ALBERT

Description: Introduction: Several studies reported combined contraceptives influence in hemostatic and lipid profile besides the concern about the thromboembolism and cardiovascular risks due to the steroids hormones use. As women with hemoglobin (Hb) variants have a pre-existing inflammatory condition and considering the high frequency of hemoglobin variant worldwide, this study aims to evaluate the association of hematological, lipid, glicemic, inflammatory and hemostatic profiles in women using combined oral contraceptives and carriers of hemoglobin variants. Methods: We performed a cross-sectional study including 591 women in reproductive-age. We investigated their hemoglobin profile and COCs use. Of them, we included 60 women with HbAA, 21 with HbAC, 25 with HbAS and 7 with HbSC profiles, all of them combined oral contraceptives users. Among those combined oral contraceptives nonusers, 9 were HbAC and 19 HbSC. We evaluated fasting serum glucose, triglycerides, total cholesterol, low-density lipoprotein cholesterol (LDL-c), high-density lipoprotein cholesterol (HDL-c), C-reactive protein (CRP), fibrinogen, D-dimer and hematological profile among the studied groups. This study was conducted in accordance and approved by the Research Ethics Committee of the Fundação Oswaldo Cruz - FIOCRUZ, Brazil; and also with the Helsinki Declaration of 1975, and its revisions. Mann-Whitney U tests were used to compare two groups of values within the same variable. Results: We observed significant differences in some hematological and cardiometabolic parameters in women carriers of different Hb variants and using COCs. We found relevant increases in CRP levels in HbSC and HbAC women that seem to be associated with different types of progestins present in combined oral contraceptives formulations. Also, combined oral contraceptives use seems to be associated with decreased HDL-c levels in HbAC women. Otherwise, D-dimer levels were increased in all women with Hb variants, independently of the contraceptive use. Conclusions: Although combined oral contraceptive remains an important method to prevent unintended pregnancy, our data suggest that contraception in women carriers of Hb variants, including those in heterozygosis, should be carefully evaluated, especially, considering the pre-existent inflammatory and pro-thrombotic conditions that together with combined oral contraceptive use may result in additional health problems, such as cardiovascular and thromboembolic diseases. Disclosures No relevant conflicts of interest to declare.

Description: Introduction Sickle cell anemia (SCA) is a genetical hemolytic disorder defined as chronic inflammatory disease. Nitrite (NO-2) in SCA (HbSS) patients may be associated with the hemolytic process while fetal hemoglobin (Hb F) presents protective effect in these patients. Serum NO-2 interferes with the role of Hb F in reducing the hemolytic process contributing to vaso-occlusive crises. Aims The aim of this study was to evaluate the correlation of the serum levels of nitrite with fetal hemoglobin, low density lipoprotein cholesterol (LDL-C) and triglycerides levels in HbSS patients in steady-state of the Centro de Referência em Doença Falciforme de Itabuna (CERDOFI), Bahia, Brazil. Methods Forty-two patients diagnosed with SCA were included at baseline. Diagnosis was confirmed by high-performance liquid chromatography (HPLC). Serum levels of nitrite were performed using Griess reaction, and fetal hemoglobin, LDL cholesterol and triglycerides levels were measured by biochemical methods. The experimental protocol was approved by the Researcher Board Committee on Human Research, Gonçalo Moniz Research Center and informed consents were signed by patients. Results Our results showed a significant increase in NO-2 (p

