2,3-Di-O-acyl-trehalose (DAT) is a glycolipid located on the outer layer of the Mycobacterium tuberculosis cell envelope. Due to its noncovalent linkage to the mycobacterial peptidoglycan, DAT could easily interact with host cells located in the focus of infection. The aim of the present work was to study the effects of DAT on the proliferation of murine spleen cells. DAT was purified from reference strains of M. tuberculosis, or M. fortuitum as a surrogate source of the compound, by various chromatography and solvent extraction procedures and then chemically identified. Incubation of mouse spleen cells with DAT inhibited in a dose-dependent manner concanavalin A-stimulated proliferation of the cells. Experiments, including the propidium iodide exclusion test, showed that these effects were not due to death of the cells. Tracking of cell division by labeling with 5,6-carboxyfluorescein diacetate succinimidyl ester revealed that DAT reduces the rounds of cell division. Immunofluorescence with an anti-CD3 monoclonal antibody indicated that T lymphocytes were the population affected in our model. Our experiments also suggest that the extent of the suppressive activity is strongly dependent on the structural composition of the acyl moieties in DATs. Finally, the inhibitory effect was also observed on antigen-induced proliferation of mouse spleen cells specific for Toxoplasma gondii. All of these data suggest that DAT could have a role in the T-cell hyporesponsiveness observed in chronic tuberculosis.Mycobacterium tuberculosis, the infectious agent of tuberculosis, is responsible for more deaths than any other single pathogen. It causes 2 to 3 million deaths annually and accounts for more than 30% of the deaths of human immunodeficiency virus-positive individuals (13). Factors affecting the pathogenesis of tuberculosis are complex and poorly defined; however, it is well established that the major common feature in chronic tuberculosis is the suppression of the T-cell immune response (3). For instance, while immune depression and immune activation are simultaneously present in tuberculosis, more profound defects in the cell-mediated immunity, which is largely responsible for protection, are clearly correlated with more extensive tissue damages (43).M. tuberculosis synthesizes both stimulatory and suppressive components for T cells. In general, it is accepted that hostmycobacterium interactions are mediated primarily by specialized molecules expressed on the mycobacterial cell envelope. The immunosuppressive capability of M. tuberculosis is attributed at least in part to lipoarabinomannan (LAM), a major cell wall-associated lipoglycan (21,28,29). However, the M. tuberculosis cell wall contains many other distinctive and chemically unusual components, with a predominance of lipid molecules (12). According to data obtained from M. tuberculosis and M. bovis BCG strains, lipid molecules from the cell wall of mycobacteria can migrate outside from the phagocytic vacuole (1, 5). As a consequence, glycolipids noncovalently lin...

The ROP2 protein of Toxoplasma gondii has previously been proposed as a vaccine candidate against toxoplasmosis. In this work we characterize the immune response induced by injection of plasmid DNA coding for this protein in three strains of mice (BALB/c, C57BL/6, and CBA/J) displaying different levels of susceptibility to toxoplasmosis and compare it with that obtained by vaccination with the live attenuated ts-4 strain of T. gondii. The ROP2 gene was cloned in the eukaryotic expression vector pcDNA3 and the resulting plasmid, named pcDNA3/ROP2, was used to immunize mice. After three immunizations with the plasmid, mice developed antibodies that could be detected by ELISA using a recombinant truncated form of ROP2; and these antibodies also recognized the natural protein by Western blot. Plasmid immunization generated antibodies against the ROP2 of both of the IgG1 and IgG2a isotypes in CBA/J and BALB/c mice and both of the IgG1 and IgG2c isotypes in C57BL/6 mice. However, animals vaccinated with the ts-4 strain generated only IgG2a (in CBA/J and BALB/c mice) or IgG2c (in C57BL/6 mice) against ROP2. Kinetic studies of the generation of isotypes indicated that both isotypes were generated at the same time. Mice immunized with the plasmid DNA did not resist a challenge with the virulent RH strain of T. gondii, while mice vaccinated with the ts-4 strain resisted the same challenge. However, in pcDNA3/ROP2-immunized BALB/c mice, death was significantly delayed with respect to the pcDNA3-immunized control group. These results suggest that plasmid immunization using the ROP2 gene generates a mixed T(H1)/T(H2) response against ROP2, which is different from that obtained by vaccination with live tachyzoites of the ts-4 strain (T(H1) response) and is not protective against the highly virulent RH strain of the parasite.

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Bacterial DNA containing immunostimulatory motifs (CpG) induces the development of a T(H1) immune response. Since protection against Toxoplasma gondii is correlated with this type of response, the aim of this work was to determine if a synthetic oligodeoxynucleotide (ODN) containing CpG sequences could be useful as adjuvant for the induction of a long-lasting protective immune response against T. gondii. BALB/c mice immunized with a total soluble antigen of T. gondii (TSA2) mixed with ODN-containing CpG sequences developed a typical TH1 response, as determined by antibody isotypes and interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) production by spleen cells. However, they did not resist a challenge with the virulent RH strain of the parasite. Absence of protection paralleled with lower levels of IFN-gamma, when compared with mice vaccinated with the live tachyzoites of the attenuated ts.4 strain of the parasite, which resisted this challenge. Intraperitoneal injection of ODN alone to mice induced a high degree of resistance to a lethal challenge inoculated by the same route. Nevertheless, this nonspecific protection was transient. Thus, the use of ODN containing CpG motifs as adjuvant is of limited value for the induction of a protective immune response against T. gondii.

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