bluuebunny/crossref_metadata_2025_split huggingface dataset:
Effectiveness of Management of Suspected Deep Vein Thrombosis in General Practice Based on a Clinical Decision Rule Including a Point of Care D-dimer Test.
Authors: Cate-Hoek Arina ten , Toll Diane , van der Velde Eit Frits , Hoes Arno , Buller Harry , Prins Martin 
Published: October 2019

Abstract
Prior studies have evaluated the safety and effectiveness of a diagnostic work up based on combined use of a clinical decision rule and a D-dimer laboratory test in patients with suspected deep vein thrombosis in secondary care facilities. The overall outcome of these studies show that approximately 30% of additional objective evaluation can be avoided without compromising the safety, resulting in failure rates of less than 1%. The objective of our study was to assess the effectiveness of a modified algorithm using a dichotomized clinical decision rule including a point of care D-dimer test in patients suspected of deep vein thrombosis in primary care. The AMUSE study is a prospective cohort study of consecutive patients with clinically suspected acute deep vein thrombosis conducted in 400 general practices coordinated by 3 university affiliated centers in the Netherlands from March 2004 through January 2007. The study population of 1029 patients included 63% women. Patients were categorized as “deep vein thrombosis unlikely” or “deep vein thrombosis likely” using a dichotomized version of a for general practice adapted clinical decision rule based on the Wells rule. Patients classified as unlikely were excluded from further testing. All other patients underwent ultrasonography. Anticoagulants were withheld from patients classified as unlikely. All patients were followed up for 3 months. The main outcome measure was symptomatic or fatal venous thromboembolism (VTE) during 3 months follow-up. Deep vein thrombosis was classified as unlikely in 500 patients (48.6%), all of whom were not treated with anticoagulants; subsequent non fatal VTE occurred in 8 patients (1.6% [95 CI 0.7–3.1%]). In 500 patients categorized as likely, deep vein thrombosis was confirmed in 124 patients (24.8%), 3 patients did not receive objective testing (0.6%) and 373 patients were tested negative (74.6%). Two out of 4 patients not receiving objective testing had confirmed deep vein thrombosis in the 3 month follow-up period. In patients that tested negative 4 non fatal events occurred during the 3 month follow-up (1.1%). The algorithm was completed and allowed a management decision in 97.2% of patients. We conclude that this management strategy is both safe and highly effective in patients with suspected deep vein thrombosis in general practice. Its use is associated with low risk for subsequent fatal and nonfatal VTE and a further increase in the yield of objective diagnostic tests.
Hoxa9 Suppresses Cellular Senescence and Sustains the Development of Leukemic Stem Cells Induced by Fusion Proteins In the Absence of Bmi1.
Authors: Smith Lan-Lan , Yeung Jenny , Zeisig Bernd B , Huijbers Ivo , Popov Nik , Barnes Josephine , Wilson Amanda J , Lugthart Sanne , Delwel Ruud , Gil Jesus , van Lohuizen Maarten , So Chi Wai Eric 
Published: October 2019

Abstract
Abstract 1578
While self renewal is an essential feature for the maintenance of both normal hematopoietic stem cells (HSCs) and leukemic stem cells (LSCs), very little is known about the underlying molecular pathways. Here we report a critical functional interplay between Bmi1 and Hox in establishment of HSCs and LSCs. Using Bmi1-/- bone marrow cells, we observe that leukemia-associated fusion proteins have distinctive Bmi1 requirements. AML1-ETO (AE) and PLZF-RARα (PR) fail to transform Bmi1-/- primary hematopoietic cells, and induce expression of p16/Arf leading to oncogene-induced senescence (OIS). In contrast, MLL-AF9 driving expression of multiple Hox genes can bypass oncogene-induced senescence and exhibits modest Bmi1-dependence for establishment of LSCs, which can induce leukemia upon serial transplants. Since members of Hox genes with proclaimed self-renewal property are specifically up-regulated by MLL fusions in patient samples and our murine models, we asked the question if these Hox genes may partly compensate the functions associated with the loss of Bmi1. To this end, we generated compound Bmi1-/-Hoxa9-/- mice, which have even more compromised hematopoietic stem cell/progenitor compartments than those of Bmi1-/- or Hoxa9-/- mice. Bmi1-/-Hoxa9-/- mice have a greater than eight-fold reduction in the absolute number of Lin-Sca+kit+ (LSK) in the bone marrow as compared to Bmi1-/- mice and a very significant forty-fold reduction for long term hematopoietic stem cells (LT-HSC). More importantly, while MAF9 is able to transform wild type, Bmi1-/- and Hoxa9-/-, it fails to transform Bmi1-/-Hoxa9-/- cells for establishment of LSCs, which can however be resurrected by re-expression of either Bmi1 or Hoxa9, indicating a critical functional interplay between these protein in development of MLL LSCs. Consistent with the known function of Bmi1 in suppressing cellular senescence and the expression of p16/Arf loci, we showed that Hoxa9 alone can also inhibit replicative senescence and Ras-induced senescence in primary human fibroblast. Forced expression of Hoxa9 can suppress p16/Arf expression, as well as cellular senescence induced by AE and PR in Bmi1-/- cells. Together, these results reveal a previously unrecognized functional interplay between Hox and Bmi1 in regulating cell senescence and development of LSCs induced by fusion proteins, which also suggests that synergistic targeting of both molecules may be required for certain LSCs.

