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Transplantation of encapsulated porcine hepatocytes improves metabolic function and survival in baboons with acute liver failure
Vol. 213, No. 3S, September 2011
Surgical Forum Abstracts
S69
weeks after allograft rejection the protesome inhibitor, Bortezomib, was injected intravenously (0.75mg/kg) twice weekly for 60 days. Serum alloantibody responses were assayed using flow cross-match. Total plasma cells numbers were enumerated using flow cytometry and ELISPOT.
CONCLUSIONS: Engrafted (tolerant) animals demonstrate significant changes in their peripheral T cell and APC compartments. Our data suggests that tolerance after IUHCTx, like self-tolerance, is an active, dynamic process that relies on both central and peripheral mechanisms to maintain engraftment.
RESULTS: All control and experimental C57BL/6 recipients of BALB/c cardiac allografts rejected their graft promptly within 16 days and demonstrated alloantibody by flow cross-match. DSA was sustained in the control mice (n⫽2) at day 60, while the experimental mice (n⫽4) treated with Bortezomib, had elimination of DSA by flow cross-match at day 60 and a marked reduction in plasma cell numbers by both flow cytometry and ELISPOT. Furthermore, a cohort of sensitized mice were followed for return of DSA and have remained cross-match negative for 4 months following treatment.
Transplantation of encapsulated porcine hepatocytes improves metabolic function and survival in baboons with acute liver failure Zurab Machaidze MD Massachusetts General Hospital/Harvard Medical School, Boston, MA
CONCLUSIONS: Within a murine model in the absence of plasmaexchange, sustained proteasome inhibition can eliminate alloreactive plasma cells, as detected by ELISPOT and flow cytometry, and reduce alloantibody to negligible levels. Furthermore, once therapy was completed DSA does not return.
Peripheral tolerance mechanisms are important for the maintenance of donor cell engraftment after in utero hematopoietic cell transplantation Amar Nijagal MD, Tom Le BS, Marta Wegorzewska BS, Andrew Tucker MS, Tippi C MacKenzie MD, FACS University of California-San Francisco, San Francisco, CA INTRODUCTION: In utero hematopoietic cell transplantation (IUHCTx) is a promising treatment strategy for inherited stem cell disorders. We and others have shown that engraftment after IUHCTx leads to donor-specific tolerance via thymic deletion of antigenspecific T cells. However, the role of peripheral mechanisms such as regulatory T cells (Tregs) remains unknown. METHODS: We performed IUHCTx of B6 hematopoietic cells into allogeneic BALB/c fetuses and analyzed conventional (CD4⫹Foxp3⫺) and regulatory (Tregs; CD4⫹Foxp3⫹) T cells in engrafted animals for activation markers by flow cytometry at 5 weeks of age. Since the initiation of a robust T cell response requires the expression of costimulatory molecules by antigen presenting cells (APC), we also characterized the expression of Class I, Class II, B7-1, B7-2, and CD40 on APC. Controls were age-matched, uninjected animals. RESULTS: Engrafted animals had a significantly higher percentage of Tregs compared to controls. Interestingly, engrafted animals also had a higher percentage of activated (CD44Hi CD62Lo) Tregs as well as activated conventional T cells. Donor-derived T cells also showed activation. Analysis of APC demonstrated significant increases in the expression of costimulatory molecules including B7-1, B7-2 and CD40 in both host- and donor-derived APC in engrafted animals compared to controls, suggesting both direct (donorderived) and indirect (host-derived) antigen presentation lead to T cell education.
