Sickle cell disease: A distinction of two most frequent genotypes (HbSS and HbSC).

Sickle cell disease (SCD) consists of a group of hemoglobinopathies in which individuals present highly variable clinical manifestations. Sickle cell anemia (SCA) is the most severe form, while SC hemoglobinopathy (HbSC) is thought to be milder. Thus, we investigated the clinical manifestations and laboratory parameters by comparing each SCD genotype. We designed a cross-sectional study including 126 SCA individuals and 55 HbSC individuals in steady-state. Hematological, biochemical and inflammatory characterization was performed as well as investigation of previous history of clinical events. SCA patients exhibited most prominent anemia, hemolysis, leukocytosis and inflammation, whereas HbSC patients had increased lipid determinations. The main cause of hospitalization was pain crises on both genotypes. Vaso-occlusive events and pain crises were associated with hematological, inflammatory and anemia biomarkers on both groups. Cluster analysis reveals hematological, inflammatory, hemolytic, endothelial dysfunction and anemia biomarkers in HbSC disease as well as SCA. The results found herein corroborate with previous studies suggesting that SCA and HbSC, although may be similar from the genetic point of view, exhibit different clinical manifestations and laboratory alterations which are useful to monitor the clinical course of each genotype.

The clinical significance of K-Cl cotransport activity in red cells of patients with HbSC disease.

HbSC disease is the second commonest form of sickle cell disease, with poorly understood pathophysiology and few treatments. We studied the role of K-Cl cotransport activity in determining clinical and laboratory features, and investigated its potential role as a biomarker. Samples were collected from 110 patients with HbSC disease and 41 with sickle cell anemia (HbSS). K-Cl cotransport activity was measured in the oxygenated (K-Cl cotransport(100)) and deoxygenated (K-Cl cotransport(0)) states, using radioactive tracer studies. K-Cl cotransport activity was high in HbSC and decreased significantly on deoxygenation. K-Cl cotransport activity correlated significantly and positively with the formation of sickle cells. On multiple regression analysis, K-Cl cotransport increased significantly and independently with increasing reticulocyte count and age. K-Cl cotransport activity was increased in patients who attended hospital with acute pain in 2011 compared to those who did not (K-Cl cotransport(100): mean 3.87 versus 3.20, P=0.009, independent samples T-test; K-Cl cotransport(0): mean 0.96 versus 0.68, P=0.037). On logistic regression only K-Cl cotransport was associated with hospital attendance. Increased K-Cl cotransport activity was associated with the presence of retinopathy, but this effect was confounded by age. This study links variability in a fundamental aspect of cellular pathology with a clinical outcome, suggesting that K-Cl cotransport is central to the pathology of HbSC disease. Increased K-Cl cotransport activity is associated with increasing age, which may be of pathophysiological significance. Effective inhibition of K-Cl cotransport activity is likely to be of therapeutic benefit.

Immune regulation in chronically transfused allo-antibody responder and nonresponder patients with sickle cell disease and ß-thalassemia major.

Red blood cell alloimmunization is a major complication of transfusion therapy. Host immune markers that can predict antibody responders remain poorly described. As regulatory T cells (Tregs) play a role in alloimmunization in mouse models, we analyzed the Treg compartment of a cohort of chronically transfused patients with sickle cell disease (SCD, n = 22) and ß-thalassemia major (n = 8) with and without alloantibodies. We found reduced Treg activity in alloantibody responders compared with nonresponders as seen in mice. Higher circulating anti-inflammatory IL-10 levels and lower IFN-γ levels were detected in non-alloimmunized SCD patients. Stimulated sorted CD4+ cells from half of the alloimmunized patients had increased frequency of IL-4 expression compared with nonresponders, indicating a skewed T helper (Th) 2 humoral immune response in a subgroup of antibody responders. All patients had increased Th17 responses, suggesting an underlying inflammatory state. Although small, our study indicates an altered immunoregulatory state in alloantibody responders which may help future identification of potential molecular risk factors for alloimmunization.

Novel permeability characteristics of red blood cells from sickle cell patients heterozygous for HbS and HbC (HbSC genotype).

