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3 Inhibitor R507 Prevents Acute Rejection and Chronic Obliteration of Transplanted Rat Tracheas
S82
The Journal of Heart and Lung Transplantation, Vol 32, No 4S, April 2013
Purpose: One promising approach for the induction of transplant tolerance is the pre-treatment of transplant recipients with donor MHC-alloantigen. Our study focuses on the oral delivery of MHC-antigen encoding genes via chitosan-DNA nanoparticles to modulate the allo-immune response in order to reduce the development of transplant arteriosclerosis, the hallmark feature of chronic rejection after heart transplantation. Methods and Materials: We performed fully allogeneic mouse abdominal aortic transplantats using C57BL/6 (H2b) mice as donors and CBA.J (H2k) mice as recipients Aortic grafts were analyzed by histology and morphometry on day 30 after transplantation, levels of circulating alloantibodies were detected by FACS analysis. Results: Pre-treatment of recipient mice with chitosan-DNA nanoparticles encoding for Kb, one of the MHC-I molecules of the donor, resulted in a significant reduction of intimal proliferation compared to untreated controls. When Ovalbumin was fed instead of Kb encoding nanoparticles (Kb-NP) or Balb/c (H2d) grafts were used instead of C57BL/6 (H2b) grafts as antigen controls, both groups showed no reduction of intimal thickness indicating an antigen-specific mechanism. In addition, analysis of peripheral blood of the transplanted mice showed significant suppression of alloantibody formation in the KbNP fed group compared to all other allogeneic transplanted groups suggesting modulation of the humoral immune response. Conclusions: These results demonstrate the potential of chitosan-DNA nanoparticles to induce Kb-specific tolerance and to reduce the development of transplant arteriosclerosis. 205 An Innovative Approach to Selectively Inhibit Mesenchymal Cells Isolated from BOS Patients E. Cova,2 M. Colombo,3 M. Morosini,2 S. Inghilleri,2 S. Miserere,2 S. Magni,2 D. Prosperi,3 F. Meloni.1 1Department Molecular Medicine, University of Pavia and IRCCS Sn Matteo Foundation, Pavia, Italy; 2 Department of Molecular Medicine, IRCCS San Matteo Foundation, Pavia, Italy; 3Department of Biotechnology and Biosciences, University of Milano Bicocca, Milan, Italy. Purpose: The presence of mesenchymal cells (MC), the primary source of fibrotic cells, has been described in BAL fluid of LTR patients as predictive of BOS onset (Badri et al, 2011). CD44 cell surface glycoprotein has been associated with an invasive fibroblast phenotype. Aim of this work was to assay the presence of CD44 in MC isolated from BAL of LTR patients with BOS to set up biocompatible functionalized drug-loaded nanoparticles (NP) and to prove their effectiveness to specifically inhibit cell proliferation. The expression of active mTOR, the target of the selected drug, everolimus h, was also determined. Methods and Materials: MC isolated from 5 BOS pts (grade 0, 1 and 2) were characterized for CD44, CD34, CD45, CD90 and CD105 surface marker expression by flow cytometry and used for the experiments. The presence of mTOR was assayed by immunocytochemistry. Fluorescent labeled NP functionalized with an anti-CD44 antibody and loaded with E (CD44NPE) have been used to assess cell uptake by confocal microscopy, cell apoptosis/death and proliferation by flow cytometry. Results: CD44 was expressed by 85-98% of cultured cells while mTOR was present in all cells. Cell lines were also positive for CD90 and, in lower percentage, for CD105, and negative for CD34 and CD45. CD44 was not expressed by normal alveolar epithelia. CD44NPE were shown to adhere and enter into the cells after 45 min incubation. Drug free NP, used as negative control, were completely inert. CD44NPE treated cells showed a significantly higher mean rate of annexin V expression at 8 h (17,4 % vs. 4,5%) and 24 h (42,15% vs. 5,3%) respect to control cells while mean 7AAD expression at 24 h was 4,1 vs. 1.3%. Likewise, cell proliferation was significantly inhibited at 24, 72, 96 and 120 h. As a corollary, we also demonstrated lack of a pro-inflammatory activity of drug-loaded NP. Conclusions: These results demonstrate the effectiveness of targeted drug-loaded NP to inhibit MC and are highly promising in the view of developing a less toxic, local treatment of BOS.
