四聚体技术检测人外周血巨细胞病毒抗原特异性CTL

为深入了解抗病毒免疫机制,探索人在健康状态及巨细胞病毒(CMV)急性感染时抗原特异性细胞毒T淋巴细胞(CTL)数量的动态变化。以CMV抗原肽、HLA-A*0201重链和轻链制备CMV四聚体;分离并检测12名健康被检者外周血单个核细胞(PBMC)中CMV抗原特异CTL数量;PBMC体外培养建立CTL细胞系,于末次刺激的不同时相进行四聚体染色,流式细胞仪(FACS)检测抗原特异CTL数量。结果发现9名被检者PBMC中均检出抗原特异CTL;细胞系中CMV特异性CTL数量急剧增多,细胞系与PBMC相比,差异有显著的统计学意义(P<0.001);研究结果提示,9名被检者感染过CMV,血液中存在少量免疫记忆性T细胞,当再次遭遇同一抗原后发生克隆扩增。     

人工抗原提呈细胞的制备和应用的研究

目的 制备人工抗原提呈细胞（artificial antigen presenting cell,aAPC）并用它从HLA-A2阳性健康个体外周血单个核细胞（PBMC）中诱导和扩增特异性细胞毒性T淋巴细胞（CTL）.方法 将HLA-A2-EBV四聚体分子和抗CD28抗体分子吸附固定在细胞大小的聚苯乙烯乳胶微球（5μm）表面制成aAPC;采用流式细胞仪表型分析;aAPC和HLA-A2阳性个体人外周血单核细胞进行混合淋巴细胞反应;用HLA-A＊0201-EBV四聚体染色法检测特异性CTL的频率;应用细胞内细胞因子染色法检测特异性CTL功能性细胞因子IFN-γ的分泌;采用LDH释放法检测特异性CTL的特异杀伤活性.结果 流式细胞仪分析显示微球表面吸附有HLA-A2-EBV四聚体分子和抗CD28抗体分子;四聚体检测及细胞内细胞因子染色法检测与经典细胞毒试验结果一致,结果表明aAPC在体外可诱导抗原特异性CTL的生成.结论 aAPC制备成功,并在体外有效地诱导抗原特异性CTL的生成.     

乙肝病毒蛋白抗原表位特异性CTL体外扩增条件优化

目的 优化乙肝蛋白抗原表位特异性CTL体外扩增条件.方法 选择急性自限性HBV感染者外周血,分离PBMC,体外用乙肝蛋白抗原表位多肽特异性刺激,特定天数后收集细胞,用CD8抗体与MHC-I类分子HBV抗原肽四聚体进行染色流式检测,检测扩增后乙肝蛋白抗原表位特异性CTL在CD8阳性细胞群中所占的频率.结果 1)在不同多肽浓度刺激下,当浓度在20 us/mL时,所测得特异性CTL占PBMC中CD8+T细胞的频率达到最高峰;2)固定刺激多肽浓度,在不同的刺激天数获得的特异性CTL频率第10天时最高;3)在不同的细胞因子培养条件中,当加入的细胞因子IL-2,IL-7,IL-15组合时所获得的特异性CTL频率最高.结论 在刺激肽浓度为20 μg/mL,并加入适量的细胞因子(IL-2,IL-7,IL-15),刺激10天的条件下,所获得的乙肝蛋白抗原特异性CTL频率最高.     

