AL amyloidosis with primary presentation of multiple serous cavity effusion and severe cholestasis: a case report and review of literature.

BACKGROUND: Immunoglobulin light chain (AL) amyloidosis commonly affects the kidney or heart, but may also involve the liver at a histopathological level. Early diagnosis of AL amyloidosis is important for proper management with desirable outcome. We reported here an unusual case of AL amyloidosis, presenting primarily with multiple serous cavity effusion, accompanied with rapidly progressive cholestasis. CASE PRESENTATION: A previously healthy 63-year-old man presented with dysuria, frequent urination, oliguria and oedema of lower extremities for one month, accompanied with jaundice and hypoproteinemia. CT demonstrated multiple serous cavity effusion, focal hypodense lesions in the liver, and focal low-density in the spleen. Laparoscopy with liver biopsy revealed liver and spleen fibrosis with congestion, no visceral rupture, following haemorrhagic ascites from abdominocentesis. This patient was transferred to our (tertiary) hospital. The diagnosis of amyloidosis was confirmed with histopathology/immunohistochemistry. Haematopoietic stem cell transplantation was not applicable, however chemotherapy was advised, due to the patient's Mayo score 3. The patient declined chemotherapy and was self-discharged back to his hometown hospital with palliative care, however only lasted a further one-month. DISCUSSION: The lesson we have learnt from this case that any patients with multiple serous cavity effusion and isolated hepatic involvement, primary amyloidosis should be considered. Multiple serous cavity effusion may serve as an indicator for poor prognosis of hepatic AL amyloidosis.

Algorithm of Golgi protein 73 and liver stiffness accurately diagnoses significant fibrosis in chronic HBV infection.

BACKGROUND & AIMS: Serum Golgi protein 73 (GP73) is a potential biomarker for fibrosis assessment. We aimed to develop an algorithm based on GP73 and liver stiffness (LS) for further improvement of accuracy for significant fibrosis in patients with antiviral-naïve chronic hepatitis B virus (HBV) infection. METHODS: Diagnostic accuracy evaluation of GP73 and development of GP73-LS algorithm was performed in training cohort (n = 267) with an independent cohort (n = 133) for validation. RESULTS: A stepwise increasing pattern of serum GP73 was observed across fibrosis stages in patients with antiviral-naïve chronic HBV infection. Serum GP73 significantly correlated (rho = 0.48, P < .001) with fibrosis stage and was an independent predictor for the presence of significant fibrosis (OR, 95%CI: 1.02, 1.01-1.03, per increase in 1 ng/mL, P < .001). Both LS (AUROC, 95%CI: 0.82, 0.77-0.87, accuracy: 74.7%) and GP73 (AUROC, 95%CI: 0.76, 0.71-0.82, accuracy: 71.5%) well-predicted significant fibrosis and outperformed APRI (AUROC, 95%CI: 0.69, 0.63-0.76, accuracy: 66%) and FIB-4 (AUROC, 95%CI: 0.66, 0.60-0.73, accuracy: 63.6%). Using GP73-LS algorithm, GP73 < 63 in agreement with LS < 8.5 provided accuracy of 81.7% to excluded significant fibrosis. GP73 ≥ 63 in agreement with LS ≥ 8.5 provided accuracy of 93.3% to confirm significant fibrosis. Almost 64% or 68% of patients in the training or validation cohort could be accurately classified. CONCLUSIONS: Serum GP73 is a robust biomarker for significant fibrosis diagnosis. GP73-LS algorithm provided better diagnostic accuracy than currently available approaches. More than 60% antiviral naïve CHB patients could use this algorithm without resorting to liver biopsy.

Fibroblast growth factor 21 attenuates hepatic fibrogenesis through TGF-ß/smad2/3 and NF-κB signaling pathways.

