bluuebunny/crossref_metadata_2025_split huggingface dataset:
Monitoring Endothelial Cell Activation In Vivo during Thrombus Formation.
Authors: Atkinson Ben T. , Nandivada Prathima , Furie Bruce , Furie Barbara C. 
Published: October 2019

Abstract
The endothelium serves as a metabolically active interface between the blood and underlying tissues and offers response to changes in its microenvironment. The endothelium is rapidly activated in response to stimuli associated with fluctuations in shear stress, physical trauma, oxidative stress, and thrombotic and inflammatory mediators. To study endothelial cell activation we have monitored calcium mobilization in vitro in cultured endothelial cells and also in situ in the living mouse, following vessel wall injury. The addition of exogenous ADP (10 μM) or thrombin (1 U/mL) to Human Umbilical Vein Endothelial Cells (HUVECs) loaded with the calcium sensitive dye, Fluo-4 AM, is followed by rapid elevation of intracellular calcium, with a sustained peak observed within 10 sec. To further investigate the ability of the endothelium to activate in response to mediators potentially localized in the microenvironment of the endothelium during thrombus formation, we investigated the ability of IL-6 (0.1 ng/mL), IL-8 (80 ng/mL), MCP-1 (15 ng/mL), NAP-2 (10 ng/mL) and SDF-α (60 ng/mL) to mobilize calcium in HUVECs. These potential agonists were selected based on their primary roles in inflammation and thrombosis, and proposed roles in atherosclerosis and tumor angiogenesis. SDF-α and NAP-2 rapidly mobilized calcium in HUVECs, with similar maximum responses, but NAP-2 initiated a more prolonged (4-fold longer) rise in intracellular calcium. IL-6, IL-8, and MCP-1 also mobilized calcium, but the rise in intracellular calcium was almost 4-fold lower than that observed with SDF-α and NAP-2. Comparable calcium mobilization occurs in HUVECs subjected to a single pulse of a nitrogen dye-tuned laser. In addition, targeting of single cells within a confluent culture of endothelial cells initiated calcium elevation in the targeted cell and was followed by a wave of calcium elevation in surrounding cells. To determine whether this endothelial cell activation, and more specifically the calcium elevation, occurs in vivo, we conducted experiments using the laser-induced model of thrombus formation to look for calcium elevation in the arteriolar endothelium in live mice. Calcium elevation was monitored by Fluo-4AM introduced intravenously into the mouse circulation via the jugular vein. Fluo-4AM is non-specific in its uptake among cell types, and endothelial cell uptake of the dye in the cremaster muscle microcirculation was observed. Upon laser-induced injury, calcium elevation in the endothelium could be monitored by intravital microscopy. The observed calcium elevation was rapid (within 30 s) and preceded detection of platelets in the developing thrombus. These results add to the evidence of a dynamic endothelium and demonstrate that the endothelium activates rapidly prior to thrombus formation in the laser induced thrombosis model.
Dasatinib-Induced Platelet Dysfunction.
