赖型钩端螺旋体溶血素基因HlyX的真核表达及基因免疫

目的 在哺乳动物细胞中表达含有赖型钩端螺旋体溶血素基因HlyX的重组质粒,并检测其作为DNA疫苗诱导小鼠体液免疫应答的效果.方法 以赖型钩端螺旋体全基因组为模板,PCR扩增出目的基因HlyX,以质粒pcDNA3.1为载体,双酶切构建重组质粒,转化大肠杆菌DH5α,双酶切、PCR及测序鉴定重组质粒.将构建成功的重组质粒转染COS-7细胞,通过RT-PCR、Western blot鉴定目的基因的表达.将重组质粒免疫BALB/c小鼠,每两周1次,共3次,ELISA法检测小鼠的体液免疫应答水平.结果 扩增出全长约1100 bp的HlyX基因,重组质粒经双酶切、PCR及测序鉴定表明重组质粒构建成功.RT-PCR检测显示重组质粒转染组能扩增出约1100 bp的目的基因片段,Western blot分析可见在相对分子质量(Mr)40×103左右出现特异性的目的条带.DNA免疫后诱导小鼠产生了较高的抗体水平(1∶6561～1∶19 683).结论 赖型钩端螺旋体溶血素基因HlyX真核表达质粒能够诱导小鼠产生高滴度的特异性抗体,为其作为钩体DNA疫苗的研究和开发奠定了基础.     

CVB3-VP1基因免疫诱导特异性抗病毒免疫应答及保护作用

目的 :构建表达柯萨奇病毒B3(CVB3)主要包膜蛋白VP1的基因疫苗 ,并研究该疫苗诱导CVB3特异性免疫应答及免疫保护的作用。方法 :抽提CVB3RNA ,以RT PCR扩增VP1基因 ,克隆于真核表达载体 pcDNA3中 ,构建质粒pcDNA3 VP1。将该质粒转染Hela细胞 ,观察其表达情况 ;以 5 0 μgpcDNA3 VP1质粒DNA肌注免疫BALB/c小鼠 3次 ,检测CVB3特异性体液和细胞免疫应答。间隔 4wk以 5×LD50 的CVB3攻击小鼠 ,观察攻击后小鼠的存活情况。结果 :构建了重组质粒 pcDNA3 VP1,并在体外获得有效表达。以该质粒肌肉免疫BALB/c小鼠 ,可诱生高水平的IgM和IgG ,VP1多肽特异性淋巴细胞增殖反应及CTL活性均显著高于 pcDNA3免疫的对照组。病毒攻击试验表明 ,pcDNA3 VP1免疫组33.3%小鼠可长期存活 ,其心肌组织未见明显的病理学改变 ;而对照小鼠平均仅存活 6 .7d ,心肌显示大量的局灶性坏死和炎性细胞浸润。结论 :pcDNA3 VP1免疫可诱生CVB3特异性体液及细胞免疫应答 ,保护免疫小鼠抵抗CVB3的致死性攻击     

丙型肝炎病毒核心蛋白DNA免疫初步研究

目的　观察HCV核心蛋白基因的DNA免疫效果。方法　将HCV核心蛋白基因插入真核表达载体pcDNA3.1( ) ,构建重组质粒pcDNA3.1 c。在证明该重组质粒可在哺乳动物COS7细胞中表达的基础上 ,用重组质粒 10 0 μg免疫小鼠 ,同时设立空白质粒组和PBS组两组对照 ,初次免疫后 4周、8周各进行一次加强免疫。小鼠体液免疫反应和T淋巴细胞增殖检测分别采用间接免疫荧光法和MTT法。结果　pcDNA3.1 c可在COS7细胞内表达HCV核心抗原 ,接种于Balb c小鼠能有效诱导体液和细胞免疫应答。结论　重组质粒pcDNA3.1 c对于丙型肝炎防治具有潜在价值     

