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Ischemia–reperfusion injury (IRI) is a complex inflammatory process that detrimentally affects the function of transplanted organs. Neutrophils are important contributors to the pathogenesis of renal IRI. Signaling by G-CSF, a regulator of neutrophil development, trafficking, and function, plays a key role in several neutrophil-associated inflammatory disease models. In this study, we investigated whether targeting neutrophils with a neutralizing mAb to G-CSFR would reduce inflammation and protect against injury in a mouse model of warm renal IRI. Mice were treated with anti–G-CSFR 24 h prior to 22-min unilateral renal ischemia. Renal function and histology, complement activation, and expression of kidney injury markers, and inflammatory mediators were assessed 24 h after reperfusion. Treatment with anti–G-CSFR protected against renal IRI in a dose-dependent manner, significantly reducing serum creatinine and urea, tubular injury, neutrophil and macrophage infiltration, and complement activation (plasma C5a) and deposition (tissue C9). Renal expression of several proinflammatory genes (CXCL1/KC, CXCL2/MIP-2, MCP-1/CCL2, CXCR2, IL-6, ICAM-1, P-selectin, and C5aR) was suppressed by anti–G-CSFR, as was the level of circulating P-selectin and ICAM-1. Neutrophils in anti–G-CSFR–treated mice displayed lower levels of the chemokine receptor CXCR2, consistent with a reduced ability to traffic to inflammatory sites. Furthermore, whole transcriptome analysis using RNA sequencing showed that gene expression changes in IRI kidneys after anti–G-CSFR treatment were indistinguishable from sham-operated kidneys without IRI. Hence, anti–G-CSFR treatment prevented the development of IRI in the kidneys. Our results suggest G-CSFR blockade as a promising therapeutic approach to attenuate renal IRI.

The complement system is a potent mediator of ischemia–reperfusion injury (IRI), which detrimentally affects the function and survival of transplanted kidneys. Human complement receptor 1 (HuCR1) is an integral membrane protein that inhibits complement activation by blocking the convertases that activate C3 and C5. We have previously reported that CSL040, a truncated form of recombinant soluble HuCR1 (sHuCR1), has enhanced complement inhibitory activity and improved pharmacokinetic properties compared to the parent molecule. Here, we compared the capacity of CSL040 and full-length sHuCR1 to suppress complement-mediated organ damage in a mouse model of warm renal IRI. Mice were treated with two doses of CSL040 or sHuCR1, given 1 h prior to 22 min unilateral renal ischemia and again 3 h later. 24 h after reperfusion, mice treated with CSL040 were protected against warm renal IRI in a dose-dependent manner, with the highest dose of 60 mg/kg significantly reducing renal dysfunction, tubular injury, complement activation, endothelial damage, and leukocyte infiltration. In contrast, treatment with sHuCR1 at a molar equivalent dose to 60 mg/kg CSL040 did not confer significant protection. Our results identify CSL040 as a promising therapeutic candidate to attenuate renal IRI and demonstrate its superior efficacy over full-length sHuCR1 in vivo.

Deficiency in the membrane-bound complement regulators CD55 and CD59 exacerbates renal ischemia-reperfusion injury (IRI) in mouse models, but the effect of increasing CD55 and CD59 activity has not been examined. In this study, we investigated the impact of overexpression of human (h) CD55 ± hCD59 or treatment with soluble rhCD55 in a mouse model of renal IRI. Unilaterally nephrectomised mice were subjected to 18 (mild IRI) or 22 min (moderate IRI) warm renal ischemia, and analyzed 24 h after reperfusion for renal function (serum creatinine and urea), complement deposition (C3b/c and C9), and infiltration of neutrophils and macrophages. Transgenic mice expressing hCD55 alone were protected against mild renal IRI, with reduced creatinine and urea levels compared with wild type littermates. However, the renal function of the hCD55 mice was not preserved in the moderate IRI model, despite a reduction in C3b/c and C9 deposition and innate cell infiltration. Mice expressing both hCD55 and hCD59, on the other hand, were protected in the moderate IRI model, with significant reductions in all parameters measured. Wild type mice treated with rhCD55 immediately after reperfusion were also protected in the moderate IRI model. Thus, manipulation of CD55 activity to increase inhibition of the C3 and C5 convertases is protective against renal IRI, and the additional expression of hCD59, which regulates the terminal complement pathway, provides further protection. Therefore, anti-complement therapy using complement regulatory proteins may provide a potential clinical option for preventing tissue and organ damage in renal IRI.

Background Complement activation plays an important role in the pathogenesis of renal ischemia-reperfusion (IR) injury (IRI), but whether this involves damage to the vasculoprotective endothelial glycocalyx is not clear. We investigated the impact of complement activation on glycocalyx integrity and renal dysfunction in a mouse model of renal IRI. Methods Right nephrectomized male C57BL/6 mice were subjected to 22 minutes left renal ischemia and sacrificed 24 hours after reperfusion to analyze renal function, complement activation, glycocalyx damage, endothelial cell activation, inflammation, and infiltration of neutrophils and macrophages. Results Ischemia-reperfusion induced severe renal injury, manifested by significantly increased serum creatinine and urea, complement activation and deposition, loss of glycocalyx, endothelial activation, inflammation, and innate cell infiltration. Treatment with the anti-C5 antibody BB5.1 protected against IRI as indicated by significantly lower serum creatinine (P = 0.04) and urea (P = 0.003), tissue C3b/c and C9 deposition (both P = 0.004), plasma C3b (P = 0.001) and C5a (P = 0.006), endothelial vascular cell adhesion molecule-1 expression (P = 0.003), glycocalyx shedding (tissue heparan sulfate [P = 0.001], plasma syndecan-1 [P = 0.007], and hyaluronan [P = 0.02]), inflammation (high mobility group box-1 [P = 0.0003]), and tissue neutrophil (P = 0.0009) and macrophage (P = 0.004) infiltration. Conclusions Together, our data confirm that the terminal pathway of complement activation plays a key role in renal IRI and demonstrate that the mechanism of injury involves shedding of the glycocalyx.

