bluuebunny/crossref_metadata_2025_split huggingface dataset:
C5a Depletion As a Novel Strategy to Inhibit T Regulatory Cells and Enhance Antigen-Specific Immune Responses
Authors: Veeramani Suresh , Weiner George J. 
Published: October 2019

Abstract
Abstract 3285

Background:
The complement system has complex activity that impacts on the immune response in a broad variety of ways. The current study was designed to assess the effect of complement components, specifically C5a, on the immune regulatory cells and on the development of an antigen-specific active immune response.

Methods:
Myeloid dendritic cells (mDCs), enriched from healthy human peripheral blood mononuclear cells, were pulsed with antigen (tetanus toxoid) and co-cultured with autologous, enriched human CD4+ T cells in the presence of various purified complement components. The percent of CD4+ T-cells that were CD25highFoxp3+ (henceforth referred to as Tregs) was determined. The presence of cytokines in supernatant of mDCs cultured with purified complement proteins was also evaluated. In murine models, the effect of C5a on in vivo induction of Tregs and on the development of immune response to ovalbumin was determined by analyzing anti-ovalbumin antibody. This was done in C5-sufficient (B10-D2-HC1) and C5-deficient (B10-D2-HC0) mice immunized with 100 μg of ovalbumin, and in wild type C57Bl/6 mice immunized with 100 μg of ovalbumin along with either irrelevant rat IgG2a (Ova+Isotype control) or rat anti-mouse C5a antibody (Ova+anti-C5a Ab).

Results:

In Vitro:

In Vivo:

Conclusions:
Presence of C5a in the immune microenvironment results in increased generation of Treg cells and leads to dampening of antigen-specific immune responses. Absence or depletion of C5a results in a drop in the Tregs and a higher antigen-specific immune response. Ongoing studies are exploring the use of C5a depletion as a novel strategy to overcome the low immunogenicity of vaccines, such as cancer vaccines.

Disclosures:
No relevant conflicts of interest to declare.
TLR3 Activation Is Involved in Venous Thrombosis Development
Authors: Lemarie Catherine A , Le Angela , Aghourian Meghedi , Wu Jianqiu , Blostein Mark D 
Published: October 2019

Abstract
Abstract 3309
Venous thromboembolism (VTE) afflicts 117 people per 100,000 each year and is an important cause of morbidity and mortality. The pathophysiology of VTE is now better understood from experimental studies. Leukocytes, chemokines, and proinflammatory cytokines are involved in VTE resolution and vein wall healing. This process resembles sterile wound healing with distinct phases of polymorphonucleic neutrophil and monocyte influx followed by fibrosis. Inflammation is emerging as a central mechanism in both the genesis and resolution of VTE. Recently, the role of toll-like receptor (TLR) signaling in modulating sterile inflammation has become better defined in experimental injury models. TLR3 senses dsRNA, a by-product of viral replication, in the endosome. In addition, TLR3 has been increasingly linked to tissue damage. Endogenous RNA released by damaged tissue or necrotic cells is able to induce TLR3 expression and signaling. Thus, we hypothesize that TLR3 might be involved in the inflammatory development of VTE. Intravenous injection of polyinosine polycytidylic acid (polyI:C), a synthetic double-stranded RNA analog, increases the size of thrombi after FeCl3-induced inferior vena cava injury (IVC) compared to mice treated with vehicle control (p<0.05). In TLR3 deficient (TLR3−/−) mice, polyI:C did not induce a further increase in thrombus size compared to vehicle control. Recently, neutrophils have been shown to initiate and propagate venous thrombosis. PolyI:C injection was associated with an increased of neutrophil infiltration in the thrombus in WT but not in TLR3−/− mice (p<0.05). We found that polyI:C injection was associated with increased neutrophil activation. In the thrombus of WT mice, immunofluorescence staining for myeloperoxidase and citrulinated H3, markers for neutrophils activation, were increased by polyI:C. Interestingly, in TLR3−/− mice, no increase of these markers was found after polyI:C injection as compared to vehicle control. In vitro incubation of endothelial cells with polyI:C induces production of pro-inflammatory cytokines IL-8 and CCL5 involved in the recruitment of neutrophils as demonstrated by a cytokine array. We found that mRNA expression of TLR3, IL-8 and CCL5 were dramatically increased with polyI:C. When endothelial cells were transfected with siRNA for TLR3, mRNA expression of TLR3, IL-8 and CCL5 was blunted in response to polyI:C. These results suggest that release of IL-8 and CCL5 from endothelial cells after TLR3 activation are involved in neutrophil recruitment. Taken together, these results strongly suggest that TLR3 stimulation after endothelial cell injury participate in thrombus formation by inducing a pro-inflammatory response leading to the recruitment and activation of neutrophils.

