Establishment of shared antigen reactive cytotoxic T lymphocyte using co-stimulatory molecule introduced autologous cancer cells

Cytotoxic T lymphocytes (CTLs) play an essential role in immunological responses for tumor rejection. In the past decade, many tumor-associated antigens (TAAs) have been identified predominantly in melanomas. Several clinical trials based on such antigenic peptides with or without adjuvants brought about partially favorable results, suggesting that identification of more immunogenic TAAs is needed. We show here the successful establishment of human leukocyte antigen (HLA)-A24-restricted CTL (TcLHK2 line1) from a pleural effusion of lung cancer patient, using B7.1 (CD80) transduced autologous lung cancer cells as an antigen-presenting cell (APC). TcLHK2 line1 recognized autologous lung adenocarcinoma cell line LHK2 in an HLA-A24-restricted fashion. Moreover, this CTL line also recognized allogeneic HLA-A24-positive lung adenocarcinoma cell line, gastric carcinoma cell line and melanoma cell line. These data raise the possibility that co-stimulatory molecule B7.1 (CD80) plays important role to overcome the immunological tolerance. Furthermore, TcLHK2 line1 is a useful tool for the identification of widely expressed shared antigens restricted by HLA-A24. Further analysis of this CTL and autologous cancer cell line will bring about novel TAAs.     

CD3和CD28双刺激活化的健康人外周血T淋巴细胞与胃泌素拮抗剂合用对结肠癌细胞的杀伤作用

目的探讨联合应用胃泌素拮抗剂和CD3/CD28单克隆抗体共刺激活化健康人外周血T淋巴细胞(PBLs)在体外对结肠癌细胞株HT-29的影响及杀伤途径。为结直肠癌的过继免疫治疗提供参考。方法外周血T淋巴细胞(PBLs)的分离与体外培养;CD3/CD28共刺激活化外周血T淋巴细胞(PBLs);MTT法检测活化细胞的体外淋巴细胞毒作用,并用流式细胞仪检测结肠癌细胞的凋亡情况。结果用CD28共刺激细胞和CI—988分别处理6d后对结肠癌细胞株HT-29抑制率为66%和47.5%,二者合用后3d和6d抑制率分别为41%和90.5%;较单用任何一种处理对结肠癌细胞株HT-29杀伤作用更强(P<0.01)。流式细胞仪FCM图像可见实验组细胞群坏死比例为30.1%,凋亡细胞占19.2%,而对照组仅有坏死细胞0.22%,凋亡细胞1.6%。结论联合应用胃泌素拮抗剂CI-988可以提高单抗协同诱导的效应细胞对结肠癌细胞株HT-29总体抑制,而坏死细胞进一步增多说明效应细胞是通过诱导肿瘤细胞坏死及细胞凋亡两条途径来实现抑制作用。     

鼻咽癌外周血单个核细胞Fas/FasL的表达与预后的相关性研究

目的:研究凋亡相关基因Fas/FasL在鼻咽癌患者诱导化疗前后外周血单个核细胞中的表达,探讨其与鼻咽癌预后的相关性。方法:采用流式细胞仪对2002年收治的38例鼻咽癌诱导化疗前后及15例健康对照组外周血单个核细胞中Fas/FasL的表达进行检测,并结合临床资料进行分析。结果:5a生存率为65.8%(25/38),死亡13例。鼻咽癌患者外周血单个核细胞中Fas/FasL的表达明显高于健康对照组(P<0.05);Ⅲ+Ⅳa期的表达明显高于Ⅱ期(P<0.05)。化疗后与化疗前比较,Fas表达明显增高(P<0.05),FasL表达稍增高(P>0.05)。化疗前死亡组Fas/FasL的表达明显高于生存组(P<0.05);化疗后死亡组Fas的表达明显高于生存组(P<0.05),FasL的表达明显低于生存组(P<0.05)。生存组化疗后Fas/FasL明显高于化疗前(P<0.01,P<0.05)。死亡组化疗后Fas明显高于化疗前(P<0.01),FasL稍低于化疗前(P>0.05)。结论:外周血单个核细胞Fas/FasL的表达及诱导化疗前后的改变与鼻咽癌分期、预后有关,在鼻咽癌患者中对其进行检测可能具有重要临床意义。     

