Manipulating dendritic cells to induce regulatory T cells.

Dendritic cells (DCs) induce and regulate T-cell responses, and tolerogenic DCs can promote the development of regulatory T cells with suppressive activity. The possibility of manipulating DCs using different pharmacological or biological agents, enabling them to exert tolerogenic activities, could be exploited to better control a variety of chronic inflammatory conditions, from autoimmune diseases to allograft rejection.

Cardiomyocytes induce endothelial cells to trans-differentiate into cardiac muscle: implications for myocardium regeneration.

The concept of tissue-restricted differentiation of postnatal stem cells has been challenged by recent evidence showing pluripotency for hematopoietic, mesenchymal, and neural stem cells. Furthermore, rare but well documented examples exist of already differentiated cells in developing mammals that change fate and trans-differentiate into another cell type. Here, we report that endothelial cells, either freshly isolated from embryonic vessels or established as homogeneous cells in culture, differentiate into beating cardiomyocytes and express cardiac markers when cocultured with neonatal rat cardiomyocytes or when injected into postischemic adult mouse heart. Human umbilical vein endothelial cells also differentiate into cardiomyocytes under similar experimental conditions and transiently coexpress von Willebrand factor and sarcomeric myosin. In contrast, neural stem cells, which efficiently differentiate into skeletal muscle, differentiate into cardiomyocytes at a low rate. Fibroblast growth factor 2 and bone morphogenetic protein 4, which activate cardiac differentiation in embryonic cells, do not activate cardiogenesis in endothelial cells or stimulate trans-differentiation in coculture, suggesting that different signaling molecules are responsible for cardiac induction during embryogenesis and in successive periods of development. The fact that endothelial cells can generate cardiomyocytes sheds additional light on the plasticity of endothelial cells during development and opens perspectives for cell autologous replacement therapies.

Regulatory T cells induced by 1 alpha,25-dihydroxyvitamin D3 and mycophenolate mofetil treatment mediate transplantation tolerance.

1alpha,25-dihydroxyvitamin D3, the active form of vitamin D3, and mycophenolate mofetil, a selective inhibitor of T and B cell proliferation, modulate APC function and induce dendritic cells (DCs) with a tolerogenic phenotype. Here we show that a short treatment with these agents induces tolerance to fully mismatched mouse islet allografts that is stable to challenge with donor-type spleen cells and allows acceptance of donor-type vascularized heart grafts. Peritransplant macrophages and DCs from tolerant mice express down-regulated CD40, CD80, and CD86 costimulatory molecules. In addition, DCs from the graft area of tolerant mice secrete, upon stimulation with CD4+ cells, 10-fold lower levels of IL-12 compared with DCs from acutely rejecting mice, and induce a CD4+ T cell response characterized by selective abrogation of IFN-gamma production. CD4+ but not CD8+ or class II+ cells from tolerant mice, transferred into naive syngeneic recipients, prevent rejection of donor-type islet grafts. Graft acceptance is associated with impaired development of IFN-gamma-producing type 1 CD4+ and CD8+ cells and an increased percentage of CD4+CD25+ regulatory cells expressing CD152 in the spleen and in the transplant-draining lymph node. Transfer of CD4+CD25+ cells from tolerant but not naive mice protects 100% of the syngeneic recipients from islet allograft rejection. These results demonstrate that a short treatment with immunosuppressive agents, such as 1alpha,25-dihydroxyvitamin D3/mycophenolate mofetil, induces tolerance to islet allografts associated with an increased frequency of CD4+CD25+ regulatory cells that can adoptively transfer transplantation tolerance.

Nonviral and viral gene transfer to the kidney in the context of transplantation.

BACKGROUND/AIMS: Local modulation of the immune response through genetic manipulation of the graft is an attractive novel approach to overcome the toxicity of immunosuppressive therapy to prevent acute graft rejection. We have previously reported that the cationic polymer polyethylenimine 25k (PEI 25k) transduced reporter genes when injected into the renal artery, but with a low transfection efficiency. Here we compare the risk/benefit profiles of such a nonviral versus a viral technique of gene transfer to the kidney in the context of renal transplantation. METHODS: Donor kidneys from Lewis rats were perfused in a closed circuit with an artificial cell-free medium containing PEI 25k complexed to an expression vector coding for the beta-galactosidase (beta-gal) gene and subsequently transplanted in a syngeneic animal. In a second set of experiments, donor kidneys were injected or perfused with a replication-deficient adenovirus encoding the beta-gal gene (AdCMV. beta-gal; 1 x 10(9) plaque-forming units) before transplantation. RESULTS: Perfusion of the kidney with PEI 25k/DNA complexes resulted in large areas of hypoperfusion characterized by injured glomeruli and tubuli, capillary thrombosis and accumulation of C3 in glomerular capillaries. Reperfusion of the kidney was achieved by lowering the PEI 25k/DNA ratio, but no detectable transfection was observed. In animals receiving adenovirus, the beta-gal activity increased with time and was localized mainly in proximal and distal tubular cells, as documented by beta-gal histochemistry and in situ hybridization. A significantly increased expression of beta-gal was achieved by perfusion of the kidney with AdCMV.beta-gal before transplantation, beta-gal staining mainly localizing in proximal and distal tubular cells. CONCLUSIONS: Unlike nonviral methods of gene delivery, adenovirus-mediated gene transfer to the kidney offers exciting perspectives for the development of molecular medicine in the field of organ transplantation.

Chronic allograft nephropathy in the rat is improved by angiotensin II receptor blockade but not by calcium channel antagonism.

