腺病毒介导的胞嘧啶脱氨酶自杀基因联合IL-2基因疗法的肿瘤治疗作用及免疫机理分析

目的以腺病毒作为载体，将大肠杆菌胞嘧啶脱氨酶（ＣＤ）基因与小鼠ＩＬ－２基因联合转移，研究其体内抗肿瘤作用及免疫机理。方法小鼠皮下接种黑色素瘤Ｂ１６Ｆ１０细胞后３天，肿瘤局部注射表达ＩＬ－２的重组腺病毒ＡｄＩＬ－２和表达ＣＤ的重组腺病毒ＡｄＣＤ，然后连续１０天给予５－氟胞嘧啶（５－Ｆｃ）３００ｍｇ／ｋｇ进行治疗。结果联合治疗组荷瘤小鼠皮下肿瘤结节的生长明显受到抑制，小鼠存活期明显长于ＡｄＩＬ－２、ＡｄＣＤ／５－Ｆｃ、ＡｄｌａｃＺ／５－Ｆｃ或ＰＢＳ组。经联合治疗后，小鼠脾细胞的ＮＫ活性和ＣＴＬ杀伤活性明显增强；肿瘤瘤体内ＣＤ４、ＣＤ８细胞浸润增加；肿瘤细胞表达Ｈ－２Ｋｂ和Ｂ７－１分子明显增加。结论联合应用自杀基因和ＩＬ－２基因治疗，一方面可以明显抑制荷瘤小鼠肿瘤生长，另一方面可以提高机体对肿瘤细胞免疫应答，增加机体的抗肿瘤作用，是肿瘤基因治疗中一条行之有效的途径。     

白细胞介素18基因修饰的树突状细胞的表型分析及其免？…

目的：研究ＩＬ－１８基因修饰后的树突状细胞（ＤＣ）的表型和功能的变化。方法;以含ＩＬ－１８基因的重组腺病毒体外转染小鼠骨髓来源的ＤＣ,ＥＬＩＳＡ检测ＩＬ－１８分泌水平,以ＲＴ－ＰＣＲ检测ＩＬ－１８ｍＲＮＡ表达,以ＦＡＣＳ法分析其ＤＣ表型变化,采用（＾３Ｈ）－ＴｄＲ检测其ＭＬＲ的变化。     

低剂量环磷酰胺增强MHC I类限制性肿瘤抗原多肽冲击致敏 …

目的：研究低剂量环磷酰胺（Ｃｙ）联合ＭＨＣ Ｉ类限制性肿瘤抗原多肽Ｍｕｔｌ致敏、白细胞介素２（ＩＬ－２）基因修饰的树突状细胞（ＤＣｓ）对转移性肺癌小鼠的治疗作用及其免疫学机理。方法：制备小鼠骨髓来源的ＤＣｓ,用转移性Ｌｅｗｉｓ肺癌特异性多肽Ｍｕｔｌ预激经ＩＬ－２基因修饰的ＤＣｓ联合低剂量Ｃｙ治疗转移性肺癌小鼠。通过ＦＡＣＳ分析其脾细胞内Ｔ淋巴细胞比例的变化,＾５１Ｃｒ释放法检测ＣＴＬ和ＮＫ细胞杀伤     

腺病毒介导的胞嘧啶脱氨酶自杀基因联合IL—2基因疗法的肿瘤治 …

以腺病毒作为载体,将大肠杆菌胞嘧啶脱氨酶基因与小鼠ＩＬ－２基因联合转移,研究其体内抗肿瘤作用及免疫机理。方法小鼠皮下接种黑色素瘤Ｂ１６Ｆ１０细胞后３天,肿瘤局部 注射表达ＩＬ－２的重要腺病毒ＡｄＩＬ－２和表达的ＣＤ的重组腺病毒ＡｄＣＤ,然后连续０天给予５－氟胞嘧啶３００ｍｇ／ｋｇ进行治疗。     

应用流式细胞仪检测大肠癌患者外周血淋巴细胞IL—2受体的表达

应用流式细胞仪检测２２例大肠癌患者外周血淋巴细胞ＩＬ－２受体（ＩＬ－２Ｒ）表达。结果表明：大肠癌患者ＩＬ－２Ｒ表达能力明显低下，低下程度与肿瘤病期早晚有关，而与肿瘤部位无关。早期大肠癌（Ｄｕｋｅｓ Ａ，Ｂ期）ＩＬ－２Ｒ表达基本正常，而中晚期大肠癌（Ｄｕｋｅｓ Ｃ，Ｄ期）ＩＬ－２Ｒ表达显著低下。ＩＬ－２Ｒ表达低下可能会削弱肿瘤宿主对ＩＬ－２治疗的反应。     

