Reduction in CD1 positive epidermal Langerhans cell numbers in leprosy lesions following epicutaneous application of 2:4 dinitrochlorobenzene.

A study was made on Langerhans cells (LC) in the dermal lesions of leprosy after epicutaneous application of 2:4 dinitrochlorobenzene (DNCB) to the lesion. LC were quantitated with OKT6 monoclonal antibody and indirect immunofluorescence. A depletion or reduction in the numbers of CD1+ epidermal LC was observed at both 4 and 24 hours after the application of DNCB in the lesions of both tuberculoid and lepromatous leprosy, compared to untreated lesions.

Clinical and histopathological correlation in the classification of leprosy.

This study reports our observations on the correlation between clinical and histopathological diagnoses of the classification of leprosy. The histopathological classification of leprosy in 1351 cases was done per Ridley-Jopling criteria and was compared with the clinical diagnoses of the same cases. These 1351 cases included 79 cases diagnosed clinically as having a "reaction." However, the histopathologists could not detect any evidence of reaction in 16 of these 79 cases (20%). Of the remaining 1272 cases, 68 (5%) were reported as "no evidence of leprosy" by the histopathologists; 37 of these 68 were found to be from the clinically indeterminate type of leprosy. Histopathological and clinical diagnoses of the classification of leprosy coincided in 69% of the cases. Concordance between the clinical and histopathological diagnoses for different types of leprosy was: indeterminate (I) = 36%, tuberculoid (TT) = 50%, borderline tuberculoid (BT) = 77%, borderline (BB) = 26%, borderline lepromatous (BL) = 43%, and lepromatous (LL) = 91%. When some of the types were combined (BT with TT, BL with LL), the overall concordance figure was 76%; concordance for the TT/BT group was 80%, for the BL/LL group it was 93%. Since both TT and BT are considered paucibacillary and LL or BL are considered multibacillary for treatment purposes, differentiating TT from BT or BL from LL is, perhaps, therapeutically irrelevant. However, for classification purposes it appears that the weight given to different signs and/or histopathological parameters for classifying leprosy cases (especially TT, BB and I) needs to be reassessed.

An ultrastructural study of Schwann cells in peripheral nerves of leprosy patients.

An ultrastructural study of peripheral nerves in leprosy patients was carried out of ascertain the changes in Schwann cells containing myelinated and nonmyelinated axons. Axonal multiplication was noticed in nonmyelinated axons in specimens from both tuberculoid and lepromatous leprosy. The Schwann cells in tuberculoid nerves were devoid of M. leprae in contrast to those in lepromatous nerves in which large number of bacilli were seen. These observations suggest that the Schwann cells containing nonmyelinated axons may be affected more frequently in either type of leprosy.

In situ demonstration of Mycobacterium leprae antigens in leprosy lesions using monoclonal antibodies.

Cryostat sections of skin and nerve lesions of leprosy were stained with monoclonal antibodies recognising Mycobacterium leprae antigens and indirect immunofluorescence. In both the tuberculoid and lepromatous lesions, PGL1, 55-65-kDa, 17-kDa protein antigens and cross-reactive non-protein antigens were present. 65-kDa antigens were seen mainly in the skin lesions of lepromatous leprosy. The infiltrates in both the skin and nerve granulomas of tuberculoid and lepromatous leprosy showed membranous staining with monoclonal antibodies recognising PGL1 and 55-65-kDa antigens. Bacilli in the lesions and the cells in the lymph node granulomas of patients with tuberculosis or the infiltrates in the lesions of tinea corporis or sections of normal skin did not show any staining with these monoclonal antibodies. These results confirm that M. leprae antigens are present and are expressed on the infiltrating cells of leprosy lesions.

Immunohistological analysis of nerve granulomas in neuritic leprosy.

