A novel bystander effect involving tumor cell-derived Fas and FasL interactions following Ad.HSV-tk and Ad.mIL-12 gene therapies in experimental prostate cancer

To enhance the NK population induced by Herpes Simplex virus thymidine kinase (HSV-tk) gene transduction and ganciclovir (GCV) treatment, adenovirus-mediated (Ad) expression of IL-12 was added to Ad.HSV-tk + GCV as combination gene therapy. This approach resulted in improved local and systemic growth suppression in a metastatic model of mouse prostate cancer (RM-1). In vitro assay of tumor infiltrating lymphocytes noted superior lysis of both RM-1 and Yac-1 targets with combination therapy, but in vivo depletion of NK cells only negatively impacted on systemic growth inhibition. TUNEL assay of primary tumors noted induction of apoptosis between two and four times higher than controls lasting for 6-8 days post-vector injection. After demonstrating that Ad.HSV-tk/GCV and Ad.mIL-12-induced IFN-gamma independently up-regulated expression of FasL and Fas, respectively, studies examined tumor cell-mediated death through Fas/FasL-induced apoptosis as a mechanism of primary tumor growth suppression. In vitro, combination therapy at low vector doses resulted in synergistic growth suppression, which could be negated by the addition of anti-FasL antibody. In vivo co-inoculation of an adenovirus expressing soluble Fas resulted in combination therapy-treated tumors, which were three times larger than expected, and a reduction in apoptosis to baseline levels. In FasL knockout mice, combination therapy maintained the superior results experienced in wild-type mice, indicating that tumor cell, not host cell FasL, was responsible for Fas transactivation. Therefore, the combination of Ad.HSV-tk/GCV + Ad.mIL-12 results in enhanced local growth control via apoptosis due to tumor cell expression of Fas and FasL and improved anti-metastatic activity secondary to a strong NK response.     

IFN-γ and Fas/FasL are required for the antitumor and antiangiogenic effects of IL-12/pulse IL-2 therapy

Systemic administration of IL-12 and intermittent doses of IL-2 induce complete regression of metastatic murine renal carcinoma. Here, we show that overt tumor regression induced by IL-12/pulse IL-2 is preceded by recruitment of CD8(+) T cells, vascular injury, disrupted tumor neovascularization, and apoptosis of both endothelial and tumor cells. The IL-12/IL-2 combination synergistically enhances cell surface FasL expression on CD8(+) T lymphocytes in vitro and induces Fas and FasL expression within tumors via an IFN-gamma-dependent mechanism in vivo. This therapy also inhibits tumor neovascularization and induces tumor regression by mechanisms that depend critically on endogenous IFN-gamma production and an intact Fas/FasL pathway. The ability of IL-12/pulse IL-2 to induce rapid destruction of tumor-associated endothelial cells and regression of established metastatic tumors is ablated in mice with a dysregulated Fas/FasL pathway. The common, critical role for endogenous IFN-gamma and the Fas/FasL pathway in early antiangiogenic effects and in antitumor responses suggests that early, cytokine-driven innate immune mechanisms and CD8(+) T cell-mediated responses are interdependent. Definition of critical early molecular events engaged by IL-12/IL-2 may provide new perspective into optimal therapeutic engagement of a productive host-antitumor immune response.     

转染外源性FasL的CD34+细胞诱导同种抗原特异性T细胞凋亡的实验研究

为探讨通过Fas/FasL途径选择性去除移植物中成熟T细胞的可能性 ,本研究借助反转录病毒途径 ,以FasL基因转染骨髓CD34+ 细胞 ,与经过富集的T细胞进行单向混合培养 (刺激细胞 /效应细胞比例为 5∶1) ,加入或不加入IFN γ ,IL 2 ,应用原位末端标记法 (TUNEL)和流式细胞术 (FCM)检测培养 5天后的细胞凋亡率 ,并比较两种细胞因子对移植物中CD34+ 细胞的影响。以转染外源FasL的CD34+ 细胞为刺激细胞 ,T细胞凋亡率为 (12 .1±1.5 ) %〔对照组为 (3.2± 1.1) % ,P <0 .0 1〕 ;经IFN γ或IL 2作用后 ,转染细胞诱导T细胞的凋亡率分别上升至(2 0 .1± 2 .3) % ,(17.6± 1.3) % (P <0 .0 1) ;且IL 2对CD34+ 细胞Fas抗原的表达无明显影响。上述结果表明 ,转染外源FasL的骨髓CD34+ 细胞可诱导同种抗原特异性T细胞凋亡 ,IFN γ和IL 2增强上述致细胞凋亡作用 ,且IL 2更有利于临床应用 ;提示该方案可选择性清除移植物中同种抗原特异性T细胞 ,可望为临床上防治移植物抗宿主病 (GVHD)提供新的途径     

