Prognostic value of anti-HBc quantification in hepatitis B virus related acute-on-chronic liver failure.

BACKGROUND AND AIM: It has been reported that serum quantification of anti-HBc (qAnti-HBc) could predict antiviral response in chronic hepatitis B (CHB) patients, while its role in hepatitis B virus-related acute-on-chronic liver failure (HBV-ACLF) remains unclear. Its implication in HBV-ACLF was evaluated in this study. METHODS: Baseline serum qAnti-HBc levels were retrospectively detected in HBV-ACLF and CHB patients using recently developed double-sandwich immunoassay. The association of qAnti-HBc level with clinical outcomes was evaluated by multiple logistic regression. Nomogram was adopted to formulate an algorithm incorporating qAnti-HBc for the prediction of survival in HBV-ACLF. The post-hospitalization of HBV-ACLF patients were followed-up for 1 year. RESULTS: Eighty-eight HBV-ACLF as training set, 80 HBV-ACLF as validation set and 216 CHB cases were included. Serum qAnti-HBc level was significantly higher in HBV-ACLF (4.95 ± 0.54 log10  IU/mL) than CHB patients (4.47 ± 0.84 log10  IU/mL) (P < 0.01). Among HBV-ACLF cases, both in training and validation set, patients with poor outcomes had lower qAnti-HBc level. Area under receiver operating characteristic curve of the novel qAnti-HBc inclusive model was 0.82, superior to 0.73 from model for end-stage liver disease scores (P = 0.018), which was confirmed in validation set. During follow-up, the qAnti-HBc level declined at month 3 and month 6, then plateaued at 3.84 log10  IU/mL. CONCLUSIONS: Serum qAnti-HBc level was associated with disease severity and might be served as a novel biomarker in the prediction of HBV-ACLF clinical outcomes. The underlying immunological mechanism warrants further investigation.

Comparison of Serum Hepatitis B Virus RNA Levels and Quasispecies Evolution Patterns between Entecavir and Pegylated-Interferon Mono-treatment in Chronic Hepatitis B Patients.

Hepatitis B virus (HBV) RNA may independently predict virological and serological response. This study aimed to compare dynamic changes in serum HBV RNA levels and HBV quasispecies evolution patterns between entecavir and pegylated-interferon mono-treatment in chronic hepatitis B patients and to determine the clinical significance during treatment. TaqMan real-time PCR was used for quantitative analysis. HBV RNA levels were retrospectively determined in serial serum samples from 178 chronic hepatitis B patients who received either entecavir or pegylated-interferon treatment. Both serum HBV DNA and RNA quasispecies were analyzed via next-generation sequencing. Receiver operating characteristics (ROC) analysis was performed to evaluate the prediction value of individual biomarkers for hepatitis B e antigen (HBeAg) seroconversion. Patients who received pegylated-interferon treatment showed stronger declines in HBV RNA levels than did those who received entecavir treatment. Serum HBV RNA levels were lower in patients with subsequent HBeAg seroconversion. At baseline, the level of HBV RNA was better than other indicators in predicting HBeAg seroconversion. Moreover, the predictive value of serum HBV RNA levels was better in the entecavir group. Baseline HBV RNA exhibited a significantly higher genetic diversity than HBV DNA and had a significant decline after 4 weeks of entecavir treatment. Higher baseline genetic diversity may result in a better outcome in pegylated-interferon-treated patients. Serum HBV RNA levels showed different decline kinetics, and HBV RNA quasispecies showed different evolution patterns in entecavir and pegylated-interferon mono-treatment. Taken together, serum HBV RNA may serve as a promising biomarker of HBeAg seroconversion in patients during antiviral treatment.

Serum M2BPGi level is a novel predictive biomarker for the responses to pegylated interferon-α treatment in HBeAg-positive chronic hepatitis B patients.