BACKGROUND Transgenic expression of human thrombomodulin (hTBM), which has the potential to solve the problem of coagulation dysregulation in pig-to-primate xenotransplantation, may have additional benefits by neutralizing the proinflammatory cytokine high-mobility group box 1 (HMGB1). The aim of this study was to investigate HMGB1-mediated effects on porcine aortic endothelial cells (PAEC) from wild-type (WT) and hTBM transgenic pigs. METHODS Porcine aortic endothelial cells were treated with HMGB1, human (h)TNFα or lipopolysaccharide (LPS). Procoagulant and proinflammatory responses were assessed by measuring expression of cell surface markers (adhesion molecules, fibrinogen-like protein 2, plasminogen activator inhibitor (PAI)-1), secretion of porcine cytokines and chemokines (HMGB1, TNFα, IL-8, monocyte chemotactic protein-1), and formation of PAI-1/tissue plasminogen activator complexes. Thrombin-mediated degradation of HMGB1 in the presence of PAEC was examined by Western blot and functional assay. RESULTS High-mobility group box 1 potently activated WT PAEC, increasing the expression of E-selectin, vascular cell adhesion molecule-1, intercellular adhesion molecule-1, fibrinogen-like protein 2, and PAI-1, the secretion of TNFα, IL-8, and monocyte chemotactic protein-1 and the formation of PAI-1/tissue plasminogen activator complexes. Human TNFα- or LPS-induced activation of WT PAEC was inhibited by treatment with rabbit anti-HMGB1 antibody. Transgenic expression of hTBM significantly reduced the activation of PAEC by HMGB1 or hTNFα, and significantly enhanced thrombin-induced HMGB1 cleavage. Chemically induced shedding of the lectin-like domain of TBM resulted in significantly increased HMGB1-induced PAEC activation. CONCLUSIONS High-mobility group box 1 exerts powerful proinflammatory and procoagulant effects on WT PAEC, and appears to be an important downstream mediator for the actions of hTNFα and LPS. Human thrombomodulin transgenic PAECs are less sensitive to activation by either HMGB1 or hTNFα, an effect that appears to be dependent on the lectin-like domain of TBM.

Background: 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. Methods: Six pig forelimbs per group, respectively, stemming from either wild-type (wt) or HLA-E/hCD46 double-transgenic (tg) animals, were perfused ex vivo with heparinized human blood for 12hours. Blood samples were collected at defined time intervals, cell numbers counted, and peripheral blood mononuclear cells analyzed for phenotype by flow cytometry. Muscle biopsies were analyzed for NK cell infiltration. In vitro NK cytotoxicity assays were performed using pEC derived from wt and tg animals as target cells. Results: Ex vivo, a strong reduction in circulating human CD45 leukocytes was observed after 60minutes of xenoperfusion in both wt and tg limb groups. NK cell numbers dropped significantly. Within the first 10minutes, the decrease in NK cells was more significant in the wt limb perfusions as compared to tg limbs. Immunohistology of biopsies taken after 12hours showed less NK cell tissue infiltration in the tg limbs. In vitro, NK cytotoxicity against hCD46 single tg pEC and wt pEC was similar, while lysis of double tg HLA-E/hCD46 pEC was significantly reduced. Finally, circulating cells of pig origin were observed during the ex vivo xenoperfusions. These cells expressed phenotypes mainly of monocytes, B and T lymphocytes, NK cells, as well as some activated endothelial cells. Conclusion: sEx vivo perfusion of pig forelimbs using whole human blood represents a powerful tool to study humoral and early cell-mediated rejection mechanisms of vascularized pig-to-human xenotransplantation, although there are several limitations of the model. Here, we show that (i) transgenic expression of HLA-E/hCD46 in pig limbs provides partial protection from human NK cell-mediated xeno responses and (ii) the emergence of a pig cell population during xenoperfusions with implications for the immunogenicity of xenografts.

BACKGROUND:Asialoglycoprotein receptor 1 (ASGR1) mediates capture and phagocytosis of platelets in pig to primate liver xenotransplantation. However thrombocytopenia is also observed in xenotransplantation or xenoperfusion of other porcine organs than liver. We therefore assessed ASGR1 expression as well as ASGR1 mediated xenogeneic platelet phagocytosis in vitro and ex vivo on porcine aortic femoral arterial and liver sinusoidal endothelial cells (PAEC/PFAEC/PLSEC). METHODS:Porcine forelimbs were perfused with whole heparinized human or autologous pig blood. Platelets were counted at regular intervals. Pig limb muscle and liver as well as PAEC/PFAEC/PLSEC were characterized for ASGR1 expression. In vitro PAEC cultured on microcarrier beads and incubated with non anticoagulated human blood were used to study binding of human platelets and platelet white blood cell aggregation. Carboxyfluorescein diacetate succinimidyl ester labeled human platelets were exposed to PAEC/PFAEC/PLSEC and analyzed for ASGR1 mediated phagocytosis. RESULTS:Human platelet numbers decreased from 102 {\textpm} 33 at beginning to 13 {\textpm} 6 {\texttimes} 10/µL (P < 0.0001) after 10 minutes of perfusion whereas no significant decrease of platelets was seen during autologous perfusions (171 {\textpm} 26 to 122 {\textpm} 95 {\texttimes} 10/µL). The PAEC PFAEC and PLSEC all showed similar ASGR1 expression. In vitro no correlation was found between reduction in platelet count and platelet white blood cell aggregation. Phagocytosis of human carboxyfluorescein diacetate succinimidyl ester labeled platelets by PAEC/PFAEC/PLSEC peaked at 15 minutes and was inhibited (P < 0.05 to P < 0.0001) by rabbit anti ASGR1 antibody and asialofetuin. CONCLUSIONS:The ASGR1 expressed on aortic and limb arterial pig vascular endothelium plays a role in binding and phagocytosis of human platelets. Therefore ASGR1 may represent a novel therapeutic target to overcome thrombocytopenia associated with vascularized pig to primate xenotransplantation.

