Validation of immunity induced by inactivated CCPP vaccine with different adjuvants

The study was conducted in the premises National Veterinary Institute (NVI) to validate the immunity induced by inactivated F38 antigen adjuvated with saponin and Montanide ISA 50 and combined with and without anthrax vaccine.Post-inoculation reactions; pyrogenic effects, safety and inocuity of the vaccines were assessed. Increased body temperature and local edematous reactions were seen in animals inoculated with saponin adjuvated CCPP vaccine (100%) while 20% of the goats in ISA 50 adjuvated group showed local reaction. Sera collected from day 0 to 10th week were tested to assess the sero-conversion using monoclonal antibody based B-ELISA technique. Saponin adjuvated groups, in both monovalent CCPP and in the combined CCPP with anthrax vaccine showed a higher mean percentage of inhibition value as compared with ISA 50 adjuvated vaccine.After 8 months of post vaccination, contact challenge trial was conducted in 66 experimentally vaccinated and 20 negative control goats combined with 15 actively CCPP sick goats. Various clinical signs were recorded daily, autopsy was done on died goats and the live goats were sacrificed after 2 months of contact. The side by side samples from thoracic exudates, lung and mediastinal and bronchial lymph nodes were collected from goats shown to have developed indicative CCPP lesion for isolation and F38 antigen detection.The present experimental study indicated that application of inactivated and adjuvated CCPP vaccine significantly reduced the morbidity and development of lesions (P < 0.001). Among vaccinated groups CCPP + anthrax + saponin showed better protection, with low rate of nasal discharge and cough at 33% and 28.6%, respectively, and protection level of 94.1% from death and 65% from lung lesion development. However, the variation in protection among the vaccinated groups was not significant (P > 0.05).These findings disclosed that inactivated CCPP vaccine adjuvated with saponin and ISA 50 significantly reduce morbidity and mortality of goats due to CCPP and also indicated the importance of utilization of ISA 50 as alternative adjuvant to minimize post-vaccinal reactions encountered in use of saponin as adjuvant.     

Combined chemotherapy of platinum and fluorouracil promotes T cell–mediated antitumor immunity

The two-drug combined chemotherapy of platinum and fluorouracil has been reported to efficiently kill tumor cells as the first-line treatment for advanced gastric cancer.However,the effect of these drugs on T cells remains unclear.Here,we showed that T cells including CD4+T cells and CD8+T cells of the patients with advanced gastric cancer after platinum and fluorouracil chemotherapy exhibited enhanced ex vivo proliferation ability as compared to that before chemotherapy.In addition,platinum and fluorouracil also promoted the differentiation of human T cells into Th1 and Th9 subtypes and cytotoxic T lymphocytes(CTLs)in vitro and in vivo.Accordingly,the combination therapy greatly suppressed tumor growth with increased tumor infiltration of Th1,Th9,and CTL cells in a mouse tumor model.Moreover,in activated T cells,long-term treatment with these two drugs further facilitates T cell activation along with promoted nuclear factor-κB(NF-κB)activation.Our findings demonstrate a previously unidentified function of platinum and fluorouracil combination chemotherapy in promoting T cell–mediated antitumor immunity.     

Caspase-8 activity prevents type 2 cytokine responses and is required for protective T cell-mediated immunity against Trypanosoma cruzi infection

During Trypanosoma cruzi infection, T cells up-regulate caspase-8 activity. To assess the role of caspase-8 in T cell-mediated immunity, we investigated the effects of caspase-8 inhibition on T cells in viral FLIP (v-FLIP) transgenic mice. Compared with wild-type controls, increased parasitemia was observed in v-FLIP mice infected with T. cruzi. There was a profound decrease in expansion of both CD4 and CD8 T cell subsets in the spleens of infected v-FLIP mice. We did not find differences in activation ratios of T cells from transgenic or wild-type infected mice. However, the numbers of memory/activated CD4 and CD8 T cells were markedly reduced in v-FLIP mice, possibly due to defective survival. We also found decreased production of IL-2 and increased secretion of type 2 cytokines, IL-4 and IL-10, which could enhance susceptibility to infection. Similar, but less pronounced, alterations were observed in mice treated with the caspase-8 inhibitor, zIETD. Furthermore, blockade of caspase-8 by zIETD in vitro mimicked the effects observed on T. cruzi infection in vivo, affecting the generation of activated/memory T cells and T cell cytokine production. Caspase-8 is also required for NF-kappaB signaling upon T cell activation. Blockade of caspase-8 by either v-FLIP expression or treatment with zIETD peptide decreased NF-kappaB responses to TCR:CD3 engagement in T cell cultures. These results suggest a critical role for caspase-8 in the establishment of T cell memory, cell signaling, and regulation of cytokine responses during protozoan infection.     

