A novel DNA/histone H4 peptide complex detects autoantibodies in systemic lupus erythematosus sera

Background: The detection of anti-dsDNA antibodies is critical for the diagnosis and follow-up of systemic lupus erythematosus (SLE) patients. The presently available assays are characterized by a non-optimal specificity (solid phase assays) or sensitivity (Crithidia Luciliae immunofluorescence test (CLIFT)). To overcome the limits of CLIFT and solid phase chromatin assays, we explored the diagnostic potential of an assay based on plasmid DNA containing a highly bent fragment of 211 bp from Crithidia Luciliae minicircles, complexed with histone peptides. Methods: Electrically neutral complexes of PK201/CAT plasmid (PK) DNA and histone 4 (H4) peptides were evaluated by electromobility shift assay. Complexes of H4 peptides and PK were absorbed to the solid phase to detect specific immunoglobulin G (IgG) in sera. Sera from 109 SLE patients, 100 normal healthy subjects, and 169 disease controls were tested. Results: H4(14-34) containing the consensus sequence for DNA binding interacts with PK, retarding its migration. H4(14-34)/PK complexes were used to test sera by ELISA. Anti-H4-PK antibodies were detected in 56 % of SLE sera (more frequently in patients with skin or joint involvement) versus 5.9 % in disease controls; inhibition assays show that sera react with epitopes present on DNA or on the complex, not on the peptide. Antibody titer is correlated with European Consensus Lupus Activity Measurement (ECLAM) score and anti-complement component 1q (C1q) antibodies, negatively with C3 levels. Anti-H4-PK antibodies compared with CLIFT and solid phase dsDNA assays display moderate concordance. Conclusions: The H4/PK assay is a simple and reliable test which is useful for the differential diagnosis and evaluation of disease activity in SLE patients

SP181PAX2+ PROGENITOR CELLS PLAY A KEY ROLE IN TUBULAR REGENERATION AFTER ACUTE KIDNEY INJURY

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Calibrating the Dynamic Energy Simulation Model for an Existing Building: Lessons Learned from a Collective Exercise

Calibration of the existing building simulation model is key to correctly evaluating the energy savings that are achievable through retrofit. However, calibration is a non-standard phase where different approaches can possibly lead to different models. In this study, an existing residential building is simulated in parallel by four research groups with different dynamic simulation tools. Manual/automatic methodologies and basic/detailed measurement data sets are used. The calibration is followed by a validation on two evaluation periods. Monitoring data concerning the windows opening by the occupants are used to analyze the calibration outcomes. It is found that for a good calibration of a model of a well-insulated building, the absence of data regarding the users’ behavior is more critical than uncertainty on the envelope properties. The automatic approach is more effective in managing the model complexity and reaching a better performing calibration, as the RMSE relative to indoor temperature reaches 0.3 C compared to 0.4–0.5 C. Yet, a calibrated model’s performance is often poor outside the calibration period (RMSE increases up to 10.8 times), and thus, the validation is crucial to discriminate among multiple solutions and to refine them, by improving the users’ behavior modeling

Podocyte Regeneration Driven by Renal Progenitors Determines Glomerular Disease Remission and Can Be Pharmacologically Enhanced

Podocyte loss is a general mechanism of glomerular dysfunction that initiates and drives the progression of chronic kidney disease, which affects 10% of the world population. Here, we evaluate whether the regenerative response to podocyte injury influences chronic kidney disease outcome. In models of focal segmental glomerulosclerosis performed in inducible transgenic mice where podocytes are tagged, remission or progression of disease was determined by the amount of regenerated podocytes. When the same model was established in inducible transgenic mice where renal progenitors are tagged, the disease remitted if renal progenitors successfully differentiated into podocytes, while it persisted if differentiation was ineffective, resulting in glomerulosclerosis. Treatment with BIO, a GSK3s inhibitor, significantly increased disease remission by enhancing renal progenitor sensitivity to the differentiation effect of endogenous retinoic acid. These results establish renal progenitors as critical determinants of glomerular disease outcome and a pharmacological enhancement of their differentiation as a possible therapeutic strategy

Essential but differential role for CXCR4 and CXCR7 in the therapeutic homingof human renal progenitor cells

Recently, we have identified a population of renal progenitor cells in human kidneys showing regenerative potential for injured renal tissue of SCID mice. We demonstrate here that among all known chemokine receptors, human renal progenitor cells exhibit high expression of both stromal-derived factor-1 (SDF-1) receptors, CXCR4 and CXCR7. In SCID mice with acute renal failure (ARF), SDF-1 was strongly up-regulated in resident cells surrounding necrotic areas. In the same mice, intravenously injected renal stem/progenitor cells engrafted into injured renal tissue decreased the severity of ARF and prevented renal fibrosis. These beneficial effects were abolished by blocking either CXCR4 or CXCR7, which dramatically reduced the number of engrafting renal progenitor cells. However, although SDF-1–induced migration of renal progenitor cells was only abolished by an anti-CXCR4 antibody, transendothelial migration required the activity of both CXCR4 and CXCR7, with CXCR7 being essential for renal progenitor cell adhesion to endothelial cells. Moreover, CXCR7 but not CXCR4 was responsible for the SDF-1–induced renal progenitor cell survival. Collectively, these findings suggest that CXCR4 and CXCR7 play an essential, but differential, role in the therapeutic homing of human renal progenitor cells in ARF, with important implications for the development of stem cell–based therapies

