Intrapleural transplantation of allogeneic pancreatic islets achieves glycemic control in a diabetic non-human primate.

Clinical islet transplantation has relied almost exclusively on intraportal administration of pancreatic islets, as it has been the only consistent approach to achieve robust graft function in human recipients. However, this approach suffers from significant loss of islet mass from a potent immediate blood-mediated inflammatory response (IBMIR) and a hypoxic environment. To avoid these negative aspects of the portal site, we explored an alternative approach in which allogeneic islets were transplanted into the intrapleural space of a non-human primate (NHP), treated with an immunosuppression regimen previously reported to secure routine survival and tolerance to allogeneic islets in NHP. Robust glycemic control and graft survival were achieved for the planned study period of >90 days. Our observations suggest the intrapleural space provides an attractive locale for islet transplantation due to its higher oxygen tension, ability to accommodate large transplant tissue volumes, and a lack of IBMIR-mediated islet damage. Our preliminary results reveal the promise of the intrapleural space as an alternative site for clinical islet transplantation in the treatment of type 1 diabetes.

Bioengineered omental transplant site promotes pancreatic islet allografts survival in non-human primates.

The transplanting islets to the liver approach suffers from an immediate posttransplant loss of islets of more than 50%, progressive graft dysfunction over time, and precludes recovery of grafts should there be serious complications such as the development of teratomas with grafts that are stem cell-derived islets (SC-islets). The omentum features an attractive extrahepatic alternative site for clinical islet transplantation. We explore an approach in which allogeneic islets are transplanted onto the omentum, which is bioengineered with a plasma-thrombin biodegradable matrix in three diabetic non-human primates (NHPs). Within 1 week posttransplant, each transplanted NHP achieves normoglycemia and insulin independence and remains stable until termination of the experiment. Success was achieved in each case with islets recovered from a single NHP donor. Histology demonstrates robust revascularization and reinnervation of the graft. This preclinical study can inform the development of strategies for ß cell replacement including the use of SC-islets or other types of novel cells in clinical settings.

FasL microgels induce immune acceptance of islet allografts in nonhuman primates.

Islet transplantation to treat insulin-dependent diabetes is greatly limited by the need for maintenance immunosuppression. We report a strategy through which cotransplantation of allogeneic islets and streptavidin (SA)-FasL-presenting microgels to the omentum under transient rapamycin monotherapy resulted in robust glycemic control, sustained C-peptide levels, and graft survival in diabetic nonhuman primates for >6 months. Surgical extraction of the graft resulted in prompt hyperglycemia. In contrast, animals receiving microgels without SA-FasL under the same rapamycin regimen rejected islet grafts acutely. Graft survival was associated with increased number of FoxP3+ cells in the graft site with no significant changes in T cell systemic frequencies or responses to donor and third-party antigens, indicating localized tolerance. Recipients of SA-FasL microgels exhibited normal liver and kidney metabolic function, demonstrating safety. This localized immunomodulatory strategy succeeded with unmodified islets and does not require long-term immunosuppression, showing translational potential in ß cell replacement for treating type 1 diabetes.

Establishment and maintenance of genomic methylation patterns in mouse embryonic stem cells by Dnmt3a and Dnmt3b.

We have previously shown that the DNA methyltransferases Dnmt3a and Dnmt3b carry out de novo methylation of the mouse genome during early postimplantation development and of maternally imprinted genes in the oocyte. In the present study, we demonstrate that Dnmt3a and Dnmt3b are also essential for the stable inheritance, or "maintenance," of DNA methylation patterns. Inactivation of both Dnmt3a and Dnmt3b in embryonic stem (ES) cells results in progressive loss of methylation in various repeats and single-copy genes. Interestingly, introduction of the Dnmt3a, Dnmt3a2, and Dnmt3b1 isoforms back into highly demethylated mutant ES cells restores genomic methylation patterns; these isoforms appear to have both common and distinct DNA targets, but they all fail to restore the maternal methylation imprints. In contrast, overexpression of Dnmt1 and Dnmt3b3 failed to restore DNA methylation patterns due to their inability to catalyze de novo methylation in vivo. We also show that hypermethylation of genomic DNA by Dnmt3a and Dnmt3b is necessary for ES cells to form teratomas in nude mice. These results indicate that genomic methylation patterns are determined partly through differential expression of different Dnmt3a and Dnmt3b isoforms.

A peroxisomal APX from Puccinellia tenuiflora improves the abiotic stress tolerance of transgenic Arabidopsis thaliana through decreasing of H2O2 accumulation.

Ascorbate peroxidase (APX, EC 1.11.1.11) is one of the major members of the ROS scavenging system that plays an important role in improving saline-alkali tolerance. Puccinellia tenuiflora, as a perennial wild grass, is able to grow in extreme saline-alkali soil environments. In this study, we investigated the relationship between the P. tenuiflora ascorbate peroxidase (PutAPX) gene and saline-alkali tolerance. A phylogenetic analysis indicated that PutAPX is closely related to AtAPX3 and OsAPX4 and that these genes are on the same branch. The PutAPX-GFP fusion protein is located in the peroxisome in onion epidermal cells. The transcriptional expression of PutAPX increased with prolonged exposure to NaCl, NaHCO3, PEG6000 and H2O2 stresses in P. tenuiflora. The overexpression of PutAPX in Arabidopsis thaliana significantly increased the tolerance of plants treated with 150 and 175mM NaCl and decreased the extent of lipid peroxidation. The transgenic seedlings presented higher chlorophyll content than wild type (WT) seedlings treated with 1, 3, and 5mM NaHCO3 and 3mM H2O2. The DAB staining results revealed that the H2O2 content in transgenic seedlings was significantly lower than that in WT plants under both normal conditions and 200mM NaCl stress. Moreover, the expression of APX proteins and enzyme activity in the transgenic seedlings increased to level that were greater than twofold higher than those found in WT plants exposed to 200mM NaCl. The saline-alkali tolerance conferred by the PutAPX gene may provide a reliable basis for the use of molecular breeding techniques to improve plant tolerance and obtain a better understanding of the physiological mechanism of anti-oxidative and ROS stresses.