A modified kynurenine bioassay for quantitative determination of human interferon-γ

Boyanova, Maya; Tsanev, Roumen; Ivanov, Iván

doi:10.1016/s0003-2697(02)00208-7

Cited by 22 publications

(16 citation statements)

References 14 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In some experiments, Eos were also cultured with IFN-␥ in the presence of 1-methyl-tryptophan (800 M), a specific IDO inhibitor, or anti-human IFN-␥ Ab (10 g/ml). A colorimetric method was used to determine the concentration of KYN in culture supernatant as previously described (13).…”

Section: Cell Isolation and Culturementioning

confidence: 99%

Cutting Edge: Human Eosinophils Regulate T Cell Subset Selection through Indoleamine 2,3-Dioxygenase

Odemuyiwa

Ghahary

Li³

et al. 2004

The Journal of Immunology

189

171

View full text Add to dashboard Cite

Allergy involves eosinophilia and Th2 polarization. Indoleamine 2,3-dioxygenase (IDO)-catalyzed conversion of tryptophan to kynurenines (KYN) regulates T cell function. We show that human eosinophils constitutively express IDO. Eosinophils treated with IFN-γ showed an 8-fold increase in IDO mRNA within 4 h; IL-3, IL-5, and GM-CSF had no effect on baseline IDO expression. IL-3 pretreatment of eosinophils reduced IFN-γ-induced IDO mRNA expression below baseline. Conversely, GM-CSF, but not IL-5, resulted in a 2-fold increase in IFN-γ-induced IDO. Treatment with IL-3, IL-5, GM-CSF, or IFN-γ alone expressed IDO enzymatic activity (the presence of KYN in supernatants 48 h postculture). CD28 cross-linking resulted in measurable KYN in culture supernatants, inhibitable by a neutralizing anti-IFN-γ. Coculture of eosinophils with an IFN-γ-producing T cell line, but not IL-4-producing T cell clone, led to apoptosis and inhibition of CD3 or CD3/CD28-induced proliferation. Eosinophils infiltrating asthmatic lung and associated lymphoid tissue exhibited intracellular IDO immunoreactivity. Eosinophils may, therefore, maintain Th2 bias through IDO.

show abstract

Section: Cell Isolation and Culturementioning

confidence: 99%

Cutting Edge: Human Eosinophils Regulate T Cell Subset Selection through Indoleamine 2,3-Dioxygenase

Odemuyiwa

Ghahary

Li³

et al. 2004

The Journal of Immunology

189

171

View full text Add to dashboard Cite

show abstract

“…Antiproliferative activity was determined by a modified kynurenine bioassay on WISH cells as described in [30]. …”

Section: Methodsmentioning

confidence: 99%

His-FLAG Tag as a Fusion Partner of Glycosylated Human Interferon-Gamma and Its Mutant: Gain or Loss?

Krachmarova

Tileva

Lilkova

et al. 2017

BioMed Research International

Self Cite

View full text Add to dashboard Cite

In order to obtain glycosylated human interferon-gamma (hIFNγ) and its highly prone to aggregation mutant K88Q, a secretory expression in insect cells was employed. To facilitate recombinant proteins purification, detection, and stability the baculovirus expression vectors were constructed to bear N-terminal His6-FLAG tag. Although the obtained proteins were glycosylated, we found that their biological activity was 100 times lower than expected. Our attempts to recover the biological properties of both proteins by tag removal failed due to enterokinase resistance of the tag. Surprisingly, the tag was easily cleaved when the proteins were expressed in E. coli cells and the tag-free proteins showed fully restored activity. To shed light on this phenomenon we performed molecular dynamics simulations. The latter showed that the tags interact with the receptor binding domains and the flexible C-termini of the fusion proteins thus suppressing their complex formation with the hIFNγ receptor. We hypothesize that in the case of glycosylated proteins the tag/C-terminal interaction positions the FLAG peptide in close proximity to the glycans thus sterically impeding the enterokinase access to its recognition site.

show abstract

“…Biological activity of the expressed in the supernatant protein was measured by hIFNγ antiproliferative activity on WISH cells following a modified kynurenine bioassay (6).…”

Section: Methodsmentioning

confidence: 99%

Expression of Human Interferon-Gamma Gene in Human Tissue Culture Cells

Leister¹,

Tileva

Krachmarova

et al. 2013

Biotechnology & Biotechnological Equipment

View full text Add to dashboard Cite

A modified kynurenine bioassay for quantitative determination of human interferon-γ

Cited by 22 publications

References 14 publications

Cutting Edge: Human Eosinophils Regulate T Cell Subset Selection through Indoleamine 2,3-Dioxygenase

Cutting Edge: Human Eosinophils Regulate T Cell Subset Selection through Indoleamine 2,3-Dioxygenase

His-FLAG Tag as a Fusion Partner of Glycosylated Human Interferon-Gamma and Its Mutant: Gain or Loss?

Expression of Human Interferon-Gamma Gene in Human Tissue Culture Cells

Contact Info

Product

Resources

About