bluuebunny/crossref_metadata_2025_split huggingface dataset:
Proteolytic Activation of the Chemokine CCL15 by Cathepsin G from Neutrophil Granulocytes Modulates Proliferation of Primitive Hematopoietic Precursor Cells.
Authors: Richter Rudolf , Bistrian Roxana , Henschler Reinhard 
Published: April 2020

Abstract
The human chemokine HCC-2 (MIP-1δ, small inducible cytokine subfamily A [Cys-Cys], member 15, hmrp-2b, MIP-5, NCC-3) is a potent chemoattractant for monocytes, lymphocytes and endothelial cells, and has been shown to inhibit proliferation of primitive hematopoietic precursor cells (HPC). CCL15 is constitutively expressed in gut and liver, and was identified to circulate in human plasma. The length of the N-terminus of CCL15 was found to control biological activity. We therefore aimed to investigate the processing of CCL15 in the hematopoietic system, and the effect of processed isoforms on HPC proliferation. Incubation of full-length CCL15 with cell-free bone marrow supernatant prepared from normal untreated mice revealed a decrease in molecular weight by approximately 3 kD. In contrast, the molecular weight of CCL15 remained unchanged when cell-free bone marrow supernatant of mice with regenerating hematopoiesis after 5 Gy gamma irradiation was analyzed. Incubation of full-length CCL15 with isolated neutrophils, but not monocytes, lymphocytes or thrombocytes revealed N-terminal deletions of 21 (Δ21), 23 (Δ23) and 26 (Δ26) amino acids, as assessed by Edman sequencing/mass spectrometry. Both (1) inhibition of the neutrophil-derived CCL15 proteolytic activity by protease inhibitors Suc-Ala-Ala-Pro-Phe-pNA, secretory leucoprotease inhibitor or elastatinal, and (2) addition of the recombinant serine proteases cathepsin G or elastase to full-length CCL15 revealed that the Δ23 and Δ26 isoforms are produced by cathepsin G, whereas formation of the Δ21isoform is dependent on elastase. In addition, the Δ23 and Δ26 isoforms of CCL15 was also directly isolated and sequenced from blood filtrate (hemofiltrate) collected from renal insufficiency patients. The Δ26 isoform was synthesized and analyzed in HPC proliferation assays. Compared to full length CCL15, the Δ26 isoform displayed a mean 2.5 fold increased potency to inhibit CFU-A colony formation from post 5-fluorouracil murine bone marrow. In addition, the Δ26 isoform efficiently abrogated cytosine arabinoside-induced suicide of murine spleen colony forming unit (CFU-S) in post 5-fluorouracil murine bone marrow, and inhibited growth of human cord blood CD34+ single cell clones induced by hematopoietic growth factors SCF, IL-3, GM-CSF and EPO after flow-cytometric cell sorting (mean 2.3 fold inhibition). Our findings indicate a role for neutrophil-derived cathepsin G in growth control of colony of primitive hematopoietic progenitor cells through the proteolytic processing of CCL15.
Analysis of the X-Linked F-Cell Production Locus.
Authors: Keefer Jeffrey R. , Purvis Shirley H. , Dover George J. , Smith Kirby D. 
Published: April 2020

Abstract
This laboratory has previously identified a locus on the X chromosome at Xp22.2–22.3 (F-cell production locus or FCP) that is responsible for approximately 40% of the genetic variability in F-cell number in patients with sickle cell disease (SCD). We have re-examined the association of this region with F-cell production by multipoint linkage analysis. We have confirmed linkage to Xp22.2–22.3 and refined the candidate locus to a region of approximately 3 cM, between markers DXS452 and DXS410, with a maximum LOD score of 3.315. Linkage to a more extended region of 11 cM with an average LOD score of 1.5 could not be excluded. In an effort to identify candidate genes within this region that influence F-cell production, we screened known genes within this region for transcription factor binding sites that had relevance to HbF production. We first searched for CREB binding sites given the involvement of cAMP in of HbF induction in CD34 cells recently identified by this laboratory. Of the 15 ESTs containing CREB sequences, several also had binding sites for NFE2 or NFE2-like transcription factors. We examined 4 genes, 3 within the refined candidate locus (KIAA1280, TBL1X, and KAL1) and one in the more extended locus region (EGFL6) for expression levels within pedigrees of known F-cell phenotypes. Results indicate that, within families, a reduced level of KIAA1280 message is associated with high F-cell phenotypes in lymphoblastoid cells from patients with sickle cell anemia. The other genes examined had no such correlation.
KIAA1280, also designated BMO42 is a gene of unknown function that is expressed in bone marrow. The predicted protein contains a region homologous to a conserved protein kinase C domain. While not yet definitive, these studies provide the first suggestive evidence for a candidate gene within the FCP locus. Further studies will attempt to define the function of this gene and explore it’s involvement in F-cell regulation.
Various Drugs with or without an Antisickling Effect in the In Vitro Tests Showed a Strong Antisickling Effect in the In Vivo Studies in Transgenic Sickle Mice.
Authors: Asakura Toshio , Abdulmalik Osheiza , Iyamu Efe , Chen Qiukan , Yang Jisheng , Abraham Donald J. , Safo Martin K. , Brugnara Carlo , Evans Greg 
Published: April 2020

