bluuebunny/crossref_metadata_2025_split huggingface dataset:
Inhibition of Activation of the Classical Pathway of Complement by Human Neutrophil Defensins
Authors: van den Berg Rocco H. , Faber-Krol Maria C. , van Wetering Sandra , Hiemstra Pieter S. , Daha Mohamed R. 
Published: October 2019

AbstractDefensins are small, cationic antimicrobial peptides that are present in the azurophilic granules of neutrophils. Earlier studies have shown that defensins may influence complement activation by specific interaction with activated C1, C1q, and C1-inhibitor. In the present study, we show that the defensin human neutrophil peptide-1 (HNP-1) is able to inhibit activation of the classical complement pathway by inhibition of C1q hemolytic activity. The binding site for HNP-1 on C1q is most likely located on the collagen-like stalks, as a clear, dose-dependent binding of HNP-1 to either intact C1q or to the collagen-like stalks of C1q was demonstrated using enzyme-linked immunosorbent assay (ELISA). Besides binding of HNP-1 to C1q, also a limited binding to C1 and to a mixture of C1r and C1s was observed, whereas no binding to C1-inhibitor was found. Because binding of HNP-1 to C1-inhibitor has been suggested in earlier studies, we also assessed the binding of HNP-1 to mixtures of C1-inhibitor with either C1r/ C1s or C1. No binding was found. Using a competition ELISA, it was found that HNP-1, but not protamine, inhibited binding of biotin-labeled HNP-1 to C1q in a dose-dependent fashion. In the fluid phase, preincubation of HNP-1 with C1q resulted in complex formation of HNP-1 and C1q and generation of stable complexes. In conclusion, HNP-1 is able to bind to C1q in the fluid phase and inhibits the classical complement pathway. This mechanism may be involved in the control of an inflammatory response in vivo.
Congenital Erythropoietic Porphyria Successfully Treated by Allogeneic Bone Marrow Transplantation
Authors: Tezcan I. , Xu W. , Gurgey A. , Tuncer M. , Cetin M. , Öner C. , Yetgin S. , Ersoy F. , Aizencang G. , Astrin K.H. , Desnick R.J. 
Published: October 2019

AbstractThe long-term biochemical and clinical effectiveness of allogenic bone marrow transplantation (BMT) was shown in a severely affected, transfusion-dependent 18-month-old female with congenital erythropoietic porphyria (CEP), an autosomal recessive inborn error of heme biosynthesis resulting from mutations in the uroporphyrinogen III synthase (URO-synthase) gene. Three years post-BMT, the recipient had normal hemoglobin, markedly reduced urinary porphyrin excretion, and no cutaneous lesions with unlimited exposure to sunlight. The patient was homoallelic for a novel URO-synthase missense mutation, G188R, that expressed less than 5% of mean normal activity in Escherichia coli, consistent with her transfusion dependency. Because the clinical severity of CEP is highly variable, ranging from nonimmune hydrops fetalis to milder, later onset forms with only cutaneous lesions, the importance of genotyping newly diagnosed infants to select severely affected patients for BMT is emphasized. In addition, the long-term effectiveness of BMT in this patient provides the rationale for future hematopoietic stem cell gene therapy in severely affected patients with CEP.
Intravascular Coagulation Activation in a Murine Model of Thrombomodulin Deficiency: Effects of Lesion Size, Age, and Hypoxia on Fibrin Deposition
Authors: Healy Aileen M. , Hancock Wayne W. , Christie Patricia D. , Rayburn Helen B. , Rosenberg Robert D. 
Published: October 2019

AbstractWe consecutively inactivated both alleles of the thrombomodulin (TM) gene in murine embryonic stem (ES) cells and generated TM-deficient (TM−/−) chimeric mice. Quantitation of an ES-cell marker and protein C cofactor activity indicates that up to 50% of pulmonary endothelial cells are ES-cell derived and therefore TM deficient. Infusions of 125I-fibrinogen into mice show a significant increase (fourfold, P &lt; .005) in radiolabeled cross-linked fibrin in TM−/− chimeric mouse lung as compared with wild-type mice. However, only chimeric mice that exhibit at least a 30% reduction in protein C cofactor activity and are at least 15 months old display this phenotype. Immunocytochemical localization of TM in chimeras shows a mosaic pattern of expression in both large and small blood vessels. Colocalization of cross-linked fibrin and neo (used to replace TM) reveals that fibrin is deposited in TM−/− regions. However, the fibrin deposits were largely restricted to pulmonary vessels with a lumenal area greater than 100 μm2. The hypercoagulable phenotype can be induced in younger chimeric mice by exposure to hypoxia, which causes a fivefold increase in β-fibrin levels in lung. Our findings show that TM chimerism results in spontaneous, intravascular fibrin deposition that is dependent on age and the magnitude of the TM deficiency.
A Missense Mutation in γ-Glutamyl Carboxylase Gene Causes Combined Deficiency of All Vitamin K-Dependent Blood Coagulation Factors
Authors: Brenner Benjamin , Sánchez-Vega Beatriz , Wu Sheue-Mei , Lanir Naomi , Stafford Darrel W. , Solera Jesus 
Published: October 2019