原发性胆汁性肝硬化HLA-A*0201限制性PDC-E2特异性CTL功能分析

目的：探讨原发性胆汁性肝硬化（PBC）中HLA-A＊ 0201限制的PDC-E2159-167特异性CTL与PDC-E2165-174特异性CTL功能。方法：分析15例HLA-A＊0201阳性（A2＋）PBC患者抗原肽PDC-E2159-167、PDC-E2165-174诱导的CTL对以表达HLA-A＊ 0201分子的T2细胞作为靶细胞的细胞毒性作用，并利用四聚体技术结合细胞内因子染色研究PDC-E2159-167特异性CTL与PDC-E2165-174特异性CTL分泌细胞因子（IFN-γ、IL-4和TNF-α）情况。结果：15例A2＋PBC患者中，12例PDC-E2159-167诱导的CTL和10例PDC-E2165-174诱导的CTL对负载相应抗原肽的靶细胞特异杀伤活性大于10%（E∶T=40∶1），而对照组则均小于10%；15例A2＋PBC患者从PBMC诱导的PDC-E2159-167特异性CTL和PDC-E2165-174特异性CTL中分泌IFN-γ细胞比值分别为0.33%±0.24%（0%～0.86%）、0.27%±0.24%（0%～0.75%），未检测到分泌TNF-α或IL-4的PDC-E2159-167或PDC-E2165-174特异性CTL。结论：结果表明，大部分PBC患者中HLA-A＊ 0201限制性PDC-E2159-167特异性CTL和PDC-E2165-174特异性CTL具有特异杀伤活性，HLA-A＊ 0201限制性PDC-E2特异性CTL主要分泌IFN-γ并非IL-4、TNF-α。     

酶联免疫斑点法检测抗原特异的CTL免疫应答试验条件的优化

目的：探讨酶联免疫斑点（ELISPOT）实验特异性及敏感性的因素，优化酶联免疫斑点法检测抗原特异的CTL免疫应答的实验条件。方法：HLA－A2阳性正常人的外周血单个核细胞在体外经流感病毒抗原肽诱导1周后，观察不同实验条件对酶联免疫斑点法检测抗原特异的CTL免疫应答的影响。结果：CTL诱导时，效应细胞先分离出CD8^＋T细胞组获得的Flu抗原特异的细胞频数要高于CTL诱导后再分离出CD8^＋T细胞组（P＜0．05），自体DC（树状细胞）作APC（抗原提呈细胞）比自体CD8^-PBMC作APC，CTL的诱导效率高且特异（P＜0．05）；使用细胞因子组合（IL－7＋IL－2＋IL－6）优于单纯使用IL－2。CTL行ELISPOT检测时，T2－2细胞较T2－1细胞具有更强抗原提呈功能，增加Flu抗原特异的CD8^＋T细胞频数（P＜0．05）。结论：优化了酶联免疫斑点法检测抗原特异的CTL免疫应答的试验条件，优化的方法在临床肿瘤疫苗的研究中具有应用价值。     

HBc-HLA-A*1101-β2m复合物单体及其四聚体的制备和鉴定

目的制备HBc-HLA-A ＊ 1101-β2m复合物单体和四聚体,并对其特异性进行了鉴定。方法原核高效表达的HLA-A ＊ 1101-BSP和β2m蛋白,在抗原肽（乙型肝炎病毒的核心蛋白HBc88-96）的存在下,体外复性折叠成可溶性的HLA-A ＊ 1101抗原肽复合物单体,经BirA酶作用并通过凝胶过滤层析法纯化复合物单体;然后将此复合物单体与藻红蛋白标记的链霉亲和素按一定比例耦合构建成四聚体;最后用流式细胞仪检测分析该四聚体结合特异性CTL的能力。结果Western blot和Dot-ELISA检测显示所制备的HLA-A ＊ 1101抗原肽复合物单体具有天然构象,且可被生物素化;流式细胞仪分析显示所制备的四聚体可与HLA-A11^＋供者的抗原特异性CTL结合。结论成功制备了具有完整构象的生物素化HLA-A ＊ 1101抗原肽复合物单体;此四聚体可以特异检测抗原特异性的细胞毒性T细胞。     

慢性乙型肝炎病人HBcAg特异性CTL的体外诱导及活性

目的 从慢性乙型肝炎病人外周血单个核细胞（PBMC）中诱导和扩增乙型肝炎病毒核心抗原（HBcAg）特异性细胞毒性T淋巴细胞（CTL）。方法 含乙型肝炎病毒重组逆转录病毒载体转染病人自身永生化B细胞作为抗原递呈细胞，联合重组HBcAg和IL-2，从病人PBMC中诱导和扩增HBcAg特异性CTL，^51Cr释放法检测其细胞毒活性。结果 此方法诱导的TCL可以有针对性的杀伤靶细胞，而不能够杀伤空载体转染及未经转染的B细胞。结论 所建立的方法可以在慢性乙型肝炎病人的PBMC中诱导出HBcAg特异性CTL活性。     

