bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification of BCR-ABL Dependent Gene Regulation by Using a Phosphoproteomics Approach.
Authors: Ziegler Patrick , Balabanov Stefan , Hartmann Ulrike , Kammer Winfried Kammer , Nordheim Alfred , Brummendorf Tim H. 
Published: April 2020

Abstract
Introduction: The selective tyrosine kinase inhibitor imatinib (formerly STI571, Glivecâ) has been shown to block phosphorylation of tyrosine residues by occupying the ATP binding site of the Abl tyrosine kinases Bcr-Abl, c-Abl, v-Abl and Abl-related gene (ARG), as well as platelet-derived growth factor receptor (PDGF) alpha and beta and of the receptor for human stem cell factor (SCF) c-kit. We chose a large scale phospho-proteomics approach to identify novel downstream targets of imatinib which could possibly be utilized for combined treatment strategies.
Material and Methods: Phospho-proteomics was performed by comparison of large scale phosphotyrosine-immunoprecipitation of imatinib (10 mM/2 hours) versus DMSO treated K562 cells separated by one-dimensional polyacrylamide gelelectrophoresis. In addition enriched CD34+ cells (&gt;70%) of a newly diagnosed Bcr-Abl positive CML patient were used immediately after purification and treated in the same way as described above. Resulting differentially immuno-precipitated proteins were analyzed using matrix-assisted laser desorption/ionization - time of flight mass spectrometry (MALDI-TOF) and nano electrospray ionization tandem mass spectrometry (ESI-MS/MS). Protein identification via peptide mass-fingerprinting and peptide sequencing was performed using Mascot search tool and NCBI nr database. Differential phosphorylation was confirmed by combined immunoprecipitation and western blot analysis of selected candidate proteins.
Results: Phospho-proteomics of K562 cells revealed 8 differentially phosphorylated proteins after a two hour treatment with imatinib including the recently identified c-cbl, and Bcr-Abl itself, the latter confirming autophosphorylation. Ship2 which was originally identified as beeing constitutively phosphorylated in chronic myelogenous leukemia progenitor cells showed reduced, imatinib sensitive phosphorylation. Remaining candidates could be classified as being involved in protein folding or in ATPase activities associated with a variety of functions (type II AAA). The analysis of primary CD34+ cells from a CML patient showed a predominance of the nonmuscular myosin heavy chain protein in different molecular weight forms.
Discussion: We detected significant imatinib-dependent differences in protein phosphotyrosine-immunoreactivity of the Bcr-Abl -dependent cell line K562. Previously identified down-stream targets of Bcr-Abl could be confirmed and novel candidate proteins were identified. Phosphorylation of Ship2, a previously identified down-stream target of Bcr-Abl, was found to be inhibited by imatinib treatment. Ongoing studies are aimed at the characterization of the role of the identified phospho-proteins, particularly type II AAAs for Bcr-Abl induced signal transduction as well as for the development of resistance to imatinib.
Critical Role of Dok-1 and Dok-2 in Leukemia Suppression.
Authors: Niki Masaru , Di Cristofano Antonio , Zhao Mingming , Hirai Hisamaru , Van Aelst Linda , Cordon-Cardo Carlos , Pandolfi Pier Paolo 
Published: April 2020

Abstract
Chronic myelogenous leukemia (CML) is characterized by the presence of the chimeric p210bcr/abl oncoprotein which shows elevated and constitutive protein tyrosine kinase activity relative to the normal c-abl tyrosine kinase. While several p210bcr/abl substrates have been identified, their relevance in the pathogenesis of the disease is unclear. We have identified a family of proteins, Dok (downstream of tyrosine kinase), coexpressed in hematopoietic progenitor cells. Members of this family such as p62dok (Dok-1) and p56dok-2 (Dok-2) associate with the p120 rasGTPase-activating protein (rasGAP) upon phosphorylation by p210bcr/abl as well as receptor and non-receptor tyrosine kinases. Here we report the generation and characterization of single and double Dok-2 or Dok-1/Dok-2 KO mutants. Single KO mice displayed normal steady state hematopoiesis. By contrast, concomitant Dok-1 and Dok-2 inactivation resulted in aberrant hemopoiesis and Ras/MAP kinase activation. Strikingly, all Dok-1/Dok-2 double KO mutants spontaneously developed transplantable CML-like leukemia due to increased cellular proliferation and reduced apoptosis. Furthermore, Dok-1 or Dok-2 inactivation markedly accelerated leukemia and blastic crisis onset in Tec-p210bcr/abl transgenic mice known to develop, after long latency, a myeloproliferative disorder resembling human CML. These findings unravel the critical and unexpected role of Dok-1 and 2 in tumor suppression and control of the hematopoietic compartment homeostasis.
Evaluation of the Profile of Thrombin Generation during the Process of Whole Blood Clotting as Assessed by Thromboelastography.
Authors: Rivard Georges E. , Kathleen Brummel , Li Fan , Angelique Hofer , Eli Cohen , Kenneth Mann G. 
Published: April 2020

