bluuebunny/crossref_metadata_2025_split huggingface dataset:
A Novel Thrombin Fluorogenic Substrate of High Affinity, Catalytic Efficiency and Selectivity.
Authors: Baird T. Regan , Jacobs Margaret , Tinklepaugh Adam , Gross Peter , Furie Barbara C. , Furie Bruce 
Published: October 2019

Abstract
Thrombin is a serine protease with multiple functions, including the conversion of fibrinogen to fibrin, platelet activation, activation of Factor VIII and Factor V. Although many low molecular weight substrates have been developed for the study of thrombin catalytic activity, our interest in analyzing thrombin activity in the blood of a living mouse required development of a new class of thrombin substrates of high affinity and high selectivity whose product upon hydrolysis could be visualized by intravital fluorescence microscopy. We have developed a novel substrate for thrombin using the fluorochrome Alexa 488 and the quencher QSY35. Alexa 488 is conjugated to the N-terminus of a 12 amino acid peptide based upon the thrombin cleavage site in the α-chain of fibrinogen and the quencher is coupled to the C-terminus of the peptide, yielding Alexa 488-KGGVR-GPRVVEA-QSY35. We term this substrate FBG-12. Through fluorescence energy resonance transfer, the emission from Alexa 488 is absorbed by the quencher QSY 35, thus minimizing fluorescence, since the two moieties have overlapping spectral properties and are separated by a Förster radius less than 44 Å. Thrombin hydrolyzes the peptide yielding the N-terminal fragment, Alexa 488-KGGVR-COOH, which is highly fluorescent, and the C-terminal fragment NH2-GPRVVEA-QSY35 peptide which, being physically separated from the Alexa 488, no longer quenches the fluorochrome. The peptide was synthesized by solid phase peptide synthesis, its N-terminus and C-terminus were modified with Alexa 488 and QSY35 respectively, and its identity confirmed by mass spectroscopy and protein sequencing. The kinetic properties of FBG-12 were determined in vitro. Hydrolysis of the substrate by thrombin resulted in a linear increase in fluorescence at 525 nm over time and was dependent on enzyme concentration. The fluorescence of the product of thrombin hydrolysis of FBG-12 was 120-fold greater than that of the substrate. Michaelis-Menten kinetic analysis of thrombin hydrolysis of the fluorogenic substrate FBG-12 revealed a Km of 2 μM, a kcat of 759 s−1, and a kcat/Km of 3795 x 106 M−1 s−1. To determine the selectivity of this substrate, other plasma serine proteases were analyzed for their ability to hydrolyze FBG-12. Factor Xa, Factor VIIa, and activated protein C did not hydrolyze FBG-12. Factor XIa (Km 11 μM, kcat 20s−1, kcat/Km = 20 x 106 M−1 s−1) did hydrolyze FBG-12, although the reaction was inefficient compared to thrombin. Our characterization of FBG-12 suggests that this substrate is hydrolyzed efficiently and selectively by thrombin. Its spectral properties and solubility in a physiologic environment make FBG-12 a suitable substrate for the detection of thrombin activity via intravital imaging during thrombus formation in vivo.
Mobilization of Stem Cells with DT-PACE Plus Pegfilgrastim vs. DT-PACE Plus Filgrastim in Previously Treated MM Patients.
Authors: Tricot Guido , Reiner Maureen , Burns Michael , Zangari Maurizio , van Rhee Frits , Barlogie Bart , Cottler-Fox Michele 
Published: October 2019

Abstract
Mobilization with chemotherapy (CT) plus hematopoietic growth factors (HGFs) is superior to mobilization with HGFs only. For stem cell mobilization after chemotherapy HGFs are typically given once or twice a day for approximately 14 days. The aim of this study was to evaluate whether mobilization of peripheral blood stem cells (PBSC) with DT-PACE plus pegfilgrastim was equivalent to mobilization with DT-PACE + filgrastim. Patients analyzed were enrolled in two consecutive studies 2001–12 (N=97) and 2003–41 (N=72) and included only patients who had received ≥ 2 cycles of prior therapy, but no prior transplant. Both protocols employed a single cycle of induction with DT-PACE (Lee et al. JCO2003;21:2732) followed by stem cell collection with either filgrastim 5μg/kg bid until completion of stem cell collection (2001–12) or pegfilgrastim 6mg on days 6 and 13 (2003–41). Patients then proceeded with tandem transplants, one consolidation cycle with DT-PACE and two years of maintenance with thalidomide (100mg daily) and dexamethasone (4 mg/day, days 1–4 q 3 weeks). Group characteristics were compared using the Kruskall-Wallis test. Time to hematopoietic recovery after transplantation was analyzed with Kaplan-Meier plots, and comparison was performed using the logrank statistic. The two studies had comparable characteristics, except that more patients ≥ 65 years were enrolled in 2003–41 (35% vs 13%; p=.001). The median number of collection days was 2 in both studies (p=.8). The median number of CD34 cells/kg (x106) collected per day was 9.7 (2001–12) vs 12.7 (2003–41) (p=.2). The total number of CD34 cell/kg (x106) collected was 20.4 (2001–12) versus 25.4 (2003–41) (p=.2). The median number of CD34 cells/kg (x106) infused after the first transplant was 4.1 (2001–12) and 4.5 (2003–41) (p=.5). The time to recover ANC > 500/μl and platelets > 20,000/μl without transfusion was shorter for 2003–41 patients (13 versus 15 days; p< .0001) (Figure 1). Mobilization of PBSC with pegfilgrastim is easier for patients because they only receive 2 injections and is as effective as filgrastim when combined with DT-PACE. Moreover, mobilization with pegfilgrastim may result in a more rapid hematologic recovery after transplantation.
