bluuebunny/crossref_metadata_2025 huggingface dataset:
Transduction and Expansion of HIV+ CD4 T Cells with an HIV-1 Based Lentiviral Vector and Immobilized CD3/CD28 Antibodies Maintains the Diversity of the TCR Vβ Repertoire.
Authors: Lemiale Franck , Pereira Mario , Humeau Laurent , Dropulic Boro
Published: October 2019

Recently, we initiated the first ex vivo HIV-based gene therapy trial in humans with HIV+ CD4+ T cells. In this protocol, a modified lentiviral vector carrying an anti-HIV payload is used to modify CD4+ T cells isolated from HIV-infected patients by apheresis and CD8 negative selection. The T cells are activated in the presence of vector and expanded using immobilized CD3/CD28 antibodies, and then infused back into the patient. T cell receptor (TCR) repertoire analysis has value for safety monitoring of adoptive T cell transfers in the detection of aberrant clonal expansions or deletions. In this study, the TCR Vβ repertoire was assessed using a flow cytometry based assay at various time points in the selection/transduction/expansion process of CD4+ T cells. PBMC isolated from whole blood of HIV+ patients were CD4-selected using a CD8 negative selection, followed by enrichment by CD3 antibody. CD4+ purified cells were transduced with the lentiviral vector, VRX496, in the presence of retronectin, and then co-cultured with CD3/CD28 coated M450 Dynabeads for ten days. The TCR Vβ repertoire was assessed in throughout the process using a FACS-based assay that employs a panel of 20 monoclonal antibodies recognizing most of the 24 Vβ families in PBMC and CD4+ T cells. Repertoires from subjects with normal polyclonal TCR profiles were conserved, as shown by the absence of any significant change in any Vβ family. Moreover, the transduction/expansion of CD4+ T cells from a patient with a previously skewed TCR profile allowed the improvement of the TCR Vβ repertoire. Finally, no significant difference was observed in the repertoire of cells transduced with VRX496 versus mock-transduced cells. These data demonstrate stability of the repertoire diversity and thus provide important support information in favor of the safety of a gene therapy approach involving lentiviral vector mediated modification and expansion of CD4+ T-cells.
Fondaparinux in the Treatment of Heparin-Induced Thrombocytopenia.
Authors: Bradner James , Hallisey Robert K. , Kuter David J.
Published: October 2019

The development of new non-heparin anticoagulant therapies has increased interest in the treatment of type II heparin-induced thrombocytopenia (HIT). Standard therapy involves administration of an intravenous direct thrombin inhibitor (DTI), either argatroban or lepirudin. Due to a lack of cross-reactivity to HIT antibodies in vitro, the synthetic pentasaccharide fondaparinux sodium is an appealing alternative, but its use in this disorder has not been comprehensively examined. Here, we report our experience with fondaparinux in HIT and an analysis of safety and efficacy in this setting. We reviewed all in-patient cases of HIT at the Massachusetts General Hospital that were confirmed by the heparin-platelet factor 4 antibody test and were treated with fondaparinux. Twenty patients satisfied diagnostic criteria for HIT. Each patient studied lacked another competing, compelling etiology for thrombocytopenia and was treated with fondaparinux for more than five days within one month of diagnosis. The average duration of therapy was 17 days and 16 patients (80%) were treated within two weeks of diagnosis. Patients were grouped according to whether they received fondaparinux after initial therapy with a DTI (n=10) or as initial treatment following the diagnosis of HIT (n=10). The initial dose of fondaparinux administered was 2.5 mg in 18 patients. Neither absolute nor relative thrombocytopenia was identified in either group following treatment. Patients with HIT treated with fondaparinux while thrombocytopenic experienced platelet recovery on average in 3.7 days, as represented in Figure 1. In addition, there were no observed thrombotic complications among patients treated with fondaparinux. In this analysis of a high-risk group of inpatients with type-II HIT, fondaparinux was tolerated without the continuation or recurrence of thrombocytopenia. This suggests that cross-reactivity between the heparin-PF4 antibody and fondaparinux is not likely to be clinically significant. Additionally, the absence of thrombotic sequelae among fondaparinux-treated patients argues that fondaparinux may be a well-tolerated, effective treatment for this morbid, prothrombotic disorder. A prospective trial of fondaparinux in the treatment of type-II HIT is planned.

Figure Figure
Does All-Transretinoic Acid (ATRA) Have a Role in Non-APL Acute Myeloid Leukaemia?: Results from 1666 Patients in Three MRC Trials.
Authors: Burnett Alan K. , Milligan Donald , Hills Robert K. , Goldstone Anthony H. , Prentice Archie G. , Wheatley Keith , Webb David C. , Gibson Brenda
Published: October 2019

ATRA has a key role in the treatment of APL where the presence of the PML-RARα protein is predictive of response. Pre-clinical data and some non-randomised clinical studies suggested that ATRA may enhance the effect of chemotherapy particularly based on its potential to reduce the Bcl2 expression which may thereby improve apoptotic responses to chemotherapy. We have evaluated this possibility in 3 randomised trials in remission induction in patients under 60 years (AML12) n=1097; in relapse (AML-HR) n=362; and as a non-intensive approach in older patients (AML14-non-intensive) n=207. In AML12 patients were randomised to receive standard induction chemotherapy options (ADE vs DAT or MAE vs DAT with Ara-C at either Ara-C 200mg/m2/day or 400mgs/m2/day). There was no significant difference in CR rate (83% vs 85%) or survival at 5 years (40% vs 36%) on ATRA or no ATRA overall or in any demographic, risk or treatment subgroup.

