bluuebunny/crossref_metadata_2025 huggingface dataset:
Thymidylate Synthase (TS) Expression In Diffuse Large B-Cell Lymphoma (DLBCL): Identification of a Potential Therapeutic Target
Authors: Racila Emilian , Syrbu Sergei I , Nasr Michel , Juweid Malik E.
Published: October 2019

Abstract 5107

Purpose:

TS is critical for the de novo synthesis of thymidine needed for tumor cell proliferation and DNA damage repair, whereas thymidine kinase 1 (TK1) is key to the salvage pathway providing thymidine recovered from plasma. While TK1 expression has been previously shown to correlate with proliferation in DLBCL, little is known about TS levels in these neoplasms. We hypothesized that, similarly to TK1, TS expression levels associate with increased proliferation and thus may represent a novel therapeutic target in large cell lymphoma.

Methods:

To address this hypothesis, we determined the expression of TS in lymph node biopsies from 43 DLBCL patients sequentially enrolled in the study with no pre-selection criteria other than the primary histological diagnosis of lymphoma, and then compared it to TK1 expression. In addition, Ki-67 (MIB1) was included as an established marker of cell proliferation. Pearson correlation along with the correlation coefficient and two-tailed P values were calculated to measure the extent of covariation.

Results:

TS expression and both MIB1 and TK1 were significantly correlated with correlation coefficients of 0.43 and 0.65, respectively (95% CI 0.13 to 0.65 and 0.44 to 0.8; P values = 0.006 and <0.0001, respectively). The correlation between TS and MIB1 expression was in fact stronger than between MIB1 and TK1 (P values 0.006 and 0.04, respectively). Mean percent positive cells and standard deviations for MIB1, TS and TK1 were 44.5% (SD=35), 51.8% (SD=33.4), and 44% (SD=28.5), respectively. Despite the general good correlation, however, six patients had relatively high TS levels (defined as % cell staining above the mean for TS) and relatively low TK1 levels (% cell staining below the mean for TK1), while the reverse was true in seven patients. Eight patients had high levels of TK1 and TS with ≥ 70% cells positive for both; seven patients had low levels of both proteins (≤30% of cells stained).

Conclusion and Significance:

This is the first report of TS expression in DLBCL. TS is frequently expressed in DLBCL and there is a significant correlation between TS and TK1 or MIB1 expression. Further studies are warranted to determine the correlation between TS or TK1 expression and patient prognosis in comparison to MIB1. Importantly, the common expression of TS in patients with DLBCL indicates that TS may be a suitable target for TS inhibitors (TSIs) in these neoplasms. Preclinical studies are underway to test this hypothesis.

Disclosures:

No relevant conflicts of interest to declare.
RNAi Identifies Survivin as a Potential Target for Therapy In Pediatric Acute Lymphoblastic Leukemia
Authors: Chang Bill H , Tyner Jeffrey W , Jemal Abdusebur , Thayer Mathew , Druker Brian J
Published: October 2019

Abstract 5108

Background:

Pediatric Acute Lymphoblastic Leukemia (ALL) remains the most common pediatric malignancy. Despite advances in treatment and outcomes, there continue to be subsets of patients that are refractory to standard intensive chemotherapy and hematopoietic stem cell transplant. Therefore, novel gene targets for therapy are needed to further advance treatment for this disease. Survivin, a member of the chromosome passenger complex and inhibitor of apoptosis has been shown to be over-expressed in malignant cells and in relapsed disease. Therefore, survivin may be a potential target for therapy in pediatric ALL. Further, RNAi technology can be used as a rapid functional screen to identify target genes crucial for viability in ALL cell lines and in primary patient samples.

Design/methods:

Pediatric lymphoblastic cell lines and fresh primary mononuclear cells from newly diagnosed patients with ALL were used for all studies. Survivin was silenced by transfecting cells (via electroporation or transductin (IDT)) with siRNA SMARTpools obtained from Dharmacon. Protein expression was identified 48 hours after treatment with siRNA. Viability was measured using a standard methane-thiosulfonate viability assay. Activation of apoptosis was identified using the Guava nexin Annexin V binding assay.

Result:

Survivin is highly expressed in multiple ALL cell lines and in many primary ALL samples. Silencing of survivin expression with siRNA decreases viability and increases apoptosis in ALL cell lines and in primary ALL samples. Decreased protein expression of approximately fifty percent was sufficient to cause an increase in cell death. Finally, inhibition of P53 expression abrogates this phenotype suggesting that the mechanism of cell death induced following survivin silencing involves activation of the P53 pathway.

Conclusion:

Gene silencing with siRNA can be used as a rapid functional screen to identify potential targets for therapy. Implementing this technique identified survivin as a potential target for therapy in pediatric ALL. The results of these experiments will be used as a foundation to develop a comprehensive understanding of the mechanisms of survivin dependence in pediatric ALL. Future studies will also be designed to develop a model system for targeting of survivin in a therapeutically-relevant manner such that these findings can be translated into improved treatments for patients with ALL.

Disclosures:

No relevant conflicts of interest to declare.
Seasonal Variation In the Occurrence of Venous Thromboembolism In Canada
Authors: Kondal Dimple , Tagalakis Vicky , Ciampi Antonio , Kahn Susan
Published: October 2019

Abstract 5120

Background.

The evidence in support of a seasonal variation in the occurrence of venous thromboembolism (VTE) is conflicting and based on studies of mostly small or moderate size, often single centered and moreover, have never included data from Canada which has clearly-defined seasons with wide temperature differences between winter and summer months.

Objectives.

