Single-center kidney paired donation: the Methodist San Antonio experience.

Many potential kidney transplant recipients are unable to receive a live donor transplant due to crossmatch or blood type incompatibility. Kidney paired donation increases access to live donor transplantation but has been significantly underutilized. We established a kidney paired donation program including consented incompatible donor/recipient pairs as well as compatible pairs with older non-human leukocyte antigen identical donors.

Kidney paired donation: a single center approach to increase living donor transplantation.

Methodist Specialty and Transplant Hospital has performed a total of 125 KPD transplants from the onset of the program in Dec. 2007. With the addition of the KPD program, live donor transplants have increased annually by 35% demonstrating the ability to substantially increase access to transplantation utilizing this modality.

Suppression by CD4+CD25+ regulatory T cells is dependent on expression of heme oxygenase-1 in antigen-presenting cells.

Heme oxygenase-1 (HO-1) has been viewed as a cytoprotective protein, ameliorating the effects of inflammatory cellular damage, and as beneficial in allograft protection from acute and chronic rejection, suggesting important functions in both innate and adaptive immune responses. Mice deficient in HO-1 exhibit defective immune regulation characterized by a proinflammatory phenotype. We examined if impaired regulatory T cell (Treg) function contributes to the immunoregulatory defects observed in HO-1(-/-) mice.

Heme oxygenase-1 deficiency promotes epithelial-mesenchymal transition and renal fibrosis.

Induction of heme oxygenase-1 (HO-1) is associated with potential antifibrogenic effects. The effects of HO-1 expression on epithelial-mesenchymal transition (EMT), which plays a critical role in the development of renal fibrosis, are unknown. In this study, HO-1(-/-) mice demonstrated significantly more fibrosis after 7 d of unilateral ureteral obstruction compared with wild-type mice, despite similar degrees of hydronephrosis.

Heme oxygenase-1 expression in macrophages plays a beneficial role in atherosclerosis.

Heme oxygenase (HO-1) is the rate-limiting enzyme in the catabolism of heme, which leads to the generation of biliverdin, iron, and carbon monoxide. It has been shown to have important antioxidant and antiinflammatory properties that result in a vascular antiatherogenic effect. To determine whether HO-1 expression in macrophages constitutes a significant component of the protective role in atherosclerosis, we evaluated the effect of decreased or absent HO-1 expression in peritoneal macrophages on oxidative stress and inflammation in vitro, and the effect of complete deficiency of HO-1 expression in macrophages in atherosclerotic lesion formation in vivo.

Adeno-associated viral vector-mediated interleukin-10 prolongs allograft survival in a rat kidney transplantation model.

Interleukin-10 (IL-10) is a pleiotropic cytokine that plays a pivotal role in the regulation of immune responses. Hence, we evaluated the effects of a recombinant adeno-associated viral vector 1 (rAAV1) encoding rat IL-10 (rAAV1-IL-10) in a rat model of kidney allograft rejection. Dark Agouti rat kidneys were transplanted into Wistar-Furth (WF) rats 8 weeks following a single intramuscular administration of either rAAV1-IL-10 or rAAV1-green fluorescence protein (GFP).

Freeze-thaw increases adeno-associated virus transduction of cells.

A combination of gene and cell-based therapies may provide significant advantages over existing treatments in terms of their effectiveness. However, long-term efficient gene delivery has been difficult to achieve in many cell types, including endothelial cells. We developed a freeze-thaw technique which significantly increases the transduction efficiency of recombinant adeno-associated virus vectors in human aortic endothelial cells (23-fold) and in human renal proximal tubular epithelial cells (128-fold) in comparison to current methods for transduction.

Induction of heme oxygenase-1 modulates the profibrotic effects of transforming growth factor-beta in human renal tubular epithelial cells.

Transforming growth factor-beta (TGF-beta) is implicated in a variety of kidney diseases where it promotes extracellular matrix (ECM) deposition and pro-inflammatory events, but it also stabilizes and attenuates tissue injury through the activation of cytoprotective proteins, including heme oxygenase-1 (HO-1). HO-1 catalyzes the conversion of heme into carbon monoxide (CO), iron, and biliverdin, which is subsequently converted to bilirubin. The beneficial effects of HO-1 induction include decreasing pro-oxidants (heme), increasing anti-oxidants (biliverdin and bilirubin), and producing a vasodilator with anti-apoptotic and anti-inflammatory properties (CO).

