bluuebunny/crossref_metadata_2025_split huggingface dataset:
Redirection of Antigen-Specific T Cells to Tumor Cells Using Nanoparticle-Based Antigen-Specific Redirectors (ATRs)
Authors: Schuetz Christian , Varela Juan C. , Perica Karlo , Haupt Carl , Oelke Mathias , Schneck Jonathan 
Published: November 2019

Abstract
Immunotherapy is the modulation of a patient’s immune system to treat illness. Unfortunately many T cell based attempts have failed due to the fact that existing tumor-specific T cells are mostly anergic or tolerized and ex vivo generated T cells are often already of exhausted phenotype. Therefore, investigators have developed alternative approaches including bispecific antibody technology to redirect fully functional non-tumor specific T cells to the tumor. This has been primarily accomplished through targeting CD3 which is expressed on all T cells to engage and redirect them towards a molecule that is expressed on the tumor cells. Here we present a novel nanoparticle based approach to selectively target cytotoxic T cells (CTL) and re-direct them to kill tumors, termed ATR (Antigen-specific T cell Redirectors).
ATR were generated by coupling either MHC-Ig dimer or clonotypic anti-TCR antibody 1B2 to target the effector T cell population and an anti-CD19 to re-direct those to CD19+ tumor target cells onto 50-100nm nanoparticles. Flow cytometry and microscope based data confirm that the described ATR phenotype efficiently and stably stain tumor and T cells in a dose dependent manner and ATR mediate antigen-specific conjugate formation of effector T cells and tumor target cells. We further developed two clinically relevant protocols to test and optimize our ATR in vitro. First a pre-treatment approach in which the effector T cells are pre-incubated with ATR mimicking an adoptive transfer approach and second a co-culture protocol that mimics an active immunotherapy approach of direct ATR injection. Antigen-specific ATR mediated re-direction of T cells to tumor target cells was demonstrated in 51Cr-release killing assays at low E:T ratios. Variation of ATR target-cell : effector-cell targeting molecule ratio could further increase efficacy. Finally, intra tumoral ATR injection induced T cell re-direction and reduced tumor growth in a s.c. Raji/SCIDbeige treatment model.
In summary this data demonstrates that ATR target and redirect antigen-specific CTL to tumor cells that would otherwise not be recognized and mediates their lysis. ATR can be used to develop new innovative immunotherapeutic approaches for all cancers that can be targeted with antibodies or antibody-like molecules. Furthermore, ATR could also be used in conjunction with virus-specific immunization to specifically increase the targeted CTL population. Ultimately, we expect ATR and their potential for clinical applications to increase our understanding of tumor immunotherapy through T cell redirection.

Disclosures
Oelke: NextImmune: Equity Ownership. Schneck:NextImmune: Equity Ownership.
ETS Transcription Factor Etsrp/Etv2 Is Required for Lymphangiogenesis in Zebrafish and Is a Potential Therapeutic Target in Lymphatic Disease
Authors: Davis Jennifer A. , Desai Sharina Palencia , Sumanas Saulius 
Published: November 2019

Abstract
Lymphangiogenesis is of great clinical interest because of its essential role in such pathologic settings as inflammation, vascular malformations, and tumor metastasis. Previous observational hurdles are now overcome by use of the optically clear and genetically modifiable zebrafish embryo, which provides a vertebrate model for understanding lymphangiogenesis and for revealing putative therapeutic targets for metastatic disease and vascular/lymphatic malformations that currently have no cure. We and others have previously demonstrated that an evolutionarily conserved ETS transcription factor Etv2 / Etsrp functions as a master regulator during embryonic vascular development. However, its role in lymphatic development is not defined. Here we performed conditional knockdown of Etv2 function using photoactivatable morpholinos (MO) in a zebrafish model system to test Etv2 requirement during lymphangiogenesis. Our data show that conditional Etv2 inhibition at 1-day-post-fertilization (dpf) resulted in specific defects in lymphatic development, while blood vessel formation was not affected. The major lymphatic vessel, the thoracic duct, was absent or discontinuous in Etv2-depleted embryos (Figure 1) and the lymphatic progenitors failed to migrate (Figure 2) in absence of Etv2 function. Expression of the lymphatic markers lyve1, prox1a and the major VegfC receptor VegfR3 was strongly down regulated in the conditional Etv2 MO knockdown embryos. We further show that Etv2 expression is enriched in the posterior cardinal vein where lymphangioblasts (lymphatic endothelial cell precursors) originate during lymphangiogenesis and that overexpression of Etv2 throughout blood vasculature perturbs lymphangiogenesis. We further show that in the absence of Etv2 function, lymphatic progenitors fail to respond to VegfC signaling. Our results suggest that Etv2 initiates lymphangiogenesis in part by regulating Vegf3 expression. These results argue that Etv2 is a novel critical factor during lymphatic development, and it may represent a new target for chemical modulation in multiple lymphatic disorders.
Figure 1 Figure 1. Figure 2 Figure 2.

