bluuebunny/crossref_metadata_2025_split huggingface dataset:
Quantitation of Non-Inhibitory Anti-Factor VIII Antibodies in human Plasma
Authors: Krudysz-Amblo Jolanta , Parhami-Seren Behnaz , Butenas Saulius , Brummel-Ziedins Kathleen E. , Gomperts Edward D. , Rivard Georges E. , Mann Kenneth G. 
Published: April 2020

Abstract
Hemophilia A is a clinically heterogeneous bleeding disorder characterized by the absence of functional factor VIII (FVIII). The most severe complication from exposure to exogenous factor VIII therapy is the development of alloantibodies that neutralize the effect of the therapeutic agent. The standard method for quantitation of FVIII inhibitors in human plasma is by the Nijmegen method, a modification of the Bethesda assay. Our objective was to develop a sensitive and specific fluorescence based immunoassay for the quantitation of anti-FVIII antibodies (FVIIIAb) in human plasma. An affinity purified human anti-FVIIIAb isolated from a hemophilia A patient was used as a calibrator, with a detectability limit of 40±1.5pM. The calibrator and the human plasma anti-FVIIIAb were captured on recombinant FVIII coupled microspheres and probed with mouse anti-human Ig conjugated to R-Phycoerythrin. Citrated human plasma samples from 150 healthy donors and 39 inhibitor-negative hemophilia A subjects were studied and compared to 4 inhibitor-positive hemophilia A plasma samples with inhibitor titers of 1BU (94.6±0.08nM), 11BU (214.3±7.1nM), 106BU (2209.4±84.9nM) and 140BU (2417.7±3.8nM) as measured by the Nijmegen method. Among the 150 normal control plasmas, 4 subjects (3%) were determined as positive (anti-FVIIIAb ≥ 40pM) with concentrations in the range of 0.6nM to 1.9nM, well below that of 1BU (94.6±0.08nM) inhibitor-positive sample. Anti-FVIIIAb was also evaluated in seven commercial inhibitor-free plasma samples reported to contain &lt;1% FVIII activity. Two of the seven plasmas were found to contain anti-FVIIIAb with concentrations of 112.9±1.9nM and 71.1±8.5nM. The concentration of the antibody in both samples was within the range of the measured molar concentration of an inhibitor-positive 1BU titer plasma sample. Thirty two inhibitor-negative hemophilia A subjects were also quantitated for anti-FVIIIAb. Eleven of the 32 samples tested positive for anti-FVIIIAb with molar concentrations in the range of 0.6–20nM. The overall prevalence of the non-inhibitory anti-FVIIIAbs in hemophilia A subjects evaluated by our immunoassay (n=39) is approximately 33% relative to healthy donor population in which 3% were determined as positive (≥40pM) for FVIIIAb.
