ALBERT

Description: Background: Individuals with chronic pain may have hyperactive microglia, which stimulate neurons to send a pain signal with little or no stimuli. Microglia are therefore a potential drug target to treat chronic pain, but drug discovery has been stymied by differences between human and animal neurobiology, and lack of healthy human CNS microglia. We cultured peripheral blood derived monocytes to develop characteristics of CNS derived microglia, termed peripheral blood derived microglia like cells (PB-MLC). We found that lipopolysaccharide (LPS) treated PB-MLCs from patients with chronic pain, from sickle cell disease (SCD) or chronic headaches, secreted more pro-inflammatory cytokines (TNF-alpha, IL-1beta, and IL-6) than PB-MLCs from normal donors, suggesting that patient pain phenotype was preserved in culture; PB-MLC from individuals with chronic pain were hyperactive in vitro as their microglia are in vivo. We hypothesize that PB-MLCs can be developed as a cell-based assay to screen compounds to treat chronic pain. To validate our model system, we compared cultured PB-MLCs to CNS derived microglia cells, using Sprague-Dawley rats, and treated human PB-MLC with microglia activation inhibitors shown to work in vivo in murine models. Methods: We isolated rat brain derived microglia (BDM) and rat peripheral blood monocytes; both were cultured with murine IL-34 (100 ng/ml) and GM-CSF (10 ng/ml). BDM and rPB-MLC were morphologically analyzed by fluorescence imaging microscopy, combined with machine learning, phenotyped by RT-qPCR and indirect immunofluorescence with anti-TMEM119, CD68, and Iba1 antibodies. Cells were treated with LPS for 24 hours, and TNF-alpha, IL-1beta, and IL-6 secretion measured by ELISA. For human PB-MLC studies, monocytes were cultured with GM-CSF (10 ng/ml) and IL-34 (100 ng/ml) for 7 days. PB-MLC morphology was analyzed as above; phenotyped with anti-CX3CR1, TMEM119, CD68, and Iba1 antibodies. PB-MLCs were treated with 100 ng/ml LPS with or without minocycline (2.5, 5, 10, 25 μg/mL), clopidogrel (1, 2, 4 μM) and MRS2395 (1, 5, 10 μM), for 24 h; TNF-alpha and IL-1beta secretion measured by ELISA. Results: We found that rPB-MLC resemble BDM morphologically, express the same microglia specific markers (TMEM119, P2RY12) and can be activated by LPS (Figure 1). Monocytes not cultured with IL-34 and GM-CSF did not express microglia specific genes (Figure 2A). To evaluate the possibility of using the PB-MLC model system to screen compounds to inhibit microglia activation, we tested PB-MLC cells with the following microglial inhibitors shown to be active in murine models in vivo: minocycline, MRS2395, and clopidogrel. MRS2395 and clopidogrel significantly suppressed the release of proinflammatory cytokine TNF-alpha from LPS-induced activated PB-MLCs in a dose-dependent manner (Figure 2B); minocycline did not. Conclusions: We validated our model system by comparing CNS derived microglia to rPB-MLCs and found they share morphology, similar cytokine secretion in response to LPS, and expression of microglia-specific genes. We confirmed that human PB-MLC expressed microglia specific genes while the original monocytes did not. Since P2Y12 is implicated in chronic pain, we tested two P2Y12 receptor agonists, clopidogrel and MRS2395, in our human PB-MLC system. When challenged with LPS, clopidogrel and MRS2395 inhibited LPS-induced PB-MLC activation in vitro as it had in vivo in a murine chronic pain model. We propose to use our human PB-MLC to screen for compounds that reduce microglia hyperactivity, to identify pharmacologic agents to treat chronic pain. Disclosures No relevant conflicts of interest to declare.

