A novel suicide gene therapy targeting the overexpression of eukaryotic initiation factor 4E improves survival in a rat peritoneal carcinomatosis model

BACKGROUND: Eukaryotic Initiation Factor 4E (eIF4E) is pivotal in translating mRNAs with complex 5' un-translated regions (UTRs). A target-specific gene therapy was developed by splicing a complex 5'UTR upstream of the herpes simplex virus thymidine kinase (TK) gene in an adenovirus vector (Ad-HSV-UTK). Translation of the suicide TK gene is restricted to cells that overexpress eIF4E. We investigated the efficacy of this novel therapy in a rat peritoneal carcinomatosis (PC) model. METHODS: A PC model was developed by implanting a syngeneic 0.25 cm(3) tumor into Fisher 344 rats' omentum. Rats were grouped as follow: No surgery (? CS), cytoreductive surgery alone (CS), and CS + Ad-HSV-UTK + gancyclovir (GCV). 10(9) Ad-HSV-UTK was injected intraperitoneally (i.p.) and GCV (50 mg/kg) was administered i.p. every other day, beginning on postoperative day 2. The Kaplan-Meier survival method and log-rank test were statistical tests used. RESULTS: Treated rats had a significantly longer median and overall survival than the ? CS and CS groups (P = .012). The median survivals for the treated rats, ? CS, CS were 18 days, 9 days, and 11 days, respectively. CONCLUSIONS: Treatment with a novel suicide gene therapy following cytoreductive surgery prolonged survival in a rat peritoneal carcinomatosis model.     

eIF4E-targeted suicide gene therapy in a minimal residual mouse model for metastatic soft-tissue head and neck squamous cell carcinoma improves disease-free survival

BACKGROUND: Translation initiation factor eIF4E unwinds long 5'-untranslated regions of certain tightly regulated mRNAs and, thereby, facilitates their translation into proteins. eIF4E has been shown to be overexpressed in a majority of solid tumors, including head and neck cancers. To exploit this dysregulation, a long 5'-untranslated region was spliced upstream of a thymidine kinase (Tk) gene to enhance translation of this "suicide" gene within cells overexpressing eIF4E. We investigated the efficacy of therapy with an adenovirus incorporating this novel suicide gene (Ad-HSV-UTk) following cytoreductive tumor surgery in improving disease-free and overall survival in a mouse soft-tissue metastasis model for head and neck squamous cell carcinoma. MATERIALS AND METHODS: SCC-7 (orally-derived mouse SCCa) cells were treated with Ad-HSV-Tk, Ad-HSV-UTk, Ad-null, or saline and characterized for eIF4E and Tk levels by Western blot analysis. Cytotoxicities for cells treated with Ad-HSV-Tk, Ad-HSV-UTk, or Ad-null were quantified by MTS assay. Mice bearing SCC-7-induced tumors received cytoreduction followed by Ad-HSV-UTk + ganciclovir (GCV) or control treatment and were followed for disease-free and overall survival. RESULTS: SCC-7 cells showed uniformly high levels of eIF4E but elevated Tk for Ad-HSV-Tk- and Ad-HSV-UTk-treated cells over Ad-null-treated cells. Cytotoxicities for Ad-HSV-Tk- and Ad-HSV-UTk-treated cells were, correspondingly, observed to be 100-fold more sensitive than Ad-null-treated cells to GCV treatment. Cytoreduced mice receiving Ad-HSV-UTk + GCV treatment showed significantly longer disease-free survival (P = 0.0045) than control arm mice. CONCLUSIONS: Ad-HSV-UTk suicide gene therapy prolonged disease-free survival in a mouse minimal residual soft-tissue head and neck squamous cell carcinoma metastasis model.     

