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We describe the incidence of early graft failure ( EGF, defined as loss of function from any cause within 3 days after transplant) in a large cohort of Gal TKO pig organs transplanted into baboons in three centers, and the effect of additional expression of a human complement pathway-regulatory protein, CD46 or CD55 (Gal TKO. hCPRP). Baboon recipients of life-supporting Gal TKO kidney (n = 7) or heterotopic heart (n = 14) grafts received either no immunosuppression (n = 4), or one of several partial or full immunosuppressive regimens (n = 17). Fourteen additional baboons received a Gal TKO. hCPRP kidney (n = 5) or heart (n = 9) and similar treatment regimens. Immunologic, pathologic, and coagulation parameters were measured at frequent intervals. EGF of Gal TKO organs occurred in 9/21 baboons (43%). hCPRP expression reduced the Gal TKO EGF incidence to 7% (1/14; P < 0.01 vs. Gal TKO alone). At 30 mins, complement deposits were more intense in organs in which EGF developed (P < 0.005). The intensity of peri-transplant platelet activation (as β-thromboglobulin release) correlated with EGF, as did the cumulative coagulation score (P < 0.01). We conclude that (i) the transgenic expression of a hCPRP on the vascular endothelium of a Gal TKO pig reduces the incidence of EGF and reduces complement deposition, (ii) complement deposition and platelet activation correlate with early Gal TKO organ failure, and (iii) the expression of a hCPRP reduces EGF but does not prevent systemic coagulation activation. Additional strategies will be required to control coagulation activation. [ABSTRACT FROM AUTHOR]

Background Scientists working in the field of xenotransplantation do not employ a uniform method to measure and report natural and induced antibody responses to non- Galα(1,3) Gal (non- Gal) epitopes. Such humoral responses are thought to be particularly pathogenic after transplantation of vascularized Gal TKO pig organs and having a more uniform assay and reporting format would greatly facilitate comparisons between laboratories. Methods Flow cytometry allows examination of antibody reactivity to intact antigens in their natural location and conformation on cell membranes. We have established a simple and reproducible flow cytometric assay to detect antibodies specific for non- Gal pig antigens using primary porcine aortic endothelial cells ( pAECs) and cell culture-adapted pAEC cell lines generated from wild type and α1,3galactosyl transferase knockout (Gal TKO) swine. Results The consensus protocol we propose here is based on procedures routinely used in four xenotransplantation centers and was independently evaluated at three sites using shared cells and serum samples. Our observation support use of the cell culture-adapted Gal TKO pAEC KO:15502 cells as a routine method to determine the reactivity of anti-non- Gal antibodies in human and baboon serum. Conclusions We have developed an assay that allows the detection of natural and induced non-Gal xenoreactive antibodies present in human or baboon serum in a reliable and consistent manner. This consensus assay and format for reporting the data should be accessible to laboratories and will be useful for assessing experimental results between multiple research centers. Adopting this assay and format for reporting the data should facilitate the detection, monitoring, and detailed characterization of non- Gal antibody responses. [ABSTRACT FROM AUTHOR]

The histopathology of cardiac xenograft rejection has evolved over the last 20 yr with the development of new modalities for limiting antibody-mediated injury, advancing regimens for immune suppression, and an ever-widening variety of new donor genetics. These new technologies have helped us progress from what was once an overwhelming anti-Gal-mediated hyperacute rejection to a more protracted anti-Gal-mediated vascular rejection to what is now a more complex manifestation of non-Gal humoral rejection and coagulation dysregulation. This review summarizes the changing histopathology of Gal- and non-Gal-mediated cardiac xenograft rejection and discusses the contributions of immune-mediated injury, species-specific immune-independent factors, transplant and therapeutic procedures, and donor genetics to the overall mechanism(s) of cardiac xenograft rejection. [ABSTRACT FROM AUTHOR]

Myeloid derived suppressor cells (MDSC) are a heterogeneous population of hematopoietic derived cell precursors that can suppress immune responses in a variety of inflammatory settings. Here we review recent studies detailing expansion of phenotypically and functionally disparate MDSC. Findings related to MDSC accumulation, activation, and mechanisms utilized in immune suppression are presented. Further, we discuss recent reports that suggest MDSC are expanded during transplantation and that modulation of MDSC can participate in preventing graft rejection. [ABSTRACT FROM AUTHOR]

Nguyen B-NH, Azimzadeh AM, Schroeder C, Buddensick T, Zhang T, Laaris A, Cochrane M, Schuurman H-J, Sachs DH, Allan JS, Pierson RN. Absence of Gal epitope prolongs survival of swine lungs in an ex vivo model of hyperacute rejection. Xenotransplantation 2011; 18: 94-107. © 2011 John Wiley & Sons A/S. Galactosyl transferase gene knock-out (GalTKO) swine offer a unique tool to evaluate the role of the Gal antigen in xenogenic lung hyperacute rejection. We perfused GalTKO miniature swine lungs with human blood. Results were compared with those from previous studies using wild-type and human decay-accelerating factor-transgenic (hDAF) pig lungs. GalTKO lungs survived 132 ± 52 min compared to 10 ± 9 min for wild-type lungs (P = 0.001) and 45 ± 60 min for hDAF lungs (P = 0.18). GalTKO lungs displayed stable physiologic flow and pulmonary vascular resistance (PVR) until shortly before graft demise, similar to autologous perfusion, and unlike wild-type or hDAF lungs. Early (15 and 60 min) complement (C3a) and platelet activation and intrapulmonary platelet deposition were significantly diminished in GalTKO lungs relative to wild-type or hDAF lungs. However, GalTKO lungs adsorbed cytotoxic anti-non-Gal antibody and elaborated high levels of thrombin; their demise was associated with increased PVR, capillary congestion, intravascular thrombi and strong CD41 deposition not seen at earlier time points. In summary, GalTKO lungs are substantially protected from injury but, in addition to anti-non-Gal antibody and complement, platelet adhesion and non-physiologic intravascular coagulation contribute to Gal-independent lung injury mechanisms. [ABSTRACT FROM AUTHOR]

Chronic rejection currently limits the long-term efficacy of clinical transplantation. Although B cells have recently been shown to play a pivotal role in the induction of alloimmunity and are being targeted in other transplant contexts, the efficacy of preemptive B cell depletion to modulate alloimmunity or attenuate cardiac allograft vasculopathy (CAV) (classic chronic rejection lesions found in transplanted hearts) in a translational model has not previously been described. We report here that the CD20-specific antibody (alphaCD20) rituximab depleted CD20+ B cells in peripheral blood, secondary lymphoid organs, and the graft in cynomolgus monkey recipients of heterotopic cardiac allografts. Furthermore, CD20+ B cell depletion therapy combined with the calcineurin inhibitor cyclosporine A (CsA) prolonged median primary graft survival relative to treatment with alphaCD20 or CsA alone. In animals treated with both alphaCD20 and CsA that achieved efficient B cell depletion, alloantibody production was substantially inhibited and the CAV severity score was markedly reduced. We conclude therefore that efficient preemptive depletion of CD20+ B cells is effective in a preclinical model to modulate pathogenic alloimmunity and to attenuate chronic rejection when used in conjunction with a conventional clinical immunosuppressant. This study suggests that use of this treatment combination may improve the efficacy of transplantation in the clinic. [ABSTRACT FROM AUTHOR]