We used discharge data from a population-level hospital registry to assess the occurrence of a seasonal pattern in hospital admissions with VTE (deep vein thrombosis (DVT) or pulmonary embolism (PE)), DVT alone and PE alone in the province of Quebec, Canada.

Methods.

Using data from the province of Quebec's hospital discharge database (Med-Echo) which systematically records information on all hospital admissions in Quebec since 1967, we constructed a retrospective cohort of all individuals who had a first-time discharge diagnosis of DVT or PE between January 1, 1996 and December 31, 2004 and no prior discharge diagnosis for DVT or PE back to 1983. DVT and PE were defined based on the International Classification of Diseases, 9th edition, Clinical Modification. VTE cases were grouped according to season and month of occurrence, and statistical significance of seasonal variation was determined using the Edwards' and Walter & Elwood test.

Results.

The cohort comprised of 45,588 (26,076 (57%) women and 19,512 (43%) men) admitted patients with incident VTE. The mean age was 62.5 years (SD 17.6) and 26,537 (58%) patients had DVT alone, 12,758 (28%) had PE alone and 6,239 (14%) had DVT with PE. Data by season showed a statistically significant difference with the lowest proportion of hospital VTE admissions in summer months (24.1%) and highest in winter (25.9%) months (p<0.0001). Seasonal variation in number of admissions by month was statistically significant for PE alone (p=0.0084; adjusted for total number of monthly hospital admissions for the Quebec province) with peak occurrence in November-December. There was no seasonal variation in monthly VTE (p=0.12) and DVT alone (p=0.87) admissions.

Conclusion.

Our large-scale population study provides evidence that in Quebec, Canada there is a seasonal variation in PE hospital admissions with an annual autumn peak. The underlying pathophysiologyic mechanisms are unknown and deserve further study.

Disclosures:

Tagalakis: Pfizer: Research Funding; Sanofi Aventis: Honoraria. Kahn:Sigvaris: Research Funding; sanofi-aventis: Advisory Board, Research Funding; Boehringer Ingelheim:.
Inherited Thrombophilia In Omani Women with Recurrent Pregnancy Loss
Authors: Farsi Khalil Al , Zadjali Shoaib Al , Falahi Karima Al , Al-Khabori Murtadha K. , Pathare Anil , Alkindi Salam
Published: October 2019

Abstract 5124

Background:

Recurrent pregnancy loss (RPL) is a common clinical problem. Inherited thrombophilia has been reported to be associated with RPL by different groups.

Methods:

We retrospectively analyzed the records of women who had thrombophilia testing for RPL, defined as 2 or more pregnancy losses, between the period of June 2006 and June 2010. The following thrombophilic disorders were included: protein C deficiency (PC), protein S deficiency (PS), anti-thrombin deficiency (AT), activated protein C resistance (APCR) and more recently, as molecular testing became available at our institution, factor V G1691A (FVL), prothrombin G20210A (PTG) and Methyl tetrahydrofolate reductase C677T (MTHRF) mutations. Women were excluded if testing was only done during pregnancy or in the immediate post-partum period.

Results:

A total of 136 women were identified. Median age was 32 (range: 18–44) with a median number of RPL of 3 (range: 2–11). Median number of first trimester losses was 2 (range 0–11). Two women had only second trimester losses and one had only third trimester losses. PS deficiency was identified in 8 women (5.8%), PC deficiency in 3 (2.2%), AT deficiency in 1 (0.7%) and APCR in 1 (0.7%). Of 30 women who had genetic analysis by PCR, 8 had abnormal results (MTHFR: 4 heterozygous, 1 homozygous; FVL: 2 heterozygous and PTG: 1 heterozygous). We did not find any correlation between the number of RPLs and the finding of a positive thrombophilia screen in the overall group or in the group of women who had molecular testing.

Conclusion:

Inherited thrombophilia is not as common in our patient population as described in other groups. However, a prospective study with a control group and a full panel of thrombophilia testing is needed to assess the prevalence and significance of such defects in women with RPL.

Disclosures:

Pathare: Sultan Qaboos University: Employment, Research Funding. Alkindi:Sultan Qaboos University: Employment, Research Funding.
PTEN Regulates Collagen-Induced Platelet Activation
Authors: Weng Zhen , Li Ding , Zhang Lin , Ruan Changgeng , Chen Guo-Qiang , Gartner T. Kent , Liu Junling
Published: October 2019

Abstract 5126

PI3K has been shown to play an important role in collagen-induced platelet activation, but the role(s) of PTEN, a major regulator of the PI3K/Akt signaling pathway, has not been examined in platelets. Here, we report that PTEN−/− mouse blood contains 25% more platelets than PTEN+/+ blood, and that PTEN deficiency significantly shortened the bleeding time, increased the sensitivity of platelets to collagen-induced activation and aggregation, and enhanced phosphorylation of Akt at Ser473 in response to collagen. Furthermore, we found that PP2, and the combination of apyrase, indomethcin+1B5, respectively inhibited collagen-induced aggregation in both PTEN+/+ and PTEN−/− platelets. In contrast, LY294002 (a PI3K inhibitor) prevented the aggregation of PTEN+/+, but not PTEN−/− platelets. Therefore, PTEN apparently regulates collagen-induced platelet activation through PI3K/Akt dependent and independent signaling pathways.

Disclosures:

No relevant conflicts of interest to declare.
Iron Deficiency Anemia and Chronic Renal Insufficiency In An Oncology Population
Authors: Boccia Ralph , Lahue Betsy , Hauser Robert , Dioguardi Jessica
Published: October 2019

Abstract 5152

Anemia in oncology patients is common and may negatively impact cancer treatment outcomes. Iron deficiency is a major contributor to anemia, which can be exacerbated by both underlying conditions and chemotherapeutic agents. Additionally, many oncology patients may have concomitant renal insufficiency or chronic kidney disease (CKD), increasing their susceptibility to iron deficiency. Therefore, recognizing renal insufficiency as well as iron deficiency may improve anemia management in oncology patients.

