Darcielle Bruna Dias Elias

Chronic inflammatory state in sickle cell anemia patients is associated with HBB(*)S haplotype.

The chronic inflammatory state in sickle cell anemia (SCA) is associated with several factors such as the following: endothelial damage; increased production of reactive oxygen species; hemolysis; increased expression of adhesion molecules by leukocytes, erythrocytes, and platelets; and increased production of proinflammatory cytokines. Genetic characteristics affecting the clinical severity of SCA include variations in the hemoglobin F (HbF) level, coexistence of alpha-thalassemia, and the haplotype associated with the HbS gene. The different haplotypes of SCA are Bantu, Benin, Senegal, Cameroon, and Arab-Indian. These haplotypes are associated with ethnic groups and also based on the geographical origin. Studies have shown that the Bantu haplotype is associated with higher incidence of clinical complications than the other haplotypes and is therefore considered to have the worst prognosis. This study aimed to evaluate the profile of the proinflammatory cytokines interleukin-6, tumor necrosis factor-α, and interleukin-17 in patients with SCA and also to assess the haplotypes associated with beta globin cluster S (HBB(*)S). We analyzed a total of 62 patients who had SCA and had been treated with hydroxyurea; they had received a dose ranging between 15 and 25 (20.0±0.6)mg/kg/day for 6-60 (18±3.4)months; their data were compared with those for 30 normal individuals. The presence of HbS was detected and the haplotypes of the beta S gene cluster were analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Our study demonstrated that SCA patients have increased inflammatory profile when compared to the healthy individuals. Further, analysis of the association between the haplotypes and inflammatory profile showed that the levels of IL-6 and TNF-α were greater in subjects with the Bantu/Bantu haplotype than in subjects with the Benin/Benin haplotype. The Bantu/Benin haplotype individuals had lower levels of cytokines than those with the Bantu/Bantu haplotype and greater levels than those of subjects with the Benin/Benin haplotype. For IL-17, a slight trend toward decreased levels was observed in the subjects with the Benin/Benin haplotype, when compared to those with the Bantu/Bantu and Bantu/Benin haplotypes; however, this difference was not statistically significant. Our results show that genetic polymorphisms in sickle cell anemia are associated with the inflammatory profile.

