bluuebunny/crossref_metadata_2025 huggingface dataset:
A Phase I Study of Tipifarnib Combined with Conventional Induction and Consolidation Therapy for Previously Untreated Patients with Acute Myeloid Leukemia Age 60 and Over.
Authors: Brandwein Joseph M. , Leber Brian F. , Howson-Jan Kang , Schimmer Aaron D. , Schuh Andre C. , Gupta Vikas , Yee Karen W.L. , Minden Mark D.
Published: October 2019

Standard induction therapy for patients over age 60 with acute myeloid leukemia (AML) has produced complete response (CR) rates of around 55%, but most patients eventually relapse. Tipifarnib (R115777, ZarnestraR) has shown activity as a single agent in AML, and is well tolerated in older patients at doses up to 600 mg BID. This agent also has additive/synergistic effects on AML cell lines when combined with daunorubicin (DNR). From 2005–2007, patients age 60 and over with previously untreated AML (de novo or secondary) were enrolled in a Phase I study combining tipifarnib with standard induction therapy. The regimen consisted of cytarabine 100 mg/m2/day continuous IV infusion on days 1–7, DNR 60 mg/m2/day IV push x 3 on days 6–8 and tipifarnib orally twice daily on days 6–15. Tipifarnib was escalated over four doses levels in successive patient cohorts (200, 300, 400 and 600 mg). Patients achieving CR were eligible to received one consolidation using the same regimen. Dose-limiting toxicity (DLT) was defined as Grade III–IV non–hematologic toxicity or hematologic recovery times > 40 days unless due to persistent leukemia. Up to 2/6 DLTs were permitted at each dose level. The following DLT’s were identified during induction: Dose level I: 2/6 (grade III hyperbilirubinemia, grade IV transient respiratory arrest), dose level II: 0/3, dose level III: 0/3 and dose level IV: 2/6 (grade III typhlitis, grade III supraventricular tachycardia). Four additional patients were enrolled at dose level IV, with one DLT (grade III diarrhea). There were no DLTs during consolidation. There were no cases of delayed hematologic recovery. Of 22 evaluable patients, there were 9 CR, 3 MLFS, 2 PR and 8 non-responders. Of 7 patients with adverse risk cytogenetics, there were 3 CR, 1 MLFS and 1 PR. In summary, this regimen was well tolerated and the DLT was not reached, although somewhat more GI toxicity was seen at dose level IV. Because of the inherent toxicity of the underlying regimen and the elderly population, it was decided not to escalate further, and tipifarnib 600 mg BID has been chosen as the recommended dose for further study using this regimen.
Inhibition of Tissue Factor (TF) Pathway Blunts Hepatic Tumor Development in a Rat Colorectal Cancer Model.
Authors: Zerbib Philippe , Grimonprez Alexandre , Corseaux Delphine , Petersen Lars , Jude Brigitte
Published: October 2019

Occurrence of hepatic metastasis (HM) is the leading cause of death in colorectal cancer, making their prevention a major challenge. Tissue factor (TF), the principal initiator of coagulation after binding to its ligand, coagulation factor FVIIa, is expressed by most colorectal cancer cells, and has been demonstrated to be associated to tumor invasion and metastasis. The aim of this study was to analyze the effects of inhibition of the TF pathway in a rat model of HM of colorectal cancer.

Methods DHDK12 Pro B cells (20.106 cells), a tumor cell line established from chemically induced colon carcinoma in BDIX rats, were injected in the portal vein of syngenic rats. Inhibition of TF was achieved in 19 rats (treat ment group) through intraperitoneal injection of active site-inactivated factor VIIa (FFR-FVIIa, Novo Nordisk, Denmark) (10μg/g, once a day from day 3 to day 8 after HM induction). Additionally, 18 rats underwent cancer cell infusion according to the same procedure, without FFR-FVIIa treatment (control group). Rats were sacrificed at day 14.

Results In the control group, infusion of cancer cells resulted in the development of macroscopical hepatic tumors in 17 out of 18 rats. In rats treated by FFR-FVIIa, no macroscopical or histological hepatic tumors were visible on the liver surface in 16 out of 19 rats (p=0.002, versus control group).

