bluuebunny/crossref_metadata_2025 huggingface dataset:
Naturally Processed Peptides Eluted from Acute Myeloid Leukemia Cells : A Potent Antigen Source for Immunotherapy.
Authors: Delluc Stephanie , Tourneur Lea , Boix Charlotte , Michallet Anne-Sophie , Varet Bruno , Guillet Jean-Gerard , Buzyn Agnes
Published: October 2019

Acute myeloid leukemia (AML) is a heterogenous group of diseases characterized by a clonal proliferation of myeloid progenitors. Its poor prognosis with conventional chemotherapy justifies seeking for adjuvant immunotherapeutic approaches to eliminate minimal residual disease. We evaluated an immunotherapeutic strategy that bypass the need for epitope identification and the limitation due to HLA restriction. Naturally processed peptides were extracted by acid elution from AML cells at diagnosis, and loaded on mature dendritic cells (mDCs) derived from autologous monocytes obtained when the patients were in complete remission (CR). We evaluated i) the feasibility to elute naturally processed peptides from AML cells at diagnosis, ii) the capacity of mDCs loaded with eluted peptides (mDC/EP) to stimulate specific T cell lines in vitro. We showed that stimulation by mDC/EP was able to generate anti-leukemic T cells lines from PBMC of 6 AML patients in CR. CD4+ and CD8+ T cells were isolated from T cell lines of 5 patients and analyzed for their proliferation, INF-γ production and cytotoxicity in response to autologous or allogeneic AML targets, or to normal autologous PBMC. We showed that both CD4+ and CD8+ leukemia-specific T cells were generated in vitro by mDC/EP stimulations since proliferation of CD4+ T cells, IFN-γ secretion by CD4+ and CD8+ T cells and cytotoxicity mediated by CD8+ T cells were induced in response to stimulation with autologous AML cells. Furthermore, we could not detect auto-immune recognition of autologous normal PBMC, consistent with the specificity of the T cell response induced by mDC/EP. These results provide the proof of concept for using mDC/EP to vaccinate patients with poor-risk AML, and will soon be evaluated in a phse I/II clinical trial.
Phase 2 Study of the Anti-Angiogenesis Agent AG-013736 in Patients with Poor Prognosis Acute Myeloid Leukemia (AML) or Myelodysplastic Syndrome (MDS).
Authors: Giles Francis J. , Steinfeldt Heidi , Bellamy William T. , Bycott Paul , Pithavala Yazdi , Reich Steven D. , List Alan F.
Published: October 2019

Background: AG-013736 is a potent and selective inhibitor of VEGF, PDGF and cKit receptor tyrosine kinases with broad preclinical activity in solid tumors and an AML model. To investigate the anti-leukemic effects of AG-013736 in patients with poor prognosis AML and MDS, we performed a multicenter Phase II Trial.

Methods: Eligible patients (pts) had poor prognosis AML (defined as age of pt ≥65 years, secondary AML, or unfavorable cytogenetics) or poor prognosis MDS (defined as ≥10% myeloblasts in marrow or blood). A 2-stage Simon design was employed with an “unimportant” response rate set at 10% and an “important” rate at 30%. The α and β rates were set at 0.1 with 1 response (complete or partial) in 11 pts in the first stage required before proceeding to the second stage. A successful trial required at least 5 responses in 25 pts. AG-013736 was dosed orally at 5 mg twice a day. Marrow and blood samples were taken for various markers of angiogenesis and microvessel density.

Results: 12 pts (8 AML; 4 MDS) were enrolled including 1 pt who replaced a patient who was withdrawn for lack of compliance after 1 dose. Average age was 77 (range 58–88) years; 7 men, 5 women; 10 white and 2 Asian. All pts were ECOG performance status 0 (5 pts) or 1 (7). 8 pts required blood and 9 platelet transfusions. 8 pts discontinued for lack of efficacy, 3 for non-compliance, and 1 for refusal to participate further. AG-013736-related Grade 3 and 4 adverse events (AEs) included hypertension or blood pressure (BP) elevation (4), mucosal inflammation (1), and deep vein thrombosis (1). Common (>10%) mild-moderate AEs included hoarseness (8 pts), diarrhea (5), BP increased or hypertension (4), proteinuria (3), and mucosal inflammation (2). 9 (75%) pts required at least 1 antihypertensive medication. There were no responses, and therefore enrollment was stopped at 12 patients. Patients received study drug for a median of 53 (range 1–190) days, and 2 pts, one with AML and one with MDS, had stable disease for 132 and 190 days, respectively. Treatment-related changes in biomarkers are being assessed.

Conclusion: AG-013736 was generally well tolerated in this small cohort of elderly pts, 2 of whom had stable disease of significant duration. The combination of AG-013736 with other targeted therapies or cytotoxic agents appears feasible and may be warranted in future studies in patients with poor prognosis AML/MDS.
Generation of a Novel Engineered Murine Apyrase in Pichia Pastoris. Enzymatic Profile and Inhibition of Human Platelet Reactivity.
Authors: Olson Kim E. , Drosopoulos Joan H.F. , Broekman Marinus J. , Islam Naziba , Pulte Dianne , Marcus Aaron J.
Published: October 2019

