抗原捕获聚合酶链式反应（AC－PCR）直接分型检测单纯疱疹病毒

用抗单纯疱疹病毒（ＨＳＶ）型共同性ｇＣ和ｇＤ羊克隆抗体（ＭｃＡｂ）,包被即Ｅｐｐｅｎｄｏｒｆ管,捕捉ＨＳＶ,同时加入３个引物：一个是ＨＳＶ─１／ＨＳＶ─２型共同性上游引物,另两个分别是ＨＳＶ─１和ＨＳＶ─２型特异性下游引物。借此建立了能直接分型检测ＨＳＶ的抗原捕获聚合酶链式反应（ＡＣ─ＰＣＲ）。ＨＳＶ─１的扩增产物为４７７ｂｐ,ＨＳＶ─２的为３９９ｂｐ两型病毒经ＡＣ─ＰＣＲ扩增后产生分子量不同的ＤＮＡ片段,致使ＡＣ─ＰＣＲ能直接分型检测ＨＳＶ。ＨＳＶ─１和ＨＳＶ─２扩增产物的克隆和序列分析表明,本方法特异性好。用本法检测Ｂａｌｂ／ｃ幼鼠中枢神经系统ＨＳＶ感染的脑标本,进一步证实本方法不仅敏感、特异,而且分型准确。     

肾综合征出血热人体组织中病毒包涵体的性质及意义的进一步研究

应用免疫组化、原位分子杂交、电镜及免疫电镜等方法进一步对肾综合征出血热人体尸检组织中病毒包涵体（ＩＢ）的抗原、核酸性质和超微结构特点作了进一步观察。结果,在３９例中的２０例尸检病例组织中显示出病毒核蛋白抗原和血凝素抗抗原阳性的ＩＢ,其中包括１６例陕西尸检病例组织中的６例休克期和１例多尿期病例,１７例上海病例中的３例休克期和９例少尿期病例及３例江西病例中的１例休克期病例。ＩＢ主要分布在呼吸道和肺泡、肾远曲小管和集合管、胃肠道、腺垂体、扁桃体、胰腺、前列腺等组织的粘膜上皮和腺上皮细胞及肝细胞和睾丸生精上皮细胞胞浆中,阳性细胞形态基本正常。应用原位分子杂交,可在该组细胞小同时检测到病毒ＲＮＡ,多为胞浆内弥漫阳性,仅少数组织中显示出病毒ＲＮＡ阳性ＩＢ结构。电镜观察阳性组织细胞中出现由大量微丝微管及颗粒样结构组成的ＩＢ结构,其小的病毒颗粒状结构、内质网及纤维丝状结构呈病毒抗原阳性,上述结构位于高尔基体区。结果说明该病毒有感染上皮细胞的特性,对其宿主细胞的致细胞病变作用是极其温和的,且多表现在亚细胞水平。ＩＢ可能是病毒过量表达抗原的堆积或病毒复制部位,而微丝微管结构可能参与病毒的感染过程。     

流行性出血热循环免疫复合物组分分析

应用ＳＤＳ－聚丙烯酰胺凝胶电泳及免疫转印技术对流行性出血热患才血清中免疫合物组分进行了分析。流行性出血热循环免疫复合物经ＳＤＳ－ＰＡＧＥ分离,考马斯亮兰染色,显色主要有７条带,分子量分别为２３ｋＤ,５０ｋＤ,５２ｋＤ,６５ｋＤ,７２ｋＤ,８０ｋＤ及１００ｋＤ。采用该病毒特异性抗血清、单克隆抗体以及人免疫球蛋白、补体成分抗血清识别,在其特环免疫复合物中可检出特异性病毒抗在及相应的免疫球状蛋白和补体成     

不同感染状态下EB病毒核抗原亚型表达的研究

本实验用免疫印迹法纯化的抗ＥＢＮＡ亚型抗体,结合显微荧光分光光度检测技术,检测在不同感染状态下,三种亚型ＥＢＮＡ抗原在细胞中的表达程度。结果表明,处于潜伏感染状态下的Ｒａｊｉ细胞ＥＢＮＡ－１表达量较大,经巴豆油和正丁酸诱导进入钝挫感染状态后ＥＢＮＡ－１表达减少,而ＥＢＮＡ－２的表达增强。Ｂ＿（９５－８）细胞也有相似的趋势。表明ＥＢ病毒的激活可能与不同ＥＢＮＡ亚型表达量的改变有关。     

