bluuebunny/crossref_metadata_2025 huggingface dataset:
Surface Sialic Acid Prevents Loss of GPIbα during Platelet Storage and Rescues In Vivo Survival of Murine Platelets.
Authors: Jansen Gerard , Josefsson Emma C. , Hartwig John H. , Hoffmeister Karin M.
Published: October 2019

Platelet processing and storage are associated with platelet lesion, e.g. shape change, activation, release reaction and apoptosis, which is partially due to loss of surface receptors. Surface sialic acid is considered to be a key determinant for the survival of circulating blood cells and glycoproteins. However, its role in platelet receptor loss and platelet survival is unclear. In this study, the relationship between surface sialic acid and platelet receptor loss was investigated in vitro and in vivo. Murine platelets stored at room temperature for 6 hours lost surface sialic acid, as evidenced by flow cytometry using FITC conjugated RCA I lectin, which recognizes exposed galactose residues. This loss correlated with a 30–60% loss of surface receptors GPIbα and GPV, but not GPIX and integrin αIIbβ3, as measured by flow cytometry. Treatment of murine platelets with the neuraminidase (NA) substrate fetuin partially decreases the loss of GPIbα and GPV to 10–20%. In vitro, sialic acid was cleaved from the platelet surface by adding NA (α2-3,6,8-NA (V. cholerae) or α2-3,6,-NA (C. perfringens)) to murine platelets. Removal of sialic acid correlated with the removal of 50–60% of surface GPIbα and GPV, but not GPIX and integrin αIIbβ3. Addition of fetuin, or the more specific NA inhibitor 2,3-dehydro-2-deoxy-, sodium salt (DANA), completely prevented this loss, as determined by both flow cytometry and Western blot analysis. Murine platelets treated with α2-3,6,8-NA (V. cholerae) ± the addition of DANA were labeled with the green dye CMFDA and transfused into age-, strain- and sex-matched C57BL/6 mice to measure platelet survival. NA-treated platelets were cleared within minutes after transfusion, whereas the addition of DANA rescued platelet survival to control-count increments. Our study shows that inhibiting the loss of surface sialic acid prevents platelet surface GPIbα and GPV loss during storage in vitro and rescues platelet survival in vivo.
Combined Inhibition of CDK and PI3K/Akt Pathways Induces Apoptosis of Mantle Cell Lymphoma.
Authors: Mohapatra Subhra , Chu Baoky , Zhao Xiuhua , Tao Jianguo , Sotomayor Eduardo , Pledger W. Jack
Published: October 2019

Mantle cell lymphoma (MCL) arises from the neoplastic transformation of naïve B cells in the mantle zone of the B cell follicle. It is an aggressive and incurable B cell neoplasm. It accounts for 5% to 8% of non-Hodgkin’s lymphomas. Patients with MCL respond initially to chemotherapy but ultimately relapse. Mean survival time is only three to four years. Thus, the need for new treatments that effectively combat MCL is obvious and imperative. Defects in cell cycle regulation and apoptosis are primary events in MCL. It is characterized by the presence of a chromosomal translocation t (11:14)(q13:q32), which results in deregulated cyclin D1 expression. Cyclin D1 overexpression in MCL is thought to play a major role in lymphomagenesis, although the precise mechanisms by which tumor formation and progression occur are not fully understood. Constitutive activation of the PI3K/Akt pathway contributes to the pathogenesis and survival of MCL. Activated AKT was found in cultured MCLs as well as in 100% of aggressive-blastoid variants of MCL tumors, compared to 30% of typical MCLs. Towards the search for novel therapies for MCLs, we examined the potential of roscovitine (rosc) or flavopiridol (flav), inhibitors of cyclin dependent kinase (CDK), as a single agent or in combination with LY294002 (LY), a PI3K/Akt inhibitor, in inducing apoptosis of various MCL lines as well as in MCL patient samples. CDK inhibitors modestly increased the percentage of apoptotic cells (∼30%), whereas LY had no effect. However, when added in combination, rosc/LY or flav/LY induced apoptosis in more than 70% of cells. In an effort to understand the mechanism of apoptosis, we identified three targets: cyclin D1, the anti-apoptotic proteins myeloid cell leukemia-1 (Mcl-1) and X-linked Inhibitor of Apoptosis (XIAP). CDK inhibitors eliminated Mcl-1 expression, slightly reduced XIAP abundance and had very little effect on abundance of cyclin D1. Conversely, LY reduced cyclin D1 expression and slightly reduced the abundance of Mcl-1 and XIAP. A larger decrease in XIAP abundance is seen in cultures treated with a combination of rosc/LY or flav/LY. On the basis of these findings, we suggest that agents that target Mcl-1, XIAP and cyclin D1 will be most effective in inducing apoptosis of human MCLs.
Efficacy of FDG-PET/CT Imaging To Guide Biopsies in the Detection of Richter’s Syndrome.
Authors: Le Gouill Steven , Bodet-Milin Caroline , Kraeber-Bodere Françoise , Moreau Anne , Campion Laurent , Dupas Benoit , Gastinne Thomas , Moreau Philippe , Harousseau Jean-Luc
Published: October 2019

Introduction: Most lymphomas show a good FDG uptake, with significant difference in intensity according to different histopathological subtypes. Richter’s syndrome  (RS) is defined as the development of aggressive lymphoma in patients with low grade histopathological subtypes. Diagnosis requires histological findings. The aim of our study was to investigate prospectively FDG-PET/CT to guide biopsy in the diagnosis of aggressive transformation of low grade lymphomas.

Material and methods: Thirty-eight patients, presenting clinical and/or biological signs of aggressive transformation were included. The 38 underwent FDG-PET/CT. FDG-PET/CT scans were analysed visually and semi-quantitatively with maximal standardised uptake value (SUVmax) normalised for body mass. Guided-biopsy was performed 4 weeks after FDG-PET/CT, in the abnormal focus showing the highest SUVmax and histopathological analysis was performed.

