Molecular mimicry between HSP 65 of Mycobacterium leprae and cytokeratin 10 of the host keratin; role in pathogenesis of leprosy.

Mycobacteria are known to induce autoimmune response in the host. Anti-host keratrin antibodies (AkAbs) might be responsible for the autoimmune phenomena in leprosy patients as majority of leprosy lesions are manifested in the skin and occurrence of keratosis is not an uncommon feature. The aim of this study was to find out the level of AkAbs in leprosy patients across the spectrum and to explore its correlation with the clinical manifestation of the disease. Further, mimicking epitopes of keratin and Mycobacterium leprae components were characterized. We screened 140 leprosy patients (27 BT, 28 BL, 41 LL, 25 T1R, 19 ENL), 74 healthy controls (HC) and 3 psoriasis patients as positive control. Highest AkAbs level was observed in the psoriasis patients followed by T1R, LL, BL, ENL, TT/BT. AkAbs level was significantly (p<0.05) higher in all the groups of leprosy patients except TT/BT in comparison to HC. Significant positive correlation was found between number of lesions and level of AkAbs in leprosy patients. Highest lympho-proliferation for keratin protein was observed in T1R, followed by BL/LL, TT/BT, ENL. Lympho-proliferation was significantly (p<0.05) higher in all groups of leprosy patients except ENL in comparison to HC. Interestingly, it was noted that hyperimmunization of inbred strains of female BALB/c mice and rabbit with M. leprae soluble antigen (MLSA) induce higher level of AkAbs. The percentage of FoxP3(+) expressing Treg cells (total CD4(+)CD25(+)FoxP3(+) andCD4(+)CD25(+hi)FoxP3(+)) in splenocytes and lymph nodes of hyperimmunized mice were declined in comparison to control mice. Further, it was found that this autoimmune response can be adoptively transferred in naïve mice by splenocytes and lymph node cells as well as T cells. Comparative molecular characterization between keratin and MLSA noted a cross-reactivity/similarity between these two antigens. The cross-reactive protein of keratin was found to be in molecular weight range ≈74-51kDa and at pI 4.5 while the cross-reactive protein of MLSA was found to be in molecular weight ≈65kDa and at pI 4-4.5. Cross-reactive protein of keratin and MLSA was identified and characterized by MALDI-TOF/TOF analysis and Mascot software. It was found that the keratin (host protein) which reacted with anti-M. leprae sera is cytokeratin-10 and MLSA which reacted with anti-keratin sera is heat shock protein 65 (HSP 65). Seven B-cell epitopes of cytokeratin-10 and HSP 65 was found to be similar by multiple sequence alignment using ClustalW server and out of which 6 B-cell epitopes were found to be on the surface of HSP 65. In conclusion, our study provides evidence for the existence of molecular mimicry between cytokeratin-10 of keratin (host protein) and 65kDa HSP (groEL2) of M. leprae. Presence of heightened CMI response of leprosy patients to keratin and positive correlation of AkAbs level with number of lesions of leprosy patients showed the clinical evidence for its role in the pathogenesis in leprosy.

Clofazimine-induced crystal-storing histiocytosis producing chronic abdominal pain in a leprosy patient.

Clofazimine-induced crystal-storing histiocytosis is a rare but well-recognized condition in the literature. Besides the common reddish discoloration of the skin, clofazimine produces gastrointestinal disturbances-sometimes severe abdominal pain, prompting exploratory laparotomy, because pathologic and radiologic findings can produce diagnostic difficulties if the pathologic changes caused by clofazimine are not recognized. The authors report such a case in a leprosy patient to emphasize the importance of history taking, the radiologic abnormalities of the small intestine, and the pathologic findings in small intestine and lymph node biopsies. Clofazimine crystals are red in the frozen section and exhibit bright-red birefringence. However, they are clear in routinely processed histologic sections because they dissolve in alcohol and organic solvents. They also appear as clear crystal spaces during electron microscopic study, but some osmiophilic bodies can be observed. Histiocytosis caused by clofazimine crystals produces infiltrative lesions in radiologic studies mimicking malignant lymphoma or other infiltrative disorders. Associated plasmacytosis in the histologic sections can simulate lymphoplasmacytic lymphoma or multiple myeloma with crystal-storing histiocytosis. With the knowledge of this rare condition caused by clofazimine, appropriate management to avoid an unnecessary laparotomy is possible.

