Chiaki Komatsu

Biopatterned CTLA4/Fc Matrices Facilitate Local Immunomodulation, Engraftment, and Glucose Homeostasis After Pancreatic Islet Transplantation

Pancreatic islet transplantation (PIT) represents a potential therapy to circumvent the need for exogenous insulin in type 1 diabetes. However, PIT remains limited by lack of donor islets and the need for long-term multidrug immunosuppression to prevent alloimmune islet rejection. Our goal was to evaluate a local immunoregulatory strategy that sustains islet allograft survival and restores glucose homeostasis in the absence of systemic immunosuppression. Nanogram quantities of murine CTLA4/Fc fusion protein were controllably delivered within human acellular dermal matrix scaffolds using an inkjet-based biopatterning technology and cotransplanted with allogeneic islets under the renal capsule to create an immunoregulatory microenvironment around the islet allograft. We achieved long-term engraftment of small loads of allogeneic islet cells with 40% of MHC-mismatched mouse recipients maintaining sustained normoglycemia following pancreatic β-cell ablation by streptozotocin. Biopatterned CTLA4/Fc local therapy was associated with expansion of Foxp3+ regulatory T cells and shifts in cytokine production and gene expression from proinflammatory to regulatory profiles, thus substantially benefiting islet allografts survival and function. This study is a new paradigm for targeted therapies in PIT that demonstrates the favorable effects of immune alterations in the transplant milieu and suggests a unique strategy for minimizing systemic immunosuppression and promoting islet allograft survival.

Abstract P9: Non-Invasive Objective Evaluation of Peripheral Neuroregeneration Using Magnetic Resonance Diffusion Tensor Imaging

ConClusion: This study confirms feasibility of application of hES conduit for restoration of peripheral nerve defects. hES supported with hMSC demonstrated comparable functional outcomes to the autograft technique. The role of local hMSC application in nerve regeneration is currently under investigation. P9 non-invasive objective evaluation of Peripheral neuroregeneration Using Magnetic resonance diffusion tensor imaging

IGF-1 and Chondroitinase ABC Augment Nerve Regeneration after Vascularized Composite Limb Allotransplantation

Impaired nerve regeneration and inadequate recovery of motor and sensory function following peripheral nerve repair remain the most significant hurdles to optimal functional and quality of life outcomes in vascularized tissue allotransplantation (VCA). Neurotherapeutics such as Insulin-like Growth Factor-1 (IGF-1) and chondroitinase ABC (CH) have shown promise in augmenting or accelerating nerve regeneration in experimental models and may have potential in VCA. The aim of this study was to evaluate the efficacy of low dose IGF-1, CH or their combination (IGF-1+CH) on nerve regeneration following VCA. We used an allogeneic rat hind limb VCA model maintained on low-dose FK506 (tacrolimus) therapy to prevent rejection. Experimental animals received neurotherapeutics administered intra-operatively as multiple intraneural injections. The IGF-1 and IGF-1+CH groups received daily IGF-1 (intramuscular and intraneural injections). Histomorphometry and immunohistochemistry were used to evaluate outcomes at five weeks. Overall, compared to controls, all experimental groups showed improvements in nerve and muscle (gastrocnemius) histomorphometry. The IGF-1 group demonstrated superior distal regeneration as confirmed by Schwann cell (SC) immunohistochemistry as well as some degree of extrafascicular regeneration. IGF-1 and CH effectively promote nerve regeneration after VCA as confirmed by histomorphometric and immunohistochemical outcomes.

The impact of Topical Immunosuppressive Treatment on Functional Characterization of Skin Dendritic Cell Subpopulations in Vascularized Composite Allotransplantation

