bluuebunny/crossref_metadata_2025 huggingface dataset:
Paxillin Becomes Tyrosine-Phosphorylated and Binds Csk in a GpIIb-IIIa-Dependent Manner Following GPVI-Induced Activation of Murine Platelets.
Authors: Rathore Vipul B. , Newman Peter J. , Newman Debra K.
Published: October 2019

Though Src family kinases (SFKs) play a critical role in collagen-induced platelet activation, little is known about how SFK activity is regulated following exposure of platelets to collagen. In resting cells, SFKs are maintained in an inactive conformation, in part, via intramolecular interactions between their SH2 domain and a C-terminal tyrosine residue whose phosphorylation state is controlled by the C-terminal Src kinase, Csk. Access of Csk to SFKs, in turn, is regulated by recruitment of Csk, via its SH2 domain, to one or more tyrosine-phosphorylated Csk-binding proteins, which include Csk binding protein (Cbp/PAG) itself, paxillin, and its closely-related paxillin family member, Hic-5. Recent studies have shown that human platelets possess only two Csk-binding proteins: Cbp/PAG and Hic-5, and that Hic-5 can become tyrosine-phosphorylated when platelets are stimulated with a variety of platelet agonists, including, thrombin, U46619, and collagen. The purpose of the present investigation was to characterize the complement of Csk-binding proteins in murine platelets and to begin to determine their role in regulating collagen-induced platelet activation. Murine platelets, like human platelets, were found to express Hic-5, and in addition contained two other Csk-binding proteins: paxillin and leupaxin. Of these, both paxillin and Hic-5 became tyrosine phosphorylated, and paxillin was shown to be able to recruit Csk in a time-dependent manner following exposure of murine platelets to collagen and the GPVI/FcRg chain collagen receptor-specific agonist, CRP. These data suggest that the function of Hic-5 in human platelets may be performed by both Hic-5 and paxillin in mice. Finally, both paxillin tyrosine phosphorylation and Csk recruitment were blocked by agents that interfered with either platelet granule secretion or with integrin engagement, consistent with the notion that members of the paxillin family function as integrin-dependent negative feedback regulators of platelet adhesion and spreading.
Propofol-Induced Bleeding Dyscrasia: A Case Report.
Authors: Fahed Rabih C. , Schulz2 Thomas K.
Published: October 2019

Propofol is one of the most commonly used anesthetic drugs, with rapid induction, maintenance and recovery times. Its relative safety has resulted in it becoming a popular choice for general anesthesia.

A 56 y o woman with no prior history of bleeding underwent laparoscopic cholecystectomy. Postoperatively she experienced bleeding to the degree that open laparotomy was required to achieve hemostasis.Two years later, she underwent open sigmoid resection under propofol anesthesia for refractory diverticulitis. Severe postoperative bleeding ensued, necessitating IV fluid resuscitation and transfusion of packed red blood cells.Template bleeding time was repeatedly greater than 20 minutes on the first postoperative day. Platelet count, coagulation studies, von Willebrand disease assays, fibrinogen level and fibrinolytic system assays were found to be normal. Platelet aggregation in response to arachidonic acid was decreased at 9% (reference 60 - 120 %). The patient received platelet transfusions; hemostasis was achieved and the template bleeding time returned to normal on the second postoperative day and remained normal on repeat testing several weeks later.

A few reports have shown an increased bleeding with propofol, which is thought to be related to inhibition of thromboxane A2 synthesis and increased synthesis of leucocyte nitric oxide. Some studies show increased bleeding even without any change in the template bleeding time.

In summary, we report a case of a propofol-induced life threatening bleeding dyscrasia associated with a prolonged template bleeding time and platelet aggregation studies consistent with decreased response to arachidonic acid. This rarely reported complication should always be in the differential diagnosis of postoperative bleeding given the widespread usage of propofol anesthesia in major surgeries.
Direct Activation of Platelets by Tissue Factor Is Amplified by FVIIa and Potentiated by Serotonin: Influence of Shear Rate.
Authors: Diaz-Ricart Maribel , Lopez-Vilchez Irene , Galan Ana M. , Tonda Raul , Ordinas Antonio , Escolar Gines
Published: October 2019

Although exposure of tissue factor (TF) at sites of disrupted atherosclerotic plaques plays a critical role in platelet-triggered ischemic complications, a direct interaction of TF with platelets has not been yet demonstrated. We have investigated the effects of TF on platelets under different shear rate conditions. Exposure of isolated washed platelets to TF derived from human placenta resulted in reversible aggregation (35-50% max at 1 min) using standard platelet aggregometry. This response was αIIb-β3 dependent and occurred in the presence of residual concentrations of calcium. TF-induced reversible aggregation was enhanced and made irreversible to 90–100% by the addition of FVIIa (5μg/ml). This second phase of activation was blocked by the presence of serine-protease inhibitors. A combination of ultrastructural and flow cytometry studies revealed that although TF induced shape change and moderate expression of P-selectin an ANV positive phospholipids, internal contraction and full platelet degranulation only occurred after addition of FVIIa. All these transformations occurred together with tyrosine phosphorylation of proteins. Platelets adhered, spread and aggregated onto synthetic membranes coated exclusively with TF that had been exposed to circulating blood. Addition of FVIIa caused a 50% enhancement on platelet interactions and a two-fold increase in fibrin deposition. Serotonin enhanced overall TF induced platelet interactions under the different experimental approaches. A selective serotonin reuptake inhibitor (citalopram) reduced the intensity and extent of the effects of TF in the two experimental settings used in our investigations. Our results are consistent with a direct role of TF on platelet-triggered ischemic complications. We demonstrate that a lipidated from of TF activates isolated platelets in suspension. In studies conducted with flowing whole blood we show that TF firmly attached to a surface acts itself as an adhesive substrate, and promotes formation of aggregates. These TF-platelet interactions are enhanced through serotoninergic mechanism and markedly potentiated in the presence of FVIIa. The possible synergism between FVIIa and serotoninergic mechanisms may well contribute to the development of severe ischemic complications triggered on ruptured atherosclerotic plaques.
Isolation of CD34+AC133+Flk1+ Cells Does Not Identify the Most Primitive Endothelial Progenitor Cells.
Authors: Ingram David A. , Fenoglio Amy , Mead Laura E. , Yoder Mervin C.
Published: October 2019

