Study of the antibody response against Mycobacterium tuberculosis antigens in Warao Amerindian children in Venezuela.

This study was aimed at investigating alternate methods for serodiagnosis of tuberculosis (TB), which are needed because bacteriologic diagnosis of childhood TB is difficult. A selection of 80 serum and saliva samples were tested from Warao indigenous children under 15 years of age; 34 high TB suspects (28 positive and 6 negative for the tuberculin skin test, TST) and 46 healthy contact children (32 positive and 14 negative for the TST). Several enzyme-linked immunosorbent assay (ELISA) serological tests were developed to test for Mycobacterium tuberculosis-specific antibodies, including serum IgA, IgG, IgE, and secretory IgA (sIgA) in saliva against 3 specific antigens (PPD, HSP60, 38 kDa). Of these, 2 antigens, PPD and 38 kDa, showed significantly higher reactivity. The sensitivity and specificity of these tests for diagnosis remained limited, between 26.5% and 38.2%, and 77.4% and 97%, respectively. Of all the samples studied and combinations realized between all isotypes and antigens combined with 3 isotypes (anti-PPD IgG, IgE, and anti-38kDa sIgA) managed to detect the largest number of patients, showing an improved sensitivity level of 64.7%, although specificity levels dropped to 81.8%. These results were compared with the Omega diagnostics commercial kit results. The commercial kits showed significantly lower reactivity (sensitivity of 20% and 13.33% to Myco G and Complex Plus, respectively) and a specificity of 100%. This study shows that in indigenous populations of Venezuela, where invasive procedures cannot be used to select samples but evaluation with a chest X-ray for radiological studies is available, the combination of 3 specific isotypes may be a useful tool to increase diagnostic accuracy with pulmonary TB in this population, when used together with clinical and epidemiological criteria.

Study of the antibody response against Mycobacterium tuberculosis antigens in Warao Amerindian children in Venezuela

This study was aimed at investigating alternate methods for serodiagnosis of tuberculosis (TB), which are needed because bacteriologic diagnosis of childhood TB is difficult. A selection of 80 serum and saliva samples were tested from Warao indigenous children under 15 years of age; 34 high TB suspects (28 positive and 6 negative for the tuberculin skin test, TST) and 46 healthy contact children (32 positive and 14 negative for the TST). Several enzyme-linked immunosorbent assay (ELISA) serological tests were developed to test for Mycobacterium tuberculosis-specific antibodies, including serum IgA, IgG, IgE, and secretory IgA (sIgA) in saliva against 3 specific antigens (PPD, HSP60, 38 kDa). Of these, 2 antigens, PPD and 38 kDa, showed significantly higher reactivity. The sensitivity and specificity of these tests for diagnosis remained limited, between 26.5 percent and 38.2 percent, and 77.4 percent and 97 percent, respectively. Of all the samples studied and combinations realized between all isotypes and antigens combined with 3 isotypes (anti-PPD IgG, IgE, and anti-38kDa sIgA) managed to detect the largest number of patients, showing an improved sensitivity level of 64.7 percent, although specificity levels dropped to 81.8 percent. These results were compared with the Omega diagnostics commercial kit results. The commercial kits showed significantly lower reactivity (sensitivity of 20 percent and 13.33 percent to Myco G and Complex Plus, respectively) and a specificity of 100 percent. This study shows that in indigenous populations of Venezuela, where invasive procedures cannot be used to select samples but evaluation with a chest X-ray for radiological studies is available, the combination of 3 specific isotypes may be a useful tool to increase diagnostic accuracy with pulmonary TB in this population, when used together with clinical and epidemiological criteria.

IFN-gamma, IL-4, IL-10 and IL-12 gene expression in BCG-Leishmania vaccination of Trypanosoma cruzi-infected mice.

We have previously shown that vaccination of BALB/c mice with a combination of BCG plus killed Leishmania promastigotes, applied by the i.p. route 10 and 3 days before Trypanosoma cruzi inoculation, prolonged their survival and decreased their parasitaemia. In the present study we show that the BCG-Leishmania vaccine induced higher levels of circulating IFN-gamma in acute and chronic infection of mice [on day 25 and 40 post-infection (p.i.) respectively], in comparison to unvaccinated animals (PBS-treated). Though the IFN-gamma mRNA content of spleen cells of vaccinated and infected mice (on day 25 p.i.) was similar to that of unvaccinated animals, the BCG-Leishmania vaccine enhanced significantly the production of IFN-gamma by spleen cells stimulated with T. cruzi antigens. This effect was observed to a lower extent in BCG- and Leishmania-treated mice. The BCG-Leishmania vaccine reduced the expression of the IL-10 mRNA of splenocytes as soon as day 12 p.i., before the peak parasitaemia. Such this effect was not observed in BCG- or Leishmania-treated animals. On day 25 p.i., the BCG plus Leishmania- or BCG-treatment of mice abolished the capacity of spleen cells to produce IL-10 in response to T. cruzi antigens. The levels of mIL-4 RNA and protein production were not modified in any group of mice. T. cruzi infection in BCG-Leishmania-vaccined mice stimulated an early and high production of IL-12 transcripts in spleen cells during the acute phase of the infection, that was prolonged during the chronic phase of infection. This effect was weaker or absent in BCG- and Leishmania-treated animals, respectively. These results indicate that the BCG-Leishmania vaccine stimulates the production of IL-12 and IFN-gamma, but inhibits that of IL-10 and is without effect on IL-4 when mice are infected with T. cruzi. This highlights the key role of endogenously produced IFN-gamma, IL-10 and IL-12 in the control of T. cruzi acute and chronic infection in mice and the favorable modulation of their balance by a vaccination combining BCG and Leishmania.

