Byoung Chol Oh

A multiphase transitioning peptide hydrogel for suturing ultrasmall vessels

Many surgeries are complicated by the need to anastomose, or reconnect, micron-scale vessels. Although suturing remains the gold standard for anastomosing vessels, it is difficult to place sutures correctly through collapsed lumen, making the procedure prone to failure. Here, we report a multi-phase transitioning peptide hydrogel that can be injected into the lumen of vessels to facilitate suturing. The peptide, which contains a photocaged glutamic acid, forms a solid-like gel in a syringe and can be shear-thin delivered to the lumen of collapsed vessels (where it distends the vessel), and the space between two vessels (where it is used to approximate the vessel ends). Suturing is performed directly through the gel. Light is used to initiate the final gel-sol phase transition that disrupts the hydrogel network, allowing the gel to be removed and blood flow to resume. This gel adds a new tool to the armamentarium for micro- and supermicrosurgical procedures.

Functional Reconstruction of Urogentital Tissue Defects Using Vascularized Composite Allotransplantation: A Novel Microsurgical Penis Transplant Model and Clinical & Histological Rejection Classification

Introduction Defects of the male urogenital structures subsequent to major trauma are associated with impaired urinary and sexual function and result in a significantly reduced quality-of-life. Modern reconstructive methods employ autologous tissue-based reconstructions and implant placement to reconstruct a functional phallus, but outcomes are frequently unsatisfactory. Penis transplantation has been successfully employed in the recent past and represents an exciting avenue for restoration of male urogenitalia and function by using “like-with-like” tissue, when no conventional options are available. However, only little is currently known about the immunological features of these unique tissue grafts. Thus, to fill this critical void, we established a new animal model and clinical & histological rejection classification. Materials and Methods In male 8-week old BN & Lewis rats the penis was dissected to design a penile graft based on the internal pudendal artery and dorsal penile vein including prepuce skin. A non-suture cuff technique was employed to perform end-to-end anastomosis of the graft vessels to the recipient inferior epigastric vessels. 30 syngeneic and allogeneic transplants were performed. Grafts were followed clinically and histologically at post-operative days (POD) 3,5,7,9,11,13,14,16 and 18. Results The graft design using anastomosis of the dorsal penile vein and the distal internal pudendal artery at the bifurcation into dorsal and deep penile arteries ensures optimal perfusion of the entire superficial and deep graft tissues. The non-suture cuff technique allows for successful microvascular anastomosis by a single surgeon in an average of 2.5 hours, at a 91% surgical success rate. Long-term graft survival (>POD 45) was observed in syngeneic transplants. Graft rejection follows a 4-stage clinical progression, indicating comparability to other skin containing VCAs. Graft rejection, however appears to be minimally delayed compared to hind limb allografts, with all untreated allografts fully rejected by POD 16. Histological analysis allowed for the development of a specific 4-grade rejection classification in analogy to the 2007 BANFF criteria for hand transplantation. Of note, graft skin and urethral lining tissue are first targets of rejection, which follows a distal to proximal pattern. Conclusion We established a robust and reproducible murine model to study the immunobiology of urogenital tissue in the context of reconstructive transplantation. The graft design ensures vascular perfusion of all penile tissues. Heterotopic inset allows for standardized visual monitoring of graft viability. We propose a novel 4-grade histological rejection scale based on graft skin and urethral lining as the main targets of rejection.

Ex vivo Expanded Regulatory T cells Combined with Short-term Costimulation Blockade Prevent Rejection of Vascularized Composite Allografts