Cumulative incidence analysis Recovery of ANC to 500 and Platelets > 20K 2003-41 vs. 2001-12, Transplant 1 Cumulative incidence analysis Recovery of ANC to 500 and Platelets > 20K 2003-41 vs. 2001-12, Transplant 1
Varying Response to Imatinib in CML Patients with BCR-ABLM244V Mutation.
Authors: Khorashad Jamshid S. , Anand Mona , Marin David , Andreasson Catharina , Ghorashian Sara , Apperley Jane F. , Goldman John M. , Kaeda Jaspal S. 
Published: October 2019

Abstract
BCR-ABL kinase domain mutation in a CML patient with clinical resistance to imatinib is generally accepted as ‘causal’. Here we report the kinetics of the mutant BCR-ABL transcripts in 6 CML patients with the M244V mutation that are at variance with the accepted view. Prior to starting imatinib, one patient in chronic phase (CP) was previously untreated, 4 patients in CP had previously received alfa-interferon, and the sixth had received an autograft but had subsequently progressed to accelerated phase (AP). Patients were started on 400 mg/day dosage which was escalated to 600, 800 or 1000 mg/day following little or no response. Philadelphia chromosome was the only cytogenetic abnormality detected at the time of diagnosis and at start of imatinib treatment. The median time from diagnosis to starting imatinib was 19 months (range: 3 – 120 months). The M224V mutations were detected initially as a result of routine screening by Sanger’s direct sequencing of cDNA from blood of patients on imatinib, at which time the mutant clone in each patient already constituted >80% of BCR-ABL transcripts, measured by QR-PCR. We then retrospectively measured levels of mutated BCR-ABL transcripts in all the stored samples, median 14.5 samples (5 – 28), by pyrosequencing. Of the 4 patients for whom there were pre-imatinib samples the BCR-ABLM244V allele was identified in one case (previously treated with IFN-alfa) at 3%, but was not detected in the other three. In the 2 patients for whom there were no pre-imatinib samples, the BCR-ABLM244V allele was present at 3.0% and 93% respectively in the earliest available sample. In the 5 patients whose earliest samples showed low or absent mutant transcripts the non-mutated BCR-ABL population was progressively replaced by BCR-ABLM244V-positive cells, eventually becoming the dominant clone with a median time from first sample to maximal level of 35 months (11 – 54 months). In three patients total BCR-ABL transcripts decreased and they achieved complete cytogenetic remission (CCyR). In the other three patients (including the newly diagnosed patient) there was little or no reduction in BCR-ABL transcript numbers and all failed to achieve CCyR. One patient in this latter group acquired trisomy 8, reported 40 months prior to blastic phase; she died following allogeneic SCT. The response to increased imatinib dose in three patients is consistent with the reported IC50 for the M244V mutation, i.e. moderate resistance, but the response in the other three patients is not. In summary, the response to imatinib in 6 patients with the same BCR-ABL mutation is variable. Furthermore, these observations imply the mutation is unlikely to be the sole cause of the resistance in at least in 3 of the patients. The observed heterogeneous phenotype of these 6 CML patients is likely to be due to a myriad of undefined contributory factors.
Imatinib and Leptomycin B Are Effective in Overcoming Imatinib-Resistance Due to Bcr-Abl Amplification or Clonal Evolution but Not Due to Bcr-Abl Kinase Domain Mutation.
