Anjan K. Bongoni

A single localized dose of enzyme-responsive hydrogel improves long-term survival of a vascularized composite allograft

Tacrolimus-encapsulated hydrogels improve long-term survival of a vascularized composite allograft. A Leg Up for Transplant A one-shot delivery can enhance transplant survival in rats. Gajanayake et al. report that tacrolimus-encapsulated hydrogels improve long-term survival of a vascularized composite allograft (VCA). VCA is a promising alternative to reconstructive surgery or prosthesis use. However, transplant recipients must undergo systemic long-term immunosuppression, which increases their susceptibility to infection, kidney disease, and certain cancers. The authors encapsulated the immunosuppressive tacrolimus in a hydrogel composed of a material FDA classified as generally recognized as safe that was degraded by inflammatory enzymes. The tacrolimus-laden hydrogel increased graft survival time with one injection in a rat hindlimb transplant model. If reproduced in people, this delivery method could increase patient compliance and potentially decrease toxicity in transplant recipients. 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.

Regulatory sequences of the porcine THBD gene facilitate endothelial-specific expression of bioactive human thrombomodulin in single- and multitransgenic pigs.

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 consisting 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 transgene expression of hTM was stably inherited to the offspring. When endothelial cells from pigs carrying the hTM transgene—either alone or in combination with an aGalTKO and a transgene encoding the human CD46—were tested in a coagulation assay with human whole blood, the clotting time was increased three- to four-fold (P<0.001) 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.

Combined Inhibition of Complement (C5) and CD14 Markedly Attenuates Inflammation, Thrombogenicity, and Hemodynamic Changes in Porcine Sepsis

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.

siRNA mediated knockdown of tissue factor expression in pigs for 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 < 0.0001) and significantly decreased thrombus formation compared to WT (mean thrombus coverage per viewing field in %; WT: 23.5 ± 13.0, TFkd: 2.6 ± 3.7, p < 0.0001). Our data show that a functional knockdown of TF is compatible with normal development and survival of pigs. TF knockdown could be a valuable component in the generation of multi‐transgenic pigs for xenotransplantation.

Development of a bead-based multiplex assay for the simultaneous detection of porcine inflammation markers using xMAP technology.

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% and <1.4% 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.

Complement dependent early immunological responses during ex vivo xenoperfusion of hCD46/HLA‐E double transgenic pig forelimbs with human blood

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.

Porcine extrahepatic vascular endothelial asialoglycoprotein receptor 1 mediates xenogeneic platelet phagocytosis in vitro and in human-to-pig ex vivo xenoperfusion

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 ± 33 at beginning to 13 ± 6 × 103/&mgr;L (P < 0.0001) after 10 minutes of perfusion, whereas no significant decrease of platelets was seen during autologous perfusions (171 ± 26 to 122 ± 95 × 103/&mgr;L). 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.

Activation of the lectin pathway of complement in pig-to-human xenotransplantation models

Background Natural IgM containing anti-Gal antibodies initiates classic pathway complement activation in xenotransplantation. 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 &agr;-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.

Effect of pressure-controlled intermittent coronary sinus occlusion (PICSO) on myocardial ischaemia and reperfusion in a closed-chest porcine model

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<0.05). Significantly more haemorrhagic lesions were seen in the ischaemic myocardium of group C, compared to groups A and B (p=0.002). The necrotic area differed non-significantly among groups. CONCLUSIONS PICSO was safe and effective in improving coronary perfusion pressure and reducing antibody deposition consistent with reduced microvascular obstruction and ischaemia/reperfusion injury.

Organ inflammation in porcine Escherichia coli sepsis is markedly attenuated by combined inhibition of C5 and CD14.

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.