Fibroblast growth factor 21 (FGF-21) is a secreted protein, which has anti-diabetic and lipocaic effects, but its ability to protect against hepatic fibrosis has not been studied. In this study, we investigated the ability of FGF-21 to attenuate dimethylnitrosamine (DMN)-induced hepatic fibrogenesis in mice and the mechanism of its action. Hepatic fibrosis was induced by injection of DMN, FGF-21 was administered to the mice once daily in association with DMN injection till the end of the experiment. Histopathological examination, tissue 4-hydroxyproline content and expressions of smooth muscle α-actin (α-SMA) and collagen I were measured to assess hepatic fibrosis. Ethanol/PDGF-BB-activated hepatic stellate cells (HSCs) were used to understand the mechanisms of FGF-21 inhibited hepatic fibrogenesis. Results showed that FGF-21 treatment attenuated hepatic fibrogenesis and was associated with a significant decrease in intrahepatic fibrogenesis, 4-hydroxyproline accumulation, α-SMA expression and collagen I deposition. FGF-21 treatment inhibited the activation of HSCs via down-regulating the expression of TGF-ß, NF-κB nuclear translocation, phosphorylation levels of smad2/3 and IκBα. Besides, FGF-21 treatment caused activated HSC apoptosis with increasing expression of Caspase-3, and decreased the ratio of Bcl-2 to Bax. In conclusion, FGF-21 attenuates hepatic fibrogenesis and inhibits the activation of HSC warranting the use of FGF-21 as a potential therapeutic agent in the treatment of hepatic fibrosis.

Recombinant Newcastle disease virus expressing P53 demonstrates promising antitumor efficiency in hepatoma model.

BACKGROUND: Numerous studies have demonstrated that the NDV-mediated gene therapy is a promising new approach for treatment of cancers. P53 plays a vital role in tumor suppression and surveillance. Therefore, we hypothesize that a recombinant NDV expressing P53 would be an ideal agent for the hepatoma therapy. RESULTS: In the essay, the human P53 gene was incorporated into the genome of a lentogenic strain (named rNDV-P53), which did not affect viral replication kinetics and magnitude in HepG2 cells. Compared to the vehicle virus, rNDV-P53 increased cell growth suppressor ratio and early apoptosis by 2 folds, and decreased the mitochondrial membrane potential in HepG2 cells. In vivo studies, treatment with rNDV-P53 reduced tumor volume of tumor-bearing mice by more than 4 folds, tumor weight by more than 5 folds comparing with rNDV. The 120-day survival rate of rNDV-P53-treated mice was 75 %, survival rate of rNDV-treated mice was 12.5 %. TUNEL analysis showed a significant increase in the apoptosis rate in the tumor tissues of rNDV-P53-treated mice than that of rNDV-treated mice. Moreover, serum chemistries revealed an insignificant change of blood urea nitrogen (BUN), creatinine levels, alanine aminotransferase (ALT) and aspartate transaminase (AST) in rNDV-P53-treated group compared to normal mice, suggesting treatment with the recombinant virus was not toxic. CONCLUSION: rNDV-P53 is a potent candidate for carcinoma therapy especially for hepatocarcinoma.

Recombinant Newcastle disease virus (NDV/Anh-IL-2) expressing human IL-2 as a potential candidate for suppresses growth of hepatoma therapy.

Newcastle disease virus (NDV) have shown oncolytic therapeutic efficacy in preclinical study and are currently approved for clinical trials. NDV Anhinga strain which is a mesogenic strain should be classified as lytic strain and has a therapeutic efficacy in hepatocellular cancer. In this study, we evaluated the capacity of NDV Anhinga strain to elicit immune reaction in vivo and the possibility for using as a vaccine vector for expressing tumor therapeutic factors. Interleukin-2 (IL-2) could boost the immune response against the tumor cells. Therefore, we use NDV Anhinga strain as backbone to construct a recombinant virus (NDV/Anh-IL-2) expressing IL-2. The virus growth curve showed that the production of recombinant NDV/Anh-IL-2 was slightly delayed compared to the wild type. The NDV/Anh-IL-2 strain could express soluble IL-2 and effectively inhibit the growth of hepatocellular carcinoma in vivo. 60 days post-treatment, mice which were completely cured by previous treatment were well protected when rechallenged with the same tumor cell. From the H&E-stained sections, intense infiltration of lymphocyte was observed in the NDV Anhinga strain treated group, especially in NDV/Anh-IL-2 group. The NDV Anhinga strain could not only kill the tumor directly, but could also elicit immune reaction and a potent immunological memory when killing tumor in vivo. In conclusion, the Anhinga strain could be an effective vector for tumor therapy; the recombinant NDV/Anh-IL-2 strain expressing soluble IL-2 is a promising candidate for hepatoma therapy.

Fibroblast growth factor (FGF21) protects mouse liver against D-galactose-induced oxidative stress and apoptosis via activating Nrf2 and PI3K/Akt pathways.

