Prevalence of arboviruses in sickle cell disease patients from two different regions of Brazil, the North and Northeast.

Sickle Cell Disease (SCD) is a hereditary disease characterized by extravascular and intravascular hemolysis and clinical variability, from mild pain to potentially life-threatening. Arboviruses include mainly Zika (ZIKV), Chikungunya (CHKV), and Dengue (DENV) virus, and are considered a public and social health problem. The present cross-sectional observational study aimed to investigate the prevalence of arbovirus infection in SCD patients from two Brazilian cities, Salvador and Manaus located in Bahia and Amazonas states respectively. A total of 409 individuals with SCD were included in the study, and 307 (75.06 %) patients tested positive for DENV-IgG, 161 (39.36 %) for ZIKV-IgG, and 60 (14.67 %) for CHIKV-IgG. Only one individual was positive for DENV-NS1 and another for DENV-IgM, both from Salvador. No individuals had positive serology for ZIKV-IgM or CHIKV-IgM. Arbovirus positivity by IgG testing revealed that the SCD group presented high frequencies in both cities. Interestingly, these differences were only statistically significant for ZIKV-IgG (p = 0.023) and CHIKV-IgG (p = 0.005) among SCD patients from Manaus. The reshaping of arbovirus from its natural habitat by humans due to disorderly urban expansion and the ease of international Mobility has been responsible for facilitating the spread of vector-borne infectious diseases in humans. We found the need for further studies on arboviruses in this population to elucidate the real association and impact, especially in acute infection. We hope that this study will contribute to improvements in the personalized clinical follow-up of SCD patients, identifying the influence of arbovirus infection in severe disease manifestations.

Prevalence of arboviruses in sickle cell disease patients from two different regions of Brazil, the North and Northeast

ABSTRACT Sickle Cell Disease (SCD) is a hereditary disease characterized by extravascular and intravascular hemolysis and clinical variability, from mild pain to potentially life-threatening. Arboviruses include mainly Zika (ZIKV), Chikungunya (CHKV), and Dengue (DENV) virus, and are considered a public and social health problem. The present cross-sectional observational study aimed to investigate the prevalence of arbovirus infection in SCD patients from two Brazilian cities, Salvador and Manaus located in Bahia and Amazonas states respectively. A total of409 individuals with SCD were included in the study, and 307 (75.06 %) patients tested positive for DENV-IgG, 161 (39.36 %) for ZIKV-IgG, and 60 (14.67 %) for CHIKV-IgG. Only one individual was positive for DENV-NS1 and another for DENV-IgM, both from Salvador. No individuals had positive serology for ZIKV-IgM or CHIKV-IgM. Arbovirus positivity by IgG testing revealed that the SCD group presented high frequencies in both cities. Interestingly, these differences were only statistically significant for ZIKV-IgG (p = 0.023) and CHIKV-IgG (p = 0.005) among SCD patients from Manaus. The reshaping of arbovirus from its natural habitat by humans due to disorderly urban expansion and the ease of international Mobility has been responsible for facilitating the spread of vector-borne infectious diseases in humans. We found the need for further studies on arboviruses in this population to elucidate the real association and impact, especially in acute infection. We hope that this study will contribute to improvements in the personalized clinical follow-up of SCD patients, identifying the influence of arbovirus infection in severe disease manifestations.

Effectiveness of Pharmacokinetic-Guided Hydroxyurea Dose Individualization in Patients with Sickle Cell Anemia: A Mini-Review.

Inconsistent therapeutic responses have been observed among patients with sickle cell anemia (SCA) undergoing hydroxyurea (HU) following the adoption of the standardized protocol. Moreover, this treatment regimen necessitates a prolonged period to reach the maximum tolerated dose in which beneficial therapeutic effects are observed in most SCA patients. To overcome this limitation, several studies have performed HU dose adjustments in SCA patients based on individualized pharmacokinetic profiles. The present systematic mini-review aims to select and analyze published data to present an overview of HU pharmacokinetics studies performed in SCA patients, as well as evaluate the effectiveness of the dose adjustment strategy. A systematic search was performed in the Embase, Pubmed, Scopus, Web of Science, Scielo, Google Scholar, and the Virtual Library of Health databases from December 2020 to August 2022, with a total of five studies included. Inclusion criteria consisted of studies in which the dose adjustment was performed in SCA patients based on pharmacokinetic parameters. Quality analyzes were performed using QAT, while data synthesis was performed according to the Cochrane Manual of Systematic Reviews of Interventions. Analysis of the selected studies revealed improved HU treatment effectiveness using personalized dosages in SCA patients. Moreover, several laboratory parameters were utilized as biomarkers of the HU response, and methods designed to simplify the adoption of this practice were presented. Despite the scarcity of studies on this topic, HU-personalized treatment based on individualized pharmacokinetic profiles represents a viable alternative for SCA patients who are candidates for HU therapy, especially for pediatric patients. Registration number: PROSPERO CRD42022344512.

