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Antibody‐mediated rejection (AMR) is a common cause of graft failure after pig‐to‐nonhuman primate organ transplantation, even when the graft is from a pig with multiple genetic modifications. The specific factors that initiate AMR are often uncertain. We report two cases of pig kidney transplantation into immunosuppressed baboons in which we identify novel factors associated with the initiation of AMR. In the first, membranous nephropathy was the initiating factor that was then associated with the apparent loss of the therapeutic anti‐CD154 monoclonal antibody in the urine when severe proteinuria was present. This observation suggests that proteinuria may be associated with the loss of any therapeutic monoclonal antibody, for example, anti‐CD154 or eculizumab, in the urine, resulting in xenograft rejection. In the second case, the sequence of events and histopathology tentatively suggested that pyelonephritis may have initiated acute‐onset AMR. The association of a urinary infection with graft rejection has been well‐documented in ABO‐incompatible kidney allotransplantation based on the expression of an antigen on the invading microorganism shared with the kidney graft, generating an immune response to the graft. To our knowledge, these potential initiating factors of AMR in pig xenografts have not been highlighted previously. [ABSTRACT FROM AUTHOR]

Antibody‐mediated rejection (AMR) is the commonest cause of failure of a pig graft after transplantation into an immunosuppressed nonhuman primate (NHP). The incidence of AMR compared to acute cellular rejection is much higher in xenotransplantation (46% vs. 7%) than in allotransplantation (3% vs. 63%) in NHPs. Although AMR in an allograft can often be reversed, to our knowledge there is no report of its successful reversal in a pig xenograft. As there is less experience in preventing or reversing AMR in models of xenotransplantation, the results of studies in patients with allografts provide more information. These include (i) depletion or neutralization of serum anti‐donor antibodies, (ii) inhibition of complement activation, (iii) therapies targeting B or plasma cells, and (iv) anti‐inflammatory therapy. Depletion or neutralization of anti‐pig antibody, for example, by plasmapheresis, is effective in depleting antibodies, but they recover within days. IgG‐degrading enzymes do not deplete IgM. Despite the expression of human complement‐regulatory proteins on the pig graft, inhibition of systemic complement activation may be necessary, particularly if AMR is to be reversed. Potential therapies include (i) inhibition of complement activation (e.g., by IVIg, C1 INH, or an anti‐C5 antibody), but some complement inhibitors are not effective in NHPs, for example, eculizumab. Possible B cell‐targeted therapies include (i) B cell depletion, (ii) plasma cell depletion, (iii) modulation of B cell activation, and (iv) enhancing the generation of regulatory B and/or T cells. Among anti‐inflammatory agents, anti‐IL6R mAb and TNF blockers are increasingly being tested in xenotransplantation models, but with no definitive evidence that they reverse AMR. Increasing attention should be directed toward testing combinations of the above therapies. We suggest that treatment with a systemic complement inhibitor is likely to be most effective, possibly combined with anti‐inflammatory agents (if these are not already being administered). Ultimately, it may require further genetic engineering of the organ‐source pig to resolve the problem entirely, for example, knockout or knockdown of SLA, and/or expression of PD‐L1, HLA E, and/or HLA‐G. [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]

Heart and lung transplant recipients require care provided by clinicians from multiple different specialties, each contributing unique expertise and perspective. The period the patient spends in the intensive care unit is one of the most critical times in the perioperative trajectory. Various organizational models of intensive care exist, including those led by intensivists, surgeons, transplant cardiologists, and pulmonologists. Coordinating timely efficient intensive care is an essential and logistically difficult goal. The present work product of the American Society of Transplantation's Thoracic and Critical Care Community of Practice, Critical Care Task Force outlines operational guidelines and principles that may be applied in different organizational models to optimize the delivery of intensive care for the cardiothoracic organ recipient. [ABSTRACT FROM AUTHOR]