Serum Mac-2-binding protein glycosylation isomer (M2BPGi) level was found to be a useful prognostic marker for hepatitis B e antigen (HBeAg)-positive chronic hepatitis B (CHB) patients treated with nucleoside/nucleotide analogs (NUCs) therapy, and the aim of our study is to evaluate the clinical implementation of M2BPGi level in the prediction of antiviral responses to pegylated-interferon-α (PEG-IFN-α) treatment in HBeAg-positive CHB patients. Ninety-six CHB patients who received PEG-IFN-α treatment for at least 48 weeks were recruited. The serum M2BPGi, alanine aminotransferase (ALT), hepatitis B surface antigen (HBsAg), HBeAg, and HBV DNA levels at baseline, weeks 4, 12, and 24 after PEG-IFN-α treatment were determined and their associations with antiviral responses were evaluated and the virological response (VR) rate and serological response (SR) rate after 48 weeks of treatment were 65.6% and 35.4%, respectively. Baseline serum M2BPGi level was significantly different between VR and non-VR (P = 0.002) or SR and non-SR groups (P = 0.012). Multivariate analyses suggested that baseline serum M2BPGi level was independently associated with VR and SR of PEG-IFN-α treatment at week 48. The area under the ROC curve (AUC) of baseline M2BPGi was 0.682 in predicting VR, which was superior to HBsAg (AUC = 0.566) or HBV DNA (AUC = 0.567). The AUC of baseline M2BPGi in predicting SR was 0.655, which was also higher than that of HBsAg (AUC = 0.548) or HBV DNA (AUC = 0.583). These results suggested that baseline serum M2BPGi level was a novel predictor of VR and SR for PEG-IFN-α treatment in HBeAg-positive CHB patients.

Protective Effects of Reduced Beta 2 Glycoprotein I on Liver Injury in Streptozotocin (STZ)-Diabetic Rats by Activation of AMP-Activated Protein Kinase.

BACKGROUND Protective effects of reduced beta 2 glycoprotein I (Rb2GPI) against vascular injury of diabetes mellitus have been extensively investigated. However, the effects of Rb2GPI on liver injury in diabetic animals have not been reported. MATERIAL AND METHODS A diabetic rat model of was produced by systemic injection of streptozotocin (STZ). Rats were divided into a normal control group, a model group, and an Rb2GPI treatment group (N=6 in each group). After treatments, blood serum and liver tissue were collected to test the protection of Rb2GPI. AMP-activated protein kinase (AMPK) was detected by immunohistochemistry and Western blotting. RESULTS Our results revealed that Rß2GPI reduced blood glucose, serum creatinine, and urea nitrogen levels, as well as serum inflammation cytokines, including interleukin (IL)-6, tumor necrosis factor (TNF)-α and C-reactive protein in the diabetic rats. Importantly, Rß2GPI prevented liver injury in the diabetic rats as confirmed by hematoxylin-eosin (H&E) staining, alanine transaminase, aspartate transaminase, and gamma-glutamyl transferase. Reactive oxygen species (ROS) were promoted by diabetic modeling and were attenuated by Rß2GPI administration. Moreover, Rß2GPI significantly reduced liver catalase, malondialdehyde, and superoxide dismutase levels in the diabetic rats. Rß2GPI reduced liver glycolipid storage in STZ diabetic rats. Both immunohistochemistry and Western blotting demonstrated that Rß2GPI promoted AMPK phosphorylation in the diabetic rats. CONCLUSIONS Our data proved that Rß2GPI prevented liver injury in diabetic rats, likely through activating the AMPK signaling pathway.

Long noncoding RNA TUG1 alleviates extracellular matrix accumulation via mediating microRNA-377 targeting of PPARγ in diabetic nephropathy.

