Monitoring adenoviral DNA delivery,using a mutant herpes simplex virus type 1 thymidine kinase gene as a PET reporter gene

Current gene therapy protocols often suffer from an inability to monitor the site, level and persistence of gene expression following somatic DNA delivery. Herpes simplex virus 1 thymidine kinase (HSV1-tk) is currently under intensive investigation as a reporter gene for in vivo imaging of reporter gene expression. The presence of the HSV1-tk reporter gene is repetitively and non-invasively monitored by systemic injection of positron-emitting, radionuclide-labeled thymidine analogues or acycloguanosine HSV1-TK substrates and subsequent detection, by positron emission tomography, of trapped, phosphorylated product. To improve the efficacy of the HSV1-tk PET reporter gene system, both alternative substrates and mutations in the HSV1-tk gene have been described. We used a replication defective adenovirus to deliver the HSV1-sr39tk mutant enzyme and the wild-type HSV1-tk enzyme to mice. HSV1-sr39TK demonstrates greater sensitivity than wild-type HSV1-TK enzyme in vivo, using 9-[(4-[(18)F]fluoro-3-hydroxymethylbutyl)guanine as probe, following adenovirus-mediated hepatic expression in mice. Using this adenoviral delivery system, the location, magnitude and duration of HSV1-sr39tk PET reporter gene expression could be non-invasively, quantitatively and repetitively monitored for over 3 months by microPET.     

MicroPET imaging of Cre-loxP-mediated conditional activation of a herpes simplex virus type 1 thymidine kinase reporter gene

Site-specific recombination tools such as the Cre-loxP system are used to create animal models where conditional gene deletion/activation studies are required. In the current proof of principle study, we have demonstrated that a PET reporter gene (PRG), the herpes simplex virus type 1 thymidine kinase (HSV1-tk), can be made to remain silent and can be activated by Cre-loxP-mediated recombination in cell culture and in living mice. An adenovirus carrying a silent HSV1-tk was tail-vein injected (1 x 10(9) PFU) in six transgenic mice that express Cre recombinase in their liver (Cre+) and in four control mice (Cre-). The liver-specific expression of the PRG in Cre+ mice was detected in the microPET following injection of the reporter probe, 9-[4-fluoro-3-(hydroxymethyl)butyl]guanine ([(18)F]-FHBG). The [(18)F]-FHBG accumulation in the liver in terms of percent-injected dose per gram of tissue was 7.72+/-1.13 for the Cre+ mice and 0.10+/-0.02 for the Cre- mice (P<0.05) 48 h after adenoviral injection. These results were further validated by quantitative RT-PCR, western blotting and by in vitro assays for herpes simplex virus type 1 thymidine kinase enzyme activity. Thus by using the Cre-loxP system it is possible to modulate a PRG and noninvasively monitor the extent of Cre-loxP-mediated gene activation by imaging in a microPET scanner.     

对肿瘤内miR-21表达进行契伦科夫光学成像的新方法:基于HSV1-tk报告基因

目的构建一种基于I型单纯疱疹病毒胸苷激酶（HSV1-tk）的新型报告基因体系,用于对肿瘤内微小核糖核酸（miR-21）的表达进行契伦科夫光学成像。方法将细胞巨病毒（CMV）启动子基因、HSV1-tk基因以及可被miR-21互补结合的三联miR-21靶基因序列,串联构建成报告基因（CMV-HSV1-tk-3×miR-21t）,即将CMV-HSV1-tk-3×miR-21t序列连接到pcDNA3.1质粒载体中并转染A549细胞。向稳定表达上述报告基因的A549细胞（A549T细胞）加入9-[4-[18F]氟-3(羟甲基)丁基]鸟嘌呤（[18F] FHBG）孵育;或采用可互补结合miR-21的梯度剂量反义寡聚miR-21（Anti-miR-21）处理A549T细胞后,加入[18F]FHBG孵育。分别对上述细胞进行契伦科夫光学成像和放射性γ计数。另构建A549T裸鼠皮下移植瘤模型,分为2组分别瘤内注射Anti-miR- 21与对照混合RNA,然后分别经尾静脉注射[18F]FHBG后进行活体契伦科夫光学成像。结果A549T细胞摄取[18F]FHBG后,其光学信号强度、γ计数分别与细胞数量之间呈线性正相关（R2=0.9962、0.9807）;加入Anti-miR-21的剂量与光学信号强度、γ计数之间分别呈剂量依赖性正相关（P < 0.05）;A549T细胞皮下瘤模型成像结果显示,瘤内注射Anti-miR-21与对照RNA的移植瘤对比,肿瘤组织信号更高且视觉对比显著。结论基于microRNA调控的示踪剂摄取相关报告基因体系,本研究成功构建了一种用于对肿瘤内miR-21表达进行契伦科夫光学成像的新方法。      

