bluuebunny/crossref_metadata_2025 huggingface dataset:
Flt3 Ligand Enhances the Yield of Primitive Cells After Ex Vivo Cultivation of CD34+ CD38dim Cells and CD34+ CD38dim CD33dim HLA-DR+ Cells
Authors: Dooley Douglas C. , Xiao Mang , Oppenlander Barbara K. , Plunkett J. Michael , Lyman Stewart D.
Published: October 2019

Flt3 ligand (FL) has been proposed as a possible modulator of early hematopoietic cell growth. The purpose of this study was to analyze the impact of FL on ex vivo expansion of hematopoietic cells obtained from adult donors. We sought to precisely identify hematopoietic populations responsive to FL and to quantitate the ability of FL to enhance the survival and/or proliferation of early hematopoietic precursors in a stroma-free culture system. Towards that end, four CD34+ subsets were isolated and their response to FL was characterized. In methylcellulose, FL significantly increased colony formation by CD34+ CD38dim cells but not CD34+ CD38+ cells. In suspension culture, the enhancement of cell expansion by FL was 10 times greater with the CD34+ CD38dim fraction than the CD34+ CD38+ fraction. FL stimulated the generation of colony-forming unit–granulocyte-macrophage (CFU-GM) from the CD34+CD38dim fraction by 14.5- ± 5.6-fold. To determine if CD34+ CD38dim cells responded uniformly to FL, the population was subdivided into a CD34+ CD38dim CD33dim HLA-DR+ (HLA-DR+) fraction and a CD34+ CD38dim CD33dim HLA-DRdim (HLA-DRdim) fraction. FL was far more effective at stimulating cell and progenitor growth from the HLA-DR+ fraction. To determine if FL enhanced or depleted the number of precommitted cells in expansion culture, CD34+ CD38dim and HLA-DR+ fractions were incubated in liquid culture and analyzed by flow cytometry. Inclusion of FL enhanced the absolute number of primitive CD34+ CD33dim cells and CD34+ HLA-DRdim cells after 5 to 12 days of cultivation. To confirm immunophenotypic data, the effect of FL on long-term culture-initiating cells (LTCIC) was determined. After 2 weeks of incubation of CD34+ CD38dim or HLA-DR+ cultures, LTCIC recoveries were significantly higher with FL in 5 of 6 trials (P < .05). For HLA-DR+ cells, LTCIC recoveries averaged 214% ± 87% of input with FL and 24% ± 16% without FL. In contrast, HLA-DRdim LTCIC could not be maintained in stroma-free culture. We conclude that less than 10% of CD34+ cells respond vigorously to FL and that those cells are contained within the HLA-DR+ fraction. FL stimulates the expansion of total cells, CD34+ cells, and CFU-GM and enhances the pool of early CD34+ CD33dim cells, CD34+ HLA-DRdim cells, and LTCIC. These data indicate that it is possible to expand hematopoietic progenitors from adult donors without losing precursors from the precommitted cell pool.
Interacting Regions in the A1 and A2 Subunits of Factor VIIIa Identified by Zero-Length Cross-Linking
Authors: O'Brien Lynn M. , Huggins Christine F. , Fay Philip J.
Published: October 2019

Factor VIIIa is a heterotrimer of A1, A2, and A3-C1-C2 subunits, the activity of which is labile due to a weak affinity interaction of the A2 subunit with the A1/A3-C1-C2 dimer. We have used the zero-length cross-linking reagent, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC), to localize regions of interaction within the A1 and A2 subunits. Reaction of factor VIIIa with EDC resulted in the formation of a cross-linked product of approximately 90 kD consisting of the A1 and A2 subunits as judged by Western blotting. Alkaline resistance of this product indicated an amide rather than ester linkage. Factor VIIIa activity decreased as the concentration of cross-linked product increased, suggesting that flexibility in the inter-subunit interaction may be required for proper cofactor function. This product was not formed in the contiguous A1-A2 domains of factor VIII, suggesting that, upon cofactor activation, a conformational change occurs that leads to the formation of a new interdomainal salt bridge(s). Reaction of the EDC-treated factor VIIIa with activated protein C (APC), which cleaves the A1 subunit at Arg336 and bisects the A2 subunit at Arg562, resulted in the formation of an approximately 30 kD product that contains the C-terminus region of A1 covalently linked to the N-terminal half of the A2. The approximately 90 kD cross-linked product was generated after reaction of A2 subunit with A1/A3-C1-C2 dimer but not with A1336/A3-C1-C2, a form of the dimer produced by APC cleavage and lacking the C-terminal acidic region of A1. A synthetic peptide corresponding to this acidic region (Met337-Arg372) was found to covalently cross-link to the isolated A2 subunit in 1:1 stoichiometry, suggesting that this region is both necessary and sufficient for the interaction of the A1 and A2 subunits. Sequence analysis of this product suggested that Glu344 in the A1 peptide may contribute to the cross-linkage. These results indicate that activation of factor VIII results in formation of a new ionic linkage(s) localized to the acidic C-terminal region of A1 and the N-terminal half of A2.
The Human Integrin β3 Gene Is 63 kb and Contains a 5′-UTR Sequence Regulating Expression
Authors: Wilhide Calvin C. , Jin Ying , Guo Qingbin , Li Lu , Li Su-Xia , Rubin Edwin , Bray Paul F.
Published: October 2019

