Abstract 3259: Leukocyte Subset Specific Gene Expression In Acute Stroke Patients

Stroke ◽  
2012 ◽  
Vol 43 (suppl_1) ◽  
Author(s):  
Mateusz G Adamski ◽  
Yan Li ◽  
Hua Yu ◽  
Erin Wagner ◽  
Sareen Amarjeet ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Stroke ◽  
Whole Blood ◽  
Peripheral Blood ◽  
Fold Increase ◽  
Specific Gene ◽  
Related Gene ◽  
Stroke Patients ◽  
Leukocyte Subset ◽  
Control Subjects

Background: Alterations in gene expression in the peripheral blood of patients with acute stroke have been demonstrated using microarray technology. Whole blood and peripheral blood mononuclear cells (PBMCs) were used in prior studies in which panels of genes diagnostic for stroke were developed. We aimed to determine the cellular sources of alterations in gene expression by studying individual leukocyte subsets. Methods: The expression of four genes previously found to be upregulated in ischemic and hemorrhagic stroke (IL1R2, S100A9, ETS2 and F5) was measured in four leukocyte subsets: CD14+ monocytes, CD4+ T cell lymphocytes, CD20+ B cell lymphocytes and PBMCs. These four genes had been reported in at least two of the previously published stroke-related gene panels. Peripheral blood was obtained from six acute stroke patients (all <48 hours from symptom onset) and 6 age, race and sex matched control subjects. Leukocytes were separated from whole blood using density gradient centrifugation and column magnetic bead cell sorting. The purity of separated leukocyte subsets exceeded 90% and was verified with flow cytometry. Messenger RNA was isolated from each leukocyte subset and analyzed by two step RT PCR and qPCR. The expression of the four stroke-related genes was compared to the expression of a housekeeping gene (GAPDH). The relative expression of individual genes and of the 4 gene panel within cellular subsets was compared between stroke patients and control subjects. Results: Individually, IL1R2 and S100A9 were significantly over-expressed in stroke patients with a 10 fold increase for IL1R2 in PBMCs (p<0.05) and a 3 fold increase for S100A9 in the CD4+ T and CD20+ B lymphocyte subsets (p<0.05). When analyzed as a panel of four genes the expression of IL1R2, S100A9, ETS2 and F5 was significantly higher in both the CD4+ T lymphocytes (p<0.05) and CD20+ B lymphocytes (p<0.05) of stroke patients but not in the monocytes or the PBMCs. Conclusion: These results show the potential diagnostic value of selected genes from panels previously found in microarray studies in stroke patients. They also emphasize the value of panel analysis over that of single gene expression and the potential cellular specificity of alterations in gene expression. Analysis of whole blood and PBMCs alone may not reflect important dynamic changes in stroke-related gene expression.

scholarly journals Comparison of whole blood and spleen transcriptional signatures over the course of an experimental malaria infection

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Carlos Talavera-López ◽  
Yaw Bediako ◽  
Jing-wen Lin ◽  
John Joseph Valletta ◽  
Mario Recker ◽  
...  
Keyword(s):  
Gene Expression ◽  
Immune Responses ◽  
Whole Blood ◽  
Peripheral Blood ◽  
Lymphoid Organ ◽  
Splenic Tissue ◽  
Specific Gene ◽  
Learning Approaches ◽  
Transcriptome Data ◽  
Cellular Immune

Abstract Although the spleen is broadly accepted as the major lymphoid organ involved in generating immune responses to the erythrocytic stages of the malaria parasite, Plasmodium, human splenic tissue is not readily available in most cases. As a result, most studies of malaria in humans rely on peripheral blood to assess cellular immune responses to malaria. The suitability of peripheral blood as a proxy for splenic immune responses is however unknown. Here, we have simultaneously analysed the transcriptomes of whole blood and spleen over 12 days of erythrocytic stage Plasmodium chabaudi infection in C57BL/6 mice. Using both unsupervised and directed approaches, we compared gene expression between blood and spleen over the course of infection. Taking advantage of publicly available datasets, we used machine learning approaches to infer cell proportions and cell-specific gene expression signatures from our whole tissue transcriptome data. Our findings demonstrate that spleen and blood are quite dissimilar, sharing only a small amount of transcriptional information between them, with transcriptional differences in both cellular composition and transcriptional activity. These results suggest that while blood transcriptome data may be useful in defining surrogate markers of protection and pathology, they should not be used to predict specific immune responses occurring in lymphoid organs.

scholarly journals Whole Blood Transcriptome Analysis in Children with Sickle Cell Anemia

