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Background: Successful preclinical transplantations of porcine hearts into baboon recipients are required before commencing clinical trials. Despite years of research, over half of the orthotopic cardiac xenografts were lost during the first 48 hours after transplantation, primarily caused by perioperative cardiac xenograft dysfunction (PCXD). To decrease the rate of PCXD, we adopted a preservation technique of cold non‐ischemic perfusion for our ongoing pig‐to‐baboon cardiac xenotransplantation project. Methods: Fourteen orthotopic cardiac xenotransplantation experiments were carried out with genetically modified juvenile pigs (GGTA1‐ KO/hCD46/hTBM) as donors and captive‐bred baboons as recipients. Organ preservation was compared according to the two techniques applied: cold static ischemic cardioplegia (IC; n = 5) and cold non‐ischemic continuous perfusion (CP; n = 9) with an oxygenated albumin‐containing hyperoncotic cardioplegic solution containing nutrients, erythrocytes and hormones. Prior to surgery, we measured serum levels of preformed anti‐non‐Gal‐antibodies. During surgery, hemodynamic parameters were monitored with transpulmonary thermodilution. Central venous blood gas analyses were taken at regular intervals to estimate oxygen extraction, as well as lactate production. After surgery, we measured troponine T and serum parameters of the recipient's kidney, liver and coagulation functions. Results: In porcine grafts preserved with IC, we found significantly depressed systolic cardiac function after transplantation which did not recover despite increasing inotropic support. Postoperative oxygen extraction and lactate production were significantly increased. Troponin T, creatinine, aspartate aminotransferase levels were pathologically high, whereas prothrombin ratios were abnormally low. In three of five IC experiments, PCXD developed within 24 hours. By contrast, all nine hearts preserved with CP retained fully preserved systolic function, none showed any signs of PCXD. Oxygen extraction was within normal ranges; serum lactate as well as parameters of organ functions were only mildly elevated. Preformed anti‐non‐Gal‐antibodies were similar in recipients receiving grafts from either IC or CP preservation. Conclusions: While standard ischemic cardioplegia solutions have been used with great success in human allotransplantation over many years, our data indicate that they are insufficient for preservation of porcine hearts transplanted into baboons: Ischemic storage caused severe impairment of cardiac function and decreased tissue oxygen supply, leading to multi‐organ failure in more than half of the xenotransplantation experiments. In contrast, cold non‐ischemic heart preservation with continuous perfusion reliably prevented early graft failure. Consistent survival in the perioperative phase is a prerequisite for preclinical long‐term results after cardiac xenotransplantation. [ABSTRACT FROM AUTHOR]

The activation of the endothelial surface in xenografts is still a poorly understood process and the consequences are unpredictable. The role of Ca2+‐messaging during the activation of endothelial cells is well recognized and routinely measured by synthetic Ca2+‐sensitive fluorophors. However, these compounds require fresh loading immediately before each experiment and in particular when grown in state‐of‐the‐art 3D cell culture systems, endothelial cells are difficult to access with such sensors. Therefore, we developed transgenic pigs expressing a Ca2+‐sensitive protein and examined its principal characteristics. Primary transgenic endothelial cells stimulated by ATP showed a definite and short influx of Ca2+ into the cytosol, whereas exposure to human serum resulted in a more intense and sustained response. Surprisingly, not all endothelial cells reacted identically to a stimulus, rather activation took place in adjacent cells in a timely decelerated way and with distinct intensities. This effect was again more pronounced when cells were stimulated with human serum. Finally, we show clear evidence that antibody binding alone significantly activated endothelial cells, whereas antibody depletion dramatically reduced the stimulatory potential of serum. Transgenic porcine endothelial cells expressing a Ca2+‐sensor represent an interesting tool to dissect factors inducing activation of porcine endothelial cells after exposure to human blood or serum. [ABSTRACT FROM AUTHOR]

Abstract: Background: In pig‐to‐human xenotransplantation, interactions between human natural killer (NK) cells and porcine endothelial cells (pEC) are characterized by recruitment and cytotoxicity. Protection from xenogeneic NK cytotoxicity can be achieved in vitro by the expression of the non‐classical human leukocyte antigen‐E (HLA‐E) on pEC. Thus, the aim of this study was to analyze NK cell responses to vascularized xenografts using an ex vivo perfusion system of pig limbs with human blood. Methods: Six pig forelimbs per group, respectively, stemming from either wild‐type (wt) or HLA‐E/hCD46 double‐transgenic (tg) animals, were perfused ex vivo with heparinized human blood for 12 hours. Blood samples were collected at defined time intervals, cell numbers counted, and peripheral blood mononuclear cells analyzed for phenotype by flow cytometry. Muscle biopsies were analyzed for NK cell infiltration. In vitro NK cytotoxicity assays were performed using pEC derived from wt and tg animals as target cells. Results: Ex vivo, a strong reduction in circulating human CD45 leukocytes was observed after 60 minutes of xenoperfusion in both wt and tg limb groups. NK cell numbers dropped significantly. Within the first 10 minutes, the decrease in NK cells was more significant in the wt limb perfusions as compared to tg limbs. Immunohistology of biopsies taken after 12 hours showed less NK cell tissue infiltration in the tg limbs. In vitro, NK cytotoxicity against hCD46 single tg pEC and wt pEC was similar, while lysis of double tg HLA‐E/hCD46 pEC was significantly reduced. Finally, circulating cells of pig origin were observed during the ex vivo xenoperfusions. These cells expressed phenotypes mainly of monocytes, B and T lymphocytes, NK cells, as well as some activated endothelial cells. Conclusions: Ex vivo perfusion of pig forelimbs using whole human blood represents a powerful tool to study humoral and early cell‐mediated rejection mechanisms of vascularized pig‐to‐human xenotransplantation, although there are several limitations of the model. Here, we show that (i) transgenic expression of HLA‐E/hCD46 in pig limbs provides partial protection from human NK cell‐mediated xeno responses and (ii) the emergence of a pig cell population during xenoperfusions with implications for the immunogenicity of xenografts. [ABSTRACT FROM AUTHOR]