For many patients with terminal/advanced cardiac failure, heart transplantation is the most effective, durable treatment option, and offers the best prospects for a high quality of life. The number of potentially life-saving donated human organs is far fewer than the population who could benefit from a new heart, resulting in increasing numbers of patients awaiting replacement of their failing heart, high waitlist mortality, and frequent reliance on interim mechanical support for many of those deemed among the best candidates but who are deteriorating as they wait. Currently, mechanical assist devices supporting left ventricular or biventricular heart function are the only alternative to heart transplant that is in clinical use. Unfortunately, the complication rate with mechanical assistance remains high despite advances in device design and patient selection and management, and the quality of life of the patients even with good outcomes is only moderately improved. Cardiac xenotransplantation from genetically multi-modified (GM) organ-source pigs is an emerging new option as demonstrated by the consistent long-term success of heterotopic (non-life-supporting) abdominal and life-supporting orthotopic porcine heart transplantation in baboons, and by a recent 'compassionate use' transplant of the heart from a GM pig with 10 modifications into a terminally ill patient who survived for 2 months. In this review, we discuss pig heart xenotransplantation as a concept, including pathobiological aspects related to immune rejection, coagulation dysregulation, and detrimental overgrowth of the heart, as well as GM strategies in pigs to prevent or minimize these problems. Additional topics discussed include relevant results of heterotopic and orthotopic heart transplantation experiments in the pig-to-baboon model, microbiological and virologic safety concepts, and efficacy requirements for initiating formal clinical trials. An adequate regulatory and ethical framework as well as stringent criteria for the selection of patients will be critical for the safe clinical development of cardiac xenotransplantation, which we expect will be clinically tested during the next few years. [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]

The clinical course of the first patient to receive a gene‐edited pig heart transplant was recently reported by the University of Maryland team. Although the pig heart functioned well for >40 days, serum anti‐pig antibodies then increased, and the patient sadly died after 60 days. Because of his debilitated pre‐transplant state, the patient never thrived despite excellent graft function for several weeks, and the cause of his demise continues to be uncertain. A few days before an increase in anti‐pig antibodies was observed, the patient had received intravenous human immunoglobulin (IVIg), and whether this played a role in his cardiac deterioration has been discussed. Furthermore, mcfDNA testing indicated an increase in pig cytomegalovirus (CMV), and its possible role in the development of cardiac dysfunction has also been considered. On the basis of the limited data provided in the publication and on our previous investigations into whether IVIg contains anti‐TKO pig antibodies and therefore might be deleterious to TKO pig organ xenografts, we suggest that the steady rise in anti‐pig antibody titer was more consistent with the failure of the immunosuppressive regimen to prevent elicited anti‐TKO pig antibody production, rather than from the passive transfusion of IVIg or the presence of pig CMV in the graft. Although the outcome of the Maryland experience was disappointing, valuable lessons were learned. Our attention was drawn to the potential risks of heart transplantation in a "deconditioned" patient, the administration of IVIg, the transmission of pig CMV, and of the difficulties in interpreting myocardial biopsy findings. [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]

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]

Background: Loss of barrier function when GalTKO.hCD46 porcine lungs are perfused with human blood is associated with coagulation pathway dysregulation, innate immune system activation, and rapid sequestration of human formed blood elements. Here, we evaluate whether genetic expression of human tissue factor pathway inhibitor (hTFPI) and human CD47 (hCD47), alone or with combined selectin and integrin adhesion pathway inhibitors, delays GalTKO.hCD46 porcine lung injury or modulates neutrophil and platelet sequestration. Methods: In a well‐established paired ex vivo lung perfusion model, GalTKO.hCD46.hTFPI.hCD47 transgenic porcine lungs (hTFPI.hCD47, n = 7) were compared to GalTKO.hCD46 lungs (reference, n = 5). All lung donor pigs were treated with a thromboxane synthase inhibitor, anti‐histamine, and anti‐GPIb integrin‐blocking Fab, and were pre‐treated with Desmopressin. In both genotypes, one lung of each pair was additionally treated with PSGL‐1 and GMI‐1271 (P‐ and E‐selectin) and IB4 (CD11b/18 integrin) adhesion inhibitors (n = 6 hTFPI.hCD47, n = 3 reference). Results: All except for two reference lungs did not fail within 480 min when experiments were electively terminated. Selectin and integrin adhesion inhibitors moderately attenuated initial pulmonary vascular resistance (PVR) elevation in hTFPI.hCD47 lungs. Neutrophil sequestration was significantly delayed during the early time points following reperfusion and terminal platelet activation was attenuated in association with lungs expressing hTFPI.hCD47, but additional adhesion pathway inhibitors did not show further effects with either lung genotype. Conclusion: Expression of hTFPI.hCD47 on porcine lung may be useful as part of an integrated strategy to prevent neutrophil adhesion and platelet activation that are associated with xenograft injury. Additionally, targeting canonical selectin and integrin adhesion pathways reduced PVR elevation associated with hTFPI.hCD47 expression, but did not significantly attenuate neutrophil or platelet sequestration. We conclude that other adhesive mechanisms mediate the residual sequestration of human formed blood elements to pig endothelium that occurs even in the context of the multiple genetic modifications and drug treatments tested here. [ABSTRACT FROM AUTHOR]

