Specific autoantibody profiles and disease subgroups correlate with circulating micro-RNA in systemic sclerosis.

OBJECTIVE: To evaluate the expression profiles of cell-free plasma miRNAs in SSc and to characterize their correlation with disease subgroups (lcSSc and dcSSc) and with autoantibody profiles. METHODS: Using quantitative RT-PCR, the abundance of 45 mature miRNAs in plasma was determined in 95 patients (lcSSc = 63; dcSSc = 32), representing the following autoantibody subgroups: ACA, anti-DNA topoisomerase I, anti-RNA polymerase III and anti-U1-ribonucleoprotein. MiRNA data were correlated with clinical and paraclinical data. Multiple regression was used to model membership of the lcSSc, dcSSc and autoantibody subgroups, based on miRNA expression profiles. RESULTS: Thirty-six miRNAs were measurable in all samples. Four (miRNA-223, -181b, -342-3p and -184) were differently expressed in lcSSc and dcSSc (false discovery rate < 0.05). Ten miRNAs exhibited statistically significantly different levels in one or more autoantibody groups, and five (miRNA-409, -184, -92a, -29a and -101) remained significant after correction for multiple comparisons. Multiple regression models accurately predicted ACA and anti-DNA topoisomerase I antibody-positive patients (area under the curve (AUC) = 0.97 and 0.93, respectively) as well as membership of the dcSSc and lcSSc groups (AUC = 0.88). CONCLUSION: Circulating miRNA profiles differ between lcSSc and dcSSc patients and between patients with different autoantibodies. This is the first time autoantibody profiles, disease phenotypes and plasma miRNA profiles have been shown to correlate in an autoimmune disease. The data support a pathobiological role of miRNAs because specific miRNAs associate with autoantibody profiles of known diagnostic and prognostic value.

Circulating microRNA expression profiles associated with systemic lupus erythematosus.

OBJECTIVE: To evaluate the specificity of expression patterns of cell-free circulating microRNAs (miRNAs) in systemic lupus erythematosus (SLE). METHODS: Total RNA was purified from plasma, and 45 different specific, mature miRNAs were determined using quantitative reverse transcription-polymerase chain reaction assays. A total of 409 plasma samples were obtained from 364 different patients with SLE, healthy control subjects, and control subjects with other autoimmune diseases. The results in the primary cohort of 62 patients with SLE and 29 healthy control subjects were validated in 2 independent cohorts: a validation cohort comprising 68 patients with SLE and 68 healthy control subjects, and a disease control cohort comprising 20 patients with SLE (19 of whom were from the other validation cohort), 46 healthy control subjects, 38 patients with vasculitis, 18 patients with rheumatoid arthritis, and 20 immunosuppressed patients. RESULTS: Seven miRNAs were statistically significantly differentially expressed in plasma from patients with SLE. The expression of miRNA-142-3p (miR-142-3p) and miR-181a was increased, and the expression of miR-106a, miR-17, miR-20a, miR-203, and miR-92a was decreased. In addition, the expression of miR-342-3p, miR-223, and miR-20a was significantly decreased in SLE patients with active nephritis. A predictive model for SLE based on 2 or 4 miRNAs differentiated patients with SLE from control subjects (76% accuracy) when validated independently (P < 2 × 10(-9) ). Use of the 4-miRNA model provided highly significant differentiation between the SLE group and disease controls, except for those with vasculitis. CONCLUSION: Circulating miRNAs are systematically altered in SLE. A 4-miRNA signature was diagnostic of SLE, and a specific subset of miRNA profiles was associated with nephritis. All of the signature miRNAs target genes in the transforming growth factor ß signaling pathways. Other targets include regulation of apoptosis, cytokine-cytokine receptors, T cell development, and cytoskeletal organization. These findings highlight possible dysregulated pathways in SLE and suggest that circulating miRNA patterns distinguish SLE from other immunoinflammatory phenotypes.