To determine the proportion of oncology patients with iron deficiency anemia (IDA), potential renal insufficiency and the current utilization of IV iron treatment in these populations, a large electronic medical records database was examined. All patients treated with chemotherapy in a physician services oncology network from January 1, 2008 to August 1, 2010 were selected in order to determine the prevalence of an iron deficiency anemia (IDA) diagnosis (expressed as an ICD-9-CM 280.1-.9), and a diagnosis of CKD (expressed as either from a diagnosis code of CKD (585.1-.6) or estimated GFR <60 mL/min).

Overall, 83,219 patients receiving a chemotherapeutic agent were selected. Of these patients, 36% (30,298) were diagnosed with IDA, 34% of whom were receiving IV iron treatment (9,971/30,298). These patients represented approximately 85% (9971/11,776) of the total IV iron treated population in the dataset.

While only 8% (2,482/30,298) of patients with a diagnosis of IDA also had a diagnosis of CKD. Of the patients that did present with a GFR level, 49% of patients had a GFR level <60 mL/min. However, the true prevalence of CKD in this population was difficult to determine because a significant percentage of patients did not present with a GFR level available in their records.

In this oncology patient population IDA is relatively common, with greater than 30% of patients diagnosed. While only one third of these patients were receiving IV iron treatment in the IDA patient population, it is unknown whether patients are not being treated for their iron deficiency or are being managed on oral iron therapy alone. Given recent controversies around ESA use, guidelines suggest that iron indices should be checked, and IV iron supplementation should be considered in patients receiving ESA therapy. Additionally, only a fraction of patients diagnosed with IDA are also diagnosed with CKD, even though analysis of GFR values suggests that a significant number of patients may have renal insufficiency. These data suggest that identification of patients with renal insufficiency may be suboptimal and that further treatment of IDA in this patient population may be warranted in order to optimize anemia management.

Disclosures:

Boccia: AMAG: Consultancy, Honoraria, Speakers Bureau. Lahue:AMAG: Employment. Hauser:AMAG: Research Funding. Dioguardi:AMAG: Employment.
Thrombocytopenia In Severe Anemia of Iron Deficiency
Authors: Ayalew Getinet D. , Mittal Juhi , Khillan Ratesh , Kim Miriam , Braverman Albert S. , Sidhu Gurinder
Published: October 2019

Abstract 5153

Introduction:

Iron deficiency suppresses hemoglobin synthesis and erythropoiesis, but the resulting anemia is frequently associated with thrombocytosis.

Methods:

The clinical and hematologic data of seven women with severe iron deficiency anemia (IDA) and thrombocytopenia were retrospectively analyzed.

Results:

All patients were African-American women with symptomatic IDA, due to bleeding from uterine fibroids in 6 and from colonic diverticulosis in 1. They were 31–70 years of age, median 38. None had palpable splenomegaly. Hemoglobin ranged from 2.9–5.5, median 4.2 g/dL. MCV ranged from 57–70 fl, median 68. Absolute reticulocyte counts ranged from 19,000 – 23, 000/mm3. The initial serum ferritin ranged from 2 to 42 ng/ml, median 4. Serum iron levels ranged from 10 to 70 mcg/dl with median 30, while iron-binding capacities ranged from 381–426 mcg/dl. Serum erythropoietin (EPO) levels were >2000U/ml in two of the patients. Serum lactic dehydrogenase, bilirubin levels and liver function tests were normal; and Coombs' test negative in all cases. White blood cell counts were normal. The platelet counts ranged from 12 to 103, with a median of 46 × 109/L. Peripheral blood smears showed microcytic hypochromic red blood cells (RBC), with no evidence of platelet clumping. Bone marrow aspiration and biopsy on two patients showed increased numbers of normal megakaryocytes, erythroid hyperplasia and absent iron stores.

Six patients were treated with packed RBC transfusions, and ferrous sulphate 325 mg orally was initiated at presentation in 7. Their thrombocytopenia was not treated with steroids or other agents. Three patients' platelet count reached normal or super-normal levels within 72 hours. Six patients were seen at ≥3 months after presentation, and all had achieved normal platelet counts and hemoglobin.

Conclusions:

These data imply that severe IDA can sometimes cause thrombocytopenia rather than thromobocytosis. We cannot be sure whether these patients' uniform normalization of platelet counts was due to treatment of their anemia by transfusion, or iron therapy. Though bone marrow megakaryocyte numbers were increased in 2 patients, there is no evidence for peripheral platelet destruction. Platelet release from megakaryocytes may have decreased in these patients. Pharmacologic EPO therapy can occasionally cause thrombocytopenia, and high endogenous EPO levels in our patients may have reduce their platelet counts. This conclusion is consistent with their apparent response to transfusion. Though the pathogenesis of IDA-associated thrombocytopenia is not known, our data suggest that the results of anemia and iron deficiency treatment should be evaluated before investigating thrombocytopenia as an independent problem.

