Clinically available immunosuppression averts rejection but not systemic inflammation after porcine islet xenotransplant in cynomolgus macaques.

A safe, efficacious, and clinically applicable immunosuppressive regimen is necessary for islet xenotransplantation to become a viable treatment option for diabetes. We performed intraportal transplants of wild-type adult porcine islets in 25 streptozotocin-diabetic cynomolgus monkeys. Islet engraftment was good in 21, partial in 3, and poor in 1 recipient. Median xenograft survival was 25 days with rapamycin and CTLA4Ig immunosuppression. Adding basiliximab induction and maintenance tacrolimus to the base regimen significantly extended median graft survival to 147 days (p < .0001), with three animals maintaining insulin-free xenograft survival for 265, 282, and 288 days. We demonstrate that this regimen suppresses non-Gal anti-pig antibody responses, circulating effector memory T cell expansion, effector function, and infiltration of the graft. However, a chronic systemic inflammatory state manifested in the majority of recipients with long-term graft survival indicated by increased neutrophil to lymphocyte ratio, IL-6, MCP-1, CD40, and CRP expression. This suggests that this immunosuppression regimen fails to regulate innate immunity and resulting inflammation is significantly associated with increased incidence and severity of adverse events making this regimen unacceptable for translation. Additional studies are needed to optimize a maintenance regimen for regulating the innate inflammatory response.

Long-term efficacy and safety of porcine islet macrobeads in nonimmunosuppressed diabetic cynomolgus macaques.

Although human islet transplantation has proven to provide clinical benefits, especially the near complete amelioration of hypoglycemia, the supply of human islets is limited and insufficient to meet the needs of all people that could benefit from islet transplantation. Porcine islets, secreting insulin nearly identical to that of human insulin, have been proposed as a viable supply of unlimited islets. Further, encapsulation of the porcine islets has been shown to reduce or eliminate the use of immunosuppressive therapy that would be required to prevent rejection of the foreign islet tissue. The goal of the current study was to determine the long-term safety and efficacy of agarose encapsulated porcine islets (macrobeads) in diabetic cynomolgus macaques, in a study emulating a proposed IND trial in which daily exogenous insulin therapy would be reduced by 50% with no loss of glucose regulation. Four of six animals implanted with macrobeads demonstrated ≥ 30% reduction in insulin requirements in year 1 of follow-up. Animals were followed for 2, 3.5, and 7.4 years with no serious adverse events, mortality or evidence of pathogen transmission. This study supports the continued pursuit of encapsulated porcine islet therapy as a promising treatment option for diabetes mellitus.

Cross-validation of commercial enzyme-linked immunosorbent assay and radioimmunoassay for porcine C-peptide concentration measurements in non-human primate serum.

