成骨肉瘤相关抗原的纯化及其特异性分析

用抗正常组织抗体制成免疫亲和层析柱，把成骨肉瘤组织提取物反复过此免疫亲和层析柱，其中的正常组织成分被吸附在层析柱上，不被吸附的成分经ＳＤＳ－ＰＡＧＥ分析，显示单一条带，是一种分子量为３２ＫＤ的抗原成分。利用免疫电泳、ＥＬＩＳＡ和琼脂双向扩散试验进行了特异性分析，结果表明，此抗原只在肿瘤组织中存在，而在正常组织中没有。用此抗原免疫ＢＡＢＬ／Ｃ小鼠制备了一株抗成骨肉瘤的单克隆抗体２Ｇ１０。用此单抗进行免疫组织化学染色，与正常组织反应阳性率为０／９，与成骨肉瘤组织反应阳性率为２０／２１。     

人精子抗原基因表达产物HSG2Ag的纯化和鉴定

本研究运用离子交换层析法分离纯化人精子抗原基因表达克隆ＨＳＧ２的溶原蛋白，并用ＥＬＩＳＡ法鉴定其中的表达产物（ＨＳＧ２Ａｇ）。结果提示，用上述方法分离和纯化的ＨＳＧ２Ａｇ纯化度高，特异性高，适用应用于抗精子抗体检测试剂盒的制备，并为进一步研制精子抗原免疫避孕疫苗提供候选成分。     

肝癌病人树突状细胞诱导高效而特异的抗肝癌免疫

目的 以肝癌病人树突状细胞（ＤＣ）体外诱导抗肝癌免疫。方法 自肝癌病人外周血中分离出单个核细胞（ＰＢＭＣ０;以人肝癌细胞系ＨｅｐＧ２肿瘤细胞的肿瘤相关抗原（ＴＡＡ）激活ＤＣ;以粒／巨噬细胞集落刺激因子（ＧＭ－ＣＳＦ）及白介素４（ＩＬ－４）联合刺激ＰＢＭＣ中ＤＣ;ＤＣ诱导自体Ｔ淋巴细胞增殖,分化为细胞毒性Ｔ细胞（ＣＴＬ）;检测ＣＴＬ及其上清液对ＨｅｐＧ２,ＢＥＬ－７４０２,ＬＯＶＯｅｙ ＨＯＳ－８６     

异种移植排斥机理的探讨

补体的激活是超急性排斥的中心环节，为了研究经典及旁路途径在这种排斥中的作用，本研究建立了体外超急性排斥模型。选择猪血管内皮细胞为人靶，人血清为天然抗体和补体源，用四唑盐法（ＭＴＴ）行补体依赖的细胞毒反应（ＣＤＣ）。人血清能溶解５８±５％的猪血管内皮细胞。加入ＥＧＴＡ阻断经典途径后人血清的溶细胞率降为５１±３％（Ｐ〈０．０１）。同样Ｃｌｑ缺乏的人血清仅溶解３７±７％猪血管内皮细胞（Ｐ〈０．００１）。     

人精子有效抗原基因表达克隆的研究

我们从人睾丸组织中提取ＲＮＡ，并进一步纯化出ｍＲＮＡ；以此为模板，在反转录酶和ＤＮＡ多聚酶的作用下，合成ｃＤＮＡ；将ｃＤＮＡ与λｇＩＩ载体重组后转染大肠杆菌，构建人睾丸ｃＤＮＡ表达文库。运用遗传显色法和噬菌斑原位杂交法鉴定表达文库后，用兔抗人精子抗体筛选人精子抗原基因表达克隆（ＨＳＧ）。对ＨＳＧ表达抗原（ＨＳＧＡｇ）和特异抗体（ＨＳＧＡｂ）进行纯化和抗生育效应的测定。结果显示：（１）人睾丸ｃＤＮＡ表达文库容量为１．８２Ｘｌ０６ｐｆｕ，遗传显色法示重组率为６７％，噬菌斑原位杂交法示重组率为５１％。（２）经兔抗人精子抗体筛选２Ｘｌ０４ｐｆｕ，得８株人精子抗原基因表达克隆。（３）人血清、兔血清ＨＳＧ２Ａｂ和兔血清ＨＳＧ８Ａｂ对人精子具有补体依赖细胞毒作用。（４）兔血清ＨＳＧ３Ａｂ能阻断人精子在顶体反应时顶体后区ＣｏｎＡ受体的暴露。结果提示，ＨＳＧ２、ＨＳＧ３和ＨＳＧ８克隆的表达抗原是精子有效抗原，能作为精子免疫避孕疫苗的候选成分。     

