bluuebunny/crossref_metadata_2025 huggingface dataset:
Asymptomatic Factor VII Padua in African Americans.
Authors: Tidd Theresa M. , Ptashkin Beverly , Pollak Eleanor S.
Published: April 2020

Background: Symptomatology in congenital human FVII deficiency with FVII:C (Factor VII Coagulant Activity) levels < 1% ranges from asymptomatic to severe hemorrhagic problems. Additionally, FVII:C differs markedly depending upon the source of tissue factor (TF) in the thromboplastin utilized for measuring FVII:C. FVII deficiency reported in 1978 in patients from Padua, Italy was later shown to be associated with an arginine (R) to glutamine (Q) mutation at FVII amino acid 304. A study by Triplett et al. from 1985 reported FVII deficiency in 26 patients including 16 Caucasians, 1 Hispanic and 9 blacks. Of these patients, all 9 black patients were asymptomatic whereas the other 17 patients had a clinically relevant bleeding disorder. Since that time there have been no reports describing the genetic cause of FVII deficiency in asymptomatic African American individuals.

Methods: Plasma was obtained from patient blood samples obtained in 3.2% sodium citrate. FVII:C levels were performed using one-stage clotting assays with either rabbit brain thromboplastin (Simplastin Excel, Biomerieux, Durham, NC) or lyophilized recombinant human TF (Innovin, Dade Behring, Inc., Newark, DE). Factor VII antigen levels (VII:Ag) were measured using an enzyme-linked immunoabsorbent assay (American Bioproducts Co, Parsippany, NJ). A normal pool was constructed by mixing equal volumes of plasma from greater than 20 healthy control subjects.

Results Patient # Prothrombin Time in seconds FVII:C Simplastin Excel FVII:C Innovin FVII:Ag Genotype Phenotype 1-African American 15.6 16% 33% 61% Heterozygote R304Q Minor Nose Bleeds 2- African American Prolonged 26% 54% 67% Heterozygote R304Q Asymptomatic for bleeding 3- African American 14.5 21% 64% NA Heterozygote R304Q Asymptomatic for bleeding

Conclusions: We show that FVII deficiency in three asymptomatic African American patients at our specialty laboratory was due to FVII-Padua (R304Q). An additional ten African American patients showed prolonged prothrombin times and FVII:C values consistent with the laboratory phenotype of FVII-Padua. This diagnosis prevented inappropriate blood transfusions prior to surgery in several of these patients.
A Novel Congenital Bleeding Disorder Related to High Plasma Heparin-Like Anticoagulant Activity.
Authors: Wang Zhaoyue , Yang Haiyen , Bai Xia , Zhang Wei , Ruan Changgeng
Published: April 2020

Heparin or heparin-like compounds present in human plasma in minute amounts. It has been reported that a very few patients with such diseases as plasma cell neoplasms, acute monoblastic leukemia and acquired immune deficincy syndrome have an increased plasma heparin-like anticoagulant activity. Recently, we found a 10-year-old girl who was physically and developmentally normal, but had recurrent episodes of prolonged bleeding and hematoma starting in her early childhood, which could be stopped by transfusion of fresh frozen plasma or prothrombin complex concentrate. The coagulation tests of her plasma were regularly repeated since she was 2 years old, and always revealed a markedly prolonged APTT (61.8–104 seconds, normal 28–40 seconds) and TT (36–50.1 seconds, normal 14–21 seconds), and a slightly prolonged PT (15.9–25 seconds, normal 11–14.5 seconds). Fibrinogen, prothrombin and other coagulation factors as well as anticoagulant and fibrinolytic systems were all normal. The results of immunologic measurements were either negative or within normal ranges. Treatment of the patient’s plasma in vitro with either protamine or heparinase could completely normalize the coagulation abnormalities, but not with normal plasma. The anticoagulant activity of her plasma corresponded to 0.2 heparin U/mL when measured by a TT assay using normal plasma as substrate and standardized with porcine heparin. Her plasma heparin concentration was 0.22 heparin U/mL when measured using a colometric assay. In ex vivo study, the abnormal coagulation tests could effectively be corrected when the patient was intravenously administed with protamine. Considering these characteristic laboratory features of the patient, we suppose it would probably represent a novel congenital bleeding disorder related to high plasma heparin-like anticoagulant activity which, to our knowledge, had not been described before.
Increased Levels of Platelet-Derived Microparticles in Patients with DIC.
Authors: Kawasugi Kazuo , Noguchi Mizuho , Tashiro Haruko , Gotoh Moritaka , Shirafuji Naoki , Matsuda Juzo , Miyazawa Yukihisa , Kinoshita Tadatoshi
Published: April 2020

Circulating microparticles of various cell types are present in healthy individuals and individuals in various disease states. In these microparticles, platelet-dreived microparticles (PMPs) may be implicated in thrombosis and haemostasis. However, little is known about the role of PMPs in patients with DIC. To investigate the role of PMPs, we measured plasma levels of PMPs in patients with sepsis-associated DIC (n=20) and acute promyelocytic leukemia (APL)-induced DIC (n=5). Plasma samples from those patients groups were assayed for PMPs levels by enzyme-linked immunosorbent assay (ELISA, Japan Immunoresearch Laboratory, Japan). Also, sP-selectin levels were determined by ELISA.

The thrombin antithrombin complexes (TAT) levels were higher in both types of DIC patients as reported by others. In the septic patients with DIC, we found significant elevation in PMPs levels as compared with normal controls. However, we did not find any difference in plasma levels of PMPs in the APL patients with DIC. The sP-selectin levels were elevated in the septic patients with DIC. In early phase of septic patients with DIC, plasma levels of PMPs tend to be higher than that in late phase of septic patients with DIC.These results suggest that plasma levels of PMPs may be candidates for a marker of septic DIC. It appears that PMPs may contribute to the processes of septic DIC.

