Leishmania aethiopica: infections in laboratory animals.

Leishmania aethiopica parasites were inoculated into 11 different strains and species of small laboratory animals. Clinical lesions were only produced following inoculation of hamster noses and thus this parasite is highly selective in both species and site for the laboratory animals tested. Parasites could, however, be recovered from draining lymph nodes 3 weeks after infection of BALB/c mice. Lesions in hamsters were progressive and nonulcerating (up to 1 year) and histologically resembled diffuse cutaneous leishmaniasis (DTH) in man. Pronounced delayed hypersensitivity responses to L. aethiopica antigens only developed in mice despite the absence of clinical lesions. Weak DTH responses were produced in hamsters with clinical lesions only after 25 weeks of infection.

Human monoclonal antibodies against Mycobacterium leprae.

Human hybridomas were constructed which produce antibodies against three different extracts of Mycobacterium leprae. A thioguanine-resistant (Thgr), ouabain-resistant (Ouar), human lymphoblastoid cell line, KR-4, was hybridized with Epstein-Barr virus-transformed cell lines from lepromatous leprosy patients with fusion frequencies of greater than 10(-5). Non-Epstein-Barr virus-transformed donor cells fused at much lower rates (less than 2 X 10(-7]. Hybrids were selected in medium containing hypoxanthine aminopterin thymidine and 10(-5) M ouabain. An enzyme-linked immunosorbent assay was used to screen for antibodies against three crude extracts of armadillo-derived M. leprae, including (i) a soluble sonic extract preparation, (ii) sodium dodecyl sulfate extract of insoluble sonicated M. leprae, and (iii) a purified phenolic glycolipid antigen. Of a total of 2,200 final clones screened, 359 were found to secrete antibody which bound to soluble sonic extracts and the sodium dodecyl sulfate extract (6.7 and 9.6%, respectively), whereas 12.5% (21 out of 168) showed positivity to the glycolipid antigen. Four selected hybridomas also reacted with the deacylated derivative of M. leprae phenolic-glycolipid antigen. The specificity of these monoclonal antibodies was partially determined by screening on a panel of crude extracts from four other mycobacteria. Nine clones of 122 showed reactivity to M. leprae only. The predominant immunoglobulin was immunoglobulin M, and quantities up to 10 micrograms/ml were produced. Antibody production by hybrid clones was stable in more than 75% of the clones grown in continuous culture. By comparison, 10,000 Epstein-Barr virus-transformed lymphocyte clones from lepromatous leprosy patients were screened for anti-M. leprae antibody production, and all of the 42 clones that were initially positive in the enzyme-linked immunosorbent assay lost their antibody-producing capabilities within 6 weeks in culture. These results suggest that a combination of Epstein-Barr virus transformation and hybridization may be an optimal method in producing human monoclonal antibodies from leprosy patients.

Human monoclonal antibodies.

The technology for the production of murine monoclonal antibodies has been refined enormously since its introduction in 1975. However, the technology for generating human monoclonal antibodies has only recently come into its own. In this review, three currently available approaches to the production of human monoclonal antibodies are described. These include the hybridoma technique, based on the fusion of antibody-producing human B lymphocytes with either mouse or human myeloma or lymphoblastoid cells; the EBV immortalization technique, based on the use of Epstein-Barr virus (EBV) to 'immortalize' antigen-specific human B lymphocytes; and the EBV-hybridoma technique, based on a combination of the first two methods. The EBV-hybridoma system retains the advantageous features of the other two systems while overcoming their pitfalls and may be the current method of choice for producing human monoclonal antibodies with a defined specificity.

Antigen-specific suppressor cells in subclinical leprosy infection.

A two-stage in-vitro culture system was used to assay cells which suppress the lympho-proliferative response to Mycobacterium leprae (ML). Responses to ML, purified protein derivative of tuberculin, and streptokinase-streptodornase were preferentially suppressed by mitomycin-treated cells which had been primed with the same antigen in a 7-day primary culture. Healthy subjects exposed to leprosy for more than 3 years showed strong suppression of the response to ML antigens (11 of 12 showed more than 40% suppression), whereas those exposed for 3 months to 3 years showed much less suppression (12 of 15 showed less than 40% suppression). The in-vitro generation of strong ML-specific suppression may reflect the maturation of a well-regulated and protective immune response. However, premature induction and in-vivo activation of these suppressor cells could predispose to disseminated (lepromatous) forms of leprosy. With this assay it would be possible to assess the ability of proposed leprosy vaccines to engage strongly the regulatory network controlling the immune response to ML in the same way as long-term exposure to the natural infection.