Disclosures
No relevant conflicts of interest to declare.
Impaired Hemostasis Caused By Deficit of Tissue Factor-Triggered Blood Coagulation Leads to Alveolar Hemorrhages and Increased Mortality in Influenza A Infection
Authors: Tatsumi Kohei , Antoniak Silvio , Milner Justin , Beck Melinda , Mackman Nigel 
Published: November 2019

Abstract
Objective: Viral infections lead to activation of coagulation which enhances inflammatory responses. For instance, influenza A is associated with activation of coagulation and the increased risk for thrombotic events, such as myocardial infarction. Here we investigated the role of the coagulation cascade and platelets in influenza A virus (IAV) infection in mice.
Methods: To investigate the role of the coagulation cascade in influenza infection, we examined the effect of either low levels of tissue factor or wild-type mice that were administered with 4 μg/mL of warfarin via drinking water. To study the role of platelets in influenza infection, we examined protease-activated receptor 4 (PAR-4, the main functional receptor on mouse platelets) deficient mice or wild-type mice treated with 0.15 mg/mL of clopidogrel or 0.4 mg/mL of aspirin via drinking water. All mice were infected intranasally with mouse adapted influenza A H1N1/PR8 (IAV). Changes in body weights and survival were analyzed up to 14 days after infection. In addition, bronchoalveolar lavage fluid (BALF) or lungs were collected and analyzed 7 or 14 days after virus challenge.
Results: LowTF mice exhibited higher mortality rate and increased loss of body weights compared to littermate control mice after IAV infection (P<0.01). At day 7, BALF of LowTF was red and had increased red blood cell numbers and hemoglobin levels compared to infected control mice (P<0.001). Thrombin-antithrombin complex (TAT) levels in the BALF of infected LowTF mice were lower than that of control mice. In addition, lung histology revealed that LowTF mice had extensive hemorrhages. Similarly, mice treated with warfarin and infected with IAV showed higher mortality compared with the warfarin-treated mice without infection or IAV-infected mice without drugs. Warfarin treated mice also had red BALF, higher red blood cell number and hemoglobin levels in the BALF, and lung bleeding. PAR-4 deficient mice exhibited visible lung hemorrhages with increased hemoglobin levels in the BAL (P<0.01) and increased mortality compared to controls (P<0.05), although the bleeding was less than LowTF mice. Treatment of mice with clopidogrel or aspirin did not increased lung bleeding or mortality.
Conclusion: Pulmonary hemostasis requires both TF-mediated activation of blood coagulation and thrombin activation of platelets during IAVinfection. Use of antithrombotics during IAV infection may increase the risk of lung bleeding.

Disclosures
No relevant conflicts of interest to declare.
Molecular Genetic Analysis in Patients with a Phenotype-Based Putative Diagnosis of Von Willebrand's Disease Type 1
Authors: Steiner Michael , Steiner Rosa , Krammer-Steiner Beate 
Published: November 2019

Abstract
The conclusive diagnosis of von Willebrand's disease (VWD) type 1 represents a challenging task. In addition to repeatedly performed phenotypic assessment, genetic analysis of the VWF gene may help establish the diagnosis. The aim of the present study was to assess the contribution of molecular genetic analysis in confirming the diagnosis in cases of suspected VWD type 1. Twenty-one patients (18 females, 3 males) with bleeding tendency were preliminary classified as likely or possible VWD type 1 based on phenotypic assays (VWF:Ag, VWF:RCoF < 50 % or 51-59 %, respectively) and further investigated by direct sequencing and MLPA analysis of the VWF gene. Ten different heterozygous mutations were identified in eleven patients (52 % confirmation rate). The mutation Y1584C (exon 28) was found in two unrelated patients. Most patients (9 of 11) demonstrated missense mutations in exons 20, 21, 28, 45 and 49. Newly identified mutations were K2566N (exon 45) and a 15 bp insertion predicting L168insX (exon 5). Large deletions and duplications were not detected. The results confirm the varying molecular pathology underlying VWD type 1. Molecular genetic analysis represents a useful approach to conclusively establish the diagnosis of VWD type 1 in approximately 50 % of patients with a phenotype-based preliminary diagnosis.