Patients with high risk disease (adverse cytogenetics or less than PR after course 1 of induction) or relapse were randomised to receive Fludarabine/Ara-C or ADE with or without G-CSF, with or without ATRA for 60 days. The addition of ATRA made no overall impact on remission rate (61% vs 61%) or survival at 3 years (23% vs 27%). There was no subgroup where an advantage was seen.

In AML14 patients not considered fit for intensive treatment were randomised to receive Hydroxyurea or Low Dose Ara-C with or without ATRA for 90 days. There was no evidence that patients who received ATRA derived any benefit either overall or within the treatment arms.

In this extensive assessment, no advantage of adding ATRA to chemotherapy - as a continuous daily dose during induction - could be detected in any trial or in any subgroup within each trial.
Reaction of Fibrin-Bound Thrombin with a Covalent Antithrombin-Heparin Complex Results in an Anticoagulant Surface.
Authors: Smith Lesley J. , Berry Leslie R. , Chan Anthony K.
Published: October 2019

Thrombin bound to fibrin is protected from inhibition by mixtures of antithrombin (AT) and heparin (H). Resistance of thrombin to reaction with AT + H when fibrin is present results from the formation of a ternary fibrin:thrombin:H complex. However, recent investigations using an antithrombin-heparin conjugate (ATH) showed that the covalent ATH complex rapidly inhibits fibrin-bound thrombin, with the thrombin-ATH product remaining attached to the fibrin. One intriguing possibility from these findings was that the heparin chain of ATH (or thrombin-ATH) on the fibrin surface might still be available to interact with fluid-phase molecules. We studied the anticoagulant activity of fibrin-bound ATH by determining the ability to catalyze the inhibition of added thrombin + AT. Excess fibrin monomer (polymerization inhibited by GPRP) was mixed with thrombin to form soluble complexes. Fibrin:thrombin was then incubated in the absence or presence of AT, H, AT + H or ATH, at various concentrations relative to the thrombin. Following treatment with anticoagulant, a vast excess of exogenous thrombin + AT was added and the ability of fibrin:thrombin-bound heparin to catalyze inhibition of the exogenous thrombin was assessed by measuring the loss of thrombin activity (chromogenic substrate assay). In the absence of heparin, reaction of exogenous thrombin + AT was essentially unaffected by the presence of soluble fibrin:thrombin. When fibrin:thrombin complex was mixed with AT + H (concentrations equimolar to thrombin), an insignificant ability to catalyze inhibition of excess thrombin by AT was observed (remaining thrombin activity = 49 +/− 4 vs 43 +/− 3 mOD/min for reaction with AT + H vs without AT + H, respectively). However, when similar concentrations of ATH were combined with fibrin:thrombin, a marked catalytic activity was seen (remaining thrombin = 34 +/− 3 mOD/min). Furthermore, the significant catalytic effect of ATH could be demonstrated over a wide range of concentrations relative to fibrin:thrombin. At these same concentrations, AT + H had little effect on exogenous thrombin + AT reactions. We conclude that although AT + H is unable to effectively inhibit fibrin-bound thrombin, covalently linked ATH not only overcomes this defect but can convert the fibrin:thrombin into a new site for anticoagulant activity to catalyze thrombin inhibition by exogenous AT. If clot-based antithrombin activity is demonstrated in vivo, we speculate that ATH may be useful for transforming thrombi from procoagulant to anticoagulant entities.
Antithrombotic and Hemorrhagic Effects of DU-176b, a Novel, Potent and Orally Active Direct Factor Xa Inhibitor: A Wider Safety Margin Compared to Heparins and Warfarin.
Authors: Furugohri Taketoshi , Honda Yuko , Matsumoto Chikako , Isobe Koji , Sugiyama Nobutoshi , Morishima Yoshiyuki , Shibano Toshiro
Published: October 2019

DU-176b is a novel potent, orally active and selective direct inhibitor of factor Xa (FXa). Direct FXa inhibitors have been reported to exert little effect on bleeding time at antithrombotic doses in animal studies. The aim of the present study was to compare the antithrombotic and hemorrhagic effects of DU-176b with unfractionated heparin (UFH), low molecular weight heparin (LMWH; dalteparin) and warfarin in rat models of thrombosis and hemorrhage. Rats were treated with DU-176b, UFH and LMWH by continuous intravenous infusion for 2 – 2.5 h, and with warfarin orally once daily for 4 days before thrombosis or hemorrhage. Thrombosis was induced by the insertion of a platinum wire into the inferior vena cava and left for 60 min. Tail template bleeding time was measured after an incision on the tail. DU-176b dose-dependently inhibited thrombus formation in the venous thrombosis model. The dose required for 50% inhibition (ED50) was 0.076 mg/kg/h. In contrast, the dose of DU-176b to double template bleeding time (BT2) was 0.75 mg/kg/h, indicating 10-fold dissociation of the doses of antithrombotic and hemorrhagic effects. UFH, LMWH and warfarin also prevented thrombus formation (ED50 = 56 U/kg/h, 66 U/kg/h and 0.16 mg/kg/day, respectively), but prolonged bleeding time at slightly higher doses (BT2 = 73 U/kg/h, 135 U/kg/h and 0.21 mg/kg/day, respectively) than the effective doses. The dissociation of the doses for these compounds was only 1.3, 2.0 and 1.3-fold, respectively. Moreover, the slope of dose-antithrombotic response curve of DU-176b was significantly slighter than those of UFH, LMWH and warfarin, indicating that the therapeutic dose range of DU-176b would be wider than those of the other anticoagulants. These results suggest that direct and selective inhibition of FXa by DU-176b is preferable for the treatment of thrombotic diseases in the aspect of lack of compromising primary hemostasis.
Relationships between Risk Factors for Coronary Artery Disease and Aspirin Resistance.
Authors: Schwartz Kenneth A. , Schwartz Dianne E. , Barber Kimberly R. , Reeves Mathew R. , Hall Janette M. , De Franco Anthony C.
Published: October 2019

