bluuebunny/crossref_metadata_2025_split huggingface dataset:
TLR-Signaling Induces Granulocytic MDSCs Generation by Directing Myeloid Lineage Commitment During Polymicrobial Sepsis
Authors: Kong Yaxian , Zhang Weimei , Liu Wei , Miao Peng , Zeng Hui 
Published: October 2019

Abstract
Abstract 2160

Introduction:
Elevated leukocyte frequency at early stage of polymicrobial sepsis is a common phenomenon, which is considered to be a result of rapid migration of neutrophils from the bone marrow (BM). However, a dramatic increase of granulocytic myeloid-derived suppressor cells (MDSCs) of sepsis usually occurs after sustained stimuli of the pathogens, which implies that emergency granulopoiesis is involved in negative regulation of immune response. In the present study, we try to reveal the mechanisms by which TLR signal regulates emergency myelopoiesis during infection.

Methods and models
:B Mice were subjected to cecal ligation and puncture to make polymicrobial sepsis model. Blood, spleens and bone marrow were harvested post operation at 1, 3, 7 and 12 day, and analyzed using flow cytometry. Hematopoietic stem cells and special progenitors were isolated using an FACS Aria sorter, and then total mRNA was extracted and reverse-transcripted. Some lineage commitment-related genes including Ikzf1,Gfi1, Cebp/a, PU.1, Csf1r, Csf2r, Csf3r, Notch1, IL-7r, Flt3 were analyzed by Real-time PCR. The methylcellulose colony assay of single-cell suspensions of bone marrow cells was used to identify myeloid progenitors in sepsis model.

Results:
We found that the elevated frequency of myeloid cells was associated with an expansion of hematopoietic stem cell (HSC) pool accompanied by augmented proportion of cells entering cell cycle. Furthermore, increased HSC numbers resulted in gradually elevated proportions of common myeloid progenitors (CMPs) and granulocyte/macrophage progenitors (GMPs), but a decrease of the frequency of common lymphoid progenitors (CLPs). In line with the decreased ratio of lymphoid/myeloid progenitors, real-time PCR revealed impaired expression levels of lymphoid differentiation-related genes in HSCs and CLPs, including Flt3, IL7R, Notch1, Ikaros and Rag1. By contrast, the expression levels of myeloid differentiation-related genes (eg., CEBP/a, Gfi1, PU.1, GM-CSFR) were up-regulated or remained unchanged in the stem cell and myeloid progenitor compartments. Moreover, consistent with elevated frequency of granulocytic MDSCs, an increase of Gfi-1 expression which made a granulocytic decision and a decrease of M-CSFR expression which played a crucial role in monopoiesis were noticed in CMPs and GMPs. More importantly, similar results were obtained in the mice challenged with LPS peritoneal injection.

Conlusion:
Our study sheds light on its importance of TLR-signaling in generation and replenishment of granulocytic MDSCs by affecting early myeloid /lymphoid lineage decision and late granulocyte /monocyte lineage decision.

Disclosures:
No relevant conflicts of interest to declare.
Surface HLA-DR Expression in Monocyte Subpopulations During Adverse Events After Hematopoietic Stem Cell Transplantation
Authors: Döring Michaela , Haufe Susanne , Erbacher Annika , Müller Ingo , Handgretinger Rupert , Hofbeck Michael , Kerst Gunter 
Published: October 2019

Abstract
Abstract 2161

Background:
Human leukocyte antigen DR (HLA-DR) surface expression in CD14++ monocytes reflects the activation state of these cells. Decreased HLA-DR expression levels have been described as an indicator of immunosuppression. According to CD14 and CD16 expression levels, monocyte subpopulations have been classified into “classical” CD14++/CD16- (M1), “non-classical” CD14+/CD16++ (M3), “intermediate” CD14++/CD16+ (M2) and CD14-/CD16++ (M4) monocytes (Figure 1). This study analyzes HLA-DR expression in these monocyte subpopulations after hematopoietic stem cell transplantation (HSCT) and in particular during transplant-related adverse events.

Patient and Methods:
30 pediatric patients (< 15 years) (n = 23) and young adults (> 15 years) (n = 7) (7 autologous, 12 allogeneic and 11 allogeneic haploidentical transplanted) with hemato-oncological malignancies and immunodeficiency disorders were consecutively included after informed written consent. Median age was 9.5 years (range 0.5–38 years). Flowcytometric assessment of HLA-DR expression was started the day before conditioning therapy and continued up to 379 days after HSCT. Normal values of HLA-DR expression in monocyte subpopulations were established from a control group of healthy children and young adults (n = 20). Surface marker expression of CD14, CD16, CD56, and HLA-DR was determined by four-color flowcytometry.

