scholarly journals Development and Characterisation of a Novel NF-κB Reporter Cell Line for Investigation of Neuroinflammation

2017 ◽  
Vol 2017 ◽  
pp. 1-10 ◽  
Author(s):  
Marie-Theres Zeuner ◽  
Thomas Vallance ◽  
Sakthivel Vaiyapuri ◽  
Graeme S. Cottrell ◽  
Darius Widera
Keyword(s):  
Cell Line ◽  
Glioma Cell Line ◽  
Neural Cells ◽  
Reporter Cell Line ◽  
Reporter Cell ◽  
Necrosis Factor Alpha ◽  
In Vitro Drug Screening ◽  
Anti Inflammatory ◽  
Cost Efficient

Aberrant activation of the transcription factor NF-κB, as well as uncontrolled inflammation, has been linked to autoimmune diseases, development and progression of cancer, and neurological disorders like Alzheimer’s disease. Reporter cell lines are a valuable state-of-the art tool for comparative analysis of in vitro drug screening. However, a reporter cell line for the investigation of NF-κB-driven neuroinflammation has not been available. Thus, we developed a stable neural NF-κB-reporter cell line to assess the potency of proinflammatory molecules and peptides, as well as anti-inflammatory pharmaceuticals. We used lentivirus to transduce the glioma cell line U251-MG with a tandem NF-κB reporter construct containing GFP and firefly luciferase allowing an assessment of NF-κB activity via fluorescence microscopy, flow cytometry, and luminometry. We observed a robust activation of NF-κB after exposure of the reporter cell line to tumour necrosis factor alpha (TNFα) and amyloid-β peptide [1-42] as well as to LPS derived from Salmonella minnesota and Escherichia coli. Finally, we demonstrate that the U251-NF-κB-GFP-Luc reporter cells can be used for assessing the anti-inflammatory potential of pharmaceutical compounds using Bay11-7082 and IMD0354. In summary, our newly generated cell line is a robust and cost-efficient tool to study pro- and anti-inflammatory potential of drugs and biologics in neural cells.

scholarly journals Counteracting Interactions between Lipopolysaccharide Molecules with Differential Activation of Toll-Like Receptors

2002 ◽  
Vol 70 (12) ◽  
pp. 6658-6664 ◽  
Author(s):  
George Hajishengallis ◽  
Michael Martin ◽  
Robert E. Schifferle ◽  
Robert J. Genco
Keyword(s):  
Cell Line ◽  
Interleukin 1 ◽  
Signal Transduction Pathway ◽  
Reporter Cell Line ◽  
Reporter Cell ◽  
Tlr2 Expression ◽  
Necrosis Factor Alpha ◽  
Inflammatory Reactions ◽  
Tnf Α ◽  
Induced Tolerance

ABSTRACT We investigated counteracting interactions between the lipopolysaccharides (LPS) from Escherichia coli (Ec-LPS) and Porphyromonas gingivalis (Pg-LPS), which induce cellular activation through Toll-like receptor 4 (TLR4) and TLR2, respectively. We found that Ec-LPS induced tolerance in THP-1 cells to subsequent tumor necrosis factor alpha (TNF-α) and interleukin 1 beta (IL-1β) induction by Pg-LPS, though the reverse was not true, and looked for explanatory differential effects on the signal transduction pathway. Cells exposed to Pg-LPS, but not to Ec-LPS, displayed persisting expression of IL-1 receptor-associated kinase without apparent degradation, presumably allowing prolonged relay of downstream signals. Accordingly, cells pretreated with Pg-LPS, but not with Ec-LPS, were effectively activated in response to subsequent exposure to either LPS molecule, as evidenced by assessing nuclear factor (NF)-κB activity. In fact, Pg-LPS primed THP-1 cells for enhanced NF-κB activation and TNF-α release upon restimulation with the same LPS. This was a dose-dependent effect and correlated with upregulation of surface TLR2 expression. Furthermore, we observed inhibition of NF-κB-dependent transcription in a reporter cell line pretreated with Ec-LPS and restimulated with Pg-LPS (compared to cells pretreated with medium only and restimulated with Pg-LPS), but not when the reverse treatment was made. Although Pg-LPS could not make cells tolerant to subsequent activation by Ec-LPS, Pg-LPS inhibited Ec-LPS-induced TNF-α and IL-6 release when the two molecules were added simultaneously into THP-1 cell cultures. Pg-LPS also suppressed P. gingivalis FimA protein-induced NF-κB-dependent transcription in the 3E10/huTLR4 reporter cell line, which does not express TLR2. This rules out competition for common signaling intermediates, suggesting that Pg-LPS may block component(s) of the TLR4 receptor complex. Interactions between TLR2 and TLR4 agonists may be important in the regulation of inflammatory reactions.

scholarly journals A new reporter cell line to monitor HIV infection and drug susceptibility in vitro