Individuals heterozygous for HbS and HbC (HbSC) represent about 1/3(rd) of sickle cell disease (SCD) patients. Whilst HbSC disease is generally milder, there is considerable overlap in symptoms with HbSS disease. HbSC patients, as well as HbSS ones, present with the chronic anaemia and panoply of acute vaso-occlusive complications that characterize SCD. However, there are important clinical and haematological differences. Certain complications occur with greater frequency in HbSC patients (like proliferative retinopathy and osteonecrosis) whilst intravascular haemolysis is reduced. Patients with HbSC disease can be considered as a discrete subset of SCD cases. Although much work has been carried out on understanding the pathogenesis of SCD in HbSS homozygotes, including the contribution of altered red blood cell permeability, relatively little pertains directly to HbSC individuals. Results reported in the literature suggest that HbSC cells, and particularly certain subpopulations, present with similar permeability to HbSS cells but there are also important differences - these have not been well characterized. We hypothesise that their unique cell transport properties accounts for the different pattern of disease in HbSC patients and represents a potential chemotherapeutic target not shared in red blood cells from HbSS patients. The distinct pattern of clinical haematology in HbSC disease is emphasised here. We analyse some of the electrophysiological properties of single red blood cells from HbSC patients, comparing them with those from HbSS patients and normal HbAA individuals. We also use the isosmotic haemolysis technique to investigate the behaviour of total red blood cell populations. Whilst both HbSS and HbSC cells show increased monovalent and divalent (Ca(2+)) cation conductance further elevated upon deoxygenation, the distribution of current magnitudes differs, and outward rectification is greatest for HbSC cells. In addition, although Gd(3+) largely abolishes the cation conductance of both HbSS and HbSC cells, only in HbSS ones are currents inhibited by the aminoglycosides like streptomycin. This distinction is retained in isosmotic lysis experiments where both HbSS and HbSC cells undergo haemolysis in sucrose solutions but streptomycin significantly inhibits lysis only in HbSS cells. These findings emphasise similarities but also differences in the permeability properties of HbSS and HbSC cells, which may be important in pathogenesis.

Impaired cytoadherence of Plasmodium falciparum-infected erythrocytes containing sickle hemoglobin.

Sickle trait, the heterozygous state of normal hemoglobin A (HbA) and sickle hemoglobin S (HbS), confers protection against malaria in Africa. AS children infected with Plasmodium falciparum are less likely than AA children to suffer the symptoms or severe manifestations of malaria, and they often carry lower parasite densities than AA children. The mechanisms by which sickle trait might confer such malaria protection remain unclear. We have compared the cytoadherence properties of parasitized AS and AA erythrocytes, because it is by these properties that parasitized erythrocytes can sequester in postcapillary microvessels of critical tissues such as the brain and cause the life-threatening complications of malaria. Our results show that the binding of parasitized AS erythrocytes to microvascular endothelial cells and blood monocytes is significantly reduced relative to the binding of parasitized AA erythrocytes. Reduced binding correlates with the altered display of P. falciparum erythrocyte membrane protein-1 (PfEMP-1), the parasite's major cytoadherence ligand and virulence factor on the erythrocyte surface. These findings identify a mechanism of protection for HbS that has features in common with that of hemoglobin C (HbC). Coinherited hemoglobin polymorphisms and naturally acquired antibodies to PfEMP-1 may influence the degree of malaria protection in AS children by further weakening cytoadherence interactions.

Oxidative status of valinomycin-resistant normal, beta-thalassemia and sickle red blood cells.

Exposure of red blood cells (RBC) to the K+ -ionophore valinomycin (val), causes loss of KCl and water, resulting in cell dehydration, manifested by increased cell density. While almost all normal val-treated RBC dehydrate, in sickle cell anemia (SCA) a portion of the RBC fail to dehydrate and maintain a light density, indicating the existence of val-resistant (val-res) RBC. In thalassemia and sickle cell disease (SCD), although the primary lesion is in the globin genes, damage to the RBC is partly mediated by oxidative stress. We previously showed that such RBC are under oxidative stress, having more reactive oxygen species (ROS) and less reduced glutathione than normal RBC. We now report a relationship between the phenomenon of val-res and the RBC oxidative status: Treatment with oxidants that increase ROS, also increased the frequency of val-res cells. Val-res cells had higher oxidative status than other RBC in the sample. Similar to SCA, thalassemic blood has more val-res cells than does normal blood. Val-res cells in thalassemic and sickle blood showed a higher oxidative status than normal val-res cells. Thus, oxidative stress might be involved in generation of val-res cells. Further studies are required to elucidate the origin and significance of these cells.