Results: Histopathological analysis concluded to aggressive transformations in 17 cases (15 NHL and 2 Hodgkin diseases included one composite lymphoma) and low grade lymphomas in 21 cases. Size of lesions on CT was not predictive of RS (p=0.094). FDG-PET/CT images showed abnormal FDG uptake in the 38 included patients. SUVmax ranged from 11.7 to 41.2 in patients with Richter’s syndrome and from 1.7 to 17.0 in patients with no sign of transformation(p<0.0001). Gradient of SUVmax was significantly higher in patients with Richter’s syndrome (p<0.0001). A SUVmax threshold of 14.0 gave the better balance with sensitivity, specificity, predictive positive and negative values around 95.0%. This threshold of 14.0 correctly classified as transformed or not transformed 36/38 patients (94.7%).

Conclusion: FDG-PET/CT imaging is a very relevant tools to detect aggressive transformation of low grade lymphomas and to guide biopsy.
Human Hematopoietic and Mesenchymal Stem Cells Are Interconnected by Cadherin-Catenin Based Junctions.
Authors: Wuchter Patrick , Saffrich Rainer , Schubert Mario , Wagner Wolfgang , Lannert Heinrich , Eckstein Volker , Ho Anthony D.
Published: October 2019

The direct cell-cell contact between human hematopoietic stem cells (HSC) and their niche has been shown to control stem cell fate by regulating self-renewal versus differentiation. In previous studies we have demonstrated gap and adherens junctions arranged in complex villiform, vermiform, intercalating protrusions and invaginations among mesenchymal stem cells (MSC). Specific cadherin-catenin-molecules, among other junction proteins, were found in these contact systems. Using MSC-feeder-layer as an in vitro surrogate model for the hematopoietic stem cell niche, we have analyzed the intercellular junctional complexes between HSC and MSC in this study. Additionally, we characterized cell-cell-connections between leukemia stem cells (LSC) and MSC. MSC were derived from bone marrow aspirates from healthy voluntary donors. HSC were isolated from umbilical cord blood. Leukemia stem cells were obtained from bone marrow aspirates from patients suffering from acute myeloid leukemia either at the time of initial diagnosis or at relapse after chemotherapy. After 24–48 hours of co-cultivation, we have stained the cell-cell contacts with a panel of antibodies specific for various components of tight, gap and adherens junctions including alpha-/beta-catenin, N-cadherin, Cadherin 11, E-Cadherin, p120, Vinculin and Connexin 43. Using advanced confocal laser scanning microscopy in combination with deconvolution and volume rendering software, we were able to produce 3D-images of intercellular junctions between HSC/MSC as well as between LSC/MSC. We have confirmed previous observations that HSC demonstrated directed locomotion towards a MSC gradient. HSC then migrated around the MSC and some of them are then interconnected by podia formation to the MSC. These podia are directly linked to the MSC feeder layer and at the intimate contact zone we have identified N-cadherin as well as alpha-/beta-catenin as the main junction proteins. The specific function of this cadherin/catenin-complex is probably associated with redistribution of beta-catenin and hence to self-renewing division, similar to the findings in stem cells of drosophila model systems. Functional analyses are currently underway to confirm this hypothesis. We have also compared these findings with a similar setting consisting of human LSC co-cultured with MSC-feeder-layer.
Human Placental Derived Mesenchymal Stromal Cells (MSC) Grown in 3D-Culture (PLX-I), Promotes Engraftment of Human Umbilical Cord Blood (hUCB) Derived CD34+ Cells in NOD/SCID Mice.
Authors: Burger Ora , Ashtamker Galit F. , Bercovich Noam , Rusanovsky Marina , Pinzur Lena , Jacov Osnat M. , Carmi Nirit D. , Meiron Moran , Lior Limor , Meretzki Shai , Prather William R. , Kadouri Avinoam , Treves Avi J. , Rowe Jacob M. , Horwitz Edwin M. , Nagler Arnon
Published: October 2019

The use of UCB for transplantation in adults is limited due to the low cell number in each unit. Engraftment rates are lower and the time for neutrophil and platelet recovery is longer following CBT compared to bone marrow (BM) transplants. Co-transplantation of human hematopoietic stem/progenitor cells (HSC) with mesenchymal stromal cells in an irradiated NOD/SCID mice model has been demonstrated to promote HSC engraftment. Using PluriX technology, we expanded human placental derived MSC on 3D-carriers in a bioreactor (PLX-I) and evaluated its potential to promote UCB engraftment in the NOD/SCID mice model. Flow cytometer analysis detected high rates of expression of CD105, CD73, CD90 and CD29 and lacked expression of CD45, CD34 and CD19 surface markers in the PLX-I cells. PLX-I cells were capable of differentiation into bone, adipose tissue and cartilage under specific inductive conditions. Additionally, they possess both immune privileged and immune suppressive characteristics in a mixed lymphocyte reaction (MLR) assay. In this study 60 – 100x103 hUCB derived CD34+ cells were injected into the tail vein of 7–8 week old NOD/SCID mice along with 0.5x106 PLX-I cells. Human cell engraftment was tested in both sublethally irradiated (350 rad) and chemotherapy (50 mg/kg busulfan) treated NOD/SCID mice. Following 5–6 weeks, mice BM FACS analysis showed a significant increase in % hCD45+ rate in mice transplanted with PLX-I cells compared to mice transplanted with CD34+ cells alone: 13.6 vs. 31.7, p=0.01, (n=6) in the irradiation setting and 28.8 vs 6.3, p<0.05 (n=7) in the chemotherapy setting. These preclinical results demonstrate the potential of human placental-derived MSCs, grown as a 3D culture (PLX-I), to promote hUCB CD34+ cell engraftment in BM. Co-transplantation of PLX-I may be considered for improving the delayed engraftment using CB as the source of HSC.
Modulation of Fox-Regulated Alternative Splicing Events during Erythropoiesis.
Authors: Gallagher Thomas L. , Gee Sherry L. , Schluepen Christina L. , Conboy John G.
Published: October 2019