Abstract: The need to assess heart failure at an early stage highlights the importance of accurate microarray analysis using small tissue samples. To test our ability to obtain high quality RNA from biopsy-sized cardiac specimens, amplification was performed on RNA from biopsy-sized samples of left ventricle (LV) tissue from one explanted failing human heart and one non-failing heart. Two methods were used: one-cycle (1C) amplification of 1.6 μg of RNA, and two-cycle (2C) amplification of 50 ng of RNA. The resulting cRNA was hybridized to Affymetrix GeneChip® arrays. Over 65% of all differentially expressed genes for failing vs non-failing hearts were concordant between 1C and 2C RNA amplification. Differentially expressed genes between 1C and 2C RNA amplification in our study were highly correlated (R 2 = 0.957 and changes in gene expression agreed with prior studies on genes and heart failure; e.g., decreased α-myosin heavy chain and α-tropomyosin, as well as increased expression of insulin-like growth factor). Two cycles of amplification from cardiac biopsies will permit accurate transcription profiling of heart failure at pre-symptomatic stages. Ability to measure gene expression from nanogram amounts of RNA will provide new opportunities to predict progression to symptomatic heart failure, and to identify potential targets for therapy. [Copyright &y& Elsevier]

Introduction: Macaque species offer a valuable model for translational allo-transplantation and tolerance studies. Cardiac allograft vasculopathy in Macaca fascicularis is associated with elaboration of anti-donor antibodies. Since T-independent pathways of B cell activation have been described, and anti-B cell strategies have proven to be a fruitful tolerogenic adjunct in rodent and xenogenic models, here we investigate whether an anti-CD20 antibody (rituximab) would be useful to deplete B-cells in a preclinical allo-transplantation setting in macaques. Methods: Three cynomolgus macaques which had previously rejected a cardiac allograft and one with concurrent subacute vascular rejection were treated weekly with rituximab 20 mg/kg IV for 4 and 2 weeks, respectively. B-cell levels (CD19 + cells) were measured by flow cytometry in peripheral blood, spleen, lymph node and bone marrow cells at various intervals after initiation of treatment. B-cells and plasma cells were also analyzed by immunohistochemistry at necropsy in spleen, lymph node, tonsil and thymus tissue sections. Anti-donor antibody titers were measured by flow cytometry. Results: B-cells expressing CD19 were not detectable in the peripheral blood in any animal within 24 h after initial treatment, or over the ensuing month. At necropsy, the germinal centers in spleen and lymph node were completely depleted of CD20 + B-cells in 2 animals, leaving a hypocellular trabecular pattern around preserved plasma cell follicles. Substantial but incomplete depletion of B-cells was demonstrated in the other 2 animals, in each instance immunohistochemical findings in spleen and lymph node exhibiting higher sensitivity for residual B-cells compared to FACS. Anti-donor antibody titers exhibited kinetics similar to untreated animals over this short follow-up. Comment: Treatment with anti-CD20 very efficiently depletes peripheral and tissue B-cells but not plasma cells in this macaque species. Biopsy of lymph node is necessary and may be sufficient to assess B-cell clearance in secondary lymphoid organs in this model. [ABSTRACT FROM AUTHOR]

The authors discuss research on the effect of pre-emptive treatment with maturation-resistant monocyte-dervied donor dendritic cells (DC) on kidney allograft outcome in nonhuman primates. They reference the study "Regulatory Dendritic Cell Infusion Prolongs Kidney Allograft Survival in Nonhuman Primates" by M. B. Ezzelerab published in the "American Journal of Transplantation" in 2013. They believe that the work provides evidence of the immunosuppressive efficacy of modified dendritic cells.

Thrombomodulin is important for the production of activated protein C (APC), a molecule with significant regulatory roles in coagulation and inflammation. To address known molecular incompatibilities between pig thrombomodulin and human thrombin that affect the conversion of protein C into APC, GalTKO.hCD46 pigs have been genetically modified to express human thrombomodulin (hTBM). The aim of this study was to evaluate the impact of transgenic hTBM expression on the coagulation dysregulation that is observed in association with lung xenograft injury in an established lung perfusion model, with and without additional blockade of nonphysiologic interactions between pig vWF and human GPIb axis. Expression of hTBM was variable between pigs at the transcriptional and protein level. hTBM increased the activation of human protein C and inhibited thrombosis in an in vitro flow perfusion assay, confirming that the expressed protein was functional. Decreased platelet activation was observed during ex vivo perfusion of GalTKO.hCD46 lungs expressing hTBM and, in conjunction with transgenic hTBM, blockade of the platelet GPIb receptor further inhibited platelets and increased survival time. Altogether, our data indicate that expression of transgenic hTBM partially addresses coagulation pathway dysregulation associated with pig lung xenograft injury and, in combination with vWF‐GP1b‐directed strategies, is a promising approach to improve the outcomes of lung xenotransplantation. [ABSTRACT FROM AUTHOR]

Introduction: Expression of human complement pathway regulatory proteins (hCPRP's) such as CD46 or CD55 has been associated with improved survival of pig organ xenografts in multiple different models. Here we evaluate the hypothesis that an increased human CD46 gene dose, through homozygosity or additional expression of a second hCPRP, is associated with increased protein expression and with improved protection from injury when GTKO lung xenografts are perfused with human blood. Methods: Twenty three GTKO lungs heterozygous for human CD46 (GTKO.heteroCD46), 10 lungs homozygous for hCD46 (GTKO.homoCD46), and six GTKO.homoCD46 lungs also heterozygous for hCD55 (GTKO.homoCD46.hCD55) were perfused with human blood for up to 4 h in an ex vivo circuit. Results: Relative to GTKO.heteroCD46 (152 min, range 5–240; 6/23 surviving at 4 h), survival was significantly improved for GTKO.homoCD46 (>240 min, range 45–240, p =.034; 7/10 surviving at 4 h) or GTKO.homoCD46.hCD55 lungs (>240 min, p =.001; 6/6 surviving at 4 h). Homozygosity was associated with increased capillary expression of hCD46 (p <.0001). Increased hCD46 expression was associated with significantly prolonged lung survival (p =.048),) but surprisingly not with reduction in measured complement factor C3a. Hematocrit, monocyte count, and pulmonary vascular resistance were not significantly altered in association with increased hCD46 gene dose or protein expression. Conclusion: Genetic engineering approaches designed to augment hCPRP activity — increasing the expression of hCD46 through homozygosity or co‐expressing hCD55 with hCD46 — were associated with prolonged GTKO lung xenograft survival. Increased expression of hCD46 was associated with reduced coagulation cascade activation, but did not further reduce complement activation relative to lungs with relatively low CD46 expression. We conclude that coagulation pathway dysregulation contributes to injury in GTKO pig lung xenografts perfused with human blood, and that the survival advantage for lungs with increased hCPRP expression is likely attributable to improved endothelial thromboregulation. [ABSTRACT FROM AUTHOR]

The phenomenon of diminishing hematocrit after in vivo liver and lung xenotransplantation and during ex vivo liver xenoperfusion has largely been attributed to action by resident liver porcine macrophages, which bind and destroy human erythrocytes. Porcine sialoadhesin (siglec‐1) was implicated previously in this interaction. This study examines the effect of porcine genetic modifications, including knockout of the CMAH gene responsible for expression of Neu5Gc sialic acid, on the adhesion of human red blood cells (RBCs) to porcine macrophages. Wild‐type (WT) porcine macrophages and macrophages from several strains of genetically engineered pigs, including CMAH gene knockout and several human transgenes (TKO+hTg), were incubated with human RBCs and "rosettes" (≥3 erythrocytes bound to one macrophage) were quantified by microscopy. Our results show that TKO+hTg genetic modifications significantly reduced rosette formation. The monoclonal antibody 1F1, which blocks porcine sialoadhesin, significantly reduced rosette formation by WT and TKO+hTg macrophages compared with an isotype control antibody. Further, desialation of human RBCs with neuraminidase before addition to WT or TKO+hTg macrophages resulted in near‐complete abrogation of rosette formation, to a level not significantly different from porcine RBC rosette formation on porcine macrophages. These observations are consistent with rosette formation being mediated by binding of sialic acid on human RBCs to sialoadhesin on porcine macrophages. In conclusion, the data predict that TKO+hTg genetic modifications, coupled with targeting of porcine sialoadhesin by the 1F1 mAb, will attenuate erythrocyte sequestration and anemia during ex vivo xenoperfusion and following in vivo liver, lung, and potentially other organ xenotransplantation. [ABSTRACT FROM AUTHOR]