Authors: Kancha Rama K. , Miething Cornelius , Bubnoff Nikolas V. , Gotze Katharina , Peschel Christian , Duyster Justus 
Published: October 2019

Abstract
Constitutive activity of the Bcr-Abl kinase has been shown to be the causative event in the molecular pathogenesis of CML and Ph+ ALL. Even though Imatinib, a small molecule inhibitor of the Abl kinase, is very effective in treating the disease, development of resistance to this drug is a common phenomenon in advanced stage disease. Using Leptomycin B (LMB), a nuclear export blocker, Vigneri et al. have shown that Bcr-Abl kinase activity trapped in the nucleus induces cell death, thus being a new potential treatment option for Bcr-Abl expressing diseases. To test whether this approach might be effective in the case of imatinib resistant leukaemia several Bcr-Abl positive, imatinib-resistant Ba/F3 clones were established and characterized: 1. Ba/F3 cells resistant due to a point mutation in the Abl kinase domain at position T315I. 2. Ba/F3 cells resistant due to amplification of the Bcr-Abl kinase. 3. Ba/F3 cell clones resistant to imatinib with clear features of clonal evolution. All four cell lines were tested for the efficacy of a combined treatment approach with imatinib and leptomycin B. This combination was very effective in inducing apoptosis in imatinib resistant Ba/F3 cells displaying Bcr-Abl amplification. One out of two cell lines with signs of clonal evolution also responded to this combination treatment. However, imatinib resistant cells expressing the Bcr-AblT315I mutant kinase were completely resistant to this approach. Thus, these results are in agreement with the proposed hypothesis of Vigneri and wang that the combined effect of leptomycin B and imatinib requires Bcr-Abl kinase inhibition (which is not achieved in cells expressing Bcr-Abl T315I) by imatinib and nuclear entrapment by LMB To check the feasibility of this combined treatment for purging purposes, in vitro colony forming assays were performed with mixed cultures of Bcr-Abl positive BaF3 cells and primary murine bone marrow. All colonies formed after combined treatment with imatinib and LMB were derived from BM cells, indicating selective toxicity towards Bcr-Abl positive cells while sparing primary murine bone marrow cells. We are currently evaluating the effectivity of the combined LMB/imatinib treatment as purging strategy in a murine bone marrow transplantation model.
Psychological Outcome after Hematopoietic Cell Transplantation for Sickle Cell Disease.
Authors: Pradere Jerome , Drain Elise , Taieb Olivier , Bernaudin Francoise , Champion Marie , Bonnet Doris , Dutray Benoit , Abbal Tahar , Chaudre Geraldine , Kuentz Mathieu , Coic Lena , Gluckman Eliane , de Montalembert Mariane , Dhedin Nathalie , Vernant Jean-Paul , Benkerrou Malika , Bertrand Yves , Vannier Jean-Pierre , Steschenko Dominique , Bordigoni Pierre , Bachir Dora , Galacteros Frederic , Casanova Jean-Laurent , Fischer Alain , Rose Marie 
Published: October 2019

Abstract
Hematopoietic cell transplantation (HCT) is the only potentially curative treatment for sickle cell disease (SCD). The probability of event-free survival is currently between 85 and 90% after matched sibling-donor HCT. This intensive treatment conducts to a major change for patients and families. The purpose of the present study was to determine the psychological impact on individuals and their families of this recovery process after HCT. The hypothesis was that the recovery is a process with different aspects (medical, psychological, individual, familial and cultural) which could be discordant. This study was supported by the Etablissement Français des Greffes (EFG) and conducted by child and adolescent psychiatrists, psychologists and a medical anthropologist. The assessment consisted in two semi-structured interviews adressed to the patients and their families for studying their illness narratives and questionnaires for quality of life, post-traumatic stress disorder (PTSD), depression and anxiety. 28 patients (17 males, 11 females) with SCD treated in France by HCT (22 by bone marrow and 6 by cord blood transplantation) and their families were evaluated at least one year (median 5 years, range, 1–14 years) after HCT at the median age of 14 years (range, 4–24 years). Among these 28 patients, 7 had prior history of severe vasculopathy and 6 of stroke before the HCT. At evaluation, 27 patients were free from SCD symptoms (but 3 had moderate chronic GVHD), 1 had graft rejection. 9 patients (4 males, 5 females) (median age 16 years, range 7–23 years) presented psychiatric symptoms as inhibition, depression, hypomaniac symptoms, anxiety, stutter. A psychiatric care was indicated for these 9 subjects. No diagnosis of current PTSD was found. Psychological distress was reported by parents. 21 patients considered themselves as cured, 4 of them in an incomplete way (none with stroke before the HCT, 3 with moderate chronic GVHD), and 3 not cured at all (none with stroke before the HCT, 1 with graft rejection). The study of the narratives illustrated the major individual, familial and cultural changes after the HCT. The recovery needed complex adjustments. Patients had to cope with new potentialities and requirements: school and relationships with peers, identity issues (disease transmission, infertility) and family changes. This study underlies the importance of psychological interventions for improving the outcome of patients with SCD treated by HCT and their families.
