bluuebunny/crossref_metadata_2025_split huggingface dataset:
FLT-3 Ligand and Marrow Stroma-Derived Factors Promote CD3γ, CD3δ, CD3ζ, and RAG-2 Gene Expression in Primary Human CD34+LIN−DR− Marrow Progenitors
Authors: Gaffney Patrick M. , Lund Jeanne , Miller Jeffrey S. 
Published: October 2019

AbstractWe hypothesize that early lymphoid commitment from primitive hematopoietic marrow progenitors is governed by signals from the marrow microenvironment leading to sequential induction of lineage-specific genes. Using expression of lymphoid genes as markers of differentiation, we characterize a highly purified population (>99.8% by double sorting) of primary human CD34+Lin−DR− progenitors. This population was then used to evaluate the effects of supplemental cytokines (interleukin-2 [IL-2], IL-3, IL-7, c-kit ligand), FLT-3 ligand (FL), and stroma-derived factors on lymphoid differentiation in vitro. CD3, RAG-1, Ikaros, CD10, and TdT transcripts were detected in the starting CD34+Lin−DR− population. By contrast, CD3γ, CD3δ, CD3ζ, and RAG-2 transcripts were not present in any samples tested. The presence of supplemental cytokines alone at culture initiation permitted stimulation of the expression of CD3ζ, but not of CD3γ or CD3δ. However, when FL and stroma-derived factors were added to cytokines, CD3 gene expression was induced in all samples. The predominant CD3 transcripts induced by optimal culture conditions were alternatively spliced isoforms lacking transmembrane sequences (CD3δ and CD3γ) and portions of the intracellular and extracellular domains (CD3γ). The combination of cytokines, FL, and stromal factors also provided a potent stimulus for RAG-2 gene expression. These findings show that FL in combination with stroma-derived factors provide important signals to promote early events required for lymphoid differentiation.
Jumping Translocations of Chromosome 1q in Multiple Myeloma: Evidence for a Mechanism Involving Decondensation of Pericentromeric Heterochromatin
Authors: Sawyer Jeffrey R. , Tricot Guido , Mattox Sandy , Jagannath Sundar , Barlogie Bart 
Published: October 2019

AbstractKaryotypes in multiple myeloma (MM) are complex and exhibit numerous structural and numerical aberrations. The largest subset of structural chromosome anomalies in clinical specimens and cell lines involves aberrations of chromosome 1. Unbalanced translocations and duplications involving all or part of the whole long arm of chromosome 1 presumably occur as secondary aberrations and are associated with tumor progression and advanced disease. Unfortunately, cytogenetic evidence is scarce as to how these unstable whole-arm rearrangements may take place. We report nonrandom, unbalanced whole-arm translocations of 1q in the cytogenetic evolution of patients with aggressive MM. Whole-arm or “jumping translocations” of 1q were found in 36 of 158 successive patients with abnormal karyotypes. Recurring whole-arm translocations of 1q involved chromosomes 5,8,12,14,15,16,17,19,21, and 22. A newly delineated breakpoint present in three patients involved a whole-arm translocation of 1q to band 5q15. Three recurrent translocations of 1q10 to the short arms of different acrocentric chromosomes have also been identified, including three patients with der(15)t(1;15)(q10;p10) and two patients each with der(21)t(1;21)(q10;p13) and der(22)t(1;22) (q10;p10). Whole-arm translocations of 1q10 to telomeric regions of nonacrocentric chromosomes included der(12)t(1;12) (q10;q24.3) and der(19)t(1;19)(q10;q13.4) in three and two patients, respectively. Recurrent whole-arm translocations of 1q to centromeric regions included der(16)t(1;16)(q10;q10) and der(19)t(1;19)(q10;p10). The mechanisms involved in the 1q instability in MM may be associated with highly decondensed pericentromeric heterochromatin, which may permit recombination and formation of unstable translocations of chromosome 1q. The clonal evolution of cells with extra copies of 1q suggests that this aberration directly or indirectly provides a proliferative advantage.
In Vitro Incorporation of GPI-Anchored Proteins Into Human Erythrocytes and Their Fate in the Membrane
Authors: Civenni Gianluca , Test Samuel T. , Brodbeck Urs , Bütikofer Peter 
Published: October 2019

