bluuebunny/crossref_metadata_2025 huggingface dataset:
Protein Phosphatase 2B Inhibitor, Cyclosporine A, Stimulates Endothelial Cell Exocytosis of Ultra-Large VWF Multimeric Strings by Regulating Munc18c Phosphorylation.
Authors: Nolasco Lettica , Gushiken Francisca C. , Patel Vimal , Turner Nancy , Moake Joel , Vijayan K. Vinod
Published: April 2020

Exocytosis of Weibel Palade bodies (WPBs) containing ultra-large Von Willebrand factor (ULVWF) plays an essential role in the processes of inflammation and thrombosis. The exocytosis of ULVWF involves the fusion of WPBs to the endothelial cell plasma membrane via vesicular trafficking families of proteins. Reversible post-translational modification, including the phosphorylation of tyrosine (Tyr) and/or serine/threonine (Ser/Thr) residues or S-nitrosylation of cysteine residues on one or more vesicular trafficking proteins tightly regulates the exocytotic process. The phosphorylation of any trafficking protein is determined by the interplay between protein kinases and protein phosphatases and any alteration in the activity of either enzyme can affect exocytosis. In comparison to agonist-induced, protein kinase-dependent signaling pathways that regulate exocytosis in endothelial cells, there is scant information about any possible role for the regulation of protein phosphatases in this process. Inhibition of protein phosphatase 2B (PP2B) also called calcineurin by the immunosuppressive drug, cyclosporine (CsA), is associated with thrombotic microangiopathy in a subset of transplant patients. Calcineurin is the target of a cyclophilin-CsA complex. We detected both calcineurin and cyclophilin A in resting human umbilical vein endothelial cells (HUVECs). Because CsA is a potent inhibitor of PP2B, we hypothesized that CsA induces ULVWF secretion and thrombotic events at least in part, by facilitating the phosphorylation of vesicle trafficking proteins that promote the exocytosis of ULVWF from endothelial cells. In vitro, we found that nanomolar concentrations of CsA or a cell permeable PP2B auto inhibitory peptide stimulated the release of ULVWF from HUVECs. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) viability assays revealed that PP2B inhibitors did not damage the HUVECs. In vivo, plasma levels of VWF were increased after intraperitoneal injection of CsA. These studies suggest that PP2B negatively regulate ULVWF exocytosis. Microscopic studies revealed that the ULVWF strings secreted by HUVECs treated with CsA or PP2B auto-inhibitory peptide induced the adhesion of platelets. CsA-treated HUVECs exhibited an increased serine phosphorylation of Munc18c, a vesicle trafficking associated protein that promotes granule exocytosis. These findings support a mechanism whereby inhibition of PP2B by CsA enhances cell secretion, including endothelial cell secretion of ULVWF by facilitating Munc18c phosphorylation.
Quantitative Analysis of Gene Expression for Vascular Endothelial Growth Factor and Its Application.
Authors: Bai Xia , Fu Jianxin , Ding Kaiyang , Cen Jiannong , Wang Zhaoyue , Ruan Changgeng
Published: April 2020