Abstract
The evaluation of thrombin generation during blood coagulation has become the preferred clinically relevant approach to assess the global integrity of this complex process. In order to give the full picture of the physiology of this multifactorial reaction, all cellular and soluble plasma participants to this reaction have to be present in their natural whole blood environment. Whole blood thrombin generation tests are best suited for this purpose but are somewhat cumbersome for use in real time clinical practice. Thrombelastography has long been recognized as a clinically useful technology for assessment of whole blood coagulation. The purpose of this study was to evaluate the possibility of linking the tracing generated by clotting of whole blood in a Thrombelastograph® (TEG®) Hemostasis System with the generation of thrombin as assessed by thrombin/antithrombin complex (TAT). 320μL of citrated whole blood (3.2% buffered citrate and 1000μg/ml of corn trypsin inhibitor; 1 part to 9 parts of blood) from volunteers was clotted in the presence of 20μL CaCl2 (200mM) and 20μL of Innovin (1/100 diluted in PBS/albumin 4%, pH 7.4). Clotting was monitored with 8 channels of the TEG® system. At different time points (4 to 40 min) the whole blood TEG® reaction cups were put in 2640μL of cold quenching solution (EDTA 50mM, L-benzamidine 10mM, NaCl 150mM, HEPES 2mM, pH 7.4, and 10μL of 10mM D-Phe-Pro-Arg CH2Cl chloromethylketone diluted in 0.01M HCl), vortexed, and centrifuged, and the supernatants were kept at −80°C until assayed for TAT by ELISA. Total Thrombus Generation (TTG) was calculated from the first derivative of the TEG® waveform and was compared to the thrombin generation as measured by TAT as described above. The two vectors of values - the TAT thrombin generation data and the corresponding TEG® TTG - were analyzed using Pearson correlation coefficients and linear, non-linear, and natural log (Ln) transformation of TAT values for least-squares goodness-of-fit curves. The best least-squares fit is the exponential curve (Fig. 1) and its corresponding natural log transformation of TAT value as shown by Fig. 2, with R2 = 0.8787 (P &lt; 0.0001). The prediction equation for TAT thrombin generation is y = 3122.7e0.0005x, where x is the TEG® TTG variable (P &lt; 0.0001). Linearizing using the natural log of the TAT thrombin generation variable (Fig. 2), produces the same R2 as the exponential curve. The prediction equation is y = 8.0465 + 0.0005x, significant at P &lt; 0.0001, where y is the TAT thrombin generation in the Ln transformation variable and x is the TEG® TTG variable. The high magnitude of R2 and the high significance of the prediction equation demonstrate the high efficacy of the prediction of TAT thrombin generation using the TEG® TTG.
Host γd T cells Exacerbate Experimental Acute Graft-Versus-Host Disease through Activation of Host Antigen Presenting Cells.
Authors: Maeda Yoshinobu , Reddy Pavan , Liu Chen , Keith Bishop D. , Ferrara James L.M. 
Published: April 2020