Free leptin index and PAPP-A: a first trimester maternal serum screening test for pre-eclampsia.

BACKGROUND: Prophylaxis with low-dose aspirin may reduce the risk of pre-eclampsia (PE) if introduced in first trimester. The performance of first trimester maternal serum screening for PE using free leptin index (fLI) and PAPP-A, where fLI = leptin/leptin soluble receptor was studied. METHODS: First trimester serum samples from 126 PE pregnancies and 289 control pregnancies were studied. fLI and PAPP-A were converted into gestational age and maternal weight independent log MoM values of PAPP-A and fLI. The screening performance of markers was studied by receiver-operator-characteristics curves. The performance of population screening was estimated by Monte Carlo simulation. RESULTS: fLI was significantly (p < 0.001) elevated [mean log MoM 0.2165 (SD: 0.2604)] compared to controls [mean log MoM -0.0368 (SD: 0.3132)] and PAPP-A was significantly (p < 0.001) reduced [mean log MoM -0.0133 (SD: 0.2661)] compared to controls [mean log MoM 0.0474 (SD: 0.2521)] in PE pregnancies. There was no correlation between fLI and PAPP-A in control or PE pregnancies. Combined fLI and PAPP-A screening for PE had estimated population detection rates of 22% and 35% for false positives rates of 6% and 12%, respectively. CONCLUSION: Combining PAPP-A and fLI improves screening performance for PE compared to single marker screening.

MicroRNA-126 and epidermal growth factor-like domain 7 predict recurrence in patients with colon cancer treated with neoadjuvant chemotherapy.

Aim: Neoadjuvant chemotherapy may represent a shift in the treatment of locally advanced colon cancer. The angiogenic couple has-microRNA-126 (miRNA-126) and epidermal growth factor-like domain 7 (EGFL7) are transcribed from the same gene and regulates all aspects of angiogenesis and may influence the ability of tumor cells to disseminate. The aim was to analyze the relationship between miRNA-126 and EGFL7 and disease recurrence in patients with locally advanced colon cancer treated with neoadjuvant chemotherapy. Methods: This study included 71 patients from a phase II study all planned for three cycles of capecitabine and oxaliplatin before surgery. Blood was sampled at baseline and right before and after the operation. Circulating miRNA-126 was analysed by RT-qPCR and a quantitative immunoassay was used for the analyses of EGFL7. Results: The rates of 5-year disease-free survival (DFS) and overall survival (OS) were 80% and 85%, respectively. The level of circulating miRNA-126 before the operation predicts recurrence, P = 0.035. In patients with values below and above the median the recurrence rate was 31% and 4%, respectively. Similar results applied to EGFL7. A combined estimate identified a subgroup of patients (25 of 71) with no recurrence and a 5-year DFS and OS rate of 100%, respectively. Conclusion: MicroRNA-126 and EGFL7 are predictors for disease recurrence in patients with locally advanced colon cancer treated with neoadjuvant chemotherapy and may assist in selection of adjuvant chemotherapy.

Diagnostic plasma miRNA-profiles for ovarian cancer in patients with pelvic mass.

BACKGROUND: Ovarian cancer is the fifth most common cancer in women worldwide. Moreover, there are no reliable minimal invasive tests to secure the diagnosis of malignant pelvic masses. Cell-free, circulating microRNAs have the potential as diagnostic biomarkers in cancer. Here, we performed and validated a miRNA panel with the potential to distinguish OC from benign pelvic masses. METHODS: The profile of plasma microRNA was determined with a panel of 46 candidates in a discovery group and a validation group, each consisting of 190 pre-surgery plasma samples from age-matched patients with malignant (n = 95) and benign pelvic mass (n = 95), by real time RT-qPCR. RESULTS: Four up-regulated (miR-200c-3p, miR-221-3p, miR-21-5p, and miR-484) and two down-regulated (miR-195-5p and miR-451a) microRNAs were discovered. From those, miR-200c-3p and miR-221-3p were further confirmed in a validation cohort. A combination of these 2 microRNAs together with CA-125 yielded an overall diagnostic accuracy of AUC = 0.96. CONCLUSIONS: We showed consistent plasma microRNA profiles that provide independent diagnostic information of late stage OC.

