DLco and echocardiographic parameters in identifying mild pulmonary hypertension in the EUSTAR cohort of systemic sclerosis patients.

BACKGROUND: The 2022 European Society of Cardiology (ESC)/European Respiratory Society (ERS) guidelines define pulmonary hypertension (PH) as a resting mean pulmonary artery pressure (mPAP) > 20 mmHg at right heart catheterization (RHC). Previously, patients with a mPAP between 21 and 24 mmHg were classified in a "grey zone" of unclear clinical significance. RESEARCH QUESTION: What is the diagnostic performance of the main parameters used for PH screening in detecting systemic sclerosis (SSc) patients with mPAP 21-24 mmHg at RHC? STUDY DESIGN AND METHODS: SSc patients from the European Scleroderma Trials and Research (EUSTAR) database with available tricuspid annular plane systolic excursion (TAPSE), systolic PAP (sPAP) and mPAP data were included. Patients with mPAP 21-24 mmHg and patients with mPAP ≤ 20 mmHg were considered for the analysis. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and accuracy were calculated. RESULTS: TAPSE/sPAP was lower in the group of SSc patients with mPAP 21-24 mmHg than in non-PH group [0.58 (0.46-0.72) mm/mmHg vs 0.69 (0.57-0.81) mm/mmHg, p < 0.01]. No difference was found in other parameters between the two groups. Diffusing capacity of the lungs for carbon monoxide (DLCO) < 80% of the predicted value had the highest sensitivity (88.9%) and NPV (80%), but the lowest specificity (18.2%) and PPV (30.8%) in detecting SSc patients with mPAP 21-24 mmHg. TAPSE/sPAP < 0.55 mm/mmHg had the highest specificity (78.9%), PPV (50%) and accuracy (68.1%); its NPV was of 75.4%, while its sensibility 45.1%. INTERPRETATION: DLCO < 80% of the predicted value is the parameter with the highest sensitivity and NPV in detecting SSc patients with mPAP 21-24 mmHg. TAPSE/sPAP < 0.55 mm/mmHg has the highest specificity, PPV and accuracy and, therefore, can be a useful additional parameter to decrease the number of unnecessary RHC.

Pathogenic autoantibody internalization in myositis.

Objectives: Myositis is a heterogeneous family of autoimmune muscle diseases. As myositis autoantibodies recognize intracellular proteins, their role in disease pathogenesis has been unclear. This study aimed to determine whether myositis autoantibodies reach their autoantigen targets within muscle cells and disrupt the normal function of these proteins. Methods: Confocal immunofluorescence microscopy was used to localize antibodies and other proteins of interest in myositis muscle biopsies. Bulk RNA sequencing was used to study the transcriptomic profiles of 668 samples from patients with myositis, disease controls, and healthy controls. Antibodies from myositis patients were introduced into cultured myoblasts by electroporation and the transcriptomic profiles of the treated myoblasts were studied by bulk RNA sequencing. Results: In patients with myositis autoantibodies, antibodies accumulated inside myofibers in the same subcellular compartment as the autoantigen. Each autoantibody was associated with effects consistent with dysfunction of its autoantigen, such as the derepression of genes normally repressed by Mi2/NuRD in patients with anti-Mi2 autoantibodies, the accumulation of RNAs degraded by the nuclear RNA exosome complex in patients with anti-PM/Scl autoantibodies targeting this complex, and the accumulation of lipids within myofibers of anti-HMGCR-positive patients. Internalization of patient immunoglobulin into cultured myoblasts recapitulated the transcriptomic phenotypes observed in human disease, including the derepression of Mi2/NuRD-regulated genes in anti-Mi2-positive dermatomyositis and the increased expression of genes normally degraded by the nuclear RNA exosome complex in anti-PM/Scl-positive myositis. Conclusions: In myositis, autoantibodies are internalized into muscle fibers, disrupt the biological function of their autoantigen, and mediate the pathophysiology of the disease.

Nailfold videocapillaroscopy patterns in systemic sclerosis: implications for cutaneous subsets, disease features and prognostic value for survival.

