Graft dysfunction in compassionate use of genetically engineered pig-to-human cardiac xenotransplantation: a case report.

BACKGROUND: A genetically engineered pig cardiac xenotransplantation was done on Jan 7, 2022, in a non-ambulatory male patient, aged 57 years, with end-stage heart failure, and on veno-arterial extracorporeal membrane oxygenation support, who was ineligible for an allograft. This report details our current understanding of factors important to the xenotransplantation outcome. METHODS: Physiological and biochemical parameters critical for the care of all heart transplant recipients were collected in extensive clinical monitoring in an intensive care unit. To ascertain the cause of xenograft dysfunction, we did extensive immunological and histopathological studies, including electron microscopy and quantification of porcine cytomegalovirus or porcine roseolovirus (PCMV/PRV) in the xenograft, recipient cells, and tissue by DNA PCR and RNA transcription. We performed intravenous immunoglobulin (IVIG) binding to donor cells and single-cell RNA sequencing of peripheral blood mononuclear cells. FINDINGS: After successful xenotransplantation, the graft functioned well on echocardiography and sustained cardiovascular and other organ systems functions until postoperative day 47 when diastolic heart failure occurred. At postoperative day 50, the endomyocardial biopsy revealed damaged capillaries with interstitial oedema, red cell extravasation, rare thrombotic microangiopathy, and complement deposition. Increased anti-pig xenoantibodies, mainly IgG, were detected after IVIG administration for hypogammaglobulinaemia and during the first plasma exchange. Endomyocardial biopsy on postoperative day 56 showed fibrotic changes consistent with progressive myocardial stiffness. Microbial cell-free DNA testing indicated increasing titres of PCMV/PRV cell-free DNA. Post-mortem single-cell RNA sequencing showed overlapping causes. INTERPRETATION: Hyperacute rejection was avoided. We identified potential mediators of the observed endothelial injury. First, widespread endothelial injury indicates antibody-mediated rejection. Second, IVIG bound strongly to donor endothelium, possibly causing immune activation. Finally, reactivation and replication of latent PCMV/PRV in the xenograft possibly initiated a damaging inflammatory response. The findings point to specific measures to improve xenotransplant outcomes in the future. FUNDING: The University of Maryland School of Medicine, and the University of Maryland Medical Center.

Increased human complement pathway regulatory protein gene dose is associated with increased endothelial expression and prolonged survival during ex-vivo perfusion of GTKO pig lungs with human blood.

INTRODUCTION: Expression of human complement pathway regulatory proteins (hCPRP's) such as CD46 or CD55 has been associated with improved survival of pig organ xenografts in multiple different models. Here we evaluate the hypothesis that an increased human CD46 gene dose, through homozygosity or additional expression of a second hCPRP, is associated with increased protein expression and with improved protection from injury when GTKO lung xenografts are perfused with human blood. METHODS: Twenty three GTKO lungs heterozygous for human CD46 (GTKO.heteroCD46), 10 lungs homozygous for hCD46 (GTKO.homoCD46), and six GTKO.homoCD46 lungs also heterozygous for hCD55 (GTKO.homoCD46.hCD55) were perfused with human blood for up to 4 h in an ex vivo circuit. RESULTS: Relative to GTKO.heteroCD46 (152 min, range 5-240; 6/23 surviving at 4 h), survival was significantly improved for GTKO.homoCD46 (>240 min, range 45-240, p = .034; 7/10 surviving at 4 h) or GTKO.homoCD46.hCD55 lungs (>240 min, p = .001; 6/6 surviving at 4 h). Homozygosity was associated with increased capillary expression of hCD46 (p < .0001). Increased hCD46 expression was associated with significantly prolonged lung survival (p = .048),) but surprisingly not with reduction in measured complement factor C3a. Hematocrit, monocyte count, and pulmonary vascular resistance were not significantly altered in association with increased hCD46 gene dose or protein expression. CONCLUSION: Genetic engineering approaches designed to augment hCPRP activity - increasing the expression of hCD46 through homozygosity or co-expressing hCD55 with hCD46 - were associated with prolonged GTKO lung xenograft survival. Increased expression of hCD46 was associated with reduced coagulation cascade activation, but did not further reduce complement activation relative to lungs with relatively low CD46 expression. We conclude that coagulation pathway dysregulation contributes to injury in GTKO pig lung xenografts perfused with human blood, and that the survival advantage for lungs with increased hCPRP expression is likely attributable to improved endothelial thromboregulation.