SARS冠状病毒S基因重组DNA诱导的体液免疫反应

目的 以严重急性呼吸综合征冠状病毒（SARS-CoV）密码子优化的S、S1和S2基因分别构建其真核表达质粒，免疫BALB／c小鼠，以初步评价其诱导特异性体液免疫的效果。方法将人工合成密码子优化的S、S1和S2基因克隆入pcDNA4／HisMax-TOPO表达载体，重组质粒转染293T细胞，Western blot和免疫组化检测其真核表达，重组质粒免疫BALB／c小鼠，酶联免疫（ELISA）检测抗S蛋白抗体，伪病毒中和试验、细胞融合抑制试验检测中和抗体。结果3种重组质粒均可在真核细胞中获得表达，免疫小鼠后可诱导针对S蛋白的特异性抗体，抗体在12周观察期内呈持续上升趋势；其中，仅S和S1蛋白重组质粒能够诱导中和抗体的产生，以S蛋白的效价为高。结论密码子优化S和S1蛋白重组质粒可以有效诱导BALB／c小鼠产生中和抗体，其抗体可能具有阻断SARS-CoV侵袭易感细胞的能力。该结果为进一步研究SARS-CoV DNA疫苗提供了参考依据。     

小鼠β防御素2与柯萨奇病毒B3 VP1融合基因疫苗的构建和免疫效果研究

目的:构建小鼠β-防御素2(Mouse beta-defensin-2,mBD2)与柯萨奇病毒B组3型(Coxsackievirus B3,CVB3)VP1融合基因疫苗,并观察其对小鼠的免疫效果.方法:克隆mBD2基因,构建重组质粒pcDNA3/mBD2和pcDNA3/mBD2-L-VP1.将4～6周龄BALB/c雄性小鼠随机分为5组,分别于股四头肌注射PBS(A组)、pcDNA3(B组)、pcDNA3/mBD2(C组)、pcDNA3/VP1(D组)、pcDNA3/mBD2-L-VP1(E组),每次接种剂量100 μg/只,3周免疫1次,共3次.每次免疫后第14天眼眶静脉取血,用微量中和试验滴定血清中和抗体效价.第三次免疫后第21天,每组随机取3只小鼠,制备脾淋巴细胞悬液,用CCK-8细胞计数法检测特异性CTL的杀伤活性;每组取3只小鼠以3LD50 CVB3病毒攻击,第7天取血处死,检测血清病毒滴度.结果:成功克隆了mBD2基因,构建了pcDNA3/mBD2和pcDNA3/mBD2-L-VP1两种重组质粒;D组和E组血清中和抗体滴度随免疫次数增加而提高(P＜0.01);E组血清中和抗体滴度和脾细胞特异性CTL杀伤活性显著高于其他组(P＜0.01),血中CVB3病毒滴度显著低于其他组(P＜0.01).结论:pcDNA3/mBD2-L-VP1能诱导小鼠对CVB3产生较强的体液免疫和细胞免疫,能有效抑制病毒在体内增殖.     

两株登革2型病毒E基因重组质粒免疫BALB/c小鼠产生特异性抗体水平的比较

目的 用含登革2型病毒(Dengue type 2 virus,DEN2)B株和NGC株E基因部分序列pcDNA3.1重组质粒初次和加强免疫BALB/c小鼠,观察免疫小鼠体液免疫应答的差异.方法 用两株含DEN2 E基因部分序列(1～476 bp)的pcDNA3.1重组质粒与含有佐剂的重组质粒共同免疫BALB/c小鼠,初次免疫后第14天、28天分别加强免疫1次,共免疫3次.收集初次免疫后第14、28、42、70和98天外周血标本,间接ELISA法测定小鼠血浆特异性IgM/IgG类抗体水平,细胞病变抑制法检测特异性抗体水平.结果 不同DEN2毒株E基因部分序列的pcDNA3.1重组质粒初次和加强免疫BALB/c小鼠诱导特异性IgM、IgG类抗体的产生存在差异,B株重组质粒加强免疫小鼠后特异性抗体效价水平较高并持续较长时间.结论 DEN2两毒株E基因部分序列重组质粒免疫小鼠后诱生的特异性抗体类别、水平存在差异.     

