Blood RNA Samples Research Articles

Whole blood gene expression analyses in patients with single versus recurrent venous thromboembolism

Introduction Venous thromboembolism may recur in up to 30% of patients with a spontaneous venous thromboembolism after a standard course of anticoagulation. Identification of patients at risk for recurrent venous thromboembolism would facilitate decisions concerning the duration of anticoagulant therapy. Objectives In this exploratory study, we investigated whether whole blood gene expression data could distinguish subjects with single venous thromboembolism from subjects with recurrent venous thromboembolism. Methods 40 adults with venous thromboembolism (23 with single event and 17 with recurrent events) on warfarin were recruited. Individuals with antiphospholipid syndrome or cancer were excluded. Plasma and serum samples were collected for biomarker testing, and PAXgene tubes were used to collect whole blood RNA samples. Results D-dimer levels were significantly higher in patients with recurrent venous thromboembolism, but P-selectin and thrombin-antithrombin complex levels were similar in the two groups. Comparison of gene expression data from the two groups provided us with a 50 gene probe model that distinguished these two groups with good receiver operating curve characteristics (AUC 0.75). This model includes genes involved in mRNA splicing and platelet aggregation. Pathway analysis between subjects with single and recurrent venous thromboembolism revealed that the Akt pathway was up-regulated in the recurrent venous thromboembolism group compared to the single venous thromboembolism group. Conclusions In this exploratory study, gene expression profiles of whole blood appear to be a useful strategy to distinguish subjects with single venous thromboembolism from those with recurrent venous thromboembolism. Prospective studies with additional patients are needed to validate these results.

Risk factors for ANA positivity in healthy persons.

IntroductionThe finding of antinuclear antibody (ANA) positivity in a healthy individual is usually of unknown significance and in most cases is benign. However, a subset of such individuals is at risk for development of autoimmune disease. We examined demographic and immunological features that are associated with ANA positivity in clinically healthy persons to develop insights into when this marker carries risk of progression to lupus.MethodsBiological samples from healthy individuals and patients with systemic lupus erythematosus (SLE) were obtained from the Dallas Regional Autoimmune Disease Registry (DRADR). Measurements carried out on serum samples included ANA, extractable nuclear antibodies (ENA) and autoantibody profiling using an array with more than 100 specificities. Whole blood RNA samples from a subset of individuals were used to analyze gene expression on the Illumina platform. Data were analyzed for associations of high ANA levels with demographic features, the presence of other autoantibodies and with gene expression profiles.ResultsOverall, ANA levels are significantly higher in females than in males and this association holds in patients with the autoimmune diseases lupus and rheumatoid arthritis (RA) as well as in healthy controls (HC). Age was not significantly associated with ANA levels and the elevated ANA values could not be explained by higher IgG levels. Another autoantibody, anti- cyclic citrullinated peptide (CCP), did not show gender dimorphism in rheumatoid arthritis (RA) or healthy individuals. The autoantigen array showed significant elevations of other autoantibodies in high ANA HCs. Some of these autoantibodies were directed to antigens in skin and others were related to autoimmune conditions of kidney, thyroid or joints. Gene expression analyses showed a greater prevalence of significantly upregulated genes in HCs with negative ANA values than in those with significant ANA positivity. Genes upregulated in high ANA HCs included a celiac disease autoantigen and some components of the Type I interferon (IFN) gene signature.ConclusionsRisks for ANA positivity include female gender and organ-specific autoimmunity. Upregulation of skin-specific autoantibodies may indicate that early events in the break of tolerance take place in cutaneous structures. Some of these changes may be mediated by Type I IFN. Blood profiling for expressed autoantibodies and genes has the potential to identify individuals at risk for development of autoimmune diseases including lupus.

