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Pathologic Findings in Explanted Pulmonary Allografts
Abstracts Conclusions: Outward vessel remodeling in CAV, assessed be several measures, was reduced compared to NVA. This is likely due to both accelerated intimal ingrowth and limited outward remodeling in CAV. Mechanisms influencing these processes (eg immunosuppressant effect on extracellular matrix metabolism and allograft endothelial injury) need further elucidation. 673 Pathologic Findings in Explanted Pulmonary Allografts L. Xu,1 C. Drachenberg,1 A. Iacono,2 A. Burke.1 1Pathology, University of Maryland, Baltimore, MD; 2Pulmonary Medicine, University of Maryland, Baltimore, MD. Purpose: The most common causes of pulmonary graft failure are rejection and infections. There are few pathologic studies of pulmonary allografts that were explanted for re-transplant. Methods and Materials: We performed histopathologic analysis of a series of explanted pulmonary allografts over a 7-year period. Results: Of 168 explanted lungs, 10 were previously transplanted allografts (6%). The indications of initial transplant were idiopathic pulmonary fibrosis (6), emphysema (2), primary arterial hypertension (1), and Hermansky-Pudlak fibrosis (1). The duration of transplant was o 1 month in 3 cases (4 days - 29 days), and 4 1 month in 7 (range 1.5 - 48 months). The causes of graft failure in the early graft failure group was acute lung injury in all cases, with 2 showing arterial thrombosis with pulmonary infarcts. All three patients died after second transplant (7 days - 5 months). The causes of graft failure in the lateral group were chronic rejection with obliterative bronchiolitis (4), cellular rejection (2), and bronchial stenosis (1). Vascular lesions were prominent in three cases, with arterial intimitis with focal thrombosis in two cases (one each with acute and chronic rejection), and diffuse polymer embolization in one case. Incidental lesions included acute and fibrinous organizing pneumonia, aspergilloma, and focal alveolar proteinosis. In the lateral group, two patients died (both at 4 months), and the remainder were alive at last follow up (mean 22 months, 4-48 months). Conclusions: The major cause of early graft failure is acute lung injury, and late graft failure is acute or chronic rejection. Vascular disease is common in both groups. In this small study, survival was poor in patients re-transplanted within one month of initial transplant. 674 Anti-IL6R Attenuates Humoral Responses to Allograft in a Mouse Model of Allosensitization G. Wu, N.-N. Chai, A. Chen, S. Jordan, A. Klein. Comprehensive Transplant Center, Cedars-Sinai Medical Center, Los Angeles, CA. Purpose: The role IL-6 plays in transplant rejection has been increasingly recognized, therefore, targeting the IL-6/IL-6R pathway may have applications in treatment of allograft rejection. This study evaluated the efficacy of a mousenized rat-anti-mouse IL-6R mAb, mMR16-1, in suppressing alloantibody responses. Methods and Materials: C57BL/6 mice were sensitized with skin allografts from a HLA.A2 transgenic mouse, and treated with intraperitoneal injections of mMR16-1 or control antibody. Donor specific antibody (DSA) responses were monitored weekly for 5 weeks by measurement of serum anti-HLA.A2 antibodies in a flow cytometric antibody binding assay. Results: mMR16-1 significantly reduced DSA responses to skin allograft, resulting in significant decreases of anti-HLA.A2 IgM (treated vs. control: 12.7þ2.3 MFI vs. 22.9þ3.2 MFI at day 14, p¼0.00022), IgG2a (99.41 þ35.31 MFI vs. 300.6þ53.04 MFI at day 28, p¼6.621E-05) and IgG1 (25.6þ9.8 MFI vs. 40.01þ7.7 MFI at day 28, p¼0.029), respectively. Anti-IL6R treatments also normalized serum amyloid A (SAA), an acute phase reactant induced by IL-6 (po0.01 vs. control). In addition, mMR16-1 treatment caused accumulation of soluble IL-6R as well as IL-6 in the blood. Thus, the alternation in IL-6/IL-6R homeostasis likely impairs both the
