Steady-state Sickle Cell Disease Research Articles

Clinical Evaluation of Extract of Cajanus cajan (Ciklavit®) in Sickle Cell Anaemia

The major pathology in sickle cell anaemia (SCA) is sickling of red cells due to the precipitation of reduced haemoglobin. We report our experience with extract of Cajanus cajan as a possible antisickling agent by determining changes, if any, in clinical and laboratory features of the disease in patients given the extract in a single-blind placebo-controlled study. One hundred patients with steady-state SCA were randomized into treatment and placebo arms. The extract/placebo were administered twice daily to the subjects. Weight, hepatosplenomegaly, blood levels of biliurubin, urea, creatinine, and packed cell volume (PCV) were monitored over a 6-month period. Recall episodes of pain 6 months before enrolment were compared with episodes of pains recorded during the treatment period. Twenty-six cases (55.3 per cent) had hepatomegaly on enrolment. This significantly reduced to 33.3 per cent at 6 months (p = 0.03); but increased in the placebo arm (p > 0.05). The total number of recall painful episodes in cases was 207 (mean 4.4 +/- 10.3 (SD), range 0-60) and fell to 191 (mean 4.2 +/- 4.4 (SD), range 0-16); p = 0.03. Episodes of pain increased from 109 in controls (mean 2.6 +/- 5.0 (SD), range 0-26) to 164 (mean 3.9 +/- 4.3 (SD), range 0-22); p = 0.01. Mean PCV in the cases showed no appreciable changes (p = 0.1) but there was a significant increase in the controls (p = 0.02). In conclusion, the extract may cause a reduction of painful crises and may ameliorate the adverse effects of sickle cell anaemia on the liver. The mechanism of action remains to be determined.

Renal Vascular Resistance in Sickle Cell Painful Crisis

Vaso-occlusive painful crisis is one of the characteristic manifestations of sickle cell disease (SCD). We aimed to investigate the state of renal vascular resistance by means of Doppler ultrasonography during vaso-occlusive painful crisis in patients with SCD. The 60 patients with homozygous SCD who entered the study were divided into 2 groups. Group 1 included 45 patients who were living in steady-state conditions and had no history of transfusions within the 3 months before the initiation of the study. Group 2 consisted of 15 patients with signs of painful vaso-occlusive crisis during evaluation. Group 2 patients had significant reductions in 3 measures of flow velocity in both main renal arteries, compared with group 1 patients (P < .04, P < .001, and P < .01). Mean and end-diastolic velocities in the segmental arteries (P < .01, and P < .001, respectively) and end-diastolic velocities in the interlobar arteries (P < .04) were lower in group 2 patients than in group 1 patients. Analysis of resistive (RI) and pulsatile (PI) indices in the investigated arteries demonstrated that the RI of the renal (P < .001; P < .0001), segmental (P < .002; P < .0001) and interlobar (P < .001; P < .0001) arteries of both kidneys in group 2 < .0001; P < .0001) for both kidneys were markedly higher in group 2 patients than in group 1 patients and the healthy subjects, respectively. Our preliminary results suggest a reduction of renal blood flow and an increase in renal vascular resistance during painful crisis compared with steady-state SCD.

Transgenic Sickle Mice Are Markedly Sensitive to Renal Ischemia-Reperfusion Injury

Ischemic injury is invoked as a mechanism contributing to end-organ damage and other complications of sickle cell disease (SCD). However, the intrinsic sensitivity of tissues in SCD to ischemic insults has never been addressed. We examined the effect of renal ischemia in a transgenic mouse expressing human sickle hemoglobin. Twenty-four hours after bilateral, total renal artery occlusion for 15 minutes, transgenic sickle mice exhibited worse renal function and more marked histological injury. With bilateral renal ischemia of greater duration (22.5 minutes), and after 6 hours, transgenic sickle mice exhibited massive vascular congestion, sickling of red blood cells, more marked histological injury in the kidney, and more prominent congestion in the capillary beds in the lungs and heart. Additionally, serum amyloid P-component, the murine homologue of C-reactive protein, was markedly increased in transgenic sickle mice as compared to wild-type mice. Twenty-four hours after bilateral renal ischemia for 22.5 minutes, transgenic sickle mice exhibited 28% mortality, with no mortality observed in any other group. With bilateral renal ischemia of short or long duration, renal expression of caspase-3 was most prominent in transgenic sickle mice subjected to ischemia. Thus, renal ischemia in this murine model induces more severe renal injury and extrarenal complications. We conclude that tissues in SCD exhibit heightened vascular congestion and sensitivity to ischemia and that clinically apparent or silent episodes of ischemia may contribute to the complications of SCD.

