bluuebunny/crossref_metadata_2025_split huggingface dataset:
Gamma-Retroviral and Lentiviral Vectors Have Different Preferences for Specific Classes of Genes and Specific Genomic Features in Human Hematopoietic Cells
Authors: Recchia Alessandra , Testa Anna , Cittaro Davide , Felice Barbara , Cattoglio Claudia , Luzi Lucilla , Mavilio Fulvio 
Published: April 2020

Abstract
Integration of gamma-retroviral (RV) and lentiviral (LV) vectors follows non-random patterns in mammalian genomes, with a common preference for active chromatin regions. The molecular basis of the interaction between retroviral pre-integration complexes and the human genome is poorly understood, particularly in the case of gamma-retroviruses. We have mapped a large number of RV and LV integrations in human CD34+ hematopoietic cells, transduced in vitro and analyzed without selection. Recurrent insertion sites (hot spots) account for &gt;20% of the RV integration events, while they are significantly less frequent in the case of LV vectors. RV but not LV hot spots are highly enriched in proto-oncogenes, cancer-associated CIS, and growth-controlling genes. Genes involved in hematopoietic and immune system development are targeted at high frequency and enriched in hot spots, suggesting that the CD34+ gene expression program is instrumental in directing RV integration. To obtain information about the genetic determinants of retroviral integration preferences, we investigated the role of transcriptional regulatory element within the vector as well as the chromatin-specific determinants of the target sites in directing RV and LV integration. Comparative analysis of the “integromes” of vectors carrying different transcriptional regulatory elements provide information on the role of transcriptional complexes bound to genomic and viral sequences in targeting retroviral pre-integration complexes (PICs). Analysis of the chromatin characteristics around integration sites provides information about specific structures, protein components or epigenetic modifications that may favor the binding of retroviral PICs. Genomic sequences flanking the integration sites (±1 kb) were annotated and analyzed using TRANSFAC and JASPAR matrix database and UCSC track. This analysis shows that the content and arrangement of putative transcription factor binding sites around the insertion points is different between RV and LV vectors, and between vectors carrying different LTR modifications. The frequency, coverage and position of the major class of repeats in our datasets were also analyzed. Hyper Geometric Distribution analysis revealed a significant abundance of SINE and LINE repeats around the RV vector integration sites. These data indicate that vector design and the target cell gene expression program have a significant impact in determining the integration characteristics of retroviral vectors.
Quantifying Capsid Peptide:MHC I Complexes Following Adeno-Associated Virus (AAV) Transduction
Authors: Pien Gary C. , Hasbrouck Nicole C. , Maus Marcela V. , Mingozzi Federico , High Katherine A. 
Published: April 2020

Abstract
Recent data from a clinical trial of adeno-associated virus (AAV) 2-mediated hepatic gene transfer of factor IX into hemophilia B subjects demonstrated the presence of AAV2-capsid-specific CD8+ T cells and identified an immunodominant AAV2 epitope. The expansion and contraction of this CD8+ T cell population coincided with the rise and fall of an asymptomatic elevation in liver transaminases, and correlated with the expression and subsequent loss of factor IX expression. These observations suggest that CD8+ T cells directed against AAV capsid antigen clear the transduced hepatocytes. Indeed, others in our laboratory have directly shown the lysis of AAV-transduced hepatocytes by CD8+ T cells in vitro. However, the stoichiometry and kinetics of AAV capsid degradation and processing for MHC class I presentation are presently unknown. We now report the generation of soluble AAV capsid-specific T cell receptor (TCR) tetramers and their use in quantifying capsid peptide:MHC I complexes presented on cell surfaces. CD8+ T cell clones specific for AAV2-derived peptide 74 (VPQYGYLTL) presented in the context of HLA-B*0702 were isolated and used to generate TCR cDNA. This cDNA was transfected into CHO cells to produce soluble TCR α and β chain fusion proteins. The soluble TCR was biotinylated, then tetramerized to streptavidin-conjugated fluorochrome for use in flow cytometric analyses. Cell lines expressing HLA-B*0702 were loaded with cognate AAV2 peptide, a cross-reactive AAV1 peptide (IPQYGYLTL), or irrelevant HLA-B*0702 epitopes derived from HIV-1 gp120 (IPRRIRGGL) or EBV nuclear antigen (RPPIFIRRL). Staining with TCR-tetramers reveals that this reagent binds AAV-derived peptides specifically. In addition, this reagent exhibits HLA-specificity, as cognate peptide loaded onto cells which express alternate HLA alleles was not detected. These results demonstrate that TCR-tetramers retain their antigen and MHC context specificities. Capsid peptide titration experiments using HLA-B*0702-bearing JYA2B7 and SK-MES-1 cell lines reveal that TCR-tetramers exhibit a limit of detection of approximately 105 cell surface peptide:MHC I complexes. Efficiency of detection is estimated at 1 peptide:MHC I complex for every 1010 peptide molecules added to cell culture. Preliminary studies indicate that the number of presenting complexes wanes by 48 hours following peptide loading. In contrast, AAV1-mediated transduction experiments demonstrate that capsid peptide:MHC I complexes are detectable after 48 hrs. In conclusion, these results are in agreement with our central hypothesis that AAV capsid is degraded upon transduction and gains access to MHC Class I antigen presentation pathways. Studies with the TCR-tetramers will allow definition of kinetics and capsid dose-dependence of antigen presentation, defining parameters of cell vulnerability to host immune-mediated clearance of transduced cells.
GABP Transcription Factor Is Required for Myeloid Cell Development In Vivo.
Authors: Yang Zhong-fa , Drumea Karen , Rosmarin Alan G. 
Published: April 2020

