bluuebunny/crossref_metadata_2025 huggingface dataset:
B7H1/B7.1 Interaction Alleviate Acute Gvhd in a PD-1 Dependent Manner
Authors: Deng Ruishu , Cassady Kaniel , Li Xiaofan , Yao Sheng , Zhang Mingfeng , Racine Jeremy , Lin Jeffrey , Chen Lieping , Zeng Defu
Published: November 2019

Interactions of B7H1 (PD-L1) on antigen presenting cells with its two ligands, PD-1 and B7.1, on T cells play a pivotal role in controlling T cell activation, proliferation, anergy, and apoptosis. However, the interactions between the two pathways remain unknown. Using a graft-versus-host disease (GVHD) model of C57BL/6 donor to MHC-mismatched BALB/c recipient, we report here that:1) Wild-type (WT) conventional alloreactive CD4+ T (Tcon) cells induces more severe acute GVHD in B7H1 deficient recipients due to lack of both B7H1/PD-1 and B7H1/B7.1 interaction, resulting in reduced Tcon proliferation and apoptosis and therefore increased expansion of Tcon cells. In contrast, PD-1 deficient Tcon cells induced less severe GVHD in B7H1 deficient recipients compared to WT recipients due to lack of B7H1/B7.1 interaction and reduced Tcon cell proliferation. 2) Specific blockade of the B7H1/B7.1 axis by use of an anti-B7H1 mAb exacerbate acute GVHD. This is because anti-B7H1 mAb reduces donor Tcon cell proliferation, IL-2 production, expression of PD-1, and activation-induced apoptosis, resulting in expansion of alloreactive Tcon cells. In contrast, in the absence of PD-1 on donor T cells, specific blockade of B7H1/B7.1 interaction ameliorates acute GVHD due to reduction of donor Tcon proliferation without impact on Tcon cell apoptosis. 3) B7H1 fused to an immunoglobulin Fc domain (B7H1-Ig), when produced in vivo by hydrodynamic injection of B7H1 plasmid, ameliorates GVHD by augmenting both proliferation and apoptosis of WT- alloreactive Tcon cells. Conversely, in the absence of PD-1 on donor T cells, B7H1-Ig treatment has no impact on apoptosis but augments donor T cell proliferation and worsens GVHD. These results indicate that interaction of B7.1 with B7H1 augments Tcon cell proliferation, IL-2 production, and expression of PD-1. Higher expression of PD-1 leads to increased donor Tcon cell apoptosis mediated by the B7H1/PD1 pathway therefore ameliorates GVHD. But in the absence of PD-1, B7H1/B7.1 interaction only augments T cell proliferation and causes more severe GVHD. Additionally, by engaging both PD-1 and B7.1, B7H1-Ig can be a powerful therapeutic reagent for down-regulating the T cell immune response (This work was supported by NIH R01 AI066008).

Disclosures

No relevant conflicts of interest to declare.
The BRAF Pseudogene Is a Proto-Oncogenic Competitive Endogenous RNA
Authors: Karreth Florian , Reschke Markus , Chapuy Bjoern , Shipp Margaret A. , Chiarle Roberto , Pandolfi Pier Paolo
Published: November 2019

