- Email: [email protected]
Technique of immunoapheresis in heterotopic and orthotopic xenotransplantation of pig hearts into cynomolgus and rhesus monkeys
Technique of Immunoapheresis in Heterotopic and Orthotopic Xenotransplantation of Pig Hearts into Cynomolgus and Rhesus Monkeys P. Brenner, M. Schmoeckel, H. Reichenspurner, T. Felbinger, M. Hinz, V. Eder, A. Rucker, C. Wimmer, S. Uchita, S. Kriegeskorte, B. Meiser, J. Mu¨ller-Ho¨cker, D. Seidel, C. Hammer, and B. Reichart
U
P TO 30% of patients awaiting heart transplantation die while on the waiting list because of a worldwide shortage of human donor organs.1 Cardiac xenotransplantation would offer an alternative. Hyperacute xenograft rejection (HXR) of vascularized organs transplanted between phylogenetically distant species (discordant xenotransplantation), such as pig and primate, is triggered by preexisting xenoreactive natural antibodies (XNAb). These XNAb of the xenogenic serum bind to Gal␣1-3Gal epitopes of glycoproteins and glycolipids of the porcine vascular endothelium activating the complement cascade by the classical and alternative pathway.2 This causes microvascular thrombosis, interstitial edema, hemorrhage, and cell necrosis.3 Several techniques have been applied to remove XNAb and to deplete complement,4 such as plasma exchange, plasmapheresis, xenogeneic organ perfusion, unspecific antibody absorbents, the use of haptens like ␣Gal13Gal-fragments, and penicillamine.5 In our experiments we investigated the elimination of immunoglobulins (Ig) IgG, IgM, and IgA by immunoapheresis (IA) with a reusable Ig-Therasorb column (Baxter/Therasorb Corp., Unterschleissheim, Germany) to prevent HXR. In addition, first technical data about IA in a heterotopic (abdominal) and orthotopic xenotransplantation model with small old world monkeys should be evaluated. In recent studies IA was effective to prevent HXR in an ex vivo working heart perfusion model.6 METHODS AND ANIMALS The IA column Ig-Therasorb consists of a glass container with Sepharose CL-4B suspension coated with polyclonal sheep antibodies. These antibodies bind specifically the immunoglobulin IgG (subclasses 1 to 4), IgM, IgA, circulating immune complexes, and fragments of immunoglobulins. Every passage of plasma through the column leads to an average IgG reduction of 60% to 70%. The Ig-Therasorb unit consists of a pair of reuseable columns. In a first step the blood of the monkeys is separated by plasmapheresis and the cellular fraction returned to the recipient. Blood flow at the separation device should reach a rate of approximately 20 to 50 mL/min to ensure a constant plasma flow. The plasma flow is directed to the first column (300 mL filling volume) and then switched to the second column. In the meantime bound immunoglobulins are lysed from the sepharose and the column is regener-
ated by glycine and PBS buffer solution. In our series we performed two cycles of immunoadsorption before exposing the pig heart to the antibody-depleted blood of the cynomolgus monkey. A total of nine xenotransplantations (XT) of landrace pig hearts were performed. Two orthotopic (oXHTx) and one heterotopic xenotransplantation (hXHTx) of Cynomolgus monkeys (4.5 to 6.4 kg) and two hXHTx of Rhesus monkeys (5.7 and 10.5 kg) were performed with IA. Four hXHTx of Cynomolgus monkeys (2.6 to 4.3 kg) without IA served as a control group. In oXHTx blood for two cycles of IA was taken from the extracorporeal circulation and in hXHTx from a central venous catheter. Before and after IA and continuously after transplantation levels of immunoglobulins, complement factors and anti-pig antibodies (hemagglutination assay) were determined. Complement components and activity (CH100, AP100), cardiac enzymes like LDH, GOT, CK, CK-MB, and troponin I were measured. Myocardial tissue samples of xenografts were examined immunohistochemically and by light and electron microscopy.
RESULTS
OXHTx and hXHTx with IA showed better myocardial function until oXHTx experiments were terminated according to protocol after 130 ⫾ 21.2 minutes and hXHTx after 140 ⫾ 35.3 minutes without histologic signs of HXR. The group without IA showed graft failure after 78 ⫾ 27.6 minutes. The tissue of xenografts without IA showed increased deposition of immunoglobulins (IgM) and complement components (C3, C4 and C5b-9) if compared to xenografts of the IA group. IA removed 86% of IgM, 80% of IgG, and 85% of IgA. Accordingly 75% of anti-pig antibodies were reduced by IA. Without IA all anti-pig antibodies were eliminated after 60 minutes by adsorption From the Department of Cardiac Surgery (P.B., M.S., H.R., M.H., V.E., A.R., C.W., S.U., S.K., B.M., B.R.), Institute for Surgical Research (P.B., C.H.), Institute of Anesthesiology (T.F.), Pathological Institute (J.M.H.), and the Institute for Clinical Chemistry (D.S.), Klinikum Grosshadern, Ludwig-MaximiliansUniversity of Munich, Munich, Germany. Supported by a grant from Bavarian Research Foundation and by Baxter/Therasorb Comp, Unterschleissheim, Germany. Address reprint requests to Paolo Brenner, MD, Department of Cardiac Surgery, Klinikum Grosshaderm, Marchioninistr. 15, D-81377 Munich, Germany.
