- Email: [email protected]
Advanced ageing of T lymphocytes in geriatric end-stage renal disease patients
238
Abstracts
human. In the present study we investigated whether circulating MSC were present in the peripheral blood of healthy individuals and patients with organ injury. We were unable to detect MSC in the blood of healthy individuals by flow cytometry and cell culture techniques. We then analyzed the presence of MSC in the blood of patients with end-stage renal disease (n = 10), end-stage liver disease (n = 10) and in heart transplant patients with biopsy proven rejection (n = 8), by culturing of mononuclear cells under MSCsupporting culture conditions. In none of these patients MSC were identified in the blood. In the stromal vascular fraction of adipose tissue and in liver transplant perfusion fluid we were able to detect MSC, indicating that the methods used enabled the detection of MSC. The conclusion of this study is that MSC are not detectable in the circulation in patients with injured solid organs and during aggressive immune responses.
doi:10.1016/j.trim.2014.11.158
B14-1049 Thymic output prior to kidney transplantation is associated with the risk for acute rejection R.W.J. Meijersa, N.H.R. Litjensa, A.W. Langerakb, C.C. Baana, W. Weimara, M.G.H. Betjesa a
Department of Internal Medicine, Section of Nephrology and Transplantation, Rotterdam, The Netherlands b Department of Immunology, Erasmus Medical Center, Rotterdam, The Netherlands
Background: The uremia-induced inflammatory environment in end-stage renal disease (ESRD) patients is associated with premature T-cell ageing, which results in defective T-cell-mediated immunity. Since T cells play a major role in acute rejection (AR), we hypothesized that an aged T-cell compartment prior to kidney transplantation (KTx) is associated with a lower risk for AR post-KTx. Methods: For this purpose, we analyzed pre-KTx samples of 20 kidney transplant recipients with a biopsy-proven AR within three months post-KTx (AR patients). For each patient with AR, two nonrejectors (NR) were included, matched for age, number of HLAmismatches and cytomegalovirus serostatus. Thymic output was assessed by determining the T-cell receptor excision circle (TREC) content and percentages of CD31+ (Ki67−) naïve T cells as recent thymic emigrants (RTEs). The relative telomere length (RTL) was determined as a measure for proliferative history and immunophenotyping was used to establish the differentiation status of circulating T cells. Results: Interestingly, the AR patients had a significantly higher thymic output (p b 0.05) as measured by TREC-content and x percentages of CD31+Ki67− naïve CD4+ and CD8+ T cells pre-KTx. The RTL of both CD4+ and CD8+ T cells did not differ between the two patient groups prior to KTx. Furthermore, the AR patients had a significantly (p b 0.05) lower percentage CD4+ memory T cells lacking CD28 (2.75 ± 0.95% vs. 5.11 ± 1.02%). For the CD8+ T-cells, AR patients had a significantly (p b 0.05) higher number of central memory cells (32.3 ± 5.00 vs. 20.62 ± 2.77 cells/ml) compared to the matched NR. Conclusion: An aged T-cell phenotype together with a lower thymic output prior to KTx is associated with a lower risk for AR. (This study was financially supported by the Dutch Kidney Foundation (KSPB.10.12)).