Introduction The high immunogenicity of skin has manifested as multiple acute rejection episodes in the majority of human vascularized composite allotransplantation (VCA) case. Skin dendritic cells (DC) are believed to play an important role in both the initiation and regulation of skin alloimmunity. Resident skin DC can migrate out of allogeneic skin into the recipient’s skin and draining lymph nodes, and may exert regulatory functions after VCA. Thus, isolating migratory skin DC and examining their effect on T-cell immune responses are parts of our continued efforts to optimize targeted immunomodulation. In the present study, we assessed the function of skin-resident and skin-migrated DC subsets on regulating the T-cell immune response and characterized their changes in VCA under topical FK506 immunosuppression. Materials and Methods 1) Hind-limb transplantation was performed from BN to Lewis rats and recipients were treated with topical FK506 (0.5mg/kg, 0.1% FK506 ointment, applied once daily). FK506 levels in blood and local skin were measured using liquid chromatography tandem-mass spectrometry. Skin-resident DC from transplanted limbs were isolated at day 8 post-transplantation and quantification of skin DC subsets was analyzed by flow cytometry. 2) Migrated skin DC from the limb of 7-day topical FK506 treated untransplanted and naïve Lewis rats were isolated in vitro after skin being cultured under a stimulation culture condition and were characterized by subsets and functional specialization using mixed lymphocyte reaction (MLR) and flow cytometric analysis. Results and Discussion 1) Limb allografts showed clinical signs of grade 2-3 rejection on day 8 post-transplantation. High skin levels (2110±177 ng/ml) and low blood levels (3.5±2.2 ng/ml) of FK506 were determined after 7-day topical treatment. Skin-resident dermal DC(DDC) declined with an elevation trend in Langerhans cells(LC) compared to no treatment group(Figure 1). Migrated LC and mature skin DC were lower, while DDC were slightly higher in FK506-treated skin compared to naïve skin(Figure 2). These data indicate the probable effect of topical immunosuppression on the modulation of skin DC subsets after VCA and skin DC migration and maturation could be inhibited by exposure to FK506. 2) In MLR, skin-migrated DC inhibited effector T cell (Teff) proliferation and exhibited synergistic effects with regulatory T cells (Treg). The addition of skin-migrated DC promoted Foxp3 expression in CD4+ T cells. In contrast, the addition of skin-resident DC decreased Foxp3 and promoted IL-17 expression, suggesting there is differential contribution of skin-migrated and skin-resident DC to the Teff response in vitro. Conclusion In vivo alterations of skin-resident DC subsets and ex vivo emigration and maturation of skin DC are affected by a short-term topical immunosuppression in VCA. The determination of skin-migrated DC in regulating T-cell immune responses will provide a target for immunomodulation. Figure. No caption available. American Association of Plastic Surgeons(AAPS)/Plastic Surgery Foundation(PSF) Academic Scholarship Award. The UPP Academic Foundation Research Grant.

Abstract P18: In Vivo Evaluation Of The Retina And Optic Nerve After Whole Eye Transplantation Using Optical Coherence Tomography, Manganese-enhanced Magnetic Resonance Imaging And Electroretinography

1Department of Plastic Surgery,University of Pittsburgh, Pittsburgh, PA, 2Department of Bioengineering,University of Pittsburgh, Pittsburgh, PA, 3Department of Ophthalmology, University of Pittsburgh, Pittsburgh, PA, 4Department of Ophthalmology, New York University School of Medicine, New York, NY, 5Neuroimaging Laboratory, University of Pittsburgh, Pittsburgh, PA, 6Veterans Administration Pittsburgh Healthcare System, Pittsburgh, PA

2561: Dynamics and correlates of skin dendritic cells with distinctive immune response profiles in vascularized composite allografts

2561: Dynamics and correlates of skin dendritic cells with distinctive immune response profiles in vascularized composite allografts Wensheng Zhang, MD, PhD, Chiaki Komatsu, MD, Firuz Gamal Feturi, BPharm, PhD, Lin He, MD, Liwei Dong, Jiaqing Wu, MD, PhD, Maxine Reedy Miller, MD, Jeffrey Mark Walch, MD/PhD, Alicia Mathers, PhD, Angus W. Thomson, Kia M. Washington, MD, Vijay Gorantla, and Mario Solari University of Pittsburgh, Pittsburgh, PA, USA Introduction Vascularized composite allografts (VCA) such as hand allografts, contains large amount of most immunogenic skin component Multiple acute rejection episodes in the majority of human VCA routinely target skin while sparing other tissue types Skin dendritic cells (DCs) are thought to play critical roles in either initiation or regulation of skin immunity However, their contribution to the unique alloimmune response and acute skin rejection of VCA is still largely unknown This study seeks to characterize the spatiotemporal dynamics of skin resident DCs at different stages of rejection after hind-limb transplantation and to better understand the role of skin DCs in immunomodulation of VCA. Materials and methods Lewis rats received allogeneic hind-limb transplants from BN rats without immunosuppressive treatment, and were inspected daily for their clinical signs of rejection using a visual scoring system Recipients were sacrificed at different time points post-transplantation Allograft skin, adjacent recipient skin, and draining lymph nodes were harvested and processed to obtain single cell suspension for flow cytometric analysis, or to extract total RNA and proteins and assessed by real-time PCR and Luminex. Results 1) We developed a new technique for isolation and characterization of skin DCs in rat hind-limb transplant model 2) Skin resident lymphocytes and distinct subsets of skin migratory DCs: Langerhans cells (LCs), dermal DCs (DDCs), and langerinC DDCs, in the transplanted limb were identified and enumerated 3) Skin migratory DCs in both allograft and recipient limb skin showed different patterns of change with increasing severity of rejection 4) The expression of Th1-, Th2-, Th17-, and Tregassociated genes and cytokines in allograft and recipient limb skin exhibited dynamic changes and temporal correlation with the quantity of skin DCs during the process of rejection. Conclusions The correlative trends between skin DC subsets and T cell-mediated alloimmune response suggest a complex cutaneous immune cell network modulated by skin DCs in VCA By understanding the dynamics of skin DCs and their influence on the T-cell response, the novel targeted immunomodulation therapy can be developed for VCA. Funding This work was supported by American Association of Plastic Surgeons Academic Scholarship. CONTACT Mario Solari solarimg@upmc.edu