bluuebunny/crossref_metadata_2025_split huggingface dataset:
PF4/H Complexes Induce Germinal Centers in Vivo
Authors: Joglekar Manali , Qi Rui , Arepally Gowthami M. 
Published: October 2019

Abstract
Abstract 269
Mice injected with ultra-large complexes (ULCs) of platelet factor-4 (PF4) and heparin (PF4/H) exhibit a strong immune response which mimics that seen in patients developing Heparin induced thrombocytopenia (HIT)(Suvarna, Blood 2005). Previous studies with this murine immunization model have shown that murine (m) PF4/H ULCs potently activate dendritic cells (DCs) (Chudasama, Blood 2010) and initiate a T-cell dependent immune response (Suvarna, Blood 2005). To examine cellular/anatomic basis of PF4/H antibody formation, we examined the role of the spleen and germinal center formation in mice injected with mPF4/H complexes. To evaluate the role of the spleen, we immunized splenectomized mice (SPL) (n=15) and wild type (WT) mice (n=15) retro-orbitally with 100 mg/mL mPF4 and 5 U/mL Hep for 5 days. Antibodies to mPF4/H complexes were detected by an ELISA developed in our laboratory (Suvarna, Blood 2005; Suvarana, Blood 2007). As shown in Figure 1, SPL animals showed a marked reduction in Ab formation compared to WT animals (mean A450nm ± SD; SPL 0.206 ± 0.253 vs. WT 0.76 ± 0.788, p<0.003). We next asked if antibody responses to PF4/H were associated with germinal center (GC) formation. The GC is a hallmark of T-cell dependent (TD) immune responses and is considered vital for development of high-affinity and isotype-switched antibodies. For these studies, mice were injected with a high dose of PF4/H (400 ug/mL PF4: 10 U/mL heparin) (n=2), standard protocol dose of PF4/H (100 ug/mL: 5 U/mL) and or buffer (Hank's Balanced Salt Solution, n=3) daily for 5 days via retro-orbital injection. As a positive control for GC formation, we injected two mice with a conventional antigen, nitrophenyl conjugated chicken gamma-globulin (NP-CGG). Mice expressing high antibody titers on Day 16 (mean A450nm± SD; high dose- 1.112 ± 0.5767; standard dose- 1.186 ± 0.7853) were sacrificed, spleens harvested and frozen in an embedding medium. Splenic sections (5m, longitudinal) were obtained as frozen sections and stained with immunohistochemical markers for GCs (GL-7 FITC; 1:400, counter-stain- FITC Alexa Fluor 488; 1:200), T-cells (TCR-β PE; 1:200) and B-cell follicles (B220 Biotin; 1:400, counter-stain- Streptavidin Alexa Fluor 350; 1:200). Images were obtained using Carl Zeiss AxioVert 200M microscope and analyzed with AxioVision Rel 4.6 software. Fluorescent images were processed with Adobe photoshop CS5.1. As shown in Figure 2, we noted that mice injected with NP-CGG, had ∼ 22 GC's, mice injected with buffer had 2 GC's and mice injected with high and standard dose of PF4/H, had ∼20–22 and 15–17 GC's respectively. These studies show that mPF4/H complexes elicit GC formation in association with secretion of mPF4/H Abs and that GC formation is comparable to that seen with conventional antigen.

Disclosures:
No relevant conflicts of interest to declare.
Long Term Safety and Efficacy Of Low Fixed Dose Hydroxyurea In Pediatric Patients With Sickle Cell Anemia: A Single Center Study From Central India
Authors: Jain Dipty L. , Krishnamurti Lakshmanan , Sarathi Vijaya , Desai Saumil , Gokhale Amruta 
Published: October 2019

Abstract

Introduction
Hydroxyurea (HU) therapy ameliorates the morbidity associated with Sickle cell anemia (SCA). HU is typically escalated to maximum tolerated doses (MTD) for treatment of SCA patients. Monitoring HU for toxicity poses potential challenges in low resource settings. We have previously reported the safety and efficacy of fixed low dose HU at 10mg/kg body weight ( Jain et al 2013). We studied the long term safety and efficacy of low dose HU in pediatric patients with SCA.

Methods
Long term observational follow up of 40 young Indian SCA patients was carried out for a period of 10 years in a single tertiary care center in central India. The number of clinical events including vasocclusive crises, acute chest syndrome, stroke, sequestration crises, avascular bone necrosis in the entire cohort was assessed prior to starting HU and subsequently every 3 months. Laboratory parameters including hemoglobin levels, Hb F levels, mean corpuscular volume, mean corpuscular hemoglobin concentration were also followed up over the same time period.

