bluuebunny/crossref_metadata_2025_split huggingface dataset:
Variants of Recombinant Factor IX with Enhanced Functional Properties.
Authors: Hartmann Rudolf , Dockal Michael , Kammlander Willibald , Panholzer Erwin , Scheiflinger Friedrich 
Published: October 2019

Abstract
Activated human factor IX (FIXa) exhibits very low catalytic activity towards its physiologic substrate factor X (FX), unless the cofactors, activated factor VIII (FVIIIa), procoagulant phospholipid membranes and Ca2+ are present. We were interested in the elucidation of the structural basis for the FVIIIa dependency of FIXa activity. The surface loop 99 of FIXa is important for regulation of FIXa activity. In FIXa not bound to FVIIIa, this loop is stabilized in an inactive conformation and limits access of substrate to the catalytic cleft. Aiming to improve the catalytic activity in the absence of FVIIIa, amino acid exchanges supposedly influencing the 99-loop were introduced into FIX’s catalytic domain (Hopfner KP et al. EMBO J1997;16:6626–35; Sichler K et al. JBC2003;278:4121–26). One of the 3 most promising mutants generated was FIX-Y94F/K98T (FIX-C), with 2 residues exchanged in the 99-loop. Both residues are known to restrict access of FX to the S2 and S4 substrate binding pockets. In FIX-Y94F/K98T/Y177F/I213V/E219G (FIX-L) two additional sites were targeted. By hydrogen bond formation with Asn97 and Asn100, Y177 locks the 99-loop in an inactive conformation, which is released by binding of FVIIIa to FIXa. I213 and E219 at the S1 site are conserved in FIX whereas V213 and G219 are conserved among most other trypsin-like proteases. In FIX-Y94F/A95aK/K98T/Y177F/I213V/E219G (FIX-M) the mutation A95aK is thought to support the interaction between loop 99 and 60, so that conformations similar to equivalent loops of FXa can be adopted. All full-length mutants and wild type FIX were expressed in HEK293 cells, purified and preactivated FIX was removed by chromatographic steps. The resulting proteins were tested for their amidolytic activity and further assessed in FX activation assays in the absence and the presence of FVIIIa. Activated partial thromboplastin time (aPTT) assays were carried out, using plasmas depleted of FVIII or FIX, and FVIII-inhibitor patient plasma. The rationally designed proteins showed not only improved catalytic properties but also some FVIIIa independent activity (table 1). In an aPTT assay, FIX-M activity was equivalent to 1.6% FVIII in FVIII-depleted plasma and to 162 mU/ml FEIBA in FVIII inhibitor patient plasma whereas the combination of mutations Y94F/K98T as exemplified in FIX-C resulted in a molecule with ∼2.5-fold higher specific activity than plasma derived FIX when assayed in FIX-depleted plasma.
Characterization of rationally designed FIX proteins. Amidolytic Activity FX activation aPTT +FVIIIa −FVIIIa Ratio FIX-DP FVIII-DP FVIII-IP kcat / KM kcat / KM +FVIIIa / −FVIIIa mM−1min−1 ×10−3 mM−1min−1 mM−1min−1 ×10−4 ×103 mU/mg mU FVIII /5μg FIX mU FEIBA / 5μg FIX (Abbreviations: DP, depleted plasma; IP, inhibitor patient plasma) pdFIX 6 10 2 52 194 0 0 FIX-WT 9 9 3 37 216 1 15 FIX-C 22 12 4 29 498 15 114 FIX-L 15 32 33 10 180 12 115 FIX-M 7 9 11 8 208 16 162
Purified MHC-Matched Hematopoietic Stem Cell Transplantation Following Total Lymphoid Irradiation and Anti-Thymocyte Globulin (TLI+ATG) Blocks EAE Pathogenesis.
Authors: Lee Li-Fen , Burge Matthew , Ho Peggy , Sobel Raymond , Shizuru Judith 
Published: October 2019