β干扰素基因修饰的人骨髓间充质干细胞治疗前列腺癌小鼠移植瘤的实验研究

目的 探讨人β干扰素(interferon-beta,IFN-β)基因修饰的人骨髓间充质干细胞(human mesenchymal stem cell,hMSC)对人前列腺癌小鼠移植瘤的治疗作用.方法 构建人IFN-β基因腺病毒表达载体Ad-IFN-β,体外转染hMSC,使用4,6-联脒-2-苯基吲哚(DAPI)标记表达IFN-β的hMSC( IFN-β-hMSC).应用人前列腺癌PC-3细胞株皮下种植建立小鼠前列腺癌动物模型,将IFN-β-hMSC经尾静脉注射入小鼠模型体内,收集肿瘤和肝、肺、脾、肾等脏器作冰冻切片和石蜡切片,荧光显微镜下观察肿瘤及各脏器中IFN-β-hMSC的分布情况.42只小鼠随机分为治疗组:IFN-β-hMSC(2×106,2×105)组;对照组:Ad-hMSC组、hMSC组、Ad-IFN-β组、重组IFN-β组、生理盐水组,每组6只.记录各组尾静脉注射后荷瘤小鼠肿瘤大小及生存期.结果 IFN-β-hMSC注射后14 d,荷瘤小鼠前列腺癌组织冰冻切片和石蜡切片中均可见DAPI标记的IFN-β-hMSC,而肝、肺、脾、肾等脏器的切片中均未见IFN-β-hMSC的存在.肿瘤质量:IFN-β-hMSC(2 × 106)治疗组(1.35±0.28)g、IFN-β-hMSC(2×105)组(1.43±0.41)g,与对照Ad-hMSC组(3.49±0.25)g、hMSC组(3.58±0.30)g、AdIFN-β组(3.30±0.24)g、重组IFN-β组(3.32±0.25)g、生理盐水组(3.32±0.47)g比较差异均有统计学意义(P ＜0.05);IFN-β-hMSC(2 × 106)治疗组中位生存时间91 d、IFN-β-hMSC(2×105)组87 d,与对照Ad-hMSC组57 d、hMSC组59 d、Ad-IFN-β组62 d、重组IFN-β组61 d、生理盐水组61 d比较差异有统计学意义(P＜0.05).结论 IFN-β-hMSC在体内具有向前列腺癌微环境聚集转移的特性,且能够抑制前列腺癌移植瘤的生长,延长荷瘤鼠的生存期.     

白细胞介素-15对小鼠胃癌的治疗作用

目的 建立小鼠胃癌模型,检测荷瘤状态对小鼠免疫细胞的影响,同时给予白细胞介素-15(IL-15)免疫基因治疗,以检测IL-15对胃癌的预防及治疗效果.方法 采用多因素联合攻击法诱导小鼠胃癌,同时采用IL-15表达质粒载体进行免疫基因治疗.20周后取胃标本行病理学检查,并取血及脾细胞检测T细胞亚群及CD4+ CD25+调节性T细胞(Treg).同时进行自然杀伤(NK)细胞的细胞毒性试验,通过统计学方法分析结果.结果 多因素联合攻击法诱导胃癌发生率为37.5％.诱癌组小鼠血清IL-15水平(9.20±2.92) ng/L明显降低(P＜0.05),且其脾NK细胞杀伤活性(216.91±117.80) U/L明显降低(P＜0.05).诱癌组小鼠外周血Treg水平(8.07±6.62)％明显升高(P＜0.05),而对照组小鼠脾细胞Treg水平(4.40±3.34)％明显降低(P＜0.05).各组小鼠之间外周血T细胞亚群变化差异无统计学意义(P＞0.05).诱癌组小鼠脾细胞CD3+T细胞水平(78.31±29.79)％明显升高(P＜0.05),而CD4+T细胞(19.98±5.77)％及CD8+T细胞水平(10.15±1.72)％明显降低(P＜0.05).结论 多因素联合攻击法为小鼠胃癌模型建立提供了良好的模型.小鼠荷瘤状态下免疫功能下降,通过IL-15免疫基因治疗可提高机免疫功能,起到抗肿瘤的效果.     

