Recombinant adeno-associated virus (rAAV) shows great promise for gene therapy, however scalability, yield and quality remain significant issues. Here we describe an rAAV manufacturing strategy using ...a 'helper' adenovirus that self-inhibits its major late promoter (MLP) to truncate its own replication. Inserting a tetracycline repressor (TetR) binding site into the MLP and encoding the TetR under its transcriptional control allowed normal adenovirus replication in the presence of doxycycline but only genome amplification and early gene expression (the 'helper' functions) in its absence. Using this self-inhibiting adenovirus we demonstrate delivery of adenoviral helper functions, AAV rep and cap genes, and the rAAV genome to yield up to 30-fold more rAAV vectors compared to the helper-free plasmid approach and significant improvements in particle infectivity for a range of serotypes. This system allows significant improvements in the production of serotypes rAAV2, rAAV6, rAAV8 and rAAV9, and enables propagation of existing rAAV without transfection, a process that improves batch quality by depleting reverse packaged DNA contaminants. We propose this as a high-yielding, contaminant-free system suitable for scalable rAAV manufacture.
Helicobacter pylori is the strongest risk factor for the development of gastric cancer. Although the specific mechanisms by which this pathogen induces carcinogenesis have not been fully elucidated, ...high-expression interleukin (IL)-1β alleles are associated with increased gastric cancer risk among H. pylori-infected persons. In addition, loss of matrix metalloproteinase 7 (MMP7) increases mucosal inflammation in mouse models of epithelial injury, and we have shown that gastric inflammation is increased in H. pylori-infected MMP7(-/-) C57BL/6 mice. In this report, we define mechanisms that underpin such responses and extend these results into a genetic model of MMP7 deficiency and gastric cancer. Wild-type (WT) or MMP7(-/-) C57BL/6 mice were challenged with broth alone as an uninfected control or the H. pylori strain PMSS1. All H. pylori-challenged mice were successfully colonized. As expected, H. pylori-infected MMP7(-/-) C57BL/6 mice exhibited a significant increase in gastric inflammation compared with uninfected or infected WT C57BL/6 animals. Loss of MMP7 resulted in M1 macrophage polarization within H. pylori-infected stomachs, as assessed by Luminex technology and immunohistochemistry, and macrophages isolated from infected MMP7-deficient mice expressed significantly higher levels of the M1 macrophage marker IL-1β compared with macrophages isolated from WT mice. To extend these findings into a model of gastric cancer, hypergastrinemic WT INS-GAS or MMP7(-/-) INS-GAS mice were challenged with H. pylori strain PMSS1. Consistent with findings in the C57BL/6 model, H. pylori-infected MMP7-deficient INS-GAS mice exhibited a significant increase in gastric inflammation compared with either uninfected or infected WT INS-GAS mice. In addition, the incidence of gastric hyperplasia and dysplasia was significantly increased in H. pylori-infected MMP7(-/-) INS-GAS mice compared with infected WT INS-GAS mice, and loss of MMP7 promoted M1 macrophage polarization. These results suggest that MMP7 exerts a restrictive role on H. pylori-induced gastric injury and the development of premalignant lesions by suppressing M1 macrophage polarization.
Abstract
Vapor transmission coefficients of building materials can be determined using the cup method, known from ASTM and ISO standards. The measurement principle is simple, but the method is known ...to be problematic. In this paper, problems concerning the discrepancy between assumed and real values of relative humidity inside the cups are investigated. This concerns especially dry cups, in which desiccants are expected to produce relative humidity close to 0%. Interestingly, there are studies which suggest a much greater value should be used. To verify these claims, experiments were conducted for cups filled with calcium chloride (CaCl
2
), and saturated aqueous solution of potassium nitride (KNO
3
), covered with hemp-lime concrete samples. The study confirms earlier reports of a discrepancy between actual and assumed values of relative humidity inside the cups and shows that they might introduce significant error in the measurement of water vapor transmission coefficients.
Aim: Interspinous process device (IPD) placement is an attractive treatment option for lumbar spinal and foraminal stenosis. The goal of the treatment is to release the stress on facets joints as ...well as decompress the nerve roots by enlarging the intervertebral foramina and narrowed canal recesses. Purpose: To evaluate possible structural changes in the lumbar spine after implantation of an IPD on operated and adjacent segments. Patients and Methods: Twenty-two patients were enrolled in the study. Preoperative MRI scans of the lumbar spine evaluated recess and foraminal stenosis prior to the application of an IPD. CT exams were performed and morphometric measurements were made to assess the size of intervertebral foramina after implantation on the operated and adjacent segments. Results: Statistically significant enlargements in diameter and surface area of the intervertebral foramen were seen at the operating level. On the right and left sides, foraminal enlargement after the procedure was 1 mm in diameter. The average enlargement of the foramina surface area at the level of implantation was 10 mm.sup.2. The median interspinous distance was significantly enlarged by 3.5 mm. No significant changes in adjacent segments were observed. Clinical improvement was confirmed by the Oswestry Disability Index (ODI) and visual analog scale (VAS). Preoperative disability was reduced (mean ODI from 70.5 (12.25) to 49.5 (23.75)), as well as back pain (mean VAS from 8.0 (1.7) to 4.4 (2.6)) and pain in lower limbs (mean VAS from 7.4 (1.9) to 3.8 (2.9)). Conclusion: Decompression surgery using an IPD is effective in the treatment of lumbar foraminal and canal stenosis. It provides relief of symptoms in short-term observation through enlargement of intervertebral foramina and decompression of neural roots. It reduces overload of facet joints of the operated segment and does not decrease the size of the intervertebral foramina and disc heights of adjacent segments. Keywords: interspinous device, low lumbar back pain, lumbar decompression, spinal canal stenosis, foraminal stenosis
The article presents the results of rheological testing of Al 5754 alloy in series 5xxx, obtained for deformation parameters corresponding to the process of extrusion of large-size sections on ...presses. The effect of deformation conditions on the variations in yield stress magnitude was determined. Then, using the least squares method., the actual values of the coefficients of the mathematical model describing the rheological properties of the material under investigation were determined, thus obtaining grounds for conducting the model studies of the extrusion process based on numerical methods.
The paper presents the results of numerical modelling of AZ61 magnesium alloy hot deformation using modified ECAE method. The temperature-velocity conditions were analysed using FEM. The extrusion ...process was realised using the die with modified angular channel containing horizontal contracting zone in the material exit direction. The channels were arranged at right angle relatively to each other. The main aim of the numerical research was to determine the most favourable parameters of the extrusion that allow obtaining the products with good mechanical properties. The final product is a round rod that could be used as a material charge to further plastic deformation process.
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