重组腺病毒载体Ad5-hBDNF-EGFP的构建与鉴定*☆

背景：基因治疗是目前脊髓损伤治疗的方向，目的基因和载体是基因治疗的关键。 目的：构建携带增强型绿色荧光蛋白基因(Enhanced green fluorescence protein, EGFP)标志人脑源性神经营养因子(human brain-derived neurotrophic factor，hBDNF)基因重组腺病毒载体。 设计、时间及地点：单一样本观察，实验于2007-09/2008-06在福建医科大学附属第一医院完成。 材料：感受态大肠杆菌DH-5α购自美国Stratagene公司；pDC316-hBDNF、载体质粒pDC316-mCMV-EGFP、腺病毒骨架质粒pBHGlox_E1,3Cre、腺病毒包装系统AdMax和包装细胞株293购自加拿大Mixcrobix-Biosystems公司。 方法：以pDC316-hBDNF为模板，聚合酶链反应扩增酶切获得hBDNF基因片段，连接到带有EGFP标记基因的载体质粒pDC316-mCMV-EGFP上，构建穿梭质粒pDC316-hBDNF-mCMV-EGFP。利用AdMax包装系统，穿梭质粒与骨架质粒pBHGlox_E1，3Cre共转染293包装细胞, 同源重组产生复制缺陷型重组腺病毒载体Ad5-hBDNF-EGFP，反复感染293细胞扩增病毒后，离子交换法纯化病毒，并测定病毒颗粒数及滴度。 主要观察指标：①hBDNF基因原始质粒聚合酶链反应鉴定。②穿梭质粒pDC316-hBDNF-mCMV-EGFP的构建及鉴定。③重组腺病毒Ad5-hBDNF-EGFP的包装、扩增及纯化。④毒种目的基因的聚合酶链反应鉴定。⑤纯化病毒的滴度测定结果。 结果：经聚合酶链反应鉴定、限制性酶切分析及序列测定，证明已正确构建重组穿梭质粒pDC316-hBDNF-mCMV-EGFP和重组腺病毒载体Ad5-hBDNF-EGFP；扩增纯化后，测得重组腺病毒颗粒数为2.4×1011VP / mL，A260/A280值约为2.0，滴度为0.8×1010 CCID50/ mL。 结论：已成功构建重组腺病毒载体Ad5-hBDNF-EGFP，为hBDNF基因功能及基因治疗的进一步研究奠定实验基础。     

大鼠黏结蛋白聚糖1基因重组腺病毒载体的构建及感染效率☆

背景：重组腺病毒质粒的构建方法主要有体外连接法和同源重组法。同源重组法有操作复杂、耗时长、效率低、纯化难的缺点。体外连接法又不可避免非特异性的基因重组及基因突变。 目的：应用改良体外连接法构建携带黏结蛋白聚糖1基因的重组腺病毒载体，测定其在心肌成纤维细胞中的感染效率。 设计、时间及地点：单一样本实验，于2007-08/2008-02在中山大学附属第二医院林百欣实验中心完成。 材料：穿梭载体pShuttle-CMV（含绿色荧光报告基因）和腺病毒骨架质粒pAdxsi购自诺赛基因组研究中心有限公司。 方法：核苷酸序列鉴定pCMV-Sport6.1-Sdc1质粒；用KpnⅠ + XhoⅠ从质粒pCMV-Sport6.1-Sdc1切出黏结蛋白聚糖1基因片段，亚克隆至pShuttle-CMV中，形成重组穿梭质粒。用I-CeuI + I-SceI双酶切出重组穿梭质粒中CMV-Sdc1片段，亚克隆至腺病毒基因组质粒中，得到重组腺病毒质粒。将重组腺病毒质粒转染293细胞包装获得重组腺病毒AdCMVSdc1，转化体外培养的心肌成纤维细胞。 主要观察指标：用DNA测序、酶切及聚合酶链反应法鉴定重组质粒和病毒，并测定重组腺病毒的滴度和感染效率。 结果：①核苷酸序列分析表明，pCMV-Sport6.1-Sdc1质粒正确携带大鼠黏结蛋白聚糖1 cDNA；黏结蛋白聚糖1基因被克隆于载体pShuttle-CMV上，以KpnⅠ + XhoⅠ双酶切可回收3 kb的克隆片段和5.1 kb的载体片段；重组腺病毒质粒用XhoⅠ酶切得到7个片段而空载体仅得到6个片段。②重组腺病毒质粒在293细胞中包装后产生的重组腺病毒对293细胞有致病作用；提取病毒DNA行聚合酶链反应鉴定可扩增出1.13 kb的特异性片段；用病毒上清多次重复感染293细胞扩增重组腺病毒后，病毒滴度检测达2.0×1011 PFU/mL。③用纯化浓缩后的重组腺病毒以感染复数为100感染心肌成纤维细胞，24 h后所有细胞均表达绿色荧光。 结论：成功构建了携带大鼠黏结蛋白聚糖1基因的重组腺病毒载体，经纯化浓缩后具有较高的滴度，能有效转染心肌成纤维细胞。     