BACKGROUND: C-peptide concentration is widely used as a marker of insulin secretion and is especially relevant in evaluating islet graft function following transplantation, because its measurement is not confounded by the presence of exogenous insulin. To address the shortage of human islet donors, the use of porcine islets has been proposed as a possible solution and the stringent pig-to-non-human primate (NHP) model is often the most relevant for pre-clinical evaluation of the potential for diabetes reversal resulting from an islet xenograft. The Millipore radioimmunoassay (RIA) was exclusively used to measure porcine C-peptide (PCP) until 2013 when the assay was discontinued and subsequently a commercially available enzyme-linked immunosorbent assay (ELISA) from Mercodia has been widely adopted. Both assays have been used in pre-clinical trials evaluating the therapeutic potential of xenograft products in reversing diabetes in the pig-to-NHP model, to interpret data in a comparable way it may be useful to perform a harmonization of C-peptide measurements. METHODS: We performed a method comparison by determining the PCP concentration in 620 serum samples collected from 20 diabetic cynomolgus macaques transplanted with adult porcine islets. All analyses were performed according to manufacturer instructions. RESULTS: With both assays, we demonstrated an acceptable detection limit, precision, and recovery. Linearity of the ELISA met acceptance criteria at all concentrations tested while linearity of the RIA only met acceptance criteria at five of the eight concentrations tested. The RIA had a detection limit of 0.16 ng/mL, and recovery ranged from 82% to 96% and met linearity acceptance criteria at 0.35 ng/mL and from 0.78 to 2.33 ng/mL. The ELISA had a detection limit of 0.03 ng/mL, and recovery ranged from 81% to 115% and met linearity acceptance criteria from 0.08 to 0.85 ng/mL. Both assays had intra-assay precision <11% and inter-assay precision <14%. PCP concentration measured by ELISA demonstrated a significant correlation with RIA (R2 =.9721, P<.0001). This strong correlation supports use of the regression equation y=2.029x+0.0897 to transform ELISA data to RIA or inversely y=0.4930x-0.0456 to convert RIA data to ELISA for direct comparison between assays in the concentration range of 0-3.0 ng/mL. Measured C-peptide concentration was lower with the ELISA than with the RIA; individual measurements plotted against the averages of the pair demonstrated that the variability from the mean strongly depended on increasing concentration. CONCLUSIONS: Porcine C-peptide can be reliably measured in NHP serum using the Mercodia ELISA, making this assay interchangeable with the Millipore RIA. Inherent differences in antibody affinity and calibration factors may explain the lower ELISA values as compared to the RIA; however without access to a traceable reference standard, it is not possible to determine which assay is most accurate. Regression modeling resulted in a correction factor appropriate for conversion of ELISA data to RIA-equivalent data facilitating comparison of assay results longitudinally and between groups.

First update of the International Xenotransplantation Association consensus statement on conditions for undertaking clinical trials of porcine islet products in type 1 diabetes--Chapter 4: pre-clinical efficacy and complication data required to justify a clinical trial.

In 2009, the International Xenotransplantation Association (IXA) published a consensus document that provided guidelines and "recommendations" (not regulations) for those contemplating clinical trials of porcine islet transplantation. These guidelines included the IXA's opinion on what constituted "rigorous pre-clinical studies using the most relevant animal models" and were based on "non-human primate testing." We now report our discussion following a careful review of the 2009 guidelines as they relate to pre-clinical testing. In summary, we do not believe there is a need to greatly modify the conclusions and recommendations of the original consensus document. Pre-clinical studies should be sufficiently rigorous to provide optimism that a clinical trial is likely to be safe and has a realistic chance of success, but need not be so demanding that success might only be achieved by very prolonged experimentation, as this would not be in the interests of patients whose quality of life might benefit immensely from a successful islet xenotransplant. We believe these guidelines will be of benefit to both investigators planning a clinical trial and to institutions and regulatory authorities considering a proposal for a clinical trial. In addition, we suggest consideration should be given to establishing an IXA Clinical Trial Advisory Committee that would be available to advise (but not regulate) researchers considering initiating a clinical trial of xenotransplantation.

A comprehensive microbiological safety approach for agarose encapsulated porcine islets intended for clinical trials.