Disclosures:
No relevant conflicts of interest to declare.
Distinct Functions of pH-Sensing Histidine Residues in the Multimer Assembly and Storage of Von Willebrand Factor in Weibel-Palade Bodies
Authors: Huang Jing , Westfield Lisa A , Sadler J. Evan 
Published: October 2019

Abstract
Abstract 3311
The hemostatic function of von Willebrand factor (VWF) depends on the formation of disulfide-linked multimers. We have previously shown that several evolutionarily conserved His residues within the VWF propeptide are critical for pH-dependent VWF multimerization. For example, the charge-neutralization mutations H395A and H460A in the VWF D2 domain prevented multimer assembly, while the charge-stabilization mutation H460K preserved it, suggesting that protonation at these sites is essential. In this study, we investigated the role of these His residues in VWF targeting to storage granules and agonist-induced secretion. Full-length VWF was expressed in transiently-transfected HEK293 cells. If Weibel-Palade body-like storage granules (WPBs) were formed, their morphology was analyzed by immunofluorescence microscopy. A WPB was defined as “long” if length/width was > 2, or “round” if length/width was ≤ 2. To analyze the assembly of VWF multimers, we stimulated VWF secretion by incubating cells with 100 ng/ml of phorbol myristate acetate for 1 hour and collected cell lysates and supernatants for multimer gel electrophoresis. Expression of wild type VWF and VWF H460K induced the formation of approximately 60% long WPBs. Treatment of cells with monensin increases the pH of the Golgi, and as expected monensin reduced the storage of wild type VWF in long WPBs. However, monensin did not impair the storage of VWF H460K or reduce the fraction of long WPBs, indicating that a positive charge at residue 460 can sustain tubular storage of VWF despite neutralization of the late secretory pathway. Both WT VWF and VWF H460K were secreted with a normal distribution of multimers. VWF H395A and VWF H460A formed predominantly round pseudo-WPBs, with only 10–20% long granules, and these mutations also disrupted VWF multimerization. In contrast, VWF H395R and VWF H395A/H460R did not assemble multimers but nevertheless formed long WPBs. Insertion of an extra Gly between the vicinal cysteines in the D1 domain (159CGLC162 to 159CGGLC162) was reported to prevent multimerization but allow storage in long WPBs in AtT-20 cells (Mayadas & Wagner, PNAS 1992; 89:3531). We found that this mutation also inhibited VWF multimerization and resulted in the formation of long WPBs in HEK293 cells. These results indicate that VWF multimerization and packing in elongated WPBs both depend on the acidic pH of the late secretory pathway. However, these processes are independent, with distinct structural requirements, and they can be dissociated by mutations of specific His residues.
VWF construct Multimers Granule Shape Granule Shape (with monensin) Wild type Yes Long Round H395A No Round H395R No Long H395A/H460R No Long H460A No Round H460K Yes Long Long 159CGGLC162 No Long

Disclosures:
No relevant conflicts of interest to declare.
Incidence of Heparin Induced Thrombocytopenia in the Transplant Population: A Large Retrospective Cohort, Single Transplant Centre Experience
Authors: Hassan Syed , Khoulani Dania , Badami Ami , Alirhayim Zaid , Younes Mohamad A. , Kuriakose Philip 
Published: October 2019

Abstract
Abstract 3320

Objective:
Heparin induced thrombocytopenia (HIT), a prothrombotic complication of heparin therapy, can lead to serious thromboembolic events and cause significant morbidity and mortality. Its occurrence has never been studies in transplant patients, where use of heparin products is very common. We aim to study its prevalence in the transplant population at our institute.