Description: Introduction: MPs are stable units that express on their surface specific proteins of the cells that were originated. In this sense, neutrophils MPs have a singular importance since they present in their surface myeloperoxidase (MPO), an enzyme that are involved with vascular inflammatory conditions. Studies have shown that the use of HU, currently the only pharmacological drug approved to treat SCA patients, leads to reduction in count neutrophils, monocytes and reticulocytes, and the slight increase in total hemoglobin concentration. However, some studies have shown that MPs levels are not affected by HU treatment. Recently, our group showed that nod-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome and interleukin (IL)-1β could contribute to the inflammatory condition in SCA patients. This study tested the hypothesis that neutrophils challenged with serum of sickle cell anemia (SCA) patients, treated with hydroxyurea (HU), decrease neutrophils microparticles (MPs) production. Therefore, the aim of this study was to evaluate the inflammatory state of neutrophils from healthy volunteers by NLRP3 and IL1B gene expression and of SCA patients treated or untreated with HU and to measure the production of MPs derived from neutrophils challenged with SCA patient's serum, treated or not with HU. Methods: We enrolled 10 SCA patients (all in steady state with age of 8.0 ± 1.9 years), from the Fundação de Hematologia e Hemoterapia do Estado da Bahia (HEMOBA). Neutrophils were isolated from one healthy donor as previously described (Pitanga et al, 2014). The protein concentration of neutrophils MPs was evaluated using the BCA method (Thermo Scientific,Waltham, MA, USA) according to the manufacturer's protocol and described by Pitanga et al (2014). NLRP3 and IL1Bgene expressions genes were performed by real time PCR with SYBR Green assays from neutrophils RNA in trizol reagent. Relative expression was estimated through the 2ddct method, with GAPDH and ACTB genes. Serum biochemical analysis was evaluated using commercially available biochemical kits. This study was conducted in accordance and approved by the Research Ethics Committee of the Fundação Oswaldo Cruz - FIOCRUZ, Brazil; and also with the Helsinki Declaration of 1975, and its revisions. The informed consents were signed by patients or official responsible. Results: Our findings showed that NLRP3 (6.95 ± 1.77)and IL1B (6.46 ± 3.31) genes are highly expressed in neutrophils of SCA patients comparing to healthy volunteers (2.43 ± 0.73, p=0.0286; 2.65 ± 1.30, p=0.0081 respectively). Additionally, neutrophils from SCA patients treated with HU decrease NLRP3 gene expression (4.06 ± 1.12; p= 0.0357), but it was not observed for IL1B gene expression (6.03 ± 3.13; p=0.3472). We demonstrated that serum from SCA patients increased neutrophils MPs production (181.90 ± 53.00) comparing to healthy volunteer's serum (37.63 ± 13.06; p=0.0011). However, HU treatment decreased this scenario (30.33 ± 13.70; p=0.0022). Conclusions: This study highlighted that HU acts decreasing NLRP3 gene expression and neutrophils MPs production suggesting that NLRP3 protein could participate of cellular activation and production of MPs, contributing to the maintenance of inflammation as well as vascular activation. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: Sickle cell anemia (SCA) presents a chronic inflammatory condition associated with vaso-occlusive painful episodes and intravascular hemolysis. For the innate response, a family of cellular receptors called Toll-like receptors (TLRs) has multiples functions, which may lead to several different pathways that can modulate the SCA pathogenesis. For example, TLR expression is not restricted to leukocytes, but has been reported in multiple cell types, including endothelial cells, implying that the role of the TLR pathway may not be limited to immune response. TLRs are thought to play important role in the maintenance of the inflammatory status observed in these patients. We aim to evaluate the role that lysed red blood cells (RBC) from SCA patients play in TLR2, TLR4, TLR5 and TLR9 gene expression in peripheral blood mononuclear cells (PBMC) in presence or absence of hydroxyurea (HU). Methods: Ten SCA patients in steady-state (age 10.3 ± 4.6 years) and 7 healthy volunteers (age 14.5 ± 5.2 years) were recruited at the Fundação de Hematologia e Hemoterapia da Bahia (HEMOBA). PBMC were isolated from one healthy donor for cell cultures challenged by lysed RBC of SCA patients (SS-RBC) and RBC of healthy volunteers (AA-RBC), in presence or absence of HU. TLRs gene expressions were performed by qPCR. Serum biochemical analysis was evaluated using commercially available biochemical kits. This study was approved by the Research Ethics Committee of the Oswaldo Cruz Foundation, and was developed in accordance with the Declaration of Helsinki of 1975 and its revisions. All subjects or their