scholarly journals BHK-21 Cell Clones Differ in Chikungunya Virus Infection and MXRA8 Receptor Expression

Viruses ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 949
Author(s):  
Peiqi Yin ◽  
Margaret Kielian
Keyword(s):  
Cell Lines ◽  
Primary Infection ◽  
Sindbis Virus ◽  
Chikungunya Virus ◽  
Mrna Level ◽  
Receptor Expression ◽  
Chikv Infection ◽  
Cell Clones ◽  
Clonal Cell Lines ◽  
Exogenous Expression

Baby hamster kidney-21 (BHK-21) cells are widely used to propagate and study many animal viruses using infection and transfection techniques. Among various BHK-21 cell clones, the fibroblast-like BHK-21/C-13 line and the epithelial-like BHK-21/WI-2 line are commonly used cell clones for alphavirus research. Here we report that BHK-21/WI-2 cells were significantly less susceptible to primary infection by the alphavirus chikungunya virus (CHIKV) than were BHK-21/C-13 cells. The electroporation efficiency of alphavirus RNA into BHK-21/WI-2 was also lower than that of BHK-21/C-13. The growth of CHIKV was decreased in BHK-21/WI-2 compared to BHK-21/C-13, while primary infection and growth of the alphavirus Sindbis virus (SINV) were equivalent in the two cell lines. Our results suggested that CHIKV entry could be compromised in BHK-21/WI-2. Indeed, we found that the mRNA level of the CHIKV receptor MXRA8 in BHK-21/WI-2 cells was much lower than that in BHK-21/C-13 cells, and exogenous expression of either human MXRA8 or hamster MXRA8 rescued CHIKV infection. Our results affirm the importance of the MXRA8 receptor for CHIKV infection, and document differences in its expression in two clonal cell lines derived from the original BHK-21 cell cultures. Our results also indicate that CHIKV propagation and entry studies in BHK-21 cells will be significantly more efficient in BHK-21/C-13 than in BHK-21/WI-2 cells.

Inducible anti-sense RNA for angiotensinogen in stably transformed hepatoma cell lines

1990 ◽  
Vol 4 (2) ◽  
pp. 107-117 ◽  
Author(s):  
W. M. Clouston ◽  
C. J. Lloyd ◽  
R. I. Richards
Keyword(s):  
Cell Lines ◽  
Reductase Activity ◽  
Hepatoma Cell ◽  
Shuttle Vector ◽  
Mrna Level ◽  
Mrna Levels ◽  
Hepatoma Cell Lines ◽  
Metallothionein Promoter ◽  
Clonal Cell Lines ◽  
Fold Induction

ABSTRACT Angiotensinogen mRNA is found in many extrahepatic tissues, where it may participate in local angiotensin-generating systems. In this study we explore the feasibility of using anti-sense RNA to decrease angiotensinogen production in rat H4IIEC3 hepatoma cells. An amplifiable shuttle vector was modified to allow the production of high levels of stable anti-sense RNA from two regions of the mouse angiotensinogen gene under the control of the inducible sheep metallothionein promoter. Stably transformed, clonal cell lines expressing anti-sense RNA for angiotensinogen were isolated after selection with the aminoglycoside G418. Subsequently, the number of chromosomally integrated copies of the angiotensinogen anti-sense constructs was coamplified by methotrexate selection for dihydrofolate reductase activity carried on the shuttle vector. With a 20- to 30-fold induction of the anti-sense RNAs, the target angiotensinogen mRNA level was reduced to 25–30% of control values. The specificity of this effect was confirmed by showing no decrease in either β-tubulin or neomycin phosphotransferase mRNA levels. Using tissue-specific promoters, it should be possible to direct these effects to specific organs in transgenic mice. However, in agreement with results from other groups, our findings suggest that it will not be possible to eradicate completely the target gene product using the anti-sense RNA strategy.

