bluuebunny/crossref_metadata_2025_split huggingface dataset:
Alternative Splicing of a Novel Glycophorin Allele GPHe(GL) Generates Two Protein Isoforms in the Human Erythrocyte Membrane
Authors: Huang Cheng-Han , Blumenfeld Olga O. , Reid Marion E. , Chen Ying , Daniels Geoff L. , Smart Elizabeth 
Published: October 2019

AbstractThe Henshaw antigen (synonym: He or MNS6) is carried by an altered form of glycophorin B (GPB), but the molecular basis for its variable expression or quantitative polymorphism remains largely undefined. We report here the identification and analysis of a novel glycophorin He allele, GPHe(GL), which gives rise to the expression of two protein isoforms in the erythrocyte membrane. In addition to the nucleotide changes defining the epitopic sequence of He, a single C-to-G nucleotide transversion in exon V coding for the membrane domain was found to cause aberrant RNA splicings by creating a new acceptor splice site. In addition, a T-to-G transversion at −6 position of the acceptor splice site for exon IV was identified. Both full-length and truncated transcripts of GPHe(GL) were detected as the result of partial activation of the new acceptor splice site and partial inactivation of the normal splice sites. The full-length cDNA encoded He, S, and U antigens, whereas the three truncated ones lacked either the sequence for S and U antigens or a large portion of the membrane domain or both. The GPB gene on the other chromosome was apparently normal and its transcript encoded N, s, and U antigens. These results correlate alternative RNA splicing with the expression of two GPHe isoforms and thus delineate a new mechanism for the phenotypic diversity of membrane glycophorins.
Prevention of Thrombocytopenia by Thrombopoietin in Myelosuppressed Rhesus Monkeys Accompanied by Prominent Erythropoietic Stimulation and Iron Depletion
Authors: Neelis Karen J. , Qingliang Luo , Thomas G. Roger , Cohen Bob L. , Eaton Dan L. , Wagemaker Gerard 
Published: October 2019

Abstract
The effectiveness of thrombopoietin (TPO) in alleviating thrombocytopenia was evaluated in a placebo-controlled study involving rhesus monkeys exposed to 5 Gy total-body irradiation (TBI) (300-kV x-rays) to result in 3 weeks of pancytopenia. Supraoptimal treatment with human recombinant TPO (10 μg/kg/d subcutaneously, days 1 to 21 after TBI) was highly effective in preventing thrombocytopenia, with nadirs for thrombocytes, on average, far higher than 100 × 109/L, a greatly accelerated recovery to normal values, and no need for thrombocyte transfusions. TPO appeared to act selectively in that neutrophil regeneration was not influenced but red blood cell lineage recovery was prominently stimulated, with reticulocyte regeneration being initiated 10 days earlier than in placebo-treated animals. The reticulocytosis was followed by a normoblastosis that occurred earlier and was more pronounced than in placebo-treated monkeys. The effect of TPO on the red blood cell lineage was also reflected in a less profound nadir for hemoglobin (Hb) and hematocrit values than in placebo controls. However, this effect was not followed by a rapid recovery to normal values, due to development of a microcytic hypochromic anemia. Iron depletion was demonstrated by measurements of total serum iron and total iron-binding capacity (TIBC) and could be prevented by prophylactic intramuscular (IM) iron before TBI or corrected by IM iron after TPO treatment. Rechallenging with TPO in week 8 after TBI demonstrated a homogenous thrombocyte response similar in magnitude to the initial response, but a greatly diminished reticulocyte response. This demonstrated that the erythropoietic response to TPO administration depends on the hemopoietic state of the animal and may reflect multiple TPO target cells. It is postulated that the extremely rapid erythropoiesis due to TPO treatment in the initial regeneration phase following myelosuppression results in iron depletion by a mechanism similar to that seen following erythropoietin treatment in patients with end-stage renal failure. It is concluded that protracted TPO therapy to counteract thrombocytopenic states may result in iron depletion and that the iron status should be monitored before, during, and after TPO treatment.
Selective Expansion of Primitive Normal Hematopoietic Cells in Cytokine-Supplemented Cultures of Purified Cells From Patients With Chronic Myeloid Leukemia
Authors: Petzer A.L. , Eaves C.J. , Barnett M.J. , Eaves A.C. 
Published: October 2019