legal guardians agreed to collect the biological sample after read and sign the informed consent. Results: We observed that TLR4 (185.0 ± 37.51, p=0.0167) and TLR9 (1.80 ± 1.51, p=0.0394) were higher expressed in PBMC culture challenged by lysed SS-RBC than by lysed AA-RBC (148.5 ± 21.43, p=0.0238; 0.38 ± 0.29, p=0.3429 respectively), whereas PBMC challenged by lysed SS-RBC had TLR2 (1.56 ± 0.34, p=0.0167) and TLR5 (1.48 ± 0.50, p=0.0043) gene expression similar to lysed AA-RBC (1.61 ± 0.30, p=0.0238; 1.55 ± 0.23, p=0.0079 respectively). Surprisingly, HU treatment did not modulate TLR expression. It is known that hemolytic byproducts such as heme and hemoglobin are able to bind TLR4 and TLR9. Conclusion: Despite the global relevance of SCA, mechanisms that contribute to the disease severity and heterogeneity are poorly understood, even though that is the same underlying genetic mechanism. In an attempt to contribute to the field, our data reinforce the hypothesis that lysed RBCs, especially lysed SS-RBCs, act as danger signals, stimulating TLR expression and contributing to inflammation. This study highlighted that HU does not prevent TLR-dependent inflammation, pointing out the need to develop new drugs that act with different mechanisms of action of those observed for HU. Disclosures No relevant conflicts of interest to declare.

Description: INTRODUCTION: Sickle cell disease (SCD) is characterized by the hemoglobin S (HbS) presence associated with other hemoglobin variants, as found in the hemoglobin SC disease (HbSC), or with globin chain synthesis defects, and sickle cell anemia (SCA) is the most severe form of SCD. Among clinical manifestations in SCD it has been highlight the stroke, which is the main cause of death among patients. Recent studies have associated the presence of gene polymorphisms with the stroke risk in SCA individuals. Several genetic markers have been previously associated with stroke risk, but many of the results remain controversial once they could not completely elucidate the effect of individuals' genetic heterogeneity in the stroke development. Thus, we sought to investigate genetic polymorphisms through genome-wide association among SCD patients with stroke risk. METHODS: We selected four SCA individuals and four HbSC individuals based on the transcranial Doppler (TCD) values in according to the velocities described by Adams et al and Deane et al respectively. Patients were matched by sex and age and are followed in the Pediatric Cerebrovascular Disease Outpatient Center at the Hospital Universitario Professor Edgard Santos of the Universidade Federal da Bahia. This study was approved by the Research Board of the Secretaria de Saúde do Estado da Bahia, and all experimental protocol were performed in accordance with the ethical standards of national research committee and with the Helsinki Declaration of 1975 and its later amendments. TCD was performed in all subjects included in the study, and the highest velocity found was assessed in the middle cerebral arteries and distal intracranial internal carotid. Genomic DNA was extracted from peripheral blood and the DNA concentration was evaluated and used for SNP analysis in an Illumina Human Omni5-4 v.1.1 BeadChip kit, and raw intensities were analyzed in GenomeStudio Software. We select SNPs showing different genotypes when individuals were pairwise compared according to TCD velocities such is showed in the Figure 1. We evaluate the SNPs using SNPnexus online tool and the UCSC, SIFT and POLYPHEN categories. The next step consisted of running these selected SNPs in the NHGRI-EBI Catalog of published genome-wide association studies with p-values 〈 1.0 x 10-5. Additionally, we used STRING database to construct a network of protein-protein interactions. RESULTS: Using the UCSC category, we selected 3698, 3851 and 3528 unique SNPs in coding regions in non-synonymous for corresponding amino acid change for the three comparisons of SCA individuals respectively: abnormal TCD vs normal TCD, abnormal TCD vs conditional TCD and abnormal TCD vs conditional high TCD. Using the SIFT and POLYPHEN categories we selected the SNPs with a highly confident prediction to be damaging and SNPs predicted to be probably damaging, and we found 551, 505 and 598 unique SNPs and 424, 393 and 425 unique SNPs, in TCD-SCA individuals respectively. The next step was