Background Reconstructive transplantation represents a valid therapeutic option after devastating tissue loss. Routine clinical application, however, is hampered by the toxicity of long-term maintenance immunosuppression. The current study investigated a novel approach using ex vivo expanded regulatory T cells combined with a short-term immunomodulatory strategy in a murine hind limb transplantation model. Methods Fully MHC-mismatched orthotopic hind limb transplants were performed from Balb/C to C57BL/6 mice. Recipients in the experimental groups received a combination regimen consisting of 0.5mg CTLA4 Ig on day 0, 2, 4 and 6 post-transplant, 20mg/kg anti-Thy 1.2 mAb on POD-1, and 1mg/kg Rapamycin (POD 0-9), and in one group, 1 wk expanded CD4+CD25+ Treg cells. Allograft survival was monitored and flow cytometric analysis was performed to evaluate mixed chimerism and clonal deletion of alloreactive T cells. Treg activity was assessed in vitro using suppression assays in order to support and supplement in vivo data. Results Combination of T cell depletion and CTLA4-Ig plus short-course of Rapamycin increased VCA survival significantly while untreated controls rejected allografts (MST 105 days; Untreated, MST 9 days; CTLA4 Ig only, MST 17 days, Rapamycin, MST 20 days; T cell depletion, 20 days; p<0.01[GB1]). [GB2] Mixed chimerism was detected in recipients receiving this combined treatment protocol with 5.013 ± 1.23 % of CD11b+ cells being donor-derived on POD 55. V&bgr; – TCR staining profiles in recipients after full treatment showed 1.570 ± 0.3700 % of &ngr;&bgr;5+CD4+ T cells, while naïve C57BL/6 express 3.567 ± 0.3690 % of &ngr;&bgr;5+CD4+ T cells, suggesting the actuation of central deletion of developing donor-reactive T cells. In order to further prolong allograft survival, one week expanded Tregs were then included in the combination therapy. The suppressive activity of the CD4+CD25+ Tregs was confirmed with in vitro suppression assays. The addition of ex vivo expanded regulatory T cells further increased VCA survival to >200 days and induced long-term stable mixed chimerism[GB3] with 16.7±1.5 % of CD11b cells being donor-derived on POD 55 after administration of expanded Treg cells. Conclusion The combination of T cell depletion, costimulation blockade, and a short-course of Rapamycin prevents VCA rejection and significantly prolongs graft survival without the need for myeloablative conditioning or maintenance therapy. Moreover, regulatory T cells added in the early post transplant period further optimize immune regulation by inducing sustained mixed chimerism.

2558: Optimization of intra-thymic transplantation of donor-derived thymic epithelial cells to promote lasting regulation of anti-donor reactivity

2558: Optimization of intra-thymic transplantation of donor-derived thymic epithelial cells to promote lasting regulation of anti-donor reactivity Sara Alfadil, Kim Mi-Jeong, Marcos Iglesias Lozano, Byoung Chol Oh, W. P. Andrew Lee, Gerald Brandacher, Thomas Serworld, and Giorgio Raimondi Johns Hopkins University School of Medicine, Vascularized Composite Allotransplantation (VCA) Laboratory, Baltimore, MD; Joslin Diabetes Center, Boston, MA, USA Background Targeting the process of central (thymic) selection of developing T lymphocytes is the key mechanism for transplant tolerance induction mediated bybone marrow transplantation (BM-Tx)-based protocols. However, they are not amenable to most transplant recipients. Thymic Epithelial Cells (TEC), a population of stromal cells residing in the thymus, exert a major contribution to central selection. Unfortunately, donor TEC do not develop following BM-Tx protocols. Therefore, we propose a new strategy that voids the need for heavy pre-conditioning and is based on generating a donor-recipient “Hybrid Thymus,” through donor TEC engraftment, to re-engineer the thymic microenvironment and promote lasting central tolerance. Methods We developed an improved protocol for isolating mouse TEC via a combination of negative and positive selection. Allogeniec (Allo)-TEC purified from BALB/c were injected into the right thymic lobe of C57BL/6, Recipients were divided in groups based on treatment with CTLA4-Ig C/¡ anti-CD40L C/¡ T depletion. 14, 28, and 45 days post-injection Allo-TEC survival was assessed and the selection and reactivity of T cells was analyzed via flow cytometry and CFSEbased mixed lymphocyte reaction. Results Our optimized purification protocol yields an average 70% TEC purity. Unmanipulated recipients promptly rejected Allo-TEC, indicating the existence of effective immunity in the thymus. However, CTLA4-Ig and MR1 co-administration exerted a significant (but not lasting) protection. In this latter group, the percentage of peripheral Vb11C T cells (inversely correlated with central selection) on d24 was significantly lower, indicating functional activity of the engrafted donor TEC. Addition of transient T depletion enabled longterm engraftment of Allo-TEC that, on d45, were in a normal architecture within the thymus integrated with recipient thymocytes. However, antidonor T cell reactivity remained unaltered, suggesting the need for engraftment in both thymic lobes for proper regulation. Conclusion Our preliminary data show that the thymic engraftment, survival, and function of allo-TEC can be promoted via costimulation blockade C T depletion. These exciting results indicate that engineering a donor-recipient Hybrid Thymus is feasible and has the potential to promote a dominant regulation of alloreactivity that could be conducive to transplant tolerance induction. CONTACT Sara Alfadil salfadi1@jhmi.edu