Abstract
Since its inception in 1997, the NHLBI Sickle Cell Disease (SCD) Reference Laboratory (Ref Lab) has received over 150 candidate drugs from over 30 researchers, universities and companies for evaluation. All candidate drugs are evaluated for both their beneficial and adverse effects in vitro using more than 10 sophisticated methods. Specifically, each candidate drug is studied to see whether it inhibits cell sickling, prolongs the delay time prior to deoxy-hemoglobin (Hb) S polymerization, increases the solubility of deoxy-Hb S, hydrates red blood cells (RBCs), prevents dehydration of RBCs, shifts the oxygen equilibrium curve (OEC) toward the left, reduces the adhesion of RBCs to endothelial cells, promotes the synthesis of Hb F, causes hemolysis, promotes formation of met-Hb and increases the denaturation of intracellular Hb S. With this new approach, we have found more than 10 new antisickling agents that showed beneficial effects without significant adverse effects in the in vitro tests. Of these, five drugs were further studied in vivo using transgenic mice that produce human Hb S, and all five drugs inhibited the formation of sickled cells in the blood and significantly prolonged their survival time under acute hypoxic conditions. The five drugs are NIPRISAN (plant extracts) [Brit. J. Haematol. (BJH) 122:1001,2003], MX-1520 (prodrug of vanillin) (BJH125: 788,2004), 5-hydroxymethyl-2-furfural (5HMF) (BJH128:552,2005), NS3623 (Blood 97: 1461,2001, in vivo results not shown), and FLOCOR (surfactant) (Blood94:420a,1999). Of these 5 drugs, FLOCOR and NS3623 did not have an antisickling effect in vitro. It is quite interesting that FLOCOR and NS3623 which showed no direct antisickling effect in vitro, showed a strong antisickling effect in vivo. Some of our results suggest that combined use of some of these drugs may have a synergistic effect in vivo. Studies on the combined use of these drugs are planned. Researchers with candidate drugs that may have beneficial effects for SCD are welcome to contact the SCD Ref Lab for free in vitro evaluation of their agents (for more information, please see our website at www.tatcom.com/sickle-cell).
A Transgenic Mouse Model of Homozygous HbE Disease.
Authors: Chen Qiuying , Kuypers Frans A. , Rybicki Anne C. , Bhagat Amrita , Suzuka Sandra M. , Fabry Mary E. , Nagel Ronald L. , Hirsch Rhoda Elison 
Published: April 2020

Abstract
Hemoglobin E (β26 Glu→Lys) is the most common abnormal hemoglobin (Hb) variant in the world. Homozygotes for HbE have a benign clinical picture (microcytic and mildly anemic) with rare clinical symptoms. However, when HbE coexists with β0-thalassemia, it is the most common severe hemoglobinopathy world-wide. National research priorities in the United States now include HbE related diseases because of the growing number of immigrants with these diseases, as well as the significant world impact of this life-threatening condition. In efforts to understand these diseases, a transgenic mouse model of homozygous HbE Disease was generated (Chen et al. Blood Cells Mol. Dis., 2004). Subsequent breeding has yielded knockout transgenic mice expressing exclusively human HbE: RBC smears show hypochromia, target cells, and stress reticulocytes. Measurement of PS exposure with Annexin V does not show a significantly increased population of PS exposing cells in the blood of these mice. Density gradients showing broader RBC density distributions correlate with increased RDW. RBC parameters determined by the ADVIA show low MCV (31.6±1.5fl vs. 46.9±2.1fl for C57) and MCH (9.8±0.1pg vs. 14.8±0.7pg for C57); and normal MCHC (32.0±2.0g/dl vs. 31.2±1.7 g/dl for C57) [n=5]. These RBC parameters mirror human HbE homozygotes. Increased levels of zinc protoporphyrin (ZPP), free protoporphyrin (PPIX), and fluorescent heme degradation products (FHDP) are found. To see if these porphyrins and FHDP arise from alterations in heme synthesis, real-time PCR technology methods were used. A comparison of the gene expression of enzymes involved in heme synthesis was made from RNA extracted from the spleens (the mouse hematopoietic organ). Upregulation in the rate-limiting enzyme erythroid delta-aminolevulinate synthase (ALAS) and other heme synthesis enzymes is suggested. These observations may be explained by increased erythropoiesis, compatible with the increase in % reticulocytes (7.2%±2.5, n=5); and/or elevated RBC porphyrins observed in these mice. This result is consistent with the effect of elevated porphyrins on ALAS in peripheral blood of erythropoietic protoporphyria-chemically induced mice (Inafuku et al., J Dermatol Sci, 1999). Considering the above, we conclude that the HbE full knockout mice are an excellent model for the benign human homozygous EE disease, and will serve as an important control for the proposed transgenic studies to generate HbE/β-thalassemia mice.
Factor VIII Inhibitors in Haemophilia A Patients after Continuous Infusion of Factor VIII Concentrates.
Authors: von Auer Charis , Oldenburg Johannes , Krause Manuela , Miesbach Wolfgang , Scharrer Inge ,   CI-Study Group in Germany
Published: April 2020

