髓鞘蛋白脂质蛋白多肽139—151诱发实验性自身免疫性脑脊髓炎小鼠模型

目的 用髓鞘蛋白脂质蛋白多肽（PLP）139-151诱发实验性自身免疫性脑脊髓炎（EAE）小鼠模型。方法 应用PLP139－151抗原加完全福（氏）佐剂免疫SWXJ小鼠。结果 免疫14－20d后小鼠即发病,发病率为80％,而且多数呈缓解－复发型。病理学证实发病小鼠脑和脊髓内具有典型的脱髓鞘改变和炎性细胞浸润。结论 以PLP139－151为抗原免疫SWXJ小鼠诱发EAE,具有模型稳定、发病率高和缓解－复发的特点,是研究多发性硬化较理想的动物模型。     

黄芩甙治疗实验性自身免疫性脑脊髓炎的实验研究

目的研究黄芩甙治疗实验性自身免疫性脑脊髓炎(EAE)的可能性及有效性。方法应用髓鞘脂质蛋白(PLP)139-151免疫SJL/J小鼠诱发慢性复发-缓解型EAE模型,研究黄芩甙对PLP139-151诱导的小鼠淋巴结细胞增殖反应及干扰素-γ(IFN-γ)、白介素-4(IL-4)分泌的作用,使用流式细胞技术检测黄芩甙对EAE小鼠淋巴结细胞CCR5表达的影响;通过神经功能评分观察黄芩甙治疗EAE的有效性。结果黄芩甙可抑制PLP139-151诱导的淋巴结细胞增殖反应及IFN-γ的分泌,提高IL-4的分泌(P<0.001),有效降低EAE小鼠淋巴结细胞CCR5的表达;显著改善EAE小鼠神经功能评分(P<0.01),使发病时间延迟(P<0.01)。EAE小鼠发病数和死亡数较对照组减少,但差异并无统计学意义(P>0.05)。结论黄芩甙可有效治疗EAE,为黄芩甙用于临床多发性硬化的治疗提供了实验依据。     

小胶质细胞在PLP_(139-151)诱导SJL小鼠EAE模型中的作用研究

目的观察髓鞘蛋白脂质蛋白139-151(proteolipid protein,PLP_(139-151))诱导SJL小鼠构建的复发缓解型的实验性自身免疫性脑脊髓炎(experimental autoimmune encephalomyelitis,EAE)模型复发期脑内小胶质细胞的病理改变。方法应用100μg、150μg PLP_(139-151)多肽片段免疫SJL小鼠构建复发缓解型EAE模型(分别简称为100μg PLP组、150μg PLP组),另设健康对照组(对照组;采用与实验组等体积溶媒处理),观察35 d。观察两组EAE小鼠的发病情况及神经功能残疾进展评分差异,选择发病快、神经功能残疾进展评分高者(计为EAE组)观察复发期小胶质细胞活化情况。EAE组于复发期疾病高峰时,对照组于相同时间点,取小鼠大脑皮层组织,应用免疫组织化学(immunohistochemical,IHC)染色及免疫荧光(immunofluorescence,IF)染色比较两组小胶质细胞的活化情况。结果 150μg PLP组小鼠在两个发病高峰(分别为免疫第13、28天)均早于100μg PLP组(分别为免疫第15天、31天),神经功能评分第2次发病高峰高于100μg PLP组(3.00±0.79 vs. 1.75±0.90,t=2.33,P0.05),第1次发病高峰与100μg PLP组差异无统计学意义(P0.05),故选择150μg PLP组(计作EAE组)观察复发期(免疫后第28天)小胶质细胞活性。与对照组相比,EAE组小鼠复发期大脑皮层中小胶质细胞的活化明显增多,组织染色上表现为Iba-1表达增高,细胞体积增大,细胞突起增多。结论 150μg PLP_(139-151)多肽片段免疫SJL小鼠构建的复发缓解型EAE模型复发期小鼠大脑皮层小胶质细胞明显活化。     

