Hybrid fibroin/polyurethane small-diameter vascular grafts: from fabrication toin vivopreliminary assessment.

To address the need of alternatives to autologous vessels for small-calibre vascular applications (e.g. cardiac surgery), a bio-hybrid semi-degradable material composed of silk fibroin (SF) and polyurethane (Silkothane®) was herein used to fabricate very small-calibre grafts (Øin= 1.5 mm) via electrospinning. Bio-hybrid grafts werein vitrocharacterized in terms of morphology and mechanical behaviour, and compared to similar grafts of pure SF. Similarly, two native vessels from a rodent model (abdominal aorta and vena cava) were harvested and characterized. Preliminary implants were performed on Lewis rats to confirm the suitability of Silkothane® grafts for small-calibre applications, specifically as aortic insertion and femoral shunt. The manufacturing process generated pliable grafts consisting of a randomized fibrous mesh and exhibiting similar geometrical features to rat aortas. Both Silkothane® and pure SF grafts showed radial compliances in the range from 1.37 ± 0.86 to 1.88 ± 1.01% 10-2mmHg-1, lower than that of native vessels. The Silkothane® small-calibre devices were also implanted in rats demonstrating to be adequate for vascular applications; all the treated rats survived the surgery for three months after implantation, and 16 rats out of 17 (94%) still showed blood flow inside the graft at sacrifice. The obtained results lay the basis for a deeper investigation of the interaction between the Silkothane® graft and the implant site, which may deal with further analysis on the potentialities in terms of degradability and tissue formation, on longer time-points.

Chronic lung allograft pathology lesions in two rat strain combinations.

BACKGROUND: Chronic lung allograft dysfunction remains an obstacle to long-term survival after lung transplantation. Two phenotypes have been described: obliterative bronchiolitis and restrictive allograft syndrome. Preclinical models are essential to analyze chronic lung allograft dysfunction pathophysiology. METHODS: Orthotopic lung transplants from 38 Lewis into Fischer 344 (Lew→F344) and 67 Brown-Norway into Lewis (BN→Lew) rats were performed in our center in the last decade. We carefully reviewed and quantified all grafts with chronic rejection (40 cases) (18 Lew→F344, 22 BN→Lew) with the aim to investigate if histological changes of chronic lung allograft dysfunction could be also detected in rat grafts. RESULTS: All animals showed human reminiscent histological lesions. Early chronic rejection lesions were detected in BN→Lew. End-stage chronic rejection with features of obliterative bronchiolitis was observed in 33% of Lew→F344; end-stage with restrictive allograft syndrome chronic rejection in 67% and 80% of Lew→F344 and BN→Lew, respectively. BN→Lew showed higher grades of endotheliitis, vascular fibrosis, and lower grades of lymphoid aggregates than Lew→F344 (P=0.007, P=0.043, P=0.004, respectively). CONCLUSIONS: Chronic rejection lesions in rat lung allografts mimic those in humans. The frequent occurrence of restrictive allograft syndrome-like lesions in BN→Lew may be related to a higher degree of mismatch in this strain combination. These animal models could allow future mechanistic studies to better understand chronic lung allograft dysfunction pathogenesis.

Amnion epithelial cells are an effective source of factor H and prevent kidney complement deposition in factor H-deficient mice.

Complement factor H (FH) is the main plasma regulator of the alternative pathway of complement. Genetic and acquired abnormalities in FH cause uncontrolled complement activation amplifying, with the consequent accumulation of complement components on the renal glomeruli. This leads to conditions such as C3 glomerulopathy (C3G) and atypical hemolytic uremic syndrome (aHUS). There is no effective therapy for these diseases. Half of the patients progress to end-stage renal disease and the condition recurs frequently in transplanted kidneys. Combined liver/kidney transplantation is a valid option for these patients, but the risks of the procedure and donor organ shortages hamper its clinical application. Therefore, there is an urgent need for alternative strategies for providing a normal FH supply. Human amnion epithelial cells (hAEC) have stem cell characteristics, including the capability to differentiate into hepatocyte-like cells in vivo.Here, we administered hAEC into the livers of newborn Cfh-/- mice, which spontaneously developed glomerular complement deposition and renal lesions resembling human C3G. hAEC engrafted at low levels in the livers of Cfh-/- mice and produced sufficient human FH to prevent complement activation and glomerular C3 and C9 deposition. However, long-term engraftment was not achieved, and eventually hAEC elicited a humoral immune response in immunocompetent Cfh-/- mice.hAEC cell therapy could be a valuable therapeutic option for patients undergoing kidney transplantation in whom post-transplant immunosuppression may protect allogeneic hAEC from rejection, while allogeneic cells provide normal FH to prevent disease recurrence.