A switch in the alternative splicing pattern in protein 4.1R pre-mRNA, involving activation of exon 16 splicing, occurs in late erythropoiesis. Because the inclusion of exon 16 leads to synthesis of protein isoforms with high affinity for spectrin and actin, and mechanical strengthening of the membrane, this switch is critical for normal erythyroid differentiation. Previous studies have shown that Fox-2 protein is a splicing regulator that binds to intronic enhancer sequences downstream of exon 16, and that this binding stimulates the inclusion of exon 16. Fox binding sites are also located downstream of other tissue-specific alternative exons, some of which exhibit variable splicing efficiencies, leading us to investigate whether splicing switch strength is influenced by variations in the sequence of the Fox binding-site motif (UGCAUG) and by the number of these protein binding sites. A series of minigenes was created in which protein 4.1R E16 splicing efficiency was measured in transfection studies. Mutation of the first U residue of the Fox binding site resulted in a significantly weaker, but still Fox dependent activation of splicing, whereas mutation of the terminal G residue dramatically reduced enhancer activity. Insertion of two wild-type UGCAUG elements enhanced splicing substantially in a Fox protein concentration-dependent manner, and four elements gave even stronger inclusion. To test whether the reduced splicing efficiency observed for minigenes containing mutated enhancer elements was due to a reduction in Fox binding, surface plasmon resonance (SPR) was employed. Our binding studies indicate that full-length mouse Fox-2a bound to the UGCAUG sequence with high affinity (KD ∼100nM). Furthermore, in agreement with our functional splicing assays, Fox binding affinity for the hexameric sequence was reduced with mutations in position one, and almost eliminated with mutations in position six. There was an excellent correlation between binding affinity of Fox protein for the enhancer motif, and strength of enhancer activity measured in functional splicing assays. Additional studies demonstrated that Fox enhancer activity can be reduced by the presence of a closely linked binding site for hnRNP A1, a known splicing inhibitor. Taken together, these studies demonstrate that the composition and number of binding sites for Fox can significantly affect the efficiency of splicing for E16. It is likely that through these combinatorial mechanisms, Fox splicing factors are able to modulate the efficiency with which tissue specific exons in the erythroid alternative splicing program can be spliced into mature mRNA.
GS-9219 - A Novel Prodrug of the Acyclic Nucleotide PMEG Has Antitumor Activity Against Naive and Melphalan-Refractory Spontaneous Myeloma in Pet Dogs.
Authors: Thamm Douglas H. , Tumas Daniel B. , Reiser Hans , Wolfgang Grushenka H.I. , Kurzman Ilene D. , Plaza Susan , Anderson Christie , Robat Cecilia , Vail David M.
Published: October 2019

Multiple myeloma is an important hematopoietic cancer in humans and pet dogs. While clinical remission can be achieved using currently available antineoplastic agents, eventual drug resistant relapse is common. GS-9219, a novel double prodrug of the anti-proliferative nucleotide analog 9-(2-phosphonylmethoxyethyl) guanine (PMEG), has been shown to have potent cytotoxic activity in vitro in human lymphoblasts and leukemia cell lines and in vivo in naturally occurring non-Hodgkin’s lymphoma in dogs (naive and refractory). We hypothesized that malignant plasma cells in multiple myeloma similarly would possess the intracellular enzymatic machinery necessary for the activation of GS-9219. To generate proof-of-concept, activity and safety data in multiple myeloma, a pilot study with GS-9219 monotherapy was initiated in pet dogs with naturally occurring chemotherapy-naive or refractory multiple myeloma. Three dogs with spontaneously occurring IgA myeloma (1 naive, 2 melphalan-refractory) have been treated with GS-9219 as a 30-minute intravenous infusion weekly for 2 weeks at 1 mg/kg, then every 3 weeks for another 3 treatments at 0.8 mg/kg (total of five GS-9219 doses). To date, major anti-tumor responses have been observed in all 3 multiple myeloma dogs treated with GS-9219. Two dogs are in complete remission as indicated by normalization of serum paraprotein and complete resolution of hypercalcemia, peripheral cytopenias and bone marrow plasmacytosis. The third currently has a strong partial response (normal marrow and >95% reduction in serum paraprotein). The only significant toxicity noted throughout the study was a single episode of transient neutropenia in one dog which resolved and, after a one week delay, treatment was continued without issue. Assessment of durability of response is currently ongoing, with all dogs remaining in remission to date; one dog has remained in complete remission for more than 5 months following completion of the treatment regimen. In conclusion, GS-9219 has significant anti-tumor activity in spontaneous melphalan-refractory or treatment-naive canine multiple myeloma.
Activated Protein C Inhibits Lipopolysaccharide-Induced Anandamide and 2-Arachidonoylglycerol Generation In Vitro and In Vivo.
Authors: Maruyama Ikuro , Wang Yin , Liu Yan , Kawahara Koichi , Yamakuchi Munekazu , Hashiguchi Teruto , Obata Toru
Published: October 2019

