Plasma levels of TGF-ß1 in homeostasis of the inflammation in sickle cell disease.

Sickle cell disease (SCD) represents a chronic inflammatory condition with complications triggered by the polymerization of hemoglobin S (Hb S), resulting in a series of cellular interactions mediated by inflammatory cytokines, as the transforming growth factor beta (TGF-ß), which plays an important role in inflammation resolution. This study assessed the relation between SCD inflammation and the plasma concentration of TGF-ß1, and also checked the influence of the presence of -509C/T polymorphism in TGFB1 gene on TGF-ß1 plasma values. The plasma levels of TGF-ß1 were quantified by ELISA in 115 patients with SCD (genotypes SS, SD-Los Angeles, Sß-thalassemia and SC) and in 58 individuals with no hemoglobinopathies (Hb AA), as the control group. The -509C/T polymorphism in TGFB1 gene was screened by PCR-RFLP. The correlation between TGF-ß1 plasma levels and the inflammation was based on its association with the count of platelets, total white blood cells (WBC) and neutrophils in the peripheral blood. Patients with SCD showed plasma levels of TGF-ß1 higher than the control group, especially the Hb SS genotype, followed by the group with Hb SD. Polymorphism investigation showed no interference in the values obtained for the cytokine in the groups evaluated. All SCD groups showed TGF-ß1 levels positively correlated to the platelets and WBC counts. The original data obtained in this study for SCD support the involvement of TGF-ß1 in regulating of the inflammatory response and suggest that this marker possibly may become a potential therapeutic target in the treatment of the disease.

Severity of Brazilian sickle cell disease patients: severity scores and feasibility of the Bayesian network model use.

The integration of the several clinical and laboratory dimensions and the influence of each parameter on the sickle cell disease (SCD)-related mortality is useful for predicting the phenotype of an individual. This study evaluated the feasibility of the SCD severity calculator use to measure disease severity in Brazilian patients. The study group was composed of 500 SCD patients (440 HbSS and 60 HbSC) diagnosed by molecular biology. We observed a decrease in severity scores in 72 SCD patients assessed before and after the hydroxyurea (HU) use. Furthermore, the HU influenced the increase of mean corpuscular volume (MCV) and HbF concentration, and the decrease of leukocytes and total bilirubin. We found 180 (36.0%) patients with intermediate phenotype, 170 (34.0%) mild phenotype and 150 (30.0%) with severe phenotype. Patients with ages >40 years had higher mean score (0.778±0.177) than patients between 18 and 40 years (0.562±0.152) and patients between 5 and 17 years (0.322±0.145). We observe that there is a tendency of individuals with leg ulcers, avascular necrosis and cardiac complications with increasing age. Correlation analysis showed relations between severity scores with leukocytes, reticulocytes, bilirubin, lactate dehydrogenase, HbS, hemoglobin and hematocrit (p<0.05). Several comparisons involving age groups, SCD genotype and phenotypic classification had satisfactory results and this classification will be used for future studies involving genetic polymorphisms, response to treatment with HU and oxidative stress markers in SCD.

Hemoglobin D-Punjab: origin, distribution and laboratory diagnosis

This review discusses hemoglobin D-Punjab, also known as hemoglobin D-Los Angeles, one of the most common hemoglobin variants worldwide. It is derived from a point mutation in the beta-globin gene (HBB: c.364G>C; rs33946267) prevalent in the Punjab region, North-western Indian. Hemoglobin D-Punjab can be inherited in heterozygosis with hemoglobin A causing no clinical or hematological alterations, or in homozygosis, the rarest form of inheritance, a condition that is commonly not related to clinical symptomatology. Moreover, this variant can exist in association with other hemoglobinopathies, such as thalassemias; the most noticeable clinical alterations occur when hemoglobin D-Punjab is associated to hemoglobin S. The clinical manifestations of this association can be similar to homozygosis for hemoglobin S. Although hemoglobin D-Punjab is a common variant globally with clinical importance especially in cases of double heterozygosis, hemoglobin S/D-Punjab is still understudied. In Brazil, for example, hemoglobin D-Punjab is the third most common hemoglobin variant. Thus, this paper summarizes information about the origin, geographic distribution, characterization and occurrence of hemoglobin D-Punjab haplotypes to try to improve our knowledge of this variant. Moreover, a list of the main techniques used in its identification is provided emphasizing the importance of complementary molecular analysis for accurate diagnosis.

Hemoglobin D-Punjab: origin, distribution and laboratory diagnosis.

