自体LAK细胞和rIL—2治疗晚期肾癌的实验与临床研究

应用自体ＬＡＫ细胞和重组白细胞介素－２（ｒＩＬ－２）治疗２０例晚期肾癌患者。自患者周围血分离到的单个核细胞体外经ｒＩＬ－２短期培养，其ＮＫ、ＬＡＫ活性明显增强并于第５、７天达高峰。当这些ＬＡＫ细胞与ｒＩＬ－２过继回输给同一患者后，病人周围血ＮＫ、ＬＡＫ活性明显增加（Ｐ＜０．０１），ＮＫ比率、ＩＬ－２受体表达明显增加（Ｐ＜０．０５），提示对肾癌患者的免疫调节作用。本组病人获部分缓解（ＰＲ）１例，轻度     

三磷酸腺苷氯化镁对兔缺血后肝组织保护作用的研究

家兔全肝缺血３０分钟后，分别给予生理盐水、三磷酸腺苷（ＡＴＰ）和三磷酸腺苷氯化镁（ＡＴＰ－ＭｇＣｌ２），术后第１，３，５天取血测定谷丙转氨酶（ＡＬＴ）、乳酸脱氢酶（ＬＤＨ）和碱性磷酸酶（ＡＬＰ）。术后第１天ＡＴＰ－ＭｇＣｌ２组ＡＬＴ明显低于对照组（Ｐ＜０．０５），而ＡＴＰ组与对照组相比无显著差别；ＡＴＰ－ＭｇＣｌ２及ＡＴＰ组的ＬＤＨ和ＡＬＰ与对照组相比无显著差别。术后２４小时超微结构示：对照组肝明显水肿、细胞器严重损伤和血窦内微小血栓；而ＡＴＰ－ＭｇＣｌ２组细胞轻度水肿，细胞器基本正常，血窦内无微小血栓。实验结果说明ＡＴＰ－ＭｇＣｌ２对缺血肝组织确有保护作用，其机制可能是通过改善微循环和减轻细胞而起作用的     

维持性血液透析病人NK－IL－2－IFNr系统的动态观察

本文检测了４０例维持性血液透析（ＭＨＤ）患者自然杀伤细胞（Ｎｋ）活性，并同时检测了患者外周血单个核细胞（ＰＢＭＣ）在植物血凝素（ＰＨＡ）刺激下白细胞介素２（ＩＬ－２）和免疫干扰素（ＩＦＮｒ）产生水平。结果发现ＭＨＤ患者ＮＫ活性、ＩＬ－２活性和ＩＦｖｒ水平均显著低于正常对照组（Ｐ＜０．０１）；单次血液透析后ＭＨＤ患者的ＮＫ活性、ＩＬ－２和ＩＦＮｒ诱生水平均有不同程度的升高；长期血液透析对ＮＫ活性、ＩＬ－２和ＩＦＶｒ诱生水平影响不大，正常对照ＮＫ活性与ＰＢＭＣ诱生ＩＬ－２和ＩＦＮｒ水平存在直线正相关，而ＭＨＤ患者相关性无显著意义，但透析后ＭＨＤ患者ＮＫ活性与ＩＬ－２活性相关性有显著意义。提示：ＭＮＤ淋巴细胞存在多方面功能受损，包括免疫调节环路的紊乱；血液透析能部分纠正这些免疫学异常；但长期血液透析无助于改善ＭＨＤ病人的免疫功能受损。     