BACKGROUND: The use of porcine islets to replace insulin-producing islet ß-cells, destroyed during the diabetogenic disease process, presents distinct challenges if this option is to become a therapeutic reality for the treatment of type 1 diabetes. These challenges include a thorough evaluation of the microbiological safety of the islets. In this study, we describe a robust porcine islet-screening program that provides a high level of confidence in the microbiological safety of porcine islets suitable for clinical trials. METHODS: A four-checkpoint program systematically screens the donor herd (Large White - Yorkshire × Landrace F1 hybrid animals), individual sentinel and pancreas donor animals and, critically, the islet macrobeads themselves. Molecular assays screen for more than 30 known viruses, while electron microscopy and in vitro studies are employed to screen for potential new or divergent (emergent) viruses. RESULTS: Of 1207 monthly samples taken from random animals over a 2-year period, only a single positive result for Transmissible gastroenteritis virus was observed, demonstrating the high level of biosecurity maintained in the source herd. Given the lack of clinical signs, positive antibody titers for Porcine reproductive and respiratory syndrome virus, Porcine parvovirus, and Influenza A confirm the efficacy of the herd vaccination program. Porcine respiratory coronavirus was found to be present in the herd, as expected for domestic swine. Tissue homogenate samples from six sentinel and 11 donor animals, over the same 2-year period, were negative for the presence of viruses when co-cultured with six different cell lines from four species. The absence of adventitious viruses in separate islet macrobead preparations produced from 12 individual pancreas donor animals was confirmed using validated molecular (n = 32 viruses), in vitro culture (cells from four species), and transmission electron microscopy assays (200 cell profiles per donor animal) over the same 2-year period. There has been no evidence of viral transmission following the implantation of these same encapsulated and functional porcine islets into non-immunosuppressed diabetic cynomolgus macaques for up to 4 years. Isolated peripheral blood mononuclear cells from all time points were negative for PCV (Type 2), PLHV, PRRSV, PCMV, and PERV-A, PERV-B, and PERV-C by PCR analysis in all six recipient animals. CONCLUSION: The four-checkpoint program is a robust and reliable method for characterization of the microbiological safety of encapsulated porcine islets intended for clinical trials.

Evaluation of commercial ELISA and RIA for measuring porcine C-peptide: implications for research.

BACKGROUND: Pre-clinical demonstration of porcine islet graft function is necessary to support the clinical transplantation of pig islets. C-peptide concentration is an especially useful marker of insulin secretion, because its measurement is not confounded by the presence of exogenous insulin. To measure porcine C-peptide (PCP), researchers in the field exclusively used the Millipore (previously Linco Research) radioimmunoassay (RIA) until 2011, when Mercodia released an alternative enzyme-linked immunosorbent assay (ELISA). (At the end of 2013, the Millipore RIA was withdrawn from the market for commercial reasons.) In our current study, to directly compare these two assays, we performed validation studies on each. We also performed interlaboratory comparisons. Then, to determine the level of agreement between the assays, we analyzed the porcine serum C-peptide concentration measurement results obtained from each assay. METHODS: Using pre-established method validation acceptance criteria, we determined and evaluated the detection limit, sensitivity, precision, linearity, and recovery of the two commercially available PCP assays described above (ELISA and RIA). After validation requirements were met, we performed a method comparison by determining C-peptide concentration in 60 serum samples collected from 31 normal, healthy adult Landrace pigs in the fasting state; a subset underwent an intravenous glucose challenge test, to stimulate the typical physiologic range of C-peptide. All analyses were performed according to manufacturer instructions. To compare the assays, we used Deming regression analysis. RESULTS: Both assays met acceptance criteria. The RIA had a sensitivity of 0.1 ng/ml; it was linear to 2.9 ng/ml. The ELISA had a detection limit of 0.03 ng/ml; it was linear to 1.2 ng/ml. Recovery ranged from 89 to 113% with both assays. The coefficient of variability was 8% in interlaboratory comparisons. Deming regression analysis directly comparing both assays revealed significant correlation between them (before log-transformation: R2=0.9803, P<0.0001; after log-transformation: R2=0.9727, P<0.0001). Measured C-peptide concentration was lower with the ELISA than with the RIA; individual measurements plotted against the averages of the pair demonstrated that the variability from the mean strongly depended on increasing concentration. To transform ELISA data, we used the standard regression equation y=2.191x+0.1119 and the log-transformed regression equation y=0.8101x+0.7502. Both the transformed and the log-transformed (exponential) values approximated the measured RIA levels with a high degree of accuracy in the concentration range of 0 to 1.0 ng/ml. CONCLUSIONS: Porcine C-peptide concentration can be reliably measured in porcine serum samples with either assay (ELISA or RIA). However, the C-peptide results generated by these two assays are not equivalent. Therefore, assay bias must be considered before directly comparing pre-clinical studies that used either of these assays. We determined that harmonization between the assays is appropriate in a specific concentration range. Outside of that range, we do not know whether a linear correction function can be more broadly applied. The variation between the two assays may be related to calibration or reagent factors. To determine which assay is truly more accurate and to effectively compare interlaboratory results, we will need a traceable reference standard.

