The role of CD4+ CD25+ Treg in the mechanism of autoimmune auditory neuropathy in SD rats / 中华耳鼻咽喉头颈外科杂志

Objective: To investigate the role of CD4+CD25+regulatory cell (CD4+CD25+Treg) in auditory neuropathy (AN) using a rat model of autoimmune auditory neuropathy. Methods: The SD rats were immunized with P0 protein emulsified in complete Freunds adjuvant for 8 weeks. The number of CD4+CD25+Treg in peripheral blood and cochlea and the expression of Foxp3 gene in cochlea were detected respectively 2, 4, 6 and 8 weeks after the immunization with P0 protein in rats. Then CD4+CD25+Treg were transferred intravenously to the AN rats at 2, 4, 6 and 8 weeks of the immunization, respectively. The change of auditory brainstem response (ABR) and distortion product otoacoustic emission (DPOAE) were detected, and the morphological changes in the inner ear were investigated. Results: The number of CD4+CD25+Treg in the peripheral blood of AN rats decreased gradually after 2, 4, 6 and 8 weeks of P0 protein immunization. The number of CD4+CD25+Treg in cochlea gradually increased with the prolongation of immunization time, but the expression of Foxp3 gene in cochlea gradually decreased over time. After intravenous transplantation of CD4+CD25+Treg in AN rats, the threshold of ABR response decreased, and DPOAE had no significant change. The number of spiral ganglion neurons in cochlea increased, and hair cells had no significant change under electron microscope. Conclusions: The decrease in the number and function of CD4+CD25+Treg reduces its inhibitory effect on autoimmune response and promotes the occurrence of autoimmune auditory neuropathy in AN rats. Adoptive transfer of CD4+CD25+Treg can reduce the autoimmune response and promote the recovery of autoimmune auditory neuropathy.

Effects of an intratympanic injection of dexamethasone combined with gentamicin on the expression level of serum p0 protein antibodies in patients with meniere's disease

OBJECTIVES: To investigate the effects of an intratympanic injection of dexamethasone combined with gentamicin on the expression level of serum P0 protein antibodies in patients with Meniere's disease (MD). METHODS: A total of 136 patients with MD treated in our hospital were enrolled in this study. Among them, 68 patients were treated with an intratympanic injection of dexamethasone combined with gentamicin (observation group). Another 68 patients were treated with gentamicin alone (control group). RESULTS: After treatment, the expression levels of IgG and IgM in the two groups significantly decreased (p<0.05); the levels in the observation group were significantly lower than those in the control group (p<0.05). The incidences of vertigo, tinnitus, and gait instability in the observation group were significantly lower than those in the control group (p<0.05). Vestibular symptom index (VSI) scores in the observation group were significantly lower than those in the control group (p<0.05). We observed no significant difference between the two groups in the number of vertigo attacks 6 months after treatment (p>0.05). CONCLUSION: For patients with MD, dexamethasone combined with gentamicin can reduce the incidence of vertigo, tinnitus, and gait instability, but it has no effect on the efficacy or number of vertigo attacks 6 months after treatment. Therefore, the levels of myelin P0 protein antibodies after treatment can be used as predictors of vertigo at 6 months after treatment.

Inhibition of KLF7-Targeting MicroRNA 146b Promotes Sciatic Nerve Regeneration / 神经科学通报·英文版

