Microfasciculation: a morphological pattern in leprosy nerve damage.

AIMS: To study Microfasciculation, a perineurial response found in neuropathies, emphasizing its frequency, detailed morphological characteristics and biological significance in pure neural leprosy (PNL), post-treatment leprosy neuropathy (PTLN) and non-leprosy neuropathies (NLN). METHODS AND RESULTS: Morphological characteristics of microfascicles were examined via histological staining methods, immunohistochemical expression of neural markers and transmission electronmicroscopy. The detection of microfasciculation in 18 nerve biopsy specimens [12 PNL, six PTLN but not in the NLN group, was associated strongly with perineurial damage and the presence of a multibacillary inflammatory process in the nerves, particularly in the perineurium. Immunoreactivity to anti-S100 protein, anti-neurofilament, anti-nerve growth receptor and anti-myelin basic protein immunoreactivity was found within microfascicles. Ultrastructural examination of three biopsies showed that fibroblast-perineurial cells were devoid of basement membrane despite perineurial-like NGFr immunoreactivity. Morphological evidence demonstrated that multipotent pericytes from inflammation-activated microvessels could be the origin of fibroblast-perineurial cells. CONCLUSIONS: A microfasciculation pattern was found in 10% of leprosy-affected nerves. The microfascicles were composed predominantly of unmyelinated fibres and denervated Schwann cells (SCs) surrounded by fibroblast-perineurial cells. This pattern was found more frequently in leprosy nerves with acid-fast bacilli (AFB) and perineurial damage while undergoing an inflammatory process. Further experimental studies are necessary to elucidate microfascicle formation.

Role of PGL-I antibody detection in the diagnosis of pure neural leprosy.

Pure neural leprosy (PNL) is difficult to diagnose because skin lesions and acid-fast bacilli (AFB) in slit smears are absent. At present, the gold standard for PNL diagnosis is the histopathological examination of a peripheral nerve biopsy. Even so, detection of bacteria is difficult and histological findings may be non-specific. Furthermore, nerve biopsy is an invasive procedure that is only possible in specialized centres. Therefore, there is a need for additional diagnostic methods that may help to confirm the clinical diagnosis of PNL. In the present study, an additional laboratory test, the ELISA for anti-phenolic glycolipid I (PGL-I) IgM antibodies, was performed on 103 individuals with clinical and neurophysiological signs of peripheral neuropathy, of which 67 were diagnosed as PNL patients and 36 remained as 'not diagnosed as PNL', as well as on a control group of 34 patients with other neurological diseases. An antibody response was present in 14/67 (21%) of the patients diagnosed as PNL as compared with 3/34 (9%) of controls. Anti-PGL-I positivity was observed in 5/8 (63%) of the AFB positive cases. Patients whose diagnosis was confirmed solely by Mycobacterium leprae PCR on the nerve sample had 4/25 (16%) seropositivity. In addition, anti-PGL-I antibodies were detected in 9/40 (23%) of the PNL patients who were PCR negative for M. leprae DNA. Moreover, two patients who showed clinical and eletrophysiological manifestations suggestive of PNL were diagnosed with the help of their positive test results in the anti-PGL-I ELISA. In conclusion, detection of antibodies against PGL-I in patients with peripheral neuropathy is useful as an additional laboratory test to help PNL diagnosis.

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.

Criteria for diagnosis of pure neural leprosy.

The clinical diagnosis of pure neural leprosy (PNL) remains a public health care problem mainly because skin lesions-the cardinal features of leprosy-are always absent.Moreover, the identification of the leprosy bacillus is not easily achieved even when a nerve biopsy can be performed. In an attempt to reach a reliable PNL diagnosis in patients referred to our Leprosy Outpatient Clinic, this study employed a variety of criteria. The nerve biopsies performed on the 67 individuals whose clinical, neurological, and electrophysiological examination findings strongly suggested peripheral neuropathy were submitted to M. leprae identification via a polymerase chain reaction (PCR). Mononeuropathy multiplex was the most frequent clinical and electrophysiological pattern of nerve dysfunction, while sensory impairment occurred in 89% of all cases and motor dysfunction in 81%. Axonal neuropathy was the predominant electrophysiological finding, while the histopathological nerve study showed epithelioid granuloma in 14% of the patients, acid fast bacilli in 16%, and nonspecific inflammatory infiltrate and/or fibrosis in 39%. PCR for M. leprae was positive in 47% of the nerve biopsy samples (n=23). PCR, in conjunction with clinical and neurological examination results, can be a powerful tool in attempting to identify and confirm a PNL diagnosis.

