Increased vascular deposition of oxidized LDL in untreated juvenile dermatomyositis.

BACKGROUND: Juvenile dermatomyositis (JDM) is a systemic vasculopathy associated with metabolic derangements and possible increased risk for premature atherosclerosis. Oxidation of low-density lipoprotein (LDL) in the endothelium is an early step in atherosclerotic plaque formation. It is not known if oxidized LDL is altered in children with untreated JDM. The deposition of oxidized LDL in the vasculature of muscle biopsies (MBx) from patients with untreated JDM and pediatric controls was assessed. FINDINGS: Frozen tissue sections of MRI-directed MBx from 20 female children with untreated JDM and 5 female controls were stained with DAPI and fluorescently labeled antibodies against von Willebrand factor (vWF) and LDL oxidized by copper (oxLDL). Blood vessels were identified by positive vWF staining, and total fluorescence of oxLDL within the vessel walls was measured. Children with untreated JDM had increased deposition of oxLDL in the walls of muscle vasculature compared to healthy children (difference in means ± SEM = 19.86 ± 8.195, p = 0.03). Within the JDM cohort, there was a trend towards increased oxLDL deposition with longer duration of untreated disease (r = 0.43, p = 0.06). There was no significant correlation found between oxLDL deposition and markers of acute JDM disease activity including disease activity scores or muscle enzymes. CONCLUSIONS: This study found increased deposition of oxLDL within blood vessels of children with untreated JDM supporting the concern that these children are at increased risk for premature atherosclerosis from chronic exposure to vascular oxLDL. This study highlights the importance of early diagnosis and treatment initiation to ameliorate cardiovascular damage.

Decreased Peripheral Blood Natural Killer Cell Count in Untreated Juvenile Dermatomyositis Is Associated with Muscle Weakness.

Juvenile Dermatomyositis (JDM) is the most common inflammatory myopathy in pediatrics. This study evaluates the role of Natural Killer (NK) cells in Juvenile Dermatomyositis (JDM) pathophysiology. The study included 133 untreated JDM children with an NK cell count evaluation before treatment. NK cell subsets (CD56low/dim vs. CD 56bright) were examined in 9 untreated children. CD56 and perforin were evaluated in situ in six untreated JDM and three orthopedic, pediatric controls. 56% of treatment-naive JDM had reduced circulating NK cell counts, designated "low NK cell". This low NK group had more active muscle disease compared to the normal NK cell group. The percentage of circulating CD56low/dim NK cells was significantly lower in the NK low group than in controls (0.55% vs. 4.6% p < 0.001). Examination of the untreated JDM diagnostic muscle biopsy documented an increased infiltration of CD56 and perforin-positive cells (p = 0.023, p = 0.038, respectively). Treatment-naive JDM with reduced circulating NK cell counts exhibited more muscle weakness and higher levels of serum muscle enzymes. Muscle biopsies from treatment-naive JDM displayed increased NK cell infiltration, with increased CD56 and perforin-positive cells.

Proof-of-concept study evaluating humoral primary immunodeficiencies via CJ:KREC ratio and serum BAFF level.

Humoral primary immunodeficiencies are the most prevalent form of primary immunodeficiency (PID). Currently, there is no convenient method to quantify newly formed B cells. The aim of this proof-of-concept study was to quantitate the ratio of coding joints (CJs) to Kappa-deleting recombination excision circles (KRECs) and serum B cell activating factor (BAFF) in patients with humoral primary immunodeficiency and assess if they correlate with disease severity. This IRB-approved study was conducted at one academic children's hospital. Patients with humoral PIDs and healthy controls were included. CJ and KREC levels were measured via qPCR. Serum BAFF levels were measured using Mesoscale. 16 patients with humoral PID and 5 healthy controls were included. The mean CJ:KREC ratio in the CVID, antibody deficiency syndromes, and controls groups, respectively were 13.04 ± 9.5, 5.25 ± 4.1, and 4.38 ± 2.5 (p = 0.059). The mean serum BAFF levels in CVID, antibody deficiency syndromes and controls were 216.3 ± 290 pg/mL, 107.9 ± 94 pg/mL and 50.9 ± 12 pg/mL, respectively (p = 0.271). When the CVID patients were subdivided into CVID with or without lymphoproliferative features, the BAFF level was substantially higher in the CVID with lymphoproliferation cohort (mean 372.4 ± 361 pg/mL, p = 0.031). Elevated CJ:KREC ratio was observed in CVID, although statistical significance was not achieved, likely due to the small sample size. Serum BAFF levels were significantly higher in CVID patients with lymphoproliferative features. We speculate that the CJ:KREC ratio and serum BAFF levels can be utilized in patients with humoral PID, once more extensive studies confirm this exploratory investigation.

