bluuebunny/crossref_metadata_2025_split huggingface dataset:
Targeting The PI3K/AKT Pathway To Inhibit Platelet Activation and Thrombus Formation
Authors: Yi Wenxiu , Li Wei , Ren Lijie , Mao Xinliang , Zhu Li 
Published: October 2019

Abstract
The phosphatidylinositol 3' –kinase (PI3K)-Akt signaling pathway has been shown to be critical in modulating platelet function and increasing number of studies have been focusing on the development of PI3K inhibitors to modulate platelet function. We recently identified a novel small molecule compound S14161, namely 8-ethoxy-2-(4-fluorophenyl)-3-nitro-2H-chromene, displaying potent antileukemia and antimyeloma activity via inhibition of the PI3K pathway (Mao et al, Blood, 2011, 117:1986). In the present study, we evaluated the effect of S14161 on platelet activation and the underlying mechanisms. Gel-filtered human platelets were isolated from venous blood of healthy adults and the effect of S14161 on platelet aggregation in response to agonists was determined. Results showed that S14161 inhibited platelet aggregation induced by collagen, convulxin, thrombin, PAR1 agonist peptide SFLLRN, and U46619 in a dose dependent manner (2.5-10μM) with the most striking inhibition for collagen by 89.8% (P<0.001, n=3) and for U46619 by 94.3% (P<0.001, n=3), respectively compared to vehicle-treated samples when 10μM S14161 was used. Flow cytometry studies showed that S14161 inhibits convulxin- or thrombin-induced P-selectin expression and fibrinogen binding of single platelet. S14161 also inhibited platelet spreading on fibrinogen and clot retraction, processes mediated by outside-in signaling. Using a microfluidic chamber we demonstrated that incubation of S14161 decreases platelet adhesion on collagen-coated surface by about 80% at various time points of blood flow in the chambers. Western blot showed that similar to LY294002, the classic PI3K inhibitor, S14161 inhibited phosphorylation of Akt Ser473 and Akt Thr308 in response to collagen, thrombin, or U46619, implying the involvement of PI3K pathway. Additionally, S14161 inhibited MAPK/ERK1/2 phosphorylation. Finally, the effects of S14161 on thrombus formation in vivo were measured using a ferric chloride-induced carotid artery injury model in mice. The intraperitoneal injection of S14161 (2mg/kg) to male C57BL6/J mice significantly extended the first occlusion time (5.05±0.99 min, N=9) compared to the vehicle controls (3.72±0.95 min, N=8) (P<0.05), but did not increase the bleeding time (P>0.05). Taken together, our data showed that S14161 inhibits platelet activation and thrombus formation, and may be developed as a novel therapeutic agent for the prevention of thrombotic disorders. (This study was supported by National Natural Science Foundation of China 81170132 to Li Zhu)

Disclosures:
No relevant conflicts of interest to declare.
Megakaryocyte Abnormalities Occur In The Absence Of Cell-Mediated Immunity In a Murine Model Of Passive Immune Thrombocytopenia (ITP)
Authors: Semple John W. , Hunt Kristin , Hou Yu , Aslam Rukhsana , Speck Edwin R. , Ni Heyu , Lazarus Alan H 
Published: October 2019

Abstract
Immune thrombocytopenia (ITP) is an autoimmune bleeding disorder characterized by increased peripheral immune platelet destruction and megakaryocyte defects in the bone marrow. Although ITP was originally thought to be primarily due to humoral mediated autoimmunity it is now evident that T cells can also play a contributing role to the thrombocytopenia. In fact, the exact interplay between platelet destruction, megakaryocyte dysfunction, and the elements of both the humoral and cell mediated immune systems still remain incompletely defined. In murine passive models of ITP, the direct administration of anti-platelet antibodies can result in severe thrombocytopenia which is evident within 24 hours of injection. While most studies have focused on immune platelet destruction in the spleen, an additional possibility is that the anti-platelet antibody also has an effect on megakaryocytes. To unequivocally determine if antiplatelet antibodies have an effect on megakaryocytes in an in vivo model, BALB/c mice were intravenously administered 2 ug of an anti-GPIIbIIIa antibody (MReg30) or 50 uL of a high tittered anti-GPIIIa (anti-β3) serum from BALB/c GPIIIa (CD61) knockout mice immunized with wild type platelets. Platelet counts were assessed over time and the bone marrow and spleens were harvested for histological examination of megakaryocytes. Both preparations of antiplatelet antibodies significantly reduced platelet numbers within 1 day of antibody or serum administration. This thrombocytopenia could be rescued by administration of 2 g/kg of IVIg ip. Compared with naïve control mice, histological (H&E staining) examination of the bone marrow and spleens revealed that megakaryocytes were significantly increased in number and all exhibited abnormalities consistent with apoptosis e.g. pyknotic nuclei. IVIg administration completely prevented these megakaryocyte abnormalities. These results show that passively administered anti-platelet antibodies not only affect platelet counts but also significantly affect megakaryocyte physiology in the absence of cell mediated immunity.

