bluuebunny/crossref_metadata_2025 huggingface dataset:
Mutation Analysis and Clinical Sprectrum Of Patients With Glanzmann’s Thrombasthenia and Bernard Soulier Syndrome
Authors: Tokgoz Huseyin , Caliskan Umran , Nejat Akar , Ak Erdem
Published: October 2019

Introduction and Aim

Glanzmann’s thrombasthenia (GT) is an inherited disorder of platelet aggregation, resulting from defective glycoprotein IIb/IIIa on platelet surface. Bernard Soulier Syndrome (BSS) is also an inherited disorder of platelet adhesion and associated with defective glycoprotein Ib-V-IX on platelet surface. Both of these disorders usually present with mucocutaneous bleedings. We aimed to evaluate the clinical and genetic characteristics of the patients diagnosed as BSS and GT in department of pediatric hematology of Meram Faculty of Medicine, retrospectively.

Method

Seven patients diagnosed with BSS and 20 patients diagnosed with GT were enrolled to the study. Medical records of patients were reviewed retrospectively. Glycoprotein IIb gen rearrangement was investigated in genetic department of Ankara University Medical School, Turkey. Mutational analysis for BSS was performed in Medicina Interna ed Oncologia Medica, Italy. The correlation between clinical outcome and genotyping was investigated.

Results

Of 20 patients of GT, 8 were male and 12 were female. Of 7 patients of BSS, all of them were female. Glycoprotein IIB gene rearrangement was detected in 7 patient of GT. 5 of 7 were newly described mutations in published literature. Mutations in GpIBB and GpIBA gene were detected in 7 patients with BSS. No correlation was observed among the clinical and genotype characteristics of patients both with GT and BSS. The most common patterns of bleeding were epistaxis and gum bleeding. Life threatening bleeding was seen in 5 of GT patients (4 gastrointestinal bleeding, 1 mediastinal hematoma) and 2 of BSS patients (1 splenic rupture and 1 gastrointestinal bleeding). No patients had died due to major bleedings. One patient with GT experienced spontaneous duodenal intramural bleeding resulting duodenal obstruction. One patient with BSS experienced spontaneous mediastinal hematoma. Although the BSS, it is interesting that the patient was able to control of mediastinal hematoma. Further investigations revealed the patient has prothrombotic mutation (heterozygous FV Leiden).

Conclusion

The most common bleeding pattern in patients with thrombocyte dysfunction is mucocutaneous bleeding. Some patients may suffer from life-threatening bleedings. Our study contributes to the literature because of five newly described mutations in GT patients. It may be hypothesed that the presence of prothrombotic mutation in patients with thrombocyte dysfunction may reduce the severity of bleedings.

Disclosures:

No relevant conflicts of interest to declare.
A Threshold Optical Density Value In Immunoassay Predicts Thrombosis In Patients with Heparin Induced Thrombocytopenia
Authors: Paul Asit K. , Fricke William , Kouides Peter A.
Published: October 2019

Heparin-induced thrombocytopenia (HIT) is an IgG mediated adverse reaction of heparin use associated with increased thrombosis. Heparin-platelet factor 4 (HPF4) enzyme immunoassay (EIA) detects all classes of antibody against HPF4 resulting in potential over-diagnosis of HIT. The objective of the current study was to determine an optimal threshold for diagnosis of HIT by EIA that would improve the diagnostic specificity and predict thrombosis. We retrospectively analyzed data of 101 consecutive patients who were tested positive based on conventional cut-off value of optical density (OD) obtained from EIA. Study patients were grouped as low (n=22), intermediate (n=26) and high (n=53) clinical probability group of HIT based on the 4T clinical probability scoring. An OD ratio (ODR) by EIA was calculated for each patient by dividing patient’s OD by the cut-off OD. Using low and high clinical probability as negative and positive diagnosis of HIT, a receiver operating characteristic curve was generated for the range of ODRs. An ODR of >2.5 provided the best trade-off between sensitivity and specificity. When a combination of clinical probability and ODR>2.5 were used to diagnose HIT, 20/26 patients with intermediate probability were reclassified as not having HIT. This reduced the overall diagnosis of HIT by 42% compared to that by EIA alone. Using this diagnostic criterion, the rate (58% vs. 10%, P<0.001) and the risk (Odds ratio 7.6, 95% confidence interval, 3.4-17.2) of thrombosis were significantly greater in patients with HIT than in patients without HIT. Thus, when patient’s OD 2.5 times greater than cut-off OD in EIA was combined with clinical probability for the diagnosis of HIT, it improved the diagnostic specificity and predicted thrombosis. This data also suggest the use of a universal ODR rather than an assay-specific cut-off for the diagnosis of HIT by EIA.

Disclosures:

No relevant conflicts of interest to declare.
Comparison Of Distant Relapse Rates Of Immune Thromocytopenia In Patients Treated With Steroids Versus Anti-Rh(D) with/Without Steroids Versus Rituximab with/Without Steroids
Authors: Varma Mala , Grossbard Michael L.
Published: October 2019

Background

Among therapies for immune thrombocytopenia (ITP) that achieve long unmaintained remissions, distant relapse rates have not been compared.

Specific Aims

To compare relapse rates following umaintained remissions exceeding 1 year in patients with ITP treated with steroids versus anti-Rh(D) with/without steroids versus rituximab with/without steroids.

Methods

Institutional Review Board approval was obtained. The charts of 52 consecutive patients with ITP followed in a hematology practice between 07/01/03 and 06/30/13 were reviewed. Remission was defined as a platelet count > 100,000/µL. Patients with the aforementioned treatment and remission characteristics were identified. Steroids were administered alone as dexamethasone 40 mg PO daily for 4 days. Anti-Rh(D) was administered as 75 mcg/kg IV with or without prednisone 60-85 mg PO daily tapered over 4 weeks or dexamethasone 40 mg PO daily for 4 days. Rituximab was administered as 375 mg/m2 IV weekly for 4 weeks or as a single dose with or without dexamethasone 40 mg PO daily for 4 days. Relapse was defined as a platelet count < 30,000/µL. Statistical calculations included ANOVA to compare demographics and chi-square contingency table analysis to compare distant relapse rates (http://www.physics.csbsju.edu/stats).