Abstract
Large numbers of T cells bearing γd T cell receptors are present in graft-versus-host disease (GVHD) target tissues. We investigated the potential role of host γd T cells during acute GVHD in a well-characterized GVHD model following full intensity conditioning (11 Gy TBI). BM and spleen T cells from BALB/c (H2d) donors were transplanted into wild type (wt) B6, aß T cell deficient B6 (aß −/−) or γd T cell deficient B6 (γd −/−) hosts. γd −/− hosts demonstrated significantly better day 35 survival (85%) than wt (40%) or aß−/− hosts (18%) (P&lt;0.05). Reconstitution of γd −/− B6 hosts with B6 type γd T cells 24 hr prior to BMT restored lethal GVHD (50 % day 35 survival). In vivo, γd −/− B6 hosts demonstrated at least a five fold reduction in donor T cell expansion and cytokine production. In vitro, T cells proliferated less when co-cultured with allogeneic γd −/− dendritic cells (DCs) than with wt DCs (40,127 ± 1634 vs. 72,503 ± 1296, P&lt;0.05). BM-derived DCs cultured with γd T cells caused greater proliferation of allogeneic T cells than DCs cultured with aß T cells (15.1 ± 21 x 104 vs. 5.1 ± 1.2 x 104, P&lt;0.05). We next tested the effect of γd T cells on host DCs in vivo using a model system in which only the DCs injected prior to BMT expressed the alloantigen that stimulated the GVHD reaction. MHC Class II −/− B6 mice that had been depleted of γd T cells were given 11 Gy TBI and injected one day prior to BMT with B6 DCs that had been co-cultured either with γd T cells or with medium. On day 0 both groups of recipient mice were injected with BM plus splenic T cells from allogeneic bm12 donors. On day +5, CD4+ donor T cells expanded four times more in recipients of DCs co-cultured with γd T cells than in recipients of control DCs and serum levels of TNF-a were significantly higher (36.7 + 6.8 vs. 21.3 + 3.7 pg/ml, P&lt;0.05). Together these data demonstrate that γd T cells amplify the stimulatory function of host DCs and increase the severity of GVHD, suggesting that a new therapeutic target for the prevention of the major BMT toxicity.
Keratinocyte Growth Factor Ameliorates Acute Graft-versus-Host Disease in a Novel Nonmyeloablative Haploidentical Transplantation Model.
Authors: Bos Gerard M.J. , Vanclee Ariane , Oving Ellis , Lutgens Ludy , Deutz Nicolaas , Schouten Harry 
Published: April 2020

Abstract
Allogeneic stem cell transplantations (SCT) are currently being used as a therapy for hematological malignancies, some solid tumors and non-malignant bone marrow deficiencies. Nevertheless, clinical applicability is limited due to toxicity of conditioning regimens, graft-versus-host disease (GVHD) and the scarcity of HLA-identical family donors. New concepts are based on nonmyeloablative conditioning to reduce toxicity, prevention or amelioration of GVHD and the use of haploidentical donors to increase donor availability.
To meet these requirements, we have developed a nonmyeloablative conditioning regimen in a haploidentical F1 → F1 mouse model. These mini-transplantations, consisting of low dose total body irradiation and cyclophosphamide-based chemotherapy, resulted in stable full donor chimerism, but also in the development of GVHD. Administration of keratinocyte growth factor (KGF) before and after SCT resulted in reduced GVHD, evident as reduced weight loss and a lesser degree of dermatitis than in saline-treated controls. KGF preserved plasma citrulline and TNF-a levels, both indicative for reduced radiation-induced injury to the gastrointestinal tract. Citruline has recently been described as a new marker for gastrointestinal toxicity. Six months after transplantation, survival rates were significantly higher in KGF-treated animals (57% as compared to PBS-treated controls (5.3%).
Figure Figure
These data indicate that nonmyeloablative haploidentical transplantations might be feasible and that KGF might contribute substantially to reduction of lethal GVHD, also after nonmyeloablative procedures. For clinical transplantation such an effect would be significant if intensive T cell depletion might be prevented in haploidentical transplantation protocols and therefore reduced infection rates can be obtained.
Pathobiology of Hemophilic Synovitis II: Synovial Cell Proliferation.
Authors: Valentino Leonard A. , Hakobyan Narine , Kazarian Tamara , Jabbar Adnan A. , Jabbar Kausar J. 
Published: April 2020

Abstract
Hemophilia is a genetic disease due to the deficiency of blood coagulation factor VIII or IX. Bleeding is common, especially into joints where a proliferative synovitis develops resulting in a debilitating arthritis, hemophilic arthropathy. The pathogenesis of blood-induced hemophilic synovitis (HS) is poorly understood. Repeated episodes of bleeding induce synoviocyte hypertrophy and hyperplasia, an intense neovascular response and inflammation of the synovial membrane. The component(s) in blood that initiates these changes is (are) not known, although iron is often proposed as one possibility. Previously, we reported that iron promotes the proliferation of human synovial fibroblasts and increases c-myc expression in vitro (Blood2002; 100(3)912–6). Here, we describe the proliferative effects of joint bleeding following traumatic hemarthrosis in a murine model of severe, human hemophilia A. Controlled, blunt trauma to the right knee joint consistently resulted in joint swelling due to a combination of bleeding and inflammation. The left, control knee was sham injured and remained normal for the duration of the experiment. Hemarthrosis resulted in acute morbidity evidenced by inactivity, weight loss and immobility. Hemosiderin was found in the synovial membrane of the right knees only. The numbers of synovial cell layers present in the membrane of the right (injured) knee increased during the experiment (figure). Expression of Ki67 in the nucleus, a marker of cell proliferation, confirmed the increase in synovial cell proliferation in membranes from the right (injured) knees. Taken together these data support the notion that iron causes aberrant gene expression and is, at least in part, responsible for the pathological findings observed in the joints of patients following bleeding and contributes to the development of hemophilic synovits.
Figure Figure
Age and the Prevalence of Bleeding Disorders in Women Presenting with Menorrhagia.
Authors: Philipp Claire S. , Faiz Ambarina , Dowling Nicole , Dilley Anne , Michaels Lisa A. , Ayers Charletta , Miller Connie H. , Bachmann Gloria , Evatt Bruce , Saidi Parvin 
Published: April 2020