Aspirin prevents or delays atherogenesis and subsequent arterial occlusions. Patients who are relatively resistant to aspirin have a higher rate of vascular occlusive events. We evaluated the hypothesis that in patients with a previous myocardial infarction (MI) relative resistance to aspirin was related to risk factors for coronary heart disease (CHD). We studied 192 patients with a history of at least 1 MI including 136 males and 64 females. Mean age was 62. Compliance was assured by studying patients 2 hours after they were observed to ingest 325mg aspirin and confirmed with a qualitative assay (arachidonic acid 1.0mM stimulated light aggregometry) that detected aspirin inhibition of platelet aggregation in all of the study patients. To assess amount of aspirin inhibition of platelet aggregation (aspirin resistance) platelet prostaglandin agonist (PPA) 30mM stimulated light aggregation was performed (Schwartz J. Lab Clin Med2002;139:227). A CHD risk assessment score that includes diabetes, HDL, total cholesterol, high blood pressure (HBP) smoking and age was used as a measure of 10 year risk for CHD. Statistical comparisons were evaluated using a univariate general linear model. A significant relationship was observed between platelet resistance to aspirin inhibition and increased CHD risk (p=0.03). Evaluation of individual CHD risk factors showed that diabetic patients were more resistant to aspirin as demonstrated by an increased slope of aggregation (p=0.02) a decreased time to 50% aggregation (p=0.04) and a linear relationship between an increase in fasting blood sugar and an increased aggregation slope (p=0.05). A higher (BMI) (p=0.05) or a lower HDL (p=0.02) were also related to aspirin resistance. Although decreased aspirin inhibition of platelets was observed in patients who smoked or had HBP, these differences were not significant. We conclude that in patients with CHD a relative resistance to aspirin inhibition of platelet aggregation correlates with a commonly used risk assessment equation and with the presence of diabetes, increasing BMI or decreasing HDL. This suggests the hypothesis that CHD patients who have a higher risk for CHD, or those who have diabetes or obesity or a decreased HDL, may benefit from additional platelet inhibiting medications.
Specific Inhibition of BCR-ABL Gene Expression in Acute Lymphoblastic Leukemia by Catalytic DNAzymes.
Authors: Taube Tillmann , Shalapour Shabnam , Seifert Georg J. , Pfau Madlen , Henze Guenter , Seeger Karlheinz
Published: October 2019

The BCR-ABL fusion protein p190 resulting from the translocation t(9;22) exhibits dysregulated tyrosine kinase activity and was shown to cause acute lymphoblastic leukemia (ALL). Detection of the BCR-ABL fusion gene in childhood ALL is associated with an adverse prognosis and defines a group of high risk patients. Because the BCR-ABL gene fusion is specific for leukemic cells it represents an ideal target for leukemia specific treatment approaches.

Catalytic DNAzymes are able to cleave mRNA in a sequence specific manner, causing inhibition of protein translation from the DNAzyme targeted mRNA both in vitro and in vivo. In order to cut off the BCR-ABL driven malignant proliferation, we designed DNAzymes to impede the expression of p190 BCR-ABL by cleaving the BCR-ABL mRNA adjacent to the fusion site. One construct was found that cleaved the target mRNA efficiently and specifically leaving BCR and ABL, relevant for normal cell survival and proliferation, unaffected. Activity and specificity of the BCR-ABL DNAzyme was investigated in cleavage assays with in vitro transcribed BCR-ABL, BCR and ABL mRNA. DNAzymes were delivered to cultured BCR-ABL+ ALL cells by lipid transfection. The efficiency of cellular delivery reached 90% as studied by flow cytometry, fluorescence microscopy and confocal microscopy after transfection of FITC labeled DNAzymes. To control for unspecific effects of DNAzyme delivery as well as for antisense effects, a catalytically inactive DNAzyme still exhibiting BCR-ABL antisense activity was designed. Fourty-eight hours after a single treatment of BCR-ABL+ ALL-cells with DNAyzmes the BCR-ABL mRNA concentration, as measured by quantitative real-time RT-PCR, was significantly reduced by 56% and 66% compared to controls treated with the inactivated DNAzyme and to untreated cells, respectively. Western blot analysis showed a decrease in p190 protein levels after DNAzyme treatment in comparison to the control treated with inactive DNAzyme as well as to the untreated cells. Most noteworthy, four days after a single DNAzyme treatment the net growth of BCR-ABL+ ALL cells treated with the active DNAzyme was inhibited by 68% compared to the untreated control.

From these data we conclude, firstly, DNAzymes targeting mRNA coding for the minor BCR-ABL variant are able to significantly reduce the amount of fusion mRNA in the cells, leading to a reduction in protein expression, followed by the inhibition of BCR-ABL driven proliferation of ALL cells. Secondly, this exemplified setting gives a hint that DNAzymes might be of therapeutic use in hematopoietic malignancies associated with specific mutations, expressing oncogenic fusion genes or overexpressing oncogenic genes.
DDAVP at a Maximal Dose of 15 ug Administered Subcutaneously Is a Safe and Effective Alternative to 20 ug IV for Patients > 50 kg with von Willebrand Disease or Mild Hemophilia A.
Authors: Mangel Joy , Laudenbach Lori , Schembri Sheila , Jardine Lawrence , Lohmann Reinhard
Published: October 2019

Background: DDAVP (desmopressin) is commonly used for the prophylaxis and treatment of bleeding in patients with mild forms of von Willebrand disease (VWD) and Hemophilia A (HemA). The standard dose is 0.3 ug/kg IV, to a maximum of 20 ug. In 1995, our Bleeding Disorders Program began to use a maximum dose of 15 ug both for DDAVP challenges and for therapeutic purposes. This dosing change was triggered by the new availability of 15 ug (1 ml) vials of DDAVP. We also switched to subcutaneous administration rather than intravenous, given the smaller volume (1 ml), and the body of evidence supporting this route of administration (De Sio et al, Thrombosis and Hemostasis1985; 54:387–9). With this strategy, patients weighing <50 kg continue to receive a weight-based dose, but those weighing ≥ 50 kg receive 15ug.

