Raw264.7细胞蛋白和PD-L1抗体在小鼠黑色素瘤(B16)中的抑瘤效果评价

目的探讨小鼠来源白血病毒诱导的肿瘤细胞(Raw264.7)蛋白和程序性死亡-配体(PD-L1)在C57BL/6小鼠黑色素瘤(B16)中的抑瘤效果。方法采用6~8周龄清洁级C57BL/6小鼠30只(雌雄各半),平均分为3组。各组小鼠分别于皮下接种B16细胞(2×10⁵个/只),待小鼠肿瘤平均体积达到100 mm³时,给予治疗。group A:腹腔注射Raw264.7细胞蛋白(50μg/只),每周1次,共2次。group B:腹腔注射PD-L1抗体(600μg/只,本课题组生产纯化),每3天1次,共4次。group C:相同时间点给予等体积生理盐水对照。各组小鼠给药后,测量肿瘤大小,末次测量后,各组小鼠无菌取脾脏,应用流式细胞术分析CD3/4/8⁺T细胞群比例。结果各组荷瘤小鼠给予相应治疗后,组C小鼠肿瘤体积增长迅速,末次测量平均体积达到4004 mm³;组B小鼠增长次之,末次平均体积达到1637 mm³;组A小鼠体积增长最慢,末次平均体积仅为882 mm³(P<0.01)。对小鼠脾脏免疫细胞分析显示,组B小鼠中CD3⁺CD8⁺T cell比例明显上调,平均达到77.65%,而CD3⁺CD4⁺T比例明显下调,仅为11.86%(P<0.01);而组A和组C两组小鼠CD3⁺CD4⁺T和CD3⁺CD8⁺T没有明显区别(P>0.05)。结论制备的RAW264.7细胞灭活蛋白和PD-L1抗体对于B16荷瘤小鼠均有抑瘤作用,且前者效果更为明显。     

结直肠癌及腺瘤患者CD4+、CD8+和CD28+T淋巴细胞的亚群变化及临床意义

背景与目的：结直肠癌（colorectal cancer,CRC）严重影响患者生存。探讨肿瘤微环境（tumor microenvironment,TME）T细胞亚群在CRC和腺瘤中的表达及意义。方法：用免疫组织化学法和流式细胞术对51例健康人（对照组）、46例结直肠腺瘤（腺瘤组）、100例CRC（癌症组）和15例CRC术后（癌术后组）患者进行T细胞亚群检测。结果：① 对照组、腺瘤组及癌症组3组中CD4⁺T细胞的阳性率分别是90.00%、43.75%及32.65%,CD8⁺T淋巴细胞的阳性率分别是30.00%、56.25%及75.51%,CD28⁺T淋巴细胞的阳性率分别是42.86%、30.00%及20.00%。② 对照组、腺瘤组及癌症组3组中CD4⁺、CD4⁺/CD8⁺、CD28⁺、CD8⁺CD28⁺和CD8^－CD28⁺逐渐降低,CD8⁺、CD8⁺CD28^－逐渐增加（P＜0.05）;癌术前术后T细胞亚群差异有统计学意义（P＜0.05）。结论：① CRC微环境T细胞亚群中CD4⁺、CD4⁺/CD8⁺、CD28⁺、CD8⁺CD28⁺和CD8^－CD28⁺呈递减趋势,CD8⁺、CD8⁺CD28^－呈递增趋势,且在癌前病变腺瘤中已逐步出现上述趋势变化。② CRC患者行肿瘤切除术后,其T细胞亚群有所恢复,故在一定程度上,CRC中T细胞亚群的变化可以早期预测结直肠疾病的发展。     

肿瘤细胞减灭术联合洛铂术中腹腔灌注治疗在老年原发性卵巢癌患者中的应用效果

目的探讨肿瘤细胞减灭术联合洛铂术中腹腔灌注治疗在老年原发性卵巢癌患者中的应用效果。方法根据治疗方式的不同将166例老年原发性卵巢癌患者分为手术治疗组和联合化疗组,每组83例,手术治疗组患者采用肿瘤细胞减灭术治疗,联合化疗组患者采用肿瘤细胞减灭术联合洛铂术中腹腔灌注治疗。比较两组患者的围手术期指标、T淋巴细胞因子水平、肿瘤标志物[人附睾蛋白4(HE4)、血管内皮生长因子(VEGF)、糖类抗原125(CA125)]水平及不良反应发生率。结果联合化疗组患者的手术时间明显长于手术治疗组,术中出血量、术中腹腔积液量均明显少于手术治疗组,住院时间明显短于手术治疗组,差异均有统计学意义(P<0.01)。治疗后,两组患者的CD3⁺CD4⁺、CD3⁺CD56⁺、CD3⁺CD4⁺/CD3⁺CD56⁺均高于本组治疗前,联合化疗组患者的CD3⁺CD4⁺、CD3⁺CD...     

