用肾综合征出血热病毒结构蛋白体外免疫正常人外周血淋巴细胞制备单克隆抗体

用亲和层析提取的肾综合征出血热病毒（ＨＦＲＳＶ）结构蛋白（５０ｋＤ和６７ｋＤ）为抗原，在体外免疫正常人外周血淋巴细胞（ＰＢＬ）。ＰＢＬ经亮氨酸甲基酯处理后，在含有ｓＰＷＭ－Ｔ、ＩＬ－２及不同浓度抗原的培养基中培养６ｄ，计数存活ＰＢＬ数量。结果表明，在上述体外免疫条件下存活ＰＢＬ的数量与抗原剂量呈正相关。将此体外免疫的ＰＢＬ与人－鼠杂交瘤细胞（Ｋ６Ｈ６／Ｂ５）融合，获得了３株分泌抗ＨＦＲＳＶ人单抗的杂交瘤细胞，表明在体外免疫条件下，正常人ＰＢＬ能够对ＨＦＲＳＶ结构蛋白发生特异性免疫应答。     

人外周血特异性抗体形成细胞的培养及检测

采用以绵羊红细胞（ＳＲＢＣ）为免疫原，两次诱导并伴有ｐｗｍ作用的免疫培养方法，对２３例健康人外周血淋巴细胞进行体外诱导，并以空斑细胞（ｐｌａｑｕｅ－ｆｏｒｍｉｎｇｃｅｌ，ＰＦＣ）技术检测培养中产生的特异性抗体形成细胞（ａｎｔｉｂｏｄｙ－ｐｒｏｄｕｃｉｎｇｃｅｌ），结果表明：该条件下的实验组产生的ＰＦＣ数最高，细胞活性好。以兔ＲＢＣ代替ＳＲＢＣ的特异性溶血试验中，几乎无ＰＦＣ产生，证实ＳＲＢＣ诱导ＰＦＣ的特异性及本实验体外免疫培养方法的可行性。此外，本文采用的改良ＰＦＣ单电子层的检测方法，操作简便，便于临床应用，可作为研究人体免疫细胞功能的手段之一。     

检测Epstein-Barr病毒特异性细胞毒性T淋巴细胞方法的建立及其初步研究

目的建立一种非放射性、简便易行的可检测特异性细胞毒性Ｔ淋巴细胞的方法，并且初步应用于Ｅｐｓｔｅｉｎ－Ｂａｒ病毒的细胞免疫应答。方法用重组的ＥＢＶ－ＬＭＰ１痘苗病毒、ＴＫ＋痘苗病毒和杆状病毒系统表达的ＥＢＶ－ＬＭＰ１蛋白分别免疫Ｂａｌｂ／Ｃ小鼠，用Ｐ８１５细胞和乳酸脱氢酶法检测ＥＢ病毒特异性细胞毒性Ｔ细胞的杀伤效应。结果重组ＥＢＶ－ＬＭＰＩ痘苗病毒免疫组原发ＣＴＬ水平和体外诱生的二次ＣＴＬ水平均高于ＴＫ＋痘苗病毒免疫组和正常组；杆状病毒系统表达的ＥＢＶ－ＬＭＰ１蛋白免疫组的ＣＴＬ水平也明显高于正常鼠。结论本法可以较好的反映ＥＢ病毒特异性细胞毒性Ｔ细胞的水平，而且再一次说明ＬＭＰ１基因能够诱发特异性的细胞免疫。     

检测Epstein—Barr病毒特异性细胞毒性T淋巴细胞方？…

目的 建立一种非放射性、简便易行的可检测特异性细胞毒性Ｔ淋巴细胞的方法，并且初步应用于Ｅｐｓｔｅｉｎ－Ｂａｒｒ病毒的细胞免疫应答。方法 用重组的ＥＢＶ－ＬＭＰ１痘苗病毒、ＴＫ＾＋痘苗病毒和杆状病毒系统表达的ＥＢＶ－ＬＭＰ！蛋白分别免疫Ｂａｌｂ／Ｃ小鼠，用Ｐ８１５细胞和乳酸脱氢酶法检测ＥＢ病毒特异性细胞毒性Ｔ细胞的杀伤效应。结果 重组ＥＢＶ－ＬＭＰ１痘苗病毒免疫组原发ＣＴＬ水平和体外诱生的二次ＣＴＬ     