Results

There were 16 unmaintained remissions exceeding 1 year in 13 patients following treatment with steroids, anti-Rh(D) with/without steroids, or rituximab with/without steroids. Mean age on presentation, sex ratio, duration of ITP prior to therapy, and duration of follow-up after therapy were similar for the 3 groups (Table 1).

Distant relapse rates were 100%, 14.2%, and 66.7% for the steroid, anti-Rh(D) with/without steroid, and rituximab with/without steroid groups, respectively, P = 0.03 (Table 2).

Conclusion

Among patients who achieve long unmaintained remissions of ITP with steroids, anti-Rh(D) with/without steroids, or rituximab with/without steroids, those treated with anti-Rh(D)-based therapy are the most likely to have extended remission.

Disclosures:

Off Label Use: Rituximab therapy of ITP.
Surgeryand Invasive Procedure With Bypassing Agents In Hemophilic Patients With Inhibitors: A Single Center Experience
Authors: Park Young Shil , Ju Hee Young
Published: October 2019

Inhibitory antibodies to factor VIII (FVIII) or factor IX (FIX) are one of the most important complications of managing patients with hemophilia A and B. For hemophiliacs with inhibitors, recent advances in the use of bypassing agents such as recombinant activated FVII (rFVIIa, NovoSeven®) and Factor VIII Inhibitor Bypassing Activity (FEIBA®) have enabled them to aggressively manage their diseases in emergency or elective surgery. This report shows an updated evaluation of the safety and effectiveness of bypassing agents in the treatment of peri-operative bleeding in this patient population.

We reviewed the cases of patients with hemophilia and inhibitors underwent surgery or other invasive procedures between May 2008 and July 2013 using bypassing agents or high dose FVIII concentrates at a single center. Thirty two procedures (23 orthopedic surgeries and 9 other surgeries and procedures) were conducted in 16 hemophilia patients with inhibitors. The median age of the patients was 30.5 years old (range, 7-52). Most patients (14/16, 87.5%) are hemophilia A and 2 patients (12.5%) were hemophilia B. The median titer of inhibitors at procedures was 15 BU (range, 0.7-1900). Patients with low responding inhibitors were 2 cases, and high dose factor VIII concentrates were used. Most patients had high responding inhibitors and one of two bypassing agents was used. Twenty one cases were covered using FEIBA®, nine with rFVIIa initially. The median duration of hospitalization was 14 days (range, 1-58). In most cases, bleeding stopped or controlled well. But, in 5 cases, patients’ bleeding was controlled by sequential bypassing therapy. 4 cases were performed at emergency conditions, and those are segmental resection of small bowel, craniectomy with intracerebral hematoma removal, exploratory thoracotomy, and angiography with embolization. Efficacy of bypassing agents at various surgeries and procedures, based on final patient outcome, was 93.8% (30/32). Two deaths occurred at emergency condition as a result of hypovolemic shock secondary to intracranial hemorrhage or retroperitoneal bleeding.

Good control of hemostasis can be achieved with bypassing agents in hemophilia patients with inhibitors undergoing invasive procedures. If surgery or procedure would perform at emergency condition, more active and aggressive management should be needed. The use of bypassing agents in peri-operative period can allow invasive procedure and surgery to be safe and successful in hemophilic patients with inhibitors.

Disclosures:

No relevant conflicts of interest to declare.
Naturally Occurring Anticoagulants and Their Association With Thromboembolism In Patients With β-Thalassemia Major
Authors: Sultan Sadia , Irfan Syed Mohammed , Zeeshan Rozina
Published: October 2019

Introduction

Hypercoagulopathy and thromboembolic manifestations are being increasing acknowledged in transfusion dependent thalassemics; both intermedia and major. Studies in preceding decade have shown that hemostatic alterations including natural anticoagulant deficiency obligate thalassemic for thromboembolism. The aim of our study is to determine the status of natural anticoagulants and their association with thromboembolism during follow up.

Method

This is a prospective case-control study, during which 40 cases and 30 controls were registered between Jan 2009 to Dec 2009. Complete blood count, protein C, protein S, antithrombin, serum ferritin, liver function test; HbsAg and Anti HCV were determined. Patients were followed till 30th June 2012 for thromboembolic disease.

Data was entered and analyzed using SPSS version 17. The results were expressed as mean ± SD for quantitative variables and qualitative variables are presented as frequency & percentages. Student‘t’ test was applied for the comparison of means. We also computed spearman correlation at 5% level of significance to identify relationship between the deficiency of natural anticoagulants with maternal characteristics, hematological parameters and biochemical markers. Chi- square test was applied for correlation of prothrombotic markers with hepatitis B & C.

Results

The mean age of patients and control was 12.30±5.5 and 13.39±4.5 years respectively. There were 21 males and 19 females in patient group. The mean protein C, protein S and antithrombin in patients and control were 58.25±22.5 versus 110.67±22.60 (P<0.001), 67.90±19.58 versus 98.70±21.54 and 89.73±18.09 versus 104.0±10.98 (P<0.001) respectively. Protein C was exceedingly deficient in 65% followed by protein S & antithrombin in 35% and 20% respectively. Protein S deficiency revealed positive correlation with protein C deficiency and hemoglobin ≤ 8 gm% was correlated with antithrombin deficiency(P<0.05). No positive correlation of prothrombotic markers were established with others parameters evaluated. Till June 2012, 7 patients were lost to follow up and 2 died owing to cardiac failure. Of the 31 patients in regular follow up none has experienced thromboembolism both clinically and radiologically.