胰腺癌新生血管密度和血管内皮生长因子表达的研究

我们采用免疫组织化学染色及形态半定量方法对胰腺癌标本中的微血管密度 (ＭＶＤ)标记后定量计数和血管内皮生长因子 (ＶＥＧＦ)表达强度进行研究 ,结果报道如下。一、材料与方法我院 1980～ 1997年术前未经抗肿瘤治疗的胰腺癌切除标本 3 1例 ,常规石蜡包埋 ,4μｍ切片 ,苏木素 伊红染色 ,按分化程度病理分型 ,ＴＮＭ分期。胰腺癌旁组织 15例 ,正常胰腺组织 7例作为对照。免疫组织化学染色采用链菌素亲生物素 过氧化物酶标免疫组织化学染色技术 (Ｓ Ｐ)法。标记血管内皮细胞兔抗人第Ⅷ因子相关抗原 (Ｆ8 ＲＡ )及免疫ＩｇＧ多抗血管内皮生…     

兔胆色素结石中Ag、特异性IgG的检测及意义

目的探讨兔胆色素结石（ＰＳ）中是否含有抗原、特异性抗体及ＰＳ成因间的关系。方法选用家兔２０只,随机分成两组（１０×２）,做胆石模型,胆道注人１２５Ｉ标记的灭活Ｅ．ｃｏｌｉ为实验组,而注人等量未标记的灭活Ｅ．ｃｏｌｉ为对照组,计算成石率,对实验组胆汁、胆石进行４０秒γ计数,胆石切片作抗３３６０Ｅ．ｃｏｌｉ特异ＩｇＧ荧光抗体染色,测定光强度。结果兔ＰＳ中有Ａｇ及特异性ＩｇＧ存在。结论ＰＳ中的Ａｇ及特异性ＩｇＧ可以形成免疫复合物（ＩＣ）,而ＩＣ可能是ＰＳ的雏形,Ａｇ－Ａｂ亲和力可能是兔ＰＳ形成的力学机制之一。     

猪到人异种移植抗原与移植免疫研究

同种异体组织和器官移植的供体来源有限，使异种移植再度成为研究热点。猪被认为是理想的供体之一。异种移植的主要障碍为异种抗原被人血清中天然存在的抗体结合、激活补体导致的超急排斥反应。现已认识的主要靶抗原为αＧａｌ，它的表达受α１，３半乳糖基转移酶（αＧＴ）的控制。针对αＧａｌ克服超急排斥反应的方法有：免疫吸附去除针对αＧａｌ的天然抗体，酶处理去除内皮细胞（ＰＡＥＣ）表面的αＧａｌ，基因工程获得不表达αＧＴ从而无αＧａｌ的动物。除αＧａｌ以外，还有一些抗原可与人血清抗体结合：如ＴＮＦ诱导ＰＡＥＣ表达的ｇｐ６５和ｇｐ１００，猪组织表达的人血型抗原Ａ，但这些抗原的作用还不清楚。猪ＭＨＣ（即ＳＬＡ）的作用尚有争论，异种移植细胞免疫中，直接途径和间接途径均存在。     

