bluuebunny/crossref_metadata_2025 huggingface dataset:
Relationship between Uteroplacental Blood Flow snd Thrombophilia in Women eith Late Complicated Pregnancies.
Authors: Pranger Delphine , Deneys Veronique , Hubinont Corinne , Bernard Pierre , Debieve Frederic , Biard Jean-Marc , Ferrant Augustin , Hermans Cedric
Published: October 2019

There is a growing body of evidence that thrombophilia is linked to several obstetrical complications, most likely through uteroplacental vascular insufficiency. Very few studies have however evaluated whether alterations of the uteroplacental blood flow, as assessed functionally by Doppler ultrasound analysis, are associated with thrombophilic abnormalities in women with late pregnancy complications. Forty-nine non-smoking women followed during their whole pregnancy in the High Risk Pregnancy Clinic of the Cliniques Universitaires Saint-Luc, Brussels, in 2003 were enrolled in this retrospective study. They all had an unexplained late pregnancy complication (intrauterine growth retardation (IUGR) (n=34), intrauterine fetal death (IUFD) (n=4), preeclampsia (n=11)). They were not treated with anti-thrombotic agents. They all had a complete thrombophilic work-up (functional antithrombin, protein C and S assays, homocystein level, antiphospholipid antibodies (lupus anticoagulant and anticardiolipin antibodies), activated protein C resistance, G20210A prothrombin gene mutation). Doppler evaluation of the uterine arteries (assessed by the presence of bilateral notches after 26 weeks) and umbilical arteries (assessed by resistance index above 90th percentile) was performed. Thrombophilic abnormalities were identified in 8 of 49 (16%) women (factor V Leiden (FVL) (n=4), G20210A prothrombin gene mutation (n=2) and isolated anticardiolipin antibodies (n=2)). Of the 49 women, 20 had an abnormal Doppler (uterine (n=5) and umbilical (n=15)) including IUGR (n=13), IUFD (n=2) and preeclampsia (n=5). Four women with thrombophilia were found to have Doppler abnormalities (4/20; 20%) while most women with Doppler alterations had no thrombophilia. In conclusion, abnormal uteroplacental Doppler findings are frequently found in women with late pregnancy complications. They do not seem to be correlated with the presence of thrombophilic abnormalities even if thrombophilia seems to be more frequent in obstetrical complications.
Thrombophilic Disorders and the Risk of Thromboembolic Events in Women on Oral Contraceptives and Hormone Replacement Therapy.
Authors: De Sancho Maria Teresa , Barauskas Tanya M. , Rand Jacob H.
Published: October 2019

Objective: To evaluate the frequency and distribution of thrombophilic conditions in women with thromboembolic events (TEE) while on oral contraceptives (OC) or hormone replacement therapy (HRT). To assess the contribution of the duration of hormone use and a positive family history for TEE in predicting thrombosis.

Methods: Retrospective analysis of all case records of women who developed a TEE while on OC or HRT and who were referred for hypercoagulable evaluation to the Thrombosis and Hemostasis Section at a teaching hospital from 1999–2002.

Results: 65 women who were taking OC or HRT and developed a TEE were identified with one or more hypercoagulable disorders. 23 women tested positive for two or more hypercoagulable disorders: 16 had Factor V Leiden 3 of them were homozygous and 13 heterozygous, 5 were heterozygous for the prothrombin gene g20210a polymorphism, 26 had positive antiphospholipid antibody tests, 10 had elevated homocysteine levels, 4 had protein C deficiency and 7 had protein S deficiency. There were 65 thromboembolic events: 16 pulmonary emboli, 17 cerebrovascular events, 11 intra-abdominal vascular thromboses, 13 extremity deep venous thrombosis (DVTs), 5 superficial thrombophlebitis, and 2 retinal vein thromboses. Of the 65 women, 37% of patients had a positive family history of thrombosis. 52% had additional risk factors for thrombosis including smoking or obesity. 51% had been taking OC or HRT for > 1 year.

Conclusions: In this population, thrombophilic conditions contribute in most cases to TEE in women on OC and HRT. Approximately 50% of TEE occurred in women on OC or HRT for > 1 year. The burden of thrombosis associated with hypercoagulability in women on OC and HRT has been underestimated in prior studies by including only DVT/PE. Family and personal history of thrombosis must be thoroughly explored before initiating or continuing OC or HRT and a positive history of thrombosis may warrant a hypercoagulable evaluation.
The Hemostatic State in Cancer Patients: The Relationship between Hypercoagulable Markers and Cancer Prognostic Markers.
Authors: Mousa Shaker A. , Alizadeh Hussain , Al-Tajer Shaikhah
Published: October 2019

Background: The most common abnormalities described in cancer patients are the elevation of the clotting factors V, VIII, IX, and XI; increased fibrinogen/fibrin degradation products; hyperfibrinogenemia; and thrombocytosis. We performed a prospective study to evaluate the relationship between changes in hemostatic parameters and cancer prognostic markers.

Methods: We enrolled patients who were diagnosed as having different types of nonhematological, solid organ tumor. The most important natural inhibitors of abnormal coagulation (protein C, protein S, and antithrombin) and D-dimer and prothrombin activation peptide F 1 + 2 as markers of the status of coagulation system were studied in these groups of patients prior to any form of therapy. These markers were repeatedly measured with each treatment course and their results were correlated with other markers of tumor prognosis. These parameters were also studied before and after any surgical intervention for the tumor. The main aim of the study was to assess the changes in hemostatic parameters and to see if these changes can be used as markers for tumor prognosis.