Method: In order to evaluate this new dosing regimen, a retrospective review was performed of 62 patients with VWD (n=36) or mild hemophilia (n=26) weighing ≥ 50kg who underwent DDAVP challenges using a 15 ug s/c dose of DDAVP. Data was also collected on the 9 patients (5 VWD, 4 HemA) tested prior to 1995 who received 20 ug IV of DDAVP.

Results: Levels of von Willebrand factor antigen (vWFag), ristocetin cofactor activity (RCof) and factor VIII (FVIII) were measured immediately prior to and 1 hour after administration of DDAVP. In the VWD patients (14M, 22F), median baseline levels of vWFag, RCof and FVIII were 31, 27 and 45 U/ml respectively, and rose to a median of 93, 90 and 177 U/ml following a 15 ug dose of DDAVP. This resulted in a median increase of 64 for vWFag (range 0–180), 55 for RCof (24–191) and 121 for FVIII (21–237). Results were comparable in the 5 pts who received 20 ug doses of DDAVP, achieving median increases of 77 (range 17–125), 53 (40–91) and 98 (79–184) respectively. In the HemA patients (19M, 7F), median baseline level of FVIII was 23 U/ml, and rose to a median of 57 U/ml following 15 ug DDAVP. Median increase in FVIII levels was 33 U/ml (range 2–108). These results compare favorably to the 4 patients who received 20 ug DDAVP and achieved a median FVIII increase of 19 U/ml (range 9–60). Chart review identified 26 patients (14 VWD, 12 HemA) who successfully received 15 ug doses of DDAVP in 44 clinical settings, either for the treatment of an acute bleeding episode or as prophylaxis prior to a planned surgical or dental procedure.

Conclusions: DDAVP given subcutaneously to a maximum dose of 15 ug appears to be a safe and effective alternative to 20 ug IV for pts > 50 kg with VWD or mild hemophilia A.
Clonal Involvement of the CD34+CD33− Progenitor Population by Leukemic Cells Harboring FLT3/ITD Correlates with High Risk of Relapse in Pediatric Acute Myeloid Leukemia.
Authors: Wright Jessica A. , Alonzo Todd A. , Gerbing Robert B. , Woods William G. , Lange Beverly J. , Radich Jerald P. , Bernstein Irwin D. , Meshinchi Soheil
Published: October 2019

Internal tandem duplication of the FLT3 gene (FLT3/ITD) has been associated with high risk of relapse in acute myeloid leukemia (AML) yet nearly 25–30% of the patients with FLT3/ITD have long-term disease free survival with conventional chemotherapy. We hypothesized that FLT3/ITD AML patients with poor clinical outcome may have disease that involves less mature hematopoietic precursors than patients with favorable outcome. To test this hypothesis, we isolated less mature, CD34+CD33− and more mature, CD34+CD33+ precursor cells from 24 pediatric AML patients enrolled on Children’s Cancer Group clinical trials CCG-2891 and 2961 previously identified as having a FLT3/ITD. Granulocyte/monocyte colonies (CFU-GM) were grown in methylcellulose, harvested, and analyzed for the presence of FLT3/ITD after 14 days of growth. Twenty patients yielded sufficient cells and growth of CFU-GM colonies for analysis. FLT3/ITD was detected in CFU-GM colonies derived from CD34+CD33+ cells in all patient samples (median 80% of colonies tested per patient, range 6–100%). In contrast, FLT3/ITD was detected in CFU-GM colonies derived from CD34+/CD33− cells in only 11 of the 20 patient samples (median 46% of colonies tested per patient, range 6–100%). Of the 9 patient samples without FLT3/ITD involvement of CD34+CD33− colonies, 8 achieved a CR, 6 of whom are long-term survivors, and one patient died of non-leukemic causes. In contrast, of the 11 patients with CD34+CD33− cell involvement, 9 either failed to achieve CR or relapsed after achieving CR, and 2 died of non-leukemic causes. Actuarial progression-free survival at 4 years from diagnosis for the patients with and without FLT3/ITD in the CD34+CD33− population was 0% vs. 68% respectively (p=0.017). As allelic ratio of the FLT3/ITD has been used to define high-risk patients within the FLT3/ITD cohort, we determined the FLT3/ITD allelic ratio in our study population and correlated it with the presence of FLT3/ITD in the CD34+CD33− population. Ten of the 11 (91%) of the patient samples with FLT3/ITD involvement of the progenitor cells had high allelic ratio compared to 5 of 9 (56%) of the patients without early cell involvement. Together these data suggest that clonal dominance of FLT3/ITD containing leukemia cells at the CD34+CD33− stage of hematopoietic development is correlated with a high risk of relapse. Further studies are required to determine whether clonal dominance at this hematopoietic stage is a variable that, independent of high allelic ratio, accounts for the poor clinical outcome seen in a subset of FLT3/ITD positive AML patients.
Homeostatic Proliferation of Chronic Lymphocytic Leukemia.
Authors: Wirths Stefan , Lanzavecchia Antonio
Published: October 2019

