ALBERT

Description: In Africa, the natural history of SCD is often assumed to be same to the African Diaspora in the US, Jamaica, Europe or Latin America. Yet the environment can be different, including different pathogen exposure, such as malaria. To help better understand this, over 2000 references were identified using the names of all current or past names of African continent countries and the truncated word sickl$, followed by secondary nested and cross reference searches. Six cases series describing causes of death were identified, representing 182 children (Ndugwa, 1973, Athale, 1994, Koko, 1998, Diagne, 2000, Rahimy, 2003, Van-Dunem, 2007). Gender was reported in 172, 73 were female (42%). Age was reported in 118, 52 were 〈 5 years (44%). Four studies described some impediment to care or arrival for care in extremis in1/4 to over ½ of patients that died. In Uganda, 9/12 (75%) patient died at home. In Gabon 6/23 (26%) patients died within 4 hours of reaching the hospital and 11/23 (48%) within 24 hours. In Benin 2/10 (20%) died of splenic sequestration diagnosed at home; 38/64 (53%) of patients in Mozambique that died, lived outside of the capital. Causes of death were identified in 146 individuals. These included: fever/sepsis: n=59 (40%), including meningitis: n=15 (10%) and pyelonephritis: n=2 (1%); acute anemia: n=43 (29%), including spleen sequestration: n=28 (19%) and aplastic anemia: n=8 (5%); pain: n=22 (15%); acute chest syndrome/pneumonia: n=18 (12%); CNS: n=8 (5%), including stroke: n=4 (3%), seizure/ coma: n=5 (3%); liver disease: n=5 (3%) including hepatitis: n=3 (2%); Other: n=19 (13%) including wasting/ malnutrition: n=7 (5%), heart failure/cardiomyopathy: n=4 (3%), diarrhea and vomiting: n=3 (2%), transfusion reaction: n=2 (1%). Infectious pathogens were identified in 26, including malaria: n=10 (38%), S. pneumoniae: n=3 (12%), Salmonella: n=2 (8%), H. influenza, Klebsiella and Citrobacter: n=1 (4%) each; viral agents were reported in n=8 (31%) including HBV: n=5 (19%), HIV: n=3 (12%). Reported general population hemoglobinopathy surveys after birth revealed the following Relative Risk (RR) of observing individuals with hemoglobin SS compared to Hardy Weinberg expected frequencies (some age cohorts overlap; Tanzania '56, Benin '09, Burkina Faso '70, Central African Republic'75, Gabon'65/'80, Gambia'56, Ghana '56/‘57/'00/'10, Kenya '04/'10, Malawi '72/'00/'04, Mozambique '86, Nigeria '56/'70/'79/'81/'84/'05, Senegal '69, Sierra Leone '56). Age 0-1 years, total n=2112 observed n=22 (1.0%), expected n=16.5 (0.8%), RR=1.3 (95% CI=0.7,2.5), p=0.441. Age 0-6 years, total n=4078; observed n=39 (1.0%); expected n=40.6 (1.0%); RR=1.0 (95% CI=0.6,1.5), p=0.925. Age 5-19 years, total n=1880; observed n= 5 (0.3%); expected n= 24.8 (1.3%); RR=0.2 (95% CI=0.1,0.5); p

Description: Abstract 4855 Sickle cell disease (SCD) results from a point mutation in the beta globin gene that leads to the replacement of glutamic acid with a valine at the sixth position of the beta globin chain of hemoglobin. Children with SCD experience delayed skeletal maturation and more than 70% of adult SCD patients' exhibit osteopenia or osteoporosis. The underlying mechanisms responsible are unclear; however in a number of disorders inflammation is established to drive bone loss. Importantly, persistent immune-activation in SCD leads to a chronic inflammatory state that may be pertinent. In this study we characterized the skeletons of four month old Berkeley SCD mice, an animal model that recapitulates key features of SCD including inflammation. We show that as in children with SCD, the skeletons of SCD mice have significantly reduced bone mineral density (BMD). Micro-computed tomography shows that homozygous sickle cell mice were also significantly denuded in cancellous and cortical bone volumes, a consequence of an enhanced pro-osteoclastogenic bone microenvironment. Similarly