Authors: Quintas-Cardama Alfonso , Han Xin , Kantarjian Hagop , Cortes Jorge 
Published: October 2019

Abstract
Dasatinib is efficacious and safe in patients (pts) with chronic myelogenous leukemia (CML) after imatinib failure. However, dasatinib therapy has been linked to bleeding in some pts, typically affecting mucosal surfaces. We analyzed the effect of dasatinib on basic coagulation tests and platelet aggregation function in 55 pts with CML in chronic phase at diagnosis (1), >8 weeks after tyrosine kinase inhibitor (TKI) discontinuation (3), or during therapy with the ABL/SRC inhibitors dasatinib (n=27) and bosutinib (n=12), or the aminopyrimidine derivatives imatinib (n=8) and nilotinib (n=4). Median age was 54 yrs (range, 22–80), WBC 5.5 ×109/L (range, 2.1–58.5), hemoglobin 12.2 g/dL (range, 9.7–15.1), and platelets 230 ×109/L (range, 80–746). Only 2 pts (both on imatinib and warfarin) had abnormal basic coagulation parameters (prolonged prothrombin time [PT]). In the remainder 53 pts, the median PT time was 11.6 sec (9.8–13), activated partial thromboplastin time (aPTT) 28.5 sec (range, 21–33.9), and fibrinogen level 495 mg/dL (range, 336–700), suggesting an intact secondary hemostasis. To evaluate the primary hemostasis status, platelet aggregation was measured by turbidometry in 1 mL of platelet-rich plasma upon stimulation with 5 μM of ADP, 1 μg/ml of collagen, 1 μM of epinephrine, 1 mM arachidonic acid, or 8 μL of ristocetin by using a Whole Blood Lumi-Aggregometer (Chrono-Log Corporation, Havertown, PA). Aggregation was expressed as the maximal percent change in light transmittance from baseline using platelet-poor plasma as a reference. Five (9%) patients (2 bosutinib, 1 nilotinib, and 2 imatinib) were on aspirin and showed an aspirin-like effect (decreased platelet aggregation with arachidonic acid [AA] and epinephrine [Epi]). Patients off-TKI or untreated had a normal platelet aggregation test. Results on the remaining 46 assessable patients demonstrate that dasatinib is strongly associated platelet aggregation defects, particularly an aspirin-like effect, when compared with other TKIs.
Platelet function analysis by PFA-100 showed that addition of dasatinib (400 nM) to fresh whole blood obtained from a healthy volunteer rapidly induced platelet dysfunction. Closure times were prolonged from 100 sec (baseline) to >300 sec in response to Epi (normal <160 sec) but not in response to ADP (from 72 to 84 sec, normal <120 sec) after 30 min incubation. In summary, our data indicate that dasatinib does not interfere with secondary hemostasis. Rather, it inhibits platelet function in an aspirin-like manner. This effect, while independent, can be additive to dasatinib-induced thrombocytopenia and may account for the bleeding diathesis observed in pts with CML receiving dasatinib.
Table 1. Effect of BCR-ABL kinase inhibitors on platelet aggregation Dasatinib Bosutinib Nilotinib or Imatinib Platelet aggregation test n=27 n=10 n=9 p value Normal (%) 4 (15) 8 (80) 6 (67) < 0.01 Abnormal (%) 23 (85) 2 (20) 3 (33) < 0.006 Only with Epi 7 1 0 With both Epi and AA 14 1 3 With all agonists 2 0 0
DNA-Based Measurement of BCR-ABL in Chronic Myeloid Leukemia (CML).
Authors: Morley Alexander , Bartley Paul , Martin-Harris Henry , Latham Sue , Ross David , Hughes Timothy 
Published: October 2019

Abstract
Aim To develop a DNA-based method for measurement of minimal residual disease (MRD) in CML
Background Currrent RNA-based methods for quantification of the BCR-ABL translocation have a number of disadvantages, including the potential for RNA degradation, requirement for a reverse transcription step, uncertain sensitivity and specificity, and only an indirect relationship between assay result and cell number.
Methods Tagged PCR primers were designed to span the common 3kb breakpoint region of BCR and corresponding 140kb breakpoint region of ABL. Primers were spaced to bind at approximately 500bp intervals in order to limit the size of the target sequence and ensure efficient amplification. PCR was performed with pools of 1-6 BCR forward primers and pools of 24–282 ABL reverse primers. The first round products were diluted and amplified in a second round using similarly sized pools of nested primers. The resulting products were sequenced, the sites of breakpoint identified and 3 pairs of PCR primers were synthesised for each patient. A 3 round nested quantitative PCR was established, in which amplification remained exponential until the third round, which was a quantitative real-time PCR using a Taqman probe or EvaGreen to measure fluorescence. Dilutions of the diagnosis DNA were used to establish a standard curve. Test samples, corresponding to different levels of MRD, were artificial mixtures made by diluting diagnosis DNA in up to 25 mcg of normal peripheral blood DNA. Specificity was determined by quantifying BCR-ABL sequences in 25 mcg of normal peripheral blood DNA.
