Establishment of a consistent L929 bioassay system for TNF‐<i>α</i> quantitation to evaluate the effect of lipopolysaccharide, phytomitogens and cytodifferentiation agents on cytotoxicity of TNF‐<i>α</i> secreted by adherent human mononuclear cells

Shiau, Ming Yuh; Chiou, Hui‐Ling; Lee, Yao Ling; Kuo, Tzer Min; Chang, Yih Hsin

doi:10.1080/09629350123139

Cited by 30 publications

(26 citation statements)

References 18 publications

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“…To ascertain the neutralizing efficacy of anti-TNF-TT fusion antibodies compared with the parental anti-TNF, we performed L929 assay as described previously 52 . Briefly, murine L929 fibrosarcoma cells in log phase were harvested by trypsinization, washed and suspended in cell growth medium (RPMI containing 10% FBS, 2 mM L -glutamine, 1% Na-pyruvate, 1% non-essential amino acids, 0.1% β-mercaptoethanol and 1% Pen/Strep).…”

Section: Methodsmentioning

confidence: 99%

Transmembrane TNF-dependent uptake of anti-TNF antibodies

Deora

Hegde

Lee

et al. 2017

mAbs

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TNF-α (TNF), a pro-inflammatory cytokine is synthesized as a 26 kDa protein, anchors in the plasma membrane as transmembrane TNF (TmTNF), and is subjected to proteolysis by the TNF-α converting enzyme (TACE) to release the 15 kDa form of soluble TNF (sTNF). TmTNF and sTNF interact with 2 distinct receptors, TNF-R1 (p55) and TNF-R2 (p75), to mediate the multiple biologic effects of TNF described to date. Several anti-TNF biologics that bind to both forms of TNF and block their interactions with the TNF receptors are now approved for the treatment of a variety of immune-mediated diseases. Several reports suggest that binding of anti-TNFs to TmTNF delivers an outside-to-inside ‘reverse’ signal that may also contribute to the efficacy of anti-TNFs. Some patients, however, develop anti-TNF drug antibody responses (ADA or immunogenicity). Here, we demonstrate biochemically that TmTNF is transiently expressed on the surface of lipopolysaccharide-stimulated primary human monocytes, macrophages, and monocyte-derived dendritic cells (DCs) and expression of TmTNF on the cell surface is enhanced following treatment of cells with TAPI-2, a TACE inhibitor. Importantly, binding of anti-TNFs to TmTNF on DCs results in rapid internalization of the anti-TNF/TmTNF complex first into early endosomes and then lysosomes. The internalized anti-TNF is processed and anti-TNF peptides can be eluted from the surface of DCs. Finally, tetanus toxin peptides fused to anti-TNFs are presented by DCs to initiate T cell recall proliferation response. Collectively, these observations may provide new insights into understanding the biology of TmTNF, mode of action of anti-TNFs, biology of ADA response to anti-TNFs, and may help with the design of the next generation of anti-TNFs.

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Section: Methodsmentioning

confidence: 99%

Transmembrane TNF-dependent uptake of anti-TNF antibodies

Deora

Hegde

Lee

et al. 2017

mAbs

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“…The supernatant (100 µl/well) of the macrophages after indicated treatment/s was added to L929 cells for 20 h [34], [35]. MTT (3-(4,5-dimethylthazol-2-yl)-2,5-diphenyltetrazolinum bromide) salt (0.25 mg/ml) was added to each well.…”

Section: Methodsmentioning

confidence: 99%

The Synthetic Melanocortin (CKPV)2 Exerts Anti-Fungal and Anti-Inflammatory Effects against Candida albicans Vaginitis via Inducing Macrophage M2 Polarization

Zou

Duan

et al. 2013

PLoS ONE

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In this study, we examined anti-fungal and anti-inflammatory effects of the synthetic melanocortin peptide (Ac-Cys-Lys-Pro-Val-NH2)2 or (CKPV)2 against Candida albicans vaginitis. Our in vitro results showed that (CKPV)2 dose-dependently inhibited Candida albicans colonies formation. In a rat Candida albicans vaginitis model, (CKPV)2 significantly inhibited vaginal Candida albicans survival and macrophages sub-epithelial mucosa infiltration. For mechanisms study, we observed that (CKPV)2 inhibited macrophages phagocytosis of Candida albicans. Meanwhile, (CKPV)2 administration inhibited macrophage pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) release, while increasing the arginase activity and anti-inflammatory cytokine IL-10 production, suggesting macrophages M1 to M2 polarization. Cyclic AMP (cAMP) production was also induced by (CKPV)2 administration in macrophages. These above effects on macrophages by (CKPV)2 were almost reversed by melanocortin receptor-1(MC1R) siRNA knockdown, indicating the requirement of MC1R in the process. Altogether, our results suggest that (CKPV)2 exerted anti-fungal and anti-inflammatory activities against Candida albicans vaginitis probably through inducing macrophages M1 to M2 polarization and MC1R activation.