A Standardized Approach to Orthotopic (Life-supporting) Porcine Cardiac Xenotransplantation in a Nonhuman Primate Model.

Cardiac xenotransplantation from swine has been proposed to "bridge the gap" in supply for heart failure patients requiring transplantation. Recent preclinical success using genetically modified pig donors in baboon recipients has demonstrated survival greater than 6 mo, with a modern understanding of xenotransplantation immunobiology and continued experience with large animal models of cardiac xenotransplantation. As a direct result of this expertise, the Food and Drug Administration approved the first in-human transplantation of a genetically engineered cardiac xenograft through an expanded access application for a single patient. This clinical case demonstrated the feasibility of xenotransplantation. Although this human study demonstrated proof-of-principle application of cardiac xenotransplantation, further regulatory oversight by the Food and Drug Administration may be required with preclinical trials in large animal models of xenotransplantation with long-term survival before approval of a more formalized clinical trial. Here we detail our surgical approach to pig-to-primate large animal models of orthotopic cardiac xenotransplantation, and the postoperative care of the primate recipient, both in the immediate postoperative period and in the months thereafter. We also detail xenograft surveillance methods and common issues that arise in the postoperative period specific to this model and ways to overcome them. These studies require multidisciplinary teams and expertise in orthotopic transplantation (cardiac surgery, anesthesia, and cardiopulmonary bypass), immunology, genetic engineering, and experience in handling large animal donors and recipients, which are described here. This article serves to reduce the barriers to entry into a field with ever-growing enthusiasm, but demands expertise knowledge and experience to be successful.

Knock-out of N-glycolylneuraminic acid attenuates antibody-mediated rejection in xenogenically perfused porcine lungs.

BACKGROUND: Antibody-mediated rejection has long been known to be one of the major organ failure mechanisms in xenotransplantation. In addition to the porcine α1,3-galactose (α1,3Gal) epitope, N-Glycolylneuraminic acid (Neu5Gc), a sialic acid, has been identified as an important porcine antigen against which most humans have pre-formed antibodies. Here we evaluate GalTKO.hCD46 lungs with an additional cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) gene knock-out (Neu5GcKO) in a xenogeneic ex vivo perfusion model METHODS: Eleven GalTKO.hCD46.Neu5GcKO pig lungs were perfused for up to 6 h with fresh heparinized human blood. Six of them were treated with histamine (H) blocker famotidine and 1-thromboxane synthase inhibitor Benzylimidazole (BIA) and five were left untreated. GalTKO.hCD46 lungs without Neu5GcKO (n = 18: eight untreated and 10 BIA+H treated) served as a reference. Functional parameters, blood, and tissue samples were collected at pre-defined time points throughout the perfusion RESULTS: All but one Neu5GcKO organs maintained adequate blood oxygenation and "survived" until elective termination at 6 h whereas two reference lungs failed before elective termination at 4 h. Human anti-Neu5Gc antibody serum levels decreased during the perfusion of GalTKO.hCD46 lungs by flow cytometry (∼40% IgM, 60% IgG), whereas antibody levels in Neu5GcKO lung perfusions did not fall (IgM p = .007; IgG p < .001). Thromboxane elaboration, thrombin generation, and histamine levels were significantly reduced with Neu5GcKO lungs compared to reference in the untreated groups (p = .007, .005, and .037, respectively); treatment with BIA+H masked these changes. Activation of platelets, measured as CD62P expression on circulating platelets, was lower in Neu5GcKO experiments compared to reference lungs (p = .023), whereas complement activation (as C3a rise in plasma) was not altered. MCP-1 and lactotransferin level elevations were blunted in Neu5GcKO lung perfusions (p = .007 and .032, respectively). Pulmonary vascular resistance (PVR) rise was significantly attenuated and delayed in untreated GalTKO.hCD46.Neu5GcKO lungs in comparison to the untreated GalTKO.hCD46 lungs (p = .003) CONCLUSION: Additional Neu5GcKO in GalTKO.hCD46 lungs significantly reduces parameters associated with antibody-mediated inflammation and activation of the coagulation cascade. Knock-out of the Neu5Gc sialic acid should be beneficial to reduce innate immune antigenicity of porcine lungs in future human recipients.

