Nuovi biomarcatori molecolari per la diagnosi e la terapia della sclerosi sistemica

Background: La Sclerosi Sistemica (SSc) è una malattia rara del tessuto connettivo caratterizzata da danno microvascolare; attivazione dei fibroblasti con aumentata produzione di collageno nella cute, vasi sanguigni e organi interni; disregolazione dell’immunità con produzione di autoanticorpi. Importante è stata la scoperta di autoanticorpi che riconoscono e attivano il recettore del PDGF in pazienti SSc ma non nei controlli, inducendo stress ossidativo e deposizione di collagene, suggerendo un loro ruolo attivo nella patogenesi della SSc. Il nostro gruppo di ricerca ha messo a punto tecniche per clonare autoanticorpi monoclonali anti-PDGFR; identificato i diversi epitopi del PDGFR riconosciuti dai vari monoclonali; dimostrato che questi autoanticorpi causano fibrosi in vivo utilizzando un nuovo modello murino. Materiali e metodi: Utilizzando un saggio ELISA già ottimizzato, sono stati analizzati i sieri di pazienti SSc. 25 sieri SSc positivi al test ELISA insieme a 25 sieri controllo sono stati spediti a Pepscan Presto, Lelystat, Olanda, e testati con una Pepscan-based ELISA. Qui è stata generata una specifica library peptidica per valutare il legame tra gli epitopi conformazionali del PDGFr e gli autoanticorpi presenti nel siero SSc e identificare quali tra questi fossero il bersaglio di una maggiore reattività. Oltre ai 60 peptidi espressione dei primi tre domini della porzione extracellulare del PDGFr, la library contiene anche 60 peptidi rappresentanti la proteina NOX-2 appartenente alla famiglia delle NADPH ossidasi. Secondo la nostra ipotesi, sia la proteina NOX-2 che il PDGFr rappresentano il bersaglio della risposta autoimmunitaria da parte dei pazienti affetti da SSc. Risultati: esiste una differente reattività dei sieri dei pazienti SSc rispetto ai sieri controllo nei confronti di alcuni peptidi contenuti nella library. Ciò dimostra che i sieri SSc contengono autoanticorpi che riconoscono epitopi conformazionali specifici: 13 appartenenti a NOX2 e 21 al PDGFR. Tra i peptidi statisticamente significativi sono stati identificati alcuni che riescono a discriminare meglio i pazienti dai controlli e che potrebbero rappresentare i possibili bersagli di test diagnostici epitope-based. Abbiamo individuato valori di cut-off, mediante curve Roc. I peptidi migliori a discriminare i pazienti SSc dai controlli sono: PEP8 e PEP9 (espressione di NOX), PEP61 e PEP75 (espressione del PDGFr). Il valore cut-off di PEP61 = 435 è quello associato alla più alta sensibilità e specificità (se= 68%, sp= 80%). Nonostante una numerosità campionaria troppo esigua per conclusioni statisticamente significative, indipendentemente dal punteggio cut off considerato, cinque individui appartenenti al gruppo controlli si posizionino sempre al di sopra del livello soglia, risultando sempre positivi ad un ipotetico test diagnostico. Conclusioni: Con questo lavoro sono stati individuati specifici epitopi conformazionali che rappresentano il sito di legame tra anticorpi anti-PDGFr con i sieri di pazienti SSc. Esiste una diversa reattività dei sieri SSc al recettore del PDGF sulla base della quale è stato possibile fare una suddivisione dei pazienti in sottogruppi in parte diversi rispetto alla tradizionale classificazione (forma diffusa vs limitata). Indipendentemente dal punteggio cut-off considerato, cinque sieri controllo presentano una reattività estremamente elevata per tutti i peptidi della library, con un comportamento paragonabile ai pazienti SSc diffusa. Ci si interroga se questa polireattività sierica può essere espressione di una familiarità verso malattie autoimmuni o di una malattia non ancora manifesta. Se queste osservazioni verranno confermate da studi su una più ampia numerosità campionaria, questi peptidi potranno rappresentare il bersaglio di specifici test diagnostici epitope-based, e soprattutto di mirate terapie molecolari.Background: Systemic Sclerosis (SSc) is a rare disorder of the connective tissue, characterized by fibrosis of the skin, blood vessels, and visceral organs; additional manifestations include alterations of the microvasculature and production of autoantibodies. An important step forward has been the demonstration in SSc patients but not in controls, the presence of agonistic autoantibodies targeting endogenous PDGF receptor in SSc patients. These data suggest that the anti-PDGFR autoantibodies play an active role in the pathogenesis of SSc. Our research group has developed techniques to clone anti-PDGFR monoclonal autoantibodies; identified the different PDGFR epitopes recognized by agonistic anti PDGFR autoantibody; demonstrated that they are pro fibrotic in vivo tested in the preclinical model of the transgenic mouse. Materials and methods: by a competitive ELISA, serums of SSc patients were analyzed. 25 SSc serums positive by ELISA with 25 control serums were shipped to Pepscan Presto, Lelystat, The Netherlands, and tested with a Pepscan-based ELISA. A specific peptide library was then generated to evaluate the binding between the conformational epitopes of PDGFr and the autoantibodies present in the SSc serum and identify which of these are the target of higher reactivity. In addition to the 60 peptides expression of the first three domains of the extracellular portion of PDGFr, the library also contains 60 peptides representing the NOX-2 protein belonging to the NADPH oxidase family. According to our hypothesis, both the NOX-2 protein and the PDGFr are the target of the autoimmune response in SSc patients. Results: a different reactivity of the serums of SSc patients compared to the control serums emerged against some peptides contained in the library. This shows that the serums of SSc patients contain autoantibodies that recognize specific conformational epitopes:13 belonging to NOX2 and 21 to PDGFR. Among the statistically significant peptides, we identified the peptides that are able to better discriminate patients from the controls and that could represent possible targets for epitope-based diagnostic tests in the future. Cut-off values were identified, using Roc curves. The better peptides able to discriminate SSc patients from controls are: PEP8 and PEP9 (NOX expression), PEP61 and PEP75 (PDGFr expression). The cut-off value of PEP61 equal to 435 is the one associated with the highest sensitivity and specificity (se= 68%, sp= 80%). Despite too small a sample size to statistically significant conclusions, regardless of the cut-off score considered, five individuals belonging to the control group always position themselves above the threshold level, always positive to a hypothetical diagnostic test. Conclusions: with this research work, specific conformational epitopes have been identified that represent the site of binding between anti-PDGFr antibodies with serums of SSc patients. There is a different reactivity of SSc serums to the PDGF receptor on the basis of which it has been possible to subdivide patients into sub-groups that are partly different from the traditional classification (diffuse vs limited form). Independently of the cut-off score considered, five control serums have an extremely high reactivity for all peptides in the library, with a behavior comparable to diffuse SSc patients. This data makes us question whether this serum polireactivity may be the expression of a familiarity with autoimmune diseases or a disease not yet manifest. If these observations are confirmed by studies on a larger sample number, these peptides could be the target of specific epitope-based diagnostic tests, and especially of targeted molecular therapies

