ATP7B R778L mutant hepatocytes resist copper toxicity by activating autophagy and inhibiting necroptosis.

Wilson's disease (WD) is an inherited disease characterized by copper metabolism disorder caused by mutations in the adenosine triphosphatase copper transporting ß gene (ATP7B). Currently, WD cell and animal model targeting the most common R778L mutation in Asia is lacking. In addition, the mechanisms by which hepatocytes resist copper toxicity remain to be further elucidated. In this study, we aimed to construct a novel WD cell model with R778L mutation and dissected the molecular basics of copper resistance. A novel HepG2 cell line stably expressing the ATP7B R778L gene (R778L cell) was constructed. The expression of necroptosis- and autophagy-related molecules was detected by PCR and Western blot (WB) in wild-type (WT) HepG2 and R778L cells with or without CuSO4 treatment. In addition, we detected and compared the levels of autophagy and necroptosis in CuSO4-treated R778L cells with the activation and inhibition of autophagy. Moreover, the mRNA and protein levels of autophagy and necroptosis signaling molecules were compared in R778L cells with the overexpression and knockdown of Unc-51 Like Autophagy Activating Kinase 1 (ULK1) and Autophagy Related 16 Like 1 (ATG16L1). We successfully constructed an R778L mutation HepG2 cell line. CuSO4 triggered the enhanced expression of autophagy and necroptosis signaling molecules in WT HepG2 cells and R778L cells. Remarkably, higher levels of autophagy and necroptosis were observed in R778L cells compared with those in WT cells. Autophagy activation led to weakened necroptosis mediated by RIPK3 and MLKL, conversely, autophagy inhibition brought about enhanced necroptosis. At the molecular level, ULK1- and ATG16L1 overexpression resulted in reduced necroptosis levels and vice versa. ULK1- and ATG16L1-mediated autophagy activation protects hepatocytes against RIPK3- and MLKL-mediated necroptosis in our new WD cell model treated with CuSO4. Targeted therapy by autophagy activation or necroptosis inhibition may be a novel and effective strategy to treat WD.

Recompensation in treatment-naïve HBV-related decompensated cirrhosis: a 5-year multi-center observational study comparing patients with ascites and bleeding.

BACKGROUND AND AIMS: Recompensation between patients with ascites and bleeding was unknown in treatment-naïve HBV-related decompensated cirrhosis. METHODS: In this retrospective multi-center study, treatment-naïve HBV-related decompensated patients were enrolled at first decompensating event of ascites and/or variceal bleeding. Further complications and clinical characteristics were collected using standard case report form every 6 months to year-5 of antiviral treatment. Recompensation was defined as maintaining free of decompensation for one year and achieving liver function within Child-Pugh A and/or MELD < 10. RESULTS: Totally, 170 (170/298, 57.0%) patients in ascites group of 298 (298/383, 77.8%) treatment-naïve decompensated patients and 33 (33/85, 38.8%) in bleeding group of 85 (85/383, 22.2%) patients, achieved recompensation. Ascites group had higher 5-year rate of recompensation than bleeding group (63.3% vs. 46.5%, p = 0.012), respectively. Patients achieving recompensation in ascites group maintained lower rate of second decompensation than these in bleeding group (at year-5: 26.7% vs. 43.3%, p = 0.032). Specifically, recompensated patients in ascites group had predominantly 5-year rate of further ascites (24.0%) and lower rate of further bleeding (6.0%), which differed from the pattern of these in bleeding group, with lower rate of further ascites (16.0%, p = 0.599) and significantly higher rate of further bleeding (33.9%, p < 0.001). Both patients had superior long-term prognosis (death/LT rate at year-5: 0.6% vs. 3.0%, p = 0.196). CONCLUSION: Ascites patients could achieve higher rate of recompensation through antiviral therapy than bleeding patients. Recompensated patients in ascites group had better prognosis in terms of preventing further bleeding.

Serum HBsAg and HBcrAg is associated with inflammation in HBeAg-positive chronic hepatitis B patients.