2022 ◽  
Vol 12 ◽  
Author(s):  
Beatrice E. Gee ◽  
Andrea Pearson ◽  
Iris Buchanan-Perry ◽  
Roger P. Simon ◽  
David R. Archer ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rna Sequencing ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Whole Blood ◽  
Differentially Expressed ◽  
Mrna Levels ◽  
Control Subjects ◽  
Non Coding Rna ◽  
And Control

Whole transcriptome RNA-sequencing was performed to quantify RNA expression changes in whole blood samples collected from steady state sickle cell anemia (SCA) and control subjects. Pediatric SCA and control subjects were recruited from Atlanta (GA)—based hospital(s) systems and consented for RNA sequencing. RNA sequencing was performed on an Ion Torrent S5 sequencer, using the Ion Total RNA-seq v2 protocol. Data were aligned to the hg19 reference genome and analyzed in the Partek Genomics studio package (v7.0). 223 genes were differentially expressed between SCA and controls (± 1.5 fold change FDR p &lt; 0.001) and 441 genes show differential transcript expression (± 1.5 fold FDR p &lt; 0.001). Differentially expressed RNA are enriched for hemoglobin associated genes and ubiquitin-proteasome pathway genes. Further analysis shows higher gamma globin gene expression in SCA (33-fold HBG1 and 49-fold HBG2, both FDR p &lt; 0.05), which did not correlate with hemoglobin F protein levels. eQTL analysis identified SNPs in novel non-coding RNA RYR2 gene as having a potential regulatory role in HBG1 and HBG2 expression levels. Gene expression correlation identified JHDM1D-AS1(KDM7A-DT), a non-coding RNA associated with angiogenesis, enhanced GATA1 and decreased JAK-STAT signaling to correlate with HBG1 and HBG2 mRNA levels. These data suggest novel regulatory mechanisms for fetal hemoglobin regulation, which may offer innovative therapeutic approaches for SCA.

scholarly journals Dysregulation of MS risk genes and pathways at distinct stages of disease

2017 ◽  
Vol 4 (3) ◽  
pp. e337 ◽  
Author(s):  
Sundararajan Srinivasan ◽  
Marco Di Dario ◽  
Alessandra Russo ◽  
Ramesh Menon ◽  
Elena Brini ◽  
...  
Keyword(s):  
Gene Expression ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Gene List ◽  
Literature Mining ◽  
Specific Gene ◽  
Healthy Population ◽  
Gene Expressions ◽  
Altered Expression ◽  
Risk Genes

Objective:To perform systematic transcriptomic analysis of multiple sclerosis (MS) risk genes in peripheral blood mononuclear cells (PBMCs) of subjects with distinct MS stages and describe the pathways characterized by dysregulated gene expressions.Methods:We monitored gene expression levels in PBMCs from 3 independent cohorts for a total of 297 cases (including clinically isolated syndromes (CIS), relapsing-remitting MS, primary and secondary progressive MS) and 96 healthy controls by distinct microarray platforms and quantitative PCR. Differential expression and pathway analyses for distinct MS stages were defined and validated by literature mining.Results:Genes located in the vicinity of MS risk variants displayed altered expression in peripheral blood at distinct stages of MS compared with the healthy population. The frequency of dysregulation was significantly higher than expected in CIS and progressive forms of MS. Pathway analysis for each MS stage–specific gene list showed that dysregulated genes contributed to pathogenic processes with scientific evidence in MS.Conclusions:Systematic gene expression analysis in PBMCs highlighted selective dysregulation of MS susceptibility genes playing a role in novel and well-known pathogenic pathways.

Microfluidic coagulation assay for monitoring anticoagulant therapy in acute stroke patients

2017 ◽  
Vol 117 (03) ◽  
pp. 519-528 ◽  
Author(s):  
Annemarie Bluecher ◽  
Sascha Meyer dos Santos ◽  
Nerea Ferreirós ◽  
Sandra Labocha ◽  
Isabel Maria Rodrigues Meyer dos Santos ◽  
...  
Keyword(s):  
Acute Stroke ◽  
Vitamin K ◽  
Whole Blood ◽  
Acoustic Waves ◽  
Oral Anticoagulants ◽  
Surface Acoustic Waves ◽  
Vitamin K Antagonist ◽  
Emergency Setting ◽  
Stroke Patients ◽  
Emergency Patients

