C3d对基因免疫诱导的HBV特异性体液免疫应答的调节作用

观察补体C3d对HBV基因免疫诱导的特异性体液免疫应答的调节作用 ,为增强HBV基因疫苗免疫效果寻求新途径。将HBV preS2 /S编码基因分别插入真核表达载体TR4 2 1和含有 3拷贝C3d编码基因的TR4 2 1 C3d3质粒 ,构建重组质粒TR4 2 1 preS2 /S和TR4 2 1 preS2 /S C3d3。采用肌肉注射法对BALB/c小鼠实施基因免疫 (10 0 μg/ 10 0 μl/只小鼠 ) ,以空质粒为对照 ,定期采集血清。ELISA法检测免疫小鼠血清特异性抗 HBs IgG及其亚型 ,并采用NaSCN竞争ELISA法检测其亲合力。结果表明 ,TR4 2 1 preS2 /S C3d3重组质粒免疫组诱导的特异性抗 HBs IgG水平明显高于TR4 2 1 preS2 /S重组质粒免疫组 (P <0 0 5 ) ;而且TR4 2 1 preS2 /S C3d3重组质粒免疫组诱导的抗 HBs IgG抗体的亲合力 (ED50 :1 375 )显著高于TR4 2 1 preS2 /S重组质粒组 (ED50 :0 875 ) ,但C3d并不改变基因免疫诱导的特异性抗HBs IgG各亚型水平的格局 ,仍以IgG2b和IgG2a为主。提示C3d可增强基因免疫诱导的HBV特异性体液免疫应答 ,并促进特异性抗体亲和力的成熟 ,这为提高HBV基因疫苗的免疫效果提供了新的途径。     

GM-CSF对抗原化抗体基因免疫TH细胞应答的调节作用

目的研究GM-CSF(粒-单核巨噬细胞集落刺激因子)与抗原化抗体联合基因免疫时,GM-CSF对TH细胞应答的调节作用。方法在编码免疫球蛋白重链的质粒中分别克隆黏蛋白1(MUC1)中特异性PDTRP抗原表位和GM-CSF编码基因,构建含PDTRP和GM-CSF编码基因的抗原化抗体表达重组体。经脾免疫和肌肉加强的方式免疫BALB／c小鼠。采用ELISA方法检测小鼠血清中特异性抗体的水平及其亚类并动态观察;RT-PCR方法检测TH细胞分化中细胞因子和转录因子的表达水平。结果抗原化抗体基因免疫能诱导机体产生免疫应答。GM-CSF增强抗原化抗体基因免疫诱生的抗PDTRP特异性IgG抗体水平并且伴随IgG1／IgG2a显著性升高,同时可增强淋巴细胞TH2型细胞因子及转录因子(IL-4、GATA-3)mRNA的表达水平。结论GM-CSF在增强抗原化抗体基因免疫诱导的免疫应答的同时使得免疫应答向TH2方向偏移。     

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.     

hCGβ-C3d3基因免疫BALB/c小鼠选择性促进B细胞克隆扩增

评价分子佐剂C3d在基因免疫中选择性促进抗原特异性B细胞增殖作用。分别用质粒pCMV4-hCG-βC3d3和pCMV4-hCGβ免疫BALB/c小鼠,免疫剂量为50 pmol。间隔3周相同剂量加强免疫1次。加强免疫后第1周及第2周,MTT法分析各组小鼠脾脏B细胞和T细胞增殖;ELISPOT法分析两免疫组小鼠脾脏hCGβ抗原特异性IgG分泌细胞。结果显示在加强免疫后第1周及第2周,hCG-βC3d3免疫组B细胞增殖能力显著高于hCGβ免疫组及正常对照组;而各组间T细胞增殖状况未见明显差异。hCG-βC3d3免疫组较hCGβ免疫组小鼠脾细胞分泌IgG的量和分泌hCGβ抗体的细胞数量均明显增加。结果表明分子佐剂C3d对于B细胞具有选择性克隆扩增作用。     