Summary: The future clinical application of animal‐to‐human transplantation (xenotransplantation) is of importance to society as a whole. Favourable preclinical data relevant to cell, tissue and solid organ xenotransplants have been obtained from many animal models utilizing genetic engineering and protocols of pathogen‐free husbandry. Findings have reached a tipping point, and xenotransplantation of solid organs is approaching clinical evaluation, the process of which now requires close deliberation. Such discussions include considering when there is sufficient evidence from preclinical animal studies to start first‐in‐human xenotransplantation trials. The present article is based on evidence and opinions formulated by members of the European Society for Organ Transplantation who are involved in the Transplantation Learning Journey project. The article includes a brief overview of preclinical concepts and biology of solid organ xenotransplantation, discusses the selection of candidates for first‐in‐human studies and considers requirements for study design and conduct. In addition, the paper emphasizes the need for a regulatory framework for xenotransplantation of solid organs and the essential requirement for input from public and patient stakeholders. [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]

As an academic, Agnès published more than 115 scientific publications in numerous top-tier journals in the fields of xenotransplantation, transplantation, coagulation, and cardiology. Keywords: cardiology; cardiothoracic surgery; immunobiology; transplantation; xenotransplantation EN cardiology cardiothoracic surgery immunobiology transplantation xenotransplantation 1 3 3 04/12/21 20210301 NES 210301 GLO:FSO/01mar21:xen12689-fig-0001.jpg PHOTO (COLOR): . gl Dr Agnès Marie Azimzadeh, BSc, PhD 1963-2021. During her post-doctoral fellowship (1993-99) while working on xenotransplantation at Strasbourg's Institute of Transplantation with Professors Cazenave and Soulillou, Agnès began to develop her extensive network of national and international collaborators. Agnès published numerous articles in Xenotransplantation, AJT, and Transplantation, with a focus on lung xenotransplantation using both ex vivo (whole blood perfusion) and in vivo (pig-to-baboon) models. [Extracted from the article]

ABSTRACT: BACKGROUND: In this study, we applied behavioral economics to optimize elementary school lunch choices via parent‐driven decisions. Specifically, this experiment tested an optimal defaults paradigm, examining whether strategically manipulating the health value of a default menu could be co‐opted to improve school‐based lunch selections. METHODS: The design was a randomized comparison of optimal versus suboptimal (standard) default lunch menus for all first‐graders in a school district for a period of 1 week. We hypothesized that making the default lunch option healthier, while providing parents the opportunity to access and choose from the standard school menu for their child, would yield more frequent selection of healthier items than when the default option was suboptimal. RESULTS: Overall, 127 (93%) first‐grade children's families participated. Among those families randomized to receive the nutritionally optimized default menu, all but one remained with these options; of those parents randomized to the standard menu (suboptimal default), all parents remained with these options (Χ2 = 123.06, df = 1, p < .001). CONCLUSIONS: The mere positioning of choices, without restricting options, significantly affected which menu items the children received during the test period. Results are proof of concept for a strategy to increase health‐promoting school lunch content, procedures, and policies. [ABSTRACT FROM AUTHOR]

Information about several papers discussed at the Joint U.S. Food and Drug Administration (FDA)-International Xenotransplantation Association (IXA) Symposium held in Baltimore, Maryland on September 20, 2017 is presented. Topics include scientific advances in xenotransplantation, infection and risk-benefit of xenotransplantation, and regulatory considerations of xenotransplantation products. The symposium featured several speakers including David Cooper, Jay Fishman, and Judith Arcidiacono.