Background: Antibody‐mediated rejection has long been known to be one of the major organ failure mechanisms in xenotransplantation. In addition to the porcine α1,3‐galactose (α1,3Gal) epitope, N‐Glycolylneuraminic acid (Neu5Gc), a sialic acid, has been identified as an important porcine antigen against which most humans have pre‐formed antibodies. Here we evaluate GalTKO.hCD46 lungs with an additional cytidine monophospho‐N‐acetylneuraminic acid hydroxylase (CMAH) gene knock‐out (Neu5GcKO) in a xenogeneic ex vivo perfusion model Methods: Eleven GalTKO.hCD46.Neu5GcKO pig lungs were perfused for up to 6 h with fresh heparinized human blood. Six of them were treated with histamine (H) blocker famotidine and 1‐thromboxane synthase inhibitor Benzylimidazole (BIA) and five were left untreated. GalTKO.hCD46 lungs without Neu5GcKO (n = 18: eight untreated and 10 BIA+H treated) served as a reference. Functional parameters, blood, and tissue samples were collected at pre‐defined time points throughout the perfusion Results: All but one Neu5GcKO organs maintained adequate blood oxygenation and "survived" until elective termination at 6 h whereas two reference lungs failed before elective termination at 4 h. Human anti‐Neu5Gc antibody serum levels decreased during the perfusion of GalTKO.hCD46 lungs by flow cytometry (∼40% IgM, 60% IgG), whereas antibody levels in Neu5GcKO lung perfusions did not fall (IgM p =.007; IgG p <.001). Thromboxane elaboration, thrombin generation, and histamine levels were significantly reduced with Neu5GcKO lungs compared to reference in the untreated groups (p =.007,.005, and.037, respectively); treatment with BIA+H masked these changes. Activation of platelets, measured as CD62P expression on circulating platelets, was lower in Neu5GcKO experiments compared to reference lungs (p =.023), whereas complement activation (as C3a rise in plasma) was not altered. MCP‐1 and lactotransferin level elevations were blunted in Neu5GcKO lung perfusions (p =.007 and.032, respectively). Pulmonary vascular resistance (PVR) rise was significantly attenuated and delayed in untreated GalTKO.hCD46.Neu5GcKO lungs in comparison to the untreated GalTKO.hCD46 lungs (p =.003) Conclusion: Additional Neu5GcKO in GalTKO.hCD46 lungs significantly reduces parameters associated with antibody‐mediated inflammation and activation of the coagulation cascade. Knock‐out of the Neu5Gc sialic acid should be beneficial to reduce innate immune antigenicity of porcine lungs in future human recipients. [ABSTRACT FROM AUTHOR]

Introduction: Platelet sequestration, inflammation, and inappropriate coagulation cascade activation are prominent in liver xenotransplant models and are associated with poor outcomes. Here, we evaluate a cassette of six additional genetic modifications to reduce anti‐pig antibody binding (α‐1,3‐galactosyl transferase knockout [GalTKO]) and target coagulation dysregulation (human endothelial protein C receptor [hEPRC] and thrombomodulin [hTBM]), complement pathway regulation (human membrane cofactor protein, hCD46), inflammation heme oxygenase 1 [hHO‐1]), and a self‐recognition receptor (integrin‐associated protein [hCD47]), as well as donor pharmacologic treatments designed to blunt these phenomena. Methods: Livers from GaltKO.hCD46 pigs ("2‐gene," n = 3) and GalTKO.hCD46 pigs also transgenic for hEPRC, hTBM, hCD47, and hHO‐1 ("6‐gene," n = 4) were perfused ex vivo with whole human blood. Six‐gene pigs were additionally pretreated with desmopressin (DDAVP) and clodronate liposomes to deplete vWF and kupffer cells, respectively. Results: The average perfusion times increased from 304 (±148) min in the 2‐gene group to 856 (±61) min in the 6‐gene group (p =.010). The average heparin administration was decreased from 8837 U/h in the 2‐gene to 1354 U/h in the 6‐gene group (p =.047). Platelet sequestration tended to be delayed in the 6‐gene group (p =.070), while thromboxane B2 (TXB2, a platelet activation marker) levels were lower over the first hour (p =.044) (401 ± 124 vs. 2048 ± 712 at 60 min). Thrombin production as measured by F1+2 levels tended to be lower in the 6‐gene group (p =.058). Conclusions: The combination of the hEPCR.hTBM.hCD47.hHO‐1 cassette along with donor pig DDAVP and clodronate liposome pretreatment was associated with prolonged function of xenoperfused livers, reduced coagulation pathway perturbations, and decreased TXB2 elaboration, and reflects significant progress to modulate liver xenograft injury in a pig to human model. [ABSTRACT FROM AUTHOR]

Platelet sequestration is a common process during organ reperfusion after transplantation. However, instead of lower platelet counts, when using traditional hemocytometers and light microscopy, we observed physiologically implausible platelet counts in the course of ex‐vivo lung and liver xenograft organ perfusion studies. We employed conventional flow cytometry (FC) and imaging FC (AMINS ImageStream X) to investigate the findings and found platelet‐sized fragments in the circulation that are mainly derived from red blood cell membranes. We speculate that this erythrocyte fragmentation contributes to anemia during in‐vivo organ xenotransplant. [ABSTRACT FROM AUTHOR]

The transplantation of organs across species offers the potential to solve the shortage of human organs. While activation of human platelets by human von Willebrand factor (vWF) requires vWF activation by shear stress, contact between human platelets and porcine vWF (pvWF) leads to spontaneous platelet adhesion and activation. This non‐physiologic interaction may contribute to the thrombocytopenia and coagulation pathway dysregulation often associated with xenotransplantation of pig organs in nonhuman primates. Pigs genetically modified to decrease antibody and complement‐dependent rejection (GTKO.hCD46) were engineered to express humanized pvWF (h*pvWF) by replacing a pvWF gene region that encodes the glycoprotein Ib‐binding site with human cDNA orthologs. This modification corrected for non‐physiologic human platelet aggregation on exposure to pig plasma, while preserving in vitro platelet activation by collagen. Organs from pigs with h*pvWF demonstrated reduced platelet sequestration during lung (p ≤.01) and liver (p ≤.038 within 4 h) perfusion ex vivo with human blood and after pig‐to‐baboon lung transplantation (p ≤.007). Residual platelet sequestration and activation were not prevented by the blockade of canonical platelet adhesion pathways. The h*pvWF modification prevents physiologically inappropriate activation of human or baboon platelets by porcine vWF, addressing one cause of the thrombocytopenia and platelet activation observed with xenotransplantation. [ABSTRACT FROM AUTHOR]

Consistent survival of life-supporting pig heart xenograft recipients beyond 90 days was recently reported using genetically modified pigs and a clinically applicable drug treatment regimen. If this remarkable achievement proves reproducible, published benchmarks for clinical translation of cardiac xenografts appear to be within reach. Key mechanistic insights are summarized here that informed recent pig design and therapeutic choices, which together appear likely to enable early clinical translation. [ABSTRACT FROM AUTHOR]

