bluuebunny/crossref_metadata_2025_split huggingface dataset:
Elevated Pulse Pressure Is Associated with Hemolysis, Proteinuria and Chronic Kidney Disease in Sickle Cell Disease
Authors: Novelli Enrico M , Hildesheim Mariana E , Rosano Caterina , Vanderpool Rebecca , Simon Marc , Kato Gregory , Gladwin Mark T 
Published: November 2019

Abstract
A seeming paradox of sickle cell disease is that patients do not suffer from a high prevalence of systemic hypertension in spite of endothelial dysfunction, chronic inflammation and vasculopathy. However, some patients do develop systolic hypertension and increased pulse pressure, an increasingly recognized major cardiovascular risk factor in other populations. Hence, we hypothesized that pulse pressure, unlike other blood pressure parameters, is independently associated with markers of hemolytic anemia and cardiovascular risk in sickle cell disease. We analyzed the correlates of pulse pressure in patients (n = 661, 53% female, 76% HbSS) enrolled in the walk-PHaSST multicenter, international sickle cell trial with data on clinical laboratory tests and echocardiographic measures. In univariate analyses, pulse pressure was consistently and positively correlated with all markers of hemolysis, including reticulocyte proportion, LDH, total bilirubin, AST and hemolytic component (a variable derived using principal component analysis from four markers of hemolysis - LDH, AST, total bilirubin, and reticulocyte percent) in HbSS patients. Pulse pressure was also positively correlated with creatinine, urine albumin-to-creatinine ratio and presence of proteinuria. Among SS patients, four risk factors were associated with pulse pressure independently of each other: reticulocyte count (beta = 2.37, p = 0.02), hemolytic index (beta = 1.53, p=0.002), serum creatinine (beta = 3.21, p = 0.02), and proteinuria (beta = 2.52, p = 0.04). By contrast, markers of hemolysis were not independently associated with systolic, diastolic and mean arterial pressure. These results from the largest sickle cell disease cohort to date since the Cooperative Study of Sickle Cell Disease show that pulse pressure is strongly and independently associated with hemolysis, proteinuria and chronic kidney disease. We propose that high pulse pressure may be associated with clinical complications of vascular dysfunction in sickle cell disease.

Disclosures
No relevant conflicts of interest to declare.
Mass Spectrometry of Urine Proteins for Early Detection of Renal Complications in Sickle Cell Disease
Authors: Adjei Elena A , Kumari Namita 
Published: November 2019

Abstract
PURPOSE: Sickle cell disease (SCD) is an autosomal recessive genetic disorder caused by a single G6V mutation in the β-globin gene. SCD patients have various complications including chronic renal failure and nephrotic syndrome which can develop in 30-50% of sickle cell patients. Currently there are no reliable methods to identify the risk for renal complications in the early stages for the subset of people who will develop renal failure. It is essential that new noninvasive prognostic biomarkers be discovered to help assess patients for risk of renal failure which may lead to early intentions and greater survival rates among SCD patients.
METHODS: Urine samples were collected from 25SCD patients and 6 healthy controls. Trypsin digests of urine proteins were analyzed by nano LC coupled in-line to LTQ Orbitrap XL tandem mass spectrometer. Proteins identified with Proteome Discoverer software were further quantified using SIEVE 2.1 (Thermo).
RESULTS: About 80 proteins were detected in urine. Among those, about 10 proteins were found at higher levels in SCD patients, including seruloplasmin, transferring and alpha-1-acid glycoprotein precursor.
CONCLUSION: Several of the detected proteins may cause early changes in glomerular permeability and be a potential biomarker for early renal manifestations in SCD. Further studies are needed to form a more conclusive relationship between renal complications and the proteins present in urine of SCD patients.
SUPPORT: NIH Research Grants 8G12MD007597 and P50HL118006-01.