To study thrombogenesis in wild-type and transgenic mice we engineered a recombinant, soluble form of murine ENTPDase1/CD39 for use as a therapeutic agent. ENTPDase1 contains N- and C-terminal intracellular and transmembrane regions flanking an extracellular domain containing the 5 enzymatically active apyrase-conserved regions. The soluble portion of murine CD39 was amplified using PCR with 5′-XhoI and 3′-XbaI linked primers and cloned into the XhoI/XbaI-digested yeast expression vector pGAPZα. The vector, which features the constitutive GAP promoter and α factor secretion signal, was modified by elimination of the Ste13 cleavage sites and by introduction of a 5′-6His tag separated from the murine solCD39 coding sequence by an enterokinase cleavage site. Stably expressing clones were selected. Aliquots of 30-fold concentrated conditioned medium, obtained following 72 h culture of a yeast clone, were assayed for ADPase and ATPase activity and for the ability to inhibit platelet reactivity. Nucleotidase activity was monitored by native gel electrophoresis and staining using ATP metabolism to phosphate and conversion of Ca-phosphate into PbS, as well as by radio-TLC analyses. Platelet aggregation was quantitated using ADP and collagen as stimuli in heparinized and citrated human platelet-rich plasma (PRP). SolCD39 produced in mammalian cells (CHO, or COS-1) yields an active enzyme that is heavily N-glycosylated. Similarly, Pichia pastoris-produced (murine) solCD39 yields a comparably active protein that is also heavily glycosylated, albeit with the yeast-specific high mannose-type N-glycans. Its ADPase to ATPase activity ratio was ~1.2. Yeast-derived murine solCD39 was treated with PNGase F, an amidase that cleaves between the innermost N-acetyl glucosamine of complex oligosaccharides and asparagine residues of glycoproteins. This removed all saccharides from solCD39, as shown by PAS staining, and by mobility shifts by Western blot analysis and Pb staining in native gels. Interestingly, the deglycosylated protein retained ~60% of both enzymatic and biological activities as compared to the parent molecule. The figures shows platelet aggregation to 5 μM ADP in citrated PRP for control (no murine solCD39), isolated solCD39, and equal quantites of solCD39 that was deglycosylated, as well as subjected to the deglycosylation reaction in the absence of PNGase F. These data are similar for the present murine yeast-produced molecule and COS-produced human solCD39. Our data indicate the possibility of high scale production of solCD39 in yeast, with excellent yield, stability and antithrombotic activity.

Figure Figure
Significant Differences in Neutralization of Heparin and Its Low Molecular Fragments by Protamine Sulfate: An In Vivo Study.
Authors: Crowther Mark A. , Petr Klement , Peng Liao , Frank Chen , Leslie Berry B. , Anthony Chan K.C.
Published: October 2019

In clinical practice, patients receiving low molecular weight heparin (LMWH) occasionally suffer bleeding. Protamine sulphate (PS) is often used to reverse the anticoagulant effect of LMWH in such cases. However, the optimal regimen of PS for complete neutralization of LMWH fragments has not been established. Results from our previous in vitro studies indicate that the ability of PS to neutralize LMWHs is inversely related to the charge of the low molecular weight heparin molecule; more heavily charged LMWHs (such as Tinzaparin) are more readily neutralized than less charged LMWHs (such as Enoxaparin). The aim of the current study was to confirm these findings using an in vivo model. Twenty minutes after administration of either saline, unfractionated heparin [UFH, 100U/kg], Tinzaparin [100U/kg] or enoxaparin [100U/kg], 50% of anesthetized rabbits received either saline or PS [1 mg/100 U of heparin or LMWH]. The efficacy of PS neutralization was assessed by serial measurements of anti-factor Xa heparin plasma levels. Results are presented as mean of the anti-factor Xa heparin activities normalized to the level at 10 minutes and summarized in the table below. As expected, PS completely neutralized the anti-factor Xa effect of UFH. However, PS was significantly less effective for neutralization of Tinzaparin (about 66%) and Enoxaparin (about 44%) at the dose tested. We conclude that when tested in an in vivo model LMWHs vary in their protamine neutralizability. More highly charged LMWHs (e.g. Tinazaparin) are more neutralizable than less highly charged products (e.g. Enoxaparin).

Residual Anti-Xa heparin effect

Time Enoxaparin Tinzaparin UFH Saline + PS Enoxaparin +PS Tinzaparin + PS UFH + PS 10 min 1.00 1.00 1.00 0 1.00 1.00 1.00 20 min 0.85 0.86 0.70 0 0.80 0.85 0.89 Protamine 25 min 0.71 0.75 0.69 0 0.45 0.29 0.01 35 min 0.68 0.64 0.48 0 0.40 0.28 0 50 min 0.48 0.32
Patients with Coronary Artery Disease Have an Increased Incidence of Aspirin Resistance: Association of PFA-100 Closure Time with Clinical Findings.
Authors: Frelinger Andrew L. , Linden Matthew D. , Furman Mark I. , Fox Marsha L. , Barnard Marc R. , Li Youfu , Barringhaus Kurt , Przyklenk Karin , Michelson Alan D.
Published: October 2019

Background: The occurrence of thrombotic events despite aspirin (ASA) therapy in patients with vascular disease has been termed ASA resistance. However, limited data are available correlating laboratory evidence of platelet function with clinical ASA resistance. The PFA-100 collagen/epinephrine (COL/EPI) cartridge can detect the ASA-induced inhibition of platelet cyclooxygenase 1 in a high shear flow environment that mimics in vivo arterial conditions.

Aim: To investigate whether there is an association of PFA-100 test results with clinical ASA resistance.

Methods: Peripheral blood was collected prior to angiography from 560 sequential ASA-treated patients (81 or 325 mg > 3 d). Patients receiving thienopyridines were not excluded but those receiving GPIIb-IIIa antagonists were. Patients with normal angiographic arteries ("No CAD", low probability of ASA resistance) were compared to those with angiographic CAD or a single prior CAD event and to those with multiple CAD events ("CAD" and "Repeat CAD" respectively, higher probability of ASA resistance). Citrate 3.8% anticoagulated blood was tested in the PFA-100 in duplicate using the COL/EPI test cartridge. Patients in the No CAD group were younger, more often female, and less often diabetic, hypertensive, and lipidemic than CAD or Repeat CAD patients.

Results: A short (< 142 s) closure time (CT) in at least one of the duplicate PFA-100 COL/EPI cartridges occurred more frequently in CAD and Repeat CAD patients compared to No CAD patients (p < 0.05, Table).