人巨细胞病毒对单纯疱疹病毒1型抗原表达的影响

我们用免疫胶体金色埋前标记技术和免疫荧光技术研究了人胚肺细胞(HEL)内,人巨细胞病毒(HCMV-AD_(169))对单纯疱疹病毒1型(HSV-ⅠSM_(44))抗原表达的影响,旨在探讨在细胞这一微生境内,一病毒对另一病毒可能发生的影响。电镜下计数HSV-1组和HCMV HSV-1组特异性结合金颗粒数得HSV-1组为657个,HCMV HSV-1组的总数为283个。t检验P<0.01,差别非常显著。并且HSV-1组细胞的胞浆中的病毒颗粒,比HCMV HSV-1组明显多。荧光显微镜下:HSV-1组阳性细胞数为689个HCMV HSV-1组只有484个,经poisson分布u检验,P<0.01,差别非常显著。免疫荧光实验还表明:HSV-1组,抗血清在1:320时仍有荧光清晰的阳性细胞,而HCMV HSV-1组,抗血清在1:160时,却无荧光阳性细胞。细胞病变效应(CPE)动态观察显示:HSV-1组8小时即有细胞病变,24小时蔓延整个单层;而HCMV HSV-1组超感染14小时才有细胞病变。24小时约有75％细胞受累。结果表明HCMV对HSV-1的抗原表达有明显的抑制作用。对抑制作用的可能机理及其在分子生态学中的意义,进行了讨论。     

Structural and serological characterisation of an O-specific polysaccharide from Serratia plymuthica

Abstract The surface polysaccharides of a strain of Serratia plymuthica were characterised and shown to consist of a linear, acidic galactoglucomannan as well as a major and a minor neutral galactan. Immunoblotting results demonstrated cross-reactions between this strain and others with similar galactans ( S. marcescens O16 and O20, Klebsiella O1, and Pasteurella haemolytica T4 and T10).     

Different Populations of Melanocytes Are Present in Hair Follicles and Epidermis

Melanocytes in human skin reside both in the epidermis and in the matrix and outer root sheath of anagen hair follicles. Comparative study of melanocytes in these different locations has been difficult as hair follicle melanocytes could not be cultured. In this study we used a recently described method of growing hair follicle melanocytes to characterize and compare hair follicle and epidermal melanocytes in the scalp of the same individual. Three morphologically and antigenically distinct types of melanocytes were observed in primary culture. These included (1) moderately pigmented and polydendritic melanocytes derived from epidermis; (2) small, bipolar, amelanotic melanocytes; and (3) large, intensely pigmented melanocytes; the latter two were derived from hair follicles. The three sub-populations of cells all reacted with melanocyte-specific monoclonal antibody. Epidermal and amelanotic hair follicle melanocytes proliferated well in culture, whereas the intensely pigmented hair follicle melanocytes did not. Amelanotic hair follicle melanocytes differed from epidermal melanocytes in being less differentiated, and they expressed less mature melanosome antigens. In addition, hair follicle melanocytes expressed some antigens associated with alopecia areata, but not antigens associated with vitiligo, whereas the reverse was true for epidermal melanocytes. Thus, antigenically different populations of melanocytes are present in epidermis and hair follicle. This could account for the preferential destruction of hair follicle melanocytes in alopecia areata and of epidermal melanocytes in vitiligo.     

Plasmodium falciparum: carbohydrates as receptor sites of invasion

Monosaccharides, disaccharides, and trisaccharides were tested as inhibitors of the in vitro growth of Plasmodium falciparum (strain FCB). While certain monosaccharides (N-acetyl-D-glucosamine, D-mannose, and 3-O-methyl-D-glucose) proved to exhibit a toxic or reversibly retarding effect on the intraerythrocytic development of the parasite, the corresponding alpha- or beta-methylglycosides did not. Several methylglycosides, synthetic di- and tri-saccharides, and artificial blood group antigens were further tested for inhibitory effects on invasion of host red blood cells in vitro. The synthetic disaccharides beta DGlcNAc(1----4) alpha DManOMe and beta DGlcNAc(1----4) DGlcNAc (chitobiose) were good inhibitors of invasion at 10 mM concentration, whereas beta DGal(1----4)beta DGlcNAcOMe was negligibly inhibitory. The inhibition rate of N-acetyl-D-glucosamine, beta-glycosidically linked to bovine serum albumin (BSA) by an alipathic spacer, -(CH2)8CO-, was not enhanced, compared to the corresponding hapten, beta DGlcNAcO(CH2)8COOCH3. The inhibition rates of blood group A- and B-trisaccharide haptens, which were inhibitors of invasion, were also not significantly enhanced when coupled to BSA by way of the corresponding amide spacer, -(CH2)2NHCO(CH2)7CO-. A remarkable enhancement of the inhibition rate was, however, observed when beta DGal(1----3) alpha DGalNAcO(CH2)2NHCO(CH2)7COOCH3 (T-hapten) was coupled to BSA. A clear-cut decrease in the inhibition rates of different beta-glycosides of N-acetyl-D-glucosamine, beta DGlcNAcOR, was observed, depending on the nature of the aglycon R(p-nitrophenyl greater than -(CH2)8COOCH3 greater than -(CH2)2NHCO(CH2)2COOCH3 greater than -CH3). Also, p-nitrophenyl-alpha-D-glucopyranoside was a much better inhibitor of invasion than the corresponding methyl glycoside, alpha DGlcOMe, which was not inhibitory. The properties of the aglycon spacer, used for the covalent attachment of the carbohydrate to the carrier protein, may thus be crucial for the outcome of the inhibition rate.     