This review discusses hemoglobin D-Punjab, also known as hemoglobin D-Los Angeles, one of the most common hemoglobin variants worldwide. It is derived from a point mutation in the beta-globin gene (HBB: c.364G>C; rs33946267) prevalent in the Punjab region, Northwestern Indian. Hemoglobin D-Punjab can be inherited in heterozygosis with hemoglobin A causing no clinical or hematological alterations, or in homozygosis, the rarest form of inheritance, a condition that is commonly not related to clinical symptomatology. Moreover, this variant can exist in association with other hemoglobinopathies, such as thalassemias; the most noticeable clinical alterations occur when hemoglobin D-Punjab is associated to hemoglobin S. The clinical manifestations of this association can be similar to homozygosis for hemoglobin S. Although hemoglobin D-Punjab is a common variant globally with clinical importance especially in cases of double heterozygosis, hemoglobin S/D-Punjab is still understudied. In Brazil, for example, hemoglobin D-Punjab is the third most common hemoglobin variant. Thus, this paper summarizes information about the origin, geographic distribution, characterization and occurrence of hemoglobin D-Punjab haplotypes to try to improve our knowledge of this variant. Moreover, a list of the main techniques used in its identification is provided emphasizing the importance of complementary molecular analysis for accurate diagnosis.

Genetic and biochemical markers of hydroxyurea therapeutic response in sickle cell anemia.

BACKGROUND: Sickle cell anemia (SCA) presents a complex pathophysiology which can be affected by a number of modifying factors, including genetic and biochemical ones. In Brazil, there have been no studies verifying ßS-haplotypes effect on oxidative stress parameters. This study evaluated ßS-haplotypes and Hb F levels effects on oxidative stress markers and their relationship with hydroxyurea (HU) treatment in SCA patients. METHODS: The studied group was composed by 28 SCA patients. Thirteen of these patients were treated with HU and 15 of them were not. We used molecular methodology (PCR-RFLP) for hemoglobin S genotype confirmation and haplotypes identification. Biochemical parameters were measured using spectrophotometric methods (Thiobarbituric-acid-reactive substances and Trolox equivalent antioxidant capacity levels, catalase and GST activities) and plasma glutathione levels by High-performance liquid chromatography coupled to electrochemical detection. RESULTS: We found the highest frequency of Bantu haplotype (48.2%) which was followed by Benin (32.1%). We observed also the presence of Cameroon haplotype, rare in Brazilian population and 19.7% of atypical haplotypes. The protective Hb F effect was confirmed in SCA patients because these patients showed an increase in Hb F levels that resulted in a 41.3% decrease on the lipid peroxidation levels (r =-0.74, p=0.01). Other biochemical parameters have not shown differential expression according to patient's haplotypes. Bantu haplotype presence was related to the highest lipid peroxidation levels in patients (p < 0,01), but it also conferred a differential response to HU treatment, raising Hb F levels in 52.6% (p = 0.03) when compared with the group with the same molecular profile without HU usage. CONCLUSIONS: SCA patients with Bantu haplotype showed the worst oxidative status. However these patients also demonstrated a better response to the treatment with HU. Such treatment seems to have presented a "haplotype-dependent" pharmacological effect.

The influence of hydroxyurea on oxidative stress in sickle cell anemia.

OBJECTIVE: The oxidative stress in 20 sickle cell anemia patients taking hydroxyurea and 13 sickle cell anemia patients who did not take hydroxyurea was compared with a control group of 96 individuals without any hemoglobinopathy. METHODS: Oxidative stress was assessed by thiobarbituric acid reactive species production, the Trolox-equivalent antioxidant capacity and plasma glutathione levels. RESULTS: Thiobarbituric acid reactive species values were higher in patients without specific medication, followed by patients taking hydroxyurea and the Control Group (p < 0.0001). The antioxidant capacity was higher in patients taking hydroxyurea and lower in the Control Group (p = 0.0002 for Trolox-equivalent antioxidant capacity and p < 0.0292 for plasma glutathione). Thiobarbituric acid reactive species levels were correlated with higher hemoglobin S levels (r = 0.55; p = 0.0040) and lower hemoglobin F concentrations(r = -0.52; p = 0.0067). On the other hand, plasma glutathione levels were negatively correlated with hemoglobin S levels (r = -0.49; p = 0.0111) and positively associated with hemoglobin F values (r = 0.56; p = 0.0031). CONCLUSION: Sickle cell anemia patients have high oxidative stress and, conversely, increased antioxidant activity. The increase in hemoglobin F levels provided by hydroxyurea and its antioxidant action may explain the reduction in lipid peroxidation and increased antioxidant defenses in these individuals.