Conclusions: Failure of ASA to inhibit platelet function, as measured by the PFA-100, is associated with CAD, even in patients without prior history of CAD. The PFA-100, a shear-dependent system, may therefore be a useful test to assess the presence of ASA resistance. This test, unlike some tests for aspirin resistance, does not include ADP and therefore can be used in patients receiving thienopyridines (a frequent co-medication with aspirin).

No CAD CAD Repeat CAD *p<0.05 vs. No CAD PFA-100 CT < 142 s (no. of patients) 4 45 26 PFA-100 CD ≥ 142 s (no. of patients) 79 251 155 Total (no. of patients) 83 296 181 % PFA-100 CT < 142 s 5% 15%* 14%*
The Expression of LI-Cadherin in Bone Marrow Is Correlated with Clinical Outcome in Children with Acute Lymphoblastic Leukemia at First Relapse.
Authors: Wu Shuling , Hernández Anay Marquetti , Dietel Corinna , von Stackelberg Arend , Henze Guenter , Seeger Karl , Gessner Reinhard
Published: October 2019

Liver and Intestine (LI)-cadherin, also termed cadherin-17, is a calcium ion dependent cell-cell adhesion molecule that is highly expressed in the intestinal epithelia but not in the upper gastric tract. Although LI-cadherin is not found in the healthy gastric mucosa, it is ectopically expressed in gastric metaplasia and adenocarcinomas. Recently it was revealed that LI-cadherin expression correlates with gastric cancer progression and lymph node metastasis. In mice LI-cadherin was found to be expressed by B cell progenitor cells. Whether LI-cadherin expression is also linked to the development and progression of malignant hematologic diseases is not known.

In this study, we for the first time showed that LI-cadherin can be detected in several leukemic B-cell lines like Raji, MHH, Nalm6, Z33, REH, and T-cell lines Jurkat, Molt3, Molt4, Molt16 by western bloting and RT-PCR. We subsequently analyzed the expression of LI-cadherin in leukemic bone marrow (BM) slide samples and correlated its impact on the clinical outcome in 119 children with first relapse acute lympnoblastic leukemia (ALL) enrolled in the Berlin-Frankfurt-Münster ALL relapse trial. LI-cadherin expression could be detected in 24 of 119 BM samples (20.2%) using immunofluorescence staining. The frequencies of LI-cadherin in B-cell progenitor (BCP)-ALL and pre-T/T-ALL were 18.4% (19/103) and 31.3% (5/16), respectively. Within BCP-ALL relapses, the maturation-linked expression of LI-cadherin was seen in 22.9% of more mature pre-B (n = 35) than in 17.2% of common (n = 64) or 0% of pro-B (n = 4) leukemic cells. Analysis was confined to BCP lineage to avoid biases related to immunophenotype. Most notably, the probability of survival (pSRV) at 5 years was significantly worse for children with first relapse BCP-ALL who expressed LI-cadherin in BM samples (n = 19) compared with patients who had no expression of LI-cadherin (n = 84) (pSRV: 0.71±0.06 vs. 0.35±0.17; p<0.05). Furthermore, the expression of LI-cadherin was also observed in BM stromal cells which are assumed to support leukemic cell growth and survival.

These results indicated that the expression of LI-cadherin in BM represents a negative prognostic factor for children with first relapse ALL. Since stromal cells also express LI-cadherin, they could bind to LI-cadherin expressing leukemic cells and thus modulate disease progression and/or response to therapy.
Determination of a HLA II Promiscuous Peptide Cocktail as Potential Vaccine Against EBV Latency II Malignancies.
Authors: Depil Stephane , Morales Olivier , Maillere Bernard , Delhem Nadira , Francois Violaine , Georges Bertrand , Morschhauser Franck , Auriault Claude , Pancre Veronique
Published: October 2019

The Epstein-Barr virus (EBV) is associated with several malignant diseases which can be distinguished by their patterns of viral latent gene expression. The latency II program is limited to the expression of the non-immunodominant antigens EBNA1, LMP1 and LMP2 and is seen in EBV-positive Hodgkin’s disease, nasopharyngeal carcinomas and peripheral T/NK-cell lymphomas. CD4+ T cells may play a crucial role in controlling these EBV latency II malignancies. The aim of this study was to determine a peptide cocktail containing highly promiscuous MHC class II epitopes derived from the EBV latency II antigens. Using the prediction software TEPITOPE, we selected 24 potential MHC II promiscuous peptides derived from the EBV latency II antigens EBNA1, LMP1 and LMP2. The predicted peptides were then submitted to peptide-binding assays on HLA II purified molecules, which allowed the selection of 6 peptides (EBNA1: 3; LMP1: 1; LMP2: 2) with a highly promiscuous capability of binding. This peptide cocktail was immunogenic in a model of HLA-DR1 transgenic mice, leading to a specific cellular and humoral Th1 response. To determine whether the peptides of the cocktail were recognized by human CD4+ T cells, we stimulated CD8-depleted PBMCs from 12 healthy EBV-seropositive donors expressing various HLA genotypes. A proliferative response was observed in all of the donors tested. Significant levels of IFN-gamma and IL-10 secretion were observed in 7/12 and 4/12 donors, respectively, using ELISA method. We also found a relatively low but significant number of IFN-gamma-secreting memory cells in 8 of the 10 donors tested using ELISPOT assay (range, 22 to 167 SFC per 106 CD8-depleted PBMCs). Finally, we analyzed by ELISPOT the responses obtained using PBMCs from 5 EBV-seropositive Hodgkin patients at different stages of the disease. No significant response was observed in the 2 patients at diagnosis. By contrast, a highly significant response was observed in a patient with EBV-associated Hodgkin lymphoma in remission.