Disclosures
No relevant conflicts of interest to declare.
The Innate Immune System Favors Emergency Monopoiesis at the Expense of DC Differentiation to Control Bacterial Infections
Authors: Bieber Kristin , Pasquevich Karina A. , Günter Manina , Grauer Matthias , Pötz Oliver , Schleicher Ulrike , Biedermann Tilo , Beer-Hammer Sandra , Bühring Hans-Jörg , Rammensee Hans-Georg , Zender Lars , Autenrieth Ingo B. , Kanz Lothar , Lengerke Claudia , Autenrieth Stella E. 
Published: November 2019

Abstract
Dendritic cells (DCs) are critical in host defense against infection, bridging the innate and adaptive immune system. Patients with sepsis display reduced circulating and splenic DCs and impaired DC function that may contribute to prolonged immune suppression and exacerbation of infection. However, the mechanisms of pathogen-induced DC depletion remain poorly understood. Here, a mouse model of systemic bacterial infection was employed to analyze the impact of different bacterial pathogens on DC development in vivo. We found that the numbers of bone marrow (BM) hematopoietic progenitors committed to the DC lineages were reduced following systemic infection with different Gram-positive and Gram-negative bacteria. In parallel, a TLR4-dependent increase of committed monocyte progenitors in the BM as well as mature monocytes in the spleen was observed. In line, adoptively transferred FLT3+ myeloid progenitors (MPs) developed preferentially to monocytes at the expense of DCs in infected animals. Analyses performed on mixed BM chimeras suggested that both the reduction of DC progenitors and the induction of monopoiesis following infection were dependent on extrinsic TLR4 signaling driving the secretion of IFN-g regulated chemokines. Consistently, these effects were completely abrogated by suppression of IFN-g signaling.
Elevated monocyte numbers in the spleen triggered by infection were due to a CCR2-dependent egress from the BM. In CCR2-deficient mice, in which monocytosis reportedly is abrogated, we observed a significantly increased bacterial load in the spleen and a reduced survival rate, highlighting the importance of monocytes for bacterial clearance. Together, our data provide evidence for a general response of myeloid progenitors upon bacterial infection to enhance monocyte production, thereby increasing the availability of innate immune cells as a first line of defense against invading pathogens. Concomitantly the development of DCs is impaired, which may be responsible for transient immunosuppression in e.g. bacterial sepsis.

Disclosures
No relevant conflicts of interest to declare.
Direct SIRPa Blockade Augments Macrophage Responses to Therapeutic Anticancer Antibodies
Authors: Weiskopf Kipp , Ring Aaron M , Guo Nan , Schnorr Peter J , Maute Roy Louis , Volkmer Jens-Peter , Weissman Irving L 
Published: November 2019

Abstract
Antibody-dependent phagocytosis is a crucial mechanism underlying the efficacy of therapeutic antibodies for cancer. However, a major barrier to phagocytosis exists in the CD47-SIRPa interaction. CD47, a molecular “don’t eat me” signal, is highly expressed on many types of cancer and acts as a myeloid-specific immune checkpoint. By engaging SIRPa, an inhibitory receptor on macrophages, CD47 limits the ability of macrophages to respond to therapeutic antibodies. In this study, we developed anti-human SIRPa antibodies that block the interaction between CD47 and SIRPa. By themselves, these antibodies were not sufficient to induce human macrophage phagocytosis. However, when combined with a variety of tumor-specific antibodies, the anti-SIRPa antibodies exhibited remarkable synergy and augmented antibody-dependent phagocytosis of human cancer cells. Addition of anti-SIRPa antibodies improved phagocytosis of cancer in response to rituximab, cetuximab, and trastuzumab, among other therapeutic antibodies. Moreover, anti-SIRPa antibodies enhanced macrophage phagocytosis of lymphoma cells in response to anti-CD20 variants produced with each subclass of human IgG or glycoengineered anti-CD20 antibodies, indicating they could be effective for the majority of therapeutic antibodies that are clinically approved or under development. Importantly, both polarized macrophages and primary tumor-associated macrophages exhibited increased responses to therapeutic antibodies when SIRPa was blocked. In contrast to therapies targeting CD47 directly, anti-SIRPa antibodies benefit from more restricted expression of SIRPa and could have more favorable pharmacokinetic and toxicity profiles. They are an important addition to the growing arsenal of therapeutics targeting the CD47-SIRPa axis for cancer and could act as universal adjuvants to therapeutic antibodies.