Acute vascular rejection (AVR), in particular microvascular thrombosis, is an important barrier to successful pig-to-primate xenotransplantation. Here, we report the generation of pigs with decreased tissue factor (TF) levels induced by small interfering (si)RNA-mediated gene silencing. Porcine fibroblasts were transfected with TF-targeting small hairpin (sh)RNA and used for somatic cell nuclear transfer. Offspring were analyzed for siRNA, TF mRNA and TF protein level. Functionality of TF downregulation was investigated by a whole blood clotting test and a flow chamber assay. TF siRNA was expressed in all twelve liveborn piglets. TF mRNA expression was reduced by 94.1 ± 4.7% in TF knockdown (TFkd) fibroblasts compared to wild-type (WT). TF protein expression in PAEC stimulated with 50 ng/mL TNF-α was significantly lower in TFkd pigs (mean fluorescence intensity TFkd: 7136 ± 136 vs. WT: 13 038 ± 1672). TF downregulation significantly increased clotting time (TFkd: 73.3 ± 8.8 min, WT: 45.8 ± 7.7 min, p

In vivo, endothelial cells are crucial for the natural anticoagulation of circulating blood. Consequently, endothelial cell activation leads to blood coagulation. This phenomenon is observed in many clinical situations, like organ transplantation in the presence of pre-formed anti-donor antibodies, including xenotransplantation, as well as in ischemia/reperfusion injury. In order to reduce animal experimentation according to the 3R standards (reduction, replacement and refinement), in vitro models to study the effect of endothelial cell activation on blood coagulation would be highly desirable. However, common flatbed systems of endothelial cell culture provide a surface-to-volume ratio of 1 - 5 cm(2) of endothelium per mL of blood, which is not sufficient for natural, endothelial-mediated anticoagulation. Culturing endothelial cells on microcarrier beads may increase the surface-to-volume ratio to 40 - 160 cm(2)/mL. This increased ratio is sufficient to ensure the "natural" anticoagulation of whole blood, so that the use of anticoagulants can be avoided. Here an in vitro microcarrier-based system is described to study the effects of genetic modification of porcine endothelial cells on coagulation of whole, non-anticoagulated human blood. In the described assay, primary porcine aortic endothelial cells, either wild type (WT) or transgenic for human CD46 and thrombomodulin, were grown on microcarrier beads and then exposed to freshly drawn non-anticoagulated human blood. This model allows for the measurement and quantification of cytokine release as well as activation markers of complement and coagulation in the blood plasma. In addition, imaging of activated endothelial cell and deposition of immunoglobulins, complement- and coagulation proteins on the endothelialized beads were performed by confocal microscopy. This assay can also be used to test drugs which are supposed to prevent endothelial cell activation and, thus, coagulation. On top of its potential to reduce the number of animals used for such investigations, the described assay is easy to perform and consistently reproducible.

Corline Heparin Conjugate (CHC), a compound of multiple unfractionated heparin chains, coats cells with a glycocalyx-like layer and may inhibit (xeno)transplant-associated activation of the plasma cascade systems. Here, we investigated the use of CHC to protect WT and genetically modified (GTKO.hCD46.hTBM) pig aortic endothelial cells (PAEC) in two pig-to-human in vitro xenotransplantation settings. Model 1: incubation of untreated or hTNFα-treated PAEC with 10% human plasma induced complement C3b/c and C5b-9 deposition, cellular activation and coagulation activation in WT and GTKO.hCD46.hTBM PAEC. Coating of untreated or hTNFα-treated PAEC with CHC (100 µg/ml) protected against human plasma-induced endothelial activation and damage. Model 2: PAEC were grown on microcarrier beads, coated with CHC, and incubated with non-anticoagulated whole human blood. Genetically modified PAEC significantly prolonged clotting time of human blood (115.0 ± 16.1 min, p

Source at: http://doi.org/10.1007/s00395-017-0610-9. Licensed CC BY-NC-ND 4.0. Inhibition of complement factor 5 (C5) reduced myocardial infarction in animal studies, while no benefit was found in clinical studies. Due to lack of cross-reactivity of clinically used C5 antibodies, different inhibitors were used in animal and clinical studies. Coversin (Ornithodoros moubata complement inhibitor, OmCI) blocks C5 cleavage and binds leukotriene B4 in humans and pigs. We hypothesized that inhibition of C5 before reperfusion will decrease infarct size and improve ventricular function in a porcine model of myocardial infarction. In pigs (sus scrofa), the left anterior descending coronary artery was occluded (40 min) and reperfused (240 min). Coversin or placebo was infused 20 min after occlusion and throughout reperfusion in 16 blindly randomized pigs. Coversin significantly reduced myocardial infarction in the area at risk by 39% (p=0.03, triphenyl tetrazolium chloride staining) and by 19% (p=0.02) using magnetic resonance imaging. The methods correlated significantly (R=0.92, p

BACKGROUND Besides α1,3-galactosyltransferase gene (GGTA1) knockout, several transgene combinations to prevent pig-to-human xenograft rejection are currently being investigated. In this study, the potential of combined overexpression of human CD46 and HLA-E to prevent complement- and NK-cell-mediated xenograft rejection was tested in an ex vivo pig-to-human xenoperfusion model. METHODS α1,3-Galactosyltransferase knockout heterozygous, hCD46/HLA-E double transgenic (transgenic) as well as wild-type pig forelimbs were ex vivo perfused with whole, heparinized human and autologous pig blood, respectively. Blood samples were analyzed for the production of porcine and/or human inflammatory cytokines as well as complement activation products. Biopsy samples were examined for deposition of human and porcine C3b/c, C4b/c, and C6 as well as CD62E (E-selectin) and CD106 (VCAM-1) expression. Apoptosis was measured in the porcine muscle tissue using TUNEL assays. Finally, the formation of thrombin-antithrombin (TAT) complexes was measured in EDTA plasma samples. RESULTS No hyperacute rejection was seen in this model. Extremity perfusions lasted for up to 12 h without increase in vascular resistance and were terminated due to continuous small blood losses. Plasma levels of porcine cytokines IL1β, IL-6, IL-8, IL-10, TNF-α, and MCP-1 as well as human complement activation markers C3a (P = 0.0002), C5a (P = 0.004), and soluble C5b-9 (P = 0.03) were lower in blood perfused through transgenic as compared to wild-type limbs. Human C3b/c, C4b/c, and C6 as well as CD62E and CD106 were deposited in tissue of wild-type limbs, but significantly lower levels (P