FGF21 is recently discovered with pleiotropic effects on glucose and lipid metabolism. However, the potential protective effect of FGF21 against D-gal-induced injury in the liver has not been demonstrated. The aim of this study is to investigate the pathophysiological role of FGF21 on hepatic oxidative injury and apoptosis in mice induced by D-gal. The 3-month-old Kunming mice were subcutaneously injected with D-gal (180 mg kg(-1) d(-1)) for 8 weeks and administered simultaneously with FGF21 (5 or 1 mg kg(-1) d(-1)). Our results showed that the administration of FGF21 significantly alleviated histological lesion including structure damage, degeneration, and necrosis of hepatocytes induced by D-gal, and attenuated the elevation of liver injury markers, serum AST, and ALP in a dose-dependent manner. FGF21 treatment also suppressed D-gal-induced profound elevation of ROS production and oxidative stress, as evidenced by an increase of the MDA level and depletion of the intracellular GSH level in the liver, and restored the activities of antioxidant enzymes SOD, CAT, GSH-Px, and T-AOC. Moreover, FGF21 treatment increased the nuclear abundance of Nrf2 and subsequent up regulation of several antioxidant genes. Furthermore, a TUNEL assay showed that D-gal-induced apoptosis in the mouse liver was significantly inhibited by FGF21. The expression of caspase-3 was markedly inhibited by the treatment of FGF21 in the liver of D-gal-treated mice. The levels of PI3K and PBK/Akt were also largely enhanced, which in turn inactivated pro-apoptotic signaling events, restoring the balance between pro- and anti-apoptotic Bcl-2 and Bax proteins in the liver of D-gal-treated mice. In conclusion, these results suggest that FGF21 protects the mouse liver against D-gal-induced hepatocyte oxidative stress via enhancing Nrf2-mediated antioxidant capacity and apoptosis via activating PI3K/Akt pathway.

Long-term lamivudine treatment achieves regression of advanced liver fibrosis/cirrhosis in patients with chronic hepatitis B.

BACKGROUND AND AIM: Antiviral therapy is important in advanced liver fibrosis/cirrhosis with chronic hepatitis B (AdLF-CHB) patients, but complete regression of cirrhosis remains to be the challenge. We aimed to investigate whether up to 10 years lamivudine treatment achieves liver fibrosis/cirrhosis regression in AdLF-CHB patients. METHODS: Improvement of hepatic fibrosis/cirrhosis, virological response and disease progression were evaluated in 28 AdLF-CHB patients with up to 10 years lamivudine treatment. Liver biopsy was performed in all of the 28 patients at baseline, but only 19 patients had second biopsy at year 10. RESULTS: There were 24 hepatitis B e antigen (HBeAg)-positive and 4 HBeAg-negative patients within the original 28 AdLF-CHB patients. At the end of 10 years lamivudine treatment, 20 of the 24 HBeAg-positive patients had HBeAg loss. HBeAg seroconversion was detected in 10 of these 20 HBeAg loss patients. HBsAg loss was observed in 4 of the original 28 patients. Among these four HBsAg loss patients, three had HBsAg seroconversion. All patients achieved hepatitis B virus DNA (HBV DNA) undetectable. Histopathology was evaluated between paired original and final liver biopsies among 19 patients as follows: 4/19 achieved complete liver fibrosis/cirrhosis regression; 9/19 improved in Ishak fibrosis score; whereas 6/19 showed no fibrosis improvement. About 75% patients achieved inflammatory/fibrotic improvement. No significant disease progression was observed in 24/28 patients. Furthermore, no significant difference in histopathology improvement, cirrhosis regression, disease progression between non-resistance and rescue for resistance was observed. CONCLUSION: Long-term lamivudine therapy achieves regression of fibrosis/cirrhosis and improvement of histological and disease progression in AdLF-CHB patients.

Hepatitis C virus core antigen, an earlier and stronger predictor on sustained virological response in patients with genotype 1 HCV infection.

