Immunological Relationships Between Hexons of Certain Human Adenoviruses

Antisera against hexons of serotypes 2, 4, 5, and 6 (subgroup III), and 15 (subgroup II) were absorbed with purified hexons of various serotypes representing the different subgroups of human adenoviruses. Group, subgroup, and type specificities of hexons could be distinguished. The subgroup specificity of type 4 hexons resembled that of hexons of subgroup I members (types 3, 11, and 16). Antihexon sera gave a type-specific inhibition of virion-associated hemagglutinin. The inhibiting activity of different sera was found to be inversely related to the length of fibers of the serotype concerned. Virions of serotypes carrying fibers shorter than about 20 nm (types 3, 4, 9, 11, and 15) were readily inhibited, whereas those of serotypes with longer fibers (types 2 and 6) were inhibited only by relatively large amounts of antibody measured in terms of homotypic complement fixation activity. The reciprocal cross-neutralization between serotypes 4 and 16 was studied separately. Hexons of both serotypes each carried a type-specific component and, in addition, a unique antigen specificity common to the two types. This common antigen specificity was interpreted to be available to a larger extent at the surface of virions (and probably also isolated hexons) of type 4 than of type 16. These results suggest an explanation for the predominantly one-sided character of the cross-neutralization between types 4 and 16.     

Hybrid of Monkey and Human Adenoviruses

Following joint replication of monkey SA7 adenovirus (C8 strain) and human adenovirus type 2 in green monkey kidney tissue culture, a virus possessing the properties of a hybrid was obtained. It was designated Ad2C8. Ad2C8 preparations contained two types of viral particles: human adenovirus type 2, and hybrid particles. The hybrid virions multiplied in green monkey kidney cells in the presence of human adenovirus types 1, 2, and 3, but not 3 and 7, and acquired the capsid of the helper adenovirus. The hybrid can serve as a helper for human adenoviruses. It can apparently induce T antigen of the C8 virus but, in contrast to the latter, does not induce tumors in hamsters.     

Capsid Mosaics of Intermediate Strains of Human Adenoviruses

Antisera against isolated capsid components of intermediate adenovirus strains, types 3-16 (the San Carlos agent) and 15-9 and of "parental" prototype strains were compared in neutralization tests, hemagglutination-inhibition (HI) tests employing soluble and virion-associated hemagglutinin as antigens, and by electron microscopy. Hexons of the intermediate strains were found to be similar to, but not identical to, those of the prototype strains with which a cross-reaction occurred in neutralization tests (types 3 and 15). In contrast, fibers of intermediate strains displayed characteristics relating them to the corresponding components of prototype strains to which a relationship has been found in HI tests. Fibers (and possibly even pentons) of types 9 and 15-9 appeared to be identical, whereas fibers of types 3-16 and 16 displayed antigen specificities of both common and unique nature.     

Hybrids of Human and Monkey Adenoviruses (Adeno-Adeno Hybrids) That Can Reproduce in Monkey Cells: Biological and Molecular Genetic Peculiarities

A highly oncogenic monkey adenovirus SA7(C8) facilitates the reproduction of human adenovirus type 2 (Ad2) in monkey cells. Upon mixed infection of monkey cells with both viruses, these viruses recombine producing defective adeno-adeno hybrids Ad2C8 serologically identical to Ad2 and capable of assisting Ad2 to reproduce in monkey cells. Ad2C8 and Ad2 form an intercomplementary pair inseparable in monkey cells. Unlike oncogenic SA7(C8), Ad2C8 is a nononcogenic virus for hamsters but is able to induce tumor antigens of this virus (T and TSTA). Molecular genetic analysis of 68 clones of adeno-adeno hybrids revealed that the left part of their genome consists of Ad2 DNA, and the right part contains no less than 40% of the viral SA7(C8) genome where E2A, E3, and E4 genes are located. Apparently, the products of these genes contribute to the composition of adenoviral tumor antigens, while the E4 gene is involved in complementation of monkey and human adenoviruses and makes a contribution to host range determination of these viruses.     

Quantification and Stability of Human Adenoviruses and Polyomavirus JCPyV in Wastewater Matrices

Human adenoviruses (HAdV) and human polyomavirus JCPyV have been previously proposed as indicators of fecal viral contamination in the environment. Different wastewater matrices have been analyzed by applying real-time quantitative PCR procedures for the presence, quantity, and stability of a wide diversity of excreted HAdV and JCPyV. High quantities of HAdV and JCPyV were detected in sewage, effluent wastewater, sludge, and biosolid samples. Both viruses showed high stability in urban sewage. These results confirm the suitability of both viruses as indicators of human fecal viral pollution.     