AbstractTo identify potential mutations in the γ-glutamyl carboxylase gene, the sequence of all exons and intron/exon borders was determined in 4 patients from a consanguineous kindred with combined deficiency of all vitamin K-dependent procoagulants and anticoagulants and results were compared with normal genomic sequence. All 4 patients were homozygous for a point mutation in exon 9 that resulted in the conversion of an arginine codon (CTG) to leucine codon (CGG) at residue 394. Screening of this mutation based on introduction of Alu I site in amplified fragment from normal allele but not from the mutated allele showed that 13 asymptomatic members of the kindred were heterozygous for the mutation. The mutation was not found in 340 unrelated normal chromosomes. The segregation pattern of the mutation which is the first reported in the γ-glutamyl carboxylase gene fits perfectly with phenotype of the disorder and confirms the suggested autosomal recessive pattern of inheritance of combined deficiency of all vitamin K-dependent procoagulants and anticoagulants in this kindred. The mutated carboxylase protein expressed in Drosophila cells was stable but demonstrated threefold reduced activity compared with WT carboxylase, confirming that the L394R mutation results in a defective carboxylase.
Moderate Dose Escalation for Advanced Stage Hodgkin’s Disease Using the Bleomycin, Etoposide, Adriamycin, Cyclophosphamide, Vincristine, Procarbazine, and Prednisone Scheme and Adjuvant Radiotherapy: A Study of the German Hodgkin’s Lymphoma Study Group
Authors: Tesch H. , Diehl V. , Lathan B. , Hasenclever D. , Sieber M. , Rüffer U. , Engert A. , Franklin J. , Pfreundschuh M. , Schalk K.P. , Schwieder G. , Wulf G. , Dölken G. , Worst P. , Koch P. , Schmitz N. , Bruntsch U. , Tirier C. , Müller U. , Loeffler M. 
Published: October 2019

Abstract
The BEACOPP (bleomycin, etoposide, adriamycin, cyclophosphamide, vincristine, procarbazine, and prednisone) regimen, a rearranged and accelerated version of the standard COPP/adriamycin, bleomycin, vinblastine, and dacarbazine (ABVD) chemotherapy, has been shown to be effective and safe in a previous pilot study for advanced stage Hodgkin’s disease (HD). The present study aimed to determine a maximum practicable dose of three drugs, ie, etoposide, adriamycin, and cyclophosphamide, for which acute toxicities were acceptable and to assess the feasibility of the escalated scheme. Sixty untreated patients with advanced stage HD were enrolled in this study. Radiotherapy was given in 44 patients (73%) after chemotherapy to initial bulk lesions and residual disease. Granulocyte-colony stimulating factor (G-CSF) was given from day 8 to prevent prolonged neutrocytopenia and severe infections. The intended doses of adriamycin, etoposide, and cyclophosphamide in the BEACOPP schedule could be substantially escalated: adriamycin from 25 to 35, cyclophosphamide from 650 to 1,200, and etoposide from 100 to 200 mg/m2. The major toxicities were leukocytopenia and thrombocytopenia with considerable heterogeneity between individual patients. Of 60 patients, 56 (93%) achieved a complete remission (CR). At a median observation of 32 months, the rates of survival and freedom from treatment failure (FFTF) were estimated to be 91% (95% confidence interval 83% to 99%) and 90% (82% to 98%). These results show that a moderate dose escalation of adriamycin, cyclophosphamide, and etoposide of the baseline BEACOPP regimen is feasible. The escalated BEACOPP regimen shows very encouraging results in advanced stage HD and is now being compared in a randomized phase III study with BEACOPP at baseline dose level.
Impaired Cotranslational Processing as a Mechanism for Type I Antithrombin Deficiency
Authors: Fitches Alison C. , Appleby Ruth , Lane David A. , De Stefano Valerio , Leone Giuseppe , Olds Robin J. 
Published: October 2019

