bluuebunny/crossref_metadata_2025_split huggingface dataset:
New Mutation in Chromosome 9 Associated with Familial Autosomal Recessive Macrothrombocytopenia
Authors: Revel-Vilk Shoshana , Shai Ela , Gashem Nivin , Hyam Esti , Spectre Galia , Elpeleg Orly , Varon David 
Published: October 2019

Abstract
We report a family with new autosomal recessive disorder associated with macrothrombocytopenia which is different from all reported hereditary macrothrombocytopenia.
Case report: The affected family members report mild bruising and epistaxis. The females’ family members report, in addition, severe menorrhagia and recurrent episodes of hemorrhagic corpus luteum. Two family members with macrothrombocytopenia and two without macrothrombocytopenia report hearing loss and cataracts.
Methods: The study protocol was approved by our local Ethics committee and written consent was obtained to prepare and store DNA. Platelet morphology, FACS analysis, protein analysis and genome-wide association studies were used to characterize the disease and locate the genetic abnormality.
Results: In a consanguineous family from Arab origin we identified 5 affected individuals (3 males and 2 females from three families) with platelet count ranging around 4-20 x109/L. Their shared grandparent died of post trauma bleeding (no data on his platelet count). Their parents have normal platelet count and platelet morphology. Blood film of effected family members show macrothrombocytopenia with occasional giant platelets. By flow cytometry CD41 and CD42b expressions were around 60% in effected family members and CD62 expression was around 20-30% in resting and thrombin-activated platelets (Figure 1). Immunoflurescence of platelets shows normal distribution of non-muscular mysoin IIA, GPIbIX, dense granules and lysosome markers Lamp-1, Lamp-2 and the dense granule CD63. Platelets diffusely stained for P-selectin and stained very low for VWF and thrombospondin. GPIIbIIIa staining revealed a ring distribution at the outer cell membrane (Prof. Andreas Greinacher, Greifswald, Germany). Transmission electron microscopy imaging shows abnormal distribution of platelet granules, with suspected protein accumulation in the cytoplasm (Figure 2). Protein analysis of platelet extracts by SDS-PAGE separation shows different band patterns in the effected individuals compared to controls (Figure 3). Mass spectrometry analysis of a representative band identified five candidate proteins that their over-expression may cause the defect in platelets organelles distribution and the defect of their function. Through whole genome analysis three large homogenous regions were found. To further narrow the search SNP analysis was added between two effected members. The search was narrowed to an area in chromosome 9p and centromere 9q with 201 candidate genes.
Discussion: We present a large consanguineous family with previously non reported autosomal recessive macrothrombocytopenia. The functional and morphological pattern show dual platelet population. Further work is warranted.
Figure 1 Figure 1. Figure 2 Figure 2. Figure 3 Figure 3.

Disclosures
No relevant conflicts of interest to declare.
Carboxypeptidase B2 Is Protective in a Mouse Model of Shiga Toxin-Induced Hemolytic Uremic Syndrome
Authors: Nishimura Toshihiko , Morser John , Shao Zhifei , Leung Lawrence L. 
Published: October 2019

