bluuebunny/crossref_metadata_2025 huggingface dataset:
Identifying Iron Overload in Pediatric Oncology Patients
Authors: Giamanco Nicole , Warwick Anne B. , Crouch Gary
Published: November 2019

Background. Intensive therapy for childhood cancer is possible in large part due to improvements in supportive care that are currently available. Blood product transfusions, including red blood cell transfusions, are supportive care measures which are important in the tolerability of this therapy, and are frequently used in the majority of patients receiving therapy for childhood cancer. Although it is well recognized that frequent red blood cell transfusions in pediatric hematological disorders, such as sickle cell disease and thalassemia major, lead to iron overload and its complications (ie, organ dysfunction), pediatric oncology patients receiving numerous red blood cell transfusions are not routinely screened or evaluated for risk of iron overload or its consequences. This study was done to identify pediatric oncology patients with iron overload in a general pediatric hematology/oncology clinic.

Methods. A retrospective blood bank records review was performed of pediatric hematology/oncology patients treated in our clinic in the last 10 years (2003-2013) to identify those patients receiving >10 packed red blood cell (PRBC) transfusions. These patients’ medical records were reviewed to determine which patients had been assessed for iron overload with a serum ferritin level. If a serum ferritin was obtained and the result was >1000 ug/L, records were reviewed to determine if patients received therapy for iron overload.

Results. For the time period 2003-2013, blood bank records were screened on 144 patients. Fifty patients (34.7%) were identified as receiving >10 PRBC transfusions. Of these patients, 22 (44%; M/F = 12/10; age range = 2-23 y/o) were patients who had received therapy or were on active therapy for childhood cancer; 14 with leukemia (5 AML, 9 ALL), 8 with solid tumors (5 sarcomas, 1 hepatoblastoma, 1 lymphoma). Of these 22 patients, 6 (27%) patients had a serum ferritin level that was obtained and the remaining 16 (73%) had not. All the patients (100%) with a serum ferritin result had a serum ferritin level >1000 ug/L (range 1048-22021). Only one patient (off-therapy sarcoma patient, serum ferritin =1788 ug/L) was on therapy for iron overload (Exjade and phlebotomy).

Conclusions. Pediatric oncology patients receiving numerous PRBC transfusions are at risk for iron overload. Routine risk screening and assessment for iron overload should be accomplished in all pediatric oncology patients as part of their off therapy follow-up management. For those identified as having iron overload, appropriate therapy should be considered as indicated.

Disclosures

No relevant conflicts of interest to declare.
Left Ventricular Global Function Index and Left Ventricular Mass Volume Ratio By CMR: Relation with Heart Failure in Thalassemia Major Patients
Authors: Meloni Antonella , De Franceschi Lucia , Maddaloni Domenico , Carollo Sabrina , Sarli Roberto , Cosmi Carlo , Ruffo Giovan Battista , Neri Maria Giovanna , Resta Maria Chiara , Vallone Antonino , Positano Vincenzo , Pepe Alessia
Published: November 2019

Introduction: Recently two novels indicators of left ventricular (LV) performance assessed by Cardiovascular Magnetic Resonance (CMR) have been introduced: the LV global function index (LVGFI) and the LV mass/volume ratio (LVMVR). The LVGFI combines LV stroke volume, end-systolic and end diastolic volumes, as well as LV mass, integrating structural as well as mechanical behaviour. Elevated LVMVR is indicative of concentric remodelling. A LVGFI <37% and a LVMVR>1 were shown to be associated with the occurrence of cardiovascular events in no-thalassemic populations.

This retrospective cohort study aimed to systematically evaluate in a large historical cohort of thalassemia major (TM) in the CMR era whether the LVGFI and the LVMVR were associated with a higher risk of heart failure.

Methods: We considered 812 TM patients (391 M, 30.4±8.6 years), consecutively enrolled in the Myocardial Iron Overload in Thalassemia (MIOT) network. LVGFI and LVMRI were quantitatively evaluated by SSFP cine images. The T2* value in all the 16 cardiac segments was evaluated and a global heart T2* value <20 ms was considered indicative of myocardial iron overload (MIO).