Transplantation-Induced Ischemia-Reperfusion Injury Modulates Antigen Presentation by Donor Renal CD11c+F4/80+ Macrophages through IL-1R8 Regulation.

BACKGROUND: In donor kidneys subjected to ischemia-reperfusion injury during kidney transplant, phagocytes coexpressing the F4/80 and CD11c molecules mediate proinflammatory responses and trigger adaptive immunity in transplantation through antigen presentation. After injury, however, resident renal macrophages coexpressing these surface markers acquire a proreparative phenotype, which is pivotal in controlling inflammation and fibrosis. No data are currently available regarding the effects of transplant-induced ischemia-reperfusion injury on the ability of donor-derived resident renal macrophages to act as professional antigen-presenting cells. METHODS: We evaluated the phenotype and function of intragraft CD11c+F4/80+ renal macrophages after cold ischemia. We also assessed the modifications of donor renal macrophages after reversible ischemia-reperfusion injury in a mouse model of congeneic renal transplantation. To investigate the role played by IL-1R8, we conducted in vitro and in vivo studies comparing cells and grafts from wild-type and IL-R8-deficient donors. RESULTS: Cold ischemia and reversible ischemia-reperfusion injury dampened antigen presentation by renal macrophages, skewed their polarization toward the M2 phenotype, and increased surface expression of IL-1R8, diminishing activation mediated by toll-like receptor 4. Ischemic IL-1R8-deficient donor renal macrophages acquired an M1 phenotype, effectively induced IFNγ and IL-17 responses, and failed to orchestrate tissue repair, resulting in severe graft fibrosis and aberrant humoral immune responses. CONCLUSIONS: IL-1R8 is a key regulator of donor renal macrophage functions after ischemia-reperfusion injury, crucial to guiding the phenotype and antigen-presenting role of these cells. It may therefore represent an intriguing pathway to explore with respect to modulating responses against autoantigens and alloantigens after kidney transplant.

Vein Suturing Results in Worse Lung Graft Outcomes Compared to the Cuff Method.

BACKGROUND: The rat orthotopic lung transplant model is not widely used yet because of the complexity of the procedure, in particular, venous anastomosis. Here, we performed a rat orthotopic lung transplantation using either the suture (ST) or cuff (CT) method for vein anastomosis. OBJECTIVES: To compare the vein ST and CT techniques in terms of operative time, success, recipient survival, and early histological outcomes was the objective of this study. METHODS: A total of 24 left lung transplants in rats were performed. Twelve syngeneic (Lewis to Lewis) and 12 allogeneic (Brown-Norway to Lewis) lung transplants were performed using either the vein ST or the CT procedure. Arterial and bronchial anastomoses were performed with the CT technique. Graft histological damage was evaluated 3-7 days post-transplant in all rat lungs. RESULTS: The surgical success rate was 75% in both the ST and CT groups. Failures related mainly to vein bleeding (n = 2 in the ST group) and thrombosis (n = 1 in the ST group; n = 2 in the CT group). Total ischemia time was longer in the ST group (122 ± 25 min in ST group vs. 83 ± 10 min in CT group, mean ± SD), due to prolonged warm ischemia time (60 ± 12 min in the ST group vs. 21 ± 5 min in the CT group, mean ± SD), reflecting the time required to complete the vein ST procedure. The prolonged warm ischemia time resulted in significantly higher vascular inflammation in syngeneic grafts (2.3 ± 1.2 ST group vs. 0 in the CT group, mean ± SD) and in increased severity of ischemia/reperfusion injury and acute graft rejection (3.6 ± 0.4 in the ST group vs. 2.6 ± 0.4 in the CT group, mean ± SD) in allogeneic lung transplants. CONCLUSIONS: The vein ST technique is a more time-consuming procedure than the CT method and the prolonged anastomosis time has a deleterious impact on transplant outcomes. These findings suggest that warm ischemia time - one of the modifiable transplant factors - should be considered a major risk factor in lung transplantation, particularly in the setting of donation after cardiac death.

