bluuebunny/crossref_metadata_2025_split huggingface dataset:
Combinatorial Antimicrobicidal Peptides Exhibit Enhanced Activity Against Bacterial Contaminants In Stored Platelet Concentrates (PCs).
Authors: Ketha Krishna Mohan V. , Rao Shilpakala Sainath , Atreya Chintamani D 
Published: October 2019

Abstract
Abstract 1124

Introduction:
Contamination of blood and blood components by bacteria, virus, fungi and parasites is a major safety risk in transfusion medicine. Novel proof-of-concepts for microbial reduction that enhance risk-to-benefit ratio of the treated products are still an unmet public health need in transfusion medicine. We have recently shown that synthetic antimicrobicidal peptides (AMPs) originating from thrombin-induced human platelet-derived antimicrobial proteins (named PD1-PD4) and repeats of Arginine-Tryptophan (RW1-RW5), demonstrate microbicidal activity against bacteria in plasma and PCs. In the present study we selected RW2, RW3 and RW4 and tested the effect of the peptides individually and in various combinations (cocktail) to see whether the cocktail approach enhances the antimicrobicidal activity of these peptides.

Methods:
The RW2, RW3 and RW4 peptides were tested on platelet samples spiked with 104 colony forming units (cfu) of Staphylococcus aureus, Staphylococcus epidermidis and Pseudomonas aeruginosa. Each spiked sample was incubated individually with 10 mM of RW2, RW3, and RW4 peptides for 1 hour at 37°C to observe their individual effect. Peptides were then combined in the following way to assess their cocktail effect: RW2+RW3; RW2+RW4; RW3+RW4; RW2+RW3+RW4. Spiked sample without any peptide was included as control. Following incubation with the peptide a ten-fold dilution of the inoculum was made and a fixed volume plated on nutrient agar plates. The plates were incubated at 37°C for 18–24 hours and colonies were counted and expressed as cfu/ml.

Results:
Our analysis revealed that individual peptides RW2, RW3 and RW4 were either ineffective or were moderately microbicidal (100-fold reduction) against S. aureus, S. epidermidis and P. aeruginosa. However, in combination (cocktail), these peptides together demonstrated enhanced microbicidal activity against bacteria in spiked platelets. The combinations, RW2+RW4, RW3+RW4 and RW2+RW3+RW4 were the most effective that resulted in 1000-fold reduction of all three bacteria tested.

Conclusion:
The present study conclusively demonstrates the synergistic effect of antimicrobicidal peptides whose combined microbicidal activity is 1000-fold higher than when the peptide is acting alone. By selecting appropriate combination of AMPs against specific bacteria of interest, new proof-of-concepts could be developed that are alternatives to the current pathogen reduction agents.
The findings and conclusions in this abstract have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any Agency determination or policy.

Disclosures:
No relevant conflicts of interest to declare.
Selective Depletion of T Cell Alloresponses Using a Photodepletion Strategy Preferentially Targets CD4+ T Cells.
Authors: McIver Zachariah A. , Grim Andrew , Naguib Nicolas , Khuu Hahn , Battiwalla Minoo , Stroncek David , Barrett A. John 
Published: October 2019

