bluuebunny/crossref_metadata_2025_split huggingface dataset:
Retinoic Acid Inhibits Monocyte to Macrophage Survival and Differentiation
Authors: Kreutz Marina , Fritsche Jana , Ackermann Ute , Krause Stefan W. , Andreesen Reinhard 
Published: October 2019

AbstractVitamin A metabolites are potent differentiation-inducing agents for myelomonocytic cell lines in vitro and are successfully used for the treatment of patients with acute promyelocytic leukemia. However, little is known about the effects of vitamin A on normal hematopoietic cells. Therefore, we investigated the effect of vitamin A on differentiation and activation of human blood monocytes (MO). Culturing MO for up to 4 days with 9-cis retinoic acid (RA) and all-trans RA but not retinol reduced MO survival, with the remaining cells being morphologically comparable to control cells. Because macrophage colony-stimulating factor (M-CSF) is a well-known survival factor for MO, we measured the M-CSF content of MO culture supernatants using enzyme-linked immunosorbent assay and found that RA suppressed the constitutive secretion of M-CSF. Northern analysis showed that the M-CSF mRNA expression was only slightly reduced by RA treatment, suggesting regulation on the posttranscriptional level. In contrast to MO, M-CSF secretion by MO-derived macrophages (MAC) was not altered by RA, suggesting a differentiation-dependent switch in the responsiveness of MO/MAC to RA. Because M-CSF is not only a survival-promoting but also a differentiation-promoting factor for myeloid cells, we analyzed the effect of RA on MO to MAC maturation. RA suppressed the expression of the maturation-associated antigen carboxypeptidase M (CPM)/MAX.1 at both the protein and mRNA levels and modulated the lipopolysaccharide-stimulated cytokine secretion of MO/MAC. The addition of exogenous M-CSF to RA-containing MO cultures fails to overcome the RA-induced inhibition of MO differentiation. However, the survival rate was improved by exogenous M-CSF. We conclude that RA acts via two different mechanisms on monocyte survival and differentiation: posttranscriptionally by controlling M-CSF secretion, which decreases MO survival, and transcriptionally regulating the expression of differentiation-associated genes. The regulation of M-CSF production may contribute to the antileukemic effect of RA in vivo by reducing autocrine M-CSF production by leukemic cells.
The Therapeutic Potential of Ribozymes
Authors: James Helen A. , Gibson Ian 
Published: October 2019

AbstractRibozymes are catalytic RNA molecules that recognize their target RNA in a highly sequence-specific manner. They can therefore be used to inhibit deleterious gene expression (by cleavage of the target mRNA) or even repair mutant cellular RNAs. Targets such as the mRNAs of oncogenes (resulting from base mutations or chromosome translocations, eg, ras or bcr-abl) and viral genomes and transcripts (human immunodeficiency virus–type 1 [HIV-1]) are ideal targets for such sequence-specific agents. The aim of this review is therefore to introduce the different classes of ribozymes, highlighting some of the chemistry of the reactions they catalyze, to address the specific inhibition of genes by ribozymes, the problems yet to be resolved, and how new developments in the field give hope to the future for ribozymes in the therapeutic field.
High Efficiency Adenovirus-Mediated Gene Transfer to Human Dendritic Cells
Authors: Dietz Allan B. , Vuk-Pavlovic Stanimir 
Published: October 2019

AbstractThe interest in the use of human dendritic cells in cancer immunotherapy calls for efficient ex vivo methods of dendritic cell education. To extend the range of methods available, we generated phenotypically characteristic dendritic cells from peripheral blood monocytes incubated with granulocyte-macrophage colony-stimulating factor and interleukin-4 and infected them with an adenovirus containing a humanized version of green fluorescent protein as a marker of gene expression. The levels of expressed protein were high, but they were further increased in combination with cationic liposomes. In comparison to transfection efficiency of the homologous expression plasmid, adenovirus-mediated gene transfer was substantially more efficient. With the aid of liposome-mediated infection, gene transfer into CD83+ dendritic cells was highly effective, resulting in more than 90% of the cells expressing the transgene.
The SH3 Domain Contributes to BCR/ABL-Dependent Leukemogenesis In Vivo: Role in Adhesion, Invasion, and Homing
Authors: Skorski Tomasz , Nieborowska-Skorska Malgorzata , Wlodarski Pawel , Wasik Mariusz , Trotta Rossana , Kanakaraj Palanisamy , Salomoni Paolo , Antonyak Mark , Martinez Robert , Majewski Miroslaw , Wong Albert , Perussia Bice , Calabretta Bruno 
Published: October 2019