编码TCR可变区的DNA免疫诱导BALB/C小鼠调节性免疫应答

目的：研究编码卵清白蛋白(OVA)T细胞克隆TCR Vα5．2或Vβ2.1的DNA免疫正常小鼠诱发调节性免疫应答。方法：用重组质粒pcDNA3．1-Vα5．2或pcDNA3．1-Vβ2.1免疫BALB／C小鼠,RT-PCR证实重组质粒能在体内和体外真核细胞中转录。^3H-TdR掺入法分析细胞增殖,^3H-TdR标记的靶细胞检测杀伤T细胞的杀伤效应,免疫荧光法分析血清中特异抗体水平和Vβ2^ T细胞数量。结果：pcDNA3．1-Vα．2或pcDNA3．1-Vβ2．1能在体内和体外真核细胞中转录,重组质粒免疫BALB／C小鼠能诱导产生特异性体液免疫应答、T细胞增殖反应及对靶细胞CTL反应,但pcDNA3．1-Vβ2．1免疫没有导致Vβ2^ T克隆清除,而是细胞活性部分封闭。结论：编码TcR Vα5．2或Vβ2．1的DNA免疫能诱导正常BALB/C小鼠调节性免疫应答。     

恙虫病东方体Karp株47kD蛋白基因的表达及DNA免疫初步研究

目的　观察恙虫病东方体Karp株相对分子质量 (Mr)为 47× 10 3 蛋白基因的DNA免疫效果。方法　将恙虫病东方体Karp株Mr 为 47× 10 3 的蛋白基因插入真核表达载体pcDNA3.1( +) ,构建重组质粒pcDNA3.1 47。在证明该重组质粒可在哺乳动物COS7细胞中表达的基础上 ,单独和将其与Mr 为 40× 10 3 重组蛋白联合免疫小鼠。在每次加强免疫之后 10d ,检测小鼠体液和细胞免疫反应。结果　质粒DNA和蛋白联合免疫组所诱导的抗体水平和脾淋巴细胞增殖反应均高于重组质粒DNA和蛋白单独免疫组 ,而第二次加强免疫后重组质粒诱导脾淋巴细胞的增殖情况则显著弱于其它免疫组 (P <0 .0 5 )。结论　重组质粒pcDNA3 .1 47和重组Mr 为 47× 10 3 蛋白在刺激小鼠免疫反应上具有相互加强作用。     

乙肝核酸疫苗诱导BALB/C小鼠的CTL免疫应答

目的：研究携带HBsAg基因的载体质粒pcDHBs诱导小鼠CTL应答效果。方法：将HBsAg基因连接到真核表达载体pcDNA3．1上，构建成载体质粒pcDHBs。将纯化后的质粒pcDRBs和pcDNA3．1肌肉注射免疫小鼠，眼眶采血检测血清中抗体水平。用质粒pcDHBs转染P815细胞制备乙肝疫苗诱导BALB／C小鼠CTL活性检测的靶细胞。免疫后，取脾细胞，按效靶比为10：1、25：1、50：1进行CIL杀伤检测。结果：免疫pcDHBs疫苗后，检测到小鼠血清中的RBsAb。用pcDHBs进行转染的P815细胞能够检测到HBsAg基因片段和蛋白质抗原的表达。用pcDHBs免疫组小鼠的CTL杀伤率均明显高于pcDNA3．1免疫组。结论：质粒pcDHBs作为核酸疫苗能够诱导小鼠体液免疫应答和CTL免疫应答。     