Abstract
In prior studies, we demonstrated that transplantation of major histocompatibility complex (MHC) matched and mismatched purified hematopoietic cells (HSCs) can block diabetes pathogenesis in pre-diabetic NOD mice. Here we applied this treatment to experimental allergic encephalomyelitis (EAE), an animal model for the human autoimmune disease multiple sclerosis (MS). C57BL/6 (B6) strains congenic for the CD45 locus were immunized with MOG 35–55 to induce disease. Similar to our studies in NOD mice we observed that disease response after transplantation of MHC-mismatched hematopoietic cells in EAE affected mice was superior to those that received congenic cells. Importantly, mice transplanted with MHC-matched HSC or BM showed no significant improvement in disease symptoms when compared with congenic recipients or disease controls. This result was markedly different from the positive response observed in NOD mice transplanted with MHC-matched HSC. We then sought to optimize translation of this approach by giving total lymphoid irradiation (TLI) plus anti-thymocyte globulin (ATG), a non-myeloablative regimen and permits allogeneic hematopoietic cell engraftment with significant protective effect against GVHD. We found that TLI/ATG treatment in wild type C57BL/6 mice permit engraftment of purified HSC isolated from MHC-matched AKR/B donors and improve disease severity significantly. The level of stable mixed chimerism correlated with the clinical score. While the prior studies demonstrated that a class of regulatory cells that express both nature killer cell and T cell markers (NK-T) are present at high levels in TLI/ATG treated mice, we determined that regulatory CD4+CD25+Foxp3+ cells are also present at a disproportionately high levels as compared to wild type animals. To study the role of regulatory T cell in disease protection, a flexible lentiviral vector platform supporting constitutive expression of a Tet-transactivating component with a selection marker and coordinated conditional expression of miR-shRNA targeting Foxp3 gene products in stem cells is being developed. The detailed mechanism will be discussed. We hope a MHC-matched hematopoietic graft in conjunction with TLI/ATG conditioning shown here to successfully ameliorate EAE can be translated to the treatment of human autoimmune disease.
Effects of a Novel, Selective Jak2 Inhibitor, AZ60, on STAT5 Signaling and Cellular Growth in Jak2 V617F Cell Lines.
Authors: Gozgit Joseph M. , Bebernitz Geraldine A. , Patil Pankaj , Ye Minwei , Wu Jiaquan , Ioannidis Stephanos , Davies Audrey , Wang Tao , Huszar Dennis , Zinda Michael 
Published: October 2019

Abstract
A role for Jak2 in the etiology of the myeloproliferative diseases (MPDs) was discovered with the identification of a single activating point mutation, V617F, in the pseudokinase domain of JAK2. We have developed a Jak2 inhibitor, AZ60, which inhibits in vitro JAK2 enzyme activity with a Ki of 0.45 nM. AZ60 demonstrates inhibition of STAT5 phosphorylation and proliferation in a Tel-Jak2 engineered cell line with IC50 values of 18 and 23 nM, respectively. To understand the selectivity versus other Jak kinase family members we engineered three additional cell lines containing Tel fusions with the kinase domains of Jak1, Jak3 and Tyk2. Under these settings, AZ60 demonstrates a 15 to 30-fold selectivity for Tel-Jak2 driven STAT5 phosphorylation when compared to other Jak kinase family members. AZ60 was also tested for its ability to inhibit STAT5 phosphorylation and cellular proliferation in two human hematological cell lines, Set-2 and Hel. Set-2 expresses both wt and V617F Jak2, while Hel is homozygous for the Jak2 V617F mutation. AZ60 decreased phospho-STAT5 levels in a dose-dependent manner in both Set-2 and Hel cells with IC50 values of 15 and 25 nM, respectively. Complete inhibition of proliferation and a marked induction of apoptosis were observed in both cell lines following treatment with AZ60. Induction of apoptosis by AZ60 was characterized by a time- and dose-dependent increase in caspase 3/7 activities and PARP-cleavage. These data demonstrate AZ60 is a potent and selective inhibitor of Jak2 and may help decipher the mechanisms underlying Jak2-driven myeloproliferative disease.
Clinical, Prognostic and Possible Therapeutic Relevance of Angiogenesis in Non-Hodgkin’s Lymphoma.
Authors: Etto Leina Y. , Lacerda Emilio C.A. , Baiocchi Otavio C.G. , Silva Vanderleia C. , Dalboni Maria Aparecida , Inaoka Riguel J. , Corbi Fernanda C. , Alves Antonio C. , Vettore Andre L. , Colleoni Gisele W.B. 
Published: October 2019