PLP不同肽段诱导EAE模型的对比研究

目的建立不仅与多发性硬化(multiple sclerosis，MS)临床表现、病理特征接近而且病程相似的较为理想的复发一缓解型实验性自身免疫性脑脊髓炎(experimental autoimmune encephalomyelitis，EAE)模型。方法应用髓鞘蛋白脂质蛋白(proteolipid potein，PLP)多肽的两种免疫优势表位肽段PLP139-151和PLP178-191免疫雌性SJL／J小鼠，制作复发缓解型EAE(relapse remitting experimental autoimmune encephalomyelitis，RR—EAE)模型，观察其体重及神经功能评分的变化，应用HE、Luxol fas tblue髓鞘染色等方法观察模型的组织形态学改变。结果两种PLP肽段免疫的小鼠发病均具有缓解一复发的特点，出现明显的神经系统体征；小鼠发病时脑和脊髓组织显示明显的血管鞘形成、卫星现象和炎性细胞浸润以及脱髓鞘改变。结论PLP两种肽段均可诱发RR-EAE模型，这与临床MS的缓解一复发病程更相似，更能表现MS的临床特点，是研究MS的较为理想的动物模型。     

MK-801对EAE动物模型轴索损伤的保护作用研究

目的通过建立实验性自身免疫性脑脊髓炎(EAE)动物模型,对EAE的发病机制和轴索损伤进行研究,并进行药物干预,寻找新的治疗方法。方法选用髓鞘蛋白脂蛋白(PLP139-151)多肽作为抗原免疫雌性SJL/J小鼠,诱发EAE小鼠模型,应用MK-801(0.30mg/kg)进行干预。发病后取脑、脊髓组织切片,进行免疫组织化学检查。结果模型组23(23/30)只小鼠发病,发病率为76.7%,平均发病时间为19±2.58天,平均神经功能评分为2.14±0.69分。药物(MK-801)治疗组16(16/30)只小鼠发病,发病率为53.3%,平均发病时间为21±1.25天(P<0.05),平均神经功能评分为1.08±0.42分(P<0.02);病理显示药物干预后病灶减少,病变减轻。结论PLP多肽诱发SJL/J小鼠的EAE动物模型的病理改变主要累及脊髓。谷氨酸的兴奋性毒性参与MS发病和轴索损伤,应用NMDA受体拮抗剂MK-801可以减少神经功能缺失,降低发病率。     

实验性自身免疫性脑脊髓炎动物模型早期轴索损伤

目的 研究实验性自身免疫性脑脊髓炎(EAE)动物模型发病早期的轴索损伤.方法 采用髓鞘蛋白脂蛋白(PLP139-151)多肽作为抗原诱发EAE小鼠模型.小鼠经免疫后每天进行神经功能评分及称体质量,于发病后剥离脑及脊髓组织进行病理学研究.结果 7只(23.3%)小鼠于免疫后15～22 d内发病,平均免疫后发病时间为(19.00±2.58)d,平均神经功能评分为(2.14±0.69)分.免疫前小鼠平均体质量[(21.85±0.94)g]与发病后体质量[(23.24±1.55)g]比较差异无统计学意义.HE染色可见发病小鼠软脊膜下脊髓组织炎性细胞浸润明显,以小血管周围为主的血管袖套形成.病变累及脊髓多见,且较大脑病变重.LFB染色可见炎细胞浸润处髓鞘有不同程度脱失,同时Bodian银染可见轴索肿胀、横断.免疫组织化学染色可见髓鞘碱性蛋白着色的相同区域淀粉样前体蛋白浓染,而星形胶质细胞GFAP染色增生不明显.结论 PLP多肽诱发SJL/J小鼠的EAE模型临床症状较轻,病变主要累及脊髓,而在大脑未发现与MS类似的典型病灶.在EAE发病早期,髓鞘还未明显脱失时即可见轴索损伤,且早期轴索损伤与临床症状并不平行.     