Quantification of Enterococci and Human Adenoviruses in Environmental Samples by Real-Time PCR

Pathogenic bacteria and enteric viruses can be introduced into the environment via human waste discharge. Methods for rapid detection and quantification of human viruses and fecal indicator bacteria in water are urgently needed to prevent human exposure to pathogens through drinking and recreational waters. Here we describe the development of two real-time PCR methods to detect and quantify human adenoviruses and enterococci in environmental waters. For real-time quantification of enterococci, a set of primers and a probe targeting the 23S rRNA gene were used. The standard curve generated using Enterococcus faecalis genomic DNA was linear over a 7-log-dilution series. Serial dilutions of E. faecalis suspensions resulted in a lower limit of detection (LLD) of 5 CFU/reaction. To develop real-time PCR for adenoviruses, degenerate primers and a Taqman probe targeting a 163-bp region of the adenovirus hexon gene were designed to specifically amplify 14 different serotypes of human adenoviruses, including enteric adenovirus serotype 40 and 41. The standard curve generated was linear over a 5-log-dilution series, and the LLD was 100 PFU/reaction using serial dilutions of purified adenoviral particles of serotype 40. Both methods were optimized to be applicable to environmental samples. The real-time PCR methods showed a greater sensitivity in detection of adenoviruses in sewage samples than the viral plaque assay and in detection of enterococci in coastal waters than the bacterial culture method. However, enterococcus real-time PCR overestimated the number of bacteria in chlorinated sewage in comparison with the bacterial culture method. Overall, the ability via real-time PCR to detect enterococci and adenoviruses rapidly and quantitatively in the various environmental samples represents a considerable advancement and a great potential for environmental applications.     

Replication and Complementation of Human Adenoviruses and Simian Papovavirus at an Elevated Temperature

The simian papovavirus SV40 replicated as well in simian cells incubated at 41 C as in cells incubated at 37 C, although the latent period was shortened at the elevated temperature. Human adenoviruses differed in their responses to the elevated temperature. Some serotypes, such as 3, 4, 5, 7, 8, 16, and 21, replicated as well, or almost as efficiently, in human cells incubated at 41 C as in cells incubated at 37 C, whereas with other serotypes, such as 1, 2, 6, 12, and 14, maximal yields in cultures incubated at 41 C were much lower than the yields from companion cultures incubated at 37 C. This difference was also detected in simian cells co-infected with SV40 and a human adenovirus; maximal complementation occurred with some serotypes at the elevated temperature but not with other serotypes. The degree of complementation observed in the simian cells at 41 C was directly correlated with the ability of the adenovirus to replicate at 41 C in human cells. Therefore, the capacity of SV40 to serve as a helper virus is not affected by the elevated temperature, showing that the complementation event supplied by the simian virus is heat-stable between 37 and 41 C. Maximal complementation appeared to depend upon a characteristic present in the adenovirus genome.     

综述与专论: 免疫突触形成的生物学特点及其光学成像研究

免疫突触(immunological synapse,IS)是抗原提呈细胞与T细胞免疫识别时,多种分子参与、分阶段不断变化的过程,涉及黏附分子、细胞因子、信号传导分子、细胞骨架蛋白等多分子的聚集或离散．其形成不仅促进T细胞和抗原提呈细胞的稳定接触,而且激活T细胞信号传导途径,促进T细胞的活化和增殖．对IS的研究可以从分子水平解释免疫激活、免疫耐受、病原微生物感染与免疫细胞相互作用的机制,为进一步揭示疾病发生的分子机制,寻求疾病防治的靶向分子提供新的思路．近年来,光学成像的发展为可视化研究IS形成与T细胞活化的关系提供了有力帮助,为研究生理病理状态下的免疫应答提供了有力工具．     

Human Adenoviruses and Coliphages in Urban Runoff-Impacted Coastal Waters of Southern California

