Morbidity In Sickle Cell Disease Research Articles

Estimated GFR Derived From Plasma Creatinine by Mass Spectrometry (MSMS) Is A Reliable Measure of Renal Function During the Hyperfiltration Phase In Sickle Cell Disease (SCD)

AbstractAbstract 2649 Aim:The hypothesis is that a height based eGFR formula using creatinine measured by mass spectrometry is a reliable measure of GFR in children with SCD. Background:The kidney is a particularly sensitive to hypoxia and renal failure is a major cause of morbidity and mortality in SCD. Children with SCD commonly hyper filtrate, thus making it difficult to establish a reliable Glomerular Filtration Rate (GFR) measure. It is essential to correctly assess the GFR, in order to identify when it begins to fall back towards normal and then starts to decline. Reporting of estimated glomerular filtration rate (eGFR) derived from plasma creatinine (pCr) measurements is now recommended for the classification and monitoring of patients with chronic kidney disease. The new MDRD formula requires the use of pCr methods traceable to stable isotope dilution mass spectrometry (MS). In children the recommendation is to use one of the formulae based on height (Ht), e.g. Schwartz et al1, Morris et al2, both of which were derived using traditional Jaffe creatinine methodology; eGFR = 40 × Ht (cm)/pCr (μmol/l). The introduction of MS traceable creatinine methods requires a review of the k factor. This correlation has never been validated in hyper filtration/or children with SCD. Methods:43 HbSS patients, age range 5–20yrs, attending the Evelina Children's Hospital were studied, including transfused and non-transfused patients. The plasma samples were also used for the measurement of plasma creatinine by stable isotope dilution mass spectrometry. eGFR was calculated using k=31. Results:Inutest GFR ranged from 70–175 ml/min/1.73m2. There was significant correlation between eGFR and inutest GFR (P<0.01, r=0.68). Conclusions:These preliminary data suggest that eGFR should prove valuable in monitoring GFR in children with SCD, even during hyper filtration, providing the constant is appropriately adjusted for the analytical method of creatinine. Disclosures:Inusa:Novartis: Research Funding.

Multi‐modal intervention for the inpatient management of sickle cell pain significantly decreases the rate of acute chest syndrome

Pain in children with sickle cell disease (SCD) is the leading cause of acute care visits and hospitalizations. Pain episodes are a risk factor for the development of acute chest syndrome (ACS), contributing to morbidity and mortality in SCD. Few strategies exist to prevent this complication. We performed a before-and-after prospective multi-modal intervention. All children with SCD admitted for pain during the 2-year study period were eligible. The multi-modal intervention included standardized admission orders, monthly house staff education, and one-on-one patient and caregiver education. A total of 332 admissions for pain occurred during the study period; 159 before the intervention and 173 during the intervention. The ACS rate declined by 50% during the intervention period 25% (39 of 159) to 12% (21 of 173); P = 0.003. Time to ACS development increased from 0.8 days (0.03-5.2) to 1.7 days (0.03-5.8); P = 0.047. No significant difference was found in patient demographics, intravenous fluid amount administered, frequency of normal saline bolus administration, or cumulative opioid amount delivered in the first 24 hr. Patient controlled analgesia-use was more common after the intervention 52% (82 of 159) versus 73% (126 of 173; P = 0.0001) and fewer patients required changes in analgesic dosing within the first 24 hr after admission (26%, 42 of 159 vs. 16%, 28 of 173; P = 0.015). A multi-modal intervention to educate and subsequently change physician's behavior likely decreased the rate of ACS in the setting of a single teaching hospital.

Severe pandemic H1N1 and seasonal influenza in children and young adults with sickle cell disease

AbstractInfluenza causes excess morbidity in sickle cell disease (SCD). H1N1 pandemic influenza has been severe in children. To compare H1N1 with seasonal influenza in SCD (patients younger than 22), we reviewed medical records (1993-2009). We identified 123 cases of laboratory-confirmed influenza (94 seasonal, 29 H1N1). Those with seasonal influenza were younger (median 4.4 vs 8.7 years old, P = .006) and had less asthma (24% vs 56%, P = .002). Those with H1N1 influenza more often had acute chest syndrome (ACS; 34% vs 13%, P = .01) and required intensive care (17% vs 3%, P = .02), including mechanical ventilation (10% vs 0%, P = .02). In multivariate analysis, older age (odds ratio [OR] 1.1 per year, P = .04) and H1N1 influenza (OR 3.0, P = .04) were associated with ACS, and older age (OR 1.1 per year, P = .02) and prior ACS (OR 3.3 per episode in last year, P < .006) with intensive care. Influenza, especially H1N1, causes critical illness in SCD and should be prevented.

