Nuria Bolaños

New immunosuppresor strategies in the treatment of murine lupus nephritis

Renal involvement in systemic lupus erythematosus is a common complication that significantly worsens morbidity and mortality. Although treatment with corticosteroids and cytotoxic drugs may be useful in many cases, morbidity associated with these drugs and the relapsing nature of the disease make it necessary to develop new treatment strategies. Five-month old female NZB/W F1 mice were divided into the following groups: CYP group (n = 10), cyclophosphamide (CYP) 50 mg/kg intraperitoneally every 10 days; RAPA 1 group (n = 10) oral daily sirolimus (SRL), 1 mg/kg; RAPA 12 group (n = 13), oral daily SRL, 12 mg/kg; FTY group (n = 10), oral fingolimod (FTY720), 2 mg/kg three times per week. An additional group of 13 non-treated mice were used as a control (control group). Follow-up was performed over four months. Animal survival, body weight, anti-DNA antibodies and proteinuria were determined. Kidneys were processed for conventional histology and immunofluorescence for IgG and complement. Total histological score (HS) was the sum of mesangial expansion, endocapillary proliferation glomerular deposits, extracapillary proliferation, interstitial infiltrates, tubular atrophy and interstitial fibrosis. All treated groups had lower proteinuria at the end of the follow-up with respect to the control group (P < 0.0001). Serum anti-DNA antibodies were appropriately controlled in RAPA 1 and CYP groups, but not in FTY or RAPA 12 groups. SRL and CYP arrested, and perhaps reversed almost all histological lesions. FTY720 ameliorated histological lesions but did not control mesangial expansion or interstitial infiltrates. SRL produces great improvement in murine lupus nephritis, while FTY720 seems a promising alternative if used in appropriate doses.

Hepatocyte growth factor gene therapy enhances infiltration of macrophages and may induce kidney repair in db/db mice as a model of diabetes

Aims/hypothesisWe previously demonstrated hepatocyte growth factor (HGF) gene therapy was able to induce regression of glomerulosclerosis in diabetic nephropathy through local reparative mechanisms. The aim of this study was to test whether bone-marow-derived cells are also involved in this HGF-induced reparative process.MethodsWe have created chimeric db/db mice as a model of diabetes that produce enhanced green fluorescent protein (EGFP) in bone marrow cells. We performed treatment with HGF gene therapy either alone or in combination with granulocyte-colony stimulating factor, in order to induce mobilisation of haematopoietic stem cells in these diabetic and chimeric animals.ResultsWe find HGF gene therapy enhances renal expression of stromal-cell-derived factor-1 and is subsequently associated with an increased number of bone-marrow-derived cells getting into the injured kidneys. These cells are mainly monocyte-derived macrophages, which may contribute to the renal tissue repair and regeneration consistently observed in our model. Finally, HGF gene therapy is associated with the presence of a small number of Bowman’s capsule parietal epithelial cells producing EGFP, suggesting they are fused with bone-marrow-derived cells and are contributing to podocyte repopulation.Conclusions/interpretationAltogether, our findings provide new evidence about the therapeutic role of HGF and open new opportunities for inducing renal regeneration in diabetic nephropathy.

CD40 gene silencing reduces the progression of experimental lupus nephritis modulating local milieu and systemic mechanisms.

Lupus nephritis (LN) is an autoimmune disorder in which co-stimulatory signals have been involved. Here we tested a cholesterol-conjugated-anti-CD40-siRNA in dendritic cells (DC) in vitro and in a model of LPS to check its potency and tissue distribution. Then, we report the effects of Chol-siRNA in an experimental model of mice with established lupus nephritis. Our in vitro studies in DC show a 100%intracellular delivery of Chol-siRNA, with a significant reduction in CD40 after LPS stimuli. In vivo in ICR mice, the CD40-mRNA suppressive effects of our Chol-siRNA on renal tissue were remarkably sustained over a 5 days after a single preliminary dose of Chol-siRNA. The intra-peritoneal administration of Chol-siRNA to NZB/WF1 mice resulted in a reduction of anti-DNA antibody titers, and histopathological renal scores as compared to untreated animals. The higher dose of Chol-siRNA prevented the progression of proteinuria as effectively as cyclophosphamide, whereas the lower dose was as effective as CTLA4. Chol-siRNA markedly reduced insterstitialCD3+ and plasma cell infiltrates as well as glomerular deposits of IgG and C3. Circulating soluble CD40 and activated splenic lymphocyte subsets were also strikingly reduced by Chol-siRNA. Our data show the potency of our compound for the therapeutic use of anti-CD40-siRNA in human LN and other autoimmune disorders.