Human EPCs are currently defined as a CD133+/Flk1+ subpopulation of CD34+ cells that can be isolated from peripheral blood, bone marrow, and fetal liver (Nature Medicine, 2003). While these cells are widely considered to be EPCs and subsequently used for angiogenic therapies and biomarkers of cardiovascular disease, their proliferative and clonogenic potential has not been compared to other populations of EPCs, which do not express the cell surface antigens, CD34 and AC133. Analogous to a paradigm established in the hematopoietic cell system, we recently developed a single cell clonogenic assay to reproducibly identify the following EPCs: (1) high proliferative potential - endothelial colony forming cells (HPP-ECFC), which form macroscopic colonies that form secondary and tertiary colonies upon replating, (2) low proliferative potential - endothelial colony forming cells (LPP-ECFC), which form colonies greater than 50 cells, but do not form secondary colonies upon replating, (3) endothelial cell clusters (EC-clusters) that contain less than 50 cells, and (4) mature terminally differentiated endothelial cells (EC), which do not divide (Blood, 2004). Utilizing this assay and EPCs derived from human umbilical cord blood mononuclear cells, we compared the clonogenic and proliferative potential of 1,000 single CD34+AC133+Flk1+ and CD34-AC133−Flk1+ umbilical cord blood derived endothelial cells. We conducted four independent experiments. We demonstrate that a complete hierarchy of EPCs can be identified in a population of CD34−AC133−Flk1+ cord blood derived ECs (Table I, n=4). Remarkably, we further show that CD34−AC133−Flk1+ cord blood derived ECs contain more proliferative EPCs (LPP-ECFCs and HPP-ECFCs) compared to CD134+AC133+Flk1+ cord blood derived ECs (Table I, n=4, *p<0.05). In fact, preliminary experiments suggest that CD34−AC133−Flk1+ cells mature into CD34+AC133+Flk1+ cells. Finally, some individual single CD34−AC133−Flk1+ HPP-ECFCs can expand rapidly to 107 cells in ex vivo culture. Retroviral marking of the cell progeny derived from the single cell confirmed that they were derived from the parent cell. Thus, these studies describe the use of a clonogenic assay to identify a novel population of CD34−AC133−Flk1+ EPCs, and preliminary experiments demonstrate that CD34 and AC133 are not universal cell surface markers of the most primitive EPCs.

Percent of 1,000 Single Cells Plated

Mature EC EC-Cluster LPP-ECFC HPP-ECFC CD34+AC133+Flk1+ ECs 70±11 16±5 10±4 3±1 CD34-AC133-Flk1+ ECs 52±12 17±6 20±3* 10±4*
Vascular Endothelial Growth Factor (VEGF) Induces Cell Survival Via an Additional Urokinase (uPA)-Dependent Mechanism.
Authors: Prager Gerald W. , Koshelnick Yuri , Mihaly Judit , Brunner Patrick , Binder Bernd R.
Published: October 2019

VEGF, the most important angiogenic growth factor, is known to induce cell-survival mainly via phosphorylation of the pro-apoptotic proteins BAD, PED/PEA-2 or pro-caspase-9 or inhibition of SAPKs (stress activated kinases). These mechanisms are all dependent on the PI3-kinase/Akt pathway. We could show recently that VEGF also induces pro-uPA activation via the PI3-kinase signaling pathway beside and independent of the transcriptional upregulation of uPA. Active uPA does not only contribute to angiogenesis via its proteolytic properties, but also effectuates itself pro-angiogenic signalling by the induction of endothelial cell migration, proliferation and differentiation. We were interested whether generation of uPA upon VEGF is inducing an additional effect on endothelial cell survival. First, we compared VEGF with urokinase in respect to cell survival in apoptosis assays and observed a pivotal anti-apoptotic effect of both stimuli, dose dependently and dependent on the type of matrix used. In addition, cell survival effects were additive, when both stimuli were added simultaneously. While VEGF-induced cell survival was PI3-kinase dependent, because it could be inhibited by the specific PI3-kinase inhibitor LY294002, the uPA-induced cell survival was PI3-kinase independent. Furthermore, uPA was able to rescue apoptosis induced by PI3-kinase inhibition in VEGF stimulated endothelial cells. From these data we conclude that uPA is indeed inducing an additional - PI3-kinase independent - cell survival mechanism. While VEGF led to a PI3-kinase dependent phosphorylation of Akt, which resulted in CDC42 activation, uPA activated CDC42 and its downstream effectors PAK leading to IKK-1 phosphorylation in a PI3-kinase/Akt independent manner. This indicates that the anti-apoptotic properties of uPA are not Akt, but NF-kappaB mediated. Indeed, when we used adenovirus overexpressing I-kappaB to block the NF-kappaB pathway, uPA was ineffective to support cell survival. In addition VEGF and uPA, both induced a transcriptional upregulation of inhibitor of apoptosis proteins (IAPs) in an NF-kappaB-dependent manner, among them most significantly the X-linked inhibitor of apoptosis protein (XIAP); again VEGF in a PI3-kinase dependent, uPA in a PI3-kinase independent manner.

