The Expression of GATA-3 and FoxP3 Genes and the Frequency of Tregs Are Correlated with Gvhd Following Allogeneic HSCT

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 5181-5181
Author(s):  
Xiuli Wu ◽  
Qifa Liu ◽  
Can Liu ◽  
Zhiping Fan ◽  
Li Xuan ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
Correlation Coefficient ◽  
Pearson Correlation ◽  
Pearson Correlation Coefficient ◽  
Spearman Correlation ◽  
Healthy Individuals ◽  
Expression Levels ◽  
Genes Expression ◽  
Gene Expression Levels

Abstract Abstract 5181 Objective Hematopoietic stem cell transplantation (HSCT) is a common therapeutic option for leukemia, but its use is limited by mortality due to graft-versus-host-disease (GVHD). Naturally occurring regulatory T cells (Tregs) are mediators of immunologic tolerance that attenuate GVHD in experimental models. Tregs were first defined by a CD4+CD25+ phenotype, but subsequent studies identified forkhead box protein 3 (FoxP3) as a highly specific marker in both mouse and human T cells with regulatory function. GATA-3 is a member of the GATA family of transcription factors, and is sufficient to induce a Th2 phenotype. GATA-3 could act as an inhibitor of FoxP3 expression in early T cell differentiation was found in transgenic mice model recently. But whether GATA-3 expression could correlate with GVHD incidence, or whether GATA-3 could influence Tregs recovery and contribute to the development of GVHD are still not clear. So we investigated the frequency of Tregs in the early period after HSCT and at the time of GVHD onset, and quantitatively measured the expression of GATA-3 and FoxP3 at GVHD onset for analyzing the correlation to the development of GVHD. Methods Sixty-seven leukemia patients underwent allogenic (allo-) HSCT were enrolled in this investigation, including 45 males and 22 females (median age: 32.3 years, range 21≂f45). Fourteen healthy individuals (7 males and 7 females, median age: 30.3 years, range 26≂f40) served as controls. Frequencies of T lymphocyte subsets were determined by flow cytometry. The expression levels of human FoxP3 and GATA-3 genes were determined by real-time quantitative PCR with SYBR Green I technique. Results The frequencies of CD4+ CD25+ T cells were lower at week 4 post-HSCT, but significant higher after week 4 and lower again after week 8 (P=0.000). The frequencies of FoxP3+ T cells were also lower at week 4 and remained low levels within 12 weeks after HSCT. As early as 4 to 8 weeks after HSCT, the frequencies of CD4+ FoxP3+ T cells were lower than that of week 4 (P=0.048), but the frequencies of CD25+ FoxP3+ T cells were higher than that of week 4 (P=0.022). Frequencies of CD4+ CD25+ cells in patients at GVHD onset were similar in patients without GVHD (P=0.262) or healthy individuals (P=0.166). But the frequencies of FoxP3+ cells, FoxP3+ CD25+ cells and FoxP3+ CD4+ cells in patients at GVHD onset were significantly lower than that in patients without GVHD (P=0.000, 0.004, and 0.001). The FoxP3 gene expression levels in patients at GVHD onset were significantly lower than that in patients without GVHD (P=0.044), but the GATA-3 gene expression levels were significantly higher in patients at GVHD onset compared to patients without GVHD (P=0.005). The FoxP3 gene expression levels in patients at aGVHD onset were significantly higher than that in patients at cGVHD onset (P=0.038), but the GATA-3 gene expression levels were significantly lower in patients at aGVHD onset compared to patients at cGVHD onset (P=0.004). A highly significant correlation between FoxP3 and GATA-3 genes expression levels existed in healthy individuals (Spearman correlation coefficient r=-0.893, P=0.007). Correlations between FoxP3 and GATA-3 genes expression levels were also significant in patients at aGVHD onset (Pearson correlation coefficient r=0.749, P=0.020) or cGVHD onset (Pearson correlation coefficient r=0.538, P=0.071) or in GVHD (Spearman correlation coefficient r=0.370, P=0.090). But there is no significant correlation between FoxP3 and GATA-3 genes expression levels in patients without GVHD (Pearson correlation coefficient r=-0.141, P=0.821). Conclusions GATA-3 and FoxP3 genes expression and the frequency of Tregs are correlated with GVHD following allogeneic HSCT. GATA-3 can influence the expression of FoxP3 in the development of GVHD. Supported by National Natural Science Foundation of China(30971300), Science and Technology Planning Project of Guangdong Province of China (2009A030200007) and China Postdoctoral Science Foundation (200902332, 20080440776). Disclosures: No relevant conflicts of interest to declare.