Conclusion

Decremented prothrombotic markers, primarily protein C, are implicated in elevated thrombotic risk in TM patients. However we did not encounter thromboembolism in our patients during follow up. We recommend prothrombotic screening and prophylactic anticoagulation in high risk group: bed bound splenectomized, cardiopulmonary complications and post operative.

Disclosures:

No relevant conflicts of interest to declare.
Thromboprophylaxis In Pregnancy: Are Current Guidelines Working?
Authors: Hodgins Mitchell , Hart Caroline , Brose Kelsey , Moodley Uthaman
Published: October 2019

Thromboprophylaxis in pregnancy poses many challenges for physicians given the potential complications to both mother and fetus. Given the difficulty of enrolling this population in appropriately designed studies, current guidelines rely on results extrapolated from other populations and observational studies. The purpose of this quality assurance project is to examine thromboprophylaxis in pregnancy and ensure that, by following current practice-based guidelines, we are meeting treatment goals by providing therapy that is both safe and efficacious.

A retrospective chart review was completed on all patients between 2008 and 2013 who were treated with prophylactic doses of anticoagulation during their antepartum/postpartum period. Each pregnancy was followed throughout gestation and postpartum to assess for thrombotic events, major bleeding (as per the International Society for Thrombosis and Haemostasis (ISTH) definition), and outcomes.

A total of 44 patients were reviewed. Of these, 21 (48%) met criteria for recurrent miscarriage and 19 (43%) had documented thrombophilia. 19 (43%) had a previous history of VTE, and 18 of the 19 (95%) previous VTE were associated with increased hormonal states. There were no antepartum or postpartum thrombotic events. 4 (9%) patients underwent Doppler ultrasound for clinical suspicion of DVT, but all were negative studies. There were no major bleeding episodes. 15 (34%) patients reported some form of minor bleeding, which did not meet ISTH criteria for major bleeds. Of the 21 pregnancies that met criteria for previous recurrent miscarriage, 15 (71%) had successful term deliveries, 1 (5%) had fetal loss, and 5 (24%) unknown outcomes.

Given the absence of recurrent thrombosis and major bleeding events in the population examined, it would appear that following current practice guidelines is both a safe and efficacious practice.

Disclosures:

No relevant conflicts of interest to declare.
Intricacies Of Treating Heparin Induced Thrombocytopenia With Preexisting Antiphospholipid Syndrome and Renal Insufficiency
Authors: Gohari Pouyan , Rubin Philip
Published: October 2019

Treatment of heparin induced thrombocytopenia (HIT) requires the use of alternatives anticoagulants, such as a direct thrombin inhibitor – argatroban. Use of argatroban relies on frequent laboratory monitoring of the activated partial thromboplastin time (aPTT) to maintain an adequate level of anticoagulation. This requirement poses a challenge in patients with prolonged baseline aPTT, such as in antiphospholipid syndrome. While other assays are being explored they require an extended turn around time because of their limited availability.

We present a case of a 63 year old male with a history of antiphospholipid syndrome, end stage renal disease on hemodialysis, hepatitis C, and on long term anticoagulation with warfarin for cerebral vein thrombosis. His hospital course required transition to unfractionated heparin and subsequently developed thrombocytopenia. A heparin-platelet factor 4 ELISA antibody assay was performed for an intermediate clinical likelihood of HIT and returned positive. Although not confirmed with the gold standard serotonin release assay (SRA), clinical suspicion for HIT obligated treatment with an alternative anticoagulant. Conventional dosing and administration of argatroban however could not be performed because of the patient’s prolonged baseline aPTT. Other agents such as fondaparinux were also not possible in the setting of renal insufficiency. Short of other treatment techniques accepted in this unique set of circumstances we practiced a fixed dose argatroban (0.5 mcg/kg/min for Child’s class B cirrhosis). The patient tolerated the empiric dosing well until discontinued because of a negative SRA. This case demonstrates the limitations of current treatment recommendations of HIT and need for further investigation in similar patients.

Disclosures:

No relevant conflicts of interest to declare.
Use Of Prothrombin Complex Concentrates In Warfarin Anticoagulation Reversal In The Emergency Department: A Quality Improvement Study Of Administration Delays
Authors: Bordeleau Simon , Marceau Daniele , Poitras Julien , Archambeault Patrick , Breton Carolle
Published: October 2019

Introduction

In some bleeding situations, quick reversal of warfarin anticoagulation is important. In the event of a major life-threatening bleeding event, the anticoagulation reversal delay can have an impact on mortality. This study aimed to improve the administration delay when using Prothrombin Complex Concentrate (PCC) for the emergent reversal of warfarin anticoagulation in the emergency department.

Methods

An audit and feedback quality improvement project was conducted in three phases: a retrospective audit phase, an analysis and feedback phase and prospective evaluation phase. The charts of all eligible patients in a single Emergency Department (ED) in Québec, Canada, who received 4-factor PCC since the introduction of this product in 2009 until October 31, 2011 were retrospectively audited with pre-planned evaluation criteria. The administration delay of PCC was calculated from the time of prescription to the time of administration. After this retrospective chart audit, we determined where improvements could be attained, gave feedback to the ED and the blood bank, and we created an action plan to ensure the timely administration of PCC. The action plan was then implemented in practice to reduce the administration delay. Finally, a six-month prospective evaluation study was conducted to determine if our action plan was followed and improved the administration delays.

