Neutrophil extracellular traps (NET) formation is part of the neutrophil response to infections, but excessive or inappropriate NETosis may trigger the production of autoantibodies and cause organ ...damage in autoimmune disorders. Spontaneously netting neutrophils are not frequent and induction of NET in vitro by selected stimuli is necessary to investigate their structure. In the present work, the protein composition and post-translational modifications of NET produced under different stimuli have been studied by means of proteomic analysis. Neutrophils from healthy donors were stimulated by PMA, A23187, Escherichia coli LPS or untreated; after three hours, cells were washed, treated with DNase and supernatants collected for mass spectrometry. Data were analyzed by unsupervised hierarchical clustering analyses. We identified proteins contained in NETs of any source or exclusive of one stimulus: LPS-induced and spontaneous NET diverge in protein composition, while PMA- and A23187-induced NET appear more similar. Among the post-translational modifications we examined, methionine sulfoxidation is frequent especially in PMA- and LPS-induced NETs. Myeloperoxidase is the protein more extensively modified. Thus, proteomic analysis indicates that NETs induced by different stimuli are heterogeneous in terms of both protein composition and post-translational modifications, suggesting that NET induced in different conditions may have different biological effects.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Uromodulin (Tamm–Horsfall protein) is the most abundant protein excreted in the urine under physiological conditions. It is exclusively produced in the kidney and secreted into the urine via ...proteolytic cleavage. Its biological function is still not fully understood. Uromodulin has been linked to water/electrolyte balance and to kidney innate immunity. Also, studies in knockout mice demonstrated that it has a protective role against urinary tract infections and renal stone formation. Mutations in the gene encoding uromodulin lead to rare autosomal dominant diseases, collectively referred to as uromodulin-associated kidney diseases. They are characterized by progressive tubulointerstitial damage, impaired urinary concentrating ability, hyperuricemia, renal cysts, and progressive renal failure. Novel in vivo studies point at intracellular accumulation of mutant uromodulin as a key primary event in the disease pathogenesis. Recently, genome-wide association studies identified uromodulin as a risk factor for chronic kidney disease (CKD) and hypertension, and suggested that the level of uromodulin in the urine could represent a useful biomarker for the development of CKD. In this review, we summarize these recent investigations, ranging from invalidation studies in mouse to Mendelian disorders and genome-wide associations, which led to a rediscovery of uromodulin and boosted the scientific and clinical interest for this long discovered molecule.
BackgroundSystemic Lupus Erythematosus (SLE) and Lupus Nephritis (LN) are prototypical autoimmune conditions. Autoantibodies (AutoAb) play a central role in disease initiation and ...progression.MethodsIn the present work, we investigated sera of SLE and LN patients obtained through the ZEUS consortium, using a high-density peptide array covering the coding sequences of 464 proteins involved in rheumatologic and immunologic processes encompassing 17,402 tiled peptides. Results were validated by ELISA in patients affected by SLE (n=113) or LN (n=210). Immunohistochemistry and electron microscopy was performed in a subset of LN patients (n=12). The prognostic value of selected identified AutoAbs was tested in LN patients with at least two paired serum and urine samples (T0–12). Predictive value of baseline anti-FMNL1 AutoAbs was tested in subset of patients treated with Belimumab.ResultsA panel of 133 proteins reactive to SLE and LN sera were identified.Among those proteins, peptides coding for Formin-like 1 Protein (FMNL1) scored with the highest probability suggestive of FMNL1 being a potential antigen in both SLE and LN patients. High levels of anti-FMNL1 IgG2 were detected by ELISA in serum of LN and SLE patients and baseline FMNL1 values predicted proteinuria at 12-month follow-up in LN patients. Patients with high circulating anti-FMNL1 IgG2 maintained high levels of proteinuria after 1-year follow- up and failed to respond to Belimumab, suggestive of potential use of anti-FMNL1 as clinical predictive biomarker.In kidney LN biopsies, FMNL1 protein co-stained with macrophage marker CD68, its high expression being associated with proliferative LN stage III-IV. In glomeruli, FMNL1 co-stained with IgG2 suggestive of specific interaction and potentially modulation of macrophages by circulating anti-FMNL1 AutoAbs.ConclusionsDeployment of peptide array