iRNA对家兔急性实验性血清病的免疫调节作用

本实验以一次性静脉注射BSA诱发的家兔急性实验性血清病(AESS)为模型。于免疫后连续4天用iRNA处理模型动物,观察了iRNA对AESS家兔的血清学和免疫病理学影响,并建立了家兔血清补体旁路活性的检测方法。结果表明:iRNA能降低AESS家兔循环特异性抗体和特异性免疫复合物水平,减轻血清补体消耗,减少肾小球内免疫复合物的沉积和炎性细胞的浸润,使肾炎病变得以缓解。提示:iRNA可能通过抑制特异性体液免疫应答,发挥对AESS的免疫调节作用,从而减轻其免疫病理损伤的程度。     

Cell participation in immune response by immune ribonucleic acid. I. The role of T lymphocytes in immune response by immune RNA against T-dependent antigens

T- and B-cell participation in the immune response induced by immune ribonucleic acid (iRNA) preparations against T-dependent antigens was studied using athymic nude, neonatally thymectomized (NT) and cyclophosphamide-treated (CY) mice. The iRNA(T + B) preparations were made from the spleen of BALB/c mice immunized with these antigens. Injection of the iRNA into nude or NT mice caused an increase in the number of specific rosette-forming cells (RFC) and of memory cells capable of responding to secondary stimulus with a small dose of the corresponding antigen. Injection with T-dependent antigens or with iRNA(T + B) did not cause any immune response in CY mice, suggesting depletion of the B-cell function. The iRNA(T) and iRNA(B) were prepared, respectively, from the thymuses of BALB/c mice and from the spleens of nude mice which had been immunized with T-dependent antigens. Injection of nude mice with both iRNA(T) and iRNA(B) caused an increase in the number of specific RFC and the secondary antibody formation response after boosting with a small dose of the corresponding antigen. Injection of iRNA(T) preparation into nude mice could induce the anamnestic response after boosting. However, neither of the iRNA(T) or iRNA(B) preparation could induce in nude mice the proliferation of the number of specific RFC. These results indicate the presence of at least two kinds of iRNA preparations against T-dependent antigens and that the cooperation of iRNA(T) and iRNA(B) was required for the induction of immune response against T-dependent antigens.     

介绍一种制备特异性TF和特异性iRNA的方法

以乙肝疫苗、人喉癌细胞膜抗原为抗原，猪脾细胞为效应细胞，经体外免疫后收集应答细胞，制备ＰＳＨＢＶ－ＴＦ ＰＳＡＣ－ｉＲＮＡ。通过抗原特异性细胞免疫功能试验证实，ＰＳＨＢＶ－ＴＦ和ＰＳＡＣ－ｉＲＮＡ都能转移特异性细胞免疫功能。采用体外免疫法制备ＰＳＨＢＶ－ＴＦ和ＰＳＡＣ－ｉＲＮＡ是可行的，并且具有诸多优点。     

Cell preparation in immune response by immune ribonucleic acid. II. Independence of T lymphocytes in immune response against T-independent antigens.

The immune response of congenitally athymic (nude) mice induced by immune ribonucleic acid (iRNA) to lipopolysaccharides of Escherichia coli 0-55 (LPS) was studied. The thymus-independent nature of the immune response of mice to LPS was confirmed and nude mice responded to LPS in a manner similar to normal mice. An iRNA preparation extracted from the spleen of nude mice immunized with LPS could induce the proliferation of rosette-forming cells (RFC) in nude mice. iRNA preparations were insensitive to treatment with deoxyribonuclease and pronase, but were inactivated by ribonuclease treatment. The active fraction of the iRNA preparation had sedimentation values in a sucrose density gradient between 7 and 16 S and comprised only a small fraction of the total RNA present in the spleen cells, thereby indicating that the active moiety was one or more species of RNA. The anamnestic response was induced by treatment with iRNA made from the spleen of nude mice immunized with LPS. An increase in the number of rosette-forming cells (RFC), plaque forming cells (PFC) and formation of humoral antibody to LPS was seen after injection with a small amount of LPS 4 weeks after iRNA treatment.     