构建人HCN4基因重组腺病毒载体及其对大鼠骨髓间充质干细胞的感染效率

背景：超极化激活及环化核苷酸门控阳离子通道(hyperpolarization-activated cyclic nucleotide-gated cation channel, HCN)基因由于具有不增加诱发心律失常的风险、能够接受自主神经系统调节等优势,成为目前最受关注的生物起搏备选基因。 目的：构建携带人HCN4基因的重组腺病毒载体,并测定其对大鼠骨髓间充质干细胞的感染效率。 设计、时间及地点：细胞-基因学体外实验,于2008-02/09在中山大学附属第二医院林百欣实验中心完成。 材料：SD大鼠10只,由中山大学实验动物中心提供。携带目的基因人HCN4 cDNA的质粒pcDNA3.1-HCN4、人胚肾293细胞、大肠杆菌DH5α由中山大学附属第二医院林百欣实验中心保存。腺病毒穿梭质粒pShuttle-CMV、骨架质粒pAdxsi购自北京诺赛基因组研究中心有限公司。 方法：质粒pcDNA3.1-HCN4用Hind Ⅲ+Xba Ⅰ双酶切后回收HCN4片段,亚克隆至pShuttle-CMV中,得到重组穿梭质粒;I-Ceu Ⅰ+I-Sce Ⅰ双酶切处理pShuttle-CMV-HCN4,回收CMV-HCN4片段,亚克隆至腺病毒骨架载体pAdxsi,得到重组腺病毒质粒;重组腺病毒质粒酶切线性化后,应用脂质体法转染293细胞进行包装扩增,得到重组腺病毒AdHCN4;应用AdHCN4转染大鼠骨髓间充质干细胞。 主要观察指标：重组腺病毒质粒载体的鉴定,重组腺病毒的鉴定及滴度测定,重组腺病毒的感染效率。 结果：构建的重组穿梭质粒pShuttle-CMV-HCN4用Hind Ⅲ+Xho Ⅰ双酶切,得到大小为3 600 bp(HCN4)和5 100 bp (pShuttle-CMV)两个片段,DNA测序结果证实人HCN4基因的全长序列已正确插入到pShuttle-CMV穿梭质粒中;重组腺病毒质粒pAdxsi-CMV-HCN4用XhoⅠ酶切得到7个片段,而作为对照的空腺病毒质粒只得到6个片段;重组腺病毒质粒在293细胞中包装后产生的重组腺病毒对293细胞有致病作用;重组腺病毒AdHCN4 PCR鉴定可见657 bp的阳性扩增条带;经多次重复感染后,病毒滴度检测达2.5×1011PFU/mL。成功转染AdHCN4的大鼠骨髓间充质干细胞可表达绿色荧光蛋白,当病毒感染复数值为800,转染效率最高,达90%。 结论：实验成功构建携带人HCN4基因的重组腺病毒载体,并可在体外有效转染大鼠骨髓间充质干细胞。     

