scholarly journals Binding to Decay-Accelerating Factor Is Not Required for Infection of Human Leukocyte Cell Lines by Enterovirus 70

2004 ◽  
Vol 78 (6) ◽  
pp. 2674-2681 ◽  
Author(s):  
Alain Haddad ◽  
M. Reza Nokhbeh ◽  
David A. Alexander ◽  
Sandra J. Dawe ◽  
Christine Grisé ◽  
...  
Keyword(s):  
Cell Lines ◽  
Hela Cells ◽  
Human Leukocyte ◽  
Myeloid Cell ◽  
Surface Expression ◽  
Virus Binding ◽  
Decay Accelerating Factor ◽  
The Central Nervous System ◽  
Enterovirus 70

ABSTRACT Enterovirus 70 (EV70) is one of several human enteroviruses that exhibit a propensity for infecting the central nervous system (CNS). The mechanisms by which neurotropic enteroviruses gain access to and invade the CNS are poorly understood. One possibility is that circulating leukocytes become infected and carry neurotropic enteroviruses to the CNS. We examined the ability of EV70 to infect cell lines derived from lymphoid, myeloid, and monocytic lineages. Most leukocyte cell lines tested bound radiolabeled EV70 and were permissive for EV70 replication, suggesting that EV70, in contrast to other enteroviruses, has an in vitro tropism that includes lymphoid, monocytic, and myeloid cell lines. For some of the cell lines, virus binding and infection correlated with surface expression of decay-accelerating factor (DAF), an attachment protein for EV70 on HeLa cells. However, EV70 also adsorbed to and infected cell lines that expressed little or no DAF. In contrast to what was observed for HeLa cells, neither DAF-specific monoclonal antibodies nor phosphatidylinositol-specific phospholipase C treatment inhibited EV70 binding to permissive leukocyte cell lines, and antibody blockade of DAF had little or no effect on EV70 replication. We also found that neither the human coxsackievirus-adenovirus receptor nor intercellular cell adhesion molecule 1, which mediate the entry of coxsackie B viruses and coxsackievirus A21, respectively, functions as a receptor for EV70. EV70 binding to all cell lines was sensitive to sialidase treatment and to inhibition of O glycosylation by benzyl N-acetyl-α-d-galactosaminide. Taken together, these results suggest that a sialylated molecule(s) other than DAF serves as a receptor for EV70 on permissive human leukocyte cell lines.

scholarly journals Tuberin levels during cellular differentiation in brain development

2021 ◽  
Author(s):  
Bashaer Abu Khatir ◽  
Gordon Omar Davis ◽  
Mariam Sameem ◽  
Rutu Patel ◽  
Jackie Fong ◽  
...  
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Brain Development ◽  
Cell Lines ◽  
Neural Development ◽  
Time Course ◽  
Embryonic Mouse ◽  
Organ Systems ◽  
The Central Nervous System

Tuberin is a member of a large protein complex, Tuberous Sclerosis Complex, and acts as a sensor for nutrient status regulating protein synthesis and cell cycle progression. Mutations in the Tuberin gene, TSC2, lead to the formation of tumors and developmental defects in many organ systems, including the central nervous system. Tuberin is expressed in the brain throughout development and levels of Tuberin have been found to decrease during neuronal differentiation in cell lines in vitro. Our current work investigates the levels of Tuberin at two stages of embryonic development in vivo, and we study the mRNA and protein levels during a time course using immortalized cell lines in vitro. Our results show that Tuberin levels remain stable in the olfactory bulb but decrease in the Purkinje cell layer during embryonic mouse brain development. We show here that Tuberin levels are higher when cells are cultured as neurospheres, and knockdown of Tuberin results in a reduction in the number of neurospheres. These data provide support for the hypothesis that Tuberin is an important regulator of stemness and the reduction of Tuberin levels might support functional differentiation in the central nervous system. Understanding how Tuberin expression is regulated throughout neural development is essential to fully comprehend the role of this protein in several developmental and neural pathologies.

scholarly journals Regulation of the human cellular glutathione peroxidase gene during in vitro myeloid and monocytic differentiation

Blood ◽  
1994 ◽  
Vol 84 (11) ◽  
pp. 3902-3908 ◽  
Author(s):  
Q Shen ◽  
S Chada ◽  
C Whitney ◽  
PE Newburger
Keyword(s):  
Steady State ◽  
Glutathione Peroxidase ◽  
Cell Lines ◽  
Leukemia Cell ◽  
Myeloid Cell ◽  
Dimethyl Formamide ◽  
Monocytic Differentiation ◽  
Granulocytic Differentiation ◽  
Regulation Of Expression