doi:10.1016/j.trim.2014.11.159
B14-1051 The effect of adipose tissue derived mesenchymal stem cells on B cell proliferation and differentiation M. Franquesaa, F. Mensaha, R. Huizingab, M.G.H. Betjesa, W. Weimara, C.C. Baana, M. Hoogduijna a
Transplantation and Nephrology Laboratory, Internal Medicine Department, Erasmus MC, Netherlands b Immunology Department, Erasmus MC, Netherlands Background: Mesenchymal stem cells (MSC) have proven immunomodulatory capacity which makes them a promising therapeutic tool in transplantation. While the immunosuppressive effect of MSC on T cellmediated effector mechanisms has been well studied, less is known about the effects of MSC on B cell-mediated immune responses. Methods: MSC were isolated from subcutaneous fat tissue from kidney transplant donors. Resting mature B cells from tonsils were obtained by CD43 negative selection with Magnetic Activated Cell Sorting (MACS). MSC were co-cultured with CFSE-labeled B cells stimulated in a T cell-like fashion (anti-IgM + anti-CD40 + IL2) or by PMA/ionomycin activated CD4 T cells. Proliferation and B cell phenotype were analyzed by Flow Cytometry, and IgG production quantified by ELISA. Results: Proliferation of B cells activated in a T cells-like manner (anti-IgM + anti-CD40 + IL2) was not affected by the presence of MSC, while MSC decrease the proliferation of B cells stimulated with activated T cells. An induction of plasmablasts (CD19+ CD27high CD38high) occurred when B cells were stimulated in a T cell dependent manner or in the presence of activated CD4 T cells. MSC abolished the differentiation into plasmablasts completely, which was correlated with decreased IgG production. Furthermore, MSCs induced an increase in the percentage of CD19+ CD27− CD38high CD24high regulatory-like B cells when stimulated in a Tcell-like fashion. Conclusion: MSC inhibit B cell differentiation while increasing the proportion of regulatory-like B cells. The reduction of B cell proliferation by MSC is T cell-dependent. These results suggest a therapeutic role of MSC for the treatment of patients suffering from B cell mediated alloreactivity. doi:10.1016/j.trim.2014.11.160
B14-1052 Advanced ageing of T lymphocytes in geriatric end-stage renal disease patients L. Huanga, N.H.R. Litjensa, R.W.J. Meijersa, A.W. Langerakb, W. Weimara, C.C. Baana, M.G.H. Betjesa a
Division of Nephrology and Transplantation, Dept of Internal Medicine, Erasmus Medical Center, The Netherlands b Division of Nephrology and Transplantation, Dept of Immunology, Erasmus Medical Center, The Netherlands Introduction: The proportion of geriatric patients (N60 years) suffering from end-stage renal disease (ESRD) has significantly increased over the last decade. ESRD is associated with premature immunological ageing of the T cell system which may explain the increased susceptibility for infections, cancer and cardiovascular diseases in these patients. This may in particular be of importance in geriatric patients but data in this age group are limited. The aim of this study was to assess the concept of immunological ageing of T lymphocytes in geriatric ESRD patients prior to KTx, compared to age-matched healthy controls.
Abstracts
Patients, materials and methods: We determined T-cell ageing parameters in circulating T cells of 91 ESRD patients and 73 age- and CMV-matched healthy controls. These parameters included thymic output by T-cell receptor excision circle (TREC)-content and number of CD31 + naive T cells, T-cell differentiation status by assessing the naive/memory, central memory (CM)/effector memory (EM) plus terminally differentiated effector (EMRA) ratios and percentage of CD28null memory T cells, and T cell proliferative history by relative telomere length (RTL). Results: Compared to age-matched healthy controls, geriatric ESRD patients had a lower thymic output according to lower TREC-content and CD31+ naive T cell numbers. Moreover, geriatric ESRD patients had a significantly lower T cell count due to decreased CD4+, but not CD8+, T-cell numbers. This T cell compartment consisted of relatively more memory T cells resulting from significant lower numbers of naive T cells. In addition, the memory T cell compartment contained relatively more differentiated T-cell subsets (EM, EMRA) and CD28null T cells. In addition, the RTL was significantly reduced in CD8+, and tended to be shorter in CD4+ T cells in geriatric ESRD patients. Conclusions: Geriatric ESRD patients have an advanced aged T cell compartment when compared to age-matched healthy controls. This advanced aged T cell compartment may impact the clinical course following kidney transplantation. doi:10.1016/j.trim.2014.11.161
B14-1057 Criteria for viability assessment of discarded human donor livers during ex-vivo normothermic machine perfusion Michael E. Suttona,b,1, Sanna op den Driesa,b,1, Negin Karimiana,b, Marieke T. de Boera, Janneke Wiersema-Buistb, Annette S.H. Gouwc, Henri G.D. Leuveninkb, Ton Lismana,b, Robert J. Portea a
Section of Hepatobiliary Surgery and Liver Transplantation, Department of Surgery, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands b Surgical Research Laboratory, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands c Department of Pathology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands 1 Both authors contributed equally to this work.