The Influence of Uridine Diphosphate Glucuronosyl Transferase 1A Promoter Polymorphism, βS-Globin Gene Haplotype, Co-Inherited α-Thalassemia Trait and Hb F on Steady-State Serum Bilirubin Levels in Sickle Cell Anemia.

The chronic hemolysis of sickle cell anemia (SCA) produces hyperbilirubinemia but serum bilirubin level varies considerably and the modulating factors have not been fully elucidated. One identified genetic factor is the uridine diphosphate glucuronosyl transferase 1A (UGT1A) gene promoter polymorphism in which homozygosity for the (AT)7 allele has been associated with increased bilirubin levels in children with SCA. The same association has been reported in apparently healthy individuals, in β-thalassemia, G6PD deficiency, neonatal jaundice and hereditary spherocytosis. In the present study, in addition to UGT1A promoter genotype, serum bilirubin level was related to other genetic modifiers - βS-globin gene haplotype, Hb F, co-inherited α-thal trait, age and gender. The patients were randomly selected from the sickle cell clinic, Medical College of Georgia. UGT1A promoter genotypes were determined using automated sequencing and spreadex gel electrophoresis. Other investigations were with standard techniques. There were 67 SCA patients (41 males, 26 females), aged 2 to 44 years (mean of 20.6±10.7). Ten (14.9%) patients were homozygous for the (AT)6 UGT1A allele, 22 (32.8%) were homozygous for the (AT)7, while 35 (52.2%) were heterozygous for the two. Serum bilirubin was significantly higher in the homozygous (AT)7 group (3.7±1.5, 5.6±2.4 and 3.8±2.3 mg/dl respectively). It was also significantly higher in males than females (4.9±2.7 vs 3.7±1.7 mg/dl) and highest in patients aged >10 years. Co-inherited α-thal trait did not significantly affect the levels. There was a significant negative linear correlation (r = −0.304, p=0.016) of serum bilirubin with Hb F. Hb F was also significantly higher among females than males and when the different age groups were compared, it was highest in those <10 years old. Apart from UGT1A (AT)7 homozygosity, Hb F significantly influences serum bilirubin level in steady-state SCA. This is probably mediated via its hemolysis-sparing effect. Our (AT)7 homozygotes, including both children and adults, had significantly higher mean serum bilirubin level as previously reported by others. Indeed, when only the adults were analyzed, there was a distinct trimodal distribution among the 6/6, 6/7 and 7/7 UGT1A genotypes with serum bilirubin levels of 3.9 ± 0.9, 4.9 ± 2.2 and 5.8 ± 2.5 mg/dl respectively. Among children (<10 years), this trimodal distribution is lost, which supports the premise that a high Hb F is probably more important in modulating serum bilirubin levels in this age group.Serum Bilirubin and Other Parameters Grouped According to UGT1A1 GenotypeUGT1A1 GenotypeAge (yrs)Hb (g/dl)Retics (%)Hb F (%)Bilirubin (mg/dl)6/6 (n=10)18.1±8.77.9±1.211.3±4.78.4±6.43.7±1.56/7 (n=35)17.9±10.98.7±1.311.4±4.210.9±8.63.8±2.37/7 (n=22)26.1±9.38.3±2.110.9±4.86.6±5.45.6±2.4

Increased GM-CSF Levels in Sickle Cell Disease Are Associated with Increased Leukocyte Counts and Are Reversed by Hydroxyurea.

