bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation by Interleukin-10 and Interleukin-4 of Cyclooxygenase-2 Expression in Human Neutrophils
Authors: Niiro Hiroaki , Otsuka Takeshi , Izuhara Kenji , Yamaoka Kunihiro , Ohshima Koichi , Tanabe Tadashi , Hara Shuntaro , Nemoto Yoshiaki , Tanaka Yosuke , Nakashima Hitoshi , Niho Yoshiyuki
Published: October 2019

Neutrophils are important effector cells of acute inflammation because of their potential capacity to synthesize various proinflammatory mediators, and inhibition of their production is expected to result in anti-inflammatory effects. In this study, we investigate the effects of the anti-inflammatory cytokines, interleukin-10 (IL-10) and IL-4, on prostanoid synthesis in human neutrophils. Neutrophils isolated from healthy donors constitutively produced a small amount of prostaglandin E2 (PGE2 ) without any stimulations, whereas they produced a large amount of PGE2 after lipopolysaccharide (LPS) stimulation. IL-10 and IL-4 selectively inhibited their LPS-induced PGE2 production. Inhibition by both cytokines occurred at an early stage of LPS stimulation. Anti–IL-10 treatment of LPS-stimulated neutrophils resulted in enhanced PGE2 production. LPS-induced PGE2 and thromboxane B2 (TXB2 ) production in aspirin-treated neutrophils was significantly inhibited by IL-10, IL-4, and NS-398. Moreover, IL-10 and IL-4 inhibited LPS-induced cyclooxygenase (COX) activity in neutrophils. Western blot and immunocytochemical analysis showed that COX-2 protein was clearly induced in LPS-stimulated neutrophils and that its induction was inhibited by both IL-10 and IL-4. Moreover, both of these cytokines inhibited COX-2 mRNA expression in LPS-stimulated neutrophils. These results raise the possibility that these two cytokines may both offer potent clinical utility as anti-inflammatory agents in the future.
Constitutive Activation of U937 Promonocytic Cell Clones Selected for Their Resistance to Parvovirus H-1 Infection
Authors: López-Guerrero José Antonio , Rayet Béatrice , Tuynder Marcel , Rommelaere Jean , Dinsart Christiane
Published: October 2019

The human promonocytic cell line U937 is highly sensitive to the lytic effect of the autonomous parvovirus H-1. Rare cell variants that resisted H-1 virus infection could be isolated, of which four (RU1, RU2, RU3, and RU4) were further characterized. In contrast to parental cells, the RU clones sustained an abortive H-1 virus infection. Three of the clones showed a significant decrease in the accumulation levels of the c-Myc oncoprotein and in their capacity for forming tumors in immunodeficient mice. Surprisingly, all RU clones resisted the suppressing effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on c-myc oncogene expression and cell proliferation. In contrast, RU clones exhibited the TPA-induced changes in membrane surface antigens and nonspecific esterase activities that are characteristic of monocytic differentiation. Studies of the activation steady-state of RU cells demonstrated the constitutive production of significant amounts of nitric oxide (NO) and superoxide anion (O−2⋅ ). Inhibitors of NO and O−2⋅ . production sensitized all RU cells to the killing effect of parvovirus H-1 and increased the production of infectious viral particles. These data argue for the participation of active oxygen species in macrophage defence mechanisms against parvovirus infection. Moreover, the use of parvovirus H-1 as a selective agent in a cell-colony formation assay allowed us to show that expression of defined markers of monocytic differentiation can be uncoupled from suppression of proliferation.
Is Kaposi's Sarcoma–Associated Herpesvirus Ubiquitous in Urogenital and Prostate Tissues?
Authors: Tasaka Taizo , Said Jonathan W. , Morosetti Roberta , Park Dorothy , Verbeek Walter , Nagai Masami , Takahara Jiro , Koeffler H. Phillip
Published: October 2019

Controversy exists as to whether Kaposi's sarcoma–associated herpesvirus (KSHV) is more widespread than originally reported. Recently, Monini et al reported that KSHV is ubiquitous in urogenital and prostate tissues and sperm of healthy Italian adults using nested polymerase chain reaction (PCR). We have examined for the presence of KSHV in 10 normal prostates from Italian men and 10 from men from the United States, as well as 32 prostatic, 30 vulvar, 24 ovarian, 20 cervical, and 30 testicular cancer specimens from patients from the United States. None of the patients had a history of human immunodeficiency virus infection. The samples were tested by nested PCR. The sensitivity of this assay was determined by a dilution study performed by diluting KSHV DNA from the KS-1 cells (a primary effusion lymphoma cell line which is estimated to have 16 copies of KSHV per cell) in DNA from a K562 myeloid cell line. The nested PCR that we used can detect 2.4 copies of KSHV sequences on a background of K562 DNA. All the samples were negative for KSHV sequences. Therefore, we cannot confirm the finding that KSHV sequences are ubiquitous in urogenital and prostate tissues. Furthermore, because our samples were from both the United States and Italy, the discrepancy between results is unlikely to be explained by either ethnic or environmental factors. False-positive results easily occur using nested primer PCR because of contamination. Our data argue that KSHV is not widely disseminated in urogenital tissues from nonimmunosuppressed individuals.
Modulation of Protein Kinase C Activity in Plasmodium falciparum – Infected Erythrocytes
Authors: Hall Belinda S. , Daramola Olalekan O. , Barden Graeme , Targett Geoffrey A.T.
Published: October 2019