Abstract
Continuous infusion (CI) of coagulation factor concentrates has been used in haemophilia treatment since the early 1990s. Recent reports of the occurrence of an inhibitor (inh.) after CI have raised concerns about this method of treatment. We conduct a retrospective study to investigate the development of inh. After CI of factor VIII (FVIII) concentrates in Germany and report on further study results. A questionnaire was sent to 100 haemophilia treating colleagues in Germany. So far 42 colleagues have answered, in 19 haemophilia treatment centers CI was used as treatment modality, 5 of them still use it today. Altogether 200 CI were conducted in 128 patients (pat.). In 14 pat. Inh. Development was detected. 5 of these pat. Were suffering from severe, 1 from moderate and 8 from mild haemophilia, their median age was 27 years. Indications for treatment before inh. Detection were major bleeds and surgical procedures. Plasmaderived (9) and recombinant (4) factor concentrates were given in various infusion sets. Data concerning gene mutation, exposure days, inhibitor characteristic, former change of product, concomitant medication, vaccination and blood transfusion were collected. Regarding the genotype, we found 4 missense mutations, 2 intron-22-inversions, 1 small deletion, 1 splice site mutation, 6 were unknown. In our study strikingly the inh. Developed very often in pat. With mild or moderate haemophilia and genotypes that exhibit an inh. Prevalence of &lt; 10 %. Our further data confirm the assumption that pat. With mild haemophilia might exhibit a much higher prevalence of inh. Development when treated with an “intensive FVIII - treatment” such as CI. On the other hand there might be an uncommon inh. pathomechanism in connection with CI, resulting in a peculiar group of pat. developing the inh. after this way of application. A prospective multicenter study to investigate the inhibitor development after CI should be conducted.
Inhibitor Epitopes Identified from Inhibitor Positive Plasma of a Hemophilia B Patient Change during ITT.
Authors: Königs Christoph , Kessel Christoph , Stumpf Susanne , Roland Janti , Klingebiel Thomas , Kreuz Wolfhart 
Published: April 2020

Abstract
Hemophilia B is an X-linked bleeding disorder. Substitution of recombinant (rFIX) or plasma derived (pdFIX) FIX preparations are required to restore coagulation. A rare but severe complication is the development of FIX inhibitors that are often accompanied by allergic reactions. Immune tolerance therapies (ITTs) for inhibitor elimination are difficult and can induce further allergic reactions. ITTs for FIX inhibitors are less established and less successful than for FVIII inhibitors.
In this study, three phage displayed random peptide libraries were screened by biopanning to isolate small peptides that mimic the epitope of FIX inhibitors. In the patient, the inhibitor was eliminated by ITT but reappeared again. Plasma samples before ITT and at reappearance of inhibitor were used to isolate inhibitor specific phage clones. Phage clones showing specific reactivity with inhibitor positive plasma compared to control plasma by ELISA were amplified and DNA was sequenced to determine the peptide sequence displayed on the phage. Before ITT, 31 isolated phage clones specific for FIX inhibitors have been sequenced so far. 10/31 contain a common motif. After ITT, 24 clones specific for patient plasma have been sequenced. 10/24 contain a different common motif. Synthetic peptides have been generated according to the phage displayed sequence to demonstrate binding of inhibitors and test for cross reactivity.
The surface of the model of FIX was screened for these motifs with a novel software tool. Different conformational epitopes could be identified on the surface of the molecule before and after ITT.
Small peptides (mimotopes) have been identified that mimic epitopes for FIX inhibitors in a hemophilia B patient. The sequences can be aligned to different conformational epitopes on FIX. It appears that the epitope has changed in the course of the ITT. The mimotopes can also be used to target inhibitor specific B cells for a novel approach in inhibitor elimination in hemophilia B.
Functional Characterization of Aspergillus Fumigatus Specific T-Cells Clones.
Authors: Lehrnbecher Thomas , Koehl Ulrike , Roilides Emmanuel , Simitsopoulou Maria , Hanisch Mitra , Tramsen Lars , Latge Jean-Paul , Topp Max S. , Einsele Hermann , Klingebiel Thomas , Beck Olaf 
Published: April 2020