Identification of effector T cells responsible for enhanced antitumor immunity induced by tumor vaccine and pyran copolymer

Summary The combination of concanavalin A-bound, but not concanavalin A-free, L1210 murine leukemia vaccine and pyran copolymer produced an enhanced therapeutic response in L1210-bearing mice. Immunoprophylactic experiments showed that the effective combination produced enhanced antitumor immunity as determined by refractoriness to the inoculation of live L1210 cells. In vitro antiproliferation and intraperitoneal adoptive transfer tests showed that antitumor effector cells were detected in the spleen and peritoneal cavity of primed mice after, but not before, live L1210 cell inoculation. These effector cells were identified as T cells on the basis of their non-adherence to plastic flasks and sensitivity to treatment with anti-mouse brain-associated T cell antigen antisera and complement. Although non-T cell populations, including macrophages of mice primed with L1210 vaccine and pyran copolymer, inhibited the in vitro proliferation of L1210 cells, we obtained evidence suggesting that they were not the primary antitumor effector cell populations responsible for the in vivo elimination of the inoculated L1210 cells.     

Mechanisms underlying immunosuppression induced by Trypanosoma cruzi

Trypansoma cruzi affects immune responsiveness in mammalian hosts. Studies with patients and infected animals have defined some of the immunological dysfunctions but not the underlying mechanisms. Recent work using an in vitro model system of T. cruzi-human lymphocyte interactions has made it possible to uncover specific alterations in human lymphocyte activation induced by this parasite. Felipe Kierzenbaum and Marcelo Sztein discuss recent advances in our understanding of the processes that lead to impaired human lymphocyte function and that might be involved in the immunosuppression seen in the acute phase of Chagas disease.     

IgA-driven T cell-mediated anti-bacterial immunity in man after live oral Ty 21a vaccine

Cellular immunity against Salmonella typhi was observed by using a direct anti-bacterial in vitro assay in volunteers orally vaccinated with the live S. typhi mutant strain Ty 21a. With this experimental approach, it was demonstrated that Ty 21a vaccine also induces cellular immunity against S. paratyphi A and B. Interestingly, the mechanism involved in cellular immunity against bacteria seems to be of an antibody-dependent cellular cytotoxicity (ADCC) type, with IgA acting as the humoral arm and CD4+ T lymphocytes as the cellular one. In accordance with the increase in IgA-driven ADCC against S. typhi, a major rise in IgA against O and H antigens was observed in the serum of vaccinees in parallel to an increase in IgG of identical specificity. Furthermore, a Ty 21 vaccine induced cellular activity against flagellar antigens. These results indicate that IgA-ADCC by T lymphocytes against bacteria can originate from local stimulation of the gut mucosal immune system. This cellular defense mechanism might be at the origin of the protection induced by Ty 21a vaccine.     

No intrinsic deficiencies in CD8+ T cell-mediated antitumor immunity with aging

Aging is associated with a decline in immune function, particularly within the T cell compartment. Because CD8(+) T cells are critical mediators of protective immunity against cancer, which arises more frequently with advancing age, it is important to understand how aging affects T cell-based antitumor responses. We used our DUC18 T cell/CMS5 tumor model system to examine the ability of both aged APCs and aged, tumor-specific CD8(+) T cells to mount protective responses to tumors in vivo. An assessment of aged DUC18 T cells in vitro showed a naive phenotype, but impaired proliferation in response to anti-CD3 and anti-CD28 stimulation. We found that DCs from young and old recipient mice are comparable phenotypically, and endogenous APCs in these mice are equally able to prime adoptively transferred young DUC18 T cells. Even when aged DUC18 T cells are transferred into aged CMS5-challenged mice, Ag-specific proliferation and CD25 expression are similar to those found when young DUC18 T cells are transferred into young mice. Although trafficking to tumor sites appears unequal, old and young DUC18 T cells reject primary CMS5 challenges to the same degree and with similar kinetics. Overall, we found no loss of endogenous APC function or intrinsic defects in CD8(+) DUC18 T cells with advanced age. Therefore, when young and old tumor-specific T cell populations are equivalently sized, CD8(+) T cell-mediated antitumor immunity in our system is not impaired by age, a finding that has positive implications for T cell-based immunotherapies.     