Adeno-associated virus vector-mediated gene delivery to the vasculature and kidney.

Relatively successful elsewhere, gene delivery aimed at the vasculature and kidney has made very little progress. In the kidney, the hurdles are related to the unique structure-function relationships of this organ and in the blood vessels to a variety of, mostly endothelial, factors making the delivery of transgenes very difficult. Among gene-therapeutic approaches, most viral gene delivery systems utilized to date have shown significant practical and safety-related limitations due to the level and duration of recombinant transgene expression as well as their induction of a significant host immune response to vector proteins.

Interleukin 10 attenuates neointimal proliferation and inflammation in aortic allografts by a heme oxygenase-dependent pathway.

Interleukin 10 (IL-10) is a pleiotropic cytokine with well known antiinflammatory, immunosuppressive, and immunostimulatory properties. Chronic allograft rejection, characterized by vascular neointimal proliferation, is a major cause of organ transplant loss, particularly in heart and kidney transplant recipients. In a Dark Agouti to Lewis rat model of aortic transplantation, we evaluated the effects of a single intramuscular injection of a recombinant adeno-associated viral vector (serotype 1) encoding IL-10 (rAAV1-IL-10) on neointimal proliferation and inflammation.

An integral role for heme oxygenase-1 and carbon monoxide in maintaining peripheral tolerance by CD4+CD25+ regulatory T cells.

Over the past decade, a great deal of interest and attention has been directed toward a population of regulatory T cells (Treg) coexpressing the markers CD4 and CD25. The hallmark phenotype of this cell population resides in its ability to dominantly maintain peripheral tolerance and avert autoimmunity. Despite robust research interest in Treg, their mechanism of action and interaction with other cell populations providing immune regulation remains unclear.

Efficient transduction of vascular endothelial cells with recombinant adeno-associated virus serotype 1 and 5 vectors.

Recombinant adeno-associated virus (rAAV) has become an attractive tool for gene therapy because of its ability to transduce both dividing and nondividing cells, elicit a limited immune response, and the capacity for imparting long-term transgene expression. Previous studies have utilized rAAV serotype 2 predominantly and found that transduction of vascular cells is relatively inefficient. The purpose of the present study was to evaluate the transduction efficiency of rAAV serotypes 1 through 5 in human and rat aortic endothelial cells (HAEC and RAEC).

Heme oxygenase-1 modulates early inflammatory responses: evidence from the heme oxygenase-1-deficient mouse.

Induction of heme oxygenase-1 (HO-1) is protective in tissue injury in models of allograft rejection and vascular inflammation through either prevention of oxidative damage or via immunomodulatory effects. To examine the specific role of HO-1 in modulating the immune response, we examined the differences in immune phenotype between HO-1 knockout (HO-1(-/-)) and wild-type (HO-1(+/+)) mice. Consistent with previous findings, marked splenomegaly and fibrosis were observed in HO-1(-/-) mice.

Pharmacology of calcineurin antagonists.

Cyclosporine and tacrolimus share the same pharmacodynamic property of activated T-cell suppression via inhibition of calcineurin. The introduction of these drugs to the immunosuppressive repertoire of transplant management has greatly improved the outcomes in organ transplantation and constitutes arguably one of the major breakthroughs in modern medicine. To this date, calcineurin inhibitors are the mainstay of prevention of allograft rejection.

Targeting recombinant adeno-associated virus vectors to enhance gene transfer to pancreatic islets and liver.

Human pancreatic islet cells and hepatocytes represent the two most likely target cells for genetic therapy of type I diabetes. However, limits to the efficiency of rAAV serotype 2 (rAAV2)-mediated gene transfer have been reported for both of these cell targets. Here we report that nonserotype 2 AAV capsids can mediate more efficient transduction of islet cells, with AAV1 being the most efficient serotype in murine islets, suggesting that receptor abundance could be limiting.

Adeno-associated virus (AAV) as a vehicle for therapeutic gene delivery: improvements in vector design and viral production enhance potential to prolong graft survival in pancreatic islet cell transplantation for the reversal of type 1 diabetes.

Most viral gene delivery syslems utilized to date have demonstrated significant limitations in practicality and safety due to the level and duration of recombinant transgene expression as well as their induction of host immunogenicity to vector proteins. Recombinant adeno-associated virus (rAAV) vectors appear to offer a vehicle for safe, long-term therapeutic gene transfer; factors afforded through the propensity of rAAV to establish long-term latency without deleterious effects on the host cell and the relative non-immunogenicity of the virus or viral expressed transgenes. The principal historical limitation of this vector system, efficiency of rAAV-mediated transduction, has recently observed a dramatic increase as the titer, purity, and production capacity of rAAV preparations have improved.

