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P70S6 kinase phosphorylation: a new site to assess pharmacodynamy of sirolimus.

Wang JY, Fan H - Chin. Med. J. (2015)

Bottom Line: The p70S6K phosphorylation in patients receiving a sirolimus (19.5 ± 7.7) was significantly lower than in HC (50.1 ± 11.3, P < 0.001), tacrolimus (37.7 ± 15.7, P < 0.001) or cyclosporine treated patients (41.7 ± 11.7, P < 0.001).The p70S6K phosphorylation in HC (50.1 ± 11.3) was significantly higher than in tacrolimus (37.7 ± 15.7, P < 0.01) or cyclosporine-treated patients (41.7 ± 11.7, P < 0.01).There was correlation between data from phospho-flow cytometry and data from Western blotting (r = 0.88, P < 0.001).

View Article: PubMed Central - PubMed

Affiliation: Department of Hepatobiliary Surgery, Beijing Chaoyang Hospital, Capital Medical University, Beijing 100020, China.

ABSTRACT

Background: The phosphorylation of p70S6 kinase (p70S6K) represents an important target for sensitive detection on pharmacodynamic effects of sirolimus, but the methods of assessing p70S6K phosphorylation are still unclear. The aim of this study was to investigate p70S6K phosphorylation located down-stream of the mammalian target of rapamycin (mTOR) pathway in peripheral blood mononuclear cells (PBMCs) of liver transplant patients through different methods.

Methods: Seventy-five liver transplant recipients from Beijing Chaoyang Hospital of the Capital Medical University were analyzed in this study. Patients were divided into three groups, patient treated with sirolimus (n = 22), patient treated with tacrolimus (n = 30), patient treated with cyclosporine (n = 23). The p70S6K phosphorylation of PBMCs in patients and healthy control (HC, n = 12) were analyzed by phospho-flow cytometry and Western blotting. A correlation analysis of data from phospho-flow cytometry and Western blotting was performed. Intra-assay variability of p70S6K phosphorylation in HC and different patients were measured.

Results: Intra-assay variability of p70S6K phosphorylation in phospho-flow cytometry was from 4.1% to 8.4% and in Western blotting was from 8.2% to 18%. The p70S6K phosphorylation in patients receiving a sirolimus (19.5 ± 7.7) was significantly lower than in HC (50.1 ± 11.3, P < 0.001), tacrolimus (37.7 ± 15.7, P < 0.001) or cyclosporine treated patients (41.7 ± 11.7, P < 0.001). The p70S6K phosphorylation in HC (50.1 ± 11.3) was significantly higher than in tacrolimus (37.7 ± 15.7, P < 0.01) or cyclosporine-treated patients (41.7 ± 11.7, P < 0.01). There was correlation between data from phospho-flow cytometry and data from Western blotting (r = 0.88, P < 0.001).

Conclusions: The degree of mTOR inhibition by assessing p70S6K phosphorylation was established by phospho-flow cytometry and Western blotting. Assessment of p70S6K phosphorylation may play an adjunct role to on pharmacodynamically guide and individualize sirolimus based on immunosuppression.

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The p70S6 kinase phosphorylation of peripheral blood mononuclear cells in patients treated with different immunosuppressive agents and health control. MFI index: Mean fluorescence intensity index (‡P < 0.001 vs. sirolimus group, †P < 0.05).
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Figure 2: The p70S6 kinase phosphorylation of peripheral blood mononuclear cells in patients treated with different immunosuppressive agents and health control. MFI index: Mean fluorescence intensity index (‡P < 0.001 vs. sirolimus group, †P < 0.05).

Mentions: P70S6 kinase phosphorylation of PBMCs was assessed in patients treated with different immunosuppressive agent and HC [Figure 2]. The phospho-flow technique detected a significant loss of p70S6K phosphorylation at Thr389 in PBMCs of patients who treated with sirolimus (n = 26, mean MFI index: 19.5 ± 7.7) compared to HC (n = 16, mean MFI index: 50.1 ± 11.3, P < 0.001) and liver transplant recipients receiving an immunosuppressive therapy based on tacrolimus (n = 35, mean MFI index: 37.7 ± 15.7, P < 0.001) or cyclosporine (n = 23, mean MFI index: 41.7 ± 11.7, P < 0.001). The p70S6K phosphorylation in HC (50.1 ± 11.3) was higher than that in tacrlimus (37.7 ± 15.7, P < 0.01) and cyclosporine (41.7 ± 11.7, P < 0.01), but no significant difference was shown between tacrolimus (37.7 ± 15.7) and cyclosporine group (41.7 ± 11.7, P = 0.29).


