Lung disease associated with systemic juvenile idiopathic arthritis (SJIA-LD) remains poorly understood. Evaluation of bronchoalveolar lavage fluid (BALF) may better reflect disease pathogenesis. The objectives of our study were to measure levels of cytokines and chemokines in BALF and their associations with clinical features and treatment in patients with SJIA-LD. Children with SJIA-LD undergoing clinically indicated diagnostic bronchoscopy were enrolled. Comparator BALF was collected from patients with other chronic inflammatory and non-inflammatory lung diseases. BALF was assayed for IL-6, 8 and 18, S100A8/9, S100A12, sCD25, CCL11, CCL17, CCL25, MMP7, and CXCL9. BALF was obtained from 21 patients with SJIA-LD and 54 comparator patients with other lung diseases. Compared to all controls, children with SJIA-LD had a significant elevation of IL-18 (median (IQR) 1406 (722.3–2812) vs 37.2 (25.3–70) pg/mL, p < 0.0001), S100A8/9 (4745.3 (3003–11506) vs. 1297.5 (260–7755) ng/mL, p = 0.02), sCD25 (31.6 (19–50.3) vs. 13.6 (8.6–37.3) pg/mL, p = 0.04), MMP-7 (18,466.7 (10,462.2–33,516.1) vs. 13.645 (8.6–37.3) pg/mL, p = 0.0384), and CCL17 (0 (0–63.1; p = 0.038) vs 0 (0–0) pg/mL. BALF IL-18 was significantly higher in children with SJIA-LD compared to all control subgroups (inflammatory and non-inflammatory airway disease, autoimmune pulmonary alveolar proteinosis, and poorly controlled asthma), in patients who were actively treated with anti-cytokine biologics (N = 9), and in patients who ultimately underwent hematopoietic stem cell transfer (N = 8). Patients receiving anti-cytokine biologics also had significant elevations in several other cytokines compared to patients without such treatments, while profiles in those treated with JAKi (N = 14) were largely similar. Linear regression analysis showed an association between BALF level of IL-18 and the number of immunosuppressive medications utilized (p = 0.0167), but not with O2 requirement, dose of anakinra or prednisone, plasma IL-18, ferritin, CRP or ESR. Patients with SJIA-LD showed a distinct BALF cytokine profile, including significant IL-18, S100A8/9 protein, sCD25, and CCL-17 elevation. The level of IL-18 was higher in patients who required more immunosuppressive medications and were actively treated with anti-cytokine biologics. The relationship between BALF cytokine profiles and anti-cytokine biologics and JAK inhibitors is unclear and should be evaluated further.

Objective To evaluate the ability of the Renal Activity Index for Lupus (RAIL) score, a urine biomarker-derived score, to capture and predict the course of active lupus nephritis (LN) in adult patients. Methods Available serial urine samples collected up to week 52 from a subset of adults with active biopsy-proven proliferative LN participating in the double-blind randomized ALLURE trial of abatacept (ClinicalTrials.gov: NCT01714817 ) were used to calculate RAIL scores from creatinine-adjusted urine biomarkers (neutrophil gelatinase-associated lipocalin [NGAL], kidney injury molecule 1 [KIM-1], monocyte chemotactic protein 1 [MCP-1], adiponectin, hemopexin, ceruloplasmin). Discriminative performance of RAIL scores alone over time were compared with urine protein/creatinine ratio (UPCR), kidney function (estimated glomerular filtration rate [eGFR]), and mixed model analysis of RAIL score adjusted for baseline UPCR, eGFR, age, weight, sex, and race, with comparisons by renal response states including complete renal response (CRR), partial renal response but not CRR (PRR-only), and nonresponse (NR). Results The analysis included 240 patients who contributed 599 samples. At weeks 12/24/52, there were 44/22/15 patients with PRR-only, 27/33/18 with CRR, and 127/61/15 NR. RAIL scores, eGFR, and UPCR improved over time irrespective of abatacept use, but were significantly lower with CRR compared to NR. The eGFR alone had poor accuracy (area under the receiver-operating characteristic curve [AUC] < 0.51) to discriminate renal response. Only after correction of baseline UPCR and eGFR, the RAIL score had excellent accuracy to reflect CRR from other renal response states at the current (AUC = 0.83-0.84) and next visit (AUC = 0.84-0.85) and performed better than UPCR; without correction, UPCR and RAIL score had similarly good accuracy. Conclusion RAIL scores identify active LN and longitudinally predict the course of adult LN. (ClinicalTrials.gov: NCT01714817 )

