bluuebunny/crossref_metadata_2025_split huggingface dataset:
Factor VIIIa Decay Due to A2 Subunit Dissociation Is Regulated by the A1 Domain.
Authors: Parker Ernest T. , Doering Christopher B. , Lollar Pete 
Published: April 2020

Abstract
FVIII contains three homologous A domains, two homologous C domains and a single B domain. The B domain and an activation peptide between the B and A3 domains are released during the activation of fVIII by thrombin. The resulting fVIIIa molecule has a heterotrimeric A1/A2/A3-C1–C2 subunit structure. FVIIIa is unstable at physiological concentrations and pH due to dissociation of the A2 subunit from the A1/A3-C1–C2 dimer, which is three-fold faster in human fVIIIa than porcine fVIIIa. Fine regulation of fVIII activity appears to be important because plasma fVIII levels are positively correlated with a risk of arterial and/or venous thrombosis. Additionally, mutations in the fVIII gene have been identified that result in fast A2 dissociation and a hemophilia phenotype. To characterize structural determinants involved in A2 subunit dissociation, we constructed and purified recombinant human, porcine and hybrid human/porcine fVIII molecules corresponding to all eight combinations of human and porcine fVIII A1–A2–A3 domains (HHH, HHP, HPH, HPP, PHH, PHP, PPH and PPP). All of the constructs contained the human C1 and C2 domains. The rate of A2 subunit dissociation was studied at pH 7.4 by measuring decay of fVIIIa activity in an assay system using purified intrinsic fXase components and displayed first-order kinetics in all cases. Constructs containing the porcine A1 domain behaved similarly to porcine fVIIIa (Table 1). In contrast, constructs containing the human A1 domain behaved similarly to human fVIII, except for the HPH construct, which displayed intermediate behavior.
Table 1 A domain hybrid fVIIIIa decay rates Construct k (min− 1)a a mean and estimated s.d. from nonlinear least-squares analysis HHP 0.317 ± 0.018 HHH (human) 0.275 ± 0.013 HPP 0.262 ± 0.011 HPH 0.193 ± 0.012 PPH 0.128 ± 0.005 PHP 0.118 ± 0.003 PPP (porcine) 0.109 ± 0.003 PHH 0.105 ± 0.002
The fVIII A domains contain two azurin-like subdomains. Additionally, the A1 domain contains a COOH-terminal 37 amino acid acidic region. To further characterize the involvement of the A1 domain in fVIIIa decay, we prepared constructs that contained the combinations hhp, phh, pph, hpp or hph of the three A1 subdomains in an otherwise human background. Comparison of their fVIIIa decay rates indicated that the acidic region is not involved in A2 dissociation (Table 2, cf. hhp versus pph). Substitution of either azurin-like porcine subdomain produced relatively stable fVIIIa molecules.
Table 1 A1 subdomain hybrid fVIIIIa decay rates Construct k (min− 1)a a mean and estimated s.d. from nonlinear least-squares analysis hhp 0.263 ± 0.012 phh 0.167 ± 0.005 pph 0.157 ± 0.008 hpp 0.133 ± 0.005 hph 0.134 ± 0.010
These results indicate that porcine-specific amino acid side chains in both azurin-like A1 subdomains are responsible for differential fVIIIa decay rates. A1 domain mutations may represent adaptations to differences in hemostatic requirements between species.
Specific Antibodies to Moesin, a Membrane-Cytoskeleton Linker Protein, Are Frequently Detected in Patients with Immune-Mediated Aplastic Anemia.