Results: Eighty (9.9%) patients had a LVGFI<37% and, compared to the patients with a normal LVGFI, they showed a significant higher frequency of heart failure (43.8% vs 4.2%; P<0.0001). Patients with a LVGFI<37% had a significant higher risk of heart failure (odds-ratio-OR=17.59, 95%CI=9.95-21.09; P=<0.001). The risk remained significant also adjusting for the presence of MIO (OR=15.54, 95%CI=8.05-26.27; P=<0.001).

Thirty (3.7%) patients had a LVMVR≥1% and, compared to the patients with a normal LVMRI, they showed a significant higher frequency of heart failure (20.0% vs 7.7%; P=0.015). Patients with a LVMVR≥1% had a significant higher risk of heart failure (OR=3.01, 95%CI=1.18-7.64; P=0.021). The risk remained significant also adjusting for the presence of MIO (OR=3.44, 95%CI=1.31-9.01; P=0.012).

In a multivariate model including LVGFI, LVMVR and heart iron, the significant predictors of heart failure were a LVGFI<37% (OR=14.05, 95%CI=7.66-25.77; P=<0.001) and a global heart T2*<20 ms (OR=1.94, 95%CI=1.08-3.47; P=0.026).

Conclusions: In TM patients a LVGFI<37% was associated with an higher risk of heart failure, independent by the presence of MIO. A widespread program using CMR exploiting its multi-parametric potential can have considerable power for the early identification and treatment of patients at risk for heart failure.

Disclosures

No relevant conflicts of interest to declare.
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

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

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

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

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

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

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

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

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

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.
Impaired Platelet Responses to Thromboxane a2 and Thrombin in Mice Lacking Receptor-Interacting Protein Kinase 3
Authors: Zhang Yiwen , Zhang Jian , Yan Rong , Zhang Jie , Chen Mengxing , Jiang Miao , Li Zhenyu , Ruan Changgeng , He Sudan , Dai Kesheng
Published: November 2019

Objective: Receptor-interacting protein 3 (RIP3) is a member of RIP family with a Ser/Thr protein kinase domain in its amino-terminus which is essential for kinase activity and autophosphorylation. The roles of RIP3 in embryonic development and different disease pathologies, such as inflammation and infections, have been reported in recent years. However, the role of RIP3 in thrombosis and hemostasis remains unknown.

Methods: Hematologic analysis was performed and tail bleeding time was monitored. Mouse platelets were isolated from anti-coagulated whole blood. Platelet aggregation and secretion were recorded at real time. Platelet P-selectin exposure and specific fibrinogen binding were detected by flow cytometry. TXA2 generation was measured with enzyme immunoassay (EIA) kit. Protein phosphorylations were detected by western blotting.

Result: RIP3-/- mice had tail-bleeding times that were significantly prolonged compared with their wild type littermates. In an in vivo model of mesenteric arteriole thrombosis, mice lacking RIP3 exhibited delayed thrombus formation, fewer accumulated platelets, smaller thrombi, and prolonged occlusion times. RIP3 was expressed in both human and mouse platelets. Deletion of RIP3 in mouse platelets caused a marked defect in aggregation and attenuated dense granule secretion in response to low doses of thrombin or a thromboxane A2 (TXA2) analogue, U46619. The defect in ADP secretion appears responsible for the impaired platelet aggregation, because addition of exogenous ADP rescued the reduced platelet aggregation. Although TXA2 generation and α-granule secretion were not impaired, integrin αIIbβ3 activation was attenuated in RIP3-/- platelets. Moreover, phosphorylation of Akt induced by U46619 or thrombin was markedly reduced in the absence of RIP3. Activation of Akt signaling restored the impaired aggregation of RIP3-/- platelets. ERK and p38 phosphorylation elicited by either U46619 or thrombin was attenuated in RIP3-/- platelets. In contrast, U46619- and thrombin-induced activation of PTEN, PDK1, or Src was not impaired in RIP3-/- platelets.