BACKGROUND: Earlier kinetics of serum HCV core antigen (HCVcAg) and its predictive value on sustained virological response (SVR) were investigated in patients with genotype 1 HCV infection during antiviral treatment. METHODS: In a multi-centered, randomized and positive drug-controlled phase IIb clinical trial on type Y peginterferon α-2b ( NCT01140997), forty-eight CHC patients who participated in pharmacokinetics were randomly divided into 4 cohorts and treated with PegIFNα (type Y peginterferon α-2b 90 µg, 135 µg, 180 µg and PegIFNα-2a 180 µg, respectively, once a week) and ribavirin (< 75 kg, 1000 mg daily and ≥ 75 kg, 1200 mg daily) for 48 weeks, and then followed up for 24 weeks. 32 patients infected with genotype 1 HCV and completed the whole process were included in this study. HCV RNAs were detected at baseline, and weeks 4, 12, 24, 48 and 72 using Cobas TaqMan. ARCHITECT HCVcAg was performed at 24, 48, 72, 96, 120 and 144 h in addition to the above time points. The receiver operating curves (ROCs) were performed to study the predictive values of HCVcAg decline on SVR. RESULTS: Following antiviral treatment, serum HCVcAg levels rapidly declined within the first week and correlated well with corresponding HCV RNA at baseline, weeks 4, 12, 24, 48 and 72 (rs = 0.969, 0.928, 0.999, 0.983, 0.985 and 0.946, respectively, P < 0.001). All of the areas under the receiver operating curves (AUROCs) were more than 0.80 and showed good predictive power on SVR at 24, 48, 72, 96, 120 and 144 h. The144 h was the best predictive time point of HCVcAg decline on SVR because of its largest AUROC (more than 0.90). CONCLUSIONS: Early kinetics of serum HCVcAg predicts SVR very well in genotype 1 CHC patients during antiviral treatment, and its reduction value at 144 h is an earlier and stronger predictor on SVR than rapid virological response and early virological response. (TRN: NCT01140997).

FGF21 treatment ameliorates alcoholic fatty liver through activation of AMPK-SIRT1 pathway.

Fibroblast growth factor 21 (FGF21), a recently identified member of the FGF superfamily, is mainly secreted from the liver and adipose tissues and plays an important role in improving metabolic syndrome and homeostasis. The aim of this study is to evaluate the role of FGF21 in alcoholic fatty liver disease (AFLD) and to determine if it has a therapeutic effect on AFLD. In this paper, we tested the effect of FGF21 on alcohol-induced liver injury in a murine model of chronic ethanol gavage and alcohol-treated HepG2 cells. Male KM mice received single dose of 5 g/kg ethanol gavage every day for 6 weeks, which induced significant fatty liver and liver injury. The alcohol-induced fatty liver cell model was achieved by adding ethanol into the medium of HepG2 cell cultures at a final concentration of 75 mM for 9 days. Results showed that treatment with recombinant FGF21 ameliorated alcoholic fatty liver and liver injury both in a murine model of chronic ethanol gavage and alcohol-treated HepG2 cells. In addition, FGF21 treatment down-regulated the hepatic expression of fatty acid synthetic key enzyme, activated hepatic AMPK-SIRT1 pathway and significantly down-regulated hepatic oxidative stress protein. Taken together, FGF21 corrects multiple metabolic parameters of AFLD in vitro and in vivo by activation of the AMPK-SIRT1 pathway.

Incorporating Network Pharmacology and Experimental Validation to Identify Bioactive Compounds and Potential Mechanisms of Digitalis in Treating Anaplastic Thyroid Cancer.

Anaplastic thyroid cancer (ATC) is one of the most lethal malignant tumors for which there is no effective treatment. There are an increasing number of studies on herbal medicine for treating malignant tumors, and the classic botanical medicine Digitalis and its active ingredients for treating heart failure and arrhythmias have been revealed to have significant antitumor efficacy against a wide range of malignant tumors. However, the main components of Digitalis and the molecular mechanisms of its anti-ATC effects have not been extensively studied. Here, we screened the main components and core targets of Digitalis and verified the relationship between the active components and targets through network pharmacology, molecular docking, and experimental validation. These experiments showed that the active ingredients of Digitalis inhibit ATC cell activity and lead to ATC cell death through the apoptotic pathway.

Ruxolitinib induces apoptosis and pyroptosis of anaplastic thyroid cancer via the transcriptional inhibition of DRP1-mediated mitochondrial fission.