Disclosures:
No relevant conflicts of interest to declare.
Characterization Of The Immature Platelet Fraction (IPF) Parameters In Children With Immune Thrombocytopenia
Authors: Ghemlas Ibrahim A. , Klaassen Robert J. , Barrowman Nick , Leung Elaine W. 
Published: October 2019

Abstract

Background
Immune thrombocytopenia (ITP) is characterized by impaired megakaryocte production and maturation, as well as premature platelet destruction. The effect of this disease on platelet production is not clear. The Immature Platelet Fraction (IPF) is a novel parameter available on the Sysmex XE-Series Hematology analyzers. It can be expressed as both IPF percent (IPF %) and absolute IPF count (AIPF#). It has been shown that IPF% is higher in Immune Thrombocytopenia (ITP) than in the normal population, and is an indicator of bone marrow platelet production or thrombopoiesis.

Objectives
To characterizes the IPF parameters in pediatric patients with ITP at diagnosis and during different phases of the disease (newly diagnosed, persistent, and chronic).

Method
This is a retrospective, single institution study performed at a tertiary care pediatric hospital. We reviewed the medical charts and electronic databases of all patients with ITP who had measured IPF parameters between June 2011 and June 2013. The standard definitions and terminology of the International Working Group were used.
Patient age, phase of ITP at the time of the blood test (newly diagnosed, persistent, and chronic), initial platelet count, IPF%, AIPF#, and type of therapy given (including observation alone) were recorded. We then compared the platelet count, IPF%, and AIPF# between the ITP phases at the time of initial CBC and looked at the relationship between the platelet count and IPF%.
Comparisons between groups were performed using Kruskal-Wallis tests or Mann-Whitney tests, as appropriate. Association between measured blood parameters was measured using the Spearman correlation coefficient.

Results

Conclusion
IPF parameters may be useful in understanding the mechanism of ITP through the evolution of the disease. From the time of diagnosis of ITP through progression to chronic ITP, there is a steady decrease in IPF% and increase in AIPF# .The high IPF% in the newly diagnosed setting suggests that megakaryocytes are still active, whereas the lower IPF% in chronic ITP indicates inhibition of megakaryopoiesis or a different underlying pathophysiology. This may have important clinical implications when considering TPO receptor agonist therapy.

Disclosures:
No relevant conflicts of interest to declare.
Alternative and Terminal Complement Pathway Biomarkers At Presentation More Precisely Define The Clinical Diagnosis Of aHUS
Authors: Cataland Spero R , Holers V. Michael , Yang Shangbin , Wu Haifeng M 
Published: October 2019

Abstract

Introduction
Recent studies demonstrating the effectiveness of eculizumab in the treatment of atypical hemolytic uremic syndrome (aHUS) have increased the need for more rapid and objective diagnostic tests to help confirm the diagnosis of aHUS over other thrombotic microangiopathies (TMA).

Methods
We performed a retrospective study of all patients enrolled in the Ohio State University TTP/aHUS Registry since 2003. From this cohort of patients diagnosed with an acute TMA (platelets<100 x 109/ with schistocytes present, and without an alternative explanation), patients with a pretreatment serum creatinine >2.25 mg/dL and measureable ADAMTS13 activity (>10%) were analyzed. The clinical and demographic data for these 19 patients are shown in Table 1. Banked plasma samples from these study subjects were used to measure complement activation factors Bb (alternative pathway (AP)), C4d (classical pathway), C3a, (generalized complement activation), C5a, and C5b-9 complex (terminal complement pathway (TCP)). These complement biomarker data were analyzed in the context of a documented mutation in complement regulatory proteins and response to therapy with PEX and/or eculizumab. Response was defined as a normal platelet count and improved serum creatinine (at 6 months) independent of PEX therapy or with continued eculizumab therapy.

