Inflammatory demyelination in a patient with CMT1A

We report a case of Charcot-Marie-Tooth disease (CMT), with identified PMP22 gene duplication (CMT type 1A), and with evidence of an inflammatory demyelinating process superimposed on the course of the chronic genetic disease. Macrophage-associated demyelination was observed on the peripheral nerve biopsy. This observation supports some experimental data from the literature and shows that there may be a genetic susceptibility to inflammatory demyelinating processes in certain CMT kindreds.     

A Family Harboring CMT1A Duplication and HNPP Deletion

Charcot-Marie-Tooth disease type 1A (CMT1A) is associated with duplication of chromosome 17p11.2-p12, whereas hereditary neuropathy with liability to pressure palsies (HNPP), which is an autosomal dominant neuropathy showing characteristics of recurrent pressure palsies, is associated with 17p11.2-p12 deletion. An altered gene dosage of PMP22 is believed to the main cause underlying the CMT1A and HNPP phenotypes. Although CMT1A and HNPP are associated with the same locus, there has been no report of these two mutations within a single family. We report a rare family harboring CMT1A duplication and HNPP deletion.     

Schwann cell differentiation in Charcot-Marie-Tooth disease type 1A (CMT1A): normal number of myelinating Schwann cells in young CMT1A patients and neural cell adhesion molecule expression in onion bulbs

Charcot-Marie-Tooth disease type 1A (CMT1A) is a common hereditary demyelinating neuropathy caused by a duplication of the gene for the myelin protein PMP22, resulting in overexpression of PMP22 in young patients. Although genetically well defined, the pathogenesis of the hereditary demyelinating neuropathy CMT1A is still unclear. Homology of PMP22 cDNA to the growth arrest-specific gene gas3 and experiments in vitro showing decreased proliferation in PMP22-overexpressing Schwann cells suggest a role of PMP22 in Schwann cell differentiation. Furthermore, overexpression of PMP22 in fibroblasts induces programmed cell death. In this report we applied morphometrical methods using electron micrographs and immunohistochemistry to further characterise Schwann cells in CMT1A nerve biopsy samples from CMT1A patients. We show that the total number of PMP22-expressing Schwann cells, i.e. Schwann cells that are in a 1:1 relationship with axons, was not reduced in sural nerve biopsy samples from six young CMT1A patients. We excluded non-specific secondary Schwann cell proliferation. Thus, in young CMT1A patients with increased PMP22 overexpression there seems to be no evidence for altered initial Schwann cell proliferation in achieving a 1:1 relationship to axons prior to the process of de- and remyelination. Further, using electron microscopy we found no evidence for apoptosis of Schwann cells in CMT1A . However, we provide additional support for an abnormal Schwann cell phenotype in CMT1A by showing the expression of neural cell adhesion molecule immunoreactivity in onion bulbs. Thus, the role of PMP22 in cell growth and differentiation does not lead to an altered number of myelinating Schwann cells but to altered Schwann cell differentiation in CMT1A. Received: 23 September 1996 / Revised: 28 November 1996, 31 January 1997, 2 April 1997 / Accepted: 3 April 1997     

GABA uptake by purified rat Schwann cells in culture

Purified rat Schwann cells maintained in culture for up to 6 months retained their ability to take up the neurotransmitter γ-aminobutyric acid (GABA) by a high-affinity mechanism. Although cultured fibroblasts also accumulated GABA, they did so by a low affinity mechanism. These results indicate that Schwann cells continue to express a high affinity GABA transport system in the absence of signals from neurons.     

Comparative study of cell culture and purification methods to obtain highly enriched cultures of proliferating adult rat Schwann cells

We present here a fast protocol that could be used to obtain highly purified cultures of maximal proliferating adult rat Schwann cells. These adult rat Schwann cells can be transfected in a nonbiological way using the physical transfection method of electroporation. Schwann cells are decisive in recovery of peripheral nerves after injury. In a clinical context, the use of enriched adult Schwann cells is necessary for autologous cell transplantation within nerve transplants for peripheral nerve repair. Different parameters such as tissue preparation, culture conditions, and protocols for enrichment, elevation of proliferation rates, and transfection were evaluated in cell cultures harvested from adult rat peripheral nerves. Cell preparation from in vivo predegenerated adult rat sciatic nerves combined with the use of melanocyte growth medium supplemented with forskolin, fibroblast growth factor (FGF)-2, and pituitary extract as a selective, serum-free culture medium, with a secondary cell-enrichment step using specific detachment, resulted in highly enriched cultures of adult rat Schwann cells (>90%) with enhanced proliferation rates (>or=40%). About 20% of these adult Schwann cells could be modified genetically using an optimized electroporation protocol.     