In conclusion, we have determined an HLA II promiscuous peptide cocktail containing epitopes from EBV latency II antigens. This promiscuous peptide cocktail may be immunogenic in the majority of the population and could be used in immunotherapeutic approaches (as peptide-based vaccine or cellular therapy) against EBV latency II malignancies.
Anaemia Management in Patients on Peritoneal Dialysis in Portugal: A Nationwide Multicenter Survey.
Authors: Branco Patrícia Q. , Abreu Cristina P. , Pessegueiro Pedro , Amoedo Manuel , Rodrigues Anabela , Cabrita Antonio , Bruges Margarida , de Almeida Edgar , Prata Mateus Martins
Published: October 2019

Introduction: The European Best Practice Guidelines for the Anaemia Management (EBPG) recently published emerged as an international standard in the management of anemia in chronic kidney disease (CKD) patients.

Objectives: To evaluate the impact of theses guidelines in the clinical practice in Portugal.

Methods: This epidemiological, multicentric and cross-sectional study included patients on peritoneal dialysis that were under erythropoietin treatment in Portugal during 2004. Etiology of CKD, prevalence and anaemia treatment, comorbidity and side effects were evaluated.

Results: 220 patients from 5 Units were evaluated. Mean haemoglobin was 12.34 g/dL and only 13,2% of patients had haemoglobin <11 g/dL. Ninety-nine percent of patients were treated with erythropoietin: 65% with beta erythropoietin (beta) and 35% with darbepoetin alfa (darbe). Subcutaneous route had been used in all cases: 5,4% with once-monthly administration (darbe), 19,55 % two times monthly (darbe and beta), 59% with once a week regimen (darbe and beta), 11,55% in two weekly doses (beta) and 4,5% in three weekly administrations (beta). Doses requirements were different according to administration frequency. More than 85% of patients had haemoglobin > 11 g/dl and no significant difference in haemoglobin levels was achieved in the two groups (beta and darbe). Erythropoietin doses were greater in the group treated with darbe (127 versus 113 UI/kg/week), but inflammation markers were significantly higher as well diabetic patients in this group.

Conclusion: These results suggest that, according to the EBPG’s, mostly patients on PD in Portugal are already being well treated presenting heamoglobin levels in the therapeutic range.
The Involvement of Reactive Oxygen Species in Polyploidization of the M-07e Human Megakaryocytic Cell Line.
Authors: Côté Serge , Dussault Nathalie , Simard Carl
Published: October 2019

Hematopoietic cells mature in the bone marrow under the control of a diversity of growth factors and the influence of various cell types producing superoxide and other reactive oxygen species (ROS). As ROS may regulate activities of redox-sensitive enzymes implicated in a wide range of cellular processes, we have exposed the human megakaryocytic cell line M-07e to hydrogen peroxide (H2O2) at concentrations that increased intracellular ROS and examined whether expression of the megakaryocytic programme could be enhanced. The growth-factor dependent M-07e cells display surface markers characteristic of both early myeloid progenitors and more committed members of the magakaryocyte (Mk) lineage, such as glycoproteins GPIIb-IIIa (CD41) and GPIb (CD42). H2O2 significantly reduced cell proliferation without affecting viability. After 4 days of exposure to this reagent, expression of the early Mk marker CD41 was 1.2 times higher than that of control cells. Although no change in the expression of the late Mk marker CD42 was detected, exposure to H2O2 was found to increase the incidence of multinucleate cells, polyploidy and abnormal microtubule organising centre numbers. Investigation of this phenomenon on synchronized M-07e cells revealed that H2O2 arrested cytokinesis at a late stage and that some nuclei were still able to incorporate bromodeoxyuridine (BrdU). Cell division was similarly impaired when M-07e cells were either exposed to botulin toxin C3 transferase or Y-27362 inhibitor, suggesting that H2O2 treatments affected members of the Rho family of small GTP-binding proteins and/or their effectors. Together, these findings indicate that endoreplication in Mk may be linked to changes in the cellular redox state of these cells and support the concept that differentiation and polyploidization are independently regulated events.
Rituximab, Fludarabine, Mitoxantrone, and Dexamethasone (R-FND) for Relapsed Indolent Lymphomas (RIL).
Authors: Hagemeister F.B. , McLaughlin P. , Fayad L. , Samaniego F. , Dang N. , Goy A. , Romaguera J. , Pro B. , Beasley V. , Cabanillas F. , Rodriguez M.
Published: October 2019