白细胞介素-7基因抗肿瘤效应的实验观察研究

目的：探讨白细胞介素－７裸ＤＮＡ质粒用于小鼠肿瘤基因治疗的可行性。方法：构建ＩＬ－７基因真核表达质粒（ｐＳＶＫ３ ＩＬ－７）,并将此质粒ＤＮＡ直接肌肉注射到Ｂａｌｂ／ｃ小鼠体内,在此之前７天、同时、之后７天背部皮下接种ＳＰ－２肿瘤细胞,观察该质粒基因的体内抗肿瘤生物学活性;并通过ＲＴ－ＰＣＲ的方法检测注射部位ＩＬ－７ｍＲＮＡ的表达情况。结果：在实验中观察到直接肌肉注射ＩＬ－７基因质粒ＤＮＡ的荷瘤鼠     

福氏完全佐剂包裹白细胞介素2和肿瘤坏死因子对小鼠S180肿瘤的抑制作用

本文报道一种新的免疫治疗方法。用福氏完全佐剂（Ｆｒｅｕｎｄ＇ｓ　ｃｏｍｐｌｅｔｅ　ａｄｊｕｖｅｎｔ，ＦＣＡ）包裹白细胞介素２（ｌｎｔｅｒｌｅｕｋｉｎ　２，ＩＬ－２）及／或肿瘤坏死因子（Ｔｕｍｏｒ　Ｎｅｃｒｏｓｉｓ　Ｆａｃｔｏｒ，ＴＮＦ）作了抗Ｓ１８Ｏ肉瘤的体内抗瘤实验。结果表明：ＩＬ－２及／或ＴＮＦ经佐剂包裹后，可明显减轻其毒副作用，并显著提高ＩＬ－２及／或ＴＮＦ的抗瘤作用。     

急性白血病凋亡基因及细胞DNA检测的临床意义

目的：研究急性白血病促、抑凋亡基因表达、白血病细胞ＤＮＡ含量及三指标间相关性。方法：用流式细胞术检测５１例急性白血病及２０例良性血液患者ＣＤ９５、ｂｃｌ－２表达率、白血病细胞ＤＮＡ含量。结果：急性白血病ＣＤ９５表达率降低且与完全缓解率无关。ｂｃｌ－２表达率、白血病细胞ＤＮＡ异位体率增高且化疗后完全缓解率低。ＣＤ９５、ｂｃｌ－２,白血病细胞ＤＮＡ指数（ＤＩ）间无相关性,三指标表达均异常相关性分析,Ｃ     

小鼠B16黑色素瘤细胞HACs的提纯及其抑瘤作用和机理的探讨

目的：从小鼠Ｂ１６黑色素瘤细胞胞浆中提纯热休克蛋白抗原肽复合物（ｈｅａｔ ｓｈｏｃｋ ｐｒｏｔｅｉｎ－ａｎｔｉｇｅｎ ｐｅｐｔｉｄｅ ｃｏｍｐｌｅｓｅｓ．ＨＡＣＣｓ）,并观察其抑瘤作用及探索其机制。方法：ＣＮＢｒ－Ｓｅｐｈａｒｏｓｅ４Ｂ亲和层析法纯纯Ｂ１６ ＨＡＣｓ,应用体内免疫法检测ＨＡＣ的抑瘤作用,结晶紫染色法检测γ－ＩＦＮ分泌活性,ＣｏｎＡ诱导的淋巴母细胞增殖法检测ＩＬ－２分泌活性,＾３Ｈ－     

bcl—2,53,cathepsin D,PSA表达与乳腺癌预后的关系

目的：探讨ｂｃｌ－２、ｐ５３、Ｃａｔｈｅｐｓｉｎ Ｄ（ＣＤ）和有列朱特异笥抗原（ＰＳＡ）表达与乳腺癌预后的关系。方法：应用免疫组化ＬＳＡＢ方法检测６４例乳腺癌及３０例乳腺良性为的表达。分析ｂｃｌ－２、ｐ５３、ＣＤ和ＰＳ七乳腺癌组织学分级、腋地转移、复发和承后的关系。结果：ｂｃｌ－２、ｐ５３表达之间有显著性差异（Ｐ〈０．０５）,呈负相关;ｂｃｌ－２与ＣＤ和ＰＳＡ之间表达之间均无关（Ｐ〉０．０５）,ｂ     