From these data we conclude that VEGF is inducing cell survival in a strictly PI3-kinase dependent manner, on the one hand via its known mitochondrial pathway, but also via the PI3-kinase dependent pro-uPA activation leading to an NFkappa B dependent upregulation of inhibitor of antiapoptosis proteins.
Sulfur- and Selenium-Containing Compounds Derived from Natural and Synthetic Sources in Angiogenesis Modulation.
Authors: Mousa Shaker A. , Mohamed Seema , O’Connor Laura , Block Eric
Published: October 2019

Increases in serum sulfate may explain some of the therapeutic effects of methylsulfonyl methane (MSM), dimethyl sulfoxide, and glucosamine sulfate. Organic sulfur, as sulfur-containing amino acids, can be used to increase synthesis of S-adenosylmethionine, glutathione, taurine, and N-acetylcysteine. MSM may be effective for the treatment of allergy, pain syndromes, athletic injuries, and bladder disorders. Epidemiological studies have suggested that brassica vegetables are protective against cancers of the lungs and alimentary tract. Cruciferous vegetables are the dietary source of glucosinolates, a large group of sulfur-containing glycosides. However, dosages, mechanisms of action, and rationales for the use of various sulfur-containing compounds from natural or food products need to be better defined. The low toxicological profiles of these sulfur compounds, combined with promising therapeutic effects, warrant continued preclinical and clinical investigations. In this current study the angiostatic (antiangiogenesis) efficacy for individual sulfur-containing compounds derived from natural or synthetic sources was determined using in vitro (endothelial cell [EC] tube formation assay) and in vivo (chick chorioallantoic membrane [CAM] model). In vitro EC tube formation studies demonstrated significant inhibition of fibroblast growth factor-2 (FGF-2) stimulated EC tube formation by the different sulfur- and selenium-containing compounds, with different potency depending on the oxidation state of the sulfur or selenium. Furthermore, utilizing the CAM model, similar inhibitory efficacy of FGF-2 induced angiogenesis. These data suggest that sulfur and selenium compounds derived from natural sources might be a useful therapy for the inhibition of angiogenesis associated with human tumor growth and other pathological angiogenesis-mediated disorders, such as ocular and inflammatory diseases.

Antiangiogenesis Efficacy of Sulfone/Sulfoxide and Selenone/Selenoxide in the Chick Chorioallantoic Membrane Model

Treatment Branch Points ± SEM % Inhibition ± SEM FGF = fibroblast growth factor; PBS = phosphate-buffered saline. PBS control 64.67 ± 3.00 ----- FGF-2 (1.5 ug/ml) 178.20 ± 5.32 ----- FGF-2 + diphenyl sulfoxide (100 uM) 145.65 ± 11.10 34.12 ± 4.8 FGF-2 + diphenyl sulfone (3 uM) 82.56 ± 4.4 84.25 ± 3.6 FGF-2 + dimethyl selenoxide (30 uM) 120.86 ± 3.1 50.74 ± 2.7 FGF-2 + dimethyl selenone (3 uM) 71.63 ± 3.8 93.87 ± 3.3
Heightened Proteolysis of Plasma ADAMTS13 Antigen in Patients with Thrombotic Thrombocytopenic Purpura.
Authors: Yagi Hideo , Matsumoto Masanori , Iwamoto Taka-aki , Ishizachi Hiromichi , Nishimura Hitomi , Isonishi Ayami , Soejima Kenji , Nakagaki Tomohiro , Mori Toshio , Fujimura Yoshihiro
Published: October 2019

A total of 140 subjects with thrombotic thrombocytopenic purpura (TTP)/hemolytic-uremic syndrome (HUS), in which 25 were Upshaw-Schulman syndrome (USS, belonged to 20 families), 107 were acquired TTP (96 idiopathic and 11 drug-induced), and 8 were acquired O157:H7-associated HUS, were analyzed on their plasma ADAMTS13 antigen (:AGN), using 3 novel anti-ADAMTS13 murine monoclonal IgG antibodies (A10, C7, and WH2-1–11). Epitope of these antibodies resided on the disintegrin-like, 7th/8th thrombospondin-1, and 4th thrombospondin-1 domains, respectively. The WH2-11–1 alone was immunoreactive on western blot (WB) under reducing conditions. Further, in a static assay system, A10 totally and C7 partially inhibited ADAMTS13 activity (:ACT), but WH2-11–1 did not. A sandwich ELISA using C7 as the 1st antibody and biotinylated A10 as the 2nd antibody revealed a significant reduction of plasma ADAMTS13:AGN level in patients with acquired TTP, but a considerable variation in those with USS, suggesting that the quantitative ELISA has a limited diagnostic value.

A quantitative and semi-qualitative WB analysis using A10 and WH2-11-1, however, demonstrated that normal plasmas had a 190/180 kD-doublet band under non-reducing conditions. A size exclusion chromatography of normal plasma was able to separate a 190 kD-protein from a 180 kD-band, and the former turned to be a single 200 kD-band on WB under reducing conditions, but in the latter such protein band was completely invisible, indicating that the 180 kD-band had undergone proteolytic modification. All the patient plasmas with USS and a roughly half of those with acquired TTP had a broad and fuzzy ~190/180 kD-band on WB before reduction, but it almost entirely disappeared after reduction. In patient plasmas with acquired HUS, however, the pattern of WB analysis was indistinguishable from that of the normal subjects.