黑色素瘤抗原基因在肝细胞癌细胞株中的表达

黑色素瘤抗原基因 (ＭＡＧＥ)是ｖａｎｄｅｒＢｒｕｇｇｅｎ等[1] 于 1991年发现的重要的肿瘤相关基因 ,在肝癌标本中ＭＡＧＥ 1表达率达 80 %左右 ,应用ＭＡＧＥ抗原肽对手术、化疗等常规方法治疗效果差的肝细胞肝癌 (ＨＣＣ)进行免疫治疗有一定的应用价值和可行性[2 ,3 ] 。应用ＭＡＧＥ抗原肽对ＨＣＣ进行免疫治疗的体外研究的关键是 ,诱导出对带有ＭＡＧＥ抗原的靶细胞具有特异性杀伤性能力的细胞毒淋巴细胞 (ＣＴＬ)。本实验对 8种ＨＣＣ细胞株ＭＡＧＥ基因的表达和人类白细胞抗原一类抗原 (ＨＬＡⅠ类抗原 )类型进行了研究 ,筛选出相…     

急进性肾炎中抗肾小球基底膜抗体的检测及其临床意义

目的 了解急进性肾炎（ＲＰＧＮ）中抗肾小球基底膜（ＧＢＭ）抗体的发生率及其临床意义。方法 自行制备可溶性人ＧＢＭ抗原，应用酶联免疫吸附方法（ＥＬＩＳＡ）对２９例ＰＲＧＮ患者进行抗ＧＢＭ抗体的检测，并对阳性血清用免疫印迹法进行鉴定、验证。结果 ２９例ＲＰＧＮ患者中５例抗ＧＢＭ抗体阳性，占１７％，其中一例伴ＡＮＣＡ阳性。免疫印迹法证实均识别２３－２７ＫＤ，４０－５４ＫＤ的蛋白条带。３／４例免疫荧光呈现     

靶向血管内皮细胞治疗肝癌的实验研究

目的 运用抗肝癌区血管内皮细胞的单克隆抗体进行抑癌实验。方法 通过建立裸小鼠人肝癌动物模型来进行,抑瘤实验。结果 抗肝癌区血管内皮细胞的单克隆抗体在肝癌动物模型的抑瘤裕有明显的抑瘤作用。结论 抗体在动物实验中明显的抑瘤作用,表明通过应用这种抗体可对肝癌进行以血管为靶向的生物治疗,从而为肝癌的治疗提供一种新的治疗方法。     

Role of the antibody to vascular endothelial cells in hyperacute rejection in patients undergoing cardiac transplantation

Traditionally, the human lymphocyte antigens have been considered to be the major barrier to successful transplantation, and lymphocytes have been used as the target cell in evaluating histocompatibility. The presence in the serum of recipients of preformed antibodies, cytotoxic to donors lymphocytes, is associated with a high probability of hyperacute rejection. We identified 11 patients in whom, despite a compatible direct lymphocytotoxic cross-match, acute failure of the cardiac homograft was associated with histologic and immunologic findings consistent with hyperacute rejection. Direct immunofluorescence and immunohistochemical staining showed the presence of antibodies on the surface of vascular endothelial cells in each of these 11 patients. The serum of these recipients was found to contain antibodies against a panel of endothelial cells. In contrast, cytotoxic antibodies to vascular endothelial cells were not present in a control group of 18 heart transplant recipients who did not experience hyperacute rejection. Thus the presence of antibodies against vascular endothelial cells seems to be related to hyperacute rejection of the cardiac allograft.     

In Vitro Evaluation of New Possible Cell Engraftment Enhancers for Cell Transplantation

Orthotopic liver transplantation (OLT) is the best treatment to restore liver function in liver failure. The low availability of organs has focused interest on the use of cell transplantation to restore liver function. However, this technique is limited because cells can not bind to liver parenchyma and die soon after perfusion. Pretransplant treatment with engraftment enhancers (EE) to increase vascular permeability may increase cell attachment. Using an endothelial cell culture to measure the loss of intercellular endothelial adhesion as a screening test, we evaluated the capacity of 15 monoclonal antibodies against adhesion molecules expressed on endothelial cells to act as EE showing that 3 antibodies (anti-CD54, efalizumab, and abciximab) act as EE by producing disruptions in the cell layer.     