Matrix metalloproteinase inhibition improves survival in an orthotopic model of human pancreatic cancer

Matrix metalloproteinases (MMPs) have been implicated in the growth and invasiveness of primary and metastatic tumors. Hypothesizing that MMP inhibition would slow cancer growth, the MMP inhibitor BB-94 (batimistat) was evaluated in an orthotopic animal model of human pancreatic carcinoma. Ten million human pancreatic cancer cells were surgically implanted into the pancreata of 30 athymic nu/nu mice. Intraperitoneal administration of 30 mg/kg BB-94 or vehicle control began 7 days after tumor implantation (13 mice with confirmed implantations in each group) and continued daily for 21 days, and then three times weekly until death or sacrifice at day 70. Representative tumors harvested from mice in each group were analyzed for presence and activity of MMP-2 and MMP-9. Animal weights were significantly higher in the BB-94-treated group at sacrifice (mean 58.4 ±7.9 g vs. 39.8 ±6.2 g; P<0.05, Student’s t test). The likelihood of survival to 70 days was significantly higher in the treated group (4 of 13 vs. 0 of 13, P <0.05, Z-test for end points) than in the control group as was overall survival (P = 0.03, Wilcoxon test). Nine mice in the control group developed metastases to the liver, peritoneum, abdominal wall, or local lymph nodes, whereas only two mice in the BB-94 group had evidence of metastatic disease (P <0.02, Fisher’s exact test), in both instances confined to the abdominal wall. Tumors from treated mice manifested lower MMP activity than those from control animals. These reports support the use of MMP inhibitors alone or as an adjunct in the treatment of pancreatic cancer. Presented in part at the Thirty-Seventh Annual Meeting of The Society for Surgery of the Alimentary Tract, San Francisco, Calif., May 19–22, 1996.     

Inhaled nitric oxide improves survival rates during hypoxia in a sickle cell (SAD) mouse model

BACKGROUND: The hallmark of sickle cell disease (SCD) is erythrocyte sickling during deoxygenation of the abnormal hemoglobin S (HbS). When HbS is deoxygenated, it aggregates into polymers, resulting in distortion of the erythrocyte structure, producing microvascular thrombosis and ischemia. The transgenic SAD mouse produces three types of human hemoglobin: S, Antilles, and D-Punjab (HbSAD) and provides an animal model for SCD. We studied the effects of nitric oxide (NO) breathing at various doses and time regimens in the presence of severe hypoxia (6% oxygen) using the SAD mouse model. METHODS: Age- and sex-matched control and SAD mice were exposed to 6% oxygen breathing in an environmental chamber and assessed for survival up to 1 h. Animals received different inhaled NO concentrations before and/or during hypoxia. Blood was obtained to evaluate the oxyhemoglobin dissociation curve and measure methemoglobinemia. RESULTS: Pretreatment by breathing NO at 20 ppm by volume in air for 30 min, and continuing to breathe 20 ppm NO during hypoxia resulted in improvement in survival rates in the SAD mouse (75%, n = 8) as compared with control SAD mice (11%, n = 9; P < 0.001). Pretreatment alone or breathing lower doses of NO were not protective. Changes in HbSAD oxygen affinity were not detected with NO breathing, and methemoglobin levels were low in all surviving mice. CONCLUSIONS: Breathing NO produced a rapid, protective effect to severe hypoxic stress in SAD mice. There appears to be a required loading period between NO breathing and its beneficial effect during hypoxic stress, possibly because of the total amount of NO delivered to SAD hemoglobin, blood cell components, and endothelium. NO breathing may be beneficial as a therapeutic intervention in SCD.     