Results
The median complement biomarker data at presentation are shown in Table 2. Complement activation products are generally sensitive biomarkers for aHUS patients. Notably, elevation of the AP (Bb) and the TCP (C5b-9) biomarkers were more prominent and were seen in 16/19 and 19/19 patients respectively. This is in contrast to previously published data from acquired TTP patients from our group that demonstrated less pronounced activation of the AP and TCP at presentation (accepted for publication, JTH). Similar levels of alternative, classical and terminal complement activation were seen among patients regardless of mutation status or response to PEX. In a preliminary analysis using a one sample T-test model, (Bb) activation was shown to be significantly lower in the non-responder to eculizumab compared to the 8 responding patients. TCP activation was also lower in the non-responder but did not show statistical significance.

Conclusions
Complement biomarkers of the alternative and terminal complement pathway may more precisely define the diagnosis of aHUS. These hypothesis generating data suggest that pre-treatment complement biomarkers provide valuable information in the diagnosis of aHUS and could be useful in the prediction of response to eculizumab

Disclosures:
No relevant conflicts of interest to declare.
Mechanism Of Anti-Myeloma Effects Due To Silencing Of Deptor
Authors: Yang Yonghui , Lichtenstein Alan 
Published: October 2019

DEPTOR is a recently described 48 kDa protein that binds to mTOR and inhibits this kinase within TORC1 and TORC2 complexes. Over-expression of DEPTOR specifically occurs in the multiple myeloma genetic sub-class that contains IgH translocations. Most interestingly, DEPTOR knock-down in over-expressing MM cell lines induces growth arrest and apoptosis, indicating DEPTOR could serve as a therapeutic target in selected patients. To investigate the mechanism of MM cytoreduction induced by DEPTOR silencing we generated a doxycycline-inducible DEPTOR shRNA. Induced DEPTOR knockdown in 8226 and OPM-2 cell lines resulted in phosphorylation of p70S6K and 4E-BP1 (due to release of TORC1 inhibition) as well as apoptosis, cell cycle arrest and senescence. RAPTOR knockdown in these MM cells prevented TORC1 stimulation and the anti-MM effects resulting from DEPTOR silencing. Thus, the acute activation of TORC1 mediated the apoptosis and growth arrest. Although DEPTOR silencing and mTORC1 activation resulted in depressed AKT activity, probably due to the TORC1-PI3K/AKT negative feedback loop, this was not relevant to anti-MM effects as over-expression of wild type or a phosphomimetic version of AKT did not prevent the adverse effects. Similarly, induction of ER stress was also not relevant to the anti-MM effects as markers for UPR activation did not occur during DEPTOR silencing. Although western blot did not show alteration of p53 expression, p21 and p27 were induced subsequent to DEPTOR knockdown. Associated RAPTOR knockdown prevented the upregulation of p21 but did not affect p27 levels. In addition, over-expression of AKT did not prevent the upregulation of p21. To test whether p21 was integral to DEPTOR knockdown effects, we silenced p21. P21-silenced MM cells were protected against apoptosis, cell cycle arrest and senescence induced by DEPTOR knockdown. Furthermore, DEPTOR knockdown in a murine xenograft model, achieved by ingestion of doxycyline, resulted in tumor regression, TORC1 activation, apoptosis and p21 induction. These data indicate a novel pathway to explain the lethal effect of DEPTOR knockdown in MM cells. The pathway is initiated by acute activation of mTORC1, with subsequent p21 induction. The data also suggest that DEPTOR over-expression in some MM clones is pro-tumoral due to its ability to suppress expression of CDK inhibitors like p21 and p27.

