bluuebunny/crossref_metadata_2025_split huggingface dataset:
Chronic Granulomatous Disease and Mcleod Syndrome: Single Center Report of Four Cases.
Authors: Mahlaoui Nizar , Dumont Marie-Dominique , Peyrard Thierry , Blanche Stephane , Fischer Alain 
Published: October 2019

Abstract
Abstract 3595
Poster Board III-532

Background
Chronic Granulomatous Disease (CGD) is an inherited disease affecting innate immunity and leading to increased susceptibility to severe invasive fungal and bacterial infections. The X linked form of CGD is the most frequent and is caused by mutations in the gp91phox subunit of the NADPH oxidase complex encoding gene CYBB. McLeod syndrome patients present with late-onset neuromuscular troubles and a mild chronic haemolytic anaemia with acanthocytes. It is defined by the lack of expression of Kell antigens on erythrocytes and caused by mutations in the KX gene located close to the CYBB gene. The association of CGD and McLeod syndrome is a rare event. It can also be associated with Duchenne muscular dystrophy (DMD), retinitis pigmentosa or ornithine transcarbamylase deficiency. Patients : Four CGD patients patients were recently diagnosed with McLeod syndrome in Necker Hospital on a 2 year period of time. Their clinical and main biological characteristics are reviewed and a review of recent litterature Observations: In 1 patient, McLeod was evoked because of association of Duchenne myopathy and CGD revealed at 10 months with major hypotonia and recurrent infections. Initially, no acanthocytes were seen but Kell antigens testings led to confirmation of McLeod. Three other infants were diagnosed with CGD because of lung infections. One had lung biopsies revealing Nocardia sp. infection and received packed blood red cells. Four months after, an allo immunisation against red cells and a weakened Kell antigen expression led to McLeod diagnosis. For other patients, diagnosis was evoked because of anaemia and acanthocytes on blood smear analysis. All had confirmed by CYBB mutations with complete deletion. None had muscle weakness or retinal abnormality. For 2 infants, iron therapy and erythropoietin led to the correction of anemia.

Conclusion
Management and evaluation of X-linked CGD patients should require the search of McLeod syndrome. Biological diagnosis is sometimes uneasy, especially because acanthocytes are sometimes hard to identify on blood smears. Kell antigen reactivity screening could be easy to perform. When McLeod syndrome is diagnosed, patients are included in the French national registry for rare erythrocyte phenotype to receive appropriate transfusion management. Erythropoietin therapy could be useful in preventing chronic anemia.

Disclosures:
No relevant conflicts of interest to declare.
Erythropoietin as a Novel Pro-Inflammatory Mediator of Macrophages.
Authors: Lifshitz Lilach , Tabak Galit , Gassman Max , Mittelman Moshe , Neumann Drorit 
Published: October 2019

Abstract
Abstract 3596
Poster Board III-533
The immunomodulatory effects of erythropoietin (EPO) on the cellular and humoral compartments of the immune system were originally described by our group in multiple myeloma patients and have been further elucidated in murine experimental models (Mittelman, 2001; Katz 2005; 2007; Prutchi-Sagiv, 2006). However, the mechanisms of action by which EPO affects lymphocyte number and function are still unknown, particularly since lymphocytes do not carry EPO receptors (EPO-R). We thus set to unravel mechanisms underlying the anti-neoplastic immunomodulatory action of EPO. These studies led us to the novel discovery that dendritic cells (DCs) express EPO-R, and that EPO enhances their survival and function (Prutchi-Sagiv, 2008; Lifshitz, 2009). Here we focus on macrophages as an additional EPO target, since in analogy to DCs, macrophages are also antigen presenting cells, and serve as key effectors of the innate immune response.
Using murine models, we first explored the in-vivo effects of EPO using recombinant human EPO (rHuEPO, EPREXR, JC)-injected mice, as well as transgenic mice over-expressing human EPO (termed tg6). EPO treatment was associated with an increased splenic macrophage population, detected by F4/80 expression, and an increased number of macrophages expressing CD11b, CD80 and MHC class II. We further explored the effect of in-vivo EPO administration in an inflammatory model exploiting thioglygollate injection to induce recruitment of peritoneal inflammatory macrophages. The inflammatory macrophages obtained from both EPO injected and from tg6 mice displayed increased expression of F4/80, CD11b, CD80 and MHC class II and augmented phagocytic activity, as compared to the control counterparts.
These results are supported by in-vitro studies in bone marrow derived macrophages (BMDMs). We show that BMDMs express EPO-R mRNA, as detected by RT-PCR. In-vitro stimulation of the BMDMs with rHuEPO activated multiple signaling pathways including STAT1, STAT5, MAPK, AKT and NFkB indicating macrophage activation via surface EPO-R. EPO treatment of the BMDMs up-regulated their surface expression of CD11b, F4/80 and CD80, as well as enhanced their phagocytic activity. EPO treatment of LPS-stimulated BMDMs augmented IL-12 secretion, and decreased IL-10 secretion.
In conclusion our results show that macrophages are direct targets of EPO and that EPO treatment enhances their pro-inflammatory activity and function. These findings point to the multifunctional role of EPO and may advance its clinical applications as an anti-neoplastic immunomodulator.