IFN对慢性乙肝患者PBMC内HBV-DNA的阴转作用

[目的]探讨IFN-α2b对慢性乙肝患者HBV-DNA的阴转作用。[方法]选择典型慢性乙肝患者160例,其中慢性乙肝轻度93例、中度67例。随机分为IFN治疗组和常规治疗组,用PCR法动态检测患者HBV-DNA载量;动态复查肝功能。[结果]IFN-α2b治疗3个疗程后,慢性乙肝轻度患者HBeAg阴转率、HBeAg血清转换率和PBMC内、血清中HBV-DNA阴转率显著高于常规治疗者(P﹤0.01);慢性乙肝中度IFN-α2b治疗者与常规亚组相比差异亦有统计学意义(P﹤0.05)。慢性乙肝轻、中度患者IFN-α治疗后,ALT降低值、AST降低值、AST/ALT均优于常规组(P﹤0.01～P﹤0.05)。[结论]IFN-α2b对PBMC、血清HBV-DNA和HBeAg均有较好阴转效果,并能促进肝功能恢复。     

CpG-ODN活化的外周血单个核细胞体外对肿瘤细胞杀伤效应的研究

目的探讨CpGODN体外活化的外周血单个核细胞（PBMC）后对肿瘤细胞的杀伤效应。方法CpG—ODN体外刺激人外周血单个核细胞（PBMC），将活化的PBMC与肿瘤细胞按50：1的比例共孵育72h后，以MTT和酶学检测活化的PBMC对肿瘤细胞的杀伤作用。结果CpC—ODN诱导活化的PBMC对肿瘤细胞的杀伤作用增强，而对正常肝细胞则无明显的杀伤作用。结论CpG—ODN可诱发机体免疫效应，提高对肿瘤细胞的杀伤作用。     

Regulatory effects of deoxycholic acid, a component of the anti-inflammatory traditional Chinese medicine Niuhuang, on human leukocyte response to chemoattractants

Niuhuang is a commonly used Chinese traditional medicine with immunoregulatory and anti-inflammatory properties. Deoxycholic acid (DCA) is a major active constituent of Niuhuang. The reaction of human leukocytes to chemoattractants is an important part of the host immune response and also plays a crucial role in the development of inflammation. We, therefore, investigated the in vitro effects of DCA on human monocyte and neutrophil responses to classic chemoattractants [fMet-Leu-Phe (fMLP), complement fraction 5a (C5a)], CC chemokine [monocyte chemoattractant protein-1 (MCP-1/CCL2)], and/or CXC chemokines [stromal cell-derived factor-1 (SDF-1alpha/CXCL12), interleukin-8 (IL-8/CXCL8)]. The results showed that DCA significantly inhibited fMLP-induced monocyte and neutrophil chemotaxis and calcium mobilization, and also blocked the binding of [3H]fMLP and anti-formyl peptide receptor (FPR) monoclonal antibodies (mAb) to the cells. The inhibitory effects of DCA on calcium mobilization and anti-FPR-mAb binding to the receptor could be abrogated by washing DCA out of the cell suspension, suggesting that DCA blocked fMLP receptors via a steric hindrance mechanism, not via receptor internalization. DCA had no significant inhibitory effects on MCP-1-, SDF-1alpha-, or C5a-induced monocyte function, or C5a- or IL-8-induced neutrophil function. Taken together, our experimental results suggest that blockade of fMLP receptors may contribute to the anti-inflammatory effects of traditional medicine containing DCA.     

圆孢子虫感染者T细胞亚群数量变化的逻辑质研究

目的：采用逻辑质方法探讨圆孢子虫病患者的T细胞亚群数量的变化特点。方法：采用生物素-链霉亲和素（Biotin-Streptavidin，BSA）法对卵囊阳性患者分别进行外周血T细胞亚群检测。结果：圆孢子虫卵囊阳性患者的CD3^＋、CD4^＋和CD4^＋／CD8^＋阳性百分率，经逻辑质的t1-4。指标分析，与正常人的T细胞亚群数量存在显著的逻辑差异性（t=0．0611，P〉0．05；t≥3．681，P〈0．05）。结论：圆孢子虫病患者的T细胞亚群数量低于正常人。     

