bluuebunny/crossref_metadata_2025_split huggingface dataset:
Upregulation of Microparticles and CD40 Ligand in Antiphospholipid Syndrome: Potential Implication in Inflammatory Responses and Thrombosis.
Authors: Fabbrini Nancy , Hoppensteadt Debra , Messmore Harry , Bick Rodger , Adiguzel Cafer , Fareed Jawed 
Published: April 2020

Abstract
Antiphospholipid syndrome (APS) represents a complex pathphysiologic state where vascular endothelium, platelet activation, coagulation and inflammatory processes are contributory to the overall outcome. Cellular apoptosis and immune mediated fragmentation results in the generation of microparticles (MP). Platelet associated CD40 Ligand contributes to the inflammatory complications, which may be further augmented by MP. To test the hypothesis that both the MP and CD40 ligand are increased in patients with APS, 90 samples received at Hines VA/Loyola Medical Center laboratories for antiphospholipid screening were profiled for MP utilizing a functional method and CD40 ligand employing an ELISA method (R&D, Minneapolis, MN). ProteinChip Array analysis was also carried out for biomarker profiling to identify a previously described inflammatory biomarker at 11.9 kDa utilizing surface enhanced laser desorption/ionization (SELDI). Fifty normal healthy male and female plasma samples were used as controls for comparative purposes. Of the 90 samples screened, 30 were found to be positive for the antiphospholipid antibody (APA) titer using an ELISA method (American Diagnostica, Stamford, CT). All of the 30 positive samples have elevated DRVVT; however, wide variations in the APTT were noted. The remainder of the 60 samples, which were negative for APA, also showed variability in the DRVVT and APTT values. In contrast, the DRVVT and APTT values in the normal were within the expected range. None of the normal samples were positive for the APA antibody. Most strikingly, the CD40 ligand in the samples positive for APA titer were markedly higher (480 ± 230 pg/ml) than the 60 which were negative (180 ± 80 pg/ml). The normal values were much lower (90 ± 30 pg/ml). The MP titer in the APA positive samples was also much higher (34 ± 14 nM) in contrast to the APA negative samples (18 ± 12 nM). The normal levels were even lower (9 ± 4 nM). In the ProteinChip Array analysis, the APA positive samples showed a higher prevalence of the unique biomarker at 11.9 kDa. This unique biomarker was absent in the normal human plasma. These findings clearly suggest that APA represents a complex syndrome where hemostatic activation involving the formation of the MP, upregulation of inflammatory process as evident by the generation of CD40 ligand and protease activation resulting in the formation of unique biomarkers contribute to its overall pathophysiologic outcome. Sequential profiling of these parameters may be helpful in the risk stratification of these patients.
Two-Hybrid Screening Identifies an Interaction between the CUB Domains of Human ADAMTS13 and Factor XII.