• Some cardiac tissues are not amenable to routine RNA isolation. • Ovation RNA-Seq V2 system preforms reliable robust amplification. • ΔΔCT and standard curve variations of RT-qPCR provide similar results. • NanoString does not correlate well with traditional RT-qPCR methods. • NanoString may be less sensitive to small variations in gene expression. Quantitative gene expression profiling of cardiac allografts characterizes the phenotype of the alloimmune response, yields information regarding differential effects that may be associated with various anti-rejection drug regimens, and generates testable hypotheses regarding the pathogenesis of the chronic rejection lesions typically observed in non-human primate heart transplant models. The goal of this study was to assess interplatform performance and variability between the relatively novel NanoString nCounter Analysis System, ΔΔCT (relative) RT-qPCR, and standard curve (absolute) RT-qPCR utilizing cynomolgus monkey cardiac allografts. Methods for RNA isolation and preamplification were also systematically evaluated and effective methods are proposed. In this study, we demonstrate strong correlation between the two RT-qPCR methods, but variable and, at times, weak correlation between RT-qPCR and NanoString. NanoString fold change results demonstrate less sensitivity to small changes in gene expression than RT-qPCR. These findings appear to be driven by technical aspects of each platform that influence the conditions under which each technique is ideal. Collectively, our data contribute to the general effort to optimally utilize gene expression profiling techniques, not only for transplanted tissues, but for many other applications where accurate rank-order of gene expression versus precise quantification of absolute gene transcript number may be relatively valuable. [ABSTRACT FROM AUTHOR]

Abstract: Background: Human neutrophils are sequestered by pig lung xenografts within minutes during ex vivo perfusion. This phenomenon is not prevented by pig genetic modifications that remove xeno‐antigens or added human regulatory molecules intended to down‐regulate activation of complement and coagulation pathways. This study investigated whether recipient and donor interleukin‐8 (IL‐8), a chemokine known to attract and activate neutrophils during inflammation, is elaborated in the context of xenogeneic injury, and whether human or pig IL‐8 promote the adhesion of human neutrophils in in vitro xenograft models. Methods: Plasma levels of pig, human or non‐human primate (NHP) IL‐8 from ex vivo pig lung perfusion experiments (n = 10) and in vivo pig‐to‐baboon lung transplantation in baboons (n = 22) were analysed by ELISA or Luminex. Human neutrophils stimulated with human or pig IL‐8 were analysed for CD11b expression, CD18 activation, oxidative burst and adhesion to resting or TNF‐activated endothelial cells (EC) evaluated under static and flow (Bioflux) conditions. For some experiments, human neutrophils were incubated with Reparixin (IL‐8/CXCL8 receptor blocker) and then analysed as in the in vitro experiments mentioned above. Results: Plasma levels of pig IL‐8 (~6113 pg/mL) increased more than human (~1235 pg/mL) between one and four hours after initiation of ex vivo lung perfusion. However, pig IL‐8 levels remained consistently low (<60 pg/mL) and NHP IL‐8 plasma levels increased by ~2000 pg/mL after four hours in a pig‐to‐baboon lung xenotransplantation. In vitro, human neutrophils’ CD11b expression, CD18 activation and oxidative burst all increased in a dose‐dependent manner following exposure to either pig or human IL‐8, which also were associated with increased adhesion to EC in both static and flow conditions. Reparixin inhibited human neutrophil activation by both pig and human IL‐8 in a dose‐dependent fashion. At 0.1 mg/mL, Reparixin inhibited the adhesion of IL‐8‐activated human neutrophils to pAECs by 84 ± 2.5%. Conclusions: Pig IL‐8 increased in an ex vivo model of pig‐to‐human lung xenotransplantation but is not detected in vivo, whereas human or NHP IL‐8 is elevated to a similar degree in both models. Both pig and human IL‐8 activate human neutrophils and increase their adhesion to pig aortic ECs, a process significantly inhibited by the addition of Reparixin to human neutrophils. This work implicates IL‐8, whether of pig or human origin, as a possible factor mediating in lung xenograft inflammation and injury and supports the evaluation of therapeutic targeting of this pathway in the context of xenotransplantation. [ABSTRACT FROM AUTHOR]

Abstract: Background: Alongside the need to develop more effective and less toxic immunosuppression, the shortage of human organs available for organ transplantation is one of the major hurdles facing the field. Research into xenotransplantation, as an alternative source of organs, has unveiled formidable challenges. Porcine lungs perfused with human blood rapidly sequester the majority of circulating neutrophils and platelets, which leads to inflammation and organ failure within hours, and is not significantly attenuated by genetic modifications to the pig targeted to diminish antibody binding and complement and coagulation cascade activation. Methods: Here, we model the interaction of freshly isolated human leukocytes with xenotransplanted vasculature under physiologic flow conditions using microfluidic channels coated with porcine endothelial cells. Both isolated human neutrophils and whole human blood were perfused over transgenic pig aortic endothelial cells that had been activated with rhTNF‐α or rhIL‐4 using the BioFlux system. Novel compounds GMI‐1271 and rPSGL1.Fc were tested as E‐ and P‐ selectin antagonists, respectively. Cellular adhesion and rolling events were tracked using FIJI (imageJ). Results: Porcine endothelium activated with either rhTNF‐α or rhIL‐4 expressed high amounts of selectins, to which isolated human neutrophils readily rolled and tethered. Both E‐and P‐selectin antagonism significantly reduced the number of neutrophils rolling and rolling distance in a dose‐dependent manner, with near total inhibition at higher doses (P < .001). Similarly, with whole human blood, selectin blocking compounds exhibited dose‐dependent inhibition of prevalent leukocyte adhesion and severe endothelial injury (Untreated: 394 ± 97 PMNs/hpf, 57 ± 6% loss EC; GMI1271+rPSGL1.Fc: 23 ± 9 PMNs/hpf, 8 ± 6% loss EC P < .01). Conclusions: Selectin blockade may be useful as part of an integrated strategy to prevent neutrophil‐mediated organ xenograft injury, especially during the early time points following reperfusion. [ABSTRACT FROM AUTHOR]

Abstract: In addition to immune barriers, molecular incompatibilities between species are predicted to limit pig liver survival in primate xenotransplantation models. Assessment and measurement of synthetic function of genetically modified porcine livers after ex vivo perfusion with human blood have not previously been described. Eight porcine livers from α1,3‐galactosyl transferase knockout and human membrane cofactor (GalTKO.hCD46), six livers from GalTKO.hCD46 and N‐glycolylneuraminic acid knockout (GalTKO.hCD46.Neu5GcKO), and six livers from GalTKO.hCD46 with humanized decay‐accelerating factor (hCD55), endothelial protein C receptor (hEPCR), tissue factor pathway inhibitor (hTFPI), and integrin‐associated protein (hCD47) (GalTKO.hCD46.hCD55.hEPCR.hTFPI.hCD47) pigs were perfused with human blood under physiologic conditions. Timed blood samples were tested for liver enzymes and for pig‐specific albumin production via Western blot. Porcine albumin levels increased with time in all experiments. By densitometry, GalTKO.hCD46.Neu5GcKO livers had the highest albumin levels, measured both as total produced, and when controlled for perfusion duration, compared to GalTKO.hCD46 (P = .068) and GalTKO.hCD46.hCD55.hEPCR.hTFPI.hCD47 livers (P = .04). Porcine livers perfused with human blood demonstrated the synthetic ability to produce albumin in all cases. GalTKO.hCD46.Neu5GcKO pig livers demonstrated the most robust albumin production. This suggests that the Neu5GcKO phenotype provides a protective effect on the graft due to decreased human antibody recognition and graft injury. [ABSTRACT FROM AUTHOR]