Introduction:We aimed to develop multiplex (MLP) assays of 6 biomarkers, namely adiponectin, neutrophil gelatinase-associated lipocalin (NGAL), monocyte chemoattractant protein-1 (MCP-1), kidney injury molecule-1 (KIM-1), ceruloplasmin, and hemopexin used in the Renal Activity Index for Lupus (RAIL) and establish MLP assays using the Milliplex MLP and the electrochemiluminescence Mesoscale Discovery (MSD) technology, to compare with the gold standard of established single immunoassays. Methods:A total of 104 banked urine samples from the CCHMC Lupus Cohort were used. RAIL biomarker concentrations were assayed using established individual immunoassays, and concentrations were compared with MLP reagents using both the MSD and MLP platforms. MLP assay development involved assessment of biomarker concentrations in 40 individual urine samples, followed by evaluation of optimal sample dilution using 14 additional samples on each platform. Then 50 samples were assayed in duplicate under optimized MLP conditions, and biomarker concentration compared with those using single assays. After correcting for urine creatinine, RAIL scores of the samples were determined and compared between testing platforms (single immunoassays, MLP). Results:Our results indicate that a 1:25 urine dilution was optimal when using the MLP platforms. Biomarker concentrations by single immunoassays correlated with those on the Milliplex platform strongly for KIM-1, MCP-1, and NGAL (r = 0.726-0.86, P < 0.0001), moderately for adiponectin (r = 0.629, P < 0.0001) and weakly for ceruloplasmin (r = 0.367, P = 0.009). Using the MSD platform, comparatively lower correlations with those by single immunoassay were observed (NGAL: r = 0.516, P = 0.0001; adiponectin and hemopexin: r ≤ 0.29, P = 0.042; ceruloplasmin, KIM-1, and MCP-1: all r < 0.2). Conclusion:Milliplex technology is suitable to measure RAIL biomarker concentrations in urine samples diluted 1:25.

Abstract Background Juvenile idiopathic arthritis (JIA) comprises a heterogeneous group of conditions that can cause marked disability and diminished quality of life. Data on predictors of clinical response are insufficient to guide selection of the appropriate biologic agent for individual patients. This study aimed to investigate the propensity of S100A8/9 and S100A12 as predictive biomarkers of abatacept response in polyarticular-course juvenile idiopathic arthritis (pJIA). Methods Data from a phase 3 trial (NCT01844518) of subcutaneous abatacept in patients with active pJIA (n = 219) were used in this exploratory analysis. Association between biomarker levels at baseline and improvements in JIA-American College of Rheumatology (ACR) criteria responses or baseline disease activity (measured by Juvenile Arthritis Disease Activity Score in 27 joints using C-reactive protein [JADAS27-CRP]) were assessed. Biomarker level changes from baseline to month 4 were assessed for disease outcome prediction up to 21 months. Results At baseline, 158 patients had available biomarker samples. Lower baseline S100A8/9 levels (≤ 3295 ng/mL) were associated with greater odds of achieving JIA-ACR90 (odds ratio [OR]: 2.54 [95% confidence interval (CI): 1.25–5.18]), JIA-ACR100 (OR: 3.72 [95% CI: 1.48–9.37]), JIA-ACR inactive disease (ID; OR: 4.25 [95% CI: 2.03–8.92]), JADAS27-CRP ID (OR: 2.34 [95% CI: 1.02–5.39]) at month 4, and JIA-ACR ID (OR: 3.01 [95% CI: 1.57–5.78]) at month 16. Lower baseline S100A12 levels (≤ 176 ng/mL) were associated with greater odds of achieving JIA-ACR90 (OR: 2.52 [95% CI: 1.23–5.13]), JIA-ACR100 (OR: 3.68 [95% CI: 1.46–9.28]), JIA-ACR ID (OR: 3.66 [95% CI: 1.76–7.61]), JIA-ACR90 (OR: 2.03 [95% CI: 1.07–3.87]), JIA-ACR100 (OR: 2.14 [95% CI: 1.10–4.17]), and JIA-ACR ID (OR: 4.22 [95% CI: 2.15–8.29]) at month 16. From baseline to month 4, decreases in S100A8/9 and S100A12 generally exceeded 50% among JIA-ACR90/100/ID responders. Conclusion Lower baseline levels of S100A8/9 and S100A12 proteins predicted better response to abatacept treatment than higher levels and may serve as early predictive biomarkers in pJIA. Decreases in these biomarker levels may also predict longer-term response to abatacept in pJIA.