L-arginine as an adjuvant drug in the treatment of sickle cell anaemia

Sickle cell disease (SCD) is an inherited disorder of haemoglobin synthesis that is determined by homozygosity of haemoglobin S (HbSS) and characterized by recurring episodes of vaso-occlusion, chronic inflammatory state, chronic haemolysis and progressive vasculopathy with resultant imbalance in the signalling mediated by nitric oxide (NO) (Sullivan et al, 2010). NO is a powerful vasodilator that acts by preventing the adhesion of leucocytes to the endothelium, inhibiting the expression of adhesion molecules and acting as an antiinflammatory substance (Kato et al 2007). In sickle cell anaemia (SCA), arginase – an enzyme present inside red blood cells, is released during haemolysis, catalysing the hydrolysis of L-arginine, the substrate for the production of NO in ornithine and urea, reducing the bioavailability of NO (Kato et al, 2007). The reduction of L-arginine also occurs following the increased consumption of NO as a result of the increase in reactive oxygen species (ROS), generated by the presence of free haemoglobin, ischaemic injury of recurrent reperfusion, pro-inflammatory state, and the high autoxidation of haemoglobin S (HbS) (Kato et al, 2007). Hydroxycarbamide (HC; also known as hydroxyurea) is a cytotoxic, mutagenic, recombinogenic and antineoplastic agent. It has been used to treat SCA by increasing the synthesis of HbF and total haemoglobin and reducing haemolysis (-Morris et al, 2008). HC also acts to reduce the expression of adhesion molecules, with anti-inflammatory and anti-aggregating properties, contributing to the decrease of vaso-occlusive episodes and reducing the need for blood transfusions, the frequency of hospitalizations, and mortality rate (-Morris et al, 2008). HC has also been attributed to affect NO metabolism, increasing production thereof via the cGMP cycle and consequently increasing HbF (Morris et al, 2003). HC therapy increases utilization of the arginine substrate, for the production of NO, by the activity of NOS (Nahavandi et al, 2000). L-arginine, a semi-essential amino acid, is a substrate for the endothelial nitric oxide synthase (eNOS) enzyme for the production of NO, and is reduced in SCA patients, thereby limiting the effectiveness of HC (Sullivan et al, 2010). The reduced overall bioavailability of arginine, measured by low plasma levels of L-arginine or L-ornithine, or L-ornithine and L-citrulline, is associated with increased mortality in SCA patients (Morris et al, 2005). The reduction of arginine levels in patients with SCA was demonstrated to be associated with endothelial damage, multiple organ injury, increased haemolysis and pulmonary hypertension, contributing to high mortality of these patients (Morris et al, 2008). The present study aimed to evaluate a therapeutic proposal for treating SCA, including supplementation with L-arginine as an adjuvant drug for treatment with HC. This was a randomized clinical trial that included 21 adult patients (9 men and 12 women, aged 20–40 years) with a clinical and laboratory diagnosis of SCD, confirmed by molecular biology, undergoing treatment with HC for more than one year at a referral university hospital in Fortaleza, Ceará, Brazil. After informed consent, the selected patients were randomly divided into Group I (Control group: HC only therapy, n = 09) and Group II (Study group; therapy with HC + L-arginine, n = 12), matched for age and sex. The patients selected for Group II were prescribed Reforgan (L-Arginine 250 mg), with a dose of one pill daily for 90 d, as a supplement to Hydréia (HC, dosage ranged from 500 to 1500 mg/day). Group II patients (HC + L-arginine) showed a significant increase in nitrite levels, HbF and reticulocytes, when compared to both baseline values and the Group I (HC only) at the various time points tested, demonstrating that supplementation with L-arginine increased the bioavailability of this substrate, leading to a better response to treatment with HC, as seen in Fig 1 (Table I). The increase in the levels of nitrite and HbF in patients after 12 weeks of use of HC + L-arginine, in relation to the baseline, demonstrates that such association induces a better therapeutic response to HC and confirms that the action of HC involves an NO-dependent pathway causing increased consumption of L-arginine, which is reduced in SCA patients and limits their response to HC. Our results support those of previous studies, which demonstrated that the in vitro induction of HF in progenitor cells occurs via NOSand soluble guanylate cyclase (Strouse et al, 2008), that there is chronic depletion of arginine levels in knockout sickle cell mice, and when treated with L-arginine, there is an increase in NO levels (Dasgupta et al, 2006). Patients using HC + L-arginine also showed an increase in reticulocyte counts at 12 weeks compared to the baseline, indicating that the arginine acts by stimulating erythropoiesis. Baliga et al (2010) evaluated the effect of the association of HC + L-arginine on the synthesis of fetal haemoglobin by erythroid colony-forming units, demonstrating excellent

DNA damage in leukocytes of sickle cell anemia patients is associated with hydroxyurea therapy and with HBB*S haplotype

Hydroxyurea (HU) is the primary pharmacologic agent for preventing the complications and improving the quality of life of sickle cell anemia (SCA) patients. Although HU has been associated with an increased risk of leukemia in some patients with myeloproliferative disorders, the mutagenic and carcinogenic potential of HU has not been established. This study used the alkaline comet assay to investigate DNA damage in peripheral blood leukocytes from 41 individuals with SCA treated with HU (SCAHU) and from 26 normal individuals. The presence of HbS and the analysis of the haplotypes of the beta S gene cluster were done by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The damage index (DI) in the SCAHU group was significantly higher than in controls (p<0.001). Neither gender nor age was associated with DNA damage in controls or SCAHU individuals. Among the SCAHU individuals, DI was significantly influenced by length of HU treatment (p=0.0039) and BMI (p=0.001). Individuals with length of HU treatment≥20 months and BMI≤20kg/m(2) had a significantly greater DI than those with length of HU treatment<20 months and BMI>20kg/m(2). No significant influence of mean HU dose was observed on DI (p=0.950). However, individuals who received a mean HU dose≥20mg/kg showed a higher DI than those who received less. Furthermore, an association was observed between DI damage and HBB*S gene haplotypes. DI values for the Bantu/Bantu haplotype was greater when compared to the Benin/Benin haplotype; and the Bantu/Benin haplotype had a DI lower than the Bantu/Bantu haplotype and greater than the Benin/Benin haplotype. Our results show that DNA damage in sickle cell anemia is associated not only with treatment with HU but also with genotype.