Disclosures
Weiskopf: Stanford University: CD47/SIRPa blocking therapies Patents & Royalties. Ring:Stanford University: CD47/SIRPa blocking therapies Patents & Royalties. Guo:Stanford University: CD47/SIRPa blocking therapies Patents & Royalties. Volkmer:Stanford University: CD47/SIRPa blocking therapies Patents & Royalties. Weissman:Stanford University: CD47/SIRPa blocking therapies Patents & Royalties.
Molecular Dynamics As a Computational Tool to Study the Immune Reactivity: Identification of Conformational Changes in the Major Histocompatibility Complex
Authors: Garcia Guerrero Estefania , Pérez Simon Jose Antonio , Sánchez Abarca Luis Ignacio , Diaz Moreno Irene , De la rosa Miguel Angel , Diaz Quintana Antonio 
Published: November 2019

Abstract
Introduction: The generation of the immune response requires the recognition of peptides presented by the major histocompatibility complex (MHC) through the T cell receptor (TCR). In the hematopoietic transplantation context, T cells (LT) from the donor recognize foreign MHC or own MHC bound to foreign peptides (pMHC), generating an alloimmune response. Currently, the molecular mechanisms of LT alloimmune activation are unknown. In order to analyze the molecular interactions between peptides, MHC and TCR, we have implemented Molecular Dynamics techniques. We have compared immunologically reactive complexes (HLA-A2/TAX/TCR-A6) to non/weakly reactive complexes (HLA-A2/V7R/TCR-A6; HLA-A2/P6A/TCR-A6; HLA-A2/Y8A/TCR-A6).
Methods: Starting structure of a reactive complex was downloaded from the PDB database and used to model mutations known to lead to different degrees of immune reactivity. Dynamics simulations were performed and analyzed using the program AMBER version 9. The simulation time was approximately 10 ns. Further analysis was carried out using the script ARO (Díaz-Moreno et al. 2009) in the VMD Tk console.
Results: A total of 10 MD trajectories have been reckoned, to simulate the behavior of isolated components of the different pMHC-TCR complexes. Analysis of the fluctuations show that pMHC binding barely restrains TCR motions, affecting mostly to CDR3 loops. Opposite, pMHC displayed substantial changes in its dynamics upon comparing its free versus ternary form (pMHC-TCR). Moreover, we studied the salt-bridges formed between peptide and MHC and we observed a significant loss of salt-bridges in non reactive complexes as compared to reactive ones. Furthermore, the loss of salt-bridges in non reactive complexes affects the electrostatic potential of pMHC complex. According to these results, we next studied the salt-bridges formed between pMHC and TCR. As observed within the binary complex p-MHC, we confirmed a significant loss of salt-bridges in non reactive as compared to reactive complex. Finally, we also analyzed the electrostatic potential of pMHC-TCR complexes and we observed differences between reactive complex and weakly/non reactive complexes.
Conclusions: The MHC shows strong changes in its molecular dynamics upon binding to TCR, decreasing its mobility. The “pattern” of salt-bridges between the peptides and the MHC varies depending on the reactivity of the complex, with a loss of salt-bridges in the non-reactive as compared to the reactive ones, which results in change in the electrostatic pattern. In addition, also the number of salt-bridges between pMHC and TCR increases in reactive complexes as compared to non-reactive ones.

Disclosures
No relevant conflicts of interest to declare.
The Impact of Nuclear Organization and Homolgous Recombination in Repair of DNA Damage Introduced By Aid during Class Switch Recombination
Authors: Rocha Pedro P , Fu Yi , Kim JungHyun , Skok Jane 
Published: November 2019

Abstract
Class Switch Recombination (CSR) involves the introduction of double stranded breaks (DSBs) at the switch regions of the immunoglulin heavy chain (Igh) locus by the enzyme Activation Cytidine Deaminse (AID). AID can also act as a general mutator targeting other loci in the genome which can then either be repaired faithfully or in an error-prone fashion introducing mutations and potentially initiating B cell lymphoma. The factors contributing to the choice of repair pathway are not fully understood. Here we tested the hypothesis that repair pathway choice is influenced by differential accessibility and expression levels of target loci across cell cycle. More specifically in the context of CSR we tested whether differential regulation of gene accessibility across cell cycle is an important determinant for AID binding and subsequent repair pathway choice as different repair pathways predominate at different stages of cell cycle. Using 3D-FISH in conjunction with Immunofluorescence we observed that AID target genes that are faithfully repaired are more accessible (found in euchromatic regions) in the G2 phase of the cell cycle then genes that are frequently mutated. In contrast, those genes which are repaired in an error prone fashion are more accessible in the G1 phase of cell cycle. Since Homologous Recombination mediated repair (HR), which is a faithful repair mechanism, occurs in G2 we speculate that accessibility of these genes at this stage of cell cycle facilitates action by this repair pathway. Conversely, genes that are more accessible during the G1 phase of cell cycle will be repaired by the non-homologous end joining (NHEJ) repair pathway and therefore are more likely to be mutated. Thus, HR could be the pathway by which faithful repair is accomplished and use of the NHEJ pathway on the other hand could contribute to the introduction of dangerous DNA mutations that might lead to B cell transformation and cancer. To connect differences in accessibility with repair pathway usage, we used a mouse model carrying a hypomorphic mutation in BRCA2, a protein involved in HR. This is the first mouse model impaired in HR that eludes embryonic lethality and allows inspection of the role of this pathway in maintaining genomic stability in splenocytes undergoing CSR. Our preliminary investigations indicate that in Brca2 mutant B cells not only is the integrity of fathfully repaired loci compromised, but the Igh locus is also damaged. Taken together these results support our hypothesis and further indicate that the HR pathway is involved in repairing Igh. Given that approximately 95% of lymphomas are of B cell origin and many of these are associated with AID mediated breaks, it is crucial for us to understand the factors that influence targeting and repair.