Role of paraoxonase 1 activity and PON1 gene polymorphisms in sickle cell disease.

Sickle cell disease (SCD) patients often exhibit a dyslipidemic sub-phenotype. Paraoxonase 1 (PON 1) is a serum glycoprotein associated with the high-density lipoproteins cholesterol (HDL-C), and variability in PON1 activity depends on the PON1 genotypes. We investigated the influence of PON1c.192Q > R and PON1c.55L > M polymorphisms on PON1 activity and laboratory parameters and the association between PON1 activity and clinical manifestations in SCD patients. We recruited 350 individuals, including 154 SCD patients and 196 healthy volunteers, which comprised the control group. Laboratory parameters and molecular analyses were investigated from the participants' blood samples. We have found increased PON1 activity in SCD individuals compared to the control group. In addition, carriers of the variant genotype of each polymorphism presented lower PON1 activity. SCD individuals carrying the variant genotype of PON1c.55L > M polymorphism had lower platelet and reticulocyte counts, C-reactive protein, and aspartate aminotransferase levels; in addition to higher creatinine levels. SCD individuals carrying the variant genotype of PON1c.192Q > R polymorphism had lower triglyceride, VLDL-c, and indirect bilirubin levels. Furthermore, we observed an association between PON1 activity history of stroke and splenectomy. The present study confirmed the association between PON1c.192Q > R and PON1c.55L > M polymorphisms and PON1 activity, in addition to demonstrate their effects on markers of dislipidemia, hemolysis and inflammation, in SCD individuals. Moreover, data suggest PON1 activity as a potential biomarker related to stroke and splenectomy.

Secretome from human adipose-derived mesenchymal stem cells promotes blood vessel formation and pericyte coverage in experimental skin repair.

Human adipose tissue-derived stem cells (hASC) secretome display various therapeutically relevant effects in regenerative medicine, such as induction of angiogenesis and tissue repair. The benefits of hASC secretome are primarily orchestrated by trophic factors that mediate autocrine and paracrine effects in host cells. However, the composition and the innate characteristics of hASC secretome can be highly variable depending on the culture conditions. Here, we evaluated the combined effect of serum-free media and hypoxia preconditioning on the hASCs secretome composition and biological effects on angiogenesis and wound healing. The hASCs were cultured in serum-free media under normoxic (NCM) or hypoxic (HCM) preconditioning. The proteomic profile showed that pro- and anti-antiangiogenic factors were detected in NCM and HCM secretomes. In vitro studies demonstrated that hASCs secretomes enhanced endothelial proliferation, survival, migration, in vitro tube formation, and in vivo Matrigel plug angiogenesis. In a full-thickness skin-wound mouse model, injection of either NCM or HCM significantly accelerated the wound healing. Finally, hASC secretomes were potent in increasing endothelial density and vascular coverage of resident pericytes expressing NG2 and nestin to the lesion site, potentially contributing to blood vessel maturation. Overall, our data suggest that serum-free media or hypoxic preconditioning enhances the vascular regenerative effects of hASC secretome in a preclinical wound healing model.

Association of laboratory markers and cerebral blood flow among sickle cell anemia children.

Background: Stroke is one of the highest complications of sickle-cell anemia (SCA). The Transcranial Doppler (TCD) has been adopted worldwide as a gold standard method for detecting alterations in the blood velocity in cerebral arteries. In this study, we investigated the association between laboratory parameters and increased cerebral blood flow velocity in Brazilian SCA pediatric patients. Methods: The study included 159 pediatric patients with SCA, submitted to TCD velocity screening, and the time-averaged maximum mean velocity (TAMMV) was determined in the middle cerebral artery (MCA), anterior cerebral artery (ACA), and distal intracranial internal carotid artery (ICA). We compared cerebral blood flow in patients stratified by the following: TCD1-defined as normal, with TAMMV inferior to 170 cm/s; TCD2-conditional, with TAMMV above 170 cm/s, but less than 199 cm/s; TCD3-altered, with TAMMV greater than or equal to 200 cm/s. Results: TAMMV was negatively correlated with age and weight (p < 0.05). Moreover, TAMMV was associated or correlated with reductions in HbF, RBC, hemoglobin, hematocrit, HDL, and haptoglobin and, increases in MCV, MCH, RDW, reticulocytes, WBC, lymphocytes, monocytes, eosinophils, total and indirect bilirubin, LDH, AST, ALT, glucose, ferritin, and AAT (p < 0.05). Conclusion: The current study highlights the importance of the investigation of hemolytic and inflammatory biomarkers for monitoring the clinical outcome of SCA pediatric patients, to avoid acute or chronic stroke. Moreover, glucose and HDL-C appear useful for predicting higher TAMMV.