Sickle-cell disease and malaria: evaluation of seasonal intermittent preventive treatment with sulfadoxine-pyrimethamine in Senegalese patients—a randomized placebo-controlled trial

Sickle-cell disease (SCD) patients are at high risk of developing malaria which is a major contributor to morbidity and mortality in this disease. In Senegal, malaria transmission is high during rainy season, between July and October, and it was noted that sickle-cell crisis are frequent during this period. Then we carried out a double-blind randomized controlled trial to compare the impact of monthly sulfadoxine-pyrimethamine (SP) during the high-transmission season versus placebo on malaria incidence and morbidity of sickle-cell anemia. Sixty (60) SCD patients were randomized either to receive three intermittent preventive treatment (ITP) with SP or placebo using the random permutation table with nine elements. The drug was administrated as follows: sulfadoxine 25 mg/kg and pyrimethamine 1.25 mg/kg and this treatment was given once during the following months: September, October, and November. Overall four episodes of malaria disease were diagnosed, all these cases in the placebo arm. Thus, overall prevalence was 6.6% and there was no other case of malaria in the SP arm during the study period. Parasitological diagnosis confirmed the presence of Plasmodium falciparum in all four cases. No patient death was encountered during the study. SP treatment was well tolerated as only one patient (1.6%) in the SP arm reported pruritus. A significant reduction of patients' complaints (p = 002) and blood requirements (p = 0.001) was noted in the SP group; whereas, no impact was observed on vaso-occlusive crisis and hospitalization occurrence. Malaria prophylaxis by monthly intake of SP during the transmission period of the parasite reduced the prevalence of malaria and was safe in SCD patients leaving in malaria endemic area.

Segmentation and quantification of pulmonary artery for noninvasive CT assessment of sickle cell secondary pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is a progressive vascular disease that results in high mortality and morbidity in sickle cell disease (SCD) patients. PAH diagnosis is invasive via right heart catheterization, but manual measurements of the main pulmonary artery (PA) diameters from computed tomography (CT) have shown promise as noninvasive surrogate marker of PAH. The authors propose a semiautomated computer-assisted diagnostic (CAD) tool to quantify the main PA size from pulmonary CT angiography (CTA). A follow-up retrospective study investigated the potential of CT and image analysis to quantify the presence of PAH secondary to SCD based on PA size. The authors segmented the main pulmonary arteries using a combination of fast marching level sets and geodesic active contours from smoothed pulmonary CTA images of 20 SCD patients with proven PAH by right heart catheterization and 20 matched negative controls. From the PA segmentation, a Euclidean distance map was calculated and an algorithm based on fast marching methods was used to compute subvoxel precise centerlines of the PA trunk (PT) and main left/right PA (PM). Maximum distentions of PT and PM were automatically quantified using the centerline and validated with manual measurements from two observers. The pulmonary trunk and main were significantly larger (p < 0.001) in PAH/SCD patients (33.73 +/- 3.92 mm for PT and 25.17 +/- 2.90 for PM) than controls (27.03 +/- 2.94 mm for PT and 20.62 +/- 3.06 for PM). The discrepancy was qualitatively improved when vessels' diameters were normalized by body surface area (p < 0.001). The validation of the method showed high correlation (mean R=0.9 for PT and R = 0.91 for PM) and Bland-Altman agreement (0.4 +/- 3.6 mm for PT and 0.5 +/- 2.9 mm for PM) between CAD and manual measurements. Quantification errors were comparable to intraobserver and interobserver variability. CAD measurements between two different users were robust and reproducible with correlations of R = 0.99 for both PT and PM and Bland-Altman agreements of -0.13 +/- 1.33 mm for PT and -0.08 +/- 0.84 mm for PM. Results suggest that the semiautomated quantification of pulmonary artery has sufficient accuracy and reproducibility for clinical use. CT with image processing and extraction of PA biomarkers show great potential as a surrogate indicator for diagnosis or quantification of PAH, and could be an important tool for drug discovery and noninvasive clinical surveillance.

Arginine Therapy for Vaso-Occlusive Pain Episodes in Sickle Cell Disease.