MCI-186治疗实验性自身免疫性脑脊髓炎的研究

目的:探讨MCI-186干预实验性自身免疫性脑脊髓炎(EAE)的疗效及机制。方法:PLP_(139-151)诱发EAE,分别在EAE发病前后予MCI-186治疗,观察病程;MRI观察病灶;免疫组化检测诱导型一氧化氮合酶(iNOS)和硝基酪氨酸(NT)表达;TUNEL法检测细胞凋亡。结果:EAE发病前MCI-186干预可延迟发病,减轻炎症,抑制iNOS、NT的表达及细胞凋亡;EAE发病后MCI-186治疗减轻炎症,抑制病情进展。结论:MCI-186治疗EAE有效,通过保护血脑屏障、抑制炎症和自由基损伤起作用。     

自身抗原冲击的树突状细胞抑制实验性自身免疫性脑脊髓炎具有抗原特异性(简报)

树突状细胞(dendritic cells，DC)不仅有免疫刺激作用．而且能通过诱导中枢和外周耐受而调节免疫反应。近年发现，在诱发自身免疫性疾病动物模型如实验性自身免疫性脑脊髓炎(EAE)之前，皮下注射致病抗原碱性髓鞘蛋白68-86(MBP68-86)体外冲击的DC，对该抗原诱导的EAE模型有明显抑制作用。此研究采用皮下注射3种不同的致脑炎肽MBP68-86、蛋白脂质蛋白139-151(PLP139-151)和髓鞘少突胶质细胞糖蛋白肽(MOG35-55)体外冲击DC，对EAE进行研究。     

清开灵有效治疗实验性自身免疫性脑脊髓炎

目的　研究清开灵治疗实验性自身免疫性脑脊髓炎 ( EAE)的可能性及有效性。方法　应用蛋白脂质蛋白1 39- 1 51 ( PLP1 39- 1 51 )免疫 SJL/ J小鼠诱发慢性复发 -缓解性 EAE模型 ,研究清开灵对 PLP1 39- 1 51 诱导的小鼠淋巴结细胞增殖反应及 γ-干扰素 ( IFN-γ)分泌的作用 ,并通过神经功能评分观察清开灵治疗 EAE的有效性。结果　清开灵可抑制 PLP1 39- 1 51 诱导的淋巴结细胞增殖反应及 IFN-γ的分泌 ( P <0 .0 0 1 ) ,有效改善 EAE小鼠神经功能评分 ( P <0 .0 1 ) ,使发病时间延迟 ( P <0 .0 1 )。EAE小鼠发病数和死亡数较对照组减少 ,但其差异并无显著性 ( P>0 .0 5 )。结论　清开灵可有效治疗 EAE,为清开灵用于临床治疗多发性硬化提供了实验依据。     

2-BFI对EAE小鼠血脑屏障通透性和AQP4 mRNA表达的影响

目的观察2-(2-苯并呋喃基)-2-咪唑啉(2-BFI)对实验性自身免疫性脑脊髓炎(EAE)小鼠血脑屏障和水通道蛋白4(AQP4)mRNA表达的影响。方法 C57BL/6小鼠39只随机分为正常对照组、EAE组和2-BFI干预组。各组每日进行神经功能缺损评分;第20天进行病理学检查;伊文思蓝荧光定量方法检测血脑屏障通透性;电子显微镜观察血脑屏障的超微结构变化;Real-time PCR法测定AQP4 mRNA的表达水平。结果与EAE组比较,2-BFI干预组小鼠神经功能缺损及血脑屏障病理损伤减轻,AQP4 mRNA的表达水平明显降低,均差异有统计学意义。结论 2-BFI对EAE小鼠的神经保护作用与减少AQP4 mRNA的表达、减轻血脑屏障通透性有关。     

Treatment of relapsing experimental autoimmune encephalomyelitis with T cell receptor peptides

Restricted T cell receptor (TCR) VB gene usage by T cells for recognition of antigens involved in the production of experimental autoimmune encephalomyelitis (EAE) offers the possibility of selective immunotherapy. We determined the preferential VB gene usage of lymph node-derived clones from SJL/J mice to recognize the encephalitogenic epitope PLP 139-151 and from PL/J mice to recognize the newly described encephalitogenic epitope PLP 43-64. In addition, the VB gene usage for recognition of PLP 139-151 by T cell lines derived from SJL/J spinal cords was analyzed. Lymph node-derived SJL/J lines and clones specific for PLP 139-151 expressed VB2, VB4, and VB17a preferentially, and PL/J lines and clones specific for PLP 43-64 expressed VB2 and VB8.2 preferentially. A VB4+ SJL/J clone and a VB8.2+ PL/J clone were encephalitogenic. Encephalitogenic SJL/J lines derived from spinal cord expressed VB2, VB10, VB16, and VB17a preferentially, with a predominance of VB2. Candidate TCR peptides were synthesized and tested from the VB gene families VB4, VB8.2, and VB17a, based on our data and previous data on BP-induced EAE in mice. Treatment of relapsing EAE (R-EAE) in SJL/J mice with VB4 and VB17a peptides reduced clinical and histological disease severity, and treatment of R-EAE in (PLxSJL)F1 mice with VB4 and VB8.2 peptides also reduced clinical and histological disease. The use of TCR peptide therapy may have applications for the treatment of human autoimmune diseases such as multiple sclerosis. © 1993 Wiley-Liss, Inc.     