Anaplastic thyroid carcinoma (ATC) has a 100% disease-specific mortality rate. The JAK1/2-STAT3 pathway presents a promising target for treating hematologic and solid tumors. However, it is unknown whether the JAK1/2-STAT3 pathway is activated in ATC, and the anti-cancer effects and the mechanism of action of its inhibitor, ruxolitinib (Ruxo, a clinical JAK1/2 inhibitor), remain elusive. Our data indicated that the JAK1/2-STAT3 signaling pathway is significantly upregulated in ATC tumor tissues than in normal thyroid and papillary thyroid cancer tissues. Apoptosis and GSDME-pyroptosis were observed in ATC cells following the in vitro and in vivo administration of Ruxo. Mechanistically, Ruxo suppresses the phosphorylation of STAT3, resulting in the repression of DRP1 transactivation and causing mitochondrial fission deficiency. This deficiency is essential for activating caspase 9/3-dependent apoptosis and GSDME-mediated pyroptosis within ATC cells. In conclusion, our findings indicate DRP1 is directly regulated and transactivated by STAT3; this exhibits a novel and crucial aspect of JAK1/2-STAT3 on the regulation of mitochondrial dynamics. In ATC, the transcriptional inhibition of DRP1 by Ruxo hampered mitochondrial division and triggered apoptosis and GSDME-pyroptosis through caspase 9/3-dependent mechanisms. These results provide compelling evidence for the potential therapeutic effectiveness of Ruxo in treating ATC.

Combination of IL-34 and AFP improves the diagnostic value during the development of HBV related hepatocellular carcinoma.

IL-34 involves in host immunity regulated carcinogenesis. Alpha-fetoprotein (AFP) is related to the development of HCC. We explored if combination of IL-34 and APF could improve the diagnostic value in HBV related hepatocellular carcinoma (HBV-HCC). Serum was obtained from HBV patients or healthy control. Liver tissue was obtained from liver biopsy in CHB, HBV related cirrhosis patients or curative resection in HBV-HCC patients. Serum IL-34 and MCSF, or intrahepatic IL-34, MCSF and CD68+ tumor associate macrophages (TAMs) were determined using ELISA or immunohistochemistry. Serum IL-34 was 1.7, 1.3 or 2.3-fold higher in HBV-HCC than that of CHB, HBV related cirrhosis or healthy control, which was inhibited following trans-hepatic arterial chemoembolization (TACE) in HBV-HCC patients. Intra-hepatic IL-34 was higher in HBV-HCC than that of the other three groups. Intra-hepatic IL-34 was associated with high HBV-DNA, HBeAg-, poor differentiation and small tumor size of HBV-HCC patients. Intra-hepatic TAMs in HBV-HCC were increased 1.7 or 1.3-fold, compared to that from CHB or HBV-cirrhosis patients. Intra-hepatic TAMs were associated with high HBV-DNA, high tumor differentiation, small tumor size, abnormal AFP and more tumor number. AFP plus serum IL-34, showed the highest AUC (0.837) with sensitivity (0.632) and highest specificity (0.931), suggesting that AFP plus IL-34 enhances the reliability for prediction of the development of HBV-HCC among CHB patients. Circulating and intra-hepatic IL-34 was upregulated gradually in HBV disease progression from CHB, cirrhosis and HCC. IL-34 may be used as a diagnostic biomarker and potential therapeutic target for the management of HBV-HCC.

Alantolactone induces concurrent apoptosis and GSDME-dependent pyroptosis of anaplastic thyroid cancer through ROS mitochondria-dependent caspase pathway.