Oxidative stress and antioxidant capacity in sickle cell anaemia patients receiving different treatments and medications for different periods of time.

To evaluate, in a longitudinal study, the profile of lipid peroxidation and antioxidant capacity markers in sickle cell anaemia patients receiving different treatments and medication over different time periods. The three groups were: patients undergoing transfusion therapy and receiving iron chelator deferasirox (DFX group, n = 20); patients receiving deferasirox and hydroxyurea (DFX + HU group, n = 10), and patients receiving only folic acid (FA group, n = 15). Thiobarbituric acid-reactive substance (TBARS) assays and trolox-equivalent antioxidant capacity (TEAC) assays were evaluated during two different periods of analysis, T0 and T1 (after ~388 days). Higher FA group TBARS values were observed compared with the DFX + HU group (p = 0.016) at T0; and at T1, higher FA group TBARS values were also observed compared with both the DFX group (p = 0.003) and the DFX + HU group (p = 0.0002). No variation in TEAC values was seen between groups, at either T0 or T1. The mean values of TBARS and TEAC for both the DFX and DFX + HU groups decreased at T1. The antioxidant effects of HU and DFX were observed by through an increase in TEAC levels in DFX and DFX + HU groups when compared with those of normal subjects. Increased TEAC values were not recorded in the FA group, and lipid peroxidation was seen to decrease after DFX and HU use.

The influence of hydroxyurea on oxidative stress in sickle cell anemia

OBJECTIVE: The oxidative stress in 20 sickle cell anemia patients taking hydroxyurea and 13 sickle cell anemia patients who did not take hydroxyurea was compared with a control group of 96 individuals without any hemoglobinopathy. METHODS: Oxidative stress was assessed by thiobarbituric acid reactive species production, the Trolox-equivalent antioxidant capacity and plasma glutathione levels. RESULTS: Thiobarbituric acid reactive species values were higher in patients without specific medication, followed by patients taking hydroxyurea and the Control Group (p < 0.0001). The antioxidant capacity was higher in patients taking hydroxyurea and lower in the Control Group (p = 0.0002 for Trolox-equivalent antioxidant capacity and p < 0.0292 for plasma glutathione). Thiobarbituric acid reactive species levels were correlated with higher hemoglobin S levels (r = 0.55; p = 0.0040) and lower hemoglobin F concentrations(r = -0.52; p = 0.0067). On the other hand, plasma glutathione levels were negatively correlated with hemoglobin S levels (r = -0.49; p = 0.0111) and positively associated with hemoglobin F values (r = 0.56; p = 0.0031). CONCLUSION: Sickle cell anemia patients have high oxidative stress and, conversely, increased antioxidant activity. The increase in hemoglobin F levels provided by hydroxyurea and its antioxidant action may explain the reduction in lipid peroxidation and increased antioxidant defenses in these individuals.

Relationship between oxidative stress, glutathione S-transferase polymorphisms and hydroxyurea treatment in sickle cell anemia.

This study evaluated the oxidative stress and antioxidant capacity markers in sickle cell anemia (SCA) patients with and without treatment with hydroxyurea. We assessed GSTT1, GSTM1 and GSTP1 polymorphisms in patients and a control group. The study groups were composed of 48 subjects without hemoglobinopathies and 28 SCA patients, 13 treated with HU [SCA (+HU)], and 15 SCA patients not treated with HU [SCA (-HU)]. We observed a significant difference for GSTP1 polymorphisms in SCA patients with the V/V genotype that showed higher glutathione (GSH) and Trolox equivalent antioxidant capacity (TEAC) (p=0.0445 and p=0.0360), respectively, compared with the I/I genotype. HU use was associated with a 35.2% decrease in the lipid peroxidation levels of the SCA (+HU) group (p<0.0001). Moreover, the SCA (+HU) group showed higher TEAC as compared to the control group (p=0.002). We did not find any significant difference in glutathione-S-transferase (GST) activity between the groups (p=0.76), but the catalase (CAT) activity was about 17% and 30% decreased in the SCA (+HU) and SCA (-HU) groups, respectively (p<0.00001). Whereas the plasma GSH levels were ~2 times higher in the SCA patients than the control group (p=0.0005). HU use has contributed to higher CAT activity and TEAC, and lower lipid peroxidation in patients under treatment. These findings may explain the influence of HU in ameliorating oxidative stress on SCA subjects.