Vascular endothelial growth factor (VEGF) is a crucial mediator of angiogenesis, and plays an important role in pathogenesis of leukemia. However, the importance of VEGF in differentiation or apoptosis of leukemic cells remains to be evaluated. A competitor DNA fragment template of VEGF gene mimic was constructed with the method of gene recombinant technologies, and a competitive quantitative reverse transcriptase-polymerase chain reaction (cQRT-PCR) for analyzing VEGF gene expression was performed to assess the regulation of VEGF gene expression in the process of all-trans retinoic acid (ATRA)-induced differentiation of an acute promyelocytic leukemia cell line NB4. In construction of a standard curve from which the amount of target cDNA was derived, serial dilutions of the target were co-amplified with a constant amount of mimic, and the intensities of bands corresponding to the target and the mimic were measured. CD11b antigen and nitroblue tetrazolium (NBT) reduction rate of NB4 cells were also assayed at different time points. cQRT-PCR was a sensitive and reliable tool for analysis of VEGF gene expression, with a detectable range from 1 to 2 times 10 the fifth power molecules. The number of VEGF gene transcripts detected by means of cQRT-PCR assay was 42.3, 12.6, 3.6, and less than 1.0 times 10 the fifth powder per microgram of NB4 total RNA at 0, 12, 24 and 48 hours after ATRA treatment, respectively. The rapid down-regulation of VEGF gene expression during ATRA-induced NB4 cell differentiation was accompanied by an upregulation of CD11b expression and an increased NBT reduction rate. In conclusion, cQRT-PCR could be used as an efficient method of qualitative analysis of VEGF gene expression. ATRA significantly depresses VEGF expression and its antileukemic effect can be brought through the two ways of differentiation induction and angiogenesis inhibition.
Pharmacogenomics Study of Biological Pathways Related to Bortezomib Activity in Myeloma.
Authors: Mulligan George , Bryant Barb , Roels Steven , Bolt Andrew , Meng Yan , Koenig Erik , Boral Anthony , Esseltine Dixie-Lee , Anderson Kenneth A. , Bergsagel Peter Leif , Sonneveld Pieter , Broyl Annemiek , Mitsiades Constantine , Chng Wee-Joo , Richardson Paul G. , Trepicchio William
Published: April 2020

Introduction: Clinical development of bortezomib in multiple myeloma (MM) included pharmacogenomic gene expression profiling (GEP) of pre-treatment tumor biopsies in order to evaluate the feasibility of developing predictive classifiers in prospective clinical trials and to better define the biological pathways related to drug sensitivity. By partitioning the samples into train (n=101) and test (n=80) datasets, statistically significant classifiers of response and survival were identified and then validated [Mulligan G, et al: Blood. 2007; 109:3177–88]. Here we describe specific genes and biological pathways associated with bortezomib efficacy in relapsed MM.

Methods: Using tumor GEP data from 188 patients treated with single-agent bortezomib we identified genes associated with overall survival (OS), as well as genes associated with response (R) or progressive disease (PD) after bortezomib therapy. These clinical endpoints were also analyzed with biological pathway algorithms including GSEA [Subramanian A, et al: Proc Natl Acad Sci2005; 102:15545–50] Ingenuity Pathway Analysis and Gene Ontology/GATHER. These results were then compared to GEP data from a patient subset (n=76) treated with single-agent dexamethasone and also cell lines selected for differential sensitivity to bortezomib.

Results: A comparison of R and PD highlighted numerous differentially expressed genes; approximately 200 were over-expressed in R and 500 were over-expressed in PD (t-test <0.01). R-associated genes include signaling molecules like TRADD, CFLAR and TANK, while PD-associated genes included oncogenes (NRAS), apoptotic regulators (DAP3) and cancer antigens (CTAG). Pathway tools helped to integrate and extend these observations, consistently identifying NFkB activation and overexpression of adhesion pathways as R-associated. Overexpression of protein synthesis and mitochondrial pathways were PD-associated. The overexpression of pathways such as NFkB, adhesion and cytokines appear linked specifically with bortezomib and not dexamethasone sensitivity. These approaches were also applied to patient survival. In contrast to analyses of single genes, the pathway tools highlighted links between response and survival, including adhesion and inflammatory cytokine pathway expression.

Conclusions: This study highlights the diversity of biological pathways expressed in MM and the utility of clinical genomics to associate pathways with activity of single-agent bortezomib.
Thrombotic Thrombocytopenic Purpura with Normal Initial Hemoglobin.
Authors: Yarlagadda Madhavi , LePar Felice
Published: April 2020

