Absence of Gal epitope prolongs survival of swine lungs in an ex vivo model of hyperacute rejection

Nguyen B‐NH, Azimzadeh AM, Schroeder C, Buddensick T, Zhang T, Laaris A, Cochrane M, Schuurman H‐J, Sachs DH, Allan JS, Pierson RN. Absence of Gal epitope prolongs survival of swine lungs in an ex vivo model of hyperacute rejection. Xenotransplantation 2011; 18: 94–107. © 2011 John Wiley & Sons A/S. Abstract: Background: Galactosyl transferase gene knock‐out (GalTKO) swine offer a unique tool to evaluate the role of the Gal antigen in xenogenic lung hyperacute rejection. Methods: We perfused GalTKO miniature swine lungs with human blood. Results were compared with those from previous studies using wild‐type and human decay‐accelerating factor‐transgenic (hDAF^+/+) pig lungs. Results: GalTKO lungs survived 132 ± 52 min compared to 10 ± 9 min for wild‐type lungs (P = 0.001) and 45 ± 60 min for hDAF^+/+ lungs (P = 0.18). GalTKO lungs displayed stable physiologic flow and pulmonary vascular resistance (PVR) until shortly before graft demise, similar to autologous perfusion, and unlike wild‐type or hDAF^+/+ lungs. Early (15 and 60 min) complement (C3a) and platelet activation and intrapulmonary platelet deposition were significantly diminished in GalTKO lungs relative to wild‐type or hDAF^+/+ lungs. However, GalTKO lungs adsorbed cytotoxic anti‐non‐Gal antibody and elaborated high levels of thrombin; their demise was associated with increased PVR, capillary congestion, intravascular thrombi and strong CD41 deposition not seen at earlier time points. Conclusions: In summary, GalTKO lungs are substantially protected from injury but, in addition to anti‐non‐Gal antibody and complement, platelet adhesion and non‐physiologic intravascular coagulation contribute to Gal‐independent lung injury mechanisms.     

A soluble chimeric inhibitor of C3 and C5 convertases, complement activation blocker-2, prolongs graft survival in pig-to-rhesus monkey heart transplantation

Complement plays a critical role in many pathologic processes and in xenograft rejection. Therefore, effective complement inhibitors are of great interest. In pig-to-primate organ transplantation, hyperacute rejection results from antibody deposition and complement activation. Complement activation blocker-2 (CAB-2), a recombinant soluble chimeric protein derived from human decay accelerating factor (DAF) and membrane cofactor protein, inhibits C3 and C5 convertases of both classical and alternative pathways. CAB-2 reduces complement-mediated tissue injury of a pig heart perfused ex vivo with human blood. Therefore, we studied the efficacy of CAB-2 when a pig heart is transplanted heterotopically into rhesus monkeys receiving no immunosuppression. Graft survival in three control monkeys was 1.26 ± 0.2 h; it was markedly prolonged in eight monkeys that received CAB-2. Of the six monkeys that received a single dose of CAB-2 (15 mg/kg i.v.), four had graft survivals of 21, 95, 96, and 108 h, and two died at 7 to11 h post-transplant with a beating graft, as a result of technical complications. The two monkeys given multiple doses of CAB-2 had graft survivals of 95 and 96 h. CAB-2 markedly inhibited complement activation, as shown by a strong reduction in generation of C3a and SC5b-9. At graft rejection, tissue deposition of iC3b, C4 and C9 was similar or slightly reduced from controls, and deposition of IgG, IgM, C1q and fibrin did not change. Thus, complement inhibition with CAB-2 abrogates hyperacute rejection of pig hearts transplanted into rhesus monkeys, but does not prevent delayed/acute vascular rejection. These studies demonstrate that the beneficial effects of complement inhibition on survival of a pig heart xenograft in rhesus monkeys are similar to those in other primate species and that CAB-2 may be useful in xenotransplantation and other complement-mediated conditions.     