Infection of human erythrocytes with the malaria parasite Plasmodium falciparum induces many morphological and biochemical changes in the host cell. Host serine/threonine protein kinases could be involved in some of these processes. The aim of this study was to determine the effect of infection on red blood cell protein kinase C (PKC) and establish the importance of this enzyme in parasite growth and sexual stage differentiation. Phorbol myristate acetate (PMA)-induced translocation of erythrocyte PKC activity is impaired in erythrocytes enriched for mature asexual stage infected cells. Western blotting shows that this is due to a relative reduction in membrane PKC protein levels rather than inhibition of enzyme activity and analysis of PKC activity isolated from whole cell lysates by DE52 chromatography suggests that total activatable PKC levels are lower in infected erythrocytes. A reduction in PMA-induced activation is also observed in PKC assays performed in situ. Downregulation of erythrocyte PKC by overnight incubation with PMA before infection causes a significant decrease in the rate of the asexual growth, suggesting that the enzyme, although lost later in infection, may be important in the earlier development of the parasite. By contrast, the lack of PKC had no effect on the production of sexual stage parasites.
Severe Combined Immunodeficiency Mice Engrafted With Human T Cells, B Cells, and Myeloid Cells After Transplantation With Human Fetal Bone Marrow or Liver Cells and Implanted With Human Fetal Thymus: A Model for Studying Human Gene Therapy
Authors: Yurasov Sergey , Kollmann Tobias R. , Kim Ana , Raker Christina A. , Hachamovitch Moshe , Wong-Staal Flossie , Goldstein Harris
Published: October 2019

To develop an in vivo model wherein human hematopoiesis occurs, we transplanted severe combined immunodeficiency (SCID) mice with either human fetal bone marrow (HFBM) or human fetal liver (HFL). After transplantation of SCID mice with cultured HFBM (BM-SCID-hu mice) or HFL cells (Liv-SCID-hu mice), significant engraftment of the mouse bone marrow (BM) and population of the peripheral blood with human leukocytes was detected. Human colony-forming unit–granulocyte macrophage and burst forming unit-erythroid were detected in the BM of the BM-SCID-hu and Liv-SCID-hu mice up to 8 months after transplantation. When the HFBM or HFL cells were transduced with a retroviral vector before transplantation, integrated retroviral sequences were detected in human precursor cells present in the SCID mouse BM and in leukocytes circulating in the peripheral blood (PB) up to 7 months after transplantation. The PB of the BM-SCID-hu mice also became populated with human T cells after implantation with human thymic tissue, which provided a human microenvironment wherein human pre-T cells from the BM could mature. When the HFBM was retrovirally transduced before transplantation, integrated retrovirus was detected in sorted CD4+CD8+ double positive and CD4+ single positive cells from the thymic implant and CD4+ cells from the PB. Taken together, these data indicated that the BM of our BM-SCID-hu and Liv-SCID-hu mice became engrafted with retrovirally transduced human hematopoietic precursors that undergo the normal human hematopoietic program and populate the mouse PB with human cells containing integrated retroviral sequences. In addition to being a model for studying in vivo human hematopoiesis, these mice should also prove to be a useful model for investigating in vivo gene therapy using human stem/precursor cells.
Long-Term Maintenance of Hematopoiesis in Irradiated Mice by Retrovirally Transduced Peripheral Blood Stem Cells
Authors: Drize Nina , Chertkov Joseph , Sadovnikova Elena , Tiessen Stefan , Zander Axel
Published: October 2019

Mobilized peripheral blood stem cells (PBSC) are used as a source of hematopoietic stem cells for transplantation and gene therapy. It is still unclear, however, whether the PBSC are fully equivalent to normal bone marrow hematopoietic stem cells and whether they are able to provide long-term function of transgene in reconstituted mice. In the present study, mobilized PBSC from male mice were transduced with human adenosine desaminase gene (hADA) and were used for reconstitution of lethally irradiated female mice. At 112, 3, 6, 9, and 12 months after reconstitution, the bone marrow cells were repeatedly collected from each mouse under light anesthesia and the number of colony-forming unit-spleen (CFU-S), spleen repopulating ability (SRA), and the integration of human ADA gene were studied in CFU-S–derived colonies by polymerase chain reaction (PCR) and Southern blot hybridization analyses. After 9 months, the proportion of donor CFU-S detected by PCR with a Y-chromosome–specific probe in mice reconstituted with mobilized PBSC was 75.3% ± 6.0%, which is similar to the concentration of donor CFU-S seen after bone marrow transplantation. Similarly, there was no difference in the concentration of CFU-S in mice reconstituted with transduced mobilized PBSC or bone marrow cells. However, in both cases the CFU-S content in the bone marrow was reduced fivefold to 10-fold compared with the concentration of CFU-S in mice transplanted with nontransduced bone marrow. The SRA of CFU-S in mice reconstituted with peripheral blood and bone marrow cells was the same 1.5 months posttransplantation, but after an additional 4 months, SRA of mice reconstituted with bone marrow cells was fivefold higher as compared with those engrafted by PBSC. The integration of the human ADA gene was observed during 9 months in about 60% of studied CFU-S. The proportion of marked colonies sharply decreased 1 year following reconstitution. One to 9 individually labeled clones could be shown simultaneously by Southern blot hybridization in the same reconstituted mice during the whole period of observation. The time of clone existence was about 3 months. We conclude that long-term marrow repopulating cells mobilized into circulation by treatment with granulocyte colony-stimulating factor (G-CSF ) and stem cell factor (SCF ) are capable of maintaining lifelong polyclonal hematopoiesis in reconstituted mice.
Vascular Cell Adhesion Molecule-1 Is Expressed by Cortical Thymic Epithelial Cells and Mediates Thymocyte Adhesion. Implications for the Function of α4β1 (VLA4) Integrin in T-Cell Development
Authors: Salomon Daniel R. , Crisa Laura , Mojcik Christopher F. , Ishii Jennifer K. , Klier George , Shevach Ethan M.
Published: October 2019