Plasma MicroRNA Profiles in Patients with Early Rheumatoid Arthritis Responding to Adalimumab plus Methotrexate vs Methotrexate Alone: A Placebo-controlled Clinical Trial.

OBJECTIVE: The aim was to identify plasma (i.e., cell-free) microRNA (miRNA) predicting antitumor necrosis and/or methotrexate (MTX) treatment response in patients enrolled in an investigator-initiated, prospective, double-blinded, placebo-controlled trial (The OPERA study, NCT00660647). METHODS: We included 180 disease-modifying antirheumatic drug-naive patients with early rheumatoid arthritis (RA) randomized to adalimumab (ADA; n = 89) or placebo (n = 91) in combination with MTX. Plasma samples before and 3 months after treatment initiation were analyzed for 91 specific miRNA by quantitative reverse transcriptase-polymerase chain reaction on microfluidic dynamic arrays. A linear mixed-effects model was used to test for associations between pretreatment miRNA and changes in miRNA expression and American College of Rheumatology/European League Against Rheumatism (ACR/EULAR) Boolean (28 joints) remission at 3 and 12 months, applying false discovery rate correction for multiple testing. Using leave-one-out cross validation, we built predictive multivariate miRNA models and estimated classification performances using receiver-operating characteristics (ROC) curves. RESULTS: In the ADA group, a higher pretreatment level of miR-27a-3p was significantly associated with remission at 12 months. The level decreased in remitting patients between pretreatment and 3 months, and increased in nonremitting patients. No associations were found in the placebo group receiving only MTX. Two multivariate miRNA models were able to predict response to ADA treatment after 3 and 12 months, with 63% and 82% area under the ROC curves, respectively. CONCLUSION: We identified miR-27a-3p as a potential predictive biomarker of ACR/EULAR remission in patients with early RA treated with ADA in combination with MTX. We conclude that pretreatment plasma-miRNA profiles may be of predictive value, but the results need confirmation in independent cohorts.

Elevation of brain-enriched miRNAs in cerebrospinal fluid of patients with acute ischemic stroke.

BACKGROUND: The purpose of this study was to investigate the potential of cerebrospinal fluid miRNAs as diagnostic biomarkers of acute ischemic stroke using three different profiling techniques in order to identify and bypass any influence from technical variation. METHODS: Cerebrospinal fluid (CSF) from patients with acute ischemic stroke (n = 21) and controls (n = 21) was collected by lumbar puncture. miRNA analysis was performed with three different methods: 1) Trizol RNA extraction followed by Illumina Next Generation Sequencing (NGS) on all small RNAs, 2) Exiqon RNA extraction protocol and miRNA qPCR assays, and 3) validation of 24 selected miRNAs with Norgen Biotek RNA extraction protocol and Applied Biosystems qPCR assays. RESULTS: NGS detected 71 frequently expressed miRNAs in CSF of which brain-enriched miR-9-5p and miR-128-3p were significantly higher in CSF of stroke patients compared to controls. When dividing stroke patients into groups according to infarct size several brain-enriched miRNAs (miR-9-5p, miR-9-3p, miR-124-3p, and miR-128-3p) were elevated in patients with infarcts >2 cm3. This trend appeared in data from both NGS, qPCR (Exiqon), and qPCR (Applied Biosystems) but was only statistically significant in some of the measurement platforms. CONCLUSIONS: Several brain-enriched miRNAs are elevated in the CSF three days after stroke onset, suggesting that these miRNAs reflect the brain damage caused by ischemia. The expression differences seem, however, limited to patients with larger ischemic brain injury, which argues against the use of CSF miRNAs as diagnostic biomarkers of stroke based on current methods.