Abstract
Considering that angiogenesis may have importance in the pathogenesis of non-Hodgkin’s lymphomas (NHL), the aims of this study were to correlate hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (FGF) serum levels and microvessel density (MVD) with cell origin, biological behavior, tumor load and prognosis in NHL.
Materials and methods: Eighty-seven consecutive previously untreated NHL patients had serum samples collected and were included in the study. Thirty-seven (42%) of them also had additional serum follow-up samples. Control group was composed by ten healthy blood donors. HGF, VEGF and FGF serum levels were determined by ELISA. MVD was measured by CD34 staining in patients with available paraffin blocks.
Results: HGF mean serum level was significantly higher in both early and advanced NHL Ann Arbor stages when compared to control group. HGF was also significantly higher in both aggressive and indolent NHL when compared to control group. Furthermore, mean serum level of HGF in aggressive NHL was significantly higher than in indolent NHL. Regarding International Prognostic Index (IPI), mean serum levels of HGF at diagnosis was significantly higher for patients with IPI > 2 when compared to IPI <=2. FGF mean serum level was also significantly higher in early and advanced NHL stages when compared to controls. We also observed that FGF mean serum level was significantly higher in indolent NHL when compared to control group. Regarding VEGF, serum levels were significantly higher in aggressive NHL when compared to control group. Sequential analyses of HGF, VEGF and FGF serum levels in NHL showed that serum HGF and VEGF levels decreased significantly after 6 months of treatment completion. When we analyzed MVD, no associations between patient’s groups were found, even when stage, biologic behavior or cell origin were taken into account. We did not find correlation among MVD and serum levels of HGF, VEGF and FGF at diagnosis either.
Conclusions: Our findings suggest that HGF seems to play an important role in NHL pathogenesis because:
its serum level is associated to tumor load and aggressiveness; it can be correlated to treatment response.
In face of these results, we could suggest that HGF is a potential target for therapeutic intervention in NHL.
The Effect of Hemoglobin (Hb) Level at ESA Initiation on Outcomes in Cancer Patients (pts) with Chemotherapy-Induced Anemia (CIA): A Patient-Based Meta-Analysis from 6 Randomized, Placebo-Controlled Trials (RCTs) of Darbepoetin alfa (DA).
Authors: Crawford J. , Henry D. , Vansteenkiste J. , Ludwig H. , Lillie T. , Fleishman A. , Glaspy J. 
Published: October 2019

Abstract
Introduction: ESAs are associated with an increased and quantifiable risk of venous thromboembolism (VTE); however, the treatment alternative, RBC transfusion, has only a transient effect and the associated risks are often more numerous and less predictable. Repeated transfusions also diminish patients’ personal independence. Transfusion risks include mistransfusions, hemolytic reactions, exposure to infectious agents, immune-mediated reactions, and iron and circulatory overload. Previous studies have suggested that in order to maximally reduce transfusion requirements, ESAs should be initiated when anemia is mild. We conducted a retrospective analysis to assess the effect of hemoglobin (Hb) level at ESA initiation on the risk of transfusions and safety outcomes.
Methods: A combined analysis of patient-level data from randomized, blinded, placebo-controlled studies in which the ESA darbepoetin alfa (DA) was administered subcutaneously in patients with nonmyeloid tumors and CIA (hemoglobin ≤11 g/dL at study screening) was performed. Patients had received at least 1 prior cycle of chemotherapy and had further chemotherapy planned with or without radiotherapy. Endpoints were analyzed by baseline Hb level and included transfusions, embolic/thrombotic events, death, and death or disease progression. Hazards ratios (HR, with 95% confidence interval limits) were used to describe the risk of these endpoints in the DA group relative to placebo.
Results: Patients (n=2112) were mostly white, with a mean age of 62 years. Nearly half were ≥65 years. Baseline characteristics were similar, with the most frequent tumor types in both treatment groups being lung and hematologic cancers, and most with stage III or higher disease. For both groups, patients with lower baseline Hb levels had a higher probability of transfusions; yet the reduced risk of transfusions in the DA group relative to placebo was apparent at each baseline Hb level. The known increased risk of VTEs in the DA group did not appear to be associated with baseline Hb level. There was no increased risk of death or progression at any baseline Hb level.
Conclusions: Results from this patient-level meta-analysis in CIA patients suggest that baseline Hb is a predictor of transfusion risk, and that earlier ESA intervention lowers the risk of future transfusions. Initiating DA at Hb ≥ 9 to < 10 g/dL resulted in a rate of transfusion of 32%; this rate was substantially lowered when DA was initiated at Hb ≥ 10 to < 11 g/dL. There did not appear to be an increased risk of death or VTEs as a result of initiating DA therapy at the higher Hb levels. Therefore, delaying the start of therapy may only increase the transfusion requirements, resulting in increased risk to the patient, and additional strain on the national blood supply.
Connexin-43 Mediates Fcγ Receptor-Induced Calcium Signaling by Acting as an NAD/Cyclic ADP-Ribose Transporter.
Authors: Song Eun-Kyung , Lee Young-Rae , Yu Hong-Nu , Rah So-Young , Kim Uh-Hyun , Lee Seun-Jin , Kim Jong-Suk , Han Myung-Kwan 
Published: October 2019

