Dawn Parsell

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

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 β2 microglobulin inhibits GalTKO.hCD46 pig cell injury or prolongs lung function in two preclinical perfusion models.

Recellularization via the bile duct supports functional allogenic and xenogenic cell growth on a decellularized rat liver scaffold

ABSTRACT Recent years have seen a proliferation of methods leading to successful organ decellularization. In this experiment we examine the feasibility of a decellularized liver construct to support growth of functional multilineage cells. Bio-chamber systems were used to perfuse adult rat livers with 0.1% SDS for 24 hours yielding decellularized liver scaffolds. Initially, we recellularized liver scaffolds using a human tumor cell line (HepG2, introduced via the bile duct). Subsequent studies were performed using either human tumor cells co-cultured with human umbilical vein endothelial cells (HUVECs, introduced via the portal vein) or rat neonatal cell slurry (introduced via the bile duct). Bio-chambers were used to circulate oxygenated growth medium via the portal vein at 37C for 5-7 days. Human HepG2 cells grew readily on the scaffold (n = 20). HepG2 cells co-cultured with HUVECs demonstrated viable human endothelial lining with concurrent hepatocyte growth (n = 10). In the series of neonatal cell slurry infusion (n = 10), distinct foci of neonatal hepatocytes were observed to repopulate the parenchyma of the scaffold. The presence of cholangiocytes was verified by CK-7 positivity. Quantitative albumin measurement from the grafts showed increasing albumin levels after seven days of perfusion. Graft albumin production was higher than that observed in traditional cell culture. This data shows that rat liver scaffolds support human cell ingrowth. The scaffold likewise supported the engraftment and survival of neonatal rat liver cell slurry. Recellularization of liver scaffolds thus presents a promising model for functional liver engineering.

Platelet sequestration and activation during GalTKO.hCD46 pig lung perfusion by human blood is primarily mediated by GPIb, GPIIb/IIIa, and von Willebrand Factor

Here, we ask whether platelet GPIb and GPIIb/IIIa receptors modulate platelet sequestration and activation during GalTKO.hCD46 pig lung xenograft perfusion.

N-glycolylneuraminic acid knockout reduces erythrocyte sequestration and thromboxane elaboration in an ex vivo pig-to-human xenoperfusion model

Wild‐type pigs express several carbohydrate moieties on their cell surfaces that differ from those expressed by humans. This difference in profile leads to pig tissue cell recognition of human blood cells causing sequestration, in addition to antibody‐mediated xenograft injury. One such carbohydrate is N‐glycolylneuraminic acid (Neu5Gc), a sialic acid molecule synthesized in pigs but not in humans. Here, we evaluate livers with and without Neu5Gc in an ex vivo liver xeno perfusion model.

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

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.

Progress in Xenogeneic Lung Transplantation Using Multi-Transgenic Donor Pigs and Targeted Supportive Drug Treatments

Purpose Significant progress in genetic engineering has enabled the generation of knock-out pigs that additionally express multiple human “transgenes” chosen to address various known xenogeneic rejection pathways. We evaluated which transgene combinations are associated with prevention of lung xenograft injury in a rigorous life-supporting pig-to-baboon lung xenograft model. Methods GTKO.hCD46 porcine donor organs with up to 4 additional genetic modifications, including hCD55, hTBM, hEPCR, hTFPI, hCD47, hCD39, hHO-1, HLA-E, A20 and humanized vWF were used in 48 left single lung transplants into baboons. A rationally designed “platform drug regimen” consisted of steroids, sC1Inh, thromboxane synthase inhibitor, histamine receptor blockers, and anti-GPIb Fab. Desmopressin (DDAVP) was given to the donor animals prior to lung procurement, to deplete pig endothelial VWF. Immunosuppression consisted of aCD20, ATG, MMF, aCD40, coupled with anti-IL6R moAb and/or Alpha-1 Antitrypsin. Xenograft function was assessed intermittently by transplant blood flow measurements and radiographs. Results Xenogeneic lungs with 6 genetic modifications (6-GE) transplanted into baboons survived for up to 8d (hCD55.hEPCR.hTBM.hCD39) and 9d (hEPCR.hTBM.hCD47.HO-1), vs <12hrs for most 3- or 4-GE lungs. Several other donor transgene-combinations resulted in 47d survival. Lungs of GTKO.hCD46.hCD55.hEPCR.hCD47.hTFPI and a few 3- or 4-GE phenotypes (eg including HLA-E and hvWF) consistently exhibited life-supporting (LS) lung function for 24-30h, with relatively normal macro- and microscopic lung appearance until 2-3 d. Lung xenograft failure was usually associated with rebounding anti-pig antibody titer and loss of lung vascular barrier function leading to alveolar flooding and consolidation, consistent with “delayed xenograft rejection”. Accumulated evidence suggests that, in addition to anti-non-Gal antibody, recipient NK cells (HLA-E) and donor macrophage activation (thromboxane) each play important roles to drive the residual inflammation. Conclusion Combining multi-transgenic donor organs with mechanism-directed drug treatments significantly prolongs life-supportive lung xenograft function and recipient survival. Aggregation of existing targeted genetic modifications, along with antibody depletion and mechanism-based drug additions appear likely to successfully control known pathogenic pathways, and further advance lung xenotransplantation towards clinical application. United Therapeutics SRA. NIH U19A1090959.

Genetic Engineering in Xenotransplantation: What Human Transgenes Should the GTKO.hCD46 Pig Lung Donor Additionally Express?