Optimal therapy for patients (pt) with B-cell RIL remains controversial, and many treatment options include rituximab (R). Based on phase I–II studies of FND (fludarabine 25 mg/m2, d1–3; mitoxantrone 10 mg/m2, d1; dexamethasone 20 mg d1–5) treatment for RIL in which we observed a 48% CR rate, we studied the combination of R (375 mg/m2 d1) and FND for pt with RIL. Pt could not have received prior fludarabine, and were eligible if they had not previoiusly received R or had a response lasting at least 6 months (mo) to prior treament with R. Cycles were repeated every 4 weeks, and required an absolute neutrophil count of 1,000 and a platelet count of 100,000 to administer each cycle. Forty-two pt were entered onto this trial; however, one never received therapy after signing consent, and two were deemed ineligible after signing consent because of low cardiac ejection fractions (EF). All pt underwent biopsy prior to therapy; histologies included follicular gr1–3 in 30 pt, small lymphocytic in 7, marginal zone in 2. The median age was 58 (range 39–84) and median prior therapies 1 (range 1–3). The median number of cycles of R-FND delivered per patient was 6 (range 1–8). Of the 39 eligible and evaluable pt, 19 entered CR and 9 CRu for a CR/CRu rate of 72%, and 9 entered PR (23%) for an overall response rate of 95%. One had stable disease (SD), and one progression (PD). Eight underwent high dose therapy followed by autologous stem cell transplant (SCT) following a response to R-FND therapy. Five of these had achieved CR/CRu with R-FND after 1–6 cycles, and 3 PR after 2–6 cycles. In all, 14 of the 37 responders to R-FND (37%) have had progression; however, none of the 8 who underwent SCT has had progression, nor has the one with SD. In, 24 (62%) still remain free of progression with a median follow-up for living pt of 32 mo. In all, 6 pt have died of PD, including 4 who were ineligible for or refused SCT. The 2-year failure-free survival (FFS) and overall survival (OS) results for all pt are 66% and 90%. The 2-year FFS for responders is 66%, and the 2-year OS for all pt, with pt undergoing SCT censored at time of SCT is 81%. As expected, the main toxicity following R-FND was hematologic: gr 4 neutropenia occurred in 15 pt and in 15 of 130 cycles for which information is available, and gr 3–4 thrombocytopenia in 5 pt and in 6 cycles. In all, 13 of the 28 responders received less than 6 cycles of therapy; reasons included prolonged thrombocytopenia in 4, early SCT in 6, asymptomatic decrease in EF in one, and physician’s choice in 2. No patients died of acute toxicity while receiving R-FND. We conclude that 1) R-FND is a very active and well tolerated regimen for relapsed indolent lymphomas 2) Results appear very favorable compared to prior studies with FND 3) In this population, patients receiving R-FND were able to undergo SCT, and had very favorable outcomes.
Immune Tolerance Induction by High Doses FVIII in Hemophilia A Patients with Inhibitor Does Not Require Deletion of FVIII-Specific T Cells.
Authors: Jacquemin Marc G. , Lavend’homme Renaud , Tinot Thérèse , Peerlinck Kathelijne , Saint-Remy Jean Marie , Pautard Brigitte
Published: October 2019

In patients with hemophilia A and FVIII inhibitor, tolerance to FVIII can frequently be restored by prolonged administration of FVIII (immune tolerance induction). We studied the cellular response to FVIII in two patients successfully desensitized with high doses of FVIII. Each patient had also transiently received glucocorticoids, when the antibody titer increased after an initial decline upon start of the desensitization. On repeated stimulations with FVIII loaded autologous dendritic cells, FVIII-specific T oligoclonal cell lines and T cell clones were derived from one of the two patients up to two years after complete elimination of the inhibitor. The interleukins produced by the T cell lines included IL-2, IL-5 and IL-13. Although ITI is frequently unsuccessful in mild/moderate haemophilia A patients, this patient remained tolerant to FVIII even after a 10 days FVIII continuous infusion, 18 months after complete elimination of the inhibitor. Several microcultures from CD4+ T cells purified after this treatment responded to FVIII specifically demonstrating the long term persistence of FVIII-specific T cells after ITI. By contrast, no FVIII-specific T cell lines could be derived from blood of control individuals without hemophilia A. Thus, although in animal models of tolerance, administration of high doses of antigen result in T cell elimination, deletion of FVIII-specific T cells is not required for successful tolerance induction to FVIII.
The Concentration of Exogenously Added Lipids and Endogenously Available Microparticles Are Major Determinants of Thrombin Generation in Plasma.
Authors: Kluft C. , Meijer P. , Kret R. , Kaufmann V. , Mager J.
Published: October 2019

Thrombin generation tests are started by adding coagulation activator (e.g. tissue factor or contact activator) together with lipids.

We evaluated the role of lipids in tests started with a fixed amount of tissue factor (7.16 pM) and addition of either 3.2 (high) or 0.32 (low) μM of lipids (Technothrombin ® TGA assays from Technoclone, Vienna) in both normal plasma and plasma that was ultracentrifuged (30 minutes at 15,000 g) to remove microparticles (MPs). The tests were performed in plasma samples of groups of apparently healthy individuals.

It was observed in 54 healthy volunteers that starting with high or low lipids substantially influenced the total amount of thrombin generated expressed by the area under the curve (AUC) (AUC median 2492, IQR 716 versus AUC median 1154, IQR 652 nM*min, respectively), the rate of thrombin formation or velocity index (VI) (median 53.4, IQR 43.6 and median 11.7, IQR 10.6 nM/min, respectively), and the lag time to the start of thrombin generation (median 10.3, IQR 2.4 versus median 17.5, IQR 5.0 minutes, respectively). It can be concluded that the VI is the most sensitive variable showing approximately a factor of 5 difference between high and low lipid.

The difference of adding high or low lipid on VI was primarily dependent upon the lipid concentration and to a limited extend influenced (univariate) by factor II levels (12%) and factor IX levels (10 %), taking into account practically all known coagulation factors (fibrinogen, II, V, VII, VIII, IX, X, XI, XII, PC, PS, PZ, TFPI, PCI) determined in the 54 plasma’s as potential determinant. In plasma of 36 volunteers microparticles were removed and VI dropped to 19% in comparison to the untreated plasma when tested with the addition of high lipid and to 3.1% with the addition of low lipid. Re-addition of MPs to a specific plasma restored VI dose dependently with an optimum at 2x104 MPs/ml. The same level of VI (63.8 and 62.0 nM/min, respectively) was reached with high and low lipid addition when 2.104 MPs/ml were added.

It is concluded that endogenous MPs play an important role in thrombin generation tests, in particular but not exclusively when the test is performed with low levels of added lipids.
Metabolic Analysis of Bone Marrow Mesenchymal Stem Cells and Adipose-Derived Stem Cells Using NMR-Based Metabolomics.
Authors: Ogawa Rei , Fujimura Juri , Hirakawa Keiko , Uekusa Kyoko , Yuta Kohtaro , Mizuno Hiroshi , Ohno Youkichi , Hyakusoku Hiko
Published: October 2019

Background: Nuclear magnetic resonance (NMR) -based metabolomics has been suggested to be useful for exhaustive analysis of metabolic pathways of cells or tissues. We report our study on bone marrow mesenchymal stem cells (BSCs) and adipose-derived stem cells (ASCs) using NMR-based metabolomics.