Results: Fifty-two patients were enrolled in this study. Two patients had esophageal adenocarcinoma, 6 had gastric cancer, 16 had colorectal cancer, 4 had exocrine pancreatic carcinoma, 2 had adenocarcinoma of gallbladder, 6 had adenocarcinomatous type of non-small cell lung cancer, 2 had small cell lung cancer, 12 had infiltrating breast carcinoma, and 2 had ovarian cancer. In all cases, the elevation of the levels of hypercoaguable markers was directly correlated with tumor stage, tumor markers, and radiological findings of cancer state. Additionally, the level of naturally occurring coagulation inhibitors were all decreased with progression of tumor. These hypercoaguable parameters were also raised after surgical resection of these tumors.

Conclusions: Based on our findings, determination of D-dimer, F1 + 2, protein C, protein S, and antithrombin levels can be used as markers to assess the tumor prognosis.
D-Dimer Assay during Menstrual Period.
Authors: Chan Howard H. , Johnson Judith A. , Panju A. , Bradley Christine A.
Published: October 2019

Introduction:

Fibrin D-dimer is the degradation products from cross-linked fibrin. It is a marker of intravascular thrombogenesis. Menstruation is associated with activation of coagulation and fibrinolytic pathways. To investigate the changes of plasma fibrin D-dimer level during menstruation cycle, we measure the D-dimer level on day 1, 3, 5, and 21 of the menstruation period for 5 consecutive female volunteers. MDA D-dimer assay is used because it is one of the most sensitive laboratory methods for quantitative measurement of D-dimer level.

Methods:

Normal female volunteers with normal regular menstruation cycle are recruited. To avoid other confronting factors, subjects with recent history of thromboembolic diseases; ischemic heart disease; pregnancy or child birth; active malignancy; active inflammatory or infectious disease; surgery or trauma; and/or use of birth control pills are excluded. All volunteers have the menstruating period identified. Day 1 of menstruating period is defined as the first day when menstruating flow occurs. On Day 1, 3, 5 and 21 of menstruating cycle, blood samples are collected in tubes precoated with 3.2% trisodium citrate-dihydrate. Specimens are centrifuged at 2500 g for 15 minutes. The MDA D-dimer assay is performed using a commercial kit as per manufacturer’s instructions. Plasma fibrin MDA D-dimer level is expressed in F.E.U./ml (normal range is <0.50 F.E.U./ml). ANOVA statistical analysis for single factor is used to compare the differences among the D-dimer levels on day 1, 3, 5 and 21 of the menstruation cycle. The study has been reviewed and approved by the committee for ethics for research.

Results:

The results are summarized in table 1 and figure 1. There is no significant variation of D-dimer level measured on day 1, 3, 5, and 21 of menstruation cycle. (p = 0.382)

Conclusion:

The MDA D-dimer level does not have significant change during the menstruation period. The clotting process during menstruation may mainly be extravascular and, therefore, not affect the D-dimer level. Clinically, D-dimer test can still be used to rule out venous thromboembolism in a menstruating woman.

D-dimer levels (F.E.U./ml) on various days of menstruation cycle for 5 volunteers

Figure Figure Subject Number 2 3 4 5 6 Day 1 0.12 0.21 0.31 0.34 0.44 Day 3 0.10 0.18 0.23 0.13 0.31 Day 5 0.11 0.17 0.16 0.27 0.22 Day 21 0.21 0.10 0.17 0.39 0.29
Sticky Platelet Syndrome (SPS) as a Cause of Unexplained Thrombosis.
Authors: Kubisz Peter , Musial Jan
Published: October 2019

Background: Sticky platelet syndrome (SPS) was first described in 1983 by Holliday. The results of recent studies suggest SPS to be a common cause of arterial and venous thrombosis. With respect to otherwise unexplained venous thrombosis, the prevalence of SPS approximates that of activated protein C resistance (APC-R). It has been found that SPS accounted for about 21% of otherwise unexplained arterial events (acute myocardial infarction, cerebrovascular thrombosis, transient cerebral ischemic attacks, retinal thrombosis, and peripheral arterial thrombosis) and accounted for about 13,2% of otherwise unexplained venous events (deep vein thrombosis, with or without pulmonary embolus).

SPS is an autosomal dominant platelet disorder associated with arterial and venous thromboembolic events, frequently recurrent under oral anticoagulant therapy. It is characterized by hyperaggregability of platelets in platelet-rich plasma with adenosine diphosphate (ADP) and epinephrine (type I), epinephrine alone (type II), or ADP alone (type III). Combination of SPS with other congenital thrombophilic defects have been described. Low-dose aspirin treatment (80–100 mg) ameliorates the clinical symptoms and normalizes hyperaggregability. The precise etiology of this defect is at present not known, but receptors on the platelet surface may be involved.

Patients and methods: We examined fifty patients with otherwise unexplained arterial or venous thrombosis. SPS evaluations were performed according to the method of Mammen. The equipment used was PACKS-4 aggregometer.

Results: SPS was detected in eight patients. Clinically SPS resulted as an arterial thrombosis in five patients (2 coronary thrombosis, 2 cerebral ischemic attacks, 1 peripheral arterial thrombosis), two individuals showed deep venous thrombosis (DVT) and one patient passed recurrent abortions. SPS was classified as type I in 2 cases, 5 patients had type II and only 1 patient was diagnosed as type III, respectively (4 of them showed a glycoprotein IIIa abnormality).