Abstract
Abstract 1467
P-glycoprotein (Pgp) is the product of the multidrug-resistance-1 (MDR1) gene and actively transports various molecules across the extracellular membrane. Previous studies demonstrated that activated lymphocytes decrease Pgp activity and preferentially retain the photosensitizing agent 4, 5-dibromorhodamine 123 (TH9402, Kiadis Pharma, NL). We evaluated a photodepletion technique to achieve selective depletion (SD) of graft-versus-host disease (GVHD) alloreacting T cells in 24 HLA-identical sibling stem cell transplants. Donor lymphocytes were activated by 72 hr exposure with irradiated in-vitro expanded recipient T lymphocytes and pulsed with TH9402. Alloactivated T cells preferentially retaining the photosensitizer were eliminated by light exposure. The transfused cell products were then analyzed for T cell subset frequency. After SD preferential CD4+ T cell depletion occurred with an inversion of the CD4+/CD8+ ratio. Within the CD4+ compartment a significant depletion of naïve (CD27+ CD45RO-), central memory (CD27+ CD45RO+), and effector (CD27-CD45RO+) populations was noted. Conversely, CD8+ naïve and effector subsets was relatively conserved with depletion occurring predominantly in the central memory population. Additionally, an 80% reduction in B cells occurred, with a 60% reduction of cytotoxic NK cells (CD56+CD16+) and a relative expansion of regulatory NK cells (CD56+CD16-). We compared outcomes of 24 SD transplants with 34 patients receiving a T-cell depleted transplant. All patients with hematological malignancies were conditioned with fludarabine, cyclophosphamide, and total body irradiation and received a CD34-selected stem cell allograft from an HLA identical sibling. SD recipients also received 5×106/kg SD donor T cells. Low-dose cyclosporine was used as the only post-transplant immunosuppression. Median follow up was 14.5 months. The probability of severe acute GvHD (grade III-IV, 13%) and relapse (25%) was low for all patients. On day 100 post-transplant SD transplant recipients had lower absolute CD4+ counts, and increased rates of CMV reactivation requiring treatment (median 2 vs 1 reactivations within 100 days, p<0.01), and more chronic GvHD (70% vs 31%, p=0.04). Previous studies have shown that Pgp efflux of rhodamine-123 in quiescent T cells varied between subsets, with a preferential retention occurring in the CD4+ and central memory compartments (Br J Haematol. 1998 Jun;101(4):722-7). Our data is in accordance with these observations. In conclusion, while SD appeared to eliminate alloactivated T cells resulting in low frequencies of severe acute GVHD, differences in Pgp activity and dye retention led to the preferential non-specific elimination of CD4+ and central memory T cells which may have contributed to greater viral reactivation and chronic GVHD.

Disclosures:
No relevant conflicts of interest to declare.
Incidence of Arterial and Venous Thrombosis in Vonwillebrand Disease
Authors: Hassan Syed , Qureshi Waqas , Amer Syed , Almahmoud Mohamed , Dabak Vrushali S. , Kuriakose Philip 
Published: October 2019

Abstract
Abstract 101

Objective:
To date, only a few case studies have reported occurrence of thrombosis in patients with VonWillebrand disease (VWD). No studies have looked at its incidence in this patient population. The aim of this study was to test our hypothesis that decreased VonWillebrand factor (VWF) levels confer a protective effect on arterial and venous thrombosis.

Methods:
This is a retrospective cohort study including patients (n = 350) with the ICD-9 code of VonWillebrand disease who were identified from our hospital database over a period of 25 years (Jan 1985 – Dec 2010). An extensive review of the patients' medical records was carried out to authenticate VonWillebrand disease, after which 198 patients were included in the analysis. A random parallel control sample without VonWillebrand disease matched for age, sex, hypertension, hyperlipidemia, atrial fibrillation and diabetes mellitus was also obtained from the hospital database. The primary outcomes were incidence of diagnosis of symptomatic arterial thrombosis [Cardiovascular events (CAD): Unstable angina, Q wave and non Q wave Myocardial infarction; Cerebrovascular events (CVD): stroke and transient ischemic attack] and venous thrombosis [deep vein (DVT), cerebral vein, portal vein, renal vein thrombosis and pulmonary embolism (PE)]. The results were computed using multivariate conditional logistic regression analysis and proportions were compared using McNemer'sChi – square test.

