从Th1、Th17细胞极化探讨iMDC负载P258-73肽段干预EAN的机制

目的 观察未成熟髓源树突状细胞负载P258-73肽段干预实验性自身免疫性神经炎的效果,及干预对IL-17、IFN-γ mRNA表达的影响,从Th1、Th17细胞极化的角度探讨其干预机制.方法 P258-73aa负载于体外培养的iMDC,获得P258-73aa-iMDC,用P253-78aa和CFA免疫Lewis大鼠制成EAN动物模型,免疫前7d各组大鼠分别给予PBS、iMDC及P258-73aa-iMDC干预.观察发病情况并作临床评分及病理改变.收集引流淋巴结细胞检测淋巴细胞增殖反应,RT-PCR技术检测大鼠脾脏、淋巴结和坐骨神经中IL-17、IFN-γ mRNA的表达.结果 P258-73aa-iMDC干预组大鼠的平均临床评分、抗原特异性淋巴细胞增殖反应和坐骨神经炎性细胞浸润均降低;IFN-γ 及IL-17 mRNA在脾脏、淋巴结和坐骨神经中的表达也明显降低.结论 P258-73aa-iMDC通过影响IL-17、IFN-γ 的分泌,影响Th1、Th17细胞极化,抑制抗原特异性淋巴细胞增殖,从而减轻EAN的发病,这可能是其诱导免疫耐受的机制之一.

Abstract：

Objective To explore the improving potential of immature myeloid dendritic cell (Imdc) pulsed with P258-73aa peptide (P258-73aa-Imdc) in experimental autoimmune neuritis (EAN) ,and to explore the role of Th1/Th17 cells polarization in this tolerance therapy by detecting the expression of IL-17 and IFN-γ mRNA. Methods P258-73aa21 was pulsed with Imdc in vitro to get P258-73aa-iMDC. Rats of each group were immunized with P253-78aa and CFA. 7 days before immunization, each group was injected with PBS or iMDC or P258-73aa-iMDC respectively. Clinical scores of each group and histopathological changes were evaluated and the lymphocyte proliferation response was assayed; IL-17 and IFN-γ mRNA in spleen,lymph node and sciatic nerves were measured by RT-PCR. Results The P258-73aa-iMDC interferred group had lower average clinical score and suppressed antigen specific lymphocyte proliferation, as well as milder infiltration by the inflammatory cells in sciatic nerves. Meanwhile, the expression of IL-17/IFN-γ mRNA in spleen, lymph node and sciatic nerves were also decreased. Conclusion The protective effect of P258-73aa-iMDC could be associated with the inhibition of lymphocyte proliferation and IL-17, IFN-γ through the polarization of Th1/Th17 cells,which is probably one of the tolerance mechanism of P258-73aa-iMDC in EAN.     

IL-17、RORγt在实验性自身免疫性神经炎中的表达

目的 观察Th17型细胞因子IL-17和Th17细胞特异性转录因子维甲酸受体相关孤儿受体γ的胸腺异构体(RORγt) mRNA在实验性自身免疫性神经炎(EAN)模型中的表达,以探讨Th17细胞在EAN中的作用.方法 用P253-78aa肽段免疫Lewis大鼠,建立EAN模型,观察大鼠发病情况和组织病理改变,并检测淋巴细胞增殖反应,用RT-PCR技术检测IL-17和RORγt在大鼠发病高峰期脾脏、淋巴结和坐骨神经中的表达.结果 EAN组大鼠在第14-16天发病高峰期时平均临床评分为(7.5±1.2),病理学检查可见明显炎性细胞浸润,对P253-78aa的刺激产生强烈淋巴细胞增殖反应,与对照组相比,IL-17和RORγt mRNA在脾脏、淋巴结和坐骨神经中的表达均显著升高(P<0.001).结论 IL-17和RORγt表达上调与EAN的发病相关.     

