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H K Prasad

Publications and source records attributed to H K Prasad.

28 records · Page 2Linked to original sources
PubMed1987

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.

Bacteriological Techniques↗
PubMed1986

Influence of delayed immune reactions on human epidermal keratinocytes.

The epidermal changes that occur in human cutaneous immune responses have been investigated in the tuberculin reaction and in the lesions of tuberculoid and lepromatous leprosy and cutaneous leishmaniasis. In each situation, there was a dermal accumulation of monocytes and T cells, and the epidermis exhibited thickening. In the tuberculin response, the thickness of the epidermis sometimes doubled in 48-72 hr, and this was attributed to increases in both size and number of keratinocytes. In addition, the phenotype of the keratinocytes changed from Ia- to Ia+. Similar changes in keratinocyte Ia-antigen expression occurred in the epidermis overlying untreated tuberculoid leprosy and cutaneous leishmaniasis lesions, but not in lepromatous leprosy. We suggest that one or more epidermal growth factors may be generated in the course of a delayed immune reaction in the dermis.

Antigens, Bacterial↗
PubMed1985

Alternate radiolabeled markers for detecting metabolic activity of Mycobacterium leprae residing in murine macrophages.

This study demonstrated the utility of using 4% NaOH as a murine macrophage cell-solubilizing agent to discriminate between host macrophage metabolism and that of intracellular Mycobacterium leprae. A 4% concentration of NaOH had no deleterious effect on labeled mycobacteria. Thereby, alternate radiolabeled indicators of the metabolic activity of intracellular M. leprae could be experimented with. Significant incorporation of 14C-amino acid mixture, [14C]leucine, [14C]uridine, and carrier-free 32P was observed in cultures containing freshly extracted ("live") strains of M. leprae as compared with control cultures containing autoclaved bacilli.

Amino Acids↗
PubMed1983

Radiometric macrophage culture assay for rapid evaluation of antileprosy activity of rifampin.

The antileprosy effect of rifampin was evaluated by a newly developed rapid in vitro assay wherein 31 human-derived strains and 1 armadillo-derived strain of Mycobacterium leprae were maintained for 2 and 3 weeks, respectively, in murine and human macrophages in the presence of [3H]thymidine. Of these strains, 27 showed significant incorporation of the radiolabel in cultures of live bacilli as compared with control cultures of heat-killed bacilli of the same strain. Consistent and significant inhibition of [3H]thymidine uptake was observed in M. leprae resident cultures with 3 to 200 ng of rifampin per ml as compared with similar cultures without the drug. In general, an increase in percent inhibition was seen from 3 to 20 ng/ml, with marginal increases at 40, 50, and 100 ng/ml. M. leprae strains appear to be remarkably susceptible to this drug in the in vitro assay.

Animals↗
PubMed1982

Rapid, radiolabeled macrophage culture method for detection of dapsone-resistant Mycobacterium leprae.

Mycobacterium leprae cells extracted from the skin biopsies of 14 bacilliferous lepromatous patients were maintained in human-murine macrophage cultures for 3 weeks in the presence of [3H]thymidine and DDS (4,4'-diaminodiphenyl sulfone). All cultures except one containing freshly extracted viable bacilli showed significant incorporation of [3H]thymidine as compared to control cultures containing heat-killed bacilli of the corresponding strain. Six susceptible strains of M. leprae obtained from untreated, freshly diagnosed patients showed significant inhibition of the uptake of the radiolabel in the presence of 3 and 10 ng of DDS per ml per culture. Eight strains of M. leprae obtained from patients clinically suspected of DDS resistance were tested in a similar manner. These strains were also concurrently inoculated in the footpads of mice given orally 10(-2), 10(-3), and 10(-4) g of DDS per 100 g of body weight for 9 months. Concordant results were obtained by both methods: five strains were found to be resistant, one was susceptible, and one was partially resistant. Strain VIII did not incorporate [3H]thymidine in the macrophage cultures and proved to be resistant in the mouse footpad. The macrophage culture system provides a sensitive, rapid screening method for the early diagnosis of DDS resistance.

Adult↗
PubMed1982

Lack of correlation between morphological index and viability as assessed by the uptake of 3H-thymidine by macrophage resident M. leprae.

Thirty strains of M. leprae derived from skin biopsies of lepromatous leprosy patients were scored for Morphological Index (MI) and concurrently maintained for 2 weeks in macrophage cultures containing 3H thymidine. Selective and significant incorporation of the radioactive label was observed in cultures containing freshly extracted M. leprae as compared to control cultures with autoclaved bacilli from the same biopsy. The percentage incorporation of 3H thymidine ranged from 103 to 1140%. Morphological Index of the bacilli from these individuals varied from zero to 8. Six cultures containing bacilli with MI of less than or equal to 1 and 3 containing bacilli with MI of zero showed significant incropration of 3H thymidine. There was no correlation between the percent of solid or beaded bacilli in the inoculum and the ability of M. leprae to incorporate 3H thymidine in the macrophage cultures.

Animals↗
PubMed1982

Radiolabelled M. leprae resident in human macrophage cultures as an in vitro indicator of effective immunity in human leprosy.

Twelve strains of human derived, freshly extracted M. leprae maintained within human macrophages showed a 2.1-13.2-fold increase in the incorporation of 3H-thymidine compared to parallel cultures containing heat-killed bacilli of the same strain. The addition of antigen stimulated lymphokines from five paucibacillary, tuberculoid leprosy patients resulted in the inhibition of the uptake of the radiolabel by 49-87%. Minimal, or no, inhibition was noted in the presence of similar culture supernatants from five bacilliferous lepromatous leprosy individuals. The results indicate that in contrast to lepromatous leprosy, tuberculoid patients possess antigen reactive lymphocytes which modulate macrophage function through soluble products. Attention is drawn to a rapid and sensitive in vitro method with potential for studying the immunological mechanisms leading to bacterial killing in human leprosy.

Cells, Cultured↗
PubMed1981

Incorporation of 3H-thymidine in Mycobacterium leprae within differentiated human macrophages.

The factors influencing the incorporation of 3H-thymidine (3H-Tdr) in the DNA of Mycobacterium leprae within macrophages derived from human blood have been evaluated. Fifty strains of M. leprae derived from skin nodules of patients with lepromatous leprosy were studied for their ability to incorporate 3H-Tdr. Control macrophages of the same donor maintained alone, or with autoclaved M. leprae, showed low levels of baseline 3H-Tdr incorporation. During a 15-day period of pulsing, 27 of the M. leprae strains incorporated 3H-Tdr at levels of 216-2834% of the incorporation by control cultures. Significant incorporation was observable by the second week of culture and cumulative increases occurred by the third week. A 24-h pulse with 3H-Tdr was inadequate for a detectable increase. A minimal duration of 4-5 days of continuous pulsing was required to obtain a significant increase in the incorporation of 3H-Tdr. Of the 50 M. leprae strains, 23 (46%) failed to incorporate the radiolabel. This failure was apparently not related to differences in the disease status of patients, to the transport conditions for the biopsies, to morphological indices of the extracted M. leprae or to the origin of the host macrophages.

Cell Differentiation↗