Comportamiento de los pacientes con diagnóstico tardío de lepra. Camaguey, Cuba, 2015 / No disponible

Se realizó un estudio descriptivo, longitudinal y retrospectivo con el objetivo de caracterizar el comportamiento de los pacientes con diagnóstico tardío de lepra notificados durante el período comprendido del 1 de enero del 2009 al 30 de septiembre del 2014 residentes en la provincia Camagüey. El universo de estudio estuvo constituido por 26 pacientes con diagnóstico tardío de lepra. Se determinó que el grupo de edad más afectado fue el de 60 años y más, con predominio del sexo masculino, escolaridad primaria y per cápita familiar regular. El mayor tiempo transcurrido entre los primeros síntomas y el diagnóstico fue de 1 a 3 años y la mancha anestésica constituyó el signo más frecuente de inicio de la enfermedad. Aproximadamente la mitad de los pacientes estuvieron evaluados por especialistas de Medicina General Integral antes del diagnóstico. Predominó la lepra lepromatosa, la discapacidad grado I, de localización en los pies y de tipo anestesia. Alrededor de las tres cuartas partes de los pacientes fueron detectados de forma espontánea y casi la mitad de los mismos presentó un tiempo de permanencia en el área de salud de entre 1 y 2 años

A descriptive, longitudinal and retrospective study was made with the aim of characterizing the behavior of the patients with a diagnosis of late leprosy notified during the period of January 1st, 2009 up to September 30th, 2014 in the county of Camagüey. The study consisted of 26 patients with a late diagnosis of leprosy. It was determined that the most affected age group was that of 60 years and above, with prevalence of the men, primary education and middle family per capita. The longest delay between first symptoms and diagnosis went from 1 to 3 years and the anesthetic patch constituted the most frequent sign as initial start of the illness. Approximately half of the patients were evaluated by specialists of Integral General Medicine before the diagnosis. The most frequent type was lepromatous leprosy, disability grade I prevailed mainly on feet. Approximately three fourths of the patients were detected in a spontaneous way and almost half of them were present in the area between 1 and 2 years

CXCL10, MCP-1, and other immunologic markers involved in neural leprosy.

Nerve damage in leprosy can be directly induced by Mycobacterium leprae in the early stages of infection, however, immunomediated mechanisms add gravity to the impairment of neural function in symptomatic periods of the disease. This study investigated the immunohistochemical expression of immunomarkers involved in the pathogenic mechanisms of leprosy nerve damage. These markers selected were CXCL10, CCL2 chemokines and immunomarkers as CD3, CD4, CD8, CD45RA, CD45RO, CD68, HLA-DR, and metalloproteinases 2 and 9 (MMP2 and MMP9) occurring in nerve biopsy specimens collected from leprosy (23) and nonleprosy patients (5) suffering peripheral neuropathy. CXCL10, CCL2, MMP2, and MMP9 immunoreactivities were found in the leprosy nerves but not in nonleprosy samples. Immunolabeling was predominantly found in recruited macrophages and Schwann cells composing the inflammatory cellular population in the leprosy-affected nerves. The immunohistochemical expression of all the markers, but CXCL10, was associated with fibrosis, however, only CCL2 was, independently from the others, associated with this excessive deposit of extracellular matrix. No difference in the frequency of the immunolabeling was detected between the AFB⁺ and AFB⁻ leprosy subgroups of nerve, exception made to some statistical trend to difference in regard to CD68⁻ and HLA-DR⁺ cells in the AFB⁻ nerves exhibiting epithelioid granuloma. MMP9 expression associated with fibrosis is consistent with previous results of research group. The findings conveys the idea that CCL2 and CXCL10 chemokines at least in advanced stages of leprosy nerve lesions are not determinant for the establishment of AFB⁺ or AFB⁻ leprosy lesions, however, CCL2 is associated with macrophage recruitment and fibrosis.

An attempt to improve pure neural leprosy diagnosis using immunohistochemistry tests in peripheral nerve biopsy specimens.