Immunohistological analysis of infiltrates of nerves in patients with neuritic leprosy was carried out using monoclonal antibodies defining T cell subsets, Langerhans cells, HLA DR antigens, and indirect immunofluorescence. In all, eight nerves were analyzed. 2 of the 8 nerves showed epithelioid cell granulomas surrounded by large numbers of lymphocytes. The predominant lymphocytes in these granulomas were activated T cells expressing CD3 and HLA DR antigens. The proportion of CD3+ and CD4+ cells was higher than that of CD8+ cells. The ratio of CD4+/CD8+ cells in these two biopsy specimens was 5.6 and 1.5, respectively. In these nerves CD4+ cells were diffusely scattered into epithelioid cell granulomas, while CD8+ cells were localized at the periphery of the granuloma. The remaining six nerves showed macrophages containing numerous bacilli, and a few lymphocytes and plasma cells diffusely distributed into the granuloma. In these nerves, only occasional lymphocytes expressing CD3 or CD4 or CD8 and HLA DR antigens were noticed. In two fo the biopsy specimens, a small proportion of CD8+ cells were visualized. Macrophages and Schwann cells were HLA DR+ in all nerves. CD1+ cells were not seen in the infiltrates of any of these nerves. A similar pattern and distribution of cells was noticed in the nerve granulomas of tuberculoid and lepromatous leprosy. These findings suggest that the mechanisms of nerve damage in the patients with neuritic leprosy could be either immunological or non-immunological, depending on the nature and characteristics of the infiltrates.

Isolation and characterization of infiltrates in the nerves of patients with neuritic leprosy.

A study was done on the characteristics of infiltrating cells in the nerves of 9 patients with pure neuritic leprosy, by preparing a single cell suspension. The patients had no skin lesion. Histopathological examination revealed that 2 of the 9 nerves showed granulomas characteristics of tuberculoid leprosy, while the remaining 7 had features of lepromatous granulomas. In the nerves showing tuberculoid granulomas, a high proportion of lymphocytes were T cells as they formed rosettes with sheep erythrocytes and only a few percent were EAC rosette forming cells. On the other hand, the nerves showing lepromatous granulomas contained only occasional lymphocytes which formed E and EAC rosettes. Macrophages from the granulomas of all the nerves were esterase positive, peroxidase negative, contained M. leprae and did not exhibit C3 surface receptors.

CD1-positive epidermal Langerhans cells in regressed tuberculoid and lepromatous leprosy lesions.

A comparison was made of the numbers of epidermal Langerhans cells in active and regressed lesions of tuberculoid and lepromatous leprosy using the OKT6 and OKIa monoclonal antibodies. A reduction in the numbers of CD1+ epidermal Langerhans cells was noticed in the regressed lesions of both the tuberculoid and lepromatous leprosy lesions unlike the active lesions. The majority of infiltrates in both types of regressed lesions were HLA-DR+ and CD1-.

An ultrastructural study of blood vessels in peripheral nerves of leprosy patients: blood vessels in peripheral nerves.

Ultrathin sections of nerves of tuberculoid and lepromatous leprosy were examined in an electron microscope for changes in endoneural blood vessels. In the tuberculoid nerves, hypertrophy of endothelial cells was the most prominent feature. This was to such an extent that the lumen of blood vessel was often closed. Endoneural blood vessels showed multilayers separated by collagen and ground substances. In contrast, in the lepromatous nerves, there was no hypertrophy of endothelial cells in the blood vessels and the lumen of the vessels was open. M. leprae were seen within the endothelial cells and these organisms were intact and probably viable. These observations suggest a possible involvement of endoneural blood vessels which may contribute to nerve damage in leprosy.

Ultrastructural characteristics of macrophages in dermal leprosy granulomas: macrophages in leprosy granulomas.