Backgrounds & aims: Liver inflammation is the main risk factor for developing liver fibrosis, cirrhosis, and even hepatocellular carcinoma in chronic hepatitis B (CHB) patients. To replace biopsy, additional non-invasive biomarkers to diagnose and grade liver necroinflammation are urgently required in clinical practice. Method: Ninety-four CHB patients, including 74 HBeAg-positive and 20 HBeAg-negative patients, were enrolled and started entecavir or adefovir therapy. Serum HBV RNA, HBV DNA, HBsAg, hepatitis B core-related antigen (HBcrAg), ALT and AST levels, as well as intrahepatic HBV DNA and cccDNA were measured at baseline and during treatment. Liver inflammation was assessed at baseline and month 60 by liver biopsy. Inflammation regression was defined as a ≥1-grade decrease according to the Scheuer scoring system. Results: In HBeAg-positive CHB patients, at baseline, serum HBsAg and HBcrAg levels negatively correlated with inflammation grade, while ALT and AST levels positively correlated with inflammation grade. AST plus HBsAg exhibited excellent diagnostic ability for significant inflammation with an AUROC of 0.896. After 60 months of antiviral treatment, almost all the patients' liver inflammation ameliorated to G1, and no patients had inflammation progression. Conclusion: Besides ALT and AST, serum HBsAg and HBcrAg correlated with inflammation grade in HBeAg-positive CHB patients before NAs treatment. Moreover, the combination of HBsAg and AST exhibited excellent diagnostic ability for significant inflammation.

Serum HBV RNA is associated with liver fibrosis regression in HBeAg-positive chronic hepatitis B patients treated with nucleos(t)ide analogues.

Noninvasive methods for assessing hepatic fibrosis are clinically necessary. This study aims to explore HBV markers correlated with liver fibrosis and capable of diagnosing significant fibrosis and predicting fibrosis regression. Seventy-four HBeAg-positive chronic hepatitis B (CHB) patients were enrolled and started on entecavir or adefovir therapy. Serum HBV RNA, HBV DNA, HBsAg and hepatitis B core-related antigen (HBcrAg) levels were measured at baseline and during treatment. Liver fibrosis was assessed at baseline and month 60 by liver biopsy. Fibrosis regression was defined as Ishak fibrosis score decreased ≥1-point. At baseline, HBsAg, HBcrAg and HBV RNA levels had a stronger correlation with Ishak fibrosis score (r = -.441, p = .002; r = -.469, p = .001; r = -.398, p = .001) than APRI and FIB-4 (r = .321 p = .006; r = .371, p = .001). HBsAg >4 log10 IU/ml plus HBcrAg >7 log10 IU/ml or HBsAg >4 log10 IU/ml plus HBV RNA >5 log10 copies/ml exhibited the same excellent diagnostic ability for significant fibrosis with the AUROC of 0.857. After 60 months of antiviral treatment, 66.7% of patients who suffered significant fibrosis at baseline achieved fibrosis regression, and an HBV RNA decline from baseline to month 6 greater than 0.63 log10 copies/ml could predict the fibrosis regression at month 60. In conclusion, serum HBsAg, HBcrAg and HBV RNA are potential markers for predicting significant liver fibrosis. HBV RNA measurement would be particularly useful for monitoring hepatic fibrosis changes in HBeAg-positive CHB patients.

Clinical study of hepatitis B vaccine in achieving hepatitis B surface antibody seroconversion in patients with functional cure

Abstract Hepatitis B Surface Antigen (HBsAg) seroclearance is the highest treatment goal recommended by the current guidelines for hepatitis B. Levels of antibodies to HBsAg (anti-HBs) are strongly associated with HBsAg recurrence, but hepatitis B vaccination may increase the anti-HBs seroconversion rate and reduce recurrence. We conducted a retrospective clinical study to ascertain the effect of this vaccination on the seroconversion rate and levels of protective anti-HBs after HBsAg. In this retrospective study, we distributed a questionnaire through an online survey platform to collect information related to hepatitis B vaccination in patients with functional cure of hepatitis B with Interferon-α (IFNα) therapy. We enrolled 320 patients who achieved functional cure from IFNα therapy. Of these, 219 patients had received hepatitis B vaccination according to their personal preference and drug accessibility after HBsAg seroclearance, whereas the remaining 101 patients did not receive hepatitis B vaccination. The anti-HBs seroconversion rate of 78.1% in the vaccinated group was significantly greater than that in the unvaccinated group (41.6%) (p < 0.001). Stratified comparisons with anti-HBs of ≥ 100 IU/L and ≥ 300 IU/L showed that both proportions in the vaccinated group were greater than those in the unvaccinated group (71.2% vs. 32.7% and 56.2% vs. 17.8%, respectively, all p-values < 0.001). Logistic regression analysis showed that the odds ratio of vaccination was 4.427, which was the strongest influencing factor for anti-HBs, reaching 100 IU/L or higher. Hepatitis B vaccination in patients after HBsAg seroclearance not only increased the anti-HBs seroconversion rate but also significantly increased antibody levels, with good safety, indicating the clinical value of vaccine therapy for patients with functional cure.