Long noncoding RNA taurine-upregulated gene 1 (lncRNA TUG1) has been reported to play a key role in the progression of diabetic nephropathy (DN). However, the role of lncRNA TUG1 in the regulation of diabetic nephropathy remains largely unknown. The aim of the present study is to identify the regulation of lncRNA TUG1 on extracellular matrix accumulation via mediating microRNA-377 targeting of PPARγ, and investigate the underlying mechanisms in progression of DN. Microarray was performed to screen differentially expressed miRNAs in db/db DN mice. Afterwards, computational prediction programs (TargetScan, miRanda, PicTar and miRGen) was applied to predict the target gene of miRNAs. The complementary binding of miRNA and lncRNA was assessed by luciferase assays. Protein and mRNA expression were detected by western blot and real time quantitate PCR. MiRNA-377 was screened by miRNA microarray and differentially up-regulated in db/db DN mice. PPARγ was predicted to be the target of miR-377 and the prediction was verified by luciferase assays. Expression of miR-377 was up-regulated in mesangial cell treated with high glucose (25 mM), and overexpression of miR-377 inhibited PPARγ expression and promoted PAI-1 and TGF-ß1 expression. The expression of TUG1 antagonized the effect of miR-377 on the downregulation of its target PPARγ and inhibited extracellular matrix accumulation, including PAI-1, TGF-ß1, fibronectin (FN) and collagen IV (Col IV), induced by high glucose. LncRNA TUG1 acts as an endogenous sponge of miR-377 and downregulates miR-377 expression levels, and thereby relieving the inhibition of its target gene PPARγ and alleviates extracellular matrix accumulation of mesangial cells, which provides a novel insight of diabetic nephropathy pathogenesis.

Serum WFA+ -M2BP levels for evaluation of early stages of liver fibrosis in patients with chronic hepatitis B virus infection.

BACKGROUND & AIMS: Accurate evaluation of liver fibrosis is crucial for predicting progression of chronic hepatitis B virus (HBV) infection. We assessed the utility of a novel fibrosis glycobiomarker Wisteria floribunda agglutinin-positive Mac-2-binding protein (WFA+ -M2BP) for evaluating liver fibrosis and disease progression in patients with chronic HBV infection. METHODS: We enrolled 774 patients with chronic HBV infection, with or without fibrosis, diagnosed by liver biopsy/FibroScan. Patients who underwent liver biopsy (n = 297) were divided into training (n = 221) and validation (n = 76) groups. Serum WFA+ -M2BP values were measured and compared with FIB-4 index, aspartate aminotransferase (AST)-to-platelet ratio (APRI) and AST-to-alanine aminotransferase ratio (AAR) using receiver-operating characteristic (ROC) analysis. RESULTS: Serum WFA+ -M2BP levels increased significantly with fibrosis progression (P < 0.0001). Area under the ROC curve of WFA+ -M2BP for diagnosing significant fibrosis was higher than that of FIB-4 (P = 0.198), APRI (P = 0.017) and AAR (P < 0.001), with sensitivity and specificity in the training set of 60.5% and 79.8% and validation set of 59.5% and 82.1%, respectively. Serum WFA+ -M2BP levels were significantly correlated with FibroScan values (P < 0.0001) and improved the accuracy of FibroScan in assessing significant fibrosis. Changes in WFA+ -M2BP levels were parallel with those in FibroScan values during nucleot(s)ide analogues therapy in patients with chronic HBV infection. CONCLUSIONS: WFA+ -M2BP is an accurate serum indicator for assessing early stages of liver fibrosis and may monitor regression of fibrosis during the treatment of chronic HBV infection. WFA+ -M2BP provides a simple and reliable alternative or complementary method to liver biopsy and FibroScan.

Association of Thyroid-Stimulating Hormone Levels with Microvascular Complications in Type 2 Diabetes Patients.

BACKGROUND Subclinical hypothyroidism (SCH) is typically featured by elevated serum concentration of thyroid-stimulating hormone (TSH). This study aimed to determine the relationship between TSH levels and microvascular complications in type 2 diabetes patients. MATERIAL AND METHODS A total of 860 type 2 diabetes patients were enrolled in this cross-sectional study. Subjects were evaluated for anthropometric measurements, thyroid function, diabetic retinopathy, and diabetic kidney disease. TSH was divided into 3 levels: 0.27-2.49 mU/l, 2.5-4.2 mU/l, and >4.2 mU/l. RESULTS Among the participants, 76 subjects (8.8%) were diagnosed with subclinical hypothyroidism (SCH) (male: 6.6% and female: 11.8%). The prevalence of diabetic retinopathy did not differ among the groups (P=0.259). Of the 860 type 2 diabetic subjects, we further excluded invalid or missing data. Therefore, 800 and 860 subjects were included in our study of diabetic retinopathy (DR) and diabetic kidney disease (DKD), respectively. The frequencies of microalbuminuria and macroalbuminuria differed significantly among the different groups. The frequency of DKD was significantly different among the 3 groups (P=0.001) and was higher in subjects with higher TSH levels. After an adjustment for confounding variables, TSH levels were significantly associated with DKD (P<0.001). When compared with subjects with TSH 0.27-2.49 mU/l, the frequency of DKD was higher in subjects with TSH >4.20 mU/l (OR 1.531, 95% CI 1.174-1.997) and with TSH 2.50-4.20 mU/l (OR 1.579, 95% CI 1.098-2.270). However, TSH levels was not significantly correlated with DR (P=0.126). CONCLUSIONS Type 2 diabetic patients with higher TSH values had a higher prevalence of DKD.