特异性乙型肝炎病毒转移因子的研制及临床应用

用乙型肝炎疫苗免疫绵羊，在细胞介导免疫应签的高峰期，放血取脾制备特异性乙型肝炎病毒转移因子。对慢性乙型病毒性肝炎病例，以ＲＨＡ法检测ＨＢｓＡｇ，１：６４以上者均列为观察对象，共８０例。随机抽样分成特异性的ＨＢＶ－ＴＦ治疗组和肝必复对照组，结果显示，特异性ＨＢＶ－ＴＦ组ＨＢｓＡｇ转阴率、下降率及有效率与肝必复比较，差异显著，ＨＢＶ－ＴＦ对血清ＡＬＴ有一定的下降及恢复作用，尤以ＨＢｓＡｇ转阴病毒为显著     

应用基因重组病毒表达产物筛选FBL3小鼠白血病肿瘤特异性抗原…

肿瘤特异性抗原是肿瘤免疫研究中心课题，众多的研究表明Ｔ细胞在肿瘤免疫中起重要作用，因而寻长Ｔ细胞所识别的肿瘤特异性表位并以为瘤苗的靶抗原有重要的研究价值，以一系列重组Ｆ－ＭｕＬＶｇａｇ病毒载体，用已建立的ＦＢＬ３－ＣＴＬ克隆筛选，并通过Ｈ－２Ｉ类分子结合实验，精确地测得Ｆ－ＭｕＬＶｇａｇ中一个能被为ＦＢＬ３－ＣＴＬ克隆所识别的表位（ＣＣＬＣＬＴＶＦＬ，ｐ８５～９３），并以此ｐ８５～９３合成肽免疫动     

T细胞受体Vβ基因在识别HSV—2过程中的表达水平和特点

本课题主要研究Ｔ细胞受体（ＴＣＲ）识别抗原后Ｖβ基因发生重排，ｍＲＮＡ表达水平改变。用紫外线处理的单纯疱疹病毒－２型（ＨＳＶ－２）、ＨＳＶ－２及ＰＨＡ分别感染或刺激正常人的ＰＢＬｓ，培养４～６天后，提取ｍＲＮＡ，并采用ＲＴ－ＰＣＲ、Ｓｏｕｔｈｅｒｎ杂交发现识别ＨＳＶ－２的ＴＣＲＶβ２、６、７、８基因在体外选择性扩增。在采用ＨＳＶ－２攻击皮肤病患者发作期、缓解期以及再发作期的ＰＢＬｓ时，发现ＴＣＲＶβ基因表达随着病程变化而改变，尤其Ｖβ７亚家族表达水平显著高于其它亚家族基因，这显示了ＴＣＲＶβ基因的变化是恒定特异性的。     

应用基因重组病毒表达产物筛选FBL3小鼠白血病肿瘤特异性抗原表位

肿瘤特异性抗原是肿瘤免疫研究的中心课题，众多的研究表明Ｔ细胞在肿瘤免疫中起重要作用，因而寻找Ｔ细胞所识别的肿瘤特异性表位并以此为瘤苗的靶抗原有重要的研究价值。以一系列重组Ｆ－ＭｕＬＶｇａｇ病毒载体，用已建立的ＦＢＬ３－ＣＴＬ克隆筛选，并通过Ｈ－２Ｉ类分子结合实验，精确地测得Ｆ－ＭｕＬＶｇａｇ中一个能被为ＦＢＬ３－ＣＴＬ克隆所识别的表位（ＣＣＬＣＬＴＶＦＬ，ｐ８５～９３）。并以此ｐ８５～９３合成肽免疫动物成功诱导出能识别亲本ＦＢＬ３肿瘤细胞。     

