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Association of Vascular Endothelial Growth Factor gene polymorphism with acute rejection after lung and heart-lung transplantation
The Journal of Heart and Lung Transplantation Volume 24, Number 2S
(PBECs) in culture to invade an artificial basement membrane was assessed using a Matrigel coated 8m filter transwell. Results: S100A4 staining was identified in 7/10 biopsy samples. A median 15% (0 – 48) of the epithelium was stained. S100A4 staining was identified in un-stimulated epithelial cultures. proMMP-2 and proMMP-9 was detectable in PBEC culture supernatant and the level of gelatinolytic activity was up regulated (⬎4x) by TGF-1 stimulation (p⫽.0028). PBECs demonstrated an invasive cell phenotype with a time dependant increase in migrating cells. Conclusions: EMT is common in stable lung allografts. These cells may be a significant source of effector fibroblasts. Matrix metalloproteinases, as a key component of the EMT proteome, offer novel therapeutic targets to inhibit EMT. 22 LONG-TERM ACCEPTANCE OF PORCINE PULMONARY ALLOGRAFTS WITHOUT CHRONIC REJECTION T. Shoji,1 A. Muniappan,1 D.A. Guenther,1 H. Sahara,1 J.C. Wain,1 S.L. Houser,1 R. Hasse,1 M. Bravard,1 J.C. Madsen,1 J.S. Allan,1 1 Department of Surgery, Massachusetts General Hospital and Harvard Medical School, Boston, MA Aims: Using a strategy developed in our laboratory to induce tolerance across strong allogeneic barriers, we have used a brief, but intensive course of immunosuppression to determine whether the induction of donor-specific hyporesponsiveness would prevent graft rejection in an MHC-inbred miniature swine lung transplantation model. Methods: Left lung grafts were performed using MHC class I-disparate donors. Recipients received a 12-day course of cyclosporine (10 –13 mg/kg/d) (n ⫽ 6) or a 12-day course of tacrolimus (0.15 mg/kg/d) (n ⫽ 3) as their only immunosuppression. Control animals received no immunosuppression (n ⫽ 3). Results: Cyclosporine-treated recipients exhibited graft survival from 67 to ⬎605 days. All six animals developed acute rejection between PODs 27 and 108. Two animals lost their grafts on PODs 67 and 69, before developing chronic rejection. The other four recipients developed chronic rejection between PODs 119 and 238. In contrast, all tacrolimustreated recipients maintained their grafts long term, without developing chronic rejection (⬎ 353, ⬎ 322, and ⬎ 245). These recipients also exhibited donor-specific hyporesponsiveness in vitro. Furthermore, after priming with donor antigen, peripheral blood lymphocytes from tacrolimus-treated recipients were able to suppress the response of naive recipient-matched lymphocytes to donor and third-party antigen, suggesting of the presence of regulatory T cells. Untreated control animals lost their grafts to acute rejection by POD 11. Conclusions: A brief post-operative course of tacrolimus was able to induce long-term graft acceptance and donor-specific hyporesponsiveness in a class I-disparate preclinical lung transplant model. Donor-specific hyporesponsiveness and the generation of regulatory T cells appear to accompany the induction of graft acceptance. These data suggest that tolerance induction is a therapeutic strategy that could abrogate chronic lung allograft rejection. 23 ASSOCIATION OF VASCULAR ENDOTHELIAL GROWTH FACTOR GENE POLYMORPHISM WITH ACUTE REJECTION AFTER LUNG AND HEART-LUNG TRANSPLANTATION S. Datta,1 C. Leonard,2 N. Yonan,2 I.V. Hutchinson,3 1Cardiothoracic Unit, Manchester Royal Infirmary, Manchester, United Kingdom; 2 Transplant Unit, Wythenshawe Hospital, Manchester, United Kingdom; 3Dept. of Immunology, School of Biological Sciences, Univ. of Manchester, Manchester, United Kingdom Statement of Purpose: Acute allograft rejection (AAR) gives rise to increased morbidity and mortality after lung and heart-lung transplan-
Abstracts
S49
tation. In most cases, AAR episodes occur in the first year after transplantation. Vascular Endothelial Growth Factor (VEGF) is a mitogen for endothelial cells and is expressed in bronchial tissue. VEGF facilitates adhesion and migration of leucocytes across the endothelium and is thought to be pro-inflammatory. This study investigates whether genetically determined variation in VEGF expression influences the development of AAR after clinical lung and heart-lung transplantation. Statement of procedures: VEGF promoter polymorphisms were examined by SSP-PCR using DNA from 62 recipients. 