Disclosures:
No relevant conflicts of interest to declare.
Inherent CD40L Expression Complements CD8+ T Cell Dependent Anti-Tumor Immunity
Authors: Thiel Andreas , Frentsch Marco , Stark Regina , Japp Alberto Sada , Listopad Joanna , Matzmohr Nadine , Meier Sarah , Busch Dirk , Schulz Axel , Blankenstein Thomas 
Published: October 2019

Introduction & Objective
Adoptive T cell therapy with tumor-specific CD8+ T cells is a promising treatment option for a variety of malignant diseases. However, it is unclear which subset of CD8+ T cells characterized by distinct functions is most suitable for achieving effective and durable anti tumor responses. So far CD8+ T cells have been considered to act predominantly as cytotoxic effector cells in cellular anti-tumor immunity. In this respect cytolytic molecules such as perforin and granzymes and apoptosis-inducing receptors of the tumor necrosis family such as FasL, TNFα and TRAIL have been regarded as major CD8+ T cell effector mechanisms.

Methods & Results
We here demonstrate in an experimental tumor model that CD40L, the key molecule for “immunological help”, is expressed by up to 50% of tumor-specific CD8+ T cells in B6 mice challenged with SV40 T antigen+ cancer cells. To study the influence of CD40L on anti-tumor CD8+ T cell immunity in vivo we challenged Rag1-/- mice with cancer cells and transferred wt or CD40L-/- CD8+ T cells. Transfer of wt CD8+ T cells prevented the establishment of a solid tumor, whereas injection of CD40L-/- CD8+ T cells alone or in addition with wt CD4+ T cells resulted in a non-controlled tumor development similar to non-treated tumors. The requirement of CD40L on CD8+ T cells for tumor rejection was further confirmed by injecting cancer cells in mice that lack CD40L expression only on mature CD8+ T cells. CD40Lflox x E8Icre mice were more susceptible to tumor formation than wt mice. Furthermore we demonstrated that CD8+ T-cell derived CD40L had to interact with CD40 on cancer cells, an eminent signal to induce apoptosis in various cancer cell types.

Conclusion
Our results reveal a crucial functional relevance of CD40L expressed by CD8+ T cells in anti-tumor immunity. Various cancer cell types express CD40 and its engagement induces pro-apoptotic or growth-inhibitory signals in a variety of cancer cells. Therefore CD40 agonists are recognized as promising agents for therapeutic interventions. We here introduce CD40L+ CD8+ memory T cells as a new major physiological source of CD40L, essential for rejection of tumors. Our data reveal that the presence or absence of CD40L+ CD8+ T cells represents a crucial element in control of CD40 expressing cancers disclosing novel treatment approaches in adoptive T-cell therapies novel treatment approaches.

Disclosures:
No relevant conflicts of interest to declare.
Allogeneic Cytokine-Induced Killer Cells Eliminate Host Dendritic Cells Due To The Enhanced Killing Activity By IFN-Gamma, Leading To Less Graft-Versus-Host Disease
Authors: Mase Shintaro , Fujiki Toshihiro , Kuroda Rie , Araki Raita , Maeba Hideaki , Koizumi Shoichi , Yachie Akihiro , Nishimura Ryosei 
Published: October 2019

Cytokine-induced killer (CIK) cells are ex vivo–expanded T lymphocytes expressing both natural killer (NK)– and T-cell markers. We have reported that adoptive transfer of allogeneic CIK cells in a murine model caused minimal graft-versus-host disease (GVHD) with retention of antitumor activity mediated by NKG2D, which is an activating receptor expressed on NK cells. It was reported previously that IFN-gamma was a critical cytokine for preventing GVHD because CIK cells generated from IFN-gamma knock out mice caused lethal GVHD. One possible mechanism of protective effect against GVHD was that a large amount of IFN-gamma secreted by CIK cells could promote Fas-mediated apoptosis of recipient antigen activated donor T cells. However the mechanism of suppression of GVHD with CIK cells is not fully understood. Host residual dendritic cells (DCs) are most important cells in initiating GVHD reaction. Therefore, we hypothesize that CIK cells eliminate host DCs as same as kill tumor cells, and then reduce GVHD severity. To test this, 51Cr release assay was performed using BM derived DCs as target cells. We showed that allogeneic CIK cells had cytolytic activity against DCs, in marked contrast killing activity of IFN-gamma deficient CIK cells was much less compared to that of WT CIK cells (p< 0.05). Expression level of surface markers including NKG2D and CD8/CD4 ratio was not different between WT and IFN-gamma KO CIK cells. To further explore the mechanism why IFN-gamma deficient CIK cells reduced killing activity against DCs, IFN-gamma neutralizing antibody was added into killing mixture with WT CIK cells. However killing activity of WT CIK cells against DCs with IFN-gamma antibody did not changed compared to that of WT CIK alone. These results suggested that IFN-gamma itself did not affect killing activity directly, but IFN-gamma was essential cytokine to enhance killing activity during the culture of CIK cells. Next concern was whether CIK cells eliminated host DCs in vivo or not, we compared the residual number of host-type splenic DCs between the mice receiving bone marrow (BM) cells plus WT CIK cells and IFN-gamma deficient CIK cells. As shown in the figure below, the number of host DCs in the spleen in the mice receiving WT CIK cells was significantly lower compared to those of the mice receiving IFN-gamma KO CIK cells (p<0.05). In conclusion, allogeneic CIK cells eliminated host DCs due to the enhanced killing activity by IFN-gamma, leading to much less GVHD.