Results:
Median data acquisiton period was 228 days (range of 43–379 days). By analysis of covariance (ANCOVA), HLA-DR expression in healthy controls varied in dependency of age. In young adults (> 15 years), HLA-DR expression was significantly (P < 0.05) lower in M2, M3 and M4 but not in M1 monocyte subpopulations (P = 0.084) compared to children and adolescents (< 15 years). After HSCT, a significant decrease of HLA-DR expression in M1 and M4 monocytes was observed during sepsis or bacteremia (P < 0.01). The onset of graft-versus-host-disease (GvHD)°III-°IV was associated with a significant increase of HLA-DR expression in M1 monocytes (P < 0.05). HLA-DR expression in monocyte subpopulations was not altered before or during veno-occlusive disease (VOD), a relapse of underlying disease or before death.

Conclusion:
The distinct HLA-DR expression pattern in monocyte subpopulations during sepsis and GvHD may facilitate identification of patients at risk after HSCT. Moreover, HLA-DR expression in monocyte subpopulations may be used to monitor treatment response in these entities.
CD14++/CD16- (M1), CD14++/CD16+ (M2), CD14+/CD16++ (M3) and CD14-/CD16++(M4)

Disclosures:
No relevant conflicts of interest to declare.
Cellular and Biochemical Properties of Cell-Free DNA: A Prognostic Marker In Severe Sepsis Patients
Authors: Gould Travis J , Dwivedi Dhruva , Pepler Laura , Swystun Laura , Lysov Zakhar , Liaw Patricia 
Published: October 2019

Abstract
Abstract 2169

Background:
Sepsis is the leading cause of morbidity and mortality in patients in the non-coronary ICU. However, the heterogeneity of patients with severe sepsis makes the identification of high-risk patients (ie. those who are sickest and thus would require more aggressive and expensive therapies) an ongoing challenge. Previously, we have shown that high levels of cell-free DNA (cfDNA) appear to be a powerful predictor of death in severe sepsis patients. Using Receiver Operating Characteristic curves, we have demonstrated that the area under the curve for cfDNA is 0.96 (95% CI=0.93–1.00). The purpose of this study is to characterize the source and biochemical properties of cfDNA in sepsis.

Methods:
Toll-like receptor (TLR) cells (which are stimulated by microbial-specific, unmethylated DNA) were used to determine whether the cfDNA found in septic patients is host- or microbe-derived. Whole blood, monocytes, and neutrophils were isolated from healthy volunteers and stimulated with either lipoteichoic acid and peptidoglycan (from Gram-positive bacteria) or lipopolysaccharide (LPS) from Gram-negative bacteria. Monocytes and neutrophils were also incubated with LPS-induced apoptosis inhibitors, cilostamide and cilostazol, prior to stimulation with LPS. DNAse enzymatic activity was quantified using Org 590 DNAse activity ELISA (Orgentec Diagnostica, GmbH), while DNAse protein was quantified by Western blot analyses.

Results:
Exposure of TLR9-reporter cells to cfDNA from sepsis patients revealed that the cfDNA is host-derived. Stimulation of whole blood, monocytes, neutrophils, and endothelial cells to microbial cell wall components revealed that monocytes and neutrophils are the major sources of cfDNA in sepsis. Of the total amount of nuclear DNA present within monocytes and neutrophils, nearly all was released into the supernatant upon stimulation. Inhibition of LPS-induced apoptosis with cilostamide and cilostazol did not completely prevent the release of cfDNA from monocytes and neutrophils. We also found that cfDNA levels correlated with levels of histones in septic patients and that the cfDNA was degraded to nucleosomal units. Finally, we observed an inverse correlation between levels of DNAse activity/DNAse protein and levels of cfDNA.

Conclusions:
These studies are the first to characterize the source and biochemical properties of cfDNA in sepsis. A portion of cfDNA release from monocytes and neutrophils appears to occur via an apoptosis-independent mechanism. Plasma levels of DNAse enzyme were reduced in septic patients compared to healthy volunteers.

