Hypophosphorylation of NF-H and NF-M subunits of neurofilaments and the associated decrease in KSPXK kinase activity in the sciatic nerves of swiss white mice inoculated in the foot pad with mycobacterium leprae.

OBJECTIVE: To study the phosphorylation state of neurofilament (NF) proteins and activity of KSPXK kinase in the sciatic nerves of Swiss white (S/W) mice inoculated in the hind foot pads with M. leprae. DESIGN: Test group includes S/W mice inoculated in the foot pads with freshly harvested human derived (viable) M. leprae. Control groups were constituted by (1) Age matched un-inoculated mice, (2) Mice similarly inoculated with M. smegmatis and (3) heat killed M. leprae. Phosphorylation state of NF was studied using Western blot analysis and phosphor-specific NF antibody (SMI 31; Sternberger Monoclonals, Inc.). The KSPXK kinase activity was assayed by using KSPXK fusion protein in a radiometric method using gamma(22)P ATP. RESULTS: Several fold increase in M. leprae numbers was seen in viable M. leprae group while M. smegmatis failed to show any fold increase in the foot pads of S/W mice. Western immunoblot analysis of cytoskeletal preparation from sciatic nerves of uninoculated mice and mice inoculated with M. smegmatis showed immunoreactivity to SMI 31 antibody and protein bands corresponding to both NF-H and NF-M at all the time points from 4-20 months post inoculation. In case of viable M. leprae; SMI 31 reactive protein bands were seen at 4 months but not at any of the later intervals, i.e., between 6-20 months. With heat killed M. leprae transient loss of immunoreactivity to SMI 31 was seen. Decrease in KSPXK kinase activity was recorded in sets inoculated with viable and heat killed M. leprae, and corroborated with loss of immunoreactivity seen in WBs reacted with SMI 31 antibody. CONCLUSIONS: Alterations in the sciatic nerve NF cytoskeleton was seen following inoculation in the hind foot pad with both viable and heat killed M. leprae. The hypophosphorylation of NF observed in this study corroborates with the earlier observations in human leprous nerves.

ErbB2 receptor tyrosine kinase signaling mediates early demyelination induced by leprosy bacilli.

Demyelination is a common pathologic feature in many neurodegenerative diseases including infection with leprosy-causing Mycobacterium leprae. Because of the long incubation time and highly complex disease pathogenesis, the management of nerve damage in leprosy, as in other demyelinating diseases, is extremely difficult. Therefore, an important challenge in therapeutic interventions is to identify the molecular events that occur in the early phase before the progression of the disease. Here we provide evidence that M. leprae-induced demyelination is a result of direct bacterial ligation to and activation of ErbB2 receptor tyrosine kinase (RTK) signaling without ErbB2-ErbB3 heterodimerization, a previously unknown mechanism that bypasses the neuregulin-ErbB3-mediated ErbB2 phosphorylation. MEK-dependent Erk1 and Erk2 (hereafter referred to as Erk1/2) signaling is identified as a downstream target of M. leprae-induced ErbB2 activation that mediates demyelination. Herceptin (trastuzumab), a therapeutic humanized ErbB2-specific antibody, inhibits M. leprae binding to and activation of ErbB2 and Erk1/2 in human primary Schwann cells, and the blockade of ErbB2 activity by the small molecule dual ErbB1-ErbB2 kinase inhibitor PKI-166 (ref. 11) effectively abrogates M. leprae-induced myelin damage in in vitro and in vivo models. These results may have implications for the design of ErbB2 RTK-based therapies for both leprosy nerve damage and other demyelinating neurodegenerative diseases.

Light and ultrastructural study of sciatic nerve lesions induced using intraneural injection of viable Mycobacterium leprae in normal and immunosuppressed Swiss white mice.