The hematopoietic cytokine, GM-CSF, stimulates the proliferation and differentiation of progenitor cells to macrophages, eosinophils and megakaryocytes. Increased levels of GM-CSF have previously been reported in sickle cell disease (SCD) patients with low fetal hemoglobin (HbF) levels (Croizat, Br J Haematol. 87:592; 1994); however, a clearcut association between GM-CSF and HbF levels in SCD has yet to be established, since there is some evidence that levels may depend on other factors (Haider et al., Acta Haematol. 102:140; 1999). To further investigate the possible role of GM-CSF in SCD, plasma samples were collected from normal individuals (AA), steady-state sickle cell anemia patients (SS) and sickle cell patients on hydroxyurea therapy (SSHU; 20-30 mg/kg/day). Plasma GM-CSF levels were measured using a specific immunoenzymatic assay. HbF levels were quantified by ion-exchange HPLC and hematological measurements obtained using an Advia Hematology System. Plasma GM-CSF levels seem to be higher in SS patients than in normal controls (0.828 ± 0.215 pg/ml, n=39; 0.230 ± 0.081 pg/ml, n=9; respectively). In contrast, GM-CSF was significantly lower in SSHU patients than in SS without HU (0.288 ± 0.104 pg/ml; n=14; P = 0.045 compared to SS). A negative correlation between plasma GM-CSF and HbF levels was seen in patients (r = −0.332; P = 0.026; n=45; SS and SSHU groups) and, notably, a positive correlation between GM-CSF and white blood cell counts was observed (r = 0.302; P = 0.037; n= 48; SS and SSHU groups). Furthermore, GM-CSF (2.5 ng/ml) abolished the production of γ globin during erythropoietin-stimulated differentiation of TF-1 hematopoietic cells. These results provide further support to the hypothesis that plasma GM-CSF correlates negatively with HbF in sickle cell disease. Interestingly, in vitro data suggest that increased GM-CSF may, in fact, diminish HbF levels rather than reflect increased haematopoietic stress as a consequence of low HbF. Importantly, GM-CSF appears to correlate with leukocyte numbers in SCD, and levels were significantly decreased in patients taking HU, indicating that this cytokine may contribute to the leukocytosis seen in some SCD patients and may play a role in leukocyte count reduction by HU.

Increased adhesive properties of eosinophils in sickle cell disease

Objective A role for leukocytes in vasoocclusion is becoming increasingly recognized. Here we investigate a possible role for the eosinophil in sickle cell anemia (SCA). Patients and methods Eosinophils were isolated from whole blood samples of 59 steady-state SCA homozygous SS and control individuals using Percoll gradient separation, followed by immunomagnetic sorting. Adhesion of cells to FN was evaluated using static adhesion assays (60 min, 37°C, 5% CO 2) and eosinophil adhesion molecular expression was observed by flow cytometry. Results SCA patients presented significantly elevated absolute eosinophil numbers. Furthermore, eosinophils isolated from these individuals demonstrated a significantly greater adhesion (∼70% increased) to fibronectin (FN) than normal eosinophils in static adhesion assays. Coincubation of eosinophils with integrin-blocking monoclonal antibodies in adhesion assays showed that an association of the VLA-4, LFA-1, and Mac-1 integrins mediate the adhesion of SCA eosinophils to FN. Flow cytometry demonstrated that the expression of these integrins, however, was unaltered on the surface of SCA eosinophils, suggesting that the increased SCA eosinophil adhesion is a consequence of increased integrin affinity or avidity. SCA eosinophil adhesion to FN was further increased by the cytokine, GM-CSF, indicating that inflammation processes may further stimulate eosinophil adhesion in these patients. Conclusion We report that eosinophil numbers may be significantly increased in SCA individuals and that these cells appear to exist in an activated state. Such alterations may indicate a role for the eosinophil in the vasooclusive process.

Increased levels of soluble ICAM-1 in the plasma of sickle cell patients are reversed by hydroxyurea.

Increased adhesive events between the blood vessel endothelium and red and white cells play a central role in the initiation of vasoocclusive crisis in sickle cell disease (SCD). Soluble VCAM-1 levels are increased in the plasma of sickle cell patients and may be reduced during hydroxyurea (HU) therapy. Reports regarding any changes in soluble ICAM-1 (sICAM-1) levels in sickle cell patients, however, are conflicting, and as yet no beneficial effect of HU upon levels has been observed. Thus, we sought to thoroughly investigate changes in sICAM-1 levels in SCD patients and the effect of HU therapy (20-30 mg/kg/day). Plasma sVCAM-1 levels were significantly higher in steady-state SCD patients than in normal controls (766 +/- 86 ng/mL vs. 325 +/- 38 ng/mL, respectively, P < 0.0001). sVCAM-1 levels were decreased in patients on HU therapy (543 +/- 69 ng/mL) compared to those not taking HU; however, this difference was not significant. Plasma sICAM-1 levels were significantly increased in steady-state SCD patients compared to normal individuals (285 +/- 20 ng/mL vs. 202 +/- 16 ng/mL, respectively, P = 0.002), and HU therapy significantly reduced sICAM-1 levels in patients (217 +/- 12, P = 0.027) to levels approaching those of healthy individuals. sVCAM-1 levels inversely correlated with fetal hemoglobin levels in SCD patients, while a nonsignificant inverse trend was observed between sICAM-1 levels and fetal hemoglobin. In conclusion, plasma sICAM-1 levels were significantly increased in SCD patients, and this increase was reversed by hydroxyurea therapy, possibly reflecting reduced endothelial activation in patients taking HU. Such an event may benefit patients by reducing adhesive interactions between white cells and the endothelium.