Disclosures
No relevant conflicts of interest to declare.
Serum Cartilage Oligometric Matrix Protein and Pettersson Scoring System As an Index for Hemophilic Arthropathy
Authors: Hassab Hoda MA , EL-Gendy Wessam M , El-Noueam Khaled I M , Abdel Ghany Hayam M , Elwan Myssoon M 
Published: November 2019

Abstract

The pathogenesis of hemophilic arthropathy is multifactorial, with changes occurring in the synovium, bone, cartilage, and blood vessels. Recurrent joint bleeding causes synovial proliferation and inflammation (hemophilic synovitis) that contributes to end stage degeneration (hemophilic arthropathy); with pain and limitation of motion that severely affects patients’ quality of life.
The aim of the present study was to evaluate the degree of joint damage in boys with hemophilia using plain x-ray, and to measure serum level of human cartilage oligomeric matrix protein, to determine its relation to the degree of joint damage.
The study was carried out on thirty boys with hemophilic arthropathy (group I) and ten healthy boys were included as control (group II).
All hemophilic patients were scored for Functional independence score (FISH score) in hemophilia and radiological changes (Pettersson’s score) using conventional frontal and lateral radiographs of the most affected joint.
Factor activity level was measured for all hemophilic patients while serum cartilage oligomeric matrix protein (COMP) was measured for hemophilic patients and control group.
Among the thirty hemophilic patients, 26 (86.7%) patients were hemophilia A and 4 (13.3%) patients were hemophilia B
All patients were receiving on demand replacement therapy using plasma derived Factor concentrate or fresh frozen plasma (FFP) according to availability.
Fifteen patients (50%) had severe hemophilia, 7 (23.3%) had moderate and 8(26.72%) had mild hemophilia.
A higher serum level of COMP with a mean of 757± 211.30 in the severe hemophiliacs, and a mean of 403.57 ± 86.49 and 211.25 ± 74.26 in the moderate and mild hemophiliacs respectively the difference was statistically significant (p < 0.001).
Serum level of COMP in group I was significantly higher than in group II (p=0.004) with significant negative correlation with FISH score (r=-0.435 p=0.016). COMP correlated positively with joint space narrowing of the Pettersson score (r=0.421 p<0.001) and with total Pettersson score (r=0.421 p=0.020).
The number of joints affected (during life) of hemophilic patients ranged between 1-12 with a mean of 5.50 ± 2.46 Joints. A significant positive correlation between serum level of COMP and number of joints affected (r = 0.487, p = 0.006).
Joint space narrowing is the most important indicator of cartilage loss. Serum COMP level is indicative of the amount of joint damage in patients with hemophilic arthropathy.
The combined scoring of functional independence and Pettersson in addition with serum levels of COMP give a good overview of the degree of hemophilic arthropathy

Disclosures
No relevant conflicts of interest to declare.
Activation of the Hypoxia-Inducible Factor Pathway Expands Early Thymic Progenitors
Authors: Hollenbeck Anita , Weber Stefanie , Händschke Kathrin , Necke Mandy , Opalka Bertram , Dührsen Ulrich , Göthert Joachim Rudolf 
Published: November 2019