Induction of IgM memory with RNA from the spleens of immunized mice.

Ribonucleic acid (immune RNA, iRNA), extracted from the spleens of mice immunized with sheep red blood cells (SRBC), induced specific immunological memory. The humoral immune responses of iRNA-treated animals were compared with in vivo and in vitro primary and secondary responses, using the haemolytic plaque-forming cell (PFC) assay. IgM antibody formation was markedly higher in iRNA-treated than in any other mice. The effect of iRNA was antigen-specific and similar materials from non-immunized mice had no activity.     

特异性iRNA对烧伤患者CD20~＋B细胞及增殖反应的影响

将２８名烧伤患者随机分成两组，对照组１３例进行常规治疗，治疗组１５例在常规治疗基础上应用特异性免疫核糖核酸（ｉＲＮＡ），同时采用鼠抗人Ｂ细胞ＣＤ２０单克隆抗体（ＭｃＡｂ），借助ＡＰＡＡＰ桥联酶标技术，观察了两组烧伤患者外周血ＣＤ２０＋Ｂ细胞数量（百分比）及增殖反应的变化。结果发现：（１）烧伤后患者外周血ＣＤ２０＋Ｂ细胞数量百分比和ＣＤ２０＋Ｂ细胞对丝裂原刺激的增殖反应均明显低于正常组（Ｐ＜００１）；（２）治疗组经特异性ｉＲＮＡ治疗后ＣＤ２０＋Ｂ细胞数量比治疗前平均增加１１．５％，而对照组患者ＣＤ２０＋Ｂ细胞数量及增殖反应无明显变化，表明特异性ｉＲＮＡ可明显提高烧伤患者外周血ＣＤ２０＋Ｂ细胞数量，并可增强烧伤患者外周血ＣＤ２０＋Ｂ细胞对丝裂原刺激的增殖反应。     

严重烧伤病人PMNCD11b/CD18受体表达及特异性iRNA对其调节作用

本文探讨了严重烧伤对病人外周血中性粒细胞 (PMN)CD11b/CD18受体表达的影响及特异性免疫核糖核酸 (iRNA)对其调节作用。结果发现 :(1)严重烧伤病人PMNCD11b/CD18受体表达率明显下降 ,至伤后第 10天时 ,分别只有正常的6 7 1%和 6 8 9% ,且其下降程度与烧伤面积成正比 ;(2 )伤后早期应用特异性iRNA可明显提高烧伤病人PMNCD11b/CD18受体表达率 ;(3)临床观察发现 ,治疗组伤后创面细菌培养阳性率 ,创面脓毒症及菌血症的发生率明显低于对照组 (P均 <0 0 5 ) ,创面愈合时间明显短于对照组 (P <0 0 1)。该结果为临床应用特异性iRNA防治烧伤后感染提供了实验依据。     

转移因子和免疫核糖核酸对LAK细胞抗肿瘤活性的影响

本研究用重组白细胞介素２（ｒＩＬ－２）联合转移因子（ＴＦ）或抗肿瘤免疫核糖核酸（ｉＲＮＡ）作用于外周血单个核细胞（ＰＢＭＣ），测定其对Ｋ５６２，Ｒａｊｉ及Ｈ７４０４细胞的杀伤活性。结果发现，高浓度ＴＦ（０．２５ｕ／ｍｌ）可抑制ＬＡＫ活性，ＴＦ进一步稀释（０．１２５ｕ／ｍｌ）可使其抑制作用消失。ＴＦ和抗肿瘤ｉＲＮＡ不能进一步提高最适剂量ｒＩＬ－２（５００ｕ／ｍｌ）诱导的ＬＡＫ活性，但能显著增强亚适剂量ｒＩＬ－２（２００ｕ／ｍｌ）诱导的ＬＡＫ活性，诱导ＬＡＫ活性增高的ＴＦ最适剂量为０．０３１ｕ／ｍｌ。ＴＦ或抗肿瘤ｉＲＮＡ单独不能诱导出ＬＡＫ活性，而当两者联合应用可诱导ＰＢＭＣ产生ＬＡＫ活性。本文为ＴＦ及抗肿瘤ｉＲＮＡ协同ＬＡＫ细胞疗法在临床应用提供实验基础。     