Blood mononuclear cell gene expression profiles characterize the oxidant, hemolytic, and inflammatory stress of sickle cell disease

AbstractIn sickle cell disease, deoxygenation of intra-erythrocytic hemoglobin S leads to hemoglobin polymerization, erythrocyte rigidity, hemolysis, and microvascular occlusion. Ischemia-reperfusion injury, plasma hemoglobin-mediated nitric oxide consumption, and free radical generation activate systemic inflammatory responses. To characterize the role of circulating leukocytes in sickle cell pathogenesis we performed global transcriptional analysis of blood mononuclear cells from 27 patients in steady-state sickle cell disease (10 patients treated and 17 patients untreated with hydroxyurea) compared with 13 control subjects. We used gender-specific gene expression to validate human microarray experiments. Patients with sickle cell disease demonstrated differential gene expression of 112 genes involved in heme metabolism, cell-cycle regulation, antioxidant and stress responses, inflammation, and angiogenesis. Inducible heme oxygenase-1 and downstream proteins biliverdin reductase and p21, a cyclin-dependent kinase, were up-regulated, potentially contributing to phenotypic heterogeneity and absence of atherosclerosis in patients with sickle cell disease despite endothelial dysfunction and vascular inflammation. Hydroxyurea therapy did not significantly affect leukocyte gene expression, suggesting that such therapy has limited direct anti-inflammatory activity beyond leukoreduction. Global transcriptional analysis of circulating leukocytes highlights the intense oxidant and inflammatory nature of steady-state sickle cell disease and provides insight into the broad compensatory responses to vascular injury.

Differential Cell Count of Bone Marrow Aspirates in Steady-state Sickle Cell Anaemia Patients

Background: Megaloblastic arrest of the bone marrow is one of the causes of anaemic crises in patients with sickle cell anaemia. The diagnosis of this condition will require that the reference level of megaloblastosis during steady state be properly documented. Objectives:To document a reference differential cell count in the bone marrow aspirates of patients with sickle cell anaemia and determine the level of megaloblastosis that can be regarded as indicating megaloblastic crisis in such patients. Design: Systematic counting of haematopoietic cells in the bone marrow of children with confirmed Hb phenotype SS who are in steady state. Subjects and Methods: The subjects were 11 of 68 children with sickle cell anaemia attending the paediatric outpatient clinic at the Lagos University Teaching Hospital, Lagos, in respect of whom parental consent for bone marrow aspiration was obtained. About 4.5 ml of blood was obtained from the antecubital vein of each child, for full blood count. Bone marrow was aspirated from the posterior superior iliac spine. Slides were stained with MayGrünwald-Giemsa stain. Proportions of erythroid, myeloid, lymphoid and megakaryocytic cells out of 250 nucleated bone marrow cells were determined. Results: Steady state mean packed cell volume (PCV) was 0.2 ± 0.017 L/L. The mean reticulocyte count was 5.9 percent (95% CI, 5.3 – 7.0%) and the mean cell volume was 91.8 ± 2.7 fl. Erythroid precursors constituted 34.5 percent of the total nucleated bone marrow cells (NBMC). Of these, polychromatic and orthochromatic erythroid blasts predominated, constituting 49 and 36percent respectively, of total erythroid precursors. Polychromatic and orthochromatic megaloblasts constituted 16.5percent (95% CI, 7-25%) of total NBMC or 47.8 percent of erythroid precursors. The myeloid erythroid ratio was 1:1. The reference range of megaloblasts was 8-26 percent of the NBMC. Conclusion: Patients with sickle cell anaemia in steady-state may show megaloblastic bone marrow changes even with routine folate supplements. Megaloblastic crisis should not be diagnosed until megaloblasts are in excess of 26 percent of the total NBMC. Nigerian Journal of Paediatrics2003;30:54-59