The human blood platelet fibrinogen receptor, integrin αIIbβ3 (glycoprotein IIb-IIIa) is an archetypal member of the integrin family of adhesive molecules and is the only integrin encoded by genes physically linked in the genome. Because studies on the normal and abnormal expression of any gene require a thorough understanding of its organization, the initial goals of the current study were to determine the size and complete the genomic organization for the β3 gene. We now report the isolation of the entire β3 gene in a single P1 plasmid and for the first time have linked the first and second exons on a contiguous fragment of DNA. Using pulsed-field gel analysis, we determined the full size of the β3 gene to be 63 kb and show a large (16.7 kb) first intron; based on this information, we propose a uniform numbering system for the β3 exons. We have completed the 5′ genomic structure and generated a long-range restriction map. The promoter and the 5′ end of the first intron were found to have approximately 50% sequence identity with a region of the avian β3 gene known to possess functional transcriptional activity. Analysis of three different homologous regions led to the identification of a sequence in the 5′-UTR of the human gene, CCGCGGGAGG, which shares 90% identity with the avian gene and which bound nuclear proteins in DNaseI and electrophoretic mobility shift assay studies. Mutating this sequence caused a 2.6-fold reduction in reporter gene activity. In these studies we have (1) determined the full length and 5′ organization of the β3 gene, (2) identified a large region of homology between the 5′ regions of the avian and human genes, and (3) identified a sequence in the 5′-UTR that augments gene expression. Knowing the genomic structure of β3 has permitted the uncovering of new mechanisms of mutagenesis causing Glanzmann thrombasthenia (Jin et al, J Clin Invest 98:1745, 1996), and our findings will be valuable for such genetic analyses as well as for studies on the transcriptional regulation of β3 and other integrin genes.
Structural and Functional Basis for JAK3-Deficient Severe Combined Immunodeficiency
Authors: Candotti Fabio , Oakes Scott A. , Johnston James A. , Giliani Silvia , Schumacher Richard F. , Mella Patrizia , Fiorini Maurilia , Ugazio Alberto G. , Badolato Raffaele , Notarangelo Luigi D. , Bozzi Fabio , Macchi Paolo , Strina Dario , Vezzoni Paolo , Blaese R. Michael , O'Shea John J. , Villa Anna
Published: October 2019

Mutations of the Janus family kinase JAK3 have been found to be responsible for autosomal recessive severe combined immunodeficiency (SCID) in humans. We report here the analysis of four new unrelated patients affected by JAK3-deficient SCID. The genetic defects were heterogeneous and included a large intragenic deletion as well as different point mutations, leading to missense substitutions, early stop codons, or splicing defects. We performed a series of studies of the biochemical events induced by cytokines on lymphoblastoid B-cell lines obtained from these patients. Abnormalities in tyrosine phosphorylation of JAK3 in response to interleukin-2 (IL-2) and IL-4 were present in all patients. Accordingly, IL-2–mediated phosphorylation of STAT5 was also absent or barely detectable. On the contrary, in all cases, we could show reduced but clear phosphorylation of STAT6 upon IL-4 stimulation. In one patient carrying a single amino acid change (Glu481Gly) in the JH3 domain of JAK3, we observed partially conserved IL-2 responses resulting in reduced but detectable levels of JAK3 and STAT5 phosphorylation. Interestingly, the patient bearing this mutation developed a substantial number of circulating CD4+/CD45RO+ activated T lymphocytes that were functionally impaired. In two cases, patients' cells expressed JAK3 proteins with mutations in the JH2 pseudo-kinase domain. A single cysteine to arginine substitution (Cys759Arg) in this region resulted in high basal levels of constitutive JAK3 tyrosine phosphorylation unresponsive to either downregulation by serum starvation or cytokine-mediated upregulation. The characterization of the genetic defects and biochemical abnormalities in these JAK3-deficient patients will help define the role of JAK3 in the ontogeny of a competent immune system and may lead to a better understanding of the JAK3 functional domains.
Markedly Reduced Expression of Platelet c-mpl Receptor in Essential Thrombocythemia
Authors: Horikawa Yoko , Matsumura Itaru , Hashimoto Koji , Shiraga Masamichi , Kosugi Satoru , Tadokoro Seiji , Kato Takashi , Miyazaki Hiroshi , Tomiyama Yoshiaki , Kurata Yoshiyuki , Matsuzawa Yuji , Kanakura Yuzuru
Published: October 2019

Thrombopoietin (TPO) is implicated as a primary regulator of megakaryopoiesis and thrombopoiesis through binding to the cytokine receptor c-Mpl (the product of the c-mpl proto-oncogene). In an effort to determine the pathophysiological role of TPO-c-Mpl system in essential thrombocythemia (ET), we have examined the levels of serum TPO and the expression and function of platelet c-Mpl in 17 patients with ET. In spite of extreme thrombocytosis, serum TPO levels were slightly elevated or within normal range in most, if not all, patients with ET (mean ± SD, 1.31 ± 1.64 fmol/mL), as compared with normal subjects (0.76 ± 0.21 fmol/mL). Flow cytometric and Western blot analyses revealed that the expression of platelet c-Mpl was strikingly reduced in all patients with ET. Furthermore, the expression of platelet c-mpl mRNA was found to be significantly decreased in the ET patients tested. In contrast, almost identical levels of GPIIb/IIIa protein and mRNA were expressed in platelets from ET patients and normal controls. In addition to expression level, activation state of platelet c-Mpl was investigated in ET patients. Immunoblotting with anti-phosphotyrosine antibody showed that no aberrant protein-tyrosine phosphorylation was observed in platelets of ET patients before treatment with TPO, and the levels of TPO-induced protein-tyrosine phosphorylation, including c-Mpl-tyrosyl phosphorylation, roughly paralleled those of c-Mpl expression, suggesting that c-Mpl–mediated signaling pathway was not constitutively activated in platelets of ET patients. These results suggested that the TPO-c-Mpl system may not be directly linked to pathogenesis of ET, and that gene(s) mutated in ET may be important in regulating the levels of c-mpl gene expression in addition to the growth and differentiation of multipotential hematopoietic stem cells.
HOXC4, HOXC5, and HOXC6 Expression in Non-Hodgkin's Lymphoma: Preferential Expression of the HOXC5 Gene in Primary Cutaneous Anaplastic T-Cell and Oro-Gastrointestinal Tract Mucosa-Associated B-Cell Lymphomas
Authors: Bijl Janet J. , van Oostveen Johan W. , Walboomers Jan M.M. , Horstman Anja , J.C. van den Brule Adriaan , Willemze Rein , Meijer Chris J.L.M.
Published: October 2019

