Vaso-occlusive Episodes Research Articles

Microparticles As Biomarkers Of Osteonecrosis Of The Hip In Sickle Cell Disease

IntroductionSickle cell disease (SCD) is the most common cause of osteonecrosis of the femoral head (ONFH) in children. ONFH is a debilitating condition that is associated with mobility limitations, chronic pain, and an impaired quality of life. While the mechanisms that cause ONFH remain unknown, ischemia from recurrent microvascular occlusion is likely to play a role. Vascular occlusion may result directly from obstruction by sickled cells, or indirectly via complex interdependent pathways characterized by sustained endothelial activation, chronic inflammation, and coagulation. Microparticles (MP) are small, cell membrane-derived vesicles generated in response to cellular activation, injury or apoptosis. MPs have emerged as potential modulators of inflammation and thrombosis and have been found to be elevated in patients with ONFH in the general population. ObjectiveThis pilot study examined whether microparticle levels in patients with SCD who have ONFH differ from SCD patients without ONFH, as well as healthy African American (AA) controls. MethodsSubjects were recruited at their baseline status and were excluded if they had been transfused within the past 30 days, hospitalized for a vaso-occlusive pain episode, acute chest syndrome, fever or surgery within the past 30 days, or had bony lesions of the femur or hip due to causes unrelated to SCD. For MP analysis, whole blood was collected in sodium citrate tubes and centrifuged for 15 minutes at 1500 x g at 20° C to generate platelet poor plasma. Aliquots of the plasma were immediately frozen and stored at -80° C until the time of MP analysis. 300 μl samples were diluted in PBS and centrifuged at 10000 x g for 1hr and the supernatant was centrifuged at 100,000 x g for 2 hr. The pellet was re-suspended in 1 mL of PBS and subjected to nanoparticle-tracking analysis to determine concentration and size. Additional laboratory biomarkers of inflammation and coagulation, including highly-sensitive C-reactive protein (hs-CRP), von Willebrand factor antigen (vWF Ag), tissue factor (TF), and D-dimer were analyzed for differences between groups. Analysis of variance was used to compare MP and biomarker levels between the three groups. The institutional review board at Children's Hospital & Research Center Oakland approved the study protocol and written informed consent was obtained from all participants. ResultsCharacteristics of the 30 subjects enrolled are shown in Table I.Total microparticle levels in ONFH(+) patients were 2.3-fold higher than in ONFH(-) patients, and 2.5-fold higher than in AA controls (Figure 1). Mean MP levels for ONFH(+) patients, ONFH(-) patients, and AA controls were 4.55 x 1010, 1.99 x 1010, and 1.85 x 1010, respectively. Microparticle levels in ONFH(-) SCD patients did not differ from AA controls. There were no statistically significant differences in hsCRP, vWF Ag, TF, or D-dimer levels between the ONFH(-) and ONFH(+) groups. [Display omitted] [Display omitted] ConclusionsThe results of this study demonstrate significantly elevated MP levels in individuals with SCD who have ONFH. Additional studies are needed to better understand the mechanistic effects of MPs on the development of ONFH and to determine whether MP levels may be useful as a predictive biomarker for early disease detection.This publication was supported by NIH/NCRR UCSF-CTSI Grant Number UL1 RR024131. Disclosures:No relevant conflicts of interest to declare.

Inhibition Of the Rho/Rho-Kinase Pathway Reduces In Vitro Adhesion Of Sickle Cell Anemia Eosinophils To Human Endothelial Cells

IntroductionSickle cell anemia (SCA) individuals present a chronic inflammatory condition that contributes to the recurrent episodes of vaso-occlusion. This process is a multi-step process that involves different cell types such as red blood cells, reticulocytes, activated endothelial cells, platelets and leukocytes. Endothelial dysfunction contributes to the vaso-occlusion process and leads to inflammation. Leukocytes may have a propagating and, possibly, initiating role in SCA vaso-occlusion. In vivo and in vitro studies using animal models show that neutrophils play a key role in vaso-occlusion; however, eosinophils (EOs) may also participate in this phenomenon in humans. We have previously shown that SCA eosinophils demonstrating greater adhesion to fibronectin. A small GTPase, Rho, and its target molecule, Rho-kinase, play an important role in the cell functions, including contractility, chemotaxis, adhesion, and migration. Y-27632 and NSC23766 are Rho-kinase inhibitors, that reduce leukocyte infiltration in several models of EOs inflammation, such as endotoxic liver and lung injury. The objective of this study was to investigate the effect of Rho kinase pathway on the in vitro adhesion of sickle EOs to activated human endothelial cell layers (umbilical vein endothelial cells, HUVEC). MethodsEosinophils were isolated from peripheral blood of healthy controls (n=5), steady-state SCA patients (SCA, n=7) and SCA patients on HU therapy (SCAHU, n=5), using Percoll gradient separation, followed by immunomagnetic sorting. EO adhesion (1x106 cell/ml in Ham's F12 K) to cultured HUVEC grown to confluence was assessed using static adhesion assays. HUVEC cells were treated, or not, with 100 µM NSC23766 or Y-27632 for 1h before incubation with an inflammatory stimulus (10nM TNF-alpha – 3 h), subsequently Eos were added to the cell layer (1 hour, 37°C, 5%CO2). For adhesion under flow conditions, biochips with microchannels coated with HUVEC were pre treated or not with Y-27632 or NSC23766 per 1 h before the TNF-alphastimulus. Purified eosinophils was added to HUVEC-coated microchannels at a flow rate of 0.5 dynes/cm2. Images of adherent eosinophils were recorded using real-time video microscopy and analysed using Duco Cell software. ResultsEOs from SCA patients demonstrated a significantly greater adhesion to HUVEC than healthy controls (14.42 ± 1.8% vs 9.35 ± 1.7 %; p<0.02); no significant difference was observed between the SCAHU group, compared to the healthy control and SCA group (10.5 ± 1.6%). Pre treatment of HUVEC with TNF-alpha significantly augmented the adhesion of healthy control, SCA, and SCAHU eosinophils to HUVEC (16.1 ± 3.2%, 33.1 ± 5.1%, 17.9 ± 3.5%, P<0.01, respectively). However, this adhesion was significantly higher for SCA EOs compared to healthy control and SCAHU EOs (p<0.05). Interestingly, when the HUVEC was pre-treated with NSC23766 then stimulated with TNF-alpha, adhesion of EOs from SCA and SCAHU was significantly reduced (22.9 ± 5,6%, 17.2 ± 6.1%, respectively, p<0.05). However, when HUVEC was pre-treated with Y-27632 no significant difference was found. In addition, EOs from SCA patients demonstrated greater adhesion to HUVEC under flow conditions when compared to healthy control and SCAHU. EOs when the HUVEC was protect with Rho inhibitors (Y-27632 and NSC23766) before stimulation with TNF-alpha, EOs adhesion under physiological shear stress was diminished in all groups investigated (healthy control, SCA and SCAHU). ConclusionRho kinase inhibitors were able to reduce endothelial activation and consequent eosinophil adhesion, in an in vitro model. Therapy with such Rho-kinase inhibitors could be used in patients with SCA, with beneficial effects on the vascular occlusion process.Financial Support: FAPESP/CNPq/INCTS Disclosures:No relevant conflicts of interest to declare.