Avascular Necrosis of the Femoral Head in Adult Kuwaiti Sickle Cell Disease Patients

While sickle cell disease (SCD) is generally mild in most Kuwaitis, because of their elevated fetal Hb levels, avascular necrosis of the femoral head (AVNFH) appears to be a common complication. It was recently documented in 26.7% of Kuwaiti children with SCD. There have, however, been no previous studies of adult patients. This is a 1-year study of consecutive, steady-state SCD patients seen in the hematology clinic of Mubarak Al-Kabeer Hospital. The patients’ charts were reviewed for frequency of hospitalizations, any documented complications and steady-state complete blood count (CBC). MRI was performed using T1- and T2-weighted FATSAT sequences in coronal and axial planes with 4-mm-thick slices on a 1.5-tesla GE super-conducting magnet. Thirty-five patients were studied, consisting of 25 SS and 10 Sβ⁰Thal patients aged between 17 and 44, with a mean age of 26.7 ± 9.3 years. Seventeen (48.6%) had varying degrees of AVNFH; among the 70 hips examined, 29 (41.1%) were affected. Of the 17 patients affected, 11 (64.7%) were SS, while 6 (35.3%) were Sβ⁰Thal. There were 14 (82.4%) males and 3 (17.6%) females (χ² = 8.6, p < 0.01). The mean age of those affected, 27.5 ± 10.7 years, was not significantly higher than that of the unaffected (26.3 ± 8.0 years). Eleven (64.7%) of those affected had a history of frequent vaso-occlusive crisis. No significant differences could be demonstrated in the mean CBC and Hb F values of the two groups; coexistent α-thal trait was not a factor in the SS group. Male gender was the only significant predisposing factor identified. While more patients with frequent vaso-occlusive crises were affected, the difference was not significant. AVNFH is, indeed, quite common among Kuwaiti SCD patients and there is a need for early institution of preventive and therapeutic protocols.

Spontaneous circulation of myeloid-lymphoid–initiating cells and SCID-repopulating cells in sickle cell crisis

The only curative therapy for sickle cell disease (SCD) is allogeneic hematopoietic stem cell (HSC) transplantation. Gene therapy approaches for autologous HSC transplantation are being developed. Although earlier engraftment is seen when cells from GCSF-mobilized blood are transplanted than when bone marrow is transplanted, administration of GCSF to patients with SCD can cause significant morbidity. We tested whether primitive hematopoietic progenitors are spontaneously mobilized in the blood of patients with SCD during acute crisis (AC-SCD patients). The frequency of myeloid-lymphoid–initiating cells (ML-ICs) and SCID-repopulating cells (SRCs) was significantly higher in blood from AC-SCD patients than in blood from patients with steady-state SCD or from normal donors. The presence of SRCs in peripheral blood was not associated with detection of long-term culture–initiating cells, consistent with the notion that SRCs are more primitive than long-term culture–initiating cells. As ML-ICs and SRCs were both detected in blood of AC-SCD patients only, these assays may both measure primitive progenitors. The frequency of ML-ICs also correlated with increases in stem cell factor, GCSF, and IL-8 levels in AC-SCD compared with steady-state SCD and normal-donor sera. Because significant numbers of ML-ICs and SRCs are mobilized in the blood without exogenous cytokine treatment during acute crisis of SCD, collection of peripheral blood progenitors during crisis may yield a source of autologous HSCs suitable for ex-vivo correction by gene therapy approaches and subsequent transplantation.

Spontaneous circulation of myeloid-lymphoid-initiating cells and SCID-repopulating cells in sickle cell crisis.

The only curative therapy for sickle cell disease (SCD) is allogeneic hematopoietic stem cell (HSC) transplantation. Gene therapy approaches for autologous HSC transplantation are being developed. Although earlier engraftment is seen when cells from GCSF-mobilized blood are transplanted than when bone marrow is transplanted, administration of GCSF to patients with SCD can cause significant morbidity. We tested whether primitive hematopoietic progenitors are spontaneously mobilized in the blood of patients with SCD during acute crisis (AC-SCD patients). The frequency of myeloid-lymphoid-initiating cells (ML-ICs) and SCID-repopulating cells (SRCs) was significantly higher in blood from AC-SCD patients than in blood from patients with steady-state SCD or from normal donors. The presence of SRCs in peripheral blood was not associated with detection of long-term culture-initiating cells, consistent with the notion that SRCs are more primitive than long-term culture-initiating cells. As ML-ICs and SRCs were both detected in blood of AC-SCD patients only, these assays may both measure primitive progenitors. The frequency of ML-ICs also correlated with increases in stem cell factor, GCSF, and IL-8 levels in AC-SCD compared with steady-state SCD and normal-donor sera. Because significant numbers of ML-ICs and SRCs are mobilized in the blood without exogenous cytokine treatment during acute crisis of SCD, collection of peripheral blood progenitors during crisis may yield a source of autologous HSCs suitable for ex-vivo correction by gene therapy approaches and subsequent transplantation.

