bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Pharmacogenetics and Inhibitor Risk (PIR) Study: Establishing the Spectrum of Factor (F)VIII Gene (F8) Mutations in African-American Hemophilia A Patients.
Authors: Howard Tom E. , Machiah Deepa K. , Viel Kevin R. , Channel Cynthia , Ameri Afshin , Iyer Rathi V. , Watts Ray G. , Lutcher Charles , Davis Joanna , Abshire Thomas C. , Almasy Laura 
Published: April 2020

Abstract
The development of neutralizing antibodies against plasma-derived or recombinant (r) molecules of wildtype (wt) factor (F)VIII protein is the most serious complication of replacement therapy for hemophilia A (hA) patients. Although the pathogenesis of these antibodies, termed inhibitors, is complex and poorly understood, ethnicity, a recently established risk determinant, is clearly involved since African-American (AA) patients experience this complication ~2-fold more often than Caucasians. In a previous study -- in which the FVIII genes (F8) from 137 unrelated healthy people, representing 7 ethnic groups, were resequenced -- we identified 4 common nonsynonymous single nucleotide polymorphisms (nsSNPs) and thereby demonstrated that FVIII is not a monomorphic protein in non-hemophiliacs. Interestingly, 5 of the 6 distinct wt proteins encoded by the allelic combinations or haplotypes (H) of these nsSNPs, designated H1, H2, ..., H5, are expressed by AA’s compared to only 2 in Caucasians (H1 and H2). Because H3, H4 and H5 are 1) partially defined by AA-restricted minor alleles of R484H and M2238V, 2 nsSNPs that substitute amino acids in the A2 and C2 major B-cell inhibitor epitopes, 2) represent the wt FVIII protein in ~27% of AA’s, and 3) differ from the rFVIII proteins used clinically, we designed the PIR study -- the 1st cross-sectional study of AA hA patients -- to determine if pharmacogenetic factors contribute to their greater inhibitor risk. Because the strongest known risk factor for inhibitors is the F8 mutation type, we are resequencing the known functional regions of F8 in the 1st 50 enrolled PIR subjects, out of the 223 total AA hA patients treated at the six participating Region IV South hemophilia treatment centers, to test the plausible alternative hypothesis that the higher inhibitor incidence is due to a different spectrum of molecular abnormalities than has been found in other ethnic subgroups of patients already examined at the DNA sequence level. We have 1) determined the plasma FVIII-activity and -antigen levels, 2) identified the causative mutation in 18 patients, several of which are novel, and 3) are currently performing RT-PCR analyses for detecting the common inversions in introns 1 and 22. Once completed, we will statistically compare the prevalence of AA F8 mutation subtypes to that in hA patients from other ethnic subgroups (detailed in the HAMSTeRS database), and the proportion of AA FVIII deficiencies that are 1) severe, moderate and mild, and 2) CRM-positive and -negative.
Human Metapneumovirus Infection in Adult Patients after Allogeneic Hemopoietic Stem Cell Transplantation.
Authors: Colombo Anna A. , Algarotti Alessandra , Debiaggi Maurizia , Parea Maurizio , Bragotti Letizia Zenone , Bernasconi Paolo , Caldera Daniela , Canducci Filippo , Lazzarino Mario , Alessandrino Emilio P. 
Published: April 2020

Abstract
The human metapneumovirus (hMPV) has been described as possible cause of respiratory disease in pediatric and adult subjects. There are not, however, data concerning the epidemiology and or the clinical impact of hMPV infection in adults receiving allogeneic hemopoietic stem cell (HSC) transplant. We report the results of a prospective study aimed to asses the incidence of hMPV infection in patients undergoing allogeneic HSC transplant. We analyzed by reverse transcription PCR assay, the presence of hMPV in nasopharingeal aspirate (NPA) collected before transplant and on day 0, +15, +30, +60, +90, +180, +360 from transplant. Detection of hMPV RNA was based on two set primers pair located in the L and N genes. Amplified hMPV RNA was sequenced for genotyping in region N.