载脂蛋白C3 转基因小鼠脾脏免疫细胞表型及其活性分析

目的：分析载脂蛋白C3（Apo C3）转基因小鼠脾脏免疫细胞的表型及活性。方法：以12只ICR背景Apo C3转基因小鼠为研究模型,选取鼠龄/性别匹配的野生型小鼠作为对照,采集小鼠球后静脉丛血液,以酶反应显色比色法联合酶标仪测定血浆甘油三酯水平,选取血浆甘油三酯含量≥ 1 000 mg/dL （11.3 mmol/L）的小鼠用于后续实验。取小鼠脾脏制备成单细胞悬液,采用细胞膜表面分子、胞内分子及核内分子染色法,通过流式细胞术检测转基因小鼠和野生型小鼠脾脏免疫细胞频率和表型。再将转基因小鼠或野生型小鼠脾脏细胞与结肠癌细胞MC38分别以1：1、1：3、3：1效靶比共培养72 h后,通过流式细胞术检测CD8⁺ T细胞CD107a的表达。结果：Apo C3转基因小鼠血浆甘油三酯含量较野生型小鼠显著升高（P<0.01）,而体质量差异无统计学意义（P>0.05）。与野生型小鼠相比,Apo C3转基因小鼠脾脏CD3⁺CD8⁺、CD3⁺CD8⁺NKG2D⁺细胞频率明显增多,髓系抑制性Gr-1⁺CD11B⁺细胞频率增加,脾脏NK1.1⁺NK、NK1.1⁺NKG2D⁺细胞的频率明显减少（P均>0.05）;脾脏CD3⁺CD4⁺NKG2D⁺、CD3⁺CD4⁺IL-17⁺细胞频率以及调节性T细胞,NKT细胞,γδT细胞,B细胞和巨噬细胞频率均无明显变化（P均>0.05）。Apo C3转基因小鼠CD8⁺CD62L^-CD44⁺、CD8⁺CD44⁺KLGR1⁺频率升高,以效应性T细胞为主,且CD8⁺ T细胞分泌IFN-γ活性增强;转基因小鼠中CD8⁺ T淋巴细胞CD107a的表达增加差异均具有统计学意义（P均<0.05）。结论：Apo C3转基因小鼠呈现高水平血浆甘油三酯,其免疫细胞表型及活性具有显著变化,其中CD8⁺ T细胞频率及生物活性上调,髓系抑制性细胞频率升高,NK细胞频率降低;并且转基因小鼠中CD8⁺T淋巴细胞对靶细胞杀伤能力增强。     

过继性免疫治疗对非小细胞肺癌患者外周血T细胞亚群的影响

目的 研究细胞因子诱导的杀伤（cytokine-induced killer,CIK）细胞治疗对非小细胞肺癌（non-small cell lung cancer,NSCLC）患者外周血T细胞亚群及一般情况的影响。方法 采集非小细胞肺癌患者外周血,常规方法体外扩增CIK细胞,分三次回输患者体内,流式细胞仪检测非小细胞肺癌患者治疗前与治疗3次后T细胞亚群的变化,分析其与健康对照组相比外周血T细胞亚群的差异,并观察CIK细胞治疗前后患者一般情况变化。结果 与对照组相比,NSCLC患者外周血中CD3⁺ T 细胞比例、CD3⁺CD4⁺T细胞比例、CD4⁺/CD8⁺ 比值显著下降（P<0.05）,CD3⁺CD8⁺ T细胞比例显著升高（P<0.05）。CIK细胞治疗后与治疗前,CD3⁺ T 细胞比例、CD3⁺CD4⁺ T细胞比例、CD4⁺/CD8⁺ T细胞比值显著升高（P<0.05）,CD3⁺CD8⁺ T细胞比例显著降低（P<0.05）。CIK细胞治疗后与对照组相比,CD3⁺ T 细胞比例、CD4⁺/CD8⁺T细胞比值显著下降（P<0.05）。CD3⁺CD4⁺ T细胞比例、CD3⁺CD8⁺ T细胞比例升高,但差异无统计学意义（P>0.05）。CIK细胞治疗前Tregs较对照组显著升高（P<0.05）。CIK细胞治疗后与治疗前相比,Tregs比例显著下降（P<0.05）。结论 CIK细胞输注治疗可以增强NSCLC患者的免疫功能,并改善患者的一般情况,提高生活质量,且未见明显不良反应。     