INTRODUCTION: Liver transplantation can be the only treatment option for some patients with acute liver failure (ALF). We evaluated the effects of encapsulated porcine hepatocyte xenotransplantation on liver metabolic function and survival in baboons with ALF. METHODS: ALF in baboons was induced by 75% hepatectomy with 0, 30, and 60 minutes of warm ischemia of the remnant liver. Pig hepatocytes were isolated from surgically procured MGHminiature swine livers by collagenase perfusion. Alginate-polylysinealginate was used to form 400 to 800 um capsules, and hepatocyte viability pre- and post-encapsulation was 92 ⫾ 2 and 82 ⫾ 10%, respectively. Encapsulated hepatocytes were infused into the baboon’s peritoneum at the end of the laparotomy. RESULTS: Baboons that underwent 0 (n⫽2) and 30 min (n⫽1) warm ischemia of the remnant liver had transient worsening of liver function before recovering. Ones that underwent 60 minutes of ischemia (n⫽3) died on post-operative days (POD) 6, 7 and 10 of liver failure, characterized by worsening coagulopathy, encephalopathy (max NH3 levels ⫽ 435 mol/L) and hyperbilirubinemia (max total bilirubin⫽9.5 mg/dL). Baboons (N⫽3) that received 60 minutes ischemia and encapsulated hepatocytes (70-120 ml of hepatocytes in 200-350 ml of capsules) showed improved liver function compared to those that did not. One died on POD 22 with features of liver failure. Two animals survived ⬎30 days after transplantation with a POD 14 biopsy showing normal liver structure and normal coagulation parameters and bilirubin level. CONCLUSIONS: Transplantation of encapsulated porcine hepatocytes provide temporary metabolic support for baboons with ALF, allowing the remaining liver tissue to regenerate.
CHOP mediates cold ischemia and reperfusion injury in steatotic hepatocytes Parvathi Balachandran MD, Bernard J Dubray MD, Richard S Hotchkiss MD, Jianluo Jia MD, Brett Knolhoff BSc, Gundumi A Upadhya PhD, Thalachallour Mohanakumar PhD, William C Chapman MD, FACS, Christopher D Anderson MD, FACS Washington University-St Louis, St Louis, MO INTRODUCTION: We have previously shown that acute endoplasmic reticulum (ER) stress is triggered following transplantation of steatotic livers. C/EPB Homologous Protein (CHOP) is recognized as the major ER stress cell death mediator. We hypothesized that cold
Surgical Forum Abstracts
S69
weeks after allograft rejection the protesome inhibitor, Bortezomib, was injected intravenously (0.75mg/kg) twice weekly for 60 days. Serum alloantibody responses were assayed using flow cross-match. Total plasma cells numbers were enumerated using flow cytometry and ELISPOT.
CONCLUSIONS: Engrafted (tolerant) animals demonstrate significant changes in their peripheral T cell and APC compartments. Our data suggests that tolerance after IUHCTx, like self-tolerance, is an active, dynamic process that relies on both central and peripheral mechanisms to maintain engraftment.
RESULTS: All control and experimental C57BL/6 recipients of BALB/c cardiac allografts rejected their graft promptly within 16 days and demonstrated alloantibody by flow cross-match. DSA was sustained in the control mice (n⫽2) at day 60, while the experimental mice (n⫽4) treated with Bortezomib, had elimination of DSA by flow cross-match at day 60 and a marked reduction in plasma cell numbers by both flow cytometry and ELISPOT. Furthermore, a cohort of sensitized mice were followed for return of DSA and have remained cross-match negative for 4 months following treatment.
Transplantation of encapsulated porcine hepatocytes improves metabolic function and survival in baboons with acute liver failure Zurab Machaidze MD Massachusetts General Hospital/Harvard Medical School, Boston, MA
CONCLUSIONS: Within a murine model in the absence of plasmaexchange, sustained proteasome inhibition can eliminate alloreactive plasma cells, as detected by ELISPOT and flow cytometry, and reduce alloantibody to negligible levels. Furthermore, once therapy was completed DSA does not return.