Sepsis associated DIC APL with DIC Controls (n=20) *(P<0.05) significantly different controls PMPs 34.1 ± 50.1* 9.6 ± 3.6 6.5 ± 0.9 U/ml TAT 25.4 ± 15.5* 22.5 ± 15.3* <;3 μ g/ml
Autoimmunity and Hypercoagulability in Anderson Fabry Patients from Argentina.
Authors: Aggio Mario C. , Martinez Pablo A.
Published: April 2020

Autoimmunity secondary to chronic immunostimulation by glucocerebrosides has been reported in Gaucher disease, as well as in Anderson Fabry disease (AFD) by others and us.

AIMS: To get further insight on this association, we looked for antinuclear and antiphospholipid-related autoantibodies in a series of AFD Argentine patients.

METHODS/PATIENTS: 35 patients, 16 hemizygous and 19 heterozygous from 4 families, were studied for: 1) autoantibodies to extractable nuclear antigens (ENAs): (SSA/RO, SSB/La, Sm/RNP, and Jo-1); 2) anti-double stranded DNA (dsDNA), and 3) antiphospholipid antibodies: lupus anticoagulant (LA), anticardiolipin antibodies (ACA), and antiphosphatidylserine antibodies, (APA).

RESULTS: At least one autoantibody was detected in 18 patients (51.4%). They were more frequent found in hemizygous (68.8%) than in heterozygous (42.1%). Elevated titers of ENAs were seen in 11.4% (n=4). Positive for LA were 34.3% (n=12) and for ACA 14.3% (n=5). Three patients shared two autoimmune markers. Anti dsDNA and APA were never present.

CONCLUSIONS:

There was an unexpected high incidence of autoimmune responses: one every two AFD patients showed at least one autoantibody; They were more frequent in hemizygous than in heterozygous cases. The pathologic implications of their presence is still not known, although it is probable that antiphospholipid antibodies contribute to hypercoagulability in AFD.

Autoimmunity seems to play an important role in some pathogenic disturbances seen in other lysosomal storage diseases. So, humoral/clinical correlations should be followed closely to detect the eventual outcome of collagen diseases or other clinical manifestations of autoimmunity in AFD.
Factor V G1691A Mutation and Factor II G20210A Variant in Children with Venous Thrombosis or Stroke. An International Database Update.
Authors: Nowak-Gottl Ulrike , Kurnik Karin , Manco-Johnson Marilyn , Young Guy
Published: April 2020

The present database pooling was performed to investigate the clinical phenotype in pediatric factor V G1691A (FV) and factor II G20210A (FII) carriers. 230 pediatric patients with deep venous thrombosis (DVT: n=143) or arterial ischaemic stroke (AIS: n=94) carrying either the FV (n=178) or the FII variant (n=52) not suffering from additional thrombophilic risk factors were consecutively enrolled from 2 US centers and one German database. Antithrombotic therapy was performed according to standard protocols either with LMWH/vitamin K-antagonists (DVT) or LMWH/ASS (AIS) for 6 months (range: 3–indefinitive). Patients were followed for a median of 5.4 years (range: 1–23). FV children with first disease onset were significantly younger compared with patients in the FII group (p=0.02), and showed more abdominal vein thrombosis than children with the FII variant (p=0.02).

Following withdrawal of antithrombotic therapy a first recurrent event was diagnosed in 12 of 178 children with the FV mutation (6.7%), whereas pediatric patients with the FII mutation showed recurrent thrombosis in 6 of 52 patients (11.5%). Median (range) relapse time was 6.5 years (0.1–15.2) in FV carriers compared with 15 years (13–16) in the FII group (p=0.06). Interestingly, recurrent thrombotic events were recorded significantly more often in children with DVT compared with AIS (p< 0.001).

In conclusion, the clinical phenotypes of FVC and FII are different with respect to first thrombotic onset, and thrombotic locations, and recurrent thrombosis in both phenotypes occurred more often in DVT compared with AIS.
IgA Anticardiolipin Antibodies as a Risk Factor for Arterial and Venous Thrombotic Events.
Authors: Duran Esther Urbaez , Kasunic Tim , Cotant Matthew , Kuriakose Philip
Published: April 2020

Background: Many studies have concluded that the presence of anticardiolipin antibodies is associated with both venous and arterial thrombotic events. The role of IgA anticardiolipin has been studied previously with conflicting results. Some of this disparity is thought to be due in part to the lack of international standards in measuring these levels.

Objective : To further evaluate the role of IgA anticardiolipin anitbody as a risk factor for thrombosis.

Patients and Methods: We conducted a single institution retrospective review of all patients with elevated IgA anticardiolipin antibodies extracted from a database of 20,000 patients for whom IgA anticardiolipin had been measured.

Results: One hundred forty-eight patients were identified with elevated IgA anticardiolipin antibodies as measured by semiquantitative indirect Elisa. Patients had a median age of 52.5; 52% were African American, 38% Caucasain and 9% of other ethnic background. Of these patients, 67 had associated arterial or venous thrombotic events: 15.9% were pulmonary embolisms, 47.5% were deep venous thromboses and 36.6% were arterial thrombotic events. Patients were further evaluated for other thrombotic risk factors including IgG and IgM anticardiolipin antibodies; lupus anticoagulant; pt 20210; activated protein C resistance/Factor V Leiden; hyperhomocysteinemia; antithrombin; protein C/protein S deficiency; active malignancy with or without concurrent chemotherapy; tamoxifen, oral contraceptive or hormone replacement therapy; catheter-related risk; pregnancy; myeloproliferative disorders and hyperviscosity syndromes; sickle cell disease; and surgery, trauma or immobilization within 6 weeks of the event. In 35.8% of evaluated patients with elevated IgA and thrombotic events, there was was no concommitant elevation of IgG or IgM anticardiolipin. In 33.3% of patients with thrombotic events, none of the other predetermined risk factors were present.