Intrinsic cytotoxicity of natural killer cells to pancreatic islets in vitro

BB rats develop spontaneous autoimmune insulin-dependent diabetes mellitus that is similar to human insulin-dependent diabetes. In this study, we used an in vitro islet cell cytotoxicity assay to study the possible role of natural killer (NK) cells and their soluble effector molecules in this disorder. First, the results demonstrated that in vivo treatment of acutely diabetic BB rats with anti-asialogangliosideM1 (an NK cell antiserum) but not with anti-T-lymphocyte antibodies reduces spleen cell cytotoxic activity to islets in vitro. Flow microfluorometry (FMF)-sorting experiments were then used to confirm that the splenic cytotoxic effector cell in acutely diabetic BB rats is a CD8+/CD5- NK cell. Further analysis demonstrated that both FMF-sorted NK cell populations from Wistar-Furth rats and unfractionated spleen cells from athymic nu/nu rats with high intrinsic NK cell activity also exhibit high islet cell cytotoxic activity in vitro. Finally, we found that the kinetics and differential cytotoxic activity of NK cells toward islets in vitro could be mimicked by NK cell culture supernatants containing high levels of NK cytotoxic factor (NKCF). The islet cytotoxic activity of these culture supernatants was specifically inhibited by the addition of anti-NKCF monoclonal antibody. These results demonstrate that NK cells from diabetic and nondiabetic rats are cytotoxic to islet cells in vitro. They further suggest that this cytotoxic effect may be mediated in part through the production and release of soluble factors such as NKCF.     

Involvement of antibody-dependent apoptosis in graft rejection

BACKGROUND: Both humoral factors and apoptosis have been recently suggested to play a role in chronic allograft rejection. However, a link between alloantibodies and grafted cell apoptosis has never been proposed. Using the aortic allograft model in the rat, we have previously demonstrated the presence of IgG associated with the disappearance of donor endothelial and medial smooth muscle cells. In the present study, we tested the interaction between recipient allosera, enriched with antibodies by presensitization, and primary culture of cardiovascular cells of donor origin. METHODS: For this purpose endothelial cells, smooth muscle cells, adventitial fibroblasts, and cardiac myocytes of donor origin were cultured. Binding of alloantisera to these cells was analyzed by flow cytometry. Apoptosis of donor cells was evaluated by Tdt-mediated d' UTP-FITC nick end labeling, 4',6-diamidino-2-phenylindole and DNA ladder techniques. The alloantisera were compared with anti-MHC class I monoclonal antibodies. Finally the colocalization of antibodies and apoptosis was investigated in vivo. RESULTS: In vitro, alloantisera bind to cardiovascular cells of donor origin. These cells expressed MHC class I but not MHC class II. There was a partial competition between anti-MHC I mouse monoclonal antibody and alloantisera mainly of the IgG isotype. Alloantisera bound to, but did not induce lysis of, donor RBC. Alloantisera induced apoptosis of donor cardiovascular cells as assessed by the typical morphological aspect of the donor cells after 24 hr of incubation. These data were confirmed by the Tdt-mediated d' UTP-FITC nick end labeling positivity of the cells and the fragmentation of the nucleus visualized by 4',6-diamidino-2-phenylindole and DNA ladder techniques. Similar apoptosis was induced by specific monoclonal antibodies directed against the MHC class I of donor cells. Primary culture of similar vascular cells of recipient origin was insensitive to alloantisera directed against donor alloantigens. Finally, in vivo, using allopresentization and aortic allografts, an association of alloantibody binding and endothelial cell apoptosis was observed at day 5, and a similar association with smooth muscle cell apoptosis on day 12 after grafting. CONCLUSION: These data demonstrate the role of humoral injury in chronic allograft rejection and suggest new therapeutical approaches focused on the induction of resistance to antibody-dependent apoptosis.     