Endothelial dysfunction in patients with sickle cell disease is related to selective impairment of shear stress–mediated vasodilation

Interactions between the endothelium and erythrocytes may contribute to the vascular complications of sickle cell disease (SCD). Endothelium-derived nitric oxide (NO) plays a major role in the regulation of vasomotor tone in response to wall shear stress (WSS) variations and pharmacologic stimuli. However, little is known about endothelial NO production in patients with steady-state SCD. We investigated endothelial NO production in response to flow or vasoactive agonists in 16 homozygous patients with steady-state SCD and 15 controls. Flow-mediated dilation (FMD), arterial diameter changes in response to 100% oxygen inhalation, blood viscosity, and calculated WSS were determined in all patients and controls. At baseline, WSS was higher in SCD patients than in controls, whereas arterial diameter was similar. In patients with SCD, FMD was impaired (1.73% +/- 0.44% vs 3.97% +/- 0.24% in the controls, P <.001) and vasoconstriction in response to 100% oxygen was abolished. Using venous occlusion plethysmography, forearm blood flow (FBF) was evaluated in response to acetylcholine, nitro-monomethyl-L-arginine (L-NMMA), and sodium nitroprusside (SNP) in subgroups of 9 controls and 7 patients with SCD. Acetylcholine induced a significantly greater FBF increase in the patients (9.7 +/- 2.9 mL/min/100 mL of forearm volume vs 2.5 +/- 1.5 mL/min/100 mL in the controls, P <.001), whereas responses to L-NMMA and SNP were similar. These results suggest that endothelial dysfunction may prevent the arterial diameter of patients with SCD from adapting to chronic or acute shear stress elevations. This may contribute to the pathophysiology of vaso-occlusive crisis in patients with SCD.

Alpha-2-Macroglobulin and Interleukin-6 Levels in Steady-State Sickle Cell Disease Patients

Endothelial activation and subclinical microvascular occlusions are an ongoing process during steady-state sickle cell disease, leading to interleukin production and an acute-phase response. Alpha-2-macroglobulin (α2M) is an acute-phase protein mainly regulated by interleukin-6 (IL-6). On the other hand, α2M acts as a carrier protein for IL-6 during inflammatory stress. The purpose of this study is to further assess the interactions between IL-6 and α2M as potent modulators of inflammatory reactions during the steady state of sickle cell disease. We measured α2M and IL-6 levels in 21 patients (12 male, 9 female; age range 12–44 years) in the steady state of sickle cell disease. Four patients had homozygous sickle cell anaemia and 17 had double heterozygous sickle cell/β-thalassaemia. Diagnostic quantification of α2M was performed by rate nephelometry. Commercial enzyme immunoassay test kits were used for the quantitative measurement of IL-6. The α2M and IL-6 levels were compared to the values obtained from healthy volunteers. Mean values (± SD) of α2M and IL-6 were found to be significantly increased (p < 0.0005) in the patients (α2M: 337.2 ± 104 mg/dl; IL-6: 4 ± 2.1 pg/ml) compared to the healthy controls (α2M: 204.2 ± 45.8 mg/dl; IL-6: 1.15 ± 2.5 pg/ml). IL-6 values were positively correlated with α2M levels (r = 0.61, p < 0.01). We observed increased α2M and IL-6 levels in steady-state sickle cell disease and a positive correlation between these two inflammatory mediators. We suggest that α2M is a potent modulator of the inflammatory reaction and tissue repair mechanism during steady-state microvascular occlusions. Elucidating the role of α2M in sickle cell disease could lead to the development of novel strategies and therapies for preventing the harmful systemic or local effects of excess cytokine production.