Abstract
GABP is an ets transcription factor that regulates genes that are required for innate immunity, including CD18 (β2 leukocyte integrin), lysozyme, and neutrophil elastase. GABP consists of two distinct and unrelated proteins. GABPα binds to DNA through its ets domain and recruits GABPβ, which contains the transactivation domain; together, they form a functional tetrameric transcription factor complex. We recently showed that GABP is required for entry into S phase of the cell cycle through its regulation of genes that are required for DNA synthesis and cyclin dependent kinase inhibitors (Yang, et al. Nature Cell Biol9:339, 2007). Furthermore, GABP is an essential component of a retinoic acid responsive myeloid enhanceosome (Resendes and Rosmarin Mol Cell Biol26:3060, 2006). We cloned Gabpa (the gene that encodes mouse Gabpα) from a mouse genomic BAC library and prepared a targeting vector in which the ets domain is flanked by loxP recombination sites (floxed allele). Deletion of both floxed Gabpa alleles causes an early embryonic lethal defect. In order to define the role of Gabpα in myelopoiesis, we bred floxed Gabpa mice to mice that bear the Mx1-Cre transgene, which drives expression of Cre recombinase in response to injection of the synthetic polynucleotide, poly I-C. Deletion of Gabpa dramatically reduced granulocytes and monocytes in the peripheral blood, spleen, and bone marrow, but myeloid cells recovered within weeks. In vitro colony forming assays indicated that myeloid cells in these mice were derived only from Gabpa replete myeloid precursors (that failed to delete both Gabpa alleles), suggesting strong pressure to retain Gabpα in vivo. We used a novel competitive bone marrow transplantation approach to determine if Gabp is required for myeloid cell development in vivo. Sub-lethally irradiated wild-type recipient mice bearing leukocyte marker CD45.1 received equal proportions of bone marrow from wild type CD45.1 donor mice and floxed-Mx1-Cre donor mice that bear CD45.2. Both the CD45.2 (floxed-Mx1-Cre) and CD45.1 (wild type) bone marrow engrafted well. Mice were then injected with pI-pC to induce Cre-mediated deletion of floxed Gabpa. The mature myeloid and T cell compartments were derived almost entirely from wild type CD45.1 cells. This indicates that the proliferation and/or differentiation of myeloid and T cell lineages requires Gabp. In contrast, B cell development was not impaired. We conclude that Gabpa disruption causes a striking loss of myeloid cells in vivo and corroborates prior in vitro data that GABP plays a crucial role in proliferation of myeloid progenitor cells.
Iron Absorption: Comparison of Oral Heme-Bound Iron with Inorganic Ferrous Sulfate.
Authors: Estrada Dexter T. , Schwartz Kenneth A. 
Published: April 2020

