Inflammatory Cytokines, Adipocytokines, and Th17/Treg Balance in Patients with Nonalcoholic Fatty Liver Disease following Administration of Dahuang Zhechong Pills.

Objectives: The occurrence and development of nonalcoholic fatty liver disease (NAFLD) is related to lipid peroxidation, imbalance of inflammatory response factors, and immune function disorder. This study was conducted with the purpose of investigating the expression levels of inflammatory cytokines and adipocytokines and Th17/Treg balance in NAFLD patients treated with Dahuang Zhechong pills (DHZCPs). Methods: The study recruited 100 NAFLD patients who were then arranged into the test group and control group. Patients in the test group were treated with DHZCPs, while patients in the control group were untreated. Peripheral TH17 and Treg cells were detected by flow cytometry, and peripheral IL-17, IL-10, hs-CRP, and TNF-α expression levels were determined by enzyme-linked immunosorbent assay (ELISA) methods. The concentrations of ghrelin, leptin, and adiponectin were quantitatively examined. Results: The levels of TC, TG, ALT, and AST were declined but the level of HDL-C was increased in NAFLD patients treated with DHZCPs compared with untreated patients (P < 0.05). The ratio of Th17/Treg in NAFLD patients treated with DHZCPs was (1.52 ± 0.21), which was significantly lower than (2.39 ± 0.45) of untreated patients (P < 0.05). The levels of IL-17, hs-CRP, and TNF-α were lower, but the level of IL-10 was higher in NAFLD patients treated with DHZCPs than that in untreated patients (P < 0.05). The expression levels of ghrelin and adiponectin in NAFLD patients treated with DHZCPs were evidently higher than those in untreated patients (P < 0.01), and the expression level of leptin in NAFLD patients treated with DHZCPs was evidently lower than that in untreated patients (P < 0.01). Conclusions: Administration of DHZCPs regulates the immune function of NAFLD patients by keeping Th17/Treg balance and affecting the levels of inflammatory cytokines and adipocytokines.

Enhancement of human plasma glucosylceramide assay sensitivity using delipidized plasma.

Glucosylceramide (GL-1) level in human has been considered as a surrogate biomarker for enzyme replacement and substrate reduction therapies (ERT and SRT) for Gaucher and Fabry patients. Due to the high endogenous level of GL-1 in human plasma, it is difficult to achieve the analytical sensitivity of plasma GL-1 below the normal endogenous level (1.7 µg/mL to 6.6 µg/mL) when using the standard addition method and regular plasma matrix for standard curve. A high sensitivity plasma GL-1 assay with LLOQ at 0.1 µg/mL was developed and validated using delipidized plasma so that patient plasma concentrations that are below normal reference range can be measured accurately. The normal reference range was established from 120 healthy donors using this developed new method. Twenty-three Fabry patient plasma samples including baseline and post-investigation drug treatment samples were measured. All post-treatment samples showed GL-1 concentration below 2.0 µg/mL, indicating the utility of the reported high sensitivity assay using delipidized plasma for monitoring the plasma GL-1 biomarker level in patients.

A novel approach for quantitation of glucosylceramide in human dried blood spot using LC-MS/MS.

BACKGROUND: Glucosylceramide, an efficacy biomarker for Gaucher Type 1 disease, exhibits poor solubility in polar solvents and whole blood which makes it difficult to prepare a homogenous blood standard. RESULTS: We developed a novel method using standard addition approach by spiking a small volume of analyte solution on the surface of prespotted dried blood spot. The whole spots were punched out for subsequent extraction and LC-MS/MS analysis. The assay performance met all validation acceptance criteria. Glucosylceramide concentrations in 50 paired plasma and dry blood spot samples obtained from Gaucher Type 1 patients were tested and the results demonstrated the feasibility of using the DBS method for clinical biomarker monitoring. CONCLUSION: The new approach greatly improves assay precision and accuracy.

A quantitative evaluation of active gelatinolytic sites in uterine endometrioid adenocarcinoma using film in situ zymography: association of stronger gelatinolysis with myometrial invasion.

Collagen matrix degradation by malignant tumor cells plays an essential role in the process of tumor invasion and metastasis. The purpose of this study was to detect in situ gelatinase activity in endometrioid adenocarcinomas of the uterine corpus. In order to carry out quantitative evaluation, autoexposure time (AET) on gelatin-coated film (film in situ zymography: FIZ) was measured. The gelatinase activity was located primarily within cancers and was prominently suppressed by the addition of a chelating agent to the film. This suggests that matrix metalloproteinases (MMPs) play an important role in the gelatinase activity. The gelatinase activity in the normal endometrium is almost negligible, despite positive immunoreactivity for MMP-2 and -9. Tumor tissues that had invaded more than half of the myometrium showed significantly higher activity than those that had invaded less than half. There was no significant difference in gelatinase activity among tumor stages, grades, vessel invasion or immunoreactivity for MMPs, with the exception that stage 2b cancers showed higher activity than stage 1a. The study suggested that the level of MMP-mediated gelatinolysis is an important factor for myometrial invasion in uterine endometrioid adenocarcinoma. Thus, a quantitative assessment of active gelatinolysis using FIZ and AET should be a useful tool in evaluating in situ matriolytic activity in local myometrial invasion by uterine endometrioid adenocarcinoma.