We have used the HL-60 and PLB-985 myeloid leukemia cell lines to examine the regulation of expression of the important intracellular antioxidant enzyme, glutathione peroxidase (GSH-Px), during phagocytic cell differentiation in vitro. Induction of differentiation along the monocytic pathway by phorbol ester results in an approximately twofold rise in enzyme activity and a parallel increase in the rate of 75Se incorporation into immunoprecipitable GSH-Px protein. Induction along the granulocytic pathway by dimethyl formamide (DMF) results in similar changes in steady-state enzyme levels and rates of GSH-Px protein synthesis. Steady-state levels of GSH-Px gene transcripts also increase more than twofold, approximately in parallel with the enzyme levels. Nuclear run-on transcription assays of GSH-Px mRNA synthesis show ratios of induced to uninduced transcript levels of 2.24 and 1.59 with phorbol myristate acetate (PMA) induction and DMF, respectively, in HL- 60 cells, and ratios of 1.34 and 3.46 with PMA and DMF, respectively, in PLB-985 cells. Half lives of GSH-Px mRNA are unchanged or slightly shorter after differentiation of HL-60 cells, and slightly longer after induction of PLB-985. Overall, the present studies show that GSH-Px activity rises during in vitro-induced monocytic or granulocytic differentiation of myeloid cell lines and that the increased expression of the cellular GSH-Px gene occurs through complex mechanisms that include transcriptional up-regulation. This pattern contrasts with the nearly complete cotranslational regulation of GSH-Px expression by exogenous selenium.

scholarly journals Expression and secretion of glycosylphosphatidylinositol-specific phospholipase D by myeloid cell lines

1994 ◽  
Vol 297 (3) ◽  
pp. 547-554 ◽  
Author(s):  
M Xie ◽  
M G Low
Keyword(s):  
Cell Lines ◽  
Phospholipase D ◽  
Myeloid Cell ◽  
Human Monocyte ◽  
Fold Increase ◽  
Surface Expression ◽  
Immunofluorescent Staining ◽  
Reaction Products ◽  
Monocytic Differentiation ◽  
Potential Contributor

Glycosylphosphatidylinositol (GPI)-specific phospholipase D (GPI-PLD) is abundant in mammalian plasma. It could potentially regulate the surface expression of GPI-anchored proteins, but it remains to be established which tissue(s) or cell type(s) are the principal sources of the circulating enzyme. Here we report that all the myeloid cell lines tested, including K562 (multipotential blast), KG-1 (human myeloblast), HL-60, NB4, PLB-985 (human promyelocyte), U937 (human promonocyte), THP-1 (human monocyte) and J774, RAW264.7 (mouse monocyte/macrophage), contained GPI-degrading activity. T.l.c. analysis of reaction products confirmed the activity as a phospholipase D. These cells also exhibited positive immunofluorescent staining with an anti-GPI-PLD monoclonal antibody. The expression of GPI-PLD activity was not substantially reduced when the cells were cultured in either serum-free medium or GPI-PLD-depleted regular medium. Both granulocytic and monocytic differentiation of myelomonoblastic lines (e.g. HL-60) induced by dimethyl sulphoxide or phorbol diester respectively was accompanied by a 2-3-fold increase in GPI-PLD activity. J774 and HL-60 cells secreted GPI-PLD into the medium constitutively. Taken together, these data suggest that myeloid cells are a potential contributor to the circulating GPI-PLD pool. As leucocytes express many important GPI-anchored surface antigens, these cells may prove to be a valuable model system for studying the physiological functions of GPI-PLD.