Materials and Methods: Five-week-old Wister rats were used for this study. BSCs were harvested from the femur, ASCs from inguinal fat pads, and control fibroblasts from the abdominal dermis. The cells were cultured in DMEM with 10% fetal bovine serum and harvested after two passages of the subculture. Then, the cells were subjected to freezing in liquid nitrogen and crushed to extract the aqueous metabolites. 1H-NMR spectra was measured and analyzed by a computer software (Alice2 for metabolome™ and ADMEWORKS/ModelbuilderTM).

Results: BSCs, ASCs and fibroblasts were clearly separated into three groups on a principal component analysis (PCA) plot.

Conclusion: ASCs, BSCs and fibroblasts were considered to have different metabolic activities, and NMR-based metabolomics will henceforth be useful for the detection, analysis, and characterization of various kinds of stem cells.
Thrombelastographic Observations on the Characteristics of Hemophilic, Thrombocytopenic and Heparinized Blood
Authors: DE NICOLA PIETRO , MAZZETTI G. M.
Published: October 2019

1. Thrombelastographic determinations were made in cases of AHG-, PTC- and PTA-deficiencies, in thrombocytopenias and in normal plasmas after the addition, in vitro, of heparin and synthetic heparin-like substances.

2. The components of the thrombelastogram (TEG) were correlated with respect to the reaction time (r), the rate of clot formations (k), and the maximal amplitude (ma).

3. The differentiation of the hemophilic and thrombocytopenic syndromes was made on the basis of the typical variations of r, k and ma: prolonged r and k in hemophilia, prolonged k and decreased ma in thrombocytopenias.

4. The behavior of heparinized blood was characterized by a hemophilia-like prolongation of r and k and a thrombocytopenic-like decrease of ma, with variations depending on the compound used.

5. The correlations between the r, k and ma values of TEG are suggested for the evaluation of thrombelastography.
The Effects of Varying Concentrations of Human Platelets and Their Stored Derivatives on the Recalcification Time of Plasma
Authors: KLEIN EDMUND , FARBER SIDNEY , FREEMAN GUSTAVE , FIORENTINO ROSEMARY
Published: October 2019

Human platelets and their stored, frozen and lyophilized derivatives do not excessively promote coagulation, as indicated by the calcium clotting time. High concentrations of platelet material inhibit coagulation, while dilution to physiologic levels returns the recalcification time to normal values.
The Effects of Various EDTA Complexes on Coagulation
Authors: LEIKIN SANFORD , BESSMAN SAMUEL P.
Published: October 2019

The one-stage prothrombin time of oxalated plasma was prolonged on incubation with disodium EDTA. This was due to the destruction of Ac-Globulin or the inhibition of its production from a precursor stage. There was a similar change with disodium calcium EDTA, indicating some metal other than calcium was probably being chelated.

A marked prolongation of the prothrombin time of oxalated plasma occurred on incubation with cupric, ferrous, stannous, cobaltous, and magnesium EDTA, but manganous, zinc and nickel EDTA produced no prolongation of the prothrombin time. This would support the concept that a metal ion may be concerned with the maintenance of Ac-Globulin or its conversion into an active form.

The cobaltous, cupric, ferrous and ferric ions produced a prolongation of the prothrombin time. This effect was corrected by the use of prothrombin free plasma except in the case of cobalt. The manganous, barium and nickel ions did not prolong the prothrombin time and nickel was effective in maintaining the prothrombin time at its original level. Its most effective concentration was .001 M. At higher concentrations it produced a prolongation which was not corrected by the addition of prothrombin-free plasma.
Studies on Copper Metabolism
Authors: CARTWRIGHT G. E. , GUBLER C. J. , BUSH J. A. , WINTROBE M. M.
Published: October 2019

1. The morphology of the erythrocytes has been studied in 22 normal swine, 65 swine deficient in copper, and 16 swine deficient in iron. The mean values (± the standard deviation) for the volumes of packed red cells in the three groups were 42 ± 2.5, 21 ± 6.6, and 21 ± 6.1 ml/100 ml., respectively. The mean values for the mean corpuscular volumes were 55 ± 3.1, 43 ± 7.1, and 36 ± 5.3, respectively. The mean values for the mean corpuscular hemoglobin concentrations were 33 ± 1.1, 29 ± 2.3, and 28 ± 3.0, respectively.

2. The addition of 11 water-soluble B vitamins, 4 fat-soluble vitamins, ascorbic acid, and minerals (with the exception of copper) to the copper-deficient milk diet did not prevent or delay the development of anemia, modify the morphology of the red cells or alter the degree of hypoferremia.

3. The normoblasts in the marrow of a copper-deficient pig were similar morphologically to those in the marrow of a pig deficient in iron.

4. The intravenous administration of one Gm. of iron, given prior to the development of copper-deficiency, did not delay or prevent the development of anemia.

5. It is concluded that the anemia of copper-deficiency in swine is morphologically similar to the anemia due to iron deficiency in swine.
A Sign of Severe Radiation Injury Observed in the Erythrocyte Sedimentation of Dogs
Authors: SACHER GEORGE A.
Published: October 2019

Erythrocyte sedimentation curves are analyzed by determining the constants of a linear relation between logarithm of sedimentation distance and logarithm of time that obtains for the initial part of the sedimentation process. The sedimentation curves obtained from dogs exposed to x-rays, or injected with radiostrontium or plutonium were analyzed in this manner. The pathologic sedimentation curves fall into two groups. Dogs that received sublethal dosages of x-ray, radiostrontium or plutonium showed a response (phase I response) in which decreased slope of the log-log line is associated with increased intercept. Dogs that received dosages in or beyond the LD50 range showed, during the period of maximum acute death rate, a further increase in intercept associated with an increase in slope (phase II response). An increased slope indicates an increased rate of rouleau growth. It is inferred that the phase I response is due to decreased hematocrit and the phase II response to an increased titer of an agglutinating principle.
Studies on the Anemia of Disseminated Malignant Neoplastic Disease. II. Study of the Life Span of the Erythrocyte
Authors: HYMAN GEORGE A. , GELLHORN ALFRED , HARVEY JANE L.
Published: October 2019

1. A study of the life span of the erythrocyte in patients with advanced neoplastic disease was performed by transfusing blood from 23 hospitalized patients into 53 healthy volunteers.