Effect of Timing and Complement Receptor Antagonism on Intragraft Recruitment and Protolerogenic Effects of Mesenchymal Stromal Cells in Murine Kidney Transplantation.

BACKGROUND: Mesenchymal stromal cells (MSCs) have protolerogenic effects in renal transplantation, but they induce long-term regulatory T cells (Treg)-dependent graft acceptance only when infused before transplantation. When given posttransplant, MSCs home to the graft where they promote engraftment syndrome and do not induce Treg. Unfortunately, pretransplant MSC administration is unfeasible in deceased-donor kidney transplantation. METHODS: To make MSCs a therapeutic option also for deceased organ recipients, we tested whether MSC infusion at the time of transplant (day 0) or posttransplant (day 2) together with inhibition of complement receptors prevents engraftment syndrome and allows their homing to secondary lymphoid organs for promoting tolerance. We analyzed intragraft and splenic MSC localization, graft survival, and alloimmune response in mice recipients of kidney allografts and syngeneic MSCs given on day 0 or on posttransplant day 2. C3a receptor (C3aR) or C5a receptor (C5aR) antagonists were administered to mice in combination with the cells or were used together to treat MSCs before infusion. RESULTS: Syngeneic MSCs given at day 0 homed to the spleen increased Treg numbers and induced long-term graft acceptance. Posttransplant MSC infusion, combined with a short course of C3aR or C5aR antagonist or administration of MSCs pretreated with C3aR and C5aR antagonists, prevented intragraft recruitment of MSCs and graft inflammation, inhibited antidonor T-cell reactivity, but failed to induce Treg, resulting in mild prolongation of graft survival. CONCLUSIONS: These data support testing the safety/efficacy profile of administering MSCs on the day of transplant in deceased-donor transplant recipients and indicate that complement is crucial for MSC recruitment into the kidney allograft.

Extracellular vesicles derived from T regulatory cells suppress T cell proliferation and prolong allograft survival.

We have previously shown that rat allogeneic DC, made immature by adenoviral gene transfer of the dominant negative form of IKK2, gave rise in-vitro to a unique population of CD4+CD25- regulatory T cells (dnIKK2-Treg). These cells inhibited Tcell response in-vitro, without needing cell-to-cell contact, and induced kidney allograft survival prolongation in-vivo. Deep insight into the mechanisms behind dnIKK2-Treg-induced suppression of Tcell proliferation remained elusive. Here we document that dnIKK2-Treg release extracellular vesicles (EV) riched in exosomes, fully accounting for the cell-contact independent immunosuppressive activity of parent cells. DnIKK2-Treg-EV contain a unique molecular cargo of specific miRNAs and iNOS, which, once delivered into target cells, blocked cell cycle progression and induced apoptosis. DnIKK2-Treg-EV-exposed T cells were in turn converted into regulatory cells. Notably, when administered in-vivo, dnIKK2-Treg-EV prolonged kidney allograft survival. DnIKK2-Treg-derived EV could be a tool for manipulating the immune system and for discovering novel potential immunosuppressive molecules in the context of allotransplantation.

Experimental Evaluation of Kidney Regeneration by Organ Scaffold Recellularization.