A previous study has indicated that Krüppel-like factor 7 (KLF7), a transcription factor that stimulates Schwann cell (SC) proliferation and axonal regeneration after peripheral nerve injury, is a promising therapeutic transcription factor in nerve injury. We aimed to identify whether inhibition of microRNA-146b (miR-146b) affected SC proliferation, migration, and myelinated axon regeneration following sciatic nerve injury by regulating its direct target KLF7. SCs were transfected with miRNA lentivirus, miRNA inhibitor lentivirus, or KLF7 siRNA lentivirus in vitro. The expression of miR146b and KLF7, as well as SC proliferation and migration, were subsequently evaluated. In vivo, an acellular nerve allograft (ANA) followed by injection of GFP control vector or a lentiviral vector encoding an miR-146b inhibitor was used to assess the repair potential in a model of sciatic nerve gap. miR-146b directly targeted KLF7 by binding to the 3'-UTR, suppressing KLF7. Up-regulation of miR-146b and KLF7 knockdown significantly reduced the proliferation and migration of SCs, whereas silencing miR-146b resulted in increased proliferation and migration. KLF7 protein was localized in SCs in which miR-146b was expressed in vivo. Similarly, 4 weeks after the ANA, anti-miR-146b increased KLF7 and its target gene nerve growth factor cascade, promoting axonal outgrowth. Closer analysis revealed improved nerve conduction and sciatic function index score, and enhanced expression of neurofilaments, P0 (anti-peripheral myelin), and myelinated axon regeneration. Our findings provide new insight into the regulation of KLF7 by miR-146b during peripheral nerve regeneration and suggest a potential therapeutic strategy for peripheral nerve injury.

Development of cell models for high-throughput screening system of Charcot-Marie-Tooth disease type 1

PURPOSE: Charcot-Marie-Tooth disease (CMT) is a peripheral neuropathy mainly divided into CMT type 1 (CMT1) and CMT2 according to the phenotype and genotype. Although molecular pathologies for each genetic causative have not been revealed in CMT2, the correlation between cell death and accumulation of misfolded proteins in the endoplasmic reticulum (ER) of Schwann cells is well documented in CMT1. Establishment of in vitro models of ER stress-mediated Schwann cell death might be useful in developing drug-screening systems for the treatment of CMT1. MATERIALS AND METHODS: To develop high-throughput screening (HTS) systems for CMT1, we generated cell models using transient expression of mutant proteins and chemical induction. RESULTS: Overexpression of wild type and mutant peripheral myelin protein 22 (PMP22) induced ER stress. Similar results were obtained from mutant myelin protein zero (MPZ) proteins. Protein localization revealed that expressed mutant PMP22 and MPZ proteins accumulated in the ER of Schwann cells. Overexpression of wild type and L16P mutant PMP22 also reduced cell viability, implying protein accumulation-mediated ER stress causes cell death. To develop more stable screening systems, we mimicked the ER stress-mediated cell death in Schwann cells using ER stress inducing chemicals. Thapsigargin treatment caused cell death via ER stress in a dose dependent manner, which was measured by expression of ER stress markers. CONCLUSION: We have developed genetically and chemically induced ER stress models using Schwann cells. Application of these models to HTS systems might facilitate the elucidation of molecular pathology and development of therapeutic options for CMT1.

A Costa Rican family affected with Charcot-Marie-Tooth disease due to the myelin protein zero (MPZ) p.Thr124Met mutation shares the Belgian haplotype / Una familia costarricense afectada por la enfermedad de Charcot-Marie-Tooth, debido a la mutación p.thr124Met en la proteína mielina cero (MPZ) compartida con el haplotipo belga

The p.Thr124Met mutation in the myelin protein zero (MPZ) causes the Charcot-Marie-Tooth disease type 2J, a peripheral neuropathy with additional symptoms as pupillary alterations and deafness. It was observed in several families around the world originating e. g. from Germany, Belgium, Japan, Italy and North America. Here we report Central American patients originating from a family in Costa Rica carrying this mutation. Clinical, electrophysiological and molecular analysis of patients and controls were performed, including gene and linked markers´ sequencing. Carriers share almost the entire haplotype with two non related Belgian CMT patients. As a result of the haplotype analysis, based on ten markers (seven SNPs, two microsatellites and an intronic polyA stretch), the founder effect hypothesis for this allele migration is suggestive. Rev. Biol. Trop. 62 (4): 1285-1293. Epub 2014 December 01.