5′-ABL Gene Deletion on Derivative Chromosome 9 Detected by FISH: Prognostic Significance in CML Patients after Allogeneic Hematopoietic Stem Cell Transplantation (HSCT).
Authors: Chaudhri Naeem A. , Aljurf Mahmoud D. , Sahovic Entezam A. , Almhareb Fahad , Al-Sharif Fahad , Al-Zahrani Hazzaa , Al-Omar Hamad , Al-Shanqeeti Ali , Zaidi Syed , Morshed Manzur , Al-Anazi Khalid , El-Hassan Ibrahim , Al-Lahham Mohammad , Iqbal Mohammad A. 
Published: October 2019

Abstract
BACKGROUND AND OBJECTIVE
Large cryptic deletions of the 5′-ABL regions on derivative chromosome 9 can be identified by FISH in 10%–15% of Ph+ CML patients. This abnormality may be associated with unfavorable prognosis with conventional therapies. There is limited data regarding the prognosis of 5′-ABL gene deletion abnormality in recipients of allogeneic HSCT. We performed an analysis of our prospectively collected database to evaluate the prognostic importance of this karyotypic abnormality in HSCT patients.
METHODS:
Between1989–2004, 24 allogeneic HSCT patients with 5′-ABL gene deletion detected by FISH using ES-BCR/ABL probe were identified. The diagnosis was CML CP1, 16(66.6%), CML CP2, 5(20.8%), CML AP 2(8.3%), CML blastic phase 1(4.1%). Sex ratio: M/F 8/17; median age: 25.06 (14.7–51.9) years. All donors were HLA matched identical sibling. Stem cell source was BM in 15(62.5%), PB in 9(37.5%). Mean CD34 cells x106/kg dose was, BM 5.0 (2.05–7.67), PB 7.18 (1.64–10.71). Conditioning regimen was BU/CY2 in 19, CY/TBI in 4 and FLU/TBI in 1 patient.
A second group of 206 Ph+ CML pts was evaluated for comparison. These were patients with CML without 5′-ABL gene deletions that underwent allogeneic SCT from 1989–2004. This group was matched for age, sex, conditioning regimen, type of SCT, stem cell source and HLA matched sibling donors. The diagnosis was CML CP 148(71.8%), CML CP2 37(18.0%) AP 21(10.2%).
RESULTS:
After a median follow up of 17.5 mths in group 1 (del 9) and 17.05 mths in group 2 (without del 9), relapse occurred in 11/24(45.8%) in group 1 and 48/206(23.3%) in group 2, indicating RFS of 54.2% vs 76.7% respectively (P= .03). A sub-group analysis was made between CML CP1 patients only, between both groups. With 16 patients in group 1 and 148 pts in group 2 with a median FU of 21.5 vs 26.5 mths, the RFS was 7/16(43.7%) vs 118/148 (79.7%) respectively (P=. 001).
CONCLUSION
Patients with 5′-ABL gene deletions on derivative chromosome 9 have a significantly higher relapse rate even after high dose chemotherapy and allogeneic HSCT as compared to patients without this abnormality. We recommend close monitoring of these patients by RT-PCR to detect early molecular relapse and treatment with DLI and/or Imatinib Mesylate.
Favorable Influence of Pretransplant Rituximab but Not of High-Dose Ara-C in Upfront Autologous Stem Cell Transplantation (SCT) for Mantle Cell Lymphoma (MCL).
Authors: Rieger Michael , Hensel Manfred , Seyfarth Baerbel , Sitter Soeren , Martin Simona , Haas Rainer , Kneba Michael , Schmitz Norbert , Ho Anthony D. , Dreger Peter 
Published: October 2019

Abstract
Recent data suggests that the prognosis of MCL might be improved by administration of rituximab and high-dose ara-C (HA) prior to upfront SCT. In order to investigate the relative contributions of rituximab and HA, respectively, we retrospectively analyzed 98 patients who received SCT as part of 1st-line treatment of advanced-stage MCL. Patients (median age 55 (29–67)) with stage II–IV MCL were treated with a sequential therapy comprising 2–8 cycles of CHOP or CHOP-like regimens +/− rituximab for remission induction, DexaBEAM (49%) or HA-containing chemotherapy (39%) for further consolidation and mobilization of stem cells, and high-dose therapy with SCT. 22% were mobilized with CHOP alone. High-dose regimens were TBI/CY (38%), TBI/CY + rituximab (R-TBI/CY; 35%), or BEAM (27%).
