bluuebunny/crossref_metadata_2025 huggingface dataset:
The Occurrence of Antibodies to Components of the Natural Coagulation Inhibitor System, Protein Z and Protheombin in Normal Pregnancy.
Authors: Ku De-Hui W. , Arkel Yale S. , Paidas Michael J. , Triche Elizabeth , Huang Shannon , Musto Jeffrey , Lockwood Charles J.
Published: October 2019

There are recent reports that indicate that antibodies to components of the natural coagulation inhibitor system and coagulation factors, such as FII, are associated with increased risk for thrombosis and adverse pregnancy outcomes (APO) in patients with autoimmune disease and the anticardiolipin syndrome. In view of the importance of the natural inhibitor system in regulating the prothrombotic process and particularly in the hypercoagulable milieu of pregnancy, we studied the occurrence of several of these antibodies in patients characterized with normal pregnancies (NP). We used 82 1st trimester (TRI), 64 2nd TRI, 78 3rd TRI, and 50 non-pregnant controls for this study. Our data are demonstrated in the tables below. The salient features; the PZ and PS IgM antibodies are greater than the non-pregnant controls throughout gestation but the PZ and PS IgG antibodies are greater than non-pregnant controls in the later phases of pregnancy. IgG and IgM antibodies to FII and PC are increased compared to non-pregnant controls throughout gestation. When the antibody levels are compared in NP in each trimester; PZ IgM increases in the 3rd trimester, PS IgG increase in each trimester and PS IgM in the first two trimesters. FII IgM increases in each trimester and IgG does not increase with gestation. PC IgM increases with each trimester and PC IgG remains unchanged. The prevalence of progressive increases in the antibody response is mainly IgM. This would tend to indicate more acute than chronic response and stimulation. These findings indicate that in NP an immune response is present in most pregnancies that can affect the hemostatic balance. Yet to be determined are the tipping points for pathological consequences.

Table 1

1st trimester (OD) 2nd trimester (OD) 3rd trimester (OD) CTR (OD) CTR=non-pregnant control Anti-PZ IgG 0.26+/−0.42 0.21 +/−0.31 0.30+/−0.29 0.19+/−0.14 Anti-PZ IgM 0.36+/−0.22 0.21+/−0.11 0.39+/−0.20 0.07+/−0.17 Anti-PS IgG 0.29+/−0.35 0.45+/−0.25 0.44+/−0.31 0.27+/−0.20 Anti-PS IgM 0.48+/−0.38 0.66+/−0.42 0.61+/−0.36 0.26+/−0.15 Anti-FII IgG 0.92+/−0.53 0.86+/−0.57 0.98+/−0.65 0.44+/−0.41 Anti-FII IgM 0.92+/−0.51 0.612+/−0.42 0.77+/−0.44 0.26+/−0.31 Anti-PC IgG 0.96+/−0.61 0.99+/−0.78 0.91+/−0.57 0.51+/−0.32 Anti-PC IgM 1.01+/−0.43 0.90+/−0.29 0.87+/−0.41 0.54+/−0.27

Table 2

1st vs 2nd 1st vs 3rd 2nd vs 3rd CTR vs 1st CTR vs 2nd CTR vs 3rd NS=non significant Anti-PZ IgG NS NS NS NS NS p<0.05 Anti-PZ IgM NS NS p<0.05 p<0.05 p<0.05 p<0.05 Anti-PS IgG P<0.05 P<0.05 NS NS p<0.05 p<0.05 Anti-PS IgM p<0.05 p<0.05 NS p<0.05 p<0.05 p<0.05 Anti-FII IgG NS NS NS p<0.05 p<0.05 p<0.05 Anti-FII IgM p<0.05 p<0.05 p<0.05 p<0.05 p<0.05 p<0.05 Anti-PC IgG NS NS NS p<0.05 p<0.05 p<0.05 Anti-PC IgM p<0.05 p<0.05 NS p<0.05 p<0.05 p<0.05
Acquired Hemophilia: Characteristics and Treatment Outcome of a Standardized Therapeutic Approach in a Singapore Series.
Authors: Ng Heng J. , Tan Daryl C.L. , Lee Lai H.
Published: October 2019

Introduction: Acquired hemophilia is a rare bleeding disorder with significant mortality risk but is yet highly treatable. Patients diagnosed with this condition in our institution have been treated with a standardized approach aimed at detecting and controlling bleeding early and inhibitor elimination.

Objective: Review the effectiveness and treatment outcome of our therapeutic approach to acquired hemophilia patients.

Methods: Consecutive patients diagnosed with acquired hemophilia over a period of 5 years from July 1998 till July 2004 were retrospectively reviewed for their underlying characteristics, responses to treatment, complications and subsequent outcome.

Results: 13 patients were recorded with age ranging from 17 to 89 years (median 66 years) and a male to female ratio of 10:3. Follow-up periods range from 5 to 56 months. All patients presented with variable degree of bleeding. Four patients were given recombinant factor VIIa (from 1 to 5 doses) for the control of life threatening bleeding. Three patients received prothrombin complexes as bypassing agents. One other patient received both agents..All patients received both cyclophosphamide (50–100mg/day) and prednisolone (1 mg/kg/day) for elimination of inhibitors at diagnosis. Six patients received intravenous immunoglobulin of 1 gm/kg/day for 2 days with this decision based on the severity of bleeding. Two early deaths were recorded from retroperitoneal bleeding and cerebral infarct following recombinant factor VIIa therapy, respectively. Another death occurred following sepsis with multiple co-morbidities, while being given immunosuppressive therapy. He was the only patient experiencing significant leucopenia while on cyclophosphamide therapy. Two patients were lost to follow-up subsequently. Of 8 patients evaluable for response to inhibitor eliminating therapy with cyclophosphamide and prednisolone, all but one achieved a complete remission within 7 to 71 days with a median of 41 days. Of these 7 patients, there was one early relapse, which occurred from non-compliance to treatment while it was being tailed down. Remission was successfully reinduced on reintroduction of both cyclophophamide and prednisolone. All other patients have remained in remission after discontinuation of therapy. The patient with failure of complete response had persistent low inhibitor levels without bleeding complications and died after 56 months of follow up from unrelated causes. Overall mortality of evaluable patients was 27% (3 out of 11).