Multiplexed Digital Quantification of mRNA Abundance: a Highly Reproducible, PCR–independent Assessment of BAALC, ERG, and MN1 mRNA Levels in Acute Myeloid Leukemia (AML) Bone Marrow (BM) and Peripheral Blood (PB) Samples.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1599-1599
Author(s):  
Jacqueline E. Payton ◽  
Guido Marcucci ◽  
Michael D. Radmacher ◽  
Kati Maharry ◽  
Christian Langer ◽  
...  
Keyword(s):  
Gene Expression ◽  
Correlation Coefficients ◽  
Mrna Abundance ◽  
White Blood Count ◽  
Spearman Correlation ◽  
Expression Levels ◽  
Rna Quality ◽  
Qrt Pcr ◽  
Digital Quantification ◽  
Gene Expression Levels

Abstract Abstract 1599 Poster Board I-625 The greatest obstacle to routine clinical testing of gene expression levels has been the lack of reproducibility of currently used methodologies, such as quantitative reverse transcriptase PCR (qRT-PCR) and microarray expression profiling. While these assays are useful for retrospective analyses of batched samples, they cannot be used for upfront evaluation of individual patients (pts) for molecular risk and treatment assignment. To overcome this barrier, we tested a recently developed, high throughput, PCR-independent, digital quantification technology, the nCounter system (Nanostring® Technologies). This system counts individual mRNA molecules, rather than measuring non-linear fluorescence generated by PCR-amplified targets (eg qRT-PCR). Using 72 AML samples and spike-in controls, we and collaborators demonstrated that the nCounter system is highly reproducible, sensitive, and accurate to femtomolar concentrations (Payton, J, et al. JCI 119:1714-26; Geiss, G, et al. Nat Biotech 26:317-25). Here we validated this technology using an independent set of 101 pts with a diagnosis of de novo cytogenetically normal AML. At diagnosis, pts presented with FAB subtypes M0, M1, M2, M4, M5(A, B), had a median age of 43 years (range 19-59), median white blood count of 28.5× 103/μL (range 1.4-273.0), median of 69% BM blasts (range 22-95) and median of 65% PB blasts (range 0-97). Paired BM and PB specimens were available for 27 pts; blast percentages were ≥ 20% for all paired specimens. We used the nCounter system to measure mRNA abundance (‘counts‘) of 27 genes whose expression correlates with clinical and/or pathological criteria, including 3 genes associated with prognosis (BAALC, ERG, MN1), and control/housekeeping genes (GAPDH, ABL, Actin). Briefly, mononuclear cells from pretreatment BM or PB were enriched on Ficoll-Hypaque gradients and RNA was isolated using Trizol reagent; 100ng of total RNA was assayed in triplicate by nCounter according to the manufacturer's protocols. The nCounter results demonstrated substantial reproducibility, with a median CV [coefficient of variation, (standard deviation/mean *100)] <6% across replicates. In addition, the nCounter counts for BAALC, ERG, and MN1 normalized to ABL were highly correlated with the ABL-normalized qRT-PCR results. Significant correlation was observed for all 3 genes, with the following Spearman correlation coefficients: BAALC r = 0.9, ERG r = 0.7, and MN1 r = 0.8 (all p<0.001). Correlation of BAALC, ERG, and MN1 nCounter counts with the expression levels measured by Affymetrix® HG-U133 plus 2.0 microarrays were also tested. Summary measures of microarray gene expression levels were computed using the Robust Multichip Average method, which incorporates quantile normalization of arrays. Significant correlation of nCounter and microarray results was observed, with Spearman correlation coefficients as follows: BAALC r = 0.96, ERG r = 0.8, and MN1 r = 0.8 (all p<0.001). For the 27 sets of paired samples, nCounter results for BM and PB were also significantly correlated, with Spearman correlation coefficients of BAALC r = 0.9, ERG r = 0.7, and MN1 r = 0.6 (all p<0.001). Because RNA quickly degrades if not promptly isolated from PB or BM, and degraded RNA often fails qRT-PCR assays, we determined whether RNA quality affected nCounter performance by assessment of standard quality parameters, including ratio of absorbance at 260 and 280 nm (260:280, a measure of RNA purity, acceptable 1.8-2.0) and RNA Quality Index (RQI, which assesses 18S:28S rRNA ratio and RNA degradation, 7-10 acceptable). Quality ranged from very high, with 260:280 ratios >1.9 and RQI scores >9, to relatively low, with 260:280 ratios <1.8, RQI scores <4, and degraded RNA visible on the Experion® RNA chip. Such a range of RNA quality is consistent with our experience with clinical specimens, which may be delayed in transit to the laboratory. Nevertheless, fewer than 3% of nCounter assays failed to generate acceptable results (11/393 assays), likely because no PCR step is required. Our results show that the nCounter system is a rapid, relatively inexpensive ($0.72/assay), and highly reproducible methodology that will be very useful for routine diagnostic testing of prognostic gene expression and upfront molecular-risk assessment for treatment guidance in AML pts. Disclosures No relevant conflicts of interest to declare.