Disclosures:
Mittelman: BioGAL- Start up (inactive): Equity Ownership, Patents & Royalties. Off Label Use: Non erythroid effects: immune, anti-cancer (all under investigation).
Glia Maturation Factor-Gamma Mediates Lipopolysaccharide-Induced Inflammation Via p38 MAPK and NF-Kappab.
Authors: Aerbajinai Wulin , Chin Kyung , Zhu Jianqiong , Hyun Lee , Kumkhaek Chutima , Rodgers Griffin P 
Published: October 2019

Abstract
Abstract 3598
Poster Board III-535
Neutrophils are the most abundant leukocytes in the circulation and provide a primary innate immune defense function against bacterial pathogens before the development of a specific immune response. Activation of TLR4 signaling pathways by lipopolysaccharide (LPS) leads to the production of a broad array of cytokines and mediators that coordinate the immune response. The TLR4-mediated inflammatory response is tightly regulated, since excessive activation of TLR4 may induce a variety of inflammatory diseases, so the negative regulation of TLR-triggered inflammatory response attracts much attention. Glia maturation factor gamma (GMFG), a novel factor in actin cytoskeleton reorganization, is preferentially expressed in inflammatory cells, but its function in leucocytes is undefined. In this study, we investigated whether GMFG participates in the molecular events underlying the inflammatory reaction and how GMFG mediated neutrophils inflammatory response to LPS. We show here that small interfering RNA-mediated knockdown of GMFG expression in leukocytes exhibited greater than 80% inhibition of the endogenous expression levels of proinflammatory cytokines and chemokines, including TNF-alpha, IL-1beta, IL-6, and IL-8 using quantitative RT-PCR. These decreased cytokine productions could be partially restored by stimulation with TLR4 agonist LPS, but exhibit significantly lower level in silencing of GMFG cells compared to non-targeting silencing control cells (inhibition by 71%+/−3.9% of TNF-alpha, 68%+/−6.9% of IL-1beta, 50%+/−4.3% of IL-6 and 40%+/−3.1% of IL-8). Knockdown of GMFG expression results in decreased phospho-p38 MAPK levels as well as suppressed NF-kappaB by Western blot analysis. These data suggest that endogenous expression of GMFG is required for basal cytokine and chemokine response and their induced response to LPS stimulation. Moreover, immunofluorescence analysis revealed that gene silencing of GMFG inhibited LPS-induced nuclear translocation of NF-kappaB in myeloid HL-60 and THP-1 cells. In contrast, transient overexpression of GMFG significantly enhanced endogenous level of proinflammatory cytokines by 1.8∼2.2 fold, phosphor-p38 MAPK and nuclear translocation of NF-kappaB in the absence of LPS stimulation. These results suggest that GMFG-mediated regulation of inflammatory cytokines occurs through activation of p38 MAPK and NF-kappaB signaling pathway. Taken together, our findings identify GMFG as a novel factor that participates in the regulation of TLR4-mediated control of cytokine and chemokine expression through the p38 MAPK and NF-kappaB pathway. These data, if confirmed in in vivo experiments, implicates GMFG as a potential target for therapeutic intervention in neutrophil-mediated autoimmune disease.