Analysis of differential gene expression profiles in Caenorhabditis elegans knockouts for the v-SNARE master protein 1.

At chemical synapses, neurons communicate information to other cells by secreting neurotransmitters or neuropeptides into the synaptic cleft, which then bind to receptors on the target cell. Preliminary work performed in our laboratory has shown that mutant nematodes lacking a protein called VSM-1 have increased synaptic density compared with the wild type. Consequently, we hypothesized that genes expressed in vsm-1 mutants mediate enhanced synaptogenesis. To identify these genes of interest, we utilized microarray technology and quantitative PCR. To this end, first we isolated the total RNA from young-adult wild-type and vsm-1 mutant Caenorhabditis elegans. Next, we synthesized cDNA from reverse transcription of the isolated RNA. Hybridization of the cDNA to a microarray was performed to facilitate gene expression profiling. Finally, fluorescently labeled microarrays were analyzed, and the identities of induced and repressed genes were uncovered in the open-source software Magic Tool. Analyses of microarray experiments performed using three independent biological samples per strain and three technical replicas and dye swaps showed induction of genes coding for major sperm proteins and repression of SPP-2 in vsm-1 mutants. Microarray results were also validated and quantified by using quantitative PCR.

Comparisons of phenotype and immunomodulatory capacity among rhesus bone-marrow-derived mesenchymal stem/stromal cells, multipotent adult progenitor cells, and dermal fibroblasts.

BACKGROUND: Potent immunomodulatory effects have been reported for mesenchymal stem/stromal cells (MSCs), multipotent adult progenitor cells (MAPCs), and fibroblasts. However, side-by-side comparisons of these cells specifically regarding immunophenotype, gene expression, and suppression of proliferation of CD4(+) and CD8(+) lymphocyte populations have not been reported. METHODS: We developed MAPC and MSC lines from rhesus macaque bone marrow and fibroblast cell lines from rhesus dermis and assessed phenotypes based upon differentiation potential, flow cytometric analysis of immunophenotype, and quantitative RT-PCR analysis of gene expression. Using allogeneic lymphocyte proliferation assays, we compared the in vitro immunomodulatory potency of each cell type. RESULTS AND CONCLUSIONS: Extensive phenotypic similarities exist among each cell type, although immunosuppressive potencies are distinct. MAPCs are most potent, and fibroblasts are the least potent cell type. All three cell types demonstrated immunomodulatory capacity such that each may have potential therapeutic applications such as in organ transplantation, where reduced local immune response is desirable.

Loss of ATP-Sensitive Potassium Channel Expression and Function in the Nervous System Decreases Opioid Sensitivity in a High-Fat Diet-Fed Mouse Model of Diet-Induced Obesity.

During diabetes progression, ß-cell dysfunction due to loss of potassium channels sensitive to ATP, known as KATP channels, occurs, contributing to hyperglycemia. The aim of this study was to investigate if KATP channel expression or activity in the nervous system was altered in a high-fat diet (HFD)-fed mouse model of diet-induced obesity. Expression of two KATP channel subunits, Kcnj11 (Kir6.2) and Abcc8 (SUR1), were decreased in the peripheral and central nervous system of mice fed HFD, which was significantly correlated with mechanical paw-withdrawal thresholds. HFD mice had decreased antinociception to systemic morphine compared with control diet (CON) mice, which was expected because KATP channels are downstream targets of opioid receptors. Mechanical hypersensitivity in HFD mice was exacerbated after systemic treatment with glyburide or nateglinide, KATP channel antagonists clinically used to control blood glucose levels. Upregulation of SUR1 and Kir6.2, through an adenovirus delivered intrathecally, increased morphine antinociception in HFD mice. These data present a potential link between KATP channel function and neuropathy during early stages of diabetes. There is a need for increased knowledge of how diabetes affects structural and molecular changes in the nervous system, including ion channels, to lead to the progression of chronic pain and sensory issues.