OBJECTIVES: To assess the associations and prognostic value of scleroderma patterns by nailfold videocapillaroscopy (NVC) in patients with systemic sclerosis (SSc) and cutaneous subsets. METHODS: At baseline, 1356 SSc patients from the RESCLE registry were compared according to the scleroderma pattern as Late pattern and non-Late pattern, which included Early and Active patterns. Patient characteristics, disease features, survival time and causes of death were analysed. RESULTS: Late pattern was identified in 540 (39.8%), and non-Late pattern in 816 (60.2%) patients (88% women; 987 lcSSc/251 dcSSc). Late pattern was associated to dcSSc (OR=1.96; p<0.001), interstitial lung disease (ILD) (OR=1.29; p=0.031), and scleroderma renal crisis (OR=3.46; p<0.001). Once the cutaneous subset was disregarded in an alternative analysis, both digital ulcers (DU) (OR=1.29; p<0.037) and anti-topoisomerase I antibodies (OR=1.39; p< 0.036) emerged associated with the Late pattern. By cutaneous subsets, associations with Late pattern were: (1) in dcSSc, acro-osteolysis (OR=2.13; p=0.022), and systolic pulmonary artery pressure >40 mmHg by Doppler echocardiogram (OR=2.24; p<0.001); and (2) in lcSSc, ILD (OR=1.38; p=0.028). Survival was reduced in dcSSc with Late pattern compared to non-Late pattern (p=0.049). Risk factors for SSc mortality in multivariate regression Cox analysis were age at diagnosis (HR=1.03; p<0.001), dcSSc (HR=2.48; p<0.001), DU (HR=1.38; p=0.046), ILD (HR=2.81; p<0.001), and pulmonary arterial hypertension (HR=1.99; p<0.001). CONCLUSIONS: SSc patients with Late pattern more frequently present dcSSc and develop more fibrotic and vascular manifestations. Advanced microangiopathy by NVC identifies dcSSc patients at risk of reduced survival due to SSc-related causes.

External clinical validation of automated software to identify structural abnormalities and microhaemorrhages in nailfold videocapillaroscopy images.

OBJECTIVES: Automated systems to analyse nailfold videocapillaroscopy (NVC) images are needed to promptly and comprehensively characterise patients with systemic sclerosis (SSc) or Raynaud's phenomenon (RP). We previously developed, and validated in-house, a deep convolutional neural network-based algorithm to classify NVC-captured images according to the presence/absence of structural abnormalities and/or microhaemorrhages. We present its external clinical validation. METHODS: A total of 1,164 NVC images of RP patients were annotated by 5 trained capillaroscopists according to the following categories: normal capillary; dilation; giant capillary; abnormal shape; tortuosity; microhaemorrhage. The images were also presented to the algorithm. Matches and discrepancies between algorithm predictions and those annotations obtained by consensus of ≥3 or ≥4 interobservers were analysed. RESULTS: Consensus among ≥3 capillaroscopists was achieved in 86.9% of images, 75.8% of which were correctly predicted by the algorithm. Consensus among ≥4 experts occurred in 52.0% of cases, in which 87.1% of the algorithm's results matched with those of the expert panel. The algorithm's positive predictive value was >80% for microhaemorrhages and unaltered, giant or abnormal capillaries. Sensitivity was >75% for dilations and tortuosities. Negative predictive value and specificity were >89% for all categories. CONCLUSIONS: This external clinical validation suggests that this algorithm is useful to assist in the diagnosis and follow-up of SSc or RP patients in a timely manner. It may also be helpful in the management of patients with any pathology presenting with microvascular changes, as the algorithm has been designed to also be useful for research aiming at extending the usage of nailfold capillaroscopy to more conditions.

Anti-U11/U12 Antibodies as a Rare but Important Biomarker in Patients with Systemic Sclerosis: A Narrative Review.

Anti-nuclear (ANA) are present in approximately 90% of systemic sclerosis (SSc) patients and are key biomarkers in supporting the diagnosis and determining the prognosis of this disease. In addition to the classification criteria autoantibodies for SSc [i.e., anti-centromere, anti-topoisomerase I (Scl-70), anti-RNA polymerase III], other autoantibodies have been associated with important SSc phenotypes. Among them, anti-U11/U12 ribonucleoprotein (RNP) antibodies, also known as anti-RNPC-3, were first reported in a patient with SSc, but very little is known about their association and clinical utility. The U11/U12 RNP macromolecular complex consists of several proteins involved in alternative mRNA splicing. More recent studies demonstrated associations of anti-anti-U11/U12 antibodies with SSc and severe pulmonary fibrosis as well as with moderate to severe gastrointestinal dysmotility. Lastly, anti-U11/U12 autoantibodies have been strongly associated with malignancy in SSc patients. Here, we aimed to summarize the knowledge of anti-U11/U12/RNPC-3 antibodies in SSc, including their seroclinical associations in a narrative literature review.