从IFN-γ／IL-33表达变化探讨EAN中的Th1／Th2细胞极化

目的探讨IFN-γ／和IL-33在实验性自身免疫性神经炎（EAN）发病机制中的作用及EAN中的Th1／Th2细胞极化。方法用P253-78肽段免疫Lewis大鼠,建立EAN模型,观察其发病情况和组织病理改变,并检测淋巴细胞增值反应,用RT-PCR技术检测干扰素γ（IFN-γ）和白介素33（IL-33）在大鼠发病高峰期脾脏、淋巴结和坐骨神经中的表达。结果EAN组大鼠临床表现明显,病理检查可见大量炎性细胞浸润;坐骨神经组织、淋巴结,脾脏中IFN-γ mRNA表达显著升高,IL-33mRNA表达明显减少,其引流淋巴结淋巴细胞对P253-78aa的刺激发生强烈的淋巴细胞增殖反应。结论IFN-γ对EAN发病起促进作用,IL-33对EAN大鼠起保护作用;EAN中Th0细胞向Th1的转化明显增强而向Th2细胞的转化则受到抵制。     

从Th1、Th17细胞除极探讨丙戊酸干预实验性自身免疫性神经炎的机制

目的从Ⅰ型辅助T细胞(Th1)、17型辅助T细胞(Th17)细胞除极角度探讨丙戊酸(VPA)干预实验性自身免疫性神经炎(EAN)的机制。方法实验大鼠随机分为VPA治疗组、EAN组、正常组,应用周围神经髓鞘抗原(P257-81)多肽与完全弗氏佐剂的混合液免疫VPA治疗组和EAN组大鼠。VPA治疗组大鼠于免疫当天至第15天每天腹腔内注射300mg·kg-1丙戊酸钠。观察发病情况,坐骨神经电生理改变及组织病理学变化,检测腹股沟淋巴结中IFN-γ、IL-17 mRNA水平。结果 VPA治疗组的最初发病时间迟于EAN组(P<0.05),其高峰期临床评分显著低于EAN组(P<0.05),坐骨神经复合肌肉动作电位(CMAP)的波幅较EAN组明显升高,潜伏期和时限显著缩短(P<0.05)。髓鞘脱失和炎性细胞浸润较EAN组明显减少(P<0.05)。淋巴结中IFN-γ、IL-17mRNA表达明显下降(P<0.05)。结论 VPA通过影响Th1、Th17细胞除极,使IFN-γ、IL-17分泌下降,从而抑制EAN大鼠的自身免疫反应。     

丙戊酸对实验性自身免疫性神经炎大鼠保护作用的机制

目的观察丙戊酸(VAP)对实验性自身免疫性神经炎(EAN)大鼠的保护作用及其机制。方法实验大鼠随机分为VAP高剂量组、VAP低剂量组、EAN模型组、正常组,应用P2 57-81多肽与完全弗氏佐剂的混合液诱导EAN模型。VAP于免疫当天至第15d每天腹腔内注射。观察各组大鼠发病情况和坐骨神经组织病理学变化,检测外周血中Th17细胞和Foxp3+Treg细胞含量,检测淋巴结中TNF-α、IFN-γ、IL-17、TGF-βmRNA表达。结果 VAP高剂量组的最初发病时间迟于EAN组(P<0.05),其高峰期临床评分显著低于EAN组(P<0.05),坐骨神经炎性细胞浸润较EAN组明显减少;VAP高剂量组和低剂量组外周血中Th17细胞比例较EAN组显著减少(P<0.05),Foxp3+Treg细胞比例较EAN组显著增加(P<0.05),淋巴结中促炎细胞因子TNF-α、IFN-γ及IL-17mRNA表达与EAN组比较明显下降(P<0.05),VAP高剂量组抑炎细胞因子TGF-βmRNA表达与EAN组比较明显升高(P<0.05)。结论 VAP对EAN有治疗作用,这种作用可能与其能够增加Foxp3+Treg细胞和抑炎细胞因子TGF-β含量、减少TH17细胞含量和促炎细胞因子的表达有关。     

