Expression of human thrombomodulin by GalTKO.hCD46 pigs modulates coagulation cascade activation by endothelial cells and during ex vivo lung perfusion with human blood.

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).

Increased human complement pathway regulatory protein gene dose is associated with increased endothelial expression and prolonged survival during ex-vivo perfusion of GTKO pig lungs with human blood.

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.

Knock-out of N-glycolylneuraminic acid attenuates antibody-mediated rejection in xenogenically perfused porcine lungs.

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.

Thromboxane and histamine mediate PVR elevation during xenogeneic pig lung perfusion with human blood.

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.

Pilot Study of Delayed ICOS/ICOS-L Blockade With αCD40 to Modulate Pathogenic Alloimmunity in a Primate Cardiac Allograft Model.

Background: Inducible costimulator (ICOS) is rapidly upregulated with T-cell stimulation and may represent an escape pathway for T-cell costimulation in the setting of CD40/CD154 costimulation blockade. Induction treatment exhibited no efficacy in a primate renal allograft model, but rodent transplant models suggest that the addition of delayed ICOS/ICOS-L blockade may prolong allograft survival and prevent chronic rejection. Here, we ask whether ICOS-Ig treatment, timed to anticipate ICOS upregulation, prolongs NHP cardiac allograft survival or attenuates pathogenic alloimmunity.

Interleukin-8 mediates neutrophil-endothelial interactions in pig-to-human xenogeneic models.

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.

Selective CD28 Inhibition Modulates Alloimmunity and Cardiac Allograft Vasculopathy in Anti-CD154-Treated Monkeys.

Background: Selective CD28 inhibition is actively pursued as an alternative to B7 blockade using cytotoxic T lymphocyte antigen 4 Ig based on the hypothesis that the checkpoint immune regulators cytotoxic T lymphocyte antigen 4 and programmed death ligand 1 will induce tolerogenic immune signals. We previously showed that blocking CD28 using a monovalent nonactivating reagent (single-chain anti-CD28 Fv fragment linked to alpha-1 antitrypsin [sc28AT]) synergizes with calcineurin inhibitors in nonhuman primate (NHP) kidney and heart transplantation. Here, we explored the efficacy of combining a 3-week "induction" sc28AT treatment with prolonged CD154 blockade.

N-glycolylneuraminic acid knockout reduces erythrocyte sequestration and thromboxane elaboration in an ex vivo pig-to-human xenoperfusion model.

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.

Comparative Evaluation of αCD40 (2C10R4) and αCD154 (5C8H1 and IDEC-131) in a Nonhuman Primate Cardiac Allotransplant Model.

Background: Specific blockade of T cell costimulation pathway is a promising immunomodulatory approach being developed to replace our current clinical immunosuppression therapies. The goal of this study is to compare results associated with 3 monoclonal antibodies directed against the CD40/CD154 T cell costimulation pathway.

Methods: Cynomolgus monkey heterotopic cardiac allograft recipients were treated with either IDEC-131 (humanized αCD154, n = 9), 5C8H1 (mouse-human chimeric αCD154, n = 5), or 2C10R4 (mouse-rhesus chimeric αCD40, n = 6) monotherapy using a consistent, comparable dosing regimen for 3 months after transplant.

Transgenic expression of human leukocyte antigen-E attenuates GalKO.hCD46 porcine lung xenograft injury.

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 (GalTKO) and express human membrane cofactor protein (hCD46). Natural killer cells rapidly disappear from the blood during perfusion of GalTKO.hCD46 porcine lungs with human blood and presumably are sequestered within the lung vasculature.

Preemptive CD20+ B cell Depletion Attenuates Cardiac Allograft Vasculopathy in CD154-Treated Monkeys.

Background: Anti-CD154 monotherapy is associated with antidonor allo-antibody (Ab) elaboration, cardiac allograft vasculopathy (CAV), and allograft failure in preclinical primate cell and organ transplant models. In the context of calcineurin inhibitors (CNI), these pathogenic phenomena are delayed by preemptive "induction" B cell depletion.

Methods: αCD154 (IDEC-131)-treated cynomolgus monkey heart allograft recipients were given peritransplant rituximab (αCD20) alone or with rabbit antihuman thymocyte globulin.

Nucleosome Positioning of Intronless Genes in the Human Genome.