AbstractIn many different cells, glycosylphosphatidylinositol (GPI)-anchored molecules are clustered in membrane microdomains that resist extraction by detergents at 4°C. In this report, we identified the presence of such domains in human erythrocytes and examined the ability of exogenously-added GPI-anchored molecules to colocalize with the endogenous GPI-anchored proteins in these detergent-insoluble complexes. We found that the addition to human erythrocytes of three purified GPI-anchored proteins having different GPI lipid moieties resulted in their efficient and correct incorporation into the membrane. The extent of membrane insertion was dependent on the intactness of the GPI lipid moiety. However, unlike the endogenous GPI-anchored proteins, the in vitro incorporated GPI molecules were not resistant to membrane extraction by Triton X-100 at 4°C. In addition, in contrast to the endogenous GPI-anchored proteins, they were not preferentially released from erythrocytes during vesiculation induced by calcium loading of the cells. These results suggest that in vitro incorporated GPI-linked molecules are excluded from pre-existing GPI-enriched membrane areas in human erythrocytes and that these microdomains may represent the sites of membrane vesicle formation.
Purging of Mammary Carcinoma Cells During Ex Vivo Culture of CD34+ Hematopoietic Progenitor Cells With Recombinant Immunotoxins
Authors: Spyridonidis A. , Schmidt M. , Bernhardt W. , Papadimitriou A. , Azemar M. , Wels W. , Groner B. , Henschler R. 
Published: October 2019

AbstractTumor cells have been found in autologous hematopoietic cell transplants used after high-dose chemotherapy. To specifically eliminate contaminating mammary tumor cells during ex vivo expansion of CD34+ hematopoietic progenitor cells, we used recombinant immunotoxins (ITs) directed against cell-surface antigens expressed on mammary carcinoma cells. ITs were expressed from fusion cDNAs combining a single-chain antibody fragment (scFv) directed against the Erb-B2 or epidermal growth factor (EGF) receptors with a truncatedPseudomonas exotoxin A fragment devoid of its cell-binding domain. CD34+ hematopoietic progenitor cells did not express Erb-B2 and EGF receptors as detected by Western blotting. Ex vivo expansion of total hematopoietic cells or of colony-forming cells from CD34+ progenitors in the presence of stem-cell factor (SCF), interleukin-1 (IL-1), IL-3, IL-6, and erythropoietin (Epo) was not affected when ITs were added to the cultures. In contrast, MDA-MB 453 and MCF-7 mammary carcinoma cells were depleted in a dose- and time-dependent manner by more than 3 log in coculture with CD34+ cells over a period of 7 days in the presence of 100 to 1,000 ng/mL of anti–Erb-B2 IT. This included elimination of the subpopulations with regrowth potential. Similarly, addition of either anti–Erb-B2 or anti-EGF receptor ITs to primary breast cancer cells isolated from patients with metastatic disease resulted in elimination of cytokeratin-positive cells in seven of seven samples. ITs are highly efficient and convenient to use for the depletion of mammary tumor cells during ex vivo expansion of hematopoietic progenitor-cell autografts.
Evaluation by Multivariate Analysis of the Differentiation Inhibitory Factor nm23 as a Prognostic Factor in Acute Myelogenous Leukemia and Application to Other Hematologic Malignancies
Authors: Yokoyama Akihiro , Okabe-Kado Junko , Wakimoto Naoki , Kobayashi Hirofumi , Sakashita Akiko , Maseki Nobuo , Nakamaki Tsuyoshi , Hino Ken-ichiro , Tomoyasu Shigeru , Tsuruoka Nobuyoshi , Motoyoshi Kazuo , Nagata Naokazu , Honma Yoshio 
Published: October 2019