Th1/Th2型细胞因子在实验性自身免疫性神经炎发病机制中的作用

目的 建立P2多肽诱导的实验性自身免疫性神经炎(EAN)大鼠模型,探讨Th1/Th2型细胞因子在EAN发病机制中的作用.方法 实验组用100 μg或200 μg P257-81多肽加完全弗氏佐剂(FCA)免疫Lewis大鼠,对照组单用FCA免疫,致敏后每日对大鼠进行临床评分,比较高峰期最高评分.致敏第14天测定淋巴结细胞培养液上清干扰素(IFN)-γ、IL-4及IL-10的含量,并进行坐骨神经病理学检查.结果 实验大鼠瘫痪高峰期最高评分P257-81 200 μg组(3.6±0.3)显著高于100 μg组(2.2±0.6,P＜0.01);P257-81 200 μg组大鼠病程显著长于100 μg组;IFN-γ含量,两组实验大鼠均显著高于对照组[分别为(530.6±91.7)、(806.3±132.4)和(35.0±5.9)pg/ml,均P＜0.01],而P257-81 200 μg组显著高于100 μg组(P＜0.01);IL-4和IL-10含量,P257-81 100 μg组均显著高于对照组(均P＜0.01),P257-81 200 μg组显著低于对照组(P＜0.05,P＜0.01);坐骨神经病理可见EAN急性期以炎性细胞浸润为主,P257-81 200 μg组慢性期无炎性细胞浸润,而表现为多发性局灶性脱髓鞘和神经纤维崩解未恢复.结论 EAN临床表现随致敏原P257-81多肽剂量增加而加重;在EAN急性期,IFN-γ水平与EAN临床表现大致平行;EAN疾病具有自限性可能与IL-4和IL-10水平增高有关,而疾病迁延可能与IL-4和IL-10水平降低有关.     

Rho激酶抑制剂对OX40及其配体mRNA在实验性变态反应性神经炎中表达的影响

目的 研究Rho激酶(ROK)抑制剂对OX40及其配体(OX40L)mRNA在实验性变态反应性神经炎(experimental allegic neuritis,EAN)大鼠坐骨神经、脾脏、外周血和淋巴结中表达的影响.方法 54只Lewis大鼠随机分为EAN模型组、EAN+ROK抑制剂干预组和完全弗氏佐剂对照(CFA)组.分别在免疫后第9、17、26天处死动物,取其坐骨神经根、脾脏、外周血单个核细胞和淋巴结,采用逆转录PCR(RT-PCR)技术检测OX40和OX40L mRNA在各组织的表达水平.结果 EAN+ROK抑制剂组大鼠OX40 mRNA在坐骨神经中第9、17、26天的表达分别为0.266±0.031、0.298±0.024和0.113±0.018;在淋巴结中第9、17、26天的表达分别为0.453±0.030、0.496±0.100和0.220±0.016;OX40L mRNA在坐骨神经中第9、17、26天的表达分别为0.247±0.018、0.298±0.026和0.165±0.013;在淋巴结中第9、17、26天的表达分别为0.283±0.027、0.306±0.011和0.161±0.012.与EAN组比较,OX40和OX40L mRNA的表达明显降低(t=2.24～4.89,P＜0.05),坐骨神经炎性细胞浸润和脱髓鞘减轻.CFA组大鼠无症状.结论 ROK抑制剂可以减轻EAN发病程度,抑制OX40/OX40L的表达可能是其作用机制之一.     

Tα146-162-iMDC对实验性自身免疫性重症肌无力中B细胞活化的影响

目的 通过测定B淋巴细胞激活因子（BLyS）及TNF家族B细胞活化因子受体（BAFF-R）在实验性自身免疫性重症肌无力（EAMG）小鼠模型脾脏及淋巴结中的表达,探讨不成熟树突状细胞（iMDC）负载免疫优势肽段Tα146-162对MG中B细胞活化的影响。方法 诱导小鼠骨髓细胞分化为iMDC并以其负载Tα146-162。将C57BL/6J小鼠随机分为发病组（A）、干预组（B）、正常对照组（C）,每30d以电鳗来源的乙酰胆碱受体（TAChR）免疫A组和B组,共3次,每次免疫后3d以Tα146-162-iMDC皮下注射B组。第90天终止实验,用RT-PCR法检测脾脏BLyS、BAFF-R mRNA的表达。结果 （1）A组模型成功率75%,平均临床评分1.67分,B组分别为25%和0.33分,差异有统计学意义（P〈0.01）。（2）脾脏中BLyS mRNA的表达水平A组较C组明显上调（P〈0.01）,B组亦高于C组（P〈0.05）,但相对A组降低（P〈0.01）。（3）脾脏中BAFF-R mRNA的表达水平A组及B组相对C组下调（P〈0.05）,且A组显著低于B组（P〈0.05）。结论 应用Tα146-162-iMDC干预能降低EAMG的发病率及发病程度,其机制可能与BLyS/BAFF-R调控的B细胞活化密切相关。     