Characterization of Full-Length Genomes of Hepatitis B Virus Quasispecies in Sera of Patients at Different Phases of Infection.

Hepatitis B virus (HBV) infection results in different clinical presentation due to different levels of immune response. Our study aimed to characterize HBV full-length genome quasispecies (QS) in patients with different phases of infection to better understand its pathogenesis. Forty treatment-naive HBV-infected patients were enrolled, including 10 cases of acute hepatitis B (AHB), 9 cases of immunotolerant (IT) HBV carriers, 11 cases of chronic hepatitis B (CHB), and 10 cases of acute-on-chronic liver failure (ACLF). The present study was conducted by clone-based sequencing. QS heterogeneity within each open reading frame was calculated. The mutation frequency index (MFI) and amino acid variations within the large HBsAg, HBcAg, and HBxAg regions were analyzed based on the different infection phases. In total, 606 HBV full-length sequences were obtained. HBV QS had higher heterogeneity in ACLF and CHB than that in IT among chronically infected individuals. AHB patients had the lower QS heterogeneity at onset than those with chronic infection. ACLF patients had the highest frequency of mutations in the core promoter and precore region. A triple mutation (A1762T/G1764A/G1896A) was observed more frequently in genotype C than in genotype B. The MFI indicated that specific peptides of the studied regions had more frequent mutations in ACLF. Furthermore, several amino acid variations, known as T- and B-cell epitopes, were potentially associated with the immunoactive phase of infection. More HBV genome mutations and deletions were observed in patients with more severe diseases, particularly in specific regions of the core and preS regions, the clinical significance and mechanism of which need to be further investigated.

Liraglutide reduces the body weight and waist circumference in Chinese overweight and obese type 2 diabetic patients.

AIM: To investigate the effects of liraglutide, a glucagon-like peptide-1 (GLP-1) receptor activator, on body weight and waist circumference in Chinese overweight and obese type 2 diabetic patients. METHODS: A total of 328 Chinese overweight and obese type 2 diabetic patients were included in this multi-center, open-labeled and self-controlled clinical study. The patients were subcutaneously injected with liraglutide once daily for 24 weeks as add-on therapy to their previous hypoglycemic treatments. Statistical analyses were performed using SPSS software package version 11.5 for Windows. RESULTS: Liraglutide treatment caused significant reduction of the mean body weight (from 86.61±14.09 to 79.10±13.55 kg) and waist circumference (from 101.81±13.96 to 94.29±14.17 cm), resulting in body weight lose of 5%-10% in 43.67% patients, and body weight loss above 10% in 34.06% patients, who had significant lower plasma creatinine levels. Baseline waist circumference, BMI and HOMA-IR were independently correlated with the body weight loss. Furthermore, liraglutide treatment significantly decreased HbA1c levels (from 8.66%±2.17% to 6.92%±0.95%) with HbA1c<7.0% in 35.37% patients, who had a significantly lower baseline level of HbA1c, but higher baseline levels of C peptide and glucagon. Moreover, liraglutide treatment resulted in greater body weight loss in patients with a long duration of diabetes, and better glycemic control in patients with a short duration of diabetes. CONCLUSION: Liraglutide significantly reduces body weight and waist circumference in Chinese overweight and obese type 2 diabetic patients. Patients with apparent visceral obesity, insulin resistance and a long duration of diabetes may have greater body weight loss; whereas patients with high insulin-secreting ability, hyperglucagonemia, and short-duration diabetes may obtain better glycemic control with liraglutide.