The rising number of patients needing renal replacement therapy, alongside the significant clinical and economic limitations of current therapies, creates an imperative need for new strategies to treat kidney diseases. Kidney bioengineering through the production of acellular scaffolds and recellularization with stem cells is one potential strategy. While protocols for obtaining organ scaffolds have been developed successfully, scaffold recellularization is more challenging. We evaluated the potential of in vivo and in vitro kidney scaffold recellularization procedures. Our results show that acellular scaffolds implanted in rats cannot be repopulated with host cells, and in vitro recellularization is necessary. However, we obtained very limited and inconsistent cell seeding when using different infusion protocols, regardless of injection site. We also obtained experimental and theoretical data indicating that uniform cell delivery into the kidney scaffolds cannot be obtained using these infusion protocols, due to the permeability of the extracellular matrix of the scaffold. Our results highlight the major physical barriers that limit in vitro recellularization of acellular kidney scaffolds and the obstacles that must be investigated to effectively advance this strategy for regenerative medicine.

Simplified Method to Measure Glomerular Filtration Rate by Iohexol Plasma Clearance in Conscious Rats.

BACKGROUND/AIMS: Glomerular filtration rate (GFR) is the best index for evaluating renal function. We aimed to develop a simplified iohexol plasma clearance procedure for GFR measurement in rats without urine collection, animal catheterization or anesthesia, with limited sampling and requiring blood instead of plasma, to further reduce the sample volume and improve animal welfare. METHODS: After iohexol injection (129.4 mg), samples were drawn according to 2-compartment kinetics and analyzed by high performance liquid chromatography. Healthy male Lewis rats were used to find a correction factor (CF) to obtain the 'reference clearance' from the simplified 1-comparment model. This approach was validated using male or female (Lewis, Sprague-Dawley) rats and animals with renal mass reduction (RMR). In additional rats, different simplified approaches were evaluated. RESULTS: Iohexol concentrations in blood and plasma strongly correlated (r = 0.9784, p < 0.0001). A CF of 0.90 enabled the calculation of the reference GFR. Validation results in male Lewis rats were 0.99 ± 0.27 for the reference GFR and 1.03 ± 0.29 ml/min/100 g for the simplified approach. Results in female Sprague-Dawley rats confirmed the suitability of the proposed method. In RMR rats, GFR was 0.14 ± 0.05 and 0.14 ± 0.04 ml/min/100 g for the reference and simplified model, respectively. CONCLUSION: The procedure we set up to measure GFR in conscious rats was proven to be reliable, required a small volume of blood at only 4 selected time points, without the need to collect urine or catheterize the animals, was applicable to rats from different strains and sexes, both healthy and with renal function impairment. Moreover, the procedure enables the monitoring of GFR changes over time in the same animal, thereby reducing the number of animals to be used.

In vivo regeneration of elastic lamina on fibroin biodegradable vascular scaffold.

PURPOSE: There is an increasing need for vascular grafts in the field of surgical revascularization. Artificial grafts offer alternative strategies to autologous tissue, however, small caliber (diameter <6 mm) vascular prosthesis are associated with a high incidence of thrombosis and early failure. Despite promising results, vascular tissue engineering is not yet a clinical reality due to the complexity of this approach. We aimed at investigating the use of fibroin, a biodegradable protein derived from silk, as an acellular vascular graft for in vivo recellularization. 
 METHODS: We produced small caliber fibroin matrices by electrospinning to replace small arterial segments. Electrospun fibroin scaffolds were implanted into the abdominal aorta of Lewis rats by end-to-end anastomosis. Seven days after implantation, fibroin matrices were recovered and processed for histological and immunohistochemical analysis.
 RESULTS: Fibroin matrices allowed host cell infiltration, extracellular matrix remodeling, and ensured good patency of the grafts in the short term. Endothelial cells and smooth muscle cells were present in the explanted construct. Development of an elastic lamina adjacent to the lumen of the scaffold was observed with organization of intima and media layers. Vasa-vasorum were also present in the outer layer of the fibroin material.
 CONCLUSIONS: Our results indicate that formation of vascular tissue containing elastin occurs already at 7 days after implantation on fibroin scaffold without in vitro cellularization. The use of an acellular electrospun silk fibroin tubular scaffold could be a promising strategy for in vivo regeneration of vascular tissue in the clinical reality.

In vivo maturation of functional renal organoids formed from embryonic cell suspensions.