scholarly journals Influences of Serotonin Hydrochloride on Adiponectin, Ghrelin and KiSS1 Genes Expression

2020 ◽  
Vol 9 ◽  
Author(s):  
Fariba Mahmoudi ◽  
Khadijeh Haghighat Gollo
Keyword(s):  
Gene Expression ◽  
Saline Group ◽  
Polymerase Chain Reaction Method ◽  
Male Rats ◽  
Expression Levels ◽  
Serum Lh ◽  
Genes Expression ◽  
Lh Release ◽  
The Mean ◽  
Gene Expression Levels

Background: Serotonin and kisspeptin stimulates gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) release while ghrelin and adiponectin inhibit them. In the present experimental study, the effects of central injection of serotonin were investigated on LH concentration, KiSS1, adiponectin, and ghrelin genes expression. Materials and Methods: Fifteen Wistar male rats in three groups received saline or serotonin hydrochloride via the third cerebral ventricle. Blood samples were collected via the tail vein. Serum LH concentration and relative gene expression were evaluated by radioimmunoassay and real-time polymerase chain reaction method, respectively. Results: Serotonin significantly increased the mean serum LH concentration and  KiSS1 gene expression levels compared to the saline group. Serotonin significantly decreased the mean ghrelin and adiponectin genes expression levels compared to the saline group. Conclusion: The serotonergic pathway may have stimulatory effects on hypothalamic kisspeptin synthesis, partly via inhibiting hypothalamic ghrelin and adiponectin neural activity.[GMJ.2020;9:e1767]

scholarly journals Methods of Estimating Correlation Coefficients in the Presence of Influential Outlier(s)

2021 ◽  
Vol 4 (3) ◽  
pp. 157-185
Author(s):  
Etaga H.O. ◽  
Okoro I. ◽  
Aforka K.F. ◽  
Ngonadi L.O.
Keyword(s):  
Correlation Coefficient ◽  
Pearson Correlation ◽  
Correlation Coefficients ◽  
Pearson Correlation Coefficient ◽  
Spearman Correlation ◽  
Correlation Methods ◽  
Linear Relationships ◽  
Median Correlation ◽  
Standing Problem ◽  
Research Analysis

Correlation methods are indispensable in the study of the linear relationship between two variables. However, many researchers often adopt inappropriate correlation methods in the study of linear relationships which usually leads to unreliable results. Recurrently, most researchers ignorantly employ the Pearson method in a dataset that contained outliers, instead of more appropriate correlation methods such as Spearman, Kendall Tau, Median and Quadrant which might be suitable in the calculation of correlation coefficient in the presence of influential outliers. It is noted that the accuracy of estimation of correlation coefficients under outliers has been a long-standing problem for methodological researchers. This is due to low knowledge of correlation methods and their assumptions which have led to inappropriate application of correlation methods in research analysis. Five different methods of estimating correlation coefficients in the presence of influential outlier (contaminated data) were considered: Pearson Correlation Coefficient, Spearman Correlation Coefficient, Kendall Tau Correlation Coefficient, Median Correlation Coefficient and Quadrant Correlation Coefficient.

scholarly journals The effect of education and sporting experience of iranian premier league football players on their awareness of sports law