2. The Ashby technic for red cell labeling was used alone in 16 instances, Cr51 technic in 52 instances, and a combination of the 2 technics in 8 instances.

3. Normal or nearly normal life spans were suggested by the slopes of curves in 15 instances where whole blood from patients was transfused into volunteers.

4. Some shortening of the life span of the patient’s red cells was noted when these cells, separated from the plasma, were transfused into volunteers or back into patients, possibly related to the handling during plasma removal.

5. Control studies using either Cr51 or Ashby labeling technic indicated a normal life span of approximately 120 days in the volunteers employed could be achieved by each method.

6. Values obtained with Cr51 were so similar to those using Ashby labeling that the easier Cr51 technic is recommended for future studies.

7. The normal erythrocyte transfused into patients with neoplastic disease may have a moderate to considerably shortened life span.

8. These studies seem to demonstrate an absence of an intrinsic defect in the erythrocytes of patients with neoplastic disease, and further favor the presence of a hemolytic plasma factor of considerable importance in the pathogenesis of the anemia of cancer.
Studies on the Biosynthesis of Heme In Vitro by Avian Erythrocytes
Authors: GOLDBERG A. , ASHENBRUCKER HELEN , CARTWRIGHT G. E. , WINTROBE M. M.
Published: October 2019

1. A method, based on the uptake of radioiron into heme, is described for the measurement of heme synthesis in a hemolysate of chicken erythrocytes.

2. The addition of glycine, δ-aminolevulinic acid, porphobilinogen or protoporphyrin 9 to the system augmented heme synthesis.

3. Citrate potentiated heme synthesis in the presence of glycine, but had no effect when porphobilinogen was added. Succinate, in the presence of glycine did not enhance heme synthesis.

4. The addition of coproporphyrin I or III, or uroporphyrin I or III, did not augnment the uptake of radioiron into heme. The addition of mesoporphyrin 9 or hematoporphyrin 9 enhanced the uptake of radioiron. These studies are iterpreted as suggesting that protoporphyrin, but not uroporphyrin or coproporphyrin, is a precursor of heme. For reasons discussed, further work will be necessary to determine if mesoporphyrin and hematoporphyrin are precursors of protoheme.

5. The synthesis of heme was inhibited by the addition of malonate or lead. Evidence is presented that both of these substances affected heme synthesis at several different levels, particularly the formation of δ-aminolevulinic acid and the incorporation of iron into protoporphyrin.

6. Plasma extracts from chickens, made anemic by bleeding or by the administration of phenylhydrazine, did not potentiate heme synthesis.

7. Normal, nonreticulated, human erythrocytes failed to synthesize heme in the presence of glycine, δ-aminolevulinic acid, porphobilinogen on protoporphyrin 9.
Hematologic Observations on Patients with Sickle Cell Anemia Sustained at Normal Hemoglobin Levels by Multiple Transfusions
Authors: CHAPLIN HUGH , KEITEL HANS G. , PETERSON RALPH E.
Published: October 2019

(1) Three sickle cell patients were sustained at normal hemoglobin levels for 3-4 months by means of repeated transfusions of fresh blood.

(2) In response to transfusions, there was a decline in reticulocytes to normal levels during the first 5-7 days of observation. During periods in which the hemoglobin was maintained at high-normal or super-normal levels, the reticulocyte values were depressed below the normal range. A distinct reticulocyte response was observed when the hemoglobin declined to approximately 11.0 Gm. per cent following cessation of transfusions.

(3) Employing anti-M differential agglutination, a simple exponential decline was observed in the number of sickle cells in each patient’s circulation during the period of sustained normal hemoglobin concentration.

(4) The continued production of new sickle cells during the first week of observation complicated the interpretation of the differential agglutination data, but provided indirect evidence for the presence of an especially short-lived proportion of the patient’s cells. Support for this concept was derived from the radioactive iron utilization studies performed in Case 1.
Long Term Disease Free Survival in Patient with Linfoplasmocytic Lymphoma and Regression of Associated Severe Renal Failure after Treatment with Bortezomib.
Authors: Abbadessa Antonio , Monaco Giuseppe , Troiano Mario , Liguori Lucia , Focarile Elisa , Vigliotti Maria Luigia , Iaccarino Salvatore
Published: October 2019

Background. We describe a case of lymphoplasmocytic lymphoma, refractory to conventional treatments and complicated with bone lesions and acute renal failure, completely regressed after therapy with bortezomib. Case report. Woman, 48 years old, in march 1994 receives diagnosis of lymphoplasmocytic lymphoma, refractory to conventional chemotherapy (chlorambucil, CHOP, fludarabine), immunotherapy (rituximab), radiotherapy. She comes to our attention in november 2005 for disease’s progression, associated to severe renal failure (sieric creatinine=3.65 mg/dl; creatinine clearance = 17.52 ml/min), caused by serious monoclonal proteinuria (21500 mg/24h). We decide to use dexamethazone (20 mg) and bortezomib at a reduced dose (1mg/m2 twice a week on days 1,4,8,11, for eight cycles). At the end of second cycle with bortezomib, there is a conspicuous reduction of proteinuria (350 mg/24h) with partial recovery of renal function (sieric creatinine = 1.7mg/dl; creatinine clearance=23.68ml/min) Because of severe neurologyc toxicity (convulsion, peripheric neuropathy) and appearance of herpetic lesions, we decide to suspend treatment with bortezomib. After eleven months from the end of treatment with bortezomib, PET total-body, shows only a moderate hypercaptation on the anterior segment of lower lobe of right lung. At a follow-up of 20 months renal function results clearly improved (sieric creatinine = 1.4 mg/dl; creatinine clearance=44.57 ml/min) with normalization of proteinuria (104 mg/24h).