2523: Vascularized composite allograft tolerance with transient high-dose tacrolimus across a full MHC mismatch in a large animal model

2523: Vascularized composite allograft tolerance with transient high-dose tacrolimus across a full MHC mismatch in a large animal model Howard D. Wang, MD, Edward W. Swanson, MD, Hsu-Tang Cheng, MD, Jeffrey Walch, MD, PhD, Jose C. Alonso-Escalante, MD, Keli Kolegraff, MD, PhD, Joseph Lopez, MD, MBA, Georg Furtmuller, MD, Byoung Chol Oh, DVM, PhD, Amy Quan, MPH, Joshua Budihardjo, Sara AlFadil, MD, Sara Mulla, MD, Samuel Fidder, MD, Paul Akre, MS, Justin M. Sacks, MD, Steven C. Bonawitz, MD, Giorgio Raimondi, PhD, Jaimie T. Shores, MD, Damon S. Cooney, MD, PhD, W. P. Andrew Lee, MD, and Gerald Brandacher, MD Johns Hopkins University School of Medicine, Vascularized Composite Allotransplantation (VCA) Laboratory, Baltimore, MD, USA Background Vascularized composite allografts (VCA) can enhance the quality of life for patients with severe facial or extremity injuries, and induction of tolerance would avoid the risk of immunosuppression and increase application of VCA The purpose of this study is to investigate strategies for tolerance induction in a large animal model Methods Heterotopic osteomyocutaneous hind limb transplantation was performed in 19 MGHminiature swine across full swine leukocyte antigen mismatch All animals received non-myeloablative conditioning with 50cGy total body and 350cGy thymic irradiation for induction Group I was treated with high-dose tacrolimus (15–20 ng/ml) maintenance therapy Group II was treated with low-dose tacrolimus (4–6 ng/ml) Group III received low-dose tacrolimus and 20 mg/kg of CTLA4-Ig administered on POD2, 7, 14, 30, 60, 90, and 120 Group IV received transient high-dose tacrolimus until POD60 Group V received transient highdose tacrolimus until POD60 and was switched to CTLA4-Ig administered on POD60, 85, 100, 120 and 150 Graft rejection was monitored by clinical assessment and protocol skin biopsies Alloreactivity against donor antigens was assessed using an optimized CFSE-based mixed lymphocyte reaction (MLR) Results Prolonged high-dose tacrolimus led to maintenance of VCA in 3/3 animals but was associated with major infectious complications 2/3 animals in group II rejected their grafts by POD46 and 217 In group III, 2/5 animals demonstrated rejection prior to POD150, while 3/5 animals achieved long-term survival of their VCA beyond POD300 3/3 animals in group IV and 4/5 animals in group V achieved indefinite graft survival beyond POD300 despite weaning of all immunosuppression The one animal in group V that rejected its graft began to show evidence of rejection on POD277 Donor specific unresponsiveness was confirmed in all long-term survivors in vitro by CFSE-MLR Conclusions Tolerance of VCA containing vascularized bone marrow can be achieved with a regimen of peritransplant high-dose tacrolimus without myeloablative conditioning. These findings describe a potential induction regimen to eliminate the need for long-term immunosuppression after reconstructive transplantation. CONTACT Howard D. Wang, MD hdwang86@gmail.com

2589: Heterotopic rat penis transplantation: A novel microsurgical model to study distinct immunologic features of urogenital tissues in the setting of reconstructive transplantation