Description: Background: High fetal hemoglobin (HbF) levels reduce mortality and morbidity in sickle cell disease (SCD). Results from a prior clinical trial to assess safety of high dose vitamin D and our unbiased genomic analyses identifying a vitamin D regulated protein as a HbF inducer suggests that vitamin D replacement may increase HbF levels in SCD patients. To test this hypothesis, we performed a single center retrospective chart review investigating the impact of vitamin D replacement on HbF levels in our pediatric SCD population. Methods: We reviewed electronic medical records of pediatric patients with SCD with one or more serum vitamin D level who used vitamin D replacement from January 2007 to March 2020. SCD patients on chronic blood transfusion were excluded. Our cohort contained 81 SCD patients (ages: 2.3-19.9 years; 40 males) 71 HbSS, 6 HbSC, 3 HbSβ0, and1 Hb S-δβ. Vitamin D deficiency was defined as vitamin D levels 〈 30 ng/ml. All subjects were on hydroxyurea, with stable MCV (mean 96 fL at baseline and 95.6 fL during the study period). The patients received a median vitamin D dose of 2002 IU/day for a median of 229 days with a median follow-up of 365 days after replacement. Statistical analyses: Vitamin D deficient and non-deficient SCD patients data was compared at baseline by t test (for continuous variables with normal distribution), Wilcoxon test (for continuous variables with non-normal distribution), chi2 test (for categorical variables), and Fisher exact test (for categorical variables with small sample size). Linear relationships between vitamin D levels and HbF, CBC, BMI, and age were assessed using scatter plots and correlation coefficients (r value). Non-normal variables were log transformed to achieve normal distribution. A linear mixed effect (MLE) models were run to analyze linear relationship of variables of time dependent variables, including variable number of clinic visits and variable follow-up time. All models had 'vitamin D level" as an exposure variable; hydroxyurea and folic acid use as covariates, and one of the following as an outcome variable: HbF, hemoglobin, MCV, MCHC, reticulocyte count, WBC count, and platelet count. We performed mediation analysis to determine if HbF or MCV were intermediate variables for the effect of vitamin D on reticulocyte count. All LME models and scatter plots were created separately for the period of vitamin D replacement and a one-year follow-up period after cessation of vitamin D replacement to capture any lag period in HbF induction and sustained effect of vitamin D replacement. Results: The LME model, adjusting for hydroxyurea and folic acid, indicated that HbF increased by 0.68 percentage points with every 10 ng/ml rise in vitamin D levels during the vitamin D replacement period (p=0.04) and continued to rise after replacement, exhibiting a 2.2 percentage point rise per 10 ng/mL increase above replacement HbF levels during a 12 month follow-up period (p=0.005) (Figure 1, Table 1). WBC and absolute reticulocyte count (ARC) decreased significantly following vitamin D replacement according to the LME models (p

Description: Pharmaceutical drugs targeting dyslipidemia and cardiovascular disease (CVD) may increase the risk of fatty liver disease and other metabolic disorders. To identify potential novel CVD drug targets without these adverse effects, we perform genome-wide analyses of participants in the HUNT Study in Norway (n = 69,479) to search for protein-altering variants with beneficial impact on quantitative blood traits related to cardiovascular disease, but without detrimental impact on liver function. We identify 76 (11 previously unreported) presumed causal protein-altering variants associated with one or more CVD- or liver-related blood traits. Nine of the variants are predicted to result in loss-of-function of the protein. This includes ZNF529:p.K405X, which is associated with decreased low-density-lipoprotein (LDL) cholesterol (P = 1.3 × 10−8) without being associated with liver enzymes or non-fasting blood glucose. Silencing of ZNF529 in human hepatoma cells results in upregulation of LDL receptor and increased LDL uptake in the cells. This suggests that inhibition of ZNF529 or its gene product should be prioritized as a novel candidate drug target for treating dyslipidemia and associated CVD.