Abstract 573 BACKGROUND:Vaso-occlusion is the principal factor in the morbidity of sickle cell disease (SCD). Vaso-occlusive painful episodes (VOE) are common, debilitating, and the leading cause of hospitalizations, emergency room visits, and are associated with an increased mortality rate. There is no effective therapy that targets the underlying mechanisms of VOE. Symptomatic relief with analgesics is the only availabel treatment. Nitric oxide (NO) is a potent vasodilator that contributes to a variety of vaso-occlusive events in SCD. We have found that an arginine deficiency and low NO bioavailability occurs during VOE in SCD. Since arginine is the obligate substrate for NO production, and an acute deficiency is associated with VOE, we hypothesized that arginine supplementation may be a safe and beneficial treatment for sickle cell pain. PATIENTS AND METHODS:Hospitalized SCD patients > 3 years diagnosed within 24 hours with VOE and without associated complications were eligible; written informed consent was obtained. A total of 56 patients completed randomization in this double-blinded, placebo controlled trial. A standardized treatment and monitoring program for VOE was followed. Average age was 13.9 ± 4 years (range 3.6-19 years), and 52% were female. Patients received intravenous (IV) or oral arginine (0.1 gram/kg TID, n=28) or placebo (n=28) for 5 days or until discharge from the hospital. Narcotic records for 2 patients (randomized to placebo arm) were incomplete and were not included in the narcotic use analysis. An intention to treat analysis was performed for narcotic use applying an unpaired t-test with Welch's correction to adjust for unequal variance. RESULTS:Age was equally distributed between treatment and placebo groups. 57% of the arginine treatment group and 46% of the placebo group were female. A significant reduction in narcotic use (defined as total morphine use over the course of the hospital stay in mg/kg) by 56% was observed in the treatment arm receiving IV or oral arginine compared to placebo (mean ± SEM: 1.8 ± 0.4 mg/kg; n=28 vs. 4.1 ± 0.8mg/kg; n=26, p=0.01). Average length of hospitalization was 4.5 ± 0.4 days, and there was no significant difference between the 2 groups (4.1 ± 0.3 vs. 4.8 ± 0.5 days, p = 0.27; arginine vs. placebo arm). Four episodes of acute chest syndrome (ACS) developed during the study, three in the treatment arm and one in the placebo arm. There was one patient who experienced clinical deterioration associated with ACS requiring emergent transfusion and a transfer to the pediatric intensive care unit (PICU) in the placebo arm. No clinical deterioration or PICU transfers occurred in the arginine arm. Five in the treatment arm received transfusion vs. four in the placebo arm. No drug-related adverse events were observed. No significant differences were observed between pre and post therapy liver or renal function, or hematological parameters in the arginine treatment group vs. placebo. Two patients admitted for pain management ultimately did not receive IV narcotics. Both had been randomized into the arginine-treatment arm and received arginine therapy per protocol throughout their hospital stay and required only oral narcotics and non-steroidal analgesia. Reduction in narcotic use in the treatment arm remained significant even when these 2 patients were excluded from the analysis (p=0.02). CONCLUSIONS:IV arginine therapy represents a novel nutritional intervention for the treatment of pain in hospitalized patients with SCD. Use of IV arginine should also be considered in the treatment of VOE in the emergency department setting prior to hospitalization, although further investigation is warranted. A reduction of narcotic use by over 50% observed in this study is remarkable, as this is the first successful intervention for sickle cell-related pain that targets the underlying mechanism of vaso-occlusion through a promising NO-based therapy. Arginine is a safe and inexpensive intervention with narcotic-sparing effects that should be considered as an adjunct to standard therapy for VOE requiring hospitalization. Disclosures:Off Label Use: Arginine for treatment of sickle cell vaso-occlusive pain episodes.

Promising Therapies in Sickle Cell Disease

Despite the fact that sickle cell anemia was one of the first diseases to have a demonstrated genetic etiology, to date there is still only one approved therapy for this disease. Recent increases in our understanding of the pathophysiology of the disease should translate into improved and more rapid development of newer therapies. This review will focus on the following current and potential therapeutic strategies to reduce the morbidity of sickle cell anemia. 1) Therapies such as decitabine, hydroxyurea, butyrate, lenalidomide and pomalidomide, which decrease the polymerization rate of HbS by increasing the concentration of Hb F; 2) Drugs that decrease relative intracellular HbS concentration by increasing total cell volume via inhibition of normal membrane ion exchange channels, such as KCL Cotransporter and Gardos Channels. These inhibitors include magnesium pidolate, imidazole antimycotics, arginine and Senicapoc; 3) Treatment of sickle cell vasoocclusion through inhibition of endothelial or cell surface adhesion molecules, such ICAM 4 and alpha(v)beta(3) integrins, by drugs related to the GPII(b)III(a) inhibitors or adhesion molecule modulators, and 4) Attempts to achieve vasodilation by nitric oxide and antioxidant therapy. This review will discuss the status of these emerging therapies in the treatment of sickle cell anemia.

Correction of Murine Sickle Cell Disease Using γ-Globin Lentiviral Vectors to Mediate High-level Expression of Fetal Hemoglobin

Increased levels of red cell fetal hemogloblin, whether due to hereditary persistence of expression or from induction with hydroxyurea therapy, effectively ameliorate sickle cell disease (SCD). Therefore, we developed erythroid-specific, gamma-globin lentiviral vectors for hematopoietic stem cell (HSC)-targeted gene therapy with the goal of permanently increasing fetal hemoglobin (HbF) production in sickle red cells. We evaluated two different gamma-globin lentiviral vectors for therapeutic efficacy in the BERK sickle cell mouse model. The first vector, V5, contained the gamma-globin gene driven by 3.1 kb of beta-globin regulatory sequences and a 130-bp beta-globin promoter. The second vector, V5m3, was identical except that the gamma-globin 3'-untranslated region (3'-UTR) was replaced with the beta-globin 3'-UTR. Adult erythroid cells have beta-globin mRNA 3'-UTR-binding proteins that enhance beta-globin mRNA stability and we postulated this design might enhance gamma-globin expression. Stem cell gene transfer was efficient and nearly all red cells in transplanted mice expressed human gamma-globin. Both vectors demonstrated efficacy in disease correction, with the V5m3 vector producing a higher level of gamma-globin mRNA which was associated with high-level correction of anemia and secondary organ pathology. These data support the rationale for a gene therapy approach to SCD by permanently enhancing HbF using a gamma-globin lentiviral vector.

