大鼠精核蛋白抗血清的制备

用大鼠精核蛋白－核糖核酸复合物免疫大鼠得到了特异的抗ＲＰ抗血清,并用Ｉｍｍｕｎｏｄｏｔｔｉｎｇ和Ｉｍｍｎｕｏｂｌｏｔｔｉｎｇ方法验证了其特异性。该抗血清和ＲＰ有特异的反应,并和哺乳动物小鼠和羊的精核蛋白有一定程度交叉反应,而与体细胞类型核蛋白无交叉反应。并对制备该抗血清的意义予以讨论。     

双链核糖核酸免疫化学研究I．双链多聚[I]：多聚[c]的抗原性

用国产多聚肌苷酸和多聚胞嘧啶核苷酸(简称多聚[I]：多聚[c])制备成双链核糖核酸特异性抗血清,用环状沉淀和’H标记的番茄花叶病毒双链核糖核酸测得抗血清效价分别为1：128和1：6400。用免疫琼脂双扩散、对流免疫电泳和放射免疫测定可测定出多聚[I]：多聚[c]的最低浓度分别为391ng、12．2／ng和10pg／ml抗血清和酵母、TMV、P。X的单链核糖核酸、小牛胸腺DNA不反应。试验证明用国产多聚[I]：多聚[C]制备的抗血清,可有效地用于双链核糖核酸的免疫化学鉴定。由于在免疫双扩散和对流免疫电泳中抗血清能和微量双链核糖核酸反应并产生可见沉淀线,从而有可能利用这两种简便灵敏的方法测定病毒的双链RNA,单链RNA病毒的RF型RNA,以及双链RNA真菌病毒的筛选。     

双链核糖核酸免疫化学研究 Ⅰ.双链多聚[I]:多聚[C]的抗原性

用国产多聚肌苷酸和多聚胞嘧啶核苷酸(简称多聚[I]:多聚[C])制备成双链核糖核酸特异性抗血清,用环状沉淀和~3H标记的番茄花叶病毒双链核糖核酸测得抗血清效价分别为1:128和1:6400。用免疫琼脂双扩散、对流免疫电泳和放射免疫测定可测定出多聚[I]:多聚[C]的最低浓度分别为391ng、12.2/ng和10pg/ml抗血清和酵母、TMV、PVX 的单链核糖核酸、小牛胸腺DNA不反应。试验证明用国产多聚[I]:多聚[C]制备的抗血清,可有效地用干双链核糖核酸的免疫化学鉴定。由于在免疫双扩散和对流免疫电泳中抗血清能和微量双链核糖核酸反应并产生可见沉淀线,从而有可能利用这两种简便灵敏的方法测定病毒的双链RNA,单链RNA病毒的RF型RNA,以及双链RNA真菌病毒的筛选。     

免疫核糖核酸研究的进展

从定型的免疫活性细胞中抽提出的核糖核酸制品称为免疫核糖核酸。各种体内或体外的试验证明免疫核糖核酸能超越种间屏障特异地传递免疫反应。现在有证据表明,免疫核糖核酸可能具有信息核糖核酸的分子特征;它的主要来源是巨噬细胞,而受其作用后产生效应的细胞是 T 淋巴细胞或 B 淋巴细胞。免疫核糖核酸研究是一项意义重大而探索性较强的工作,本文着重讨论了关于其化学本质、细胞来源和作用机理等方面的新近进展,并且指出了今后工作中有待解决的若干关键问题。     

外泌体形成分泌调控及其在肿瘤发生发展中的作用

外泌体(exosome)是起源于细胞内体的双层膜小泡。作为细胞间重要的介质,外泌体转运脂质、蛋白质、RNA等物质,从而发挥重要的生物学功能。外泌体的形成与分泌需要经过一个多因素、多阶段参与的精细调控过程。供体细胞分泌负载内含物的外泌体进入肿瘤微环境,随后受体细胞以不同的作用方式摄取其中的内含物,引起肿瘤的侵袭转移、免疫逃逸、治疗抵抗等。近年来,外泌体已成为医学研究的热点之一。现就外泌体形成分泌调控及其在肿瘤发生发展中的作用进展予以综述。     