Methods:
This is a retrospective cohort, single center study which looked into the clinical and laboratory database of all the patients that has undergone any kind of transplant at our institution over a period of 25 years (January 1985 - December 2010). In patients with clinical suspicious of HIT, a pre-test probability was calculated using the 4T scoring system. Results of the laboratory test like the ELISA HIT antibody (HIT ab) test and the functional serotonin release assay test (SRA) along with clinical manifestation of skin necrosis or thromboembolic events were reviewed.

Results:
Medical records of 2800 patients that has undergone transplant from January 1985- December 2010 were reviewed. HIT antibody assay was performed in 262 patients in which HIT was suspected. Of these, only 48 (18%) patients (mean age 57 ± 11 years, 71% women) had HIT ab positive, 9 were pre transplant recipient and remaining 39 were post transplant recipients. Baseline characteristics of the transplant population are illustrated in Table.1. Confirmatory test, SRA was performed in 8 HIT antibody positive patients, of whom only 4 were positive. The mean 4T score in HIT suspected patients was 3.7 ±1.3, while the score in HIT ab positive patients was 4.2 ± 1.2. Thrombotic complications were seen in 11(0.4%) patients, with the highest incidence rate of 1% in heart transplant recipients. No transplant patient had skin manifestations. Direct Thrombin inhibitor (DTI) was used only in 5 patients who had thrombotic events. No other complication or mortality was reported in any of the HIT ab positive transplant patients.

Conclusion:
To our knowledge, this is the first study of its kind that has shown very low incidence of HIT in transplant population. In conclusion, transplant patients can safely undergo any type of organ transplant, without having any peri or post operative complications or immediate mortality related to HIT.

Disclosures:
No relevant conflicts of interest to declare.
Primary Immune Thrombocytopenia Guidelines for Bone Marrow Aspirate Reduce the Diagnostic Rate of Myelodysplastic Syndromes
Authors: Vincelli Iolanda , Cufari Patrizia , Roda' Filippo , D'Errigo Maria Grazia , Oliva Esther Natalie , Nobile Francesco 
Published: October 2019

Abstract
Abstract 3328

Introduction:
About one quarter of patients with low risk myelodysplastic syndromes may present with isolated thrombocytopenia, defined by International Prognostic Scoring System (IPSS) as a platelet (PLT) count < 100 Gi/L. Primary immune thrombocytopenia (ITP) is defined as an acquired immune-mediated disorder characterized by isolated thrombocytopenia, PLT count <100 Gi/L, and the absence of any obvious initiating and/or underlying cause of the thrombocytopenia (Rodeghiero et al, Blood, 2009). Recent guidelines consider bone marrow (BM) aspirate informative in patients > 60 years of age, those with systemic symptoms or abnormal signs or in some cases where splenectomy is considered (Provan et al, Blood, 2009). We performed a retrospective chart analysis between January 2008 and July 2012 of all patients referred to our clinic for thrombocytopenia but with a PLT count 100– 149 Gi/L. The aim of the study was to evaluate whether BM aspirates in these patients be informative.

Results:
Twenty-six cases (18 males/8 females) of mean age 59 ± SD 17 years were evaluated at our clinic for a PLT count below normal lab range values, > 100 Gi/L. At the time of evaluation none had bleeding symptoms. PLT counts ranged from 101 to 149 Gi/L, mean 122 Gi/L. Five patients had an enlarged spleen. BM aspirates identified 20 patients with low IPSS risk MDS. Cytogenetics were obtained in 15 cases: 7 normal, 2 del20q, 6 –Y. Twelve patients were aged < 60; 8 (66%) were diagnosed with MDS. Amongst the 14 patients with an indication for BM aspirate (age > 60), 12 (86%) had MDS.

Conclusion:
The thresholds of PLT count 100 Gi/L and age 60 years for BM aspirate in patients with isolated thrombocytopenia reduces the diagnostic rate of MDS. In our retrospective review, 66% of patients (younger age and PLT > 100 Gi/L) would not have had MDS diagnosed. BM aspirate should be considered irrespective of age, since more than half of the patients in our case review had MDS with PLT > 100 Gi/L as a single cytopenia and age under 60.

