Myoblasts Survive Intracardiac Transfer and Divide Further After Transplantation

The Heart Surgery Forum. 2002  |  Pubmed ID: 12538115

Skeletal myoblasts have been proven to survive transplantation into myocardial scar tissue. The objective of this study was to evaluate whether these cells can also be transferred into vital myocardium and maintain their ability for cell division after transplantation. In addition, an intravital fluorescence dye for marking these cells was evaluated.

New Technique for Chest Opening in Mice: U-sternotomy

Microsurgery. 2003  |  Pubmed ID: 12833331

In cardiac xenotransplantation, transfections with recombinant adeno-associated viruses could be a promising way of modulating the cardiomyocyte genome. Proteins like TRAIL, hDAF, Il-10, Il-4, and beta-galactosidase may be expressed on cardiomyocyte surfaces in order to prevent cellular rejection processes. However, after intracoronary perfusion with transfecting viruses, it takes up to 4 weeks before expression of the transgene can be detected. Mouse models have been established to create reliable transfection protocols. Currently, the major problem involves performing the intraaortic injection in mice without causing a lethal pneumothorax. Here we describe a new technique for chest opening to safely reach the mouse ascending aorta without opening the pleural space. This U-sternotomy reduces the risk of animal death and therefore the amount of quite expensive virus solutions needed.

Successful Resection of a Symptomatic Right Ventricular Lipoma

The Annals of Thoracic Surgery. Oct, 2003  |  Pubmed ID: 14530040

We report the case of a 31-year-old woman with a 4-year history of recurrent palpitations, presenting with an increased frequency of paroxysms caused by ventricular tachycardias during pregnancy. A cardiac tumor of unknown origin infiltrating the right ventricle was diagnosed. Three weeks after prophylactic abrasion the tumor was totally excised with the use of cardiopulmonary bypass including restoration of the right ventricular wall and the tricuspid valve. Histology confirmed diagnosis of a benign cardiac lipoma. The postoperative course was uneventful and the patient was discharged 7 days after surgery. There was no episode of ventricular tachycardias during the 6-month follow-up.

Immunosuppression with FK778 and Mycophenolate Mofetil in a Rat Cardiac Transplantation Model

Transplantation. Dec, 2003  |  Pubmed ID: 14702537

FK778 blocks the dihydro-orotate dehydrogenase, necessary for pyrimidine synthesis, and mycophenolate mofetil (MMF) inhibits the inosine monophosphate dehydrogenase, a crucial enzyme for purine biosynthesis. Beneficial immunosuppressive effects have been suggested for the combination of both drugs. The Brown Norway-Lewis rat heterotopic heart transplantation model was used. FK778 (5 and 20 mg/kg/day), MMF (10 and 40 mg/kg/day), or a combination of both drugs for 10 days was used for prevention of acute graft rejection. Grafts of untreated animals were rejected after 6.2 +/- 0.4 days. Low-dose FK778 and low-dose MMF administration did not result in a significantly prolonged graft survival (6.7 +/- 0.8 and 8.7 +/- 1.4 days; P=not significant). Grafts of rats treated with high-dose FK778 or high-dose MMF survived significantly longer (17.0 +/- 2.8 and 20.7 +/- 3.8 days; P<0.01). Concomitant use of low-dose FK778 with low-dose MMF produced synergistic interactions (mean survival time 12.3 +/- 2.9 days; P<0.01; combination index=0.85). High-dose drug combination (mean survival time 24.0 +/- 1.4 days) showed antagonistic drug interaction (combination index=1.55) with increased toxic side effects.

The Interaction Between FK778 and Tacrolimus in the Prevention of Rat Cardiac Allograft Rejection is Dose Dependent

Transplantation. Feb, 2004  |  Pubmed ID: 15084926

The synthetic malononitrilamide FK778 inhibits T- and B-cell responsiveness, phagocyte effector function, exerts inhibitory activity against cytomegalovirus, and is thus one of the most promising new immunosuppressive drugs. The aim of this study was to evaluate the combination of FK778 and tacrolimus in a high-responder rat cardiac transplantation model.