The diagnosis of pure neural leprosy (PNL) is based on clinical and laboratory data, including the histopathology of nerve biopsy specimens and detection of Mycobacterium leprae DNA by polymerase chain reaction (PCR). Given that histopathologic examination and PCR methods may not be sufficient to confirm the diagnosis, immunolabeling of lipoarabinomanan (LAM) and/or phenolic glycolipid 1 (PGL-1) M. leprae wall components was utilized in the present investigation in an attempt to detect any vestigial presence of M. leprae in acid-fast bacilli (AFB) nerve samples. Twenty-three PNL nerve samples (6 AFB and 17 AFBPCR) were cryosectioned and subjected to LAM and PGL-1 immunohistochemical staining by immunoperoxidase. Five nonleprosy nerve samples were used as controls. The 6 AFB samples showed LAM/PGL-1 immunoreactivity. Among the 17 AFB samples, 8 revealed LAM and/or PGL-1 immunoreactivity. In 17 AFBPCR patients, just 7 yielded LAM and/or PGL-1 nerve results. In the PNL cases, the detection of immunolabeled LAM and PGL-1 in the nerve samples would have contributed to an enhanced diagnostic efficiency in the absence of molecular diagnostic facilities.

Gene expression profiling specifies chemokine, mitochondrial and lipid metabolism signatures in leprosy.

Herein, we performed microarray experiments in Schwann cells infected with live M. leprae and identified novel differentially expressed genes (DEG) in M. leprae infected cells. Also, we selected candidate genes associated or implicated with leprosy in genetic studies and biological experiments. Forty-seven genes were selected for validation in two independent types of samples by multiplex qPCR. First, an in vitro model using THP-1 cells was infected with live Mycobacterium leprae and M. bovis bacillus Calmette-Guérin (BCG). In a second situation, mRNA obtained from nerve biopsies from patients with leprosy or other peripheral neuropathies was tested. We detected DEGs that discriminate M. bovis BCG from M. leprae infection. Specific signatures of susceptible responses after M. leprae infection when compared to BCG lead to repression of genes, including CCL2, CCL3, IL8 and SOD2. The same 47-gene set was screened in nerve biopsies, which corroborated the down-regulation of CCL2 and CCL3 in leprosy, but also evidenced the down-regulation of genes involved in mitochondrial metabolism, and the up-regulation of genes involved in lipid metabolism and ubiquitination. Finally, a gene expression signature from DEG was identified in patients confirmed of having leprosy. A classification tree was able to ascertain 80% of the cases as leprosy or non-leprous peripheral neuropathy based on the expression of only LDLR and CCL4. A general immune and mitochondrial hypo-responsive state occurs in response to M. leprae infection. Also, the most important genes and pathways have been highlighted providing new tools for early diagnosis and treatment of leprosy.

Gene expression profiling specifies chemokine, mitochondrial and lipid metabolism signatures in leprosy

Herein, we performed microarray experiments in Schwann cells infected with live M. leprae and identified novel differentially expressed genes (DEG) in M. leprae infected cells. Also, we selected candidate genes associated or implicated with leprosy in genetic studies and biological experiments. Forty-seven genes were selected for validation in two independent types of samples by multiplex qPCR. First, an in vitro model using THP-1 cells was infected with live Mycobacterium leprae and M. bovis bacillus Calmette-Guérin (BCG). In a second situation, mRNA obtained from nerve biopsies from patients with leprosy or other peripheral neuropathies was tested. We detected DEGs that discriminate M. bovis BCG from M. leprae infection. Specific signatures of susceptible responses after M. leprae infection when compared to BCG lead to repression of genes, including CCL2, CCL3, IL8 and SOD2. The same 47-gene set was screened in nerve biopsies, which corroborated the down-regulation of CCL2 and CCL3 in leprosy, but also evidenced the down-regulation of genes involved in mitochondrial metabolism, and the up-regulation of genes involved in lipid metabolism and ubiquitination. Finally, a gene expression signature from DEG was identified in patients confirmed of having leprosy. A classification tree was able to ascertain 80% of the cases as leprosy or non-leprous peripheral neuropathy based on the expression of only LDLR and CCL4. A general immune and mitochondrial hypo-responsive state occurs in response to M. leprae infection. Also, the most important genes and pathways have been highlighted providing new tools for early diagnosis and treatment of leprosy.

Cytokine and protein markers of leprosy reactions in skin and nerves: baseline results for the North Indian INFIR cohort.