Aforementioned WB analysis was then performed on a girl of acquired TTP with high-titer inhibitors against ADAMTS13, using the sequential plasma samples obtained during the past 3 years, and confirmed complete normalization of the abnormal ADAMTS13:AGN patterns in accord to the increase of plasma ADAMTS13:ACT, together with her clinical improvements. Protease(s) responsible for this heightened cleavage is currently under investigation.
Generation of Floxed Tissue Factor Mice.
Authors: Pawlinski Rafal , Pedersen Brian , Tencati Michael , Mackman Nigel
Published: October 2019

Tissue factor (TF) is the primary cellular initiator of blood coagulation. Since TF Deficiency leads to embryonic lethality, we generated floxed TF mice to allow analysis of the effect of deleting the TF gene in different cell types. A targeting vector was constructed containing two loxP sites spanning the promoter and intron 1 together with a neomycin resistance gene (NEO) in intron 1 that is flanked by two frt sites. The floxed TF gene targeting plasmid was transfected into ES cells and clones were selected using G418 and gancyclovir. We screened 144 clones by Southern blotting and found six targeted clones. Three clones were injected into blastocysts to generate chimeric mice. Only one chimera demonstrated germline transmission when bred with C57BL/6 mice. TFflox/flox mice were generated at the expected frequency (15 out of 58; 25.8%) from TF+/flox intercrosses, indicating that the floxed TF allele is functional. To detemine if the DNA between the loxP site can be efficiently excised from the floxed TF allele to create an inactive gene, we crossed TFflox/flox mice with mice expressing the Cre transgene under the control of the adenovirus EIIa promoter. This transgene is expressed in one cell zygotes and results in deletion of DNA at the earliest stage of development. From this breeding, we generated TF+/flox− offspring and intercrossed them. To date, analysis of 14 mice at wean has not identified a TFflox−/flox− mouse, suggesting efficient inactivation of the TF gene. To remove the NEO cassette from intron 1 of the floxed TF allele, we crossed TFflox/flox with transgenic mice expressing flipase under the control of the human β-actin promoter (FLP). TF+/flox/FLP+ mice were intercrossed. To date, we have successfully generated 9 TFflox/flox/NEO− mice. These mice will be crossed with tissue-specific deleter strains to investigate the contribution of TF expression in various cell types to a variety of biological processes, including hemostasis, inflammation, angiogenesis, metastasis and cell migration.
F VIII Inhibitors in Haemophilia A Patients who Are Considered as Previously Treated Patients.
Authors: von Auer Ch. , Krause M. , Miesbach W. , Scharrer I. , Asmelash G. , Stier-Brueck I. ,   PTP Study Group in Germany
Published: October 2019

Previously treated patients (PTP), who have not developed an inhibitor (inh) so far, are considered to be tolerant to factor VIII and at low risk for inh development. Therefore inh detection in a PTP should raise concerns about the concomitant variables such as product neo-antigenicity or way of application.

In our own center we recently detected the new development of a high responding inh to factor VIII in a 58 year old patient with severe haemophilia A. To find out about the current situation regarding inh development in PTP in Germany, we conducted a retrospective study.

A questionnaire was sent to 99 haemophilia treating physicians, so far 46 of them answered.

24 PTP-inh were registered during the last 5 years. Patients had at least 20 ED and/or one change of factor concentrate. Age (9 months to 70 years, median 35), severity of haemophilia A (16 severe, 2 moderate, 6 mild), exposure days (ED 6 to >1500, median 37) and genotype (4 intron-22-inversions, 3 large deletions, 2 missense mutations, 1 stop mutation, 1 insertion, 1 small deletion, 11 unknown) were recorded.

8 different factor VIII concentrates were given during inh development (5 plasma derived, 3 recombinant). Way of application (16 bolus infusion, 3 continuous infusion, 5 times both), infused amount until inh development (3000 IU to >1 mio IU), inh characteristic (15 HR, 9 LR), concomitant diseases and medication were registered.

In conclusion it became obvious that inh in PTP are still a serious and underestimated problem in haemophilia treatment today.

Our patient numbers are still too small to draw conclusions concerning given F VIII products or way of application. Secondly data showed that there is a variety of PTP definitions in Germany, referring to age of pat, number of ED and former change of product. A definition from the SSC of the ISTH for PTP would be helpful. The continuous use of the German register for drug side effects would make it easier to evaluate data in the future. A prospective, not product related study should be conducted.
Nonpuerperal Ovarian Vein Thrombosis: A Report of Seven Cases.
Authors: Ferman Debra , Bradley Thomas P. , Warshawsky Robin , Allen Steven L.
Published: October 2019

Background: Nonpuerperal ovarian vein thrombosis (OVT) is a rare clinical entity. Therapy is not well defined. OVT is usually asymptomatic. Improved CT and MR imaging technology enables OVT to be diagnosed with greater frequency. Nonpuerperal OVT may be a distinct clinical entity.

Methods: The medical records of women with nonpuerperal OVT were reviewed and their clinical course and treatment recorded. Cases were identified by scanning a CT computerized database over the past 3 years with the key words ovarian vein thrombosis.

Results: 7 patients (pts) were identified. Age range was 38–61, median 51 years. 3 pts had OVT on the left and 4 on the right. OVT followed a procedure in 2 pts. 2 pts with breast cancer developed OVT, 1 during adjuvant tamoxifen and 1 receiving chemotherapy for metastatic disease. 1 pt had AML in CR. 3 pts presented with lower abdominal pain on the involved side and 1 pt had lower abdominal pain on the opposite side. The 3 pts with cancer were asymptomatic. 4 pts had uterine fibroids. 1 developed OVT in the setting of an acute diarrheal illness. 1 pt had prior DVT and 1 a family history of DVT. Only 1 pt had a hypercoagulable evaluation; negative. 2 pts had clot extending to the junction with the inferior vena cava (IVC) and both were anticoagulated with enoxaparin followed by warfarin. 1 pt was anticoagulated with enoxaparin alone. No embolic complications occurred.