免疫核糖核酸

免疫核糖核酸(immune RNA简称iRNA)是七十年代出现的一种新的特异性免疫制剂,由于它能够超越种间界限传递特异性的免疫应答能力,而受到越来越多的重视,现已应用到肿瘤及某些传染病及感染的治疗方面,并已取得一些可观的成果。本文介绍了关于iRNA研究的近况。     

大鼠精核蛋白抗血清的制备

用大鼠精核蛋白（Ｒａｔ　Ｐｒｏｔａｍｉｎｅ,ＲＰ）－核糖核酸（ＲＮＡ）复合物（ＲＰ－ＲＮＡ　Ｃｏｍｐｌｅｘｅｓ）免疫大鼠,得到了特异的抗ＲＰ抗血清,并用Ｉｍｍｕｎｏｄｏｔｔｉｎｇ和Ｉｍｍｕｎｏｂｌｏｔｔｉｎｇ方法验证了其特异性。该抗血清和ＲＰ有特异的反应,并和哺乳动物小鼠和羊的精核蛋白有一定程度交叉反应,而与体细胞类型核蛋白（Ｈ１,Ｈ２ａ,Ｈ２ｂ,Ｈ３,Ｈ４）无交叉反应。并对制备该抗血清的意义予以讨论。     

基于核酸适配体的肿瘤免疫治疗进展

肿瘤免疫治疗是通过调节机体的免疫功能来控制和杀伤肿瘤的一种治疗手段。针对免疫检查点的治疗等一系列临床突破使得肿瘤的免疫治疗受到了广泛重视。目前,抗体治疗和过继性细胞治疗是肿瘤免疫治疗的主要方式,但是这些方法仍具有副作用较强,实体瘤治疗难以实现,治疗费用高昂等缺点。因此改进和发展更加高效、安全、低成本的新技术仍十分必要。适配体是利用指数富集的配体系统进化技术筛选得到的单链寡核苷酸,有核酸"抗体"之称。适配体具有低免疫原性、组织穿透力强、易于化学合成与修饰等优势,且与其靶标的结合具有较好亲和力和特异性,可像抗体一样实现肿瘤的免疫治疗。对适配体在肿瘤免疫治疗相关技术中的新应用作一综述,主要包括基于免疫检查点的抗肿瘤作用、双特异性适配体的肿瘤免疫治疗、适配体靶向递送siRNA的肿瘤免疫治疗和适配体联合抗体的肿瘤免疫治疗等方面。     

羊口疮病毒蛋白ORFV035的表达、纯化和多克隆抗体的制备

克隆表达羊口疮病毒蛋白ORFV035,并制备其多克隆抗体,为后续对病毒复制、装配、形态发生和成熟过程的研究奠定基础。PCR扩增羊口疮病毒ORFV035基因,将其与质粒pET-30a(+)经Bam HⅠ和HindⅢ双酶切后连接,构建重组质粒pET30a-035。重组质粒经双酶切和测序鉴定,转化感受态大肠杆菌BL21,IPTG诱导表达,SDS-PAGE鉴定蛋白表达情况。表达产物进行超声破碎和Ni柱纯化,纯化后目的蛋白免疫小鼠,制备多克隆抗体并对其进行鉴定。成功构建了重组质粒pET30a-035,在大肠杆菌BL21中以包涵体形式高效表达。包涵体洗涤、溶解后进行Ni柱纯化,得到纯度较高的ORFV035-his融合蛋白。以纯化蛋白免疫小鼠获得多克隆抗体。Western blot检测显示该多抗可以识别天然ORFV035蛋白。成功诱导表达、纯化ORFV035蛋白并制备ORFV035多克隆抗体。     

Transfer Agent of Immunity

Detection of the cells which contain antibody was accomplished by a method of immune adherence of human erythrocytes to a single cell, termed SCIA (single cell immune adherence) reaction. Peritoneal exudate cells were collected from mice immunized with flagella of either Salmonella enteritidis or S. tennessee. Serologically specific antibody was detectable in some of the peritoneal exudate cells of such mice. An immune ribonucleic acid (RNA) was extracted from the peritoneal exudate cells of mice immunized with salmonella flagella. When mice were injected intraperitoneally with this preparation, serologically specific antibody was found in some of their peritoneal exudate cells by the SCIA method. This preparation was inactivated by treatment with ribonuclease, but was resistant to proteinases, deoxyribonuclease and anti-flagella antibody, suggesting that this agent is of RNA nature and does not contain antigen or fragment thereof.     