T-cell development requires a series of discrete selection and activation signals delivered to maturing progenitors in the thymic cortex and medulla. We have previously shown the constitutive activity of the integrin, α4β1 (VLA4), on a unique subpopulation of immature cortical thymocytes and proposed a role for integrin-mediated adhesion in positive selection by cortical epithelium. In the present report we show that thymic epithelial cell lines express vascular cell adhesion molecule-1 (VCAM-1) a high-affinity ligand for α4β1, and that VCAM-1 mediates thymocyte binding to these lines. Immunohistochemistry and confocal microscopy show that VCAM-1 is selectively expressed in situ by thymic epithelium in the cortex and corticomedullary junction, two locations at which VCAM-1 could determine the interaction between immature thymocytes and selecting elements on epithelial cells. In parallel, we confirmed that fibronectin (FN), the alternative ligand for α4β1, is expressed predominantly in the medulla. These results suggest that VCAM-1 is an adhesive ligand in the thymic cortex for the activated form of α4β1 constitutively expressed during development by immature double positive thymocytes. The structural segregation of the alternative ligand, FN, to the medulla suggests that medullary FN may regulate the migration, development, and export of more mature thymocytes.
Splenectomy in Far-Advanced Hodgkin’s Disease
Authors: SYKES MARGUERITE P. , KARNOFSKY DAVID A. , MCNEER GORDON P. , CRAVER LLOYD F.
Published: October 2019

Five patients with far-advanced Hodgkin’s disease and with evidence of hematopoietic failure were treated by splenectomy in order to determine whether: (1) the course of the disease could be modified, (2) the hematologic picture improved, and (3) responsiveness to nitrogen mustard or x-ray therapy restored. These patients showed a transient slight improvement in their hematologic status, but the course of the disease possibly was accelerated, and the patients all died within thirteen weeks, without showing renewed suitability or increased responsiveness to therapy.

On the basis of our data and a review of thirty cases from the literature, it is concluded that splenectomy is not a useful procedure its Hodgkin’s disease, except for certain specific indications. These may be: (1) an apparently solitary splenic tumor; (2) acquired hemolytic anemia, although this process may be better controlled in some cases by treating the underlying Hodgkin’s disease with x-rays, nitrogen mustard, or triethylene melamine ; (3) thrombocytopenic purpura, which appears to be more profound than is to be expected from the severity and extent of Hodgkin’s disease; and (4) hypersplenism. Hematopoietic depression in the vast majority of patients with Hodgkin’s disease, however, cannot be attributed to splenic overactivity or malfunction.
Characterization of Cell Cycle Status and E2F Complexes in Mobilized CD34+ Cells Before and After Cytokine Stimulation
Authors: Williams Catherine D. , Linch David C. , Watts Mike J. , Thomas N. Shaun B.
Published: October 2019

Mobilized peripheral blood progenitors (CD34+ cells) have been shown to be either in the G0 or G1 phase of the cell cycle. In this study, it is shown that they are small cells with low protein content suggestive of G0. Support for this is provided by showing that the principal E2F complex consists of hypophosphorylated p130, E2F-4, and DP-1. The E2F-4 is more highly phosphorylated than in quiescent T cells. In response to cytokines in vitro, the CD34+ cells start to enter G1 within 8 hours and enter S-phase at about 48 hours. As cells enter G1, E2F-4 is dephosphorylated to several hypophosphorylated forms and three new DNA-binding complexes appear, including one containing E2F-4, DP-1, and p107. We suggest that mobilized CD34+ cells may be maintained in G0 by p130, E2F-4, and DP-1 and the coordinate dephosphorylation of E2F-4 and hyperphosphorylation of p130 may be central to the initiation of proliferation.
HLA-DR–Mediated Signals for Hematopoiesis and Induction of Apoptosis Involve But Are Not Limited to a Nitric Oxide Pathway
Authors: Lee Jong Wook , Beckham Cassandra , Michel Bryce R. , Rosen Henry , Deeg H. Joachim
Published: October 2019

Cross-linking of major histocompatibility complex (MHC) class II antigens by anti-HLA-DR monoclonal antibody (MoAb; H81.9; IgG2a) results in inhibition of hematopoiesis in canine and human models. Inhibition of hematopoiesis is associated with apoptosis in a proportion of marrow cells. Since in murine macrophages class II cross-linking triggers nitric oxide (NO) production, and NO is thought to affect regulation of hematopoiesis, we investigated whether NO was involved in our models. In murine J774 monocytes/macrophages, MoAb H81.9 did induce NO. NO production was blocked by NG-monomethyl-L-arginine (NMMA), an inhibitor of NO synthase (NOS), and by the antioxidant N-acetylcysteine (NAC). In human and canine long-term marrow cultures (LTMCs) and in enriched marrow monocytes, however, no measurable increase in NO production was noted after H81.9 exposure. Nevertheless, NAC protected LTMCs against H81.9 induced inhibition of hematopoiesis. Therefore, we determined the effect of an exogenous NO donator, sin-1 (3-morpholinosydnonimine), on canine and human LTMCs and on apoptosis. Sin-1 at concentrations ≥100 μg/mL inhibited LTMCs and induced apoptosis; at low concentrations (1 μg/mL), however, sin-1 stimulated the generation of colony-forming unit granulocyte-macrophage. Combined treatment with sin-1 at 100 μg/mL and MoAb H81.9 resulted in profound inhibition of hematopoiesis in both canine and human LTMCs, and had an additive effect on apoptosis. At 1 μg/mL sin-1 counteracted the effect of H81.9 on hematopoiesis. The effect of sin-1 on apoptosis and hematopoiesis in LTMC was largely prevented by NAC. These results are consistent with the hypothesis that HLA-DR mediated apoptosis and inhibition of hematopoiesis involve oxidative stress. However, the biphasic response of hematopoiesis to sin-1 suggests a complex regulatory network possibly related to differences in NO sensitivity of distinct subpopulations of cells. Signals in addition to NO appear to be involved in the effect of anti-HLA-DR MoAb on hematopoiesis.
Prognostic Significance of Bcl-2 Protein Expression and Bcl-2 Gene Rearrangement in Diffuse Aggressive Non-Hodgkin's Lymphoma
Authors: Gascoyne Randy D. , Adomat Sheryle A. , Krajewski Stanislaw , Krajewska Maryla , Horsman Douglas E. , Tolcher Anthony W. , O'Reilly Susan E. , Hoskins Paul , Coldman Andrew J. , Reed John C. , Connors Joseph M.
Published: October 2019