BACKGROUND: Anaplastic thyroid cancer (ATC) is one of the fatal cancers and has not effective treatments. Alantolactone (ATL), a terpenoid extracted from traditional Chinese medicinal herb Inula helenium L., confers significant anti-inflammatory, antibacterial and antitumor activity. However, the activity and mechanisms of ATL in ATC remain unclear. PURPOSE: To investigate the potential anti-ATC effects in vitro and in vivo and the mechanisms involved. METHODS: The anti-proliferative activity of Alantolactone (ATL) against ATC cells was analyzed through CCK-8 and colony formation assays. Flow cytometry assay was performed to assess the cell cycle, cell apoptosis, ROS, and mitochondrial membrane potential (ΔΨm), whereas the cellular localization of cytochrome c and calreticulin were determined using cellular immunofluorescence assays. The lactate dehydrogenase (LDH) enzyme activity in the cell culture medium was measured using a commercial LDH kit, whereas ELISA was conducted to assess the secretory function of IL-1ß. Western blot assays were conducted to determine the expression or regulation of proteins associated with apoptosis and pyroptosis. Subcutaneous tumor model of nude mice was established to evaluate the anticancer activity of ATL in vivo. The expression of Ki67, cyclin B1, cleaved-PARP, cleaved-caspase 3, and IL-1ß in the animal tumor tissues was profiled using immunohistochemistry analyses. RESULTS: Our data showed that ATL significantly inhibited the proliferation and colony formation activity of ATC cells. ATL induced ATC cell cycle arrest at G2/M phase, and downregulated the expression of cyclin B1 and CDC2. Furthermore, ATL induced concurrent apoptosis and pyroptosis in the ATC cells, and the cleavage of PARP and GSDME. It also significantly increased the release of LDH and IL-1ß. Mechanically, ATL-mediated increase in ROS suppressed the Bcl-2/Bax ratio, downregulated the mitochondrial membrane potential and increased the release of cytochrome c, leading to caspase 9 and caspase 3 cleavage. We also found that ATL induced the translocation of an immunogenic cell death marker (calreticulin) to the cell membrane. In addition, it inhibited the growth of the ATC subcutaneous xenograft model, and activated proteins associated with apoptosis and pyroptosis, with a high safety profile. CONCLUSION: Taken together, these results firstly demonstrated that ATL exerted an anti-ATC activity by inducing concurrent apoptosis and GSDME-dependent pyroptosis through ROS-mediated mitochondria-dependent caspase activation. Meanwhile, these cell deaths exhibited obvious characteristics of immunogenic cell death, which may synergistically increase the potential of cancer immunotherapy in ATC. Further studies are needed to explore deeper mechanisms for the anti- ATC activity of ATL.

TESC promotes differentiated thyroid cancer development by activating ERK and weakening NIS and radioiodine uptake.

PURPOSE: Most differentiated thyroid cancer (DTC) patients have a good prognosis after surgery, but radioiodine refractory differentiated thyroid cancer (RAIR-DTC) patients have a significantly reduced 5-year survival rate (<60%) and a significantly increased recurrence rate (>30%). This study aimed to clarify the tescalcin (TESC) role in promoting the malignant PTC progression and providing a potential target for RAIR-DTC treatment. METHODS: We analyzed TESC expression and clinicopathological characteristics using the Cancer Genome Atlas (TCGA) and performed qRT-PCR on tissue samples. TPC-1 and IHH-4 proliferation, migration, and invasion were detected after transfection with TESC-RNAi. Using Western blot (WB), several EMT-related indicators were detected. Moreover, iodine uptake of TPC-1 and IHH-4 after transfection with TESC-RNAi was detected. Finally, NIS, ERK1/2, and p-ERK1/2 levels were determined by WB. RESULTS: TESC was significantly upregulated in DTC tissues and positively correlated with BRAF V600E mutation based on data analysis from TCGA and our center. Reduced expression of TESC in both IHH-4 (BRAF V600E mutation) and TPC-1 (BRAF V600E wild type) cells significantly inhibited cell proliferation, migration, and invasion. It downregulated the EMT pathway markers Vimentin and N-cadherin, and increased E- cadherin. Moreover, TESC knockdown significantly inhibited ERK1/2 phosphorylation and decreased NIS expression in DTC cells, with a remarkably increased iodine uptake rate. CONCLUSIONS: TESC was highly expressed in DTC tissues and may have promoted metastasis through EMT and induced iodine resistance by downregulating NIS in DTC cells.

Silencing of AJAP1 expression by promoter methylation activates the Wnt/ß-catenin signaling pathway to promote tumor proliferation and metastasis in salivary adenoid cystic carcinoma.