A sex-informed approach to improve the personalised decision making process in myelodysplastic syndromes: a multicentre, observational cohort study

Background Sex is a major source of diversity among patients and a sex-informed approach is becoming a new paradigm in precision medicine. We aimed to describe sex diversity in myelodysplastic syndromes in terms of disease genotype, phenotype, and clinical outcome. Moreover, we sought to incorporate sex information into the clinical decision-making process as a fundamental component of patient individuality. Methods In this multicentre, observational cohort study, we retrospectively analysed 13 284 patients aged 18 years or older with a diagnosis of myelodysplastic syndrome according to 2016 WHO criteria included in the EuroMDS network (n=2025), International Working Group for Prognosis in MDS (IWG-PM; n=2387), the Spanish Group of Myelodysplastic Syndromes registry (GESMD; n=7687), or the Dusseldorf MDS registry (n=1185). Recruitment periods for these cohorts were between 1990 and 2016. The correlation between sex and genomic features was analysed in the EuroMDS cohort and validated in the IWG-PM cohort. The effect of sex on clinical outcome, with overall survival as the main endpoint, was analysed in the EuroMDS population and validated in the other three cohorts. Finally, novel prognostic models incorporating sex and genomic information were built and validated, and compared to the widely used revised International Prognostic Scoring System (IPSS-R). This study is registered with ClinicalTrials.gov, NCT04889729. Findings The study included 7792 (58middot7%) men and 5492 (41middot3%) women. 10 906 (82middot1%) patients were White, and race was not reported for 2378 (17middot9%) patients. Sex biases were observed at the single-gene level with mutations in seven genes enriched in men (ASXL1, SRSF2, and ZRSR2 p<0middot0001 in both cohorts; DDX41 not available in the EuroMDS cohort vs p=0middot0062 in the IWG-PM cohort; IDH2 p<0middot0001 in EuroMDS vs p=0middot042 in IWG-PM; TET2 p=0middot031 vs p=0middot035; U2AF1 p=0middot033 vs p<0middot0001) and mutations in two genes were enriched in women (DNMT3A p<0middot0001 in EuroMDS vs p=0middot011 in IWG-PM; TP53 p=0middot030 vs p=0middot037). Additionally, sex biases were observed in co-mutational pathways of founding genomic lesions (splicing-related genes, predominantly in men, p<0middot0001 in both the EuroMDS and IWG-PM cohorts), in DNA methylation (predominantly in men, p=0middot046 in EuroMDS vs p<0middot0001 in IWG-PM), and TP53 mutational pathways (predominantly in women, p=0middot0073 in EuroMDS vs p<0middot0001 in IWG-PM). In the retrospective EuroMDS cohort, men had worse median overall survival (81middot3 months, 95% CI 70middot4-95middot0 in men vs 123middot5 months, 104middot5-127middot5 in women; hazard ratio [HR] 1middot40, 95% CI 1middot26-1middot52; p<0middot0001). This result was confirmed in the prospective validation cohorts (median overall survival was 54middot7 months, 95% CI 52middot4-59middot1 in men vs 74middot4 months, 69middot3-81middot2 in women; HR 1middot30, 95% CI 1middot23-1middot35; p<0middot0001 in the GEMSD MDS registry; 40middot0 months, 95% CI 33middot4-43middot7 in men vs 54middot2 months, 38middot6-63middot8 in women; HR 1middot23, 95% CI 1middot08-1middot36; p<0middot0001 in the Dusseldorf MDS registry). We developed new personalised prognostic tools that included sex information (the sex-informed prognostic scoring system and the sex-informed genomic scoring system). Sex maintained independent prognostic power in all prognostic systems; the highest performance was observed in the model that included both sex and genomic information. A five-to-five mapping between the IPSS-R and new score categories resulted in the re-stratification of 871 (43middot0%) of 2025 patients from the EuroMDS cohort and 1003 (42middot0%) of 2387 patients from the IWG-PM cohort by using the sex-informed prognostic scoring system, and of 1134 (56middot0%) patients from the EuroMDS cohort and 1265 (53middot0%) patients from the IWG-PM cohort by using the sex-informed genomic scoring system. We created a web portal that enables outcome predictions based on a sex-informed personalised approach. Interpretation Our results suggest that a sex-informed approach can improve the personalised decision making process in patients with myelodysplastic syndromes and should be considered in the design of clinical trials including low-risk patients. Copyright (c) 2022 Published by Elsevier Ltd. All rights reserved