Disclosures:
No relevant conflicts of interest to declare.
Activated Protein C Targets PI3K-p85/XBP1 Pathway to Inhibit Hyperglycemia Induced Endoplasmic Reticulum Stress in Diabetic Nephropathy
Authors: Isermann Berend H. , Wang Hongjie , Nawroth Peter P. , Madhusudhan Thati 
Published: October 2019

Abstract
Abstract 3354
Diabetic nephropathy (DN) is a multifactorial disease associated with substantial changes in the haemostatic system. A hallmark of diabetes induced haemostatic dysfunction is impaired thrombomodulin (TM) dependent-protein C (PC) activation. Impaired PC activation triggers glomerular podocyte and endothelial cell dysfunction, thus promoting DN. The intracellular mechanism through which loss of TM and PC activation contributes to DN is not known. Here we show that the haemostatic mediator activated PC (aPC) regulates cellular homeostasis by inhibiting hyperglycemia induced endoplasmic reticulum (ER)-stress in DN.
Hyperglycaemia was induced in wild-type (wt) mice or mice with altered activity of the TM-PC system (loss of function secondary to impaired PC activation (TMPro/LacZ) or gain of function with high aPC plasma levels (APChigh)). Subsets of diabetic mice were treated with the chemical ER-chaperone TUDCA. After 26 weeks of persistent hyperglycaemia markers of DN were determined and tissue samples were isolated for ex vivo analysis. Supplementary in vitro assays were performed in podocytes and endothelial cells.
Persistent hyperglycaemia in wt mice caused severe ER-stress and DN. Ex vivo analysis of transcription factors regulating the ER-stress response showed an increase of the ER-stress markers CHOP and ATF6, while nuclear translocation of the highly conserved transcription factor X-box binding protein-1 (XBP1) was reduced in DN. These changes were aggravated in diabetic TMPro/LacZ mice. Conversely, in a mouse model with constitutively higher aPC levels (APChigh mice) nuclear levels of XBP1 were normalized and expression of ATF6 and CHOP was reduced despite persistent hyperglycaemia. In addition, aPC reverses the pathological ER-stress alterations in diabetic TMPro/LacZ mice. Pharmacological inhibition of ER-stress by TUDCA normalized nuclear levels of XBP1, inhibited CHOP/ATF6 expression, and protected against DN in diabetic wt and TMPro/LacZ mice. In vitro hyperglycaemia inhibited nuclear translocation of XBP1 in endothelial cells and podocytes, the two cellular components of glomerular filtration barrier. Activated PC directly promotes the nuclear translocation of XBP1 in these cells, which is required to inhibit hyperglycaemia induced ER-stress. Deletion of XBP1 in podocyte or endothelial cells abolished the cytoprotective effect of aPC. Furthermore, aPC regulates the interaction of PI3K regulatory subunit p85α with XBP1 which is known to mediate its nuclear translocation and diminished hyperglycaemia induced ER-stress. These studies demonstrate that hyperglycemia induced ER-stress is causally linked to DN and establish a novel link between haemostatic system and ER function in regulating cellular homeostasis in chronic kidney disease.

Disclosures:
No relevant conflicts of interest to declare.
Crystal Structure of a Proteolytic Fragment of ADAMTS13 Reveals Alternative Disulfide Pairings in the Cysteine-Rich Domain
Authors: Skipwith Christopher G. , Stayrook Steven , Rottensteiner Hanspeter , Scheiflinger Friedrich , Zheng X. Long 
Published: October 2019

