Gheorghe Braileanu

DNA Microarray Analysis

UV-B regulated clones identified with DNA microarray analysis. RNA was isolated from leaves of Arabidopsis thaliana Columbia 0 plants irradiated with UV-BBE= 0.137 W/m 2 for 3 hours. A positive value indicates induction (up-regulation) whereas a negative value indicates repression (down-regulation) of mRNA transcripts. Genes marked in red were used for northern blot analysis. The annotation is based on sequencing from Monsanto (in black) or in our department (in red).

Expression of human CD46 modulates inflammation associated with GalTKO lung xenograft injury

Evaluation of lungs from GalTKO.hCD46 pigs, genetically modified to lack the galactose‐α(1,3)‐galactose epitope (GalTKO) and to express human CD46, a complement regulatory protein, has not previously been described. Physiologic, hematologic and biochemical parameters during perfusion with heparinized fresh human blood were measured for 33 GalTKO.hCD46, GalTKO (n = 16), and WT pig lungs (n = 16), and 12 pig lungs perfused with autologous pig blood. Median GalTKO.hCD46 lung survival was 171 min compared to 120 for GalTKO (p = 0.27) and 10 for WT lungs (p < 0.001). Complement activation, platelet activation and histamine elaboration were significantly reduced during the first 2 h of perfusion in GalTKO.hCD46 lungs compared to GalTKO (ΔC3a at 120′ 812 ± 230 vs. 1412 ± 1047, p = 0.02; ΔCD62P at 120′ 9.8 ± 7.2 vs. 25.4 ± 18.2, p < 0.01; Δhistamine at 60′ 97 ± 62 vs. 189 ± 194, p = 0.03). We conclude that, in addition to significant down‐modulation of complement activation, hCD46 expression in GalTKO lungs diminished platelet and coagulation cascade activation, neutrophil sequestration and histamine release. Because GalTKO.hCD46 lung failure kinetics correlated directly with platelet and neutrophil sequestration, coagulation cascade activation and a rise in histamine levels within the first hour of perfusion, further progress will likely depend upon improved control of these pathways, by rationally targeted additional modifications to pigs and pharmacologic interventions.

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

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. Here we asked whether porcine expression of the human NK cell inhibitory ligand HLA‐E and β2 microglobulin inhibits GalTKO.hCD46 pig cell injury or prolongs lung function in two preclinical perfusion models.

Live Colonocytes in Newborn Stool: Surrogates for Evaluation of Gut Physiology and Disease Pathogenesis

Studies of gastrointestinal pathophysiology are not feasible by biopsies in human neonates. We examined the utility of live colonocytes in stool in studying cellular markers during early neonatal life. Expression of IgA, IgG, cluster of differentiation-45 cells (CD45), and toll-like receptors-2 and 4 (TLR2 and TLR4) were analyzed by flow cytometry. Colonocyte RNA extracts were used in quantitative real-time PCR (qRT-PCR) to examine the expression of cytokeratin-19, ribosomal protein-24, and tight-junction (Tj) protein zonula occludens-1 (ZO-1). Colonocyte yield varied between 5 × 104 to 2 × 106 cells/g of stool. Meconium samples yielded a highly enriched population of viable cells. Although low, all samples showed CD45-positive cells during the initial weeks of life. Starting as early as d 2, IgA expression was observed in 69% of the cells. Low to moderate expression of IgG was observed with a linear increase as the infants grew. There was an almost total lack of TLR2 staining; however, >55% of the colonocytes showed TLR4 expression. Although high levels of IgA in gut cells may serve as a natural protectant during neonatal period, increased TLR4 may provide a niche for lipopolysaccharide (LPS)-mediated epithelial damage. Use of stool colonocytes can be a valuable noninvasive approach for studying gut pathophysiology in the neonatal period.

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

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.

Platelet sequestration and activation during GalTKO.hCD46 pig lung perfusion by human blood is primarily mediated by GPIb, GPIIb/IIIa, and von Willebrand Factor

Here, we ask whether platelet GPIb and GPIIb/IIIa receptors modulate platelet sequestration and activation during GalTKO.hCD46 pig lung xenograft perfusion.

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

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.

Genetic Engineering in Xenotransplantation: What Human Transgenes Should the GTKO.hCD46 Pig Lung Donor Additionally Express?

Background Recent advances in genetic engineering have enabled generation of pigs expressing multiple transgenes as potential donors for xenotransplantation. What transgenes contribute to protect xenogeneic lungs from being injured by known xenorejection mechanisms, has not previously been systematically evaluated. Here we summarize results directly testing several individual transgenes on a platform GTKO.hCD46 in a xenogeneic lung perfusion model. Methods GTKO.hCD46 pig lungs, additionally expressing either hEPCR, HLAE, hvWF, hTBM, hCD55 or including Neu5GC knock-out were perfused with fresh heparinized human blood for up to 8 hours. Functional parameters, as well as blood and tissue samples were analyzed and compared to lungs that did not have the respective additional genetic modification. Results Lung “survival” was significantly increased in lungs with hEPCR and HLA-E. While HLA-E, hEPCR and hTBM expression led to reduced BTG elaboration, platelet sequestration was only markedly reduced by the expression of humanized vWF. Pulmonary vascular resistance as well as histamine elaboration showed significantly lower values in lungs with HLA-E or Neu5GcKO. The knock-out of the Neu5Gc epitope also significantly reduced thromboxane levels and activation of the coagulation cascade (F1+2). Conclusion Several mechanisms associated with GTKO.hCD46 xenolung injury are modulated by additional expression of individual pathway-targeting human transgenes. We conclude that each of the pathways targeted by the transgenes tested, including Neu5GcKO, provide protection from non-Gal-antibody-mediated lung rejection mechanisms. We hypothesize that combinations of the beneficial genetic modifications tested here will result in further improvement of xenogeneic lung function and extension of graft survival. United Therapeutics SRA. NIH U19A1090959.

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 &agr;CD154, n = 9), 5C8H1 (mouse-human chimeric &agr;CD154, n = 5), or 2C10R4 (mouse-rhesus chimeric &agr;CD40, 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 (35 ± 31 days) was significantly prolonged in both 5C8H1-treated (142 ± 26, P < 0.002) and 2C10R4-treated (124 ± 37, P < 0.020) allografts. IDEC-131-treated grafts had higher cardiac allograft vasculopathy 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 alloantibody (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 day 14 after transplant (−457 ± 152 cells/&mgr;L) compared with 5C8H1-treated animals (16 ± 25, P = 0.037), and the resurgent B cells were primarily of a naive phenotype. Conclusions 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 cardiac allograft vasculopathy development and antidonor alloAb production during treatment.