Synthesis and Characterization of 9-(4-[18F]Fluoro-3-(hydroxymethyl)butyl)-2-(phenylthio)-6-oxopurine as a Novel PET Agent for Mutant Herpes Simplex Virus Type 1 Thymidine Kinase Reporter Gene Imaging

Molecular Imaging and Biology - [18F]FHBG has been used as a positron emission tomography (PET) imaging tracer for the monitoring of herpes simplex virus type 1 thymidine kinase (HSV1-tk), a...     

Noninvasive imaging of ex vivo intracoronarily delivered nonviral therapeutic transgene expression in heart.

We developed a clinically applicable approach for noninvasive monitoring of reporter-therapeutic linked gene expression in the whole heart of large animals using PET imaging and further validated the efficacy and cardiac adverse effects of reporter-therapeutic linked gene transfer in a rabbit cervical heterotopic functional heart transplant model. Cationic liposome complexed with a vector containing a herpes simplex virus type 1 mutant thymidine kinase (HSV1-sr39tk) as the reporter gene and a recombinant human immunosuppressive cytokine, interleukin-10 (hIL-10), as the therapeutic gene was ex vivo intracoronarily delivered into cardiac allografts before implantation. Long-term HSV1-sr39tk and hIL-10 transgene and protein overexpression associated with myocardial PET reporter probe 9-(4-[18F]fluoro-3-hydroxymethylbutyl)guanine ([18F]FHBG) accumulation was observed in the allografts. The expression of the HSV1-sr39tk gene was significantly correlated with the hIL-10 gene expression and the total myocardial [18F]FHBG accumulation quantified as a percentage of intravenously injected [18F]FHBG dose. A homogeneous distribution of [18F]FHBG accumulation was seen in the whole heart similar to the distribution of [18F]fluorodeoxyglucose, a PET glucose metabolism probe. The immunosuppressive therapeutic efficacy remained the same in allografts treated with reporter-therapeutic linked gene and therapeutic gene only. No cardiac adverse effect was found. Our results demonstrate for the first time that PET reporter-therapeutic linked gene imaging is applicable for noninvasively monitoring ex vivo intracoronarily delivered therapeutic transgene expression in the whole heart.     

A fully automated one pot synthesis of 9-(4-[18F]fluoro-3-hydroxymethylbutyl) guanine for gene therapy studies.

PURPOSE: To develop a new fully-automated method for the synthesis of 9-(4-[18F]fluoro-3-hydroxymethylbutyl)guanine ([18F]FHBG) amenable for its routine use in gene therapy monitoring studies.PROCEDURES: A nuclear interface commercial synthesizer was substantially modified and adapted to the synthesis of the referred compound. After the fluorination reaction of the tosylate precursor, the intermediate product was purified by Solid Phase Extraction (SPE) before the hydrolysis. The final product was purified by semi-preparative high performance liquid chromatography (HPLC).RESULTS: [18F]FHBG was obtained in 10-15% yield in 65 minutes including HPLC purification. The radiotracer was > 99% chemically and radiochemically pure, sterile and free from pyrogens. The synthesized compound was shown to accumulate in thymidine kinase (tk) expressing cells both in cell culture, and in laboratory animals infected with an adenoviral vector containing the herpes simplex virus type 1 thymidine kinase (HSV1-tk) gene.CONCLUSIONS: This new procedure facilitates the compliance with the applicable regulatory guidelines for positron emission tomography (PET) radiopharmaceuticals and will assist the clinical application of [18F]FHBG-PET as a noninvasive way to monitor gene therapy in humans.     