犬腺病毒DNA疫苗的免疫实验研究

目的研究犬腺病毒DNA疫苗pVAX1-CpG-Loop刺激机体产生免疫应答的效果。方法采用2种不同的免疫途径和免疫方案免疫Balb/c小鼠;免疫后每周断尾采血,分离血清测定血清IgG抗体效价;采用MTT和CCK-8方法检测免疫小鼠的T淋巴细胞增殖活性,EIA试剂盒测定IFN-γ的浓度。结果所有接种DNA疫苗的小鼠均产生了针对抗原病毒的特异性IgG抗体。细胞学试验提示构建的犬腺病毒DNA疫苗也可诱导小鼠产生细胞性免疫应答,重组质粒-蛋白联合的免疫方案在刺激机体细胞免疫应答方面优于单一重组质粒。结论以上结果提示犬腺病毒DNA疫苗pVAX1-CpG-Loop既能诱导小鼠产生特异性的体液免疫应答,也诱导小鼠产生了细胞免疫应答,对于机体具有一定的免疫保护效果。     

Schizophrenia in a North Swedish geographical isolate, 1900–1977. Epidemiology, genetics and biochemistry

Over 200 schizophrenic patients belonging to three major and interrelated pedigree complexes have been investigated over the past 30 years in a North Swedish geographically isolated population, presently numbering about 6,000. An intensive investigation of a number of biochemical correlates and genetic markers in a few selected families belonging to one of the major pedigrees has indicated new strategies for the current research program.
Schizophrenia, as defined operationally, is significantly associated with decreased activities of two enzymes (1) blood platelet monoamine oxidase, (2) plasma dopamine-β-hydroxylase, and (3) with the genetic marker Gc² (group specific antigen). Both enzymes are subject to genetic variation. A positive score for linkage between schizophrenia and low plasma DBH activity has been calculated, but, so far, available data are insufficient for discrimination between linkage and partial contribution of genetically controlled low plasma DBH to the pathogenesis of the disease. Alternatively, both mechanisms could be involved.
As a model for continued research, schizophrenia is explained as based on a double dominant-recessive genotype (Aabb), representing a vulnerability which in about 50 % of cases develops into clinical schizophrenia. It is suggested that the dominant mutation (A) operates on or affects MAO activity, and that the recessive genotype (bb) is instrumental in low variates of DBH activity and very likely such variates within the normal range of physiological variation. Moreover, it is suggested that the combined effects of MAO- and DBH-reduced efficiency on the metabolism of e.g. dopamine could be an essential pathogenic mechanism for the schizophrenic illness which is segregating in this population.     

Renal dysplasia and asplenia in two sibs

A family is reported in which two sibs, one male and the other female, both died within 24 hours of birth with enlarged polycystic kidneys. Postmortem histology in the second child showed gross renal dysplasia. In both children the pancreas was enlarged, nodular and cystic but the liver appeared macroscopically normal. In the second child, histological examination confirmed pancreatic fibrosis with cystic dilation of ducts, but showed portal fibrosis with bile duct proliferation in the liver.
This combination of findings is very reminiscent of those in a girl and her brother reported by Ivemark et al. (1959). The children reported here also showed absence or hypoplasia of the spleen, cardiac anomalies and other features of the Ivemark syndrome (Ivemark 1955), a quite different, usually sporadic, congenital disorder. It is suggested that the children described here have a distinct lethal congenital disorder, probably inherited in an autosomal recessive manner.     

The dominant diseases of modernized societies as omega-3 essential fatty acid deficiency syndrome: Substrate beriberi