The shortage of transplantable organs provides an impetus to develop tissue-engineered alternatives. Producing tissues similar to immature kidneys from simple suspensions of fully dissociated embryonic renal cells is possible in vitro, but glomeruli do not form in the avascular environment. Here, we constructed renal organoids from single-cell suspensions derived from E11.5 kidneys and then implanted these organoids below the kidney capsule of a living rat host. This implantation resulted in further maturation of kidney tissue, formation of vascularized glomeruli with fully differentiated capillary walls, including the slit diaphragm, and appearance of erythropoietin-producing cells. The implanted tissue exhibited physiologic functions, including tubular reabsorption of macromolecules, that gained access to the tubular lumen on glomerular filtration. The ability to generate vascularized nephrons from single-cell suspensions marks a significant step to the long-term goal of replacing renal function by a tissue-engineered kidney.

Prolonged cold ischemia accelerates cellular and humoral chronic rejection in a rat model of kidney allotransplantation.

One of the leading causes of long-term kidney graft loss is chronic allograft injury (CAI), a pathological process triggered by alloantigen-dependent and alloantigen-independent factors. Alloantigen-independent factors, such as cold ischemia (CI) may amplify the recipient immune response against the graft. We investigated the impact of prolonged cold ischemia and the subsequent delayed graft function on CAI in a fully MHC-mismatched rat model of kidney allotransplantation. Prolonged CI was associated with anticipation of proteinuria onset and graft function deterioration (ischemia: 90d; no ischemia: 150d), more severe tubular atrophy, interstitial fibrosis, and glomerulosclerosis, and increased mortality rate (180d survival, ischemia: 0%; no ischemia: 67%). In ischemic allografts, T and B cells were detected very early and were organized in inflammatory clusters. Higher expression of BAFF-R and TACI within the ischemic allografts indicates that B cells are mature and activated. As a consequence of B cell activity, anti-donor antibodies, glomerular C4d and IgG deposition, important features of chronic humoral rejection, appeared earlier in ischemic than in non-ischemic allograft recipients. Thus, prolonged CI time plays a main role in CAI development by triggering acceleration of cellular and humoral reactions of chronic rejection. Limiting CI time should be considered as a main target in kidney transplantation.

Both darbepoetin alfa and carbamylated erythropoietin prevent kidney graft dysfunction due to ischemia/reperfusion in rats.

BACKGROUND: Ischemia/reperfusion (I/R) injury is an important cause of renal graft dysfunction. Increases in cold and warm ischemia times lead to a higher risk of early posttransplant complications including delayed graft function and acute rejection. Moreover, prolonged cold ischemia is a predictor of long-term graft loss in kidney transplant patients. METHODS: Darbepoetin alfa (DA) and carbamylated nonerythropoietic derivative of erythropoietin (CEPO) protective effects were evaluated in a model of I/R injury after kidney transplantation in both syngeneic and allogeneic combinations. The effects of wortmannin (phosphorylated Akt [p-Akt] inhibitor) administration were also investigated. Serum creatinine was evaluated at 16, 24, 48 hr and at 4 and 7 days posttransplant. Animals were killed 24 hr or 7 days after transplant and kidneys were processed for histological analysis, immunohistochemistry assessment of erythropoietin receptor (EPOR) and ß-common chain receptor expression, granulocyte infiltration, nitrotyrosine staining, p-Akt expression, peritubular capillary (PTC) density, apoptosis, antioxidant, and antiapoptotic gene expression. RESULTS: DA and CEPO significantly reduced serum creatinine, tubular injury, tubular nitrotyrosine staining, and prevented I/R-induced tubular apoptosis, but only when given both to the donor and to the recipient. DA and CEPO cytoprotection was associated with prevention of I/R-induced drop of p-Akt expression in tubuli, and almost complete preservation of capillary density in the tubulointerstitium of the graft. CEPO was more effective than DA in reducing tubular oxidative stress and preserving PTCs. CONCLUSION: DA and CEPO when given both to the donor and to the recipient, prevented renal graft dysfunction, tubular oxidative stress, and apoptosis after I/R injury in kidney transplantation. Their cytoprotection was mediated by tubular p-Akt activation and PTC density preservation.