Twenty patients with a median age of 37 yrs (range 25–58), receiving allogeneic HSC for hematological disorders (AML 11 pts, ALL 5 pts, MDS 2 pts, NHL and MM 1 pt each), entered the study. Six pts received transplant from an HLA identical sibling and 14 from an unrelated donor (MUD). As preparative regimen, 15 pts had a standard myeloablative conditioning regimen, and 5 received reduced intensity conditioning with Thiotepa and Fludarabine. Acute graft-versus-host-disease prophylaxis consisted of Cyclosporine A and Methotrexate. In 6 pts receiving MUD transplant, in vivo T depletion by ATG was performed. At transplant, all patients were in good performance status and none had signs of active acute respiratory disease. Of 20 patients, four had a prior history of pulmonary infection from agents different from hMPV.
Of 49 NPA samples analyzed so far, 29 (59%) resulted hMPV-positive. Sequence analysis performed in 12 patents detected a virus genotype-A. Of 12 pts in whom 3 or more NPA samples were collected, 9 (45%) resulted positive for hMPV RT-PCR, with persistence of infection for a period ranging from 14 to 84 days. No patient, however, showed clinical signs of respiratory disease. These data indicate an high prevalence and a long persistence of asymptomatic hMPV infection in adults receiving allogeneic HSC transplant.
A Randomized Crossover Study of High- and Standard-Dose Recombinant Factor VIIa for Treatment of Hemarthroses in Hemophiliacs with Inhibitors.
Authors: Santagostino Elena , Mancuso Maria E. , Rocino Angiola , Mancuso Giacomo , Scaraggi Francesco A. , Morfini Massimo , Daniele Filomena , Barillari Giovanni , Mannucci Pier M. 
Published: April 2020

Abstract
A multicenter randomized crossover trial was designed to compare efficacy, safety, feasibility and cost associated with standard- and high-dosages of rFVIIa for home treatment of hemarthroses in hemophiliacs with inhibitors. Enrolled patients were instructed to treat within 6 hours from the onset of bleeding 4 consecutive haemarthroses of ankles, knees or elbows either with the rFVIIa standard dose of 90 mcg/kg (repeated as necessary every 3 hours) or with a single high dose of 270 mcg/kg. Patients who did not achieve a success within 9 hours continued rFVIIa treatment with repeated standard doses. The response to treatment was assessed for up to 48 hours by patients/caregivers who reported on a visual analogic scale (VAS) graded from 0 to 100 the improvement in symptoms and also rated the response as effective, partially effective or ineffective. Success was defined a treatment rated as effective and VAS score ≥70 and failure a treatment rated as ineffective and VAS score ≤30, responses that did not fulfil these criteria being considered a partial response. Of 20 hemophiliacs with inhibitors enrolled (median age: 27 years), 18 treated 32 hemarthroses assigned to the standard- and 36 to the high-dose regimen during the study period of 18 months. Forty-eight hemarthroses (71%) occurred in target joints. Success rates for standard- and high-dose regimens were similar: 31% and 25% at 9 hours, 53% and 50% at 24 hours, 66% and 64% at 48 hours. Recurrence rates were identical (3%). The median number of rFVIIa boluses needed to achieve a successful course was 3 for the standard-dose and 1 for the high-dose regimen, and the median amount of rFVIIa used per successful course was identical (270 mcg/kg). Our results indicate that the use of a high-dose regimen with rFVIIa for home treatment of hemarthroses is effective, safe, does not imply an increased consumption of rFVIIa and requires the infusion of a smaller number of boluses. Its convenience is particularly relevant in cases with difficult venous access and in hemorrhages into target joints.
Quantum Dot Based Duplex In Situ Hybridisation for Gene Expression Profiling.