Conclusion: IgA anticardiolipin may be an independent risk factor for development of venous and arterial thrombotic events. A large case-controlled or prospective, randomized trial with standardized measurement will likely be needed to further clarify this issue.
Sticky Platelet Syndrome: The Clinical Spectrum of Thrombosis.
Authors: Bick Rodger L. , Fareed Jawed
Published: April 2020

Sticky Platelet Syndrome (SPS) was first described by Mammen, E., et al. In 1984, is inherited as an autosomal dominant and is associated with both arterial and venous thrombosis. It is also associated with placental vascular thrombosis and resultant recurrent miscarriage syndrome (RMS). It was thought to be of interest to assess the prevalence and types of thrombosis associated with SPS in a strictly thrombosis hemostasis referral practice over a one year period. During 2004, 599 first-time new thrombosis patients were seen, excluding recurrent miscarriage syndrome (RMS). Diagnosis of SPS was by the method of Mammen. Of 599 pts. 20.5% had SPS. The most common defect in the population was antiphospholipid syndrome and Factor V Leiden was seen in 8.5% of all pts. Of these SPS patients 60% suffered arterial (AA) and 39% suffered venous thrombosis. 4 pts. Had both. The types of thrombosis in descending order of prevalence were as follows: DVT = 27.6%, cerebrovascular thrombosis (CVT) = 19.5%, transient ischemic attacks (TIA) = 18.6%, PE = 12.1%, retinal artery thrombosis (RT) = 8.1%, mesenteric AA thrombosis = 7.3%, microvascular peripheral AA = 7.3%, coronary AA = 3.2% and popliteal AA = 0.8%. All pts. Were treated with low-dose ASA and those with CVT or TIA also received clopidogrel (75 mg. /day). At one-year follow-up, no recurrences were seen.

In summary: as has been noted by Mammen and others, SPS is a common hereditary thrombophilia associated with arterial and venous thrombosis. The prevalence in this population of first-time thrombosis pts. Was 2-fold that of Factor V Leiden mutation. Thus, a work-up for SPS should always be considered in pts. With unexplained thrombosis.
Warfarin Resistance Due to a Val29Leu Mutation in the VKORC1 Protein.
Authors: Huff Jason D. , Lawson Heather L. , Harish V. C. , Zhang Lin , Owen John
Published: April 2020

Warfarin resistance is a common clinical problem encountered by physicians of all subspecialities. Medical noncompliance and dietary indiscretions are felt to be the usual causes of a subject’s “resistance” to anticoagulation with coumarin derivatives. Warfarin’s target is the vitamin K epoxide reductase complex (VKOR). This complex functions within the vitamin K dependent γ-carboxylase system to recycle oxidized vitamin K. Until recently, the genetic sequence of components of this complex remained a mystery (Li et al., Nature 2004). Mutations in a subunit of this complex named vitamin K epoxide reductase complex subunit 1 (VKORC1) were isolated in human subjects with warfarin resistance (Rost et al., Nature 2004). Knowledge of this coding sequence has enabled us to study individual subjects suspected to have a genetic source for their warfarin resistance. Our study subject is a 24-year-old Caucasian female who received warfarin after developing bilateral pulmonary emboli. She required 30 mg of warfarin daily to achieve an international normalized ratio greater than 2.0. After institutional review board approval and with the patient’s informed consent, genomic DNA was extracted from the subject’s peripheral blood. The three exons comprising the VKORC1 gene were amplified by polymerase chain reaction (PCR). PCR primers were designed using the published sequence and online Primer3 software. Prolonged extension times were required to obtain good amplification of all three exons. After purification, the PCR products were sequenced and analyzed. The sequence analysis demonstrated a heterozygous G→T mutation in exon 1 at base pair 85. This results in a Val29Leu substitution, which has been described in a warfarin resistant patient by Rost et al. The second and third exons matched completely with the published sequence. The gene frequency of the mutation was then determined by examining 400 random, discarded DNA samples. The HypCH4 IV restriction enzyme cleaves at 2 positions in exon 1 in the wild-type genotype, but at just one position if our known mutation is present. The mutation was not identified in any sample.

In conclusion, we have identified a second patient with a Val29Leu substitution in vitamin K epoxide reductase resulting in warfarin resistance. Our analysis of 400 random samples has not identified any other example of this mutation, indicating that this is a rare occurrence. Genetic analysis of patients requiring more than 20 mg of warfarin daily would likely reveal more mutations in the VKORC1 protein. The identification of such mutations would provide valuable insight into the structure-function relationships of this protein complex.
Rituximab Immunotherapy for the Antiphospholipid Syndrome.
Authors: Bern Murray M.
Published: April 2020

The antiphospholipid syndrome (APS) is a heterogeneous disease expressed in a variety of ways, including thromboembolic disease. Therapy for this phase of APS is primarily anticoagulation. When the disease is aggressive and resistant to anticoagulants immunotherpay has been applied with variable success. Steroids, cyclophosphamide, plasma exchange, plasmapheresis, and immunoglobulin infusion have been used. This is a report of a patient with very aggressive APS treated successfully with rituximab immunotherapy. This 60 year old female had four episodes of deep vein thrombosis and two episodes of pulmonary embolus. An inferior vena cava filter had been placed. She came to hematologic attention in 2001, when the APS diagnosis was established based upon positive lupus anticoagulant and antibodies to cardiolipin and phosphatidyl serine. Long term steroid therapy was considered potentially too toxic. Azathioprine therapy was initiated at 100 mg daily and then increased to 150 mg daily without reduction of antibody titers. Thereafter she developed thrombosis in-situ in the pulmonary artery, requiring surgical thrombectomy. Therapy with rituximab was begun at 375 mg/m2 weekly times four, with repeat infusions every 6 months. Infusions were well tolerated. Warfarin therapy was continued throughout. Antibody titers have fallen.

