Contact-dependent demyelination by Mycobacterium leprae in the absence of immune cells.

Demyelination results in severe disability in many neurodegenerative diseases and nervous system infections, and it is typically mediated by inflammatory responses. Mycobacterium leprae, the causative organism of leprosy, induced rapid demyelination by a contact-dependent mechanism in the absence of immune cells in an in vitro nerve tissue culture model and in Rag1-knockout (Rag1-/-) mice, which lack mature B and T lymphocytes. Myelinated Schwann cells were resistant to M. leprae invasion but undergo demyelination upon bacterial attachment, whereas nonmyelinated Schwann cells harbor intracellular M. leprae in large numbers. During M. leprae-induced demyelination, Schwann cells proliferate significantly both in vitro and in vivo and generate a more nonmyelinated phenotype, thereby securing the intracellular niche for M. leprae.

Immunohistochemical detection of PGL-1, LAM, 30 kD and 65 kD antigens in leprosy infected paraffin preserved skin and nerve sections.

A panel of lipid, carbohydrate and protein antibodies were optimized for use in detecting M. leprae antigens in paraffin embedded material. Skin and nerve biopsies from 13 patients across the leprosy spectrum were studied. All antibodies detected antigen in tissues with a BI > 1. Phenolic-glycolipid was not detected in bacteriologically negative tissue but lipoarabinomanan (LAM) and protein antigens were detected. Staining with LAM was strongest and gave least background. The transfer of this immunohistochemical technique to paraffin embedded material will allow examination of tissue with better morphology and from clinics without access to tissue freezing facilities.

[Mycobacterium leprae: behavior in the adipocyte undergoing differentiation]. / Mycobacterium leprae: comportement dans l'adipocyte en voie de différenciation.

We have investigated the behaviour of M. leprae in murine preadipocyte cells (clone Ob17) undergoing the adipose cell conversion process in vitro. Actively differentiating Ob17 cells were infected with M. leprae. The morphological index (MI) of the acid-fast bacteria (AFB) present at day 12 and day 25 after infection was compared to the MI of the AFB inoculated. An increase of the MI was consistently observed. This increase is suppressed by rifampicin. Due to important cell loss, an increase of the number of the AFB per culture could not be obtained in monolayer tissue cultures. In order to prevent cell loss, we used a three-dimensional culture system. This cell culture system is an in vitro reconstitution of the human dermis, a main target organ for the leprosy bacillus. Adipocytes infected with M. leprae are incorporated in a condensed collagen lattice together with skin fibroblasts. Under such conditions, both an increase of the MI and an increase of the number of the AFB are obtained. This suggests that cellular functions related to the adipose cell differentiation process might complement the defective bacterial genome, leading to transient multiplication in vitro.

Attempts to cultivate Mycobacterium leprae in fat tissue.

The behaviour of M. leprae in fat tissue was studied. Preadipocyte cells were infected with M. leprae and injected intradermally (I.D.) into nude mice. Adipose nodules obtained by in vivo differentiation of infected cells were maintained in vivo for 3 months and subsequently incubated in vitro for 3 months. Counts of bacilli showed no increase over this 6 months period. It is concluded that undifferentiated preadipocyte and mature fat cells are not permissive for M. leprae. The morphological changes observed following passage of M. leprae into adipose nodules might be related to the process of adipose cell differentiation.

In vitro synthesis of antimycobacterial antibodies with different specificities in various tissues of leprosy patients.

For the detection of the synthesis in vitro of anti-Mycobacterium leprae antibodies in various tissues of leprosy patients, biopsy specimens of skin lesions, nasal mucosa, larynx, lymph nodes, and bone marrow were cultured in a medium containing 14C-labeled lysine and isoleucine. The culture fluids were analyzed by crossed immunoelectrophoresis with intermediate gel and autoradiography. The results show that synthesis of anti-M. leprae antibodies occurs at the investigated sites of leprosy patients and that the specificities of the synthesized antibodies differ between sites in individual patients. It is conceivable that these antibodies play a role in the local defense against M. leprae.

