SARS相关冠状病毒M蛋白基因重组核酸疫苗的实验研究

目的：构建针对SAILS相关冠状病毒M蛋白基因的重组核酸疫苗，观察其免疫小鼠后肌体的抗体产生情况，探讨其作为抗SAILS病毒疫苗的可能性。方法：通过分子生物学的方法构建SARS相关冠状病毒M蛋白基因真核表达质粒pcDNA3．1／M。经肌注免疫健康BALB／c小鼠，在免疫后的2．4、6周取小鼠血清，用ELISA法检测其中的抗体。结果：接种含M基因的真核表达质粒pcDNA3．1／M的低剂量组和高剂量组的小鼠血清在接种2周后就可检测出SARS-CoV特异性IgG，第4周时，这种特异性IgG水平有升高趋势，第6周时，血清中的抗体含量与4周时无大的差异。高剂量组产生抗体与低剂量组产生抗体无显著差异。pcDNA3．1空载体接种的对照组未检测出特异性抗体（其OD值低于0．18）。结论：构建的真核表达质粒pcDNA3．1／M能诱导小鼠产生了针对SARS的抗体。     

Chitosan-DNA基因免疫诱导粘膜特异性免疫应答及其抗CVB3感染作用

目的：构建新型粘膜基因疫苗，诱生CVB3VP1特异性粘膜免疫应答，探讨粘膜免疫抗CVB3感染的作用，为病毒性心肌炎的特异性防治奠定基础。方法：抽提CVB3 RNA，以RT-PCR扩增得VP1基因，插入真核表达载体pcDNA3中，构建质粒pcDNA3-VP1，以Chitosan多糖包裹形成Chitosan-DNA基因疫苗。将该质粒转染Hela细胞，观察其体外表达情况。以50辉DNA剂量的Chitosan-DNA疫苗滴鼻免疫BALB／C小鼠3次，检测CVB3特异性体液和细胞免疫应答；隔4周以5LD50活CVB3致死攻击小鼠，观察攻击后存活情况。结果：制备了直径为80-100nm的Chitosan-DNA复合物颗粒，体外转染证实Chitosan-DNA复合物中VPl的表达高于pcDNA3-VP1质粒-Lipofectamine的表达水平。该疫苗滴鼻3次免疫后，不仅诱生了高水平的IgG，而且诱生了高水平的粘膜IgA抗体，第6周抗体P/N值分别达3．5和3．2。特异性细胞免疫应答研究发现，该疫苗诱生了较强的VP1特异性CTL杀伤作用，并显著高于pcDNA3-VP1和pcDNA3组。CVB3攻击后，可保护33．3％小鼠长期存活，而pcDNA3和pcDNA3-VP1质粒滴鼻免疫对照组的平均存活天数分别为8．5和10．8天。病理学研究显示：Chitosan-DNA免疫小鼠心肌组织基本正常，而对照小鼠死亡前心肌显示大量的灶性坏死和炎性浸润。结论：Chitosan-DNA基因疫苗滴鼻免疫可诱生CVB3特异性粘膜IgA应答及CTL反应，具有一定的抗CVB感染的免疫保护力。     

重组乙型肝炎病毒变异s基因疫苗诱导小鼠产生特异性体液免疫应答

目的：构建乙型肝炎病毒(HBV)变异s基因真核表达载体，检测其诱导小鼠产生特异性体液免疫应答。方法：利用限制性内切酶定位克隆构建s基因nt587 G→A的真核表达载体pcMV-S2．S 145R(PR).用其转染人肝癌细胞系Hep G2后，用EIA、EILISA及免疫细胞化学法，观察其抗原性。以重组变异型s基因真核表达载体(PR)和载体pcDNA3．0分别免疫C57BL／6小鼠各5只。每只小鼠各肌肉注射纯化质粒100μg.用ELISA法检测血清抗-HBs及抗-HBs2抗体的效价。结果：体外实验证实，变异型HBs矩可与抗-HBs结合；PR免疫小鼠可诱导其产生抗-HBs抗体及抗．HBs2抗体，但抗-HBs2抗体的出现早于抗-HBs抗体约1～2wk。结论：HBV变异s基因(nt587G→A)的真核表达载体的表达产物具有良好的抗原性，能够诱导C57BL／6小鼠产生体液免疫应答。     