Role of mitochondria in the myopathy of juvenile dermatomyositis and implications for skeletal muscle calcinosis.

OBJECTIVES: To elucidate mechanisms contributing to skeletal muscle calcinosis in patients with juvenile dermatomyositis. METHODS: A well-characterized cohorts of JDM (n = 68), disease controls (polymyositis, n = 7; juvenile SLE, n = 10, and RNP + overlap syndrome, n = 12), and age-matched health controls (n = 17) were analyzed for circulating levels of mitochondrial (mt) markers including mtDNA, mt-nd6, and anti-mitochondrial antibodies (AMAs) using standard qPCR, ELISA, and novel-in-house assays, respectively. Mitochondrial calcification of affected tissue biopsies was confirmed using electron microscopy and energy dispersive X-ray analysis. A human skeletal muscle cell line, RH30, was used to generate an in vitro calcification model. Intracellular calcification is measured by flow cytometry and microscopy. Mitochondria were assessed for mtROS production and membrane potential by flow cytometry and real-time oxygen consumption rate by Seahorse bioanalyzer. Inflammation (interferon-stimulated genes) was measured by qPCR. RESULTS: In the current study, patients with JDM exhibited elevated levels of mitochondrial markers associated with muscle damage and calcinosis. Of particular interest are AMAs predictive of calcinosis. Human skeletal muscle cells undergo time- and dose-dependent accumulation of calcium phosphate salts with preferential localization to mitochondria. Calcification renders skeletal muscle cells mitochondria stressed, dysfunctional, destabilized, and interferogenic. Further, we report that inflammation induced by interferon-alpha amplifies mitochondrial calcification of human skeletal muscle cells via the generation of mitochondrial reactive oxygen species (mtROS). CONCLUSIONS: Overall, our study demonstrates the mitochondrial involvement in the skeletal muscle pathology and calcinosis of JDM and mtROS as a central player in the calcification of human skeletal muscle cells. Therapeutic targeting of mtROS and/or upstream inducers, such as inflammation, may alleviate mitochondrial dysfunction, leading to calcinosis. AMAs can potentially identify patients with JDM at risk for developing calcinosis.

Skin disease is more recalcitrant than muscle disease: A long-term prospective study of 184 children with juvenile dermatomyositis.

BACKGROUND: Persistent skin manifestations, especially calcinoses, contribute to morbidity in children with juvenile dermatomyositis. OBJECTIVE: To compare the course of skin and muscle involvement and document frequency of calcinosis in juvenile dermatomyositis. METHODS: Prospective cohort study of 184 untreated children with juvenile dermatomyositis (July 1971 to May 2019) at a single children's hospital. RESULTS: Disease Activity Scores (DASs) were persistently higher for skin versus muscle at all points; clinical inactivity (DAS ≤2) occurred earlier for muscle than skin. Among vascular features for DAS for skin, eyelid margin capillary dilatation was most frequent (54.3%) and persisted longest. Intravenous methylprednisolone reduced DAS for skin more than oral prednisone at 12 months (P = .04). Overall, 16.8% of patients (n = 31) had calcifications, with 4.9% at enrollment. Despite therapy, 25.0% of calcifications recurred and 22.6% failed to resolve; of the latter, 71.4% (n = 5) were present at enrollment. Children with persistent calcifications had longer duration of untreated disease than those whose calcifications resolved (mean 12.5 months) (P < .001). Hydroxychloroquine did not improve DAS for skin (P = .89). LIMITATIONS: DAS does not quantify nailfold capillary dropout. CONCLUSIONS: In juvenile dermatomyositis, skin disease presents with greater activity and is more recalcitrant to therapies than muscle disease. Early and aggressive treatment can limit the severity and persistence of calcifications identified later in the disease course.

Comparison of Lesional Juvenile Myositis and Lupus Skin Reveals Overlapping Yet Unique Disease Pathophysiology.

