Antibody array-based proteomic screening of novel biomarkers in malignant biliary stricture.

BACKGROUND: Distinguishing between benign and malignant bile duct strictures has long been a diagnostic challenge in clinical practice. OBJECTIVE: This study aimed to discover novel biomarkers in bile to improve the diagnostic accuracy of malignant biliary strictures. METHODS: Bile samples were collected from 6 patients with malignant or benign biliary stricture, respectively. Protein profiles of the bile were analyzed with a semi-quantitative human antibody array of 440 proteins. Then the differential expressed proteins were screened by Venn diagram analysis. Following this, the accuracy of these potential biomarkers for discriminating between malignant and non-malignant biliary strictures was validated in a larger (n= 40) group of patients using ROC analysis and the best biomarker combination was further selected by lasso analysis. RESULTS: Twenty proteins were found differentially expressed in malignant versus benign biliary strictures, 6 of which were identified by Venn diagram analysis to be up-regulated regardless of the location of biliary strictures. Among the 6 biomarkers, bile lipocalin-2, P-cadherin, and adipsin showed better diagnostic utility than that of bile CA19-9. Lasso analysis identified that lipocalin-2, P-cadherin and CA19-9 as a group of makers best distinguished malignant from benign strictures. CONCLUSIONS: Lipocalin-2 and P-cadherin measurements in bile could be clinically useful for the detection of malignant biliary strictures.

High Serum Levels of Cholesterol Increase Antitumor Functions of Nature Killer Cells and Reduce Growth of Liver Tumors in Mice.

BACKGROUND AND AIMS: The relationship between serum cholesterol level and development of hepatocellular carcinoma (HCC) remains unclear. We investigated the effects of serum cholesterol level on development of liver tumors in mice. METHODS: We performed studies with C57BL/6J mice, mice with disruption of the low-density lipoprotein receptor gene (Ldlr-/-mice), and mice with conditional deletion of nature killer (NK) cells (NKdele mice). Some C57BL/6J and NKdele mice were given injections of diethylinitrosamine to induce liver tumor formation. Mice were placed on a normal diet (ND) or high-cholesterol diet (HCD) to induce high serum levels of cholesterol. We also studied mice with homozygous disruption of ApoE (ApoE-/- mice), which spontaneously develop high serum cholesterol. C57BL/6J and NKdele mice on the ND or HCD were implanted with Hep1-6 (mouse hepatoma) cells and growth of xenograft tumors and lung metastases were monitored. Blood samples were collected from mice and analyzed by biochemistry and flow cytometry; liver and tumor tissues were collected and analyzed by histology, immunohistochemistry, and RNA-sequencing analysis. NK cells were isolated from mice and analyzed for cholesterol content, lipid raft formation, immune signaling, and changes in functions. We obtained matched tumor tissues and blood samples from 30 patients with HCC and blood samples from 40 healthy volunteers; levels of cholesterol and cytotoxicity of NK cells were measured. RESULTS: C57BL/6J mice on HCD and ApoE-/- mice with high serum levels of cholesterol developed fewer and smaller liver tumors and lung metastases after diethylinitrosamine injection or implantation of Hep1-6 cells than mice on ND. Liver tumors from HCD-fed mice and ApoE-/- mice had increased numbers of NK cells compared to tumors from ND-fed mice. NKdele mice or mice with antibody-based depletion for NK cells showed similar tumor number and size in ND and HCD groups after diethylinitrosamine injection or implantation of Hep1-6 cells. NK cells isolated from C57BL/6J mice fed with HCD had increased expression of NK cell-activating receptors (natural cytotoxicity triggering receptor 1 and natural killer group 2, member D), markers of effector function (granzyme B and perforin), and cytokines and chemokines compared with NK cells from mice on ND; these NK cells also had enhanced cytotoxic activity against mouse hepatoma cells, accumulated cholesterol, increased lipid raft formation, and immune signaling activation. NK cells isolated from HCD-fed Ldlr-/- mice did not have increased cholesterol content or cytotoxic activity against mouse hepatoma cells compared with ND-fed Ldlr-/- mice. Serum levels of cholesterol correlated with number and activity of NK cells isolated from human HCCs. CONCLUSIONS: Mice with increased serum levels of cholesterol due to an HCD or genetic disruption of ApoE develop fewer and smaller tumors after injection of hepatoma cells or a chemical carcinogen. We found cholesterol to accumulate in NK cells and activate their effector functions against hepatoma cells. Strategies to increase cholesterol uptake by NK cells can be developed for treatment of HCC.