T细胞淋巴瘤DNA疫苗的实验研究

目的：研究T细胞淋巴瘤TCR Vβ DNA疫苗诱发小鼠体液免疫反应。方法：将：BALB／C小鼠随机分为pcDNA3．1组、pcDNA3．1／TCR Vβ8组、pcDNA3．1／TCR Vβ8 CpG 质脂体组和全硫代CpC组，共4组(每组6只)，双侧股四头肌注射疫苗免疫，于第0、2、4周各免疫1次，共3次。每次免疫前及免疫开始后至第8周，取鼠血，应用间接免疫荧光法检测小鼠抗体生成情况。结果：pcDNA3．1／TCR Vβ组、pcDNA3．1／TCR Vβ CpC 脂质体组小鼠血清中均产生了特异性抗体，抗体滴度在第4周开始增高，第6周时达到高峰。同一时间段相比，特异性抗体滴度后者显著高于前者。结论：证明了DNA重组质粒pcDNA3．1/TCR Vβ可诱导小鼠产生特异性抗T细胞淋巴瘤TCR Vβ8抗体；CpG和脂质体增强了TCR Vβ8 DNA疫苗诱导的体液免疫反应。     

IL-15真核表达质粒的构建及对乙肝疫苗诱导的体液免疫应答的影响

目的 研究两种不同的IL-15真核表达质粒对乙肝蛋白疫苗诱导的免疫应答的影响。方法：构建IL-15真核表达质粒(简称pIL-15)和含有IL-12信号肽的IL-15真核表达质粒(简称pIL-2s-15)，CTLL-2细胞增殖实验验证两种质粒真核表达产物的生物学活性。将这两种质粒分别与HBsAg共免疫BALB／C小鼠，用ELISA法检测小鼠血清抗-HBs IgG及IgGl、IgG2a亚类的效价。结果：与HBsAg蛋白疫苗共免疫时，pIL-15可使HBsAg诱导的抗-HBsIgG效价升高，显著高于载体pcDNA3．1与HB—sAg共免疫对照组，pIL-2s-15对HBsAg诱导抗-HBsIgC效价没有明显影响。与HBsAg pcDNA3．1组相比，HBsAg pIL-2s-15组和HBsAg pIL-15组诱生的抗HBsIgG2a亚类均升高，但前者IgG2a／IgG1比值最高，与HBsAg pcDNA3．1组相比差别有显著性；HBsAg pIL-15组IgG2a／IgG1比值与HBsAg pcDNA3．1组相比差别无显著性。结论 pIL-15真核表达质粒可增强蛋白疫苗诱导的体液免疫应答，pIL-2s-15真核表达质粒则主要使免疫应答趋向Th1型。     

插入IL-2基因优化HBV DNA疫苗的研究

目的：观察IL-2基因的插入对DNA疫苗免疫效应的影响，探讨优化DNA疫苗设计、提高DNA疫苗兔疫效应的途径。方法：采用PCR产物直接克隆和重组DNA技术构建了人IL-2和HBsAg融合基因的真核表达载体pcDNA3．1 -S/IL-2，通过脂质体基因转移技术导入Cos-7细胞中检测其瞬间表达，并经肌肉注射免疫C57Bl/6小鼠，以检测和比较它们的细胞和体液免疫应答。结果：通过酶切、PCR及测序证实已正确完整地插入 IL-2基因，成功构建了 pcDNA3．1 -S/IL-2重组质粒。体外转染Cos-7后可见基因的表达和分泌，pcDNA3．1 -S/IL-2可以提高免疫小鼠的抗 HBs抗体滴度和脾淋巴细胞诱生的IL-2的生物活性水平，增加 HBsAg特异性的脾淋巴细胞增殖指数。结论：IL-2和 HBsAg基因的融合表达对 DNA疫苗的免疫反应起协同和增强作用，提示IL-2基因插入可能是增强DNA疫苗的免疫效应的可行途径。     