The prognostic significance of Bcl-2 protein expression and bcl-2 gene rearrangement in diffuse large cell lymphomas (DLCL) is controversial. Bcl-2 protein expression prevents apoptosis and may have an important role in clinical drug resistance. The presence of a bcl-2 gene rearrangement in de novo DLCL suggests a possible follicle center cell origin and perhaps a distinct clinical behavior more akin to low-grade non-Hodgkin's lymphoma (NHL). The purpose of this study was to determine the impact of Bcl-2 protein expression and bcl-2 gene rearrangement (mbr and mcr) on survival of a cohort of patients with DLCL who were uniformly evaluated and treated with effective chemotherapy. Patients included the original MACOP-B cohort (n = 121) and the initial 18 patients treated with the VACOP-B regimen (total = 139). All patients had advanced-stage disease, were 16 to 70 years old, and corresponded to Working Formulation categories F, G, or H. No patients had prior treatment, discordant lymphoma, or human immunodeficiency virus seropositivity. Paraffin sections from diagnostic biopsies were analyzed for bcl-2 gene rearrangement including mbr and mcr breakpoints by polymerase chain reaction and Bcl-2 protein expression by immunohistochemistry. With a median follow-up of 81 months, overall (OS), disease-free (DFS), and relapse-free survival (RFS) were measured to determine the prognostic significance of these parameters. Analyzable DNA was present in 118 of 139 (85%) cases, with 14 demonstrating a bcl-2 rearrangement (11 mbr, 3 mcr). All 14 of these bcl-2 gene rearrangement-positive cases were found in the 102 patients with a B-cell immunophenotype, but the presence of this rearrangement had no significant influence on survival. Bcl-2 protein expression was interpretable in 116 of 139 (83%) cases, with immunopositivity detected in 54 of 116 (47%). Using a cut-off of greater than 10% Bcl-2 immunopositive tumor cells for analysis, positive Bcl-2 protein expression was seen in 28 of 116 (24%) patients and the presence of this expression correlated with decreased 8-year OS (34% v 60%, P < .01), DFS (32% v 66%, P < .001), and RFS (25% v 59%, P < .001). Bcl-2 protein expression remained significant in multivariate analysis that included the clinical international prognostic index factors and immunophenotype (P < .02). In conclusion, although bcl-2 gene rearrangement status could not be shown to have an impact on outcome, Bcl-2 protein expression is a strong significant predictor of OS, DFS, and RFS in DLCLs.
RARα1/RARα2-PML mRNA Expression in Acute Promyelocytic Leukemia Cells: A Molecular and Laboratory-Clinical Correlative Study
Authors: Li Yun-Ping , Andersen Janet , Zelent Arthur , Rao Sreenivas , Paietta Elisabeth , Tallman Martin S. , Wiernik Peter H. , Gallagher Robert E.
Published: October 2019

In addition to the major fusion gene PML-RARα, the t(15; 17) in acute promyelocytic leukemia (APL) produces the reciprocal fusion gene RARα-PML. To determine the scope of RARα-containing mRNA expression in APL cells, we tested PML-RARα–positive APL cells for the presence of mRNAs initiated from two distinct RARα gene promoters, α1 and α2. From the normal allele, both RARα1 and RARα2 mRNAs were expressed in all APL cases (N = 24). From the translocated allele, RARα1-PML mRNA was expressed in 77% and RARα2-PML mRNA in 28% of cases (N = 98). RARα2-PML mRNA was not observed in the absence of RARα1-PML mRNA. There was no association between RARα1-PML or RARα2-PML mRNA expression and the type of PML-RARα mRNA formed by either 5′ or 3′ breaksites in the PML gene. RARα1-PML mRNAs and RARα2-PML mRNAs from 5′ PML breaksite cases coded for full-length RARα-PML proteins but RARα2-PML mRNAs from 3′ PML breaksite cases encoded a truncated RARα2 peptide. RARα1/α2-PML mRNA expression was not associated with differences in APL cell sensitivity to all-trans retinoic acid(tRA)-induced differentiation in vitro or in clinical outcome after tRA or chemotherapy induction therapy (protocol E2491). Our analysis indicated that RARα1/α2-PML mRNA expression markedly differs from normal RARα1/α2 mRNA expression, that the difference in RARα1-PML and RARα2-PML mRNA expression frequency is primarily related to the genomic separation of the RARα1 and RARα2 coding exons, and that variations in RARα1/α2-PML mRNA expression likely have no clinically relevant function in APL cells.
Sensitivity to a Metabolite of Diclofenac as a Cause of Acute Immune Hemolytic Anemia
Authors: Bougie D. , Johnson S.T. , Weitekamp L.A. , Aster R.H.
Published: October 2019