Reversal of T cell unresponsiveness by augmentation of antigen presenting cell function.

We have previously described epitopes of the 18 kDa protein of Mycobacterium leprae which stimulate T and B cell responses in different strains of mice. A series of overlapping 20-mer peptides that span the 18 kDa protein were used as immunogens to examine T and B cell recognition of different epitopes. Strain-specific variation in the epitopes which induce the strongest responses was affected by genes linked to the H-2 complex and the T cell responses revealed by re-challenge with antigen were at least partially controlled by factors other than T cell specificity. We have examined the responses to one such antigen, peptide 1-20, which contains strongly immunogenic epitopes for T and B cells. T cells from draining lymph nodes of peptide 1-20 immunized B10.BR, but not BALB/c mice, proliferated in vitro in response to rechallenge with peptide 1-20 or whole protein. Immunization with the same peptide also induced specific antibody only in B10.BR mice. However, immunization of BALB/c mice results in 'silent' priming of T cells since these can be induced to respond in vitro to this antigen when cultured with activated macrophages as antigen presenting cells (APC). The failure of APC from mBALB/c mice primed with peptide 1-20 to stimulate CD4+ proliferation when re-challenged in vitro and the failure to elicit antibody responses to peptide 1-20 are presumably due to the same defect in antigen-presenting cell function, since presentation of peptide 1-20 by activated macrophages is sufficient to restore both responses.(ABSTRACT TRUNCATED AT 250 WORDS)

Protection of mice against mycobacterial infection by lymphoid cell vaccination.

Intracellular parasites may thrive by inducing the host's immune system to suppress the effector immune response that otherwise limits multiplication. Hosts are traditionally immunized with the parasite antigens that induce effector immunity. Alternatively, one might vaccinate the host with the host lymphoid cells involved in suppression. Multiplication of Mycobacterium marinum was prevented by vaccinating mice with cells prepared from the popliteal lymph nodes of mice in which the organisms were multiplying logarithmically, that were inactivated by fixation with glutaraldehyde. Cells obtained later during infection, when the donor mice manifest immunity, did not protect against infection. Thus, it may be possible to influence the course of a microbial infection by immunizing the host not only with components of the organisms, but also with the host components that are exploited by the organism.

Distribution in tissue sections of the human groEL stress-protein homologue.

A monoclonal antibody (ML30) raised against the 65 kDa heat-shock protein of mycobacteria showed widespread staining of sections from standard paraffin-embedded human tissues. The staining had a granular pattern and was particularly marked in cells with abundant mitochondria. Increased staining was observed in the synovial lining, histocytes and in the endothelium of reactive and rheumatoid synovium; it was also increased in the reactive lung alveolar lining. It is suggested that the antibody identifies an epitope in mitochondria of a protein homologous with the groEL heat-shock protein of bacteria.

Presence of Mycobacterium leprae-reactive lymphocytes in lymph nodes of lepromatous leprosy patients.

A critical problem in leprosy is the relative deficiency of antigen-specific T cell-mediated immunity. We were successful in detecting a significant response to viable M. leprae in mononuclear cells isolated from the lymph nodes of lepromatous leprosy patients in contrast to the apparent M. leprae-specific energy seen in the peripheral blood. This observation suggests that antigen-reactive lymphocytes are generated in the lymph nodes of lepromatous patients but the inability to detect them in the circulation may be due either to a different processing and presentation of mycobacterial antigens within the peripheral blood and lymph node compartments or to a selective sequestration of lymphocytes within the lymph node.