Study on the Retreatment, Outcome, and Potential Predictors of Recurrence in Patients With Recurrence of Hepatitis B After Functional Cure.

Background: Studies about the retreatment and predictors for patients with hepatitis B recurrence after functional cure are rare. This study aimed to evaluate the effect of retreatment, outcome, and potential predictors of recurrence in patients with recurrence after functional cure. Methods: A long-term follow-up was conducted with 32 cumulatively obtained patients who relapsed after cessation of pegylated interferon (Peg-IFN)-based antiviral treatment. The decision of whether to treatment or which therapeutic method to use [Peg-IFN or nucleos(t)ide analogs (NAs)] was based on the patient's preferences and wishes. The rate of achieving functional cure and the clinical outcomes of different therapeutic methods were analyzed. Hepatitis B surface antibody (anti-HBs) and hepatitis B core antibody (anti-HBc) levels were detected in patients with blood samples during follow-up to evaluate the predictive ability of recurrence. Results: The follow-up time of 32 recurrence cases was 42-532 weeks after recurrence (median 226 weeks). In the 20 patients who received retreatment (15 received Peg-IFN and 5 received NAs only), the rate of functional cure was 65.0% (13/20); it was 86.7% (13/15) in the patients retreated with Peg-IFN. Three cases experienced recurrence again. Five patients received NA treatment, and no functional cure was achieved. No drug intervention was administered for 12 patients, 2 of them with hepatitis B virus (HBV) DNA spontaneous clearance, and one patient achieved spontaneous hepatitis B surface antigen (HBsAg) clearance during follow-up. Patients who relapsed after functional cure with Peg-IFN treatment did not have liver cirrhosis or hepatocellular carcinoma during the follow-up, regardless of whether they received retreatment. Anti-HBs and anti-HBc levels at the end of therapy were predictors of recurrence (p < 0.001, p = 0.023). The value of combining the above two indicators in predicting recurrence was further improved, the areas under the receiver operating characteristic curves were 0.833, at combining predictors >-0.386, the predictive sensitivity and specificity for recurrence were 86.67% and 90.62%. Conclusion: The functional cure rate was above 80% for patients with recurrence treated by Peg-IFN. During the follow-up, liver cirrhosis and hepatocellular carcinoma were not observed in all recurrence cases. High levels of anti-HBs and anti-HBc at the time of drug discontinuation are less likely to relapse.

A Glycolysis-Related Gene Signature Correlates With the Characteristics of the Tumor Immune Microenvironment and Predicts Prognosis in Patients With Hepatocellular Carcinoma.

Aim: To develop a glycolysis-related gene signature that correlated with the characteristics of the tumor immune microenvironment and had good predictive power for overall survival (OS) in hepatocellular carcinoma (HCC). Methods: Gene expression profiles, RNA sequencing data, clinical characteristics and survival information for 407 patients with HCC and 58 healthy controls were downloaded from the TCGA database. GSEA 4.1.0 software was used to evaluate the glycolysis-related pathways enriched in HCC compared to normal liver tissue. Univariate Cox, Least Absolute Shrinkage, Selection Operator, and two-step multivariate Cox analyses were used to construct a glycolysis-related gene signature for prognostic prediction. The glycolysis-related gene signature was combined with clinical characteristics to generate a nomogram. Tumor-infiltrating immune cell profiles and PD-L1 protein expression in HCC tissues were investigated. Results: The gene expression profiles of HCC tissues were enriched in glycolysis-related pathways. A glycolysis-related gene signature was used to categorize patients as high-risk or low-risk, where high-risk patients had significantly worse OS. Receiver operating characteristic curves confirmed the predictive capability of the glycolysis-related gene signature for OS (AUC >0.80). There was a significant difference in M0 macrophage (p = 0.017), dendritic cell (p = 0.043), B cell (p = 0.0018), CD4 T cell (p = 0.003), Treg (p = 0.01) and mast cell (p = 0.02) content and PD-L1 protein expression (p = 0.019) between HCC tissues in patients in the high-risk and low-risk groups. Conclusion: We established a glycolysis-related gene signature for OS in HCC that was predictive in training and test TCGA cohorts and correlated with the characteristics of the HCC tumor immune microenvironment. The glycolysis-related gene signature may guide clinical decision-making concerning patient selection for immunotherapy in HCC.