Conclusion: Mortality remains significant in this condition with control of early bleeding risks and treatment complications paramount to survival. For the great majority of patients, our strategy affords effective inhibitor elimination and sustained remission of this condition
Monosomy 7 and Deletion 7q in Childhood AML. A Collaborative Study of 20 Study Groups.
Authors: Hasle Henrik , Alonzo Todd , Avrignon Anne , Behar Cathrine , Chang Myron , Creutzig Ursula , Fischer Alexandra , Forestier Erik , Fynn Alicira , Harbott Jochen , Harrison Christine J. , van den Heuvel-Eibrink Marry , Kaspers Gertjan , Locatelli Franco , Nöllke Peter , Polychronopoulou Sophia , Ravindranath Yaddanapudi , Razzouk Bassem , Reinhardt Dirk , Savva Natalia N. , Stark Batia , Suciu Stefan , Trebo Monika , Tsukimoto Ichiro , Webb David , Wojcik Dorota , Woods William G. , Zimmermann Martin , Niemeyer Charlotte M. , Raimondi Susana
Published: October 2019

Monosomy 7 (−7) and deletion 7q [del(7q)] are rare in childhood AML and are considered to be associated with a poor outcome. We retrospectively analyzed data on 267 children with de novo AML or RAEB-T (PB or BM blasts > 20%) with −7 or del(7q) with or without additional cytogenetic aberrations (other). Patients were diagnosed between 1985 and 2003. Karyotypes showed −7 (n=90), −7 other (n=82), del(7q) (n=22), and del(7q) other (n=73). All 24 patients with RAEB-T had −7 +/− other. The median age at diagnosis was 7.6 years (range; 0–18.1) with no difference in age distribution between cytogenetic subgroups. The most common additional cytogenetic aberrations were inv(3)/t(3;3) in 19 patients (−7, n=17; del(7q), n=2) and favorable aberrations [t(8;21), inv(16), and t(15;17)] observed in 25 patients (−7, n=3; del(7q), n=22). The 5-year overall probability of survival for the whole group was 39%, SE=3%, and survival was higher in patients with del(7q) +/− other compared with −7 +/− other (52% vs. 30%). The survival of patients with del(7q) and favorable cytogenetic aberrations (n=22) was higher than that of other patients with del(7q) (75% vs. 46%, p=0.026). There was no significant difference in survival for patients with −7 and −7 other. Complete remission (CR) was obtained in 188 patients (−7 +/− other 62%; del(7q) +/− other 88%), of these, 67 received hematopoietic stem cell transplantation (HSCT) in CR1. There was no significant difference (Mantel-Byar test) in survival for patients who received HSCT in CR1 (49%, SE= 7%) and those who received chemotherapy only (51%, SE=5%, 54% for patients surviving at least the median time to HSCT). In conclusion childhood AML subgroups −7 and del(7q) differ in their associated cytogenetic aberrations and response to therapy.
Comparison of Phenotypic Assessment and Mutation Detection in the Diagnosis of Carrier State in Hemophilia: Identification of 10 Novel Mutations.
Authors: Chuansumrit Ampaiwan , Sasanakul Werasak , Williams Ian , Goodeve Anne , Kadegasem Praguywan , Peake Ian
Published: October 2019

The carrier state in 54 females (A38, B16) at risk from 35 moderate and severe hemophilia families (A25, B10) in Thailand, was determined. They were classified as obligate (A17, B8) and possible (A21, B8) carriers by history taking. The phenotypic assessment was performed in two subsequent blood samples taken one week apart when they were not pregnant or on birth control pills. Then, molecular genetics among hemophiliac patients were performed. Inversion of intron 22 among 25 hemophilia A patients was initially performed. Then, the mutations were intensively detected by using conformation sensitive gel electrophoresis (CSGE). Coding region, intron/exon boun-daries, and 5′ and 3′ regions of the factor VIII and IX genes were amplified in 33 separate reactions in hemophilia A patients without inversion of intron 22 (n=10) and 9 separate reactions in hemophilia B patients (n=10). Samples displaying abnormal CSGE profiles were numbered according to Wood et al and Yoshitake et al. The results revealed that mutation were found at 88% (22/25, 15 inversion of intron 22 and 7 mutations) in hemophilia A and 90% (9/10) in hemophilia B patients. Ten were novel mutations as shown in Table 1. Also, the carrier state assessed by the phenotypic study and mutation detections are shown in Table 2. As a result, the phenotypic assessment alone showed a limitation in the diagnosis of carrier state especially hemophilia B carrier while the mutation detections provide an absolute diagnosis over phenotypic assessment.

Table 1. The identified ten novel mutations among Thai hemophilia A and B patients.

Table 2. Carrier state assessed by phenotypic study and mutation detections among females at risk.