A Description of the Hemolytic Component in Sickle Leg Ulcer: The Role of Circulating miR-199a-5p, miR-144, and miR-126.

Sickle leg ulcers (SLU) are malleoli lesions with exuberant hemolytic pathophysiology. The microRNAs are potential genetic biomarkers for several pathologies. Thereby, we aimed to assess the expression of circulating miR-199a-5p, miR-144, and miR-126 in association with hemolytic biomarkers in SLU. This cross-sectional study included 69 patients with sickle cell disease, 52 patients without SLU (SLU-) and 17 patients with active SLU or previous history (SLU+). The results demonstrated elevated expression of circulating miR-199a-5p and miR-144 in SLU+ patients while miR-126 expression was reduced. Circulating miR-199a-5p and miR-144 were associated with hemolytic biomarkers such as LDH, indirect bilirubin, AST, GGT, iron, ferritin, RBC, hemoglobin, and NOm, in addition to association with impaired clinical profile of SLU. Furthermore, in silico analyses indicated interactions of miR-199a-5p with HIF1A, Ets-1, and TGFB2 genes, which are associated with vasculopathy and reduced NO. In contrast, miR-126 was associated with an attenuating clinical profile of SLU, in addition to not characterizing hemolysis. In summary, this study demonstrates, for the first time, that hemolytic mechanism in SLU can be characterized by circulating miR-199a-5p and miR-144. The circulating miR-126 may play a protective role in SLU. Thus, these microRNAs can support to establish prognosis and therapeutic strategy in SLU.

Inclusion Complexes of Non-Steroidal Anti-Inflammatory Drugs with Cyclodextrins: A Systematic Review.

Non-steroidal anti-inflammatory drugs (NSAIDs) are one of the most widely used classes of medicines in the treatment of inflammation, fever, and pain. However, evidence has demonstrated that these drugs can induce significant toxicity. In the search for innovative strategies to overcome NSAID-related problems, the incorporation of drugs into cyclodextrins (CDs) has demonstrated promising results. This study aims to review the impact of cyclodextrin incorporation on the biopharmaceutical and pharmacological properties of non-steroidal anti-inflammatory drugs. A systematic search for papers published between 2010 and 2020 was carried out using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) protocol and the following search terms: "Complexation"; AND "Cyclodextrin"; AND "non-steroidal anti-inflammatory drug". A total of 24 different NSAIDs, 12 types of CDs, and 60 distinct inclusion complexes were identified, with meloxicam and ß-CD appearing in most studies. The results of the present review suggest that CDs are drug delivery systems capable of improving the pharmacological and biopharmaceutical properties of non-steroidal anti-inflammatory drugs.

Priapism in sickle cell disease: Associations between NOS3 and EDN1 genetic polymorphisms and laboratory biomarkers.

Priapism is a urologic emergency characterized by an uncontrolled, persistent and painful erection in the absence of sexual stimulation, which can lead to penile fibrosis and impotence. It is highly frequent in sickle cell disease (SCD) associated with hemolytic episodes. Our aim was to investigate molecules that may participate in the regulation of vascular tone. Eighty eight individuals with SCD were included, of whom thirty-seven reported a history of priapism. Priapism was found to be associated with alterations in laboratory biomarkers, as well as lower levels of HbF. Patients with sickle cell anemia using hydroxyurea and those who received blood products seemed to be less affected by priapism. Multivariate analysis suggested that low HbF and NOm were independently associated with priapism. The frequency of polymorphisms in genes NOS3 and EDN1 was not statistically significant between the studied groups, and the presence of the variant allele was not associated with alterations in NOm and ET-1 levels in patients with SCD. The presence of the variant allele in the polymorphisms investigated did not reveal any influence on the occurrence priapism. Future studies involving larger samples, as well as investigations including patients in priapism crisis, could contribute to an enhanced understanding of the development of priapism in SCD.

