Pig-to-Nonhuman Primates Pancreatic Islet Xenotransplantation: An Overview

The therapy of type 1 diabetes is an open challenging problem. The restoration of normoglycemia and insulin independence in immunosuppressed type 1 diabetic recipients of islet allotransplantation has shown the potential of a cell-based diabetes therapy. Even if successful, this approach poses a problem of scarce tissue supply. Xenotransplantation can be the answer to this limited donor availability and, among possible candidate tissues for xenotransplantation, porcine islets are the closest to a future clinical application. Xenotransplantation, with pigs as donors, offers the possibility of using healthy, living, and genetically modified islets from pathogen-free animals available in unlimited number of islets. Several studies in the pig-to-nonhuman primate model demonstrated the feasibility of successful preclinical islet xenotransplantation and have provided insights into the critical events and possible mechanisms of immune recognition and rejection of xenogeneic islet grafts. Particularly promising results in the achievement of prolonged insulin independence were obtained with newly developed, genetically modified pigs islets able to produce immunoregulatory products, using different implantation sites, and new immunotherapeutic strategies. Nonetheless, further efforts are needed to generate additional safety and efficacy data in nonhuman primate models to safely translate these findings into the clinic

A local glucose-and oxygen concentration-based insulin secretion model for pancreatic islets

<p>Abstract</p> <p>Background</p> <p>Because insulin is the main regulator of glucose homeostasis, quantitative models describing the dynamics of glucose-induced insulin secretion are of obvious interest. Here, a computational model is introduced that focuses not on organism-level concentrations, but on the quantitative modeling of local, cellular-level glucose-insulin dynamics by incorporating the detailed spatial distribution of the concentrations of interest within isolated avascular pancreatic islets.</p> <p>Methods</p> <p>All nutrient consumption and hormone release rates were assumed to follow Hill-type sigmoid dependences on local concentrations. Insulin secretion rates depend on both the glucose concentration and its time-gradient, resulting in second-and first-phase responses, respectively. Since hypoxia may also be an important limiting factor in avascular islets, oxygen and cell viability considerations were also built in by incorporating and extending our previous islet cell oxygen consumption model. A finite element method (FEM) framework is used to combine reactive rates with mass transport by convection and diffusion as well as fluid-mechanics.</p> <p>Results</p> <p>The model was calibrated using experimental results from dynamic glucose-stimulated insulin release (GSIR) perifusion studies with isolated islets. Further optimization is still needed, but calculated insulin responses to stepwise increments in the incoming glucose concentration are in good agreement with existing experimental insulin release data characterizing glucose and oxygen dependence. The model makes possible the detailed description of the intraislet spatial distributions of insulin, glucose, and oxygen levels. In agreement with recent observations, modeling also suggests that smaller islets perform better when transplanted and/or encapsulated.</p> <p>Conclusions</p> <p>An insulin secretion model was implemented by coupling local consumption and release rates to calculations of the spatial distributions of all species of interest. The resulting glucose-insulin control system fits in the general framework of a sigmoid proportional-integral-derivative controller, a generalized PID controller, more suitable for biological systems, which are always nonlinear due to the maximum response being limited. Because of the general framework of the implementation, simulations can be carried out for arbitrary geometries including cultured, perifused, transplanted, and encapsulated islets.</p

Focus on collagen: in vitro systems to study fibrogenesis and antifibrosis _ state of the art

Fibrosis represents a major global disease burden, yet a potent antifibrotic compound is still not in sight. Part of the explanation for this situation is the difficulties that both academic laboratories and research and development departments in the pharmaceutical industry have been facing in re-enacting the fibrotic process in vitro for screening procedures prior to animal testing. Effective in vitro characterization of antifibrotic compounds has been hampered by cell culture settings that are lacking crucial cofactors or are not holistic representations of the biosynthetic and depositional pathway leading to the formation of an insoluble pericellular collagen matrix. In order to appreciate the task which in vitro screening of antifibrotics is up against, we will first review the fibrotic process by categorizing it into events that are upstream of collagen biosynthesis and the actual biosynthetic and depositional cascade of collagen I. We point out oversights such as the omission of vitamin C, a vital cofactor for the production of stable procollagen molecules, as well as the little known in vitro tardy procollagen processing by collagen C-proteinase/BMP-1, another reason for minimal collagen deposition in cell culture. We review current methods of cell culture and collagen quantitation vis-à-vis the high content options and requirements for normalization against cell number for meaningful data retrieval. Only when collagen has formed a fibrillar matrix that becomes cross-linked, invested with ligands, and can be remodelled and resorbed, the complete picture of fibrogenesis can be reflected in vitro. We show here how this can be achieved. A well thought-out in vitro fibrogenesis system represents the missing link between brute force chemical library screens and rational animal experimentation, thus providing both cost-effectiveness and streamlined procedures towards the development of better antifibrotic drugs