to animal models of postmenopausal osteoporosis we found a significant increase in T cell activation state and TNFa production in SCD mice, and an elevated expression of receptor activator of NF-kB ligand (RANKL), the key osteoclastogenic cytokine in the bone marrow. Finally, we show a significantly elevated number of monocytes (early osteoclast precursors) that likely further amplifies osteoclastogenesis in the context of elevated RANKL in SCD. Taken together our data validate the Berkeley sickle mouse as a suitable model to explore the mechanisms of bone loss in SCD, and suggest a disruption in the immuno-skeletal interface resulting from chronic inflammation as key to bone loss in SCD. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4852 Sickle Cell Disease (SCD) is a complex disease with various complications such as stroke, vaso-occlusive crisis (VOC), acute chest syndrome and leg ulcers. Sickle cell anemia (SCA; homozygous hemoglobin SS) is the most common form of SCD. Genetic variations and/or environmental modifiers may modulate clinical presentation of SCD. Few studies have examined hemoglobinopathies in Tunisia, North Africa. However, recently, frequencies of beta-thalassemia and sickle cell trait were estimated at 2.21% and 1.89%, respectively (Fattoum, 2006). In order to identify genetic factors that may predispose patients to SCD complications in this population, a pilot case control study was designed to assess polymorphisms in Human Platelet Antigen (HPA) Genes. HPA polymorphisms were recently associated with severe coronary artery disease in the general population in Tunisia (Abboud et al, 2010) and VOC presentation in SCA patients from Bahrain (Al-Subaie et al, 2009). We present here a study conducted in collaboration with the Department of Clinical Hematology at the Aziza Othmana Hospital in Tunis (Tunisia). The National Medical Ethics committee of Tunisia as well as the Institutional Review Board (IRB) of Morehouse School of Medicine (MSM) approved the study. Blood samples, clinical history and DNA samples were collected from SCD adult patients and healthy controls after informed consent. Previously validated questionnaires for genetic risks in patients with SCD (courtesy of Dr Telen, Duke University) were adapted to French. The Helena test kit was used to generate hemoglobin variant data in conjunction with cellulose acetate electrophoresis. Blood samples were collected in EDTA vacutainer tubes and genomic DNA was isolated,stored at −80°C and then shipped to MSM. Single nucleotide polymorphisms, SNPs (Table 1) were genotyped using PCR-RFLPs and compared with different clinical sub-phenotypes such as, onset age, strokes, cardiac problems, splenectomies, etc. as defined in the questionnaire. Pearson Chi-Square was used for comparison and a PCLeu33ProMspIHPA2rs606517GP1BA (2811)524 C〉TMet145ThrSfaNIHPA3rs591117ITGA2B (3674)2622 T〉GIle843SerFokITable 2:Tunisian cohort collection took place from January to December 2009. This table summarizes the number and genotypes of patients enrolled and questionnaires collectedSexMaleFemaleTotal (%)TOTAL455196 (100)SCD Patients403575 (78)Healthy Controls51621 (22)Hemoglobin Genotype of SCD patientsSS141529 (38.6)Sβ010919 (25.3)Sβ+112 (2.6)SC112 (2.6)SO101 (1.3)unknown13922 (29.3)Total (%)403575 (100)Questionnaires filled282654No questionnaires111021 Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 244 Stroke is a major cause of morbidity and mortality among children with sickle cell anemia (SCA). Children with SCA at risk for stroke can be identified by transcranial Doppler (TCD) ultrasound screening for abnormally high cerebral artery blood flow velocity and strokes can be prevented by chronic packed red blood cell (RBC) transfusion. However, the mechanisms that lead to cerebral vasculopathy and stroke in SCA and that explain the beneficial effects of chronic RBC transfusions in stroke prevention are