Peripheral immune cell gene expression predicts survival of patients with non-small cell lung cancer

Prediction of cancer recurrence in patients with non-small cell lung cancer (NSCLC) currently relies on the assessment of clinical characteristics including age, tumor stage, and smoking history. A better prediction of early stage cancer patients with poorer survival and late stage patients with better survival is needed to design patient-tailored treatment protocols. We analyzed gene expression in RNA from peripheral blood mononuclear cells (PBMC) of NSCLC patients to identify signatures predictive of overall patient survival. We find that PBMC gene expression patterns from NSCLC patients, like patterns from tumors, have information predictive of patient outcomes. We identify and validate a 26 gene prognostic panel that is independent of clinical stage. Many additional prognostic genes are specific to myeloid cells and are more highly expressed in patients with shorter survival. We also observe that significant numbers of prognostic genes change expression levels in PBMC collected after tumor resection. These post-surgery gene expression profiles may provide a means to re-evaluate prognosis over time. These studies further suggest that patient outcomes are not solely determined by tumor gene expression profiles but can also be influenced by the immune response as reflected in peripheral immune cells.     

Specific MIF release by rat lymphocytes following incubation with syngeneic anti-tumor “Immune” RNA

Other investigators have previously shown that normal nonimmune lymphoid cells, after incubation with “Immune” RNA, will release MIF when these cells are incubated with the specific antigen used to immunize the RNA donor. This conversion can be detected with the macrophage migration inhibition assay. These observations have been confirmed in a system involving the transfer of immune response to tumor associated antigens with syngeneic “Immune” RNA. Syngeneic “Immune” RNA was extracted from the spleens of Fischer 344/N rats bearing growing transplants of one or another of two syngeneic chemically induced sarcomas. Normal, nonimmune Fischer 344/N spleen cells were incubated with these RNA preparations. When these RNA-incubated spleen cells were exposed to solubilized antigens from that particular tumor used to immunize the RNA donor, MIF was released. RNAse treatment of the “Immune” RNA abrogated the response, while DNAse or pronase treatment did not.     

Gene Expression Analysis of Peripheral Cells for Subclassification of Pediatric Inflammatory Bowel Disease in Remission

Objective

In current clinical practice, optimal treatment of inflammatory bowel disease (IBD) aims at the induction and maintenance of clinical remission. Clinical remission is apparent when laboratory markers of inflammation are normal and clinical symptoms are absent. However, sub-clinical inflammation can still be present. A detailed analysis of the immune status during this inactive state of disease may provide a useful tool to categorize patients with clinical remission into subsets with variable states of immune activation.

Design

By using Affymetrix GeneChips, we analysed RNA gene expression profiles of peripheral blood leukocytes from pediatric IBD patients in clinical remission and controls. We performed (un)supervised clustering analysis of IBD-associated genes and applied Ingenuity® pathway software to identify specific molecular profiles between patients.

Results

Pediatric IBD patients with disease in clinical remission display heterogeneously distributed gene expression profiles that are significantly distinct from controls. We identified three clusters of IBD patients, each displaying specific expression profiles of IBD-associated genes.

Conclusion

The expression of immune- and IBD-associated genes in peripheral blood leukocytes from pediatric IBD patients in clinical remission was different from healthy controls, indicating that sub-clinical immune mechanisms are still active during remission. As such, RNA profiling of peripheral blood may allow for non-invasive patient subclassification and new perspectives in treatment regimes of IBD patients in the future.     