Accessory cell function of cells isolated from Mycobacterium leprae-induced granulomas.

The large cells from Mycobacterium leprae-induced granulomas in guinea pig lymph nodes were separated by Percoll discontinuous density gradient centrifugation and on a fluorescence-activated cell sorter (FACS) using cross-reacting monoclonal antibody to human MHC Class II antigens. Large Percoll-separated cells (83% Class II antigen positive and 52% macrophage-specific antigen positive) and FACS-separated cells are able to act as antigen-presenting cells for T-cell proliferation to PPD. In previous studies, macrophage antigen-positive cells consistently failed to act as accessory cells. This indicates that there is a population of accessory cells which are macrophage antigen negative and MHC Class II antigen positive present in these M. leprae-induced granulomas.

Immunity to leprosy. III. The in vitro induction of B lymphocyte proliferation by mycobacteria.

The development of murine proliferative response assays has been initiated to begin to evaluate T-lymphocyte responses to the antigens of Mycobacterium leprae. In this study, M. leprae and 13 related strains of mycobacteria have been tested for stimulatory effects in proliferation assays using murine spleen, thymus or lymph node cultures. A number of mycobacteria were found to directly stimulate the proliferation of spleen and lymph node cells of all mouse strains tested including C3H/HeJ mice. Thymocyte cultures showed no response. The mitogenic effects of mycobacteria in spleen cultures were not dependent upon the presence of T cells or adherent cells, and resulted in the production of antibody-forming cells. Thus, these bacteria acted as polyclonal B-cell mitogens and could be readily distinguished from the lipopolysaccharide of Gram-negative bacteria by their mitogenic activity on C3H/HeJ spleen cells. The species of mycobacteria which exhibit direct mitogenic effects in spleen and lymph node cultures are a particular problem when specific immune responses to the antigens of these bacteria are compared. Such comparisons are necessary if in vitro assays are to be used to determine the nature of crossreactive antigens between M. leprae and other mycobacteria.

Characterization and functional studies of the murine T-lymphocyte response to Mycobacterium leprae antigen.

Mice were immunized with Mycobacterium leprae in incomplete Freund's adjuvant, and sensitized lymphocytes were obtained from draining lymph nodes. The lymphocytes thus obtained proliferated specifically in vitro in the presence of M. leprae antigen, and this response was shown to be both T-cell and macrophage dependent. T-cell blasts generated in vitro in response to M. leprae antigen were grown in the presence of interleukin-2 (IL-2). The proliferative response of these blasts to M. leprae antigen was strictly dependent on the presence of syngeneic spleen cells as antigen-presenting cells. M. leprae-immune F1 blasts responding to the antigen in the context of either parental H-2 haplotype-bearing accessory cell could be obtained by positive selection from an F1 hybrid-responding cell population. By means of flow microfluorometry the T-cell phenotype of the M. leprae-specific T-cell blasts was found to be Thy-1+ and to be composed of Lyt-1+ and Lyt-2+ subpopulations. Functionally, the blasts were shown to transfer delayed-type hypersensitivity locally to non-immunized recipients and to have cytolytic activity. Limiting dilution analysis showed the frequency of M. leprae-responding cells from blasts grown in IL-2 to be approximately 1/333.

Specific antimononuclear phagocyte monoclonal antibodies. Application to the purification of dendritic cells and the tissue localization of macrophages.