Durability of hepatitis B surface antigen seroclearance and subsequent risk for hepatocellular carcinoma: A meta-analysis.

Hepatitis B surface antigen (HBsAg) seroclearance is regarded as the ideal endpoint for antiviral treatment. However, reports on the durability of and outcomes after HBsAg seroclearance are few, which has become a focus in clinical practice. This meta-analysis was performed to evaluate the durability and hepatocellular carcinoma (HCC) incidence after HBsAg seroclearance after treatment cessation. We searched PubMed, Embase, Medline and Web of Science for studies that reported the durability and HCC incidence after HBsAg seroclearance published between 1 January 2000 and 31 January 2020. Data were analysed by a random-effects model. Thirty-eight studies and 43,924 patients were finally included. The results showed that HBsAg seroclearance was durable, with a pooled recurrence rate of 6.19% (95% CI: 4.10%-8.68%). There was no significant difference in recurrence rates after different seroclearance methods or among recurrence types and different regions. Anti-HBs seroconversion resulted in a significantly reduced recurrence rate (RR = 0.25, p < .001). Patients who experienced HBsAg seroclearance had significantly lower HCC incidence than HBsAg-positive (RR = 0.41, p < .001). The pooled HCC incidence after HBsAg seroclearance was 1.88%; this rate was reduced to 0.76% among patients without baseline cirrhosis. In conclusion, the analysis during an average follow-up of 4.74 years suggested that in patients who experienced sustained HBsAg seroclearance and anti-HBs seroconversion, this was associated with low HCC incidence. Patients without baseline cirrhosis benefited even more. We emphasize the importance of gaining HBsAg seroclearance while highlighting the benefits of achieving this as early as possible.

Pathological features of COVID-19-associated lung injury: a preliminary proteomics report based on clinical samples.

The COVID-19 pandemic has emerged as a global health emergency due to its association with severe pneumonia and relative high mortality. However, the molecular characteristics and pathological features underlying COVID-19 pneumonia remain largely unknown. To characterize molecular mechanisms underlying COVID-19 pathogenesis in the lung tissue using a proteomic approach, fresh lung tissues were obtained from newly deceased patients with COVID-19 pneumonia. After virus inactivation, a quantitative proteomic approach combined with bioinformatics analysis was used to detect proteomic changes in the SARS-CoV-2-infected lung tissues. We identified significant differentially expressed proteins involved in a variety of fundamental biological processes including cellular metabolism, blood coagulation, immune response, angiogenesis, and cell microenvironment regulation. Several inflammatory factors were upregulated, which was possibly caused by the activation of NF-κB signaling. Extensive dysregulation of the lung proteome in response to SARS-CoV-2 infection was discovered. Our results systematically outlined the molecular pathological features in terms of the lung response to SARS-CoV-2 infection, and provided the scientific basis for the therapeutic target that is urgently needed to control the COVID-19 pandemic.

Clinical Characteristics and Treatment Outcomes in Human Immunodeficiency Virus (HIV)-Infected Patients with Liver Abscess: A Retrospective Study of 53 Patients.