Abstract
Cyclic ADP-ribose (cADPR) is produced from NAD by CD38, a leukocyte receptor and ectoenzyme with its catalytic domain positioned outside of cells, and mobilizes Ca2+ from ryanodine sensitive Ca2+ stores. It has long been questioned how NAD approach CD38 and extracellularly generated cADPR reaches its intracellular target Ca2+ stores. Here we address this question by identifying connexin 43 hemichannels (Cx43) as a transporter of cyclic ADP-ribose as well as NAD in Fcg receptor (FcgR) triggering-induced calcium signaling. FcgR clustering triggers a Ca2+ transient via three sequential steps, Cx43-mediated NAD export, CD38-mediated cADPR production and subsequent Cx43-mediated cADPR import in murine macrophages. FcgR clustering induces Cx43 phosphorylation, thereby opening Cx43 and transporting NAD and cADPR. After cADPR-mediated Ca2+ transient, Cx43 is immediately dephosphorylated and closed, blocking further cADPR transport. This finding is the first evidence showing the involvement of Cx43 in cADPR-mediated Ca2+ release induced by physiological ligand where the apparent topological paradox of extracellular cADPR synthesis and its intracellular action exist. Our results provide new insight into the mechanism of intracellular calcium release triggered by the extracellularly generated calcium mobilizer cADPR, which is involved in (FcgR)-mediated calcium signaling in murine macrophages.
Lymphocyte Depletion in Breast Cancer Patients Receiving Adjuvant Dose-Dense Chemotherapy.
Authors: Angitapalli Revathi , Mashtare Terry L. , Janarthanan Bagirathan 
Published: October 2019

Abstract
Background: Dose-intense chemotherapy regimens are reportedly known to cause lymphocyte depletion. Increased incidence of pneumocystis carinii pneumonia (PCP) was reported in patients with diffuse large B-cell lymphoma treated with dose-dense CHOP plus rituximab. No studies have been conducted to date to assess the lymphocyte depletion in breast cancer patients receiving dose-dense chemotherapy. We retrospectively compared the lymphocyte recovery in breast cancer patients treated with dose-dense (q 2 weekly) doxorubicin/cyclophosphamide (AC) to those treated with AC in a non-dose-dense (q 3 weekly) method.
Methods: Newly diagnosed breast cancer patients treated at our institution between 2002 and 2006, with at least 4 cycles of AC in an adjuvant/neoadjuvant setting were analyzed. Complete blood count (CBC) with differential including white blood cells (WBC), hemoglobin (Hg), platelets, neutrophils, lymphocytes, monocytes, eosinophils and basophils were collected at baseline before the start of chemotherapy, before each ensuing cycle of chemotherapy and after the 4th cycle of chemotherapy.
Results: A total of 103 patients were analyzed of which 50 patients were treated with dose-dense AC and 53 patients with non-dose-dense AC. After the 4th cycle of chemotherapy, mean absolute lymphocyte count (ALC) was 780 ± 440/mm3 in the dose-dense group versus 950 ± 600/mm3 in the non-dose-dense group (p = .4617), mean Hg was 11.4 ± 1.2g/dL in the dose-dense group versus 12.2 ± 0.9g/dL in the non-dose-dense group, mean platelet count was 241 ± 85 x109/L in the dose-dense group versus 391 ± 128 x109/L in the non-dose-dense group. No opportunistic infections were noted in the dose-dense group.
Conclusions: These results demonstrate that dose-dense AC results in slightly increased lymphocyte depletion compared to non-dose-dense AC, not statistically significant. This didn’t result in any increased infectious complications. In both groups, baseline ALC was significantly positively related to ALC after the 4th cycle of chemotherapy (p < 0.0001). Dose-dense AC did result in statistically significant platelet depletion.
Regulation of αIIbβ3-Signaling by Myomesin Family of Proteins.
Authors: Reddy Kumar B. 
Published: October 2019

