New intranasal and injectable gene therapy for healthy life extension.

As the global elderly population grows, it is socioeconomically and medically critical to provide diverse and effective means of mitigating the impact of aging on human health. Previous studies showed that the adeno-associated virus (AAV) vector induced overexpression of certain proteins, which can suppress or reverse the effects of aging in animal models. In our study, we sought to determine whether the high-capacity cytomegalovirus vector (CMV) can be an effective and safe gene delivery method for two such protective factors: telomerase reverse transcriptase (TERT) and follistatin (FST). We found that the mouse cytomegalovirus (MCMV) carrying exogenous TERT or FST (MCMVTERT or MCMVFST) extended median lifespan by 41.4% and 32.5%, respectively. We report CMV being used successfully as both an intranasal and injectable gene therapy system to extend longevity. Specifically, this treatment significantly improved glucose tolerance, physical performance, as well as preventing body mass loss and alopecia. Further, telomere shortening associated with aging was ameliorated by TERT and mitochondrial structure deterioration was halted in both treatments. Intranasal and injectable preparations performed equally well in safely and efficiently delivering gene therapy to multiple organs, with long-lasting benefits and without carcinogenicity or unwanted side effects. Translating this research to humans could have significant benefits associated with quality of life and an increased health span.

HOW TO MEASURE THE LARGEST BASAL DIMENSION OF CHOROIDAL MELANOMA: A MATHEMATICAL STUDY.

PURPOSE: To identify the most accurate ultrasonographic technique to measure the largest basal dimension (LBD) of choroidal melanoma. METHODS: B-scan ultrasound images were retrospectively reviewed in 99 eyes of 99 choroidal melanoma patients. The LBD was measured using one, two, and three straight lines along the inner and outer sclera. Theoretical arc length, calculated using trigonometry formulas based on the spherical model with the axial length as the sphere diameter, was used for comparisons with the actual measurements using straight lines. RESULTS: For straight-line measurements in the inner sclera, the lowest error was found when using two straight-line measurements (P = 0.118). Differences in measurement using one-segment or three-segment measurements as compared with the theoretical arc length were found to be statistically significant (P < 0.001 in both cases). For tumors with LBD smaller than 12 mm, the absolute error, compared with the theoretical arc length, was smaller than 1 mm. In the outer sclera, the smallest errors were also found for measurements using two straight-line segments; however, it was statistically different than the theoretical inner arc length (P < 0.001). CONCLUSION: When using ultrasound to estimate LBD of ocular tumors, 2 straight-line measurements should be used when LBD is larger than 12 mm. For tumors with LBD smaller than 12 mm, measurements using 1 straight-line segment can provide accurate estimates.

Gene expression in mouse ovarian follicle development in vivo versus an ex vivo alginate culture system.

Ovarian follicle maturation results from a complex interplay of endocrine, paracrine, and direct cell-cell interactions. This study compared the dynamic expression of key developmental genes during folliculogenesis in vivo and during in vitro culture in a 3D alginate hydrogel system. Candidate gene expression profiles were measured within mouse two-layered secondary follicles, multi-layered secondary follicles, and cumulus-oocyte complexes (COCs). The expression of 20 genes involved in endocrine communication, growth signaling, and oocyte development was investigated by real-time PCR. Gene product levels were compared between i) follicles of similar stage and ii) COCs derived either in vivo or by in vitro culture. For follicles cultured for 4 days, the expression pattern and the expression level of 12 genes were the same in vivo and in vitro. Some endocrine (cytochrome P450, family 19, subfamily A, polypeptide 1 (Cyp19a1) and inhibin ßA subunit (Inhba)) and growth-related genes (bone morphogenetic protein 15 (Bmp15), kit ligand (Kitl), and transforming growth factor ß receptor 2 (Tgfbr2)) were downregulated relative to in vivo follicles. For COCs obtained from cultured follicles, endocrine-related genes (inhibin α-subunit (Inha) and Inhba) had increased expression relative to in vivo counterparts, whereas growth-related genes (Bmp15, growth differentiation factor 9, and kit oncogene (Kit)) and zona pellucida genes were decreased. However, most of the oocyte-specific genes (e.g. factor in the germline α (Figla), jagged 1 (Jag1), and Nlrp5 (Mater)) were expressed in vitro at the same level and with the same pattern as in vivo-derived follicles. These studies establish the similarities and differences between in vivo and in vitro cultured follicles, guiding the creation of environments that maximize follicle development and oocyte quality.

Biosimilar Gene Therapy: Investigational Assessment of Secukinumab Gene Therapy.

OBJECTIVE: Tumor necrosis factor-alpha (TNF-α), checkpoint inhibitors, and interleukin-17 (IL-17) are critical targets in inflammation and autoimmune diseases. Monoclonal antibodies (mAbs) have a successful portfolio in the treatment of chronic diseases. With the current progress in stem cells and gene therapy technologies, there is the promise of replacing costly mAbs production in bioreactors with a more direct and cost-effective production method inside the patient's cells. In this paper we examine the results of an investigational assessment of secukinumab gene therapy. MATERIALS AND METHODS: In this experimental study, the DNA sequence of the heavy and light chains of secukinumab antibodies were cloned in a lentiviral vector. Human chorionic villous mesenchymal stem cells (CMSCs) were isolated and characterized. After lentiviral packaging and titration, part of the recombinant viruses was used for transduction of the CMSCs and the other part were applied for systemic gene therapy. The engineered stem cells and recombinant viruses were applied for ex vivo and in vivo gene therapy, respectively, in different groups of rat models. In vitro and in vivo secukinumab expression was confirmed with quantitative real-time polymerase chain reaction (qRT-PCR), western blot, and ELISA by considering the approved secukinumab as the standard reference. RESULTS: Cell differentiation assays and flow cytometry of standard biomarkers confirmed the multipotency of the CMSCs. Western blot and qRT-PCR confirmed in vitro gene expression of secukinumab at both the mRNA and protein level. ELISA testing of serum from treated rat models confirmed mAb overexpression for both in vivo and ex vivo gene therapies. CONCLUSION: In this study, a lentiviral-mediated ex vivo and in vivo gene therapy was developed to provide a moderate dose of secukinumab in rat models. Biosimilar gene therapy is an attractive approach for the treatment of autoimmune disorders, cancers and other chronic diseases.