FK778 Attenuates Lymphocyte-endothelium Interaction After Cardiac Transplantation: in Vivo and in Vitro Studies

Transplantation. Jul, 2004  |  Pubmed ID: 15257041

The malononitrilamide FK778 is a novel derivate of leflunomide and interacts with T- and B-cell function by inhibiting de novo pyrimidine synthesis. We investigated the effects of FK778 upon acute cardiac allograft rejection and upon adhesion molecule upregulation in experimental transplantation and by using in vitro cell culture.

FK778, a Novel Immunosuppressive Agent, Reduces Early Adhesion Molecule Up-regulation and Prolongs Cardiac Allograft Survival

Transplant International : Official Journal of the European Society for Organ Transplantation. Feb, 2005  |  Pubmed ID: 15691275

The adhesion molecules, P-selectin, ICAM-1, and VCAM-1 are important mediators of T-cell adhesion and T-cell co-stimulation. We investigated the effect of the malononitrilamide FK778 on cardiac allograft survival, acute allograft rejection, and adhesion molecule up-regulation in a heterotopic, cardiac transplantation model. Rats received low- or high-dose FK778 or no treatment. Grafts were harvested on the fifth postoperative day for histologic examinations. To assess allograft survival, recipients were treated for a maximum of 10 days and grafts were harvested after cessation of the contractile activity. FK778 low dose showed a mild but significant decrease in mononuclear infiltration but failed to markedly reduce histologic rejection, adhesion molecule up-regulation, or to prolong allograft survival. However, high-dose FK778 treatment significantly reduced early up-regulation of P-selectin, ICAM-1, and VCAM-1, abolished infiltration, reduced histologic rejection and resulted in prolonged cardiac allograft survival. Therefore, FK778 is a novel, highly desirable immunosuppressive drug for transplantation medicine.

The Phytoestrogen BiochaninA Weakens Acute Cardiac Allograft Rejection Without Affecting the Reproductive System

Transplant Immunology. Oct, 2005  |  Pubmed ID: 16223672

Since the two estrogen receptor isoforms ERalpha and ERbeta have been discovered it is unclear by which receptor immunomodulating or feminizing effects are mediated. In this study, the effects of the two selective ERalpha- and ERbeta-agonists ethinylestradiol and biochaninA, respectively, on acute cardiac allograft rejection, uterus growth, vascular adhesion molecule and MHC-II expression were investigated and verified using in vitro cell culture.

FK778 and Tacrolimus Prevent the Development of Obliterative Airway Disease After Heterotopic Rat Tracheal Transplantation

The Journal of Heart and Lung Transplantation : the Official Publication of the International Society for Heart Transplantation. Nov, 2005  |  Pubmed ID: 16297791

The effectiveness of the novel immunosuppressive agent FK778 and of tacrolimus to prevent the development of obliterative airway disease (OAD) was investigated in an animal model.

Sirolimus and FK778: a Comparison of Two Anti-proliferative Immunosuppressants for Prevention of Experimental Obliterative Airway Disease

Transplant International : Official Journal of the European Society for Organ Transplantation. Apr, 2006  |  Pubmed ID: 16573547

This study examined the efficacies of sirolimus and the novel immunosuppressive agent FK778 to prevent obliterative airway disease (OAD). Tracheae from Brown-Norway donors were heterotopically transplanted in the greater omentum of Lewis rats. Recipients were treated for 28 days with sirolimus (0.5 or 2 mg/kg), FK778 (5 or 20 mg/kg), or combination regimens (0.5 + 5 mg/kg, 2 + 20 mg/kg). Tracheal segments were evaluated for degree of luminal obliteration, percentage of luminal epithelial cell coverage, and peritracheal infiltration. In vitro smooth muscle cell (SMC) proliferation and migration assays were performed to assess direct nonimmune-related effects of the drugs. Sirolimus 2 mg/kg and FK778 20 mg/kg effectively reduced graft infiltration and prevented airway obliteration, whereas FK778 5 mg/kg was insufficient. Sirolimus 0.5 mg/kg at least showed moderate inhibitory effects on luminal obliteration and graft infiltration. Combination regimens revealed no significant beneficial effects. Both sirolimus and FK778 barely showed preserved epithelial coverage. Within the range of relevant concentrations, FK778 showed more potent anti-proliferative and anti-migratory effects on SMC in vitro than sirolimus. Both agents proved effective to prevent OAD development without preserving relevant amounts of epithelium. The anti-proliferative potency on SMCs seems to be an especially important mechanism for FK778. De novo combination regimens revealed no beneficial interaction and thus remain doubtful.

