Lack of evidence for a role of the myelin basic protein gene in multiple sclerosis susceptibility in Sardinian patients

A link between myelin basic protein (MBP) polymorphism and multiple sclerosis (MS) has been reported in some populations but not in others. We analysed two polymorphisms in the 5' flanking region of the MBP exon 1 gene in MS patients from the founder population of Sardinia. Using the transmission disequilibrium test (TDT), MBP polymorphisms were analysed in 363 singleton MS families. No distortion in transmission of the tetranucleotide repeat (ATGG)12 and of the 1116–1540 nt alleles was found. Moreover, we discovered no epistatic effect of the MBP gene on the HLA/MHC DRB1,DQB1, DPB1 loci or on alleles defined by D6S1683 marker found to be associated with MS in Sardinians. We concluded that the MBP gene does not play a role in MS susceptibility in Sardinians. Received: 11 March 2002, Accepted: 18 June 2002 Correspondence to Professor Maria Giovanna Marrosu     

Evaluation of a rat model of experimental autoimmune encephalomyelitis with human MBP as antigen

Experimental autoimmune encephalomyelitis(EAE) is a good model for human multiple sclerosis(MS)research.However,there are some defects in the traditional models.Here,we improved the model by using thehuman myelin basic protein(MBP) as antigen.EAE was induced by immunization of female Wistar rats withhuman MBP.Compared with the traditional models,the new model was evaluated by clinical signs topathological changes.The immune state of the model was assessed by the lymphocyte infiltrative response andlevels of TNF-α,IFN-γ,IL-10.It was found that most of rats exhibited tail tone loss and hind-limb paralysis,also there were demyelination,infiltrative lymphocyte foci,“neuronophagia”in the cortex of cerebra and thewhite matter of spinal cords.PBMCs and spleen lymphocytes were strongly responsive to the stimulation ofMBP and PHA.The levels of TNF-α and IFN-γ were altered with the severity of EAE.In the remitting phase,IL-10 was increased significantly.This study demonstrates that the animal model of EAE induced by humanMBP bears resemblance to the features of human multiple sclerosis and promises to be a better model than everbefore for the study of MS.Cellular & Molecular Immunology.2004;1(5):387-391.     

表达人HGF／MBP融合蛋白的pMAL—MBP／HGF重组质粒的构建及鉴定

将人ＨＧＦ完整编码区ｃＤＮＡ片段插入ＭＢＰ表达型ｐＭＡＬ－Ｃ２载体质粒中，构建了表达人ＨＧＦ／ＭＢＰ融合蛋白的ｐＭＡＬ－ＭＢＰ／ＨＧＦ重质粒。表达的ＨＧＦ／ＭＢＰＧＥ分析分子量约为１１０ｋＤ，Ｗｅｓｔｅｒｎｂｌｏｔｔｉｎｇ表明能被兔抗ＭＢＰ抗体和抗人ＨＧＦＭｃＡｂ所识别。     

人源抗狂犬病毒二硫键稳定抗体在大肠杆菌中的融合表达

目的在大肠杆菌pMAL-P2x系统中表达anti-rabiesMBP-ScdsFv融合蛋白,酶切获得目的蛋白ScdsFv,进行纯化、鉴定及简单活性分析。方法利用DNA重组技术,将全长的ScdsFv蛋白基因克隆至原核表达载体pMAL-p2x中,重组质粒转化大肠杆菌E.coliER2566,IPTG诱导表达。表达产物经Xa酶切后,经Ni柱、AmyloseResin亲和层析进行纯化,SDS-PAGE和Western blot对其进行鉴定,ELISA检测其结合活性。结果成功地构建了重组质粒pMAL-ScdsFv,经诱导表达、蛋白酶切、亲和纯化后获得目的蛋白。ELISA检测其具有抗原特异结合活性,其热稳定性有显著提高。结论抗狂犬病毒ScdsFv蛋白具有良好的结合活性和生物学活性稳定性,可作为候选分子用于狂犬病的防治研究。     