Results: With a median follow-up of 35 (1–135) months, estimated median progression-free survival (PFS) of all 98 patients was 95 months from diagnosis and 82 months from SCT(median overall survival not reached). Pretransplant rituximab was given to 40 patients and improved PFS (3-year PFS from SCT 93% (95%CI 83–100%) vs 67% (54–79%) although this was still not statistically significant (hazard ratio (HR) .37; p .07). In contrast, pretransplant HA had no impact on PFS even after adjusting for rituximab (HR 1.9; p .61). PFS after R-TBI/CY tended to be superior to that after TBI/CY only if the 58 patients without pretransplant rituximab were considered (HR .39; p .07). Age, sex, time from diagnosis, and remission status after cytoreduction or immediately prior to SCT did not affect PFS.
Conclusions: Pretransplant rituximab but not high-dose ara-C seems to improve the outcome of early SCT for MCL. Addition of rituximab to myeloablative treatment with TBI/CY might be beneficial only in the absence of pretransplant rituximab. Pretransplant rituximab with upfront SCT provides long-term disease control in a large proportion of patients with MCL.
Morphine Exaggerates Retinopathy in Transgenic Sickle Mice.
Authors: Gupta Kalpna , Chen Chunsheng , Lutty Gerald A. , Hebbel Robert P. 
Published: October 2019

Abstract
Proliferative retinopathy is a complication of sickle cell disease (SCD). Promotion of angiogenesis by opioids (Cancer Res62: 4491, 2002) suggests that opioid analgesics used to treat pain may exaggerate retinopathy in patients with SCD. To examine the hypothesis that morphine augments retinopathy, we used sickle mice with transgenes for human α and βS globin (NY1DD model) and their wild type (WT) C57/BL6 controls. Simulating the analgesic doses used in humans, we injected morphine subcutaneously (50 mg / day / 70 Kg with 10 mg increments every 2 weeks and / or equimolar amount of naloxone) for 6 to 15 months into 9 – 11 months old mice. Retinal vasculature was visualized with darkfield illumination using adenosine diphosphatese reaction. Morphine induced neovascularization, corkscrew formation resembling microangiopathy in human, arterio-venous anastomoses and several vascular tufts in the inner 1/3 and whole retina after 10 and 15 months of treatment, respectively in all NY1DD but not in WT mice. Only 1/5 or 2/7 PBS-treated NY1DD mice showed early neovascular changes at 20 (treated for 10 mo) or 26 (treated for 15 mo) months of age, respectively. Retinopathy scores by ICROP system used in neonatal intensive care units for abnormal vessels, blood vessel tortuosity and tufts in NY1DD mice were: 10 mo treatment, PBS, 1.5±0.5; morphine, 10±0.7 (p<0.0001 vs PBS); morphine + naloxone, 4±1; naloxone, 3.8±1.2 and 15 mo treatment, PBS, 5.9±2 and morphine, 13±0.5 (p<0.003 vs PBS). Naloxone significantly inhibited (p<0.005, morphine vs morphine +naloxone) morphine-induced retinopathy, suggesting an opioid receptor-mediated mechanism. To elucidate the mechanism of morphine-induced retinopathy we isolated retinal endothelial cells (REC) by panning with anti-CD31 from WT and NY1DD mice. Similar to VEGF induced proliferation and survival, morphine stimulated 5–6 fold proliferation and increased survival by about 75% in both WT and NY1DD REC, in a dose dependent manner. Both VEGF164 and morphine stimulated time-dependent MAPK/ERK and STAT3 phosphorylation in WT REC. VEGF and morphine stimulate nitric oxide synthase pathway and stimulate angiogenesis. This raised the possibility of a cross-talk between VEGF receptor(s) and opioid receptor(s). We found that morphine stimulates the phosphorylation of VEGFR2/Flk1 in WT REC which co-immunoprecipitated with mu opioid receptor (MOR) using anti-MOR antibodies. Both, morphine- or VEGF-induced phospho STAT3 also co-immunoprecipitated with MOR, suggesting that activation of either MOR or Flk1 transactivates the other. Thus, addition of morphine to the VEGF replete microenvironment in SCD can have an exaggerating effect on the progression of retinopathy. Since patients with SCD receive opioids for long periods of time, we speculate that retinopathy in these patients may in part be due to opioid use.