© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
Xenotransplantation 2014: 21: 482–484 doi: 10.1111/xen.12144
XENOTRANSPLANTATION
Literature Update
Xenotransplantation literature update, July– August 2014 Burlak C, Taylor RT. Xenotransplantation literature update, July–August 2014. Xenotransplantation 2014; 21: 482–484. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
Christopher Burlak1 and R. Travis Taylor2 1
Department of Surgery, Schultz Diabetes Institute, University of Minnesota, Minneapolis, MN, 2 Department of Medical Microbiology and Immunology, University of Toledo Medical Center, Toledo, OH, USA Key words: coagulation disorder – mesenchymal stem cells – pig – xenotransplantation Abbreviations: NHP, Non-human primate; GTKO, galactosyl transferase knockout; aGal, Galactose alpha 1,3 galactose; Neu5Gc, N-Glycolyl neuraminic acid; rhAT, recombinant human antithrombin; Sur1, sulfonylurea receptor 1; Kir6.2, inward rectifying potassium channel; DSA, donor-specific antibody; IBMIR, instant blood mediated inflammatory reaction. Address reprint requests to Christopher Burlak, Department of Surgery, Schultz Diabetes Institute, University of Minnesota, 420 Delaware St. SE, Minneapolis, MN 55455, USA (E-mail: [email protected]) Received 2 September 2014; Accepted 4 September 2014
Zoonosis
Dr. Jay Fishman is an expert in the study of transplantation-acquired diseases and is the Director of Transplant Infectious Diseases and Compromised Host Program at Massachusetts General Hospital. He is therefore one of the most qualified people to conduct a comparison of the known infectious risks observed in allotransplantation to the potential risks of xenotransplantation. This editorial distills the literature of zoonosis to the conclusion that while much of what has been done suggests that the risk of transmission is low or negligible, the risk of xenotransplantation to humans is unknown [1]. The author points us to the current work of Dr. Wynyard in an attempt to describe our infections risk and summarized those findings [2]. Briefly, pigs screened for 26 microorganisms associated with allotransplantation or thought to be important to xenotransplantation were used as 482
islet donors in a clinical trial. After 1 yr, those patients had no detectable levels of any of the microorganisms. Islet cell transplantation may be unique and the use of solid organ xenotransplantation may create a different set of risks, but the data from the trial are encouraging nonetheless. Taveira et al. [3] highlight a risk of human cytomegalovirus (HCMV) infection of pig xenografts in a recent study to evaluate cross-species virus entry and gene expression. Their study shows that infection with what is considered a strictly human pathogen can indeed have a profound impact on xeno cells and potentially xenografts. This work proposes that HCMV infection should be a consideration prior to xenotransplantation. Interestingly, Fishman points out that if human-tohuman organ donation were held to the infectious risk expectations we have described for xenotransplantation, allotransplantation would be impossible. Admitting that we cannot screen for all
Xenotransplantation literature update pathogens (known or unknown), we must come to an understanding of the acceptable risk based on detectable syndromes and incorporate developing technologies. Humoral immunity to xenografts
When we discuss the role of anti-pig antibodies in xenotransplantation, we are typically referring to the ability of bound antibodies to attract and fix complement. Stewart et al. [4] hypothesized that after xenotransplantation, we may be immunologically educated to make antibodies toward porcine factor VIII, thereby creating a blocking antibody which could inhibit normal coagulation. The authors immunized NHP with pig cells and tested serum samples for the development of antibody that could block factor VIII function in the presence of injected GTKO pig cells. A blocking antibody was produced that bound the C1 domain of factor VIII resulting in a significant reduction in coagulation as measured Bethesda units. It is unclear how this relates to clinical pathology but suggests that inhibitory antibodies my play a part in chronic xenograft disfunction. The creation of pigs lacking both the aGal and Neu5Gc carbohydrate antigens significantly reduced human antibody binding as compared to pigs lacking only the aGal epitope. The previous study described the generation of the aGal and Neu5Gc deficient pigs and IgG/IgM binding from 10 individuals. Most recently, Burlak et al. [5] have compared the reactivity of 121 human serum samples against GGTA1 knockout and GGTA1/ CMAH knockout pig cells. The authors found that nearly everyone tested had an improved crossmatch analysis against the GGTA1/CMAH knockout pigs. Antibody binding did not vary by age or gender. Surprisingly, people with O blood type produced slightly more anti-pig antibodies. This study highlights the tremendous power in genetic engineering to push our field ever closer to pig cells and organs that could be used clinically. Similarly, Wang et al. [6] hypothesized that because less human antibody bound to GGTA1/ CMAH pig PBMCs, pig erythrocytes would also bind less human antibody and therefore, fix less complement. The authors convincingly demonstrated an improvement in GGTA1/CMAH erythrocyte survival when challenged by human antibody and complement. Interestingly, serum from baboons was more reactive to the GGTA1/ CMAH knockout pig erythrocytes than erythrocytes bearing the GGTA1 knockout alone. Unfortunately, antibodies from baboon serum were more reactive to the same genetically modified pig