Abstract
Oral iron supplementation is an effective method for treating iron deficiency anemia. However, current preparations of inorganic iron have significant gastrointestinal adverse effects, that sometimes prevent patients from complying with their prescribed iron. Gut absorption of heme bound iron is via a different pathway than inorganic iron. Heme-bound iron may also have fewer gastrointestinal side effects as it is present in red meat and other specially prepared foods. If it can be shown that oral heme-bound iron is absorbed as readily as oral inorganic iron, then patients who cannot tolerate inorganic iron would have a less expensive alternative to parenteral iron which is costly and can cause life-threatening risk of anaphylaxis. The purpose of this study was to determine if there is a significant difference between the oral absorption of heme-bound iron, in the form of cooked bovine blood soup, and inorganic ferrous sulfate. Ten normal adult subjects were recruited for participation in this study and informed consents were obtained. All subjects had normal baseline fasting serum iron studies and ferritin. After blood for a fasting serum iron was obtained, subjects ingested a cooked bovine blood soup, containing 18 mg of elemental heme iron. Repeat iron studies were taken 2 hours after ingestion of the heme iron preparation. After a 2 week washout period, the same 10 subjects had baseline serum iron drawn followed by ingestion of 18 mg of elemental iron in the form of inorganic ferrous sulfate elixir. Serum iron was again measured 2 hours after ingestion of ferrous sulfate. The mean increase in serum iron after ingestion of bovine blood preparation was 18.5 micrograms/dL. In comparison, the mean increase in serum iron after ingestion of ferrous sulfate was 37.9 micrograms/dL. A paired T test analysis did not show any significant difference in the increase of serum iron from baseline to 2 hours between the two iron preparations (P&gt;0.1112). No adverse effects were reported by any of the subjects after ingestion of either preparation. In conclusion, heme iron ingested in the form of a cooked bovine blood soup is well tolerated, produces increases in the concentrations of serum iron that are comparable to those produced by ferrous sulfate, and supports the principle that reasonable amounts of iron can be absorbed from heme.
Prevalence and Trend of Hematopoietic Stem Cell Transplantation for Adult Aplastic Anemia/Pure Red Cell Aplasia in Korea.
Authors: Kim Hawk , Baek Jin Ho , Shin Su Jin , Min Young Joo , Park Jae-Hoo , Lee Je-Hwan , Lee Kyoo-Hyung 
Published: April 2020

Abstract
Aplastic anemia (AA) and pure red cell aplasia (PRCA) seems more frequent in Asian countries including Korea. However, there is no exact data regarding its prevalence and frequency of allogeneic hematopoietic stem cell transplantation (HSCT) in Korea. Here we present the demographic data of AA/PRCA in Korea. Data were prepared by retrieving computerized database of National Health Insurance Corporation and Korea National Statistical Office. HSCT data were collected from all HSCT centers in Korea through Korea Hematopoietic Stem Cell Transplantation Nurses’ Society. The selection criteria of PRCA and AA were D60 and D61 in ICD-10 code. Study years for crude incidence/prevalence of AA/PRCA and trend of HSCT for AA/PRCA were from 2000 to 2005, and from 1997 to 2006, respectively. All patients were 10 or more years old. In terms of prevalence, PRCA was decreasing annually from 2000 to 2005 with 0.97, 1.12, 0.72, 0.61, 0.67, and 0.47 per 100,000 persons, respectively. On the contrary, the prevalence of AA was almost same from 2000 to 2005 with 11.28, 12.93, 12.36, 11.07, 11.09, and 10.54 per 100,000 persons, respectively. Female was more affected to both AA and PRCA. The average female to male ratios in AA and PRCA were 1.38 and 1.82. The peak decades of AA were fourth and fifth decades. On the other hand, the peak decades of PRCA were second and sixth decades. The frequency of HSCT for AA/PRCA was increasing in spite of the decreasing crude incidence during a decade from 1997 to 2006, in that 48, 56, 78, 88, 69, 68, 76, 85, 86, and 92, respectively. The frequency of HSCT was similar in both genders. In spite of the possibility of over-estimation, the prevalence of AA and PRCA was very higher in Korea than in western countries.
The Comparison of the Cytobiological Role of Bone Marrow CD4+ T Cells in Aplastic Anemia Patients and Healthy Subjects.
Authors: Zheng Miao , Liu Wenli , Zhou Jianfeng , Sun Hanying , Zhang Yicheng 
Published: April 2020