Antibody titers after 3 cycles of therapy Anticardiolipin Baseline (mg/dl) 3 cycles of Therapy Followup (mg/dl) IgG 1032 854 IgM 255 <20 IgA 645 495 Antiphosphatidyl serine IgG 1620 775 IgM <25 <25 IgA 720 315

There have been no further episodes of TED. Therapy with rituximab has been well tolarted and appears to be effective. Therapy is continuing with additional antibody titers to be obtained.
Predictors of Red Cell Transfusion in Medically III Patients.
Authors: Yuen Maggie M. , Anderson David R. , Rose Caren
Published: April 2020

Although red cell transfusion is a relatively common treatment in medically ill patients, much is not known about the clinical determinants used to guide its use. We conducted a retrospective, case-controlled chart review of admissions to the medical teaching units (MTU) at this center (Queen Elizabeth II Health Sciences Centre, Halifax, Nova Scotia). Forty-two patients who received red blood cell transfusion during admission to the MTU from January to March 31, 2004. Clinical data collected included: age, gender, indication for MTU admission, co-morbid disease, smoking, admission hemoglobin, pre- and post-transfusion hemoglobin, arterial pressure of oxygen, lactic acidosis, parameters of shock, presence of active bleeding, and need for surgery. Logistic regression analysis was used to identify significant clinical determinants of transfusion. Red blood cell transfusion rate was 8%. Of admission diagnoses and co-morbidities, only cancer was associated with a trend for transfusion (p=0.07). Those who received red blood cell transfusion had significantly lower admission hemoglobin levels (p=0.005), higher serum creatinine (p=0.02), and lower mean arterial pressure (p=0.01). Significant predictive clinical factors of red blood cell transfusion included congestive heart failure (OR=3.72, CI 1.034, 27.160), admission hemoglobin level (d/L, OR=0.956, CI 0.927, 0.986), mean arterial pressure (mm Hg, OR=0.942, p=0.005) and serum creatinine (ml/min, OR=1.008, CI 1.003, 1.014). In conclusion, predictors of red blood cell transfusion for medically ill patients include low admission hemoglobin, renal insufficiency, and low blood pressure.
The Temporal Gene Expression Patterns of Embryonic Hematopoiesis after Hemangioblast Commitment.
Authors: Chen Dong , Lewis Rachel L. , Daigh Christine A. , Wang Ping , Kendzioski Christina M. , Chen Xin , Brown Patrick O. , Thomson James A.
Published: April 2020

Human embryonic stem (ES) cells provide a unique model for studying the development and function of human tissues. To better understand early development of the hematopoietic lineage, here we use microarray expression analysis to examine the temporal patterns of gene expression in embryoid bodies derived from human ES cells around the time when the definitive hematopoiesis peaks. We use an empirical Bayes hierarchical modeling approach, called EBarrays, to assess each of the possible temporal patterns of gene expression for the 5 time points examined, and correlate those patterns with the emergence of definitive hematopoiesis. We find that several genes known to be associated with early hematopoietic commitment, such as Flk-1 and TAL-1/SCL, are expressed 4-6 days before the peak colony formation cells (CFCs) production. The expression of a distinct group of genes previously identified as important in adult hematopoietic self-renewal (such as PIK3R1, ABCB1/MDR-1, RGS18, IRS1, SENP6/SUMO-1, and Wnt5A etc. correlated with the emergence of definitive hematopoiesis. Novel genes demonstrating the same expression pattern as this group are likely to have critical roles in the development and self-renewal of definitive hematopoietic precursors.
Cocultures of Umbilical Cord Blood Hematopoietic Progenitor Cells and Umbilical Cord Endothelium.
Authors: Moldenhauer Anja , Selders Lisa , Genter Gesche , Hieronymus T. , Ruau D. , Lun Andreas , Futschik Matthias , Kiesewetter Holger , Zenke Martin , Salama Abdulgabar
Published: April 2020

Purpose. The expansion of hematopoietic progenitor cells (HPC) from cord blood is limited so far. For HPC expansion and differentiation we evaluated a coculture model of cord blood-derived HPC and endothelial cells (EC) isolated from the umbilical cord.

Methods. CD34(+) HPC were cultured directly in contact with the endothelial monolayer or indirectly using a 0.4μm microporous transmembrane. EC were stimulated by various cytokines including TPO, interleukin (IL) 1β, IL4, and IL6. After 1 and 2 weeks, the HPC were counted and analyzed by flow cytometry. Their plating efficiencies were determined by methylcellulose assays, and their reconstitutive potential was analyzed by cobblestone assays. DNA microarrays on the expanding cells as well as on cytokine-stimulated EC were also performed.