抗人黑素瘤双特异性抗体增强LAK细胞毒效应的实验研究

抗肿瘤单克隆抗体的高度特异性与杀伤细胞对肿瘤的细胞毒效应相互结合起来，是增强ＬＡＫ细胞抗肿瘤作用的重要途径。本研究用化学连接法制备了既可识别淋巴细胞表面ＣＤ３抗原又可识别ＬｉＢｒ黑素瘤细胞表面肿瘤相关抗原的双特异性抗体ＣＤ３－ＨＢ８７５９。ＦＡＣＳ分析证实，ＣＤ３－ＨＢ８７５９具有结合两种抗原的活性。４ｈ５１Ｃｒ释放细胞毒实验结果表明，ＣＤ３－ＨＢ８７５９在诱导相可显著增强ＬＡＫ对ＬｉＢｒ细胞的细胞毒效应，也能使非ＬＡＫ细胞获得细胞毒性；在杀伤相加入ＣＤ３－ＨＢ８７５９对ＬＡＫ细胞的细胞毒作用也具有促进作用。上述结果表明，ＣＤ３－ＨＢ８７５９是一种抗人黑素瘤的双特异性抗体，为体内应用提供了良好的实验基础。     

合成多肽体外诱导乙肝病毒抗原特异性CTL杀伤机制研究

为探讨合成多肽体外诱导乙肝病毒抗原特异性Ｔ细胞杀伤机制，应用免疫组化检测ＨＧＶ－ＣＴＬｓＦａｓ配体表达情况，燕分析培养上清一氧化氮含量与细胞毒活性的关系。结果显示，ＨＢＶ－ＣＴＬｓ对转染ＨＢＶ ＤＮＡ的肝癌细胞存在明显的特异性细胞毒活性，其Ｆａｓ配体表达阳性率分别为５８％，５６％，明显高于ＣＤ３－ＡＫ细胞组，细胞培养上清ＮＯ含量动态分析显示，ＨＢＶ－ＣＴＬｓ细胞培养上清ＮＯ含量与细胞毒活性存在正相     

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。     

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.     

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.     

Interaction of Herpesvirus with Spleen Cell Subpopulations: Comparison of a Neurotropic and a Lymphotropic Virus

We studied the interaction of a neurotropic herpesvirus, herpes simplex virus type 1 (HSV-1) or type 2 (HSV-2), and a lymphotropic herpesvirus, guinea pig herpes-like virus (HLV), with guinea pig spleen cells. Both HSV-1 and HSV-2 and HLV can attach to and penetrate into B- or T-enriched cells. Less than 1.4% of the total B- or T-enriched cell populations were susceptible to infection by HLV and to some degree to HSV-1 or HSV-2 as determined by infectious center assays. After specific antiserum treatment, higher titers of intracellular virus were detected in HLV-infected cells than in HSV-1- or HSV-2-infected cells. Both B-enriched and T-enriched cells could support HLV replication, but not that of HSV-1 or HSV-2. The replication of HSV-1 was demonstrated in guinea pig spleen cells pretreated with lipopolysaccharide but not with phytohemagglutinin. Furthermore, when cells were separated into B- and T-enriched cells, the B- enriched cells prestimulated with lipopolysaccharide were susceptible to HSV-1 replication, whereas the T-enriched cells prestimulated with phytohemagglutinin were not. The differences observed in vitro in the interactions of these two herpesviruses with guinea pig spleen cell subpopulations may provide a basis for understanding the differences observed in vivo in the pathogenesis of these two viruses; i.e., HLV is capable of infecting and persisting in guinea pig lymphocytes, whereas HSV is not.     

Demonstration and characterization of a serum factor produced by activated T cells.