Disclosures:
No relevant conflicts of interest to declare.
Daily G-CSF (filgrastim) and a Single Dose Of Pegylated G-CSF (pegfilgrastim) After Chemotherapy Result In Different Kinetics Of Circulating Hematopoetic Progenitors: Implication For Potential Stem Cell Harvesting
Authors: Schaub Felix , Federmann Birgit , Kanz Lothar , Möhle Robert 
Published: October 2019

Several studies have shown that a single dose of pegylated G-CSF (pegfilgrastim, Neulasta®) administered after chemotherapy allows sufficient collection of hematopoietic progenitor cells (HPC) for autologous transplantation. Pegfilgrastim has not been approved for HPC mobilization, but the decision for stem cell harvesting may be made after pegG-CSF has already been given. In this case, particular kinetics of circulating HPC after pegG-CSF must be taken into account to guide stem cell harvesting. In an intra-individual analysis, we compared mobilization of HPC in 12 lymphoma patients undergoing two identical cycles of salvage chemotherapy (etoposide, ifosfamide, cisplatin, ± epirubicin, ± rituximab) within a clinical trial (ClinicalTrials.gov: NCT00306111). G-CSF (filgrastim, Neupogen® 5 ug/kg/d) was given daily after the 1st cycle starting at day 3, while a single dose of pegfilgrastim (Neulasta®, 6 mg) was administered after the 2nd cycle. Circulating HPC were analyzed by flow cytometry and colony assays. We found that neutrophil recovery was remarkably pronounced after pegG-CSF resulting in a significant higher white blood count at day 12 compared with filgrastim (WBC: 6.8 ± 1.9 G/l vs. 3.2 ± 0.8 G/l, mean ± SEM), but a similar WBC at day 14 and day 16. However, kinetics of circulating HPC were markedly different: A trend to higher numbers of CD34+ progenitors was observed after pegfilgrastim at day 12 compared to filgrastim, followed by a sharp drop at day 14: 25 ± 12 /μl (pegfilgrastim) vs. 69 ± 22 /μl (filgrastim). Particularly the number of more primitive CD34+CD38- progenitors was significantly reduced at day 14: 3.7 ± 3.0 /μl (pegfilgrastim) vs. 11.7 ± 6.3 /μl (filgrastim). Similar results were found for the more primitive CD34+CD133+ und CD34+CD90+ populations. The colony formation (cloning efficiency) of the HPC was not different. Also, coexpression of homing-related receptors (CXCR4, CD62L/L-selectin) was similar in both groups. We conclude that after chemotherapy and pegG-CSF, sufficient numbers of HPC are circulating which allows for HPC collection during the early recovery phase. However, the HPC count is rapidly dropping once the WBC has recovered, in line with the known elimination of pegGCSF by binding to neutrophils. Thus, when a stem cell harvest is scheduled after neutrophil recovery in these patients, daily addition of non-pegylated G-CSF should be considered.

Disclosures:
Kanz: Amgen GmbH: Research Funding. Möhle:Amgen GmbH: Research Funding. Off Label Use: Pegfilgrastim is not approved for stem cell mobilization.