The role of TAPSE/sPAP ratio in predicting pulmonary hypertension and mortality in the systemic sclerosis EUSTAR cohort.

OBJECTIVES: The study aim was to evaluate the predictive role of the echocardiography-derived tricuspid annular plane systolic excursion/systolic pulmonary artery pressure (TAPSE/sPAP) ratio for pulmonary hypertension (PH) diagnosis and mortality in the European Scleroderma Trials and Research (EUSTAR) cohort. METHODS: Eligible patients were systemic sclerosis (SSc) patients registered in the EUSTAR database with at least one visit recording TAPSE and sPAP data. Individual centres were required to provide TAPSE and sPAP data at 12 ± 3 months before right heart catheterization (RHC). Logistic regression analysis was applied to analyse the predictive ability of TAPSE/sPAP ratio for PH diagnosis. Cox regression analysis was performed to evaluate TAPSE/sPAP ratio as a predictive factor for all-cause mortality. RESULTS: 2555 SSc patients met the inclusion criteria for this study with 355 SSc patients having available RHC data at baseline. PH was confirmed by RHC in 195 SSc patients (54.9%). TAPSE/sPAP ratio < 0.55 mm/mmHg [OR 0.251 (95% CI 0.084-0.753), p < 0.05] and FVC/DLCO [OR 2.568 (95% CI 1.227-5.375), p < 0.05] were significantly associated with PH diagnosis. In logistic regression analysis with echocardiographic parameters at 12 ± 3 months before RHC, TAPSE/sPAP ratio < 0.55 mm/mmHg [OR 0.265 (95% CI 0.102-0.685), p < 0.01] and FVC/DLCO [OR 2.529 (95% CI 1.358-4.711), p < 0.01] were associated with PH diagnosis. In multivariate Cox regression analysis, TAPSE/sPAP ratio ≤ 0.32 mm/mmHg [HR 0.310 (0.164-0.585), p < 0.001] was the most significant predictive factor for death. CONCLUSIONS: TAPSE/sPAP ratio < 0.55 mm/mmHg is a predictive risk factor for PH. TAPSE/sPAP ratio ≤ 0.32 mm/mmHg is a predictive risk marker for all-cause mortality.

Clinico-pathological phenotypes of systemic sclerosis-associated myopathy: analysis of a large multicentre cohort.

OBJECTIVE: The objective of this study was to analyse the clinico-serological and histological phenotypes of patients with SSc with associated myopathy. METHODS: From November 2002 to September 2020, 52 patients with SSc underwent a muscle biopsy for suspected myopathy. We established two subgroups according to the histological findings based on the presence of isolated fibrosis or fibrosis together with significant inflammation. These patterns were designated as fibrosing and inflammatory, respectively. Clinical data, antibody profile, electrophysiologic studies, muscle biopsy findings and data regarding treatment, mortality and survival were compared between the two groups. RESULTS: Fourteen biopsies had a fibrosing pattern, whereas 26 showed an inflammatory pattern that could be classified (according to the predominant pattern) into DM (n = 7), necrotizing myopathy (n = 4) and non-specific myositis (n = 15). Additionally, 12 muscle biopsies were reported as neurogenic atrophy (n = 2), or normal muscle or minimal changes (n = 10). Compared with the inflammatory group, SSc patients with the fibrosing pattern presented a higher prevalence of ischaemic heart disease (38.5% vs 3.8%, P = 0.011), conduction abnormalities or arrhythmias (61.5% vs 26.9%, P = 0.036), anti-topo I antibodies (42.9% vs 11.5%, P = 0.044), greater median ESR (53.5 mm/h vs 32.5 mm/h, P = 0.013), with poor response to treatment and a higher mortality (42.9% vs 3.8%, P = 0.004) and lower cumulative survival (P = 0.035). CONCLUSIONS: Patients with SSc-associated myopathy require a comprehensive approach that encompasses clinical, serological and histopathological aspects, given their outcome predictive capacity. At least two different phenotypes can be drawn, considering clinico-pathological features. Significant differences are delineated between both a fibrotic and an inflammatory phenotype.