Improving Tumor Immunotherapy by Using Distinct Subpopulations of the γδT Cell Repertoire with High Anti-Tumor Reactivity.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4504-4504
Author(s):  
Suzanne van Dorp ◽  
Samantha Hol ◽  
Victoria Marcu-Malina ◽  
Nicole Thuss ◽  
Henk Lokhorst ◽  
...  
Keyword(s):  
Tumor Cell ◽  
Cell Lines ◽  
Tumor Immunotherapy ◽  
Receptor Expression ◽  
Tumor Cell Lines ◽  
Variable Regions ◽  
Γδt Cells ◽  
Expression Levels ◽  
Cell Clones ◽  
Functional Analyses

Abstract Abstract 4504 Introduction Currently innate immune cells such as γ9δ2T cells are explored in tumor immunotherapy e.g. by adoptive transfer of in vitro expanded bulk γ9δ2T cells. Objective We speculated that γ9δ2T cells are highly variable in function and specificity due to differences in γ9δ2TCR, NKG2D and KIR expression and that efficacy of adoptively transferred γ9δ2T cells can be increased by transfer of defined subpopulations or clones rather than bulk γ9δ2T cells. Methods A variety of γ9δ2T cell clones, derived from a healthy donor, were tested for expression levels of γ9δ2TCR, NKG2D and KIRs by flow cytometry analysis. The sequence of the γ9δ2TCR of different clones was further analyzed. Reactivity of γ9δ2T cell clones to a panel of tumor and normal cell lines was tested and these functional analyses were correlated to receptor expression levels and compared with bulk γ9δ2T cells of the same donor. Results Functional analyses revealed a high interclonal variability in recognizing leukemia or solid tumor cell lines. Consequently, γ9δ2T cell clones with high anti-tumor reactivity were superior in killing tumor cells when compared to bulk γ9δ2T cells. Different variable regions of γ9δ2-chains and different expression levels of NKG2D and KIRs were detected in multiple clones. No correlations could be found between TCR, NKG2D, and KIR expression on γ9δ2T cell clones and their response to different tumor cell lines when clones expressed different γ9δ2TCRs. However, analysis of γ9δ2T-cell clones with identical γ9δ2TCRs revealed that a clone with higher reactivity against cancer cells expresses higher amounts of NKG2D and lower inhibitory KIRs when compared to a clone with lower reactivity. Conclusion γ9δ2TCR, NKG2D and KIR expression in γ9δ2T cells is highly variable and cannot be directly correlated to an effective anti-tumor response. Only, when T-cells express one defined γ9δ2TCR, a modulating activity of NKG2D and KIRs can be observed. Thus, we conclude that anti-tumor reactivity is fine tuned by all three receptors and thereby we speculate that the γ9δ2TCR defines tumor-specificity and activity is further modulated by NKG2D and KIRs. These results support the application of distinct subpopulations or of genetically engineered γδT cells with defined receptors rather than bulk γ9δ2T cells in the context of anti-tumor immunotherapies. Disclosures: No relevant conflicts of interest to declare.

Stimulation of prostate cancer cell proliferation by bone-derived factors.

2011 ◽  
Vol 29 (7_suppl) ◽  
pp. 32-32
Author(s):  
P. Sluka ◽  
G. Whitty ◽  
I. D. Davis
Keyword(s):  
Prostate Cancer ◽  
Cell Proliferation ◽  
Cell Survival ◽  
Cell Lines ◽  
Mrna Level ◽  
Receptor Expression ◽  
Cancer Cell Proliferation ◽  
Lncap Cells ◽  
Du145 Cells ◽  
And Migration

