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Effectiveness of rituximab in pediatric idiopathic nephrotic syndrome suggests that B cells play a pathogenic role. We tested safety and efficacy of the B-cell-modulating agent belimumab in frequently relapsing nephrotic syndrome (FRNS). An open-label, prospective, single-arm pilot study (EUDRACT 2017-003839-11) was designed to treat 10 children with FRNS with i.v. belimumab for 12 months. Prednisone was tapered/stopped. Safety, number of relapses, cumulative prednisone dose and B-cell subset "levels" are referred to both B cell subset and immunoglobulin. Five patients were enrolled, and four reached the primary 6-month endpoint. Of these, two completed the 12-month endpoint. Three patients experienced ≥2 relapses while on belimumab, requiring additional immunosuppression. Compared to the 6 months before belimumab treatment, the mean number of relapses (1.4 vs. 2, p=0.21) and the mean cumulative prednisone dose (1.86 vs. 2.62 g/m2, p=0.17) were not significantly reduced during the 6 months on belimumab. This study was terminated by the steering committee after the interim evaluation because belimumab failed to show clear benefits to counterbalance the inconvenience of monthly i.v. infusion. During follow-up, total and mature-naive B cells decreased, while no change in memory B-cells was observed. Serum immunoglobulins remained stable. No infusion reaction was observed. Short-term treatment with belimumab in pediatric FRNS was well tolerated. The number of patients was too small to draw conclusions on efficacy. Nonetheless, we did not observe clear improvements. The burden of monthly in-hospital i.v. infusions outweighed potential benefits. Persistence of circulating memory B cells supports their pathogenic role in the disease. A higher resolution version of the Graphical abstract is available as Supplementary information.

Type 1 diabetes and thyroid disease are T cell-dependent autoimmune endocrinopathies. The standard substitutive administration of the deficient hormones does not halt the autoimmune process; therefore, development of immunotherapies aiming to preserve the residual hormonal cells, is of crucial importance. PTPN22 C1858T mutation encoding for the R620W lymphoid tyrosine phosphatase variant, plays a potential pathophysiological role in autoimmunity. The PTPN22 encoded protein Lyp is a negative regulator of T cell antigen receptor signaling; R620W variant, leading to a gain of function with paradoxical reduced T cell activation, may represent a valid therapeutic target. We aimed to develop novel wild type PTPN22 short interfering RNA duplexes (siRNA) and optimize their delivery into Jurkat T cells and PBMC by using liposomal carriers. Conformational stability, size and polydispersion of siRNA in lipoplexes was measured by CD spectroscopy and DLS. Lipoplexes internalization and toxicity evaluation was assessed by confocal microscopy and flow cytometry analysis. Their effect on Lyp expression was evaluated by means of Western Blot and confocal microscopy. Functional assays through engagement of TCR signaling were established to evaluate biological consequences of down-modulation. Both Jurkat T cells and PBMC were efficiently transfected by stable custom lipoplexes. Jurkat T cell morphology and proliferation was not affected. Lipoplexes incorporation was visualized in CD3+ but also in CD3- peripheral blood immunotypes without signs of toxicity, damage or apoptosis. Efficacy in affecting Lyp protein expression was demonstrated in both transfected Jurkat T cells and PBMC. Moreover, impairment of Lyp inhibitory activity was revealed by increase of IL-2 secretion in culture supernatants of PBMC following anti-CD3/CD28 T cell receptor-driven stimulation. The results of our study open the pathway to future trials for the treatment of autoimmune diseases based on the selective inhibition of variant PTPN22 allele using lipoplexes of siRNA antisense oligomers.

A simple, accurate and sensitive high-performance liquid chromatographic method with UV detection was carried out to measure plasma concentrations of mycophenolic acid. Following a simplified acid hydrolysis of the sample, the separation was carried out in 4 min using a Zorbax Eclipse C(8) reversed-phase column with a flow-rate of 1.5 ml/min, and monitoring the absorbance at 250 nm. Throughput was up to 100 samples in 24 h. Within the investigated concentration ranges of mycophenolic acid (0-100 mg/l), good linearity (r>0.99) was obtained. The method is sensitive (the limit of detection was about 20 microg/l) and precise (for 0.49 mg/l added to plasma, within-run C.V. was 2% and between-run was 4.2%; for 2.88 mg/l, within-run C.V. was 0.35% and between-run C.V. was 0.69%; for 24.38 mg/l, within-run C.V. was 0.77% and between-run C.V. was 3.1%). Analytical recoveries were 96% for 0.5 mg/l mycophenolic acid added to plasma, 100% for 12 mg/l and 102.5% for 24 mg/l.