Fasciola hepatica and Schistosoma mansoni: immunofluorescent antigen localization and cross-reactivity

In an attempt to identify the tissue sources of biochemically purified antigenic fractions of Fasciola hepatica and Schistosoma mansoni, antisera were tested against plastic-embedded sections of worms of various ages by an indirect fluorescent-antibody-labeling technique. Antibodies prepared against antigens purified by chromatography of F. hepatica whole worm extract through concanavalin A-Sepharose 4B labeled the parenchyma and tegument of adult F. hepatica strongly while antibodies developed against antigens purified by antibody-affinity chromatography against antibodies of S. mansoni labeled only the parenchyma. Antigens common to these two groups clearly originated from F. hepatica parenchyma. Certain of these common antigens are known to provide significant protection in mice to challenge with S. mansoni cercariae, and in the present study antisera against F. hepatica extracts cross-labeled S. mansoni adult male parenchyma. Reciprocal cross-reactions between antisera against S. mansoni and the parenchyma of adult F. hepatica were also noted. FhFIIb, an extract of F. hepatica which Tailliez described as not cross-reacting with S. mansoni, was found to contain no F. hepatica parenchymal antigens. Antigenic fractions of F. hepatica and S. mansoni collected from the surface of worms after incubation in nonionic detergent were unexpectedly found to contain much parenchymal antigen, suggesting leakage of internal components into the supernatant during preparation. Antisera to F. hepatica developed during a natural infection in rabbits labeled tegumental components and gut strongly but did not react with parenchymal tissue. Antisera against extracts of adult schistosomes labeled the parenchyma of male worms and the glycocalyx of the cercarial tegument, indicating the presence of common antigens in the adult and the cercarial stage. Reciprocal reactions between anticercarial sera and adult sections provided further evidence of shared antigenicity. Antisera against S. mansoni egg antigens strongly labeled sections of eggs in liver tissue and cross-reacted with cercarial glycocalyx, indicating the existence of common antigens between these two stages. The antisera also cross-reacted with what appeared to be non-membrane-bound protein in the tegument of F. hepatica. The soluble egg antigen extract shared antigenicity with the parenchyma of both S. mansoni and F. hepatica but circumoval precipitin had no cross-reactivity with this tissue. Thus S. mansoni eggs contain nondiffusable components sharing antigenic specificity with adult parenchymal tissue.(ABSTRACT TRUNCATED AT 400 WORDS)     

Patterns of nucleotide substitutions inferred from the phylogenies of the class I major histocompatibility complex genes

Summary Patterns of nucleotide substitutions in human major histocompatibility complex (MHC) class I genes were estimated by using phylogenetic trees of DNA sequences. The pattern is defined as a set of 12 parameters, each of which represents the relative frequency of substitutions from a particular nucleotide to another. The pattern at the antigen recognition sites (ARS) in functional MHC genes was remarkably different from that at the remaining coding region (non-ARS). In particular, the proportion of transitions among all the nucleotide substitutions (P _s) was extremely low at the third codon positions of ARS. In the HLA-A genes, P _s at the third codon positions was only 6% in ARS, whereas it was 69% in non-ARS. In HLA-B, the corresponding values were 30% in ARS and 80% in non-ARS, respectively. On the other hand, P _s in a class I pseudogene (HLA-H) was 57%, which was in good agreement with P _s in other pseudogenes. Because pseudogenes are selectively neutral, the pattern in pseudogenes is regarded as the pattern of spontaneous substitution mutations. In general, the pattern in functional genes that are subject to selective forces deviates from the pattern in pseudogenes. At the third codon positions in coding regions, transitions scarcely cause amino acid replacements, whereas about half of transversions do cause replacements. Accordingly, P _s at the third codon positions decreases if amino acid replacements are accelerated by natural selection but increases if amino acids are conserved by functional constraint. Our observations imply that the ARS region is subject to natural selection favoring amino acid replacements, whereas the non-ARS region is subject to functional constraint. Offprint requests to: T. Gojobori