贝伐珠单抗联合放疗治疗肺癌脑转移的临床疗效及对患者免免疫疫功功能能、肿瘤标志物的影响

目的 探讨贝伐珠单抗联合放疗治疗肺癌脑转移的临床疗效及对患者免疫功能、肿瘤标志物的影响。方法 依据治疗方案的不同将80例肺癌脑转移患者分为观察组(n=44)和对照组(n=36),对照组患者给予单纯放疗,观察组患者给予贝伐珠单抗联合放疗。比较两组临床疗效、免疫功能指标(CD3⁺、CD4⁺、CD8⁺,计算CD4⁺/CD8⁺)、肿瘤标志物[癌胚抗原(CEA)、甲胎蛋白(AFP)、神经元特异性烯醇化酶(NSE)]水平、不良反应发生情况及远期疗效。结果 观察组患者的客观缓解率和疾病控制率分别为81.82%、95.45%,均高于对照组患者的58.33%、75.00%,差异均有统计学意义(P﹤0.05)。治疗后,两组患者CD3⁺、CD4⁺水平和CD4⁺/CD8⁺均低于本组治疗前,CD8⁺水平均高于本组治疗前,且观察组患者CD3⁺、CD4⁺水...     

EGFR单抗及小分子酪氨酸激酶抑制剂联合放疗对非小细胞肺癌细胞放疗敏感性的影响

目的探讨表皮生长因子受体(EGFR)单抗及EGFR小分子酪氨酸激酶抑制剂(EGFR-TKI)联合放疗对非小细胞肺癌(NSCLC)细胞放疗敏感性的影响,并分析其作用机制。方法将83例非小细胞肺癌患者作为研究对象,按照随机数字表法分为对照组41和观察组42例。对照组给予EGFR-TKI联合放疗,观察组在对照组治疗基础上给予EGFR单抗治疗,比较2组治疗前后外周血T淋巴细胞亚群水平(CD4⁺、CD8⁺、CD4⁺/CD8⁺)、血清基质金属蛋白酶-9(MMP-9)、癌胚抗原(CEA)水平,不良反应,NSCLC细胞放疗敏感性及平均无疾病进展生存时间。结果治疗8周后,对照组CD4⁺、CD4⁺/CD8⁺水平较治疗前明显下降,观察组CD4⁺、CD4⁺/CD8⁺水平显著高于对照组(P<0.05)。治疗8周后,2组血清MMP-9、CEA水平均显著下降(P<0.05),且观察组血清MMP-9、CEA水平均低于对照组(P<0.05)。观察组不良反应发生率与对照组无显著差异(P>0.05),放疗敏感性、平均无疾病进展生存时间均高于对照组(P<0.05)。结论EGFR单抗与EGFR-TKI具有协同作用,联合放疗可增强NSCLC细胞放疗敏感性,提高疗效。     

小鼠衰老所致“正虚”过程中免疫功能衰退表征的特点

目的：基于小鼠渐进衰老模型探讨衰老所致“正虚”的免疫功能衰退表征的特点。方法：使用不同月龄(2、6、15月龄)C57BL/6小鼠,通过流式细胞术检测并比较小鼠外周血和脾组织中T细胞、髓源性抑制细胞(MDSC)及其亚群的丰度变化。结果：外周血中T细胞亚群表型为CD3⁺CD4⁺CD44-CD62L⁺的幼稚CD4⁺T细胞(2 vs 6月龄,P=0.137;2 vs 15月龄,P=0.004;6 vs15月龄,P=0.105)和表型为CD3⁺CD8⁺CD44-CD62L⁺的幼稚CD8⁺T细胞(2 vs 6月龄,P=0.179;2 vs 15月龄,P=0.001;6 vs15月龄,P=0.015)出现与衰老有关的细胞比例降低,差异具有统计学意义。表型为CD3⁺CD4⁺CD44⁺CD62L⁺的中央记忆CD8⁺T细胞出...     