Upregulation of CD20 on Human Acute Lymphoblastic Leukemia Cells by IL-4 and CpG Motif Containing Oligonucleotides Increases Susceptibility to Rituximab.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1879-1879
Author(s):  
Bart Nijmeijer ◽  
Marianke L.J. Van Schie ◽  
Roelof Willemze ◽  
J.H. Frederik Falkenburg
Keyword(s):  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Surface Expression ◽  
Chimeric Antibody ◽  
Transcriptional Level ◽  
Apparent Lack ◽  
Expression Levels ◽  
Cd20 Expression ◽  
Cpg Motif

Abstract Monoclonal antibodies are emerging modalities in the treatment of hematologic malignancies. Rituximab (RTX), a chimeric antibody that recognizes CD20, shows therapeutic efficacy in non-Hodgkin lymphoma (NHL). Precursor-B ALL (pB-ALL) may also express CD20. However, little is known on the activity of RTX in this disease. We determined CD20 surface expression levels on primary pB-ALL cells by flow cytometry following a standardized protocol in which cells are stained with RTX and secondary antibody. CD20 on primary ALL cells was generally absent or expressed at lower levels than on primary NHL cells (median mean fluorescence intensity (MFI): 35.1, range 5–423 in 17 pB-ALL samples, and median MFI 1082, range 440–1818 in 6 NHL samples). The ALL cells that expressed the highest levels of CD20 were similarly susceptible to RTX mediated complement dependent cytotoxicity (CDC) as NHL cells (median 17.8% lysis, range 9.5–98% and 41% lysis, range 13–72%, respectively). However, in pB-ALL cells, RTX activity was strongly limited by CD20 expression levels as lysis strictly correlated with CD20 surface expression (P=0.89), and CD20 negative pB-ALL cells were not killed. Despite apparent lack of surface expression, quantitative RT-PCR (qPCR) demonstrated that all samples expressed CD20 mRNA. Transcriptional expression levels correlated with surface CD20 expression (P=0.72). Therefore we explored upregulation of CD20 as a strategy to augment activity of RTX in ALL. We previously established an in vitro culturing system that supports long-term proliferation of ALL cells. Using the LeidenALL cell lines that were generated in this system, we evaluated the effect of various agents on expression of CD20. Ten LeidenALL pB-ALL cell lines were cultured in the presence or absence of IL-2, IL-3, IL-5, IL-7, IL-15, a CpG motif containing oligonucleotide (CpG), TNFa or IFNg. Culture of LeidenALL cells in the presence of IL-4 resulted in upregulation of CD20 in 4 out of 10 samples, 24 hours after incubation. Culture of LeidenALL cells in the presence of CpG resulted in upregulation of CD20 in another 4 out of 10 samples, 48 hours after incubation. None of the other cytokines affected expression of CD20. IL-4 and CpG displayed synergistic action as co-incubation of LeidenALL cells with IL-4 and CpG resulted in further increased expression of CD20 in 9 out of the 10 samples after 48 hours of co-incubation. Mean CD20 expression increased from MFI 406 (range 14–2668) to MFI 1572 (range 70–4394). qPCR revealed that CD20 was upregulated on a transcriptional level in all of the 10 cell lines. Upregulation of CD20 augmented RTX-mediated CDC (from 41% lysis, range 8–95% to 78% lysis, range 3–93%). Three CD20-negative LeidenALL cell lines that initially were not susceptible to RTX-mediated CDC were lysed after upregulation. In an in vitro antibody-dependent cellular cytotoxicity (ADCC) assay, using peripheral blood mononuclear cells from healthy donors, rituximab-mediated ADCC was increased in 7 out of the 10 samples (from 18% lysis, range 4–48% to 25% lysis, range 6–51%). In conclusion, these results demonstrate that RTX may possess activity in pB-ALL. Activity of RTX in ALL can be potentiated by modulation of the leukemic cells. These insights may allow successful application of rituximab in ALL.

A Humanized Anti-Ganglioside GM2 Antibody, BIW-8962, Exhibits ADCC/CDC Activity against Multiple Myeloma Cells and Potent Anti- Tumor Activity in Mouse Xenograft Models.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1718-1718 ◽  
Author(s):  
Toshihiko Ishii ◽  
Asher Alban Chanan-Khan ◽  
Jazur Jafferjee ◽  
Noreen Ersing ◽  
Takeshi Takahashi ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Phase 1 ◽  
Xenograft Model ◽  
Surface Expression ◽  
Scid Mice ◽  
Subcutaneous Xenograft ◽  
Anti Tumor Activity