Use Of Hydroxyurea For Sickle Cell Disease In An Academic Center

Introduction The sole disease-modifying agent approved by the FDA for sickle cell disease (SCD) is hydroxyurea (HU), but it may be underutilized in the management of this condition. Not all patients respond to or can tolerate treatment with HU and it is not clear a priori who will respond. We evaluated the utilization of HU in a cross-sectional study of adults with SCD at the University of Illinois at Chicago (UIC) to determine how often HU has been attempted in all individuals who fulfill the criteria for the use of this disease modifying agent. Methods HU status was noted at the time of blood draw for enrollment in the UIC registry of SCD. Those not on HU were assessed as to whether or not they met criteria for being on HU in particular three or more vaso-occlusive episodes requiring presentation to a health care facility during the preceding 12 months or a lifetime history of one or more episodes of acute chest syndrome. The subjects who met these criteria for HU treatment were then divided into two groups, those who had been treated with HU in the past and those who had never received treatment. Those previously treated with HU were further assessed as to whether or not treatment was discontinued due to an adverse drug reaction, development of a contraindication, non-response, refusal to continue treatment, or noncompliance with treatment and/or clinic visits. Those who had never been on HU were categorized into whether or not treatment was not provided because of a contraindication, patient refusal of treatment, or noncompliance with clinic visits. Results Of 290 subjects enrolled in the study, 171 (59%) were either on HU (N=124, 43%), did not meet criteria for HU (N=35, 12%), or were on a chronic transfusion program (N=12, 4%). The remaining 119 (41%) were not on HU although they met the criteria. Among these 119 subjects HU had been attempted in almost half (N=58) but 37 were no longer on HU because of an adverse drug reaction, development of a contraindication, or non-response and 18 were noncompliant or refused to continue treatment. For those subjects in whom HU had never been attempted (N=61), almost half (N=30) had refused treatment or had been noncompliant with clinic visits. It was not possible to establish a valid reason for non-treatment in only 25 of 119 subjects not receiving HU (4 subjects in the group in which HU had been attempted in the past and 21 in the group in which HU had never been prescribed). Conclusion A cross-sectional survey at our sickle cell center clarified the picture for utilization of HU in individuals with SCD at UIC. Among those fulfilling criteria for HU therapy, 75% were currently receiving the medication or had received it in the past. A valid reason for non-treatment could be established in the majority of patients not receiving HU. However, 25 individuals (8% of all subjects included in the study) fulfilled criteria for treatment with HU but were not receiving the agent and the reason for non-treatment could not be established. Disclosures: No relevant conflicts of interest to declare.

Targeting BCL11A and KLF1 For The Treatment Of Sickle Cell Disease and β-Thalassemia In Vitro using Antisense Oligonucleotides

