The detection of Mycobacterium leprae protein and carbohydrate antigens in skin and nerve from leprosy patients with type 1 (reversal) reactions.

Type 1 (reversal) reactions are the most common immunological complications of leprosy. These episodes of delayed hypersensitivity produce severe local immunopathology and ultimately nerve damage. To date, the Mycobacterium leprae antigens associated with type 1 reactions have not been identified. Using monoclonal antibodies to defined protein and carbohydrate M. leprae epitopes (65, 35 and 28 kd and lipoarabinomannan [LAM]) in a two-step immunoperoxidase staining technique, M. leprae antigens were demonstrated in skin and nerve biopsies from patients in reversal reaction. Antigen presence and staining patterns were similar in skin and nerve lesions, implying that the pathological processes are similar in the two sites. Antigens were present both in macrophages and Schwann cells but also as a diffuse extracellular infiltrate associated with the inflammatory infiltrate. The 28-kd antigen was present most strongly and may be a potential candidate antigen for initiating type 1 reactions. LAM also stained strongly and persisted after treatment. The possible roles of LAM and 65 kd in the cellular events of type 1 reactions are discussed.

Immunohistochemical analysis of cellular infiltrate and gamma interferon, interleukin-12, and inducible nitric oxide synthase expression in leprosy type 1 (reversal) reactions before and during prednisolone treatment.

The effects of prednisolone treatment on the cellularity and cytokine (gamma interferon, interleukin-12, and inducible nitric oxide synthase) profiles of leprosy skin type 1 (reversal) reactions were studied using immunohistochemistry. Skin biopsies were taken from 15 patients with leprosy type 1 (reversal) reactions at days 0, 7, 28, and 180 after the start of steroid treatment. Prednisolone treatment had little effect at day 7, but by day 28 significant decreases were found in cytokine levels. Some patients maintained cytokine production at days 28 and 180. These results illustrate the strong Th1 profile of type 1 reactional lesions, the slow response to steroid therapy, and continuing activity at 180 days.

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.

Immunocytochemical localization of inducible nitric oxide synthase and transforming growth factor-beta (TGF-beta) in leprosy lesions.

Inducible nitric oxide synthase (iNOS) and TGF-beta were localized by immunocytochemistry in skin lesions from patients across the leprosy spectrum, and from patients undergoing reversal reaction. iNOS expression was highest at the tuberculoid pole of the spectrum, and increased during reversal reaction. TGF-beta was observed throughout the leprosy spectrum, but was highest at the lepromatous pole. Levels of TGF-beta decreased during reversal reaction. Reduced levels of TGF-beta may contribute to unregulated inflammatory responses during reactional episodes.

Tumour necrosis factor-alpha (TNF-alpha) synthesis is associated with the skin and peripheral nerve pathology of leprosy reversal reactions.

Leprosy may be complicated by episodes of increased cell-mediated immunity towards Mycobacterium leprae (reversal reactions) which result in severe local immunopathology in skin lesions and peripheral nerves. Using in situ hybridization and MoAb techniques we have demonstrated TNF-alpha mRNA and TNF-alpha protein in macrophages infiltrating leprosy skin and peripheral nerve. Levels of TNF-alpha mRNA are significantly increased in reactional skin and nerve, particularly in borderline tuberculoid patients. TNF-alpha mRNA and TNF-alpha protein levels are higher in reactional nerves then reactional skin. In both reactional skin and nerve TNF-alpha mRNA is more abundant than TNF-alpha protein; this may reflect the rapid turnover of TNF-alpha protein in an immunologically dynamic situation, such as is seen in reversal reaction. Our findings emphasize the importance of documenting both mRNA and protein production when assessing the role of cytokines in pathology. The leprosy reversal reaction may be regarded as a useful model of tissue immunopathology in which TNF-alpha is generated as part of the host response to infection, but also produces local tissue damage.

Nerve and skin damage in leprosy is associated with increased intralesional heat shock protein.

Leprosy is frequently complicated by the development of reversal reactions in which peripheral nerve and skin lesions become inflamed and irreversible nerve damage may ensue. Increased expression of proteins belonging to the 70-kD heat shock family (hsp 70) occurs in cells of the central nervous system exposed to hyperthermia, physical damage or drug-induced trauma. In the present study we have used immunocytochemical staining to monitor hsp70 levels in peripheral nerves infected by Mycobacterium leprae. Hsp70 was detected in skin and nerve lesions from all leprosy patients, but was particularly prominent in lesions from patients undergoing reversal reactions. Hsp70 immunocytochemistry can thus be used as a marker of neural injury in the peripheral as well as in the central nervous system. The cellular dynamics of nerve damage in leprosy are currently poorly understood, and we postulate that the immunopathology of leprosy may be partly due to an autoimmune response to heat shock proteins.

Results of the third immunology of leprosy/immunology of tuberculosis antimycobacterial monoclonal antibody workshop.

An international workshop was sponsored by the World Health organization to screen new antimycobacterial monoclonal antibodies and to identify antibodies which could be recommended as standard reagents giving consistent results under differing assay conditions. Fifty-eight antibodies were submitted to the workshop by eight independent laboratories. Nineteen of the antibodies recognized antigens distinct from those identified in earlier workshops, defining at least 10 new protein antigens. Monoclonal antibodies characterized in the workshop provide a set of convenient reagents for further characterization of mycobacterial antigens.