AbstractThe differentiation inhibitory factor nm23 can inhibit the differentiation of murine and human myeloid leukemia cells. We recently reported that nm23 genes were overexpressed in acute myelogenous leukemia (AML), and a higher level of nm23-H1expression was correlated with a poor prognosis in AML, especially in AML-M5 (acute monocytic leukemia). To evaluate the importance ofnm23 expression as a prognostic factor in AML, we compared it with other putative prognostic factors in AML. An analysis of the correlation between nm23 expression and the clinical parameters of 110 patients with AML demonstrated that increased nm23-H1mRNA levels were associated with resistance to initial chemotherapy and with reduced overall survival. Multivariate analysis using Cox's proportional hazard model also showed that elevated nm23-H1mRNA levels significantly contributed to the prognosis of patients with AML. Especially in AML-M5, nm23-H1 status was the most important prognostic factor. Furthermore, to determine whether we can apply the results observed in AML to other hematologic malignancies, we investigated the relative levels of nm23-H1 and nm23-H2transcripts in 149 patients with hematologic neoplasms, including 110 with de novo AML, 9 with de novo acute lymphoblastic leukemia, 14 with myelodysplastic syndrome, 16 with chronic myelogenous leukemia (CML), and 5 normal subjects by the reverse transcriptase-polymerase chain reaction. Expression of nm23-H1 was significantly higher in all the hematologic neoplasms, except CML in chronic phase, than in normal blood cells. nm23 may have a prognostic effect in these hematologic malignancies as well as in AML.
Characterization and Use of an Antibody Detecting the CBFβ-SMMHC Fusion Protein in inv(16)/t(16;16)-Associated Acute Myeloid Leukemias
Authors: Viswanatha David S. , Chen I.-Ming , Liu Pu Paul , Slovak Marilyn L. , Rankin Cathy , Head David R. , Willman Cheryl L. 
Published: October 2019

AbstractThe inv(16)(p13q22) and t(16;16)(p13;q22) cytogenetic abnormalities occur commonly in acute myeloid leukemia (AML), typically associated with French-American-British (FAB) AML-M4Eo subtype. Reverse transcriptase-polymerase chain reaction (RT-PCR) techniques have been recently developed to detect the presence of several variants of the resultant CBFB-MYH11 fusion gene that encodes a CBFβ-smooth muscle myosin heavy chain (SMMHC) fusion protein. We have now determined the clinical use of a polyclonal antibody [anti-inv(16) Ab] directed against a junctional epitope of the most common type of CBFβ-SMMHC fusion protein (type A), which is present in 90% of inv(16)/t(16;16) AML cases. Using flow cytometry, reproducible methods were developed for detection of CBFβ-SMMHC proteins in permeabilized cells; flow cytometric results were then correlated with cytogenetics and RT-PCR detection methods. In an analysis of 42 leukemia cases with various cytogenetic abnormalities and several normal controls, the anti-inv(16) Ab specifically detected all 23 cases that were cytogenetically positive for inv(16) or t(16;16), including a single AML case that was RT-PCR–negative. In addition to detecting all type A fusions, the anti-inv(16) Ab also unexpectedly identified the type C and type D CBFβ-SMMHC fusion proteins. Molecular characterization of one RT-PCR–positive and Ab-positive t(16;16) case with a non-type A product showed a novel previously unreported CBFB-MYH11 fusion (CBFB nt 455-MYH11 nt 1893). Flow cytometric results were analyzed using the Kolmogorov-Smirnov statistic D-value and the median value for positive samples was 0.65 (range, 0.35 to 0.77) versus 0.07 (range, −0.21 to 0.18) in the negative group (P < .0001). The overall concordance between cytogenetics and RT-PCR was 97%, whereas the concordance between flow cytometry and cytogenetics was 100%. Thus, using the anti-inv(16) Ab, all cytogenetically positive and RT-PCR–positive AML cases with inv(16) or t(16;16) could be rapidly identified. This study demonstrates the use of this antibody as an investigational tool in inv(16)/t(16;16) AML and suggests that the development of such reagents may have potential clinical diagnostic use.
Flt3high and Flt3low CD34+Progenitor Cells Isolated From Human Bone Marrow Are Functionally Distinct
Authors: Götze Katharina S. , Ramı́rez Manuel , Tabor Kelly , Small Donald , Matthews William , Civin Curt I. 
Published: October 2019