Extended ex vivo normothermic perfusion for preservation of vascularized composite allografts

Ischemia and reperfusion injury remains a significant limiting factor for the successful revascularization of amputated extremities. Ex vivo normothermic perfusion is a novel approach to prolong the viability of the amputated limbs by maintaining physiologic cellular metabolism. This study aimed to evaluate the outcomes of extended ex vivo normothermic limb perfusion (EVNLP) in preserving the viability of amputated limbs for over 24 hours. A total of 10 porcine forelimbs underwent EVNLP. Limbs were perfused using an oxygenated colloid solution at 38°C containing washed RBCs. Five forelimbs (Group A) were perfused for 12 hours and the following 5 (Group B) until the vascular resistance increased. Contralateral forelimbs in each group were preserved at 4°C as a cold storage control group. Limb viability was compared between the 2 groups through assessment of muscle contractility, compartment pressure, tissue oxygen saturation, indocyanine green (ICG) angiography and thermography. EVNLP was performed for 12 hours in group A and up to 44 hours (24-44 hours) in group B. The final weight increase (−1.28 ± 8.59% vs. 7.28 ± 15.05%, P = .548) and compartment pressure (16.50 ± 8.60 vs. 24.00 ± 9.10) (P = .151) were not significantly different between the two groups. Final myoglobin and CK mean values in group A and B were: 875.0 ± 325.8 ng/mL (A) versus 1133.8 ± 537.7 ng/mL (B) (P = .056) and 53 344.0 ± 16 603.0 U/L versus 64 333.3 ± 32 481.8 U/L (P = .286). Tissue oxygen saturation was stable until the end in both groups. Infra-red thermography and ICG-angiography detected variations of peripheral limb perfusion. Our results suggest that extended normothermic preservation of amputated limbs is feasible and that the outcomes of prolonged EVNLP (>24 hours) are not significantly different from short EVNLP (12 hours).     

Protease inhibitor nafamostat mesilate attenuates complement activation and improves function of xenografts in a discordant lung perfusion model

Tagawa T. Protease inhibitor nafamostat mesilate attenuates complement activation and improves function of xenografts in a discordant lung perfusion model. Xenotransplantation 2011; 18: 315–319. © 2011 John Wiley & Sons A/S. Abstract: Background: Anti‐complement activity of nafamostat mesilate (FUT‐175) is strong including its variety of pharmacological effects. The effect of FUT‐175 for xenografts in an ex vivo guinea pig‐to‐rat lung perfusion model was evaluated. Methods: Heparinized Lewis rat blood was used to perfuse the lungs in three groups (n = 6 each). Group I used Lewis rat left lung for donor, Group X used guinea pig left lung for donor, and Group XF used guinea pig left lung for donor, which was perfused with Lewis rat blood with 0.2 mg/ml of FUT‐175. Complement activity causing 50% hemolysis (CH50) in the perfusion blood and pulmonary function either before or during perfusion were serially measured. Pathological assessments of the lungs were also carried out after perfusion. Results: The duration of satisfactory pulmonary function was significantly increased in Group XF. Complement activity causing 50% hemolysis in Group XF decreased more significantly compared to Group X. FUT‐175 suppressed both the increase in pulmonary arterial pressure and airway resistance, and the decrease in dynamic lung compliance. In Group X, pathology showed intra‐alveolar hemorrhage, perivascular edema, and medial thickening with endothelial swelling of the pulmonary arteries. In Group XF, less changes were observed compared to Group X. Group X showed deposition of IgM, IgG, and C3 at the endothelium of arteries, which was fewer in Group XF, and even fewer in Group I. Conclusions: This study suggests that FUT‐175 inhibited complement activation and improved lung xenograft function. FUT‐175 ameliorates hyperacute rejection in a guinea pig‐to‐rat ex vivo xenogeneic lung perfusion model.     