TNF is essential for the cell-mediated protective immunity induced by the radiation-attenuated schistosome vaccine.

C57BL/6 mice exposed to the radiation-attenuated schistosome vaccine exhibit high levels of protective immunity. The cell-mediated pulmonary effector mechanism involves IFN-gamma-producing CD4+ T cells in a focal response around challenge larvae. IFN-gamma can promote production of TNF and can synergize with this cytokine in its actions on responder cells. We have examined whether TNF plays a role in lung phase immunity to schistosomes using mice with a disrupted gene for TNFRI (TNFRI-/-). The most dramatic finding was that the schistosome vaccine elicited no protection whatsoever in these mice. However, this could not be attributed to a lack of responder cells, because more lymphocytes were lavaged from the airways of TNFRI-/- than wild-type mice. Furthermore, CD4+ T cells were equally represented in airway populations from the two groups and produced IFN-gamma upon Ag stimulation in vitro. In contrast, pulmonary macrophage function was defective in TNFRI-/- mice, as indicated by a failure to up-regulate inducible NO synthase mRNA. Histopathological analysis revealed that focal infiltrates were of similar size and cell composition in the two groups but that more parasites were free of foci in the TNFRI-/- mice. These animals had a greatly impaired IgG response to schistosomes, which may explain their lack of residual protection due to Ab in a situation where cell-mediated immunity is disabled. We suggest that the absence of protective immunity could result from a retarded build-up of leukocytes around migrating lung worms and/or a deficit in accessory cell function within a focus, both of which would permit parasite escape.     

Trypanosoma cruzi: early resistance induced by culture-derived trypomastigotes

Previous observations in this laboratory showed that injection of culture-derived trypomastigotes (CT), in CBA/J mice, induced an early increased resistance that was detected 24-72 hr after antigen injection and permitted mice to survive a challenge of 10(5) blood trypomastigotes (BT) corresponding to 2000 LD50%. Present experiments were conducted to determine the optimal conditions for inducing this early resistance and to investigate the early morphological changes which occurred in blood and lymphoid organs of mice infected with either BT or CT. Among nine antigens tested, only living CT showed a protective effect permitting most of mice to survive 30 days after BT challenge, while control mice injected with PBS or other antigens died at 10 +/- 1 days. A dose-response relationship was seen when different doses of CT were tested, higher doses of CT inducing higher survival and lower parasitemia. Injection of CT by either an im or ip route induced similar degrees of resistance but significantly different results were obtained when mice were challenged by using ip or im routes. Higher parasitemia and lower survival were always obtained when animals were challenged by the ip route. Within 72 hr, mice injected with BT presented a lymphopenia which reached a maximum at 48 hr, a depletion of thymic cortical zone, and splenomegaly with hyperplasia of the white pulp and congestion of the red pulp. No gross alterations were observed in animals infected with CT. Overall data suggest that the early resistance is a specifically induced phenomenon and that BT and CT induce different early reactions in the CBA/J lymphoid organs.(ABSTRACT TRUNCATED AT 250 WORDS)     

TARC and RANTES enhance antitumor immunity induced by the GM-CSF-transduced tumor vaccine in a mouse tumor model

INTRODUCTION: Transduction of the granulocyte-macrophage colony stimulating factor (GM-CSF) gene into mouse tumor cells abrogates their tumorigenicity in vivo. Our previous report demonstrated that gene transduction of GM-CSF with either TARC or RANTES chemokines suppressed in vivo tumor formation. In this paper, we examined whether the addition of either recombinant TARC or RANTES proteins to irradiated GM-CSF-transduced tumor vaccine cells enhanced antitumor immunity against established mouse tumor models to examine its future clinical application. MATERIALS AND METHODS: Three million irradiated WEHI3B cells retrovirally transduced with murine GM-CSF cDNA in combination with either recombinant TARC or RANTES were subcutaneously inoculated into syngeneic WEHI3B-preestablished BALB/c mice. RESULTS: Vaccinations were well tolerated. Mice treated with GM-CSF-transduced cells and the chemokines demonstrated significantly longer survival than mice treated with GM-CSF-transduced cells alone. Splenocytes harvested from mice treated with the former vaccines produced higher levels of IL-4, IL-6, IFN-gamma, and TNF-alpha, suggesting enhanced innate and adaptive immunity. Immunohistochemical analysis of tumor sections after vaccination revealed a more significant contribution of CD4+ and CD8+ T cells to tumor repression in the combined vaccine groups than controls. CONCLUSIONS: TARC and RANTES enhance the immunological antitumor effect induced by GM-CSF in mouse WEHI3B tumor models and may be clinically useful.     