Embryonic stem cells, derived either after in vitro fertilization or nuclear transfer, prolong survival of semiallogeneic heart transplants.

Tolerance induction toward allogeneic organ grafts represents one of the major aims of transplantation medicine. Stem cells are promising candidates for promoting donor-specific tolerance. In this study, we investigated the immunomodulatory properties of murine embryonic stem cells (ESCs), obtained either by in vitro fertilization (IVF-ESCs) or by nuclear transfer (NT-ESCs), in heart transplant mouse models. IVF-ESCs did not prolong the survival of fully allogeneic cardiac transplants but significantly prolonged the survival of semiallogeneic hearts from the same ESC donor strain for >100 d in 44% of the animals. However, 28% of transplanted animals infused with IVF-ESCs experienced development of a teratoma. NT-ESCs similarly prolonged semiallogeneic heart graft survival (>100 d in 40% of the animals) but were less teratogenic. By in vitro studies, IVF-ESC and NT-ESC immunoregulation was mediated both by cell contact-dependent mechanisms and by the release of soluble factors. By adding specific inhibitors, we identified PGE(2) as a soluble mediator of ESC immunoregulation. Expansion of regulatory T cells was found in lymphoid organs and in the grafts of IVF-ESC- and NT-ESC-tolerized mice. Our study demonstrates that both IVF-ESCs and NT-ESCs modulate recipient immune response toward tolerance to solid organ transplantation, and that NT-ESCs exhibit a lower tendency for teratoma formation. Because NT-ESCs are obtained by NT of a somatic cell from living individuals into an enucleated oocyte, they could represent a source of donor-derived stem cells to induce tolerance to solid organ allograft.

The Toll-IL-1R member Tir8/SIGIRR negatively regulates adaptive immunity against kidney grafts.

Members of the TLR/IL-1R superfamily mediate ischemia/reperfusion injury and initiate immune response in transplanted organs. In this study, we tested the hypothesis that Toll-IL-1R8 (TIR8), a negative regulator of TLR/IL-1R highly expressed in the kidney, modulates immune cell activation underlying kidney rejection. In a mouse model of fully mismatched kidney allotransplantation in which the graft is spontaneously accepted, intragraft Tir8 expression was enhanced compared with naive kidneys. Targeted deletion of Tir8 in the graft exerted a powerful antitolerogenic action leading to acute rejection. Similarly, in a mouse model of kidney graft acceptance induced by costimulation blockade, most Tir8(-/-) grafts were acutely rejected. Despite similar levels of TLR4, IL-1R, and their ligands, the posttransplant ischemia/reperfusion-induced inflammatory response was more severe in Tir8(-/-) than in Tir8(+/+) grafts and was followed by expansion and maturation of resident dendritic cell precursors. In vitro, Tir8(-/-) dendritic cell precursors acquired higher allostimulatory activity and released more IL-6 upon stimulation with a TLR4 ligand and TNF-alpha than Tir8(+/+) cells, which may explain the increased frequency of antidonor-reactive T cells and the block of regulatory T cell formation in recipients of a Tir8(-/-) kidney. Thus, TIR8 acts locally as a key regulator of allogeneic immune response in the kidney. Tir8 expression and/or signaling in donor tissue are envisaged as a novel target for control of innate immunity and amelioration of graft survival.

Propionyl-L-carnitine prevents early graft dysfunction in allogeneic rat kidney transplantation.