Heparins Increase Endothelial Nitric Oxide Bioavailability by Liberating Vessel-immobilized Myeloperoxidase

Circulation. Apr, 2006  |  Pubmed ID: 16606792

Neutrophils and monocytes are centrally linked to vascular inflammatory disease, and leukocyte-derived myeloperoxidase (MPO) has emerged as an important mechanistic participant in impaired vasomotor function. MPO binds to and transcytoses endothelial cells in a glycosaminoglycan-dependent manner, and MPO binding to the vessel wall is a prerequisite for MPO-dependent oxidation of endothelium-derived nitric oxide (NO) and impairment of endothelial function in animal models. In the present study, we investigated whether heparin mobilizes MPO from vascular compartments in humans and defined whether this translates into increased vascular NO bioavailability and function.

The Selective Estrogen Receptor-beta Agonist Biochanin A Shows Vasculoprotective Effects Without Uterotrophic Activity

Menopause (New York, N.Y.). May-Jun, 2006  |  Pubmed ID: 16735947

Current hormone therapy in postmenopausal women is associated with uterotrophic activity and cancer-promoting effects. In this experimental study, we compared the effects of the selective estrogen-receptor (ER) beta agonist biochanin A, and the selective ERalpha agonist ethinylestradiol, on the development of intimal hyperplasia after balloon injury and on uterus morphology.

Inhibition of Restenosis Development After Mechanical Injury: a New Field of Application for Malononitrilamides?

Cardiology. 2007  |  Pubmed ID: 17028423

To investigate the efficacy of the malononitrilamide FK778 to prevent vascular smooth muscle cell (SMC) migration/proliferation, and vascular fibrosis, the key events in restenosis development using in vivo and in vitro studies.

FK778 in Experimental Xenotransplantation: a Detailed Analysis of Drug Efficacy

The Journal of Heart and Lung Transplantation : the Official Publication of the International Society for Heart Transplantation. Jan, 2007  |  Pubmed ID: 17234520

This study examines the efficacy of FK778 regimens for prevention of different phases of xenograft rejection.

Coincidence of Aortic Valve Stenosis and Regurgitation and Multiple Cardiac Papillary Fibroelastomas in a Young Male Adult

The Journal of Thoracic and Cardiovascular Surgery. Feb, 2007  |  Pubmed ID: 17258602

Experimental Orthotopic Tracheal Transplantation: The Stanford Technique

Microsurgery. 2007  |  Pubmed ID: 17326196

The rat heterotopic tracheal transplantation model is widely used as an experimental model to study the development of obliterative airway disease (OAD) and to assess immunosuppressive strategies for chronic rejection. Despite its widespread application, the heterotopic transplantation model does have a number of limitations like the lack of air flow and mucociliary clearance. The present article provides a detailed description of the surgical technique for orthotopic tracheal transplantations, which may share more similarities with lung transplants in humans. The technique is easy to learn, the procedure is well tolerated by the animals, and the grafts develop OAD lesions similar to those of human obliterative bronchiolitis.