干扰素-β对实验性自身免疫性神经炎治疗作用的组织病理学和免疫学研究

目的:探讨干扰素β-(interferonβ-,IFNβ-)对实验性自身免疫性神经炎(experimentalautoimmuneneuritis,EAN)的治疗作用。方法:从牛坐骨神经提取髓鞘碱性蛋白(myelinbasicprotein,MBP)制备免疫原注射入豚鼠双后足垫诱导EAN模型。将90只健康、成年、雄性豚鼠随机分为正常对照组、IFN-β治疗EAN组、磷酸盐缓冲生理盐水(phosphatebuffedsaline,PBS)治疗EAN组,每组30只。免疫后第12天给予IFN-β或PBS腹腔注射,分别在治疗后7和14d对各组大鼠进行组织病理学和免疫学指标的检测。结果:与PBS治疗组相比,接受IFN-β治疗的豚鼠临床评分较低,坐骨神经干脱髓鞘程度轻,炎细胞浸润减少;脾脏单个核细胞IFN-γ、TNF-α的分泌量减少,IL-4、TGF-β的分泌增多。结论:IFN-β能改善EAN临床症状,促进疾病恢复,具有一定的治疗作用。     

树突状细胞负载Tα146-162对EAMG中Crry、CD59a、CD59b mRNA表达的影响

目的观察实验性自身免疫性重症肌无力(EAMG)小鼠肌肉中Crry、CD59a、CD59b mRNA的表达和Tα146-162-iMDC诱导免疫耐受对三者的影响。方法用GM-CSF、IL-10诱导骨髓细胞分化、增殖为不成熟的髓源性树突状细胞(iMDC),之后将Tα146-162负载其上(Tα146-162-iMDC)。健康雌性C57BL/6小鼠30只,随机分为模型组(A组,12只)、干预组(B组,12只)和正常对照组(C组,6只),A、B组小鼠用电鳗乙酰胆碱受体(T-AChR)免疫诱导EAMG,B组小鼠用Tα146-162-iMDC干预。按照Christadoss标准对小鼠进行EAMG评分,3H-TdR标记检测淋巴细胞增殖反应,RT-PCR方法检测Crry、CD59a、CD59b mRNA表达。结果A、B组小鼠平均临床评分分别是1.67±1.15vs0.33±0.65(P<0.01),发病率分别是75%vs25%(P<0.05)。B组较A组小鼠抗原特异性淋巴细胞增殖反应明显降低(P<0.01)。A组较C组小鼠Crry mRNA、CD59a mRNA表达明显降低(P<0.05);B组较A组Crry mRNA、CD59a mRNA表达明显升高(P<0.05),B组CD59a mRNA与C组相比无显著性差异(P>0.05)。3组小鼠肌肉中均检测不到CD59b mRNA。结论Crry mRNA、CD59a mRNA表达减少可能参与EAMG发生,CD59b可能与EAMG发病无关;Tα146-162-iMDC上调小鼠肌肉中Crry、CD59a mRNA表达及抑制抗原特异性淋巴细胞增殖反应可能是其阻抑EAMG发生的机制之一。     

Epilepsy and anomalies of neuronal migration: MRI and clinical aspects

Neuronal migration disorders are the result of disturbed brain development. In such disorders, neurons are abnormally located. In diagnosing these conditions, magnetic resonance imaging is superior to any other imaging technique. This enables us to improve our knowledge of the clinical correlates of neuronal migration. With reference to migrational disorder, a retrospective study of all 303 patients with epileptic seizures referred for magnetic resonance imaging during a 3-year period was performed, 13 patients (aged 12-41, mean age 27) were identified. They represent 4.3% of the entire study group. Of the patients with known epilepsy, 6.7% and of the mentally retarded, 13.7% had migrational disorders. Four patients had schizencephaly as the dominant finding, one was classified as hemimegalencephaly, 2 had isolated heterotopias, and 6 had localized pachy- and/or poly-microgyria. The clinical pictures are complex. Ectopias of grey matter are recognised foci of epilepsy, but from an epileptological and a clinical viewpoint little attention has been given to these disorders. The present study shows that malmigration is not rare in epilepsy patients, especially not in the mentally retarded.     

