Plasma levels of TGF-β1 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-β), which plays an important role in inflammation resolution. This study assessed the relation between SCD inflammation and the plasma concentration of TGF-β1, and also checked the influence of the presence of −509C/T polymorphism in TGFB1 gene on TGF-β1 plasma values. The plasma levels of TGF-β1 were quantified by ELISA in 115 patients with SCD (genotypes SS, SD-Los Angeles, Sβ-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-β1 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-β1 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-β1 levels positively correlated to the platelets and WBC counts. The original data obtained in this study for SCD support the involvement of TGF-β1 in regulating of the inflammatory response and suggest that this marker possibly may become a potential therapeutic target in the treatment of the disease.     

HPLC determination of hemoglobins to establish reference values with the aid of statistics and informatics

The purpose of the present study was to establish reference values for hemoglobins (Hb) using HPLC, in samples containing normal Hb (AA), sickle cell trait without alpha-thalassemia (AS), sickle cell trait with alpha-thalassemia (ASH), sickle cell anemia (SS), and Hb SC disease (SC). The blood samples were analyzed by electrophoresis, HPLC and molecular procedures. The Hb A2 mean was 4.30 +/- 0.44% in AS, 4.18 +/- 0.42% in ASH, 3.90 +/- 1.14% in SS, and 4.39 +/- 0.35% in SC. They were similar, but above the normal range. Between the AS and ASH groups, only the amount of Hb S was higher in the AS group. The Hb S mean in the AS group was 38.54 +/- 3.01% and in the ASH it was 36.54 +/- 3.76%. In the qualitative analysis, using FastMap, distinct groups were seen: AA and SS located at opposite extremes, AS and ASH with overlapping values and intermediate distribution, SC between heterozygotes and the SS group. Hb S was confirmed by allele-specific polymerase chain reaction. The Hb values established will be available for use as a reference for the Brazilian population, drawing attention to the increased levels of Hb A2, which should be considered with caution to prevent incorrect diagnoses.     

Sickle-cell disease in a British urban community.

Seventy cases of sickle-cell disease were identified in the London Borough of Brent from records dating back to 1962. All but three were still alive and, with one exception, were recalled for confirmation of the diagnosis and to provide personal and family histories. The group consisted of 22 individuals with homozygous sickle-cell anaemia (Hb SS), 12 with sickle-cell/beta-thalassaemia double heterozygosity, 34 with sickle-cell/haemoglobin C disease (Hb SC), and two with the combination of haemoglobin S and hereditary persistence of fetal haemoglobin. They were predominantly of West Indian origin, more than half had been born in Britain, and most were aged under 25. The records for 304 patient admissions between 1962 and 1979 were analysed. There were 199 sickle-cell-disease-related admissions, 61 unrelated to sickle-cell disease, and 44 for pregnancy or its complications. Admissions per patient-year averaged less than one, except for children with Hb SS under the age of 5 years, who were admitted more frequently. The commonest reasons for admission were painful crises (74% of all admissions) and the "chest syndrome" (21%). There were four pneumococcal infections, all in children with Hb SS under the age of 8 years; all recovered. Three patients, aged 10, 15, and 50 years, died. The two children with Hb SS died in their sleep without gross evidence of sickling at necropsy. Multiple brain infarcts were found at necropsy in the 50-year-old woman with Hb SC who, having survived nine uneventful pregnancies, succumbed to an infection after cryosurgery to the cervix. Obstetric records were available for 18 term pregnancies in 11 women. Three antenatal sickling crises and three postpartum thromboembolic complications were encountered. There were no maternal or perinatal deaths. Fifteen asymptomatic individuals with sickle-cell disease were diagnosed as a result of routine screening procedures. There are likely to be many such individuals currently undiagnosed in the community. They urgently need identification because of their increased risks from pregnancy, surgery, and infection.     

Genetic heterogeneity of thalassemias in Mexican mestizo patients with hemolytic anemia

Twenty-eight Hb abnormalities (16 thalassemias, 10 Hb variants and 2 hereditary persistences of fetal hemoglobin) were detected in an etiopathogenic search in 131 patients with hemolytic anemia. The observed thalassemic genotypes [6 beta(0)/beta, 2 (delta beta) (0)/beta(0), 2 beta(0)/beta(+), 2 beta(0)/beta(s), 2 beta(+)/beta(+), 1 beta(+)/beta(s) and 1 (delta beta)(0)/beta] showed heterogeneity in the population studied. A native origin of some of these mutations is considered.     