Cold ischaemia, innate immunity and deterioration of the glomerular filtration barrier in antibody-mediated acute rejection

BACKGROUND In renal transplantation, cold ischaemia (CI) determines acute rejection through innate immunity among others. Acute rejection episodes are a risk factor for late allograft dysfunction and proteinuria. This implies some alteration of the glomerular filtration barrier (GFB). Besides its effects on acute rejection, we hypothesized that CI might somehow damage the GFB being directly responsible for late proteinuria. METHODS On rat kidney allografts suffering from antibody-mediated acute rejection with or without CI and compared with syngeneic grafts, we quantified the gene expression of innate and adaptive immune mediators and assessed the capillary glomerular basement membranes (CapBM) by immunostaining collagen-IV (ColIV). ColIV was also assessed in equivalent groups from a previous chronic study followed up for 24 weeks. RESULTS CI up-regulated enzymes critical in the stabilization of collagen chains, increasing ColIV deposition and thickening the CapBM. CI increased the C4d and IgG deposits within grafts, amplified innate immunity (heat shock protein 70, fibronectin, Toll-like-receptor-4 and MyD88) and synergized with alloreactivity in triggering adaptive response through CD40. CONCLUSIONS Initial CI increased the ColIV deposition in CapBM, damaging the GFB and being responsible for part of the proteinuria associated with late allograft dysfunction. This deterioration of the GFB is related to the early innate immunity activation and subsequent up-regulation of CD40 in acute rejected grafts. In chronic rejected allografts, thickened CapBM may be a consequence of an unresolved immune-inflammatory response worsened by CI.

Penetration of polymeric nanoparticles loaded with an HIV-1 inhibitor peptide derived from GB virus C in a vaginal mucosa model

ABSTRACT Despite the great effort to decrease the HIV infectivity rate, current antiretroviral therapy has several weaknesses; poor bioavailability, development of drug resistance and poor ability to access tissues. However, molecules such as peptides have emerged as a new expectative to HIV eradication. The vaginal mucosa is the main spreading point of HIV. There are natural barriers such as the vaginal fluid which protects the vaginal epithelium from any foreign agents reaching it. This work has developed and characterized Nanoparticles (NPs) coated with glycol chitosan (GC), loaded with an HIV‐1 inhibitor peptide (E2). In vitro release and ex vivo studies were carried out using the vaginal mucosa of swine and the peptide was determined by HPLC MS/MS validated method. Moreover, the peptide was labeled with 5(6)‐carboxyfluoresceine and entrapped into the NPs to carried out in vivo studies and to evaluate the NPs penetration and toxicity in the vaginal mucosa of the swine. The mean size of the NPs, &xgr; and the loading percentage were fundamental features for to reach the vaginal tissue and to release the peptide within intercellular space. The obtained results suggesting that the fusion inhibitor peptides loaded into the NPs coated with GC might be a new way to fight the HIV‐1, due to the formulation might reach the human epithelial mucosa and release peptide without any side effects.

Erratum to: JAK3-STAT pathway blocking benefits in experimental lupus nephritis

Unfortunately, after publication of this article [1], it was noticed that the name of Juliana Draibe was listed incorrectly. The corrected name can be seen in the author list above and the original article has been updated to reflect this change.

Does Timng of MSC Infusion Determines Optimal Immunomodulation in a Renal Transplant Model in Rat?: Abstract# C1576