206 The Selective JAK1/3 Inhibitor R507 Prevents Acute Rejection and Chronic Obliteration of Transplanted Rat Tracheas T. Deuse,1 X. Hua,1 M. Stubbendorff,1 V. Taylor,2 Y. Chen,2 G. Park,2 H. Reichenspurner,1 R.C. Robbins,3 S. Schrepfer.1,3 1TSI-Lab, Cardiovascular Surgery, University Heart Center Hamburg, Hamburg, Germany; 2Rigel Pharmaceuicals, San Francisco; 3CT Surgery, Stanford University School of Medicine, Stanford. Purpose: Despite advances in the field of immunosuppression, obliterative airway disease remains an unsolved problem after clinical lung transplantation and the incidence of chronic renal failure remains high. The efficacy of the highly selective JAK1/3 inhibitor R507 on preclinical airway transplantation was investigated. Methods and Materials: Orthotopic Lew-BN trachea transplantations were performed. Treatment groups received oral R507 (60mg/kg BID) or everolimus (4mg/kg QD). Mechanistic studies were performed in vitro using full-thickness human airway epithelium and R507enriched medium. Results: On postoperative day 6, mononuclear CD3 and CD68 graft infiltration were significantly reduced in both treatment groups compared to the control group, but significantly more potently with R507. IFN-g Elispot frequencies were significantly lower with R507 and everolimus treatment. At 60 days, airway obliteration was significantly reduced from 32⫾5% in the control group to 24⫾6% (p¼0.004) and 14⫾5% (po0.001) in the everolimus and R507 groups, respectively. Using luciferase-positive Lewis donors, we could demonstrate that the obliterated tissue was of donor-origin. The respiratory epithelium was only preserved in the R507 group (88⫾5%), but widely destroyed in the control and everolimus groups. There was a surge in donor-specific IgG antibody production in the control group that was effectively abrogated with both R507 and everolimus. Side effect screening showed that only everolimus significantly increased creatinine, BUN, cholesterol and triglyceride levels over the study period. In vitro data showed that R507 was not cell toxic. Conclusions: R507 is a potent lymphocyte-targeting immunosuppressant without kidney and airway toxicity. It effectively prevents acute graft rejection as well as the development of obliterative airway disease. We believe it will be a valuable drug for lung transplantation. 207 Inhaled Immunosuppression Using the Novel JAK1/3 Inhibitor R507 T. Deuse,1 X. Hua,1 M. Stubbendorff,1 V. Taylor,2 Y. Chen,2 G. Park,2 H. Reichesnpurner,1 R.C. Robbins,3 S. Schrepfer.1,3 1TSI-Lab, Cardiovascular Surgery, University Heart Center, Hamburg, Germany; 2 Rigel, San Francisco; 3CT Surgery, Stanford University School of Medicine, Stanford. Purpose: Local immunosuppression by inhalation is a novel strategy after lung transplantation. Here we investigate the feasibility of R507 delivery via aerosol, assess its immunosuppressive efficacy, and evaluate its airway toxicity on human airway epithelial cells. Methods and Materials: Orthotopic rat tracheal transplantations were performed in the Lew-to-BN model and recipients were left untreated or treated with R507 (60mg/kg BID) orally (PO) or via aerosol (AER) for 60 days. Drug distribution after inhalation, graft histopathology, host immune responsiveness, and side effects were closely monitored over time. Full-thickness human airway epithelium (AE) was grown in vitro at air-liquid interface to study cell biology and equal R507 doses were either added to the bottom chamber (MED) or aerosolized for gas phase exposure. Cell toxicity and the epithelial integrity were studied. Results: SPECT imaging demonstrated a linear tracer accumulation within the transplanted trachea during R507 inhalation. After 60 days, obliterative airway disease (OAD) was similarly inhibited with R507 PO (14.1⫾4.8%) and AER (21.7⫾9.8%) vs. untreated animals (37.1⫾7.2%, po0.05). Epithelial cell viability was preserved even after local administration of R507 AER as demonstrated by PAS histopathology and confocal immunofluorescence for cytokeratin.
