To grow Mycobacterium leprae in cultured mouse peritoneal macrophages, studies were made on 1) the purification of M. leprae from lepromatous nodules by trypsinization, 2) growth experiment of purified M. leprae in cu1tured macrophages by in vivo infection-in vitro cultivation technique and 3) the observation of pathological changes in sp1eens of mice induced by intraperitoneal inoculation of purified M. leprae. Results are summarized as follows. 1. A simple and effective procedure is described for purification of M. leprae from biopsied nodules of lepromatous leprosy patients by trypsinization and high speed centrifugation. The procedure resulted in a good yie1d of homogeneous preparation of M. leprae with a negligible contamination of tissue debris. 2. Significant decreases were observed in the numbers of acid-fast bacilli in cultured macrophages and of macrophages harboring acid-fast bacilli by the length of intervals between the time of intraperitoneal inoculation of purified M. leprae and the time of initiation of macrophage cultures. 3. Microscopic examination of stained preparations of macrophages cultured by in vivo infection-in vitro cultivation technique indicated that an apparent increase in the number of acid-fast bacilli in the macrophages occurred when the cultures made at 24 hours and 1 week after inoculation were maintained in vitro up to 2 months or more. 4. Pathological changes in the spleens of mice inoculated with purified M. leprae were of mainly degenerative nature in the red pulp. No multiplication of M. leprae was observed in the spleens of mice up to 5 months after inoculation.
Adolescent ; Adult ; Animal ; Cells, Cultured ; Female ; Human ; Leprosy/microbiology* ; Macrophages/microbiology* ; Male ; Mice ; Mycobacterium leprae/growth & development* ; Mycobacterium leprae/isolation & purification ; Peritoneum/microbiology*

Efforts have been made to accomplish a long term in vitro cultivation of mouse peritoneal macrophage as a host cell for growth of Mycobacterium lepraemurium. Following the inoculation with live or heat-killed Myco. lepraemuriuum of cultured macrophages either on a cover-slip in Leighton tube or in a small petri dish, microscopic observations of acid-fast (AF) stained slide preparation, and total counts of AF bacilli that were released by ultrasonic treatment from the macrophages in small petri dish have been followed in order to present microscopic and quantitative evidence of the actual multiplication of Myco. lepraemurium in cultured mouse peritoneal macrophage. The results are summarized and conclusions are as follows; 1. Successful long term in vitro cultivation of mouse peritoneal macrophage has been accomplished. The growth medium for tissue culture consisted of NCTC 109;50% heat-inactivated calf serum; 40% and beef embryo extract (diluted 1 : 5); 10% and the medium was renewed every 3 to 4 days. The incubation temperature was 37 degrees C; before and at 30 degrees C; after the inoculation with Myco. lepramurium. The CO2 content inside the CO2 humidity incubator for the cultivation of macrophage was kept at 5%. 2. In cultures of macrophage inoculated with live Myco. lepraemurium, clear features of increases in the number of AF bacilli inside individual cell, of elongation of bacill and of increased solidity in AF staining were observed. However, these features were absent in cultlires of macrophage inoculated with heat-killed Myco. lepraemurium. 3. The ultrasonic treatment of macrophage inoculated with 1ive Myco. lepraemurium, and the quantitative assessment of total number of AF bacilli through the course of 6 to 8 weeks after inoculation has provided partial but substantial evidence of actual multiplication of Myco. lepraemurium in cultured macrophages.
Animal ; Female ; Macrophages/microbiology* ; Mice ; Mycobacterium leprae/growth & development* ; Peritoneum/cytology ; Tissue Culture

Various primary cells and an established cell line were cultured in roller tubes and in suspension to evaluate their potential roles as host cells to support the growth of M. leprae in vitro. The primary cells originated from the organs of chipmunks, mice and humans. Phagocytic ability of those cells except for macrophages was found to be low and did not vary much according to their origin. However, when macrophages from mice peritonial exudate were exposed to the bacteria, the phagocytic efficiency was higher than 47%. In spite of those good primary results, the macrophages are not cells which can adapt well in vitro for long term culture, which is essential for the growth of such a slow growing M. leprae. Thus, somatic cell hybridization between the macrophages and HeLa was made by fusing them with polyethylene glycole. Those hybrids appeared to have both the characteristics of the parent cells, which can provide a natural intracellular environment such as the macrophages and the infinite growth capability of the HeLa cells in vitro.
Animal ; Culture Media ; Hela Cells ; Human ; Hybrid Cells ; Macrophages ; Mice ; Mycobacterium leprae/growth & development* ; Sciuridae