Description: The Trans-Omics for Precision Medicine (TOPMed) programme seeks to elucidate the genetic architecture and biology of heart, lung, blood and sleep disorders, with the ultimate goal of improving diagnosis, treatment and prevention of these diseases. The initial phases of the programme focused on whole-genome sequencing of individuals with rich phenotypic data and diverse backgrounds. Here we describe the TOPMed goals and design as well as the available resources and early insights obtained from the sequence data. The resources include a variant browser, a genotype imputation server, and genomic and phenotypic data that are available through dbGaP (Database of Genotypes and Phenotypes)1. In the first 53,831 TOPMed samples, we detected more than 400 million single-nucleotide and insertion or deletion variants after alignment with the reference genome. Additional previously undescribed variants were detected through assembly of unmapped reads and customized analysis in highly variable loci. Among the more than 400 million detected variants, 97% have frequencies of less than 1% and 46% are singletons that are present in only one individual (53% among unrelated individuals). These rare variants provide insights into mutational processes and recent human evolutionary history. The extensive catalogue of genetic variation in TOPMed studies provides unique opportunities for exploring the contributions of rare and noncoding sequence variants to phenotypic variation. Furthermore, combining TOPMed haplotypes with modern imputation methods improves the power and reach of genome-wide association studies to include variants down to a frequency of approximately 0.01%.

Description: Biomarker development is a key clinical research need in sickle cell disease (SCD). Hemorheological parameters are excellent candidates as abnormal red blood cell (RBC) rheology plays a critical role in SCD pathophysiology. Here we describe a microfluidic device capable of evaluating RBC deformability and adhesiveness concurrently, by measuring their effect on perfusion of an artificial microvascular network (AMVN) that combines microchannels small enough to require RBC deformation, and laminin (LN) coating on channel walls to model intravascular adhesion. Each AMVN device consists of three identical capillary networks, which can be coated with LN (adhesive) or left uncoated (non-adhesive) independently. The perfusion rate for sickle RBCs in the LN-coated networks (0.18 ± 0.02 nL/s) was significantly slower than in non-adhesive networks (0.20 ± 0.02 nL/s), and both were significantly slower than the perfusion rate for normal RBCs in the LN-coated networks (0.22 ± 0.01 nL/s). Importantly, there was no overlap between the ranges of perfusion rates obtained for sickle and normal RBC samples in the LN-coated networks. Interestingly, treatment with poloxamer 188 decreased the perfusion rate for sickle RBCs in LN-coated networks in a dose-dependent manner, contrary to previous studies with conventional assays, but in agreement with the latest clinical trial which showed no clinical benefit. Overall, these findings suggest the potential utility of the adhesive AMVN device for evaluating the effect of novel curative and palliative therapies on the hemorheological status of SCD patients during clinical trials and in post-market clinical practice.

Description: (1) Background: The aim of the present study was to compare oxygen gradient ektacytometry parameters between sickle cell patients of different genotypes (SS, SC, and S/β+) or under different treatments (hydroxyurea or chronic red blood cell exchange). (2) Methods: Oxygen gradient ektacytometry was performed in 167 adults and children at steady state. In addition, five SS patients had oxygenscan measurements at steady state and during an acute complication requiring hospitalization. (3) Results: Red blood cell (RBC) deformability upon deoxygenation (EImin) and in normoxia (EImax) was increased, and the susceptibility of RBC to sickle upon deoxygenation was decreased in SC patients when compared to untreated SS patients older than 5 years old. SS patients under chronic red blood cell exchange had higher EImin and EImax and lower susceptibility of RBC to sickle upon deoxygenation compared to untreated SS patients, SS patients younger than 5 years old, and hydroxyurea-treated SS and SC patients. The susceptibility of RBC to sickle upon deoxygenation was increased in the five SS patients during acute complication compared to steady state, although the difference between steady state and acute complication was variable from one patient to another. (4) Conclusions: The present study demonstrates that oxygen gradient ektacytometry parameters are affected by sickle cell disease (SCD) genotype and treatment.