Progressive genetic modifications of porcine cardiac xenografts extend survival to 9 months.

We report orthotopic (life-supporting) survival of genetically engineered porcine cardiac xenografts (with six gene modifications) for almost 9 months in baboon recipients. This work builds on our previously reported heterotopic cardiac xenograft (three gene modifications) survival up to 945 days with an anti-CD40 monoclonal antibody-based immunosuppression. In this current study, life-supporting xenografts containing multiple human complement regulatory, thromboregulatory, and anti-inflammatory proteins, in addition to growth hormone receptor knockout (KO) and carbohydrate antigen KOs, were transplanted in the baboons. Selective "multi-gene" xenografts demonstrate survival greater than 8 months without the requirement of adjunctive medications and without evidence of abnormal xenograft thickness or rejection. These data demonstrate that selective "multi-gene" modifications improve cardiac xenograft survival significantly and may be foundational for paving the way to bridge transplantation in humans.

Human erythrocyte fragmentation during ex-vivo pig organ perfusion.

Platelet sequestration is a common process during organ reperfusion after transplantation. However, instead of lower platelet counts, when using traditional hemocytometers and light microscopy, we observed physiologically implausible platelet counts in the course of ex-vivo lung and liver xenograft organ perfusion studies. We employed conventional flow cytometry (FC) and imaging FC (AMINS ImageStream X) to investigate the findings and found platelet-sized fragments in the circulation that are mainly derived from red blood cell membranes. We speculate that this erythrocyte fragmentation contributes to anemia during in-vivo organ xenotransplant.

Thromboxane and histamine mediate PVR elevation during xenogeneic pig lung perfusion with human blood.

BACKGROUND: Elevated pulmonary vascular resistance (PVR), platelet adhesion, coagulation activation, and inflammation are prominent features of xenolung rejection. Here, we evaluate the role of thromboxane and histamine on PVR, and their contribution to other lung xenograft injury mechanisms. METHODS: GalTKO.hCD46 single pig lungs were perfused ex vivo with fresh heparinized human blood: lungs were either treated with 1-Benzylimidazole (1-BIA) combined with histamine receptor blocker famotidine (n = 4) or diphenhydramine (n = 6), 1-BIA alone (n = 6) or were left untreated (n = 9). RESULTS: Six of the nine control experiments (GalTKO.hCD46 untreated), "survived" until elective termination at 4 hours. Without treatment, initial PVR elevation within the first 30 minutes resolved partially over the following hour, and increased progressively during the final 2 hours of perfusion. In contrast, 1-BIA, alone or in addition to either antihistamine treatment, was associated with low stable PVR. Combined treatments significantly lowered the airway pressure when compared to untreated reference. Although platelet and neutrophil sequestration and coagulation cascade activation were not consistently altered by any intervention, increased terminal wet/dry weight ratio in untreated lungs was significantly blunted by combined treatments. CONCLUSION: Combined thromboxane and histamine pathway blockade prevents PVR elevation and significantly inhibits loss of vascular barrier function when GalTKO.hCD46 lungs are perfused with human blood. Platelet activation and platelet and neutrophil sequestration persist in all groups despite efficient complement regulation, and appear to occur independent of thromboxane and histamine antagonism. Our work identifies thromboxane and histamine as key mediators of xenolung injury and defines those pathways as therapeutic targets to achieve successful xenolung transplantation.

