Human erythrocyte fragmentation during ex‐vivo pig organ perfusion

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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.

Section: Resultsmentioning

confidence: 85%

Section: Discussionmentioning

confidence: 99%

Section: Platelet Sequestration and Activationmentioning

confidence: 96%

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

Chaban

Habibabady

Hassanein

et al. 2022

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“…The effect of 1F1 is extremely consistent, reducing rosetting to around 4% regardless of pig macrophage phenotype, very near that of pig macrophages with pig RBCs (∼2%) and pig macrophages with desialated human RBCs (∼1%). Thus, targeting porcine sialoadhesin with an antibody Fab or Fc‐silenced antibody could be useful as a therapeutic agent or potential solution to the problem of human erythrocyte sequestration and destruction by pig livers 29,30 and other organs 7,31,32 . Treatment with the 1F1 antibody decreased the rate of loss of human RBCs during extracorporeal perfusion of WT pig livers over a 72‐h period, but did not prevent loss completely 17 .…”

Section: Discussionmentioning

confidence: 99%

“…Thus, targeting porcine sialoadhesin with an antibody Fab or Fc-silenced antibody could be useful as a therapeutic agent or potential solution to the problem of human erythrocyte sequestration and destruction by pig livers 29,30 and other organs. 7,31,32 Treatment with the 1F1 antibody decreased the rate of loss of human RBCs during extracorporeal perfusion of WT pig livers over a 72-h period, but did not prevent loss completely. 17 Because NA treatment results in lower binding than 1F1 blockade, and the latter is not increased by higher concentrations of 1F1 (not illustrated), we conclude that the residual rosetting after 1F1 treatment is mediated by sialic acid interacting with some other ligand on pig macrophages macrophages that bind one or two RBCs represent a physiologically important phenomenon is unknown, but seems unlikely, since pig macrophages and pig RBCs also exhibit this behavior in the in vitro rosette assay.…”

Section: Sialic Acid Mediates Adhesion Of Human Erythrocytes To Porci...mentioning

confidence: 99%

Genetic modifications designed for xenotransplantation attenuate sialoadhesin‐dependent binding of human erythrocytes to porcine macrophages

Petitpas¹,

Habibabady²,

Ritchie³

et al. 2022

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The phenomenon of diminishing hematocrit after in vivo liver and lung xenotransplantation and during ex vivo liver xenoperfusion has largely been attributed to action by resident liver porcine macrophages, which bind and destroy human erythrocytes. Porcine sialoadhesin (siglec‐1) was implicated previously in this interaction. This study examines the effect of porcine genetic modifications, including knockout of the CMAH gene responsible for expression of Neu5Gc sialic acid, on the adhesion of human red blood cells (RBCs) to porcine macrophages. Wild‐type (WT) porcine macrophages and macrophages from several strains of genetically engineered pigs, including CMAH gene knockout and several human transgenes (TKO+hTg), were incubated with human RBCs and “rosettes” (≥3 erythrocytes bound to one macrophage) were quantified by microscopy. Our results show that TKO+hTg genetic modifications significantly reduced rosette formation. The monoclonal antibody 1F1, which blocks porcine sialoadhesin, significantly reduced rosette formation by WT and TKO+hTg macrophages compared with an isotype control antibody. Further, desialation of human RBCs with neuraminidase before addition to WT or TKO+hTg macrophages resulted in near‐complete abrogation of rosette formation, to a level not significantly different from porcine RBC rosette formation on porcine macrophages. These observations are consistent with rosette formation being mediated by binding of sialic acid on human RBCs to sialoadhesin on porcine macrophages. In conclusion, the data predict that TKO+hTg genetic modifications, coupled with targeting of porcine sialoadhesin by the 1F1 mAb, will attenuate erythrocyte sequestration and anemia during ex vivo xenoperfusion and following in vivo liver, lung, and potentially other organ xenotransplantation.

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

Chaban

McGrath

Habibabady

et al. 2023

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IntroductionExpression 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.MethodsTwenty 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.ResultsRelative 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.ConclusionGenetic 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.