Results

Seventy-seven charts were reviewed in the retrospective chart audit. The mean administration delay was 73.6 minutes (STD [34.1]) with a median of 70.0 minutes (25-75% IQR [45.0-95.0]). We found that this delay was principally due to the following barriers that prevented timely administration of PCCs: communication problems between the ED and the blood bank and reconstitution and delivery inefficiencies. In order to address these barriers, we developed an action plan that involved the following elements: a flowchart to remind all clinicians how to order PCCs and a new delivery method from the blood bank to the ED. During the 6 months following the implementation of our action plan, 39 patients received PCCs and the mean administration time decreased to 33.2 minutes (STD [14.2]) (p<.0001) with a median of 30.0 minutes (25-75% IQR [24.3-38.8]).

Conclusion

This audit and feedback quality improvement project involving the development and the implementation of an action plan comprising of a flowchart and a new delivery process reduced the administration time of PCC by more than half. Future studies to measure the impact of implementing a similar audit and feedback process involving an action plan in other centers should be conducted before this type of improvement process is implemented on wider scale.

Disclosures:

No relevant conflicts of interest to declare.
Induction of protein kinase C mRNA in cultured lymphoblastoid T cells by iron-transferrin but not by soluble iron
Authors: Alcantara O , Javors M , Boldt DH
Published: October 2019

Iron-transferrin (FeTF) is an essential growth factor required for proliferation of lymphoid cells. FeTF activates protein kinase C (PKC) in the lymphoblastoid T-cell line, CCRF-CEM. We have treated CEM cells with human FeTF, then examined levels of PKC mRNA by hybridization analysis using cDNA probes specific for alpha-, beta-, and gamma-PKC subspecies. CEM cell mRNA hybridized with the beta-subspecies probe but not with probes for alpha- or gamma-subspecies. After exposure to FeTF an increase in PKC-beta mRNA was detectable at 10 minutes, peaked at 12 hours, and was sustained for 72 hours. Nuclear transcription assays demonstrated that rates of PKC-beta mRNA transcription were increased in FeTF-treated cells. By contrast, steady state levels of PKC-beta mRNA did not increase after treatment of cells with apotransferrin or gallium TF. Similarly, treatment with soluble iron as ferric ammonium citrate did not increase steady state levels of PKC-beta mRNA, despite producing a marked increase in cellular ferritin content. Ferritin increased from a baseline value of 63 ng/10(6) cells to 98 and 100 ng/10(6) cells in CEM cells treated for 1 hour with ferric ammonium citrate or FeTF, respectively. FeTF did not increase cytoplasmic-free calcium in CEM cells loaded with fura-2, indicating that binding of FeTF to transferrin receptors did not open membrane Ca2+ channels or release intracellular Ca2+. In addition, pretreatment of cells with desferrioxamine, but not ferrioxamine, blocked the FeTF-induced increase in PKC-beta transcripts. Therefore, iron as FeTF (not soluble iron or nonferric TF) stimulates transcription of the CEM cell PKC-beta gene. Transcriptional rate of the PKC-beta gene does not correlate with cellular iron content as judged by ferritin measurements. Furthermore, the requirement for FeTF does not appear to reflect activation of a classic agonist pathway as judged by stable cellular Ca2+. These data suggest that delivery of iron by FeTF to one or more specific cellular compartments may stimulate PKC-beta gene transcription in CEM cells.
Expression of the c-myc proto-oncogene in multiple myeloma and chronic lymphocytic leukemia: an in situ analysis
Authors: Greil R , Fasching B , Loidl P , Huber H
Published: October 2019

The c-myc gene plays a pivotal role in mediating the competence state for cell cycle transversion. This biologic role is in contradiction to reports of elevated expression of the gene in multiple myeloma, a tumor with restricted self-renewal capacity. To more clearly define the role of this gene in plasma cells of myeloma patients, c-myc messenger RNA (mRNA) and/or oncoprotein expression were semiquantitatively analyzed on the single cell level in 19 cases of multiple myeloma, among them 1 biclonal case and 1 case with coexistent chronic lymphocytic leukemia (CLL). Performing anti-sense/mRNA in situ hybridization, mature c-myc gene transcripts were detected in 92% (12 of 13) of cases and could definitely be attributed to the plasma cells by our study. The number of Ki 67-positive plasma cells actively passing the cell cycle was less than 1% and independent of c-myc gene expression. However, because the presence of the 152-c-MYC epitope was correlated to extent of marrow plasmacytosis (r = .64; P = .043) and content of plasmablasts (P = .09), the c-myc gene might serve a function different from proliferative activity, but also associated with tumor cell mass. In CLL cells (21 of 22 cases) and their benign counterparts, ie, bone marrow and peripheral blood lymphocytes, the anti-sense/c-myc mRNA hybridization signals remained below the threshold considered as cutpoint between negative and positive. The low amounts of c-myc transcripts were correlated to neither stage of disease (P = .52) nor lymphocyte counts (P = .24). Because the numbers of peripheral blood lymphoma cells were independent of tumor mass and of c-myc gene transcripts expressed, peripheral blood lymphocytosis might more likely reflect homing processes than proliferative activity in CLL.
Hematologic effects of stem cell factor in vivo and in vitro in rodents
Authors: Ulich TR , del Castillo J , Yi ES , Yin S , McNiece I , Yung YP , Zsebo KM
Published: October 2019