Most of the 39 members of the homeobox (HOX) gene family are believed to control blood cell development. HOXC4 and HOXC6 gene expression levels increase with differentiation of lymphoid cells. In contrast, HOXC5 is not expressed in the lymphoid lineage, but was found in lymphoid cell lines, representing the neoplastic equivalents of various differentiation stages of T and B lymphocytes. In the present study, we investigated the HOXC4, HOXC5, and HOXC6 gene expression pattern in 89 non-Hodgkin's lymphomas (NHLs) of different histologic subtypes and originating from different sites. Using RNA in situ hybridization and semiquantitative reverse transcription-polymerase chain reaction, we found expression of HOXC4 in 83 of 88 and HOXC6 in 77 of 88 NHLs and leukemias investigated. In contrast, HOXC5 expression was found in only 26 of 87 NHLs and appeared to be preferentially expressed by two specific subsets of lymphomas, ie, primary cutaneous anaplastic T-cell lymphomas (9 of 9) and extranodal marginal zone B-cell lymphomas (maltomas; 7 of 9). These results indicate that, in contrast to HOXC4 and HOXC6, HOXC5 shows a type- and site-restricted expression pattern in both T- and B-cell NHLs.
Murine Macrophage Mannose Receptor Promoter Is Regulated by the Transcription Factors PU.1 and SP1
Authors: Eichbaum Quentin , Heney David , Raveh David , Chung Michael , Davidson Mark , Epstein Judith , Ezekowitz R. Alan B.
Published: October 2019

The mannose receptor (MR) is a transmembrane protein that functions primarily as a phagocytic receptor for a wide range of microorganisms. Its expression appears to be restricted to tissue macrophages and Langerhans cells. To gain an understanding of the regulation of the gene, we have isolated the 5′ flanking sequence of the murine MR gene and have analyzed a 536-bp sequence upstream of the ATG start site for transcriptional activity. This sequence lacks a TATA box but contains an initiator (Inr) consensus element overlapping the single transcriptional start site. Transcription factor binding sites contained within this sequence include PU.1, Sp1, ETS, GATA, and MYB motifs. Serial 100-bp deletions of this promoter fragment fused to a luciferase reporter gene showed various patterns of activity when transfected into different cell types. In myeloid cells, sequence elements upstream of bp −300 appeared to have a silencing effect on promoter activity. Of the four potential PU.1 binding sites contained within the fragment, one site (at −164) bound the PU.1 factor most strongly, whereas the adjacent PU.1 site (at −177 bp) bound PU.1 to a lesser degree. Mutations of these sites decreased transcriptional activity but did not abolish it. However, promoter activity was abrogated when both the −164 bp PU.1 site and the adjacent −177 bp PU.1 site were mutated. In addition, mutation of the Sp1 site also significantly reduced promoter activity. Cotransfection studies in Drosophila Schneider cells indicated that PU.1 and Sp1 may function synergistically in transactivating the murine MR. This study indicates that MR gene expression is regulated in part by the interaction between the ubiquitously expressed factor Sp1 and the lymphoid/myeloid factor PU.1 and provides a basis for studying the regulation of this gene.
Expression of Functional TrkA Receptor Tyrosine Kinase in the HMC-1 Human Mast Cell Line and in Human Mast Cells
Authors: Tam See-Ying , Tsai Mindy , Yamaguchi Masao , Yano Koji , Butterfield Joseph H. , Galli Stephen J.
Published: October 2019

Nerve growth factor (NGF ) can influence mast cell development and function in murine rodents by interacting with its receptors on mast cells. We now report the identification of mRNA transcripts of full-length tyrosine kinase-containing trkA, trkB, and trkC neurotrophin receptor genes in HMC-1 human mast cell leukemia cells. Although HMC-1 cells lacked p75 mRNA, they expressed transcripts for the exon-lacking splice variant of trkA (trkAI), truncated trkB (trkB.T1), and truncated trkC. By flow cytometry, HMC-1 cells exhibited expression of TrkA, TrkB, and TrkC receptor proteins containing full-length tyrosine kinase domains. NGF stimulation of HMC-1 cells induced tyrosine phosphorylation of TrkA protein, increased expression of the early response genes c-fos and NGF1-A, and activation of ERK-mitogen–activated protein (MAP) kinase, results which indicate that TrkA receptors in HMC-1 cells are fully functional. Highly purified populations of human lung mast cells expressed mRNAs for trkA, trkB and trkC, whereas preparations of human umbilical cord blood-derived mast cells expressed mRNAs for trkA and trkC, but not trkB. Moreover, preparations of human umbilical cord blood-derived immature mast cells not only expressed mRNA transcript and protein for TrkA, but exhibited significantly higher numbers of chymase-positive cells after the addition of NGF to their culture medium for 3 weeks. In addition, HMC-1 cells expressed mRNAs for NGF, brain-derived neurotrophic factor (BDNF ), and neurotrophin-3 (NT-3), the cognate ligands for TrkA, TrkB, and TrkC, whereas NGF and BDNF transcripts were detectable in human umbilical cord blood mast cell preparations. Taken together, our findings show that human mast cells express a functional TrkA receptor tyrosine kinase and indicate that NGF may be able to promote certain aspects of mast cell development and/or maturation in humans. Our studies also raise the possibility that human mast cells may represent a potential source for neurotrophins.
Lineage-Specific Regulation of Hematopoiesis by HOX-B8 (HOX-2.4): Inhibition of Granulocytic Differentiation and Potentiation of Monocytic Differentiation
Authors: Krishnaraju Kandasamy , Hoffman Barbara , Liebermann Dan A.
Published: October 2019