Conclusion These results suggest that blockage of TF with proteolytically inactive FFR-VIIa inhibits hepatic tumor development at an early stage of tumoral development and TF is a target for adjuvant therapy in the prevention of HM.
A Chromatin-Associated Histone H3 Dementhylase Promotes the Immortalization of MEFs and the Cycling of HSC-Like Cells in Culture.
Authors: Pfau Raymond , Tzatsos Alexandros , Bear Susan E. , Polytarchou Christos , Kampranis Sotirios , Van Etten Richard A. , Tsichlis Philip N.
Published: October 2019

A common integration site, cloned from MoMuLV-induced rat T cell lymphomas was mapped immediately upstream of Ndy1, a gene expressed primarily in ES cells, hematopoietic stem cells, testis, spleen and thymus. Ndy1 encodes a chromatin-associated nuclear protein. Ndy1 and its homolog Ndy2, which is also a target of provirus integration in retrovirus-induced lymphomas, encode proteins that were recently shown to possess JmjC-dependent histone H3 demethylase activity. Mouse embryo fibroblasts (MEFs) engineered to express Ndy1 or Ndy2 undergo immortalization in the absence of replicative senescence via a JmjC domain-dependent process. Knock down of endogenous Ndy1 or expression of JmjC domain mutants of Ndy1 promotes senescence, suggesting that Ndy1 is a physiological inhibitor of senescence in dividing cells. Infection of bone marrow cells with a high titer MigR1/Ndy1 retrovirus led to the reproducible isolation of immortalized cultures that have the phenotypic characteristics of hematopoietic stem cells. We conclude that Ndy-mediated demethylation of histone H3, promotes cell immortalization, oncogenesis and the cycling of stem cells.
Study of Patients Having Splenectomy Due to Trauma for Long Term Infectious and Vascular Complications.
Authors: Purcell Patricia L. , Crary Shelley E. , Adix Leah , Alder Adam , Buchanan George R.
Published: October 2019

Splenectomy, commonly performed for hematologic conditions, is followed by an increased risk of septicemia and arterial and deep venous thrombosis and pulmonary arterial hypertension. Most recent studies of post-splenectomy vascular complications focus primarily on patients with sickle cell disease, thalassemia, or hereditary stomatocytosis. However, the incidence and pathophysiology of complications in these patients is confounded by persistent hemolysis. Therefore, study of long term post-splenectomy infectious and vascular complications in patients without hemolysis or other serious medical conditions is indicated. Patients having splenectomy for trauma are often young and otherwise healthy so might be good candidates for investigation of post-splenectomy sequelae. To determine the feasibility of conducting a study of infectious and vascular complications following splenectomy for trauma, we retrospectively studied such patients admitted to Parkland Memorial Hospital, a 950 bed public hospital and the site of the Dallas metropolitan area’s primary trauma center. Using appropriate ICD-9 and CPT codes, three separate databases (hospital medical records, trauma registry, and professional billing compilation) were reviewed in order to identify patients ≥ age 18 yr. having total splenectomy for trauma during the past 15 years and to determine whether follow-up data were available in the records regarding subsequent vascular events or surrogate laboratory markers suggesting an increased risk of thrombosis. Between 1992 and 2006, 855 individuals had splenectomy, including 643 (75%) following trauma. The annual number of patients having splenectomy due to trauma did not decline during this period. A comprehensive chart review was performed on the 77 patients having splenectomy for trauma during 2003 and 2004. Records were unavailable in four patients, and 20 died of their injuries shortly after admission. Follow-up records of the remaining 53 patients - 26 (49%) of whom appeared to be discharged in good condition without serious sequelae - were reviewed to attempt to gather data regarding clinical features and laboratory test results. Post hospitalization follow-up occurred in only 9 (17%) of these 53 patients. Six had serious post-trauma complications (e.g., paraplegia); only three seen in follow-up more than six months following splenectomy appeared well. We conclude that complete splenectomy following trauma remains a common procedure. Retrospective review of these patients’ records does not permit evaluation of post-splenectomy complications. However, many patients were discharged shortly following splenectomy without other serious injuries. These patients could potentially be studied prospectively to assess the frequency, timing, and pathophysiology of infection, thrombosis, pulmonary arterial hypertension, and other complications of the post-splenectomy state without the confounder of hemolytic anemia.
Impaired Natural Killer Cells Immune Synapse Formation in Acute Myeloid Leukemia.
Authors: Xing Dongxia , Ramsay Alan G. , Decker William , Li Sufang , Robinson Simon , Yang Hong , Gribben John G. , Shpall Elizabeth J.
Published: October 2019