维生素A和锌联合在体外诱导人外周血单核细胞DNA损伤的研究

目的　通过碱性单细胞电泳凝胶技术和流式细胞术,研究维生素A和锌联合在体外诱导人的外周血单核细胞(PBMC)DNA的损伤。方法　分离健康人PBMC在体外培养,分别加入不同剂量的维生素A和锌改变培养基环境,通过单细胞电泳凝胶技术和流式细胞术,分别检测各个剂量组PBMC的细胞凋亡率与DNA断裂情况。结果　体外培养时补充适量的锌和维生素A尚未使PBMC凋亡率增加,没有加重DNA损伤,但同时补充过量的锌和维生素A就会引起PBMC的广泛凋亡和严重的DNA损伤(P <0 .0 5 ) ,且比由于维生素A或锌单独过量引起的损伤更严重。当维生素A过量时,补充适量锌对已损伤的细胞有一定的修复作用,但可能会扩大受损的细胞范围;当锌过量时,补充适量维生素A对正常细胞有一定的保护作用,但可能会加剧已受损细胞的损伤程度。结论　在体外试验中,过量的补充维生素A和锌可能会造成人的PBMC的DNA单链严重损伤,具体机理尚须进一步研究。     

金黄色葡萄球菌肠毒素C2基因定点突变分析

目的 利用金黄色葡萄球菌肠毒素C2(SEC2)基因His118定点突变,以获得超抗原性保持不变的减毒肠毒素.方法 利用PCR介导的定点突变技术,对SEc2基因中的His118密码子进行定点突变,得到的带有突变基因的表达质粒在大肠埃希菌中用异丙基硫代-β-D-半乳糖苷(IPTG)诱导其表达.采用镍离子金属整合(NiNTA)亲和层析和凝胶过滤层析纯化突变体蛋白,并测定突变体蛋白超抗原性及体外抑瘤效果.结果 应用点突变试剂盒,PCR扩增得到SEc2突变体表达质粒pET-28a-SEC2(H118Y)在1nmol.L IPTG诱导下高效表达.突变体蛋白SEC(H118Y)在2～200ng时刺激人体细胞增殖效果与sEC2基本一致,体现二者具有相同的超抗原性.SEC(H118Y)时,在2～200ng与SEC2抑制大肠癌细胞Cx-1效果基本相同,而在500ng时,SEC(H118Y)抑瘤效果明显优于与SEC2.结论 SEC(H118Y)超抗原性和抑瘤活性没有因His118位点的突变而改变.     

Fetal peripheral blood mononuclear cell proliferative responses to mitogenic and allergenic stimuli during gestation

Blood samples were obtained from fetuses and premature babies (n=51) (15-34 weeks gestation) to determine at what stage the fetal immune system was able to produce a positive proliferative response to common allergens. Peripheral blood mononuclear cells (PB MC) were stimulated with the mitogen, phytohaemagglutinin (PHA), and the allergens, house dust mite, cat fur. birch tree pollen, β-lactoglobulin, ovalbumin and bee venom (mellitin). Results were expressed as ratios of stimulated to unstimulated ³H thymidine incorporation, and as percent positive responders. There was an increase in proliferation ratio which correlated with increasing gestational age for PHA (p < 0.0001), cat fur (p=0.042), birch pollen (p=0.022) and β-lactoglobulin (p=0, 006). The point in gestation when cells from some individuals began responding to the allergens with a ratio of 2. 0 was at approximately 22 weeks. PBMC proliferative response ratios were higher from samples from babies > 22 weeks gestation compared to < 22 weeks for the mitogen and all allergens, except mellitin. There was also a greater proportion of positive responders from samples > 22 weeks compared to < 22 weeks for the mitogen and all allergens, except mellitin. Maternal exposure to birch pollen, which has a discrete season, was assessed to determine whether exposure had occurred at 22 weeks gestation or beyond. Results showed a higher prolifera tive response in infant cells stimulated with birch pollen (p=0.005) and higher proportion of positive responders (p=0.01) in the group of babies whose mothers had been exposed to hirch pollen beyond 22 weeks, compared to those whose mothers had not been so exposed. These results suggest that in utero fetal exposure to an allergen from around 22 weeks gestation may result in primary sensitisation to that allergen, leading to positive proliferative responses, at birth.