hCGβ-03d3基因免疫BALB／c小鼠选择性促进B细胞克隆扩增

评价分子佐剂C3d在基因免疫中选择性促进抗原特异性B细胞增殖作用。分别用质粒pCMV4-hcGβ-C3d3和pCMV4-hOGβ免疫BALB／c小鼠，免疫剂量为50pmol。间隔3周相同剂量加强免疫1次。加强免疫后第1周及第2周，MTT法分析各组小鼠脾脏B细胞和T细胞增殖；ELISPOT法分析两免疫组小鼠脾脏hcGβ抗原特异性IgG分泌细胞。结果显示在加强免疫后第1周及第2周，hCGβ-C3d3免疫组B细胞增殖能力显著高于hCGβ免疫组及正常对照组；而各组间T细胞增殖状况未见明显差异。hcGβ-C3d3免疫组较hCGβ免疫组小鼠脾细胞分泌IgG的量和分泌hCGβ抗体的细胞数量均明显增加。结果表明分子佐剂C3d对于B细胞具有选择性克隆扩增作用。     

C3d-P28对基因免疫诱导的HBV特异性细胞免疫应答的调节作用

目的：观察补体C3d-P28对基因免疫诱导的HBV特异性细胞免疫应答的调节作用，为增强基因免疫效果寻求新方法。方法：将质粒pVAON33-S2／S质粒(仅含HBV-preS2／S编码基因)和pVAON33-S2／S-P28．4质粒(含HBV-prS2/S和4拷贝C3d-P28的编码基因)以肌肉注射法对BALB／C小鼠实施基因免疫(100μg/100μl只)，并以空载质粒为对照。定期采集免疫小鼠血清、ELISA法检测其中特异性抗HBs-IgG及其亚型的水平；免疫小鼠脾细胞经特异性抗原(HBsAg)刺激后，采用，H-TdR掺入法、半定量RT-PCR法、同位素释放法分别检测其特异性淋巴细胞增殖活性、IL-4和IFN-γ基因表达的水平、CTL杀伤活性。结果：pVAON33-S2／S-P28．4质粒基因免疫诱导的特异性淋巴细胞增殖活性、抗HBs-IgG水平以及特异性CTL杀伤活性均明显高于pVAON33-S2／S质粒；C3d-P28未改变基因免疫诱导的抗HBs-IgG各亚型水平的格局，同时增强IgG1、IgG2a、IgG2b的水平；pVAON33-S2／S-P28．4质粒诱导的IL-4和IFN-γ的基因表达水平均明显高于pVAON33-S2／S质粒。结论：C3d-P28可在提高体液免疫应答的同时增强基因免疫诱导的HBV特异性细胞免疫应答。     

分子佐剂C3d3与hCGβ基因融合增强hCGβ基因疫苗体液免疫应答

目的：提高hCGβ在真核细胞中的表达效率，观察分子佐剂C3d3增强hCGβ基因疫苗的体液免疫应答。方法：构建带有增强目的基因表达效率的狗胰岛素前原信号肽(DPPISS)的高效真核表达质粒pCMV4-hCGβ-C3d3、pCMV4-hCGβ和pCMV4-C3d3。分别用pCMV4-DPPLSS-hCGβ-C3d3、pcMV4-DPPLSS-hCGβ和pcMV4-DPPISS-hCGβ联合pcMV4-DPPISS-C3d3免疫BALB／c小鼠，各组免疫剂量分别为5、10、50、100、500μmol，间隔3周相同剂量加强免疫1次。间接ELISA法分析免疫小鼠外周血抗hCGβ抗体动态变化以及IgG亚型。结果：含有DPPISS的pCMV4-hCGβ-C3d3较无DPPISS的pCMV4-hCGβ-C3d3在COS-7细胞中的表达效率明显提高。50μmol是BALB／c小鼠hCGβ基因免疫的最佳免疫剂量。C3d3使得BALB／c小鼠hCGβ的免疫原性增强了400倍。hCGβ-C3d3免疫组IgG1／IgG2a比值明显高于hCGβ免疫组和hCGβ与C3d3联合免疫组。结论：分子佐剂C3d3与hCGβ基因融合能够显著增强hCGβ基因避孕疫苗的体液免疫应答及抗体类型转换。     

