bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Role of the cGMP-Dependent Protein Kinase II in Platelet Activation and In Vivo Thrombosis.
Authors: Li Zhenyu , Zhang Guoying , Yin Hong , Feil Robert , Hofmann Franz , Du Xiaoping 
Published: April 2020

Abstract
Although it was previously believed that the intracellular secondary messenger cGMP inhibits platelets, we have recently shown that cGMP-dependent protein kinase I (PKG I) in fact plays a stimulatory role in platelet activation. However, there are apparent differences between the PKG inhibitors and PKG I knockout in their effects on platelet activation. PKG inhibitors are more potent in inhibiting platelet activation than PKG I knockout. More importantly, although platelet secretion and aggregation induced by collagen were inhibited by PKG inhibitors, they are not significantly affected in PKG I knockout platelets. There are two types of PKG, PKG I and PKG II. PKG II has not been previously described in platelets. Here we show that PKG II mRNA is expressed in platelets using RT-PCR with primers specific for a C-terminal fragment of human PKG II cDNA. We further cloned the complete cDNA of human PKG II by RT-PCR using the purified human platelet mRNA as a template. Furthermore, PKG II from platelet lysates was pulled down by cGMP conjugated agarose beads and detected by western blot using a polyclonal antibody against PKG II. These data indicate that PKG II is expressed in platelets. To investigate the role of PKG II in platelet activation, washed wild type or PKG II knockout (PKG II−/−) mouse platelets in tyrode’s solution were exposed to platelet agonists. Platelet aggregation and ATP secretion induced by low concentrations of collagen were significantly reduced in PKG II deficient mice, indicating that PKG II plays important roles in collagen-induced platelet activation. PKG II−/− platelets also showed reduced aggregation and secretion to low dose of a thromboxane A2 (TXA2) analog, U46619. However, low dose thrombin-induced platelet activation was not negatively affected in PKG II−/− platelets, but was inhibited in PKG I−/− platelets. To evaluate the in vivo role of PKG II, we compared in vivo thrombus formation of wild type and PKG II knockout mice using the FeCl3-injured carotid artery thrombosis model. The time to the formation of stable thrombus in PKG II−/− mice (median, 420.0 seconds, n=15) is significantly prolonged compared to wild type mice (median, 321.0 seconds, n=15) (p=0.031). Tail-bleed time analysis also indicated a remarkably prolonged bleeding time in PKG II−/− mice (the median bleeding time was 73.50 seconds (n=18) in wild type mice, 454.50 seconds (n=20) in PKG II knockout mice) (p=0.0008). Thus, PKG II plays an important role in promoting platelet activation, thrombosis and hemostasis. PKG I and PKG II have differential roles in platelet activation induced by different platelet agonists.
FcRγ-Chain Signals in GPVI-Deficient Platelets.
Authors: Liu Junling , Ware Jerry , Jackson Carl W. , Gartner T. Kent 
Published: April 2020

Abstract
The receptor glycoprotein VI (GPVI) is required for hemostasis and collagen-induced platelet aggregation. GPVI and FcRγ-chain form a noncovalent signaling complex. Genetic ablation of murine FcRγ-chain prevents expression of GPVI, and immunodepletion of GPVI removes FcRγ chain from platelet membranes. The GPVI/FcRγ-chain relationship has confounded elucidation of the individual role(s) of GPVI and FcRγ-chain in signal transduction. For example, botrocetin (bt)/von Willebrand factor (vWf) and γ-thrombin induced aggregation of washed platelets each appear to be FcRγ-chain-dependent. However, since FcRγ-chain−/− platelets also lack GPVI, the independent role(s), if any, of these signaling molecules in this aggregation could not be established. But, genetic ablation of murine GPVI does not exclude expression of the FcRγ-chain. Therefore, based on analyses of genetically deficient GPVI platelets that express FcRγ-chain, we demonstrate here that bt/vWf and thrombin-induced aggregation of washed platelets are each dependent on FcRγ-chain thereby demonstrating that FcRγ-chain can function in the absence of GPVI. Presumably, another receptor or receptors is/are able to activate FcRγ-chain in GPVI −/− and GPVI +/+ platelets. GPIb may not directly activate FcRγ-chain because GPIb elicited activation of αIIbβ3 is not FcRγ-chain dependent. The integrin αIIbβ3 appears to be common to the signaling described here because both the non-agglutination elicited bt/vWf-induced, and the γ-thrombin-induced TxA2 production and ATP secretion were β3-dependent. Therefore, αIIbβ3 may have activated this FcRγ-chain-dependent signaling. This hypothesis was tested by investigating the role of FcRγ-chain in αIIbβ3-mediated outside-in signaling induced by a palmitoylated derivative of the peptide KVGFFKR, a peptide which directly activates αIIbβ3 causing β3-dependent signaling that elicits TxA2 production, ATP secretion and platelet aggregation. As with γ-thrombin-treated FcRγ-chain−/− platelets, pKVGFFKR-treated FcRγ-chain−/− platelets did not produce TxA2, secrete ATP or aggregate. But, pKVGFFKR-treated GPVI−/− platelets produced the wild type level of TxA2, secreted the wild type level of ATP, and aggregated normally demonstrating that pKVGFFKR-induced αIIbβ3-mediated outside-in signaling is FcRγ-chain dependent. These results support the hypothesis that activation of FcRγ-chain is αIIbβ3-dependent in the signaling described here. The results of ELISA studies support this conclusion.