Thrombotic thrombocytopenic purpura (TTP) is a life threatening multisystem disorder considered a true medical emergency. Usually patients are said to present with low hemoglobin as well as low platelets. This is a case of patient with early TTP having normal hemoglobin at presentation. A 37 year old male with history of idiopathic thrombocytopenic purpura (ITP) about 20 years ago, Ulcerative Colitis s/p colon resection, Graves disease s/p ablation, Lupus anticoagulant, and TTP about 3 years prior to admission which was treated with plasma exchange, was in his usual state of health until the morning of admission when he developed red urine and petechiae on his shoulder after playing football with his son the day before. He also reported a small amount of blood when he blew his nose. He denied fevers, chills, headache, confusion, anuria or any recent viral illness. Physical exam on admission revealed stable vital signs without fever, mild scleral icterus, and diffuse petechiae on shoulders/chest and ankle. He was alert and oriented × 3. Lab data showed platelets 5 k/ul, hemoglobin 14.3 g/dl, white blood cells 7.7 k/ul, BUN 29 mg/dl, creatinine 1.0 mg/dl, total bilirubin 6.6 mg/dl, direct bilirubin 0.5 mg/dl, reticulocytes 1.3%, LDH 1245 u/l, and haptoglobin < 7 mg/dl. Direct Coombs was negative. Peripheral smear showed 5–6 schistocytes/HPF. DIC work up was negative. Urinalysis showed large occult blood, 83 RBCs and 300mg/dl of protein. We suspected an early presentation of recurrent TTP. He was treated with methylprednisolone 1mg/kg and FFP 2–3 units then therapeutic plasma exchange with initial good response. His hemoglobin dropped to 10.9 within the next 36 hrs. He required plasma exchange over 3 months as well as other treatments including steroids, vincristine, splenectomy, and rituximab. Eventually he remained in remission. This case is notable for the normal initial hemoglobin. Isolated thrombocytopenia is often said to be ITP, but we believe that in this case the patient presented so soon after the development of symptoms that the hemoglobin had not yet declined. As TTP can be fatal if not recognized and treated promptly, it may be prudent to evaluate all patients with acute profound thrombocytopenia for TTP.
Hemophilia Treatment - A Model for Appropriate Cost Control.
Authors: Aledort Louis M. , Coates James G.
Published: April 2020

Hemophilia patients consume large quantities of biologicals. Many are on prophylactic regimens which cost substantially more and have better outcomes as compared to on-demand treatment. In either situation, 95% of costs of care are biologics. As costs are high, third-party payers frequently challenge various treatment regimens. Aetna Inc., working with nurses and a hemophilia treater, initiated a systematic evaluation program of 400 members with hemophilia enrolled in HMO plans to investigate whether these costs could be appropriately controlled. Several caveats were agreed to:

No therapeutic modality would be changed unless medically indicated. Patients would be supported to have an annual visit to a comprehensive center, if they were not already followed by one. Changes where biologics were purchased were only done with physician and client agreement.

Over the past three years, substantial savings have been accomplished PMPM (per member / per month fully-insured HMO member).

The annual spending PMPM for biologics PMPM Total dollars Before program 2003 $0.66 25,650,021 Year 1 2004 $0.60 23,832,539 Year 2 2005 $0.54 21,309,444 Year 3 2006 $0.54 19,395,661

The savings were attributable, in the main, to simply shifting how and where biologics were purchased and could not have been accomplished without the federal PHS pricing program. In three years’ time, annual savings per patient were substantial. The savings per subscriber encouraged Aetna, Inc. to continue this program.
Glanzmann Thromboasthenia with Isolated Amegakaryocytic Thrombocytopenia.
Authors: Corinaldesi Giorgio , Corinaldesi Christian
Published: April 2020