AbstractWe have previously reported that primitive normal hematopoietic cells detectable as long-term culture-initiating cells (Ph-LTC-IC) are present at high levels in the blood of some patients with chronic myeloid leukemia (CML). We now show that this population can be expanded several-fold when highly purified CD34+CD38− cells isolated from the blood of such patients are cultured for 10 days in a serum-free medium containing 100 ng/mL of Flt3-ligand and Steel factor and 20 ng/mL of interleukin-3 (IL-3) and IL-6, and granulocyte colony-stimulating factor. In similar cultures initiated with CD34+CD38− cells from CML blood samples in which all of the LTC-IC were leukemic (Ph+), Ph+ LTC-IC activity was rapidly lost both in the presence and absence of admixed CD34+CD38− cells isolated from normal marrow. Conversely, the ability of normal LTC-IC to expand their numbers was shown to be independent of the presence of Ph+LTC-IC and later types of Ph+colony-forming cell (CFC) progenitors. In contrast to the LTC-IC, CFC were consistently -a m p l i f i e d  i n  c u l t u r e s  i n i t i a t e d  w i t h  C M L - d e r i v e d -CD34+CD38− cells and the additional CFC present after 10 days were, like the starting population of CFC, almost exclusively Ph+ regardless of the genotype(s) of the LTC-IC in the original CML samples. Amplification of the Ph+CFC population in these cultures showed the same factor dependence as previously demonstrated for the in vitro expansion of CFC from normal marrow CD34+CD38− cells. Ph+LTC-IC disappeared regardless of the cytokines present. Taken together these findings support a model of CML in which the leukemic stem cells are characterized by a decreased probability of self-renewal and an increased probability of differentiation. In addition, they suggest new opportunities for improving the treatment of CML using strategies that require autologous stem cell rescue.
The Common Marmoset as a Target Preclinical Primate Model for Cytokine and Gene Therapy Studies
Authors: Hibino Hitoshi , Tani Kenzaburo , Ikebuchi Kenji , Wu Ming-Shiuan , Sugiyama Hajime , Nakazaki Yukoh , Tanabe Tsuyoshi , Takahashi Satoshi , Tojo Arinobu , Suzuki Shuzo , Tanioka Yoshikuni , Sugimoto Yoshikazu , Nakahata Tatsutoshi , Asano Shigetaka 
Published: October 2019

Abstract
Nonhuman primate models are useful to evaluate the safety and efficacy of new therapeutic modalities, including gene therapy, before the inititation of clinical trials in humans. With the aim of establishing safe and effective approaches to therapeutic gene transfer, we have been focusing on a small New World monkey, the common marmoset, as a target preclinical model. This animal is relatively inexpensive and easy to breed in limited space. First, we characterized marmoset blood and bone marrow progenitor cells (BMPCs) and showed that human cytokines were effective to maintain and stimulate in culture. We then examined their susceptibility to transduction by retroviral vectors. In a mixed culture system containing both marmoset stromal cells and retroviral producer cells, the transduction efficiency into BMPCs and peripheral blood progenitor cells (PBPCs) was 12% to 24%. A series of marmosets then underwent transplantation with autologous PBPCs transduced with a retroviral vector carrying the multidrug resistance 1 gene (MDR1) and were followed for the persistence of these cells in vivo. Proviral DNA was detectable by polymerase chain reaction (PCR) in peripheral blood granulocytes and lymphocytes in the recipients of gene transduced progenitors up to 400 days posttransplantation. To examine the function of the MDR1 gene in vivo, recipient maromsets were challenged with docetaxel, an MDR effluxed drug, yet the overall level of gene transfer attained in vivo (&lt;1% in peripheral blood granulocytes) was not sufficient to prevent the neutropenia induced by docetaxel treatment. Using this model, we safely and easily performed a series of in vivo studies in our small animal center. Our results show that this small nonhuman primate, the common marmoset, is a useful model for the evaluation of gene transfer methods targeting hematopoietic stem cells.
High-Level Expression of Mpl in Platelets and Megakaryocytes Is Independent of Thrombopoietin
Authors: Cohen-Solal Karine , Vitrat Natacha , Titeux Monique , Vainchenker William , Wendling Françoise 
Published: October 2019