Results Samples from 23 patients are being studied and the translocation breakpoint has thus far been isolated and sequenced in 15. Study of the remaining patients is in progress. No favoured breakpoint site or sequence has as yet become evident, although the numbers are small. The results of assays of mixtures of DNA from 9 patients are shown in Fig 1.
There results follow a 1:1 linear relationship (line shown) between expected and measured MRD levels down to approximately 10−6. Use of a Taqman probe or EvaGreen for quantification gave similar results DNA samples from 15 normal individuals were tested against each of the set of BCR-ABL primers from 3 patients. No BCR-ABL translocations were detected.
Conclusions
Isolation and sequencing of the BCR-ABL translocation breakpoint is feasible in the majority of patients with CML. Short-range multiplex PCR is a promising strategy for doing this. Sensitive and specific DNA-based measurement of BCR-ABL sequences is possible for CML patients in whom the BCR-ABL breakpoint sequence can be determined. The clinical utility of this approach to measurement of MRD in CML remains to be determined.
Figure Figure
A 3,239 -Patient Combined Eastern Cooperative Oncology Group (ECOG), M.D. Anderson Cancer Center (MDA) Analysis of the Effect of CR vs. Responses < CR on Long-Term Survival in Newly-Diagnosed AML Treated with Ara-C-Containing Regimens: Implications for Targeted Drug Development.
Authors: Estey Elihu , Sun Zhuoxin , Rowe Jacob , Faderl Stefan , Cassileth Peter , Sartiano George , Tartaglia Anthony , Garcia-Manero Guillermo , Cripe Larry , Bennett John , Kantarjian Hagop , Tallman Martin 
Published: October 2019

Abstract
New definitions of response for patients with AML have been suggested, e.g. CRp, marrow CR, hematologic improvement. Although the criteria for these are less demanding than those for CR, some have proposed that their attainment can convert AML to a “chronic disease” associated with long-term survival. To test this hypothesis, we used ECOG and MDA data to assess the effect of the achievement of CR rather than responses < CR following initial Rx on probabilities of survival 3 and 5 years after diagnosis. 2,061 pts treated on 6 ECOG trials 1976–1999 and 1,651 pts given various ara-C-containing regimens at M.D. Anderson 1980–1999 formed our database. We excluded from the survival analyses the 473 pts (11% ECOG,16% MDA)who died within 4 weeks of beginning treatment. CR rates were 62% and 60% for ECOG and MDA pts, respectively. 11% of ECOG and 54% of MDA pts were age ≥ 60. CR rates in these pts were 46% ECOG and 49% MDA. Survival probabilities according to site and response to initial Rx are as follows:
Group Site/Response to Initial Rx Patients Probability Survival at 3 Years (%+/−SE) Probability Survival at 5 Years (%+/− SE) All Pts ECOG/CR 1277 38+/− 1 33+/−1 MDA/CR 996 30+/−1 24+/−1 ECOG/<CR 567 11+/1 9+/−1 MDA/<CR 399 4+/−1 2+/−0 Age ≥ 60 ECOG/CR 106 25+/4 15+/−3 MDA/CR 374 19+/2 12+/−2 ECOG/<CR 62 2+/2 0+/−0 MDA/<CR 221 2+/−1 0+/−0 Age < 60 ECOG/CR 1171 39+/−1 34+/−1 MDA/CR 622 38+/−2 31+/2 ECOG/<CR 505 12+/−1 10+/−1 MDA/<CR 178 6 +/−2 4+/−2
We are assessing whether pts who survived 3 or 5 years despite not achieving CR with initial Rx attained CR with salvage Rx (e.g. allogeneic transplant) or had CRp or marrow CR rather than complete resistance after initial Rx. Nonetheless, our data indicate that in older pts given ara-C-containing regimens, long-term survival is essentially impossible without achievement of CR. Similarly, very few younger pts not achieving CR survive at 3–5 years. Therefore, it appears likely that newer “targeted agents” will have to contribute more than producing responses < CR and must fundamentally change the biology of the disease to improve long-term survival in AML.