Pilot Study of Delayed ICOS/ICOS-L Blockade With αCD40 to Modulate Pathogenic Alloimmunity in a Primate Cardiac Allograft Model.

BACKGROUND: Inducible costimulator (ICOS) is rapidly upregulated with T-cell stimulation and may represent an escape pathway for T-cell costimulation in the setting of CD40/CD154 costimulation blockade. Induction treatment exhibited no efficacy in a primate renal allograft model, but rodent transplant models suggest that the addition of delayed ICOS/ICOS-L blockade may prolong allograft survival and prevent chronic rejection. Here, we ask whether ICOS-Ig treatment, timed to anticipate ICOS upregulation, prolongs NHP cardiac allograft survival or attenuates pathogenic alloimmunity. METHODS: Cynomolgus monkey heterotopic cardiac allograft recipients were treated with αCD40 (2C10R4, d0-90) either alone or with the addition of delayed ICOS-Ig (d63-110). RESULTS: Median allograft survival was similar between ICOS-Ig + αCD40 (120 days, 120-125 days) and αCD40 (124 days, 89-178 days) treated animals, and delayed ICOS-Ig treatment did not prevent allograft rejection in animals with complete CD40 receptor coverage. Although CD4+ TEM cells were decreased in peripheral blood (115 ± 24) and mLNs (49 ± 1.9%) during ICOS-Ig treatment compared with monotherapy (214 ± 27%, P = 0.01; 72 ± 9.9%, P = 0.01, respectively), acute and chronic rejection scores and kinetics of alloAb elaboration were similar between groups. CONCLUSIONS: Delayed ICOS-Ig treatment with the reagent tested is probably ineffective in modulating pathogenic primate alloimmunity in this model.

Selective CD28 Inhibition Modulates Alloimmunity and Cardiac Allograft Vasculopathy in Anti-CD154-Treated Monkeys.

BACKGROUND: Selective CD28 inhibition is actively pursued as an alternative to B7 blockade using cytotoxic T lymphocyte antigen 4 Ig based on the hypothesis that the checkpoint immune regulators cytotoxic T lymphocyte antigen 4 and programmed death ligand 1 will induce tolerogenic immune signals. We previously showed that blocking CD28 using a monovalent nonactivating reagent (single-chain anti-CD28 Fv fragment linked to alpha-1 antitrypsin [sc28AT]) synergizes with calcineurin inhibitors in nonhuman primate (NHP) kidney and heart transplantation. Here, we explored the efficacy of combining a 3-week "induction" sc28AT treatment with prolonged CD154 blockade. METHODS: Cynomolgus monkey heterotopic cardiac allograft recipients received sc28AT (10 mg/kg, d0-20, n = 3), hu5C8 (10-30 mg/kg, d0-84, n = 4), or combination (n = 6). Graft survival was monitored by telemetry. Protocol biopsies and graft explants were analyzed for International Society of Heart and Lung Transplantation acute rejection grade and cardiac allograft vasculopathy score. Alloantibody, T-cell phenotype and regulatory T cells were analyzed by flow cytometry. Immunochemistry and gene expression (NanoString) characterized intra-graft cellular infiltration. RESULTS: Relative to modest prolongation of median graft survival time with sc28AT alone (34 days), hu5C8 (133 days), and sc28AT + hu5C8 (141 days) prolonged survival to a similar extent. CD28 blockade at induction, added to hu5C8, significantly attenuated the severity of acute rejection and cardiac allograft vasculopathy during the first 3 months after transplantation relative to hu5C8 alone. These findings were associated with decreased proportions of circulating CD8 and CD3CD28 T cells, and modulation of inflammatory gene expression within allografts. CONCLUSIONS: Induction with sc28AT promotes early cardiac allograft protection in hu5C8-treated NHPs. These results support further investigation of prolonged selective CD28 inhibition with CD40/CD154 blockade in NHP transplants.