Non-coding RNAs have long been viewed as non-functional genomic relicts of evolution, but recetn findings have implicated their importance in physiology and disease. Recently, in vitro experiments demonstrated that the pseudogenes of PTEN and KRAS operate as natural miRNA decoys (competitive endogenous RNAs or ceRNAs) that regulate the expression of their parental genes. However, in vivo evidence for a causal role of pseudogenes in cancer development is lacking. To investigate whether the BRAF pseudogene (BRAFps) possesses oncogenic properties we generated transgenic mice carrying a Tet-inducible BRAF pseudogene allele. Global BRAFps overexpression resulted in the development of aggressive B-cell lymphoma after 6-12 months. These tumors were characterized by a profound expansion of B-lymphocytes in the spleen, as well as splenomegaly, lymphadenopathy and infiltration of the kidneys, lungs, and liver by neoplastic cells. The BRAFps-induced lymphoma was polyclonal, transplantable, dependent on continuous BRAFps expression, and cooperated with heterozygous loss of PTEN to accelerate disease onset. Mechanistically, we propose that BRAFps functions as a ceRNA that sequesters miRNAs from BRAF and possibly other targets. Indeed, overexpression of BRAFps results in elevated levels of BRAF in a Dicer-dependent manner. This, in turn, increased BRAF-dependent MAPK signaling and proliferation. To further validate the ceRNA activity of BRAFps, we engineered mice to express only the 3’UTR or CDS of BRAFps as each portion of the pseudogene may individually engage in miRNA-mediated crosstalk with BRAF. Notably, both BRAFps-CDS and BRAFps-3’UTR increased spleen and lymph node weights 6 months after induction. Interestingly, BRAFps-3’UTR elicited a lymphoma phenotype similar to full length BRAFps, while mice expressing BRAFps-CDS developed a more indolent form of this phenotype, suggesting that lymphomagenesis is primarily mediated by the BRAFps 3’UTR. BRAFps transcript was undetectable in primary human B-cells, but was aberrantly expressed in primary human DLBCL and human DLBCL cell lines. Expression of BRAF and BRAFps was positively correlated in human primary DLBCL and human DLBCL cell lines. In addition, gains or amplifications of the genomic locus containing BRAFps were found in various human cancer types. Overexpression of BRAFps in human lymphoma cells elevated BRAF levels, MAPK activation, proliferation and growth in xenografts. Our results demonstrate for the first time the oncogenic potential of a pseudogene in an engineered mouse model and indicate that ceRNA- mediated regulation is an important regulatory mechanism of gene expression in vivo.

Disclosures

No relevant conflicts of interest to declare.
High Ferritin Level Is High Risk Factor of II-IV Acute Gvhd or Severe Cytopenia Post-Transplant in Thalassemia HSCT
Authors: Liao Jianyun , Wen Jianyun , Li Chunfu
Published: November 2019

Purpose: To find out causes of II-IV acute graft-versus-host disease (GVHD) or severe cytopenia after hematopoietic stem cell transplantation (HSCT).

Methods: Sixty-four children with thalassemia major underwent HSCT from fifty-seven 7/8 (at allele of A, B, C, or DRB1) matched donors and seven 7/8 match associated with a DQB1 or DPB1 allele mismatched donors from January 2009 to September 2013. Thirty-three patients suffered from II-IV acute GVHD or severe cytopenia beyond day 28 post-transplant (SCB-28PT, WBC < 2G/L for 4 sequence weeks after HSCT, Group 1). The remains (n=31) associated with well status (Group 2). Current study focus on effect of ferritin, recipient age and donor age on the Group 1.

Results: Ferritin was significant higher in Group1 than Group 2 (4178.3 vs.3033.8 ng/ml, p=0.042). Ferritin was also proved as the dangerous cause by multi-factor binary regression analysis (p=0.017). There were insignificant in comparison of Group 1 and Group 2 in donor’s age (30.2 vs.30.4, p=0.939) and receptor’s age (6.3 vs. 6.4,p=0.889), respectively.

Conclusions: The current study showed high ferritin level is a high risk factor of II-IV acute GVHD or SCB-28PT.

Disclosures

No relevant conflicts of interest to declare.
In Human Beta-Globin Gene Locus, ERV-9 LTR Retrotransposon Interacts with and Activates Beta- but Not Gamma-Globin Gene
Authors: Tuan Dorothy , Pi Wenhu
Published: November 2019

Retrotransposons including endogenous retroviruses and their solitary long terminal repeats (LTRs) comprise over 40% of the human genome. Many of them are located in intergenic regions far from genes. Whether these intergenic retrotransposons serve beneficial host functions was not known. In the human b-globin gene locus, an ERV-9 LTR retrtransposon is located near the 5’ end of the locus control region (LCR) at 40-70 kb upstream of the human fetal g- and adult b-globin genes. To address the functional significance of the intergenic ERV-9 LTR, we generated transgenic (Tg) mice carrying a 100 Kb BAC clone spanning the entire human b-globin gene locus from the ERV-9 LTR to b-globin gene and showed that the LTR retrotransposon serves long-range, beneficial host function (Pi et al., PNAS 2010): The ERV-9 LTR containing multiple CCAAT and GATA motifs competitively recruits high concentration of NF-Y and GATA-2 present in low abundance in adult erythroid cells to assemble an LTR/RNA polymerase II complex. Deletion of the ERV-9 LTR by Cre-loxP mediated in situ recombination in the BAC Tg mice suppresses transcription of b-globin gene but activates transcription of g-globin gene. The results indicate that the ERV-9 LTR activates transcription of b-globin gene in erythroid cells during development. Therefore, LTR deletion drastically suppressed b-globin gene and re-activated g-globin gene through a competitive mechanism of globin gene switching.