SummaryReliable detection of anticoagulation status in patients treated with non-vitamin K antagonist oral anticoagulants (NOACs) is challenging but of importance especially in the emergency setting. This study evaluated the potential of a whole-blood clotting time assay based on Surface Acoustic Waves (SAW-CT) in stroke-patients. The SAW-technology was used for quick and homogenous recalcification of whole blood inducing a surface-activated clotting reaction quantified and visualised by real-time fluorescence microscopy with automatic imaging processing. In 20 stroke or transient ischaemic attack (TIA)-patients taking NOACs kinetics of SAW-CT were assessed and correlated to other coagulation parameters (PT, aPTT) and NOAC-plasma concentration measured by tandem mass spectrometry (LC-MS/MS). In 225 emergency patients with suspicion of acute stroke or TIA, SAW-CT values were assessed. Mean (± SD) SAW-CT in non-anticoagulated stroke patients (n=180) was 124 s (± 21). In patients on dabigatran or rivaroxaban, SAW-CT values were significantly higher 2 and 8 hours (h) after intake rising up to 267 seconds (s) (dabigatran, 2 h after intake) and 250 s (rivaroxaban, 8 h after intake). In patients on apixaban, SAW-CT values were only moderately increased 2 h after intake (SAW-CT 153 s). In emergency patients, SAW-CT values were significantly higher in NOAC and vitamin K antagonist (VKA)-treated as compared to non-anticoagulated patients. In conclusion, the SAW-CT assay is capable to monitor anticoagulant level and effect in patients receiving dabigatran, rivaroxaban and the VKA phenprocoumon. It has a limited sensitivity for apixaban-detection. If specific SAW-CT results were used as cut-offs, SAW-CT yields high diagnostic accuracy to exclude relevant rivaroxaban and dabigatran concentrations in strokepatients.

IL-2 Therapy Promotes the Expansion of Human CD4+CD25+ Regulatory T Cells and Selectively Upregulates the Expression of FOXP3 in T Cells In Vivo.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1257-1257
Author(s):  
Emmanuel Zorn ◽  
Erik A. Nelson ◽  
Mehrdad Mohseni ◽  
Despina Litsa ◽  
Haesook Kim ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
Regulatory T Cells ◽  
Nk Cells ◽  
Quantitative Pcr ◽  
Peripheral Blood ◽  
Fold Increase ◽  
Foxp3 Expression ◽  
Prolonged Administration

Abstract Recombinant IL-2 has been used extensively in clinical trials to enhance a wide range of immune responses. Overall this strategy has had limited efficacy. Recent evidence suggests that IL-2 plays a key role in the generation and maintenance of CD4+CD25+ regulatory T cells (Treg) in vivo. In our study, we investigated the effect of prolonged administration of recombinant IL-2 on Treg in vivo. In a retrospective analysis, we first examined CD4+CD25+ Treg in blood samples collected from 21 cancer patients before and after they started continuous treatment with IL-2 at a dose of 2 X 105 U/m2/day for 3 months. Nine patients received IL-2 beginning 3 months after CD6 T cell depleted allogeneic bone marrow transplantation (BMT) for CML. The remaining 12 patients received IL-2 as treatment for advanced solid tumors. Overall toxicity was minimal and none of the transplant patients developed GVHD following IL-2 administration. Previous reports demonstrated that this prolonged treatment with low-dose IL-2 resulted in the expansion of CD56+CD3− NK cells in peripheral blood. Further analysis showed that 15 patients exhibited an expansion of Treg in peripheral blood 26 to 77 days after beginning IL-2 as demonstrated by an increase in the CD4+CD25+/CD3+ ratio (median fold increase 2.68; range 1.3 to 59). Three patients had no significant change and 3 patients demonstrated a decreased Treg/CD3 ratio. Using RNA from the same samples we also measured the expression of the Treg specific transcription factor FOXP3 by quantitative PCR. Nineteen of 21 patients showed a marked increase in FOXP3 expression following IL-2 treatment (8.38 median fold increase; range 1.4 to 41.5). Only 2 of 21 patients had lower FOXP3 expression after IL-2 administration. Since IL-2 treatment resulted in the expansion of NK cells as well as Treg, we purified CD56+CD3− NK cells and CD4+ T cells from patient samples collected post-IL-2 treatment, and measured FOXP3 gene expression in both subsets. In 4 analyzed cases, FOXP3 was selectively expressed in CD4+ T cells. Further analysis of purified Treg and NK cells incubated with IL-2 in vitro confirmed that FOXP3 expression was selectively induced in Treg, and also suggested that the in vivo increase in FOXP3 expression resulted from both Treg expansion and up-regulation of gene expression at the single cell level. To study the duration of the IL-2 effect, we analyzed additional samples collected 2 to 8 months after IL-2 treatment was completed. Nine of 10 patient samples tested showed a decrease in the CD4+CD25+/CD3+ ratio (1.39 median fold decrease; range 1.13 to 15.02). Using quantitative PCR, expression of FOXP3 decreased for 6 of 8 patients tested (10.76 median fold decrease; range 1.22 to 88.31). These results indicate that prolonged administration of IL-2 promotes the expansion of CD4+CD25+ Treg in vivo and also has a direct effect on FOXP3 expression. Although administration of IL-2 has previously been used to enhance T and NK cell responses, this study demonstrates that IL-2 therapy predominantly reinforces the regulatory component of the immune response, and may provide a means for controlling immune reactions in vivo.