Transfer Agent of Immunity

Using erythrocytes as antigen particles, number of antibody-forming cells was enumerated by immunocytoadehesion technique, in which formation of rosette was shown to be inhibited by anti-mouse immunoglobulin sera. This number increased in vitro after treatment of spleen cells of mice for 60 min with RNA fraction extracted from spleen of mice immunized with erythrocytes used in the enumeration, and incubation of cells for 12 hr at 37 C. Response of cells treated with immune RNA fraction was immunologically specific and was inhibited by puromycin or cycloheximide. The activity of immune RNA capable of converting nonimmune cells to antibody-forming cells was shown to be sensitive to ribonucleases but resistant to deoxyribonuclease and proteolytic enzyme.     

Effect of RNA from brain of immunized mice on the specific immune response

The objective of the investigation was to study the influence of RNA isolated from the brains of immunized mice on the immune response in syngeneic recipients. (CBAxC57BL/6)F1 male mice immunized by SRBC and RRBC were used for isolation of experimental RNA. Mice injected with medium 199 were used for isolation of control RNA. Experimental and control RNA was injected into syngeneic mice recipients immunized by SRBC, RRBC or rat RBC preliminary. It was shown that experimental RNA stimulate the specific immune response only.     

Use of I125 labelled poly(I). poly(C) for determination of antibodies to double-stranded RNA by immune complex absorption to nitrocellulose filters]

125I-labeled double-stranded polyribonucleotide complex was used for detection of antibodies to double-stranded RNA in sera from people and immunized animals by the method of immune complex adsorption by the nitrocellulose filters. The technique is simple and sensitive. Antibodies to double-stranded RNA WERE detected in sera from patients with different diseases and from normal individuals. Systemic lupus erythematosus sera contain as a rule higher amounts of antibodies to double-stranded RNA. Often these antibodies were measured together with those directed towards native and denatured DNA. Anti-double-stranded RNA antibodies from sera of immunised animals and patients with systemic lupus erythematosus are highly specific.     

T-cell responses to multiple antigens presented by RNA-transfected APCs: a possible immunomonitoring tool

The increasingly deeper understanding of how the immune system recognizes and destroys tumors promises to enable the development of new approaches for gene therapy and immunotherapy. However, a treatment that induces safe and potentially beneficial antitumor responses is expected to require stepwise refinements. As part of this challenge, assays are needed to measure specific antitumor immune responses in patients. This becomes problematic because most tumors express unknown tumor antigens and it is often difficult to obtain sufficient amounts of viable tumor material for in vitro assays. Recently it was demonstrated that RNA derived from tumor cells stimulated T cells in an antigen-specific manner. These studies have formed the basis for the development of dendritic cell vaccines that express tumor antigens following translation of tumor RNA. Therefore, it occurred to us that antigen-presenting cells transfected with total tumor RNA might also be valuable in monitoring the antitumor responses induced in patients who participate in clinical trials. To test this hypothesis, we developed a model in which Epstein-Barr virus (EBV)–transformed lymphoblastoid cell lines were used as a source of RNA. Since this RNA encodes for known EBV antigens, it was possible to determine whether the expected responses were observed. Our results show for the first time that T cells primed to APC transfected with RNA isolated from EBV-infected lymphocytes exhibited a fine specificity that enabled them to recognize individual EBV antigens.This work was supported by a Developmental Research Grant from the Derald H. Ruttenberg Cancer Center, Mount Sinai School of Medicine.     

Some perspectives on the transfer of cell-mediated immunity by immune-RNA

Summary Ribonucleic acid extracts of lymphoid cells from immune hosts were used to transfer in vivo and in vitro cell-mediated immune reactivity to a variety of antigens. The in vivo immune responses transferred by RNA included the delayed cutaneous hypersensitivity reaction to fungal and chemically-defined antigens and the tumor-rejection reaction to guinea pig hepatoma antigens. The in vitro immune responses transferred by RNA included macrophage migration inhibition by fungal, chemically-defined, and tumor antigens. The transfer activity of RNA preparations was contained in the 8 s to 18 s species of RNA and was sensitive to RNAse but not to DNase or trypsin. Antigen was not detectable in the RNA preparations and appeared to have no role in the transfer activity. Syngeneic, allogeneic, or xenogeneic sources of RNA could transfer immune reactivity. In each system tested, the transfer of cell-mediated reactivity by RNA was specific for the antigen used to sensitize the RNA donor. The potential use of RNA-mediated transfer of immunity is discussed.