Role of CD18-dependent and CD18-independent mechanisms in the increased leukocyte adhesiveness and in the variations of circulating white blood cell populations induced by anti-CD3 monoclonal antibodies

Anti-CD3 antibodies induce a quick and profound depletion of peripheral blood mononuclear cells (PBMCs) that is not well understood. We studied the effect of OKT3, a mouse monoclonal antibody againts the human CD3 complex, on the in vitro adhesion of human PBMCs to monolayers of fresh and fixed human umbilical vein endothelial cells (HUVECs). OKT3 induced an increased adhesiveness of PBMCs. This phenomenon was blocked with anti-CD18 antibodies, indicating the participation of 2 integrins. As this increased adhesiveness could explain the lymphopenia by adhesion of PBMCs to endothelial cells and their sequestration in some peripheral vascular beds, we studied the effect of anti-CD18 antibodies in vivo on mice injected with 145/2C11, a hamster monoclonal antibody against murine CD3. Mice treated with 145/2C11 presented with a transient granulocytopenia and a sustained reduction in PBMCs. A monoclonal anti-CD18 antibody prevented the granulocytopenia but had no effect the drop in PBMCs. Consequently, the in vivo depletion of PBMCs after administration of an anti-CD3 monoclonal antibody involves CD18-independent mechanisms, while the transient drop in polymorphonuclear cells appears to be CD18-dependent.     

Identification and characterization of monoclonal antibodies that partially block human natural antibody binding to pig endothelial cell xenoantigens

Abstract: The shortage of human donors for clinical transplantation has led to a serious consideration of the use of non-human species as organ donors. The major barrier to the clinical use of xenografts from species such as the pig in human transplantation has been the aggressive nature of the immune-mediated rejection of the graft. We have recently identified the molecular weights of several endothelial cell surface proteins that may be targets of human antibody-mediated responses to pig aortic endothelial cells (PAEC). In this series of experiments, we produced a panel of rat monoclonal antibodies (Mabs) to PAEC in an effort to identify Mabs that detect pig xenoantigens. Mabs were selected based on flow cytometric binding to PAEC, pig platelets, and various pig cell lines, including a pig kidney cell line (LLC-PK1) reported to react with human natural antibodies (HNA). Eleven of the eighty-three antibodies produced were cytotoxic for PAEC. Six of the cytotoxic clones recognized a 44 kDa protein and two of the clones recognized a 115 kDa protein expressed on the surface of PAEC. Since PAEC target antigens recognized by human natural antibodies include both 115 and 44 kDa antigens, these Mab clones were selected for further study. Several distinct patterns of tissue reactivity were demonstrated within this group of antibodies by immunohistochemical analysis; however all monoclonal antibodies were highly reactive with endothelial cells in all tissues examined. Two monoclonal antibodies recognizing antigens that are highly expressed on pig endothelial cells (92–98%) and pig platelets (74–92%), but moderately expressed on pig splenocytes (33–38%), were capable of reproducibly blocking 48–53% of human IgM binding to pig endothelial cells when analyzed with flow cytometry. This data suggests that these Mabs may recognize epitopes of potential significance in the human-to-pig xenograft reaction.     

Study on adoptive immunotherapy with tumor-infiltrating lymphocytes (TIL) for renal cell carcinoma--amplification of IL-2-elicited TIL proliferation by OKT3-monoclonal antibody

Human autologous peripheral blood lymphocytes (PBL) and lymphocytes infiltrating renal cell carcinoma (TIL) were cultured with medium containing 1000 IU/ml of human interleukin 2 (IL-2). A high cytotoxic activity against fresh autologous as well as cultured allogenic tumor cells was developed. By culturing these lymphocytes with OKT3 monoclonal antibody during the initial 2 days of long-term culture, in terms of T cell activation signal, IL-2-driven lymphocyte proliferation was remarkably accelerated with maintenance of appreciable level of cytotoxic activity. The same culture method also induced an increase in OKT3 and IL-2 receptor positive lymphocyte population in LAK cells and TIL. This method may enable us to gain more autologous TIL in vitro for adoptive immunotherapy of renal cell carcinoma than the usual culture method with IL-2 alone. Five patients with metastatic renal cell carcinoma were treated with adoptive immunotherapy with TIL, LAK and IL-2. One patient with pulmonary metastasis has had a minor response which has lasted for 3 months so far. We have not experienced any serious side effects during the treatment.     