The Journal of Heart and Lung Transplantation, Vol 32, No 4S, April 2013
Purpose: One promising approach for the induction of transplant tolerance is the pre-treatment of transplant recipients with donor MHC-alloantigen. Our study focuses on the oral delivery of MHC-antigen encoding genes via chitosan-DNA nanoparticles to modulate the allo-immune response in order to reduce the development of transplant arteriosclerosis, the hallmark feature of chronic rejection after heart transplantation. Methods and Materials: We performed fully allogeneic mouse abdominal aortic transplantats using C57BL/6 (H2b) mice as donors and CBA.J (H2k) mice as recipients Aortic grafts were analyzed by histology and morphometry on day 30 after transplantation, levels of circulating alloantibodies were detected by FACS analysis. Results: Pre-treatment of recipient mice with chitosan-DNA nanoparticles encoding for Kb, one of the MHC-I molecules of the donor, resulted in a significant reduction of intimal proliferation compared to untreated controls. When Ovalbumin was fed instead of Kb encoding nanoparticles (Kb-NP) or Balb/c (H2d) grafts were used instead of C57BL/6 (H2b) grafts as antigen controls, both groups showed no reduction of intimal thickness indicating an antigen-specific mechanism. In addition, analysis of peripheral blood of the transplanted mice showed significant suppression of alloantibody formation in the KbNP fed group compared to all other allogeneic transplanted groups suggesting modulation of the humoral immune response. Conclusions: These results demonstrate the potential of chitosan-DNA nanoparticles to induce Kb-specific tolerance and to reduce the development of transplant arteriosclerosis. 205 An Innovative Approach to Selectively Inhibit Mesenchymal Cells Isolated from BOS Patients E. Cova,2 M. Colombo,3 M. Morosini,2 S. Inghilleri,2 S. Miserere,2 S. Magni,2 D. Prosperi,3 F. Meloni.1 1Department Molecular Medicine, University of Pavia and IRCCS Sn Matteo Foundation, Pavia, Italy; 2 Department of Molecular Medicine, IRCCS San Matteo Foundation, Pavia, Italy; 3Department of Biotechnology and Biosciences, University of Milano Bicocca, Milan, Italy. Purpose: The presence of mesenchymal cells (MC), the primary source of fibrotic cells, has been described in BAL fluid of LTR patients as predictive of BOS onset (Badri et al, 2011). CD44 cell surface glycoprotein has been associated with an invasive fibroblast phenotype. Aim of this work was to assay the presence of CD44 in MC isolated from BAL of LTR patients with BOS to set up biocompatible functionalized drug-loaded nanoparticles (NP) and to prove their effectiveness to specifically inhibit cell proliferation. The expression of active mTOR, the target of the selected drug, everolimus h, was also determined. Methods and Materials: MC isolated from 5 BOS pts (grade 0, 1 and 2) were characterized for CD44, CD34, CD45, CD90 and CD105 surface marker expression by flow cytometry and used for the experiments. The presence of mTOR was assayed by immunocytochemistry. Fluorescent labeled NP functionalized with an anti-CD44 antibody and loaded with E (CD44NPE) have been used to assess cell uptake by confocal microscopy, cell apoptosis/death and proliferation by flow cytometry. Results: CD44 was expressed by 85-98% of cultured cells while mTOR was present in all cells. Cell lines were also positive for CD90 and, in lower percentage, for CD105, and negative for CD34 and CD45. CD44 was not expressed by normal alveolar epithelia. CD44NPE were shown to adhere and enter into the cells after 45 min incubation. Drug free NP, used as negative control, were completely inert. CD44NPE treated cells showed a significantly higher mean rate of annexin V expression at 8 h (17,4 % vs. 4,5%) and 24 h (42,15% vs. 5,3%) respect to control cells while mean 7AAD expression at 24 h was 4,1 vs. 1.3%. Likewise, cell proliferation was significantly inhibited at 24, 72, 96 and 120 h. As a corollary, we also demonstrated lack of a pro-inflammatory activity of drug-loaded NP. Conclusions: These results demonstrate the effectiveness of targeted drug-loaded NP to inhibit MC and are highly promising in the view of developing a less toxic, local treatment of BOS.