Induction of Caspase-Independent Apoptosis by Sickle Cell Disease (SCD) Serum in Non-SCD Leukocytes

Increased white cell counts are associated with augmented mortality and morbidity in sickle cell disease (SCD) and leukocytes play a central role in the vaso-occlusive process of sickle cell disease by adhering to the vascular endothelium and participating in mechanisms of oxidative stress and inflammation. The relevance of neutrophil death to the pathogenesis of inflammatory disease is now recognized, since alterations in the apoptotic processes of leukocytes may affect their cellular function and inflammatory processes. As such, this study investigated the effect of serum from control individuals (CON), SCD patients in steady state (SCD) and SCD patients on hydroxyurea therapy (SCDHU; 20–30 mg/kg/day HU) on the apoptotic processes of neutrophils (Neu). Neu were separated from whole blood of CON individuals using a ficoll-paque gradient; serum was also separated and filtered through 0.22μm ultra-filters. Pools of serum were prepared by mixing serum from 10 individuals from each subject group. Neutrophils (4×106 cells/ml) were cultured in the presence of pooled filtered serum (10% v/v) for 16h (37°C, 5%CO2) in DMEM. Apoptosis was evaluated by detection of annexin V binding, and viability was determined by MTS assay. CON Neu cultured in the presence of pooled SCD serum demonstrated a significantly higher percentage of apoptotic cells (67.6 ± 4.3%; n=4) compared to Neu cultured with CON serum (52.3 ± 3.6%; n=4; P<0.01 comp. SCD, paired t-test) or SCDHU serum (55.4 ± 4.2%; n=4; P<0.01 comp. SCD). In contrast, Caspase-3 activity was not significantly different in CON Neu following 16h of culture in the presence of CON and SCD serum (2.46±0.13 and 2.50±0.19-fold increased caspase-3 activity at 16h compared to basal activity, respect., n>5; P>0.05); Neu cultured with SCDHU serum demonstrated a higher caspase-3 activity (4.23±0.70-fold increased activity, n=8, P>0.05 Wilcox. matched pairs), but this increase was not statistically different to that of Neu cultured with CON serum. Viability did not differ significantly following 16h-culture of CON Neu with CON, SCD, or SCDHU serum (0.70±0.05; 0.80±0.06; 0.86±0.08 OD490nm, respect., n>9, P>0.05); furthermore co-incubation of the Neu with serums in the presence of the programmed necrosis inhibitor, Necrostatin (10μM), had no significant effect on cell viability (0.80±0.05; 0.83±0.05; 0.86±0.05 OD490nm, for CON, SCD and SCDHU respect., n>9, P>0.05), indicating that SCD serum does not appear to induce a process of necroptosis in Neu. Neutrophil death in SCD, like other inflammatory diseases, appears to be regulated by a complex network of both intracellular death and survival signaling pathways that may be modulated by extracellular stimuli. We demonstrate, under the conditions described herein, that filtered SCD serum can induce an apoptotic process in non-SCD neutrophils that does not appear to be mediated by alterations in caspase-3 activity. High levels of circulating TNF-α, a cytokine that has been previously found elevated in the serum of our population of SCD patients, are known to elicit neutrophil death via a caspase-independent mechanism; likewise, intracellular reactive oxygen species (ROS) production also induces caspase-independent neutrophil apoptosis. Interestingly, activity levels of superoxide dismutase (SOD), were found in significantly lower levels in SCD serum compared to CON serum (data not shown), suggesting that SCD serum may have a greater capacity to induce ROS production in neutrophils. Notably, serum from SCDHU individuals did not stimulate increased apoptosis in control neutrophils, indicating a reduction in the serum factor(s) that elicit apoptosis in these cells. As such, Neu may be subject to both anti-and pro-apoptotic stimuli in the circulation of SCD individuals. Since alterations in Neu apoptotic processes may have significant effects on Neu function and number, in turn contributing to inflammatory processes and cellular damage at sites of inflammation, studies to understand the complex balance of these mechanisms in SCD are necessary.

Histopathology of experimentally induced asthma in a murine model of sickle cell disease

AbstractAsthma is a comorbid condition associated with increased rates of pain, acute chest syndrome, and premature death in human sickle cell disease (SCD). We developed an experimental asthma model in SCD and control mice expressing either normal human or murine hemoglobin to determine its effect on mortality and lung pathology. To induce lung inflammation, experimental mice were sensitized to ovalbumin (OVA) by subcutaneous OVA implantation (Sen), allowed 2 weeks to recover, and then divided into 2 groups, each receiving over a subsequent 10-day period the same dosage of aerosolized OVA but 2 different levels of exposure: 15 minutes (LoSen) and 30 minutes (HiSen). During recovery, 10% of SCD mice died compared with no deaths in control mice. An additional 30% of HiSen SCD mice died during aerosolization compared with 10% in LoSen SCD. Histologic indices of lung inflammation (eg, eosinophil recruitment, airway and vessel wall thickening, and immunoreactive TGFβ and fsp-1) and bronchial alveolar lavage fluid eosinophil peroxidase activity differentially increased in sensitized mice compared with unsensitized mice. Our findings indicate SCD mice with experimentally induced asthma are more susceptible to death and pulmonary inflammation compared with control mice, suggesting that asthma contributes significantly to morbidity and mortality in SCD.