Complement component C4 structural variation and quantitative traits contribute to sex-biased vulnerability in systemic sclerosis.

Copy number (CN) polymorphisms of complement C4 play distinct roles in many conditions, including immune-mediated diseases. We investigated the association of C4 CN with systemic sclerosis (SSc) risk. Imputed total C4, C4A, C4B, and HERV-K CN were analyzed in 26,633 individuals and validated in an independent cohort. Our results showed that higher C4 CN confers protection to SSc, and deviations from CN parity of C4A and C4B augmented risk. The protection contributed per copy of C4A and C4B differed by sex. Stronger protection was afforded by C4A in men and by C4B in women. C4 CN correlated well with its gene expression and serum protein levels, and less C4 was detected for both in SSc patients. Conditioned analysis suggests that C4 genetics strongly contributes to the SSc association within the major histocompatibility complex locus and highlights classical alleles and amino acid variants of HLA-DRB1 and HLA-DPB1 as C4-independent signals.

Standardized incidence ratios and risk factors for cancer in patients with systemic sclerosis: Data from the Spanish Scleroderma Registry (RESCLE).

AIM: Patients with systemic sclerosis (SSc) are at increased risk of cancer, a growing cause of non-SSc-related death among these patients. We analyzed the increased cancer risk among Spanish patients with SSc using standardized incidence ratios (SIRs) and identified independent cancer risk factors in this population. MATERIAL AND METHODS: Spanish Scleroderma Registry data were analyzed to determine the demographic characteristics of patients with SSc, and logistic regression was used to identify cancer risk factors. SIRs with 95% confidence intervals (CIs) relative to the general Spanish population were calculated. RESULTS: Of 1930 patients with SSc, 206 had cancer, most commonly breast, lung, hematological, and colorectal cancers. Patients with SSc had increased risks of overall cancer (SIR 1.48, 95% CI 1.36-1.60; P < 0.001), and of lung (SIR 2.22, 95% CI 1.77-2.73; P < 0.001), breast (SIR 1.31, 95% CI 1.10-1.54; P = 0.003), and hematological (SIR 2.03, 95% CI 1.52-2.62; P < 0.001) cancers. Cancer was associated with older age at SSc onset (odds ratio [OR] 1.22, 95% CI 1.01-1.03; P < 0.001), the presence of primary biliary cholangitis (OR 2.35, 95% CI 1.18-4.68; P = 0.015) and forced vital capacity <70% (OR 1.8, 95% CI 1.24-2.70; P = 0.002). The presence of anticentromere antibodies lowered the risk of cancer (OR 0.66, 95% CI 0.45-0.97; P = 0.036). CONCLUSIONS: Spanish patients with SSc had an increased cancer risk compared with the general population. Some characteristics, including specific autoantibodies, may be related to this increased risk.

Left ventricular diastolic dysfunction in systemic sclerosis: Clinical, immunological and survival differences in the Spanish RESCLE registry.

OBJECTIVES: Left ventricular diastolic dysfunction (LVDD) remains poorly studied in Systemic Sclerosis (SSc). To determine the prevalence and to define factors associated with LVDD and survival in a large cohort of patients with SSc. METHODS: An observational study was conducted with data from the multicentre Spanish Scleroderma Registry (RESCLE) to identify factors associated with LVDD and estimate survival. RESULTS: Out of 1517 patients, 319 (21.0%) had LVDD. The subset of sine scleroderma SSc was associated to LVDD (14.7% vs. 10.6%, p =0.048), whilst diffuse cutaneous SSc was more prevalent in non-LVDD (16.0 % vs. 21.2%, p =0.041). Multivariable analysis identified that LVDD was associated with older age at diagnosis of SSc (OR 1.05; 95% CI 1.04 to 1.06), longer time from diagnosis (OR 1.04; 95% CI 1.03 to 1.06), presence of telangiectasia (OR 1.42; 95% CI 1.08 to 1.88), treatment with calcium channel blockers (CCB) (OR 1.51; 95% CI 1.16 to 1.96), and inversely related to angiotensin-converting-enzyme inhibitors (ACEi) use (OR 0.59; 95% CI 0.44 to 0.80). SSc patients with LVDD had increased mortality (23.8 vs. 17.4%, p =0.010) and shortened survival from the first SSc symptom (p =0.040), even though it was not found to be an independent risk factor for death. CONCLUSIONS: LVDD is relatively common in SSc patients, and it is associated with worst prognosis, older age, longer time from diagnosis of SSc, presence of telangiectasia and vasodilator treatment.