重组腺病毒携带的人GHS-R1a基因在人胚肾293细胞中的表达(英文)

目的构建携带GHS-R1a基因的重组腺病毒,为转基因做准备。方法采用PCR法扩增GHS-R1a基因片段,随后亚克隆至腺病毒穿梭质粒pAdTrack-CMV,形成重组质粒pAdTrack-CMV-GHS-R1a。线性化的pAdTrack-CMV-GHS-R1a和腺病毒骨架质粒pAdEasy1在感受态大肠杆菌BJ5183内同源重组。将线性化重组质粒pAdGHS-R1a转染进HEK293细胞,并用激光共聚焦显微镜、RT-PCR和Western blot技术检测绿色荧光蛋白及GHS-R1a的表达。结果重组质粒pAdGHS-R1a经Pac I酶切后得到一个大于30kb的大片段和一个4.5 kb的小片段,证明重组腺病毒质粒pAdGHS-R1a构建成功。Ad-GHS-R1a转染293细胞后24h能观察到绿色荧光蛋白的表达。RT-PCR和Westernblot均证实GHS-R1a基因能在293细胞中有效表达。结论携带GHS-R1a基因的重组腺病毒构建成功,为进一步研究GHS-R1a的功能提供了基础。     

应用AdEasy腺病毒载体系统构建鼠白细胞介素10基因重组腺病毒★

目的：AdEasy系统可在原核细胞E.coli BJ5183中完成穿梭质粒与骨架质粒之间的高效同源重组，经抗生素培养板筛选重组体，然后转染293细胞获得重组病毒，极大简化了载体的构建过程。实验利用AdEasy腺病毒载体系统构建鼠白细胞介素10基因重组腺病毒。 方法：实验于2006-08/2007-05在青岛大学医学院病毒学实验室完成。①实验材料：清洁级SD大鼠5只，实验过程中对动物的处置符合动物伦理学标准。腺病毒穿梭质粒pAdTrack-CMV，骨架质粒pAdeasy-1，大肠杆菌BJ5183和DH10B，人胚肾293细胞均由青岛大学医学院微生物教研室罗兵教授惠赠。②实验方法：从脂多糖刺激培养的大鼠脾细胞中提取细胞总RNA，应用反转录多聚酶链反应技术扩增白细胞介素10 cDNA，定向亚克隆至pAdtrack-CMV中构建腺病毒穿梭质粒pAdtrack-CMV-IL-10，酶切线性化后转化到含腺病毒骨架质粒pAdEasy的BJ5183大肠杆菌中，获得重组腺病毒质粒pAd-IL-10，Lipofectamin包装转染293细胞，获得重组腺病毒vAd-IL-10，荧光显微镜下观察绿色荧光蛋白的表达。将293细胞接种于96孔板中，每孔1×104个细胞，浓缩后的病毒贮存液按不同比例稀释加至培养板中，测定重组腺病毒滴度。用重组腺病毒vAd-IL-10感染293细胞3 d后，以TRIzol一步法提取细胞总RNA，所获RNA加入DNaseⅠ消化处理可能污染的DNA后，PCR产物行琼脂糖电泳检测白细胞介素10 mRNA的表达。 结果：①重组腺病毒的包装及滴度测定：经限制性内切酶转染293细胞16 h后，荧光显微镜观察到细胞中有GFP荧光，可间接反映目的基因的表达。将病毒上清反复感染293细胞扩增重组腺病毒，通常传至第4~5代于接种病毒后24~48 h，几乎可观察到所有细胞出现荧光，此时收获病毒，重组病毒命名为vAd-IL-10。vAd-IL-10滴度为5.5×108 pfu/mL，vAd-GFP滴度为9.0×108 pfu/mL。②目的基因RT-PCR产物的鉴定：提取重组腺病毒感染293细胞总RNA，RT-PCR扩增后琼脂糖电泳显示特异性580 bp预计大小的片段，表明该重组腺病毒可在293细胞中有效转录。 结论：利用AdEasy腺病毒载体系统成功构建了携带白细胞介素10基因的重组腺病毒载体，并制备出高滴度的重组腺病毒vAd-IL-10，可在293细胞中有效转录。     