Sickle cell anemia (SCD) is a hereditary blood disorder in which red blood cells (RBC) become sickle-shaped and block blood vessels, leading to painful vaso-occlusive episodes. Sickling occurs because of a point-mutation in the β-globin gene of hemoglobin. Fetal hemoglobin (HbF, α2γ2) is the main oxygen transport protein with greater oxygen binding affinity in the fetus during the last months of embryonic development and the first few months of life after birth. HbF inhibits sickling by interfering with the polymerization of hemoglobin S. Higher HbF levels in SCD correlate with better survival and because HbF production can be reactivated pharmacologically in adults, it can be used for the treatment of SCD as well as β-thalassemia. In β-thalassemia, there is reduced or absent synthesis of the β-globin gene, causing ineffective erythropoiesis.B-cell lymphoma/leukemia 11A (BCL11A) is a transcription factor in the zinc-finger protein family and is expressed in B cells and erythroid cells. BCL11A represses fetal hemoglobin expression by binding to the GGCCCGG motif in the β-globin promoter region. Erythroid Kruppel-like factor (KLF1) is an erythroid-specific transcription factor that regulates β-globin expression through direct interaction with its promoter and indirectly regulates γ-globin expression through the regulation of BCL11A. By reducing the expression of BCL11A and KLF1, we can promote production of HbF through the upregulation of γ-globin expression.To demonstrate upregulation of γ-globin mRNA expression in vitro, we used MEL-h-b-BAC line#7 cells, a murine erythroleukemic cell line harboring the entire human beta globin locus and expressing mouse BCL11A and KLF1 (Tim Townes, Univ. of Alabama at Birmingham). Antisense oligonucleotides (ASOs) targeting mouse BCL11A or mouse KLF1 were added to the cells in a dose-dependent manner. Seven days later, with free uptake of the ASOs into the cells, we observed dose-dependent reduction of mouse BCL11A mRNA (IC50 = 0.7 μM) and mouse KLF1 mRNA (IC50 = 3 μM). Consequently, we observed a 300 +/- 8% upregulation of human γ-globin mRNA expression after achieving ∼90% reduction in BCL11A mRNA expression after ASO treatment compared to the untreated control cells. Similarly, KLF1 ASO treatment caused a 500 +/- 58% up regulation of human γ-globin mRNA expression after achieving ∼80% mRNA reduction in KLF expression. These data indicate that targeting BCL11A and/or KLF1 with ASO treatment can cause an increase in γ-globin expression that is necessary for the upregulation of fetal hemoglobin and may be used for the treatment of sickle-cell anemia and β-thalassemia. Disclosures:Peralta: Isis Pharmaceuticals, Inc.: Employment. Low: Isis Pharmaceuticals, Inc.: Employment. Kim: Isis Pharmaceuticals, Inc.: Employment. Murray: Isis Pharmaceuticals, Inc.: Employment. Guo: Isis Pharmaceuticals, Inc.: Employment. Freier: Isis Pharmaceuticals, Inc.: Employment. Townes: University of Alabama at Birmingham: Employment. Hung: Isis Pharmaceuticals, Inc.: Employment.

Sickle Cell Disease: Management options and challenges in developing countries

Sickle Cell Disease (SCD) is the most common genetic disorder of haemoglobin in sub-Saharan Africa. This commentary focuses on the management options available and the challenges that health care professionals in developing countries face in caring for patients with SCD.In a developing countries like Ghana, new-born screening is now about to be implemented on a national scale. Common and important morbidities associated with SCD are vaso-occlusive episodes, infections, Acute Chest Syndrome (ACS), Stroke and hip necrosis. Approaches to the management of these morbidities are far advanced in the developed countries. The differences in setting and resource limitations in developing countries bring challenges that have a major influence in management options in developing countries.Obviously clinicians in developing countries face challenges in managing SCD patients. However understanding the disease, its progression, and instituting the appropriate preventive methods are paramount in its management. Emphasis should be placed on early counselling, new-born screening, anti-microbial prophylaxis, vaccination against infections, and training of healthcare workers, patients and caregivers. These interventions are affordable in developing countries.

Neurodevelopmental Deficits Among Infants and Toddlers with Sickle Cell Disease

Neurodevelopmental deficits are among the serious complications of sickle cell disease (SCD). However, few studies have prospectively evaluated neurodevelopmental deficits in very young children with SCD. We analyzed baseline neurodevelopmental data from a cohort of 80 infants and toddlers with SCD to identify primary disease-related events and sociodemographic risk factors associated with early developmental delay. This is an analysis of baseline date of a 4-year mixed, cross-sectional/longitudinal study. Full-term children at age 3.5 years or younger with SCD (any genotype) were eligible. Neurodevelopmental evaluations (Bayley II) were conducted at ages 9, 15, 21, 30, and 40 months. Demographics, hematologic variables, and medical events were obtained. Significant neurodevelopmental deficits were evident: 17.5% scoring >2SD below the mean on Bayley Mental Index or Motor Index. Odds ratio of significant developmental delay was >9 times more likely among those who had experienced vaso-occlusive pain episodes, after controlling for socioeconomic status (SES), gender, pneumonia/acute chest syndrome, and hemoglobin concentration. Male gender was also a risk factor for developmental delay. Early cognitive and motor delays were present in young children with SCD, with higher prevalence among those who had experienced pain crises. Increased vulnerability of male gender is consistent with other at-risk populations but has not been previously addressed in SCD research. Furthermore, these delays are not sufficiently explained by lower SES. Significant developmental delay in children with SCD may go unrecognized by primary care practices, medical specialty clinics, or parents. The importance of routine neurodevelopmental assessment for children with chronic medical conditions is clear.

A Pilot Study

This pilot study was conducted to determine the effectiveness of Healing Touch on anxiety, stress, pain, pain medication usage, and selected physiological measures of hospitalized adults with sickle cell disease experiencing a vaso-occlusive pain episode. Healing Touch sessions were administered for 30 minutes on four consecutive days, and the self-reported data on anxiety, stress, pain, and the selected physiological data were collected while controlling for music and presence. A parallel-group randomized control trial comparing the effects of Healing Touch with Music (HTM) to Attention Control with Music (ACM). Due to the small sample size, there were no statistically significant changes in any between-group comparisons, except for present pain on Day 4 for the ACM group. For both groups, the within-group comparison showed a nonsignificant reduction in physiological parameters, a statistically significant reduction in anxiety and stress for the ACM group after Day 4, and a statistically significant reduction in stress in the HTM group after Days 2 and 4. The pre- to postintervention reductions in present pain were greater in the HTM group across all 4 days, but the only statistically significant within groups findings were in the HTM group (p < .01) on Day 1. Further research is needed.