206 The Selective JAK1/3 Inhibitor R507 Prevents Acute Rejection and Chronic Obliteration of Transplanted Rat Tracheas T. Deuse,1 X. Hua,1 M. Stubbendorff,1 V. Taylor,2 Y. Chen,2 G. Park,2 H. Reichenspurner,1 R.C. Robbins,3 S. Schrepfer.1,3 1TSI-Lab, Cardiovascular Surgery, University Heart Center Hamburg, Hamburg, Germany; 2Rigel Pharmaceuicals, San Francisco; 3CT Surgery, Stanford University School of Medicine, Stanford. Purpose: Despite advances in the field of immunosuppression, obliterative airway disease remains an unsolved problem after clinical lung transplantation and the incidence of chronic renal failure remains high. The efficacy of the highly selective JAK1/3 inhibitor R507 on preclinical airway transplantation was investigated. Methods and Materials: Orthotopic Lew-BN trachea transplantations were performed. Treatment groups received oral R507 (60mg/kg BID) or everolimus (4mg/kg QD). Mechanistic studies were performed in vitro using full-thickness human airway epithelium and R507enriched medium. Results: On postoperative day 6, mononuclear CD3 and CD68 graft infiltration were significantly reduced in both treatment groups compared to the control group, but significantly more potently with R507. IFN-g Elispot frequencies were significantly lower with R507 and everolimus treatment. At 60 days, airway obliteration was significantly reduced from 32⫾5% in the control group to 24⫾6% (p¼0.004) and 14⫾5% (po0.001) in the everolimus and R507 groups, respectively. Using luciferase-positive Lewis donors, we could demonstrate that the obliterated tissue was of donor-origin. The respiratory epithelium was only preserved in the R507 group (88⫾5%), but widely destroyed in the control and everolimus groups. There was a surge in donor-specific IgG antibody production in the control group that was effectively abrogated with both R507 and everolimus. Side effect screening showed that only everolimus significantly increased creatinine, BUN, cholesterol and triglyceride levels over the study period. In vitro data showed that R507 was not cell toxic. Conclusions: R507 is a potent lymphocyte-targeting immunosuppressant without kidney and airway toxicity. It effectively prevents acute graft rejection as well as the development of obliterative airway disease. We believe it will be a valuable drug for lung transplantation. 207 Inhaled Immunosuppression Using the Novel JAK1/3 Inhibitor R507 T. Deuse,1 X. Hua,1 M. Stubbendorff,1 V. Taylor,2 Y. Chen,2 G. Park,2 H. Reichesnpurner,1 R.C. Robbins,3 S. Schrepfer.1,3 1TSI-Lab, Cardiovascular Surgery, University Heart Center, Hamburg, Germany; 2 Rigel, San Francisco; 3CT Surgery, Stanford University School of Medicine, Stanford. Purpose: Local immunosuppression by inhalation is a novel strategy after lung transplantation. Here we investigate the feasibility of R507 delivery via aerosol, assess its immunosuppressive efficacy, and evaluate its airway toxicity on human airway epithelial cells. Methods and Materials: Orthotopic rat tracheal transplantations were performed in the Lew-to-BN model and recipients were left untreated or treated with R507 (60mg/kg BID) orally (PO) or via aerosol (AER) for 60 days. Drug distribution after inhalation, graft histopathology, host immune responsiveness, and side effects were closely monitored over time. Full-thickness human airway epithelium (AE) was grown in vitro at air-liquid interface to study cell biology and equal R507 doses were either added to the bottom chamber (MED) or aerosolized for gas phase exposure. Cell toxicity and the epithelial integrity were studied. Results: SPECT imaging demonstrated a linear tracer accumulation within the transplanted trachea during R507 inhalation. After 60 days, obliterative airway disease (OAD) was similarly inhibited with R507 PO (14.1⫾4.8%) and AER (21.7⫾9.8%) vs. untreated animals (37.1⫾7.2%, po0.05). Epithelial cell viability was preserved even after local administration of R507 AER as demonstrated by PAS histopathology and confocal immunofluorescence for cytokeratin.