Real-time resolution of point mutations that cause phenovariance in mice.

With the wide availability of massively parallel sequencing technologies, genetic mapping has become the rate limiting step in mammalian forward genetics. Here we introduce a method for real-time identification of N-ethyl-N-nitrosourea-induced mutations that cause phenotypes in mice. All mutations are identified by whole exome G1 progenitor sequencing and their zygosity is established in G2/G3 mice before phenotypic assessment. Quantitative and qualitative traits, including lethal effects, in single or multiple combined pedigrees are then analyzed with Linkage Analyzer, a software program that detects significant linkage between individual mutations and aberrant phenotypic scores and presents processed data as Manhattan plots. As multiple alleles of genes are acquired through mutagenesis, pooled "superpedigrees" are created to analyze the effects. Our method is distinguished from conventional forward genetic methods because it permits (1) unbiased declaration of mappable phenotypes, including those that are incompletely penetrant (2), automated identification of causative mutations concurrent with phenotypic screening, without the need to outcross mutant mice to another strain and backcross them, and (3) exclusion of genes not involved in phenotypes of interest. We validated our approach and Linkage Analyzer for the identification of 47 mutations in 45 previously known genes causative for adaptive immune phenotypes; our analysis also implicated 474 genes not previously associated with immune function. The method described here permits forward genetic analysis in mice, limited only by the rates of mutant production and screening.

Improving allogeneic islet transplantation by suppressing Th17 and enhancing Treg with histone deacetylase inhibitors.

Islet transplantation is a new treatment for achieving insulin independence for patients with severe diabetes. However, major drawbacks of this treatment are the long graft survival, the necessity for immunosuppressive drugs, and the efficacy of transplantation. Donor-specific transfusion (DST) has been shown to reduce rejection after organ transplantation, potentially through enhanced regulatory T-cell (Treg) activity. However, recent findings have shown that activated Treg can be converted into Th17 cells. We focused on histone deacetylase inhibitors (HDACi) because it was reported that inhibition of HDAC activity prevented Treg differentiation into IL17-producing cells. We therefore sought to enhance Treg while suppressing Th17 cells using DST with HDACi to prolong graft survival. To stimulate Treg by DST, we used donor splenocytes. In DST with HDACi group, Foxp3 mRNA expression and Treg population increased in the thymus and spleen, whereas Th17 population decreased. qPCR analysis of lymphocyte mRNA indicated that Foxp3, IL-10, and TGF-b expression increased. However, interleukin 17a, Stat3 (Th17), and IFN-g expression decreased in DST + HDACi group, relative to DST alone. Moreover, DST treated with HDACi prolonged graft survival relative to controls in mice islet transplantation. DST with HDACi may therefore have utility in islet transplantation.

Low temperature condition prevents hypoxia-induced islet cell damage and HMGB1 release in a mouse model.

One of the major issues in clinical islet transplantation is the poor efficacy of islet isolation. During pancreas preservation and islet isolation, islets suffer from hypoxia as islets are highly sensitive to hypoxic conditions.Cold preservation has been applied to minimize hypoxia-induced cell damage during organ preservation.However, the studies related to hypoxia-induced islet cell damage during islet isolation are limited. Recently,we demonstrated that mouse islets contain high levels of high-mobility group box 1 protein (HMGB1), and during proinflammatory cytokine-induced damage, islets release HMGB1 outside the cell. The released HMGB1 is involved in the initial events of early islet loss. In the present study, we hypothesize that low temperature conditions could prevent both hypoxia induced islet cell damage and HMGB1 release from islets in a mouse model. Isolated mouse islets underwent normoxic condition (95% air and 5% CO(2)) at 37°C or hypoxic conditions (1% O(2), 5% CO(2), and 94% N(2)) at 37°C (hypoxia-37°C islets), 22°C (hypoxia-22°C islets), or 4°C (hypoxia-4°C islets) for 12 h. In vitro and in vivo viability and functionality tests were performed. HMGB1, IL-6, G-CSF, KC, RANTES, MCP-1, and MIP-1α levels in the medium were measured. Low temperature conditions substantially reduced hypoxia-induced necrosis (p < 0.05) and apoptosis (p < 0.05). In addition, low temperature islet culture significantly increased the insulin secretion from islets by high glucose stimulation (p < 0.05). All of the recipient mice reversed diabetes after receiving the hypoxia-4°C islets but not after receipt of hypoxia-37°C or 22°C islets. The amounts of released HMGB1, IL-6, G-CSF, KC, RANTES, MCP-1, and MIP-1α were significantly reduced in the hypoxia-4°C islets compared to those of the hypoxia-37°C islets (p < 0.05). In conclusion, low temperature conditions could prevent hypoxia-induced islet cell damage, inflammatory reactions in islets, and HMGB1 release and expression. Low temperature conditions should improve the efficacy of isolated islets.

Improving efficacy of clinical islet transplantation with iodixanol-based islet purification, thymoglobulin induction, and blockage of IL-1ß and TNF-α.

Poor efficacy is one of the issues for clinical islet transplantation. Recently, we demonstrated that pancreatic ductal preservation significantly improved the success rate of islet isolation; however, two transplants were necessary to achieve insulin independence. In this study, we introduced iodixanol-based purification, thymoglobulin induction, and double blockage of IL-1ß and TNF-α as well as sirolimus-free immunosuppression to improve the efficacy of clinical islet transplantation. Nine clinical-grade human pancreata were procured. Pancreatic ductal preservation was performed using ET-Kyoto solution in all cases. When the isolated islets met the clinical criteria, they were transplanted. We utilized two methods of immunosuppression and anti-inflammation. The first protocol prescribed daclizumab for induction, then sirolimus and tacrolimus to maintain immunosuppression. The second protocol used thymoglobulin for induction and tacrolimus and mycophenolate mofetil to maintain immunosuppression. Eternacept and anakinra were administered as anti-inflammatory drugs. The total amount of insulin required, HbA1c, and the SUITO index were determined to analyze and compare the results of transplantation. All isolated islet preparations (9/9) met the criteria for clinical transplantation, and they were transplanted into six type 1 diabetic patients. All patients achieved insulin independence with normal HbA1c levels; however, the first protocol required two islet infusions (N = 3) and the second protocol only required a single infusion (N = 3). The average SUITO index, at 1 month after a single-donor islet transplantation, was significantly higher in the second protocol (49.6 ± 8.3 vs. 19.3 ± 6.3, p < 0.05). Pancreatic ductal preservation, iodixanol-based purification combined with thymoglobulin induction, and blockage of IL-1ß and TNF-α as well as sirolimus-free immunosuppression dramatically improved the efficacy of clinical islet transplantations. This protocol enabled us to perform successful single-donor islet transplantations. Further large-scale studies are necessary to confirm these results and clarify the mechanism of each component.