Disclosures:

No relevant conflicts of interest to declare.
A T2* MRI Prospective Survey on Heart and Liver Iron In Thalassemia Major Patients Treated with Sequential Deferipron–Desferrioxamine versus Deferasirox
Authors: Pepe Alessia , Rossi Giuseppe , Meloni Antonella , Maria Chiara Dell'Amico , Domenico Giuseppe D'Ascola , Capra Marcello , Filosa Aldo , Cianciulli Paolo , Maggio Aurelio , Salvatori Cristina , Restaino Gennaro , Lombardi Massimo , Borgna-Pignatti Caterina
Published: October 2019

Abstract 5165

Introduction:

Most deaths in thalassemia major (TM) result from cardiac complications due to iron overload. No data are available in literature about possible different changes in cardiac and liver iron in TM patients treated with sequential deferiprone–deferoxamine (DFP-DFO) versus deferasirox (DFX). Magnetic Resonance (MR) is the unique non invasive suitable technique to evaluated quantitatively this issue. The aim of this multi-centre study was to assess prospectively in the clinical practice the efficacy of the DFP-DFO vs DFX in a cohort of TM patients by quantitative MR.

Methods:

Among the first 739 TM patients enrolled in the MIOT (Myocardial Iron Overload in Thalassemia) network, 253 patients performed a MR follow up study at 18 ± 3 months according to the protocol. We evaluated prospectively the 25 patients treated with DFP-DFO versus the 44 patients treated with DFX between the 2 MR scans. Myocardial and liver iron concentrations were measured by T2* multislice multiecho technique.

Results:

The doses of the sequential treatment were DFP 70±14 mg/kg/d for 4 d/w and DFO 42±8 mg/kg/d for 3 d/w, the dose of DFX was 26±6 mg/kg/d. Excellent/good levels of compliance were similar in the 2 groups (DFP-DFO 96% vs DFX 100%; P = 0.36). At baseline the 2 groups were homogeneous for cardiac and liver iron. Among the patients with no significant myocardial iron overload at baseline (global heart T2* 3 20 ms), there were no significant differences between groups to maintain the patients without myocardial iron overload (DFP-DFO 95% vs DFX 96%; P = 1.0). Among the patients with myocardial iron overload at baseline (global heart T2* < 20 ms), only in the DFX group there was a significant improvement in the global heart T2* value (11 ± 5 ms at baseline versus 16 ± 8 at 18 ± 3 months, P = 0.0001) and in the number of segment with a normal T2* value (P = 0.003). The improvement in the global heart T2* was not significantly difference in the DFP-DFO versus the DFX group (mean difference global heart T2* 2.2 ± 4.1 ms versus 4.6 ± 4.8 P = 0.2). The changes in the mean serum ferritin level were not significantly different between groups. In patients with liver iron overload at baseline (liver T2* < 5.1 ms), the change in the liver T2* was not significant between groups (mean difference liver T2* 0.9 ± 2.1 ms vs 2.4 ± 5.2; P = 0.3).

Conclusions:

Prospectively in the clinical setting over 15 months we did not find significant differences on cardiac and liver iron by quantitative MRI in TM patients treated with sequential DFP–DFO versus the TM patients treated with DFX.

Disclosures:

No relevant conflicts of interest to declare.
Precision of Multi-Echo CMR for Myocardial Iron Overload Evaluation Is Dependent From MR Sequence Design
Authors: Meloni Antonella , Positano incenzo , Pepe Alessia , Maria Chiara Dell'Amico , Menichetti Luca , De Marchi Daniele , Favilli Brunella , Peluso Angelo , Spasiano Anna , Landini Luigi , Maggio Aurelio , Lombardi Massimo
Published: October 2019

Abstract 5169

Introduction.

T2* multiecho cardiovascular magnetic resonance (CMR) is largely used to assess iron overload in heart because of the established inverse relationship between the T2* value and the iron concentration in tissues (Wood JC et al, Hemoglobin 2008). The decay of CMR signal is sampled at several echo times (TEs) and the T2* is inferred by fitting the decay curve to an appropriate model (Positano V et al, NMR in Biomed 2007). Our aim was to quantify the reliance on TEs of the expected error in T2* value determination.

Methods.

The Cramer-Rao lower bounds theory (CRLB) was used. CRLB provide a fundamental limit to the accuracy in determination of the T2* value from experimental data in dependence of SNR at signal samples. CRLB were evaluated for a commonly used multi-echo sequence with the first TE equal to the minimum achievable (1.4-2 ms), ΔTE of about 2.3 ms to minimize the fat-water interface artifacts, 10 echoes to assure acquisition in a single breath-hold.

Results.

Percent error in T2* values assessment was lower than 10% in the range of clinical interest, with the exception of very low T2* values. Precision in measurement of low T2* values is strongly dependent on the value of the first TE, that is limited by the used scanner. T2* values greater than 1.8 ms and 1.5 ms can be assessed with an error below 20% using a first TE of 2 ms and 1.5 ms, respectively (see Figure).

Conclusions.

T2* multiecho sequences used in clinical practice assure an acceptable precision for T2* values ≥ 2 ms, depending from the used hardware. This limit includes almost all patients with hemochromatosis or hemosiderosis in country where the patients can be well managed. For patients with very high myocardial iron overload sequences with lower minimum echo time and/or lower echoes interval may be useful.