人骨髓间充质干细胞分离培养方法的改进*

背景：当前分离培养骨髓间充质干细胞方法的有多种,但在外科手术中进行获取的方法仍然较少,而外科分离所得细胞对于相应疾病的具有揭示原因和提供研究线索的价值。 目的：在骨科手术中以直接贴壁差速贴壁分离培养纯化与鉴定骨髓间充质干细胞。 方法：在骨科关节置换等大手术中吸取少量人骨髓血,采用直接贴壁法分离骨髓间充质干细胞,在贴壁后36~48 h行洗盘处理,并通过长期体外培养,特定培养基自身的筛选作用对细胞进行筛选,当细胞生长达到足量时进行鉴定。 结果与结论：采用直接贴壁法能够在长骨髓腔内混合血中分离培养骨髓间充质干细胞并形成集落,其形态学表现和表面分子经鉴定符合骨髓间充质干细胞特点,能够获得符合要求的人骨髓间充质干细胞,从而进行后续实验。     

Analysis of the benefits of vitamin cocktails in treating Charcot-Marie-Tooth disease type 1A

We recently proposed that the use of high doses of ascorbic acid (AA) could constitute the first potential treatment for Charcot-Marie-Tooth disease type 1A (CMT1A).4 We investigated the potential benefits of using cocktails of vitamins for CMT1A therapy. We used transient transfection of Schwann cells with a construction placing the expression of a reporter gene under the control of the Schwann cell-specific promoter of PMP22. Transfected cells were cultured with or without addition of ascorbic acid, vitamin A, vitamin E, or a cocktail of these vitamins. Adding vitamin A or E counteracts the effect of ascorbic acid in inhibiting PMP22 expression. We thus recommend that vitamins A and E should not be included in combination with AA in clinical trials.     

周围髓鞘蛋白22基因重复突变致夏科-马里-图斯病1A亚型的临床和神经电生理特征

目的探讨周围髓鞘蛋白22(PMP22)基因重复突变阳性的夏科-马里-图斯病(CMT)lA亚型患者临床和神经电生理改变特点。方法总结21例PMP22基因重复突变阳性的CMTlA患者的临床特点,并分析其神经电生理特征。结果 21例患者中,10例临床特征符合四肢远端萎缩无力的典型CMTl型表现,另外11例呈不典型性,如仅有头晕、合并听力障碍、上肢姿势性震颤、反复发作性肢体无力、伴有小脑性共济失调及癫疒间等。10例患者肌电图出现纤颤电位和(或)正锐波,15例患者运动单位电位时限延长。神经传导存在广泛异常,所有患者被检的运动或感觉神经传导速度存在不同程度的减慢或消失。结论 PMP22重复突变阳性的CMTlA患者具有较高的临床异质性,其电生理特点为肌电图呈神经源性损害,感觉神经病变重于运动神经,下肢受累程度重于上肢,神经电生理检查对CMT1A的诊断很重要。     

Tissue culture methods to study neurological disorders: Establishment of immortalized Schwann cells from murine disease models

Previously, the authors have established spontaneously immortalized cell lines from long‐term cultures of normal adult mouse Schwann cells. Establishment of such Schwann cell lines derived from murine disease models may greatly facilitate studies of the cellular mechanisms of their peripheral nervous system lesions in the relevant diseases. Recently, the authors have established immortalized Schwann cell lines derived from Niemann–Pick disease type C mice (NPC; spm/spm) and globoid cell leukodystrophy mice (twitcher). In the present study, long‐term cultures were maintained of Schwann cells derived from dorsal root ganglia and consecutive peripheral nerves of another NPC mouse (npc^nih/npc^nih, npc^nih/+), myelin P0 protein‐deficient mice (P0–/–, P0+/–) with their wild‐type littermates (P0+/+), and neurofibromatosis type 1 gene (NF1)‐deficient mice (Nf1^Fcr/+) for 8–10 months, and immortalized cell lines from all these animals established spontaneously. These cell lines had spindle‐shaped Schwann cell morphology and distinct Schwann cell phenotypes and retained genomic and biochemical abnormalities, sufficiently representing the in vivo pathological features of the mutant mice. These immortalized Schwann cell lines can be useful in studies of nervous system lesions in these mutant mice and relevant human disorders.     