Authors: Takamatsu Hiroyuki , Feng Xingmin , Lu Xuzhang , Sugimori Chiharu , Chuhjo Tatsuya , Takami Akiyoshi , Okawa Katsuya , Nakao Shinji 
Published: April 2020

Abstract
Although there is a large body of evidence that immune mechanisms play an essential role in the development of acquired aplastic anemia (AA), knowledge of autoantigens eliciting the immune system attack is limited. In an immune-mediated subset of AA patients characterized by the presence of HLA-DRB1*1501 and an increase in the proportion of paroxysmal nocturnal hemoglobinuria (PNH)-type blood cells, not only T-cell response but also B-cell response to antigens derived from hematopoietic stem cells may take place. To test this hypothesis, we screened sera of such immune-mediated AA patients for the presence of IgG antibodies recognizing a megakaryoblastic leukemia cell line, UT-7, using immunofluorescence staining. Since the sera from several patients stained cytoplasm of UT-7 cells, lysates of the cell line were subjected to Western blotting using the same sera. A clear band of 80 kDa protein was revealed by the sera from several patients. Peptide mass finger printing of the protein extracted from the single band identified this protein as moesin. Moesin is a membrane-cytoskeleton linker protein in cytoplasm and involved in the formation of microvilli, cell adhesion sites, and ruffling membranes. When sera of a large number of AA patients were screened for the presence of antibodies specific to recombinant moesin protein with Western blotting and ELISA, significantly high titer of the antibodies was detected in 35 of 110 (32%) AA patients. The prevalence of anti-moesin antibodies in AA patients showing increased PNH-type cells (41%) was significantly higher than those (12%) without PNH-type cells ( p =0.002). Anti-moesin antibodies were detectable in 15 of 39 (38%) recently diagnosed AA patients before therapy and in 20 of 71 (28%) patients who had a long history of AA. Four of 6 newly diagnosed patients showing anti-moesin antibodies responded to ATG+cyclosporine therapy and restored good hematopoietic function. Since B lymphocytes reportedly secrete moesin as a form of exosome (J Biol Chem, 2003), we examined supernatants of various leukemia cell lines incubated for 1 hour for the presence of moesin. The membrane-linker protein was detectable in the culture supernatant of 4 (UT-7, K562, OUN-1 and NB4) of 10 myeloid leukemia cell lines. These findings indicate that B-cell response to moesin may represent immune pathophysiology of AA and that moesin secreted from hematopoietic progenitor cells as a form of exosome may be efficiently processed by antigen-presenting cells, leading to induction of antibodies and possibly T cells specific to moesin in immune-mediated AA.
Pharmacogenetic Analysis of Polymorphisms in Pharmacological Pathway of Vincristine, Doxorubicine and Dexamethasone (VAD Regimen) To Predict Response in Patients with Multiple Myeloma.
Authors: Jamroziak Krzysztof , Balcerczak Ewa , Panczyk Mariusz , Piaskowski Sylwester , Janus Agnieszka , Cebula Barbara , Mirowski Marek , Smolewski Piotr , Urbanska-Rys Halina , Cichocki Piotr , Robak Tadeusz 
Published: April 2020

Abstract
Combination of vincristine, doxorubicin and dexamethasone (VAD regimen) is a widely-used, effective and economic initial treatment of multiple myeloma (MM). However, between 30 and 50% of MM patients do not respond to VAD, and exposure to VAD may induce multi-drug resistance. Therefore, in the era of emerging novel therapies for MM, there is a need for pre-treatment identification of potential responders to VAD. Importantly, all three VAD components have common steps in their pathways of transport and metabolism, thus even modest inherited alteration caused by single nucleotide polymorphisms (SNP) may influence VAD efficacy an toxicity. The aim of this study was to investigate whether analysis of common functional SNP in drug transporter P-glycoprotein gene (MDR1 or ABCB1), phase I cytochrome P450A metabolizing enzymes genes (CYP3A4 and CYP3A5), phase II glutathione S-transferase P metabolizing enzyme gene (GSTP1) as well as in glucocorticoid receptor gene NR3C1 can be useful to predict response to VAD in MM. Fifty-two uniformly treated MM patients were investigated for 8 common SNPs including MDR1 exon 12 C1236T, MDR1 exon 21 G2677T/A, MDR1 exon 26 C3435T, CYP3A4*1B, CYP3A5*3, GSTP1 A313G as well as NR3C1 bcl I and NR3C1 N363S polymorphisms. Genotypes were identified using direct sequencing or RFLP methods. The response to VAD was estimated after administration of 3 courses of VAD, and patients who achieved complete or partial remission of MM were assigned as responders to VAD. In the cohort of included MM patients there were 39/52 (75%) responders to VAD. The analysis of the prognostic impact of the genotyped SNPs showed that carriers of P-glycoprotein gene MDR1 2677G allele had a significantly greater probability of achievement of the response to VAD as compared to the non-carriers, odds ratio(OR)=8.0, 95% confidence interval (95%CI)= 1.2–52.9, Fisher exact test p=0.031). Moreover, the predictive effect of MDR1 2677G allele was further increased by the presence of glucocorticoid receptor NR3C1 gene 363N allele. The carriers of both MDR1 2677G and NR3C1 363N alleles had 8.4-fold greater probability to achieve complete or partial remission with 3 courses of VAD (OR=8.40, 95% CI=1.8–40.0, p=0.008). Taken together, these data support the hypothesis that analysis of common functional polymorphisms in drug transporters, metabolizing enzymes and receptors may be useful for individualization of the initial therapy of MM. Particularly MDR1 G2677T/A and NR3C1 N363S SNPs can be considered as determinants of response to VAD therapy.