白细胞介素10抑制核因子кB介导的肾小管上皮细胞表达细胞间黏附分子1

目的研究白细胞介素１０（ＩＬ－１０）对人近端肾小管上皮细胞（ＨＫ－２细胞）在促炎症因子肿瘤坏死因子α（ＴＮＦα）作用下表达细胞间黏附分子１（ＩＣＡＭ－ｌ）及其相关核转录途径的影响。方法 用ＨＫ－２细胞作靶细胞，用细胞酶联免疫吸附法（ＥＩＪＳＡ）和Ｎｏｒｔｈｅｒｎ杂交观察ＩＣＡＭ－１的蛋白和基因的表达，以电泳迁移率变动法测定转录因子核因子ｋＢ （ＮＦｋＢ）和激活蛋白 Ｉ（ＡＰ－１）的活性。结果ＴＮＦα呈剂量依赖地诱导ＨＫ－２细胞ＮＦｋＢ的活化及ＩＣＡＭ－１的基因和蛋白表达，这些作用可以被ＮＦｋＢ的抑制剂对甲苯磺－Ｌ－苯丙氨酸氯甲基甲酮（ＴＰＣＫ）所抑制，但ＴＮＦα对ＨＫ－２细胞的ＡＰ－１活性无影响。 ＩＬ－１０（１～ ２０ｎｇ／ｍｌ）可抑制 ＴＮＦα诱导的 ＨＫ－２细胞 ＩＣＡＭ－ｌ基因和蛋白表达及 ＮＦｋＢ的活化。结论ＴＮＦα诱导人肾小管上皮细胞ＨＫ－２的ＮＦｋＢ活化，进而促进ＩＣＡＭ－ｌ基因和蛋白表达，ＩＬ－１０可抑制ＴＮＦα诱导的上述炎症效应。     

静脉营养支持对胃癌患者免疫功能的影响

伴营养不良的进展期胃癌（ＡＧＣ）２４例，随机发为对照组和静脉营养支持组。治疗组于术前给予静脉营养支持５－７天。治疗前后，分别测定周围静脉血的前白蛋白、转铁蛋白和ＮＫ、ＬＡＫ细胞活性。发现经营养支持后，上述指标显著提高Ｐ＜０．０２５－０．００１）。提示术前静脉营养支持能改善ＡＧＣ患者的营养状况和免疫功能。     

直肠粘膜下注射重组白细胞介素-2治疗直肠癌的临床研究

目的 观察不同用途应用重组白细胞介素－２（ｒＩＬ－２）对患者全身及肿瘤局部免疫的影响。方法 对４５直肠癌患者给予肌肉或直肠粘膜下注射ｒＩＬ－２。结果 直肠粘膜下应用ｒＩＬ－２不仅提高患者全身免疫功能，而且能激活肿瘤区域淋巴结天然杀伤细胞（ｎａｔｕｒａｌ ｋｉｌｌｅｒ，ＮＫ）和淋巴因子激活杀伤细胞（ｌｙｍｐｈｏｋｉｎｅ－ａｃｔｉｖａｔｅｄ ｋｉｌｌｅｒ，ＬＡＫ）活性，促进淋巴结生发中心增生、浆细胞反     

大肠癌患者硒与免疫功能关系的研究

作者将大肠癌患者４４例分为治疗组与对照组，观察投药前后血清硒浓度与Ｔ淋巴细胞亚群、ＮＫ、ＬＡＫ细胞活性的变化，同时测定３５例大肠癌及正常组织内硒含量，结果显示大肠癌患者血硒水平（０．８１±０．１４μｍｏｌ／Ｌ）低于正常，补硒后血硒明显升高，与对照组差别显著（Ｐ＜０．０１）。治疗组的ＣＤ３、ＣＤ４、ＣＤ４／ＣＤ８及ＮＫ、ＬＡＫ细胞活性投药后有明显升高，与对照组相比差别显著，表明适量补硒能促进人体细胞免疫功能。此外，大肠癌肿组织的硒含量为２２．１３±１．７６μｍｏｌ／ｇ，明显低于周围正常大肠组织的硒含量２４．３０±１．９６μｍｏｌ／ｇ（Ｐ＜０．０１），提示大肠癌可能与局部低硒以致免疫力降低有关     

维持性血液透析病人NK—IL—2—IFNr系统的动态观察

本文检测了４０例维持性血液透析（ＭＨＤ）患者自然杀伤细胞（ＮＫ）活性，并同时检测了患者外周血单个核细胞（ＰＢＭＣ）在植物血凝素（ＰＨＡ）刺激下白细胞介素２（ＩＬ－２）和免疫干扰素（ＩＦＮｒ）产生水平。结果发现ＭＨＤ患者ＮＫ活性、ＩＬ－２活性和ＩＦＮｒ水平均显著低于正常对照组（Ｐ〈０．０１）；单次血液透析后ＭＨＤ患者的ＮＫ活性、ＩＬ－２和ＩＦＮｒ诱生水平均有不同程度的升高；长期血液透析对ＮＫ活性、Ｉ     

