bluuebunny/crossref_metadata_2025 huggingface dataset:
A Phase II Trial of Bexarotene, a Retinoid X Receptor Agonist, in Non-M3 Acute Myeloid Leukemia
Authors: Tsai Donald E. , Potuzak Melissa , Mato Anthony , Loren Alison Wakoff , Porter David , Stadtmauer Edward A. , Goldstein Steven , Perl Alexander , Swider Cezary , Bagg Adam , Youssef Emile , Luger Selina
Published: April 2020

In vitro, bexarotene inhibits the proliferation of non-M3 AML cell lines and induces differentiation of leukemic blasts. Our previous phase I study in non-M3 AML showed evidence of leukemic response as manifested by reduction in bone marrow blast counts (15% response rate), improved platelet counts (41%) and improved neutrophil counts (26%). Based on these results, a phase II trial in non-M3 AML was initiated at the phase I MTD. In the current phase II trial, bexarotene (300mg/m2) was administered daily as monotherapy until disease progression or unacceptable side effects occurred. Fourteen patients have been enrolled: 8M/6F, median age 74 (range 20–83), 9 secondary AML (MDS or prior chemotherapy), 9 primary refractory or relapsed < 1 year after induction, 5 no prior induction chemotherapy, 5 requiring hydroxyurea at the time of enrollment for leukemic blast control, 4 prior allogeneic stem cell transplant, 12 blood transfusion dependent, 11 platelet transfusion dependent, and 8 neutropenic. Overall, no significant adverse events were noted. All patients received prophylactic antihyperlipidemic agents and achieved good lipid control. Two patients developed mild hypothyroidism related to bexarotene. Five patients were evaluable with bone marrow biopsy at 2 months: 1 50% reduction in absolute blasts, 1 SD and 3 PD. Similar to data from our prior phase I study, evidence of clinical activity was manifested as platelet count response in 1 patient and neutrophil increases attributable to bexarotene in 2 patients. When combining the results of our phase I experience (27 patients) with our phase II data (14 patients), there is a suggestion of increased activity in patients with 5q minus abnormalities with 4/7 (57%) benefiting (2 BM response, 4 neutrophil improvements and 1 platelet response). Conversely rates of clinical benefit were lower in patients with multiple (>3) cytogenetic abnormalities (2/13), relapse after stem cell transplant (1/9) or requiring hydroxyurea for peripheral blast control at the time of study enrollment (0/10). Bexarotene is very well tolerated at the dose level studied. Early evidence for clinical activity has been seen as exemplified by improvement in platelet count, increased neutrophil counts and decreased bone marrow blasts. In summary, we conclude that bexarotene is an active agent in a subgroup of patients with AML. Study enrollment continues with amended inclusion criteria to focus on patients more likely to benefit from treatment.
AVE5026: A New Hemisynthetic Ultra Low Molecular Weight Heparin with Enriched Anti-Xa Activity and Enhanced Antithrombotic Activity for Management of Cancer Associated Thrombosis
Authors: Hoppensteadt Debra , Jeske Walter , Walenga Jeanine M. , Fareed Jawed
Published: April 2020

AVE5026 is an ULMWH with a high anti-Xa activity associated with residual anti-IIa activity (ratio anti-Xa/IIa>30). This ratio may be favorable for use in cancer patients as it may have a higher therapeutic window. This agent is a polydisperse mixture of oligomeric heparin fragments (molecular weight 2000–3000 daltons) prepared by partial and controlled depolymerization of porcine mucosal heparin (UFH).. The anticoagulant profile of AVE5026 has been previously reported (Hoppensteadt, et al. Blood 2007). To validate the hypothesis that AVE5026 is an appropriate antithrombotic in cancer, studies were carried out to determine its antithrombotic, bleeding, tumor growth, angiogenesis and apoptosis effects in animal models. AVE5026 produced a dose-dependent antithrombotic response in the rat laser model of microvascular thrombosis (2.5 mg/kg IV, 9±1.7 laser shots; 5.0 mg/kg SC, 8±1.6 laser shots; control 3±0.8 laser shots). The relative bleeding effects of AVE5026 in a rat tail bleeding model were negligible in comparison to enoxaparin (E) and UFH (all 5 mg/kg IV; UFH >1000 sec; E 360±57 sec; AVE5026 209±29 sec; Control 215±32 seconds). Repeated administration of AVE5026 in dose ranges of 1–5 mg/kg od, SC for 14 days did not result in accumulation of the anti-Xa activity. Furthermore, in contrast to E, there was no enhancement of the hemorrhagic profile. Both E and AVE5026 inhibited tumor growth by regulating angiogenesis and apoptosis in a Lewis Lung carcinoma (14% vs. 22% decrease) rat model and in a Walker 256 carcinosarcoma model (21% vs. 30% decrease) Although comparable results to E were demonstrated in animal models of arterial thrombosis, the bleeding effect of AVE5026 were disproportionately lower. This superior safety-efficacy ratio may translate into improved antithrombotic efficacy with decreased bleeding risk, suggesting that AVE5026 may provide the optimal treatment strategy for the management of cancer associated thrombosis.
Prevention of Venous Thromboembolism in Medically Ill Patients: Global and Individual Adherences Adjust to Risk Groups
Authors: Grand Beatriz , Orosco Jorge , Cacchione Roberto , Duarte Patricio , Riveros Dardo
Published: April 2020

Introduction: Venous thromboembolism (VTE) is an important problem for medical patients. In spite of the existence of evidence-based guidelines for prevention of VTE, prophylaxis for venous thromboembolism is underutilized.