Abstract
Menorrhagia occurring during adolescence and perimenopause is presumed to be associated with anovulation, and during perimenopause, with uterine pathology, such as subserosal uterine myoma, as well. The frequency of bleeding disorders in women presenting with menorrhagia at the extremes of menstruating age, ie adolescence and perimenopause, are not known. We conducted a study to evaluate the frequency and types of hemostatic defects found in adolescent and perimenopausal age women diagnosed with menorrhagia. 115 women with a physician diagnosis of menorrhagia, including 25 adolescent women, 25 perimenopausal age women, and 65 women between the ages of 20 and 44, underwent comprehensive hemostatic testing for possible bleeding disorders. There were no significant differences between the three age groups in mean hemoglobin, percentage of women with anemia, duration of menses, mean pictorial blood assessment scores, or race. Overall, 48% of women were found to have hemostatic abnormalities.
Hemostatic abnormalities among women with menorrhagia
Overall ≤ 19 yrs 20–44 yrs ≥ 45 yrs p-value N=115 N=25 N=65 N=25 *Platelet aggregation, Von Willebrand factor, and/or coagulation factor Platelet Aggregation 44% 44% 48% 32% 0.48 Von Willebrand Factor 7% 4% 8% 8% 0.78 Coagulation Factor 5% 8% 6% 0% 0.34 Any Abnormality* 48% 48% 54% 32% 0.32
Adolescents and perimenopausal age women diagnosed with menorrhagia were as likely as women presenting between age 20 to 44 to have underlying hemostatic defects. Among the adolescents, neither age at presentation nor time from menarche were predictive of having a bleeding disorder. The platelet aggregation defects observed were similar among the three age groups with the exception of ADP induced platelet aggregation defects which were seen significantly more frequently in adolescents compared to older women (p≤ 0.01). Ristocetin and epinephrine induced platelet aggregation defects were the most common aggregation defects in women 20 years and older and ADP induced platelet aggregation defects were the most common among adolescents. There were no significant differences in platelet ATP release abnormalities between the three age groups. Our results demonstrate that a high proportion of adolescents and perimenopausal age women presenting with menorrhagia have bleeding disorders. These results suggest that menorrhagia in the adolescent, even if presenting soon after onset of menarche, and in perimenopausal age women, even if fibroids are present, may warrant hemostatic evaluation. Further age-specific studies evaluating treatment benefits and outcomes in women with menorrhagia diagnosed with bleeding disorders are warranted.
Survival of HIV Infected Individuals with Hemophilia after 20 Years: From the Canadian Hemophilia Registry.
Authors: Walker Irwin R. , Arnold Donald M. , Julian Jim A. 
Published: April 2020