Interleukin 12 gene transfer into skin distant from the tumor site elicits antimetastatic effects equivalent to local gene transfer

We have reported that particle-mediated interleukin 12 (IL-12) gene transfer into the skin overlying the local tumor inhibits systemic metastases. To further characterize this effect, we compared the antitumor and antimetastatic effects of IL-12 cDNA delivered at the local tumor site versus at a site distant from the primary tumor, in a spontaneous metastasis model of LLC-F5 tumor. Local IL-12 gene delivery into the skin overlying the intradermal tumor (local IL-12 treatment) on days 7, 9, and 11 after tumor implantation resulted in the most suppression of the growth of the primary LLC-F5 tumor, whereas IL-12 gene transfer into the skin distant from the tumor (distant IL-12 treatment) was less effective. In contrast, both local IL-12 and distant IL-12 treatment, followed by tumor excision, inhibited lung metastases to a similar extent, resulting in significantly extended survival of test mice. The results of in vivo studies using depleting anti-asialo GM1 antibody and anti-CD4/anti-CD8 monoclonal antibodies, or neutralizing anti-interferon gamma (IFN-gamma) monoclonal antibody demonstrated that natural killer (NK) cells, CD8(+) T cells, and IFN-gamma contributed to the antimetastatic effects in both treatment groups. Furthermore, the levels of mRNA expression of vascular endothelial growth factor and matrix methalloproteinase 9 at the tumor microenvironment were suppressed after both local and distant IL-12 treatment. These results suggest that the current particle-mediated IL-12 gene delivery in the spontaneous LLC-F5 metastasis model can confer antimetastatic activities, irrespective of the gene transfection site, via a combination of several mechanisms involving CD8(+) T cells, NK cells, IFN-gamma, and antiangiogenesis.     

Direct in vivo transfection of antisense Fas-ligand reduces tumor growth and invasion

The expression of Fas ligand (FasL) by tumor cells has been reported to have multiple, conflicting effects on tumor growth. The majority of the data support the theory that FasL expressing tumor cells evade immune surveillance by killing T cells expressing Fas. However, the role of the humoral immune-blockade by FasL expressing tumor cells has not been assessed. Using immune-competent mice, we observed that FasL expressing tumor cells reduced the antitumor antibody production together with the T and B cell content of the spleen in these mice. Further, to determine if the expression of FasL in the environment of the tumor suppresses the humoral antitumor immune response and influences tumor growth, a mouse model lacking T cells was used. To assess whether a local reduction of FasL could reduce tumor progression, a plasmid encoding antisense FasL cDNA was delivered directly into a growing tumor (SW620 colon carcinoma). Intratumoral delivery of the plasmid was able to transfect tumor cells, stromal cells, and peritumoral muscle cells. This antisense FasL tumor tissue transfection persisted for at least 25 days, produced a systemic decrease in soluble FasL, and resulted in a 50% reduction in the rate of tumor growth when compared with tumor tissue of the control groups. These results suggest that direct transfection of antisense FasL cDNA impairs FasL translation in tumor and stromal cells, and can inhibit tumor progression by impairing the FasL-mediated, stromal cell-assisted, tumor counter-attack.     

Fas ligand expression by astrocytoma in vivo: maintaining immune privilege in the brain?

Astrocytomas are among the most common brain tumors that are usually fatal in their malignant form. They appear to progress without significant impedance from the immune system, despite the presence of intratumoral T cell infiltration. To date, this has been thought to be the result of T cell immunosuppression induced by astrocytoma-derived cytokines. Here, we propose that cell contact-mediated events also play a role, since we demonstrate the in vivo expression of Fas ligand (FasL/CD95L) by human astrocytoma and the efficient killing of Fas-bearing cells by astrocytoma lines in vitro and by tumor cells ex vivo. Functional FasL is expressed by human, mouse, and rat astrocytoma and hence may be a general feature of this nonlymphoid tumor. In the brain, astrocytoma cells can potentially deliver a death signal to Fas+ cells which include infiltrating leukocytes and, paradoxically, astrocytoma cells themselves. The expression of FasL by astrocytoma cells may extend the processes that are postulated to occur in normal brain to maintain immune privilege, since we also show FasL expression by neurons. Overall, our findings suggest that FasL-induced apoptosis by astrocytoma cells may play a significant role in both immunosuppression and the regulation of tumor growth within the central nervous system.     