Background: Salivary adenoid cystic carcinoma (SACC) is a unique malignant tumor of the salivary gland with poor prognosis, which is not effective with chemotherapy and targeted drugs. Therefore, it is important to explore the molecular mechanism underlying SACC invasion and metastasis to develop novel therapeutic strategies and targets in clinical research. Methods: Real-time quantitative polymerase chain reaction (RT-qPCR) and western blot (WB) were performed to detect the expression of Adherens Junctions Associated Protein 1 (AJAP1). Methylation-specific PCR was used to evaluate the methylation of the AJAP1 promoter. AJAP1 was overexpressed or knocked down by lentivirus-mediated transfection. Kaplan-Meier analysis was conducted to create a survival curve and the log-rank test was used to analyze the overall survival (OS). The prognostic correlation was assessed using univariate and multivariate Cox regression analyses. Co-immunoprecipitation (Co-IP) was utilized to pull down the possible binding protein of AJAP1 and laser scanning confocal microscopy was applied to detect the subcellular localization of AJAP1, E-cadherin, and ß-catenin. Cell viability, colony formation, wound healing, and Transwell invasion assays were performed to evaluate the function of AJAP1 in vitro. A subcutaneous xenograft assay in nude mice was performed to verify the function of AJAP1 in vivo. Results: AJAP1 was downregulated in SACC tumors and was closely related to SACC lymph node/distant metastasis, which was an independent risk factor for SACC prognosis. Methylation-specific PCR confirmed that high methylation of the AJAP1 promoter was the main cause of its silencing. Overexpression or knockdown of AJAP1 in SACC cells could significantly inhibit or promote the proliferation, invasion, and metastasis of SACC cells, respectively, in both the in vitro and in vivo experiments. Mechanically, we found that AJAP1 binds to E-cadherin and ß-catenin to form a complex in cytomembrane, reducing the nuclear translocation of ß-catenin and blocking the Wingless/Integrated/ß-catenin (Wnt/ß-catenin) signaling pathway to play a suppressive role in cancer. Conclusions: In conclusion, these results suggest that the downregulation of AJAP1 protein expression may play a certain role in progression and metastasis of SACC. Our study indicates that AJAP1 may be a potential prognostic molecular marker and therapeutic target for SACC.

The immune enhancement effects of recombinant NDV expressing chicken granulocyte-macrophage colony-stimulating factor on the different avian influenza vaccine subtypes.

Avian influenza is an acute and highly contagious infectious disease that is caused by the influenza virus. Avian influenza has been widely spread all over the world, has caused property loss and has threatened human life and security. In this study, the recombinant plasmid rClone30-chGM-CSF was constructed and rescued to the recombinant virus rClone30-chGM-CSF successfully. After 8 days of immunization with the recombinant virus, the titre of NDV HI (haemagglutination inhibition) antibodies in SPF chickens reached its peak. The average titre of the rClone30-chGM-CSF group reached 6 log2 and was significantly higher than the protection critical value of 4 log2 ; the titres of the rClone30 group and the blank group were 2.86 log2 and 1 log2 , respectively, indicating that the recombinant virus can effectively improve the NDV antibody titre. Then, SPF chickens were co-immunized with the recombinant virus and with three different vaccine subtypes of inactivated avian influenza. The results indicated that the SPF chickens that were immunized with the vaccine plus rClone30-chGM-CSF showed significantly higher avian influenza antibody levels than those in the single vaccine groups. Furthermore, the SPF chickens in the vaccine plus rClone30-chGM-CSF group elicited stronger CD4+ and CD8+ T-cell proliferative responses and also had upregulated transcriptional levels of interleukin-1ß (IL-1ß), IL-4, IL-6 and IL-17 compared with those in the single vaccine groups. This study has shown that the recombinant virus expressing chicken granulocyte-macrophage colony-stimulating factor (chGM-CSF) can be used not only as an NDV vaccine to effectively improve the titre of NDV antibodies but also as a biological adjuvant to enhance the immune effects of the avian influenza vaccine. Therefore, this recombinant virus can also be used as a biological adjuvant for other poultry vaccines.

[The effect of N-acetyl-L-cysteine on endoplasmic reticulum stress mediated apoptosis of HepG2 cells].