AbstractWe generated monoclonal antibodies against the human Flt3 receptor and used them to study the characteristics of normal human bone marrow cells resolved based on Flt3 expression. Human CD34+ or CD34+lin− marrow cells were sorted into two populations: cells expressing high levels of Flt3 receptor (Flt3high) and cells with little or no expression of Flt3 receptor (Flt3low). Flt3 receptor was detected on a subset of CD34+CD38− marrow cells, as well as on CD34+CD19+ B lymphoid progenitors and CD34+CD14+CD64+ monocytic precursors. Flt3 receptor was also present on more mature CD34−CD14+ monocytes. In colony-forming assays, Flt3high cells gave rise mainly to colony-forming unit–granulocyte-macrophage (CFU-GM) colonies, whereas Flt3low cells produced mostly burst-forming unit-erythroid colonies. There was no difference in the number of multilineage CFU-Mix colonies between the two cell fractions. Cell cycle analysis showed that a large number of the Flt3low cells were in the G0 phase of the cell cycle, whereas Flt3highcells were predominantly in G1. Cell numbers in the suspension cultures initiated with Flt3high cells were maintained in the presence of Flt3 ligand (FL) alone, and increased in response to FL plus kit ligand (KL). In contrast, cell numbers in the suspension cultures started with Flt3low cells did not increase in the presence of FL, or FL plus KL. Upregulation of Flt3 receptor on Flt3low cells was not detected during suspension culture. CD14+ monocytes were the major cell type generated from CD34+lin−Flt3high cells in liquid suspension culture, whereas cells generated from CD34+lin−Flt3low cells were mainly CD71+GlycA+ erythroid cells. These results show clear functional differences between CD34+Flt3high and CD34+Flt3low cells and may have implications concerning the in vitro expansion of human hematopoietic progenitor cells.
The Assessment of the Hematopoietic Reservoir After Immunosuppressive Therapy or Bone Marrow Transplantation in Severe Aplastic Anemia
Authors: Podestà Marina , Piaggio Giovanna , Frassoni Francesco , Pitto Anna , Zikos Panagiotis , Sessarego Mario , Abate Monica , Teresa Van Lint Maria , Berisso Giovanni , Bacigalupo Andrea 
Published: October 2019

AbstractWe investigated the hematopoietic reservoir in 43 severe aplastic anemia (SAA) patients following immunosuppression (IS) (n = 15) or bone marrow transplantation (BMT) (n = 28), at a median interval of 5 years (range, 2-20) from treatment. All patients had normal blood counts, good marrow cellularity, and normal numbers of colony forming unit-granulocyte macrophages (CFU-GM). Burst forming unit-erythroid (BFU-E) and colony forming unit-granulocyte erythroid megakaryocyte macrophages (CFU-GEMM) numbers were reduced when compared with normal controls. However, the most pronounced defect was observed at the level of long-term culture-initiating cells (LTC-IC), which significantly differed from controls (P < .00001) both for IS and BMT patients. Their number did not improve with time and was not affected by transplant or treatment-related variables. When IS patients were compared with BMT we found comparable numbers of CFU-GEMM (P = .8) and LTC-IC (P = .9), but lower numbers of BFU-E and CFU-GM (P = .05 and P = .004, respectively), suggestive of a persistent suppressive mechanism. These data indicate that LTC-IC numbers are severely reduced in BMT and IS patients, contradicting the common belief that the former are fully reconstituted as compared with the latter. In addition, the number of mature cells and committed progenitors does not seem to reflect the real size of the hematopoietic reservoir and few stem cells may be sufficient to guarantee normal hematopoiesis long term.
A Common Genetic Polymorphism (46 C to T Substitution) in the 5′-Untranslated Region of the Coagulation Factor XII Gene Is Associated With Low Translation Efficiency and Decrease in Plasma Factor XII Level
Authors: Kanaji Taisuke , Okamura Takashi , Osaki Koichi , Kuroiwa Mika , Shimoda Kazuya , Hamasaki Naotaka , Niho Yoshiyuki 
Published: October 2019