Background Recent advances in genetic engineering have enabled generation of pigs expressing multiple transgenes as potential donors for xenotransplantation. What transgenes contribute to protect xenogeneic lungs from being injured by known xenorejection mechanisms, has not previously been systematically evaluated. Here we summarize results directly testing several individual transgenes on a platform GTKO.hCD46 in a xenogeneic lung perfusion model. Methods GTKO.hCD46 pig lungs, additionally expressing either hEPCR, HLAE, hvWF, hTBM, hCD55 or including Neu5GC knock-out were perfused with fresh heparinized human blood for up to 8 hours. Functional parameters, as well as blood and tissue samples were analyzed and compared to lungs that did not have the respective additional genetic modification. Results Lung “survival” was significantly increased in lungs with hEPCR and HLA-E. While HLA-E, hEPCR and hTBM expression led to reduced BTG elaboration, platelet sequestration was only markedly reduced by the expression of humanized vWF. Pulmonary vascular resistance as well as histamine elaboration showed significantly lower values in lungs with HLA-E or Neu5GcKO. The knock-out of the Neu5Gc epitope also significantly reduced thromboxane levels and activation of the coagulation cascade (F1+2). Conclusion Several mechanisms associated with GTKO.hCD46 xenolung injury are modulated by additional expression of individual pathway-targeting human transgenes. We conclude that each of the pathways targeted by the transgenes tested, including Neu5GcKO, provide protection from non-Gal-antibody-mediated lung rejection mechanisms. We hypothesize that combinations of the beneficial genetic modifications tested here will result in further improvement of xenogeneic lung function and extension of graft survival. United Therapeutics SRA. NIH U19A1090959.

Feasibility of basic transesophageal echocardiography in hemorrhagic shock: potential applications during resuscitative endovascular balloon occlusion of the aorta (REBOA)

BackgroundThere are numerous studies in the cardiovascular literature that have employed transesophageal echocardiography (TEE) in swine models, but data regarding the use of basic TEE in swine models is limited. The primary aim of this study is to describe an echocardiographic method that can be used with relative ease to qualitatively assess cardiovascular function in a porcine hemorrhagic shock model using resuscitative endovascular balloon occlusion of the aorta (REBOA).MethodsMultiplane basic TEE exams were performed in 15 during an experimental hemorrhage model using REBOA. Cardiac anatomical structure and functional measurements were obtained. In a convenience sample (two animals from each group), advanced functional cardiovascular measurements were obtained before and after REBOA inflation for comparison with qualitative assessments.ResultsBasic TEE exams were performed in 15 swine. Appropriate REBOA placement was confirmed using TEE in all animals and verified with fluoroscopy. Left ventricular volume was decreased in all animals, and left ventricular systolic function increased following REBOA inflation. Right ventricular systolic function and volume remained normal prior to and after hemorrhage and REBOA use. Mean ejection fraction (EF) decreased from 64% (S.D. 9.6) to 62.1 (S.D. 16.8) after hemorrhage and REBOA inflation (p = 0.76); fractional area of change (FAC) decreased from 49.8 (S.D. 9.0) to 48.5 (S.D. 13.6) after hemorrhage and REBOA inflation (p = 0.82).ConclusionBasic TEE, which requires less training than advanced TEE, may be employed by laboratory investigators and practitioners across a wide spectrum of experimental and clinical settings.

2582: Bone marrow regulated local protective mechanisms of vascularized composite allografts in non-human primates

2582: Bone marrow regulated local protective mechanisms of vascularized composite allografts in non-human primates Mehmet C. Uluer, MD, ScM, Arthur J. Nam, Jhade D. Woodall, MD, Wessam Hassanein, MD, David A. Bruno, MD, Dawn Parsell, BS, Urmil Drhu, BS, Branko Bojovic, Stephen T. Bartlett, MD, and Rolf N. Barth, MD University of Maryland School of Medicine, Baltimore, MD, USA; University of Maryland Medical Center, Baltimore, MD, USA Background Vascularized composite allografts (VCA) containing vascularized bone marrow (VBM) prolongs graft survival Cell populations within the co-transplanted VBM are believed to protect the skin and muscle graft components We performed experiments to define these mechanisms in our established non-human primate model of facial transplantation. Methods Three experimental groups were performed of facial VCA Group 1: VCACVBM segments to MHCmismatched cynomolgus macaques after donor irradiation (n D 4) Group 2: VCA without bone C heterotopic VBM without skin (n D 4) Group 3: VCACVBM after donor lymphodepletion with ATGAM (n D 2) Immunosuppression consisted of tacrolimus and mycophenolate mofetil End points were graft rejection or PTLD Chimerism was assayed using donor specific anti-MHC antibodies. Results Group 1 recipients underwent early graft loss (mean 32 § 21 d) from rejection (Banff IV) Chimerism was undetectable in 3 animals Donor VBM demonstrated replacement with recipient cells (50%, 70%, 100%, and 100%) Group 2 recipients also experienced early skin rejection (mean 37§18d), while one animal had PTLD Chimerism was likewise undetectable and heterotopic VBM demonstrated fibrosis and recipient replacement Of the 2 preliminary group 3 recipients one rejected early at day 12, while the other developed PTLD Complete replacement of the bone marrow was seen in the early rejecting animal These data compared to historical VCACVBM grafts with mean survival of 348§ 86 days (rejection only after immunosuppression withdrawal), 75% chimerism, and viable donor VBM. Conclusions Facial VCACVBM contain cell populations that protect the graft from early rejection and graft loss Our data support that these cell populations are radiosensitive, and do not confer systemic protective effects These results support a hypothesis that regulatory cell populations within VBM have local down-regulatory functions toward graft infiltrating alloreactive lymphocytes The finding that cells are noted to be susceptible to depletion or downregulation of their protective effect by irradiation should be kept in consideration when choosing pre-operative conditioning regimens and treating rejection in VCA. CONTACT Mehmet C. Uluer, MD, ScM mculuer@gmail.com