poorly understood. We have previously shown that pre-treatment serum levels of brain derived neurotropic factor (BDNF) and platelet derived growth factor (PDGF) subtypes, biomarkers of cerebral ischemia and arterial remodeling, were associated with both high TCD velocity and development of stroke. We hypothesized that frequency of RBC transfusion would be associated with altered serum levels of neurodegenerative, inflammatory and angiogenic markers in SCA children with high TCD velocity and tested this hypothesis by assaying levels of these markers in post-STOP serum samples. Frozen serum samples drawn one year after subject's exit from the STOP clinical trial phase were utilized. Given the positive trial results, all but 9 subjects had been on chronic transfusion regimen for at least one year at the time of sample collection. Eighty samples were assayed using multiplex antibody immobilized beads (Millipore Corp, Billerica, MA). The mean fluorescent intensity was measured using the Milliplex xMAP system powered by Luminex (Bio-Rad, Hercules, CA). Ten biologically related neurodegenerative, inflammatory and angiogenic biomarkers were tested. The total number (frequency) of RBC transfusions recorded over the study period (4 years) for each participant was categorized into High (≥ 40), Moderate (20 – 39) or Low (〈 20) frequency of transfusion. Median distribution with 10 – 90th percentile of the levels of biomarkers and TCD velocity were expressed using box-plots and the differences in median distribution between groups based on frequency of transfusion was estimated using Kruskal-Wallis test. A principal component analysis (PCA) loading plot was used to demonstrate the biological relationships between the biomarkers, taking into consideration linear correlations and spatial relationships between them. There were no significant differences in the hematological and anthropometric measures between groups. Overall, our result showed that low transfusion frequency was associated with high serum levels of biomarkers and vice versa, despite no significant difference in hemoglobin level between groups. The high frequency transfusion group had lower serum levels of BDNF (p = 0.02), sVCAM-1 (p 〈 0.001), PDGF-AA (p 〈 0.001), CCL5 (p 〈 0.01), tPAI-1 (p 〈 0.01) and NCAM (p 〈 0.01) levels compared with the low frequency transfusion group (figure 1 a – e). Although not shown in the figures, the same pattern was observed with TCD velocity which was lower (160, 115.7 – 204.9 cm/s) in the low compared with the high (195, 154 – 272 cm/s) frequency transfusion group. In addition, the medium frequency transfusion group had significantly lower serum sVCAM-1 (p 〈 0.01) compared with the low frequency transfusion group and higher PDGF-AA (p 〈 0.01) compared with the high frequency transfusion group. A PCA loading plot (figure 2) shows clustering of the biomarkers that are most closely biologically related, these are also the biomarkers that were significantly affected by the frequency of transfusion. Red blood cell transfusions in the STOP study were associated with reduced serum levels of biomarkers of angiogenesis (PDGF-AA and sVCAM-1), cerebral ischemia/neuronal survival adaptation (BDNF and NCAM) and inflammation (RANTES/CCL5), and this effect was most pronounced in the group with the highest frequency of transfusions (equivalent to most chronic transfusion regimen). This suggests that the protective effects of chronic RBC transfusions on stroke development in children with SCA may be attributable to improved cerebral perfusion, reduced inflammation and down-regulation of hypoxia-induced angiogenic responses that promote arterial remodeling. One or more in this group of biologically-related and relevant markers may be useful for monitoring children with SCA receiving stroke prevention therapies and for designing treatment targets. Disclosures: No relevant conflicts of interest to declare.