Objective: To assess the global and individual adherences (adjust to risk groups) to venous thromboembolism prophylaxis guidelines (VTEPG) in medical ill patients.

Desing: Prospective observational study.

Setting: The 50-bed medical floors of a university hospital.

Material and Methods: Patients: All consecutively medically ill patients (pt) admitted during a 6-month period (n: 452).

VTEPG: the guide divided medical patients in risk groups (G); G1: No need of VTEP (35 pt); G2: myocardial infarction (9 pt); G3: Stroke (21 pt); G4: high risk with thrombophilia, previous VTE, cancer (113pt); G5: respiratory and congestive heart failure, pulmonary infection (137 pt); G6: trauma (5 pt); G7: not included in previous groups, required an individual evaluation to asses VTE risk (112pt).

Prophylaxis methods included: low dose unfractionated heparin (UFH) or low molecular weight heparin (enoxaparin 40 mg/d) and mechanical methods.

Main outcome measurements: 1- Global: correct application of the guide (defined by a coincidence between the prophylaxis received and the recommendation); 2-Adjust to risk group.

Results: Global adherence: Correct application of the guide was noted in 252 pts (56%);

Adjust to risk group: GR1:71%; GR2:44%; GR3:62%; GR4:76%; GR5:55%; GR6:60% and GR7:26%.

Conclusion:

In our experience in medical patients global adherence to guides was 56%, the best adherence was observed in high risk groups 76% and the lowest 26% in those patients that need an individual risk assessment. This study and follow up permitted us to detect inaccurate prophylaxis uses and take corrective measures.
Warfarin-Induced Skin Necrosis in Vasculitis
Authors: Corinaldesi Giorgio , Corinaldesi Christian
Published: April 2020

Warfarin-induced skin necrosis (WISN) is a dangerous and rare complication 0.01–0.1% of patients, typically occurs in middle-age between the 3rd and 10th days after initiation of therapy, it is characterised by the lack of vascular or perivascular inflammation; early histology shows cutaneous infarcts, haemorrhages of precapillary arterioles, fibrin deposit in the postcapillary venules within the walls, absence of arterioral thrombosis which are distinctive features. We have studied two patients: a 48-years old women with AF, and a 59 years old women with deep vein thrombosis treated with warfarin (INR of 2.5–3.0 was achieved). Clinically the patients developed pain and paresthesia, erythematous flush and hemorrhagic bullae and full-thickness with skin necrosis, the patients developed ecchymoses and purpura 2 weeks later the beginning of therapy, There were multiple and symmetric lesions reported over lower abdomen and upper extremities, buttocks, thighs, breast and areas of increased subcutaneous fat, painful lesions occurred suddenly. The disorder occurs more commonly in individual who are overweight, and/or with an underlying thrombophilia. The laboratory screen demonstrated ATIII, antinuclear antibody, ANCA, absence of anticardiolipin antibodies, and C- and S-protein deficiency, one patients had factor V-Leiden mutation. The differential diagnosis is with necrotizing fascitis, venous gangrene, heparin induced thrombocytopenia, Fournier’s gangrene, purple toe syndrome, cryofibrinogenemia. The management was conservative, warfarin was discontinued, intravenous heparin was started (2mg/Kg/day), fresh frozen plasma was given with corticosteroids; as an alternative, long term therapy included fondaparinux. Surgical debridement of the necrotic tissue is needed usually, followed by secondary healing, split thickness skin autograft or flap.
Lack of Impact on Platelet Increments by Number of Apheresis Platelet Units Transfused
Authors: Patel Anush , Bernstein Zale P. , Kepner James , Patel Anil , Ahluwalia Manmeet S , Becker Joanne L
Published: April 2020

Introduction: Prophylactic platelet transfusions are often given when a predetermined platelet count is reached and the amount given is that necessary to achieve a desired post count. This method of transfusion ignores the question of whether the patient receives increased benefit from transfusion when more than one unit is given. To determine what effect this practice has had on transfusion outcome and platelet utilization, we have reviewed 5 years of platelet transfusion data to look specifically at transfusion events when more than one single donor platelet was given during a 24 hour period.

Methods: Transfusion data was obtained from that used for utilization review 2002 –2006. The data was sorted to identify records indicating that 2 or more units of apheresis platelets were received in a 24 hour period. This information was further selected to include only those with hematologic diseases. The platelet counts identified for inclusion were the counts prior to the release of the first unit given during the day and the first count after the release of the last unit. The change in the platelet count for the number of units was recorded and averaged for each unit increment for each record. Statistical analysis was performed and was not statistically significant.

Results For the 5 year period, 86% of all platelet transfusions at our institution were given to patients with hematologic diseases and 27% of these were given as 2, 3, or 4 units within a 24 hour period. 716 records had 2377 days with multiple transfusions; 1802, 428, and 147 episodes of 2, 3, and 4 units respectively. The increase in platelet count for a single unit transfusion at our institution averages 32.3 ×109/L. The platelet increments for 2, 3, and 4 units showed no statistical increase from this number or each other. There is no trend for the final platelet count to be greater when more units are given. These results do not take patient condition or the use of specialized products into consideration.

Discussion: Our experience suggests that transfusions of multiple platelet units may not have a greater impact on platelet increments than that when fewer units are transfused. To fully understand this data and its potential impact on patient care, further subgroup analysis within the platelet increments and correlation with patient condition and the prescribed transfusion parameters is needed.