© 2000 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
0041-1345/00/$–see front matter PII S0041-1345(00)01135-0
Transplantation Proceedings, 32, 1087–1088 (2000)
1087
1088
onto the xenograft during HXR. Activity of the complement system was no more detectable after IA while in the untreated group a low activity was observed. Only in monkeys without IA LDH, GOT, and CK/CK-MB were significantly higher than in the IA group. IA in small primates caused a significant decrease of the hemoglobin level by hemodilution (NaCl-solution content of the column: 300 mL) to a hemoglobin level of 5 mg/dL in all recipients. White blood cells were reduced at the end of the extracorporeal circulation after oXHTx, but also in hXHTx after IA. In the control group white blood cell count remained constant. Another serious side-effect of IA was the massive decrease of platelets. DISCUSSION
Hemodilution proved to be a typical problem of IA in small monkeys, because normally an Ig-Therasorb column with 300 mL plasma volume is specified for adult patient treatment. Thus for IA of small monkeys with less than 10 kg an
BRENNER, SCHMOECKEL, REICHENSPURNER ET AL
IA column with a decreased filling volume would be more suitable. Removal of antibodies and complement by IA proved to be an effective method to prevent HXR after XT of Cynomolgus and Rhesus monkeys. We were able to demonstrate that IA can also be performed in small nonhuman primates. IA could be a useful treatment to prevent HXR in future clinical infant and children XT. REFERENCES 1. Markmann JF, Baker CF: Curr Probl Surg 31:390, 1994 2. Lu CY, Khair-El-Din TA, et al: FASEB J 8:1122, 1994 3. Platt JL, Fischel RJ, Matas AJ, et al: Transplantation 52:214, 1991 4. Shapiro R, Tzakis AG, Scantlebury V, et al: J Invest Surg 3:39, 1990 5. Bach FH, Platt JL, Cooper DKC: In Cooper DKC et al (eds): Xenotransplantation. Heidelberg: Springer; 1991, p 81 6. Brenner P, Hinz M, Huber H, et al: Eur J Cardiothorac Surg 15:672, 1999
U
P TO 30% of patients awaiting heart transplantation die while on the waiting list because of a worldwide shortage of human donor organs.1 Cardiac xenotransplantation would offer an alternative. Hyperacute xenograft rejection (HXR) of vascularized organs transplanted between phylogenetically distant species (discordant xenotransplantation), such as pig and primate, is triggered by preexisting xenoreactive natural antibodies (XNAb). These XNAb of the xenogenic serum bind to Gal␣1-3Gal epitopes of glycoproteins and glycolipids of the porcine vascular endothelium activating the complement cascade by the classical and alternative pathway.2 This causes microvascular thrombosis, interstitial edema, hemorrhage, and cell necrosis.3 Several techniques have been applied to remove XNAb and to deplete complement,4 such as plasma exchange, plasmapheresis, xenogeneic organ perfusion, unspecific antibody absorbents, the use of haptens like ␣Gal13Gal-fragments, and penicillamine.5 In our experiments we investigated the elimination of immunoglobulins (Ig) IgG, IgM, and IgA by immunoapheresis (IA) with a reusable Ig-Therasorb column (Baxter/Therasorb Corp., Unterschleissheim, Germany) to prevent HXR. In addition, first technical data about IA in a heterotopic (abdominal) and orthotopic xenotransplantation model with small old world monkeys should be evaluated. In recent studies IA was effective to prevent HXR in an ex vivo working heart perfusion model.6 METHODS AND ANIMALS The IA column Ig-Therasorb consists of a glass container with Sepharose CL-4B suspension coated with polyclonal sheep antibodies. These antibodies bind specifically the immunoglobulin IgG (subclasses 1 to 4), IgM, IgA, circulating immune complexes, and fragments of immunoglobulins. Every passage of plasma through the column leads to an average IgG reduction of 60% to 70%. The Ig-Therasorb unit consists of a pair of reuseable columns. In a first step the blood of the monkeys is separated by plasmapheresis and the cellular fraction returned to the recipient. Blood flow at the separation device should reach a rate of approximately 20 to 50 mL/min to ensure a constant plasma flow. The plasma flow is directed to the first column (300 mL filling volume) and then switched to the second column. In the meantime bound immunoglobulins are lysed from the sepharose and the column is regener-
ated by glycine and PBS buffer solution. In our series we performed two cycles of immunoadsorption before exposing the pig heart to the antibody-depleted blood of the cynomolgus monkey. A total of nine xenotransplantations (XT) of landrace pig hearts were performed. Two orthotopic (oXHTx) and one heterotopic xenotransplantation (hXHTx) of Cynomolgus monkeys (4.5 to 6.4 kg) and two hXHTx of Rhesus monkeys (5.7 and 10.5 kg) were performed with IA. Four hXHTx of Cynomolgus monkeys (2.6 to 4.3 kg) without IA served as a control group. In oXHTx blood for two cycles of IA was taken from the extracorporeal circulation and in hXHTx from a central venous catheter. Before and after IA and continuously after transplantation levels of immunoglobulins, complement factors and anti-pig antibodies (hemagglutination assay) were determined. Complement components and activity (CH100, AP100), cardiac enzymes like LDH, GOT, CK, CK-MB, and troponin I were measured. Myocardial tissue samples of xenografts were examined immunohistochemically and by light and electron microscopy.