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]

Renal failure causes morbidity and mortality after lung transplantation and is aggravated by exposure to nephrotoxic immunosuppressant ( IS) drugs. We report an off-label experience using belatacept for lung transplant recipients with severe renal insufficiency to reduce nephrotoxic IS exposure. We analyzed data retrospectively from a consecutive series of lung transplant patients with renal insufficiency in whom belatacept treatment was initiated between June 2012 and June 2014 at the University of Maryland Medical Center. Eight patients received belatacept because of acute or chronic renal insufficiency (median) GFR 24 ( IQR 18-26). Glomerular filtration rate ( GFR) remained stable in two patients and increased in five. One patient with established renal and respiratory failure received only the induction dose of belatacept and died 4 months later of respiratory and multisystem organ failure. Calcineurin inhibitor or sirolimus exposure was safely withheld or reduced without moderate or severe acute rejection during ongoing belatacept in the other seven patients. FEV1 remained stable over the 6-month study interval. Belatacept use appears to permit safe transient reduction in conventional immunosuppressive therapy and was associated with stable or improved renal function in a small retrospective series of lung transplant recipients with acute or chronic renal insufficiency. [ABSTRACT FROM AUTHOR]

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 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]

Background Dysregulation of coagulation is considered a major barrier against successful pig organ xenotransplantation in non-human primates. Inflammation is known to promote activation of coagulation. The role of pro-inflammatory factors as well as the relationship between inflammation and activation of coagulation in xenograft recipients is poorly understood. Methods Baboons received kidney (n = 3), heart (n = 4), or artery patch (n = 8) xenografts from α1,3-galactosyltransferase gene-knockout ( GTKO) pigs or GTKO pigs additionally transgenic for human complement-regulatory protein CD46 ( GTKO/ CD46). Immunosuppression ( IS) was based on either CTLA4Ig or anti- CD154 costimulation blockade. Three artery patch recipients did not receive IS. Pro-inflammatory cytokines, chemokines, and coagulation parameters were evaluated in the circulation after transplantation. In artery patch recipients, monocytes and dendritic cells ( DC) were monitored in peripheral blood. Expression of tissue factor ( TF) and CD40 on monocytes and DC were assessed by flow cytometry. C-reactive protein ( C- RP) levels in the blood and C- RP deposition in xenografts as well as native organs were evaluated. Baboon and pig C- RP m RNA in heart and kidney xenografts were evaluated. Results In heart and kidney xenograft recipients, the levels of INFγ, TNF-α, IL-12, and IL-8 were not significantly higher after transplantation. However, MCP-1 and IL-6 levels were significantly higher after transplantation, particularly in kidney recipients. Elevated C- RP levels preceded activation of coagulation in heart and kidney recipients, where high levels of C- RP were maintained until the time of euthanasia in both heart and kidney recipients. In artery patch recipients, INFγ, TNF-α, IL-12, IL-8, and MCP-1 were elevated with no IS, while IL-6 was not. With IS, INFγ, TNF-α, IL-12, IL-8, and MCP-1 were reduced, but IL-6 was elevated. Elevated IL-6 levels were observed as early as 2 weeks in artery patch recipients. While IS was associated with reduced thrombin activation, fibrinogen and C- RP levels were increased when IS was given. There was a significant positive correlation between C- RP, IL-6, and fibrinogen levels. Additionally, absolute numbers of monocytes were significantly increased when IS was given, but not without IS. This was associated with increased CD40 and TF expression on CD14+ monocytes and lineageneg CD11c+ DC, with increased differentiation of the pro-inflammatory CD14+ CD11c+ monocyte population. At the time of euthanasia, C- RP deposition in kidney and heart xenografts, C- RP positive cells in artery patch xenograft and native lungs were detected. Finally, high levels of both pig and baboon C- RP m RNA were detected in heart and kidney xenografts. Conclusions Inflammatory responses precede activation of coagulation after organ xenotransplantation. Early upregulation of C- RP and IL-6 levels may amplify activation of coagulation through upregulation of TF on innate immune cells. Prevention of systemic inflammation in xenograft recipients ( SIXR) may be required to prevent dysregulation of coagulation and avoid excessive IS after xenotransplantation. [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]

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]

