Talyta Ellen de Jesus dos Santos

Impact of iron overload on interleukin-10 levels, biochemical parameters and oxidative stress in patients with sickle cell anemia

Objective The aim of this study was to evaluate the impact of iron overload on the profile of interleukin-10 levels, biochemical parameters and oxidative stress in sickle cell anemia patients. Methods A cross-sectional study was performed of 30 patients with molecular diagnosis of sickle cell anemia. Patients were stratified into two groups, according to the presence of iron overload: Iron overload (n = 15) and Non-iron overload (n = 15). Biochemical analyses were performed utilizing the Wiener CM 200 automatic analyzer. The interleukin-10 level was measured by capture ELISA using the BD OptEIAT commercial kit. Oxidative stress parameters were determined by spectrophotometry. Statistical analysis was performed using GraphPad Prism software (version 5.0) and statistical significance was established for p-values < 0.05 in all analyses. Results Biochemical analysis revealed significant elevations in the levels of uric acid, triglycerides, very low-density lipoprotein (VLDL), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), urea and creatinine in the Iron overload Group compared to the Non-iron overload Group and significant decreases in the high-density lipoprotein (HDL) and low-density lipoprotein (LDL). Ferritin levels correlated positively with uric acid concentrations (p-value < 0.05). The Iron overload Group showed lower interleukin-10 levels and catalase activity and higher nitrite and malondialdehyde levels compared with the Non-iron overload Group. Conclusion The results of this study are important to develop further consistent studies that evaluate the effect of iron overload on the inflammatory profile and oxidative stress of patients with sickle cell anemia.

Tumor suppressor p53 protein expression: prognostic significance in patients with low-risk myelodysplastic syndrome

Background At the time of diagnosis, more than 50% of patients with myelodysplastic syndrome have a normal karyotype and are classified as having a favorable prognosis. However, these patients often show very variable clinical outcomes. Furthermore, current diagnostic tools lack the ability to look at genetic factors beyond karyotyping in order to determine the cause of this variability. Objective To evaluate the impact of p53 protein expression at diagnosis in patients with low-risk myelodysplastic syndrome. Methods This study enrolled 38 patients diagnosed with low-risk myelodysplastic syndrome. Clinical data were collected by reviewing medical records, and immunohistochemical p53 staining was performed on bone marrow biopsies. Results Of the 38 participants, 13 (34.21%) showed p53 expression in their bone marrow. At diagnosis, this group of patients also presented clinical features characteristic of a poor prognosis more often than patients who did not express p53. Furthermore, patients expressing p53 had a shorter median survival time compared to those without p53 expression. Conclusion This study shows that the expression of p53 at diagnosis is a useful indicator of distinct clinical characteristics and laboratory profiles found in low-risk myelodysplastic syndrome patients. These data indicate that the immunohistochemical analysis of p53 may be a prognostic tool for myelodysplastic syndrome and should be used as an auxiliary test to help determine the best therapeutic choice.

The role of iron overload on oxidative stress in sickle cell anemia

UNLABELLED Repeated blood transfusions in patients with sickle cell anemia (SCA) increases the risk of iron overload (IO), contributing to oxidative stress. MATERIALS & METHODS Blood samples of 15 SCA patients without IO (group 1) and 15 SCA patients with IO (group 2) and 30 healthy individuals were collected to investigate oxidative stress. IO was categorized using repeated measures of serum ferritin. The biomarkers evaluated were plasmatic malondialdehyde (MDA), nitrite and erythrocyte catalase. RESULTS MDA and nitrite were higher in group 2 than in group 1 and the healthy group (p < 0.001 for MDA and nitrite). Catalase presented lower in group 2 than group 1 and the healthy group (p < 0.001). We obtained a positive correlation between ferritin and MDA (r = 0.40; p < 0.02), and between ferritin and nitrite (r = 0359; p = 0.023). CONCLUSION The results demonstrated that IO is an important risk factor for enhanced oxidative stress in SCA.

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.