Critical Role of the Th1 Transcription Factor T-Bet in An Animal Model of Immune-Mediated Bone Marrow Failure
Authors: Tang Yong , Chen Jichun , Young Neal 
Published: April 2020

Abstract
Patients with aplastic anemia have elevated T-bet, a Th1 transcription factor, in peripheral blood CD4 and CD8 T cells, suggesting that T-bet over-expression and dysregulated Th1 immune response contributes to pathophysiology of marrow failure (Solomou EE et al., Blood. 2006; 107:3983). In the present study, we studied the role of T-bet in inducing bone marrow failure in a mouse model of immune-mediated BM failure, employing mice engineered with a germline T-bet deletion as lymphocyte donors. Compared with T-bet +/+ wild-type controls, T-bet−/− mice have similar cellular composition in various lymphohematopietic tissues including peripheral blood, spleen, thymus, lymph nodes (LN), and BM. Incubation of effector T-bet−/− LN cells with MHC-mismatched target CByB6F1 (F1) BM cells in an in vitro cytotoxicity assay resulted in a significantly lower proportion of apoptotic target cells than did wild-type T-bet+/+ LN effector cells, suggesting that T-bet−/− effector LN cells are functionally defective. While infusion of 5×106 wild-type T-bet+/+ LN cells into sublethally-irradiated F1 mice led to severe pancytopenia and aplastic bone marrow in recipient mice, infusion of the same number of T-bet−/− LN cells failed to result in marrow failure, and recipients had relatively normal blood counts and bone marrow cellularity. By flow cytometry, both expansion of CD4+ and CD8+ T cells and elevation in intracellular Th1 cytokine gamma interferon (IFN-γ), which are characteristic of marrow cells in recipients received B6 LN cells, were absent in recipients receiving T-bet −/− LN cells. Serum IFN-γ concentration in F1 mice infused with T-bet −/− LN cells was similar to the level in F1 control mice received TBI alone, and both were significantly lower than serum IFN-γ in recipients of wild-type B6 LN cells. In contrast, serum TGF-γ concentration was higher in F1 mice that received TBI alone or TBI plus T-bet −/− LN cell infusion, compared with mice that received TBI plus B6 LN cells. An increase of T-bet −/− LN cell infusion to 10×106 cells per recipient led to very mild BM failure. Contrary to the markedly increased number of CD4+ and CD8+ T cells and elevated IFN-γ level in the BM of F1 mice which have received wild type B6 LN cells, F1 mice infused with T-bet −/− LN have low CD4+ and CD8+ cells and low IFN-γ level in the BM similar to F1 mice received TBI alone, but they show increased IL4 and IL17 levels within bone marrow T cells, indicating that the diminished Th1 immune response due to T-bet deficiency was partially compensated by up-regulated Th2 and Th17 responses. Our data demonstrated that T-bet plays a critical role in immune mediated bone marrow failure. Approaches targeting to T-bet signal pathway may lead to novel treatment for bone marrow failure and other autoimmune diseases.
The Fanconi Anemia Protein, FANCG, Binds Directly to the SH3 Domain of aII Spectrin and This Interaction May Play An Important Role in the Stability of aII Spectrin and Its Function in Repair of DNA Interstrand Cross-Links
Authors: Lefferts Joel A , Wang Chuan , Baralt Melissa , Lambert Muriel W 
Published: April 2020

Abstract
Fanconi anemia (FA) is a genetic disorder characterized by bone marrow failure, a predisposition to cancer, congenital abnormalities and a cellular hypersensitivity to DNA interstrand cross-linking agents. This hypersensitivity in FA cells correlates with a defect in ability to repair interstrand cross-links. We have shown that the structural protein, nonerythroid a spectrin (aIISp), plays an important role in the repair of DNA interstrand cross-links and that there is a deficiency in aIISp in FA cells. The reduced levels of aIISp in FA cells are due to reduced stability of this protein. We propose that the stability of aIISp is dependent upon one or more of the FA proteins. The present study was undertaken in order to get a clearer understanding of the proposed role of FA proteins in maintaining the stability of aIISp in the cell and the functional importance of this relationship in the repair of DNA interstrand cross-links and the repair defect in FA cells. For these studies, FA proteins were examined for their ability to directly interact with aIISp and, if so, to map the sites of interaction. Four overlapping regions of aIISp were constructed and binding of FA proteins to each of these regions was examined using yeast two-hybrid analysis. Of the proteins examined, FANCG was found to interact with one of these regions of aIISp and specifically with the SH3 domain within this region. The site of interaction in FANCG was mapped to a SH3 domain binding motif, which contains a consensus sequence with preference for the SH3 domain of aIISp. The sites of interaction in both FANCG and aIISp were confirmed using site-directed mutagenesis. Two FA proteins that did not contain any SH3 binding motifs, FANCC and FANCF, did not interact with the SH3 domain of aIISp or any of the other regions of aIISp. These results thus demonstrate that one of the FA proteins, FANCG, contains a class of motifs that has specificity for binding to SH3 domains and binds to the SH3 domain of aIISp via this motif. This binding is important in the DNA repair process and in the FA repair defect, as is shown by our in vivo studies in which FA-G cells, transformed with the FANCG cDNA and stably expressing FANCG, showed normal levels of aIISp and a correction of the defect in ability to repair DNA interstrand cross-links. We therefore propose that the binding of FANCG to aIISp is not only important for the stability of aIISp in cells but also for the role aIISp plays in the DNA repair process. Thus a deficiency in FANCG, such as occurs in FA-G cells, could lead to reduced stability of aIISp which in turn could be an important factor in the defective DNA repair pathway in FA cells.