Abstract
Hemolytic uremic syndrome (HUS) is characterized by a triad of microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure. The most common cause of HUS is Shiga toxin (STX)-producing E. coli, and eculizumab, a monoclonal antibody against complement C5, has shown clinical efficacy in some patients. Carboxypeptidase B2 (CPB2) is a metalloprotease activated by the thrombin/thrombomodulin complex that inactivates a number of inflammatory mediators, including complement C3a, and C5a by removing their C-terminal arginine. We hypothesized that in a murine model of STX-induced HUS, Cpb2-/- mice would have exacerbated disease compared to wild type (WT) mice due to excessive C3a and/or C5a in the absence of CPB2. A mouse model of STX-induced HUS was established by giving STX and LPS toxins intraperitoneally. Cpb2-/- mice had worse survival than WT (37% survival vs. 87% at 48h, p=0.0156). At 48h, severe thrombocytopenia developed in both WT and Cpb2-/- mice (WT: 0.096x106/μL; Cpb2-/-: 0.054x106/μL) compared to controls (1.2x106/μL; p>0.0001 vs. either WT or Cpb2-/-), with Cpb2-/- mice showing worse thrombocytopenia. Renal insufficiency was worse in Cpb2-/- mice than WT mice (BUN at 48h: 85 mg/dL vs. 37 mg/dL, p=0.0074; creatinine: 1.33 mg/dL vs. 0.23 mg/dL; p=0.0112, for Cpb2-/- and WT mice respectively, compared with normal baseline BUN and creatinine of 19 mg/dL and 0.1 mg/dL). Cpb2-/- mice developed worse anemia than WT (hemoglobin 9.8 g/dL vs. 12.4 g/dL, p=0.001 in Cpb2-/- vs. WT mice respectively). At 48h, liver function was worse in Cpb2-/- mice than WT mice, while plasma LDH was increased in Cpb2-/- mice more than WT mice. Using a standardized health score, the Cpb2-/- mice were worse than WT mice at all time points. Thus this model recapitulates STX-induced HUS with the Cpb2-/- mice having worse disease than WT. If the animals were treated with STX alone, there were no deaths in either genotype at 48h and only 37.5% mortality in Cpb2-/- mice by 60h compared with no deaths in WT mice. BUN, creatinine, liver enzymes and LDH were increased in both genotypes treated with STX alone compared to untreated mice, but there was no significant difference between the genotypes. Treatment with LPS alone caused thrombocytopenia in both WT and Cpb2-/- mice and LDH, BUN and creatinine levels were higher in Cpb2-/- mice than in WT mice, but there was no death at 48h and no drop in hemoglobin. Thus while either STX alone or LPS alone caused pathological conditions in the mice, the typical triad of HUS was only present when STX and LPS were given in combination. The Cpb2-/- mice had worse disease than WT mice consistent with our hypothesis on the role of CPB2 in inactivating C3a and/or C5a in STX-induced HUS. The potential efficacy of C3a and/or C5a blockade and anti-thrombotic agents will be tested in this model.

Disclosures
No relevant conflicts of interest to declare.
Resistance to Thrombomodulin but Not to Activated Protein C in Cirrhotic Patients
Authors: Sinegre Thomas , Abergel Armand , Duron Cedric , Berger Marc G , Lebreton Aurélien 
Published: October 2019

Abstract
Introduction: Cirrhotic patients have an impairment of haemostasis. They are exposed to both thrombotic and bleeding events. Global coagulation tests as thrombin generation assays (TGA) could be informative to evaluate the coagulation step in this context. When the protein C (PC) pathway is sensibilised using thrombomodulin (TM), a « resistance » to the PC pathway appears, increasing with the severity of the hepatopathy. Nevertheless, patients with cirrhosis have a decrease protein C level which is probably involved in this « resistance ». The aim of this study was to compare the TGA with thrombomodulin or activated PC (aPC) to by-pass the PC defect.
Materials and Methods: Patients prospectively included were confirmed cirrhotic patients (prothrombin time <70% and/or liver dysmorphia and/or fibroscan> 20 kPa and/or histology and/or association of portal hypertension and liver failure). Patients were exclusively male, free of hepatocellular carcinoma and were not anticoagulated. Patients with on-going infection or inflammatory complication were excluded. None of them had a thromboembolic event or a familial history of thromboembolism.
TGA were performed in the presence/absence of TM and in the presence/absence of aPC, with previously determined concentrations inhibiting 50% of endogeneous thrombin potential (ETP) of healthy plasmas. Results were expressed as ratios with/without TM or with/without aPC. Statistical analysis was based on Kruskal-Wallis test and Dunn's post test. All tests were performed in triplicate. The study met all ethical autorizations.
Results: We enrolled 15 Child-Pugh A, patients 11 Child-Pugh B, 10 Child-Pugh C and 14 healthy controls. Comparatively to controls, PC, protein S (PS) and FVIII levels were significantly decreased in cirrhotic patients. ETP ratio with TM were statistically different between controls vs Child-Pugh B (p<0.05) and controls vs Child-Pugh C (p<0.01). Peak height ratio with TM were statistically different between controls vs Child-Pugh A (p<0.05), controls vs Child-Pugh B (p<0.01) and controls vs Child-Pugh C (p<0.01). All these significant differences disappear when we used aPC instead of TM, suggesting that the « resistance » to the soluble TM is due, at least partially, to the acquired deficiency in PC.
Discussion and Conclusions: The main finding of this study is that the acquired protein C deficiency actively participate to the « resistance » to soluble thrombomodulin in cirrhotic patients. These data are in line with studies of the Tripodi group who have also demonstrated a participation of the protein C deficiency in the resistance to thrombomodulin and Protac. Further studies are needed to evaluate the association between this biological phenotype and thrombotic events.