Simplified Protocol to Isolate, Purify, and Culture Expand Mesenchymal Stem Cells

Stem Cells and Development. Feb, 2007  |  Pubmed ID: 17348808

Mesenchymal stem cells (MSCs) are widely used for experimental regenerative strategies. Due to their differentiation capacity into mesenchymal lineages, they are a potential cellular source for tissue regeneration. Because there is no specific antigen that can be used to define MSCs directly, there is no consensus about how to isolate them. Here we describe a simple protocol to isolate, purify, and culture expand murine bone marrow MSCs using magnetic cell sorting and plastic adherence. We further show that cytokine supplementation enhances MSC proliferation without jeopardizing their pluripotency.

Techniques for Experimental Heterotopic and Orthotopic Tracheal Transplantations - When to Use Which Model?

Transplant Immunology. Jun, 2007  |  Pubmed ID: 17493528

Different animal models have been developed to study the pathogenesis and treatment of obliterative airway disease (OAD). Here we describe the techniques of heterotopic and orthotopic tracheal transplantations in the rat, comparing the kinetics of systemic host immune response and of histopathologic OAD development.

Right Atrial Mass After Primary Repair of an Atrial Septal Defect: Thrombus Masquerading As a Myxoma

The Annals of Thoracic Surgery. Nov, 2007  |  Pubmed ID: 17954102

Atrial septal defects are among the most common congenital anomalies requiring surgical repair. Thrombus formation after patch-based repair is a recognized complication, usually manifested by an embolic event. However, thromboembolic complications after primary repair of atrial septal defects are exceedingly rare. We present a 38-year-old woman found to have a right atrial mass diagnosed as a myxoma by echocardiography and magnetic resonance imaging 3 years after primary atrial septal defect repair. However, final pathology revealed an organized thrombus. A review of the literature and clinical management of postoperative atrial thrombi are discussed.

Inhibition of Aldehyde Dehydrogenase Type 2 Attenuates Vasodilatory Action of Nitroglycerin in Human Veins

FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology. Jul, 2008  |  Pubmed ID: 18272654

Recent studies suggest that the mitochondrial aldehyde dehydrogenase (ALDH)2 is involved in vascular bioactivation of nitroglycerin (GTN). However, neither expression of ALDH2 nor its functional role in GTN bioactivation has been reported for the main drug target in humans, namely capacitance vessels. We investigated whether ALDH2 is expressed in human veins and whether inhibition of the enzyme attenuates nitroglycerin effects in these vessels. We determined expression of ALDH2 and dehydrogenase activity in human veins by reverse transcriptase-polymerase chain reaction, Western blotting, and immunofluorescence microscopy. In vitro contraction experiments were performed in the presence or absence of the ALDH inhibitors chloral hydrate, cyanamide, and ethoxycyclopropanol. Concentration response curves were determined for the alpha-agonist phenylephrine, nitroglycerin, and the direct NO donor diethylamine NONOate (DEA-NONOate). ALDH2 expression was largely confined to smooth muscle cells as determined by confocal immunofluorescence microscopy. Contractile responses to phenylephrine were unaffected by all ALDH inhibitors tested. In clear contrast, the ALDH inhibitors significantly reduced the potency of nitroglycerin by approximately 1 order of magnitude (P < or = 0.01). Neither of the inhibitors affected the potency of the direct NO donor DEA-NONOate, which ruled out nonspecific effects on the NO signaling cascade. In human capacitance vessels, ALDH2 is a key enzyme in the biotransformation of the frequently used antianginal drug nitroglycerin.

Failed Induction of Heme Oxygenase 1 in Endothelial Cells Exposed to the Hemoglobin Based Oxygen Carrier Oxyglobin

Artificial Cells, Blood Substitutes, and Immobilization Biotechnology. 2008  |  Pubmed ID: 18293159

We investigated the effect of the bovine hemoglobin based oxygen carrier HBOC-200 (Oxyglobin) in rat aortic endothelial cells (RAEC) on the activation of heme oxygenase 1 (HO-1). RAEC were incubated in the presence of 75 microM (G1) or 225 microM (G2) HBOC-200. The positive control (G3) was performed by incubation with 50 microM Hemin. For negative control (G4) cells were incubated with medium alone. G2 and G3 reached a significant increase of bilirubin concentration compared to G4. A positive HO-1 signal in the Western Blot was seen in G3 12 and 24 h after incubation. The Western Blot of G1, G2 and G4 showed no HO-1 signal. These data suggest that HBOC-200 in the applied dosage cannot induce HO-1 expression in RAEC, and may be degraded by isoenzymes at a lower level.