erythrocytes suggesting that pre-clinical models in non-human primates (NHP) may be challenging. Adaptive immunity to xenografts
Chai et al. [7] have used a rat to mouse nerve xenotransplantation model to explore the influence of regulatory T cells on acute rejection. The authors pre-treated mice with anti-IFN-gamma, anti-IL17, and anti-IL-22 and evaluated the presence of IFN-gamma, IL-17, and IL-22 positive cells in the spleen at numerous time points after transplantation. IFN-gamma, IL-17, and IL-22 positive cells and circulating cytokines were significantly reduced in treated mice. The authors conclude that reduction in effector T cells increased the relative proportion of regulatory T cells present which resulted in prolongation of peripheral nerve xenograft survival. This study is among others that suggest that genetic modification of pigs to reduce T-cell interaction may improve xenografts without additional immunosuppressive drugs. Pre-clinical studies
Ramackers et al. [8] have produced a recombinant human antithrombin (rhAT) that if administered early in transplantation could reduce thrombotic microangiopathy and prolong graft survival. Wildtype pig kidneys were perfused with whole human blood in an ex vivo model of kidney xenotransplantation. Recombinant antithrombin introduction to the perfusion circuit reduced vascular resistance and prevented a loss of platelet function in a dose dependent manner. Perfusion time was doubled in the presence of rhAT when pig kidneys were perfused with human blood. The authors point out that the effective dose correlated to a potential clinically tolerable concentration. This study suggests that rhAT as a therapy or perhaps transgene will prolong solid organ xenotransplantation. Perfusion studies with genetically modified kidneys are likely outside the duration capable in an ex vivo perfusion model but will be exciting to observe in NHP or human clinical trials. Steroid-free immune suppression based on antibody therapy blocks or removes cells producing the immune response is highly effective in na€ıve patients but less so when donor-specific antibodies are present. The presence of preformed or DSA may contribute to IBMIR especially in those cases where more than one pancreas or islet graft have been transplanted. Costimulation as described for NHP or mouse models can dramatically reduce the T-cell response but it is unclear if the anti-islet antibodies are important to graft rejection. Diab 483
Burlak and Taylor et al. [9] used the alloxan-induced diabetes model in mice to test the importance of DSA to islet rejection in the presence of costimulatory blockade including anti-CD154, CTLA4Ig, and/or antiLFA-1. Mice that received costimulation blockade had almost no cellular infiltration and surprisingly showed no difference in graft survival when injected with na€ıve serum or xeno-educated serum. Intraperitoneal glucose tolerance tests were similar between treatments and at 1 and 4 months. The composition of the xeno serum is unclear, and therefore, further study of the specificity of those antibodies may provide insight into the accuracy of this model and targets of importance to islet rejection. Because of the similarity of costimulation blockade between this mouse model and that used in NHP models it would be interesting if these groups could compare data on cell populations affected and targets of the DSA response. The authors present data that in combination with their previous studies suggest that antibodies may not contribute to a decreased graft function in islet xenografts. Kelly et al. [10] sought to define surface receptors on isolated pig islets that could be targeted with contrast agents for clinical imaging studies and potentially control of insulin production. Their goal is to develop methods for monitoring transplanted pig islets. The authors characterized the expression and function of the sulfonylurea receptor 1 (Sur1), inward rectifying potassium channel (Kir6.2), glucagon-like peptide 1 receptor, and adrenergic receptor alpha 2A on cultured islets. The islets tested contained mRNA transcript for the target receptors. The stimulators glibenclamide and exendin-4 enhanced glucose-stimulated insulin secretion more than 2-fold. In contrast, epinephrine inhibited insulin secretion by approximately 72%. The authors succeeded in blocking the effect of epinephrine with yohimbine, an adrenergic receptor alpha 2A agonist. These studies suggest that these receptors could be targeted for imaging. Intraportal injection of islets in a rodent