携带双报告基因真核表达载体pHSV1-TK-IRES2-EGFP在小鼠骨髓间充质干细胞内的表达

背景:单纯疱疹病毒胸腺嘧啶核苷激酶(herpes simplex virus 1-thymidine kinase,HSV1-TK)与绿色荧光蛋白(EGFP)双报告基因共表达载体构建及其在骨髓间充质干细胞内的表达研究报道较少。目的:构建pHSV1-TK-IRES2-EGFP真核表达载体,并检测其在体外培养的小鼠骨髓间充质干细胞内的表达。方法:应用聚合酶链式反应从质粒pHSV106中扩增出HSV1-TK基因后与pMD18-T载体连接,构成重组质粒pHSV1-TK/18T。重组质粒以限制性内切酶BglⅡ和SalⅠ进行双酶切,将其插入同样双酶切处理的pIRES2-EGFP载体内;并将新构成的重组质粒pHSV1-TK-IRES2-EGFP以脂质体法转染小鼠骨髓间充质干细胞。结果与结论:酶切鉴定结果表明,扩增的HSV1-TK基因序列正确,大小为1130bp;重组质粒载体内的HSV1-TK序列与GeneBank报告的序列完全一致;骨髓间充质干细胞转染后在荧光显微镜下可以观察到有特异性绿色荧光出现,HSV1-TK的mRNA有表达。实验成功构建了pHSV1-TK-IRES2-EGFP真核表达载体,在体外培养的小鼠骨髓间充质干细胞内能有效的表达。     

携带双报告基因真核表达载体pHSV1-TK-IRES2-EGFP在小鼠骨髓间充质干细胞内的表达

背景：单纯疱疹病毒胸腺嘧啶核苷激酶（herpes simplex virus 1-thymidine kinase,HSV1-TK）与绿色荧光蛋白（EGFP）双报告基因共表达载体构建及其在骨髓间充质干细胞内的表达研究报道较少。目的：构建pHSV1-TK-IRES2-EGFP真核表达载体,并检测其在体外培养的小鼠骨髓间充质干细胞内的表达。方法：应用聚合酶链式反应从质粒pHSV106中扩增出HSV1-TK基因后与pMD18-T载体连接,构成重组质粒pHSV1-TK/18T。重组质粒以限制性内切酶BglⅡ和SalⅠ进行双酶切,将其插入同样双酶切处理的pIRES2-EGFP载体内;并将新构成的重组质粒pHSV1-TK-IRES2-EGFP以脂质体法转染小鼠骨髓间充质干细胞。结果与结论：酶切鉴定结果表明,扩增的HSV1-TK基因序列正确,大小为1130bp;重组质粒载体内的HSV1-TK序列与GeneBank报告的序列完全一致;骨髓间充质干细胞转染后在荧光显微镜下可以观察到有特异性绿色荧光出现,HSV1-TK的mRNA有表达。实验成功构建了pHSV1-TK-IRES2-EGFP真核表达载体,在体外培养的小鼠骨髓间充质干细胞内能有效的表达。     

Efficient suicide gene therapy of transduced and distant untransduced ovary tumors is correlated with significant increase of intratumoral T and NK cells

Gene therapy using herpes simplex type 1 thymidine kinase gene (HSV1-TK) transfer followed by ganciclovir (GCV) treatment has revealed an important intratumoral and regional bystander effect that is at least partly immune-mediated. The aim of this work was to study the modifications of T lymphocyte subpopulations in a model of distant bystander effect occurring between ovary tumors. Bilateral ovarian tumors were generated in 21 WKY rats by injection in the ovarian pouch of either parental or HSV1-TK-expressing DWA-OC-1 ovarian cancer cells. After 14 days, rats were treated for two weeks with GCV (75 mg/kg x 2/d) or saline. All rats were killed at day 29 for pathological examination. The tumor-infiltrating mononuclear cells were analyzed by semi-quantitative immunohistochemistry. As compared to rats receiving saline, GCV-treated animals exhibited a complete disappearance of the HSV1-TK+ tumors with residual fibrotic scars (ovary weights: 0.46 +/- 0.4 g vs 10.11 +/- 1.5 g, P < 0.001). Interestingly, the contralateral HSV1-TK negative tumor showed a significant regression (12.39 +/- 1.93 g vs 22.24 +/- 237 g, P < 0.014). Furthermore, a lower incidence of tumoral ascitis was found in the GCV-receiving group (20% vs 90% P < 0.02). Within both TK- and TK+ tumors, there was a significant increase of CD4+, CD8+ and NK cells in the GCV-treated group compared to the saline-treated group. This study thus indicates that a distant bystander effect not only acts between close tumors within a given organ such as the liver, but also between more distant tumors in the peritoneal cavity. This effect is associated with significant infiltration of the tumor by immune system cells, supporting the notion that the distant bystander effect is immune-mediated.     