G183(P) Cost Effectiveness of Hydroxyurea Therapy in Preventing Inpatient Admissions Due to Vaso-Occlusive Crisis in Sickle Cell Children – a District General Hospital Experience

AimTo study the effectiveness of hydroxyurea in reducing the number of inpatient admissions due to vaso-occlusive crisis in children with sickle cell disease and estimation of potential cost savings for...

A randomized, placebo-controlled trial of arginine therapy for the treatment of children with sickle cell disease hospitalized with vaso-occlusive pain episodes

Painful episodes of vaso-occlusion are the leading cause of hospitalizations and emergency department visits in sickle cell disease, and are associated with increased mortality. Low nitric oxide bioavailability contributes to vasculopathy in sickle cell disease. Since arginine is the obligate substrate for nitric oxide production, and an acute deficiency is associated with pain, we hypothesized that arginine may be a beneficial treatment for pain related to sickle cell disease. Thirty-eight children with sickle cell disease hospitalized for 56 episodes of pain were randomized into this double-blinded placebo-controlled trial. Patients received L-arginine (100 mg/kg tid) or placebo for 5 days or until discharge. A significant reduction in total parenteral opioid use by 54% (1.9 ± 2.0 mg/kg versus 4.1 ± 4.1 mg/kg, P=0.02) and lower pain scores at discharge (1.9 ± 2.4 versus 3.9 ± 2.9, P=0.01) were observed in the treatment arm compared to the placebo one. There was no significant difference in hospital length of stay (4.1 ± 01.8 versus 4.8 ± 2.5 days, P=0.34), although a trend favored the arginine arm, and total opioid use was strongly correlated with the duration of the admission (r=0.86, P<0.0001). No drug-related adverse events were observed. Arginine therapy represents a novel intervention for painful vaso-occlusive episodes. A reduction of narcotic use by >50% is remarkable. Arginine is a safe and inexpensive intervention with narcotic-sparing effects that may be a beneficial adjunct to standard therapy for sickle cell-related pain in children. A large multi-center trial is warranted in order to confirm these observations.

Acute care of pediatric patients with sickle cell disease: A simulation performance assessment

Sickle cell disease (SCD) is a rare disorder with cardinal features including hospitalization for vaso-occlusive pain episodes, acute pulmonary injury, and increased infection rates. For physician-trainees, learning optimal SCD management is challenging because of limited exposure to life threatening complications requiring timely interventions. To create, demonstrate reliability, and validate simulation-based, acute care SCD scenarios for physician-trainees, seven scenarios were derived from SCD patient cases. For each scenario, participants had 5 minutes to complete diagnostic and treatment interventions. Participants were divided into two groups based on clinical experience: interns or residents/fellows. Two raters scored performances using diagnostic and therapeutic checklists--indicating whether specific actions were performed and a global, 1 (poor) to 9 (excellent), rating. Scenario scores were calculated by averaging rater scores on each metric. Reliability was defined through uniformity in rater scoring and consistency of participant performance over scenarios. Validity was demonstrated by the performance gradient where the more experienced trainees outperform those early in training. Twenty-eight pediatric residents and hematology fellows took part in the study. Reliability for assessment scores overall was moderate. Performance on all but one scenario was moderately predictive of overall performance. Senior resident/fellows performed significantly better than interns. Positive associations existed between overall performance scores (P < 0.01) and months of postgraduate training (P < 0.01). Mannequin-based simulation is a novel method for teaching pediatric residents SCD-specific acute care skills. The assessment provided reliable and valid measures of trainees' performance. Further studies are needed to determine simulation's utility in education and evaluation.

Low‐dose ketamine as a potential adjuvant therapy for painful vaso‐occlusive crises in sickle cell disease

The hallmark of sickle cell disease (SCD) is the acute painful vaso-occlusive crisis (VOC). Among SCD patients, vaso-occlusive pain episodes vary in frequency and severity. Some patients rarely have painful crises, while others are admitted to the hospital multiple times in a year for parenteral analgesics. Opioids are the mainstay of therapy for SCD-related pain. However, a subset of patients report continued pain despite escalating doses of opioids. Tolerance and opioid-induced hyperalgesia (OIH) have been considered as possible explanations for this phenomenon. The activation of the N-methyl-d-aspartate (NMDA) receptor has been implicated in both tolerance and OIH. As a NMDA receptor agonist, ketamine has been shown to modulate opioid tolerance and OIH in animal models and clinical settings. Low-dose ketamine, by virtue of its NMDA receptor agonist activity, could be a useful adjuvant to opioid therapy in patients with refractory SCD-related pain. Based on limited studies of adjuvant ketamine use for pain management, low-dose ketamine continuous infusion appears safe. Further clinical investigations are warranted to fully support the use of low-dose ketamine infusion in patients with SCD-related pain.

