An actinomycete was isolated during a soil screening programme to obtain L-3,4-dihydroxyphenylalanine producers. A mutant of this organism was isolated by chemical mutagenesis and it accumulated 1 g/litre L-dihydroxyphenylalanine when grown on L-tyrosine. Resting cells converted 30% of tyrosine in the reaction mixture. The use of resting cells for dihydroxyphenylalanine production is advantageous as it eliminates interfering substances which accumulate during fermentation.

The production of L-asparaginase by two mutants ofSerratia marcescens grown on 14 different media was studied. The enzyme content increased from trace levels to 2.4 international units per ml when the organisms were grown in glycerol-peptone yeast extract medium. Glucose was the best carbon source under aerobic conditions. The enzyme content increased when L-asparagine was present in the growth medium.

Macrophages phagocytoseMycobacterium leprae and live bacilli inside such macrophages alter the lipid metabolism. There is increased accumulation of cholesterol ester in the bacteria infected cells. This increase appears to be due to the decreased level of esterase enzyme that could hydrolyse cholesterol esters. Associated with decreased level of this enzyme is the reduced amount of protein synthesis. Increased cholesterol ester may be responsible for conversion of macrophages into foamy cells in the presence ofM. leprae.

Macrophages from lepromatous leprosy patients showed poor adherence toMycobacterium leprae. The phagocytic activity of the macrophages was not correlated to the influence on the adherence ability. Based on the phagocytic behaviour of macrophages from normal individuals and from lepromatous leprosy, patients as well as the action of neuraminidase in reversing the extent of adherence, it is suggested that macrophages from lepromatous leprosy patients differ from those from normal individuals in regard to their surface properties. There was no relationship between the degree of adherence and the concentration of Fc receptors of the macrophages. It was also shown that an extract of lysed macrophages from lepromatous leprosy patient was able to reduce the adherence ofMycobacterium leprae to normal macrophages. This study shows that adherence is a good indicator of the surface property of macrophages which in turn could play an important role in the cell mediated immunity of the patient. The observations suggest altered macrophage membrane structure in the long term-treated, otherwise normal, lepromatous leprosy patients.

A reliable screening technique for assessing the sensitivity ofMycobacterium leprae to drugs has been developed. The method is based on the susceptibility or otherwise ofM. leprae- infected tissues from lepromatous leprosy patients to the action of diaminodiphenyl sulphone (dapsone) or rifampicin on the incorporation of [¹⁴C]-acetate into lipids. The extent of inhibition or lack of inhibition correlated very well with the drug sensitivity or resistance of the bacteria isolated from the patients to the above drugs. A similar trend was observed when the incorporation into individual fractions of neutral lipids was measured. There was no incorporation by heat-killed tissues. This method correlates well with the 3,4-dihydroxyphenylalanine uptake studies.

Reduction inF_c receptor expression as assayed by ‘erythrocyte’ rosetting of macrophage cultures from long term treated lepromatous leprosy patients (bactereologically negative) was seen in the presence of viableMycobacterium leprae. Macrophages with and without intracellular bacilli demonstrated this reduction. On the basis of this observation the conditioned medium ofMycobacterium leprae infected macrophage cultures of lepromatous patients, were tested on macrophages from normal individuals for [³H]-leucine incorporation and antigen specific physical interaction with lymphocytes. Both these parameters showed decreased values as compared to the controls which were not exposed to this conditioned medium. Lymphocyte transformation toMycobacterium leprae in leucocyte cultures of normal individuals was also reduced in the presence of the conditioned medium from lepromatous patients’ macrophages. The indication that this factor may be a prostaglandin was suggested by the observation that its synthesis was inhibited by indomethacin. Its importance in the non-specific depression in cell-mediated immunity seen in lepromatous patients is discussed.