Abstract 2663

Poster Board II-639

Natural killer (NK) cells are an important component of the innate immune surveillance of tumor cells. Defective NK cell function has been correlated with poor prognosis in acute myeloid leukemia (AML). It is well established that NK cell-mediated cytolytic activity is significantly diminished in AML patients; the mechanisms of this hypo-function are not well understood. Identifying mechanisms of tumor-induced immune suppression of lymphocytes function will aid the development of effective immunotherapeutic strategies. In the present study we examined the molecular basis for impaired NK cell responses in AML and demonstrate impaired NK cell immunological synapse formation. Confocal microscopy was used to visualize F-actin polymerization at the immune synapse between CD56+ CD3- NK cells and autologous AML blasts. We identified a significant reduction in formation of the NK cell immune synapse (NKIS) (p<0.001) from AML patients compared healthy donors (> 70% reduction). This defect was induced by direct tumor contact since NK cell defects were induced in healthy NK cells when they were co-cultured (in direct contact) for 48 hr with allogeneic AML blasts, but not with healthy allogeneic monocytes (P < 0.01). In control transwell co-culture experiments, where the NK cells and AML blast were not in direct contact, we did not observe the induced defect. We examined the molecular nature of the AML blast induced defect by quantifying recruitment of a number of these NK cell adhesion and cytoskeletal signaling proteins to the immune synapse. Following primary co-culture with AML blasts, healthy NK cells showed significantly reduced recruitment of integrin LFA-1, CD2, Lck, WASP, and tyrosine-phosphorylated protein to the NK-AML target interactions synapse (P < 0.001). These studies demonstrate a role for the tumor induced immune suppression of NK cells and will aid in the development of immunotherapeutic approaches targeting AML.

Disclosures:

No relevant conflicts of interest to declare.
Risk of EBV-Positive Hodgkin Lymphoma Varies Over 30-Fold by HLA Class I Genotype and History of Infectious Mononucleosis.
Authors: Jarrett Ruth , Johnson Paul CD , Rostgaard Klaus , Lake Annette , Shield Lesley , Little Ann-Margaret , Ekstrom-Smedby Karin , Adami Hans Olov , Glimelius Bengt , Dutoit Stephen Hamilton , Kane Eleanor , Taylor G. Malcolm , McConnachie Alex , Ryder Lars P , Sundstrom Christer , Andersen Paal Skytt , Chang Ellen T , Alexander Freda , Melbye Mads , Hjalgrim Henrik
Published: October 2019

Abstract 269

Introduction:

Markers in the human leukocyte antigen (HLA) class I locus and infectious mononucleosis (IM) have both been associated with Epstein-Barr (EBV)-positive Hodgkin lymphoma (HL). IM is caused by late infection with EBV. To further explore associations between HLA class I genes and EBV-positive HL and to investigate potential interactions between genetic (HLA) and environmental risk factors in the development of HL, we performed a case-only study of 934 HL cases.

Methods:

HLA-A and B typing were performed by PCR sequence specific oligonucleotide assay. Logistic regression was used to estimate odds ratios (ORs) of EBV-positive HL relative to EBV-negative HL for HLA-A and B genotypes and other known risk factors.

Results:

Increasing age, male sex, HLA-A*01 and a previous history of IM were all associated with a statistically significant increased odds of EBV-positive HL while HLA-A*02 and HLA-B*07 were associated with a protective effect. Significant interactions were detected between HLA-A*01 and HLA-B*07 and between prior IM and HLA-A*02, such that HLA-B*07 abolished the increased odds associated with HLA-A*01 and HLA-A*02 abrogated the increased odds associated with prior IM. In our final model of EBV-positive HL, HLA-B*07 was associated with a dominant protective effect (OR=0.57, 95% confidence interval (CI): 0.34–0.94); HLA-A*02 was associated with a protective effect which was additive (for individuals with no prior IM, ORper allele =0.69, 95% CI: 0.50–0.97); and HLA-A*01 was associated with an increased odds in patients who were HLA-B*07-negative (ORper allele=2.15, 95% CI: 1.54–3.01). Prior IM was independently associated with EBV-positive HL (OR=3.35, 95% CI: 1.67–6.73). This resulted in a >32-fold higher odds of EBV-positive HL in HLA-A*01 homozygotes with IM compared to HLA-A*02 homozygotes without IM.

Conclusions:

We have identified strong associations between both genetic and environmental risk factors and EBV-positive HL. Our findings suggest that HLA class I-restricted, cell-mediated responses to EBV and a prior history of IM play critical roles in the pathogenesis of EBV-positive HL.

Disclosures:

No relevant conflicts of interest to declare.
Nucleostemin Gene Expression in Acute Promyelocytic Leukemia Patients.
Authors: Ashrafi Farzaneh , Nadali Fatemeh , Ghavamzadeh Ardeshir , Alimoghaddam Kamran , Rostami Shahrbano , Ashrafi Mahdi , Keyhani Manoutchehr
Published: October 2019

Abstract 4430

Background

Nucleostemin (NS), a novel p53-binding protein has been shown essential for stem and cancer cell proliferation and implicated in oncogenesis. Nucleostemin expression had been shown in gastric cancer (SGC-7901) cells, human hepatocarcinoma (HepG2) cells, human cervical cancer (Hela) cells, human osteosarcoma (OS-732) cells.