Metabolomic profile of systemic sclerosis patients

Systemic sclerosis (SSc) is an autoimmune disease of unknown aetiology characterized by vascular lesions, immunological alterations and diffuse fibrosis of the skin and internal organs. Since recent evidence suggests that there is a link between metabolomics and immune mediated disease, serum metabolic profile of SSc patients and healthy controls was investigated by1H-NMR and GC-MS techniques. The results indicated a lower level of aspartate, alanine, choline, glutamate, and glutarate in SSc patients compared with healthy controls. Moreover, comparing patients affected by limited SSc (lcSSc) and diffuse SSc (dcSSc), 6 discriminant metabolites were identified. The multivariate analysis performed using all the metabolites significantly different revealed glycolysis, gluconeogenesis, energetic pathways, glutamate metabolism, degradation of ketone bodies and pyruvate metabolism as the most important networks. Aspartate, alanine and citrate yielded a high area under receiver-operating characteristic (ROC) curves (AUC of 0.81; CI 0.726-0.93) for discriminating SSc patients from controls, whereas ROC curve generated with acetate, fructose, glutamate, glutamine, glycerol and glutarate (AUC of 0.84; CI 0.7-0.98) discriminated between lcSSc and dcSSc. These results indicated that serum NMR-based metabolomics profiling method is sensitive and specific enough to distinguish SSc from healthy controls and provided a feasible diagnostic tool for the diagnosis and classification of the disease

Computed tomography assessment of evolution of interstitial lung disease in systemic sclerosis: Comparison of two scoring systems

BACKGROUND: The aim of this study was to evaluate and compare the internal and external responsiveness of a computed-aided method (CaM) with a conventional visual reader-based score (CoVR) to measure interstitial lung disease (ILD) in patients with systemic sclerosis (SSc) on high resolution computed tomography (HRCT). METHODS: Forty-five patients were evaluated in this retrospective cohort. HRCTs were collected at baseline and after 1 year. HRCT abnormalities were evaluated according to a CoVR (Warrick's method) and a quantitative CaM. Internal 1-year responsiveness was tested with a standardized mean response (SRM). Analyses of the receiver operating characteristic curves (ROCs) evaluated the sensitivity and specificity of the two methods to discriminate between clinically relevant progression and no relevant progression, using expert judgment as the gold standard (external responsiveness). RESULTS: In one year, lung involvement was stable/improved in 17 of the 45 patients (37.8%) and worsened in 28 patients (62.2%). HRCT scores changed moderately over the follow-up period. Using SFM, CaM was significantly more responsive in detecting changes due to treatment than the CoVR method. Likewise, in the analysis of the ROC curve, CaM scores showed the highest performance (AUC ROC CaM vs. CoVR, 0.951 vs. 0.807; p = 0.0065). CONCLUSION: Quantitative analysis of CaM was more responsive than the CoVR method to accurately evaluate and monitor SSc-ILD progression or response to therapy

Agonistic anti-PDGF receptor autoantibodies from patients with systemic sclerosis impact human pulmonary artery smooth muscle cells function in vitro