BACKGROUND: Previous studies investigating the role of cytokines in the pathogenesis of leprosy have either been on only small numbers of patients or have not combined clinical and histological data. The INFIR Cohort study is a prospective study of 303 new multibacillary leprosy patients to identify risk factors for reaction and nerve damage. This study characterised the cellular infiltrate in skin and nerve biopsies using light microscopic and immunohistochemical techniques to identify any association of cytokine markers, nerve and cell markers with leprosy reactions. METHODOLOGY/PRINCIPAL FINDINGS: TNF-α, TGF-ß and iNOS protein in skin and nerve biopsies were detected using monoclonal antibody detection immunohistochemistry techniques in 299 skin biopsies and 68 nerve biopsies taken from patients at recruitment. The tissues were stained with hematoxylin and eosin, modified Fite Faraco, CD68 macrophage cell marker and S100. CONCLUSIONS/SIGNIFICANCE: Histological analysis of the biopsies showed that 43% had borderline tuberculoid (BT) leprosy, 27% borderline lepromatous leprosy, 9% lepromatous leprosy, 13% indeterminate leprosy types and 7% had no inflammation. Forty-six percent had histological evidence of a Type 1 Reaction (T1R) and 10% of Erythema Nodosum Leprosum. TNF-α was detected in 78% of skin biopsies (181/232), iNOS in 78% and TGF-ß in 94%. All three molecules were detected at higher levels in patients with BT leprosy. TNF-α was localised within macrophages and epithelioid cells in the granuloma, in the epidermis and in dermal nerves in a few cases. TNF-α, iNOS and TGF-ß were all significantly associated with T1R (p<0.001). Sixty-eight nerve biopsies were analysed. CD68, TNF-α and iNOS staining were detectable in 88%, 38% and 28% of the biopsies respectively. The three cytokines TNF-α, iNOS and TGF-ß detected by immunohistochemistry showed a significant association with the presence of skin reaction. This study is the first to demonstrate an association of iNOS and TGF-ß with T1R.

Usage of signaling in neurodegeneration and regeneration of peripheral nerves by leprosy bacteria.

Multiple signaling pathways play key regulatory roles during the development of peripheral nervous system (PNS) and also in neuroregeneration process following nerve degeneration. Schwann cells, the glial cells of the PNS, by interacting with neuronal (axonal) ligands, mainly neuregulins via receptor tyrosine kinase (RTK) complex, ErbB2/ErbB3, initiate intracellular signaling pathways to drive proliferation and differentiation of Schwann cells, both during development and the process of regeneration and re-myelination after nerve injury. One of the major signaling kinases, extracellular signal-regulated kinase-1/2 (ERK1/2), that is also a downstream signaling pathway of neuregulin-ErbB2/ErbB3 activation, has been identified as a key regulator of Schwann cell proliferation, differentiation, demyelination and nerve regeneration. Recent studies have provided evidence that the bacterium that causes human leprosy, Mycobacterium leprae that has a unique capacity to invade Schwann cells of the adult PNS, utilizes the neuregulin-ErbB2/ErbB3 associated signaling network to the bacterial advantage. M. leprae directly bind to ErbB2 on myelinated Schwann cells and activate the RTK by a novel route that bypasses the classical neuregulin/growth factor-induced ErbB2-ErbB3 heterodimerization, and subsequently induce downstream the canonical Erk1/2 signaling, leading to myelin breakdown and subsequent axonal damage. This initial injury provides a survival advantage for M. leprae as it induces de-differentiation and generates myelin-free cells, which are highly susceptible to M. leprae invasion and promote bacterial survival. Once invaded M. leprae activate Erk1/2 via a non-canonical pathway and subsequently increase the cell proliferation and maintain the infected cells in de-differentiated state, thereby preventing remyelination. Therefore, by subverting major RTKs and signaling pathways in adult Schwann cells M. leprae appear to propagate the bacterial niche and maintain survival within the PNS. These studies may also provide new insights into our understanding of signaling mechanisms involve in both neurodegeneration and neuroregeneration.

Expression of metalloproteinases (MMP-2, MMP-9, and TACE) and TNF-alpha in the nerves of leprosy patients.

Matrix metalloproteinases (MMPs) and tumor necrosis factor alpha (TNF-alpha) play important and related roles in the pathogenesis of nerve injury. MMP-dependent and TNF-alpha-dependent processes of neurodegeneration, such as blood-nerve breakdown and immune cell recruitment, are characteristic of leprosy nerve damage. Our work has contributed to the understanding of the role of cytokines in the process, but the role of MMPs in the pathogenesis of neuritic leprosy has not been investigated. This study analyzed the changes in mRNA expression and immunodistribution of MMP-2, MMP-9, TNF-alpha-converting enzyme (TACE), TNF-alpha in nerves of 27 pure neuritic leprosy (PNL) patients, both acid-fast bacilli positive (AFB(+)) and acid-fast bacilli negative (AFB(-)), and 8 non-leprosy patients with control peripheral neuropathic conditions. MMP-2, MMP-9, and TNF-alpha mRNA expression was significantly induced in the AFB(-) relative to the AFB(+) neuritic leprosy group and nonlepritic controls; TACE levels were also elevated in the AFB(-) group, but this change was not statistically significant. Immunoreactive profiles for TNF-alpha and MMPs demonstrated strong reactivity of myelinated axons, infiltrating macrophages, Schwann cells, endothelial cells, and perineurial cells in neuritic leprosy biopsies. This study provides the evidence of the involvement of MMPs in the pathogenesis of PNL neuropathy.