C1575 Adipose Tissue-Derived Stem Cells Suppress Acute Cellular Rejection Via TSG-6 and CD44 Interaction in Rat Kidney Transplantation. T. Kato,1 M. Okumi,1 Y. Kakuta,1 K. Yamanaka,1 S. Takahara,2 N. Nonomura.1 1Urology, Osaka University Graduate School of Medicine, Suita, Osaka, Japan; 2Advanced Technology for Transplantation, Osaka University Graduate School of Medicine, Suita, Osaka, Japan. Objective: ]Mesenchymal stem cells have exhibited immunomodulatory effects in vitro. Notably, adipose tissue yields far more stem cells (adipose tissue-derived stem cells, ADSCs) than bone marrow. However, the benefi cial effects of ADSCs on alloreactivity in organ transplant models are not well known. In this study, we evaluated the benefi cial effects of ADSCs in a fully MHC-mismatched rat kidney transplantation and analyzed the underlying molecular mechanism. Methods: Autologous ADSCs (2×106) were injected via the left renal artery of the donors prior to the nephrectomy (ADSCs group). Graft survival, histological changes, and the expression of cytokines/proteins were assessed. In vitro experiment, the immunosuppressive capacity of ADSCs was tested in a mixed lymphocyte reaction (MLR). Results: Histology of the ADSCs group revealed a reduced rejection grade, while the mean number of infi ltrated CD4+/CD8+T cells was also signifi cantly decreased as compared to the control (p=0.028 and p=0.016). Injection of ADSCs led to a signifi cantly prolonged graft survival compared with the control (p=0.013). In vitro, ADSCs dose-dependently suppressed alloreactive lymphocytes and increased the level of TNF-stimulated gene 6 protein (TSG-6) in MLR, which has antiinfl ammatory capacity. Recombinant TSG-6 markedly suppressed alloreactive T cells through downregulating CD44 on CD4+Tcells, which may lead to the suppression of T cell activation and infi ltration into allografts. Conclusions: Our fi ndings showed that ADSCs attenuated acute rejection by secreting TSG-6 plus direct cell interaction. These fi ndings contribute to the clinical application of these cells in organ transplantation. Abstract# C1576 Does Timng of MSC Infusion Determines Optimal Immunomodulation in a Renal Transplant Model in Rat? A. Merino,1 E. Ripoll,1 N. Bolanos,1 M. Goma,2 L. de Ramon,1 N. Lloberas,1 O. Bestard,1,3 J. Grinyo,1,3,4 J. Torras.1,3,4 1Laboratory of Nephrology, IDIBELL, L’Hospitalet, Barcelona, Spain; 2Pathology, Hospital Universitari Bellvitge, L’Hospitalet, Barcelona, Spain; 3Nephrology, Hospital Universitari Bellvitge, L’Hospitalet, Barcelona, Spain; 4Ciencies Cliniques, Facultat De Medicina. Universitat Barcelona, L’Hospitalet, Barcelona, Spain. Aim: To investigate the effectiveness and timing of the immunomodulatory response produced by mesenchymal stromal cells (MSCs) in a rat model of allogeneic kidney transplantation. Methods:To study the immunologic/immunosupressor effects of MSCs (donor), Lewis rats received an allogeneic kidney from Wistar rats. The infusion pattern of MSCs was determined in a previous experimental design where naïve rats were injected with 106cells and the principal cellular blood subpopulations were analyzed each 24 h.Rats were randomized into the four groups listed below and followed for 21 days. 1. NT, in which rats received infusion of PBS (n =7) 2. CsA, in which rats received a single daily dose of 5 mg/kg cyclosporine by oral gavages for 21 days (n =7) 3. MSC4, in which rats received two infusion of MSCs (106cells), Day -4 before renal transplantation and 1 hour after the surgery (n = 5) 4. MSC7, in which rats received two infusion of MSCs (106cells), Day -7 before renal transplantation and 1 hour after the surgery (n = 5). To study the principal cellular blood subpopulations and the renal function, samples were collected before the surgery, on day 7 post-transplant, and on the day of sacrifi ce. Results: Analyzing the three major subpopulations of lymphocytes in peripheral blood (T cells, B cells and NK cells), we observed that both MSC treatments decrease the percentage of the three lymphocyte subpopulations at 7 days post-transplantation. But the MSC7 group showed a percentage of lymphocytes similar to the CsA group. The renal function, measured by creatinine clearance, in the MSC7 group was signifi cantly lower than the MSC4 and NT. The MSC7 group had signifi cantly increased the survival respect to MSC4 and NT groups. Conclusions: MSC have a benefi cial effect on the immune system of transplanted rats which improved graft function. However, the infusion pattern of MSCs plays a crucial role in the immune system modulation. C1576 Does Timng of MSC Infusion Determines Optimal Immunomodulation in a Renal Transplant Model in Rat? A. Merino,1 E. Ripoll,1 N. Bolanos,1 M. Goma,2 L. de Ramon,1 N. Lloberas,1 O. Bestard,1,3 J. Grinyo,1,3,4 J. Torras.1,3,4 1Laboratory of Nephrology, IDIBELL, L’Hospitalet, Barcelona, Spain; 2Pathology, Hospital Universitari Bellvitge, L’Hospitalet, Barcelona, Spain; 3Nephrology, Hospital