Green fluorescent protein-adenoviral construct as a model for transient gene therapy for human cultured keratinocytes in an athymic mouse model

BACKGROUND: The goal of gene therapy for cultured keratinocyte grafts is to accelerate growth and wound healing following engraftment without producing long-term complications from the delivered gene. We studied a Green Fluorescent Protein-Adenoviral construct (GFP-ADV) to determine the characteristics of gene expression in human cultured keratinocyte grafts. METHODS: Twelve GFP-ADV grafts and twelve control grafts were transplanted to the flanks of 24 athymic mice. Mouse flanks were monitored with fluorescence-filtered microscopy and, on Day 21, were sectioned and stained with anti-human MHC Class I with H&E counterstaining. Real-time PCR was performed on graft biopsies for adenoviral DNA. RESULTS: Fluorescence decreased from Days 3 to 5 resulting in no difference between GFP-ADV and control grafts from days 5 to 10. All grafts were positive for human MHC Class I with an epithelial architecture by H&E. Day 21 GFP-ADV grafts were negative for adenoviral DNA. CONCLUSION: The delivered gene was transiently expressed without the persistence of viral DNA, demonstrating the potential of adenoviral gene delivery for the improvement of wound healing without long-term adverse effects to the graft.     

Effect of antiadhesive agents on peritoneal carcinomatosis in an experimental model

BACKGROUND: Auto-crosslinked polysaccharide hyaluronan-based solution (Hyalobarrier-gel) prevents postoperative adhesions. However, its effect on tumour growth is still unknown. The aim of the present study was therefore to investigate the impact on survival of intra-abdominally administered Hyalobarrier-gel, native hyaluronan (HA) and hyaluronan/carboxymethylcellulose (HA/CMC), after intraperitoneal tumour implantation. METHODS: After receiving an intraperitoneal inoculum of the human HT29 colorectal cell line, 615 athymic nude mice were assigned randomly to five groups: groups 1 and 2 received Hyalobarrier-gel 20 mg/ml (n = 124) and 40 mg/ml (n = 126) respectively; groups 3 and 4 received HA (n = 120) and HA/CMC film (Seprafilm) (n = 123) respectively. The survival of each treated group was compared with that of group 5, the control, which had no treatment (n = 122). RESULTS: As 34 of the 615 mice were not eligible, 581 animals were considered for the analysis. At 120 days, 136 animals (23.4 per cent) were still alive. At autopsy there was macroscopic absence of tumour in 75 cases (12.9 per cent). No statistically significant differences were found between the treatment and the control groups with respect to postoperative death and absence of tumour implantation. There was no difference in survival rate between the control group and groups treated with Hyalobarrier-gel, HA or HA/CMC. CONCLUSION: Hyalobarrier-gel, HA and HA/CMC had no negative impact on the survival rate in mice that received an intraperitoneal implantation of HT29 colorectal human tumour cells.     

FTY720 application following isolated warm liver ischemia improves long-term survival and organ protection in a mouse model

BACKGROUND: Ischemia-reperfusion-Injury (I/RI) is a common complication in transplant-, liver-, and heart surgery. The I/RI is mediated and aggravated by different types of leukocytes such as lymphocytes, monocytes, and neutrophil granulocytes, with consecutive enlargement of the expression of adhesion molecules. This study shows an organ-protective effect of an intraoperative FTY720 administration following warm liver ischemia (Pringle's maneuver). METHODS: Male c57BL6/J mice (n = 46, body weight [BW] 25 to 30 g) were used. Either FTY720 (1 mg/kg BW), steroids (5 mg/kg BW), or physiological saline solution was administered intraperitoneally. Liver-ischemia was applied for 30 minutes with subsequent follow-up for 48 hours. At termination, all surviving animals were sacrificed. The impact of the drugs administered on long-term survival, time of death, and development of blood T-lymphocyte concentration was determined. Follow-up of T-lymphocyte concentration in peripheral blood was examined throughout FACS-analysis. RESULTS: Following 30 minutes of ischemia, FTY720, but not steroid or vehicle treatment, showed a significant protective effect on long-term survival. FACS-analysis showed significant T-lymphocyte depletion in peripheral blood following FTY720 but not steroids or vehicle treatment. CONCLUSION: The improved long-term survival following FTY720 application shown in this study might be due to a protective effect of FTY720 in prevention of I/RI. This might be mediated by the T-lymphocyte depletion shown in the FACS-analysis.     