Size Matters for Interplicata Diameter: A Case-Control Study of Plateau Iris.

PURPOSE: Ultrasound biomicroscopy (UBM) has been used to characterize anterior segment dimensions in plateau iris configuration (PIC), but transverse measurements between the recesses of the ciliary sulcus (sulcus-to-sulcus diameter [STSD]) and the ciliary body processes (interplicata diameter [IPD]) have not been reported. We measured STSD and IPD and compared these among eyes with PIC, primary angle closure (PAC), and control eyes with open angles. DESIGN: Retrospective, cross-sectional clinical study. PARTICIPANTS: Sixty-nine participants, 37 PIC, 13 PAC, and 19 controls. METHODS: We searched our clinical UBM database for PAC and PIC cases. Controls were assembled by reviewing images obtained for surveillance of ocular surface lesions. Anterior segment measurements were performed using the UBM digital caliper tool. Robust-fit ANOVA identified among-group differences. Pairwise t tests were used to test the significance of between-group differences. MAIN OUTCOME MEASURES: Anterior chamber depth (ACD), angle opening distance (AOD), ciliary body area and thickness, iris area, horizontal and vertical STSD, and horizontal and vertical IPD. RESULTS: Fifty-five left eyes were analyzed (30 PIC, 10 PAC, and 15 controls). ACD was smaller in PAC than in PIC and control eyes (P < 0.05 for PIC vs. PAC; P < 0.01 for control vs. PAC). Mean AOD was smaller in PIC than controls (P < 0.05) and smaller in PAC than PIC (P < 0.001). Vertical STSD was smaller in both PAC and PIC than controls (P = 0.04 for PIC vs. control; P < 0.01 for PAC vs. control). Horizontal STSD was smaller in PIC than controls (P = 0.02). Vertical IPD was smaller in PIC than controls (P = 0.04) and smaller in PAC than PIC eyes (P = 0.02). Horizontal IPD was smaller in PIC and PAC than controls (P = 0.03 for PIC vs. control; P < 0.01 for PAC vs. control). CONCLUSIONS: STSD and IPD are narrower in PIC and PAC than in healthy eyes. Further studies that examine the ratio of white-to-white cornea diameter to the IPD may provide a mechanism for reported cases of in-the-bag uveitis-glaucoma-hyphema syndrome in PIC.

Mechanisms for in-the-bag uveitis-glaucoma-hyphema syndrome.

We propose 2 mechanisms of uveitis-glaucoma-hyphema (UGH) syndrome in 2 patients with intracapsular or in-the-bag single-piece acrylic intraocular lenses (IOLs). In the first case, pseudophacodonesis secondary to zonular laxity from pseudoexfoliation syndrome caused chafing of the posterior iris by the square-edged haptic. In the second case, focal capsular fibrosis around the square-edged haptics combined with anteriorly rotated ciliary processes in plateau iris configuration caused points of chafing. Extensive capsular fibrosis of the haptic in both cases precluded IOL exchange. In the first case, a capsular tension ring redistributed zonular tension and reduced symptoms. In the second case, endoscopic cyclophotocoagulation relieved areas of chafing and resolved symptoms. In-the-bag square-edged haptics of single-piece acrylic IOLs are a potential source of iridociliary chafing in certain situations. The mechanisms observed here should be considered to promptly diagnose and treat UGH.

Spatial analysis of 3' phosphoinositide signaling in living fibroblasts, III: influence of cell morphology and morphological Polarity.

Activation of phosphoinositide (PI) 3-kinase is a required signaling pathway in fibroblast migration directed by platelet-derived growth factor. The pattern of 3' PI lipids in the plasma membrane, integrating local PI 3-kinase activity as well as 3' PI diffusion and turnover, influences the spatiotemporal regulation of the cytoskeleton. In fibroblasts stimulated uniformly with platelet-derived growth factor, visualized using total internal reflection fluorescence microscopy, we consistently observed localized regions with significantly higher or lower 3' PI levels than adjacent regions (hot and cold spots, respectively). A typical cell contained multiple hot spots, coinciding with apparent leading edge structures, and at most one cold spot at the rear. Using a framework for finite-element modeling with actual cell contact area geometries, we find that although the 3' PI pattern is affected by irregular contact area shape, cell morphology alone cannot explain the presence of hot or cold spots. Our results and analysis instead suggest that these regions reflect different local 3' PI dynamics, specifically through a combination of mechanisms: enhanced PI 3-kinase activity, reduced 3' PI turnover, and possibly slow/constrained 3' PI diffusion. The morphological polarity of the cell may thus bias 3' PI signaling to promote persistent migration in fibroblasts.