ATP—MgC12对缺血后肝组织保护作用的研究

研究ＡＴＰ－ＭｇＣｌ２对缺血后肝组织的作用。方法：家兔全肝缺血３０分钟后，分别给予生理盐水、ＡＴＰ和ＡＴＰ－ＭｇＣｌ２处理。术一第１天、第３天和第５天取血测定谷丙转氨酶（ＡＬＴ０、乳酸脱氢酶（ＬＤＨ０和碱性磷酸酶（ＡＬＰ）并分别检测各 肝组织学的改变。结果：术后第１天ＡＴＰ－ＭｇＣｌ２组和ＡＬＴ明显低于对照组（Ｐ〈０．０５），而ＡＴＰ组与对照组相比无显著差别：ＡＴＰ－ＭｇＣｌ２及ＡＴＰ组的ＬＤＨ和     

进展期胃癌患者脾脏的细胞免疫状态

本文检测了进展期胃癌患者（ｎ＝１６）脾静脉血淋巴细胞（ＳＶＬ）和脾细胞（ＳＣ）的自然杀伤细胞（ＮＫ）、淋巴因子激活的杀伤细胞（ＬＡＫ）的杀伤活性及Ｔ细胞（ＴＣ）亚群，与胃良性病变（ｎ＝７）的ＳＶＬ及外伤性脾破裂（ｎ＝６）的ＳＣ对照比较，探讨进展期胃癌（ＡＧＣ）患者脾脏的细胞免疫状态，为胃癌术中合理的处理脾脏提供免疫学依据。结果表明：ＡＧＣ患者脾脏的ＮＫ、ＬＡＫ细胞杀伤活性显著降低（Ｐ＜０．０２，Ｐ＜０．０１）；ＣＤ４显著减少，ＣＤ８显著增加，ＣＤ４／ＣＤ８比值显著降低（Ｐ＜０．０２～０．０１）。提示进展期胃癌患者脾脏的细胞免疫功能受到明显抑制。     

Assessment of in vitro lymphokine activated killer (LAK) cell activity against renal cancer cell lines and its suppression by serum factor using crystal violet assay

Summary Lymphokine activated killer (LAK) cell activity against renal cancer cell lines was assessed in vitro using a crystal violet assay. A standard 4-h ⁵¹chromium release assay and a 48-h crystal violet assay showed that both natural killer-susceptible (NC65) and-resistant (ACHN) renal cancer cell lines were sensitive to LAK cells which had been generated by a 3-day incubation of peripheral blood mononuclear cells (PBMC) with recombinant interleukin 2 (rIL-2). Optimal LAK activity was generated by a 5-day culture of PBMC with 1 U rIL-2/ml. LAK activity was enhanced by the presence of IL-2 in the crystal violet assay system, while it was suppressed by fresh autologous serum. The suppressive effect was found in serum from both normal donors and patients with metastatic renal cell carcinoma, suggesting that non-specific suppressive factor(s) affecting LAK cell activity were present in human sera.     

Augmentation of the lymphokine-activated killer cell response in head and neck cancer patients by combination interleukin-2 and interferon-alpha

