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.

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.

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.

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.

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.

Species incompatibilities in the pig-to-macaque islet xenotransplant model affect transplant outcome: a comparison with allotransplantation.

BACKGROUND: Porcine islet transplantation into diabetic non-human primates is considered most relevant in translational research supporting a clinical application. Most studies have focused on immunosuppressive protocols, while metabolic aspects have mainly been utilized in graft monitoring. We evaluated data from our group regarding human and non-human primate (NHP) allotransplantation and pig-to-NHP xenotransplantation to identify incompatibilities in metabolic factors and their consequences for transplant outcomes. METHODS: Basic gluco-metabolic parameters (fasting blood glucose, C-peptide, and response to stimulation with arginine or glucose) were derived from literature (humans), 72 macaques, and 47 adult Landrace pigs. Islet preparations from 15 human deceased donors, 61 macaques, and 23 adult pigs were compared with respect to yield, fractional viability assessed by oxygen consumption normalized for DNA, and in vitro glucose-induced insulin release. Metabolic parameters at day 75 after a single islet transplantation in the liver were compared for 19 patients and 9 macaques receiving an allotransplant and 11 macaques receiving a porcine xenotransplant: recipients received chronic immunosuppression. RESULTS: Pigs differ from NHPs and humans by a much lower C-peptide level (0.42 vs. 1.3 to 2.0 ng/ml, respectively) and a 2- to 7-fold lower C-peptide response to arginine stimulation. In contrast, NHPs have the highest metabolic demand as evidenced by a high C-peptide and high C-peptide response to arginine stimulation; values are about twice higher than in humans. For manufactured islet preparations, these differences are reflected by glucose-stimulated insulin release (the stimulation index for pigs is 1.5, for humans 3.8, and for macaques 7.7), but not by fractional viability, which was in the same range. The day 75 outcome after transplantation assessed by C-peptide was similar for allotransplanted humans and NHPs (80 to 90% good graft function) and lower in xenografted NHPs (36% good graft function); gluco-metabolic parameters were in accordance with graft function, albeit different between species because normoglycemia under exogenous insulin is maintained more aggressively in patients than in NHPs. In xenografted NHPs, the shift in glycemic control with respect to normal values, combined with low values of circulating porcine C-peptide, resembled more the normal condition in a pig than that in a macaque. CONCLUSIONS: The substantially lower glucose-induced insulin response in adult porcine islet preparations as opposed to islets manufactured from humans or macaques combined with the much higher need for insulin in macaques than in humans creates an imbalance between the metabolic demand and the engrafted islet mass in the pig-to-NHP xenograft recipient. Engrafted islet mass is affected by dose, suggesting that a much higher dose level of islets is necessary in the xenogeneic setting than in human or NHP allotransplantation, and pig islets need to be given at a higher dose in macaques than the anticipated effective dose in humans. To cope with differences in metabolic demand and presumably also metabolic dynamics, a liberal regime in supportive exogenous insulin might be essential to achieve long-term survival. These intrinsic characteristics of the NHP model deserve consideration to optimally design experimental studies with the perspective of translational value of results.

Meeting Report of the 35th Annual Meeting of the Academy of Surgical Research: Summary of Presentations, Labs, and Workshops, Focusing on Experimental Surgery, Clearwater Beach, FL, 25-27 September 2019.

The 35th Annual Meeting of the Academy of Surgical Research took place in Clearwater Beach, Florida on 25-27 September 2019. This meeting brings together the experimental surgery community to share the latest in research and surgical techniques and fosters professional development through education, training, and certification. The Academy is made up of a diverse group of technicians, veterinarians, medical doctors, and biomedical researchers from industry, academia, and complimentary disciplines supporting discovery and translational research. Over 165 participants from 30 different states and 6 countries were in attendance for the scientific program and social events. Four keynotes were presented together with breakout and poster session tracks. Participants were able to work on applied skills in practical courses that included hands-on surgical technique wet-labs complimented with dry-lab suture technique and surgical anesthesia, plus workshops and roundtables focused on improving study design and publication best practices. Attendees were able to enjoy sun and fun while connecting with potential mentors and collaborators during the social program. We present the highlights from this meeting in this report together with selected abstracts that illustrate the diverse scientific expertise of the Academy and promising new surgical research.

Serum cytokine profiles in healthy nonhuman primates are blunted by sedation and demonstrate sexual dimorphism as detected by a validated multiplex immunoassay.