Comparative Evaluation of αCD40 (2C10R4) and αCD154 (5C8H1 and IDEC-131) in a Nonhuman Primate Cardiac Allotransplant Model.

BACKGROUND: Specific blockade of T cell costimulation pathway is a promising immunomodulatory approach being developed to replace our current clinical immunosuppression therapies. The goal of this study is to compare results associated with 3 monoclonal antibodies directed against the CD40/CD154 T cell costimulation pathway. METHODS: Cynomolgus monkey heterotopic cardiac allograft recipients were treated with either IDEC-131 (humanized αCD154, n = 9), 5C8H1 (mouse-human chimeric αCD154, n = 5), or 2C10R4 (mouse-rhesus chimeric αCD40, n = 6) monotherapy using a consistent, comparable dosing regimen for 3 months after transplant. RESULTS: Relative to the previously reported IDEC-131-treated allografts, median survival time (35 ± 31 days) was significantly prolonged in both 5C8H1-treated (142 ± 26, P < 0.002) and 2C10R4-treated (124 ± 37, P < 0.020) allografts. IDEC-131-treated grafts had higher cardiac allograft vasculopathy severity scores during treatment relative to either 5C8H1 (P = 0.008) or 2C10R4 (P = 0.0002). Both 5C8H1 (5 of 5 animals, P = 0.02) and 2C10R4 (6/6, P = 0.007), but not IDEC-131 (2/9), completely attenuated IgM antidonor alloantibody (alloAb) production during treatment; 5C8H1 (5/5) more consistently attenuated IgG alloAb production compared to 2C10R4 (4/6) and IDEC-131 (0/9). All evaluable explanted grafts experienced antibody-mediated rejection. Only 2C10R4-treated animals exhibited a modest, transient drop in CD20 lymphocytes from baseline at day 14 after transplant (-457 ± 152 cells/µL) compared with 5C8H1-treated animals (16 ± 25, P = 0.037), and the resurgent B cells were primarily of a naive phenotype. CONCLUSIONS: In this model, CD154/CD40 axis blockade using IDEC-131 is an inferior immunomodulatory treatment than 5C8H1 or 2C10R4, which have similar efficacy to prolong graft survival and to delay cardiac allograft vasculopathy development and antidonor alloAb production during treatment.

Transgenic expression of human leukocyte antigen-E attenuates GalKO.hCD46 porcine lung xenograft injury.

BACKGROUND: Lung xenografts remain susceptible to loss of vascular barrier function within hours in spite of significant incremental advances based on genetic engineering to remove the Gal 1,3-αGal antigen (GalTKO) and express human membrane cofactor protein (hCD46). Natural killer cells rapidly disappear from the blood during perfusion of GalTKO.hCD46 porcine lungs with human blood and presumably are sequestered within the lung vasculature. Here we asked whether porcine expression of the human NK cell inhibitory ligand HLA-E and ß2 microglobulin inhibits GalTKO.hCD46 pig cell injury or prolongs lung function in two preclinical perfusion models. METHODS: Lungs from pigs modified to express GalTKO.hCD46 (n=37) and GalTKO.hCD46.HLA-E (n=5) were harvested and perfused with human blood until failure or elective termination at 4 hours. Airway pressures and pulmonary artery hemodynamics were recorded in real time. Blood samples were also collected throughout the experiment for analysis. Porcine aortic endothelial cells (PAECs) from each genotype were cultured in monolayers in microfluidic channels and used in fluorescent cytotoxicity assays using human NK cells. RESULTS: HLA-E expression on GalTKO.hCD46 PAECs was associated with significantly decreased antibody-dependent and antibody-independent NK-mediated cytotoxicity under in vitro conditions simulating physiologic shear stress. Relative to GalTKO.hCD46 pig lungs perfused with human blood on an ex vivo platform, additional expression of HLA-E increased median lung survival (>4 hours, vs 162 minutes, P=.012), and was associated with attenuated rise in pulmonary vascular resistance, and decreased platelet activation and histamine elaboration. As expected, HLA-E expression was not associated with a significant difference in NK cell adhesion to endothelial cells in vitro, or NK cell and neutrophil sequestration during organ perfusion. CONCLUSIONS: We conclude human NK cell activation contributes significantly to GalTKO.hCD46 pig endothelial injury and lung inflammation and show that expression of HLA-E is associated with physiologically meaningful protection of GalTKO.hCD46 cells and organs exposed to human blood.