Although normothermic machine perfusion of donor livers may allow assessment of graft viability prior to transplantation, there is currently no data on what would be a good parameter of graft viability. To determine whether bile production is a suitable biomarker that can be used to discriminate viable from non-viable livers we have studied functional performance as well as biochemical and histological evidence of hepatobiliary injury during ex vivo normothermic machine perfusion of human donor livers. After a median duration of cold storage of 6.5 hours, twelve extended criteria human livers that were declined for transplantation were ex vivo perfused for 6 h at 37 °C with an oxygenated solution based on red blood cells and plasma, using pressure controlled pulsatile perfusion of the hepatic artery and continuous portal perfusion. During perfusion, two patterns of bile flow could be identified: 1) steadily increasing bile production, resulting in a cumulative output of ≥30 g after 6 h (high bile output group), and 2) a cumulative bile production b20 g in 6 h (low bile output group). Concentrations of transaminases and potassium in the perfusion fluid were significantly higher in the low bile output group, compared to the high bile output group. Biliary concentrations of bilirubin and bicarbonate were 4-times and 2-times higher in the high bile output group. Livers in the low bile output group displayed more signs of hepatic necrosis and venous congestion, compared to the high
239
bile output group. In conclusion, bile production is an easy assessable biomarker of hepatic viability during ex vivo machine perfusion of human donor livers. It could potentially be used to identify extended criteria livers that are suitable for transplantation. doi:10.1016/j.trim.2014.11.162
B14-1059 Gradual warming-up of kidneys reduces injury compared to immediate reperfusion P. Mahbouba, P.J. Ottensa, R.J. Ploegb, H.G. Leuveninka a
Dept of Surgery, Groningen Transplant Center, University Medical Center, Groningen, The Netherlands b Nuffield Department of Surgical Sciences, University of Oxford, Oxford, United Kingdom Background: Reperfusion injury after cold storage (CS) of organs is an inevitable consequence of a transplant procedure. We hypothesize that the sudden warm reperfusion after CS is detrimental for the graft. We therefore evaluated different warming-up temperatures of the organ before reperfusion in order to improve organ quality. Methods: Rat left kidneys were retrieved and stored in University of Wisconsin solution for 24 hours at 4 °C followed by immediate reperfusion at 38 °C or gradually warming-up (WU) to 10 °C, 25 °C or 38 °C, using isolated perfused kidney machine (IPK). Renal function and renal injury were assessed by IPK with an oxygenated modified Williams Medium E solution for a total of 90 minutes. Results: The increases in biochemical markers of injury such as AST and LDH in perfusate in the control group were significantly greater compared with WU groups (P b 0.05). In the 10 °C and 38 °C WU groups NAG was significantly lower than the control group. Glomerular filtration rate (GFR) at the end of the reperfusion was similar in all the groups while ultra filtrate output was significantly higher in the control group. Sodium re-absorption improved in the WU groups and it was significantly greater in the 25 °C WU group. Parallel to this, level of total protein was significantly lower in all the WU groups after 90 min of reperfusion. KIM-1 and HSP-70 gene expression was reduced in the 10 °C and 25 °C WU groups compared to the control group. There were no differences in renal blood flow(RBF) and intra renal resistance(IRR) between the four groups after 90 min of reperfusion. ATP level did not change in all the four groups. Histological evaluation did not show significant differences between the groups. Conclusion: Gradually warming up is associated with less renal injury and better renal function indicating that reperfusion injury might be reduced. doi:10.1016/j.trim.2014.11.163
B14-1060 Monocyte profiles in kidney transplant recipients: Stable grafts vs. rejection E.J.F. Vereykena,1, M.D. Kraaija,1, L.B. Hilbrandsb, P.J.M. Leenenc, D.A. Hesselinka, T.P.P. van den Boscha, C.C. Baana, M.G.H. Betjesa, A.T. Rowshania a
Dept of Nephrology and Transplantation, Erasmus University Medical Center, Rotterdam, the Netherlands b Dept of Nephrology, Radboud University, Nijmegen Medical Center, Nijmegen, The Netherlands c Dept of Immunology, Erasmus University Medical Center, Rotterdam, The Netherlands 1 Vereyken EJF and Kraaij MD contributed equally to this work.