BACKGROUND Although episodes of liver abscess (LA) have been reported in patients infected with the human immunodeficiency virus (HIV), specific symptoms in these patients remain unclear. MATERIAL AND METHODS The clinical characteristics, laboratory findings, treatments, and final clinical outcomes of LA in 53 HIV-infected patients were analyzed. RESULTS The most common clinical manifestations were fever (92.5%), chills (41.5%), and abdominal pain (37.7%). The mean CD4⁺ T cell count in these HIV-infected patients at admission was 328.09±236.192 cells/µL. LA and blood cultures were positive in six (17.6%) and two (5.4%) patients, respectively. Thirteen strains of pathogens, including Staphylococcus, Corynebacterium, and Candida, were detected in LA cultures. Forty-four (95.7%) of 46 patients were successfully treated with antibiotics plus image-guided percutaneous aspiration, drainage, or surgery, whereas four (57.1%) of the remaining seven patients were successfully treated with antibiotics alone. Septic shock [odds ratio (OR)=8.970; 95% confidence interval (CI)=0.840-92.110; p=0.014] and ascites (OR=7.057; 95% CI=0.683-72.957; p=0.016) were found to be independent risk factors for poor prognosis. The clinical characteristics of LA in HIV-infected patients were nonspecific, with bacteria being the primary pathogens. CONCLUSIONS Antibiotics plus image-guided percutaneous drainage can effectively improve treatment outcomes in HIV-infected patients with LA.

Longitudinal COVID-19 profiling associates IL-1RA and IL-10 with disease severity and RANTES with mild disease.

BACKGROUND: Identifying immune correlates of COVID-19 disease severity is an urgent need for clinical management, vaccine evaluation, and drug development. Here, we present a temporal analysis of key immune mediators, cytokines, and chemokines in blood of hospitalized COVID-19 patients from serial sampling and follow-up over 4 weeks. METHODS: A total of 71 patients with laboratory-confirmed COVID-19 admitted to Beijing You'an Hospital in China with either mild (53 patients) or severe (18 patients) disease were enrolled with 18 healthy volunteers. We measured 34 immune mediators, cytokines, and chemokines in peripheral blood every 4-7 days over 1 month per patient using a bioplex multiplex immunoassay. RESULTS: We found that the chemokine RANTES (CCL5) was significantly elevated, from an early stage of the infection, in patients with mild but not severe disease. We also found that early production of inhibitory mediators including IL-10 and IL-1RA were significantly associated with disease severity, and a combination of CCL5, IL-1 receptor antagonist (IL-1RA), and IL-10 at week 1 may predict patient outcomes. The majority of cytokines that are known to be associated with the cytokine storm in virus infections such as IL-6 and IFN-γ were only significantly elevated in the late stage of severe COVID-19 illness. TNF-α and GM-CSF showed no significant differences between severe and mild cases. CONCLUSION: Together, our data suggest that early intervention to increase expression of CCL5 may prevent patients from developing severe illness. Our data also suggest that measurement of levels of CCL5, as well as IL-1RA and IL-10 in blood individually and in combination, might be useful prognostic biomarkers to guide treatment strategies.

Transplantation of ACE2- Mesenchymal Stem Cells Improves the Outcome of Patients with COVID-19 Pneumonia.

A coronavirus (HCoV-19) has caused the novel coronavirus disease (COVID-19) outbreak in Wuhan, China. Preventing and reversing the cytokine storm may be the key to save the patients with severe COVID-19 pneumonia. Mesenchymal stem cells (MSCs) have been shown to possess a comprehensive powerful immunomodulatory function. This study aims to investigate whether MSC transplantation improves the outcome of 7 enrolled patients with COVID-19 pneumonia in Beijing YouAn Hospital, China, from Jan 23, 2020 to Feb 16, 2020. The clinical outcomes, as well as changes of inflammatory and immune function levels and adverse effects of 7 enrolled patients were assessed for 14 days after MSC injection. MSCs could cure or significantly improve the functional outcomes of seven patients without observed adverse effects. The pulmonary function and symptoms of these seven patients were significantly improved in 2 days after MSC transplantation. Among them, two common and one severe patient were recovered and discharged in 10 days after treatment. After treatment, the peripheral lymphocytes were increased, the C-reactive protein decreased, and the overactivated cytokine-secreting immune cells CXCR3+CD4+ T cells, CXCR3+CD8+ T cells, and CXCR3+ NK cells disappeared in 3-6 days. In addition, a group of CD14+CD11c+CD11bmid regulatory DC cell population dramatically increased. Meanwhile, the level of TNF-α was significantly decreased, while IL-10 increased in MSC treatment group compared to the placebo control group. Furthermore, the gene expression profile showed MSCs were ACE2- and TMPRSS2- which indicated MSCs are free from COVID-19 infection. Thus, the intravenous transplantation of MSCs was safe and effective for treatment in patients with COVID-19 pneumonia, especially for the patients in critically severe condition.