Results. EC stimulation with thrombopoietin and IL4 had no significant effect on mononuclear cell expansion. In contrast, IL1β stimulation resulted in a 10 and 100fold increase of the cell number after one and two weeks, respectively. More than 90% of these cells were CD33(+) and had a plating efficiency equivalent to that of HPC post isolation (7.3 ±1.2 versus 8.3±0.68%). The number of 5-wk cobblestone-area forming cells among these expanded cells was diminished, whereas the number of 2-wk cobblestone areas did not change (10.6 versus 8.5). The expanded cells could further mature into functional granulocytes. Time-course DNA microarrays of IL1β-stimulated EC demonstrated an increased expression of genes encoding tumor necrosis factor-induced proteins, interleukins, and endothelial adhesion molecules. When IL1β was replaced by IL6, a 7-fold expansion of morphologically progenitor-like cells took place. 10–13% of these cells were positive for CD133, had a plating efficiency of 10.2±1.7%, and showed on average 14.5 five-week cobblestone areas.

Conclusion. Cocultures of cord blood-derived HPC with umbilical cord-derived EC offer alternative methods for the expansion and differentiation of hematopoietic cells.
Granulocyte-Colony Stimulating Factor Decreases Angina and Improves Exercise Capacity in Patients with Chronic Refractory ‘No Option’ Ischemic Heart Disease.
Authors: Ma David D.F. , Kovacic Jason C. , Dodds Anthony , Tao Helen , McDonald Peter , Feneley Michael P. , Muller David W. , Fruend Judith , Milliken Sam , Moore John , Itescu Silviu , Graham Robert M.
Published: April 2020

Granulocyte-Colony stimulating factor (G-CSF) is widely used in the treatment of hematological malignancies. The administration of G-CSF is a novel therapy for ischemic heart disease (IHD), but current data are controversial and the safety and efficacy of G-CSF in acute or chronic IHD is unclear.

Aim: to assess safety and efficacy of G-CSF administration and stem cell mobilization in patients with chronic refractory ‘no option’ IHD.

Methods: After baseline cardiac assessment (CA) [Seattle Angina Questionnaire (SAQ), Bruce exercise stress test (EST), persantin-Sestamibi and dobutamine-echocardiographic imaging], stable ‘no option’ IHD patients received open-label G-CSF 10μg/kg for 5 days, with an EST (to facilitate myocardial cytokine generation and stem cell trafficking) on the 4th and 6th days. After 3 months, CA and the same regimen of G-CSF and ESTs was repeated, but in addition, leucopheresis and a randomized double-blinded intracoronary infusion of either CD133+ or unselected cells was performed (randomized data remains blinded). Final CA was 3 months thereafter.

Results: Thirteen patients (12 male, 1 female, mean age 62) received 21 cycles of G-CSF. There were no deaths, Q-wave AMIs or any complications with long-term sequelae, although, transient troponin-I elevation (n = 3) and thrombocytopenia (n = 2) were observed. Mean CD133+ cell count rose from 1.28 to 56.12 x1012/L (p = 0.001). There was no age-related trend towards lower numbers of G-CSF mobilized CD34+ cells in the IHD patients, as compared to a group of younger (<60 y.o) normal male donors (n = 66). After the first cycle of G-CSF, SAQ angina frequency and physical limitation scores improved, and EST time increased (all p < 0.01). There was further but less marked improvement in each of these parameters after the second cycle of G-CSF and cell infusion. Overall, SAQ angina frequency score improved 46 points (95% CI +22 to +70, p = 0.003). This was reflected by reduced anginal frequency and nitrate use (both p < 0.005). Overall, SAQ physical limitation score improved 26 points (95% CI +17 to +35, p = 0.0003), EST time improved 97 seconds (95% CI +41 to +153, p = 0.005) and Duke Treadmill Score improved 4.3 points (95% CI −0.2 to +8.8, p = 0.058).

Conclusions: G-CSF and intracoronary cell infusion is safe in chronic ‘no option’ IHD patients. In this phase I study G-CSF improved anginal frequency, nitrate use and EST performance. A placebo controlled phase II trial investigating G-CSF with an appropriate cytokine stimulus is warranted.
Involvement of Lipid Raft Domain Signaling in Ex Vivo Maintenance of Hematopoietic Progenitor Cells.
Authors: Wirthlin Louisa , Meyerrose Todd E. , Creer Michael H. , Nolta Jan A.
Published: April 2020

Ex vivo manipulation of hematopoietic stem cell populations has been well documented to result in loss of pluripotentiality, proliferative deficits, and a decreased ability to repopulate post-transplantation. These defects result from a multitude of factors, and here we examine the contribution of lipid raft-localized signaling complexes and pro-survival growth factors, such as hepatocyte growth factor (HGF), in mediation of these defects. Specifically, we have examined the effects of diffused oxygen and hepatocyte growth factor (HGF) on ex vivo culture conditions for preservation of hematopoietic stem cell potentiality. We hypothesize that the high level of diffused oxygen in traditional cell culture systems represents a cellular stress leading to differentiation and loss of potential. We further hypothesize that this effect can be mitigated by the pro-survival factor HGF. Human umbilical cord blood derived mononuclear cells were first isolated by a ficoll density gradient followed by immunomagnetic depletion of mature cell lineages. These lineage depleted cells (Lin-) were cultured on fibronectin-coated plates in serum free media and kept in a 37° humidified atmosphere. We supplemented the cultures with stem cell factor (SCF), Flt-3, thrombopoietin (TPO), and HGF, and then manipulated the available oxygen for the cultured cells between 18% (ambient) and 2–3% (hypoxic). The cells were harvested at various time points (24h, 48h, 72h, and 96h) and assayed for lineage cell markers in conjunction with surrogate markers of lipid raft formation by flow cytometry. Preservation of phenotypic cell surface markers such as CD34, CD133, and CD31 were assayed, as well as the presentation of c-met, the receptor for HGF. Results were validated by immunohistochemistry and confocal microscopy for colocalization of receptor-raft complexes. We found that total lipid raft expression was upregulated in hypoxic conditions versus ambient, with a significant increase in a population of cells that highly expressed lipid raft markers. In addition, we found that cells cultured in the presence of HGF in ambient conditions showed a decrease in the presence of lipid rafts with a trend toward lower expression in those cells cultured with HGF. This population of cells demonstrated a specific preservation of CD34+ expressing cells, and decreased incidence of apoptosis in all compartment surveyed. Functional assays of colony-forming ability, SCID-repopulating activity, and competitive repopulation were also assayed, to correlate the upregulation of lipid raft domain presentation with relevant stem cell ability. Current focus of this investigation centers on the identification of the specific receptor signaling complexes associated with these raft domains.
Isolation and Characterization of Murine Novel cDNA Clone Using Newly Constructed Gene Trap Vector.
Authors: Shirasaki Ryosuke , Tashiro Haruko , Noguchi Mitsuho , Goto Moritaka , Kawasugi Kazuo , Shirafuji Naoki
Published: April 2020