硼替佐米联合地塞米松、来那度胺化疗方案治疗多发性骨髓瘤的疗效和安全性评价

目的 探讨硼替佐米联合地塞米松、来那度胺化疗方案治疗多发性骨髓瘤的疗效及安全性。方法 将多发性骨髓瘤患者96例按随机数字表法分为观察组和对照组。对照组采用硼替佐米联合环磷酰胺、地塞米松化疗方案，观察组采用硼替佐米联合地塞米松、来那度胺化疗方案，均连续给予3个疗程的治疗(28 d为1个疗程)。将2组患者的各项指标进行统一的对比分析。结果 治疗后观察组患者的临床总有效率(93.75%)高于对照组(79.17%);治疗后2组患者的血清白细胞介素(IL)-6、IL-17、C反应蛋白(CRP)、M蛋白、β2微球蛋白水平、骨髓瘤细胞比例、外周血CD3⁺CD8⁺水平与治疗前比较均降低，观察组较对照组均处于较低水平；治疗后2组患者外周血CD3⁺CD4⁺、CD3⁺CD4⁺/CD3⁺CD8⁺、Treg、血清血红蛋白(Hb)水平与治疗前比较均升高，观察组较对照组均处于较高水平，有显著性差异(P均<0.05);治疗期间2组不良反应...     

CD+T细胞及Treg细胞表面PD-1水平与肺癌患者术后预后的关系

目的 探讨CD⁺T细胞及调节性T(Treg)细胞表面程序性死亡受体1(PD-1)水平与肺癌患者术后预后的关系。方法 选取行肺癌根治术的肺癌患者126例,统计术后2年内预后不良发生率,并根据其预后情况将其分为良好组和不良组,单因素分析肺癌患者术后预后不良的影响因素,Logistic回归分析肺癌患者术后预后不良的影响因素,受试者工作特征曲线(ROC)分析CD⁺T细胞及Treg PD-1水平对肺癌患者术后预后的预测价值。结果 两组肿瘤淋巴结转移(TNM)分期、CD3⁺、CD3⁺CD4⁺、CD3⁺CD8⁺及Treg PD-1水平比较,均有显著差异(P<0.05);Logistic回归分析显示,CD3⁺、CD3⁺CD4⁺是肺癌患者术后预后不良的独立保护因素,CD3⁺CD8⁺、Treg PD-1是肺癌患者术后预后不良的独立危险因素(...     

Thymic anti-tumor effectors in mice cured of lymphoma by cyclophosphamide plus TNF-α therapy: Phenotypic and functional characterization up to 20 months after initial tumor inoculation

As reported previously, cyclophosphamide plus tumor necrosis factor-α treatment of C57BL/6 mice bearing advanced EL4 lymphoma induced approx. 60% long-term (i.e., >60 days) survivors. These mice developed protective immunity, as evidenced by 1) rejection (100% survival) of EL4 tumor re-implanted on day 60 (day 0 = initial tumor implantation); and 2) development of significant levels of specific EL4 tumor cell killing activity by both splenocytes and thymocytes. Using this model, age-related changes in functionally and phenotypically definable thymocyte subsets were assessed. In thymocytes from 90 to 308 day survivors, specific immune memory was long term; both CD4⁺ and CD8⁺ cells were required for the ex vivo stimulation of lytic activity, but the specific anti-EL4 cytotoxic effector was CD4⁻CD8⁺. On day 520, the surviving mice were randomized into 2 groups. One group received a second re-challenge with EL4 tumor cells and all survived. The other group was sacrificed on day 520. Their thymocytes, exposed to X-irradiated EL4, developed anti-EL4 lytic activity and, in comparison with thymocytes of young and age-matched control mice, were markedly enriched in CD4⁻CD8⁺CD44⁺ cells. On day 625, thymocytes from the survivors of the day 520 re-challenge were evaluated and were found to have developed specific anti-EL4 lytic activity. Phenotypically, they had returned toward the pattern seen in age-matched control mice although CD4⁻CD8⁺CD44⁺ cells remained increased. These mice were ≥2 years old, the median life span of C57BL/6 mice. Thus, mice cured of tumor by an immuno-modulating regimen rejected re-implanted primary tumor and maintained specific thymic anti-tumor immune memory for life. Int. J. Cancer 76:579–586, 1998.© 1998 Wiley-Liss, Inc.     

