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We conclude human NK cell activation contributes significantly to GalTKO.hCD46 pig endothelial injury and lung inflammation and show that expression of HLA-E is associated with physiologically meaningful protection of GalTKO.hCD46 cells and organs exposed to human blood.

The roles of the large membrane surface of the oxygenator and the high mechanical shear stress (HMSS) of the pump in the extracorporeal membrane oxygenation (ECMO) circuit were examined under a pediatric support setting. A clinical centrifugal pump and a pediatric oxygenator were used to construct the ECMO circuit. An identical circuit without the oxygenator was constructed for comparison. Fresh human blood was circulated in the two circuits for 4 hours under the identical pump speed and flow. Blood samples were collected hourly for blood damage assessment, including platelet activation, generation of platelet‐derived microparticles (PDMP), losses of key platelet hemostasis receptors (glycoprotein (GP) Ibα (GPIbα) and GPVI), and high molecular weight multimers (HMWM) of von Willebrand factor (VWF) and plasma free hemoglobin (PFH). Platelet adhesion on fibrinogen, VWF, and collagen was further examined. The levels of platelet activation and generation of PDMP and PFH exhibited an increasing trend with circulation time while the expression levels of GPIbα and GPVI receptors on the platelet surface decreased. Correspondingly, the platelets in the blood samples exhibited increased adhesion capacity to fibrinogen and decreased adhesion capacities on VWF and collagen with circulation time. Loss of HMWM of VWF occurred in both circuits. No statistically significant differences were found in all the measured parameters for blood damage and platelet adhesion function between the two circuits. The results indicate that HMSS from the pump played a dominant role in blood damage associated with ECMO and the impact of the large surface of the oxygenator on blood damage was insignificant.

Administration of donor-derived immature dendritic cells (DC) can prolong the survival of MHC-mismatched cardiac allografts. Genetic modification of DC by immunosuppressive molecules can enhance their potential tolerogenicity. In this study bone marrow derived immature DC were genetically modified by transforming growth factor (TGF) beta1 by recombinant Ad. TGF-beta(1) gene modified immature DC (TGF-beta-DC) displayed a characteristic phenotype of immature DC, decreased ability to secrete interleukin 12, and reduced allostimulatory ability. TGF-beta-DC induced alloantigen-specific T cell hyporesponsiveness in vitro and in vivo, and Th2 cytokine polarization. mRNA expression of donor MHC class II (Ia(b)) and human TGF-beta(1) was detected in spleen and lymph nodes of the allogeneic recipients for 3 weeks after TGF-beta-DC infusion, indicating that microchimerism of TGF-beta-DC is exhibited in allogeneic recipients. In this murine cervical heterotopic heart transplantation model, the survival of the allograft in recipients intravenously infused with TGF-beta-DC 7 days before transplantation was greatly prolonged, and about 67% of cardiac grafts survived more than 40 days. Histological analysis of the allografts showed that the normal myocardial architecture was well preserved, accompanied by very little necrotic cells, but interstitial fibrosis replaced myocytes, and moderate collagen suffused the whole cardiac allograft in the recipients infused with TGF-beta-DC. mRNA expression of type III procollagen was markedly increased in the allografts of the recipients infused with TGF-beta-DC. Our results suggest that infusion of TGF-beta(1) gene modified immature DC prolongs the survival of the allograft through the effective induction of donor-specific T cell hyporesponsiveness. However, TGF-beta(1) expressed by gene modified immature DC can cause the fibrosis of the allografts, which may limit the application of this approach in the allograft transplantation.

Natural killer T (NKT) cells play a critical role in the host's innate immune response. CD1d-mediated presentation of glycolipid antigens to NKT cells has been established; however, the mechanisms by which NKT cells recognize infected or cancerous cells remain unclear. 5(')-AMP activated protein kinase (AMPK) is a master regulator of lipogenic pathways. We hypothesized that activation of AMPK during infection and malignancy could alter the repertoire of antigens presented by CD1d and serve as a danger signal to NKT cells. In this study, we examined the effect of alterations in metabolism on CD1d-mediated antigen presentation to NKT cells and found that an infection with lymphocytic choriomeningitis virus rapidly increased CD1d-mediated antigen presentation. Hypoxia inducible factors (HIF) enhance T-cell effector functions during infection, therefore antigen presenting cells pretreated with pharmacological agents that inhibit glycolysis, induce HIF and activate AMPK were assessed for their ability to induce NKT-cell responses. Pretreatment with 2-deoxyglucose, cobalt chloride, AICAR and metformin significantly enhanced CD1d-mediated NKT-cell activation. In addition, NKT cells preferentially respond to malignant B cells and B-cell lymphomas express HIF-1α. These data suggest that targeting cellular metabolism may serve as a novel means of inducing innate immune responses.

acids on glycoconjugates play a pivotal role in many biological processes. In the airways, sialylated glycoproteins and glycolipids are strategically positioned on the plasma membranes of epithelia to regulate receptor-ligand, cell-cell, and host-pathogen interactions at the molecular level. We now demonstrate, for the first time, sialidase activity for ganglioside substrates in human airway epithelia. Of the four known mammalian sialidases, NEU3 has a substrate preference for gangliosides and is expressed at mRNA and protein levels at comparable abundance in epithelia derived from human trachea, bronchi, small airways, and alveoli. In small airway and alveolar epithelia, NEU3 protein was immunolocalized to the plasma membrane, cytosolic, and nuclear subcellular fractions. Small interfering RNA-induced silencing of NEU3 expression diminished sialidase activity for a ganglioside substrate by Ͼ70%. NEU3 immunostaining of intact human lung tissue could be localized to the superficial epithelia, including the ciliated brush border, as well as to nuclei. However, NEU3 was reduced in subepithelial tissues. These results indicate that human airway epithelia express catalytically active NEU3 sialidase.

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 chimericαCD40, n=6) monotherapy using a consistent, comparable dosing regimen for 3 months after transplant. Results Relative to the previously reported IDEC-131 treated allografts, median survival time (MST 35±31 days) was significantly prolonged in both 5C8H1 (142±26, p<0.002) and 2C10R4 (124±37, p<0.020) treated allografts. IDEC-131 treated grafts had higher CAV severity scores during treatment relative to either 5C8H1 (p=0.008) or 2C10R4 (p=0.0002). Both 5C8H1(5 of 5 animals, p=0.02) and 2C10R4 (6/6, p=0.007), but not IDEC-131 (2/9), completely attenuated IgM antidonor alloAb production during treatment;5C8H1 (5/5) more consistently attenuated IgG alloAb production compared to 2C10R4 (4/6) and IDEC-131 (0/9). All evaluable explanted grafts experienced antibody-mediated rejection. Only 2C10R4 treated animals exhibited a modest, transient drop in CD20+ lymphocytes from baseline at d14 after transplant (-457±152 cells/μL) compared to 5C8H1 treated animals (16±25, p=0.037), and the resurgent B cells were primarily of a naïve phenotype. Conclusion In this model, CD154/CD40 axis blockade using IDEC-131 is an inferior immunomodulatory treatment than 5C8H1 or 2C10R4, which have similar efficacy to prolong graft survival and to delay CAV development and antidonor alloAb production during treatment.

Blockade of CD40 pathway with anti-CD40L mAb potentiates the tolerogenic potential of DC-CTLA4Ig and enhances the induction of antigen-specific immune tolerance more effectively.