Abstract BIW-8962 is a humanized anti-ganglioside GM2 (GM2) monoclonal antibody, produced by Poteligent technology to enhance ADCC activity. GM2 is expressed on many cancer cells including multiple myeloma (MM), small cell lung cancer and glioma cells. In this study, we evaluated the anti-myeloma activity of BIW-8962 in preclinical myeloma models both in vitro and in vivo. Expression of GM2 was analyzed in 15 human MM cell lines by FCM. Eleven out of 15 MM cell lines had positive surface expression of GM2. GM2 as a potential target was then verified in primary MM samples obtained from patients. Eleven out of 15 samples were positive for GM2. We then used two GM2 positive MM cell lines (U266B1 and KMS-11) and evaluated ADCC and CDC activity of BIW-8962 in vitro. BIW-8962 exhibited a potent ADCC and less potent CDC activity. In vivo anti-tumor activity of BIW-8962 was then examined using the standard subcutaneous xenograft model; KMS-11 was inoculated in the flank of SCID mice. BIW-8962 (intravenously administered biweekly for 3 weeks) exhibited a potent anti-tumor activity from as low a dose level as 0.1 mg/kg. Furthermore, in a more clinically relevant model, in which OPM-2/GFP (GM2 positive MM cell line) cells were intravenously inoculated into SCID mice with preferentially tumor growth within the bone marrow microenvironment, BIW-8962 (intravenously administered biweekly for 4 weeks, 10 mg/kg) suppressed OPM-2/GFP cell growth and serum M protein elevation, demonstrating in vivo anti-myeloma effect of BIW-8962. Our preclinical investigations rationalize clinical evaluation of BIW-8962 in patients with MM. Currently BIW-8962 is being investigated in a Phase 1 study in patients with multiple myeloma.

scholarly journals Cholangiocyte expression of α2β1-integrin confers susceptibility to rotavirus-induced experimental biliary atresia

2008 ◽  
Vol 295 (1) ◽  
pp. G16-G26 ◽  
Author(s):  
Mubeen Jafri ◽  
Bryan Donnelly ◽  
Steven Allen ◽  
Alex Bondoc ◽  
Monica McNeal ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Cell Surface ◽  
Biliary Atresia ◽  
Cell Lines ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Newborn Mice ◽  
A Cell

Inoculation of BALB/c mice with rhesus rotavirus (RRV) in the newborn period results in biliary epithelial cell (cholangiocyte) infection and the murine model of biliary atresia. Rotavirus infection of a cell requires attachment, which is governed in part by cell-surface expression of integrins such as α2β1. We hypothesized that cholangiocytes were susceptible to RRV infection because they express α2β1. RRV attachment and replication was measured in cell lines derived from cholangiocytes and hepatocytes. Flow cytometry was performed on these cell lines to determine whether α2β1 was present. Cholangiocytes were blocked with natural ligands, a monoclonal antibody, or small interfering RNA against the α2-subunit and were infected with RRV. The extrahepatic biliary tract of newborn mice was screened for the expression of the α2β1-integrin. Newborn mice were pretreated with a monoclonal antibody against the α2-subunit and were inoculated with RRV. RRV attached and replicated significantly better in cholangiocytes than in hepatocytes. Cholangiocytes, but not hepatocytes, expressed α2β1 in vitro and in vivo. Blocking assays led to a significant reduction in attachment and yield of virus in RRV-infected cholangiocytes. Pretreatment of newborn pups with an anti-α2 monoclonal antibody reduced the ability of RRV to cause biliary atresia in mice. Cell-surface expression of the α2β1-integrin plays a role in the mechanism that confers cholangiocyte susceptibility to RRV infection.

scholarly journals Opposite replication polarity of the germ line c-myc gene in HeLa cells compared with that of two Burkitt lymphoma cell lines.

1989 ◽  
Vol 9 (2) ◽  
pp. 586-593 ◽  
Author(s):  
M Leffak ◽  
C D James
Keyword(s):  
Cell Lines ◽  
Hela Cells ◽  
Burkitt Lymphoma ◽  
Germ Line ◽  
Lymphoma Cell ◽  
Cell Type Specificity ◽  
Intact Cells ◽  
Burkitt Lymphoma Cell ◽  
Myc Gene

To study the cell type specificity of the direction of replication of the human c-myc genes and the relationship of replication polarity to transcriptional activity, we analyzed the directions of replication of the c-myc genes in two Burkitt lymphoma cell lines, CA46 and ST486, and in HeLa cells. On the basis of in vitro runoff replication of forks initiated in intact cells, we found that transcribed c-myc genes in the germ line configuration in HeLa cells were replicated in the direction of transcription from origins in the 5'-flanking DNA, while the repressed, unrearranged c-myc genes of CA46 and ST486 cells were replicated in the antitranscriptional direction. In contrast, the transcribed c-myc genes of CA46 cells were replicated in the transcriptional direction, while the translocated, amplified c-myc genes of ST486 cells showed no preferred polarity of replication. The data also provided evidence for the existence of an endogenous barrier to DNA polymerases in the flanking DNA immediately 5' to the HeLa c-myc genes.