Clinical characteristics of liver failure with hemophagocytic lymphohistiocytosis.

Liver failure with hemophagocytic lymphohistiocytosis (HLH) is a life-threatening syndrome with high mortality. The aim of this study was to decipher clinical and laboratory characteristics of hemophagocytic lymphohistiocytosis after definite diagnosis of liver failure and to provide clues for early diagnosis and treatment of HLH in patients with liver failure. Eleven patients diagnosed with liver failure and HLH were retrospectively investigated in this study. All patients presented with jaundice, persistent high-grade fever, pancytopenia, splenomegaly, evidence of hemophagocytes in the bone marrow and laboratory abnormalities indicating HLH. The average interval from the earliest diagnosis of liver failure to a definitive diagnosis of HLH was 17.27 days. Six (54.55%) patients died during follow-up. For patients with liver failure after admission and subsequently definitively diagnosed with HLH, bilirubin and INR were significantly decreased. HLH is definitely diagnosed at an intermediate or late stage when patients have already suffered from liver failure. The initial dose of glucocorticoid (methylprednisolone) was decreased to 1-1.5 mg/kg/d and gradually reduced thereafter. In conclusion, for patients with liver failure, HLH should be screened as early as possible upon persistent fever, splenomegaly and unexplained pancytopenia. For patients with liver failure and HLH, the dosage of glucocorticoid should be reduced to avoid serious side effects.

Kaempferol induces hepatocellular carcinoma cell death via endoplasmic reticulum stress-CHOP-autophagy signaling pathway.

Kaempferol is a flavonoid compound that has gained widespread attention due to its antitumor functions. However, the underlying mechanisms are still not clear. The present study investigated the effect of kaempferol on hepatocellular carcinoma and its underlying mechanisms. Kaempferol induced autophagy in a concentration- and time-dependent manner in HepG2 or Huh7 cells, which was evidenced by the significant increase of autophagy-related genes. Inhibition of autophagy pathway, through 3-methyladenine or Atg7 siRNA, strongly diminished kaempferol-induced apoptosis. We further hypothesized that kaempferol can induce autophagy via endoplasmic reticulum (ER) stress pathway. Indeed, blocking ER stress by 4-phenyl butyric acid (4-PBA) or knockdown of CCAAT/enhancer-binding protein homologous protein (CHOP) with siRNA alleviated kaempferol-induced HepG2 or Huh7 cells autophagy; while transfection with plasmid overexpressing CHOP reversed the effect of 4-PBA on kaempferol-induced autophagy. Our results demonstrated that kaempferol induced hepatocarcinoma cell death via ER stress and CHOP-autophagy signaling pathway; kaempferol may be used as a potential chemopreventive agent for patients with hepatocellular carcinoma.

Cell death biomarker M65 is a useful indicator of liver inflammation and fibrosis in chronic hepatitis B: A cross-sectional study of diagnostic accuracy.

Cell death markers, M65 and M30, have been suggested to be sensitive markers of liver inflammation and fibrosis in nonalcoholic fatty liver disease and chronic hepatitis C. Our aim was to investigate whether these markers were useful in diagnosing liver inflammation and fibrosis in chronic hepatitis B (CHB).We examined 186 patients with CHB; 18 sex- and age-matched healthy subjects were controls. The blood samples were collected from CHB patients within 1 week before or after liver biopsy. According to METAVIR score system, liver inflammation was graded from A0 to A3, and fibrosis from F0 to F4.Serum M65 and M30 levels were in parallel with the grades of liver inflammation. M65, not M30, increased significantly in patients with severe inflammation and normal alanine aminotransferase. M65 is one of the independent predictors of severe liver inflammation (≥A2). The levels of M65 and M30 levels significantly increased in parallel with the degree of inflammation in F1 patients, whereas they showed no statistical difference between different stages of fibrosis in A1 patients.Serum M65 is a useful indicator of liver inflammation in CHB patients. Serum M65, not M30, is valuable in the grading of liver fibrosis.