Endothelial cell signalling induced by trans-sialidase from Trypanosoma cruzi

The protozoan responsible for Chagas' disease, Trypanosoma cruzi , expresses on its surface an unusual trans -sialidase enzyme thought to play an important role in host–parasite interactions. Trans -sialidase is the product of a multigene family encoding both active and inactive proteins. We have demonstrated that despite lacking enzymatic activity due to a single mutation, Tyr342-His, inactive trans -sialidase displays sialic acid binding activity, with identical specificity to that of its active analogue. In this work we demonstrate that binding of a recombinant inactive trans -sialidase to molecules containing α2,3-linked sialic acid on endothelial cell surface triggers NF-κB activation, expression of adhesion molecules and upregulation of parasite entry into host cells. Furthermore, inactive recombinant trans -sialidase blocks endothelial cell apoptosis induced by growth factor deprivation. These results suggest that inactive members of the trans -sialidase family play a role in endothelial cell responses to T. cruzi infection.     

Blockade of B7-H1 improves myeloid dendritic cell-mediated antitumor immunity

Suppression of dendritic cell function in cancer patients is thought to contribute to the inhibition of immune responses and disease progression. Molecular mechanisms of this suppression remain elusive, however. Here, we show that a fraction of blood monocyte-derived myeloid dendritic cells (MDCs) express B7-H1, a member of the B7 family, on the cell surface. B7-H1 could be further upregulated by tumor environmental factors. Consistent with this finding, virtually all MDCs isolated from the tissues or draining lymph nodes of ovarian carcinomas express B7-H1. Blockade of B7-H1 enhanced MDC-mediated T-cell activation and was accompanied by downregulation of T-cell interleukin (IL)-10 and upregulation of IL-2 and interferon (IFN)-gamma. T cells conditioned with the B7-H1-blocked MDCs had a more potent ability to inhibit autologous human ovarian carcinoma growth in non-obese diabetic-severe combined immunodeficient (NOD-SCID) mice. Therefore, upregulation of B7-H1 on MDCs in the tumor microenvironment downregulates T-cell immunity. Blockade of B7-H1 represents one approach for cancer immunotherapy.     

Induction of T cell-mediated immunity using a c-Myb DNA vaccine in a mouse model of colon cancer

Overexpression of the proto-oncogene c-Myb occurs in more than 80% of colorectal cancer (CRC) and is associated with aggressive disease and poor prognosis. To test c-Myb as a therapeutic target in CRC we devised a DNA fusion vaccine to generate an anti-CRC immune response. c-Myb, like many tumor antigens, is weakly immunogenic as it is a "self" antigen and subject to tolerance. To break tolerance, a DNA fusion vaccine was generated comprising wild-type c-Myb cDNA flanked by two potent Th epitopes derived from tetanus toxin. Vaccination was performed targeting a highly aggressive, weakly immunogenic, subcutaneous, syngeneic, colon adenocarcinoma cell line MC38 which highly expresses c-Myb. Prophylactic intravenous vaccination significantly suppressed tumor growth, through the induction of anti-tumor immunity for which the tetanus epitopes were essential. Vaccination generated anti-tumor immunity mediated by both CD4(+) and CD8(+) T cells and increased infiltration of immune effector cells at the tumor site. Importantly, no evidence of autoimmune pathology in endogenous c-Myb expressing tissues was detected as a consequence of breaking tolerance. In summary, these results establish c-Myb as a potential antigen for immune targeting in CRC and serve to provide proof of principle for the continuing development of DNA vaccines targeting c-Myb to bring this approach to the clinic.     

