| 1. |
- Bongoni, Anjan K., et al.
(författare)
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Surface modification of pig endothelial cells with a branched heparin conjugate improves their compatibility with human blood
- 2017
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Ingår i: Scientific Reports. - : NATURE PUBLISHING GROUP. - 2045-2322. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- Corline Heparin Conjugate (CHC), a compound of multiple unfractionated heparin chains, coats cells with a glycocalyx-like layer and may inhibit (xeno) transplant-associated activation of the plasma cascade systems. Here, we investigated the use of CHC to protect WT and genetically modified (GTKO. hCD46. hTBM) pig aortic endothelial cells (PAEC) in two pig-to-human in vitro xenotransplantation settings. Model 1: incubation of untreated or hTNFa-treated PAEC with 10% human plasma induced complement C3b/c and C5b-9 deposition, cellular activation and coagulation activation in WT and GTKO. hCD46. hTBM PAEC. Coating of untreated or hTNFa-treated PAEC with CHC (100 mu g/ml) protected against human plasma-induced endothelial activation and damage. Model 2: PAEC were grown on microcarrier beads, coated with CHC, and incubated with non-anticoagulated whole human blood. Genetically modified PAEC significantly prolonged clotting time of human blood (115.0 +/- 16.1 min, p < 0.001) compared to WT PAEC (34.0 +/- 8.2 min). Surface CHC significantly improved the human blood compatibility of PAEC, as shown by increased clotting time (WT: 84.3 +/- 11.3 min, p < 0.001; GTKO. hCD46. hTBM: 146.2 +/- 20.4 min, p < 0.05) and reduced platelet adhesion, complement activation, coagulation activation and inhibition of fibrinolysis. The combination of CHC coating and genetic modification provided the greatest compatibility with human blood, suggesting that pre-transplant perfusion of genetically modified porcine organs with CHC may benefit post-transplant xenograft function.
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| 2. |
- Diswall, Mette, 1979, et al.
(författare)
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Structural characterization of alpha1,3-galactosyltransferase knockout pig heart and kidney glycolipids and their reactivity with human and baboon antibodies.
- 2010
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Ingår i: Xenotransplantation. - : Wiley. - 1399-3089 .- 0908-665X. ; 17:1, s. 48-60
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: alpha1,3-galactosyltranferase knockout (GalT-KO) pigs have been established to avoid hyperacute rejection in GalT-KO pig-to-human xenotransplantation. GalT-KO pig heart and kidney glycolipids were studied focusing on elimination of Gal-antigens and whether novel antigens would appear. Non-human primates are used as pre-clinical transplantation experimental models. Therefore, sera from baboons transplanted with GalT-KO hearts were compared with human serum regarding reactivity with pig glycolipids. METHODS: Neutral and acidic glycolipids were isolated from GalT-KO and WT pig hearts and kidneys. Glycolipid immune reactivity was tested on TLC plates using human affinity-purified anti-Gal Ig, anti-blood group monoclonal antibodies, lectins, and human serum as well as baboon serum collected before and after GalT-KO pig heart transplantations. Selected glycolipid fractions, isolated by HPLC, were structurally characterized by mass spectrometry and proton NMR spectroscopy. RESULTS: GalT-KO heart and kidney lacked alpha3Gal-terminated glycolipids completely. Levels of uncapped N-acetyllactosamine precursor compounds, blood group H type 2 core chain compounds, the P1 antigen and the x(2) antigen were increased. Human serum antibodies reacted with Gal-antigens and N-glycolylneuraminic acid (NeuGc) in WT organs of which only the NeuGc reactivity remained in the GalT-KO tissues. A clear difference in reactivity between baboon and human antibodies with pig glycolipids was found. This was most pronounced for acidic, not yet identified, compounds in GalT-KO organs which were less abundant or lacking in the corresponding WT tissues. CONCLUSIONS: GalT-KO pig heart and kidney completely lacked Gal glycolipid antigens whilst glycolipids synthesized by competing pathways were increased. Baboon and human serum antibodies showed a different reactivity pattern to pig glycolipid antigens indicating that non-human primates have limitations as a human pre-clinical model for immune rejection studies.
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| 3. |
- Längin, Matthias, et al.
(författare)
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Cold non-ischemic heart preservation with continuous perfusion prevents early graft failure in orthotopic pig-to-baboon xenotransplantation
- 2021
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Ingår i: Xenotransplantation. - : Wiley. - 0908-665X .- 1399-3089. ; 28:1
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 4. |
- Längin, Matthias, et al.
