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Rosado M.M.,Immunology Research Area | Bernardo M.E.,Ospedale Pediatrico Bambino Gesu | Scarsella M.,Immunology Research Area | Conforti A.,Ospedale Pediatrico Bambino Gesu | And 12 more authors.
Stem Cells and Development | Year: 2015

Bone marrow (BM)-derived mesenchymal stromal cells (MSCs), endowed with immunosuppressive and anti-inflammatory properties, represent a promising tool in immunoregulatory and regenerative cell therapy. Clarifying the interactions between MSCs and B-lymphocytes may be crucial for designing innovative MSC-based strategies in conditions in which B cells play a role, including systemic lupus erythematosus (SLE) and rejection of kidney transplantation. In this study, we show that, both in healthy subjects and in patients, in vitro B-cell proliferation, plasma-cell differentiation, and antibody production are inhibited by BM-derived MSCs when peripheral blood lymphocytes are stimulated with CpG, but not when sorted B cells are cultured with MSCs+CpG. Inhibition is restored in CpG+MSC cocultures when sorted T cells are added to sorted B cells, suggesting that this effect is mediated by T cells, with both CD4+ and CD8+ cells playing a role. Moreover, cell-cell contact between MSCs and T cells, but not between MSCs and B cells, is necessary to inhibit B-cell proliferation. Thus, the presence of functional T cells, as well as cell-cell contact between MSCs and T cells, are crucial for B-cell inhibition. This information can be relevant for implementing MSC-based therapeutic immune modulation in patients in whom T-cell function is impaired. © 2015 Mary Ann Liebert, Inc.

PubMed | Unit of Nephrology and Dialysis., University of Calabria, University of Chieti Pescara and CoreQuest
Type: | Journal: Drug design, development and therapy | Year: 2016

The use of glucose as the only osmotic agent in peritoneal dialysis (PD) solutions (PDSs) is believed to exert local (peritoneal) and systemic detrimental actions, particularly in diabetic PD patients. To improve peritoneal biocompatibility, we have developed more biocompatible PDSs containing xylitol and carnitine along with significantly less amounts of glucose and have tested them in cultured Human Vein Endothelial Cells (HUVECs) obtained from the umbilical cords of healthy (C) and gestational diabetic (GD) mothers.Primary C- and GD-HUVECs were treated for 72 hours with our PDSs (xylitol 0.7% and 1.5%, whereas carnitine and glucose were fixed at 0.02% and 0.5%, respectively) and two glucose-based PDSs (glucose 1.36% or 2.27%). We examined their effects on endothelial cell proliferation (cell count), viability (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide assay), intracellular nitro-oxidative stress (peroxynitrite levels), Vascular Cell Adhesion Molecule-1 and Intercellular Adhesion Molecule-1 membrane exposure (flow cytometry), and HUVEC-monocyte interactions (U937 adhesion assay).Compared to glucose-based PDSs, our in vitro studies demonstrated that the tested PDSs did not change the proliferative potential both in C- and GD-HUVECs. Moreover, our PDSs significantly improved endothelial cell viability, compared to glucose-based PDSs and basal condition. Notably, glucose-based PDSs significantly increased the intracellular peroxynitrite levels, Vascular Cell Adhesion Molecule-1 and Intercellular Adhesion Molecule-1 membrane exposure, and endothelial cell-monocyte interactions in both C- and GD-HUVECs, as compared with our experimental PDSs.Present results show that in control and diabetic human endothelial cell models, xylitol-carnitine-based PDSs do not cause cytotoxicity, nitro-oxidative stress, and inflammation as caused by hypertonic glucose-based PDSs. Since xylitol and carnitine are also known to favorably affect glucose homeostasis, these findings suggest that our PDSs may represent a desirable hypertonic solution even for diabetic patients in PD.

PubMed | University of Turin, Irccs Instituto Of Ricerche Farmacologiche Mario Negri, Unit of Nephrology and Dialysis and Regina Margherita Hospital
Type: Case Reports | Journal: Pediatric nephrology (Berlin, Germany) | Year: 2016

The risk of disease recurrence after a kidney transplant is high in patients with atypical hemolytic uremic syndrome (aHUS) and mutations in the complement factor H (FH) gene (CFH). Since FH is mostly produced by the liver, a kidney transplant does not correct the genetic defect. The anti-C5 antibody eculizumab prevents post-transplant aHUS recurrence, but it does not cure the disease. Combined liver-kidney transplantation has been performed in few patients with CFH mutations based on the rationale that liver replacement provides a source of normal FH.We report the 9-year follow-up of a child with aHUS and a CFH mutation, including clinical data, extensive genetic characterization, and complement profile in the circulation and at endothelial level. The outcome of kidney and liver transplants performed separately 3years apart are reported.The patient showed incomplete response to plasma, with relapsing episodes, progression to end-stage renal disease, and endothelial-restricted complement dysregulation. Eculizumab prophylaxis post-kidney transplant did not achieve sustained remission, leaving the child at risk of disease recurrence. A liver graft given 3years after the kidney transplant completely abrogated endothelial complement activation and allowed eculizumab withdrawal.Liver transplant may definitely cure aHUS and represents an option for patients with suboptimal response to eculizumab.