Recombinant rat stem cell factor (rrSCF) administered to rats as a single intravenous injection causes a dose-dependent neutrophilia and lymphocytosis as well as the appearance of immature myeloid cells and occasional blast cells in the circulation. Neutrophilia begins at 2 hours, peaks at 4 to 6 hours, and subsides between 12 and 24 hours. Lymphocytosis occurs at 0.5 hours and has subsided by 2 hours. rrSCF- induced neutrophilia and lymphocytosis are abrogated by boiling, demonstrating that endotoxin-contamination of the rrSCF preparation is not responsible for the observed hematologic effects. The bone marrow at 6 hours after injection of rrSCF shows a left-shifted myeloid and erythroid hyperplasia as evidenced by significant increases in the absolute numbers of morphologically recognizable early myeloid and erythroid precursors. A concurrent decrease in the absolute numbers of mature marrow neutrophils is noted, suggesting that the release of marrow neutrophils contributes to the peripheral neutrophilia. After 2 weeks of daily injections of rrSCF, bone marrow smears demonstrate a remarkable mast cell hyperplasia accompanied by a decrease in total marrow cellularity and by a striking erythroid and lymphoid hypoplasia. rrSCF also causes mast cells to appear in the circulation and causes a systemic increase in embryonic connective tissue-type, but not mucosal- type, mast cells. In vitro long-term culture of lineage-depleted mouse bone marrow cells with rrSCF results in an almost pure outgrowth of mast cells.
Effect of chemical conjugation of recombinant single-chain urokinase- type plasminogen activator with monoclonal antiplatelet antibodies on platelet aggregation and on plasma clot lysis in vitro and in vivo
Authors: Dewerchin M , Lijnen HR , Stassen JM , De Cock F , Quertermous T , Ginsberg MH , Plow EF , Collen D
Published: October 2019

The murine monoclonal antiplatelet antibodies MA-TSPI-1 (directed against human thrombospondin) and MA-PMI-2, MA-PMI-1, and MA-LIBS-1 (directed against ligand-induced binding sites [LIBS] on human platelet glycoprotein IIb/IIIa) were conjugated with recombinant single-chain urokinase-type plasminogen activator (rscu-PA) using the cross-linking reagent N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP). The conjugates (rscu-PA/MA-TSPI-1, rscu-PA/MA-PMI-2, rscu-PA/MA-PMI-1, and rscu-PA/MA-LIBS-1), purified by immunoadsorption and gel filtration, were obtained with recoveries of 34% to 45%, with an average stoichiometry of 1.6 to 1.8 IgG molecules per rscu-PA molecule, and with unaltered specific activities and affinities. Preincubation of human platelet-rich plasma with rscu-PA/MA-PMI-2, rscu-PA/MA-PMI-1, or unconjugated rscu-PA resulted in partial inhibition of ADP-induced aggregation; 25% inhibition was obtained with 63 micrograms/mL rscu-PA and with 6 micrograms u-PA/mL rscu-PA/MA-PMI-2 or 1.2 micrograms u- PA/mL rscu-PA/MA-PMI-1. In an in vitro system composed of a 125I-fibrin- labeled platelet-rich human plasma clot immersed in normal human plasma, the conjugates had threefold to greater than 15-fold less fibrinolytic potency than unconjugated rscu-PA. The thrombolytic potency of rscu-PA/MA-PMI-1 and rscu-PA/MA-LIBS-1 was compared with that of rscu-PA and that of a control conjugate rscu-PA/MA-1C8 in a pulmonary embolism model in the hamster, using clots prepared from platelet-poor or platelet-rich human plasma. Lysis was measured 30 minutes after the end of a 60-minute intravenous infusion of the thrombolytic agents. rscu-PA, rscu-PA/MA-PMI-1, rscu-PA/MA-LIBS-1, as well as rscu-PA/MA-1C8 had comparable thrombolytic potencies (percent lysis per dose administered) towards platelet-poor human plasma clots. In contrast, the thrombolytic potency of rscu-PA/MA-PMI-1 and of rscu- PA/MA-LIBS-1 towards platelet-rich clots was 2.3- to 3-fold higher than that of rscu-PA (P less than .005) and fivefold to sevenfold higher than that of the control conjugate (P less than .01).
Investigation of the mechanisms of monoclonal antibody-induced platelet activation
Authors: Horsewood P , Hayward CP , Warkentin TE , Kelton JG
Published: October 2019

Antiplatelet antibodies can activate platelets causing platelet aggregation and the release reaction. However, the pathway of activation by these antibodies is unknown and several potential mechanisms are possible. In this report, we describe studies investigating potential pathways of platelet activation by IgG antibodies. We tested 16 different IgG monoclonal antibodies (MoAbs) against a variety of platelet surface components and found that six antibodies were capable of causing platelet aggregation and release. These included MoAbs against glycoprotein (GP) IIb/IIIa, CD9, GPIV, and two other not well-characterized platelet components. There was no relationship between the number of platelet binding sites and the ability of an MoAb to activate the platelets. By adding intact and F(ab')2 preparations of the MoAb to control or Fc receptor-blocked platelets, we found that in all instances the MoAbs initiated platelet activation via interacting with the platelet Fc receptors. Clustering of the platelet protein components using a secondary antibody did not cause activation. Studies into the pathway of Fc-dependent activation demonstrated that the MoAbs were capable of activating platelets by occupying Fc receptors on adjacent platelets (interplatelet activation), as well as on the same platelet (intraplatelet activation).
Neutrophils express the high affinity receptor for IgG (Fc gamma RI, CD64) after in vivo application of recombinant human granulocyte colony- stimulating factor
Authors: Repp R , Valerius T , Sendler A , Gramatzki M , Iro H , Kalden JR , Platzer E
Published: October 2019