Functional and structural changes of chronic renal allograft failure share similarities with other chronic nephropathies with low nephron number. In models of reduced nephron number, angiotensin-converting enzyme inhibitors or angiotensin II receptor blockers prevented proteinuria and retarded renal lesions. This study investigates whether blockade of angiotensin II activity prevented chronic allograft injury in the Fisher 344 --> Lewis rat kidney transplant model, and compares its effect with that of calcium channel blockers, the main antihypertensive agents used in transplant patients to control BP. Transplanted rats received either no treatment (control), the type 1 angiotensin II receptor antagonist losartan, or the calcium channel blocker lacidipine. Rats received cyclosporine for the first 10 d posttransplant to prevent acute rejection. Doses of antihypertensive drugs were adjusted to achieve a comparable level of BP control throughout the study. Awake systolic BP was comparable in animals given losartan or lacidipine during the 6-mo observation period. Daily treatment with losartan but not lacidipine resulted in a significant decrease in the amount of proteinuria, preserved glomerular and tubulointerstitial structure, and improved graft survival compared with corresponding parameters in control untreated rats. GFR, measured as inulin and p-aminohippurate clearances, respectively, in rats surviving the 6-mo follow-up, was numerically but not significantly higher in losartan-treated animals than in all other groups. Thus, at comparable levels of BP control, losartan but not lacidipine effectively protects animals from chronic allograft injury and allows long-term survival.

The kidney triggers graft-versus-host disease in experimental combined transplantation of kidney and stem cell-enriched peripheral leukocytes.

As a preclinical step to human studies with combined stem cell-enriched peripheral leukocytes and organ transplantation from the same donor, a series of studies in rats was undertaken. These studies indicated that Lewis rats infused intravenously with major histocompatibility complex-incompatible (from Brown-Norway rats), stem cell-enriched peripheral leukocyte preparation alone never developed graft-versus-host disease (GHVD). However, GVHD invariably manifested in all animals a few days after the kidney was transplanted in rats that had been previously primed with stem cell-enriched peripheral leukocytes from the same kidney donor strain. GVHD was prevented by substituting the crude preparation of stem cell-enriched peripheral leukocytes with a purified preparation that was almost completely free of T lymphocytes. However, in these latter experiments all rats rejected their kidney graft within 10 days from the surgery. In rats previously given the crude stem cell-enriched peripheral leukocyte preparation, perioperative administration of the fusion protein CTLA4lg also prevented GVHD and prolonged kidney graft survival up to 106 to 175 days. By contrast, animals with kidney transplants, which were given CTLA4lg without stem cells, rejected their grafts within 35 days. All together, these findings may possibly contribute to the creation of rationally designed strategies of combining organ and bone marrow from the same donor to enhance mixed chimerism and prolong survival after organ transplantation.

Evidence that an angiotensin-converting enzyme inhibitor has a different effect on glomerular injury according to the different phase of the disease at which the treatment is started.

In rats with streptozotocin-induced diabetes, the effect of an angiotensin-converting enzyme (ACE) inhibitor on the evolution of glomerular injury according to the time at which the treatment is started with respect to the onset of the disease was studied. Three groups of animals were used, a control Group 1 and two groups of diabetic rats treated with insulin (Groups 2 and 3). The latter were monitored until urinary protein excretion reached 40 to 50 mg/24 h (on average, 23 wk after the induction of the diabetes). At this time, Group 2 continued to receive insulin alone, whereas Group 3 was also given the ACE inhibitor moexipril for 8 more wk. Untreated diabetic rats showed a moderate increase in systolic blood pressure that was normalized by moexipril administration. Urinary protein excretion progressively increased during the 8-wk follow-up in untreated diabetics that, at the end of the study, developed moderate glomerular sclerosis. Moexipril treatment lowered urinary protein excretion to a normal range and completely prevented glomerular injury. Three other groups of rats were similarly treated, except that moexipril treatment was started later on (when proteinuria reached 100 to 200 mg/24 h, on average, 32 wk after the induction of diabetes), and were monitored for another 8 wk. Untreated and treated diabetics had comparable blood glucose levels throughout. Systolic blood pressure, significantly increased in untreated diabetic rats, was effectively controlled by moexipril administration.(ABSTRACT TRUNCATED AT 250 WORDS)

Renal cyclophilin-like protein gene expression parallels changes in sodium excretion in experimental nephrosis and is positively modulated by atrial natriuretic peptide.

Experimental evidence is available to indicate that intrarenal mechanisms play a role in the impaired salt excretion of nephrotic syndrome by multiple and still incompletely defined mediators. It is documented herein that the gene encoding for cyclophilin-like protein (Cy-LP) is up-regulated in renal medulla from adriamycin (ADR)-treated rats as compared with control animals. In the cortex of rats with ADR nephrosis, no change in Cy-LP as compared with that in controls was found for the entire observation period. By contrast, in the medulla of nephrotic rats, Cy-LP gene expression was significantly higher than in controls. Values of urinary Na excretion were inversely correlated to Cy-LP mRNA expression levels. Because in ADR nephrosis a blunted natriuretic response to ANP has been previously reported, it was investigated whether ANP infusion modulated Cy-LP mRNA in the renal medulla. ADR-treated rats, but not control rats, infused for 1 h with ANP (1 microgram/kg.min) had a significant (P < 0.05) increase in medullary Cy-LP mRNA as compared with nephrotic animals receiving the vehicle alone. These findings might be taken to suggest that renal Cy-LP gene expression is positively modulated in nephrotic syndrome and parallels changes in sodium excretion.