Glanzmann thromboasthenia is an autosomal recessive bleeding disorder caused by a quantitative and/or qualitative defect of the platelet glycoprotein GPalfaIIb/betaIIIa (IIb/IIIa integrin ITGA2B-ITGB3) complex. We have studied a 24 years old patient (exon 2113 T-C substitution missense C705F/C674R) with refractory bleeding platelet count above 25.000, and a very prolonged bleeding time, extensive mucocutaneous petechiae, nosebleeds, and bleeding gums, marked spenomegaly, and conjunctival hemorrage with a prevalence of HLA-DRw2 and DRB1-0410 and a severely decreased platelet production. Immunophenotyping of lymphocytes showed a relative increase of the subset of gamma/delta T-cell receptor (TCR) positive, and CD4 CD8 negative T lymphocytes; expression of c-Mpl and TPO were down-regulated. The bone marrow examination revealed extremely scarce and small megakaryocytes (with a great reduction of CD34+ and CD41+ and a small fraction of CD42+) and with normal marrow cellularity regarding other cellular lines; chromosomal analysis was also normal, while there was an associated severe anaemia with elevated HbF without any history of alcohol use or drugs ingestion. The important observation in this case was the need of many platelet trasfusions, and of fresh frozen plasma (FFP); the functional consequences of this kind of therapy include the induction of abnormalities of the hormones or their receptors (TPO/c-Mpl) that can be decreased in these patients; in addition, platelet mass blocks the biologic effects of SDF-1/CXCR4 receptor and the degree of this inhibition is correlated inversely with platelet count, and supports the evidence that the thrombocytopenia may be related to the inhibition of transcription factors (GATA-1, NF-E2, TAL-1, ETS family), and it directly correlates with the excess of platelet transfusions as an inducible knock-out in MK differentiation. Moreover, this findings indicate that when the receptors (c-Mpl) is saturated, the JAK-2 activation is neutralized and subsequent phosphorylation of STAT-3 and STAT-5 are down-regulated. This patient responded to treatment with danazol 200 mg daily and desmopressin (DDAVP) 0.3 mg/kg, while an additional therapy with traxenamic acid and recombinant FVIIa reduced mucosal bleeding.
The Effectiveness of Recombinant Factor VIIa in the Treatment of Bleeding in Pediatric Patients: A Ten-Year Experience from 1997 to 2007.
Authors: Chuansumrit Ampaiwan , Teeraratkul Sumate , Tripongkaruna Suporn , Wanichkul Suthep , Sirachainan Nongnuch , Pakakasama Samart , Nuntnarumit Pracha , Hongeng Suradej
Published: April 2020

Introduction: Recombinant factor VIIa (rFVIIa) has been used in the treatment of bleeding in Thailand since 1997.

Methods: 125 patients receiving rFVIIa at the Faculty of Medicine, Ramathibodi Hospital and Regional Hospitals from 1997 to 2007 were retrospectively evaluated. Their age group ranged from newborn (n=11), infants (n=21), children (n=86) to adolescents (n=7). They were divided into 2 groups. Group 1 included 11 hemophiliacs with high inhibitor (A10, B1) exhibiting 90 episodes of bleeding or undergoing surgeries. The rFVIIa 100–200 μg/kg at 1–2 h interval for 1–4 doses combined with prednisolone, transamine, fibrin glue and dental splint were given. In cases of surgery, 100 μg/kg at 1–2 h interval was given in the first 24 h and extended to 3–4 h until the removal of stitches or 7–14 days after the surgical procedures. Sequential use of rFVIIa with non-activated or activated prothrombin complex concentrates were given to 9 patients. Group 2 included 114 non-hemophiliacs with or without pre-existing hemostatic disorder for controlling excessive bleeding unresponsive to conventional blood components or for preventing bleeding in invasive procedures. There were 122 episodes involving patients with Dengue Hemorrhagic Fever (n=38), massive bleeding (n=49), invasive procedure (n=23), Glanzmann thrombasthenia (n=6), congenital factor VII deficiency (n=5) and acquired aplastic anemia (n=1). For controlling excessive bleeding, 40–100 μg/kg of rFVIIa was given at 15–30 min interval for 1–4 doses until the bleeding significantly reduced, followed by 40–100 μg/kg at 2–4 h interval for 24–48 h until the bleeding was completely ceased. For preventing bleeding in invasive procedures, the similar regimen but with a lower fixed dose of 40–100 μg/kg was used. One to two doses were usually used. An exceptional high dose of 200–300 μg/kg was used in patients with Glanzmann thrombasthenia and a lower dose of 20–30 μg/kg at 6 h interval was used in patients with congenital factor VII deficiency.