Alternatively, the primary effect of the ERV-9 LTR could be to suppress g-globin gene during development. Therefore, LTR deletion re-activated g-globin gene, which then suppressed transcription of b-globin gene. To differentiate between these two possibilities, we utilized Mx1-Cre mice to conditionally delete the ERV-9 LTR in the erythroid cells of the adult BAC transgene mice, in which g-globin gene was already silenced and b-globin gene fully activated. If the primary target of the ERV-9 LTR enhancer complex was g-globin gene, deletion of the ERV-9 LTR should not be able to activate the already silenced g-globin gene nor to suppress the fully active b-globin gene. However, the same effect on transcriptional suppression of b-globin gene and re-activation of g-globin gene was observed. These results indicate that the primary target of the ERV-9 LTR is the b-globin gene and not the g-globin gene. The molecular factors underlying the preferential interaction between the ERV-9 LTR and the b-globin gene are under investigation and will be presented

Disclosures

No relevant conflicts of interest to declare.
Pericardial Effusion Is a Marker of Increased Mortality in Thalassemia Major Patients
Authors: Meloni Antonella , Toia Patrizia , Sardella Leonardo , Serra Giuseppe , Chiari Roberta , D'Ambrosio Alfonso , De Franceschi Lucia , Neri Maria Giovanna , Resta Maria Chiara , Preziosi Paolo , Positano Vincenzo , Benni Monica , Pepe Alessia
Published: November 2019

Introduction. In different types of not-hematological diseases the presence of a small pericardial effusion (PE) was associated with worse survival even after adjustment for patient characteristics, suggesting that it is a marker of underlying disease.In thalassemia major (TM) pericardial effusion was shown to be one of the manifestations of heart disease but its potential prognostic importance has never been investigated in the modern era. Cardiovascular Magnetic Resonance (CMR) by cine SSFP sequences was demonstrated to be extremely sensitive to even a small amount of PE.

This is the first prospective study evaluating if the presence of pericardial effusion is associated with increased mortality in TM.

Methods. 1259 patients (648 females, mean age 31.02 ± 8.64 years) enrolled in the Myocardial Iron Overload in Thalassemia (MIOT) were prospectively followed from their first Magnetic Resonance Imaging (MRI) scan. CMR was used to quantify myocardial iron (MIO) overload by a multislice multiecho T2* approach and to assess biventricular function parameters and to detect PE by cine SSFP sequences.

Results. PE was present in 25 (2.0%) patients.Patients with and without PE were comparable for age and ratio of men/women. At the baseline, the percentage of patients with MIO (global heart T2* value < 20 ms) was comparable between patients with and without PE (12.0 % vs 28.7%; P=0.074) and left ventricular and right ventricular ejection fractions were not significantly different between the two groups.

Mean follow-up (FU) time was 44.55 ± 20.35 months and there were 15 deaths.

Mortality was greater for patients with PE compared to those without an effusion (8.0% vs 1.1%, P=0.034).

PE was a significant predictive factor for death (hazard ratio-HR=12.64, 95%CI=2.78-57.42, P=0.001). PE remained a significant prognosticator for death also in a multivariate model including MIO ms (PE: HR=17.36, 95%CI=3.65-82.62, P<0.0001and global heart T2* < 20 ms: HR=3.07, 95%CI=1.07-8.75, P=0.036).

Conclusions. PE is quite rare in TM patients and it is not related to myocardial iron overload. An important role in the development of PE could be played by the 'iron-induced' pericardial siderosis but, due to the limitations of the current non-invasive CMR techniques, we were not able to address this issue.