Anandamide(ANA) and 2-arachidonoylglycerol(2-AG), two lipid mediators existing in the central nervous system and various peripheral tissues, have been shown to exert their diverse biological effects through cannabinoid receptors CB1 and CB2, and are therefore defined as endogenous cannabinoids. Recently, ANA and 2-AG have been shown to be generated by macrophages and platelets, respectively, by the stimulation with lipopolysaccharide(LPS) in vitro and in vivo in rats. We showed the endocannabinoids are increased in septic shock patients. Furthermore, the increased endocannabinoids are efficiently adsorbed onto polymyxin B immobilized column resulting the improvement of hypotensive shock. On the other hand it has been showed that activated protein C (APC) can ameliorate endotoxin induced-shock and -disseminated intravascular coagulation in experimental animals and patients, although the mechanisms governing its action are unknown. In the present study we found that APC inhibited the generation of ANA and 2-AG in vitro by lipopolysaccharide (LPS)-activated macrophages and platelets, respectively. Furthermore, APC significantly decreased the levels of ANA and 2-AG in the sera from rats preinfused with APC followed by LPS infusion. Consistent with the decrease of them, diminishment of hypotension and an increase in survival rate were confirmed in the APC pretreatment group. These findings demonstrate that APC can inhibit the generation of ANA and 2-AG in vitro and in vivo, and thereby suppress their induced hypotension. This action of APC may explain its efficiency in the treatment of septic shock in humans and experimental animals.
Unrelated Cord Blood Transplantation Using Myeloablative Regimen for Acute Leukemia Patients Aged between 50 and 55 Years: Single Institutional Retrospective Comparison with Patients Less Than 50 Years of Age.
Authors: Konuma Takaaki , Takahashi Satoshi , Ooi Jun , Tomonari Akira , Tsukada Nobuhiro , Kato Seiko , Oiwa-Monna Maki , Uchimaru Kaoru , Tojo Arinobu , Yamaguchi Takuhiro , Asano Shigetaka
Published: October 2019

Background: Increasing age has been well-known as an obvious risk factor for graft-versus-host disease (GVHD) and transplant-related mortality (TRM) resulting negative impact on allogeneic transplantation including cord blood transplant (CBT). The incidence of sever GVHD after CBT, on the other hand, is lower than that after transplants using graft from adult cells, so we have expected the better results of CBT in older patients.

Objectives and Methods: This study aimed to evaluate safety and efficacy of CBT using myeloablative regimen for older patients with acute leukemia.

Patients and Methods: We retrospectively compared outcomes of older patients with acute leukemia with younger adults in our institute. Nineteen elderly patients (median age 52, range 50–55) and 81 young patients (median 49, range 16–49) received myeloablative conditioning regimen including 12 Gy of total body irradiation. GVHD prophylaxis comprised cyclosporine with (N=96) or without (N=4) methotrexate.

Results: Comparisons of characteristics in the 2 age groups showed similar distributions for weight, gender ratio, diagnosis [de novo acute myeloid leukemia (AML), myelodysplastic syndrome related secondary AML, or acute lymphoblastic leukemia], disease status at transplantation, total nucleated cell dose and CD34+ cell dose in the graft before cryopreservation and proportions of HLA and sex compatibility between donors and recipients. The median period of follow-up for survivors after CBT was 730 days for older group and 1331 days for younger group, respectively. Grade II to IV acute GVHD occurred in 10 of 17 evaluable older patients and 49 of 75 evaluable younger patients (P = 0.61), while no older patients, but 6 younger patients developed grade III to IV acute GVHD. Extensive-type chronic GVHD occurred in 4 of 15 evaluable older patients and 18 of 69 evaluable younger patients (P = 0.96), respectively. The cumulative incidence of TRM at 100 days was 5% versus 6% (P = 0.70), and of relapse at 3 years was 29% versus 20% (P = 0.33) and the estimated disease-free survival at 3 years was 67% and 71% (P = 0.53) for older or younger patients, respectively. There was no significant difference in GVHD, TRM, relapse, and DFS between 2 age groups.

Conclusion: The use of cord blood as a stem cell source might contribute to be decreased in the incidence of acute and chronic GVHD resulting in decreased TRM in older patients. Our results suggest that myeloablative CBT might be as safe and effective in patients with acute leukemia aged between 50 and 55 years as in younger patients.
Human Antibodies with Specificity for the Spacer Domain of ADAMTS13 Are Derived from VH1-69 Germline Genes.
Authors: Pos Wouter , Luken Brenda M. , Kremer-Hovinga Johanna , Turenhout Ellen A.M. , Jefferis Roy , Dong Jing-fei , Fijnheer Rob , Voorberg Jan
Published: October 2019

Acquired thrombotic thrombocytopenic purpura (TTP) is characterized by the presence of circulating antibodies directed towards ADAMTS13. This results in the formation of large platelet aggregates that occlude the microvasculature giving rise to thrombocytopenia and micro-angiopathic hemolytic anemia in patients with TTP who lack functional ADAMTS13. Antibodies directed towards the spacer domain of ADAMTS13 are present in the majority of patients with acquired TTP. We have previously isolated a panel of human monoclonal antibodies directed towards ADAMTS13 from the immunoglobulin repertoire of one patient with acquired TTP employing phage display. To further extend these observations we obtained a panel of human anti-ADAMTS13 antibodies from a second patient with acquired TTP. Sequence analysis revealed that the variable heavy chain domains of the majority of anti-ADAMTS13 antibodies were encoded by germline gene segment VH1–69. The VH1–69 encoded antibody II-1 and the previously described antibody I-9, which was also encoded by VH1–69 were selected for further study. Epitope-mapping studies employing a hybrid ADAMTS13 variant containing the spacer domain of ADAMTS1 showed that both antibodies were directed towards the spacer domain. Both antibodies inhibited ADAMTS13 activity as measured by cleavage of FRETS-VWF73 substrate and also inhibited cleavage of VWF multimers under static conditions as well as under flow on the surface of endothelial cells. We subsequently developed an assay to monitor the presence of VH1-69 encoded antibodies in plasma of patients with acquired TTP using the anti-idiotypic monoclonal antibody G8 which specifically reacts with human antibodies that use the VH1-69 gene segment. Analysis of a large cohort of patients with acquired TTP revealed that antibodies reactive with the spacer domain of ADAMTS13 express the VH1-69 derived idiotype that is recognized by monoclonal antibody G8. These results support the notion that VH1-69 derived human antibodies directed towards the spacer domain of ADAMTS13 develop in the majority of patients with acquired TTP.
Identification of a Stroma-Mediated Wnt Signal Promoting Self-Renewal of Hematopoietic Stem Cells in the Stem Cell Niche.
Authors: Kang Young-Ju , Jho Eek-hoon , Kim Hanjun , Park Gyeongsin , Shim Jae-Seung , Oh Il-Hoan
Published: October 2019

