Riccardo Sfriso

Release of pig leukocytes and reduced human NK cell recruitment during ex vivo perfusion of HLA-E/human CD46 double-transgenic pig limbs with human blood.

In pig‐to‐human xenotransplantation, interactions between human natural killer (NK) cells and porcine endothelial cells (pEC) are characterized by recruitment and cytotoxicity. Protection from xenogeneic NK cytotoxicity can be achieved in vitro by the expression of the non‐classical human leukocyte antigen‐E (HLA‐E) on pEC. Thus, the aim of this study was to analyze NK cell responses to vascularized xenografts using an ex vivo perfusion system of pig limbs with human blood.

Multiple genetically modified GTKO/hCD46/HLA‐E/hβ2−mg porcine hearts are protected from complement activation and natural killer cell infiltration during ex vivo perfusion with human blood

In pig‐to‐human xenotransplantation, early cellular rejection reactions are mediated by natural killer cells (NK cells). Human NK cells are inhibited by HLA‐E via CD94/NKG2A receptors. To protect porcine grafts against human NK cell responses, transgenic GTKO pigs expressing hCD46 and HLA‐E have been generated. The aim of this study was to test the effect of this genetic modification on xenogeneic, and in particular human NK cell response, using an ex vivo perfusion model of pig hearts with human blood.

Real Time High-Resolution Imaging of Porcine Endothelial Glycocalyx Shedding by Human Serum in an in Vitro Microfluidic Model of Pig-to-Human Xenotransplantation

Background The glycocalyx, a complex layer of membrane-bound proteoglycans, sialic acid-containing glycoproteins and glycolipids is a key element for the maintenance of homeostasis in blood vessels. Pathophysiological conditions such as ischemia/reperfusion injury and vascular diseases lead to damage of the glycocalyx impairing endothelial functions. Increased capillary permeability, adhesion of immune cells, thrombosis and vascular inflammation are typical consequences of glycocalyx shedding. In xenotransplantation, donor graft endothelium interfaces with the recipient blood, resulting in glycocalyx degradation and consequent switch of the graft endothelium to a pro-coagulant and pro-inflammatory phenotype. In the present study a microfluidic perfusion system was used to assess the shedding of heparan sulfate (HS), the main component of the endothelial glycocalyx, after perfusion of porcine aortic endothelial cells (PAEC) with normal human serum (NHS). Methods Wild type PAEC were seeded in microchannels of 550 &mgr;m diameter with round cross-sections and left to reach confluence overnight under static conditions. A peristaltic pump was then connected and a flow of 600 &mgr;l/min, corresponding to a shear stress of 10 dyn/cm2, applied for 48 hours. Flow-adapted PAEC as well as PAEC grown under static conditions were incubated with fluorescent wheat germ agglutinin (WGA), which binds to N-acetyl-D-glucosamine (GlcNAc) and N-acetyl neuraminic acid (NeuAc) components of the glycocalyx, or fluorescence-labeled anti-HS antibody. Subsequently, PAEC microchannels were perfused for 2 hours with 10% NHS or cell culture medium. During NHS perfusion, real-time fluorescence imaging was performed using high-speed confocal microscopy. Results PAEC exposed to physiological flow for 48 hours considerably upregulated the expression of HS as compared to static culture conditions (43% ± 3% increase of HS-staining) whereas GlcNAc / NeuAc staining was reduced (61% ± 10% reduction). Perfusion of PAEC-microchannels with 1:10 diluted NHS at 10 dyn/cm2 led to a significant, time-dependent reduction of both HS (38% ± 6% reduction after 10 minutes) and GlcNAc / NeuAc staining (43% ± 9% reduction). Longer perfusion times led to increasing detachment of PAEC, which could be visualized by confocal microscopy. Conclusion Our preliminary data confirm that flow and shear stress are important factors affecting the composition of the endothelial glycocalyx in terms of expression of HS, GlcNAc, and NeuAc. The reduced staining of these sugar molecules during NHS perfusion supports the idea that in a pig-to-human xenotransplantation setting the porcine endothelial glycocalyx is shed after contact with human serum. Experiments with several different types of transgenic porcine endothelial cells are currently ongoing to provide insight into the role of the different plasma cascade systems in the shedding of the glycocalyx.