Authors: Yu Zheyong , Sadler J. Evan 
Published: April 2020

Abstract
The metalloprotease ADAMTS13 regulates the size of released von Willebrand factor (VWF) multimers by cleaving a peptide bond in the VWF A2 domain. Unlike the other ADAMTS family members, ADAMTS13 contains two C-terminal CUB domains, and mutations in this region have been found in patients with congenital thrombotic thrombocytopenic purpura (TTP). In vitro studies indicate that CUB domains are dispensable for ADAMTS13 catalytic activity under static conditions with denatured VWF substrate, but they may be crucial for recognition of native VWF under flow. CUB domains in many proteins mediate protein-protein interactions, and to identify potential binding partners we conducted yeast two-hybrid screening using MATCHMAKER GAL4 two-hybrid system 3 (Clontech Laboratories Inc., CA, USA). A cDNA fragment encoding both CUB domains was inserted into the pGBKT7 vector. The resulting GAL4BD-CUB fusion protein was used as bait to screen a human liver cDNA library. Both bait and library vectors were simultaneously introduced into Saccharomyces cerevisiae AH109 and double transformants were selected on minimal synthetic dropout media lacking Trp, Leu and His. A total of approximately 3 × 106 clones were screened. Putative positive colonies were re-streaked and tested for β-galactosidase activity. DNA fragments from positive colonies were sequenced and 26 proteins were identified, 10 of which were plasma proteins including factor XII (FXII), β2-glycoprotein I, and fibrinogen β chain. Interactions were further characterized with a binding assay employing purified ADAMTS13 immobilized on microtiter plates. The FXII-ADAMTS13 interaction was saturable and reversible, with Kd = 17 nM, which is significantly lower than the plasma concentration of FXII (∼ 375 nM). Deletion of CUB domains in ADAMTS13(ΔCUB) reduces its affinity toward FXII by ∼10-fold (Kd = 164 nM). Activated FXII (FXIIa) bound saturably and reversibly to ADAMTS13 with Kd = 19 nM. Binding of FXIIa to ADAMTS13(ΔCUB) was undetectable, suggesting that the FXIIa-ADAMTS13 interaction is more specific than the FXII-ADAMTS13 interaction. This interaction of FXIIa and ADAMTS13 was totally blocked by a heat and acid stable inhibitor in plasma, whereas the interaction of FXII and ADAMTS13 was decreased only 50% by plasma. These data suggest that FXII can bind either to ADAMTS13 CUB domains or with lower affinity to a distinct site that does not involve the CUB domains; FXIIa, a two-chain enzyme formed by a single cleavage of FXII, only retains the structure involved in high affinity binding to ADAMTS13 CUB domains. Fibrinogen and β2-glycoprotein 1 also bound to ADAMTS13 in vitro, but with much lower affinity. However, the plasma concentrations of both proteins (about 9 μM and 4 μM, respectively) are at least 10 times higher than their apparent Kd values. Whether these CUB domain-interacting proteins are important for the regulation of ADAMTS13 activity in vivo remains to be established.
A Small Fragment of ADAMTS13 Enhances Cleavage of von Willebrand Factor by the Intact Enzyme.
Authors: Majerus Elaine M. 
Published: April 2020

Abstract
ADAMTS13 regulates hemostasis by cleaving von Willebrand Factor (VWF) to smaller less thrombogenic fragments. Absence of ADAMTS13 activity leads to thrombotic thrombocytopenic purpura, an often fatal disease that is characterized by microangiopathic hemolytic anemia, thrombocytopenia and microvascular thrombosis especially in renal and central nervous system vasculature. ADAMTS13 like other ADAMTS family members has a characteristic domain structure consisting of a metalloprotease domain, a disintegrin domain, a thrombospondin type 1 repeat (TSR), a cysteine-rich domain, a spacer domain, 7 additional TSRs and 2 carboxyl terminal CUB domains. Regions in the spacer and C-terminal thrombospondin type 1 repeats (TSR) and CUB domains have been shown previously to be important for the interaction of ADAMTS13 and VWF. To further characterize the interaction of ADAMTS13 and VWF, we generated a construct consisting of the spacer domain and the second TSR of ADAMTS13. This construct is called ST2 for Spacer and Tsr2. Although many constructs of ADAMTS13 fail to be secreted because of poor folding, ST2 is folded since it is secreted from human embryonic kidney 293 cells. Previous work has shown that the spacer domain alone can bind VWF under static conditions, therefore ST2 was included in VWF cleaving assays. Unexpectedly, ST2 was found to enhance the cleavage of VWF by ADAMTS13 by approximately 10-fold in assays using either guanidine or urea-denatured VWF. To understand how ST2 enhanced the cleavage of VWF by ADAMTS13, interactions of ST2 with ADAMTS13 and VWF were studied. ST2 was found to specifically co-immunoprecipitate with VWF but not with ADAMTS13. A Kd calculated from these co-immunoprecipitation experiments was 8.7 nM and the stoichiometry of binding was approximately 1 mole/mole. One explanation for the enhanced cleavage of VWF by ADAMTS13 in the presence of ST2 is that ST2 may induce a conformational change in VWF that makes the cleavage site more accessible. To test this hypothesis, VWF in the presence and absence of ST2 was subjected to trypsin digestion at different concentrations of trypsin. Differential cleavage of VWF was seen in the presence and absence of ST2 at all concentrations of trypsin tested. This is consistent with induction of a conformational change in VWF by ST2. These studies provide additional information on the structure-function relationship of these two key proteins in the control of hemostasis.