Reduced Intensity Conditioning (RIC) Allogeneic Stem Cell Transplantation (allo-SCT) for Patients with Acute Lymphoblastic Leukemia (ALL): A Survey from the European Group for Blood and Marrow Transplantation (EBMT).
Authors: Mohty Mohamad , Labopin Myriam , Boiron Jean-Michel , Theorin Niklas , Fauser Axel , Rambaldi Alessandro , Boogaerts Marc , Slavin Shimon , Majolino Ignazio , Blaise Didier , Rocha Vanderson 
Published: April 2020

Abstract
The role of RIC allo-SCT for adult patients with ALL is still under considerable debate. While the use of such so-called nonmyeloabaltive or RIC regimens has emerged as an attractive modality to decrease transplant-related toxicity, it is still unclear whether such regimens are associated with a graft-vs.-leukemia (GVL) effect in adult ALL. This report describes the results of 91 adult ALL patients (62 M and 29 F) treated in different centers affiliated with the EBMT. Median age was 40 y. (range, 17–66). Twenty nine (32%) patients were transplanted in first complete remission (CR), 28 (31%) beyond first CR, and 34 (37%) in advanced disease. In this cohort, 39 patients (51%) had a Ph+ or t (4;11) ALL. The majority of patients received a PBSC graft (78%) from an HLA-matched sibling donor (n=62; 68%). The RIC regimen included Fludarabine (80%) associated to low dose TBI in 22 patients (24%), and/or low dose Bussulfan (&lt;8mg/kg). ATG was used in 29 patients (33%), mainly in unrelated HSCT. With a median follow-up of 34 months (range, 7–76), the incidences of grade II-IV and grade III-IV acute GVHD were 35% and 13% respectively. At 2 years, chronic GVHD occurred in 49% of patients. Cumulative incidence of transplant-related mortality at 3 years (TRM; 11 fatal infections, 5 refractory GVHD, 6 other) was 24% (15% in patients transplanted in first CR). Cumulative incidence of relapse at 3 years was 58%+/−5 in this high risk group. Three years leukemia-free survival (LFS) was 18% in the whole study group, with this being significantly higher (34%) in patients transplanted in first CR. Patients transplanted in advanced refractory disease had the worst LFS (5%). In multivariate analysis, disease status at time of allo-SCT (first CR vs. other) was the strongest factor associated with an improved LFS (P=0.04; RR=2.1; 95%CI, 1.02–4.20).
The results of this retrospective registry based study suggest that RIC allo-SCT for adult ALL may represent a valid therapeutic option when a conventional standard conditioning is not possible. Of note, results obtained in patients transplanted in first CR (LFS and TRM) compare favorably with previous results achieved after standard myeloablative allo-SCT, warranting further investigations in this subgroup. Also, better understanding of the putative GVL effect in adult ALL, in addition to designing maintenance strategies (imatinib in Ph+ ALL for eg.) after RIC allo-SCT, may further improve the outcome of adult ALL.
Contrasting Effects of Post-Transplant Lymphopenia on Proliferation and Degranulation in Antigen-Specific CD8 Memory T Cells.
Authors: Shatry Alwi M. , Levy Robert B. 