Disclosures:
No relevant conflicts of interest to declare.
TRAF2 Ring Domain Plays An Essential Role in Inhibition of TNFα-Induced Cell Death Independent of NF-kB Activation.
Authors: Blackwell Ken , Zhang Laiqun , Shi Zhaohui , Habelhah Hasem 
Published: October 2019

Abstract
Abstract 3621
Poster Board III-557
The proinflammatory cytokine TNFα regulates immune responses, inflammation and programmed cell death by activating the JNK/c-Jun and IKK/NF-κB signaling pathways. TRAF2 and RIP1 play critical roles in TNFα-induced activation of JNK and IKK, and inhibition of apoptosis. In the field, TRAF2 RING domain-mediated RIP1 ubiquitination is believed to play an essential role in TNFα-induced IKK activation, and the RING domain-deleted TRAF2 (TRAF2-ΔN) has been widely used as a dominant negative to block TNFα-induced JNK and IKK activation in transient overexpression systems. Here, we report that stable expression of TRAF2-Δ[N in TRAF2 and TRAF5 double knockout (T2/5 DKO) cells at a physiological level almost completely restores TNFα-induced IKK activation, but does not restore RIP1 ubiquitination. In addition, stable expression of TRAF2-ΔN in T2/5 DKO cells efficiently inhibited TNFα-induced later-phase prolonged JNK activation, but failed to inhibit TNFα-induced cell death. The basal and inducible expression of antiapoptotic proteins in TRAF2-ΔN-expressing cells is totally normal, yet these cells are still sensitive to TNFα-induced cell death due to the inefficient recruitment of cIAP1 to the TNFR1 complex. Moreover, stable expression of TRAF2-WT, but not TRAF2-ΔN, in T2/5 DKO cells suppressed constitutive p100 processing. On the other hand, TRAF2-ΔN-expressing cells exhibited significant resistance to oxidative stress-induced cell death compared to TRAF2-WT-expressing cells. These data suggest that: i) the TRAF2 RING domain plays a critical role in the inhibition of cell death induced by TNFα, and is essential for suppression of p100 processing in unstimulated cells; ii) RIP1 polyubiquitination is not required for TNFα-induced IKK activation, but may play a role in inhibition of TNFα-induced cell death; and iii) prolonged JNK activation has no obligate role in TNFα-induced cell death.

Disclosures:
No relevant conflicts of interest to declare.
Functional Analysis of Pegasus: The ‘Atypical’ Member of the Ikaros Gene Family.
Authors: John Liza B , Ward Alister C 
Published: October 2019

Abstract
Abstract 3645
Poster Board III-581
The Ikaros family of zinc finger transcription factors are an important class of hematopoietic regulators involved in the development of the immune system. However, the in vivo role of its most divergent ‘atypical’ member, Pegasus, remains unknown. Therefore, we have utilised zebrafish to explore the function of Pegasus. Bioinformatics analysis identified a single pegasus orthologue in zebrafish. Wholemount in situ hybridisation revealed an initial broad distribution of maternally-derived pegasus transcripts during early zebrafish embryogenesis, which was replaced by specific expression in the brain, eye, thymus, gut and pancreas during late embryogenesis. Morpholino-mediated gene knockdown of zebrafish pegasus resulted in a reduction in cells expressing markers for hematopoietic stem cells (lmo2), granulocytes (mmp9), macrophages (fms), as well as early (ikaros) and late (rag1) lymphoid cells while, conversely, those expressing early (spi1) and late (l-plastin) myeloid markers were increased. No difference was noted in the early erythroid (gata1) compartment, although mature red blood cells appeared reduced, as judged by o-dianisidine staining. During emergency hematopoiesis (elicited by LPS injection), numbers of granulocytes (mpo+) and macrophages (lyz+) were both significantly further reduced. In addition, morphants also showed anterior defects, including reduced forebrain, smaller eyes and otoliths, which corresponded with a reduction of expression of the brain markers pax2.1, krox20 and dlx1. Gut expression of ifabp also appeared affected. Collectively, these experiments suggest a clear role for pegasus in hematopoiesis, as well as novel functions in brain and gut development.

Disclosures:
No relevant conflicts of interest to declare.
Loss of the Gata1 Gene IE Exon Leads to Variant Transcript Expression and the Production of GATA1 Protein Lacking the N-Terminal Domain.
Authors: Kobayashi Eri , Shimizu Ritsuko , Kikuchi Yuko , Takahashi Satoru , Yamamoto Masayuki 
Published: October 2019

