Background-The noninvasive, tissue-specific delivery of therapeutic agents to the heart would be a valuable clinical tool.This study addressed the hypothesis that albumin-coated microbubbles could be used to effectively deliver an adenoviral transgene to rat myocardium by ultrasound-mediated microbubble destruction. Methods and Results-Recombinant adenovirus containing ␤-galactosidase and driven by a constitutive promoter was attached to the surface of albumin-coated, perfluoropropane-filled microbubbles. These bubbles were infused into the jugular vein of rats with or without simultaneous echocardiography. Additional controls included ultrasound of microbubbles that did not contain virus, virus alone, and virus plus ultrasound. One group underwent ultrasoundmediated destruction of microbubbles followed by adenovirus infusion. Rats were killed after 4 days and examined for ␤-galactosidase expression. The hearts of all rats that underwent ultrasound-mediated destruction of microbubbles containing virus showed nuclear staining with 5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside substrate, indicating expression of the transgene. None of the control animals showed myocardial expression of the ␤-galactosidase transgene. By quantitative analysis, ␤-galactosidase activity was 10-fold higher in the treated group than in controls (PϽ0.0001). Conclusions-Ultrasound-mediated destruction of albumin-coated microbubbles is a promising method for the delivery of bioactive agents to the heart.

Background-Noninvasive, tissue-specific delivery of therapeutic agents would be a valuable clinical tool. We have previously shown that ultrasound-targeted microbubble destruction can direct expression of an adenoviral reporter to the heart. The present study shows that this method can be applied to selectively deliver plasmid vectors to the heart. Methods and Results-We used albumin and lipid microbubbles containing plasmids with a luciferase transgene to target the heart in rats. After 4 days, organs were harvested and analyzed for reporter gene expression. In a second set of experiments, the hearts of rats treated with plasmids were harvested at various time points during a 4-week period. Both luciferase activity and mRNA concentrations were measured. Luciferase transfection with plasmids showed highly specific gene expression in the heart, with hardly any activity in control organs. Time course evaluation showed high transgene expression in the first 4 days, with a rapid decline thereafter. Repeated treatment produced a second peak of transgene expression with similar decay. Conclusions-Ultrasound-mediated destruction of microbubbles directs plasmid transgene expression to the heart with much greater specificity than viral vectors and can be regulated by repeated treatments. This noninvasive technique is a promising method for cardiac gene therapy. Key Words: echocardiography Ⅲ gene therapy Ⅲ ultrasonics Ⅲ contrast media T he molecular understanding of disease has accelerated in recent years, producing many new potential therapeutic targets. A noninvasive delivery system that can target specific anatomical sites would be a great boon for many of these therapies, particularly those based on manipulation of gene expression. Viral vectors, which have been commonly used for gene therapy, 1 are limited by hepatic toxicity, immunogenic properties, inflammation, and low tissue specificity, as well as the difficulty and expense of producing large amounts of pure virus. The use of naked DNA or liposome carriers has the disadvantage of low transfection efficiency and the requirement for invasive delivery techniques. Many strategies have been developed for augmenting tissue specificity. These include decorating viral vectors with cell-specific ligands, 2 the use of cell-specific 3 or pathology-specific 4 promoters in the transgene construction, and physical placement of the vector in the vicinity of the target by catheter-based methods 5,6 or by direct injection. [7][8][9] We have investigated ultrasound-targeted microbubble destruction (UTMD) as a method for delivering drugs or genes to specific tissues. 10 This method involves the attachment of drugs or genes to gas-filled microbubbles, which are then circulated through the intravascular space and mechanically destroyed within the target organ by ultrasound. [11][12][13] Theoretically, one should be able to target any anatomic site that is accessible to ultrasound, including selected diseased or damaged regions of an organ. Moreover, it should be possible to combine ...

Ultrasound-targeted microbubble destruction can be used to deliver adenoviral or plasmid DNA to the myocardium. This technique holds great promise in applying the rapidly expanding repertoire of gene therapies being developed for cardiac disease.