to select the common SNPs to perform Genome-wide association study (GWAS) classification. The same strategy used for TCD-SCA was performed to TCD-HbSC groups of individuals. We identified 3654, 3722 and 3847 as unique non-synonymous SNPs predicted by UCSC to be located in genomic coding regions respectively. Using the SIFT category 429, 447 and 418 unique SNPs were found in the TCD-HbSC groups of individuals respectively. PolyPhen classification was used and we found 517, 539 and 508 unique SNPs in the TCD-HbSC groups respectively. The next step was to perform GWAS classification. Results obtained utilizing the GWAS showed that each pair of individuals had a different number of SNPs identified (Table 1). The network of protein-protein interactions for the three conditions of SCA individuals and HbSC individuals are shown in the Figure 2. CONCLUSIONS: We suggest that the DOCK6 rs2278426, TYR rs1042602, CYP4F2 rs2108622, MST1 rs3197999, OR51B5/6 rs5006884, THADA rs7578597, FUT2 rs602662, MTHFR rs1801133, TSEN15 rs1046934, CFB rs12614 and ABCG5 rs6756629SNPs may be candidate variants to be further investigated in HbSC individuals with high speed of cerebral blood flow. We suggest that the SLCO1B1 rs4149056, PRIM1 rs2277339, APOB rs676210, TYK2 rs12720356, TSEN15 rs1046934, CYP4F2 rs2108622 and MST1 rs3197999 as candidate SNPs to be further investigated in SCA with high cerebral blood flow velocities. Disclosures No relevant conflicts of interest to declare.

Description: INTRODUCTION: Clinical complications in hemoglobin SC disease (HbSC) are mild compared to sickle cell anemia (SCA), the most severe type of sickle cell disease. However, HbSC individuals have 100 times greater risk of stroke compared to the healthy population. The lack of studies to determine reference values for transcranial Doppler (TCD) velocities in HbSC is still a limitation considering that we have been used TCD velocities previously determined for the SCA and having as standard the one described by Adams et al. (1992). Other studies described cerebral blood flow velocities (CBFV) for HbSC individuals, and considered as abnormal values that exceed 128 cm/s and 143.5 cm/s respectively. Studies show the association of abnormal TCD with distinct laboratorial biomarkers, despite the controversial results. To our knowledge, there are no published studies based on specific biomarkers and abnormal CBFV for HbSC individuals. Thus, we aim to investigate the association of genetic, hematological and biochemical data with CBFV in HbSC. METHODS: A cross-sectional study comprising 68 HbSC individuals, with an average age of 6.96 ± 3.90 years, whom 40 (58.82 %) were female, attended the Pediatric Cerebrovascular Disease Outpatient Center at the Hospital Universitario Professor Edgar Santos (HUPES) of the Universidade Federal da Bahia (UFBA) was conducted. All procedures were in accordance with the 1964 Helsinki declaration and its later amendments. The CBFV was estimated with TCD, and the time-averaged maximum mean velocity (TAMMV) in the middle cerebral arteries, anterior cerebral artery and distal intracranial internal carotid was assessed. Hematological and biochemical parameters, nitric oxide (NO) measurement and genetic polymorphisms in methylenetetrahydrofolate reductase (MTHFR) 677C〉T (rs1801133), vascular cell adhesion molecule (VCAM)-833T〉C (rs1041163)and VCAM 1238G〉C genes, α3.7Kb thalassemia (-α3.7-thal) and Beta S (βS) haplotypes were investigated. Statistical analyses were performed using SPSS version 18.0 software, Graphpad Prism version 6.0 and JMP software. P values 15.55%) and NO (〈 29.86 uM) were independently associated with TAMMV values higher than 125.75 cm/s (75th percentile) (Table 2). CONCLUSIONS: We suggest that RBC, Hb, Ht, RDW, monocyte, direct bilirubin, NO, ferritin, polymorphism on the MTHFR 677C〉T gene and the absence of α3.7Kb thalassemia are associated with TAMMV on HbSC individuals. The TAMMV 125.75cm/s is lower than the those proposed by Deane et al. (2008) and Vieira et al. (2014), but have shown to be associated with hematological and biochemical changes found in both velocities 128 cm/s and 143.50 cm/s respectively. Thus, we also suggest that TAMMV of 125.75 cm/s should be investigated in additional studies as a cut-off point for stroke risk in HbSC individuals. Disclosures No relevant conflicts of interest to declare.