Nucleosomes, the basic units of chromatin, are involved in transcription regulation and DNA replication. Intronless genes, which constitute 3 percent of the human genome, differ from intron-containing genes in evolution and function. Our analysis reveals that nucleosome positioning shows a distinct pattern in intronless and intron-containing genes.

Early graft failure of GalTKO pig organs in baboons is reduced by expression of a human complement pathway-regulatory protein.

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 GalTKO pig organs transplanted into baboons in three centers, and the effect of additional expression of a human complement pathway-regulatory protein, CD46 or CD55 (GalTKO.hCPRP). Baboon recipients of life-supporting GalTKO 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).

Four-dimensional characterization of thrombosis in a live-cell, shear-flow assay: development and application to xenotransplantation.

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.

Development of a consensus protocol to quantify primate anti-non-Gal xenoreactive antibodies using pig aortic endothelial cells.

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 GalTKO 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.

Nucleosome organization in the vicinity of transcription factor binding sites in the human genome.

Background: The binding of transcription factors (TFs) to specific DNA sequences is an initial and crucial step of transcription. In eukaryotes, this process is highly dependent on the local chromatin state, which can be modified by recruiting chromatin remodelers. However, previous studies have focused mainly on nucleosome occupancy around the TF binding sites (TFBSs) of a few specific TFs.

Pig-to-baboon liver xenoperfusion utilizing GalTKO.hCD46 pigs and glycoprotein Ib blockade.

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.

Preemptive CD20+ B cell depletion attenuates cardiac allograft vasculopathy in cyclosporine-treated monkeys.

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.

Preliminary findings on the use of pulsatile machine reperfusion of a placenta to improve the cord blood collection yield including primitive hematopoietic stem cell fractions.

Background: Umbilical cord blood (CB) use is limited in most adults since the mean amount of hematopoietic stem cells (HSCs) collected is generally insufficient to engraft successfully. This study demonstrates for the first time the feasibility of pulsatile machine placenta reperfusion (PMPR), which significantly improves CB collection quantity and quality compared to standard collection methods.

Study Design And Methods: PMPR was performed on eight delivered placentas up to 39 hours after venipuncture-based collection.

RCAS/SCL-TVA animal model allows targeted delivery of polyoma middle T oncogene to vascular endothelial progenitors in vivo and results in hemangioma development.

Purpose: To recapitulate the generation of cancer stem cells in the context of an intact animal using a retroviral vector capable of in vivo delivery of oncogenes to primitive endothelial and hematopoietic stem cells.

Experimental Design: Targeting of these progenitors was achieved using transgenic mice in which the avian TVA retroviral receptor was placed under the control of the stem cell leukemia (scl/tal-1) gene promoter and SCL +19 enhancer.

Results: Injection of an avian retrovirus encoding polyoma middle T (PyMT), an oncogene that transforms endothelial cells, caused rapid lethality in all SCL-TVA mice but not in control TVA(-) littermates.

Human brain endothelial cells (HUBEC) promote SCID repopulating cell expansion through direct contact.

The objective of this study was to re-evaluate the previously published hematopoietic stem cell (HSC) expansion work using human brain endothelial cells (HUBEC). The expansion effect of contact and non-contact conditions was reported to be equivalent by others. However, we report here different results that the expansion can be achieved only with direct contact.

IMP dehydrogenase inhibitor mycophenolate mofetil induces caspase-dependent apoptosis and cell cycle inhibition in multiple myeloma cells.

Multiple myeloma is an incurable disease for the majority of patients, therefore requiring new biological targeted therapies. In primary myeloma cells, IMP dehydrogenase (IMPDH) was shown to be consistently overexpressed. We therefore tested the IMPDH inhibitor mycophenolate mofetil (MMF) currently available as a clinical therapeutic agent for its antimyeloma activity in vitro.

Phase I clinical trial of the inosine monophosphate dehydrogenase inhibitor mycophenolate mofetil (cellcept) in advanced multiple myeloma patients.

Purpose: Inosine monophosphate dehydrogenase (IMPDH) inhibitors have been used to induce leukemia blast cell differentiation but have not been tested in multiple myeloma for activity. Currently, available IMPDH inhibitor, mycophenolate mofetil (MMF), which is known as an immunosuppressant, was shown to induce apoptosis in myeloma cell lines. On the basis of our preclinical studies, we designed a clinical study to test our hypothesis that MMF has antimyeloma activity.