Thrombin-Cleaved Osteopontin Is Regulated by Thrombin-Activatable Carboxypeptidase B in Rheumatoid Arthritis.
Authors: Myles Timothy , Sharif Shadi A. , Du Xiao-Yan , Song Jason J. , Elizabeth Price , Robinson William H. , Leung Lawrence L. 
Published: April 2020

Abstract
The pro-inflammatory cytokine osteopontin (OPN) is present in rheumatoid arthritis (RA) synovial fluids (SF). OPN can be cleaved by thrombin exposing the cryptic C-terminal α4β1/α9β1 integrin-binding motif (SVVYGLR). We have previously shown that thrombin-activated proCPB (CPB) can remove the C-terminal arginine from the SVVYGLR motif abrogating its α4β1 binding. Using peptides based on the C-terminus of thrombin-cleaved OPN (OPN-R) and thrombin/CPB-cleaved OPN (OPN-L), rabbit anti-OPN-R and anti-OPN-L antibodies were generated. Specificity of these antibodies was demonstrated by western blot analysis. Specific ELISAs for OPN-R and OPN-L showed significant elevation of OPN-R and OPN-L levels in SF from patients with RA (n=26) as compared to osteoarthritis (OA, n=18) and psoriatic arthritis (PA, n=10). (OPN-R median values: 138.6 ng/mL (RA), 10.6 ng/mL (OA), and 2.2 ng/mL (PA) p &lt;0.001; OPN-L median values 205.3 ng/mL (RA), 25.9 ng/mL (OA), and undetectable in PA p &lt;0.003). Multiplex cytokine analysis showed that elevated OPN-R and OPN-L levels in RA were significantly correlated with multiple inflammatory cytokines including IL-6, IL-12p40 and FGF-2, while OPN-FL levels only exhibited a statistical correlation with IL-6. Synovial lining cells expressed functional TM that supported the activation of proCPB to CPB by thrombin. ProCPB activation by thrombin, which may occur either systemically or locally within the inflamed joint space, inactivates thrombin-cleaved OPN and C5a and represents a homeostatic response in modulating the inflammatory process in RA.
The Severity of Bleeding Tendency in 5 Patients with Factor V Deficiency Due to Distinct Kinds of Factor V Mutations.
Authors: Shinozawa Keiko , Amano Kagehiro , Suzuki Takashi , Inaba Hiroshi , Fukutake Katsuyuki 
Published: April 2020

Abstract
Coagulation factor V (FV) deficiency is a rare autosomal recessive bleeding disorder. It is poor correlation between FV levels in plasma and the severity of bleeding tendency. In the present study, we identified 5 mutations in the FV gene (F5) in 5 unrelated Japanese patients with reduced plasma FV activities associated with inherited FV deficiency. Their bleeding tendencies varied in severity from asymptomatic to severe. We hypothesized that the severity of bleeding symptoms with severe FV deficiency is correlated with FV levels in platelets, and performed recombinant mutant proteins expression experiments and analyzed of platelet FV. The data concerning 5 patient’s FV levels in plasma and the bleeding symptoms are given in Table. The F5 mutations in 5 Japanese patients were identified by direct sequencing. One of the 5 patients was a compound heterozygote for FV mutations and carried a 5-base pair (bp) deletion in exon 22 (del.ACCCT) and V1813M. The other 4 patients were homozygous for a D68H, N468S, V1813M, and R2174L mutations, respectively. Four mutations except V1813M are newly identified mutations. These mutations were introduced independently by site-directed mutagenesis into a pMT2/FV mammalian expression plasmid containing the full-length FV cDNA, and the wild-type and mutant FV proteins were expressed in HEK293 cells. In the conditioned media, FV specific activities of the FV-D68H, FV-N468S, FV-V1813M, FV-R2174L, and 5bp del. mutants were an approximately 22%, 81%, 28%, 40%, and 19% of wild-type, respectively. On the other hand, analysis of platelet from patient by using RT-PCR showed that platelet F5 mRNA of FV-R2174L and FV-N468S was equal amount to