Evaluation of parameters for efficient purification and long-term storage of herpes simplex virus-based vectors.

Replication competent oncolytic herpes simplex virus (HSV) vectors have been used extensively to treat solid tumors with promising results. However, highly defective HSV vectors will be needed for applications that require sustained therapeutic gene expression in the absence of vector-related toxicity or inflammation. These vectors require complementing cell lines for their manufacture, creating significant challenges to achieve high yields of infectious virus particles. We recently described an improved upstream process for the production of a non-cytotoxic HSV vector for gene therapy applications. Here, we sought to optimize the downstream conditions for purification and long-term storage of the same vector, JΔNI5. We compared different methods to remove cellular impurities and concentrate the vector by monitoring both physical and biological titers, resulting in the establishment of optimal conditions for vector production. To optimize the long-term storage parameters for non-cytotoxic HSV vectors, we evaluated vector stability at low temperature and sensitivity to freeze-thaw cycles. We report that suboptimal purification and storage methods resulted in loss of vector viability. Our results describe effective and reproducible protocols for purification and storage of HSV vectors for pre-clinical studies.

A Comparative Study of Aluminium and Titanium Warm Sprayed Coatings on AZ91E Magnesium Alloy.

Aluminium (Al) and titanium (Ti) coatings were applied on AZ91E magnesium alloy using a low-pressure warm spray (WS) method. The deposition was completed using three different nitrogen flow rates (NFR) for both coatings. NFR effects on coating microstructure and other physical properties were systematically studied. Microstructural characterization was performed using scanning electron microscopy (SEM), and the porosity was estimated using two methods-image analysis and X-ray microtomography. The coating adhesion strength, wear resistance, and hardness were examined. The protective properties of the coatings were verified via a salt spray test. Decreasing NFR during coating deposition produced more dense and compact coatings. However, these conditions increased the oxidation of the powder. Al coatings showed lower hardness and wear resistance than Ti coatings, although they are more suitable for corrosion protection due to their low porosity and high compactness.

Protocol Optimization for the Production of the Non-Cytotoxic JΔNI5 HSV Vector Deficient in Expression of Immediately Early Genes.

Non-toxic herpes simplex virus (HSV) vectors can be generated by functional deletion of all immediate-early (IE) genes, providing a benign vehicle with potential for gene therapy. However, deletion of multiple IE genes raises manufacturing concerns and thus limits clinical application of these vectors. To address this issue, we previously developed a novel production cell line, called U2OS-ICP4/27, by lentiviral transduction of human osteosarcoma U2OS cells with two essential HSV IE genes, ICP4 and ICP27. To optimize the process of vector manufacturing on this platform, we evaluated several cell culture parameters of U2OS-ICP4/27 for high-titer and -quality production of non-toxic HSV vectors, revealing that the yields and functionality of these vectors can be significantly influenced by culturing conditions. We also found that several chemical compounds can enhance the replication of non-toxic HSV vectors and their release from producer cells into the supernatants. Notably, the vector produced by our optimized protocol displayed a greatly improved vector yield and quality and showed elevated transgene expression in cultures of primary dorsal root ganglion neurons. Taken together, our optimized production approach emerges as a relevant protocol for high-yield and high-quality preparation of non-toxic HSV-based gene therapy vectors.

A case of nonfunctioning pancreatic endocrine tumor with atypical imaging findings due to prominent fibrosis of the tumor stroma.

The patient, a 56-year-old woman, was found during routine checkup to have a disorder of hepatic function. Abdominal ultrasonography showed an ill-defined hypoechoic mass in the head and body of the pancreas; however, no blood-flow signal was observed within the tumor on Doppler ultrasonography. Abdominal computed tomography showed a low-density area in the arterial and portal venous phases. The lesion was visualized as an area of low signal intensity on both T1- and T2-weighted magnetic resonance images, whereas fluorodeoxyglucose positron emission tomography showed fluorodeoxyglucose accumulation in the tumor. Although a preoperative diagnosis was difficult to make, a rapid cytologic examination revealed evidence of a pancreatic endocrine tumor, and subtotal stomach-preserving pancreaticoduodenectomy with portal vein resection was performed. Histopathological examination showed tumor cell nests scattered in abundant fibrotic tissue; the tumor cells had proliferated in a cord-like fashion and showed immunostaining for chromogranin A. Staining for fibroblast activation protein α was seen in the fibroblastic cells contained within the fibrous stroma surrounding the tumor cell nests, whereas both the fibroblastic cells in the tumor and those in the stroma showed a high rate of staining for thrombospondin. We presume that tumor-associated fibroblasts were involved in the fibrosis of the tumor stroma.

Detection of segmentation cracks in top coat of thermal barrier coatings during plasma spraying by non-contact acoustic emission method.

Numerous cracks can be observed in the top coat of thermal barrier coatings (TBCs) deposited by the atmospheric plasma spraying (APS) method. These cracks can be classified into vertical and horizontal ones and they have opposite impact on the properties of TBCs. Vertical cracks reduce the residual stress in the top coat and provide strain tolerance. On the contrary, horizontal cracks trigger delamination of the top coat. However, monitoring methods of cracks generation during APS are rare even though they are strongly desired. Therefore, an in situ, non-contact and non-destructive evaluation method for this objective was developed in this study with the laser acoustic emission (AE) technique by using laser interferometers as a sensor. More AE events could be detected by introducing an improved noise reduction filter and AE event detection procedures with multiple thresholds. Generation of vertical cracks was successfully separated from horizontal cracks by a newly introduced scanning pattern of a plasma torch. Thus, generation of vertical cracks was detected with certainty by this monitoring method because AE events were detected only during spraying and a positive correlation was observed between the development degree of vertical cracks and the total AE energy in one experiment.

Warm spraying-a novel coating process based on high-velocity impact of solid particles.

In recent years, coating processes based on the impact of high-velocity solid particles such as cold spraying and aerosol deposition have been developed and attracting much industrial attention. A novel coating process called 'warm spraying' has been developed, in which coatings are formed by the high-velocity impact of solid powder particles heated to appropriate temperatures below the melting point of the powder material. The advantages of such process are as follows: (1) the critical velocity needed to form a coating can be significantly lowered by heating, (2) the degradation of feedstock powder such as oxidation can be significantly controlled compared with conventional thermal spraying where powder is molten, and (3) various coating structures can be realized from porous to dense ones by controlling the temperature and velocity of the particles. The principles and characteristics of this new process are discussed in light of other existing spray processes such as high-velocity oxy-fuel spraying and cold spraying. The gas dynamics of particle heating and acceleration by the spraying apparatus as well as the high-velocity impact phenomena of powder particles are discussed in detail. Several examples of depositing heat sensitive materials such as titanium, metallic glass, WC-Co cermet and polymers are described with potential industrial applications.