An Eight-Gene Blood Expression Profile Predicts the Response to Infliximab in Rheumatoid Arthritis

BackgroundTNF alpha blockade agents like infliximab are actually the treatment of choice for those rheumatoid arthritis (RA) patients who fail standard therapy. However, a considerable percentage of anti-TNF alpha treated patients do not show a significant clinical response. Given that new therapies for treatment of RA have been recently approved, there is a pressing need to find a system that reliably predicts treatment response. We hypothesized that the analysis of whole blood gene expression profiles of RA patients could be used to build a robust predictor to infliximab therapy.Methods and FindingsWe performed microarray gene expression analysis on whole blood RNA samples from RA patients starting infliximab therapy (n = 44). The clinical response to infliximab was determined at week 14 using the EULAR criteria. Blood cell populations were determined using flow cytometry at baseline, week 2 and week 14 of treatment. Using complete cross-validation and repeated random sampling we identified a robust 8-gene predictor model (96.6% Leave One Out prediction accuracy, P = 0.0001). Applying this model to an independent validation set of RA patients, we estimated an 85.7% prediction accuracy (75–100%, 95% CI). In parallel, we also observed a significantly higher number of CD4+CD25+ cells (i.e. regulatory T cells) in the responder group compared to the non responder group at baseline (P = 0.0009).ConclusionsThe present 8-gene model obtained from whole blood expression efficiently predicts response to infliximab in RA patients. The application of the present system in the clinical setting could assist the clinician in the selection of the optimal treatment strategy in RA.

In vitro and in vivo modulation of the equine immune response by parapoxvirus ovis

While immune modulators are used routinely in equine medicine, their mechanism of action is not always known. To determine the effect of a commercial preparation of inactivated parapoxvirus ovis (Orf virus; PPVO) on cytokine gene expression by equine peripheral blood mononuclear cells (PBMC) both in vitro and in vivo. PBMC were prepared from 6 mixed-breed yearlings and cultured in vitro with PPVO with or without Concanavalin A (Con A) for 24 h. Effects on the expression of IFNalpha, IFNbeta IFNgamma, TNFalpha and IL-18 were analysed by real time quantitative PCR (RT-PCR). In addition, 12 yearling horses were treated with PPVO and whole blood RNA samples were prepared at regular intervals to assess effects on in vivo cytokine gene expression. Six of those yearlings were later treated with saline and served as treatment controls. Nine additional yearlings were injected intradermally with a single dose and their injection sites biopsied at 24 and 48 h for cytokine expression. In vitro culture of PBMC with PPVO led to a significant increase in IFNalpha and IFNbeta gene expression compared to mock-stimulated cultures. In addition, expression of IFNgamma and TNFalpha was significantly higher in PBMC stimulated with PPVO and Con A, than those stimulated with Con A alone. No changes were observed in IL-18 gene expression in vitro. Treatment of horses with a 3-dose regimen of PPVO resulted in elevation of IFNgamma gene expression, which was detected 24 h after the first dose and declined thereafter. Intradermal inoculation led to increased expression of IFNgamma along with IFNbeta, IL-15 and IL-18. Together these results indicate that PPVO stimulated IFNgamma production both in vitro and in vivo. Increased cytokine expression could account for its immunomodulatory activity. The absence of adverse reactions and clear indications of increased expression of cytokine gene expression supports previous clinical uses for this immune modulator in those situations when increased expression of IFNgamma is warranted.

Circulating Markers Reflect Both Anti- and Pro-Atherogenic Drug Effects in ApoE-Deficient Mice

BACKGROUND: Current drug therapy of atherosclerosis is focused on treatment of major risk factors, e.g. hypercholesterolemia while in the future direct disease modification might provide additional benefits. However, development of medicines targeting vascular wall disease is complicated by the lack of reliable biomarkers. In this study, we took a novel approach to identify circulating biomarkers indicative of drug efficacy by reducing the complexity of the in vivo system to the level where neither disease progression nor drug treatment was associated with the changes in plasma cholesterol. RESULTS: ApoE-/- mice were treated with an ACE inhibitor ramipril and HMG-CoA reductase inhibitor simvastatin. Ramipril significantly reduced the size of atherosclerotic plaques in brachiocephalic arteries, however simvastatin paradoxically stimulated atherogenesis. Both effects occurred without changes in plasma cholesterol. Blood and vascular samples were obtained from the same animals. In the whole blood RNA samples, expression of MMP9, CD14 and IL-1RN reflected pro-and anti-atherogenic drug effects. In the plasma, several proteins, e.g. IL-1beta, IL-18 and MMP9 followed similar trends while protein readout was less sensitive than RNA analysis. CONCLUSION: In this study, we have identified inflammation-related whole blood RNA and plasma protein markers reflecting anti-atherogenic effects of ramipril and pro-atherogenic effects of simwastatin in a mouse model of atherosclerosis. This opens an opportunity for early, non-invasive detection of direct drug effects on atherosclerotic plaques in complex in vivo systems.