Quantitation of cell number by a positron emission tomography reporter gene strategy.

PURPOSE: An important potential of positron emission tomography (PET) is the capacity for quantitation of cell signals in an anatomic regions of interest. However, little is known about the constraints and parameters for using PET signal detection to establish cell numbers in regions of interest. In this study, we determined the correlation of PET signal to cell number, and characterized the cellular limit of detection for PET imaging. PROCEDURES: Cells expressing the herpes simplex virus type I thymidine kinase PET reporter gene (HSV1-sr39TK) were detected following accumulation of [(18)F]FHBG (9-[4-[(18)F]-fluoro-3-(hydroxymethyl) butyl]guanine) by microPET scanning and quantitation. RESULTS: When cells were cultured with [(18)F]FHBG in vitro, and then transferred to a model vascularized site, 73% retention was observed one hour post-transfer. Using this information, and the measured attenuation of PET signal in whole mouse scans, we assessed the per-cell uptake of [(18)F]FHBG in the vascularized site following standard parenteral administration of the substrate. We observed a cell number-dependent signal, with a limit of detection calculated as 10(6) cells in a region of interest of 0.1 mL volume. Quantitatively similar parameters were observed with stably tranfected N2a glioma cells and retrovirally transduced primary T lymphocytes. CONCLUSION: These methods and findings provide a strategy for quantitation of cellularity using PET imaging that has implications for both experimental models and clinical diagnosis.     

Direct correlation between positron emission tomographic images of two reporter genes delivered by two distinct adenoviral vectors

Biodistribution, magnitude and duration of a therapeutic transgene's expression may be assessed by linking it to the expression of a positron emission tomography (PET) reporter gene (PRG) and then imaging the PRG's expression by a PET reporter probe (PRP) in living animals. We validate the simple approach of co-administering two distinct but otherwise identical adenoviruses, one expressing a therapeutic transgene and the other expressing the PRG, to track the therapeutic gene's expression. Two PET reporter genes, a mutant herpes simplex virus type 1 thymidine kinase (HSV1-sr39tk) and dopamine-2 receptor (D(2)R), each regulated by the same cytomegalovirus (CMV) promoter, have been inserted into separate adenoviral vectors (Ad). We demonstrate that cells co-infected with equivalent titers of Ad-CMV-HSV1-sr39tk and Ad-CMV-D(2)R express both reporter genes with good correlation (r(2) = 0.93). Similarly, a high correlation (r(2) = 0.97) was observed between the expression of both PRGs in the livers of mice co-infected via tail-vein injection with equivalent titers of these two adenoviruses. Finally, microPET imaging of HSV1-sr39tk and D(2)R expression with 9-(4-[(18)F]fluoro-3-hydroxymethylbutyl) guanine ([(18)F]FHBG) and 3-(2-[(18)F]fluoroethyl)spiperone ([(18)F]FESP), utilizing several adenovirus-mediated delivery routes, illustrates the feasibility of evaluating relative levels of transgene expression in living animals, using this approach.     

Imaging of hypoxia-driven gene expression in an orthotopic liver tumor model

The purpose of this study was to monitor hypoxia in an orthotopic liver tumor model using a hypoxia-sensitive reporter imaging system and to image enhanced gene expression after clamping the hepatic artery. C6 and RH7777 Morris hepatoma cells were transduced with a triple reporter gene (HSV1-tk/green fluorescent protein/firefly luciferase-triple fusion), placed under the control of a HIF-1-inducible hypoxia responsive element (HRE). The cells showed inducible luciferase activity and green fluorescent protein expression in vitro. Isolated reporter-transduced Morris hepatoma cells were used to produce tumors in livers of nude rats, and the effect of hepatic artery clamping was evaluated. Tumor hypoxia was shown by immunofluorescence microscopy with the hypoxia marker EF5 [2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl acetamide)] and the fluorescent perfusion marker Hoechst 33342, and by pO(2) electrode measurements. For tumor hypoxia imaging with the HRE-responsive reporter, both luciferase bioluminescence and [(18)F]2'-fluoro-2'-deoxyarabinofuranosyl-5-ethyluracil positron emission tomography was done, and the presence of hypoxia in Morris hepatoma tumors were successfully imaged by both techniques. Transient clamping of the hepatic artery caused cessation of tumor perfusion and severe hypoxia in liver tumors, but not in adjacent liver tissue. These results show that the orthotopic reporter-transduced RH7777 Morris hepatomas are natively hypoxic and poorly perfused in this animal model, and that the magnitude of hypoxia can be monitored using a HRE-responsive reporter system for both bioluminescence and positron emission tomography imaging. However, the severity of tumor ischemia after permanent ligation of the hepatic artery limits our ability to image severe hypoxia in this animal model.     