Disparity in the management of iron overload between patients with sickle cell disease and thalassemia who received transfusions

Transfusion therapy is frequently used to prevent morbidity in sickle cell disease (SCD), and subsequent iron overload is common. The objective of this study was to evaluate the current standard of care in monitoring iron overload and related complications in patients with SCD compared to thalassemia (Thal). A cross-sectional study was conducted at 31 hematology clinics in the United States, Canada, or the United Kingdom. Patients who received transfusions with a mean serum ferritin level of least 2000 ng per mL were eligible. A total of 199 patients with SCD (113 female; 24.9 +/- 13.2 years) and 142 with Thal (66 female; 25.8 +/- 8.1 years) were recruited, and data were collected between 2001 and 2003 by interview and medical record review. Although both groups were recruited on the basis of significant iron overload, the likelihood of performing a liver biopsy for routine iron monitoring was significantly higher (odds ratio [OR], 3.4; 95% confidence interval [CI], 2.2-5.3) in Thal than SCD. Thal patients were also more likely to be screened for iron-related organ injury including an echocardiograph for cardiomyopathy (OR, 2.6; p < 0.001; 95% CI, 1.6-4.2), alanine aminotransferase for liver function (OR, 8.3; CI, 1.05-64.4), and thyroid-stimulating hormone for hypothyroidism (OR, 12.3; CI, 7.0-21.5). For adult SCD patients, those maintained on simple transfusion with a serum ferritin level of greater than 2500 ng per mL were the least likely to have a liver biopsy (p < 0.03). These data highlight the unsystematic monitoring of iron and related organ injury in SCD. Until the relationship between iron and related comorbidities is better understood, routine monitoring of iron overload in SCD patients who receive transfusions should be considered a standard part of clinical care.

Methacholine challenge in children with sickle cell disease: A case series

Lung disease is a major cause of morbidity in children with sickle cell disease (SCD). Asthma in children with SCD is associated with a twice greater rate of pain and acute chest syndrome (ACS) episodes when compared to children with SCD but without asthma. Provocation challenges with methacholine are used to diagnose asthma when spirometry is normal, bronchodilator reactivity is absent, or the clinical picture is ambiguous. There have been only limited descriptions of use of methacholine challenge in individuals with SCD. We conducted a retrospective cohort study of 21 children with SCD and recurrent respiratory tract symptoms who were challenged with methacholine to determine if airway hyper responsiveness (AHR) was present. Fourteen (67%) of the children had a positive challenge. Of the 14 patients, four were given a new diagnosis of asthma based on the presence of chronic chest symptoms and the newly determined AHR and started on inhaled corticosteroids (ICS). In each positive challenge, forced expiratory volume in one second (FEV(1)) was reversed to at least 90% of baseline 15 min after bronchodilator treatment. Oxygen saturation decreased in 93% of those with a positive challenge, but returned to baseline values 15 min after bronchodilator treatment. No patient developed a pain or ACS episode within at least 1 month after the challenge. Evaluation of AHR with methacholine challenge in patients with SCD appears to be well tolerated and may elucidate a cause of SCD morbidity.

The Effects of Chronic Opiates Pain Therapy in Sickle Cell Anemia.

Sickle cell disease (SCD) patients experience high rates of morbidity and early mortality. Hydroxyurea (HU) therapy is associated with decreased morbidity and mortality as well as improved patient health outcomes (Charache et al, 1995 and Steinberg et al, 2003). However, the effect of chronic opiate pain management on HU treatment outcomes is unknown. In the current study, we evaluated the effects of HU and chronic long acting opiate therapy-OxyContin, methadone, fentanyl and MS Contin-on the following laboratory parameters: white blood cell count (WBC), hemoglobin (Hb), and mean corpuscular volume (MCV). Changes in these variables are known to be linked with HU effects on morbidity and mortality in SCD. 142 homozygous sickle cell anemia patients (75 females and 67 males) from the outpatient clinic at Duke University Medical Center were included in this study. Clinical data were collected by patient interview as well as review of medical records after informed consent. Patients were classified in 4 groups based on report of chronic daily use of long acting opiates and/or HU (current therapy with HU) as follows: No HU or opiates (None); opiates only; HU only; and HU and opiates. ANOVAs were used to identify combined effects of opiate therapy, HU therapy and the 3 laboratory parameters. Overall, we found that patients on HU (n=76, 54%), had lower WBC and higher Hb and MCV than the no HU patients (n=66, 46%), with p<.01 for all associations, as could be expected. In contrast to the positive effects of HU, patients on opiates had higher WBC than non opiate patients (p<.01). Surprisingly, when adjusting for gender and age, opiate therapy appeared to moderate the effect of HU treatment on WBC (p=.05) and MCV (p=.04). Patients who were on HU and opiates concurrently exhibited 17% higher WBC and 6% lower MCV than patients who were on HU alone, suggesting that opiate therapy may be attenuating the positive effects of HU. The findings from the current study confirm that HU treatment is associated with better patient health indices, i.e. lower WBC, higher Hb and MCV. In contrast, chronic opiate pain management, often a concomitant therapy with HU, was associated with poorer patient laboratory indices, both with or without concurrent HU treatment. Overall, these data indicate that the chronic use of opiates may decrease the efficacy of HU treatment in this patient population. Further studies are needed to study the mechanism of drug interaction between opiate and HU in SCD patients. The previously suggested inhibitory effect of HU on the cytochrome P450 CYP3A, an important cytochrome in the metabolism of some opiates (Reardon et al, 2005), should be considered for future studies of drug-drug interaction in SCD. [Display omitted]

Decitabine Improves Clinical Outcomes in Severely Ill Sickle Cell Disease Patients Who Have Exhausted Standard of Care Options.