Conclusion: Our data demonstrate a novel role for RIP3 in amplifying U46619- and thrombin-induced platelet activation by mediating Akt-dependent ADP secretion, and in supporting hemostasis and thrombus formation in vivo. RIP3 may represent a novel target to modulate PARs and TP signaling and a potential new target for antithrombotic strategy.

Disclosures

No relevant conflicts of interest to declare.
Pharmacological and Genetic Inhibition of Autophagy Decreases the Secretion of High Molecular Weight Von Willebrand Factor from Endothelial Cells in Vitro and in Vivo
Authors: Hanby Hayley A. , Zheng X. Long
Published: November 2019

The regulation of von Willebrand factor (VWF) processing, packaging, and secretion is important for primary hemostasis. Recently, autophagy has been implicated in modulating VWF maturation and Weibel-Palade body (WPB) morphology. Additionally, treatment of mice and humans with chloroquine (CQ), an anti-malarial agent and pharmacological inhibitor of autophagy, is shown to increase bleeding time. Therefore, we hypothesize that targeting autophagic flux may be therapeutic for arterial thrombotic disorders such as thrombotic thrombocytopenic purpura (TTP). To test this hypothesis, we first treated human umbilical vein endothelial cells (HUVECs) with CQ in culture and showed that CQ dose-dependently decreased VWF antigen levels and multimer sizes in the conditioned medium of histamine-stimulated cells (not shown). Knockdown of an autophagy-related protein (Atg7) with shRNA in HUVECs had similar effect to CQ treatment in VWF secretion and multimer distribution (not shown). More interestingly, daily injections (i.p.) of CQ at 60 mg/kg into Adamts13-/- mice (CAST/Ei strain) for 7 days reduced plasma VWF concentration by more than 60% compared to vehicle control (Fig. 1). Interestingly, VWF secreted from CQ-treated mice remained functional as collagen binding activity/antigen ratio was comparable to vehicle control mice. However, multimer analysis demonstrated the selective lack of ultra large VWF in plasma of Adamts13-/- mice treated with CQ as compared with those treated with vehicle alone (Fig. 2). These results indicate that targeting autophagy pathway with a pharmacological agent, such as CQ, may modulate VWF secretion and, thereby, arterial thrombosis. Our ongoing experiments are to determine the therapeutic efficacy of CQ and other autophagy inhibitors in murine models of arterial thrombosis and TTP.

Figure 1 Figure 1.

Figure 2 Figure 2.

Disclosures

No relevant conflicts of interest to declare.
Association Between a Pro-Inflammatory Axis Comprised By TSP1-TGFβ-CTGF with Three microRNAs, Mir-19a, miR122 and Mir-133a in the Pathophysiology of Diabetic Retinopathy: A Therapeutic Novel Modality
Authors: Dela Cadena Raul A , Lester Melissa , Del Carpio-Cano Fabiola , Shevchenko Alina , St. Angelo John , Boakye Jeffery , Deutsch Leonardo , Alvarez de la Cadena Philippe , Foster William
Published: November 2019

Introduction: Diabetic retinopathy (DR) is a severe ocular complication of type 2 diabetes (T2DM). Our laboratory has been studying the role of a pro-inflammatory axis comprised by TSP1-TGFb and CTGF in T2DM. The axis is differentially expressed in patients with T2DM and non-proliferative DR (NPDR) when compared with patients with T2DM and proliferative DR (PDR). The differential expression of such axis is accompanied by interesting cytokine profiles as well. The purpose of this study was to evaluate potential microRNAs associated with the above changes in order to understand better the pathophysiology of T2DM. Recent studies indicate that miRNAs transported to exosomes or HDL can be transferred in an active form to recipients cells potentially involving them in cell-to-cell communication.

