Kinetics of accumulation of gamma delta receptor-bearing T lymphocytes in mice infected with live mycobacteria.

The kinetics of accumulation of T cells bearing the gamma delta heterodimer form of the T-cell receptor in mice infected with live Mycobacterium bovis BCG or M. tuberculosis was studied. Substantial numbers of gamma delta T cells accumulated in mice given primary mycobacterial infections, although this accumulation was in parallel to, but not preferential to, that of alpha beta receptor-bearing T cells. In contrast, no accumulation of gamma delta cells was observed in memory immune mice upon rechallenge, thus suggesting that gamma delta cells play no role in the anamnestic response. The results of the study show, further, that large accumulations of gamma delta T cells can also be induced by inoculation with oil adjuvant vehicles containing heat-killed mycobacteria, although not by inoculation of the heat-killed bacteria alone.

In vivo depletion of natural killer cell activity leads to enhanced multiplication of Mycobacterium avium complex in mice.

Earlier investigations have shown that murine natural killer (NK) cells inhibit the growth of fungal and bacterial pathogens in vivo and in vitro. In order to define the role of NK cells in Mycobacterium avium complex infection, in vivo depletion of NK cells by using anti-NK1.1 monoclonal antibody and conventional anti-asialo-GM1 antibody has been attempted. Repeated injection of 200 micrograms of anti-NK1.1 or 50 micrograms of anti-asialo-GM1 antibody effectively depleted NK activity in the spleens of C57BL/6 mice. The growth kinetics of M. avium complex over a period of 4 weeks showed that the colony counts in the spleens of the antibody-treated group were significantly (P less than 0.01) higher than those of the control group and compared well with those of the genetically NK cell-deficient C57BL/6 bg/bg mutant. The alternate strategy of in vivo stimulation of NK activity by poly(I:C) administration did not show a similar reduction in CFU in the spleen compared with the untreated control. The in vivo antibody depletion of NK activity provides direct evidence on the role of NK cells in the control of intracellular mycobacterial pathogens such as M. avium complex.

Uptake of purine and pyrimidine nucleosides by macrophage-resident Mycobacterium leprae: 3H-adenosine as an indicator of viability and antimicrobial activity.

Freshly extracted human- and armadillo-derived Mycobacterium leprae maintained within murine macrophages incorporated significant levels (p less than 0.05 to p less than 0.001) of 3H-adenosine and 3H-hypoxanthine by 6 and 9 days of the culture period. The incorporation of 3H-adenosine was twofold or more higher than 3H-thymidine in 10 out of 15 human-derived M. leprae isolates. Macrophage-adapted bacilli incorporated 10-14-fold higher levels of 3H-adenosine compared to the same bacilli maintained in axenic cultures. The incorporation of these two labels was inhibited by dapsone and rifampin, indicating the utility of in vitro radiometric assays for screening antileprosy drugs and drug sensitivity/resistance in patients.

Comparison of radiometric macrophage assay and fluorescein diacetate/ethidium bromide staining for evaluation of M. leprae viability.

Earlier studies from our laboratory reported that a radiometric Mycobacterium leprae resident macrophage assay was a useful in vitro indicator of bacillary viability with good correlation with the established mouse foot pad model. The present study compares our assay with the recently described fluorescein diacetate/ethidium bromide (FDA/EB) method. M. leprae extracted from the dermal lesions of 73 bacilliferous leprosy patients were tested concurrently by both techniques. Good correlation (r = 0.52, p less than 0.001) was found between the radiometric assay evaluating DNA synthesis and the FDA/EB staining reflecting the presence of active esterase enzyme. In addition, the utility of the FDA/EB staining in the monitoring of therapy was established. Twenty-two patients treated for greater than 1 year showed lower numbers of green fluorescing bacilli when compared to 19 untreated or short-term-treated individuals.