We undertook this study to validate the Pediatric Arthritis Ultrasound Scoring System for the knee joint (PAUSS-knee) in children with juvenile idiopathic arthritis (JIA). Children with JIA were enrolled to prospectively receive a musculoskeletal ultrasound (MSUS) examination of the knee and a physical examination to determine presence/absence of clinical arthritis. MSUS images were scored using the PAUSS-knee, a semiquantitative MSUS scoring system (0–3, normal to severe) for B-mode and power Doppler mode. In addition to MSUS, a subset of participants also received magnetic resonance imaging (MRI) of the knee, which was scored according to the combined Juvenile Arthritis MRI Scoring (JAMRIS) system. Spearman's correlations (r s ) were used to calculate associations between variables. Test characteristics of the PAUSS-knee were calculated with MRI as the reference standard. Inflammatory biomarkers were assessed in synovial fluid from involved knees. Eighty children with JIA contributed 112 MSUSs and 25 MRIs of the knee. Of the knees, 41% (n = 46) had clinical evidence of arthritis. The B-mode PAUSS-knee score moderately correlated with clinically determined arthritis (r s = 0.54, P < 0.001) and strongly correlated with the JAMRIS score (r s = 0.75, P < 0.001). Compared with MRI, the area under the curve for the B-mode PAUSS-knee was 0.92. For a cutoff of >1, the B-mode PAUSS-knee had a sensitivity of 83% and specificity of 82%. Biomarker analysis indicates that interleukin-2R levels correlate with PAUSS score. Our data indicate that the PAUSS-knee has excellent accuracy for the diagnosis of arthritis when compared with MRI. The PAUSS-knee has the potential to effectively inform JIA medical decision-making in real time.

Study Objective:To determine if baseline cytokines and their changes over postoperative days 0-2 (POD0-2) predict acute and chronic postsurgical pain (CPSP) after major surgery.Design:Prospective, observational, longitudinal nested study.Setting:University-affiliated quaternary children's hospital.Patients:Subjects (≥8 years old) with idiopathic scoliosis undergoing spine fusion or pectus excavatum undergoing Nuss procedure.Measurements:Demographics, surgical, psychosocial measures, pain scores, and opioid use over POD0-2 were collected. Cytokine concentrations were analyzed in serial blood samples collected before and after (up to two weeks) surgery, using Luminex bead arrays. After data preparation, relationships between pre- and post-surgical cytokine concentrations with acute (% time in moderate-severe pain over POD0-2) and chronic (pain score>3/10 beyond 3 months post-surgery) pain were analyzed. After adjusting for covariates, univariate/multivariate regression analyses were conducted to associate baseline cytokine concentrations with postoperative pain, and mixed effects models were used to associate longitudinal cytokine concentrations with pain outcomes.Main Results:Analyses included 3,164 measures of 16 cytokines from 112 subjects (median age 15.3, IQR 13.5-17.0, 54.5% female, 59.8% pectus). Acute postsurgical pain was associated with higher baseline concentrations of GM-CSF (β=0.95, SE 0.31; p=.003), IL-1β (β=0.84, SE 0.36; p=.02), IL-2 (β=0.78, SE 0.34; p=.03), and IL-12 p70 (β=0.88, SE 0.40; p=.03) and longitudinal postoperative elevations in GM-CSF (β=1.38, SE 0.57; p=.03), IFNγ (β=1.36, SE 0.6; p=.03), IL-1β (β=1.25, SE 0.59; p=.03), IL-7 (β=1.65, SE 0.7, p=.02), and IL-12 p70 (β=1.17, SE 0.58; p=.04). In contrast, CPSP was associated with lower baseline concentration of IL-8 (β= -0.39, SE 0.17; p=.02), and the risk of