Maxine R. Miller

Whole-eye transplantation: a look into the past and vision for the future.

Blindness afflicts ~39 million people worldwide. Retinal ganglion cells are unable to regenerate, making this condition irreversible in many cases. Whole-eye transplantation (WET) provides the opportunity to replace diseased retinal ganglion cells, as well as the entire optical system and surrounding facial tissue, if necessary. Recent success in face transplantation demonstrates that this may be a promising treatment for what has been to this time an incurable condition. An animal model for WET must be established to further enhance our knowledge of nerve regeneration, immunosuppression, and technical aspects of surgery. A systematic review of the literature was performed to evaluate studies describing animal models for WET. Only articles in which the eye was completely enucleated and reimplanted were included. Study methods and results were compared. In the majority of published literature, WET can result in recovery of vision in cold-blooded vertebrates. There are a few instances in which mammalian WET models demonstrate survival of the transplanted tissue following neurovascular anastomosis and the ability to maintain brief electroretinogram activity in the new host. In this study we review in cold-blooded vertebrates and mammalian animal models for WET and discuss prospects for future research for translation to human eye transplantation.

Splenectomy Promotes Indirect Elimination of Intraocular Tumors by CD8+ T Cells That Is Associated with IFNγ- and Fas/FasL-Dependent Activation of Intratumoral Macrophages

Miller and colleagues demonstrate a mechanism by which splenectomy promotes rejection of intraocular tumors, which involves an IFNg-and-Fas/FasL-dependent interaction between CD8+ T cells and intratumoral macrophages eliciting severe ocular inflammation that indirectly eliminates intraocular tumors by inducing phthsis. Ocular immune privilege (IP) limits the immune surveillance of intraocular tumors as certain immunogenic tumor cell lines (P815, E.G7-OVA) that are rejected when transplanted in the skin grow progressively when placed in the anterior chamber of the eye. As splenectomy (SPLNX) is known to terminate ocular IP, we characterized the immune mechanisms responsible for rejection of intraocular tumors in SPLNX mice as a first step toward identifying how to restore tumoricidal activity within the eye. CD8+ T cells, IFNγ, and FasL, but not perforin, or TNFα were required for the elimination of intraocular E.G7-OVA tumors that culminated in destruction of the eye (ocular phthisis). IFNγ and FasL did not target tumor cells directly as the majority of SPLNX IFNγR1−/− mice and Fas-defective lpr mice failed to eliminate intraocular E.G7-OVA tumors that expressed Fas and IFNγR1. Bone marrow chimeras revealed that IFNγR1 and Fas expression on immune cells was most critical for rejection, and SPLNX increased the frequency of activated macrophages (Mφ) within intraocular tumors in an IFNγ- and Fas/FasL-dependent manner, suggesting an immune cell target of IFNγ and Fas. As depletion of Mφs limited CD8 T cell–mediated rejection of intraocular tumors in SPLNX mice, our data support a model in which IFNγ- and Fas/FasL-dependent activation of intratumoral Mφs by CD8+ T cells promotes severe intraocular inflammation that indirectly eliminates intraocular tumors by inducing phthisis, and suggests that immunosuppressive mechanisms that maintain ocular IP interfere with the interaction between CD8+ T cells and Mφs to limit the immunosurveillance of intraocular tumors. Cancer Immunol Res; 2(12); 1175–85. ©2014 AACR.

Clinical Considerations for Vascularized Composite Allotransplantation of the Eye.

AbstractVascularized composite allotransplantation represents a potential shift in approaches to reconstruction of complex defects resulting from congenital differences as well as trauma and other acquired pathology. Given the highly specialized function of the eye and its unique anatomical components, vascularized composite allotransplantation of the eye is an appealing method for restoration, replacement, and reconstruction of the nonfunctioning eye. Herein, we describe conventional treatments for eye restoration and their shortcomings as well as recent research and events that have brought eye transplantation closer to a potential clinical reality. In this article, we outline some potential considerations in patient selection, donor facial tissue procurement, eye tissue implantation, surgical procedure, and potential for functional 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

2564: Total human eye allotransplantation (THEA): Protocol optimization of imaging modalities in a non-human primate model

2564: Total human eye allotransplantation (THEA): Protocol optimization of imaging modalities in a non-human primate model Edward H. Davidson, MA, (Cantab), MBBS, Jonathan Carney, Brian Lopresti, Maxine Reedy Miller, MD, and Kia M. Washington University of Pittsburgh, Pittsburgh, PA, USA Background To pioneer vascularized composite allotransplantation (VCA) of the human eye as a clinical reality, our group has developed the first orthotopic model for eye transplantation in the rat for testing of immunomodulation and neuroregeneration therapies. We have also performed human cadaveric studies to design surgical protocols for donor and recipient procedures ahead of advancing to a non-human primate surgical model of eye transplantation Non-invasive methodologies of graft monitoring are paramount to development of this model as well as for clinical practice in future transplantation programs.

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

Total Human Eye Allotransplantation: Developing Surgical Protocols for Donor and Recipient Procedures

Background: Vascularized composite allotransplantation of the eye is an appealing, novel method for reconstruction of the nonfunctioning eye. The authors’ group has established the first orthotopic model for eye transplantation in the rat. With advancements in immunomodulation strategies together with new therapies in neuroregeneration, parallel development of human surgical protocols is vital for ensuring momentum toward eye transplantation in actual patients. Methods: Cadaveric donor tissue harvest (n = 8) was performed with orbital exenteration, combined open craniotomy, and endonasal approach to ligate the ophthalmic artery with a cuff of paraclival internal carotid artery, for transection of the optic nerve at the optic chiasm and transection of cranial nerves III to VI and the superior ophthalmic vein at the cavernous sinus. Candidate recipient vessels (superficial temporal/internal maxillary/facial artery and superficial temporal/facial vein) were exposed. Vein grafts were required for all anastomoses. Donor tissue was secured in recipient orbits followed by sequential venous and arterial anastomoses and nerve coaptation. Pedicle lengths and calibers were measured. All steps were timed, photographed, video recorded, and critically analyzed after each operative session. Results: The technical feasibility of cadaveric donor procurement and transplantation to cadaveric recipient was established. Mean measurements included optic nerve length (39 mm) and caliber (5 mm), donor artery length (33 mm) and caliber (3 mm), and superior ophthalmic vein length (15 mm) and caliber (0.5 mm). Recipient superficial temporal, internal maxillary artery, and facial artery calibers were 0.8, 2, and 2 mm, respectively; and superior temporal and facial vein calibers were 0.8 and 2.5 mm, respectively. Conclusion: This surgical protocol serves as a benchmark for optimization of technique, large-animal model development, and ultimately potentiating the possibility of vision restoration transplantation surgery. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, V.