Objective: To understand better the mechanism of the maintenance of immature state of stem cells, murine novel cDNA clones are to be isolated by the gene trap method.

Method: The trap vector was constructed with En-2 intron sequence followed by splicing acceptor sequence, IRES signal sequence and b-galactosidase cDNA. NeoR gene was also contained without poly-A additional signal. After electroporation into Embryonic stem (Es) cell lines (D3, and E14.1 Köln), neomycin-resistant Es clones were picked up, and were selected in which the expression of b-gal was detected in the undifferentiated condition cultured with leukemia inhibitory factor (LIF), and feeder layer cells by X-gal staining, but were not detected in the differentiated condition with ATRA, and without LIF nor feeder layers. Using 5′ RACE method, the transcripts from the selected clones were identified. One clone was further analyzed. Full-length cDNA was isolated from a library constructed from Es D3 cells, and its characterization was determined.

Result and Discussion: one unique full-length cDNA clone was isolated for the further characterization. It was constructed with 2225 nucleotides, and putative 466 amino acids were encoded in a single long open reading frame. The transcript was detected in the undifferentiated Es cells but not in differentiated Es cells nor in various kinds of organs by northern blotting analysis. When this clone was over-expressed in murine 10T1/2 cells, RT-PCR analysis demonstrated the down-regulation of Id-1 gene. These data suggested that the isolated cDNA clone was possibly related to keeping Es cells to be immature multipotent state. We now try to isolate the related cDNA clones which are expressed mainly in the hematopoietic stem cells.
Splicing Variants of Osteocalcin-mRNA in Haematological Malignancies.
Authors: Wihlidal Peter , Karlic Heidrun , Varga Franz , Nosslinger Thomas , Pittermann Elisabeth , Pfeilstocker Michael
Published: April 2020

Osteocalcin (OCN) is defined as a marker for osteoblasts but incompletely spliced variants as characterised by intron retention may also occur in non-osseous tissues including tumour cells. Bone marrow of haematological diseases was shown to harbour a stem cell candidate common to MSC (mesenchymal stem cells) and HSC (haematopoietic stem cells), which expressed OCN. In this study, we analysed cells from bone marrow samples obtained from 4 patients with AMLs, one patient with CML at blast crisis, one CML patient in chronic phase and 7 patients with myeloproliferative disease (MPD), as well as stem cells from peripheral blood and bone marrow obtained from healthy donors. In attempt to find out whether the presence of such stem cells is associated with disease status, sequential bone marrow samples from a patient with acute myeloid leukaemia (AML/M2Eo) were analysed at 6 time points including diagnosis, remission (= months 4, 5 and 17 after diagnosis) and relapse (months 29 and 41 after diagnosis). RT-PCR analyses were performed to characterise spliced and unspliced mRNA variants of OCN and to quantify mRNA levels of c-KIT and the Runt-transcription factors AML1 and AML3, known to regulate gene expression of c-KIT and OCN. Results of immunostaining for c-KIT and OCN showed that positive signals for OCN were detected only in those bone marrow smears, which expressed spliced OCN-mRNA, i.e. the bone marrow samples from the AML/M2Eo-patient in the relapse phase, the 2 samples of AML/M2, the sample of CML at blast crisis. The same samples and all others, which were positive for the unspliced OCN-mRNA, were also positive for c-KIT (both at the protein and at the mRNA level) as well as for AML1- and AML3-mRNA, however, at lower quantities. Our data indicate an association of OCNs expression with haematological malignancies at disease stages with activated bone marrow stem cells.
Cultured CD41a+ Megakaryocytes Derived from Cord Blood CD34+ Cells Are Activated Via STAT5 but Not STAT3.
Authors: Smith Stephen
Published: April 2020