N-glycosylation mutations within hepatitis B virus surface major hydrophilic region contribute mostly to immune escape.

BACKGROUND & AIMS: HBV immune escape represents a challenge to prevention, diagnosis, and treatment of hepatitis B. Here, we analyzed the molecular and clinical characteristics of HBV immune escape mutants in a Chinese cohort of chronically infected patients. METHODS: Two hundred sixteen patients with HBsAg and anti-HBs were studied, with one hundred eighty-two HBV carriers without anti-HBs as a control group. Recombinant HBsAg bearing the most frequent N-glycosylation mutations were expressed in CHO and HuH7 cells. After confirming N-glycosylation at the most frequent sites (129 and 131), together with inserted mutations, functional analysis were performed to study antigenicity and secretion capacity. RESULTS: One hundred twenty-three patients had the wild-type HBs gene sequence, 93 patients (43%) had mutants in the major hydrophilic region (MHR), and 47 of the 93 patients had additional N-glycosylation mutations, which were transmitted horizontally to at least 2 patients, one of whom was efficiently vaccinated. The frequency of N-glycosylation mutation in the case group was much higher than that of the control group (47/216 vs. 1/182). Compared with wild-type HBsAg, HBsAg mutants reacted weakly with anti-HBs using a chemiluminescent microparticle enzyme immunoassay. Native gel analysis of secreted virion in supernatants of transfected HuH7 cells indicated that mutants had better virion enveloping and secretion capacity than wild-type HBV. CONCLUSIONS: Our results suggest that specific HBsAg MHR N-glycosylation mutations are implicated in HBV immune escape in a high endemic area.

Inherent lipid metabolic dysfunction in glycogen storage disease IIIa.

We studied two patients from a nonconsanguineous family with life-long abnormal liver function, hepatomegaly and abnormal fatty acid profiles. Abnormal liver function, hypoglycemia and muscle weakness are observed in various genetic diseases, including medium-chain acyl-CoA dehydrogenase (MCAD) deficiency and glycogen storage diseases. The proband showed increased free fatty acids, mainly C8 and C10, resembling fatty acid oxidation disorder. However, no mutation was found in ACADM and ACADL gene. Sequencing of theamylo-alpha-1, 6-glucosidase, 4-alpha-glucanotransferase (AGL) gene showed that both patients were compound heterozygotes for c.118C > T (p.Gln40X) and c.753_756 del CAGA (p.Asp251Glufsx29), whereas their parents were each heterozygous for one of these mutations. The AGL protein was undetectable in EBV-B cells from the two patients. Transcriptome analysis demonstrated a significant different pattern of gene expression in both of patients' cells, including genes involving in the PPAR signaling pathway, fatty acid biosynthesis, lipid synthesis and visceral fat deposition and metabolic syndrome. This unique gene expression pattern is probably due to the absence of AGL, which potentially accounts for the observed clinical phenotypes of hyperlipidemia and hepatocyte steatosis in glycogen storage disease type IIIa.

Clinical implications of evolutionary patterns of homologous, full-length hepatitis B virus quasispecies in different hosts after perinatal infection.

Chronic hepatitis B virus (HBV) infection via perinatal transmission is common in the Asia-Pacific region, but related quasispecies (QS) characteristics are not yet defined. To investigate the homologous, full-length HBV QS after perinatal infection and their clinical implications, five pairs of mother-daughter patients with chronic HBV infection (one patient with liver cirrhosis, one with immune tolerance, and eight with chronic hepatitis) were included. Full-length HBV were amplified by PCR from serum samples before antiviral treatment and cloned; an average of 17 clones per sample were sequenced, and the QS complexities, diversities, and evolution patterns were analyzed. Full-length HBV sequence similarities within mother-daughter pairs were 91.3 to 98.3%. The QS complexities of full-length HBV were similar between mothers and daughters (median of 0.9734 compared to 0.9688, P>0.05), as were the diversities (median of 3.396×10(-3) compared to 4.617×10(-3) substitutions/site, P>0.05). However, the distribution patterns of QS complexities and diversities within specific genes were different, and QS genetic distances of the mothers were higher than those of daughters, both more evident in pairs with different antiviral responses and different immune phases or stages. The nucleotide substitution rate of full-length HBV was 14.388×10(-5) substitutions/site/year, whereas the preC/C gene rate was the highest. Mutations and indels were more common in clones from the mothers, which decreased the affinity of epitopes by 6- to 89-fold. The various genes from homologous HBV genomes evolved in different patterns despite numerically comparable full-length QS complexities and diversities. The different patterns may correlate with the immune stages of chronic HBV infection, which warrants further study.