Targeted mapping and utilization of the perihepatic surface for therapeutic beta cell replacement and retrieval in diabetic non-human primates.

Introduction: Successful diabetes reversal using pancreatic islet transplantation by various groups illustrates the significant achievements made in cell-based diabetes therapy. While clinically, intraportal islet delivery is almost exclusively used, it is not without obstacles, including instant blood-mediated inflammatory reaction (IBMIR), relative hypoxia, and loss of function over time, therefore hindering long-term success. Here we demonstrate the perihepatic surface of non-human primates (NHPs) as a potential islet delivery site maximizing favorable characteristics, including proximity to a dense vascular network for adequate oxygenation while avoiding IBMIR exposure, maintenance of portal insulin delivery, and relative ease of accessibility through minimally invasive surgery or percutaneous means. In addition, we demonstrate a targeted mapping technique of the perihepatic surface, allowing for the testing of multiple experimental conditions, including a semi-synthetic hydrogel as a possible three-dimensional framework to improve islet viability. Methods: Perihepatic allo-islet cell transplants were performed in immunosuppressed cynomolgus macaques using a targeted mapping technique to test multiple conditions for biocompatibility. Transplant conditions included islets or carriers (including hydrogel, autologous plasma, and media) alone or in various combinations. Necropsy was performed at day 30, and histopathology was performed to assess biocompatibility, immune response, and islet viability. Subsequently, single-injection perihepatic allo-islet transplant was performed in immunosuppressed diabetic cynomolgus macaques. Metabolic assessments were measured frequently (i.e., blood glucose, insulin, C-peptide) until final graft retrieval for histopathology. Results: Targeted mapping biocompatibility studies demonstrated mild inflammatory changes with islet-plasma constructs; however, significant inflammatory cell infiltration and fibrosis were seen surrounding sites with the hydrogel carrier affecting islet viability. In diabetic NHPs, perihepatic islet transplant using an autologous plasma carrier demonstrated prolonged function up to 6 months with improvements in blood glucose, exogenous insulin requirements, and HbA1c. Histopathology of these islets was associated with mild peri-islet mononuclear cell infiltration without evidence of rejection. Discussion: The perihepatic surface serves as a viable site for islet cell transplantation demonstrating sustained islet function through 6 months. The targeted mapping approach allows for the testing of multiple conditions simultaneously to evaluate immune response to biomaterials at this site. Compared to traditional intraportal injection, the perihepatic site is a minimally invasive approach that allows the possibility for graft recovery and avoids IBMIR.

A bioengineered artificial interstitium supports long-term islet xenograft survival in nonhuman primates without immunosuppression.

Cell-based therapies hold promise for many chronic conditions; however, the continued need for immunosuppression along with challenges in replacing cells to improve durability or retrieving cells for safety are major obstacles. We subcutaneously implanted a device engineered to exploit the innate transcapillary hydrostatic and colloid osmotic pressure generating ultrafiltrate to mimic interstitium. Long-term stable accumulation of ultrafiltrate was achieved in both rodents and nonhuman primates (NHPs) that was chemically similar to serum and achieved capillary blood oxygen concentration. The majority of adult pig islet grafts transplanted in non-immunosuppressed NHPs resulted in xenograft survival >100 days. Stable cytokine levels, normal neutrophil to lymphocyte ratio, and a lack of immune cell infiltration demonstrated successful immunoprotection and averted typical systemic changes related to xenograft transplant, especially inflammation. This approach eliminates the need for immunosuppression and permits percutaneous access for loading, reloading, biopsy, and recovery to de-risk the use of "unlimited" xenogeneic cell sources to realize widespread clinical translation of cell-based therapies.