Disclosures
No relevant conflicts of interest to declare.
Notch Signaling By Either Notch1 or Notch2 Mediates Expansion of AGM-Derived Long-Term HSC Populations in Vitro
Authors: Hadland Brandon K , Varnum-Finney Barbara , Moon Randall T , Poulos Michael Gustave , Butler Jason M. , Rafii Shahin , Bernstein Irwin D. 
Published: October 2019

Abstract
Long-term, adult-engrafting hematopoietic stem cells (HSC) first emerge from hemogenic endothelial (HE) precursors in the context of embryonic arterial vessels such as the dorsal aorta of the AGM (Aorta-Gonad-Mesonephros region), a process which requires Notch1 receptor signaling. However, a possible subsequent role for Notch receptor activation during the unique period of substantial HSC expansion in embryonic development remains less well defined. Here, we show that endothelial cells derived from the murine embryonic AGM region (AGM-EC) or fetal liver (FL-EC) provide an in vitro substrate for generation/maturation of HSC from HE/HSC-precursor populations derived from early stage murine embryos, which lack adult-engraftment capacity prior to co-culture. Notably, these EC substrates, endogenously expressing Notch ligands of the Jagged and Delta families, also support subsequent numerical expansion of AGM-derived HSC, expressing Notch1 and Notch2 receptors, as determined by limit dilution transplantation analysis. Consistent with a requirement for Notch activation in this process, phenotypic HSC expansion during EC co-culture is blocked by gamma-secretase mediated Notch pathway inhibition. Furthermore, we show that in vitro Notch activation by immobilized ligand Delta1, together with cytokines and inhibition of the TGF-beta pathway, is sufficient to increase the number of AGM-derived HSC in the absence of EC stroma. Expansion of phenotypic hematopoietic stem/progenitor cells generated by culture on Delta1 is inhibited by antibody-mediated blockade of the combination of Notch1 and Notch2, but not by either Notch1 or Notch2 alone. Consistent with this, Notch receptor-specific activation by either immobilized Notch1 or Notch2 antibody is sufficient to support AGM-derived HSC in vitro in preliminary experiments. Altogether, these studies suggest a role for Notch pathway activation by either Notch1 or Notch2 in supporting embryonic-stage HSC expansion subsequent to initial Notch1-mediated HSC specification.

Disclosures
Moon: Fate Therapeutics: co-founder Other. Rafii:Angiocrine Biosciences: Founder Other.
BCL-2 and c-MYC Rearrangements in Leukemic Phase of Diffuse Large B Cell Lymphoma Predicts Central Nervous System Involvement
Authors: Shuhua Yi , Zhong Shizheng , Zou Dehui , Li Chengwen , Li Zengjun , Liu Wei , Lv Rui , Zhang Peihong , Chen Huishu , Wang Huijun , Qiu Lugui 
Published: October 2019

Abstract
Introduction
Diffuse large B-cell lymphomas (DLBCL) are the most common lymphoid neoplasms but rarely with leukemic phase. Few studies had described the clinical characteristics of DLBCL with leukemic phase and found that these patients had high incidence of centre nervous system (CNS) involvement but with no knowledge of the reason.
Methods
We reviewed the clinical characteristics of 40 DLBCL patients in leukemic phase during 2001 and 2012. The DNA probes were LSI BCL2 and MYC Dual Color, Break Apart Rearrangement Probe were usedto detect the abnormalities of 18q21 and 8q24 by FISH.We also used LSI IGH/BCL2 Dual Color, Dual Fusion Translocation Probe and LSI IGH/MYC/CEP 8 Tri-Color Dual Fusion Probesto detect the partner fusion genes of BCL2 or MYC.
Results
The median age of these 40 patients was 46 years(range, 15-75) with 19 men patients. The median WBC was 12.32×109/l(range, 1.2-66.57)with a median 35.74% (range, 1–90) of circulating lymphomatous cells identified by flow cytometry.14 of these 40 patients (35%)had CNS involvement.There were gender differences between CNS involvement or not. Ten of the fourteen patients (71.4%) with CNS involvement were male, while in non CNS involvement group only 9 of 26 (34.6%) cases were male (p=0.046).57.1% patients with CNS involvement had IPI scores 4-5, which was higher than those without CNS involvement (11.5%, p=0.001).All of these 14 patients had elevated LDH was and was significantly higher than those without (61.5%, p=0.007).
There were 14 patients (33.5%) had the BCL2 or MYC aberrationrespectively and nine of them simultaneously had both of c-MYC and BCL2 rearrangement, which we defined as double-hit DLBCL patients here. No IGH/BCL2 fusion gene was detected and only one patient with IGH/MYC fusion with c-MYC rearrangement. Eight of the nine double-hit patients had CNS involvement, while only six of the other 31 patients with either BCL2 or MYC or none rearrangements had CNS involvement (88.9% vs. 19.4%; p＜0.001). By logistic regression analysis, we found that double-hit with BCL2 and c-MYC rearrangement was the only one independent factor correlated with CNS involvement with relative risk 15.3 (95% confidence interval, 1.4-171.1).
Conclusion
DLBCL patients in leukemic phase had higher incidence of CNS involvement and double-hit with BCL2 and c-MYC gene rearrangements might contribute to CNS involvement in this population.