A nested-PCR method was used to detect the occurrence of human adenovirus in coastal waters of Southern California. Twenty- to forty-liter water samples were collected from 12 beach locations from Malibu to the border of Mexico between February and March 1999. All sampling sites were located at mouths of major rivers and creeks. Two ultrafiltration concentration methods, tangential flow filtration (TFF) and vortex flow filtration (VFF), were compared using six environmental samples. Human adenoviruses were detected in 4 of the 12 samples tested after nucleic acid extraction of VFF concentrates. The most probable number of adenoviral genomes ranged from 880 to 7,500 per liter of water. Coliphages were detected at all sites, with the concentration varying from 5.3 to 3332 PFU/liter of water. F-specific coliphages were found at 5 of the 12 sites, with the concentration ranging from 5.5 to 300 PFU/liter. The presence of human adenovirus was not significantly correlated with the concentration of coliphage (r = 0.32) but was significantly correlated (r = 0.99) with F-specific coliphage. The bacterial indicators (total coliforms, fecal coliforms, and enterococci) were found to exceed California recreational water quality daily limits at 5 of the 12 sites. However, this excess of bacterial indicators did not correlate with the presence of human adenoviruses in coastal waters. The results of this study call for both a reevaluation of our current recreational water quality standards to reflect the viral quality of recreational waters and monitoring of recreational waters for human viruses on a regular basis.     

人尿源性干细胞及人脐带间充质干细胞的生物学性状比较

该研究探讨人尿源性干细胞(human urine-derived stem cells,hUSCs)及人脐带间充质干细胞(human umbilical cord mesenchymal stem cells,hUC-MSCs)的生物学性状差异。分离培养hUSCs及hUC-MSCs,显微镜下观察细胞形态,流式细胞术检测干细胞表面标记物,锥虫蓝拒染实验及克隆形成实验检测细胞增殖能力,划痕实验及Transwell迁移实验检测细胞迁移能力,碱性磷酸酶(alkaline phosphatase,ALP)染色、茜素红染色、油红O染色及阿利新蓝染色评估多向分化潜能。hUSCs为米粒状贴壁生长细胞,hUC-MSCs为长梭形贴壁细胞,呈旋涡状排列生长,两种细胞表型分析相似,均表达多种间充质干细胞标志物,但CD24在hUC-MSCs表达阳性,而CD105在hUSCs表达阳性。hUC-MSCs的增殖及迁移能力优于hUSCs,但后者的克隆形成能力更强。hUSCs及hUCMSCs都具有成骨、成脂、成软骨分化能力,hUC-MSCs的成骨能力强而hUSCs的成脂能力强。该研究成功分离培养出增殖能力强并具有多向分化潜能的hUSCs,该细胞与hUC-MSCs相比具有相似的生物学性状,可作为再生医学自体移植的理想种子细胞来源。     

人胚髁状突软骨细胞的改良培养及生物学特性的研究

目的:探讨行之有效的人胚髁状突软骨细胞体外分离培养方法及研究其生物学特性.方法:用0.25%胰蛋白酶和0.2%Ⅱ型胶原酶分阶段联合消化法分离髁状突软骨细胞,将分离的软骨细胞与未消化完的小片软骨共同置入预涂多聚赖氨酸的培养瓶中培养,通过倒置显微镜,免疫组织化学染色、电镜观察等方法与传统培养方法对比,检测软骨细胞的分离后存活率、贴壁生长速度及传代7次内髁状突软骨细胞表型改变相伴随的形态学及生物学特征.结果:本方法培养的人髁突软骨细胞存活率可达98%以上,细胞贴壁能力强,与传统培养方法形成细胞单层的速度相比具有统计学意义(p<0.05).7代以内培养的髁状突软骨细胞免疫组织化学染色阳性,透射电镜可见细胞内有丰富的粗面内质网及线粒体.而传统培养方法所获得的人髁状突软骨细胞存活率为90%左右,培养3代以后免疫组织化学染色显示其软骨细胞表型逐渐减弱,培养至第7代其免疫组织化学染色为阴性.结论:本方法培养的髁状突软骨细胞存活率高,且能维持软骨细胞的特有表型,至少能保持软骨细胞7代稳定,是一种简便、有效的培养方法.     

Biological and Immunological Properties of Recombinant Human, Rat, and Chicken Nerve Growth Factors: A Comparative Study