Authors: Tholouli Eleni , Di Vizio Dolores , O’Connell Fionnuala , Loda Massimo , Twomey David , Golub Todd , Levenson Richard , Hoyland Judith A. , Yin John A.L. , Byers Richard 
Published: April 2020

Abstract
Quantum dots (QDs) are fluorescent semiconductor nanocrystals (2–10-nm core diameter) possessing the unique properties of extremely high fluorescence efficiency, lack of photobleaching and long fluorescence lifetime, making them an ideal tool for bioimaging. We have developed a novel technique for in situ hybridisation (ISH) using biotinylated oligonucleotides conjugated to streptavidin coated QD, and used them in this study to label bone marrow trephine samples.
50-mer long oligonucleotide probes were conjugated to QDs prior to ISH and conjugation efficiency was demonstrated by gel electrophopresis. ISH conditions and molar ratio of QDs to probe were optimised using a polyT probe. Images were captured using a CRI Nuance spectral imaging system and signal intensity was semi-quantitated using IPLab software. Specific oligonucleotide hybridisation was demonstrated using a probe for myeloperoxidase (MPO) in 40 bone marrow sections infiltrated by acute myeloid leukaemia (AML), acute lymphoblastic leukaemia (ALL), chronic myeloid leukaemia (CML) as well as reactive bone marrow. In each case hybridisation signal was consistent with the distribution of MPO by standard immunohistochemistry - MPO was strongly expressed by myeloid blasts and absent in lymphoid blasts; in CML, most, but not all, cells were positive for MPO, in comparison to many fewer positive cells in reactive marrow. Duplex ISH was demonstrated using a probe for bcl-2 together with MPO in 5 bone marrow sections infiltrated by follicular lymphoma (FL). Strong hybridisation signal for bcl-2 was detected in all cells of the paratrabecular aggregates of FL but showed only scattered positivity in the remainder of the bone marrow. Conversely, MPO was absent in the paratrabecular aggregates and present in the myeloid cells in the remainder of the marrow. This pattern was present in both single and duplex ISH for MPO and bcl-2 in the marrow infiltrated by FL. Duplex ISH was performed both by sequential hybridisation with bcl-2 followed by MPO, and simultaneously with a mix of bcl-2 and MPO probes. As negative controls, scrambled oligonucleotide probes for the corresponding genes were used in each case and did not show hybridisation.
In summary, we have developed a generic method for QD labelling and semi-quantitative detection of oligonucleotide ISH in routinely processed clinical tissue samples. Although, in this study we primarily used bone marrow trephine samples, this technique can be applied to any tissue. It has the potential to facilitate transfer of microarray-identified gene signatures to clinical research and diagnostics, whilst the ability of spectral imaging to resolve multiple signals offers the possibility of multiplexed probe detection.
Prepared and Application of New Microarray-Based Method for Detecting SNP in the Risk of Hematological Malignancy.
Authors: Chen Bao-An , Jiang Ni , Ji Mei-Ju , Hou Peng , Lu Zu-Hong , Gao Chong , Ding Jia-Hua , Sun Yun-Yu , Wang Jun , Cheng Jian , Zhao Gang , Fei Fei , Chen Ning-Na 
Published: April 2020

Abstract
Objective To investigate a new microarray-based method for single nucleotide polymorphisms (SNP) genotyping, and to study the role of 5,10-methylenetetrahydrofolate reductase(MTHFR) SNP in hematological malignancy pathogenesis in the Chinese population.
Methods Prepared a aimed gene microarray based on cDNA microarray theory, dual-color fluorescenece hybridization was used for detecting SNP loci, sequencing was performed to confirm the results. In order to prepare the function SNP detection microarray, three different functionalized glass slides were selected, which were modified with 3-aminopropyltriethoxysilane (APTES), poly-l-lysine and glutaraldehyde, respectively. In order to obtain satisfied signal intensity, the ultraviolet crosslinking energy and the immobilized DNA concentration were optimized. By using the above SNP microarray, MTHFR C677T and A1298C SNP loci of 157 controls and 127 patients with hematological malignancy[30 multiple myeloma(MM),28 non-Hodgkin’s lymphoma(NHL), 22 acute lymphoblastic leukemia(ALL),40 acute myeloid leukemia(AML),7 chronic myeloid leukemia (CML)] from Jiangsu province were detected.