Disclosures:

No relevant conflicts of interest to declare.
Study of Two Subjects with Absence of Severe Anemia with Homozygous β0 Thalassemia Due to Disrupted γ-to-β Switch
Authors: Yoon Donghoon , Kim Soo Jin , Hickman Kimberly , Hussey Dottie , Prchal Josef T.
Published: October 2019

Abstract 5172

Sickle cell disease (SCD) and β thalassemia due to defects of the HBB (β globin gene) are among the most common inherited genetic disorders. At birth, there is a switch of γ globin transcription to β and d, with replacement of HbF by HbA and HbA2 virtually completed by six months of age. At that time, serious inherited disorders of the β gene, such as sickle cell disease and Cooley's anemia (homozygosity for β0 thalassemia mutations), become clinically apparent. Cooley's anemia is a life-threatening disorder wherein, in most patients, chronic transfusions or bone marrow transplantations are needed to sustain life. Rare patients with homozygosity or compound heterozygosity for β0 have no or only mild anemia. These patients maintain a high level of γ globin synthesis, apparently from a disrupted γ-to-β switch, thus attenuating their disease state. Recent work has demonstrated that BCL11A plays an important role in the suppression of γ-globin expression, as do polymorphisms of the gene that remain to be fully elucidated at a functional level. We recruited two unrelated subjects with homozygous β0 thalassemia mutations with no or only mild anemia (Patient #1, IVS2+1 G>A; Hb 14.2 Gm%; 97.2% HbF, 2.8% HbA2, Patient #2, IVS 2 G-T; Hb 11.2 Gm%; 92/5% HbF, 6.8% HbA2, 0.7% HbA). We sequenced transcripts and genomic loci of BCL11A from these patients. No mutations or splicing variants on transcripts were found. However, when the ≂f102 kb of genomic material from patient #1 was sequenced, 5 single nucleotide changes at intron II were found (2 known and 3 previously unpublished), while no genomic changes were found in patient #2. We then performed in vitro erythroid expansion from peripheral blood utilizing high erythropoietin concentrations and analyzed the cell proliferation and expression of globin and BCL11A genes. Interestingly, detectable amounts of β-globin transcripts were present in both patients during expansion, although protein levels were not detectable by the conventional HPLC method, probably due to limited sensitivity of this assay. Patient #1 showed mild in vitro induction of β-globin expression, which is lower than the control group, but no apparent cell proliferation. Patient #2 showed no induction of β-globin expression and hyperproliferation at a later stage of expansion (See Figure); however, the levels of BCL11A and γ-globin transcripts were indistinguishable from controls.

Although we were unable to detect any abnormality of the BCL11A transcript as a cause of high fetal hemoglobin expression in these patients, we cannot rule out the possibility that the intronic mutations in patient #1 may interfere with BCL11A gene translation, perhaps by interference with non-coding RNA. The potential molecular mechanism of γ-to-β switch is being explored by gene expression profiling and microRNA analyses.

Disclosures:

No relevant conflicts of interest to declare.
Differential Modulation of Cytokines by VEGF In End Stage Renal Disease
Authors: Bansal Vinod , Hoppensteadt Debra , Litinas Evangelos , Thethi Indermohan , Fareed Jawed
Published: October 2019

Abstract 5187

Vascular endothelial growth factor (VEGF) mediates nitric oxide dependent angiogenic effects contribute to the inflammatory responses. VEGF is upregulated in ESRD and contributes to the cardiovascular dysfunction in ESRD. VEGF is also upregulated by erythropoietin which is commonly used in the management of ESRD patients. With the availability of BioChip Array Technology, a multiparametric screening of inflammatory mediators/cytokines can be measured and relevance of VEGF can be established. Utilizing the Randox Biochip technology, we profiled the plasma samples from 53 ESRD patients to determine their relevance to VEGF levels. Pre-dialysis samples from 53 male and female ESRD patients on maintenance hemodialysis were analyzed using a cytokine biochip for VEGF, IL2, IL4, IL6, IL8, IFNG1, TNF, IL1, MCP-1 and EGF. In addition, NO, asymmetric dimethyl arginine (ADMA), serotonin and functional microparticles levels were also measured. A group of 50 normal healthy males and females constituted the control group. The circulating levels of VEFG and mediators were compared with the normals. In addition, VEGF levels were compared with the other mediators. In comparison to the normals, ESRD patients exhibited decreased levels of IL4, IL8, TNF and EGF, which ranged from 20–210%. Increased levels of IL2, IL6, IL10, IFNG1, IL1 and MCP-1 were noted (20-350%). NO levels were also increased, however ADMA remained unchanged. Both the serotonin and microparticle levels were also elevated in comparison to the normals (p>0.05) VEGF levels showed wide variation, however on a cumulative basis a 30–210% increase in VEGF was noted. Except for the MCP-1, the correlation of VEGF with other inflammatory markers was relatively poor (r2=<0.25). These results show that ESRD patients can be profiled for various inflammatory mediators. VEGF levels were markedly increased, however only MCP-1 levels correlated well with the increase in VEGF levels. Additional studies are warranted to clarify the regulatory role of VEGF and other mediators on inflammatory cytokines and cellular activation in ESRD.

Disclosures:

No relevant conflicts of interest to declare.
Nox2 siRNA Transfection Down Regulates VEGF Induced-Angiogenesis on Endothelial Progenitor Cells Which Are Derived From HCB
Authors: Yoo Eun-Sun , Mun Yeung-Chul , Nam Eun Mi , Lee Kyoung Eun , Huh Jung Won , Lee Soon Nam , Cho Jung Yeon , Ahn Jee Young , Lee Solip , Seong Chu-Myong
Published: October 2019

Abstract 5188

Background:

Reactive oxygen species (ROS) such as superoxide and H2O2 have roles signaling for molecules on angiogenesis. NADPH oxidase Nox2 (gp91phox) is a major source of ROS. Previous, we had found that Nox2-based NADPH oxidase (gp91phox)-induced ROS may play important roles on EPCs migration and proliferation by VEGF (Blood. 2009;114:Abstract 1445). In the present study, we studied the impact of down-regulation of Nox2 on intracellular ROS level, proliferation, transmigration, and in vitro tube formation of HCB derived EPCs via Nox2 siRNA transfection.