La mutación p.thr124Met en la proteína mielina cero (MPZ) causa la enfermedad de Charcot-Marie-Tooth tipo 2J, una neuropatía periférica con síntomas adicionales como alteraciones pupilares y sordera. Se ha observado en varias familias alrededor del mundo, originarias de Alemania, Bélgica, Japón, Italia y Norteamérica, entre otras. Aquí reportamos a pacientes centroamericanos provenientes de Costa Rica que acarrean esta mutación. Se realizaron análisis clínico, electrofisiológico y molecular de pacientes y controles, incluyendo secuenciación del gen y de marcadores ligados a éste. Estos pacientes comparten casi por completo el haplotipo con dos pacientes belgas no emparentados. Como resultado del análisis de los haplotipos, basado en diez marcadores (siete SNPs, dos microsatélites y un elemento poli-A intrónico), se sugiere que se ha dado un efecto fundador en la migración de este alelo.

Myelin protein zero and its antibody in serum as biomarkers of n-hexane-induced peripheral neuropathy and neurotoxicity effects / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Chronic exposure to n-hexane can lead to peripheral neuropathy that no effective treatment regimen could be applied presently. This study investigated whether myelin protein zero (P0) protein and its antibody could be used to distinguish n-hexane intoxication and protect workers from peripheral neuropathy.</p><p><b>METHODS</b>We compared P0 protein and its antibody among three levels of n-hexane-exposed groups, which included 18 patients with n-hexane-induced peripheral neuropathy as case group, 120 n-hexane-exposed workers as n-hexaneexposed control group, and 147 non-hexane-exposed participants used as control group. ELISA method was applied to detect P0 protein and its antibody.</p><p><b>RESULTS</b>P0 protein in serum was significantly higher in the case group and n-hexane-exposed control group in comparison with the control group (P < 0.01). Compared with the n-hexane-exposed control group, the case group also had significant increase of P0 protein (P < 0.01). After 6 months therapy, P0 protein was observed to decrease significantly in the case group (P < 0.01). The P0 antibody in serum was significantly higher in the n-hexane-exposed control group than in the control group (P < 0.01), but not significantly different between cases and controls.</p><p><b>CONCLUSIONS</b>P0 antibodies in serum may be a short-term effect biomarker for n-hexane exposure. P0 protein in serum may be an early effective biomarker for peripheral nerve neuropathy and its biological limit value needs investigation in the future study.</p>

Morphological study of Schwann cells remyelination in contused spinal cord of rats / 中华创伤杂志(英文版)

<p><b>OBJECTIVE</b>To study the role and effect of Schwann cells (SCs) remyelination in contused spinal cord.</p><p><b>METHODS</b>Green fluorescence protein expressing-SCs were transplanted into the epicenter, rostral and caudal tissues of the injury site at 1 week after the spinal cords were contused. At 6 weeks, the spinal cords were removed for cryosections, semithin sections and ultrathin sections, and then immunocytochemical staining of myelin basic protein (MBP), P0 protein (P0) and S100 protein (S100) was carried out on the cryosections. Qualitative and semiquantitative analyses were performed on the cryosections and semithin sections. Ultrastructure of myelinated fibers was observed on the ultrathin sections under electron microscope.</p><p><b>RESULTS</b>Transplanted SCs and myelinated fibers immunocytochemically labeled by MBP, P0 as well as S100 distributed in whole injured area. The quantity of myelinated fibers labeled by the three myelin proteins showed no statistical difference, however, which was significantly larger than that of controls. On the semithin sections, the experimental group demonstrated more myelinated fibers in the injured area than the controls, but the fibers had smaller diameter and thinner myelin sheath under electron microscope.</p><p><b>CONCLUSION</b>SCs can promote regeneration of injured nerve fibers and enhance remyelination, which may be histological basis of SCs-mediated functional repair of injured spinal cords.</p>

