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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: 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.

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

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

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 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.

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.