Encephalitogenic T lymphocytes develop from SJL/J hematopoietic cells transplanted into severe combined immimodeficient ( SCID) mice

Previously, we constructed chimeras by injecting hematopoietic cells from experimental autoimmune encephalomyelitis (EAE)-susceptible SJL (H-2^S) strain mice into severe combined immunodeficient (SCID) C.B-17scid /scid (H-2^d) mice. These SCID mouse-SJL mouse hematopoietic cell chimeras developed passive EAE following adoptive transfer of PLP S139–151-specific SJL T lymphocyte line cells, but were resistant to active EAE induced by primary immunization with PLP S139–151. In order to gain an understanding of the encephalitogenic potential of transplanted hematopoietic progenitors in SCID mouse-SJL mouse chimeras, we attempted to induce EAE in hematopoietic chimeras constructed with or without an additional SJL fetal thymus implant. Chimeras with the thymus implant were susceptible to passive and active EAE while chimeras without the thymus implant were susceptible to passive but not active EAE. Encephalitogenic, CD4⁺, TCR⁺ T lymphocytes were selected in vitro from PLP S139-151-immunized, thymus-implanted chimeras. These results showed that hematopoietic SJL progenitors developed into antigen-presenting accessory cells and immunocompetent encephalitogenic T lymphocytes following transplantation into SCID mice. The development of primary immune reactivity depended on a fetal thymus implant for expression in SCID mouse-SJL mouse chimeras.     

Sequence 104–117 of myelin proteolipid protein is a cryptic encephalitogenic T cell determinant for SJL/J mice

Immunization of animals with myelin proteolipid protein (PLP) causes experimental autoimmune encephalomyelitis (EAE), a disease model that shares many features with human multiple sclerosis (MS). The SJL/J (H-2⁵) mouse is widely used in EAE studies because of its high disease susceptibility. Previous studies have shown that sequences 139–151 HCLGKWLGHPDKF and 178–191 NTWTTCQSIAFPSK represent distinct co-immunodominant encephalitogenic determinants of PLP for SJL/J mice. In the present study, we identify a third distinct PLP encephalitogenic peptide for SJL/J mice. Following immunization with PLP 104–117 KTTICGKGLSATVT, 10/14 SJL/J mice developed clinical and histological EAE with a mean time of onset of 38 days (18–65 days). T cell lines generated from SJL/J mice immunized with p104–117 were predominantly (> 90%) CD3⁺, CD4⁺, αβTCR⁺, CD8^dim, γδTCR^dim T cells and responded in an Ag-specific, I-As-restricted manner to p104–117. Upon adoptive transfer of 16−40 × 10⁶ T line cells, EAE was produced in naive SJL/J recipients 20–34 days after transfer. The delayed onset of both active and passive disease may be related to the non-immunodominant, cryptic nature of p104–117 in SJL/J mice. Lymph node cells from SJL/J mice immunized with either whole PLP or with pooled encephalitogenic PLP peptides responded to challenge with the immunodominant PLP determinants p139–151 and p178–191 but did not respond to p104–117. The existence of three distinct PLP encephalitogenic T cell determinants for SJL/J mice suggests that susceptibility to EAE and perhaps MS may be related to promiscuous T cell recognition of multiple myelin protein determinants.     

Induction of active and adoptive relapsing experimental autoimmune encephalomyelitis (EAE) using an encephalitogenic epitope of proteolipid protein.