PE was found to be a strong predictor for death, independently by the presence of myocardial iron overload. The non-invasive diagnosis of pericardial effusion is important for a more complete definition of the cardiac involvement of TM patients. The increased risk of death associated with PE may be used along with other clinical characteristics when estimating a patient's prognosis and monitoring.

Disclosures

Pepe: Chiesi: Speakers Bureau; ApoPharma Inc.: Speakers Bureau; Novartis: Speakers Bureau.
Antibody Mediated Depletion of iNKT Cells Protects Against Hypoxia-Induced Pulmonary Injury in a Murine Model of Sickle Cell Disease
Authors: Pei Hong , Schaub Robert , Scheuplein Felix , Mashal Robert , Linden Joel
Published: November 2019

Background: Painful vaso-occlusive crises (pVOC) are caused by the polymerization of sickle deoxyhemoglobin, red cell sickling and vaso-occlusion. Vaso-occlusion is exacerbated by an inflammatory cascade that is initiated by iNKT cell activation. Townes sickle cell mice have mouse globins replaced by human globins, including the mutant human sickle b-globin gene (SS). NKT-14 is a monoclonal antibody directed specifically to the mouse invariant T cell receptor (iTCR). The antibody is a chimeric mouse IgG2a that, when bound to the mouse iNKT cell, promotes a rapid, specific and long lasting (> 14 days) depletion.

Aims: Our primary goal was to determine if selective iNKT cell depletion with NKT-14 reduces lung inflammation and injury in response to hypoxia-induced RBC sickling in Townes SS mice.

Methods: Hypoxic pulmonary injury was induced by placing mice in 8% O2 (Coy Lab products)followed by reoxygenation. Some mice were subject to a second period of hypoxia and continuously monitored to determine carotid Hb-saturation (Starr MouseOx). Total cell numbers and activation state of iNKT cells and neutrophils in lung were determined by FACS. Pulmonary vascular leak was quantified with Evans Blue dye.

Results: Twelve hours of 8% hypoxia followed 4 hours of reoxygenation (H/R) was found to produce non-lethal lung inflammation and injury in SS mice. Compared to isotype Ab controls, injection of four mice with 100µg/mouse of NKT-14 Ab three days prior to HR was found to decrease the number of pulmonary iNKT cells by 91% (P < 0.01). Following H/R, NKT-14 Ab treatment resulted in a significant 65% decrease in lung neutrophil accumulation (Fig. A, P < 0.01), and a 54% decrease in pulmonary vascular leak (Fig. B, P < 0.05). In addition, arterial Hb-Sat during a second exposure of SS mice to hypoxia was improved (Fig. C).

Figure 1 Figure 1.

Conclusions: Selective depletion of mouse iNKT cells by injection of NKT-14 Ab three days prior to H/R results in substantial reductions in pulmonary inflammation, vascular leak and injury as measured by Hb-Sat in SS mice. The results suggest that prophylactic Ab-mediated depletion of iNKT cells in humans with SCD may be useful for reducing the incidence and severity of pVOC and acute chest syndrome.

Disclosures

Schaub: NKT Therapeutics Inc: Employment, Equity Ownership. Scheuplein:NKT Therapeutics Inc: Employment, Equity Ownership. Mashal:NKT Therapeutics Inc: Employment, Equity Ownership.
KLF1 Null Neonates Display Hydrops Fetalis and a Deranged Erythroid Transcriptome
Authors: Perkins Andrew C , Magor Graham , Tallack Michael R , Gillinder Kevin R , Bell Charles , Williams Bronwyn
Published: November 2019