Growth and drug sensitivity of M. lepraemurium by tissue culture applying monolayer and agar suspension technique.

M. lepraemurium grow well in a Balb/c 3T3 recloned cell line (A31). In monolayer culture, the average generation time of M. lepraemurium in A31 cells was 5.3 to 9.4 days at 37 degrees C. A31 cells are very sensitive to infection with M. lepraemurium. Bacterial increases were readily apparent 30 days after inoculating 2 X 10(5) A31 cells in monolayer culture with only six bacilli. The intracellular bacilli were well transferred without apparent losses by host cell transfer. The growth of intracellular bacilli was inhibited by streptomycin 100 micrograms/ml, clindamycin 25 micrograms/ml, INH 5 micrograms/ml, and rifampin 5 micrograms/ml. When streptomycin or clindamycin was removed from the culture medium after 41 days of treatment and the cultivation continued in drug-free medium, the intracellular bacilli began to multiply once more without a lag period. When the intracellular bacilli were treated with INH for 35 days or rifampin for ten days, growth resumed, but only after lag periods after removal of these drugs. We utilized agar suspension techniques for the cultivation of host cells M. lepraemurium because normal cells or transformed cells ceased undergoing cell division and remained healthy for long periods of time in agar medium. M. lepraemurium grew well in A31, A31 transformed by polyoma virus, nude mouse foot pad, chick embryo, and human neuroblastoma cells, utilizing the agar suspension technique. The agar suspension cell culture method should provide useful clues for the cultivation of M. leprae.

Organized nerve culture. I. A technique to study the effect of M. leprae infection.

Neonatal dorsal root ganglia were cultivated in vitro by the technique of Murray. Within a week bundles of organized nerve fibers containing proliferating Schwann cells in different phases of axon association and fibroblast cells destined to become peri- or endoneural cells were obtained, peri- or endoneural cells were obtained. Many of these nerve fibers were myelinated within 3-4 weeks. Such 1 or 2 week old cultures were inoculated with M. leprae, and bacilli were found in the cytoplasm of Schwann cells and fibroblasts, demonstrating that these cells are phagocytic in nature and that it is possible to infect them with M. leprae, Schwann cells, mostly in the free or early association phase, engulfed the bacilli, and this affected their further interaction with the axons and subsequent myelin synthesis.

Organized nerve culture. II. DNA synthesis in Schwann cells in the presence of M. leprae.

Schwann cells that contain M. leprae fail to incorporate DNA precursor, indicating blockage of DNA synthesis. Such a block could lead to no proliferation of Schwann cells, an essential requirement for successful association with axons and consequent myelination.

In vitro cultivation and characterization of Mycobacterium lepraemurium.

Three different strains of M. lm were regularly grown in vitro from suspensions of mouse organs if at least 10(5) organisms were inoculated on Ogawa egg yolk medium and incubated at 35 degrees C in a humidified, CO2 enriched atmosphere. Growth is slow and requires 2-3 months. Colonies are 1-2 mm in diameter, white to pale yellow. Microscopically the bacteria are acid-alcohol-fast pleomorphic rods with branchings and beaded filamentous forms. Mice inoculated with in vitro grown subcultures develop an infection indistinguishable from the one observed after injection with mouse passage strains of M. lm. The in vitro characteristics of the strains are identical and different from all other known mycobacteria.

In vitro synthesis of anti-mycobacterial antibodies in biopsies from skin lesions of leprosy patients.

To demonstrate local synthesis of anti-Mycobacterium leprae antibodies, biopsies from skin lesions of leprosy patients were cultured in vitro in a medium containing 14C-labeled lysine and isoleucine, and the culture fluids were analyzed by crossed immunoelectrophoresis with intermediate gel and autoradiography. The results show that anti-M. leprae antibodies were synthesized in vitro in the biopsies from the skin lesions of leprosy patients and that the specificity of the locally produced antibodies varied from patient to patient.