A 75-year-old woman taking the nonsteroidal anti-inflammatory drug diclofenac (DCF ) presented with acute Coombs-positive hemolytic anemia and subsequently developed renal failure. A drug-dependent antibody specific for red blood cells (RBCs) could not be demonstrated by in vitro testing with DCF. However, her serum was found to contain an IgM antibody that reacted strongly with RBCs in the presence of urine from any of four subjects who had ingested DCF. The active substance in urine was isolated, subjected to high-performance liquid chromatographic (HPLC) analysis, and found to be a glucuronide conjugate of a known DCF metabolite, 4′-hydroxydiclofenac (4′-OH DCF ). Negative results were obtained with four other DCF metabolites. Two 4′-OH DCF glucuronides were synthesized in vitro using a liver microsomal system. One promoted agglutination of normal RBCs by the patient's serum and was identified as the glucuronide ester of 4′-OH DCF by proton nuclear magnetic resonance (NMR) analysis. Studies with a panel of RBCs showed that the patient's antibody reacted preferentially with the e antigen of the Rh system. Acute immune hemolytic anemia in this patient appears to have been caused by sensitization to DCF modified by 4′ hydroxylation and glucuronidation. This is the first reported example of immune cytopenia caused by sensitivity to a glucuronide conjugate of a drug metabolite. Since glucuronidation is a common pathway of drug metabolism, studies of the frequency with which glucuronide derivatives of primary medications cause immune cytopenia seem warranted.
Selective Expansion of Primitive Normal Hematopoietic Cells in Cytokine-Supplemented Cultures of Purified Cells From Patients With Chronic Myeloid Leukemia
Authors: Petzer A.L. , Eaves C.J. , Barnett M.J. , Eaves A.C.
Published: October 2019

We have previously reported that primitive normal hematopoietic cells detectable as long-term culture-initiating cells (Ph-LTC-IC) are present at high levels in the blood of some patients with chronic myeloid leukemia (CML). We now show that this population can be expanded several-fold when highly purified CD34+CD38− cells isolated from the blood of such patients are cultured for 10 days in a serum-free medium containing 100 ng/mL of Flt3-ligand and Steel factor and 20 ng/mL of interleukin-3 (IL-3) and IL-6, and granulocyte colony-stimulating factor. In similar cultures initiated with CD34+CD38− cells from CML blood samples in which all of the LTC-IC were leukemic (Ph+), Ph+ LTC-IC activity was rapidly lost both in the presence and absence of admixed CD34+CD38− cells isolated from normal marrow. Conversely, the ability of normal LTC-IC to expand their numbers was shown to be independent of the presence of Ph+LTC-IC and later types of Ph+colony-forming cell (CFC) progenitors. In contrast to the LTC-IC, CFC were consistently -a m p l i f i e d  i n  c u l t u r e s  i n i t i a t e d  w i t h  C M L - d e r i v e d -CD34+CD38− cells and the additional CFC present after 10 days were, like the starting population of CFC, almost exclusively Ph+ regardless of the genotype(s) of the LTC-IC in the original CML samples. Amplification of the Ph+CFC population in these cultures showed the same factor dependence as previously demonstrated for the in vitro expansion of CFC from normal marrow CD34+CD38− cells. Ph+LTC-IC disappeared regardless of the cytokines present. Taken together these findings support a model of CML in which the leukemic stem cells are characterized by a decreased probability of self-renewal and an increased probability of differentiation. In addition, they suggest new opportunities for improving the treatment of CML using strategies that require autologous stem cell rescue.
Mutations in the Fas Antigen in Patients With Multiple Myeloma
Authors: Landowski Terry H. , Qu Ning , Buyuksal Ibrahim , Painter Jeffrey S. , Dalton William S.
Published: October 2019

Programmed cell death, or apoptosis, is well documented as a physiological means of eliminating activated lymphocytes and maintaining immune homeostasis. Apoptosis has also been implicated in the targeting of tumor cells by cytotoxic T lymphocytes and natural killer cells. One of the two primary mechanisms used in cell-mediated cytotoxicity is the Fas/FasLigand system. Activated or transformed cells expressing the Fas antigen on their surface are susceptible to killing by immune effector cells that express the Fas ligand. Many neoplastic cells, including those derived from patients with multiple myeloma, express Fas antigen on their surface, but do not undergo apoptosis in response to antigen crosslinking. One possibility for the lack of Fas-mediated apoptosis includes mutations in the Fas antigen. Loss of function mutations in the Fas antigen have been associated with congenital autoimmune disease in humans, and have been defined as the genetic defect the in lpr mice. Mutations in the Fas antigen have not been previously described in cancer patients. In this study, we show that mutations occur in the Fas antigen which may cause loss of function and contribute to the pathogenesis of the neoplastic disease, multiple myeloma. Using reverse transcriptase-polymerase chain reaction (RT-PCR), single-stranded conformation polymorphism (SSCP) analysis, and DNA sequencing, we examined the cDNA structure of the Fas antigen in 54 bone marrow (BM) specimens obtained from myeloma patients. Six patient specimens (11%) did not express detectable levels of Fas antigen mRNA. Of the 48 BM specimens which did express Fas antigen, 5 (10%) displayed point mutations. All of the mutations identified were located in the cytoplasmic region of the Fas antigen known to be involved in transduction of an apoptotic signal. Two separate individuals demonstrated an identical mutation at a site previously shown to be mutated in the congenital autoimmune syndrome, ALPS. One patient exhibited a point mutation at a site only two amino acids removed from the documented lesion of the lprcg mouse. Although the functional status of these point mutations remains to be determined, we propose that Fas antigen mutations may contribute to the pathogenesis and progression of myeloma in some patients.
The Bmx Tyrosine Kinase Induces Activation of the Stat Signaling Pathway, Which Is Specifically Inhibited by Protein Kinase Cδ
Authors: Saharinen Pipsa , Ekman Niklas , Sarvas Krista , Parker Peter , Alitalo Kari , Silvennoinen Olli
Published: October 2019