Ninjurin 1 asp110ala single nucleotide polymorphism is associated with protection in leprosy nerve damage.

Leprosy is the major cause of non-traumatic neuropathy. Herein, we investigated the role of ninjurin 1, an adhesion molecule involved in nerve regeneration in leprosy. Our results demonstrated that M. leprae stimulates in vitro up-regulation of ninjurin mRNA in cultured Schwann and blood cells as well as in vivo mRNA and protein expression in leprosy nerve biopsies. A polymorphism (asp110ala) was investigated in a case-control study (1123 individuals) and no association was found with leprosy per se or with disseminated forms. Nevertheless, ala110 was associated with functional nerve impairment (OR=2.42; p=0.02 for ala/ala) and with lower mRNA levels. Our data suggests that asp110ala could be a valuable genetic marker of nerve damage in leprosy.

Use of fine needle aspiration cytology in leprotic lesions: a report of 4 cases.

BACKGROUND: Although a few studies have shown fine needle aspiration cytology (FNAC) to be a sensitive diagnostic tool in the detection of nerve involvement, its role as an initial diagnostic procedure in pure neuritic leprosy (PNL) and in the detection of skeletal lesions with unusual findings has not been documented before. CASES: Three patients who presented with thickened nerves and a fourth with biopsy-proven lepromatous leprosy with lesions in hand bones underwent FNAC. Of the 3 patients with nerve thickening, 2 had a clinical suspicion or diagnosis of neuritic leprosy, whereas in the third patient a clinical differential diagnosis of a soft tissue tumor or parasitic cyst was considered. FNAC in all 3 cases revealed epithelioid cell granulomas, Langhans giant cells and caseous necrosis. Fites and Ziehl-Neelsen stains were negative for acid-fast bacilli. Cytologic diagnosis of pure neuritic leprosy was made in all 3 cases and confirmed by histopathologic examination. FNAC of skeletal lesions from the fourth patient confirmed involvement of bone with unusual cytologic findings of epithelioid cell granulomas and giant cells along with a significant proportion of foamy macrophages and strong Fites stain positivity. CONCLUSION: FNAC is a simple, useful, minimally traumatic and routinely applicable procedure in the diagnosis of pure neuritic leprosy and leprous osteitis.

S-100 as a useful auxiliary diagnostic aid in tuberculoid leprosy.

BACKGROUND: The diagnosis of tuberculoid leprosy is often difficult on hematoxylin and eosin (H&E) due to the absence of demonstrable nerve destruction. This study evaluates the utility of S-100 staining in identifying nerve fragmentation and differentiation of tuberculoid leprosy from other cutaneous granulomatous diseases. METHODS: Fifty cases of leprosy including 38 borderline tuberculoid (BT), two tuberculoid (TT), and 10 indeterminate leprosy (IL) were studied. Eleven controls of non-lepromatous cutaneous granulomatous lesions were included. S-100 was used for identifying the following dermal nerve patterns: infiltrated (A), fragmented (B), absent (C), and intact (D) nerves. RESULTS: On H&E, only 18/38 (47.4%) BT cases and 1/2 (50%) TT cases revealed neural inflammation. On S-100 staining of BT cases, 28/38 (73.7%) showed pattern B followed by patterns C and A in 8/38 (21.1%) and 2/38 (5.3%) cases, respectively. Both the TT cases showed pattern B. Only intact nerves (D) were seen in all the control cases. S-100 identified nerve damage in 4/10 (40%) IL cases. The patterns A, B, and C had sensitivity, specificity, and positive and negative predictive values of 100% in diagnosing tuberculoid (BT + TT) leprosy. CONCLUSIONS: S-100 is superior to H&E in identifying nerve fragmentation (p < 0.01). It also aids the differential diagnosis of tuberculoid leprosy.

Insights into regulation of human Schwann cell proliferation by Erk1/2 via a MEK-independent and p56Lck-dependent pathway from leprosy bacilli.