Figure Figure
Stability of Activated Recombinant Human Factor VII (rFVIIa) after 28 Days Storage at 24° C and 43° C
Authors: Dubick Michael A , Dorfman Ryan , Klemcke Harold G , Kheirabadi Bijan S , Holcomb John B
Published: April 2020

The stability of rFVIIa (Novo Seven) was evaluated over a 28 day period. Lyophilized recombinant factor VIIa (1.2 mg/vial configuration) powder was tested at recommended storage conditions of 4°C or accelerated stability testing conditions of 24°C and 65% ± 5% relative humidity (RH) and 43°C plus 65% ± 5% RH. No significant change was noted in the appearance of any of the rFVIIa samples based on turbidity and visual appearance of the lyophilized cake and reconstituted liquid product. rFVIIa activity was determined by bioassay using a modified prothrombin time (PT). Chemical integrity of the rFVIIa product was determined by reverse-phase HPLC and by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). No significant changes were observed in the activity of any of the 28 day-stored samples based on the modified PT as compared to the initial baseline 4°C samples. Reverse Phase-HPLC detected a single primary peak that accounted for 97% of the material. No significant degradation of this peak was observed in any of the 28 day-stored samples compared to the initial baseline 4°C samples. Non-reduced SDS-PAGE revealed a 50,000 molecular weight band and reduced SDS-PAGE showed 30,000 and 20,000 molecular weight bands. No significant changes were observed in the protein bands in any of the 28 day-stored samples analyzed by reduced and non-reduced SDSPAGE as compared to the initial baseline 4°C samples. Taken together, these data suggest that in vitro rFVIIa activity and chemical integrity of the lyophilized powder remain intact for at least 28 days even at 43°C with 65% relative humidity.
Increased Levels of Anaphylatoxin (C5a) and Bradykinin in End-Stage Renal Disease Patients on Maintenance Hemodialysis. Potential Relevance to Heparin Mediated Hemodynamic Responses
Authors: Adiguzel Cafer , Bansal Vinod , Cunanan Josephine , Litinas Evangelos , Hoppensteadt Debra , Fareed Jawed
Published: April 2020

Besides the upregulation of inflammatory mediators, end stage renal disease (ESRD) patients maintained on hemodialysis are subjected to periodic exposure to heparin and contact activation due to procedural settings. Recently the presence of a heparin contaminant, namely hypersulfated chondroitin sulfate was linked with the adverse reactions and deaths observed in these patients (Kishmoto, et al. N J Med 2008). To validate this report we measured both the C5a anaphlatoxin and bradykinin levels in ESRD patients prior to and after maintenance hemodialysis. The control group comprised of 40 normal healthy individuals were included to establish the normal level of these mediators. A sandwich ELISA method utilizing a monoclonal antibody which is specific for human C5a and bradykinin were used in these studies. Both the C5a and bradykinin were elevated in pre-dialysis samples from ESRD patients (C5a: 3.2±0.6 ng/ml vs 14.2± 4.6 ng/ml, bradykinin: 6.4±1.8 ng/ml vs 9.3±2.4 ng/ml). Moreover, dialysis itself produced an increase in both the C5a and bradykinin levels. Moreover, the postdialysis samples were further increased, suggesting that dialysis and heparinization itself result in the up-regulation of these mediators. Supplementation of heparin to the plasma also resulted in the generation of both C5a and bradykinin. The plasma samples included in these studies represents patients who were not treateded with the contaminant heparin. Additional studies on in-vitro generation of these markers with contaminated heparin and the isolated contaminant showed that both of these triggered the generation of C5a and bradyknin. These results suggest that both C5a and bradykinin are up-regulated in ESRD patients and this level can be further augmented by dialysis and heparinization. Therefore, additional factors may have contributed to the complex adverse reaction profiles and deaths in patients administrated with contaminated heparin.
Analysis of Children with Coagulation Test Abnormality in Pre-Surgical Evaluation
Authors: Kwon Seung Yeon , Han Jung Woo , Won Sung Chul , Song Jaewoo , Lyu Chuhl Joo
Published: April 2020

Prothrombine time (PT) and activated prothrombine time (aPTT) are common tests used for screening of coagulation function before surgical procedures. We analyzed underlying causes of abnormal coagulation test results which were incidentally found during pre-surgical evaluation in healthy patients without definite bleeding history. Total 58 children referred to pediatric hematoloy service for abnormal PT and aPTT results in pre-surgical evaluation between June 2006 and May 2008 were analyzed retrospectively by review of medical records. 50 patients showed aPTT prolongation, 5 patients PT prolongation, 2 patients PT and aPTT prolongation and another three patients showed normal PT and aPTT. Among 55 patients with abnormal results, 25 patients (43%) were recovered spontaneously during their follow up tests, 17 patients (29%) showed lower level of certain coagulation factor than reference range and the other 13 patients were lost during follow up despite of recommendation for further evaluation. Mean value of international normalized ratio (INR) for PT and aPTT of the patients recovered spontaneously were 1.05±0.11, 44.53±5.01seconds(s), and 1.12±0.11, 47.0±5.36s in patients with lower level of coagulation factor, showing significant increase of PTT in patients with lower factor levels (p<0.05). Median time required for spontaneous recovery was four weeks and 18 patients (72%) were recovered within this time. Among 17 patients with lower level of certain coagulation factor then reference level, there were 11 patients with low factor XII level, three patients with low factor VIII level, three patients with low von Willebrand factor, two patients each for low factor VII and factor XI and one patient with low factor V level. Among them three patients with low level in von Willebrand factor, one patient with low factor VII and two patients with low factor XI showed deficient level of coagulation factors requiring factor replacement for the surgical procedures. From this analysis of patients with incidentally found PT or aPTT prolongation, 43% of patients were spontaneously recovered during follow up period within 4 weeks in median. However, we also found that 29% of patients had relatively lower level of coagulation factor than reference range. Even though most of them were factor XII decrease which is not closely related with bleeding tendency, six patients had significant deficiencies of coagulation factors requiring factor replacement during surgical procedures. These results suggest that we should keep following up and undergo adequate evaluation for underlying coagulation factor deficiencies in patients who have sustaining PT and aPTT prolongation abnormalities despite of absence of any bleeding history.
Involvement of Heparanase in Early Pregnancy Losses
Authors: Nadir Yona , Henig Israel , Vlodavsky Israel , Brenner Benjamin
Published: April 2020