Clinical Trypanosoma cruzi Disease after Cardiac Transplantation in a Cynomolgus Macaque (Macaca fascicularis).

A cynomolgus macaque received a heterotopic cardiac allograft as part of a transplant study, with monoclonal antibodies targeted to specific immune costimulation molecules (CD154, CD28) but no traditional immunosuppressive therapy after surgery. Clinical anemia was detected on postoperative day (POD) 35 and had worsened (Hgb, 2.3 g/dL; Hct = 7.3%) by POD 47, despite type-matched whole-blood transfusions. After a total of 4 blood transfusions, hematologic parameters were improved (Hgb, 5.9 g/dL; Hct, 18.7%). On POD 50, a peripheral blood smear revealed trypomastigotes, and qualitative RT-PCR of whole blood identified the organism as Trypanosoma cruzi. Although clinically stable initially, the macaque soon developed sufficient weight loss to necessitate euthanasia on POD 64. The final diagnosis was clinical anemia due to T. cruzi infection. This study represents the first reported case of Chagas disease after heart transplant in a NHP.

Development of a consensus protocol to quantify primate anti-non-Gal xenoreactive antibodies using pig aortic endothelial cells.

BACKGROUND: Scientists working in the field of xenotransplantation do not employ a uniform method to measure and report natural and induced antibody responses to non-Galα(1,3)Gal (non-Gal) epitopes. Such humoral responses are thought to be particularly pathogenic after transplantation of vascularized GalTKO pig organs and having a more uniform assay and reporting format would greatly facilitate comparisons between laboratories. METHODS: Flow cytometry allows examination of antibody reactivity to intact antigens in their natural location and conformation on cell membranes. We have established a simple and reproducible flow cytometric assay to detect antibodies specific for non-Gal pig antigens using primary porcine aortic endothelial cells (pAECs) and cell culture-adapted pAEC cell lines generated from wild type and α1,3galactosyl transferase knockout (GalTKO) swine. RESULTS: The consensus protocol we propose here is based on procedures routinely used in four xenotransplantation centers and was independently evaluated at three sites using shared cells and serum samples. Our observation support use of the cell culture-adapted GalTKO pAEC KO:15502 cells as a routine method to determine the reactivity of anti-non-Gal antibodies in human and baboon serum. CONCLUSIONS: We have developed an assay that allows the detection of natural and induced non-Gal xenoreactive antibodies present in human or baboon serum in a reliable and consistent manner. This consensus assay and format for reporting the data should be accessible to laboratories and will be useful for assessing experimental results between multiple research centers. Adopting this assay and format for reporting the data should facilitate the detection, monitoring, and detailed characterization of non-Gal antibody responses.

Live colonocytes in newborn stool: surrogates for evaluation of gut physiology and disease pathogenesis.

Studies of gastrointestinal pathophysiology are not feasible by biopsies in human neonates. We examined the utility of live colonocytes in stool in studying cellular markers during early neonatal life. Expression of IgA, IgG, cluster of differentiation-45 cells (CD45), and toll-like receptors-2 and 4 (TLR2 and TLR4) were analyzed by flow cytometry. Colonocyte RNA extracts were used in quantitative real-time PCR (qRT-PCR) to examine the expression of cytokeratin-19, ribosomal protein-24, and tight-junction (Tj) protein zonula occludens-1 (ZO-1). Colonocyte yield varied between 5 × 104 to 2 × 106 cells/g of stool. Meconium samples yielded a highly enriched population of viable cells. Although low, all samples showed CD45-positive cells during the initial weeks of life. Starting as early as d 2, IgA expression was observed in 69% of the cells. Low to moderate expression of IgG was observed with a linear increase as the infants grew. There was an almost total lack of TLR2 staining; however, >55% of the colonocytes showed TLR4 expression. Although high levels of IgA in gut cells may serve as a natural protectant during neonatal period, increased TLR4 may provide a niche for lipopolysaccharide (LPS)-mediated epithelial damage. Use of stool colonocytes can be a valuable noninvasive approach for studying gut pathophysiology in the neonatal period.