Discussion. Renal faillure in course of lymphoplasmocytic lymphoma is a not frequent event. It is due to amount of paraprotein on endothelial side of glomerular basal membrane. Generally standard treatments not improves the renal failure. The therapeutic activity of the antiproteosome in myeloma and in different lymphomas is currently well documented (O’Connor et al, JCO2005, 23 (4):676–684; K. Anderson et al, JCO2006 Annual Meeting Proceedings Part I. Vol 24 (18S): 7504). Other authors have demonstrated the renal repair (A. K. Malani et al, Acta Haematologica2006;116 (4):255–258).

Conclusion. bortezomib, proteosome inhibitor, can be used to improve not only myeloma and lymphoma course but also to repair renal failure, when caused by paraprotenemia/paraproteinuria.
Systematic Profiling and Novel Targets of the Bcr-Abl Kinase Inhibitors Imatinib, Nilotinib and Dasatinib.
Authors: Hantschel Oliver , Rix Uwe , Buerckstuemmer Tilmann , Schmidt Uwe , Kneidinger Michael , Bennett Keiryn L. , Kaupe Ines , Ellmeier Wilfried , Valent Peter , Superti-Furga Giulio
Published: October 2019

The BCR-ABL tyrosine kinase inhibitor imatinib represents the current frontline therapy in chronic myeloid leukemia (CML). Since imatinib resistance has become a major clinical problem in recent years, two second-generation drugs, nilotinib and dasatinib, displaying increased potency against BCR-ABL and targeting imatinib-resistant CML clones were developed. To predict potential side effects and novel medical uses we generated comprehensive drug-protein interaction profiles for all three drugs. We have used an unbiased chemical proteomics approach to detect drug-binding proteins directly from lysates of CML cells. Our studies yielded four major findings:

The interaction profiles of the three drugs displayed strong differences and only a small overlap covering the ABL kinases. Dasatinib bound in excess of thirty Tyr− and Ser−/Thr−kinases, including major regulators of the immune system suggesting that dasatinib might have a particular impact on immune function. Despite the high specificity of nilotinib the receptor tyrosine kinase DDR1 was identified and validated as an additional major target. The oxidoreductase NQO2 was bound and inhibited by imatinib and nilotinib at physiologically relevant drug concentrations representing the first non-kinase target of these drugs.

In addition, we have concentrated on the promiscuous binding profile of dasatinib: Besides Abl and Src kinases, we have identified the Tec kinases Btk and Tec, but not Itk, as major binders of dasatinib. The kinase activity of Btk and Tec, but not of Itk, was inhibited by nanomolar concentrations of dasatinib in vitro and in cultured cells. We identified the gatekeeper residue as the critical determinant of dasatinib susceptibility. Mutation of Thr-474 in Btk to Ile and Thr-442 in Tec to Ile conferred resistance to dasatinib, while mutation of the corresponding residue in Itk (Phe-435) to Thr sensitized the otherwise insensitive Itk to dasatinib. The configuration of this residue may be a predictor for dasatinib sensitivity across the kinome. Analysis of mast cells derived from Btk-deficient mice suggested that inhibition of Btk by dasatinib may be responsible for the observed reduction in histamine release upon dasatinib treatment. Furthermore, dasatinib inhibited histamine release in primary human basophils and secretion of pro-inflammatory cytokines in immune cells. The observed inhibition of Tec kinases by dasatinib predicts immunosuppressive (side) effects of this drug and may offer new therapeutic opportunities for inflammatory and immunological disorders.
Activity of Circulating Proteasomes Correlates with Clinical Behavior in Patients with Chronic Myeloid Leukemia.
Authors: Ma Wanlong , Kantarjian Hagop , Donahue Amber , Zhang Xi , O’Brien Susan , Jilani Iman , Estey Elihu , Estrov Zeev , Cortes Jorge , Keating Michael , Giles Francis , Albitar Maher
Published: October 2019

The ubiquitin-proteasome pathway is implicated in the pathogenesis of many malignancies and is an important regulator of cell proliferation, apoptosis, DNA repair, and the stress response. Free circulating proteasomes have been reported in the plasma and sera of cancer patients. We measured proteasome peptidase activity in the plasma of patients with chronic myeloid leukemia (CML) and correlated these findings with clinical behaviour. Plasma samples from apparently healthy volunteers (n = 92) and patients in the chronic (n = 104) or accelerated/blast phase (n = 56) of CML were analysed with fluorogenic kinetic assays. Using peptide-AMC (7-amino-4-methylcoumaran) substrates, we measured the three reported proteasome enzymatic activities: chymotrypsin-like (Ch-L), trypsin-like (Tr-L), and caspase-like (Cas-L). All proteasome activities were significantly higher in CML patients than in healthy volunteers. Extensive characterization of proteasome activities revealed correlations between levels or ratios of specific peptidase activities and disease phase, progression, aggressiveness, and survival. High Ch-L activity correlated with shorter survival in both CP (P=0.037) and Acc/Bl phase (P=0.047). Patients in CP and a ratio of Cas-L: Tr-L activity >1.48 had significantly shorter survival (P=0.03). Cas-L: Tr-L activity ratio was predictor of survival in Acc/Bl patients as a continuous variable. These findings suggest that proteasome activity in the plasma of CML patients reflects disease activity and can be used as a biomarker for predicting clinical behavior. Studies exploring the role of proteasome inhibitors in CML are therefore warranted.