bluuebunny/crossref_metadata_2025 huggingface dataset:
Tryptophan 650 of human granulocyte colony-stimulating factor (G-CSF) receptor, implicated in the activation of JAK2, is also required for G- CSF-mediated activation of signaling complexes of the p21ras route
Authors: Barge RM , de Koning JP , Pouwels K , Dong F , Lowenberg B , Touw IP
Published: October 2019

Granulocyte colony-stimulating factor (G-CSF) induces rapid phosphorylation of JAK kinases as well as activation of the p21ras route through interaction with its specific receptor (G-CSF-R). The cytoplasmic membrane-proximal region of G-CSF-R (amino acids 631 to 684) is necessary for proliferation induction and activation of JAK2. In contrast, activation of Shc and Syp, signaling molecules implicated in the p21ras signaling route, depends on the phosphorylation of tyrosine residues located in the membrane-distal region (amino acids 685 to 813) of G-CSF-R. We investigated whether G-CSF-induced activation of signaling complexes of the p21ras route depends on the function of the membrane-proximal cytoplasmic region of G-CSF-R. A G- CSF-R mutant was constructed in which tryptophan 650 was replaced by arginine and expressed in BAF3 cells (BAF/W650R). In contrast to BAF3 cell transfectants expressing wild-type G-CSF-R, BAF/W650-R cells did not proliferate and did not show activation of JAK2, STAT1, or STAT3 in response to G-CSF. Immunoprecipitations with anti-Shc and anti-Grb2 antisera showed that mutant W650R also failed to activate Syp and Shc. These data indicate that the membrane-proximal cytoplasmic domain of G- CSF-R is not only crucial for proliferative signaling and activation of JAK2 and STATs, but is also required for activation of the p21ras route, which occurs via the membrane-distal region of G-CSF-R.
Regulation of thrombopoietin levels by c-mpl-mediated binding to platelets
Authors: Fielder PJ , Gurney AL , Stefanich E , Marian M , Moore MW , Carver-Moore K , de Sauvage FJ
Published: October 2019

The involvement of platelets and the c-mpl receptor in the regulation of thrombopoietin (TPO) plasma concentrations and tissue mRNA levels was investigated in both normal mice and mice defective in c-mpl (c-mpl- /-). Although c-mpl-/- mice have fewer platelets and higher plasma TPO activity than normal mice, there was no increase in TPO mRNA levels as measured by an S1 nuclease protection assay. After the intravenous injection of 125I-TPO, specific uptake of radioactivity by the spleen and blood cells was present in the normal mice, but absent in the c-mpl- /- mice. Platelet-rich plasma (PRP) from normal mice was able to bind and internalize 125I-TPO, whereas PRP from c-mpl-/- mice lacked this ability. Analysis of 125I-TPO binding to normal PRP indicated that binding was specific and saturable, with an approximate affinity of 560 pmol/L and 220 receptors per platelet. PRP from normal mice was also able to degrade 125I-TPO into lower molecular weight fragments. After the intravenous injections, c-mpl-/- mice cleared a dose of 125I-TPO at a much slower rate than did normal mice. Injection of washed platelets from normal mice into c-mpl-/- mice resulted in a dramatic, but transient, decrease in plasma TPO levels. These data provide evidence that platelets regulate plasma TPO levels via binding to the c-mpl receptor on circulating platelets.
Allogeneic cell therapy with donor peripheral blood cells and recombinant human interleukin-2 to treat leukemia relapse after allogeneic bone marrow transplantation
Authors: Slavin S , Naparstek E , Nagler A , Ackerstein A , Samuel S , Kapelushnik J , Brautbar C , Or R
Published: October 2019

Allogeneic bone marrow transplantation (BMT) is the only effective treatment for hematologic malignancies resistant to conventional chemotherapy. Until recently, no cure existed for patients who relapsed post-BMT. We present our long-term observations on remission induction, after relapse post-BMT, by allogeneic cell therapy (allo-CT) and the feasibility of remission induction in allo-CT-resistant patients by activation of antileukemia effector cells with recombinant human interleukin-2 (rhIL-2) in vitro and in vivo. The longest observation of successful allo-CT (event-free survival, greater than 8 years) was made in a patient with resistant pre-B lymphoblastic leukemia who received infusions with graded increments of donor (female) peripheral blood lymphocytes (PBL) as soon as bulky hematologic and extramedullary relapse was noticed early post-BMT. The patient is currently without evidence of residual host (male) cells as determined by polymerase chain reaction (PCR). Of 17 patients with acute and chronic leukemia in relapse after BMT, 10 were reinduced into complete remission. Four patients with cytogenetic relapse responded to allo-CT alone, while five of six patients with overt hematologic relapse responded only after additional activation of donor with rhIL-2. Allo-CT can, therefore, successfully reverse chemoradiotherapy-resistant relapse of both acute and chronic leukemia. Moreover, in patients resistant to donor lymphocyte infusion, remission can be accomplished by additionally activating donor PBL in vitro and/or in vivo with rhIL-2. Based on our observations, after BMT, allo-CT should be considered the treatment of choice for patients with hematologic malignancies resistant to conventional anticancer modalities. Allogeneic activated cell therapy (allo ACT) should be considered for patients with tumor cells resistant to allo-CT. Although allo-CT, followed if indicated by allo-ACT, can be effective for patients with overt hematologic relapse, reversal of persistent minimal residual disease or documented molecular/cytogenetic relapse early after BMT may also be considered as a possible indication for allo-CT.
Inhibition of factor XII in septic baboons attenuates the activation of complement and fibrinolytic systems and reduces the release of interleukin-6 and neutrophil elastase
Authors: Jansen PM , Pixley RA , Brouwer M , de Jong IW , Chang AC , Hack CE , Taylor FB Jr , Colman RW
Published: October 2019