With contrasting results recently reported on the effects of b-catenin on hematopoietic stem cells (HSCs), the precise role of Wnt on HSC regulation remains in question. Here, we show that Wnt-b-catenin signaling triggers distinct biological effects on HSCs depending on the target of activation within the hematopoietic microenvironment. Retroviral transduction of a stable form of b-catenin into HSCs caused a loss of competitive repopulating units (CRUs) in a limiting-dilution assay, whereas stabilized b-catenin in stromal cells CRU frequencies of co-cultured HSCs with higher preservation of undifferentiated state and caused enhanced levels of reconstitution in a manner dependent on direct contact between HSC and stroma. The enhancing effect of b-catenin stabilized stroma on HSC was also observed for human HSCs exhibiting higher frequencies of lympho-myeloid repopulating cells after transplantation into NOD/SCID mice. Interestingly, gene expression patterns of Wnt signaling molecules revealed compartmentalization in a manner that canonical Wnt ligands were preferentially expressed in the hematopoietic cells while molecules for reception of the signal such as Frizzled receptors or their co-receptors are preferentially expressed in stromal component, suggesting the role of stromal component as a target of Wnt signals in the niche. Furthermore, b-catenin accumulated selectively in the endosteal stroma of the trabecule region in “stressed” marrows, but not in “steady-state” marrows. Taken together, these results suggest stroma-mediated Wnt signals may function as microenvironmental cues for HSC self-renewal in the stem cell niche.
Co-Development of Intravital, Laser-Induced Experimental and Computational Models of Thrombogenesis.
Authors: Kamocka Malgorzata , Chen Nan , Xu Xhiliang , Alber Mark , Rosen Elliot D.
Published: October 2019

We have developed modified protocols for confocal intravital monitoring of thrombus development in mouse mesenteric vessels following laser-induced injury. By including tetramethyrhodamine-labeled dextran (70,000 mw) in the blood, unlabeled cells (leukocytes and reticulocytes) form black silhouettes where the red fluorescent dye is excluded in the confocal image. In addition, platelets were labeled with rat monoclonal anti-mouse CD41/61 (Emfret Analytics, Germany) mixed with secondary goat anti-rat IgG antibody labeled with Alexa Fluor 488 and human fibrinogen was labeled with Alexa Fluor 350. Thus, high resolution confocal images of the developing thrombus included platelets in green, fibrin in blue, plasma in red and cellular components in black. Near real-time video recordings indicate the importance of flow parameters on the developing clot. In particular, turbulent flow patterns around the developing thrombus contribute to the incorporation of blood cells in the thrombus leading to the development of thrombi with a heterogeneous domain structure. Conceivably, different mechano-elastic properties of the heterogeneous domains might contribute to thrombus instability as the thrombus is subject to varying stresses and strains. In parallel with the modified experimental vascular injury model, we have begun development of a computational model of thrombus development. The modeling framework consists of a stochastic and discrete Cellular Potts Model (CPM) to describe platelet and cellular interactions and continuous submodels to describe hydrodynamic and biochemical reactions. The components of the computational model include the vessel wall, platelets (in resting and activated states), blood cells, coagulation reactions, fibrin formation, and hydrodynamic parameters. By varying input variables (such as flow rate, blood viscosity, coagulant activities) and monitoring properties of the developing thrombus, one can modify and validate the computational model. While our experimental systems enable us to determine essential components required for thrombus development, a validated model could help identify threshold levels of key components involved in thrombogenesis. This may be particularly useful in identifying hemostatic risk factors if these threshold levels are within the normal range found in the human population.
Functional and Structural Characterization of Factor Xa Dimer in Solution.
Authors: Chattopadhyay Rima , Iacob Roxana , Majumder Rinku , Tomer Kenneth B. , Lentz Barry R.
Published: October 2019

Previous studies of blood coagulation factor Xa (bovine) showed that binding of water soluble phosphatidylserine (C6PS) to factor Xa (FXa) leads to Ca2+ dependent inactive (∼1000-fold inactivation) dimer formation. We show now that human factor Xa activity is also regulated by C6PS-induced dimerization in the presence of 5 mM Ca2+ We also report that the FXa dimer is inactive:

despite the fact dimerization does not block the active site; in part because it does block a substrate exosite; the dimer interface involves lysine residues that boarder the active site and exosites, and the structure of FXa in the dimer is altered relative to the monomer.

We have measured initial rates of prothrombin activation (using synthetic thrombin substrate S-2238) at varying FXa and substrate concentrations to show that the kcat/Km decreased (kcat decreased significantly and Km increased slightly) with an increase in FXa dimer formation. The observed significant decrease in kcat indicates that dimerization affects the alignment of substrate with the active site, perhaps through altering substrate binding or through altering the structure of the active site. Amidolytic activity of monomeric FXa (using synthetic substrate S-2765) decreased in response to C6PS binding, while that of the dimer increased slightly. This indicates that dimerization did not block the active site but may alter its conformation. CD and mass spectrometry showed that both Ca2+ and C6PS binding alter FXa structure and that dimerization further alters structure. Acetylation of exposed lysine residues and analysis of MS patterns obtained under conditions that favor either monomer or dimer FXa revealed that the dimerization buries lysines residues 222 and 224 (chymotrypsin numbering) that boarder the active site and are in putative exocytes. We used MS data, fluorescence energy transfer data for active site labeled FXa, to model the FXa dimer structure based on a FXa monomer model (from Gla-domainless Xa X-ray structure and Gla-EGFn with Ca2+) but the requirement that known membrane binding sites or paired FXa molecules would be located in plane was failed. Our lack of success supports our other measurements suggesting that the structure of FXa in a dimer is very different from that in a monomer.