EFFECT OF ANTI-IL-6 AND ANTI-10 MONOCLONAL ANTIBODIES ON THE SUPPRESSION OF THE NORMAL T LYMPHOCYTE MITOGENIC RESPONSE BY STEADY STATE SICKLE CELL DISEASE SERA

Previously published work has shown that sera from healthy sickle cell disease (SCD) patients inhibits normal lymphocyte mitogenic response to phytohemagglutinin (PHA) in vitro. The current study is to attempt to ascertain what effect antibody to type 2 cytokines, interleukin (IL)-6 and 10, have on the suppression of lymphocyte PHA response by SCD sera. Peripheral blood mononuclear cells (PBMC), separated by density gradient were obtained from 2 healthy normal donors. Sera from 50 healthy SCD patients, 50 normal healthy controls and pooled normal O, Rh+ (O+) sera were utilized in standard in vitro PHA stimulation of PBMC cultures. Mitogenic responses were expressed as mean counts per minute (cpm) of triplicate cultures. Fifty triplicate cultures of PHA stimulated normal PBMC were done with 10% normal pooled O+, normal control and SCD steady state sera only. In addition 50 cultures were done with SCD sera containing 1 μg/ml of anti-IL-6 monoclonal antibody, as well as 28 SCD serum cultures containing 1 μg/ml of anti-IL-10 monoclonal antibody. The final 11 SCD serum culture experiments contained a combination of both anti-IL-6 and anti-IL-10 antibody, each at the concentration of 1 μg/ml. Results revealed > 15% suppression of mitogenic response in the SCD sera supplemented cultures as compared to control sera in 47/50 (94%) and in 40/50 (80%) of normal pooled O+, as calculated by mean cpm. The degree of suppression ranged from 17% to 98% in individual experiments. The addition of anti-IL-6 antibody alone significantly improved mean cpm (> 20%) in 19/50 (38%) of SCD serum responses compared to O+ sera and 23/50 (46%) of control sera. Complete correction occurred in 9/50 (18%) of all SCD serum suppressions as compared to O+ sera and 6/50 (12%) when compared to control sera. Similarly, anti-IL-10 antibody decreased suppression of the mean cpm of SCD serum cultures in 18/28 (64%) and completely corrected 3/18 (11%). The combined antibody data revealed > 20% increase in mean cpm in 10/11(91%) experiments. Inhibitors of mitogenic response were present in a significant percentage of the SCD sera utilized in the present study. The significant corrective effects of both monoclonal antibodies would seem to support the original hypothesis that high circulating levels of type 2 cytokines may represent the cell-mediated dependent inhibitory factors expressed in the sera of many healthy SCD patients.

Arginine therapy: a novel strategy to induce nitric oxide production in sickle cell disease.

To determine the effects of L-arginine (L-Arg) supplementation on nitric oxide metabolite (NOx) production, oral L-Arg was given to normal controls, sickle cell disease (SCD) patients at steady state and SCD patients hospitalized with a vaso-occlusive crisis (VOC). L-Arg (0.1 g/kg) increased NOx formation by 18.8 +/- 68% in normal controls, whereas steady-state SCD patients demonstrated a paradoxical decrease in NOx of -16.7 +/- 4% (P = 0.004). In contrast, patients with VOC demonstrated a dramatic increase in NOx production by +77.7 +/- 103%, a response that was dose dependent. L-Arg appears to be the rate-limiting step in NOx production during VOC. Oral arginine may therefore benefit SCD patients by inducing an increase in NO production during VOC.

Arginine therapy: a novel strategy to induce nitric oxide production in sickle cell disease

To determine the effects of l‐arginine (l‐Arg) supplementation on nitric oxide metabolite (NOx) production, oral l‐Arg was given to normal controls, sickle cell disease (SCD) patients at steady state and SCD patients hospitalized with a vaso‐occlusive crisis (VOC). l‐Arg (0·1 g/kg) increased NOx formation by 18·8 ± 68% in normal controls, whereas steady‐state SCD patients demonstrated a paradoxical decrease in NOx of −16·7 ± 4% (P = 0·004). In contrast, patients with VOC demonstrated a dramatic increase in NOx production by +77·7 ± 103%, a response that was dose dependent. l‐Arg appears to be the rate‐limiting step in NOx production during VOC. Oral arginine may therefore benefit SCD patients by inducing an increase in NO production during VOC.

Tumor necrosis factor-alpha is undetectable in the plasma of SS patients with elevated Hb F.