Pain, mood, and functional status in youth with sickle cell disease hospitalized for pain

Youth with sickle cell disease (SCD) are frequently hospitalized for severe episodes of pain. As part of a validation study of a new acute pain functional assessment tool we collected data at a single time point during hospitalization on a sample consisting of 159 unique SCD patients (55.3% female, 86% African American, 67% Hgb SS), ages 7-21 years (M=15.73, SD=3.63) who were admitted for vaso-occlusive episode at four urban children hospitals in the eastern United States. In this sample we describe current pain intensity, functional motor status (Functional Independence Measure; FIM), and mood (Positive Affect-Negative Affect Scale for Children; PANAS-C) as well as pain burden (Sickle Cell Pain Burden Interview-Youth; SCBPI-Y), functional status (Child Activity Limitations Interview; CALI), and quality of life (Pediatric Quality of Life Inventory; PedsQL) over the previous four weeks.

Daily Changes in Pain, Mood and Physical Function in Youth Hospitalized for Sickle Cell Disease Pain

Youth with sickle cell disease (SCD) are commonly hospitalized for treatment of painful vaso-occlusive episodes (VOE). However, limited data are available concerning the course of hospitalization for these children and adolescents and, in particular, whether daily changes occur in pain, emotional status and physical function. To characterize changes in daily pain intensity, physical function and mood over the course of hospitalization, and to determine whether specific clinical characteristics were associated with these changes. Daily ratings of pain (0 to 10 numerical rating scale) and mood (Positive and Negative Affect Scale for Children) were completed by 25 youth (11 to 20 years of age) with SCD over a total of 152 days (mean [± SD] = 6.7±5.6 days) of hospitalization. Trained raters determined each youth's daily physical function. Linear mixed modelling was used to examine changes in pain, mood and physical function during hospital stay. The rate of change over the course of hospitalization was significant for reductions in pain intensity (P<0.001) and improvements in physical (motor) function (P=0.001). Positive affect over time was significantly associated with subjects' physical function scores (B 0.24 [95% CI 0.12 to 0.35]) but not with their pain scores. In contrast, negative affect was positively associated with pain and inversely associated with physical function scores (B 1.58 [95% CI 0.23 to 2.93]). The results of the present study demonstrated that children made daily improvements in physical function and pain over hospitalization for VOE. Mood was related to changes in pain and physical recovery. Assessment of physical function and mood during hospitalization may help guide strategies to better understand the pain experience in youth with SCD hospitalized with VOE.

Organic Anion Transporting Polypeptide 1B (OATP1B) Transporters Modulate Hydroxyurea Pharmacokinetics

Abstract 1008 Background:Sickle cell is the most common inherited blood disorder affecting millions worldwide. Hydroxyurea is currently the only FDA approved drug for the treatment of sickle cell anemia (SCA) that alters disease pathophysiology. Known for inducing fetal hemoglobin production, hydroxyurea can decrease hospitalizations, vaso-occlusive pain episodes, and mortality associated with SCA. Although efficacious for many patients, a large degree of inter-patient variability in pharmacokinetic (PK) and pharmacodynamic (PD) properties of hydroxyurea has been noted, but sources of this variation remain largely undefined. Plasma transmembrane transporters have been shown to play an important role in the determination drug PK and PD of various xenobiotics. Our recent in vitro studies have demonstrated that hydroxyurea is a substrate for specific solute carrier (SLC) transporters including members of the organic anion transporting polypeptide 1B (OATP1B) subfamily which consists of OATP1B1, and OATP1B3 in humans, and one mouse orthologue Oatp1b2. These transporters are highly expressed on the sinusoidal membrane of hepatocytes, and function as uptake transporters bringing substrates from the blood into the liver. Limited reports have suggested that Oatp1b2 transcript is also found in kidney and intestine as well. In the present study we evaluated the uptake of the hydroxyurea by different haplotypes of hOATP1B3 transporter in vitro, as well as the influence of the Oatp1b2 transporter on hydroxyurea PK in vivo using a transporter knock-out mouse model. Methods:Transporter-mediated cellular uptake of hydroxyurea was determined in vitro by measuring [14C]-hydroxyurea accumulation in Xenopus laevis oocytes that over express two common haplotypes of OATP1B3. hOATP1B3*1 and the functional variant hOATP1B3*2 that contains the 334T>G and 699G>A SNPs were assessed. In vivo PK Studies were performed using a knockout model mouse model in which mOATP1b2 was eliminated from the DBA/LcaJ1 background. Mice were dose with 50mg/kg of [14C]-hydroxyurea via oral gavage or intravenous (i.v.) injection. Serial blood samples were collected at defined timepoints and concluded with terminal blood and tissue collections at 2 hours post injection. Hydroxyurea levels were determined based on radioactivity in plasma and liver, and kidney homogenate. Accumulation in the urine was also measured over a 3 day period. Results:In vitro kinetic studies did not show any differences between OATP1B3*1 and OATP1B3*2 haplotypes in time-, or concentration-dependent accumulation of hydroxyurea. The inhibitor substrates naringen and rifampin decreased hydroxyurea accumulation by at least 50% in OATP1B3*1 but did not decrease accumulation in OATP1B3*2. These in vitro studies suggest that the genetic mutations associated with these two haplotypes do not have a functional impact on the transport of hydroxyurea, but may have significant implications for potential drug-drug interaction. In vivo, Hydroxyurea PK was altered in the absence of the Oatp1b2 transporter. Plasma levels of hydroxyurea were similar after i.v. injections, but total exposure (AUC) in Oatp1b2-KO mice was decreased by 20% with an AUC of 2257 mg-min/ml compared to 2872 mg-min/ml observed in the wildtype mice (p=0.04). Hydroxyurea levels in the kidneys of Oatp1b2-KO mice were approximately 50% of wildtype with a mean accumulation of 360 pmol/g tissue (p= 0.0025). In addition, 30% less hydroxyurea was excreted in urine of Oatp1b2-KO mice by 72 hours (p=0.023). Accumulation of hydroxyurea in liver of Oatp1b2-KO mice and wildtype mice were 107 and 132 pmol/g tissue respectively, but this difference was not statistically significant. These results suggest that hydroxyurea PK is altered in the absence of Oatp1b2 transporter after oral administration. Conclusions:These studies provide the first in vivo evidence that specific transporters may be key determinants of hydroxyurea PK. Future studies are warranted to investigate the influence of human OATP1B and other transporters that modulate hydroxyurea PK. Identification of such transporters may help to elucidate factors such as genetic variations in the transporters and/or drug-drug interactions that may contribute to PK and PD variability observed in sickle cell patients. Disclosures:No relevant conflicts of interest to declare.