Abstract
Abstract 3648
Poster Board III-584
GATA1 is a transcription factor essential for the differentiation of erythroid cells and megakaryocytes. Since GATA1 regulates genes related to the survival, proliferation and differentiation of hematopoietic cells, regulation of the Gata1 gene expression is critically important for the understanding of hematopoiesis. The Gata1 locus contains multiple untranslated first exons plus five common coding exons. Of these first exons, erythroid first exon (IE exon) is important for the Gata1 gene expression in the hematopoietic lineages. However, due to the embryonic lethality of this IE exon knockdown mice, less is understood about the contribution of the IE exon to adult hematopoiesis. Here, we achieved specific deletion of the IE exon in adulthood by crossing the IE-floxed mice with the interferon-inducible Mx1-Cre transgenic mice. This conditional IE-deletion mouse (ΔIE mouse) showed severe thrombocytopenia with increased premature megakaryocytes similarly to the phenotypes reported in the conditional Gata1 knockout mice in which the entire Gata1 gene was deleted in adulthood. In addition, the ΔIE mice showed severe anemia with skewed erythroid maturation, and importantly this erythroid phenotypes substantially differed from those observed in the conditional Gata1 knockout mice. Further analyses revealed that the Gata1 mRNA level in the megakaryocytic lineage was significantly downregulated. By contrast, in the erythroid lineage, Gata1 mRNA was retained at a comparable level to that in control mice utilizing two alternative first exons; one was the IEb/c, which was previously reported as a first exon rarely used in hematopoietic cells, and the other was newly identified IEd exon located within the second intron. Surprisingly, in the ΔIE mice these transcripts failed to produce full-length GATA1 protein, but instead inefficiently yielded GATA1 lacking the N-terminal 83 amino acids. This form of GATA1 is often observed in Down syndrome-associated transient myeloproliferative disorder and acute megakaryoblastic leukemia. Of note, the transcript derived from exon IEb/c preserved the first translation initiation codon in exon 2 but lost the potential to select the first translation initiation codon or failed to produce full-length GATA1. The present study demonstrates that the IE exon is instrumental to adult erythropoiesis by regulating the proper level of transcription and by selecting the correct translation start site for production of adequate full-length GATA1 protein.

Disclosures:
No relevant conflicts of interest to declare.
Myeloid-Derived Suppressor Cells in Patients with Hodgkin Lymphoma.
Authors: Parrinello Nunziatina , La Cava Piera , Tibullo Daniele , Giallongo Cesarina , Di Bartolo Orazio , Spina Paolo , Fiumara Paolo , Schinocca Luciana , Conticello Concetta , Chiarenza Annalisa , Palumbo Giuseppe A , Di Raimondo Francesco 
Published: October 2019

Abstract
Abstract 3662
Poster Board III-598

Background
Immune suppression and angiogenesis are mechanisms key to tumour growth and progression. Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of cells of myeloid origin and include immature macrophages, dendritic cells (DC) and other myeloid cells. In mice are phenotypically characterized as CD11b+Gr-1+ cells, while in human they have an immature phenotype, including lineage negative (Lin-), CD14-, HLA-DR-, CD15+, CD34+, CD11b+, CD33+, and CD13+ cells. MDSC reduce activated T-cell number and inhibit their function through different mechanisms including: L-arginine metabolism, nitric oxide (NO), up-regulation of reactive oxygen species (ROS), and secretion of immunosuppressive cytokines. MDSC also promote tumor-dependent angiogenesis as well as tumor metastasis.
Their accumulation has been described in patients affected by some solid tumors but information on haematological neoplasms are lacking. Our study investigated by flow cytometry the presence of MSDC in the peripheral blood of patients affected by Hodgkin Lymphoma (HL).

Methods
We studied 14 patients with HL at diagnosis and 10 age-matched healthy controls (HC). Peripheral blood mononuclear cells were stained with the following monoclonal antibodies:CD11b, CD13, CD14, CD34, CD45, for 20 minutes at room temperature. After lysing red cells, cells were analyzed by flow cytometry.

Results
we observed a increased number of MDSC (CD11b+,CD13+,CD34+,CD14-, CD45+) in the peripheral blood of patients with HL compared to HC (13,37 ± 17,77 ×109/l vs 1,45± 0,98 ×109/l, p=0,0007). We also found that patients with advanced-stage Hodgkin disease (III and IV) have higher number of MDSC, compared to patients stage I and II (p= 0,04).

Conclusion
These data suggest a role for myeloid-derived suppressor cells in promoting tumor cell proliferation in hodgkin lymphoma.