Intratumoral injection of interleukin-12 plasmid DNA,either naked or in complex with cationic lipid,results in similar tumor regression in a murine model

Effective eradication of established tumors and generation of a lasting systemic immune response is an important goal for cancer gene immunotherapy. The method of gene delivery may also be critical for the generation of an effective antitumor response. We compared the level of transgene expression and antitumor activity of two different interleukin (IL)-12 DNA preparations (naked DNA versus DNA lipid complex). Established murine adenocarcinoma (CT26) and renal cell carcinoma (Renca) tumors in BALB/c mice were treated by direct intratumoral injection of a nonviral plasmid DNA vector encoding the murine IL-12 (mIL-12) gene, either alone (naked) or in complexes with cationic lipid. Both treatments resulted in the same percentage (87%) of mice undergoing a complete tumor regression of the CT26 tumor. For the Renca tumor model, complete tumor regression was observed in 67 and 75% of animals treated with naked mIL-12 DNA and mIL-12 DNA plus lipid, respectively. Mice that were rendered tumor free for > 50 days by mIL-12 gene therapy rejected a subsequent challenge of parental tumor cells but not of an unrelated, syngeneic tumor. The marked reduction of tumor growth in tumor-bearing mice treated with mIL-12 cDNA was associated with the augmentation of tumor-specific cytotoxic T cells, enhanced production of IFN-gamma in spleen and lymph node cells, and increased splenomegaly and lymphadenopathy. The CD8+: CD4+ ratio in tumor-infiltrating lymphocytes was significantly increased in the tumor-bearing mice treated with mIL-12 DNA alone and mIL-12 cDNA plus lipid as compared with a control vector-treated group. These results indicate that direct intratumoral gene transfer with naked nonviral IL-12 DNA provides an effective and simple method for the treatment of murine tumors, suggesting an approach for clinical application.     

Pulmonary contusion induces alveolar type 2 epithelial cell apoptosis: role of alveolar macrophages and neutrophils

Alveolar type 2 (AT-2) cell apoptosis is an important mechanism during lung inflammation, lung injury, and regeneration. Blunt chest trauma has been shown to activate inflammatory cells such as alveolar macrophages (AMs) or neutrophils (polymorphonuclear granulocytes [PMNs]), resulting in an inflammatory response. The present study was performed to determine the capacity of different components/cells of the alveolar compartment (AMs, PMNs, or bronchoalveolar lavage [BAL] fluids) to induce apoptosis in AT-2 cells following blunt chest trauma. To study this, male Sprague-Dawley rats were subjected to either sham procedure or blunt chest trauma induced by a single blast wave. Various time points after injury (6 h to 7 d), the lungs were analyzed by immunohistochemistry, for AT-2 cells, or with antibodies directed against caspase 3, caspase 8, Fas, Fas ligand (FasL), BAX, and BCL-2. Bronchoalveolar lavage concentrations of TNF-alpha, IL-1beta, and soluble FasL were determined by enzyme-linked immunosorbent assay. Furthermore, cultures of AT-2 cells isolated from healthy rats were incubated with supernatants of AMs, PMNs, or BAL fluids obtained from either trauma or sham-operated animals in the presence or absence of oxidative stress. Annexin V staining or TUNEL (terminal deoxynucleotidyl transferase) assay was used to detect apoptotic AT-2 cells. Histological evaluation revealed that the total number of AT-2 cells was significantly reduced at 48 h following trauma. Fas, FasL, active caspase 8, and active caspase 3 were markedly up-regulated in AT-2 cells after chest trauma. BAX and BCL-2 did not show any significant changes between sham and trauma. IL-1beta, but not TNF-alpha, levels were markedly increased at 24 h after the injury, and soluble FasL concentrations were significantly enhanced at 6, 12, 24, and 48 h after the insult. Apoptosis of AT-2 cells incubated with supernatants from cultured AMs, isolated at 48 h following chest trauma was markedly increased when compared with shams. In contrast, no apoptosis was induced in AT-2 cells incubated with supernatants of activated PMNs or BAL fluids of traumatized animals. In summary, blunt chest trauma induced apoptosis in AT-2 cells, possibly involving the extrinsic death receptor pathway. Furthermore, mediators released by AMs appeared to be involved in the induction of AT-2 cell apoptosis.     

