Project description:Patients with sickle cell anemia (SCA) show higher levels of circulating pro-inflammatory cytokines and endothelial activation markers compared to healthy peers and altered blood transcriptome profiles. However, the characterization of the whole-blood transcriptome profile in response to exercise has not been evaluated in children with SCA. Twenty-three children/adolescents with SCA (10-19 years old) and 17 healthy controls (10-22 years old) performed eight 2-min bouts of cycle ergometry interspersed with 1-min rest intervals (50% or 70% of the peak workload achieved during the cardiopulmonary exercise test). Whole-blood transcriptome profile (RNA-seq) was performed before and after the exercise protocol. Following exercise, gene pathways associated with innate immunity were altered in both children with SCA and controls. Our results shed light on the common and different immune responses to acute exercise at the molecular level in children with SCA and healthy controls.
Project description:We hypothesized that miRNA regulation may be invloved in hydroxyurea-mediated fetal hemoglobin induction. Microarray analysis was utilized as an initial screening tool to determine differential miRNA expression in CD71+ erythroid cells comparing cells from control individuals without sickle cell anemia to patients with sickle cell anemia prior to treatment with hydroxyurea and patients receiving the maximum tolerated dose (MTD) of hydroxurea. CD71+ cells were isolated from whole blood of control individuals (n=2), pediatric patients without hydroxyurea treatment (n=3) and pediatric patients at hydroxyurea MTD (n=3). All 8 samples were analyzed for miRNA expression.
Project description:We hypothesized that miRNA regulation may be invloved in hydroxyurea-mediated fetal hemoglobin induction. Microarray analysis was utilized as an initial screening tool to determine differential miRNA expression in CD71+ erythroid cells comparing cells from control individuals without sickle cell anemia to patients with sickle cell anemia prior to treatment with hydroxyurea and patients receiving the maximum tolerated dose (MTD) of hydroxurea.
Project description:Genetic differences in endothelial biology could underlie development of phenotypic heterogeneity amongst individuals afflicted with vascular diseases. We obtained BOEC (blood outgrowth endothelial cells) from 20 subjects with sickle cell anemia (age 4-19) shown to be either at-risk (n=11) or not-at-risk (n=9) for ischemic stroke due to, respectively, having or not having occlusive disease at the Circle of Willis (CoW). Gene expression profiling identified no significant single gene differences between the two groups, as expected. However, analysis of Biological Systems Scores, using gene sets that were pre-determined to survey each of nine biological systems, showed that only changes in inflammation signaling are characteristic of the at-risk subjects, as supported by multiple statistical approaches Experiment Overall Design: We obtained BOEC (blood outgrowth endothelial cells) from 20 subjects with sickle cell anemia (age 4-19) shown to be either at-risk (n=11) or not-at-risk (n=9) for ischemic stroke due to, respectively, having or not having occlusive disease at the Circle of Willis (CoW). To allow power calculations to be done, we performed microarray analysis on BOEC from 27 normal subjects of diverse ages. Gene expression profilings were obtained by using Affymetrix U133A chips
Project description:To analyze expression of inflammatory cytokines in Exhaled Breath Condensates from pediatric patients with sickle cell disease, asthma, sickle cell disease and asthma, and controls
Project description:Sickle cell disease (SCD) is a red blood cell disorder associated with hemolysis and inflammation affecting hematopoietic system homeostasis. While both innate and T cell driven adaptive immune responses are involved in driving SCD-linked inflammation, the involvement of the B cell compartment in the pathophysiology of SCD, especially in pediatric patients, is much less understood. To determine the range of immune changes, we assessed the frequency of twelve immune populations via flow cytometry and levels of B cell related cytokines of the peripheral blood (PB) of pediatric (n=13) and adult (n=12) patients with SCD on hydroxyurea as compared to pediatric (n=5) and adult (n=10) race-matched controls. We also evaluated bulk RNA sequencing of the PB of pediatric SCD patients and controls. The majority of SCD patients carried the hemoglobin SS genotype (92.3% pediatrics and 66.7% adults). Mean Hydroxyurea dose was 23.4 mg/kg and 20.0 mg/kg for pediatric SCD (PSCD) and adult SCD (ASCD), respectively. Laboratory data revealed anemia with mean hemoglobin 9.4 g/dl in PSCD and 9.7 g/dl in ASCD with elevated hemolysis in pediatric and adult SCD with mean reticulocyte count of 7.1% in PSCD and 8.8% in ASCD with mean absolute reticulocyte count (ARC) 203 ± 94 for pediatric SCD and 214 ± 169 for adult SCD. Flow cytometry data revealed 1.9-fold increase (p value 0.012) of CD3+CD4+ cells in pediatric SCD versus pediatric controls; this change was not seen in adult SCD versus adult controls (decrease 17%, p value 0.302). Pediatric SCD patients had 3.5-fold increase (p=0.005) in CD19+ B cells without further increase in differentiated B cells, and this change was not seen in adult SCD versus adult controls (increase 7%, p value 0.815). We performed bulk RNA sequencing on the PB of pediatric patients with SCD as compared to the pediatric controls. Obtained transcriptomic data indicated significant changes in transcripts linked to humoral immunity. Over Representation Analysis (ORA) of cell type revealed upregulation of transcripts linked to pro-B cells (27 genes, top 3:E2F2,RAD51,ASPM) and plasma cells (37 genes, top 3:IGF1,TNFRSF17,DERL3). Analysis of B cell related cytokines revealed significant increase in IL2 (p value 0.013), IL4 (p value 0.026), TNFβ (p value 0.039), and IL13 (p value 0.034) inpediatric SCD versus pediatric controls withno significance for these cytokines in adult SCD versus adult controls. BAFF was elevated in both pediatric and adult SCD versus age-matched controls (p value0.041 and p value 0.005, respectively). In sum, our data indicate significant changes in CD4+ and CD19+ cells andsuggest significant alteration of the B cell maturation in pediatric SCD samples. Our findings point to previously unreported effect of SCD on the humoral immune system in children with SCD treated with hydroxyurea, warranting further investigation.
Project description:The heterozygous inheritance of the sickle cell gene, called sickle cell trait (HbAS), was previously thought to be benign. HbAS is now associated with an increased risk for chronic kidney disease, pulmonary embolisms, and exertional rhabdomyolysis. The role of gene expression, as regulated by microRNAs, in these clinical HbAS associations has not been investigated. We explored the association between HbAS and the differential expression of circultaing (plasma) microRNAs when compared to the normal hemoglobin phenotype (HbAA). In stark contrast, the homozygous inheritance of the sickle cell gene, called sickle cell anemia (HbSS) is a severe disease that was previously associated with early mortality and severe morbidity. With appropriate care and early interventions, people with HbSS have improved life expectancy but still experience significant morbidity including cardiac, kidney, and pulmonary disease. The role of gene expression as regulated by microRNA in these clinical HbSS associations has not been investigated. We also explored whether the presence of HbSS is associated with the differential expression of circultaing (plasma) microRNAs as compared to the normal hemoglobin phenotype (HbAA).