Homeobox proteins comprise a major class of transcription factors, which have been implicated in normal hematopoiesis and leukemogenesis. Notable in this context is the homeobox gene HOX-B8 (formerly known as HOX-2.4), which was shown to cooperate with hematokines to induce leukemia, and to enhance self-renewal of immature myeloid progenitors when expressed alone. How HOX-B8 may affect lineage specific development of hematopoietic progenitor cells is unknown. Here it is shown that ectopic expression of HOX-B8 specifically inhibited dimethyl sulfoxide (DMSO)-induced granulocytic differentiation of autonomously proliferating HL-60 myeloid progenitor cells. HOX-B8 also inhibited the granulocyte colony-stimulating factor (G-CSF )–induced granulocytic developmental program of factor dependent 32Dcl3 hematopoietic progenitors, including survival, proliferation, and differentiation, as evident by rapid apoptosis of the cells following removal of interleukin-3 (IL-3) and addition of G-CSF. In sharp contrast, HOX-B8 had no effect on macrophage differentiation of M1 and HL-60 cells induced by IL-6 and phorbol-12-myristate-13-acetate, respectively. Moreover, HOX-B8 expression endowed the 32Dcl3 cells with the ability to be induced by granulocyte-macrophage colony-stimulating factor (GM-CSF ) for terminal differentiation exclusively along the macrophage lineage; this effect was at least partially mediated via expression of the zinc finger transcription factor Egr-1. Thus, ectopic expression of HOX-B8 in hematopoietic progenitor cells appears to differentially affect lineage specific development, negatively regulating granulocyte development and positively regulating macrophage development.
The β Chain of the Interleukin-3 Receptor Functionally Associates With the Erythropoietin Receptor
Authors: Jubinsky Paul T. , Krijanovski Oleg I. , Nathan David G. , Tavernier Jan , Sieff Colin A.
Published: October 2019

Interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF ) receptors share a common β chain (βc), and both cytokines enhance erythropoietin (Epo)-dependent in vitro erythropoiesis by primary hematopoietic progenitors and factor-dependent cells. These data suggest that the Epo receptor and βc may functionally interact. To determine whether such interactions can be documented, we studied a murine factor-dependent cell line (Ba/F3), which endogenously expresses IL-3R. First, Ba/F3 cells were transfected with murine EpoR, making them responsive to both IL-3 and Epo. Next, the EpoR expressing cells were transfected with murine βc. This resulted in an enhanced sensitivity of these cells to Epo, which was especially pronounced at low Epo concentrations. Ba/F3-EpoR were then treated with antisense oligodeoxynucleotides to the murine β. Control sense and nonsense had no effect on Epo-dependent growth, but the antisense markedly and specifically inhibited Epo-dependent growth. In contrast, the antisense did not affect β-globin message levels (another Epo-responsive effect in these cells) detectable by Northern blot. Finally, Western blot analysis of proteins immunoprecipitated from cells expressing both receptors with antibody against β and blotted with antibody against EpoR, or immunoprecipitated with antibody against EpoR and blotted with antibody against β, showed that EpoR and β coimmunoprecipitate. These data show that the β chain functionally and physically associates with the EpoR. This suggests that these cytokine receptors exist as a large supercomplex and offers the first molecular explanation for the synergistic effects of IL-3 and GM-CSF with Epo during erythropoiesis.
Functional Interactions Between the Thrombin Receptor and the T-Cell Antigen Receptor in Human T-Cell Lines
Authors: Joyce David E. , Chen Yan , Erger Rochelle A. , Koretzky Gary A. , Lentz Steven R.
Published: October 2019

The proteolytically activated thrombin receptor (TR) is expressed by T lymphocytes, which suggests that thrombin may modulate T-cell activation at sites of hemostatic stress. We examined the relationship between TR function and T-cell activation in the Jurkat human T-cell line and in T-cell lines with defined defects in T-cell antigen receptor (TCR) function. Stimulation with thrombin or the synthetic TR peptide SFLLRN produced intracellular Ca2+ transients in Jurkat cells. As the concentration of TR agonist was increased, peak Ca2+ mobilization increased, but influx of extracellular Ca2+ decreased. TR signaling was enhanced in a TCR-negative Jurkat line and in T-cell lines deficient in the tyrosine kinase lck or the tyrosine phosphatase CD45, both of which are essential for normal TCR function. TCR cross-linking with anti-CD3 IgM desensitized TR signaling in Jurkat cells, but not in CD45-deficient cells. A proteinase-activated receptor (PAR-2)–specific agonist peptide, SLIGKV, produced small Ca2+ transients in both MEG-01 human megakaryocytic cells and Jurkat cells, but was less potent than the TR-specific agonist TFRIFD in both cell types. Like TR signaling, PAR-2 signaling was enhanced in TCR-negative or lck-deficient Jurkat clones. These findings provide evidence for functional cross-talk between proteolytically activated receptors and the TCR.
Seroreactivity to an Envelope Protein of Human T-Cell Leukemia/Lymphoma Virus in Patients With CD3− (Natural Killer) Lymphoproliferative Disease of Granular Lymphocytes
Authors: Loughran Thomas P. , Hadlock Kenneth G. , Yang Qing , Perzova Raisa , Zambello Renato , Semenzato Gianpietro , Foung Steven K.H. , Poiesz Bernard J.
Published: October 2019