2589: Heterotopic rat penis transplantation: A novel microsurgical model to study distinct immunologic features of urogenital tissues in the setting of reconstructive transplantation Samuel A. J. Fidder, BS, Georg J. Furtmuller, MD, Byoung Chol Oh, DVM, PhD, Barbara Kern, MD, Denver M. Lough, MD, PhD, W. P. Andrew Lee, MD, Damon S. Cooney, MD, PhD, Richard J. Redett, MD, and Gerald Brandacher, MD Johns Hopkins University School of Medicine, Vascularized Composite Allotransplantation (VCA) Laboratory, Baltimore, MD, USA Background Defects of the male urogenital structures are associated with impaired sexual function and a significantly reduced quality-of-life. Modern reconstructive methods employ autologous tissue-based reconstructions and implant placement to reconstruct a functional phallus. Penis transplantation has been successfully employed in the recent past and represents an exciting avenue for restoration of male urogenitalia and function by using “likewith-like” tissue. This animal model is designed to fill a critical void as only little is known on the immunology of these unique tissue grafts. Methods In male 8-week old BN & Lewis rats the penis was dissected to design a penile graft based on the internal pudendal artery and dorsal penile vein including the skin of the prepuce. The non-suture cuff technique was employed to perform end-toend anastomosis of the graft vessels to the recipient inferior epigastric vessels. Syngeneic and allogeneic transplants were performed Native and graft penile tissue were obtained at various time points for histologic analysis. Results Graft design yields suitable caliber and length of graft vessels at the radix of the penis. Anastomosis of the dorsal penile vein and the distal internal pudendal artery at the bifurcation into dorsal and deep penile arteries ensures optimal perfusion of the entire superficial and deep graft tissues. The non-suture cuff technique allows for successful microvascular anastomosis by a single surgeon in an average of 25 hours. Longterm graft survival (>30 days) was observed in syngeneic transplants (N D 3). To date, allogeneic transplant combinations from BN to Lewis rats have been performed and tissues have been harvested for analysis at various time points (ie POD 3, 5, 7, 9, 11; ND 5). Conclusion We have been able to establish a robust and reproducible murine model to study the unique immunobiology of urogenital tissue in the context of reconstructive transplantation. The design of the graft ensures optimal vascular perfusion of superficial and deep penile tissues. Heterotopic inset further allows for visual monitoring of graft viability, while the native penis serves an optimal control. The graft design includes the dorsal penile nerve and may thus be a platform for studies on erectile tissues and function. CONTACT Gerald Brandacher, MD gbrandacher@jhmi.edu

2587: Vascularized composite allotransplantation combined with donor bone marrow and post transplantation high-dose cyclophosphamide (PTCy) induces immune tolerance after reconstructive transplantation

2587: Vascularized composite allotransplantation combined with donor bone marrow and post transplantation high-dose cyclophosphamide (PTCy) induces immune tolerance after reconstructive transplantation Georg J. Furtmuller, MD, Byoung Chol Oh, DVM, PhD, Madeline Fryer, BS, Jeffrey Dodd-o, MD, PhD, Sudipto Ganguly, PhD, Giorgio Raimondi, PhD, Damon Cooney, MD, PhD, W. P. Andrew Lee, MD, Leo Luznik, MD, and Gerald Brandacher, MD Johns Hopkins University School of Medicine, Vascularized Composite Allotransplantation (VCA) Laboratory, Baltimore, MD, USA; Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA Background Reconstructive transplantation has become an enthusiastically employed means for reconstruction of devastating tissue defects However, conventional immunosuppression required to maintain allograft survival hinders its widespread application The treatment concept presented here is designed to induce immunosuppression-free allograft survival Methods: Murine skin, heart, and hindlimb transplants were performed across a full MHC mismatch barrier Recipients were treated with PTCy (ie non-myeloablative TBI, T-cell depletion and a single dose of post-transplant cyclophosphamide) with and without donor BM and splenocytes (DBM) Mixedand regulatory T cell chimerism as well as Vb-TCRstaining was performed Donor-specific unresponsiveness was tested via mixed lymphocyte reactions (MLR) and by secondary skin and heart transplant challenge. Results Untreated animals rejected allogeneic skin grafts, hearts and hindlimbs acutely within 14 § 1 days, 9 §2 days, and 8 § 1 days, respectively The PTCy extended skin and heart graft survival (32 § 8; 65 § 4 days, respectively) Additional DBM augmentation lead to allograft survival of >150 days in skin and heart Indefinite graft survival of >250 days was observed in all animals receiving the induction regimen and a VCA § DBM In groups receiving a VCA with and without DBM, donor chimerism was detected at 3017% § 872% and 2251% § 596%, respectively Regulatory T cell chimerism showed recipient-derived Tregs predominantly contributing to the Treg pool (926C/¡ 42%) in the early phase after transplantation (POD 14–30) whereas at later time points (POD 60-100) donor-derived Foxp3C cells contributed equally (462 C/¡ 113%). Gradual depletion of developing T cell clones by donor-derived cells, shown by progressive reduction in circulating Vb5/11C T cells, indicates actuation of central mechanism for tolerance induction All long-term survivors showed donor-specific T cell unresponsiveness invitro (MLR) while demonstrated proliferation against 3rd party stimulators In-vivo, tolerant animals rejected 3rd party skin while donor-matched tissues were accepted long-term (skin and heart). Conclusion Robust tolerance and immunosuppression-free long-term allograft survival can be induced combining DBM transplantation with PTCy in a fully MHC-mismatched murine models of skin, heart, and VCA The intragraft vascularized bone marrow component of a hind limb provides sufficient amounts of DBM to induce stable multi-lineage mixed chimerism and immune tolerance. CONTACT Gerald Brandacher, MD gbranda2@jhmi.edu