Heparanase activity is implicated in cell invasion, tumor metastasis and angiogenesis. Recently, we have reported that heparanase stimulates tissue factor (TF) expression in endothelial and cancer cells, resulting in elevation of coagulation activity (Nadir et al. JTH, 2006). We also found that heparanase releases tissue factor pathway inhibitor (TFPI) from endothelial and tumor cells, contributing to the local procoagulant balance (Nadir et al. TH, 2008). Heparanase was cloned from and is abundant in the placenta. We investigated the role of heparanase in the placenta, focusing on its effect on TF, TFPI, TFPI-2, and vascular endothelial growth factor-A (VEGF-A). Twenty formalin embedded samples of early pregnancy losses (weeks 5–10) were studied. Ten cases were miscarriages of women with thrombophilia and recurrent fetal losses, and ten control cases were pregnancy terminations. Applying real time RT-PCR and immunostaining for heparanase, TF, TFPI, TFPI-2, and VEGF-A it was found that in fetal losses related to thrombophilia the levels of heparanase, VEGF-A and TFPI-2 were significantly increased (2–3 fold) compared to placentas from controls. Next, JAR (human choriocarcinoma throphoblast) cells were transfected to overexpress the full length (65 kDa) heparanase or incubated with active (50+8 kDa) recombinant heparanase. Immunoblotting and real time RT-PCR showed that throphoblasts overexpressing or incubated with exogenous heparanase exhibited a 2–3 fold increase in TFPI and TFPI-2 in cell lysates both at the protein and mRNA levels, with no detectable effect on VEGF-A and TF. Accumulation of TFPI and TFPI-2 in the cell culture medium was increased 5–6 fold, exceeding the observed induction of TFPI and TFPI-2 gene transcription. Extracellular accumulation of TFPI and TFPI-2 was already evident 60 min following heparanase addition, prior to protein induction, indicating an active release of preformed proteins. These results indicate a regulatory effect of heparanase on TFPI and TFPI-2 in throphoblasts, suggesting a potential involvement in early miscarriages.
Coagulation Induced by Dilute Tissue Factor Depends More Critically on Factor II and X Concentration Than on FVII or FIX
Authors: Gudmundsdottir Brynja R. , Bjornsdottir Alexia M , Onundarson Pall T.
Published: April 2020

Prothrombin time based tests used to monitor coumarin anticoagulation measure the initiation phase of fibrin formation (clotting time, CT) in platelet poor plasma. Additional information can be obtained using computerized rotational thromboelastometry (ROTEM) which also measures the consequent propagation phase (measured as maximum velocity, Vmax), and the stabilization phase (maximum clot firmness, MCF).

We used ROTEM to study the effect of individual vitamin K dependent (VK) coagulation factor (F) concentration on clotting induced by dilute thromboplastin in platelet poor and platelet rich plasma (PPP and PRP).

As expected, FII, FVII and FX in PPP equally affected the prothrombin time whereas FIX did not. In PPP the ROTEM CT, however, was affected more by the concentration of FII and FX than by FVII (data not shown). To imitate physiological clotting better, factor deficient platelet poor plasma was repleted by adding frozen platelets in an optimal concentration corresponding to 100 × 109/L.. In the PRP the ROTEM CT was more dependent on the concentration of FII than of FVII, more dependent on FX than on FII and not dependent on FIX at all (left figure). The Vmax (reflecting the propagation phase) was also more dependent on FII and factor X than on the concentration of FVII or FIX which both may demonstrate a threshold effect (right figure). The stabilization phase or maximum clot firmness (MCF, not shown) was influenced similarly by FII and FX but was not influenced by FVII or FIX.

Figure Figure

Conclusion: During deficiency of vitamin K dependent coagulation factors the concentration of FII and FX may be more critical for hemostasis than FVII or FIX concentration. This may have practical implications for the appropriate choice of monitoring assays during coumarin administration.
Human Hepatic Stellate Cells Carry a New Splice Variant of the Human ADAMTS13
Authors: Kimchi-Sarfaty Chava , Blaisdell Adam , Marple Andrew , Roma Isabella , Newell Jordan , Dobkin Alexandra , Allen Courtni , Friedman Scott , Burge Christopher , Shomron Noam
Published: April 2020

The ADAMTS13 gene plays an integral role in vascular homeostasis. A severe deficiency in ADAMTS13 triggers a rare disorder known as thrombotic thrombocytopenic purpura (TTP). Various missense and nonsense point mutations have been described in exonic regions of ADAMTS13 along with several frameshift deletions and one frameshift insertion. Additionally, several intronic mutations have been discovered in USA patients that result in aberrant mRNA splicing forms. A tissue based screen of ADAMTS13 alternative splicing has never been assessed. Besides the known mRNA splice isoforms, others are evident from the Expressed Sequence Tag (EST) databases. We analyzed alternative splicing of ADAMTS13 in several tissues in order to assess whether alternative splicing might play a role during its gene expression. We identified a novel isoform of ADAMTS13 that retains intron 25, evident in human, but not in mouse, hepatic cells. Moreover, the retained intron is not detected in mice possibly because they do not possess regulatory sites inserted into the human intron at a later evolutionary stage. This might suggest that ADAMTS13 is post-transcriptionally regulated at the splicing level. It also shows the need for future analysis of transcript levels of this new isoform in TTP patients. We hypothesize that irregular balance between the two splice isoforms might affect ADAMTS13 functionality, thus, it is possible that our findings might be of clinical significance.