Results:
Out of 198 patients (mean age 44.2 ± 17.5, women 72%) with VWD, the incidence of arterial and venous thrombosis is reported in table 1.VWD was found to be an independent protective predictor from arterial thrombosis (OR 0.28, 95% CI 0.14 – 0.54, p <0.0001), more so in CAD (OR 0.28, 95% CI 0.12 – 0.64, p = 0.002) than in CVD (OR 0.28, 95% CI 0.10 – 0.77, p = 0.01). However this was not the case in venous thrombosis as seen in DVT (OR 0.62, 95% CI 0.20 – 1.93, p = 0.41) or PE (OR 0.50, 95% CI 0.09 – 2.75, p = 0.423).One case of portal vein thrombosis was also seen. Also the arterial thrombosis in VWD was not a predictor of mortality; 10 deaths were reported in VWD patients, while 4 in control population (OR 1.8, 95% CI 0.49 – 6.76, p = 0.37).

Conclusion:
In a population of relatively younger individuals with VonWillebrand disease, our study suggests a reduced incidence of arterial thrombosis but not in venous thrombosis. This brings up the possibility that there are could be other pathways or factors involved in arterial and venous thrombosis. To our knowledge, this is the first large observational study that has provided insight into the thrombotic disease in this group of patients.

Disclosures:
No relevant conflicts of interest to declare.
Leptin and Adiponectin Blood Levels in Patients with Steady State Sickle Cell Disease Are Related to Chronic Hemolytic and Inflammatory Biomarkers
Authors: Makis Alexandros , Challa Anna , Koutsouka Freideriki , Hatzimichael Eleftheria , Pliakou Xanthi , Briasoulis Evangelos , Siamopoulou Antigone , Chaliasos Nikolaos 
Published: October 2019

Abstract
Abstract 1012

Background.
Vascular endothelial cell activation and chronic inflammatory processes along with concomitant chronic energy deficiency characterize the steady state of sickle cell disease (SCD). Adipose tissue besides its lipid storage has also an endocrine function producing adipokines most important of which are leptin and adiponectin. These two hormones regulate food intake and energy expenditure but they are also involved in the regulation of inflammatory responses, hematopoiesis and angiogenesis. We evaluated leptin and adiponectin blood levels and determined any possible correlations with disease severity and inflammation markers in steady state SCD patients.

Methods.
We measured leptin and adiponectin using ELISA as well as high sensitivity C-reactive protein (hs-CRP) using nephelometry, in blood serum of 42 patients (24 male-18 female, median age 34 years, range 29–52 years) with steady state sickle cell/beta thalassemia and 40 healthy individuals matched for age, sex and body mass index (BMI) z-score who served as controls. Steady state was defined as a crisis-free period for at least 4 weeks after the previous clinical event and 3 or more months since the last blood transfusion. After natural logarithmic transformation, the measured values were investigated for correlations against hemoglobin, white blood cells, bilirubin, lactate dehydrogenase (LDH), ferritin, insulin levels, fetal hemoglobin (HbF), number of events/year (severe painful crises, transfusions, severe infections or other complications) and treatment with iron-removing agents or hydroxurea.

Results.
Leptin was found significantly lower in patients compared to controls (median±SD, 2.9±1.9 vs 8.5±4.9 ng/ml, p=0.006), whereas adiponectin and hs-CRP were found significantly elevated (median±SD 11.3±6.1 vs 7.5±3.1 μg/ml, p=0.003 and 2.8±1.1 mg/L vs 1.4±0.9; p=0.018, respectively). No correlations were found between adipokines and age, gender, chelation or hydroxurea treatment, ferritin, insulin, HbF, hemoglobin or clinical events. Leptin, but not adiponectin, levels were positively correlated with BMI z-score (r= 0.71, p=0.018) and negatively correlated with LDH levels (r=0.69, p=0.012). Concerning adiponectin, significant positive correlation was noted with hs-CRP levels and white blood cell number (r=0.644, p=0.015 and r=0.68, p=0.028).

Conclusions.
Adipokine blood levels are altered in steady state SCD possibly reflecting the effect of chronic hemolytic and inflammatory state on adipose tissue dysfunction. This could be considered as another endocrinopathy that affects SCD patients.