Methods:

Outgrowing endothelial progenitor cells were established from mononuclear cells of human cord blood (Yoo et al, Stem cells. 2003;21:228-235) using EGM-2 media in a fibronectin-coated dish. EPCs were transfected with HiPerFection transfection reagent plus Nox2 siRNA or non-targeting control siRNA and cultured for 5 hours. 100ng/ml of VEGF was added to the transfected cells and cultured for overnight. Expression of Nox2 and pERK in the Nox2 siRNA transfected EPCs were detected by western blot analysis. Intracellular ROS level was analyzed by staining with 2, 7-dichlrodihydro-fluorescein-diacetate (H2DCF-DA) and flow cytometry. Transmigration against VEGF was performed using transwell system (Costar) and in vitro tube formation was assayed using In vitro angiogenesis kit (Chemicon).

Results:

Intracellular ROS level was increased during endothelial progenitor cell culture which were derived from HCB by VEGF treatment. Proliferation, in vitro tube formation matrigel assay and migration assay on endothelial progenitor cells using VEGF were decreased with Nox2 siRNA transfection when compared with that of control group. In western blot data, Nox2-based NADPH oxidase (gp91phox) was increased by VEGF and decreased by Nox2 siRNA transfection. VEGF induced pERK expression was also decreased by Nox2 siRNA transfection as well.

Conclusions:

Based on our studies, Nox2-based NADPH oxidase (gp91phox)-induced ROS may have important roles on proliferation in HCB induced EPCs by VEGF stimulation. Furthermore, Nox2 siRNA transfection into HCB derived EPC down-regulated intracellular ROS production and pERK expression. Our data may be useful finding the new therapeutic targets for ischemic heart and ischemic limb diseases by manipulating the level of intracellular ROS via Nox2.

Disclosures:

No relevant conflicts of interest to declare.
Genome-Wide Array-Based Methylation Profiling Reveals Preferential Methylation of Homeobox Transcription Factor Genes In Mantle Cell Lymphoma and Pro-Apoptotic Genes In Chronic Lymphocytic Leukemia
Authors: Halldorsdottir Anna M , Kanduri Meena , Marincevic Millaray , Göransson Hanna , Isaksson Anders , Mansouri Mahmoud , Stamatopoulos Kostas , Sander Birgitta , Ehrencrona Hans , Rosenquist Richard
Published: October 2019

Abstract 536

Introduction:

Mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL) are B-cell malignancies of different postulated origin, genetics, clinical presentation and prognosis. Several studies have reported that both MCL and CLL individually exhibit aberrant methylation in comparison to normal B-cells. However, a comprehensive comparison of the methylation profiles of these two B-cell disorders has not been performed yet. This strategy has the potential to identify cellular pathways and genes that are specifically targeted in each disease.

Methods:

We applied the genome-wide Illumina Infinium HumanMethylation27 BeadChip array (Illumina, San Diego, USA) which measures methylation levels at 27,578 CpG dinucleotides covering 14,495 genes, to compare the methylation profiles in: (i) 20 MCL cases; and, (ii) 30 CLL cases, 15 each with unmutated stereotyped subset #1 (IGHV1-5-7/IGKV1(D)-39) B cell receptors (BCRs) or mutated stereotyped subset #4 (IGHV4-34/IGKV2-30) BCRs, where these two subsets represent prototypes of unmutated and mutated CLL. The methylation status for each detected CpG site ranged between 0.1 (completely unmethylated) to 1 (completely methylated).

Results:

As expected, major differences in methylation patterns between MCL and CLL were observed. When the methylation profiles of the two entities were compared, 51 genes were identified as differentially methylated in all comparisons (MCL versus both CLL subsets combined and each subset separately). Among the 19 genes highly methylated in MCL were six (32%) homeobox or homeodomain-containing transcription factors (e.g. POU4F1, PITX3), whereas genes enhancing cell proliferation and tumor progression such as MERTK and CAMP were hypomethylated in MCL. Of the 32 genes hypermethylated in CLL were six pro-apoptotic genes, including DYRK2 and CYFIP2, the tumor suppressor PRDM2 and the cell cycle regulator CCND1.

Conclusions:

We report for the first time disease-biased methylation profiles for different functional classes of genes in MCL or CLL. Homeobox genes were highly methylated in MCL, whereas CLL was characterized by methylation of apoptosis-related genes. The identified differences in global methylation profiles between MCL and CLL may assist in unfolding distinct epigenetic silencing mechanisms involved in the pathogenesis of these B-cell malignancies.

Disclosures:

No relevant conflicts of interest to declare.
A Two-Year Prospective Study on Bleeding Tendency In 105 Patients with Type 2 A and M Von Willebrand Disease
Authors: Castaman Giancarlo , Federici Augusto B. , Baronciani Luciano , Mannucci Pier M. , Rodeghiero Francesco
Published: October 2019

Abstract 543

Background:

Type 2 A and 2 M von Willebrand Disease (VWD) are both characterized by the presence of a dysfunctional von Willebrand factor (VWF) and a variable bleeding tendency. The bleeding incidence and possible clinical differences between these two types have never been investigated prospectively in a large number of patients.

Aims and design of the study:

To investigate clinical history and determinants of bleeding in a cohort of patients (pts) with type 2 A and M VWD characterized by mutations over 24 months of follow-up.

Patients and Methods:

10 families with type 2 A VWD (47 patients) and 15 with type 2 M (58 patients) with type 2 M VWD were diagnosed according to the recommendations of the ISTH-SSC subcommittee on VWF and prospectively followed-up from April 2007 to March 2009. Bleeding score (BS) was calculated in all the patients at enrollment by the administration of the same questionnaire tested in type 1 VWD. In case of VWF:RCo levels < 10 U/dL, all patients were tested with a more sensitive ELISA assay to calculate ratios between VWF:RCo and VWF:Ag. VWF mutations were identified in all the patients.