[18F]FHBG PET/CT Imaging of CD34-TK75 Transduced Donor T Cells in Relapsed Allogeneic Stem Cell Transplant Patients: Safety and Feasibility

Described herein is a first-in-man attempt to both genetically modify T cells with an imagable suicide gene and track these transduced donor T cells in allogeneic stem cell transplantation recipients using noninvasive positron emission tomography/computerized tomography (PET/CT) imaging. A suicide gene encoding a human CD34-Herpes Simplex Virus-1-thymidine kinase (CD34-TK75) fusion enabled enrichment of retrovirally transduced T cells (TdT), control of graft-versus-host disease and imaging of TdT migration and expansion in vivo in mice and man. Analysis confirmed that CD34-TK75-enriched TdT contained no replication competent γ-retrovirus, were sensitive to ganciclovir, and displayed characteristic retroviral insertion sites (by targeted sequencing). Affinity-purified CD34-TK75⁺-selected donor T cells (1.0–13 × 10⁵)/kg were infused into eight patients who relapsed after allogeneic stem cell transplantation. Six patients also were administered 9-[4-(¹⁸F)fluoro-3-hydroxymethyl-butyl]guanine ([¹⁸F]FHBG) to specifically track the genetically modified donor T cells by PET/CT at several time points after infusion. All patients were assessed for graft-versus-host disease, response to ganciclovir, circulating TdT cells (using both quantitative polymerase chain reaction and [¹⁸F]FHBG PET/CT imaging), TdT cell clonal expansion, and immune response to the TdT. This phase 1 trial demonstrated that genetically modified T cells and [¹⁸F]FHBG can be safely infused in patients with relapsed hematologic malignancies after allogeneic stem cell transplantation.     

Quantitative imaging of gene induction in living animals.

Methods to repeatedly, non-invasively, and quantitatively image gene expression in living animals are rapidly emerging and should fundamentally change studies of gene expression in vivo. We previously developed assays utilizing positron emission tomography (PET) to image reporter gene expression. In this paper we: (1) describe a new bi-directional, tetracycline-inducible system that can be used to pharmacologically induce target gene expression and to quantitatively image induced expression by using a PET reporter gene; (2) demonstrate the potential of this system in transient and stable cell transfection assays; and (3) demonstrate the ability to repetitively and quantitatively image tetracycline and tetracycline analog induction of gene expression in living animals. We utilize the dopamine type-2 receptor (D(2)R) and the mutant herpes-simplex virus type 1 thymidine kinase (HSV1-sr39tk) reporter genes to validate this system. We utilize microPET technology to show that quantitative tomographic imaging of gene induction is possible. We find a high correlation (r(2) = 0.98) between 'target' and reporter gene expression. This work establishes a new technique for imaging time-dependent variation of gene expression both from vectors with inducible promoters and in transgenic animals in which pharmacologic induction of gene expression must be monitored. These techniques may be applied both in gene therapy and for the study of gene expression in transgenic animals.     

The bystander effect mediated by the new murine gammaherpesvirus 72--thymidine kinase/5'-fluoro-2'-deoxyuridine (MHV72-TK/5-FUdR) system in vitro