Fc receptors are important effector molecules of neutrophilic granulocytes (polymorphonuclear neutrophils [PMN]), connecting phagocytic cells and the specific immune response. Neutrophils from healthy donors express the low-affinity receptors for IgG Fc gamma RII (CD32) and Fc gamma RIII (CD16), but not the high-affinity receptor Fc gamma RI (CD64). The latter has been found on neutrophils from patients with certain bacterial infections and can be induced in vitro after incubation with interferon-gamma. We show here that neutrophils strongly express Fc gamma RI after in vivo application of recombinant human granulocyte colony-stimulating factor (rhG-CSF). PMN from patients receiving rhG-CSF displayed higher cytotoxicity against Daudi lymphoma cells in vitro compared with control patients and with healthy donors. Fab fragments against Fc gamma RII (monoclonal antibody [MoAb] IV.3) inhibited neutrophil-mediated cytotoxicity of healthy donors but not of patients during rhG-CSF therapy. Therefore, expression of Fc receptors by PMN was investigated by flow cytometry and the mean fluorescence intensity (MFI) was compared. After staining with MoAb 32.2 against Fc gamma RL, the median MFI of neutrophils from G-CSF patients (median, 4.78; range, 2.40 to 8.50; n = 5) was significantly higher (P = .002 and P = .001, respectively) than the median MFI of patients not receiving G-CSF (median, 1.23; range, 1.01 to 1.58; n = 6) and the median MFI of healthy donors (median, 1.04; range, 0.67 to 1.12; n = 6). Fc gamma RI disappeared after the discontinuing of the G- CSF injections, but was reinduced during the next treatment cycle with rhG-CSF. The high expression of Fc gamma RI during rhG-CSF therapy correlated with enhanced cytotoxicity. In vitro incubation with rhG-CSF also enhances cytotoxicity, but only minor increments in Fc gamma RI expression were observed. Thus, during in vivo application of rhG-CSF neutrophils acquire an additional potent receptor for mediating tumor cell killing in vitro by induction of the high-affinity receptor for IgG (Fc gamma RI, CD64).
Progression and survival studies in early chronic lymphocytic leukemia
Authors: Molica S
Published: October 2019

To investigate the natural history of stage A chronic lymphocytic leukemia (CLL) we reviewed 84 such patients. Among 74 cases evaluable for disease progression, 22 (29.6%) progressed to more advanced clinical stages (9 to stage B, 13 to stage C); the actuarial estimation of such an event at 4 years was 30% (95% CI: 26.3% to 33.6%). Despite a linear trend toward an increasing risk (r = .92), the hazard function analysis showed a constant pattern of progression, suggesting a lack of correlation of such an event with time (r = .04). Furthermore, disease progression when analyzed as a time-dependent variable had a clear-cut impact on survival (P less than .001). With the aim of identifying a subgroup of patients with low probability of disease progression and death, we applied to our set of patients four different proposals for subclassifying stage A. All methods were similar in terms of sample size, 5-year survival rate, and disease progression risk, suggesting that the choice between different proposals is somewhat arbitrary. Whatever the criteria are for defining “smoldering” CLL, such patients (accounting in the present study for 20.5% of overall series and 46.7% of stage A patients) should not be treated until progression occurs.
Functional characterization of an abnormal factor XII molecule (F XII Bern)
Authors: Wuillemin WA , Huber I , Furlan M , Lammle B
Published: October 2019

An 18-year-old healthy woman was found to have cross-reacting material (CRM)-positive factor XII (F XII) deficiency, F XII clotting activity was less than 0.01 U/mL, whereas F XII antigen was 0.11 U/mL. An F XII inhibitor was excluded. To partially characterize the molecular defect of the abnormal F XII, immunologic and functional studies were performed on the proposita's plasma. The abnormal F XII was a single chain molecule with the same molecular weight (80 Kd) and the same isoelectric points (pl, 5.9 to 6.8) as normal F XII. Dextran sulfate activation of the proposita's plasma showed no proteolytic cleavage of F XII even after 120 minutes, whereas F XII in pooled normal plasma, diluted 1:10 with CRM-negative F XII-deficient plasma, was completely cleaved after 40 minutes. Adsorption to kaolin was identical for both abnormal and normal F XII. In the presence of dextran sulfate and exogenous plasma kallikrein, the abnormal F XII was cleaved with the same rate as normal F XII. However, kallikrein-cleaved abnormal F XII was not able to cleave factor XI and plasma prekallikrein, in contrast to activated normal F XII. Thus, these studies show that the functional defect of this abnormal F XII, denoted as F XII Bern, is due to the lack of protease activity of the kallikrein-cleaved molecule. Therefore, the structural defect is likely to be located in the light chain region of F XII, containing the enzymatic active site.
Platelet activation by a synthetic hydrophobic polymer, polymethylmethacrylate
Authors: Ware JA , Kang J , DeCenzo MT , Smith M , Watkins SC , Slayter HS , Saitoh M
Published: October 2019

Platelets adhere to artificial surfaces in the initial stage of thrombus formation, but the subsequent steps in signal transduction that lead to platelet activation by artificial surfaces are not understood. When 0.325-micron diameter beads composed of a hydrophobic polymer, polymethylmethacrylate (PMMA), were added to gel-filtered aequorin-loaded platelets suspended in media containing Ca2+, the platelets aggregated; addition of fibrinogen was not required. Platelet aggregation was preceded by an increase in cytoplasmic Ca2+ and was accompanied by phosphorylation of the 47-Kd substrate of protein kinase C (PKC), 5-hydroxytryptamine (5-HT) release, and accumulation of phosphatidic acid. All these effects were partially inhibited by apyrase and aspirin. Monoclonal antibodies (MoAbs) 7E3 and M148 and the synthetic peptides RGDS and fibrinogen gamma chain fragment 400–411, all of which bind to the platelet fibrinogen receptor glycoprotein IIb- IIIa (GPIIb-IIIa) and inhibit fibrinogen binding, prevented PMMA- induced aggregation but did not inhibit the Ca2+ increase. Chymotrypsin- treated platelets aggregated after addition of fibrinogen, but not PMMA. We conclude that platelets interact initially with PMMA at membrane sites other than those required for fibrinogen binding, leading to activation of membrane phospholipases and PKC, an increase in cytoplasmic Ca2+, release of 5-HT, ADP, and fibrinogen from storage granules, and to platelet aggregation.
Role of AUUU sequences in stabilization of granulocyte-macrophage colony-stimulating factor RNA in stimulated cells
Authors: Akashi M , Shaw G , Gross M , Saito M , Koeffler HP
Published: October 2019