AbstractWe studied the Hga I polymorphism (46 C/T) in the 5′-untranslated region of the coagulation factor XII (FXII) gene corresponding to four bases upstream from the ATG translation initiation codon. By using allele-specific restriction analysis with restriction endonuclease Hga I, the allele frequency of 46C/T was estimated to be 0.27/0.73 in Orientals (allele number =152), and conversely, 0.8/0.2 in Caucasians (allele number =40). Because it has been reported that plasma levels of FXII were lower in Orientals than in Caucasians, we investigated the relationship between this polymorphism and plasma levels of FXII. As a result, there were significant differences in plasma FXII levels between these three allele types: C/C,170±38% (178±27%); C/T, 141±29% (123±34%); and T/T, 82±19% (61±11%) [FXII activity (FXII antigen levels)]. In heterozygotes of 46 C/T both alleles were equally transcribed in hepatocytes, as determined by reverse transcription polymerase chain reaction (RT-PCR), suggesting little influence of the polymorphism at the level of transcription or on the stability of mRNA. In in vitro transcription/translation analysis, less FXII was produced from cDNA containing 46 T than from that containing 46 C. Therefore, it is highly likely that the 46 T polymorphism in the FXII gene decreased the translation efficiency and led to low plasma levels of FXII activity and antigen, probably due to the creation of another ATG codon and/or impairment of the consensus sequence for the translation initiation scanning model.
Adenovirus-Mediated Human Immunodeficiency Virus-1 Nef Expression in Human Monocytes/Macrophages and Effect of Nef on Downmodulation of Fcγ Receptors and Expression of Monokines
Authors: De Swapan K. , Venkateshan Chettemgere N.S. , Seth Prem , Carleton Gajdusek D. , Gibbs Clarence J. 
Published: October 2019

AbstractTo characterize the effect of human immunodeficiency virus-1 (HIV-1)nef expression in human monocytes/macrophage (HMØ) and U937 on the levels of FcγRs, HLA antigens, and monokines, elutriated HMØs and U937 cells were transfected with an adenovirus-mediated Nef expression system. Nef-expressing cells downmodulated FcγRI, FcγRII, and upregulated HLA class I molecules. Nef-expressing HMØs, treated with lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate (PMA), overexpressed tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-10. However, IL-6 was induced by LPS and inhibited by PMA. Additionally, a subpopulation of Nef-expressing HMØs underwent apoptosis. Our data suggest that HIV-1 nefdownmodulated FcγRs in myeloid cells in a manner similar to that previously reported for its effect on CD4+ in T cells.
A20 Inhibits NF-κB Activation in Endothelial Cells Without Sensitizing to Tumor Necrosis Factor–Mediated Apoptosis
Authors: Ferran Christiane , Stroka Deborah M. , Badrichani Anne Z. , Cooper Jeffrey T. , Wrighton Christopher J. , Soares Miguel , Grey Shane T. , Bach Fritz H. 
Published: October 2019