Analysis of oxidative status and biochemical parameters in adult patients with sickle cell anemia treated with hydroxyurea, Ceara, Brazil.

Background Sickle cell anemia is a hemoglobinopathy caused by a mutation that results in the production of an abnormal hemoglobin molecule, hemoglobin S (Hb S). This is responsible for profound physiological changes, such as the sickling of red blood cells. Several studies have shown that hydroxyurea protects against vaso-occlusive crises. Objective The aim of this study was to evaluate the oxidative stress associated with biochemical parameters in patients with sickle cell anemia treated with hydroxyurea. Methods The study was conducted with 20 male and 25 female patients at the Hospital Universitário Walter Cantídio. The patients were divided into two groups: a study group (n = 12), patients with sickle cell anemia who were receiving hydroxyurea and a control group (n = 33) of sickle cell anemia patients not submitted to hydroxyurea treatment. The biochemical parameters analyzed were ferritin, transferrin, and serum iron. Glutathione was measured in its reduced form to analyze the oxidative state. Results The results showed insignificant increases in the levels of serum iron, transferrin and ferritin in patients treated with hydroxyurea when compared with those who did not take the medication. However, the glutathione levels were significantly higher in patients taking hydroxyurea than in controls. Conclusions These results indicate that hydroxyurea possibly acts as an antioxidant by increasing glutathione levels.

The Effect of a Selective Inhibitor of Phosphodiesterase-9 on Oxidative Stress, Inflammation and Cytotoxicity in Neutrophils from Patients with Sickle Cell Anaemia

The aim of the study was to investigate the possible anti‐inflammatory and antioxidant effects of BAY 73‐6691 on neutrophils from SCA patients. This study included 35 patients with a molecular diagnosis of SCA, whose neutrophils were isolated and treated with BAY 73‐6691 at the concentrations 100, 10, 1.0 and 0.1 μg/mL. LDH release and MTT assays were performed to verify cell viability. To evaluate oxidative stress, the following parameters were determined by spectrophotometric assays: NO and malondialdehyde (MDA) levels and activity of catalase, superoxide dismutase (SOD) and glutathione peroxidase (GPx). As inflammatory markers, myeloperoxidase (MPO) levels were evaluated by colorimetric assay and TNF‐α by enzyme immunoassay. The results showed that neutrophils from SCA patients not treated with hydroxyurea (HU) had significantly lower NO levels and catalase and SOD activity, as well as significantly higher MDA, MPO and TNF‐α levels when compared with neutrophils from SCA patients treated with HU and neutrophils from control group. Treatment of SCA neutrophils with BAY 73‐6691 resulted in 94%, 200% and 168% increase in NOx levels, SOD and catalase activity, respectively. In addition, there was a reduction of approximately 46% and 45% in TNF‐α and MPO levels, respectively. In SCAHU neutrophils, there was a 30% and 44% increase in NOx levels and SOD activity, respectively, and a 28% and 37% decrease in TNF‐α and MPO levels, respectively. However, these effects were observed at cytotoxic doses only. The results of this study are original and demonstrate that inhibition of phosphodiesterase‐9 in neutrophils from SCA patients with BAY 73‐6691 was able to increase the NO bioavailability and attenuate oxidative stress and inflammation in neutrophils from patients not treated with HU.

Correlation of low levels of nitrite and high levels of fetal hemoglobin in patients with sickle cell disease at baseline