Background. Among other mismatches between human and pig incompatibilities in the blood coagulation systems hamper the xenotransplantation of vascularized organs. The provision of the porcine endothelium with human thrombomodulin (hTM) is hypothesized to overcome the impaired activation of protein C by a heterodimer con sisting of human thrombin and porcine TM. Methods. We evaluated regulatory regions of the THBD gene optimized vectors for transgene expression and generated hTM expressing pigs by somatic cell nuclear transfer. Genetically modified pigs were characterized at the molecular cellular histological and physiological levels. Results. A 7.6 kb fragment containing the entire upstream region of the porcine THBD gene was found to drive a high expression in a porcine endothelial cell line and was therefore used to control hTM expression in transgenic pigs. The abundance of hTM was restricted to the endothelium according to the predicted pattern and the trans gene expression of hTM was stably inherited to the offspring. When endothelial cells from pigs carrying the hTM transgeneVeither alone or in combination with an aGalTKO and a transgene encoding the human CD46Vwere tested in a coagulation assay with human whole blood the clotting time was increased three to four fold (PG0.001) compared to wild type and aGalTKO/CD46 transgenic endothelial cells. This for the first time demonstrated the anticoagulant properties of hTM on porcine endothelial cells in a human whole blood assay. Conclusions. The biological efficacy of hTM suggests that the (multi )transgenic donor pigs described here have the potential to overcome coagulation incompatibilities in pig to primate xenotransplantation. Keywords: Xenotransplantation Coagulation incompatibility Thrombomodulin Transgenic donors Human thrombomodulin Transgenic donor pigs.

Background. Natural IgM containing anti Gal antibodies initiates classic pathway complement activation in xenotrans plantation. However in ischemia reperfusion injury IgM also induces lectin pathway activation. The present study was therefore focused on lectin pathway as well as interaction of IgM and mannose binding lectin (MBL) in pig to human xenotransplantation models. Methods. Activation of the different complement pathways was assessed by cell enzyme linked immunosorbent assay using human serum on wild type (WT) and > galactosyl transferase knockout (GalTKO)/hCD46 transgenic porcine aortic endothelial cells (PAEC). Colocalization of MBL/MASP2 with IgM C3b/c C4b/c and C6 was investigated by immunofluorescence in vitro on PAEC and ex vivo in pig leg xenoperfusion with human blood. Influence of IgM on MBL binding to PAEC was tested using IgM depleted/repleted and anti Gal immunoabsorbed serum. Results. Activation of all the three complement pathways was observed in vitro as indicated by IgM C1q MBL and factor Bb deposition on WT PAEC. MBL deposition colocalized with MASP2 (Manders’ coefficient [3D] r2=0.93) C3b/c (r2=0.84) C4b/c (r2=0.86) and C6 (r2=0.80). IgM colocalized with MBL (r2=0.87) and MASP2 (r2=0.83). Human IgM led to dose dependently increased deposition of MBL C3b/c and C6 on WT PAEC. Colocalization of MBL with IgM (Pearson’s coefficient [2D] rp2=0.88) C3b/c (rp2=0.82) C4b/c (rp2=0.63) and C6 (rp2=0.81) was also seen in ex vivo xenoperfusion. Significantly reduced MBL deposition and complement activation was observed on GalTKO/hCD46 PAEC. Conclusion. Colocalization of MBL/MASP2 with IgM and complement suggests that the lectin pathway is activated by human anti Gal IgM and may play a pathophysiologic role in pig to human xenotransplantation. Keywords: Mannose binding lectin Complement pathways Natural antibody Xenotransplantation Porcine aortic endothelial cells.

Objective Ischemia-reperfusion (I/R) injury is a major clinical problem linked to vascular surgery. Currently, no drugs to prevent or to treat I/R injury are approved for clinical use. C1 inhibitor (C1 INH) is known to reduce activation of the plasma cascade systems that are involved in the pathophysiologic process of I/R injury. The aim of this study was therefore to investigate the effect of C1 INH on complement deposition and endothelial cell activation in a rat model of hind limb I/R injury. Methods Male Wistar rats (wild type, bred at the central animal facility, University of Bern), weighing 250 to 320 g, were used. The rats underwent 2-hour ischemia and 24-hour reperfusion by unilateral clamping of the femoral artery and additional use of a tourniquet. Five groups were divided according to intravenous treatment 5 minutes before ischemia: 50 IU/kg C1 INH (n = 5); 100 IU/kg C1 INH (n = 7); vehicle control (n = 5); nontreated control (n = 7); and normal, healthy control without intervention (n = 4). At the end, muscle edema, tissue viability, and histologic features were assessed. Deposition of immunoglobulin M, C1r, C4d, and fibrin and expression of plasminogen activator inhibitor 1, heparan sulfate (HS), E-selectin, and vascular cell adhesion molecule 1 were evaluated by fluorescence staining. In addition, high-mobility group box 1 protein was measured in plasma. Results Edema formation was reduced by C1 INH at two dosages, mirrored by improved histologic injury scores and preserved muscle viability. Deposition of immunoglobulin M, C4d, and fibrin was significantly decreased by 100 IU/kg C1 INH compared with nontreated controls. Pretreatment with 100 IU/kg C1 INH also significantly reduced HS shedding and expression of plasminogen activator inhibitor 1 as well as plasma levels of high-mobility group box 1 protein. Conclusions Pretreatment with both 50 and 100 IU/kg C1 INH attenuated reperfusion injury of rat hind limbs. Pretreatment with 100 IU/kg also preserved the endothelial HS layer as well as the natural, profibrinolytic phenotype of the endothelium. Prevention of endothelial cell activation by C1 INH may therefore be a promising strategy to prevent I/R injury in the clinical setting of peripheral vascular diseases and elective surgery on extremities.

AIMS To investigate a pressure-controlled intermittent coronary sinus occlusion (PICSO) system in an ischaemia/reperfusion model. METHODS AND RESULTS We randomly assigned 18 pigs subjected to 60 minutes ischaemia by left anterior descending (LAD) coronary artery balloon occlusion to PICSO (n=12, groups A and B) or to controls (n=6, group C). PICSO started 10 minutes before (group A), or 10 minutes after (group B) reperfusion and was maintained for 180 minutes. A continuous drop of distal LAD pressure was observed in group C. At 180 minutes of reperfusion, LAD diastolic pressure was significantly lower in group C compared to groups A and B (p=0.02). LAD mean pressure was significantly less than the systemic arterial mean pressure in group C (p=0.02), and the diastolic flow slope was flat, compared to groups A and B (p=0.03). IgG and IgM antibody deposition was significantly higher in ischaemic compared to non-ischaemic tissue in group C (p

