El Tor型霍乱弧菌口服活疫苗候选株IEM108的构建

目的 构建能够诱导抗菌抗毒素免疫和抵抗CTXΦ转染的霍乱弧菌生物安全性口服活疫苗候选株。方法 以不产毒的EI Tor型霍乱弧菌疫苗候选株IEMl01为出发菌株，以管家基因thyA为选择压力，在IEMl01的thyA基因缺失株IEMl01-T中，通过质粒．染色体致死平衡系统表达霍乱毒素B亚单位基因ctxB和介导CTXΦ噬菌体免疫的rstR基因。在家兔模型中，通过检测血清杀弧菌抗体和抗CTB的IgG抗体来评价该新建疫苗候选株的免疫原性；以对家兔攻毒试验后肠段积液量来评价其保护力。结果 含ctxB、rstR和大肠杆菌来源的thyA基因的重组质粒在：IEMl01-T中稳定存在，GMI-ELISA检测ctxB基因能够很好表达。动物试验表明，该新建疫苗候选株能刺激机体产生高滴度的血清杀弧菌抗体和抗CTB IgG抗体，能较好抵抗至少4μg纯品CT和不同毒株的攻击。结论 应用质粒．染色体致死平衡系统构建了能抵抗CTXΦ转染和稳定表达ctxB的生物安全性抗菌抗毒口服活疫苗候选株，其在家兔模型中有较好的免疫原性和保护力。     

霍乱弧菌减毒活疫苗候选株IEM109的构建

目的构建保护性抗原基因整合于染色体上的霍乱弧菌活疫苗候选株，减弱毒力和增强其遗传稳定性。方法以我们筛选的天然不含霍乱毒素的疫苗候选株IEM101为出发株，通过基因重组技术将IEM101染色体上与噬菌体复制整合有关的TLC因子、attRS位点及具细胞毒作用的RTX基因簇同时进行缺失，并插入具免疫原性的ctxB基因及介导噬菌体免疫的rstR基因，构建疫苗候选株IEM109。GM1-ELISA法检测1EM109中ctxB基因的表达情况。细胞培养观察候选株对哺乳动物细胞Hep-2的毒性作用。结果PCR及原位杂交均证实霍乱弧菌疫苗候选株IEM109中TLC因子、attRS位点、rtxA、rtxC基因被缺失，而ctxB、rstR基因整合人染色体DNA。GM1-ELISA示ctxB基因表达良好。IEM109对Hep-2细胞的毒性作用消失。结论构建了出发株染色体上毒力相关基因簇被保护性抗原基因替换的重组改造菌株，可作为候选株进行进一步的免疫效果考核。     

7株O139霍乱弧菌CT基因阴性菌的生物学性状研究

目的拟从毒力基因ｃｔｘ阴性的Ｏ１３９霍乱弧菌中筛选疫苗候选株。方法用常规鉴定技术、Ｓｏｕｔｈｅｒｎｂｌｏｔｉｎｇ技术和兔肠段结扎试验检测了７株霍乱肠毒素（ＣＴ）基因阴性的Ｏ１３９霍乱弧菌的一般特性、毒性和毒性基因;通过小肠免疫家兔,用ＰＢＬ－ＥＬＩＳＡ、ＥＬＩＳＡ和杀弧菌抗体试验评价抗原性;用家兔主动保护试验和乳鼠被动保护试验研究保护效果。结果７株菌都具有Ｏ１３９霍乱弧菌的一般特性,均不引起肠段积液,毒力基因ｃｔｘ、ｚｏｔ、ａｃｅ和ＲＳ１探针杂交结果均为阴性。Ｂ１３和９４００１免疫后０～２８天血清抗体ＩｇＧ效价一直上升,而９４００２免疫后１４天最高;９４００２的血清杀弧菌抗体峰值最高。Ｂ１３免疫家兔后可保护１０５ＣＦＵ毒株的攻击,９４００１和９４００２免疫后可保护１０５～７ＣＦＵ毒株的攻击,９４００２加强免疫后能保护１０７ＣＦＵ毒株的攻击。Ｂ１３免疫血清１４稀释后对乳鼠的保护率为２０％,９４００１和９４００２的免疫血清１１６稀释后的保护率为５０％。结论７株Ｏ１３９霍乱弧菌属于毒力基因遗传单元缺失,无毒或毒性低,其中９４００２的免疫原性和保护效果较好,且初次免疫后存在免疫记忆     