Background Sickle cell disease is a hemoglobinopathy characterized by hemolytic anemia, increased susceptibility to infections and recurrent vaso-occlusive crises that reduces the quality of life of sufferers. Objective To evaluate the correlation of the levels of lactate dehydrogenase, malonaldehyde and nitrite to fetal hemoglobin in patients with sickle cell disease not under treatment with hydroxyurea in outpatients at a university hospital in Fortaleza, Ceará, Brazil. Methods Forty-four patients diagnosed with sickle cell disease were enrolled at baseline. Diagnosis was confirmed by evaluating the beta globin gene using polymerase chain reaction-restriction fragment length polymorphism. The concentration of fetal hemoglobin was obtained by high-performance liquid chromatography. Serum levels of nitrite, malonaldehyde and lactate dehydrogenase were measured by biochemical methods. Results Significantly higher levels of lactate dehydrogenase, nitrite and malonaldehyde were observed in patients with sickle cell disease compared to a control group. The study of the correlation between fetal hemoglobin levels and these variables showed a negative correlation with nitrite levels. No correlation was found between fetal hemoglobin and malonaldehyde or lactate dehydrogenase. When the study population was stratified according to fetal hemoglobin levels, a decrease in the levels of nitrite was observed with higher levels of fetal hemoglobin (p-value = 0.0415). Conclusion The results show that, similar to fetal hemoglobin levels, the concentration of nitrite can predict the clinical course of the disease, but should not be used alone as a modulator of prognosis in patients with sickle cell disease.

Avaliação das concentrações de malonaldeído e nitrito em pacientes com anemia falciforme em tratamento ou não com hidroxiureia

Objective: To determine the serum levels of malondialdehyde and nitrite in patients with sickle cell anemia treated or not with hydroxyurea in outpatient?s setting. Methods: Of the 65 patients with sickle cell anemia selected for the study, 51 of them were not treated with hydroxyurea (Group 1), 14 made chronic use of hydroxyurea (Group 2) and 20 individuals had no hemoglobinopathies (Control Group). Results: The Control Group had a lower and more homogeneous concentration of malondialdehyde levels as compared to the other groups. The results of Groups 1 and 2 showed increased values of malondialdehyde levels when compared to the Control Group. Considering the values of Groups 1 and 2, there were no significant changes in the malondialdehyde levels. There was no significant difference in the serum levels of nitrite between the groups. Group 2 presented a statistically significant correlation between serum malondialdehyde levels and the clinical variables investigated. In turn, Group 1 showed correlation only with occurrence of three or more vaso-occlusive crises. There was no correlation between nitrite levels and the clinical variables. Conclusion: The results revealed that during the pathogenesis of sickle cell anemia, an increase in lipid peroxidation was observed. On the other hand, no changes in oxidative parameters were detected during treatment with hydroxyurea, probably due to the short period of treatment of the patients studied. Objetivo: Determinar os niveis sericos de malonaldeido e de nitrito em pacientes com anemia falciforme em tratamento ou nao com hidroxiureia e em acompanhamento ambulatorial. Metodos: Dos 65 pacientes com diagnostico de anemia falciforme selecionados para o estudo, 51 nao fizeram tratamento com hidroxiureia (Grupo 1) e 14 fizeram uso cronico de hidroxiureia (Grupo 2), sendo que 20 individuos nao tinham hemoglobinopatias (Grupo Controle). Resultados: O Grupo Controle possuia menor e mais homogenea concentracao dos niveis de malonaldeido em relacao aos outros grupos. Os resultados do Grupo 1 e do Grupo 2 mostraram valores aumentados dos niveis de malonaldeido quando comparados ao Grupo Controle. Quando comparados os valores dos Grupos 1 e 2, nao foram observadas alteracoes significativas nos niveis de malonaldeido. Nao houve diferenca significativa nos niveis sericos de nitrito entre os grupos. Verificou-se que, no Grupo 2, houve uma correlacao estatisticamente significativa dos niveis sericos de malonaldeido com as variaveis clinicas investigadas. Por sua vez, o Grupo 1 mostrou correlacao somente com a ocorrencia de tres ou mais crises vaso-oclusivas. Nao se verificou nenhuma correlacao nos niveis de nitrito com as variaveis clinicas. Conclusao: Os resultados revelaram que, durante o estabelecimento da patogenese da anemia falciforme, pode ser observado um aumento na peroxidacao lipidica. Por outro lado, durante o tratamento com a hidroxureia, nao foi detectada nenhuma alteracao nos parâmetros oxidativos, provavelmente devido ao curto periodo de tratamento dos pacientes em estudo.