2548: Tolerance induction to vascularized composite allografts by costimulation blockade

2548: Tolerance induction to vascularized composite allografts by costimulation blockade Byoung Chol Oh, DVM, PhD, Georg Furtmuller, MD, Madeline Fryer, BS, Johanna Grahammer, Stefan Schneeberger, Damon Cooney, MD, PhD, W. P. Andrew Lee, MD, Giorgio Raimondi, PhD, and Gerald Brandacher, MD Johns Hopkins University School of Medicine, Vascularized Composite Allotransplantation (VCA) Laboratory, Baltimore, MD, USA; Dept. of Visceral, Transplant and Thoracic Surgery, Innsbruck Medical University, Innsbruck, Austria Background Vascularized composite allotransplantation (VCA) has become a valid therapeutic option after devastating tissue loss While costimulation blockade (CoB) has shown considerable efficacy in preventing rejection, its efficacy in VCA remains poorly explored Here we investigated the immunoregulatory potential of CoB in a novel murine model of hind limb transplantation. Methods Fully MHC-mismatched allogeneic, orthotopic hind limb transplants were performed from Balb/c to C57BL/6 mice Recipient animals received combinations of total body irradiation (TBI), CTLA4-Ig, and anti-CD154 mAb (CoB) Mixed chimerism, clonal deletion of alloreactive T cells, and cytokine production were assessed by flow cytometry. Results CoB treated recipients showed increased survival compared to untreated and CTLA4-Ig only groups (Mean survival time [MST] 82 days) Adding non-myeloablative TBI to CoB enabled indefinite graft survival (MST,>210 days) Mixed chimerism induced by donorderived bone marrow (BM) was detected in the CoB treated group, and was even higher in TBICCoB treated recipients Decreased v̂I211C and v̂I25CCD4C T cells were detected in both groups treated with either CoB or TBICCoB, suggesting central thymic deletion of donor reactive T cells Donor specific tolerance was confirmed in long-term survivors (TBICCoB group) by acceptance of donor matched secondary skin grafts and rejection of third party ones In long term survivors treated with TBICCoB, decreased T cell responsiveness and increased graft-infiltrating regulatory T cells were detected on POD50. Conclusion Our results show that CoB enables the tolerogenicity of BM components carried by VCA, but requires TBI to establish durable donor-specific tolerance. CONTACT Byoung Chol Oh, DVM, PhD boh3@jhmi.edu

2546: Non-cytoreductive immunosuppression prevents rejection of vascularized composite allografts