Pre-miR-146a (rs2910164 G>C) single nucleotide polymorphism is genetically and functionally associated with leprosy.

Mycobacterium leprae infects macrophages and Schwann cells inducing a gene expression program to facilitate its replication and progression to disease. MicroRNAs (miRNAs) are key regulators of gene expression and could be involved during the infection. To address the genetic influence of miRNAs in leprosy, we enrolled 1,098 individuals and conducted a case-control analysis in order to study four miRNAs genes containing single nucleotide polymorphism (miRSNP). We tested miRSNP-125a (rs12975333 G>T), miRSNP-223 (rs34952329 *>T), miRSNP-196a-2 (rs11614913 C>T) and miRSNP-146a (rs2910164 G>C). Amongst them, miRSNP-146a was the unique gene associated with risk to leprosy per se (GC OR = 1.44, p = 0.04; CC OR = 2.18, p = 0.0091). We replicated this finding showing that the C-allele was over-transmitted (p = 0.003) using a transmission-disequilibrium test. A functional analysis revealed that live M. leprae (MOI 100:1) was able to induce miR-146a expression in THP-1 (p<0.05). Furthermore, pure neural leprosy biopsies expressed augmented levels of that miRNA as compared to biopsy samples from neuropathies not related with leprosy (p = 0.001). Interestingly, carriers of the risk variant (C-allele) produce higher levels of mature miR-146a in nerves (p = 0.04). From skin biopsies, although we observed augmented levels of miR-146a, we were not able to correlate it with a particular clinical form or neither host genotype. MiR-146a is known to modulate TNF levels, thus we assessed TNF expression (nerve biopsies) and released by peripheral blood mononuclear cells infected with BCG Moreau. In both cases lower TNF levels correlates with subjects carrying the risk C-allele, (p = 0.0453 and p = 0.0352; respectively), which is consistent with an immunomodulatory role of this miRNA in leprosy.

Transforming growth factor-ß1 may be a key mediator of the fibrogenic properties of neural cells in leprosy.

Fibrosis is the main cause of irreversible nerve damage in leprosy. Phenotypic changes in Mycobacterium leprae (ML)-infected Schwann cells (SCs) have been suggested to mediate this process. We found that SC line cultures stimulated with ML upregulated transforming growth factor-ß1 (TGF-ß1), and that TGF-ß1 or ML induced increased numbers of α-smooth muscle actin (α-SMA)-positive cells with characteristic stress fibers. Mycobacterium leprae and TGF-ß1 also induced increased type I collagen and fibronectin mRNA and secretion and augmented mRNA levels of SOX9 and ZEB1, which are involved in the epithelial-mesenchymal transition. These effects could be inhibited by the TGF-ß1 type I receptor (ALK5) inhibitor, SB-431542. In nerve biopsies from leprosy-infected patients with varying grades of fibrosis (n = 11), type I and III collagen and fibronectin were found in the endoneurium and perineurium, α-SMA-positive cells filled the fibrotic perineurium but not the endoneurium, and CD34-positive fibroblasts predominated in the endoneurium. Results of transcriptional studies of 3 leprosy nerves and 5 controls were consistent with these data, but α-SMA and other mRNA levels were not different from those in the control samples. Our findings suggest that TGF-ß1 may orchestrate events, including reprogramming of the SC phenotype, leading to transdifferentiation, connective tissue cell expansion, and fibrogenesis in the evolution of leprosy nerve lesions during some evolutionary stages.

Primary neural leprosy: systematic review.

The authors proposed a systematic review on the current concepts of primary neural leprosy by consulting the following online databases: MEDLINE, Lilacs/SciELO, and Embase. Selected studies were classified based on the degree of recommendation and levels of scientific evidence according to the "Oxford Centre for Evidence-based Medicine". The following aspects were reviewed: cutaneous clinical and laboratorial investigations, i.e. skin clinical exam, smears, and biopsy, and Mitsuda's reaction; neurological investigation (anamnesis, electromyography and nerve biopsy); serological investigation and molecular testing, i.e. serological testing for the detection of the phenolic glycolipid 1 (PGL-I) and the polymerase chain reaction (PCR); and treatment (classification criteria for the definition of specific treatment, steroid treatment, and cure criteria).