Ischemia-reperfusion injury is an important cause of graft failure. Because carnitine regulates substrate flux and energy balance across membranes which may be deranged in ischemia we determined whether its use was effective in preventing kidney injury in an allogeneic transplant model. Brown Norway rats received a Lewis rat kidney transplant and were then treated with cyclosporine A to avoid rejection. The grafts were stored in Belzer solution supplemented with propionyl-L-carnitine during the cold ischemia period. Compared to rats receiving untreated kidneys but with equal cold ischemia times, the post-transplant serum creatinine values of the carnitine-treated transplants were significantly lower. Histological evaluation 16 h after transplant showed that propionyl-L-carnitine significantly inhibited tubular necrosis and neutrophil infiltration of the allografts and improved the 3 month graft survival. Treated transplants also had decreased lipid peroxidation, inducible nitric oxide synthase expression and protein nitration compared to the untreated grafts. Post-transplant serum creatinine levels were significantly reduced and graft survival was slightly prolonged in rats not receiving cyclosporine A treatment and transplanted with a kidney treated with propionyl-L-carnitine. The efficacy of propionyl-L-carnitine to modulate ischemia-reperfusion injury during transplantation suggests that its use in human transplantation is worth testing.

Pretransplant infusion of mesenchymal stem cells prolongs the survival of a semiallogeneic heart transplant through the generation of regulatory T cells.

In this study, we investigated whether mesenchymal stem cells (MSC) had immunomodulatory properties in solid organ allotransplantation, using a semiallogeneic heart transplant mouse model, and studied the mechanism(s) underlying MSC tolerogenic effects. Either single (portal vein, day -7) or double (portal vein, day -7 and tail vein, day -1) pretransplant infusions of donor-derived B6C3 MSC in B6 recipients induced a profound T cell hyporesponsiveness and prolonged B6C3 cardiac allograft survival. The protolerogenic effect was abrogated when donor-derived MSC were injected together with B6C3 hematopoietic stem cells (HSC), suggesting that HSC negatively impact MSC immunomodulatory properties. Both the induction (pretransplant) and the maintenance phase (>100 days posttransplant) of donor-derived MSC-induced tolerance were associated with CD4(+)CD25(+)Foxp3(+) Treg expansion and impaired anti-donor Th1 activity. MSC-induced regulatory T cells (Treg) were donor-specific since adoptive transfer of splenocytes from tolerant mice prevented the rejection of fully MHC-mismatched donor-specific secondary allografts but not of third-party grafts. In addition, infusion of recipient-derived B6 MSC tolerized a semiallogeneic B6C3 cardiac allograft, but not a fully MHC-mismatched BALB/c graft, and expanded Treg. A double i.v. pretransplant infusion of recipient-derived MSC had the same tolerogenic effect as the combined intraportal/i.v. MSC infusions, which makes the tolerogenic protocol applicable in a clinical setting. In contrast, single MSC infusions given either peritransplant or 1 day after transplant were less effective. Altogether these findings indicate that MSC immunomodulatory properties require HSC removal, partial sharing of MHC Ags between the donor and the recipient and pretransplant infusion, and are associated with expansion of donor-specific Treg.

Effect of seliciclib (CYC202, R-roscovitine) on lymphocyte alloreactivity and acute kidney allograft rejection in rat.

BACKGROUND: T cell stimulation by alloantigens is followed by cell cycle progression, an event that is critically dependent on cyclin-dependent kinases. METHODS: We conducted a study to evaluate whether the cyclin-dependent kinase inhibitor seliciclib affected rat lymph node cells (LNc) activation and proliferation induced by either concanavalin A or allogeneic splenocytes in vitro and studied the mechanisms underlying the suppressive effect. We also investigated the immunosuppressive properties of seliciclib in vivo. RESULTS: Seliciclib completely inhibited in vitro proliferation of LNc and CD8 T cells, in response to either concanavalin A or allogeneic splenocytes. The percentage of activated LNc was lower in mixed leukocyte reactions (MLR) added with seliciclib than in MLR added with vehicle. The percentages of viable and apoptotic cells at the end of MLR with seliciclib were comparable to those of MLR with vehicle. LNc pre-exposed in MLR to seliciclib did not respond to further stimulation with alloantigens, and neither IL-2 nor IL-15 restored proliferation. These data indicate that the inhibitory effect of seliciclib on T cell alloreactivity is not because of cytotoxic effect but is associated with induction of profound T cell anergy. LNc harvested at the end of the primary MLR with seliciclib did not suppress the proliferation of syngeneic LNc cells toward allogeneic splenocytes, thus excluding that seliciclib induced the formation of regulatory cells. Finally, seliciclib partially prolonged grafted animal survival in a rat model of fully major histocompatibility complex-mismatched kidney transplantation. CONCLUSIONS: Altogether these results document that seliciclib regulates lymphocyte reactivity and may exert an immunosuppressive effect in vivo in the setting of transplantation.

