Nakul Rao

Abstract 103: primed mesenchymal stem cells prevent endothelial activation and improve allograft perfusion following transplantation.

PurPose: Endothelial activation following ischemia-reperfusion injury (IRI) in transplantation triggers the inflammatory cascade, compromising allograft perfusion. Additionally, IRI is a critical factor that contributes to the incidence and severity of both acute and chronic rejection. We have previously demonstrated that mesenchymal stem cells (MSCs) can be seeded into allografts ex vivo where they take up residence in the perivascular space. While conventional expansion of MSCs produces an innate immunomodulatory phenotype, conditions that enhance this phenotype may be utilized to attenuate endothelial failure following ischemic insult during transplantation. We hypothesized that expansion under hypoxic conditions or with inflammatory cytokines primes the immunosuppressive functions of MSCs and improves allograft perfusion subsequent to ex vivo delivery.

Abstract 4: Site Specific Targeting of PUMA Induced ROS Prevents Radiation Injury via a Smad3 Independent Mechanism.

PurPose: Although radiation therapy is an instrumental tool in the treatment of numerous cancers, it is hampered by its detrimental effects on the skin. Namely, impaired wound healing, fibrosis and scarring often requiring surgical intervention. Previously, we have demonstrated that radiation induced PUMA expression and subsequent ROS overproduction is a critical factor in the development of cutaneous fibrosis. However, it is unclear whether this effect is dependent on SMAD3 expression, a central fibrosis pathway. Here, we investigate the downstream molecular mechanism for radiation protection with PUMA knock down in cutaneous radiation injury.

Ex vivo allotransplantation engineering: Delivery of mesenchymal stem cells prolongs rejection-free allograft survival

Current pharmacologic regimens in transplantation prevent allograft rejection through systemic recipient immunosuppression but are associated with severe morbidity and mortality. The ultimate goal of transplantation is the prevention of allograft rejection while maintaining recipient immunocompetence. We hypothesized that allografts could be engineered ex vivo (after allotransplant procurement but before transplantation) by using mesenchymal stem cell–based therapy to generate localized immunomodulation without affecting systemic recipient immunocompetence. To this end, we evaluated the therapeutic efficacy of bone marrow–derived mesenchymal stem cells in vitro and activated them toward an immunomodulatory fate by priming in inflammatory or hypoxic microenvironments. Using an established rat hindlimb model for allotransplantation, we were able to significantly prolong rejection‐free allograft survival with a single perioperative ex vivo infusion of bone marrow–derived mesenchymal stem cells through the allograft vasculature, in the absence of long‐term pharmacologic immunosuppression. Critically, transplanted rats rejected a second, nonengineered skin graft from the same donor species to the contralateral limb at a later date, demonstrating that recipient systemic immunocompetence remained intact. This study represents a novel approach in transplant immunology and highlights the significant therapeutic opportunity of the ex vivo period in transplant engineering.

2576: Optimization of a differential cytokine profile-based non-invasive diagnostic and predictive tool for reliable diagnosis of acute rejection in VCA