Commercially available assays for the simultaneous detection of multiple inflammatory and cardiac markers in porcine blood samples are currently lacking. Therefore, this study was aimed at developing a bead-based, multiplexed flow cytometric assay to simultaneously detect porcine cytokines [interleukin (IL)-1β, IL-6, IL-10, and tumor necrosis factor alpha], chemokines (IL-8 and monocyte chemotactic protein 1), growth factors [basic fibroblast growth factor (bFGF), vascular endothelial growth factor, and platelet-derived growth factor-bb], and injury markers (cardiac troponin-I) as well as complement activation markers (C5a and sC5b-9). The method was based on the Luminex xMAP technology, resulting in the assembly of a 6- and 11-plex from the respective individual singleplex situation. The assay was evaluated for dynamic range, sensitivity, cross-reactivity, intra-assay and interassay variance, spike recovery, and correlation between multiplex and commercially available enzyme-linked immunosorbent assay as well as the respective singleplex. The limit of detection ranged from 2.5 to 30,000 pg/ml for all analytes (6- and 11-plex assays), except for soluble C5b-9 with a detection range of 2-10,000 ng/ml (11-plex). Typically, very low cross-reactivity (

Background Revascularization of amputated extremities after prolonged ischemia is complicated by reperfusion injury. We assessed ischemia/reperfusion (I/R) injury of porcine extremities after prolonged preservation using extracorporeal circulation (ECC). Methods Forelimbs of 32 pigs were divided into four groups based on ischemia times: group I: 6 h, group II: 12 h, group III: 0 h plus replantation, and group IV: 6 h plus replantation. Limbs were perfused with autologous blood using ECC for 12 h except group II with only 5 h perfusion. Limbs from groups III and IV were heterotopically replanted with a 7-d follow-up. Contralateral limbs served as controls in all groups. Tissue, plasma, and serum were analyzed for the extent of I/R injury. Results No significant differences in tissue wet/dry ratios were found within or between groups. This finding was confirmed by histology, except for an increased damage in group IV muscles compared with baseline (P = 0.016). Complement C3 deposition was only increased in group IV muscle (P = 0.031), group II nerves (P = 0.046), and group II vessels (P = 0.037). Group IV muscle and nerve tissues were the only ones with significant IgM antibody deposition (P = 0.031) at end of perfusion. Values were normal again after replantation. Reduced complement activity and elevated IL-6, IL-8, MCP-1, VEGF, PDGF-bb, bFGF, and complement split products were found during perfusion but were normal again after replantation. Staining for heparin sulfate proteoglycans and von Willebrand factor confirmed minimal activation of endothelial cells. Conclusion The results demonstrate that prolonged limb preservation using ECC has minimal impact on I/R-induced tissue injury. Extracorporeal perfusion is a potential limb-preserving technique encouraging further studies for use in limb revascularization.

Ischemia/reperfusion (I/R) injury has been extensively studied in organs such as heart, brain, liver, kidney, and lung. As a vascularized organ, bone is known to be susceptible to I/R injury too, but the respective mechanisms are not well understood to date. We therefore hypothesized that, similar to other organs, plasma cascade-induced inflammation also plays a role in bone I/R injury. Reperfusion injury in rat tibia was induced by unilateral clamping of the femoral artery and additional use of a tourniquet, while keeping the femoral vein patent to prevent venous congestion. Rats were subjected to 4h ischemia and 24h reperfusion. Deposition of complement fragment C3b/c and fibrin as well as expression of tissue factor (TF), tissue plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1), and E-selectin was detected by immunohistochemistry. In plasma, the levels of high mobility group box1 (HMGB1) were measured by ELISA. The total level of complement in serum was assessed by the CH50 test. Our results show that deposition of C3b/c was significantly increased with respect to healthy controls in cortical bone as well as in marrow of reperfused limbs. C3b/c deposition was also increased in cortical bone, but not in bone marrow, of contralateral limbs. Deposition of fibrin, as well as expression of PAI-1, was significantly increased in bone after ischemia and reperfusion, whereas expression of tPA was reduced. These differences were most prominent in vessels of bone, both in marrow and cortical bone, and both in reperfused and contralateral limbs. However, PAI-1, was only increased in vessels of reperfused cortical bone and there were no significant changes in expression of E-selectin. With respect to solid bone tissue, a significant increase of C3b/c and fibrin deposition was shown in osteocytes, and for fibrin also in the bone matrix, in both contralateral and reperfused cortical bone compared with normal healthy controls. A slight expression of TF was visible in osteocytes of the normal healthy control group, while TF was not present in the experimental groups. Moreover, CH50 values in serum decreased over time and HMGB1 was significantly increased in plasma of animals at the end of reperfusion. We conclude that ischemia and reperfusion of bone leads to activation of the complement and coagulation systems and a downregulation of the fibrinolytic cascade. In the acute phase, a vascular inflammation induced by activation of the plasma cascade systems also occurs in the bone. This is similar to I/R injury of other vascularized organs and tissues.

Calcium and iron overload participate in the mechanisms of ischemia/reperfusion (I/R) injury during myocardial infarction (MI). Calcium overload induces cardiomyocyte death by hypercontraction, while iron catalyses generation of reactive oxygen species (ROS). We therefore hypothesized that dexrazoxane, an intracellular metal chelator, would attenuate I/R injury. MI was induced in pigs by occlusion of the left anterior descending artery for 1 hour followed by 2 hours reperfusion. Thirty minutes before reperfusion either 5 mg/ml dexrazoxane (n = 5) or saline (n = 5) was infused intravenously. Myocardial necrosis as percentage of the area at ischemic risk was found to be similar in both groups (77.2 ± 18% for dexrazoxane and 76.4 ± 14% for saline group) as determined by triphenyl tetrazolium chloride staining of the ischemic myocardium. Also, serum levels of troponin-I were similar in both groups. A conductance catheter was used to measure left ventricular pressure and volume at all times. Markers for tissue damage due to ROS (HNE), endothelial cell activation (CD31) and inflammation (IgG, C3b/c, C5b9, MCP-1) were assessed on tissue and/or in serum. No significant differences were observed between the groups for the parameters analyzed. To conclude, in this clinically relevant model of early reperfusion after acute myocardial ischemia, dexrazoxane lacked attenuating effects on I/R injury as shown by the measured parameters.