We describe a case of severe neonatal anemia with kernicterus due to compound heterozygosity for null mutations in KLF1, each inherited from asymptomatic parents. One of the mutations is novel. This is the first described case of a KLF1 null human. The phenotype of severe DAT-negative non-spherocytic hemolytic anaemia (NSHA), jaundice, hepato-splenomegaly, and marked erythroblastosis is more severe than that present in CDA type IV due to dominant mutations in the second zinc-finger of KLF1. There was a very high level of HbF expression into childhood (>70%), consistent with a key role for KLF1 in human hemoglobin switching. We performed RNA-seq on circulating erythroblasts and found human KLF1 acts like mouse Klf1 to coordinate expression of >500 genes required to build a red cell including those encoding globins, cytoskeletal components, AHSP, heme synthesis enzymes, cell cycle regulators, and blood group antigens. We interrogated recently published KLF1 ChIP-seq to determine directly regulated KLF1 genes in man. We identify novel KLF1 target genes including KIF23 and KIF11 which are required for proper cytokinesis. KIF23 mutations result in CDA type III so our finding provides a direct link btween CDA IV adn CDA III. We also identify new roles for KLF1 in autophagy, global transcriptional control and RNA splicing. We suggest loss of KLF1 should be considered in otherwise unexplained cases of severe neonatal NSHA or hydrops fetalis.

Disclosures

No relevant conflicts of interest to declare.
Expression of Core-Binding Factor-β Is Regulated By Mir-143 and Mir-223 during Granulopoiesis
Authors: Larsen Maria Torp , Borgkilde Anette , Clemmensen Stine Novrup , Borregaard Niels , Cowland Jack B.
Published: November 2019

Granulopoiesis is a strictly regulated process governed to a large extent by an ordered temporal expression of transcription factors. One of the earliest expressed transcription factors, obligatory for correct granulopoiesis, is the Runx1:CBF-beta heterodimer that induces cell division and transcription of genes encoding azurophile granule proteins. The expression level of Runx1:CBF-beta peaks in myeloblasts (MBs) and promyelocytes (PMs) and declines when cells enter the myelocyte (MC) stage, where cell proliferation ceases and terminal differentiation commences. Runx1 binds DNA directly and provides the transcriptional activation domain whereas CBF-beta binds to Runx1 and increases its DNA-binding affinity. CBF-beta is in most cases required for Runx1-mediated transcriptional activity.

microRNAs (miRNAs) post-transcriptionally regulate protein expression and are important for proper granulopoiesis. Using a combined density gradient and immunomagnetic purification protocol, we have isolated neutrophil precursors from human bone marrow and identified 135 differentially regulated miRNAs. Two of these, miR-143 and miR-223, were predicted to target CBFB mRNA that encodes the CBF-beta subunit. Both miRNAs are expressed at low levels in MBs and PMs and increases 5-fold when the cells mature to MCs and remain high in the following stages of neutrophil differentiation. This expression pattern supports a role for miR-143 and miR-223 as translational inhibitors of CBF-beta that diminish the transcriptional capacity of Runx1 and thus aid in terminating cell proliferation in MCs.

Transient transfection of HEK293 cells, that express CBF-beta endogenously, with pre-miR-143 and pre-miR-223 demonstrated a strong suppression of CBF-beta synthesis when both miRNAs were present. A luciferase reporter assay where the 3’-UTR of CBFB mRNA is inserted in the 3’-UTR of the firefly luciferase transcript confirmed that both miRNAs affect translation. Mutation of either the miR-143 or miR-223 miRNA recognition element (MRE) partly relieved repression of the luciferase mRNA and mutation of both MREs completely abolished translational repression. shRNA against CBFB mRNA was used to detect transcriptional targets affected by downregulation of CBF-beta in a murine promyelocyte cell line (MPRO) and in the myeloblastic cell line 32Dcl3 and expression of a number of genes encoding azurophilic granule proteins were found to be significantly diminished.

Together these data demonstrate that miR-143 and miR-223 affect CBF-beta expression and indicate that correct timing of expression of these two miRNAs is a prerequisite for proper granulopoiesis.

Grant acknowledgments: The Danish Medical Research Council, Novo Nordisk Foundation, Brøchner Mortensen Foundation, and the Danish Cancer Society.