Disclosures:
No relevant conflicts of interest to declare.
G6PC3 Deficiency Associated with Congenital Neutropenia and Enterocolitis
Authors: Diestelhorst Jana , Kotlarz Daniel , Appaswamy Giridharan , Beier Rita , Krawitz Peter M , Robinson Peter N , Hecht Jochen , Puchalka Jacek , Schäffer Alejandro A. , Pfeifer Dietmar , Baumann Ulrich , Pfister Eva-Doreen , Brandes Gudrun , Palla Gabriella , Jacobs Roland , Kreipe Hans H. , Mundlos Stefan , Klein Christoph 
Published: October 2019

Abstract
Abstract 2170
Glucose-6-phosphatase catalytic subunit 3 (G6PC3) deficiency causes congenital neutropenia in conjunction with various cardiac or urogenital developmental aberrations.
We here describe a consanguineous pedigree of maroccan descent. Four patients presented with early-onset inflammatory bowel diseases associated with severe congenital neutropenia. Two children died in early childhood, two patients (19-years old female, 17-years old male) are alive with signs of recurrent infections (mouth ulcera, otitis, upper and lower respiratory tract infections) and refractory Crohn's like enterocolitis. Mutations in the Glucose-6-phosphate translocase (SLC37A4) gene were excluded.
In an attempt to discover the underlying molecular pathophysiology, we performed SNP-based homozygosity mapping followed by in depth next-generation exome sequencing. We identified two genes with potentially deleterious homozygous sequence variations: P2RY11 (exon 13; c.C2160G, p.Y720X), encoding a protein with antiapoptotic function, and G6PC3 (exon 2; c.C323T, p.P108L). The G6PC3 mutation segregates with the disease phenotype, whereas the P2RY11 variant was also found in a healthy sibling.
Functional experiments in patient's neutrophil granulocytes showed an accelerated oxidative burst capacity (OxyBurst Assay, Phagoburst Assay). Furthermore, G6PC3-deficient individuals showed a rapid dissipation of the mitochondrial membrane potential upon treatment with valinomycin, whereas the inner mitochondrial membrane potential in neutrophils from healthy individuals was maintained. We observed increased apoptosis in G6PC3-deficient neutrophils upon exposure to staurosporine. Interestingly, increased apoptosis was also seen in granulocytes from the healthy sibling with the P2RY11 sequence variant, suggesting that P2RY11 deficiency may aggravate the phenotype of G6PC3 deficiency.
In summary, our study indicates that the clinical spectrum of G6PC3 deficiency may be more diverse than previously appreciated and that P2RY11 may have a modifying effect on G6PC3-deficient neutrophil granulocytes.
J.D. and D. K. contributed equally to this work and should be considered aequo loco.

Disclosures:
Schäffer: National Institues of Health: Employment; Intramural Research Program of the National Institues of Health, NLM: Research Funding.
The Significance of mTOR Inhibitor, Everolimus in TGF-β-Induced Regulatory T cells from Cord Blood
Authors: Nagamura-Inoue Tokiko , Kobayashi Seiichiro , Ogami Kazuo , Yamamoto Yuki , Izawa Kiyoko , Tojo Arinobu 
Published: October 2019

Abstract
Abstract 2180

Background:
Regulatory T cells (Tregs) play an important role in immune-tolerance to allograft. Cord blood (CB) is rich in naïve T cells and is a promising source of inducible Tregs (iTregs), since it was reported that stable iTregs may be derived exclusively from naïve T cells. However, the standard method for iTregs has not yet been established. Here we studied the impact of mTOR inhibitors, rapamycin (Rap) and everolimus (Eve), on ex vivo expansion of iTregs from CB-CD4+ T cells. Methods: CB-CD4+ T cell were isolated using anti-CD4 monoclonal antibody (MAb)-conjugated magnetic beads, and cultured in a flask coated with anti-CD3/CD28 MAbs and supplemented with IL-2 and TGF-β in the presence or absence of Rap or Eve. After two weeks of culture, the total number of CD4+ T cells was calculated, and the incidence of CD25+Foxp3+ cell population among those was estimated by FACS. Results and Discussions: Both Rap and Eve significantly increased the incidence of CD25+Foxp3+ cell population in CD4+ T cells. However, Rap apparently inhibited their growth and did not increase the absolute number of CD25+Foxp3+ cells in comparison to the control. On the other hand, Eve contributed to efficient expansion of iTregs at the concentration between 1 and 50ng/ml without no significant inhibition of their growth. Expansion of CD4+ T cells with TGF-β and Eve yielded 71.5 ±23.5% purity of CD25+Foxp3+ cells which also expressed CTLA-4 as well as the memory phenotype, while the purity obtained with TGF-β only was 47.4±30.0% and that without TGF-β/Eve was 7.3±4.5%. Thus, an average of 2.95±2.8 x107 iTregs were obtained from the initial input of 5×104 CD4+ T cells. The resulting iTregs with TGF-β, TGF-β/Rap and TGF-β/Eve inhibited the proliferation of CFSE-labeled T cells stimulated with allogeneic dendritic cells. The precise mechanism for Foxp3 induction by mTOR inhibitors still remains to be elucidated. Furthermore, we found that expression of CD26 (DPP-IV) was significantly down-regulated in CD4+ T cells expanded with TGF-β and profoundly with TGF-β/Eve, while CD127 was negative after culture in all the conditions. Mean fluorescence intensity of CD26 indicated 67.5 in CD4+ T cells without TGF-β, 1.58 with TGF-β, 0.18 with TGF-β/Rap and 0.12 with TGF-β/Eve, respectively. Accordingly, CD26 negativity may be an indicator of iTregs together with Foxp3. Conclusion: mTOR inhibitor, Eve, is an efficient co-inducer of iTregs and applicable to ex vivo expansion of iTregs in a clinical setting.