Classification of methods in transcranial Electrical Stimulation (tES) and evolving strategy from historical approaches to contemporary innovations

Transcranial Electrical Stimulation (tES) encompasses all methods of non-invasive current application to the brain used in research and clinical practice. We present the first comprehensive and technical review, explaining the evolution of tES in both terminology and dosage over the past 100 years of research to present day. Current transcranial Pulsed Current Stimulation (tPCS) approaches such as Cranial Electrotherapy Stimulation (CES) descended from Electrosleep (ES) through Cranial Electro-stimulation Therapy (CET), Transcerebral Electrotherapy (TCET), and NeuroElectric Therapy (NET) while others like Transcutaneous Cranial Electrical Stimulation (TCES) descended from Electroanesthesia (EA) through Limoge, and Interferential Stimulation. Prior to a contemporary resurgence in interest, variations of transcranial Direct Current Stimulation were explored intermittently, including Polarizing current, Galvanic Vestibular Stimulation (GVS), and Transcranial Micropolarization. The development of these approaches alongside Electroconvulsive Therapy (ECT) and pharmacological developments are considered. Both the roots and unique features of contemporary approaches such as transcranial Alternating Current Stimulation (tACS) and transcranial Random Noise Stimulation (tRNS) are discussed. Trends and incremental developments in electrode montage and waveform spanning decades are presented leading to the present day. Commercial devices, seminal conferences, and regulatory decisions are noted. We conclude with six rules on how increasing medical and technological sophistication may now be leveraged for broader success and adoption of tES.     

Hepatic Considerations in the Use of Antiepileptic Drugs

Summary: Virtually all of the major antiepileptic drugs (AEDs) can cause hepatotoxicity, although fatal hepatic reactions are rare. The mechanisms, incidences, and risk profiles for such reactions differ from drug to drug. With carbamazepine and phenytoin, hepatotoxicity may be due to drug hypersensitivity. Although the profiles of patients at risk have not been well-defined for these two antiepileptic drugs, it would appear from reports in the literature that older adolescents and adults are at higher risk than children of developing serious or fatal hepatotoxicity. Once hepatotoxicity develops, mortality rates are 10–38% with phenytoin and 25% for carbamazepine. The risk profile for valproate fatal hepatotoxicity has been more clearly defined. Those at primary risk of fatal hepatic dysfunction are children under the age of 2 years who are receiving multiple anticonvulsants and also have significant medical problems in addition to severe epilepsy. The risk is considerably lower for patients over the age of 2 years on valproate monotherapy. In contrast to the risk profile with other AEDs, adults receiving valproate as monotherapy have the lowest risk of hepatotoxicity. Fatal hepatic dysfunction coincident with valproate may be the result of aberrant drug metabolism. Concomitant use of AEDs that induce microsomal P450 enzymes (e.g., phenytoin and phenobarbital) may enhance the production of a toxic metabolite, and hence the greater risk of hepatotoxicity with polypharmacy.     

Vascular Malformations and Epilepsy: Clinical Considerations and Basic Mechanisms

Summary: Vascular malformations (VMs) are associated with epilepsy. The natural history of the various VMs, clinical presentation, and tendency to provoke epilepsy determine treatment strategies. Investigations have probed the mechanisms of epileptogenesis associated with these lesions. Electrophysiologic changes are associated with epileptogenic cortex adjacent to VMs. Putative pathophysiologic mechanisms of epileptogenesis include neuronal cell loss, glial proliferation and abnormal glial physiology, altered neurotransmitter levels, free radical formation, and aberrant second messenger physiology.     

