Melanie L. Graham

Inducing stable tolerance to transplants remains a challenge in immunology. Previously, we induced tolerance to allogeneic islets in nonhuman primates by preemptive alloantigen delivery to antigen-presenting cells in situ. Here, mass cytometry phenotyping with incorporated donor-derived MHC-I peptide–loaded MHC-II tetramers revealed accumulation of allospecific CD4 ⁺ T cell clusters in the spleen of tolerant recipients. Areg ⁺ Tr1 regulatory and terminally exhausted EGFR ^hi T (Tex) cells represented the predominant allospecific subsets. Trajectory analysis showed that antigen-experienced effector memory T cells differentiated into suppressive Areg ⁺ Tr1 and EGFR ⁺ TOX ⁺ Nur77 ⁺ TCF-1 ⁻ Tex subsets. Cell-cell communication mapping showed that exhausted and effector memory T cells engaged with allospecific Tr1 cells via the Areg-EGFR axis. Gene silencing studies confirmed that Tr1 cells use Areg-EGFR signaling to drive the metabolic suppression and epigenetic reprogramming of CD4 ⁺ T cells through a Nur77-dependent pathway. These findings point to the splenic Areg ⁺ Tr1 cell-EGFR ⁺ Teff cell axis as a critical immunoregulatory pathway in peripheral transplant tolerance.

Background/Objectives: Adipose tissue (AT) plays significant roles in energy storage, metabolite signaling, and immunomodulation. The understanding of its underlying mechanisms of dysregulation can provide insight into complex disease processes through analysis with histology, flow cytometry, metabolomics, and proteomics. Tissue sampling in the clinical setting has largely shifted towards minimally invasive approaches to improve factors such as patient satisfaction, post-operative recovery, and procedure length. In contrast, preclinical animal models continue to rely on more invasive methods until refined, minimally invasive techniques are developed and systematically assessed. To improve animal welfare and enhance clinical translatability, there is a critical need to reverse translate these approaches into animal models. Methods: Our study evaluated the feasibility and performance of a commercially available vacuum-assisted biopsy (VAB) device for AT sampling in a preclinical nonhuman primate (NHP) model. Six rhesus NHPs successfully underwent three serial AT biopsies with a VAB device (n = 18). Results: All animals recovered without any serious or unexpected adverse events. The amount of adipose tissue collected per biopsy (0.5–2.7 g) was proportional to the number of individual tracks. Isolation of the stromal vascular fraction (SVF) from a subset of samples (n = 6) yielded 0.41 ± 0.12 × 10⁶ cells/g of tissue. Conclusions: The minimally invasive VAB technique is a safe and reliable method of AT collection in NHPs. This feasibility study demonstrated adequate volumes of tissue cores that are suitable for typical, downstream research applications including immunologic studies and pathology, while improving animal welfare.

Emesis is one of the most common side effects of ketamine sedation; however, predictors like species and sex have not been studied in macaques. This study aimed to determine the incidence and predictors of emesis in macaques undergoing ketamine sedation and to assess the efficacy of orally administered antiemetics in preventing emesis. A retrospective analysis was performed using medical records from rhesus and cynomolgus macaques who received ketamine sedation for veterinary examination events from August 2023 to April 2024. Among the 70 sedations performed using IM ketamine, the overall incidence of emesis was 26%. Cynomolgus macaques had a significantly higher risk of emesis (odds ratio (OR) 46.14, 95% confidence interval (CI): 7.08–493.9; p < 0.0001), with an emesis incidence of 55%. Females showed a higher incidence of emesis (75%) with an odds ratio of 3.72 (95% CI: 0.55–17.91; p = 0.24), though this difference was not statistically significant. In a prospective, randomized study, we compared the prophylactic use of oral maropitant citrate at a dose of 2 mg/kg or oral ondansetron at a dose of 1 mg/kg versus placebo for the prevention of emesis in cynomolgus macaques undergoing veterinary examination. Emesis was reduced from 58% in the control group to 50% in the maropitant group and 33% in the ondansetron group. However, the reduction in relative risk with either treatment did not reach statistical significance. Prophylactic oral administration of maropitant appears ineffective in preventing emesis in cynomolgus macaques. In contrast, prophylactic oral ondansetron is a more effective alternative in preventing emesis in these animals.