Background Wild-type pigs express several carbohydrate moieties on their cell surfaces that differ from those expressed by humans. This difference in profile leads to pig tissue cell recognition of human blood cells causing sequestration, in addition to antibody-mediated xenograft injury. One such carbohydrate is N-glycolylneuraminic acid (Neu5Gc), a sialic acid molecule synthesized in pigs but not in humans. Here, we evaluate livers with and without Neu5Gc in an ex vivo liver xeno perfusion model. Methods Livers from pigs with an α1,3-galactosyl transferase gene knockout (Gal TKO) and transgenic for human membrane cofactor ( hCD46) with (n = 5) or without (n = 7) an additional Neu5Gc gene knock out (Neu5Gc KO) were perfused ex vivo with heparinized whole human blood. A drug regimen consisting of a histamine inhibitor, thromboxane synthase inhibitor, and a murine anti-human GPIb-blocking antibody fragment was given to half of the experiments in each group. Results Liver function tests ( AST and ALT) were not significantly different between livers with and without the Neu5Gc KO. Gal TKO. hCD46.Neu5Gc KO livers had less erythrocyte sequestration as evidenced by a higher mean hematocrit over time compared to Gal TKO. hCD46 livers ( P = .0003). The addition of Neu5Gc KO did not ameliorate profound thrombocytopenia seen within the first 15 minutes of perfusion. TXB2 was significantly less with the added drug regimen ( P = .006) or the presence of Neu5Gc KO ( P = .017). Conclusions The lack of Neu5Gc expression attenuated erythrocyte loss but did not prevent profound early onset thrombocytopenia or platelet activation, although TXB2 levels were decreased in the presence of Neu5Gc KO. [ABSTRACT FROM AUTHOR]

Presentation of sialic acid (Sia) varies among different tissues and organs within each species, and between species. This diversity has biologically important consequences regarding the recognition of cells by 'xeno' antibodies (Neu5Gc vs Neu5Ac). Sia also plays a central role in inflammation by influencing binding of the asialoglycoprotein receptor 1 ( ASGR-1), Siglec-1 (Sialoadhesin), and cellular interactions mediated by the selectin, integrin, and galectin receptor families. This review will focus on what is known about basic Sia structure and function in association with xenotransplantation, how changes in sialylation may occur in this context (through desialylation or changes in sialyltransferases), and how this fundamental pathway modulates adhesive and cell activation pathways that appear to be particularly crucial to homeostasis and inflammation for xenografts. [ABSTRACT FROM AUTHOR]

Background Lung xenografts remain susceptible to loss of vascular barrier function within hours in spite of significant incremental advances based on genetic engineering to remove the Gal 1,3-αGal antigen (Gal TKO) and express human membrane cofactor protein ( hCD46). Natural killer cells rapidly disappear from the blood during perfusion of Gal TKO. hCD46 porcine lungs with human blood and presumably are sequestered within the lung vasculature. Here we asked whether porcine expression of the human NK cell inhibitory ligand HLA-E and β2 microglobulin inhibits Gal TKO.hCD46 pig cell injury or prolongs lung function in two preclinical perfusion models. Methods Lungs from pigs modified to express Gal TKO.h CD46 (n=37) and Gal TKO.h CD46. HLA-E (n=5) were harvested and perfused with human blood until failure or elective termination at 4 hours. Airway pressures and pulmonary artery hemodynamics were recorded in real time. Blood samples were also collected throughout the experiment for analysis. Porcine aortic endothelial cells ( PAECs) from each genotype were cultured in monolayers in microfluidic channels and used in fluorescent cytotoxicity assays using human NK cells. Results HLA-E expression on Gal TKO.h CD46 PAECs was associated with significantly decreased antibody-dependent and antibody-independent NK-mediated cytotoxicity under in vitro conditions simulating physiologic shear stress. Relative to Gal TKO. hCD46 pig lungs perfused with human blood on an ex vivo platform, additional expression of HLA-E increased median lung survival (>4 hours, vs 162 minutes, P=.012), and was associated with attenuated rise in pulmonary vascular resistance, and decreased platelet activation and histamine elaboration. As expected, HLA-E expression was not associated with a significant difference in NK cell adhesion to endothelial cells in vitro, or NK cell and neutrophil sequestration during organ perfusion. Conclusions We conclude human NK cell activation contributes significantly to Gal TKO.h CD46 pig endothelial injury and lung inflammation and show that expression of HLA-E is associated with physiologically meaningful protection of Gal TKO.h CD46 cells and organs exposed to human blood. [ABSTRACT FROM AUTHOR]

Background: Porcine xenografts are a promising source of scarce transplantable organs, but stimulate intense thrombosis of human blood despite targeted genetic and pharmacologic interventions. Current experimental models do not enable study of the blood/endothelial interface to investigate adhesive interactions and thrombosis at the cellular level under physiologic conditions. The purpose of this study was to develop and validate a live-cell, shear-flow based thrombosis assay relevant to general thrombosis research, and demonstrate its potential in xenotransplantation applications. Methodology/Principal Findings: Confluent wild-type (WT, n = 48) and Gal transferase knock-out (GalTKO, which resist hyperacute rejection; n = 11) porcine endothelia were cultured in microfluidic channels. To mimic microcirculatory flow, channels were perfused at 5 dynes/cm2 and 37°C with human blood stained to fluorescently label platelets. Serial fluorescent imaging visualized percent surface area coverage (SA, for adhesion of labeled cells) and total fluorescence (a metric of clot volume). Aggregation was calculated by the fluorescence/SA ratio (FR). WT endothelia stimulated diffuse platelet adhesion (SA 65 ± 2%) and aggregation (FR 120 ± 1 a.u.), indicating high-grade thrombosis consistent with the rapid platelet activation and consumption seen in whole-organ lung xenotransplantation models. Experiments with antibody blockade of platelet aggregation, and perfusion of syngeneic and allo-incompatible endothelium was used to verify the biologic specificity and validity of the assay. Finally, with GalTKO endothelia thrombus volume decreased by 60%, due primarily to a 58% reduction in adhesion (P < 0.0001 each); importantly, aggregation was only marginally affected (11% reduction, P < 0.0001). Conclusions/Significance: This novel, high-throughput assay enabled dynamic modeling of whole-blood thrombosis on intact endothelium under physiologic conditions, and allowed mechanistic characterization of endothelial and platelet interactions. Applied to xenogeneic thrombosis, it enables future studies regarding the effect of modifying the porcine genotype on sheer-stress-dependent events that characterize xenograft injury. This in-vitro platform is likely to prove broadly useful to study thrombosis and endothelial interactions under dynamic physiologic conditions. [ABSTRACT FROM AUTHOR]