Macrophages that have ingested liveMycobacterium leprae show a preferential accumulation of cholesterol ester. Such an accumulation is not seen, on the ingestion of dead bacteria. Among the macrophages that ingest liveMycobacterium leprae, the presence of dapsone or rifampicin prevents largely the alteration in the anticipated increase in the cholesterol ester indicating the sensitivity of the bacteria to the drug. In the small number of relapsed patients, the presence of dapsone did not reduce the cholesterol ester increase, suggesting that theMycobacterium leprae present are either resistant or escaped detection. The method provides a rapid drug screening system foranti-Mycobacterium leprae activity of known and unknown compounds

The observation that liveMycobacterium leprae on entry into macrophages from lepromatous leprosy patients reduced the number of EA rosetting macrophages, was extended to macrophages from Swiss white mice also. Further, the fact that deadMycobacterium leprae do not bring about such a change in macrophages from mice, allowed us to develop this into a bacterial viability testing system. Thus drug treated macrophages in the presence ofMycobacterium leprae showed normal rosetting ability ifMycobacterium leprae are inactivated by the drug, but showed reduced level of rosetting when bacteria were not susceptible to the drug. It was shown that a drug like dapsone, does act onMycobacterium leprae based on its permeability, quantity available inside the macrophages and inhibition of its action by Para amino benzoic acid. The inactivation ofMycobacterium leprae by sulphone and rifampicin was also proved by the flourescence diacetate method, which showed poorly viable bacteria after exposure to drugs. Thus it has been possible to develop a rapid drug screening method for testing the activity of unknown compound againstMycobacterium leprae.

The observations that liveMycobacterium leprae after entry into cultured peritoneal macrophages from mice, reduced the EA rosetting macrophages, have been exploited to determine the minimum inhibitory concentration of diamino diphenyl sulphone and rifampicin. Diamino diphenyl sulphone showed a minimum inhibitory concentration of 0.028 μg/ml and rifampicin 0.11μg/ml when given externally. However, there was accumulation of diamino diphenyl sulphone inside the macrophages. At an external concentration of 0.028 μg/ml the concentration inside the macrophage was 0.5μg/ml. The minimum inhibitory concentration for diamino diphenyl sulphone in this assay system is higher by several folds and that for rifampicin is slightly lower, than what is reported earlier with mice foot pad experiments. The minimum inhibitory concentration reported in this assay system is quite close to what is observed forin vitro inhibition ofMycobacterium lufu with both the drugs

It has been demonstrated thatMycobacterium leprae, are caPable of taking uP uracil and incorPorating it into trichloroacetic acid-insoluble materials, both as free susPension of bacteria, as well as when they are inside the macrophages, a host cell for theirin vivo survival. Same amount of bacteria show better incorPoration inside macroPhages than as free bacterial susPension. Both tyPes of incorPoration are inhibited by rifamPicin an antileProsy drug and an RNA synthesis inhibitor. Thus uracil uPtake byMycobacterium lePrae inside macroPhages has been used for standardising a raPidin vitro viability assay for the leProsy causing bacteria.

The macrophages from peripheral blood of normal healthy individuals respond to live or killedMycobacterium leprae by producing superoxide. On the other hand, the macrophages from bacteriologically positive (B +LL) or long term treated bacteriologically negative (B -LL) and tuberculoid leprosy patients are unable to produce superoxide when stimulated with liveMycobacterium leprae. While killedMycobacterium leprae induce superoxide with the cells from tuberculoid andB(-)LL patients, cells fromB(+)LL patients fail to respond. The deficiency inB(-)LL patients to produce superoxide appears to be specific withMycobacterium leprae and the defect can be counteracted by the addition of colchicine. These observations indicate a preexisting membrane disposition which does not favour superoxide production. A similar situation is seen in the cells from tuberculoid leprosy patients. Thus it appears that both cured and active lepromatous leprosy patients have defective macrophages, unable to respond to liveMycobacterium leprae to produce superoxide anion, in contrast to the situation with the cells from normal healthy individuals.

Macrophages cultured from the peripheral blood of normal individuals, tuberculoid leprosy patients and long-term-treated, bacteriologically negative lepromatous leprosy patients are able to release hydrogen peroxide on stimulation withMycobacterium leprae. Macrophages from lepromatous leprosy patients who are bacteriologically positive produce considerably lower levels of hydrogen peroxide, even though stimulation of these cells withMycobacterium leprae is definitely demonstrable. This differential stimulation of macrophages appears to be largely specific toMycobacterium leprae. There is also a good indication that decreased stimulation of macrophages from positive patients could be due to an after-effect of infection. It is possible that while other factors aid survival ofMycobacterium leprae in the macrophages, hydrogen peroxide may not be as effective in the killing of the bacteria in infected patients as it would be, perhaps, in other infections.