Freshly harvested M. leprae were microinjected into the sciatic nerves of nonimmunosuppressed (non-TR) and immunosuppressed (TR) mice using the technique described by Wisniewski and Bloom. The lesions thus induced, on bypassing the blood-nerve barrier, were biopsied at regular intervals beginning 24 hr and followed up to one year. The fate of M. leprae and the ensuing inflammation and nerve damage were studied using light and electron microscopy. The lesions in both non-TR and TR mice at 24 hr showed an influx of polymorphonuclear leukocytes and an increase in mast cells. The influx and peaking of lymphocytes were delayed by two weeks and 6 weeks, respectively, in TR mice, but the density of lymphocytes at the peak intervals was comparable in both. The plasma cells denoting the humoral response were seen in both, but there was a delay of 3 weeks in non-TR mice. The lesions in non-TR mice showed differentiation of macrophages into epithelioid cells and the formation of giant cells depicting borderline tuberculoid leprosy (BT), Whereas in TR mice, the macrophages showed foamy cytoplasmic changes depicting borderline lepromatous leprosy (BL). Other significant observations common to both non-TR and TR mice were: a) The lesions remained highly localized and showed signs of regression at the 6th and the 12th month intervals. b) The characteristic segmental demyelination and some attempt at remyelination were seen at the site. c) The influx of lymphocytes concorded well with demyelination. d) Bacteria were only seen in the macrophages and never in the Schwann cells or endothelial cells. e) Bacteria persisted in the macrophages, but appeared progressively degenerate at the 6th and 12th post-inoculation months, suggesting loss of viability. The study shows that there was a very effective containment of the infection and that the Schwann cells were resistant to M. leprae infection in the neural milieu. Nerve damage and Schwann cell bacillation do not go hand-in-hand.

Effect of T-cell depletion on bacterial multiplication and pattern of nerve damage in M. leprae-infected mice.

Various mechanisms for nerve damage in tuberculoid leprosy have been proposed. A common feature amongst them is the crucial role played by T-cells. Therefore, the present study was designed to determine the role of T-cells in the induction of nerve damage in leprosy using two different protocols for obtaining graded levels of T-cell depletion: (i) Cyclosporine A, for depletion of T-helper cells and (ii) Anti Thy 1.2, for total depletion of T-cells. The findings indicate that the early changes seen in the unmyelinated fibres may not involve T-cells. However, the later stages of nerve damage associated with demyelination are dependent on T-cell responses.

Animal model to study the mechanism of nerve damage in leprosy--a preliminary report.

Intraneural injection of 10-20 x 10(6) viable Mycobacterium leprae into the sciatic nerve of normal, unsensitized, Swiss white mice gives rise to a tuberculoid type of granulomatous response in 2 weeks. The same dose of viable M. leprae when injected into the sciatic nerves of unsensitized immunosuppressed mice (T200 x 5R) elicited a macrophage response. When macrophages were systemically immobilized using an intraperitoneal injection of silica quartz dust in normal mice, the lesion produced was of the lepromatous type, suggesting a role for the macrophage in the induction of the tuberculoid type of granulomatous response. In all of these in situ experiments, M. leprae failed to enter the Schwann cells.

Reaction of peripheral nerves to vascular and bacterial injuries.

Mouse sciatic nerves were subjected to devascularization, M. leprae inoculation, and combined insult of devascularization + footpad inoculation (FPI). Changes were seen in FPI nerves only after eight months, but in cases of combined insult, changes were evident in hours. Both the groups showed initial loss of small myelinated fibres. No proliferation of Schwann cells was in FPI nerves, but in combined insult it was maximum after two weeks. Presence of M. leprae seems to be arresting Schwann cell activity after two weeks. Blood vessels showed increased endothelial cell cytoplasm, basement membrane proliferation and villi formation. These changes seem to be specific of endoneurial blood vessels of leprosy nerves. Increased number of mast cells seems to be specific of devascularized and FPI nerves. Increased number of macrophages expressed low immunity of devascularized nerves. Eosinophils migrated to endoneurium as a result of leakage of axoplasm.

An immunoelectronmicroscopical study of the expression of major histocompatibility complex (MHC) class II antigens in guinea pig sciatic nerves following induction of intraneural mycobacterial granulomas.

A guinea pig model of nerve damage in leprosy has been used to investigate the expression of major histocompatibility complex (MHC) class II antigens in granulomatous lesions in nerves. Using an immunoelectronmicroscopical technique, infiltrating mononuclear cells and endoneural fibroblast-like cells are shown to be class II-positive in the experimental neural lesions. Schwann cells are not class II-positive under these conditions, although at the light microscope level Schwann cell-like cells appear to be positively stained. This illustrates the value of immunoelectronmicroscopy in the investigation of cell surface proteins in situ as compared with conventional light immunohistochemistry.

Nerve damage induced by mycobacterial granulomas in guinea pig sciatic nerves.

A possible model for nerve damage in leprosy has been developed in the sciatic nerve of the guinea pig. Intraneural injection of 10(7) BCG organisms into an unsensitized animal induces an epithelioid cell granuloma in 2 weeks similar to that found in tuberculoid leprosy patients. In contrast, intraneural injection of 10(9) cobalt-irradiated Mycobacterium leprae organisms induces a macrophage granuloma in 5 weeks, similar to that found in lepromatous leprosy patients. Histological, immunohistochemical, electron microscopical and electrophysiological studies have demonstrated that the lesions induced in the experimental animals show many of the features documented in studies of nerve damage in leprosy patients.