Noris M.,Irccs Instituto Of Ricerche Farmacologiche Mario Negri | Galbusera M.,Irccs Instituto Of Ricerche Farmacologiche Mario Negri | Gastoldi S.,Irccs Instituto Of Ricerche Farmacologiche Mario Negri | Macor P.,University of Trieste | And 12 more authors.
Blood | Year: 2014

Atypical hemolytic-uremic syndrome (aHUS) is associated with genetic complement abnormalities/anti-complement factor H antibodies, which paved the way to treatment with eculizumab. We studied 44 aHUS patients and their relatives to (1) test new assays of complement activation, (2) verify whether such abnormality occurs also in unaffected mutation carriers, and (3) search for a tool for eculizumab titration. An abnormal circulating complement profile (low C3, high C5a, or SC5b-9) was found in 47% to 64% of patients, irrespective of disease phase. Acute aHUS serum, but not serum from remission, caused wider C3 and C5b-9 deposits than control serum on unstimulated human microvascular endothelial cells (HMEC-1). In adenosine 5'-diphosphate-activated HMEC-1, also sera from 84% and 100% of patients in remission, and from all unaffected mutation carriers, induced excessive C3 and C5b-9 deposits. At variance, in most patients with C3 glomerulopathies/immune complex-associated membranoproliferative glomerulonephritis, serum-induced endothelial C5b-9 deposits were normal. In 8 eculizumab-treated aHUS patients, C3/SC5b-9 circulating levels did not change posteculizumab, whereas serum-induced endothelial C5b-9 deposits normalized after treatment, paralleled or even preceded remission, and guided drug dosing and timing. These results point to efficient complement inhibition on endothelium for aHUS treatment. C5b-9 endothelial deposits might help monitor eculizumab effectiveness, avoid drug overexposure, and save money considering the extremely high cost of the drug. © 2014 by The American Society of Hematology.

Sharma S.K.,BP Koirala Institute of Health science | Zou H.,Southern Medical University | Togtokh A.,University of Mongolia | Ene-Iordache B.,Mario Negri Institute for Pharmacological Research | And 9 more authors.
American Journal of Kidney Diseases | Year: 2010

Background In 2007, the International Society of Nephrology funded the Kidney Disease Data Center database to house data from sponsored programs aimed at preventing chronic kidney disease and its complications in developing nations. This study compares baseline characteristics and burden of illness among participants from centers in China, Mongolia, and Nepal. An important secondary objective is to show the feasibility of screening for chronic kidney disease and its major risk factors in a diverse group of lower income settings. Study Design Cross-sectional screening study. Setting & Participants Participants from Nepal (n = 8,398), China (n = 1,999), and Mongolia (n = 997). Screening was open to the public for participants in China and Nepal; referral from a general practitioner was required for participants in Mongolia. Outcomes Estimated glomerular filtration rate (eGFR), proteinuria, hypertension, diabetes, obesity, cardiovascular risk. Measurement Demographic and clinical data were collected prospectively using a standard format. Blood and urine specimens were provided according to local protocol. Results Of 11,394 participants, decreased eGFR (<60 mL/min/1.73 m2) was present in 7.3%-14% of participants across centers; proteinuria (<1+) on dipstick (2.4%-10%), hypertension (26%-36%), diabetes (3%-8%), and obesity (body mass index <30 kg/m2; 2%-20%) were all common. Predicted 5-year cardiovascular risk <10% ranged from 20%-89%. Numbers needed to screen to detect a new case of eGFR <60 mL/min/1.73 m2, hypertension, or diabetes were 2.6 (95% CI, 2.5-2.7), 3.4 (95% CI, 3.1-3.7), and 4.7 (95% CI, 3.3-8.0) for Nepal, China, and Mongolia, respectively. Limitations May not be representative of the general population. Conclusions The acceptable diagnostic yield of abnormalities across these 3 diverse settings suggests that trials of targeted screening and intervention are feasible and warranted in such countries. © 2010 National Kidney Foundation, Inc.