Conclusion: 4 pts with OVT were symptomatic and 3 were detected incidentally. 2 developed OVT following procedures, 3 had predisposing underlying conditions, and 2 had a personal or family history of thrombosis. Optimum therapy is not defined. Symptomatic disease alone is not an indication for anticoagulation. Anticoagulation may be indicated if clot extends to the IVC.
Hyperhomocysteinemia in Cancer Patients with Thrombosis Is Independent of Methylene Tetrahydrofolate Reductase Gene Mutation.
Authors: Fareed Jawed , Tobu Mahmut , Hoppensteadt Debra A. , Cunanan Josephine , Iqbal Omer , Demir Muzaffer , Deitcher Steven
Published: October 2019

Plasma homocysteine levels are considered to be an important surrogate marker of vasculopathy in thrombotic disorders. Methylene tetrahydrofolate reductase (MTHFR) gene mutations, nutritional factors (folic acid/ B12) and anticancer drugs are primarily responsible for the observed increase in this surrogate marker. In addition, it is well known that cancer patients exhibit increased prevalence of thrombotic events. The aim of these studies was to demonstrate that an upregulation of homocysteine plays a role in cancer associated thrombosis. However, initial screening of cancer patients showed that the malignancy related thrombotic state was independent of molecular defects in Factor V Leiden, Prothrombin 20210 and MTHFR ( 677 and 1298) variants (Hoppensteadt et al. Proceedings ASCO2003. 22:346;861). In a group of 102 patients who were diagnosed with cancer and thrombosis and in a group of medical patients (cancer free) with thrombosis enrolled in a treatment study with low molecular weight heparin (CORTES, n=50), blood levels of homocysteine were measured using a highly specific ELISA method (Diazyme Laboratories, San Diego, CA). In addition, molecular methods utilizing PCR were used to measure the MTHFR (677 and 1298) variants. On a cumulative basis the cancer patients with thrombosis exhibited a relatively higher level of homocysteine (11.8± 4.7 uM/L) in comparison to normal (n=140, 4.6± 2.9 uM/L) and medical patients with thrombosis (5.3± 3.1 uM/L). Of the 102 cancer patients profiled for the molecular analysis 18% were positive for the 677 variant and 12% were positive for the 1298 variant. Similar results were obtained in the medical patients and normal volunteers. These results clearly suggest that, cancer patients with thrombosis exhibited a relatively higher level of circulating homocysteine levels which are unrelated to a molecular defect in MTHFR. Medical (cancer free) patients with thrombosis do not exhibit this increase in circulating homocysteine levels. Thus, other relevant factors such as the use of chemotherapeutic agents like methotrexate and purine analogs may contribute to this apparently acquired upregulation of homocysteine. This observation warrants periodic monitoring of homocysteine in patients with thrombosis and cancer. Nutritional supplementation of folic acid and B12 may be helpful in the overall management of these patients including those who are treated with methotrexate and purine analogues.
4g/5g Prevalence in 223 Patients with Thrombosis and in 162 Healthy Controls, from the Centre Region of Portugal.
Authors: Maia Rosa , Cortesao Emilia , Geraldes Catarina , Simoes Luis , Simoes Carla , Castro Graca , Anacleto Gabriel , Barbosa Vieira , Teixeira Adriana
Published: October 2019

A deletion/insertion polymorphism (4G or 5G) in the promoter of the PAI-1 gene has been suggested to be involved in regulation of the synthesis of the inhibitor, the 4G allele being associated with enhanced gene expression, and therefore related with thrombosis. In the present work we studied the prevalence of 4G/5G polymorphism in 223 unrelated patients with history of objectively confirmed thromboembolism, and in 162 healthy unrelated controls, both groups natural from all centre regions of Portugal. In this normal cohort, the prevalence of 4G/4G is 23%, 4G/5G is 38% and 5G/5G is 39%; in the affected population is, respectively, 47%, 21.5% and 30%, which means that 4G/4G is twice more frequent in the patients with thrombosis. When we relate the age of the first thrombosis episodes in the three groups, we find no significative difference, as the respective media is 36.8; 38.6 and 35.5 years in the 4G/4G, 4G/5G and 5G/5G group, respectively. This data suggest that this polymorphism by itself, even in homozygosity, is not associated with earlier thrombosis. In our patients, we studied the presence of Lupus Anticoagulant, Factor V Leiden, Factor IIG20210A, MTHFR C677T, and also Antithrombin III, Protein S and Protein C levels. We analyse the prevalence of the three mutations in patients with DVP, PTE, ischemic and venous CVA and we only find a significative difference in the 4G/4G group: 46.2% patients with DVP and 48.2% patients with PTE (23% in normal cohort). In conclusion, in the centre region of Portugal, the prevalence of 4G/4G is 23%, 4G/5G is 38% and 5G/5G is 39%; in our cohort of unrelated patients the only significative difference is in the 4G/4G group (47%); this variation maintain in the DVP and PTE group. We did not find difference at the age of the first thrombotic episode, in the three groups.
Management of Anticoagulation in Patients Who Requiere Colonoscopy or Gastroscopy: Safety and Efficacy of Sodic Bemiparin (Hibor®.
Authors: Santamaria Maria Amparo , Mateo Jose , Pujol Nuria , Souto Juan C. , Constans Mireia , Ortin Rosa , Llobet Dolores , Fontcuberta Jordi
Published: October 2019

Introduction

Nowadays, most patients under oral anticoagulation treatment (OAT) require invasive procedures such as colonoscopy or gastroscopy. The goals of the management OF OAT are to minimize the risk of thromboembolism and bleeding. Few studies have analyzed the efficacy and safety of low-molecular-weight-heparin (LMWH) in the periprocedure management. We have performed a prospective, observational study to evaluate these parameters using high-dose thromboprophylactic therapy with sodic Bemiparin. (Hibor ®)

Methods

From January 2004 since June 2004, patients under OCT were included before colonoscopy or gastroscopy procedures. Periprocedure prophylaxis consisted on: Acenocoumarol patients: Day -3: withdrawal acenocumarol. Days -2,-1,0: Hibor ®3500 UI/d sc and days +1,+2,+3: Hibor ®3500 U/I + acenocumarol. And day +5: acenocumarol only. Warfarin patients: Días -5,-4: withdrawal warfarin, −3, −2, −1, 0; Hibor ® 3500 UI/d sc, days +1,+2,+3,+4: Hibor ®3500 UI/d sc and warfarin and day +5; warfarin only. Thromboembolic complications and bleeding were recorded in a 3 months follow-up.