Combined nasal administration of encephalitogenic myelin basic protein peptide 68-86 and IL-10 suppressed incipient experimental allergic encephalomyelitis in Lewis rats

Mucosal administration of low doses of myelin basic protein (MBP) peptide 68-86 (MBP 68-86) or anti-inflammatory cytokine IL-10 effectively prevented experimental allergic encephalomyelitis (EAE), but failed to suppress the disease if given after 7 days postimmunization (p.i.), i.e., after T cell priming had occurred. We anticipated that combined administration of autoantigen and IL-10 can treat incipient EAE. Lewis rats with EAE actively induced with MBP 68-86 and complete Freund's adjuvant received 120 microg MBP 68-86 + 200 ng IL-10 per rat per day from day 7 p.i. and for 5 consecutive days. These rats showed later onset, lower clinical scores, less body weight loss, and shorter duration of EAE than rats receiving MBP 68-86 or IL-10 only or PBS. EAE amelioration was associated with decreased infiltration of ED1(+) macrophages and CD4(+) T cells within the central nervous system and with decreased proliferative responses of lymph node cells, indicating that combined administration of MBP 68-86 and IL-10 induced immune hyporesponsiveness. IFN-gamma secretion as well as IFN-gamma, TNF-alpha, IL-4, and IL-10 mRNA expression by lymph node MNC was down-regulated in the treated rats. Immune hyporesponsiveness, rather than immune deviation or regulatory mechanisms, seems to be responsible for the protection of EAE after autoantigen + IL-10 administration by the nasal route.     

补肾活血中药对糖尿病大鼠视路突触素1和髓鞘硷性蛋白的影响

目的：探讨补肾活血中药对糖尿病大鼠视路突触素1和髓鞘硷性蛋白（myelin basic protein,MBP)的影响。方法：以链佐菌素造成大鼠糖尿病模型后，给予补肾活血中药加降糖灵混悬液连续6月，采用免疫组化结合图像分析半定量检测视网膜，外侧膝状体，视皮质17例区三级神经元轴突突触素Ⅰ和神经纤维髓鞘硷性蛋白的含量。结果：与正常组相比，模型组大鼠三级神经元轴突突触素Ⅰ和神经纤维MBP的含量明显降低（P＜0．001），补肾活血中药组大鼠三级神经元轴突突触素Ⅰ和神经纤维MBP的含量明显增加（P＜0．001）。结果：补肾活血中药能够增加糖尿病大鼠视路三级神经元突触的密度，并可提高神经纤维MBP合成，恢复髓鞘，有利于神经纤维传导功能的恢复。     

HIV-gp120 affects the functional activity of oligodendrocytes and their susceptibility to complement

The aim of this study was to assess whether the HIV protein gp120 can induce direct or/and indirect damage to oligodendrocytes (OL). Using highly purified cultures of rat OL, we report that gp120 binds to OL and induces functional alterations in these cells. Indeed, the percentage of cells expressing myelin basic protein (MBP) and the levels of all four MBP isoforms were substantially reduced after a 3-day treatment with 10 nM gp120. As gp120 depressed the ability of OL to reduce the tetrazolium salt MTT (a sign of mitochondrial impairment), the alteration of MBP production may be a consequence of decreased metabolic activity. The above effects were accompanied by a small increase in the number of apoptotic nuclei (from 4.3% in controls to 17.6% in cells treated for 3 days with gp120). As complement can lyse OL and gp120 is known to activate complement, we also studied the interaction between these two factors using OL cultures. The viral protein potentiated (by about 25%) the lytic effect of complement, when administered to the cultures 5 hr after complement, and depressed it (by about 30–40%), when added 5 hr before complement. Heat denaturation and anti-gp120 antibodies prevented the direct effect of gp120 on OL, but did not influence the interactions between gp120 and complement. Some gp120 non glycosylated peptides (V3 loop, 254-274 and 415-435 peptides) mimicked the ability of gp120 to antagonize the lytic effect of complement, but not that of potentiating complement lytic activity. In conclusion, our study indicates that gp120 can alter OL functional activity directly and can interfere with OL susceptibility to complement mediated lysis. J. Neurosci. Res. 50:946–957, 1997. © 1997 Wiley-Liss, Inc.     