Conclusion: Because treatment with heparin or warfarin will not alleviate the thrombotic tendency of SPS, but simple aspirin therapy almost always will correct the defect and protect the individual from second event, it is particularly important to define the presence of this defect.
Synthetic Anti-Xa Drugs Can Be Used for Parenteral Anticoagulation but Not Fondaparinux.
Authors: Iqbal Omer , Hoppensteadt Debra , Fareed Daniel , Fareed Jawed
Published: October 2019

Targeting at the level of factor Xa and/or prothrombinase complex by synthetic anti-Xa agents such as JTV-803 (Akros Pharma, Princeton, NJ), DX-9065a (Daiichi Pharmaceuticals, Tokyo, Japan), and Fondaparinux (Sanofi-Synthelabo, Toulouse, France) represents an important approach in anticoagulant therapy. Factor Xa is an essential component of the prothrombinase complex and leads to the generation of thrombin. Synthetic heparin pentasaccharide, Fondaparinux (Arixtra®) has been approved by the United States Food and Drug Administration for the prophylaxis of deep vein thrombosis in patients undergoing hip and knee replacement surgeries. There is a rapid stride in the development of newer synthetic inhibitors of factor Xa such as DX-9065a, and JTV-803 in various thrombotic indications. In order to evaluate the parenteral anticoagulant potential of these synthetic factor Xa inhibitors, we used the Hemochron (celite) activated clotting time (ACT) assay. DX-9065a at a final concentration of 5 and 10 μg/ml increased the ACTs to 250 and 300 seconds, respectively. Similar anticoagulant potential was noticed with JTV-803. These findings suggest that these agents may be useful as parenteral anticoagulants during interventional cardiologic procedures, surgical anticoagulation and for the prevention of vascular access occlusion. Fondaparinux requiring antithrombin (AT) for its anticoagulant effect does not increase the celite ACT to the extent as other synthetic factor Xa inhibitors. Fondaparinux even at a final concentration of 100 μg/ml increase the ACT to about 200 seconds and hence are not suitable to be used as parenteral anticoagulants. The concentrations of JTV-803, DX-9065a and fondaparinux required to increase the ACT to near 250 seconds are 6.2, 5 and 125 μg/ml, respectively. While fondaparinux is AT-dependent, the synthetic anti-Xa agents are AT-independent in their actions. The antithrombin sparing effect of direct anti-Xa agents may contribute to an additional anticoagulant effect as reflected by increased ACT levels. Furthermore, there are some other advantages of direct anti-Xa agents when compared to fondaparinux. While direct anti-Xa agents may be used for patients with AT deficiency, fondaparinux being AT-dependent, may not. The inhibition of the clot-bound and prothrombinase-bound factor Xa are additional advantages of direct Xa inhibitors when compared to fondaparinux. Fondaparinux being AT-dependent and upon complexation with AT is not capable of inhibiting the clot-bound factor Xa. Oral Xa inhibitors are being developed and when available patients may have an ideal transition from a parenteral anti-Xa agent to an oral Xa inhibitor. The results clearly suggest that synthetic factor Xa inhibitors except fondaparinux may be used as parenteral anticoagulants. Large-scale clinical studies are warranted to evaluate these findings.
Evaluation of Stability of a Sucrose-Formulated, Full-Length rFVIII for Use in Continuous Infusion Therapy.
Authors: Martinowitz Uri , Lubetsky Aaron , Tamarin Ilia , Luboshitz Jacob
Published: October 2019

Introduction: Stability of a FVIII preparation in mini-pumps is an important consideration for its use in a continuous infusion manner. Our goal was to evaluate the stability of a specific recombinant FVIII (Kogenate®FS; KOGENATE®Bayer) in two commonly used infusion mini-pumps, with and without anticoagulant additives that may be used in clinical practice to prevent local thrombophlebitis.

Methods: Kogenate® FS (250 IU/vial and 1000 IU/vial) was reconstituted using aseptic technique according to the manufacturer’s instructions with normal water for injection (100 IU/ml and 400 IU/ml, respectively); an additional dilution of 100 IU/ml was made from the 1000 IU/vial size. Reconstituted material was spiked with unfractionated heparin (UFH) or low molecular weight heparin (LMWH; Enoxaparin/Clexane) to a final concentration ~5 U/ml, or left untreated and then transferred to reservoirs of 2 mini-pumps (Walkmed, Medfusion Inc., Duluth, GA and CADD, Deltec, Inc., St. Paul, MN). All bags were stored in a light-protected environment at room temperature (22°C) for 7 days. Samples were drawn at baseline (immediately post reconstitution), 3h, 6h, 12h, 24h, and days 2–7, frozen at −30°C, and assayed for FVIII activity by one-stage assay. On Day 7, the residual volume of the mini-pump containers was cultured for bacteria.

Results: All samples except one* had FVIII activity >90% of baseline.

Conclusions: Kogenate® FS appears to retain excellent stability at room temperature during 7 days in both CADD and Walkmed infusion pumps with and without addition of UFH or LMWFH. No bacterial growth was observed. These results indicate that Kogenate® FS may be useful in continuous infusion therapy.

Figure Figure
KLF4 as a Mediator of Quiescence in Hematopoietic Stem/Progenitor Cells.
Authors: Alder Jonathan K. , Georgantas Robert W. , Yu Xiaobing , Civin Curt I.
Published: October 2019