霍乱弧菌毒力表达调控基因toxR缺失株的构建及其功能的研究

目的　通过构建霍乱弧菌toxR基因缺失株来研究toxR基因对霍乱弧菌减毒菌株IEM101和高产毒株569B毒力表达的调控作用。方法　采用自杀性质粒和接合转移技术，将2个中间含有四环素基因的toxR基因分别与霍乱弧菌减毒株IEM101和高产毒株569B染色体toxR基因重组，从而获得toxR基因缺失株IEM101-4和569B-43，并对2个toxR基因缺失株和其原出发菌株的霍乱肠毒素的产率和主要外膜蛋白图谱进行比较。结果　采用GM1-ELISA检测受测菌CT基因表达，toxR基因缺失株569B-43的P/N值为1.82，而其原出发菌株569B的P/N为4.52，而IEM101和其toxR基因缺失株的P/N值均低于2。采用SDS-PAGE对受试菌外膜蛋白进行分析，toxR基因缺失株569B和IEM101的外膜蛋白图谱相比，均多出2条相对分子质量（Mr）为40×103和43×103外膜蛋白区带。结论　toxR蛋白是霍乱肠毒素基因ctx表达的正调控因子，是霍乱弧菌主要外膜蛋白（Mr为40×103和43×103)编码基因的负调控因子。     

宋内痢疾菌无毒株S7表达霍乱弧菌O抗原及霍乱毒素B亚单位工程菌的构建

以宋内氏痢疾菌无毒株S7作为受体,将含有编码霍乱弧菌O抗原及霍乱毒素B亚单位基因的质粒转移入其中,构建成重组菌株S7COB。质粒电泳图谱显示重组株中存在被转移的外源质粒。重组株的生化特性和毒力表型与受体相同。菌体凝集和全菌体酶联免疫吸附试验(ELISA)证明在重组株的菌体表面同时表达了霍乱弧菌和宋内氏痢疾菌的O抗原。GM1-ELISA表明该重组株能在细胞内表达霍乱毒素B亚单位。以5亿、10亿的重组株免疫LIBP品系小鼠,免疫小鼠对宋内氏毒株S63攻击的保护率为70％～100％,对霍乱弧菌毒株569B攻击的保护率为30％～54％。     

重组霍乱毒素B亚基与恶性疟原虫多表位融合蛋白的免疫原性研究

目的　评价霍乱毒素B亚基与恶性疟原虫多价抗原的融合蛋白的免疫原性。方法　在大肠杆菌中表达重组霍乱毒素与恶性疟原虫多价抗原的融合蛋白 ,通过免疫小鼠评价融合蛋白的免疫原性。结果　通过亲和层析纯化霍乱毒素B亚基与恶性疟原虫多价抗原的融合蛋白 ,不加任何佐剂 ,以 5 0 μg/只的剂量免疫C5 7BL/ 6J小鼠 ,3次免疫后 ,CTB抗体滴度达 1∶6 40 0 ,抗疟原虫抗体滴度 1∶16 0 0 ,其CTL活性为 2 0 7%。结论　霍乱毒素具有较好的佐剂作用 ,经融合蛋白免疫的小鼠能产生良好的体液和细胞免疫 ,为评价该抗原的免疫保护作用打下了基础     

甲型副伤寒杆菌ompA基因分布及重组表达产物的免疫学鉴定

目的 构建甲型副伤寒杆菌ompA基因原核表达系统,确定其重组表达产物rOmpA免疫原性和保护作用,检测甲型副伤寒杆菌临床菌株ompA基因携带率.方法 采用PCR从甲型副伤寒杆菌临床株JH01中扩增ompA基因,T-A克隆后测序并构建ompA基因原核表达系统.采用SDS-PAGE和Bio-Rad凝胶图像分析系统检测rOmpA表达情况及其产量,采用免疫扩散法、微量肥达试验和Western blot鉴定其抗原性和免疫反应性.采用PCR检测98株甲型副伤寒杆菌临床菌株ompA基因携带率.采用小鼠感染模型,了解rOmpA对甲型副伤寒杆菌50001株感染的免疫保护作用.结果 与报道的相关序列比较,所克隆的ompA基因核苷酸和氨基酸序列相似性均为100%.rOmpA表达量约为细菌总蛋白的65%.rOmpA能与其兔抗血清和甲型副伤寒杆菌全菌抗血清产生免疫反应(Western blot),并可在家兔中诱导产生抗体.94.9%(93/98)甲型副伤寒杆菌临床菌株含有ompA基因.100 μg和200 μg rOmpA对感染小鼠的免疫保护率分别为41.7%(5/12)和58.3%(7/12).rOmpA免疫小鼠或保护试验存活小鼠血清对各副伤寒杆菌H抗原的凝集效价为1:5～1:40.结论 ompA基因在甲型副伤寒杆菌临床菌株中分布广泛.rOmpA有良好的免疫原性和一定的免疫保护作用,可考虑为甲型副伤寒杆菌基因工程疫苗候选抗原.     