Supported by grant from the NHBL (HL 072827 to BRL).
Time to Complete Cytogenetic Response (CCyR) Does Not Affect Long-Term Outcomes for Patients on Imatinib Therapy.
Authors: Guilhot Francois , Larson Richard A. , O’Brien Stephen G. , Gathmann Insa , Druker Brian J.
Published: October 2019

Background: Results from the International Randomized Study of Interferon and STI571 (IRIS) trial showed that achievement of a CCyR is prognostically relevant for long-term survival in patients (pts) with chronic myeloid leukemia in chronic phase (CML-CP). With the advent of next generation tyrosine kinase inhibitors (TKIs), there is a need to understand factors that may influence long term outcomes. Here we analyze whether time to achievement of a CCyR affects long term outcomes.

Methods: The relationship between time to CCyR and long-term outcomes was examined for 551 imatinib-treated patients with newly diagnosed CML-CP at the 6-year follow up of the IRIS trial. As evaluations included non-responders, landmark analyses were conducted in which only pts who were treated for ≥1 year were included (n=509). Patients were stratified according to time to achieving a CCyR as follows: ≤6 months (n=265), >6≤12 months (n=99), >12≤18 months (n=34), >18 months (n=49), or no CCyR during imatinib treatment (n=62). In each category patients were assessed for duration of cytogenetic response, event free survival (EFS; any event while on study), freedom from progression to accelerated phase (AP) or blast crisis (BC), and overall survival (OS).

Results: In the 447 patients who were treated for at least 1 year and achieved a CCyR (<1% Ph+) during therapy, the durability of major cytogenetic response (1%–35% Ph+) did not differ significantly regardless of when CCyR was achieved (P=0.76). For the overall population, estimated 6-year rates were 88% for OS, 83% for EFS, and 93% for freedom from progression to AP/BC. No statistically significant difference was observed between the responders when categorized according to time to response. However, patients who did not achieve a CCyR had significantly worse outcomes than those who achieved CCyR (P<0.001). Estimated 6-year OS rates were 94%, 95%, 91% and 98% for patients who first achieved a CCyR within 6, 12, 18 months and after 18 months, respectively (P=0.55 for overall comparison of the response categories), compared with 63% for pts without CCyR during imatinib therapy. Estimated EFS rates at 6 years were 93%, 90%, 87% and 89% (P=0.58) and 33% for patients who do not achieve a CCyR, respectively. At 6 years the estimated rates of freedom from progression to AP/BC were 97%, 97%, 97% and 98% (P=0.98), respectively, but only 63% for pts who do not achieve a CCyR. Overall Survival by Time to CCyR.

Conclusion: Long-term outcomes on imatinib for patients in CML-CP are independent of time to achieve CCyR. Therefore, achievement of a “late” CCyR does not increase the potential for progression or portend a worse overall survival for patients treated with imatinib.

Figure Figure
Factor XI Homodimer Formation Is Mediated by Electrostatic (Lys331 with Glu287) and Hydrophobic (Ile290 with Leu284 and Tyr329 with Tyr329) Interactions in the Apple 4 Domain and Is Essential for Normal Proteolytic Activation of Factor XI by Factor XIIa, Thrombin and Factor XIa.
Authors: Wu Wenman , Sinha Dipali , Lear James D. , Billings Paul C. , Walsh Peter N.
Published: October 2019

Factor XI (FXI), a coagulation protein essential for normal hemostasis, is a homodimer consisting of two identical subunits of 80 KDa linked by a disulfide bond formed by Cys321 within the Apple 4 (A4) domain of each subunit. Prekallikrein (PK), in spite of its high homology with FXI both in amino acid sequence and domain structure, is a monomer. Cys321 in PK forms an intrachain disulfide bond with Cys326. However FXI/C321S (in which interchain disulfide bond formation is precluded) is a noncovalent dimer. Thus, there are interacting residues between the two subunits of FXI that are responsible for mediating its unique homodimeric structure. Examination of the crystal structure of FXI (Papagrigoriou E, McEwan P, Walsh PN, Emsley J. Nature Structural & Molecular Biology. 2006;13:557–8) shows salt bridges between Lys331 of one subunit with Glu287 of the other subunit as well as hydrophobic interactions at the interface of the A4 domains involving Ile290, Leu284 and Tyr329. FXI/C321S, FXI/C321S/K331A, FXI/C321S/E287A, FXI/C321S/I290A, FXI/C321S/Y329A, FXI/C321S/L284A and FXI/C321S/K331R were expressed in HEK293 cells and characterized using size exclusion chromatography (SEC), analytical ultracentrifugation (AUC), and functional assays. Whereas FXI/C321S existed in a monomer/dimer equilibrium (Kd ∼40 nM) all other mutants were predominantly monomers by SEC with impaired dimer formation by AUC (Kd 3.4–38 μM). All the monomeric mutants when converted to the active enzyme, FXIa, were able to hydrolyze the small chromogenic substrate S-2366 with normal values of Km and Vmax and cleaved the macromolecular substrate FIX at both its scissile bonds at rates similar to those observed with wtFXIa strongly suggesting that all mutant proteins were properly folded. However all the monomeric mutants displayed impaired clotting activity in an APTT assay and displayed markedly decreased rates of activation by FXIIa or thrombin and autoactivation in the presence or absence of dextran sulfate. We conclude that salt bridges formed between Lys331 of one subunit and Glu287 of the other together with hydrophobic interactions of residues Ile290 with Leu284 and Tyr329 with Tyr329 and are essential for normal homodimer formation, which is essential for normal proteolytic activation of FXI by FXIIa, thrombin and FXIa either in solution or on an anionic surface.
Identification of ADAMTS13 Epitopes Required for Binding to von Willebrand Factor Using Lambda Phage Surface Display.
Authors: Moriki Takanori , Maruyama Ichiro N. , Yamaguchi Yusuke , Igari Atsuko , Ikeda Yasuo , Murata Mitsuru
Published: October 2019