抗狂犬病免疫核糖核酸的分离纯化及部分理化性质和生物学活性初探

目的 探索一种抗狂犬病免疫核糖核酸(iRNA)的制备方法 ,为狂犬病病毒暴露后的免疫治疗开辟途径.方法 用狂犬病病毒免疫马,从抗体阳性血清的马匹中分离肝脏,依次采用十二烷基磺酸钠、苯酚、三氯甲烷、乙二醇单甲醚、十六烷基三甲基溴化铵、乙醇等提取总RNA.结果 提纯品经理化性质检测,得到的iRNA占肝脏总重的0.15%,其中DNA含量为2.86%,蛋白质含量为1.26%.最大吸收峰位于258 nm;A_(258)/A_(280)为2.0;RNA鉴别试验呈阳性反应.用高效液相色谱法测定,相对分子质量为13.7×10~3;增色效应50.67%.生物学活性测定结果 显示,白细胞黏附抑制率为41.73%.动物保护率为50%,生命延长率为31.62%.结论 实验提取的抗狂犬病iRNA,经用国家颁发的iRNA质控标准对照检测,结果 相同.为狂犬病治疗药物的研究奠定了实验室基础.     

High-efficiency polyethylene glycol-mediated transformation of mammalian cells

A new, high-efficiency method for transformation of mammalian cells with nucleic acids is described which yields 10⁵–10⁶ plaques/g poliovirus infectious RNA (iRNA). The optimized procedure consists of two steps: (1) exposure of cells to iRNA in a high ionic-strength buffer followed by (2) a brief exposure to a 35% polyethylene glycol (PEG) solution. Optimized conditions for each variable in the procedure are described. Under optimized conditions for PEG-mediated transformation with RNA, large numbers of transformants are recovered with plasmid DNA as well. The procedure presented is similar to other high-efficiency PEG-mediated methods previously described for the genetic transformation of both nonprotoplasted Escherichia coliand yeast.     

Down-regulation of viral replication by adenoviral-mediated expression of siRNA against cellular cofactors for hepatitis C virus

Small interfering RNA (siRNA) is currently being evaluated not only as a powerful tool for functional genomics, but also as a potentially promising therapeutic agent for cancer and infectious diseases. Inhibitory effect of siRNA on viral replication has been demonstrated in multiple pathogenic viruses. However, because of the high sequence specificity of siRNA-mediated RNA degradation, antiviral efficacy of siRNA directed to viral genome will be largely limited by emergence of escape variants resistant to siRNA due to high mutation rates of virus, especially RNA viruses such as poliovirus and hepatitis C virus (HCV). To investigate the therapeutic feasibility of siRNAs specific for the putative cellular cofactors for HCV, we constructed adenovirus vectors expressing siRNAs against La, polypyrimidine tract-binding protein (PTB), subunit gamma of human eukaryotic initiation factors 2B (eIF2Bgamma), and human VAMP-associated protein of 33 kDa (hVAP-33). Adenoviral-mediated expression of siRNAs markedly diminished expression of the endogenous genes, and silencing of La, PTB, and hVAP-33 by siRNAs substantially blocked HCV replication in Huh-7 cells. Thus, our studies demonstrate the feasibility and potential of adenoviral-delivered siRNAs specific for cellular cofactors in combating HCV infection, which can be used either alone or in combination with siRNA against viral genome to prevent the escape of mutant variants and provide additive or synergistic anti-HCV effects.     