Results: The efficacy of controlling bleeding in hemophiliacs revealed effective (80/90, 88.9%), partially-effective (6/90, 6.7%) and ineffective (4/90, 4.4%) responses while the non-hemophiliacs revealed effective (81/122, 88.5%), partially-effective (14/122, 11.5%) and ineffective (27/122, 22.1%) responses. The case-fatality rates were 0% and 27.2% (31/114) in hemophiliacs and non-hemophiliacs, respectively. Three episodes of thrombosis (3/125, 2.4%) were found 3 children underwent cardiac surgery.

Conclusion: rFVIIa has shown an effectiveness in the treatment of bleeding in pediatric patients with hemophiliacs and non-hemophiliacs.
Management of High Haemorragic Risk Surgery: A Case of Acquired von Willebrand Disease (AVWD).
Authors: Mostarda Giovanni , Cafro Anna Maria , Caimi Teresa Maria , Redaelli Rosaria , Corno Anna Rosa , Sambruni Ivano , Morra Enrica
Published: April 2020

Introduction: Acquired von Willebrand Disease (AVWD) is a rare bleeding disorder occurring in patients with haematological malignancies, solid tumours, immunological and cardiovascular disorders and monoclonal gammopathy of undetermined significance (MGUS). Several pathogenetic mechanisms have been proposed to explain acquired von Willebrand factor (VWF) defect including autoantibodies. Muco-cutaneous bleeding and laboratory findings are similar to those of the congenital VWD. Several approaches have been attempted to correct haemostatic abnormalities with the aim to control bleeding diathesis or to prevent bleeding during surgery. Desmopressin, plasma-derived FVIII/VWF concentrates, intravenous high dose immunoglobulin (HDIg) are the most commonly used. The efficacy of therapy is not predictable and has to be tested before surgery or invasive procedures. In several cases more than one therapeutic option is required. Case report. The patient was a 64-years-old woman with IgGλ-MGUS. Because of persistent uterine bleeding a neoplasia was suspected. The pre-operative coagulative screening showed a prolonged aPTT with markedly reduced levels of FVIII and VWF-related activities (Table 1). VWF multimeric analysis evidenced loss of high and intermediate molecular weight multimers. Patient’s personal and familial history was negative for bleeding and VWF level was normal in other family members. Desmopressin administration (s.c.0.3 μg/kg) obtained a rapid but transient correction of plasma VWF-related parameters, with a return to baseline levels within 6 hours. HDIg intravenous infusion (1 g/kg/day for two consecutive days) led to stable normalization of VWF:RCo, VWF:Ag and FVIII for at least 7 days (Table 1). A laparoscopic hystero-annessiectomy was performed after two-day HDIg infusion: no bleeding complications were observed in the peri-operative period. Histological diagnosis was endometrial hyperplasia. During the two-months follow-up no haemorrhagic episodes were reported.

Comments: In AVWD the correction of laboratory abnormalities is crucial to prevent bleeding in patient undergoing high haemorrhagic risk surgery. The choice among therapeutic options is empirical and depends on underlying disease and clinical setting. The efficacy has to be verified. In our patient HDIg infusion obtained a sustained normalisation of VWF-related parameters and prevented bleeding during surgery and in the post-operative period.

Table 1. Patient’s laboratory parameters before and after treatment with HDIg VWF-related parameters Reference interval basal +1* +5* +7* *days after HDIg infusion aPTT (R) 1.73 1.21 1.21 1.27 <1.19 VWF:RCo (U/dL) <8 59 56 50 46-118 VWF:Ag (U/dL) 11 66 91 85 43-145 FVIII (U/dL) 14 96 85 96 51-164
von Willebrand Disease in Chronic Immune Thrombocytopenic Purpura Children.
Authors: Lee Ji Hye , Yoon Hwi-Joong , Lee Kun Soo
Published: April 2020

Purpose: von Willebrand disease (vWD) is the most common hereditary bleeding disorder and type 2B combines thrombocytopenia. So it must be considered in patients found to have low platelet counts, particularly if there is a family history of mucocutaneous hemorrhage. We performed this study to diagnose the type 2B vWD in chronic immune thrombocytopenic purpura (ITP) children.

