Hepatitis C Virus Infection Involves CD34+Hematopoietic Progenitor Cells in Hepatitis C Virus Chronic Carriers

Blood ◽  
1998 ◽  
Vol 92 (9) ◽  
pp. 3328-3337 ◽  
Author(s):  
Domenico Sansonno ◽  
Claudio Lotesoriere ◽  
Vito Cornacchiulo ◽  
Massimo Fanelli ◽  
Pietro Gatti ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Progenitor Cells ◽  
Hcv Infection ◽  
Hematopoietic Progenitor Cells ◽  
Hcv Rna ◽  
Rt Pcr ◽  
Hematopoietic Progenitor ◽  
Cd34 Cells

Although hepatitis C virus (HCV) mainly affects hepatocytes, infection is widespread and involves immunologically privileged sites. Whether lymphoid cells represent further targets of early HCV infection, or whether other cells in the hematopoietic microenvironment may serve as a potential virus reservoir, is still unclear. We studied whether pluripotent hematopoietic CD34+ cells support productive HCV infection and can be used to establish an in vitro infection system for HCV. Six patients were selected as part of a cohort of HCV chronic carriers who developed a neoplastic disease. Reverse transcriptase-polymerase chain reaction (RT-PCR) and branched DNA signal amplification assays were used to detect and quantitate HCV RNA in extracted nucleic acids from purified bone marrow and peripheral blood CD34+ cells. Direct in situ RT-PCR, flow cytometry analysis, and immunocytochemistry were applied to demonstrate specific viral genomic sequences and structural and nonstructural virus-related proteins in intact cells. Results indicated that both positive and negative HCV RNA strands and viral proteins were present in CD34+ cells from all HCV-positive patients and in none of the controls. Additional experiments showed that a complete viral cycle took place in CD34+ cells in vitro. Spontaneous increases in viral titers indicated that virions were produced by infected hematopoietic progenitor cells. To further define the cellular tropism, we attempted to infect CD34+ cells in vitro. We were unable to demonstrate viral uptake by cells. These findings suggest that HCV replication can occur in the early differentiation stages of hematopoietic progenitor cells, and that they may be an important source of virus production. © 1998 by The American Society of Hematology.

Hepatitis C Virus Infection Involves CD34+Hematopoietic Progenitor Cells in Hepatitis C Virus Chronic Carriers

Blood ◽  
1998 ◽  
Vol 92 (9) ◽  
pp. 3328-3337 ◽  
Author(s):  
Domenico Sansonno ◽  
Claudio Lotesoriere ◽  
Vito Cornacchiulo ◽  
Massimo Fanelli ◽  
Pietro Gatti ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Progenitor Cells ◽  
Hcv Infection ◽  
Hematopoietic Progenitor Cells ◽  
Hcv Rna ◽  
Rt Pcr ◽  
Hematopoietic Progenitor ◽  
Cd34 Cells

Abstract Although hepatitis C virus (HCV) mainly affects hepatocytes, infection is widespread and involves immunologically privileged sites. Whether lymphoid cells represent further targets of early HCV infection, or whether other cells in the hematopoietic microenvironment may serve as a potential virus reservoir, is still unclear. We studied whether pluripotent hematopoietic CD34+ cells support productive HCV infection and can be used to establish an in vitro infection system for HCV. Six patients were selected as part of a cohort of HCV chronic carriers who developed a neoplastic disease. Reverse transcriptase-polymerase chain reaction (RT-PCR) and branched DNA signal amplification assays were used to detect and quantitate HCV RNA in extracted nucleic acids from purified bone marrow and peripheral blood CD34+ cells. Direct in situ RT-PCR, flow cytometry analysis, and immunocytochemistry were applied to demonstrate specific viral genomic sequences and structural and nonstructural virus-related proteins in intact cells. Results indicated that both positive and negative HCV RNA strands and viral proteins were present in CD34+ cells from all HCV-positive patients and in none of the controls. Additional experiments showed that a complete viral cycle took place in CD34+ cells in vitro. Spontaneous increases in viral titers indicated that virions were produced by infected hematopoietic progenitor cells. To further define the cellular tropism, we attempted to infect CD34+ cells in vitro. We were unable to demonstrate viral uptake by cells. These findings suggest that HCV replication can occur in the early differentiation stages of hematopoietic progenitor cells, and that they may be an important source of virus production. © 1998 by The American Society of Hematology.

scholarly journals In vitro expansion of hematopoietic progenitor cells induces functional expression of Fas antigen (CD95)