AbstractThrombopoietin (TPO) is a hematopoietic growth factor that regulates megakaryocytopoiesis and platelet production through binding to its receptor, Mpl, encoded by the c-mpl proto-oncogene. Circulating levels of TPO are regulated by receptor-mediated uptake and degradation. To better understand this mode of TPO regulation, we examined whether expression of Mpl was regulated by its ligand. Using RNase protection analysis, we found no differences in the levels ofc-mpl transcripts in megakaryocytes (MKs) produced in vitro either in the presence or absence of TPO and in platelets (PLTs) obtained from mice hyperstimulated in vivo by ectopic secretion of TPO. Similarly, Western blot analysis of MKs produced in the presence or absence of TPO showed no difference in Mpl levels. Levels of Mpl, GpIIb, or P-selectin were virtually identical in platelet lysates obtained from normal, TPO knockout and mildly TPO-stimulated mice. In contrast, the expression of Mpl was significantly reduced in PLTs from severely thrombocythemic mice. These results show that TPO does not have a major effect on the transcription or translation of Mpl. However, they do suggest that an excess of circulating TPO can lead to the disappearance of Mpl from PLTs via catabolism.
Direct Evidence of Endothelial Injury in Acute Myocardial Infarction and Unstable Angina by Demonstration of Circulating Endothelial Cells
Authors: Mutin Murielle , Canavy Isabelle , Blann Andrew , Bory Michel , Sampol José , Dignat-George Françoise 
Published: October 2019

AbstractCirculating endothelial cells (CECs) have been detected in association with endothelial injury and therefore represent proof of serious damage to the vascular tree. Our aim was to investigate, using the technique of immunomagnetic separation, whether the pathological events in unstable angina (UA) or acute myocardial infarction (AMI) could cause desquamation of endothelial cells in circulating blood compared with effort angina (EA) and noncoronary chest pain. A high CEC count was found in AMI (median, 7.5 cells/mL; interquartile range, 4.1 to 43.5, P &lt; .01 analysis of variance [ANOVA]) and UA (4.5; 0.75 to 13.25 cells/mL, P &lt; .01) within 12 hours after chest pain as compared with controls (0; 0 to 0 cells/mL) and stable angina (0; 0 to 0 cells/mL). CEC levels in serial samples peaked at 15.5 (2.7 to 39) cells/mL 18 to 24 hours after AMI (P &lt; .05 repeated measures ANOVA), but fell steadily after UA. Regardless of acute coronary events, the isolated cells displayed morphologic and immunologic features of vascular endothelium. The CECs were predominantly of macrovascular origin. They did not express the activation markers intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, and E-selectin, although some were positive for tissue factor. CECs failed to exhibit characteristics of apoptosis (TUNEL assay) excluding this event as a possible mechanism of cell detachment. The presence of CECs provides direct evidence of endothelial injury in AMI and UA, but not in stable angina, confirming that these diseases have different etiopathogenic mechanisms.
Characterization of T-Cell Repertoire of the Bone Marrow in Immune-Mediated Aplastic Anemia: Evidence for the Involvement of Antigen-Driven T-Cell Response in Cyclosporine-Dependent Aplastic Anemia
Authors: Zeng Weihua , Nakao Shinji , Takamatsu Hideyuki , Yachie Akihiro , Takami Akiyoshi , Kondo Yukio , Sugimori Naomi , Yamazaki Hirohito , Miura Yuji , Shiobara Shintaro , Matsuda Tamotsu 
Published: October 2019

AbstractTo determine whether the antigen-driven T-cell response is involved in the pathogenesis of aplastic anemia (AA), we examined the complementarity-determining region 3 (CDR3) size distribution of T-cell receptor (TCR) β-chain (BV) subfamilies in the bone marrow (BM) of untreated AA patients. AA patients who did not respond to immunosuppressive therapy and those who obtained unmaintained remission early after cyclosporine (CyA) or antithymocyte globulin (ATG) therapy exhibited essentially a normal CDR3 size pattern. In contrast, five patients who needed continuous administration of CyA to maintain remission exhibited a skewed CDR3 size pattern in a number (&gt;40%) of BV subfamilies suggestive of clonal predominance. The skewing of CDR3 size distribution became less pronounced in one of the CyA-dependent patients when the patient achieved unmaintained remission after a 4-year therapy with CyA, whereas it persisted longer than 7 years in the other patient requiring maintenance therapy. Sequencing of BV15 cDNA for which the CDR3 size pattern exhibited apparent clonal predominance in all CyA-dependent patients showed high homology of the amino acid sequence of the CDR3 between two different patients. These findings indicate that antigen-driven expansion of T cells is involved in the pathogenesis of AA characterized by CyA-dependent recovery of hematopoiesis.
TCR Gene Rearrangements and Expression of the Pre-T Cell Receptor Complex During Human T-Cell Differentiation
Authors: Blom Bianca , Verschuren Martie C.M. , Heemskerk Mirjam H.M. , Bakker Arjen Q. , van Gastel-Mol Ellen J. , Wolvers-Tettero Ingrid L.M. , van Dongen Jacques J.M. , Spits Hergen 
Published: October 2019