At any given time, only a very small fraction of the hematopoietic stem cells (HSCs) in an organism are actively dividing; the vast majority of these cells remain in a quiescent state. HSCs can escape this quiescent state to self-renew and give rise to progenitor cells (HPCs), which expand rapidly to form all of the mature progeny cells of the lympho-hematopoietic system. Despite the centrality of quiescence in hematopoiesis, the mechanisms by which quiescence is regulated are largely unknown. Several studies have shown that p21CIP/WAF1 is required for cell cycle arrest of HSCs, and deletion of p21 results in an increase in HSC pool size. However, little is known about the pathways upstream of p21. Recently, we identified 81 genes that were highly over-expressed by normal HSC-enriched CD34+/CD38− cells compared to HPC-enriched CD34+/CD38+ cells. One of the HSC-over-expressed genes was Krupple-Like Factor 4 (KLF4), a member of the Kruppel-like transcription factor family. KLF4 has been associated with a quiescent phenotype in T and B lymphocytes, and exogenous expression of KLF4 induces growth arrest in a colon cancer cell line. It has been shown that KLF4 is an important mediator of p21 in the G1/S cell cycle arrest following DNA damage. Thus, we hypothesized that KLF4 is an important upstream regulator of HSPC quiescence via p21. To begin to test this, we designed a dual promoter self-inactivating (SIN) lentiviral vector that expressed KLF4 under the control of the EF1α promoter and GFP under the CMV promoter. We transduced primary mouse bone marrow (BM) Lin− or human cord blood (CB) CD34+ HSPCs, and purified the resulting successfully transduced GFP+ cells by fluorescence-activated cell sorting. KLF4-transduced human or mouse HPCs displayed a major reduction in colony forming capacity, as compared to either GFP− HPCs transduced with the vector containing KLF4 and GFP or HPCs transduced with the parental control vector expressing only GFP (Fig 1 shows representative experiments). In suspension cultures, KLF4-transduced cell populations generated fewer total cells, accompanied by an increase in apoptic/necrotic cells. In the KLF4-transduced cells, a higher percent were CD34+. These results are consistent with KLF4 inhibiting HPC differentiation and inducing a p21-like pathway with cell cycle arrest and increased apoptosis. Further investigation of KLF4 cellular and molecular actions may not only lead to enhanced understanding of the molecular basis of HSPC quiescence, but also to novel methods for expansion of normal HSPCs.

Figure Figure
Translational Control of the Hematopoiesis-Specific RhoH Gene Is Mediated by Upstream Open Reading Frames.
Authors: Lahousse Sébastien , Denis Claude , Kerckaert Jean-Pierre , Galiègue-Zouitina Sylvie
Published: October 2019

The hematopoiesis-specific RhoH gene was identified in our laboratory, as a BCL6 partner gene (1), in follicular B-non Hodgkin’s lymphoma associated with the recurring t(3;4) chromosomal translocation (2). The RhoH gene encodes a Rho small G protein supposed to be a Rac antagonist (3). The RhoH protein is always in a GTP-bound state (i.e. constitutively active), which suggests a strongly regulated synthesis. This work is aimed at elucidating molecular mechanisms that may regulate this RhoH protein synthesis.

The RhoH gene contains one coding exon only (exon 2) preceded by six uncoding exons, and the RhoH transcripts exhibit an important 5′ UTR heterogeneity, especially in the B-lymphoid lineage. This heterogeneity both reflects (i) an use of multiple transcription start sites and (ii) alternative splicing events of some 5′ uncoding exons (4). The present study is devoted to assess the functional relevance of these 5′ uncoding exons in the RhoH protein synthesis. Some of them (exons 1a, 1b and X4) contain an upstream ORF (uORF) sequence. Ten percent of eukaryotic mRNAs, 2/3 of which are involved in the control of cellular growth and differentiation, contain such uORFs within their 5′-UTR sequence. These uORFs are involved in the translational control of these genes (5). RhoH thus provides an additional opportunity to assess the role of uORFs in the translation of a cellular mRNA. Moreover, uORFs have never been implicated in the regulation of any small G family member gene.

The data show that the uORFs sequences from the RhoH gene repress the translation of a downstream reporter gene, in transfected hematopoietic cell lines (erythroid, B- and T- lymphoid). The initiation codon from 1a-, 1b-, and X4- uORFs is strongly involved in this phenomenon, as the inhibitory effect was suppressed by an uAUG invalidation. Targeted mutations (premature Stop, silent or mis-sens) within these uORFs sequences were functionally analyzed in the same reporter LUC system. The results led us to distinguish two uORF-mediated translational regulatory mechanisms: (i) an “uAUG dependent / uORF-peptide dependent” mechanism for the 1a- and 1b- uORFs sequences, and (ii) an “uAUG dependent / uORF-peptide independent” mechanism for the X4- uORF.

The RhoH transcripts exhibit a 5′ UTR heterogeneity, which might modulate the protein synthesis. Three main RhoH transcripts are expressed in the different hematopoietic lineages: “1a-X4-2”, “1a-1b-X4-2” and “1b-X4-2” (4), therefore the effects of the 1a-, 1b-, and X4- uORFs were investigated in combination (1a+X4; 1a+1b+X4; 1b+X4), as in the RhoH transcripts. The results convinced us to propose an uORF-mediated translational regulatory mechanism, which is based on a combinatory effect (synergy or antagonism) of three uORFs. This mechanism is called: “translational lock-unlock” model of the RhoH gene. In vitro translation experiments aimed at validating this model will be presented.
Effects of Constitutive Production of Colony-Stimulating Factors by Human Lung Cancer Cells on Invasive Capacity.
Authors: Uemura Yoshiki , Kobayashi Makoto , Nakata Hideshi , Kubota Tetsuya , Taguchi Hirokuni
Published: October 2019

In advanced clinical stage, many cases of lung cancer produce colony-stimulating factors (CSFs), including granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage-colony stimulating factor (GM-CSF), and macrophage-colony stimulating factor (M-CSF). The biological properties of the overproduction of CSFs by tumor cells are not well understood. We previously found that CSFs produced by two lung cancer cell lines, OKa-C-1 and MI-4, which constitutively produce abundant amounts of G-CSF, GM-CSF, and M-CSF, stimulate the tumor’s own growth through an autoctine mechanism. In this study, we examined whether CSFs produced by tumor cells contribute to the invasion of the tumor itself.

