Tacrolimus fk506

The efflux pump inhibitors (EPIs), Phe-L-Arg-β-naphthylamine dihydrochloride (Phe-Arg) and tacrolimus (FK506) (Sigma-Aldrich, USA), were used in combination with FLC or VRC in order to determine if the antifungal activity could be enhanced [28 (link)]. MICs were developed in the presence of azole (64 to 0.125 mg/L) with and without the presence of each EPI at a concentration of 64 mg/L. All the systems were incubated for 48 h at 37 °C and the results were expressed as the lowest azole concentration that resulted in the total inhibition of visible fungal growth.

Splenic T cells of WT mice were cultured to examine the major stimuli and their signaling pathways to induce LSP1 expression. Briefly, T cells were isolated from the spleens and prepared as single-cell suspensions. CD4⁺ T cells or CD8⁺ T cells were purified by magnetic separation using anti-CD4 beads or anti-CD8 beads (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. Purified CD4⁺ T cells or CD8⁺ T cells were stimulated with recombinant IFN-γ (10 ng/mL, R&D Systems), transforming growth factor-β (TGF-β, 2 ng/mL, R&D Systems), IL-10 (10 ng/mL, R&D Systems) or antimouse CD3ε Ab (1 µg/mL, 145-2 C11, Invitrogen) plus antimouse CD28 Ab (1 µg/mL, 37.51, Invitrogen) in complete media for 72 hours. In some experiments, ciclosporin A (Sigma), tacrolimus (FK506, Sigma) and rapamycin (Sigma) were treated to the T cells stimulated with anti-CD3/anti-CD28 Abs for 72 hours to determine whether the calcineurin pathway is involved in LSP1 expression. The cultured cells were harvested and stained to detect intracellular LSP1 expression by flow cytometry and/or western blot analysis.

Procedures involving living animals were approved by local ethics committees and were performed according to the Guidelines of the Italian National Institutes of Health (Art. 31 D.lgs 26/2014, 4 March 2014). Animals used in the study were 3-month-old dystrophic C57BL/ 10ScSn Dmd^mdx and age-matched wild-type control mice provided by Charles River (Calco, Lecco, Italy). Postoperatively, animals were administered by intraperitoneal injection of the clinically approved immunosuppressive drug tacrolimus (FK-506; Sigma-Aldrich St. Louis, MO, USA) 2 mg/kg per day [32 (link)]. Acute hindlimb ischemia was induced by removal of the femoral artery, as described previously [33 (link)]. Measure of the blood flow in the ischemic hindlimb compared to the contralateral control was performed by laser Doppler perfusion imaging (Lisca Inc., North Brunswick, NJ, USA).