Abstract
Background : Patients with hemophilia A and B have been tracked by the Canadian Hemophilia Registry since 1980. The total number on the Registry is 3307 (A=2721, B=586) of which 662 were/are HIV-positive. New HIV and HCV infections ceased after 1985 and 1988 respectively with the introduction of safer blood products; HIV-infected individuals with hemophilia therefore resemble a cohort with HIV infection that was acquired in a relatively narrow time period about 20 years ago. Current status of HIV-positive patients is Active 250 (37.8%), Deceased 406 (61.3%), Lost to Follow Up 9 (0.9%). Follow up data is available for 3135/3307 (95% of all patients), and for 656/662 (99% of HIV-positive patients). We previously reported (Haemophilia1998;4:714) a peak in the death rate of HIV-positive individuals during the years 1991 to 1993 with a subsequent decline.
Objective : To update the rate and causes of death among HIV positive hemophilia patients in Canada with current information.
HIV-Related Mortality : The annual number of deaths in the HIV-positive group has been [year-#(deaths per 100 person-years)], 1980-0(0), 81-0(0),82-0(0),83-3(0.45),84-1(0.15),85-2(0.30),86-12(1.8),87-12(1.9),88-21(3.3),89-25(4.1),90-24(4.1),91-45(8.0),92-38(7.4),93-Currently, 256/662 (38.7%) are alive.
Causes of Death (HIV+) : The commonest primary causes of death in HIV-positive individuals have been: [cause #(%)] AIDS 284(70), liver failure 45(11), bleeding 18(4.4) and infections 15(3.7). Twenty-eight patients had liver disease listed as a secondary or contributing cause of death; thus liver disease was the primary or contributory cause of death in 45+28=73 individuals (18% of HIV-positive deaths).
HCV-Related Deaths : HCV status is positive in 1151 of the total population of 3307 (35%) individuals in the registry, negative in 1260 (38%) and unknown in 896 (27%). Of HIV-positive individuals, 448 (68%) are co-infected with HCV, 53 of whom have died with liver failure (12% overall, 18% of deaths). HIV/HCV co-infected individuals have higher overall mortality than those not co-infected (46% vs. 12%, p&lt;0.0001). In addition, HCV singly-infected individuals have lower overall mortality (7%). 703 HIV-negative individuals are infected with HCV, of whom 7 have died with liver failure (1% overall, 13% of deaths).
Conclusions : The death rate of HIV-positive individuals peaked in the years 1991-3 and decreased thereafter with 38% of individuals still living at 20 years follow up. However deaths still occur at an annual rate of 3–5%. HIV/HCV co-infected individuals have the highest mortality and HCV single-infected individuals have the lowest mortality of those with transfusion-transmitted infection. Liver failure is a common mode of death in both HIV- and HCV-infected individuals.
Interferon-Gamma (IFN-γ) Secretion Defect in Peripheral Blood Mononuclear Cells (PBMCs) from Severe Hemophilia A (HA) Patients Prior to Therapeutic Exposure To Factor VIII (FVIII) Concentrates.
Authors: Gill Joan Cox , Stephany Karen , Helsell Craig 
Published: April 2020

Abstract
The recent availability of highly purified recombinant coagulation factor concentrates to treat hemorrhages in hemophilic patients is thought to circumvent perturbations in immune function by preventing chronic intravenous exposure to multiple foreign protein antigens. We previously reported a PHA-induced IFN-γ secretion defect in severe hemophilia A patients who had been exposed exclusively to highly purified coagulation factor VIII concentrates. We now report the results of cytokine secretion in response to PHA in cultured PBMCs from severe hemophilia A patients never exposed to FVIII concentrates. PBMCs were isolated from whole blood from three severe HA patients, 3 age-matched normal boys, 3 boys with severe hemophilia B and an adult control. Cells were stained with PKH26 (Sigma), a fluorescent dye with aliphatic tails that intercalate into membrane lipids; cells were than washed and cultured in complete media in wells of microtiter plates with and without PHA. Cells were harvested at 5 days and proliferation detected by loss of membrane PKH26 staining detected by flow cytometry. Cytokines secreted into the supernatants of 48 hour and 5 day cultures were measured by ELISA assays (BD Pharmingen). PBMCs from severe HA patients proliferated normally in response to PHA but failed to secrete IFN-γ when compared to normal boys and boys with severe HB:
PBMC Proliferation and Secretion of IFN-γ in Response to PHA
Subject Group HA HB Normal Boys Adult Control PHA Proliferation (% unstained cells) M± 1SD 79.3 ±3.7 78.2±13.3 95.4±1.5 89.3±5.3 INF-γ Secretion (pg/mL) M± 1 SD 6.7±4.7 429.0±464 69.1±32.4 770.1±148.7
The severe HA patients did not have improvement in INF-γ secretion following therapeutic exposure to FVIII concentrates. Addition of physiologic concentrations of FVIII or FIX to cultures with or without PHA did not alter the results of proliferation or cytokine secretion regardless of the inhibitor history of the patients. (1 HA and 1 HB patient developed high responder inhibitors.) Secretion of IL-4, IL-5 and IL-10 were moderately decreased in some HA patients as well. In summary, we have confirmed an INF-γ secretion defect in PBMCs from patients with HA in spite of normal proliferation in response to PHA. The defect is not present in HB or normal age-matched control subjects and is not corrected by addition of FVIII to cultures. We conclude that the defect is not due to exposure to intravenous foreign antigens. The precise clinical significance and any possible relation to the higher risk of inhibitor development in HA will require further investigation.
PBMC Proliferation and Secretion of IFN-γ in Response to PHA
Subject Group HA HB Normal Boys Adult Control PHA Proliferation (% unstained cells) M± 1SD 79.3 ±3.7 78.2±13.3 95.4±1.5 89.3±5.3 INF-γ Secretion (pg/mL) M± 1 SD 6.7±4.7 429.0±464 69.1±32.4 770.1±148.7