Long-term survival after partial hepatectomy for sub-stage patients with intermediate stage hepatocellular carcinoma.

BACKGROUND: The current study aimed to examine the long-term survival after partial hepatectomy for patients with BCLC intermediate stage hepatocellular carcinoma (HCC) stratified by the Bolondi's sub-staging model. MATERIALS AND METHODS: This cohort consisted of 360 patients with BCLC intermediate stage HCC who underwent partial hepatectomy between January 2008 and February 2010. Patients were stratified into 3 subgroups (B1-B3) based on the Bolondi's sub-staging model. The last follow-up was conducted at February 2014. RESULTS: Of these patients, 166, 171 and 23 patients had B1, B2, and B3 sub-stage HCC, respectively. The postoperative 5-year Overall survival (OS) rate for patients with these three sub-stages was 49.5%, 33.7% and 12.9%, respectively (P < 0.001). Compared with the reported survival outcomes from previous studies which used transarterial chemoembolization (TACE) as first-line treatment, hepatectomy had a better median survival than TACE in B1 and B2 patients. On multivariable analysis, presence of esophageal and gastric varices, higher NDR score, presence of microvascular invasion, differentiation grade III-IV, and patterns of AFP decreases after surgery were the independent risk factors of OS in the sub-stages B1 and B2 patients. A nomogram which integrated all these independent risk factors was developed, with a C-index of 0.71 for OS prediction. The calibration curve showed an optimal agreement between prediction by the nomogram and actual observation. CONCLUSIONS: The patients with intermediate stage HCC clarified as sub-stages B1 and B2 according to Bolondi's model had an optimal long-term survival following partial hepatectomy than TACE. Their postoperative prognosis could be accurately predicted by our proposed nomogram.

Application of multiplex PCR combined with invasive reaction and chromogenic reaction catalyzed by gold nanoparticles in detection of encephalitis and meningitis virus / 中国人兽共患病学报

We developed a method for detecting encephalitis and meningitis virus by using multiplex PCR combined with invasive reaction and a chromogenic reaction catalyzed by gold nanoparticles.Primers were designed based on the conservative regions of encephalitis and meningitis virus (Eastern equine encephalitis virus,EEEV;Western equine encephalomyelitis virus,WEEV;West Nile virus,WNV;Nipah virus,NiPA;Japanese encephalitis virus,JEV).Multiplex PCR system,invasive reaction and a chromogenic reaction catalyzed by gold nanoparticles were established to detect different encephalitis and meningitis virus in one reaction.Tick-borne encephalitis virus (TBEV),St Louis encephalitis virus (StLEV),Chikungunya virus (CHIKV) and Dengue virus(DV) were used to test its specificity.Quantitative RNA transcribed in vitro and PCR fragments were used to assess its sensitivity.Clinical specimens collected from JEV patients were detected by this method.A method for detecting encephalitis and meningitis virus by using multiplex PCR,invasive reaction and a chromogenic reaction catalyzed by gold nanoparticles were successfully established.This method can detect targeted pathogens specifically,and it has no cross reaction with TBEV,StLEV,CHIKV and DV.The detecting limitation for different targets was 103 copies/μL.Clinical samples were positive for JEV nucleic acids for above assay.The method presented here has characteristic of high specificity,sensitivity and throughput.The results can be observed by visual inspection.This method has broad application prospects in pathogen detection.