2576: Optimization of a differential cytokine profile-based non-invasive diagnostic and predictive tool for reliable diagnosis of acute rejection in VCA Piul S. Rabbani, PhD, Rohini L. Kadle, MD, Nakul Rao, Chin Park, and Daniel Ceradini New York University School of Medicine, New York, NY, USA Background Current methods of detection of early acute transplant rejection relies on invasive tissue biopsies and time-consuming histological analysis We propose an alternative method using adhesive discs to analyze molecular changes in cells sampled from the epidermis of a vascular composite allotransplant (VCA), to detect markers of acute rejection We aim to validate efficiency of skin stripping as a non-invasive predictor and sensitive diagnostic test for acute rejection in VCA. Methods Using an established VCA rat model, we transplanted composite flaps from donor Brown-Norway rats to age-matched recipient Lewis rats Following cyclosporine for 5 days, we inspected daily for clinical signs of rejection and sampled transplanted skin with adhesive CuDerm-discs at each time point up to rejection We performed QRT-PCR on sampled cells for cytokines associated with early rejection We sampled flaps for biopsies and histology to corroborate the disc data. Results CuDerm-disc-sourced PCR revealed that expressions of MCP1, MIP1̂I§, MIP1̂I and CXCL10 increased progressively with mild and advanced rejection, compared to the immunosuppressed stage (p < 005) MIP3̂I§ and CXCL9 showed significant upregulation at mild rejection (19-fold, 70-fold, respectively), but a downregulation during advanced rejection (4fold,20-fold, respectively, p < 005) Comparison of mild and advanced rejection showed highly significant differential cytokine expression (p < 001) We verified the sensitivity and validity of the CuDerm-disc method by comparison of mRNA expression in VCA biopsies and found cytokine detection comparable between both methods The mild and advanced rejection cytokine profiles from discs also corresponded with the Banff classification of cellular allograft rejection of the respective flap histologies. Conclusions Skin stripping, when compared to traditional tissue biopsy, is a comparable and reliable analytical tool The cytokine profiles gathered from skin stripping are distinct and capable of detecting the earliest stages of acute rejection, as well as distinguishing from advanced rejection, stages which are difficult to analyze definitively using traditional histology Our results clearly demonstrate the promise of skin stripping as a noninvasive tool in predicting and diagnosing early rejection, prior to onset of advanced rejection.

Abstract 119: Development of a Non-invasive Diagnostic and Predictive Tool for Acute Rejection in Vca Using Differential Cytokine Profiling

86 RESULTS: Herceptin administration increased the rate of motoneuron regeneration by 3x and sensory neuron regeneration by 3.7x compared to saline treated animals after the first week. However, the extent of motor and sensory regeneration was nearly complete in both groups by the end of the second week. In addition, the total number of myelinated fibers growing distally beyond the repair site was significantly increased in rats receiving Herceptin (2488 ± 154) compared to rats that received saline (1896 ± 251) (p < 0.05) four weeks after repair. When delayed repair was performed after a 3-month period of chronic denervation, Herceptin increased the number of acutely, but not chronically, axotomized motoneurons after two weeks. Interestingly, Western blot analysis revealed no change in ErbB2 activation with Herceptin administration. However, immunofluorescent imaging revealed decreased levels of activated EGFR on regenerating neurons, a factor known to be inhibitory to axon regeneration.

Ex Vivo Primed Mesenchymal Stem Cell Therapy Prevents Endothelial Failure and Enhances Allograft Perfusion

Endothelial failure following ischemia-reperfusion injury (IRI) in transplantation triggers the inflammatory cascade, compromising allograft perfusion and contributing to acute rejection. Culture conditions that activate the innate immunomodulatory phenotype of mesenchymal stem cells (MSCs) may attenuate IRI-mediated endothelial failure. We hypothesized that expansion in hypoxia or with inflammatory cytokines primes immunosuppressive functions of MSCs and improves allograft perfusion after ex-vivo delivery.

Abstract 129: Phosphodiesterase Type 5 Inhibition Enhances The Angiogenic Profile Of Adipose-derived Stem Cells.

ConClusion: Our findings suggest that incorporating paclitaxel in fat grafts for breast reconstruction following primary breast surgery is a viable option for decreasing the risk of local recurrence. 4OH tamoxifen can also be incorporated in fat grafting with ER-positive breast cancer patients. An in-vivo model employing lipoaspirate and cancer cells to test encapsulated chemotherapeutics is currently being developed with encouraging results. Taken together, incorporating encapsulated chemotherapeutic drugs in autologous fat grafts for breast reconstruction procedures is a feasible therapeutic option for breast cancer survivors. 129 Phosphodiesterase type 5 inhibition enhances the angiogenic Profile of adipose-derived Stem cells