100% of mice of group 4 (controls). and overall survival were significantly improved in SERPINB3-treated groups vs. controls. Histological analysis demonstrated a lower prevalence of severe tubular lesions in kidneys of group 5 vs. group 6. SERPINB3-treated mice showed an overall trend toward a reduced prevalence of severe lesions in both strains. Th17:Treg ratio was significantly decreased in splenocytes of MRL/mice treated with SERPINB3, compared to untreated control mice. Conclusions: SERPINB3 significantly improves disease course and delays the Takinib onset of severe glomerulonephritis in lupus-prone mice, possibly inducing a more Takinib tolerogenic immune phenotype. mice MRL/mice display an impaired Fas function due to a recessive autosomal mutation named (standing for lymphoproliferation). Descending abnormalities in the apoptotic process lead to diverse clinical features, mostly depending on dysregulated CD4+ T cell and B cell function including widespread lymphadenopathy with double negative T cell infiltrates increasing with disease severity, early severe proliferative nephritis leading to death between 3 and 7 months of age, severe necrotizing arteritis, neuropsychiatric symptoms and erosive polyarthritis (16). MRL/mice were treated with recombinant SERPINB3 before the development of proteinuria in order to explore the preventive approach in a multiorgan system. Twenty 8-week-old MRL/female mice (Harlan Laboratories) were subdivided into 2 groups of 10 mice each and were intraperitoneally injected with a total volume of 100 l consisting of 7.5 g of SERPINB3 in 100 l of vehicle (group 5) or 100 l of vehicle (group 6), as controls. Mice were injected twice a week, starting from the 9th to the 18th week of age. Urine samples were collected and proteinuria was evaluated weekly. Blood samples were collected from the caudal vein 3 weeks apart, starting from the 9th week of age, until mice sacrifice at week 13 (6 mice) and 16C18 (6 mice). Time for sacrifice was chosen mice and from week 17 in NZB/W F1 mice. Creatinine assessment was carried out on Cobas 8000 (Roche Diagnostics) using an enzymatic method, traceable to Isotope Dilution Mass Spectrometry (IDMS) reference procedure. Disease-free and overall survival were evaluated in all mice. Proteinuria-free survival was defined as 300 mg/dl, according to manufacturer’s instruction, as the threshold of 300 mg/dl designates a frank positivity. Measurement of serum autoantibodies Serum levels of mouse IgG anti-C1q and anti-dsDNA antibodies were evaluated by standardized home-made ELISA tests as previously described (17). Briefly, for anti-C1q antibodies, plates were coated with C1q at a concentration of 5 g/ml. Sera were added in duplicate diluted 1:4 in 1% BSA/PBS with 1 M NaCl, to prevent immunocomplexes (ICs) formation. Alkaline phosphatase-conjugated goat anti-mouse IgG was added at the dilution of Rabbit Polyclonal to MYT1 1 1:10,000 in 1% Takinib BSA/PBS with 1 M NaCl. Finally, mice SERPINB3-treated and vehicle-treated MRL/mice were sacrificed at 13 and 16C18 weeks of age. The spleens were removed and dissociated in RPMI medium supplemented with 50 mM HEPES and 10% fetal bovine serum. The cell suspension was passed through a 70-m strainer, and cells were collected by centrifugation at 300 for 5 min. Erythrocytes were lysed by incubating the cells in red blood cell lysis buffer (eBiosciences) at room temperature for 5 min. T lymphocytes were then isolated using the EasySep? mouse T cells isolation kit (StemCell? technologies) following the manufacturer’s instructions. Flow cytometry analysis CD4 + CD25 + Foxp3 + regulatory T (Treg) cells are important regulators of immune response and the imbalance between Treg cells and T helper (Th)17 cells has been already described in a number of different inflammatory and autoimmune diseases (19). The Th17:Treg ratio in T cells isolated.