3C10 and 1D9 are two related monoclonal antibodies that specifically identify human mononuclear phagocytes in a large number of sites, including blood monocytes, alveolar macrophages, and macrophages in tissue sections of spleen, lymph node, and skin. The antigen persists on monocytes cultured for greater than 4 wk, but it is not found on giant cells. The 3C10-1D9 determinant is carried by a 55 kD polypeptide, is expressed at approximately 40,000 copies per monocyte, and is protease sensitive. The antigen is clearly different from HLA-class II or Ia-like antigens that have been studied with a new monoclonal 9.3F10. The 9.3F10 antigen is found on B cells, dendritic cells and monocytes; is protease resistant, and occurs on a 33-29 kD doublet typical of class II products. The 3C10 monoclonal provides a clear distinction between human mononuclear phagocytes and dendritic cells. First, monocytes and lymphocytes can be eliminated from plastic-adherent mononuclear cells using 3C10, complement, and two previously described cytotoxic antibodies, BA-1 (anti-B cell) and Leu-1 (anti-T cell). As a result, the trace dendritic cell component of blood can be enriched to considerable purity (65-75%) and yield. Second, immunocytochemical staining of tissue sections reveals that 3C10+ macrophages are anatomically segregated from dendritic cells. Large numbers of 3C10+ cells are found in red pulp of spleen and in regions surrounding lymphatic channels of lymph node. However, 3C10+ macrophages are scarce in white pulp of spleen and the lymphocyte-rich cortex of node that are the sites where dendritic cells are localized. 3C10+ cells in skin are found in the dermis, particularly in leprosy infiltrates, but the Langerhans' cells of epidermis are 3C10-. The distinctive localization of macrophages and dendritic cells is consistent with their respective functions as effector and accessory cells in the immune response.

Correlaçoes entre histopatologia cutânea e de linfonodos nas fases evolutivas da hanseníase virchoviana

Com o fito de avaliar as modificaçoes evolutivas dos linfonodos na hanseníase virchowiana, bem como as correlaçoes entre os estádios evolutivos da infecçao cutânea e nos linfonodos, estudados, do ponto de vista histológico e baciloscópico: Biópsia de lesao cutânea representativa e linfonodo inguinal de dez pacientes virchowianos em atividade e de dez pacientes virchowianos em atividade e de dez pacientes virchowianos em regressao. Fragmentos de pele e linfonodos axilar colhidos em necrópsias de dez pacientes virchowianos residuais (branqueados). Tanto nos virchowianos em atividade como nos virchovianos em regressao observou-se marcada heterogeneidade na constituiçao histiocitária dos infiltrados especificos presentes nos linfonodos em contraste com a constituiçao homogenea dos infiltrados específicos cutâneos. Os granulomas macrofágicos nos linfonodos assumem uma estratificaçao bem caracteristica com histiócitos jovens em localizaçao superficial e histiócitos progressivamente mais vacuolados e regressivos situando-se na área paracortical profunda

Immunological characteristics of the armadillo, Dasypus sabanicola.

The immunological responses of the armadillo are of interest because of its susceptibility to generalized lepromatoid infection with Mycobacterium leprae. In this study, specimens of Dasypus sabanicola were found to have a typical mammalian distribution of lymphoid cells in thymus, spleen, lymph nodes and blood. Their complement was active in bactercidal, protozoan immobilization and haemolytic systems. Blood lymphocytes responded to phytohaemagglutinin and to pokeweed mitogen. Sensitization with ovalbumin in CFA resulted in the production of circulating precipitins; strong Arthus reactions were detectable in the sensitized animals. Responses of cell-mediated immunity to DNCB and to M. tuberculosis were very discrete. Heat-killed M. leprae elicited granulomatous reactions characterized by microscopic necrosis, but without abundant lymphocytic infiltration; skin tests and lymphocytic transformation were generally negative in the animals injected with M. leprae.

Growth of macrophages obtained from various sources.

Techniques to obtain macrophages from various sources of the mouse were reported. The following sources were included: peritoneal exudate, alveolar lavage, blood leucocytes, bone marrow, spleen, liver, lungs, lymph nodes, thymus, thyroid, heart muscle, kidney, and subcutaneous cover glass implants. Human blood macrophages were also included. Long-term cinemicrographic studies revealed sustained good growth of these macrophages. Cell multiplication was detected in all of these cultures except those obtained from the peritoneal exudate. Pure cultures of macrophages were obtained from blood of the mouse and human. Macrophages obtained from other sources were accompanied by some growth of fibroblasts. Methods to eliminate the fibroblasts in cultures were discussed.