P70S6 kinase phosphorylation: a new site to assess pharmacodynamy of sirolimus.

Wang JY, Fan H - Chin. Med. J. (2015)

The p70S6 kinase phosphorylation of peripheral blood mononuclear cells in patients treated with different immunosuppressive agents and health control. MFI index: Mean fluorescence intensity index (‡P < 0.001 vs. sirolimus group, †P < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4834780&req=5

Figure 2: The p70S6 kinase phosphorylation of peripheral blood mononuclear cells in patients treated with different immunosuppressive agents and health control. MFI index: Mean fluorescence intensity index (‡P < 0.001 vs. sirolimus group, †P < 0.05).
Mentions: P70S6 kinase phosphorylation of PBMCs was assessed in patients treated with different immunosuppressive agent and HC [Figure 2]. The phospho-flow technique detected a significant loss of p70S6K phosphorylation at Thr389 in PBMCs of patients who treated with sirolimus (n = 26, mean MFI index: 19.5 ± 7.7) compared to HC (n = 16, mean MFI index: 50.1 ± 11.3, P < 0.001) and liver transplant recipients receiving an immunosuppressive therapy based on tacrolimus (n = 35, mean MFI index: 37.7 ± 15.7, P < 0.001) or cyclosporine (n = 23, mean MFI index: 41.7 ± 11.7, P < 0.001). The p70S6K phosphorylation in HC (50.1 ± 11.3) was higher than that in tacrlimus (37.7 ± 15.7, P < 0.01) and cyclosporine (41.7 ± 11.7, P < 0.01), but no significant difference was shown between tacrolimus (37.7 ± 15.7) and cyclosporine group (41.7 ± 11.7, P = 0.29).

Bottom Line: The p70S6K phosphorylation in patients receiving a sirolimus (19.5 ± 7.7) was significantly lower than in HC (50.1 ± 11.3, P < 0.001), tacrolimus (37.7 ± 15.7, P < 0.001) or cyclosporine treated patients (41.7 ± 11.7, P < 0.001).The p70S6K phosphorylation in HC (50.1 ± 11.3) was significantly higher than in tacrolimus (37.7 ± 15.7, P < 0.01) or cyclosporine-treated patients (41.7 ± 11.7, P < 0.01).There was correlation between data from phospho-flow cytometry and data from Western blotting (r = 0.88, P < 0.001).

View Article: PubMed Central - PubMed

Affiliation: Department of Hepatobiliary Surgery, Beijing Chaoyang Hospital, Capital Medical University, Beijing 100020, China.

ABSTRACT

Background: The phosphorylation of p70S6 kinase (p70S6K) represents an important target for sensitive detection on pharmacodynamic effects of sirolimus, but the methods of assessing p70S6K phosphorylation are still unclear. The aim of this study was to investigate p70S6K phosphorylation located down-stream of the mammalian target of rapamycin (mTOR) pathway in peripheral blood mononuclear cells (PBMCs) of liver transplant patients through different methods.

Methods: Seventy-five liver transplant recipients from Beijing Chaoyang Hospital of the Capital Medical University were analyzed in this study. Patients were divided into three groups, patient treated with sirolimus (n = 22), patient treated with tacrolimus (n = 30), patient treated with cyclosporine (n = 23). The p70S6K phosphorylation of PBMCs in patients and healthy control (HC, n = 12) were analyzed by phospho-flow cytometry and Western blotting. A correlation analysis of data from phospho-flow cytometry and Western blotting was performed. Intra-assay variability of p70S6K phosphorylation in HC and different patients were measured.

Results: Intra-assay variability of p70S6K phosphorylation in phospho-flow cytometry was from 4.1% to 8.4% and in Western blotting was from 8.2% to 18%. The p70S6K phosphorylation in patients receiving a sirolimus (19.5 ± 7.7) was significantly lower than in HC (50.1 ± 11.3, P < 0.001), tacrolimus (37.7 ± 15.7, P < 0.001) or cyclosporine treated patients (41.7 ± 11.7, P < 0.001). The p70S6K phosphorylation in HC (50.1 ± 11.3) was significantly higher than in tacrolimus (37.7 ± 15.7, P < 0.01) or cyclosporine-treated patients (41.7 ± 11.7, P < 0.01). There was correlation between data from phospho-flow cytometry and data from Western blotting (r = 0.88, P < 0.001).

Conclusions: The degree of mTOR inhibition by assessing p70S6K phosphorylation was established by phospho-flow cytometry and Western blotting. Assessment of p70S6K phosphorylation may play an adjunct role to on pharmacodynamically guide and individualize sirolimus based on immunosuppression.

Show MeSH
Related in: MedlinePlus