S245 classic signaling and trans-signaling pathways, which would interfere with a wide range of IL-6 dependent immune activation events. Conclusions: The data indicate that antibody therapy targeting the IL6/IL-6R pathway may serve as a strategy to suppress DSA generation. Future mechanistic studies to dissect various cellular compartments targeted by anti-IL-6R in the alloimmunity will shed more light on our understanding of the therapeutic properties of anti-IL-6R on rejection suppression. 675 In Vitro Modeling of Duchenne Muscular Dystrophy (DMD) Cardiomyopathy Using Human Induced Pluripotent Stem Cells (hiPSC) F. Kamdar,1,2 M.J. Doyle,2 C. Chapman,2 J. Lohr,2 N. Koyano Nakagawa,2 D.J. Garry.1,2 1Division of Cardiology, University of Minnesota, Minneapolis; 2Lillehi Heart Institute, University of Minnesota, Minneapolis. Purpose: DMD is the most common an debilitating X-linked muscular dystrophy that leads to devastating skeletal and cardiac muscle damage. DMD cardiomyopathy has now emerged as the leading cause of death in DMD, however there is limited knowledge of pathophysiology of DMD cardiomyopathy. The purpose of this is to utilize patient-specific hiPSC derived cardiomyocytes (CM) to model DMD cardiomyopathy. Methods and Materials: Dermal fibroblasts were obtained from a DMD patient with severe cardiomyopathy and a normal healthy control. The fibroblasts were reprogrammed to hiPSC with retrovirus containing the human transcription factors, Oct4, Sox2, Klf4, and c-Myc. The DMD and control hiPSC lines were differentiated to CM using a monolayer, extra cellular matrix, growth factor directed differentiation. Molecular and physiologic assays were performed at day 30 of differentiation. Results: The DMD and control hiPSC lines were fully characterized and demonstrated pluripotent markers with qRT-PCR and immunostaining, formed teratomas with all three germ layers, and were karyotypically normal. DMD hiPSC were shown to have absence of dystrophin exons 4-43, consistent with the patient’s mutation. DMD hiPSC differentiated to beating CM sheets and initiation of beating was observed between days 10-14 in both DMD and control hiPSC CM. Increased membrane fragility was seen in d30 DMD hiPSC CM compared to control CM as assayed by hypo-osmotic stress and LDH release. Initial gene expression, demonstrated significantly lower dystrophin expression in DMD hiPSC CM as compared to control hiPSC CM, while there was significantly higher induction of beta-1adrenergic receptor gene expression in DMD hiPSC CM. Conclusions: Our results suggest that DMD hiPSC CM can be differentiated to cardiomyocytes and have a unique phenotype for disease investigation. Further evaluation of the molecular and physiologic phenotype of DMD hiPSC CM will serve as a platform for developing novel therapeutic regimens for patients with muscular dystrophy cardiomyopathies. 676 T Cell Responses in Lung Transplantion – Role of Alloantigen Priming and Regulation on Development of Transplant Arteriosclerosis in a Humanized Mouse Model A.-K. Knofel, ¨ 1 N. Frank,1 N. Madrahimov,1 J. Salman,1 W. Sommer,1 K. Jansson,1 P. Ziehme,1 D. Jonigk,2 M. Avsar,1 A. Haverich,1 G. Warnecke.1 1HTTV, Medical School of Hannover, Hannover, Germany; 2Pathology, Medical School of Hannover, Hannover, Germany. Purpose: In animal models, both na¨ıve and alloantigen-primed Treg inhibit rejection. In clinical recipients of transplants, however, not much is known with regard to the actual function of those Treg that can be found in vivo. Here, we studied the functional importance of both na¨ıve and alloantigen-primed T cell responses in a humanized mouse model, utilizing transplant arteriosclerosis as the experimental readout. Methods and Materials: The pericardiophrenic artery was procured from leftover tissue of lung grafts transplanted within our clinical