Peripheral tolerance mechanisms are important for the maintenance of donor cell engraftment after in utero hematopoietic cell transplantation Amar Nijagal MD, Tom Le BS, Marta Wegorzewska BS, Andrew Tucker MS, Tippi C MacKenzie MD, FACS University of California-San Francisco, San Francisco, CA INTRODUCTION: In utero hematopoietic cell transplantation (IUHCTx) is a promising treatment strategy for inherited stem cell disorders. We and others have shown that engraftment after IUHCTx leads to donor-specific tolerance via thymic deletion of antigenspecific T cells. However, the role of peripheral mechanisms such as regulatory T cells (Tregs) remains unknown. METHODS: We performed IUHCTx of B6 hematopoietic cells into allogeneic BALB/c fetuses and analyzed conventional (CD4⫹Foxp3⫺) and regulatory (Tregs; CD4⫹Foxp3⫹) T cells in engrafted animals for activation markers by flow cytometry at 5 weeks of age. Since the initiation of a robust T cell response requires the expression of costimulatory molecules by antigen presenting cells (APC), we also characterized the expression of Class I, Class II, B7-1, B7-2, and CD40 on APC. Controls were age-matched, uninjected animals. RESULTS: Engrafted animals had a significantly higher percentage of Tregs compared to controls. Interestingly, engrafted animals also had a higher percentage of activated (CD44Hi CD62Lo) Tregs as well as activated conventional T cells. Donor-derived T cells also showed activation. Analysis of APC demonstrated significant increases in the expression of costimulatory molecules including B7-1, B7-2 and CD40 in both host- and donor-derived APC in engrafted animals compared to controls, suggesting both direct (donorderived) and indirect (host-derived) antigen presentation lead to T cell education.
INTRODUCTION: Liver transplantation can be the only treatment option for some patients with acute liver failure (ALF). We evaluated the effects of encapsulated porcine hepatocyte xenotransplantation on liver metabolic function and survival in baboons with ALF. METHODS: ALF in baboons was induced by 75% hepatectomy with 0, 30, and 60 minutes of warm ischemia of the remnant liver. Pig hepatocytes were isolated from surgically procured MGHminiature swine livers by collagenase perfusion. Alginate-polylysinealginate was used to form 400 to 800 um capsules, and hepatocyte viability pre- and post-encapsulation was 92 ⫾ 2 and 82 ⫾ 10%, respectively. Encapsulated hepatocytes were infused into the baboon’s peritoneum at the end of the laparotomy. RESULTS: Baboons that underwent 0 (n⫽2) and 30 min (n⫽1) warm ischemia of the remnant liver had transient worsening of liver function before recovering. Ones that underwent 60 minutes of ischemia (n⫽3) died on post-operative days (POD) 6, 7 and 10 of liver failure, characterized by worsening coagulopathy, encephalopathy (max NH3 levels ⫽ 435 mol/L) and hyperbilirubinemia (max total bilirubin⫽9.5 mg/dL). Baboons (N⫽3) that received 60 minutes ischemia and encapsulated hepatocytes (70-120 ml of hepatocytes in 200-350 ml of capsules) showed improved liver function compared to those that did not. One died on POD 22 with features of liver failure. Two animals survived ⬎30 days after transplantation with a POD 14 biopsy showing normal liver structure and normal coagulation parameters and bilirubin level. CONCLUSIONS: Transplantation of encapsulated porcine hepatocytes provide temporary metabolic support for baboons with ALF, allowing the remaining liver tissue to regenerate.
CHOP mediates cold ischemia and reperfusion injury in steatotic hepatocytes Parvathi Balachandran MD, Bernard J Dubray MD, Richard S Hotchkiss MD, Jianluo Jia MD, Brett Knolhoff BSc, Gundumi A Upadhya PhD, Thalachallour Mohanakumar PhD, William C Chapman MD, FACS, Christopher D Anderson MD, FACS Washington University-St Louis, St Louis, MO INTRODUCTION: We have previously shown that acute endoplasmic reticulum (ER) stress is triggered following transplantation of steatotic livers. C/EPB Homologous Protein (CHOP) is recognized as the major ER stress cell death mediator. We hypothesized that cold