Results Based on the immobilization efficiency, APTES modified glass slides were selected to fabricate DNA microarrays. The optimal ultraviolet crosslinking energy was 400 mJ and the optimal concentration of immobilized DNA was 300 mg/ml. Homozygous wild type, heterozygote type and homozygous mutant type yielded green, yellow and red fluorescence, respectively. Sequencing validated the results.The allele frequencies of MTHFR677C, 677T were 58.7%, 41.3% in patients and 66.9%, 33.1% in controls, respectively; the frequencies of 677CC, 677CT, 677TT genotype were 37.8%, 41.7%, 20.5% in patiens and 45.9%, 42.0%, 12.1% in controls, respectively. The allele frequencies of MTHFR1298A, 1298C were 83.1%, 16.9% in patients and 84.1%, 15.9% in controls, respectively; the frequencies of 1298AA, 1298AC, 1298CC genotype were 70.9%, 24.4%, 4.7% in patiens and 69.4%, 29.3%, 1.3% in controls, respectively. In multivariate analysis, significant difference was observed in the allele frequencies of C677T(χ2=4.08, P=.043) and in the genotype frequency of 677TT(OR 2.09, 95%CI 1.04–4.20, P=.039). The frequencies of 677TT (33.3%) and 1298CC(6.7%) in MM group were found significantly higher than in control group(OR 8.92, 95%CI 1.06–75.24, P=.006; OR=4.80, 95%CI 1.56–14.73, P=.044), resectively. No associations were found between any polymorphisms and susceptibilities to ALL, AML and NHL.
Conclusions This microarray-based method is accurate, high-throughput and cheap, suitable for SNP genotyping in a large of individuals. MTHFRC677T polymorphisms influence the risk of hematological malignancy in Chinese population. Both MTHFR677TT and MTHFR1298CC genotype are susceptive to MM.
AF1q Is a Novel Poor Prognostic Marker for Adult Acute Myelogenous Leukemia (AML) Patients with Normal Cytogenetics.
Authors: Strunk Crawford J. , Platzbecker Uwe , Thiede Christian , Schaich Markus Andreas , Illmer Thomas , Kang Zizhen , Leahy Patrick , Li Chunbiao , Reiner Sunee , Laughlin Mary J. , Lazarus Hillard M. , Bunting Kevin D. , Gerson Stanton L. , Ehninger Gerhard , Tse William W. 
Published: April 2020

Abstract
Adult AML with normal cytogenetics accounts for 45–55% of de novo AML patients, who usually are categorized as intermediate-risk but have wide 5-yr overall survival (OS) rates between 24–42%. The optimal post-remission treatment strategies, especially hematopoietic stem cell transplantation (HSCT), are yet to be defined. Recent molecular studies suggested that clinical outcome may be predicted by presence or absence of mutation or changes in expression of specific genes such as FMS-like tyrosine 3 (FLT3). The AF1q gene, an MLL fusion partner, originally was identified in an AML patient’s leukemic cells with t(1; 11) (q21; q23) abnormalities. We previously found that high AF1q expression is an independent poor-prognosticator for OS and disease-free survival (DFS) in pediatric AML patients. Data indicated that allogeneic HSCT in AML patients with high AF1q expression might improve survival. To test this hypothesis in adults, we studied leukemia marrow AF1q expression by real-time polymerase chain reaction (Q-PCR) in 290 normal cytogenetic adult AML patients treated within a trial of the German AML Study Initiative Leukemia (DSIL). Patients were induced with two cytarabine-based cycles followed by consolidation therapy. Forty-eight patients received allogeneic and 58 patients received autologous HSCT after initial induction. Over 95% of AML patients had elevated AF1q expression compared to control with highest up to 409-fold. Quartile analysis of the study population showed that the highest AF1q expression trended toward worse OS (p=0.09) and DFS (p=0.1) compared to low AF1q expression despite similar complete remission rates after induction (low 66% vs. high 69%). High AF1q expression was significantly associated with the presence of an FLT3 ITD mutation (p=0.013). Patients with high AF1q expression given allogeneic HSCT had a similar OS and DFS compared to pts with low AF1q expression (62% & 56% vs 64% & 62% at 3 yr, respectively). In fact, autologous HSCT showed an inferior OS (median 25 mo vs. median not reached, p=0.04) and DFS (p=0.09) in the high AF1q group compared to the low expression group. These data suggest that high AF1q expression may be a useful molecular prognostic marker for poor prognosis adult AML patients with normal cytogenetics and allogeneic HSCT may improve OS. Further studies are focusing on whether AF1q is interactive with FLT3 in leukemogenesis as well as serving as a simultaneous therapeutic target for AML with FLT3 mutation.
