Breonics (United States)

Introduction: We have previously demonstrated the feasibility of bioengineering the luminal surface within renal allografts without adversely affecting function. The result was prolonged canine renal allograft survival in the absence of systemic immunosuppression or immunologic manipulation of the recipient. Referred to as “immunocloaking” our approach entails coating the vascular surfaces within kidneys with a nano-barrier membrane, NB-LVF4, that is composed of laminin, vitrogen, fibronectin, and type IV collagen. Several integrin complexes are involved in the attachment of NB-LVF4 to the surface of vascular endothelial cells (VEC). NB-LVF4 provides an apical surface that remains non-thrombogenic and non-immunogenic. The goal is to interrupt the normal interface by providing a physical barrier between recipient immune cells in circulation and the renal allograft vasculature. We now report the underlying immunological mechanism involved in immunocloaking. Methods: Proliferation assays (n=5) were performed using responding peripheral blood mononuclear cells (MNCs) stimulated with autologous lymphocytes and untreated confluent VEC (controls) and with confluent immunocloaked VEC (test). Following 72 hours of stimulation, supernatants were collected from each triplicate well and tested for cytokine and chemokine responses using the Luminex xMap platform. Early T cell activation was measured using CD4+, CD69+ detection by flow cytometry. Results: Immunocloaking resulted in statistically significantly inhibition of the cytokines/chemokines: IL-1β, IL-6, γ-IFN, IL-2, TNF-α, CD-69, MIG and MIP-1α (p< 0.5). The inhibition of the cytokines produced by antigen presenting cells, IL-1β, IL-1β, MIP-1α and γ-IFN suggest that antigen presentation was prevented. Similarly, the inhibition of markers of T cell activation, IL-6, IL-2, CD-69 and MIG suggest the blockade of T cell mediated responses.Figure: [Immunocloak Cytokine/APC Analysis]Conclusions: Immunocloaking with the NB-LVF4 provides a mechanism to eliminate the allo-recognition that normally occurs immediately on reperfusion. The lack of allo-responses upon reimplantation could provide a new window of opportunity to introduce adjunct therapies to support tolerance induction.

We previously reported that a bioengineered interface consisting of a nano-barrier membrane (NB-LVF4) used as an artificial interface between skin allografts and wound surfaces significantly prolonged graft survival without immunosuppression. We now evaluated whether NB-LVF4 could serve as a targeted drug delivery system to further improve outcomes. Fibroblast growth factor-1 (FGF-1) was selected for its known function as a wound hormone. Full-thickness 8-mm skin grafts were cross-transplanted between out-bred mice. Controls were transplanted without treatment. In test groups, the NB-LVF4, with or without FGF-1, was applied to both basolateral skin and wound surfaces with polymerization resulting in a tridimensional transparent membrane. The mice were evaluated for T-cell activation and development of donor-specific antibody. Rejection occurred in controls by 7 days. NB-LVF4 treatment, with or without FGF-1, was found to significantly prolong allograft survival (27 and 28 days, respectively [p < 0.05]). Untreated controls stimulated 10-fold shift in the number of circulating CD4+ cells while test groups exhibited substantially reduced shifts in CD4+ cells. The group treated with FGF-1 did not develop donor-specific antibody. Treatment with the NB-LVF4 membrane delays the onset of allograft rejection in the absence of systemic immunosuppression. FGF-1 appears to prevent the development of a humoral response by preventing B cell activation.

1BREONICS, Inc., Albany/NY/UNITED STATES OF AMERICA, 2BREONICS, Inc., Albany/New York/UNITED STATES OF AMERICA, 3Dept Of Carniofacial And Plastic Surgery, University of Utrecht, Utrecht/NETHERLANDS

Stubenitsky, Bart M.1; Booster, Maurits H.1; Brasile, Lauren2; Araneda, Dorian3; Haisch, Carl E.3; Kootstra, Gauke1 Author Information

Future approaches to expand the organ donor pool with marginal and nonheartbeating donors, will be dependent upon prospective organ evaluation. Restoration of metabolism by preservation at warmer temperatures could potentially provide the window for such evaluation. Using a small bovine model, kidneys were subjected to either < 15, < 30 or < 60 minutes of warm ischemia (WI) followed by cold ischemia (CI) in ViaSpan. After WI and CI, kidneys were transitioned to a warm temperature perfusion (30 degrees C to 32 degrees C) using exsanguinous metabolic support (EMS) technology. Restored renal metabolism and function was assessed by oxygen consumption, glucose consumption, urine production, glomerular filtration rate, and hemodynamic characteristics. The results of this study suggest that it is feasible to distinguish viable from nonviable organs ex vivo by assessing renal metabolism and function during warm preservation using EMS technology.