A novel<Emphasis Type="Italic"> PMP22</Emphasis> mutation Ser22Phe in a family with hereditary neuropathy with liability to pressure palsies and CMT1A phenotypes

We describe a Cypriot family in which some family members presented with episodes of pressure palsies, while other family members had a slowly progressive chronic polyneuropathy typical of the Charcot-Marie-Tooth type 1 phenotype. All family members were evaluated clinically, with nerve conduction studies, and with genetic testing. In all affected individuals there was clinical and electrophysiological evidence of diffuse demyelinating sensorimotor polyneuropathy and a novel point mutation in the PMP22 gene (Ser22Phe) was identified.     

Overexpression of ErbB2 and ErbB3 receptors in Schwann cells of patients with Charcot-Marie-tooth disease type 1A

Axon-derived neuregulins (NRGs) are a family of growth factors whose binding to ErbB tyrosine kinase receptors promotes the maturation, proliferation and survival of Schwann cells (SCs). Correct NRG/ErbB signaling is essential for the homeostasis of axonal-glial complexes and seems to play a role in peripheral nerve repair. The potential involvement of ErbB receptors in human peripheral neuropathies has not been clarified. Therefore, we assessed the immunoreactivity for EGFR (ErbB1), ErbB2, and ErbB3 in nerve biopsies from patients with different forms of Charcot-Marie-Tooth disease, type 1, (CMT1), as compared to others with inflammatory neuropathies and controls. The most notable changes consisted in the overexpression of ErbB2 and ErbB3 by SCs of nerves from CMT1A patients. These findings are consistent with an impairment of SC differentiation and expand the molecular phenotype of CMT1A. The upregulation of these receptors may play a role in the inhibition of myelination or in the promotion of recurrent demyelination and axonal damage.     

Purification of rat Schwann cells from cultures of peripheral nerve: an immunoselective method using surfaces coated with anti-immunoglobulin antibodies

We report a method for deriving purified rat Schwann cells by immunoselective removal of fibroblasts. Contaminating fibroblasts labeled with antibody against specific surface marker Thy 1.1 are bound on plastic surfaces coated with a second antibody. The efficacy of the method is demonstrated by flow cytometry and by specific Schwann cell Ran-1 immunofluorescence.     

Inhibition of glial proliferation in vitro by serum from patients with multiple sclerosis

Primary cell cultures from fetal rat CNS have been employed to evaluate the effects caused by the addition of serum from patients affected by multiple sclerosis (MS). MS-serum supplemented media caused a decrease in [3H]-thymidine incorporation into the cultures, thus indicating an inhibitory effect on proliferating glial cells. Sera from patients in remission stage of the disease showed an inhibitory effect not significatively lower than those from patients in acute stage. These results suggest that glial cells may be a target of circulating factors present in MS.     

副肿瘤性神经系统综合征患者外周血单个核细胞的增殖反应及其对小细胞肺癌细胞株的抑制作用

目的研究副肿瘤性神经系统综合征(PNS)患者外周血单个核细胞(PBMC)的增殖反应及其对小细胞肺癌(SCLC)细胞株(H446)的抑制作用。方法将7例PNS患者和6例SCLC患者PBMC加入白介素-2(IL-2)体外培养后,再分别与H446单独、混合培养。用流式细胞术分析H446、总PBMC、CD4+、CD8+T细胞增殖指数(即增殖后的细胞总数与增殖前的细胞总数的比值)及CD4+、CD8+、CD4+/CD8+比例,并与正常对照组比较。结果PNS组与SCLC组PBMC单独培养与混合培养后H446、总PBMC、CD4+、CD8+T细胞增殖指数差异无统计学意义。经IL-2刺激后,PNS患者CD4+T细胞比例[(76.54±3.96)%]较正常对照组[(51.75±17.3)%]显著升高(P<0.05)。结论PNS患者PBMC增殖反应以CD4+T细胞为主,其对SCLC细胞没有抑制作用。     

A simple method for the separation of retinal sublayers from the entire retina with special reference to application for cell culture

We developed a simple method for the separation of rat retinal sublayers. The rat retina was carefully removed from the sclera and treated in trypsin solution. After this treatment, the retinal sublayers could be easily separated by using Millipore filter paper into an outer nuclear layer, an inner nuclear layer, an inner plexiform layer, and a ganglion cell layer + nerve fiber layer. The viable cells of each of the sublayers could be cultured on a poly-L-lysine coated chamber for at least several weeks in Eagle's minimum essential medium supplemented with calf serum and glucose.     