Abstract
Invasive aspergillosis (IA) remains a major cause of morbidity and mortality in patients with hematological malignancies, in particular in patients who have undergone allogeneic hematopoietic stem cell transplantation (SCT). There is a growing body of evidence that T-cells play an important role in the immunological response to Aspergillus fumigatus. Using the Aspergillus fumigatus antigen extract EC SAB and the IFN-γ secretion assay (Miltenyi Biotec, Germany), we generated Aspergillus fumigatus specific T-cell clones by limiting dilution (n=4). Flow cytometry revealed a cell population of CD3+/CD4+ cells (mean±SEM, 98.2±1.2%). Functional assessment by ICC revealed that an average of 8.7% of these cells (range, 6.6%–18.5%) specifically secreted IFN-γ on stimulation with EC SAB, which supports the TH1 response of the generated cells to Aspergillus fumigatus antigens. The antigenic components of EC SAB are one or more proteins, since the addition of proteinase completely suppressed the stimulating effect of this preparation. The percentage of IFN-γ producing CD3+/CD4+ cells was less than 1% upon activation with antigen extracts from Aspergillus flavus, Aspergillus niger, Alternaria alternata, Mucor racemosus, Penicillium notatum and Candida albicans, indicating that the generated T-cell clones are specific for Aspergillus fumigatus. A strong proliferation of the generated Aspergillus fumigatus specific T-cells was seen after re-stimulation with EC SAB, whereas alloreactivity was reduced compared to CD4+ T-cells of the original fraction. Hyphal damage of Aspergillus fumigatus was assessed by means of an XTT assay. Polymorphonuclear leukocytes (PMNs) showed a similar hyphal damage when tested alone (mean±SEM, 14.2±2.1%), in combination with antigen presenting cells (APCs) (15.1±1.4%), or in combination with Aspergillus fumigatus specific T-cells (15.0±2.0%). A comparable hyphal damage was seen when Aspergillus fumigatus specific T-cells were co-incubated with APCs (14.2±1.7%). In contrast, the combination of APCs and Aspergillus fumigatus specific T-cells with PMNs resulted in a significantly higher hyphal damage compared to all other settings (23.3±2.8%; P&lt;.0001). Interestingly, APCs alone or Aspergillus fumigatus specific T-cells alone showed a weak, but significant capacity to induce hyphal damage (7.4±1.1% and 11.3±1.8%, respectively). Before considering a clinical application, however, further studies need to focus on defining the optimal antigen(s) which reproducibly induce a TH1 response and elicit high antifungal activity, as well as to characterize the subpopulation of patients undergoing allogeneic SCT who ultimately benefit from either a prophylactic or a therapeutic adoptive transfer of Aspergillus fumigatus specific T-cells.
CMV Immunity Status in Older (&gt;50 Years Old) Subjects after Non-Myeloablative or Reduced Intensity Regimen for Hematopoietic Cell Transplantation (HCT): A Comparison with a Younger Cohort after Ablative HCT.
Authors: Gallez-Hawkins Ghislaine , Thao Lia , Lacey2 Simon F. , Martinez Joybelle , Franck Anne E. , Li Xiuli , Lomeli Norma A. , Spielberger Ricardo , Nakamura Ryotaro , Forman Stephen J. , Diamond Don J. , Zaia John A. 
Published: April 2020