大鼠蛋白酪氨酸磷酸酶1B基因靶向RNA干扰重组腺病毒的构建

背景：研究者们早已发现蛋白酪氨酸磷酸酶1B（protein tyrosine phosphatase，PTP1B）可负性调节胰岛素信号转导，但目前制约PTP1B功能研究的重要环节是缺乏有效的工具。 目的：拟构建PTP1B基因靶向RNA干扰重组腺病毒。 设计、时间及地点：体外细胞学基因转染实验，于2007-05/12在解放军第二军医大学长海医院中心实验室完成。 材料：293细胞由中科院上海细胞研究所提供，真核表达质粒psiRNA-PTP1B由上海吉凯生物公司合成，AdEasy-1菌、穿梭载体pAdTrack、大肠杆菌DH5α由本室保存，重组腺病毒PCR鉴定引物由上海生工生物公司设计合成。 方法：由生物公司合成针对大鼠PTP1B mRNA的特异性siRNA真核表达质粒psiRNA-PTP1B，双酶切得到siRNA-PTP1B表达片段，插入pAdTrack载体上，获得转移质粒pAdTrack-siRNA-PTP1B。后者经线性化后，转化含有腺病毒骨架质粒pAdEasy-1的大肠杆菌BJ5183进行同源重组，PacⅠ酶切鉴定，筛选出正确重组腺病毒质粒pAdEasy-siRNA-PTP1B，酶切线性化后转染293细胞包装成重组病毒颗粒。通过反复感染扩增病毒，采用氯化铯密度梯度离心法纯化重组腺病毒。 主要观察指标：荧光显微镜观察绿色荧光蛋白的表达，鉴定重组腺病毒穿梭载体及重组腺病毒是否构建成功。 结果：PacⅠ酶切鉴定证实重组腺病毒质粒pAd-siRNA-PTP1B构建成功，转染293细胞后有绿色荧光蛋白表达。PCR鉴定表明重组腺病毒中含有siRNA-PTP1B片断，成功构建了携带PTP1B干扰RNA的重组腺病毒Ad-siRNA-PTP1B，293细胞扩增纯化后，获得约3.6×109 efu/mL滴度的重组腺病毒。 结论：应用AdEasy-1系统可以高效快速制备表达PTP1B干扰RNA的重组腺病毒。     

以海马神经元为靶细胞Ad-Easy系统快速构建携带PTEN基因的重组腺病毒表达载体

摘要：目的：应用Ad-Easy腺病毒载体系统快速构建携带PTEN (Phosphatase and tensin homolog deleted on chromosome ten)基因的重组腺病毒表达载体,为应用基因技术研究PTEN对缺血性脑损伤后的神经保护作用奠定基础。方法：采用RT-PCR法从大鼠海马神经元扩增获得目的基因PTEN, 克隆入pAdTrack-CMV穿梭质粒（含绿色荧光蛋白（Green fluorescence protein,GFP）基因）形成转移载体pAdTrack-CMV-PTEN,将其在大肠杆菌BJ5183内与腺病毒骨架质粒pAdEasy-1同源重组;重组子酶切鉴定正确后转染HEK293细胞,PCR分析鉴定扩增情况及Western blot检测受腺病毒感染的海马神经元内PTEN蛋白的表达。结果：荧光显微镜检测到受腺病毒感染的HEK293细胞表达GFP,出现明显的细胞病变效应（Cytopathic effect,CPE）,经3轮扩增得到了病毒所需滴度。PTEN蛋白在受感染腺病毒神经元内表达显著增强。结论：利用Ad-Easy系统成功快速的构建了携带PTEN基因的腺病毒表达载体,为研究基因治疗缺血性脑损伤奠定了基础。     