Abstract
Early thymic progenitors enter the thymus and are exposed to regional hypoxia while they develop in a step-wise manner to mature functional T-cells. Therefore, hypoxia might represent an important component of the highly specialized thymic microenvironment. On the molecular level the hypoxia-inducible factor pathway controls the cellular response to hypoxia. In this pathway, the von-Hippel-Lindau protein (pVHL) continuously mediates the destruction of the transcription factor hypoxia-inducible factor-1α (HIF-1α) under normoxic conditions. Under hypoxia HIF-1α degradation is inhibited leading to the activation of HIF-1α target genes.
Others used lck-Cre transgene-mediated conditional in vivo deletion of the Vhl gene to study the role of the oxygen-sensing pathway in developing thymocytes and found normal numbers of early double-negative (DN; CD4-CD8-) thymocytes (Biju et al., Mol Cell Biol, 2004). However, lck-Cre deletion initiates at the DN3 (CD25+CD44-) stage leaving the Vhl locus of very early DN1 (CD25-CD44+), DN2 (CD25+CD44+) and DN3 thymocytes unaltered. Therefore, we here used the ubiquitous hematopoietic deleter strain vav-Cre to investigate the role of pVHL in very early thymocytes (vav-Cre;VhlloxP;loxP mice). Using a PCR-based strategy we confirmed complete deletion of the Vhl gene in this model. We observed unaltered DN1 and DN2 progenitor numbers, however in contrast to the published lck-cre-mediated system we consistently observed an up to twofold expansion of the DN3 cellular compartment. As the hypoxia-inducible factor pathway was shown to modulate NOTCH1 signaling we studied Notch1 expression on Vhl-deficient thymocytes. Strikingly, Notch1 expression was significantly increased on expanded Vhl null DN3 thymocytes. At the DN3 developmental stage selection of cells with an accurately re-arranged T-cell receptor β-locus occurs. Thus, we analyzed pre- and post-β-selection DN3 cells by CD28 staining. Interestingly, we found both pre- and post-β-selection DN3 subpopulations expanded. In order to investigate whether the progenitor expansion is mediated by the lack of HIF-1α inhibition in the Vhl-deficient context we studied DN3 thymocytes in a conditional hematopoietic HIF-1α gain-of-function model (vav-Cre;HIF1dPA). Overexpression of HIF-1α, which is insensitive to pVHL-mediated degradation in vav-Cre;HIF1dPAmice, also resulted in expanded DN3 thymocytes.
In summary, we describe novel conditional models to genetically alter the hypoxia-inducible factor pathway within very early thymic progenitors. Genetic Vhl loss led to an expansion of DN3 thymocytes. This DN3 expansion is most likely due to the absence of HIF-1α-inhibition, because HIF-1α overexpression phenocopied the Vhl-deficient DN3 thymocyte expansion.

Disclosures
Dührsen: Celgene: Honoraria, Research Funding.
Development of RA101348, a Potent Cyclic Peptide Inhibitor of C5 for Complement-Mediated Diseases
Authors: Ricardo Alonso , Arata Michelle , DeMarco Steven J , Dhamnaskar Ketki , Hammer Robert , Josephson Kristopher , Seyb Kathleen , Tobe Sylvia , Wang Zhaolin , Zheng Hong , Treco Douglas 
Published: November 2019

Abstract
The involvement of complement in the pathogenesis of serious, life-threatening diseases underscores the importance in developing therapeutics to intervene in this cascade. Specifically, dysregulation of the complement system is central in multiple hematologic conditions. Antibody blockade of the C5 component of complement has been approved as a treatment for paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic-uremic syndrome (aHUS), validating C5 as a therapeutic target for these conditions.
Ra Pharma has developed a macrocyclic peptide platform capable of yielding potent inhibitors of protein-protein interactions. Recently, we developed a short cyclic peptide that binds complement C5 with high affinity (KD = 2.6 nM) and inhibits its cleavage into C5a and C5b by a novel mechanism. At low nanomolar concentrations, RA101348 blocks the formation of the terminal complement complex (TCC) in vitro, as demonstrated in RBC lysis assays for both alternative and classical complement activation pathways.
Inhibition of in vivo complement activity was evaluated in cynomolgus monkeys following single- and multi-dose subcutaneous (SC) administration. RA101348 exhibited high SC bioavailability, and low, single doses inhibited complement activity by 90-100%. The effect is durable owing to an in vivo terminal half-life of approximately 72 hrs. Repeat dosing for 7 days was well tolerated in monkeys at up to 100-fold above projected human therapeutic doses and resulted in sustained inhibition of complement activity.
RA101348 is a novel therapeutic approach for inhibiting the activity of C5 for the treatment of disorders caused or associated with complement dysregulation. It offers the potential to enable self-administration of therapeutics for patients with PNH and other complement disorders.

Disclosures
Ricardo: Ra Pharmaceuticals: Employment. Arata:Ra Pharmaceuticals: Employment. DeMarco:Ra Pharmaceuticals: Employment. Dhamnaskar:Ra Pharmaceuticals: Employment. Hammer:Ra Pharmaceuticals: Employment. Josephson:Ra Pharmaceuticals: Employment. Seyb:Ra Pharmaceuticals: Employment. Tobe:Ra Pharmaceuticals: Employment. Wang:Ra Pharmaceuticals: Employment. Zheng:Ra Pharmaceuticals: Employment. Treco:Ra Pharmaceuticals: Employment.
Rapamycin Can Reduce Apoptosis of CD4+CD25+ Regulatory T Cells from Mouse Model of Severe Aplastic Anemia
Authors: Lin Zenghua , Liu Hong , Zhu Li , Wang Xuli , Qian Yu , Yang Xi , Cao Xin , Cai Yifeng , Zhang Yaping , Song Guoqi , Qian Juan 
Published: November 2019