Quantitative assessment of human whole blood RNA as a potential biomarker for infectious disease

Infection remains a significant cause of morbidity and mortality especially in newborn infants. Analytical methods for diagnosing infection are severely limited in terms of sensitivity and specificity and require relatively large samples. It is proposed that stringent regulation of the human transcriptome affords a new molecular diagnostic approach based on measuring a highly specific systemic inflammatory response to infection, detectable at the RNA level. This proposition raises a number of as yet poorly characterised technical and biological variation issues that urgently need to be addressed. Here we report a quantitative assessment of methodological approaches for processing and extraction of RNA from small samples of infant whole blood and applying analysis of variation from biochip measurements. On the basis of testing and selection from a battery of assays we show that sufficient high quality RNA for analysis using multiplex array technology can be obtained from small neonatal samples. These findings formed the basis of implementing a set of robust clinical and experimental standard operating procedures for whole blood RNA samples from 58 infants. Modelling and analysis of variation between samples revealed significant sources of variation from the point of sample collection to processing and signal generation. These experiments further permitted power calculations to be run indicating the tractability and requirements of using changes in RNA expression profiles to detect different states between patient groups. Overall the results of our investigation provide an essential first step toward facilitating an alternative way for diagnosing infection from very small neonatal blood samples, providing methods and requirements for future chip-based studies.

Effects of Globin mRNA Reduction Methods on Gene Expression Profiles from Whole Blood

Excessive globin mRNA in whole blood RNA decreases transcript detection sensitivity and increases signal variation on microarrays. Hence, methods based on peptide nucleic acid inhibitory oligos and biotinylated DNA capture oligos have been developed to reduce globin mRNA. However, there is limited information about the effects of these two methods on gene expression profiles. Thus, we systematically compared the facility and effects of the two globin reduction methods on profile measurements from Jurkat cell line RNA with or without spiked globin mRNA and human blood RNA isolated using PAXgene collection tubes. We showed that the methods were efficient at increasing the sensitivity of transcript detection without loss of specificity, but neither method could recover a profile equivalent to that of an identical RNA sample without globin mRNA excesses. The capture oligo method had slightly better transcript detection sensitivity for cell line RNA, lowered signal variation for PAXgene RNA, and more similar profiles to controls than the inhibitory method. However, the capture method required larger amounts of initial high-quality RNA to yield sufficient cRNA amounts, and its procedures were more complex and time consuming than the inhibitory method. These results inform the selection of methods suitable for multicenter surveillance of gene expression profiles.

Asbestos content of lung tissue in asbestos associated diseases: a study of 110 cases.

Diseases associated with asbestos exposure include asbestosis, malignant mesothelioma, carcinoma of the lung, and parietal pleural plaques. In this study the asbestos content of lung tissue was examined in groups of cases representing each of these diseases and in several cases with non-occupational idiopathic pulmonary fibrosis. Asbestos bodies (AB), which are the hallmark of asbestos exposure, were present in the lungs of virtually everyone in the general population and present at increased levels in individuals with asbestos associated diseases. The highest numbers of AB occurred in individuals with asbestosis, all of whom had levels greater than or equal to 2000 ABs/g wet lung tissue. Every case with a content of 100,000 ABs/g or higher had asbestosis. Intermediate levels occurred in individuals with malignant mesothelioma and the lowest levels in patients with parietal pleural plaques. There was no overlap between the asbestos content of lung tissue from patients with asbestosis and those with idiopathic pulmonary fibrosis. Lung cancer was present in half the patients with asbestosis, and the distribution of histological patterns did not differ from that in patients with lung cancer without asbestosis. The asbestos body content in patients with lung cancer was highly variable. Control cases had values within our previously established normal range (0-20 ABs/g). There was a significant correlation (p less than 0.001) between AB counted by light microscope and AB and uncoated fibres counted by scanning electron microscopy. The previous observation that the vast majority of asbestos bodies isolated from human tissues have an amphibole core was confirmed.

