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.

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.

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.

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.

Laboratory and Genetic Biomarkers Associated with Cerebral Blood Flow Velocity in Hemoglobin SC Disease.

Reference values for cerebral blood flow velocity (CBFV) in hemoglobin SC disease (HbSC) have not been established. We aimed to investigate associations between laboratory and genetic biomarkers associated with CBFV in HbSC children. Sixty-eight HbSC children were included; CBFV was analyzed by transcranial Doppler, and the time-averaged maximum mean velocity (TAMMV) was estimated. Hematological, biochemical, immunological, and genetic analyses were performed. TAMMV was negatively correlated with red blood cell count (RBC) count, hemoglobin, hematocrit, and direct bilirubin (DB), yet positively correlated with monocytes and ferritin. We found that children with TAMMV ≥ 128 cm/s had decreased red blood cell distribution width (RDW) and nitric oxide metabolite (NOx) concentration. Children with TAMMV ≥ 143.50 cm/s had decreased hemoglobin and hematocrit, as well as increased ferritin levels. Decreased hemoglobin, hematocrit, RDW, and NOx and increased ferritin were detected in children with TAMMV ≥ 125.75 cm/s. The CAR haplotype was associated with higher TAMMV. In association analyses, RBC, hemoglobin, hematocrit, RDW, monocyte, DB, NOx, and ferritin, as well as the CAR haplotype, were found to be associated with higher TAMMV in HbSC children. Multivariate analysis suggested that high TAMMV was independently associated with hematocrit, RDW, and NOx. Additional studies are warranted to validate the establishment of a cutoff value of 125.75 cm/s associated with elevated TAMMV in HbSC children.

Association of classical markers and establishment of the dyslipidemic sub-phenotype of sickle cell anemia.

BACKGROUND: Sickle cell anemia (SCA) patients exhibit sub-phenotypes associated to hemolysis and vaso-occlusion. The disease has a chronic inflammatory nature that has been also associated to alterations in the lipid profile. This study aims to analyze hematological and biochemical parameters to provide knowledge about the SCA sub-phenotypes previously described and suggest a dyslipidemic sub-phenotype. METHODS: A cross-sectional study was conducted from 2013 to 2014, and 99 SCA patients in steady state were enrolled. We assessed correlations and associations with hematological and biochemical data and investigated the co-inheritance of -α3.7Kb-thalassemia (-α3.7Kb-thal). Correlation analyses were performed using Spearman and Pearson coefficient. The median of quantitative variables between two groups was compared using t-test and Mann-Whitney. P-values <0.05 were considered statistically significant. RESULTS: We found significant association of high lactate dehydrogenase levels with decreased red blood cell count and hematocrit as well as high levels of total and indirect bilirubin. SCA patients with low nitric oxide metabolites had high total cholesterol, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol and reduced very low-density cholesterol, triglycerides, direct bilirubin level and reticulocyte counts. In SCA patients with high-density lipoprotein cholesterol greater than 40 mg/dL, we observed increased red blood cell count, hemoglobin, hematocrit, and fetal hemoglobin and decreased nitric oxide metabolites levels. The presence of -α3.7Kb-thal was associated with high red blood cell count and low mean corpuscular volume, mean corpuscular hemoglobin, platelet count and total and indirect bilirubin levels. CONCLUSIONS: Our results provide additional information about the association between biomarkers and co-inheritance of -α3.7Kb-thal in SCA, and suggest the role of dyslipidemia and nitric oxide metabolites in the characterization of this sub-phenotype.

CIPROFLOXACIN RESISTANCE PATTERN AMONG BACTERIA ISOLATED FROM PATIENTS WITH COMMUNITY-ACQUIRED URINARY TRACT INFECTION.

OBJECTIVE: To identify the main bacterial species associated with community-acquired urinary tract infection (UTI) and to assess the pattern of ciprofloxacin susceptibility among bacteria isolated from urine cultures. METHODS: We conducted a retrospective study in all the patients with community-acquired UTI seen in Santa Helena Laboratory, Camaçari, Bahia, Brazil during five years (2010-2014). All individuals who had a positive urine culture result were included in this study. RESULTS: A total of 1,641 individuals met the inclusion criteria. Despite the fact that participants were female, we observed a higher rate of resistance to ciprofloxacin in males. The most frequent pathogens identified in urine samples were Escherichia coli, Klebsiella pneumoniae and Staphylococcus saprophyticus. Antimicrobial resistance has been observed mainly for ampicillin, sulfamethoxazole + trimethoprim and ciprofloxacin. Moreover, E. coli has shown the highest rate of ciprofloxacin resistance, reaching 36% of ciprofloxacin resistant strains in 2014. CONCLUSION: The rate of bacterial resistance to ciprofloxacin observed in the studied population is much higher than expected, prompting the need for rational use of this antibiotic, especially in infections caused by E. coli. Prevention of bacterial resistance can be performed through control measures to limit the spread of resistant microorganisms and a rational use of antimicrobial policy.