Distinct Characteristics of t(8;21) Acute Myeloid Leukemia with Active KIT Mutation.
Authors: Lorenzo Felipe , Nishii Kazuhiro , Monma Fumihiko , Katayama Naoyuki , Yagasaki Fumiharu , Miwa Hiroshi , Miyawaki Shuich , Shiku Hiroshi 
Published: April 2020

Abstract
The t(8;21) translocation, which generates a chimeric gene, AML1/ETO, is one of the most common chromosomal abnormalities in acute myeloid leukemia (AML) and is associated with a good clinical outcome. However, the clinical features of t(8;21)AML with additional genetic aberrations have not been well elucidated. In this study, we screened for KIT mutations in 44 patients with t(8;21)AML. While 2 cases had a silent type mutation at codon 546 in the juxtamembrane domain, Lyn546Lyn, 8 cases (17.8%) had Asp816 mutations in the tyrosine kinase domain, Asp&gt;Asn in 2 cases, Asp&gt;Tyr in 4 cases, Asp&gt;Val in 1 case and Asp&gt;His in 1 case. Patients with Asp816 mutations showed leukocytosis (P = 0.001) and poor overall survival (P &lt; 0.0001), when compared to those without Asp816 mutations. When the Asp816 mutation was transfected into a stem cell factor-dependent cell line, Mo7e, the cells grew rapidly and displayed stem cell factor-independent proliferation. These results suggest that t(8;21)AML with Asp816 mutations in KIT may be a distinctive subtype of t(8;21)AML and that this genetic aberration has an adverse effect on the clinical outcome for t(8;21)AML.
Differential Effects of HOXB4 Overexpression on Short and Long-Term Repopulating Cells in Nonhuman Primates.