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“…L929 cytotoxicity bioassay. A modified L929 cytotoxic bioassay system for TNF-␣ quantification was employed to evaluate secreted TNF-␣ in treated ASTs (37). Briefly, 2 ϫ 10 4 L929 cells per well were seeded into a 96-well tissue culture plate and cultured in RPMI medium supplemented with 5 g/ml streptomycin, 5 U/ml penicillin, and 10% FCS.…”

Section: Methodsmentioning

confidence: 99%

Shiga Toxin 1-Induced Inflammatory Response in Lipopolysaccharide-Sensitized Astrocytes Is Mediated by Endogenous Tumor Necrosis Factor Alpha

Landoni

Campos-Nebel

Schierloh³

et al. 2010

Infect Immun

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Hemolytic-uremic syndrome (HUS) is generally caused by Shiga toxin (Stx)-producingThe epidemic form of hemolytic-uremic syndrome (HUS) has been associated with enterohemorrhagic infections caused by Shiga toxin (Stx)-producing Escherichia coli (STEC) organisms (33). HUS is the most common cause of acute renal failure in children and is related to the endothelial damage of glomeruli and/or arterioles of the kidney and epithelial cell damage induced by Stx through the interaction with its globotriaosylceramide (Gb 3 ) receptor (35). Although Stx is the main pathogenic factor and is necessary for epidemic HUS development, clinical and experimental evidence suggests that the inflammatory response is able to potentiate Stx toxicity. In fact, both bacterial lipopolysaccharide (LPS) and polymorphonuclear neutrophils (PMN) play a key role in the full development of HUS (15). Moreover, PMN leukocytosis in patients correlates with a poor prognosis (17).Endothelial cell damage is not limited to the kidney but extends to other organs; in severe cases, the brain can be affected. In fact, central nervous system (CNS) complications indicate severe HUS, and brain damage involvement is the most common cause of death (14).However, the pathogenesis of CNS impairment is not yet fully understood. Although it has been demonstrated that human brain endothelial cells (BECs) are relatively resistant to Stx, inflammatory mediators, such as tumor necrosis factor alpha (TNF-␣), markedly increase human BEC sensitivity to Stx cytotoxicity (11).BECs are part of the blood-brain barrier (BBB), which protects the brain from potentially harmful substances and leukocytes present in the bloodstream. Thus, the integrity of BBB function is theorized to be a key component in CNS-associated pathologies, and BEC damage is thought to be one of the possible mechanisms involved in the disruption of the BBB in HUS. In fact, LPS from bacterial infections leads to the release of TNF-␣, interleukin-1␤ (IL-1␤), and reactive oxygen species (ROS), all of which have the ability to open the BBB.Several in vivo studies demonstrated previously that Stx is able to impair BBB function, increasing its permeability (21). Moreover, Stx itself is able to cross the endothelial barrier and enter into the CNS, since Stx activity in cerebrospinal fluid was previously observed (19,23), and Stx was previously immunodetected in many brain cells including astrocytes (ASTs) and neurons (44).

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Establishment of a consistent L929 bioassay system for TNF‐α quantitation to evaluate the effect of lipopolysaccharide, phytomitogens and cytodifferentiation agents on cytotoxicity of TNF‐α secreted by adherent human mononuclear cells

Cited by 30 publications

References 18 publications

Transmembrane TNF-dependent uptake of anti-TNF antibodies

Transmembrane TNF-dependent uptake of anti-TNF antibodies

The Synthetic Melanocortin (CKPV)2 Exerts Anti-Fungal and Anti-Inflammatory Effects against Candida albicans Vaginitis via Inducing Macrophage M2 Polarization

Shiga Toxin 1-Induced Inflammatory Response in Lipopolysaccharide-Sensitized Astrocytes Is Mediated by Endogenous Tumor Necrosis Factor Alpha

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