Plasmid DNA- and messenger RNA-based anti-cancer vaccination

Tumor cells (over-) express specific antigens which allow them to be recognized and destroyed by the immune system. Triggering anti-tumor immunity in cancer patients by specific vaccination is foreseen as a safe and versatile method to control cancer. As a source of antigen, whole tumor cells, nucleic acids, proteins or derived peptides have been used. This review focuses on the utilization of vaccines based on plasmid DNA (pDNA) and messenger RNA (mRNA) coding for tumor associated antigens. Both vectors (pDNA and mRNA) are grouped under the designation "minimal nucleic acid vector" or MNAV. The current knowledge on anti-tumor vaccination based on MNAV-encoded tumor antigens, methods of delivery, principles of production and optimization is discussed. Furthermore, an up-to-date summary of published clinical trials using MNAV for the vaccination against solid tumors is given. Recent preclinical and early phase clinical trials demonstrate promising synergies between vaccination and other treatments such as chemotherapy or non-specific immune enhancement regimens. Combining optimized MNAV formulations and parallel adjuvant treatments could allow to turn MNAV-based vaccines into efficient anti-tumor immunotherapies in humans.     

Viral and non-viral approaches for transient delivery of mRNA and proteins

The transient delivery of gene products (RNA or proteins) is not a biotechnological invention but rather an evolutionarily conserved process underlying and regulating a variety of biological functions. On the basis of insights into the underlying mechanisms, several viral and cell-based approaches have been developed for the delivery of RNA or proteins. Prominent applications include the induction of major biological or therapeutic effects on the basis of "hit-and-run" mechanisms, such as vaccination, cell fate modification (reprogramming, differentiation), control of cell trafficking, enhancement of cell regeneration, and genome engineering using sequence-specific recombinases or nucleases. Ideally, procedures for delivery of RNA or proteins should be targeted to specific cells, overcome biophysical hurdles without harming cellular integrity, circumvent the various alarm signals of the innate immune system, allow dose-controlled delivery of functional biomacromolecules, and avoid the induction of an adaptive immune response. Here we review the current state of approaches for the delivery of mRNA and proteins with a focus on RNA viruses, virus-like particles including retrovirus particle-mediated transfer of mRNA or proteins, extracellular vesicles, and cell-penetrating peptides. The basic concepts and recent advances are put into perspective in the context of potential limitations of the technologies and strategies to overcome cellular barriers and defense mechanisms.     

Single-stranded small interfering RNA are more immunostimulatory than their double-stranded counterparts: a central role for 2'-hydroxyl uridines in immune responses

It has recently become apparent that certain small interfering RNA (siRNA) sequences stimulate the innate immunity through endosomal Toll-like receptors (TLR), particularly TLR7 and TLR8. However, it remains unclear whether siRNA duplexes act as specific ligands for these receptors. To address this question and to overcome the problem of immune activation by siRNA, several RNA sequences were chemically synthesized and their effects were investigated. Results indicate that human peripheral blood mononuclear cells (PBMC) recognize and respond to a large number of sense or antisense single-stranded (ss) siRNA. In most cases immunostimulatory RNA motifs are more effectively recognized by innate immunity in the context of ss siRNA as compared to siRNA duplexes. Novel immunostimulatory RNA motifs were identified and their replacement with adenosines abrogated immune activation. Most notably, replacement of the 2'-hydroxyl uridines with either 2'-fluoro, 2'-deoxy or 2'-O-methyl uridines abrogated immune activation. Thus, immune recognition of RNA by TLR can be evaded by 2'-ribose modifications of only uridines. Collectively, the data should facilitate the development of siRNA therapeutics and expand the understanding of how RNA is sensed by innate immunity.     