Appearance of Stress-Inducible Membrane Proteins ULBP on Blood Cells of Patients with PNH, Aplastic Anemia, and MDS: Possible Implication of ULBP in Immune-Mediated Marrow Injury.
Authors: Hanaoka Nobuyoshi , Horikawa Kentaro , Tsuzuki Yasuchika , Nagakura Shoichi , Ishihara Sonoko , Mitsuya Hiroaki , Nakakuma Hideki , Kawaguchi Tatsuya 
Published: April 2020

Abstract
Although it has been suggested that paroxysmal nocturnal hemoglobinuria (PNH), idiopathic aplastic anemia (AA), and myelodysplastic syndromes (MDS) share immune-mediated bone marrow injury, the molecular events in the injury is still unclear. Currently we have shown that leukemic K562 cells became less sensitive to natural killer (NK) cells in vitro when the leukemic cells acquired PIG-A mutations (Nagakura S et al, Blood2002; 100: 1031–1037). The decreased sensitivity was explained by a deficiency of stress-inducible and GPI-linked membrane proteins ULBP (Hanaoka N et al, Blood2004; 104: 774a). In general, the exposure of ULBP on cell membrane leads to activation of both NK and T cells expressing NKG2D, a receptor for ULBP. It is then conceivable that the ULBP-NKG2D engagement causes blood cell injury. In support of this idea, we detected both ULBP-expressing (ULBP+) blood cells (granulocytes or marrow cells) in 9 of 17 (53%) patients with PNH and killing of granulocytes of 4 the 9 patients by autologous lymphocytes. Granulocytes of healthy donors were negative for ULBP and intact in the killing assay. ULBP+ granulocytes were further detected in 24 of 45 (53%) patients with AA, and 10 of 21 (48%) patients with MDS, whereas ULBP+ granulocytes were detected in neither patients with autoimmune hemolytic anemia nor those with drug-induced marrow injury. Our study suggests not only that there was pathogenic stress to induce ULBP on blood cell membrane but also that the ULBP-NKG2D engagement was implicated in the pathogenesis of marrow failure, at least in a part, of patients with PNH and its related disorders. We thus propose that membrane ULBP would be of potential probe for the diagnosis of immune-mediated marrow injury and the evaluation of its treatment.
Risk Factors for Survival and Thrombosis in Patients with Paroxysmal Nocturnal Hemoglobinuria (PNH): A Single Institution Study of 68 Patients.
Authors: Smith Sean B. , Tefferi Ayalew , Hoyer James D. , Wolanskyj Alexandra P. 
Published: April 2020

Abstract
Background:
Paroxysmal Nocturnal Hemoglobinuria (PNH) is a clonal stem cell disorder characterized by intravascular hemolysis, thrombosis, bone marrow failure and reduced survival. In this current single institution study of patients with PNH, we investigate the risk factors predictive of survival and thrombosis.
Methods:
Data were abstracted from medical records of a consecutive cohort of patients with PNH seen at the Mayo Clinic. Diagnosis was confirmed by the presence of intravascular Hemolysis and either a positive Ham’s test, and/or by the presence of a GPI-deficient hematopoietic clone. Follow-up data were obtained by contacting the patients and by reviewing the social security death index.
Results:
i. Patients and treatment:
The study cohort included 68 patients with PNH (median age 32 years, range 10–77; 33 females) with a median follow up of 84.5 months (range, 1–538 months). Presenting features included infection (n=9), thrombosis (n=9), neutropenia (absolute neutrophil count (ANC) &lt; 1000) (n=32) severe intravascular hemolysis (n=16) and splenomegaly (n=11). Prior to the diagnosis of PNH, 6 patients (8.8%) carried a diagnosis of aplastic anemia (AA) and 2 (2.9%) myelodysplastic syndrome (MDS). Complications documented after diagnosis included major thrombosis (n=11), major hemorrhage (n=4), and development of AA (n=7), MDS (n=4), and non-hematologic cancer (n=4) but none experienced leukemic transformation. Treatment included prednisone (n=30), other immunosuppressive agents (n=6), androgens (n=10), and bone marrow transplantation (n=4). Review of treatment with systemic anticoagulation disclosed only 2 patients that were receiving prophylactic warfarin therapy and both patients remain free of thrombosis after 1 and 2 years of therapy.
ii. Thrombotic events and risk factors:
Overall, 20 patients (29.4 %) experienced one or more episodes of major thrombosis. Among several clinical and laboratory parameters evaluated at presentation, only the detection of urine hemoglobin and infection were found to be significantly associated with the occurrence of thrombosis (p=0.05)
iii. Risk factors for survival
To date, 18 patients (27%) have died and median survival for the study cohort is projected at 21.1 years. Multivariate analysis identified thrombosis (p=0.003) and an ANC of &gt; 1000 (p= 0.05) at presentation as independent risk factors for inferior survival. In addition, the occurrence of major hemorrhage was significantly associated with poor survival (p= 0.04).