Published: April 2020

Abstract
Lymphopenia following bone marrow transplant (BMT) can induce expansion of donor T cells which help contribute to reconstitution in recipients. We are interested in elucidating the responses of transplanted CD8 T memory (TM) to lymphopenic signals in the recipient splenic and marrow environments. To compare proliferative responses of marrow and spleen populating antigen-specific CD8 TM to lymphopenic signals, purified (95% pure) CFSE labeled B6 CD8 TM were co-transplanted with syngeneic or allogeneic TCD-BM to ablatively conditioned (9.0 Gy) syngeneic B6 recipients. Antigen-specific CD8 TM were obtianed following priming B6 mice against BALB.B antigens. An H60 tetramer (LTFNYRNL/H2-Kb) conjugated to PE was used to identify immunodominant H60-specific CD8+ cells which expressed a central memory (TM) phenotype (CD44+, Ly6C+). Three days post-transplant of these CD8 TM, recipient spleen and marrow cells were analyzed for CFSE dilution in gated H60+CD8+ T cells. Transferred H60+ CD8+ cells in the marrow compartment consistently showed greater (5 – 6 cell divisions) proliferative activity compared to those populating the spleen (1 – 2 divisions) in recipients of syngeneic (i.e. homeostatic) as well as allogeneic TCD-BM. Thus, the marrow compartment apparently provided greater support for the early homeostatic proliferation of these MiHA-specific CD8 TM. Furthermore, the presence of antigen in B6 recipients of allogeneic BM did not significantly enhance the proliferation of transferred CD8+ TM, indicating that homeostatic signals induced greater overall proliferation than antigen activation in the simultaneous presence of both signals early post-transplant. To examine for non-proliferative functional activity in transplanted antigen-specific CD8 TM, degranulation was assessed by surface expression of LAMP-1 (CD8 107a) which was analyzed 3 days after the transfer of purified splenic CD8 T cells from B6BALB.B into lethally irradiated (9.0 Gy) syngeneic B6 mice. Analysis of recipient spleeen and marrow showed that transferred antigen-specific in these tissues markedly upregulated surface LAMP-1 expression. Interestingly, there was greater frequency (~90%) of CD8+ TM in the spleen expressing LAMP-1 than in the bone marrow (56%). To determine if this degranulation response was limited to transplanted TM, a homogeneous population of naive T cells (TN) from OT-1 RAG−/− TCR tg mice was prepared and transplanted in an identical manner. LAMP-1 expression was marginally detectable in these cells. In total, these results suggest that transplanted CD8 TM and TN respond differently in the homeostatic environment post-transplant. Moreover the observations may suggest that CD8 TM can exhibit effector-like activity in the lymphopenic milieu immediately post-transplant. These findings have implications for the relative contribution of hematopoietic compartments during immune reconstitution after HCT.
The Menin Tumor Suppressor Protein Is an Essential Oncogenic Cofactor for MLL-Associated Leukemogenesis.
Authors: Yokoyama Akihiko , Somervaille Tim , Cleary Michael L. 
Published: April 2020

Abstract
The Mixed Lineage Leukemia (MLL) protein is a histone methyltransferase that is mutated in clinically and biologically distinctive subsets of acute leukemia. MLL normally associates with a cohort of highly conserved cofactors to form a macromolecular complex that includes menin, a product of the MEN1 tumor suppressor gene, which is mutated in heritable and sporadic endocrine tumors. We demonstrate here that oncogenic MLL fusion proteins retain an ability to stably associate with menin through a high-affinity, amino-terminal, conserved binding motif and that this interaction is required for the initiation of MLL-mediated leukemogenesis. Furthermore, menin is essential for maintenance of MLL-associated but not other oncogene induced myeloid transformation. Acute genetic ablation of menin reverses aberrant Hox gene expression mediated by MLL-menin promoter-associated complexes, and specifically abrogates the differentiation arrest and oncogenic properties of MLL-transformed leukemic blasts. These results demonstrate that a human oncoprotein is critically dependent on direct physical interaction with a tumor suppressor protein for its oncogenic activity, validate a potential target for molecular therapy, and suggest central roles for menin in altered epigenetic functions underlying the pathogenesis of hematopoietic cancers.