About 1900, modern food selection and processing caused widespread $\text{epidemics}$ of the B vitamin deficiency diseases of beriberi and pellagra which, for genetic reasons, often expressed as different diseases ranging from bowel and heart disease to dermatoses and psychoses. But the B vitamins merely help convert essential fatty acids (EFA) into the prostaglandin (PG) tissue regulators and it now turns out that, through hydrogenation, milling and selection of w3-poor southern foods, we have also been systematically depleting, by as much as 90%, a newly discovered trace Nordic EFA (w3) of special importance to primates and sole precursor of the PG3(4) series, even as a concurrent fiber deficiency increases body demand for EFA. Since substrate EFA is processed by many B vitamin catalysts, an EFA deficiency will mimic a $\text{panh}$ypovitaminosis B, i.e., a mixture of $\text{substrate}$ beriberi and $\text{substrate}$ pellagra resembling vitamin beriberi and pellagra but exhibiting as even more diverse $\text{endemic}$ disease. This would consitute a second stage of the Modern Malnutrition and explain why some workers now hold the dominant diseases of modermized societies to be new, nutritionally based, pellagraform yet lipid-related and to range, once again, from heart disease to psychosis. It is an assumption that our dominant diseases are unrelated to each other or are merely revealed by our diagnostic acumen and therapeutic success; and that hydrogenating millions of tons of food oils annually, to destroy the rancidity producing w3-EFA, is safe for $\text{primates}$. Extensive beriberiform disease is reported here in 32 typical cases taken from medical practice which responds strikingly to linseed oil supplements (60% w3-EFA) in confirmation of identical results in Capuchins.     

HLA in North Colombia Chimila Amerindians

HLA-A,-B,-C,-DRB1 and -DQB1 alleles have been studied in Chimila Amerindians from Sabana de San Angel (North Colombian Coast) by using high resolution molecular typing. A frequent extended haplotype was found:HLA-A*24:02-B*51:10-C*15:02-BRB1*04:07-DQB1*03:02 (28.7%) which has also been described in Amerinndian Mayos Mexican population (Mexico, California Gulf, Pacific Ocean). Other haplotypes had already been found in Amerindians from Mexico (Pacific and Atlantic Coast), Peru (highlands and Amazon Basin), Bolivia and North USA. A geographic pattern according to HLA allele or haplotype frequencies is lacking in Amerindians, as already known. Also, five new extended haplotypes were found in Chimila Amerindians. Their HLA-A*24:02 high frequencies characteristic is shared with aboriginal populations of Taiwan; also, HLA-C*01:02 high frequencies are found in New Zealand Maoris, New Caledonians and Kimberly Aborigines from Australia. Finally, this study may show a model of evolutionary factors acting and rising one HLA allele frequency (-A*24:02), but not in others that belong to the same or different HLA loci.     

'Very sore nights and days': the child's experience of illness in early modern England, c.1580-1720

Sick children were ubiquitous in early modern England, and yet they have received very little attention from historians. Taking the elusive perspective of the child, this article explores the physical, emotional, and spiritual experience of illness in England between approximately 1580 and 1720. What was it like being ill and suffering pain? How did the young respond emotionally to the anticipation of death? It is argued that children’s experiences were characterised by profound ambivalence: illness could be terrifying and distressing, but also a source of emotional and spiritual fulfilment and joy. This interpretation challenges the common assumption amongst medical historians that the experiences of early modern patients were utterly miserable. It also sheds light on children’s emotional feelings for their parents, a subject often overlooked in the historiography of childhood. The primary sources used in this article include diaries, autobiographies, letters, the biographies of pious children, printed possession cases, doctors’ casebooks, and theological treatises concerning the afterlife.     

Guidelines for the Validation and Use of Immunoassays for Determination of Introduced Proteins in Biotechnology Enhanced Crops and Derived Food Ingredients

Recent advancements in agricultural biotechnology have created a need for analytical techniques to determine introduced proteins in crops enhanced through modern biotechnology techniques. These proteins are expressed in plant tissues and may be present in food ingredients. Immunoassays are ideally suited for protein detection and may be used as both quantitative and threshold methods. Microplate ELISA and lateral flow devices are two of the most commonly used immunoassay formats for agricultural biotechnology applications. This paper provides general background information and a discussion of criteria for the validation and application of immunochemical methods to the analysis of proteins introduced into plants and food ingredients using biotechnology methods. It is the result of a collaborative effort of members of the Analytical Environmental Immunochemical Consortium. This collaborative effort represents the combined expertise of several organizations to reach consensus on establishing guidelines for the validation and use of immunoassays. Further, the paper offers developers and users a consistent approach to adopting the technology as well as aid in producing accurate and meaningful results.     