Sepsis is an infection-induced systemic inflammatory syndrome, potentially causing organ failure. We previously showed attenuating effects on inflammation, thrombogenicity and haemodynamics by inhibiting the Toll-like receptor co-factor CD14 and complement factor C5 in a porcine Escherichia coli-induced sepsis model. The present study explored the effect on organ inflammation in these pigs. Tissue samples were examined from the combined treatment group (n = 8), the positive (n = 8) and negative (n = 6) control groups after 4h of sepsis. Inflammatory biomarkers were measured using ELISA, multiplex and qPCR analysis. Combined inhibition of C5 and CD14 markedly attenuated IL-1β by 31-66% (P < 0.05) and IL-6 by 54-96% (P < 0.01) in liver, kidney, lung and spleen; IL-8 by 65-100% in kidney, lung, spleen, and heart (P < 0.05) and MCP-1 by 46-69% in liver, kidney, spleen and heart (P < 0.05). Combined inhibition significantly attenuated tissue factor mRNA upregulation in spleen (P < 0.05) and IP-10 mRNA upregulation in four out of five organs. Finally, C5aR mRNA downregulation was prevented in heart and kidney (P < 0.05). Combined inhibition of C5 and CD14 thus markedly attenuated inflammatory responses in all organs examined. The anti-inflammatory effects observed in lung and heart may explain the delayed haemodynamic disturbances observed in septic pigs receiving combined inhibition of C5 and CD14.

BACKGROUND Dysregulation of the coagulation system due to inflammatory responses and cross-species molecular incompatibilities represents a major obstacle to successful xenotransplantation. We hypothesized that complement inhibition mediated by transgenic expression of human CD46 in pigs might also regulate the coagulation and fibrinolysis cascades and tested this in ex vivo human-to-pig xenoperfusions. METHODS Forelimbs of wild-type and hCD46/HLA-E double transgenic pigs were ex vivo xenoperfused for 12 hours with whole heparinized human blood. Muscle biopsies were stained for galactose-α1,3-galactose, immunoglobulin M, immunoglobulin G, complement, fibrin, tissue factor, fibrinogen-like protein 2, tissue plasminogen activator (tPA), and plasminogen activator inhibitor (PAI)-1. The PAI-1/tPA complexes, D-dimers, and prothrombin fragment F1 + 2 were measured in plasma samples after ex vivo xenoperfusion. RESULTS No differences of galactose expression or deposition of immunoglobulin M and immunoglobulin G were found in xenoperfused tissues of wild type and transgenic limbs. In contrast, significantly lower deposition of C5b-9 (P < 0.0001), fibrin (P = 0.009), and diminished expression of tissue factor (P = 0.005) and fibrinogen-like protein 2 (P = 0.028) were found in xenoperfused tissues of transgenic limbs. Levels of prothrombin fragment F1 + 2 (P = 0.031) and D-dimers (P = 0.044) were significantly lower in plasma samples obtained from transgenic as compared to wild-type pig limb perfusions. The expression of the fibrinolytic marker tPA was significantly higher (P = 0.009), whereas PAI-1 expression (P = 0.022) and PAI-1/tPA complexes in plasma (P = 0.015) were lower after transgenic xenoperfusion as compared to wild-type xenoperfusions. CONCLUSIONS In this human-to-pig xenoperfusion model, complement inhibition by transgenic hCD46 expression led to a significant inhibition of procoagulant and antifibrinolytic pathways.

Currently, systemic immunosuppression is used in vascularized composite allotransplantation (VCA). This treatment has considerable side effects and reduces the quality of life of VCA recipients. We loaded the immunosuppressive drug tacrolimus into a self-assembled hydrogel, which releases the drug in response to proteolytic enzymes that are overexpressed during inflammation. A one-time local injection of the tacrolimus-laden hydrogel significantly prolonged graft survival in a Brown Norway-to-Lewis rat hindlimb transplantation model, leading to a median graft survival of >100 days compared to 33.5 days in tacrolimus only-treated recipients. Control groups with no treatment or hydrogel only showed a graft survival of 11 days. Histopathological evaluation, including anti-graft antibodies and complement C3, revealed significantly reduced immune responses in the tacrolimus-hydrogel group compared with tacrolimus only. In conclusion, a single-dose local injection of an enzyme-responsive tacrolimus-hydrogel is capable of preventing VCA rejection for >100 days in a rat model and may offer a new approach for immunosuppression in VCA.

Background Besides α1,3 galactosyltransferase (Gal) gene knockout several transgene combinations to prevent pig-to-human xenograft rejection are being investigated. hCD46/HLA-E double transgenic pigs were tested for prevention of xenograft rejection in an ex vivo pig-to-human xenoperfusion model. In addition, expression of human thrombomodulin (hTM-) on wild-type and/or multi-transgenic (GalTKO/hCD46) background was evaluated to overcome pig-to-human coagulation incompatibility. Methods hCD46/HLA-E double transgenic as well as wild-type pig forelimbs were ex vivo perfused with whole, heparinized human blood and autologous blood, respectively. Blood samples were analyzed for production of porcine and/or human inflammatory cytokines. Biopsy samples were examined for deposition of complement proteins as well as E-selectin and VCAM-1 expression. Serial blood cell counts were performed to analyze changes in human blood cell populations. In vitro, PAEC were analyzed for ASGR1 mediated human platelet phagocytosis. In addition, a biochemical assay was performed using hTM-only and multi-transgenic (GalTKO/hCD46/hTM) pig aortic endothelial cells (PAEC) to evaluate the ability of hTM to generate activated protein C (APC). Subsequently, the anti-coagulant properties of hTM were tested in a microcarrier based coagulation assay with PAEC and human whole blood. Results No hyperacute rejection was seen in the ex vivo perfusion model. Extremity perfusions lasted for up to 12 h without increase of vascular resistance and had to be terminated due to continuous small blood losses. Plasma levels of porcine IL1β (P < 0.0001), and IL-8 (P = 0.019) as well as human C3a, C5a and soluble C5b-9 were significantly (P < 0.05–