Recovery of the NK Cells and Its Intending Clinical Roles after Unmanipulated Haploidentical Blood and Marrow Transplantation.
Authors: Huang Xiao Jun , Zhao Xiang-yu , Chang Ying-jun 
Published: October 2019

Abstract
Objectives: The goal of this study was to investigate the association of natural killer (NK) cell recovery with clinical outcomes after unmanipulated haploidentical blood and marrow transplantation.
Methods: We sequentially monitored the reconstitution kinetics of circulating NK cells, CD56bright and CD56dim, in 43 patients by flow cytometry, and the functionality recovery of cytokine or cytotoxicity of NK cells by flow cytometry or LDH release assay after transplantation.
Results: Reconstitution of NK cells was rapid but accompanied by skewing of cell subsets mainly in CD56bright, which had a greater growing speed. Linear regression analysis demonstrated that dose of CD34+ cells in the allografts was inversely correlated with the ratio of T/NK cells (β=−0.506, P=0.003) and CD56dim/CD56bright cell (β=−0.403, P=0.018) by day 14 after HSCT, and dose of CD3+ T cells in the allografts was also inversely correlated with the ratio CD56dim/CD56bright cells by day 14 after HSCT (β=−0.474, P=0.005). Compared with non-acute graft-versus-host disease (aGVHD) patients, patients with aGVHD had a higher level of NK subsets during week 2 post-transplantation. Cox regression analysis revealed that the patients with more CD56bright NK cells in the recovery stage had a higher survival rate (hazard risk [HR], 0.353; p=0.019) and the patients with higher ratio of T/NK (>1.0) had a higher chance of getting aGVHD (HR, 3.889; p=0.034) and cGVHD (HR, 3.925; p=0.028).
Conclusions: Our results suggest that the recovery of NK cells is and can be used as an indicator to predicate the clinical outcomes after unmanipulated haploidentical transplantation.
Figure 1. High number of circulating CD56bright NK cells correlated with better overall survival, and less TRM. Patients were subgrouped into ‘High’, ‘Middle’, and ‘Low’, group based on 33% and 67% percentage of circulating CD56bright NK cell count by day 14 after transplantation. Kaplan-Meier survival estimates OS (Figure 1A) in these three groups and cumulative incidence estimates TRM (Figure 1B). Figure 1. High number of circulating CD56bright NK cells correlated with better overall survival, and less TRM. Patients were subgrouped into ‘High’, ‘Middle’, and ‘Low’, group based on 33% and 67% percentage of circulating CD56bright NK cell count by day 14 after transplantation. Kaplan-Meier survival estimates OS (Figure 1A) in these three groups and cumulative incidence estimates TRM (Figure 1B).
IL23R Arg381Gln Variant in the Donor Confers Strong Protection Against Acute Graft-Versus Host Disease after Hematopoietic Stem Cell Transplantation in Children with Hematological Malignancies.