Spleen rosette forming cells (RFC) from adult thymectomized mice have a low sensitivity to inhibition by anitheta serum (AOS) and azathioprine (AZ) in comparison with normal spleen or thymus RFC. Thymus extracts and normal mouse serum (but not spleen extracts or thymectomized mouse serum) correct this abnormality after a 30 min in vitro incubation with spleen cells. We report here the existence of a serum factor produced in allogeneic reactions with the same activity on rosettes as thymic factor (TF). This 'allogeneic' factor (AF) is detectable in mice undergoing a graft versus host reaction (GVHR), rejecting skin allografts or allogeneic cells or responding to thymus-dependent antigens such as heterologous red blood cells or BSA. The T-cell origin of AF is indicated by AF presence in nude mice submitted to the same allogeneic stimuli as listed above and in normal mice injected with PVP or LPS. AF is distinct from the thymic factor as shown by differences in electric charge. Moreover, in contrast with TF there is no specific high molecular weight inhibitor of AF. Preliminary biochemical studies indicate that AF is probably a peptide of low molecular weight (greater than 5000 daltons). Its target cell is probably a T-cell precursor.     

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。     

Studies on thymus products: III. Epithelial origin of the serum thymic factor

Serum thymic factor (TF) has been tested by its action on spleen rosette-forming cells from adult thymectomized mice. It has been confirmed in a blind study using coded serum samples that TF disappeared early after adult thymectomy and reappeared after grafting a thymus, either as a free graft or enclosed in cell impermeable diffusion chambers. Similar reconstitution was also obtained by grafting a non-lymphoid epithelial thymoma or pure epithelial thymus, obtained by in vivo incubation of a thymus within a diffusion chamber in an intermediate host. Conversely, TF levels were not restored in thymectomized animals treated with dispersed spleen cells or with dispersed thymic lymphocytes.     

Concentration of iron and distribution of iron and transferrin after experimental iron overload in rat tissues in vivo: study of the liver, the spleen, the central nervous system and other organs

The purpose of this study was to estimate the iron concentration in the liver, spleen and brain of control rats and rats overloaded with iron and to determine the distribution of iron and of transferrin (TF). Iron was administered to Wistar rats by food supplemented with 3% carbonyl iron for 3 months, or intraperitoneally, or intraveneously as iron polymaltose for 4 months (total administered dose: 300 or 350 mg/rat, respectively). Iron concentration was estimated by atomic absorption spectrophotometry and iron- and TF-distribution histochemically and immunohistochemically, respectively. In control rats the organ with the highest iron content was the spleen, followed by the liver and brain. After iron loading the increase of iron in the liver was greater than that of the spleen; iron concentration in the brain did not change significantly. Distribution of iron in the liver was in Kupffer cells throughout the lobule and in hepatocytes at its periphery. No difference in the number of positive cells or staining intensity for TF was observed between control rats and iron overloaded animals in the liver or central nervous system (CNS); the spleen was negative for TF. Distribution of TF in the liver showed a centrilobular localisation in hepatocytes. TF reaction in the brain occurred in oligodendrocytes, vessel walls, choroid plexus epithelial cells and some neurons. In conclusion, experimental iron overload in rats leads to iron uptake mainly by reticuloendothelial (RE) cells and hepatocytes, indicating that hepatocytes are of particular importance for iron metabolism. Iron uptake by the brain was not significant, probably because the brain is protected against iron overload. Iron overload did not influence location and quantity of TF in the liver and CNS, whereas the visualisation of iron and TF did not coincide. This indicates that TF may have other functions beyond iron transport.     

B cells as accessory cells in a Con A response of a T cell clone

Accessory cell (AC) function of B cells was examined in Con A response of a cloned T cell line, 22-9D, which is Thy 1+,L3T4+,Lyt2-,H-2KbDb+ and I-Ab-.22-9D cells produced IL 2 in the presence of Con A without participation of AC. For the initiation of a proliferative response to Con A, the addition of spleen cells or spleen adherent cells was required. B cells as AC were unable to induce the proliferative response. In the presence of culture supernatant of spleen cells stimulated with Con A (CAS), 22-9D cells showed proliferative response to Con A with B cell AC. The response was inhibited by a relevant monoclonal anti-I-A antibody. Although irradiated spleen cells as AC induced IL 2 receptor expression of 22-9D cells in the presence of Con A, B cells were shown to require the addition of unknown factor(s) in CAS, which was suggested to be different from IL 1, IL 2, IL 3, or IFN-gamma, for the induction of the receptor expression on 22-9D cells.