Abstract
Immunity declines with age as demonstrated by cell-mediated and humoral responses to alloantigens. The susceptibility of these elderly subjects to endogenous virus infection, such as human cytomegalovirus (HCMV) reactivation, is a particular concern during the process of hematopoietic stem cell transplantation (HCT) and immune reconstitution. In this report, the host contribution to stem cell engraftment and differentiation was evaluated by comparing the HCMV immune response in older subjects (&gt; 50 y.o.) to a younger (&lt; 50 y.o.) transplant population. This was a retrospective analysis of a subset of data collected prospectively and with IRB approval for characterization of the CMV immune response of allogeneic transplant patients. Within the dataset, two groups of patients were compared. Group 1 consisted of 10 patients &gt;50 y.o. who had received reduced intensity or non-myeloablative conditioning regimen, and Group 2 consisted of 13 patients &lt;50 y.o., most of whom had received a myeloablative regimen. Because 9 of 10 in Group 1 had had CMV reactivation, Group 2 was selected from the subset of younger patients with known post-transplant CMV infection. CMV infection was defined as either a positive CMV blood culture using shell vial assay or a positive CMV PCR on plasma. Subjects were assessed on days 40, 90, 120, 150, 180, and 360 post-HCT by CMV-specific tetramer-binding assay using CD8 cells, assays for intracellular INF-g response of CD4 and CD8 cells, and a T-cell receptor excision circle (TREC) assay. There were no significant differences observed in the CD4+/IFN-g+ cell responses to CMV antigen nor were the rates of activated CD4+/CD69+/IFN-g+ cells different between the groups. Group 1 was also characterized by a robust CD8+/IFN-g+ response to HLA-specific CMV peptides, and all subjects had ≥ 2cells/μl by day 150 post-HCT. The frequency of CMV tetramer positive cells (≥ 2cells/μl) was 50% in Group 1 by day 90 post-HCT and was not statistically different from Group 2. The T cell renewal in the thymus as measured by the TREC spanned over 0 -- 92 copies/μg of total cellular DNA in Group 1 and from 0 – 129 copies/μg in Group 2 during the first year post-HCT (n.s.). In conclusion, CMV immune reconstitution in older transplant subjects, who undergo a reduced intensity or non-myeloablative regimen, is robust and, in this small sampling, did not differ from that observed in a younger adult group.
Genetic Polymorphisms in Chemokine Receptor 5, Interleukin 10 and Monocyte Chemoattractant Protein 1 Contribute to Cytomegalovirus Reactivation and Disease after Allogeneic Stem Cell Transplantation.
Authors: Loeffler Juergen , Steffens Michael , Arlt Eva-Maria , Toliat Mohammad-Reza , Wienker Thomas , Hebart Holger , Boeckh Michael , Ljungman Per , Trenschel Rudolf , Einsele Hermann 
Published: April 2020

Abstract
Human Cytomegalovirus (HCMV) infection is still one of the most challenging complications after allogeneic stem cell transplantation (alloSCT). Here, we demonstrate that defined single nucleotide polymorphisms (SNPs) are associated with the susceptibility to HCMV infection after alloSCT.
Genotyping of 83 patients (with HCMV reactivation) and 71 controls (no HCMV reactivation) was performed. All donors and SCT recipients were Caucasians. Patients suffered from chronic myeloproliferative disorders (n = 16) and AML /ALL /SAA /NHL /MDS (n = 117). For 21 patients, underlying disease could not be determined retrospectively. Genotyping was performed by PyroseqencingTM on the PSQTM HS96A System (Biotage AB, Uppsala Sweden) or on ABI 7900 ABI PRISM® Sequence Detection System using TaqMan® Assay-By-DesignTM and Assay-On-Demand TM (Applied Biosystems).
By Armitage’s-Trend-Test, no significant association for 78 /87 markers and the occurrence of HCMV reactivation or disease (p &gt; 0.05) was found. However, 9 markers showed a significant association with HCMV reactivation and /or disease. These markers are located in CCR5, MCP 1and IL10. If comparing individuals with HCMV-DNAemia versus no reactivation regardless of disease, 5 markers showed a significant association (in MCP 1and IL10). Two markers of MCP 1were significantly associated with HCMV reactivation, but none of the markers analyzed in CCR5 and IL10. Thus, we presume that HCMV reactivation is associated with polymorphisms in MCP1, whereas disease might be related to polymorphisms in CCR5 and IL10. Analyzing the clinical parameters, we found an association between CD34 selection and HCMV reactivation (p = 0.0378) or HCMV disease (p = 0.001). Furthermore, severe acute GvHD was significantly related to HCMV disease (p = 0.0015), chronic GvHD was significantly associated to HCMV disease only if reactivation versus disease was compared (p = 0.034). Treatment with corticosteroids led to HCMV reactivation (p = 0.036). In conclusion, screening of alloSCT patients for the presence of defined polymorphisms may help to predict the individual risk of HCMV reactivation and disease, and thus justifying additional antiviral prophylaxis.