Methemoglobin measure in adult patients with sickle-cell anemia: influence of hydroxyurea therapy

Introduction: Hemoglobin S (HbS) is unstable hemoglobin that easily oxidizes, causing methemoglobin (MetHb) increased production in patients with sickle-cell anemia (SCA). Objectives: To determine MetHb levels and the influence of hydroxyurea (HU) therapy on this marker in patients with SCA. Materials and methods: Blood samples from 53 patients with SCA at the steady-state, with and without HU therapy, and 30 healthy individuals were collected to evaluate MetHb levels. The MetHb measurement was performed by spectrophotometry. Complete blood count, HU measurements, and fetal hemoglobin (HbF) and HbS concentrations were taken from medical records. Results: MetHb levels were statically higher in patients with SCA when compared to control group (p < 0.001). There was no statistical difference in MetHb level between SCA patients, either using or not HU. We obtained a positive correlation between MetHb measurements and HbS concentration (r = 0.2557; p = 0.0323). Conclusion: HbS presence favored hemoglobin breaking down, and consequently increased MetHb production. Treatment with HU, however, did not influence the levels of this marker.

Bone marrow fibrosis at diagnosis is associated with TP53 overexpression and adverse prognosis in low‐risk myelodysplastic syndrome

Myelodysplastic syndrome (MDS) is a heterogeneous group of stem cell clonal alterations, culminating in a high risk of progression to acute myeloid leukaemia (AML) (Greenberg et al, 2012). The prognosis of these patients is commonly determined by the International Prognostic Score System (IPSS), which considers the number of cytopenias, cytogenetic alterations and number of blasts in the bone marrow at diagnosis. Patients with a lower risk of progression to AML are classified as low risk; however, a subgroup of these patients develops a disease with an aggressive course and a lower survival rate (Mittelman et al, 2010). This clinical heterogeneity reinforces the need to identify auxiliary markers for prognostic stratification systems. Bone marrow fibrosis is a histological finding in approximately 10–20% of patients with de novo MDS; however, MDS with fibrosis is not yet recognized as a distinct entity and its prognostic role is still under debate (Ramos et al, 2016). The immunohistochemical expression of TP53 has been used as a predictive marker for mutations in the TP53 gene in several haematological diseases due to the low-cost and easy to perform technique (Kulasekararaj et al, 2013). Moreover, studies have demonstrated that TP53 expression is an independent adverse prognostic factor in patients with highrisk MDS, being associated with complex karyotype and therapy-related MDS (Kulasekararaj et al, 2013). Therefore, the present study investigated the impact of bone marrow fibrosis and TP53 expression at diagnosis on the clinical characteristics and overall survival of patients with low-risk MDS. Seventy-three low-risk MDS patients treated at a reference University Hospital in the northeast of Brazil participated in the study. Patients were stratified according to the IPSS and revised IPSS (IPSS-R) criteria (Greenberg et al, 2012). Clinical data at diagnosis, such as karyotype, complete blood count, number of blasts, presence of fibrosis and evolution to death or AML, were collected from medical records. TP53 expression was determined by immunohistochemistry, according to Shah et al (2012). Briefly, 5-lm sections of bone marrow were incubated with the p53-DO7 monoclonal antibody (Dako, Santa Clara, CA, USA). TP53 protein was identified by the ABC – peroxidase technique, staining with 3,30-Diaminobenzidine (DAB; Dako), and counterstaining with haematoxylin. The percentage and intensity of TP53 staining was assessed based on a total manual count of 1000 granulocytic precursors. TP53 expression was defined as positive for a strong nuclear staining pattern, according to the Modified Quick Score (Kulasekararaj et al, 2013) in at least 1% of the cells analysed (Saft et al, 2014). Patients were stratified for the presence of bone marrow fibrosis at diagnosis according to the criteria used by the European Myelofibrosis Network (Thiele et al, 2005). The study was approved by the Research Ethics Committee of the Federal University of Cear a (protocol #129/12) and all patients agreed to participate. Statistical analysis was performed using the GraphPad Prism 5.0 software (GraphPad Software Inc., La Jolla, CA, USA), using chi-square and Student’s t or Mann–Whitney test, according data normality, verified by Kolmogorov–Smirnov test. The Kaplan–Meier curve was performed to verify differences in overall survival. Significance was set at P < 0 05. The median age of the study patients was 65 years, with a predominance of females (64 3% females and 35 7% males). Most of the patients (49 32%) were classified as single-lineage dysplasia, followed by multi-lineage dysplasia (41 09%), MDS with ringed sideroblasts (6 85%) and MDS with isolated del(5q) (2 74%). Patients with bone marrow fibrosis (MF-2 and MF-3) had significantly lower haemoglobin and haematocrit values at diagnosis when compared to patients without fibrosis (MF-0 and MF-1) (P = 0 001). High TP53 expression was associated with the presence of bone marrow fibrosis (P = 0 008). There was no difference between the other analysed parameters (Table I). Patients with bone marrow fibrosis [P = 0 0007, hazard ratio (HR) = 0 02, 95% confidence interval (CI) = 0 002– 0 20] and high TP53 expression (P = 0 0039, HR = 14 34, 95% CI = 2 0–102 8) had lower overall survival when compared to patients without these findings. Overall survival in patients with bone marrow fibrosis (MF-2 and MF-3) did not demonstrate a significant difference regarding TP53 expression (P = 0 3794, HR = 2 62, 95% CI = 0 34–17 40) (Fig 1). Some studies have reported on the importance of bone marrow fibrosis in prognostic characteristics and survival of patients with de novo MDS; however, the role of these Correspondence