Exon Nucleotide Amino acid FVIII:C/FIX:C (%) Promotor to exon 22 large deletion - FVIII:C < 1 6 680 G>A W208X FVIII: C = 2 8 1264 G>C D403H FVIII:C < 1 12 1820_1821 del CA 588 frame shift FVIII:C < 1 13 2066 T>G L670R FVIII:C = 1 14 4794 A>T E1579D FVIII:C = 1 21 6266 G>A W2070X FVIII:C < 1 A 57_58 del GA −37 frame shift FIX:C < 1 H 31127 T>G C336G FIX:C < 1 H 31171 T>G C350W FIX:C = 3.6 Type of carrier N FVIII:C or FIX:C < 50% FVIII:C/vWF:Ag < 0.6 Inversion intron 22 CSGE Total mutation Obligate hemophilia A 17 35.3% 70.6% 64.7% 17.6% 82.3% Obligate hemophilia B 8 12.5% - - 87.5% 87.5% Possible hemophilia A 21 19.0% 42.8% 33.3% 14.3% 47.6% Possible hemophilia B 8 25.0% - - - 62.5%
Thrombin, Fibrin Monomer and Factor XIII in Patients with Unexplained Intraoperative Bleeding.
Authors: Korte Wolfgang C. , Gabi Konrad , Gähler Anita , Schnider Thomas , Riesen Walter
Published: October 2019

To explore relevant changes in unexplained intraoperative bleeding, we evaluated elements of the final steps of the coagulation cascade in 226 consecutive patients undergoing elective surgery. Patients were stratified for the occurrence of unexplained intraoperative bleeding according to predefined criteria.

Twenty patients (8.8%) developed unexplained bleeding. Median intraoperative blood loss was 1350 ml (bleeders) and 400 ml (non-bleeders), p<0.001. Fibrinogen and factor XIII were more rapidly consumed in bleeders (p<0.001). Soluble fibrin formation (fibrin monomer) was elevated in bleeders throughout surgery (p < 0.014, table 1). However, F. XIII availability per unit thrombin generated was significantly decreased in bleeders pre-, intra- and postoperatively (p < 0.051). Computerized thrombelastography showed a parallel, significant reduction in clot firmness.

We suggest that a mild, pre-existing coagulopathy is not rare in surgical patients and probably can result in clinically relevant intraoperative bleeding. This haemostatic disorder shows impaired clot firmness, probably secondary to decreased cross-linking (due to a loss of F. XIII, both in absolute measures and per unit thrombin generated).

We are currently conducting a double blind, randomized trial on the use of F. XIII early during surgery. We suggest that the preoperative measurement of fibrin monomer concentration might allow preoperative risk stratification for intraoperative blood loss.

Fibrin Monomer is increased in “bleeders”

Non-Bleeder (FM median μg/l) Bleeder (FM median μg/l) p (Rank Sum test) T1 (preop) 7 18 <0.01 T2 (intraop) 6 14 <0.01 T3 (intraop) 9 20 0.014 T4 (postop) 26 52 <0.01 T5 (postop) 29 86 <0.01
Cost of Major Bleeding Following Major Orthopedic Surgery.
Authors: Oster Gerry , Vera-Llonch Montserrat , Hagiwara May
Published: October 2019

Rationale . The risk of major bleeding in major orthopedic surgery (MOS) patients who receive antithrombotics as prophylaxis against venous thromboembolism (VTE) may be higher for some agents than others. The cost of this complication is unknown.

Objective . To assess the impact of major bleeding following MOS on length of stay (LOS) and inpatient charges.

Methods . Using a database with information on ~750,000 admissions annually to 100+ US acute-care hospitals (MQProfile, Cardinal Information Corp.), we identified all patients who underwent MOS between 1/1/98 and 12/13/00. We stratified patients according to whether or not they experienced major postoperative bleeding prior to hospital discharge, defined as: (a) fatal bleeding; (b) nonfatal bleeding at critical site; (c) re-operation due to bleeding; and (d) overt bleeding with bleeding index (BI) ≥ 2, where BI = number of blood units transfused plus pre-bleeding minus post-bleeding hemoglobin (g/dL) values. LOS and total inpatient charges were compared between patients with and without major bleeding.

Results . 23,518 patients were identified who underwent MOS. Incidence of major bleeding was 2.6%. In multivariate analyses controlling for differences in baseline characteristics between patients with and without major bleeds, adjusted mean LOS was 1.8 days longer (95% CI: 1.5, 2.0) among the former (6.1 days vs 4.3 days for those without bleeds); adjusted mean total inpatient charges were $7,593 higher (95% CI: $6,622, $8,646) ($25,669 vs $18,076).

Conclusion . Major bleeding following MOS significantly increases LOS and hospital charges. The cost of major bleeding following MOS should factor into choice of VTE prophylaxis.
Clopidogrel Resistance and Aspirin Resistance - a Cause of Coronary Artery Stent Thrombosis?.
Authors: Klamroth Robert , Hoffmann Stefan , Andresen Dietrich , Landgraf Helmut
Published: October 2019

Background: Occlusions of coronary artery stents are a seldom but often fatal complication after coronary artery intervention. With aggressive platelet inhibition therapy (combination of Aspirin (ASS) and clopidogrel) a reduction of stent thrombosis to less than 2% was achieved. The inhibition of platelet function has great interindividual variability and resistance against ASS and/or clopidogrel were previously described. Therefore we studied the relation between coronary artery stent thrombosis and insufficient inhibition of platelet function in patients after coronary artery intervention.

Methods: Between 2000 and 2003 we examined 20 patients (pts) with coronary artery stent thrombosis within 4 weeks after coronary artery intervention. All patients received a clopidogrel loading dose of 300mg followed by 75mg/die and ASS 100mg/die. Platelet inhibition was studied by light transmittance aggregometry in platelet rich plasma (Aggregometer PAP 4, moelab inc.). Aggregation was recorded as the maximum percentage change in light transmittance from baseline using platelet poor plasma as a reference. We defined ASS resistance as platelet aggregation > 30% after induction with 0,5mg/ml arachidonic acid and clopidogrel resistance as platelet aggregation > 30% after induction with 20 μmol ADP. We compared these pts with 20 pts without stent thrombosis within 4 weeks after coronary artery intervention.

Results: In the group with coronary artery stent thrombosis we found 13/20 patients with insufficient platelet inhibition. 2/20 pts in this group were resistent against both ASS and clopidogrel. In 2/20 pts we observed ASS resistance and in 9/20 pts clopidgrel resistance. In the group of patients without stent thrombosis we found only 1/20 pt with ASS resistance and 1/20 pt with clopidogrel resistance.