Sickle Cell Anemia: Variants in the CYP2D6, CAT, and SLC14A1 Genes Are Associated With Improved Hydroxyurea Response.

Differences in hydroxyurea response in sickle cell anemia may arise due to a series of factors with genetic factors appearing to be predominant. This study aims to investigate the effects of single nucleotide polymorphisms in genes encoding drug-metabolizing enzymes and solute carriers on hydroxyurea response, in patients with sickle cell anemia. For that purpose, a total number of 90 patients with sickle cell anemia were recruited, 45 were undergoing hydroxyurea treatment, while 45 were not under the treatment. Association analyses were performed between CYP3A4 (rs2740574), CYP2D6 (rs3892097), CAT (rs7943316 and rs1001179), and SLC14A1 (rs2298720) variants and laboratory parameters. According to our findings, patients with hydroxyurea treatment demonstrated higher HbF levels and a significant improvement in hemolytic, hepatic, inflammatory, and lipid parameters in comparison to those without the treatment. We also found significant associations between the CYP2D6 (rs3892097), CAT (rs7943316 and rs1001179), and SLC14A1 (rs2298720) variants and an improvement of the therapeutic effects, specifically the hemolytic, hepatic, inflammatory, lipid, and renal parameters. In conclusion, our results highlight the importance of the investigated variants, and their strong association with hydroxyurea efficacy in patients with sickle cell anemia, which may be considered in the future as genetic markers.

TGFBR3 Polymorphisms (rs1805110 and rs7526590) Are Associated with Laboratory Biomarkers and Clinical Manifestations in Sickle Cell Anemia.

Individuals with sickle cell anemia (SCA) present chronic anemia, hemolysis, an exacerbated inflammatory response, and heterogeneous clinical complications, which may be modulated by the transforming growth factor beta (TGF-ß) pathway. Thus, we aimed to investigate polymorphisms (rs1805110 and rs7526590) of the transforming growth factor beta receptor III gene (TGFBR3) with regard to laboratory biomarkers and clinical manifestations in individuals with SCA. Hematological, biochemical, immunological, and genetic analyses were carried out, as well as serum endothelin-1 measurements. The minor allele (A) of the TGFBR3 rs1805110 polymorphism was associated with increased hemoglobin, hematocrit, reticulocyte counts, total cholesterol, low-density lipoprotein, uric acid, and endothelin levels, as well as decreased platelet distribution width (PDW) and the occurrence of bone alterations. The minor allele (T) of TGFBR3 rs7526590 was associated with increased red cell distribution width, PDW, alkaline phosphatase, aspartate aminotransferase, total and indirect bilirubin, and lactate dehydrogenase levels, as well as lower ferritin levels and the occurrence of leg ulcers. Our data suggest that the minor allele (A) of TGFBR3 rs1805110 is associated with inflammation and bone alterations, while the minor allele (T) of TGFBR3 rs7526590 is related to hemolysis and the occurrence of leg ulcers.

Investigation of Lipid Profile and Clinical Manifestations in SCA Children.

INTRODUCTION: Clinical complications in sickle cell anemia (SCA) are heterogeneous and involve several molecules. It has been suggested that SCA individuals present a dyslipidemic phenotype and that lipid parameters are associated with severe clinical complications, such as pulmonary hypertension. We sought to investigate associations between lipid parameters and clinical manifestations, as well as other laboratory parameters in a population of pediatric SCA patients. METHODS: Our cross-sectional evaluation included 126 SCA patients in steady state and who were not undergoing lipid-lowering therapy. Hematological and biochemical parameters were characterized, and previous clinical manifestations were investigated. RESULTS: Total cholesterol and low-density lipoprotein cholesterol (LDL-C) levels were increased in patients with a previous history of pneumonia, which also positively correlated with HbS levels. Decreased LDL-C levels were also associated with leg ulcers and anemia. Elevated high-density lipoprotein cholesterol (HDL-C) levels were associated with pain crises, increased viscosity, and decreased hemolysis. Several studies have determined that lipids play a role in the vascular impairment seen in SCA, which was corroborated by our findings. CONCLUSIONS: In sum, our results suggest that total cholesterol, HDL-C, and LDL-C levels are associated with hemolysis and anemia markers and, most importantly, with clinical complications related to vasculopathy in SCA.

An evaluation of ticagrelor for the treatment of sickle cell anemia.