Results
The total number of patients in this study was 40 (17 females & 23 males). Mean age was 12.95 years ± 9.62. All patients had HbSS. There was an increase in mean hemoglobin, HbF, MCV and MCH over time in the patient population ( Table 1). There was a decrease in the mean number of hospitalizations, vasocclusive crises, acute chest syndromes, stroke and severe anemia ( Table 2). HU was well tolerated with minimal hematological or other toxicity (Table3). Patients who experienced transient hematological, liver or renal toxicity were able to resume HU at the same dose. HU was permanently discontinued in 2 patients who had manifestations of HIV/AIDS. HU was temporarily interrupted in one patient who was receiving treatment for tuberculosis and in one patient for the duration of a normal pregnancy. There was one death attributable to severe septicemia without evidence of neutropenia after 2 years follow up. There were 3 subjects who were lost to follow up, one after 2 years follow up and remaining 2 after 5 years of follow up. Of note, patients had a high HbF level at baseline. High HbF levels at baseline, the variable relationship of HbF to disease severity and amelioration of disease in Indian patients with high HbF has been previously reported ( Jain et al 2012, Patel et al 2012, Italia et al 2009).

Conclusions
Low fixed dose HU is well tolerated and is efficacious in reducing incidence of VOC, hospitalizations, severe anemia, stroke and acute chest syndrome. Low fixed dose HU may be advantageous in low resource settings because of lower toxicity and consequently less need for monitoring. There is a need for multi-center randomized comparisons of low fixed dose HU with conventional escalation of HU dose to MTD.

Disclosures:
Krishnamurti: GlycoMimetics, Inc.: Research Funding.
Hyperfiltration Is a Risk Factor For The Development Of Microalbuminuria In Sickle Cell Anemia
Authors: Vazquez Benjamin , Saraf Santosh L. , Molokie Robert E. , Gowhari Michel , Hassan Johara , Lash James P. , Gordeuk Victor R. 
Published: October 2019

Abstract
Hyperfiltration, which has been implicated in the early stages of development of chronic kidney disease (CKD) in diabetes mellitus, is a common finding in sickle cell anemia (SCA) patients with normal kidney function. We hypothesized that it may be a risk factor for the development of microalbuminuria, an early indicator of glomerular injury that is observed in 40-60% of adult patients with sickle cell anemia (SCA).
In the cross-sectional analysis, 233 adult SCA patients treated at the University of Illinois at Chicago Sickle Cell Center with baseline estimated glomerular filtration rates (eGFR) determined by the MDRD equation and albuminuria concentrations were screened. Data was collected from steady-state visits defined as four weeks from a vaso-occlusive pain episode or red blood cell transfusion. Eight-two patients with normoalbuminuria (urine albumin concentration < 30mg/g creatinine) and an eGFR ≥ 90mL/min/1.73m2 were evaluated of which 38 (46%) had normal eGFR (defined as eGFR 90-150mL/min/1.73m2) and 44 (54%) had hyperfiltration (defined as eGFR >150mL/min/1.73 m2). On univariate analysis, the median age of patients with hyperfiltration was 23 years compared to 34 years in patients with a normal eGFR (p<0.0001). Additionally, patients with hyperfiltration had lower hemoglobin F concentrations (3.9 vs. 7.3%, p=0.01) and higher absolute reticulocyte counts (390 vs. 288 x103/µL, p=0.03) than patients with normal eGFR, respectively. On multivariate analysis, hyperfiltration was independently associated with age (OR 0.9, 95% CI: 0.8-0.9; p=0.0001) and the natural log of hemoglobin F concentration (OR 0.5, 95% CI: 0.2-1.0; p=0.04).
Forty-six patients with normoalbuminuria and eGFR ≥ 90mL/min/1.73m2 were followed longitudinally for progression to microalbuminuria. At a median follow up of 30 months (range 1-81 months), 35% (16/46) of patients developed microalbuminuria with a mean time to progression of 49 months (range 11-57 months). The average time to develop microalbuminuria in patients with hyperfiltration was 33 months (42%, 11/26) compared to 62 months in patients with normal eGFR (25%, 5/20) (age-adjusted HR 4.0, 95% CI 1.5 – 10.4; log-rank p=0.016) (Figure 1).
In summary, hyperfiltration in SCA patients with normoalbuminuria was associated with younger age and lower hemoglobin F concentration on cross sectional analysis and with higher rates of progression to microalbuminuria on longitudinal analysis. Future research to better understand the development of hyperfiltration and its contribution to kidney damage may help guide future therapies for sickle cell nephropathy.

