Cecilia Guillen-Vargas

Analysis of Toxoplasma gondii antigens with sera from toxoplasmosis patients

Some proteins of the Toxoplasma gondii are recognized by IgG, IgM and IgA antibodies in patients with acute and chronic toxoplasmosis, depending on the strain and stage of the Toxoplasma. Sixty-nine sera from immunocompetent individuals were studied through the Western-Blot Test: 20 has an acute infection, 29 has a chronic toxoplasmosis infection and 20 were healthy (seronegatives). The protein analysis revealed by IgG and IgM antibodies were performed through the immunoplot method in order to know their recognition frequency (f) and be valued as infection markers. In the acute phase, the IgM antibodies showed a recognition frequency (f = 0.60) for the 60 kDa protein, and in the chronic phase the IgG antibodies showed a recognition frequency (f = 0.68) for the 12 kDa protein. Seronegatives revealed no type of band. The protein of 12 kDa can be a diagnostic marker of the chronic phase while protein 60 kDa of the acute phase of toxoplasmosis.

Addition of Anti-CD28 Antibodies Restores PBMC Proliferation and IFN-gamma Production in Lepromatous Leprosy Patients

During antigen recognition, T lymphocytes are primed by a physical interaction with antigen-presenting cells (APC). At least two signals are needed to activate T cells. One is provided by T cell receptor (TCR)/CD3 in the context of the mayor histocompatibility complex (MHC), and another signal is mediated by antigen-independent molecules, that is T cell membrane-bound CD28 and its specific ligand B7-1 (CD80) present in APC. Both signals trigger a series of metabolic events initiating right at the cell membrane and ending with activation and proliferation of T cells as well as specific cytokines synthesis. Our main goal was to determine whether deficiency in interferon-gamma (IFN-gamma) production shown by peripheral blood mononuclear cells (PBMC) from lepromatous leprosy (LL) patients, could be overcome by reconstituting in vitro the appropriate signals (by means of addition of anti-CD28 and anti-CD80 monoclonal antibodies). We also determined the stimulation index (SI) in the same PBMC. Our results demonstrated no significant differences in CD80 expression monocytes and B lymphocytes from LL patients when compared with healthy subjects. Nonetheless, CD28 expression significantly decreased in lymphocytes from LL patients (p < 0.01). Regarding IFN-gamma levels and SI, LL-PBMC failure before mitogenic stimuli could be reversed by further incubation with anti-CD28 antibody, but stimulation by specific antigen of Mycobacterium leprae was not changed. Addition of anti-CD80 antibody significantly increased IFN-gamma levels in phytohemagglutinin (PHA)-stimulated PBMC, although proliferation deficiency persisted. Cells stimulated with specific antigen did not modify either their proliferation or IFN-gamma levels.

Abnormalities in Intracellular Processing and Expression of Interferon-γ Receptor in Adherent Cells From Lepromatous Leprosy Patients

Peripheral blood mononuclear cells in lepromatous leprosy (LL) patients produce low levels of interferon-gamma (IFN-gamma) and interleukin-12 (IL-12), and these cells exhibit partial or complete deficiency in the IL-12 receptor. The behavior of the IFN-gamma receptor (IFN-gamma R) has not been described in cells from people with leprosy. We found higher levels of mRNA for IFN-gamma R1 and IFN-gamma R2 in adherent cells stimulated with IFN-gamma and Mycobacterium leprae membrane proteins from LL patients compared with healthy subjects. Flow cytometry showed no significant difference in IFN-gamma R1 expression between LL patients and healthy subjects. Immunoblotting detected only the mature glycosylated form of the 61-67 kDa IFN-gamma R2 protein in healthy subjects. In contrast, cells from LL patients showed three different expression patterns: (1) the immature deglycosylated form of the 34.8 kDa IFN-gamma R2 protein, (2) the mature glycosylated 61-67 kDa form, and (3) both forms. Our data indicate the existence of abnormalities in the intracellular processing and protein expression of the IFN-gamma R in response to specific stimuli such as IFN-gamma and M. leprae membrane proteins in adherent cells of LL patients.

Deficiency in the incorporation of labeled thymidine and inhibition in the biosynthesis of interleukin-2 in lymphocytes obtained from Histoplasma capsulatum infected mice

The incorporation of (3H) thymidine and the biosynthesis of interleukin-2(IL-2) were investigated in Concanavalin A (ConA) and histoplasmin stimulated lymphocytes from spleen of infected Balb/c mice with the yeast phases of Histoplasma capsulatum. The ability to incorporate (3H) thymidine of Con A stimulated lymphocytes in culture from spleen of Histoplasma capsulatum infected mice, as well as the IL-2 content present in the supernatants of that cultures, were depressed along the first three weeks of the experiments, but starting week five, normal values were restored or even discretly increased. Incorporation of (3H) thymidine in histoplasmin stimulated lymphocytes remained inhibited along the seven weeks the experiment lasted. Results showed that inoculation of H. capsulatum yeast in mice provoked a temporary immunosuppression on cell mediated immunity, that can be explained by means of the inability of T cells to produce enough IL-2 necessary for the proliferation of T cells in culture.