To investigate the potential of murine gamma-herpesvirus 72 thymidine kinase (MHV-72-TK) to act as a suicide gene, we used a mammalian expression vector on rat fibroblastoid cells deficient in the cellular TK gene. Substrate specificity was assessed in vitro in cells with stable expression of MHV-72-TK. The Herpes simplex virus 1-TK (HSV-1-TK) was used as a reference suicide gene. Unlike HSV-1-TK modified cells, which were sensitive to ganciclovir (GCV) (IC50=9.7 microM), cells modified by MHV-72-TK did not show sensitivity to this drug. The use of 3'-azido-3'-deoxythymidine (AZT) and (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) did not affect the growth of cells expressing either MHV-72-TK or HSV-1-TK in the range of concentration used for AZT (0-375 microM) and for BVDU (0-50 microM). In contrast, 5'-fluoro-2'-deoxyuridine (5-FUdR) was extremely cytotoxic and effectively killed MHV-72-TK expressing cells (IC50 value 2.1 microM). This value was 16 times lower than that required to kill cells expressing HSV1-TK. To test whether the bystander effect between two heterologous cell types could be mediated by the MHV-72-TK/5-FUdR system in vitro, cells expressing MHV-72-TK were co-cultured with the tumour fibroblastoid cell line NAD for 48 hours before the drug (10.8 microM) was added. The cell mixtures contained various ratios of cells expressing MHV-72-TK (0 to 50% of total cells). Only 1% of MHV-72-TK-expressing cells were needed to enhance mouse tumour cell killing and to decrease the survival rate to 25.6%. The bystander effect was more pronounced when 10% of cells expressing MHV-72-TK were used, decreasing survival to 17.4%. In parallel, the same concentration of 5-FUdR dose only marginally inhibited tumour cell growth in the absence of exogenous TK activity (84% survival). These results demonstrate the efficiency of MHV-72-TK as a suicide gene when 5-FUdR is used as a prodrug. When sequenced, MHV-72-TK proved to be identical to MHV-68 strain TK.     

Central nervous system toxicity of two adenoviral vectors encoding variants of the herpes simplex virus type 1 thymidine kinase: reduced cytotoxicity of a truncated HSV1-TK

Herpes simplex virus type 1-thymidine kinase (HSV1-TK) in combination with ganciclovir is an efficient and widely used strategy in brain tumour gene therapy. Recently, we have shown effective inhibition of glioma growth in a syngeneic rat model using recombinant adenoviruses expressing the full-length HSV1-TK and an N-terminus truncated variant, HSV1-DeltaTK in the presence of ganciclovir. We also showed active chronic brain inflammation in the long-term survivors (3 months) treated with HSV1-TK plus GCV. Furthermore, our results indicated loss of myelinated fibres, oedema and indices of ongoing axonal degeneration. In this study, we assessed the cytotoxicity of both HSV1-TK variants in the presence or absence of ganciclovir, in primary cultures of neurones and glia, and in the rat brain in vivo. Our results indicate that, at viral doses where tumour cells are sensitive to the enzyme/prodrug system, (1) there is no major cytotoxicity for either neurones or glial cells grown in primary cultures, (2) on its own the full-length HSV1-TK is more cytotoxic than its truncated version HSV1-DeltaTK for a population of non-neuronal and non-glial cells within neocortical primary cultures, and (3) in vivo, when delivered into the striatum, RAds encoding HSV1-TK are more cytotoxic than RAds encoding HSV1-DeltaTK, after administration of ganciclovir. The effectiveness of HSV1-DeltaTK in preventing brain tumour growth in vivo, combined with its reduced cytotoxicity, both in vivo and in primary cultures of CNS cells, could represent an advantage for treatment of brain tumours using gene therapy.     

Effects of plasminogen activator inhibitor-1 on ischemic brain injury in permanent and thrombotic middle cerebral artery occlusion models in mice

BACKGROUND AND OBJECTIVES: Tissue plasminogen activator (t-PA) improves the outcome of ischemic stroke by recanalization of occluded vessels, but has neurotoxic side effects in experimental stroke models. Here, the effect of plasminogen activator inhibitor-1 (PAI-1), an endogenous inhibitor of t-PA, on ischemic infarct volume was studied. METHODS: After either permanent ligation or thrombotic occlusion of the middle cerebral artery (MCA), infarct volume, spontaneous reperfusion of thrombosed MCA, t-PA/PAI-1 complex level, and blood-brain barrier (BBB) permeability in the ischemic region was studied in transgenic mice with overexpression of PAI-1 and wild-type littermate controls and in mice with intracerebroventricular injection of human PAI-1. RESULTS: Infarct volume was smaller in PAI-1 transgenic mice (2.9 +/- 3.7 mm3, mean +/- SD) than in controls (8.9 +/- 5.0 mm3, P < 0.05) after permanent MCA ligation (plasma PAI-1 level 39 +/- 23 ng mL(-1) in transgenic mice vs. 1.5 +/- 0.6 ng mL(-1) in controls), whereas after MCA thrombosis it was larger in transgenics (13.1 +/- 3.1 mm3) than in controls (8.0 +/- 3.2 mm3, P < 0.05). Spontaneous reperfusion of the thrombosed MCA was significantly delayed in transgenic vs. control mice. In the ligation model, t-PA/PAI-1 complex levels were higher and BBB disruption was more pronounced in the ischemic region. Human PAI-1 injection reduced infarct volume by about 50% in wild-type mice but not in t-PA gene deficient mice. CONCLUSIONS: High PAI-1 levels reduced infarct volume in the permanent MCA ligation model, but enhanced it in the MCA thrombosis model.     