Complement and the TLR family constitute two important branches of innate immunity. We previously showed attenuating effects on inflammation and thromogenicity by inhibiting the TLR coreceptor CD14 in porcine sepsis. In the present study, we explored the effect of the C5 and leukotriene B4 inhibitor Ornithodoros moubata complement inhibitor (OmCI; also known as coversin) alone and combined with anti-CD14 on the early inflammatory, hemostatic, and hemodynamic responses in porcine Escherichia coli–induced sepsis. Pigs were randomly allocated to negative controls (n = 6), positive controls (n = 8), intervention with OmCI (n = 8), or with OmCI and anti-CD14 (n = 8). OmCI ablated C5 activation and formation of the terminal complement complex and significantly decreased leukotriene B4 levels in septic pigs. Granulocyte tissue factor expression, formation of thrombin–antithrombin complexes (p < 0.001), and formation of TNF-α and IL-6 (p < 0.05) were efficiently inhibited by OmCI alone and abolished or strongly attenuated by the combination of OmCI and anti-CD14 (p < 0.001 for all). Additionally, the combined therapy attenuated the formation of plasminogen activator inhibitor-1 (p < 0.05), IL-1β, and IL-8, increased the formation of IL-10, and abolished the expression of wCD11R3 (CD11b) and the fall in neutrophil cell count (p < 0.001 for all). Finally, OmCI combined with anti-CD14 delayed increases in heart rate by 60 min (p < 0.05) and mean pulmonary artery pressure by 30 min (p < 0.01). Ex vivo studies confirmed the additional effect of combining anti-CD14 with OmCI. In conclusion, upstream inhibition of the key innate immunity molecules, C5 and CD14, is a potential broad-acting treatment regimen in sepsis as it efficiently attenuated inflammation and thrombogenicity and delayed hemodynamic changes.

Commercially available assays for the simultaneous detection of multiple inflammatory and cardiac markers in porcine blood samples are currently lacking. Therefore, this study was aimed at developing a bead-based, multiplexed flow cytometric assay to simultaneously detect porcine cytokines [interleukin (IL)-1β, IL-6, IL-10, and tumor necrosis factor alpha], chemokines (IL-8 and monocyte chemotactic protein 1), growth factors [basic fibroblast growth factor (bFGF), vascular endothelial growth factor, and platelet-derived growth factor-bb], and injury markers (cardiac troponin-I) as well as complement activation markers (C5a and sC5b-9). The method was based on the Luminex xMAP technology, resulting in the assembly of a 6- and 11-plex from the respective individual singleplex situation. The assay was evaluated for dynamic range, sensitivity, cross-reactivity, intra-assay and interassay variance, spike recovery, and correlation between multiplex and commercially available enzyme-linked immunosorbent assay as well as the respective singleplex. The limit of detection ranged from 2.5 to 30,000 pg/ml for all analytes (6- and 11-plex assays), except for soluble C5b-9 with a detection range of 2-10,000 ng/ml (11-plex). Typically, very low cross-reactivity (3% and1.4% by 11- and 6-plex, respectively) between analytes was found. Intra-assay variances ranged from 4.9 to 7.4% (6-plex) and 5.3 to 12.9% (11-plex). Interassay variances for cytokines were between 8.1 and 28.8% (6-plex) and 10.1 and 26.4% (11-plex). Correlation coefficients with singleplex assays for 6-plex as well as for 11-plex were high, ranging from 0.988 to 0.997 and 0.913 to 0.999, respectively. In this study, a bead-based porcine 11-plex and 6-plex assay with a good assay sensitivity, broad dynamic range, and low intra-assay variance and cross-reactivity was established. These assays therefore represent a new, useful tool for the analysis of samples generated from experiments with pigs.

The heterotopic thoracic pig-to-baboon heart transplantation has been established by our group as a safe preclinical model. Since the recipient's own heart remains in place, it is possible to evaluate immunological reactions of various types and the symptoms of the thrombotic microangiopathy under working heart conditions. However, these experiments are complex and costly. In order to limit the number of transplants, we tested our multi-transgenic porcine donor hearts in an ex-vivo perfusion model first. Ex-vivo model: Both beating ventricles were perfused with heparinized freshly drawn human whole blood using a centrifugal pump and a membrane oxygenator. During 3 h of observation, cardiac function parameters were obtained continuously; specimens from the perfusate, coronary sinus blood, and myocardial biopsies were assessed. Various genetically modified porcine donor hearts were tested; we are currently evaluating the impact of human thrombomodulin on the porcine microcirculation; the efficacy of complement regulation is another work package. Pig-to-baboon heterotopic cardiac xenotransplantation: In our latest group, seven baboons received five double (Gal-KO/hCD46), and two triple (Gal-KO/hCD46/hTM resp./HLA-E) transgenic pig hearts. Our immunosuppressive regimen included preoperative anti-CD20-antibody, bortezomib, dexamethasone and cyclophosphamide; postoperatively, ATG, tacrolimus, MMF, anti-CD20-antibody, bortezomib and dexamethasone were administered. Total lymph node irradiation (6 Gy) was applied on postoperative day five. The triple transgenic hearts survived 35 and 37 days respectively. Both hearts maintained function throughout the experiment. Both recipients succumbed to fungal infections. Humoral rejection was seen only once. The next barrier appears to be the complex event of thrombotic microangiopathy that needs to be addressed with the additional expression of human thrombomodulin strategies.