that of normal subjects, although the amount of FV-V1813M platelet F5 mRNA was reduced. Platelet FV protein from patients was analyzed by western blotting and ELISA. Although the amount of platelet FV-R2174L protein was equal to that of normal platelets, platelet FV-V1813M protein was considerably reduced. In addition, the amount of FV-N468S protein in platelet was observed between that of normal subjects and FV-V1813M. These results indicate that the fact that sufficient amounts of FV are stored in platelet is required for local hemostasis. The results of FV-R2174L suggest that both functionality and amount of FV-R2174L in platelet is enough to cope with local bleeding, resulting very mild bleeding tendency. On the basis of present findings, we conclude that the severity of bleeding due to severe FV deficiency is correlated with not only plasma FV level, but also platelet FV.
Five patient’s FV levels in plasma and the bleeding symptoms Patient No. Mutation FV activity (%) FV antigen (%) Bleeding symptoms 1 D68H 4 4 asymptomatic 2 N468S 3 3 asymptomatic 3 V1813M & 5-bp deletion &lt;1 9 severe 4 V1813M &lt;1 4 moderate 5 R2174L 1 5 very mild
Effective Factor VIII Endocytosis Is under Conformational Control of von Willebrand Factor.
Authors: Meijer Alexander B. , Roosendaal Sigrid D. , de Laat Bas , van den Biggelaar Maartje , Limburg Vincent , van der Zwaan Carmen , Rodenburg Kees W. , Mertens Koen 
Published: April 2020

Abstract
Von Willebrand Factor (VWF) protects Factor VIII (FVIII) from rapid clearance of the cofactor from the circulations. This notion may agree with the observation that VWF blocks FVIII binding to LDL receptor (LDLR) and LDL receptor-related protein (LRP) in vitro. In spite of the presence of VWF, however, we have previously demonstrated that LRP and LDLR are involved in the catabolism of FVIII in vivo. This suggests that dissociation of the FVIII-VWF complex in plasma or at the cell surface drives the LRP/LDLR dependent clearance of FVIII. We therefore first assessed the effect of VWF on the binding of FVIII to the cell surface employing confocal microscopy. To this end, a functional FVIII derivative containing the yellow fluorescent protein (FVIIIYFP) was incubated with LRP-expressing or LDLR-expressing CHO cells at 4 °C in the presence and absence of VWF. The result showed that there was a distinct yellow fluorescent staining of the cell surface irrespective of the presence of VWF. When the same experiment was performed at 37 °C, however, yellow fluorescent focal spots rapidly appeared inside the cells but only in the absence of VWF. Co-localization studies demonstrated that these spots originated from FVIIIYFP present in early endosomes. These findings suggest that VWF blocks the transfer of FVIII to its endocytic receptors but not the binding of FVIII to the cell surface. Intriguingly, in the presence of ristocetin, FVIIIYFP was localized inside the cells not only in the absence of VWF but also in its presence. In agreement with this finding, flow cytometric analysis confirmed that VWF was no longer able to prevent endocytosis of FVIIIYFP in the presence of ristocetin. These results suggest that switching VWF in the VWF-FVIIIYFP complex into its active conformation triggers the endocytic uptake of FVIII. The restored endocytosis of FVIII was not the consequence of a reduced affinity of “active-VWF” for FVIII. The latter was concluded from solid phase binding studies that showed that FVIII-VWF complex formation is indistinguishable in the presence and absence of ristocetin. We propose that switching VWF in the VWF-FVIII complex into its active conformation initiates at the cell surface a sequence of molecular events, which ultimately lead to the endocytic uptake of FVIII by cells expressing LRP or LDLR.