抗骨桥蛋白抗体抑制HCCLM3裸鼠移植瘤的生长并增强其对放疗敏感度的实验

目的 探讨抗骨桥蛋白抗体对人肝癌HCCLM3细胞裸鼠移植瘤生长、转移和血管生成的抑制作用及对放疗的增敏作用.方法 74只裸鼠右侧腹皮下接种肝癌细胞株HCCLM3,次日随机取50只裸鼠随机分为5组,每组10只:A组尾静脉注射0.9％氯化钠溶液,B组尾静脉注射非特异性抗体,C、D、E组分别尾静脉注射抗OPN抗体5、10、20 mg/kg,每周2次.每两天观察肿瘤体积变化,第10周末处死裸鼠,收集肿瘤组织、肺组织进行病理组织学检查,用免疫组织化学法测肿瘤微血管密度.其余24只裸鼠随机分为4组:F组给予尾静脉注射0.9％氯化钠溶液、G组给予荷瘤鼠肿瘤局部以6 MeV电子线照射、H组给予尾静脉注射抗OPN抗体20 mg/kg、Ⅰ组给予尾静脉注射抗OPN抗体20 mg/kg+肿瘤局部6 MeV电子线照射.观察肿瘤体积变化,比较各组的肿瘤体积、抑瘤率,利用增敏系数(enhancement factor,EF)评价抗OPN抗体的增敏作用.结果 与A、B对照组相比,抗OPN抗体均能明显抑制肿瘤的生长(P＜0.01)、降低肿瘤组织中血管密度(P＜0.05)、减少肺转移(P＜0.05),其中以20 mg/kg剂量组明显.在放疗的实验中,抗体联合放疗组抑瘤率明显高于单纯放疗组和抗体组(P均＜0.01).EF＞1,提示抗OPN抗体对移植瘤有放疗增敏作用.结论 抗OPN抗体明显抑制了肿瘤的生长、转移、血管生成并能提高肝癌对放疗的敏感度,它有望成为治疗肝癌的又一武器.     

Specific antitumour immunity of HIFU-activated cytotoxic T lymphocytes after adoptive transfusion in tumour-bearing mice

Purpose: The aim of this study was to investigate the specific anti-tumour immunity of cytotoxic T lymphocytes (CTL) activated by high-intensity focused ultrasound (HIFU) after adoptive transfer in a murine tumour model. Materials and methods: H22 tumour-bearing mice were treated by either HIFU or sham-HIFU, while naïve syngeneic mice were used as controls. They were sacrificed and the spleens were harvested 14 days after HIFU. T lymphocytes were obtained from the spleens, and then adoptively transferred into 40 mice each bearing a 3-day implanted H22 tumour. On day 14 after adoptive transfer, 10 mice were sacrificed in each group for assessment of the number of tumour-infiltrating T lymphocytes and interferon-gamma (IFN-γ) secreting cells. The remaining 30 mice were continuously observed for 60 days, and tumour growth, progression and survival were recorded. Results: HIFU significantly increased peripheral blood CD3⁺, CD4⁺ levels and CD4⁺/CD8⁺ ratio (P?<?0.05), CTL cytotoxicity (P?<?0.01) and IFN-γ and TNF-α secretion (P?<?0.01) in H22 tumour-bearing mice. Adoptive transfer of HIFU-activated T lymphocytes into the autologous tumour-bearing mice induced a significant increase of tumour-infiltrating T lymphocytes and IFN-γ-secreting cells (P?<?0.001). Compared to the control and sham-HIFU groups, HIFU-activated lymphocytes elicited significant inhibition of in vivo tumour growth (P?<?0.01) and progression (P?<?0.0001), and longer survival time in the tumour-bearing mice (P?<?0.001). Conclusions: HIFU could enhance CTL’s specific antitumour immunity. Adoptive transfer of HIFU-activated T lymphocytes could increase local antitumour immunity, and elicit stronger inhibition on tumour growth and progression, with more survival benefit in the autologous tumour-bearing mice.     