Telomerase Gene Mutation Screening and Telomere Length Detection in Chinese Patients with Bone Marrow Failure Syndrome.
Authors: Han Bing , Liu Bo , Zhao Yongqiang 
Published: April 2020

Abstract
Background Acquired bone marrow failure syndrome (BMF) is a group of diseases include aplastic anemia(AA), melodysplastic syndrome (MDS) and paraoxymal nocturnal hemoglobinuria (PNH). Some BMF patients have short telomeres in their peripheral nucleated cells. The length of telomere is maintained by a group of enzymes called telomerase complex. The core components of this complex are a RNA template and a reverse transcriptase, called TERC and TERT, respectively. Recently several studies in the west and Japan have disclosed the presence of telomerase complex gene mutation in a small group of patients with acquired bone marrow failure. They speculated that this small group of patients might represent a subset of cryptogenic Dyskeratosis Congenita (DKC), in which the premature exhaustion of hematopoietic reservoir is caused by mutations in the telomerase gene. This group of patients, though very small in number, would benefit from early bone marrow transplantation instead of traditional immunosuppressive therapy. The incidence of aplastic anemia in Chinese people is relatively high compared with that in the western country. But there has so far been no study in China about the incidence of telomerase gene mutation in acquired bone marrow failure and its relationship with telomere length.
Objectives To study the incidence of telomerase gene (namely TERC and TERT ) mutation in Chinese patients with acquired bone marrow failure and explore its relationship with telomere shortening.
Methods Blood samples from 90 patients with AA, MDS, and PNH in northern China were collected and performed TERC and TERT mutation analysis. Telomere length was measured by Southern blotting and compared with their normal counterparts.
Results 2 TERC mutations (n37 A→G, reported previously ; n66G→C) and 2 TERT mutations (n1870G→T (E/*); n1780G→T (S/I) ) were identified in 90 BMF patients. Among them, 3 mutations are reported first time. 1 patient with TERT mutation, however, was finally diagnosed as DKC instead of acquired AA, making the incidence of telomerase gene mutation in Chinese people with acquired bone marrow failure 3.4%, similar to that of the western people. Southern Blot analysis showed the small group of patients carrying TERC and TERT mutations has very short telomeres, compared with normal controls and with their aplastic counterparts.
Conclusions The incidence of telomerase gene mutation in Chinese people with acquired bone marrow failure is 3.4%, similar to that of the western people. This small group of patients has very short telomeres, it is thus clinically important to screen for this small group of patients.
Xeroderma Pigmentosum Group B Binding Contributes to p210 BCR/ABL Mediated Transformation in CML.