Abstracts
human. In the present study we investigated whether circulating MSC were present in the peripheral blood of healthy individuals and patients with organ injury. We were unable to detect MSC in the blood of healthy individuals by flow cytometry and cell culture techniques. We then analyzed the presence of MSC in the blood of patients with end-stage renal disease (n = 10), end-stage liver disease (n = 10) and in heart transplant patients with biopsy proven rejection (n = 8), by culturing of mononuclear cells under MSCsupporting culture conditions. In none of these patients MSC were identified in the blood. In the stromal vascular fraction of adipose tissue and in liver transplant perfusion fluid we were able to detect MSC, indicating that the methods used enabled the detection of MSC. The conclusion of this study is that MSC are not detectable in the circulation in patients with injured solid organs and during aggressive immune responses.
doi:10.1016/j.trim.2014.11.158
B14-1049 Thymic output prior to kidney transplantation is associated with the risk for acute rejection R.W.J. Meijersa, N.H.R. Litjensa, A.W. Langerakb, C.C. Baana, W. Weimara, M.G.H. Betjesa a
Department of Internal Medicine, Section of Nephrology and Transplantation, Rotterdam, The Netherlands b Department of Immunology, Erasmus Medical Center, Rotterdam, The Netherlands
Background: The uremia-induced inflammatory environment in end-stage renal disease (ESRD) patients is associated with premature T-cell ageing, which results in defective T-cell-mediated immunity. Since T cells play a major role in acute rejection (AR), we hypothesized that an aged T-cell compartment prior to kidney transplantation (KTx) is associated with a lower risk for AR post-KTx. Methods: For this purpose, we analyzed pre-KTx samples of 20 kidney transplant recipients with a biopsy-proven AR within three months post-KTx (AR patients). For each patient with AR, two nonrejectors (NR) were included, matched for age, number of HLAmismatches and cytomegalovirus serostatus. Thymic output was assessed by determining the T-cell receptor excision circle (TREC) content and percentages of CD31+ (Ki67−) naïve T cells as recent thymic emigrants (RTEs). The relative telomere length (RTL) was determined as a measure for proliferative history and immunophenotyping was used to establish the differentiation status of circulating T cells. Results: Interestingly, the AR patients had a significantly higher thymic output (p b 0.05) as measured by TREC-content and x percentages of CD31+Ki67− naïve CD4+ and CD8+ T cells pre-KTx. The RTL of both CD4+ and CD8+ T cells did not differ between the two patient groups prior to KTx. Furthermore, the AR patients had a significantly (p b 0.05) lower percentage CD4+ memory T cells lacking CD28 (2.75 ± 0.95% vs. 5.11 ± 1.02%). For the CD8+ T-cells, AR patients had a significantly (p b 0.05) higher number of central memory cells (32.3 ± 5.00 vs. 20.62 ± 2.77 cells/ml) compared to the matched NR. Conclusion: An aged T-cell phenotype together with a lower thymic output prior to KTx is associated with a lower risk for AR. (This study was financially supported by the Dutch Kidney Foundation (KSPB.10.12)).