Binding of ovarian steroids to erythrocytes in patients with sickle cell disease; effects on cell sickling and osmotic fragility.

Ovarian steroids appear to influence the manifestations of sickle cell disease (SCD); oestrogens can adversely affect erythrocyte function, whereas progestogens may inhibit sickling and decrease the osmotic fragility of erythrocytes. The aims of the present studies were: (i) to characterise the binding of oestradiol and progesterone to erythrocytes from women with HbSS, HbSC and HbAA genotypes; (ii) to investigate whether steroids modulate susceptibility to sickling or osmotic fragility of HbSS and HbAA erythrocytes. Erythrocytes were incubated for 1h with [3H]-steroids at 4 and 37 degrees C. Binding of both oestradiol and progesterone was independent of temperature and steroid concentration, but was decreased by sequential "washing" of erythrocytes in fresh incubation buffer. Binding capacity was 80 +/- 6% greater for oestradiol (versus progesterone) in all three genotypes, and binding of both steroids was decreased by > or = 70% in HbSS erythrocytes compared to HbSC or HbAA erythrocytes. Pre-incubation of erythrocytes with 35 microM oestradiol or 30 microM progesterone had no significant effect on susceptibility of HbSS and HbAA erythrocytes to sickling, or on osmotic fragility. We conclude that both oestradiol and progesterone bind in a low affinity, non-saturable manner to erythrocytes with decreased binding in cells from women with HbSS. However, steroid binding does not affect susceptibility to sickling or osmotic fragility irrespective of haemoglobin genotype.

Cardiorespiratory and metabolic responses to exercise in HbSC sickle cell patients.

PURPOSE: Relative to healthy control individuals with normal hemoglobin (Hb), patients carrying the double heterozygous form of sickle cell disease (HbSC) display an impaired oxygen transport capacity. The present study was undertaken to determine the influence of the decreased oxygen availability associated with the presence of HbSC on the cardiorespiratory and metabolic responses to endurance exercise. METHODS: Eleven black men affected by the double heterozygous form of the sickle cell disease (HbSC group) and seven healthy subjects with normal Hb (HbAA group) of the same ethnic origin submitted successively to an incremental exercise test to exhaustion on a cycle ergometer for the determination of their maximal tolerated power and to a 20-min endurance exercise. RESULTS: The HbSC had a significantly lower exercise tolerance than the HbAA. During the endurance exercise, they exhibited furthermore significantly lower VO2, VCO2, and minute ventilation V(E) than the HbAA. Despite the fact that the HbSC exercised at a significantly lower mean absolute work rate than the HbAA, except for the ventilatory equivalent for CO2 (V(E)/VCO2), which was higher (P < 0.001) in the HbSC group, the other parameters recorded during the 20-min endurance exercise (heart rate, arterial PaO2, PaCO2, pH, lactate, and VE/VO2, the ventilatory equivalent for O2) and during the subsequent recovery (blood lactate) were similar for both groups. CONCLUSION: The study underscores the importance of considering relative work rate as well as absolute work rate to arrive at a correct interpretation of exercise and recovery data. The results give evidence that the modifications of homeostasis brought into play by exercise were shifted toward distinctly lower absolute work rates in HbSC patients.

The correlation between phase shifts in gradient-echo MR images and regional brain iron concentration.