AbstractTo determine the possible role of the BCR/ABL oncoprotein SH3 domain in BCR/ABL-dependent leukemogenesis, we studied the biologic properties of a BCR/ABL SH3 deletion mutant (▵SH3 BCR/ABL) constitutively expressed in murine hematopoietic cells. ▵SH3 BCR/ABL was able to activate known BCR/ABL-dependent downstream effector molecules such as RAS, PI-3kinase, MAPK, JNK, MYC, JUN, STATs, and BCL-2. Moreover, expression of ▵SH3 BCR/ABL protected 32Dcl3 murine myeloid precursor cells from apoptosis, induced their growth factor-independent proliferation, and resulted in transformation of primary bone marrow cells in vitro. Unexpectedly, leukemic growth from cells expressing ▵SH3 BCR/ABL was significantly retarded in SCID mice compared with that of cells expressing the wild-type protein. In vitro and in vivo studies to determine the adhesive and invasive properties of ▵SH3 BCR/ABL-expressing cells showed their decreased interaction to collagen IV- and laminin-coated plates and their reduced capacity to invade the stroma and to seed the bone marrow and spleen. The decreased interaction with collagen type IV and laminin was consistent with a reduced expression of α2 integrin by ▵SH3 BCR/ABL-transfected 32Dcl3 cells. Moreover, as compared with wild-type BCR/ABL, which localizes primarily in the cytoskeletal/ membrane fraction, ▵SH3 BCR/ABL was more evenly distributed between the cytoskeleton/membrane and the cytosol compartments. Together, the data indicate that the SH3 domain of BCR/ABL is dispensable for in vitro transformation of hematopoietic cells but is essential for full leukemogenic potential in vivo.
Retrovirally Transduced CD34++ Human Cord Blood Cells Generate T Cells Expressing High Levels of the Retroviral Encoded Green Fluorescent Protein Marker In Vitro
Authors: Verhasselt Bruno , De Smedt Magda , Verhelst Rita , Naessens Evelien , Plum Jean 
Published: October 2019

AbstractHuman umbilical cord blood (UCB) hematopoietic stem cells (HSC) receive increased attention as a possible target for gene-transfer in gene therapy trials. Diseases affecting the lymphoid lineage, as adenosine deaminase (ADA) deficiency and acquired immunodeficiency syndrome (AIDS) could be cured by gene therapy. However, the T-cell progenitor potential of these HSC after gene-transfer is largely unknown and was up to now not testable in vitro. We show here that highly purified CD34++ Lineage marker-negative (CD34++Lin−) UCB cells generate T, natural killer (NK), and dendritic cells in a severe combined immunodeficient mouse fetal thymus organ culture (FTOC). CD34++Lin− and CD34++CD38−Lin− UCB cells express the retroviral encoded marker gene Green Fluorescent Protein (GFP) after in vitro transduction with MFG-GFP retroviral supernatant. Transduced cells were still capable of generating T, NK, and dendritic cells in the FTOC, all expressing high levels of GFP under control of the Moloney murine leukemia virus (MoMuLV) long terminal repeat promotor. We thus present an in vitro assay for thymic T-cell development out of transduced UCB HSC, using GFP as a marker gene.
Lipopolysaccharide Activates Caspase-1 (Interleukin-1–Converting Enzyme) in Cultured Monocytic and Endothelial Cells
Authors: Schumann Ralf R. , Belka Claus , Reuter Dirk , Lamping Norbert , Kirschning Carsten J. , Weber Joerg R. , Pfeil Dagmar 
Published: October 2019

AbstractInterleukin-1β (IL-1β) is a pleiotropic proinflammatory cytokine. Mechanisms leading to its secretion include not only release of newly synthesized protein, but also cleavage of a preformed immature precursor protein into an active secretory form by the intracellular protease caspase-1 (formerly termed IL-1–converting enzyme [ICE]). Caspase-1 belongs to a rapidly growing family of cysteine proteases with substrate specificity for aspartate involved in cellular apoptosis. We have used an assay determining the caspase-1 activity based on cleavage of a fluorogenic peptide substrate to elucidate its role in lipopolysaccharide (LPS)-induced secretion of IL-1β. We show that LPS induces moderate caspase-1 activity in the monocytic cell line THP-1, in freshly isolated peripheral blood monocytes, and in human umbilical vein endothelial cells (HUVECs) in a time- and dose-dependent fashion. Caspase-1 activation by LPS was associated with cleavage of the IL-1β precursor protein that was followed by release of the mature IL-1β protein in monocytic cells. In contrast, subsequent release of IL-1β by HUVECs was not significant. LPS-induced caspase-1 activation appeared not to result from modulation of caspase-1 transcript accumulation and inhibition of caspase-1 activity was accomplished by two specific inhibitors, YVAD-CHO and YVAD-CMK, capable of alleviating the release of mature IL-1β. Taken together, these results show that LPS moderately activates caspase-1 and that caspase-1 activation contributes to LPS induction of IL-1β secretion.
Dominantly Transmitted β-Thalassemia Arising From the Production of Several Aberrant mRNA Species and One Abnormal Peptide
Authors: Faustino Paula , Osório-Almeida Leonor , Romão Luı́sa , Barbot José , Fernandes Berta , Justiça Benvindo , Lavinha João 
Published: October 2019