Use of proteoliposome as a vaccine against Trypanosoma cruzi in mice

We have generated proteoliposomes carrying proteins of Trypanosoma cruzi for use as immunogens in BALB/c mice. T. cruzi trypomastigote and amastigote forms were sonicated and mixed with SDS, with 94% recovery of soluble proteins. To prepare proteoliposomes, we have used a protocol in which dipalmitoylphosphatidylcholine, dipalmitoyl-phosphatidylserine and cholesterol were incubated with the parasite proteins. BALB/c mice immunized with 20microg were able to generate antibodies which, in Western blotting, reacted with the proteins of T. cruzi. We further investigated the ability of peritoneal cells from immunized mice to arrest the intracellular replication of trypomastigotes, in vitro. After 72h of culture, the number of intracellular parasites in immunized macrophages decreased significantly, as compared to controls. Despite the fact that exposure of mice to T. cruzi proteins incorporated into proteoliposomes generate antibodies and activate macrophages, the immunized mice were not protected against T. cruzi intraperitoneal challenge.     

Phagocytosis of Trypanosoma cruzi by Human Polymorphonuclear Leukocytes1

Phagocytosis of culture forms of Trypanosoma cruzi was assayed by a radioisotopic method. Purified polymorphonuclear leukocytes (PMN) were mixed with ³H-uridine-labeled T. cruzi epimastigotes in the presence or absence of anti-T. cruzi antibodies. The reaction was stopped by adding N-ethyl-maleimide, and noningested parasites were lysed by complement. The percentage of radioactivity incorporated into the PMN pellet was recorded. The phagocytosis reaction was rapid, yielding maximum incorporation at 30 min at which point the radioactivity associated with the PMN cells decreased through release of the isotope to the supernatant. The degree of incorporation of radio-labeled parasites was a function of the effector/target cell ratio and the antibody concentration. The method is suitable for the quantitative determination of phagocytosis of T. cruzi by normal PMN.     

Programmed cell death in Trypanosoma cruzi induced by Bothrops jararaca venom

Cells die through a programmed process or accidental death, know as apoptosis or necrosis, respectively. Bothrops jararaca is a snake whose venom inhibits the growth of Trypanosoma cruzi epimastigote forms causing mitochondrion swelling and cell death. The aim of the present work was to determine the type of death induced in epimastigotes of T. cruzi by this venom. Parasite growth was inhibited after venom treatment, and 50% growth inhibition was obtained with 10 microg/ml. Ultrastructural observations confirmed mitochondrion swelling and kinetoplast disorganization. Furthermore, cytoplasmic condensation, loss of mitochondrion membrane potential, time-dependent increase in phosphatidylserine exposure at the outer leaflet plasma membrane followed by permeabilization, activation of caspase like protein and DNA fragmentation were observed in epimastigotes throughout a 24 h period of venom treatment. Taken together, these results indicate that the stress induced in epimastigote by this venom, triggers a programmed cell death process, similar to metazoan apoptosis, which leads to parasite death.     

Different signaling pathways are involved in cardiomyocyte survival induced by a Trypanosoma cruzi glycoprotein

We have recently reported that Trypanosoma cruzi infection protects cardiomyocytes against apoptosis induced by growth factor deprivation. Cruzipain, a major parasite antigen, reproduced this survival effect by a Bcl-2-dependent mechanism. In this study, we have investigated the molecular mechanisms of cruzipain-induced cardiomyocyte protection. Neonatal BALB/c mouse cardiac myocytes were cultured under minimum serum conditions in the presence of cruzipain or T. cruzi (Tulahuen strain). Some cultures were pretreated with the phosphatidylinositol 3-kinase (PI3K) inhibitor Ly294002 or specific inhibitors of the mitogen-activated protein kinase (MAPK) family members such as the mitogen-activated protein kinase kinase (MEK1) inhibitor PD098059, Jun N-terminal kinase (JNK) inhibitor SP600125, p38 MAPK inhibitor SB203580. Inhibition of PI3K and MEK1 but not JNK or p38 MAPK increased the apoptotic rate of cardiomyocytes treated with cruzipain. Phosphorylation of Akt, a major target of PI3K, and ERK1/2, MEK1-targets, was achieved at 15 min and 5 min, respectively. In parallel, these kinases were strongly phosphorylated by T. cruzi infection. In cultures treated with cruzipain, cleavage of caspase 3 was considerably diminished after serum starvation; Bcl-2 overexpression was inhibited by PD098059 but not by Ly294002, whereas Bad phosphorylation and Bcl-xL expression were increased and differentially modulated by both inhibitors. The results suggest that cruzipain exerts its anti-apoptotic property in cardiac myocytes at least by PI3K/Akt and MEK1/ERK1/2 signaling pathways. We further identified a differential modulation of Bcl-2 family members by these two signaling pathways.