Diurnal Variations of Human Circulating Cell-Free Micro-RNA.

A 24-hour light and dark cycle-dependent rhythmicity pervades physiological processes in virtually all living organisms including humans. These regular oscillations are caused by external cues to endogenous, independent biological time-keeping systems (clocks). The rhythm is reflected by gene expression that varies in a circadian and specific fashion in different organs and tissues and is regulated largely by dynamic epigenetic and post-transcriptional mechanisms. This leads to well-documented oscillations of specific electrolytes, hormones, metabolites, and plasma proteins in blood samples. An emerging, important class of gene regulators is short single-stranded RNA (micro-RNA, miRNA) that interferes post-transcriptionally with gene expression and thus may play a role in the circadian variation of gene expression. MiRNAs are promising biomarkers by virtue of their disease-specific tissue expression and because of their presence as stable entities in the circulation. However, no studies have addressed the putative circadian rhythmicity of circulating, cell-free miRNAs. This question is important both for using miRNAs as biological markers and for clues to miRNA function in the regulation of circadian gene expression. Here, we investigate 92 miRNAs in plasma samples from 24 young male, healthy volunteers repeatedly sampled 9 times during a 24-hour stay in a regulated environment. We demonstrate that a third (26/79) of the measurable plasma miRNAs (using RT-qPCR on a microfluidic system) exhibit a rhythmic behavior and are distributed in two main phase patterns. Some of these miRNAs weakly target known clock genes and many have strong targets in intracellular MAPK signaling pathways. These novel findings highlight the importance of considering bio-oscillations in miRNA biomarker studies and suggest the further study of a set of specific circulating miRNAs in the regulation and functioning of biological clocks.

Circulating Extracellular microRNA in Systemic Autoimmunity.

MicroRNAs (miRNAs) are differentially regulated in healthy, activated, inflamed, neoplastic, or otherwise pathological cells and tissues. While their main functions are executed intracellularly, many miRNAs can reproducibly be detected extracellularly in plasma and serum. This circulating, extracellular miRNA is protected against degradation by complexation with carrier proteins and/or by being enclosed in subcellular membrane vesicles. This, together with their tissue- and disease-specific expression, has fuelled the interest in using circulating microRNA profiles as harbingers of disease, i.e., as diagnostic analytes and as clues to dysregulated pathways in disease. Many studies show that inflammation and immune dysregulation, e.g., in autoimmune diseases, are associated with distinct miRNA expression changes in targeted tissues and in innate and adaptive immunity cells such as lymphocytes, natural killer cells, neutrophil granulocytes, and monocyte-macrophages. Exploratory studies (only validated in a few cases) also show that specific profiles of circulating miRNAs are associated with different systemic autoimmune diseases including systemic lupus erythematosus (SLE), systemic sclerosis, and rheumatoid arthritis. Even though the link between cellular alterations and extracellular profiles is still unpredictable, the data suggest that circulating miRNAs in autoimmunity may become diagnostically useful. Here, we review important circulating miRNAs in animal models of inflammation and in systemic autoimmunity and summarize some proposed functions of miRNAs in immune regulation and dysregulation. We conclude that the studies suggest new hypotheses and additional experiments, and that further diagnostic development is highly dependent on analytical method development and on obtaining sufficient numbers of uniformly processed samples from clinically well-characterized patients and controls.

The circulating cell-free microRNA profile in systemic sclerosis is distinct from both healthy controls and systemic lupus erythematosus.