Conclusions: Insufficient platelet inhibition could be one cause of early stent thrombosis after coronory artery intervention. Platelet function testing is able to detect insufficient platelet inhibition and early testing could be an instrument to prevent coronary artery stent thrombosis.
Down Regulation of Inflammatory Markers after Treatment with Topically Administered Mucopolysaccharide Polysulfate.
Authors: Raake Wolfram , Hoppensteadt Debra , Schultz Christopher , Neville Brian , Fareed Jawed
Published: October 2019

Mucopolysaccharide polysulfate (MPS) represents a mammalian tracheal preparation, which mimics many of the properties of heparins including its anti-inflammatory actions. MPS is used topically for the treatment of inflammatory disorders. This study was designed to test the hypothesis that topical administration of MPS mobilizes many substances to mediate its therapeutic effects. A group of four non-human primates (Macaca Mulatta) were administered 4.5% MPS ointment in a dosage of 45 mg/kg, another four monkeys were administered placebo ointment at a dosage of 1.0 g/kg for 10 days. Citrated blood samples were drawn at baseline, 2, 4, 8 and 24 hours on days 1, 2, 5, 7 and 10. Markers of inflammation including thrombin activatable fibrinolytic inhibitor (TAFI), C-reactive protein (CRP), CD-40 Ligand (CD-40L), plasminogen activator inhibitor (PAI-1) and monocyte chemotactic protein (MCP-1) utilizing ELISA based methods, were used to measure the anti-inflammatory response. In the MPS treated group, a down regulation of all of these markers was observed over the 10 day period. CRP (baseline, 10.2 ±3.6; day 10, 5.1 ± 2.4 μg/ml), CD-40L (baseline, 298 ± 52; day 10, 138 ± 38 pg/ml) and MCP-1 (baseline, 350 ±68; day 10, 178 ± 42 pg/ml) showed a 50% decrease in the levels of these markers at day 10 in comparison to baseline. The PAI-1 (baseline, 65.1 ±21.2; day 10, 48.2 ±12.1 ng/ml) and TAFI (baseline, 138 ±25; day 10, 99 ± 12 % NHP) levels showed a slightly lesser effect. These results suggest that the topical administration of MPS leads to a decrease in the markers of inflammation that may result in promotion of the healing of inflammatory lesions.
The Risk of Deep Venous Thrombosis and Sepsis in Cancer Patients Due to PICC Lines.
Authors: Lin Lee , Walker Stanley
Published: October 2019

Context

Upper extremity deep venous thrombosis (DVT) represents 8–10% of total DVT of the limb. Long term central venous catheters represent a major cause of upper extremity DVT, especially in cancer patients. This study looks specifically at the risk posed by peripherally inserted central catheters (PICC).

Purpose of the Study:

To assess the risk of DVT and sepsis due to PICC lines in cancer patients as compared to non-cancer patients. Furthermore, to assess the risk of PICC line related DVT and sepsis among different cancer types.

Design, Setting, and Patients:

Retrospective chart review of all 190 patients (representing n=244 procedures) who had undergone a PICC line procedure in Union Memorial Hospital (Baltimore, MD) from August 2003 to December 2003. Based on their medical records, it was determined whether or not the patient had experienced PICC line related sepsis or thrombosis. Cancer as a potential risk factor was included in the information collected. 33 of 190 patients were cancer patients. Average patient age 56.7 years.

Results:

Preliminary results show that from August 1, 2003 to December 5, 2003, among the non-cancer patients 6 (3.8%) patients with DVT and 6 (3.8%) patients with sepsis were identified. Among the cancer patients, 3 (9.1%) patients with DVT and 3 (9.1%) patients with sepsis were identified. Though cancer patients made up only 17.4% of the patient population, they represented 33.3% of the cases of DVT and sepsis.

Conclusion:

Cancer patients are at a greater risk of thromboses and sepsis due to PICC lines as compared to non-cancer patients. Cancer patients, therefore, represent a unique population of PICC line patients who may require a different protocol of prophylaxis and complication prevention. While different types of cancers may pose different levels of risk (Sorensen et al), this could not be determined with confidence with the preliminary findings due to the small population evaluated so far.

Non-cancer patients

Cancer patients

DVT 6 3.8% Sepsis 6 3.8% DVT 3 9.1% Sepsis 3 9.1%
MUTZ-3, a Human Cell Line Modell for Osteoclast Differenziation.
Authors: Berardi Anna C. , Parafioriti Antonina , Bottazzo Ginfranco , Ciraci Elisa
Published: October 2019

No human myeloid leukemia-derived cell lines possess the ability to acquire a osteoclstic cell phenotype. In contrast, the CD34+ human acute myeloid leukemia cell line MUTZ-3 responds to macrophage-colony-stimulating-factor (M-CSF), receptor activator NF-kB ligand (RANK-L) and tumor necrosis factor-a (TNF-a), cytokines known to be pivotal both in vivo and in vitro for osteoclasts generation from monocytes and CD34+ stem cells. In all respects, MUTZ-3 cells behave as the immortalized equivalent of CD34+ osteoclasts precursors. Upon stimulation with specific cytokine cocktails, they formed numerous multinucleated osteoclasts expressing tartrate resistent acid phosphatase (TRAP) and calcitonin receptors (CTR) within 12 days of culture. MUTZ-3-osteoclasts formed extensive lacunae mineral resorption when cultured on mineralized surface after 21 day of cultures. These findings demonstrate the unique suitability of MUTZ-3 cells as an unlimited source of human CD34+ osteoclasts progenitors for the study of cytokine-induced osteoclast differentiation.
Ex Vivo Expansion of Mobilized Peripheral Blood Myeloid Progenitor Cells: Pretreated Patients vs Normal Donors.
Authors: De Bruyn Cécile , Delforge Alain , Bron Dominique
Published: October 2019

Background: Additional infusion of ex vivo differentiated myeloid progenitor may reduce or abrogate severe neutropenia following high-dose chemotherapy and peripheral blood (PB) transplantation. Normal HPC as well as HPC from patients in clinical remission can be used as source of cells. The aims of our study were to compare the ex vivo expansion of myeloid progenitor cells starting from pretreated patients (PP) and normal donors (ND) and to evaluate if the cells collected from poor mobilizers -for which the ex vivo expansion is particularly interesting- present similar ex vivo expansion capacities than cells from good mobilizers.