Universitari Bellvitge, L’Hospitalet, Barcelona, Spain; 4Ciencies Cliniques, Facultat De Medicina. Universitat Barcelona, L’Hospitalet, Barcelona, Spain. Aim: To investigate the effectiveness and timing of the immunomodulatory response produced by mesenchymal stromal cells (MSCs) in a rat model of allogeneic kidney transplantation. Methods:To study the immunologic/immunosupressor effects of MSCs (donor), Lewis rats received an allogeneic kidney from Wistar rats. The infusion pattern of MSCs was determined in a previous experimental design where naïve rats were injected with 106cells and the principal cellular blood subpopulations were analyzed each 24 h.Rats were randomized into the four groups listed below and followed for 21 days. 1. NT, in which rats received infusion of PBS (n =7) 2. CsA, in which rats received a single daily dose of 5 mg/kg cyclosporine by oral gavages for 21 days (n =7) 3. MSC4, in which rats received two infusion of MSCs (106cells), Day -4 before renal transplantation and 1 hour after the surgery (n = 5) 4. MSC7, in which rats received two infusion of MSCs (106cells), Day -7 before renal transplantation and 1 hour after the surgery (n = 5). To study the principal cellular blood subpopulations and the renal function, samples were collected before the surgery, on day 7 post-transplant, and on the day of sacrifi ce. Results: Analyzing the three major subpopulations of lymphocytes in peripheral blood (T cells, B cells and NK cells), we observed that both MSC treatments decrease the percentage of the three lymphocyte subpopulations at 7 days post-transplantation. But the MSC7 group showed a percentage of lymphocytes similar to the CsA group. The renal function, measured by creatinine clearance, in the MSC7 group was signifi cantly lower than the MSC4 and NT. The MSC7 group had signifi cantly increased the survival respect to MSC4 and NT groups. Conclusions: MSC have a benefi cial effect on the immune system of transplanted rats which improved graft function. However, the infusion pattern of MSCs plays a crucial role in the immune system modulation. Abstract# C1577 A Strategy for Reducing the Immunogenicity of Human Renal Allografts. L. Brasile,1 P. Glowacki,1 B. Stubenitsky.2 1BREONICS, Inc., Albany, NY; 2U of Alabama, Birmingham, AL. Background-Transplantation is unique immunologically due to both donor and recipient antigen presenting cells being present at reperfusion. As part of immune surveillance, Dendritic Cells (DC)/passenger leukocytes (PL) are trapped in the renal parenchyma at organ procurement. On reperfusion, DC migrate into secondary lymphatics where host immune cells encounter donor antigens via direct antigen presentation. We tested the feasibility of minimizing immunogenicity by removal of DC during ex vivo warm perfusion. Methods/Materials-We used an acellular Exsanguinous Metabolic Support (EMS) perfusion technology at 32°C with human renal allografts. The human kidneys were received on ice, fl ushed with EMS solution (32°C) and placed on perfusion. Biopsies were taken preand post-EMS perfusion (24H). Frozen sections were made from representative sections of the kidneys. The number and location of the DC within the kidneys were determined using an indirect immunofl uorescence assay with CD209 (DC-sign) antibody and a secondary antibody of Alexafl uor488, with fl uoroshield plus DAPI. The results were obtained using ImageJ software measuring positive fl uorescence per fi eld. Results-There was a signifi cant reduction in the number of resident DC following 24H of EMS perfusion. C1577 A Strategy for Reducing the Immunogenicity of Human Renal Allografts. L. Brasile,1 P. Glowacki,1 B. Stubenitsky.2 1BREONICS, Inc., Albany, NY; 2U of Alabama, Birmingham, AL. Background-Transplantation is unique immunologically due to both donor and recipient antigen presenting cells being present at reperfusion. As part of immune surveillance, Dendritic Cells (DC)/passenger leukocytes (PL) are trapped in the renal parenchyma at organ procurement. On reperfusion, DC migrate into secondary lymphatics where host immune cells encounter donor antigens via direct antigen presentation. We tested the feasibility of minimizing immunogenicity by removal of DC during ex vivo warm perfusion. Methods/Materials-We used an acellular Exsanguinous Metabolic Support (EMS) perfusion technology at 32°C with human renal allografts. The human kidneys were received on ice, fl ushed with EMS solution (32°C) and placed on perfusion. Biopsies were taken preand post-EMS perfusion (24H). Frozen sections were made from representative sections of the kidneys. The number and location of the DC within the kidneys were determined using an indirect immunofl uorescence assay with CD209 (DC-sign) antibody and a secondary antibody of Alexafl uor488, with fl uoroshield plus DAPI. The results were obtained using ImageJ software measuring positive fl uorescence per fi eld. Results-There was a signifi cant reduction in the num