Background Genetically modified pigs are a promising potential source of lung xenografts. Ex vivo xenoperfusion is an effective platform for testing the effect of new modifications, but typical experiments are limited by testing of a single genetic intervention and small sample sizes. The purpose of this study was to analyze the individual and aggregate effects of donor genetic modifications on porcine lung xenograft survival and injury in an extensive pig lung xenoperfusion series. Methods Data from 157 porcine lung xenoperfusion experiments using otherwise unmodified heparinized human blood were aggregated as either continuous or dichotomous variables. Lungs were wild type in 17 perfusions (11% of the study group), while 31 lungs (20% of the study group) had one genetic modification, 40 lungs (39%) had 2, and 47 lungs (30%) had 3 or more modifications. The primary endpoint was functional lung survival to 4 h of perfusion. Secondary analyses evaluated previously identified markers associated with known lung xenograft injury mechanisms. In addition to comparison among all xenografts grouped by survival status, a subgroup analysis was performed of lungs incorporating the GalTKO.hCD46 genotype. Results Each increase in the number of genetic modifications was associated with additional prolongation of lung xenograft survival. Lungs that exhibited survival to 4 h generally had reduced platelet activation and thrombin generation. GalTKO and the expression of hCD46, HO-1, hCD55, or hEPCR were associated with improved survival. hTBM, HLA-E, and hCD39 were associated with no significant effect on the primary outcome. Conclusion This meta-analysis of an extensive lung xenotransplantation series demonstrates that increasing the number of genetic modifications targeting known xenogeneic lung injury mechanisms is associated with incremental improvements in lung survival. While more detailed mechanistic studies are needed to explore the relationship between gene expression and pathway-specific injury and explore why some genes apparently exhibit neutral ( hTBM, HLA-E) or inconclusive (CD39) effects, GalTKO, hCD46, HO-1, hCD55, and hEPCR modifications were associated with significant lung xenograft protection. This analysis supports the hypothesis that multiple genetic modifications targeting different known mechanisms of xenograft injury will be required to optimize lung xenograft survival. [ABSTRACT FROM AUTHOR]

Xenotransplantation has undergone important progress in controlling initial hyperacute rejection in many preclinical models, with some cell, tissue, and organ xenografts advancing toward clinical trials. However, acute injury, driven primarily by innate immune and inflammatory responses, continues to limit results in lung xenograft models. The purpose of this article is to review the current status of lung xenotransplantation-including the seemingly unique challenges posed by this organ-and summarize proven and emerging means of overcoming acute lung xenograft injury. [ABSTRACT FROM AUTHOR]

Background Although transplantation of genetically modified porcine livers into baboons has yielded recipient survival for up to 7 days, survival is limited by profound thrombocytopenia, which becomes manifest almost immediately after revascularization, and by subsequent coagulopathy. Porcine von Willebrand's factor ( VWF), a glycoprotein that adheres to activated platelets to initiate thrombus formation, has been shown to constitutively activate human platelets via their glycoprotein Ib ( GPIb) receptors. Here, we report our pig-to-primate liver xenoperfusion model and evaluate whether targeting the GPIb-VWF axis prevents platelet sequestration. Methods Twelve baboons underwent cross-circulation with the following extracorporeal livers: one allogeneic control with a baboon liver, 4 xenogeneic controls with a GalTKO.hCD46 pig liver, 3 GalTKO.hCD46 pig livers in recipients treated with αGPIb antibody during perfusion, and 4 GalTKO.hCD46 pig livers pre-treated with D-arginine vasopressin ( DDAVP) in recipients treated with αGPIb antibody during perfusion. Results All perfused livers appeared grossly and macroscopically normal and produced bile. Xenograft liver perfusion experiments treated with αGPIb antibody may show less platelet sequestration during the initial 2 h of perfusion. Portal venous resistance remained constant in all perfusion experiments. Platelet activation studies demonstrated platelet activation in all xenoperfusions, but not in the allogeneic perfusion. Conclusion These observations suggest that primate platelet sequestration by porcine liver and the associated thrombocytopenia are multifactorial and perhaps partially mediated by a constitutive interaction between porcine VWF and the primate GPIb receptor. Control of platelet sequestration and consumptive coagulopathy in liver xenotransplantation will likely require a multifaceted approach in our clinically relevant perfusion model. [ABSTRACT FROM AUTHOR]

Background: Cardiac allograft vasculopathy (CAV) remains the main cause of long-term transplant rejection. CAV is characterized by hyperproliferation of vascular smooth muscle cells (VSMCs). Canonical β-catenin signaling is a critical regulator of VSMC proliferation in development; however, the role of this pathway and its regulation in CAV progression are obscure. We investigated the activity of β-catenin signaling and the role for a putative activating ligand, transglutaminase 2 (TG2), in chronic cardiac rejection. Methods: Hearts from Bm12 mice were transplanted into C57BL/6 mice (class II mismatch), and allografts were harvested 8 weeks after transplantation. Accumulation and sub-cellular distribution of β-catenin protein and expression of several components of β-catenin signaling were analyzed as hallmarks of pathway activation. In vitro, platelet-derived growth factor treatment was used to mimic the inflammatory milieu in VSMC and organotypic heart slice cultures. Results: Activation of β-catenin in allografts compared with isografts or naïve hearts was evidenced by the augmented expression of β-catenin target genes, as well as the accumulation and nuclear localization of the β-catenin protein in VSMCs of the occluded allograft vessels. Expression of TG2, an activator of β-catenin signaling in VSMCs, was dramatically increased in allografts. Further, our ex vivo data demonstrate that TG2 is required for VSMC proliferation and for β-catenin activation by platelet-derived growth factor in cardiac tissue. Conclusions: β-Catenin signaling is activated in occluded vessels in murine cardiac allografts. TG2 is implicated as an endogenous activator of this signaling pathway and may therefore have a role in the pathogenesis of CAV during chronic allograft rejection. [Copyright &y& Elsevier]

Cooper DKC, Ekser B, Burlak C, Ezzelarab M, Hara H, Paris L, Tector AJ, Phelps C, Azimzadeh AM, Ayares D, Robson SC, Pierson RN III. Clinical lung xenotransplantation - what donor genetic modifications may be necessary? Xenotransplantation 2012; 19: 144-158. © 2012 John Wiley & Sons A/S. Abstract: Barriers to successful lung xenotransplantation appear to be even greater than for other organs. This difficulty may be related to several macro anatomic factors, such as the uniquely fragile lung parenchyma and associated blood supply that results in heightened vulnerability of graft function to segmental or lobar airway flooding caused by loss of vascular integrity (also applicable to allotransplants). There are also micro-anatomic considerations, such as the presence of large numbers of resident inflammatory cells, such as pulmonary intravascular macrophages and natural killer (NK) T cells, and the high levels of von Willebrand factor (vWF) associated with the microvasculature. We have considered what developments would be necessary to allow successful clinical lung xenotransplantation. We suggest this will only be achieved by multiple genetic modifications of the organ-source pig, in particular to render the vasculature resistant to thrombosis. The major problems that require to be overcome are multiple and include (i) the innate immune response (antibody, complement, donor pulmonary and recipient macrophages, monocytes, neutrophils, and NK cells), (ii) the adaptive immune response (T and B cells), (iii) coagulation dysregulation, and (iv) an inflammatory response (e.g., TNF-α, IL-6, HMGB1, C-reactive protein). We propose that the genetic manipulation required to provide normal thromboregulation alone may include the introduction of genes for human thrombomodulin/endothelial protein C-receptor, and/or tissue factor pathway inhibitor, and/or CD39/CD73; the problem of pig vWF may also need to be addressed. It would appear that exploration of every available therapeutic path will be required if lung xenotransplantation is to be successful. To initiate a clinical trial of lung xenotransplantation, even as a bridge to allotransplantation (with a realistic possibility of survival long enough for a human lung allograft to be obtained), significant advances and much experimental work will be required. Nevertheless, with the steadily increasing developments in techniques of genetic engineering of pigs, we are optimistic that the goal of successful clinical lung xenotransplantation can be achieved within the foreseeable future. The optimistic view would be that if experimental pig lung xenotransplantation could be successfully managed, it is likely that clinical application of this and all other forms of xenotransplantation would become more feasible. [ABSTRACT FROM AUTHOR]