Members of the hematopoietically expressed Tec tyrosine kinase family have an important role in hematopoietic signal transduction, as exemplified by the crucial role of Btk for B-cell differentiation and activation. Although a variety of cell surface receptors have been found to activate Tec tyrosine kinases, the specific signaling pathways and substrate molecules used by Tec kinases are still largely unknown. In this study a Tec family kinase, Bmx, was found to induce activation of the Stat signaling pathway. Bmx induced the tyrosine phosphorylation and DNA binding activity of all the Stat factors tested, including Stat1, Stat3, and Stat5, both in mammalian and insect cells. Bmx also induced transcriptional activation of Stat1- and Stat5-dependent reporter genes. Other cytoplasmic tyrosine kinases, Syk, Fyn, and c-Src, showed no or only weak ability to activate Stat proteins. Expression of Bmx in mammalian cells was found to induce activation of endogenous Stat proteins without activation of endogenous Jak kinases. We further analyzed the Bmx-mediated activation of Stat1, which was found to be regulated by protein kinase C δ (PKCδ) isoform, but not β 1, ε, or ζ isoforms, leading to inhibition of Stat1 tyrosine phosphorylation. In conclusion, these studies show that Bmx, a Tec family kinase, can function as an activator of the Stat signaling pathway and identify a role for PKCδ in the regulation of Bmx signaling.
Synergistic Action of Flt3 and gp130 Signalings in Human Hematopoiesis
Authors: Ebihara Yasuhiro , Tsuji Kohichiro , Lyman Stewart D. , Sui Xingwei , Yoshida Makoto , Muraoka Kenji , Yamada Kaoru , Tanaka Ryuhei , Nakahata Tatsutoshi
Published: October 2019

We recently showed that c-kit signal synergizes with glycoprotein (gp)130 signal mediated by a complex of interleukin (IL)-6 and soluble IL-6 receptor (IL-6/sIL-6R) to stimulate the expansion of human primitive hematopoietic progenitor cells and erythropoietin-independent erythropoiesis. In the present study, we examined the effect of a ligand for Flt3 (FL), whose receptor tyrosine kinase is closely related to c-kit, in combination with IL-6/sIL-6R on human hematopoiesis in vitro. In serum-containing methylcellulose clonal culture of cord blood CD34+ cells, whereas FL alone stimulated only granulocyte-macrophage (GM) colony formation, erythroid bursts and mixed colonies in addition to GM colonies were induced by FL with IL-6/sIL-6R, but not IL-6/sIL-6R alone. In suspension culture, CD34+ cells generated a small number of myeloid cells in the presence of FL or IL-6/sIL-6R alone. However, the addition of IL-6/sIL-6R to the culture with FL induced the generation of a significant number of erythroid cells and megakaryocytes in addition to myeloid cells. The combination of FL and IL-6/sIL-6R also induced a remarkable expansion of GM colony- and erythroid burst-forming cells and multipotential progenitors, although FL or IL-6/sIL-6R alone induced the generation of only a small number of progenitors for GM colonies. The synergistic effects of FL and IL-6/sIL-6R were confirmed in serum-free clonal and suspension cultures. In addition, the addition of anti-human gp130 monoclonal antibodies abrogated the synergistic action. These results indicate that Flt3 signal, as well as c-kit signal, synergizes with gp130 signal to stimulate human myelopoiesis, erythropoiesis and megakaryopoiesis, and the expansion of primitive multipotential hematopoietic progenitor cells.
Functional Heterogeneity of Human CD34+ Cells Isolated in Subcompartments of the G0 /G1 Phase of the Cell Cycle
Authors: Gothot André , Pyatt Robert , McMahel Jon , Rice Susan , Srour Edward F.
Published: October 2019

Using simultaneous Hoechst 33342 (Hst) and Pyronin Y (PY) staining for determination of DNA and RNA content, respectively, human CD34+ cells were isolated in subcompartments of the G0 /G1 phase of the cell cycle by flow cytometric cell sorting. In both bone marrow (BM) and mobilized peripheral blood (MPB) CD34+ cells, primitive long-term hematopoietic culture-initiating cell (LTHC-IC) activity was higher in CD34+ cells isolated in G0 (G0CD34+ cells) than in those residing in G1 (G1CD34+ cells). However, as MPB CD34+ cells displayed a more homogeneous cell-cycle status within the G0 /G1 phase and a relative absence of cells in late G1 , DNA/RNA fractionation was less effective in segregating LTHC-IC in MPB than in BM. BM CD34+ cells belonging to four subcompartments of increasing RNA content within the G0 /G1 phase were evaluated in functional assays. The persistence of CD34 expression in suspension culture was inversely correlated with the initial RNA content of test cells. Multipotential progenitors were present in G0 or early G1 subcompartments, while lineage-restricted granulomonocytic progenitors were more abundant in late G1 . In vitro hematopoiesis was maintained for up to 6 weeks with G0CD34+ cells, whereas production of clonogenic progenitors was more limited in cultures initiated with G1CD34+ cells. To test the hypothesis that primitive LTHC-ICs would reenter a state of relative quiescence after in vitro division, BM CD34+ cells proliferating in ex vivo cultures were identified from their quiescent counterparts by a relative loss of membrane intercalating dye PKH2, and were further fractionated with Hst and PY. The same functional hierarchy was documented within the PKH2dim population whereby LTHC-IC frequency was higher for CD34+ cells reselected in G0 after in vitro division than for CD34+ cells reisolated in G1 or in S/G2 + M. However, the highest LTHC-IC frequency was found in quiescent PKH2bright CD34+ cells. Together, these results support the concept that cells with distinct hematopoietic capabilities follow different pathways during the G0 /G1 phase of the cell cycle both in vivo and during ex vivo culture.
Analysis of the Involvement of the von Willebrand Factor–Glycoprotein Ib Interaction in Platelet Adhesion to a Collagen-Coated Surface Under Flow Conditions
Authors: Moroi Masaaki , Jung Stephanie M. , Nomura Shosaku , Sekiguchi Sadayoshi , Ordinas Antonio , Diaz-Ricart Maribel
Published: October 2019