The metalloprotease ADAMTS13 cleaves multimeric von Willebrand factor (VWF) to regulate VWF-mediated thrombus formation. We planned to search core epitopes of ADAMTS13 that is required for its binding to VWF. We constructed a random cDNA fragment library expressing various peptides of ADAMTS13 on the surface of lambda phage and screened the library using immobilized VWF as a probe. After the first screening, the C-terminus of the spacer domain from Arg670 to Glu684 (termed as epitope-1) and the middle of the cysteine-rich domain from Arg484 to Arg507 (epitope-2) were determined as epitopes. When we added the synthetic epitope-1 peptide to the second screening, a new site, from Pro618 to Glu641 (epitope-3), was found in the middle of spacer domain. While the presence of synthetic epitope-2 peptide did not affect the subsequent screening, the presence of epitope-3 peptide enhanced the isolation of clones encoding epitope-1. These results suggest that ADAMTS13 epitopes-1, -2 and -3 may interact with each other for their binding to VWF. From screening in the presence of any combination or all of the three synthetic peptides, however, no new VWF binding site was uncovered. To examine the effect of divalent metal cations on the binding of ADAMTS13 epitopes to immobilized VWF, screening was carried out in the presence or absence of 5 mM of EDTA. No new epitope site was found. We next explored inhibitory effect of the synthetic epitope peptides on ADAMTS13 protease activity using recombinant ADAMTS13 and FRETS-VWF73 as a substrate. Synthetic epitopes-2 and -3 peptides markedly inhibited the cleavage of VWF by ADAMTS13, while the synthetic epitope-1 peptide did not as efficiently as epitopes-2 and -3. The stronger inhibitory effect of epitope-3 peptide than that of epitope-1 peptide was confirmed by SDS-agarose gel electrophoresis analysis of cleavage products of denatured multimeric VWF molecules by recombinant ADAMTS13. This was consistent with the dissociation constants for the three synthetic peptides with immobilized VWF determined by surface plasmon resonance, in which epitopes-2 and -3 have higher affinities for VWF than that of epitope-1. The results described above suggest that ADAMTS13 may initially bind to immobilized VWF through the sites of epitope-1 and epitope-2 with relatively weak affinity. The binding of epitope-1 to VWF may subsequently induce the conformational change of VWF, thereby exposing a binding site for epitope-3 for the efficient catalytic cleavage of VWF by ADAMTS13.
Prolonged Progression Free Survival Does Not Relate to Quality of Response to Treatment with Thalidomide in Patients with Relapsed Multiple Myeloma (MM).
Authors: Quach Hang , Prince Miles H. , Mileshkin Linda , Seymour John F. , Westerman David , Milner Alvin D.
Published: October 2019

Introduction: Quality of response to treatment in relapse/refractory MM, especially complete response (CR) or near complete response (nCR), has been reported to correlate with better PFS and OS in the past (Hussein MA, et al. Mayo Clin Proc2006;81:889–95). In this report, we provide an update on long-term survival from two multicentre phase II trials using thalidomide +/- IFNα2B (MM-thal) and combination celecoxib-thalidomide (Cel-thal) in relapsed or refractory MM, and identify predictors of progression-free survival (PFS) beyond 24 months (m).

Method: In 1998 and 2001, two prospective multicentre phase-II trials in relapsed or refractory MM were performed to assess the efficacy of thalidomide +/- IFNα2B (MM-thal), and combination celecoxib-thalidomide (Cel-thal), respectively. Both studies were previously reported (Blood2003;102:69; Clin Can Res2005;11:5504). The analysis of progression free survival (PFS) has been updated using the Kaplan-Meier method. Baseline characteristics were compared between patients having PFS ≥ 24m and < 24m using fisher’s exact test or the Cochran-Armitage test, to identify predictors of long-term disease control.

Result: Median follow up for MM-thal (n=75) and Cel-thal (n=66) trials were 73m and 47m respectively. Median PFS in the MM-thal trial was 5.5m, with estimated PFS of 9% at 3 years (95%, CI:5-18%), and 5% at 5 years (95%, CI:2-13%). In the Cel-thal trial, median PFS was 6.8m, with estimated PFS of 21% at 3 years (95% CI: 13-33%) and 16% at 5 years (95% CI:9-27%). Overall, 27 out of 141 patients (10 from MM-thal, 17 from Cel-thal) had PFS beyond 24m. The majority of these long term responders (70%) achieved only a PR as the best response to thalidomide-based treatment;15% achieved complete response (CR), and 15% had stable disease (SD). The most significant predictors for prolonged PFS of ≥24m was β2M ≥3mg/l (p<0.0005), stage ≥2 disease (p=0.001), and non-refractory disease to previous therapy (p=0.03). Bone marrow plasma cell infiltrate following thalidomide did not predict for outcome.