Disclosures
No relevant conflicts of interest to declare.
Osteoactivin Mediates the Inhibitory Effects of Myeloid Derived Suppressor Cells
Authors: Held Stefanie Andrea Erika , Heine Annkristin , Daecke Solveig , Bringmann Anita , Brossart Peter 
Published: November 2019

Abstract
Myeloid derived suppressor cells (MDSC) play an important role in the regulation of immune responses by suppressing the function of antigen presenting cells (APC) and T cells. However, the origin and development of these cells in humans is not well understood. We observed that incubation of peripheral blood monocytes with IL-10 during their differentiation to dendritic cells (DC) results in the generation of an APC population with dramatically reduced stimulatory capacity and a CD14+ and HLA-DR low phenotype (IL-10-APC) similar to the recently described human MDSC subpopulation. In coincubation experiments we found that the addition of these cells resulted in an inhibition of DC induced T cell proliferation in a mixed lymphocyte reaction. Furthermore, these IL-10-APCs reduced the expression of CD1a and costimulatory molecules on DC as well as their activation by LPS characterized by diminished expression of maturation markers including CD83, CD80, CD86 or CD40. Interestingly, addition of IL-10-APC to mature or immature DC induced an increased expression of osteoactivin and its corresponding receptor syndecan 4 on DC thus demonstrating that osteoactivin mediates its effects by upregulating its own receptor. IL-10-APC almost completely abolished the secretion of cytokines and chemokines by mature and immature DC involved in T cell stimulation and migration such as TNF-a, IL-6, MIP-1a or Rantes. These effects were not due to induction of IL-10 production and were not observed when purified CD14+ monocytes were used as a control in the experiments. In the next set of experiments we isolated MDSC with the CD14+ HLA-DR low cell phenotype from buffy coats of healthy donors. We found, that these cells similar to the IL-10-APCs express high levels of osteoactivin that can be further upregulated by the addition of IL-10.
In order to analyze the possible mechanisms and molecules involved in these inhibitory effects we found that IL-10-APC and isolated MDSC expressed higher levels of PD-1, PD-L1, GITR, GITRL and osteoactivin, an immunosuppressive molecule that was shown to inhibit the function of T cells. Inhibition of osteoactivin on MDSC by utilizing blocking antibodies restored the function of DC in co-incubation experiments. Recently, it was shown that ß-glucans can decrease the suppressive function of MDSCs via Dectin-1. In our experiments, activation of MDSC with Dectin-1 ligand curdlan reduced the expression of osteoactivin and PD-L1 thus explaining how ß-glucans reduce the inhibitory effects of MDSC. In contrast, incubation of MDSC with TLR 2,3,4 and 7/8 ligands upregultaed osteoactivin, PD-1 and PD-L1 on the cell surface. Our results demonstrate that osteoactivin is a key molecule that mediates the inhibitory effects of MDSC on DC function.