Glycosylated hemoglobins in a diabetic patient with sickle cell anemia

Results of analysis of blood samples from a diabetic sickle cell anemia (SS) patient and 4 nondiabetic SS patients for glycosylated hemoglobins by Bio-Rex 70 chromatography, high-pressure liquid chromatography, and affinity chromatography are presented. Glycosylated components of Hb S and Hb A2 and total glycosylated hemoglobins were quantitated in this manner. The levels of the various glycosylated hemoglobins were increased twofold in the diabetic patient compared to nondiabetic SS patients. The glycosylated hemoglobin levels in the diabetic SS patient and in the nondiabetic SS patients, however, were significantly lower than the levels normally seen in nonsickle diabetics and normal adults, respectively. In contrast to a previously reported diabetic SS patient, the present case appears to be not severely affected by sickle cell disease.     

Sickle cell disease vasculopathy: a state of nitric oxide resistance

Sickle cell disease (SCD) is a hereditary hemoglobinopathy characterized by microvascular vaso-occlusion with erythrocytes containing polymerized sickle (S) hemoglobin, erythrocyte hemolysis, vasculopathy, and both acute and chronic multiorgan injury. It is associated with steady state increases in plasma cell-free hemoglobin and overproduction of reactive oxygen species (ROS). Hereditary and acquired hemolytic conditions release into plasma hemoglobin and other erythrocyte components that scavenge endothelium-derived NO and metabolize its precursor arginine, impairing NO homeostasis. Overproduction of ROS, such as superoxide, by enzymatic (xanthine oxidase, NADPH oxidase, uncoupled eNOS) and nonenzymatic pathways (Fenton chemistry), promotes intravascular oxidant stress that can likewise disrupt NO homeostasis. The synergistic bioinactivation of NO by dioxygenation and oxidation reactions with cell-free plasma hemoglobin and ROS, respectively, is discussed as a mechanism for NO resistance in SCD vasculopathy. Human physiological and transgenic animal studies provide experimental evidence of cardiovascular and pulmonary resistance to NO donors and reduced NO bioavailability that is associated with vasoconstriction, decreased blood flow, platelet activation, increased endothelin-1 expression, and end-organ injury. Emerging epidemiological data now suggest that chronic intravascular hemolysis is associated with certain clinical complications: pulmonary hypertension, cutaneous leg ulcerations, priapism, and possibly stroke. New therapeutic strategies to limit intravascular hemolysis and ROS generation and increase NO bioavailability are discussed.     

Glucose-6-phosphate dehydrogenase deficiency and sickle cell disease in Brazil.

The frequency of glucose-6-phosphate dehydrogenase (G-6-PD) deficiency was determined in 54 male patients with sickle cell diseases: 31 sickle cell anemia (SS), 14 sickle cell hemoglobinopathy (SC) and 9 HbS/beta-thalassemia (S/B-thal) by a combination of quantitative assay, fluorescent spot test and electrophoresis. Of the 54 patients tested, 7 were found to be G-6-PD deficient (G-6-PD-) (3 SS, 3 SC and 1 S/B-thal) and 47 G-6-PD normal (G-6-PD+) (6 G-6-PD A and 41 G-6-PD B). All the deficient patients were G-6-PD A-. The frequency of G-6-PD deficiency did not differ significantly from that observed in the general population. Compared to patients who were not G-6-PD-, there were no significant differences in the hemoglobin concentration and reticulocyte count in patients with sickle cell diseases who were G-6-PD-.     

The interaction of hemoglobin O Arab with Hb S and β+ thalassemia among Israeli Arabs

Summary We have studied 105 individuals in the village of Jasser El Zarka in the Northern Coast of Israel of whom 59% had at least one abnormal hemoglobin. Of the individuals studied 41% were AA, 13.3% AS, 28.6% AO_Arab, 10.5% SO_Arab, 0.9% SS, 38% Arab-⁺ Thal, and 1.9% Thal trait. The SO_Arab double heterozygotes were characterized by a normal mean corpuscular volume (MCV) and mean corpuscular hemoglobin concentration (MCHC), and an increase of Hb F (11.7±4.3%) and 2,3-diphosphoglycerate levels (27.8 m/g Hb). The increase of Hb F is higher than the one seen among O_Arabs of other ethnic backgrounds. Their clinical course was moderately severe and osteoporosis was quite frequent. The interactions of Hb O_Arab and Hb S were studied in vitro and it was confirmed the Hb O_Arab lowers the minimal gelling concentration of mixtures with Hb S (as compared to mixtures of Hb S and Hb A), but that this effect is ionic-strength dependent. Our data are in conflict with previous claims that Hb O_Arab mixtures with Hb S polymerized almost as much as pure S. Oxygen association curves show a significant displacement of the p50 to the right, but the effect of oxygen dissociation is less apparent. The displacement was not nearly as significant as with SS cells, confirming our gelation data. Blood group determinations establish that these Arab populations had black African admixture.The Hb O_Arab/⁺ Thal double heterozygotes exhibit moderate anemia (10.3g% of Hb) and the percentage of Hb A was 17.2±1.8%. The fetal Hb was 5.4±2.1% and the 2,3-diphosphoglycerate level in two cases was 17.4 mol/g Hb. The only case of a homozygote SS had moderate anemia (10.3 g Hb%), 25.7% of Hb F, and a very benign course.     