The requisite initial reaction for in vivo thrombus formation in flowing blood is platelet adhesion to the exposed surface of the extracellular matrix. The contribution of von Willebrand factor (vWF ) in plasma and glycoprotein (GP) Ib on the platelet membrane to platelet adhesion has been well-documented. We have recently developed a procedure (the “flow adhesion assay”) for measuring platelet adhesion under flow conditions that allowed us to characterize platelet adhesion to a collagen-coated surface. Here, we apply our method to analyze platelet adhesion to a vWF-coated surface to determine how this might differ from adhesion to a collagen-coated surface. Platelet adhesion to the vWF-coated surface was monitored as the linear increase in the area occupied by adherent platelets. The fluorescence image showed that platelets adhering to the vWF surface were mainly single platelets, and if any were present, the platelet aggregates were small, this being the primary difference from the adhesion to a collagen surface, where adherent platelets were mostly in aggregates. The flow adhesion assay detected the movement of platelets on the vWF surface, suggesting the reversible binding of vWF with platelets. The velocity of the platelets increased at higher shear rates or at lower vWF densities on the surface. Treatment of the vWF-coated surface with the aggregating agent botrocetin before initiation of blood flow increased platelet adhesion while dramatically decreasing the velocity of platelet movement. The present observations on the adhesion of platelets to the vWF-pretreated collagen surface and measurements of the velocity of platelets moving on the collagen surface suggest that the first interaction on the collagen-coated surface is the binding of vWF molecules to the collagen surface. This small number of vWF molecules would serve to attract and slow platelets flowing near the surface. This would facilitate the actual adhesion to the collagen surface that is mainly generated by the interaction between platelet collagen receptors, including GP Ia/IIa and GP VI, with collagen.
Neoexpression of the c-met/Hepatocyte Growth Factor-Scatter Factor Receptor Gene in Activated Monocytes
Authors: Beilmann Mario , Odenthal Margarete , Jung Waltraud , Vande Woude George F. , Dienes Hans-Peter , Schirmacher Peter
Published: October 2019

Hepatocyte growth factor-scatter factor (HGF-SF ) mediates mito-, moto-, and morphogenic effects through the MET receptor, a membrane bound tyrosine kinase. HGF-SF/MET signaling is mitogenic for a large number of epithelial and endothelial cells and activates organ regeneration. HGF-SF transcripts have been detected in various myeloid cell lines. Therefore, the potential role of HGF-SF/MET signaling for circulating cells of the immune system, especially under conditions of inflammation, was evaluated. Several B-lymphoid and myeloid cell lines were found to express HGF-SF or c-met transcripts, while activity of both genes was mutually exclusive with the exception of low level coexpression in two B-cell lines. HGF-SF transcripts were present in low quantities in freshly isolated peripheral blood mononuclear cells (PBMNCs). In contrast, c-met expression was not detected in freshly isolated cells from peripheral blood, but was induced in monocytes by activation of monocytic or T-cell function. HGF-SF incubation led to an increased c-fos steady state transcript level in myeloblastic K562 cells and moderately promoted cell viability of freshly isolated preactivated monocytes. c-met expression is thus established in activated monocytes, in particular under conditions resembling inflammation, making these cells accessible to functional effects of HGF-SF.
Methylation of the Epstein-Barr Virus Genome in Normal Lymphocytes
Authors: Robertson Keith D. , Ambinder Richard F.
Published: October 2019

Epstein-Barr virus (EBV) latent infection in B cells persists over years or decades despite a sustained cytotoxic immune response to viral antigens. We present data that methylated EBV DNA can be detected in the normal lymphocytes of healthy volunteers. Whereas methylation of foreign DNA has been recognized as a potential cellular defense mechanism, methylation of EBV DNA may be an essential part of the virus life cycle in vivo, explaining the persistence of virus-infected B cells in the face of immune surveillance. Methylation of the C promoter helps to prevent expression of the immunodominant antigens expressed from this promoter. First recognized in tumors, methylation-associated evasion of immune surveillance is not an aberration restricted to tumor tissue but is detected in normal EBV-infected lymphocytes. Methylation of the viral genome in latency also provides an explanation for the CpG suppression associated with EBV but not other large DNA viruses.
Gene Transfer into Marrow Repopulating Cells: Comparison Between Amphotropic and Gibbon Ape Leukemia Virus Pseudotyped Retroviral Vectors in a Competitive Repopulation Assay in Baboons
Authors: Kiem Hans-Peter , Heyward Scott , Winkler Aaron , Potter Jennifer , Allen James M. , Miller A. Dusty , Andrews Robert G.
Published: October 2019

Many diseases might be treated by gene therapy targeted to the hematopoietic system, but low rates of gene transfer achieved in humans and large animals have limited the application of this technique. We have developed a competitive hematopoietic repopulation assay in baboons to evaluate methods for improving gene transfer and have used this method to compare gene transfer rates for retroviral vectors having an envelope protein (pseudotype) from amphotropic murine retrovirus with similar vectors having an envelope protein derived from gibbon ape leukemia virus (GALV). We hypothesized that vectors with a GALV pseudotype might perform better based on our previous work with cultured human hematopoietic cells. CD34+ marrow cells from each of four untreated baboons were divided into two equal portions that were cocultivated for 48 hours with packaging cells producing equivalent titers of either amphotropic or GALV pseudotyped vectors containing the neo gene. The vectors contained small sequence differences to allow differentiation of cells genetically marked by the different vectors. Nonadherent and adherent cells from the cultures were infused into animals after they received a myeloablative dose of total body irradiation. Polymerase chain reaction (PCR) analysis for neo gene-specific sequences in colony-forming unit–granulocyte-macrophage from cell populations used for transplant showed gene transfer rates of 2.7%, 7.1%, <15%, and 3.9% with the amphotropic vectors and 7.1%, 11.3%, <15%, and 26.4% with the GALV pseudotyped vector. PCR analysis of peripheral blood and marrow cells after engraftment showed the neo gene to be present in all four animals analyzed at levels between 0.1% and 5%. Overall gene transfer efficiency was higher with the GALVpseudotyped vector than with the amphotropic vectors. Southern blot analysis in one animal confirmed a gene transfer efficiency of between 1% and 5%. The higher gene transfer efficiency with the GALV-pseudotyped vector correlated with higher levels of GALV receptor RNA compared with the amphotropic receptor in CD34+ hematopoietic cells. These results show that GALV-pseudotyped vectors are capable of transducing baboon marrow repopulating cells and may allow more efficient gene transfer rates for human gene therapy directed at hematopoietic cells. In addition, our data show considerable differences in gene transfer efficiency between individual baboons, suggesting that a competitive repopulation assay will be critical for evaluation of methods designed to improve gene transfer into hematopoietic stem cells.
Inhibition of Interferon Regulatory Factor-1 Expression Results in Predominance of Cell Growth Stimulatory Effects of Interferon-γ Due to Phosphorylation of Stat1 and Stat3
Authors: Sato Tadatsugu , Selleri Carmine , Young Neal S. , Maciejewski Jaroslaw P.
Published: October 2019