转化生长因子β1及其Ⅱ型受体在原发性肝癌中的表达

目的：研究重组ＦＮ多态ＣＨ５０在体内激活巨噬细胞及其抗肿瘤作用的特点。方法：体内注射ＣＨ５０和／或转染ＩＦＮ－γ基因,检测巨噬细胞产生的各种因子,测定荷瘤体内肿瘤生长速度。结果 ＣＨ５０单独作用可促进巨噬细胞产生ＮＯ,ＴＮＦ,Ｈ－１等因子,但激活作用较慢,注射ＣＨ５０和转梁ＩＮＦ－γ基因不分先后,均可在体内协同作用,迅速激活巨噬细胞。单独注射ＣＨ５０能够抑制肿瘤结节的形成,其抑制作用呈剂量依赖关系     

血管生成抑制素Endostatin研究新进展

目的：探讨抗ＰＭＬ－ＲＡＲα反义核酸对早幼粒白血病细胞生长、细胞周期、细胞凋亡的作用。方法：以ＮＢ４细胞株为研究对象;细胞计数采用台盼兰排除、计数板计数法;白血病细胞集落采用甲基纤维素半固体培养;流式细胞仪（ＦＣＭ）用于检测细胞凋亡和细胞周期。结果：以６０μｇ／ｍｌ的终浓度处理细胞,抗 ＰＭＬ－ＲＡＲα融合部位反义核酸（ＦＵＡＳ）能明显抑制ＮＢ４细胞增殖及白血病细胞集落（ＡＭＬ－ＣＦＵ）形成,最大抑制率分别为４６．８％,５１．６％;ＧＭ－ＣＳＦ能减弱ＦＵＡＳ的作用,ＦＵＡＳ作用时间越短,ＧＭ－ＣＳＦ促ＡＭＬ－ＣＦＵ增加越明显;ＦＵＡＳ处理第７,９天,出现明显的凋亡细胞群,凋亡细胞百分比分别为４１．０％,３４．２％;ＦＵＡＳ处理第５天Ｓ期下降,Ｇ２／Ｍ期升高,第７天Ｃ０／Ｇ１期明显升高,Ｓ期回升。结论：抗ＰＭＬ－ＲＡＲα反义核酸具有抑制早幼粒白血病细胞增殖、诱导细胞凋亡的作用。     

抗APO—1单抗诱导人膀胱癌多药耐药细胞凋亡的研究

目的：探讨人膀胱癌多药耐药细胞凋亡诱导途径。方法：应用免疫细胞化学、原位ＤＮＡ末端转移酶标记法、相差显微镜及电镜技术,观察ＢＩＵ－８７／ＡＤＭ细胞表面Ｆａｓ抗原的表达和抗ＡＰＯ－１单抗对该细胞存活的影响及其特征。结果：Ｆａｓ抗原在ＢＩＵ－８７／ＡＤＭ中有高表达,经抗ＡＰＯ－１单抗作用后,ＢＩＵ－８７／ＡＤＭ细胞存活率明显降低,光镜及电镜下观察到凋亡细胞的形态学改变,原位ＤＮＡ末端转移酶标记阳性。结     

人实体瘤BUdR离体标记BUdR／DNA双参数FMC测定Tpot的初步研究

探索人实体瘤标本ＢＵｄＲ离体标记ＢＵｄＲ／ＤＮＡ双参数ＦＭＣ测定Ｔｐｏｔ的方法。方法选用头颈肿瘤活检标本及直肠癌手术标本行离体溴脱氧尿嘧啶核苷标记双参数流式细胞术,测定肿瘤潜在培养增时间。结论Ｔｏｐｔ测定应尽可能采用在体ＢＵｄＲ标记,人实体瘤合适的离体标记方法需进一步摸索。     

完整肽聚糖对裸鼠腹腔巨噬细胞分泌和杀瘤功能的影响

目的：探索靶向性非病毒载体系统在人脑腕质瘤基因治疗中的应用。方法：组了ＥＧＦ－Ｒ介导的ＧＥ７基因转移系统,分别与β－ａｌ报道基因和Ｐ２１基因构成载体复合物,体外转染Ｕ２５１细胞,通过Ｘ－ｇａｌ染色、Ｗｅｓｔｅｒｎ ｂｌｏｔ分析,原位木端标记Ｄ梯带检测等,观察了外源基因的导入及对肿瘤细胞的抑制作用。结果：Ｘｇａｌ染色表明,外源基因的导入率可达８０％。转染Ｐ２１基因后,细胞生长受到明显抑制第７天生长抑     