technology was pivotal to identify novel autoantibodies present in in SLE and LN; among those AutoAbs, high circulating levels of anti-FMNL1 IgG2 were detected in both SLE and LN. Anti-FMNL1 AutoAbs target a protein expressed in glomeruli by macrophages and associated with proliferative LN. That evidence is potentially suggestive of a role of anti-FMNL1 AutoAbs in modulating cells involved in tissue repair determining the disease outcome. Zeus Consortium: Gabriella Moroni8, Renato Alberto Sinico9, Franco Franceschini10, Micaela Fredi100, Augusto Vaglio11, Lorenzo Cavagna12, Federico Pratesi13, Paola Migliorini13, Francesco Locatelli11, Giulia Pazzola14, Giampaola Pesce15, Angelo Manfredi16, Giuseppe A Ramirez16, Pasquale Esposito17, Giuseppe Murdaca18, Simone Negrini18, Leda Cipriani17, BarbaraTrezzi8, Giacomo Emmi19, Ilaria Cavazzana9, Valentina Binda9, Paride Fenaroli20, Isabella Pisani20, Giacomo Garibotto17, Carlomaurizio Montecucco12, Domenico Santoro21, Francesco Scolari22, Stefano Volpi23, Marta Mosca 23, Angela Tincani10, Andrea Petretto24 8 Department of Biomedical Sciences, Humanitas University and IRCCS Humanitas Research Hospital,Milan, Italy9 Department of Medicine and surgery, University of Milan, Bicocca, Italy;10 Rheumatology and Clinical Immunology, ASST SpedaliCivili and Università of Brescia, Italy;11Department of Biomedical, Experimental and Clinical Sciences ‘Mario Serio’, University of Firenze, and Nephrology and Dialysis Unit, Meyer Children’s Hospital, Firenze, Italy;12 Division of Rheumatology, University and IRCCS Policlinico S. Matteo, Pavia, Italy;13 Clinical Immunology Unit, Department of Internal Medicine, University of Pisa, Italy;14 Nephrology and Dialysis, Arciospedale Santa Maria nuova, Reggio Emilia, Italy;
B cell depleting therapies permit immunosuppressive drug withdrawal and maintain remission in patients with frequently relapsing nephrotic syndrome (FRNS) or steroid–dependent nephrotic syndrome ...(SDNS), but lack of biomarkers for treatment failure. Post-depletion immune cell reconstitution may identify relapsing patients, but previous characterizations suffered from methodological limitations of flow cytometry. Time-of-flight mass cytometry (CyTOF) is a comprehensive analytic modality that simultaneously quantifies over 40 cellular markers. Herein, we report CyTOF-enabled immune cell comparisons over a 12-month period from 30 children with SDNS receiving B cell depleting therapy who either relapsed (n = 17) or remained stable (n = 13). Anti-CD20 treatment depleted all B cells subsets and CD20 depleting agent choice (rituximab
vs
ofatumumab) did not affect B cell subset recovery. Despite equal total numbers of B cells, 5 subsets of B cells were significantly higher in relapsing individuals; all identified subsets of B cells were class-switched. T cell subsets (including T follicular helper cells and regulatory T cells) and other major immune compartments were largely unaffected by B cell depletion, and similar between relapsing and stable children. In conclusion, CyTOF analysis of immune cells from anti-CD20 antibody treated patients identifies class-switched B cells as the main subset whose expansion associates with disease relapse. Our findings set the basis for future studies exploring how identified subsets can be used to monitor treatment response and improve our understanding of the pathogenesis of the disease.
Background
Rituximab, a chimeric anti-CD20 monoclonal antibody, is an effective treatment in steroid-dependent nephrotic syndrome (SDNS). However, some patients develop adverse reactions.
...Case-Diagnosis/Treatment
Patient 1, a 14-year-old boy with SDNS since the age of 2, was treated with oral prednisone, cyclosporine A (CsA) and mycophenolate mofetil. A first infusion of rituximab at age 12 years was well tolerated, but this was followed by a prolonged relapse unresponsive to oral prednisone, mycophenolate mofetil and CsA. A second rituximab infusion was attempted, but treatment was interrupted due to severe dyspnea. Treatment with a humanized anti-CD20 monoclonal antibody, ofatumumab, was then attempted. The patient experienced a mild allergic reaction and maintained remission despite interruption of all treatment at >12 months of follow-up. Patient 2, a 3-year-old boy who presented at 18 months with nephrotic syndrome initially resistant to treatment with oral prednisone, was given with three intravenous boluses of methylprednisolone followed by CsA and achieved remission. Upon steroid discontinuation, the NS relapsed. Prednisone was restarted and treatment with a single dose of rituximab was never completed due to a severe allergic reaction. Ofatumumab infusion was uneventful, and he maintained remission during the follow-up period (>12 months) despite interruption of prednisone therapy. B cells reappeared at 7 months in both patients.