Results.

We included 65 patients, 33 women and 32 men. Mean age of women was 72 (range: 50–87) years-old and 72 (range: 39–86) in men. 53 were taken acenocoumarol, and 12 warfarin. OAT indications were: 9 prosthethic mechanical mitral valves 12 prosthetic mechanical aortic valves, 4 with mechanical mitral and aortic replacement, 7 with thrombophilia, 7 with embolic atrial fibrilation/flutter (AF) 18 with isolated AF,1 ischemic cardiomyopathy (IM), 3 with IM and AF and 5 with mitral valvulopathy. 24 gastroscopies and 41 colonoscopies were performed. Mean follow-up was 3 months.

No thromboembolic complications were observed. Only one episode of minor bleeding no related with Bemiparin thromboembolic therapy was recorded. One patient developed pruritus at the punction site. No heparin-related thrombopenia cases were observed.

ConclusionsHigh-dose thromboprophilactic therapy with Bemiparin (Hibor ®) is a safe and effective in periprocedures management in patients under OAT who require gastroscopy or colonoscopy
Role of Protein Kinase C in Expression of Granulocyte Colony Stimulating Factor and Granulocyte-Macrophage Colony Stimulating Factor in Lung Cancer Cells.
Authors: Uemura Yoshiki , Kobayashi Makoto , Nakata Hideshi , Kubota Tetsuya , Taguchi Hirokuni
Published: October 2019

Many cases of tumors that produce granulocyte-colony stimulating factor (G-CSF) or granulocyte macrophage-colony stimulating factor (GM-CSF) have been reported. However, the biological properties regulatory mechanisms of the overproduction of G-CSFor GM-CSF by tumor cells are not well known. We present the role of protein kinase C (PKC) pathways in the constitutive expression of G-CSF and GM-CSF by lung cancer cells. We previously established two lung cancer cell lines, OKa-C-1 and MI-4, that constitutively produce an abundant dose of G-CSF and GM-CSF. We showed that the PKC activator; phorbol 12-myristate 13-acetate (PMA) stimulated the production of GM-CSF in a dose-dependent manner and inversely reduced G-CSF in the cell lines. These effects of PMA were antagonized by PKC inhibitor; staurosporine. The induction of GM-CSF expression by PMA was mediated through the activations of nuclear factor (NF)-kB activation. The induction of G-CSF expression by staurosporine was mediated through p44/42 mitogen-activated protein kinase (MAPK) pathway signaling. PMA accelerated cell growth and inhibited cell death in the cell line. Whereas staurosporine acted inversely. GM-CSF induced by PMA might stimulate cell growth and suppress cell death. G-CSF expression by staurosporine appears to be related to the activation of p44/42 MAPK, and GM-CSF by PMA to NF-kB in OKa-C-1 and MI-4 cells.

Figure Figure
Gene Expression of Insulin-Like Growth Factor Binding Proteins (IGFBP-2 and IGFBP-3) in Childhood Acute Myeloblastic Leukemia. Intr. by Herbert Sayer.
Authors: Dawczynski Kristin , Gruhn Bernd , Pfaffendorf Nadine , Susan Wittig , Voigt Astrit , Steinbach Daniel , Zintl Felix
Published: October 2019

Insulin-like growth factor (IGF) system plays an important role in regulating cellular proliferation. Alterations to the Insulin-like growth factor system have been reported for different tumors. They are of particular interest in the search for new prognostic and therapeutic approaches to cancer treatment. This study aimed to investigate the expression of IGFBP-2, IGFBP-3, IGF-I and IGF-II genes in leukemic cells from children with previously untreated acute myeloblastic leukemia (AML).The expression of IGF-I and IGF-II genes as well as IGFBP-2 and IGFBP-3 genes were measured using TaqMan real-time PCR in 50 children (mean age 10.8±4.8 years). All four genes were expressed with a great variability. RNA samples were extracted from leukemic cells enriched by Ficoll-Hypaque density gradient separation of bone marrow or peripheral blood mononuclear cells (MNC). MNC samples from peripheral blood and bone marrow of healthy children were used as controls. The median expression of IGFBP-2 was 25 times higher in AML cells than in peripheral MNC (p<0.001) and 10 times higher in bone marrow of healthy children (p<0.01). Increased IGFBP-2 gene expression at time of diagnosis was associated with a poor prognosis and a shortened survival time. Leukemic cells of patients who remained in continuous complete remission during the follow up showed a significant decreased IGFBP-2 gene expression at time of diagnosis compared to patients who suffered from relapse (p=0.05).On the other hand, AML cells showed a significantly lower IGFBP-3 gene expression than controls. The median expression of IGFBP-3 was 30 times lower in AML cells than in peripheral MNC (p<0.001) and 20 times lower than in bone marrow of healthy children (p<0.001). No significant difference could be found in IGF-I and IGF-II expression between leukemic and normal cells. Leukemic cells from children with previously untreated AML express components of IGF system. Especially IGFBP-2 seems to play an important role. By this mechanism, IGFBP-2 promotes their own growth in an autocrine, paracrine or endocrine manner. Whether these components will be useful as prognostic factors in stratification of AML treatment in children needs to be evaluated.
Overexpression of Jak Kinases Promotes In Vitro Transformation.
Authors: Knoops Laurent , Royer Yohan , Constantinescu Stefan , Renauld Jean-Christophe
Published: October 2019