AbstractRecent studies have identified several populations of progenitor cells in the human thymus. The hematopoietic precursor activity of these populations has been determined. The most primitive human thymocytes express high levels of CD34 and lack CD1a. These cells acquire CD1a and differentiate into CD4+CD8+ through CD3−CD4+CD8− and CD3−CD4+CD8+β− intermediate populations. The status of gene rearrangements in the various TCR loci, in particular of TCRδ and TCRγ, has not been analyzed in detail. In the present study we have determined the status of TCR gene rearrangements of early human postnatal thymocyte subpopulations by Southern blot analysis. Our results indicate that TCRδ rearrangements initiate in CD34+CD1a− cells preceding those in the TCRγ and TCRβ loci that commence in CD34+CD1a+ cells. Furthermore, we have examined at which cellular stage TCRβ selection occurs in humans. We analyzed expression of cytoplasmic TCRβ and cell-surface CD3 on thymocytes that lack a mature TCRβ. In addition, we overexpressed a constitutive-active mutant of p56lckF505 by retrovirus-mediated gene transfer in sequential stages of T-cell development and analyzed the effect in a fetal thymic organ culture system. Evidence is presented that TCRβ selection in humans is initiated at the transition of the CD3−CD4+CD8− into the CD4+CD8+β− stage.
CCAAT/Enhancer Binding Protein ɛ Is Critical for Effective Neutrophil-Mediated Response to Inflammatory Challenge
Authors: Lekstrom-Himes Julie , Xanthopoulos Kleanthis G. 
Published: October 2019

AbstractTargeted mutation of CCAAT/enhancer binding protein (C/EBP) ɛ in mice results in early death, primarily due to spontaneous infection with Pseudomonas aeruginosa. Functional analysis of C/EBPɛ-deficient neutrophils, in an in vivo model of peritoneal inflammation, shows multiple defects. Reduction of phagocytotic killing by C/EBPɛ-deficient neutrophils is a result of decreased uptake of opsonized bacteria as well as little to no expression of secondary granule proteins. Abnormalities in neutrophil migration detected in a chemical peritonitis model are likely secondary to abnormal CD11b integrin and L-selectin expression on C/EBPɛ-deficient neutrophils. Alterations in neutrophil cytokine expression in response to inflammation show decreased levels of interleukin-1 receptor antagonist (IL-1Ra) and increased levels of tumor necrosis factor- (TNF-) expression by C/EBPɛ-deficient neutrophils. Additionally, TNF- expression is increased in nonactivated, circulating C/EBPɛ-deficient neutrophils. Overall, C/EBPɛ-deficient neutrophils are severely functionally impaired, evoking an abnormal microenvironment, which may contribute to the loss of normal responses to inflammatory stimuli. Similarities between the C/EBPɛ-deficient mouse model and the human disease, specific granule deficiency, will be discussed.
Impairment of Plasmodium falciparum Growth in Thalassemic Red Blood Cells: Further Evidence by Using Biotin Labeling and Flow Cytometry
Authors: Pattanapanyasat Kovit , Yongvanitchit Kosol , Tongtawe Pongsri , Tachavanich Kalaya , Wanachiwanawin Wanchai , Fucharoen Suthat , Walsh Douglas S. 
Published: October 2019

AbstractCertain red blood cell (RBC) disorders, including thalassemia, have been associated with an innate protection against malaria infection. However, many in vitro correlative studies have been inconclusive. To better understand the relationship between human RBCs with thalassemia hemoglobinopathies and susceptibility to in vitro infection, we used an in vitro coculture system that involved biotin labeling and flow cytometry to study the ability of normal and variant RBC populations in supporting the growth of Plasmodium falciparum malaria parasites. Results showed that both normal and thalassemic RBCs were susceptible to P falciparum invasion, but the parasite multiplication rates were significantly reduced in the thalassemic RBC populations. The growth inhibition was especially marked in RBCs from -thalassemia patients (both -thalassemia1/-thalassemia2 and -thalassemia1 heterozygote). Our observations support the contention that thalassemia confers protection against malaria and may explain why it is more prevalent in malaria endemic areas.
Involvement of Interleukin-10 (IL-10) and Viral IL-6 in the Spontaneous Growth of Kaposi’s Sarcoma Herpesvirus-Associated Infected Primary Effusion Lymphoma Cells
Authors: Jones Karen D. , Aoki Yoshiyasu , Chang Yuan , Moore Patrick S. , Yarchoan Robert , Tosato Giovanna 
Published: October 2019