Study of the involvement of the sciatic nerve following inoculation with M. leprae and other mycobacteria in the mouse foot pad.

In order to determine whether Mycobacterium leprae alone produce the typical damage in the sciatic nerves of foot pad inoculated mice as demonstrated earlier, a comparative study was undertaken using various other mycobacteria inoculated into the hind foot pads of normal Swiss white mice. The findings indicate that FMR isolates No. 51 and No. 75 and M. avium showed multiplication in the foot pads of the mice throughout the 4th, 6th or 8th postinoculation months and these infections were associated with neural changes in the sciatic nerves. The type of nerve involvement in the case of M. avium differs from M. leprae in being predominantly an axonal degeneration at the 8th post-inoculation month, that is, degeneration of the complete axon and myelin debris remnants; whereas in M. leprae infection, where segmental demyelination predominates, the axons are intact and it is the Schwann cell that is affected. The neural changes in the case of FMR isolates No. 51 and No. 75 were similar to those seen in mice inoculated with M. leprae obtained directly from human biopsies. Other mycobacteria, HI-75 (Skinsnes) and M. scrofulaceum, showed growth in the foot pad initially which persisted in the case of M. scrofulaceum until the 20th post-inoculation month, but no ultrastructural changes were observed in the sciatic nerves of these mice. In ICRC-inoculated mice, nerve lesions were seen much later (at the 16th post-inoculation month) and the changes were similar to those seen with M. leprae. M. vaccae, M. smegmatis, M. phlei, and M. intracellulare showed almost no growth in the foot pads of the mice, and there were no detectable changes in the sciatic nerves. M. lepraemurium showed growth in the foot pad but no lesions were seen in the sciatic nerve.(ABSTRACT TRUNCATED AT 250 WORDS)

Ultrastructure of sciatic nerve of armadillo infected with Mycobacterium leprae.

Ultrastructural observation of sciatic nerves from eight Armadillos were made. Six animals had intravenous inoculation of M. leprae, one had of foot pad, while one had natural leprosy. The available nerves were biopsied at various time sequence ranging from five weeks to twenty four months. Semithin sections did not reveal any neuropathy. Ultrastructurally perineurium was thick and endoneurial collagen was increased. Initially demyelination of non-myelinated fibres was seen in all nerves irrespective of mode of infection. This was followed by demyelination of small myelinated fibres. Active remyelination was predominantly after 17 months. Schwann cell activity was increased and various stages of division were seen. Bacilli were extracellular, intraxonal, in endothelium and in perineurium. Significant observations were on blood vessels. These observations are discussed.

Evaluation of the neurotoxicity of dapsone in the mouse model.

Effect of DDS was studied using the mouse model. It was observed that DDS did not have any neurotoxic effect. On the contrary it showed a protective action towards the nerve, when administered in the early stages following definite establishment of nerve lesions.

Vascular changes in nerves in experimental leprosy--an ultrastructural study.

Ultrastructural observations made on the blood vessels of nerves in CBA/J mice with experimentally produced M. leprae infection revealed cytoplasmic filamentous structures along with large sized vacuoles in the endothelial cells and reduplication of peripericytal basement membranes in the 6-9 months of infection. In animals with late infection of 9-15 months duration, the endothelial cells presented foamy appearance caused by an increase in the number of vacuoles. These findings are suggestive of degenerative changes in the endothelial cells probably due to circulation of noxious substance in the blood stream.

Study of evolution of nerve damage in leprosy. Part III--Sciatic nerve lesions in mice inoculated with M. leprae with nerve conduction velocity correlates.

Non-immunosuppressed Swiss mice inoculated with 5000 M. leprae in each hind foot pad were subjected to nerve conduction velocity studies followed by light and electron microscopy and fibre tease of both sciatic nerves at sequential time intervals from the 4th to the 24th month. The conduction velocity was standardized for basal temperature of 35 degrees C and uninoculated mice were used as controls. Progressive changes were noted in conduction velocity from the 6th post inoculation month and correlated with the ultrastructural changes which were first observed at the 4th month. Fibre teasing showed predominent segmental demyelination.

Study of early nerve lesions in mice infected with M. leprae.

The present study is of quantitative histology in immunologically intact mice inoculated with M. leprae. Total twelve sciatic nerves are studied. The fibres are grouped as large, medium and small sized fibres. Initially there is loss of small size fibres. At later stages there is involvement of all sized fibres and ultimately Wallerian type of degeneration sets in. The process of regeneration is more active than that of human leprosy of tuberculoid type. This study adds a new dimension in understanding the pathogenesis of leprosy.