Fas engagement induces the maturation of dendritic cells (DCs), the release of interleukin (IL)-1beta, and the production of interferon gamma in the absence of IL-12 during DC-T cell cognate interaction: a new role for Fas ligand in inflammatory responses

Ligation of the Fas (CD95) receptor leads to an apoptotic death signal in T cells, B cells, and macrophages. However, human CD34(+)-derived dendritic cells (DCs) and mouse DCs, regardless of their maturation state, are not susceptible to Fas-induced cell death. This resistance correlates with the constitutive expression of the Fas-associated death domain-like IL-1beta-converting enzyme (FLICE)-inhibitory protein (FLIP) ligand. We demonstrate a new role of Fas in DC physiology. Engagement of Fas on immature DCs by Fas ligand (FasL) or by anti-Fas antibodies induces the phenotypical and functional maturation of primary DCs. Fas-activated DCs upregulate the expression of the major histocompatibility complex class II, B7, and DC-lysosome-associated membrane protein (DC-LAMP) molecules and secrete proinflammatory cytokines, in particular interleukin (IL)-1beta and tumor necrosis factor alpha. Mature DCs, if exposed to FasL, produce even higher amounts of IL-1beta. Importantly, it is possible to reduce the production of IL-1beta and interferon (IFN)-gamma during DC-T cell interaction by blocking the coupling of Fas-FasL with a Fas competitor. Finally, during cognate DC-T cell recognition, IL-12 (p70) could not be detected at early or late time points, indicating that Fas-induced, IFN-gamma secretion is independent of IL-12.     

Cooperative effects of adenoviral vector-mediated interleukin 12 gene therapy with radiotherapy in a preclinical model of metastatic prostate cancer

We investigated the potential benefits of combining adenoviral vector mediated in situ interleukin-12 (AdmIL-12) gene therapy with radiation therapy (XRT) to enhance therapeutic efficacy. In a metastatic mouse prostate cancer cell line, 178-2 BMA, AdmIL-12+XRT demonstrated enhanced therapeutic activities in vitro as determined by clonogenic survival, apoptosis, and mIL-12 levels. At the molecular level, increased expression of tumor necrosis factor-alpha mRNA was specific for the combined therapy. In a subcutaneous 178-2 BMA in vivo model, the combination of AdmIL-12+XRT produced statistically significant tumor growth suppression compared to control vector Adbetagal, Adbetagal XRT, or AdmIL-12 as monotherapy. In addition, significant prolongation of survival was demonstrated for the combination of AdmIL-12+XRT. The combination of AdmIL-12+XRT significantly suppressed both spontaneous and pre-established lung metastases, and led to a prolonged elevation of serum IL-12 and significantly increased natural killer (NK) activities. Importantly, in vivo depletion of NK cells resulted in significant attenuation of the antimetastatic activities of AdmIL-12 alone or AdmIL-12+XRT. These combined effects suggest that AdIL-12 gene therapy together with radiotherapy may achieve maximal tumor control (both local and systemic) in selected prostate cancer patients via radio-gene therapy induced local cytotoxicity and local and systemic antitumor immunity.     

Strengthening of antitumor immune memory and prevention of thymic atrophy mediated by adenovirus expressing IL-12 and GM-CSF

