Protective immunity against Mycobacterium leprae is dependent on M. leprae-reactive T lymphocytes. M. lepare-directed T cell reactivity is high in the localized tuberculoid form of leprosy but specifically absent in the disseminated lepromatous type of the disease. Two important questions that are relevant for the understanding of the immune response in leprosy as well as for the design of rational immunoprophylaxis and -therapy strategies are: (a) what are the antigens that trigger T cell responses in tuberculoid patients and thus protect these individuals from developing lepromatous leprosy and (b) is it possible to restore T cell responsiveness to M. leprae in lepromatous patients by rechallenging the immune system with selected antigens that will trigger help but not suppression? We have addressed these question by directly probing the peripheral T cell repertoire of 10 tuberculoid and 18 lepromatous patients with large numbers of different M. leprae and BCG antigenic components that had been separated on the basis of their relative molecular mass (Mr) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotted onto nitrocellulose. This technique allows the identification of T cell-stimulating antigens independent of the expression of B cell epitopes by these antigens. So far T cell epitopes have only been mapped on M. leprae proteins that had previously been defined by antibodies. Our results show that: (a) tuberculoid patients' T cells responded preferentially to M. leprae and BCG antigens in the lower (i.e. less than 70 kDa) Mr range with a peak in the 10-25 kDa range; (b) 6 out of 18 lepromatous patients that did not respond to whole M. leprae responded strongly to isolated M. leprae components; antigens in the lower Mr. range were recognized by five out of these six patients and thus commonly seen by both tuberculoid and lepromatous patients' T cells; however, antigens in the higher Mr range, in particular greater than 150 kDa, were only recognized by lepromatous patients' T lymphocytes; (c) furthermore, the T and B cell repertoires in leprosy patients are skewed towards different antigenic fractions.
Introduction Because of its restricted distribution in normal tissues and its high expression on tumors of neuroectodermal origin, GD2 ganglioside is an excellent target for active specific immunotherapy. However, GD2 usually elicits low-titered IgM and no IgG or cellular immune responses, limiting its usefulness as a vaccine for cancer patients. We have previously shown that anti-idiotypic monoclonal antibody mimics of GD2 can induce antigen-specific humoral and cellular immunity in mice, but inhibition of tumor growth by the mimics could not be detected. Methods and results Here, we isolated two peptides from phage display peptide libraries by panning with GD2-specific mAb ME361. The peptides inhibited binding of the mAb to GD2. When coupled to keyhole limpet hemocyanin (KLH) or presented as multiantigenic peptides in QS21 adjuvant, the peptides induced in mice antibodies binding specifically to GD2 and delayed-type hypersensitive lymphocytes reactive specifically with GD2-positive D142.34 mouse melanoma cells. Induction of delayed-type hypersensitivity (DTH) reaction was dependent on CD4-positive lymphocytes. The immunity elicited by the peptides significantly inhibited growth of GD2-positive melanoma cells in mice. Conclusion Our study suggests that immunization with peptides mimicking GD2 ganglioside inhibits tumor growth through antibody and/or CD4-positive T cell-mediated mechanisms. Cytolytic T lymphocytes most likely do not play a role. Our results provide the basis for structural analysis of carbohydrate mimicry by peptides.
The purpose of this study was to determine the cytokine profile in 21 dogs with canine immune-mediated polyarthritis (IMA) and 15 dogs with osteoarthritis (OA) caused by cranial cruciate ligament rupture (CCLR). The mRNA expression of interleukin (IL)-1alpha, IL-1beta, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-18, interferon (IFN)-gamma, transforming growth factor (TGF)-beta, and tumour necrosis factor (TNF)-alpha were analysed in synovial fluid by semi-quantitative RT-PCR, while TNF-alpha protein was determined by L929 cytotoxicity assay. The frequency of lymphocytes was analysed using FACScan. Both disorders reveal a similar cytokine expression pattern, except for significant lower IL-1beta expression in OA. Th1 cytokines IL-2 and IFN-gamma were detected, while IL-4 was nearly absent in IMA and OA. Furthermore, the bioassay demonstrates a significantly higher production of TNF-alpha in synovial fluid of dogs with IMA, compared to dogs with OA (p < 0.05). The frequency of CD4+, CD8+ and MHC class II+ cells was relatively higher in synovial fluids compared to peripheral blood in IMA. These findings reveal that the difference between the cytokine pattern of canine IMA and OA seems to be rather quantitative than qualitative. Both joint disorders show predominance of pro-inflammatory cytokines and absence of TH2 cytokine expression, indicating the potential of IL-4 for a gene therapeutic approach.
Biologically active membrane gangliosides, expressed and released by many human tumors, are hypothesized to significantly impact tumor progression. Lack of a model of complete and specific tumor ganglioside depletion in vivo, however, has hampered elucidation of their role. Here we report the creation of a novel, stable, genetically induced tumor cell system resulting in specific and complete blockade of ganglioside synthesis. Wild type (WT) and GM3 synthase/GM2 synthase double knockout (DKO) murine embryonic fibroblasts were transformed using amphotropic retrovirus-transduced oncogenes (pBABE-c-MycT58A+H-RasG12V). The transformed cells, WTt and DKOt respectively, evidenced comparable integrated copy numbers and oncogene expression. Ganglioside synthesis was completely blocked in the DKOt cells, importantly without triggering an alternate pathway of ganglioside synthesis. Ganglioside depletion (to <0.5 nmol/107 cells from 9-11 nmol/107 WTt or untransfected normal fibroblasts) did not adversely affect cell proliferation kinetics but did reduce cell migration on fibronectin-coated wells, consistent with our previous observations in ganglioside-depleted normal human fibroblasts. Strikingly, despite similar oncogene expression and growth kinetics, DKOt cells evidenced significantly impaired tumor growth in syngeneic immunocompetent mice, underscoring the pivotal role of tumor cell gangliosides and providing an ideal system for probing their mechanisms of action in vivo.
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