“The findings and conclusions in this presentation have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any Agency determination or policy.”
Use of Amphipathic Helical Peptides as An Anticoagulant
Authors: Charbonneau Sophie , Peng Henry , Shek Pang N , Blostein Mark
Published: April 2020

We have previously shown that an ideal amphipathic helical peptide of K7L15 composition (IAP) accelerates factor IXa-mediated factor X turnover and factor Xamediated prothrombin turnover in a phospholipid free system (Biochem J., 2008, 412:545). Under these conditions, IAP behaves as a phospholipid membrane allowing coagulation factors to bind and exert their actions. However, when IAP is used with in vitro assays that employ phospholipids such as an active partial thromboplastin time (aPTT), IAP paradoxically behaves as an anticoagulant by prolonging clotting times. We hypothesize that this anticoagulant effect occurs by blocking binding sites for coagulation factors on phospholipids membranes. To test this hypothesis, we employed three phopholipid-dependant coagulation assays, the aPTT, dilute PT and dilute RVV, with both low and high concentrations of phospholipids. We show that these coagulation times are prolonged by IAP in a concentration dependent manner and that this prolongation is abrogated by adding excess phospholipid, demonstrating phospholpid dependence for this inhibition. In purified tenase and prothrombinase assays, in the presence of phospholipids, IAP inhibits substrate turnover consistent with our hypothesis. To show direct binding between IAP and phospholipids, we conducted fluorescence spectroscopy experiments and show direct binding between IAP and phospholipid membranes. In summary, the above data demonstrate that IAP acts as an anticoagulant by blocking the interaction of coagulation factors with phospholipids membranes.
Effect of Human Recombinant VWF Administration on Plasma ADAMTS13 Activities in Non-Human Primates and Rabbits
Authors: Varadi Katalin , Rottensteiner Hanspeter , Muchitsch Eva M , Weber Alfred , Gritsch Herbert , Turecek Peter L. , Ehrlich Hartmut J , Schwarz Hans Peter
Published: April 2020

Von Willebrand factor (VWF) is composed of a series of multimers, the sizes of which are regulated by the plasma metalloprotease ADAMTS13. Reports suggest that a transient increase in VWF levels, triggered for instance by DDAVP treatment, will result in a decrease in ADAMTS13 activity. This phenomenon is widely believed to be due to ADAMTS13 being exhausted when confronted with an excess of substrate. Treating patients who have type 3 von Willebrand disease with a recombinant human von Willebrand factor (rVWF) that has not yet been exposed to ADAMTS13 might thus cause a drop in ADAMTS13 levels. We therefore tested whether a rise in VWF concentrations to supraphysiological levels caused by administration of rVWF could influence endogenous ADAMTS13 levels in animal models. Various doses of human rVWF (300, 600, and 1200 RCo IU/kg BW) were injected into rabbits and cynomolgus monkeys and plasma samples were collected at a range of time points. As expected, VWF antigen rose sharply in a dose-dependent manner (~25 IU/ml VWF:Ag for the highest dose, 15 min after injection) and then declined gradually (~7 IU/ml VWF:Ag for the highest dose, 18 hours after injection). When these samples were tested for ADAMTS13 activity using the FRETS assay, no relevant changes were observed throughout the entire test period in the rabbit or in the monkey samples, indicating that rVWF, even at high doses, did not compromise ADAMTS13 activity. Both rabbit and cynomolgus ADAMTS13 recognized human rVWF as the specific cleavage products were detectable by electrophoresis at all doses administered. These animal studies thus indicate that an excess of intravenously administered rVWF leading to supraphysiological levels does not exhaust ADAMTS13.
Aplastic Anemia, Pesticide Exposure and Response to Cyclosporine Monotherapy: A Single Institution Case Controlled Study
Authors: Malhotra Hemant , Singh Ajay Pal , Rishi Jai Prakash , Khandelwal Purshottam D
Published: April 2020

Introduction:The present study is a prospective, hospital-based, case-controlled study of 19 patients of acquired Aplastic Anaemia (AA) from the SMS Medical College Hospital, Jaipur, India.

Patients & Methods:All newly diagnosed patients of Aplastic Anaemia over a one year period starting from Jan 2005 were included in the study. Detailed demographic parameters including a careful evaluation for pesticide exposure, haematological profile, serum organochlorine levels and response to oral Cyclosporine-A (Cyc-A) were recorded as per a preset Performa. Patients were followed for a minimum of 12 months. Patients receiving stem cell transplantation (SCT) and anti-thymocyte globulin (ATG) were excluded from the study. Serum quantitative and qualitative estimation of organochlorine pesticide levels by gas chromatography were done in all patients at study entry and compared with age and sex matched normal control.