In previous studies, we have shown that administration of monoclonal antibody (MoAb) C6B7 against human factor XII to baboons challenged with a lethal dose of Escherichia coli abrogates activation of the contact system and modulates secondary hypotension. To evaluate the contribution of activated contact proteases to the appearance of other inflammatory mediators in this experimental model of sepsis, we studied the effect of administration of MoAb C6B7 on activation of complement and fibrinolytic cascades, stimulation of neutrophil degranulation, and release of the proinflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6). Activation of the complement system, as reflected by circulating C3b/c and C4b/c levels, was significantly reduced in five animals that had received MoAb C6B7 before a lethal dose of E coli as compared with five control animals that had been given a lethal challenge only. Inhibition of contact activation also modulated the fibrinolytic response, since the release of tissue-type plasminogen activator (t-PA) and the appearance of plasmin-alpha2-antiplasmin (PAP) complexes into the circulation was significantly attenuated upon pretreatment with anti-factor XII MoAb. In contrast, plasma levels of plasminogen activator inhibitor (PAI) were modestly enhanced in the treatment group. Degranulation of neutrophils, as assessed by circulating elastase-alpha1-protease inhibitor complexes, and release of IL-6 but not of TNF-alpha was decreased in anti-factor XII-treated animals. Observed differences in the inflammatory response between treatment and control groups were not likely due to different challenges, since the number of E coli that had been infused, as well as circulating levels of endotoxin after the challenge, were similar for both groups. These data suggest that activation of the contact system modulates directly or indirectly various mediator systems involved in the inflammatory response during severe sepsis in nonhuman primates.
Interleukin-3 cooperates with tumor necrosis factor alpha for the development of human dendritic/Langerhans cells from cord blood CD34+ hematopoietic progenitor cells
Authors: Caux C , Vanbervliet B , Massacrier C , Durand I , Banchereau J
Published: October 2019

We have previously shown that tumor necrosis factor (TNF)alpha strongly potentiates the granulocyte-macrophage colony-stimulating factor (GM- CSF)/interleukin (IL)-3-dependent proliferation of CD34+ hematopoietic progenitor cells (HPC) through the recruitment of early progenitors with high proliferative potential. Furthermore, the combination of GM- CSF and TNFalpha allows the generation of large numbers of dendritic/Langerhans cells (D-Lc). Herein, we analyzed whether IL-3, when combined to TNFalpha would, as does GM-CSF, allow the generation of CD1a+ D-Lc. Accordingly, cultures of cord blood CD34+ HPC with IL-3 + TNFalpha yielded 20% to 60% CD14+ cells and 11% to 17% CD1a+ cells, while IL-3 alone did not generate significant numbers of CD1a+ cells. Although the percentage of CD1a+ cells detected in IL3 + TNFalpha was lower than that observed in GM-CSF + TNFalpha (42% to 78%), the strong growth induced by IL-3 + TNFalpha generated as many CD1a+ cells as did GM-CSF + TNFalpha. The CD14+ and CD1a+ cells generated with IL-3 + TNFalpha are similar to CD14+ and CD1a+ cells generated in GM-CSF alone and GM-CSF + TNFalpha, respectively. CD1a+ cells differed from CD14+ cells by (1) dendritic morphology, (2) higher expression of CD1a, CD1c, CD4, CD40, adhesion molecules (CD11c, CD54, CD58), major histocompatibility complex (MHC) class II molecules and CD28 ligands (CD80 and CD86), (3) lack of Fc receptor FcgammaRI (CD64) and complement receptor CR1 (CD35) expression, and (4) stronger induction of allogeneic T-cell proliferation. Thus, in combination with TNFalpha, IL-3 is as potent as GM-CSF for the generation of CD1a+ D-Lc from cord blood CD34+ HPC. The dendritic cell inducing ability of IL-3 may explain why mice with inactivated GM-CSF gene display dendritic cells.
ILA, a member of the human nerve growth factor/tumor necrosis factor receptor family, regulates T-lymphocyte proliferation and survival
Authors: Schwarz H , Blanco FJ , von Kempis J , Valbracht J , Lotz M
Published: October 2019

ILA, a gene induced by lymphocyte activation, is a member of the human nerve growth factor tumor necrosis factor receptor family and the human homologue of murine 4–1BB. The present study analyzed the role of ILA in the regulation of human peripheral blood T-lymphocyte function. Antibodies raised against different fusion proteins recognized ILA on activated lymphocytes. These antibodies significantly increased anti- CD3--induced T-lymphocyte proliferation. When anti-CD3--stimulated cells were incubated on ILA-expressing CHO cells, proliferation was inhibited. CHO cells transfected with a control construct and not expressing ILA did not reduce T-cell proliferation. A purified fusion protein containing the extracellular domain of ILA and the constant domain of human IgG (ILA-IgG) also inhibited lymphocyte proliferation. Results obtained by 3H-thymidine incorporation were confirmed by cell cycle analysis that showed a decrease in the number of lymphocytes in S phase. Lymphocyte morphology in cultures with ILA-expressing CHO cells was suggestive of apoptosis. Flow cytometry on propidium iodide-stained cells showed a time-dependent increase in the number of hypodiploid apoptotic cells when lymphocytes were cultured on ILA-expressing CHO cells. Internucleosomal DNA cleavage was seen in these cultures, but not in the presence of ILA-negative CHO cells. Studies on the mechanism by which ILA regulates T-cell function showed that ILA-IgG inhibited anti-CD3-induced T-cell proliferation when presented in immobilized but not in soluble form. These results suggest that ILA may act by cross- linking its ligand and thereby inhibit T-cell proliferation.
Expression of granulocyte colony-stimulating factor and granulocyte colony-stimulating factor receptor genes in partially overlapping monoclonal B-cell populations from chronic lymphocytic leukemia patients
Authors: Corcione A , Corrias MV , Daniele S , Zupo S , Spriano M , Pistoia V
Published: October 2019