Noris M.,Irccs Instituto Of Ricerche Farmacologiche Mario Negri | Remuzzi G.,Unit of Nephrology and Dialysis
Nature reviews. Nephrology | Year: 2014

Haemolytic uraemic syndrome (HUS) is characterized by nonimmune haemolytic anaemia, thrombocytopenia and renal impairment-most incidents in childhood are caused by shiga toxin-producing bacteria. Atypical HUS (aHUS) accounts for 10% of cases and has a poor prognosis. About 60% of patients with aHUS have dysregulation of the alternative complement pathway (complement-mediated aHUS). The kidney is the main target organ, but other organs might also be affected. Cardiac complications occur in 3-10% of patients with complement-mediated aHUS, as a consequence of microangiopathic injury in the coronary microvasculature, and can cause sudden death. Emerging evidence also suggests that either thrombosis or stenosis of the medium and large arteries might complicate disease course, and such disorders occur even after renal function is lost. In this Perspectives article we discuss the impact of cardiovascular involvement in complement-mediated aHUS, the role of acute and chronic complement hyperactivation in such events and the implications for treatment.

Caroli A.,Mario Negri Institute for Pharmacological Research | Caroli A.,Laboratory of Epidemiology | Antiga L.,Mario Negri Institute for Pharmacological Research | Conti S.,Mario Negri Institute for Pharmacological Research | And 7 more authors.
American Journal of Pathology | Year: 2011

Total kidney and cyst volumes have been used to quantify disease progression in autosomal dominant polycystic kidney disease (ADPKD), but a causal relationship with progression to renal failure has not been demonstrated. Advanced image processing recently allowed to quantify extracystic tissue, and to identify an additional tissue component named "intermediate," appearing hypoenhanced on contrast-enhanced computed tomography (CT). The aim of this study is to provide a histological characterization of intermediate volume, investigate its relation with renal function, and provide preliminary evidence of its role in long-term prediction of functional loss. Three ADPKD patients underwent contrast-enhanced CT scans before nephrectomy. Histological samples of intermediate volume were drawn from the excised kidneys, and stained with hematoxylin and eosin and with saturated picrosirius solution for histological analysis. Intermediate volume showed major structural changes, characterized by tubular dilation and atrophy, microcysts, inflammatory cell infiltrate, vascular sclerosis, and extended peritubular interstitial fibrosis. A significant correlation (r = -0.69, P < 0.001) between relative intermediate volume and baseline renal function was found in 21 ADPKD patients. Long-term prediction of renal functional loss was investigated in an independent cohort of 13 ADPKD patients, followed for 3 to 8 years. Intermediate volume, but not total kidney or cyst volume, significantly correlated with glomerular filtration rate decline (r = -0.79, P < 0.005). These findings suggest that intermediate volume may represent a suitable surrogate marker of ADPKD progression and a novel therapeutic target. © 2011 American Society for Investigative Pathology.

Ruggenenti P.,Irccs Instituto Of Ricerche Farmacologiche Mario Negri | Ruggiero B.,Irccs Instituto Of Ricerche Farmacologiche Mario Negri | Cravedi P.,Irccs Instituto Of Ricerche Farmacologiche Mario Negri | Vivarelli M.,Childrens Hospital Bambino Gesu | And 12 more authors.
Journal of the American Society of Nephrology | Year: 2014

The outcome of steroid-dependent or frequently relapsing nephrotic syndrome of minimal change disease (MCD), mesangial proliferative GN (MesGN), or FSGS may be poor and with major treatment toxicity. This academic,multicenter, off-on trial (ClinicalTrials.gov #NCT00981838) primarily evaluated the effects of rituximab therapy followed by immunosuppression withdrawal on disease recurrence in 10 children and 20 adults with MCD/MesGN (n=22) or FSGS who had suffered ≥2 recurrences over the previous year and were in steroidinduced remission for ≥1 month. Participants received one dose (n=28) or two doses of rituximab (375 mg/m2 intravenously).At 1 year, allpatientswere in remission: 18were treatment-free and 15 never relapsed.Compared with the year before rituximab treatment, total relapses decreased from 88 to 22 and the per-patient median number of relapses decreased from 2.5 (interquartile range [IQR], 2-4) to 0.5 (IQR, 0-1; P<0.001) during 1 year of follow-up. Reduction was significant across subgroups (children, adults,MCD/MesGN, and FSGS; P<0.01). After rituximab, the per-patient steroid maintenance median dose decreased from 0.27 mg/kg (IQR, 0.19-0.60) to 0mg/kg (IQR, 0-0.23) (P,0.001), and the median cumulative dose to achieve relapse remission decreased from 19.5 mg/kg (IQR, 13.0-29.2) to 0.5 mg/kg (IQR, 0-9.4) (P<0.001). Furthermore, the mean estimated GFR increased from 111.3±25.7 to 121.8±29.2ml/min per 1.73m2 (P=0.01),with the largest increases in children and in FSGS subgroups. The mean height z score slope stabilized in children (P<0.01). Treatment was well tolerated. Rituximab effectively and safely prevented recurrences and reduced the need for immunosuppression in steroiddependent or frequently relapsing nephrotic syndrome, and halted disease-associated growth deficit in children. Copyright © 2014 by the American Society of Nephrology.