Natural killer (NK) cells are CD3− large granular lymphocytes (LGL) responsible for immunity against viral infections. A chronic lymphoproliferative disorder of NK cells has been described in which the expanded NK cells display a restricted phenotype and cytotoxic activity. These data raise the hypothesis that proliferating LGL in these patients result from discrete expansions of NK cells responding to an unknown, perhaps viral, antigen. Recently, it was found that mice transgenic for the tax gene of human T-cell leukemia/lymphoma virus (HTLV) develop NK leukemia. Therefore, we studied 15 patients with chronic NK lymphoproliferative disorder for evidence of HTLV infection. Sera were tested using an HTLV-I/II-enzyme linked immunosorbent assay and a modified Western blot assay containing recombinant env proteins. None of the sera met conventional criteria for HTLV seroreactivity. However, sera from 11 patients (73%) reacted with the recombinant HTLV env protein p21E. The anti-p21E reactivity of these sera was then mapped employing the recombinant proteins GD21 and BA21. No reactivity to the immunodominant HTLV epitope GD21 was observed, suggesting that prototypical HTLV infection is unlikely in these patients. This was confirmed by finding no evidence for HTLV nucleic acids by PCR analyses employing primers specific for conserved regions in the env, pol, and pX genes. In contrast, 10 of the 15 sera reacted with the epitope BA21, documenting for the first time an association between a unique seroreactivity and disease. The high incidence of BA21 seroreactivity in these patients suggests that exposure to a protein containing homology to BA21 may be important in the pathogenesis of this lymphoproliferative disorder.
In Vivo Removal of Malaria Parasites From Red Blood Cells Without Their Destruction in Acute Falciparum Malaria
Authors: Angus Brian J. , Chotivanich Kesinee , Udomsangpetch Rachanee , White Nicholas J.
Published: October 2019

During acute falciparum malaria infection, red blood cells (RBC) containing abundant ring-infected erythrocyte surface antigen (Pf 155 or RESA), but no intracellular parasites, are present in the circulation. These RESA-positive parasite negative RBC are not seen in parasite cultures in vitro. This indicates that in acute falciparum malaria there is active removal of intraerythrocytic parasites by a host mechanism in vivo (probably the spleen) without destruction of the parasitized RBC. This may explain the observed disparity between the drop in hematocrit and decrease in parasite count in some hyperparasitemic patients. The fate of these “once-parasitized” RBC in vivo is not known.
Presence in Human Erythrocyte Membranes of a Novel Form of Sialidase Acting Optimally at Neutral pH
Authors: Venerando Bruno , Fiorilli Amelia , Croci Gian Luigi , Tettamanti Guido
Published: October 2019

The feature of intact human erythrocytes and erythrocyte white ghosts is a unique sialidase activity with acidic optimal pH (acidic sialidase). The treatment of white ghosts with mildly alkaline isotonic solutions at 37°C, like that used to produce resealed ghosts, is accompanied by the expression, together with the acidic sialidase, of a novel sialidase with a pH optimum of 7.2 (neutral sialidase) that remained masked in the inside-out vesicles prepared from white ghosts. Exhaustive treatment of resealed ghosts with Bacillus Thuringiensis phosphatidylinositol-specific phospholipase C causes an almost complete release of the acidic sialidase, with the neutral enzyme remaining totally unaffected. The treatment of resealed ghosts with 1.2% Triton X-100 resulted in the solubilization of only the neutral sialidase, whereas 3.6% octylglucoside also solubilized the acidic sialidase. The neutral enzyme affected not only the artificial substrate but also any sialoderivatives of a ganglioside, glycoprotein, and oligosaccharide nature; the acidic enzyme did not affect sialoglycoproteins. Erythrocyte endogenous gangliosides were hydrolyzed by both sialidases, whereas the endogenous sialoglycoproteins responded to only the neutral enzyme. It was definitely proved that the acidic sialidase is located on the outer erythrocyte membrane surface, so presumably the neutral enzyme has the same location. It could be that the newly discovered neutral sialidase has a physiologic role in the releasing of sialic acid from erythrocytes during the erythrocyte aging process, leading to eventual phagocytosis by macrophages.
Expression of SCL Is Normal in Transfusion-Dependent Diamond-Blackfan Anemia But Other bHLH Proteins Are Deficient
Authors: Zhang Min-Ying , Clawson Gary A. , Olivieri Nancy F. , Bell Laurie L. , Begley C. Glenn , Miller Barbara A.
Published: October 2019

Basic helix-loop-helix proteins, which are tissue specific (SCL) or broadly expressed (E proteins), interact positively to regulate erythroid specific genes. Here, expression of SCL and two broadly expressed E proteins, E47 and HEB, was high early in erythroid differentiation and declined during maturation. Stimulation of erythroid progenitors/precursors with stem cell factor (SCF ) enhanced SCL and E protein levels, one mechanism by which SCF may increase erythroid proliferation. Interactions between SCL and E proteins are competed by Id2, which binds and sequesters E proteins. Upregulation of Id2, demonstrated here late in erythroid differentiation, may downregulate genes involved in erythroid proliferation/differentiation. We examined expression of bHLH proteins in transfusion-dependent patients with Diamond-Blackfan anemia (DBA) to determine if these interactions are disrupted. In erythroblasts from patients, expression of SCL protein and mRNA was normal and SCL increased in response to SCF. However, E47 and HEB protein levels were significantly decreased. Id2 was strongly expressed in patients. Through reduction of SCL/E protein heterodimer formation, abnormal levels of bHLH transcription factors may affect expression of erythroid specific genes, such as β globin. Stimulation of Diamond-Blackfan cells with SCF partially compensated for this defect, enhancing expression of E47, HEB, and SCL. SCF may function to increase SCL/E protein heterodimer formation, which may be one of the mechanisms through which SCF stimulates erythroid proliferation/ differentiation in DBA.
Bone Marrow Contains Virus-Specific Cytotoxic T Lymphocytes
Authors: Slifka Mark K. , Whitmire Jason K. , Ahmed Rafi
Published: October 2019