B lymphocytes were purified from the peripheral blood of 30 B-cell chronic lymphocytic leukemia (B-CLL) patients and tested for the ability to produce granulocyte colony-stimulating factor (G-CSF) in vitro. Fifteen Staphylococcus aureus Cowan I (SAC)-stimulated, but not unstimulated, B-cell suspensions produced G-CSF in short-term cultures. Accordingly, G-CSF mRNA was detected only in SAC-stimulated B cells. Five CLL B-cell fractions that released G-CSF following exposure to SAC were also incubated with CD40 or anti-mu antibodies in the presence or absence of recombinant (r) interleukin-2 (IL-2) or IL-4. The 5 cell suspensions produced G-CSF only on culture with CD40 monoclonal antibody in combination with rIL-2 or rIL-4. CD5+ B lymphocytes, which represent the normal counterparts of most B-CLL proliferations, did not produce G-CSF under any of the above culture conditions. G-CSF produced by leukemic B lymphocytes was biologically active, because conditioned media of SAC-stimulated cells supported the in vitro growth of myeloid colonies from normal bone marrow progenitors. The colony stimulating activity of CLL B-cell supernatants was ascribed to both G-CSF and granulocyte-macrophage colony stimulating factor. G-CSF receptors (G- CSFRs) were detected on freshly isolated B lymphocytes from 7 of 11 B- CLL patients; 5 of these cell suspensions produced G-CSF in culture, whereas 2 did not. rG-CSF rescued 3 of the 7 G-CSFR+ cell fractions from spontaneous apoptosis but had no effect on their in vitro proliferation.
Growth inhibition of human leukemic cells by WT1 (Wilms tumor gene) antisense oligodeoxynucleotides: implications for the involvement of WT1 in leukemogenesis
Authors: Yamagami T , Sugiyama H , Inoue K , Ogawa H , Tatekawa T , Hirata M , Kudoh T , Akiyama T , Murakami A , Maekawa T
Published: October 2019

We have previously reported expression of WT1 in acute leukemia. To elucidate its biological significance, we examined the effect of the suppression of the WT1 expression by WT1 antisense oligomers on the growth of the leukemic cells expressing WT1. When 20 different WT1 antisense (AS) oligomers covering from the 5′ cap sites of the WT1 gene to the 3′ end were examined for the inhibitory effect on the growth of K562 cells expressing WT1, four WT1 AS oligomers inhibited the cell growth, whereas WT1 sense and random sequence oligomers had no effect on the cell growth of K562. Moreover, WT1 AS oligomers significantly inhibited the growth of the clonogenic cells of fresh leukemic cells in six of 14 patients with acute myeloid leukemia, in one of two patients with chronic myelogenous leukemia (CML) chronic phase, and in one of one patient with CML blastic crisis. However, these oligomers did not inhibit normal colony-forming unit-granulocyte-macrophage. Western blot analysis clearly demonstrated the significant reduction in the WT1 protein levels in the K562 and fresh leukemic cells that were treated with the WT1 AS oligomers, confirming that the inhibitory effect of the WT1 AS oligomers on the cell growth operates via the reduction in the WT1 protein levels. These results show that WT1 plays an important role in leukemogenesis.
A novel mutation found in the 3' domain of NADH-cytochrome B5 reductase in an African-American family with type I congenital methemoglobinemia
Authors: Jenkins MM , Prchal JT
Published: October 2019

Congenital methemoglobinemia caused by an erythrocytic deficiency of cytochrome b5 reductase (b5R; type I) in African-American individuals was first reported by this laboratory. The rarity of this observation is possibly due to the difficulty detecting cyanosis that is masked by naturally occurring dark skin pigment. Since previous biochemical studies on the African-American family with variant enzyme b5R- Shreveport showed enzyme instability, we focused on molecular analysis of its transcript. The transcript size was the same as that of a normal control. The nucleotide sequence of both normal and variant transcripts were examined by directly sequencing reverse transcriptase-polymerase chain reaction (RT-PCR) product. The propositus was found to be homozygous for a G to A transition at codon 212 in exon 8, changing a glutamate to a lysine (E212K). In addition, a C to G transversion was found at codon 116 in exon 5, changing a threonine to a serine (T116S). Using allele-specific PCR, we determined that E212K was found only in the propositus and her heterozygous mother. Furthermore, E212K is predicted to disrupt an alpha-helix peptide structure of b5R, suggesting that this is likely the disease-causing mutation. In contrast, T116S was found to be a high-frequency polymorphism specific for the African-American population. The E212K mutation is uniquely present in the 3′ end of the b5R gene (exon 8), which differs from those b5R mutations found among Japanese subjects (exons 3 and 5) and in an Italian subject (exon 4) and, thus, further contributes to our understanding of the structure/function relationship of this housekeeping enzyme.
Pure red cell aplasia: association with large granular lymphocyte leukemia and the prognostic value of cytogenetic abnormalities [see comments]
Authors: Lacy MQ , Kurtin PJ , Tefferi A
Published: October 2019