Description: Background: A higher than normal basal inflammatory state is characteristic of sickle cell disease (SCD). Hydroxyurea (HU) is the only FDA approved drug for SCD. However, HU is a chemotherapy drug and is therefore naturally cytotoxic, often inducing apoptosis. Chronic inflammation in sickle cell patients is invariably associated with injury to the vascular endothelium. Quercetin is a dietary flavonoid found ubiquitously in plants and foods that have anti-oxidative and anti-inflammatory characteristics. Hypothesis: The dietary flavonoid quercetin will decrease cytotoxic effects of Lipopolysaccharide and HU induced cell damage to vascular endothelial cells. Aims: 1. to develop an in vitro inflammatory model simulating chronic baseline inflammation observed in sickle cell disease. 2. to test the effect of the physiological dose of hydroxyurea (HU), on the inflammatory model. 3. to examine the role of quercetin (QCT), a dietary flavonoid with anti-oxidative and anti-inflammatory characteristics, for reducing the inflammation. Methods: Lipopolysaccharide (LPS) was used to induce inflammation in immortalized mouse aortic endothelial cells (iMAECs), providing an in vitro model of inflamed endothelial cells. The cells were exposed to LPS throughout the entire experiment. Interventions included treating the LPS exposed cells with QCT, HU, or QCT + HU over 50 hours. The 50-hour period included 24 hours of varying treatments, followed by two hours of hypoxic exposure and then 24 hours under normal aerobic exposure. Untreated cells (controls) provided a comparison. Data analyses included comparisons of control cells with the inflammatory model, and pairwise comparisons between the inflammatory model and the different treatments. In this experiment lactate dehydrogenase (LDH) was measured by colorimetric assay, as an indication of cell damage. Results: At the end of the experiment, the LDH level for the inflammatory model was significantly higher than LDH for the control cells (P = 0.0005) fig 1. Treatment with 30 micromoles QCT gleaned a trend toward reduced LDH compared with the inflammatory model (p = 0.1) fig 2. LDH was significantly higher after treatment with 100 micromoles HU compared with the inflammatory model (p = 0.0005) Fig 3. However, LDH was significantly reduced after treatment with a combination of 30 micromoles QCT/100 micromoles HU, compared with 100 micromoles HU alone (p = 0.0008) fig 3. Conclusions: These results suggest that quercetin may be effective against vascular endothelial cell damage for iMAECs in vitro. It also shows promise in preventing HU-induced cytotoxicity, which was a surprising finding from these results. This latter finding is interesting, and should be given more consideration, since HU is the only FDA-approved drug for treating sickle cell patients, and its use is rapidly increasing. Figure 1 Figure 1. Figure 2 Figure 2. Figure 3 Figure 3. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 2123 Little is known about the relative rate of pubertal development among children with different phenotypes of sickle cell disease (SCD). This study proposes, for the first time, to compare the influence of SCD phenotypes on pubertal development (PD) and body mass. We hypothesized that differences in pubertal development and body mass index (BMI) are modulated by SCD phenotype and treatment modality. We therefore conducted a cross-sectional study of SCD children and adolescents aged 8 – 18 years old (yo) to determine the impact of SCD phenotype and treatments on pubertal development (according to sexual maturity rating by Tanner stages, TS) and BMI. Participants were recruited from Children's Healthcare of Atlanta and enrolled after written informed consent was obtained. Subjects' age, height, weight, self-reported TS (via questionnaire), physician-reported TS, and major therapies were recorded. We investigated two major phenotypes, hemoglobin SC disease (SC, n= 16) and sickle cell anemia (SS, n=58). Within the SS group there were three treatment sub-groups, no-treatment (SS, n=18), hydroxyurea (S HU, n=21), and red blood cell transfusion (S Tx, n=19). Since age is associated with achieving a certain level of sexual maturity we divided the participants according to Nelson's Textbook of Pediatrics. A Tanner stage of