Authors: Pannucci Nicole L , Sahay Sutapa , Thomas Emily K , Chen Ru , Megjugorac Nicholas J , Adams Homer C , Rodriguez Pedro L , Fitzpatrick Ethan R , Williams David A. , Whitehead Ian P 
Published: April 2020

Abstract
Patients with chronic myelogenous leukemia (CML) typically carry a balanced reciprocal translocation between chromosomes 9 and 22, resulting in the production of an in-frame fusion protein known as p210 BCR/ABL. Expression of p210 BCR/ABL in myeloid cells is associated with a variety of transformed cellular phenotypes including changes in nucleotide excision repair (NER). Consistent with this, previous studies have demonstrated that p210 BCR/ABL interacts directly with xeroderma pigmentosum group B (XPB), a protein necessary for both transcription and NER. In the current study we have mapped the docking site for XPB within the RhoGEF domain of BCR, and have constructed a p210 BCR/ABL mutant that is deficient in XPB binding. The mutant has normal kinase activity, and retains Grb2 binding ability, but can no longer phosphorylate XPB. When expressed in 32Dc13 cells, the mutant still confers IL-3 independent growth, but abrogates the resistance to ultraviolet C (UVC)-induced apoptosis seen in wild-type p210 BCR/ABL expressing cells. In a bone marrow transplantation model for CML, mice that express the mutant have significantly extended lifespans. Whereas p210 BCR/ABL transplanted mice became moribund within 28–35 days of transplantation, mutant mice survived for an average of 78 days. Necropsies performed at death revealed splenomegaly and clear evidence of myeloproliferation in the mutant mice, however tissue infiltration and lung hemorrhage was less extensive than in the p210 BCR/ABL expressing mice. These results suggest that the interaction between p210 BCR/ABL and XPB regulates disease progression, and may represent a novel target for therapeutic intervention.
Inhibiting Alternative Non Homologus Endjoining (NHEJ) Pathways: Therapeutic Targets in Chronic Myeloid Leukemia (CML).
Authors: Sallmyr Annahita , Tobin Lisa , Tomkinson Alan E. , Rassool Feyruz V. 
Published: April 2020

Abstract
BCR-ABL fusion tyrosine kinase in chronic myeloid leukemia (CML), induces high levels of ROS that generate DNA double strand breaks (DSBs). We previously showed that CML cells repair DSBs by aberrant non homologous end-joining (NHEJ) that is characterized by large DNA deletions. The generation of DNA deletions represents a mechanism by which genomic alterations may be acquired in the progression of chronic phase CML to blast crisis. Recently, we demonstrated that a “back-up” or alternative NHEJ pathway is involved in aberrant repair of DSBs in CML. Proteins in this pathway include, DNA ligase IIIα, XRCC1 and poly(-ADP) ribose polymerase (PARP). We have identified that NHEJ proteins, DNA ligase IIIα and WRN are overexpressed in CML. This increased expression appears to be dependent on the presence of BCR-ABL. “Knockdown” of these proteins leads to an accumulation of unrepaired DSBs, demonstrating their essential involvement in DSB repair in CML cells. The goal of the current study is to evaluate the effect of inhibiting “back-up” DNA repair proteins in proliferation and apoptosis of BCR-ABL-positive CML compared with standard Imatinib therapy. To evaluate whether “back-up” repair proteins may be therapeutic targets, we used siRNA down-regulation and small molecule inhibition of DNA ligase IIIα and PARP in BCR-ABL positive cell lines. Importantly, we have recently identified small molecule inhibitors of DNA Ligases by computer aided drug design (CADD). Inhibition of DNA ligases and PARP result in a significant increase in apoptosis of CML cells (K562, Kasumi 4, MEG01 and KU812 and P210 MO7e), comparable with the cell death observed with imatinib treatment. Importantly, CML cell lines resistant to imatinib treatment demonstrate similar apoptotic levels in response to “back-up” repair protein inhibition. These inhibitors are in the process of being tested in CML xenografts and mouse models for therapeutic efficacy in vivo. Our data suggest that the survival of CML cells is at least in part maintained by repair of DSBs using “Back-up” NHEJ. The main proteins involved in this pathway, which include DNA ligase IIIα, XRCCI, DNA Ligase I, PARP and WRN have the potential to be novel therapeutic targets in CML patients that have acquired resistance to imatinib.
Clinical Relevance of Vascular Endothelial Growth Factor (VEGFA) and VEGF Receptor (VEGFR2) Gene Single Nucleotide Polymorphism on the Treatment Outcomes Following Imatinib Mesylate Therapy.
Authors: Kim Dong Hwan , Ahn Hee Kyung , Xu Wei , Kamel-Reid Suzanne , Liu Xiangdong , Siminovitch Katherine , Lipton Jeffrey H. 