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 (Gal TKO) and express human membrane cofactor protein ( hCD46). Natural killer cells rapidly disappear from the blood during perfusion of Gal TKO. 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 β2 microglobulin inhibits Gal TKO.hCD46 pig cell injury or prolongs lung function in two preclinical perfusion models. Methods Lungs from pigs modified to express Gal TKO.h CD46 (n=37) and Gal TKO.h CD46. 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 Gal TKO.h CD46 PAECs was associated with significantly decreased antibody-dependent and antibody-independent NK-mediated cytotoxicity under in vitro conditions simulating physiologic shear stress. Relative to Gal TKO. 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 Gal TKO.h CD46 pig endothelial injury and lung inflammation and show that expression of HLA-E is associated with physiologically meaningful protection of Gal TKO.h CD46 cells and organs exposed to human blood. [ABSTRACT FROM AUTHOR]

Mutations in the insulin (INS) gene may cause permanent neonatal diabetes mellitus (PNDM). Ins2 mutant mouse models provided important insights into the disease mechanisms of PNDM but have limitations for translational research. To establish a large animal model of PNDM, we generated INSC94Y transgenic pigs. A line expressing high levels of INSC94Y mRNA (70-86% of wild-type INS transcripts) exhibited elevated blood glucose soon after birth but unaltered β-cell mass at the age of 8 days. At 4.5 months, INSC94Y transgenic pigs exhibited 41% reduced body weight, 72% decreased β-cell mass (253% relative to body weight), and 60% lower fasting insulin levels compared with littermate controls. β-cells of INSC94Y transgenic pigs showed a marked reduction of insulin secretory granules and severe dilation of the endoplasmic reticulum. Cataract development was already visible in 8-day-old INSC94Y transgenic pigs and became more severe with increasing age. Diabetes-associated pathological alterations of kidney and nervous tissue were not detected during the observation period of 1 year. The stable diabetic phenotype and its rescue by insulin treatment make the INSC94Y transgenic pig an attractive model for insulin supplementation and islet transplantation trials, and for studying developmental consequences of maternal diabetes mellitus. [ABSTRACT FROM AUTHOR]

The purpose of this study was to establish inducible transgene expression in pigs, a model organism with great promise for experimental physiology and translational medicine, using the binary tet-on system. This expression system is activated by doxycycline (dox) via the tet-controlled transactivator (TA). Binding of TA to the transactivator response element (TRE) results in transcription of downstream genes. First, we cloned transgenic founder pigs expressing TA under the control of the CMV enhancer/chicken β-actin promoter (CAG). Then, cells from CAG-TA transgenic founders were nucleofected with TRE-controlled expression vectors for either porcine cytotoxic T-lymphocyte associated antigen 4-Fc domain of immunoglobulin G1 (CTLA-4Ig) or soluble receptor activator of NF-KB ligand (RANKL), and double-transgenic offspring were cloned. Dox administration resulted in a dose-dependent increase in expression of CTLA-4Ig or RANKL, in nucleofected cells and in transgenic pigs, while in the absence of dox, the levels of both proteins were below the detection limit. Inducible transgene expression was reproduced in double-transgenic offspring generated by cloning or breeding. Our strategy revealed the first two examples of inducible transgene expression in pigs. The CAG-TA transgenic pigs generated in this study constitute an interesting basis for future pig models with inducible transgene expression. [ABSTRACT FROM AUTHOR]

Most MUC5B mucin polymers in the upper airways of humans and pigs are produced by submucosal glands. MUC5B forms N-terminal covalent dimers that are further packed into larger assemblies because of low pH and high Ca2+ in the secretory granule of the mucin-producing cell. We purified the recombinant MUC5B N-terminal covalent dimer and used single- particle electron microscopy to study its structure under intracellular conditions. We found that, at intragranular pH, the dimeric MUC5B organized into head-to-head noncovalent tetramers where the von Willebrand D1-D2 domains hooked into each other. These N-terminal tetramers further formed long linear complexes from which, we suggest, the mucin domains and their C termini project radially outwards. Using conventional and video microscopy, we observed that, upon secretion into the submucosal gland ducts, a flow of bicarbonate- rich fluid passes the mucin-secreting cells. We suggest that this unfolds and pulls out the MUC5B assemblies into long linear threads. These further assemble into thicker mucin bundles in the glandular ducts before emerging at the gland duct opening. We conclude that the combination of intracellular packing of the MUC5B mucin and the submucosal gland morphology creates an efficient machine for producing linear mucin bundles. [ABSTRACT FROM AUTHOR]