Conclusion: Thalidomide, and in particular combination celecoxib-thalidomide has substantial activity in relapsed MM with prolonged PFS beyond 24m in approximately 19% of patients. The strongest predictor of prolonged PFS is β2M. The depth of response to thalidomide had little influence on predicting remission duration.
Low Molecular Weight Heparin in Sufficient Dose of ≥70 IU/kg as an Effective and Safe Thromboprophylaxis in Patients with Newly Diagnosed Multiple Myeloma during Conventional VAD Induction Therapy.
Authors: Pour Ludek , Kessler Petr , Gregora Evzen , Zemanova Marketa , Penka Miroslav , Gumulec Jaromir , Adam Zdenek , Jaroslav Bacovsky , Frankova Hana , Havlikova Kamila , Lehanka Frantisek , Maisnar Vladimir , Meluzinova Ivana , Stavarova Yvetta , Straub Jan , Svobodnik Adam , Scudla Vlastimil , Sumna Elena , Walterova Lenka , Hajek Roman
Published: October 2019

Background: The patients with multiple myeloma (MM) have an increased risk of venous thromboembolism (VTE), particularly during the induction phase of therapy. Low molecular weight heparins (LMWH) seem to be useful for thromboprophylaxis in these patients. We analyzed prophylactic efficiency of LMWH based on dose either lower or higher than 70 IU/kg of body weight.

Patients and Methods: 223 patients with newly diagnosed MM were treated with induction chemotherapy consisting of vincristine, doxorubicin and dexamethasone (VAD) or vincristine, idarubicin and dexamethasone (VID) as a part of multicenter CMG 2002 trial. The first cohort of 137 patients received no systematic thromboprophylaxis. After a high incidence of thrombotic complications has been reported, the thromboprophylaxis with different doses of LMWHs was used in 86 consecutive patients for 4 months of induction therapy. Finally, 101 patients enrolled to the CMG 2002 trial in single centre were analyzed retrospectively, based on dose either lower or higher than 70 IU/kg of body weight. From this 101 patients 34% (34/101) received no systematic thromboprophylaxis, 38% (39/101) received LMWH (dalteparin) daily dose lower than 70 IU/kg, and 28% (28/101) received LMWHs daily dose higher than 70 IU/kg. The efficacy and safety of this treatment were analyzed. The chi-square test was used for statistical analysis.

Results: Incidence of VTE was significantly reduced in the group of 86 patients receiving the recommended thromboprophylaxis, as compared to 137 patients without prophylaxis (1.2% vs. 12.4%, p=0.003, risk reduction 11.25%). Single centre analysis showed that no VTE was developed in the group of 28 patients receiving more than 70 IU/kg of LMWH daily. It was significantly different from the 34 patients without prophylaxis (0% vs. 15%, p=0.002). The incidence of VTE in 39 patients with LMWHs daily dose lower than 70 IU/kg was reduced to 7.6%, which is still clinicaly significant as compared to cohort of 28 patients receiving more than 70 IU/kg of LMWH daily (0% vs. 7.6%, p=0.05). No case of major bleeding was developed in any group.

Conclusions: Thromboprophylaxis with LMWHs is effective in patients with newly diagnosed multiple myeloma during the induction chemotherapy only if the LMWH dose is sufficient. Our date shown that minimal sufficient dose seems to be more than 70 IU/kg of LMWH daily, as in this cohort of patients no case of VTE was developed.
Treatment of Patients with Multiple Myeloma Complicated by Renal Failure with Bortezomib - Based Regimens.
Authors: Roussou Maria , Kastritis Efstathios , Anagnostopoulos Athanasios , Psimenou Erasmia , Grapsa Irini , Matsouka Charis , Barmparousi Despina , Terpos Evangelos , Dimopoulos Meletios Athanasios
Published: October 2019

Introduction: Renal failure is a common feature of multiple myeloma and a major management problem. There is limited data regarding the reversibility of renal failure, the kinetics of serum creatinine and the safety of novel agents, such as bortezomib, when administered to newly diagnosed or relapsed/refractory patients with renal failure. The purpose of our analysis was to assess the frequency of renal failure improvement and kinetics of serum creatinine in patients who received bortezomib-based regimens.

Patients and methods: We evaluated 20 consecutive patients with newly diagnosed (n=7) or relapsed/refractory (n=13) multiple myeloma and renal failure, defined as a serum creatinine ≥ 2mg/dl. Patients’ median age was 66 years (range 43–88 years). Median serum creatinine was 3.8 mg/dl (range 2–11.9 mg/dl) and median creatinine clearance was 15.3 ml/min (range 6.4–33.3). Other features included hemoglobin <10gr/dl in 12 patients, platelets <100 × 109/l in 3 patients and elevated serum LDH in 9 patients. All patients received bortezomib plus dexamethasone alone or in combination with other agents, such as thalidomide, doxorubicin or melphalan. Reversibility of renal failure was defined as a sustained decrease of serum creatinine to <1.5 mg/dl and renal response was defined as ≥50% decrease of serum creatinine from its peak value.

Results: Reversal of renal failure was documented in 35% of all patients and the median time to reversal was 23 days. Moreover, 9 patients (45%) had 50% decrease in serum creatinine and the median time to decrease was 34 days. Some decrease of creatinine was documented in 88% of patients. Among four patients who were on renal dialysis, 2 became independent of this procedure after the second and the third cycle of treatment. The objective response rate was 61% and the median progression free survival for responders was 12 months. Toxicities were similar to those seen in myeloma patients without renal failure who were treated with bortezomib-based regimens. Grade 3–4 neutropenia and thrombocytopenia were seen in 28% and 22% of patients respectively. One patient died of infection and bortezomib had to be discontinued in 4 patients due to grade III neurotoxicity.

Conclusions: When bortezomib-based regimens are administered to myeloma patients with renal impairment their toxicity and efficacy are similar to those observed in patients with normal renal function. Moreover, these regimens are associated with rapid improvement of renal function in most patients and with reversal of renal failure in one-third of them.