Proteolipid protein (PLP) is a major component of the central nervous system (CNS) myelin membrane and has been shown to induce acute experimental autoimmune encephalomyelitis (EAE) in genetically susceptible animals. Here we describe conditions by which a relapsing-remitting form of EAE can be reliably induced in SJL/J mice either actively immunized with the major encephalitogenic PLP peptide, PLP13-151(S), or following adoptive transfer of PLP139-151(S)-specific T cells. The disease follows a reliable relapsing-remitting course with acute clinical signs first appearing 6-20 days after priming or transfer and relapses first appearing at 30-45 days. The initial onset of disease correlates with delayed-type hypersensitivity (DTH) reactivity specific for PLP139-151(S), in the apparent absence of T cell reactivity to the major myelin basic protein (MBP) peptide. Histologically, both the active and adoptive forms of the disease are characterized by extensive mononuclear cell infiltration and severe demyelination of the CNS. These results suggest that T cell responses specific for PLP139-151(S) are sufficient to induce clinical and histological R-EAE in SJL/J mice. This model should prove useful for examination of the cellular and molecular events involved in clinical relapses and perhaps in determining the role of PLP-specific T cell responses in multiple sclerosis (MS).     

Novel monoclonal antibodies against proteolipid protein peptide 139-151 demonstrate demyelination and myelin uptake by macrophages in MS and marmoset EAE lesions

Experimental autoimmune encephalomyelitis (EAE) induced by immunization of mice with epitopes of the proteolipid protein (PLP), a major myelin constituent, forms a useful model for the study of multiple sclerosis (MS). In addition, MS patients display PLP-specific T- and B-cell responses, suggesting that PLP reactivity is relevant to pathogenesis.Here, the generation and characterization of a panel of mouse monoclonal antibodies (Mab) against PLP139-151, the prominent encephalitogenic sequence in SJL/J mice is described. Five Mab were generated by conventional immunization of an SJL/J mouse and hybridoma generation. These Mab reacted well with the PLP139-151 peptide in ELISA and belonged to the IgG2a and IgG2b subclasses, consistent with CD4+ T helper 1-cell-supported antibody formation. The Mab also efficiently detected PLP peptide-BSA conjugates in Western blot, confirming their multi-assay applicability.The Mab were subsequently used to determine the occurrence of demyelination in brains of MS patients and marmoset monkeys with EAE. Immunohistochemistry on both paraffin and frozen sections demonstrated a homogeneous expression of PLP139-151 in normal myelin, and a complete absence in lesions containing demyelinated areas, confirming that the Mab can be used as a general myelin marker. In active demyelinating MS lesions, the Mab visualized the peptide in the cytoplasm of macrophages containing phagocytosed myelin.In conclusion, this panel of Mab against the encephalitogenic PLP139-151 epitope forms a useful tool for further study of autoantigen expression, demyelination/remyelination and the staging of lesional activity in MS patients, as well as in EAE models in distinct animal species.     

Myelin proteolipid protein: minimum sequence requirements for active induction of autoimmune encephalomyelitis in SWR/J and SJL/J mice

Proteolipid protein (PLP) is the major protein constituent of mammalian central nervous system myelin. We have previously identified two different PLP encephalitogenic T cell epitopes in two mouse strains. Murine PLP peptides 103-116 YKTTICGKGLSATV and 139-151 HCLGKWLGHPDKF are encephalitogenic determinants in SWR/J (H-2q) and SJL/J (H-2s) mice, respectively. The purpose of the present study was to determine the minimum sequence requirements for each of these PLP encephalitogens. In SWR/J mice, at least two distinct overlapping peptides can induce experimental autoimmune encephalomyelitis (EAE). The eleven residue sequences PLP 105-115 TTICGKGLSAT and PLP 106-116 TICGKGLSATV are encephalitogenic in SWR/J mice, but PLP 106-115 TICGKGLSAT, the decapeptide indigenous to both sequences, is non-encephalitogenic. In contrast, the shortest PLP sequence capable of inducing EAE in SJL/J mice is the nonapeptide 141-149 LGKWLGHPD. These data indicate that encephalitogenic determinants of PLP are short contiguous peptide sequences similar in length and diversity to those of MBP.     