The effect of 2,5-hexanedione on myelin protein zero expression, and its mitigation using Ginkgo biloba extract / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To investigate the role of myelin protein zero (P(0)) in 2,5-hexanedione (2,5-HD)-induced peripheral nerve injury, and the protective effect of Ginkgo biloba extract (Egb761) on 2,5-HD-induced toxic peripheral neuropathy.</p><p><b>METHODS</b>After 4 weeks of treatment with 2,5-HD at different doses (50, 100, 200, 400 mg/kg) in rats, changes in the levels of P(0) in rat sciatic nerves was investigated, and the effect of Egb761 on 2,5-HD-induced toxic peripheral neuropathy was studied.</p><p><b>RESULTS</b>The blood-nerve barrier (BNB) permeability of the sciatic nerve increased, and the expression of P(0) mRNA and P(0) protein decreased in a dose-dependent manner after treatment with 2,5-HD for 4 weeks. Pretreatment with Egb761 protected against BNB interruption, and inhibited P(0) mRNA and protein reduction during 2,5-HD treatment. Pretreatment with Egb761 significantly reduced loss of body weight (P<0.01) and mitigated gait abnormalities (2.85±0.22) induced by 400 mg/kg 2,5-HD (P<0.01). It also reduced the signs of neurotoxicity induced by 2,5-HD.</p><p><b>CONCLUSION</b>2,5-HD inhibited the expression of P(0) in a dose-dependent manner, and this may be an important mechanism by which toxic peripheral neuropathy is induced by 2,5-HD. Egb761 has a protective effect against 2,5-HD-induced peripheral neurotoxicity in rats.</p>

The auditory study of guinea pig immunized with P0 protein / 临床耳鼻咽喉头颈外科杂志

OBJECTIVE@#To investigate the auditory characteristics of guinea pigs immunized with purified P0 protein from inner ear of guinea pigs.@*METHOD@#Purified inner ear P0 protein was made by Preparative SDS-PAGE. To evaluate the results of Auditory Brainstem Response (ABR), Compound Action Potention (CAP), and Distortion Product Oto-acoustic Emissions (DPOAE) in guinea pigs immunized with purified guinea pigs inner ear myelin protein P0.@*RESULT@#Seven ears of the guinea pigs immunized with purified inner ear P0 protein developed hearing loss. In the ABR study, peak latencies of wave I, III and the interpeak latency of I -III, I-IV were elevated in this group of guinea pigs compared with the control group (P 0.05). Even though the contra-lateral suppressive effect had the depressed tendency, but there was no significant different when compared with the controls (P > 0.05).@*CONCLUSION@#Purified inner ear P0 protein is an important autoimmune inner ear antigen and can develop autoimmune disease of the auditory nerve.

Construction of recombinant adenovirus and mediated reported gene expression in the guinea pig cochlea / 临床耳鼻咽喉头颈外科杂志

OBJECTIVE@#To purify P0 protein from guinea pig's inner ear by preparative SDS-PAGE and study the possible role it may play in the etiology of autoimmune inner ear disease.@*METHOD@#A mixture of membraneous proteins of inner ear was separated by preparative SDS-PAGE. The corresponding band at 30kd was cut and electrically eluted. The protein collected was identified by analytical SDS-PAGE and Western blot assay. A group of 20 guinea pigs were immunized with P0 protein emulsified in complete Freund's adjuvant, another 10 guinea pigs were immunized with complete Freund 's adjuvant only as control. The guinea pigs' hearing thresholds, serum IgG level and morphological changes in the inner ear were investigated. The distribution of P0 protein in the cochlear was detected by immunohistochemical technique.@*RESULT@#The purity of the protein was demonstrated by a single band at the 30 kD site in SDS-PAGE, which was identified as P0 protein by western blot analysis assay. About 17.5% P0-immunized guinea pigs showed increased hearing thresholds, elevated IgG level (F =6.48, P <0. 01), as well as a decreased number of spiral ganglion cells and inflammatory cell infiltration in the cochlear nerve region. The P0 protein is distributed in the cochlear nerve and spiral ganglion only.@*CONCLUSION@#P0 protein from guinea pig's inner ear can be successfully purified by preparative SDS-PAGE and an animal model of experimental autoimmune inner ear disease induced by P0 protein is successfully established.