Disclosures:
No relevant conflicts of interest to declare.
Irradiation-Mediated Thymic Plasticity Is An Age-Related Phenomenon in Immunodeficient Mice.
Authors: Singh Kamaldeep , Myers Aaron , Prockop Susan E 
Published: October 2019

Abstract
Abstract 3677
Poster Board III-613
Successful T lymphopoiesis is essential for maintaining resistance to infection, mediating graft versus tumor activity, and as a platform for some immune based therapies. In mouse models of T cell immunodeficiency syndromes successful T cell reconstitution requires robust cellular reconstitution as well as remodeling of the thymic stroma. We have previously demonstrated that robust thymic lymphopoiesis in young IL-7 receptor deficient (IL7R−/−) mice can occur without prior irradiation and have linked this to the presence of intact stromal niches for DN3 precursors. In contrast to IL7R−/− mice, RAG−/− mice generate wild type T lymphocytes via thymic T lymphopoiesis after transfer of wild type precursors, but the thymus of transplanted RAG−/− mice remains hypocellular. As has been shown before, thymic cellularity of young RAG−/− mice can be rescued by irradiation followed by transfer of wild type precursors. We now demonstrate a profound difference between young and old RAG−/− mice in the thymic remodeling that occurs after irradiation and transfer of wild type hematopoietic stem cells. While the thymi of young RAG−/− mice undergo profound remodeling and develop normal cortico-medullary areas with segregation of single positive cells to the medulla, older RAG−/− mice do not undergo this remodeling and the cortical and medullary stroma remains less organized and more interdigitated. In addition, while the thymi of these older RAG−/− mice do develop small nests of medullary thymic epithelial cells (identified by flow cytometry and immunohistochemistry) not visualized in the untreated RAG−/− mice, these cells do not appropriately express MHC and these thymi do not appropriately segregate single positive developing T lymphocytes to these medullary areas. In vitro testing of sorted single positive thymocytes from these mice demonstrates failure of appropriate negative selection. Together these studies identify a specific defect in the thymic stroma of aging immunodeficient mice preventing appropriate de novo T lymphopoiesis.

Disclosures:
No relevant conflicts of interest to declare.
RIAM and RapL Regulate Distinct Signaling Events and Functional Outcomes Upon TCR-Mediated Activation.
Authors: Patsoukis Nikolaos , Lafuente Esther M , Boussiotis Vassiliki A 
Published: October 2019

Abstract
Abstract 3683
Poster Board III-619
Rap1, a small GTPase of the Ras superfamily, originally identified by its ability to reverse Ras-mediated transformation, is now known to regulate cytoskeletal reorganization, cell morphology, adherens junction positioning and adhesion. The best-studied function of Rap1 is inside-out activation of integrins and cell adhesion. Two newly identified Rap1 effectors, RapL and RIAM, have been implicated in Rap1 mediated in inside-out activation of integrins and cell adhesion. However, significant differences in the structure and interactions of these molecules indicate that they may mediate distinct signaling events. RIAM has a N-terminal coiled-coil region, central RA and pleckstrin homology (PH) domains, and proline-rich N-terminal and C-terminal regions, with multiple FPPPP motifs capable of interacting with the EVH1 domains of the actin regulatory proteins Ena/VASP, multiple XPPPP motifs interacting with Profilin and multiple PXXP motifs capable of interacting with SH3 domain containing proteins. Because of these properties RIAM is a regulator of actin polymerization but also interacts with components of the T cell signaling machinery. In contrast, RapL has an RBD (Ras binding domain-structurally similar to RA domain) and a C-terminal coiled-coil region and interacts with Rap1 via its RBD domain and its N-terminal region, and with the aL subunit of LFA-1 via its C-terminal domain. In the present study we investigated the role of RIAM and RapL in regulating signaling and functional events activated via the TCR. For this purpose we used RIAM-knockdown (KD) and RapL-KD Jurkat T cells in which endogenous RIAM and RapL, respectively, had been depleted by siRNA. Whereas activation of the extracellular signal regulated kinases MEK1/2 and Erk1/2 was impaired by depletion of RIAM, activation of these kinases was unaffected by depletion of RapL. In contrast, activation of p38 was unaltered in RIAM-KD cells but was abrogated in RapL-KD cells. Moreover, RIAM knockdown resulted in impaired activation of Ras and Rap1 due to defective activation of the calcium and diacylglycerole-dependent GEFs, RasGRP1 and CalDAG-GEFI. In contrast, RapL knockdown had no effect on these events compared to control Jurkat T cells. Strikingly, RIAM-KD cells displayed impaired IL-2 production in response to stimulation with SEE-loaded APC or to TCR/CD3-plus-CD28 crosslinking, whereas RapL-KD cells displayed a dramatic increase in IL-2 production upon stimulation under the same conditions. These results indicate that although both RIAM and RapL regulate Rap1-dependent LFA-1 activation, these molecules have distinct roles in regulating signaling and functional outcomes of T cell responses after T cell receptor triggering.

Disclosures:
No relevant conflicts of interest to declare.