Disclosures:
No relevant conflicts of interest to declare.
Intermittent Use Of Sufadoxine-Pyrimethamine To Prevent Malaria In Patients With SCD In Malaria Endemic Regions: Findings From The Sickle Cell Disease Research and Control Center Of Bamako, Mali
Authors: Diallo Dapa Aly , Guindo Aldiouma , Touré Boubacari Ali , Sarro Yaya , Fané Baba , Dorie Alain 
Published: October 2019

Abstract
Sickle cell disease (SCD) is a chronic disorder affecting erythrocytes and is especially prevalent throughout Sub-Saharan Africa where malaria is thought to be a significant cause of morbidity and mortality in affected individuals. In the absence of effective malaria vaccine, one of the affordable alternatives to prevent malaria at present is chemoprophylaxis. In order to evaluate the effectiveness of a monthly intermittent prophylaxis treatment (IPT) with sulfadoxine-pyrimethamine (SP) during high malaria transmission season in patients with sickle cell disease, two groups of SCD patients from two different sites were compared. The first group constituted of patients followed at the Sickle Cell Disease Research and Control Center of Bamako where IPT is routinely administered while the second group consisted of individuals enrolled in the health district in the same locality but no malaria prophylaxis. In this area, the incidence of resistance of P.falciparum to SP is estimated < 10%. SP combination was administrated as follows: sulfadoxine 25mg/kg and pyrimethamine 1.25mg/kg. For both groups, diagnosis of malaria was performed by using the rapid diagnosis test for the presence of P.falciparum. From 2011 to 2012, 687 SCD patients (457 from the Sickle Cell Disease Research and Control Center and 115 from the health district) were enrolled. The observed prevalence of malaria infection in the group receiving a monthly IPT by SP (1.5%) was much lower than the group (6%) for which IPT was not offered (P=0.01). When data was stratified by hemoglobin genotypes, malaria was found to occur entirely in SS and SC patients and no malaria cases were observed in S/β-thalassemia patients. SP was well tolerated since no patient in SP arm reported pruritus and no serious adverse events including death were recorded during the study. In malaria endemic areas where the incidence of resistance to SP is low, anti-malarial prophylaxis with this combination therapy significantly reduced the incidence of malaria in SCD patients with good safety and a lower cost.

Disclosures:
No relevant conflicts of interest to declare.
Assessing B Lymphocyte Clonal Diversity, Expansion, and Convergent Evolution By High-Throughput Sequencing Of Rearranged IGH Segments From Naïve and Memory Repertoires
Authors: DeWitt William S , Lindau Paul , Emerson Ryan O , Sherwood Anna , Williamson David , Rieder Mark J , Chung Moon , Carlson Christopher S , Robins Harlan 
Published: October 2019

Abstract
Diversity in B lymphocyte antigen specificity, necessary for effective adaptive immunity, arises from structural diversity of the B cell receptor generated by random somatic rearrangement of the immunoglobin heavy (IgH) and light chain loci during lymphocyte development. Antigen-experienced (memory) B cells are stimulated to proliferate upon successful binding of antigen, whereas the extent to which unexperienced (naïve) cells proliferate, if it all, is unclear. During memory clonal expansion, daughter cells undergo somatic hypermutation, which introduces random single nucleotide variants to the rearranged gene segments, selectively optimizing antigen affinity. Among memory B cells that express identical receptor proteins, the extent to which the cells are identical by descent, or rather have convergently evolved from different naïve clones to the same specificity, is also unknown.
We performed high-throughput sequencing of rearranged IgH gene segments for B cells sorted into memory (CD19+, CD27+) and naïve (CD19+, CD27-,IgM+, IgD+) samples (∼5 million cells each) from each of three adults. Each sample was split among 188 libraries for multiplex PCR amplification of IgH sequences at ∼10x coverage, allowing us to estimate clonal abundance by measuring the number of libraries occupied by each sequence.
Using a maximum entropy inverse model, all three memory samples evince three distinct subpopulations, corresponding to memory cell types of differing abundance. Data from naïve samples suggest extreme diversity with more than 98% of clones occupying one library (i.e., almost surely present in only one cell in the starting material). A measure-theoretic upper bound on mean clone abundance in the naive repertoire was computed based on Monte Carlo simulation, indicating that naïve B cells typically undergo little, if any, expansion. Additionally, somatic hypermutation in the memory samples was investigated to detect convergent evolution of distinct naïve B cell clones toward common amino acid sequence (hence common antigen specificity) in the memory B cell compartment.