OBJECTIVE: To evaluate the expression profile of cell-free circulating microRNA (miRNA) in systemic sclerosis (SSc), healthy controls (HC), and systemic lupus erythematosus (SLE). METHODS: Total RNA was purified from plasma and 45 different, mature miRNA were measured using quantitative PCR assays after reverse transcription. Samples (n = 189) were from patients with SSc (n = 120), SLE (n = 29), and from HC (n = 40). Expression data were clustered by principal components analysis, and diagnostically specific miRNA profiles were developed by leave-one-out cross-validation. Diagnostic probability scores were derived from stepwise logistic regression. RESULTS: Thirty-seven miRNA specificities were consistently detected and 26 of these were unaffected by SSc sample age and present in more than two-thirds of SSc samples. SSc cases showed a distinct expression profile with 14/26 miRNA significantly decreased (false discovery rate < 0.05) and 5/26 increased compared with HC. A 21-miRNA classifier gave optimum accuracy (80%) for discriminating SSc from both HC and SLE. The discrimination between HC and SSc (95% accuracy) was strongly driven by miRNA of the 17 ∼ 92 cluster and by miR-16, -223, and -638, while SLE and SSc differed mainly in the expression of miR-142-3p, -150, -223, and -638. Except for a weak correlation between anti-Scl-70 and miR-638 (p = 0.048), there were no correlations with other patient variables. CONCLUSION: Circulating miRNA profiles are characteristic for SSc compared with both HC and SLE cases. Some of the predicted targets of the differentially regulated miRNA are of relevance for transforming growth factor-ß signaling and fibrosis, but need to be validated in independent studies.

Associations between gastrointestinal toxicity, micro RNA and cytokine production in patients undergoing myeloablative allogeneic stem cell transplantation.

Allogeneic hematopoietic stem cell transplantation (HSCT) is a procedure with a high risk of treatment related mortality. The primary aim of the present study was to examine associations between markers of gastrointestinal toxicity, markers of systemic inflammation, and plasma levels of microRNA (miRNA) -155 and -146a during the first month after HSCT. The secondary aim was to characterize the impact of the toxic-inflammatory response on the function of circulating leukocytes during immune recovery. Thirty HSCT patients were included. Gastrointestinal injury was monitored by toxicity scores, lactulose-mannitol test and plasma citrulline, as a measure of the enterocyte population. Nadir of citrulline and maximum of oral toxicity scores, intestinal permeability, CRP and plasma levels of IL-6 and IL-10 was seen at day +7 post-HSCT. miRNA-155 and mi-RNA-146a showed an inverse relation with significantly elevated miRNA-155 and decreased miRNA-146a levels, from day 0 to day +28 compared with pre-conditioning levels. Citrulline levels below the median at day +7 were associated with higher spontaneous production of IL-6 and TNF-α as well as higher in vitro stimulated production of IL-17A at day +21. This study is the first to demonstrate that toxic responses to chemotherapy are accompanied by differential regulation of miRNAs with opposing effects on immune regulation. We find that a proinflammatory miRNA profile is sustained during the first three weeks after the transplantation, indicating that these miRNAs may play a role in the regulation of the inflammatory environment during immune reconstitution after HSCT.

Cell-free plasma microRNA in pancreatic ductal adenocarcinoma and disease controls.

OBJECTIVES: There are no tumor-specific biochemical markers for pancreatic ductal adenocarcinoma (PDAC). Tissue-specific gene expression including microRNA (miRNA) profiling, however, identifies specific PDAC signatures. This study evaluates associations between circulating, cell-free plasma-miRNA profiles and PDAC in a disease and disease-control cohort. METHODS: We performed a microarray profiling of 847 different mature miRNAs from plasma in an exploratory cohort of 20 patients with PDAC or other pancreatic diseases, profiling of 45 miRNAs in plasma samples from PDAC (n = 48) and disease controls (n = 47), and evaluation of associations of data with diagnosis, survival, and CA-19-9. RESULTS: We find 7 significantly deregulated miRNAs in PDAC using univariate statistics. At a false-discovery rate of 5%, miRNA-375 remained significantly elevated in PDAC. MicroRNA-375 did not improve diagnosis of PDAC in this cohort (70% accuracy) and did not correlate with survival. However, 3 controls (other gastrointestinal cancers) with increased CA-19-9 did not show increased miRNA-375. CONCLUSIONS: In the plasma-miRNA population, we find miRNA-375, which is selectively expressed in the endocrine pancreas under normal conditions, increased in PDAC cases compared with patients with other pancreatic or gastrointestinal diseases. The miRNA-375 does not outperform CA-19-9 diagnostically in the present cohort. However, it shows promising specificity and should be examined in larger prospective studies.