The majority of patients with head and neck cancer present with advanced disease (stage III and IV), for which current chemotherapeutic regimens offer dismal results. Although known to have defects in their cell-mediated immunity, their poor performance status makes them unlikely candidates for aggressive immunotherapeutic protocols because of associated severe toxicities. This study evaluates the effect of subthreshold recombinant interferon-alpha (rIFN-alpha) and interleukin-2 (rIL-2) on the generation of lymphokine-activated killer (LAK) cells from the peripheral blood of patients with head and neck cancers. In vitro treatment of patients' lymphocytes consisted of incubation in 1,000 U/mL rIL-2, 100 U/mL rIL-2, 100 U/mL rIFN-alpha, and 100 U/mL rIFN-alpha plus 100 U/mL rIL-2 for 4 to 5 days. Cytotoxicity was measured using a standard 4-hour chromium-51 (51Cr)-release assay with Raji (B lymphoblastoid) tumor target cells. LAK activity was arbitrarily defined as greater than 20% cytolysis of Raji target cells. LAK activity was generated in a smaller percentage of the head and neck cancer patients by 1,000 U/mL rIL-2 compared with normal adult donors: 54% versus 100%, p less than 0.05; IFN-alpha (100 U/mL) induced LAK activity in approximately 50% of the cancer patients. The addition of rIFN-alpha (100 U/mL) to rIL-2 (100 U/mL) resulted in LAK generation in a higher percentage of patients (83% versus 54%), as well as increased levels of cytotoxicity, p = 0.05. This combination also resulted in cytotoxicity levels equivalent to high-dose (1,000 rIL-2 U/mL). These in vitro data support a clinical trial to assess the therapeutic efficacy of combined low-dose rIL-2 and rIFN-alpha in vivo in head and neck cancer patients.     

Clinical efficacy of recombinant interleukin-2 for renal cell carcinoma and the effect of blood transfusion upon the immune response of these patients

We studied the efficacy and safety of recombinant interleukin-2 (rIL-2: S-6820) for the treatment of renal cell carcinoma as well as the effect of blood transfusion upon the immune response of these patients. Among 14 cases of renal cell carcinoma treated by i.v. infusion of rIL-2, a partial response (PR) was achieved in one patient, 10 patients had no change, and in 3 had the disease progressed. The overall efficacy rate was 7.1%. However, the rate increased to 12.5% in cases with pulmonary metastases and to 14.3% in cases without any blood transfusion within a year before treatment with rIL-2. No severe side effects were observed, except for central nervous system disturbance in one case. During the rIL-2 therapy, LAK activity was suppressed in the transfused patients. On the other hand, NK activity was augmented in transfused patients to the same degree as in non-transfused cases. No significant changes of lymphocyte count and the subsets of peripheral blood lymphocytes were observed in either group treated with rIL-2. Anemia and radical nephrectomy did not affect the immune response in these patient. Thus, it appeared that blood transfusion altered the immune response in patients treated with rIL-2. However, it could not be concluded that transfusion definitely had an adverse effect on the clinical efficacy of rIL-2 for renal cell carcinoma.     

The characterization of peritoneal and pleural exudate cells from malignant effusions

The immune function of peripheral blood cells and cells from the pleural and abdominal effusions of patients with advanced cancer was compared to that of peripheral blood cells from controls. The parameters examined included lymphocyte subsets, natural killer (NK) cell activity, and anti-Daudi and lymphokine-activated killer (LAK) cell activity. The percentage of CD4⁺ pleural and peritoneal exudate cells (PEC) was significantly higher than the percentage of peripheral blood mononuclear cells (PBMC) in the patients. The percentage of CD8⁺CD11⁺ PEC and PBMC, being the suppressor T-cells, of the patients was increased compared with controls, while the percentage of CD8⁺CD11^– PEC, being the cytotoxic T-cells, was identical to the PBMC of both patients and controls. The NK activity of PEC was significantly lower than that of PBMC in both patients and controls, and there was no correlation between the NK activity of PBMC and PEC. Although the anti-Daudi activity of PEC was markedly low, LAK cells with high activity could be induced by culture with interleukin-2 for 4 days. These results suggest that the immune function of cells in malignant effusions may be depressed due to a low population of cytotoxic T cells, low NK activity and increased suppressor T cells, while the local administration of interleukin-2 may induce LAK cells. Therefore, effective local immunotherapy for malignant effusions should not only augment effector cells, but also inhibit supprssor cells.     