OBJECTIVE: Skin inflammation heralds systemic disease in juvenile myositis, yet we lack an understanding of pathogenic mechanisms driving skin inflammation in this disease. We undertook this study to define cutaneous gene expression signatures in juvenile myositis and identify key genes and pathways that differentiate skin disease in juvenile myositis from childhood-onset systemic lupus erythematosus (SLE). METHODS: We used formalin-fixed paraffin-embedded skin biopsy samples from 15 patients with juvenile myositis (9 lesional, 6 nonlesional), 5 patients with childhood-onset SLE, and 8 controls to perform transcriptomic analysis and identify significantly differentially expressed genes (DEGs; q ≤ 5%) between patient groups. We used Ingenuity Pathway Analysis (IPA) to highlight enriched biologic pathways and validated DEGs by immunohistochemistry and quantitative real-time polymerase chain reaction. RESULTS: Comparison of lesional juvenile myositis to control samples revealed 221 DEGs, with the majority of up-regulated genes representing interferon (IFN)-stimulated genes. CXCL10, CXCL9, and IFI44L represented the top 3 DEGs (fold change 23.2, 13.3, and 13.0, respectively; q < 0.0001). IPA revealed IFN signaling as the top canonical pathway. When compared to childhood-onset SLE, lesional juvenile myositis skin shared a similar gene expression pattern, with only 28 unique DEGs, including FBLN2, CHKA, and SLURP1. Notably, patients with juvenile myositis who were positive for nuclear matrix protein 2 (NXP-2) autoantibodies exhibited the strongest IFN signature and also demonstrated the most extensive Mx-1 immunostaining, both in keratinocytes and perivascular regions. CONCLUSION: Lesional juvenile myositis skin demonstrates a striking IFN signature similar to that previously reported in juvenile myositis muscle and peripheral blood. Further investigation into the association of a higher IFN score with NXP-2 autoantibodies may provide insight into disease endotypes and pathogenesis.

Endothelial and Inflammation Biomarker Profiles at Diagnosis Reflecting Clinical Heterogeneity and Serving as a Prognostic Tool for Treatment Response in Two Independent Cohorts of Patients With Juvenile Dermatomyositis.

OBJECTIVE: Juvenile dermatomyositis (DM) is a heterogeneous systemic immune-mediated vasculopathy. This study was undertaken to 1) identify inflammation/endothelial dysfunction-related biomarker profiles reflecting disease severity at diagnosis, and 2) establish whether such biomarker profiles could be used for predicting the response to treatment in patients with juvenile DM. METHODS: In total, 39 biomarkers related to activation of endothelial cells, endothelial dysfunction, and inflammation were measured using multiplex technology in serum samples from treatment-naive patients with juvenile DM from 2 independent cohorts (n = 30 and n = 29). Data were analyzed by unsupervised hierarchical clustering, nonparametric tests with correction for multiple comparisons, and Kaplan-Meier tests with Cox proportional hazards models for analysis of treatment duration. Myositis-specific antibodies (MSAs) were measured in the patients' serum using line blot assays. RESULTS: Severe vasculopathy in patients with juvenile DM was associated with low serum levels of intercellular adhesion molecule 1 (Spearman's rho [rs ] = 0.465, P = 0.0111) and high serum levels of endoglin (rs = -0.67, P < 0.0001). In the discovery cohort, unsupervised hierarchical clustering analysis of the biomarker profiles yielded 2 distinct patient clusters, of which the smaller cluster (cluster 1; n = 8) exhibited high serum levels of CXCL13, CCL19, galectin-9, CXCL10, tumor necrosis factor receptor type II (TNFRII), and galectin-1 (false discovery rate <0.0001), and this cluster had greater severity of muscle disease and global disease activity (each P < 0.05 versus cluster 2). In the validation cohort, correlations between the serum levels of galectin-9, CXCL10, TNFRII, and galectin-1 and the severity of global disease activity were confirmed (rs = 0.40-0.52, P < 0.05). Stratification of patients according to the 4 confirmed biomarkers identified a cluster of patients with severe symptoms (comprising 64.7% of patients) who were considered at high risk of requiring more intensive treatment in the first 3 months after diagnosis (P = 0.0437 versus other cluster). Moreover, high serum levels of galectin-9, CXCL10, and TNFRII were predictive of a longer total treatment duration (P < 0.05). The biomarker-based clusters were not evidently correlated with patients' MSA serotypes. CONCLUSION: Results of this study confirm the heterogeneity of new-onset juvenile DM based on serum biomarker profiles. Patients with high serum levels of galectin-9, CXCL10, TNFRII, and galectin-1 may respond suboptimally to conventional treatment, and may therefore benefit from more intensive monitoring and/or treatment.

Neutrophil Extracellular Traps in Tissue and Periphery in Juvenile Dermatomyositis.