doi:10.1016/j.trim.2014.11.159
B14-1051 The effect of adipose tissue derived mesenchymal stem cells on B cell proliferation and differentiation M. Franquesaa, F. Mensaha, R. Huizingab, M.G.H. Betjesa, W. Weimara, C.C. Baana, M. Hoogduijna a
Transplantation and Nephrology Laboratory, Internal Medicine Department, Erasmus MC, Netherlands b Immunology Department, Erasmus MC, Netherlands Background: Mesenchymal stem cells (MSC) have proven immunomodulatory capacity which makes them a promising therapeutic tool in transplantation. While the immunosuppressive effect of MSC on T cellmediated effector mechanisms has been well studied, less is known about the effects of MSC on B cell-mediated immune responses. Methods: MSC were isolated from subcutaneous fat tissue from kidney transplant donors. Resting mature B cells from tonsils were obtained by CD43 negative selection with Magnetic Activated Cell Sorting (MACS). MSC were co-cultured with CFSE-labeled B cells stimulated in a T cell-like fashion (anti-IgM + anti-CD40 + IL2) or by PMA/ionomycin activated CD4 T cells. Proliferation and B cell phenotype were analyzed by Flow Cytometry, and IgG production quantified by ELISA. Results: Proliferation of B cells activated in a T cells-like manner (anti-IgM + anti-CD40 + IL2) was not affected by the presence of MSC, while MSC decrease the proliferation of B cells stimulated with activated T cells. An induction of plasmablasts (CD19+ CD27high CD38high) occurred when B cells were stimulated in a T cell dependent manner or in the presence of activated CD4 T cells. MSC abolished the differentiation into plasmablasts completely, which was correlated with decreased IgG production. Furthermore, MSCs induced an increase in the percentage of CD19+ CD27− CD38high CD24high regulatory-like B cells when stimulated in a Tcell-like fashion. Conclusion: MSC inhibit B cell differentiation while increasing the proportion of regulatory-like B cells. The reduction of B cell proliferation by MSC is T cell-dependent. These results suggest a therapeutic role of MSC for the treatment of patients suffering from B cell mediated alloreactivity. doi:10.1016/j.trim.2014.11.160
B14-1052 Advanced ageing of T lymphocytes in geriatric end-stage renal disease patients L. Huanga, N.H.R. Litjensa, R.W.J. Meijersa, A.W. Langerakb, W. Weimara, C.C. Baana, M.G.H. Betjesa a
Division of Nephrology and Transplantation, Dept of Internal Medicine, Erasmus Medical Center, The Netherlands b Division of Nephrology and Transplantation, Dept of Immunology, Erasmus Medical Center, The Netherlands Introduction: The proportion of geriatric patients (N60 years) suffering from end-stage renal disease (ESRD) has significantly increased over the last decade. ESRD is associated with premature immunological ageing of the T cell system which may explain the increased susceptibility for infections, cancer and cardiovascular diseases in these patients. This may in particular be of importance in geriatric patients but data in this age group are limited. The aim of this study was to assess the concept of immunological ageing of T lymphocytes in geriatric ESRD patients prior to KTx, compared to age-matched healthy controls.
Abstracts
Patients, materials and methods: We determined T-cell ageing parameters in circulating T cells of 91 ESRD patients and 73 age- and CMV-matched healthy controls. These parameters included thymic output by T-cell receptor excision circle (TREC)-content and number of CD31 + naive T cells, T-cell differentiation status by assessing the naive/memory, central memory (CM)/effector memory (EM) plus terminally differentiated effector (EMRA) ratios and percentage of CD28null memory T cells, and T cell proliferative history by relative telomere length (RTL). Results: Compared to age-matched healthy controls, geriatric ESRD patients had a lower thymic output according to lower TREC-content and CD31+ naive T cell numbers. Moreover, geriatric ESRD patients had a significantly lower T cell count due to decreased CD4+, but not CD8+, T-cell numbers. This T cell compartment consisted of relatively more memory T cells resulting from significant lower numbers of naive T cells. In addition, the memory T cell compartment contained relatively more differentiated T-cell subsets (EM, EMRA) and CD28null T cells. In addition, the RTL was significantly reduced in CD8+, and tended to be shorter in CD4+ T cells in geriatric ESRD patients. Conclusions: Geriatric ESRD patients have an advanced aged T cell compartment when compared to age-matched healthy controls. This advanced aged T cell compartment may impact the clinical course following kidney transplantation. doi:10.1016/j.trim.2014.11.161
B14-1057 Criteria for viability assessment of discarded human donor livers during ex-vivo normothermic machine perfusion Michael E. Suttona,b,1, Sanna op den Driesa,b,1, Negin Karimiana,b, Marieke T. de Boera, Janneke Wiersema-Buistb, Annette S.H. Gouwc, Henri G.D. Leuveninkb, Ton Lismana,b, Robert J. Portea a
Section of Hepatobiliary Surgery and Liver Transplantation, Department of Surgery, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands b Surgical Research Laboratory, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands c Department of Pathology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands 1 Both authors contributed equally to this work.