Description: INTRODUCTION: Sickle cell disease (SCD), a complex genetic blood disorder involving multicellular interactions between blood and endothelial cells, is often accompanied by central nervous system (CNS) complications. Effects range from silent cerebral infarct (SCI) to abnormal blood flow, and consequent overt stroke. This study assessed the humanistic and economic burden associated with CNS complications in patients with SCD and identified patient-reported outcome (PRO) instruments for future research. METHODS: MEDLINE, Embase, Cochrane CENTRAL/CDSR and 11 congresses were searched to identify English language studies published from January 2000 to May 2020 and screened with predefined criteria by two independent researchers. Clinical trials (CT) or observational studies assessing humanistic burden, economic burden, or instruments used to measure burden in patients with SCD and CNS complications (N≥15) were included. Humanistic burden was broadly defined to include quality of life (QoL), symptoms and function. RESULTS: Of the 3194 articles identified, 34 were included. Study designs were 29% retrospective observational (10/34), 29% cross-sectional (10), 26% prospective (9), 9% randomized CT (3), and 6% systematic reviews (2). Study size varied widely (16-4,485 patients with SCD and CNS complications). 77% (26) focused on pediatric patients. Separately, 77% (26) were in a US setting. Overt stroke (12), stroke and SCI (11), or SCI only (7) were the most frequently described CNS complications. Twenty-five studies reported on humanistic burden, 18 of which measured cognitive function using the Wechsler Intelligence Scales. A significant decrease was reported in full scale (FS) (stroke vs. no stroke: 73.5 vs. 84.7; P=.04), verbal (abnormal vs. normal MRI: 74.1 vs. 84.6; P=.02), and performance IQ (stroke vs. no stroke: 69.5 vs. 81.5; P=.02) for patients with overt strokes or SCI compared to non-stroke SCD controls. Greater impairment was reported for overt stroke compared with SCI (multivariate meta-analysis of mean IQ difference: -10.3; P=.0013). In addition to stroke/SCI, socio-environmental factors (i.e., family income level, lack of college education) were significantly associated with a decrease in IQ (P=.005 and P=.023, respectively). Five studies assessed motor function, reporting significantly impaired function for patients with stroke compared to non-stroke SCD controls (Purdue Pegboard both hands: 7.5 vs. 10.1; P=.0001). Among children with SCD who had experienced their first stroke, those receiving hydroxyurea (HU) for prevention of recurrent stroke had significantly less moderate to severe motor disability (physician assessed) than children not receiving HU (23.1 vs. 88.9%; P

Description: Background: In sickle cell disease (SCD), hemoglobin S (HbS) polymerizes upon deoxygenation, reducing red blood cell (RBC) deformability. RBC deformability can be measured over a gradient of oxygen tensions (pO2) with the Laser Optical Rotational Red Cell Analyzer (Lorrca) ektacytometer (RR Mechatronics). Oxygen gradient ektacytometry generates 3 key parameters: 1) EImax, RBC deformability at normoxia; 2) EImin, minimum RBC deformability upon deoxygenation; and 3) the point of sickling (PoS): the oxygen tension at which a 5% decrease in deformability is observed during deoxygenation, reflecting the patient-specific pO2 at which sickling begins (Figure 1A). Previously we showed that oxygen gradient ektacytometry-derived biomarkers correlate with measures of SCD disease severity and hemolytic rate (Rab et al. Blood 2018), and is associated with vaso-occlusive crisis (VOC) frequency (Rab et al, Blood 2019). In this study, we confirm these observations in 2 independent cohorts and extend it to occurrence of acute chest syndrome (ACS), stroke including silent cerebral infarction (SCI), and transcranial Doppler (TCD) outcome. Methods: We analyzed 2 cohorts of SCD patients; an adult patient cohort of 53 SCD patients, enrolled at either University Medical Center Utrecht, The Netherlands (UMCU, n=25) or Hospital Lyon France (LIBM, n=28), and a pediatric patient cohort of 190 SCD patients enrolled at Texas Children's Hospital, USA (TCH). Subjects were HbSS or HbS/β-thalassemia, with a substantial number of subjects on hydroxyurea (HU) therapy (adult cohort 66% and pediatric cohort 86%), and not on chronic transfusion therapy. Correlations between oxygen gradient ektacytometry-derived biomarkers and the clinical complications of stroke or silent infarcts (SCI), ACS, VOC were assessed in both pediatric and adult patients. Patient groups generally did not significantly differ significantly by age, gender or HU treatment in the adult cohort except for age, which was lower in the ACS+ group (25.3years (y) compared to 32.0y) and also lower in the VOC+ group (27.1y compared to 35.8y). In the pediatric cohort, patient groups differed significantly in the ACS+ group compared to the ACS- group by age (ACS- group 8.37, ACS+ group 10.9y) and HU treatment (ACS- group 76%, ACS+ group 93%). Similarly, age was significantly higher in the Stroke+ group compared to the Stroke- group (14.0y compared to 9.3y), which was also found when studying VOC (VOC+ group 11.6y, VOC- group 8.2y). Results: In the pediatric cohort, PoS was significantly higher in patients with ACS (mean 40.3 compared to 34.9 mmHg, p=0.0001, Figure 1B). In the adult cohort, PoS was also higher in those with ACS although this did not reach significance (p=0.053, Figure 1C). In the pediatric cohort, PoS was higher in patients with stroke or SI (mean 43.0 mmHg compared to mean 37.3 mmHg, p

