IL-12与粒细胞-巨噬细胞集落刺激因子基因联合治疗小鼠肝癌的研究

目的 研究L-12与粒细胞-巨噬细胞集落刺激因子(granalocyte-macrophage-colong scimulatingfactor,GM-CSF)联合基因治疗对小鼠皮下肝癌的治疗作用。方法 将皮下接种1×106BNL肝癌细胞的小鼠分四组于肿瘤接种3、6d后分别经尾静脉高压注射总量为25μg的细胞因子编码质粒:(1)pXX-GM-CSF、pXX-IL-12各 12.5μg;以pXX-IL-12 25μg;(3)pXX-GM-CSF 25μg;(4)pXX-Neo 25μg,每组6只小鼠。观察小鼠皮下肿瘤生长情况及所诱导的细胞免疫反应。同时研究了经尾静脉高压注射后小鼠血清IL-12、GM-CSF和IFN-γ浓度变化。结累IL-12与GM-CSF联台基因治疗可以诱导更强的抗肝癌免疫和细胞免疫反应。GM-CSF可以增强IL-12分泌并减少其诱导的IFN-γ分泌。结论IL-12和GM-CSF联台基因治疗可以产生较强的抗瘤作用并能减少IL-12的副作用。     

水流动力学注射介导白细胞介素-12基因治疗小鼠原位移植性肝癌

以细胞因子白细胞介素(IL)-12为基础的抗肿瘤免疫疗法有望成为治疗肝脏肿瘤的有效手段之一.以往的IL-12抗肿瘤基因治疗研究中多采用皮下接种肝癌模型,肿瘤的生长环境与临床实际情况相差甚远.     

Oral adeno-associated virus-sTRAIL gene therapy suppresses human hepatocellular carcinoma growth in mice

The extracellular domain of the tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL) may function as a soluble cytokine to selectively kill various cancer cells without toxicity to most normal cells. We constructed a series of recombinant adeno-associated virus (AAV) vectors expressing the extracellular domain of human TRAIL fused with signal peptides of human insulin, interferon, human growth hormone, and serum albumin and designated them as AAV-ISN-T, AAV-IFN-T, AAV-HGH-T, and AAV-Alb-T, respectively. Transduction of human SMMC-7721 liver cancer cells with AAV-ISN-T led to higher levels of TRAIL(95-281) protein expression in the cell culture media and produced more apoptosis of the cells in vitro than those with AAV-IFN-T, AAV-HGH-T, and AAV-Alb-T. The therapeutic potential of AAV-ISN-T was then evaluated in a transplanted mouse model established by injection of human liver cancer SMMC-7721 cells subcutaneously. Subsequent oral or intraperitoneal administration of AAV-ISN-T resulted in a rapid, high level and long time expression of soluble TRAIL in the sera and livers of the animals, as well as effective suppression of tumor growth, with no toxicity to normal hepatocytes. These data strongly suggest that it is possible to increase soluble TRAIL expression to make full use of tumoricidal activity of TRAIL as a therapeutic strategy. In conclusion, we provide evidence that oral administration of AAV-TRAIL might be an important alternative route with practical significance for cancer gene therapy.     

Combined treatment of liver failure and hepatorenal syndrome with orthotopic liver transplantation.

Hepatorenal syndrome (HRS) is a severe complication of liver failure with high mortality. The pathogenesis of this reversible functional renal failure is not yet clearly understood. Diagnosis is based upon the association of clinical and biological criteria. A patient was admitted to our institution for severe liver failure secondary to an exacerbation of cirrhosis, where he developed a fulminant hepatorenal syndrome. Both, the renal and hepatic failure were successfully treated by orthotopic liver transplantation. Special attention was paid to the immunosuppressive treatment with Cyclosporine whose use, we believe, should be delayed until function has partially recovered.     