Blood ◽  
1996 ◽  
Vol 88 (8) ◽  
pp. 2871-2877 ◽  
Author(s):  
K Takenaka ◽  
K Nagafuji ◽  
M Harada ◽  
S Mizuno ◽  
T Miyamoto ◽  
...  
Keyword(s):  
Growth Factors ◽  
Progenitor Cells ◽  
Calf Serum ◽  
Functional Expression ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Growth Factors ◽  
Hematopoietic Progenitor ◽  
Fas Antigen ◽  
Cd34 Cells

Fas antigen (Fas Ag; CD95) is a cell surface molecule that can mediate apoptosis. Bcl-2 is a cytoplasmic molecule that prolongs cellular survival by inhibiting apoptosis. To investigate the role of both molecules in hematopoiesis, we evaluated the expression of Fas Ag and Bcl-2 on CD34+ hematopoietic progenitor cells expanded in vitro. CD34+ cells isolated from bone marrow were cultured in iscove's modified Dulbecco's medium supplemented with 10% fetal calf serum, 1% bovine serum albumin, 50 ng/mL stem cell factor, 50 ng/mL interleukin-3 (IL-3), 50 ng/mL IL-6, 100 ng/mL granulocyte colony-stimulating factor, and 3 U/mL erythropoietin for 7 days. Colony-forming unit of granulocytes/macrophages (CFU-GM) and burst-forming unit of erythroids (BFU-E) were expanded 6.9-fold and 8.8-fold in number at day 5 of culture, respectively. Freshly isolated CD34+ cells did not express Fas Ag, whereas approximately half of them expressed Bcl-2. CD34+ cells cultured with hematopoietic growth factors gradually became positive for Fas Ag and rapidly lost Bcl-2 expression. Furthermore, apoptosis was induced in the cultured CD34+ population when anti-Fan antibody (IgM; 1 microgram/mL) was added, as shown by significant decrease in the number of viable cells, morphologic changes, induction of DNA fragmentation, and significant decrease in the number of clonogenic progenitor cells including CFU. GM and BFU-E. These results indicate that functional expression of Fas Ag is induced on CD34+ cells expanded in vitro in the presence of hematopoietic growth factors. Induction of Fas Ag and downregulation of Bcl-2 may be expressed as part of the differentiation program of hematopoietic cells and may be involved in the regulation of hematopoiesis.

scholarly journals Intracellular Immunization of Rhesus CD34+ Hematopoietic Progenitor Cells With a Hairpin Ribozyme Protects T Cells and Macrophages From Simian Immunodeficiency Virus Infection

Blood ◽  
1997 ◽  
Vol 90 (12) ◽  
pp. 4822-4831 ◽  
Author(s):  
Michael Rosenzweig ◽  
Douglas F. Marks ◽  
Donna Hempel ◽  
Marina Heusch ◽  
Günter Kraus ◽  
...  
Keyword(s):  
T Cells ◽  
Progenitor Cells ◽  
Hematopoietic Progenitor Cells ◽  
Hairpin Ribozyme ◽  
Hematopoietic Progenitor ◽  
Cd34 Cells ◽  
Immunodeficiency Virus ◽  
Cell Gene ◽  
Stem Cell Gene Therapy

Abstract Evaluation of candidate genes for stem cell gene therapy for acquired immunodeficiency syndrome (AIDS) has been limited by the difficulty of supporting in vitro T-cell differentiation of genetically modified hematopoietic progenitor cells. Using a novel thymic stromal culture technique, we evaluated the ability of a hairpin ribozyme specific for simian immunodeficiency virus (SIV) and human immunodeficiency virus type 2 (HIV-2) to inhibit viral replication in T lymphocytes derived from transduced CD34+ progenitor cells. Retroviral transduction of rhesus macaque CD34+ progenitor cells with a retroviral vector (p9456t) encoding the SIV-specific ribozyme and the selectable marker neomycin phosphotransferase in the presence of bone marrow stroma and in the absence of exogenous cytokines resulted in efficient transduction of both colony-forming units and long-term culture-initiating cells, with transduction efficiencies ranging between 21% and 56%. After transduction, CD34+ cells were cultured on rhesus thymic stromal culture (to support in vitro differentiation of T cells) or in the presence of cytokines (to support differentiation of macrophage-like cells). After expansion and selection with the neomycin analog G418, cells derived from transduced progenitor cells were challenged with SIV. CD4+ T cells derived from CD34+ hematopoietic cells transduced with the ribozyme vector p9456t were highly resistant to challenge with SIV, exhibiting up to a 500-fold decrease in SIV replication, even after high multiplicities of infection. Macrophages derived from CD34+ cells transduced with the 9456 ribozyme exhibited a comparable level of inhibition of SIV replication. These results show that a hairpin ribozyme introduced into CD34+ hematopoietic progenitor cells can retain the ability to inhibit AIDS virus replication after T-cell differentiation and support the feasibility of intracellular immunization of hematopoietic stem cells against infection with HIV and SIV. Protection of multiple hematopoietic lineages with the SIV-specific ribozyme should permit analysis of stem cell gene therapy for AIDS in the SIV/macaque model.