Neonatal Seizures: Problems in Diagnosis and Classification

Summary: The clinical identification of neonatal seizures is critical for the recognition of brain dysfunction; however, diagnosis is often difficult because of the poorly organized and varied nature of these behaviors. Current classification systems are limited in their ability to communicate motor, autonomic, and electroencephalo-graphic features of seizures precisely and to provide a basis for uniform effective diagnosis, therapy, and determination of prognosis. Recent investigations of neonates, utilizing bedside electroencephalographic/polygraphic/ video monitoring techniques, have provided the basis for improved diagnosis and classification of seizures in the newborn. These studies have demonstrated that not all clinical phenomena currently considered to be seizures require electrocortical epileptiform activity for their initiation or elaboration. In addition, the specific clinical character of the phenomena considered to be seizures, the clinical state of the infant, and the character of the EEG indicate the probable pathophysiological mechanisms involved and suggest probable etiologies, prognosis, and therapy. Similarities between animal models that demonstrate reflex physiology and neonates with motor automatisms and tonic posturing suggest that these clinical behaviors may not be epileptic in origin but, rather, primitive movements of progression and posture mediated by brainstem mechanisms. Although not all clinical behaviors currently considered to be neonatal seizures may have similar pathophysiological mechanisms, they are clinically significant because they all indicate brain dysfunction.     

Valproate Monotherapy in the Management of Generalized and Partial Seizures

Summary: For decades, therapeutic tradition has promoted the concept of polypharmacy in the management of epilepsy. In recent years, however, studies have shown that, for most patients, monotherapy can provide comparable or better seizure control than administration of multiple anticonvulsants, while diminishing the potential for adverse reactions, drug interactions, and poor compliance. Valproate is an important monotherapeutic agent that is highly effective in the control of idiopathic primary and secondarily generalized epilepsies, and partial seizures that do not generalize. Comparative studies have found that valproate is at least as effective as phenytoin and carbamazepine in the treatment of generalized and partial seizures. Given the similar efficacy, other factors such as pharmacokinetics and side effects may therefore determine anticonvulsant selection for monotherapy.     

Carbamazepine Efficacy and Utilization in Children

Summary: Carbamazepine is effective for preventing partial and generalized tonic-clonic seizures in children. Although absence epilepsies are more common in children than adults, an estimated 80% of children with epilepsy have seizure types or epilepsies that are potentially responsive to carbamazepine. The differential diagnosis of ictal staring is an especially important issue in children because absence and atypical absence seizures are more prevalent in children than adults. Age-related pharmacokinetic differences and drug interactions are major considerations in children. On average, children have higher clearance rates of carbamazepine, shorter half-lives, and higher ratios of carbamazepine-10, 11-epoxide to carbamazepine than adults. In addition, children with severe epilepsy are more likely to require multiple-drug therapy, which can lead to complex drug interactions. When carbamazepine is administered along with valproate, drug protein binding interactions can cause intermittent side effects.     

Un nouveau cadre conceptuel de travail pour la psychiatrie

In an attempt to place psychiatric thinking and the training of future psychiatrists more centrally into the context of modern biology, the author outlines the beginnings of a new intellectual framework for psychiatry that derives from current biological thinking about the relationship of mind to brain. The purpose of this framework is twofold. First, it is designed to emphasize that the professional requirements for future psychiatrists will demand a greater knowledge of the structure and functioning of the brain than is currently available in most training programs. Second, it is designed to illustrate that the unique domain which psychiatry occupies within academic medicine, the analysis of the interaction between social and biological determinants of behavior, can best be studied by also having a full understanding of the biological components of behavior.     

Special Pharmacokinetic Considerations in Children

Summary: Pediatric patients have greater degrees of pharmacokinetic variability and unpredictability than adults. This variability results from the effects of pharmacogenetics, age and growth, prior and current comedication, and disease. Newborns with seizures have the least predictable dosage requirements, and their needs change as drug-eliminating mechanisms mature in the neonatal period. Infants have the highest relative capacities to eliminate antiepileptics of any age group and require the largest relative doses. In addition to age-related trends, children demonstrate the same drug-specific, pharmacokinetic phenomena that adults do, including nonlinear phenytoin elimination, nonlinear valproate binding, and autoinduction of carbamazepine. Intercurrent illness and drug interactions further modify the age-related pharmacokinetic patterns in children and make dosage requirements even more unpredictable. Recent studies have shown that febrile illness can affect drug elimination, sometimes decreasing drug levels by 50% or more. Intermittent treatment with benzodiazepines administered either orally or rectally can be an important adjunct and help minimize this type of problem for children with marginally controlled epilepsy. Intermittent benzodiazepines are also helpful for children who have febrile seizures and who need only occasional antiepileptic protection.