Ray K. Chihara

Double knockout pigs deficient in N-glycolylneuraminic acid and Galactose α-1,3-Galactose reduce the humoral barrier to xenotransplantation

Clinical xenotransplantation is not possible because humans possess antibodies that recognize antigens on the surface of pig cells. Galα‐1,3‐Gal (Gal) and N‐glycolylneuraminic acid (Neu5Gc) are two known xenoantigens.

Comparison of histidine-tryptophan-ketoglutarate solution and University of Wisconsin solution in extended criteria liver donors

Liver, pancreas, and kidney allografts preserved in histidine‐tryptophan‐ketoglutarate (HTK) and University of Wisconsin (UW) solutions have similar clinical outcomes. This study compares HTK and UW in a large number of standard criteria donor (SCD) and extended criteria donor (ECD) livers at a single center over 5 years. All adult, cadaveric liver and liver‐kidney transplants performed between July 1, 2001 and June 30, 2006 were reviewed (n = 698). There were 435 livers (62%) categorized as ECD for severe physiologic stress and 70 (10%) because of old age. Recipient outcomes included perioperative death or graft loss and overall survival. Liver enzymes were analyzed for the first month post‐transplant. Biliary complications were assessed through chart review. Overall, 371 donor livers were preserved in HTK (53%), and 327 were preserved in UW (47%). There were no statistically significant differences in any of the primary outcome measures comparing HTK and UW. The HTK group overall had a higher day 1 median aspartate aminotransferase and alanine aminotransferase, but the two groups were similar in function thereafter. HTK was superior to UW in protection against biliary complications. Kaplan‐Meier graft survival curves failed to demonstrate a significant difference in SCD or ECD livers. In conclusion, HTK and UW are not clinically distinguishable in this large sample of liver transplants, although HTK may be protective against biliary complications when compared to UW. These findings persisted for both SCD and ECD livers. Given the lower cost per donor for HTK, this preservation solution may be preferable for general use. Liver Transpl 14:365–373, 2008.

The fate of human platelets perfused through the pig liver: implications for xenotransplantation

Burlak C, Paris LL, Chihara RK, Sidner RA, Reyes LM, Downey SM, Tector AJ. The fate of human platelets perfused through the pig liver: implications for xenotransplantation.
Xenotransplantation 2010; 17: 350–361.

ASGR1 expressed by porcine enriched liver sinusoidal endothelial cells mediates human platelet phagocytosis in vitro.

Paris LL, Chihara RK, Reyes LM, Sidner RA, Estrada JL, Downey SM, Milgrom DA, Joseph Tector A, Burlak C. ASGR1 expressed by porcine enriched liver sinusoidal endothelial cells mediates human platelet phagocytosis in vitro. Xenotransplantation 2011; 18: 245–251.

Differences in human and porcine platelet oligosaccharides may influence phagocytosis by liver sinusoidal cells in vitro

Paris LL, Chihara RK, Sidner RA, Tector AJ, Burlak C. Differences in human and porcine platelet oligosaccharides may influence phagocytosis by liver sinusoidal cells in vitro. Xenotransplantation 2012; 19: 31–39.

Laparoscopic colectomy: does the learning curve extend beyond colorectal surgery fellowship?

Colorectal fellowship training adequately surpasses the learning curve with regard to safety and outcome; however, the surgeon continues to increase operative efficiency during the first year of practice.

Identification of human preformed antibody targets in GTKO pigs

Burlak C, Wang ZY, Chihara RK, Lutz AJ, Wang Y, Estrada JL, Tector AJ. Identification of human preformed antibody targets in GTKO pigs. Xenotransplantation 2012; 19: 92–101.

Primary porcine Kupffer cell phagocytosis of human platelets involves the CD18 receptor.

Background. Hepatic failure has been treated successfully with clinical extracorporeal perfusions of porcine livers. However, dog-to-pig and pig-to-baboon liver xenotransplant models have resulted in severe bleeding secondary to liver xenograft-induced thrombocytopenia. Kupffer cells (KC) are abundant phagocytic cells in the liver. KC express the CD11b/CD18 receptor, which has been implicated in chilled platelet binding and phagocytosis through interaction with platelet surface proteins and carbohydrates. We sought to identify the role of KC CD18 in liver xenograft-induced thrombocytopenia. Methods. Primary pig KC were characterized by flow cytometry, immunoblots, and quantitative polymerase chain reaction. Pig KC were used in inhibition assays with fluorescently labeled human platelets. The CD18 receptor was targeted for siRNA knockdown. Results. Domestic and &agr;1,3-galactosyltransferase double knockout porcine KC cultures were approximately 92% positive for CD18 as detected by quantitative polymerase chain reaction and flow cytometry. Use of CD18 blocking antibodies resulted in reduction of human platelet binding and phagocytosis. Additionally, asialofetuin, not fetuin, inhibited platelet phagocytosis suggesting the involvement of an oligosaccharide-binding site. Furthermore, reduced CD18 expression by siRNA resulted in decreased human platelet binding. Conclusions. Our data suggest that primary pig KC bind and phagocytose human platelets with involvement of CD18. Further understanding and modification of CD18 expression in pigs may result in a liver xenograft with reduced thrombocytopenic effects, which could be used as a bridge to allogeneic liver transplantation.

Fibronectin from alpha 1,3-galactosyltransferase knockout pigs is a xenoantigen.

BACKGROUND Antibody-mediated rejection continues to be an obstacle for xenotransplantation despite development of α1,3-galactosyltransferase knockout (GTKO) pigs. Fibronectin (Fn) from GTKO pigs was identified as a xenoantigen in baboons. N-glycolylneuraminic acid (Neu5Gc), similar to galactose α1,3-galactose, is an antigenic carbohydrate found in pigs. We evaluated human antibody reactivity and performed initial antigenic epitope characterization of Fn from GTKO pigs. MATERIALS AND METHODS GTKO pig aortic endothelial cells (AEC) were isolated and assessed for antibody-mediated complement-dependent cytotoxicity (CDC). Human and GTKO pig Fn were purified and analyzed using immunoblots. GTKO pig and human AEC absorbed human sera were assessed for CDC and anti-GTKO pig Fn antibodies. GTKO pig proteins were assessed for Neu5Gc. Immunoaffinity-purified human IgG anti-GTKO pig (hIgG-GTKOp) Fn using a GTKO pig Fn column were evaluated for cross-reactivity with other proteins. RESULTS GTKO pig AEC had greater human antibody binding, complement deposition and CDC compared with allogeneic human AEC. Human sera absorbed with GTKO pig AEC resulted in diminished anti-GTKO pig Fn antibody. Neu5Gc was identified on GTKO pig Fn and other proteins. The hIgG-GTKOp Fn cross-reacted with multiple GTKO pig proteins and was enriched with anti-Neu5Gc antibody. CONCLUSIONS Removal of antigenic epitopes from GTKO pig AEC would improve xenograft compatibility. GTKO pig Fn has antigenic epitopes, one identified as Neu5Gc, which may be responsible for pathology and cross-reactivity of hIgG-GTKOp Fn. Genetic knockout of Neu5Gc appears necessary to address significance and identification of non-Neu5Gc GTKO pig Fn antigenic epitopes.

Immortalized porcine liver sinusoidal endothelial cells: an in vitro model of xenotransplantation‐induced thrombocytopenia

Wang Z‐Y, Paris LL., Chihara RK., Tector AJ., Burlak C. Immortalized porcine liver sinusoidal endothelial cells: an in vitro model of xenotransplantation‐induced thrombocytopenia. Xenotransplantation 2012; 19: 249–255..