Invasive behaviors of the cancer cells were stimulated by exogenous CSFs but were suppressed by neutralizing antibodies against the CSFs. Matrix-degrading proteinases also are essential for successful tumor cell metastasis. Gelatin zymographs of conditioned media revealed two major bands of gelatinase activity at 68 and 92 kDa. The enzyme activities at 68 and 92 kDa were significantly enhanced in the presence of each CSF in the cell lines but were suppressed by the antibodies against the CSFs. The two gelatinase bands were characterized as matrix metalloproteinase (MMP)-2 and MMP-9, respectively. Our findings suggest that CSFs produced by tumor cells might be closely associated with the tumor invasion through gelatinase activation in CSF-producing lung cancer cells. In addition, an increase of invasion capacity and gelatinase activity by the CSFs might be mediated through the p44/42 mitogen-activated protein kinase (MAPK) signaling pathway.

Figure Figure
Expression of BCR-ABL Does Not Inhibit Apoptosis In Vitro, on a B Lymphoblastoid Cell Line.
Authors: Castro Fabiola A. , Brumatti Gabriela , Amarante-Mendes Gustavo P.
Published: October 2019

The bcr-abl oncogene is generated by the Philadelphia chromosome (Ph) translocation, fusing the BCR gene to the ABL gene and occurs mainly in two different forms. In chronic myelogenous leukemia (CML) a 210kDa Bcr-Abl protein is associated with proliferation and accumulation of myeloid cells and their precursors, whereas a 185kDa form is responsible for the pathogenesis of acute lymphocytic leukemia (ALL). Bcr-Abl not only induces cellular transformation but also regulates cell proliferation and confers resistance to a variety of apoptosis-inducing agents. Much attention has been focused on the development of novel therapies based on mechanistic understanding of Bcr-abl signaling in CML cells. However, we still do not fully understand how this molecule interferes with apoptotic machinery. The aim of this study was to investigate which step of the apoptotic machinery was most influenced by ectopic expression of Bcr-Abl myeloblastic (HL-60) and B lymphoblastic (SKW6.4) cell lines.

Ectopic expression of Bcr-Abl conferred resistance to apoptosis induced by staurosporine and anti-Fas in HL-60 cells, but not in SKW6.4 cells, although increased phosphotyrosine containing proteins could be easily observed in SKW6.4.Bcr-Abl cells. The protein levels of Bcl-xL, Mcl-1 and Flip detected by western-blot were greater in HL-60.Bcr.Abl, when compared to wild type HL-60 cells. In contrast, the proapoptotic protein Bid was considerably reduced in HL-60. Bcr-abl cells. Two independent lines of SKW6.4.Bcr-Abl cells had a small reduction of in Bid levels, but no difference was observed in the expression of the anti-apoptotic proteins Bcl-xL, Mcl-1 and Flip. At the mRNA level SKW6.4 and SKW.Bcr-Abl presented similar expression of Bcl-xL, Mcl-1 and Flip. In addition a greater expression of anti-apoptotitc proteins A1 (IDV A1/actin=0.6) and Bcl-w (IDV Bcl-w/actin=1.6) was found in SKW6.4.Bcr-Abl compare to SKW (0.2 and 0.01, respectively) wild type. Interestingly, the upregulated levels of these anti-apoptotic genes were not sufficient to prevent apoptosis in these cells. Taken together these results suggest that Bcr-abl is not capable of protecting SKW6.4 cells from apoptosis induced by staurosporine and anti-Fas, perhaps because it interacts differently with the apoptotic machinery in B lymphoblasts compared to myeloblast cell lines. Further stydies are underway in our laboratory in order to better understand the Bcr-abl-mediated anti-apoptotic signaling in B lymphoblast cells.
Differential Regulation of Pro- and Anti-Apoptotic Genes by Bcr-Abl in an In Vitro Experimental Model of Chronic Myelogenous Leukemia.
Authors: Ulbrich Axel Gustavo , Bueno-da-Silva Ana Elisa B. , Amarante-Mendes Gustavo P.
Published: October 2019

Leukemic cells from patients with Philadelphia chromosome-positive chronic myelogenous leukemia (CML) are very resistant to apoptosis induced by DNA-damaging agents and other chemotherapeutic drugs, due to the presence of Bcr-Abl, a chimeric cytoplasmic tyrosine-kinase that confers both malignancy and resistance to apoptosis. Efficient treatment of CML can be achieved with a normal bone marrow transplant, which induces a graft-versus-leukemia response, and more recently by the use of the specific inhibitor imatinib mesylate (glivec. Novartis). Glivec blocks Bcr-Abl kinase activity and, as a consequence, the malignant cell dies by apoptosis. However most glivec-treated patients, mainly in the acute and blast phases, develop resistant forms of the disease. Since resistance to apoptosis in Bcr-Abl+ cells is probably related to the inhibition of mitochondrial release of cytochrome c, an obligatory step in most apoptotic pathways, we sought to investigate expression of Bcl-2 family genes in Bcr-Abl+, glivec-treated cells. By semi-quantitative RT-PCR we analyzed the gene expression of several pro- and anti-apoptotic molecules in the transduced cell line HL-60.Bcr-Abl and the wild-type HL-60, after a 1, 4 and 8h treatment with 10μM glivec. Bcr-Abl′s kinase activity is promptly inhibited by glivec (within 5 to 15min) and HL-60.Bcr-Abl cells begin to show mitochondrial depolarization 24h after treatment with the drug, dying 48h later, whereas no effects are observed in HL-60. Soon after glivec addition some genes are transcriptionally regulated in HL-60.Bcr-Abl cells. The major differences were observed for bcl-xL (2-fold reduction), c-flip (2-fold increase), bcl-w (30% increase) and mcl-1 (20% reduction). Some pro-apoptotic molecules such as noxa also displayed differential regulation in HL-60.Bcr-Abl cells. No differences were observed in HL-60 cells. In conclusion we describe a complex transcriptional regulation mechanism dependent on Bcr-Abl tyrosine-kinase activity, which has not been previously described by the use of microarrays, and could contribute to the understanding of the mechanisms involved in protection of apoptosis and drug resistance of Bcr-Abl+ cells.
Human Fetal Liver Derived Stem Cells Can Be Support the Maintenance of Human Embryonic Stem Cells.
Authors: Baek Jin-Young , Rhee Yun-Hee , Cha Kwang-Yul , Chung Hyung-Min
Published: October 2019