Probiotic bacteria change Escherichia coli-induced gene expression in cultured colonocytes: Implications in intestinal pathophysiology.

AIM: To investigate the change in eukaryotic gene expression profile in Caco-2 cells after infection with strains of Escherichia coli and commensal probiotic bacteria. METHODS: A 19,200 gene/expressed sequence tag gene chip was used to examine expression of genes after infection of Caco-2 cells with strains of normal flora E. coli, Lactobacillus plantarum, and a combination of the two. RESULTS: The cDNA microarray revealed up-regulation of 155 and down-regulation of 177 genes by E. coli. L. plantarum up-regulated 45 and down-regulated 36 genes. During mixed infection, 27 genes were up-regulated and 59 were down-regulated, with nullification of stimulatory/inhibitory effects on most of the genes. Expression of several new genes was noted in this group. CONCLUSION: The commensal bacterial strains used in this study induced the expression of a large number of genes in colonocyte-like cultured cells and changed the expression of several genes involved in important cellular processes such as regulation of transcription, protein biosynthesis, metabolism, cell adhesion, ubiquitination, and apoptosis. Such changes induced by the presence of probiotic bacteria may shape the physiologic and pathologic responses they trigger in the host.

A novel peroxisome proliferator--activated receptor alpha/gamma dual agonist ameliorates dyslipidemia and insulin resistance in prediabetic rhesus monkeys.

TAK-559, a newly developed non-thiazolidinedione, activates both peroxisome proliferator-activated receptors alpha and gamma. We investigated the effects of TAK-559 on dyslipidemia and insulin resistance in nonhuman primates. Five adult male obese prediabetic rhesus monkeys were studied on vehicle and after TAK-559 treatment (0.3, 1.0, 3.0 mg/kg per day) for a total of 12 weeks. No significant changes were observed in body weight and fasting plasma glucose, total plasma cholesterol, very low-density lipoprotein-triglyceride, and low-density lipoprotein cholesterol levels. TAK-559 treatment resulted in significant elevation of circulating high-density lipoprotein (HDL) cholesterol levels, consisting of an increase in large HDL particles and a decrease in small dense HDL particles. Nuclear magnetic resonance data exhibited a less atherogenic lipoprotein profile with treatment. Plasma triglyceride and apolipoprotein B-100 levels decreased, whereas apolipoprotein A-I increased during TAK-559 treatment. Hyperinsulinemia and insulin resistance (quantitative insulin sensitivity check index and homeostasis model assessment) were significantly corrected with the highest dose of 3.0 mg/kg per day in these prediabetic monkeys. In addition, no adverse effects on representative liver function parameters were observed during the study period. These results suggest that TAK-559 had beneficial effects on lipoprotein profiles and insulin sensitivity, without any side effect on body weight, which suggests that TAK-559 may provide a potentially safe approach for delaying the onset of type 2 diabetes mellitus and may reduce the risk of cardiovascular disease. The positive effects of TAK-559 in nonhuman primates have led to further clinical trials of TAK-559 in Europe and the United States.

Effect of ovarian steroids on basal and oxytocin-induced prostaglandin F2alpha secretion from pig endometrial cells.