Description: Introduction Red cell rheology is abnormal in sickle cell disease (SCD); red blood cells (RBC) are rigid, dense, and the sickle hemoglobin (HbS) polymerizes with deoxygenation. There are several devices commercially available and under development to assess RBC rheology. One is an oxygen gradient ektacytometer (Lorrca with Oxygenscan, RR Mechatronics) which measures RBC deformability under oxygenated conditions (EImax) and deoxygenated conditions (EImin), and the oxygen concentration at which deformability begins to worsen, point of sickling (PoS). A commercially available hematology analyzer, the ADVIA (Siemens), measures hemoglobin (Hb) by flow and colorimetric methods, permitting automated calculation of the percent dense red blood cells (%DRBC). Allogeneic hematopoietic stem cell transplant (alloHSCT) can provide a cure for SCD, and viable gene-based therapy options are under investigation to serve the many patients without a matched related donor. However, the level of HbS correction or functional Hb induction necessary to achieve a cure is still unknown. Clinical endpoints such as prevention of pain events are important, but it is possible to be pain free for several years while still experiencing organ damage. As gene-based therapy clinical trials move forward, we must assess the level of functional improvement beyond Hb profile and conventional clinical labs. We propose that the goal of any gene-based SCD therapy should be to normalize blood rheology to the level of an individual with sickle cell trait (HbAS), and that EImax, EImin, PoS, and %DRBC may be used to distinguish between HbAS and HbSS/Sβ0 genotypes. Methods Subjects: Blood samples were collected from 257 unique patients (17 HbAS, and 240 HbSS/Sβ0) under IRB-approved protocols at Texas Children's Hospital and University Medical Center Utrecht. Patients were 56% male, ages 9 months to 22 years. Some HbSS/Sβ0 subjects were on transfusion and hydroxyurea (HU) (Table 1). Fetal hemoglobin (HbF) levels ranged from 0-41%. Oxygen gradient ektacytometry: Blood collected in EDTA and standardized to a fixed RBC count was suspended in 5 mL polyvinylpyrrolidone at room temperature. 1.5 mL of the sample solution was injected into test cup. Each sample was run in duplicate. ADVIA: 250µL of blood collected in EDTA at room temperature was aspirated to measure the %DRBC, defined as the percentage of RBCs with a Hb concentration 〉1.11 mg/mL. Analysis: Patient characteristics were summarized using median with 25th and 75th percentiles, and frequency with percentage. Characteristics and labs were compared by group with t-test, Wilcoxon rank sum test, or Fisher's exact test. Receiver operating characteristics (ROC) analyses were performed to identify HbAS versus HbSS/Sβ0 for each biomarker. All analyses were performed using Stata 15. Results The EImin, EImax, PoS, and %DRBC differed significantly between the HbAS and HbSS/Sβ0 groups, despite including transfused, very young, and HU-treated samples in the HbSS/Sβ0 cohort (p