Authors: Gruhn Bernd , Intek Juliane , Pfaffendorf Nadine , Wittig Susan , Wolff Ilona , Schiller Isabella , Voigt Astrid , Häfer Ralf , Fuchs Dietlinde , Zintl Felix 
Published: October 2019

Abstract
The IL23R gene on chromosome 1p31 encodes a subunit of the receptor for the proinflammatory cytokine interleukin-23. Recently, IL23R could be identified as a novel inflammatory bowel disease susceptibility gene. The exchange of arginine with glutamine at position 381 of IL23R causes a blockade of the IL-23 signaling pathway which is associated with a reduced risk of both Crohn’s disease and ulcerative colitis. IL-23 is a pivotal cytokine in the differentiation of T cells into inflammatory, IL-17-producing T cells. Because of the requirement for IL-23 in autoimmune disease we hypothesized that IL23R Arg381Gln reduces the risk of graft-versus host disease (GVHD) after hematopoietic stem cell transplantation (HSCT). 141 donors and 141 children (median age, 12 years) with acute lymphoblastic leukemia (n=63), acute myeloid leukemia (n=40), myelodysplastic syndrome (n=26) or chronic myeloid leukemia (n=12) who underwent allogeneic bone marrow (n=93) or peripheral blood stem cell transplantation (n=48; T-cell depleted: n=22) in a single center were genotyped of IL23R for rs11209026 (c.1142G>A, p.Arg381Gln) using TaqMan real-time polymerase chain reaction. The donor was HLA-matched unrelated in 60% of transplants and HLA-identical related in 30% of transplants. Conditioning regimen was myeloablative in all cases. Two forms of post-transplant immunosuppression predominated, cyclosporine A and methotrexate in 65% of transplants and cyclosporine A alone in 24% of transplants. The IL23R Arg381Gln variant was present in 16 of the 141 donors (11.3%) and in 15 of the 141 patients (10.6%). Interestingly, we found a significantly reduced incidence of acute GVHD grade II–IV in patients who were transplanted from a donor with this specific variant (6.2% versus 33.6%; p=0.025). Furthermore, there was no severe acute GVHD grade III–IV if the variant occurred in the donor (0% versus 14.4%). In three of the donor-patient pairs the variant was present in both individuals and no acute GVHD was observed. The occurrence of the IL23R variant, in either donors or recipients, had no significant impact on chronic GVHD, relapse rate, treatment related mortality, and overall survival. In conclusion, IL23R Arg381Gln variant in the donor confers strong protection against acute GVHD after HSCT in children with hematological malignancies. Therefore, the IL23 signaling pathway seems to play an important role in the pathogenesis of acute GVHD. Blockade of the IL23 receptor by a monoclonal antibody could be a rational therapeutic strategy for acute GVHD.
Phase I/II Study of the P-Glycoprotein (Pgp) Inhibitor Zosuquidar Administered by Continuous Infusion (CIV) with Daunorubicin (DNR) and Cytarabine (ARA-C) as Primary Therapy in Older Patients with Pgp-Positive Acute Myeloid Leukemia (AML).
Authors: Lancet Jeffrey E. , Gotlib Jason , Wetzler Meir , Luger Selina , Cripe Larry D. , Tallman Martin S. , Gojo Ivana , Mulford Deborah , Baer Maria R. , List Alan F. , Multani Pratik S. , Sikic Branimir I. 
Published: October 2019

Background: Pgp expression in AML increases with age and correlates with inferior therapeutic response and survival. Zosuquidar is a potent and highly specific Pgp inhibitor with minimal pharmacokinetic (PK) interaction with conventional xenobiotic antineoplastics. Prolonged Pgp blockade is believed necessary to optimize antineoplastic sensitization in resistant cells in vitro. We previously reported that 72-hour CIV Zosuquidar at 700 mg/d resulted in rapid and sustained Pgp inhibition during the entire period of anthracycline administration in Pgp+ AML (Lancet, et al. ASH 2006).
Methods: We report interim results from a Phase I/II trial of 72-hr CIV zosuquidar in older adults with newly diagnosed Pgp+ AML. Primary objective: to determine the response (CR + CRp) rate in Pgp+ patients. Eligibility included ages 55–75, ECOG PS 0-2, adequate end-organ function, and Pgp+ by functional assay. Zosuquidar was initiated 4 hrs prior to the first doses of DNR (45 mg/m2/d x 3d) and ARA-C (100 mg/m2/d CIV x 7d) and continued for 72 hrs. Reinduction with the same dosing schedule was permitted in patients with significant cytoreduction without aplasia. Patients who achieved a CR/CRp received up to 2 cycles of consolidation with the same agents using an abbreviated schedule.