OBJECTIVE: To analyze the mechanisms of NAC on endoplasmic reticulum (ER) stress mediated cells apoptosis of HepG2 cells and to evaluate the potential role of NAC in the treatment of liver injury. METHODS: HepG2 cells were treated with H2O2 to make a model of oxidative ER stress mediated apoptosis. To evaluate the apoptosis, various methods such as MTT, DNA ladder, Western blot and flow cytometry were used. Then the optimal dosage and incubation time of NAC intervention in apoptosis were ascertained, and the differences between induction and intervention of apoptosis, including the percentage of apoptosis, the expression of apoptotic protein (GRP78, Caspase-12, PARP) and the production of reactive oxygen species (ROS) were compared. RESULTS: The activity of the cells decreased by H2O2 (0, 1, 3, 5 mmol/L) treatments in a dose-dependent manner. The ratio of apoptotic cells increased (0.7%+/-0.5%, 26.4%+/-1.8%, 29.7%+/-1.2% and 51.2%+/-9.4%, respectively) as did the production of ROS (14.0%+/-0.5%, 95.2%+/-0.1%, 97.5%+/-0.2% and 98.3%+/-0.2%, respectively). The HepG2 cells showed typical morphologic change of ER stress 6 hr after they were treated with 3 mmol/L H2O2. ER stress mediated-apoptosis was confirmed by Western blot. NAC (10 mmol/L and 20 mmol/L) protected cells from apoptosis. Typical features of ER stress apoptosis were seen accompanied by diminishing the ratio of apoptotic cells from 29.7%+/-1.2% to 23.3%+/-4.7% and 14.3%+/-1.2%. The production of ROS also decreased from 97.5%+/-0.2% to 52.2%+/-0.8% and 51.2%+/-2.9%. The effect was related to the concentration: 20 mmol/L NAC was more effective than 10 mmol/L. CONCLUSIONS: As an oxidizing agent, H2O2 may induce ROS in cells and induce oxidative stress, causing ER stress and apoptosis. NAC can inhibit the procession of ROS directly and prevent injuries to the hepatocytes.

Interferon-based treatment is superior to nucleos(t)ide analog in reducing HBV-related hepatocellular carcinoma for chronic hepatitis B patients at high risk.

BACKGROUND: The effect of nucleos(t)ide analogs (NAs) versus interferon (IFN) on the occurrence of hepatocellular carcinoma (HCC) in chronic hepatitis B (CHB) is controversial. We assessed whether antiviral strategy affected HCC development in CHB patients at different HCC risks. METHODS: 1112 CHB patients with antiviral therapy were included in this retrospective study. Patients treated with NAs only were classified into NAs group (n = 682) while those received IFN treatment with or without NAs were defined as IFN group (n = 430). Propensity score matching (PSM) was applied to minimize baseline differences. RESULTS: Totally, 31 patients developed HCC during follow-up (median 5.41 years). The cumulative HCC incidence at 10 years was significantly lower in the IFN group than NAs group (2.7% vs 8.0%, p < 0.001). Similar results were obtained in the PSM-cohort. Patients with IFN-based treatment were less likely to develop HCC than those with NAs (Hazard ratio = 0.15; 95% CI 0.04-0.66; p = 0.012). Subgroup analyses demonstrated that this superiority of IFN in reducing HCC development was obvious in patients at high- but not low-risk of HCC. CONCLUSIONS: Reduction of HCC development was more significant in CHB patients at higher HCC risk with IFN-based therapy than NAs treatment.

The pharmacological efficacy of the anti-IL17 scFv and sTNFR1 bispecific fusion protein in inflammation mouse stimulated by LPS.

Acute lung injury (ALI) is still a leading cause of morbidity and mortality in critically ill patients. Recently, our study found that a bispecific fusion protein treatment can ameliorate the lung injury induced by LPS. However, the molecular mechanisms which bispecific fusion protein ameliorates acute lung injury remain unclear. In this study, we found that the bispecific fusion protein treatment inhibited the nuclear transcription of NF-κB in confocal laser scanning fluorescence microscopy, the bispecific fusion protein exert protective effects in the cell model of ALI induced by lipopolysaccharide (LPS) via inhibiting the nuclear factor κB (NF-κB) signaling pathway and mediate inflammation. Moreover, the treatment of the bispecific fusion protein show its efficacy in animal models stimulated by LPS, the results of real-time PCR and ELISA demonstrate that bispecific fusion protein treatment effectively inhibited the over-expression of inflammatory cytokines(tumor necrosis factor α, interleukin 1ß and interleukin 17). In addition, LPS-challenged mice exhibited significant lung injury characterized by the deterioration of histopathology, which was meliorated by bispecific fusion protein treatment. Collectively, these results demonstrate that bispecific fusion protein treatment ameliorates LPS-induced ALI through reducing inflammatory cytokines and lung inflammation, which may be associated with the decreased the nuclear transcription of NF-κB. The bispecific fusion protein may be useful as a novel therapy to treat ALI.

Correction: Identification of Optimal Insertion Site in Recombinant Newcastle Disease Virus (rNDV) Vector Expressing Foreign Gene to Enhance Its Anti-Tumor Effect.

[This corrects the article DOI: 10.1371/journal.pone.0164723.].