Methods: In this study, 11 normal donors (ND) and 17 patients (PP) were evaluated (5 NHL, 2 HK, 3 MM, 3 lymphoma, 1AML, 1 CLL and 2 solid tumor). The CD34+ cells were plated for 7 days in serum-free medium with SCF, IL-3, G-CSF and FLT3- ligand. The expression of CD34, CD13, CD15 and CD16 Ags and the presence of primary (myeloperoxydase, MPO) and secondary granules (lactoferrin, LF) were used to evaluate maturation into myeloid lineage. Functional capacities of the ex vivo generated cells were also analysed using oxydative burst activity and clonogenic capacities (CFU-GM).

Results: (1) the proliferation capacities of total leucocytes were higher in ND than in PP (28.8±5.6 vs 17.2±4.0 fold, p<0.04) with a comparable CD34+ cell expansion (8.7±3.0 vs 7.8±2.7 fold, NS); (2) in patients, 65.3±5.8% of the expanded cells were CD13+ and 23.2±4.1% remained CD34+ with 7.8±2.5 % of immature CD34+CD13− cells; these results were similar to that observed in ND (72.7±7.5% CD13+, 23.0±5.3% CD34+, 9.0±3.2% CD34+CD13−); (3) the number of myeloid progenitors produced per seeded CD34+ cell consisted of 19.0±3.7 CD13+, 11.8±3.7 CD15+, 10.2±3.2 CD16+ and 15.4±4.4 MPO+/LF+cells in PP and 25.3±5.4 CD13+, 12.5±3.8 CD15+, 7.6±2.1 CD16+ and 21.7±7.2 MPO+/LF+ cells in ND (NS); (4) these cells, essentially promyelocytes and metamyelocytes, were able to proliferate in clonogenic assay and presented oxydative burst activity; (5) we failed to observe a correlation between the cell expansions (total leucocytes, CD34+ cells and myeloid progenitor cells) and the diagnosis; (6) no correlation was observed between the mobilization of CD34+ cells into PB and the capacities of cells to be expanded in vitro.

Conclusions: in our culture system, the generation of cells committed to the myeloid lineage from CD34+ cells is feasible from ND as well as from PP, whatever is the diagnosis. The ex vivo expanded cell population consisted of cells at all stages of differentiation including immature hematopoietic progenitors. The mobilisation rate doesn’t correlate with the proliferative capacities of cells, suggesting that ex vivo expansion can be performed even in subjects with poor mobilization.
Regulated Expression of Activated Notch 1 during Embryonic Stem Cell Differentiation Preserves Multipotential Progenitors and Promotes Erythroid Cell Fate.
Authors: Ganapati Uma , Bui Lynne A. , Lynch Maureen , Dolezal Milana , Tan Hongying Tina , deVos Sven , Gasson Judith C.
Published: October 2019

Hematopoietic stem cells pass sequentially through a series of developmental decision points regulating self-renewal and lineage-specific differentiation. In normal hematopoiesis proliferation is tightly linked to differentiation in ways that are poorly understood. The Notch gene family has been shown to be evolutionarily conserved and to play an important role in determining cell fate, survival, and proliferation in multiple organisms. Numerous in vitro and in vivo studies strongly support a role for Notch signaling in the regulation of stem cell signaling and hematopoiesis. To define the function of Notch in the earliest stages of hematopoiesis, a Tetracycline-inducible system regulating expression of a ligand-independent, constitutively active form of Notch1 was introduced into murine E14Tg2a embryonic stem cells. (Era and Witte, PNAS, 97;1737–1742,2000). During co-culture, OP9 stromal cells induce the embryonic stem cells to differentiate first to hemangioblasts and subsequently to hematopoietic cells. Our studies indicate that activation of Notch signaling in flk+ hemangioblasts dramatically reduces their proliferative capacity without inducing apoptosis. Furthermore, Notch1 activation significantly reduces the levels of hematopoietic stem cell markers CD34, c-Kit and the myeloid marker CD11b. These reversible effects suggest that Notch signaling maintains the hemangioblasts in an immature state and blocks hematopoietic differentiation. When activated Notch is induced in committed hematopoietic progenitors, a shift towards definitive erythroid differentiation and decreased myeloid differentiation is observed. Microarray analysis of day8 hematopoietic progenitors following Notch activation in hemangioblasts indicates upregulation of known downstream targets of Notch signaling. Based on these results, we propose that Notch signaling plays a critical role in the earliest events regulating hematopoiesis.
Implication of HIF-1α but Not HIF-2α in the Hypoxic Response of Human Hematopoietic Progenitors.
Authors: Zhang Yanyan , Foudi Adlen , Berthebaud Magali , Buet Dorothee , Jarrier Peggy , Vainchenker William , Louache Fawzia
Published: October 2019