Multitransgenic Porcine Fibroblasts are Protected from Immunoglobulin Binding and Complement Deposition in a Xeno-Microfluidic Model

Background One of the main issues that hamper xenotransplantation to be clinically feasible is humoral rejection. Xenoreactive antibodies, both natural or elicited, contribute significantly to rejection of xenotransplants by activation of complement and interactions with a variety of effector cells. Sugars – namely Gal-&agr;1,3-Gal, Neu5Gc and &bgr;4GalNT2 – expressed on the surface of endothelial cells are thought to play an important role. In this study, we tested the potential xenoprotective role of human complement regulators – hCD46, hCD55, hCD59 – combined with A20 (NF-&kgr;B inhibitor and anti-apoptotic protein) and HO-1 (oxidative stress-response protein) using a microfluidic model where porcine kidney fibroblasts (PKF) grown on artificial 3D microvessels were perfused with normal human serum (NHS). Cell lines lacking the expression of Gal-&agr;1,3-Gal (GGTA1 KO), Neu5Gc (CMAH KO) and &bgr;4GalNT2 (&bgr;4GalNT2 KO) sugars were also tested in order to assess the effects on complement activation and immunoglobulin binding. Methods PKF WT and transgenic were seeded on artificial microvessels with diameters of 550 &mgr;m fabricated in PDMS. The cells were cultured overnight under static condition to allow them to reach confluence. A peristaltic pump was then connected and the cells were perfused for 2 hours in a close loop with 5mL of 1:10 diluted NHS. Control samples were perfused with medium without NHS. The channels were washed with PBS, fixed and stained to detect complement deposition (C3b/c and C4b/c) and immunoglobulin binding (IgG and IgM). Microfluidic chips were analyzed under laser scanning confocal microscope. Five pictures/microchannel were acquired and the fluorescence intensity was quantified and plotted in a graph. Results Data showed that the absence of sugars from the cell surface of transgenic fibroblasts resulted in a significant decrease of antibody binding and antibody dependent complement deposition. Furthermore, the addition of 5 transgenes (hCD46, hCD55, hCD59, A20 and HO-1) to the GGTA1 KO background significantly improved the protection from complement deposition as shown by the immunofluorescence pictures and by the quantification. Experiments with PKF with triple KO together with the expression of the 5 trangenes are currently ongoing. Conclusions This study shows that immunoglobulin binding and complement deposition on transgenic PKF are strongly reduced when xenoantigens (GGTA1, CMAH, &bgr;4GalNT2) are removed from the cell surface. The addition of the 5 human transgenes further enhances the protection of the graft from complement. Transgenic pigs carrying such genetic modification might represent suitable donors for future pig to baboon preclinical xenotransplantation experiments. Furthermore, the study reveals the potential of our in vitro microfluidic system in screening the effects of different transgenes prior to the production of the respective pigs for in vivo experiments.

3D artificial round section micro-vessels to investigate endothelial cells under physiological flow conditions

In the context of xenotransplantation, in ischemia/reperfusion injury as well as in cardiovascular research, the study of the fascinating interplay between endothelial cells (EC) and the plasma cascade systems often requires in vitro models. Blood vessels are hardly reproducible with standard flat-bed culture systems and flow-plate assays are limited in their low surface-to-volume ratio which impedes the study of the anticoagulant properties of the endothelial cells. According to the 3R regulations (reduce, replace and refine animal experimentation) we developed a closed circuit microfluidic in vitro system in which endothelial cells are cultured in 3D round section microchannels and subjected to physiological, pulsatile flow. In this study, a 3D monolayer of porcine aortic EC was perfused with human serum to mimic a xenotransplantation setting. Complement as well as EC activation was assessed in the presence or absence of complement inhibitors showing the versatility of the model for drug testing. Complement activation products as well as E-selectin expression were detected and visualized in situ by high resolution confocal microscopy. Furthermore, porcine pro-inflammatory cytokines as well as soluble complement components in the recirculating fluid phase were detected after human serum perfusion providing a better overview of the artificial vascular environment.