Kaempferol induces apoptosis in HepG2 cells via activation of the endoplasmic reticulum stress pathway.

Kaempferol is a flavonoid compound that has gained importance due to its antitumor properties; however, the underlying mechanisms remain to be fully understood. The present study aimed to investigate the molecular mechanisms of the antitumor function of kaempferol in HepG2 hepatocellular carcinoma cells. Kaempferol was determined to reduce cell viability, increase lactate dehydrogenase activity and induce apoptosis in a concentration­ and time­dependent manner in HepG2 cells. Additionally, kaempferol­induced apoptosis possibly acts via the endoplasmic reticulum (ER) stress pathway, due to the significant increase in the protein expression levels of glucose­regulated protein 78, glucose­regulated protein 94, protein kinase R­like ER kinase, inositol­requiring enzyme 1α, partial activating transcription factor 6 cleavage, caspase­4, C/EBP homologous protein (CHOP) and cleaved caspase­3. The pro­apoptotic activity of kaempferol was determined to be due to induction of the ER stress­CHOP pathway, as: i) ER stress was blocked by 4­phenyl butyric acid (4­PBA) pretreatment and knockdown of CHOP with small interfering RNA, which resulted in alleviation of kaempferol­induced HepG2 cell apoptosis; and ii) transfection with plasmid overexpressing CHOP reversed the protective effect of 4­PBA in kaempferol­induced HepG2 cells and increased the apoptotic rate. Thus, kaempferol promoted HepG2 cell apoptosis via induction of the ER stress­CHOP signaling pathway. These observations indicate that kaempferol may be used as a potential chemopreventive treatment strategy for patients with hepatocellular carcinoma.

Kinetic response of wild and mutant core codon 70 strains of HCV genotype 1b to pegylated interferon-α and ribavirin therapy.

BACKGROUND: Amino acid (aa) 70 substitution (R70Q/H) in the core protein of hepatitis C virus (HCV) genotype 1b has been shown to be one of the key factors in determining resistance for pegylated interferon-α plus ribavirin combination therapy (PEG-IFNα/RBV). But the exact mechanisms remain unclear. The aim of this study was to investigate the dynamic response of wild and mutant core codon 70 strains to PEG-IFNα/RBV treatment. METHODS: One hundred twelve Chinese patients with chronic HCV 1b infection were enrolled and received a standard protocol of 48 weeks of PEG-IFNα/RBV therapy and 24 consecutive weeks of follow-up. Serial blood samples were obtained at pretreatment baseline, and again at weeks 2, 4, 8, 12, and 24 during therapy for the quantification of 70R and 70Q/H strains. Dynamic characteristics and association with early virological response (EVR), sustained virological response (SVR) and IL28B genotypes were analyzed. RESULTS: Of the 112 patients enrolled in this study, 93.8% (105/112) were infected with mixture of 70R and 70Q/H strains before treatment. The 70Q/H strain was dominant in 20.5% of patients. 42.9% of patients with dominant 70Q/H exhibited EVR versus 88.6% of patients with dominant 70R (P < 0.001). Furthermore, 35.0% of patients with dominant 70Q/H exhibited SVR versus 77.4% with dominant 70R (P < 0.001). However, regardless of the dominant strain, virological response types or the IL28B SNP genotypes, 70Q/H strains always exhibited the same response to treatment as the 70R strains and the percentage of HCV harboring the 70Q/H substitution did not change significantly during treatment. CONCLUSIONS: Although the ratio of 70Q/H to 70R is related to the virological response, 70Q/H strains always exhibited the same response as the 70R strains during PEG-IFNα/RBV treatment. Substitution of R70Q/H alone is not enough to lead to resistance to therapy. Positive selection for 70Q/H induced by IFNα was not observed.

Reversal of tumor growth by gene modification of mesenchymal stem cells using spermine-pullulan/DNA nanoparticles.