Maturing hematopoietic cells are exposed to hypoxia as they develop and migrate within the bone marrow microenvironment. Previous studies using non hematopoietic cell lines and monocytes showed that CXCR4 is strongly induced by hypoxia but little is known on the regulation of CXCR4 by hypoxia in the other hematopoietic cells and during hematopoietic development. We analyzed the expression and regulation of hypoxia-inducible transcription factor-1a (HIF1a) and 2a (HIF2a), the master regulators of metabolic adaptation to hypoxia, during hematopoiesis. Real time quantitative RT-PCR showed that HIF-1a mRNA was present on all the non hematopoietic and hematopoietic cells lines including HL-60, HEL, TF1, K562, KG1, U937, Jurkat and Mo7e. In contrast, HIF-2a mRNA expression was variable among the cell lines and was detected only at very low level in some cells such as KG1, Jurkat and HEL. Hypoxia exposure rapidly induced VEGF mRNA expression in the cells that expressed HIF-1a mRNA and exhibited HIF-1a protein accumulation. Interestingly, CXCR4 induction was observed only in the cells that exhibit significant expression of HIF-2a mRNA and HIF-2a protein accumulation. A strong correlation between HIF-2a mRNA levels and the induction of CXCR4 mRNA expression by hypoxia was found. Human CD34+ cells also expressed high levels of HIF-1a mRNA, whereas HIF-2a mRNA was barely detected. Interestingly, as observed for several myeloid cell lines, CD34+ cells exhibited a strong induction of VEGF expression in response to hypoxia and hypoxia mimetic agents cobalt chloride and desferrrioxamine whereas CXCR4 receptor expression was not induced suggesting that CXCR4 mRNA induction is related to the expression of HIF-2a. Altogether these data indicated that the hypoxic responses of human hematopoietic progenitors are independent of HIF-2a. Moreover, they establish that CXCR4 regulation by hypoxia is linked to HIF-2a protein expression.
An SCL +19 Core Enhancer Targets Three Mesoderm-Derived Cell Lineages - Blood, Endothelium and Smooth Muscle.
Authors: Silberstein Lev , Sanchez Maria Jose , Socolovsky Merav , Hoffman Gary J. , Piltz Sandie , Bowen Mark , Green Anthony R. , Gottgens Berthold
Published: October 2019

The stem cell leukaemia (SCL) gene encodes a basic helix-loop-helix transcription factor with a critical role in normal haematopoiesis and angiogenesis. The SCL gene is normally expressed in haematopoietic stem cells, mast cells, megakaryocytes, endothelium and smooth muscle. Aberrant expression of the SCL gene leads to T-cell acute lymphoblastic leukaemia, whereas SCL−/− mice die due to the absence of haematopoiesis. Hence, temporal and spatial regulation of SCL expression is essential. Our laboratory has previously characterised a 5.5 kb enhancer located 3′ of the SCL transcription start site, which is capable of targeting expression of b-galactozidase (LacZ) reporter gene to haematopoietic stem cells in the foetal liver and the bone marrow, as well as embryonic endothelium. Subsequent experiments showed that a 641-base pair core enhancer gave an identical pattern of lacZ expression in the embryo. However, it was unclear if the same element (later referred to as +19 core enhancer) was capable of maintaining reporter gene expression into the adulthood, since no lacZ activity was observed in postnatal mice. Using a transgenic construct containing a eukaryotic reporter gene, human placental alkaline phosphatase, we show that in the haematopoietic system, the +19 core enhancer is sufficient to target foetal liver and bone marrow HSCs, as well as mast cells and megakaryocytes. In the erythroid lineage, the enhancer is active only during the earlier stages of erythropoiesis, despite high level of SCL expression throughout erythroblast maturation, suggesting that an additional element is likely to be required to maintain SCL expression. The enhancer also targets embryonic and adult endothelium, together with vascular and visceral smooth muscle. Taken together, our results demonstrate that the 641-bp +19 core enhancer is sufficient to integrate signals upstream of SCL in blood, endothelium and smooth muscle. Our data also suggest that developmental relationship between these three mesoderm-derived lineages could be defined through a common transcriptional environment, and indicate that SCL may play a wider role in mesodermal development than previously thought.
Interaction of AF4 Wildtype and AF4•MLL Fusion Protein with SIAH Proteins: Indication for T(4;11) Pathobiology?.
Authors: Bursen Adelheid , Sven Moritz , Anne Gaussmann , Theo Dingermann , Rolf Marschalek
Published: October 2019

The human AF4 gene (4q21) is recurrently involved in recipro cal translocations to the MLL gene (11q23), correlated with high-risk acute lymphoblastic leukemia (ALL) in infants and early childhood. The t(4;11) translocation is one of the most frequent MLL translocations known today. In general, MLL translocations are the result of an illegitimate recombination process leading to reciprocal fusions of unrelated translocation partner (TP) genes with the MLL gene. Due to the constant presence of the derivative(11) product, the hypothesis was posed that only MLL•TP fusion genes are responsible for the leukemogenic process. This concept has been successfully tested for some known MLL fusions, while some other MLL fusions failed. Here, we demonstrate growth transforming potential of AF4 wildtype and the AF4•MLL fusion protein. The underlying oncogenic mechanism involves the two E3 ubiquitin ligases SIAH1 and SIAH2, the N-terminal portion of AF4 and the protection of the AF4•MLL fusion protein against proteosomal degradation. Supported by grant Ma 1786/4-1 from the DFG.
Shared and Differential Gene Expression Profiles between Anaplastic Large Cell Lymphoma and Hodgkin Lymphoma Revealed by Supervised Clustering Analyses.
Authors: Maesako Yoshitomo , Nishikori Momoko , Ohno Hitoshi , Uchiyama Takashi
Published: October 2019