Study of cytotoxicity of recombinant interleukin-2 (rIL-2) expanded tumor infiltrating lymphocytes (TIL) in renal cell cancer

We studied subsets and cytotoxicity of recombinant interleukin-2 (rIL-2) expanded tumor infiltrating lymphocytes (TIL) from renal cell cancer (RCC) patients. TIL were successfully expanded in 13 of 14 RCC cases using anti-CD3 during initial 48 hours of culture. Percentages of CD8 positive cells among rIL-2 expanded TIL at 1 tp 4 week(s) of culture were 56.2 +/- 15.1% (range 26.2 to 79.8%, N = 13) and not necessarily predominant over CD4 positive cells. NK and LAK activities of TIL at 3 to 6 weeks of culture were 31.6 +/- 15.8% (range 1.4 to 57.4%, N = 9) and 16.6 +/- 11.6% (range 3.8 to 35.6%, N = 6), respectively. Autologous and allogeneic RCC cytotoxicity of TIL at 3 to 4 weeks of culture were 17.9 +/- 19.7% (range 0 to 47.6%, N = 4) and 18.9 +/- 14.8% (range 0 to 47.3%, N = 12), respectively. Since there was no statistical difference between them, autologous specific cytotoxicity was not demonstrated. From these results of present study, it is unlikely that most of effector cells of rIL-2 expanded TIL in autologous RCC lysis are major histocompatibility complex restricted cytotoxic T cells. And we concluded that it is doubtful that TIL is significantly superior over LAK cells in immunotherapy of human RCC.     

Intralesional infusion of lymphokine-activated killer (LAK) cells and recombinant interleukin-2 (rIL-2) for the treatment of patients with malignant brain tumor

Twenty patients with supratentorial, intracerebral lesions defined by computed tomographic scan or magnetic resonance imaging were treated by surgery and adoptive immunotherapy with lymphokine-activated killer (LAK) cells and recombinant Interleukin-2 (rIL-2, Cetus). Seventeen patients had glioblastoma, two had high-grade oligodendroglioma, and one patient had two metastatic sarcoma lesions. LAK cells were produced from blood mononuclear cells (MNC) obtained by 2 to 3 leukapheresis procedures and cultured (2.5 x 10(6) MNC/ml) 3 to 5 days with 1000 units rIL-2/ml. Although LAK cells could be produced from MNC of all patients, those taking steroids or with a low Karnofsky functional status generated, on average, suboptimal LAK cell activity. Age, sex, and serum anticonvulsant levels do not seem to influence a patient's ability to produce LAK cells in vitro. For therapy, cultured MNC (1-15 x 10(9] containing LAK cells were suspended in saline containing 10(6) units rIL-2 and injected into tissue surrounding the tumor cavity during craniotomy. For 3 days after their operations, patients received 10(6) units rIL-2 into the tumor cavity through an Ommaya reservoir. The treatment protocol was tolerated well by all patients, although they all experienced some degree of headache, fever, or lethargy that cleared within a few days of the last rIL-2 injection. When computed tomographic (CT) scans were obtained soon after treatment, areas of low density suggested a greater-than-normal extent of edema around the operative site. At the present time, CT scans indicate that the tumors of seven patients have recurred with an average disease-free interval of 25 +/- 6 weeks.(ABSTRACT TRUNCATED AT 250 WORDS)     

Improved therapeutic effect of sequential immunotherapy with cyclophosphamide, large doses of OK-432 and recombinant interleukin-2 in breast cancer patients with disseminated metastatic liver tumors

It has been generally agreed that the prognosis of widely spreaded "surgically unresectable" metastatic liver tumor originated from breast cancer is very poor. We reported here the result of clinical efficacy of sequential immunotherapy with intra-tumoral injection of large dose OK-432, after oral administration of cyclophosphamide during 7-10 days, and continuous perfusion of purified human recombinant interleukin-2 (rIL-2) from hepatic artery for the breast cancer patients with unresectable metastatic liver tumors. In all of 3 cases, metastatic liver tumor revealed overwhelming tumor reduction more than 50% of preoperative total tumor burden evaluated by computed tomography. Only 1 day after operation, large doses of OK-432 was injected intratumorally, both activity of Natural Killer (NK) cells and lymphokine activated killer (LAK) cells in peripheral blood lymphocytes were 5-20 folds augmented in all clinical trials. Serum tumor markers, i.e., Carcinoembryonic Antigen (CEA) and CA15-3, were rapidly decreased in all cases, respectively. Our clinical data indicate that intratumoral injection of large dose OK-432 and continuous administration of rIL-2 via hepatic artery, pretreated with cyclophosphamide, were clinically effective immunotherapy for reduction of metastatic liver tumor.     