Introducing the First Polymer-free Leflunomide Eluting Stent

Atherosclerosis. Sep, 2008  |  Pubmed ID: 18295768

We here describe the pharmacological characteristic, in vivo efficacy, and in vitro mechanisms of a polymer-free leflunomide eluting stent in comparison to its rapamycin-coated equivalent.

Prevention and Inhibition but Not Reversion of Chronic Allograft Vasculopathy by FK778

Transplantation. Mar, 2008  |  Pubmed ID: 18360270

This study aimed at investigating the efficacy of the novel immunosuppressant FK778 to prevent the development and progression of chronic allograft vasculopathy (CAV).

Novel Immunosuppression: R348, a JAK3- and Syk-inhibitor Attenuates Acute Cardiac Allograft Rejection

Transplantation. Mar, 2008  |  Pubmed ID: 18360272

Janus kinase (JAK)3 is crucial for signal transduction downstream of various cytokine receptors in immune cells. This is the first report on the novel JAK3 inhibitor R348.

In Vivo Imaging of Embryonic Stem Cells Reveals Patterns of Survival and Immune Rejection Following Transplantation

Stem Cells and Development. Dec, 2008  |  Pubmed ID: 18491958

Embryonic stem cell (ESC)-based transplantation is considered a promising novel therapy for a variety of diseases. This is bolstered by the suggested immune-privileged properties of ESCs. In this study, we used in vivo bioluminescent imaging (BLI) to non-invasively track the fate of transplanted murine ESCs (mESCs), which are stably transduced with a double fusion reporter gene consisting of firefly luciferase (FLuc) and enhanced green fluorescent protein (eGFP). Following syngeneic intramuscular transplantation of 1 x 10(6) mESCs, the cells survived and differentiated into teratomas. In contrast, allogeneic mESC transplants were infiltrated by a variety of inflammatory cells, leading to rejection within 28 days. Acceleration of rejection was observed when mESCs were allotransplanted following prior sensitization of the host. Finally, we demonstrate that the mESC derivatives were more rapidly rejected compared to undifferentiated mESCs. These data show that mESCs do not retain immune-privileged properties in vivo and are subject to immunological rejection as assessed by novel molecular imaging approaches.

Immunosuppressive Therapy Mitigates Immunological Rejection of Human Embryonic Stem Cell Xenografts

Proceedings of the National Academy of Sciences of the United States of America. Sep, 2008  |  Pubmed ID: 18728188

Given their self-renewing and pluripotent capabilities, human embryonic stem cells (hESCs) are well poised as a cellular source for tissue regeneration therapy. However, the host immune response against transplanted hESCs is not well characterized. In fact, controversy remains as to whether hESCs have immune-privileged properties. To address this issue, we used in vivo bioluminescent imaging to track the fate of transplanted hESCs stably transduced with a double-fusion reporter gene consisting of firefly luciferase and enhanced GFP. We show that survival after transplant is significantly limited in immunocompetent as opposed to immunodeficient mice. Repeated transplantation of hESCs into immunocompetent hosts results in accelerated hESC death, suggesting an adaptive donor-specific immune response. Our data demonstrate that transplanted hESCs trigger robust cellular and humoral immune responses, resulting in intragraft infiltration of inflammatory cells and subsequent hESC rejection. Moreover, we have found CD4(+) T cells to be an important modulator of hESC immune-mediated rejection. Finally, we show that immunosuppressive drug regimens can mitigate the anti-hESC immune response and that a regimen of combined tacrolimus and sirolimus therapies significantly prolongs survival of hESCs for up to 28 days. Taken together, these data suggest that hESCs are immunogenic, trigger both cellular and humoral-mediated pathways, and, as a result, are rapidly rejected in xenogeneic hosts. This process can be mitigated by a combined immunosuppressive regimen as assessed by molecular imaging approaches.