Rat CNS cell culture. Enhancement of neuronal survival and delay of glial proliferation by serum from patients with multiple sclerosis. A morphological study

The addition of serum from multiple sclerosis (MS) patients to the culture medium of dissociated cells from cerebral hemispheres of rat embryos caused a delay in glial proliferation and an enhancement of neuronal survival. Sera from normal individuals and patients with other neurological diseases failed to show this effect. These morphological observations are interpreted as the outcome of inhibition of in vitro gliogenesis.

---

Sommario L'aggiunta di siero prelevato da pazienti affetti da sclerosi multipla (SM) in colture primarie di cellule da S.N.C. di feto di ratto, determina un ritardo della proliferazione gliale ed un incremento della sopravvivenza dei neuroni. Tale effetto non si verifica se alle colture viene aggiunto siero prelevato da soggetti normali od affetti da altre malattie neurologiche. I dati presentati vengono interpretati come il risultato di una inibizione della gliogenesi in vitro.

     

Accumulation of peripheral myelin protein 22 in onion bulbs and Schwann cells of biopsied nerves from patients with Charcot-Marie-Tooth disease type 1A

Peripheral myelin protein 22 (PMP-22) is a glycoprotein expressed in the myelin sheath of myelinated Schwann cells. Duplication of the PMP-22 gene and its gene dosage effect have been postulated to be involved in the pathogenesis in the majority of individuals with Charcot-Marie-Tooth disease type 1A (CMT1A). Northern blot analysis has demonstrated that the mean relative ratio of PMP-22 mRNA/β-actin mRNA in biopsied nerves of patients with CMT1A is significantly higher than that in disease controls. To investigate whether the elevated expression of PMP-22 mRNA is reflected in the amount and the localization of PMP-22, we analyzed PMP-22, myelin basic protein (MBP), protein zero (P0), and S-100 immunoreactivities in biopsied nerves from six patients with CMT1A, five patients with other types of CMT, five patients with acquired demyelinating neuropathies, and two normal subjects. In all patients with CMT other than CMT1A and acquired demyelinating neuropathy, as well as in normal subjects, the myelin sheath was immunoreactive for PMP-22, MBP, and P0, while the Schwann cell cytoplasm was immunoreactive only for S-100. In five out of six patients with CMT1A, however, the PMP-22 immunoreactivity was present not only on the myelin sheath but also in the Schwann cell cytoplasm and onion bulbs (OBs). Although OBs are nonspecific and also seen in other inherited or acquired demyelinating neuropathies, the PMP-22-positive OBs were seen exclusively in CMT1A.The finding suggested that the expression of PMP-22 was abnormal for its localization and probably for the amount in patients with CMT1A carrying duplication of the PMP-22 gene. Received: 5 February 1996 / Revised, accepted: 20 May 1996     

Effect of infection with rubella virus on the development of rat cerebellar cells in culture

Immunogold-silver staining of mixed rat cerebellar neuron/glial cell cultures, using Ranscht monoclonal antibody, resulted in labelling of neuronal processes and oligodendrocytes. This antibody was originally believed to react with galactocerebroside alone, and it was concluded that labelling by it resulted from the presence of myelin [2]. Electron microscopic examination of the cultures did not reveal myelination. Infection of such cultures with rubella virus resulted in a cytopathic effect. This effect was initially characterized as a disaggregation of neuronal processes, and the disruption of a layer of membranous material covering both cell bodies and processes. The virus causes disruption of cultures of pure glial cells, but there is no evidence of virus multiplication or cytopathic effect in pure neuron cultures. The cytopathic effect in mixed neuron/glial cell cultures is concluded to be glial cell mediated.     

Transplanted cultured type-1 astrocytes can be used to reconstitute the glia limitans of the CNS: the structure which prevents Schwann cells from myelinating CNS axons

Transplantation of different glial cells into areas of demyelination made in the adult rat spinal cord allows insights into the cell-cell interaction necessary to reconstruct a glial environment around demyelinated axons. Such studies have shown that type-1 astrocytes are central to the exclusion of Schwann cells from areas of glia-free demyelination. However, for these cells to be established in a manner which prevents Schwann cell remyelination of CNS axons, cells of the O-2A lineage are also required. If cultures of isogeneic rat type-1 astrocytes and mouse O-2A cells are transplanted into lesions made in non-immunosuppressed animals. Schwann cell remyelination is limited and extensive oligodendrocyte remyelination is achieved. This paradigm creates a model of immune mediated demyelination in which the immune response is not primarily directed at oligodendrocyte specific epitopes.