AbstractMost secretory proteins, including antithrombin (AT), are synthesized with a signal peptide, which is cleaved before the mature protein is exported from the cell. The signal peptide is important in the process whereby nascent protein is recognized as requiring subsequent modification within the endoplasmic reticulum (ER). We have identified a novel mutation, 2436T→C L(-10)P, which affects the central hydrophobic domain of the AT signal peptide, in a proband presenting with venous thrombotic disease and type I AT deficiency. We investigated the basis of the phenotype by examining expression in mammalian cells of a range of variant AT cDNAs with mutations affecting the –10 residue. Glycosylated AT was secreted from COS-7 cells transfected with wild-type AT, –10L deletion, -10V or -10M variants, but not variants with P, T, R, or G at -10. Cell-free expression of wild-type and variant AT cDNAs was then performed in the presence of canine pancreatic microsomes, as a substitute for ER. Variant AT proteins with P, T, R, or G at residue –10 did not undergo posttranslational glycosylation, and their susceptibility to trypsin digestion suggested they had not been translocated into microsomes. Our results suggest that the ability of AT signal peptide to direct the protein to ER for cotranslational processing events appears to be critically dependent on maintaining the hydrophobic nature of the region including residue –10. The investigations have defined impaired cotranslational processing as a hitherto unrecognized cause of hereditary AT deficiency.
Differential Leukocyte Recruitment From Whole Blood Via Endothelial Adhesion Molecules Under Shear Conditions
Authors: Reinhardt Paul H. , Kubes Paul 
Published: October 2019

AbstractThe objective of this study was to determine if vascular cell adhesion molecule (VCAM-1), E-selectin, and P-selectin could selectively recruit leukocyte subpopulations, and whether this was affected by shear force or adhesion molecule concentration. Cover slips coated with purified adhesion molecules were incorporated into laminar flow chambers. Whole human blood was perfused for 5 minutes over these cover slips at relative shear forces of 2 to 40 dynes/cm2. Chasing the whole blood with buffer permitted visualization of leukocyte-substratum interactions. Leukocytes were observed to roll on and adhere to VCAM-1 at shears between 2 and 15 dynes/cm2. As assessed by cover slip staining, the majority of these cells were lymphocytes, but eosinophils, monocytes, and, surprisingly, neutrophils were also recruited, events inhibitable by anti–4-integrin antibody (HP1/2). Neutrophils were effectively recruited onto the selectins, with interactions occurring at shears as high as 30 and 40 dynes/cm2 for E- and P-selectin respectively. Eosinophils had high affinity for P- but not E-selectin. Mononuclear cells did not have high affinity for either selectin, but interacted avidly with VCAM-1. Antibodies against P-selectin (G1) and E-selectin (ES-1) completely blocked interactions on these substrates. Reducing the concentration of adhesion molecules did not appreciably change recruitment patterns except for VCAM-1, where neutrophils were no longer recruited. The novel use of whole blood in flow chambers shows a partial selectivity of selectins and VCAM-1 for certain subpopulations of leukocytes under varying physiologic shear conditions.
Prevalence of Antibodies Against Proteins Derived From Leukemia Cells in Patients With Chronic Myeloid Leukemia
Authors: Ling Min , Wen Yue-Jin , Lim Seah H. 
Published: October 2019

Abstract
Although various studies supported the notion that leukemia cells in chronic myeloid leukemia (CML) may be recognized by the immune system, direct evidence showing the immunogenicity in vivo of proteins derived from the leukemia cells is lacking. In this study, we have constructed an expression cDNA library from the leukemia cells of a patient with CML and used the autologous serum to screen for high-titer IgG antibodies directed at the leukemia-derived proteins. We isolated eight distinct clones from the library, suggesting that multiple immune responses were elicited in the autologous host. Sequence analysis showed high degrees of homology to known gene sequences in six of the eight clones. Neither bcr-abl nor proteinase 3 sequences were isolated. Using Northern blot analysis, seven of the eight clones showed ubiquitous expression in normal bone marrow, leukemia cell lines, fresh leukemia cells, and normal tissues. However, clone no. 4 showed restricted mRNA expression, being only detected in some fresh leukemia cells, K562 cells, and normal testicular RNA. Using bacterial lysates in dot blot analysis, a panel of sera from normal individuals and patients with CML and other hematological malignancies were screened for high-titer antibodies against these eight clones. There were, among the CML patients, signficantly higher prevalence of antibodies against seven of the eight clones. They were observed even after omitting from the analysis patients with multiple myeloma whose associated immune paresis may impair immune responses to these proteins. Interestingly, antibodies against these proteins were also detected in a small number of normal individuals. Although the precise clinical significance of our findings remains to be determined, this study provides evidence in support of the potential immunogenicity of leukemia-derived proteins in the autologous host. It also provides basis for further investigations to characterize these proteins, especially clone no. 4, and determine their potential for immune targeting in CML.
Incidence of TEL/AML1 Fusion in Children With Relapsed Acute Lymphoblastic Leukemia
Authors: Loh Mignon L. , Silverman Lewis B. , Young Mary L. , Neuberg Donna , Golub Todd R. , Sallan Stephen E. , Gilliland D. Gary 
Published: October 2019