The antioxidant tempo! attenuates pressure overload-induced cardiac hypertrophy and contractile dysfunction in mice fed a high-fructose diet. Am J Physiol Heart Circ Physiol 295: H2223-H2230, 2008; doi: 10.!! 52/ajpheart.00563.2008.-We have previously shown that high-sugar diets increase mortality and left ventricular (LV) dysfunction during pressure overload. The mechanisms behind these diet-induced alterations are unclear but may involve increased oxidative stress in the myocardium. The present study examined whether high-fructose feeding increased myocardial oxidative damage and exacerbated systolic dysfunction after transverse aortic constriction (TAC) and if this effect could be attenuated by treatment with the antioxidant tempo!. Immediately after surgery, TAC and sham mice were assigned to a high-starch diet (58% of total energy intake as cornstarch and 10% fat) or high-fructose diet (61% fructose and 10% fat) with or without the addition of tempol [0.1% (wt/wt) in the chow] and maintained on the treatment for 8 wk. In response to TAC, fructose-fed mice had greater cardiac hypertrophy (55.1% increase in the heart weight-to-tibia length ratio) than starch- fed mice (22.3% increase in the heart weight-to-tibia length ratio). Treatment with tempol significantly attenuated cardiac hypertrophy in fructose-fed TAC mice (18.3% increase in the heart weight-to-tibia ratio). Similarly, fructose-fed TAC mice had a decreased LV area of fractional shortening (from 38 ± 2% in sham to 22 ± 4% in TAC), which was prevented by tempol treatment (33 ± 3%). Markers of lipid peroxidation in fructose-fed TAC hearts were also blunted by tempo!. In conclusion, tempol significantly blunted markers of cardiac hyper- trophy, LV remodeling, contractile dysfunction, and oxidative stress in fructose-fed TAC mice. [ABSTRACT FROM AUTHOR]

Abstract: Objective: Epidemiological studies suggest that consumption of ω-3 polyunsaturated fatty acids (ω-3 PUFA) decreases the risk of heart failure. We assessed the effects of dietary supplementation with ω-3 PUFA from fish oil on the response of the left ventricle (LV) to arterial pressure overload. Methods: Male Wistar rats were fed a standard chow or a ω-3 PUFA-supplemented diet. After 1 week rats underwent abdominal aortic banding or sham surgery (n =9–12/group). LV function was assessed by echocardiography after 8 weeks. In addition, we studied the effect of ω-3 PUFA on the cardioprotective adipocyte-derived hormone adiponectin, which may alter the pro-growth serine–threonine kinase Akt. Results: Banding increased LV mass to a greater extent with the standard chow (31%) than with ω-3 PUFA (18%). LV end diastolic and systolic volumes were increased by 19% and 105% with standard chow, respectively, but were unchanged with ω-3 PUFA. The expression of adiponectin was up-regulated in adipose tissue, and the plasma adiponectin concentration was significantly elevated. Treatment with ω-3 PUFA increased total Akt protein expression in the heart, but decreased the fraction of Akt in the active phosphorylated form, and thus did not alter the amount of active phospho-Akt. Conclusion: Dietary supplementation with ω-3 PUFA attenuated pressure overload-induced LV dysfunction, which was associated with elevated plasma adiponectin. [Copyright &y& Elsevier]

Little is known about the effects of the composition of dietary carbohydrate on the development of left ventricular (LV) hypertrophy (LVH) and heart failure (HF) under conditions of pressure overload. The objective of this study was to determine the effect of carbohydrate composition on LVH, LV function, and mortality in a mouse model of chronic pressure over- load. Male C57BL/6J mice of 6 wk of age (n = 14-16 mice/group) underwent transverse aortic constriction (TAC) or sham surgery and were fed either standard chow (STD; 32% corn starch, 35% sucrose, 3% maltodextrin, and 10% fat expressed as a percent of the total energy), high-starch chow (58% corn starch, 12% maltodextrin, and 10% fat), or high-fructose chow (9% corn starch, 61% fructose, and 10% fat). After 16 wk of treatment, mice with TAC fed the STD or high-fructose diets exhibited increased LV mass, larger end-diastolic and end-systolic diameters, and decreased ejection fraction compared with sham. The high-starch diet, in contrast, prevented changes in LV dimensions and contractile function. Cardiac mRNA for myosin heavy chain-13 was increased dramatically in the fructose-fed banded animals, as was mortality (54% compared with 8% and 29% in the starch and STD banded groups, respectively). In conclusion, a diet high in simple sugar was deleterious, resulting in the highest mortality and expression of molecular markers of cardiac dysfunction in TAC animals compared with sham, whereas a high-starch diet blunted mortality, increases in cardiac mass, and contractile dysfunction. [ABSTRACT FROM AUTHOR]

Background: Hyperacute rejection (HAR) and early graft failure (EGF) have been described in a minority of pig-to-baboon heart transplants using organs transgenic for human complement regulatory proteins (hCRP). Here we investigate the role of coagulation cascade activation in the pathogenesis of HAR and EGF in a consecutive series where a high incidence of these outcomes was observed. Methods: Twenty-eight naïve wild-caught Papio anubis baboons received heterotopic heart transplants from pigs transgenic for hDAF (n = 23) or hMCP (n = 5). Immunosuppression consisted of cyclosporine A, cyclophosphamide and MMF (n = 18) or anti-CD154 mAb (IDEC-131) and ATG (n = 10). Eleven received anti-Gal carbohydrates (GAS914, n = 8, or NEX1285, n = 3), of which four also underwent extracorporeal immunoadsorption (EIA), and 12 also received pharmacologic complement inhibitors (C1 INH, n = 9, or APT070, n = 3). Results: Excluding one technical failure, 14 of 27 transplants (11 hDAF, 3 hMCP) exhibited either HAR (n = 10) or EGF (n = 4). Surprisingly, neither complement inhibition (with C1 INH or APT070) nor anti-Gal antibody depletion with GAS914, NEX1285, or additional EIA consistently prevented HAR or EGF despite low or undetectable complement deposition. Strikingly, most grafts with HAR/EGF exhibited prominent fibrinogen and platelet deposition associated with systemic coagulation cascade activation, consistent with non-physiologic intravascular coagulation, in many instances despite little evidence for antibody-mediated complement activation. Conclusion: We conclude that dysregulated coagulation correlates closely with and probably causes primary failure of pig hearts transgenic for hCRP. These data support efforts to define effective strategies to prevent dysregulated coagulation in pig organ xenografts. [ABSTRACT FROM AUTHOR]

Wu GS, Pfeiffer S, Schröder C, Zhang TS, Nguyen BN, Lea W, Kelishadi S, Atkinson JB, Schuurman H-J, White DJG, Azimzadeh AM, and Pierson III RN. Co-stimulation blockade targeting CD154 and CD28/B7 modulates the induced antibody response after a pig-to-baboon cardiac xenograft. Xenotransplantation 2005; 12: 197–208.© Blackwell Munksgaard, 2005.The induced antibodies against Galα1,3Gal (Gal) and non-Gal epitopes may contribute to delayed xenograft rejection (DXR). We asked whether blockade of the CD40/CD154 and CD28/B7 co-stimulatory pathways modulates the baboon elicited antibody response to pig Gal and non-Gal antigens.Eighteen baboons received heterotopic heart transplants from pigs transgenic for human decay-accelerating factor (n = 13) or membrane cofactor protein (n = 5). Ten reference‘‘conventional therapy’’ animals received cyclosporin A, cyclophosphamide and mycophenolate mofetil, with (n = 4) or without (n = 6) anti-CD20. Eight‘‘co-stimulation blockade’’ animals received anti-CD154 mAb (IDEC-131) and anti-thymocyte globulin, with (n = 4) or without (n = 4) anti-CD20; two of these animals also received CTLA4-Fc. Anti-αGal IgG and IgM, anti-non-Gal antibodies and graft histology were assessed serially.Excluding two early graft failures, median graft survival with conventional therapy was 15 days (range 6 to 36 days, n = 8). Anti-Gal IgG antibody remained low through day 6 to 10, only one graft failure was accompanied by significant rise in anti-Gal IgG, and the anti-non-Gal response was weak (n = 2) or absent (n = 7). However many recipients succumbed with infection (n = 4) or coagulopathy (n = 2); DXR and ICOS+ T cells were prevalent in long-surviving grafts. With co-stimulation blockade, excluding three early graft failures, median graft survival was 7 days (range 6 to 11 days, n = 5). This regimen was very well tolerated, but increased anti-Gal antibody titer within 14 days was associated with graft failure in four of six animals. Although an anti-non-Gal response was present in three of six animals during IDEC-131 monotherapy (one strong, two weak), it was absent in both cases with additional CTLA4-Fc treatment.As used here, CD154 blockade alone does not completely prevent induction of Gal and non-Gal anti-pig antibodies. Our preliminary data suggest that other co-stimulation pathways, including CD28/B7 and ICOS, are sufficient to mediate high-titer anti-non-Gal antibody to porcine antigens in baboons, and contribute significantly to the pathogenesis of DXR. [ABSTRACT FROM AUTHOR]