Description: Sickle cell anemia is a severe monogenic disorder characterized by the homozygous state of a single beta globin gene mutation, with heterogeneous clinic characteristics, associated with pro-inflammatory profile, oxidant state and hypercoagulable state. Vessels occlusion is likely initiated by intimal proliferation and amplified by inflammation, excessive adhesion of cells to activated endothelium and vascular tone dysregulation, normally modulated by NFkB pathway both endothelium cells as leukocytes. Herein, we investigated the gene expression of tissue factor (Factor III), oxide nitric synthase (NOS) and endothelial protein C receptor (EPCR) associating with biomarkers of prognosis like hemolysis markers, pro-inflammatory and anti-inflammatory profile. Forty two steady-state sickle cell disease (SCD) patients (16.5 ± 13.5 years, 20 female) from Northeast Brazil were enrolled in this study and were diagnosed in attendance of the outpatients’ clinic of the Sickle Cell Disease Center of Itabuna (CERDOFI). The control group was compound by 20 healthy Brazilian individuals with hemoglobin AA pattern matched by age, years and ethnic origin. The study was approved by the UESC ethical committee and informed consents were signed by patients or official responsible. Using real time quantitative PCR, we analyzed tissue factor, NOS and EPCR gene expression. We also measured hematological and hemoglobin parameters by electronic cell counter and HPLC respectively, biochemical profile was evaluated by colorimetric methodology and cytokine by flow cytometry. The statistical analysis was performed using the Kolmogorov–Smirnov test to access distribution of quantitative variables. Mean values of quantitative variables between groups were compared using an unpaired t-test for data distributed normally and a Mann–Whitney test for non-normal data. Oxide nitric synthase gene expression was increased in SCD patients 1.58-fold compared with healthy controls and higher tissue factor and EPCR gene expression were detected in patients than healthy controls, 4.82-fold and 5.46-fold respectively. The SCD cohort comprised pediatric and adult patients, and the medical history data was search from patient’s records where 95% of patients presented painful crisis at least once. Tissue factor gene expression was positive correlated with expression of NOS (p=0.005) and EPCR (p=0.0001). The increase tissue factor gene expression was detected in patients with high serum levels of bilirubin (p=0.026). Tissue factor gene expression above 75th percentile was associated high concentration of serum creatine kinase and serum calcium (p

Description: Introduction: The pathophysiology of sickle cell anemia (HF) is characterized by hemolytic and intermittent vasoconstrictive events with increased redox status in the vascular microenvironment that favors the chronic inflammation. Objectives: To investigate whether hydroxyurea (HU) acts in the inhibition/minimization of reactive oxygen/nitrogen species (ROS/RNS) and in the modulation of in vitro the antioxidant genes expression. Methods: DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging assay was performed to determine the antioxidant activity of HU using L-ascorbate (L-Asc) and butylated hydroxytoluene (BHT) as antioxidant controls. Human umbilical vein endothelial cell (HUVEC) and peripheral blood mononuclear cells (PBMC) were challenged with HU in the presence or absence of hemin for evaluation the cytotoxicity, inhibition of superoxide anions, nitrate/nitrite production and expression of antioxidant enzymes SOD1 (superoxide dismutase-1); GPx (glutathione peroxidase); GSR (glutathione-disulfide reductase); and HMOX1 (heme oxygenase-1 by RT-qPCR. Microarray analyses were performed on HUVEC stimulated with HU. Results: HU showed scavenging activity (similar to BHT) at concentrations equivalent to that found in the plasma of patients taking the drug (~200 μM). Treatments with HU alone or in combination with hemin did not induce toxicity in PBMC and HUVEC. HU decreases the accumulation of superoxide anions in PBMC in the presence or absence of hemin and the combined treatment of HU with hemin stimulated nitrate/nitrite production in PBMC. HU increases expression