OBJECTIVE: Neutrophils are key immune cells participating in host defense through several mechanisms, including the formation of neutrophil extracellular traps (NETs). This study was undertaken to investigate the role of neutrophils in juvenile dermatomyositis (JDM). METHODS: Electron microscopy was used to identify neutrophils in tissue. NETs were also imaged using fluorescence microscopy and quantified using a myeloperoxidase-DNA enzyme-linked immunosorbent assay (ELISA) in plasma obtained from healthy children (n = 20), disease controls (n = 29), JDM patients (n = 66), and JDM patients with history of calcifications (n = 20). Clinical data included disease activity scores and complement C4 levels. Levels of immune complexes (ICs) and calprotectin were analyzed using ELISA. RESULTS: Using electron microscopy, neutrophils were found to infiltrate affected muscle tissue, engulfing deposited calcium crystals. Uptake of the crystals led to neutrophil activation (P < 0.01) and subsequent phosphatidylinositol 3-kinase- and NADPH oxidase-dependent but peptidylarginine deiminase 4-independent formation of NETs, which contained mitochondrial DNA (P < 0.05), as confirmed in vivo (P < 0.001) and in vitro (P < 0.01). Peripheral NET levels were associated with calcinosis (P = 0.01), ICs (P = 0.008), and interleukin-8 levels (P = 0.004). Children with JDM had impaired NET clearance (P = 0.01), associated with autoantibody profiles including melanoma differentiation-associated protein 5 (P = 0.005), and depressed complement C4 levels (r = -0.72, P = 0.002). Furthermore, children with JDM showed evidence of neutrophil activation, with elevated levels of peroxidase activity (P = 0.02) and calprotectin (P < 0.01), which were associated with disease activity (P = 0.007), and dyslipidemia (odds ratio 4.7, P < 0.05). CONCLUSION: We found novel mechanisms of both calcium crystal-mediated neutrophil activation and cell death in JDM pathophysiology. Targeting this pathway may reduce the frequency and extent of calcinosis, as well as prevent long-term development of comorbidities, including atherosclerosis.

Environmental factors associated with disease flare in juvenile and adult dermatomyositis.

Objective: The aim was to assess environmental factors associated with disease flare in juvenile and adult dermatomyositis (DM). Methods: An online survey of DM patients from the USA and Canada examined smoking, sun exposure, infections, medications, vaccines, stressful life events and physical activity during the 6 months before flares, or in the past 6 months in patients without flares. Differences were evaluated by χ 2 and Fisher's exact tests, and significant univariable results were examined in multivariable logistic regression. Residential locations before flare were correlated with the National Weather Service UV index. Results: Of 210 participants (164 juvenile and 46 adult DM), 134 (63.8%) experienced a disease flare within 2 years of the survey. Subjects more often reported disease flare after sun exposure [odds ratio (OR) = 2.0, P = 0.03], although use of photoprotective measures did not differ between those with and without flare. Urinary tract infections (OR = 16.4, P = 0.005) and gastroenteritis (OR = 3.2, P = 0.04) were more frequent in the preceding 6 months in those who flared. Subjects who flared recently used NSAIDS (OR = 3.0, P = 0.0003), blood pressure medicines (OR = 3.5, P = 0.049) or medication for depression or mood changes (OR = 12.9, P = 0.015). Moving to a new house (OR = 10.3, P = 0.053) was more common in those who flared. Only sun exposure (OR = 2.2) and NSAIDs (OR = 1.9) were significant factors in multivariable analysis. Conclusion: Certain classes of environmental agents that have been associated with the initiation of DM, including sun exposure and medications, may also play a role in disease flares.

Monitoring change in volume of calcifications in juvenile idiopathic inflammatory myopathy: a pilot study using low dose computed tomography.

BACKGROUND: Dystrophic calcifications may occur in patients with J uvenile Idiopathic Inflammatory Myopathy (JIIM) as well as other connective tissue and metabolic diseases, but a reliable method of measuring the volume of these calcifications has not been established. The purpose of this study is to determine the feasibility of low dose, limited slice, Computed Tomography (CT) to measure objectively in-situ calcification volumes in patients with JIIM over time. METHODS: Ten JIIM patients (eight JDM, two Overlap) with calcifications were prospectively recruited over a 2-year period to undergo two limited, low dose, four-slice CT scans. Calculation of the volume of calcifications used a CT post processing workstation. Additional patient data included: Disease Activity Scores (DAS), Childhood Myositis Assessment Scale (CMAS), myositis specific antibodies (MSA), and the TNFα-308 promoter region A/G polymorphism. Statistical analysis utilized the Pearson correlation coefficient, the paired t-test and descriptive statistics. RESULTS: Ten JIIM, mean age 14.54 ± 4.54 years, had a duration of untreated disease of 8.68 ± 5.65 months  MSA status: U1RNP (1), PM-Scl (1), Ro (1, 4 indeterminate), p155/140 (2), MJ (3), Mi-2 indeterminate (1), negative (3). 4/8 JDM (50%) were TNF-α-308 A+. Overall, the calcification volumes tended to decrease from the first to the second CT study by 0.5 cm3 (from 2.79 ± 1.98 cm3 to 2.29 ± 2.25 cm3). The average effective radiation dose was 0.007 ± 0.002, 0.010 ± 0.005, and 0.245 mSv for the upper extremity, lower extremity and chest, respectively (compared to a standard chest x-ray-- 0.02mSV effective dosage). CONCLUSION: We conclude: 1) the limited low dose CT technique provides objective data about volume of the calcifications in JIIM; 2) measuring the volume of calcifications in an extremity is associated with minimal radiation exposure; 3) This method may be useful to evaluate the efficacy of therapies for JIIM dystrophic calcification.

