- Email: [email protected]
139
S92
Abstracts
139 FK778: NEW CELLULAR AND MOLECULAR MECHANISMS OF ACTION S. Schrepfer,1,5 T. Deuse,1 F. Koch-Nolte,2 H. Schaefer,3 E. Schwedhelm,4 R. Boeger,4 H. Reichenspurner,1 1Cardiovascular Surgery, University Heart Center Hamburg, Hamburg, Germany; 2 Department of Immunology, University Hamburg, Hamburg, Germany; 3Department of Pathology, University Hamburg, Hamburg, Germany; 4Department of Pharmacology, University Hamburg, Hamburg, Germany; 5Department of Cardiothoracic Surgery, Stanford University School of Medicine, Palo Alto, CA Purpose: This report reveals new mechanisms of action of FK778 on different cell types who are involved in acute and chronic allograft rejection. Methods/Materials: Purified rat aortic endothelial cell (EC) cultures were pre-treated with low or high concentrations of FK778. Endothelial adhesion molecule expression (ICAM-1/VCAM-1) was stimulated with TNF-alpha and quantified by FACS analysis/immunofluorescence/western blotting. Purified rat lymphocytes were incubated with the same FK778 concentrations, were stimulated via TCR/CD28 signals, and CD25-expression was quantified using FACS-analysis. Uridine addition was used in all assays to reverse the pyrimidine synthesis blockade. Lymphocyte-EC-interaction was assessed by cellculture-adhesion-assays. TNF-alpha-receptor binding assays were performed using radiolabelled TNF. SMC proliferation and migration were analysed in comparison to tacrolimus and sirolimus. Results: TNF-alpha stimulation and TCR/CD28 costimulation significantly increased EC ICAM-1/ VCAM-1-expression and lymphocyte CD25-surface-expression, respectively. These effects were dose-dependently inhibited by FK778 incubation and were not reversed by the addition of uridine. FK778 dose-dependently attenuated lymphocyte adhesion to allogeneic EC in both adhesion assays. FK778 did not interfere with TNF-receptor binding. The dose-dependent inhibition of SMC proliferation by FK778 was abolished by uridine addition, whereas the inhibitory effect on SMC migration was not affected by uridine supplementation. The latter effect was comparable to the sirolimus potency. Conclusion: FK778 directly reduces endothelial adhesion molecule up-regulation, inhibits lymphocyte activation, and attenuates lymphocyte-endothelium interaction, a critical early step in graft rejection. These effects can be separated from its blockade on pyrimidine synthesis and are not mediated via TNF-receptor blockade. The antiproliferative potency of FK778 on SMC may be an important mechanism to inhibit the fibroproliferative lesions of chronic organ rejection. 140 POLYMORPHISMS OF THE MDR1 GENE AND FREEDOM FROM BIOPSY PROVEN REJECTION IN HEART TRANSPLANTATION J.B. Barnard,1 S. Sheldon,2 S. Richardson,1 J. Fildes,1 N. Khasati,1 V. Pravica,3 I.V. Hutchinson,3 C.T. Leonard,1 N. Yonan,1 1The Transplant Centre, South Manchester University Hospitals NHS Trust, Manchester, United Kingdom; 2Tissue Typing Laboratory, Manchester Royal Infirmary, Manchester, United Kingdom; 3 Department of Immunology, Manchester University, Manchester, United Kingdom Background: Variations in the expression levels and in the activity of the multidrug-resistance MDR1 encoded P-Glycoprotein (P-gp) have a major impact on the therapeutic efficacy of many drugs including immunosuppressants. We observed a significant association of a polymorphisms in exon 26 (C3435T) and exon 21 (G2677T) of the MDR1 gene with freedom from endomyocardial biopsy proven rejection (EBPR) in a cohort of 176 heart transplant patients.
The Journal of Heart and Lung Transplantation February 2006
Methods: Using a previously described PCR method we characterized the C3435T polymorphism in exon 26 and the G2677T polymorphism in exon 21 of the MDR1 gene in 176 heart transplant patients. We examined the relationship between MDR1 polymorphisms and EBPR determined by biopsy performed at set intervals according to a standard protocol. Results: No significant differences in clinical variables were found between the 3 groups according to univariate analysis. A significant relationship was found between patient’s exon 26 and 21 genotype and their freedom from first grade 3A rejection episode by Kaplan Meier analysis Figure 1. The effect of the MDR1 polymorphisms on increased likelyhood of rejection is shown in Table 1 and effects of HLA mismatching are also shown for comparison.