From 1980 through 1994, we identified 47 adult patients with acquired pure red cell aplasia (median age, 64 years; range, 22 to 84 years). Associated clinical disorders included T-cell large granular lymphocytic (LGL) leukemia, thymoma, chronic lymphocytic leukemia, and non-Hodgkin's lymphoma. Review of bone marrow findings in 40 patients showed absence of erythroid precursors in 14 patients and rare pronormoblasts in 26. None had morphologic evidence of myelodysplasia. T-cell receptor gene rearrangement studies with Southern blot technique in 14 patients showed clonal rearrangements in nine. Karyotypic analyses performed in 28 patients showed clonal abnormalities in four. Overall, 28 of 47 patients (60%) responded to immunosuppressive therapy, but none were the patients with cytogenetic abnormalities. There was a trend toward superior response to immunosuppressive agents in the patients with T-cell LGL leukemia. Cyclophosphamide, with or without corticosteroids, was the most useful treatment agent. Cyclosporine A was effective for refractory disease. Neither the presence of an associated clinical disorder nor the existence of detectable erythroid precursors affected overall survival. We conclude that (1) T-cell LGL leukemia is the disorder most commonly associated with pure red cell aplasia, (2) the presence of clonal cytogenetic abnormality predicts poor response to immunosuppressive therapy, and (3) oral cyclophosphamide and cyclosporine A are effective treatment regimens.
Genetic basis of hypo-responsiveness of A/J mice to interleukin-3
Authors: Leslie KB , Jalbert S , Orban P , Welham M , Duronio V , Schrader JW
Published: October 2019

Hematopoietic progenitor cells of the A/J strain of mice show a pronounced defect in the ability to form colonies or proliferate in response to interleukin-3 (IL-3). Comparison of immunoblots of A/J mast cells and of mast cells from the C57BL/6 strain that respond normally to IL-3 showed that, in both strains, a 125-kD band of the expected size was recognized by an antibody against the beta chain of the IL-3 receptor, the AIC2A molecule. However, in the C57BL/6 cells, there was an additional 110-kD species not seen in cells of the A/J strain. Analyses using bone marrow-derived mast cells from a panel of A/J x C57BL/6 and A/J x C57BL/6 recombinant inbred (RI) mice showed that the hypo-responsiveness to IL-3 is governed by a single gene. However, the absence of this 110-kD species in the A/J strain did not co-map with IL- 3 hypo-responsiveness but did indeed map to the AIC2A genetic locus. These data show that this trait in the A/J strain was due to a polymorphism of the AIC2A gene unrelated to IL-3 hypo-responsiveness. Typing of the RI strains for the markers D14Mit98, D14Mitl4, and D14Mit133 mapped the locus determining hypo-responsiveness to IL-3 to the subtelomeric region of chromosome 14, the region that also bears the gene encoding the alpha chain of the IL-3 receptor (lL-3Ralpha). Immunofluorescence analyses indicated that IL-3Ralpha protein was undetectable on fresh bone marrow cells from A/J mice, although clearly detectable on cells from the responder C57BL/6 strain. However, IL- 3Ralpha was readily detectable at normal levels on A/J mast cells generated by culture of A/J bone marrow cells in a combination of IL-3 and steel factor. Moreover, IL-3Ralpha on these A/J mast cells appears to be functional in that IL-3 stimulation of these cells results in tyrosine phosphorylation events characteristic of IL-3 signaling, including tyrosine phosphorylation of the beta chain of the IL-3 receptor, Jak-2 kinase, and SHPTP2. Collectively, these data indicate that the hypo-responsiveness of A/J mice to IL-3 is due to a defect in the gene encoding IL-3Ralpha and that, although this defect gives rise to reduced expression of alpha chain on primary bone marrow cells, this defect is not absolute and that, under certain circumstances, A/J cells can express functional receptors.
Xenogeneic expression of human stem cell factor in transgenic mice mimics codominant c-kit mutations
Authors: Majumdar MK , Everett ET , Xiao X , Cooper R , Langley K , Kapur R , Vik T , Williams DA
Published: October 2019

Mutations of c-kit, which encodes a transmembrane receptor tyrosine kinase, have been identified in mice by abnormal coat color, anemia, and germ cell defects. Mice heterozygous for mutations of c-kit have a white forehead blaze and a white ventral spot, leading these mutants to be termed dominant White spotting (W). We have previously demonstrated that the membrane-associated isoform of human stem cell factor (hSCF220, the ligand for c-kit) is inefficiently processed in murine stromal cell transfectants. Thus, in murine cell lines analyzed in vitro, hSCF220 transfectants present SCF as a membrane restricted protein in contrast to the murine SCF220 cDNA protein product, which is slowly cleaved and secreted. We show here that transgenic mice expressing the human SCF220 isoform in vivo display a phenotype indistinguishable from some alleles of W. Specifically, hSCF220- expressing transgenic mice display a prominent forehead blaze and a white ventral spot. Generations of doubly heterozygous animals that carry both a mutated c-kit allele and the hSCF220 transgene display a more severe coat color abnormality. This phenotype appears to be due to occupancy of murine c-kit by human SCF and diminished cell surface expression of endogenous murine SCF. Normal signaling events that lead to cell survival or proliferation appear to be disrupted in vivo in these transgenic mice.
Neutrophil deactivation by influenza A viruses: mechanisms of protection after viral opsonization with collectins and hemagglutination-inhibiting antibodies
Authors: Hartshorn KL , Reid KB , White MR , Jensenius JC , Morris SM , Tauber AI , Crouch E
Published: October 2019