Abstract
CD4+CD25+ regulatory T cells (Tregs) are believed to control the development and progression of autoimmunity by suppressing autoreactive T cells and decreased numbers of these cells are associated with impaired immune homeostasis and development of aplastic anemia (AA). We have developed a novel mouse model of severe aplastic anemia (SAA) induced by interferon-gamma in combination with Busulphan, in which excessive apoptotic CD4+CD25+ Treg cells was found in our previous experiments. It might be one of the reasons for reduction of CD4+CD25+ Treg cells in patients with AA. Rapamycin (RAPA) is a macrolide antibiotic produced by streptomyces hygroscopicus and is considered as a potential immunosuppressant in the treatment of AA, while the effect of RAPA on apoptosis of CD4+CD25+ Tregs from patients with AA is not clear. In this study, the SAA model female mice were intraperitoneal injection with RAPA at daily dose of 0.5 mg/kg for 5 days (the RAPA-treated group, n=60), the SAA group (n=60) and the un-treated group (n=60) were as control. All mice selected to set up the SAA model developed the typical clinical and pathological patterns of SAA from day 10 post dosing. Most of them presented bleedings in association with anemia and infections. In order to explore the effect of RAPA on apoptosis of CD4+CD25+ Tregs from the novel mouse SAA model, the mononuclear cells of the peripheral blood and spleen were subjected to assess the intracellular Foxp3 expression in CD4+CD25+ Tregs by flow cytometry (FCM). In the SAA group, the FCM analysis showed down-expression of Foxp3 in CD4+CD25+ Tregs compared with the un-treated group (P<0.05). These results were in accordance with our previous observations. However, after treatment with RAPA, the expression of Foxp3 in CD4+CD25+ Tregs was increased (P<0.05). In addition, after being pured by immunomagnetic beads, the splenic CD4+CD25+ Tregs was subjected to assess apoptosis by FCM and the Akt and Stat3 phosphorylation using western blot. Compared with the un-treated group, increased CD4+CD25+ Tregs apoptosis with increased Akt phosphorylation accompanied by increased Stat3 phosphorylation was found in SAA group (P<0.05, respectively). On the contrary, RAPA-treated group exhibited CD4+CD25+ Tregs with a reduction in apoptosis rate, Akt phosphorylation and Stat3 phosphorylation compared with the SAA group (P<0.05, respectively). These results indicated that RAPA may increase the expression of Foxp3 by down-regulation the levels of Akt and Stat3 phosphorylation in splenic CD4+CD25+ Tregs from the mice model of SAA, through which might RAPA reduce apoptosis of the CD4+CD25+ Tregs from this model.

Disclosures
No relevant conflicts of interest to declare.
Oxymetholone Therapy of Fanconi Anemia Induces Hematopoietic Stem Cell Cycling By Suppressing Osteopontin Transcription
Authors: Zhang Qingshuo , Benedetti Eric , Deater Matthew , Schubert Kathryn , Major Angela , Pelz Carl , Impey Soren , Marquez-Loza Laura , Rathbun Keaney , Kato Shigeaki , Bagby Grover C. , Grompe Markus 
Published: November 2019

Abstract
Androgens are widely used for treating Fanconi anemia and other human bone marrow failure syndromes, but their mode of action remains incompletely understood. Aged Fancd2-/- mice were used to assess the therapeutic efficacy of oxymetholone and its mechanism of action. 18-month old Fancd2-/- mice recapitulated key human Fanconi anemia phenotypes including reduced bone marrow cellularity, red cell macrocytosis, and peripheral pancytopenia. As in humans, chronic oxymetholone treatment significantly improved these hematological parameters by stimulating the proliferation of hematopoietic stem and progenitor cells. RNAseq analysis implicated down-regulation of osteopontin as an important mechanism for the drug’s action. Consistent with the increased stem cell proliferation, competitive repopulation assays demonstrated that chronic oxymetholone therapy eventually resulted in stem cell exhaustion. These results expand our knowledge of the regulation of hematopoietic stem cell proliferation and have direct clinical implications for the treatment of bone marrow failure.

Disclosures
No relevant conflicts of interest to declare.