Induction of antitumor response to fibrosarcoma by Newcastle disease virus-infected tumor vaccine

Fibrosarcoma is a locally aggressive malignant tumor with a high recurrence rate, so that wide excisional surgery is necessary for treatment. However, it is often difficult to resect with a sufficient margin of excision at the site of tumor infiltration. Recombinant tumor vaccine therapy is a useful method to induce specific immunity. In this study, we have shown its utility as a candidate for therapy by applying a recombinant Newcastle disease virus (rNDV) tumor vaccine (rNDV-TV). Although the therapeutic effect of similar viruses has been examined in several tumors, the vaccination efficacy against fibrosarcoma has not been demonstrated until now. In this study, we showed the induction of an antitumor response by rNDV-TV against murine fibrosarcoma and investigated the role of lymphocytes in tumor elimination. Intraperitoneal inoculation of murine fibrosarcoma (WEHI164) cells showed increased lethality in C.B.17scid/scid (scid) mice within 2 weeks of inoculation. The survival rate increased to 80% when the mice were transfused with CD3⁺ cells from BALB/c mice previously immunized with rNDV-TV. However, all mice died from tumor growth after inoculation with non-immunized CD3⁺ cells. Although the survival rate was around 50% in mice receiving only immunized CD4⁺ and CD8⁺ cells, the survival rate was not decreased in mice receiving CD3⁺CD4^?CD8^? (natural killer T; NKT) cells together with immunized CD4⁺ and CD8⁺ cells. This study showed rNDV-TV induced an antitumor T cell response to WEHI164 cells, and major subsets of cells involved in tumor exclusion were CD4⁺ and CD8⁺ cells, together with NKT cells.     

Changes in T lymphocyte subsets in mice with CT26 colon tumors after treatment with donor lymphocyte infusion

The objective of this study was to detect changes in T lymphocyte subpopulations in mice with CT26 subcutaneous colon cancer after treatment with donor lymphocyte infusion (DLI) and cyclophosphamide (CP) chemotherapy. A colon cancer model was established by subcutaneous injection of CT26 carcinoma cells into BALB/C mice. The mice were randomized into different treatment groups. We recorded survival times, tumor growth inhibition rates, histopathological changes, and T lymphocyte subsets in peripheral blood of the mice. Mice treated with DLI and CP survived 33.5?±?5.02 days, which was significantly longer than the survival time of untreated control mice (16.7?±?2.98 days, P?<?0.01). In addition, the tumor inhibitory rate was higher in mice treated with DLI and CP (89.3 %) than that in mice treated with CP or DLI alone (67.1 and 34.5 %, respectively). There were higher levels of T lymphocytes that were CD3⁺ and CD4⁺ in mice treated with DLI alone or the combination of CP and DLI (P?<?0.05), and the ratio of CD4⁺/CD8⁺ cells was significantly improved in these mice (P?<?0.05). DLI combined with chemotherapy significantly prolonged survival and inhibited tumor growth in mice with CT26 colon cancer. This treatment might also improve immune function in these mice. Donor spleen cells that include high numbers of allogeneic lymphocytes and a few stem cells could induce a graft-versus-tumor effect, leading to elimination of residual cancer cells. This indicates that it is potentially a feasible adoptive cellular immunotherapy strategy for the management of solid tumors.     

三维适形放疗联合高强度聚焦超声治疗前列腺癌临床疗效观察

Objective: Prostate cancer is a form of cancer that develops in the prostate, a gland in the male reproductive system.Prostate cancer tends to develop in men over the age of fifty; it is one of the most prevalent types of cancer in men.This article introduced a new method of prostate cancer treatment with the combination of three dimensional conformal radiation therapy (3D-CRT) and high-intensity focused ultrasound (HIFU), its efficacy was evaluated.Methods: From January 2004 to December 2009, 95 patients were diagnosed with prostate cancer, among them, 48 patients were received combined therapy with total irradiation of TD 60 Gy/30 Fx and 5 fractions of HIFU treatment, while 47 patients were received with pure 3D-CRT with total irradiation of TD (66-72) Gy/(33-36) Fx.Various indicators were evaluated, such as the local control rate and distant metastasis rate, the changes in blood PSA and fPSA, changes in T-lymphocyte subsets and NK cells, as well as acute adverse reaction of normal tissue.Results: The local response rate difference between the two groups had statistical significance (P < 0.05); the changes in blood PSA and fPSA were significant (P < 0.05); CD3+, CD4+, CD8+, CD4+/CD8+ and NK cells of the combined group increased obviously (P < 0.01), while the latter group had no increase (P > 0.05); the combined group had lower blood cells reduction and II-level acute adverse reaction of rectum, bladder and caput humeri than the pure group, but the II-level acute adverse reaction of urogenital canal in the combined group was higher (P < 0.05).Conclusion: The combined therapy with 3D-CRT and HIFU is a good way for the treatment of aged-related prostate cancer.It can ease the symptoms, control the disease and lengthen the survival time.     