scholarly journals Synergistic Action of Flt3 and gp130 Signalings in Human Hematopoiesis

Blood ◽  
1997 ◽  
Vol 90 (11) ◽  
pp. 4363-4368 ◽  
Author(s):  
Yasuhiro Ebihara ◽  
Kohichiro Tsuji ◽  
Stewart D. Lyman ◽  
Xingwei Sui ◽  
Makoto Yoshida ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Receptor Tyrosine Kinase ◽  
Synergistic Effects ◽  
Myeloid Cells ◽  
Synergistic Action ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Cd34 Cells ◽  
Cord Blood Cd34

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.

scholarly journals The Chemokine SDF-1 Is a Chemoattractant for Human CD34+ Hematopoietic Progenitor Cells and Provides a New Mechanism to Explain the Mobilization of CD34+ Progenitors to Peripheral Blood

1997 ◽  
Vol 185 (1) ◽  
pp. 111-120 ◽  
Author(s):  
A. Aiuti ◽  
I.J. Webb ◽  
C. Bleul ◽  
T. Springer ◽  
J.C. Gutierrez-Ramos
Keyword(s):  
Progenitor Cells ◽  
Peripheral Blood ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Stromal Cell Derived Factor ◽  
Transient Elevation

Hematopoietic progenitor cells migrate in vitro and in vivo towards a gradient of the chemotactic factor stromal cell-derived factor-1 (SDF-1) produced by stromal cells. This is the first chemoattractant reported for human CD34+ progenitor cells. Concentrations of SDF-1 that elicit chemotaxis also induce a transient elevation of cytoplasmic calcium in CD34+ cells. SDF-1-induced chemotaxis is inhibited by pertussis toxin, suggesting that its signaling in CD34+ cells is mediated by seven transmembrane receptors coupled to Gi proteins. CD34+ cells migrating to SDF-1 include cells with a more primitive (CD34+/CD38− or CD34+/DR−) phenotype as well as CD34+ cells phenotypically committed to the erythroid, lymphoid and myeloid lineages, including functional BFU-E, CFU-GM, and CFU-MIX progenitors. Chemotaxis of CD34+ cells in response to SDF-1 is increased by IL-3 in vitro and is lower in CD34+ progenitors from peripheral blood than in CD34+ progenitors from bone marrow, suggesting that an altered response to SDF-1 may be associated with CD34 progenitor mobilization.

Antibody testing and RT-PCR results in hepatitis C virus (HCV) infection: HCV-RNA detection in PBMC of plasma viremia-negative HCV-seropositive persons

Infection ◽  
1998 ◽  
Vol 26 (3) ◽  
pp. 151-154 ◽  
Author(s):  
C. Caudai ◽  
M. G. Padula ◽  
I. Bastianoni ◽  
P. E. Valensin ◽  
V. Shyamala ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Hcv Infection ◽  
Hcv Rna ◽  
Plasma Viremia ◽  
Rt Pcr ◽  
Antibody Testing ◽  
Rna Detection

scholarly journals Intracellular Immunization of Rhesus CD34+ Hematopoietic Progenitor Cells With a Hairpin Ribozyme Protects T Cells and Macrophages From Simian Immunodeficiency Virus Infection

Blood ◽  
1997 ◽  
Vol 90 (12) ◽  
pp. 4822-4831
Author(s):  
Michael Rosenzweig ◽  
Douglas F. Marks ◽  
Donna Hempel ◽  
Marina Heusch ◽  
Günter Kraus ◽  
...  
Keyword(s):  
T Cells ◽  
Progenitor Cells ◽  
Hematopoietic Progenitor Cells ◽  
Hairpin Ribozyme ◽  
Hematopoietic Progenitor ◽  
Cd34 Cells ◽  
Immunodeficiency Virus ◽  
Cell Gene ◽  
Stem Cell Gene Therapy