毒素源性大肠杆菌CFA/Ⅰ、CS3重组菌株粘附力、免疫原性和保护性研究

采用可逆性肠结扎成兔腹泻(RITARD)动物模型检测了毒素源性大肠杆菌(ETEC)定居因子抗原Ⅰ(CFA/I)和表面抗原3(CS3)重组菌各1株的肠道粘附力,免疫原性和保护性。经肠道及口服接种重组菌株对肠道均有较强的粘附力,与CFAs阴性宿主菌相比P<0.0025。口服免疫家兔时,第4周血清抗体滴度达到高峰,滴度分别为1:9000和1:80000;肠道免疫家兔后血清效价在第2周达到高峰,滴度分别为1:8000和1:60000。在抗体滴度高峰时进行ETEC野生株攻毒,结果显示口服组抗腹泻保护率为66.7％～75％,肠道组为50.1％～71.4％。攻毒后免疫家兔的排菌天数较对照组明显减少。     

甲型副伤寒杆菌nmpC基因原核表达及表达产物免疫保护作用

目的 构建甲型副伤寒杆菌外膜蛋白基因nmpC的原核表达系统,确定其重组表达产物rNmpC免疫原性和保护作用,了解甲型副伤寒杆菌临床菌株nmpC基因携带及表达率.方法 采用PCR和T-A克隆法从甲型副伤寒杆菌临床株JH01中获得nmpC基因克隆并构建其原核表达系统.采用SDS-PAGE和Bio-Rad凝胶图像分析系统检测rNmpC表达情况及其产量,采用免疫扩散法、Western blot和微量肥达试验鉴定其抗原性和免疫应答性.采用PCR和ELISA分别检测98株甲型副伤寒杆菌临床菌株nmpC基因携带及表达率.采用小鼠感染模型了解rNmpC对甲型副伤寒杆菌致死性感染的免疫保护作用.结果 与报道的相关序列比较,所克隆的nmpC基因核苷酸和氨基酸序列相似性均为100%.rNmpC表达量约为细菌总蛋白的30%.rNmpC免疫家兔可产生抗体并能与甲型副伤寒杆菌全菌抗血清产生阳性Western杂交信号.所有甲型副伤寒杆菌菌株均携带nmpC基因并表达NmpC蛋白,但伤寒杆菌、乙型及丙型副伤寒杆菌未检出nmpC基因.100μg和200μgrNmpC对感染小鼠的免疫保护率分别为41.7%(5/12)和66.7%(8/12).rNmpC免疫小鼠或保护试验存活小鼠血清仅对甲型副伤寒杆菌H抗原产生1∶5～1∶40的凝集效价.结论 NmpC是甲型副伤寒杆菌独有的序列保守、分布广泛且自然表达的外膜蛋白抗原,该外膜蛋白具有良好的免疫原性和一定的免疫保护作用,可作为多价甲型副伤寒杆菌基因工程疫苗候选抗原.     

幽门螺杆菌ureB及ureB-hspA融合基因在减毒鼠伤寒沙门菌中的表达及小鼠的免疫应答

目的　构建H .pyloriureB单基因及ureB和hspA融合基因的减毒鼠伤寒沙门菌疫苗候选株 ,并进行初步的免疫原性分析。方法　将H .pyloriureB单基因及ureB和hspA融合基因分别克隆入pTrc99A asd质粒的多克隆位点之内。挑取单菌落质粒鉴定后 ,分别将其电击经中间宿主X3730转入最终宿主X4 0 72 ,SDS PAGE和Westernblot检测蛋白表达。将疫苗候选株经口或鼻免疫BALB c小鼠。结果　疫苗候选株能够分别表达出相对分子质量 (Mr)为 6 4× 10 3的UreB蛋白及 77× 10 3的UreB HspA融合蛋白。在免疫BALB c小鼠的肠液和血清中可以分别检测到针对H .pylori的特异性分泌型IgA和IgG抗体。结论　构建了能够表达幽门螺杆菌UreB及UreB HspA融合蛋白的活菌疫苗候选株 ,为探索制备H .pylori口服活菌疫苗奠定了基础。     

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.     

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.     

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.     

The case for allele‐specific recognition by the TCR

There is a sharp difference in how one views TCR structure–function–behaviour dependent on whether its recognition of major histocompatibility complex‐encoded restriction elements (R) is germline selected or somatically generated. The generally accepted or Standard model is built on the assumption that recognition of R is by the V regions of the αβ TCR, which is not driven by allele specificity, whereas the competing model posits that recognition of R is allele‐specific. The establishing of allele‐specific recognition of R by the TCR would rule out the Standard model and clear the road to a consideration of a competing construct, the Tritope model. Here, the case for allele‐specific recognition (germline selected) is detailed making it obvious that the Standard model is untenable.