(författare)
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Consistent success in life-supporting porcine cardiac xenotransplantation
- 2018
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Ingår i: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 564:7736, s. 430-433
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Tidskriftsartikel (refereegranskat)abstract
- Heart transplantation is the only cure for patients with terminal cardiac failure, but the supply of allogeneic donor organs falls far short of the clinical need1–3. Xenotransplantation of genetically modified pig hearts has been discussed as a potential alternative4. Genetically multi-modified pig hearts that lack galactose-α1,3-galactose epitopes (α1,3-galactosyltransferase knockout) and express a human membrane cofactor protein (CD46) and human thrombomodulin have survived for up to 945 days after heterotopic abdominal transplantation in baboons5. This model demonstrated long-term acceptance of discordant xenografts with safe immunosuppression but did not predict their life-supporting function. Despite 25 years of extensive research, the maximum survival of a baboon after heart replacement with a porcine xenograft was only 57 days and this was achieved, to our knowledge, only once6. Here we show that α1,3-galactosyltransferase-knockout pig hearts that express human CD46 and thrombomodulin require non-ischaemic preservation with continuous perfusion and control of post-transplantation growth to ensure long-term orthotopic function of the xenograft in baboons, the most stringent preclinical xenotransplantation model. Consistent life-supporting function of xenografted hearts for up to 195 days is a milestone on the way to clinical cardiac xenotransplantation7.
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| 5. |
- Längin, Matthias, et al.
(författare)
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Xenografts Show Signs of Concentric Hypertrophy and Dynamic Left Ventricular Outflow Tract Obstruction after Orthotopic Pig-to-baboon Heart Transplantation
- 2023
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Ingår i: Transplantation. - 0041-1337. ; 107:12, s. 328-338
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Tidskriftsartikel (refereegranskat)abstract
- Background. Orthotopic cardiac xenotransplantation has seen substantial advancement in the last years and the initiation of a clinical pilot study is close. However, donor organ overgrowth has been a major hurdle for preclinical experiments, resulting in loss of function and the decease of the recipient. A better understanding of the pathogenesis of organ overgrowth after xenotransplantation is necessary before clinical application. Methods. Hearts from genetically modified (GGTA1-KO, hCD46/hTBM transgenic) juvenile pigs were orthotopically transplanted into male baboons. Group I (control, n = 3) received immunosuppression based on costimulation blockade, group II (growth inhibition, n = 9) was additionally treated with mechanistic target of rapamycin inhibitor, antihypertensive medication, and fast corticoid tapering. Thyroid hormones and insulin-like growth factor 1 were measured before transplantation and before euthanasia, left ventricular (LV) growth was assessed by echocardiography, and hemodynamic data were recorded via a wireless implant. Results. Insulin-like growth factor 1 was higher in baboons than in donor piglets but dropped to porcine levels at the end of the experiments in group I. LV mass increase was 10-fold faster in group I than in group II. This increase was caused by nonphysiological LV wall enlargement. Additionally, pressure gradients between LV and the ascending aorta developed, and signs of dynamic left ventricular outflow tract (LVOT) obstruction appeared. Conclusions. After orthotopic xenotransplantation in baboon recipients, untreated porcine hearts showed rapidly progressing concentric hypertrophy with dynamic LVOT obstruction, mimicking hypertrophic obstructive cardiomyopathy in humans. Antihypertensive and antiproliferative drugs reduced growth rate and inhibited LVOT obstruction, thereby preventing loss of function.
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| 6. |
- Reichart, Bruno, et al.
(författare)
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Pig-to-non-human primate heart transplantation : The final step toward clinical xenotransplantation?
- 2020
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Ingår i: Journal of Heart and Lung Transplantation. - : Elsevier BV. - 1053-2498. ; 39:8, s. 751-757
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Tidskriftsartikel (refereegranskat)abstract
- Background: The demand for donated human hearts far exceeds the number available. Xenotransplantation of genetically modified porcine organs provides an alternative. In 2000, an Advisory Board of the International Society for Heart and Lung Transplantation set the benchmark for commencing clinical cardiac xenotransplantation as consistent 60% survival of non-human primates after life-supporting porcine heart transplantations. Recently, we reported the stepwise optimization of pig-to-baboon orthotopic cardiac xenotransplantation finally resulting in consistent success, with 4 recipients surviving 90 (n = 2), 182, and 195 days. Here, we report on 4 additional recipients, supporting the efficacy of our procedure. Results: The first 2 additional recipients succumbed to porcine cytomegalovirus (PCMV) infections on Days 15 and 27, respectively. In 2 further experiments, PCMV infections were successfully avoided, and 3-months survival was achieved. Throughout all the long-term experiments, heart, liver, and renal functions remained within normal ranges. Post-mortem cardiac diameters were slightly increased when compared with that at the time of transplantation but with no detrimental effect. There were no signs of thrombotic microangiopathy. The current regimen enabled the prolonged survival and function of orthotopic cardiac xenografts in altogether 6 of 8 baboons, of which 4 were now added. These results exceed the threshold set by the Advisory Board of the International Society for Heart and Lung Transplantation. Conclusions: The results of our current and previous experimental cardiac xenotransplantations together fulfill for the first time the pre-clinical efficacy suggestions. PCMV-positive donor animals must be avoided.
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