Bacterial superinfections are a major cause of morbidity and mortality during influenza A virus (IAV) epidemics. Depression of phagocyte functions resulting from attachment of the IAV hemagglutinin (HA) to cell surface sialo-glycoproteins is a likely contributory cause of these infections. We have proposed that the group of collagenous lectins (termed collectins) present in blood and pulmonary surfactant play a role in initial host defense against IAV. We used here several recombinant human surfactant protein D (RhSP-D) preparations to determine the mechanism through which opsonization of IAV with collectins protects neutrophils against the deactivating effects of IAV on cellular respiratory burst responses in vitro. RhSP-D was markedly more potent than antibodies that inhibited viral hemagglutination activity (anti-HA antibodies) at protecting neutrophils in this assay. Unlike the anti-HA antibodies, RhSP-D was protective at concentrations that minimally inhibited viral hemagglutination activity. Two related features of SP-D--the degree of multimerization and the ability to cause aggregation of IAV particles--were critical determinants of the ability of SP-D to protect neutrophils against deactivation. Similarly SP-D-induced viral aggregate formation resulted in enhanced IAV binding to neutrophils and potentiated the ability of the virus itself to trigger neutrophil respiratory burst responses. In contrast to the case of IAV-antibody complexes, SP-D-IAV complexes attached to and activated neutrophils through a neuraminidase-sensitive mechanism (ie, similar to unopsonized IAV). These results indicate that collectin-mediated viral aggregation per se may be an important host defense mechanism not only by virtue of reducing the number of infectious viral particles, but also by promoting phagocyte responsiveness.
Cholesterol-loading of membranes of normal erythrocytes inhibits phospholipid repair and arachidonoyl-CoA:1-palmitoyl-sn-glycero-3- phosphocholine acyl transferase. A model of spur cell anemia
Authors: Allen DW , Manning N
Published: October 2019

Spur cell anemia may occur in severe liver disease including alcoholic cirrhosis. Spur cell anemia red blood cells (RBCs) have a characteristic morphology, with irregular projections, an increased ratio of membrane cholesterol (Ch) to phospholipid, evidence of oxidative damage, and shortened survival resulting in hemolytic anemia. Normal RBCs may acquire many of the features of spur cells either by transfusion into a spur cell patient or in an in vitro model system that loads the RBC membrane with Ch relative to phospholipid by means of Ch-rich, phospholipid-Ch sonicates. We found evidence of abnormal phospholipid repair metabolism in spur cell anemia RBCs characterized by decreased arachidonate (Ar) uptake into phospholipids and by increased uptake into a fatty acid membrane repair intermediate, acylcarnitine (AcylCn). To study the possible modulation of phospholipid repair metabolism in spur cells by Ch-loading, we compared the Ar metabolism of RBCs loaded with Ch in vitro with that of control cells incubated in autologous serum. Ar, a polyunsaturated fatty acid, is especially sensitive to peroxidation and, thus, is likely to be involved in phospholipid repair. Ch-loading decreased the incorporation of [14C]Ar into total lipids (Ch-loaded, 1,113 +/- 48 pmol/10(10) RBCs; control, 1,525 +/- 48 pmol/10(10) RBCs) including phosphatidylethanolamine, phosphatidylserine, and phosphatidylcholine. Uptake of [14C]Ar into AcylCn increased (control AcylCn, 169 +/- 31 pmol/10(10) RBCs; Ch-loaded AcylCn, 196 +/- 35 pmol/10(10) RBCs; P = .0012). Thimerosal, an inhibitor of arachidonoyl- CoA:l-palmitoyl-sn- glycero-3-phosphocholine acyl transferase or lysophosphocholine acyl transferase (LAT), produced a similar pattern of metabolic abnormality, with decreased incorporation into phospholipid but relative increase into AcylCn. We assayed LAT in RBC membranes from Ch-loaded RBCs, using [14C]arachidonoyl CoA as precursor, and found similar decreased LAT activity at concentrations of 1-palmitoyllysophosphatidylcholine (LPC) from 1 to 30 micromol/L. Similar LAT assay results were obtained using [14C]palmitoyl LPC as the precursor. We conclude that Ch-loading of RBC membranes results in inhibition of LAT in the cell-free system in vitro and may account for the inhibited phospholipid repair in Ch-loaded intact RBCs in vitro and in spur cell anemia RBCs in vivo. Decreased ability to replace peroxidized membrane fatty acid by this metabolic pathway may contribute to the hemolytic process in spur cell anemia.
Glycosylphosphatidylinositol-anchor-deficient mice: implications for clonal dominance of mutant cells in paroxysmal nocturnal hemoglobinuria
Authors: Kawagoe K , Kitamura D , Okabe M , Taniuchi I , Ikawa M , Watanabe T , Kinoshita T , Takeda J
Published: October 2019

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hematopoietic stem cell disorder characterized by complement-mediated hemolysis. Abnormal hematopoietic cells from patients with PNH are deficient in glycosylphosphatidylinositol (GPI)-anchored proteins and clonally dominate various hematopoietic lineages in the bone marrow and the peripheral blood. Analysis of many patients with PNH has showed that somatic mutation in the X-linked gene PIG-A is responsible for the GPI- anchor deficiency in PNH. The PIG-A mutation must also be relevant to the clonal dominance of GPI-anchor deficient (GPI-) blood cells because two or more PIG-A mutant clones become dominant in many patients. However, whether the PIG-A mutation alone is sufficient for clonal dominance is not known. To address this question, we generated chimeric mice using Pig-a (the murine homologue of PIG-A) disrupted embryonic stem (ES) cells, in which the animals are chimeric with respect to the surface expression of GPI-anchored proteins. The chimerism of hematopoietic and nonhematopoietic tissues in such mice was always low, suggesting that the higher contribution of Pig-a disrupted GPI- cells had a lethal effect on the chimera. GPI- cells appeared in the peripheral blood of some of the chimeric mice. However, the percentage of GPI- erythrocytes did not increase for 10 months after birth, implying that the Pig-a mutation alone does not immediately cause the clonal dominance of GPI- blood cells; another pathologic or physiologic change(s) in the hematopoietic environments or in the clone itself may be necessary.
JAK2 is associated with the c-kit proto-oncogene product and is phosphorylated in response to stem cell factor
Authors: Weiler SR , Mou S , DeBerry CS , Keller JR , Ruscetti FW , Ferris DK , Longo DL , Linnekin D
Published: October 2019