Cyclophosphamide plus tumor necrosis factor-α chemoimmunotherapy cured mice: Life-long immunity and rejection of re-implanted primary lymphoma

Changes in functionally and phenotypically definable spleno-cyte subsets in aging mice which had been rendered tumor-free in early life by immunochemotherapy (cyclophosphamide plus tumor necrosis factor-α) were studied in the syngeneic EL4 lymphoma-C57BL/6 murine model. Treatment-induced long-term survivors (LTS) surviving rechallenge are termed “immune-LTS”. On day 120 (day 0, initial tumor inoculation), splenocytes from day 60 rechallenged immune-LTS developed significantly greater specific anti-EL4 cytolytic activity in an ex vitro assay than those from non-rechallenged LTS. Splenocytes from combination-treated groups developed significantly higher activity than those from cyclophosphamide-induced immune-LTS. The splenic effector precursor was a CD8⁺ T cell. The specific anti-EL4 effector cell from the cyclophosphamide-induced immune-LTS was CD4^? CD8⁺; however, approximately 50% of those from combination-treated immune-LTS appeared to be CD4⁺CD8⁺. On day 520 immune-LTS were randomized into 2 groups. One group was re-implanted with EL4 tumor; all mice survived. The other group was killed and, even though their splenocytes developed considerable anti-EL4 activity, their allogeneic responsiveness was as reduced as that of age* matched controls. Phenotypic analysis, compared with splenocytes from young and age-matched controls, revealed changes in the makeup of each T-cell subset, except the CD4⁺CD8⁺, and all subsets, except the CD4^? CD8^?, had increases in CD44 positivity. On day 625, the age of these mice was equivalent to the median life-span of C57BL/6 mice; nevertheless, their splenocytes developed high anti-EL4 activity. Phenotypic analysis indicated that, compared to day 520, there was a major decrease in CD4^? CD8⁺ splenocytes; we suggest that these cells had migrated to the site of tumor eradication.     

Adoptive transfer of ex vivo-activated memory T-cell subsets with cyclophosphamide provides effective tumor-specific chemoimmunotherapy of advanced metastatic murine melanoma and carcinoma

Autolymphocyte therapy (ALT) is adoptive cellular therapy of neoplastic disease using ex vivo activation of autologous (human) or syngeneic (murine) lymphocytes from tumor-bearing hosts (TBH) by low doses of anti-CD3 monoclonal antibody (MAb) and a mixture of previously prepared autologous cytokines (T3CS). Ex vivo activation by T3CS without tumor antigen results in expansion of CD44⁺ (memory) T cells. These memory T cells (ALT cells) mediate in vivo anti-tumor specificity and with cyclophosphamide (CY) are capable of curing metastatic disease in murine TBH. To determine whether CY could enhance the effectiveness of CD4⁺ or CD8⁺ subsets of ALT cells, C57BL/6J TBH with B16 melanoma or Lewis lung (3LL) carcinoma were treated with adoptive chemoimmunotherapy (ACIT) using CD4-depleted or CD8-depleted ALT cells and CY. ALT cells were derived from splenocytes of B16 or 3LL-TBH and activated ex vivo with T3CS. Depletion of CD4⁺ or CD8⁺ T cells was performed before or after activation with T3CS. B16-TBH or 3LL-TBH that received ACIT using CY with B16-derived or 3LL-derived CD8-depleted ALT cells, respectively, demonstrated cure of metastatic disease regardless of whether CD8⁺ T cells were depleted before or after T3CS activation. B16 or 3LL-TBH that received ACIT using CY with B16 or 3LL-derived CD4-depleted ALT cells also cured metastatic disease but only if CD4⁺ T cells were depleted after T3CS activation. Interleukin (IL)-2 added to pre-T3CS CD4-depleted ALT cells cultured with T3CS restored anti-tumor activity when combined with CY. TBH cured by ACIT using CY and ALT-cell subsets derived from syngeneic TBH with the identical tumor displayed tumor-specific immunity in rejecting a lethal challenge of identical but not reciprocal tumor. TBH given ACIT using CY and ALT-cell subsets derived from splenocytes of syngeneic TBH with reciprocal tumors rejected lethal challenges of both tumors. Tumor specificity measured by interferon (IFN)-γ and ⁵¹Cr-release assays was demonstrated in pre- or post-T3CS/CD8-depleted, post-T3CS/CD4-depleted and pre-T3CS + IL-2/CD4-depleted ALT-cell subsets. Our data demonstrate that ACIT using CY combined with ex vivo T3CS-activated CD44⁺ memory T-cell subsets conveys long-term tumor-specific immunity. © 1995 Wiley-Liss, Inc.     