One of the earliest events in the pathogenesis of systemic sclerosis (SSc) is microvasculature damage with intimal hyperplasia and accumulation of cells expressing PDGF receptor. Stimulatory autoantibodies targeting PDGF receptor have been detected in SSc patients and demonstrated to induce fibrosis in vivo and convert in vitro normal fibroblasts into SSc-like cells. Since there is no evidence of the role of anti-PDGF receptor autoantibodies in the pathogenesis of SSc vascular lesions, we investigated the biologic effect of agonistic anti-PDGF receptor autoantibodies from SSc patients on human pulmonary artery smooth muscle cells and the signaling pathways involved. The synthetic (proliferation, migration, and type I collagen gene α1 chain expression) and contractile (smooth muscle-myosin heavy chain and smooth muscle-calponin expression) profiles of human pulmonary artery smooth muscle cells were assessed in vitro after incubation with SSc anti-PDGF receptors stimulatory autoantibodies. The role of reactive oxygen species, NOX isoforms, and mammalian target of rapamycin (mTOR) was investigated. Human pulmonary artery smooth muscle cells acquired a synthetic phenotype characterized by higher growth rate, migratory activity, gene expression of type I collagen α1 chain, and less expression of markers characteristic of the contractile phenotype such as smooth muscle-myosin heavy chain and smooth muscle-calponin when stimulated with PDGF and autoantibodies against PDGF receptor, but not with normal IgG. This phenotypic profile is mediated by increased generation of reactive oxygen species and expression of NOX4 and mTORC1. Our data indicate that agonistic anti-PDGF receptor autoantibodies may contribute to the pathogenesis of SSc intimal hyperplasia

Celiac Disease Prevalence Is Increased in Primary Sjögren’s Syndrome and Diffuse Systemic Sclerosis: Lessons from a Large Multi-Center Study

Association of celiac disease (CD) with systemic autoimmune diseases (ADs) remains controversial. Awareness of CD in these patients is important to prevent complications, including lymphoproliferative disorders. We evaluated previously diagnosed CD prevalence in systemic lupus erythematosus (SLE), primary Sjögren’s syndrome (pSS) and systemic sclerosis (SSc) patients in comparison to 14,298 matched controls. All patients were screened for subclinical CD. Data from 1458 unselected consecutive SLE (580), pSS (354) and SSc (524) patients were collected. Previously biopsy-proven CD diagnosis and both CD- and AD-specific features were registered. All patients without previous CD were tested for IgA transglutaminase (TG). Anti-endomysium were tested in positive/borderline IgA TG. Duodenal biopsy was performed in IgA TG/endomysium+ to confirm CD. CD prevalence in AD was compared to that observed in 14,298 unselected sex- and age-matched adults who acted as controls. CD was more prevalent in pSS vs controls (6.78% vs 0.64%, p < 0.0001). A trend towards higher prevalence was observed in SLE (1.38%, p = 0.058) and SSc (1.34%, p = 0.096). Higher CD prevalence was observed in diffuse cutaneous SSc (4.5%, p ≤ 0.002 vs controls). Subclinical CD was found in two SLE patients and one pSS patient. CD diagnosis usually preceded that of AD. Primary SS and SSc–CD patients were younger at AD diagnosis in comparison to non-celiac patients. Autoimmune thyroiditis was associated with pSS and CD. CD prevalence is clearly increased in pSS and diffuse SSc in comparison to the general population. The association of CD with diffuse but not limited SSc may suggest different immunopathogenic mechanisms characterizing the two subsets. CD screening may be considered in pSS and diffuse SSc in young patients, particularly at the time of diagnosis

Long-term Outcome of Children Born to Women with Autoimmune Rheumatic Diseases: A Multicentre, Nationwide Study on 299 Randomly Selected Individuals

The concern about the offspring's health is one of the reasons for a reduced family size of women with rheumatic diseases (RD). Increased risk of autoimmune diseases (AD) and neurodevelopmental disorders (ND) has been reported in children born to patients with RD. Within a nationwide survey about reproductive issues of women with RD, we aimed at exploring the long-term outcome of their children. By surveying 398 patients who received their diagnosis of RD during childbearing age (before the age of 45), information about the offspring were obtained from 230 women who declared to have had children. A total of 148 (64.3%) patients were affected by connective tissue diseases (CTD) and 82 (35.7%) by chronic arthritis. Data on 299 children (156 males, 52.1%; mean age at the time of interview 17.1\ub19.7years) were collected. Twelve children (4.0%), who were born to patients with CTD in 75% of the cases, were affected by AD (8 cases of celiac disease). Eleven children had a certified diagnosis of ND (3.6%; 6 cases of learning disabilities); 9 of them were born to mothers with CTD (5 after maternal diagnosis). No association was found between ND and prenatal exposure to either maternal autoantibodies or anti-rheumatic drugs. Absolute numbers of offspring affected by AD and ND were low in a multicentre cohort of Italian women with RD. This information can be helpful for the counselling about reproductive issues, as the health outcomes of the offspring might not be an issue which discourage women with RD from having children