Animal model of experimental autoimmune inner ear disease induced by P0 protein purified from inner ear / 临床耳鼻咽喉头颈外科杂志

OBJECTIVE@#To purify P0 protein from guinea pig's inner ear by preparative SDS-PAGE and to study the possible role it may play in the etiology of autoimmune inner ear disease.@*METHOD@#A mixture of membraneous proteins of inner ear was separated by preparative SDS-PAGE. The corresponding band at 30,000 was cut and electrically eluted. The protein collected was identified by analytical SDS-PAGE and Western blot assay. A group of 20 guinea pigs were immunized with P0 protein emulsified in complete Freunds adjuvant, another 10 guinea pigs were immunized with complete Freunds adjuvant only as control. The guinea pigs' hearing thresholds, serum IgG level and morphological changes in the inner ear were investigated. The distribution of P0 protein in the cochlear was detected by immunohistochemical technique.@*RESULT@#The purity of the protein was demonstrated by a single band at the 30000 site in SDS-PAGE, which was identified as P0 protein by western blot analysis assay. About 17.5% P0-immunized guinea pigs showed increased hearing thresholds, elevated IgG level (F = 6.48, P<0.01), as well as a decreased number of spiral ganglion cells and inflammatory cell infiltration in the cochlear nerve region. The P0 protein is distributed in the cochlear nerve and spiral ganglion only.@*CONCLUSION@#P0 protein from guinea pigs in ner ear can be successfully purified by preparative SDS-PAGE and an animal model of experimental autoimmune inner ear disease induced by P0 protein was successfully established.

Myelin Protein Zero (MPZ) Gene Analysis in Korean Patients with Charcot-Marie-Tooth: Clinical and Electrophysiological Characteristics

BACKGROUND: Mutations in the myelin protein zero (MPZ) gene, which is located on chromosome 1q21-q22, is present in Charcot-Marie-Tooth disease type 1B (CMT1B), CMT type 2, Dejerine-Sottas syndrome, and congenital hypomyelination neuropathy. It is proposed that the nature and position of the MPZ mutations mainly determine the axonal and demyelinating phenotypes. In this study, we investigated to determine the clinical and electrophysiological characteristics in CMT patients with mutations in the MPZ gene. METHODS: We examined mutations of MPZ, in 62 Korean families diagnosed as having CMT disease. Mutations were confirmed by through both strands sequencing. Nerve conduction studies were carried out in CMT patients having each mutation. RESULTS: The three mutations (Asp118Asn, c.449-1G>T (3'-splice site), Lys236Glu), determined to be novel, were not detected in the 105 healthy controls. The mutation frequency of MPZ was similar as those found in several European populations. Electrophysiologically, 3'-splice site mutation (449-1G>T) showed the conduction block and moderate slowing nerve conduction velocities like that of CMT1B. However, the other mutations represented the electrophysiological features of CMT type 2. CONCLUSIONS: We report the identified three novel MPZ mutations in Korean CMT patients and the phenotype-genotype correlations based on nerve conduction studies.

Genetics of hereditary motor and sensory neuropathy and the Costa Rican contribution: [review]

Hereditary motor and sensory neuropathy (HMSN) or Charcot-Marie-Tooth disease (CMT) is the most common hereditary illness of the peripheral nervous system. The genetics and the physiopathological aspects of the disease clarified until know, are here summarized. More than twenty genes and ten additional loci have been related with HMSN. These findings contribute to understand the metabolism of peripheral nerves and give the basis for molecular diagnostics and future therapy. Several Costa Rican families with CMT have been identified, specially with axonal forms. Two families present mutations in the myelin protein zero gene (IMPZ). In addition, linkage have been found between the disease and locus 19q13.3 in an extended family, and a mutation segregating with the disease is present in a candidate gene of the critical interval. Costa Rica has several advantages for genetical studies, that can contribute importantly in the generation of knowledge in the neurogenetical field.