S245 classic signaling and trans-signaling pathways, which would interfere with a wide range of IL-6 dependent immune activation events. Conclusions: The data indicate that antibody therapy targeting the IL6/IL-6R pathway may serve as a strategy to suppress DSA generation. Future mechanistic studies to dissect various cellular compartments targeted by anti-IL-6R in the alloimmunity will shed more light on our understanding of the therapeutic properties of anti-IL-6R on rejection suppression. 675 In Vitro Modeling of Duchenne Muscular Dystrophy (DMD) Cardiomyopathy Using Human Induced Pluripotent Stem Cells (hiPSC) F. Kamdar,1,2 M.J. Doyle,2 C. Chapman,2 J. Lohr,2 N. Koyano Nakagawa,2 D.J. Garry.1,2 1Division of Cardiology, University of Minnesota, Minneapolis; 2Lillehi Heart Institute, University of Minnesota, Minneapolis. Purpose: DMD is the most common an debilitating X-linked muscular dystrophy that leads to devastating skeletal and cardiac muscle damage. DMD cardiomyopathy has now emerged as the leading cause of death in DMD, however there is limited knowledge of pathophysiology of DMD cardiomyopathy. The purpose of this is to utilize patient-specific hiPSC derived cardiomyocytes (CM) to model DMD cardiomyopathy. Methods and Materials: Dermal fibroblasts were obtained from a DMD patient with severe cardiomyopathy and a normal healthy control. The fibroblasts were reprogrammed to hiPSC with retrovirus containing the human transcription factors, Oct4, Sox2, Klf4, and c-Myc. The DMD and control hiPSC lines were differentiated to CM using a monolayer, extra cellular matrix, growth factor directed differentiation. Molecular and physiologic assays were performed at day 30 of differentiation. Results: The DMD and control hiPSC lines were fully characterized and demonstrated pluripotent markers with qRT-PCR and immunostaining, formed teratomas with all three germ layers, and were karyotypically normal. DMD hiPSC were shown to have absence of dystrophin exons 4-43, consistent with the patient’s mutation. DMD hiPSC differentiated to beating CM sheets and initiation of beating was observed between days 10-14 in both DMD and control hiPSC CM. Increased membrane fragility was seen in d30 DMD hiPSC CM compared to control CM as assayed by hypo-osmotic stress and LDH release. Initial gene expression, demonstrated significantly lower dystrophin expression in DMD hiPSC CM as compared to control hiPSC CM, while there was significantly higher induction of beta-1adrenergic receptor gene expression in DMD hiPSC CM. Conclusions: Our results suggest that DMD hiPSC CM can be differentiated to cardiomyocytes and have a unique phenotype for disease investigation. Further evaluation of the molecular and physiologic phenotype of DMD hiPSC CM will serve as a platform for developing novel therapeutic regimens for patients with muscular dystrophy cardiomyopathies. 676 T Cell Responses in Lung Transplantion – Role of Alloantigen Priming and Regulation on Development of Transplant Arteriosclerosis in a Humanized Mouse Model A.-K. Knofel, ¨ 1 N. Frank,1 N. Madrahimov,1 J. Salman,1 W. Sommer,1 K. Jansson,1 P. Ziehme,1 D. Jonigk,2 M. Avsar,1 A. Haverich,1 G. Warnecke.1 1HTTV, Medical School of Hannover, Hannover, Germany; 2Pathology, Medical School of Hannover, Hannover, Germany. Purpose: In animal models, both na¨ıve and alloantigen-primed Treg inhibit rejection. In clinical recipients of transplants, however, not much is known with regard to the actual function of those Treg that can be found in vivo. Here, we studied the functional importance of both na¨ıve and alloantigen-primed T cell responses in a humanized mouse model, utilizing transplant arteriosclerosis as the experimental readout. Methods and Materials: The pericardiophrenic artery was procured from leftover tissue of lung grafts transplanted within our clinical