Disclosures
No relevant conflicts of interest to declare.
B-Cell Receptors of Primary Central Nervous System Lymphoma Recognize Antigens in the Brain
Authors: Trepel Martin , Müller Fabian , Illerhaus Gerald , Glatzel Markus , Binder Mascha , Spies Elmar 
Published: October 2019

Abstract
INTRODUCTION: The pathobiology mediating the development of primary central nervous system lymphoma (PCNSL) and its exclusive manifestation in the brain and spinal cord is still poorly understood. The monoclonal B-cell receptor expressed in PCNSL cells have a germinal center typology, suggesting previous antigen contact and antigen-based selection during B-cell development. Furthermore, sequence analysis of B-cell receptors in various patients revealed a bias in the immunoglobulin repertoire in PCNSL, potentially indicating that distinct epitopes might elicit such an immune response. Nevertheless, the nature of potential antigens containing such epitopes is unknown. Here, we provide the first evidence that such antigens may be located within the central nervous system itself, which might contribute to the specific organ tropism of PCNSL.
METHODS: Cryopreserved tissue of five randomly chosen PCNSL cases and three control cases with mantle cell lymphoma were used for this study. The variable heavy and light chains of the B-cell receptors were amplified from extracted RNA and cloned in IgG1 expression vectors. Recombinant B-cell receptors were expressed and purified as soluble human immunoglobulins. To explore the potential recognition of central nervous system antigens, murine cryo- and paraffin-embedded brain tissue sections were immunostained using these PCNSL or mantle cell lymphoma-derived immunoglobulins as primary antibodies.
RESULTS: Four out of five recombinant B-cell receptors from PCNSL but none of the MCL-derived control immunoglobulins recognized distinct anatomical structures in the brain which resulted in unambiguous immunostaining especially in regions with high neuronal density. The staining of brain tissue was associated with neuronal cells in all four positive cases. Staining was stronger in the cerebellum and the hippocampus compared to other areas of the brain and staining intensity varied broadly among the four positive cases. In the cerebellum, all four positive B-cell receptors recognized the cytoplasm of Purkinje cells, the largest neurons in the brain (therefore, staining could be readily identified). Two of the B-cell receptors also recognized the Purkinje cell’s dendrites. Three of the PCNSL-derived immunoglobulins also stained the granule cells, the most frequent neuronal cell type in the brain. Two of those stained in the cytoplasm and one showed a patchy but strong staining pattern in the nucleus of these cells. All of these stainings including negative controls were reproduced multiple times in independent experiments.
CONCLUSION: B-cell receptors of primary central nervous system lymphomas recognize neuronal structures in the brain in an individually case-dependent pattern. This might point towards an important role of antigen recognition in the pathobiology and specific organ tropism of this lymphoma.