MicroRNA-10a Regulation of Proinflammatory Mediators: An Important Component of Untreated Juvenile Dermatomyositis.

OBJECTIVE: To identify differentially expressed microRNA (miRNA) in muscle biopsies (MBx) from 15 untreated children with juvenile dermatomyositis (JDM) compared with 5 controls. METHODS: Following MBx miRNA profiling, differentially expressed miRNA and their protein targets were validated by quantitative real-time PCR (qRT-PCR) and immunological assay. The association of miRNA-10a and miRNA-10b with clinical data was evaluated, including Disease Activity Score (DAS), von Willebrand factor antigen (vWF:Ag), nailfold capillary end row loops, duration of untreated disease, and tumor necrosis factor (TNF)-α-308A allele. RESULTS: In JDM, 16/362 miRNA were significantly differentially expressed [false discovery rate (FDR) < 0.05]. Among these, miRNA-10a was the most downregulated miRNA in both FDR and ranking of fold change: miRNA-10a = -2.27-fold, miRNA-10b = -1.80-fold. Decreased miRNA-10a and miRNA-10b expressions were confirmed using q RT-PCR: -4.16 and -2.59 fold, respectively. The qRT-PCR documented that decreased miRNA-10a expression was related to increased vascular cell adhesion molecule 1 in 13 of these JDM cases (correlation -0.67, p = 0.012), unlike miRNA-10b data (not significant). Concurrent JDM plasma contained increased levels of interleukin (IL) 6 (p = 0.0363), IL-8 (p = 0.0005), TNF-α (p = 0.0011), and monocyte chemoattractant proteins 1 (p = 0.0139). Decreased miRNA-10a, but not miRNA-10b, was associated with the TNF-α-308A allele (p = 0.015). In the 15 JDM, a trend of association of miRNA-10a (but not miRNA-10b) with vWF:Ag and DAS was observed. CONCLUSION: MiRNA-10a downregulation is an important element in untreated JDM muscle pathophysiology. We speculate that muscle miRNA expression in adult dermatomyositis differs from muscle miRNA expression in untreated childhood JDM.

A mouse model of human primitive neuroectodermal tumors resulting from microenvironmentally-driven malignant transformation of orthotopically transplanted radial glial cells.

There is growing evidence and a consensus in the field that most pediatric brain tumors originate from stem cells, of which radial glial cells constitute a subtype. Here we show that orthotopic transplantation of human radial glial (RG) cells to the subventricular zone of the 3rd ventricle--but not to other transplantation sites--of the brain in immunocompromised NOD-SCID mice, gives rise to tumors that have the hallmarks of CNS primitive neuroectodermal tumors (PNETs). The resulting mouse model strikingly recapitulates the phenotype of PNETs. Importantly, the observed tumorigenic transformation was accompanied by aspects of an epithelial to mesenchymal transition (EMT)-like process. It is also noteworthy that the tumors are highly invasive, and that they effectively recruit mouse endothelial cells for angiogenesis. These results are significant for several reasons. First, they show that malignant transformation of radial glial cells can occur in the absence of specific mutations or inherited genomic alterations. Second, they demonstrate that the same radial glial cells may either give rise to brain tumors or differentiate normally depending upon the microenvironment of the specific region of the brain to which the cells are transplanted. In addition to providing a prospect for drug screening and development of new therapeutic strategies, the resulting mouse model of PNETs offers an unprecedented opportunity to identify the cancer driving molecular alterations and the microenvironmental factors that are responsible for committing otherwise normal radial glial cells to a malignant phenotype.

Pilot study of etanercept in patients with refractory juvenile dermatomyositis.