Disclosures:
No relevant conflicts of interest to declare.
Group B Streptococcus Induce Platelet Activation Via Toll-Like Receptor 2
Authors: Ma Liping , Xianming Luo , Li Hongyu , Li Yiqing , Shuangfen Xie , Hui Wu 
Published: October 2019

Abstract
Abstract 1066

Objective:
Group B Streptococcus (GBS), or Streptococcus agalactiae, is one of the most common cause of life-threatening sepsis in human newborns and immunosuppressed population. Patients suffering from sepsis often display low platelet counts characterized by thrombocytopenia as a result of platelet activation. Platelets, best known for their maintenance of hemostasis, have retained features of archetypal inflammatory cells corresponding to multiple roles in antimicrobial host defense. Nine members of the Toll-like receptors (TLRs) have been reported in human platelets, and TLRs in platelets play a fundamental role in both the initiation and propagation of the inflammatory response to microbial infection. We reported previously that LPS induces platelets activation and enhances platelets to release tumor necrosis factor-α by TLR 4. Since few confirm that platelets were activated by TLRs in directly binding to bacteria, the mechanism of activation of platelets in the interaction with pathogens remains unclear. Our research aimed to verify the hypothesis that the platelets selectively bind or adhere strains of GBS and aggregate, and to investigate the mechanism through which platelets become activated in sepsis upon binding to GBS and platelet TLR 2 involves in the process.

Methods:
Six strains of GBS were separated from septic patients. We determined GBS inducible platelet reactivity by using light transmission aggregometry, platelet bacterial adhesion assay, FCM and scanning electron microscopy. TLR2 expression was determined by RT-PCR and western blot assay.

Results:
After incubating platelets with strains of GBS separately, only three strains of GBS could induce platelets activation (platelets aggregation, adhesion and upgrated the expression of CD62P on platelet, Figure 1), meanwhile the activating ability and lag time were strain-dependent. Strains of GBS that were able to induce platelets activation also upregulated the expression of platelets TLR2 (in levels of RT-PCR and protein, Figure 2). However incubated with TLR 2 antibody, platelets no longer aggregated by the stimulation of GBS (Figure 3), suggesting TLR2 was involved in GBS induced platelets activation

Disclosures:
No relevant conflicts of interest to declare.
Rare Coding Single Nucleotide Variants of ADAMTS13 Are Associated with Deep Vein Thrombosis in a Next-Generation Sequencing Association Study
Authors: Lotta Luca Andrea , Tuana Giacomo , Yu Jin , Martinelli Ida , Wang Mark , Yu Fuli , Passamonti Serena Maria , Pappalardo Emanuela , IV Walker Hale , Muzny Donna M. , Rosendaal Frits R. , Gibbs Richard A , Peyvandi Flora 
Published: October 2019