Light therapy by blue LED improves wound healing in an excision model in rats

Background

Low level light therapy (LLLT) is an attractive alternative to enhance wound healing. So far most studies are performed with red or infrared irradiation. However, we recently showed that blue light (470 nm) can significantly influence biological systems, improving perfusion by release of nitric oxide from nitrosyl complexes with haemoglobin in a skin flap model in rats. Here, we compared the effects of blue and red low level light by light-emitting diodes (LEDs) on in vivo wound healing in an excision wound model in rats.

Methods

Circular excision wounds were surgically created on the dorsum of each rat. Excisions on either the left or right side were illuminated post-OP and on five consecutive days for 10 min by LED at 470 nm or 630 nm with an intensity of 50 mW/cm², while protecting the contralateral side from exposure. In the control group, neither side was illuminated. On day 7 post-OP, we analysed planimetric and histological parameters, as well as expression of keratin-1, keratin-10 and keratin-17 on mRNA level.

Results

Illumination substantially influenced wound healing. Blue light significantly decreased wound size on day 7, which correlated with enhanced epithelialisation. Light also affected mRNA expression. Both wavelengths decreased keratin-1 mRNA on day 7 post-OP, while keratin-10 mRNA level was elevated in both light treated group compared to control. Keratin-17 mRNA was also elevated in the red light group, but was unchanged in the blue light group.

Conclusion

In contrast to previous studies, we showed that also blue light significantly influences wound healing. Furthermore, our data suggest that light therapy can play an important role in normotrophic wound healing by affecting keratin expression. Illumination would provide an easily applicable, safe and cost-effective treatment of surface wounds.     

Lysis-deficient bacteriophage therapy decreases endotoxin and inflammatory mediator release and improves survival in a murine peritonitis model

BACKGROUND: Lysis-deficient (LyD) bacteriophages (phages) kill bacteria without endotoxin (Et) release. This may minimize systemic cytokine responses and limit inflammation in bacterial sepsis. We determined the effects of t amber A3 T4 LyD and virulent wild-type (WT) phages on mouse bacterial peritonitis. METHODS: Balb/c mice were injected with B40sul Escherichia coli, treated intraperitoneally with LyD, WT, or a beta-lactam antibiotic [latamoxef sodium (LMOX)], and followed for survival. We measured Et release, tumor necrosis factor (TNF)-alpha and interleukin (IL)-6, as well as bacterial counts and peritoneal exudative cells (PECs) in peritoneal lavage fluid at 6 and 12 hours after infection. RESULTS: LyD mice showed significantly greater survival compared with other groups. Et levels were significantly lower in the LyD mice at 6 and 12 hours after infection. TNF-alpha and IL-6 levels were lower in LyD mice compared with control (untreated) mice at 12 hours. Compared with controls, bacteria counts in peritoneal lavage fluid were lower in all treatment groups (LyD, WT, or LMOX) at 6 and 12 hours. PEC counts were highest in LyD mice at 6 hours but significantly lower than that in WT phage- and LMOX-treated mice at 12 hours. CONCLUSIONS: LyD phage therapy significantly improves survival and attenuates the systemic effects of bacterial sepsis by minimizing Et release and pro-inflammatory mediators in murine bacterial peritonitis. Further studies may find phage therapy useful in treating peritonitis and multidrug-resistant bacterial infections.     