OBJECTIVE: To evaluate the efficacy of etanercept in patients with juvenile dermatomyositis (DM) refractory to standard treatment. METHODS: Nine patients with juvenile DM prospectively received etanercept 0.4 mg/kg subcutaneous twice weekly concurrently with baseline medications for 12 weeks. Patients were reevaluated 12 weeks (week 24) after stopping etanercept. Outcome measures included a validated Disease Activity Score (DAS), serum muscle enzymes, Childhood Myositis Assessment Scale (CMAS), and nailfold capillaroscopy (NFC). RESULTS: Six patients completed all visits; 2 patients completed through week 12 and 1 patient stopped after the fifth etanercept dose due to marked worsening of a rash. At week 12, 7 patients had a mild decrease in DAS and 1 patient noted worsening of the DAS. At week 24, 1 patient remained stable, 2 patients had worsening of the DAS, and 3 patients had improvement of the DAS (1 patient with inactive disease), including the patient who worsened while receiving etanercept. This patient and the patient who stopped (worsening rash) both had the tumor necrosis factor α (TNFα) 308A allele. There was a trend of worsening NFC at week 12, while at week 24 improvement of NFC was noted. There was no appreciable change in serum muscle enzymes or CMAS throughout the study. CONCLUSION: In this trial of patients with refractory juvenile DM, etanercept did not demonstrate appreciable improvement and some patients noted worsening of disease. Caution should be taken when recommending TNF receptor inhibitors to patients with active symptoms of juvenile DM, and close followup is warranted. Further investigation of the interaction of the TNFα-308A polymorphism and type I interferon is needed to define the mechanism of TNF blockade in juvenile DM.

Interferon-α induction and detection of anti-ro, anti-la, anti-sm, and anti-rnp autoantibodies by autoantigen microarray analysis in juvenile dermatomyositis.

OBJECTIVE: To evaluate serum interferon-α (IFNα) activity in the context of autoantibody profiles in patients with juvenile dermatomyositis (JDM). METHODS: Sera from 36 patients with JDM were analyzed. Autoantibody profiles were determined by probing microarrays, which were fabricated with ∼80 distinct autoantigens, with serum and a Cy3-conjugated secondary antibody. Arrays were scanned and analyzed to determine antigen reactivity. Serum IFNα activity was measured using a functional reporter cell assay. Sera were assayed alone or in combination with cellular material released from necrotic U937 cells to stimulate peripheral blood mononuclear cells from healthy donors in vitro, and IFNα production in culture was measured by a dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA). RESULTS: Reactivity against at least 1 of 41 autoantigens on the microarray, including Ro 52, Ro 60, La, Sm, and RNP, was observed in 75% of the serum samples from patients with JDM. IFNα activity was detected in 7 samples by reporter cell assay. The reporter cell assay showed a significant association of reactivity against Ro, La, Sm, and proliferating cell nuclear antigen with serum IFNα activity (P = 0.005). Significance Analysis of Microarrays (SAM) identified increased reactivity against Sm, RNP, Ro 52, U1-C, and Mi-2 in these sera. Sixteen samples induced IFNα production as measured by DELFIA, and there was a significant association of reactivity against Ro, La, Sm, and RNP with the induction of IFNα by serum and necrotic cell material (P = 0.034). SAM identified increased reactivity against Ro 60 in these sera. CONCLUSION: These data support the hypothesis that nucleic acid-associated autoantibodies, including the Ro/La and Sm/RNP complexes, may stimulate the production of active IFNα in children with JDM.

Increased expression of vascular cell adhesion molecule 1 in muscle biopsy samples from juvenile dermatomyositis patients with short duration of untreated disease is regulated by miR-126.

OBJECTIVE: To evaluate the effect of duration of untreated disease on vascular cell adhesion molecule 1 (VCAM-1) and microRNA (miRNA) expression in muscle biopsy samples from children with juvenile dermatomyositis (DM) as well as its effect on soluble VCAM-1 (sVCAM-1) and tumor necrosis factor α (TNFα) concentrations in sera from these children. METHODS: We enrolled 28 untreated children with juvenile DM and 8 pediatric controls. Eleven children with juvenile DM had short duration of untreated disease (symptoms for ≤2 months before muscle biopsy), and 17 had long duration of untreated disease (symptoms for >2 months before muscle biopsy). Vascular structures, characterized by immunofluorescence using antibodies against von Willebrand factor, VCAM-1, and α-smooth muscle actin, were measured for total area and intensity. Circulating sVCAM-1 and TNFα levels were determined in patients with short duration of untreated disease, patients with long duration of untreated disease, and controls. Differential expression of microRNA-126 (miR-126) in muscle biopsy samples from the 2 patient groups and the control group was detected by miRNA expression profiling and confirmed by quantitative reverse transcription-polymerase chain reaction in muscle biopsy samples from the 3 groups. RESULTS: Juvenile DM patients with short duration of untreated disease had significantly higher total positive area and intensity/high power field of VCAM-1 expression than did juvenile DM patients with long duration of untreated disease (P = 0.043 and P = 0.015, respectively) or controls (P = 0.004 and P = 0.001, respectively). Von Willebrand factor antigen-positive vasculature displayed greater VCAM-1 intensity in patients with short duration of untreated disease than in patients with long duration of untreated disease (P = 0.001). Circulating levels of sVCAM-1 and TNFα were significantly higher in patients with short duration of untreated disease than in controls (P = 0.013 and P = 0.048, respectively). The miRNA miR-126, a negative regulator of VCAM-1 expression, was significantly decreased (3.39-fold; P < 0.006) in patients with short duration of untreated disease compared to controls, while miR-126 expression in patients with long duration of untreated disease did not differ significantly compared to controls. CONCLUSION: In patients with short duration of untreated disease, miR-126 down-regulation is associated with increased VCAM-1 in both muscle and blood, suggesting that VCAM-1 plays a critical role early in juvenile DM disease pathophysiology, augmented by TNFα.