2020 ◽  
Vol 13 (32) ◽  
pp. 1-15
Author(s):  
Mahdi Azimi ◽  
Seyed Amir Reza Hosseinipour Rafsanjani ◽  
Mona Torkaman
Keyword(s):  
Correlation Coefficient ◽  
Pearson Correlation ◽  
Percent Level ◽  
Football Players ◽  
Pearson Correlation Coefficient ◽  
Spearman Correlation ◽  
Sports Law ◽  
Premier League ◽  
History Of ◽  
Kendall Correlation

The purpose of this study was to investigate the relationship between education and athletic background of Premier League football players on their awareness of sports Law. In this study, descriptive-analytical method and Spearman correlation coefficient were used. Ninety-five percent level was used as the criterion for rejecting the hypothesis. Questionnaires about variables were used to identify the sample group and the main questions about players' legal awareness were used for data collection. The results showed that Pearson correlation coefficient was 0.107, Spearman correlation coefficient was 0.204 and Kendall correlation coefficient was 0.139 and significant was 0.88. There is no relationship between awareness of sports law and the history of sports in the Premier League. Pearson correlation coefficient of 0.388, Spearman correlation coefficient of 0.204 and Kendall correlation coefficient of 0.139 and significant value of 0.001 indicated that there is a relationship between sport legal awareness and education of the Iranian Premier League players.

scholarly journals Molecular Signature of Persistent Histological Inflammation in Ulcerative Colitis with Mucosal Healing

2020 ◽  
Vol 29 (2) ◽  
pp. 159-166
Author(s):  
Mircea Manuc ◽  
Elena Mirela Ionescu ◽  
Elena Milanesi ◽  
Maria Dobre ◽  
Ioana Tieranu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Ulcerative Colitis ◽  
Pearson Correlation ◽  
Mucosal Healing ◽  
Formyl Peptide Receptor ◽  
Molecular Signature ◽  
Expression Levels ◽  
Endoscopic Remission ◽  
Formyl Peptide ◽  
Gene Expression Levels

Background and Aims: Therapeutic targets in ulcerative colitis (UC) have evolved over time from clinical remission to biological and endoscopic remission. Histologic remission remains a debatable outcome due to lack of data regarding its impact on long-term evolution. The development of histologic activity scores has brought standardization. We aimed to identify mucosal markers differentiating histological inflammation from histological remission in UC patients. Methods: The gene expression levels of 84 genes associated with inflammatory bowel diseases have been analyzed in 43 colonic mucosa samples from 30 patients with UC. The gene expression levels have been correlated with histological inflammation score of Geboes. Patients with endoscopic remission were divided by histological activity into two groups and molecular results were compared in order to identify differences in the mucosal gene expression. Results: We found a significant Pearson correlation (p<0.001 and r>0.5) between the Geboes score and the expression of 29 genes, whereas negative correlation (p<0.001 and r<-0.50) was observed with two genes in the entire UC cohort. In the subgroup of patients with endoscopic remission three transcripts: formyl-peptide receptor 1 (FPR1), matrix metalloproteinases 1 (MMP1) and mucine 1 (MUC1) were significantly up-regulated in patients with histological inflammation compared to patients with histologic remission. Conclusion: Our study further emphasizes the importance of histological assessment when endoscopic mucosal healing is present, as FPR1, MMP-1 and MUC1 were all significantly upregulated in patients with histological alterations.

scholarly journals Real-time Prediction of the Daily Incidence of COVID-19 in 215 Countries and Territories Using Machine Learning: Model Development and Validation (Preprint)

2020 ◽  
Author(s):  
Yuanyuan Peng ◽  
Xinjian Chen ◽  
Yibiao Rong ◽  
Chi Pui Pang ◽  
Xinjian Chen ◽  
...  
Keyword(s):  
Machine Learning ◽  
Real Time ◽  
Correlation Coefficient ◽  
Pearson Correlation ◽  
Google Trends ◽  
Pearson Correlation Coefficient ◽  
Spearman Correlation ◽  
Time Prediction ◽  
Search Volume ◽  
The Mean