Disclosures:
No relevant conflicts of interest to declare.
MixL1 Expression Identifies Putative Hematopoietic Stem Cells In the Murine Allantois.
Authors: Wolfe Adam D , Downs Karen 
Published: October 2019

Abstract
Abstract 1591
MixL1, a paired-type homeodomain transcription factor, is implicated in pre-hematopoietic commitment of stem cell populations. In poorly-differentiated human lymphoma and leukemia lines, MixL1 is inappropriately over-expressed (Drakos et al., Human Pathol 2007:38:500). When constitutively expressed in mice, MixL1 is sufficient to induce Acute Myeloid Leukemia (Glaser et al., PNAS 2006:103:16460). On the basis of these observations, we hypothesize that MixL1 plays an important role in gating the cellular decision to remain in a poorly differentiated and proliferative phase rather than proceeding to definitive hematopoietic stem cell (HSC) identity.
Several years ago, the placenta was shown to be a major site of hematopoiesis (Gekas et al., Dev Cell 2005:8:365; Ottersbach and Dzierzak, Dev Cell 2005:8:377). The placenta is composed of the chorionic disc and the allantois, the latter of which matures into the umbilical component of the placenta. The allantois exhibits definitive hematopoietic potential (Ziegler et al., Development 2006:133:4183; Corbel et al., Dev Biol 2007:301:478), and has recently been demonstrated to contain a core of stem cells referred to as the Allantoic Core Domain, or ACD, where potential placental hematopoietic activity may originate (Downs et al., Dev Dyn 2009:238:532).
Our immediate goal is to evaluate whether MixL1 is expressed in the allantois, and to establish its precise spatiotemporal whereabouts with respect to early markers of hematopoietic cells, such as Runx1 (Chen et al., Nature 2009:457:887). Using immunohistochemistry in conjunction with the LacZ/Runx1 reporter mouse (North et al., Development 1999:126:2563), we have demonstrated that MixL1 is strongly expressed in the blood islands of the yolk sac, as well as in a broad, contiguous posterior domain of the embryo that extends to include the ACD stem cell core of the allantois. This domain does not include Runx1, and MixL1 expression temporally precedes that of Runx1 in the allantois.
As development proceeds, the MixL1 signal becomes most prominent in putative nascent blood cells budding off from the poorly-described blood vessel common to the allantois, yolk sac and dorsal aortae, which we have called the “Vessel of Confluence” (VOC). Here, fetal blood is shuttled into the umbilical cord to the chorion for exchange with the mother. Shortly thereafter, Runx1 begins to appear within VOC, and is co-expressed with MixL1.
These findings provide preliminary evidence that MixL1 is expressed within the allantois, and within nascent blood cells derived from a specific arterial site common to the allantois, yolk sac, and fetus. Moreover, MixL1 expression appears to precede that of Runx1. Thus, MixL1 may identify one of the earliest hematopoietic precursor cell populations thus far known in mammals. Further, these data provide additional evidence that the allantois is a promising model system for the study of definitive hematopoiesis.