Observations & Results:

Of the 19 patients included in the study, 15 (78%) were males and the mean age was 23 years (range 7 to 64 years) in the study. Thirteen of the 19 patients (68.4%) were from rural versus urban habitat and 9/19 (47.4%) gave a history of pesticide exposure. All these were from rural background. Six patients (31.6%) had non-severe AA, 9/19 (47.4%) had severe AA and 4/19 (21%) had very severe AA. Overall response to Cyc-A at 3 months was 47.4% [complete response (CR) 10.6%] and at 6 months was 47.4% (CR 21.1%). Non-severe AA patients had a response rate of 83.3% versus 33.3% in the severe AA and 25% in the very severe AA group. Cyc-A was generally well tolerated with mild toxicity. No patient was taken off the drug because of toxicity. No patient required serum Cyc-A level monitoring. Serum organocholorine levels were significantly higher (p<0.005) in the AA patients (mean 0.35 ppm) versus controls (0.22 ppm). Mean serum organoclorine levels appear to correlate with the severity of AA. Patients with higher serum organochlorine levels were less likely to respond to Cyc-A therapy.

Conclusions:Even though this is a relatively small, single-institution study, the following conclusions can be drawn:

Single agent Cyc-A can produce a response in close to half of AA patients within 3 months of starting treatment. Considering the ease of administration and the low toxicity, Cyc-A could be considered as initial therapy in all patients of AA in a resource-limited setting (as compared to ATG and SCT). Pesticide exposure may play a role in the aetiopathogenesis of AA, particularly in patients with occupational exposure to the agent. Pesticide exposure and elevated serum pesticide levels may identify patients of AA who will not respond to immunotherapy (Cyc-A).
Is the Early Cyclosporine a Level Predictive to the Outcome of Immunosuppressive Therapy in Severe Aplastic Anemia?
Authors: Song Moo-kon , Chung Jooseop , Choi Young-Jin , Seol Young-Mi
Published: April 2020

Although there are many recent studies of efficacy of antithymoglobulin (ATG) combined with cyclosporine A (CsA), there has been no study to clarify the correlation between variability of CsA level and the response rate of immunosuppressive therapy (IST). Therefore, we assessed whether the early CsA level would be important or not for IST in acquired severe aplastic anemia (SAA), retrospectively. Thirty one patients were treated with ATG combined with CsA for 6 months. The patient group that achieved higher mean CsA level (≥200 ng/ml, higher CsA group) during first 2 weeks, was shown to more higher response rate than the group, lower mean CsA level (< 200ng/ml, lower CsA group)(response rate of 82.3% in higher CsA group, 21.4% in lower CsA group, p=0.0023). There was also a significant difference in survival rate during median 785 days between higher CsA group and lower CsA group (survival rate of 64.7% in higher CsA group, 28.6% in lower CsA group, p=0.0115). Therefore, we suggest that early higher CsA level during first 2 weeks would be an important factor to achieve the overall response of IST in severe aplastic anemia.
Long-Term Clinical Outcome in Children with Very Severe Aplastic Anemia
Authors: Chung Nack Gyun , Jeong Dae-Chul , Lee Jae-Wook , Jang Pil-Sang , Cho Bin , Kim Hack-Ki
Published: April 2020

Introduction: The clinical course of pediatric patients with severe aplastic anemia (SAA) treated with immunosuppressive therapy (IST) or hematopoietic cell transplantation (HCT) is well known. However, reports on the course and prognosis of patients with very severe aplastic anemia (vSAA) are few. Here, we report on the characteristics and outcome of children with vSAA.

Methods: We reviewed the medical records of patients diagnosed with vSAA at the Department of Pediatrics, St. Mary’s Hospital, The Catholic University of Korea between January, 1995 to December, 2007. We analyzed the clinical characteristics of the patients at diagnosis, and the effects of IST, HSCT and other supportive treatment measures on patient outcome.

Results: Forty three patients were diagnosed with vSAA during the study period, of whom 9 received supportive treatment measures only, 21 received IST consisting of cyclosporine A and either ATG or ALG, and 15 underwent HCT (including 2 patients who showed no response to initial IST). Of the 9 children who received supportive measures only, one patient recovered spontaneously and is surviving event-free for 22 months, 6 patients died from either severe infection or bleeding from 1 to 10 months after diagnosis (median 5 months), and the other two children are continuing to receive supportive transfusions. Of the patients with IST, two children showed a complete response (10.5%), 6 patients showed a partial response (31.6%), and eleven failed to respond (57.9%). Of the 11 patients who did not respond, 8 children died from infection or bleeding within 9 months after IST respectively, 2 patients were lost to follow-up, and 1 patient is receiving supportive transfusions. Of the 8 patients who showed a response, 2 have shown relapse and are being maintained with transfusions. Of the 15 HCT’s, 12 were from HLA-matched sibling donors and 3 were from matched unrelated donors. Except for the 2 patients who died from serious infections at 5 and 9 months post-HSCT respectively, all are currently surviving disease-free. The 10-year survival of the patients who received supportive measures only, IST, or HCT is 33.3±15.7%, 47.4±14.0 and 86.7±8.8% respectively (P=0.009).

Conclusion: As with SAA, HCT is the best treatment option for vSAA. If IST is undertaken, measures should be undertaken to prevent death from early infection. With only supportive care, median survival approximates only 6 months, calling for more intensive treatment strategies such as IST or HCT in the care of vSAA patients.
Viability of CD34+ Cells in Cryopreserved Cord Blood
Authors: Eom Young-woo , Jeong Seong Hyun , Choi Jin-Hyuk , Kang Seok Yun , Lee Hyun Woo , Yang Mal Sook , Kim Hyo Eun , Jang In Keun , Lee Jong Eun , Kim Yeong Jin , Kim Hugh Chul , Hwang Yoon Ho , Ahn Mi Sun , Park Joon Seong
Published: April 2020

Purpose: On performing umbilical cord blood (UCB) transplantation, faster engraftment may lead better clinical outcome. Because transplanted viable cell count in UCB is related to the engraftment, we examined cryopreserved UCB cells with several methods after thawing.