INTRODUCTION: Ticagrelor is an antiplatelet agent approved for the treatment of patients with an acute coronary syndrome or a history of myocardial infarction. Considering the evidence demonstrating that ticagrelor-mediated inhibition of platelet activation and aggregation have beneficial effects in the treatment of thrombotic conditions, clinical studies have been conducted to evaluate the use of this drug for the treatment of sickle cell disease (SCD), demonstrating satisfactory tolerability and safety. AREAS COVERED: Clinical investigation has characterized the pharmacokinetic and pharmacodynamical profile, as well as the efficacy and safety of ticagrelor to prevent painful vaso-occlusive crisis (painful episodes and acute chest syndrome) in SCD patients. EXPERT OPINION: While phase 1 and 2 clinical trials demonstrated satisfactory tolerability and safety, the conclusion of phase 3 clinical trials is crucial to prove the efficacy of ticagrelor as a therapeutic option for the treatment of SCD. Thus, it is expected that ticagrelor, especially in combination with other drugs, will improve the clinical profile and quality of life of patients with SCD.

Hydroxyurea Scavenges Free Radicals and Induces the Expression of Antioxidant Genes in Human Cell Cultures Treated With Hemin.

The excessive release of heme during hemolysis contributes to the severity of sickle cell anemia (SCA) by exacerbating hemoglobin S (HbS) autoxidation, inflammation and systemic tissue damage. The present study investigated the effect of hydroxyurea (HU) on free radical neutralization and its stimulation of antioxidant genes in human peripheral blood mononuclear cells (PBMC) and human umbilical vein endothelial cells (HUVEC) in the presence or absence of hemin. HU (100 and 200 µM) significantly reduced the production of intracellular reactive oxygen species (ROS) induced by hemin at 70 µM in HUVEC. HUVECs treated with HU+hemin presented significant increases in nitric oxide (NO) production in culture supernatants. HU alone or in combination with hemin promoted the induction of superoxide dismutase-1 (SOD1) and glutathione disulfide-reductase (GSR) in HUVECs and PBMCs, and glutathione peroxidase (GPX1) in PBMCs. Microarray analysis performed in HUVECs indicated that HU induces increased expression of genes involved in the antioxidant response system: SOD2, GSR, microsomal glutathione S-transferase (MGST1), glutathione S-transferase mu 2 (GSTM2), carbonyl reductase 1 (CBR1) and klotho B (KLB). Significant increases in expression were observed in genes with kinase activity: protein kinase C beta (PRKCB), zeta (PRKCZ) and phosphatidylinositol-4-phosphate 3-kinase catalytic subunit type 2 beta (PIK3C2B). HU also induced a significant increase in expression of the gene p62/sequestosome (p62/SQSTM1) and a significant decrease in the expression of the transcriptional factor BACH1 in HUVECs. Upstream analysis predicted the activation of Jun, miR-155-5p and mir-141-3p. These results suggest that HU directly scavenges free radicals and induces the expression of antioxidant genes via induction of the Nrf2 signaling pathway.

Sickle cell disease: A distinction of two most frequent genotypes (HbSS and HbSC).

Sickle cell disease (SCD) consists of a group of hemoglobinopathies in which individuals present highly variable clinical manifestations. Sickle cell anemia (SCA) is the most severe form, while SC hemoglobinopathy (HbSC) is thought to be milder. Thus, we investigated the clinical manifestations and laboratory parameters by comparing each SCD genotype. We designed a cross-sectional study including 126 SCA individuals and 55 HbSC individuals in steady-state. Hematological, biochemical and inflammatory characterization was performed as well as investigation of previous history of clinical events. SCA patients exhibited most prominent anemia, hemolysis, leukocytosis and inflammation, whereas HbSC patients had increased lipid determinations. The main cause of hospitalization was pain crises on both genotypes. Vaso-occlusive events and pain crises were associated with hematological, inflammatory and anemia biomarkers on both groups. Cluster analysis reveals hematological, inflammatory, hemolytic, endothelial dysfunction and anemia biomarkers in HbSC disease as well as SCA. The results found herein corroborate with previous studies suggesting that SCA and HbSC, although may be similar from the genetic point of view, exhibit different clinical manifestations and laboratory alterations which are useful to monitor the clinical course of each genotype.

Effect of N(Epsilon)-(carboxymethyl)lysine on Laboratory Parameters and Its Association with ß S Haplotype in Children with Sickle Cell Anemia.