Description: Sickle cell disease (SCD) is associated with global activation of procoagulant and proinflammatory molecules and an increased tendency to thrombosis and development of stroke. High TCD velocity is associated with increased risk for stroke and we have shown that high TCD velocity positively correlates with high serum levels of platelet derived growth factor (PDGF)–AA. Further, high frequency of packed red blood cell (PRBC) transfusion was associated with lower serum levels of PDGF-AA, sVCAM-1 and total plasminogen activator inhibitor (tPAI)–1. Lower baseline serum levels of PDGF-AA, sVCAM-1 and tPAI-1was associated with significantly higher stroke free survival. Other studies have shown that high levels of thrombin-antithrombin (TAT) complex and von Willebrand factor (vWF) is associated with acute chest syndrome and other SCD complications. There is paucity of information in their relationship with SCD associated stroke or the effect of chronic PRBC transfusion these markers of coagulopathy in SCD. We hypothesized that serum levels of VWF, TAT and other markers of coagulopathy will decrease in response to chronic PRBC transfusion in subjects with high TCD velocity on transfusion for primary stroke prevention. Our hypothesis was tested using stored serum samples from the STOP study. Stored serum samples (at baseline, study exit and one year post-trial) of subjects who were part of STOP trial, 40 from each arms (standard care [SC] and transfusion [Tx]) and at each time point of the trial (i.e. 80 at baseline, 80 at study exit and 80 at one year post-trial) were obtained and analyzed using enzyme linked immunosorbent assay (Abcam PLC., Cambridge, MA) for serum vWF and TAT complex levels. In addition, 10 samples each from children with SCD and Normal TCD velocity (SNTCD) and healthy controls (HbAA) were assayed for comparison. Briefly, samples and reagents were allowed to warm up to room temperature and assayed according to the manufacturer's protocols. Absorbance was read using a Softmax Pro (Molecular Devices LLC, Sunnyvale, CA) and concentration calculated based on a linear curve with an R2 of 0.95 or greater, generated using manufacturer provided standards. Data were presented in median with ranges. Bar graphs of median concentration with range against treatment arm at each time point were plotted. Similar bar graph of median TCD velocity against treatment arm was plotted for comparison with vWF and TAT complex at baseline and study exit respectively. Finally, scatter plots with regression line were used to show linear relationship between serum levels of vWF and TAT and number of blood transfusions from baseline to post-trial time point. The characteristics of these subjects have already been published elsewhere. At baseline (figure 1A-C), there was no significant difference in median serum levels of vWF, TAT complex or median TCD velocity between SC and Tx groups, p〉0.05. But vWF and TAT levels were significantly higher among SC or Tx than for either HbAA or SNTCD, p

Description: Abstract 516 Children with sickle cell anemia (SCA) are at increased risk for stroke due to abnormal cerebral blood flow (CBF). The Stroke Prevention (STOP I) study showed that abnormal CBF could be detected using Trans-cranial Doppler (TCD), and may predict the risk for stroke. High TCD may be an indicator of main or branch cerebral artery vascular dysfunction, stenosis or fibrosis. Abnormal CBF is associated with cerebral ischemia and ultimately stroke. Brain derived neurotropic factor (BDNF) is a nerve growth factor with anti-apoptotic and neuro-protective properties. It is released in the central nervous system in response to ischemia. Platelet derived growth factor (PDGF) is an endothelial and smooth muscle mitogen. We hypothesized that circulating BDNF and PDGF levels will be elevated and may be associated with abnormal CBF as indicated by high TCD velocity measurements. Three groups of children, i.e. 1) SCA with abnormal TCDs (SATCD) - abnormal TCD ≥200 cm/sec, 2) SCA with normal TCDs (SNTCD) -normal TCD ≤170 cm/sec and 3) healthy controls were investigated. Stored, baseline plasma samples from the Stroke Prevention (STOP I) study and from an ongoing study of nutritional effects on inflammation in SCA children (NUTSCD) were analyzed in duplicate, using a multiplex antibody immobilized bead assay. Plasma levels of BDNF, PDGF AA and AB/BB and 10 other pro- and anti-inflammatory cytokines were assessed. A total of 39 samples (11 SATCD, 21 SNTCD and 7 controls) were analyzed. Of the 11 SATCD, 8 were eventually randomized to the standard care (SC), while 3 were in the transfusion (Tx) arm of STOP I. Since some of the SCA patients subsequently developed stroke, the levels of BDNF and PDGF in those who did vs. did not develop stroke were compared. Except where not applicable, values are presented as mean±SD. Chi-square was used to access distribution, while means were compared using t-test and analysis of variance (ANOVA). A Receiver Operator Characteristic (ROC) curve was used to access the sensitivity of BDNF and PDGF in predicting abnormal CBF as indicated by abnormal TCD measurement. The TCD velocity for SATCD group was 250±32 cm/s. There was no statistically significant difference in age or gender distribution among the groups. The mean body mass index was significantly lower for SCA subjects compared with controls (15±1kg/m2 vs. 23±7 kg/m2, p= 0.026). There was a statistically significant difference in mean BDNF levels, which was highest in SATCD compared with SNTCD (234±53 vs. 166±110 pg/ml, p=0.036) and controls (34±20 pg/ml, p