Autoanticorpi patogeni nel lupus sistemico

Dal momento che gli anticorpi anti-dsDNA rivestono un ruolo centrale nel lupus eritematoso sistemico, sia come anticorpi marker, quindi importanti dal punto di vista diagnostico, sia per la loro correlazione con l’attività di malattia e il danno d’organo, e viste le problematiche inerenti alle diverse metodiche diagnostiche impiegate per la loro ricerca, questo lavoro sperimentale è stato sviluppato con l’obiettivo di ricercare nuove metodiche per rilevarli. In una precedente fase di questo lavoro, sono stati ricercati gli anticorpi anti-dsDNA nei sieri di un elevato numero di pazienti affetti da LES, utilizzando come antigeni per il riconoscimento 3 tipi diversi di DNA, lineare e circolare, di origine rispettivamente genomica e plasmidica (plasmide sperimentale pK201/CAT e di controllo pUC19). E’ stata, inoltre, ricercata una correlazione tra gli anticorpi misurati tramite le metodiche sopra citate e diversi parametri clinici e laboratoristici, tra cui l’indice ECLAM, i livelli di anticorpi anti-C1q, di grande rilievo per la diagnosi, la valutazione del livello di attività di malattia77-79 e la correlazione col danno d’organo, e i livelli del frammento C3 del complemento, essendo l’ipocomplementemia un importante parametro correlato con proporzionalità inversa al livello di attività di malattia. Nel complesso, i 3 test per misurare gli anti-dsDNA si sono dimostrati tutti in grado di discriminare nettamente pazienti LES da soggetti normali e tutti e tre hanno mostrato una correlazione positiva con l’indice ECLAM e il titolo di anticorpi anti-C1q, mentre negativa con i livelli di C3, come atteso per un test che misuri anticorpi prodotti essenzialmente nelle fasi di attività di malattia. Dal confronto fra i titoli di anticorpi anti-dsDNA misurati con i 3 metodi, si è osservo che esisteva un’elevata correlazione ma non una completa sovrapposizione, suggerendo che i diversi substrati misurino popolazioni non completamente sovrapposte di anticorpi anti-dsDNA. Questo dato è risultato ancor più evidente dal confronto dei risultati del dosaggio di anti-dsDNA mediante CLIF e mediante ELISA con i tre tipi di DNA, poiché la “sovrapposizione” fra CLIF e il test ELISA che utilizza il DNA di plasmide è risultata superiore rispetto al test che utilizza DNA genomico. La conclusione di queste osservazioni è stata che gli anticorpi misurati col test che utilizza DNA plasmidico sperimentale pK201/CAT potrebbero essere almeno in parte specifici per una particolare conformazione del dsDNA, comune al plasmide, circolare e ricco di purine, e al DNA di cinetoplasto di Crythidia Luciliae. In questa fase del lavoro sperimentale si è cercato, pertanto, di caratterizzare meglio il potenziale diagnostico di questi test, in particolare la loro specificità, applicandoli all’analisi, oltre che di soggetti sani e di LES, anche di altre malattie autoimmuni sistemiche, in cui gli anti-dsDNA non dovrebbero essere presenti. La ricerca degli anticorpi anti-dsDNA è stata eseguita con test ELISA home made, kit commerciali e CLIF test, ed i risultati sono stati confrontati tra loro in termini di sensibilità e specificità. Partendo, inoltre, dall’ipotesi secondo la quale gli anticorpi anti-dsDNA si leghino a un complesso DNA-peptide piuttosto che al dsDNA libero, un altro interessante obiettivo che questo studio si è prefisso è quello di testare la reattività di sieri di soggetti LES, di malattie di controllo e di soggetti sani nei confronti di complessi DNA-peptidi differenti. Dal confronto tra i diversi risultati si possono ottenere informazioni sulla sensibilità e sulla specificità dei test ELISAs così strutturati e, in aggiunta, evidenziare se complessi con peptidi diversi sono in gradi discriminare sottopopolazioni anticorpali diverse, che potrebbero correlarsi a sottotipi clinici differenti, ad esempio a un certo tipo d’interessamento d’organo. Nell’eventualità in cui questo fosse confermato, si potrebbe pensare di costituire un test ELISA utilizzando come antigeni diversi tipi di complessi DNA-peptide, in modo tale da poter così rilevare anticorpi diversi, aumentando perciò la sensibilità e la specificità del test

Dynamic simulation of existing buildings: considerations on the model calibration

Dynamic energy simulation is increasingly used to design retrofit interventions in existing buildings. Energy savings are correctly predicted if the simulation model is carefully calibrated against measured data. In this study the same building storey is simulated by four different research groups with different dynamic simulation tools (EnergyPlus, TRNSYS, IDA ICE). The building envelope is simulated in free floating and a parametric analysis to envelope properties and users’ behaviour is carried out. A set of basic information and measurements on the building is made available to three groups, while detailed information and measured data are provided to the fourth one. From the comparison among calibrated models it is concluded that, for a good calibration of a well-insulated building model, the absence of data regarding users’ behaviour can be more critical than detailed measurements on the envelope properties