Are all nonsolid Mycobacterium leprae dead? Does a negative finding in the mouse foot pad indicate that there is actually no growth of M. leprae in the animals?

Ever since the discovery of the foot pad technic for growth of M. leprae in mice, investigators have overemphasized the laboratory results in clinical applications. Overenthusiasm has led to some dire results in the leprosy field. Two well-known examples can be cited, which are based on the presumption that: a) all the nonsolidly stained M. leprae are dead, and b) that a negative finding in the mouse foot pad indicates no growth of M. leprae in the animals. The former led clinical investigators to claim a false emergence of drug resistance after one year's treatment with a potent antileprosy drug, B663, which was almost abandoned for later clinical use. The latter led investigators to introduce a low-dose drug treatment, which resulted in a worldwide appearance of DDS resistance in leprosy. This paper outlines the reasoning that not all nonsolid M. leprae are dead, and that not all the organisms in the foot pads are detectable by the present standard foot pad/ M. leprae technic.

Growth of macrophages obtained from various sources.

Techniques to obtain macrophages from various sources of the mouse were reported. The following sources were included: peritoneal exudate, alveolar lavage, blood leucocytes, bone marrow, spleen, liver, lungs, lymph nodes, thymus, thyroid, heart muscle, kidney, and subcutaneous cover glass implants. Human blood macrophages were also included. Long-term cinemicrographic studies revealed sustained good growth of these macrophages. Cell multiplication was detected in all of these cultures except those obtained from the peritoneal exudate. Pure cultures of macrophages were obtained from blood of the mouse and human. Macrophages obtained from other sources were accompanied by some growth of fibroblasts. Methods to eliminate the fibroblasts in cultures were discussed.

Morphological changes of Mycobacterium lepraemurium grown in cultures of mouse peritoneal macrophages.

Studies were made on morphological changes of Mycobacterium lepraemurium grown in cultures of mouse peritoneal macrophages. Two types of nonsolid or irregularly stained M. lepraemurium were observed. One type occurred in the growth phase of the organisms during the stage of preparation for bacillary multiplication. The nonsolid bacilli appeared as elongated organisms having pointed ends, isolated acid-fast dots, or faintly stained areas at the ends of the bacilli. It is possible that this irregularity in staining is due to a very gradual, versus an instantaneous, acquisition of acid-fast material during bacillary multiplication and maturation. Solid forms were again observed upon maturation. Nonsolid bacilli were also observed in macrophage cultures infected with autoclave-killed M. lepraemurium. Under these conditions there was an emergence of organisms which showed irregularly stained areas and various forms of deformity unaccompanied by elongation or multiplication. These irregularities were most probably due to the destructive process of digestion of bacillary protoplasm. The present study does not support the current hypothesis that all nonsolid acid-fast organisms are nonviable.

Chemotherapy of murine leprosy. VII. The effect of cycloserine (seromycin) on mouse leprosy

Cycloserine (Seromycin)was found to be moderately, effective in the suppression of intraperitoneally infected mouse leprosy. The smallest dosages used, 0.2 per cent in the food, showed minimal effects. Higher doses showed greater activity, of about the same order as that exhibited by streptomycin, but much less than that of isoniazid. Cycloserine has been reported to be beneficial in human tuberculosis but to have no effect in experimental tuberculosis of mice and guinea-pigs. The discovery of its suppressive action in murine leprosy suggests the desirability of the extension of screening tests to other mycobacterial infections in the search for more active antituberculosis agents.

Chemotherapy of murine leprosy. VI. The effects of isonicotinylhydrazone of 2-carboxymethoxy 3-methoxybenzaldehyde (compound 373) and isonicotinylhydrazone of 2-carboxymethoxybenzaldehyde (compound 377) on mouse leprosy

The activity of isoniazid, isonicotinylhydrazone of 2-carboxymethoxy-3-methoxybenzaldehyde (compound 373), and isonicotinylhydrazone of 2-carboxymethoxybenzaldehyde (compound 377) was compared, in equimolar doses, in mice infected intraperitoneally with M. leprae murium. All three compounds were found to be highly effective in the suppression of the murine lleprosy infection in the three-months experiment. They were most effective when treatment was started immediately after inoculation and continued for three months, and proportionally less effective when treatment was delayed for one month and then continued for two months. The degree of activity of the three compounds was more or less equal. When mice were treated for three months and allowed to live, all three compounds suppressed the infection for a period; then the disease remanifested itself, killing the animals. The suppressive activity of isoniazid and Compound 373 was similar, while that of Compound 377 was the highest. When mice were treated with either isoniazid or Compound 373 continuosly for 15 months from thhe time of inoculation, the survival time was definitely longer than when they were treated for only three monmths. Eventually, however, the infection led to death regardless of the duration of treatment. Isoniazid revealed activity similar to that of Compound 373. On the other hand, the continuous administration of Compound 377 for 15 months suppressed the infection in the majority of the mice. This appears to be the first drug to hold this infection in check for as long as 15 months.

Chemotherapy of murine leprosy

The effects of various combinations of small doses of 4,4'-diaminodiphenyl sulfone (DDS), streptomycin and isoniazid (isonicotinic acid hydrazide) have been studied in intraperitoneally-infected mouse leprosy. The suppressive effects of various combinations of these drugs were definitely superior to those obtained with the individual drugs alone. The order of activity of the drugs and combinations used, based on the decreasing total indices of chemotherapeutic effectiveness, may be expressed as follows: (1) streptomycin, isoniazid, and DDS combined; (2) isoniazid and DDS; (3) streptomycin and isoniazid; (4) streptomycin and DDS; (5) streptomycin alone; (6) isoniazid; (7) DDS.

Chemotherapy of murine leprosy. IV- The effects of amithiozone (TB1/698), p-aminosalicylic acid (PAS), B283 (a phenazine pigment), five antibiotics and three diphenylthiourea compounds on mouse leprosy

Studies have been made of the therapeutic effects on murine leprosy of several drugs and antibiotics, employing the intraperitoneally-infected mopuse. The following results were obtained: Amithiozone (TB1/698) seemed to cause some stimulation of the infection. PAS (p-aminosalicylic acid), B283 (a phenazine pigment), chlortetracycline (Aureomycin), oxytetracycline (Terramycin), Chloramphenicol (Chloromycetin), penicillin, erythromycin (Ilotycin), and three derivatives of diphenylthiourea, i. e., SU-1795, Su-1906 and SU-2358, revealed no significant activity.

Chemotherapy of murine lleprosy. III. The effects of nicotinamide and pyrazinamide (aldinamide) on mouse leprosy

A brief review of the pharmacological actions of nicotinamide and allied compounds in relation to the mycobacterial infections has been presented. The activity of nicotinamide and pyrazinamide (Aldinamide) in mouse leprosy has been studied, employing the intraperitoneal route for infection. Comparative studies of the activity of these compounds with that of three known effective drugs- isoniazid, streptomycin and DDS - were made in animals treated immediately after inoculation or after delays of one or two months. The duration of the experiments was three months. Both nicotinamide and pyrazinamide were found to be highly effective in the suppression of the leprous infection. Nicotinamide was found to have a degree of activity similar to that of pyrazinamide and of isoniazid, and superior to that of streptomycin; DDS was the least active. All these five compounds were most effective when the treatment was delayed for one or two months. Only nicotinamide, pyrazinamide and isoniazid possessed any significant activity when the treatment was delayed for two months and then carried out for only one months.