The present study aimed to investigate the association of N ε -carboxymethyllysine (CML) with laboratory parameters and ß S haplotypes in pediatric sickle cell anemia (SCA) patients with or without hydroxyurea (HU) therapy. We included 55 children with SCA (SCAtotal), where 27 were on HU treatment (SCA-HU+) and 28 without HU treatment (SCA-HU-). Laboratory characteristics were determined using electronic methods while CML was measured using competitive ELISA. ß S haplotypes were determined by RFLP-PCR. Significant increases in MCV and MCH and significant decreases in leukocytes, eosinophils, basophils, atypical lymphocytes, lymphocytes, and monocytes were found in SCA-HU+ compared to SCA-HU-. SCA-HU+ presented significant reduction in aspartate transaminase and lactate dehydrogenase and increase in creatinine levels compared to SCA-HU-. CML levels were significantly higher in both SCA-HU+ and SCA-HU- compared to the healthy control. In addition, a negative correlation was found between CML and alanine transaminase in SCA-HU+ and SCAtotal (p < 0.01). A significant association was found between CML levels and ß S haplotypes. The results suggest that CML has a role to play in SCA complications, independent of HU therapy.

Hydroxyurea alters hematological, biochemical and inflammatory biomarkers in Brazilian children with SCA: Investigating associations with ßS haplotype and α-thalassemia.

This study investigated the effects of hydroxyurea (HU) on hematological, biochemical and inflammatory parameters in children with sickle cell anemia (SCA) in association with ßS haplotype and α-thalassemia. We included 22 children with SCA who were followed for an average of 14.5 months. Laboratory parameters were assessed by electronic methods, and molecular analysis was investigated by PCR-RFLP and allele-specific PCR. Results showed significant increases in hemoglobin, HbF, hematocrit, MCV, MCH, glucose, HDL-C and albumin levels, as well as significant decreases in MCHC and AST levels, WBC, neutrophils, eosinophils, lymphocytes and reticulocytes, in children during HU therapy. HbF levels were positively correlated with hemoglobin, hematocrit, MCV and total protein, yet negatively correlated with MCHC, RDW, AAT and AST during HU therapy (p<0.05). Children who carried the Central African Republic haplotype, in response to HU therapy, presented significant increases in hemoglobin, hematocrit, triglycerides and uric acid levels, as well as significant decreases in MCHC, AST and direct bilirubin levels, WBC, neutrophils, eosinophils, lymphocytes and reticulocytes. Those with the Benin haplotype presented increases in HbF and albumin levels, and a reduction in platelet counts (p<0.05). Children with α-thalassemia presented decreased ALT during HU use, while those without this deletion presented increases in hemoglobin, hematocrit, MCV, MCH, HDL-C and albumin, as well as decreases in MCHC, neutrophils, lymphocytes, reticulocytes and AST (p<0.05). Hence, regardless of its use in association with ßS haplotypes or α-thalassemia, HU seems to be linked to alterations in hemolytic, inflammatory, hepatic, lipid and glycemic profiles.

Differential gene expression analysis of sickle cell anemia in steady and crisis state.

Sickle cell anemia is one of the most prevalent genetic diseases worldwide, showing great clinical heterogeneity. This study compared the gene expression patterns between sickle cell anemia pediatric patients in steady state and in crisis state, as compared to age-paired, healthy individuals. RNA sequencing was performed from these groups of patients/controls using Illumina HiSeq 2500 equipment. The resulting differentially expressed genes were loaded into QIAGEN's ingenuity pathway analysis. The results showed that EIF2 pathway and NRF2-mediated oxidative stress-response pathways were more highly activated both in steady state and in crisis patients, as compared to healthy individuals. In addition, we found increased activation of eIF4 and p70S6K signaling pathways in crisis state compared to healthy individuals. The transcription factor GATA-1 was found exclusively in steady state while SPI was found exclusively in crisis state. IL6 and VEGFA were found only in crisis state, while IL-1B was found exclusively in steady state. The regulator effects analysis revealed IgG1 as an upstream regulator in steady state compared to healthy individuals, resulting in invasion of prostate cancer cell lines as the disease/function outcome. For crisis-state patients versus healthy individuals, two networks of regulator effects revealed STAT1, CD40LG, TGM2, IRF7, IRF4, and IRF1 acting as upstream regulators, resulting in disease/function outcomes, including engulfment of cells and aggregation of blood cells and inflammation of joints. Our results indicated genes and pathways that can provide clues on the molecular events involved in the severity of sickle cell disease.