RNAs for transiently expressed genes such as oncogenes and cytokines, including granulocyte-monocyte colony-stimulating factor (GM-CSF), have a short half-life (T1/2). A cluster of AUUU sequences identified in the 3′ untranslated (UT) region of these RNAs has been implicated in controlling stability of these transcripts. We examined the role of AUUU sequences in mRNA stability of GM-CSF after stimulation of cells. Human fibroblasts (W138) were stably transfected with chimeric constructs containing the beta-globin gene linked to a 52-bp tail of GM- CSF containing either eight ATTTT (pNEOR beta G-AT) or eight repeats in which the AT sequences have been changed to GC sequences (pNEOR beta G- GC). Data confirmed that AUUU sequences in 3′UT region of GM-CSF play a major role in GM-CSF RNA instability. Stimulators of protein kinase C (PKC), cycloheximide (CHX), sodium fluoride (NaF), and, to a more limited extent, interleukin-1 beta (IL-1 beta), appear to stabilize GM- CSF RNA through these AUUU sequences, but tumor necrosis factor-alpha (TNF-alpha) induces stabilization of GM-CSF RNA through a mechanism independent of their AUUU sequences.
High expression of the mdr3 multidrug-resistance gene in advanced-stage chronic lymphocytic leukemia
Authors: Sonneveld P , Nooter K , Burghouts JT , Herweijer H , Adriaansen HJ , van Dongen JJ
Published: October 2019

Chronic lymphocytic leukemia (CLL) is characterized by an often indolent course with a poor therapeutic response at advanced stage. We investigated the expression of the human multidrug resistance genes mdr1 and mdr3 in 31 patients with CLL. Using specific probes for mdr1 and mdr3 mRNA, respectively, expression of both genes could be found in 29 of 31 patients. Of those, nine had high expression of mdr1 and 13 of mdr3. Although 29 of 31 patients showed coexpression of mdr1 and mdr3, the mRNA levels were not interrelated. Prior treatment did not significantly influence the level of mdr1 or mdr3 expression. In patients with advanced CLL (Rai stage 3 + 4) the mdr3 expression was significantly higher than in early-stage CLL (Rai stage 0 to 2) (mean +/- SEM, 25.4 +/- 4.2 U v 4.2 +/- 1.1 U; P less than .0001). Such a difference was not present for mdr1 expression (21.5 +/- 4.3 U v 10.7 +/- 3.1 U; P = .09). These data indicate that advanced-stage CLL is associated with an increased mdr3 expression, which may concur with a decreased sensitivity to chemotherapy.
Hereditary Hyperferritinemia-Cataract Syndrome: Relationship Between Phenotypes and Specific Mutations in the Iron-Responsive Element of Ferritin Light-Chain mRNA
Authors: Cazzola Mario , Bergamaschi Gaetano , Tonon Laura , Arbustini Eloisa , Grasso Maurizia , Vercesi Elena , Barosi Giovanni , Bianchi Paolo Emilio , Cairo Gaetano , Arosio Paolo
Published: October 2019

Recent reports have described families in whom a combination of elevated serum ferritin not related to iron overload and congenital nuclear cataract is transmitted as an autosomal dominant trait. We have studied the molecular pathogenesis of hyperferritinemia in two families showing different phenotypic expression of this new genetic disorder. Serum ferritin levels ranged from 950 to 1,890 μg/L in affected individuals from family 1, and from 366 to 635 μg/L in those from family 2. Cataract was clinically manifested in family 1 and asymptomatic in family 2. By using monoclonal antibodies specific for the H and L ferritin subunits, serum ferritin was found to be essentially L type in both normal and affected individuals. The latter also showed normal amounts of H-type ferritin in circulating mononuclear cells; on the contrary, L-type ferritin contents were 13 times normal in family 1 and five times normal in family 2 on average. Serum ferritin was glycosylated in both normal and affected individuals. There was a close relationship between mononuclear cell L-type ferritin content and serum ferritin concentration (r = 0.95, P < .00001), suggesting that the excess production of ferritin in cells was directly responsible for the hyperferritinemia. The dysregulated L-subunit synthesis was found to result from different point mutations in a noncoding sequence of genomic L-subunit DNA, which behaves as an mRNA cis-acting element known as iron regulatory element (IRE). Affected individuals from family 1 were heterozygous for a point mutation (a single G to A change) in the highly conserved, three-nucleotide motif forming the IRE bulge. Affected members from family 2 were heterozygous for a double point mutation in the IRE lower stem. Using a gel retardation assay, the observed molecular lesions were shown to variably reduce the IRE affinity for an iron regulatory protein (IRP), which inhibits ferritin mRNA translation. The direct relationship between the degree of hyperferritinemia and severity of cataract suggests that this latter is the consequence of excessive ferritin production within the lens fibers. These findings provide strong evidence that serum ferritin is a byproduct of intracellular ferritin synthesis and that the L-subunit gene on chromosome 19 is the source of glycosylated serum ferritin. From a practical standpoint, this new genetic disorder should be taken into account by clinicians when facing a high serum ferritin in an apparently healthy person.
Tat–Human Immunodeficiency Virus-1 Induces Human Monocyte Chemotaxis by Activation of Vascular Endothelial Growth Factor Receptor-1
Authors: Mitola Stefania , Sozzani Silvano , Luini Walter , Primo Luca , Borsatti Alessandro , Weich Herbert , Bussolino Federico
Published: October 2019