The characteristics of the cells of mononuclear phagocyte series (MPS) in the dermal granulomas of tuberculoid and lepromatous leprosy were studied by elereron microscopy. In the tuberculoid granulomas, majority of the cells of MPS (epithelioid cells) showed a distinct appearance. They were seen in close association with a large number of activated lymphocytes. The cells of MPS had a prominent nucleus. Their cytoplasm contained mainly rough endoplasmic reticulum, developed golgi and was free of M. leprae or its products. In contrast, the cells of MPS (macrophages) in lepromatous leprosy had a large nucleus. Their cytoplasm contained mainly whole M. leprae organisms/its phagocytosed products and rough endoplasmic reticulum. There was paucity of lymphocytes in these granulomas. These observations support the view that the cells of MPS in the tuberculoid granulomas may be secretory while those in the lepromatous granulomas were phagocytic.

Immunotherapy of treated BL/LL cases with BCG: histopathological, immunohistological and bacteriological assessments.

The persistence of dead as well as viable bacteria is an important therapeutic problem in multibacillary leprosy. In highly bacillated patients, viable bacteria are detectable in 10-15% of cases after 2 years of treatment and none of these cases became smear negative by 2 years of recommended multidrug therapy (MTD). Immunotherapy trials using BCG (0.1 mg) by intradermal route have been undertaken in cases who had MDT for 2 years and who had viable bacilli by ATP photometry and/or FDA-EB staining. Biopsies and smears were taken from local as well as distant sites at 0.4 weeks and 6 months after BCG vaccination. Biopsies were processed for ATP counts, FDA-EB staining, histopathology and immunohistology for cell types at 0.4 weeks. There was transient effect on BI, ATP counts, FDA-EB staining at local as well as distant sites in some cases. Histopathology and immunohistological findings suggest that there is tendency to form epithelioid cell granuloma at local site in all cases and at distal sites in some. There was infiltration of subepidermal zone in one case, 4 weeks after vaccination. BCG may be of use as potential immunotherapeutic agent but its usefulness needs to be investigated in depth preferably in the beginning or early phases of chemotherapy and with also repeated inoculations.

Comparison of the characteristics of infiltrates in skin and nerve granulomas of leprosy.

The characteristics of infiltrates in the dermal and neural granulomas from the same leprosy patients were compared by preparing a single cell suspension. Skin and nerve biopsies from 10 patients, 5 with tuberculoid and 5 with lepromatous leprosy were analysed. The granulomas contained lymphocytes and macrophages. Lymphocytes were the predominant infiltrating cells in the tuberculoid dermal and neural granulomas. A high proportion of lymphocytes in both the skin and nerve granulomas in these cases were activated T cells as they formed rosettes with sheep erythrocytes and expressed HLA-DR antigens. In contrast, lepromatous dermal and neural granulomas contained very few of these lymphocytes. Dermal and neural granulomas from both the types of leprosy contained mature macrophages as they were esterase positive, did not exhibit peroxidase activity and expressed HLA-DR antigens. These macrophages did not possess C3 surface receptors either. These findings suggest that the infiltrates in the skin and nerve granulomas of a given type of leprosy have similar characteristics.

Isolation and characterization of cells in granulomas of nerves of leprosy patients.

Single cell suspension from the granulomas in nerves of leprosy patients were prepared for an in vitro study of the properties of infiltrating cells. Nerve biopsies from 17 patients with tuberculoid (n = 9) and lepromatous (n = 8) leprosy cases were analysed. The granulomas were found to contain lymphocytes and macrophages. Lymphocytes were the predominant infiltrating cells in the tuberculoid nerves. In contrast, lepromatous nerves contained very few of these cells. The majority of lymphocytes in tuberculoid granulomas were activated T cells as they formed rosettes with sheep erythrocytes, exhibited esterase dots in the cytoplasm and expressed HLA-DR antigens. A small proportion of the lymphocytes also formed rosettes with EAC. Most macrophages from both the granulomas were mature macrophages as they were esterase positive, did not exhibit peroxidase activity and expressed HLA-DR antigens. The macrophages did not possess C3 surface receptors.

CD1-positive epidermal Langerhans cells in skin reactions to autologous peripheral-blood-derived mononuclear cells in leprosy patients.