: BackgroundThe role of complement in hyperacute lung xenograft rejection has not been fully elucidated. The present study evaluates the effect of complement (C) 1 esterase inhibition on hyperacute rejection of human decay-accelerating factor (hDAF)-positive pig lung by human blood.: MethodsUsing a modification of an established ex vivo model, right and left lungs from individual animals were surgically isolated and separately perfused. Pigs homozygous for hDAF were perfused with fresh human blood that was either untreated or treated with complement 1 esterase inhibitor (C1-Inh) at doses of 1 U/ml (n = 5), 5 U/ml (n = 3) or 10 U/ml plasma (n = 5).: ResultsOnly C1-Inh at 10 U/ml prolonged survival time (230 ± 48.3 minutes) as compared with controls (65.6 ± 26.5 minutes, p < 0.05) and diminished complement activation (C3a and C5a, p < 0.05). Interestingly, a low concentration of C1-Inh increased the pulmonary vascular resistance (PVR; 1 U/ml: 0.54 ± 0.3; 10 U/ml: 0.19 ± 0.08). Sequestration of neutrophils (92 ± 3%) and platelets (64 ± 13%) was not prevented by any concentration of C1-Inh. Tissue deposition of C3b and C5b-9 were diminished by hDAF expression, and further blunted by treatment, with 10 U/ml C1-Inh.: ConclusionsComplement plays a critical role in early events of lung hyperacute rejection (HAR). However, even potent inhibition of C1 esterase and C3/C5 convertase, using serum C1-Inh in pig lungs homozygous for hDAF expression, does not prevent rapid lung injury. Our findings implicate innate immune pathways resistant to efficient complement regulation, and suggest a role for neutrophils and platelets in the lung’s particular vulnerability. [Copyright &y& Elsevier]

Background: Elevated pulmonary vascular resistance (PVR), platelet adhesion, coagulation activation, and inflammation are prominent features of xenolung rejection. Here, we evaluate the role of thromboxane and histamine on PVR, and their contribution to other lung xenograft injury mechanisms. Methods: GalTKO.hCD46 single pig lungs were perfused ex vivo with fresh heparinized human blood: lungs were either treated with 1‐Benzylimidazole (1‐BIA) combined with histamine receptor blocker famotidine (n = 4) or diphenhydramine (n = 6), 1‐BIA alone (n = 6) or were left untreated (n = 9). Results: Six of the nine control experiments (GalTKO.hCD46 untreated), "survived" until elective termination at 4 hours. Without treatment, initial PVR elevation within the first 30 minutes resolved partially over the following hour, and increased progressively during the final 2 hours of perfusion. In contrast, 1‐BIA, alone or in addition to either antihistamine treatment, was associated with low stable PVR. Combined treatments significantly lowered the airway pressure when compared to untreated reference. Although platelet and neutrophil sequestration and coagulation cascade activation were not consistently altered by any intervention, increased terminal wet/dry weight ratio in untreated lungs was significantly blunted by combined treatments. Conclusion: Combined thromboxane and histamine pathway blockade prevents PVR elevation and significantly inhibits loss of vascular barrier function when GalTKO.hCD46 lungs are perfused with human blood. Platelet activation and platelet and neutrophil sequestration persist in all groups despite efficient complement regulation, and appear to occur independent of thromboxane and histamine antagonism. Our work identifies thromboxane and histamine as key mediators of xenolung injury and defines those pathways as therapeutic targets to achieve successful xenolung transplantation. [ABSTRACT FROM AUTHOR]

Previous studies of pig-to-non-human primate (NHP) islet xenotransplantation have provided important insights into the immune recognition and effector pathways operative in this relevant preclinical model. The specifics of the xenograft product, microenvironment at the implantation site, and the immunosuppressive regimen significantly influence the mechanisms underlying the rejection of xenogeneic islets. Our current understanding of the immunological barriers to survival of pig islets in NHPs is largely based on studies on intraportal islet xenografts and on comparisons with islet allografts. The demonstration of cell-mediated rejection of intraportal porcine islet xenografts at about 1 month posttransplant in monkeys immunosuppressed with the same protocols that prevent monkey islet allograft rejection indicates that islet xenograft rejection involves cellular mechanisms that are not present in acute islet allograft rejection. While these mechanisms remain poorly defined the demonstration of long-term diabetes reversal after intraportal islet xenotransplantation in non-human primates immunosuppressed with anti-CD40L but not with anti-CD40 antibody-based protocols suggests that the therapeutic efficacy of anti-CD40L in this transplantation setting likely involves the depletion of donor-reactive, activated T cells besides CD40:CD40L costimulation blockade. Rejection of intraportal islet xenografts in NHPs immunosuppressed with CTLA4-Ig and rapamycin was mediated largely by IL-15-primed, CXCR3+CD8+ memory T cells recruited by IP-10 (CXCL10) positive pig islets and macrophages that showed staining for IL-12 and iNOS. Adding basiliximab induction and tacrolimus maintenance therapy to this protocol prevented rejection in 24 of 26 recipients followed for up to 275 days. Comparison of both groups suggests, though by no means conclusive, that prolongation of graft survival in this large cohort was associated with reduced direct T cell responses to xenoantigens, reduced proportion of intrahepatic (intragraft) B cells and IFN-γ+ and IL-17+ CD4 and CD8 T cells, and increased local production of immunoregulatory molecules linked with Tregs, including TGF-β, Foxp3, HO-1, and IL-10. Anti-pig non-Gal IgG antibody elicitation was suppressed in both groups. We are currently exploring the concept of negative vaccination to markedly minimize the need for immunosuppression in islet xenotransplantation. Peritransplant administration of donor apoptotic cells extended pig-to-mouse islet xenograft survival to >250 days when combined with peritransplant B cell-depletion and rapamycin. This costimulation blockade-sparing, antigen-specific immunotherapy is expected to cause rapid pretransplant clonal deletion of indirect and anergy of direct xenospecific T cells while inducing regulatory T cells. As anti-CD40L antibodies, B cell depleting antibodies are expected to interfere with indirect antigen presentation, costimulation, and cytokine production required for optimal T cell proliferation, memory formation, and intragraft CD8+ effector function. It is conceivable that additional strategies must be employed in NHPs and eventually in diabetic patients to achieve - as previously with anti-CD40L antibodies - more complete, yet selective depletion of donor-reactive, activated T-cells for the purpose of stable xenograft acceptance. [ABSTRACT FROM AUTHOR]