algal cell extract containing the antioxidant astaxanthin reduces bacterial load and gastric inflammation. These changes are associated with a shift of the T-lymphocyte response from a predominant Th1-response dominated by IFN-γ to a Th1/Th2-response with IFN-γ and IL-4. To our knowledge, a switch from a Th1-response to a mixed Th1/Th2-response during an ongoing infection has not been reported previously.     

Differential tissue distribution of diverse clones of Trypanosoma cruzi in infected mice.

Chagas disease, caused by the protozoan Trypanosoma cruzi, presents variable clinical course but the phenomena underlying this variability remain largely unknown. T. cruzi has a clonal population structure and infecting strains are often multiclonal. T. cruzi genetic variability could be a determinant of differential tissue tropism or distribution and consequently of the clinical forms of the disease. We tested this hypothesis by using low-stringency single specific primer polymerase chain reaction (LSSP-PCR) to type genetically the parasites in tissues of experimental infected mice. BALB/c mice were simultaneously inoculated with two different T. cruzi populations (JG strain and Col1.7G2 clone). Doubly infected animals showed clear differential tissue distribution for the two populations (chronic phase). Our results indicate a significant influence of the genetic polymorphism of infecting T. cruzi populations in the pathogenesis of chronic Chagas disease.     

Effect of Endotoxin on Cytokine Production and Cell Dynamics in Mice

Previous studies have shown that endotoxin potentiates immune responses by direct stimulation of B cells and macrophages. In the present study, we assessed the ability of endotoxin to stimulate cells from different lymphoid tissue compartments to release cytokines. The in vitro stimulation of macrophages with endotoxin resulted in the production of IL-1 and TNF-$aL in a dose and time-dependent manner. Endotoxin also induced the production of IL-2, IFN-γ and IL-4 secretion in a dose dependent manner in cultured spleen, mesenteric lymph nodes and Peyer's patches cells. The intraperitoneal administration of endotoxin in mice resulted in the accumulation of leucocytes in the peritoneal cavity and in the increase of IL-1, IL-6, TNF-$aL and IFN-γ concentration in serum. In conclusion, this study confirmed that endotoxin possesses immunomodulatory activities capable of stimulating immune functions both in vitro and in vivo.     

Effect of foot shock stress on the interferon-gamma production of murine intestinal intraepithelial lymphocytes

To investigate the effect of stress on interferon (IFN)-γ production by intestinal intraepithelial lymphocytes (IEL), we exposed male C3H/HeN mice to electric foot shock for 30 min a day for 5 consecutive days. Immediately after the final foot shock stress, IEL from small intestine were isolated by Percoll density gradient. The stress induced a marked suppression of IFN-γ production by IEL stimulated with immobilized anti-CD3 mAb and a marked decrease in the proportion of IFN-γ-producing CD3⁺ IEL or αβTCR⁺ IEL stimulated with PMA + ionomycin. The αβTCR⁺ subset was the major cause of stress-induced suppression of IFN-γ production by IEL. Glucocorticoid induced the suppression of IFN-γ production by IEL in vitro, which was reversed by mifepristone (RU486), a glucocorticoid receptor antagonist. In vivo administration of RU486 reversed the stress-induced suppression of IFN-γ production by IEL. In conclusion, repeated foot shock stress suppressed IFN-γ production of IEL by stress-induced elevation of endogenous glucocorticoid. Substantial suppression of the αβTCR⁺ subset was the major cause of the suppression.     

Immune activation by a sterile aqueous extract of Cordyceps sinensis: mechanism of action

Cordyceps sinensis is a fungus that has been used for over 2,000 years in China as a treatment for a variety of conditions including infectious diseases. The available evidence suggests a hypothesis that any efficacy of C. sinensis as an anti-infective therapeutic would be related to a role as an activator of innate immune responses. The objectives of this study were first to investigate the ability of C. sinensis to activate pro-inflammatory responses in macrophages in vitro and induce protective responses against intracellular pathogens in vivo, and second to characterize a method of action. We found that C. sinensis activates murine macrophages to produce a variety of pro-inflammatory cytokines. IFN-γ synergizes with C. sinensis to amplify this response. Bacterial endotoxin contamination was ruled out as a potential artefact. The evidence presented in this study supports a hypothesis that C. sinensis activates macrophages by engaging Toll-like receptors and inducing mitogen-activated protein kinase (MAPK) pathways characteristic of inflammatory stimuli.