Although anaplastic large cell lymphoma (ALCL) and Hodgkin’s lymphoma (HL) are distinctive disease entities, there is a considerable overlap in morphological and immunophenotypical features between the two lymphoma subtypes. We performed a supervised clustering analysis comparing gene expression profiles of ALCL/HL cell lines with those of a variety of B-cell tumor cell lines. The result showed that the genes, expressions of which in the ALCL/HL cluster were lower than those in B-cell tumors, included a set of genes for protein tyrosine kinases. It is suggested that in ALCL/HL, genes encoding for signal transduction molecules downstream of T- and B- cell receptor are down-regulated, even though both diseases are of lymphoid cell origin. On the other hand, we found a total of 21 genes whose expressions were shared with ALCL and HL, including JUN, ems 1 oncogene (EMS1), cyclin-dependent kinase 6 (CDK6), and integrin alpha 4 (ITGA4). We next performed a second supervised clustering to effectively separate ALCL and HL. Of 87 genes differentially expressed between ALCL and HL, 54 genes were expressed to a higher degree in ALCL than HL, whereas expression levels of 33 genes were higher in HL than ALCL. The ALCL-associated genes included cdk inhibitors, p19INK4d and p21WAF1/CIP1, suggesting that genes involved in cell cycle-regulating pathways are differentially expressed between ALCL and HL. Our study provided potentially interesting molecules that can account for the similarity and difference of ALCL and HL.
GATA1 Mutations in Clonal Disorders of Children with and without Down Syndrome.
Authors: Magalhaes Isis Q. , Splendore Alessandra , Emerenciano Mariana , Ferrari Iris , Pombo-de-Oliveira Maria S.
Published: October 2019

Down Syndrome (DS) children are 10–20 times folder likely to develop acute leukemia (AL) within the first four years of life compared to general pediatric population. Recently acquired somatic mutations in GATA1 gene on chromosome X have been described in most cases of DS AML and congenital TMD of DS carry the same type of mutations in exon 2 of GATA1. Here we report the preliminary results of GATA1 mutation in AL with and without DS children. The aim of this study is to provide insights in the relationships of GATA1 mutations and trisomy 21 in leukemogenesis process. GATA1 mutations were assayed in genomic DNA in 34 children with DS and AL, 2 with transient myeloproliferative disorder (TMD), 3 with myelodisplastic syndrome. Sequential sample including 2 pre-diagnosis in neonate period and 1 year before diagnosis were available in two children and 40 randomly selected DS children without known hematological disorder. A rare case of a non-DS neonate with TMD and clonal trisomy 21 were also examined. Genomic DNA was extracted and the exon 2 of GATA1 was PCR amplified as described by Wechsler et al. PCR products were sequenced in both directions and analyzed in a MegaBACE 1000 automated sequencer. Presently, GATA 1 mutations were found in 7 cases of AL, in all TMD cases with DS and none MDS case of DS. Interesting, a neonate girl with no phenotypic features of DS, but TMD features whose karyotype revealed 47, XX, +21/46, XX mosaics. A G-to-T transversion was detected which is predicted to result in a premature stop codon (c.119G>T; p.Glu67X) at the time of onset of TMD. However this same mutation was not detcted at 5 years of age.To our knowledge, this is the first reported case of TMD without DS with a detected GATA1 mutation. The presence of both somatically acquired abnormalities probably confers a proliferative advantage to the cell, resulting in TMD. We postulated in this case that both genetic abnormalities were temporary because of the non self-renewing nature of the progenitor that first had a non-disjunction event and this progenitor and the proliferative clone eventually disappeared. Therefore, even the proliferative advantage that the combination of trisomy 21 and GATA1 mutation confer, maintenance of these genetic changes are necessary for full leukemic transformation and persistence.
Effect of hTR Antisense Oligodeoxynucleotides on Telomerase Acitvity and Ciplatin -Sensitivity of Primary Acute Leukemia Cells.
Authors: Xiao Yang
Published: October 2019

BACKGROUND: Between the three key components part of human telomerase, human telomerase RNA (hTR) and human telomerase reverse transcriptase (hTERT) have significant correlation with telomerase activity. The previous study has identified that the telomerase activity of K562 and HL-60 cells was special suppresssed significantly by phosphorothoate antisense oligodeoxynucleotide (ASODN) complementary to the initiator codon of hTR. This study was designed to evalutae the effection of hTR ASODN on telomerase activity and apoptosis of primary acute leukemic cells. To research whether hTR ASODN could enhance apoptosis rates of primary leukemic cells to cisplatin.

METHODS: Primary leukemic cells were treated with phosphorothoate ASODN complementary to the initiator codon of hTR in vivo. The changing of telomerase activity was assayed by telomeric repeat amplification protocol(TRAP) and polymerase chain reaction enzyme-linked immunoassay(PCR-ELISA). The survival rates of cells was measured by trypan blue exclusion. Apoptosis was assayed by morphological observation (Giemsa and PI), DNA gel electrophoresis and flow cytometry analysis technology.

RESULTS: Primary acute leukemic cells expressed high level of telomerase activity which decreased as the cells treated by hTR ASODN. The telomerase activity was suppresssed significantly by 10umol/L ASODN and the effecttion was most significantly at 72h; Apoptotic bodies of primary leukemic cells were observed easily by fluorescence micrscope when cisplatin was added 48h after ASODN treatment for 24h; Agarose gel electrophoresis of genomic DNA from primary leukemic cells treated with ASODN and cisplatin combination for 72h showed typical DNA ladder; neither did DNA from primary leukemic cells treated with sense oligodeoxynucleotide (SODN) plus cisplatin nor cisplatin alone. In addition, apoptosis rates of primary leukemic cells treated with ASODN for 24h and then with cisplatin for 72h were 41.36±9.28%. There were statistically significant difference in the percentage of apoptotic cells between hTR ASODN plus cisplatin and SODN plus cisplatin (14.51±4.78%) or cisplatin alone group (12.61±2.56)% (P<0.01).