1997 ◽  
Vol 94 (9) ◽  
pp. 4653-4658 ◽  
Author(s):  
A. Gervaix ◽  
D. West ◽  
L. M. Leoni ◽  
D. D. Richman ◽  
F. Wong-Staal ◽  
...  
Keyword(s):  
Hiv Infection ◽  
Cell Line ◽  
Drug Susceptibility ◽  
Reporter Cell Line ◽  
Reporter Cell

scholarly journals S-Layer Protein Mediates the Stimulatory Effect of Lactobacillus helveticus MIMLh5 on Innate Immunity

2012 ◽  
Vol 79 (4) ◽  
pp. 1221-1231 ◽  
Author(s):  
Valentina Taverniti ◽  
Milda Stuknyte ◽  
Mario Minuzzo ◽  
Stefania Arioli ◽  
Ivano De Noni ◽  
...  
Keyword(s):  
Cell Line ◽  
Ex Vivo ◽  
Lactobacillus Helveticus ◽  
Human Macrophage ◽  
Mouse Bone Marrow ◽  
Similar Response ◽  
Bacterial Cells ◽  
Necrosis Factor Alpha ◽  
Anti Inflammatory

ABSTRACTThe ability to positively affect host health through the modulation of the immune response is a feature of increasing importance in measuring the probiotic potential of a bacterial strain. However, the identities of the bacterial cell components involved in cross talk with immune cells remain elusive. In this study, we characterized the dairy strainLactobacillus helveticusMIMLh5 and its surface-layer protein (SlpA) usingin vitroandex vivoanalyses. We found that MIMLh5 and SlpA exert anti-inflammatory effects by reducing the activation of NF-κB on the intestinal epithelial Caco-2 cell line. On the contrary, MIMLh5 and SlpA act as stimulators of the innate immune system by triggering the expression of proinflammatory factors tumor necrosis factor alpha and COX-2 in the human macrophage cell line U937 via recognition through Toll-like receptor 2. In the same experiments, SlpA protein did not affect the expression of the anti-inflammatory cytokine interleukin-10. A similar response was observed following stimulation of macrophages isolated from mouse bone marrow or the peritoneal cavity. These results suggest that SlpA plays a major role in mediating bacterial immune-stimulating activity, which could help to induce the host's defenses against and responses toward infections. This study supports the concept that the viability of bacterial cells is not always essential to exert immunomodulatory effects, thus permitting the development of safer therapies for the treatment of specific diseases according to a paraprobiotic intervention.

scholarly journals An In Vitro Test to Screen Skin Sensitizers Using a Stable THP-1–Derived IL-8 Reporter Cell Line, THP-G8

2011 ◽  
Vol 124 (2) ◽  
pp. 359-369 ◽  
Author(s):  
Toshiya Takahashi ◽  
Yutaka Kimura ◽  
Rumiko Saito ◽  
Yoshihiro Nakajima ◽  
Yoshihiro Ohmiya ◽  
...  
Keyword(s):  
Cell Line ◽  
In Vitro Test ◽  
Reporter Cell Line ◽  
Reporter Cell ◽  
Vitro Test

scholarly journals Molecular Characterization of a Nondemyelinating Variant of Daniel’s Strain of Theiler’s Virus Isolated from a Persistently Infected Glioma Cell Line

1998 ◽  
Vol 72 (2) ◽  
pp. 1262-1269 ◽  
Author(s):  
Xiaoqi Lin ◽  
Shigeru Sato ◽  
Amy K. Patick ◽  
Larry R. Pease ◽  
Raymond P. Roos ◽  
...  
Keyword(s):  
Cell Line ◽  
Glioma Cell ◽  
Point Mutations ◽  
Glioma Cell Line ◽  
Neural Cells ◽  
Immune Recognition ◽  
Theiler's Virus

ABSTRACT Wild-type Daniel’s strain of Theiler’s virus (wt-DA) induces a chronic demyelination in susceptible mice which is similar to multiple sclerosis. A variant of wt-DA (designated DA-P12) generated during the 12th passage of persistent infection of a G26-20 glioma cell line failed to persist and induce demyelination in SJL/J mice. To identify the determinants responsible for this change in phenotype, we sequenced the capsid coding sequence (nucleotides [nt] 2991 to 3994) and found three mutations in VP1: residues 99 (Gly to Ser), 100 (Gly to Asp), and 103 (Asn to Lys). To study the role of these mutations in neurovirulence and demyelination, we prepared a recombinant virus, DAP-1C-2A/DA, with replacement of wt-DA nt 2991 to 3994 with the corresponding region of DA-P12, and viruses with individual point mutations at VP1 residues 99(Ser), 100(Asp), and 103(Lys). DAP-1C-2A/DA and viruses with a mutation at VP1 residue 99 or 100 (but not 103) completely attenuated the ability of wt-DA to induce demyelination. Failure to induce demyelination was not due to a general failure in growth, since DA-P12 and other mutant viruses lysed L-2 cells in vitro as effectively as wt-DA. The change in disease phenotype was independent of the specific B- or T-cell immune recognition because a decrease in the neurovirulence of mutant viruses was observed in neonatal mice and immune-deficient RAG1 −/− mice. This difference in neurovirulence is not the complete explanation for the failure of DA-P12 to demyelinate, since virus with a mutation at residue 103(Lys) had decreased neurovirulence but did induce demyelination. Therefore, point mutation at VP1 residue 99 or 100 altered the ability of wt-DA to demyelinate, perhaps related to a disruption in interaction between virus and receptor on certain neural cells.