AbstractPrimary effusion lymphoma (PEL) is a distinct type of lymphoma associated with Kaposi’s sarcoma-associated herpesvirus (KSHV) infection. To determine the factors responsible for the unrestrained proliferation of PEL, we have studied the growth factor requirements of the PEL-derived BCBL-1 and BC-1 cell lines. Both cell lines were found to be autocrine growth factor dependent and to release human interleukin-6 (IL-6), viral IL-6 (vIL-6), and human IL-10 in the culture supernatant. To establish whether these cytokines contribute to autocrine growth, neutralizing antibodies against human IL-6, vIL-6, human IL-10, and soluble IL-10 receptor were used. These experiments showed that human IL-10 and, to a lesser degree, vIL-6 serve as autocrine growth factors for BCBL-1 and BC-1 cells. Thus, human IL-10 and vIL-6 are growth factors released and used by PEL cells for autonomous proliferation and may be critical to the development and progression of PEL.
Interferon gamma delays apoptosis of mature erythroid progenitor cells in the absence of erythropoietin
Authors: Choi Ilseung , Muta Koichiro , Wickrema Amittha , Krantz Sanford B. , Nishimura Junji , Nawata Hajime 
Published: October 2019

AbstractBased on the hypothesis that interferon gamma (IFN-γ) may have stimulating effects on survival of hematopoietic progenitor cells, we examined the effect of IFN-γ on apoptosis of mature erythroid colony-forming cells (ECFCs) derived from human peripheral blood obtained from normal, healthy volunteers. When the cells were cultured in the presence of IFN-γ, even without erythropoietin (EPO), the viability of the cells was maintained for at least 36 hours. When apoptosis of ECFCs was assessed by flow cytometric analysis', using annexin V, IFN-γ reduced the extent of apoptosis of the cells, as well as EPO. DNA fragmentation of ECFCs was also reduced by IFN-γ. In cells cultured with IFN-γ alone, expression of Bcl-x was detected but the level of expression decreased gradually during incubation for 36 hours, and the expression level was lower than incubation with EPO. Fas expression and activation of downstream caspases were assessed by flow cytometric analysis or fluorometric protease assay. IFN-γ induced Fas expression of the cells without the activation of caspase8 or caspase3 during 16 hours of incubation, while deprivation of EPO induced expression of Fas and the activation of both caspase8 and caspase3. We propose that IFN-γ produces a stimulating signal for the survival of mature erythroid progenitor cells by reducing apoptosis through a mechanism other than modulating Fas and one related to the expression of Bcl-x.
Constitutive Stat3, Tyr705, and Ser727 phosphorylation in acute myeloid leukemia cells caused by the autocrine secretion of interleukin-6
Authors: Schuringa Jan-Jacob , Wierenga Albertus T. J. , Kruijer Wiebe , Vellenga Edo 
Published: October 2019

AbstractTo explore the activation patterns of signal transducer and activator of transcription 3 (Stat3) in acute myeloid leukemia (AML), we examined whether the phosphorylation of tyrosine705 (Tyr705) and serine727 (Ser727) residues was abnormally regulated in cells from patients with AML. In 5 of 20 (25%) patients with AML, Stat3 was constitutively phosphorylated on Tyr705 and Ser727, which were not further up-regulated by treatment with IL-6. Furthermore, Stat3 was constitutively bound to the IRE response element in these cells as determined by electrophoretic mobility shift assay, and stimulation with IL-6 did not result in increased DNA binding. Interestingly, AML cells with constitutive Stat3 activation also secreted high levels of IL-6 protein. Treating these AML cells with anti-IL-6 resulted in restored IL-6–inducible Stat3 phosphorylation on both Tyr705 and Ser727 with low or undetectable basal phosphorylation levels in unstimulated cells. In contrast, treatment with anti-IL-1 did not result in altered Stat3 phosphorylation patterns. The constitutive IL-6 expression was associated with elevated levels of suppressor of cytokine signaling-1 (SOCS-1) and SOCS-3 mRNA expression, which were not down-regulated by anti-IL-6. These data indicate that the constitutive Stat3 activation in the investigated AML blasts is caused by high IL-6 secretion levels, thus stimulating the Jak/Stat pathway in an autocrine manner, a paracrine manner, or both.