Ikaros Drives Human Haemoglobin Switching by Facilitating Active Chromatin Hub Formation.
Authors: Perkins Andrew C. , Tallack Michael , Zhan Ye , Papathanasiou Peter , Goodnow Crhistopher , Gaensler Karin , Crossley Paul M. , Dekker Job , Keys Janelle R. 
Published: April 2020

Abstract
The human β globin locus consists of an upstream locus control region (LCR) and five functional genes arranged sequentially in the order of their expression during development: 5′-ε-Gγ-Aγ- δ- β-3′. Haemoglobin switching entails the successive recruitment of these genes into an active chromatin hub (ACH). Although much is known about the cis elements and transcription factors involved in globin gene regulation, less is known about ACH formation. Here we show that the transcription factor Ikaros plays an essential role in both the formation of the β-globin ACH, and in haemoglobin switching. In Plastic mice, where the DNA-binding region of Ikaros is disrupted by a point mutation (H191R), there is concomitant marked (10 fold) down-regulation of human β-globin, and up-regulation of γ-globin gene expression. We show Ikaros binds to a critical cis elements in the LCR near the HS3 core and upstream of the δ-globin gene in the β-globin locus by electormobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) and that this DNA binding activity is lost in Plast mice. This latter site is implicated in deletional hereditary persistence of fetal haemoglobin (HPFH). Furthermore, chromatin conformation capture (3C) data suggest Ikaros facilitates long range looping between the LCR and a region upstream of the δ-globin gene. This study provides new insights into the mechanism of adult stage-specific assembly of the β-globin ACH. In addition the findings could lead to the development of novel drugs to reactivate HbF in adults with β-thalassemia and sickle cell disease.
TARC and RANTES Enhance Immunological Antitumor Effect Induced by GM-CSF Transduced Leukemia Vaccine in Mouse Model.
Authors: Inoue Hiroyuki , Iga Mutsunori , Xin Meng , Kurita Ryo , Nakayama Masaharu , Suehiro Yoko , Nakazaki Yukoh , Takayama Koichi , Nakanishi Yoichi , Tani Kenzaburo 
Published: April 2020

Abstract
Granulocyte-macrophage colony stimulating factor (GM-CSF) has been postulated to be a critical mediator in the initial tumor immune response and obvious antitumor immunomodulationg activity of autologous GM-CSF gene-transduced tumor vaccine therapy for various tumors including leukemia has been reported. Previously we demonstrated GM-CSF gene transduction into murine monocytic leukemia cell line of WEHI3B eliminated the tumorigenesity in vivo and the tumor regression started reproducibly around 10 days after tumor injection followed by disappearance within a week. We analyzed the gene expression of the transiently established tumor masses by the serial analysis of gene expression (SAGE) method to identify molecules associated with the antitumor effect. We identified that chemokine genes of TARC and RANTES were preferentially expressed in the GM-CSF-transduced tumors and the contribution of the two genes to the regression of GM-CSF-transduced tumors was suggested. To confirm the finding, we observed in vivo tumor formation of WEHI-3B cells transduced with a retroviral vector expressing murine GM-CSF, TARC, RANTES, GM-CSF+TARC or GM-CSF+RANTES, respectively. One million cells of each transduced cell group and non-transduced cell group were inoculated subcutaneously into syngeneic BALB/c mice. Then tumorigenecity and survival rate were observed. Both of the combination of GM-CSF with TARC or RANTES gene transduction profoundly inhibited tumor formation. In addition, the 2 groups demonstrated significantly longer survival than GM-CSF transduced group. To examine their therapeutic potency, 3×10^6 of irradiated WEHI3B cells retrovirally transduced with murine GM-CSF cDNA in combination with either recombinant TARC or RANTES were subcutaneously inoculated every 4 days for 4 times into syngeneic BALB/c mice preliminarily inoculated subcutaneously with 1×10^5 of wild type WEHI3B cells. Vaccinations were well tolerated without any major adverse events. The mice treated with the GM-CSF transduced cells with chemokines demonstrated significantly longer survival than the mice treated with GM-CSF transduced cells. Splenocytes harvested from mice treated with the former vaccines produced higher levels of IL-4, IL-6, IFN-γ, and TNF-α, suggesting a reinforcement of innate immunity as well as adaptive immunity. Immunohistochemical findings of the tumor section after vaccine treatment revealed more significant contribution of CD4+, CD8+ T cells to tumor repression in the combined vaccine groups than GM-CSF vaccine group. In coclusion, TARC and RANTES enhance immunological antitumor effect induced by GM-CSF in mouse WEHI3B tumor models and would be useful as the second generation GM-CSF immune gene therapy targeting leukemia.