Complement-mediated dysfunction of glomerular filtration barrier accelerates progressive renal injury.

Intrarenal complement activation leads to chronic tubulointerstitial injury in animal models of proteinuric nephropathies, making this process a potential target for therapy. This study investigated whether a C3-mediated pathway promotes renal injury in the protein overload model and whether the abnormal exposure of proximal tubular cells to filtered complement could trigger the resulting inflammatory response. Mice with C3 deficiency were protected to a significant degree against the protein overload-induced interstitial inflammatory response and tissue damage, and they had less severe podocyte injury and less proteinuria. When the same injury was induced in wild-type (WT) mice, antiproteinuric treatment with the angiotensin-converting enzyme inhibitor lisinopril reduced the amount of plasma protein filtered, decreased the accumulation of C3 by proximal tubular cells, and protected against interstitial inflammation and damage. For determination of the injurious role of plasma-derived C3, as opposed to tubular cell-derived C3, C3-deficient kidneys were transplanted into WT mice. Protein overload led to the development of glomerular injury, accumulation of C3 in podocytes and proximal tubules, and tubulointerstitial changes. Conversely, when WT kidneys were transplanted into C3-deficient mice, protein overload led to a more mild disease and abnormal C3 deposition was not observed. These data suggest that the presence of C3 increases the glomerular filtration barrier's susceptibility to injury, ultrafiltered C3 contributes more to tubulointerstitial damage induced by protein overload than locally synthesized C3, and local C3 synthesis is irrelevant to the development of proteinuria. It is speculated that therapies targeting complement combined with interventions to minimize proteinuria would more effectively prevent the progression of renal disease.

DnIKK2-transfected dendritic cells induce a novel population of inducible nitric oxide synthase-expressing CD4+CD25- cells with tolerogenic properties.

BACKGROUND: We previously documented that rat bone marrow-derived dendritic cells (DCs), transfected with an adenovirus encoding a dominant negative form of IKK2 (dnIKK2), have impaired allostimulatory capacity and generate CD4 T cells with regulatory function. Here we investigate the potency, the phenotype, and the mechanism of action of dnIKK2-DC-induced regulatory cells and we evaluated their tolerogenic properties in vivo. METHODS: Brown Norway (BN) transfected dnIKK2-DCs were cultured with Lewis (LW) lymphocytes in primary mixed lymphocyte reaction (MLR). CD4 T cells were purified from primary MLR and incubated in secondary coculture MLR with LW lymphocytes. Phenotypic characterization was performed by fluorescence-activated cell sorting and real-time polymerase chain reaction. The tolerogenic potential of CD4 T cells pre-exposed to dnIKK2-DCs was evaluated in vivo in a model of kidney allotransplantation. RESULTS: CD4 T cells pre-exposed to dnIKK2-DCs were CD4CD25 and expressed interleukin (IL)-10, transforming growth factor-beta, interferon-gamma, IL-2, and inducible nitric oxide synthase (iNOS). These cells (dnIKK2-Treg), cocultured (at up to 1:10 ratio) with a primary MLR, suppressed T-cell proliferation to alloantigens. The regulatory effect was cell-to-cell contact-independent since it was also observed in a transwell system. A nitric oxide synthase inhibitor significantly reverted dnIKK2-Treg-mediated suppression, whereas neutralizing antibodies to IL-10 and TGF-beta had no significant effect. DnIKK2-Treg given in vivo to LW rats prolonged the survival of a kidney allograft from BN rats (the donor rat strain used for generating DCs). CONCLUSIONS: DnIKK2-Treg is a unique population of CD4CD25 T cells expressing high levels of iNOS. These cells potently inhibit T-cell response in vitro and induce prolongation of kidney allograft survival in vivo.