BACKGROUND Advanced prediction of the daily incidence of COVID-19 can aid policy making on the prevention of disease spread, which can profoundly affect people's livelihood. In previous studies, predictions were investigated for single or several countries and territories. OBJECTIVE We aimed to develop models that can be applied for real-time prediction of COVID-19 activity in all individual countries and territories worldwide. METHODS Data of the previous daily incidence and infoveillance data (search volume data via Google Trends) from 215 individual countries and territories were collected. A random forest regression algorithm was used to train models to predict the daily new confirmed cases 7 days ahead. Several methods were used to optimize the models, including clustering the countries and territories, selecting features according to the importance scores, performing multiple-step forecasting, and upgrading the models at regular intervals. The performance of the models was assessed using the mean absolute error (MAE), root mean square error (RMSE), Pearson correlation coefficient, and Spearman correlation coefficient. RESULTS Our models can accurately predict the daily new confirmed cases of COVID-19 in most countries and territories. Of the 215 countries and territories under study, 198 (92.1%) had MAEs &lt;10 and 187 (87.0%) had Pearson correlation coefficients &gt;0.8. For the 215 countries and territories, the mean MAE was 5.42 (range 0.26-15.32), the mean RMSE was 9.27 (range 1.81-24.40), the mean Pearson correlation coefficient was 0.89 (range 0.08-0.99), and the mean Spearman correlation coefficient was 0.84 (range 0.2-1.00). CONCLUSIONS By integrating previous incidence and Google Trends data, our machine learning algorithm was able to predict the incidence of COVID-19 in most individual countries and territories accurately 7 days ahead.

scholarly journals PD-1H Expression Associated With CD68 Macrophage Marker Confers an Immune-Activated Microenvironment and Favorable Overall Survival in Human Esophageal Squamous Cell Carcinoma

2021 ◽  
Vol 8 ◽  
Author(s):  
Yuangui Chen ◽  
Rui Feng ◽  
Bailin He ◽  
Jun Wang ◽  
Na Xian ◽  
...  
Keyword(s):  
Gene Expression ◽  
Overall Survival ◽  
Squamous Cell Carcinoma ◽  
T Cells ◽  
Esophageal Squamous Cell Carcinoma ◽  
Cell Carcinoma ◽  
Squamous Cell ◽  
Expression Levels ◽  
Tumor Tissues ◽  
Gene Expression Levels

Esophageal squamous cell carcinoma (ESCC) is the most common type of esophageal carcinoma (EC) in China. Although the PD-1 inhibitor pembrolizumab has been approved to treat patients with EC, its therapeutic efficacy is limited. Thus, additional immunotherapeutic targets for EC treatment are needed. Programmed Death-1 Homolog (PD-1H) is a negative checkpoint regulator that inhibits antitumor immune responses. Here, PD-1H expression in 114 patients with ESCC was evaluated by immunohistochemistry. Next, 12 randomly selected tumor tissue sections were used to assess the colocalization of PD-1H protein and multiple immune markers by multiplex immunohistochemistry. Our results demonstrated that PD-1H was expressed at high frequency in ESCC tumor tissues (85.1%). PD-1H protein was predominantly expressed in CD68+ tumor-associated macrophages and expressed at low levels in CD4+ T cells and CD8+ T cells in ESCC tumor tissues. Furthermore, based on ESCC data in The Cancer Genome Atlas (TCGA), the gene expression levels of PD-1H were positively associated with the infiltration levels of immune-activated cells especially CD8+ cytotoxic T cells. In contrast, the gene expression levels of PD-1H were negatively correlated with myeloid-derived suppressor cells (MDSCs). Importantly, PD-1H expression in tumor sites was significantly correlated with favorable overall survival in patients with ESCC. Collectively, our findings first provided direct information on the PD-1H expression pattern and distribution in ESCC, and positive correlation of PD-1H expression with overall survival suggested PD-1H expression levels could be a significant prognostic indicator for patients with ESCC. Future studies need to explore the immunoregulatory of PD-1H in the tumor microenvironment of ESCC.

scholarly journals T Cell Transcriptomes from Paroxysmal Nocturnal Hemoglobinuria Patients Reveal Novel Signaling Pathways

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1042-1042
Author(s):  
Kohei Hosokawa ◽  
Sachiko Kajigaya ◽  
Keyvan Keyvanfar ◽  
Qiao Wangmin ◽  
Yanling Xie ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
T Cell ◽  
Research Funding ◽  
Memory T Cells ◽  
Healthy Controls ◽  
Rna Seq ◽  
Expression Levels ◽  
Cd8 Memory T Cells ◽  
Gene Expression Levels