Mesenchymal stem cells (MSCs) are a promising tool for delivering of therapeutic agents in cancer treatment. In the present study, our findings suggested that both i.v. and intratumoral injection of MSCs could favor tumor growth under physiologic conditions. However, the anti-tumor effects of MSC-IL-12 were achieved using our strategy. Unlike the previously reported method, the genetic engineering of MSCs was conducted by non-viral transfection using the new vector, spermine-pullulan. The transfection, cytotoxicity, and the cellular internalization of this vector were evaluated. Then, the therapeutical gene, IL-12, was delivered to the MSCs using this vector. The in vitro secretions of IL-12 by MSC-IL-12 confirmed the success of using spermine-pullulan/DNA nanoparticles for the gene transfection. We used the MSC-IL-12 for the in vivo treatment of both B16F10 metastasis tumor and the established subcutaneous B16BL6 tumor. For the B16F10 metastasis tumor, treatment with MSC-IL-12 significantly reduced lung metastases. For the established subcutaneous B16BL6 tumor, intratumoral injected MSC-IL-12 cells considerably retarded tumor growth. Prolonged survival was observed when MSC-IL-12 cells were injected through the tail vein or intratumorally, indicating that the MSCs engineered with the therapeutic gene could reverse the tumor-promoting effects of MSCs using the nonviral transduction method. However, the intravenous injected MSC-IL-12 did not prevent the tumor growth of the established subcutaneous B16BL6 tumor. Thus, we examined the the in vivo distribution of MSCs in different organs and it was found that MSCs were mainly distributed in the lungs, which may explain the inability of intravenously injected MSC-IL-12 to inhibit the growth of the established subcutaneous tumor.

Impregnation of plasmid DNA into three-dimensional PLGA scaffold enhances DNA expression of mesenchymal stem cells in vitro.

Current efforts had been made to undertake a three-dimensional (3-D) reverse transfection of bone marrow-derived mesenchymal stem cells (BM-MSCs) in PLGA scaffolds. As a kind of multipotent stem cells, BM-MSCs show great potential and tremendous capacity in the gene transfection field and PLGA 3-D scaffold has been shown to be a biomaterial that provides structural support to cells proliferation and tissue engineering. The objective of this study was to assess the transfection efficiency of BM-MSCs with a 3-D reverse transfection method by using PLGA scaffold and observe the SEM photographs of BM-MSCs cultured on PLGA scaffold. BM-MSCs were cultured in 3-D PLGA scaffold which was incorporated with pullulan-spermine/pGL3. It was shown that the gene expression duration of BM-MSCs transfected using 3D reverse method with pullulan-spermine/DNA in the presence of serum maintained 12 days at high levels as compared with the plasmid DNA in medium, and scanning electronic microscopy (SEM) photographs of BM-MSCs cultured on PLGA scaffold exhibited robust cell attachment and viability when cultured in PLGA scaffold in vitro. This study demonstrates that the 3-D reverse transfection method of BM-MSCs using PLGA scaffold could achieve long gene expression in a relatively high level, therefore this transfection system is promising in gene transfection and tissue engineering.

TGF-ß1 gene-engineered mesenchymal stem cells induce rat cartilage regeneration using nonviral gene vector.

This study evaluated the potential of utilizing transfected pTGFß-1 gene-engineered rat mesenchymal stem cells (MSCs) using nonviral vector to promote cartilage regeneration. Pullulan-spermine was used as the nonviral gene vector and gelatin sponge was used as the scaffold. MSCs were engineered with TGF-ß1 gene with either the three-dimensional (3D) reverse transfection system or the two-dimensional (2D) conventional transfection system. For the 3D reverse transfection system, pullulan-spermine/pTGF-ß1 gene complexes were immobilized to the gelatin sponge, followed by the seeding of MSCs. Pullulan-spermine/pTGF-ß1 gene complexes were delivered to MSCs cultured in the plate to perform the 2D conventional transfection system, and then MSCs were seeded to the gelatin sponge. Then, TGF-ß1 gene-transfected MSC seeded gelatin sponge was implanted to the full-thickness cartilage defect. Compared with the control group, both groups of TGF-ß1 gene-engineered MSCs improved cartilage regeneration through optical observation and histology staining. So, with pullulan-spermine as the nonviral vector, TGF-ß1-gene engineered MSCs can induce cartilage regeneration in vivo.