Lengthy periods of thrombocytopenia and prolonged platelet recovery (typically, a side-affect of high-dose chemotherapy and/or radiation) have been associated with cord blood transplants. It maybe possible to alleviate the period of thrombocytopenia using cultured megakaryocytes derived from CD34+ stem cells however, platelet development invitro is delayed. Using immuno-magnetic beads, CD34+ stem cells were isolated from Cord Blood (CB) units and then cultured in serum-free culture medium (X-VIVO-10®, BioWhittaker Co. Walkersville, MD) supplemented with Thrombopoietin Peptide Agonist (TpoA), Stem Cell Factor (SCF), Flt-3L, and Vascular Endothelial Growth Factor (VEGF). A series of culture experiments were setup to investigate the activation of the JAK-STAT pathway following cytokine stimulation. Using flow cytometry, CB CD34+ cell cultures were evaluated weekly for the expression of CD41a, STAT3, STAT5, and Proliferating Cell Nuclear Antigen (PCNA) at week 1–6 of culture. Typically, CD41a expression peaked at week 3 of culture. At week 3, the total cells were 14.1 ±4.9 x106 cells per culture with 48.8 ±12.5% CD41a+ cells or 6.4 ±1.2 x106 CD41a+ cells per culture. Similarily, PCNA activation peaked at week 3–4 (64.1 and 62.4%, respectively). The level of PCNA activation increased incrementally from weeks 1, 2 and 3 of culture however declined at 5 and 6 weeks of culture. However, there was an incremental increase in the activation of STAT5 during the 6 weeks of culture. The STAT5 expression did not peak until week 5–6 (80.9 and 73.8%, respectively). Conversely, STAT3 expression declined between week 1 and 6 from 3.2. to 0.4%, respectively. With the exception of weeks 1 and 2, there was only minor activation or no activation of STAT3. These results suggest that STAT5 but, not STAT3, is activated during CB megakarycyte differentiation. These results may provide incite as to CB megakaryocyte development and might explain the delayed production of platelets following transplant. The PCNA results suggest that the cultured cells reach their greatest proliferative potential at week 3 and 4 of culture. This might provide useful information as to the optimal time period for the transplant of the cultured cells and suggest that cells transplanted from 5 and 6-week cultures have less proliferative capacity post-transplant. Additional studies are needed to determine the differentiation of megakaryocytes and platelet development from CB CD34+ cells invitro.
Expression of Runx1 Isoforms in Sub-Populations of Human Blood and Bone Marrow Cells.
Authors: Yankelevich Maxim , Margolin Judith F. , Goodell Margaret A.
Published: April 2020

Runx1 is a pivotal regulator of hematopoiesis. Deficiency of Runx1 leads to complete block of definitive hematopoiesis in mouse embryos. Runx1 protein forms a heterodimer with CBFβ and binds to the specific DNA sequence TGT/cGGT to regulate the expression of a number of hematopoietic genes. Runx 1 has been recognized to have three different isoforms (a, b, and c) due to differential splicing and promoter utilization. The distal (Runx1c) promoter region was shown to have binding sites mediating repression of Runx1 transcription. Data on the pattern of Runx1 isoforms expression in different hematopoietic lineages are scant. The functional impact of different Runx1 isoforms on commitment and differentiation of blood cells is also essentially unknown. To further explore this, we studied Runx1 isoform expression in different subpopulations of human hematopoietic cells. Total RNA was extracted from peripheral blood and unstimulated bone marrow. Also, Lin-CD34+CD38+, and Lin-CD34+CD38− cell populations were sorted from G-CSF-stimulated bone marrow and total RNA extracted. Expression of Runx1 isoforms was assessed by semiquantitative RT-PCR analysis, using GAPDH as a control. All 3 isoforms were present in all studied cell populations. Runx1a was uniformly a minor form with lowest expression. Runx1c isoform was a dominant one in peripheral blood as opposed to Runx1b in bone marrow. Most interestingly, the ratio of Runx1b to other isoforms was significantly increased in Lin-CD34+CD38- subpopulation compared to Lin-CD34+CD38+ cells. The prevalence of Runx1c isoform in peripheral blood as compaired to bone marrow is most likely related to a different cellular composition of these tissues and suggests that Runx 1c isoform may be predominantly expressed in some of the mature cell lineages. Since Lin-CD34+CD38− cells represent most primitive progenitor cell population with multi-lineage repopulating ability, the observed overexpression of Runx1b may be related to transcriptional accessibility of these cells. Upregulation of Runx 1b with G-CSF in this subpopulation can not be ruled out as well. These preliminary data suggest that the expression of Runx1 alternative splicing isoforms is both lineage and differentiation stage-specific in human hematopoietic cells. We will also test whether Runx1 isoform expression has a consistent pattern in different types of leukemia and if it is resembles such of mature blood counterparts or primitive progenitors. These studies, as well as the studies of differential expression of Runx1 isoforms in different mature blood lineages and progenitor cell sub-populations are underway and results will be presented. These data may further shed a light on functional differences in Runx1 isoforms and mechanisms of hematopoiesis.
PMA Responsive Sequence (s) in Gαq Promoter during Megakaryocytic Differentiation.
Authors: Jalagadugula Gauthami S. , Dhanasekharan Danny , Rao A.Koneti
Published: April 2020

Human erthroleukemia cells (HEL) differentiate towards megakaryocytic (MK) phenotype when stimulated with phorbol 12-myristate-13-acetate (PMA). We observed that the expression of Gq, a protein that plays a major role in platelet signal transduction, is increased in PMA-treated HEL cells. Western blotting revealed that Gq is upregulated in PMA-treated cells relative to untreated cells. Gq gene induction by PMA treatment was investigated with respect to transcriptional control. Serial 5′-truncations of the upstream region (upto 2727 bp from the ATG) of Gq gene were fused to a luciferase (Luc) reporter gene vector, PGL-3 Basic, and were transiently transfected into HEL cells in the absence and presence of PMA (10 nM). After 24 h, reporter gene activities were measured using Dual Luciferase Reporter Assay System (Promega). A reporter plasmid −1042 bp-Luc with a genomic region −1042/−1 showed a 12 fold activity in PMA treated cells and 4 fold activity in untreated cells. Its truncated plasmid with the genomic region −1036/−1 showed a decrease in luciferase activity by 50% in treated cells; and the activity became identical to that in untreated cells. Further truncation between −1036 and −1011 caused a complete loss of activity in both the cells. Thus, a PMA responsive element was localized to a region between −1042 and −1037 bp. Transcription factor data base search (TFSEARCH) predicted two consensus sites for early growth response factor EGR-1 at -1042/−1031 and −1026/−1015. Gel shift studies were performed with two oligos, −1042/−1012 and −1036/−1012, and nuclear extracts from PMA- treated and untreated cells. The studies with −1042/−1012 probe and extracts from treated cells showed that there was nuclear protein binding, which was abolished by competition with the consensus EGR-1 sequence. In extracts from untreated cells, the protein binding was observed but was not competed with consensus EGR-1 sequence. This suggests EGR-1 binding to the region −1042/−1012 in PMA-treated cells and role for this transcription factor in inducing Gq promoter activity. Moreover, studies on the region −1036/−1012 showed nuclear protein binding that was identical between extracts of untreated and treated cells, and it was not competed with consensus EGR-1 sequence. These findings suggest that, EGR-1 binding is localized to −1042/−1037, but not to −1036/−1012.