Interventions such as immunization, penicillin prophylaxis, hydroxyurea and transfusion have extended life in patients with sickle cell disease (SCD). Nonetheless, these interventions are limited by toxicity or effectiveness; continued substantial morbidity and mortality in SCD indicates the need for better disease modification. In previous phase I/II clinical trials, 13 of 13 patients treated with the DNA hypomethylating agent decitabine responded with clinically significant fetal hemoglobin and total hemoglobin elevation and improvement in surrogate clinical end-points. However, in these early studies, no clinical end-points were measured and further studies have been delayed by funding issues. We describe an off-label experience in four patients with severe SCD that suggestsi.remarkable clinical effectiveness in patients who have exhausted standard of care and are severely ill;ii.tolerability even in the severely ill;iii.a mechanism of action based on increased reticulocytosis in addition to increased fetal hemoglobin.All four patients had multiple alloantibodies and red-cell auto-antibodies that limited availability and increased risks of transfusion, and had previously been treated with hydroxyurea with continued clinical deterioration. Three of the four patients had relative reticulocytopenia (absolute reticulocyte count <250x109/L and hemoglobin <9g/dl) and were receiving erythropoietin or darbopoietin for more than 8 weeks with continued progressive anemia and progressive congestive heart failure. All four patients were ECOG performance status 3 and ineligible for available clinical trials. Based on the clinical trial experience conducted at our institution, decitabine therapy at 0.1–0.2 mg/kg 1–2X/week was initiated in these patients not for research purposes but with the intent to produce direct clinical benefit. The limited clinical data and potential for unanticipated toxicity was discussed in full with each patient and family members. IRB approval was obtained for a retrospective chart review. No decitabine related adverse events occurred. All patients demonstrated >2g/dl increases in hemoglobin levels with an associated improvement in clinical status - decrease in pain, improvement in performance status, improvement in congestive heart failure symptoms/signs. Upward trends in the platelet and reticulocyte counts concurrent with downward trends in the neutrophil counts were consistent with previously observed effects of low dose decitabine or the related compound 5-azacytidine. Clinically significant neutropenia was avoided by dose reductions that did not reverse the improved hemoglobin levels. The differentiation altering effects of low dose decitabine relieve SCD anemia by decreasing hemolysis (through elevated HbF) and increasing reticulocytosis. Previous clinical trials, and this off-label experience, suggest that decitabine holds remarkable promise as a disease modifying agent for SCD and β-thalassemia. Further clinical trials to confirm this impression should be supported.

The Laminin 511/521–binding site on the Lutheran blood group glycoprotein is located at the flexible junction of Ig domains 2 and 3

AbstractThe Lutheran blood group glycoprotein, first discovered on erythrocytes, is widely expressed in human tissues. It is a ligand for the α5 subunit of Laminin 511/521, an extracellular matrix protein. This interaction may contribute to vaso-occlusive events that are an important cause of morbidity in sickle cell disease. Using x-ray crystallography, small-angle x-ray scattering, and site-directed mutagenesis, we show that the extracellular region of Lutheran forms an extended structure with a distinctive bend between the second and third immunoglobulin-like domains. The linker between domains 2 and 3 appears to be flexible and is a critical determinant in maintaining an overall conformation for Lutheran that is capable of binding to Laminin. Mutagenesis studies indicate that Asp312 of Lutheran and the surrounding cluster of negatively charged residues in this linker region form the Laminin-binding site. Unusually, receptor binding is therefore not a function of the domains expected to be furthermost from the plasma membrane. These studies imply that structural flexibility of Lutheran may be essential for its interaction with Laminin and present a novel opportunity for the development of therapeutics for sickle cell disease.

The Novel Hemoglobin-based Oxygen Carrier HRC 101 Improves Survival in Murine Sickle Cell Disease

Erythrocyte transfusion decreases morbidity in sickle cell disease, but is not without risk. Use of a hemoglobin-based oxygen carrier could offer the benefits of erythrocyte transfusion while reducing related complications. The authors tested the hypothesis that the novel hemoglobin-based oxygen carrier, HRC 101, would improve survival during exposure to acute hypoxia in a murine model of sickle cell disease, the transgenic mouse expressing hemoglobin SAD (alpha2beta2). Wild-type (n = 30) and transgenic SAD (n = 36) mice received 0.02 ml/g HRC 101 (hemoglobin concentration, 10 g/dl) or an equal volume of 5% albumin. Thirty percent or 6% oxygen was administered to spontaneously breathing mice during halothane anesthesia (inspired concentration, 0.5%). The time to cessation of cardiac electrical activity was recorded. Survival was compared using Kaplan-Meier analysis. Control mice survived the 60-min study period, whether breathing 30% or 6% oxygen. In contrast, all SAD mice given albumin and 6% oxygen died, with a median survival time of 9.0 min (interquartile range, 6.9-11.6 min; P < 0.0001). HRC 101 significantly increased survival in SAD mice breathing 6% oxygen. Of 12 SAD mice given HRC 101 and 6% oxygen, 4 survived the entire study period and 8 died, with a median survival time of 48 min (19-60 min; P < 0.0001 vs. albumin). HRC 101 significantly decreased sickle-related mortality during exposure to acute hypoxic stress in transgenic mice expressing hemoglobin SAD. HRC 101 warrants further evaluation as a therapeutic modality in sickle cell disease.