白细胞介素-10与减体积大鼠肝移植后肝再生的关系

目的 探讨白细胞介素-10(IL-10)与减体积大鼠肝移植术后移植肝再生的关系。方法 建立减体积大鼠肝移植模型,实验分为:肝切除组、全肝移植组和减体积肝移植组,分别于术后1、2、4、7d取肝组织,免疫组织化学检测各组IL-10的表达,流式细胞仪检测移植肝的增殖活性。结果 肝切除组、全肝移植组和减体积肝移植组肝细胞增生活跃,术后4d增殖高峰分别为26.3±0.9、35.8±2.2、32.4±1.8。IL-10与移植后肝再生呈负相关(r=-0.58,P<0.01)。结论 减体积肝移植和全肝移植术后肝脏具有同样的增殖活性,但增殖峰值较肝切除延迟。IL-10对移植肝肝再生具有明显的调控作用,同时受免疫系统产生的其它细胞因子和激素的影响。     

Hepatocyte growth factor gene therapy prevents radiation-induced liver damage

AIM: To transfer human HGF gene into the liver of rats by direct electroporation as a means to prevent radiation-induced liver damage. METHODS: Rat whole liver irradiation model was accomplished by intra-operative approach. HGF plasmid was injected into liver and transferred by electroporation using a pulse generator. Control rats (n =8) received electrogene therapy (EGT) vehicle plasmid and another 8 rats received HGF-EGT 100 μg 48 h before WLIR. Expression of HGF in liver was examined by RT-PCR and ELISA methods. Apoptosis was determined by TUNEL assay. Histopathology was evaluated 10 wk after whole liver irradiation. RESULTS: Marked decrease of apoptotic cells and down-regulation of transforming growth factor-beta 1 (TGF-β1) mRNA were observed in the HGF-EGT group 2 d after liver irradiation compared to control animals. Less evidence of radiation-induced liver damage was observed morphologically in liver specimen 10 wk after liver irradiation and longer median survival time was observed from HGF-EGT group (14 wk) compared to control rats (5wk).(P=0.031). CONCLUSION: For the first time it has been demonstrated that HGF-EGT would prevent liver from radiation-induced liver damage by preventing apoptosis and down-regulation of TGF-β1.     

Tumor-targeted IL-2 amplifies T cell-mediated immune response induced by gene therapy with single-chain IL-12.

Induction, maintenance, and amplification of tumor-protective immunity after cytokine gene therapy is essential for the clinical success of immunotherapeutic approaches. We investigated whether this could be achieved by single-chain IL-12 (scIL-12) gene therapy followed by tumor-targeted IL-2 using a fusion protein containing a tumor-specific recombinant anti-ganglioside GD(2) antibody and IL-2 (ch14.18-IL-2) in a poorly immunogenic murine neuroblastoma model. Herein, we demonstrate the absence of liver and bone marrow metastases after a lethal challenge with NXS2 wild-type cells only in mice (five of six animals) vaccinated with scIL-12-producing NXS2 cells and given a booster injection of low-dose ch14.18-IL-2 fusion protein. This tumor-protective immunity was effective 3 months after initial vaccination, in contrast to control animals treated with a nonspecific fusion protein or an equivalent mixture of antibody and IL-2. Only vaccinated mice receiving the tumor-specific ch14.18-IL-2 fusion protein revealed a reactivation of CD8(+) T cells and subsequent MHC class I-restricted tumor target cell lysis in vitro. The sequential increase in the usage of TCR chains Vbeta11 and -13 in mouse CD8(+) T cells after vaccination and amplification with ch14.18-IL-2 suggests that the initial polyclonal CD8(+) T cell response is effectively boosted by targeted IL-2. In conclusion, we demonstrate that a successful boost of a partially protective memory T cell immune response that is induced by scIL-12 gene therapy could be generated by tumor-specific targeting of IL-2 with a ch14.18-IL-2 fusion protein. This approach could increase success rates of clinical cancer vaccine trials.     

Dual inhibition of cyclooxygenase-2 and soluble epoxide hydrolase synergistically suppresses primary tumor growth and metastasis