Conclusions
Ofatumumab may be a therapeutic option in severe forms of NS with allergy to rituximab.
The Forkhead box protein P3 (FOXP3) is a transcription factor central to the function of regulatory T cells (Treg). Mutations in the
gene lead to a systemic disease called immune dysregulation, ...polyendocrinopathy, and enteropathy, an X-linked syndrome (IPEX) characterized by the triad of early-onset intractable diarrhea, type 1 diabetes, and eczema. An atypical presentation of IPEX has been reported.
We report rare cases with equivocal clinical associations that included inflammatory, kidney, and hematologic involvements screened with massively parallel sequencing techniques.
Two patients with hemizygous mutations of
c.779T>A (p.L260Q) and c.1087A>G (p.I363V) presented clinical manifestations not included in typical cases of IPEX: one was a 16-year-old male patient with an initial clinical diagnosis of autoimmune lymphoproliferative syndrome (ALPS) and who developed proteinuria and decreased kidney function due to membranous nephropathy, an autoimmune renal condition characterized by glomerular sub-epithelial antibodies. The second patient was a 2-year-old child with bone marrow failure who developed the same glomerular lesions of membranous nephropathy and received a bone marrow transplantation. High levels of IgG4 in serum, bone marrow, and kidney led to the definition of IgG4-related kidney disease (IgG4 RKD) in this young boy. The circulating Treg levels were normal in the former case and very low in the second.
Two atypical associations of functional mutations of
that include ALPS and IgG4 RKD are described. Membranous nephropathy leading to renal failure completed in both cases the clinical phenotypes that should be included in the clinical panorama of
failure.
Nephronophthisis (NPHP)-related ciliopathies are recessive, single-gene disorders that collectively make up the most common genetic cause of CKD in the first three decades of life. Mutations in 1 of ...the 15 known NPHP genes explain less than half of all cases with this phenotype, however, and the recently identified genetic causes are exceedingly rare. As a result, a strategy to identify single-gene causes of NPHP-related ciliopathies in single affected families is needed. Although whole-exome resequencing facilitates the identification of disease genes, the large number of detected genetic variants hampers its use. Here, we overcome this limitation by combining homozygosity mapping with whole-exome resequencing in a sibling pair with an NPHP-related ciliopathy. Whole-exome capture revealed a homozygous splice acceptor site mutation (c.698G>T) in the renal Mg(2+) transporter SLC41A1. This mutation resulted in skipping of exon 6 of SLC41A1, resulting in an in-frame deletion of a transmembrane helix. Transfection of cells with wild-type or mutant SLC41A1 revealed that deletion of exon 6 completely blocks the Mg(2+) transport function of SLC41A1. Furthermore, in normal human kidney tissue, endogenous SLC41A1 specifically localized to renal tubules situated at the corticomedullary boundary, consistent with the region of cystogenesis observed in NPHP and related ciliopathies. Last, morpholino-mediated knockdown of slc41a1 expression in zebrafish resulted in ventral body curvature, hydrocephalus, and cystic kidneys, similar to the effects of knocking down other NPHP genes. Taken together, these data suggest that defects in the maintenance of renal Mg(2+) homeostasis may lead to tubular defects that result in a phenotype similar to NPHP.
Renal tubulo-interstitial fibrosis is a non-specific process, representing the final common pathway for all kidney diseases, irrespective of their initial cause, histological injury, or etiology, ...leading to gradual expansion of the fibrotic mass which destroys the normal structure of the tissue and results in organ dysfunction and, ultimately, in end-stage organ failure. Proteomic studies of the fibrotic pathophysiological mechanisms have been performed in cell cultures, animal models and human tissues, addressing some of the key issues. This article will review proteomic contribution to the raising current knowledge on renal fibrosis biology and also mention seminal open questions to which proteomic techniques and proteomists could fruitfully contribute.