Methods: Viability of cryopreserved cells were examined with trypan blue, DNA contents analysis, caspase-3 activation test, intracellular esterase activity and Annexin-V/PI staining.

Results: A total of 60 samples were used in this study. After thawing, 89% of the total MNCs and 84% of CD34+ cells were viable as identified by trypan blue exclusion assay. In the CD34+ cell population, the cell death rate was found to be 47 % by Annexin-V/PI staining and less than 5 % by DNA contents analysis. Caspase-3 activity failed to document apoptosis. The intracellular esterase activity test also showed a cell death rate of about 10–20 % at 2, 4, and 6 hours after thawing.

Conclusion: Viable cells in UCB should be measured by several compensatory techniques rather than a single method. Discordance among Annexin-V/PI staining versus trypan blue exclusion, DNA contents analysis, and the caspase-3 activation test or intracellular esterase activity should be clarified in order to apply these techniques for actual cord blood transplantation.
Peripheral Blood Stem Cell Collection in Elderly Patients
Authors: Tempescul Adrian , Ianotto Jean-Christophe , Eveillard Jean-Richard , Hardy Elisabeth , Guillerm Gaelle , Dalbies Florence , Berthou Christian
Published: April 2020

Introduction Intensive treatments like autologous stem cells transplantations are final standard treatment in lymphoma and myeloma for young people (1,2). In the context of population’s ageing, those treatments should be eligible for older patients. One of the major causes which did not allowed these procedures in elderly patients is the reports of the impact of the age on the process of mobilisation of peripheral stem cell (3). We proceed a retrospective study to explore the feasibility of stem cells collection in patients older than 65 years, compared to younger people. Material and patients During the period of 1999–2006, we identified all the patients older than 65 years, with myeloma and lymphoma, who performed peripheral stem cells collection in Brest’s blood collection centre. We excluded patients for bone marrow collection. Patients were eligible if they achieved more than 2.106 CD34/kg. Then we compared the results obtained in older patients with the results obtained in younger people. Results We identified 100 patients older than 65 years between the 359 patients who underwent peripheral stem cell harvest in Brest’s harvest centre. We excluded 37 others patients for non-conformity. The elderly patients were significantly older (age 68.9 vs. 49.3 y, p<10−6). No differences were seen for the number of collection between the older and the younger patients (1.59 vs. 1.50, p>0.05). The number of CD34/kg per patient was sufficient to realise more than one autologous transplantation in both groups of patients (5.3 vs. 6.67, p=0.004). Samples were sufficiently rich for CD34/kg (4.36 vs. 5.54, p=0.02). Patients were harvested in one (60), two (25) or more than 2 collections (15). Among the older patients, 71% achieved a sufficient collection of more than 2.106 CD34/kg in one sample only. Conclusion We identified one hundred patients older than 65 years, during a period of 8 years, who performed peripheral stem cells collection, with sufficient wealth (>2.106 CD34/kg). The majority of patients, 60%, need only one stem cells harvesting. The study confirmed that old patients could be harvested like young people, and eligible for stem cells autologous transplantation.
The beta1-Tubulin Binding Protein RanBP10 Plays a Critical Role for Platelet Discoid Shape and Hemostasis
Authors: Schulze Harald , Fleischhauer Silke , Meyer Imke , Wannack Martin , Kunert Stefan
Published: April 2020

Blood platelets in the circulation are released from megakaryocytes (MKs), their precursor cells in the bone marrow. Mature MKs are located at the blood vessels and release platelets into the blood stream by intermediate structures referred to as proplatelets. Every MK can release up to a thousand virtually identical platelets. During this fragmentation process every single platelet is equipped with a rather constant number of assorted granules and a peripheral microtubule ring consisting of several coiled filaments. 8–12 microtubule filaments are found on average in the coil, which is essential for maintaining the platelet discoid shape. Generating these dynamic microtubule filaments at the very cell periphery in the absence of classical microtubule nucleating factors is strictly dependent on de novo nucleation of filaments. Beta1-tubulin is a MK/platelet-specific and the predominant isoform present in proplatelets and platelets. Mice lacking beta1-tubulin are thrombocytopenic and reveal platelet spherocytosis. In humans, a Q43P mutation in the beta1-tubulin gene Tubb1 also leads to large and spheric platelets. We have recently identified RanBP10 as a cytoplasmic beta1-tubulin binding protein that is selectively expressed in hematopoietic tissues like bone marrow and MKs. RNA interference (RNAi)- mediated RanBP10 ablation in primary MKs resulted in the collapse of long microtubule filaments whereas retrovirally forced overexpression of RanBP10 led to a marked increase in microtubule filaments, most likely by number or augmented bundling. RanBP10 harbors guanine nucleotide exchange factor activity toward Ran by exchanging GDP with GTP. As a local increase in Ran-GTP precedes nucleation of non-centrosomal microtubules in other cell types this mechanism might explain how positional information about new microtubule filaments is provided in the cell periphery of mature and proplatelet elaborating MKs. We generated a gene trap-mediated mouse model for RanBP10 deficiency. MKs derived from nullizygous animals phenocopied the collapse of microtubule filaments found by RNAi. Although RanBP10 was dispensable for overall platelet biogenesis with counts about 90–95% of normal, the ultrastructural analysis revealed that loss of RanBP10 protein resulted in a significant decline in the elliptic coefficient: Mutant platelets were more spherical and electron micrographs showed a wider variety in both microtubule filament number and coil localization including additional and incomplete coiling. Most intriguingly, RanBP10- deficient mice demonstrated a markedly prolonged bleeding time compared to littermate controls in a standardized tail bleeding assay. Platelets from nullizygous animals failed to respond normally to ADP by CD62P expression in flow cytometry. Our data thus suggest that RanBP10 plays a critical role in maintaining the tightly controlled platelet shape and function and that RanBP10 is essential for hemostasis.
Myeloid Cell Differentiation Arrest by Mir-125b-1 in Myelodysplasic Syndrome and Acute Myeloid Leukemia with the T(2;11)(p21;q23) Translocation
Authors: Bousquet Marina , Quelen Cathy , Rosati Roberto , Mas Véronique Mansat-De , Bastard Christian , Lippert Eric , Talmant Pascaline , Lafage-Pochitaloff Marina , Leroux Dominique , Gervais Carine , Viguie Franck , Lai Jean-Luc , Terre Christine , Delsol Georges , Dastugue Nicole , Mecucci Cristina , Brousset Pierre
Published: April 2020