Slow accumulation of chronic lymphocytic leukemia cells in vivo was considered due to defective apoptosis rather than proliferation. However, recent data, based on isotope incorporation studies, suggested significant continuous proliferation of CLL cells in vivo and similar observations has been made for human memory B cells. As previous gene expression studies revealed close relation of CLL cells to memory B cells we asked, whether the mechanisms driving homeostatic proliferation of human memory B cells were stimulating CLL cells as well. Comparison of CLL samples (n=20) and memory B cells of healthy donors showed high expression levels of Toll-like receptor (TLR) 7 and TLR9 and of IL2 and IL15 cytokine receptors compared to naive B cells. Proliferation of CLL cells compared to their normal counterparts was analyzed after FACS sorting and CFSE-labeling.Naive B cells did neither respond to TLR7 ligand resiquimod, TLR9 ligand CpG2006, IL2 and IL15 nor to their combination. In contrast, CLL cells and memory B cells showed comparable patterns of proliferation. Remarkably, under these conditions, terminal differentiation to plasma cells was observed for memory B cells only, while proliferation of CLL ceased after the 4th division without differentiation into Ig-secreting cells. To estimate in vivo turnover rates, B cells of healthy donors and CLL cells were stained for intracellular Ki67, that identifies recently divided cells, and were analyzed by flow cytometry. Memory B cells showed high in vivo turnover rates (2–5% Ki67+) while naive B cells where largely quiescent - results that are in line with previous isotope incorporation studies with healthy donors. Turnover rates of CLL ex vivo ranged from 0.025–1.4% Ki67+ cells, which in absolute numbers (per mL of blood) was comparable to memory B cells. These results are consistent with a concept of homeostatic proliferation of both memory B cells and CLL cells, the latter being arrested without terminal differentiation, which might account for their accumulation in vivo.
NK Cells Efficiently Prevent Engraftment of Donor Stem Cells after Tolerigenic Bone Marrow Transplantation.
Authors: Kean Leslie , Hamby Kelly , Pearson Thomas , Larsen Christian
Published: October 2019

Introduction: Immunologic tolerance remains an elusive goal of transplantation. In mice, mixed-chimerism and donor-specific tolerance can be induced by blocking the CD28/CD40L T-cell costimulatory pathways after bone marrow transplant (BMT). However, large doses of marrow (~1x109 cells/kg) are required, and these regimens have not yet been successfully translated to clinical practice. There is a growing body of evidence that NK cells may play a central role in the failure of low doses of donor bone marrow to engraft, but the mechanisms underlying NK alloreactivity remain to be determined.

Methods: (1) BMT in the presence of CD28/CD40L T cell costimulation blockade was performed using C57BL/6 (B6) recipients and Balb/C donor bone marrow. The role of host-anti-donor NK alloreactivity in preventing engraftment was determined by specifically depleting B6 NK cells. The contribution of the NK cell-surface receptor, LFA1 to NK alloreactivity was determined with the anti-LFA1 blocking antibody M17/5.2. (2) An in vivo NK alloreactivity assay was developed that should allow the investigation of the mechanism of NK alloreactivity and the molecular mediators of this process. In this assay, CFSE-labeled B6 splenocytes were adoptively transferred into B6xBalbC F1 progeny. As such, alloreactivity was specifically mediated by NK cells. NK alloreactivity was measured flow-cytometrically by the disappearance of the CFSE-labeled B6 population.

Results: Transient depletion of recipient NK cells resulted in increased donor stem cell survival and the induction of stable mixed-chimerism and tolerance despite BMT with low doses (≤2x106 cells) of donor bone marrow. This effect was specific to allogeneic donor cells: depletion of NK cells did not increase engraftment of syngeneic bone marrow. Blocking the adhesion molecule, LFA-1 recapitulated the effects of whole-scale NK depletion. Newly emergent NK cells exhibited significantly lower expression of the donor-specific activating receptor, Ly49D, and these NK cells did not exhibit in vivo alloreactivity. These results suggest that the NK repertoire in the mixed-chimeric setting exhibited donor-specific tolerance.

Using the in vivo hybrid resistance NK alloreactivity assay, we measured 80% NK-specific target killing 8 days after adoptive transfer. Significantly less killing occurred at 2, 4, and 6 days. Pre-sensitizing the recipient for 4 days increased the efficiency of killing—from 50% to 80%, suggesting a potent activation phenomenon required for efficient NK allorecognition and/or cytotoxicity.

Implications: These results reveal the importance of NK alloreactivity in the acquisition of mixed-chimerism after BMT at limiting stem cell doses, and suggest that clinical approaches to tolerance-induction transplantation may require mechanisms to control NK alloreactivity.
Serum Soluble Syndecan-1 Levels at Diagnosis Constitute an Independent Prognostic Factor of Survival in Myeloma That May Further Differentiate Patients within the ISS Stages.
Authors: Kyrtsonis Marie-Christine , Vassilakopoulos Theodoros P. , Papadogiannis Argyro , Tzenou Tatiana , Kalpadakis Christina , Antoniadis Antoine , Dimopoulou Maria N. , Siakantaris Marina P. , Kokoris Stella I. , Dimitriadou Evangelia M. , Angelopoulou Maria K. , Plata Ellen , Tsaftaridis Panagiotis , Pangalis Gerassimos A.
Published: October 2019