Cytokine profiling is a valuable tool for monitoring immune responses associated with disease and treatment. This study assessed the impact of sex and sedation on serum cytokines in healthy nonhuman primates (NHPs). Twenty-three cytokines were measured from serum using a bead-based multiplex assay. Assay validation for precision, sensitivity, recovery, linearity, and stability was performed. Samples from male and female cynomolgus and rhesus macaques either cooperating or sedated were compared. All cytokines except TNFα demonstrated acceptable sensitivity and precision, with variable recovery and linearity. IFNγ, IL-2, IL-5, IL-6, IL-8, IL-12/23 (p40), IL-13, IL-15, MCP-1, TGFα, VEGF met acceptance criteria; G-CSF, IL-4, IL-10, MIP1α, sCD40L were marginal. Higher cytokine levels were observed in females and cytokine levels were blunted in sedated NHPs when compared to awake cooperating NHPs. Significant differences observed in cytokines related to sex, species, or imposed by handling highlight the importance of model design on translational relevance for clinical settings.

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.

Characterization of Different Commercial Dietary Supplements in the Peri-Weaning Period on Consumption and Growth Performance in C57Bl/6J Mice.

This experiment was conducted to investigate the effects of common commercially available dietary supplementation in the peri-weaning period on feed intake, growth, and survival in C57Bl/6J mouse pups and lactating dams. A total of 96 pups and their dams were randomized to the control group or one of three nutritional supplement treatment groups: (i) control group without supplementation, or (ii) weanling-targeted Clear H2O gel (Gel), (iii) transgenic-targeted Bio-Serv dough (Dough), or (iv) dam diet as a mash (Chow), in the peri-weaning period (from 11 to 28 days). Stool was observed daily for a dye marker indicating supplement consumption. Pups were weaned at 21 days and followed for a total of 42 days. No pup morbidity or mortality was observed. There was a higher proportion of pups consuming dough and gel earlier than chow (p = 0.0091). The majority of treated pups (>95%) were consuming the supplement by day 23 (range 15-23), suggesting interplay between organoleptic properties of the supplement and pup maturity. All groups gained weight, with typical sexual dimorphism observed in the growth curves. Dough treatment led to significantly higher average daily gain in male pups (0.64 ± 0.03 g/d) as compared with controls (0.58 ± 0.03 g/d). The highest average daily gain in all groups was observed pre-weaning between days 21 and 28. Compared with controls, the weight gain slope was significantly higher in the Dough and Chow treatment groups and lower in Gel treatment groups, with a more pronounced effect in males. In this study, the composition of nutritional supplementation was the dominant factor in increasing the growth trend as opposed to energy density. Peri-weaning supplementation with Dough and Chow treatments improved pre- and post-growth performance in a comparable way and was more effective than Gel treatment during adaptation to solid feeding. Proper application of supplements to support weanlings can directly improve welfare and limit unintended experimental variability.

Long-Term Management of Vascular Access Ports in Nonhuman Primates Used in Preclinical Efficacy and Tolerability Studies.

Vascular access ports (VAPs) are an essential tool for long-term vascular access in preclinical studies and disease modeling in non-human primates (NHPs). We retrospectively reviewed central (inferior vena cava, IVC) and portal VAP implantation with the maintenance at our center from 15 January 2010 to 31 January 2018. In total, 209 VAPs were implanted for long-term drug administration and sampling. Patency was >95% at 6 months and >80% at 1 year for IVC VAPs and >90% at 6 months and >85% at 1 year for portal VAPs. The majority of animals had no complications and access was generally durable with device use ranging up to 7 years. In IVC, VAPs loss of patency occurred in 13% (0.035/100 d), surgical site infection in 2.9% (0.097/100 d), port pocket infection in 2.2% (0.004/100 d), erosion in 2.9%, 0.008/100 d), and mechanical failure in 4.3% (0.012/100 d). In portal, VAPs loss of patency occurred in 11.3% (0.028/100 d) and port pocket infection in 1.4% (0.003/100 d). About 12% of VAPs were removed as a result of complications.This study confirms VAP implant and maintenance is a beneficial and safe practice in NHPs resulting in favorable outcomes. High patency rates and low complication rates are comparable to the clinical setting. In addition to enabling comprehensive data collection, VAPs increase satisfaction and well-being by minimizing interference with daily routines and fostering cooperation. VAP implantation, together with an effective maintenance regimen and co-operative handling, is a reliable and convenient refined method for drug administration and blood sampling.Keywords: Vascular access port; nonhuman primates; refinement; central vascular access; portal vascular access; surgical technique; experimental surgery; animal model.