Abstract Background. Paroxysmal nocturnal hemoglobinuria (PNH) is a rare acquired blood disease, characterized by hemolytic anemia, bone marrow (BM) failure, and venous thrombosis. The etiology of PNH is a somatic mutation in the phosphatidylinositol glycan class A gene (PIG-A) on the X chromosome, which causes deficiency in glycosyl phosphatidylinositol-anchored proteins (GPI-APs). The involvement of T cells in PNH is strongly supported by clinical overlap between PNH and aplastic anemia (AA); the presence of GPI-AP deficient cells in AA associated with favorable response to immunosuppressive therapy; and an oligoclonal T cell repertoire in PNH patients. However, the molecular mechanisms responsible for the aberrant immune responses in PNH patients are not well understood. To identify aberrant molecular mechanisms involved in immune targeting of hematopoietic stem cells in BM, RNA sequencing (RNA-seq) was applied to examine the transcriptome of T cell subsets from PNH patients and healthy controls. Method. Blood samples were obtained after informed consent from 15 PNH patients and 15 age-matched healthy controls. For RNA extraction, freshly isolated peripheral blood mononuclear cells were sorted on the same day of blood draw to obtain four different T cell (CD3+ CD14- CD19- ViViD-) populations [CD4+ naïve (CD45RA+ CD45RO-), CD4+ memory (CD45RA- CD45RO+), CD8+ naïve (CD45RA+ CD45RO-), and CD8+ memory (CD45RA- CD45RO+) T cells] by fluorescence-activated cell sorter . RNA-Seq analysis from three PNH and three healthy controls was performed using the Illumina HiSeq™ 2000 platform. The Ingenuity® Pathway Analysis and Gene set enrichment analysis (GSEA) were employed to elucidate transcriptional pathways. RNA-seq data were validated by flow cytometry and quantitative real-time RT-PCR (RT-qPCR). Results and Discussion . Differentially expressed gene analysis of four T cell subsets showed distinct gene expression signatures in individual T cell subsets. In CD4+ naïve T cells, 11 gene expression levels were significantly different: five upregulated (including SRRM2 and TNFSF8) and six downregulated genes (including GIMAP6) (> 2 fold change, false discovery rate [FDR] < 0.05). In CD4+ memory T cells, 25 gene expression levels were significantly different: 15 upregulated (including JUND and TOB1) and 10 downregulated genes (including GIMAP4). In CD8+ naive T cells, only two gene expression levels were significantly different: upregulated CTSW and downregulated RPL9. In CD8+ memory T cells, seven gene expression levels were significantly different: two upregulated (CTSW and DPP4) and five downregulated genes (including SLC12A7). Further, differentially expressed gene analysis was performed by combining CD4+ naïve, CD4+ memory, CD8+ naïve, and CD8+ memory T cells from PNH or healthy controls, respectively. Out of 55 gene expression levels that were significantly different, 41 were upregulated (including TNFAIP3, JUN, JUND, TOB1, TNFSF8, and CD69) and 14 downregulated (including GIMAP4). By canonical pathways analysis, putative gene network interactions of differentially expressed genes were significantly enriched for canonical pathways of TNFR1, TNFR2, IL-17A, and CD27 signaling. By GSEA, the most significantly upregulated gene sets in CD4+ naïve, CD4+ memory, CD8+ naïve, and CD8+ memory T cells from PNH patients displayed gene signatures related to the "IGF1 pathway", "Pre-NOTCH expression and processing", "AP-1 pathway", and "ATF2 pathway", respectively. For validation of the RNA-seq data, we chose seven genes (TNFAIP3, JUN, JUND, TOB1, TNFSF8, CD69, and CTSW) because these are important mediators involved in regulation for T cells and dysregulation of these genes is associated with autoimmune diseases. Differential expression levels of TNFAIP3, JUN, and TOB1 were validated by RT-qPCR. By flow cytometry, higher expression of CD69 and TNFSF8 was confirmed in CD4+ and CD8+T cells from PNH compared to healthy controls. Conclusion. Using RNA-seq, we identified novel molecular mechanisms and pathways which may underlie the aberrant T cell immune status in PNH. Specific dysregulation of T cell intracellular signaling may contribute to BM failure and the inflammatory environment in PNH. Understanding these pathways may provide new therapeutic strategies to modulate T cell immune responses in BM failure. Disclosures Hosokawa: Aplastic Anemia and MDS International Foundation: Research Funding. Rios:GSK/Novartis: Research Funding. Weinstein:GSK/Novartis: Research Funding. Townsley:GSK/Novartis: Research Funding.