Immunizing bone marrow donors prior to bone marrow transplant (BMT) has the potential for adoptively transferring specific immunity against opportunistic pathogens. Studies have shown that long-term antibody production occurs in the bone marrow and that specific humoral immunity may be transferred from donor to recipient following BMT. However, the magnitude and duration of T-cell memory in the bone marrow compartment has not been adequately investigated. In this study, virus-specific CD8+ T-cell responses in the bone marrow were compared with those observed in the spleen of mice acutely infected with lymphocytic choriomeningitis virus (LCMV). During the acute stages of infection, most CD8+ T cells in the spleen and bone marrow showed upregulated surface expression of the activation/memory marker, LFA-1 (LFA-1hi). After clearing LCMV infection, the antiviral immune response subsided to homeostatic levels and the ratio of CD8+/LFA-1hi to CD8+/LFA-1lo T cells in the spleen and bone marrow of LCMV immune mice returned to the value observed in naive mice. Virus-specific ex vivo effector cytotoxic T-lymphocyte (CTL) responses could be identified in both spleen and bone marrow compartments at 8 days postinfection. LCMV-specific CTL precursor (CTLp) frequencies peaked in the bone marrow at 8 days postinfection and averaged one in 200 to one in 650 CD8+ T cells, a frequency similar to that observed in the spleen. After clearing the acute infection, potent LCMV-specific CTL memory responses could be demonstrated in the bone marrow for at least 325 days postinfection, indicating long-term persistence of antiviral T cells at this site. Adoptive transfer of LCMV-immune bone marrow into severe combined immunodeficiency (SCID) mice provided protection against viral challenge, whereas SCID mice that received naive bone marrow became chronically infected upon challenge with LCMV. These results indicate that after acute viral infection, virus-specific memory T cells can be found in the bone marrow compartment and are maintained for an extended period, and when adoptively transferred into an immunodeficient host, they are capable of conferring protection against chronic viral infection.
Retrovirus-Mediated Gene Transfer of NPM-ALK Causes Lymphoid Malignancy in Mice
Authors: Kuefer Martin U. , Look A. Thomas , Pulford Karen , Behm Frederick G. , Pattengale Paul K. , Mason David Y. , Morris Stephan W.
Published: October 2019

Approximately 5% to 10% of all non-Hodgkin's lymphomas contain a t(2; 5)(p23; q35) chromosomal rearrangement, which we have previously shown results in the generation of the fusion protein nucleophosmin-anaplastic lymphoma kinase (NPM-ALK). To assess the transforming potential of NPM-ALK in an animal model, we infected 5-fluorouracil–treated murine bone marrow using retroviral stocks and transplanted this infected marrow into lethally irradiated BALB/cByJ mice. Male mice were transplanted with bone marrow from female donors at 10 weeks of age, with 7 of the animals receiving marrow infected with a retroviral construct, pSRαMSVtkneo-NPM-ALK, that contains the human NPM-ALK cDNA, and 4 serving as a control group, receiving “empty” pSRαMSVtkneo-infected marrow. Whereas all mice in the control group were alive and well up to 11 months after transplantation, 4 of the 7 mice transplanted with marrow containing the NPM-ALK construct developed lymphoma within 4 to 6 months. Tumors arose in the mesenteric lymph nodes, with metastases to the lungs, kidneys, liver, spleen, and the paraspinal area. When cells from the tumors and bone marrow were transplanted into sublethally irradiated secondary recipients, 10 of these 13 mice developed tumors within 9 months. Immunoblot analysis of cell lysates using an ALK polyclonal antibody showed NPM-ALK expression in all tumors examined. Histologically, the tumors were composed of a uniform population of large immunoblastic cells with basophilic cytoplasm, centrally placed nuclei, and distinct nucleoli. Genotypic analysis showed that the tumors were B-lineage and clonal, with rearrangements of the Ig heavy- and κ light-chain loci and no rearrangements of the T-cell receptor β locus. Immunocytochemical studies confirmed the presence of IgM heavy chains and κ light chains within the tumor cells. Thus, in this retroviral gene transfer model, NPM-ALK expression in mice causes B-lineage large-cell lymphoma, suggesting a direct causative role for this activated fusion tyrosine kinase in human lymphoma.
An In Vitro Analysis of the Combination of Hemophilia A and Factor VLEIDEN
Authors: van ‘t Veer Cornelis , Golden Neal J. , Kalafatis Michael , Simioni Paolo , Bertina Rogier M. , Mann Kenneth G.
Published: October 2019