Biological and immunological properties of recombinant human, rat, and chicken nerve growth factors (NGFs) were studied and compared. Recombinant NGF proteins were produced in a transient expression system using COS cells and levels of secreted NGF protein were assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of conditioned media from in vivo [³⁵S]cysteine-labeled cell cultures. Antigenic differences among the three NGFs were studied by immunoblotting and immunoprecipitation of secreted cell products using a rabbit polyclonal antiserum against purified mouse NGF, and by a two-site enzyme im-munoassay (EIA) with a monoclonal antibody against mouse NGF. Although all three NGFs were recognized equally well in the immunoblotting, only one-third of the chicken NGF protein could be detected by immunoprecipitation or by the EIA as compared to the rat and human NGFs. Thus, changes in the three-dimensional structure of the NGF molecule are most likely responsible for the antigenic differences between avian and mammalian NGFs. The three NGF proteins were also compared in their ability to displace ¹²⁵I-mouse NGF from low-affinity NGF receptors on rat pheochromocytoma PC12 cells. Similar displacement curves and values were obtained for each NGF protein, indicating that structural differences among these molecules do not affect low-affinity binding to NGF receptors. Biological activities were studied by the ability of the conditioned media to promote neurite outgrowth from explants of 9 chick sympathetic ganglia and from PC12 ceils. Although the rat system showed a slight preference for the homologous molecule, the morphological changes, dose-response curves, and maximal stimulation values obtained with the different NGFs were practically indistinguishable in the chicken bioassay. The observed differences and similarities among the three NGF proteins are discussed in the context of their evolutionary relationship and their potential therapeutic applications.     

人滑膜间充质干细胞与人脐带间充质干细胞的生物学性状比较

该文旨在比较人滑膜间充质干细胞(human synovial mesenchymal stem cells,hSMSCs)与人脐带间充质干细胞(human umbilical cord mesenchymal stem cells,hUC-MSCs)的生物学性状.流式细胞仪鉴定hSMSCs和hUC-MSCs.比较两种间...     

Immunological Studies of Human Monoamine Oxidases

Antisera have been raised against monoamine oxidase preparations from human placenta and platelets. These antisera have been employed to characterize membrane-bound enzyme from a variety of human sources including liver, heart, and brain. The comparisons were based on a displacement radioimmunoassay system with soluble placental monoamine oxidase, previously labelled specifically with [3H]pargyline, as antigen. All forms of enzyme investigated demonstrated immunological cross reaction; however, the placental enzyme appeared to possess determinants not exhibited by the enzyme from the platelets or other tissues examined.     

Occurrence of Human Adenoviruses at Two Recreational Beaches of the Great Lakes

Human adenoviruses (HAdVs) have been related to several waterborne diseases such as acute gastroenteritis, conjunctivitis, and respiratory illness, and it has been shown that an important human exposure pathway is through recreational waters. However, HAdV occurrence at recreational freshwater beaches has not been previously investigated. In this study, a total of 58 water samples were collected from two recreational beaches on Lake Michigan (i.e., Silver Beach and Washington Park Beach) during the summer of 2004. Occurrences of HAdVs in these lake samples were determined using two hexon-based real-time PCR assays (one for monitoring all 51 serotypes of HAdVs and another for specifically detecting F species HAdVs, i.e., serotypes 40 and 41) and compared to an integrated cell culture (ICC) PCR method. The real-time PCR results showed that 8 of 30 Silver Beach samples and 6 of 28 Washington Park Beach samples contained HAdVs, and F species HAdVs were detected in three of these positive samples. The concentrations of HAdVs ranged from (1.7 ± 0.7) × 10¹ to (3.4 ± 0.8) × 10² and from (7 ± 2) × 10⁰ to (3.8 ± 0.3) × 10³ virus particles/liter for Silver Beach and Washington Park Beach, respectively. F species HAdVs were detected at levels ranging from (4.8 ± 0.8) × 10¹ to (4.6 ± 1.5) × 10² virus particles/liter. Approximately 60% of the ICC-PCR analyses agreed with the real-time PCR results. This study revealed the occurrence of HAdVs at Lake Michigan recreational beaches. Given the potential health risks, further assessment regarding sources, virus transport, and survival is needed to improve the safety of the region.     

人Flt3配体在酵母中的表达及其生物学特性

Ｆｌｔ３配体（ＦＬ）是一种具有促进早期造血功能的细胞因子,能够促进造血干／祖细胞的增殖和分化。为了获得高效表达的人可溶性Ｆｌｔ３配体（ｒｈＦＬ）重组蛋白,采用酵母偏爱的密码子,人工合成ｒｈＦＬ基因,构建表达载体ｐＰＩＣＺａＡ－ＦＬ,电转化毕氏酵母９Ｐｉｃｈｉａ ｐａｓｔｏｒｉｓ）,得到稳定分泌ｒｈＦＬ的基因工程菌株,在培养上清中,其表达量达３０ｍｇ／Ｌ。生物学活性检测提示,毕氏酵母系统表达的ｒｈＦ