Abstract
Integrins are the type-I transmembrane proteins. They function as heterodimers of α and β subunits. The platelet integrin αIIbβ3 (GPIIb-IIIa) served as the model system in understanding the signaling across integrin receptors. αIIbβ3 exists in a resting state on circulating platelets and can be activated via several inside-out signaling pathways resulting in binding to its ligands, such as fibrinogen (Fg). The inside-out signaling appears to be regulated by several cytoskeletal and signaling proteins, which directly associate with the cytoplasmic tails of αIIbβ3. We have previously identified skelemin/myomesin-I as the cytoskeletal protein that interacted with the cytoplasmic tail of β3-integrins. Skelemin/myomesin-I is very closely related to two other members: myomesins-II and III. Myomesins I-III, along with other proteins such as titin and myosin light chain kinases, are members of a much larger superfamily of proteins. The hallmark of this family of proteins is the presence of a variable number of fibronectin (Fn)-like and immunoglobulin (Ig)-like motifs. Myomesin-I contains five Fn-motifs flanked by a total of seven Ig-like motifs. The interaction between β3-integrin and myomesin-I occurs via the C-terminal Ig motifs 3–7 of myomsin-I. In order to understand the functional consequence of this interaction between myomesin-I and β3-integrin, we measured the ligand binding properties of αIIbβ3 in a CHO cell model system. We found that expression of Ig motifs 3–7 increased Fg binding to the αIIbβ3, comparable to talin-head domain, used as the positive control. However, the expression of smaller and individual Ig motifs 4 and 5 resulted in still significantly higher Fg-binding to αIIbβ3. Ig motifs from myomesin-II also behaved in a similar fashion. We also found that myomesin-II is ubiquitously expressed in all the cell types tested, including platelets, suggesting myomesin-II is the likely candidate among various myomesin family members for the regulation of signaling across αIIbβ3. Our results suggest that the Ig motifs of the myomesin represent novel domains, which can regulate inside-out signaling across αIIbβ3.
Evaluation of Inherited Prothrombotic Risk Factors in Children with Acute Lymphoblastic Leukemia.
Authors: Te Valerie Li Thiao , Favier Remi , Borg Jeanne-Yvonne , Cadet Estelle , Reynaud Jacqueline , Micheli Julie , Rachieru Petronela , Berger Claire , Auvrignon Anne , Schneider Pascale , Leverger Guy , Muchemble Brigitte Pautard 
Published: October 2019

Abstract
This retrospective study was designed to determine the prevalence of inherited prothrombotic risk factors (Factor V Leiden (FV) G1691A and prothrombin G20210A mutations, TT677 genotype of the methylenetetrahydrofolate reductase (MTHFR), protein C, protein S, antithrombin deficiencies) in a population of children with ALL treated according to the FRALLE 2000 study Protocol (High Risk and Standard Risk groups). The study was performed in 5 French Centers including Amiens, Angers, Paris Trousseau, Rouen and Saint-Etienne. From December 2000 to March 2006, 354 children aged 1 to 18 years old were consecutively admitted for ALL and were enrolled in the FRALLE 2000 Protocol. Among them, 281 patients were investigated for hereditary prothrombotic defects at the time of ALL diagnosis. Informed parental consent was required for gene analysis. Abnormal test results for protein S (functional activity and free protein S antigen concentration), protein C and antithrombin were controlled on a second blood sample after induction. In the population studied, the prevalence of one established prothrombotic risk factor was 19,2%: the FV G1691A mutation was diagnosed in 10 patients (3.6%), all heterozygous, 10 patients (3.6%) showed the heterozygous prothrombin G20210A mutation, the TT677 MTHFR genotype was found in 34 children (12.7%), 1 patient showed protein C deficiency (0.4%). No antithrombin deficiency was detected. The prevalence of inherited protein S deficiency could not be evaluated because of missing data in the family medical history. Combined prothrombotic defects were found in 2 patients (0.71%): heterozygous FV G1691A mutation combined with heterozygous prothrombin G20210A mutation in 1 patient and combined with TT677 MTHFR genotype in the second patient. Except for TT677 MTHFR genotype, the prevalence of hereditary prothrombotic risk factors in children with ALL in France were found within the prevalence reported for children treated for ALL (table 1) and comparable to the prevalence in healthy Europeans (Junker et al. 1999, Margaglione et al 2001, Mueller et al. 2005).
Comparison of the prevalence of inherited prothrombotic risk factors in children with ALL Country Population FV G1691A +/− ++ PT G20210A +/− +/+ MTHFR TT677 AT PC AT: antithrombin deficiency ; PC: protein C deficiency ; NE : non evaluated NowakGöttl et al 1999 (n=301) Germany ALL children 5.3% 0.3% 2% 0% 7.7% 0.7% 2.3% Mauz-Körholz et al. 2000 (n=108) Germany ALL children 5.6% 0% 2.8% 0% 5.6% 0% 2.7% Mitchell et al. 2002 (n=60) Canada ALL children 3.3% 0% 2% 0% NE NE NE Present study (n=281) France ALL children 3.6% 0% (n=277) 3.6% 0%(n=279) 2.7% (n=268) 0% 0.4%