The classification of factor VIII deficiency, generally used based on plasma levels of factor VIII, consists of severe (<1% normal factor VIII activity), moderate (1% to 4% factor VIII activity), or mild (5% to 25% factor VIII activity). A recent communication described four individuals bearing identical factor VIII mutations. This resulted in a severe bleeding disorder in two patients who carried a normal factor V gene, whereas the two patients who did not display severe hemophilia were heterozygous for the factor VLEIDEN mutation, which leads to the substitution of Arg506 → Gln mutation in the factor V molecule. Based on the factor VIII level measured using factor VIII–deficient plasma, these two patients were classified as mild/moderate hemophiliacs. We studied the condition of moderate to severe hemophilia A combined with the factor VLEIDEN mutation in vitro in a reconstituted model of the tissue factor pathway to thrombin. In the model, thrombin generation was initiated by relipidated tissue factor and factor VIIa in the presence of the coagulation factors X, IX, II, V, and VIII and the inhibitors tissue factor pathway inhibitor, antithrombin-III, and protein C. At 5 pmol/L initiating factor VIIa⋅tissue factor, a 10-fold higher peak level of thrombin formation (350 nmol/L), was observed in the system in the presence of plasma levels of factor VIII compared with reactions without factor VIII. Significant increase in thrombin formation was observed at factor VIII concentrations less than 42 pmol/L (∼6% of the normal factor VIII plasma concentration). In reactions without factor VIII, in which thrombin generation was downregulated by the addition of protein C and thrombomodulin, an increase of thrombin formation was observed with the factor VLEIDEN mutation. The level of increase in thrombin generation in the hemophilia A situation was found to be dependent on the factor VLEIDEN concentration. When the factor VLEIDEN concentration was varied from 50% to 150% of the normal plasma concentration, the increase in thrombin generation ranged from threefold to sevenfold. The data suggested that the analysis of the factor V genotype should be accompanied by a quantitative analysis of the plasma factor VLEIDEN level to understand the effect of factor VLEIDEN in hemophilia A patients. The presented data support the hypothesis that the factor VLEIDEN mutation can increase thrombin formation in severe hemophilia A.
Defective Actin Reorganization and Polymerization of Wiskott-Aldrich T Cells in Response to CD3-Mediated Stimulation
Authors: Gallego Marı́a Dolores , Santamarı́a Manuel , Peña José , Molina Ignacio J.
Published: October 2019

The Wiskott-Aldrich syndrome (WAS) is a severe immunodeficiency and platelet deficiency disease arising from mutation(s) in the WASP gene, which in normal cells encodes an intracellular protein able to interact with other proteins relevant to the control of cytoskeleton organization. Immunodeficiency is mainly due to T-cell progressive malfunction. Salient defects of WAS T cells are a CD3-restricted impairment in proliferative responses and cytoskeletal abnormalities, including the frequent appearance of T cells with atypical morphology. We have investigated the possibility that the CD3-restricted defect and some of the cytoskeletal defects of WAS T cells are linked. For this purpose, we immortalized by means of infection with Herpesvirus Saimiri a number of previously described allospecific WAS T-cell lines. The resulting cells preserve the surface, molecular, and functional phenotypes of their parental lines, including a negligible WASP mRNA expression as well as the CD3-restricted defect and cytoskeleton abnormalities. Results show that, in CD3-stimulated WAS T cells, the pattern of temporal changes in cell shape and F-actin distribution is substantially different from that of control cells. Furthermore, polymerization of actin, a critical step in the CD3-mediated cytoskeleton reorganization, does not occur in WAS T-cell lines in response to OKT3 stimulation. In conclusion, our data link both CD3 and cytoskeletal defects in WAS T cells, strongly suggesting that cytoskeleton abnormalities are an underlying cause for WAS immunodeficiency.
Natural Killer (NK) Cells Are Functionally Abnormal and NK Cell Progenitors Are Diminished in Granulocyte Colony-Stimulating Factor–Mobilized Peripheral Blood Progenitor Cell Collections
Authors: Miller Jeffrey S. , Prosper Felipe , McCullar Valarie
Published: October 2019

Granulocyte colony-stimulating factor (G-CSF)–mobilized peripheral blood progenitor cell (PBPC) collections are increasingly emerging as the graft of choice in many centers for autologous transplantation, and with increasing frequency for allogeneic transplantation. However, the role of myeloid cytokines in lymphoid function, lymphoid progenitors, and immune-mediated antitumor responses is not known. We studied PBPC collections from normal donors mobilized with G-CSF (10 μg/kg). CD56+/CD3− natural killer (NK) cells sorted from PBPC products exhibited a diminished ability to kill tumor targets, were less responsive in acquiring increased cytolysis with interleukin-2 (IL-2), and proliferated less than NK cells from normal unprimed peripheral blood. This abnormality was not explained by a change in phenotype of NK cells normally circulating in the blood after G-CSF administration. We could not demonstrate any direct suppressive effect on normal unprimed NK cell proliferation or cytotoxicity by culture with pharmacologic concentrations of G-CSF. We next evaluated the effects of G-CSF on CD34+ NK cell progenitors. CD34+/CD2+, CD34+/CD7+, and CD34+/CD10+ progenitors were markedly diminished in G-CSF–mobilized PBPC products. CD34+ cells cultured in limiting dilution assays showed a sixfold decrease in NK cell progenitors when derived from G-CSF–mobilized CD34+ PBPCs compared with CD34+ cells derived from unprimed marrow. The finding of decreased NK cell function, inhibited proliferation, and diminished cloning frequency after treatment with G-CSF could be mimicked in vitro by culture of primitive marrow progenitors (CD34+, lineage-negative, HLA-DR−) on stromal layers in the presence of exogenous G-CSF. The findings presented here show that G-CSF administration to normal donors decreases NK cell function and the relative frequency of NK cell progenitors within the CD34+ progenitor population. Overcoming this diminished lymphoid capacity may be important to facilitate early posttransplant immunotherapy. Our in vitro model will be used in future studies to determine the mechanism of the G-CSF–induced suppression of NK cell progenitors, which may occur early in the differentiation process.
Abnormalities of Chromosome Band 8p11 in Leukemia: Two Clinical Syndromes Can Be Distinguished on the Basis of MOZ Involvement
Authors: Aguiar Ricardo C.T. , Chase Andrew , Coulthard Stephanie , Macdonald Donald H.C. , Carapeti Melina , Reiter Andreas , Sohal Jastinder , Lennard Anne , Goldman John M. , Cross Nicholas C.P.
Published: October 2019