The Jak-STAT pathway is responsible for signal transduction by a large number of cytokine receptors. While this pathway is normally tightly regulated, constitutive STAT3 and STAT5 activation is frequent in hematologic malignancies and contributes to oncogenesis. We took advantage of the IL-3-dependent pro-B Ba/F3 cell line to analyze the mechanisms of constitutive STAT activation in vitro. Ba/F3 cells transfected with a mutated hIL-9R (Ba/F3 Phe116) poorly respond to IL-9. However, after selection with this cytokine, we obtained cell lines that proliferated well in IL-9. After cytokine withdrawal, those cells, in contrast to the parental cells, gave rise to autonomous Ba/F3 cell lines showing constitutive STAT5 activation, and that were highly tumorigenic in vivo. This process mimics the multistep process of oncogenesis occurring in vivo. To dissect the mechanisms involved in this cellular transformation, we analyzed, using cDNA microarrays, genes expressed in one of these autonomous lines. Jak1, the Jak kinase associated to the IL-9 receptor, was found to be upregulated compared to parental cells. It was the key genetic event involved in this transformation since ectopic Jak1 overexpression increased the sensitivity to IL-9 and allowed, after a second selection step, for spontaneous progression towards autonomous cell lines with constitutive STAT activation. Using Jak1 mutants, we showed that the generation of autonomous lines was dependent on the tyrosine kinase activity of Jak1 and on a functional FERM domain, the domain that mediates association with the receptor. These results were extended to the other Jak family members. Therefore, we propose that Jak overexpression can be considered as one of the oncogenic events leading to the selection of cells highly responsive to growth factors and, after a second selection step, to autonomous cells with constitutive activation of the Jak-STAT pathway. This process could be extended to human malignancies and might explain, for instance, the constitutive STAT6 activation in primary mediastinal large B cell lymphomas where Jak2 overexpression was described.
Application of Risk-Adapted Therapy for Acute Myeloid Leukemia to a Population. Effects of Stem Cell Transplantation on Outcome.
Authors: Wahlin Anders , Brune Mats L. , Billstrom Rolf
Published: October 2019

We introduced a risk-adapted treatment program for non-APL AML in four Swedish health regions. The aim was to optimise treatment results by the use of risk group stratification, mainly based on cytogenetic findings at diagnosis. All patients received induction therapy with idarubicin-cytarabine 3+7 and consolidation cycles containing high-dose cytarabine. Stem cell transplantation was done in CR1 in selected patients, sparing patients with low/intermediate risk of relapse the risks associated with transplantation. 279 patients, 77% of all AML patients 18–60 years (median 51 yrs), in the population were included in the program. Cytogenetics was performed in 98%. Excluding APL, 19 patients had low-risk. The intermediate-risk group consisted of 165 patients, 96 with a normal karyotype. 95 patients were allocated to the high-risk group. 6% died < 30 days after diagnosis. CR rate was 80%. 111 transplants, 78 allogeneic/URD and 33 autologous, were performed in CR1. 40% of all patients were alive after five years. Median overall survival time was 887 days in low-risk, 611 days in intermediate risk, 345 days in high-risk patients. Relapse-free survival times were also significantly (p<0.001) different between the three risk groups. 43% of responding patients were alive in first remission after four years. 4-year relapse-free survival was significantly better for both intermediate risk (67%) and high-risk (41%) with allogeneic/URD transplantation than with autologous transplant or chemotherapy alone. Relapse was observed more often among patients treated with chemotherapy alone (42%, p=0.03) or with autologous transplants (42%, p=0.09) than among patients receiving allogeneic/URD transplants in CR1, 22%. Our results do not support the use of autologous transplantation in AML in first remission.
A Study of the Toxicity of Gemtuzumab Ozogamicin in Primary and Relapsed AML, Administered Alone or Simultaneously with Intensive Chemotherapy.
Authors: Larsen Thomas Stauffer , Schmidt Kai G.
Published: October 2019

Introduction: Gemtuzumab Ozogamicin (Mylotarg®) is a humanized anti CD33 antibody linked to the cytotoxic drug calicheamicin, which has shown considerable antileukaemic effect in the treatment of relapsed AML. With special regard to its toxicity, we present our preliminary results of Mylotarg treatment when administered on compassionate basis as consolidating monotherapy, or combined with intensive chemotherapy.

Patients and methods: A total of 86 doses, corresponding to 65 courses were administered to 49 patients. Indications for Mylotarg treatment ( mono ~ monotherapy; comb ~ combination therapy. No.of courses are shown in parentheses): Primary AML: Reinduction ( mono:1; comb:3). Cytogenetic reinduction ( mono:3; comb:1). Consolidation ( mono:24; comb:2). Relapsed AML: Induction ( mono:4; comb:11). Reinduction ( mono:1; comb:2). Cytogenetic reinduction ( mono:1). Consolidation ( mono:10; comb:2).

Results: Up-front consolidation with Mylotarg as monotherapy ( 30 doses; 24 patients; mean Mylotarg dose 5.7 mg/m2). Fever: 51% of doses. Focal infections: 17%. Bacteremia 7%. Biochemical findings: Platelet nadir at day 10 ( < 20 bill./l: 25% doses). Neutrophil nadir at day 11 ( mean count = 0.12 bill./l). Hepatic toxicity ( grade 1; % of doses): Alkaline phosphatase 7%; ALAT: 13%; Bilirubin 0. A similar pattern was seen in the 10 relapsed patients consolidated with Mylotarg as monotherapy. One patient receiving an allogeneic transplant 3 months after Mylotarg treatment developed fatal VOD. Three other patients who undervent allogeneic stem cell transplantation 2, 2 and 3 months, respectively, after the administration of Mylotarg did not experience severe hepatic complications.