Interleukin (IL)-12 and granulocyte-monocyte colony-stimulating factor (GM-CSF) have recently been used as immunotherapeutic agents in cancer gene therapy. IL-12 and GM-CSF have differential roles in the antitumor immune response, as IL-12 targets T, NK and natural killer T (NKT) cells and GM-CSF principally targets antigen-presenting cells (APCs). To strengthen the therapeutic efficacy of these two cytokines, we generated an oncolytic adenovirus (Ad), Ad-ΔB7/IL12/GMCSF, coexpressing IL-12 and GM-CSF. Using a murine B16-F10 syngeneic tumor model, we show that Ad-ΔB7/IL12/GMCSF promoted antitumor responses and increased survival compared with an oncolytic Ad expressing IL-12 or GM-CSF alone (Ad-ΔB7/IL12 or Ad-ΔB7/GMCSF, respectively). By measuring cytotoxic T lymphocyte activity and interferon-γ production, we show that the enhanced therapeutic effect was mediated by the induction of immune cell cytotoxicity. In situ delivery of Ad-ΔB7/IL12/GMCSF resulted in massive infiltration of CD4(+) T cells, CD8(+) T cells, NK cells and CD86(+) APCs into the tissue surrounding the necrotic area of the tumor. Moreover, GM-CSF effectively promoted antitumor immune memory, which was significantly augmented by IL-12. Lastly, IL12-expressing oncolytic Ads prevented tumor-induced thymic atrophy and was associated with reduced apoptosis and increased proliferation in the thymus. Taken together, these data demonstrate that an oncolytic Ad coexpressing IL-12 and GM-CSF is a potential therapeutic tool for the treatment of cancer.     

Nonviral genetic transfer of Fas ligand induced significant growth suppression and apoptotic tumor cell death in prostate cancer in vivo

To accomplish efficient nonviral gene therapy against prostate cancer (PC), Epstein-Barr virus (EBV)-based plasmid vectors containing EBNA1 gene and oriP were employed and combined with a cationic polymer or cationic lipid. When EBV-plasmid/poly-amidoamine dendrimer complex was injected into PC-3-derived tumors established in severe combined immunodeficiency mice, a considerable expression of marker gene was obtained in the tumors, and the expression level was more than eight-fold higher than that achieved by conventional plasmid vector/dendrimer. Since most PC cells express the apoptotic signal molecule Fas (Apo-1/CD95) on their surface, Fas ligand (FasL) gene was transferred into PC cells to kill the tumor cells. In vitro transfection with pGEG.FasL (an EBV-plasmid with the FasL gene) significantly reduced the viability of PC cells, which subsequently underwent apoptosis. Intratumoral injections of pGEG.FasL into PC induced significant growth suppression of the xenograft tumors, in which typical characteristics of apoptosis were demonstrated by TUNEL staining and electron microscopic observations. When pGEG.FasL transfer was accompanied by systemic administrations of cisplatin, the tumors were inhibited even more remarkably, leading to prolonged survival of the animals. FasL gene transfection by means of EBV-based plasmid/cationic macromolecule complexes may provide a practical therapeutic strategy against PC.     

凋亡相关基因Fas和FasL在糖尿病大鼠下颌下腺内的表达

目的：观察糖尿病大鼠下颌下腺内凋亡相关基因Fas和FasL的表达变化。方法：SD雄性大鼠随机分为4周、12周模型组及对照组。用链脲佐菌素复制出糖尿病动物模型，分别于第4、12周后测体重和血糖．并取下颌下腺组织，应用免疫组织化学方法检测Fas及FasL表达变化，计算机图像分析系统测平均光密度值（MOD）。结果：大鼠下颌下腺内有Fas及FasL表达，分布于颗粒曲管、纹状管及小叶间导管上皮细胞胞质及部分细胞核内。对照组呈Fas及FasL弱阳性，4周模型组多呈中等阳性，部分呈强阳性；12周模型组大多呈强阳性。与对照组比较，4周、12周模型组各级导管上皮细胞内Fas及FasLMOD值均增高（P〈0．01）。结论：大鼠下颌下腺内Fas及FasL表达随糖尿病病程延长而增强，可能在糖尿病时下颌下腺组织凋亡中起重要作用。[著者文摘]     

Adenovirus gene transfer vectors inhibit growth of lymphatic tumor metastases independent of a therapeutic transgene