Comparison of Different Adult Stem Cell Types for Treatment of Myocardial Ischemia

Circulation. Sep, 2008  |  Pubmed ID: 18824743

A comparative analysis of the efficacy of different cell candidates for the treatment of heart disease remains to be described. This study is designed to evaluate the therapeutic efficacy of 4 cell types in a murine model of myocardial infarction.

Comparison of Transplantation of Adipose Tissue- and Bone Marrow-derived Mesenchymal Stem Cells in the Infarcted Heart

Transplantation. Mar, 2009  |  Pubmed ID: 19295307

Mesenchymal stem cells hold promise for cardiovascular regenerative therapy. Derivation of these cells from the adipose tissue might be easier compared with bone marrow. However, the in vivo fate and function of adipose stromal cells (ASC) in the infarcted heart has never been compared directly to bone marrow-derived mesenchymal cells (MSC).

A Novel JAK3 Inhibitor, R348, Attenuates Chronic Airway Allograft Rejection

Transplantation. Mar, 2009  |  Pubmed ID: 19295308

This study aimed at investigating the role of a novel JAK3 inhibitor, R348, in the prevention of chronic airway allograft rejection.

Hepatocyte Growth Factor or Vascular Endothelial Growth Factor Gene Transfer Maximizes Mesenchymal Stem Cell-based Myocardial Salvage After Acute Myocardial Infarction

Circulation. Sep, 2009  |  Pubmed ID: 19752375

Mesenchymal stem cell (MSC)-based regenerative strategies were investigated to treat acute myocardial infarction and improve left ventricular function.

Poor Functional Recovery After Transplantation of Diabetic Bone Marrow Stem Cells in Ischemic Myocardium

The Journal of Heart and Lung Transplantation : the Official Publication of the International Society for Heart Transplantation. Nov, 2009  |  Pubmed ID: 19782602

Autologous bone marrow mononuclear cell (BMMC) therapy has shown promise for improving cardiac function after myocardial infarction. The efficiency of such therapy for diabetic patients remains unknown.

Mechanisms Behind Local Immunosuppression Using Inhaled Tacrolimus in Preclinical Models of Lung Transplantation

American Journal of Respiratory Cell and Molecular Biology. Oct, 2010  |  Pubmed ID: 19880819

Inhaled immunosuppression with tacrolimus (TAC) is a novel strategy after lung transplantation. Here we investigate the feasibility of tacrolimus delivery via aerosol, assess its immunosuppressive efficacy, reveal possible mechanisms of action, and evaluate its airway toxicity. Rats received 4 mg/kg TAC via oral or inhaled (AER) administration. Pharmacokinetic properties were compared, and in vivo airway toxicity was assessed. Full-thickness human airway epithelium (AE) was grown in vitro at an air-liquid interface. Equal TAC doses (10-1,000 ng) were either added to the bottom chamber (MED) or aerosolized for gas-phase exposure (AER). Airway epithelium TAC absorption, cell toxicity, and interactions of TAC with NFκB activation were studied. Single-photon emission computed tomography demonstrated a linear tracer accumulation within the lungs during TAC inhalation. The AER TAC generated higher lung-tissue concentrations, but blood concentrations that were 11 times lower. Airway histology and gene expression did not reveal drug toxicity after 3 weeks of treatment. In vitro AE exposed to TAC at 10-1,000 ng, orally or AER, maintained its pseudostratified morphology, did not show cell toxicity, and maintained its epithelial integrity, with tight junction formation. The TAC AER-treated AE absorbed the drug from the apical surface and generated lower-chamber TAC concentrations sufficient to suppress activated lymphocytes. Tacrolimus AER was superior to TAC MED at preventing AE IFN-γ, IL-10, IL-13, monocyte chemoattractant protein-1 chemokine (C-C motif) ligand 5 (RANTES) and TNF-α up-regulation. Tacrolimus inhibited airway epithelial cell NFκB activation. In conclusion, TAC can be delivered easily and effectively into the lungs without causing airway toxicity, decreases inflammatory AE cytokine production, and inhibits NFκB activation.