Prostaglandins derived from the cyclooxygenase (COX) pathway and epoxyeicosatrienoic acids (EETs) from the cytochrome P450/soluble epoxide hydrolase (sEH) pathway are important eicosanoids that regulate angiogenesis and tumorigenesis. COX-2 inhibitors, which block the formation of prostaglandins, suppress tumor growth, whereas sEH inhibitors, which increase endogenous EETs, stimulate primary tumor growth and metastasis. However, the functional interactions of these two pathways in cancer are unknown. Using pharmacological inhibitors as probes, we show here that dual inhibition of COX-2 and sEH synergistically inhibits primary tumor growth and metastasis by suppressing tumor angiogenesis. COX-2/sEH dual pharmacological inhibitors also potently suppress primary tumor growth and metastasis by inhibiting tumor angiogenesis via selective inhibition of endothelial cell proliferation. These results demonstrate a critical interaction of these two lipid metabolism pathways on tumorigenesis and suggest dual inhibition of COX-2 and sEH as a potential therapeutic strategy for cancer therapy.Lipid signaling in the arachidonic acid (ARA) cascade is an important therapeutic target for many human disorders (1–3). Nonsteroidal anti-inflammatory drugs (NSAIDs) and cyclooxygenase (COX)-2–selective inhibitors (coxibs), which block COX-2–mediated conversion of ARA to prostaglandin E₂ (PGE₂), are widely used to treat inflammation and pain (4). Besides the COX pathway, ARA is also a substrate of cytochrome P450 (CYP) epoxygenases (largely CYP2C and CYP2J), which convert it to epoxyeicosatrienoic acids (EETs) (3). EETs have been investigated as autocrine and paracrine mediators with antihypertensive, anti-inflammatory, analgesic, and cardioprotective effects (5). Although chemically stable, EETs are unstable in vivo due to their rapid metabolism by soluble epoxide hydrolase (sEH) to form dihydroxyeicosatrienoic acids (DHETs), which are usually less active or inactive (5). Pharmacological inhibitors of sEH (sEHIs) that stabilize endogenous EETs are currently being explored as therapeutics (6).Our previous studies in murine models demonstrated powerful interactions of COX-2 and sEH pathways on pain and inflammation. Pharmacological inhibition of sEH or mice with global disruption of the gene that encodes sEH (sEH-null) synergized with multiple COX inhibitors (including NSAIDs, coxibs, and aspirin) to suppress inflammation and pain with reduced cardiovascular toxicity (7, 8). Due to the potent synergistic interactions, we recently designed and synthesized the first-in-class, to our knowledge, COX-2/sEH dual pharmacological inhibitors, which concurrently inhibit both COX-2 and sEH enzymes (9). A COX-2/sEH dual inhibitor, 4-(5-phenyl-3-{3-[3-(4-trifluoromethyl-phenyl)-ureido]-propyl}-pyrazol-1-yl)-benzenesulfonamide (PTUPB), as illustrated in Fig. S1, is more efficacious in attenuating inflammatory pain in vivo than celecoxib (a coxib) alone, trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (t-AUCB) (a selective sEHI) alone, or the combination of both celecoxib and t-AUCB (9). Coadministration of COX-2 and sEH inhibitors or administration of a dual inhibitor acts to reduce proinflammatory eicosanoids, such as PGE₂, and to increase anti-inflammatory and cardioprotective eicosanoids, such as EETs (Fig. S1B). Together, these results support the potent interactions of these two lipid metabolism pathways.Recent studies have demonstrated that both COX-2 and sEH play critical roles in angiogenesis and tumorigenesis (5, 10–13). Epidemiological and clinical evidence supports that COX-2 inhibitors inhibit multiple cancers (13). Contrary to the antitumor activity of COX-2 inhibitors, we and others have shown that when administered at the high dose of 10 mg⋅kg⁻¹⋅d⁻¹, sEHIs stimulate primary tumor growth and metastasis via EETs (11, 12, 14). Unexpectedly, we now demonstrate that a combination of a low-dose COX-2 inhibitor and a low-dose sEHI (3 mg⋅kg⁻¹⋅d⁻¹) synergistically inhibits primary tumor growth and metastasis. We extend this result by showing that the COX-2/sEH dual inhibitors also potently inhibit primary tumor growth and metastasis via suppressing tumor angiogenesis. These results support a key interaction of these two pathways on cancer progression.     