Syndecan-1 (CD138) is a heparin sulphate proteoglycan overexpressed on the surface of both normal and malignant plasma cells. Its role in multiple myeloma (MM) pathogenesis remains unclear. It may be shed from plasma cell surface and released in its soluble form (soluble syndecan-1: s-synd-1). Serum s-synd-1 levels have been shown to be a powerful prognostic factor of survival in myeloma patients. The role of s-synd-1 in the current prognostic classification systems requires further investigation. We have retrospectively determined soluble syndecan-1 in frozen sera of 99 MM patients, 18 MGUS patients, and 25 healthy individuals (HI). In all patients sera samples were collected at diagnosis. Serum s-synd-1 levels were determined by ELISA (Diaclone Research, Besanson, France) according to the manufacturer’s instructions. MM patients median age was 69 y (44–82), 51 were female, 48 male, 26 were Durie-Salmon (DS) stage I, 41 stage II, and 32 stage III while, according to the International Scoring System for MM (ISS), 32 were in stage 1, 25 in stage 2 and 42 in stage 3. Median serum s-synd-1 was 15 ng/ml (7–40) in HI, 16.5 ng/ml (8–220) in MGUS and 64 ng/ml (7–3500) in MM. Differences between HI and MM patients levels and between MGUS and MM patients levels were both significant (0.01 and 0.05 respectively). Overall survival was decreased in patients with elevated (mean + 2SD) serum s-synd-1 (5-year overall survival 35±9 vs 80±13, p=0.004). S-synd-1 levels were an independent prognostic factor of survival compared to DS and ISS staging systems. In multivariate analysis, only ISS and s-synd-1 were significant parameters of prognosis (the prognostic significance of DS, ISS and s-synd-1 was 0.06, 0.006 and 0.02 respectively). Further analysis of survival of patients stratified according to the ISS system showed a difference in survival within groups according to s-synd-1 levels. The 5-year overall survival was 100 vs 58±16 in ISS stage 1 patients with low or elevated s-synd-1 respectively, 100 vs 18±16 in stage 2 and 50±25 vs 14±13 in stage 3. The difference was statistically significant for stage 2 only (p=0.04) and there was only a trend due to the low number of patients in stages 1 and 3. In conclusion serum s-synd-1 levels at diagnosis constitute an independent prognostic factor of survival, able to further differentiate MM patients within the ISS stages.
A Phase I Study To Investigate the Pharmacokinetics (PK) and Pharmacodynamics (PD) of an Oral Platelet Growth Factor (SB-559448) in Healthy Subjects.
Authors: Su Sheng-Fang , Peng Bin , Williams Daphne , Park Jung Wook , Handel Malcolm L. , Jenkins Julian , Ho Peter
Published: October 2019

Introduction: SB-559448, is an orally bioavailable, non peptide, small molecule thrombopoietin receptor agonist that induces proliferation and differentiation of megakaryocytes and progenitor cells, ultimately increasing the production of platelets.

Method: In a randomized, single blind, placebo-controlled, parallel group, phase I study in healthy subjects, SB-559448 was administered as oral capsules once daily for 1 day and, after a 7-day washout, for 10 days at doses of 160 to 640 mg. In this dose escalation study, subjects were randomized into 4 groups of 9 subjects to receive either active or placebo medication in a ratio of 6:3. Serial blood samples were collected to determine SB-559448 concentrations and estimate PK parameters following single- and repeat-dosing. PD assessments were performed every 4 to 5 days up to 31 days following initiation of repeat dosing.

Results: The preliminary results showed that SB-559448 was well tolerated in the study, there were no serious adverse events, no significant changes in laboratory or cardiovascular safety parameters and there was no observed relationship between the incidence or severity of adverse events and dose. Most adverse events were mild in intensity and self-limiting. SB-559448 was also shown to be orally bioavailable in humans with a pharmacokinetic profile suitable for a once daily oral dosing. When administered at oral doses of 160 mg and above for 10 days, a dose dependent increase in the platelet count was observed with maximum platelet count observed on days 14 to 15 following initiation of repeat dosing.

Conclusion: The results suggest that SB-559448 is an orally administered agent that increased platelet counts in healthy subjects. Additional studies will be needed to evaluate its effects in individuals with thrombocytopenia.
Inter-Laboratory Variation and the Effect of the Type of Fluorimeter on the Results of the Technothrombin® Thrombin Generation Assay.
Authors: Meijer Piet , Kluft Cornelis
Published: October 2019

Thrombin generation is an essential step in the clotting system. Therefore the measurement of thrombin generation is a desirable parameter to elucidate deviations in the clotting system. However till recently the measurement of thrombin generation was a difficult and laborious laboratory method. Nowadays commercial tests are available at the market. We evaluated in a multicenter trial the Technothrombin® TGA assay from Technoclone GmbH Vienna, Austria with a focus on inter-laboratory variability. The Technothrombin® TGA assay is a fluorogenic method. To our knowledge this was the first large-scale inter-laboratory comparison of a thrombin generation test. A set of 8 different samples were distributed to 45 clinical centers in Europe. Thirty-six returned results (80%). Samples were selected on a different level of thrombin generation as indicated by the thrombin concentration at peak height (PTC). The PTC in the samples used varied from 4 nM to 335 nM. The inter-laboratory variability varied from 16% for high PTC (~335 nM) to about 100% for low PTC (~4 - 15 nM).

Using a normal citrated pooled plasma as a reference the other samples can be significantly discriminated by all participants (P < 0.03) using an activator with a low phospholipid concentration (0.32 μM) in the test. With a high phospholipid concentration (3.2 μM) two of the samples can not be significantly discriminated from normal pooled plasma.