developing CPSP was elevated in patients with lower longitudinal postoperative concentrations of IL-6 (β= -0.57, SE 0.26; p=.03), IL-8 (β= -0.68, SE 0.24; p=.006), and IL-13 (β= -0.48, SE 0.22; p=.03). Furthermore, higher odds for CPSP were found for females (vs. males) for IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, and TNFα, and for pectus (vs. spine) surgery for IL-8 and IL-10.Conclusion:We identified pro-inflammatory cytokines associated with increased acute postoperative pain and anti-inflammatory cytokines associated with lower CPSP risk, with potential to serve as predictive and prognostic biomarkers.

Background: Changes in the biochemical and protein composition of ocular fluid may reflect inflammation due to disruption of the blood-retinal barrier (BRB), as in uveitis. Tear fluid is studied as a source of biomarker discovery in ocular and non-ocular diseases. Tear sampling is non-invasive and tolerated by children. Objectives: We aim to compare proteins in tears and aqueous humor (AH) of children with and without uveitis. Methods: Our cross-sectional study collected tears and AH in 4 patients with uveitis (1 JIA-uveitis, 1 chronic anterior uveitis, 2 ADNIV) and 3 pediatric non-inflammatory controls. Tears were collected by Schirmer strip, and AH during routine eye surgery. Advanced proteomic strategies (iTRAQ labeling and nanoLC-MS/MS) quantified proteins and normalized by total peptide amount. Log of the mean of the ratios was derived from 2 abundance readings per protein/subject. We used Wilcoxon rank sum exact test to compare proteins in tears and AH of uveitis patients and controls (P-value <0.05). Results: Seven patients (71% females, median age 17 y [R 2-59] at collection) contributed 10 paired tear and AH samples. 145 proteins were identified at medium 95% or higher FDR confidence with at least 1 high confidence peptide. Protein abundance was significantly different in tears vs AH of children with uveitis (median 607.8 vs 5012.0, P value 0.009), but not in controls (median 634 vs 121, P value 0.175) (Figure 1A/1B). Further analysis only included proteins detected in 100% of samples. Proteomic analysis showed significantly different expression of 31 of 63 (49%) proteins in tears and AH of children with uveitis, and of 10 of 18 proteins (45%) in controls (Figure 2A/2B). Some of these proteins have already been reported to be associated with the immune response or as inflammatory markers (Immunoglobulins heavy constant alpha 1, protein S100A9, and lysozyme), in the retinal pigment epithelium (Pigment epithelium derived factor), and in inflammatory uveitis (as vitamin D-binding protein, lactotransferrin, ceruloplasmin or apoliprotein I). Conclusion: Uveitis is a vision-threatening disease that warrants exploration of techniques for early detection. The eye is an immune-privileged organ immunologically shielded by the BRB. Around 50% of proteins overlapped in tear fluid and AH. Of those that were differentially expressed, mammoglobin-B, cystatin-S, and secretoglobin family 1D member have been reported in pediatric uveitis tear biomarker studies. Lysozyme, lactoferrin, lipocalin, albumin, and lactotransferrin are common major tear proteins. As tear sampling is feasible in children compared to AH, use of tears in uveitis biomarker studies is promising. REFERENCES: NIL. Acknowledgements: NIL. Disclosure of Interests: None declared.Figure 1Differences in protein abundance in children with uveitis 1A and control group 1B Figure 2Significant different protein expression in tears and aqueous humor in children with and without uveitis.