Tumor infiltrating T lymphocytes expressing FoxP3, CCR7 or PD-1 predict the outcome of prostate cancer patients subjected to salvage radiotherapy after biochemical relapse

Tumor immunologic microenvironment is strongly involved in tumor progression and the presence of tumor infiltrating lymphocytes (TIL) with different phenotypes has been demonstrated to be of prognostic relevance in different malignancies. We investigated whether TIL infiltration of tumor tissues could also predict the outcome of prostate cancer patients. To this end, we carried out a retrospective analysis correlating the outcome of locally advanced prostate cancer patients undergone salvage radiotherapy upon relapse after radical surgery with the infiltration by different TIL populations. Twenty-two patients with resectable prostate cancer, with a mean age of 67 (+/?3.93) years, who received salvage radiotherapy with a mean of 69.66 (+/? 3.178) Gy in 8 weeks, between June 1999 and January 2009 and with a median follow up of 123 (+/? 55.82) months, were enrolled in this study. We evaluated, by immunohistochemistry, the intratumoral (^t) and peripheral stroma (^p) infiltration by CD45, CD3, CD4, CD8, CCR7, FoxP3 or PD-1-positive cells on tumor samples taken at the diagnosis (^d) and relapse times (^R). We correlated these variables with patients' biochemical progression free survival (bPFS), post-radiotherapy progression free survival (PFS), and overall survival (OS). Substantial changes in the rate of TIL subsets were found between the first and the second biopsy with progressive increase in CD4, CCR7, FoxP3, PD-1⁺ cells. Our analysis revealed that higher CD8^p,R+ and lower PD-1^R+ TIL scores correlated to a longer bPFS. Higher CD8^p,R+ and CCR7^t,R+ TIL scores and lower CD45^p,R+ and FoxP3^p,R+ TIL scores correlated to a prolonged PFS and OS. These results suggest that the immunological microenvironment of primary tumor is strictly correlated with patient outcome and provide the rationale for immunological treatment of prostate cancer.     

胸苷酸合成酶、胸苷酸磷酸化酶的表达与培美曲塞时辰化疗的疗效分析

目的构建肺腺癌A549细胞的裸鼠皮下移植瘤模型,研究按昼夜不同时辰给予培美曲塞是否能提高疗效和降低不良反应,分析胸苷酸合成酶（TS）mRNA和胸苷酸磷酸化酶（TP）mRNA表达高低与培美曲塞疗效以及不良反应的关系。方法 常规方法体外培养肺腺癌A549细胞。待肿瘤体积生长至0.5~1.5 cm³,随机将荷瘤鼠分为四组,每组5只,其中三组严格按照每昼夜24 h的三个时间点给药即:早晨7点（光照后0 h）、中午15点（光照后8 h）、晚间23点（光照后16 h）进行腹腔给药,第四组为对照组,腹腔给予同体积的0.9%氯化钠溶液。培美曲塞按照150 mg/kg应用,每天一次,连续4天。以后每周测量瘤体积,裸鼠体重,观察裸鼠的行为状态、精神以及食欲变化。实验结束时称量剥离的裸瘤重量。应用实时荧光定量PCR（Real-Time PCR）检测肿瘤组织中TS mRNA和TPmRNA的表达水平。结果7 h给药组裸鼠体重增长较快（P=0.041）,化疗不良反应较小,瘤体积生长最慢（P=0.032）,抑瘤率最大（P=0.000）。7 h给药组的肿瘤组织中TS mRNA相对表达量最低,TP mRNA的相对表达量对高,差异有统计学意义（P<0.01）。结论培美曲塞对肺腺癌裸鼠移植瘤有明显的抑制作用,疗效及不良反应与时辰给药有关,7 h给药组疗效最佳,化疗不良反应最小;TS和TP可以作为培美曲塞的疗效预测因子,为培美曲塞的个体化临床用药提供参考。