Furthermore, we evaluated the effect of the type of fluorimeter used by the clinical laboratories. The most frequently used fluorimeters in this study were the Biotek FLx 800 (n=11), Thermo Fluoroskan (n=8), Tecan Genios (n=5) and the BMG Fluostar (n=4). Overall there are no significant differences between the measurement of the PTC by the different fluorimeters. However remarkable differences were observed for the inter-laboratory variability between the different fluorimeters. The average coefficient of variation of all samples increased from BMG (19.3% ; range 11.1 - 26.9%) < Biotek (28.8% ; range 15.9 - 59.3%) < Tecan (38.5% ; range 12.6 - 81.5%) < Thermo (49.7% ; 19.1 - 96.6%).

This data shows the impact the selection of a type of fluorimeter may have on the comparability of test results of clinical samples. Selection of a measurement system with the lowest inter-laboratory variation in clinical studies shows a benefit for the comparability of study results.
Efficacy and Safety of Tinzaparin Sodium as a Bridging Agent for Patients on Oral Anticoagulant Undergoing General Surgery: Clinical Experience with the First 100 Consecutive Patients.
Authors: Volteas Spiridon K. , Babos Anastasios
Published: October 2019

The aim of the present prospective study was to test the efficacy and safety of a bridging protocol in patients under oral anticoagulants (OA) who need to undergo general surgery. One hundred consecutive patients receiving oral anticoagulant accenocoumarol for various cardiovascular disorders entered the study. Patients underwent 107 scheduled operations (among them 41 cholecystectomies, 24 colectomies, 13 hernia repairs and 9 ERCPs, the 7 followed by cholecystectomy), receiving the low molecular weight heparin (LMWH) tinzaparin sodium as the bridging agent. At least 4 days prior to the operation the OA was replaced with tinzaparin at a daily dose of 175 anti-Xa IU per kg of body weight. Patients received no LMWH on the day of the operation and the OA was co-administered with tinzaparin on the second post-operative day and for at least 2 days. When target INR (2.5 to 3) was achieved, tinzaparin administration was stopped. Lower limbs color duplex was performed pre-operatively and on the 2nd, 8th–10th and 20th–30th post-operative day. Complete anticoagulation profile, intra-operative bleeding and post-operative blood loss, large haematoma development (over 5 cm in diameter), deep vein thrombosis (DVT) and 30-day morbidity and mortality were thoroughly recorded. During the 30-day period 1 patient died (from sepsis), 9 developed wound infections and three underwent re-operations (two for anastomotic leaks and one for duodenal perforation following ERCP). None of these events was related to the bridging process. There were no proximal DVTs. Three asymptomatic distal vein thromboses (2 posterior tibial and one peroneal ) were identified on post-operative duplex scan. No large wound haematomas were seen. One colorectal surgery patient had a drain blood loss of 460 ml on the first and 140 ml on the second post-operative day, requiring blood transfusion; tinzaparin administration was delayed for three days in this case. Finally, in 17 cases we had difficulties in adjusting INR levels to the desired range; in these cases tinzaparin administration was prolonged for 2–5 days. We conclude that our bridging protocol, using therapeutic doses of the LMWH tinzaparin sodium as the bridging agent, is both safe and effective for replacing OAs in patients undergoing general surgery operations.
Thalassemia in a Hawaiian Family of Filipino Extraction
Authors: LAZAR HAROLD P.
Published: October 2019

Hematologic data are presented on thalassemia in a Hawaiian family of Filipino extraction, and support is given the recent trend to broaden the ethnic concept of thalassemia. Because of recent reappraisals of older reported cases by other investigators in the light of newer electrophoretic technics, the need for continued re-evaluation is suggested.
The Effect of Repeated Doses of Urethane (Ethyl Carbamate) on the Mitotic Activity and Cellular Composition of the Bone Marrow of Normal Mice
Authors: ROSIN A. , GOLDHABER G.
Published: October 2019

A single dose of urethane produced a rise in the mitotic index, due essentially to a large increase in the metaphase index in the bone marrow of normal mice. Repeated daily injections (three and six doses) produced an elevation of the mitotic index, which was, however, smaller than that observed after a single dose of urethane. After nine doses of urethane the mitotic index was found approximately at the same level as in the controls. The mitotic index in the bone marrow of urethane-treated mice never decreased below the normal level found in untreated mice.

After three, six and nine doses of urethane a rise in the percentage of ana- and telophases was found, in contrast to the increase in the percentage of metaphases after administration of a single dose of urethane. The percentage of prophases was decreased after six and nine doses of urethane.

The morphological changes in the mitotic figures were the same as those described after a single dose of urethane. They consisted mainly in bridge formation and chromosome aberrations in anaphase.

Very large promyelocytes, often with prematurely lobated or bizarre shaped nuclei, were observed.

There was damage to the nuclei of the erythroid precursors and consequently an increase in the myeloid-erythroid ratio in the bone marrow after repeated doses of urethane, especially after nine doses.

The maturation rate of the segmented leukocytes in the bone marrow of the urethane treated mice was augmented after repeated doses.
Studies on Leukemia
Authors: SANDBERG A. A. , CARTWRIGHT G. E. , WINTROBE M. M.
Published: October 2019

1. The urinary excretion of uric acid was studied in 17 normal subjects and in 38 patients with leukemia. The mean excretion of uric acid by the normal subjects was 6.5 mg./Kg. of body weight /24 hrs. The mean excretion of uric acid in 14 patients with acute lymphoblastic leukemia was 30.3 mg./Kg. /24 hrs.; in 13 patients with acute myeloblastic leukemia, 13.0 mg.; in 6 patients with chronic lymphocytic leukemia, 5.2 mg.; and in 5 patients with chronic myelocytic leukemia, 13.5 mg.