Clinical Efficacy and Safety of Erythroid Stimulating Agents in Sickle Cell Disease

AbstractAbstract ▪3218▪This icon denotes a clinically relevant abstractEndogenous production of erythropoietin (epo) can be decreased due to renal injury as well as by the decreased oxygen affinity of sickle hemoglobin in sickle cell disease (SCD). Erythroid stimulating agents (ESAs) are used when the endogenous production of epo is compromised and there is evidence that they may augment HbF production. However, there are potential adverse effects when using ESAs in SCD, possibly related to increased dense reticulocyte production, platelet activation, or other mechanisms. We present data on patients with SCD initiated on ESA therapy between January 2003 and January 2011. A total of 26 patients were initiated on ESA therapy during this period and eight are excluded from our analysis due to concomitant initiation of hydroxyurea. Of the 18 patients, all have HbSS disease and 14 received recombinant erythropoietin (median dose 40,000 units weekly) and four received darbopoietin (median dose 200mcg every two weeks). The median age was 44 years and eight of the 18 patients had been on a stable dose of HU (median dose 10mg/kg) prior to initiation of the ESA. Laboratory data was collected from a baseline clinical visit (defined as greater than four weeks from a vaso-occlusive pain episode or three weeks from a red blood cell transfusion) prior to starting the ESA and at one year after ESA therapy. Despite patients having a lower GFR after a year of follow up, hemoglobin values were improved and a significant proportion of patients were able to be transfusion-free. Although the increase in HbF was not statistically significant, a significant decrease in the LDH was observed. After one year of ESA therapy, there was no increased frequency of pain crises requiring medical attention or thrombotic events including stroke and the systolic and diastolic blood pressures were unchanged. Five of the patients had a prior seizure disorder and only one of the five patients developed a seizure in the year after initiating ESA therapy which was attributed to noncompliance with anti-epileptic therapy. Although limited by its retrospective nature, this study represents the largest observational cohort of SCD patients receiving ESA therapy thus far and suggests that ESA therapy may be effective in raising the hemoglobin and decreasing transfusion requirements without significant additional toxicity.VariablenPre-ESAnPost-ESAp-valueSystolic blood pressure18129 (118–139)18130 (112–136)0.56*Diastolic blood pressure1862 (60–79)1870 (60–80)0.37*Mean arterial pressure1888 (79–97)1889 (80–99)0.44*Hemoglobin185.9 (5.4–6.9)186.8 (6.1–7.7)0.001*Absolute reticulocyte Count18235 (133–356)18263 (199–419)0.32*Hemoglobin F152.3 (1.3–8.8)124.1 (2.4–9.0)0.18*LDH14417 (336–685)15359 (296–593)0.02*Indirect bilirubin182.0 (1.2–3.2)182.1 (1.2–3.3)0.67*AST1841 (35–50)1841 (30–57)0.64*BUN1818 (10–37)1819 (11–31)0.29*Serum Creatinine ≥1mg/dL1812 (66.7%)1814 (77.8%)0.46#GFR1883.3 (49.3–136.8)1878.6 (47.4–102.1)0.004*Proportion of patients requiring transfusions1818 (100%)1811 (61%)0.003#Annual pain crisis frequency181 (0.5–3)181 (0–3)0.25*Estimated systolic pulmonary artery pressure840 (34.5–45)438 (34.5–44.5)0.88**Analyses performed using the Wilcoxon matched pairs test.#Analyses performed using the Chi squared method. Disclosures:No relevant conflicts of interest to declare.