Published: April 2020

Abstract
Imatinib mesylate (IM) could reverse marrow angiogenesis and decrease the plasma level of vascular endothelial growth factor (VEGF) in chronic myeloid leukemia (CML) patients. The current study investigated the impact of 4 VEGF (VEGFA) and 3 VEGF receptor (VEGFR2) gene polymorphisms on the outcomes of 228 CML patients following IM therapy (male:female 96:132; median age at start of IM, 55 years-old; chronic phase: accelerated phase: blastic crisis, 205/17/2). The DNAs from blood samples were genotyped using MALDI-TOF-based method. VEGFA genotypes such as -2578C&gt;A (rs699947), -460T&gt;C (rs833061), +405G&gt;C (rs2010963) and +936 C&gt;T (rs3025039) loci, and VEGFR2 genotypes including rs1531289, rs1870377 and rs2305948 were analyzed. In single marker analyses, strong correlations were noted between complete cytogenetic response (CCyR) and VEGFR2 genotypes (rs1531289 and rs1870377), treatment failure and VEGFR2 genotype (rs1870377), dose escalation and VEGFR2 genotype (rs1870377), complete molecular response (CMoR) and VEGFA genotype (rs3025039), progression to advanced disease stage and VEGFA genotypes (rs699947 and rs833061). Three haplotypes of VEGFR2 gene were generated as follows: GT (46.1%), AT (27.9%) and GA (25.7%). Haplotype analyses showed good correlations between VEGFR2 haplotype and CCyR, treatment failure, and dose escalation of IM. Multivariate analyses confirmed strong correlations of VEGFR2 polymorphisms (especially rs1531289, rs1870377 or VEGFR2 haplotype) with CCyR, treatment failure, dose escalation and of VEGFA genotype (rs699947) with progression to advanced disease stage. The VEGFR2 genotypes and haplotype correlate well with cytogenetic response, treatment failure and dose escalation of IM therapy in CML patients, while VEGFA genotype correlates with progression to advanced disease stage.
The Role of LIM Kinase-1 in the Glycoprotein Ib-IX Complex-Mediated Platelet Activation and Thrombus Formation
Authors: Stojanovic Aleksandra , Gorovoy Matvey , Voyno-Yasenetskaya Tatyana , Du Xiaoping 
Published: April 2020

Abstract
LIM Kinase (LIMK)-1 is a member of the LIMK family of serine-threonine protein kinases that phosphorylates actin-binding protein cofilin and regulates actin cytoskeleton organization. LIMK1 is expressed in many cell types including platelets but the exact role of LIMK1 in platelet function remains unclear. To determine the role of LIMK1 in platelet activation, wild type or LIMK1 knockout mouse platelets were stimulated with platelet agonists. Platelet aggregation and granule secretion were analyzed. Integrin-dependent second wave of platelet aggregation induced by von Willebrand factor (VWF) in the presence of VWF activator botrocetin was abolished in LIMK1 knockout platelets. In contrast, platelet aggregation in response to the agonist peptide of protease-activated receptor-4 (PAR4, thrombin receptor), ADP and collagen was either not affected or enhanced in LIMK1 knockout platelets in comparison with wild type mouse platelets. Thus, LIMK appears to play an important role in platelet activation stimulated by VWF binding to its platelet receptor, glycoprotein Ib-IX complex (GPIb-IX) but had no stimulatory effect on or negatively regulate the GPIb-IX-independent platelet activation pathways mediated by PAR-4, ADP receptors and collagen receptors. To determine whether ligand binding to GPIb-IX stimulates LIMK activation and function, platelets were stimulated with VWF in the presence of either ristocetin or botrocetin, and immunoblotted with antibodies specifically recognizing phosphorylated LIMK1 (Serine 505) or cofilin (Serine 3). VWF induced phosphorylation of LIMK1 and LIMK substrate cofilin. Thus, VWF indeed stimulates LIMK1 activation and function. An important physiological role of GPIb-IX in platelets is to mediate platelet adhesion to subendothelial-bound VWF under shear stress at sites of vascular injury. To determine whether LIMK1 is important in platelet adhesion, we investigated whether LIMK1 knockout affected platelet adhesion to VWF-coated surfaces. LIMK1 knockout platelets are defective in mediating stable platelet adhesion to vWF under shear stress, suggesting that LIMK1 plays an important role in GPIb signaling and GPIb-IX-mediated integrin activation that is required for stable platelet adhesion under shear stress. Importantly, LIMK1 knockout mice showed significant delay in the formation of occlusive thrombus following FeCl3-induced carotid artery injury in comparison with wild type mice, indicating that the role of LIMK1 in GPIb-IX-mediated platelet activation is important in in vivo thrombosis. Together, our study reveals that LIMK1 plays an important role in GPIb-IX-mediated platelet activation and arterial thrombosis in vitro and in vivo.
