Randomised prospective trial to assess the clinical utility of multianalyte assay panel with complement activation products for the diagnosis of SLE.

Objective: We compared the physician-assessed diagnostic likelihood of SLE resulting from standard diagnosis laboratory testing (SDLT) to that resulting from multianalyte assay panel (MAP) with cell-bound complement activation products (MAP/CB-CAPs), which reports a two-tiered index test result having 80% sensitivity and 86% specificity for SLE. Methods: Patients (n=145) with a history of positive antinuclear antibody status were evaluated clinically by rheumatologists and randomised to SDLT arm (tests ordered at the discretion of the rheumatologists) or to MAP/CB-CAPs testing arm. The primary endpoint was based on the change in the physician likelihood of SLE on a five-point Likert scale collected before and after testing. Changes in pharmacological treatment based on laboratory results were assessed in both arms. Statistical analysis consisted of Wilcoxon and Fisher\u27s exact tests. Results: At enrolment, patients randomised to SDLT (n=73, age=48±2 years, 94% females) and MAP/CB-CAPs testing arms (n=72, 50±2 years, 93% females) presented with similar pretest likelihood of SLE (1.42±0.06 vs 1.46±0.06 points, respectively; p=0.68). Post-test likelihood of SLE resulting from randomisation in the MAP/CB-CAPs testing arm was significantly lower than that resulting from randomisation to SDLT arm on review of test results (-0.44±0.10 points vs -0.19±0.07 points) and at the 12-week follow-up visit (-0.61±0.10 points vs -0.31±0.10 points) (p Conclusion: Our data suggest that MAP/CB-CAPs testing has clinical utility in facilitating SLE diagnosis and treatment decisions

Micro-RNA analysis of renal biopsies in human lupus nephritis demonstrates up-regulated miR-422a driving reduction of kallikrein-related peptidase 4

Aberrancies in gene expression in immune effector cells and in end-organs are implicated in lupus pathogenesis. To gain insights into the mechanisms of tissue injury, we profiled the expression of micro-RNAs in inflammatory kidney lesions of human lupus nephritis (LN). Kidney specimens were from patients with active proliferative, membranous or mixed LN and unaffected control tissue. Micro-RNAs were quantified by TaqMan Low Density Arrays. Bioinformatics was employed to predict gene targets, gene networks and perturbed signaling pathways. Results were validated by transfection studies (luciferase assay, real-time PCR) and in murine LN. Protein expression was determined by immunoblotting and immunohistochemistry. Twenty-four micro-RNAs were dysregulated (9 up-regulated, 15 down-regulated) in human LN compared with control renal tissue. Their predicted gene targets participated in pathways associated with TGF-beta, kinases, NF-kappa B, HNF4A, Wnt/beta-catenin, STAT3 and IL-4. miR-422a showed the highest upregulation (17-fold) in active LN and correlated with fibrinoid necrosis lesions (beta = 0.63, P = 0.002). In transfection studies, miR-422a was found to directly target kallikrein-related peptidase 4 (KLK4) mRNA. Concordantly, KLK4 mRNA was significantly reduced in the kidneys of human and murine LN and correlated inversely with miR-422a levels. Immunohistochemistry confirmed reduced KLK4 protein expression in renal mesangial and tubular epithelial cells in human and murine LN. KLK4, a serine esterase with putative renoprotective properties, is down-regulated by miR-422a in LN kidney suggesting that, in addition to immune activation, local factors may be implicated in the disease

Genome-wide identification of transcripts stabilized by TNF in RA FLS.

<p>Two biologic replicates of RA FLS (derived from two different RA patients) were exposed to a single dose of TNF (10 ng/ml) for 1 or 72h. Subsequently, Act D was added for 3h and gene expression was measured by RNA sequencing. The degree of TNF-induced mRNA stabilization was calculated as the log₂ difference of TNF+Act D/TNF ratio between 1 and 72h of TNF stimulation and the adjusted p values of TNF-induced stabilization were calculated by RiboDiff. (a), Scatter-plot of the genes displaying TNF-induced mRNA stabilization comparing the degree of mRNA stabilization (y axis) to the adjusted p values of the stabilizing effect of TNF (x-axis). (b), The top 40 genes displaying the highest TNF-induced mRNA stabilization ranked by the degree of stabilization. (c), Enriched biological processes identified by GSEA/MSigDB pathway analysis of the top 10% of the genes (n = 593) displaying the highest degree of TNF-induced mRNA stabilization.</p

Scatterplots comparing the expression levels to the mRNA stability states of the expressed genes in RA FLS.

<p>Two biological replicates of RA FLS (derived from two different RA patients) were exposed to a single dose of TNF (10 ng/ml) for 1, 3, 24, or 72 hours. Subsequently, actinomycin D (Act D, 10μg/ml) was added for 3 hours to block active transcription and gene expression was measured by RNA sequencing. RPKM values were generated using CuffDiff2. The mRNA stability status was calculated genome-wide as the ratio of RPKM levels at the TNF+Act D condition divided to the RPKM levels at the TNF condition. This ratio ranges from 0 to 1 and classifies genes to a spectrum from very unstable to very stable transcripts. The genes expressed at 1 (a), 3 (b), 24 (c), and 72 (d) hours of TNF stimulation were plotted based on their expression levels and the mRNA stability states. Shades of blue represent the region of unstable genes, and shades of red represent the zone of stable genes.</p

TNF induces late stabilization of IL-6 mRNA in RA FLS.

<p>RA FLS were exposed to a single dose of TNF (10 ng/ml) for 1-72h. Subsequently, actinomycin D (Act D; 10 μg/ml) or triptolide (1 μM), or flavopiridol (Flav; 0.5 μM) was added for 1 or 3h and qPCR was used to measure the mRNA levels of IL-6 (a-c), the primary transcripts (PT) of <i>IL6</i> (d-e), and the mRNA levels of CCL5 (f). For (b-c), the remaining expression of IL-6 after exposure to inhibitors of transcription (Act D, triptolide, and flavopiridol) was calculated as % of the IL-6 mRNA expressed in the absence of inhibitor at the corresponding TNF-stimulated condition. For (a-b) and (f), cumulative results from 7 independent experiments are shown. For (d-e), FLS were exposed to a single dose of TNF (10 ng/ml) for 72 hours and then inhibitors (Act D or flavopiridol) were added for the indicated time points to block active transcription. Primers specific for the fourth intronic region of <i>IL6</i> were designed to capture primary transcripts of <i>IL6</i>.Values were normalized relative to GAPDH mRNA and presented as mean ±SEM. GAPDH was considered an appropriate internal control for normalizing qPCR results since TNF stimulation had no impact on expression levels and stability status of GAPDH mRNA (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179762#pone.0179762.s001" target="_blank">S1 Table</a>). <i>P</i> values were calculated by one-way ANOVA and Tukey post-test analysis (* = <i>p</i><0.05, ** = <i>p</i><0.01, *** = <i>p</i><0.001, ns = not significant, and ND = not detected).</p