Abstract
Abstract 107
The considerable genetic predisposition to deep vein thrombosis (DVT) is only partially accounted for by known genetic risk variants. Genome-wide association studies investigating hundreds of thousands of common single nucleotide variants (SNVs) identified a number of associations, but missing heritability remains. Rare (i.e. minor allele frequency below 1%) and low-frequency (i.e. minor allele frequency below 5%) SNVs of the coding area may be responsible for at least part of this missing heritability. In order to investigate this, we sequenced 700,000 base pairs of genomic DNA including the protein coding exons and intron-exon boundaries of 186 hemostatic/pro-inflammatory genes. Indexed genomic DNA libraries were co-captured on NimbleGen HD2 2.1M-probe chips and capture products were sequenced on SOLiD 4 platforms. More than 70 billion base-pairs of raw sequence data were produced to sequence the target area with a median redundancy of 45X in 94 thrombophilia-negative patients with DVT and 98 controls. Most of the 4366 SNVs identified were rare, novel and nonsynonymous indicating pathogenetic potential. We tested the association of coding SNVs in the ADAMTS13 and VWF genes, encoding two interconnected proteins with fundamental roles in hemostasis. Sequencing of the two genes yielded 109 variants, 108 SNVs and a c.8241_8442del frameshift deletion in exon 51 of VWF. Being a carrier of rare coding (prevalence in DVT: 17% [n=16]; prevalence in controls 4% [n=4]; odds ratio [OR]: 4.8; 95% confidence interval [CI]: 1.6–15.0), rare nonsynonymous (prevalence in DVT: 11% [n=10]; prevalence in controls 3% [n=3]; OR: 3.8; 95% CI: 1.0–14.2) or low-frequency coding (prevalence in DVT: 36% [n=34]; prevalence in controls 23% [n=23]; OR: 1.9; 95% CI: 1.0–3.5) SNVs of ADAMTS13 was associated with DVT. Carrying rare or low-frequency SNVs of VWF was not associated with DVT. The 11 different rare missense variants of ADAMTS13 found in DVT patients had never been described in association with congenital thrombotic thrombocytopenic purpura. Patients carrying at least one ADAMTS13 mutation of the categories associated with DVT had lower plasmatic levels of ADAMTS13 activity compared to patients without mutations. The change in ADAMTS13 activity was −7% (95% CI: −24 to 10%) for patients with rare coding ADAMTS13 mutations, −12% (95% CI: −30 to 6%) for patients with rare nonsynonymous mutations and −29% (95% CI: −52 to −6%) for patients with missense mutations predicted to be damaging for protein function by SIFT. Our results uncover for the first time a link between ADAMTS13, an important regulator of hemostasis implicated in microvascular thrombosis, and DVT.

Disclosures:
No relevant conflicts of interest to declare.
Shiga Toxin Upregulates Tissue Factor Gene Expression in and Functional Tissue Factor On Endothelium in the Presence of Complement Activation
Authors: Grabowski Eric F. , Kushak Rafail I. , Ingelfinger Julie R. 
Published: October 2019

Abstract
Abstract 1075
The pathophysiology of Shiga toxin (Stx)-related hemolytic uremic syndrome remains poorly understood. We hypothesize that tissue factor (TF) expression on vascular endothelium is central to the genesis of this disorder and is driven both by direct effects of Stx on endothelium activated by inflammatory cytokines derived from the action of Stx in the gastrointestinal tract, and by Stx-induced complement activation, which further augments TF expression. We used human umbilical vein endothelial cell monolayers to examine 1) gene expression of TF and TF pathway inhibitor (TFPI) with TNFα (20 ng/ml) ± Stx-1 (10 pM) compared to control, 2) total cellular and cell surface antigenic TF and TFPI, 3) TFPI secretion into supernatant, 4) factor Xa production, and 5) soluble levels of C5b-9 in complete media in response to 3–30 pM Stx-1 in the absence of cells. TF mRNA increased 2.82 ± 0.92-fold (N=13; p <0.0005) with TNFα alone vs. 1.25 ± 0.32-fold (N= 9; p =0.041) for Stx-1 alone (Fig. 1). TNFα plus Stx-1 yielded a 6.51 ± 3.48-fold increase (N=17; p <0.0005; Fig. 1). TFPI mRNA decreased with TNFα (p <0.001) andTNFα plus Stx-1 (p < 0.0005). Total cellular (p = 0.048) and cell surface (p = 0.027) TF antigen increased with TNFα, and no further with TNFα plus Stx-1. Total TFPI cellular (p = 0.0017) and cell surface (p = 0.030) antigen levels, and secretion of TFPI (p = 0.018) decreased with TNFα plus Stx-1. Median factor Xa production for TNFα plus Stx-1 vs. TNFα alone increased (p < 0.001) 3.24-fold. In the absence of cells, M199 with 10% human serum to which was added Stx-1 yielded levels of C5b-9 of 648 and 1102 ng/ml vs. 106 and 313 ng/ml in the presence of 25 mM EDTA (to block the classical and alternative pathways of complement), and intermediate levels in the presence of 15 mM EGTA and 7.5 mM Mg++ (to block the classical pathway). Similar results were found with M199 with 10% human plasma, collected using 0.2 U/ml FC in media of dalteparin. C5b-9 levels increased between 3 and 10 pM Stx-1, but did not increase further at 30 pM Stx-1 (Fig. 2). Finally, by means of immunofluorescence we also detected and measured C5b-9 on fixed cell monolayers, whether treated prior to fixation with TNFα alone or TNFα plus Stx-1. We conclude that Stx-1 augments functional TF on endothelium by increasing TF mRNA, and impairing synthesis, cell-surface association, and secretion of TFPI, while at the same time activating both the alternative and classical complement pathways. These findings support the development of therapeutic approaches to epidemic HUS that incorporate inhibitors of the TF pathway, inhibitors of the alternative and classical complement pathways, or both.