Abstract
Recent studies indicate that immune-associated aplastic anemia (AA) resembles such autoimmune diseases as insulin-dependent diabetes and chronic autoimmune thyroiditis that belong to organ-specific autoimmune diseases. Many independent investigation groups have successfully isolated the pathopoiesis-associated T cell clone causing hematopoiesis failure with a CD4 phenotype from AA patients peripheral blood and bone marrow (BM). In the current study, BM CD4+T cells were isolated from both AA patients and healthy subjects with immunomagnetic beads sorting and were compared with regard to proliferation capability, apoptosis features and the impacts of their secreted cytokines on hematopoiesis stem/progenitor cells. With the improved MTT method, BM CD4+T cells of AA group presented 1.6 times more enhanced reproductive activity than those of normal group. Induced by high concentrational CD3 monoclonal antibody for 18h, evident apoptosis cells could be seen under the electron microscope in both normal group and AA group. Quantified by flow cytometry using Annexin-V FITC/PI dual staining method, apoptosis rates in the early and advanced stages of AA group were more than those of normal group (P&lt;0.01). Apoptosis rate in early stage: normal group(7.03±0.86)%, AA group(16.11±1.37)%; apoptosis rate in advanced stage: normal group(2.07±0.42)%, AA group(8.05±0.36)%. The influence of BM CD4+T cell culture supernatant on CFU-GM formation of cord blood CD34+ hematopoiesis stem/progenitor cells: the CFU-GM count in AA group was: (74.50±9.50)/104 cells; the CFU-GM count in normal group was: (124.25±19.80)/104 cells, observing under an inverted microscope. A significant difference was present in the comparison between the two groups (P&lt;0.01). CyclinD3 mRNA and protein expression levels of cord blood CD34+ cells were both down-regulated induced by BM CD4+ T cell culture supernatant of AA patients. These results indicate a strong likelihood of an abnormally proliferative and activated state of BM CD4+T cells among AA patients and its intimate correlation with AA hematopoiesis failure by secreting some soluble humoral agents that are able to inhibit CyclinD3 and consequently suppress hematopoietic stem cell in proliferation.
Ectopic Expression of HDAC9 in Murine Lymphoid System Leads to Altered Lymphocyte Numbers and Proliferation as Well as Predisposition to Tumorigenesis.
Authors: Gil Veronica S. , Howell Louise M.C. , Yeung Jenny , Petrie Kevin R. , Smith Adrian , Sebire Neil , Matutes Estela , Wotherspoon Andy , Enver Tariq , So Eric , Zelent Arthur 
Published: April 2020

Abstract
Reversible acetylation of lysine residues on histone tails is associated with changes to chromatin structure and plays a key role in regulation of gene expression. In this process, histone hypoacetylation is generally associated with gene silencing and pharmacological inhibition of histone deacetylases (HDACs) leads usually to activation of gene expression. Decreased histone acetylation is a hallmark of cancer cells and increased HDAC expression or their mistargetting to specific gene promoters has been associated with a variety of tumors. In the past we have identified and cloned class IIa HDAC9. The HDAC9 gene is located in chromosome 7p21, which is frequently amplified in B-cell tumours such as mantle cell lymphoma (MCL) and in B-cell non-Hodgkin’s lymphoma cell lines. Consistently, initial analysis of patient samples and/or publicly available microarray data highlighted high levels of HDAC9 expression in chronic lymphocytic leukemia, folicullar lymphoma and MCL. Within the normal lymphoid system, HDAC9 is co-expressed with BCL-6 in germinal center B-cells (∼60% of cells). HDAC9 is also expressed in marginal zone B cells and a fraction of CD38 or CD27 positive subepithelial tonsilar cells. In order to examine the role of HDAC9 in the lymphoid development and pathogenesis of lymphoid malignancies we used Ig heavy chain enhancer (Eμ), which drives gene expression from early stages of B-cell development, to ectopically express HDAC9 in transgenic mice. Hemizygous and homozygous mice expressing Flag epitope tagged human HDAC9 (fHDAC9) transgene display throughout their lifespan altered B-cell development. Immunophenotypic analysis of B-cells isolated from bone marrow (BM) revealed an absence of cells expressing the pre-B/immature-B cell markers normally associated with C-E Hardy’s fractions. In vitro functional clonogenic assays for IL-7 responsive BM-derived B-cell progenitors demonstrated an increase (∼50%) in colony numbers in the transgenic BM. Moreover, morphologic and flow cytometric analyses of the transgenic colonies, but not those derived from normal BM, revealed the presence of granulocyte/macrophage colony forming units expressing the HDAC9 transgene, suggesting a lympho-myeloid lineage switch. This correlates with the finding that extramedullary myelopoiesis occurs in a fraction of mice presenting splenomegaly (44%). Furthermore, a subgroup of homozygous Eμ-fHDAC9 mice (n=16) developed tumours (81%) at middle age, and present with enlarged lymph nodes (6%) and abnormal hematopoietic elements in peripheral blood and BM. Taken together these data suggest that HDAC9 plays a role in B-cell maturation and its ectopic expression in early B-cells leads to perturbation of normal B-cell development, possibly predisposing transgenic mice to tumorigenesis.
Expression Pattern of TCR-zeta Chain in Patients with Aplastic Anemia and Polycythemia Vera.
Authors: Yang Lijian , Li Yangqiu , Li Chuang , Chen Shaohua , Chen Si , Lin Chen 
Published: April 2020