Semiautomated Radiosynthesis and Biological Evaluation of [<Superscript>18</Superscript>F]FEAU: A Novel PET Imaging Agent for <Emphasis Type="Italic">HSV1-tk/sr39tk</Emphasis> Reporter Gene Expression

2′-Deoxy-2′-[¹⁸F]fluoro-5-ethyl-1-β-d-arabinofuranosyluracil ([¹⁸F]FEAU) is a promising radiolabeled nucleoside designed to monitor Herpes Simplex Virus Type 1 thymidine kinase (HSV1-tk) reporter gene expression with positron emission tomography (PET). However, the challenging radiosynthesis creates problems for being able to provide [¹⁸F]FEAU routinely. We have developed a routine method using a commercial GE TRACERlab FX-FN radiosynthesis module with customized equipment to provide [¹⁸F]FEAU. All radiochemical yields are decay corrected to end-of-bombardment and reported as means ± SD. Radiofluorination (33 ± 8%; n = 4), bromination (85 ± 8%; n = 4), coupling reaction (83 ± 6%; n = 4), base hydrolysis steps, and subsequent high-performance liquid chromatography purification afforded purified [¹⁸F]FEAU β-anomer in 5 ± 1% overall yield (n = 3 runs) after ~5.5 h and a β/α-anomer ratio of 7.4. Radiochemical/chemical purities and specific activity exceeded 99% and 1.3 Ci/μmol (48 GBq/μmol), respectively. In cell culture, [¹⁸F]FEAU showed significantly (P < 0.05) higher accumulation in C6 cells expressing HSV1-tk/sr39tk as compared to wild-type C6 cells. Furthermore, [¹⁸F]FEAU showed slightly higher accumulation than 9-[4-[¹⁸F]fluoro-3-(hydroxymethyl)butylguanine ([¹⁸F]FHBG) in cells expressing HSV1-tk (P < 0.05), whereas [¹⁸F]FHBG showed significantly higher (P < 0.05) accumulation than [¹⁸F]FEAU in HSV1-sr39tk-expressing cells. micro-PET imaging of mice carrying tumor xenografts of C6 cells stably expressing HSV1-tk or HSV1-sr39tk are consistent with the cell uptake results. The [¹⁸F]FEAU mouse images also showed very low gastrointestinal signal with predominant renal clearance as compared to [¹⁸F]FHBG. The routine radiosynthesis of [¹⁸F]FEAU was successfully semiautomated using a commercial module along with customized equipment to provide the β-anomer in modest yields. Although further studies are needed, early results also suggest [¹⁸F]FEAU is a promising PET radiotracer for monitoring HSV1-tk reporter gene expression.     

Reduced aortic lesions and elevated high density lipoprotein levels in transgenic mice overexpressing mouse apolipoprotein A-IV.

Transgenic mouse lines carrying several copies of the mouse apo A-IV gene were produced. Lipoprotein composition and function, and aortic lesion development were examined. Apo A-IV levels in the plasma of transgenic mice were elevated threefold compared with nontransgenic littermates on a chow diet, and sixfold in mice fed an atherogenic diet. Plasma concentrations of total cholesterol, HDL cholesterol, triglycerides, and free fatty acids were similar in transgenic and control mice fed a chow diet. However, with the atherogenic diet, male transgenic mice exhibited significantly higher levels of plasma triglycerides (P < 0.05), total cholesterol (P < 0.01), HDL cholesterol (P < 0.0001), and free fatty acids (P < 0.05), and lower levels of unesterified cholesterol (P < 0.05), than nontransgenic littermates. Expression of the apo A-IV transgene had a protective effect against the formation of diet-induced aortic lesions, with transgenics exhibiting lesion scores of approximately 30% those seen in control mice. HDL-sized lipoproteins isolated from transgenic mice fed the atherogenic diet promoted cholesterol efflux from cholesterol-loaded human monocytes more efficiently than comparable lipoproteins from nontransgenic counterparts. Plasma from transgenics also exhibited higher endogenous cholesterol esterification rates. Taken together, these results suggest that apo A-IV levels influence the metabolism and antiatherogenic properties of HDL.