Steady-state sickle cell disease (SCD) patients may have increased plasma levels of acute phase reactants and pro-inflammatory cytokines because of subclinical inflammation. We have estimated TNF-alpha levels in the plasma and in supernatants following peripheral blood mononuclear cell (PBMC) activation with phytohemagglutinin (PHA) in a group of Kuwaiti SCD patients using ELISA. The group consisted of 28 SS, 8 Sbeta-thal, and 2 SD patients all in steady state; 5 SS patients were studied during 7 episodes of painful crisis. The subjects were aged 2 to 16 years, with a mean of 7.3 +/- 3.5 years. The beta(S)-globin gene cluster haplotype, alpha-tha1 status, and spleen function were determined in the SS group using standard techniques. Most (82%) were homozygous for the Saudi Arabia/India haplotype and had elevated Hb F levels ranging from 15% to 35%. There were 24 controls (Hb AA or AS), of whom 14 were healthy and 10 were acutely ill at the time of the study. None of the children with SCD (either in steady state or crisis) had detectable plasma TNF-alpha, but four controls (3 acutely ill and one healthy) had levels ranging from 61.7 to 249.8 pg/mL. Following PHA stimulation most subjects responded with high levels of TNF-alpha, with the median level among the steady-state SS patients being significantly higher than that in the controls (both the acutely ill or healthy). It therefore appears that because of the mild disease among our Arab SS children, TNF-alpha is not detectable in their plasma in steady state; these children, however, had a significantly higher response than controls following PBMC activation.

GM-CSF in Sickle Cell Anemia Patients with Elevated Hb F

We estimated plasma GM-CSF levels in a group of 28 steady-state sickle cell anemia (SS) patients in Kuwait, using an ELISA technique. There were 24 age-matched Hb AA controls, 14 of whom were healthy while 10 were acutely ill at the time of the study. Five SS patients were also studied during 6 episodes of painful crisis. Among the SS patients, 82.1% were homozygous for the Saudi Arabia/India (SAI) haplotype with Hb F ranging from 15 to 35% and total Hb from 8.5 to 11 g/dl. Three patients (siblings) were SAI/Benin compound heterozygotes with Hb F of 9–23% and total Hb >10 g/dl. One patient each was homozygous for the Benin or the Bantu haplotype; they had Hb F <2% and total Hb of 6.6 and 7.2 g/dl, respectively. Four (14.3%) steady-state SS patients had detectable plasma GM-CSF ranging from 75 to 1,817.6 pg/ml. These included the 2 patients with Hb F <2.0% and 2 with the SAI/Benin compound heterozygotes with Hb F of 11 and 9%, respectively. Four (66.7%) SS patients in crisis, 6 (42.9%) healthy controls and 6 (60%) acutely ill controls had detectable plasma GM-CSF. A clearcut association of GM-CSF with Hb F level or degree of anemia in steady-state SS patients could not be established. The appearance of GM-CSF in the plasma of patients in crisis and also among control subjects raises the possibility that other factors are involved in the production of this cytokine in the subjects studied.

Prothrombotic changes in children with sickle cell disease: relationships to cerebrovascular disease and transfusion.

Vascular occlusion has a central role in the pathophysiology of sickle cell disease (SCD) and, although there is little evidence that thrombosis alone is responsible, patients with sickle cell disease are known to have an ill-defined but increased thrombotic risk. The most serious complication of this in childhood is stroke which occurs in 7-10% of children and a further 14% have asymptomatic cerebrovascular disease (CVD) on imaging. We have performed a comprehensive profile of coagulation inhibitors and markers of thrombin generation in 96 children (83 nontransfused [NTx] and 13 transfused [Tx]) with steady-state SCD and 18 healthy sibling controls. The levels of protein S (free and total) and heparin cofactor II were reduced in both the NTx and Tx groups compared to controls and protein C and APC resistance ratios were reduced in the NTx group only. Antithrombin levels were not different from controls. Thrombin-antithrombin complexes and prothrombin fragment F1+2 were increased in both patient groups. In the NTx subgroups with or without CVD there were no differences for any of the parameters measured except for lower haemoglobin levels and higher white cell counts in those with asymptomatic CVD. We conclude that children with SCD have a reduction in levels of the majority of the coagulation inhibitors and increased thrombin generation in the steady-state and these are only partially reversed by transfusion. However, these abnormalities do not appear to play a primary role in the development of cerebrovascular disease.