In vitro, interactions between human NK cells and porcine endothelial cells (pEC) are characterized by NK cell recruitment and cytotoxicity. NK cells play a role in allo- and xenotransplantation due to the incompatibility of the MHC class I molecules expressed on transplanted cells and tissues on one hand, and the MHC class I specific inhibitory NK receptors of the recipient on the other hand. In this context we published previously that: (i) the expression of human HLA-E on pEC partially protects from polyclonal huNK cytotoxicity and completely protects from killing by NKG2Abright NK clones in vitro; but does not affect the adhesion of huNK cells to pEC or the heteroconjugate formation between huNK and porcine cells (1, 2); (ii) lymphoblasts and pEC derived from HLA-E/human beta2microglobulin transgenic pigs are effectively protected against huNK cell-mediated cytotoxicity and inhibit the secretion of IFN-gamma by co-cultured huNK cells in vitro, depending on CD94/NKG2A expression on the NK cells (3); (iii) HLA-G expression on pEC protects partially against direct huNK cytotoxicity but not against ADCC (4); (iv) HLA-G expression inhibits rolling adhesion of activated huNK cells on pEC (5, 6); (v) HLA-Cw3 expression on pEC protects against NK cell-mediated cytotoxicity (7), HLA-Cw4 reduced NK cell adhesion and cytotoxicity; HLA-Cw4 and HLA-Cw3 co-expression is not sufficient to completely overcome NK cytotoxicity via recognition of the KIR CD158a (KIR2DL1) and CD158b (KIR2DL2/3) receptors (8). The aim of this study was to evaluate NK cell recruitment and infiltration of porcine tissues using an ex vivo perfusion system of HLA-E transgenic pig legs with human blood. Over the past year, a pilot study has been set up in order to establish an experimental protocol that would allow for the study and the evaluation of the rejection mechanisms occurring during xenotransplantation and especially following reperfusion of pig organs with human blood (9). Amputated pig legs were perfused with heparinised human blood over a period of 12h. Blood samples were collected at several time points and blood cell populations were analyzed by flow cytometry (FACS) analysis. Two wild type and 2 HLA-E/CD46 transgenic pig legs were perfused ex vivo. A strong diminution of NK cells was observed after 7 h of perfusion in both the wild type and the transgenic legs. However, this decrease occurred earlier in the wild type leg than in the transgenic. In addition, the apparition of a SLA-I+ cells population of pig origin of higher granularity than the lymphocyte population over time was observed. We hypothesized that these cells could be pig endothelial cells detached from the vessel wall due to xenorejection, or alternatively originating from the pig bone marrow. These results confirmed previous in vitro studies demonstrating that pEC damage was mediated by human NK cells. Moreover, the expression of HLA-E/CD46 provided a partial protection with regard to NK cell recruitment and tissue infiltration. From these preliminary experiments the following conclusions were drawn: Pig leg perfusion with human blood was feasible for up to 12 h with stable perfusion parameters thus extending the observation time compared to previously described pig kidney, lung and beating heart perfusion systems. pH and potassium were maintained at normal levels and muscle stimulation resulted in contraction throughout the entire perfusion. Thus the pig leg reperfusion system appears to be a stable and optimized system to study rejection mechanisms over several hours in the pig-to-human xenotransplantation setting. This system allows for the study of the dynamics of the different cell populations in the blood. Indeed, a strong diminution of NK, NKT and T cells was observed. In addition, this system enables to study the apparition of cells of pig origin in the circulating blood. In conclusion, this system represents a powerful tool to study the basic molecular mechanisms taking place in a setting of xenotransplantation, and in particular to evaluate the protection from early cell-mediated rejection mechanisms in tissues originating from transgenic pigs. References: Forte P, Lilienfeld BG, Baumann BC, Seebach JD. Human NK cytotoxicity against porcine cells is triggered by NKp44 and NKG2D. J Immunol 2005; 175: 5463–5470. Lilienfeld BG, Crew MD, Forte Pet al.Transgenic expression of HLA-E single chain trimer protects porcine endothelial cells against human natural killer cell-mediated cytotoxicity. Xenotransplantation 2007; 14: 126–134. Weiss EH, Lilienfeld BG, Muller Set al. HLA-E/human beta2-microglobulin transgenic pigs: protection against xenogeneic human anti-pig natural killer cell cytotoxicity. Transplantation 2009; 87: 35–43. Seebach JD, Pazmany L, Waneck GLet al. HLA-G expression on porcine endothelial cells protects partially against direct human NK cytotoxicity but not against ADCC. Transplant Proc 1999; 31: 1864–1865. Forte P, Matter-Reissmann UB, Strasser Met al.Porcine aortic endothelial cells transfected with HLA-G are partially protected from xenogeneic human NK cytotoxicity. Hum.Immunol 2000; 61:1066–1073. Forte P, Pazmany L, Matter-Reissmann UBet al. HLA-G inhibits rolling adhesion of activated human NK cells on porcine endothelial cells. J Immunol. 2001; 167: 6002–6008. Seebach JD, Comrack C, Germana Set al. HLA-Cw3 expression on porcine endothelial cells protects against xenogeneic cytotoxicity mediated by a subset of human NK cells. J Immunol 1997; 159: 3655–3661. Forte P, Baumann BC, Schneider MKet al. HLA-Cw4 expression on porcine endothelial cells reduces cytotoxicity and adhesion mediated by CD158a+ human NK cells. Xenotransplantation 2009; 16: 19–26. Constantinescu MA, Knall E, Xu Xet al. Preservation of amputated extremities by extracorporeal blood perfusion; a feasibility study in a porcine model. J Surg.Res 2010.

Background: The classical pathway is the dominant initiator of complement activation in xenotransplantation. By amplification of C3b generation, the alternative pathway is also critical. However, little attention has been paid up to date to the involvement of the lectin pathway in xenograft rejection. Natural IgM, containing anti-Gal, is a major initiator of classical pathway complement activation, but recently it has been shown that during ischemia/reperfusion injury, IgM also induces lectin pathway activation. Thus, the present study was focused on lectin pathway as well as interaction of IgM and MBL in a pig-to-human in vitro xenotransplantation model. Methods: Cell ELISA using porcine aortic endothelial cells (PAEC) and normal human serum (NHS) was used to assess activation of the different complement pathways. To confirm activation of the lectin pathway and to analyze the role of natural IgM in it's activation, co-localized deposition of MBL/MASP2 with C3b/c, C4b/c & C6 and IgM with MBL & MASP2 was investigated by immunofluorescence (IF)/confocal microscopy on PAEC. Influence of IgM presence on MBL binding to PAEC was tested using IgM depleted/repleted and anti-Gal immunoabsorbed NHS. Finally, tissue samples from ex vivo xenoperfusion of pig limbs with whole human blood were tested for IgM mediated lectin pathway activation by IF staining. Results: Activation of all the three pathways of complement system was observed in vitro as indicated by IgM, C1q, MBL and Factor Bb binding on PAEC. MBL deposition was co-localized with MASP2, C3b/c, C4b/c and C6, suggesting a predominant role of the lectin pathway in xenograft rejection. IgM co-localization with MBL and MASP2 as well as dose-dependently increased deposition of MBL on PAEC in the presence of human polyclonal IgM, further supports the idea that upon deposition of IgM a binding site for MBL is exposed. In addition, co-localized deposition of MBL with IgM, C4b/c and C6 was also observed on ex vivo xenoperfusion samples. Conclusion: The lectin pathway of complement activation was shown to be involved in xenotransplantation. Co-localization of MBL / MASP2 with IgM and complement proteins indicate that lectin pathway activation in xenotransplantation is dependent on antigen recognition by naturalIgM. These findings suggest that, similar to ischemia/reperfusion injury, the lectin pathway has a functional role in endothelial damage in xenotransplantation.