MTL and AB contributed equally to the work

Disclosures

No relevant conflicts of interest to declare.
An Fc-Engineered CD133 Antibody for Induction of NK Cell Reactivity Against Cancer Cells
Authors: Rothfelder Kathrin , Leibold Julia , Grosse-Hovest Ludger , Buehring Hans-Joerg , Jung Gundram , Kanz Lothar , Salih Helmut R
Published: November 2019

CD133 is expressed in various types of solid tumors where it predicts an unfavourable clinical outcome (e.g. Linuma et al., J Clin Oncol. 2011). Moreover, CD133 has been shown to be preferentially expressed by cancer stem cells (CSC), as e.g. demonstrated by its usability to enrich CSCs containing populations within cancer cell preparations (e.g. Zeppernick et al., Nature 2008). As CSC display marked resistance to chemo- and radiation therapy, great efforts are presently made to develop immunotherapeutic approaches to specifically target CSC without harming healthy tissue. We here report on the preclinical characterization of a monoclonal antibody (mAb) directed towards CD133 for induction of NK cell reactivity against cancer cells. Two different anti-CD133 antibody clones (W6B3 and 293C3) were used to study expression levels of CD133 in different cell lines derived from solid tumors (A172 and U87MG, glioblastoma; CaCo-2, Colo-205, HCT-8, HT-29 and SW48, colorectal cancer; MDA-MB-231, MDA-MB-468 and BT474, breast cancer; WERI-Rb1, retinoblastoma). CD133 was recognized by W6B3 and 293C3 in 4 and 5 out of the 11 cell lines, respectively. The latter mAb was then chosen for the development of chimeric mAbs with either a wildtype human Fc part (293C3-WT) or a variant containing amino acid exchanges (S239D/I332E) to enhance its affinity to the activating Fc receptor CD16 (293C3-SDIE) and thereby its capacity to mediate antibody dependent cellular cytotoxicity (ADCC), an important antibody property that largely contributes to the clinical success of antitumor mAbs. After confirming the binding specificity of both 293C3-WT and 293C3-SDIE we employed cytotoxicity assays to comparatively analyze their ability to induce NK cell ADCC against different solid tumor cell lines. In all cases, profound induction of tumor cell lysis upon application of 293C3-SDIE was observed, and the therapeutic effect of the Fc-optimized mAb clearly exceeded that of its counterpart containing a wild type Fc part. Notably, colony forming unit assays did not reveal any toxicity of 293C3-SDIE at the level of committed hematopoietic progenitor cells. Thus, treatment with 293C3-SDIE constitutes a promising novel strategy for immunotherapy of cancer, in particular considering its ability to preferentially target CSC which is presently under study.

Disclosures

No relevant conflicts of interest to declare.
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

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

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

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

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

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

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

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

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

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.
Clinical Impact of Immunophenotype-CD15/CD30 on Outcome of Hodgkin Lympoma
Authors: Zafar Huma , Riaz Shazia , Badar Farhana
Published: November 2019

Introduction

In classical Hodgkin Disease, Reed Sternberg cells reacts with antibodies against a granulocyte associated antigen, CD15/CD30. So expression of immunochemistries CD15 and CD 30 in patients,suspecting for HD, has quite significant diagnostic value.

Aim

To find out relevance of CD15/CD30 expression in relation to clinical outcome in patients of Hodgkin's disease.

Methods and Material

A total of 301 patients of Hodgkin' disease from stage I to stage IV treated in Shaukat Khanum hospital during last 8 years, were reviewed. Data was analyzed concerning age, gender, histopathology, staging, immunochemistry and outcome.

Results:

Among these 301 patients, male to female ratio was 4.3:1.The mean age was 10.01years Maximum patients presented in stage III i.e 40.2 %. Total 55 (18.2%) patients were reported positive for CD 15 and 261 (86.7 %) for CD 30. Amongst CD 15 positive patients, 1.8% received palliation, 16.3% showed Primary progressive Hodgkin's lymphoma ( PPHL ),16.3% showed relapse. Amongst CD 15 negative patients, no patients received palliation, 9.47% showed PPHL, 15.7% showed relapse.

Amongst CD 30 positive patients, 0.38% pts received palliation, 7.66% showed PPHL, 12.2% showed relapse.

Conclusion:

There is diagnostic significance of CD15/CD 30 in Hodgkin's Disease but the prognostic significance of these immunochemistries could not be proven.

Disclosures

No relevant conflicts of interest to declare.