Risk Factors for Grade 3A EBPR Risk Factor
HR
95% CI
p-value
Exon 26/21 CC/CG Haplotype Exon 26 CC Exon 21 GG HLA 5 or 6 Mismatches HLA 0,1 or 2 Mismatches
2.18 1.8 1.68 1.18 0.76
(1.21–4.26) (1.05–3.09) (1.01–2.92) (0.075–1.86) (0.028–2.08)
0.02 0.03 0.05 0.05 0.60
Conclusions: These results highlight the importance of the MDR1 gene and P-gp in transplant rejection.
141 EXTRACORPOREAL PHOTOPHERESIS (ECP) CAUSES INTRAMYOCARDIAL DELETION OF GRAFT-SPECIFIC T CELLS IN MURINE CARDIAC TRANSPLANTS J.F. George,1 L.L. Guo,1 C. Gooden,1 D.C. Naftel,1 D. Peritt,2 J.K. Kirklin,1 1Department of Surgery, University of Alabama at Birmingham, Birmingham, AL; 2THERAKOS Corporation, Exton, PA ECP is effective in reducing the hazard for rejection resulting in death or hemodynamic compromise. Since rejection is T cell dependent, we hypothesized that ECP causes deletion or alters function of graftspecific T cells. Method: CBA/Ca mice were infused with syngeneic T cells bearing T cell receptor transgenes specific for the Kb alloantigen found in C57BL/6 mice. The fate and function of the Kb-specific infused cells were followed using CFSE label and a clonotypic antibody specific for the expressed transgenes, clone Ti98. These infused mice received a heterotopic cardiac allograft (Ctx) from a C57BL/6 mouse and were immediately infused with 107 ECP-treated CBA/Ca splenocytes (200ng/ml 8-methoxypsoralen and UV irradiation (2J/cm2). At 3 and 5 days post-transplantation, lymphoid cells from the Ctx and peripheral lymphoid organs were analyzed by flow cytometry to determine the abundance and viability of labeled Kb-specific (anti-donor) T cells.
Abstracts
139 FK778: NEW CELLULAR AND MOLECULAR MECHANISMS OF ACTION S. Schrepfer,1,5 T. Deuse,1 F. Koch-Nolte,2 H. Schaefer,3 E. Schwedhelm,4 R. Boeger,4 H. Reichenspurner,1 1Cardiovascular Surgery, University Heart Center Hamburg, Hamburg, Germany; 2 Department of Immunology, University Hamburg, Hamburg, Germany; 3Department of Pathology, University Hamburg, Hamburg, Germany; 4Department of Pharmacology, University Hamburg, Hamburg, Germany; 5Department of Cardiothoracic Surgery, Stanford University School of Medicine, Palo Alto, CA Purpose: This report reveals new mechanisms of action of FK778 on different cell types who are involved in acute and chronic allograft rejection. Methods/Materials: Purified rat aortic endothelial cell (EC) cultures were pre-treated with low or high concentrations of FK778. Endothelial adhesion molecule expression (ICAM-1/VCAM-1) was stimulated with TNF-alpha and quantified by FACS analysis/immunofluorescence/western blotting. Purified rat lymphocytes were incubated with the same FK778 concentrations, were stimulated via TCR/CD28 signals, and CD25-expression was quantified using FACS-analysis. Uridine addition was used in all assays to reverse the pyrimidine synthesis blockade. Lymphocyte-EC-interaction was assessed by cellculture-adhesion-assays. TNF-alpha-receptor binding assays were performed using radiolabelled TNF. SMC proliferation and migration were analysed in comparison to tacrolimus and sirolimus. Results: TNF-alpha stimulation and TCR/CD28 costimulation significantly increased EC ICAM-1/ VCAM-1-expression and lymphocyte CD25-surface-expression, respectively. These effects were dose-dependently inhibited by FK778 incubation and were not reversed by the addition of uridine. FK778 dose-dependently attenuated lymphocyte adhesion to allogeneic EC in both adhesion assays. FK778 did not interfere with TNF-receptor binding. The dose-dependent inhibition of SMC proliferation by FK778 was abolished by uridine addition, whereas the inhibitory effect on SMC migration was not affected by uridine supplementation. The latter effect was comparable to the sirolimus potency. Conclusion: FK778 directly reduces endothelial adhesion molecule up-regulation, inhibits lymphocyte activation, and attenuates lymphocyte-endothelium interaction, a critical early step in graft rejection. These effects can be separated from its blockade on pyrimidine synthesis and are not mediated via TNF-receptor blockade. The antiproliferative potency of FK778 on SMC may be an important mechanism to inhibit the fibroproliferative lesions of chronic organ rejection. 