Cell mediated immunity to Plasmodium vivax infection: in vitro inhibition of parasite growth by monocyte derived macrophages.

OBJECTIVE: To study the ability of soluble blood stage or cell associated antigens of Plasmodium vivax to stimulate human peripheral blood mononuclear cells (PBMC) and produce factors capable of causing inhibition of parasite growth in vitro was the objective of this investigation. METHOD: A local isolate of P vivax was either synchronized by triple sorbitol lysis for antigen preparation or used as unsynchronized culture for parasite inhibition, employing a macrophage inhibition assay. The soluble or cell associated antigens of P vivax were added to human monocyte derived macrophages with P vivax parasitized red blood cells. The percent inhibition of parasite growth was examined after 72 hrs by microscopy of Giemsa stained smears of red blood cells from the experimental and control groups. RESULTS: The differences in parasite inhibition were compared using Wilcoxon rank sum test for paired differences. Unstimulated PBMC supernatants did not inhibit parasite growth. Significant inhibition of parasite growth (90%) was seen after incubating P vivax infected erythrocytes with PBMC supernatants resulting from stimulation with soluble antigens (T = 3; P < 0.05). However, the cell associated antigens of P vivax did not stimulate PBMC to activate macrophages for parasite killing in vitro (T = 14, P < 0.05). CONCLUSION: We conclude that the soluble blood stage antigens of P vivax can stimulate human PBMC to produce factors capable of activating macrophages to function as effector cells in P vivax malaria.

Detection of Mycobacterium tuberculosis by polymerase chain reaction in 301 biological samples--a comparative study.

A repetitive target sequences of Mycobacterium tuberculosis DNA was amplified by polymerase chain reaction (PCR) in a total of 301 clinical samples. Sputum, blood, pleural fluid, and bronchial lavage specimen were taken from clinically suspected causes of tuberculosis and processed for the diagnosis of tuberculosis using a simplified procedure of DNA extraction. PCR was positive in a total of 58 samples (58/301--19.3%). A significant number of smear and culture negative cases of tuberculosis were PCR positive (37/174--21.26%). This finding, combined with the absence of either false positive or false negative results reflects the greater usefulness of this technique.

Studies on glutamine synthetase. Purification of the enzyme from mung bean (Phaseolus aureus) seedlings and modulation of the enzyme-antibody reaction by the substrates.

Glutamine synthetase (L-glutamate : ammonia ligase, EC 6.3.1.2) from Phaseolus aureus (mung bean) seedlings was purified to homogeneity by ammonium sulphate fractionation, DEAE-cellulose chromatography, Sephadex G-200 gel filtration and affinity chromatography on histidine-Sepharose. The enzyme had a molecular weight of 775,000 ± 25,000. The enzyme consisted of identical subunits with an approximate subunit molecular weight of 50,000. Hyperbolic saturation curves were obtained with the substrates, glutamate, ATP and hydroxylamine. Antibody, raised in the rabbit, against mung bean glutamine synthetase, completely inhibited the activity of the enzyme. Preincubation of the enzyme with glutamate and ATP, prior to the addition of the antibody, partially protected the enzyme against inhibition. The Km values of this enzyme-antibody complex and the native enzyme were identical (glutamate, 2.5mM; ATP, 1 mM; hydroxylamine, 0·5 mM). The Km values of the partially inhibited enzyme (the enzyme pretreated with antibody prior to the addition of substrates) were 2-fold higher than those of the native enzyme. These results suggested that the substrate-induced conformational changes in the enzyme were responsible for the protection against inhibition of the enzyme activity by the antibody.