A comparison was made on the characteristics of the infiltrates, the number and distribution of CD1-positive epidermal Langerhans cells (LC) at the sites of skin reaction induced by autologous peripheral-blood-derived mononuclear cells (PBMC) in leprosy patients. Clinically and histologically, the skin reaction was well expressed in tuberculoid patients as compared to lepromatous patients, erythema nodosum leprosum (ENL) patients and contacts. The quantum of lymphocytes in the infiltrates was maximal in the tuberculoid patients and it was minimal in lepromatous and ENL patients. The number and distribution of LC in the tuberculoid patients was significantly higher in the PBMC-inoculated sites as compared to control sites over 24 h. In contrast, no difference in the number and distribution of LC was noticed in the lepromatous and ENL patients. These observations indicate that the lymphocytes of tuberculoid patients in contrast to lepromatous leprosy patients are capable of sustenance in the local micro-environments of the skin and an effective interaction may be possible between LC and PBMC.

In vitro studies on dermal leprosy granulomas: assessment of division and protein synthesis of cells.

Single cell suspension from dermal leprosy granulomas (10 tuberculoid and 10 lepromatous) was prepared and an assessment of the division and protein synthesis by the cells was made. The cells of tuberculoid granulomas showed a high incorporation of 3H-thymidine and 14C-leucine. On the contrary, the cells of the lepromatous granulomas exhibited poor division but their protein synthesis remained unimpaired. These observations suggest that the epithelioid cell granuloma of tuberculoid leprosy appears to be more active and secretory than the macrophage granuloma of lepromatous leprosy.

Ultrastructural studies of peripheral nerves in lepromatous leprosy patients.

Ultrathin sections of the peripheral nerves taken from three lepromatous leprosy patients (One untreated, other treated and third in ENL reaction) was examined in the electronmicroscope. In the untreated patient, solid M. leprae organism inside the schwann cell and the degeneration of schwann cell was seen. In contrast, the treated patient showed the degeneration of bacilli and myelinated fibres. However, the characteristics of cells in the ENL reaction showed close similarities with the untreated case.

T and B cells in borderline (BB) leprosy.

The response to standard Dharmendra lepromin and the circulating T, B cell numbers in the peripheral blood were quantitated in 15 patients with Borderline (BB) Leprosy. On the basis of lepromin response, the patients fall into three groups (a) negative (b) +/- reaction (c) rarely positive. No significant difference in the numbers of E-rosette and EAC rosette forming cells was observed in the BB patients in comparison to controls.

Effect of supernatants of dermal leprosy granulomas on lymphocyte morphology and function.

A comparison has been made of the characteristics of dermal granulomas of tuberculoid and lepromatous leprosy by culturing them in vitro. The granulomas were derived from lesions of untreated patients and their effect was assessed on the morphology and function of lymphocytes derived from peripheral blood of normal individuals. The concentration of proteins released in the supernatants was similar in both the type of granulomas. However, the supernatants from the lepromatous granulomas markedly diminished the viability of lymphocytes when compared with supernatants derived from the tuberculoid granulomas. The supernatants from both the tuberculoid and lepromatous granulomas, contained soluble factors which depressed the 14C-leucine and 3H-thymidine incorporation by lymphocytes. The depression in 3H-thymidine uptake was more pronounced with the supernatants from the lepromatous granulomas while the diminution of 14C-leucine incorporation was more marked with supernatants from the tuberculoid granulomas. The supernatants did not show any migratory inhibitory activity in vitro. When the cells from the granulomas were dispersed and cultured in vitro, only very low concentration of proteins was detectable.

CD1 positive epidermal Langerhans cells in indeterminate leprosy.

Langerhans cells (LC) in the skin lesions of 10 untreated indeterminate leprosy patients were defined by indirect immunofluorescence using monoclonal antibodies. No difference was observed in the numbers and distribution of epidermal LC in the indeterminate lesions and controls. However, the infiltrates of these lesions contained CD1+ cells. Most cells in the infiltrates HLA DR antigens.