Although normothermic machine perfusion of donor livers may allow assessment of graft viability prior to transplantation, there is currently no data on what would be a good parameter of graft viability. To determine whether bile production is a suitable biomarker that can be used to discriminate viable from non-viable livers we have studied functional performance as well as biochemical and histological evidence of hepatobiliary injury during ex vivo normothermic machine perfusion of human donor livers. After a median duration of cold storage of 6.5 hours, twelve extended criteria human livers that were declined for transplantation were ex vivo perfused for 6 h at 37 °C with an oxygenated solution based on red blood cells and plasma, using pressure controlled pulsatile perfusion of the hepatic artery and continuous portal perfusion. During perfusion, two patterns of bile flow could be identified: 1) steadily increasing bile production, resulting in a cumulative output of ≥30 g after 6 h (high bile output group), and 2) a cumulative bile production b20 g in 6 h (low bile output group). Concentrations of transaminases and potassium in the perfusion fluid were significantly higher in the low bile output group, compared to the high bile output group. Biliary concentrations of bilirubin and bicarbonate were 4-times and 2-times higher in the high bile output group. Livers in the low bile output group displayed more signs of hepatic necrosis and venous congestion, compared to the high
239
bile output group. In conclusion, bile production is an easy assessable biomarker of hepatic viability during ex vivo machine perfusion of human donor livers. It could potentially be used to identify extended criteria livers that are suitable for transplantation. doi:10.1016/j.trim.2014.11.162
B14-1059 Gradual warming-up of kidneys reduces injury compared to immediate reperfusion P. Mahbouba, P.J. Ottensa, R.J. Ploegb, H.G. Leuveninka a
Dept of Surgery, Groningen Transplant Center, University Medical Center, Groningen, The Netherlands b Nuffield Department of Surgical Sciences, University of Oxford, Oxford, United Kingdom Background: Reperfusion injury after cold storage (CS) of organs is an inevitable consequence of a transplant procedure. We hypothesize that the sudden warm reperfusion after CS is detrimental for the graft. We therefore evaluated different warming-up temperatures of the organ before reperfusion in order to improve organ quality. Methods: Rat left kidneys were retrieved and stored in University of Wisconsin solution for 24 hours at 4 °C followed by immediate reperfusion at 38 °C or gradually warming-up (WU) to 10 °C, 25 °C or 38 °C, using isolated perfused kidney machine (IPK). Renal function and renal injury were assessed by IPK with an oxygenated modified Williams Medium E solution for a total of 90 minutes. Results: The increases in biochemical markers of injury such as AST and LDH in perfusate in the control group were significantly greater compared with WU groups (P b 0.05). In the 10 °C and 38 °C WU groups NAG was significantly lower than the control group. Glomerular filtration rate (GFR) at the end of the reperfusion was similar in all the groups while ultra filtrate output was significantly higher in the control group. Sodium re-absorption improved in the WU groups and it was significantly greater in the 25 °C WU group. Parallel to this, level of total protein was significantly lower in all the WU groups after 90 min of reperfusion. KIM-1 and HSP-70 gene expression was reduced in the 10 °C and 25 °C WU groups compared to the control group. There were no differences in renal blood flow(RBF) and intra renal resistance(IRR) between the four groups after 90 min of reperfusion. ATP level did not change in all the four groups. Histological evaluation did not show significant differences between the groups. Conclusion: Gradually warming up is associated with less renal injury and better renal function indicating that reperfusion injury might be reduced. doi:10.1016/j.trim.2014.11.163
B14-1060 Monocyte profiles in kidney transplant recipients: Stable grafts vs. rejection E.J.F. Vereykena,1, M.D. Kraaija,1, L.B. Hilbrandsb, P.J.M. Leenenc, D.A. Hesselinka, T.P.P. van den Boscha, C.C. Baana, M.G.H. Betjesa, A.T. Rowshania a
Dept of Nephrology and Transplantation, Erasmus University Medical Center, Rotterdam, the Netherlands b Dept of Nephrology, Radboud University, Nijmegen Medical Center, Nijmegen, The Netherlands c Dept of Immunology, Erasmus University Medical Center, Rotterdam, The Netherlands 1 Vereyken EJF and Kraaij MD contributed equally to this work.