Impact of interstitial lung disease on the survival of systemic sclerosis with pulmonary arterial hypertension.

To assess severity markers and outcomes of patients with systemic sclerosis (SSc) with or without pulmonary arterial hypertension (PAH-SSc/non-PAH-SSc), and the impact of interstitial lung disease (ILD) on PAH-SSc. Non-PAH-SSc patients from the Spanish SSc registry and PAH-SSc patients from the Spanish PAH registry were included. A total of 364 PAH-SSc and 1589 non-PAH-SSc patients were included. PAH-SSc patients had worse NYHA-functional class (NYHA-FC), worse forced vital capacity (FVC) (81.2 ± 20.6% vs 93.6 ± 20.6%, P < 0.001), worse tricuspid annular plane systolic excursion (TAPSE) (17.4 ± 5.2 mm vs 19.9 ± 6.7 mm, P < 0.001), higher incidence of pericardial effusion (30% vs 5.2%, P < 0.001) and similar prevalence of ILD (41.8% vs. 44.9%). In individuals with PAH-SSc, ILD was associated with worse hemodynamics and pulmonary function tests (PFT). Up-front combination therapy was used in 59.8% and 61.7% of patients with and without ILD, respectively. Five-year transplant-free survival rate was 41.1% in PAH-SSc patients and 93.9% in non-PAH-SSc patients (P < 0.001). Global survival of PAH-SSc patients was not affected by ILD regardless its severity. The multivariate survival analysis in PAH-SSc patients confirmed age at diagnosis, worse NYHA-FC, increased PVR, reduced DLCO, and lower management with up-front combination therapy as major risk factors. In conclusion, in PAH-SSc cohort risk of death was greatly increased by clinical, PFT, and hemodynamic factors, whereas it was decreased by up-front combination therapy. Concomitant ILD worsened hemodynamics and PFT in PAH-SSc but not survival regardless of FVC impairment.

Anti-Polymyositis/Scl Antibodies in Systemic Sclerosis: Clinical Associations in a Multicentric Spanish Cohort and Review of the Literature.

OBJECTIVES: To assess the clinical profile of patients with anti-polymyositis/Scl (PM/Scl) antibodies in a cohort of Spanish patients with systemic sclerosis. METHODS: From the Spanish Scleroderma Study Group database, we selected patients in whom PM/Scl antibodies had been tested. We compared demographic, clinical, laboratory, and survival data between patients with and without PM/Scl antibodies. RESULTS: Seventy-two of 947 patients (7.6%) tested positive for PM/Scl antibodies. Patients with PM/Scl antibodies presented initially with more puffy fingers and arthralgias but less Raynaud phenomenon. Regarding cumulative manifestations, myositis and arthritis were more prevalent in patients with PM/Scl antibodies, as well as pulmonary fibrosis. On the contrary, patients with PM/Scl antibodies had less pulmonary hypertension. No difference in terms of survival at 5 and 10 years was noticed between the 2 groups. CONCLUSIONS: In systemic sclerosis patients from Spain, PM/Scl antibodies are associated with a distinct clinical profile. However, PM/Scl antibodies did not influence survival.

The challenge of comprehensive nailfold videocapillaroscopy practice: a further contribution.