Laparoscopic colon surgery is performed frequently in the clinical setting for a multitude of reasons including cancer, infection, and autoimmune disease. As a result, extensive research has been conducted in relation to clinical outcomes after surgery, but more recently, in relation to the impact of surgery and other patient factors on physiologic homeostasis including the host microbiome. Despite this, experimental surgical models for laparoscopic colon surgery are scarce in the literature with most studies utilizing rodents. While rodent studies provide valuable insights into basic mechanistic processes, the translation of novel therapeutic approaches to clinical practice often requires the use of large animal models. In exploring the intricate systems biology linking surgery and medicine, sophisticated models such as nonhuman primates (NHPs) play a pivotal role. By closely resembling human anatomical, physiological, and behavioral characteristics, NHPs facilitate the development and refinement of complex surgical techniques and peri-operative practices. Furthermore, they enable longitudinal studies that comprehensively assess both immediate and long-term outcomes. The availability and utilization of multiple robust models enhance the validity of surgical research, leading to more successful translation to human clinical practice. Here we describe our technique for performing a laparoscopic sigmoid colectomy with a primary anastomosis in an NHP. The entire procedure was well tolerated without significant ventilation or hemodynamic issue. To our knowledge, this represents the first laparoscopic sigmoid colectomy with primary anastomosis performed in an NHP. Furthermore, this demonstrates the feasibility of the technique and provides a relevant, preclinical model for the study of surgical colon disease. Although the surgical colectomy model in NHPs closely resembles the clinical scenario, it is crucial to recognize that a ‘model’ inherently comes with limitations. The intended use of any model should be carefully evaluated concerning the target patient population with the consideration of potential disparities in anatomy, physiology, environmental factors, and disease to properly interpret results. This model provides an opportunity to study mechanisms, from a systems biology perspective, underlying both innovative surgical treatments and their effects on diseases such as colon cancer, as well as benign conditions like inflammatory bowel disease, diverticulitis, and anastomotic leak, offering high predictive value.

Medical procedures necessary for routine care can induce stress in both the veterinary and human clinical situations. In the research environment, nonhuman primates undergo procedures like physical examination, blood sampling, and intravenous drug or fluid administration either as a part of routine veterinary care or during the modeling of clinical disease and interventions under study. Behavioral management techniques, such as training for cooperation, allow caregivers to train primates to voluntarily engage in various medical procedures. This approach reduces stress and anxiety associated with necessary procedures, thereby enhancing efficiency and minimizing the invasiveness of medical care. Consequently, veterinary evaluation and care can be provided without compromise, resulting in enhanced clinical outcomes and overall better health. In this study, we explored the impact of the behavioral management program implemented at our center on a subset of animals undergoing routine veterinary care, focusing on the overall experience, including animal welfare, scientific rigor, and efficiency in terms of economics and time. We investigated its impact on key factors, such as the total procedure and recovery time, incidence of side effects, and welfare indicators, revealing a significant positive influence on animal care. Furthermore, through case studies, we illustrate how behavioral management facilitates timely medical care and monitoring, effectively mitigating stressors that could otherwise impair health and welfare, enabling the provision of care that would have otherwise been unachievable. A thoughtfully designed primate behavioral management program, integrating cooperation and participation with veterinary care, forms the cornerstone of superior animal welfare, enhanced clinical care, and more accurate scientific outcomes.

Anticoagulant and antiplatelet therapies are used to prevent life-threatening complications associated with thrombosis. While there are numerous clinical guidelines for antithrombotic medications, there is an incomplete understanding of whether these interventions yield similar effects in preclinical models, potentially impacting their predictive value for translational studies on the development of medical devices, therapies, and surgical techniques. Due to their close physiologic similarities to humans, we employed nonhuman primates (NHPs) using a reverse translational approach to analyze the response to clinical regimens of unfractionated heparin, low-molecular-weight heparin (LMWH) and aspirin to assess concordance with typical human responses and evaluate the predictive validity of this model. We evaluate activated clotting time (ACT) in nine rhesus and six cynomolgus macaques following the intraoperative administration of intravenous unfractionated heparin (100–300 U/kg) reflecting the clinical dose range. We observed a significant dose-dependent effect of heparin on ACT (low-dose average = 114.1 s; high-dose average = 148.3 s; p = 0.0011). LMWH and aspirin, common clinical antithrombotic prophylactics, were evaluated in three rhesus macaques. NHPs achieved therapeutic Anti-Xa levels (mean = 0.64 U/mL) and ARU (mean = 459) via VerifyNow, adhering to clinical guidance using 1.0 mg/kg enoxaparin and 81 mg aspirin. Clinical dosing strategies for unfractionated heparin, LMWH, and aspirin were safe and effective in NHPs, with no development of thrombosis or bleeding complications intraoperatively, postoperatively, or for prophylaxis. Our findings suggest that coagulation studies, performed as an integrative part of studies on biologics, bioengineered devices, or transplantation in NHPs, can be extrapolated to the clinical situation with high predictive validity.

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.

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.

During diabetes, β-cell dysfunction due to loss of potassium channels sensitive to ATP, known as K _ATP channels occurs progressively over time contributing to hyperglycemia. K _ATP 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 K _ATP 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, K _ATP 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 K _ATP channel antagonist delivery. Expression of K _ATP 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 K _ATP 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.