Results:

The 105 patients were characterized by the following mutations: type 2 A R202W/R1583Q 1 pt, S1506L 11 pts, S1543F 1 pt, R1596W 3 pts, V1607D 8 pts, I1628T 1 pt, G1629R 1 pt, G1631D 9 pts, V1665E 8 pts, Q2520P 2 pts, multiple changes 1 pt. Type 2 M: L1278P 6 pts, R1315L 3 pts, R1315C 9 pts, Y1321C 9 pts, L1361W 4 pts, R1374H 23 pts, C1927/c.8155+6C>T 1 pt, c.3831del-3 3 pts. The table summarizes the main demographic and laboratory characteristics. All the patients had a VWF:RCo/VWF:Ag ratio <0.6. The mean BS and VWF:Ag were significantly higher in type 2 A (P < 0.01). No correlation between VWF:RCo levels and the severity of BS was observed.

During follow-up, the bleeding episodes in patients with type 2 M and R1374H or R1315C mutations were very few and mild while they were recurrent and severe in those with type 2 A and V1665E or G1631D mutations, the only laboratory difference being the lack of high molecular weight multimers in type 2 A VWD. Furthermore, 44 gastrointestinal bleeding episodes occurred in 15 patients with type 2 A (range 1–7) compared to 8 episodes in 2 patients with type 2 M (range 2–6). Older age was strongly related to the risk of recurrence of this type of bleeding.

Conclusions:

Bleeding tendency in type 2 A VWD is higher than that of type 2 M VWD and it is not related to the reduction of FVIII:C and VWF activity. The risk of gastrointestinal bleeding is greater in type 2 A and might be related to the lack of high molecular weight multimers and older age. Further analysis are required to evaluate the definite impact of some mutations on the different bleeding tendency.

Disclosures:

No relevant conflicts of interest to declare.
Antigen-Specific Transfer of Functional Programmed Death Ligand 1 From Antigen Presenting Cells Onto Human CD8+ T Cells Via Trogocytosis
Authors: Gary Regina , Voelkl Simon , Palmisano Ralf , Mackensen Andreas
Published: October 2019

Abstract 584

Specific T-cell responses are initiated by T-cell receptor (TCR) recognition of peptide-MHC-complexes on antigen presenting cells (APCs). Upon specific interaction of T cells with APCs T cells capture membrane fragments and surface molecules of APCs in a process termed trogocytosis. Exchange of membrane molecules/antigens between immune cells has been observed for a long time, but the mechanisms and functional consequences of these transfers remain unclear. Here, we demonstrate that human antigen-specific CD8+ T cells do acquire the co-inhibitory molecule programmed death ligand 1 (PD-L1) from mature monocyte-derived dendritic cells (mDC) and tumor cells in an antigen-specific manner. The kinetics of PD-L1 transfer revealed a maximal PD-L1 expression on antigen-specific T cells within 3–4 hours after co-incubation with antigen-pulsed APCs, being detectable up to 72 hours. Antigen-pulsed immature DCs were less effective in transfering surface molecules such as PD-L1 onto CD8+ T cells after antigen-specific recognition. Using a transwell system we could show that the acquisition of PD-L1 requires cell-cell contact. Furthermore, PD-L1 cannot be acquired by T cells from a lysate of mDCs. The transfer process is impaired after pretreatment of T cells with concanamycin A, a specific inhibitor of vacuolar ATPases, playing an important role in membrane trafficking. Moreover, fixation of DCs with glutaraldehyde completely abrogated the acquisition of PD-L1 on T cells suggesting that an active interaction between APCs and T cells is required for trogocytosis. Of importance, CD8+ T cells which acquired PD-L1 complexes, were able to induce apoptosis of neighbouring PD-1 expressing CD8+ T cells, that could be completely blocked by an anti-PD-L1 antibody. In summary our data demonstrate for the first time that human antigen-specific CD8+ T cells take up functionally active PD-L1 from APCs in an antigen-specific fashion, leading to apoptosis of PD-1 expressing T cells. The transfer of functionally active co-inhibitory molecules from APCs onto human CD8+ T cells may serve to limit clonal expansion of antigen-specific T-cell responses but may also play a major role for T-cell exhaustion in chronic infection and tumor immunosurveillance.

Disclosures:

No relevant conflicts of interest to declare.
Chromatin Boundaries Require the Functional Cooperation Between hSET1 and NURF Complexes
Authors: Li Xingguo , Wang Shaohua , Li Ying , Qiu Yi , Huang Suming
Published: October 2019

Abstract 646

Chromatin modification and remodeling activities play a central role in organizing nuclear functions in eukaryotic genome. Chromatin domains with characteristic epigenetic marks are organized by chromatin insulator. The chicken b-globin insulator, 5′HS4, is an excellent model system to study how insulator maintains gene function and prevents the encroachment of repressive heterochromatin. We showed previously that USF1/2 bound 5′HS4 insulator mediates chromatin barrier activity by recruiting and organizing active histone modifications in the chicken b-globin locus. However, it is not clear how insulator establishes such a barrier. To further understand the physical function of USF1, we purified the USF1-associated protein complexes. We found that USF1 forms a multiprotein complex with hSET1 and NURF exhibiting histone H3K4 methyltransferase and ATP-dependent chromatin remodeling activities, respectively. Both hSET1 and NURF complexes are specifically recruited to the 5′HS4 insulator by USF1 to retain the active chromatin structure. Knock-down of BPTF, a component of NURF complex, resulted in a rapid loss of barrier activity that protects a transgene from chromatin silencing. The loss of barrier by the BPTF knock-down is accompanied by an alternation of nucleosome positioning that expands the nucleosome to the nucleosome free linker region in the HS4 insulator site and increases repressive H3K9 dimethylation. Furthermore, the suppression of hSET1, a H3K4 methyltransferase, leads to not only a loss of barrier activity, but also a loss of H3K4 dimethylation and H3K9/K14 acetylation, as well as the recruitment of BPTF at chromatin insulator sites. Thus, our data reveal a molecular mechanism in which histone modifying enzyme hSET1 and chromatin remodeling complex NURF collaborate together to maintain the 5′HS4 chromatin barrier activity and to prevent the encroachment of adjacent heterochromatin.

