Notch Signaling Pathway Expression in the Skin of Leprosy Patients: Association With Skin and Neural Damage.

Introduction: Leprosy is an infectious disease caused by Mycobacterium leprae, a debilitating disease that affects the skin and peripheral nerves. It is possible that tissue changes during infection with leprosy are related to alterations in the activity of the Notch signaling pathway, an innate signaling pathway in the physiology of the skin and peripheral nerves. Methods: This is a descriptive observational study. Thirty skin biopsies from leprosy patients and 15 from individuals with no history of this disease were evaluated. In these samples, gene expressions of cellular components associated with the Notch signaling pathway, Hes-1, Hey-1, Runx-1 Jagged-1, Notch-1, and Numb, were evaluated using q-PCR, and protein expression was evaluated using immunohistochemistry of Runx-1 and Hes-1. Results: Changes were observed in the transcription of Notch signaling pathway components; Hes-1 was downregulated and Runx-1 upregulated in the skin of infected patients. These results were confirmed by immunohistochemistry, where reduction of Hes-1 expression was found in the epidermis, eccrine glands, and hair follicles. Increased expression of Runx-1 was found in inflammatory cells in the dermis of infected patients; however, it is not related to tissue changes. With these results, a multivariate analysis was performed to determine the causes of transcription factor Hes-1 reduction. It was concluded that tissue inflammation was the main cause. Conclusions: The tissue changes found in the skin of infected patients could be associated with a reduction in the expression of Hes-1, a situation that would promote the survival and proliferation of M. leprae in this tissue.

Nerve biopsy in Indian patients with mononeuropathy multiplex of undetermined etiology.

INTRODUCTION: A diagnosis of mononeuropathy multiplex (MM) requires detailed evaluation to determine etiology. We performed nerve biopsy on patients with MM in whom the etiology could not be established via other investigations. METHODS: Sixty-eight patients with MM seen between January 2013 and June 2014 underwent detailed diagnostic evaluation. Those in whom the investigations failed to establish an etiology underwent nerve biopsy. RESULTS: A diagnosis of leprosy was confirmed in 14 patients and was highly probable in 17 others. Eleven patients had vasculitic neuropathy, and in 1 patient there were amyloid deposits on nerve biopsy. CONCLUSIONS: In 43 of 68 Indian patients (63%) with MM, nerve biopsy identified a definite (26 patients) or probable (17 patients) etiology. Nerve biopsy is a valuable investigation in MM that frequently results in a diagnosis of leprosy in India. Muscle Nerve, 2016 Muscle Nerve 55: 23-27, 2017.

Skin innervation: important roles during normal and pathological cutaneous repair.

The skin is a highly sensitive organ. It is densely innervated with different types of sensory nerve endings, which discriminate between pain, temperature and touch. Autonomic nerve fibres which completely derive from sympathetic (cholinergic) neurons are also present. During all the phases of skin wound healing (inflammatory, proliferative and remodelling phases), neuromediators are involved. Several clinical observations indicate that damage to the peripheral nervous system influences wound healing, resulting in chronic wounds within the affected area. Patients with cutaneous sensory defects due to lepromatous leprosy, spinal cord injury and diabetic neuropathy develop ulcers that fail to heal. In addition, numerous experimental observations suggest that neurogenic stimuli profoundly affect wound repair after injury and that delayed wound healing is observed in animal models after surgical resection of cutaneous nerves. All these observations clearly suggest that innervation and neuromediators play a major role in wound healing. Interactions between neuromediators and different skin cells are certainly crucial in the healing process and ultimately the restoration of pain, temperature, and touch perceptions is a major challenge to solve in order to improve patients' quality of life.

Degree of skin denervation and its correlation to objective thermal sensory test in leprosy patients.

BACKGROUND: Leprosy is an infectious disease affecting skin and peripheral nerves resulting in increased morbidity and physical deformities. Early diagnosis provides opportune treatment and reduces its complications, relying fundamentally on the demonstration of impaired sensation in suggestive cutaneous lesions. The loss of tactile sensitivity in the lesions is preceded by the loss of thermal sensitivity, stressing the importance of the thermal test in the suspicious lesions approach. The gold-standard method for the assessment of thermal sensitivity is the quantitative sensory test (QST). Morphological study may be an alternative approach to access the thin nerve fibers responsible for thermal sensitivity transduction. The few studies reported in leprosy patients pointed out a rarefaction of thin dermo-epidermal fibers in lesions, but used semi-quantitative evaluation methods. METHODOLOGY/PRINCIPAL FINDINGS: This work aimed to study the correlation between the degree of thermal sensitivity impairment measured by QST and the degree of denervation in leprosy skin lesions, evaluated by immunohistochemistry anti-PGP 9.5 and morphometry. Twenty-two patients were included. There were significant differences in skin thermal thresholds among lesions and contralateral skin (cold, warm, cold induced pain and heat induced pain). The mean reduction in the density of intraepidermal and subepidermal fibers in lesions was 79.5% (SD = 19.6) and 80.8% (SD = 24.9), respectively. CONCLUSIONS/SIGNIFICANCE: We observed a good correlation between intraepidermal and subepidermal fibers deficit, but no correlation between these variables and those accounting for the degree of impairment in thermal thresholds, since the thin fibers rarefaction was homogeneously intense in all patients, regardless of the degree of sensory deficit. We believe that the homogeneously intense denervation in leprosy lesions should be objective of further investigations focused on its diagnostic applicability, particularly in selected cases with only discrete sensory impairment, patients unable to perform the sensory test and especially those with nonspecific histopathological finds.