Disclosures:
No relevant conflicts of interest to declare.
MHC Class II and T Cell Receptor Signals Are Dispensable for IL-2-Induced Regulatory T Cell Proliferation In Vivo
Authors: Zou Tao , Satake Atsushi , Maltzman Jonathan , Kambayashi Taku 
Published: October 2019

Abstract
Abstract 2181
Regulatory T cells (Tregs) protect the host from autoimmunity and inappropriate immune activation. Thus, to ensure immune tolerance in the steady state, an adequate number of peripheral Tregs must be constantly maintained. Prior work has suggested that major histocompatibility class II (MHC II) and interleukin-2 (IL-2) are both necessary to maintain peripheral Treg homeostasis and proliferation in vivo. However, we have recently reported that Treg proliferation may not strictly depend on MHC II, as the provision of IL-2 was sufficient to drive proliferation of Tregs in an MHC II-independent manner in vitro, as long as the Tregs interacted with dendritic cells (DC)s.
Here, extending our previous in vitro observations, we tested the dependence of Treg proliferation on IL-2, DCs, and TCR signaling in vivo. Proliferation of adoptively transferred Tregs was detected in wildtype (WT) mice. This proliferation was markedly enhanced when the mice were injected with IL-2 immune complexes (IC)s but not when the IL-2 IC-injected mice lacked DCs, suggesting that IL-2-induced Treg proliferation was dependent on DCs in vivo. As previously reported, adoptively transferred Tregs did not proliferate in MHC II-deficient hosts. However, the injection of IL-2 ICs into these mice induced Treg proliferation comparable to those transferred into IL-2 IC-injected WT mice, suggesting that IL-2 signaling by Tregs obviated the need of MHC II for their proliferation. Furthermore, while the ablation of TCR signaling by conditional deletion of the adaptor protein SH2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) rendered Tregs unable to proliferate by themselves, IL-2 IC treatment partially rescued this deficiency.
We next examined the signaling pathways involved in Treg proliferation downstream of the IL-2 receptor. Despite the importance of the Stat5 pathway in IL-2 receptor signaling during Treg development in the thymus, activation of Stat5b alone was insufficient to rescue proliferation of SLP-76-deficient Tregs, indicating that alternative pathways must also be activated for Treg proliferation. Additional studies investigating the role of other signaling molecules downstream of the IL-2 receptor are currently underway.
In summary, we have demonstrated for the first time that Tregs do not require TCR signaling through interaction with MHC II for their proliferation in vivo. We propose that this MHC II-independent mode of Treg proliferation allows Tregs with multiple antigen specificities to proliferate, which ensures that a diverse TCR repertoire is continuously maintained in the Treg pool. Furthermore, we believe that exploitation of these pathways may be therapeutically beneficial in autoimmunity and in transplantation.

Disclosures:
No relevant conflicts of interest to declare.
Fibronectin Isoform Containing the Alternatively-Spliced Extra Domain A in Circulation Accelerates the Development of Atherosclerosis in Mice
Authors: Gandhi Chintan , Khan Mohammad Moshahid , Chauhan Anil K 
Published: October 2019

Abstract
Abstract 2205

Background and Objective:
The fibronectin isoform containing the alternatively-spliced extra domain A (EDA+-FN) is normally absent from the circulation, but plasma levels of EDA+-FN can become markedly elevated in several pathological conditions including atherosclerosis. It remains unclear in humans whether these elevated levels of EDA+-FN are actively contributing to disease pathogenesis, or rather simply serving as a marker associated with vascular stress and/or injury. Several in vitro studies suggest that EDA+-FN can activate toll-like receptor 4 (TLR4), an innate immune receptor that triggers pro-inflammatory responses We hypothesize that presence of EDA+-FN in plasma promotes inflammation and accelerates atherosclerotic plaque formation.