Autonomic nervous system dysfunction: Implication in sickle cell disease

Sickle cell disease is an inherited hemoglobinopathy caused by a single amino acid substitution in the β chain of hemoglobin that causes the hemoglobin to polymerize in the deoxy state. The resulting rigid, sickle-shaped red cells obstruct blood flow causing hemolytic anemia, tissue damage, and premature death. Hemolysis is continual. However, acute exacerbations of sickling called vaso-occlusive crises (VOC) resulting in severe pain occur, often requiring hospitalization. Blood rheology, adhesion of cellular elements of blood to vascular endothelium, inflammation, and activation of coagulation decrease microvascular flow and increase likelihood of VOC. What triggers the transition from steady state to VOC is unknown. This review discusses the interaction of blood rheological factors and the role that autonomic nervous system (ANS) induced vasoconstriction may have in triggering crisis as well as the mechanism of ANS dysfunction in SCD.     

Effect of Chronic Blood Transfusion on Biomarkers of Coagulation Activation and Thrombin Generation in Sickle Cell Patients at Risk for Stroke

Hypercoagulability in sickle cell disease (SCD) is associated with multiple SCD phenotypes, association with stroke risk has not been well described. We hypothesized that serum levels of biomarkers of coagulation activation correlate with high transcranial Doppler ultrasound velocity and decreases with blood transfusion therapy in SCD patients. Stored serum samples from subjects in the Stroke Prevention in Sickle Cell Anemia (STOP) trial were analyzed using ELISA and protein multiplexing techniques. 40 subjects from each treatment arm (Standard Care [SC] and Transfusion [Tx]) at three time points—baseline, study exit and one year post-trial and 10 each of age matched children with SCD but normal TCD (SNTCD) and with normal hemoglobin (HbAA) were analyzed. At baseline, median vWF, TAT and D-dimer levels were significantly higher among STOP subjects than either HbAA or SNTCD. At study exit, median hemoglobin level was significantly higher while median TCD velocity was significantly lower in Tx compared to SC subjects. Median vWF (409.6 vs. 542.9 μg/ml), TAT (24.8 vs. 40.0 ng/ml) and D-dimer (9.2 vs. 19.1 μg/ml) levels were also significantly lower in the Tx compared to the SC group at study exit. Blood levels of biomarkers coagulation activation/thrombin generation correlated positively with TCD velocity and negatively with number of blood transfusions. Biomarkers of coagulation activation/thrombin generation were significantly elevated in children with SCD, at high risk for stroke. Reduction in levels of these biomarkers correlated with reduction in stroke risk (lower TCD velocity), indicating a possible role for hypercoagulation in SCD associated stroke.     

Variations in the relative activities of erythrocyte membrane ATPase with changes in severity of sickle cell anemia

Red blood cells from 31 patients with sickle cell anemia whose hemoglobins were ascertained as SS were assayed for Mg-, Ca-, Na-, and total ATPase activities. The ATPase activities were correlated with the various stages of severity in each patient as determined by clinical parameters. The results demonstrate that increases in ATPase activities were associated with increases in the percentage severity of sickle cell anemia. Severity correlated inversely with fetal hemoglobin levels in the sickle cell patients. ATPase activities were generally higher in SS genotypes than in AS and AA normal individuals.     

Monocyte and macrophage activation by lipoteichoic Acid is independent of alanine and is potentiated by hemoglobin

Lipoteichoic acids (LTAs) are Gram-positive bacterial cell wall components that elicit mononuclear cell cytokine secretion. Cytokine-stimulating activity is thought to be dependent on retaining a high level of ester-linked D-alanine residues along the polyglycerol phosphate backbone. However, Streptococcus pyogenes LTA essentially devoid of D-alanine caused human and mouse cells to secrete as much IL-6 as LTA with a much higher D-alanine content. Furthermore, hemoglobin (Hb) markedly potentiates the stimulatory effect of various LTAs on mouse macrophages or human blood cells, regardless of their d-alanine content. LTA and Hb appear to form a molecular complex, based on the ability of each to affect the other's migration on native acrylamide gels, their comigration on these gels, and the ability of LTA to alter the absorption spectra of Hb. Because S. pyogenes is known to release LTA and secrete at least two potent hemolytic toxins, LTA-Hb interactions could occur during streptococcal infections and might result in a profound alteration of the local inflammatory response.     