Disclosures
No relevant conflicts of interest to declare.
Combination Mitigators, GS-Nitroxide JP4-039 and water Soluble Oxetanyl Sulfoxide MMS350 Improve Survival of Lethally Irradiated Mice
Authors: Epperly Michael W. , Dixon Tracy M. , He Siping , Shields Donna , Wipf Peter , Li Song , Greenberger Joel S. 
Published: November 2019

Abstract
New small molecule radiation mitigator drugs are needed to administer 24 hr or later after a mass casualty radiation event. We have developed two small molecule mitigators with applicability as clinical radiation protectors. The mitochondrial targeted GS-nitroxide, JP4-039, acts as a superoxide dismutase mimic. A novel oxetanyl sulfoxide, MMS350, acts as a hydroxyl radical scavenger and mitigates both acute total body irradiation damage and prevents late radiation pulmonary fibrosis (Kalash, Epperly, Cao et. al., Radiation Research 180:474-490, 2013). We tested the effect of both compounds together in the C57BL/6NTac mouse TBI irradiation model. Each drug was a radiation protector and mitigator with murine hematopoietic progenitor cell line 32D cl3 in vitro. JP4-039 (10 μM) or MMS350 (100 μM) were incubated with the cells one hour before irradiation or added 30 min following irradiation. The irradiated cells were plated in methylcellulose, incubated for 7 days at 37oC, and colonies of greater than 50 cells counted. The data was analyzed using linear quadratic or single hit, multi-target models. In vivo total body irradiated C57BL/6NTac mice received 9.25 Gy and were injected intravenously 24 hr later with JP4-039 (20 mg/kg) in F14 emulsion, MMS350 (20 mg/kg) or both JP4-039 (20 mg/kg) + MMS350 (20 mg/kg). Mice were followed for development of the hematopoietic syndrome (irradiation induced bone marrow failure). In vitro radiation survival curves demonstrated that both JP4-039 and MMS350 protected and mitigated 32D cl 3 cells by increasing the shoulder on the clonagenic survival curves. The shoulder on survival curve for 32D cl 3 cells was 2.0 ± 0.3 compared to 3.8 ± 0.5 when JP4-039 was administered pre irradiation (p = 0.0092), 5.4 ± 0.5 when JP4-039 was administered post irradiation (p = 0.0002), 3.5 ± 0.3 for cells incubated with MMS350 pre irradiation (p = 0.0104) or 5.3 ± 1.5 when MMS350 is administered post irradiation (p = 0.0096). Mice treated with JP4-039 had increased survival at 60 days following 9.25 Gy from 20% to 65% (p = 0.0092). Mice treated with MMS350 showed an increased survival of 55% (p = 0.0097). The combination of JP4-039 and MMS350 was additive and increased survival to 95% (p < 0.0001). The combination of JP4-039 and MMS350 increased mitigation of irradiation damage better than one drug alone.

Disclosures
No relevant conflicts of interest to declare.
A Signaling Mechanism By Which Platelet Glycoprotein Ib-IX Promotes Thrombin-Induced Platelet Activation
Authors: Estevez Brian , Delaney Michael Keegan , Stojanovic-Terpo Aleksandra , Du Xiaoping 
Published: November 2019

Abstract
Numerous reports indicate that the platelet glycoprotein (GP) Ib-IX complex (GPIb-IX) binds directly to the potent platelet agonist thrombin and is important for promoting thrombin-induced platelet activation. However, how GPIb-IX contributes to thrombin-induced platelet activation is unclear. It has been suggested that thrombin binding to GPIb facilitates the cleavage, and thus activation, of the protease-activated receptors (PAR). Our data indicate that GPIb-IX promotes thrombin signaling through a GPIb-IX signaling mechanism. We reconstituted GPIb-IX (GPIb) /Protease-activated receptor (PAR) cooperativity in response to thrombin in Chinese Hamster Ovary (CHO) cells expressing PAR1. Thrombin-induced PAR1-dependent calcium signaling was significantly enhanced by GPIb expression, and this effect of GPIb appears to require GPIb signaling, as deletion of the cytoplasmic binding site for an intracellular signaling molecule, 14-3-3, in GPIbα abolished the stimulatory effect of GPIb. The importance of GPIb-14-3-3 interaction in promoting thrombin-induced platelet activation was also shown in human platelets, in which pretreatment with MPαC, an inhibitory peptide based on a critical 14-3-3 binding site in the C-terminus of the GPIbα, inhibited thrombin-induced platelet activation. Furthermore, 14-3-3 binding site deletion in GPIba or MPαC-pretreatment inhibited thrombin-induced activation of Rac1 and phosphorylation of LIMK1, both of which have been shown to mediate von Willebrand factor-induced GPIb signaling, and the role of GPIb in promoting thrombin signaling was abolished with a Rac-inhibitor, NSC23766 or in Rac1-/- platelets. Importantly, LIMK1-/- platelets display defective thrombin-induced platelet activation but enhanced PAR4-activating peptide induced platelet activation.

Disclosures
No relevant conflicts of interest to declare.