32 Background: Interactions between cancer cells and their microenvironment affect the establishment and metastasis of cancer. The most common site of prostate cancer (PC) metastasis is bone. This study examined the effects of selected factors known to be produced by bone stroma (EGF, aFGF, HGF, β-NGF, TGF-β, and TNFα) on the proliferation of PC cells. The effect of cell culture medium (CM) conditioned by osteoblasts (OBCM) was also examined. Methods: The PC-derived cell lines PC3, LNCaP, and DU145 were used. Expression of receptors for the above-mentioned cytokines was assessed using real-time RT-PCR. After 5 days of continuous cytokine treatment, proliferation was assessed by MTS conversion, and cell survival and apoptosis was assessed by 7-AAD staining and flow cytometry. OBCM was generated using the HOS, MG63, and SaOs2 cell lines. CM from the HT1080 fibrosarcoma cell line was used as a non-bone control. Results: The PC cell lines expressed receptors for all of the cytokines examined at the mRNA level. LNCaP cell proliferation was increased by aFGF and decreased by TGF-β. Treatment with TNFα decreased proliferation of all PC cell lines. These effects were not due to apoptosis. EGF, HGF, and β-NGF did not affect proliferation of any line despite receptor expression. OBCM increased proliferation of PC3 and DU145 cells but not LNCaP cells, while HT1080 CM did not affect proliferation of any line. Conclusions: PC cells are able to respond to defined bone-derived factors and that the nature of this response varies between individual cancers. Acidic FGF increased proliferation of LNCaP cells while TGF-β and TNFα decreased proliferation of LNCaP and all cell lines, respectively; an effect not mediated by apoptosis. Current studies are examining the effect of these cytokines on other functional parameters (cell survival, adhesion and migration) and on primary PC epithelial cells. No significant financial relationships to disclose.

scholarly journals Antagonism of the Sodium-Potassium ATPase Impairs Chikungunya Virus Infection

mBio ◽  
2016 ◽  
Vol 7 (3) ◽  
Author(s):  
Alison W. Ashbrook ◽  
Anthony J. Lentscher ◽  
Paula F. Zamora ◽  
Laurie A. Silva ◽  
Nicholas A. May ◽  
...  
Keyword(s):  
Virus Infection ◽  
High Throughput ◽  
Cardiac Glycoside ◽  
High Throughput Screening ◽  
Sindbis Virus ◽  
Chikungunya Virus ◽  
Nonstructural Proteins ◽  
Chikv Infection ◽  
Sodium Potassium ◽  
Potassium Atpase

ABSTRACTChikungunya virus (CHIKV) is a reemerging alphavirus that has caused epidemics of fever, arthralgia, and rash worldwide. There are currently no licensed vaccines or antiviral therapies available for the prevention or treatment of CHIKV disease. We conducted a high-throughput, chemical compound screen that identified digoxin, a cardiac glycoside that blocks the sodium-potassium ATPase, as a potent inhibitor of CHIKV infection. Treatment of human cells with digoxin or a related cardiac glycoside, ouabain, resulted in a dose-dependent decrease in infection by CHIKV. Inhibition by digoxin was cell type-specific, as digoxin treatment of either murine or mosquito cells did not diminish CHIKV infection. Digoxin displayed antiviral activity against other alphaviruses, including Ross River virus and Sindbis virus, as well as mammalian reovirus and vesicular stomatitis virus. The digoxin-mediated block to CHIKV and reovirus infection occurred at one or more postentry steps, as digoxin inhibition was not bypassed by fusion of CHIKV at the plasma membrane or infection with cell surface-penetrating reovirus entry intermediates. Selection of digoxin-resistant CHIKV variants identified multiple mutations in the nonstructural proteins required for replication complex formation and synthesis of viral RNA. These data suggest a role for the sodium-potassium ATPase in promoting postentry steps of CHIKV replication and provide rationale for modulation of this pathway as a broad-spectrum antiviral strategy.IMPORTANCEMitigation of disease induced by globally spreading, mosquito-borne arthritogenic alphaviruses requires the development of new antiviral strategies. High-throughput screening of clinically tested compounds provides a rapid means to identify undiscovered, antiviral functions for well-characterized therapeutics and illuminate host pathways required for viral infection. Our study describes the potent inhibition of Chikungunya virus and related alphaviruses by the cardiac glycoside digoxin and demonstrates a function for the sodium-potassium ATPase in Chikungunya virus infection.

scholarly journals Inhibition of Chikungunya Virus Replication by Harringtonine, a Novel Antiviral That Suppresses Viral Protein Expression

2012 ◽  
Vol 57 (1) ◽  
pp. 155-167 ◽  
Author(s):  
Parveen Kaur ◽  
Meerra Thiruchelvan ◽  
Regina Ching Hua Lee ◽  
Huixin Chen ◽  
Karen Caiyun Chen ◽  
...  
Keyword(s):  
Protein Expression ◽  
Viral Entry ◽  
Viral Protein ◽  
Sindbis Virus ◽  
Chikungunya Virus ◽  
Early Stage ◽  
Antiviral Treatment ◽  
Chikv Infection ◽  
Viral Protein Synthesis ◽  
Viral Protein Expression