30 recipients had single lung, 23 had double lungs and nine had heart-lung transplant respectively. Summary of Results: VEGF ⫺2578CC and CA genotypes were associated with increased risk of AAR in the first one year after transplantation (p⬍0.007, odds ratio (OR) 6, 95% CI 1– 40 and p⬍0.004, OR 6.2, 95% CI 1– 41 respectively). The ⫺2578C allele showed an increased risk of AAR compared to the ⫺2578A allele (p⬍0.003, OR 6.2, 95% CI 1.2–39). Similarly, VEGF ⫺1154GG and GA genotypes were also associated with increased risk of AAR (p⬍0.005, OR 2.8, 95% CI 1.2– 6.8 and p⬍0.02, OR 1.9, 95% CI 1– 4.8 respectively). The ⫺1154G allele showed an increased risk of AAR when compared to the ⫺1154A allele (p⬍0.001, OR 2.3, 95% CI 2.1–5.6). Conclusion: These results indicate that polymorphisms at ⫺2578*C and ⫺1154*G gene are strongly associated with AAR after lung and heart-lung transplantation and may be useful markers of acute allograft rejection. 24 THE ROLE OF NKR-P1A FOLLOWING LUNG TRANSPLANTATION J.E. Fildes,1 A.H. Walker,2 J. Thekkudan,3 K. Polster,1 N. Yonan,1 I.V. Hutchinson,4 C.T. Leonard,1 1The Transplant Centre, South Manchester University Hospitals NHS Trust, Wythenshawe Hospital, Manchester, United Kingdom; 2Department of Cardiothoracic Surgery, Northern General Hospital, Sheffield, United Kingdom; 3Department of Cardiothoracic Surgery, University Hospital of Wales, Cardiff, United Kingdom; 4 Department of Immunology, School of Biological Sciences, University of Manchester, Manchester, United Kingdom Background: Therapeutic reduction of T cell competence following lung transplantation is well established. However, natural killer (NK) cells may contribute to the rejection process. NK and NKt cells infiltrate grafts during rejection. Candidate-activating receptors include NKR-P1A (or CD161), which initiates NK cytotoxicity. However, the role of CD161⫹ NK cells during lung graft rejection is unknown. This study investigated peripheral blood NK cell subsets for correlations with clinical outcome following lung transplantation. Methods: 41 consented lung transplant recipients were used in this study. Whole blood was analysed for CD3, CD16, CD56 and CD161 using flow cytometry. Results: Controlling for time, we found a significant negative correlation between CD3⫹ CD161⫹ (Co-ef ⫺0.5749, p⫽0.006), CD56⫺ CD161⫹ (co-ef ⫺0.4536, p⫽0.039) and CD56⫹ CD161⫹ (co-ef ⫺0.5495, p⫽0.010) with decline in lung function (FEV1) since transplant. There was a significant negative correlation between bronchiolitis obliterans syndrome (BOS) grade and the percentage of CD161 cells (p⫽0.006). Conclusions: We observed a decline in peripheral blood CD161 NK and NKt cell subsets in patients with reduced lung function and BOS. We also observed a decline in CD161 NK cell subsets in patients with acute rejection. This is the first association between peripheral blood NK and NKt cell subsets and clinical outcome following human lung transplantation.
(PBECs) in culture to invade an artificial basement membrane was assessed using a Matrigel coated 8m filter transwell. Results: S100A4 staining was identified in 7/10 biopsy samples. A median 15% (0 – 48) of the epithelium was stained. S100A4 staining was identified in un-stimulated epithelial cultures. proMMP-2 and proMMP-9 was detectable in PBEC culture supernatant and the level of gelatinolytic activity was up regulated (⬎4x) by TGF-1 stimulation (p⫽.0028). PBECs demonstrated an invasive cell phenotype with a time dependant increase in migrating cells. Conclusions: EMT is common in stable lung allografts. These cells may be a significant source of effector fibroblasts. Matrix metalloproteinases, as a key component of the EMT proteome, offer novel therapeutic targets to inhibit EMT. 22 LONG-TERM ACCEPTANCE OF PORCINE PULMONARY ALLOGRAFTS WITHOUT CHRONIC REJECTION T. Shoji,1 A. Muniappan,1 D.A. Guenther,1 H. Sahara,1 J.C. Wain,1 S.L. Houser,1 R. Hasse,1 M. Bravard,1 J.C. Madsen,1 J.S. Allan,1 1 Department of Surgery, Massachusetts General Hospital and Harvard Medical School, Boston, MA Aims: Using a strategy developed in our laboratory to induce tolerance across strong allogeneic barriers, we have used a brief, but intensive course of immunosuppression to determine whether the induction of donor-specific hyporesponsiveness would prevent graft rejection in an MHC-inbred miniature swine lung transplantation model. Methods: Left lung grafts were performed using MHC class I-disparate donors. Recipients received a 12-day course of cyclosporine (10 –13 mg/kg/d) (n ⫽ 6) or a 12-day course of tacrolimus (0.15 mg/kg/d) (n ⫽ 3) as their only immunosuppression. Control animals received no immunosuppression (n ⫽ 3). Results: Cyclosporine-treated recipients exhibited graft survival from 67 to ⬎605 days. All six animals developed acute rejection between PODs 27 and 108. Two animals lost their grafts on PODs 67 and 69, before developing chronic rejection. The other four recipients developed chronic rejection between PODs 119 and 238. In contrast, all tacrolimustreated recipients maintained their grafts long term, without developing chronic rejection (⬎ 353, ⬎ 322, and ⬎ 245). These recipients also exhibited donor-specific hyporesponsiveness in vitro. Furthermore, after priming with donor