Presence of new mutations in the TP53 gene in patients with low-risk myelodysplastic syndrome: two case reports

BackgroundMyelodysplastic syndromes are heterogeneous disorders. Patients with myelodysplastic syndrome disease often have ineffective hematopoiesis, cytopenias, blood cell dysplasia in one or more cell types, and are at high risk for developing acute myeloid leukemia. In myelodysplastic syndrome, mutations of TP53 gene are usually associated with complex karyotype and confer a worse prognosis. In the present study, two mutations in this gene are presented and discussed with the clinical evolution of the patients.Case presentationThe first case is a 77-year-old Brazilian woman diagnosed as having multiple lineage dysplasia myelodysplastic syndrome according to World Health Organization 2016 and classified as very low-risk by Revised International Prognostic Scoring. The second case is an 80-year-old Brazilian man also diagnosed as having multiple lineage dysplasia myelodysplastic syndrome and classified as low risk. The mutation described in the first case was already identified in some neoplasias and it is associated with a poor prognosis, but it had never been reported before in myelodysplastic syndrome. The second mutation has never been described.ConclusionsThis is a novel report for the scientific community and may be very helpful as we can better understand the disease and the impact of mutations through the follow-up of these patients and others in the future. Both patients are in a good clinical condition, suggesting that these mutations may not alter the clinical course of the disease or may be associated with a good prognosis, but their role in the disease must be investigated more deeply in a larger population.

Influence of βS-Globin Haplotypes and Hydroxyurea on Arginase I Levels in Sickle Cell Disease.

Introduction. Sickle cell disease (SCD) is characterized by hemoglobin S homozygosity, leading to hemolysis and vasoocclusion. The hemolysis releases arginase I, an enzyme that decreases the bioavailability of nitric oxide, worsening the symptoms. The different SCD haplotypes are related to clinical symptoms and varied hemoglobin F (HbF) concentration. The aim of this study was to evaluate the impact of the βS gene haplotypes and HbF concentration on arginase I levels in SCD patients. Methods. Fifty SCD adult patients were enrolled in the study and 20 blood donors composed the control group. Arginase I was measured by ELISA. The βS haplotypes were identified by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Statistical analyses were performed with GraphPad Prism program and the significance level was p < 0.05. Results. Significant increase was observed in the arginase I levels in SCD patients compared to the control group (p < 0.0001). The comparison between the levels of arginase I in three haplotypes groups showed a difference between the Bantu/Bantu × Bantu/Benin groups; Bantu/Bantu × Benin/Benin, independent of HU dosage. An inverse correlation with the arginase I levels and HbF concentration was observed. Conclusion. The results support the hypothesis that arginase I is associated with HbF concentration, also measured indirectly by the association with haplotypes.