Conclusion
The current study provides natural history information in a relatively large cohort of patients with PNH and identifies certain disease features that could be further explored for their potential as risk factors for both thrombosis and survival.
Ex Vivo Fludarabine Selectively Depletes Human Naive T-Cell Subsets: A Step Toward Modifying Donor Lymphocyte Infusion.
Authors: Smith Melody M. , Giver Cynthia R. , Waller Edmund K. , Flowers Christopher R. 
Published: April 2020

Abstract
Ex vivo modification of donor lymphocytes with purine analogs (mDL) may help to minimize graft versus host disease (GvHD) while providing beneficial graft versus leukemia (GvL) effects. In a murine model system, we have shown that allogeneic donor splenocytes, treated with fludarabine ex vivo have significantly reduced GvHD activity when transferred to irradiated recipient mice, and retain anti-viral and GvL activities (Giver, 2003). This effect appears to be mediated by relative depletion of donor CD4 CD44low, “naive” T-cells. As a first step toward developing mDL for use in patients, we sought to evaluate the effects of ex vivo fludarabine exposure on human T-cell subsets, and to determine the minimum dose of fludarabine required to achieve this effect.
Methods: Peripheral blood mononuclear cell samples from 6 healthy volunteers were evaluated at 0, 24, 48, and 72 hour time points after ex vivo incubation in varying dosages of fludarabine: 2, 5, and 10(n=3) mcg/ml. Fludarabine incubated samples were compared to samples that received no fludarabine (untreated). The total viable cell number was determined and the fractions and absolute numbers of viable CD4 and CD8 naïve and memory T-cells were determined using flow cytometry after incubation with 7-AAD (dead cell stain), CD4, CD8, CD45RA, CD62L, and CCR7 antibodies, and measuring the total viable cells/ml.
Results: The numbers of viable CD4 and CD8 T-cells remained relatively stable in control cultures. Without fludarabine, the average viability at 72 hr of naive and memory T-cells were 92% and 77% for CD4 and 86% and 63% for CD 8 (Fig. 1A). Naive CD4 T-cells were more sensitive to fludarabine-induced death than memory CD4 cells. At 72 hr, the average viability of fludarabine-treated naive CD4 T-cells was 33% at 2 mcg/ml (8.2X the reduction observed in untreated cells) and 30% at 5 mcg/ml, while memory CD4 T-cells averaged 47% viability at 2 mcg/ml (2.3X the reduction observed in untreated cells) (Fig. 1B) and 38% at 5 mcg/ml. The average viability of naive CD8 T-cells at 72 hr was 27% at 2 mcg/ml and 20% at 5 mcg/ml, while memory CD8 T-cell viability was 22% at 2 mcg/ml and 17% at 5 mcg/ml. Analyses on central memory, effector memory, and Temra T-cells, and B-cell and dendritic cell subsets are ongoing. The 5 and 10 mcg/ml doses also yielded similar results in 3 initial subjects, suggesting that 2 mcg/ml or a lower dose of fludarabine is sufficient to achieve relative depletion of the naive T-cell subset.
Conclusions: Future work will determine the minimal dose of fludarabine to achieve this effect, test the feasibility of using ex vivo nucleoside analog incubation to reduce alloreactivity in samples from patient/donor pairs, and determine the maximum tolerated dose of mDL in a phase 1 clinical trial with patients at high risk for relapse and infectious complications following allogeneic transplantation.
Figure Figure
Nonmyeloablative Stem Cell Transplantation with CD8-Depleted or Unmanipulated Peripheral Blood Stem Cells: A Prospective Randomized Trial.
Authors: Willems Evelyne , Castermans Emilie , Baron Frédéric , Baudoux Etienne , Wanten Nadine , Bonnet Christophe , Hafraoui Kaoutar , Frère Pascale , Fillet Georges , Beguin Yves 
Published: April 2020

Abstract
Background: In a previous pilot study, we demonstrated that CD8-depletion of the graft apparently reduced the severity of AGvHD without impairing the GvL effect after peripheral blood stem cell (PBSC) transplantation with a nonmyeloablative conditioning (NMSCT).
Aim of the study: To evaluate the effect of CD8-depletion on graft rejection, AGvHD and CGvHD, and relapse.