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。     

基因疫苗脾内免疫制备抗人c-kit单克隆抗体及其初步鉴定

目的：用基因脾内免疫方法制备抗人c-kil单克隆抗体，并通过对抗体生物学特性的初步研究，确定该方法制备抗体的可行性。方法：利用分子克隆方法构建重组质粒pcDNA3．1／c-kit胞外区，脾内免疫BALB／c小鼠一次，通过杂交瘤技术制备抗人c-kit单克隆抗体。采用流式细胞术、Western blot方法初步鉴定制备的抗体。结果：目的基因片段c-kit外区正确插入质粒pcDNA3．1，重组质粒免疫小鼠后通过杂交瘤技术获得了三株分泌抗人c-kit胞外区的杂交瘤细胞株6CA、2C5和5D5。其亚类均为IgM类，识别不同的抗原表位，其特异性与抗人c-kit抗体一致。结论：可用基因脾内一次免疫法制备抗人c-kit单克隆抗体。     

CpG ODN对rHBsAg免疫小鼠Th1/Th2型免疫应答的影响

目的：初步探讨CpC寡脱氧核苷酸(CpG ODN)与重组乙型肝炎表面抗原(rHBsAg)联合免疫小鼠的Th1／Th2型免疫应答效应。方法：BALB／c小鼠经后腿胫骨前肌免疫2次，ELISA法检测血清乙型肝炎表面抗体(抗-HBs)IgG亚类IgG2a/IgG1的比值；生物活性法检测脾细胞诱生上清中的IFN-γ和IL-2含量；ABC-ELISA法检测小鼠血清中IL-4、IL-10及IL-12含量。结果：加CpG ODN组与单独注射rHBsAg组相比：抗-HBs IgG亚类IgG2a／IgG1比值明显高；Th1型细胞因子IFN-γ和IL-2的表达增强，抑制Th2型细胞因子IL-4和IL-10的产生。结论：CpCODN能够明显增强rHBsAg免疫小鼠Th1型抗体亚类IgG2a的产生，并且诱导Th1型细胞因子的表达，抑制Th2型细胞因子的表达。     

pcDNA3.1/MAGE-3-HSP70融合蛋白诱导特异性抗肿瘤免疫应答的研究

目的构建重组表达质粒pcDNA3．1／MAGE-3-HSP70，观察其诱导免疫应答的能力和抗肿瘤免疫治疗的作用。方法通过RT-PCR扩增MAGE-3和HSP70 cDNA，以pcDNA3．1为载体，构建pcDNA3．1／MAGE-3-HSP70重组质粒，肌肉注射接种小鼠，间隔7d，共3次，以pcDNA3．1／MAGE-3、pcD—NA3．1／HSP70、pcDNA3．1和PBS为对照，检测脾淋巴细胞CTL杀伤活性、脾细胞培养上清IL-2、IFN-γ浓度、外周血淋巴细胞亚群变化及鼠血清中抗MAGE-3抗体水平。pcDNA3．1／MAGE-3-HSP70重组质粒免疫已负荷B16／MAGE-3的小鼠，观察小鼠成瘤时间、生存时间。结果pcDNA3．1／MAGE-3-HSP70质粒免疫的小鼠，其脾淋巴细胞对MAGE-3阳性靶细胞表现出明显的杀伤活性，与各对照组相比，P〈0．01，差异有统计学意义；外周血中CD4^＋、CD8^＋T细胞和脾细胞培养上清中TH1类细胞因子IFN-γ、IL-2水平明显升高。pcDNA3．1／MAGE-3-HSP70重组质粒免疫已负荷B16／MAGE-3的小鼠后，小鼠成瘤时间明显延迟，生存期明显延长。结论pcDNA3．1／MAGE-3-HSP70DNA疫苗在体内能诱导出显著的MAGE-3特异性抗肿瘤免疫应答，且能抑制体内已经存在的少量肿瘤细胞的成瘤。     

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.     

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.     

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.     

What will studies of Fulani individuals naturally exposed to malaria teach us about protective immunity to malaria?

There are an estimated over 200 million yearly cases of malaria worldwide. Despite concerted international effort to combat the disease, it still causes approximately half a million deaths every year, the majority of which are young children with Plasmodium falciparum infection in sub-Saharan Africa. Successes are largely attributed to malaria prevention strategies, such as insecticide-treated mosquito nets and indoor spraying, as well as improved access to existing treatments. One important hurdle to new approaches for the treatment and prevention of malaria is our limited understanding of the biology of Plasmodium infection and its complex interaction with the immune system of its human host. Therefore, the elimination of malaria in Africa not only relies on existing tools to reduce malaria burden, but also requires fundamental research to develop innovative approaches. Here, we summarize our discoveries from investigations of ethnic groups of West Africa who have different susceptibility to malaria.     

'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.     

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.     

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.