Stem cell factor (SCF) is a hematopoietic growth factor that interacts with the receptor tyrosine kinase, c-kit. We have found that SCF- stimulates rapid and transient tyrosine phosphorylation of JAK2 in human and murine cell lines, as well as in normal human progenitor cells. JAK2 and c-kit were associated in unstimulated cells with further recruitment of JAK2 to the c-kit receptor complex after SCF stimulation. Treatment of cells with JAK2 antisense oligonucleotides resulted in a 46% decrease in SCF-induced proliferation. These data demonstrate that SCF induces tyrosine phosphorylation of JAK2 and suggest that JAK2 is a component of the SCF signal transduction pathway.
Subcellular localization of transforming growth factor-alpha in human eosinophil granulocytes
Authors: Egesten A , Calafat J , Knol EF , Janssen H , Walz TM
Published: October 2019

Eosinophils are involved in the inflammatory response seen in allergy and helminthic infestations. Eosinophils synthesize transforming growth factor-alpha (TGF-alpha), which may play a role in the development of the characteristic fibrosis seen in longstanding high eosinophilia. Using immunoelectron microscopic techniques, eosinophils from peripheral blood of healthy individuals and from one patient with high eosinophilia showed presence TGF-alpha in matrix of the specific crystalloid-containing granules. In cryosections, TGF-alpha was also visualized in a vesicular compartment of the cytoplasm. In double- labeling experiments, the TGF-alpha of this latter compartment did not colocalize with CD63, a marker for lysosomes, nor with albumin of secretory vesicles. In extracts from eosinophils, obtained from healthy donors, immunoreactive TGF-alpha could be detected by enzyme-linked immunosorbent assay-technique. In addition, sera from two patients with high eosinophilia showed TGF-alpha concentrations of 1.5 ng/mL and 164 pg/mL, respectively, whereas TGF-alpha could not be detected in serum from healthy controls. In conclusion, TGF-alpha is present in the specific granules, and in an additional vesicular compartment of the cytoplasm of eosinophils.
Induction of the chemotactic S100 protein, CP-10, in monocyte/macrophages by lipopolysaccharide
Authors: Hu SP , Harrison C , Xu K , Cornish CJ , Geczy CL
Published: October 2019

The murine S100 protein CP-10 is a potent chemotactic factor for murine and human myeloid cells in vivo and in vitro. This is the first report describing regulations of the CP-10 gene by a proinflammatory stimulus, lipopolysaccharide (LPS), in cells of the monocyte/macrophage lineage. Murine monocyte/macrophage-like WEHI 265 and RAW 264.7 cells preexposed to 5 to 50 ng/mL LPS expressed significant levels of CP-10 mRNA 4 hours, and maximal at 20 hours, after a secondary LPS challenge. This was accompanied by increasing levels of cell-associated and released CP- 10 protein. In contrast, a single dose of LPS upregulated CP-10 mRNA in elicited peritoneal macrophages, whereas mRNA and protein levels decreased following LPS challenge. The state of macrophage differentiation may control responsiveness as LPS had no effect on CP- 10 basal levels in bone marrow derived macrophages. LPS-induced CP-10 expression was controlled at the transcriptional level and nuclear run- on and protein synthesis inhibition assays indicated that LPS priming and challenge of RAW cells occurred via distinct pathways. MRP14, another S100 protein generally coordinately expressed with human MRP8, was not induced by LPS under the same conditions. We propose that CP-10 may play a key role in recruitment of leukocytes into tissues in response to gram-negative bacterial infection.
Analysis of T-cell repopulation after allogeneic bone marrow transplantation: significant differences between recipients of T-cell depleted and unmanipulated grafts
Authors: Roux E , Helg C , Dumont-Girard F , Chapuis B , Jeannet M , Roosnek E
Published: October 2019

We have studied the repopulation of the T-cell compartment in 27 patients transplanted with bone marrow from an (HLA)-identical sibling. Significant differences were found between recipients of unmanipulated and T-cell depleted grafts. Analysis of the T cells by a method based on amplification of minisatellite DNA regions showed that without depletion > 99.9% of the clones responding to a mitogenic stimulus after transplantation were of donor origin. In contrast, when the graft had been depleted with Campath-1M plus complement, a significant part of the T cells cloned during the first weeks after transplantation comprised of recipient T cells that had survived the preconditioning. This mixed population of low numbers donor and recipient T cells (19 +/- 31/mm3 at day 14) expanded rapidly (predominantly CD8+ T cells) during the first 2 months, without a significant change of the ratio of recipient/donor T cells. In 11 of 17 evaluable patients a late wave ( > 9 months) of donor T cells occurred. As a consequence, T-cell chimerism changed in favor of donor T cells and the CD4/CD8 ratio that had been reversed ( < 0.5) after the first expansion, normalized (1.5 +/- 0.51). Analysis of the T-cell receptor repertoire showed that in recipients of a T-cell depleted graft, the recipient as well as the donor T cells that repopulated the peripheral T-cell pool during the first month, were the progeny of a limited number of precursors. Because without depletion, when larger numbers of donor T cells had been cotransfused with the marrow, the repertoire was much more diverse, these data show that immediately after transplantation, the peripheral pool is repopulated primarily through expansion of circulating T cells.
Erythropoiesis and vasculogenesis in embryoid bodies lacking visceral yolk sac endoderm
Authors: Bielinska M , Narita N , Heikinheimo M , Porter SB , Wilson DB
Published: October 2019