Human immunodeficiency virus-1 (HIV-1) Tat protein can be released by infected cells and activates mesenchymal cells. Among these, monocytes respond to Tat by migrating into tissues and releasing inflammatory mediators. In the present study, we have examined the molecular mechanism of monocyte activation by Tat, showing that this viral protein signals inside the cells through the tyrosine kinase receptor for vascular endothelial growth factor encoded by fms-like tyrosine kinase gene (VEGFR-1/Flt-1). Subnanomolar concentrations of Tat induced monocyte chemotaxis, which was inhibited by cell preincubation with vascular-endothelial growth factor-A (VEGF-A). This desensitisation was specific for VEGF-A, because it not was observed with FMLP. In addition, the soluble form of VEGFR-1 specifically inhibited polarization and migration induced by Tat and VEGF-A, thus confirming the common use of this receptor. Binding studies performed at equilibrium by using radiolabeled Tat showed that monocytes expressed a unique class of binding site, with a kd of approximately 0.2 nmol/L. The binding of radiolabeled Tat to monocyte surface and the cross-linking to a protein of 150 kD was inhibited specifically by an excess of cold Tat or VEGF-A. Western blot analysis with an antibody anti–VEGFR-1/Flt-1 performed on monocyte phosphoproteins immunoprecipitated by an monoclonal antibody antiphosphotyrosine showed that Tat induced a rapid phosphorylation in tyrosine residue of the 150-kD VEGFR-1/Flt-1. Taken together, these results suggest that biologic activities of HIV-1 Tat in human monocytes may, at least in part, be elicited by activation of VEGFR-1/Flt-1.
flt3 Ligand in Cooperation With Transforming Growth Factor-β1 Potentiates In Vitro Development of Langerhans-Type Dendritic Cells and Allows Single-Cell Dendritic Cell Cluster Formation Under Serum-Free Conditions
Authors: Strobl Herbert , Bello-Fernandez Concha , Riedl Elisabeth , Pickl Winfried F. , Majdic Otto , Lyman Stewart D. , Knapp Walter
Published: October 2019

Using a recently described serum-free culture system of purified human CD34+ progenitor cells, we show here a critical cooperation of flt3 ligand (FL) with transforming growth factor-β1 (TGF-β1) in the induction of in vitro dendritic cell/Langerhans cell (DC/LC) development. The addition of FL to serum-free cultures of CD34+ cells supplemented with TGF-β1, granulocyte-macrophage colony-stimulating factor, tumor necrosis factor α, and stem cell factor strongly increases both percentages (mean, 36% ± 5% v 64% ± 4%; P = .001) and total numbers (4.4- ± 0.8-fold) of CD1a+ dendritic cells. These in vitro-generated CD1a+ cells molecularly closely resemble a particular type of DC known as an epidermal Langerhans cell. Generation of DC under serum-free conditions was found to strictly require supplementation of culture medium with TGF-β1. Upon omission of TGF-β1, percentages of CD1a+ DC decreased (to mean, 10% ± 8%; P = .001) and, in turn, percentages of granulomonocytic cells (CD1a− cells that are lysozyme [LZ+]; myeloperoxidase [MPO+]; CD14+) increased approximately threefold (P < .05). Furthermore, in the absence of TGF-β1, FL consistently promotes generation of LZ+, MPO+, and CD14+ cells, but not of CD1a+ cells. Serum-free single-cell cultures set up under identical TGF-β1– and FL-supplemented culture conditions showed that high percentages of CD34+ cells (mean, 18% ± 2%; n = 4) give rise to day-10 DC colony formation. The majority of cells in these DC-containing colonies expressed the Langerhans cell/Birbeck granule specific marker molecule Lag. Without TGF-β1 supplementation, Lag+ colony formation is minimal and formation of monocyte/macrophage-containing colonies predominates. Total cloning efficiency in the absence and presence of TGF-β1 is virtually identical (mean, 41% ± 6% v 41% ± 4%). Thus, FL has the potential to strongly stimulate DC/LC generation, but has a strict requirement for TGF-β1 to show this costimulatory effect.
Adenovirus-Mediated Transfer of Tissue-Type Plasminogen Activator Augments Thrombolysis in Tissue-Type Plasminogen Activator–Deficient and Plasminogen Activator Inhibitor-1–Overexpressing Mice
Authors: Carmeliet Peter , Stassen Jean-Marie , Van Vlaenderen Ilse , Meidell Robert S. , Collen Désiré , Gerard Robert D.
Published: October 2019

Impaired fibrinolysis, resulting from increased plasminogen activator inhibitor-1 (PAI-1) or reduced tissue-type plasminogen activator (t-PA) plasma levels, may predispose the individual to subacute thrombosis in sepsis and inflammation. The objective of these studies was to show that adenovirus-mediated gene transfer could increase systemic plasma t-PA levels and thrombolytic capacity in animal model systems. Recombinant adenovirus vectors were constructed that express either human wild type or PAI-1–resistant t-PA from the cytomegalovirus (CMV) promoter. Both t-PA-deficient (t-PA−/−) and PAI-1–overexpressing transgenic mice were infected by intravenous injection of these viruses. Intravenous injection of recombinant adenovirus resulted in liver gene transfer, t-PA synthesis, and secretion into the plasma. Virus dose, human t-PA antigen, and activity concentrations in plasma and extent of lysis of a 125I-fibrin–labeled pulmonary embolism were all closely correlated. Plasma t-PA antigen and activity were increased approximately 1,000-fold above normal levels. Clot lysis was significantly increased in mice injected with a t-PA–expressing virus, but not in mice injected with saline or an irrelevant adenovirus. Comparable levels of enzyme activity and clot lysis were obtained with wild type and inhibitor-resistant t-PA viruses. Adenovirus-mediated t-PA gene transfer was found to augment clot lysis as early as 4 hours after infection, but expression levels subsided within 7 days. Adenovirus-mediated transfer of a t-PA gene can effectively increase plasma fibrinolytic activity and either restore (in t-PA–deficient mice) or augment (in PAI-1–overexpressing mice) the thrombolytic capacity in simple animal models of defective fibrinolysis.