Treatment of relapsing autoimmune encephalomyelitis with T cell receptor Vβ-specific antibodies when proteolipid protein is the autoantigen

Monoclonal antibodies (mAbs) directed against the Vβ chain of the T cell receptor (TCR) of pathogenic T cells have been used to treat acute murine experimental autoimmune encephalomyelitis (EAE) induced by myelin basic protein (BP). We evaluated anti-Vβ mAb for the treatment of relapsing EAE (R-EAE) induced in SJL/J mice by the myelin proteolipid protein (PLP) peptide 139–151. Spinal cord mononuclear cells isolated from mice immunized for R-EAE with PLP 139–151 were shown to express a predominance of Vβ2 and Vβ17 during acute and relapsing disease. T cell lines specific for PLP 139–151 were magnetically sorted to express 80–90% Vβ2. These Vβ2-enriched lines induced typical relapsing demyelinating EAE in naive recipient mice. SJL/J mice with R-EAE induced by a PLP 139–151-specific T cell line expressing 88% Vβ2 were treated with anti-Vβ2 mAb. Anti-Vβ2 mAb markedly reduced clinical and histological disease severity when given at the time of cell transfer or when given at clinical disease onset. In contrast, anti-Vβ mAbs showed only a mild clinical effect on R-EAE induced by immunization with PLP 139–151 or R-EAE induced by immunization with PLP 139–151 or R-EAE transferred by a PLP 139–151-specific T cell line expressing multiple Vβs. A cocktail of mAbs directed against Vβ2, Vβ4, and Vβ17 significantly reduced the numbers of spinal cord T cells expressing these Vβs during acute EAE but had little effect on disease course, suggesting that pathogenic T cells expressing other Vβs were producing disease. These findings may have implications for the treatment of multiple sclerosis with Vβ-selective therapy. © 1996 Wiley-Liss, Inc.     

PSGL-1 is dispensible for the development of active experimental autoimmune encephalomyelitis in SJL/J mice

The adhesion molecule P-selectin glycoprotein ligand (PSGL)-1 has been suggested to be involved in the immunopathogenesis of multiple sclerosis (MS). However, in C57BL/6 mice PSGL-1 was found to be dispensible for the development of MOG(aa35-55)-induced experimental autoimmune encephalomyelitis (EAE), an animal model for MS. To study, if involvement of PSGL-1 to EAE pathogenesis can be observed in another common mouse model, we backcrossed PSGL-1(-/-) mice for at least 12 generations into the SJL/J background and compared PLP(aa139-151) induced EAE in PSGL-1(-/-) SJL/J mice versus wild-type SJL/J mice. Here, we demonstrate that PSGL-1(-/-) SJL/J mice exhibited EAE pathogenesis indistinguishable from wild-type SJL/J mice. Our present study underscores and emphasizes previous observations that PSGL-1 is dispensible for EAE pathogenesis.     

Gender differences in experimental autoimmune encephalomyelitis develop during the induction of the immune response to encephalitogenic peptides

Multiple sclerosis (MS) strikes women more often than men. Gender differences in experimental autoimmune encephalomyelitis (EAE) parallel those seen in MS. We utilized the adoptive transfer model of EAE to determine the role of gender on the induction and effector phases of disease. PLP 139–151-sensitized spleen cells from female SJL mice were more effective at transferring disease than male cells. However, there were no gender differences in the frequency of PLP 139–151-specific T cells. PLP 139–151-specific female T cell lines induced more severe disease than male T cell lines. Disease severity was more strongly linked to the sex of the donor T cells, indicating that gender influences the immune response primarily during the induction phase. J. Neurosci. Res. 52:420–426, 1998. © 1998 Wiley-Liss, Inc.     

Functional maturation of proteolipid protein(139-151)-specific Th1 cells in the central nervous system in experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is a widely adopted animal model system for studying human multiple sclerosis that affects the central nervous system (CNS). To understand the underlying pathogenic mechanisms of the autoimmune T cell response, localization, enumeration and characterization of autoreactive T cells are essential. We assessed encephalitogenic proteolipid protein epitope (PLP(139-151))-specific T cells in the periphery and CNS of SJL/J mice using MHC class II I-As multimers during both pre-clinical and clinical phases of PLP-induced EAE in conjunction with T cell function. Our results strongly suggest that PLP(139-151)-specific CD4+ T cells first expand primarily in the CNS-draining cervical lymph nodes and then migrate to the CNS. In the CNS, these PLP-specific CD4+ T cells accumulate, become activated and differentiate into effector cells that produce IFN-gamma in response to the self-peptide.