Disclosures:
No relevant conflicts of interest to declare.
Kruppel-Like-Factor 5 (Klf-5) Controls Hematopoietic Stem Cell/Progenitor Bone Marrow Homing and Lodging Through Rab5-Mediated Expression of Active β1 Integrin
Authors: Ishikawa Eri Taniguchi , Chang Kyung-Hee , Olsson H. Andre , Nayak Ramesh C , Ficker Ashley , Dunn Susan K , Sengupta Amitava , Whitsett Jeffrey , Grimes H. Leighton , Cancelas Jose A 
Published: October 2019

Abstract
Abstract 113
The Kruppel-like factor 5 (Klf5) is a transcription factor that regulates cellular signaling and is involved in cell proliferation and oncogenesis. Recent studies have implicated Klf5 in the self-renewal and maintenance of pluripotent stem cells. In this project, we examined the role of Klf5 in hematopoietic stem/progenitor cells (HSC/P) activity. Using a murine model of inducible deficiency of Klf5 in hematopoietic tissue (Mx1-Cre/Klf5flox/flox, Klf5-deficient), we observed a severe engraftment deficiency in competitive repopulation assays in primary and secondary recipients, although it did not cause any significant hematopoietic defects basally or after chemotherapy stress in primary mice. Engraftment failure of Klf5-deficient HSC was associated with a severe defect in homing (1.8±0.8% vs 6.0±2.0% for Klf5-deficient and WT HSC/P, respectively, p<0.001), endosteal lodging (9.09±12.58% vs 44.74±12.58% for Klf5-deficient and WT HSC/P, respectively, p<0.02) and adhesion to the fibronectin C-terminus (28.7±7.3% vs 44.4±3.2% of CFU-C adhesion for Klf5-deficient and WT HSC/P, respectively, p<0.001), without altering the adhesion to laminin or collagen, or CXCL12-directed chemotaxis. The homing defect associated with increased (3.5-fold) basal circulating of HSC/P and decreased HSC/P membrane β1, but not of the β1-integrin transcript or intracellular protein. In vivo gain-of-function of Klf5 in HSC in a stem cell leukemia (Scl) promoter-driven, tetracycline-inducible (ScltTA/TRE-Klf5) binary transgenic mouse model, resulted in increased adhesion, but not migration, of Lin−/c-kit+/Sca-1+ BM cells to fibronectin (32.1±7.8% vs 20.8±4.8% of CFU-C homing for WT HSC/P). The expression of Rab5 family members, mediators of β1-integrin recycling in the early endosome, was ∼50% decreased in Klf5-deficient HSC/P. Klf5 was shown to bind directly to the promoter of Rab5A and Rab5B, and lentiviral reintroduction of Rab5B rescued the expression of active membrane β1-integrin, adhesion to fibronectin, and BM homing of Klf5-deficient HSC/P. Our data indicate that Klf5 expression is indispensable for adhesion, homing, lodging and retention of HSC/P in the BM through Rab5-dependent regulation of the expression of active β1-integrin in the cell membrane.

Disclosures:
No relevant conflicts of interest to declare.