scholarly journals A Novel Fibroblast Reporter Cell Line for in vitro Studies of Pulmonary Fibrosis

2020 ◽  
Vol 11 ◽  
Author(s):  
Julia Nemeth ◽  
Annika Schundner ◽  
Karsten Quast ◽  
Veronika E. Winkelmann ◽  
Manfred Frick
Keyword(s):  
Pulmonary Fibrosis ◽  
Cell Line ◽  
In Vitro Studies ◽  
Reporter Cell Line ◽  
Reporter Cell

scholarly journals New Reporter Cell Line To Evaluate the Sequential Emergence of Multiple Human Cytomegalovirus Mutations during In Vitro Drug Exposure

2005 ◽  
Vol 49 (12) ◽  
pp. 4860-4866 ◽  
Author(s):  
C. Gilbert ◽  
G. Boivin
Keyword(s):  
Cell Line ◽  
Human Cytomegalovirus ◽  
Drug Exposure ◽  
Luciferase Reporter ◽  
Reporter Cell Line ◽  
Reporter Cell ◽  
Luciferase Reporter Gene ◽  
Selection Of

ABSTRACT We developed a new reporter cell line for human cytomegalovirus (HCMV) drug susceptibility testing. This cell line was obtained by incorporating the luciferase reporter gene under the control of an HCMV-specific promoter into the genome of astrocytoma cells (U373MG). We then used our reporter cell line to evaluate phenotypic changes conferred by the sequential emergence of HCMV UL54 and UL97 mutations following long-term drug exposure. The laboratory strain AD169 was passaged in the presence of increasing concentrations of ganciclovir (one viral line) or foscarnet (two viral lines). Resistant viruses were plaque purified at five different concentrations of ganciclovir and at three different concentrations of foscarnet. In addition to the previously described M460I and L595S UL97 mutations and the L545S and V812L UL54 mutations, exposition to ganciclovir (up to 3,000 μM) resulted in the selection of two unreported UL54 mutations (P829S and D879G). Passages in the presence of foscarnet (up to 3,000 μM) resulted in the selection of seven not previously described UL54 mutations (K500N, T552N, S585A, N757K, L802V, L926V, and L957F) in addition to the N408D mutation that has been associated with ganciclovir and cidofovir resistance. Long-term exposure of HCMV to either ganciclovir or foscarnet ultimately resulted in the selection of multiple UL54 mutations that conferred high levels of resistance to all approved HCMV DNA polymerase inhibitors, i.e., ganciclovir, cidofovir, and foscarnet. Emergence of each viral mutation conferred stepwise increases in drug 50% inhibitory concentrations that could be objectively measured with the new reporter cell assay.

scholarly journals Engineering a reporter cell line to mimic the high oligomannose presenting surface immunoglobulin of follicular lymphoma B cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Butaek Lim ◽  
LeNaiya Kydd ◽  
Justyn Jaworski
Keyword(s):  
Follicular Lymphoma ◽  
B Cell ◽  
Cell Line ◽  
Reporter Cell Line ◽  
Reporter Cell ◽  
Variable Heavy ◽  
Hodgkin's Lymphomas ◽  
Follicular Lymphomas

AbstractSubtypes of B cell non-Hodgkin’s lymphomas, including follicular lymphomas, have shown a unique high oligomannose presentation on their immunoglobulins that will interact with natural receptors of the innate immunity, reportedly causing stimulation and proliferation. From deep sequencing of the variable heavy and light chain sequences of follicular lymphoma involved tissue sections, we identified the consensus variable sequences possessing glycosylation sites at the complementarity determining region. Using this information, we developed a cell line, referred to here as BZ, which displays the consensus variable segments as part of a surface antibody (IgM) and confirmed its presentation of high oligomannose on the heavy chain both in vitro and in vivo. An mCherry expressing variant provided a reporter cell line displaying the high oligomannose surface biomarker while affording clear fluorescent signals for FACS screening as well as for fluorescent in vivo imaging of ectopic xenograft tumors. In developing this reporter cell line that displays the biomarker glycan of follicular lymphoma, we provide a tool that may be used for future screening and validation of receptive moieties for selectively binding high oligomannose for development of targeted diagnostics or therapeutics to such B cell malignancies that display this unique glycan.

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