Frequency of glucose-6-phosphate dehydrogenase deficiency in sickle-cell disease. A study in Saudi Arabia

The frequency of glucose-6-phosphate dehydrogenase (G-6-PD) deficiency in 50 Hb S homozygotes (SS) and 98 Hb S heterozygotes (AS) was determined and compared with the frequency obtained in individuals with normal haemoglobin (AA). The observed number of SS patients with G-6-PD deficiency was significantly greater than the expected value (p less than 0.05). The frequency of G-6-PD deficiency in AA, AS and SS was found to be 0.172, 0.214 and 0.420, respectively. A statistically significant increase of G-6-PD deficiency was apparent in the Saudi sicklers. The possibility that G-6-PD deficiency and Hb S gene interact, influencing the survival of the carriers of these genetic abnormalities, is discussed.     

Prevalence of βS-globin gene haplotypes, α-thalassemia (3.7 kb deletion) and redox status in patients with sickle cell anemia in the state of Paraná, Brazil

The aim of this study was to determine the frequency of beta S-globin gene (β^S globin) haplotypes and alpha thalassemia with 3.7 kb deletion (−α^3.7kb thalassemia) in the northwest region of Paraná state, and to investigate the oxidative and clinical-hematological profile of β^S globin carriers in this population. Of the 77 samples analyzed, 17 were Hb SS, 30 were Hb AS and 30 were Hb AA. The β^Sglobin haplotypes and −α^3.7kb thalassemia were identified using polymerase chain reaction.Trolox equivalent antioxidant capacity (TEAC) and lipid peroxidation (LPO) were assessed spectophotometrically. Serum melatonin levels were determined using high-performance liquid chromatography coupled to coulometric electrochemical detection. The haplotype frequencies in the SS individuals were as follows: Bantu- 21 (62%), Benin - 11 (32%) and Atypical- 2 (6%). Bantu/Benin was the most frequent genotype. Of the 47 SS and AS individuals assessed, 17% (n = 8) had the −α^3.7kb mutation. Clinical manifestations, as well as serum melatonin, TEAC and LPO levels did not differ between Bantu/Bantu and Bantu/Benin individuals (p > 0.05). Both genotypes were associated with high LPO and TEAC levels and decreased melatonin concentration. These data suggest that the level of oxidative stress in patients with Bantu/Bantu and Bantu/Benin genotypes may overload the antioxidant capacity.     

Regulation of iron absorption in hemoglobinopathies

Beta-thalassemia and sickle cell anemia (SCD) represent the most common hemoglobinopathies caused, respectively, by deficient production or alteration of the beta chain of hemoglobin (Hb). Patients affected by the most severe form of thalassemia suffer from profound anemia that requires chronic blood transfusions and chelation therapies to prevent iron overload. However, patients affected by beta-thalassemia intermedia, a milder form of the disease that does not require chronic blood transfusions, eventually also show elevated body iron content due to increased gastrointestinal iron absorption. Even SCD patients might require blood transfusions and iron chelation to prevent deleterious and painful vaso-occlusive crises and complications due to iron overload. Although definitive cures are presently available, such as bone marrow transplantation (BMT), or are in development, such as correction of the disease through hematopoietic stem cell beta-globin gene transfer, they are potentially hazardous procedures or too experimental to provide consistently safe and predictive clinical outcomes. Therefore, studies that aim to better understand the pathophysiology of the hemoglobinopathies might provide further insight and new drugs to dramatically improve the understanding and current treatment of these diseases. This review will describe how recent discoveries on iron metabolism and erythropoiesis could lead to new therapeutic strategies and better clinical care of these diseases, thereby yielding a much better quality of life for the patients.     