HBV-preS2/S-C3d联合基因疫苗诱导的特异性免疫应答及其意义

目的：研究HBV—preS2／S—C3d联合基因疫苗诱导的特异性体液和细胞免疫应答及其意义。方法：CR2结合实验检测C3d融合蛋白的结合特性；分别用TR421、TR421-preS2／S和TR421—preS2／S—C3d3重组质粒免疫小鼠．ELISA法检测特异性抗HBs—IgG的亲合力，^3H-TdR掺入法检测其特异性淋巴细胞增殖活性（SI），半定量RT—PCR法检测IL-4、IFN-γ、T—bet和GATA-3基因表达的水平。结果：C3d融合蛋白可有效地结合CR2；TR421—preS2／S—C3d3质粒基因免疫诱导的抗HBs—IgG水平、亲合力以及SI均明显高于TR421-preS2／S组，并且前者诱导的IL-4、IFN-γ、T—bet和GATA-3基因表达水平也均高于后者。结论：C3d通过结合CR2、上调IL-4和转录因子的表达、促进抗体亲合力成熟来增强基因免疫诱导的HBV特异性免疫应答。     

真核化的原核表达系统增强HBV preS2/S基因免疫的效果

目的：研究真核化的原核表达系统——pCMV-T7poL／pT7IRES-HBs双质粒共表达体系与常规真核表达质粒相比在真核细胞中的表达差异；基因免疫小鼠诱生特异性体液免疫应答的能力及二者所诱生的抗体亚型的差异。方法：运用分子生物学方法构建真核化的原核表达系统，将构建的质粒进行基因免疫，应用DOT-EIA和ELISA等检测其所产生的抗体水平。结果：①真核化的原核表达系统在真核细胞中的表达能力明显强于常规真核表达质粒。②真核化的原核表达系统基因免疫小鼠不仅可诱生特异性体液免疫应答，且具有明显增强效应（P〈0．05）。③真核化的原核表达系统与常规真核表达质粒相比不仅可诱生同等程度的Th1型免疫应答，而且能够诱生更强的Th2型免疫应答（P〈0．01）。结论：真核化的原核表达系统——pCMV-T7pol／pT7IRES-HBs双质粒能诱导较强的体液免疫应答。     

粘膜佐剂LTB优化的抗淋病ltB-nspA融合基因疫苗鼻饲免疫效果研究

目的:用真核重组质粒pcDNA3.1(-)/ltB-nspA鼻饲免疫小鼠,探索粘膜佐剂LTB辅佐NspA所诱发的特异性体液免疫应答和细胞免疫应答水平。方法:对真核重组质粒行PCR及双酶切鉴定后大量制备,经鼻饲途径免疫雌性BALB/c小鼠,间接ELISA法检测血清中NspA特异性IgG抗体水平及小鼠生殖道灌洗液中NspA特异性sIgA抗体水平;MTT法检测脾淋巴细胞增殖水平,ELISA双抗体夹心法检测脾淋巴细胞培养上清IFN-γ含量。结果:融合基因pcDNA3.1(-)/ltB-nspA组小鼠生殖道灌洗液中sIgA(A450:0.316±0.045)明显高于pcDNA3.1(-)/nspA单基因组(P0.05)和其它对照组(P0.01);小鼠血清中IgG(A450:0.643±0.156)水平、脾淋巴细胞刺激指数(SI:1.65±0.32)和脾淋巴细胞诱生的IFN-γ(160.56±25.67pg/ml)水平均明显高于pcDNA3.1(-)/ltB、空质粒pcDNA3.1(-)和PBS对照组(P0.01)。结论:pcDNA3.1(-)/ltB-nspA融合基因疫苗经鼻饲免疫,能够诱导小鼠产生较强的特异性体液免疫应答和细胞免疫应答;粘膜佐剂LTB可辅佐NspA诱导小鼠产生更高水平的生殖道粘膜免疫。     

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.