Changes in liraglutide-induced body composition are related to modifications in plasma cardiac natriuretic peptides levels in obese type 2 diabetic patients.

BACKGROUND AND AIMS: Liraglutide treatment can improve glycemic control with a concomitant weight loss, but the underlying mechanism on weight loss is not completely understood. Cardiac natriuretic peptides (NPs) can resist body fat accumulation through increasing adipocytes lypolysis. In this study, we tested the hypothesis that liraglutide-induced weight loss was associated with increased plasma NPs concentrations. METHODS: Thirty-one outpatients with type 2 diabetes (T2D) treated with metformin and other oral antidiabetic drugs except for thiazolidinediones (TZDs) were subcutaneously administered with liraglutide for 12 weeks. Body composition, abdominal visceral adipose tissue areas (VAT) and subcutaneous adipose tissue areas (SAT) were assessed at pre- and post-treatment by dual-energy X-ray absorptiometry (DXA) scanning and abdominal computerized tomography (CT). Plasma atrial natriuretic peptides (ANP) and B-type ventricular natriuretic peptides (BNP) concentrations were tested by commercial ELISA Kit quantitatively. RESULTS: Following 12-week liraglutide treatment, body weight, waist circumference, total fat and lean mass, fat percentage, SAT and VAT areas were significantly reduced from baseline. Concurrently, plasma ANP and BNP levels were significantly increased following 12-week liraglutide treatment. There were significant correlations between the reductions in body compositions and the increases in both plasma ANP and BNP levels. CONCLUSIONS: There were significant correlations between increases in both plasma ANP and BNP levels and changes in liraglutide-induced body composition. Our data implied that increases in plasma NPs may add a novel dimension to explain how liraglutide induces weight loss.

Comparison of next-generation sequencing and clone-based sequencing in analysis of hepatitis B virus reverse transcriptase quasispecies heterogeneity.

We previously reported that, based on clone-based sequencing (CBS), hepatitis B virus (HBV) heterogeneity within the reverse transcriptase (RT) region was a predictor of antiviral efficacy. Here, by comparing ultradeep pyrosequencing (UDPS), i.e., next-generation sequencing (NGS), with CBS in characterizing the genetic heterogeneity of HBV quasispecies within the RT region, we evaluated the performance of UDPS in the analysis of HBV viral populations. HBV genomic DNA was extracted from serum samples from 31 antiviral treatment-naive patients with chronic hepatitis B. The RT region quasispecies were analyzed in parallel using CBS and UDPS. Characterization of quasispecies heterogeneity was conducted using bioinformatics analysis. Quasispecies complexity values were calculated with the formula Sn = -Σi(pilnpi)/lnN. The number of qualified strains obtained by UDPS was much larger than that obtained by CBS (P < 0.001). Pearson analysis showed that there was a positive correlation of quasispecies complexity values at the nucleotide level for the two methods (P < 0.05), while the complexity value derived from UDPS data was higher than that derived from CBS data (P < 0.001). Study of the prevalences of variations within the RT region showed that CBS detected an average of 9.7 ± 1.1 amino acid substitutions/sample and UDPS detected an average of 16.2 ± 1.4 amino acid substitutions/sample. The phylogenetic analysis based on UDPS data showed more genetic entities than did that based on CBS data. Viral heterogeneity determination by the UDPS technique is more sensitive and efficient in terms of low-abundance variation detection and quasispecies simulation than that by the CBS method, although imperfect, and thus sheds light on the future clinical application of NGS in HBV quasispecies studies.