Disclosures
No relevant conflicts of interest to declare.
Rcor1 Deficiency Disrupts Myeloerythroid Lineage Differentiation and Causes Severe Myelodysplasia
Authors: Goldman Devorah C , Yao Huilan , Fan Guang , Mandel Gail , Fleming William H. 
Published: October 2019

Abstract
Previously, we showed that the co-repressor CoREST (Rcor1) is essential for the maturation of definitive erythroid cells in the mouse fetal liver. To elucidate Rcor1’s function in multilineage hematopoiesis in the adult, we conditionally deleted Rcor1 using Mx1-Cre. The loss of Rcor1 expression in hematopoietic cells led to the rapid onset of a severe anemia due to a complete block of erythropoiesis downstream of committed erythroid progenitors. By contrast, the production of megakaryocyte progenitors, megakaryocyte maturation and platelets were maintained. In the myelomonocytic lineages, although neutrophil differentiation was completely abrogated, the number of monocytic cells was significantly increased, resulting in a peripheral blood leukocytosis and monocytic infiltrations in the liver. Rcor1-deficient monocytes showed a 66% reduction in apoptosis and possessed ~100-fold more CFU-M activity than control cells. The CFU-M derived from Rcor1 deficient bone marrow could be serially replated up to 5 times; however, replating activity was entirely cytokine dependent. Defective myelomonocytic differentiation persisted following transplantation into wild type hosts for up to 12 months but did not progress to leukemia. To begin to understand at the molecular level how Rcor1 regulates monocyte expansion, we evaluated the expression levels of genes whose ectopic expression is associated with myeloid neoplasia. In Rcor1-deficient monocytes, Gata2, Meis1 and Hoxa9 were overexpressed by 8- to 200-fold. Taken together, these data demonstrate that Rcor1 is essential for both erythroid and myelomonocytic differentiation and that its loss of function causes significant myelodysplasia.

Disclosures
No relevant conflicts of interest to declare.
IL-17A-Mediated Notch Signaling in Multiple Myeloma
Authors: Prabhala Rao H. , Hideshima Teru , Fulciniti Mariateresa , Adamia Sophia , Bandi Rajya Lakshmi , Nanjappa Puru , Anderson Kenneth C. , Munshi Nikhil C. 
Published: October 2019

Abstract
Multiple myeloma (MM) is a plasma cell malignancy, however, significant abnormalities in T cell function are considered to provide help in uncontrolled growth and survival of MM cells. We have previously reported that IL-17A-producing Th17 cells are elevated in MM, that MM cells express IL-17 receptor, and IL-17A promotes MM cell growth and survival. We have reported that MM cells themselves produce IL-17A as confirmed by RT-PCR, Western blotting and immunostaining providing a possibility of both autocrine and paracrine role for IL-17A in MM. As Notch activation has been implicated in Th17 cell differentiation and IL17A production, we have here investigated the role of Notch pathway activation in IL-17A-mediated MM cell growth within the BM microenvironment. Notch consists of 4 proteins (1-4) and has 5 ligands (DLL-1,3,4 and jagged-1, 2). We analyzed RNA-Seq data from 117 newly-diagnosed MM patients and 18 normal plasma cells and observed high expression of Notch 1, and 2 and Notch target genes Hes-1 and Hey-1 but not Notch 3 and 4 in MM. For Notch 2, isoform 2 was highly predominant. Notch expression on MM cells was further confirmed by flow cytometric analysis (Notch1-84%, Notch2-86% and Notch3-3%). Evaluating functional role of Notch in MM, when MM cells were co-cultured with Notch ligand jagged 2-expressing 3T3 cells, IL-17A was able to further induce Notch target gene Hes-1 by 45%. Interestingly, increase in the expression of Notch 2 was also observed during this interaction (increased full-length protein by 65% and active intra-cellular protein by 145%). We next evaluated effect of both anti-IL-17 antibody and Notch inhibitors on MM cells. Anti-IL-17A monoclonal antibody inhibited full-length Notch2 protein expression by 54% and active intra-cellular protein by 85%, as determined by western blot analysis. The antibody inhibitory activity was confirmed with quantitative PCR. Importantly, IL-17A mAb inhibited Hes-1 protein expression by 83%. With the observed impact of Notch signaling in MM, we next evaluated notch inhibitors MRK003, and compound E, a γ-secretase inhibitors, to determine their impact on MM cell growth and survival. We observe that Notch inhibitors affect MM cell growth (inhibition by 43%%, N=5) and IL-6 production (inhibition by 60%, N=3) in co-culture with bone marrow stromal cells. These preclinical data establish the role of IL-17 as well as Notch signaling in myeloma and provides the rationale to evaluate anti-MM activity of anti-IL-17A monoclonal antibody and Notch inhibitors in MM.

Disclosures
No relevant conflicts of interest to declare.