Methods: Institutional approved IRB and informed consent allowed this prospective study to recruit a total of 40 individuals afflicted by T2DM (n=20 NPDR and n=20 PDR) from the Ophthalmology Department Diabetes Clinic at Temple Hospital, normal controls (n=20) were recruited from the Sol Sherry Thrombosis Research Center at Temple. Western-blotting technique was utilized to document the presence of fragments from CTGF in plasma, which have been documented in vitreous fluid to be angiogenic. Multiplexed protein profiling on microarrays by rolling-circle amplification was employed to determine IL-4 and MIP-1b. Commercially available ELISA was the method to determine the plasma concentration of TSP1, TGFb and CTGF. Total RNA was extracted from human plasma using the miRCURY TM RNA isolation kit by Exiqon at their facility in Denmark.

Results and Discussion: TSP1, TGFb, CTGF, IL4, MIP-1b were significantly elevated in NPDR patients when compared to PDR patients. Fragments of CTGF were identified more abundant in patients with PDR when compared to NPDR. Only three microRNAs were differentially expressed in the normal group when compared with either NPDR (decreased), namely miR-133a (p<0.005), miR-19a (p<0.008), miR-122 (p<0.02) or PDR (decreased), miR-19a p<0.000096) and miR-122 (p<0.005). miR19a has been implicated with expression of TSP1 and CTGF, glucose levels, insulin resistance and diabetes-associated pancreatic cancer. miR-133a has been associated with cardiac hyperthrophy, fibrosis and heart failure in diabetes. miR-122 has been identified as a potential biomarker for non-alcoholic fatty liver. In summary our results link for the first time in the literature three circulating microRNAs with a the above axis in the pathophysiology of NPDR and PDR.

Disclosures

No relevant conflicts of interest to declare.
Maternal Anti-Platelet β3 Integrin Antibodies Impair Angiogenesis and Cause Intracranial Hemorrhage in Fetal and Neonatal Alloimmune Thrombocytopenia
Authors: Issaka Yougbare , Lang Sean , Yang Hong , Chen Pingguo , Zhao Xu , Tai Wei-She , Zdravic Darko , Vadasz Brian , Li Conglei , Piran Siavash , Marshall Alexandra H. , Zhu Guangheng , Tiller Heidi , Killie Mette Kjaer , Boyd Shelley , Leong-Poi Howard , Skogen Bjorn , Adamson S. Lee , Freedman John , Ni Heyu
Published: November 2019

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a life-threatening disease in which intracranial hemorrhage (ICH) is the major risk. Although thrombocytopenia caused by maternal antibodies against β3 integrin and occasionally against other platelet antigens (e.g. GPIbα) has long been assumed to be the cause of bleeding, the mechanism of ICH has never been adequately explored. Utilizing murine models of FNAIT and a high frequency ultrasound imaging system, we found that ICH only occurred in fetuses and neonates with anti-β3 integrin- but not anti-GPIbα-mediated FNAIT, despite similar thrombocytopenia in both groups. Only anti-β3 integrin-mediated FNAIT reduced brain and retina vessel density, impaired angiogenic signalling, and increased endothelial cell apoptosis; which were abrogated by maternal administration of intravenous immunoglobulin (IVIG). ICH and impairment of retinal angiogenesis was further reproduced in neonates by injection of anti-β3 integrin- but not anti-GPIbα-antisera. Utilizing cultured human endothelial cells, we found that cell proliferation, network formation, and Akt phosphorylation were inhibited only by murine anti-β3 integrin-antisera and human anti-HPA-1a IgG purified from mothers with FNAIT children. Our data suggest fetal hemostasis is unique in that impairment of angiogenesis rather than thrombocytopenia is likely the cause of ICH; importantly maternal IVIG therapy can effectively prevent this devastating disorder.