RESULTS
OXHTx and hXHTx with IA showed better myocardial function until oXHTx experiments were terminated according to protocol after 130 ⫾ 21.2 minutes and hXHTx after 140 ⫾ 35.3 minutes without histologic signs of HXR. The group without IA showed graft failure after 78 ⫾ 27.6 minutes. The tissue of xenografts without IA showed increased deposition of immunoglobulins (IgM) and complement components (C3, C4 and C5b-9) if compared to xenografts of the IA group. IA removed 86% of IgM, 80% of IgG, and 85% of IgA. Accordingly 75% of anti-pig antibodies were reduced by IA. Without IA all anti-pig antibodies were eliminated after 60 minutes by adsorption From the Department of Cardiac Surgery (P.B., M.S., H.R., M.H., V.E., A.R., C.W., S.U., S.K., B.M., B.R.), Institute for Surgical Research (P.B., C.H.), Institute of Anesthesiology (T.F.), Pathological Institute (J.M.H.), and the Institute for Clinical Chemistry (D.S.), Klinikum Grosshadern, Ludwig-MaximiliansUniversity of Munich, Munich, Germany. Supported by a grant from Bavarian Research Foundation and by Baxter/Therasorb Comp, Unterschleissheim, Germany. Address reprint requests to Paolo Brenner, MD, Department of Cardiac Surgery, Klinikum Grosshaderm, Marchioninistr. 15, D-81377 Munich, Germany.
© 2000 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
0041-1345/00/$–see front matter PII S0041-1345(00)01135-0
Transplantation Proceedings, 32, 1087–1088 (2000)
1087
1088
onto the xenograft during HXR. Activity of the complement system was no more detectable after IA while in the untreated group a low activity was observed. Only in monkeys without IA LDH, GOT, and CK/CK-MB were significantly higher than in the IA group. IA in small primates caused a significant decrease of the hemoglobin level by hemodilution (NaCl-solution content of the column: 300 mL) to a hemoglobin level of 5 mg/dL in all recipients. White blood cells were reduced at the end of the extracorporeal circulation after oXHTx, but also in hXHTx after IA. In the control group white blood cell count remained constant. Another serious side-effect of IA was the massive decrease of platelets. DISCUSSION
Hemodilution proved to be a typical problem of IA in small monkeys, because normally an Ig-Therasorb column with 300 mL plasma volume is specified for adult patient treatment. Thus for IA of small monkeys with less than 10 kg an
BRENNER, SCHMOECKEL, REICHENSPURNER ET AL
IA column with a decreased filling volume would be more suitable. Removal of antibodies and complement by IA proved to be an effective method to prevent HXR after XT of Cynomolgus and Rhesus monkeys. We were able to demonstrate that IA can also be performed in small nonhuman primates. IA could be a useful treatment to prevent HXR in future clinical infant and children XT. REFERENCES 1. Markmann JF, Baker CF: Curr Probl Surg 31:390, 1994 2. Lu CY, Khair-El-Din TA, et al: FASEB J 8:1122, 1994 3. Platt JL, Fischel RJ, Matas AJ, et al: Transplantation 52:214, 1991 4. Shapiro R, Tzakis AG, Scantlebury V, et al: J Invest Surg 3:39, 1990 5. Bach FH, Platt JL, Cooper DKC: In Cooper DKC et al (eds): Xenotransplantation. Heidelberg: Springer; 1991, p 81 6. Brenner P, Hinz M, Huber H, et al: Eur J Cardiothorac Surg 15:672, 1999