Glycocalyx Perturbation in Patients with Sickle Cell Disease: Association with Disease Morbidity.

Activated endothelium plays a pivotal role in the pathogenesis of sickle cell disease (SCD). The activation of the endothelium is caused by hypoxia reperfusion damage, high shear rates and pro-inflammatory mediators, like thrombin and TNF-alpha. The central role of the glycocalyx (a layer of hyaluronan and proteoglycans covering the endothelium) has been established as an antithrombotic and antiadhesive endothelial barrier. Recently, a technique to determine the total systemic volume of the endothelial glycocalyx in humans has been validated. Using this technique, we demonstrated that the glycocalyx volume is strongly diminished in diabetic patients and even more in diabetic patients with microangiopathy. In the present study, we measured the glycocalyx volume in 20 sickle cell patients (14 HbSS/Sβ0 and 6 HbSC) and 10 carriers of sickle cell disease. To assess whether the glycocalyx perturbation may also contribute to disease morbidity in SCD, disease morbidity score (DMS) was calculated according to predefined criteria. In addition, we determined plasma levels of the matrix metalloproteases (MMP-2 and MMP-9) and the tissue inhibitors of MMP (TIMP-1 and TIMP-2) and compared these levels with the glycocalyx volume. The total systemic glycocalyx volume was measured by subtracting the intravascular distribution volume of a glycocalyx impermeable tracer (autologous labelled erythrocytes) from that of a glycocalyx permeable tracer (dextran 40). DMS was calculated by adding up individual prevalence (present or absent) of organ damage (microalbuminuria, pulmonary hypertension, retinopathy, perceptive hearing loss, iron overload, avascular osteonecrosis, renal failure and leg ulcers) and history of sickle cell related complications (acute chest syndrome, stroke, > 2 vaso-occlusive crises/year, and cholelithiasis ). Patients with SCD (HbSS/Sβ0 and HbSC) demonstrated to have a significant reduced glycocalyx volume compared to sex and age-matched sickle cell traits 0.48 ± 0.14 and 0.5 ± 0.36 versus 1.26 ± 0.27 liters (mean ± SEM), p=0.025. Interestingly, the glycocalyx volume was associated with DMS. Patients with high (severe)DMS had a significant lower glycocalyx volume than patients with a low(mild) DMS (0.81 ± 0.26 versus 0.18 ± 0.26 liters, p=0.047). No associations between glycocalyx volume and serum levels of MMP-2, MMP-9, TIMP-1 or TIMP-2 were found. The strongly diminished glycocalyx volume in sickle cell patients resembles the chronic state of activation of the endothelium in SCD that may also may be responsible for the enhanced adhesion of leukocytes and erythrocytes as well as the prothrombotic state of these patients Since the glycocalyx layer serves as an important barrier between the endothelium and the circulating blood cells to prevent the adhesion of leukocytes, therapies that may restore or preserve glycocalyx function are warranted in SCD. [Display omitted]

CAMP-PKA Signaling Plays an Important Role in Augmented Neutrophil Adhesion and Chemotaxis in Sickle Cell Disease.

A chronic inflammatory state is now thought to underlie much of the pathophysiology of sickle cell disease (SCD). Increased leukocyte numbers are associated with increased morbidity and mortality in SCD and the adhesion of leukocytes to the vascular endothelium and sickle red cells may play an initiating role in the vaso-occlusive process. We sought to better understand the signaling events that lead to alterations in SCD neutrophil adhesive and chemotactic abilities. Neutrophils were isolated from the peripheral blood of healthy controls and SCD individuals in steady state by separation over a ficoll-paque gradient. Following washing and lysis of contaminating red cells, adhesion to fibronectin (FN) and chemotaxis of control and SCD neutrophils (4×106 cells/ml in RMPI medium) were assessed using static adhesion assays and a 96-well chemotaxis chamber assay (ChemoTX, Neuroprobe). Cell adhesion to FN (20mg/ml; 30 min, 37°C, 5% CO2) or IL-8-induced migration (2h, 37°C, 5% CO2) was calculated using a standard curve of the original cell suspension and expressed as the percentage of cells adhered or the number of cells migrated. As previously shown, SCD neutrophils demonstrate a greater ability to adhere to FN-coated plates than control individual leukocytes (13.1±1.48 % neutrophils adhered compared to 5.0±0.6 %; n=11; P<0.001 Mann-Whitney test). Interestingly, SCD neutrophils demonstrate significantly higher levels of the secondary messenger, cAMP, than control neutrophils (4.76±0.38 pMol/106 cells compared to 1.91±0.34 pMol/106 cells; n≥12; P<0.001). When neutrophils were incubated on FN-coated plates in the presence of the cAMP-dependent kinase (PKA) inhibitor, KT5720 (3μM), increased SCD neutrophil adhesion was significantly reversed to levels approaching those of control neutrophils (7.73±1.3 %; n=11; P=0.001). In contrast, control neutrophil adhesion to FN was not significantly affected by the PKA inhibitor (P>0.05). Furthermore, activation of primed SCD neutrophils by the chemokine, IL-8 (500ng/ml), resulted in a significant increase in SCD neutrophil adhesion to FN (19.3±2.2 %; P=0.02; n=7) with a concomitant increase in intracellular cAMP levels (335.8±17.1 % increase, P<0.05, n=3) that could also be reverted by co-incubation with the PKA inhibitor, KT5720 (11.8 ± 0.8 %; P=0.02; n=7). Interestingly, in vitro chemotaxis of SCD neutrophils in response to a stimulus of IL-8 (100 ng/ml) was found to be significantly increased when compared to control neutrophil IL-8-stimulated chemotaxis (14.3±1.1 x105 cells/ml migrated compared to 8.8±0.8x105 cells/ml; P=0.005; n=8). Once again, KT5720 (3 μM, pre-incubation for 15 min, 37°C), was able to significantly reverse (8.5±1.1 x105/ml; P=0.03; n=6) this enhanced IL-8-dependent SCD neutrophil chemotaxis. Results suggest that an up-regulated PKA-dependent pathway may play an important role in the altered adhesive and chemotactic properties of SCD neutrophils and their further activation by cytokines/chemokines such as IL-8, found in increased levels in the serum of SCD patients. Thus, upregulation of the cAMP-PKA pathway in SCD neutrophils may have important consequences for the chronic inflammatory state in SCD patients and may characterize a novel target for the control of altered neutrophil function in SCD.