Disclosures:

No relevant conflicts of interest to declare.
Crosstalk Between Inflammation and Thrombosis: The Surprising Role of Extracellular Histones
Authors: Xu Jun , Lupu Florea , Esmon Charles T.
Published: October 2019

Abstract MRG-1

Activated protein C (APC) is effective in treating septic patients who have already experienced multiorgan failure. The mechanisms responsible for the efficacy remain unclear. Macrophages were activated with endotoxin and gamma interferon, the supernatant harvested and subjected to incubation with activated protein C. The toxicity of the supernatant toward endothelium was blocked by APC incubation. APC incubation generated 3 new peptides which sequence determination demonstrated were derived from histones including H3 and H4. Histones incubated with endothelium were cytotoxic. APC cleaved and eliminated this cytotoxicity. Histones were identified in the circulation of mice challenged with endotoxin, baboons challenged with E. coli and some septic human patients. In mice, blocking the histone toxicity with a monoclonal antibody effectively reduced endotoxin-mediated death. Histone injection resulted in death with pathologies resembling those of sepsis including fibrin deposition and neutrophil infiltration. APC could also prevent death in a histone-mediated model. The histones elicit an inflammatory response, suggesting some recognition by the immune system. Using 293 cells expressing individual toll receptor reporter constructs, we found that the histones activated toll 2 and toll 4. Mice with either of these tolls deleted were resistant to histone-mediated death. This data, coupled with earlier studies demonstrating that histones can kill bacteria, suggest that histones play a role in innate immunity but are also toxic. The results suggest that the inflammatory response in trauma may be triggered by the cellular destruction that releases histones and further suggest that histone antagonists might be an effective mechanism to block progressive organ failure.

Disclosures:

No relevant conflicts of interest to declare.
A Dual Proto-Oncogenic and Tumor Suppressive Role of LRF/POKEMON In Hemopoietic Malignancies through Control of Cell Fate Decision
Authors: Pandolfi Pier Paolo
Published: October 2019

Abstract SCI-14

LRF (Leukemia/lymphoma-related factor, also known as POKEMON) is a member of the POZ and Kruppel (POK) family of transcription factors. LRF has been shown to play an essential role in embryonic development and to act as a master regulator of cellular differentiation in virtually any tissue where it is found expressed, including the hemopoietic compartment. As we will discuss, LRF inactivation in the mouse blocks cellular differentiation in both myeloid/erythroid and lymphoid compartments. On the other hand, LRF has been shown to possess a potent proto-oncogenic activity both in vitro and in vivo. In fact, LRF itself can transform primary cells in combination with known oncogenes and is also essential for cellular transformation of mouse embryonic fibroblasts. In addition, overexpression of LRF in immature B and T progenitor cells in vivo in the mouse lead to lethal precursor T-cell lymphoblastic lymphoma/leukemia. In agreement with this notion, LRF is aberrantly expressed in a variety of human cancers, including diffuse large B cell and follicular lymphomas, but also ovarian and breast cancers. Further, the LRF gene is found amplified in a subset of non-small cell lung cancers (NSCLCs), illustrating a direct role in human cancer. However, we speculated that due to the key role of LRF in cell fate decisions, LRF/POKEMON loss could also contribute to tumorigenesis by blocking cellular differentiation. We will discuss provocative in vivo data in support of the notion that LRF/POKEMON can indeed act as a bona fide tumor suppressor representing a compelling example of two-faced cancer genes.

Disclosures:

No relevant conflicts of interest to declare.
Does Declining Mitochondrial NAD+ and Unscheduled Opening of Mitochondrial Transition Pore Promote Mammalian Aging?
Authors: Sinclair David , Hafner Angela V. , Gomes Ana , Liang Jing , Cen Yana , Lanza Ian , Nair Sree , Sauve Anthony , Rozenzweig Anthony
Published: October 2019

Abstract SCI-3

The processes that lead to the susceptibility of post-mitotic tissues to diseases of aging are poorly understood, but mitochondria are considered a key regulator. SIRT3 is mitochondrial sirtuin, a member a family of NAD+-dependent deacetylases. We have previously shown that increased levels of NAD+ in mitochondria promote cell survival in a SIRT3-dependent manner, but how this protection is mediated is unknown. Here we show that mitochondrial NAD+ levels decline dramatically with age and that SIRT3 is a critical component and regulator of the mitochondrial transition pore (MTP). Mice lacking SIRT3 exhibit hyperacetylation of the MTP with age, and as a result, develop multiple defects in post-mitotic tissues as they age, including mitochondrial defects, susceptibility to cardiac failure, and learning and memory deficits. In a mouse model of Alzheimer's disease (AD), SIRT3 knockout mice show striking phenotypes including small size, kyphosis, and a markedly reduced lifespan. Similar to mice, human brain samples show that acetylation of the MTP increases with age and with AD. These findings indicate that declining NAD levels with age lead to memory deficits and accelerate age-related diseases in post-mitotic tissues due to a decline in SIRT3 activity and unscheduled opening of the MTP.

Disclosures:

Sinclair: GlaxoSmithKline: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.