Interferon-γ (IFN-γ) is a potent inhibitor of hematopoiesis in vitro and has been implicated in the pathophysiology of human bone marrow failure syndromes. IFN-γ both inhibits cell cycling and induces expression of the Fas-receptor, resulting in subsequent apoptosis of hematopoietic progenitor cells. IFN regulatory factor-1 (IRF-1) mediates some of these suppressive effects by activation of downstream inducible genes, such as double-stranded RNA-activatable protein kinase and inducible nitric oxide synthase. However, under certain experimental conditions, IFN-γ appears to stimulate proliferation of hematopoietic cells. Based on the hypothesis that IFN-γ–receptor triggering may activate diverse signaling cascades, we designed experiments to determine which intracellular mechanisms (in addition to the IRF-1 transduction pathway) influence the biologic effects of IFN-γ. Using antisense technique, we inhibited the IRF-1–mediated pathway in KG1a cells stimulated with IFN-γ. In contrast to the suppressive effects of IFN-γ observed in control cells, untreated and IFN-γ–treated KG-1a cells that were transduced with retroviral vectors expressing IRF-1 antisense mRNA showed enhanced proliferation. The increased growth rate was associated with decreased levels of IRF-1 mRNA and protein but unchanged levels of IRF-2. We inferred that IFN-γ could also activate a stimulatory transduction pathway that, under specific conditions, may control the cellular response to this cytokine. The family of Stat proteins is involved in signal transduction of hematopoietic growth factors. We showed that, in KG-1a cells, IFN-γ also induced phosphorylation of Stat1 and Stat3, whereas p42 MAP kinase was phosphorylated regardless of the presence of IFN-γ. Using electrophoresis mobility shift assays, IFN-γ enhanced Stat1-Stat1 homodimer and Stat1-Stat3 heterodimer formation, suggesting that, in addition to inhibitory signals mediated by IRF-1, IFN-γ may activate proliferative signals by phosphorylation of Stat1 and Stat3 proteins. The observations made in experiments with KG-1a cells were confirmed in primary hematopoietic cells. After inhibition of the IRF-1 pathway by transduction of an antisense IRF-1 retrovirus into human CD34+ cells, IFN-γ produced an aberrant stimulatory effect on hematopoietic colony formation. Conversely, in control vector-transduced CD34+ cells, the typical inhibitory response to IFN-γ was seen. Our results indicate that inhibitory cytokines such as IFN-γ may exhibit diverse biologic effects depending on the intracellular balance of transcriptional regulators, in turn influenced by the activation and differentiation status of the target cells.
Signaling Functions of the Tyrosine Residues in the βc Chain of the Granulocyte-Macrophage Colony-Stimulating Factor Receptor
Authors: Okuda Keiko , Smith Lorie , Griffin James D. , Foster Rosemary
Published: October 2019

The granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor (GMR) is a heterodimeric receptor expressed by myeloid lineage cells. Binding of GM-CSF activates at least one receptor-associated tyrosine kinase, JAK2, and rapidly induces tyrosine phosphorylation of the GMR βc-chain (GMRβ), but not the GMR α-chain (GMRα). To examine the role of GMRβ tyrosine phosphorylaiton, each of the 8 tyrosine residues in the cytoplasmic domain of the human GMRβ was mutated to phenylalanine (GMRβ-F8), and this mutant receptor was expressed with wild-type GMRα in the interleukin-3–dependent murine hematopoietic cell line, Ba/F3. GM-CSF induced tyrosine phosphorylation of multiple cellular proteins in cells expressing GMRβ-F8 , including JAK2 and STAT5. However, GM-CSF–induced tyrosine phosphorylation of both SHP2 and SHC was reduced or absent compared with wild-type. Next, a series of 8 receptors were generated, each containing only a single, restored, tyrosine residue. Tyrosine 577 was found to be sufficient to regenerate GM-CSF–dependent phosphorylation of SHC, and any of Y577, Y612, or Y695 was sufficient to regenerate GM-CSF–inducible phosphorylation of SHP2. Despite the signaling defect to SHC and SHP2, Ba/F3 cells expressing GMRβ-F8 were still able to proliferate in response to 10 ng/mL of human GM-CSF, although mitogenesis was impaired compared with wild-type GMRβ, and this effect was even more prominent at lower concentrations of GM-CSF (1 ng/mL). Overall, these results indicate that GMRβ tyrosine residues are not necessary for activation of the JAK/STAT pathway or for proliferation, viability, or adhesion signaling in Ba/F3 cells, although tyrosine residues significantly affect the magnitude of the response. However, specific tyrosine residues are needed for activation of SHC and SHP2.