Prolonged propagation of human embryonic stem (ES) cells is currently achieved by co-culture with primary or immortalized mouse embryonic fibroblast (MEF) cells. In order to replace the heterologous with homologous co-culture systems, an attempt was made using mononuclear cells derived from human fetal liver. Human fetal liver-derived mesenchymal-like stem cells (FL-MLSC) can be maintained for the prolonged period of time. They showed the characteristics of mesenchymal stem cells in various aspects. They retained a normal diploid karyotype and growth characteristics over the successive culture. Human ES cells cultured on human FL-MLSC cells up to 8 passages displayed the unique morphology and molecular markers characteristic for undifferentiated human ES cells as cultured on MEF cells. Alkaline phosphatase activity was detected in human ES cells co-cultured on human FL-MLSC. Immunocytochemical analyses showed that expressions of stage-specific embryonic antigen-3, -4 and Oct-4 were not altered on human ES cells cultured on human FLDSC. Reverse-transcriptase PCR analyses showed that similar expressions of Oct-4 and Nanog genes, markers for undifferentiated ES cells, were also observed in human ES cells cultured on both human FL-MLSC and MEF cells. Furthermore, human ES cells cultured on human FL-MLSC retained unique differentiation potentials in culture when allowed to form embryoid body. Results of this study suggest that human FL-MLSC can support the maintenance of human ES cell in vitro.
HTLV-1 Enhances Epstein-Barr Virus (EBV) Infectivity and EBV Infection Attributes to an Aggressive Organ Involvement in Adult T-Cell Leukemia.
Authors: Ueda Satomi , Maeda Yasuhiro , Yamaguchi Terufumi , Hijikata Yasuki , Kanamaru Akihisa
Published: October 2019

The frequent involvement of adult T-cell leukemia (ATL) cells in organs such as the skin and lymph nodes is observed in ATL. Epstein-Barr virus (EBV) infection has been observed in the clinical course of ATL, and ATL cells in all skin cases were strongly positive for EBV nuclear antigen (EBNA)-1 and latent membrane protein (LMP)-1 by immunohistochemical staining. Interestingly, ATL cells in all skin cases were positive for CD45R0 (UCHL1) and CD20 (L26), indicating that they have both T and B cells characterization. We suggested that HTLV-1 enhances the entry of EBV into endothelial cells, epithelial cells and EBV presents for an active role of HTLV-1 in the development of ATL. Here, we established two B-cell lines from peripheral blood of patients with ATL. EBV DNA, proviral DNA for HTLV-1 and Tax mRNA were detected in both B cell lines. On characterization of the cell lines, enhanced expression of intercellular adhesion molecule-1 or-3 (CD54, CD50), lymphocyte function-associated antigen-1 (CD11a/CD18) and Mac-1 (CD11b/CD18) was observed compared to an EBV-infected B cell line, Raji. Moreover, EBV oncogenes such as EBV-encoded small RNA (EBER)-1, EBNA-1,-2 and LMP-2A were also detected. To investigate the role of the interaction of these viruses, we transfected EBV and/or HTLV-1 into a healthy donor’s lymphocytes, Raji cells and a HTLV-1 negative T-cell line, Jurkat. Enhanced expression of adhesion molecules was observed in double transfectants (EBV and HTLV-1). Furthermore, we detected high production levels of interleukin-4 (IL-4) in those B-cell lines and transfectants. These results indicated co-expression of HTLV-1 and EBV may induce aggressive organ involvement by enhanced expression of adhesion molecules via IL-4 signaling. Furthermore, HTLV-1 could greatly enhance EBV infectivity and attribute to increased replication of EBV. EBV also could be a necessary cofactor for ATL development in immunodeficiency states. ATL patients who have been coinfected with EBV and HTLV-1 have a high risk of worse progress of disease. ATL patients have a poor treatment outcome, even with strong chemotherapy, especially who have severe skin involvements have a resistance for chemotherapy. Viewed from a different angle, our observations suggest that EBV infection may be used as one of bad prognostic factors and an identification of bad-risk patients who need to benefit from early high dose chemotherapy. Collectively, a reactivation of EBV infection and EBV oncogenes can provide an acceleration of organ involvement in ATL.These results can explain why EBV-associated ATL is more frequent and clinically more aggressive.
Berbamine Induces Apoptosis and Lowers Expression Levels of Bcr/Abl and Survivin Gene and Bcr/Abl-Related P210 in Different Types of Leukemic Cell Lines Cells.
Authors: Lin Maofang , Wu Dong
Published: October 2019