Aberrant Expression in Human Epithelial Cancers of the P2X5-Encoded Minor Histocompatibility Antigen LRH-1: Implications for Graft-Versus-Tumor Immunity Against Solid Tumors.
Authors: Dolstra Harry , Levenga Henriette , Overes Ingrid , Vos Johanna C.M. , van Horssen-Zoetbrood Agnes , van der Voort Robbert , de Mulder Pieter , de Witte T.M. 
Published: April 2020

Abstract
Allogeneic stem cell transplantation (SCT) in combination with donor lymphocyte infusion (DLI) is an experimental treatment for patients with metastatic solid tumors. The therapeutic efficacy is attributed to the graft-versus-tumor (GVT) response during which donor-derived T cells eliminate malignant cells via recognition of minor histocompatibility antigens (MiHA). To reduce accompanying GVHD, it is crucial to identify MiHA which are selectively expressed on hematopoietic cells and solid tumor cells. Previously, we identified a hematopoietic cell-restricted MiHA, designated LRH-1, which is presented by HLA-B7 and encoded by the P2X5 purinergic receptor gene (J. Clin. Invest.2005:115:3506–3516). Here, we report that LRH-1, in addition to its hematopoietic cell-restricted expression, is aberrantly expressed on epithelial tumor cell lines. We observed that P2X5 mRNA is significantly expressed in 14 out of 42 (33%) solid tumor cell lines tested by real-time quantitative RT-PCR. We detected P2X5 transcripts in 3 out of 11 renal cell carcinoma cell lines, 2 out of 4 melanoma cell lines, 3 out of 7 colorectal carcinoma cell lines, 4 out of 10 brain tumor cell lines and 2 out of 10 breast cancer cell lines. To determine whether P2X5 mRNA expression in solid tumor cell lines results in susceptibility to lysis by LRH-1-specific CTL, we performed flow cytometry-based cytotoxicity assays using P2X5-expressing tumor cell lines. Based on LRH-1 genotyping analysis, we selected six solid tumor cell lines for the cytotoxicity studies. Remarkably, LRH-1-specific CTL efficiently lysed and inhibited the growth of DAOY brain tumor cells up to 3 days of co-culture. The renal cell carcinoma cell lines SKRC-33 and SKRC-18 and the melanoma cell line BLM were also susceptible to LRH-1 CTL-mediated lysis, although less effectively. However, pre-incubation of these tumor cell lines with IFNγ and TNFα significantly increased the susceptibility to LRH-1-specific CTL and resulted in complete target cell lysis. Furthermore, these cytokine-stimulated cell lines induced higher levels of CTL degranulation as determined by CD107a staining. No cytotoxicity was observed against LRH-1-negative FM3 melanoma and SKRC-24 renal cell carcinoma cell lines. These findings illustrate that the GVT reactivity observed in solid tumors after allogeneic SCT may be selectively enhanced by LRH-1-specific immunotherapy.