Cell death mediated by Fas-FasL interaction between glial cells and MBP-reactive T cells

Apoptosis in T cells that have penetrated into the central nervous system (CNS) may be important for the physiological control of T cells with potentially dangerous reactivities to CNS antigens; such control may be dysfunctional in animals suffering from experimental autoimmune encephalomyelitis (EAE). In this study we examined the expression of Fas and FasL genes both in myelin basic protein (MBP)-reactive T cells and in glial cells and the susceptibility of these cells to death induced by Fas/FasL interaction. Both Fas and FasL gene expression is detectable in glial cells and MBP-reactive T cells. Cell death is not unidirectional: when T cells interact with glial cells death can be induced in the former or in the latter population. The ability to induce death of Fas-expressing cells varies greatly among different lines of MBP-reactive T cells, as does resistance to death induction by cells expressing FasL. Moreover, the ability of T cells both to deliver and to resist death signals is a function of their activation status: T cells freshly activated transmit a stronger apoptotic signal to Fas-positive target cells and are also more resistant to FasL-induced suicide. Soluble form of FasL provides a convenient titratable means of delivering death signals via Fas. However, comparison of the susceptibility of different targets to soluble FasL and to FasL expressed on the surface of a transfected glial line revealed differences, suggesting that signals arising from Fas/FasL interaction may be modulated by additional cell-surface molecules. J. Neurosci. Res. 52:458–467, 1998. © 1998 Wiley-Liss, Inc.     

Cytokine phenotype of human autoreactive T cell clones specific for the immunodominant myelin basic protein peptide (83–99)

Experimental allergic encephalomyelitis (EAE), an animal model resembling multiple sclerosis (MS), is mediated by myelin antigen-specific CD4+ T cells secreting cytokines such as interferon-γ (IFN-γ), tumor necrosis factor-β (TNF-β), and the proinflammatory cytokine TNF-α—all associated with the T-helper-1 (Th1) T cell subset. Based on numerous similarities between MS and EAE, it has been postulated that Th1-like T cells are involved in the pathogenesis of MS. Production of proinflammatory cytokines such as IFN-γ and, in particular, TNF-α/β by autoreactive T cells is considered crucial for the initiation and amplification of inflammatory brain lesions and possibly also for direct myelin damage. In contrast, regulatory cytokines such as interleukin-4 (IL-4), IL-10, and IL-13, which are associated with the Th2-like phenotype, may play a role in the resolution of relapses. Although the human T cell response to myelin basic protein (MBP) is well characterized in terms of antigen specificity, HLA restriction, and T cell-receptor (TCR) usage, little is known about the cytokine pattern of these autoreactive T cells. To gain such information, conditions for studying cytokine secretion by human autoreactive T cell clones (TCC) were established. The cytokine secretion profile of human autoreactive CD4+ TCC, specific for myelin basic protein peptide (83–89) [MBP(83–99)], a candidate autoantigen in MS, was investigated. Our results show that TCC cytokine production in long-term culture was stable. In addition, the correlation of various cytokines within specific TCC revealed differences compared to murine T cells. The comparison of 30 human MBP(83–99)-specific TCC demonstrated heterogeneity in cytokine secretion, with a continuum between Th1- and Th2-like cells rather than distinct Th1 or Th2 subsets. These data are important for further investigation of the potential role of cytokines in the inflammatory process of MS, and provide a powerful tool to investigate therapeutic interventions with respect to their influence on cytokine secretion of autoreactive T cells. © 1996 Wiley-Liss, Inc.