OBJECTIVE:We examine levels of candidate blood-based biomarkers (CBBs) in patients with juvenile idiopathic arthritis (JIA) treated with tofacitinib. METHODS:Patients with JIA who participated in clinical trial NCT02592434 received tofacitinib from baseline to week 18. Serial serum samples were assayed for CBBs (S100A8/9, S100A12, interleukin-18 [IL-18], serum amyloid A, resistin, vascular endothelial growth factor, angiopoietin-1, angiopoietin-2, matrix metalloproteinase 8 [MMP8], MMP2, tissue inhibitor of metalloproteinases 1, leptin, chemokine [C-X-C motif] ligand 9, soluble IL-2 receptor, intercellular adhesion molecule 1, soluble tumor necrosis factor receptor, IL-6, IL-23, monocyte chemotactic protein 1, chemokine [C-C motif] ligand 18 [CCL18], and CCL20). Association of CBBs with JIA response to treatment from baseline to week 18 were assessed. RESULTS:This study included 166 patients with polyarticular-course JIA. Paired serum samples from 143 patients were available at both baseline and week 18. Thirty-five percent (50 of 143) of patients had a JIA-American College of Rheumatology 90 (JIA-ACR90) level improvement, whereas 90, 121, and 137 (63%, 85%, and 96%) achieved JIA-ACR70, 50, and 30 improvement at week 18. Despite small numerical differences by JIA category, there were no baseline CBB values that independently predicted a decrease in Juvenile Arthritis Disease Activity Score (JADAS-27) or JIA-ACR90 response by week 18. Decrease in resistin level (baseline to week 18) was significantly associated with week 18 improvement in JADAS-27 and JIA-ACR90 response after adjusting for age, sex, JIA disease duration, and baseline resistin (r2 0.79, SE 0.070, P < 0.01, and odds ratio [95% confidence interval] 1.134 [1.018-1.264]). HLA-B27 positivity was significantly associated with not achieving a JIA-ACR90 response at week 18 (P = 0.0097). CONCLUSION:Among the CBBs included, only resistin was significantly associated with treatment response, and no CBB was identified that forecasts JIA improvement after initiation of tofacitinib. The association of HLA-B27 positivity with lower response to tofacitinib in JIA is intriguing and merits further study.

BackgroundUveitis is an inflammatory ocular disease secondary to disruption of the retinal pigmented epithelium (RPE) and blood retinal barrier (BRB). Known clinical factors do not accurately predict uveitis risk in Juvenile Idiopathic Arthritis (JIA). Tear fluid is easily obtained for biomarker study. We aim to identify tear-based markers associated with the presence of uveitis in children with JIA.MethodsIn a cross-sectional comparative cohort study, tears were collected by Schirmer strips from children with oligoarticular JIA-associated uveitis (JIA-U) and JIA without uveitis (JIA-no-U). A tandem isotope tagging (iTRAQ and TMT) strategy was used for relative quantitation via nanoLC-MS/MS to quantify proteins in the affected eye. Log transformed relative protein abundance of protein levels was compared between groups using Wilcoxon exact test. We explored the influence of arthritis activity and topical corticosteroids (CS) use on protein levels. STRING analysis was performed.ResultsTear samples of 14 JIA-U and 14 JIA-no-U patients were analyzed. Thirteen proteins were differentially expressed between both groups. Stratified analysis based on arthritis activity (inactive arthritis) and topical CS (off CS) showed that alpha-2-macroglobulin (p = 0.012), apolipoprotein A1 (p = 0.036), S100A9 (p = 0.05), haptoglobin (p = 0.066), and transthyretin (p = 0.066) consistently differentiated between both groups. On STRING analysis, these proteins were associated with the RPE, BRB, and inflammation.ConclusionImportantly, we identified proteins involved in the RPE, BRB, and immune response that were differentially abundant in the tears of children with JIA-U compared to JIA-no-U, regardless of arthritis activity or topical CS. Candidate tear-based biomarkers may represent a non-invasive means to detect uveitis.