Blockade of cell adhesion molecules enhances cell engraftment in a murine model of liver cell transplantation

AimOLT is the best alternative for patients with end-stage liver diseases. However, as the need for organs surpasses donor availability, alternatives to OLT are required. LCT could be a useful option versus OLT in several patients even though its low cell-engraftment hampers its efficiency. Endothelial cell barrier is the main obstacle for the implantation of cells into the parenchyma. Our study has focused on the modification of the endothelial barrier with monoclonal antibodies against adhesion molecules in order to increase cell engraftment in a mouse model of liver cell transplantation.MethodsAnti-mouse CD54 and anti-mouse CD61 antibodies were administered intrasplenically to healthy mice within 60 min prior to stem cell transplantation. Animals were sacrificed either short term at 2 h or middle term seven days after transplantation. Immunohistochemical techniques to detect alkaline phosphatase activity were used to identify the transplanted cells within the liver parenchyma.ResultsAnti-CD54 and anti-CD61 administration increases vascular patency and cell engraftment. This represents a 32% and 45% increase, respectively, of engrafted cells compared to the control (p < 0.05).ConclusionModification of the vascular wall with monoclonal antibodies against endothelial adhesion molecules before cell transplantation enhances cell engraftment into the mouse liver.     

Depletion of T cells from human bone marrow using monoclonal antibodies and rabbit complement. A quantitative and functional analysis

Graft-versus-host-disease (GVHD) remains the principal complication of allogeneic bone marrow transplantation. In animal models mature T lymphocytes have been shown to be responsible for GVHD and, therefore, in vitro treatment of donor bone marrow using monoclonal T cell specific antibodies and complement is currently being investigated as a strategy for the prevention of GVHD. In the present studies anti-T12 and anti-T11 monoclonal antibodies and rabbit complement were used to remove T lymphocytes from normal bone marrow. The efficacy of depletion was investigated by immunofluorescence assays and by in vitro culture of the residual cells using nonspecific mitogens or allogeneic B cells as the proliferative stimulus in the presence of lymphocyte-conditioned medium containing interleukin 2 (IL-2). Immunofluorescence analysis showed complete depletion of T12+ and T11+ cells after treatment with the respective antibodies and with the combination. Nevertheless, culture of treated bone marrow with phytohemagglutinin (PHA) or concanavalin A (Con A) and conditioned media containing IL-2 resulted in the proliferation of mature T cells (T3+, T4+ or T8+, T11+). Stimulation of treated marrow with allogeneic cells (Laz 388) resulted in the growth of a population with natural killer (NK) cell phenotype (T3-, T11+, NKH1+). The latter population was found to be strongly cytotoxic against K562 cells, a standard NK target. As expected, NK cells that are T11+ and T12- appeared to be more effected by in vitro treatment with anti-T11 than with anti-T12. A clonogenic assay was then used to quantitate the efficacy of target cell depletion in vitro. Three sequential incubations of bone marrow with either anti-T12 or anti-T11 plus complement resulted in depletion of 1-2 logs of clonogenic cells. Treatment with both antibodies concurrently resulted in elimination of 2-3 logs of clonogenic target cells. Although multiple treatments with both anti-T12 and anti-T11 were more effective than similar treatment with only one antibody, it remains to be established whether such combinations will be necessary in the clinical setting or whether more selective depletion of T cells without removal of NK cells might be optimal.