2546: Non-cytoreductive immunosuppression prevents rejection of vascularized composite allografts Byoung Chol Oh, DVM, PhD, Georg Furtmuller, MD, Marcos Iglesias, PhD, Madeline Fryer, BS, Giorgio Raimondi, PhD, Damon Cooney, MD, PhD, W. P. Andrew Lee, MD, and Gerald Brandacher, MD Johns Hopkins University School of Medicine, Vascularized Composite Allotransplantation (VCA) Laboratory, Baltimore, MD, USA Background Reconstructive transplantation represents a valid therapeutic option after devastating tissue loss such as an extremity or face Immunosuppression-free donorspecific immunological tolerance has been successfully achieved in the setting of solid organ transplantation through mixed chimerism Routine clinical application of this approach, however, is hampered by the toxicity of the cytoreductive recipient conditioning The current study investigated a novel non-cytoreductive immunosuppressive strategy in a murine model of hind limb transplantation. Methods Fully MHC-mismatched allogeneic, orthotopic hind limb transplants were performed from Balb/C to C57BL/6 mice Recipient animals in the experimental groups received no treatment (Group 1); 025 mg CTLA4 Ig on postoperative days (POD) 0, 2, 4, and 6 (Group 2); 20 mg/kg anti-T cells (anti-Thy 12 mAb) on POD-1 plus CTLA4-Ig and 1 mg/kg Rapamycin (POD0-9) (Group 3) Flow cytometric analysis was performed to evaluate mixed chimerism and clonal deletion of alloreactive T cells. Results The CTLA4 Ig treated group showed increased graft survival compared to non-treated controls (mean survival time [MST] 17 d and 8 d, respectively, p < 001) Interestingly, combination of T cell depletion and CTLA4 Ig plus short-course of Rapamycin increased VCA survival significantly (MST 105 days; p < 001) Donor derived mixed chimerism was detected in recipients receiving the combined treatment protocol with 50 § 12 % of donor derived CD11bC cells on POD 55 VÎ – TCR staining profiles in recipients after combined treatment showed 16 § 04 % of Î=2Î25CCD4C T cells, while nae Ave C57BL/6 express 36 § 04 % of Î=2Î 25CCD4C T cells, suggesting central deletion of donor-reactive T cells Also, we found infiltrating Foxp3C regulatory T cells expressing donor derived marker (H-2K) at POD60 on combined treatment representing donor derived T reg cells were infiltrated. Conclusion This study shows that combination of T cell depletion and costimulation blockade and short-course of Rapamycin prevents VCA rejection and significantly prolongs graft survival Also, these data show that a clinically acceptable non-cytoreductive strategy could be applied in VCA to avoid long-term maintenance immunosuppression. CONTACT Gerald Brandacher, MD gbranda2@jhmi.edu

2526: Non-invasive monitoring of allograft rejection using thermal imaging in a large animal model of vascularized composite allotransplantation

2526: Non-invasive monitoring of allograft rejection using thermal imaging in a large animal model of vascularized composite allotransplantation Keli Kolegraff, Amy Quan, Nicole Crane, Howard Wang, Joseph Lopez, Georg Furtmuller, Sara Alfadil, Sara Mermulla, Byoung Chol Oh, Paul Akre, Jose C. Alonso-Escalante, Jeffrey Walch, Jaimie Shores, Damon Cooney, Steven Bonawitz, Eric Elster, Gerald Brandacher, and W. P. Andrew Lee Johns Hopkins University School of Medicine, Baltimore, MD, USA; Naval Medical Research Center, Bethesda, MD, USA; Uniformed Services University of the Health Sciences and Walter Reed National Military Medical Center, Bethesda, MD, USA Background Vascularized composite allotransplantation (VCA), including hand and face transplantation, is increasingly utilized for reconstruction of disfiguring injuries. Immune rejection is a problem inherent to allotransplantation and must be recognized early to prevent graft injury/loss. Skin biopsy is required to confirm rejection however this approach may further promote rejection through tissue injury. Thermal imaging has been used to non-invasively detect inflammation and ischemia in other disease states but has not been evaluated in VCA rejection. The purpose of this study was to evaluate the utility of non-invasive thermal imaging in the detection of rejection in a swine VCAmodel. Methods Osteomyocutaneous hindlimb transplantation was performed in 9 MGH miniature swine across a full SLA mismatch. The positive rejection control group (3 pigs) did not receive any form of immunosuppression. The treated group (6 pigs) received non-myeloablative conditioning with 50cGy total body and 350cGy thymic irradiation prior to transplantation, daily tacrolimus (20–25 ng/ml) until POD 30, and IV steroids on POD 4–6. Allografts were assessed daily for erythema, edema, blistering, and ulceration. Daily infrared images were acquired using a thermal camera (FLIR E8 #63903-0303) and thermal emission intensities from graft and contralateral flank control were analyzed using Image. Results In the untreated group, erythema was observed by POD 4 and progressed to epidermolysis by POD 8 in 3/3 animals. In the treated group, rejection was observed at POD 5–7 in 6/6 animals and was reversed by POD 10 with steroids in 5/6 animals. The sixth animal developed severe rejection on POD 5 that progressed to necrosis despite steroids. Interestingly, infrared imaging did not distinguish these episodes of rejection in the early post-operative period (<POD 30) despite clinical signs of inflammation and biopsyproven rejection (Banff II–IV). At later post-operative periods (>POD 60), there was a trend toward cooler graft temperatures even in the absence of clinical rejection. Conclusions Despite signs of inflammation, infrared imaging did not reliably detect graft rejection. The finding that grafts become cooler over time suggests there may be long-term changes in graft perfusion. This may represent chronic graft injury and is the subject of on-going studies. CONTACT Keli Kolegraff keli@jhmi.edu