ABSTRACTChikungunya virus (CHIKV) is a mosquito-transmitted virus that has reemerged as a significant public health threat in the last decade. Since the 2005-2006 chikungunya fever epidemic in the Indian Ocean island of La Réunion, millions of people in more than 40 countries have been infected. Despite this, there is currently no antiviral treatment for chikungunya infection. In this study, an immunofluorescence-based screening platform was developed to identify potential inhibitors of CHIKV infection. A primary screen was performed using a highly purified natural product compound library, and 44 compounds exhibiting ≥70% inhibition of CHIKV infection were identified as positive hits. Among these, four were selected for dose-dependent inhibition assays to confirm their anti-CHIKV activity. Harringtonine, a cephalotaxine alkaloid, displayed potent inhibition of CHIKV infection (50% effective concentration [EC50] = 0.24 μM) with minimal cytotoxicity and was selected for elucidation of its antiviral mechanism. Time-of-addition studies, cotreatment assays, and direct transfection of viral genomic RNA indicated that harringtonine inhibited an early stage of the CHIKV replication cycle which occurred after viral entry into cells. In addition, quantitative reverse transcription-PCR (qRT-PCR) and Western blot analyses indicated that harringtonine affects CHIKV RNA production as well as viral protein expression. Treatment of harringtonine against Sindbis virus, a related alphavirus, suggested that harringtonine could inhibit other alphaviruses. This study suggests for the first time that harringtonine exerts its antiviral effects by inhibiting CHIKV viral protein synthesis.

Modulation of EGF Receptor Expression by Differentiating Agents in Human Colon Carcinoma Cell Lines

1990 ◽  
Vol 2 (10) ◽  
pp. 345-355 ◽  
Author(s):  
Lizabeth Deutsch Murphy ◽  
Eva M. Valverius ◽  
Maria Tsokos ◽  
Lyn A. Mickley ◽  
Neal Rosen ◽  
...  
Keyword(s):  
Colon Carcinoma ◽  
Cell Lines ◽  
Egf Receptor ◽  
Carcinoma Cell ◽  
Human Colon ◽  
Receptor Expression ◽  
Human Colon Carcinoma Cell ◽  
Carcinoma Cell Lines ◽  
Human Colon Carcinoma ◽  
Differentiating Agents

scholarly journals The Phosphatidylserine Receptor TIM-1 Enhances Authentic Chikungunya Virus Cell Entry

Cells ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1828
Author(s):  
Jared Kirui ◽  
Yara Abidine ◽  
Annasara Lenman ◽  
Koushikul Islam ◽  
Yong-Dae Gwon ◽  
...  
Keyword(s):  
Chikungunya Virus ◽  
Molecular Mechanisms ◽  
Rna Virus ◽  
Ectopic Expression ◽  
Point Mutations ◽  
Cell Entry ◽  
Target Cells ◽  
Human Hepatoma Cells ◽  
Chikv Infection ◽  
Phosphatidylserine Receptor

Chikungunya virus (CHIKV) is a re-emerging, mosquito-transmitted, enveloped positive stranded RNA virus. Chikungunya fever is characterized by acute and chronic debilitating arthritis. Although multiple host factors have been shown to enhance CHIKV infection, the molecular mechanisms of cell entry and entry factors remain poorly understood. The phosphatidylserine-dependent receptors, T-cell immunoglobulin and mucin domain 1 (TIM-1) and Axl receptor tyrosine kinase (Axl), are transmembrane proteins that can serve as entry factors for enveloped viruses. Previous studies used pseudoviruses to delineate the role of TIM-1 and Axl in CHIKV entry. Conversely, here, we use the authentic CHIKV and cells ectopically expressing TIM-1 or Axl and demonstrate a role for TIM-1 in CHIKV infection. To further characterize TIM-1-dependent CHIKV infection, we generated cells expressing domain mutants of TIM-1. We show that point mutations in the phosphatidylserine binding site of TIM-1 lead to reduced binding, entry, and infection of CHIKV. Ectopic expression of TIM-1 renders immortalized keratinocytes permissive to CHIKV, whereas silencing of endogenously expressed TIM-1 in human hepatoma cells reduces CHIKV infection. Altogether, our findings indicate that, unlike Axl, TIM-1 readily promotes the productive entry of authentic CHIKV into target cells.