sH-2K~d-HBc四聚体的构建及其初步的检测应用

目的:sH-2Kd-HBc复合物四聚体的构建与检测,为进一步检测抗原特异性T细胞,探讨免疫发病机制奠定基础。方法:将四个生物素化的可溶性复合物单体与荧光标记的亲和素结合形成四聚体。采用HBcAg真核表达质粒(pcDNA3-C)通过不同的途径免疫小鼠,获得针对核心抗原的特异性CTL,与制备的四聚体共孵育,结合流式细胞仪术进行检测。结果:三种免疫方法所得到的细胞中,抗原特异性CTL频数较对照组都有明显提高(0.24%,0.26%,0.36%vs 0.07%,P≤0.05)。显示制备的四聚体能与抗原特异性T细胞结合,具有检测功能。三种不同的免疫途径所引起的抗原特异性的体液免疫应答和细胞免疫应答强度各有不同。与传统的肌肉注射组相比,基因枪免疫组的体液免疫应答水平较弱而细胞免疫应答水平较强。高压水注射(HDI)组的体液免疫应答和细胞免疫应答水平都要明显高于肌肉注射组结论:获得了功能性的sH-2Kd-HBc复合物四聚体,为进一步检测抗原特异性T细胞奠定基础。     

同种特异T细胞疫苗诱导移植物存活期延长的研究——Ⅰ.同种特异T细胞疫苗诱导的细胞免疫水平低下

在体外实验中观察了同种特异T细胞疫苗（TCV）免疫鼠T淋巴细胞对同种抗原和有丝分裂原（ConA）的反应能力。B6同种抗原特异TCV免疫的BALB/c小鼠对B6同种抗原的反应（MLR）能力明显变抑制,对无关第三者同种抗原AKR的反应能力也显著下降,表明存在抗原非特异性的抑制作用。在BALB/c同种抗原特异TCV免疫的B6小鼠中,获得一致的结果。在观察两组TCV免疫动物T细胞对Cond诱导的淋巴细胞增殖实验中,与正常小鼠 T细胞反应性比较,TCV免疫动物 T细胞的增殖能力显著下降,表现为抗原非特异作用。此与MLR中ConA-T细胞免疫动物的结果相一致。在CML实验里,同种抗原特异TCV免疫小鼠的脾细胞体外诱导同种CTL活性明显受到抑制,CTL 活性十分低下。体外实验结果表明:同种特异TCV免疫小鼠可诱发同种抗原反应和对ConA诱发的增殖反应能力显著低下,表现为抗原非特异性作用。     

人脐带血抗巨细胞病毒和EB病毒的特异性细胞毒性T细胞的建立

目的:探讨用人的脐带血单个核细胞体外同时扩增对抗EB病毒(EBV)和巨细胞病毒(CMV)的特异性细胞毒性T淋巴细胞的可行性。方法:利用人的脐带血单个核细胞(CBMC),通过EBV感染转化成B成淋巴细胞细胞株(BLCL),再通过逆转录病毒载体,将CMV蛋白基因pp65导入BLCL,用这种细胞体外刺激同一供者脐带血的CBMC产生细胞毒性T细胞(CTL),经[⁵¹Cr]释放实验(CRA)检测产生的CTL的杀伤功能。结果: 经免疫印迹 (Western Blot)检测,我们获得了同时表达EBV和CMV特异性抗原的抗原递呈细胞BLCLpp65,免疫组化结果表明,BLCLpp65细胞表达CMVpp65抗原的阳性率高达95%。CRA结果证实,用BLCLpp65刺激产生的CTL同时对EBV和CMV都有细胞毒作用。用免疫磁珠法将CD₄⁺和CD₈⁺ T细胞分离后再进行CRA,表明特异性的细胞毒性作用主要是CD₈⁺ CTL介导的。结论:BLCLpp65是很好的抗原递呈细胞,在体外能同时表达EBV和CMV蛋白抗原,用其刺激血清病毒抗体阴性的CBMC,能够扩增出同时针对EBV和CMV的特异性CTL,其中CD₈⁺CTL起主要作用。     

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.     

'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 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.