Identification of Novel Signalling Pathways in Myeloblasts from Patients with Acute Myeloid Leukemia Using a Phosphoproteomic Strategy.
Authors: Craddock Charles , Tauro Sudhir , Griffiths Michael , Cook Mark , Singh Harprit , Wakelam Michael 
Published: April 2020

Abstract
Acquired somatic mutations which result in constitutive activation of signal transduction pathways are increasingly recognised in acute myeloid leukemia (AML). Reasoning that since protein tyrosine phosphorylation is an important mechanism mediating the transduction of proliferative and survival signals we have utilised a proteomic strategy to identify dysregulated phosphoproteins in myeloblasts from patients with AML. Using an anti-phosphotyrosine antibody we have immunoprecipitated proteins from AML blasts, separated proteins by SDS PAGE and identified proteins within distinct bands by mass spectrometry. Results in primary AML blasts have been compared with CD34+ hemopoietic progenitors harvested from GCSF mobilised normal donors. The methodology was validated using vanadate-stimulated HL60 cells. 10 patients with a median age of 51 (range 16–90 yrs) were studied. One patient had favorable risk cytogenetics, three adverse risk cytogenetics and six a normal karyotype. Mutations in the flt-3 tyrosine kinase were present in four patients. Blasts from every patient demonstrated phosphorylation of MAP kinase (MAPK) implying activation of the ras-MAPK cascade. In contrast CD34+ cells from three normal donors demonstrated weak or no MAPK phosphorylation. Since each AML sample demonstrated MAPK phosphorylation irrespective of flt-3 status we next examined their phosphotyrosine-proteome. This identified a number of phosphorylated and non-phosphorylated proteins in signalling complexes in anti-phosphotyrosine immunoprecipitates. Notable examples amongst these were receptors (Ephrin type-A3, interleukin-13, GPCR43), signalling intermediates (TAPP1, RASA1, GDI-2, LARG, SGEF, cortactin, Cd-2 associated protein) and transcription factors (ELK-1, HFK-1). These proteins identify three functionally distinct groups in AML blasts which were not detected in normal CD34+ progenitors: (i) intermediates of a tyrosine kinase mediated ras-MAPK signalling cascade (ii) intermediates in PtdIns-3-kinase mediated signalling presumably suppressing apoptosis and (iii) regulators of the actin cytoskeleton and thus cell movement. Surprisingly the strategy also identified tyrosine phosphorylation of the 5-HT3A receptor and the MRCP1-ABC transporter in a number of patients suggesting a role for these proteins in the pathogenesis of certain subtypes of AML. The phosphorylation of a number of these identified proteins was confirmed immunochemically. Thus adoption of a phosphoproteomic methodology has identified novel phosphoproteins in AML which require further validation. Our data also demonstrate a common intracellular signalling pathway (ras-MAPK) in distinct AML patients regulated by differing primary events. These observations provide information of value in the rational development of targeted therapies in AML.
Assessment of PML-RARa Real-Time Quantitative PCR for Definition of Molecular Relapse in APL Patients (Results of the APL Study Group).
Authors: Cassinat Bruno , De Botton Stephane , Ades Lionel , Raffoux Emmanuel , Escoffre-Barbe Martine , Baruchel Andre , Miclea Jean-Michel , Reman Oumedaly , Gardembas Martine , Brault Philippe , Milpied Noel , Legrand Olivier , Dombret Herve , Fenaux Pierre , Chomienne Christine 
Published: April 2020

Abstract
Although patients with Acute Promyelocytic Leukaemia (APL or AML M3) and the classical t(15;17) translocation can now benefit from efficient treatments, some 10 to 20% of patients still relapse at 5 years. In order to improve the clinical outcome of APL patients, the early detection of disease reactivation should allow treatment at molecular relapse rather than at complete haematological relapse. To this end accurately defining molecular relapse is compulsory. Recent studies suggest that RQ-PCR methods applied to the detection of minimal residual disease (MRD) could provide such criteria. Our laboratory has established a RQ-PCR method (Cassinat et al., 2001) using Taqman technology. Standard curves are established and the PML-RARa copy number is normalized according to the expression of the housekeeping gene PBGD. A total of 260 APL patients (APL study Group) have been followed from 2000 to 2004 with a total of 970 samples analyzed. Median follow up for all these patients was 24 mths (range: 