scholarly journals Insights into Antibody-Mediated Alphavirus Immunity and Vaccine Development Landscape

2021 ◽  
Vol 9 (5) ◽  
pp. 899
Author(s):  
Anthony Torres-Ruesta ◽  
Rhonda Sin-Ling Chee ◽  
Lisa F.P. Ng
Keyword(s):  
Diagnostic Tests ◽  
Inflammatory Arthritis ◽  
Chikungunya Virus ◽  
Vaccine Development ◽  
Antibody Responses ◽  
Chikv Infection ◽  
Wide Range ◽  
Susceptible Populations ◽  
Humoral Responses

Alphaviruses are mosquito-borne pathogens distributed worldwide in tropical and temperate areas causing a wide range of symptoms ranging from inflammatory arthritis-like manifestations to the induction of encephalitis in humans. Historically, large outbreaks in susceptible populations have been recorded followed by the development of protective long-lasting antibody responses suggesting a potential advantageous role for a vaccine. Although the current understanding of alphavirus antibody-mediated immunity has been mainly gathered in natural and experimental settings of chikungunya virus (CHIKV) infection, little is known about the humoral responses triggered by other emerging alphaviruses. This knowledge is needed to improve serology-based diagnostic tests and the development of highly effective cross-protective vaccines. Here, we review the role of antibody-mediated immunity upon arthritogenic and neurotropic alphavirus infections, and the current research efforts for the development of vaccines as a tool to control future alphavirus outbreaks.

scholarly journals Combination Regimens of Favipiravir Plus Interferon Alpha Inhibit Chikungunya Virus Replication in Clinically Relevant Human Cell Lines

2021 ◽  
Vol 9 (2) ◽  
pp. 307
Author(s):  
Evelyn J. Franco ◽  
Xun Tao ◽  
Kaley C. Hanrahan ◽  
Jieqiang Zhou ◽  
Jürgen B. Bulitta ◽  
...  
Keyword(s):  
Combination Therapy ◽  
Antiviral Activity ◽  
Viral Replication ◽  
Cell Lines ◽  
Interferon Alpha ◽  
Chikungunya Virus ◽  
Plaque Assay ◽  
Human Infection ◽  
Viral Burden ◽  
Combination Regimens

Chikungunya virus (CHIKV) is an alphavirus associated with a broad tissue tropism for which no antivirals or vaccines are approved. This study evaluated the antiviral potential of favipiravir (FAV), interferon-alpha (IFN), and ribavirin (RBV) against CHIKV as mono- and combination-therapy in cell lines that are clinically relevant to human infection. Cells derived from human connective tissue (HT-1080), neurons (SK-N-MC), and skin (HFF-1) were infected with CHIKV and treated with different concentrations of FAV, IFN, or RBV. Viral supernatant was sampled daily and the burden was quantified by plaque assay on Vero cells. FAV and IFN were the most effective against CHIKV on various cell lines, suppressing the viral burden at clinically achievable concentrations; although the degree of antiviral activity was heavily influenced by cell type. RBV was not effective and demonstrated substantial toxicity, indicating that it is not a feasible candidate for CHIKV. The combination of FAV and IFN was then assessed on all cell lines. Combination therapy enhanced antiviral activity in HT-1080 and SK-N-MC cells, but not in HFF-1 cells. We developed a pharmacokinetic/pharmacodynamic model that described the viral burden and inhibitory antiviral effect. Simulations from this model predicted clinically relevant concentrations of FAV plus IFN completely suppressed CHIKV replication in HT-1080 cells, and considerably slowed down the rate of viral replication in SK-N-MC cells. The model predicted substantial inhibition of viral replication by clinical IFN regimens in HFF-1 cells. Our results highlight the antiviral potential of FAV and IFN combination regimens against CHIKV in clinically relevant cell types.

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