EXCLI Journal

Original article - Effect of glucose and insulin supplementation on the isolation of primary human hepatocytes

2019-11-18T08:12:19+01:00

Primary human hepatocytes (PHHs) remain the gold standard for in vitro investigations of xenobiotic metabolism and hepatotoxicity. However, scarcity of liver tissue and novel developments in liver surgery has limited the availability and quality of tissue samples. In particular, warm ischemia shifts the intracellular metabolism from aerobic to anaerobic conditions, which increases glycogenolysis, glucose depletion and energy deficiency. Therefore, the aim of the present study was to investigate whether supplementation with glucose and insulin during PHH isolation could reconstitute intracellular glycogen storage and beneficially affect viability and functionality. Furthermore, the study elucidated whether the susceptibility of the tissue’s energy status correlates with body mass index (BMI). PHHs from 12 donors were isolated from human liver tissue obtained from partial liver resections using a two-step EDTA/collagenase perfusion technique. For a direct comparison of the influence of glucose/insulin supplementation, we modified the setup, enabling the parallel isolation of two pieces of one tissue sample with varying perfusate. Independent of the BMI of the patient, the glycogen content in liver tissue was notably low in the majority of samples. Furthermore, supplementation with glucose and insulin had no beneficial effect on the glycogen concentration of isolated PHHs. However, an indirect improvement of the availability of energy was shown by increased viability, plating efficiency and partial cellular activity after supplementation. The plating efficiency showed a striking inverse correlation with increasing lipid content of PHHs. However, 60 h of cultivation time revealed no significant impact on the maintenance of albumin and urea synthesis or xenobiotic metabolism after supplementation. In conclusion, surgical procedures and tissue handling may decrease hepatic energy resources and lead to cell stress and death. Consequently, PHHs with low energy resources die during the isolation process without supplementation of glucose/insulin or early cell culture, while their survival rates are improved with glucose/insulin supplementation.

Original article - E-selectin as a prognostic factor of patients hospitalized due to acute inflammatory respiratory diseases: a single institutional study

2019-11-11T12:26:00+01:00

When examining patients with acute inflammatory respiratory diseases, it is difficult to distinguish between infectious pneumonia and interstitial pneumonia and predict patient prognosis at the beginning of treatment. In this study, we assessed whether endothelial selectin (E-selectin) predicts the outcome of patients with acute inflammatory respiratory diseases. We measured E-selectin serum levels in 101 patients who were admitted to our respiratory care unit between January 2013 and December 2013 because of acute inflammatory respiratory diseases that were eventually diagnosed as interstitial pneumonia (n = 38) and lower respiratory tract infection (n = 63). Seven of these patients (n = 101) died. The pneumonia severity score was significantly higher and the oxygen saturation of arterial blood measured by pulse oximeter (SpO₂)/fraction of inspiratory oxygen (FiO₂) was significantly lower in the deceased patients than in the surviving patients. There were significantly fewer peripheral lymphocytes and significantly higher E-selectin serum levels in the deceased patients than in the surviving patients. In the multiple logistic regression analysis, the E-selectin serum levels and SpO₂/FiO₂ ratio were independent predictive factors of prognosis. The risk of death during acute respiratory disease was determined using a receiver operating characteristic (ROC) curve analysis. The area under the curve (AUC) was 0.871 as calculated from the ES, and the cutoff value was 6453.04 pg/ml, with a sensitivity of 1.00 and a specificity of 0.72 (p = 0.0027). E-selectin may be a useful biomarker for predicting the prognosis of patients with acute inflammatory respiratory diseases.

Original article - MicroRNA-9-5p functions as a tumor suppressor in prostate cancer via targeting UTRN

2019-11-07T08:56:16+01:00

Accumulating evidence indicates that miR-9-5p plays an important role in several diseases, especially tumor progression. In this study, we investigated the clinical significance and biological function of miR-9-5p in prostate cancer (PCa). Using quantitative real time PCR (qRT-PCR) analysis, we found miR-9-5p level was significantly down-regulated in PCa tissues and cell lines. The decreased miR-9-5p expression was associated with tumor size, preoperative PSA, Gleason score and lymph node metastasis. Kaplan-Meier survival analysis showed patients with low level of miR-9-5p had significantly decreased rates of overall survival (OS). Multivariate analyses showed that miR-9-5p was an independent predictor of PCa patients’ prognosis. Through CCK-8 and Transwell assays, miR-9-5p overexpression by miR-9-5p mimics transfection was demonstrated to suppress the proliferation, migration and invasion of PCa cells. Mechanistically, luciferase reporter assay, qRT-PCR and Western blot demonstrated that Utrophin (UTRN) is a direct target of miR-9-5p in PCa cells. The status of UTRN protein in PCa tissues was much higher than that in adjacent tissues by immunohistochemical staining and its mRNA levels were inversely correlated with miR-9-5p in PCa tissues. Importantly, UTRN knockdown by siUTRN imitated the suppressive effects of miR-9-5p on cell proliferation, migration and invasion in PCa. In summary, miR-9-5p might novel prognostic biomarker in and targeting UTRN by miR-9-5p could be potential therapeutic candidates for PCa.