Aim

This work designed to study the NS gene expression in bone marrow cells in acute promyelocytic leukemia (APL) patients and in normal bone marrow specimens.

Materials &Methods

We examined NS gene expression by Quantitative Real Time PCR in bone marrow specimens of 15 cases of APL patients, before treatment and in 4 bone marrow specimens of healthy donors of bone marrow transplantation. In the same samples of bone marrow aspiration morphology of smears was evaluated. Diagnosis of APL was based on morphology and positive PML/RARA in PCR. RT-PCR used to amplify the NS mRNA, and the GAPDH primer sets used for normalizing. For comparison of NS gene expreesion in 2 groups Mann-Whitney U test was used.

Results

15 patients enrolled in this study, 11(73%) newly diagnosed APL and 4(27%) relapsed cases. Mean age of patients was 28.67±9.56 year. NS gene expressed in all bone marrow samples of APL patients. NS gene expressed in normal bone marrow specimens too. NS gene expression in bone marrow of APL patients was significantly higher than normal bone marrows(p value =0.002) Fig 1. There was no significant difference in NS gene expression between newly diagnosed and relapsed APL cases.

Discussion

According to the results of this study it seems that NS gene expressed in normal marrow. NS expression in adult bone marrow hematopoietic stem cells had been reported in previous reports and it had been shown that NS does not express in granulocytes and B lymphocytes. It seems that stem cells and proliferating cells in the normal marrow are the source of NS expression detected in normal marrow. NS expreesion in bone marrow of APL patients was significantly higher than normal marrow. In these patients before treatment marrow is replaced by undifferentiated blasts and promyelocytes. We concluded that NS expression in these cells were high. It had been shown that NS down regulation may lead to cell cycle exit. High expression of NS in APL patients can be used in future researches for finding new targeted therapies in this disease.

Disclosures:

No relevant conflicts of interest to declare.
An Essential Role of the RNA Editing Enzyme ADAR1 in T Cell Development.
Authors: XuFeng Richard , Yang Qiong , Yuan Youzhong , Lu Binfeng , Cheng Tao , Wang Qingde
Published: October 2019

Abstract 917

Post-transcriptional regulation such as RNA editing in hematopoiesis and lymphopoiesis is poorly understood. ADAR1 (adenosine deaminase acting on RNA-1) is a RNA editing enzyme essential for embryonic development. Disruption of the ADAR1 gene was shown to cause defective embryonic hematopoiesis (Wang Q et al, Science 2000). Moreover, we have recently obtained direct evidence for the preferential effect of ADAR1 deletion on adult hematopoietic progenitor cells as opposed to the more primitive cells via a RNA-editing dependent mechanism by different conditional gene deletion strategies (Xufeng R et al PNAS 2009, in press). To further determine the role of ADAR1 in T cell development, we generated a mouse model in which ADAR1 was deleted specifically in T lymphocytes by interbreeding ADAR1lox/lox mice with Lck-Cre transgenic mice. In our current study, we report that ADAR1 is essential for T cell differentiation at the late progenitor stage in the thymus, coincident with T cell receptor-α/β expression. In ADAR1lox/loxLck-Cre mice, mature T cells decreased dramatically in peripheral blood, spleen and lymph nodes in comparison to littermate controls. In the thymus, the production of CD4+/CD8+ double positive cells was severely impaired and massive cell death was observed in pre-T cell populations. Within the pro-T cells, ADAR1 deletion resulted in a significant decrease of late progenitor cells but not early progenitor subsets. In both pro-T and pre-T cell stages, defective T cell development preferentially occurred in the beta chain positive cells, but was not apparent in gamma/delta T cells. Our data demonstrated an indispensable role of ADAR1 in early T cell differentiation that correlated with T cell receptor beta chain expression, thereby indicating that RNA editing by ADAR1 is an essential event in T cell development.

Disclosures:

No relevant conflicts of interest to declare.
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 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 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 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 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 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 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 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 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.
Incidence of Thrombosis in Children with Tunneled Central Venous Access Devices Versus Peripherally Inserted Central Catheters (PICC lines)
Authors: Kanin Maralee , Young Guy
Published: October 2019

Abstract 1146

Background:

The recent proliferation in deep vein thrombosis (DVT) in pediatric patients has been attributed to the improved care of children with serious and life-threatening disorders as well as the prolonged survival of children with chronic disease. Often such treatment involves the insertion of central venous catheters. Yet the incidence of DVT associated with peripherally-inserted central catheters (PICC) and tunneled Lines (TL) has not been investigated in depth. Thus we report a retrospective database study to determine the incidence of DVT in pediatric patients with PICC and TL lines.