These studies were undertaken to determine how treatment with 100 nM progesterone and/or 10 nM oestradiol-17beta acutely (3 h; Experiment 1) or chronically (72 h; Experiments 2-4) influenced basal and oxytocin (OT)-stimulated prostaglandin (PG) F(2alpha) secretion, in enriched cultures of pig endometrial luminal epithelial, glandular epithelial and stromal cells obtained on Day 16 (Experiments 1, 2 and 4) or Day 12 (Experiment 3) after oestrus. In Experiment 1, acute treatment with progesterone stimulated PGF(2alpha) secretion from each cell type on Day 16, whereas acute oestradiol treatment inhibited the stimulatory action of progesterone on PGF(2alpha) secretion only in glandular epithelial cells. In Experiment 2, OT stimulated phospholipase (PL) C activity in luminal epithelial cells on Day 16 only in the presence of chronic oestradiol treatment. For glandular epithelial cells on Day 16, OT stimulated PLC activity only in the presence of chronic treatment with steroid. In stromal cells on Day 16, OT stimulated PLC activity in the absence of steroids and the response to OT was further enhanced by oestradiol. In the absence of chronic treatment with steroid, OT did not stimulate PGF(2alpha) secretion from luminal epithelial cells, but oestradiol induced a response to OT. For glandular epithelial cells, OT-induced PGF(2alpha) secretion was not altered by steroids, whereas the stimulatory response to OT was inhibited by oestradiol or progesterone in stromal cells. For endometrial cells obtained on Day 12 after oestrus in Experiment 3, OT only stimulated PGF(2alpha) release from glandular epithelial and stromal cells. For luminal epithelial cells obtained on Day 16 after oestrus and cultured under polarizing conditions in Experiment 4, secretion of PGF(2alpha) occurred preferentially from the basolateral surface and was stimulated by OT more from the basolateral surface than from the apical surface. Oxytocin-induced PGF(2alpha) secretion from the apical surface was enhanced by chronic treatment with oestradiol, whereas that from the basolateral surface was enhanced by chronic treatment with progesterone. In summary, oestradiol enhanced OT-induced PGF(2alpha) secretion from the apical surface of luminal epithelial cells and reduced the response of stromal cells to OT, actions that may contribute to the reorientation of PGF(2alpha) from endocrine secretion (i.e. towards the uterine vasculature) to exocrine secretion (i.e. towards the uterine lumen) during pregnancy recognition in pigs.

Angiotensin II increases intracellular calcium concentration in pig endometrial stromal cells through type 1 angiotensin receptors, but does not stimulate phospholipase C activity or prostaglandin F2alpha secretion.

Although the presence of endometrial receptors for angiotensin (Ang) II has been demonstrated, a specific function for AngII in the uterus has not been identified. Cytosolic free Ca2+ concentration [Ca2+]i, phospholipase C (PLC) activity and prostaglandin (PG) F2alpha secretion in response to AngII and oxytocin (OT) were measured in pig endometrial stromal cells collected 16 days after oestrus. Treatment with 100 nM OT or AngII increased (P<0.001) [Ca2+]i in stromal cells similarly (720 +/- 34 v. 690 +/- 33 pM, respectively). Subsequent administration of OT or AngII to the same cells induced smaller [Ca2+]i increases (25% or 35% of the initial responses, respectively) that occurred only if the second exposure to the same agent took place at least 5 min after the first. When administered sequentially, OT and AngII each induced a full response within 1 min of the previous treatment, regardless of which peptide was applied first. Whereas OT increased PLC activity and PGF2alpha secretion in stromal cells (P<0.01), AngII did not increase either PLC activity or PGF2alpha secretion. Type I AngII (AT1) receptors were present on stromal cells, whereas AT2 receptors were absent. Therefore, the effect of AngII in stromal cells was mediated via AT1 receptors. That AngII increased [Ca2+]i in stromal cells, but did not increase PLC or PGF2alpha secretion, indicates that either AngII releases a pool of Ca2+ through a mechanism that is not mediated by PLC and is not involved in PGF2alpha secretion or that a mechanism for PGF2alpha production other than one involving Ca2+ may exist.