Correlation between Topographic Vessel Density and Retinal Thickness Changes in Patients with Diabetic Macular Edema Treated with Anti-VEGF Therapy: Is It a Suitable OCTA Biomarker?

The objective of this study was to determine the correlation between topographic vessel density (VD) and retinal thickness (RT) reductions induced by vascular endothelial growth factor inhibitors (anti-VEGF) in patients with diabetic macular edema (DME) using optical coherence tomography angiography (OCTA). This was a prospective, interventional case series. VD and RT measurements were separately taken in four parafoveal subfields at baseline and after six months of treatment. This correlation was statistically assessed using Spearman's rho correlation coefficient after adjustment for multiple comparisons. The study included a total of 48 eyes in the final analysis. Mean VD decreased from baseline to month 6 (from 45.2 (+/- 3.5) to 44.6% (+/- 3.2) in the superficial capillary plexus and from 50 (+/- 3.3) to 49% (+/- 3.9) in the deep capillary plexus). Statistically significant reductions in RT were observed in all ETDRS sectors (p < 0.0001). No significant association was found between RT and VD, even when analyzing responders and non-responders separately. After six months of anti-VEGF treatment, no significant correlation was observed between the topographic VD and RT values. These findings suggest that reductions in VD values may not solely result from a reduction in microaneurysms, also being affected by the repositioning of displaced vessels due to edema and a reduction in their caliber. Therefore, VD changes may not be a suitable indirect OCTA biomarker of microaneurysm turnover and treatment response

Intravoxel Incoherent Motion Metrics as Potential Biomarkers for Survival in Glioblastoma.

Intravoxel incoherent motion (IVIM) is an MRI technique with potential applications in measuring brain tumor perfusion, but its clinical impact remains to be determined. We assessed the usefulness of IVIM-metrics in predicting survival in newly diagnosed glioblastoma. Fifteen patients with glioblastoma underwent MRI including spin-echo echo-planar DWI using 13 b-values ranging from 0 to 1000 s/mm2. Parametric maps for diffusion coefficient (D), pseudodiffusion coefficient (D*), and perfusion fraction (f) were generated for contrast-enhancing regions (CER) and non-enhancing regions (NCER). Regions of interest were manually drawn in regions of maximum f and on the corresponding dynamic susceptibility contrast images. Prognostic factors were evaluated by Kaplan-Meier survival and Cox proportional hazards analyses. We found that fCER and D*CER correlated with rCBFCER. The best cutoffs for 6-month survival were fCER&gt;9.86% and D*CER&gt;21.712 x10-3mm2/s (100% sensitivity, 71.4% specificity, 100% and 80% positive predictive values, and 80% and 100% negative predictive values; AUC:0.893 and 0.857, respectively). Treatment yielded the highest hazard ratio (5.484; 95% CI: 1.162-25.88; AUC: 0.723; P = 0.031); fCER combined with treatment predicted survival with 100% accuracy. The IVIM-metrics fCER and D*CER are promising biomarkers of 6-month survival in newly diagnosed glioblastoma

Combined Small Interfering RNA Therapy and In Vivo Magnetic Resonance Imaging in Islet Transplantation

OBJECTIVE Recent advances in human islet transplantation are hampered by significant graft loss shortly after transplantation and inability to follow islet fate directly. Both issues were addressed by utilizing a dual-purpose therapy/imaging small interfering RNA (siRNA)-nanoparticle probe targeting apoptotic-related gene caspase-3. We expect that treatment with the probe would result in significantly better survival of transplanted islets, which could be monitored by in vivo magnetic resonance imaging (MRI). RESEARCH DESIGN AND METHODS We synthesized a probe consisting of therapeutic (siRNA to human caspase-3) and imaging (magnetic iron oxide nanoparticles, MN) moieties. In vitro testing of the probe included serum starvation of the islets followed by treatment with the probe. Caspase-3 gene silencing and protein expression were determined by RT-PCR and Western blot, respectively. In vivo studies included serial MRI of NOD-SCID mice transplanted with MN-small interfering (si)Caspase-3–labeled human islets under the left kidney capsule and MN-treated islets under the right kidney capsule. RESULTS Treatment with MN-siCaspase-3 probe resulted in decrease of mRNA and protein expression in serum-starved islets compared with controls. In vivo MRI showed that there were significant differences in the relative volume change between MN-siCaspase-3–treated grafts and MN-labeled grafts. Histology revealed decreased caspase-3 expression and cell apoptosis in MN-siCaspase-3–treated grafts compared with the control side. CONCLUSIONS Our data show the feasibility of combining siRNA therapy and in vivo monitoring of transplanted islets in mice. We observed a protective effect of MN-siCaspase-3 in treated islets both in vitro and in vivo. This study could potentially aid in increasing the success of clinical islet transplantation

Expression of the NH2-Terminal Fragment of RasGAP in Pancreatic β-Cells Increases Their Resistance to Stresses and Protects Mice From Diabetes