Ultracryotomy: development and application (with examples from the yeast cytology) (author's transl)

The preparation steps usually necessary for obtaining ultrathin frozen sections of biological material (chemical prefixation, enclosing, cryoprotective treatment, freezing, sectioning, and post-staining the sections for transmission electron microscopy) are submitted to a critical analysis. The application of cryo-ultramicrotomy, in particularly for cytochemical purposes, is reviewed. Fundamental considerations of chemical prefixation and poststaining are supported by examples from yeast cytology. Furthermore, the efficiency of the cryo-ultramicrotomy (electron optical resolution of ultrastructural details) is demonstrated on yeast cells and protoplasts.     

The universal muscular chamber. A basic unit of the genitourinary, cardiovascular, gastro-intestinal, skeletal, cutaneous, respiratory and other systems, endocameral hypertension; and spasm, the key to their idiopathic diseases and arthropathies

Starting with the integument, we see many organs are contractile sacs or multiples thereof, which tubes or bags constitute the major part of the entire body. Recognition of this basic unit and its characteristics sheds new light, individually and collectively, on many disorders previously considered unrelated. Muscular tears and perforations develop in the walls of these chambers, being no way peculiar to those organs, wherein, hydrochloric acid occurs. So, it is not necessary to explain the absence of excessive acid from patients who exhibit holes in the gastric, uterine, aortic, duodenal, rectal, pulmonary, retina, and other walls. Muscle, not acid is the great common factor relating idiopathic disorders in the gastrointestinal tract to each other and to similar diseases in other systems. When the units are linked together, the lesions tend to appear as arthropathies, i.e. at the joints. Rephrasing common-place observations, frees us from conventional, conceptual cul-de-sacs. An observation is only as good as its interpretation, so all possibilities must be considered, otherwise, we will remain blinded by our misconceptions.     

Der Einfluß von Nahrungsmitteln und Rauchen auf die klinisch-chemische Diagnostik von Phäochromozytom,Neuroblastom und Karzinoid-Syndrom

Zusammenfassung Der Einfluß von verschiedenen Nahrungsmitteln auf Methoden zur Bestimmung von Adrenalin (AD), Noradrenalin (NA), Vanillinmandelsäure (VMS), Metanephrinen (MN), Homovanillinsäure (HVS) und 5-Hydroxyindolessigsäure (5-HIE) im 24 h-Harn zur Diagnose des Phäochromozytoms bzw. Karzinoid-Syndroms wurde untersucht. Die in die Untersuchung einbezogenen Nahrungsmittel waren: Tee, Kaffee, Mandeln, Ananas, Käse, Walnüsse, Vanillepudding, Bananen, Tomaten und Milchschokolade. Außerdem wurde der Einfluß des Zigarettenrauchens auf die Bestimmung von AD, NA, VMS und MN untersucht.Walnüsse führten zu einer starken Erhöhung der 5-HIE-Ausscheidung. Bananen erhöhten die Ausscheidung von AD, NA, VMS, MN und 5-HIE. Kaffee und Ananas bewirkten eine geringe Zunahme der MN-Werte. Rauchen von 20–30 Zigaretten/Tag beeinflußte keine der vier Variablen.Wenn die beschriebenen Methoden benutzt werden, sollte lediglich auf den Verzehr von Bananen und Walnüssen vor und während der Harnsammelperioden verzichtet werden, da die oberen Normgrenzen im Harn überschritten werden könnten. Ein Verzicht auf Kaffee und Ananas in normalen Mengen ist nicht erforderlich. Es besteht kein Anlaß, weiterhin die bisherigen umfangreichen Restriktionen der übrigen Nahrungsmittel beizubehalten.