Adenovirus (Ad) gene transfer vectors traffic to regional lymph nodes (RLNs) after footpad injections in mice, resulting in localized production of interferon gamma (IFN-gamma). With this background, we evaluated the hypothesis that Ad vector administration may inhibit RLN tumor metastasis independent of the transgene in the expression cassette. Tumors of MM48, a cell line with a propensity toward lymphogenous metastasis, were established in the footpads of syngeneic C3H mice, and E1(-)E3(-) Ad vectors encoding no transgene (AdNull) or encoding an irrelevant transgene (AdCD; Escherichia coli cytosine deaminase with no 5-fluorocytosine administration) were administered (10(10) particles) in a peritumoral location. Both vectors suppressed the growth of tumor in the regional (popliteal) lymph node. This effect was localized to the regional, but not distant, lymph nodes (p < 0.05). Heat inactivation of the vector or decreasing the dose of the vector to 10(9) particles did not suppress RLN growth of the tumor when compared with 10(10) particles of active AdNull (p < 0.05 and p < 0.01, respectively). The ability of an E1(-)E4(-) vector expressing beta-galactosidase (AdRSVbetagal.11) to suppress RLN tumor growth showed that the E4 region of the Ad vector was not responsible for the effect. Blocking either IFN-gamma or natural killer (NK) cells with systemic antibody treatment in immunocompetent mice allowed rapid growth of RLN metastases despite Ad vector administration, and Ad vector injection into the footpads of tumor-free mice induced the accumulation of NK cells in the RLN. These data demonstrate that, in a metastatic murine tumor model, a low dose (10(10) particles) of replication-deficient Ad vectors inhibits RLN metastases independent of a therapeutic transgene, an effect that is mediated, at least in part, by IFN-gamma and NK cells.     

雷公藤内酯醇诱导T淋巴细胞凋亡时Fas/FasL的表达

目的 检测Fas/FasL在雷公藤内酯醇诱导的T淋巴细胞凋亡中的表达。 方法 培养人T淋巴细胞，10、20、30μg/L的雷公藤内酯醇分别刺激细胞4、8、16 h，流式细胞仪DNA分析、FITC-Annexin Ⅴ binding/PI染色检测细胞凋亡，Western blot分析检测Fas/FasL蛋白的表达。 结果 雷公藤内酯醇以剂量和时间依赖的形式诱导T细胞凋亡，雷公藤内酯醇诱导T细胞凋亡的同时伴随有Fas和FasL表达的上调，同样，雷公藤内酯醇诱导Fas和FasL表达呈现剂量和时间依赖性。结论 雷公藤内酯醇以剂量和时间依赖的形式诱导人T细胞凋亡，雷公藤内酯醇诱导T细胞凋亡的信号通路可能是通过Fas/FasL途径激活的。     

特发性肺纤维化患者支气管/肺泡上皮细胞凋亡和Fas/FasL基因表达

目的：检测细胞凋亡、Fas/FasL mRNA及其蛋白在特发性肺纤维化患者肺泡/支气管上皮细胞的表达,探讨细胞凋亡和促凋亡信号在此疾病中的意义。方法：应用末端原位杂交（TUNEL）、原位杂交、免疫组化技术,对12例特发性肺纤维化患者的肺活检组织（IPF组）及10例正常肺组织（对照组）检测了细胞凋亡、Fas/FasL mRNA及其蛋白表达的变化。结果：特发性肺纤维化组肺泡/支气管上皮细胞凋亡指数上调,明显高于对照组（P〈0.01）;IPF组肺泡/支气管上皮细胞中Fas/FasL mRNA及其蛋白表达上调,高于对照组（P〈0.01）;IPF组肺泡/支气管上皮细胞凋亡指数与其Fas/FasL mRNA及蛋白表达之间均无明显相关（P〉0.05）。结论：肺纤维化时肺泡/支气管上皮细胞凋亡上调,Fas/FasL mRNA及其蛋白表达增强,在肺纤维化发生、发展中起重要作用。     

A targeted DNA vaccine encoding fas ligand defines its dual role in the regulation of experimental autoimmune encephalomyelitis

This study used naked DNA vaccination to induce breakdown of tolerance to self and thus elicit immunological memory to native, membrane-bound Fas ligand (FasL). Upon induction of experimental autoimmune encephalomyelitis (EAE), this memory was turned on to provide protective immunity. FasL-specific autoantibodies isolated from protected animals differentially downregulated the in vitro production of TNF-alpha, but not IFN-gamma, by cultured T cells. These autoantibodies were highly protective when they were administered to rats at the onset of EAE. In contrast, administration of these FasL-specific Ab's to EAE rats after the peak of the acute phase of disease prevented spontaneous recovery from disease. This extended illness is partially explained by inhibition of mononuclear cell apoptosis at the target organ, which resulted in increased accumulation of T cells and macrophages at the site of inflammation. Hence, FasL exerts two distinct, stage-specific regulatory functions in the control of this T-cell mediated autoimmune disease of the central nervous system.     

Enhancement of Fas ligand-induced inhibition of neointimal formation in rabbit femoral and iliac arteries by coexpression of p35.