OBJECTIVE: Our laboratory has previously established in vitro that a caspase-generated RasGAP NH(2)-terminal moiety, called fragment N, potently protects cells, including insulinomas, from apoptotic stress. We aimed to determine whether fragment N can increase the resistance of pancreatic beta-cells in a physiological setting. RESEARCH DESIGN AND METHODS: A mouse line, called rat insulin promoter (RIP)-N, was generated that bears a transgene containing the rat insulin promoter followed by the cDNA-encoding fragment N. The histology, functionality, and resistance to stress of RIP-N islets were then assessed. RESULTS: Pancreatic beta-cells of RIP-N mice express fragment N, activate Akt, and block nuclear factor kappaB activity without affecting islet cell proliferation or the morphology and cellular composition of islets. Intraperitoneal glucose tolerance tests revealed that RIP-N mice control their glycemia similarly as wild-type mice throughout their lifespan. Moreover, islets isolated from RIP-N mice showed normal glucose-induced insulin secretory capacities. They, however, displayed increased resistance to apoptosis induced by a series of stresses including inflammatory cytokines, fatty acids, and hyperglycemia. RIP-N mice were also protected from multiple low-dose streptozotocin-induced diabetes, and this was associated with reduced in vivo beta-cell apoptosis. CONCLUSIONS: Fragment N efficiently increases the overall resistance of beta-cells to noxious stimuli without interfering with the physiological functions of the cells. Fragment N and the pathway it regulates represent, therefore, a potential target for the development of antidiabetes tools

Dissecting conformational contributions to glycosidase catalysis and inhibition

Glycoside hydrolases (GHs) are classified into >100 sequence-based families. These enzymes process a wide variety of complex carbohydrates with varying stereochemistry at the anomeric and other ring positions. The shapes that these sugars adopt upon binding to their cognate GHs, and the conformational changes that occur along the catalysis reaction coordinate is termed the conformational itinerary. Efforts to define the conformational itineraries of GHs have focussed upon the critical points of the reaction: substrate-bound (Michaelis), transition state, intermediate (if relevant) and product-bound. Recent approaches to defining conformational itineraries that marry X-ray crystallography of enzymes bound to ligands that mimic the critical points, along with advanced computational methods and kinetic isotope effects are discussed

Human Mesenchymal Stem Cells Protect Human Islets from Pro-Inflammatory Cytokines

Transplantation of human islets is an attractive alternative to daily insulin injections for patients with type 1 diabetes. However, the majority of islet recipients lose graft function within five years. Inflammation is a primary contributor to graft loss, and inhibiting pro-inflammatory cytokine activity can reverse inflammation mediated dysfunction of islet grafts. As mesenchymal stem cells (MSCs) possess numerous immunoregulatory properties, we hypothesized that MSCs could protect human islets from pro-inflammatory cytokines. Five hundred human islets were co-cultured with 0.5 or 1.0×106 human MSCs derived from bone marrow or pancreas for 24 hours followed by 48 hour exposure to interferon-γ, tumor necrosis factor-α and interleukin 1β. Controls include islets cultured alone (± cytokines) and with human dermal fibroblasts (± cytokines). For all conditions, glucose stimulated insulin secretion (GSIS), total islet cellular insulin content, islet β cell apoptosis, and potential cytoprotective factors secreted in the culture media were determined. Cytokine exposure disrupted human islet GSIS based on stimulation index and percentage insulin secretion. Conversely, culture with 1.0×106 bMSCs preserved GSIS from cytokine treated islets. Protective effects were not observed with fibroblasts, indicating that preservation of human islet GSIS after exposure to pro-inflammatory cytokines is MSC dependent. Islet β cell apoptosis was observed in the presence of cytokines; however, culture of bMSCs with islets prevented β cell apoptosis after cytokine treatment. Hepatocyte growth factor (HGF) as well as matrix metalloproteinases 2 and 9 were also identified as putative secreted cytoprotective factors; however, other secreted factors likely play a role in protection. This study, therefore, demonstrates that MSCs may be beneficial for islet engraftment by promoting cell survival and reduced inflammation

Fusing simulation and experiment: The effect of mutations on the structure and activity of the influenza fusion peptide

During the infection process, the influenza fusion peptide (FP) inserts into the host membrane, playing a crucial role in the fusion process between the viral and host membranes. In this work we used a combination of simulation and experimental techniques to analyse the molecular details of this process, which are largely unknown. Although the FP structure has been obtained by NMR in detergent micelles, there is no atomic structure information in membranes. To answer this question, we performed bias-exchange metadynamics (BE-META) simulations, which showed that the lowest energy states of the membrane-inserted FP correspond to helical-hairpin conformations similar to that observed in micelles. BE-META simulations of the G1V, W14A, G12A/G13A and G4A/G8A/G16A/G20A mutants revealed that all the mutations affect the peptide's free energy landscape. A FRET-based analysis showed that all the mutants had a reduced fusogenic activity relative to the WT, in particular the mutants G12A/G13A and G4A/G8A/G16A/G20A. According to our results, one of the major causes of the lower activity of these mutants is their lower membrane affinity, which results in a lower concentration of peptide in the bilayer. These findings contribute to a better understanding of the influenza fusion process and open new routes for future studies