Berbamine is a kind of bis-benzylisoquinoline extracted from Chinese herb. Berbamine and its derivatives have been proven to be a kind of calmodulin antagonist which could obviously inhibit the proliferation of different kinds of cell lines, such as hepatoma, Hela cells etc. Berbamine has been shown to induce apoptosis of HL-60 cells in vitro in a cell type-specific manner. However, main components of Berbamine induced apoptosis pathway remain to be identified. We studied Berbamine induced apoptosis of different types of leukemic cell lines cells and its possible molecular mechanisms in vitro. The results revealed that Berbamine could significantly induce apoptosis in K562, NB4 and Jurkat leukemia cell lines cells in a time- and dosage-dependent manner. With electron microscope and DNA electrophoresis, the typical apoptosis morphologic changes and DNA ladder were clearly observed. As detected by flow cytometry, the percentage of the apoptotic cells in K562, Jurkat and NB4 cells treated with 8.0μg.L−1 Berbamine increased to 49.07±4.73%, 35.57±5.49%, 54.40±4.56% (P value<0.01), respectively. Using semi-quantity RT-PCR assay, the expression level of survivin gene was lower. in K562 cells treated with 8.0μg.L−1 Berbamine than that in untreated cells (0.69±0.02 vs 1.09±0.01, P<0.01),in a time-dependent manner; as well as the expression level of survivin gene was lower. in Jurkat cells treated with 16.0μg.L−1 Berbamine than that in untreated cells (1.34±0.02 vs 2.18±0.03, P<0.01). Similarly, bcr/abl expression was weaker in K562 cells treated with 8.0μg.L−1 Berbamine than that in untreated cells (0.91±0.02 vs 1.19±0.02, P<0.01), while no difference was found between Ara-C treated K562 cells and untreated treated cells. In the other hand, Using Western Blot assay, the level of bcr/abl-related P210 was lower in K562 cells treated with 8μg.L−1 Berbamine than that in untreated cells (0.63±0.01 vs 1.04±0.02, P<0.01). AS a comparison, the level of P210 was also lower in same cells treated with 1.0μg.L−1 STI571 than that in untreated cells (0.58±0.02 vs 0.93±0.03, P value P<0.01).

In summary, Berbamine extracted from Chinese herb can obviously induce apoptosis in different kinds of leukemic cell lines including K562, Jurkat, NB4 in a time-and dosage-dependent manner in vitro. The down-regulation expressions of survivin gene, bcr/abl gene and bcr/abl -related P210 may play an important role in the apoptotic effect of Berbamine in leukemic cells.
Arsenic Trioxide Induces Apoptosis in Molt-4 Cell Lines: Caspase 8-Dependent and Caspase 3-Independent.
Authors: Qian Wenbin , Ni Wanmao , Liu Junqing
Published: October 2019

Arsenic Trioxide (As2O3) has been used successfully in the treatment of patients with relapsed or refractory acute promyelocytic leukemia (APL) without severe marrow suppression. Currently, the action of As2O3 on many other hematopoietic malignancies is under investigation. Much evidence has shown that caspase-3 plays essential executing role in apoptosis of many leukemia cell lines. But, the exact mechanism of As2O3-induced apoptosis in Molt-4 cell line which is originated from acute lymphoblastic leukemia is not well understood. Here, we investigate the action of As2O3 on Molt-4 cells and involved mechanism. Significant dose- and time-dependent inhibition of cell growth was observed by MTT assay. Following the treatment of As2O3 for 72 h, As2O3 at 4 μM exhibited 50% inhibition of growth in Molt-4 cells. The effect of As2O3 on the cell cycle was determined in Molt-4 cells by FACS analysis. DNA flow cytometric analysis with three independent experiments indicated that As2O3 induced a G1 and a G2-M phase arrest in Molt-4 cells following 6μM of exposure. Similar results were observed in Molt-4 cells following 2μM and 4μM exposure. These results indicated that As2O3 inhibited the cellular proliferation of Molt-4 cells via a G1 and a G2-M phase arrest of the cell cycle. To confirm and evaluate the induction of apoptosis, we performed the staining of cells with annexin V and PI. As with the percentages of sub-G1 group by FACS analysis, the proportion of apoptotic cells was increased in a dose-and -time dependent manner. Taken together, these results indicate that induction of apoptosis can be another mechanism of the antiproliferative effect of As2O3 besides G1 and G2-M phase arrests of the cell cycle in Molt-4 cells. We subsequently studied the activation of initiator caspase-8 and executioner caspase-3 in Molt-4 cells by Western blotting. Molt-4 cells that had undergone apoptosis on culturing with As2O3 displayed the initial activation of caspase-8 with the appearance of the large cleavage fragment of 43 to 41 kd. Despite the higher basal level of procaspase-3 expression in the Molt-4 cells prior to As2O3 treatment, we were unable to detect cleaved, activated caspase-3 following As2O3 treatment. Next, we checked whether inhibition of caspases-3 could abrogate the proapoptotic effects of As2O3. For this purpose the caspase-3 inhibitor, z-DEVD-fmk, was used. The results shown that addition of z-DEVD-fmk did not rescue Molt-4 cells from apoptosis induced by As2O3. These results clearly differ from other observations made with other leukemia cells and might explain, at least in part, that As2O3 induces apoptosis in Molt-4 cells is caspase 8-Dependent and caspase 3-Independent.
Telomeres and Telomerase in Pediatric Patients with T-Cell Acute Lymphoblastic Leukemia (T-ALL).
Authors: Greil Johann , Kleideiter Elke , Schwab Matthias , Boukamp Petra , Koscielniak Ewa , Bangerter Ute , Klotz Ulrich , Niethammer Dietrich
Published: October 2019