Alterations in neurofilament protein(s) in human leprous nerves: morphology, immunohistochemistry and Western immunoblot correlative study.

Using a specific antibody (SMI 31), the state of phosphorylation of high and medium molecular weight neurofilaments (NF-H and NF-M) was studied in 22 leprous and four nonleprous human peripheral nerves by means of immunohistochemistry, sodium dodecyl sulfate-poly acrylamide gel electrophoresis (SDS-PAGE) and Western immunoblot (WB). The results thus obtained were compared with morphological changes in the respective nerves studied through light and electron microscopy. Many of the leprous nerves showing minimal pathology revealed lack of or weak staining with SMI 31, denoting dephosphorylation. Remyelinated fibres stained intensely with SMI 31 antibody. The WB analysis of Triton X-100 insoluble cytoskeletal preparation showed absence of regular SMI 31 reactive bands corresponding to 200 and 150 kDa molecular weight (NF-H and NF-M, respectively) in 10 nerves. Three of the 10 nerves revealed presence of NF protein bands in SDS-PAGE but not in WB. Presence of additional protein band (following NF-M) was seen in four nerves. Two nerves revealed NF-H band but not NF-M band and one nerve showed trace positivity. In the remaining five nerves presence of all the three NF bands was seen. Thus, 77.3% (17/22) of human leprous nerves studied showed abnormal phosphorylation of NF protein(s). The ultrastructural study showed abnormal compaction and arraying of NF at the periphery of the axons in the fibres with altered axon to myelin thickness ratio (atrophied fibres) as well as at the Schmidt-Lantermann (S-L) cleft region. Such NF changes were more pronounced in the severely atrophied axons suggesting a direct correlation. The observed well-spaced NF in the remyelinated fibres under ultrastructural study was in keeping with both intense SMI 31 staining and presence of NF triplet bands seen in WBs in four of leprous nerves that showed a large number of regenerating fibres suggesting reversal of changes with regeneration. Findings in the present study suggest that atrophy, that is, the reduction in axonal calibre and paranodal demyelination, seen in leprous nerves may result from dephosphorylation of NF-H and NF-M proteins.

Ultrastructural study of Schwann cells and endothelial cells in the pathogenesis of leprous neuropathy.

Peripheral nerve biopsies from 4 borderline tuberculoid (BT) and 4 lepromatous (LL) patients who were on multidrug therapy were investigated by light and electron microscopic studies. The variation of diameters and distribution of myelinated and unmyelinated fibers between BT and LL patients were not significant. This study has shown significant changes in peripheral nerves and endoneural blood vessels. It was revealed that besides Schwann cells (SC), the endothelial cells (EC) of endoneural blood vessels frequently harbor M. leprae. In BT, peripheral nerves in addition to the degenerative changes of SC and presence of perineural and perivascular cuffing by mononuclear cells, the endoneurial blood vessels showed thickening of basement membrane with hypertrophy of EC leading to narrowing or complete occlusion of lumen. On the other hand, peripheral nerves of LL patients were infiltrated with large number of M. leprae shown to be present in the electron transparent zone (ETZ) of the SC. The EC of endoneurial blood vessels were found to be loaded with M. leprae, and this bacillary loaded EC was found to release M. leprae into the lumen through its ruptured membrane.

Neuritic Hansen's disease: an unusual presentation and a diagnostic challenge on fine-needle aspirate.

Fine-needle aspiration cytology is now routinely used in the diagnosis of cervical lumps. We report on a case of leprous neuritis which presented as a cervical swelling. A diagnosis of leprosy was suggested on the basis of globi within histiocytes. These histiocytes differed from those classically described in leprosy. Retrospective review demonstrated the presence of occasional nerve fragments, and some of these closely resembled granulomas. The possible close resemblance of this lesion to tuberculosis and a histiocytic proliferative disorder is highlighted. Recognition of nerve fragments along with histiocytes should suggest the diagnosis.

Correlation of quantitative tests of nerve and target organ dysfunction with skin immunohistology in leprosy.