Association between apolipoprotein ε4 allele,factor V Leiden,and plasma lipid and lipoprotein levels with sickle cell disease in southern Iran

To investigate whether there is any association between various APOE alleles and factor V Leiden (FVL) with lipid profiles and sickle cell disease (SCD) in Southern Iran. 65 SCD patients consisting of 35 sickle cell anemia homozygous (SS), 15 sickle cell heterozygous (AS) and 15 sickle cell/βThalassemia (S/βthal) patients and 68 healthy individuals with normal hematological indices were studied. APOE and FVL polymorphisms were detected by PCR–RFLP and serum lipid level was measured enzymatically. The frequencies of FVL and APOE-ε4 allele were significantly higher in SCD patients than in control (15.4 vs. 4.4 and 13.7% vs. 3.3%, respectively). The distributions of APOE ε3ε3, ε2ε3 and ε2ε4 + ε3ε4 alleles in SCD patients were significantly different from those in the control group. The SCD subjects particularly SS/S βthal (SS + S/βthal) and SS patients have significantly lower frequency of APOE ε3ε3 allele (P < 0.05) whereas SCD, SS patients and AS individuals have a significantly higher frequency of APOE ε4 allele (ε2ε4 + ε3ε4; P = 0.003, P = 0.011 and P = 0.035, respectively) compared to the control group. The LDL-C (P = 0.006) and total cholesterol (P < 0.001) levels in SCD subjects were found to be significantly lower than those in the control group. In addition, the presence of non-APOE ε4 allele (ε2ε3 + ε3ε3) resulted in a significant decrease in the level of LDL-C and total cholesterol in SCD subjects in general and in SS and SS/S βthal patients in particular compared to controls. Furthermore, the presence of APOE ε4 allele (ε2ε4 + ε3ε4) was found to be associated with the risk of sickle cell anemia [OR = 4.1, P = 0.04]. The presence of either FVL mutation (OR = 4.6; CI: 0.91–24, P = 0.07) or APOE-ε4 allele (OR = 4.07; CI: 1.01–16.4, P = 0.048) is associated with the risk of sickle cell disease in Southern Iran. This data suggest that the activation of coagulation system enhances thrombus generation and decreases antioxidant activity in SCD patients from Southern Iran.     

Transcriptional activity of paired homeobox Pax6 is enhanced by histone acetyltransferase Tip60 during mouse retina development

The pathophysiology of oxidative hemolytic anemia is closely associated with hemoglobin (Hb) stability; however, the mechanism of how Hb maintains its stability under oxidative stress conditions of red blood cells (RBCs) carrying high levels of oxygen is unknown. Here, we investigated the potential role of peroxiredoxin II (Prx II) in preventing Hb aggregation induced by reactive oxygen species (ROS) using Prx II knockout mice and RBCs of patients with hemolytic anemia. Upon oxidative stress, ROS and Heinz body formation were significantly increased in Prx II knockout RBCs compared to wild-type (WT), which ultimately accelerated the accumulation of hemosiderin and heme-oxygenase 1 in the Prx II knock-out livers. In addition, ROS-dependent Hb aggregation was significantly increased in Prx II knockout RBCs. Interestingly, Prx II interacted with Hb in mouse RBCs, and their interaction, in particular, was severely impaired in RBCs of patients with thalassemia (THAL) and sickle cell anemia (SCA). Hb was bound to the decameric structure of Prx II, by which Hb was protected from oxidative stress. These findings suggest that Prx II plays an important role in preventing hemolytic anemia from oxidative stress by binding to Hb as a decameric structure to stabilize it.     

Rational design of pyridyl derivatives of vanillin for the treatment of sickle cell disease

Hypoxia-induced polymerization of sickle hemoglobin (Hb S) is the principal phenomenon that underlays the pathophysiology and morbidity associated with sickle cell disease (SCD). Opportunely, as an allosteric protein, hemoglobin (Hb) serves as a convenient and potentially critical druggable target. Consequently, molecules that prevent Hb S polymerization (Hb modifiers), and the associated erythrocyte sickling have been investigated–and retain significant interest–as a viable therapeutic strategy for SCD. This group of molecules, including aromatic aldehydes, form high oxygen affinity Schiff-base adducts with Hb S, which are resistant to polymerization. Here, we report the design and synthesis of novel potent antisickling agents (SAJ-009, SAJ-310 and SAJ-270) based on the pharmacophore of vanillin and INN-312, a previously reported pyridyl derivative of vanillin. These novel derivatives exhibited superior in vitro binding and pharmacokinetic properties compared to vanillin, which translated into significantly enhanced allosteric and antisickling properties. Crystal structure studies of liganded Hb in the R2 quaternary state in complex with SAJ-310 provided important insights into the allosteric and antisickling properties of this group of compounds. While these derivatives generally show similar in vitro biological potency, significant structure-dependent differences in their biochemical profiles would help predict the most promising candidates for successful in vivo pre-clinical translational studies and inform further structural modifications to improve on their pharmacologic properties.