Evaluation of candidate genes for stem cell gene therapy for acquired immunodeficiency syndrome (AIDS) has been limited by the difficulty of supporting in vitro T-cell differentiation of genetically modified hematopoietic progenitor cells. Using a novel thymic stromal culture technique, we evaluated the ability of a hairpin ribozyme specific for simian immunodeficiency virus (SIV) and human immunodeficiency virus type 2 (HIV-2) to inhibit viral replication in T lymphocytes derived from transduced CD34+ progenitor cells. Retroviral transduction of rhesus macaque CD34+ progenitor cells with a retroviral vector (p9456t) encoding the SIV-specific ribozyme and the selectable marker neomycin phosphotransferase in the presence of bone marrow stroma and in the absence of exogenous cytokines resulted in efficient transduction of both colony-forming units and long-term culture-initiating cells, with transduction efficiencies ranging between 21% and 56%. After transduction, CD34+ cells were cultured on rhesus thymic stromal culture (to support in vitro differentiation of T cells) or in the presence of cytokines (to support differentiation of macrophage-like cells). After expansion and selection with the neomycin analog G418, cells derived from transduced progenitor cells were challenged with SIV. CD4+ T cells derived from CD34+ hematopoietic cells transduced with the ribozyme vector p9456t were highly resistant to challenge with SIV, exhibiting up to a 500-fold decrease in SIV replication, even after high multiplicities of infection. Macrophages derived from CD34+ cells transduced with the 9456 ribozyme exhibited a comparable level of inhibition of SIV replication. These results show that a hairpin ribozyme introduced into CD34+ hematopoietic progenitor cells can retain the ability to inhibit AIDS virus replication after T-cell differentiation and support the feasibility of intracellular immunization of hematopoietic stem cells against infection with HIV and SIV. Protection of multiple hematopoietic lineages with the SIV-specific ribozyme should permit analysis of stem cell gene therapy for AIDS in the SIV/macaque model.

Direct and Indirect Effects of Oxymetholone on Growth and Apoptosis of Hematopoietic Progenitor Cells In Vitro.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4127-4127
Author(s):  
Deog-Yeon Jo ◽  
Seong-Woo Kim ◽  
Jin-Hee Hwang ◽  
Hwan-Jung Yun ◽  
Samyong Kim
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Indirect Effects ◽  
Clonal Growth ◽  
Hematopoietic Progenitor Cells ◽  
Direct And Indirect Effects ◽  
Hematopoietic Progenitor ◽  
Colony Forming Unit ◽  
Cd34 Cells

Abstract Despite a tarnished reputation, androgens remain a common treatment for aplastic anemia, especially in the Orient. Increased apoptosis of hematopoietic cells is characteristic of aplastic anemia; however, it has not been established whether androgens affect apoptosis of hematopoietic progenitor cells. In most previous studies regarding the in vitro effects of androgens on hematopoietic cells, whole bone marrow cells were used, rather than purified hematopoietic progenitor cells such as CD34+ cells. With these questions in mind, we investigated the direct and indirect effects of oxymetholone and other androgens on apoptosis and growth of normal hematopoietic progenitor cells (HPCs) in vitro. Oxymetholone did not rescue normal BM CD34+ cells and colony-forming cells (CFCs), other than mature erythroid CFCs, from apoptosis induced by growth factor deprivation. Unexpectedly, both testosterone and 5-dihydrotestosterone (5-DHT) at a concentration of 10−5 M, but not oxymetholone, increased the percentage of annexin-positive apoptotic cells (62.2 ± 5.9%, P < 0.05; 61.7 ± 6.4%, P < 0.05, respectively) compared with the controls (52.6 ± 5.6%). The addition of either stromal cell-derived factor-1 (SDF-1) or stem cell factor (SCF) partially relieved the increase in apoptosis induced by 5-DHT, and the addition of both SDF-1 and SCF completely reversed it. Oxymetholone did not rescue CFCs from interferon-gamma (IFN-g)-induced inhibition of clonal growth of BM CD34+ cells in methylcellulose cultures. Furthermore, oxymetholone did not mitigate IFN-g-induced suppression of CD34+ cell survival in the presence of growth factors. In a methylcellulose clonogenic assay, oxymetholone stimulated the clonal growth of colony-forming unit-erythroid at low concentrations, while not affecting colony-forming unit-granulocyte/macrophage or burst-forming unit-erythroid. Oxymetholone did not reverse the IFN-g-induced inhibition of colony formation by CD34+ cells. Interestingly, oxymetholone stimulated the production of SCF and thrombopoietin in normal human bone marrow stromal cells (BMSCs) through transcriptional regulation while inhibiting the production of interleukin-6. In agreement with this, oxymetholone-treated BMSCs better supported the survival and growth of HPCs. These results suggest that oxymetholone exerts most of its myelostimulatory effects via the regulation of cytokine production in BMSCs, rather than by direct action on hematopoietic progenitor cells.