Digitoxin mimics gene therapy with CFTR and suppresses hypersecretion of IL-8 from cystic fibrosis lung epithelial cells

Cystic fibrosis (CF) is a fatal, autosomal, recessive genetic disease that is characterized by profound lung inflammation. The inflammatory process is believed to be caused by massive overproduction of the proinflammatory protein IL-8, and the high levels of IL-8 in the CF lung are therefore believed to be the central mechanism behind CF lung pathophysiology. We show here that digitoxin, at sub nM concentrations, can suppress hypersecretion of IL-8 from cultured CF lung epithelial cells. Certain other cardiac glycosides are also active but with much less potency. The specific mechanism of digitoxin action is to block phosphorylation of the inhibitor of NF-kappa B (I kappa B alpha). I kappa B alpha phosphorylation is a required step in the activation of the NF-kappa B signaling pathway and the subsequent expression of IL-8. Digitoxin also has effects on global gene expression in CF cells. Of the informative genes expressed by the CF epithelial cell line IB-3, 58 are significantly (P < 0.05) affected by gene therapy with wild-type (CFTR CF transmembrane conductance regulator). Of these 58 genes, 36 (62%) are similarly affected by digitoxin and related active analogues. We interpret this result to suggest that digitoxin can also partially mimic the genomic consequences of gene therapy with CF transmembrane conductance regulator. We therefore suggest that digitoxin, with its lengthy history of human use, deserves consideration as a candidate drug for suppressing IL-8-dependent lung inflammation in CF.     

体外垂体生长激素腺瘤转录靶向性基因治疗的实验研究

目的评价人生长激素启动子调控的基因治疗系统对垂体生长激素腺瘤的靶向性治疗作用。方法构建生长激素启动子调控的基因系统，体外转染垂体生长激素腺瘤GH3细胞；观察治疗基因HSV—TK在细胞中的表达靶向性，以及该系统杀伤细胞的能力和杀伤靶向性。结果HSV—TK蛋白能够在GH3细胞中靶向性表达，予以更昔洛韦后，GH3细胞增殖速度明显减慢，而对照细胞无明显杀伤作用（P〈0．01）。结论生长激素启动子调控的基因治疗系统能有效地、靶向性地抑制GH3细胞增殖。     

Regional therapy of malignant liver tumors

The highlights of important symposia and seminars of the 19th German Cancer Congress are reported by the respective chairmen or moderators in the present and forthcoming issues     

Ribavirin up-regulates IL-12 p40 gene expression and restores IL-12 levels in Leishmania-treated PBMCs

Ribavirin, a nucleoside analogue that interferes with viral mRNA synthesis and inhibits the replication of RNA and DNA viruses, has been recently proposed as an effective immune response modulator. In the present report, we studied the effect of ribavirin on IL-12 p40 gene expression in peripheral blood mononuclear cells (PBMCs) of healthy subjects. We also studied ribavirin effects on PBMCs activated with lipopolysaccharide (LPS) and phytohaemagglutinin (PHA) and treated with Leishmania donovani antigens. We provide evidence that ribavirin was able to up-regulate IL-12 p40 gene expression and to restore levels of IL-12 p40 gene expression and IL-12 secretion in fully activated PBMCs that were strongly inhibited by L. donovani antigens. Because effective management of leishmanial disease is usually associated with a prevalent T-helper 1 immune response with elevated production of IL-12,our preliminary results may be of particular interest, provided that they will be confirmed by further in vitro and in vivo studies, when considering a possible use of ribavirin as adjuvant in severe leishmanial disease.     

Synergistic inhibition of tumor growth in a murine mammary adenocarcinoma model by combinational gene therapy using IL-12, pro-IL-18, and IL-1beta converting enzyme cDNA

We report here that a cancer gene therapy protocol using a combination of IL-12, pro-IL-18, and IL-1beta converting enzyme (ICE) cDNA expression vectors simultaneously delivered via gene gun can significantly augment antitumor effects, evidently by generating increased levels of bioactive IL-18 and consequently IFN-gamma. First, we compared the levels of IFN-gamma secreted by mouse splenocytes stimulated with tumor cells transfected with various test genes, including IL-12 alone; pro-IL-18 alone; pro-IL-18 and ICE; IL-12 and pro-IL-18; and IL-12, pro-IL-18, and ICE. Among these treatments, the combination of IL-12, pro-IL-18, and ICE cDNA resulted in the highest level of IFN-gamma production from splenocytes in vitro, and similar results were obtained when these same treatments were delivered to the skin of a mouse by gene gun and IFN-gamma levels were measured at the skin transfection site in vivo. Furthermore, the triple gene combinatorial gene therapy protocol was the most effective among all tested groups at suppressing the growth of TS/A (murine mammary adenocarcinoma) tumors previously implanted intradermally at the skin site receiving DNA transfer by gene gun on days 6, 8, 10, and 12 after tumor implantation. Fifty percent of mice treated with the combined three-gene protocol underwent complete tumor regression. In vivo depletion experiments showed that this antitumor effect was CD8(+) T cell-mediated and partially IFN-gamma-dependent. These results suggest that a combinatorial gene therapy protocol using a mixture of IL-12, pro-IL-18, and ICE cDNAs can confer potent antitumor activities against established TS/A tumors via cytotoxic CD8(+) T cells and IFN-gamma-dependent pathways.     