The usefulness and limitations of the diabetic macaque model in evaluating long-term porcine islet xenograft survival.

BACKGROUND: Various groups have reported prolonged diabetes reversal and graft function after porcine islet transplantation into diabetic macaques using different experimental designs (macaque source, islet source, type of immunosuppression): subsequently, the International Xenotransplantation Association has published recommendations for entering a clinical trial. Our experiments showed limitations that affected consistent achievement of long-term survival. We aimed to identify these limitations and underlying causes to emphasize the translational value of this highly relevant type 1 diabetic macaque model. METHODS: We reviewed data from our institution and literature data on long-term porcine islet xenograft survival in the diabetic macaque model, especially focusing on aspects of incomplete diabetes reversal relative to macaque normal values. This phenomenon was compared with diabetes reversal in an allo-islet transplant model in macaques and with chronic insulin treatment of diabetic macaques, all with 180-day follow-up. This comparison enabled to identify potential model limitations and underlying causative factors. RESULTS: Especially in the xenograft model, the achievement of long-term graft survival revealed limitations including chronic, mild hyperglycemia and absence of body weight (BW) gain or even progressive BW loss. Metabolic incompatibilities in glycemic control (i.e., insulin kinetics) between the pig and macaque species underlie chronic, mild hyperglycemia. This phenomenon might not bear relevance for the pig-to-human species combination because the glycemic control in pigs and humans is similar and differs from that in nonhuman primates (NHP). Weight loss could be related to changes in the gastrointestinal tract related with local high exposure to orally administered immunosuppressants; these must be given at higher dose levels because of low bioavailability in macaques to achieve systemic exposure at therapeutic levels. This is aggravated by insufficient graft insulin production in proportion to the needs of macaques: this model limitation has no translational value to the pig-to-human setting. Nutritional deficits can result in incorrect interpretation of blood glucose levels and C-peptide levels regarding graft function. Likewise, nutritional status alters physiologic responses, influencing susceptibility to infectious and noninfectious complications. CONCLUSION: THE model-induced confounding described interferes with accurate interpretation of safety and efficacy studies, which affects the translational value of pig-to-NHP islet cell transplant studies to the pig-to-human transplant condition.

Differences in glucose-stimulated insulin secretion in vitro of islets from human, nonhuman primate, and porcine origin.

Porcine islet xenotransplantation is considered a potential cell-based therapy for type 1 diabetes. It is currently being evaluated in diabetic nonhuman primates (NHP) to assess safety and efficacy of the islet product. However, due to a variety of distinct differences between the respective species, including the insulin secretory characteristics of islets, the suitability and predictive value of the preclinical model in the extrapolation to the clinical setting remain a critical issue. Islets isolated from human (n = 3), NHP (n = 2), adult pig (AP, n = 3), and juvenile pig (JP, n = 4) pancreata were perifused with medium at basal glucose (2.5 mm) followed by high glucose (16.7 mm) concentrations. The total glucose-stimulated insulin secretion (GSIS) was calculated from generated insulin secretion profiles. Nonhuman primate islets exhibited GSIS 3-fold higher than AP islets, while AP and JP islets exhibited GSIS 1/3 and 1/30 of human islets, respectively. The insulin content of NHP and AP islets was similar to that of human islets, whereas that of JP islets was 1/5 of human islets. Despite the fact that human, NHP, and AP islets contain similar amounts of insulin, the much higher GSIS for NHP islets than for AP and JP islets suggests the need for increased dosing of islets from JP and AP in pig-to-NHP transplantation. Porcine islet xenotransplantation to humans may require significantly higher dosing given the lower GSIS of AP islets compared to human islets.