Model and Method:
We generated EDA+/+/ApoE−/− mice, which contain optimized spliced sites at both splicing junctions of the EDA exon and constitutively express only EDA+-FN, and EDA−/−/ApoE−/− mice, which contain an EDA-null allele of the EDA exon and express only FN lacking EDA. ApoE−/−, EDA+/+/ApoE−/− and EDA−/−/ApoE−/− were fed a high-fat Western diet (21% fat and 0.2% cholesterol) beginning at 6 weeks until they were sacrificed at 5 months of age (i.e., 14 weeks on high-fat Western diet). We compared the extent of atherosclerosis in whole aortae, stained with Oil Red O and en face lesion area measured by morphometry, and in the cross section area of the aortic sinus using the VerHoeffs/Van Gieson stain.

Results:
We report that atherosclerotic plaque (% of total aorta) formation in the aorta of EDA+/+/ApoE−/− mice was increased by two-fold compared to control ApoE−/− mice (P<0.0001). Deletion of the alternatively spliced EDA domain in the ApoE−/− mice (EDA−/−/ApoE−/−) significantly reduced atherosclerotic plaque formation in the aorta (P<0.05) compared to ApoE−/− mice. Total cholesterol and triglycerides levels were similar in ApoE−/−, EDA+/+/ApoE−/− and EDA−/−/ApoE−/− mice. Similarly, atherosclerotic plaque formation was significantly increased in the aortic sinus of EDA+/+/ApoE−/− mice, intermediate in control ApoE−/− mice and reduced in EDA−/−/ApoE−/− mice (P<0.05). Additionally, we found that macrophage content, as analyzed by immunohistochemistry, was significantly elevated in the aortic root lesions of EDA+/+/ApoE−/− mice and reduced in EDA−/−/ApoE−/− mice compared to ApoE−/− mice (P<0.05). Moreover, EDA+-FN did not affect the sex-dependent regulation of atherosclerosis in ApoE−/− mice. Future experiments using EDA+/+/ApoE−/−/TLR4−/− are under progress to determine whether EDA+-FN exacerbate atherosclerosis via upregulating TLR4 signaling.

Conclusions:
Our findings reveal that EDA+-FN is pro-inflammatory and promotes atherosclerotic lesion formation and that monitoring plasma EDA+-FN might have prognostic value in patients at high risk for atherosclerosis.

Disclosures:
No relevant conflicts of interest to declare.
The Von Willebrand Factor D'D3 Domains Modulate the Interacton of Platelet Receptor Glycoprotein Ib with the VWF-A1 Domain Under Conditions of Shear Stress
Authors: Nowak Agata Anna , Shah Amar , Laffan Michael , McKinnon Thomas A J 
Published: October 2019

Abstract
Abstract 2212
The VWF A1 domain contains the binding site for the platelet receptor glycoprotein (GP)Ib receptor, and this interaction forms the initial tethering event to capture platelets at sites of injury under high shear stress. The VWF-A1:GPIbα interaction has a high association and dissociation rate and as such platelets translocate along the VWF surface until stable attachment can occur. It has been previously shown that under static conditions the VWF D'D3 domains shield the A1 from interaction with GPIbα, however it has not been established if this occurs under conditions of shear stress. To this end we constructed VWF fragments spanning the D'-A3 domains and the A1A2A3 domains. These were expressed and purified and the ability of these constructs to interact with platelets was analysed in an in vitro flow assays system. Purified and fluorescently labelled platelets resuspended in plasma free blood were perfused over immobilised VWF at a range of shear rates (400–2000s−1), and real time movies of the interactions captured. Interestingly, platelets translocated slower across VWF-A1A2A3, with lower rolling velocities compared to VWF-D'A3 at all shear rates tested. Significantly the dissociation rate constant (koff) of the transient tethers formed between GPIbα and VWF was similar for both constructs, indicating that that the observed difference in platelet rolling velocity is not due to an alteration in bond lifetime. Frame-by-frame analysis of platelet translocation demonstrated that platelets paused more often on VWF lacking the D'D3 domains and therefore translocated a shorter distance in a set time when compared to VWF D'A3 resulting in decreased rolling velocities. We hypothesise that the accessibility of the GPIb binding site in the A1 domain is increased in the absence of the D'D3 domains, resulting in more interactions. Together these data demonstrate the D'D3 domains are able to shield the A1 domain under shear stress conditions.

Disclosures:
No relevant conflicts of interest to declare.