CONCLUSIONS: ASODN complementary to the region of hTR could significantly inhibit the telomerase activity of primary acute leukemic cells, and increased the cisplatin-induced apoptosis and enhanced cisplatin-sensitivity in vivo, indicating telomerase may be a new target of treatment to leukemia.
HHT-Induced Cell Apoptosis Is Associated with Reduction of Survivin mRNA Expression.
Authors: Cai Zhen , Bao Hanying
Published: October 2019

The inhibitor of apoptosis (IAP) gene family, which was discovered less than a decade ago, encodes a group of structurally related proteins that, in addition to their ability to suppress apoptotic cell death, are involved in an increasing number of seemingly unrelated cellular functions. The IAP families have evolved as highly conserved regulators of cell death. Homoharringtonine(HHT) is a plant alkaloid with antileukemia activity which is currently tested for treatment of acute and chronic leukemia. Our previous study suggested that HHT could induced apoptosis of MUTZ-1 in vitro, but little is know about the possible molecular mechanisms of HHT induced cell apoptosis. The study on the mechanisms of HHT in regulation of apoptosis will give us insight into further understanding of the role HHT. The data generated from this study will also provide theoretical ground for making use of HHT in clinical treatment. In this study, five cell lines including MUTZ-1, K562, Jurkat, RMPI and HL60 were used in the experiments to detect the effects of HHT on the induction of apoptosis and to study the possible mechanisms of HHT in regulation of apoptosis of the cells. Cell apoptosis were observed by flow cytometry (FCM). Cell apoptotic morphology was observed by transmission electron microscope. Semi-quantitative RT-PCR was used to evaluate the mRNA expression of survivin, XIAP, Bcl-2 and Bax in these cells. Our results demonstrated that HHT was capable of inducing apoptosis in all the five cell lines, with an increasing apoptotic rate in the order of K562, MUTZ-1/RMPI, and Jurkat/HL60, at a HHT concentration of 0.1 μg/ml for 12 hours (p<0.05). Survivin mRNA was not detected without HHT treatment but was detected in all the five cell lines after induction with HHT. Interestingly, the level of survivin mRNA expression was detected in the same but reversed order as with apoptotic rate: cell lines showed lowest apoptotic rate had highest survivin mRNA level, indicating a negative correlation between the two (p=0.003). The expression of Bcl-2 and Bax mRNA showed no significant correlation to HHT-induced apoptotic rate. There was no significant difference in XIAP expression in the 5 cell lines. We conclude that HHT might act via reduction of survivin expression and the level of survivin mRNA may serve as a predictor for chemotherapy sensitivity to HHT.
Study on the Resistant Mechanisms of K562 Cells Induced by STI571.
Authors: Liu Xiaoli , Zhang Song , Du Qingfeng , Fan Wei , Li Rong
Published: October 2019

STI571 is a highly effective drug for the therapy of CML, but there is still drug resistance, especially in blast crisis. To study the possible mechanisms of resistance to STI571, we established the BCR/ABL+ cell line with resistance to STI571 (K562-R) in vitro by culturing a wild-type K562 cells (K562-W) in gradually increased concentrations of STI571 over a period of months. Trypan blue staining, MTT assay and Hoechst 33342 staining confirmed that K562-R can live steadily at 0.5umol/L STI571. Furthermore MDR-1 expression assay, sequence analysis, fluorescence in situ hybridization(FISH) and cDNA array were used to study the potential mechanisms of acquired resistance. The MDR-1 expression percentages of K562-W and K562-R with FASC analysis were 2.68% and 1.39% respectively. No point mutant in the BCR/ABL ATP-binding site was detected and the copies of BCR/ABL fusion gene were found increased in K562-R by FISH analysis. By a expression profile of cDNA microarray, 327 genes’ expression were found down-regulating including one of homo sapiens protein tyrosine phosphatase genes(PTPRF) and 335 genes up-regulating including homo sapiens hematopoietic cell-specific Lyn substrate 1 gene(HCLS1). Our studies proved the possible mechanism of K562-R resistance involved amplification of BCR/ABL fusion gene and increase of phosphorylation activity in this cell line.
MDR Proteins Expression in AIDS-Related Non-Hodgkin’s Lymphoma.
Authors: de Faria Giselle Pinto , de Souza Barreto Luciana , Romano Sérgio , Faria Paulo Antônio , Ferreira Carlos José , Dobbin Jane , Klumb Claudete Esteves , Maia Raquel Ciuvalschi
Published: October 2019

Multidrug resistance (MDR) to chemotherapeutic drugs is a major obstacle in the treatment of patients with cancer. One of the best characterized resistance mechanisms of resistance in tumors cells is the MDR mediated by P-glycoprotein (Pgp) and multidrug resistant related protein (MRP). MDR expression in non-Hodgkin lymphoma (NHL) range widely being dependent on factors as the different antibodies, detection methods used, and clinical status (untreated patients or after failure of chemotherapy). AIDS-related NHL is associated with high rate of relapse and short overall median survivals. Previous study demonstrated that MDR positive AIDS-related LNH patients had a lower complete remission compared with MDR negative patients. In this study we analysed expression of Pgp and MRP by immunohistochemistry (IHC) in 62 high grade B-cell types NHL in an attempt to determine if MDR expression is dependent on previous anti-HIV therapy, a group of drugs well-known substrates for MDR expression. IHC detection of Pgp and MRP was performed using monoclonal antibodies according avidin-biotin method. Expression of Pgp was observed in 30 of 62 (48.4%) cases of AIDS-related NHL. MRP expression was examined in 27 specimens being 7 cases considered positives (25.9%). Co-expression of Pgp and MRP was detected in 5 out of 27 (18.5%) samples, whose patients had not previous NHL diagnosis. Anti-HIV therapy was used in 31/62 patients prior NHL diagnosis. Sixteen out of 30 patients that exhibited positive Pgp cells had not received anti-HIV therapy before NHL. Our study shows that expression of Pgp did not correlate to previous anti-HIV therapy and can be also observed in newly diagnosed AIDS-related NHL. The biological linkages between NHL and MDR phenotype are not fully understood. However, our results showed an intrinsic resistance in these patients indicating that the MDR modulation during chemotherapy could be a new approach for treatment in AIDS-related NHL.