1–83 mths) with a median number of samples per patient of 6 (range: 1–28). During this period, 38 patients presented at least one positive result after a median negative period of 13 mths and 13 of them relapsed (median follow-up of 17 mths, range from 3 to 43mths). To evaluate the value of this re-positivity, we tried to determine whether a predictive threshold of PML-RARa copy as assessed by RQ-PCR method could allow detecting patients with molecular relapse. At the time of the first positive result, patients were in hematological CR. Patients were grouped according to the normalized PML-RARa copy number and the risk of relapse was studied in each group. No relapse (n= 0/17; 0%) was noted in patients with less than 10 copies (group A). 38% relapses (n= 5/13) in patients with 10 to 102 copies (group B) and 100% relapses (n=8/8) in patients with more than 102 normalized copies (group C). Negative RQ-PCR was reached again in all patients that did not relapse. Interestingly in groups B and C, interval to hematological relapse appeared to be related to the number of PML-RARa copies at first positivity (median interval to relapse was 6 months in group B and 2 months in group C). For 3 positive patients of group B, 3 consecutive samples were analysed until relapse: copy numbers increased exponentially. From these results we conclude that RQ-PCR is a powerful tool to define the molecular relapse in APL patients as it is able to accurately identify two subgroups of patients with either a 100% or no risk of relapse wilts median follow up of 24 mths. These patients with true molecular relapse should probably receive salvage therapy without waiting for the hematological relapse.
Primary CNS Lymphoma - High-Dose-Chemotherapy with Autologous PBSCT and Hyperfractionated Radiotherapy as First-Line-Therapy - Results of a Multicenter Phase II Study.
Authors: Illerhaus Gerald , Marks Reinhard , Derigs Guenther , Peschel Christan , Frickhofen Norbert , Guttenberger Roland , Kubin Thomas , Ostertag Christoph B. , Finke Juergen 
Published: April 2020

Abstract
Primary NHL of the CNS (PCNSL) carries a poor prognosis despite initial response to steroids and radiotherapy (RT). Addition of methotrexate (MTX) to RT has improved the prognosis of patients (pts) with PCNSL, but a significant proportion of patients are still not cured. To improve relapse free survival and to reduce neurotoxicity we initiated a multicenter phase II study with early dose intensified chemotherapy (CT) and PBSCT followed by hyperfractionated whole-brain radiation (WBRT) for pts aged under 65yrs. The use of high-dose (HD) lipophilic blood-brain-barrier-penetrating agents (BCNU, Thiotepa) in addition to maximum doses of water-soluble agents (MTX, AraC) is a novel approach in the treatment of PCNSL. To reduce the risk of delayed neurotoxicity, intrathecal chemotherapy was completely avoided and CT was administered before RT. Induction treatment included 3 repetitive cycles of HD-MTX (8g/m2). AraC (2x 3g/m2) and thiotepa (40mg/m2) followed by rG-CSF were used for stem-cell-mobilisation. The conditioning regimen included BCNU (400mg/m2) and thiotepa (2x5mg/kgBW) prior PBSCT. Additional hyperfractionated WBRT (45 Gy, 2x1Gy/d) was administered as consolidation. From 1999 to 2003 thirty pts (age under 65y) have been enrolled in the study (median age 54, range 30–64y). 21 of 30 pts responded to HD-MTX (6 CR, 15 PR). 22/30 pts received high dose CT and autologous PBSCT according to the protocol. Four pts refractory to MTX proceeded to RT directly and 1 patient died from PD subsequently. One patient died because of treatment-related liver toxicity after High-Dose MTX. Beside cytopenia no severe acute toxicities [WHO Grade 3 or 4] were observed after high-dose chemotherapy. After intention-to-treat analysis the response-rate was 89%. All patients that completed the protocol obtained CR (22/22). Three of these pts died, two died during follow-up due to relapse after 1 or 5 years respectively, the third patient died after 25 mo due to progressive heart failure. With a median follow-up of 42 months (range 3–84 mo) the overall survival of all pts included and pts that fulfilled the protocol is 71,3% and 86,5%, respectively. We conclude that sequential systemic application of high-dose differential acting cytostatic agents with consolidating hyperfractionated radiotherapy is very effective and well tolerable. In a new multicenter phase II study, pts will be treated with more intensive high dose CT and PBSCT omitting consolidating radiotherapy.