We compared twelve courses of Mylotarg combined with DaunoXome and high dose cytarabine with ( n=10) or without ( n=2) added fludarabine and etoposide, administered to10 patients in 1st relapse with 8 similar courses but without Mylotarg, administered to 7 patients with similar characteristics regarding age, CR1 duration and cytogenetics ( mean Mylotarg dose 4.4 mg/m2). More cases of grade 3 diarrhea ( 33% vs 13%) and bacteremia ( 67% vs 38%) were seen in the Mylotarg group, in which three patients suffered hypoplastic deaths. One of these patients developed intestinal perforation following postremission treatment. The two other patients, with an early refractory relapse, and a late relapse with high risk chromosomal aberation, respectively, died from infectious complications following 20 and 36 days of profound neutropenia. These 3 patients recieved 5.9, 8.3 and 5.0 mg/m2 respectively, compared to the average Mylotarg dose of 4.4 mg/m2.

Conclusion: Mylotarg seems to increase toxicity when added to intensive chemotherapy in relapsed AML. The feasibility of this approach must await further studies evaluating toxicity, appropriate dosing and response pattern. When administered as consolidating monotherapy toxicity is modest.
Evaluation of Humoral Response to Trivalent Influenza Vaccination in Patients with Non-Hodgkin Lymphoma Previously Treated or Untreated with Chemotherapy.
Authors: Centkowski Piotr , Brydak Lidia , Machala Magdalena , Kalinka Ewa , Blasinska-Morawiec Maria , Wegrzyn Joanna , Skotnicki Aleksander , Federowicz Irena , Walewski Jan , Seferynska Ilona , Bilinski Przemyslaw , Warzocha Krzysztof ,   for the Polish Lymphoma Research Group
Published: October 2019

The aim of this study was to assess humoral response to influenza vaccination in 47 patients with non-Hodgkin lymphoma (NHL) in comparison with 45 healthy individuals (age 12–76 years, median 41 years; 13 males and 32 females). Patients (age 36–82, median 60 years; 24 males and 23 females) consisted of two groups, previously treated with chemotherapy (group A, n=32) or untreated (group B, n=15). In autumn 2003, all subjects were vaccinated with influenza vaccine ([Isquo]influvac’, Solvay Pharmaceuticals B.V.). Antibody responses to influenza hemagglutinin and neuraminidase were determined in sera collected before and 1 month after vaccination by hemagglutination and neuraminidase inhibition tests. Statistical analysis was performed using the Mann-Whitney unpaired and Wilcoxon paired tests. After vaccination geometric mean antibody titers (GMTs) of antihemagglutinin (HI) antibodies significantly increased (p<0.05) and mean fold increases (MFIs) from 10.3 to 26.7 in group A, 5.9 to 48.9 in group B and from 9.1 to 24.4 in the controls. Pre-vaccination protection rate, i.e. the number of subjects with the protective HI antibody titers >1:40, ranged from 0% to 9.4% in group A, 0% to 13.3% in group B and from 2.2% to 20% in the controls. After vaccination protection rates increased and ranged from 78.1% to 87.5%, 73.3% to 93.3% and 66.7% to 82.2%, respectively. Response rates, i.e. the number of subjects with at least 4-fold increase of HI antibody titers after vaccination, ranged from 59.4% to 84.4%, 73.3% to 86.7% and from 51.1% to 66.7%, respectively. In both study groups and in the controls, post-vaccination antineuraminidase (NI) antibody titers were higher (p<0.05) than pre-vaccination titers. MFIs of NI antibody levels ranged from 9.8 to 17.3 in group A, 6.5 to 10.9 in group B and 4.8 to 10.3 in the controls. After vaccination, only hemagglutinin H1 antibody titers were (p<0.05) higher in the controls than in the study group A and B. In the other cases, there were no statistically significant differences between groups or there were higher values in patients with NHL than in the control group. The results of this study indicate that influenza vaccine is immunogenic in patients with NHL and stimulates production of HI and NI antibodies in the titers comparable to healthy subjects.
Clinical Significance of Bcl-2 and Ki67 Protein Expression in Agrressive Non-Hodgkin Lymphoma.
Authors: Stavric Sonja S. Genadieva , Zografski Gore G. , Ivkovski Ljube L.J. , Siljanoski Nikola N. , Georgievski Borce B. , Stojanoski Zlate Z. , Pivkova Aleksandra A. , Cevreska Lidija L.
Published: October 2019

Aggressive Non-Hodgkin lymphoma (NHL) is heterogeneous group with respect to clinical, histopatological and evaluative features. The International Prognostic Index (IPI) has provided a widely accepted prognostic set of criteria to design therapy. However, IPI dose not determine whether routine phenotypic features, beside IPI, may influence survival. We evaluate the prognostic significance of Bcl-2 and Ki67 protein expression in relation to clinical presentation and outcome.

In the period 1989–2002,two hundred eleven patients with newly diagnosed aggressive NHL were recorded in our Department. In this study we included patients with available biopsy sample. Protein expression was analyzed on paraffin embedded tumor tissue by imunohistochemistry in relation to clinical factor and outcome. The expression of more than 20% of neoplastic cell was considered positive for bcl-2, and more than 60% for Ki-67. Comparison was made by x2 test. Survival curves were considered by Kaplan-Meyer method and compared by the long-rank test. Sixty seven patients were recorded and their characteristic were: median age 53 years, stage III and IV, 67%; present B symptoms 44%,; bone marrow infiltration 29%, elevated LDH 58%, performas status more then 1– 26%; extra nodal sites more than 2 –34%. All 62 patients received antracyclin based combination chemotherapy, 72% CHOP regiment.

Overall survival was 52% with a follow up 6–183 months. Bcl-2 expression was observed in 35,5% (22pts) and Ki67 positivity over 60% in 20 pts (30%). Overall survival was influenced by bcl-2 and Kli67 expression. Bcl-2 positive cases were significantly associated with lower overall survival (20% vs. 58%; p<0.01). Overall survuval was significantly worse in patients with Ki67 positivity over 60% (27% vs. 49%; p<0.05)

Bcl-2 and Ki67 monoclonal antibody immunostaging appears to be a simple and reproducible method of determining biological potential of tumor cells and provides useful prognostic information in patients with aggressive NHL.