Tissue Transglutaminase Enhances Fibrin-Dependent Angiogenesis and Extracellular Matrix Formation During Tissue Repair by Altering Gene Expression and Is Inhibited by Aspirin.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3055-3055
Author(s):  
Thung S. Lai ◽  
Christopher Davies ◽  
Charles Greenberg
Keyword(s):  
Gene Expression ◽  
Extracellular Matrix ◽  
Wound Healing ◽  
Growth Factor ◽  
Granulation Tissue ◽  
Tissue Transglutaminase ◽  
Fold Increase ◽  
Related Gene ◽  
Wound Healing Response ◽  
Angiogenesis Related Gene

Abstract Abstract 3055 Poster Board II-1031 Fibrin deposition triggers an injury response that involves the migration of inflammatory cells, formation of new blood vessels and the synthesis of extracellular matrix (ECM). Tissue transglutaminase (TGM2) is a calcium dependent enzyme that covalently crosslinks a wide variety of ECM proteins producing a protease resistant matrix. TGM2 is secreted by inflammatory and endothelial cells, involved in activating transforming growth factor beta-1 (TGFbeta-1) and expressed during tissue injury. In this study, we investigated how TGM2 modulated fibrin-dependent wound healing and the associated angiogenic response. We used an animal model consisting of fibrin Z-chambers (F-ZC, dual porous plexiglass chambers containing fibrin) implanted into the subcutaneous tissue of rats and harvested subsequently for quantitative assessment of granulation tissue formation (wound healing) and microvessel density (angiogenesis). We found that local administration of recombinant TGM2 into F-ZC resulted in a dose-dependent, 2-fold increase in granulation tissue thickness by day 6 of wound healing (p<0.001), an effect similar in magnitude to 25 ng/ml of TGFbeta1 administered in the F-ZC. The pro-healing effect of TGM2 was associated with a 2-fold increase in microvessel density in granulation tissue at day 6 of wound healing response (p<0.001). As a negative control, inactive recombinant C277A/TGM2 mutant did not exhibit increased wound healing response or proangiogenic effect. The data suggested that TGM2 enhanced the transition from the inflammatory stage of wound healing to proliferation stage. The two areas where TGM2 enhanced wound healing were 1) angiogenesis and 2) deposition of ECM. To investigate TGM2-induced angiogenesis-related gene expression, total RNAs were isolated from control- and TGM2-treated F-ZCs (at Day 6). Biotin-labeled cDNA probes were synthesized, and hybridized to nylon membranes containing angiogenesis-related gene arrays (Superarray, MD). The signals were detected using streptavidin-peroxidase and quantitated. We identified increased expression of VEGF receptors Flk-1 (2-fold), Flt1 and neuropilin (1.4-fold), angiopoietin-1 (2-fold) and ephrin B2 (1.8-fold). There were decreased levels (5-fold) of matrix metalloproteinases (MMPs) and increased TGFbeta-1 receptors (1.5-fold) and connective tissue growth factor (CTGF)(1.4-fold) levels. The gene expression profile suggests that TGM2 promotes angiogenesis and enhances deposition of ECM. We then investigated whether Aspirin (Acetylsalicylic Acid, ASA) a potent anti-inflammatory agent would inhibit TGM2. ASA and another chemical acetylating agent, sulfosuccinimidyl acetate (SNA), were used to investigate whether acetylation would alter the crosslinking activity of TGM2. We found acetylation by either SNA or ASA resulted in a loss of >90% of crosslinking activity. The Lys residues that were critical for inhibition were identified by mass spectrometry as Lys468 and Lys663. Molecular modeling indicates that these Lys residues play an important role in the conformation change that occurs in TGM2 from a closed-to-open shape, i.e. inactive-to-active, transitions. In conclusion, we show that TGM2-fibrin crosslinking accelerates angiogenesis and promotes ECM deposition. This suggests that TGM2-fibrin interactions mediates outside-in signaling events that aides wound healing. Furthermore aspirin can acetylate and inhibit critical residues in TGM2 that regulate TGM-2 function. Disclosures No relevant conflicts of interest to declare.

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