AbstractWe describe a dominantly inherited β-thalassemia intermedia phenotype observed in a five-generation Portuguese family. Carriers are characterized by moderate anemia, hypochromia, microcytosis, elevated hemoglobin (Hb)A2 and HbF levels, splenomegaly, hepatomegaly, and inclusion bodies in pheripheral red blood cells after splenectomy. The molecular basis of this condition is a small deletion within the 5′ consensus splicing sequence of the second intron of the β-globin gene, IVS-II-4,5 (-AG). Reticulocyte RNA studies performed by reverse transcription-polymerase chain reaction (RT-PCR) and primer extension analysis showed three abnormally processed transcripts, which, upon sequencing, were shown to correspond to (1) skipping of exon 2, and (2) activation of two cryptic splice sites (between codons 59/60, and at IVS-II-47). In vitro translation studies of these patients' reticulocyte RNA have shown that at least one of these aberrant mRNA species is translated into an abnormally elongated peptide whose cytotoxic properties could, in part, be causing the atypical dominant mode of inheritance observed in this family. We suggest that this elongated β chain is unable to combine with an α-globin chain to form a functional Hb molecule. Its degradation would, then, exhaust the proteolytic defense mechanism of the erythroid precursors, leading to inefficient proteolysis of the free α chains in excess.
Fetal Hemoglobin in Starvation Ketosis of Young Women
Authors: Peters Achim , Rohloff Dagmar , Kohlmann Thomas , Renner Florian , Jantschek Günther , Kerner Wolfgang , Lorenz Fehm Horst 
Published: October 2019

AbstractKetones can reactivate the production of fetal hemoglobin (HbF) in vitro and in vivo. A reactivation of HbF by ketones, which are generated during starvation, remains largely speculative. Therefore, we investigated HbF in 31 women with anorexia nervosa or bulimia, using both of these as models of intermittent starvation ketosis. For comparison, we also studied 42 female control subjects matched for age. β-Hydroxybutyrate levels were higher in patients than in controls (460 ± 90 v 110 ± 20 μmol/L; P < .0001). We correlated β-hydroxybutyrate, metabolic, and hematologic parameters with HbF. HbF was measured with high pressure liquid chromatography. The data were analyzed with logistic regression analysis. An elevated HbF fraction (>0.87%) was observed four times as often in patients than in controls (29%v 7%, P = .01). After adjustment for age, we found HbF elevations associated with β-hydroxybutyrate levels (P= .005). No other correlations between the various metabolic/hematologic parameters and HbF were significant. In conclusion, β-hydroxybutyrate generated in starvation is associated with increased levels of HbF. Thus, unrestrained lipolysis can produce β-hydroxybutyrate in sufficient quantities to induce a clinically measurable amount of HbF. These findings suggest that intermittent ketosis might also explain some increases of HbF in type 1 diabetes and pregnancy.
Cyclosporin A Induces Apoptosis in Childhood Acute Lymphoblastic Leukemia Cells
Authors: Ito Chikako , Ribeiro Raul C. , Behm Frederick G. , Raimondi Susana C. , Pui Ching-Hon , Campana Dario 
Published: October 2019