Clinical and Laboratory Predictors for Renal Damage in Sickle Cell Disease

AbstractAbstract ▪3252▪This icon denotes a clinically relevant abstractRenal failure is an independent marker for developing other forms of chronic organ damage and for early mortality in sickle cell disease (SCD). We conducted a retrospective, cross-sectional chart review of 292 adults with SCD (type SS or S-beta thal) treated at the University of Illinois Medical Center to assess the prevalence and factors related to kidney damage. Data was recorded from a clinic visit at least four weeks from a vaso-occlusive pain episode or red blood cell transfusion. The glomerular filtration rate (GFR) was calculated using the modification of diet in renal disease formula. Hemoglobinuria was defined by dipstick urinalysis showing trace, small, or moderate blood and <2 RBC/high power field. Urine albumin to creatinine ratios of 30 to 300 mg/g and >300 mg/g were categorized as microalbuminuria and macroalbuminuria, respectively. The median age was 33 years and 46% of patients were on hydroxyurea. We observed microalbuminuria in 41%, macroalbuminuria in 20%, hemoglobinuria in 41% and GFR <90 mL/min in 20% of adults with SCD. Univariate associations with GFR <90 mL/min are summarized in Table 1. By logistic regression, macroalbuminuria (OR 7.5, 95% CI: 2.5–22.2; p<0.0001) and age (OR 1.11, 95% CI: 1.06–1.16; p <0.0001) were independent predictors of GFR <90 mL/min (r2 = 0.23). Given the strong correlation of macroalbuminuria with GFR <90 mL/min, we explored factors related to degree of albuminuria. Univariate associations with degree of albuminuria are shown in Table 2. Logistic regression showed that independent predictors for macroalbuminuria were the presence of hemoglobinuria (OR 93.7, 95% CI: 15.2–576.5; p<0.0001) and age (OR 1.07, 95% CI: 1.01–1.14; p=0.03) (r2=0.28, p<0.0001). We further explored predictors of hemoglobinuria given its independent association with macroalbuminuria. By logistic regression, the strongest predictor of hemoglobinuria was the natural log LDH (OR 32.4; 95% CI: 8.4–124.9; p<0.0001) (r2=0.22), but other markers of hemolysis including higher absolute reticulocyte count, greater indirect bilirubin concentration and lower hemoglobin concentration were also associated with hemoglobinuria in univariate analyses. In summary, increasing age and macroalbuminuria were independent factors associated with GFR < 90mL/min in this cohort of adults with SCD, and intravascular hemolysis as reflected in hemoglobin-positive urine dipstick with negative microscopy was associated with macroalbuminuria. Further research is needed to determine if measures to decrease intravascular hemolysis and to prevent the development of macroalbuminuria can preserve renal function in patients with SCD. Investigation is also needed to identify genomic and genetic markers that put patients at risk for kidney disease and might serve as targets for preventive and therapeutic interventions.Table 1:Correlation of GFR to clinical and laboratory variables.VariableN>90N<90p-valueAge (years)23430 (25–39)5845 (37–54)<0.0001Female gender234141 (60%)5838 (65%)0.63MAP23186 (90–93)5891 (86–98)<0.0001HU therapy11048 (44%)3016 (53%)0.35Hemoglobin (g/dL)2348.9 (7.9–10)588.1 (7.4–8.9)<0.0001LDH (u/L)146309 (235–425)41375 (251–481)0.11Absolute Reticulocyte Count (×103/μL)111332 (234–444)29302 (197–368)0.001Hgb F (%)2185.5 (2.9–9.8)525.5 (2.5–9.8)0.98Albuminuria: Microalbuminuria Macroalbuminuria11246 (41%)3011 (36.7%)0.00115 (13%)13 (43.4%)Table 2:Correlation of Albuminuria to clinical and laboratory variables.VariableNNormalNMicro-albuminuriaNMacro-albuminuriap-valueAge (years)5732 (26–42)5838 (28–48)2835 (27–45)0.20Female gender5743 (75%)5834 (59%)2818 (64%)0.16MAP (mmHg)5686 (80–97)5888 (84–92)2891 (84–98)0.24HU therapy5624 (42.9%)5724 (42.1%)2817 (60.7%)0.22Hemoglobin (g/dL)578.9 (8.1–10.3)588.1 (7.5–9.4)288.5 (6.9–9.4)0.008LDH (u/L)54270 (212–358)49363 (251–434)27377 (355–488)<0.0001Indirect bilirubin (mg/dL)561.7 (1.2–2.5)572.4 (1.4–3.3)282.0 (1.3–4.3)0.03Absolute reticulocyte count (×103/μL)57256 (213–399)56332 (243–448)28369 (276–466)0.06Hgb F (%)526.6 (2.7–9.7)565.1 (2.8–11.7)267.8 (3.7–13)0.54Hemoglobinuria533 (5.7%)5429 (53.7%)2622 (84.6%)<0.0001 Disclosures:No relevant conflicts of interest to declare.

Heme oxygenase-1 gene promoter polymorphism is associated with reduced incidence of acute chest syndrome among children with sickle cell disease

AbstractSickle cell disease is a common hemolytic disorder with a broad range of complications, including vaso-occlusive episodes, acute chest syndrome (ACS), pain, and stroke. Heme oxygenase-1 (gene HMOX1; protein HO-1) is the inducible, rate-limiting enzyme in the catabolism of heme and might attenuate the severity of outcomes from vaso-occlusive and hemolytic crises. A (GT)n dinucleotide repeat located in the promoter region of the HMOX1 gene is highly polymorphic, with long repeat lengths linked to decreased activity and inducibility. We examined this polymorphism to test the hypothesis that short alleles are associated with a decreased risk of adverse outcomes (hospitalization for pain or ACS) among a cohort of 942 children with sickle cell disease. Allele lengths varied from 13 to 45 repeats and showed a trimodal distribution. Compared with children with longer allele lengths, children with 2 shorter alleles (4%; ≤ 25 repeats) had lower rates of hospitalization for ACS (incidence rate ratio 0.28, 95% confidence interval, 0.10-0.81), after adjusting for sex, age, asthma, percentage of fetal hemoglobin, and α-globin gene deletion. No relationship was identified between allele lengths and pain rate. We provide evidence that genetic variation in HMOX1 is associated with decreased rates of hospitalization for ACS, but not pain. This study is registered at www.clinicaltrials.gov as #NCT00072761.