Increased Gene Expression Of The Nr4A Family In Bone Marrow T Cells Of Patients With Early Stage Acquired Aplastic Anemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1238-1238 ◽  
Author(s):  
Kohei Hosokawa ◽  
Takumi Nishiuchi ◽  
Takamasa Katagiri ◽  
Chizuru Saito ◽  
Hiroyuki Maruyama ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Aplastic Anemia ◽  
Gene Expression Profiling ◽  
Expression Profiling ◽  
Early Stage ◽  
Healthy Individuals ◽  
Expression Levels

Abstract Background Although T-cell cytokines are believed to play an essential role in the development of acquired aplastic anemia (AA), it remains unclear which cytokines or cytokine signals trigger hematopoietic stem cell (HSC) suppression in AA. Gene expression profiling of bone marrow (BM) T cells may be useful for identifying inciting molecules, but the BM T cells in patients with severe AA are inappropriate subjects for such studies, because their gene expression profile suffers changes due to the T-cell exposure to various cytokines. Some patients with moderate AA present with profound thrombocytopenia and an increase in glycosylphophatidylinositol-anchored protein (GPI-AP)-deficient cells without significant neutropenia and anemia, and eventually progress to pancytopenia that fulfills the criteria of AA. They usually show a rapid and complete response to cyclosporine (CsA) if treated early after the thrombocytopenia is manifested. Such patients with the “pre-AA” state may serve as ideal subjects to clarify which genes are involved in the development of AA. Objectives/methods To identify the genes closely associated with the development of AA responsive to CsA, we carried out gene expression profiling of the BM mononuclear cells (BMMCs) obtained from 6 un-transfused patients with BM failure with increased GPI-AP-deficient cells (two with the pre-AA state and four with moderate AA) and three healthy individuals using a microarray analysis with Agilent Microarrays, and identified genes that were highly expressed in patients' BMMCs at the diagnosis. All patients responded to CsA monotherapy and achieved platelet recovery≥50 x 109/L within one year. Results A total of 1119 genes, including IL17C, CD40LG and NR4A1, were upregulated in the BMMCs of the six patients at onset by at least two-fold the level of healthy individuals. The extent of gene overexpression was more pronounced in two patients with the pre-AA state than in those with MAA (Fig. 1A). A comparison of the gene expression levels in BMMCs at onset with those in BMMCs obtained after achieving the platelet count≥100 x 109/L in two rapid responders to CsA revealed 1858 genes, including TNFSF9, NFATC2 and CCL4, that were upregulated. Of note, all three Nr4A family members, including Nr4a1 (Nur77), Nr4a2 (Nurr1) and Nr4a3 (NOR-1), were upregulated at onset (17.4-, 46.5- and 106.4-fold increases compared to those after CsA therapy; 37.7-, 17.9- and 72.9-fold increases compared to healthy controls, respectively) in patients who obtained the good platelet response within three months of CsA therapy (rapid responders). The expression levels of Nr4A genes in four AA patients who showed a slow response to CsA (slow responders) were comparable to those of healthy individuals (Fig. 1B). Conclusions Genes in the Nr4a subfamily are immediate early genes that can be induced by a wide array of stimuli, including growth factors and inflammatory signals. Nr4a2 is reportedly upregulated in T cells of patients with multiple sclerosis via the calcineurin–NFAT pathway, and plays a pivotal role in mediating cytokine production, such as the production of IL-17 from pathogenic T cells. In patients with early stage AA, some inflammatory stimuli may induce the overexpression of Nr4A, leading to the development of BM failure. Quantification of the Nr4A expression of BMMCs may therefore be useful for diagnosing thrombocytopenia that can progress to AA and also predicting a rapid response to CsA therapy in patients with the early stage AA. Disclosures: Nakao: Alexion: Honoraria, Research Funding.

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