AbstractExpression of the NF-κB–dependent gene A20 in endothelial cells (EC) inhibits tumor necrosis factor (TNF)–mediated apoptosis in the presence of cycloheximide and acts upstream of IκBα degradation to block activation of NF-κB. Although inhibition of NF-κB by IκBα renders cells susceptible to TNF-induced apoptosis, we show that when A20 and IκBα are coexpressed, the effect of A20 predominates in that EC are rescued from TNF-mediated apoptosis. These findings place A20 in the category of “protective” genes that are induced in response to inflammatory stimuli to protect EC from unfettered activation and from undergoing apoptosis even when NF-κB is blocked. From a therapeutic perspective, genetic engineering of EC to express an NF-κB inhibitor such as A20 offers the mean of achieving an anti-inflammatory effect without sensitizing the cells to TNF-mediated apoptosis.
Erythroid Krüppel-Like Factor Is Essential for β-Globin Gene Expression Even in Absence of Gene Competition, But Is Not Sufficient to Induce the Switch From γ-Globin to β-Globin Gene Expression
Authors: Guy Louis-Georges , Mei Qi , Perkins Andrew C. , Orkin Stuart H. , Wall Lee 
Published: October 2019

AbstractDifferent genes in the β-like globin locus are expressed at specific times during development. This is controlled, in part, by competition between the genes for activation by the locus control region. In mice, gene inactivation of the erythroid Krüppel-like factor (EKLF) transcription factor results in a lethal anemia due to a specific and substantial decrease in expression of the fetal/adult-stage–specific β-globin gene. In transgenic mice carrying the complete human β-globin locus, EKLF ablation not only impairs human β-globin–gene expression but also results in increased expression of the human γ-globin genes during the fetal/adult stages. Hence, it may appear that EKLF is a determining factor for the developmental switch from γ-globin to β-globin transcription. However, we show here that the function of EKLF for β-globin–gene expression is necessary even in absence of gene competition. Moreover, EKLF is not developmental specific and is present and functional before the switch from γ-globin to β-globin–gene expression occurs. Thus, EKLF is not the primary factor that controls the switch. We suggest that autonomous repression of γ-globin transcription that occurs during late fetal development is likely to be the initiating event that induces the switch.
Mutation Analysis of the Rearranged Immunoglobulin Heavy Chain Genes of Marginal Zone Cell Lymphomas Indicates an Origin From Different Marginal Zone B Lymphocyte Subsets
Authors: Tierens A. , Delabie J. , Pittaluga S. , Driessen A. , DeWolf-Peeters C. 
Published: October 2019

AbstractMarginal zone cell lymphoma is a recently described entity among the non-Hodgkin's lymphomas. It likely originates from the marginal zone B cells in the spleen and equivalent cells in the lymph node and extranodal tissues. Recent evidence indicates that marginal zone B cells are functionally heterogeneous and may differ with respect to the pattern of somatic hypermutation in their Ig variable genes. To test whether marginal zone lymphomas may originate from different subsets of marginal zone B cells, we performed a sequence and mutation analysis of the rearranged Ig heavy chain (IgH) variable genes (Vh) of a series of 14 cases of marginal zone lymphoma, occurring in the spleen (4), the lymph node (4), the stomach (2), the orbit (2), the tongue (1), and the skin (1). Our data show that marginal zone cell lymphomas preferentially rearrange the Vh4, Vh3, and Vh1 family genes, without preference for any particular Vh gene. Somatic mutations are present in 13 cases; one case of marginal zone cell lymphoma of the skin showed a germline configuration of the rearranged Vh gene. Mutation analysis shows evidence of antigen selection in three cases of marginal zone cell lymphoma, one of the spleen, stomach, and orbit, respectively. No evidence of antigen selection was present in the other cases. These data indicate that marginal zone cell lymphomas may arise from different subsets of marginal zone B cells. In addition, lymphomagenesis may not be triggered by antigen in all cases of marginal zone cell lymphoma.