484
or NHP model for the purpose of testing the resolution of the targeted contrast would be compelling. Additionally, the ability to effect insulin secretion as the authors suggest would be considerable progress toward addressing this potential issue. References 1. FISHMAN JA. Assessment of infectious risk in clinical xenotransplantation: the lessons for clinical allotransplantation. Xenotransplantation 2014; 21: 307–308. 2. WYNYARD S, NATHU D, GARKAVENKO O, DENNER J, ELLIOTT R. Microbiological safety of the first clinical pig islet xenotransplantation trial in New Zealand. Xenotransplantation 2014; 21: 309–323. 3. TAVEIRA A, PONROY N, MUELLER NJ, MILLARD A-L. Entry of human cytomegalovirus into porcine endothelial cells depends on both the cellular vascular origin and the viral strain. Xenotransplantation 2014; 21: 324–340. 4. STEWART JM, TARANTAL AF, HAWTHORNE WJ et al. Rhesus monkeys and baboons develop clotting factor VIII inhibitors in response to porcine endothelial cells or islets. Xenotransplantation 2014; 21: 341–352. 5. BURLAK C, WANG Z-Y, CHIHARA RK et al. Identification of human preformed antibody targets in GTKO pigs. Xenotransplantation 2012; 19: 92–101. 6. WANG Z-Y, BURLAK C, ESTRADA JL et al. Erythrocytes from GGTA1/CMAH knockout pigs: implications for xenotransfusion and testing in non-human primates. Xenotransplantation 2014; 21: 376–384. 7. CHAI H, YANG L, GAO L et al. Decreased percentages of regulatory T cells are necessary to activate Th1-Th17-Th22 responses during acute rejection of the peripheral nerve Xenotransplantation in mice. Transplantation 2014; Epub ahead of print. 8. RAMACKERS W, KLOSE J, VONDRAN FWR et al. Speciesspecific regulation of fibrinogen synthesis with implications for porcine hepatocyte xenotransplantation. Xenotransplantation 2014; 21: 444–453. 9. DIAB RAH, HASSAN M, TIBELL A, HOLGERSSON J, KUMAGAI-BRAESCH M. Rat islets are not rejected by anti-islet antibodies in mice treated with costimulation blockade. Xenotransplantation 2014; 21: 353–366. 10. KELLY AC, STEYN LV, KITZMANN JP et al. Function and expression of sulfonylurea, adrenergic, and glucagon-like peptide 1 receptors in isolated porcine islets. Xenotransplantation 2014; 21: 385–391.
Xenotransplantation 2014: 21: 482–484 doi: 10.1111/xen.12144
XENOTRANSPLANTATION
Literature Update
Xenotransplantation literature update, July– August 2014 Burlak C, Taylor RT. Xenotransplantation literature update, July–August 2014. Xenotransplantation 2014; 21: 482–484. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
Christopher Burlak1 and R. Travis Taylor2 1
Department of Surgery, Schultz Diabetes Institute, University of Minnesota, Minneapolis, MN, 2 Department of Medical Microbiology and Immunology, University of Toledo Medical Center, Toledo, OH, USA Key words: coagulation disorder – mesenchymal stem cells – pig – xenotransplantation Abbreviations: NHP, Non-human primate; GTKO, galactosyl transferase knockout; aGal, Galactose alpha 1,3 galactose; Neu5Gc, N-Glycolyl neuraminic acid; rhAT, recombinant human antithrombin; Sur1, sulfonylurea receptor 1; Kir6.2, inward rectifying potassium channel; DSA, donor-specific antibody; IBMIR, instant blood mediated inflammatory reaction. Address reprint requests to Christopher Burlak, Department of Surgery, Schultz Diabetes Institute, University of Minnesota, 420 Delaware St. SE, Minneapolis, MN 55455, USA (E-mail: [email protected]) Received 2 September 2014; Accepted 4 September 2014
Zoonosis
Dr. Jay Fishman is an expert in the study of transplantation-acquired diseases and is the Director of Transplant Infectious Diseases and Compromised Host Program at Massachusetts General Hospital. He is therefore one of the most qualified people to conduct a comparison of the known infectious risks observed in allotransplantation to the potential risks of xenotransplantation. This editorial distills the literature of zoonosis to the conclusion that while much of what has been done suggests that the risk of transmission is low or negligible, the risk of xenotransplantation to humans is unknown [1]. The author points us to the current work of Dr. Wynyard in an attempt to describe our infections risk and summarized those findings [2]. Briefly, pigs screened for 26 microorganisms associated with allotransplantation or thought to be important to xenotransplantation were used as 482