2. Following therapy with cortisone, 6-mercaptopurine or Amethopterin, the urinary excretion of uric acid increased in the cases of acute leukemia as the leukocyte count declined. As the leukocyte count approached normal levels, the uric acid excretion decreased. The urinary excretion of xanthine and guanine paralleled the excretion of uric acid.

3. In association with the administration of an aromatic nitrogen mustard derivative to one patient with chronic lymphocytic leukemia and Myleran to one patient with chronic myelocytic leukemia, there was only a slight increase in uric acid excretion.
Time Pattern of Vitamin B12Co60 Urinary Excretion in Man After Oral Administration and Parenteral "Flushing"
Authors: BEST WILLIAM R. , LANDMANN WENDELL A. , LIMARZI LOUIS R.
Published: October 2019

Serial urine collections in a number of patients with pernicious anemia given 2 µg B12Co60 orally followed in two hours by 1000 µg nonradioactive vitamin B12 showed little urinary radioactivity at any time. When these tests were repeated together with a potent oral dose of intrinsic factor concentrate, there was little activity during the first four hours. Peak excretion rates occurred most commonly between 6 and 12 hours after ingestion of radioactive B12, sometimes even later. The time of peak excretion was fairly characteristic for the individual. Secondary peaks occasionally occurred, and only slight radioactivity usually remained after 24 hours. It is postulated that the delayed peak is related to the time it takes for B12 to be transported in the intestine to the point of absorption or to the duration of the intracellular metabolic processes of absorption. For most purposes the use of fractional urinary collections is not necessary.
A Modification of the Benzidine Method for Measurement of Hemoglobin in Plasma and Urine
Authors: CROSBY WILLIAM H. , FURTH FRANK W. , THIBEAULT CAROL E.
Published: October 2019

A modification of the benzidine method for measuring hemoglobin in plasma and urine is described. When a known amount of hemoglobin is added to plasma or urine more than 95 per cent can be recovered with this procedure.
Studies on the Fibrinogen, Dextran and Phytohemagglutinin Methods of Isolating Leukocytes
Authors: SKOOG WILLIAM A. , BECK WILLIAM S.
Published: October 2019

Studies are presented on the dextran, fibrinogen, and phytohemagglutinin methods of leukocyte separation. Optimal conditions, obtainable yields, detailed directions, advantages and disadvantages of each procedure are presented. It is shown that by using these methods in combinations, leukocyte suspensions of extreme purity may be obtained.
The Effect of Certain Glycolytic Inhibitors on Aerobic Lactic Acid Production by Human Leukocytes in Vitro
Authors: MCKINNEY GORDON R. , MARTIN SAMUEL P.
Published: October 2019

Oxamic acid depressed aerobic lactic acid formation by normal, human leukocytes by only 20-25 per cent. Glyceraldehyde increased lactic acid production. Sorbose-1-phosphate was without effect. Iodacetate inhibited markedly, but fluoride depressed only in high concentrations. The significance of these findings regarding the mechanism of aerobic lactic acid formation by mature leukocytes is discussed.
Array CGH Analysis for PTCL-U Revealed a Distinct Subgroup with Pathological and Clinical Relevance.
Authors: Nakagawa Masao , Nakagawa-Oshiro Aya , Karnan Sivasundaram , Tagawa Hiroyuki , Nakamura Shigeo , Takeuchi Ichiro , Ohshima Koichi , Seto Masao
Published: October 2019

Peripheral T-cell lymphoma, unspecified (PTCL-U) is one of the most commonly encountered group among peripheral T-cell lymphomas (PTCLs). This category consists of the cases which do not belong to any of the recognized subtypes of PTCLs in the World Health Organization classification. PTCL-U comprises histologically and clinically heterogeneous groups, which makes it difficult to assess conventional therapies accurately and to develop new therapeutic targets. The first priority is thus to identify subgroups of PTCL-U. However, the molecular basis of their clinical heterogeneity is still poorly understood. Here we analyzed 51 cases of PTCL-U by means of array CGH consisting of 2304 artificial chromosome clones that cover the whole genome at a 1.3 mega base resolution. DNA gains and losses were detected in 29 cases (total regions: average 15.6, range 1–31; gains: average 8.0 regions, range 0–24; losses: average 7.6 regions, range 0–21). Regions of frequent gain(>15%; ≥5cases) were detected on 1q24.2, 1q25.1–25.2, 3q25–26.1, 3q26.2–27.1, 3q27.3–29, 4p16.2–15.2, 4q21.1–21.21, 4q21.23–28.1, 4q28.3–31.23, 4q32.1, 4q32.3–35.1, 7p, 7q, 9q31.3–32, 9q33.2–34.2, 11q14.1, 11q14.3-tel, and 16p13.2–12.2, and regions of frequent loss on 1p31.2–13.1, 2q37.3, 6q14.3, 9p21.3, 10p14–13, 10p12.31–11.22, 10q11.21–21.1, 10q23.2–23.31, 10q24.2–26.3, 13q14.11–14.2, 13q21.1–34, 16q12.1–21, 17p13.3–11.2, and 18p11.31d–11.22b. Three consecutive BAC clones at 14q32.2 and at 9p21.3 were identified as regions of recurrent high-level amplification and homozygous loss, respectively. Our array CGH analysis could reveal a distinct subgroup consisted of 29 cases which have genomic alterations. Regarding to tumor burden, histopathology, the expression of chemokine receptors, and prognosis, this subgroup displayed significantly different features compared with 22 cases showing no genomic alteration. Our findings showed that PTCL-U with genomic alteration has pathological and clinical relevance distinct from those without, implying that distinct mechanisms underlay in molecular pathogenesis of PTCL-U.