Methods:

Children <18 years of age who were admitted to Children's Hospital Los Angeles from January 1, 2003- December 31, 2009 were eligible for inclusion. Data were extracted from the CHLA hospital discharge database which includes data on all procedures and up to 20 diagnoses per admission. Diagnoses and procedures were identified by International Classification of Disease, Ninth Revision (ICD-9) coding in the hospital discharge database. PICC codes used were: 365.69 and TL codes used were: 365.57, 365.58, 365.60, 365.61, 365.63, 365.65, 365.66. DVT codes were: 415.11, 415.12, 415.19, 452, 453.0, 453.1, 453.3, 453.41, 453.42, 453.8, 453.9. Patient diagnoses other than DVT were grouped according to ICD. 9 coding into categories termed Complex Chronic Conditions (CCC) as previously described. To ensure that the DVT event was related to the individual line insertion, patients were excluded if their lines were placed after three days of hospital admissions and if they received more than one line or more than one type of line. A limitation of the data is the lack of information regarding the length of time the catheters remained in place.

Results:

Over the 6 year period of this study, 1449 eligible subjects were identified of whom 29 had DVTs (2%). There were 947 PICC insertions and 502 TL insertions. The mean age of PICC patients was 12±4 years and 56% were male. The mean age of TL patients was 8±6 years and 54% were male. There were 9 (0.95%) DVTs in PICC patients, and 20 (3.9%) DVTs in TL patients. The odds ratio for developing a DVT with a TL was 3.6 (p-value.002; confidence interval 1.6–8.1) which remained significant at 3.8 (p=.001; confidence value 1.7–8.55) when controlling for the presence of CCCs.

Conclusions:

Despite the proliferation in the utilization of PICC in recent years, it appears that they are not associated with a higher risk for DVT than TL, and in fact, the data from this study suggests that TL were associated with more DVT than PICC. Although it is possible that the results could be explained by the fact that TL in general remain in place for a longer period of time than PICC, this study nonetheless provides important reassurance that PICC appear to have relatively low thrombotic potential when compared to TL.

Disclosures:

No relevant conflicts of interest to declare.
The Balance of Gfi-1 and Gfi-1b Maintains the Undifferentiated, Multipotent State of Hematopoietic Stem Cells and Regulates Lineage-Commitment
Authors: Foudi Adlen , Hock Hanno
Published: October 2019

Abstract 1210

Gfi-1 and Gfi-1b are homologous transcriptional repressors that are expressed in hematopoietic stem cells (HSCs). Gfi-1 is crucial for the terminal maturation of neutrophils, and Gfi-1b is critical for erythropoiesis and thrombopoiesis. HSCs give rise to all mature blood lineages through a tightly regulated multistep differentiation process, but the mechanism of their early lineage specification remains largely elusive. Here, we have dissected the role of the Gfi-family factors in HSC maintenance and early lineage-commitment. To this end, we generated conditional targeted alleles for Gfi-1 and Gfi-1b that allowed for time controlled induced disruption of their genes. Acute disruption of Gfi-1 resulted in a rapid, severe decrease of HSCs numbers in the bone marrow and ablated their function in competitive repopulation assays. Surprisingly, and sharply contradicting recent claims to the opposite, acute disruption of Gfi-1b also led to decreased numbers of long-term repopulating HSCs in the bone marrow and decreased fitness in competitive transplantation. After induced, combined disruption of both factors, no HSC and progenitor cells were maintained in the bone marrow for more than 2 weeks. To elucidate the molecular mechanisms of the Gfi-family mediated HSC maintenance we performed global gene expression profiling of Gfi-1−/− and Gfi-1b−/− HSCs. Unexpectedly, both factors regulate highly distinct gene sets involved in differentiation of alternative lineages. Thus, surprisingly, their action in HSCs is not redundant but synergistic. Consistent with this, disruption of individual Gfi-family factors renders HSCs prone to differentiation to specific alternative lineages, while combined disruption is entirely incompatible with HSCs maintenance, in large part due to unchecked differentiation. Together, our data reveal that balanced expression of Gfi-1 and Gfi-1b is required for maintaining the undifferentiated, multipotent state of HSCs, while altering the balance is sufficient for inducing commitment to specific lineages.

Disclosures:

No relevant conflicts of interest to declare.