Impairment of B-lymphocyte differentiation induced by dual triggering of the B-cell antigen receptor and CD40 in advanced HIV-1-disease.

OBJECTIVE: This study was performed to investigate the hyporeactivity of purified B lymphocytes from HIV-1-infected patients. DESIGN: Given the importance of the B-cell Ag receptor (BCR) and CD40 in B-lymphocyte activation, we assessed the capacity of purified peripheral blood B lymphocytes from HIV-1-infected patients to differentiate into Ig-secreting cells in a T-cell- and accessory-cell-independent system of BCR and CD40 costimulation. METHODS: B lymphocytes from 21 HIV-1-infected patients were purified by immunomagnetic cell separation and costimulated with immobilized anti-CD40 monoclonal antibodies and Staphylococcus aureus Cowan I particles in the presence of interleukin (IL)-2 and IL-10. Homotypic aggregate formation, apoptosis, cell cycle entrance, proliferation and Ig secretion of B cells were analysed. RESULTS: Costimulation by the BCR and CD40 induced proliferation and differentiation of B lymphocytes into Ig-secreting cells in 13 patients (group I) but not in eight patients (group II). For three patients in group II, the dual triggering induced apoptosis of B cells. The unexpected inability of these cells to differentiate was associated with a high CD38 expression and a weak spontaneous production of Ig or anti-HIV-1 antibodies in patients with a high viral load and a low CD4+ lymphocyte count. Despite this anomaly, the B cells from group II were able to progress through the cell cycle after stimulation with a combination of phorbol ester and ionomycin in complete medium, suggesting an impairment in BCR and CD40 early signal transduction. CONCLUSION: Intrinsic in vitro hyporeactivity of B lymphocytes to dual triggering of BCR and CD40 was observed in advanced HIV-1 disease and appeared to be related to in vivo hyperactivation of B cells.

Virus isolation, polymerase chain reaction and in vitro antibody production for the diagnosis of pediatric human immunodeficiency virus infection.

Viral culture (VC), polymerase chain reaction (PCR) and in vitro antibody production (IVAP) by peripheral blood mononuclear cells were compared for the early diagnosis of HIV-1 infection in 46 infants born to HIV-1 seropositive mothers. The ten children considered infected on the basis of clinical signs and persistence of anti-HIV-1 antibodies had at least one positive viral culture and seven were always positive in both PCR and IVAP tests. PCR and IVAP tests were occasionally negative in three infected children. Among 30 healthy children who became seronegative and were always negative for viral culture, 22 (73.3%) were also repeatedly negative in PCR and IVAP. We report 6 cases of children classified as P2A at the term of this study but who had lost anti-HIV-1 antibodies. They presented at least one positive viral culture and occasional positive PCR and/or IVAP results. The results indicate that the combination of viral culture, PCR and IVAP tests improves the early diagnosis of pediatric HIV infection.

[Use of flow cytometry in the study of a non-specific immunodeficiency in children. A case]. / Utilisation de la cytométrie en flux dans l'étude des déficits de l'immunité non spécifique de l'enfant. Une observation.

Using flow cytometry, we explored a case of nonspecific immunodeficiency in a seven month old girl with repeated infections. This method showed evidence of granulocyte phagocytosis and oxidative metabolism abnormalities suggesting the diagnosis of a variant form of chronic granulomatous disease (CGD). Findings also showed that flow cytometry can be useful to study phagocytic cells during the neonatal period as it allows rapid multiparametric analysis with a very small amount of blood.

In vitro anti-Toxoplasma gondii antibody production by peripheral blood mononuclear cells in the diagnosis and the monitoring of toxoplasmic encephalitis in AIDS-related brain lesions.

The spontaneous secretion in vitro of anti-Toxoplasma gondii antibodies by peripheral blood mononuclear cells was assessed in 69 patients with AIDS-related brain lesions. The sensitivity and specificity of this assay in the diagnosis of toxoplasmic encephalitis (TE) were found to be 85.4% and 92.8%, respectively. Twenty-four patients with TE were observed during 1-year follow-up after initiation of anti-Toxoplasma treatment and classified on the basis of their clinical and radiologic responses as sustained responders (SR; n = 11), incomplete responders (IR; n = 7) or transient responders (TR; n = 6). In vitro anti-T. gondii antibody secretion decreased as early as the first month after initiation of treatment and disappeared within the year in SRs, persisted in IRs, and decreased but rebounded at relapse in the TR patients. In vitro anti-T. gondii antibody, which reflects immune system activation by parasitic antigens, could be a surrogate marker of TE in AIDS patients.