antigen, peripheral blood lymphocytes from tacrolimus-treated recipients were able to suppress the response of naive recipient-matched lymphocytes to donor and third-party antigen, suggesting of the presence of regulatory T cells. Untreated control animals lost their grafts to acute rejection by POD 11. Conclusions: A brief post-operative course of tacrolimus was able to induce long-term graft acceptance and donor-specific hyporesponsiveness in a class I-disparate preclinical lung transplant model. Donor-specific hyporesponsiveness and the generation of regulatory T cells appear to accompany the induction of graft acceptance. These data suggest that tolerance induction is a therapeutic strategy that could abrogate chronic lung allograft rejection. 23 ASSOCIATION OF VASCULAR ENDOTHELIAL GROWTH FACTOR GENE POLYMORPHISM WITH ACUTE REJECTION AFTER LUNG AND HEART-LUNG TRANSPLANTATION S. Datta,1 C. Leonard,2 N. Yonan,2 I.V. Hutchinson,3 1Cardiothoracic Unit, Manchester Royal Infirmary, Manchester, United Kingdom; 2 Transplant Unit, Wythenshawe Hospital, Manchester, United Kingdom; 3Dept. of Immunology, School of Biological Sciences, Univ. of Manchester, Manchester, United Kingdom Statement of Purpose: Acute allograft rejection (AAR) gives rise to increased morbidity and mortality after lung and heart-lung transplan-
Abstracts
S49
tation. In most cases, AAR episodes occur in the first year after transplantation. Vascular Endothelial Growth Factor (VEGF) is a mitogen for endothelial cells and is expressed in bronchial tissue. VEGF facilitates adhesion and migration of leucocytes across the endothelium and is thought to be pro-inflammatory. This study investigates whether genetically determined variation in VEGF expression influences the development of AAR after clinical lung and heart-lung transplantation. Statement of procedures: VEGF promoter polymorphisms were examined by SSP-PCR using DNA from 62 recipients. 30 recipients had single lung, 23 had double lungs and nine had heart-lung transplant respectively. Summary of Results: VEGF ⫺2578CC and CA genotypes were associated with increased risk of AAR in the first one year after transplantation (p⬍0.007, odds ratio (OR) 6, 95% CI 1– 40 and p⬍0.004, OR 6.2, 95% CI 1– 41 respectively). The ⫺2578C allele showed an increased risk of AAR compared to the ⫺2578A allele (p⬍0.003, OR 6.2, 95% CI 1.2–39). Similarly, VEGF ⫺1154GG and GA genotypes were also associated with increased risk of AAR (p⬍0.005, OR 2.8, 95% CI 1.2– 6.8 and p⬍0.02, OR 1.9, 95% CI 1– 4.8 respectively). The ⫺1154G allele showed an increased risk of AAR when compared to the ⫺1154A allele (p⬍0.001, OR 2.3, 95% CI 2.1–5.6). Conclusion: These results indicate that polymorphisms at ⫺2578*C and ⫺1154*G gene are strongly associated with AAR after lung and heart-lung transplantation and may be useful markers of acute allograft rejection. 24 THE ROLE OF NKR-P1A FOLLOWING LUNG TRANSPLANTATION J.E. Fildes,1 A.H. Walker,2 J. Thekkudan,3 K. Polster,1 N. Yonan,1 I.V. Hutchinson,4 C.T. Leonard,1 1The Transplant Centre, South Manchester University Hospitals NHS Trust, Wythenshawe Hospital, Manchester, United Kingdom; 2Department of Cardiothoracic Surgery, Northern General Hospital, Sheffield, United Kingdom; 3Department of Cardiothoracic Surgery, University Hospital of Wales, Cardiff, United Kingdom; 4 Department of Immunology, School of Biological Sciences, University of Manchester, Manchester, United Kingdom Background: Therapeutic reduction of T cell competence following lung transplantation is well established. However, natural killer (NK) cells may contribute to the rejection process. NK and NKt cells infiltrate grafts during rejection. Candidate-activating receptors include NKR-P1A (or CD161), which initiates NK cytotoxicity. However, the role of CD161⫹ NK cells during lung graft rejection is unknown. This study investigated peripheral blood NK cell subsets for correlations with clinical outcome following lung transplantation. Methods: 41 consented lung transplant recipients were used in this study. Whole blood was analysed for CD3, CD16, CD56 and CD161 using flow cytometry. Results: Controlling for time, we found a significant negative correlation between CD3⫹ CD161⫹ (Co-ef ⫺0.5749, p⫽0.006), CD56⫺ CD161⫹ (co-ef ⫺0.4536, p⫽0.039) and CD56⫹ CD161⫹ (co-ef ⫺0.5495, p⫽0.010) with decline in lung function (FEV1) since transplant. There was a significant negative correlation between bronchiolitis obliterans syndrome (BOS) grade and the percentage of CD161 cells (p⫽0.006). Conclusions: We observed a decline in peripheral blood CD161 NK and NKt cell subsets in patients with reduced lung function and BOS. We also observed a decline in CD161 NK cell subsets in patients with acute rejection. This is the first association between peripheral blood NK and NKt cell subsets and clinical outcome following human lung transplantation.