Extracellular Vesicles Play an Important Role in Intercellular Communication Between Bone Marrow Stroma and Hematopoietic Progenitor Cells in Myeloproliferative Neoplasms

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1957-1957
Author(s):  
Teresa L. Ramos ◽  
Luis Ignacio Sánchez-Abarca ◽  
Beatriz Rosón ◽  
Alba Redondo ◽  
Concepción Rodríguez ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Board Of Directors ◽  
Intercellular Communication ◽  
Research Funding ◽  
Hematopoietic Progenitor Cells ◽  
Rt Pcr ◽  
Hematopoietic Progenitor ◽  
Advisory Committees ◽  
Cd34 Cells

Abstract The complex interplay between bone marrow-derived mesenchymal stromal cells (BM-MSC) and neoplastic hematopoietic cells is involved in the progression of myeloproliferative neoplastic (MPN) diseases. Extracellular vesicles (EV) have emerged as a complex cell-to-cell communication system within the neoplastic microenvironment. EV are able to reprogram recipient cells by transferring proteins, mRNA and microRNA from their cell of origin. We aimed to analyze the microRNA content of EV obtained from MPN BM-MSC, as well as the changes induced when these EV are incorporated into hematopoietic progenitor cells (HPC). EV were isolated from BM-MSC of MPN patients (n=22) and healthy donors (HD) (n=19) by ultracentrifugation. Characterization of EV by transmission electron microscopy (TEM), immunoblot, multiparametric flow cytometry (MFC) and NanoSight analysis revealed vesicles with a typical bilayer-membrane characteristic morphology with a size inferior to 500 nm, which were positive for various EV markers as CD63 and CD81, and for MSC markers as CD73, CD90 and CD44 (Figure 1). MicroRNA profiling by 384-well microfluidic cards (TaqMan® MicroRNA Array A) showed an overall increase in the microRNA expression in the MPN-MSC-derived EV, when compared to EV from donor MSC. Using RT-PCR, we observed that miR-155 was selectively enriched in EV released by MPN-MSC. An overexpression of this microRNA was observed in EV (p=0.032), while a downregulation was observed in BM-MSC (p=0.0078) (Figure 2). EV incorporation was demonstrated by fluorescence microscopy and MFC, where HPC (CD34+ cells obtained by immunomagnetic selection) were co-cultured with Vybrant Dil-labeled EV. For functional studies apoptosis and clonogenic assays (CFU-GM) were performed. We observed an increase in CD34+ cell viability after incorporating EV from BM-MSC (HD and MPN). Moreover, an increase (p=0.04) in miR-155 expression was observed when HD HPC incorporated EV from MPN-MSC. When neoplastic CD34+ cells incorporated the EV derived from MPN-MSC an increase of CFU-GM number was also observed. We suggest that EV released from MPN-MSC represent a mechanism of intercellular communication between malignant stromal and hematopoietic cells, through the transfer of genetic information that may be relevant in the pathophysiology of these diseases. Funding: GRS 1034/A/14 (C. Sanidad, JCYL) and FCT (SFRH/BD/86451/2012) Figure 1 EV characterization by TEM (A), Immunobloting - CD63 (B) and MFC (C). Scale bar: 200 and 500 nm. Figure 1. EV characterization by TEM (A), Immunobloting - CD63 (B) and MFC (C). Scale bar: 200 and 500 nm. Figure 2 Expression of miR-155. RT-PCR from EV released from HD and MPN-MSC (A), and the expression of miR-155 in BM-MSC (B). Figure 2. Expression of miR-155. RT-PCR from EV released from HD and MPN-MSC (A), and the expression of miR-155 in BM-MSC (B). Disclosures Sánchez-Guijo: Bristol-Myers-Squib: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Incyte: Consultancy, Honoraria. Del Cañizo:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Jansen-Cilag: Membership on an entity's Board of Directors or advisory committees, Research Funding; Arry: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding.

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