scholarly journals Quantitative Expression and Virus Transmission Analysis of DC-SIGN on Monocyte-Derived Dendritic Cells

2002 ◽  
Vol 76 (18) ◽  
pp. 9135-9142 ◽  
Author(s):  
Frédéric Baribaud ◽  
Stefan Pöhlmann ◽  
George Leslie ◽  
Frank Mortari ◽  
Robert W. Doms
Keyword(s):  
Dendritic Cells ◽  
Cell Lines ◽  
Ebola Virus ◽  
Virus Transmission ◽  
Virus Binding ◽  
Expression Levels ◽  
Quantitative Expression ◽  
Immunodeficiency Virus

ABSTRACT The C-type lectins DC-SIGN and DC-SIGNR efficiently bind human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) strains and can transmit bound virus to adjacent CD4-positive cells. DC-SIGN also binds efficiently to the Ebola virus glycoprotein, enhancing Ebola virus infection. DC-SIGN is thought to be responsible for the ability of dendritic cells (DCs) to capture HIV and transmit it to T cells, thus promoting HIV dissemination in vitro and perhaps in vivo as well. To investigate DC-SIGN function and expression levels on DCs, we characterized a panel of monoclonal antibodies (MAbs) directed against the carbohydrate recognition domain of DC-SIGN. Using quantitative fluorescence-activated cell sorter technology, we found that DC-SIGN is highly expressed on immature monocyte-derived DCs, with at least 100,000 copies and often in excess of 250,000 copies per DC. There was modest variation (three- to fourfold) in DC-SIGN expression levels between individuals and between DCs isolated from the same individual at different times. Several MAbs efficiently blocked virus binding to cell lines expressing human or rhesus DC-SIGN, preventing HIV and SIV transmission. Interactions with Ebola virus pseudotypes were also blocked efficiently. Despite their ability to block virus-DC-SIGN interactions on cell lines, these antibodies only inhibited transmission of virus from DCs by approximately 50% or less. These results indicate that factors other than DC-SIGN may play important roles in the ability of DCs to capture and transmit HIV.

scholarly journals Dynamin- and Lipid Raft-Dependent Entry of Decay-Accelerating Factor (DAF)-Binding and Non-DAF-Binding Coxsackieviruses into Nonpolarized Cells

2009 ◽  
Vol 83 (21) ◽  
pp. 11064-11077 ◽  
Author(s):  
Kunal P. Patel ◽  
Carolyn B. Coyne ◽  
Jeffrey M. Bergelson
Keyword(s):  
Hela Cells ◽  
Tyrosine Kinases ◽  
Dominant Negative ◽  
Early Event ◽  
Small Interfering Rnas ◽  
Virus Binding ◽  
Decay Accelerating Factor ◽  
Endosomal Acidification ◽  
Group B ◽  
Adenovirus Receptor

ABSTRACT Group B coxsackieviruses (CVB) use the CVB and adenovirus receptor (CAR) to enter and infect cells. Some CVB also bind to decay-accelerating factor (DAF), but that interaction alone is insufficient for infection. We previously found that CVB3 entry into polarized human intestinal cells (Caco-2) occurs by a caveolin-dependent but dynamin-independent mechanism that requires DAF-mediated tyrosine kinase signals. In this study, we examined how CVB enter and infect nonpolarized HeLa cells and how DAF binding affects these processes. Using immunofluorescence microscopy and a combination of dominant-negative proteins, small interfering RNAs, and drugs targeting specific endocytic pathways, we found that both DAF-binding and non-DAF-binding virus isolates require dynamin and lipid rafts to enter and infect cells. Unlike what we observed in Caco-2 cells, CVB3 entered HeLa cells with CAR. We found no role for clathrin, endosomal acidification, or caveolin. Inhibition of tyrosine kinases blocked an early event in infection but did not prevent entry of virus into the cell. These results indicate that CVB3 entry into nonpolarized HeLa cells differs significantly from entry into polarized Caco-2 cells and is not influenced by virus binding to DAF.

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