Excessive lung inflammation and airway epithelial damage are hallmarks of human inflammatory lung diseases, such as cystic fibrosis (CF). Enhancement of innate immunity provides protection against pathogens while reducing lung-damaging inflammation. However, the mechanisms underlying innate immunity-mediated protection in the lung remain mysterious, in part because of the lack of appropriate animal models for these human diseases. TLR5 (Toll-like receptor 5) stimulation by its specific ligand, the bacterial protein flagellin, has been proposed to enhance protection against several respiratory infectious diseases, although other cellular events, such as calcium signaling, may also control the intensity of the innate immune response. Here, we investigated the molecular events prompted by stimulation with flagellin and its role in regulating innate immunity in the lung of the pig, which is anatomically and genetically more similar to humans than rodent models. We found that flagellin treatment modulated NF-κB signaling and intracellular calcium homeostasis in airway epithelial cells. Flagellin pretreatment reduced the NF-κB nuclear translocation and the expression of proinflammatory cytokines to a second flagellin stimulus as well as to Pseudomonas aeruginosa infection. Moreover, in vivo administration of flagellin decreased the severity of P. aeruginosa -induced pneumonia. Then we confirmed these beneficial effects of flagellin in a pathological model of CF by using ex vivo precision-cut lung slices from a CF pigz model. These results provide evidence that flagellin treatment contributes to a better regulation of the inflammatory response in inflammatory lung diseases such as CF.

Myocardial infarction is a major clinical challenge for interventional, pharmacological, and potential molecular treatment of the ischemic insult. A large animal model with clinic-derived instrumentation allows for detailed imitation of interventional catheterization routines and application routes, whereas similar anatomy and heart proportions raise the possibility to precisely evaluate the efficacy of application modes, e.g., antegrade or retrograde intracoronary application of locally acting pharmaceutical agents, viruses, and cells. Here, we describe the techniques of left ventricular catheterization and induction of ischemia and reperfusion, as well as hemodynamic monitoring and regional application of therapeutic agents in pigs.

The activation of the endothelial surface in xenografts is still a poorly understood process and the consequences are unpredictable. The role of Ca 2+ -messaging during the activation of endothelial cells is well recognized and routinely measured by synthetic Ca 2+ -sensitive fluorophors. However, these compounds require fresh loading immediately before each experiment and in particular when grown in state-of-the-art 3D cell culture systems, endothelial cells are difficult to access with such sensors. Therefore, we developed transgenic pigs expressing a Ca 2+ -sensitive protein and examined its principal characteristics. Primary transgenic endothelial cells stimulated by ATP showed a definite and short influx of Ca 2+ into the cytosol, whereas exposure to human serum resulted in a more intense and sustained response. Surprisingly, not all endothelial cells reacted identically to a stimulus, rather activation took place in adjacent cells in a timely decelerated way and with distinct intensities. This effect was again more pronounced when cells were stimulated with human serum. Finally, we show clear evidence that antibody binding alone significantly activated endothelial cells, whereas antibody depletion dramatically reduced the stimulatory potential of serum. Transgenic porcine endothelial cells expressing a Ca 2+ -sensor represent an interesting tool to dissect factors inducing activation of porcine endothelial cells after exposure to human blood or serum., (© 2020 The Authors. Xenotransplantation published by John Wiley & Sons Ltd.)

Alongside the obesity epidemic, the prevalence of maternal diabetes is rising worldwide, and adverse effects on fetal development and metabolic disturbances in the offspring's later life have been described. To clarify whether metabolic programming effects are due to mild maternal hyperglycemia without confounding obesity, we investigated wild-type offspring of INS C93S transgenic pigs, which are a novel genetically modified large-animal model expressing mutant insulin (INS) C93S in pancreatic β-cells. This mutation results in impaired glucose tolerance, mild fasting hyperglycemia and insulin resistance during late pregnancy. Compared with offspring from wild-type sows, piglets from hyperglycemic mothers showed impaired glucose tolerance and insulin resistance (homeostatic model assessment of insulin resistance: +3-fold in males; +4.4-fold in females) prior to colostrum uptake. Targeted metabolomics in the fasting and insulin-stimulated state revealed distinct alterations in the plasma metabolic profile of piglets from hyperglycemic mothers. They showed increased levels of acylcarnitines, gluconeogenic precursors such as alanine, phospholipids (in particular lyso-phosphatidylcholines) and α-aminoadipic acid, a potential biomarker for type 2 diabetes. These observations indicate that mild gestational hyperglycemia can cause impaired glucose tolerance, insulin resistance and associated metabolic alterations in neonatal offspring of a large-animal model born at a developmental maturation status comparable to human babies., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)