Improvement of bone allograft recolonization by adipose stem cells: impact of bone graft demineralization

Bone allografts combined with mesenchymal stem cells are proposed for bone tissue engineering but remains limited by a low degree of stem cells spreading and cellular recolonization. We postulate to demineralize bone allografts in view to improve adipose stem cells (ASCs) colonization and the bioactivity of the graft. Bone allografts (n=16) were treated for decellularization (4 groups of demineralization time: 0, 4, 8, 12 hours). The implants were compared in terms of residual calcium, mineral density and bioactivity (for BMP-2/VEGF contents). Each implant was scanned by microtomography to analyze macroporosity and open porosity. Helium pycnometry and Hg porosimetry were performed to assess the absolute density and microporosity. Bone surface analysis was assessed by X-Ray photoelectron spectroscopy and SEM. The bone graft recolonization by ASCs was studied in vitro by SEM, histology and DNA extraction at 24 hours/day 15 post-cellular seeding. Finally, ASCs combined with non-/demineralized bone matrix were implanted into the lumbar muscles of 10 nude rats (in comparison to bone grafts w/o cells) to study the osteoinductivity/angiogenicity by imagery/histology 29 days after implantation. A significant reduction of the calcium concentration (>-90%) was found in demineralized bone in comparison to native grafts as revealed by ionometry (0.27 vs. 4.1 g/L) and pQCT (0 vs. 0.39 g/ cm?). Demineralization significantly increase the macroporosity (>100μm by +13%) and the open porosity (>4 cm?/g vs. 2.1±1.0 cm?/g in comparison to the native graft,p<0.05). A significant increase of microporosity (>10 μm by +158% and <100 nm by 558%) was also found after demineralization. Helium pycnometry confirmed the correlation between the decrease of absolute density and demineralization of the bone graft (R?=0.81). A positive linear correlation between the decrease of calcium/increase of nitrogen atoms (at the bone surface) and the time of demineralization was found (R?=0.99,p<0.001). At day 15 post-incubation, a significant higher ASCs colonization of the bone graft was found for tissue demineralized during 12 hours (p<0.05). In conclusion, the demineralization of cancellous bones significantly improves the colonization by ASCs in view to return the bioactivity for bone regeneration

Geochemistry, geochronology, and fluid inclusion study of the Late Cretaceous Newton epithermal gold deposit, British Columbia

Newton is an intermediate-sulfidation epithermal gold deposit related to Late Cretaceous continental arc magmatism in south-central British Columbia. Disseminated gold mineralization occurs in quartz-sericite-altered Late Cretaceous felsic volcanic rocks, and feldspar-quartz-hornblende porphyry and quartz-feldspar porphyry intrusions. The mineralization can be divided into 3 stages: (1) disseminated pyrite with microscopic gold inclusions, and sparse quartz-pyrite ± molybdenite veins; (2) disseminated marcasite with microscopic gold inclusions and minor base metal-sulfides; and (3) polymetallic veins of pyrite-chalcopyrite-sphalerite-arsenopyrite. Re-Os dating of molybdenite from a stage 1 vein yielded an age of 72.1 ± 0.3 Ma (McClenaghan 2013). The age of the host rocks has been constrained by U-Pb dating of zircon: Late Cretaceous felsic volcanic rocks: 72.1 ± 0.6 Ma (Amarc Resources Ltd., unpublished data, reported in McClenaghan 2013); feldspar-quartz-hornblende porphyry: 72.1 ± 0.5 Ma; quartz-feldspar porphyry: 70.9 ± 0.5 Ma (Amarc Resources Ltd., unpublished data, reported in McClenaghan 2013). The mineralized rocks are intruded by a barren diorite, with an age of 69.3 ± 0.4 Ma. Fluid inclusions in quartz–pyrite ± molybdenite ± gold veins yielded an average homogenization temperature of 313° ± 51°C (n = 82) and salinity of 4.8 ± 0.9 wt.% NaCl equiv. (n = 46), suggesting that a relatively hot and saline fluid likely of magmatic origin was responsible for the first stage of mineralization. Some evidence for boiling was also observed in the veins. However, the bulk of the gold mineralization occurs as disseminations in the wallrocks, suggesting that wallrock reactions were the main control on ore deposition. Keywords: Newton, Intermediate-sulfidation, Epithermal, Gold, British ColumbiaThe accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