Methylation alterations of WT1 and homeobox genes in inflamed muscle biopsy samples from patients with untreated juvenile dermatomyositis suggest self-renewal capacity.

OBJECTIVE: To determine the effect of methylation alteration in inflamed muscles from children with juvenile dermatomyositis (DM) and other idiopathic inflammatory myopathies (IIMs). METHODS: Magnetic resonance imaging-directed diagnostic muscle biopsies yielded samples from 20 children with juvenile DM, which were used for genome-wide DNA methylation profiling, as were muscle biopsy samples from 4 healthy controls. Bisulfite treatment followed by pyrosequencing confirmed methylation status in juvenile DM and other IIMs. Immunohistochemistry defined localization and expression levels of WT1. RESULTS: Comparison of genome-wide DNA methylation profiling between juvenile DM muscle and normal control muscle revealed 27 genes with a significant methylation difference between the groups. These genes were enriched with transcription factors and/or cell cycle regulators and were unrelated to duration of untreated disease. Six homeobox genes were among them; ALX4, HOXC11, HOXD3, and HOXD4 were hypomethylated, while EMX2 and HOXB1 were hypermethylated. WT1 was significantly hypomethylated in juvenile DM (Δß = -0.41, P < 0.001). Bisulfite pyrosequencing verification in samples from 56 patients with juvenile DM confirmed the methylation alterations of these genes. Similar methylation alterations were observed in juvenile polymyositis (n = 5) and other IIMs (n = 9). Concordant with the other findings, WT1 protein was increased in juvenile DM muscle, with average positive staining of 11.6%, but was undetectable in normal muscle (P < 0.001). CONCLUSION: These results suggest that affected muscles of children with juvenile DM and IIMs have the capacity to be repaired, and that homeobox and WT1 genes are epigenetically marked to facilitate this repair process, potentially suggesting new avenues of therapeutic intervention.

Ovarian teratoma mimicking features of juvenile dermatomyositis in a child.

An 8-year-old girl complained for 4 months of right arm pain, weakness in both legs, difficulty in arising from a seated or squatting position, and 1 month of pain in her hips, ankles, and knees. On physical examination, she had weak neck flexors, weak proximal and abdominal muscles, and an assisted Gower maneuver; both knees and ankles were painful. Erythematous macules on her elbows, knees, and medial ankles were present without heliotrope rash or dilated eyelid capillaries. She had nail-fold erythema and decreased numbers of nail-fold capillary end-row loops (ERLs) (5.42 ERLs per mm [normal: ≥6.8 ERLs per mm]) without digital ulcers or tight skin. Laboratory testing revealed slightly elevated creatine phosphokinase (440 IU/L [normal: ≤199 IU/L]) and aldolase (11.7 U/L [normal: ≤8.6 U/L]) levels. Her eosinophilia (7.2%) was not characteristic of juvenile dermatomyositis. Rheumatologic evaluation included a positive antinuclear antibody test result (1:5120 titer), speckled pattern (normal: <80 titer), myositis-associated and -specific antibodies that showed indeterminate Mi-2, with the others negative, including p155/140, elevated immunoglobulin G (IgG) (1440 mg/dL [normal range: 608-1229]) and IgE (409 kU/L [normal: <160 kU/L]) levels, and normal levels of IgM and IgA. She had an increased neopterin level (20 nm/L [normal: <10 nm/L]) and decreased absolute count of CD3-CD56/16(+) natural killer cells (89 [lower normal limit: 138]). MRI of her thigh muscles revealed serpiginous increased T-2 signals consistent with inflammation and a complex round mass in the left pelvis. A muscle biopsy did not indicate juvenile dermatomyositis. Pelvic ultrasound confirmed a solid mass of the left ovary consistent with a mature teratoma. After surgical removal of the teratoma, the myositis, synovitis, and cutaneous findings resolved over 4 months without further therapy.