Non-volume-loaded Heart Provides a More Relevant Heterotopic Transplantation Model

Transplant Immunology. May, 2010  |  Pubmed ID: 20403439

We aimed to compare two techniques of heterotopic heart transplantation in rats. Non-volume-loaded (NL) and volume-loaded (VL) models were tested for their physiologic and immunologic properties to assess their suitability for transplant studies.

Sustained Inhibition of Epsilon Protein Kinase C Inhibits Vascular Restenosis After Balloon Injury and Stenting

Circulation. Sep, 2010  |  Pubmed ID: 20837910

ε protein kinase C (εPKC) is involved in vascular smooth muscle cell (VSMC) activation, but little is known about its function in vascular pathology. We aimed at assessing the role of εPKC in the development of restenosis.

Immunogenicity and Immunomodulatory Properties of Umbilical Cord Lining Mesenchymal Stem Cells

Cell Transplantation. 2011  |  Pubmed ID: 21054940

We here present an immunologic head-to-head comparison between human umbilical cord lining mesenchymal stem cells (clMSCs) and adult bone marrow MSCs (bmMSCs) from patients >65 years of age. clMSCs had significantly lower HLA class I expression, higher production of tolerogenic TGF-β and IL-10, and showed significantly faster proliferation. In vitro activation of allogeneic lymphocytes and xenogeneic in vivo immune activation was significantly stronger with bmMSCs, whereas immune recognition of clMSCs was significantly weaker. Thus, bmMSCs were more quickly rejected in immunocompetent mice. IFN-γ at 25 ng/ml increased both immunogenicity by upregulation of HLA class I/ HLA-DR expression and tolerogenicity by increasing intracellular HLA-G and surface HLA-E expression, augmenting TGF-β and IL-10 release, and inducing indoleamine 2,3-dioxygenase (IDO) expression. Higher concentrations of IFN-γ (>50 ng/ml) further enhanced the immunosuppressive phenotype of clMSCs, more strongly downregulating HLA-DR expression and further increasing IDO production (at 500 ng/ml). The net functional immunosuppressive efficacy of MSCs was tested in mixed lymphocyte cultures. Although both clMSCs and bmMSCs significantly reduced in vitro immune activation, clMSCs were significantly more effective than bmMSCs. The veto function of both MSC lines was enhanced in escalating IFN-γ environments. In conclusion, clMSCs show a more beneficial immunogeneic profile and stronger overall immunosuppressive potential than aged bmMSCs.

Prevention of Transplant Coronary Artery Disease by Prenylation Inhibitors

The Journal of Heart and Lung Transplantation : the Official Publication of the International Society for Heart Transplantation. Jul, 2011  |  Pubmed ID: 21458297

In this study we systematically dissect the prenylation pathway to better define the mechanism behind statin inhibition in chronic allograft rejection in heart transplants, or transplant coronary artery disease (TCAD).

Peptide Ligands Incorporated into the Threefold Spike Capsid Domain to Re-direct Gene Transduction of AAV8 and AAV9 in Vivo

PloS One. 2011  |  Pubmed ID: 21850255

Efficiency and specificity of viral vectors are vital issues in gene therapy. Insertion of peptide ligands into the adeno-associated viral (AAV) capsid at receptor binding sites can re-target AAV2-derived vectors to alternative cell types. Also, the use of serotypes AAV8 and -9 is more efficient than AAV2 for gene transfer to certain tissues in vivo. Consequently, re-targeting of these serotypes by ligand insertion could be a promising approach but has not been explored so far. Here, we generated AAV8 and -9 vectors displaying peptides in the threefold spike capsid domain. These peptides had been selected from peptide libraries displayed on capsids of AAV serotype 2 to optimize systemic gene delivery to murine lung tissue and to breast cancer tissue in PymT transgenic mice (PymT). Such peptide insertions at position 590 of the AAV8 capsid and position 589 of the AAV9 capsid changed the transduction properties of both serotypes. However, both peptides inserted in AAV8 did not result in the same changes of tissue tropism as they did in AAV2. While the AAV2 peptides selected on murine lung tissue did not alter tropism of serotypes 8 and -9, insertion of the AAV2-derived peptide selected on breast cancer tissue augmented tumor gene delivery in both serotypes. Further, this peptide mediated a strong but unspecific in vivo gene transfer for AAV8 and abrogated transduction of various control tissues for AAV9. Our findings indicate that peptide insertion into defined sites of AAV8 and -9 capsids can change and improve their efficiency and specificity compared to their wild type variants and to AAV2, making these insertion sites attractive for the generation of novel targeted vectors in these serotypes.