Disclosures:
No relevant conflicts of interest to declare.
the Utility of CD200 Expression In the Differentiation of Mantle Cell Lymphoma and B-Cell Chronic Lymphocytic Leukaemia: A Single Centre Experience.
Authors: Bhatnagar Neha , Wright Faith , McAleese Dimpna , McNamara Christopher J 
Published: October 2019

Abstract
Abstract 1673

Introduction:
Mature B-cell lymphoproliferative disorders that present with a leukemic phase may have overlapping morphological and immunophenotypic features. Accurate diagnosis is essential to determine prognosis and therapy. B-cell chronic lymphocytic leukaemia (B-CLL) and mantle cell lymphoma (MCL) both express CD5 but usually have divergent clinical courses. Conventional markers are able to discriminate in many cases but a significant proportion of cases require more detailed analysis. CD200 is a membrane glycoprotein that has been reported to differentiate between B-CLL and MCL. It is expressed on resting and activated T and B cells, but not on NK cells, monocytes, granulocytes or platelets. We prospectively assessed the utility of this antibody in a single centre, servicing haematology units in North London.

Methods:
Over a 12 month period we prospectively assessed the expression of CD200 (Clone MRC OX-104, BD Pharmingen) on neoplastic cells of patients with presenting with mature B-cell lymphoproliferative disorders. EDTA anti-coagulated peripheral blood and bone marrow underwent a lyse/wash technique before being acquired on a BD FACS Canto II flow cytometer equipped with DIVA 6.1.2 software. A lymphoid gating strategy was used to analyse the data and expression of more than 20% of nucleated cells referenced to a negative internal control was considered positive. A diagnosis was made using all available data according to the WHO classification of haematopoietic tumours, including the 5 point CLL score. The predictive value of CD200 expression was subsequently assessed.

Results:
There were 100 patients (58M: 42F) with a median age of 70 years (range 29–92). CD200 was present on the neoplastic cells of all 78 patients with B-CLL; in all 7 MCL patients CD200 was negative; 15 patients had CD5 negative B-cell lymphoproliferative disorder with variable expression of CD200. The specificity of CD200 negativity in the setting of CD5 positive B-cell clone was 100% for MCL. The positive predictive value of CD200 for CLL was 97.5% with a sensitivity of 100% and a Pearson's correlation coefficient of 1.

Conclusion:
Our results confirm CD200 expression in the neoplastic cells of B-CLL patients but not in MCL. This antibody may contribute to the accurate differentiation between B-CLL and MCL in patients presenting with the diagnostic problem of a CD5- positive lymphoproliferative disorder. We propose to add CD200 to our diagnostic B-cell lymphoma panel. Further studies are needed to determine whether CD200 could replace other immunophenotypic strategies currently used in this situation, such as CD23, FMC7 and intensity of CD22/CD79b expression.

Disclosures:
No relevant conflicts of interest to declare.
Vitamin D Triggers Killing of Burkitt Lymphoma Cells by Human Macrophages
Authors: Bruns Heiko , Mougiakakos Dimitrios , Fabri Mario , Pasemann Shirin , Jitschin Regina , Amann Kerstin , Büttner Maike , Mackensen Andreas , Gerbitz Armin 
Published: October 2019

Abstract
Abstract 1035

Introduction:
Distinct macrophage subsets have been linked with either protective or pathogenic roles in cancer. M2 macrophages have been shown to suppress adaptive tumour-specific immune responses and promote tumour growth, while M1 macrophages eliminate neoplastic cells and activate tumour-killing mechanisms. Macrophages isolated from solid tumours (tumor-associated macrophages, TAM) reveal a suppressive and tumor promoting M2-like phenotype. However, it remains unclear which tumoricidal effector mechanisms are compromised in theses immune cells. While infilatration of macrophages is a characteristic morphological hallmark in Burkitts' lymphoma (BL), the phenotype and functional properties of TAM as part of the stroma are poorly understood. In this study we investigated the phenotype of lymphoma associated macrophages (LAM) and the mechanisms by which macrophages are able to modulate the growth of tumor cells.

Methods:
We included 19 patients diagnosed with BL and 20 patients with benign reactive lymphadenopathy as a control group. Phenotypic characterizations of LAM were evaluated by immunohistochemistry and by qPCR in paraffin embedded tissues. To investigate anti lymphoma cytotoxicity of distinct macrophage subsets we generated macrophages from PBMC either by GM-CSF to create the M1 phenotype, or by M-CSF to obtain the M2 phenotype. M1 and M2 macrophages were coincubated them with several BL cell lines and cytotoxicity was analyzed by flow cytometry, qPCR and immunofluorescence.

Results:
First, we could demonstrate that BL infiltrating macrophages displayed an anti-inflammatory M2-phenotype characterized by expression of surface markers such as CD68 and CD163. Second, we identified impaired vitamin D metabolism in LAM and M2 macrophages as demonstrated by low expression of vitamin D receptor and Cyp27B1, and increased expression of Cyp24A1. Third, macrophages revealed a reduced cytotoxic potential towards lymphoma cells which was dependent on the expression of the vitamin D dependent peptide cathelicidin. Finally, we could demonstrate that excess supplementation of calcitriol led to increased cytotoxicity of M1- and M2 macrophages towards BL cells.