Familial aggregation of autoimmune disease in juvenile dermatomyositis.

OBJECTIVE: Familial aggregation of autoimmune diseases likely reflects shared pathogenic factors between different diseases. Familial aggregation of autoimmunity has not been examined in juvenile dermatomyositis. Interferon-α is thought to be a pathogenic factor in both systemic lupus erythematosus and juvenile dermatomyositis, and we have previously demonstrated familial aggregation of serum interferon-α. METHODS: Family histories were obtained from 304 families of children with juvenile dermatomyositis via 3-generation structured interviews performed by the same person. Rates of autoimmune disease in families of children with juvenile dermatomyositis were compared with published population rates. Serum interferon-α, tumor necrosis factor-α, and neopterin were measured using standard techniques. RESULTS: A total of 51% of families of children with juvenile dermatomyositis reported at least 1 additional member affected by an autoimmune disease. In particular, both type 1 diabetes and systemic lupus erythematosus were significantly more common than would be expected (odds ratio >5, P ≤ 1 × 10(-7) for both). Pedigree analysis showed particularly strong familial clustering of systemic lupus erythematosus with little decrease in incidence across generations, suggesting the possibility of rare causal genes with large effect. Untreated subjects with juvenile dermatomyositis with a family history of systemic lupus erythematosus had higher serum interferon-α than those who did not (P = .047). CONCLUSIONS: We find strong familial aggregation of specific autoimmune diseases in families of children with juvenile dermatomyositis, suggesting that these conditions share pathogenic factors. Higher serum interferon-α in juvenile dermatomyositis patients with a family history of systemic lupus erythematosus suggesting that interferon-α is one such shared factor.

Double trouble: therapeutic challenges in patients with both juvenile dermatomyositis and psoriasis.

BACKGROUND: Juvenile dermatomyositis (JDM) and psoriasis are inflammatory disorders that share interferon-α induced responses and dysregulation of cytokines, including tumor necrosis factor alpha. Although 13% of patients with JDM have a family history of psoriasis there is little information concerning children with JDM and psoriasis. OBSERVATIONS: We identified 3 children with both JDM and psoriasis. In 2 cases, psoriatic lesions occurred after the child's JDM symptoms had diminished following effective immunosuppressive therapy (high-dose intermittent intravenous methylprednisolone, methotrexate, and low dose oral corticosteroids). Patient 2, initially diagnosed as having psoriasis, was treated with prednisone and methotrexate but then developed classic JDM, which worsened following use of tumor necrosis factor alpha inhibitor and reduction of prednisone and methotrexate dosage. For each child, their history of JDM complicated the choice of therapy for psoriasis. CONCLUSIONS: Two therapies commonly used to treat psoriasis-phototherapy and tumor necrosis factor-alpha antagonists-must be used with caution in patients with both JDM and psoriasis owing to their potential to exacerbate clinical manifestations of JDM. We discuss the implications affecting treatment of children with these dual diagnoses and consider the pathophysiology linking these 2 conditions.

Serum neopterin levels as a diagnostic marker of hemophagocytic lymphohistiocytosis syndrome.

The objective of this study was to retrospectively evaluate the utility of serum neopterin as a diagnostic marker of hemophagocytic lymphohistiocytosis (HLH). The medical records of patients diagnosed with HLH (familial and secondary) between January 2000 and May 2009 were reviewed retrospectively, and clinical and laboratory information related to HLH criteria, in addition to neopterin levels, was recorded. A group of 50 patients with active juvenile dermatomyositis (JDM) (who routinely have neopterin levels assessed) served as controls for the assessment of the accuracy, sensitivity, and specificity of neopterin as a diagnostic test for HLH. The Pearson correlation was used to measure the association between serum neopterin levels and established HLH-related laboratory data. Serum neopterin levels were measured using a competitive enzyme immunoassay. During the time frame of the study, 3 patients with familial HLH and 18 patients with secondary HLH were identified as having had serum neopterin measured (all HLH patients were grouped together). The mean neopterin levels were 84.9 nmol/liter (standard deviation [SD], 83.4 nmol/liter) for patients with HLH and 21.5 nmol/liter (SD, 10.13 nmol/liter) for patients with JDM. A cutoff value of 38.9 nmol/liter was 70% sensitive and 95% specific for HLH. For HLH patients, neopterin levels correlated significantly with ferritin levels (r = 0.76, P = 0.0007). In comparison to the level in a control group of JDM patients, elevated serum neopterin was a sensitive and specific marker for HLH. Serum neopterin has value as a diagnostic marker of HLH, and prospective studies are under way to further evaluate its role as a marker for early diagnosis and management of patients.