Peripheral vascular response to inspiratory breath hold in paediatric homozygous sickle cell disease

New Findings• What is the central question of this study?Autonomic nervous dysfunction is implicated in complications of sickle cell anaemia (SCA). In healthy adults, a deep inspiratory breath hold (IBH) elicits rapid transient SNS- mediated vasoconstriction detectable using Laser Doppler Flux (LDF) assessment of the finger-tip cutaneous micovasculature.• What is the main finding and its importance?We demonstrate significantly increased resting peripheral blood flow and sympathetic activity in African children with SCA compared to sibling controls and increased sympathetic stimulation in response to vasoprovocation with DIG.This study is the first to observe an inverse association between resting peripheral blood flow and haemoglobin oxygen saturation (SpO2). These phenomena may be an adaptive response to the hypoxic exposure in SCA.There is increasing evidence that autonomic dysfunction in adults with homozygous sickle cell (haemoglobin SS) disease is associated with enhanced autonomic nervous system-mediated control of microvascular perfusion. However, it is unclear whether such differences are detectable in children with SS disease. We studied 65 children with SS disease [38 boys; median age 7.2 (interquartile range 5.1–10.6) years] and 20 control children without symptoms of SS disease [8 boys; 8.7 (5.5–10.8) years] and recorded mean arterial blood pressure (ABP) and daytime haemoglobin oxygen saturation (). Cutaneous blood flux at rest (RBF) and during the sympathetically activated vasoconstrictor response to inspiratory breath hold (IBH) were measured in the finger pulp of the non-dominant hand using laser Doppler fluximetry. Local factors mediating flow motion were assessed by power spectral density analysis of the oscillatory components of the laser Doppler signal. The RBF measured across the two study groups was negatively associated with age (r=−0.25, P < 0.0001), ABP (r=−0.27, P= 0.02) and daytime (r=−0.30, P= 0.005). Children with SS disease had a higher RBF (P= 0.005) and enhanced vasoconstrictor response to IBH (P= 0.002) compared with control children. In children with SS disease, higher RBF was associated with an increase in the sympathetic interval (r=−0.28, P= 0.022). The SS disease status, daytime and age explained 22% of the variance in vasoconstrictor response to IBH (P < 0.0001). Our findings suggest that blood flow and blood flow responses in the skin of young African children with SS disease differ from those of healthy control children, with increased resting peripheral blood flow and increased sympathetic stimulation from a young age in SS disease. They further suggest that the laser Doppler flowmetry technique with inspiratory breath hold manoeuvre appears to be robust for use in young children with SS disease, to explore interactions between , ABP and autonomic function with clinical complications, e.g. skin ulceration.

Role of Polymorphisms in Factor V (FV Leiden), Prothrombin, Plasminogen Activator Inhibitor Type-1 (PAI-1), Methylenetetrahydrofolate Reductase (MTHFR) and Cystathionine &amp;#946;-Synthase (CBS) Genes as Risk Factors for Thrombophilias

Thrombophilias are defined as a predisposition to thrombosis due to hematological changes which induce blood hypercoagulability; they can be inherited or acquired. They are individually characterized by a large phenotypic variability, even when they occur within the same family. Hereditary thrombophilias are, in most cases, due to changes related to physiological coagulation inhibitors or mutations in the genes of coagulation factors. High levels of plasma homocysteine may also be responsible for vaso-occlusive episodes and may have acquired (nutritional deficiencies of folate and vitamins B6 and B12) and/or genetic causes (mutations in the genes responsible for expression of enzymes involved in the intracellular metabolism of homocysteine). Considering that: (1) thromboses are events of multigenic and multifactorial etiopathology; (2) the presence of mutations in several genes significantly increases the risk of their occurrence; (3) the vascular territory (venous and/or arterial) affected involves different pathophysiological mechanisms and treatments, knowledge of genetic variants that may contribute to the risk and variability of the phenotypic manifestations of these diseases is extremely important. This understanding may provide support for a more individualized and therefore more effective treatment for thrombophilia carriers. Thus, this mini-review aims to address a comprehensive summary of thrombophilias and thrombosis, and discuss the role of polymorphisms in Factor V (FV Leiden), Prothrombin, Plasminogen activator inhibitor type-1 (PAI-1), Methylenetetrahydrofolate reductase (MTHFR) and Cystathionine β-synthase (CBS) genes as risk factors for thrombophilias.

Impact of PCA strategies on pain intensity and functional assessment measures in adults with sickle cell disease during hospitalized vaso‐occlusive episodes

Clinical trials of sickle cell disease (SCD) pain treatment usually observe only small decrements in pain intensity during the course of hospitalization. Sub-optimal analgesic management and inadequate pain assessment methods are possible explanations for these findings. In a search for better methods for assessing inpatient SCD pain in adults, we examined several pain intensity and interference measures in both arms of a randomized controlled trial comparing two different opioid PCA therapies. Based upon longitudinal analysis of pain episodes, we found that scores from daily average Visual Analogue Scales (VAS) and several other measures, especially the Brief Pain Inventory (BPI), were sensitive to change in daily improvements in pain intensity associated with resolution of vaso-occlusive pain. In this preliminary trial, the low demand, high basal infusion (LDHI) strategy demonstrated faster, larger improvements in various measures of pain than the high demand, low basal infusion (HDLI) strategy for opioid PCA dosing, however, verification in larger studies is required. The measures and statistical approaches used in this analysis may facilitate design, reduce sample size, and improve analyses of treatment response in future SCD clinical trials of vaso-occlusive episodes.