Induction of potent antitumor immunity by intratumoral injection of interleukin 23-transduced dendritic cells

Dendritic cells (DCs) are potent antigen-presenting cells that play a critical role in priming immune responses to tumor. Interleukin (IL)-23 can act directly on DC to promote immunogenic presentation of tumor peptide in vitro. Here, we evaluated the combination of bone marrow-derived DC and IL-23 on the induction of antitumor immunity in a mouse intracranial glioma model. DCs can be transduced by an adenoviral vector coding single-chain mouse IL-23 to express high levels of bioactive IL-23. Intratumoral implantation of IL-23-expressing DCs produced a protective effect on intracranial tumor-bearing mice. The mice consequently gained systemic immunity against the same tumor rechallenge. The protective effect of IL-23-expressing DCs was comparable with or even better than that of IL-12-expressing DCs. IL-23-transduced DC (DC-IL-23) treatment resulted in robust intratumoral CD8(+) and CD4(+) T-cell infiltration and induced a specific TH1-type response to the tumor in regional lymph nodes and spleen at levels greater than those of nontransduced DCs. Moreover, splenocytes from animals treated with DC-IL-23 showed heightened levels of specific CTL activity. In vivo lymphocyte depletion experiments showed that the antitumor immunity induced by DC-IL-23 was mainly dependent on CD8(+) T cells and that CD4(+) T cells and natural killer cells were also involved. In summary, i.t. injection of DC-IL-23 resulted in significant and effective systemic antitumor immunity in intracranial tumor-bearing mice. These findings suggest a new approach to induce potent tumor-specific immunity to intracranial tumors. This approach may have therapeutic potential for treating human glioma.     

Tumour microenvironment of both early- and late-stage colorectal cancer is equally immunosuppressive

Background

Tumour microenvironment (TME) of advanced colorectal cancer (CRC) suppresses dendritic cell (DC) maturation. Here, our aim was to determine how the microenvironment of early-stage tumours influences DCs.

Methods:

Tumour-conditioned media (TCM) was generated by culturing explant tumour tissue in vitro (n=50). Monocyte-derived DCs (MDDCs) of healthy donors or cancer patients were pretreated with TCM and stimulated with lipopolysaccharide (LPS). DC maturation was assessed by flow cytometry and cytokine production measured by ELISA.

Results:

TCM from both early- and late-staged tumours abrogated LPS-induction of IL-12p70 secretion, while increasing IL-10. The profile of inflammatory mediators in TCM was similar across stages, and all increased pSTAT3 expression by DCs.CRC patient DCs (n=31) secreted low levels of IL-12p70 and failed to upregulate expression of maturation markers in response to LPS. Furthermore, in vitro culture of autologous DCs with TCM did not change the hypo-responsiveness of patient DCs.

Conclusion:

Our data demonstrates that the TME of all stages of CRC contains inflammatory mediators capable of suppressing local DCs. MDDCs obtained from CRC patients are hyporesponsive to stimuli such as LPS. Measures to reverse the negative influence of the TME on DCs will optimise cancer vaccines in both early- and late-stage CRC.     

Interleukin-15 enhances T-cell responses by stimulation with dendritic cells

Introduction

Cytokines play important roles in regulating immune responses. Interleukin-2 (IL-2) has usually been used as an adjuvant to enhance antitumour immune responses. However, its crucial role in activation-induced cell death, inhibition of homeostatic proliferation of CD8+ memory T cells and its notable biological side effects impair its prospect of application. IL-15 has several similar functions to IL-2 and shows potential advantages over IL-2, and is being investigated to enhance antitumour dendritic cell (DC) vaccine strategies in our ongoing studies.

Objective

In this preliminary study, we evaluated the ability of IL-15, compared with IL-2, to act as an adjuvant to enhance T-cell responses activated by DCs in vitro.

Materials and methods

Bone marrow-derived DCs (BMDCs) were pulsed with tumour antigens and used to stimulate lymphocyte responses in the presence of IL-15 or IL-2. The activated T lymphocytes were examined by flow cytometric analysis, and interferon-γ (IFN-γ) enzyme-linked immunospot and cytotoxicity assays.

Results

IL-15 was observed to activate lymphocytes with comparable phenotype characteristics of activated/memory CD8+ lymphocytes, compared with IL-2. Both in primary and secondary stimulation with DCs, when using IL-15 as an adjuvant, activated lymphocytes showed higher proportions of IFN-γ-secreting subsets. In secondary stimulation with BMDCs in the presence of IL-15, the activated lymphocytes showed a stronger cytotoxicity to antigen-specific tumour target cells.