islet donors in a clinical trial. After 1 yr, those patients had no detectable levels of any of the microorganisms. Islet cell transplantation may be unique and the use of solid organ xenotransplantation may create a different set of risks, but the data from the trial are encouraging nonetheless. Taveira et al. [3] highlight a risk of human cytomegalovirus (HCMV) infection of pig xenografts in a recent study to evaluate cross-species virus entry and gene expression. Their study shows that infection with what is considered a strictly human pathogen can indeed have a profound impact on xeno cells and potentially xenografts. This work proposes that HCMV infection should be a consideration prior to xenotransplantation. Interestingly, Fishman points out that if human-tohuman organ donation were held to the infectious risk expectations we have described for xenotransplantation, allotransplantation would be impossible. Admitting that we cannot screen for all
Xenotransplantation literature update pathogens (known or unknown), we must come to an understanding of the acceptable risk based on detectable syndromes and incorporate developing technologies. Humoral immunity to xenografts
When we discuss the role of anti-pig antibodies in xenotransplantation, we are typically referring to the ability of bound antibodies to attract and fix complement. Stewart et al. [4] hypothesized that after xenotransplantation, we may be immunologically educated to make antibodies toward porcine factor VIII, thereby creating a blocking antibody which could inhibit normal coagulation. The authors immunized NHP with pig cells and tested serum samples for the development of antibody that could block factor VIII function in the presence of injected GTKO pig cells. A blocking antibody was produced that bound the C1 domain of factor VIII resulting in a significant reduction in coagulation as measured Bethesda units. It is unclear how this relates to clinical pathology but suggests that inhibitory antibodies my play a part in chronic xenograft disfunction. The creation of pigs lacking both the aGal and Neu5Gc carbohydrate antigens significantly reduced human antibody binding as compared to pigs lacking only the aGal epitope. The previous study described the generation of the aGal and Neu5Gc deficient pigs and IgG/IgM binding from 10 individuals. Most recently, Burlak et al. [5] have compared the reactivity of 121 human serum samples against GGTA1 knockout and GGTA1/ CMAH knockout pig cells. The authors found that nearly everyone tested had an improved crossmatch analysis against the GGTA1/CMAH knockout pigs. Antibody binding did not vary by age or gender. Surprisingly, people with O blood type produced slightly more anti-pig antibodies. This study highlights the tremendous power in genetic engineering to push our field ever closer to pig cells and organs that could be used clinically. Similarly, Wang et al. [6] hypothesized that because less human antibody bound to GGTA1/ CMAH pig PBMCs, pig erythrocytes would also bind less human antibody and therefore, fix less complement. The authors convincingly demonstrated an improvement in GGTA1/CMAH erythrocyte survival when challenged by human antibody and complement. Interestingly, serum from baboons was more reactive to the GGTA1/ CMAH knockout pig erythrocytes than erythrocytes bearing the GGTA1 knockout alone. Unfortunately, antibodies from baboon serum were more reactive to the same genetically modified pig
erythrocytes suggesting that pre-clinical models in non-human primates (NHP) may be challenging. Adaptive immunity to xenografts
Chai et al. [7] have used a rat to mouse nerve xenotransplantation model to explore the influence of regulatory T cells on acute rejection. The authors pre-treated mice with anti-IFN-gamma, anti-IL17, and anti-IL-22 and evaluated the presence of IFN-gamma, IL-17, and IL-22 positive cells in the spleen at numerous time points after transplantation. IFN-gamma, IL-17, and IL-22 positive cells and circulating cytokines were significantly reduced in treated mice. The authors conclude that reduction in effector T cells increased the relative proportion of regulatory T cells present which resulted in prolongation of peripheral nerve xenograft survival. This study is among others that suggest that genetic modification of pigs to reduce T-cell interaction may improve xenografts without additional immunosuppressive drugs. Pre-clinical studies
Ramackers et al. [8] have produced a recombinant human antithrombin (rhAT) that if administered early in transplantation could reduce thrombotic microangiopathy and prolong graft survival. Wildtype pig kidneys were perfused with whole human blood in an ex vivo model of kidney xenotransplantation. Recombinant antithrombin introduction to the perfusion circuit reduced vascular resistance and prevented a loss of platelet function in a dose dependent manner. Perfusion time was doubled in the presence of rhAT when pig kidneys were perfused with human blood. The authors point out that the effective dose correlated to a potential clinically tolerable concentration. This study suggests that rhAT as a therapy or perhaps transgene will prolong solid organ xenotransplantation. Perfusion studies with genetically modified kidneys are likely outside the duration capable in an ex vivo perfusion model but will be exciting to observe in NHP or human clinical trials. Steroid-free immune suppression based on antibody therapy blocks or removes cells producing the immune response is highly effective in na€ıve patients but less so when donor-specific antibodies are present. The presence of preformed or DSA may contribute to IBMIR especially in those cases where more than one pancreas or islet graft have been transplanted. Costimulation as described for NHP or mouse models can dramatically reduce the T-cell response but it is unclear if the anti-islet antibodies are important to graft rejection. Diab 483