AbstractIn an effort to identify novel antileukemic agents that can bypass the mechanisms of multidrug resistance, we found that cyclosporin A ([CyA] 5 μmol/L) produced a median cell kill of 69% (range, 47% to 85%) in seven B-lineage acute lymphoblastic leukemia (ALL) cell lines (OP-1, SUP-B15, KOPN-55bi, RS4;11, NALM6, REH, and 380) and three T-lineage ALL cell lines (MOLT4, CCRF-CEM, and CEM-C7) after 4 days of culture. At 10 μmol/L, median CyA toxicity was 99% (range, 88% to >99%). CyA was equally toxic to both a multidrug-resistant cell line, CEM-VLB100, which overexpresses gp-170 P-glycoprotein, and one resistant to topoisomerase II inhibitors, CEM-VM1-5, which has a mutation in the topoisomerase II gene. CyA was also toxic to primary leukemic cells maintained in stroma-based culture, a system that substantially prolongs in vitro cell survival. Against lymphoblasts from 21 patients with B-lineage ALL, the compound (at 5 μmol/L) reduced the leukemic cell number by a median of 87% (range, 27% to >99%) compared with results for parallel control cultures lacking CyA. Seven of these samples were from cases with unfavorable genetic features (eg, Philadelphia-chromosome orMLL gene rearrangements); three were obtained at relapse. Against T lymphoblasts (from six patients), the median reduction in cell number was 79% (range, 30% to >99%). At 10 μmol/L, the cell kill exceeded 97% in all cases studied. The mechanism of CyA cytotoxicity was found to be the activation of apoptosis, which was suppressed by phorbol myristate acetate but not by inhibitors of ceramide-mediated apoptosis, phosphatidyl inositol-3 kinase activity, or tyrosine kinase activity. These findings demonstrate high levels of CyA-induced toxicity against ALL cells at concentrations achievable in vivo, thus providing a strong rationale for clinical testing of this agent in patients with ALL.
Expression of Granulocyte-Macrophage Colony-Stimulating Factor Receptors in Human Prostate Cancer
Authors: Rivas Coralia I. , Carlos Vera Juan , Delgado-López Fernando , Heaney Mark L. , Guaiquil Victor H. , Zhang Rong H. , Scher Howard I. , Concha Ilona I. , Nualart Francisco , Cordon-Cardo Carlos , Golde David W. 
Published: October 2019

AbstractWe studied the expression and function of the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor in the human prostate carcinoma cell line LNCaP and looked for its presence in normal and neoplastic human prostatic tissue. The GM-CSF receptor is composed of two subunits, α and β. While the isolated α subunit binds GM-CSF at low-affinity, the isolated β subunit does not bind GM-CSF by itself; but complexes with the α subunit to form a high-affinity receptor. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) showed expression of mRNAs encoding the α and β subunits of the GM-CSF receptor in LNCaP cells, and the presence of the α and β proteins was confirmed by immunolocalization with anti-α and anti-β antibodies. Receptor binding studies using radiolabeled GM-CSF showed that LNCaP cells have about 150 high-affinity sites with a kd of 40 pmol/L and approximately 750 low-affinity sites with a kd of 2 nmol/L. GM-CSF signaled, in a time- and dose-dependent manner, for protein tyrosine phosphorylation and induced the proliferation of the LNCaP cells. Immunolocalization studies showed low level expression of GM-CSF α and β subunits in normal prostate tissue, with substantial expression in benign prostatic hyperplasia and prominent expression in neoplastic prostate tissue. Maximal expression of both subunits was observed in prostatic carcinomas metastatic to lymph node and bone. Tumor cells that stained positively with anti-α subunit antibodies were also reactive with anti-β subunit antibodies, indicating that they express high-affinity GM-CSF receptors. Our data show that the LNCaP cells express functional GM-CSF receptors and that prostatic carcinomas have prominent GM-CSF receptor expression. These findings imply that both hyperplastic and neoplastic prostatic tissues may be responsive to GM-CSF.
Deficient Heme and Globin Synthesis in Embryonic Stem Cells Lacking the Erythroid-Specific δ-Aminolevulinate Synthase Gene
Authors: Harigae Hideo , Suwabe Naruyoshi , Weinstock Peter H. , Nagai Mayumi , Fujita Hiroyoshi , Yamamoto Masayuki , Sassa Shigeru 
Published: October 2019

AbstractThe erythroid-specific isoform of δ-aminolevulinate synthase (ALAS-E) catalyzes the first step of heme biosynthesis in erythroid cells, and ALAS-E gene mutations are known to be responsible for x-linked sideroblastic anemia. To study the role of ALAS-E in erythroid development, we prepared mouse embryonic stem (ES) cells carrying a disrupted ALAS-E gene and examined the effect of the lack of ALAS-E gene expression on erythroid differentiation. We found that mRNAs for erythroid transcription factors and TER119-positive cells were increased similarly both in the wild-type and mutant cells. In contrast, heme content, the number of benzidine-positive cells, adult globin protein, and mRNA for β-major globin were significantly decreased in the mutant cells. These results were confirmed using another ES differentiation system in vitro and suggest that ALAS-E expression, hence heme supply, is critical for the late stage of erythroid cell differentiation, which involves hemoglobin synthesis.
Antibodies From Patients With Heparin-Induced Thrombocytopenia/Thrombosis Recognize Different Epitopes on Heparin: Platelet Factor 4
Authors: Suh Jang-Soo , Aster Richard H. , Visentin Gian P. 
Published: October 2019