Prolonged diabetes reversal after intraportal xenotransplantation of wild-type porcine islets in immunosuppressed nonhuman primates.

Cell-based diabetes therapy requires an abundant cell source. Here, we report reversal of diabetes for more than 100 d in cynomolgus macaques after intraportal transplantation of cultured islets from genetically unmodified pigs without Gal-specific antibody manipulation. Immunotherapy with CD25-specific and CD154-specific monoclonal antibodies, FTY720 (or tacrolimus), everolimus and leflunomide suppressed indirect activation of T cells, elicitation of non-Gal pig-specific IgG antibody, intragraft expression of proinflammatory cytokines and invasion of infiltrating mononuclear cells into islets.

Multiplexing AAV Serotype-Specific Neutralizing Antibodies in Preclinical Animal Models and Humans.

The accurate assessment of AAV-specific pre-existing humoral immunity due to natural viral infection is critical for the efficient use of clinical gene therapy. The method described in the present study applies equivalent infection conditions to each AAV serotype (AAV1, AAV2, AAV3, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAVAnc80L65). In the current study, we validated the assay by assessing AAV-neutralizing antibody titers in a limited cohort of random human donors and well-established preclinical large animal models, including dogs and non-human primates (NHPs). We achieved a rapid and accurate evaluation of neutralizing titers for each individual subject that can be used for clinical enrollment based on specific AAV serotypes and individualized selection of the most suitable AAV serotype for each specific patient.

Loss of ATP-sensitive channel expression and function decreases opioid sensitivity in a mouse model of type 2 diabetes.

During diabetes, ß-cell dysfunction due to loss of potassium channels sensitive to ATP, known as KATP channels occurs progressively over time contributing to hyperglycemia. KATP channels are additionally present in the central and peripheral nervous systems and are downstream targets of opioid receptor signaling. The aim of this study is to investigate if KATP channel expression or activity in the nervous system changes in diabetic mice and if morphine antinociception changes in mice fed a high fat diet (HFD) for 16 weeks compared to controls. Mechanical thresholds were also monitored before and after administration of glyburide or nateglinide, KATP channel antagonists, for four weeks. HFD mice have decreased antinociception to systemic morphine, which is exacerbated after systemic treatment with glyburide or nateglinide. HFD mice also have lower rotarod scores, decreased mobility in an open field test, and lower burrowing behavior compared to their control diet counterparts, which is unaffected by KATP channel antagonist delivery. Expression of KATP channel subunits, Kcnj11 (Kir6.2) and Abcc8 (SUR1), were decreased in the peripheral and central nervous system in HFD mice, which is significantly correlated with baseline paw withdrawal thresholds. Upregulation of SUR1 through an adenovirus delivered intrathecally increased morphine antinociception in HFD mice, whereas Kir6.2 upregulation improved morphine antinociception only marginally. Perspective: This article presents the potential link between KATP channel function and neuropathy during diabetes. There is a need for increased knowledge in how diabetes affects structural and molecular changes in the nervous system to lead to the progression of chronic pain and sensory issues.

Establishment of Skeletal Myogenic Progenitors from Non-Human Primate Induced Pluripotent Stem Cells.

Pluripotent stem (PS) cells enable the scalable production of tissue-specific derivatives with therapeutic potential for various clinical applications, including muscular dystrophies. Given the similarity to human counterparts, the non-human primate (NHP) is an ideal preclinical model to evaluate several questions, including delivery, biodistribution, and immune response. While the generation of human-induced PS (iPS)-cell-derived myogenic progenitors is well established, there have been no data for NHP counterparts, probably due to the lack of an efficient system to differentiate NHP iPS cells towards the skeletal muscle lineage. Here, we report the generation of three independent Macaca fascicularis iPS cell lines and their myogenic differentiation using PAX7 conditional expression. The whole-transcriptome analysis confirmed the successful sequential induction of mesoderm, paraxial mesoderm, and myogenic lineages. NHP myogenic progenitors efficiently gave rise to myotubes under appropriate in vitro differentiation conditions and engrafted in vivo into the TA muscles of NSG and FKRP-NSG mice. Lastly, we explored the preclinical potential of these NHP myogenic progenitors in a single wild-type NHP recipient, demonstrating engraftment and characterizing the interaction with the host immune response. These studies establish an NHP model system through which iPS-cell-derived myogenic progenitors can be studied.