During mouse embryogenesis the first hematopoietic and endothelial cells form in blood islands located between layers of visceral endoderm and mesoderm in the yolk sac. The role of visceral endoderm in primitive hematopoiesis and vasculogenesis is not well understood. We have assessed the consequences of a lack of visceral endoderm on blood cell and vessel formation using embryoid bodies derived from mouse embryonic stem (ES) cells deficient in GATA-4, a transcription factor expressed in yolk sac endoderm. When differentiated in vitro, these mutant embryoid bodies do not develop an external visceral endoderm layer. We found that Gata4-/-embryoid bodies, grown either in suspension culture or attached to a substratum, are defective in primitive hematopoiesis and vasculogenesis as evidenced by a lack of recognizable blood islands and vascular channels and a reduction in the expression of the primitive erythrocyte marker epsilon y-globin. Expression of the endothelial cell transcripts FIk-1, FIt-1, and platelet-endothelial cell adhesion molecule (PECAM) was not affected in the mutant embryoid bodies. Gata4-/-ES cells retained the capacity to differentiate into primitive erythroblasts and endothelial cells when cultured in methylcellulose or matrigel. Analysis of chimeric mice, generated by injecting Gata4-/-ES cells into 8-cell stage embryos of ROSA26 transgenic animals, showed that Gata4-/-ES cells can form blood islands and vessels when juxtaposed to visceral endoderm in vivo. We conclude that the visceral endoderm is not essential for the differentiation of primitive erythrocytes or endothelial cells, but this cell layer plays an important role in the formation and organization of yolk sac blood islands and vessels.
Selective induction of a glycoprotein IIIa ligand-induced binding site by fibrinogen and von Willebrand factor
Authors: Mondoro TH , Wall CD , White MM , Jennings LK
Published: October 2019

Ligand-induced binding sites (LIBS) are neoantigenic regions of glycoprotein (GP)IIb-IIIa that are exposed upon interaction of the receptor with the ligand fibrinogen or the ligand recognition sequence (RGDS). LIBS have been suggested to contribute to postreceptor occupancy events such as full-scale platelet aggregation, adhesion to collagen, and clot retraction. This study examined the induction requirements of a GPIIIa LIBS with regard to ligand specificity. Through the use of the anti-LIBS D3, we report that this complex- activating antibody induces fibrinogen-and von Willebrand factor-binding to GPIIb-IIIa on intact platelets. Bound ligand was detected by flow cytometric analysis and platelet aggregation assays. These bound ligands increased the number of D3-binding sites and altered the affinity of D3 for GPIIb-IIIa on platelets. In contrast, activation of platelet GPIIb-IIIa by D3 did not increase the binding of another RGD- containing ligand, vitronectin. Furthermore, bound vitronectin on thrombin-stimulated platelets did not cause the expression of the D3 LIBS epitope. We conclude direct activation of GPIIb-IIIa in the absence of platelet activation results in selective ligand interaction and that D3 LIBS induction requires the binding of the multivalent ligands, fibrinogen or von Willebrand factor. Thus, the region of GPIIIa recognized by D3 may be an important regulatory domain in ligand- receptor interactions that directly mediate platelet aggregation.
Interleukin-4 induces association of the c-fes proto-oncogene product with phosphatidylinositol-3 kinase
Authors: Izuhara K , Feldman RA , Greer P , Harada N
Published: October 2019

We have previously demonstrated that interleukin-4 (IL-4) induces tyrosine phosphorylation of a protein closely related or identical to the c-fes proto-oncogene product (FES) and association of this protein with the IL-4 receptor alpha chain (IL-4R alpha). IL-4 is known to induce association of phosphatidylinositol-3 (PI3) kinase with the IL-4R alpha. Since FES contains the consensus motifs for PI3 kinase binding, we tested the possibility that FES may associate with PI3 kinase upon IL-4 stimulation. We demonstrate herein that IL-4 stimulation induced rapid association of FES or a related protein with PI3 kinase in mouse T-cell lines. We also show an association of human FES (hFES) with the src homology 2 (SH2) domain of PI3 kinase in a COS7 cell expression system. The in vitro PI3 kinase assay using COS7 cells suggested that hFES partly contributes to the association between the hIL-4R alpha and PI3 kinase. We have further identified the important region in the cytoplasmic domain of the hIL-4R alpha for association of tyrosine-phosphorylated hFES with the hIL-4R alpha and SH2 domain of PI3 kinase using a COS7 cell expression system. These results suggest that FES or a related protein/PI3 kinase pathway may play a role in the pleiotropic effects of IL-4.
Identification of mutations in seven Chinese patients with X-linked chronic granulomatous disease
Authors: Hui YF , Chan SY , Lau YL
Published: October 2019

X-linked chronic granulomatous disease (CGD) is due to mutations in the gp91phox gene on Xp21.1. Studies in white and Japanese X-linked CGD patients have shown mutations in nearly every exon. We studied the molecular defect of seven Chinese patients with X-linked CGD from six unrelated families. Mutations were located by single-strand conformation polymorphism and then defined by sequence analysis. The mutations were two different amino acid substitutions, a nonsense mutation, an in-frame trinucleotide deletion, a single A insertion causing a frameshift, and a premature stop. Lastly, a rare splice site mutation caused by G to A transition at the terminal nucleotide of exon 3, resulting in the skipping of exon 3, was found. The possible effects of these mutations on protein structure-function or splicing were discussed. Together with previous reports, the A insertion in the run of six As from nucleotide 749 to 754 and the G to A transition at the terminal position of exon 3 may be mutation hotspots of the gp91phox gene. The extreme heterogeneous mutations found in our patients suggest the absence of ethnic group-specific mutation.
Physical and functional interactions between Stat5 and the tyrosine- phosphorylated receptors for erythropoietin and interleukin-3
Authors: Chin H , Nakamura N , Kamiyama R , Miyasaka N , Ihle JN , Miura O
Published: October 2019