Systematic Evaluation of Sickle Cell-Related Organ Damage and Complications: Implications for Sickle Cell Disease Management.

Sickle cell disease (SCD) is characterised by acute complications such as the painful vaso-occlusive crisis, acute chest syndrome and stroke. Although these acute complications are well known and mostly easy recognised, the impact of sickle cell-related chronic end-organ damage on outcome may well be underappreciated. In fact, often SCD severity is assessed. by the frequency of acute vaso-occlusive crises. However, accumulating chronic organ damage (such as renal failure and pulmonary hypertension) is nowadays recognized as an important determinant of disease morbidity as well. The aim of this study is to evaluate the prevalence of sickle cell-related complications and organ damage and their relation to SCD genotype and to established laboratory parameters of SCD morbidity in a tertiary teaching hospital. Furthermore, relations between complications and different forms of organ damage will also be determined. Sickle cell patients were consecutively and systematically screened for manifestations of chronic organ damage and their history of complications related to SCD and/or SCD-related treatment was retrospectively studied from their medical records. Organ damage and the history of sickle cell-related complications were expressed as a cumulative disease morbidity score (DMS). Every form of organ damage or complication scores 1 point and is divided by the number of screened items resulting in a maximal DMS of 1. Correlations were analysed with the Chi-Square test. All adult sickle cell patients (HbSS, HbSβ and HbSC) were included in our study (n=99). Six patients were excluded from analysis because of incomplete data (less than 7 of the 12 items).Prevalence of specific organ damage and complicationsTotalHbSSHbSβHbSCP valueN93491628Age (year)28 (22–39)27 (21–41)35 (25–41)28 (20–34)0.324**Female (%)656256710.567Disease Morbidity Score0.11 (0–0.25)0.18 (0.09–0.33)0.09 (0–0.190.10 (0–0.170.010**Organ damage/ complications (%)Microalbuminuria22330150.063Renal failure1015070.201Pulmonary hypertension2540070.006Retinopathy412830650.016Perceptive hearing loss1292760.205Iron overload1017040.063Cholelithiasis516643270.009Avascular osteonecrosis12171340.242Leg ulcers511000.089Acute chest syndrome243125110.128Frequent vaso-occlusive crises (>2/yr)14171940.374Stroke613000.053Numbers are percentages or medians (iqr). * Two-sided Chi-square tests **Two sided Kruskal-Wallis testThe DMS correlated significantly with c-reactive protein (R= 0.27, P=0.013), blood urea nitrogen (R=0.422, P>0.001) and hemoglobin (R= −0.326, P=0.003). No statistically significant correlation with leukocyte counts or HbF% was demonstrated. These data underscore the importance of systematic evaluation of all patients with SCD in the outpatient setting since many of the sickle cell-related complications may otherwise go unnoticed, thereby delaying the institution of potential therapeutic measures. Importantly, the lack of any significant association between the frequency of vaso-occlusive crises and manifest sickle cell-related organ damage illustrates its limited value as a marker of disease severity.

Sickle cell disease as a neurodevelopmental disorder

Sickle cell disease (SCD) is a blood disorder; however, the central nervous system (CNS) is one of the organs frequently affected by the disease. Brain disease can begin early in life and often leads to neurocognitive dysfunction. Approximately one-fourth to one-third of children with SCD have some form of CNS effects from the disease, which typically manifest as deficits in specific cognitive domains and academic difficulties. We discuss SCD as a neurodevelopmental disorder by reviewing the mechanisms of neurological morbidity in SCD, the timing of these mechanisms, the types of cognitive and behavioral morbidity that is typical, and the interaction of social-environmental context with disease processes. The impact of the disease on families shares many features similar to other neurodevelopmental disorders; however, social-environmental factors related to low socioeconomic status, worry and concerns about social stigma, and recurrent, unpredictable medical complications can be sources of relatively higher stress in SCD. Greater public awareness of the neurocognitive effects of SCD and their impact on child outcomes is a critical step toward improved treatment, adaptation to illness, and quality of life.