Conclusions:
These results suggest a mechanism in which vitamin D is required for innate immunity to overcome the ability of lymphoma cells to evade macrophage-mediated antitumoral responses. The present findings underscore the importance of vitamin D for sustaining innate immunity and imply that the therapeutic activation of the vitamin D pathway may even result in triggering tumoricidal effector mechanisms of LAM.

Disclosures:
No relevant conflicts of interest to declare.
HIV Triggers Interleukin 21-Mediated Induction of Granzyme B-Secreting B Cells with Regulatory and Antiviral Potential
Authors: Kaltenmeier Christof , Gawanbacht Ali , Lindner Stefanie , Beyer Thamara , Haerter Georg , Gruener Beate , Kern Peter , Kirchhoff Frank , Schrezenmeier Hubert , Jahrsdörfer Bernd 
Published: October 2019

Abstract
Abstract 1042
Certain regulatory lymphocyte subpopulations including plasmacytoid dendritic cells and regulatory T cells secrete granzyme B (GrB), thereby suppressing T cell expansion. Recently, we found that B cells can also produce GrB and acquire regulatory potential in response to interleukin (IL-)21. Since HIV has been shown to be associated with elevated serum IL-21 levels, we hypothesized that GrB-expressing B cells may be induced during HIV infection. Here, we show that infection of CD4+ T cells with HIV 1 (NL4-3), but not mock infection, induces strong expression of IL-21 without upregulation of CD40 ligand. Importantly, we further demonstrate that such IL-21+CD40Llow T cells induce GrB in cocultured B cells in an IL-21-dependent fashion, rather than supporting their differentiation into plasma cells. In support of these findings, serum levels of both IL-21 and GrB are significantly higher in HIV-infected patients before HAART as compared to healthy controls. Up to 60% of freshly isolated B cells (36.2% ± 12.9%) from patients infected with HIV, but not B cells isolated from healthy individuals, express GrB. Of note, coculture of HIV-infected CD4+ T cells with GrB+ B cells result in GrB transfer, and strongly suppresses both, proliferation of T cells and viral replication as indicated by significantly reduced p24 levels in coculture supernatants. The observed effects are enhanced by IL-21, and reduced by GrB inhibition. In summary, we demonstrate that HIV infection induces IL-21 in CD4+ T cells, thereby indirectly triggering the development of GrB-secreting B cells with antiretroviral properties. GrB-secreting B cells may play a so far unappreciated role in decelerating HIV expansion, particularly in the early phase of infection. On the other hand, induction of GrB in HIV-specific B cells may interfere with their terminal differentiation into plasma cells, which may explain the lack of an efficient anti-HIV humoral immune response in HIV-infected patients.

Disclosures:
No relevant conflicts of interest to declare.
Inhibition of PI3K/Akt and Erk Signaling Pathways by BCR-ABL Tyrosine Kinase Inhibitors up-Regulates the Immunosuppressive Receptor Osteoactivin in Monocyte-Derived Dendritic Cells
Authors: Gutknecht Michael , Joas Simone , Kanz Lothar , Salih Helmut R , Rittig Susanne M , Gruenebach Frank 
Published: October 2019