Activation of extracellular signal-regulated kinase (Erk) 1/2, which plays a critical role in diverse cellular processes, including cell proliferation, is known to be mediated by the canonical Raf-mitogen-activated protein kinase kinase (MEK) kinase cascade. Alternative MEK-independent signaling pathways for Erk1/2 activation in mammalian cells are not known. During our studies of human primary Schwann cell response to long-term infection of Mycobacterium leprae, the causative organism of leprosy, we identified that intracellular M. leprae activated Erk1/2 directly by lymphoid cell kinase (p56Lck), a Src family member, by means of a PKCepsilon-dependent and MEK-independent signaling pathway. Activation of this signaling induced nuclear accumulation of cyclin D1, G1/S-phase progression, and continuous proliferation, but without transformation. Thus, our data reveal a previously unknown signaling mechanism of glial cell proliferation, which might play a role in dedifferentiation as well as nerve regeneration and degeneration. Our findings may also provide a potential mechanism by which an obligate intracellular bacterial pathogen like M. leprae subverts nervous system signaling to propagate its cellular niche for colonization and long-term bacterial survival.

Nitrotyrosine localization to dermal nerves in borderline leprosy.

BACKGROUND: Nerve damage is a common and disabling feature of leprosy, with unclear aetiology. It has been reported that the peroxidizing agents of myelin lipids-nitric oxide (NO) and peroxynitrite-are produced in leprosy skin lesions. OBJECTIVES: To investigate the localization of nitrotyrosine (NT)-a local end-product of peroxynitrite-in leprosy lesions where dermal nerves are affected by a granulomatous reaction. METHODS: We investigated by immunohistochemistry and immunoelectron microscopy the localization of the inducible NO synthase (iNOS) and NT in biopsies exhibiting dermal nerves from patients with untreated leprosy. RESULTS: There were abundant NT-positive and iNOS-positive macrophages in the borderline leprosy granulomas infiltrating peripheral nerves identified by light microscopy, S-100 and neurofilament immunostaining. Immunoelectron microscopy showed NT reactivity in neurofilament aggregates and in the cell wall of Mycobacterium leprae. CONCLUSIONS: Our results suggest that NO and peroxynitrite could be involved in the nerve damage following borderline leprosy.

Protein phosphorylation in human peripheral nerve: altered phosphorylation of a 25-kDa glycoprotein in leprosy.

Protein phosphorylation in a low speed supernatant of human peripheral nerve (tibial and sural) homogenate was investigated. The major phosphorylated proteins had molecular mass in the range of 70, 55, 45, and 25 kDa. Mg2+ or Mn2+ was essential for maximum phosphorylation although Zn2+, Co2+, and Ca2+ could partially support phosphorylation. External protein substrates casein and histone were also phosphorylated. The protein phosphatase inhibitor orthovanadate enhanced the phosphorylation of the 45 and 25 kDa proteins significantly. Concanavalin A-Sepharose chromatography of the phosphorylated peripheral nerve proteins showed that the 25 kDa protein was a glycoprotein. Protein phosphorylation of peripheral nerves from leprosy affected individuals was compared with normals. The phosphorylation of 25 kDa protein was decreased in most of the patients with leprosy.

Intra-neural ceroid-like pigment following the treatment of lepromatous leprosy with clofazimine (B663; Lamprene).

A 33 year old male Nigerian presented with widespread involvement of peripheral nerves, several of which were tender and painful. Nerve biopsies confirmed the presence of Mycobacterium leprae in both endoneurial and perineurial areas, mainly in foamy macrophages (Virchow cells), but there were also large accumulations of an amorphous, acid-fast and alcohol-fast material which was not obviously of bacterial origin. Appropriate stains indicated that this had many characteristics of lipofuscin. Although not previously known, it was at this stage discovered that the patient had received treatment with anti-leprosy drugs nearly three years before presentation in this country. One of these was clofazimine, an aniline aposafranine derivative known to produce a ceroid-like pigment in the tissues of patients treated with this drug or lepromatous leprosy.

The penetration of dapsone, rifampicin, isoniazid and pyrazinamide into peripheral nerves.

1 Dapsone, rifampicin, isoniazid and pyrazinamide were shown to penetrate readily into the sciatic nerves of the dog and sheep. 2 These findings suggest that the continued persistence of viable drug-sensitive leprosy bacilli in the peripheral nerves of patients treated for long periods with either dapsone or rifampicin is not due to inadequate intraneural drug penetration.

Penetration of C14-labelled rifampicin into primate peripheral nerve.

The penetration of C14 Rifampicin into various tissues, but particularly peripheral nerve, has been studied in the monkey. Penetration into the substance of peripheral nerve internal to the epineurial covering was demonstrated and the significance of this in relation to the treatment of leprosy is discussed.