Methods: Seventeen cases among chronic ITP children over 6 months at the Department of Pediatrics, Kyungpook National University Hospital, Daegu, Korea from October, 1995 to June, 2007 were participated in this study. We performed screening coagulation tests such as platelet counts, activated partial thromboplastin time (aPTT), and bleeding time (BT) and specific tests for vWD such as von Willebrand factor related antigen (vWF: Ag), vWF ristocetin cofactor (vWF: RCo), factor VIII, and vWF multimer on these patients. These tests were also performed their family when the patient was diagnosed vWD. And we reviewed their past and family histories about bleeding tendency.

Results: There were four boys and thirteen girls and their mean age was 11.6 years (range: 2.8∼18.5 years). Five cases (5/17, 29.4%) were diagnosed vWD: one had lower level of vWF: RCo and factor VIII with normal level of vWF: Ag and others had lower level of vWF: RCo and vWF: Ag with normal level of factor VIII. Among these, two cases showed abnormal screening test results, prolongation of aPTT or BT. We could perform the vWF multimer test in two cases, but two had normal pattern. Among five vWD children, we could obtain the past and family bleeding tendency histories except one case and three families showed bleeding tendency. But all families showed normal screening and specific test results.

Conclusions: von Willebrand disease was combined in 5 cases (29.4%) among 17 chronic thrombocytopenic children. More evaluation such as vWF multimer and ristocetin induced platelet aggregation test (RIPA) is needed to confirm the subtype. And we should repeat the evaluation to the family who had bleeding history but showed normal results for diagnosis or exclusion of vWD.
Redistribution of Recombinant Activated Factor VIIA into Platelets and Vascular Bed: Implications on Bioavailability and Hemostatic Mechanisms.
Authors: Galan Ana Maria , Lopez-Vilchez Irene , Gracia Juan , Escolar Gines
Published: April 2020

Background: Clinical evidence suggests that hemostatic action of recombinant activated factor VII (rFVIIa) exceeds its predicted plasma half life (around 3 hours). Mechanisms involved in the long lasting effects of rFVIIa for prophylactic treatment of patients with hemophilia and inhibitors have not been fully elucidated. Previous studies from our group have demonstrated that platelets internalize tissue factor preparations containing small amounts of FVII. We have investigated the possible redistribution of rFVIIa in different intracellular compartments focussing more specifically on platelets, endothelium and subendothelial matrix.

Methods: We exposed platelet rich plasma, isolated platelets, human umbilical veins and endothelial cells in culture (HUVECs) to rFVIIa. Samples were incubated for up to 2 hours at rFVIIa concentrations from 2-60 mg/ml (normal plasma concentration: 0.5 mg/ml). Flow cytometry techniques were applied to detect surface and intracellular antigens, including FVIIa, in platelets before and after exposure to membrane permeabilizing agents. Immunocytochemical techniques were applied to detect FVIIa in cross sections of umbilical veins and confocal microscopy was used to detect FVIIa in HUVECs. In additional studies, aliquots of washed platelets previously exposed to rFVIIa were incorporated into whole blood anticoagulated with low molecular weight heparin (LMWH) and perfused through a collagen rich damaged vascular surface at a shear rate equivalent to 600/sec.

Results: Flow cytometry studies revealed a significantly enhanced presence of intracellular FVIIa in platelets previously exposed to rFVIIa. Fluorescence intensity related to FVIIa was dependent on the concentration of rFVIIa used during the incubation. A more intense labeling for FVIIa was observed in the subendothelial matrix of umbilical veins exposed to rFVIIa. In perfusion studies, presence of platelets previously exposed to rFVIIa improved platelet thrombus formation and enhanced fibrin generation with respect to parallel experiments using platelets not exposed to rFVIIa.

Conclusion: Our results indicate that rFVIIa can be internalized into platelets and redistributed into subendothelial compartments. Redistribution of rFVIIa into other cellular compartments may explain the prolonged prophylactic action of rFVIIa in some clinical conditions. The existence of extravascular sources of FVIIa may provide new insights into the physiological and pathological implications of FVIIa on hemostasis mechanisms.
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

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 <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

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

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

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

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

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

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>A (rs699947), -460T>C (rs833061), +405G>C (rs2010963) and +936 C>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

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.