Disclosures

No relevant conflicts of interest to declare.
Von Willebrand Factor-Dependent Inflammatory Responses in Mouse Septic Model By Cecal Ligation and Puncture
Authors: Kasuda Shogo , Matsui Hideto , Ono Shiro , Matsunari Yasunori , Nishio Kenji , Shima Midori , Hatake Katsuhiko , Sugimoto Mitsuhiko
Published: November 2019

Sepsis is a serious inflammatory response syndrome, in which systemic activation of both inflammatory and coagulation pathways are provoked by severe microbial infection. In addition to the known hemostatic functions, von Willebrand factor (VWF) is recently assumed to participate in a cross-talk between inflammation and thrombosis. Indeed, recent mouse model studies demonstrated that proper functional regulation of VWF-dependent inflammatory responses by ADAMTS13 considerably improved the disease progression of brain stroke or myocardial infarction. However, little is known about the detailed mechanisms or relevant role of VWF functions in inflammation. We therefore studied the physiologic relevance of VWF-dependent inflammatory responses in a mouse model of experimental sepsis by cecal ligation and puncture (CLP). The mouse CLP was performed according to the established standard protocol (Rittirsch D, et al., Nat Protoc, 2009). Briefly, mouse cecum was ligated distal to the ileocecal valve under laparotomy, punctured with 18 gauge needle and gently pressed until a small drop of stool appeared. The cecum was returned to the peritoneal cavity and 200 μL of saline was injected into the cavity to avoid dehydration before body wall and skin incision were closed with a 4-0 Sofsilk. We compared 17 wild-type (WT) and 17 VWF-gene deleted (knock-out; KO) mice (from The Jackson Laboratory, Bar Harbor, ME), all of which were 10-12 weeks of age, healthy and fertile. Excess blood loss was not observed in all (WT or KO) mice during the CLP operation. Kaplan-Meier analysis revealed the significantly (p < 0.05) lower survival rate of KO mice than that of WT (KO; 23.5% vs. WT; 58.8% at the Day 7 of CLP). The impaired survival rate of KO mice was restored by the bolus administration of human VWF (n=21, 2.5 U/mouse) to an extent comparable to that of WT. Peripheral blood analysis of KO mice at 24 hours after CLP showed the severe leukocytopenia with sharp decrease of neutrophils in blood, as compared to WT. In addition, formation of walled-off abscess was confirmed at the peri-cecal space in all the WT and KO mice alive even at the Day 7 of CLP, while such focal abscess formation was not found in mice died before the Day 3 of CLP. Thus, our results altogether indicate that VWF could play a role on the recruitment or accumulation of neutrophils for microbial killing at the local inflammatory focus. VWF-mediated platelet aggregate formation in peripheral capillary vessels then could shut down the local microcirculation, thereby blocking systemic microbial expansion as a crucial biological defense mechanism.

Disclosures

Shima: Chugai Pharmaceutical Co., Ltd.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.
Bilirubin Directly Inhibits Proteolytic Cleavage of a Fluorogenic VWF73 Peptide and Plasma Von Willebrand Factor
Authors: Lu Ruinan , Zheng X. Long
Published: November 2019

Bilirubin is a yellow breakdown product of heme catabolism. Increased levels of plasma bilirubin are a condition commonly seen in neonates and adults with hemolysis. The unconjugated bilirubin consists of an open chain of four pyrrole-like rings. Previous studies have shown that bilirubin decreases the cleavage of a commercially available FRETS­­­­­-VWF73 assay, resulting in falsely low plasma ADAMTS13 activity. However, the mechanism underlying such a reduction of ­­­­ADAMTS13 activity is not fully understood. Plasma ADAMTS13 activity is critical for differential diagnosis of thrombotic microangiopathy in which low ADAMTS13 activity (less than 5-10%) is more consistent with thrombotic thrombocytopenic purpura (TTP), while normal to mildly reduced ADAMTS13 activity (greater than 10-20%) is the common findings of hemolytic uremic syndrome (HUS). Here, we show that bilirubin directly inhibits the proteolytic cleavage of a fluorogenic VWF73 peptides labeled with fluorescein-5-maleimide (FL485-VWF73) (ext. 485nm/emi.530nm) or DyLight-633-maleimide (DL633-VWF73) (ext.638nm/emi.658 nm) in a concentration-dependent manner (Fig. 1). The IC50 for inhibiting the cleavage of FL485-VWF73 and DL633-VWF73 was estimated to be 13 micro mol/L and 70 micro mol/L, respectively. To confirm the direct inhibitory activity, bilirubin at various concentrations was incubated with plasma-derived multimeric VWF and recombinant ADAMTS13 in the presence of 1.5 M urea, 5 mM Tris-HCl, pH 8.0 for four hours. The cleavage of multimeric VWF was determined by 1% agarose and SDS-polyacrylamide gel electrophoreses, followed by Western blotting with rabbit anti-VWF IgG. The results demonstrated that bilirubin also inhibited recombinant ADAMTS13-mediated cleavage of VWF in a concentration-dependent manner with an IC50 ~0.43 milli mol/L (Fig. 2). The inhibitory activity of bilirubin on the cleavage of a fluorogenic peptide or a multimeric VWF substrate was largely eliminated by addition of bilirubin oxidase that converts bilirubin to biliverdin (not shown). Our results demonstrate for the first time that unconjugated bilirubin directly inhibits plasma and recombinant ADAMTS13 activity in addition to its known interference with certain fluorogenic assays for ADAMTS13 activity. These findings are clinically important concerning diagnosis and differential diagnosis of thrombotic microangiopathy. Additionally, our findings may shed new light on the mechanism of thrombotic complications pertinent to acute hemolysis and liver diseases.

