Maritza Cavalcante Barbosa

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.

Effect of different doses of zoledronic acid in establishing of bisphosphonate-related osteonecrosis

OBJECTIVES To establish osteonecrosis of the jaws in rats treated with different doses of zoledronic acid (ZA). METHODS Male Wistar rats (n=6-7) received three consecutive weekly intravenous ZA infusions at doses of 0.04, 0.20 or 1.00mg/kg ZA or saline (control). Four weeks after the last administration, the animals were submitted to simple extraction of the lower left first molar. An additional dose of ZA was administered seven days later, and the animals were sacrificed 28 days after exodontia. Weight was measured and blood was collected weekly for analysis. The jaw was radiographically and microscopically examined along with the liver, spleen, kidney and stomach. RESULTS All ZA doses showed a higher radiolucent area than the control (p<0.0001), but the dose of 0.04mg/kg did not show BRONJ. Doses of 0.20 and 1.00mg/kg ZA showed histological evidence of bone necrosis (p=0.0004). Anaemia (p<0.0001, r(2)=0.8073) and leucocytosis (p<0.0001, r(2)=0.9699) are seen with an increase of lymphocytes (p<0.0001, r(2)=0.6431) and neutrophils and monocytes (p=0.0218, r(2)=0.8724) in all the animals treated with an increasing dose of ZA. Haemorrhage and ectasia were observed in the spleen (p=0.0004) and stomach (p=0.0168) in a dose-dependent manner, and the animals treated with ZA showed a lower rate of weight gain (p<0.0001). CONCLUSIONS We designed a bisphosphonate-related osteonecrosis of the jaw model that reproduces radiographic and histological parameters and mimics clinical alterations such as leucocytosis, anaemia and idiosyncratic inflammatory post infusion reactions.

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

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.

Serum concentrations of nitrite and malondialdehyde as markers of oxidative stress in chronic myeloid leukemia patients treated with tyrosine kinase inhibitors

Background: Chronic myeloid leukemia is a neoplasm characterized by clonal expansion of hematopoietic progenitor cells resulting from the (9:22)(q34,11) translocation. The tyrosine kinase abl fusion protein,the initial leukemogenic event in chronic myeloid leukemia, is constitutively activated thus inducing the production of reactive oxygen species. Of particular relevance is the fact that an increase in reactive oxygen species can facilitate genomic instability and may contribute to disease progression. Objetive: To evaluate oxidative stress by determining the levels of malondialdehyde and nitrite in chronic myeloid leukemia patients under treatment with 1st and 2nd generation tyrosine kinase inhibitors monitored at a referral hospital in Fortaleza, Ceará. Methods: A cross-sectional study was performed of 64 male and female adults. Patients were stratified according to treatment. The levels of malondialdehyde and nitrite were determined by spectrophotometry. Statistical differences between groups were observed using the Student t-test and Fishers exact test. The results are expressed as mean ± standard error of mean. The significance level was set for a p-value < 0.05 in all analyses. Results: The results show significantly higher mean concentrations of nitrite and malondialdehyde in chronic myeloid leukemia patients using second-generation tyrosine kinase inhibitors compared to patients on imatinib. Conclusion: It follows that chronic myeloid leukemia patients present higher oxidative activity and that the increases in oxidative damage markers can indicate resistance to 1st generation tyrosine kinase inhibitors.

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.

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.

Pattern of hemolysis parameters and association with fetal hemoglobin in sickle cell anemia patients in steady state

Objective This study aimed to evaluate the influence of fetal hemoglobin (Hb F) on hemolysis biomarkers in sickle cell anemia patients. Methods Fifty adult sickle cell anemia patients were included in the study. All patients were taking hydroxyurea for at least six months and were followed at the outpatient clinic of a hospital in Fortaleza, Ceará, Brazil. The control group consisted of 20 hemoglobin AA individuals. The reticulocyte count was performed by an automated methodology, lactate dehydrogenase and uric acid were measured by spectrophotometry and arginase I by enzyme-linked immunosorbent assay (ELISA). The presence of Hb S was detected by high-performance liquid chromatography. The level of significance was set for a p-value <0.05. Results A significant increase was observed in the reticulocyte count and lactate dehydrogenase, uric acid and arginase I levels in sickle cell anemia patients compared to the control group (p-value <0.05). Patients having Hb F levels greater than 10% showed a significant decrease in the reticulocyte count, arginase I and lactate dehydrogenase. A significant decrease was observed in arginase I levels in patients taking hydroxyurea at a dose greater than 20 mg/kg/day. Conclusion The results of this study show that sickle cell anemia patients have increases in the hemolysis biomarkers, lactate dehydrogenase, reticulocyte count, arginase I, uric acid and increases in Hb F can reduce the reticulocyte count and arginase I and lactate dehydrogenase levels.