The purpose of this study was to investigate the relationship between the magnetic susceptibility of brain tissue and iron concentration. Phase shifts in gradient-echo images (TE = 60 ms) were measured in 21 human subjects, (age 0.7-45 years) and compared with published values of regional brain iron concentration. Phase was correlated with brain iron concentration in putamen (R2 = 0.76), caudate (0.72), motor cortex (0.68), globus pallidus (0.59) (all p < 0.001), and frontal cortex (R2 = 0.19, p = 0.05), but not in white matter (R2 = 0.05,p = 0.34). The slope of the regression (degrees/mg iron/g tissue wet weight) varied over a narrow range from -1.2 in the globus pallidus and frontal cortex to -2.1 in the caudate. These results suggest that magnetic resonance phase reflects iron-induced differences in brain tissue susceptibility in gray matter. The lack of correlation in white matter may reflect important differences between gray and white matter in the cellular distribution and the metabolic functions of iron. Magnetic resonance phase images provide insight into the magnetic state of brain tissue and may prove to be useful in elucidating the relationship between brain iron and tissue relaxation properties.

Laudanosine and atracurium concentrations in a patient receiving long-term atracurium infusion.

OBJECTIVE: Atracurium is sometimes used for muscle relaxation in patients undergoing mechanical ventilation. Use of atracurium in high doses or for a long period of time has raised the possibility of the accumulation of laudanosine, a breakdown product known to cause seizure activity in animals. The objective of this report was to see if laudanosine accumulation and seizure activity had occurred in a patient who had received a long-term, relatively high-dose infusion of atracurium. DESIGN: Case report. The patient received atracurium for 38 days, at rates ranging from 0.3 to 0.96 mg/kg/hr. An electroencephalogram (EEG) was done before the discontinuation of the infusion, and plasma concentrations of atracurium and laudanosine were measured at, and after, the termination of the atracurium infusion. The laudanosine elimination half-life was calculated. SETTING: Intensive care unit. PATIENT: A 23-yr-old woman admitted with sickle cell crisis, complicated by acute chest syndrome, acute respiratory distress syndrome, and hepatic and renal failure. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: As expected, laudanosine concentrations were increased but were below the level reported to cause seizure activity in animals. Laudanosine elimination half-life was prolonged to 617 mins, which was consistent with previously reported values. The patient's EEG was normal, with no ictal pattern. CONCLUSIONS: Despite long-term use of high doses of atracurium infusion and the increased elimination half-life of laudanosine, only moderate accumulation of laudanosine occurred, and the EEG was normal. Hence, it appears unlikely that toxic concentrations of laudanosine would be reached, even in a critically ill patient.

Localization of vascular endothelial growth factor in human retina and choroid.

OBJECTIVE: To examine the distribution and relative levels of vascular endothelial growth factor (VEGF) in the nondiabetic and preproliferative diabetic human retina and choroid. METHODS: Immunohistochemical localization was performed on frozen sections from cryopreserved postmortem human tissue using a polyclonal antibody against VEGF and a streptavidin peroxidase system. Eyes from 5 subjects without diabetes and 8 subjects with diabetes were examined and analyzed using a 7-point immunohistochemical grading system. RESULTS: In subjects without diabetes, weak or no VEGF immunoreactivity was associated with retinal blood vessels. In subjects with diabetes, we found significantly increased immunoreactivity in the retinal vascular endothelium and blood vessel walls. Vascular endothelial growth factor immunoreactivity was also associated with intravascular leukocytes in subjects with and without diabetes. In the choroid of subjects without diabetes, immunoreactivity was almost exclusively associated with intravascular leukocytes, whereas in diabetic subjects, immunoreactivity was localized within choriocapillaris endothelium, choroidal neovascular endothelium, and migrating retinal pigment epithelium cells. CONCLUSIONS: The observed increase in VEGF immunoreactivity in the diabetic retina and choroid suggests that VEGF may contribute to 2 well-documented events during retinopathy: increased vascular permeability and angiogenesis.

Molecular basis of sickle cell-endothelial cell interactions.