Improved survival in mice given systemic gene therapy in a gram negative pneumonia model

BACKGROUND: We previously demonstrated an essential role for lipopolysaccharide binding protein (LBP) in the pulmonary immune response to Gram-negative bacterial infection. LBP knockout mice had significantly higher mortality, greater rates of bacteremia, and higher counts of viable bacteria in their lungs at sacrifice compared with wild-type controls. We postulate that systemic LBP gene therapy will reconstitute a protective innate immune response in LBP knockout mice and that overexpression of LBP in wild-type mice may offer a survival advantage. METHODS: 12-16 week old female C57BL/6 wild-type mice and age matched LBP knockout mice were given 5 x 10(9) PFU of recombinant adenovirus containing either the gene for LBP or the irrelevant control protein beta-galactosidase by tail vein injection. 72 hours later each mouse was administered 1 x 10(3) CFU of Klebsiella pneumoniae by intratracheal injection. RESULTS: Administration of LBP by systemic gene therapy to LBP knockout mice improved survival from Klebsiella pneumonia to a level equivalent or better than wild-type mice exposed to the same dose of bacteria (36 versus 25%). Wild-type mice given the LBP gene therapy demonstrated increased 7 day survival from Klebsiella pneumonia when compared with controls treated with beta-galactosidase (68 versus 30%, p = 0.03). CONCLUSIONS: Systemic gene therapy with intravenous adenoviral vector transfer of LBP significantly improves survival in LBP knockout mice. Overexpression of LBP in wild-type mice improves survival from Klebsiella pneumonia. Raising levels of LBP in the setting of Gram-negative pneumonia may be of therapeutic benefit.     

Modulation of macrophage hyperactivity improves survival in a burn-sepsis model.

Macrophage hyperactivity with increased production of tumor necrosis factor, interleukin 6, interleukin 1, and prostaglandins has been demonstrated in the injured patient, but the effect of this on the clinical outcome is unclear. We studied the effect of combination interleukin 1 beta and indomethacin sodium therapy on macrophage hyperactivity and survival after sepsis in a murine burn model. Macrophage interleukin 1, interleukin 6, and tumor necrosis factor alpha production were all significantly increased 10 days after thermal injury. Treatment with recombinant human interleukin 1 beta in combination with indomethacin significantly reduced this overproduction of cytokines to normal levels, and this was associated with an improvement in survival after septic challenge (52% survival in interleukin 1 beta-indomethacin-treated group compared with 22% in burned vehicle control mice). Burned mice that received either interleukin 1 beta or indomethacin alone demonstrated tumor necrosis factor and interleukin 6 production and survival intermediate between the interleukin 1 beta-indomethacin-treated group and the vehicle control group. Control of macrophage hyperactivity is associated with improved survival from subsequent sepsis and offers a potential new strategy for the treatment of immune dysfunction in thermally injured patients.     

Right ventricular gene therapy with a beta-adrenergic receptor kinase inhibitor improves survival after pulmonary artery banding.

BACKGROUND: Increased right ventricular (RV) afterload results in RV hypertrophy and dysfunction, as well as increased levels of intracellular beta-adrenergic receptor kinase (betaARK1). We hypothesize that gene transfer of a betaARK1 inhibitor (betaARKct) may improve RV performance, morbidity, and mortality early after pulmonary artery (PA) banding. METHODS: Rabbits underwent PA banding 3 days after right coronary artery injection of an adenovirus containing the gene encoding the betaARKct peptide (n = 14), beta-galactosidase (n = 10), or an empty adenovirus (n = 19). After banding, hemodynamic instability and maximal rate of increase in right ventricular pressure (RV dP/dt(max)) were documented. For 7 days after banding, animals were monitored for mortality, activity, and appetite. RESULTS: When compared with controls, animals receiving the betaARKct transgene showed improvement in survival at 7 days (92.8% +/- 7% vs 48.3% +/- 9%, p = 0.01), less lethargy, a trend toward greater RV dP/dt(max) (NS), and increased hemodynamic stability at the time of banding (78% vs 41%, p = 0.03). CONCLUSIONS: Selective RV expression of betaARKct improves survival and morbidity after PA banding. This represents a novel therapeutic modality for clinical situations involving increased RV afterload.