Perico N.,Mario Negri Institute for Pharmacological Research | Antiga L.,Mario Negri Institute for Pharmacological Research | Caroli A.,Mario Negri Institute for Pharmacological Research | Ruggenenti P.,Mario Negri Institute for Pharmacological Research | And 18 more authors.
Journal of the American Society of Nephrology | Year: 2010

Activation of mammalian target of rapamycin (mTOR) pathways may contribute to uncontrolled cell proliferation and secondary cyst growth in patients with autosomal dominant polycystic kidney disease (ADPKD). To assess the effects of mTOR inhibition on disease progression, we performed a randomized, crossover study (The SIRENA Study) comparing a 6-month treatment with sirolimus or conventional therapy alone on the growth of kidney volume and its compartments in 21 patients with ADPKD and GFR ≥40 ml/min per 1.73 m2. In 10 of the 15 patients who completed the study, aphthous stomatitis complicated sirolimus treatment but was effectively controlled by topical therapy. Compared with pretreatment, posttreatment mean total kidney volume increased less on sirolimus (46 ± 81 ml; P = 0.047) than on conventional therapy (70 ± 72 ml; P = 0.002), but we did not detect a difference between the two treatments (P = 0.45). Cyst volume was stable on sirolimus and increased by 55 ± 75 ml (P = 0.013) on conventional therapy, whereas parenchymal volume increased by 26 ± 30 ml (P = 0.005) on sirolimus and was stable on conventional therapy. Percentage changes in cyst and parenchyma volumes were significantly different between the two treatment periods. Sirolimus had no appreciable effects on intermediate volume and GFR. Albuminuria and proteinuria marginally but significantly increased during sirolimus treatment. In summary, sirolimus halted cyst growth and increased parenchymal volume in patients with ADPKD. Whether these effects translate into improved long-term outcomes requires further investigation. Copyright © 2010 by the American Society of Nephrology.

PubMed | Irccs Instituto Of Ricerche Farmacologiche Mario Negri, Bambin Gesu Pediatric Hospital, University of Milan, Unit of Nephrology and Dialysis and 2 more.
Type: | Journal: Molecular immunology | Year: 2016

Membranoproliferative glomerulonephritis (MPGN) is an uncommon cause of chronic nephropathy recently reclassified into immunoglobulin-associated MPGN (Ig-MPGN) and C3 glomerulopathy (C3G). In this study we aimed: (1) to evaluate the complement genetic and biochemical profile in patients with Ig-MPGN/C3G; (2) to investigate whether genetic variants and different patterns of complement activation (i.e., fluid versus solid phase) correlate with disease manifestations and outcomes.In 140 patients with idiopathic Ig-MPGN or C3G we performed complement biochemical and genetic screening and correlated genetic, biochemical and histology data with clinical features.Mutations in genes encoding alternative pathway complement proteins were found in both Ig-MPGN and C3G, and mutations in the two components of the C3 convertase are the most prevalent. We also report a mutation in THBD encoding thrombomodulin in a C3G patient. The presence of mutations alone does not significantly increase the risk of Ig-MPGN or C3G, but it does so when combined with common susceptibility variants (CD46 c.-366A in Ig-MPGN; CFH V62 and THBD A473 in C3G). Finally, patients without complement gene mutations or C3NeFs--autoantibodies that stabilize the alternative pathway C3 convertase--have a higher risk of progressing to end-stage renal disease than patients with identified mutations and/or C3NeFs, suggesting the existence of different pathogenetic mechanisms that lead to renal disease.We provide new insights into the pathogenesis of Ig-MPGN/C3G that underscore the complex nature of these diseases and suggest that the current C3G classification may miss many cases associated with abnormalities of the complement alternative pathway.

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