Sheep lung as xenograft oxygenator of human blood: An ex vivo study

Abstract: In order to study whether sheep lung could be used as a suitable oxygenator for human blood, the left lungs of two groups of five sheep were used in an "isolated perfused working lung model system." A desaturator was inserted in the circuit and the lung was ventilated with ambient air. The lung was perfused in group 1 with heparinated autologous sheep blood and in group 2 with washed human erythrocytes suspended in buffered gelatin. Blood oxygenation as well as hemodynamics and airway parameters followed similar time course in both groups.     

Aurintricarboxylic acid inhibits endothelial activation, complement activation, and von Willebrand factor secretion in vitro and attenuates hyperacute rejection in an ex vivo model of pig-to-human pulmonary xenotransplantation

Abstract: Background: In the xenotransplantation of vascularized organs, such as the lung, a large area of endothelial cell layer is a big hurdle to be overcome. We investigated the potential protective effect of aurintricarboxylic acid (ATA), a known inhibitor of platelet adhesion, on endothelial damage induced by xenogeneic serum. We also assessed its role in hyperacute xenograft rejection using a porcine ex vivo lung perfusion model. Methods: Porcine endothelial cells were incubated with human serum and other inflammatory stimuli. For the evaluation of von Willebrand factor (vWF) secretion and tissue factor (TF) expression, we used human endothelial cells. E‐selectin expression, complement activation, TF expression and platelet activation were investigated by flow cytometry. In an ex vivo porcine lung perfusion model, the porcine lungs were perfused with fresh human whole blood: unmodified blood (n = 5), ATA‐treated blood (n = 5), and ATA and lepirudin‐treated blood (n = 5). Results: Aurintricarboxylic acid significantly inhibited TNF‐α‐ or lipopolysaccharide‐induced endothelial E‐selectin expression in a dose‐dependent manner. ATA also prevented human serum induced‐E‐selectin expression and human monocytic cell adhesion to porcine endothelial cells. Moreover, ATA abolished thrombin‐induced vWF secretion as well as complement activation. However, ATA induced endothelial TF expression and platelet activation in vitro. In ex‐vivo experiments, ATA treatment improved pulmonary function and attenuated sequestration of leukocytes. Although ATA did not influence thrombin generation, we were able to minimize its activity by adding lepirudin to the blood with ATA. Conclusions: Our study demonstrated in vitro protective effect of ATA on the inhibition of endothelial activation and vWF secretion and confirmed detrimental effect of ATA on induction of endothelial TF and platelet activation. The combination of ATA and lepirudin may act beneficially by preventing coagulation perturbation while maintaining improved xenograft survival.     

Dissemination of a novel organ perfusion technique: ex vivo normothermic perfusion of deceased donor kidneys

Ex vivo normothermic perfusion (EVNP) technology is a promising means of organ preservation, assessment, and preconditioning prior to kidney transplantation, which has been pioneered by a single group. We describe the challenges of setting up clinical EVNP programs in 2 new centers, as well as early patient outcomes. Governance, training, and logistical pathways are described. In order to demonstrate safety and proficiency in this new technique, early patient outcomes are also described. Patient outcomes included the incidence of primary nonfunction, delayed graft function, graft and patient survival at 1 year. Contralateral kidneys undergoing static cold storage alone were used as a comparator group. Between March 2016 and July 2017, EVNP was performed on 14 kidneys from 12 donors (11 kidneys in center 1, 3 kidneys in center 2). Of the 14 kidneys that underwent EVNP, 12 organs were implanted into 10 recipients. Two pairs of kidneys were implanted as dual grafts and 1 kidney was implanted simultaneously with a pancreas. The remaining 7 kidneys were transplanted as single allografts. Seven pairs of kidneys were available for paired analysis comparing EVNP versus static cold storage. Graft and patient outcomes were comparable between the 2 preservation techniques. The introduction of a clinical EVNP service requires a careful multimodal approach, drawing on the expertise of specialists in transplantation, hematology, and microbiology. Both new clinical EVNP programs demonstrated proficiency and safety when a structured dissemination process was followed.