Dynamic Monitoring of Intracellular Tacrolimus and Mycophenolic Acid Therapy in Renal Transplant Recipients Using Magnetic Bead Extraction Combined with LC-MS/MS.

BACKGROUND: Tacrolimus (TAC) and mycophenolic acid (MPA) are commonly used immunosuppressive therapies after renal transplant. Our objective was to quantify TAC and MPA concentrations in peripheral blood mononuclear cells (PBMCs) using liquid chromatography tandem mass spectrometry (LC-MS/MS) and to evaluate and validate the performance of the methodology. A prospective follow-up cohort study was conducted to determine whether intracellular concentrations were associated with adverse outcomes in renal transplants. METHODS: PBMCs were prepared using the Ficoll separation technique and purified with erythrocyte lysis. The cells were counted using Sysmex XN-3100 and then packaged and frozen according to a 50 µL volume containing 1.0 × 106 cells. TAC and MPA were extracted using MagnaBeads and quantified using an LC-MS/MS platform. The chromatography was run on a reversed-phase Waters Acquity UPLC BEH C18 column (1.7 µm, 50 mm × 2.1 mm) for gradient elution separation with a total run time of 4.5 min and a flow rate of 0.3 mL/min. Mobile phases A and B were water and methanol, respectively, each containing 2 mM ammonium acetate and 0.1% formic acid. Renal transplant recipients receiving TAC and MPA in combination were selected for clinical validation and divided into two groups: a stable group and an adverse outcome group. The concentrations were dynamically monitored at 5, 7, 14, and 21 days (D5, D7, D14, and D21) and 1, 2, 3, and 6 months (M1, M2, M3, and M6) after operation. RESULTS: Method performance validation was performed according to Food and Drug Administration guidelines, showing high specificity and sensitivity. The TAC and MPA calibration curves were linear (r2 = 0.9988 and r2 = 0.9990, respectively). Both intra-day and inter-day imprecision and inaccuracy were less than 15%. Matrix effects and recoveries were satisfactory. The TAC and MPA concentrations in 304 "real" PBMC samples from 47 renal transplant recipients were within the calibration curve range (0.12 to 16.40 ng/mL and 0.20 to 4.72 ng/mL, respectively). There was a weak correlation between PBMC-C0TAC and WB-C0TAC (p < 0.05), but no correlation was found for MPA. The level of immunosuppressive intra-patient variation (IPV) was higher in PBMC at 77.47% (55.06, 97.76%) than in WB at 34.61% (21.90, 49.85%). During the dynamic change in C0TAC, PBMC-C0TAC was in a fluctuating state, and no stable period was found. PBMC-C0TAC did not show a significant difference between the stable and adverse outcome group, but the level of the adverse outcome group was generally higher than that of the stable group. CONCLUSIONS: Compared with conventional therapeutic drug monitoring, the proposed rapid and sensitive method can provide more clinically reliable information on drug concentration at an active site, which has the potential to be applied to the clinical monitoring of intracellular immunosuppressive concentration in organ transplantation. However, the application of PBMC-C0TAC in adverse outcomes of renal transplant should be studied further.

Influence of naringenin on the biofilm formation of Streptococcus mutans.

OBJECTIVES: To evaluate the effect of naringenin on the biofilm formation of Streptococcus mutans (S. mutans), and to investigate its mechanisms of action and biological toxicity. METHODS: Minimum inhibitory concentrations, growth curves, and biofilm inhibition rates of naringenin were determined to assess its antimicrobial effect on S. mutans. The morphology of S. mutans and the structure of biofilm were observed by FESEM and CLSM. Bacterial aggregation, bacterial surface hydrophobicity, and real-time PCR for gtfB, gtfC, comD, comE, and luxS mRNA expression were assessed to preliminarily investigate the mechanisms of action. MTT test using human dental pulp cells (HDPCs) was also performed to investigate cytotoxicity. RESULTS: The S.mutans growth curves, FESEM, CLSM showed that both 100 and 200 µg/mL of naringenin obviously inhibited S. mutans growth and biofilm formation, increased S. mutans surface hydrophobicity, reduced bacterial aggregation, and downregulated the mRNA expression of gtfB, gtfC, comD, comE, and luxS. However, naringenin at 200 µg/mL slightly decreased the growths of HDPCs compared with 100 µg/mL. CONCLUSION: Naringenin at 100 and 200 µg/mL suppressed the second (bacterial adhesion) and third stages (biofilm maturation) of S. mutans biofilm formation. CLINICAL SIGNIFICANCE: Naringenin is promising for dental clinic promotion to prevent the biofilm formation of S. mutans, serving as a safe anti-caries agent at an appropriate concentration.

Nanoleakage evaluation at adhesive-dentin interfaces by different observation methods.

The purpose of this study was to evaluate the capability and characteristics of different nanoleakage observation methods, including light microscope (LM), field-emission scanning electron microscope (FESEM), transmission electron microscope (TEM), and confocal laser scanning microscope (CLSM). Dentin specimens were bonded with either an etch-and-rinse adhesive (SBMP) or a self-etch adhesive (GB), and prepared for nanoleakge evaluation according to different observation methods. LM, FESEM and CLSM results demonstrated that the SBMP group showed more interfacial nanoleakage than the GB group (p<0.05); by contrast, no significant difference was found in TEM results (p>0.05), however, TEM illustrated concrete nanoleakage forms or patterns. The results suggested that different observation methods might exhibit distinct images and a certain degree of variations in nanoleakage statistical results. Researchers should carefully design and calculate the optimum assembly in combination with qualitative and quantitative approaches to obtain objective and accurate nanoleakage evaluation.