Conclusion: A PMA responsive sequence (−1042/−1037) was identified in the Gq promoter. Our studies suggest that EGR-1 binding to this sequence confers the PMA responsive activity. These studies provide further evidence that EGR-1 plays an important role in the upregulation of Gq expression during PMA induced megakaryocytic differentiation.
Morbid Risks of Unnecessary Red Blood Cell Transfusion in Stable Coronary Artery Bypass Graft Patients.
Authors: Moehnle Patrick , Snyder-Ramos Stephanie A. , Weng Yi-Shin , Kulier Alexander , Boettiger Bernd W. , Wang Shirley , Levin Jack , Mangano Dennis T.
Published: April 2020

The persistent variability in red blood cell transfusion practice in coronary artery bypass graft (CABG) patients, despite established guidelines, suggests inappropriate use. Our objective was to determine the impact of postoperative red blood cell (RBC) transfusion in entirely stable CABG patients. We investigated a cohort of 940 stable CABG patients from the 5,065 patients enrolled in the Multicenter Study of Perioperative Ischemia Epidemiology II (EPI II) Study with (1) low to moderate risk profile; (2) postoperative hemoglobin levels ≥ 10 g/dL; (3) minimal postoperative blood loss, and (4) no evidence of any morbid event on the day of surgery. RBC transfusion was assessed during the first 24 postoperative hours and multiorgan outcomes as well as markers of resource utilization from postoperative day one to hospital discharge. Transfused patients (N =190, 20.2 %) were more likely to suffer myocardial infarction (OR, 1.89; 95 % CI, 1.08 to 3.29; P = 0.02); renal dysfunction (OR, 3.35; 95 % CI, 1.01 to 11.1; P = 0.04); renal failure requiring dialysis (OR, 4.01; 95 % CI, 0.99 to 16.2; P = 0.05); and/or harvest site wound infection (OR, 5.45; 95 % CI, 1.87 to 15.9; P = 0.001). RBC transfusion was shown to be an independent predictor of composite morbidity outcome, cardiac morbidity, and harvest site wound infection in multivariate analysis. In transfused patients, the mean duration of hospitalization was increased by 2.5 days (14.4 days vs. 11.9 days; P < 0.001). In CABG patients with low to moderate mortality risk profiles, adequate hemoglobin values and low bleeding rates, postoperative RBC transfusion is associated with an increased risk for cardiac, renal and infectious morbidity, as well as increased health care expenses, without any detectable benefit.
Role of Fas / FasL in Regulation of Basophillic Erythroblast Homeostasis.
Authors: Capocasale Renold J. , Makropoulos Dorie A. , Arlen Jeffrey , Achuthanandam Ram , Quinn John , Volk Amy L. , Bugelski Peter J.
Published: April 2020

Many studies have shown Fas- Fas Ligand (FasL) mediated apoptosis to be important in maturation and differentiation of erythroid precursors in vitro. To determine if there is a similar process regulating erythropoietic homeostasis in vivo, we studied erythropoiesis in Fas(lpr) and FasL (gld) deficient mice. We postulated that deficiency of Fas or FasL should result in changes in red blood cell (RBC) parameters and/or decreased levels of apoptosis of erythroblasts. To test this hypothesis under steady state conditions, blood and bone marrow were collected from 10-week old C57Bl/6 control mice, B6.MRL-Tnfrsf6 lpr /J CD95 deficient mice, and B6Smn.C3-Tnfsf6 gld /J CD95L deficient mice. Hematology was studied using a Bayer Advia 120 and femoral bone marrow was analyzed by 6-color flow cytometry using a Becton Dickinson FACSAria. Hematologic analysis revealed no differences in reticulocyte counts, RBC counts or hemoglobin (Hgb) in either lpr or gld mice compared to C57Bl/6 controls. Similarly, analysis of bone marrow revealed no differences in % of Ter-119bright CD71bright basophilic erythroblast (BEB), % apoptotic BEB (annexin V+, 7-AADdim) or % FasL+ BEB in either gld or lpr mice compared to control. As expected, lpr mice expressed 10 fold fewer Fas+ BEB while similar levels were observed in gld mice compared to controls. To test our hypothesis under stimulated conditions, control, lpr and gld mice received a single s.c. dose of 10,000 units of recombinat human erythropoietin (rhEPO). Bone marrow samples were collected 48 hours after dosing and blood samples 4, 8 and 16 days after dosing. Hematologic analysis revealed no differences in the erythropoietic response among the three strains of mice tested. Moreover, treatment with rhEPO had no effect on % Fas+ BEB in any strain, but induced a 2–5 fold increase in the % FasL+ BEB and a 2–3 fold increase in apoptotic BEB in all three strains. Based on our observations, we conclude Fas/FasL is unlikely to play a pivotal role in regulating erythroid homeostasis.