Expression and modulation of IL-3 and GM-CSF receptors in human growth factor dependent leukaemic cells

From the interleukin-3 (IL-3) responsive human myeloid cell line M-07 we derived a subclone, named M-07e, which is fully dependent for growth and survival on either granulocyte-macrophage colony stimulating factor (GM-CSF) or IL-3. In this paper the expression, specificity and modulation of GM-CSF and IL-3 receptors on M-07e cells are described. The specificity of the binding was demonstrated by the failure of other cytokines to compete, at 4 degrees C, with GM-CSF or IL-3 receptors. In addition, IL-3 was found to compete as well as GM-CSF for GM-CSF receptors while GM-CSF was a weak competitor for IL-3 receptors. Quantitative binding studies and Scatchard plot analysis revealed the presence of a single class of high-affinity GM-CSF binding sites (405 +/- 27.4 sites per cell, dissociation constant at the equilibrium 52 +/- 20 pM) and the presence of high and low-affinity regions for IL-3 binding sites (27 +/- 12 and 416 +/- 92 sites per cell for the high and low affinity regions respectively, dissociation constant at the equilibrium, 58 +/- 22 pM and 5.7 +/- 2.0 nM respectively). Finally, in agreement with the hierarchical down-modulation model, we found that IL-3 can down-modulate both IL-3 and GM-CSF receptors while GM-CSF can down-modulate only its own receptors. The present results suggest that M-07e cells, in spite of their neoplastic nature, share, with murine bone marrow cells, similar growth factor receptor regulatory mechanisms. This cell line may be regarded as a candidate model to investigate the physiological events triggered by growth factors binding to human haemopoietic cells.     

Combined gene therapy with suicide gene and interleukin-12 is more efficient than therapy with one gene alone in a murine model of hepatocellular carcinoma

BACKGROUND/AIMS: Gene therapy has emerged as a new form of treatment for unresectable hepatocellular carcinoma (HCC). We evaluate here the effect of IL-12 and the suicide gene thymidine kinase as single agents and in combination to treat experimental liver cancer. METHODS: Recombinant adenoviruses expressing mouse interleukin-12 (AdCMVIL-12) or thymidine kinase of herpes simplex virus (AdCMVtk) or lacZ reporter gene (AdCMVlacZ) were constructed. The efficacy of the treatment was evaluated in a murine HCC model based on subcutaneous implantation of liver tumor cells (BNL). RESULTS: Transduction of BNL cells after in vitro infection with AdCMVlacZ was very low at multiplicity of infection (moi) of 100, whereas 10-15% of cells were transduced when using moi 1,000. Similarly, production of IL-12 was detectable only in BNL cells infected with AdCMVIL-12 at moi 1,000. In vitro infection of BNL cells with AdCMVIL-12 at moi 100 did not abrogate tumorigenicity, whereas moi 1,000 resulted in inhibition of tumor growth in all mice as well as in abrogation of tumor formation in 3 out of 8 animals. In vivo studies showed that intratumor injection of AdCMVIL-12 induced a dose-dependent effect on tumor regression. However, none of the animals exhibited complete tumor elimination with this treatment. We observed that suppression of tumor growth was more intense in animals treated with the combination of AdCMVIL-12 plus AdCMVtk than in animals which received AdCMVtk or AdCMVIL-12 alone. The combined treatment resulted in a significant increase in animal survival, and 25% of treated animals were free of tumor for over 100 days without recurrence of the disease. CONCLUSIONS: Combination of AdCMVIL-12 and AdCMVtk is more efficient than either of the two vectors alone for the treatment of the murine model of HCC used in this study.     