Patients: 53 patients were randomised between CD8-depletion (group 1) (n=25) and no manipulation (group 2) (n=28). Two patients in the CD8 group were excluded for poor CD34+ cell count collected. Diagnoses were: AML (n=3), CML-AP (n=2), MDS (n=14), MPD (n=3), CLL (n=5), NHL (n=14), MM (n=8) and RCC (n=2). Median age was 57 (range 36–69) yrs. After conditioning with 2 Gy TBI with (n=39) or without (n=12) fludarabine, patients received PBSC from family (n=21) or unrelated (n=30) HLA-matched donors. CD8-depletion was carried out using the Eligix system and GvHD prophylaxis consisted in CyA and MMF.
Results: CD8 depletion removed 96% of CD8+ cells so that the number of CD8+ cells infused was 6.8 vs 136.8 x108 cells/Kg in group 2 (p&lt;0.0001). AGvHD of any grade was observed in 13 (56%) patients in group 1 and 17 (61%) in group 2 (NS); it was grade 3–4 in 1 (4%) and 5 (18%) patients in groups 1 and 2, respectively (NS). Limited and extensive CGvHD developed in 3 and 1 patients in group 1 and in 7 and 2 patients in group 2, respectively (NS). Nine patients in group 1 and 12 in group 2 received unmanipulated DLI for poor chimerism or disease progression. Eight (3 initial and 5 late) graft failures were observed in group 1 and one (late) in group 2. Full donor chimerism was achieved in 57% (group 1) and 50% (group 2) at day 100, and in 73% (group 1) and 59% (group 2) (NS) at 1 yr. The 2-yr OS and PFS rates are 55% and 43 % in group 1 vs 59% and 46% in group 2, respectively (NS). Four (17%) patients died of their disease in group 1 vs 3 (11%) in group 2 (NS). Two patients died of severe AGvHD in group 2 vs none in group 1.
Conclusion: In vitro CD8-depletion results in higher rates of graft failure after NMSCT. The incidence of acute and chronic GvHD is not reduced but there is a trend towards reduced severity of AGvHD. Relapse and survival rates are not changed by this strategy.
Absolute Number of Transplanted CD34+ Cells Expressing c-mpl (CD110) Correlates with Speed of Platelet Engraftment Following Autologous Stem Cell Transplantation.
Authors: Sartor Mary , Garvin Frances , Antonenas Vicki , Webb Melina , Bradstock Kenneth F. , Gottlieb David J. 
Published: April 2020

Abstract
Recovery of neutrophil numbers after peripheral blood stem cell transplantation (PBSCT) is closely associated with graft CD34+ cell dose. Predicting the speed of platelet recovery is more difficult but would be of value given that a significant minority of patients experience delayed platelet recovery and bleeding complications after transplantation.
In this study we retrospectively analysed the graft composition of 29 patients who underwent autologous transplantation, using blood stem cells mobilized with cyclophoshamide and G-CSF, to assess the utility of c-mpl expression on CD34+ cells as a predictor of platelet engraftment (ie, time to platelet count greater than 20 x 109/L for three consecutive days without the need for platelet transfusion). Absolute CD34+ cells and CD34 subsets expressing c-mpl were enumerated using a published single platform viable CD34 flow cytometry assay (BMT, 36: 199–204,2005).
Of the 29 patients, 7 required at least 21 days for platelet engraftment. These patients received a median graft dose of 5.7 x 104 CD34+CD110+ cells/kg compared with a median dose of 13.4 x 104 cells/kg received by patients who experienced platelet engraftment within 21 days of transplant (p=0.013). In contrast, there was no difference in the number of CD34+ cells/kg infused (4.0 v 4.9 x 106/kg for &gt; or &lt; 21 days for platelet engraftment respectively, p=0.23). There was a poor correlation between the absolute number of CD34+ cells and the number of CD34+CD110+ cells in the graft (r2 = 0.48). Similarly there was no correlation between the percentage of CD34+ cells expressing c-mpl and the speed of platelet engraftment (8.1 v 5.8%) for &gt; or &lt; 21 days for platelet engraftment respectively, p=0.39). Patients with &gt;21 days for platelet engraftment received platelet transfusions more often than those with &lt;21 days for platelet engraftment (median 9 v 2 transfusions, p &lt;0.001).
The absolute number of CD34+/CD110+ cells/kg infused at time of transplantation appears to be an important factor identifying patients at risk of delayed (&gt;21 days) platelet engraftment. Those with &lt;6 x 104 CD34+/CD110+ cells/kg are at particularly high risk of delayed platelet engraftment, requiring multiple transfusions after transplantation.