Burlak and Taylor et al. [9] used the alloxan-induced diabetes model in mice to test the importance of DSA to islet rejection in the presence of costimulatory blockade including anti-CD154, CTLA4Ig, and/or antiLFA-1. Mice that received costimulation blockade had almost no cellular infiltration and surprisingly showed no difference in graft survival when injected with na€ıve serum or xeno-educated serum. Intraperitoneal glucose tolerance tests were similar between treatments and at 1 and 4 months. The composition of the xeno serum is unclear, and therefore, further study of the specificity of those antibodies may provide insight into the accuracy of this model and targets of importance to islet rejection. Because of the similarity of costimulation blockade between this mouse model and that used in NHP models it would be interesting if these groups could compare data on cell populations affected and targets of the DSA response. The authors present data that in combination with their previous studies suggest that antibodies may not contribute to a decreased graft function in islet xenografts. Kelly et al. [10] sought to define surface receptors on isolated pig islets that could be targeted with contrast agents for clinical imaging studies and potentially control of insulin production. Their goal is to develop methods for monitoring transplanted pig islets. The authors characterized the expression and function of the sulfonylurea receptor 1 (Sur1), inward rectifying potassium channel (Kir6.2), glucagon-like peptide 1 receptor, and adrenergic receptor alpha 2A on cultured islets. The islets tested contained mRNA transcript for the target receptors. The stimulators glibenclamide and exendin-4 enhanced glucose-stimulated insulin secretion more than 2-fold. In contrast, epinephrine inhibited insulin secretion by approximately 72%. The authors succeeded in blocking the effect of epinephrine with yohimbine, an adrenergic receptor alpha 2A agonist. These studies suggest that these receptors could be targeted for imaging. Intraportal injection of islets in a rodent
484
or NHP model for the purpose of testing the resolution of the targeted contrast would be compelling. Additionally, the ability to effect insulin secretion as the authors suggest would be considerable progress toward addressing this potential issue. References 1. FISHMAN JA. Assessment of infectious risk in clinical xenotransplantation: the lessons for clinical allotransplantation. Xenotransplantation 2014; 21: 307–308. 2. WYNYARD S, NATHU D, GARKAVENKO O, DENNER J, ELLIOTT R. Microbiological safety of the first clinical pig islet xenotransplantation trial in New Zealand. Xenotransplantation 2014; 21: 309–323. 3. TAVEIRA A, PONROY N, MUELLER NJ, MILLARD A-L. Entry of human cytomegalovirus into porcine endothelial cells depends on both the cellular vascular origin and the viral strain. Xenotransplantation 2014; 21: 324–340. 4. STEWART JM, TARANTAL AF, HAWTHORNE WJ et al. Rhesus monkeys and baboons develop clotting factor VIII inhibitors in response to porcine endothelial cells or islets. Xenotransplantation 2014; 21: 341–352. 5. BURLAK C, WANG Z-Y, CHIHARA RK et al. Identification of human preformed antibody targets in GTKO pigs. Xenotransplantation 2012; 19: 92–101. 6. WANG Z-Y, BURLAK C, ESTRADA JL et al. Erythrocytes from GGTA1/CMAH knockout pigs: implications for xenotransfusion and testing in non-human primates. Xenotransplantation 2014; 21: 376–384. 7. CHAI H, YANG L, GAO L et al. Decreased percentages of regulatory T cells are necessary to activate Th1-Th17-Th22 responses during acute rejection of the peripheral nerve Xenotransplantation in mice. Transplantation 2014; Epub ahead of print. 8. RAMACKERS W, KLOSE J, VONDRAN FWR et al. Speciesspecific regulation of fibrinogen synthesis with implications for porcine hepatocyte xenotransplantation. Xenotransplantation 2014; 21: 444–453. 9. DIAB RAH, HASSAN M, TIBELL A, HOLGERSSON J, KUMAGAI-BRAESCH M. Rat islets are not rejected by anti-islet antibodies in mice treated with costimulation blockade. Xenotransplantation 2014; 21: 353–366. 10. KELLY AC, STEYN LV, KITZMANN JP et al. Function and expression of sulfonylurea, adrenergic, and glucagon-like peptide 1 receptors in isolated porcine islets. Xenotransplantation 2014; 21: 385–391.