甲胎蛋白特异性小干扰RNA重组腺病毒载体的构建

背景：RNA干扰技术的应用，关键在于能够采用1个有效的基因转移系统将小干扰RNA转入至靶细胞，目前广泛应用的是构建小干扰RNA表达载体。 目的：利用AdMax腺病毒载体系统构建表达人甲胎蛋白-小干扰RNA的腺病毒载体。 设计、时间及地点：开放性实验，于2007-03/10在山东大学附属济南市中心医院中心实验室完成。 材料：穿梭质粒pDC316-EGFP-U6为本元正阳基因技术公司产品；AdMax KitD试剂盒与低代数HEK293细胞为Microbix Biosystems Inc.（Canada）公司产品。 方法：选择针对甲胎蛋白 mRNA的特异性小干扰RNA靶序列，设计合成为相应的双链DNA，并将其与酶切线性化的pDC316-EGFP-U6载体片段连接，构建好的穿梭质粒pDC316-EGFP-U6-AFP-siRNA和腺病毒骨架质粒pBHGlox_E1,3Cre共转染HEK293细胞，同源重组产生重组腺病毒。 主要观察指标：对重组腺病毒进行聚合酶链反应鉴定及扩增、纯化、滴度测定。 结果：构建的穿梭质粒载体经聚合酶链反应鉴定和测序分析，证实与设计一致。重组腺病毒Ad-AFP-siRNA经聚合酶链反应和绿色荧光蛋白表达检测证实构建成功，测定滴度为1.4×109 nfu/L。 结论：实验成功构建了Ad-EGFP-U6-AFP-siRNA重组腺病毒。     

DMT1第四功能区腺病毒表达载体的构建及其功能的初步研究

目的:构建携带DMT1第四功能区基因的重组腺病毒表达载体,并对其功能进行初步探讨。方法:采用AdEasy系统构建携带DMT1第四功能区基因的重组腺病毒载体pAd-DMT1-4,脂质体法转染人胚肾293细胞,包装产生重组腺病毒vAd-DMT1-4。vAd-DMT1-4感染C6胶质瘤细胞后,采用RT-PCR检测目的基因表达,用铁孵育感染重组腺病毒的靶细胞后,测定C6胶质瘤细胞内OH.和MDA含量。结果:PCR、序列测定以及限制性酶切证实vAd-DMT1-4基因正确插入穿梭质粒,并与病毒骨架质粒pAdEasy-1重组,重组腺病毒表达载体构建成功。经293细胞包装获得具有稳定感染性的重组腺病毒vAd-DMT1-4,感染重组腺病毒的C6胶质瘤细胞DMT1-4表达增加。用铁孵育感染重组腺病毒的靶细胞后,C6胶质瘤细胞内OH.和MDA含量有增高的趋势。结论:重组腺病毒vAd-DMT1-4可有效感染C6胶质瘤细胞。     

人AWP1-RFP融合基因重组腺病毒的快速构建及其鉴定

目的 构建人的AWP1与红色荧光蛋白(RFP)融合基因的重组腺病毒载体，为研究AWP1的生物功能提供工具。方法将克隆的AWP1 cDNA插入高效表达RFP的载体pDsRedl-N1的多克隆位点，与RFP序列形成AWP1-RFP融合基因，将该融合基因亚克隆到穿梭载体pAdTrack-CMV和pAdShuttle-CMV中：经酶切鉴定正确后Pine I线形化，与骨架质粒pAdEasy-1共转染大肠杆菌BJ5183以同源重组：用lipofectAMINE将Pac I线形化的同源重组腺病毒载体转染293细胞以包装腺病毒：通过同源重组病毒载体和病毒颗粒基因组的DNA测序及PCR扩增和在293细胞中的表达鉴定重组腺病毒。结果同源重组载体和病毒颗粒的DNA测序和PCR分析显示AWPIcDNA序列正确，感染293细胞获得了AWP1-RFP融合蛋白的高效表达。结论成功构建和包装了人AWP1-RFP融合基因重组腺病毒。     

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.     

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.     

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.     

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.     

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.     

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.