of SOD1 and GPx in PBMC and HUVEC. The increase in GSR expression was observed in PBMC and HUVEC submitted to the combined treatment of HU with hemin. HU did not induce HMOX1 expression and did not decrease its expression in combination with hemin in both, PBMC and HUVEC (Figure 1). The microarray assay showed that HU induces the expression of cellular antioxidant components, such as SOD2, GSR, GST1 (glutathione S-transferase-1, GSTM2 (glutathione S-transferase mu 2), MGST1 (microsomal glutathione S-transferase 1), CBR1 (carbonyl reductase 1), protein kinases phosphatidylinositol 3-phosphate C (PRKCB, PRKCZ, PIK3C2B) and p62/SQSTM1 (sequestosome 1) (Table 1). In contrast, a decrease in BACH1 (BTB domain and CNC homolog 1) transcriptional factor expression was observed. Upstream analysis demonstrated prediction of activation for the transcriptional factor JUN and miR-155-5p. Conclusion: The findings indicate that HU can act (i) directly inhibiting on the direct inhibition of ROS/RNS; (ii) inducing nitrite/nitrate production in the presence of hemin; (iii) besides stimulating the antioxidant cellular response by inducing the antioxidant/electrophile response element (ARE/EpRE) mediated by Nrf2 under p62/SQSTM1 regulation. Disclosures No relevant conflicts of interest to declare.

Description: Introduction Chronic and acute inflammatory phenomenon’s can contribute to activate several cells types and may play important role in the steady- and crisis-states of sickle cell anemia (HbSS) patients. Under normal circumstances, the nitric oxide synthase (NOS) constitutively produces low levels of nitric oxide (NO) that are important in the maintenance of vascular homeostasis, but in HbSS this balance is disrupted, mainly because the lack of arginine and the depletion of NO. This molecule also has an anti-inflammatory role in the vascular system and the state of resistance of NO can lead to deregulation of several other factors and interaction with many molecules. The aim of this study was correlate the expression of NOS, vascular cell adhesion molecule (VCAM), c-reactive protein receptor (rCRP) genes and biochemical profile of HbSS patients. Patients and Methods We studied 21 HbSS patients from Northeast Brazil, at steady state and were transfusion free. Blood samples were obtained during regular clinical visits. The expression of NOS, VCAM and rCRP genes were performed by real time PCR with Taqman assays from leucocytes RNA in trizol reagent. Serum biochemical analysis was determined using commercially available biochemical kits. The study was approved by the FIOCRUZ ethical committee and informed consents were signed by patients or official responsible. Results Our results showed a positive correlation among the expression of NOS and VCAM (p= 0.031, r=0.525), NOS and rCRP (p〈 0.001, r=0.816) and VCAM and rCRP (p=0.003, r=0.659). Analyses of biochemical profile of HbSS individuals showed a correlation of NOS and serum calcium (p=0.044, r=0.456) as well as NOS and alkaline phosphatase (p=0.036, r=0.472). Discussion and Conclusion In this study, the correlation among genetic expression of NOS, VCAM and rCRP molecules suggests how factors involved to inflammation in HbSS are linked. In response to inflammation, the liver releases a variety of acute phase proteins. One such product, CRP, serves as a dominating diagnostic marker of inflammation. Chronic inflammatory reflects in the up-regulation of VCAM and VOC susceptibility, can increase the release of alkaline phosphatase, as well as deregulation of NOS expression due to impaired constitutive NOS activity with loss of endothelial NOS dimerization, increased NO scavenging by plasma hemoglobin and superoxide, increased arginase activity, and depleted intravascular nitrite reserves. Also, NOS couples electron flux for NO production by binding calmodulin, which is calcium-dependent, which explain the positive correlation between NOS and serum calcium. Furthermore, additional studies will elucidate the mechanism involved with the interaction of this complex network of molecule and the endothelial activation and dysfunction described in the pathophysiology of HbSS chronic and acute inflammatory. Disclosures: No relevant conflicts of interest to declare.