Tacrolimus

For determination of insulin release the following buffer solution was used (mM): 122 NaCl, 4.7 KCl, 1.1 MgCl₂, 2.5 CaCl₂, 0.5% BSA, and 10 HEPES (pH 7.4). [Ca²⁺]_c experiments were performed at 37 °C in a solution of pH 7.4 containing (mM): 140 NaCl, 5 KCl, 1.2 MgCl₂, 2.5 CaCl₂, and 10 HEPES. Glucose was added as indicated. Test compounds were either added directly to bath solution (acute effects of FGFs on insulin secretion) or to the culture medium for a period of 48 h or 7 days (all other experiments). Glucolipotoxic medium contained 25 mM glucose and 100 μM palmitate or 33 mM glucose and 500 μM palmitate. Briefly, palmitate was dissolved in 0.1 N NaOH and further diluted in double-distilled water containing 0.56% or 2.8% fat-free bovine serum albumin. This solution was added to the culture medium in a proportion of 1:10 to obtain the final palmitate concentration of 100 μM or 500 μM. The control medium contained the respective amount of fat-free bovine serum albumin and 0.1 N NaOH. Fura-2 AM, rat insulin and mouse recombinant FGF-15 (referred to as FGF-15/19 in the text), FGF-21, and FGF-23 were ordered from Biotrend. SP600125 and tacrolimus were from Selleckchem, PD-161570 from Sigma-Aldrich and PD-173074 from Biomol. All other chemicals were from Sigma-Aldrich or Diagonal.

To assess the effect of immunosuppressive drugs and immune activation on CD59 expression, we used whole blood and PBMCs of six healthy volunteers. Whole blood samples were spiked with: tacrolimus 10 ng/ml (Selleckchem, Houston, TX, USA), mycophenolic acid 2.5 µg/ml (Sigma-Aldrich), prednisolone 21-acetate 150 ng/ml (Santa Cruz Biotechnology, Dallas, TX, USA), a combination of these drugs or PBS as a negative control. Samples were incubated for 24 h at 37°C, 5% CO₂. This incubation time was selected based on preliminary time course experiments, showing a maximum effect after 24 h of incubation. Following incubation, the erythrocytes were lysed with lysing solution (BD Biosciences) and stained for flow cytometry analysis. PBMCs of the same donors were cultured for 24 h at 37°C, 5% CO₂ in the presence or absence of human T activator CD3/CD28 dynabeads (Invitrogen, Waltham, MA, USA). Subsequently, CD59 expression on T cells was measured with flow cytometry analyses.

Poly (ethylene glycol)-block-poly (d, l)-lactic-co-glycolic acid (mPEG-b-PLGA, PEG 2000 Da, PLGA 8000 Da, LA/GA = 75/25) was provided by the laboratory of Professor Fangming Zhu at the School of Chemistry, Sun Yat-sen University, and tacrolimus was obtained from Selleck Chemicals (Shanghai, China). Dialysis bags were purchased from Spectrapor (MWCO 14000, Waltham, MA, USA). Isoflurane inhaled anesthesia (Yipin pharmaceutical Co., LTD, Hebei, China) and 3% sodium pentobarbital solution (Merck, Darmstadt, German) were used to anesthesia animals. Proparacaine hydrochloride eye drops (S.A. ALCON, Fort Worth, TX, USA), Iviz (Bausch Lomb, Shandong, China), and 10-0 black mono nylon sutures (Alcon, Fort Worth, TX, USA) were used in corneal transplantation of rats. Tobramycin eye ointment purchased from Zhongshan Ophthalmic Center (Guangzhou, China). 0.1% tacrolimus eye drops were purchased from Senju Pharmaceutical Co., Ltd. (Osaka, Japan). Rabbit polyclonal anti-CD4/CD8 antibody and horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG H&L were purchased from Abcam (Cambridge, MA, USA), the Milliplex^® xMAP Rat Cytokine/Chemokine kit was purchased from EMD Millipore Corporation (Billerica, MA, USA).

Calcineurin inhibitor tacrolimus (TAC) was purchased from Selleck Chemicals (Houston, TX, USA, Code S5003). tacrolimus was dissolved in ethanol, and then the solution was poured to the surface of the NGM plates seeded with OP50. Later, synchronized L1 worms were placed and cultured on these plates for further analysis, including visulization of fat accumulation by post-fixed Nile Red staining, determination of fat content by TLC/GC, and growth rate observation, etc.

Drug combinations were tested using the EUCAST guidelines for the antifungal susceptibility testing of molds with modifications for a broth microdilution checkerboard procedure, using Nunclon™ delta surface 96-wells microtiter plates for adherent cells (Thermo Fisher Scientific, Darmstadt, Germany). The included drugs were isavuconazole (Pfizer, Berlin, Germany), tacrolimus (Selleck Chemicals, Munich, Germany), cyclosporin A (Selleck), and sirolimus (Selleck). Stock solutions of drugs were prepared in DMSO. Drug dilutions were performed to four times the final concentrations in double strength RPMI medium. All the combinations were studied on a two-dimensional checkerboard with two-fold dilutions. The final concentrations for isavuconazole were 0.03 to 16 µg/mL. The final concentrations for the immunosuppressors were 0.125 to 8 µg/mL. Fifty microliters of each concentration were distributed from Rows 1 to 8 for isavuconazole and from Columns 1 to 11 for the immunosuppressive agents. Column 12 was used a as growth control and contained 100 µL of double strength RMPI medium with DMSO.