To develop an international consensus regarding the appropriate conditions for undertaking clinical trials in xenocorneal transplantation, here we review specific ethical, logistical, scientific, and regulatory issues regarding xenocorneal transplantation, and propose guidelines for conduct of clinical xenocorneal transplantation trials. These proposed guidelines are modeled on the published consensus statement of the International Xenotransplantation Association regarding recommended guidelines for conduct of clinical islet xenotransplantation. It is expected that this initial consensus statement will be revised over time in response to scientific advances in the field, and changes in the regulatory framework based on accumulating clinical experience. [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]

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]

Based on cross-sectional analyses, it was suggested that hip circumference divided by height(1.5) -18 (the body adiposity index (BAI)), could directly estimate percent body fat without the need for further correction for sex or age. We compared the prediction of percent body fat, as assessed by dual-energy X-ray absorptiometry (PBF(DXA)), by BAI, BMI, and circumference (waist and hip) measurements among 1,151 adults who had a total body scan by DXA and circumference measurements from 1993 through 2005. After accounting for sex, we found that PBF(DXA) was related similarly to BAI, BMI, waist circumference, and hip circumference. In general, BAI underestimated PBF(DXA) among men (2.5%) and overestimated PBF(DXA) among women (4%), but the magnitudes of these biases varied with the level of body fatness. The addition of covariates and quadratic terms for the body size measures in regression models substantially improved the prediction of PBF(DXA), but none of the models based on BAI could more accurately predict PBF(DXA) than could those based on BMI or circumferences. We conclude that the use of BAI as an indicator of adiposity is likely to produce biased estimates of percent body fat, with the errors varying by sex and level of body fatness. Although regression models that account for the nonlinear association, as well as the influence of sex, age, and race, can yield more accurate estimates of PBF(DXA), estimates based on BAI are not more accurate than those based on BMI, waist circumference, or hip circumference. [ABSTRACT FROM AUTHOR]

Based on cross-sectional analyses, it was suggested that hip circumference divided by height1.5 −18 (the body adiposity index (BAI)), could directly estimate percent body fat without the need for further correction for sex or age. We compared the prediction of percent body fat, as assessed by dual-energy X-ray absorptiometry (PBFDXA), by BAI, BMI, and circumference (waist and hip) measurements among 1,151 adults who had a total body scan by DXA and circumference measurements from 1993 through 2005. After accounting for sex, we found that PBFDXA was related similarly to BAI, BMI, waist circumference, and hip circumference. In general, BAI underestimated PBFDXA among men (2.5%) and overestimated PBFDXA among women (4%), but the magnitudes of these biases varied with the level of body fatness. The addition of covariates and quadratic terms for the body size measures in regression models substantially improved the prediction of PBFDXA, but none of the models based on BAI could more accurately predict PBFDXA than could those based on BMI or circumferences. We conclude that the use of BAI as an indicator of adiposity is likely to produce biased estimates of percent body fat, with the errors varying by sex and level of body fatness. Although regression models that account for the nonlinear association, as well as the influence of sex, age, and race, can yield more accurate estimates of PBFDXA, estimates based on BAI are not more accurate than those based on BMI, waist circumference, or hip circumference. [ABSTRACT FROM AUTHOR]

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]

Xenotransplantation is one promising approach to bridge the gap between available human cells, tissues, and organs and the needs of patients with diabetes or end-stage organ failure. Based on recent progress using genetically modified source pigs, improving results with conventional and experimental immunosuppression, and expanded understanding of residual physiologic hurdles, xenotransplantation appears likely to be evaluated in clinical trials in the near future for some select applications. This review offers a comprehensive overview of known mechanisms of xenograft injury, a contemporary assessment of preclinical progress and residual barriers, and our opinions regarding where breakthroughs are likely to occur. [ABSTRACT FROM AUTHOR]