Abstract
Abstract 1050
Osteoactivin (GPNMB, DC-HIL) is a type I transmembrane glycoprotein that is expressed in dendritic cells (DC). Osteoactivin/syndecan-4 (SD-4) interaction was previously shown to inhibit T cell activation by antigen-presenting cells. We recently demonstrated that exposure of human peripheral blood monocytes to IL-10 or pharmacological levels of the BCR-ABL tyrosine kinase inhibitors (TKI) imatinib or nilotinib during differentiation into monocyte-derived DC (moDC) causes up-regulation of osteoactivin at the transcript and protein level in vitro (Schwarzbich et al., 2012). Here we aimed to elucidate the molecular mechanisms responsible for osteoactivin up-regulation in moDC upon exposure to IL-10 or TKI (imatinib, nilotinib). moDC were generated from blood monocytes by plastic adherence and exposure to GM-CSF and IL-4. Every second day, starting from the first day of culture, IL-10 or TKI were added to the culture medium. Alternatively, specific inhibitors of PI3K, Akt, Erk and/or C-Raf signal transduction pathways were added to the cell cultures. Cells were harvested on day 7 of culture for immunophenotyping and osteoactivin expression analysis using FACS, western-blotting and real-time qRT-PCR. The expression and activity of signal transduction molecules was monitored by western-blotting and pathway protein arrays. Analysis of moDC function was performed using mixed lymphocyte reactions (MLR). Our in vitro analysis revealed that IL-10 and BCR-ABL TKI inhibit the PI3K/Akt and, to a lesser extent, the Erk pathway in moDC. Specific inhibition of these signal transduction cascades resulted in profound up-regulation of osteoactivin expression in moDC confirming the involvement of PI3K/Akt and Erk in the regulation of osteoactivin expression. In line, TKI treatment and inhibition of PI3K/Akt and Erk comparably affected the phenotype of moDC. Moreover, the inhibition of these signalling cascades resulted in reduced stimulatory capacity of moDC in MLR with allogenic T cells, and this could be restored by addition of blocking osteoactivin antibody. Our data reveal that TKI exert immunosuppressive effects in moDC by interfering with pathways involved in IL-10 receptor signaling. Inhibition of osteoactivin expression or function may thus constitute a promising strategy in combinatory approaches using TKI and DC-based immunotherapy and may enhance the efficacy of immunotherapeutic interventions in cancer patients.

Disclosures:
No relevant conflicts of interest to declare.
Human-Cl Rhfviii Effectively and Safely Prevents Bleeding Episodes in Previously Treated Adult Patients with Severe Haemophilia A
Authors: Knaub Sigurd , Lissitchkov Toshko , Hampton Kingsley , Von Depka Mario , Rangarajan Savita , Hay Charles R.M. , Tuddenham Edward GD , Collins Peter , Holstein Katharina , Huth-Kühne Angela , Pabinger Ingrid , Oldenburg Johannes , Bichler Johann 
Published: October 2019

Abstract
Abstract 1132
The main purpose of this prospective, multi-center, open-label phase 3 study was to assess the efficacy of prophylactic treatment with Human-cl rhFVIII, the first human cell-line derived recombinant FVIII, in previously treated patients (PTPs) with severe haemophilia A.
Patients were to receive 30–40 international units (IU) FVIII of Human-cl rhFVIII per kg every other day for 6 months. Efficacy of preventing and treating bleeds were judged using objective criteria taking the monthly bleeding rate and the number of infusions needed to manage a break-through bleed into account. In-vivo recovery (IVR) was determined at the beginning of the study and after 3 and 6 months. FVIII:C was measured by validated chromogenic (CHR) and one-stage (OS) assays in a central laboratory, which also assigned drug potencies. Inhibitor activity was determined using the Nijmegen modification of the Bethesda assay before the first administration and at defined intervals thereafter. Thirty-two patients between 18 and 75 years of age were enrolled from 11 centres in Europe and treated prophylactically for 6.0±0.9 months (mean ± SD) with a mean prophylactic dose of 32.8 IU/kg. Sixteen patients never bled, 11 patients bled once and 5 more than once. The mean total and spontaneous monthly bleeding rate was 0.188±0.307 and 0.095±0.211, respectively. Efficacy of the prophylactic treatment was “excellent” in all patients for spontaneous BEs and “excellent” or “good” in all patients but one for all types of bleeds. All treatments of bleeds were rated as “excellent” (71.4%) or “good” (28.6%). The IVR at baseline was 2.6±0.5 % per IU/kg for the CHR and 2.2±0.5 % per IU/kg for the OS assay and remained stable during the study. A total of 2921 infusions were given in the study. Human-cl rhFVIII was well tolerated and no patient experienced a related serious adverse event. No FVIII inhibitors were detected.

Conclusion:
The data indicate that Human-cl rh FVIII is safe and efficacious in preventing and treating bleeds in PTPs with severe haemophlia A.

Disclosures:
Knaub: Octapharma AG: Employment. Lissitchkov:Octapharma AG: PI Other. Tuddenham:College London: Consultancy, Employment, Gene therapy for hemophilia A, Gene therapy for hemophilia A Patents & Royalties, Research Funding. Collins:Octapharma AG: Consultancy. Oldenburg:d and e: Baxter, Bayer, Biotest, CSL-Behring, Grifols, Inspiration, NovoNordisk, Octapharma, Pfizer e: Biogen IDec, Swedish Orphan Biovitrum: Honoraria, Research Funding. Bichler:Octapharma AG: Employment.