Abstract
The TEL/AML1 fusion associated with t(12;21)(p13;q22) is the most common gene rearrangement in childhood leukemia, occurring in approximately 25% of pediatric acute lymphoblastic leukemia (ALL), and is associated with a favorable prognosis. For example, a cohort of pediatric patients with ALL retrospectively analyzed for theTEL/AML1 fusion treated on Dana-Farber Cancer Institute (DFCI) ALL Consortium protocols between 1980 to 1991 demonstrated a 100% relapse-free survival in TEL/AML1-positive patients with a median of 8.3 years of follow-up. However, two recent studies analyzing pediatric patients with relapsed ALL have reported the same incidence of the TEL/AML1 rearrangement as in patients with newly diagnosed ALL, suggesting that TEL/AML1 positivity is not a favorable prognostic indicator. To clarify this apparent discrepancy, 48 pediatric patients treated on Dana-Farber Cancer Institute (DFCI) protocols with ALL at first or second relapse were tested forTEL/AML1 using reverse transcriptase-polymerase chain reaction (RT-PCR). The TEL/AML1 fusion was identified in only 1 of 32 analyzable relapsed ALL patients, in concordance with our previous reports of improved disease-free survival in TEL/AML1-positive patients. The low frequency of TEL/AML1-positive patients at relapse is significantly different than that reported in other studies. Although there are several potential explanations for the observed differences in TEL/AML1-positive patients at relapse, it is plausible that relapse-free survival in TEL/AML1-positive patients may be changed with different therapeutic approaches. Taken together, these results support the need for prospective analysis of prognosis in TEL/AML1-positive patients.
An Activating Mutation in the Kit Receptor Abolishes the Stroma Requirement for Growth of ELM Erythroleukemia Cells, But Does Not Prevent Their Differentiation in Response to Erythropoietin
Authors: Leslie Nick R. , O’Prey Jim , Bartholomew Chris , Harrison Paul R. 
Published: October 2019

AbstractWe have previously shown that murine ELM erythroleukemia cells can only be grown in vitro in the presence of a stromal feeder layer, or alternatively stem cell factor (SCF), without which they differentiate. When grown in the presence of SCF, ELM cells can still differentiate in response to erythropoietin (Epo), but growth on stroma prevents this. We previously isolated a stroma-independent ELM variant, ELM-I-1, that is also defective in Epo-induced differentiation. We show here that this variant has an activating mutation in the Kit receptor, converting aspartic acid 814 to histidine. Expression of the mutant receptor in stroma-dependent ELM-D cells causes growth factor-independent proliferation and also gives the cells a selective advantage, in terms of proliferation rate and clonegenicity, compared with ELM-D cells grown in optimal amounts of SCF. Expression of the mutant receptor in ELM-D cells also prevents spontaneous differentiation, but not differentiation induced by Epo. Analysis of mitogenic signaling pathways in these cells shows that the mutant receptor induces constitutive activation of p42/p44 mitogen-activated protein kinases. It also selectively inhibits the expression of p66Shc but not the p46/p52 Shc isoforms (as did treatment of ELM cells with SCF), which is of interest, because p66Shc is known to play an inhibitory role in growth factor signaling.
Mutations in the E-Domain of RARα Portion of the PML/RARα Chimeric Gene May Confer Clinical Resistance to All-transRetinoic Acid in Acute Promyelocytic Leukemia
Authors: Imaizumi Masue , Suzuki Hoshiro , Yoshinari Miyako , Sato Atsushi , Saito Toshiaki , Sugawara Akira , Tsuchiya Shigeru , Hatae Yoshiro , Fujimoto Takeo , Kakizuka Akira , Konno Tasuke , Iinuma Kazuie 
Published: October 2019