Transduction of human and mouse pancreatic islet cells using a bicistronic recombinant adeno-associated viral vector.

Recent reports indicate successful transduction of pancreatic islets using recombinant adeno-associated viral (rAAV) vectors. This advance offers new possibilities in rendering islets resistant to rejection and recurrence of autoimmune destruction in the setting of islet transplantation as treatment of type 1 diabetes. Most gene delivery approaches using islets have thus far involved transduction with a single gene.

Adeno-associated virus vector-mediated IL-10 gene delivery prevents type 1 diabetes in NOD mice.

The development of spontaneous autoimmune diabetes in nonobese diabetic (NOD) mice provides for their use as a model of human type 1 diabetes. To test the feasibility of muscle-directed gene therapy to prevent type 1 diabetes, we developed recombinant adeno-associated virus (rAAV) vectors containing murine cDNAs for immunomodulatory cytokines IL-4 or IL-10. Skeletal muscle transduction of female NOD mice with IL-10, but not IL-4, completely abrogated diabetes.

Efficient ex vivo transduction of pancreatic islet cells with recombinant adeno-associated virus vectors.

The ability to transfer immunoregulatory, cytoprotective, or antiapoptotic genes into pancreatic islet cells may allow enhanced posttransplantation survival of islet allografts and inhibition of recurrent autoimmune destruction of these cells in type 1 diabetes. However, transient transgene expression and the tendency to induce host inflammatory responses have limited previous gene delivery studies using viral transfer vectors. We demonstrate here that recombinant adeno-associated virus (rAAV) serotype 2, a vector that can overcome these limitations, effectively transduces both human and murine pancreatic islet cells with reporter genes as well as potentially important immunoregulatory cytokine genes (interleukin-4, interleukin-10), although a very high multiplicity of infection (10,000 infectious units/islet equivalent) was required.

Angiotensin II-stimulated nitric oxide release from porcine pulmonary endothelium is mediated by angiotensin IV.

In this study, a nitric oxide (NO) sensor was used to examine the ability of angiotensin II (AngII), AngIV, and bradykinin (Bk) to stimulate NO release from porcine pulmonary artery (PPAE) and porcine aortic endothelial (PAE) cells and to explore the mechanism of the AngII-stimulated NO release. Physiologic concentrations of AngII, but not Bk, caused release of NO from PPAE cells. In contrast, Bk, but not AngII, stimulated NO release from PAE cells.

Cytokine-inducible nitric oxide synthase (iNOS) expression in cardiac myocytes. Characterization and regulation of iNOS expression and detection of iNOS activity in single cardiac myocytes in vitro.

Cellular constituents of heart muscle contain both constitutive and inducible nitric oxide (NO) signaling pathways that modulate the contractile properties of cardiac myocytes. The identities of the inducible NO synthase (iNOS) isoform(s) expressed in cardiac muscle, and of the specific cell types expressing iNOS activity, remain poorly characterized. We amplified a 217-base pair cDNA by reverse transcriptase-polymerase chain reaction from primary cultures of inflammatory cytokine-pretreated adult rat ventricular myocytes (ARVM) that was nearly identical to other iNOS cDNA sequences.

Nitric oxide synthase activity in genetic hypertension.

A porphyrinic sensor was used to monitor nitric oxide released from cultured endothelial and vascular smooth muscle cells obtained from genetically hypertensive rats and from a normotensive reference strain of rats. Endothelial cell nitric oxide synthase (the constitutive enzyme) was stimulated with bradykinin, and vascular smooth muscle cell nitric oxide synthase (the inducible enzyme) was induced with interleukin-1 beta. Both types of cells from hypertensive rats released less nitric oxide than did cells from normotensive rats.

Diffusion of nitric oxide in the aorta wall monitored in situ by porphyrinic microsensors.

Porphyrinic sensors were used for the in situ monitoring of nitric oxide release and diffusion in the endothelial cell, as well as its subsequent diffusion from the endothelial cell through the muscle cells found in the rabbit aorta. The experimental data was compared with that predicted based on Fick's equation for linear diffusion. A time delay of 1.