OBJECTIVES: Although classification systems and scores for capillaroscopy interpretation have been published, there is a lack of homogenization for the procedure, especially in the way and place the images are taken, the counting of the capillaries and the measuring of their size. Our objective is to provide a deep learning-based software to obtain objective and exhaustive data for the whole nailfold without increasing the time or effort needed to do the examination, or requiring expensive equipment. METHODS: An automated software to count nailfold capillaries has been designed, through an exploratory image dataset of 2,713 images with 18,000 measurements of 3 different types. Subsequently, application rules have been created to detect the morphology of nailfold videocapillaroscopy images, through a training set of images. The software reliability has been evaluated with standard metrics used in the machine learning field for object detection tasks, comparing automatic and manual counting on the same NVC images. RESULTS: A mean average precision (mAP) of 0.473 is achieved for detecting and classifying capillaries and haemorrhages by their shape, and a mAP of 0.515 is achieved for detecting and classifying capillaries by their size. A precision of 83.84% and a recall of 92.44% in the identification of capillaries was estimated. CONCLUSIONS: Deep learning is a useful tool in nailfold videocapillaroscopy that allows to analyse objectively and homogeneously images taken with multiple devices. It should make the assessment of the capillary morphology in nailfold video capillaroscopy easier, quicker, more complete and accessible to everyone.

Circulating Cell Biomarkers in Pulmonary Arterial Hypertension: Relationship with Clinical Heterogeneity and Therapeutic Response.

BACKGROUND: Endothelial dysfunction is central to PAH. In this study, we simultaneously analysed circulating levels of endothelial microvesicles (EMVs) and progenitor cells (PCs) in PAH and in controls, as biomarkers of pulmonary endothelial integrity and evaluated differences among PAH subtypes and as a response to treatment. METHODS: Forty-seven controls and 144 patients with PAH (52 idiopathic, 9 heritable, 31 associated with systemic sclerosis, 15 associated with other connective tissue diseases, 20 associated with HIV and 17 associated with portal hypertension) were evaluated. Forty-four patients with scleroderma and 22 with HIV infection, but without PAH, were also studied. Circulating levels of EMVs, total (CD31+CD42b-) and activated (CD31+CD42b-CD62E+), as well as circulating PCs (CD34+CD133+CD45low) were measured by flow cytometry and the EMVs/PCs ratio was computed. In treatment-naïve patients, measurements were repeated after 3 months of PAH therapy. RESULTS: Patients with PAH showed higher numbers of EMVs and a lower percentage of PCs, compared with healthy controls. The EMV/PC ratio was increased in PAH patients, and in patients with SSc or HIV without PAH. After starting PAH therapy, individual changes in EMVs and PCs were variable, without significant differences being observed as a group. Conclusion: PAH patients present disturbed vascular homeostasis, reflected in changes in circulating EMV and PC levels, which are not restored with PAH targeted therapy. Combined measurement of circulating EMVs and PCs could be foreseen as a potential biomarker of endothelial dysfunction in PAH.

Comprehensive analysis of the major histocompatibility complex in systemic sclerosis identifies differential HLA associations by clinical and serological subtypes.

OBJECTIVE: The greatest genetic effect reported for systemic sclerosis (SSc) lies in the major histocompatibility complex (MHC) locus. Leveraging the largest SSc genome-wide association study, we aimed to fine-map this region to identify novel human leucocyte antigen (HLA) genetic variants associated with SSc susceptibility and its main clinical and serological subtypes. METHODS: 9095 patients with SSc and 17 584 controls genome-wide genotyped were used to impute and test single-nucleotide polymorphisms (SNPs) across the MHC, classical HLA alleles and their composite amino acid residues. Additionally, patients were stratified according to their clinical and serological status, namely, limited cutaneous systemic sclerosis (lcSSc), diffuse cutaneous systemic sclerosis (dcSSc), anticentromere (ACA), antitopoisomerase (ATA) and anti-RNApolIII autoantibodies (ARA). RESULTS: Sequential conditional analyses showed nine SNPs, nine classical alleles and seven amino acids that modelled the observed associations with SSc. This confirmed previously reported associations with HLA-DRB1*11:04 and HLA-DPB1*13:01, and revealed a novel association of HLA-B*08:01. Stratified analyses showed specific associations of HLA-DQA1*02:01 with lcSSc, and an exclusive association of HLA-DQA1*05:01 with dcSSc. Similarly, private associations were detected in HLA-DRB1*08:01 and confirmed the previously reported association of HLA-DRB1*07:01 with ACA-positive patients, as opposed to the HLA-DPA1*02:01 and HLA-DQB1*03:01 alleles associated with ATA presentation. CONCLUSIONS: This study confirms the contribution of HLA class II and reveals a novel association of HLA class I with SSc, suggesting novel pathways of disease pathogenesis. Furthermore, we describe specific HLA associations with SSc clinical and serological subtypes that could serve as biomarkers of disease severity and progression.