Abstract
Antibodies associated with heparin-induced thrombocytopenia/thrombosis (HITT) are now thought to be specific for complexes formed between heparin and platelet factor 4 (PF4), a basic protein found normally in platelet alpha granules. How these antibodies cause thrombocytopenia and, in some patients, thrombosis, is not fully understood, in part because purified antibodies that could be labeled and used as probes to characterize target epitopes have not been available. We developed a novel method for antibody purification involving binding to and elution from PF4 complexed to heparin immobilized by end-linkage (EL) to a solid phase. Isolated antibodies were functional and after biotinylation, reacted with heparin: PF4 complexes in the same manner as unlabeled antibodies. Using these probes, we found that antibodies from 11 patients with HITT recognized two, and probably three, distinct sites on heparin: PF4 complexes. The antibodies did not bind to PF4 complexed with heparin immobilized by multiple chemical cross-linkages, suggesting that the heparin molecule must be in a flexible, relatively unconstrained state to react with PF4 in such a way as to create sites for HITT antibody binding.
Thrombopoietin and Its Receptor, c-mpl, Are Constitutively Expressed by Mouse Liver Endothelial Cells: Evidence of Thrombopoietin as a Growth Factor for Liver Endothelial Cells
Authors: Cardier José E. , Dempsey Jack 
Published: October 2019

Abstract
Present data suggest that the primary site of thrombopoietin (TPO) mRNA is the liver. Previously, we reported that specific murine liver endothelial cells (LEC-1) located in the hepatic sinusoids support in vitro megakaryocytopoiesis from murine hematopoietic stem cells suggesting that these cells may be a source of TPO. We report here that TPO and its receptor, c-mpl, are coexpressed on cloned LEC-1. Enzyme-linked immunosorbent assay (ELISA), biological assay, and flow cytometry studies confirmed the expression of both TPO and its receptor, respectively, at the protein level. TPO activity was enhanced in supernatants from LEC-1 treated with tumor necrosis factor (TNF)-α and γ-interferon (INF). Our results show that TPO through its receptor stimulated the growth of LEC-1 in vitro. These observations establish LEC-1 as a novel source of TPO in the liver. To our knowledge, this is the first report that liver endothelial cells express both TPO and its receptor, c-mpl, and our findings indicate that this cytokine constitutes a growth factor for liver endothelial cells in vitro.
Development of Autologous, Oligoclonal, Poorly Functioning T Lymphocytes in a Patient With Autosomal Recessive Severe Combined Immunodeficiency Caused by Defects of the Jak3 Tyrosine Kinase
Authors: Brugnoni Duilio , Notarangelo Luigi D. , Sottini Alessandra , Airò Paolo , Pennacchio Marta , Mazzolari Evelina , Signorini Simona , Candotti Fabio , Villa Anna , Mella Patrizia , Vezzoni Paolo , Cattaneo Roberto , Ugazio Alberto G. , Imberti Luisa 
Published: October 2019

Abstract
Defects of the common gamma chain subunit of the cytokine receptors (γc) or of Jak3, a tyrosine kinase required for γc signal transduction, result in T−B+ severe combined immunodeficiency (SCID). However, atypical cases, characterized by progressive development of T lymphocytes, have been also reported. We describe a child with SCID caused by Jak3 gene defects, which strongly but not completely affect Jak3 protein expression and function, who developed a substantial number (>3,000/μL) of autologous CD3+CD4+ T cells. These cells showed a primed/activated phenotype (CD45R0+ Fas+HLA-DR+ CD62Llo), defective secretion of T-helper 1 and T-helper 2 cytokines, reduced proliferation to mitogens, and a high in vitro susceptibility to spontaneous (caused by downregulation of bcl-2 expression) as well as activation-induced cell death. A restricted T-cell receptor repertoire was observed, with oligoclonal expansion within each of the dominant segments. These features resemble those observed in γc-/y and in Jak3−/−mice, in which a population of activated, anergic T cells (predominantly CD4+) also develops with age. These results suggest that residual Jak3 expression and function or other Jak3-independent signals may also permit the generation of CD4+ T cells that undergo in vivo clonal expansion in humans; however, these mechanisms do not allow development of CD8+ T cells, nor do they fully restore the functional properties of CD4+ T lymphocytes.