Adherence of sickle erythrocytes to microvascular endothelium is posited to initiate or contribute to sickle cell vaso-occlusive pain episodes. Adherence and occlusion in vivo may depend on hemodynamics interacting with plasma, erythrocyte, and endothelial cell factors. Four receptor-mediated adherence pathways have been described to date: adherence mediated by high molecular weight von Willebrand factor multimers bridging glycoprotein lb-like and integrin receptors on sickle cells and similar receptors on endothelial cells; thrombospondin bridging CD36 on sickle reticulocytes and the alpha v beta 3 integrin on large-vessel endothelial cells or alpha v beta 3 and CD36 on microvascular endothelium; binding of sickle reticulocyte alpha 4 beta 1 receptors to vascular cell adhesion molecule 1 expressed on endothelial cells stimulated by cytokine or double-stranded RNA viruses; and binding of sickle cells to endothelial cell-associated fibronectin via sickle reticulocyte alpha 4 beta 1 activated by phorbol ester or interleukin-8. The significance of these adherence pathways in sickle cell vaso-occlusion is discussed.

Altered muscle metabolism shown by magnetic resonance spectroscopy in sickle cell disease with leg ulcers.

We performed 31P magnetic resonance spectroscopy of gastrocnemius muscle at rest in 7 normal volunteers and 12 patients with sickle cell disease (7 with leg ulcers and 5 without leg ulcers but with painful crises). We measured intracellular pH and ratios of Pi to ATP, PCr to ATP and PCr to Pi (Pi = inorganic phosphate, ATP = adenosine triphosphate, and PCr = phosphocreatine). Magnetic resonance arteriograms were also performed. Significant differences were found for PCr/Pi ratios between normals and sickle cell disease patients with leg ulcers (p < 0.008). Magnetic resonance arteriograms were normal in volunteers and patients with sickle cell disease. The altered high energy phosphate metabolism in sickle cell disease with leg ulcers is consistent with muscle ischemia or hypoxia.

Serial evaluation of iron stores in pregnant Nigerians with hemoglobin SS or SC.

Iron status of nonpregnant and pregnant Nigerian patients with hemoglobin SS or SC were assessed using serial hematological parameters, measured by Coulter counter, and serial serum ferritin concentrations measured by radioimmunoassays. The median value of 393 micrograms/L (range, 175 to 900 micrograms/L) for serum ferritin in nonpregnant patients with Hb SS and SC was significantly higher than that found in nonpregnant patients with Hb AA (median, 89.8 micrograms/L; range, 13 to 250 micrograms/L). Apart from packed cell volume values, there were no other significant differences between patients with Hb SS or SC and Hb AA in the other parameters assessed: mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration. In both the normal pregnant patients (Hb AA) and pregnant patients with Hb SS and SC the serum ferritin values decreased as pregnancy advanced to 28 weeks and rose gradually thereafter. At similar stages of gestation serum ferritin values were significantly higher in patients with Hb SS or SC than in those with Hb AA. Pregnancy seems to have induced a significant rise in mean corpuscular volume and mean corpuscular hemoglobin values in the patients with Hb SS or SC, especially in the third trimester, than in patients with Hb AA. The pattern of change in mean corpuscular hemoglobin concentration values was similar in both groups of patients. From the data obtained, it seems the iron status in the patients with Hb SS or SC was good, and pregnancy did not push the patients into an iron deficiency state. The use of prophylactic iron supplementation in pregnant patients with Hb SS or SC appears unjustified.

Whole body protein turnover and resting metabolic rate in homozygous sickle cell disease.

1. Whole body protein turnover and resting metabolic rate were measured in six adults with homozygous sickle cell disease (genotype HbSS) and in six normal adults (genotype HbAA) of similar age. 2. Turnover was measured with prime/intermittent oral doses of [15N]glycine over 18 h and resting energy expenditure was measured by indirect calorimetry. 3. In HbSS, nitrogen flux (0.9 +/- 0.08 g day-1), protein synthesis (6.0 +/- 0.5 g day-1 kg-1) and protein degradation (5.6 +/- 0.5 g day-1 kg-1) were significantly increased compared with HbAA nitrogen (flux 0.5 +/- 0.02 g day-1 kg-1, protein synthesis 3.2 +/- 0.2 g day-1 kg-1 and protein degradation 2.8 +/- 0.2 g day-1 kg-1). 4. Resting energy expenditure was significantly higher in HbSS compared with HbAA when expressed per unit of body weight (115 +/- 3 and 94 +/- 4 kJ day-1 kg-1, respectively) or weight 0.75(317 +/- 6 and 269 +/- 8 kJ day-1 kg-0.75, respectively). 5. The increase in protein turnover and energy expenditure suggest that patients with HbSS exist in a hypermetabolic state that requires greater dietary energy compared with HbAA.