Conclusions

Our study suggested that IL-15 might be a prospective adjuvant for a DC vaccine strategy against cancers. The further observation that IL-15 acts as an adjuvant for an antitumour DC vaccine strategy is worth investigating.     

Additive growth inhibitory effects of ibandronate and antiestrogens in estrogen receptor-positive breast cancer cell lines

Introduction

Dendritic cells (DCs) are key antigen-presenting cells that play an essential role in initiating and directing cellular and humoral immunity, including anti-tumor responses. Due to their critical role in cancer, induction of DC apoptosis may be one of the central mechanisms used by tumors to evade immune recognition.

Methods

Spontaneous apoptosis of blood DCs (lineage negative HLA-DR positive cells) was assessed in peripheral blood mononuclear cells (PBMCs) using Annexin-V and TUNEL assays immediately after blood collection. The role of tumor products was assessed by culturing cells with supernatants derived from breast cancer cell lines (TDSN) or PBMCs (PBMC-SN, as a control). The capacity of DC stimulation to prevent apoptosis was assessed by incubating DC with inflammatory cytokines, poly I:C, IL-12 or CD40 ligand (CD40L) prior to culture with TDSN. Apoptosis was determined by flow cytometry and microscopy, and Bcl-2 expression determined by intracellular staining.

Results

In this study we document the presence of a significantly higher proportion of apoptotic (Annexin-V⁺ and TUNEL⁺) blood DCs in patients with early stage breast cancer (stage I to II; n = 13) compared to healthy volunteers (n = 15). We examined the role of tumor products in this phenomenon and show that supernatants derived from breast cancer lines induce apoptosis of blood DCs in PBMC cultures. Aiming to identify factors that protect blood DC from apoptosis, we compared a range of clinically available maturation stimuli, including inflammatory cytokines (tumor necrosis factor-α, IL-1β, IL-6 and prostaglandin (PG)E₂ as a cytokine cocktail), synthetic double-stranded RNA (poly I:C) and soluble CD40 ligand. Although inflammatory cytokines and poly I:C induced robust phenotypic maturation, they failed to protect blood DCs from apoptosis. In contrast, CD40 stimulation induced strong antigen uptake, secretion of IL-12 and protected blood DCs from apoptosis through sustained expression of Bcl-2. Exogenous IL-12 provided similar Bcl-2 mediated protection, suggesting that CD40L effect is mediated, at least in part, through IL-12 secretion.

Conclusion

Cumulatively, our results demonstrate spontaneous apoptosis of blood DCs in patients with breast cancer and confirm that ex vivo conditioning of blood DCs can protect them from tumor-induced apoptosis.     

Phenotype and function of dendritic cells and T-lymphocyte polarization in the human colonic mucosa and adenocarcinoma

Aim

To evaluate the status of activation of the intestinal dendritic cells (DCs) and T lymphocytes (T cells) from surgical specimens of human colon and adenocarcinoma, and the potential effect of administration of interleukin 2 (IL-2).

Methods

Patients undergoing colectomy for cancer were randomized to receive subcutaneous IL-2 (12 million UI/day) (treated group; n = 10) for 3 days before operation or no treatment (control group, n = 10). DCs and T cells were isolated and purified from the lamina propria (LP) of segments of normal colon and adenocarcinoma of both groups. Cell phenotype was determined by expression of membrane receptors. Interaction between DC and T cells was assesses by a mixed leukocyte reaction using naïve T cells co-cultured with DCs. CD4+ T-cell polarization was studied by intracellular staining with monoclonal antibodies for interleukin-4 and interferon-γ.

Results

CD4+ T cells were significantly less in tumour than in LP (p < 0.05) in both treated and control groups. IL-2 did not modify the number of any of the T-cell subsets analysed. In contrast, T cells isolated from LP and neoplasm of treated patients produced more interferon-γ and less interleukin-4 (p < 0.05 vs. controls). IL-2 administration significantly increased (p < 0.05) the number of mature, myeloid and plasmocytoid DCs compared to controls. Allogeneic naïve T cells were polarized toward a Th1 type of response which appeared to be mediated by IL-2 activated DCs.

Conclusions

systemic IL-2 treatment may have immunomodulatory properties on intestinal DC maturation and drive a Th1 mediated anti-neoplastic response.