Since glutathionyl-hemoglobin has been suggested to be a clinical marker of oxidative stress in human blood and given the growing biological relevance of oxidative stress as a pathogenic factor in several diseases, we describe a method to measure glutathionyl-hemoglobin concentration in erythrocytes, by using cation-exchange high-pressure liquid chromatography with UV detection. The glutathionyl-hemoglobin peak has been identified on the basis of the following findings: (a) the peak increased when the sample was incubated with oxidized glutathione; (b) the peak disappeared when the sample was reduced with dithiothreitol, with the simultaneous increase of that corresponding to hemoglobin A0; (c) the peak could be detected by incubating hemoglobin A0 with reduced glutathione; (e) deconvoluted mass spectrum of the glutathionyl-hemoglobin peak showed a 16172.0-Da molecular mass, corresponding to hemoglobin β bound to glutathione. Glutathionyl-hemoglobin concentration has been determined in erythrocytes of 40 healthy subjects, with a mean value of 2.58 ± 0.7%, calculated as the percentage of its peak area ratio to that of total hemoglobin (HbA0+HbA2+HbA1C + glutathionyl-hemoglobin). The availability of a simple and reproducible method to detect glutathionyl-hemoglobin concentration in blood could be useful in monitoring oxidative stress, and for investigating the efficacy of antioxidant therapies in clinical trials.

Cystinosis is an autosomal recessive disease caused by impaired transport of cystine across lysosomal membranes. The subsequent lysosomal storage of the poorly soluble cystine produces crystal formation and cellular damage in many tissues. The earliest involvement occurs in the renal tubules and causes Fanconi syndrome, with polyuria, dehydration, acidosis, rickets, and failure to thrive. In untreated cystinosis, the progression of renal glomerular dysfunction leads to uremia and death by 9–10 years of age unless dialysis or renal transplantation is initiated (1). The therapy of nephropathic cystinosis involves treatment with the cystine-depleting agent cysteamine (2). The most direct diagnostic method for cystinosis is measurement of leukocyte cystine content by an Escherichia coli cystine-binding protein assay. This assay is time-consuming and involves competition between nonradioactive cystine and [14C]cystine for a cystine-binding protein. The protein-bound radioactivity is then trapped on nitrocellulose filters and is inversely correlated to nonradioactive cystine (3). Here we present a method for measuring the cystine content of polymorphonuclear (PMN) leukocytes that is based on HPLC with fluorescence detection (FD) (4)(5); this method is reproducible, sensitive, and requires no radioactive reagents. Blood (4.5 mL) was drawn by venipuncture and was collected into a heparin-containing tube. PMN leukocyte separations were carried out as described by Smolin et al. (6). After sonication of the PMN leukocytes three times for 2 s (each) in 0.1 mL of 0.1 mol/L phosphate buffer, pH 7.2, containing 5 mmol/L N -ethylmaleimide (NEM), 50 μL of 120 g/L sulfosalicylic acid was added, and the cystine content in the acid-soluble fraction was determined. The protein pellet was then dissolved in 150 μL of 0.1 mol/L NaOH, and the protein concentration was …

We describe a 6-min HPLC method to measure the total concentrations of the most important thiols in plasma and urine–cysteine, homocysteine, cysteinylglycine, and glutathione–as well as the concentrations in plasma and urine, respectively, of cysteamine and 2-mercaptopropionylglycine, two compounds used to treat disorders of cysteine metabolism. Precolumn derivatization with bromobimane and reversed-phase HPLC were performed automatically by a sample processor. Throughput was up to 100 samples in 24 h. The within-run CV ranged from 0.9% to 3.4% and the between-run CV ranged from 1.5% to 6.1%. Analytical recovery was 97–107%, with little difference between plasma and urine samples. The detection limit was ∼50 nmol/L for all the analytes studied. Thiol concentrations were determined in the plasma of 206 healthy donors and in the urine of 318 healthy donors distributed for age and sex. Mean values of plasma cysteine and homocysteine were significantly lower in infants (ages

Glutathione (l-γ-glutamyl-l-cysteinylglycine), which is present in virtually all mammalian tissues, provides reducing capacity for several reactions and plays an important role in detoxification of hydrogen peroxide, other peroxides, and free radicals (1). The synthesis and degradation of glutathione are controlled by reactions of the γ-glutamyl cycle; a decrease in blood reduced glutathione (GSH) has been reported in patients affected by deficiencies of the enzymes involved in the synthesis of glutathione (1). In cells, total glutathione can be free or bound to proteins; measurement of free glutathione in blood samples is essential for evaluation of the redox and detoxification status of cells in relation to its protective role against oxidative and free radical-mediated cell injury; moreover, GSH measurement is important for the diagnosis of γ-glutamyl cycle disorders. Recently, several methods to measure glutathione in blood have been described, but little is known about the concentrations of various forms of blood glutathione in pediatric subjects (2)(3)(4)(5)(6). We report a rapid and fully automated HPLC method for determining total (tGSH), reduced (GSH), and oxidized glutathione (GSSG) in whole blood. Moreover, we report values for the different forms of blood glutathione in pediatric subjects. The reference sample group included 227 healthy, randomly selected children who attended the outpatient clinic of our hospital. The children were selected to provide approximately equal numbers of each sex across the age range of 0–15 years. The study was performed according to the recommendations of the Ethics Committee of Children’s Hospital and Research Institute “Bambino Gesu”. Blood was collected into a Vacutainer Tube (Becton Dickinson) containing EDTA. The hemoglobin …