BACKGROUND: Aspirin is the only drug proven to reduce saphenous vein graft (SVG) failure, but aspirin resistance (ASA-R) frequently occurs after off-pump coronary artery bypass grafting (OPCAB). The factors, mechanism, and best means for preventing and/or treating ASA-R have not been established. This study hypothesizes that thrombin production during OPCAB stimulates this acquired ASA-R. STUDY DESIGN AND METHODS: A nonrandomized prospective cohort of 255 patients (n = 465 SVG) who underwent OPCAB with varied use of aprotinin (21%) and different SVG preparation techniques (standard, 56% vs. low-pressure, 44%) was analyzed. A surplus SVG segment was obtained to assess endothelial integrity. ASA-R was determined at baseline, after surgery, and on Days 1 and 3 by three assays. The effects of aprotinin on thrombin responsiveness were analyzed by means of whole-blood aggregometry, SVG tissue factor (TF) activity, and transcardiac thrombin production (i.e., F1.2 levels in aorta versus coronary sinus). SVG patency was assessed on Day 5 with multichannel CT angiography. RESULTS: ASA-R developed in 42 percent of patients after OPCAB. Multivariate analysis showed that ASA-R, endothelial integrity, and target size independently predicted early SVG failure. Aprotinin use was associated with: 1) reduced postoperative ASA-R (15%); 2) decreased platelet (PLT) response to thrombin; 3) reduced TF activity within SVG segments; 4) decreased transcardiac thrombin gradient; and 5) improved SVG patency. CONCLUSION: ASA-R is a common post-OPCAB event whose frequency may be reduced by intraoperative use of aprotinin, possibly via TF and thrombin suppression. Improved perioperative PLT function after OPCAB may also inadvertently enhance the clinical relevance of these potential antithrombotic effects. [ABSTRACT FROM AUTHOR]

Background: Although the body mass index (BMI, kg m-2) is widely used as a measure of adiposity, it is a measure of excess weight, rather than excess body fat. It has been suggested that skinfold thicknesses be measured among overweight children to confirm the presence of excess adiposity. Objective: The present study examined the additional information provided by skinfold thicknesses on body fatness, beyond that conveyed by BMI-for-age, among healthy 5- to 18-years old (n = 1196). Methods and procedures: Total body dual-energy X-ray absorptiometry (DXA) provided estimates of % body fat, and the sum of two skinfolds (triceps and subscapular) was used as an indicator of the overall skinfold thickness. Results: As assessed by the multiple R2s and the residuals of various regression models, information on the skinfold sum significantly ( p < 0.001) improved the prediction of body fatness beyond that obtained with BMI-for-age. For example, the use of the skinfold sum, in addition to BMI-for-age, increased the multiple R2s for predicting % body fat from 0.81 to 0.90 (boys), and from 0.82 to 0.89 (girls). The use of the skinfold sum also reduced the overall prediction errors (absolute value of the residuals) for % body fat by 20-30%, but these reductions varied substantially by BMI-for-age. Among overweight children, defined by a BMI-for-age ≥95th percentile, the skinfold sum reduced the predication errors for % body fat by only 7-9%. Conclusions: Although skinfold thicknesses, when used in addition to BMI-for-age, can substantially improve the estimation of body fatness, the improvement among overweight children is small. [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]

Objectives: The aim of this study was to examine the relationship of serum Mg with stage of diabetes measured by fasting serum glucose in a cohort of 485 African American and Hispanic adults.Methods: The cross sectional Rosetta study was designed to assess body composition in a multi-racial cohort of healthy adults living in New York City. The data utilized for the current analyses were collected during the years 1990 to 2000. Serum Mg and glucose were measured after a 10-12 hour fast. Dual-energy x-ray absorptiometry was used to measure fat mass (FM) and fat free mass (FFM).Results: The mean age of the cohort was 53 +/- 16 years. Hispanics had significantly lower (p < 0.05) mean serum Mg levels (0.82 +/- 0.07 mmol/L vs. 0.85 +/- 0.07 mmol/L) and FFM (48.8 +/- 10.9 kg vs. 50.9 +/- 10.3 kg) compared to African Americans. In both race/ethnic groups, individuals classified as having diabetes had significantly (p < 0.001) lower serum concentrations of Mg (0.80 +/- 0.07 mmol/L) compared to the normal group (0.84 +/- 0.07 mmol/L).Conclusions: These results show that in African American and Hispanic adults, those with diabetes have lower serum Mg levels compared to those classified as pre-diabetic or those with normal fasting glucose levels. [ABSTRACT FROM AUTHOR]

The article comments on the article "Primate T-cell Responses to Porcine Antigens: Implications for Clinical Xenotransplantation," by E. Davila, G. W. Bryne and colleagues. According to the author, Davila and colleagues have contirbuted a number of interesting new findings regarding the responses of T-cell to porcine antigens. Extensive evidence has shown that the xenogeneic T-cell response is particularly vulnerable to interruption.