Authors: Zhang Xiao-Bing , Beard Brian C. , Beebe Katherine , Humphries R. Keith , Kiem Hans-Peter 
Published: April 2020

Abstract
The inability to expand hematopoietic stem cells (HSCs) has been a significant limitation for clinical transplantation and gene therapy applications. Here we examined in a clinically relevant nonhuman primate model the ability of HOXB4 to expand HSCs and thus potentially overcome this limitation. Using a competitive repopulation assay we directly compared engraftment of HOXB4-transduced and control-transduced CD34+ cells. In 3 animals, cells were infused after a 3-day transduction and in 2 animals after an additional 6 to 9 days ex vivo expansion. Follow-up for these animals is up to 15-months. In the 3 animals that received HOXB4GFP-transduced cells without additional ex vivo culture, gene transfer efficiencies in CD34+ cells were similar between HOXB4GFP and YFP transduced cells: 45% (range 36–55%) vs. 38% (range 36– 40%). We observed a dramatic increase in HOXB4GFP marked cells from 20–30% to 52–62% during the early engraftment period, resulting in an up to 10-fold difference in granulocyte marking between HOXB4GFP and YFP marked cells at 5 weeks post-transplantation. Although gene-marking levels declined over time, HOXB4 marking was still about 2 to 3-fold higher than marking in control cells even at 15 months post-transplantation. A more pronounced effect was observed in the 2 animals that received HOXB4-overexpressing cells after an additional 6 to 9 days of ex vivo culture. Again, no difference in transduction efficiency was observed between YFP (range 34–49%) and HOXB4GFP (range 39–43%) marked cells before transplantation. However, HOXB4 marking was higher than YFP marking 1 week after transplantation, with up to a 34-fold difference in granulocyte marking at 2 weeks post-transplantation. Although the difference was decreased thereafter, a 4 to 10-fold difference was maintained in granulocyte marking after 3 months post-transplantation, suggesting a potential effect on the expansion of long-term repopulating cells. Subset analysis by flow cytometry and Taqman PCR showed HOXB4GFP and YFP marking in all subsets. Marking in CD13+ granulocytes and CD14+ monocytes was higher with HOXB4GFP-transduced cells and marking in CD3+ T cells was higher with YFP-transduced cells, suggesting that HOXB4 overexpression may have a more pronounced effect on engraftment and differentiation of myeloid than T-lymphoid precursors. LAM-PCR analysis demonstrated multiple clones of HOXB4GFP+ cells and control YFP+ cells. Our results demonstrate that HOXB4 overexpression in CD34+ cells has a very dramatic effect on expansion and engraftment of short-term repopulating cells with a less pronounced effect on long-term repopulating cells. These data should have important implications for the expansion and transplantation of HSCs, in particular for cord blood transplantations.
Isolation and Characterization of Human Antigen-Specific TCRαβ+ CD4−CD8− Double Negative Regulatory T Cells.
Authors: Fischer Karin , Voelkl Simon , Przybylski Grzegorz K. , Schmidt Christian A. , Andreesen Reinhard , Mackensen Andreas 
Published: April 2020

Abstract
Compelling evidence indicate that regulatory T (Treg) cells play an important role in the maintenance of immune tolerance to self and foreign antigens (Ag). Various subsets of T lymphocytes have been isolated in mice and humans that have the ability to down-regulate the proliferation of autoimmune effector cells. Recently, a novel subset of Ag-specific T-cell receptor (TCR)αβ+ CD4−CD8− (double negative, DN) Treg cells has been found to be able to prevent the rejection of skin and heart allografts by specifically inhibiting the function of anti-graft-specific CD8+ T cells. Here we demonstrate that peripheral DN Treg cells are present in humans, where they constitute about 1% of total CD3+ T cells, and consist of both naïve and Ag-experienced cells. Furthermore, analysis of T-cell receptor excision circles (TRECs) indicate that DN T cells are not recent thymic emigrants, but rather an expanded T-cell subset. MHC multimer staining revelaed a distinct population of DN T cells recognizing common MHC class I-restricted CMV and EBV antigens. DN T cells exhibited a strong proliferative response upon stimulation with allogeneic antigen presenting cells (APC) and secreted high amounts of IFN-γ but no IL-2, with some IL-5, and marginal levels of IL-4 and IL-10. Similar to murine DN Treg cells, human DN Treg cells are able to acquire peptide-HLA-A2 complexes from APCs by cell contact-dependent mechanisms. Furthermore, such acquired peptide-HLA complexes appear to be functionally active, in that CD8+ T cells specific for the HLA-A2-restricted self peptide, Melan-A, became sensitive to apoptosis by neighboring DN T cells after acquisition of Melan-A-HLA-A2 complexes and revealed a reduced proliferative response. These results demonstrate for the first time that a sizeable population of peripheral DN Treg cells exists in humans that are able to suppress Ag-specific T cells. DN Treg cells may serve to limit clonal expansion of allo-Ag-specific T cells after transplantation.