Association of homocysteine and inflammatory-related molecules in sickle cell anemia.

OBJECTIVE: Investigate the role of homocysteine (Hcy), Th17-related cytokines, and adhesion molecules in the inflammatory state seen in the sickle cell anemia (SCA). METHODS: We studied the Hcy, interleukin (IL)-17, and transforming growth factor ß (TGF-ß) cytokine levels of 62 SCA patients, as well as the expression levels of inflammatory and endothelial activation markers. RESULTS: We found significant associations between Hcy levels and increased expression of IL-17 and TGF-ß among SCA patients, and a positive significant correlation between Hcy and soluble vascular cellular adhesion molecules (sVCAM). SCA individuals had raised IL-17 levels when compared with controls. DISCUSSION: These results suggest a possible role of Hyc in the induction of TGF-ß and IL-17. Other authors proposed that Hcy may contribute to the initiation and progression of vascular disease by monocyte activation, resulting in the secretion of cytokines that amplify the inflammatory response. The role of Hcy in cytokine production and oxidative stress in the endothelium may explain the increase of sVCAM expression and, the vascular activation currently described among the SCA individuals with the highest Hcy serum levels. The chronic inflammation was observed in hyperhomocysteinemic mice, with an increased expression of VCAM-1 and plasma levels of tumor necrosis factor-alpha, showing an association of this inflammatory molecule and vascular changes. CONCLUSION: Our findings suggest that the increased levels of IL-17,Hcy and sVCAM contributes contributes to the vascular inflammation and activation presented by SCA patients, which probably have an important role in vaso-occlusion. On the basis of the presented data, IL-17 and Hcy might be considered as important components in the pathogenesis of SCA.

Neutrophil-derived microparticles induce myeloperoxidase-mediated damage of vascular endothelial cells.

BACKGROUND: Upon activation neutrophil releases microparticles - small plasma membrane vesicles that contain cell surface proteins and cytoplasmic matter, with biological activities. In this study we investigated the potential role of myeloperoxidase in the endothelial cell injury caused by neutrophil-derived microparticles. RESULTS: Microparticles were produced by activating human neutrophils with a calcium ionophore and characterized by flow cytometry and transmission and scanning electron microscopy. Myeloperoxidase activity was measured by luminol-dependent chemiluminescence. Neutrophil microparticles-induced injuries and morphological alterations in human umbilical vein endothelial cells (HUVECs) were evaluated by microscopy and flow cytometry. Neutrophil microparticles were characterized as structures bounded by lipid bilayers and were less than 1 µm in diameter. The microparticles also expressed CD66b, CD62L and myeloperoxidase, which are all commonly expressed on the surface of neutrophils, as well as exposition of phosphatidylserine. The activity of the myeloperoxidase present on the microparticles was confirmed by hypochlorous acid detection. This compound is only catalyzed by myeloperoxidase in the presence of hydrogen peroxide and chloride ion. The addition of sodium azide or taurine inhibited and reduced enzymatic activity, respectively. Exposure of HUVEC to neutrophil microparticles induced a loss of cell membrane integrity and morphological changes. The addition of sodium azide or myeloperoxidase-specific inhibitor-I consistently reduced the injury to the endothelial cells. Taurine addition reduced HUVEC morphological changes. CONCLUSIONS: We have demonstrated the presence of active myeloperoxidase in neutrophil microparticles and that the microparticle-associated myeloperoxidase cause injury to endothelial cells. Hence, the microparticle-associated myeloperoxidase-hydrogen peroxide-chloride system may contribute to widespread endothelial cell damage in conditions of neutrophil activation as observed in vasculitis and sepsis.

Mycobacterium tuberculosis-induced neutrophil ectosomes decrease macrophage activation.

BACKGROUND: The existence of ectosome-like microvesicles released by neutrophils was proposed a few decades ago. Other studies revealed that the innate immune response during mycobacterial infection is accompanied by an intense migration of neutrophils to the site of infection, which may be important during the acute phase of tuberculosis. We found that the ectosomes derived from infected neutrophils are biologically active and can influence the survival of Mycobacterium tuberculosis within macrophages. METHODS: Mycobacteria were cultured on supplemented Middlebrook-7H9 broth. All strains were grown to the exponential phase and quantitated by serial dilution. Human neutrophils and macrophages were infected with mycobacteria. Ectosomes from neutrophils were isolated post-infection and characterized by transmission electron microscopy and flow cytometry. To determine whether these microvesicles influenced mycobactericidal activity, mycobacteria-infected macrophages were treated with isolated ectosomes. RESULTS: Ectosomes were released from neutrophils infected with mycobacteria. These ectosomes were derived from neutrophil plasma membrane and a small proportion stained with PKH26. These microvesicles, when incubated with infected macrophages, influenced antimycobacterial activity. CONCLUSIONS: This is the first study to demonstrate that ectosomes that are shed from infected neutrophils influence mycobactericidal activity in macrophages in vitro, suggesting that these microvesicles have biological significance. Nevertheless, major gaps in our knowledge of microvesicle biology remain.