白介素18和胞嘧啶脱氨酶基因对小鼠肝癌的联合基因治疗

目的观察白介素18和胞嘧啶脱氨酶基因对小鼠肝癌MM45T.Li联合基因治疗的效果.方法将小鼠IL-18基因克隆入pGCEN中,构建成pGCEN/IL-18.包装成逆转录病毒,感染小鼠肝癌细胞MM45T.Li,制出具有生物活性的MM45T.Li/IL-18瘤苗,灭活后对荷瘤小鼠进行皮下接种.同时利用AdCD/5FC对小鼠肝癌原位注射,进行联合基因治疗.结果在接受治疗30d后,MM45T.Li对照组肿瘤体积1580～1625mm3,MM45T.Li/IL-18治疗组(366±159)mm3,AdCD/5FC治疗组(438±65)mm3,IL-18和AdCD/5FC联合治疗组体积(15±7)mm3(P＜0.05).MM45T.Li对照组中位生存期50.0～51.5d,MM45T.Li/IL-18治疗组65d,AdCD/5FC治疗组57d,联合治疗组76d,和单独治疗组相比,联合治疗组中位生存期明显延长(P＜0.05).联合基因治疗组肿瘤组织周围有更多CD4+及CD8+淋巴细胞浸润.结论IL-18和CD基因的联合治疗组,其疗效要明显好于IL-18或CD基因单独治疗组.进行联合基因治疗,既有效地减少了肿瘤负荷,又充分调动了机体的抗肿瘤免疫,提高了疗效.     

Activation of human eosinophil and neutrophil functions by haematopoietic growth factors: comparisons of IL-1, IL-3, IL-5 and GM-CSF.

We compared the effect of haematopoietic growth factors granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-1, IL-3, and IL-5 on the functional activation of human eosinophils and neutrophils from the same donor. All four colony-stimulating factors (CSF) enhanced the phagocytosis of Candida albicans by eosinophils and increased staphylococcal, but not Candida, killing. GM-CSF and IL-5 had a profound stimulating effect on eosinophil staphylocidal activity. GM-CSF and IL-3 enhanced the generation of leukotriene C4 (LTC4) induced by calcium ionophore A23187 and the release of arylsulphatase and beta-glucuronidase from specific and small granules of eosinophils. In contrast, IL-1 and IL-5 had no effect on degranulation. GM-CSF and IL-1 enhanced phagocytosis of C. albicans by neutrophils, and GM-CSF stimulated degranulation and the release of the enzymes beta-glucuronidase and arylsulphatase from neutrophils while IL-1 stimulated the release of arylsulphatase only. This study indicates that the eosinophil-active colony-stimulating factors can markedly enhance the host defence function of the eosinophil and even make it the equal of the neutrophil in staphylocidal activity. The CSF-activated eosinophil, however, may cause inappropriate inflammation and normal tissue damage.     

Molecular organization of the cytokine gene cluster, involving the human IL-3, IL-4, IL-5, and GM-CSF genes, on human chromosome 5

The human genes for the hematopoietic growth factors interleukin-3 (IL-3), IL-5, and granulocyte-macrophage colony-stimulating factor (GM-CSF) have been mapped to 5q23-31. We present in situ hybridization evidence that the human IL-4 gene is located at 5q23.3-31.2, suggesting that the four cytokine genes may be closely linked. We used pulsed-field gel electrophoresis to prepare subchromosomal restriction maps surrounding these genes to define this possible linkage more precisely. The IL-4 and IL-5 genes are tightly linked, being 90 to 240 kilobases (kb) apart, as has been shown for the IL-3 and GM-CSF genes, which are only 9 kb apart. Possible overlap of the map containing the IL-4 and IL-5 genes with restriction sites 5' to the IL-3 gene suggests that the four cytokine genes may be localized within 500 kb of each other. The endothelial cell growth factor gene (ECGF), which has also been localized to the 5q31 region, did not appear to be close to the cytokine genes. Linkage of the IL-3, IL-4, IL-5, and GM-CSF genes has important implications in the evolutionary origin and regulation of expression of these genes. The four cytokine genes are located in the region of the long arm of chromosome 5, which is deleted in the 5q- anomaly. The present study provides a basis for further investigations of this disorder.