Most chromosomal translocations in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) involve oncogenes which are either up-regulated or form part of new chimeric genes. The t(2;11)(p21;q23) translocation has been cloned in 19 cases of MDS and AML. In addition to this, we have shown that this translocation is responsible for a strong up-regulation of miR-125b (6 to 90 fold). In vitro experiments revealed that miR-125b was able to block monocytic and granulocytic differentiation of leukemic cells and primary CD34+ human blasts. Therefore, miR-125b up-regulation may represent a new mechanism of myeloid cell transformation and myeloid neoplasms carrying the t(2;11) translocation define a new clinico-pathological entity.
Acute Lymphoblastic Leukemia in a Case of Ring Chromosome
Authors: Türkmen Seval , Muller Sebastian , Mundlos Stefan , Henze Günter , Lode Holger
Published: April 2020

Ring chromosome 13 is a rare chromosomal abnormality. The classic mode of formation of a ring chromosome is breakage of the ends of both arms of a chromosome wit fusion of the broken ends resulting in the loss of the distal fragments. The clinical phenotype will depend on loss of genetic material.. Here, we report for the first time the development of an acute lymphoblastic leukaemia (ALL) in a patient with a constitutional ring chromosome 13. The chromosome analysis of the bone marrow revealed a large deletion that involved the Retinblastoma (RB1) gene. The possible role of (RB1) gene for leukemogenesis is discussed in this patient
Increased Expression of PD-1 on CD4+ T Cells from Patients with Chronic Lymphocytic Leukemia
Authors: Sartor Mary M , Gottlieb David J
Published: April 2020

Although the predominant finding in patients with chronic lymphocytic leukemia (CLL) is an expansion of monoclonal B lymphocytes, a polyclonal expansion of T cells co-exists in CLL patients. Allogenic stem cell transplants for CLL suggest that a significant graft versus leukaemia effect mediated through recognition of minor MHC or leukaemia specific antigens can be achieved. Since it appears that the immune system and probably T cells recognise CLL cells, it is possible that one or more T cell defects might contribute to the initiation or maintenance of a clone of CLL lymphocytes. PD-1 is a coinhibitory molecule that is expressed on T cells in patients with chronic viral infections. It has been suggested that PD-1 expression might be a marker of cell exhaustion due to antigenic overstimulation. We examined the expression of PD-1 and its naturally occurring ligands PD-L1 and PD-L2 on both B and T cells in patients with CLL and compared this with expression on normal peripheral blood mononuclear cells. We found that PD-1 was expressed on over 10% of CD4+ T cells in 7 of 9 cases of CLL (mean 22±16%) but not on CD4+ T cells in any of 9 normal donors (mean 0±0%), p=0.0009. There was no difference in PD-1 expression on CD8+ or CD14+ PBMCs from CLL patients and normal donors (for CD8+ 24±21% and 19±16% for CLL and normals; for CD14+ 58±16% and 71±31% for CLL and normals). More than 10% of CD5+/19+ CLL cells expressed PD-1 in 7 of 10 cases (mean 18±18%) while more than 10% of normal B cells from 6 of 7 donors also expressed PD-1 (mean 49±30%). We examined the expression of PD-1 on naïve, central memory, effector memory and terminally differentiated subsets of CD4+ cells (CD62L+CD45RA+, CD62L+CD45RA−, CD62L−CD45RA− and CD62L−CD45RA+ respectively) from CLL patients and normal donors. The expression of PD-1 was higher on CD4+ cells from CLL patients in all subsets. The effect was most prominent in the effector memory subset (mean 54±4% for CLL patients versus 26±17% for normal donors, p=0.02). We looked for expression of PD-L1 and PD-L2 on T cells, B cells, monocytes and NK cells from CLL patients and normal donors. PD-L1 was only expressed on monocytes (mean 30±23%) and NK cells (mean 14±19%) from CLL patients and on monocytes from normal donors (mean 35±26%). There was no expression of PD-L2 on any cell type in either CLL patients or normal donors. We conclude that there is increased expression of the co-inhibitory molecule PD-1 on CD4+ T cells in patients with CLL. Ligation of PD-1 by PD-L1 expressed on monocytes or NK cells could inhibit immune responses to tumor and infectious antigens leading to persistence of clonally expanded cells and predisposition to opportunistic pathogens.