Autologous Bone Marrow-Derived Stem Cell Therapy: A Promising and Prospective Approach in the Treatment of Patients with Severe Buerger’s Disease.
Authors: Boda Zoltan , Udvardy Miklos , Razso Katalin , Szarvas Mariann , Olah Zsolt , Ilonczai Peter , Toth Judit , Jambor Laszlo , Hunyadi Janos , Sipos Tamas , Kovacs Gabriella , Kappelmayer Janos , Farkas Katalin , Vereb Zoltan , Rajnavolgyi Eva 
Published: April 2020

Abstract
No effective blood-flow enhancement therapies are available for patients with severe peripheral arterial disease (SPAD), thus amputation remains the only option for relief of rest pain or gangrene. Autologous bone marrow-derived stem cell therapy (ABMSCT) is an emerging modality to induce angiogenesis from endothelial progenitors. Eight lower limbs of seven patients with SPAD (seven limbs of six patients with Buerger’s disease) were treated by ABMSCT using isolated CD34+ cells with characterized phenotype and administered by intramuscular injections. The follow-up before and 1-, 3-, 6-, 9- and 12 months after ABMSCT was based on clinical (rest pain, walking distance without pain, non-healing ulcers, ABI) and laboratory (DS-angiography, duplex- and laser ultrasonography, TcPO2) measurements. Therapeutic benefit was demonstrated by complete regression of rest pain in all of the 7 patients, and by the significant improvement of pain-free walking distance (40 m vs 500 m). The average of ABI improved significantly on the treated (before:0.41, after twelve months: 0.83), but did not change on the contralateral limb. Non-healing ischemic ulcers disappeared in five, and became smaller and thinner in two lower limbs, the ulcer remained unchanged in one case, only. The clinical improvement started one month after ABMSCT, it became more prominent after at three months, and the best clinical results were observed after twelve months of the stem cell therapy. In all of the seven cases we observed improvement on the treated limb only, the contra-lateral symptoms and ulcers remained unchanged or worsened. Confirmed by posttrial observations obtained at 18 months after ABMSCT the clinical improvement was evaluated as stable and long lasting. New collaterals were detected by angiography in three patients, but duplex ultrasonography detected improvement in two patients only. Laser ultrasonography did not show significant changes whereas TcPO2 values improved on the foot from 18.80/16.78/23.83 mmHg, and on the calf from 36.66/31.25/45.00 mmHg. These laboratory parameters did not show improvement after 1 month, however, after 6 and 12 months improved values were recorded. Severe adverse events, complications were not observed. We conclude that ABMSCT with isolated CD34+ cells is safe, effective, localized and results in local and sustained clinical benefit for patients with severe forms of Buerger’s disease. We show for the first time that low number of isolated bone marrowderived CD34+ stem cells that involve CD34+CD133+, CD34+CD133−, CD34+CD45− and CD34+CD45+ cells with different vessel forming ability confers vasculogenesis upon intramuscular implantation to patients with Buerger’s disease.