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.

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.

Loss of ATP-sensitive potassium channel expression and function in the nervous system decreases opioid sensitivity in 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 is to investigate if KATP channel expression or activity in the nervous system was altered in a high-fatdiet-( 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 in HFD mice, which is significantly correlated with mechanical paw withdrawal thresholds. HFD mice had decreased antinociception to systemic morphine compared to control diet (CON) mice, which was expected as 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 in 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.

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.

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.

Boosting of SARS-CoV-2 immunity in nonhuman primates using an oral rhabdoviral vaccine.

An orally active vaccine capable of boosting SARS-CoV-2 immune responses in previously infected or vaccinated individuals would help efforts to achieve and sustain herd immunity. Unlike mRNA-loaded lipid nanoparticles and recombinant replication-defective adenoviruses, replicating vesicular stomatitis viruses with SARS-CoV-2 spike glycoproteins (VSV-SARS2) were poorly immunogenic after intramuscular administration in clinical trials. Here, by G protein trans-complementation, we generated VSV-SARS2(+G) virions with expanded target cell tropism. Compared to parental VSV-SARS2, G-supplemented viruses were orally active in virus-naive and vaccine-primed cynomolgus macaques, powerfully boosting SARS-CoV-2 neutralizing antibody titers. Clinical testing of this oral VSV-SARS2(+G) vaccine is planned.

A nonhuman primate model of vertical sleeve gastrectomy facilitates mechanistic and translational research in human obesity.

The obesity epidemic significantly contributes to overall morbidity and mortality. Bariatric surgery is the gold standard treatment for obesity and metabolic dysfunction, yet the mechanisms by which it exerts metabolic benefit remain unclear. Here, we demonstrate a model of vertical sleeve gastrectomy (VSG) in nonhuman primates (NHP) that mimics the complexity and outcomes in humans. We also show that VSG confers weight loss and durable metabolic benefit, where equivalent caloric intake in shams resulted in significant weight gain following surgery. Furthermore, we show that VSG is associated with early, weight-independent increases in bile acids, short-chain fatty acids, and reduced visceral adipose tissue (VAT) inflammation with a polarization of VAT-resident immunocytes toward highly regulatory myeloid cells and Tregs. These data demonstrate that this strongly translational NHP model can be used to interrogate factors driving successful intervention to unravel the interplay between physiologic systems and improve therapies for obesity and metabolic syndrome.

The streptozotocin-induced diabetic nude mouse model: differences between animals from different sources.

Diabetes is induced in mice by using streptozotocin (STZ), a compound that has a preferential toxicity toward pancreatic ß cells. We evaluated nude male mice from various sources for their sensitivity to a single high dose (160 to 240 mg/kg) of STZ. Diabetes was induced in male mice (age: median, 12 wk; interquartile range, 11 to 14 wk; body weight, about 30 g) from Taconic Farms (TAC), Jackson Laboratories (JAX), and Charles River Laboratories (CRL). Mice were monitored for 30 d for adverse side effects, blood glucose, and insulin requirements. In CRL mice given 240 mg/kg STZ, more than 95% developed diabetes within 4 to 5 d, and loss of body weight was relatively low (mean, 0.4 g). In comparison, both TAC and JAX mice were more sensitive to STZ, as evidenced by faster development of diabetes (even at a lower STZ dose), greater need for insulin after STZ, greater body weight loss (mean: TAC, 3.5 g; JAX, 3.7 g), and greater mortality. We recommend conducting exploratory safety assessments when selecting a nude mouse source, with the aim of limiting morbidity and mortality to less than 10%.