Various primary cells and an established cell line were cultured in roller tubes and in suspension to evaluate their potential roles as host cells to support the growth of M. leprae in vitro. The primary cells originated from the organs of chipmunks, mice and humans. Phagocytic ability of those cells except for macrophages was found to be low and did not vary much according to their origin. However, when macrophages from mice peritonial exudate were exposed to the bacteria, the phagocytic efficiency was higher than 47%. In spite of those good primary results, the macrophages are not cells which can adapt well in vitro for long term culture, which is essential for the growth of such a slow growing M. leprae. Thus, somatic cell hybridization between the macrophages and HeLa was made by fusing them with polyethylene glycole. Those hybrids appeared to have both the characteristics of the parent cells, which can provide a natural intracellular environment such as the macrophages and the infinite growth capability of the HeLa cells in vitro.
Animal ; Culture Media ; Hela Cells ; Human ; Hybrid Cells ; Macrophages ; Mice ; Mycobacterium leprae/growth & development* ; Sciuridae

No abstract available.
Animal ; Child ; Clinical Trials ; Dapsone/therapeutic use* ; Haplorhini ; Human ; Leprosy/drug therapy* ; Mycobacterium leprae/growth & development ; Sciuridae/microbiology ; Time Factors

Leprosy is a chronic granulomatous disease caused by Mycobacterium leprae, which is an obligate intracelluar pathogen. It presents broad spectrum of clinical manifestations depending on the host's specific cell-mediated immune response to M. leprae. Especially, type I Th cells and macrophages are important in defense mechanism to M. leprae, and the immune response is regulated by cytokines secreted by immune cells. Recent investigations showed nitric oxide(NO) was the key molecule in the killing activity of macrophages, which was enhanced by IFN-gamma but suppressed by TGF-beta1 and IL-10. Since cytokine is secreted by activated immune cells with antigenic stimulation, decreased antigens by treatment modulates the expression of cytokines in leprosy. In this study, we observed the dynamics of cytokines expression using RT-PCR, such as TGF-beta1 and IL-10, which suppress the activity of macrophages, IFN-gamma, which activates macrophages, and iNOS, which represents the killing activity of macrophages, in the lesions taken from fifteen borderline lepromatous leprosy patients before and after multiple drug therapy for 4 weeks. The results are summarized as follows: 1. Before treatment, cytokines were expressed in order of IL-10, iNOS, TGF-beta1 and IFN-gamma(p>0.05). 2. After 4 weeks treatment, cytokines were expressed in order of iNOS, IL-10, TGF-beta1 and IFN-gamma(p<0.05). 3. Fifty-four percent of patients showed a non-polarized Th 0 pattern, 33% a polarized Th 1 pattern, and 20% Th-negative. Th 2 pattern was not observed. 4. The changes of cytokines expression after 4 weeks treatment were not significant, although mRNA of IL-10, TGF-beta1 and IFN-gamma were somewhat decreased. 5. There was negative correlation between TGF-beta1 and iNOS(gamma(2)=0.499, p<0.05, before treatment), positive correlation between TGF-beta1 and IFN-gamma(gamma(2)= 0.622, p<0.05, before treatment), and positive correlation between IFN-gamma and IL-10(gamma(2)= 0.935, p<0.05, before treatment; gamma(2)= 0.937, p<0.05, after treatment). In conclusion, these results suggest that TGF-beta1 and IL-10 may contribute to immune suppression in multibacterial leprosy patients, and that TGF-beta1 suppresses iNOS expression in macrophages. With 4 weeks treatment, the significant changes in cytokines expression were not observed. Interestingly, the majority of BL patients showed Th 0 pattern of cytokine, and none of Th 2 pattern.
Cytokines ; Drug Therapy ; Granulomatous Disease, Chronic ; Homicide ; Humans ; Interleukin-10* ; Leprosy* ; Leprosy, Multibacillary* ; Macrophages ; Mycobacterium leprae ; RNA, Messenger* ; Transforming Growth Factor beta1*