Primary neural leprosy: systematic review / Hanseníase neural primária: revisão sistemática

The authors proposed a systematic review on the current concepts of primary neural leprosy by consulting the following online databases: MEDLINE, Lilacs/SciELO, and Embase. Selected studies were classified based on the degree of recommendation and levels of scientific evidence according to the “Oxford Centre for Evidence-based Medicine”. The following aspects were reviewed: cutaneous clinical and laboratorial investigations, i.e. skin clinical exam, smears, and biopsy, and Mitsuda's reaction; neurological investigation (anamnesis, electromyography and nerve biopsy); serological investigation and molecular testing, i.e. serological testing for the detection of the phenolic glycolipid 1 (PGL-I) and the polymerase chain reaction (PCR); and treatment (classification criteria for the definition of specific treatment, steroid treatment, and cure criteria).

Os autores propuseram-se a realizar uma revisão sistemática em conceitos atuais sobre a hanseníase neural primária, consultando as seguintes bases bibliográficas on-line: MEDLINE, Lilacs/SciELO e Embase. Os estudos selecionados foram classificados conforme o grau de recomendação e o nível de evidência científica de acordo com o “Oxford Centre for Evidence-based Medicine”. Os seguintes temas foram revisados: investigações clínica e laboratorial cutâneas, ou seja, exame, esfregaço e biópsia de pele e reação de Mitsuda; investigação neurológica (anamnese, eletroneuromiografia e biópsia de nervo); investigação sorológica e testes moleculares, ou seja, testes sorológicos para detecção de um glicolipídio fenólico e reação de cadeia de polimerase (PCR) e tratamento (critérios de classificação para definição de tratamento específico, tratamento com esteroides e critérios de cura).

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.

Pre-miR 146a (rs2910164 G>C) single nucleotide polymorphism is genetically and functionally associated with leprosy

Mycobacterium leprae infects macrophages and Schwann cells inducing a gene expression program to facilitate its replication and progression to disease. MicroRNAs (miRNAs) are key regulators of gene expression and could be involved during the infection. To address the genetic influence of miRNAs in leprosy, we enrolled 1,098 individuals and conducted a case-control analysis in order to study four miRNAs genes containing single nucleotide polymorphism (miRSNP). We tested miRSNP-125a (rs12975333 G>T), miRSNP-223 (rs34952329 *>T), miRSNP-196a-2 (rs11614913 C>T) and miRSNP-146a (rs2910164 G>C). Amongst them, miRSNP-146a was the unique gene associated with risk to leprosy per se (GC OR = 1.44, p = 0.04; CC OR = 2.18, p = 0.0091). We replicated this finding showing that the C-allele was over-transmitted (p = 0.003) using a transmission-disequilibrium test. A functional analysis revealed that live M. leprae (MOI 100:1) was able to induce miR-146a expression in THP-1 (p<0.05). Furthermore, pure neural leprosy biopsies expressed augmented levels of that miRNA as compared to biopsy samples from neuropathies not related with leprosy (p = 0.001). Interestingly, carriers of the risk variant (C-allele) produce higher levels of mature miR-146a in nerves (p = 0.04). From skin biopsies, although we observed augmented levels of miR-146a, we were not able to correlate it with a particular clinical form or neither host genotype. MiR-146a is known to modulate TNF levels, thus we assessed TNF expression (nerve biopsies) and released by peripheral blood mononuclear cells infected with BCG Moreau. In both cases lower TNF levels correlates with subjects carrying the risk C-allele, (p = 0.0453 and p = 0.0352; respectively), which is consistent with an immunomodulatory role of this miRNA in leprosy.

Criteria for diagnosis of pure neural leprosy

The clinical diagnosis of pure neural leprosy (PNL) remains a public health care problem mainly because skin lesions-the cardinal features of leprosy-are always absent.Moreover, the identification of the leprosy bacillus is not easily achieved even when a nerve biopsy can be performed. In an attempt to reach a reliable PNL diagnosis in patients referred to our Leprosy Outpatient Clinic, this study employed a variety of criteria. The nerve biopsies performed on the 67 individuals whose clinical, neurological, and electrophysiological examination findings strongly suggested peripheral neuropathy were submitted to M. leprae identification via a polymerase chain reaction (PCR). Mononeuropathy multiplex was the most frequent clinical and electrophysiological pattern of nerve dysfunction, while sensory impairment occurred in 89% of all cases and motor dysfunction in 81%. Axonal neuropathy was the predominant electrophysiological finding, while the histopathological nerve study showed epithelioid granuloma in 14% of the patients, acid fast bacilli in 16%, and nonspecific inflammatory infiltrate and/or fibrosis in 39%. PCR for M. leprae was positive in 47% of the nerve biopsy samples (n=23). PCR, in conjunction with clinical and neurological examination results, can be a powerful tool in attempting to identify and confirm a PNL diagnosis.