140 POLYMORPHISMS OF THE MDR1 GENE AND FREEDOM FROM BIOPSY PROVEN REJECTION IN HEART TRANSPLANTATION J.B. Barnard,1 S. Sheldon,2 S. Richardson,1 J. Fildes,1 N. Khasati,1 V. Pravica,3 I.V. Hutchinson,3 C.T. Leonard,1 N. Yonan,1 1The Transplant Centre, South Manchester University Hospitals NHS Trust, Manchester, United Kingdom; 2Tissue Typing Laboratory, Manchester Royal Infirmary, Manchester, United Kingdom; 3 Department of Immunology, Manchester University, Manchester, United Kingdom Background: Variations in the expression levels and in the activity of the multidrug-resistance MDR1 encoded P-Glycoprotein (P-gp) have a major impact on the therapeutic efficacy of many drugs including immunosuppressants. We observed a significant association of a polymorphisms in exon 26 (C3435T) and exon 21 (G2677T) of the MDR1 gene with freedom from endomyocardial biopsy proven rejection (EBPR) in a cohort of 176 heart transplant patients.
The Journal of Heart and Lung Transplantation February 2006
Methods: Using a previously described PCR method we characterized the C3435T polymorphism in exon 26 and the G2677T polymorphism in exon 21 of the MDR1 gene in 176 heart transplant patients. We examined the relationship between MDR1 polymorphisms and EBPR determined by biopsy performed at set intervals according to a standard protocol. Results: No significant differences in clinical variables were found between the 3 groups according to univariate analysis. A significant relationship was found between patient’s exon 26 and 21 genotype and their freedom from first grade 3A rejection episode by Kaplan Meier analysis Figure 1. The effect of the MDR1 polymorphisms on increased likelyhood of rejection is shown in Table 1 and effects of HLA mismatching are also shown for comparison.
Risk Factors for Grade 3A EBPR Risk Factor
HR
95% CI
p-value
Exon 26/21 CC/CG Haplotype Exon 26 CC Exon 21 GG HLA 5 or 6 Mismatches HLA 0,1 or 2 Mismatches
2.18 1.8 1.68 1.18 0.76
(1.21–4.26) (1.05–3.09) (1.01–2.92) (0.075–1.86) (0.028–2.08)
0.02 0.03 0.05 0.05 0.60
Conclusions: These results highlight the importance of the MDR1 gene and P-gp in transplant rejection.
141 EXTRACORPOREAL PHOTOPHERESIS (ECP) CAUSES INTRAMYOCARDIAL DELETION OF GRAFT-SPECIFIC T CELLS IN MURINE CARDIAC TRANSPLANTS J.F. George,1 L.L. Guo,1 C. Gooden,1 D.C. Naftel,1 D. Peritt,2 J.K. Kirklin,1 1Department of Surgery, University of Alabama at Birmingham, Birmingham, AL; 2THERAKOS Corporation, Exton, PA ECP is effective in reducing the hazard for rejection resulting in death or hemodynamic compromise. Since rejection is T cell dependent, we hypothesized that ECP causes deletion or alters function of graftspecific T cells. Method: CBA/Ca mice were infused with syngeneic T cells bearing T cell receptor transgenes specific for the Kb alloantigen found in C57BL/6 mice. The fate and function of the Kb-specific infused cells were followed using CFSE label and a clonotypic antibody specific for the expressed transgenes, clone Ti98. These infused mice received a heterotopic cardiac allograft (Ctx) from a C57BL/6 mouse and were immediately infused with 107 ECP-treated CBA/Ca splenocytes (200ng/ml 8-methoxypsoralen and UV irradiation (2J/cm2). At 3 and 5 days post-transplantation, lymphoid cells from the Ctx and peripheral lymphoid organs were analyzed by flow cytometry to determine the abundance and viability of labeled Kb-specific (anti-donor) T cells.