RISH V. Application to monoclonal antibody production

RISH considers that cell surface components involved in like cell identification are not involved in the structure of the plasma membrane per se and are attached to a part of their mRNA. The mRNA then acts as a template for the synthesis of DNA. Thus the component at the cell surface is attached to an RNA/DNA receptor. If there is a conformational change in the component (antigen) this will cause a distortion in its RNA/DNA receptor. This distortion is then detected by a tissue specific T lymphocyte which removes all or part of the RNA/DNA receptor from the aberrant cell and the lymphocyte then undergoes replication. During this process receptor RNA/DNA is incorporated into the daughter lymphocyte which becomes a B lymphocyte/plasma cell producing immunoglobulin. The initial tissue specific T lymphocyte becomes a dual functional helper/suppressor cell. The plasma cell after the initial immune response becomes a circulating memory B cell displaying IgM or IgD. If this cell complexes an antigen with its surface IgM or IgD a humoral immune response will be developed as previously described, but in this case the antibodies produced will be anti-idiotypic antibodies. The anti-idiotypic antibodies will regulate the production of the antibody directed against the antigen per se. The anti-idiotypic antibodies will in turn be regulated by a second anti-idiotypic antibody. In RISH five such anti-idiotypic systems may be involved in regulating the immune response to the initial non-immunoglobulin antigen. Based on the RISH anti-idiotypic mechanism a system is briefly described whereby human memory B cells, to a particular antigen, may be isolated. These B cells may then be activated to secrete immunoglobulin with autologous isolated anti-idiotypic antibodies. These activated cells may then be infected with E.B. virus to establish an immortal cell line of B cells secreting the immunoglobulin of interest.     

RNA-based viral immunity initiated by the Dicer family of host immune receptors

Summary:  Suppression of viral infection by RNA in a nucleotide sequence homology-dependent manner was first reported in plants in early 1990s. Studies in the past 15 years have established a completely new RNA-based immune system against viruses that is mechanistically related to RNA silencing or RNA interference (RNAi). This viral immunity begins with recognition of viral double-stranded or structured RNA by the Dicer nuclease family of host immune receptors. In fungi, plants and invertebrates, the viral RNA trigger is processed into small interfering RNAs (siRNAs) to direct specific silencing of the homologous viral genomic and/or messenger RNAs by an RNaseH-like Argonaute protein. Deep sequencing of virus-derived siRNAs indicates that the immunity against viruses with a positive-strand RNA genome is induced by Dicer recognition of dsRNA formed during the initiation of viral progeny (+)RNA synthesis. The RNA-based immune pathway in these organisms overlaps the canonical dsRNA-siRNA pathway of RNAi and may require amplification of viral siRNAs by host RNA-dependent RNA polymerase in plants and nematodes. Production of virus-derived small RNAs is undetectable in mammalian cells infected with RNA viruses. However, infection of mammals with several nucleus-replicating DNA viruses induces production of virus-derived microRNAs capable of silencing host and viral mRNAs as found for viral siRNAs. Remarkably, recent studies indicate that prokaryotes also produce virus-derived small RNAs known as CRISPR RNAs to guide antiviral defense in a manner that has yet to be defined. In this article, we review the recent progress on the identification and mechanism of the key components including viral sensors, viral triggers, effectors, and amplifiers, of the small RNA-directed viral immunity. We also highlight some of the many unresolved questions.     

In vivo application of RNA leads to induction of specific cytotoxic T lymphocytes and antibodies

To study the efficiency of RNA-based vaccines, RNA coding for the model antigen beta-galactosidase (beta-gal) was transcribed in vitro from a lacZ gene flanked by stabilizing Xenopus laevis beta-globin 5' and 3' sequences and was protected from RNase degradation by condensation with the polycationic peptide protamine. The liposome-encapsulated condensed RNA-peptide complex, the condensed RNA-peptide complex without liposome or naked, unprotected RNA, was injected into BALB/c (H-2(d)) mice. All preparations led to protein expression in the local tissue, activation of L(d)-restricted specific cytotoxic T lymphocytes (CTL) and production of IgG antibodies reactive against beta-gal. RNA-triggered CTL were as efficient in the lysis of lacZ-transfected target cells as CTL triggered by a lacZ-DNA eukaryotic expression vector. Immunization with RNA transcribed from a cDNA library from the beta-gal-expressing cell line P13.1 again led to beta-gal-specific CTL and IgG induction. Thus, both naked and protected RNA can be used to elicit a specific immune response in vivo, whereby the protected RNA is stable in vitro for a longer period of time. RNA vaccines can be produced in high amounts and have the same major advantages as DNA vaccines but lack the potentially harmful effect of DNA integration into the genome.