In vitro antibody secretion by peripheral blood mononuclear cells as an expression of the immune response to Brucella spp. in humans.

We developed an assay to detect antibodies spontaneously secreted in vitro by peripheral blood mononuclear cells (PBMC) against Brucella spp. High levels of anti-Brucella immunoglobulin G (IgG) and/or IgM and/or IgA antibodies were detected in the cell supernatant solution of PBMC cultures for 12 patients suffering from acute or focalized brucellosis and for 5 patients recently vaccinated against brucellosis. This spontaneous in vitro antibody production disappeared 5 to 20 months after onset of clinical signs and 20 to 27 days after vaccination. The transient character of this anti-Brucella antibody production by PBMC is consistent with a temporary in vivo stimulation of the immune system by Brucella antigens. Detection of this secretion could improve the diagnosis of evolutive brucellosis.

Secretion of Toxoplasma gondii-specific antibody in vitro by peripheral blood mononuclear cells as a new marker of acute toxoplasmosis.

Antigen-specific antibody secretion in vitro by peripheral blood mononuclear cells (PBMC) reflects an in vivo stimulation of the immune system by the antigen. Primary infection of immunocompetent patients with T. gondii causes an acute infection followed by chronic toxoplasmosis. We examined in vitro anti-Toxoplasma antibody production by PBMC during the acute and chronic phases of toxoplasmosis. PBMC from patients with acute or chronic toxoplasmosis and seronegative subjects were cultured for up to 6 days. Anti-Toxoplasma antibodies were assayed in supernatants by ELISA and immunoblotting. Anti-Toxoplasma antibodies were detected in supernatants of PBMC from 29 pregnant women who seroconverted during gestation. PBMC from 17 patients who had chronic toxoplasmosis and PBMC from 10 seronegative healthy controls did not secrete Toxoplasma-specific antibodies. This in vitro antibody secretion was spontaneous, active and transient since it disappeared between 11 and 24 weeks after seroconversion. Anti-Toxoplasma antibody secretion by PBMC from patients with acute toxoplasmosis is consistent with an in vivo stimulation of the immune system by T. gondii antigens. Our results represent a new approach for studying the immunological response during T. gondii infection and could have important implications for the diagnosis of acute and re-activated toxoplasmosis.

Spontaneous in vitro anti-human immunodeficiency virus type 1 antibody secretion by peripheral blood mononuclear cells is related to disease progression in zidovudine-treated adults.

As part of a continuous search for surrogate markers of therapeutic efficacy in AIDS, spontaneous in vitro production by peripheral blood mononuclear cells of antibody to human immunodeficiency virus type 1 (HIV-1) was investigated in 50 HIV-1-infected adults. It was independent of CD4+ cell counts, p24 antigenemia, serum beta 2-microglobulin concentration, and clinical status of the patients. The effect of zidovudine on this antibody secretion and the appearance of signs or symptoms of HIV-1 disease progression were evaluated in 20 patients over 24 weeks. Anti-HIV-1 antibody secretion decreased significantly (P = .002) as of the first month of zidovudine treatment only in the 13 HIV-1-infected patients without disease progression. This is earlier than the occurrence of variations in CD4+ cell count and serum beta 2-microglobulin concentration. These results suggest that in vitro antibody production could be a surrogate marker for evaluation of the in vivo antiretroviral efficacy of zidovudine, even in p24 antigen-negative patients.

Hepatitis B virus (HBV)-specific in vitro antibody production by peripheral blood mononuclear cells (PBMC) after vaccination by recombinant hepatitis B surface antigen (rHBsAg).

To study the immunization induced by rHBsAg, we analysed the in vitro antibody production (IVAP) to HBsAg by PBMC from 18 subjects vaccinated by two injections on days 0 and 30. HBsAg-specific IVAP was detectable in all subjects after both the first and the second injection, and lasted for about 10 days and then disappeared. However, when the spontaneous HBsAg-specific IVAP became negative, HBsAg stimulation of PBMC cultures induced again a specific HBsAg IVAP. Cultures of cell populations separated by erythrocyte rosetting or Percoll density centrifugation showed that the cells responsible for spontaneous secretion, after in vivo stimulation, were low-density B lymphocytes. High-density B lymphocytes were involved in anti-HBs production induced by in vitro stimulation when spontaneous secretion disappeared. These data suggest that the IVAP test could be a source of important information along with serologic analysis for exploration of the immune response to hepatitis B vaccine.