Hypoxia within subcutaneously implanted macroencapsulation devices limits the viability and functionality of densely loaded islets.

Introduction: Subcutaneous macroencapsulation devices circumvent disadvantages of intraportal islet therapy. However, a curative dose of islets within reasonably sized devices requires dense cell packing. We measured internal PO2 of implanted devices, mathematically modeled oxygen availability within devices and tested the predictions with implanted devices containing densely packed human islets. Methods: Partial pressure of oxygen (PO2) within implanted empty devices was measured by noninvasive 19F-MRS. A mathematical model was constructed, predicting internal PO2, viability and functionality of densely packed islets as a function of external PO2. Finally, viability was measured by oxygen consumption rate (OCR) in day 7 explants loaded at various islet densities. Results: In empty devices, PO2 was 12 mmHg or lower, despite successful external vascularization. Devices loaded with human islets implanted for 7 days, then explanted and assessed by OCR confirmed trends proffered by the model but viability was substantially lower than predicted. Co-localization of insulin and caspase-3 immunostaining suggested that apoptosis contributed to loss of beta cells. Discussion: Measured PO2 within empty devices declined during the first few days post-transplant then modestly increased with neovascularization around the device. Viability of islets is inversely related to islet density within devices.

Successful implementation of cooperative handling eliminates the need for restraint in a complex non-human primate disease model.

BACKGROUND: Streptozotocin-induced diabetic non-human primates are used to study efficacy and safety of innovative immunosuppression after islet transplantation. We implemented a training program for medical management of a chronic disease state. METHODS: Cooperation with hand feeding and drinking, shifting, and limb presentation were trained utilizing predominately positive but also negative reinforcement in 52 animals compared with 28 macaques subjected to conventional physical and/or chemical restraint. The success and timing of behavior acquisition was evaluated in a representative subset of 14 animals. RESULTS: Over 90% of animals were successful in behavior acquisition. Programmatically this resulted in complete elimination of chair restraint and negligible requirement for sedation. About half of the trained animals had no-to-moderate thymic involution, indicative of a substantial reduction in stress. CONCLUSION: Cooperative handling enhances animal well-being. This contributes to validity of scientific results and eliminates model-induced confounding that can obstruct interpretation of safety and efficacy data.

Behavioral Management as a Coping Strategy for Managing Stressors in Primates: The Influence of Temperament and Species.

Primates involved in biomedical research experience stressors related to captivity, close contact with caregivers, and may be exposed to various medical procedures while modeling clinical disease or interventions under study. Behavioral management is used to promote behavioral flexibility in less complex captive environments and train coping skills to reduce stress. How animals perceive their environment and interactions is the basis of subjective experience and has a major impact on welfare. Certain traits, such as temperament and species, can affect behavioral plasticity and learning. This study investigated the relationship between these traits and acquisition of coping skills in 83 macaques trained for cooperation with potentially aversive medical procedures using a mixed-reinforcement training paradigm. All primates successfully completed training with no significant differences between inhibited and exploratory animals, suggesting that while temperament profoundly influences behavior, training serves as an important equalizer. Species-specific differences in learning and motivation manifested in statistically significant faster skill acquisition in rhesus compared with cynomolgus macaques, but this difference was not clinically relevant. Despite unique traits, primates were equally successful in learning complex tasks and displayed effective coping. When animals engage in coping behaviors, their distress decreases, improving welfare and reducing inter- and intra- subject variability to enhance scientific validity.