Antiretroviral changes during the first year of therapy

INTRODUCTION The Brazilian HIV/AIDS management and treatment guideline (PCDT), published in 2013, recommends and standardizes the use of highly active antiretroviral therapy (HAART) in all adult patients, in spite of LTCD4 count. This study aimed to analyze the first year of HAART use in patients from a reference center on HIV/AIDS management in Fortaleza, Ceará. METHOD This descriptive study reviewed all prescription forms of antiretroviral regimens initiation and changes from January to July 2014. All antiretroviral regimen changes that occurred during the first year of therapy were evaluated. Data were analyzed with SPSS version 20. Mean, standard deviation and frequency, Students t and Mann-Whitney tests calculations were used, with significance at p<0.05. RESULTS From 527 patients initiating HAART, 16.5% (n=87) had a regimen change in the first year. These patients were mostly male (59.8%; n=52), aged 20 to 39 years, with only one HAART change (72.4%; n=63). Efavirenz was the most often changed drug, followed by tenofovir, zidovudine and lopinavir/ritonavir. Mean time of HAART changes was 120 days, with adverse reactions as the most prevalent cause. HAART was effective in decreasing viral load since second month of treatment (p=0.003) and increasing LTCD4 lymphocytes since fifth month (p<0.001). CONCLUSION The main cause of initial HAART changes was adverse reaction and most patients had only one change in the HAART regimen. HAART prescription was in accordance to the PCDT from 2013.

Standardization method for measurement of hydroxyurea by Ultra High Efficiency Liquid Chromatography in plasma of patients with sickle cell disease

Sickle cell anemia (SCA) is a recessively inherited disease characterized by chronic hemolytic anemia, chronic inflammation, and acute episodes of hemolysis. Hydroxyurea (HU) is widely used to increase the levels of fetal hemoglobin (HbF). The objective of this study was to standardize and validate a method for the quantification of HU in human plasma by using ultra high performance liquid chromatography (UPLC) in order to determine the plasma HU levels in adult patients with SCA who had been treated with HU. We used an analytical reverse phase column (Nucleosil C18) with a mobile phase consisting of acetonitrile/water (16.7/83.3). The retention times of HU, urea, and methylurea were 6.7, 7.7, and 11.4 min, respectively. All parameters of the validation process were defined. To determine the precision and accuracy of quality controls, HU in plasma was used at concentrations of 100, 740, and 1600 µM, with methylurea as the internal standard. Linearity was assessed in the range of 50-1600 µM HU in plasma, obtaining a correlation coefficient of 0.99. The method was accurate and precise and can be used for the quantitative determination of HU for therapeutic monitoring of patients with SCA treated with HU.

Kidney dysfunction and beta S-haplotypes in patients with sickle cell disease.

Objetive To investigate the association between kidney dysfunction and haplotypes in sickle cell disease. Methods A cohort of 84 sickle cell disease patients, treated in a public health service in Fortaleza, Brazil, was studied. Hemoglobin S haplotypes were obtained from 57 patients as they had recently received blood transfusions with 18 of them agreeing to undertake urinary concentrating ability and acidification tests. The glomerular filtration rate was estimated using the Modification of Diet in Renal Disease Study equation. Urinary concentration was evaluated utilizing the urinary and serum osmolality ratio (U/Posm) after 12 hours of water deprivation. Urinary acidification was evaluated by measuring the urinary pH before and after the administration of oral CaCl2. The analysis of the haplotypes of the beta S gene cluster was carried out by polymerase chain reaction-restriction fragment length polymorphism. The analysis of variance (ANOVA) test was used for multiple comparisons of means and the Newman-Keuls test was used to identify which groups were significantly different. Results The mean age of the patients was 33 ± 13 years with 64.2% being females. The glomerular filtration rate was normal in 25 cases (30%) and a rate > 120 mL/min was seen in 52 cases (62%). Urinary concentration deficit was found in all patients who underwent the test and urinary acidification in 22%. There was no significant difference when comparing patients with the Bantu/Bantu and Benin/Benin haplotypes. On comparing patients with the Central African Republic-haplotype however, a higher number had glomerular filtration rates between 60 and 120 mL/min. Conclusion There was no significant difference among sickle cell disease patients regarding the haplotypes and kidney dysfunction.