Shortened telomeres and elevated levels of telomerase activity are apparently characteristic features of hematologic neoplasias such as high-grade lymphomas and relapsing leukemia. Thus, their measurement might be useful for monitoring disease conditions or predicting clinical outcome. In order to investigate the potential of telomere length (TRF) and telomerase activity (TA) as prognostic indicator in pediatric patients with T-cell acute lymphoblastic leukemia (T-ALL) we analyzed TRF and TA in samples from 20 patients (age range 2–17.5 years). In addition, as TA is limited by the expression of the telomerase catalytic subunit (hTERT) we analyzed hTERT expression. We found that TRF varied widely (3.5 – 8.1 kb; mean ± SD: 6.4 +/− 1.3 kb) in leukemic cells and was significantly shorter (p<0.0001) than that of age-matched controls (8.3 ± 0.4 kb; n=19). Elevated levels of TA were present in 95% of the leukemic samples. Furthermore, expression of hTERT demonstrated a wide interindividual variability (range 141–424,000 normalized units). A statistically significant association between TA and hTERT expression was not found and TRF, TA and hTERT expression was not associated with the clinical outcome in pediatric T-ALL, thereby limiting their prognostic significance.
Incidence of ZAP70 Positivity in Fresh Samples from Unselected Patients Presenting with a B-Cell Lymphoproliferative Disorder.
Authors: Petrie James A. , Milne Tim , Ebrahim Fatema , Cavenagh Jamie D. , Agrawal Samir G.
Published: October 2019

The expression of ZAP70 has been shown to strongly correlate with unmutated immunoglobulin VH in chronic lymphocytic leukaemia (CLL) cells, which in turn has been shown to distinguish a CLL subgroup of poor prognosis. Retrospective studies have linked ZAP70 expression to a more aggressive clinical course and poorer overall survival in CLL. Previous studies on ZAP70 have largely utilised frozen material. We wanted to avoid the biases of using stored material, potential artefacts of the freeze/thawing process and expand our study beyond CLL. Hence, we analysed ZAP70 by flow cytometry in 184 consecutive cases of B-cell lymphoproliferative disorder (LPD). Only fresh peripheral blood (PB) samples were used. A combination of ZAP 70 (Upstate Biotechnology), CD3 PE and CD56 PE (Coulter Immunotech) antibodies (Ab) were used to exclude the contaminating effects of T and NK cells. CD3 and CD56 staining was followed by fixation and permeabilisation (Dako Intracellular Antigen Kit) and addition of anti-ZAP70 Ab. Indirect labelling of ZAP70 was performed using a secondary conjugate IgG(H+L)-FITC (Southern Biotechnology). Positivity was established at the 20% threshold. 143 cases of CLL were examined for ZAP70 expression: 47 (33%) were positive, with the level of positivity ranging from 20 to 91% (median 41%). Three cases initially ascribed as negative, were subsequently found to be positive upon retesting at a later date. The ZAP70 negative cases of CLL had a median value of 1% (range, 0 – 14%). 41 non CLL cases were examined for ZAP70 expression, mantle cell lymphoma (MCL, 16 cases), hairy cell leukaemia (HCL, 3), diffuse large B-cell lymphoma (2) and 20 cases of B-cell LPD that could not be categorised further. Of these non CLL cases, only 4 were positive for ZAP 70 expression and all of these were cases of MCL (24 – 39% positivity.) The ZAP70 negative cases of MCL had a median value of 0% (range 0–17%). No other non CLL B-cell LPD showed levels of expression >10%. This study of flow cytometric analysis of ZAP70 expression in fresh, presentation PB of all LPDs referred to our laboratory revealed that 33% of CLL and 25% of MCL cases were positive. ZAP70 expression in CLL is bimodal (clearly positive or negative using the 20% threshold) with very few cases in the 10–19% range. No other B-cell LPDs were positive for ZAP70. As ZAP70 testing is now part of our routine LPD panel, ZAP70 positivity provides both diagnostic and prognostic information in CLL, helping to distinguish difficult CLL cases from non CLL (other than MCL). Our 3 cases of changing status may reflect improvements in our protocol or may indicate that ZAP70 status is variable over the course of the disease.
Acute Biphenotypic Leukemia: Single Center Experience of Laboratory Presentation and Review of the Diagnostic Criteria.
Authors: Owaidah Tarek M. , Behani Amal Al , Iqbal Mohammed Anwar , El Kum Nasser , Roberts George T.
Published: October 2019

Biphenotypic Acute Leukemia (BAL) is a rare, newly defined disease in the WHO classification of hematological neoplasms. There is little information about the laboratory presentation, and considerable difficulties exist in reaching a definite diagnosis. In this study, designed to examine the laboratory presentation of BAL, we report a total of 23 (3.4%) out of 676 adult and pediatric leukemia cases referred to our center during the last 4 years.

The mean age at presentation was (21.57 ± 15.59 years; range 1 year – 66 years). There were 14 males. The mean hematological and biochemical parameters were as follows: WBC 68.76 ± 65.18 x 10 9/L, Hemoglobin 94.13 ± 15.13 g/L, Platelets 105.7 ± 104.23 x 10 9/L, and LDH 1351.44 ± 1118.29 u/L. Fourteen patients (60.8%) had mixed morphology of small and large blast cells and seven were M1 and M2 according to FAB criteria.

Using the European Group for Immunological Classification of Leukemia (EGIL) scoring system, all patients had positive Myeloid markers with EGIL scores that ranged from 2.0 – 5.5. Eighteen patients were positive for B-cell markers with scores ranging from 2.0 – 8.5 but only 8 patients were positive for T-cell markers with scores of 0.5 – 4.0. Three patients were positive for markers of the three cell lines. In 7 patients myeloid lineage commitment was also demonstrable by ultrastructural myeloperoxidase. Five patients (22%) had normal cytogenetics. Philadelphia chromosome was positive by karyotype in 3 patients (13%) and MLL gene in 2 (8.7%). Three patients (13%) demonstrated trisomies involving chromosomes 19, 22 and 4, respectively.

Out of the 23 patients, 11 (48%) are alive without disease, 4 had died with leukemia, 3 are alive with disease and 5 had lost follow up.