Whole body protein turnover and resting metabolic rate in homozygous sickle cell disease

Whole body protein turnover and resting metabolic rate were measured in six adults with homozyguous sickle cell disease (genotype HbSS) and in six normal adults (genotype HbAA) of similar age. Turnover was measured with prime/intermittent oral doses of [15N]glycine over 18 h and resting energy expenditure was measured by indirect calorimetry. In HbSS, nitrogen flux (0.9 ñ 0.08 g day-1 kg-1), protein synthesis (6.0 ñ 0.5 g day-1 kg-1) and protein degradation (5.6 ñ 0.5 g day-1 kg-1) were significantly increased compared with HbAA nitrogen (flux 0.5 ñ 0.02g day-1 kg-1, protein synthesis 3.2 ñ 0.2 g day-1 kg-1 and protein degradation 2.8 ñ 0.2 g day-1 kg-1). Resting energy expenditure was significantly higher in HbSS compared with HbAA when expressed per unit of body weight (115 ñ 3 and 94 ñ 4 kj day-1 kg-1 respectively) or weight 0.75(317 ñ 6 and 269 ñ 8 kj day-1kg-0.75, respectively). The increase in protein turnover and energy expenditure suggest that patients with HbSS exist in a hypermetabolic state that requires greater dietary energy compared with HbAA. (AU)

Urea kinetics in sickle cell disease

When dietary nitrogen intake is adequate to satisfy the body's metabolic demand for nitrogen, adaptive mechanisms are involved whereby nitrogen losses are minimised. The most significant reduction in nitrogen loss is effected by reduced urea production and excretion and there is a concomitant increase in the proportion of urea hydrolysed and recycled in the body. This work was designed to explore the mechanisms by which nitrogen equilibrium is maintained in the whole body when the metabolic demands for nitrogen exceed the supply. Urea kinetics was measured in normal healthy controls (HbAA) and patients with homozygous sickle cell disease (HbSS). HbSS is characterised by an increased metabolic demand for nitrogen for erythropoesis, which is significantly higher than normal. Urea production (P), excretion (Eu), hydrolysis (T) and recycling (Pr) of hydrolysed urea to urea synthesis, were determined using isotopic urea(urea-30) orally or intravenously, and the two methods compared. The reliability of the methods was examined and demonstrated to be satisfactory. All aspects of urea metabolism were increased in HbSS compared with HbAA, except Eu which tended to be lower in HbSS. The proportion of urea hydrolysed in the colon was raised in HbSS, regardless of the route by which the isotope was given and the kinetics was measured. Individuals with sickle cell trait (HbAS), were also studied and exhibited rates of urea hydrolysis that were either similar to the HbAA values or raised to values corresponding to the HbSS. Urea nitrogen incorporated into haemoglobin in both the HbAA and HbSs. This finding confirmed the utilisation of endogenous urea nitrogen for protein synthesis in the body. The proportion of hydrolysed urea nitrogen is small (10 percent) compared to a larger pool of enteric nitrogen to which it contributes, suggesting that there may be a more significant contribution of enteric nitrogen metabolism to the nitrogen economy, than of hydrolysed urea alone. The possibility that glycine may be limited in HbSS has been indicated and warrants more detailed investigation. The metabolism of urea has been shown to be affected by depleting the body glycine pool and alternatively by glycine supplements. Results from this work support the idea that urea kinetics is modulated by dietary nitrogen intake (AU)

Hemoglobin S-mediated membrane oxidant injury: protection from malaria and pathology in sickle cell disease.

It is suggested here that a Hemoglobin S (HbS)-mediated membrane oxidant injury is responsible for both the protection from malaria infection in the heterozygous sickle cell state, and for a critical pathologic process in homozygous sickle cell disease. This suggestion is arrived at by applying to the HbS condition the oxidant stress model for malaria resistance. Such a model had been developed to explain the protection from malaria in thalassemia and in glucose-6-phosphate dehydrogenase (G6PD) deficiency (9).