Disclosures:
DeWitt: Adaptive Biotechnologies: Employment, Equity Ownership. Emerson:Adaptive Biotechnologies: Employment, Equity Ownership. Sherwood:Adaptive Biotechnologies: Employment, Equity Ownership. Williamson:Adaptive Biotechnologies: Employment, Equity Ownership. Rieder:Adaptive Biotechnologies: Employment, Equity Ownership. Chung:Adaptive Biotechnologies: Employment, Equity Ownership. Carlson:Adaptive Biotechnologies: Consultancy, Equity Ownership, Patents & Royalties. Robins:Adaptive Biotechnologies: Consultancy, Equity Ownership, Patents & Royalties.
Patients With End Stage Congestive Heart Failure Have Decreased High Molecular Weight Von Willebrand Factor Multimers and Higher Plasma Levels Of Total TGF-β1: Implantation Of a Left Ventricular Assist Devices (LVAD) Exacerbates Both Abnormalities
Authors: Ahamed Jasimuddin , Monteagudo Juan , Kinkhabwala Mona , Suarez-Farinas Mayte , Garan Arthur Reshad , Coller Barry S. , Mancini Donna 
Published: October 2019

Abstract
Left ventricular assist devices (LVADs) have improved survival of patients with end stage congestive heart failure (HF), but patients are at high risk of both hemorrhage and thrombosis. Acquired von Willebrand (vW) disease is observed in most patients after continuous flow LVAD insertion, presumably due to shear-dependent vW factor (vWf) cleavage, which may contribute to hemorrhage, but there are no validated thrombosis biomarkers. Since shear can also activate platelets, and platelets contain high concentration of transforming growth factor β1 (TGF-β1), we analyzed plasma TGF-β1 levels in patients before and after LVAD implantation and correlated the data with the loss of high molecular weight (HMW) vWf multimers.
Blood samples were collected from 14 HF patients enrolled either as a destination therapy (n=2) or as a bridge to transplantation (n=12) at the Columbia University before and after LVAD implantation. Plasma was prepared by centrifuging blood at 12,000 rpm for 5 min at 4°C within 5 min of blood drawing. Total TGF-β1 levels were measured after acidification of samples using a 2-antibody ELISA (R&D Systems). The platelet α-granule proteins thrombospondin 1 (TSP-1) and platelet factor 4 (PF4) were detected by immunoblotting. vWf multimers were analyzed by electrophoresis of plasma after running in 1.2% discontinuous agarose gel followed by in-gel immunoreactivity band quantification using the LI-COR imaging system. HMW-vWf multimers were defined as the percentage of total vWf antigen contributed by multimers above the 11th identifiable band, starting from the cathodal position.
Plasma total TGF-ß1 levels were higher in HF patients before LVAD implantation than in 16 healthy controls (3.76 ± 1.55 vs. 1.0 ± 0.60 ng/ml; p <0.001). After LVAD insertion, the levels in HF patients increased further to 5.20 ± 2.30 ng/ml (p=0.014). Plasma TGF-ß1 levels in HF patients before and after LVAD implantation were positively correlated with TSP-1 levels (r=0.86; p<0.0001), but PF4 was not detectable, suggesting in vivo release of TGF-β1 from platelets. A reduced percentage of HMW-vWf multimers was observed in HF patients before LVAD insertion compared to healthy controls (18.0 ± 10.0 vs. 29.5 ± 2.5 %; p=0.018), and the percentage was further reduced after LVAD implantation (11.0 ± 9.0 %; p=0.027). There was a weak negative correlation between the percentage of HMW-vWf multimers and TGF-ß1 after LVAD implantation (r= - 0.40; p=0.05).
We conclude that HF itself, even before LVAD insertion, is associated with both loss of HMW-vWf multimers and elevation of plasma TGF-ß1 levels and both abnormalities are exacerbated by LVAD implantation.