Figure 1 Figure 1.

Figure 2 Figure 2.

Disclosures

No relevant conflicts of interest to declare.
β7 Integrin Regulates Intra-Marrow Trafficking of Hematopoietic Stem Cells
Authors: Murakami Jodi , Xu Baohui , Franco Christopher B. , Hu Xingbin , Galli Stephen J. , Weissman Irving L , Chen Ching-Cheng
Published: November 2019

α4β7 integrin is a cell adhesion receptor that is crucial for the migration of hematopoietic progenitors and mature effector cells in the periphery, but its role in adult hematopoiesis remains controversial. To investigate this, we conducted studies using a mouse model in which β7 integrin is absent. Hematopoietic stem cells (HSCs) that lacked β7 integrin (β7KO) had significantly reduced engraftment potential. Intriguingly, the survival of β7KO mice was enhanced and their hematopoietic recovery after 5-fluorouracil-induced myeloablative stress was better compared to wild type (WT) mice, indicating that the decreased engraftment of β7KO HSCs was not caused by a defect in HSC hematopoietic activity. Next we examined the homing abilities of HSCs and we observed that β7KO HSCs had impaired migration abilities in vitro and BM homing capabilities in vivo. Lethal irradiation induced expression of the α4β7 integrin ligand - mucosal addressin cell adhesion molecule-1 (MAdCAM-1) on bone marrow (BM) endothelial cells. Moreover, blocking MAdCAM-1 reduced the homing of HSCs and impaired the survival of recipient mice. Altogether, these data indicate that β7 integrin, when expressed by HSCs, interacted with MAdCAM-1 in the BM microenvironment, thereby promoting HSC homing and engraftment. Interestingly, we also found that β7KO HSCs were retained in the BM, suggesting that β7 integrin may influence the localization of HSCs within different stem cell niches through interaction with MAdCAM-1. To examine the localization of HSCs within the BM, we used the hypoxyprobe pimonidazole to correlate oxygen status with niche localization. We observed that both β7KO and MAdCAM-1KO HSCs were more hypoxic compared to WT HSCs, demonstrating that the absence of either β7 integrin or MAdCAM-1 in mice causes HSCs to be localized in a more hypoxic region of the BM. To confirm these findings, we performed single-cell RT-PCR using Fluidigm Dynamic Array Chips and we discovered that β7KO HSCs differentially expressed genes associated with niche localization and cell cycle status compared to WT HSCs. Since hypoxia correlates with quiescence, we next assessed the cell cycle status of HSCs using Ki67 staining and in vivo BrdU assay and we found that β7KO HSCs may have reduced cell cycle activity. Collectively, these studies suggest that expression of β7 integrin on HSCs may promote exit from quiescence and influence HSC localization within the BM niche.

Disclosures

No relevant conflicts of interest to declare.