Purpose: Most hepatocellular carcinomas (HCC) are diagnosed at an advanced stage. Hypermethylation of CpG islands in promoter regions is now recognized as an important early event in carcinogenesis and detection of methylated DNA has been suggested as a potential biomarker for early detection of cancer. There are no studies on epigenetic changes in samples from HCC patients before diagnosis. We explored the possible diagnostic value of aberrant promoter hypermethylation of three tumor suppressor genes in serum DNA for early detection of HCC. Experimental Design: Aberrant promoter hypermethylation was investigated in DNA isolated from the serum of 50 HCC patients who provided repeated blood samples before diagnosis and 50 controls enrolled in a cancer screen program inTaiwan. Methylation-specific PCR was used to determine the methylation status of p16, p15, and ras association domain family 1A (RASSF1A). Results: Among cases, aberrant methylation was found in serum DNA 1to 9 years before clinical HCC diagnosis. RASSF1A had the highest frequency of hypermethylation with 35 (70%) cases having at least one positive sample compared with 22 (44%) for p16 and 12 (22%) for p15. Six subjects were hypermethylation negative for all three genes. For the 50 controls, promoter hypermethylation was found in three and two subjects for RASSF1A and p16, respectively; none had methylation of p15. A receiver operating characteristic curve that included clinical risk factors (age, HBsAg status, anti^hepatitis C virus status, smoking, and alcohol status) and hypermethylation biomarkers gave an overall predictive accuracy of 89% with sensitivity and specificity 84% and 94%, respectively. Conclusions: The analysis of epigenetic changes on RASSF1A, p16, and p15 tumor suppressor genes in serum DNA may be a valuable biomarkers for early detection in populations at high risk of HCC.

Purpose – Given the importance of high performance work systems (HPWS) with respect to firm competitive advantage, this paper holds that the contribution of HPWS toward the desired outcomes for organizations may depend significantly on employee job involvement. Underpinning the argument of happy workers being productive, the purpose of this paper is to propose the critical mediator of employee well-being to explain the hypothesized multilevel relationship between HPWS and job involvement. Design/methodology/approach – The authors distributed questionnaires to the target participants. Data collected from 451 employees and 50 HR managers/professionals of 50 firms in the three major industrial categories of manufacturing, finance, and service in Taiwan. Findings – This study identifies the significance of employee well-being by incorporating the theories of planned behavior and positive psychology and provides empirical evidence for the cross-level influence of HPWS on employee well-being and job involvement. Originality/value – This study incorporates the perspective of positive psychology as an important addition to research on SHRM and performance by highlighting employee well-being as a key mediator of SHRM and job involvement.

This study describes a method of gene delivery to pancreatic islets of adult, living animals by ultrasound targeted microbubble destruction (UTMD). The technique involves incorporation of plasmids into the phospholipid shell of gas-filled microbubbles, which are then infused into rats and destroyed within the pancreatic microcirculation with ultrasound. Specific delivery of genes to islet beta cells by UTMD was achieved by using a plasmid containing a rat insulin 1 promoter (RIP), and reporter gene expression was regulated appropriately by glucose in animals that received a RIP-luciferase plasmid. To demonstrate biological efficacy, we used UTMD to deliver RIP-human insulin and RIP-hexokinase I plasmids to islets of adult rats. Delivery of the former plasmid resulted in clear increases in circulating human C-peptide and decreased blood glucose levels, whereas delivery of the latter plasmid resulted in a clear increase in hexokinase I protein expression in islets, increased insulin levels in blood, and decreased circulating glucose levels. We conclude that UTMD allows relatively noninvasive delivery of genes to pancreatic islets with an efficiency sufficient to modulate beta cell function in adult animals.diabetes ͉ gene therapy ͉ ultrasound

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Enzyme replacement therapy (ERT) is an effective treatment for several lysosomal storage disorders (LSDs). Intravenously infused enzymes are taken up by tissues through either the mannose 6-phosphate receptor (M6PR) or the mannose receptor (MR). It is generally believed that M6PR-mediated endocytosis is a key mechanism for ERT in treating LSDs that affect the non-macrophage cells of visceral organs. However, the therapeutic efficacy of MR-mediated delivery of mannose-terminated enzymes in these diseases has not been fully evaluated. We tested the effectiveness of a non-phosphorylated α-galactosidase A produced from moss (referred to as moss-aGal) in vitro and in a mouse model of Fabry disease. Endocytosis of moss-aGal was MR-dependent. Compared to agalsidase alfa, a phosphorylated form of α-galactosidase A, moss-aGal was more preferentially targeted to the kidney. Cellular localization of moss-aGal and agalsidase alfa in the heart and kidney was essentially identical. A single injection of moss-aGal led to clearance of accumulated substrate in the heart and kidney to an extent comparable to that achieved by agalsidase alfa. This study suggested that mannose-terminated enzymes may be sufficiently effective for some LSDs in which non-macrophage cells are affected, and that M6P residues may not always be a prerequisite for ERT as previously considered.Electronic supplementary materialThe online version of this article (doi:10.1007/s10545-015-9886-9) contains supplementary material, which is available to authorized users.