Two distinct leukemia syndromes are associated with abnormalities of chromosome band 8p11. First, a myeloproliferative disorder with features characteristic of both chronic myeloid leukemia and non-Hodgkin's lymphoma and second, an acute myeloid leukemia (AML) with French-American-British (FAB) M4/5 morphology and prominent erythrophagocytosis. The two syndromes are exemplified by a t(8; 13)(p11; q12) and a t(8; 16)(p11; p13), respectively, but cytogenetic variants of both have been described. Recently, the t(8; 16) has been cloned and shown to fuse the MOZ gene at 8p11 to the CBP gene at 16p13. We have used fluorescence in situ hybridization (FISH), Southern blotting, and reverse transcriptase-polymerase chain reaction (RT-PCR) to refine the 8p11 breakpoint in three cases with t(8; 13)(p11; q12) and in a single case of AML-M5 with a clinical picture apparently identical to that found in patients with a t(8; 16), but characterized by an inv(8)(p11q13). FISH analysis was performed with several 8p11 CEPH yeast artificial chromosome (YAC) clones. YAC 782H11 was centromeric to the one case with t(8; 13) tested, but was telomeric to the inv(8). YAC 847B12 was telomeric to both the t(8; 13) and the inv(8), whereas YAC 829D12 was centromeric to the t(8; 13), but split by the inv(8). Southern blotting and PCR of YAC 829D12 showed that it contained the MOZ gene. A 900-bp MOZ fragment encompassing the published t(8; 16) breakpoint was amplified by PCR from normal peripheral blood leukocyte cDNA and used to probe Southern blots of patient DNA. A rearrangement was detected in the case with inv(8), but not in any of the three cases with t(8; 13). Southern blotting with a CBP probe and RT-PCR with MOZ and CBP primers suggested that the inv(8) does not result in a cryptic MOZ-CBP fusion. It is likely, therefore, that MOZ is fused to a novel gene at 8q13 in this case. We conclude that the t(8; 13) breakpoint is flanked by YACs 782H11 and 847B12 and is at least 1 Mb telomeric to MOZ. MOZ is involved, however, in a new variant of the t(8; 16).
Cyclin D1 Expression in Mantle Cell Lymphoma Is Accompanied by Downregulation of Cyclin D3 and Is Not Related to the Proliferative Activity
Authors: Ott M. Michaela , Bartkova Jirina , Bartek Jiri , Dürr Alexander , Fischer Lars , Ott German , Müller-Hermelink Hans Konrad , Kreipe Hans
Published: October 2019

The cell cycle regulatory protein cyclin D1 is essential for G1-S phase transition in several epithelial and mesenchymal tissues but is apparently not essential in normal mature B cells. An overexpression of cyclin D1 is induced by the chromosomal translocation t(11; 14)(q13; q32), which characterizes non-Hodgkin's lymphomas (NHLs) of mantle cell type. We studied 26 cases of mantle cell lymphoma (MCL) for the expression of cyclins D1 and D3. A total of 23 lymphomas showed a nuclear staining for cyclin D1, whereas reactive B cells of residual germinal centers were constantly negative. When compared with cyclin D3, an inverse staining pattern emerged. Whereas the B cells of residual germinal centers reacted strongly positive for cyclin D3, there was low or missing expression of cyclin D3 in MCL cells. In other B-cell lymphomas (n = 55), including chronic lymphocytic leukemia, low-grade lymphomas of mucosa-associated lymphatic tissue, follicular lymphomas, and diffuse large B-cell lymphomas, no cyclin D1 expression could be detected and 89% of these cases displayed cyclin D3 positivity. Lymphoma cell lines harboring the t(11; 14) showed cyclin D1 protein but no or very low levels of cyclin D3; three other B-cell lines, a T-cell line, and peripheral blood lymphocytes strongly expressed cyclin D3 and reacted negatively for cyclin D1. We conclude that the chromosomal translocation t(11; 14) leads to an abnormal protein expression of cyclin D1 in the tumor cells of MCL and induces a consecutive downregulation of cyclin D3. In contrast to other B-NHLs, cyclin D1 and D3 expression in MCL is not related to the growth fraction.
A High Pretreatment Serum Vascular Endothelial Growth Factor Concentration Is Associated With Poor Outcome in Non-Hodgkin's Lymphoma
Authors: Salven Petri , Teerenhovi Lasse , Joensuu Heikki
Published: October 2019

Vascular endothelial growth factor (VEGF ) is a secreted endothelial cell-specific mitogen, which is induced by hypoxia and is angiogenic in vivo. Recently, elevated serum concentrations of VEGF (S-VEGF ) have been reported in patients with cancers of various histologies. However, the prognostic significance of S-VEGF in human cancer is unknown and the origin of S-VEGF remains unsettled. We measured S-VEGF by enzyme-linked immunosorbent assay from sera taken from 82 patients with non-Hodgkin's lymphoma before treatment and stored for 9 to 15 years at −20°C. All but one of the patients had been followed-up for at least 5 years or until death. S-VEGF ranged from 15 to 964 pg/mL; median, 228 pg/mL; mean, 291 pg/mL. A higher than the median S-VEGF level was associated with a poor World Health Organization performance status, a high International Prognostic Index, a high serum lactate dehydrogenase level, and a large cell histology. Patients with lower than the median S-VEGF at diagnosis had a 71% 5-year survival rate in comparison with only 49% among those with a higher than the median S-VEGF. We conclude that a high pretreatment S-VEGF level is associated with poor outcome in non-Hodgkin's lymphoma.