The duplicated human α-globin genes: Their relative expression as measured by RNA analysis

The human α-globin genes are duplicated and encode identical polypeptides. Recently we detected in cloned genomic DNAs characteristic sequence differences between the 3′ untranslated regions of the 5′ (α2) and 3′ (α1) genes, not previously recognized by direct analysis of mRNA and cDNA transcripts. Based on these untranslated region differences, we have now used S1 nuclease mapping of RNA to detect and quantitate the two predicted α-mRNA species. With this assay we have examined the relative expression of the α-globin genes during normal development and in α-thalassemia syndromes. In normal adult reticulocytes, α2 RNA is slightly more abundant than the α1 species (ratio 60:40). This relative abundance of the a RNAs was consistently observed in fetal blood and liver RNA samples from 10 weeks of gestation to birth. In both deletion and nondeletion forms of α thalassemia, only the α1 RNA species was present in erythroid RNA. These studies resolve prior conflicts regarding the heterogeneity of α RNA and establish the normal pattern of relative α-gene expression during development independent of protein variants. RNA analysis also permits for the first time identification of the mutant genes in nondeletion forms of a thalassemia.

Urinary candidiasis after renal transplantation.

<h3>Background</h3> Lymphoma development is a serious complication of Primary Sjögren9s syndrome (pSS). To date, the biological processes that may be involved in pSS-associated lymphoma are not fully understood. <h3>Objectives</h3> The aim of our study is to use microarray gene expression data from a well-defined cohort of pSS patients to identify biological processes that may be relevant to pSS-associated lymphoma. <h3>Methods</h3> pSS patients and healthy controls from the UK primary Sjögren9s syndrome registry (UKPSSR) were used in this study. All patients fulfilled the AECG criteria. Whole genome gene expression data from whole blood RNA samples (n=144) stratified into five clinical subsets (pSS=61, pSS with lymphoma=16, pSS with other cancers=21, pSS with paraproteinemia=23 and healthy controls=23) were used for the pathway analysis. A list of 68 differentially expressed genes in pSS-associated lymphoma compared with pSS (non lymphoma) were uploaded into the Ingenuity Pathway Analysis (IPA) analytic tool. Similar approach was also used for comparison between the lymphoma and other pSS subject groups. Finally, we also examined the regulators and pathways involved in the genes from the gene expression signature in pSS-associated lymphoma we have previously described (<i>BMS1</i>, <i>NUDT14</i> and <i>MGST3</i>) [1]. <h3>Results</h3> In pSS-associated lymphoma the top canonical pathway was “Aryl Hydrocarbon Receptor (AHR) signaling,” which includes <i>MGST3</i>. Several other canonical pathways also included the genes of the 3-gene biosignature of pSS-associated lymphoma. The downstream effects and gene-gene interactions were explored through molecular networks analysis. Furthermore, important upstream regulators of the 3 biosignature genes include <i>NFE2L2</i>, <i>PPARA</i> and <i>TOCF1</i> were identified. The pathway analyses of the other pSS subgroups showed 67 common canonical pathways showed among all the pSS subgroups. Focusing on pSS-associated lymphoma versus healthy controls, 94.9% of the canonical pathways in this comparison were in common with the canonical pathways identified when comparing pSS-associated lymphoma with pSS. The “Interferon Signaling pathway” was the top pathway for all pSS subgroups comparing with healthy controls. In addition, all the non-lymphoma pSS subgroups showed similar patterns in the downstream analysis which differ from the pSS-associated lymphoma group. <h3>Conclusions</h3> The pathway analysis revealed different possible pathways that might be involved in lymphoma development in pSS and indicates a unique gene expression signature exist in pSS-associated lymphoma. These results might provide a deeper understanding and a direction for future studies to investigate lymphoma development in pSS patients. <h3>References</h3> Al-Ali S, et al. Whole blood transcriptomic signature of primary Sjögren9s syndrome associated lymphoma. Biomarkers and Targeted Therapeutics in Sjögren9s (BATTS) Conference, 2016. <h3>Disclosure of Interest</h3> None declared