Abstract
TCR zeta chain of TCR-CD3 receptor complex is though to be critical for T cells development and T cell effector function. The absence of TCR zeta chain not only influences the TCR expression on the cell membrane, but also impairs the proliferative response and the level of activation of mature T cells. Recently, the abnormal expression of TCR zeta chain was found different disease, for example in hematological malignancy. In the present study, we analyzed the expression level of TCR zeta chain gene in T cells from patients with aplastic anemia (AA) and polycythemia vera (PV), thereby to estimate the feature of T cells activation status. Real-time PCR with SYBR Green I technique was used for detecting the expression level of TCR zeta chain gene in peripheral blood mononuclear cells of patients with AA (25 cases),PV (12 cases), and 30 normal individuals. β2-microglobulin gene was used as an endogeneous reference. Evaluation of TCR zeta chain gene expression level were used the 2−ΔΔCt method. Compared with the normal control, TCR zeta chain gene expression level could be divided into two groups: 10 patients with AA (40%) and 5 patients with PV (41.7%) were over expression, while 15 patients with AA (60%) and 7 patients with PV (58.3%) were down expression. The expression level of TCR zeta gene is nonsignificat age-associated in both AA and PV patients. Moreover, the expression level of TCR zeta gene is nonsignificant age-associated or gender-link in T cells from patients with AA. The signaling pathway of T cells activation might display some disorder in patients with AA or PV. The effect of the change of TCR zeta chain gene expression pattern in AA and PV patients should be further confirmed.
A Retrospective Chart Review of the Hematologic Consequences of Roux-en-y Gastric Bypass Surgery.
Authors: Costantino Christina L. , Kruse Susan , Henry David H. 
Published: April 2020

Abstract
Roux-en-y gastric (RYGB) bypass surgery has become increasingly common as a procedure to control overweight/obesity. Due to the resulting bypass of significant amounts of gastric and duodenal mucosa, patients may develop anemia. We reviewed charts of 28 patients who had undergone RYGB surgery in the past 5 years who were referred to us for evaluation. Eighteen of these patients had no hematologic consequences as a result of the surgery. Five patients were anemic but were excluded because they had other causes for anemia than the bypass surgery: myeloma (1), pregnancy (1), breast cancer (1), and heavy menstruation (2). The remaining 5 patients however revealed hematologic abnormalities an average of approximately 23 months following RYGB surgery. In our retrospective chart review, we found anemia most commonly caused by iron deficiency although 1 patient had B12 deficiency, and we suggest periodic follow up of patients months to years after such surgery to screen for anemia from potential malabsorption of iron or B12 from this surgical alteration of gastrointestinal anatomy.
Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 Months since RYGB & first heme evaluation 24 months 28 months 39 months 11 months 11 months Pre/Post Menopausal pre pre pre pre pre Hemoglobin 8 12.9 9.8 6.7 9.9 B12 165 501 233 340 660 Ferritin 14 9 7 &lt;1 5 Folate 20.8 &gt;24.0 16.3 16.4 &gt;24.0 Creatinine 0.6 0.9 0.9 1.0 0.8 WBC 7.7 8.5 9 6.9 5.6 Platelets 364 300 473 378 462 Reticulocyte Count 1.00% 1.30% 1.20% 1.30% 1.30%
Succesful Treatment with Sirolimus in a Refractory Case of Pure Red Cell Aplasia (PRCA).
Authors: Ovilla Roberto 
Published: April 2020

Abstract
A patient with severe anemia since her birth was established a diagnosis of PRCA. Bone marrow aspirate showed an absence of red cell lineage, with lymphocyte hyperplasia. She was initiated with high dose of steroids without response. Two cycles of antithymocyte globulin (ATG) was administered, the first ATG 30mg/kg/5d with short response mainly evidenced by reticulocyte count, the second was established as ATG 15mg/kg/5d plus cyclosphosphamide 120mg/kg with a very transitory response. In November 2002 a Cord Blood Transplant was done, with a conditioning regimen based in Busulphan plus Cyclophosphamide and a prophylaxis for GVHD based on Cyclosporine plus Methylprednisolone. After a very rapid hematopoyesis recovery was discharged, but with no evidence of engraftment and autologous hematopoietic recovery, she received red cell transfusions monthly for about 36 months until December 2004 with desferroxamine as chelating drug. In December 2004 she started with Sirolimus 1mg a day orally with a very impressive response, red cells, hemoglobin levels, hematocrit, reticulocyte count improvement until normal levels. Red Cell transfusions were discontinued and two years later Sirolimus was also discontinued. She is in a very good Health Condition without any treatment in the last 8 months. We report this case in order to inform this successful treatment and in the order to use the same therapy in other cases.