Loss of nociception and hypohidrosis in skin are hallmarks of leprosy, attributed to early invasion by Mycobacterium leprae of Schwann cells related to unmyelinated nerve fibres. We have studied skin lesions and contralateral clinically unaffected skin in 28 patients across the leprosy spectrum with a range of selective quantitative sensory and autonomic tests, prior to biopsy of both sites. Unaffected sites showed normal skin innervation, when antibodies to the pan-neuronal marker PGP (protein gene product) 9.5 were used, with the exception of intraepidermal fibres which were not detected in the majority of cases. Elevation of thermal thresholds and reduced sensory axon-reflex flare responses in affected skin correlated with decreased nerve fibres in the subepidermis, e.g. axon-reflex flux units (means+/-SEM) for no detectable innervation; decreased innervation; and clinically unaffected skin, were 23+/-3.1; 41.2+/-7.3; and 84.5+/-4.0, respectively. Reduced nicotine-induced axon-reflex sweating was correlated with decreased innervation of sweat glands. Where methacholine-induced direct activation of sweat glands was affected, there was inflammatory infiltrate and loss of sweat gland structure. This study demonstrates a correlation between selective nerve dysfunction on clinical tests and morphological changes in skin, irrespective of the type of leprosy, and is the first to show that loss of sweating in leprosy may result either from decreased innervation and/or involvement of the sweat glands. The findings have implications for the selection and monitoring of patients with leprosy in clinical trials which aim to restore cutaneous function.

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.

Periodic acid-ethanol gelatin methenamine silver stain for demonstrating M. leprae and myelin in peripheral nerve fibers of leprosy patients.

We have introduced the periodic acid-ethanol gelatin methenamine silver stain for the simultaneous demonstration of Mycobacterium leprae and myelin in the peripheral nerves of leprosy patients.

Nerve damage in leprosy-a teased fibre study of sciatic nerves in experimental mice.

The extent of nerve damage was assessed in the sciatic nerves of CBA/J mice with experimental leprosy by the teased single fibre preparations. Morphological changes in the fibres were observed upto 15 months of infection and compared with control animals of the same age groups. During 4 to 9 months period, the fibres showed paranodal enlargements, widening of the Schmidt-Lantermann incisures, paranodal and whole segmental demyelination. In addition, fragmentation of axons with the formation of myelin ovoids in the schwann tubes was also observed in animals with 9 to 15 months of infection. While segmental demyelination was predominant early in infection, axonal degeneration was more evident in advanced infection. These findings may possibly, explain the underlying mechanism of nerve damage in human leprosy.

Tuberculoid leprosy and tuberculosis skin: a comparative histopathological study.

Since it has been found hard to differentiate histopathologically tuberculoid leprosy from tuberculosis of the skin, a study of 20 biopsies from each of those conditions was undertaken to identify if possible some of their characteristic features. In tuberculoid leprosy along with tuberculoid granulomata there is always selective involvement and destruction of nerves, lack of fibrosis, absence of caseous necrosis and often epidermal atrophy. In cutaneous tuberculosis, on the other hand, in addition to tuberculous granuloma, there is often a proliferation reaction of the epidermis, areas of ulceration, absence of nerve destruction, marked increase in the reticulin, significant fibrosis and occasionally caseous necrosis.

Aspectos cirurgicos da caseóse dos nervos na lepra / Surgical aspects of caseous nerves in leprosy

Baseado em 10 casos de observa‡„o pessoal, o A. faz um estudo, sob o ponto de vista cirurgico, da caseose dos nervos na lepra. Come‡a salientando que focaliza o assumpto apenas debaixo dos seus aspectos cirurgicos, pois que os mesmos casos e outros mais que n„o lhe foi dado operar ser„o apreciado, na sua fei‡„o clinica e anatomo-patologica, pelos Drs Nelson de Souza Campos e Jos‚ Oria. Acha necessario, por‚m, dizer em poucas palavras o que ‚ a afec‡„o. Na nevrite leprosa, os nervos perifericos aumentam de volume, dando  s vezes engorgitamentos em rosario; em geral sobrevem um processo de fibrose; raras vezes, entretanto, talvez em consequencia de um maior poder defensivo do organismo, com destrui‡„o dos germes e decorrente forma‡„o de substƒncias toxicas, o que se verifica ‚ a necrose, seguida de caseose de coagula‡„o e posterior liquefac‡„o, processo esse circundade por tecido granulomatoso, com fibroblastos, fibrilas colagenas, gigantocitos, linfocitos, plasmocitos, histiocitos e vasos neoformados, havendo, de regra, ausˆncia deMycobacterium leprae. Esse f¢com de caseose, que se forma de prefˆrencia no endonervo, faz saliencia no espinonervo e da¡ progride em dire‡„o aos tecidos superficiais, procurando a pele, por onde em tempo, mais ou menos remoto, se esvasia espontaneamente...