Peptide nucleic acid antisense prolongs skin allograft survival by means of blockade of CXCR3 expression directing T cells into graft.

CXCR3, predominantly expressed on memory/activated T cells, is a receptor for both IFN-gamma-inducible protein 10/CXC chemokine ligand (CXCL)10 and monokine induced by IFN-gamma/CXCL9. It was reported that CXC chemokines IFN-gamma-inducible protein 10/CXCL10 and monokine induced by IFN-gamma/CXCL9 play a critical role in the allograft rejection. We report that CXCR3 is a dominant factor directing T cells into mouse skin allograft, and that peptide nucleic acid (PNA) CXCR3 antisense significantly prolongs skin allograft survival by means of blockade of CXCR3 expression directing T cells into allografts in mice. We found that CXCR3 is highly up-regulated in spleen T cells and allografts from BALB/c recipients by day 7 of receiving transplantation, whereas CCR5 expression is moderately increased. We designed PNA CCR5 and PNA CXCR3 antisenses, and i.v. treated mice that received skin allograft transplantations. The PNA CXCR3 at a dosage of 10 mg/kg/day significantly prolonged mouse skin allograft survival (17.1 +/- 2.4 days) compared with physiological saline treatment (7.5 +/- 0.7 days), whereas PNA CCR5 (10 mg/kg/day) marginally prolonged skin allograft survival (10.7 +/- 1.1 days). The mechanism of prolongation of skin allograft survival is that PNA CXCR3 directly blocks the CXCR3 expression in T cells, which is responsible for directing T cells into skin allograft to induce acute rejection, without interfering with other functions of the T cells. These results were obtained at mRNA and protein levels by flow cytometry and real-time quantitative RT-PCR technique, and confirmed by chemotaxis, Northern and Western blot assays, and histological evaluation of skin grafts. The present study indicates the therapeutic potential of PNA CXCR3 to prevent acute transplantation rejection.

CCR3 expression induced by IL-2 and IL-4 functioning as a death receptor for B cells.

We report that CCR3 is not expressed on freshly isolated peripheral and germinal B cells, but is up-regulated after stimulation with IL-2 and IL-4 (approximately 98% CCR3(+)). Ligation of CCR3 by eotaxin/chemokine ligand (CCL) 11 induces apoptosis in IL-2- and IL-4-stimulated primary CD19(+) (approximately 40% apoptotic cells) B cell cultures as well as B cell lines, but has no effect on chemotaxis or cell adhesion. Freshly isolated B cells express low levels of CD95 and CD95 ligand (CD95L) (19 and 21%, respectively). Expression is up-regulated on culture in the presence of a combination of IL-2, IL-4, and eotaxin/CCL11 (88% CD95 and 84% CD95L). We therefore propose that ligation of such newly induced CCR3 on peripheral and germinal B cells by eotaxin/CCL11 leads to the enhanced levels of CD95 and CD95L expression. Ligation of CD95 by its CD95L expressed on neigboring B cells triggers relevant death signaling pathways, which include an increase in levels of Bcl-2 expression, its functional activity, and the release of cytochrome c from the mitochondria into the cytosol. These events initiate a cascade of enzymatic processes of the caspase family, culminating in programmed cell death. Interaction between CCR3 and eotaxin/CCL11 may, besides promoting allergic reactions, drive activated B cells to apoptosis, thereby reducing levels of Ig production, including IgE, and consequently limit the development of the humoral immune response. The apoptotic action of eotaxin/CCL11 suggests a therapeutic modality in the treatment of B cell lymphoma.