Vaccination of mice with a combination of BCG and killed Leishmania promastigotes reduces acute Trypanosoma cruzi infection by promoting an IFN-gamma response.

The combination of BCG with killed Leishmania promastigotes, demonstrated to be efficient in the cure of patients suffering American cutaneous leishmaniasis and in the induction of a long-term immune response in healthy vaccinated volunteers, was tested in BALB/c mice infected with Trypanosoma cruzi, in comparison to BCG or Leishmania alone, and a vehicle (PBS) control. BCG-Leishmania vaccination, applied intra-peritoneally 10 and 3 days before T. cruzi trypomastigote inoculation, prolonged the survival, and reduced blood parasitaemia of infected animals. Proliferation studies indicated that splenocytes of mice vaccinated with BCG-Leishmania and harvested in the acute phase of T. cruzi infection displayed stimulation indices higher than cells from PBS-treated mice when stimulated with PHA mitogen, PPD, Leishmania or T. cruzi antigens. Injections of a monoclonal antibody able to neutralise IFN-gamma into BCG-Leishmania vaccinated mice increased parasitaemia to levels similar to those of control animals (treated with PBS) and reversed the beneficial effect of vaccination on the proliferative response to T. cruzi antigen. These results show that vaccination of mice with BCG plus killed Leishmania promastigotes delayed acute T. cruzi infection, stimulated a T-cell response to T. cruzi antigen and promoted IFN-gamma production.

Effect of zinc on the proliferative response of human lymphocytes: mechanism of its mitogenic action.

To study the effect of Zn on the proliferative response of normal human lymphocytes, ZnCl2 at a final concentration of 10(-4) M was added to cultures of peripheral blood mononuclear cells (PBMC) stimulated with concanavalin A (Con A) and to autologous mixed lymphocyte cultures of responder T lymphocytes and irradiated autologous non-T cells. Addition of Zn increased by about 50% the synthesis of DNA in cultures stimulated with either 10 or 20 micrograms/ml of Con A and markedly enhanced the autologous mixed lymphocyte reaction, which increased about 5-fold in the presence of Zn. In a narrow dose range, Zn induced per se the incorporation of [3H]thymidine by PBMC, with maximal effects in cultures stimulated with 10(-4) M ZnCl2. The percentage of cells expressing receptors for IL-2 and transferrin as assessed by immunofluorescence with the monoclonal antibodies (mAb) anti-Tac and OKT9, respectively, significantly increased when PBMC were stimulated with 10(-4) M ZnCl2 alone. Maximal [3H]thymidine incorporation and maximal percentage of cells bearing those activation markers were observed on day 6 of culture. Thus, the increase in the uptake of [3H]thymidine induced by Zn is not artifactual but due to progression in the cell cycle. Incubation with the mAb anti-Tac significantly inhibited the proliferative response to Zn, indicating that this requires binding of IL-2 to its receptor. However, addition of human recombinant IL-2 did not increase [3H]thymidine incorporation by PBMC cultured in the presence of ZnCl2.

Mitogen induced lymphoproliferative responses and lymphocyte sub-populations in patients with sickle cell disease.

Mitogen induced lymphoproliferative responses and lymphocyte sub-populations were studied in a group of sickle cell disease (SCD) patients with homozygous SS hemoglobinopathy. Even though the response to a sub-optimal dose of Con A (0.5 microgram/ml of culture) was significantly decreased in patients with SCD, the proliferative responses to optimal doses of Con A, to PHA and to PWM were preserved in the patients. Addition of indomethacin to the cultures increased to a more significant degree the response to Con A of lymphocytes from patients than from the normal controls. Study of the mononuclear cell subsets indicated that the relative and absolute numbers of B lymphocytes as well as those of monocytes were significantly increased in the patients' group. The percentage of T3+ lymphocytes was found decreased in SCD. However, a rise in the number of T11+ and T4+ lymphocytes as well as in the helper/suppressor cell ratio was observed in the patients as compared to controls.

Differences in the effect of indomethacin and preincubation on the lymphoproliferative response to concanavalin A of spleen cells from low responder C57BL/6 and high responder BALB/c mice.

In vivo treatment with 100 micrograms of indomethacin each 48 h for 2 weeks enhanced the proliferative response to concanavalin A (Con A) of spleen cells from mice of the C57BL/6 (B6) strain, low responder to T cell mitogens, but did not modify the response of spleen cells from mice of the high responder strain BALB/c (C). The enhancing effect of in vivo indomethacin treatment was more marked in cultures of B6 splenocytes stimulated with high, moderately supraoptimal doses of Con A than in cultures stimulated with optimal mitogen doses. Addition of indomethacin to cultures of spleen cells from untreated donors induced greater increase of the lymphoproliferative response of cells from low responder B6 than from high responder C mice. The enhancing effect of indomethacin added in vitro was observed in cultures stimulated by optimal but not by supraoptimal doses of Con A. The addition of indomethacin did not enhance the response of B6 spleen lymphocytes depleted of adherent cells. Preincubation for 24 h prior to mitogen stimulation increased the response to high Con A doses of spleen cells from low responder B6 mice whereas this procedure did not enhance lymphocyte proliferation in cultures of spleen cells from high responder C mice. Supplementation with indomethacin in vitro combined with preincubation induced additive enhancing effects on DNA synthesis by B6 spleen lymphocytes, suggesting that each treatment acts through different mechanism(s). The results indicated that spleen cells from low responder B6 strain mice are more sensitive than cells from high responder C mice to the potentiating effect of indomethacin and preincubation on the proliferative response to Con A. These observations suggest that mechanisms sensitive to indomethacin and to preincubation contribute to the depression of mitogen induced DNA synthesis in low responder B6 mice.