Results: 67 patients with Pgp+ AML were enrolled on the study; 80 patients were included in the safety analysis. Sixty-two of the 67 Pgp+ patients received Zosuquidar at 700 mg/day, while 5 received 800 mg/day (during phase I). Median age was 66; M/F was 46/21; cytogenetics: adverse (19), intermediate (29), and unknown/not done (19); de novo/secondary AML: 36/31. Mean percentage of planned Zosuquidar actually administered was 94%. CR/CRp was achieved in 32 of 66 (48%) evaluable Pgp+ patients (CR=25, CRp=7). Fifteen of 31 (48%) patients with secondary AML, and 6 of 19 (32%) with adverse-risk cytogenetics achieved CR/CRp. Eight of 14 (57%) patients age ≥ 70 responded. Induction-related death (< 30 days) occurred in 10% of patients. Other common toxicities included infection/febrile neutropenia (89%), tremor (42%), hallucinations (11%), nausea (52%), and diarrhea (51%). Median times to neutrophil (≥ 1000/μL) and platelet (≥ 100,000/μL) recovery were 34 and 33 days, respectively. With a median followup of 7.5 months, the median overall survival was 8.9 months and the median relapse-free survival was 9.3 months.
Conclusions: CIV Zosuquidar 700 mg/d with DNR and ARA-C is well tolerated, with signs of clinical benefit in poor-risk older patients with Pgp+ AML, warranting continued study of this combination. Accrual to the current trial is ongoing.
Pre-Transplant Test Dose vs. PK Studies during Conditioning Regimen To Target iv Busulfan in Allogeneic Hematopoietic Stem Cell Transplantation.
Authors: Chunduri Sandeep , Beri Rakesh , Dobogai Lisa C. , Hurter Elizabeth , Mactal-Haaf Christina , Sweiss Karen I. , Shord Stacy , Peace David , Rondelli Damiano 
Published: October 2019

Abstract
In this study we tested the efficacy of a test dose of iv busulfan in targeting blood levels of this drug during the conditioning regimen prior to an allogeneic hematopoietic stem cell transplant. We analyzed blood samples of 22 patients undergoing allogeneic hematopoietic stem cell transplantation with a busulfan-based conditioning regimen. Patients received a test dose of busulfan at 0.8 mg/kg as a 60 minute intravenous infusion. Serial blood samples were drawn at eight time points: 15 minutes before dose, at end of infusion, 15 minutes after completion, 30 minutes after completion, 60 minutes after completion, 2 hours after completion, 4 hours after completion, and 6 hours after completion. Pharmacokinetics (PK) studies were then performed at the Seattle Cancer Care pharmacokinetics laboratory. The AUC was determined using WinNonlin Professional software. The conditioning dose of busulfan was calculated by multiplying the test dose in mg/AUC × 4800. After the first dose of busulfan was administered, the same protocol was used to test busulfan PK. If the Busulfan AUC was therapeutic (between 4800 μM*min and 5200 μM*min) then the same dose was continued. If the Busulfan AUC was low or high then the third and fourth doses of busulfan were adjusted. The test dose of 0.8 mg/kg intravenous did not have any hematological side effects. The mean historic dose (solely based on weight) was 3.2 ± 0.1 mg/kg and the mean dose based on the test dose was 3.5 ± 0.5 mg/kg (p=0.02). In 12 patients where we also analyzed PK after the first day of conditioning regimen, AUC values of busulfan obtained during test dose and after day 1 dose were not different (p=0.7). The mean dose of busulfan based on test dose was 3.5 ± 0.6 mg/kg while the final dose based on day 1 busulfan PK was 3.6 ± 0.7 mg/kg (p=0.9). Nevertheless, in 2 CML patients who were on treatment with dasatinib or nilotinib at the time of the test dose, a higher AUC was observed (AUC 6065 and 6200, respectively). A pre-transplant busulfan test dose can be safely performed anytime prior to transplant and allows targeting the dose of busulfan efficiently, thus avoiding the requirement of PK studies during the conditioning regimen.