Abstract
Abstract 3363
Proteolytic cleavage of von Willebrand factor (VWF) by ADAMTS13 depends on its non-catalytic domain (or exosite) interactions. These exosites, particularly the residues between Tyr659 and Tyr665 and their adjacent residues Arg568 and Phe592 in the ADAMTS13 spacer domain, are the major targets of anti-ADAMTS13 autoantibodies in patients with acquired TTP. In an effort to further determine the potential exosite interactions between ADAMTS13 and VWF, we carried out crystal screening of a recombinant full-length ADAMTS13 purified from stably transfected Chinese hamster ovary cells. After over 900 conditions were tested, we obtained multiple well-formed and diffractable crystals. Upon X-ray diffraction, we discovered that only a proteolytic fragment of full-length ADAMTS13 (Met432-Arg670) had been consistently crystallized. Here, we report the structure of this non-catalytic ADAMTS13 fragment at 2.2Å. The fragment encompasses 8 residues from the first TSP-1 repeat, an entire cysteine-rich domain (CA and CB), and a majority of the spacer domain. While recombinant ADAMTS13 in the present study is fully glycosylated, the overall structure agrees very well with the analogous portion of the ADAMTS13-DTCS structure previously reported (RMSD = 0.02Å, 1817 atoms, 232 residues). A region within the CA domain does vary slightly from that in the ADAMTS13-DTCS (RMSD = 0.08Å, 379 atoms, 51 residues). As with the ADAMTS13-DTCS structure, our structure shows 3 intermolecular disulfide bonds in CA, in addition to 2 free cysteine residues. In this region, our structure shows differences in the disulfide bonding pairing, and a slight shift in the α4 helix from Gly479-Cys483. Specifically, our structure reveals an intermolecular disulfide bond between the residues Cys483 and Cys527 and Cys508 and Cys522. These disulfide pair patterns are different from those reported in the ADAMTS13-DTCS structure, in which disulfide bonds are formed between the residues Cys483 and Cys522 (4.6Å apart in our structure) and the residues Cys508 and Cys527 (3.8Å apart in our structure). Our results indicate that, despite the difference in conditions under which crystals of ADAMTS13 fragments are formed, the overall structure of the Cys-rich and spacer domains are quite similar. The difference in the disulfide bond pattering may provide a novel insight into the mechanism regarding the ADAMTS13 exosite interaction with VWF. Our ongoing work is aimed to determine the complex formation between ADAMTS13 and its substrate or autoantibodies.

Disclosures:
Rottensteiner: Baxter Innovations GmbH: Employment.
Prognostic Factors for Post-Thrombotic Syndrome in Children with Limb DVT: A Bi-Institutional Cohort Study
Authors: Lyle Courtney A. , Gibson Elizabeth , Lovejoy Amy , Goldenberg Neil A 
Published: October 2019

Abstract
Abstract 3401

Background:
Early identification of children with deep venous thrombosis (DVT) of the limb, whoare at heightened risk for post-thrombotic syndrome (PTS), is important in order to evaluate therapeutic interventions aimed at decreasing the risk and severity of PTS.

Objective:
We sought to evaluate prognostic factors for PTS in children following DVT of the limbs.

Methods:
In this bi-institutional mixed cohort study with prospective ascertainment of PTS using a validated pediatric instrument, we collected data on patient/thrombus characteristics, thrombophilia testing results, and outcomes in children (<21 years at event) diagnosed with acute limb DVT at Rady Children's Hospital of San Diego and Children's Hospital Colorado.

Results:
Median age at presentation was 13 years (range, 0–18 years). Cumulative incidence (i.e., risk of PTS) was 23%, at a median follow-up duration of 33 months (range: 13.2–65 months). The presence of a lupus anticoagulant by dilute Russell Viper venom time (dRVVT) testing within two weeks of DVT diagnosis was associated with markedly increased odds of developing clinically-significant PTS (OR 16.8, 95%CI (1.60–176.2); P=0.02). The presence of an infectious or inflammatory condition at DVT presentation was neither associated with PTS risk nor dRVVT positivity.

Conclusions:
An acutely positive dRVVT following diagnosis of limb DVT appears to be a significant prognostic factor for development of clinically significant PTS in children. Larger collaborative cohort studies are required to substantiate these findings, evaluate other prognostic factors, and determine whether the present association is modulated by persistent dRVVT positivity or beta-2-glycoprotein-I dependence.

Disclosures:
Goldenberg: Eisai Inc: Research Funding; Pfizer Inc: Membership on an entity's Board of Directors or advisory committees; CPC Clinical Research: Consultancy.