Disclosures:
No relevant conflicts of interest to declare.
Platelet Sequestered Proteins Mediate Pre-Metastatic Communication Between Tumors and Bone
Authors: Kerr Bethany A. , Byzova Tatiana V. 
Published: October 2019

Abstract
Tumors can be considered parasites that exploit the host’s resources in order to promote their own growth. Tumors secrete growth factors and cytokines (tumor secretome) which recruit platelets, blood vessels, and bone marrow-derived cells (BMDCs) to promote angiogenesis and tumor progression. We have recently demonstrated that distant tumor growth can stimulate bone formation and BMDC mobilization prior to metastasis. However, the mechanism of communication between the primary tumor and the bone microenvironment remains unknown. Signals from the tumor to the bone microenvironment are likely transmitted through the circulation; nevertheless, these signals need to be protected from degradation or utilization prior to reaching their target. We hypothesized that platelets could function as regulated storage compartments for the tumor secretome. Using a murine platelet depletion model during tumor growth, we assessed the role of platelets in BMDC homing and bone remodeling. We demonstrate that platelet depletion prevents BMDC mobilization and recruitment into tumors. Further, platelet depletion inhibited tumor-induced bone formation. To determine which tumor-derived proteins in platelets were responsible for changes in BMDC mobilization and bone remodeling, we analyzed the tumor secretome components and found that 77% of proteins are enriched within platelets over plasma, and that these proteins regulate BMDC mobilization and bone remodeling. In addition, we discovered that tumor-derived proteins, specifically transforming growth factor (TGF)-β1 and matrix metalloproteinases, sequestered within platelets could stimulate osteoblast differentiation and mineralization in vitro. We also found that thrombospondin (TSP)-1 levels are increased in the platelets of tumor-bearing mice. Finally, we assessed the role of the TSP-1/TGF-β1 signaling axis in tumor-induced bone formation and BMDC mobilization. Tumors in TSP-1 null mice were larger, however tumor-induced bone remodeling decreased. Interestingly, in tumor-bearing mice, high levels of TGF-β1 were being retained in the platelets due to a lack of TSP-1 to activate and release TGF-β1. To examine the role of platelet TGF-β1 in the process of tumor-induced bone remodeling, tumors were implanted in platelet-specific TGF-β1 conditional knockout mice and the changes in tumor size and the bone microenvironment were assessed. In summary, our data show that platelet sequestration and secretion of tumor-derived proteins represent a cellular mechanism mediating communication between the bone microenvironment and the primary tumor.

Disclosures:
No relevant conflicts of interest to declare.
Adenosine Inhibits Pro-Inflammatory Thrombin Signaling In Endothelial Cells
Authors: Hassanian Seyed Mahdi , Rezaie Alireza R. 
Published: October 2019

Abstract
Adenosine is an important regulatory metabolite which attenuates inflammation when it binds and activates the adenosine A2A receptor subtype on immune cells. However, the effect of adenosine on inflammatory responses in endothelial cells is mostly unknown. Thrombin as a known pro-inflammatory protease is involved in a variety of pathophysiological processes associated with inflammation in stimulated endothelial cells. The present study investigated the effect of adenosine on thrombin-mediated modulation of pro-inflammatory responses in human umbilical vein endothelial cells (HUVECs). Adenosine, in a concentration-dependent manner (1-100µM), inhibited the barrier disruptive effect of thrombin (20nM) on endothelial monolayer. The expression level of adenosine receptors, A1, A2A, A2B and A3 was examined in HUVECs and it was found that A2A and A2B are the highest expressing receptors among the four subtypes (A2B> A2A>A1>A3) in endothelial cells. Further studies revealed that the barrier protective effect of adenosine on thrombin-induced hyperpermeability in HUVECs was abrogated by the A2A receptor specific siRNA or the A2A receptor specific antagonist, ZM-241385, but not by siRNAs targeting the other adenosine receptor subtypes. To further determine the molecular mechanisms of the barrier protective effect of adenosine, its effect on thrombin-induced RhoA activation was assessed. Pretreatment of endothelial cells with adenosine prevented both thrombin-induced RhoA activation (Rho-GTP) and its membrane translocation as evidenced by cell fractionation and Pull-down assays, respectively. Moreover, preincubation of endothelial cells with adenosine down-regulated the expression of cell surface adhesion molecules (VCAM-1 and ICAM-1) and thrombin-mediated activation of nuclear factor-kappaB (NF-kB) pathway. Adenosine also inhibited secretion of the early chemokine, MCP-1, and the late-acting pro-inflammatory cytokine, HMGB-1, by thrombin-stimulated endothelial cells. Taken together, these results suggest that adenosine can inhibit pro-inflammatory thrombin signaling responses in endothelial cells by specifically activating the A2A receptor subtype, thereby protecting endothelium during the activation of coagulation and inflammatory pathways.

Disclosures:
No relevant conflicts of interest to declare.