Anomeric specificity of hexokinase and glucokinase activities in liver and insulin-producing cells.

Conflicting data have been reported concerning the anomeric specificity of glucokinase. In the present study, liver hexokinase (Km for D-glucose 0.4 mM) displayed a higher affinity for but lower Vmax. with alpha- than with beta-D-glucose. The velocity of the reaction catalysed by liver glucokinase was higher with with beta- than with alpha-D-glucose, whatever the glucose concentration. The apparent Km of glucokinase was somewhat lower, however, with alpha- than with beta-D-glucose. Comparable results were obtained for the high-Km glucokinase-like enzymic activity present in normal pancreatic islets or insulin-producing tumoral cells. These results suggest that the anomeric specificity of glucokinase cannot account for the higher rate of glycolysis found in islets exposed to alpha- as distinct from beta-D-glucose

The coupling of metabolic to secretory events in pancreatic islets. The cytosolic redox state.

NADP-linked isocitrate dehydrogenase and malic enzyme [malate dehydrogenase (decarboxylating) (NADP)] activities were characterized in the cytosol of pancreatic islet cells. D-Glucose and L-leucine augmented the cytosolic NADPH/NADP+ ratio, as judged from the isocitrate/2-oxoglutarate and malate/pyruvate islet contents. The flow rate through the malic enzyme was judged from the output of labelled pyruvate by islets exposed to either L-[U-14C]glutamine or L-[U-14C]leucine. The cytosolic generation of NADPH, e.g. at the level of the malic enzyme, may play a role in the coupling of metabolic to secretory events in the process of nutrient-stimulated insulin release

Bioactivity of a tissue-engineered product: bridging the gap between academic and clinical studies

The translation of cell-based therapeutics from academic and fundamental sciences to clinical trial settings follows a preclinical pathway with rigorous regulatory oversights to ensure the cellmediated therapeutic effect. Although international guidelines thoroughly describe single-dose toxicity and biodistribution studies, the quantitative evaluation of the biological activity remains a major challenge for biotechnology companies and authorities. Here we discuss a strategy to demonstrate the bioactivity of an osteogenic tissue-engineered product intended to promote angiogenesis and osteogenesis in a bone defect. Osteogenic 3-dimensional (3D) grafts were manufactured as a pharmaceutical batch. A sequence of chemical treatment was applied on the native graft in view to obtained a decellularized 3D-matrix as confirmed by a reduction of >90% and 40-80% of the cellular and growth factors (VEGF, IGF1) contents, respectively. Decellularized and native grafts were implanted intra-muscularly (after cauterization of the lumbar region, n=10 nude rats) to assess the mineralization (X-ray microCT and histomorphometry on Von Kossa staining) and angiogenesis (histomorphometry on Masson’s Trichrome and Von Willebrand Factor immunostaining) at day 29 postimplantation. Human cells detection (in the explanted tissue) were also quantified after KU80/HLA type I immunostaining at the implantation site. A significant higher mineralization was found for explanted samples from the native osteogenic 3D grafts in comparison to the decellularized tissues (Bone volume/tissue volume of 2.92 ± 1.12% vs. 0.38 ± 0.59%, respectively