Adenovirus-mediated gene transfer of Fas ligand (FasL) inhibits neointimal formation in balloon-injured rat carotid arteries. Vascular smooth muscle (VSM) cells coexpressing murine FasL and p35, a baculovirus gene that inhibits caspase activity, are not susceptible to FasL-mediated apoptosis in vitro but are capable of inducing apoptosis of VSM cells that do not express p35. We reasoned that coexpression of p35 in FasL-transduced VSM cells in vivo would promote their survival, enhance FasL-induced apoptosis of adjacent VSM cells, and thereby facilitate a greater inhibition of neointimal formation. In balloon-injured rabbit femoral arteries, either Ad2/FasL/p35 or Ad2/FasL was infused into the injured site and withdrawn 20 min later. Both vectors induced a dose-dependent reduction (p < 0.05) of the neointima-to-media ratio when assessed 14 days later. However, Ad2/FasL/p35 exhibited a significantly greater inhibition of neointimal formation than Ad2/FasL. In a more clinically relevant model of restenosis, rabbit iliac arteries were injured with an angioplasty catheter under fluoroscopic guidance. Adenoviral vectors were delivered locally to the injured site over a period of 2 min, using a porous infusion balloon catheter. Twenty-eight days after gene transfer angiographic and histologic assessments indicated a significant (p < 0.05) inhibition of iliac artery lumen stenosis and neointimal formation by Ad2/FasL/p35 (5 x 10(11) particles per artery). The extent of inhibition was comparable to that achieved with Ad2/TK, an adenoviral vector encoding thymidine kinase (5 x 10(11) particles per artery) and coadministration of ganciclovir for 7 days. These data suggest that coexpression of p35 in FasL-transduced VSM cells is more potent at inhibiting neointimal formation and as such represents an improved gene therapy approach for restenosis.     

急性肺损伤大鼠肺组织细胞凋亡及Fas/FasL系统表达改变及其意义

目的：探讨细胞凋亡与急性肺损伤（ＡＬＩ）发生、发展的关系以及Ｆａｓ和／ＦａｓＬ系统表达改变的意义。方法：复制内毒素性大鼠ＡＬＩ模型;采用ＴＵＮＥＬ法、原位杂交、半定量逆转录聚合酶链反应（ＳｑＲＴ ＰＣＲ）及免疫组化等技术观察大鼠ＡＬＩ发生过程中,肺组织细胞凋亡变化以及Ｆａｓ／ＦａｓＬ系统蛋白质和ｍ ＲＮＡ表达的改变。结果：内毒素性ＡＬＩ早期（＜ ２４ 小时）,大鼠肺泡上皮细胞和肺血管内皮细胞凋亡明显增加;肺组织Ｆａｓ／ＦａｓＬｍ ＲＮＡ和蛋白质表达明显上调,且与肺组织细胞凋亡的增加相一致。结论：肺组织细胞凋亡以及Ｆａｓ／ＦａｓＬ系统表达明显上调可能参与大鼠ＡＬＩ的发病机制。     

Fas配体在髓系白血病细胞中的表达及功能研究

为了解Fas配体 (FasL)在髓系白血病细胞中的表达水平及其诱导Fas+敏感细胞发生凋亡的功能 ,用流式细胞术检测 38例髓系白血病患者 (外周血白细胞 >10 0× 10 9/L)的白血病细胞在新鲜分离及IL 2和IFN γ刺激后表面膜FasL的表达水平 ,将白血病细胞 ( 1× 10 6/ml)与Jurkat细胞 ( 1× 10 5/ml)混合培养 2 4小时后 ,用DNA电泳和流式细胞术双标法检测Jurkat细胞发生凋亡的情况。结果表明 :白血病细胞表面FasL表达量 ( 3.5 9± 1.0 5 ) %高于正常人 ( 0 .36± 0 .16 ) % ,P <0 .0 0 1,经IL 2和IFN γ刺激后其表达量明显增加 ( 7.78± 3.4 0 ) % ,P <0 .0 1;白血病细胞刺激前后诱导Jurkat细胞发生的凋亡率分别为 ( 8.2 8± 1.6 1) % ,( 10 .73± 2 .16 ) % ,差异具有显著性意义。结论 :FasL在髓系白血病细胞表面高表达且对Fas+的敏感细胞具有杀伤功能 ,推测Fas/FasL途径可能在白血病的“免疫逃逸”中发挥作用