The intrapleural administration of recombinant interleukin-2 (rIL-2) to patients with malignant pleural effusion: Clinical trials

Recombinant IL-2 (rIL-2) was administered intra-pleurally according to an original protocol to 11 patients with malignant pleural effusion, 7 of whom suffered from breast cancer and 4 from esophageal cancer. The pleural effusions either disappeared or decreased roentgenographically, and malignant cells disappeared from all 13 pleural cavities in the 11 patients, confirming the validity of this therapy to be 100%. The mean survival time from the initial administration of rIL-2 was 15.9 months. We ensured that the concentration of IL-2 in the effusion was maintained at a high level for a sufficient period of time, and that the lymphokine-activated killer (LAK) activity of lymphocytes in the effusion was augmented. Fever, eosinophilia, and a transient increase in the pleural effusion were the main side effects, but the symptoms were temporary and not serious. The results of this study therefore suggest the efficacy of intrapleural rIL-2 for patients with malignant pleural effusion.     

Adoptive immunotherapy using lymphokine-activated killer cells and recombinant interleukin-2 in preventing and treating spontaneous pulmonary metastases of syngeneic Dunning rat prostate tumor

Lymphokine-activated killer (LAK) cells were generated from splenocytes of rats bearing a weakly immunogenic Dunning prostate tumor (R-3227 AT-3) and activated with recombinant interleukin-2 (rIL-2). The maximal LAK activity was obtained from splenocytes of rats bearing tumors for 10 to 14 days after incubation with 1000 U/ml./day of rIL-2 for five to eight days. The majority of these LAK cells expressed high levels of asialo GM1 (89%), laminin (83%), OX-19 (80%) and OX-8 (88%) surface markers. LAK cells exhibited higher cytotoxicity to rat prostate tumor cells and mouse lymphoma in vitro than to other non-prostate tumor cells or normal rat splenocytes and thymocytes. Splenocytes of rats bearing prostate tumors have higher LAK activity than normal splenocytes. The Winn type assay showed that Dunning prostate tumor growth was inhibited effectively by LAK cells at a tumor cell:LAK cell ratio of 1:50. The therapeutic efficacy of LAK cells in the treatment of primary solid prostate tumors and pulmonary metastases of Dunning rats was evaluated. LAK cells in combination with rIL-2 showed a greater therapeutic benefit in 1) prevention of prostate tumor metastases to lung, 2) retardation of the primary tumor growth, 3) regression of spontaneously established pulmonary metastases, and 4) prolongation of survival as compared to untreated controls or those groups treated with LAK cells or rIL-2 alone. The results of this study indicate that the conjunctive therapeutic approach of using surgical therapy to remove primary solid tumors followed by adoptive immunotherapy with LAK cells plus in vivo administration of IL-2 may be potentially valuable in the treatment of prostate tumors, particularly for the spontaneous pulmonary metastases.     

前列腺素E2对膀胱癌患者LAK细胞增殖及抗肿瘤细胞毒作用的影响

目的探讨前列腺素E_2（PGE_2）在膀胱癌患者淋巴因子激活的杀伤细胞（LAK）增殖及细胞毒的作用。方法LAK细胞培养于含不同浓度的PGE_2;的培养基中,并用细胞计数法测定其细胞增殖率,以BIU87、EJ及患者自体肿瘤细胞为靶细胞,用MTT法测定LAK细胞对膀胱癌细胞的细胞毒作用。结果0.05～5μg／L。PGE_2对LAK细胞的增殖呈浓度依赖性抑制。PGE_2LAK细胞对膀胱癌细胞的杀伤则影响不明显。膀胱癌细胞系BIU87的条件培养基的PGE_2含量明显高于PBMC的条件培养基。结论膀胱癌患者由IL－2诱导的LAK增殖可被由PBMC和膀胱癌细胞产生的PGE_2所抑制。