Erythropoietin (Epo) and interleukin-3 (IL-3) stimulate activation of the Jak2 tyrosine kinase and induce tyrosine phosphorylation and activation of Stat5. In the present study, we have shown that Epo or IL-3 stimulation induces binding of Stat5 to the tyrosine-phosphorylated Epo receptor (EpoR) or IL-3 receptor beta subunit (betaIL3), respectively, in IL-3-dependent 32D cells expressing the EpoR. The binding of Stat5 to these cytokine receptors was shown to be rapid and transient, occurring within 1 minute of stimulation of cells and significantly decreasing after 5 minutes of cell treatment. In vivo binding experiments in COS cells showed that binding of Stat5 to the EpoR was mediated through the Stat5 Src homology 2 (SH2) domain. In vitro binding studies further showed that Stat5, but not other Stats examined, bound specifically to tyrosine-phosphorylated recombinant EpoR fusion proteins. In these in vivo and in vitro binding studies, Stat5 bound, albeit to a lesser degree, to truncated EpoR mutants in which all the intracellular tyrosines except Y-343 were removed. Furthermore, EpoR-derived synthetic phosphotyrosine peptides corresponding to Y-343, Y-401, Y-431, and Y-479 inhibited the in vitro binding of Stat5. When expressed in 32D cells, a mutant EpoR in which all the intracellular tyrosines were removed by carboxy-terminal truncation showed a significantly impaired ability to induce tyrosine phosphorylation of Stat5, particularly at low concentrations of Epo, but exhibited an increased sensitivity to Epo for growth signaling as compared with the wild-type EpoR. These results indicate that Stat5 specifically and transiently binds to the EpoR through the interaction between the Stat5 SH2 domain and specific phosphorylated tyrosines, including Y-343, in the EpoR cytoplasmic domain. It was implied that betaIL3 may also have similar Stat5 docking sites. The Stat5 docking sites in the EpoR were shown to facilitate specific activation of Stat5, which, however, may not be required for the EpoR-mediated growth signaling.
Differentially regulated expression of the G-protein-coupled receptor kinases, betaARK and GRK6, during myelomonocytic cell development in vitro
Authors: Loudon RP , Perussia B , Benovic JL
Published: October 2019

G-protein-coupled receptor kinases (GRKs) mediate agonist-specific phosphorylation and desensitization of G-protein-coupled receptors. Previous studies have shown that several of these kinases are expressed in hematopoietic cells and that GRK expression is modulated during T-lymphocyte activation. Here, we analyzed the regulation of beta-adrenergic receptor kinase (betaARK) and GRK6 expression and activity in myelomonocytic and lymphoid cells. In the promyelocytic cell line HL- 60, GRK6 protein levels and activity rose twofold to fourfold over the course of treatment with agents that induce differentiation toward either the myeloid (dimethyl sulfoxide and retinoic acid) or monocytic [1alpha,25(OH)2-vitamin D3] lineage, whereas betaARK protein levels and activity were only slightly altered. In contrast, treatment with phorbol 12,13-myristic acetate (PMA) led to a reduction in steady-state levels and activity of both betaARK and GRK6. Treatment of human lymphocytes with several polyclonal activators (phytohemagglutinin, anti-CD3 antibody and interleukin-2) resulted in enhanced betaARK and GRK6 mRNA and protein levels and increased activity of both kinases. In contrast, PMA and calcium ionophore treatment significantly elevated GRK6 protein levels, while decreasing betaARK expression. These data demonstrate that betaARK and GRK6 expression are differentially regulated during myelomonocytic cell development and lymphocyte activation.
Adenovirus vector infection of chronic lymphocytic leukemia B cells
Authors: Cantwell MJ , Sharma S , Friedmann T , Kipps TJ
Published: October 2019

Adenovirus vectors have several features that make them attractive for potential use in gene therapy, including a broad tissue tropism and an ability to infect quiescent or postmitotic cells. In light of this, we examined whether recombinant adenovirus vectors could transfer genes into neoplastic cells of patients with chronic lymphocytic leukemia (CLL), a leukemia of “resting” B cells. Using high-titer recombinant adenovirus vectors, we found we could transfer genes encoding beta-galactosidase or murine CD80 (B7–1) into the CLL B cells of all patients tested (n = 10). The efficiency of gene transduction into CLL B cells was approximately 100 to 1,000-fold lower than into HeLa cells at any given multiplicity of infection (MOI). At a MOI of 500, 10% to 70% of the CLL B cells from different patients were made to express the transgene, as assessed by multiparameter flow cytometric analysis. Sustained levels of expression with little loss in the percentage of infected cells were maintained for up to 9 days, at which point the analysis was stopped. We found that CLL B cells have markedly lower expression levels of integrins that facilitate internalization of adenovirus particles into target cells, perhaps accounting, in part, for the reduced efficiency of adenovirus-mediated gene transfer compared with that in HeLa cells. Although HeLa cells express high levels of alpha(v)beta5, and detectable amounts of alpha(v)beta3, we find CLL cells from all patients tested express only low amounts of alpha(v)beta3, and no detectable alpha(v)beta5. Activation of CLL cells via CD40 cross-linking enhances expression of alpha(v)beta3, and induces expression of alpha(v)beta5. This phenotypic change is associated with a fivefold increase in the efficiency of adenovirus-mediated gene transfer into such activated CLL B cells. This study demonstrates that adenovirus vectors can transduce genes into CLL B cells and that the efficiency of gene transduction is enhanced by activation via CD40 cross-linking. This is the first demonstration that high proportions of CLL B cells can be made to express a selected transgene, suggesting that such gene transfer methods may become useful for the study of the pathogenesis and/or treatment of this disease.