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► Proteomics studies of the fibrotic pathophysiological mechanisms have been performed in cell cultures, animal models and human tissues, addressing some of the key issues. ► This article review proteomics contribution to the raising current knowledge on renal fibrosis biology. ► This article mention seminal open questions to which proteomic techniques and proteomists could fruitful contribute.
Associated anomalies have been reported in around 20% of Hirschsprung patients but many Authors suggested a measure of underestimation. We therefore implemented a prospective observational study on ...106 consecutive HSCR patients aimed at defining the percentage of associated anomalies and implementing a personalized and up-to-date diagnostic algorithm.
After Institutional Ethical Committee approval, 106 consecutive Hirschsprung patients admitted to our Institution between January 2010 and December 2012 were included. All families were asked to sign a specific Informed Consent form and in case of acceptance each patient underwent an advanced diagnostic algorithm, including renal ultrasound scan (US), cardiologic assessment with cardiac US, cerebral US, audiometry, ENT and ophthalmologic assessments plus further specialist evaluations based on specific clinical features.
Male to female ratio of our series of patients was 3,4:1. Aganglionosis was confined to the rectosigmoid colon (classic forms) in 74,5% of cases. We detected 112 associated anomalies in 61 (57,5%) patients. The percentage did not significantly differ according to gender or length of aganglionosis. Overall, 43,4% of patients complained ophthalmologic issues (mostly refraction anomalies), 9,4% visual impairment, 20,7% congenital anomalies of the kidney and urinary tract, 4,7% congenital heart disease, 4,7% hearing impairment or deafness, 2,3% central nervous system anomalies, 8,5% chromosomal abnormalities or syndromes and 12,3% other associated anomalies.
Our study confirmed the underestimation of certain associated anomalies in Hirschsprung patients, such as hearing impairment and congenital anomalies of the kidney and urinary tract. Subsequently, based on our results we strongly suggest performing renal US and audiometry in all patients. Conversely, ophthalmologic assessment and cerebral and heart US can be performed according to guidelines applied to the general population or in case of patients with suspected clinical features or chromosomal abnormalities. This updated diagnostic algorithm aims at improving overall outcome thanks to better prognostic expectations, prevention strategies and early rehabilitation modalities. The investigation of genetic background of patients with associated anomalies might be the next step to explore this intriguing multifactorial congenital disease.
Background:
A great majority of children with idiopathic nephrotic syndrome will relapse after successful treatment of the initial episode. The possibility that different steroid dosing regimens at ...onset, adjusted for risk factors, can reduce the rate of relapse represents an interesting option to investigate.
Objectives:
To evaluate the effect of the initial steroid regimen, adjusted for time to remission (TTR), on the frequency of relapses and steroid dependence, and to verify the influence of prognostic factors on disease course.
Methods:
A multicentre, prospective, cohort study. Children with nephrotic syndrome, with TTR ≤ 10 days (Group A), were given a 20-week prednisone regimen (2,828 mg/m
2
) and those with a TTR >10 days, a 22-week regimen (3,668 mg/m
2
) (Group B). Previously published retrospective data from the same centers were also evaluated. Main outcomes were: relapse rate, number of frequent relapsers + steroid dependent children and total prednisone dose after induction.
Results:
143 children were enrolled. Rate of relapsed subjects (77 vs. 79%) and frequent relapsers + steroid dependent subjects (40 vs. 53%) did not differ between Groups A and B, or between the retrospective and prospective cohorts. The cumulative prednisone dose taken after the induction treatment was similar in both groups and in the retrospective and prospective cohorts. TTR was not associated with relapse risk. Age at onset and total serum protein were significantly lower in relapsing patients. At ROC analysis, the best cut-off was 5.3 years for age at onset and 4.2 g/dL for total serum protein. According to these cut-offs, older children with higher total serum protein had a higher relapse free survival rate (58%) than younger children with lower total serum protein (17%).
Conclusions:
TTR was not found to be a prognostic factor of relapse; because of this, different steroid regimens, adjusted for TTR, did not modify the relapse rate in any relevant measure. Conversely, younger age and low total serum protein were independent predictors of relapse risk, however this outcome was not modified by higher prednisone regimens.
Clinical Trial Registration:
https://www.ClinicalTrials.gov/
, identifier: NCT01386957 (
www.nefrokid.it
).
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