Abstract
The binding of all-trans retinoic acid (ATRA) to the ligand-binding region in the E-domain of retinoic acid receptor-α (RARα) modifies the transcriptional activity of RARα protein. ATRA probably induces differentiation of acute promyelocytic leukemia (APL) cells by binding to the E-domain of the RARα portion (RARα/E-domain) of PML/RARα chimeric protein. Therefore, molecular alteration in the RARα/E-domain of the chimeric gene is one mechanism by which patients with APL may acquire resistance to ATRA therapy. In this study using reverse transcription-polymerase chain reaction and single-strand conformation polymorphism, DNA segments amplified from the RARα/E-domain in fresh APL cells of 23 APL patients (8 males and 15 females from 4 to 76 years of age) were screened for mutations. Of those patients, 3 patients (1 with de novo and 2 with relapse) had clinical resistance to ATRA therapy. We found mutations in the RARα/E-domain of PML/RARα chimeric gene exclusively in the 2 patients who exhibited ATRA-resistance at relapse, whereas the mutations were not detected at their initial onset. Interestingly, these patients received a prolonged or intermittent administration of ATRA before relapse with ATRA-resistance. The mutations lead to the change of amino acid in the ligand-binding region of RARα/E-domain, Arg272Gln, or Met297Leu according to the amino acid sequence of RARα, respectively. Further study demonstrated that the in vitro ligand-dependent transcriptional activity of the mutant PML/RARα protein was significantly decreased as compared with that of wild-type PML/RARα. These findings suggest that mutations in the RARα/E-domain of the PML/RARα chimeric gene may confer clinical resistance to ATRA therapy in patients with APL.
Binding of Factor VIII to von Willebrand Factor Is Enabled by Cleavage of the von Willebrand Factor Propeptide and Enhanced by Formation of Disulfide-Linked Multimers
Authors: Bendetowicz Ana Victoria , Morris Jill A. , Wise Robert J. , Gilbert Gary E. , Kaufman Randal J. 
Published: October 2019

Abstractvon Willebrand factor (vWF) is a multimeric adhesive glycoprotein with one factor VIII binding site/subunit. Prior reports suggest that posttranslational modifications of vWF, including formation of N-terminal intersubunit disulfide bonds and subsequent cleavage of the propeptide, influence availability and/or affinity of factor VIII binding sites. We found that deletion of the vWF propeptide produced a dimeric vWF molecule lacking N-terminal intersubunit disulfide bonds. This molecule bound fluorescein-labeled factor VIII with sixfold lower affinity than multimeric vWF in an equilibrium flow cytometry assay (approximate KDs, 5 nmol/L v 0.9 nmol/L). Coexpression of propeptide-deleted vWF with the vWF propeptide in trans yielded multimeric vWF that displayed increased affinity for factor VIII. Insertion of an alanine residue at the N-terminus of the mature vWF subunit destroyed binding to factor VIII, indicating that the native mature N-terminus is required for factor VIII binding. The requirement for vWF propeptide cleavage was shown by (1) a point mutation of the vWF propeptide cleavage site yielding pro-vWF that was defective in factor VIII binding and (2) correlation between efficiency of intracellular propeptide cleavage and factor VIII binding. Furthermore, in a cell-free system, addition of the propeptide-cleaving enzyme PACE/furin enabled factor VIII binding in parallel with propeptide cleavage. Our results indicate that high-affinity factor VIII binding sites are located on N-terminal disulfide-linked vWF subunits from which the propeptide has been cleaved.
Telomerase Activity in Hodgkin's Disease
Authors: Norrback Karl-Fredrik , Enblad Gunilla , Erlanson Martin , Sundström Christer , Roos Göran 
Published: October 2019

AbstractTelomere maintenance executed by the action of telomerase seems to be a prerequisite for immortalization. Telomerase is found in most cell lines and malignant tumors. A telomerase-independent mechanism for telomere maintenance in Hodgkin's disease has been proposed in the absence of detectable telomerase activity. In this study, telomerase activity was detected in 31 of 77 Hodgkin's disease samples and a strong correlation between eosinophilia and absence of detectable telomerase activity was found. Purified eosinophils and specifically eosinophil-derived neurotoxin and eosinophilic cationic protein, both ribonucleases, were found to degrade telomerase. Purified neutrophils also exhibited weak telomerase degradative activity. Reanalysis of previously telomerase-negative Hodgkin's disease samples with eosinophilia using ribonuclease inhibitors resulted in the detection of telomerase activity. Ribonuclease-containing cells in vivo thus have a considerable impact on the detectability of telomerase. In Hodgkin's disease samples without eosinophilia, 24 of 27 exhibited telomerase activity at decreased levels compared with non-Hodgkin's lymphomas and at increased levels compared with reactive nodes indicative of a telomerase positive tumor component in Hodgkin's disease. Telomerase positivity of the Hodgkin's and Reed-Sternberg cells in vivo was also supported by high levels of telomerase expression in Hodgkin's disease cell lines. Based on our data, Hodgkin's lymphomas are potential targets for antitelomerase therapy.