Immunobiology of Naïve and Genetically Modified HLA-class-I-knockdown Human Embryonic Stem Cells

Journal of Cell Science. Sep, 2011  |  Pubmed ID: 21878509

Human embryonic stem cells (hESCs) can serve as a universal cell source for emerging cell or tissue replacement strategies, but immune rejection of hESC derivatives remains an unsolved problem. Here, we sought to describe the mechanisms of rejection for naïve hESCs and upon HLA class I (HLA I) knockdown (hESC(KD)). hESCs were HLA I-positive but negative for HLA II and co-stimulatory molecules. Transplantation of naïve hESC into immunocompetent Balb/c mice induced substantial T helper cell 1 and 2 (Th1 and Th2) responses with rapid cell death, but hESCs survived in immunodeficient SCID-beige recipients. Histology revealed mainly macrophages and T cells, but only scattered natural killer (NK) cells. A surge of hESC-specific antibodies against hESC class I, but not class II antigens, was observed. Using HLA I RNA interference and intrabody technology, HLA I surface expression of hESC(KD) was 88%-99% reduced. T cell activation after hESC(KD) transplantation into Balb/c was significantly diminished, antibody production was substantially alleviated, the levels of graft-infiltrating immune cells were reduced and the survival of hESC(KD) was prolonged. Because of their very low expression of stimulatory NK ligands, NK-susceptibility of naïve hESCs and hESC(KD) was negligible. Thus, HLA I recognition by T cells seems to be the primary mechanism of hESC recognition, and T cells, macrophages and hESC-specific antibodies participate in hESC killing.

Human Leukocyte Antigen I Knockdown Human Embryonic Stem Cells Induce Host Ignorance and Achieve Prolonged Xenogeneic Survival

Circulation. Sep, 2011  |  Pubmed ID: 21911816

Although human embryonic stem cells (hESC) have enormous potential for cell replacement therapy of heart failure, immune rejection of hESC derivatives inevitably would occur after transplantation. We therefore aimed to generate a hypoantigeneic hESC line with improved survival characteristics.

Assessment of Physiologic Natural Killer Cell Cytotoxicity in Vitro

Human Immunology. Nov, 2011  |  Pubmed ID: 21924314

Here, we describe an improved (51)chromium release assay (CRA) to compare donor natural killer (NK) cell activity. To validate the assay, we analyzed sample preparation, incubation, and cryopreservation of NK cells. The effector-to-target ratio was corrected for the percentage of NK cells. A logarithmic curve was fitted to the data of the CRA for calculation of the maximum activity. The specific lysis was standardized to a reference sample and normalized to the mean specific lysis of the reference. We found that a longer time span involved with both the addition and the removal of DMSO increased the recovery of NK cell activity. Freezing and thawing reduced the cytotoxicity of NK cells but sustained the relative differences that were seen between freshly prepared NK cells. In contrast, medium incubation of thawed cells markedly increased the cytotoxic potential but also deranged these relative differences. Those were widely equalized when cells were stimulated with IL-2. In conclusion, we established a standardized assay with cryopreserved peripheral blood mononuclear cells as an appropriate tool for investigation of individual physiologic NK cell activity. This assay may help to predict donor NK cell activity in vivo, to reconcile conflicting data about NK cells obtained in transplantation studies.