Reduced beta2-glycoprotein I protects macrophages from ox-LDL-induced foam cell formation and cell apoptosis.

BACKGROUND: Reduced beta2-glycoprotein I (beta2-GPI) is a free thiol-containing form of beta2-GPI that displays a powerful effect in protecting endothelial cells from oxidative stress-induced cell death. The present study aims to investigate the effect of beta2-GPI or reduced beta2-GPI on ox-LDL-induced foam cell formation and on cell apoptosis and to determine the possible mechanisms. METHODS: The RAW264.7 macrophage cell line was selected as the experimental material. Oil red O staining and cholesterol measurement were used to detect cholesterol accumulation qualitatively and quantitatively, respectively. Flow cytometry was used to detect cell apoptosis. Real-time quantitative PCR was used to detect the mRNA expression of the main proteins that are associated with the transport of cholesterol, such as CD36, SRB1, ABCA1 and ABCG1. Western blot analysis was used to detect the protein expression of certain apoptosis-related proteins, such as caspase-9, caspase-3, p38 MAPK/p-p38 MAPK and JNK/p-JNK. RESULTS: Beta2-GPI or reduced beta2-GPI decreased ox-LDL-induced cholesterol accumulation (96.45 ± 8.51 µg/mg protein vs. 114.35 ± 10.38 µg/mg protein, p < 0.05;74.44 ± 5.27 µg/mg protein vs. 114.35 ± 10.38 µg/mg protein, p < 0.01) and cell apoptosis (30.00 ± 5.10% vs. 38.70 ± 7.76%, p < 0.05; 20.66 ± 2.50% vs. 38.70 ± 7.76%, p < 0.01), and there are significant differences between beta2-GPI and reduced beta2-GPI (p < 0.05). Reduced beta2-GPI decreased the ox-LDL-induced expression of CD36 mRNA and ABCA1 mRNA (p < 0.05), as well as CD36, cleaved caspase-9, cleaved caspase-3, p-p38 MAPK and p-JNK proteins (p < 0.05 or p < 0.01). Beta2-GPI did not significantly decrease the expression of ABCA1 mRNA and the p-p38 MAPK protein. CONCLUSIONS: Both beta2-GPI and reduced beta2-GPI inhibit ox-LDL-induced foam cell formation and cell apoptosis, and the latter exhibits a stronger inhibition effect. Both of these glycoproteins reduce the lipid intake of macrophages by downregulating CD36 as well as protein expression. Reduced beta2-GPI inhibits cell apoptosis by reducing the ox-LDL-induced phosphorylation of p38 MAPK and JNK, and the amount of cleaved caspase-3 and caspase-9. Beta2-GPI does not inhibit the ox-LDL-induced phosphorylation of p38 MAPK.

C-reactive protein/oxidised low-density lipoprotein/ß2-glycoprotein I complex promotes atherosclerosis in diabetic BALB/c mice via p38mitogen-activated protein kinase signal pathway.

BACKGROUND: The aim of this study was to investigate the effect of C-reactive protein/oxidised low-density lipoprotein/ß2-glycoprotein I (CRP/oxLDL/ß2GPI) complex on atherosclerosis (AS) in diabetic BALB/c mice. METHODS: BALB/c mice were fed high-fat and normal diet. Eight weeks later, the mice fed with high-fat diet were injected with streptozotocin (STZ) to induce diabetes. The diabetic mice were respectively injected twice monthly with 20 µg oxLDL, 20 µg ß2GPI, 40 µg oxLDL/ß2GPI complex, 44 µg CRP/oxLDL/ß2GPI complex, and PBS. Aortas were stained with Sudan IV to investigate lipid plaque formation. The infiltration condition of smooth muscle cells (SMCs), macrophages, and T cells in the aortas were determined by immunohistochemistry (IH). The mRNA expressions of receptors associated with lipid metabolism were quantified by real-time PCR. The phosphorylation of p38 mitogen-activated protein kinase (p38MAPK) and MKK3/6 in aorta tissues were assessed by Western blot. The expression of inflammation cytokines was evaluated by protein chip. RESULTS: The lipid plaques were more extensive, the lumen area was obviously narrower, the ratio of intima and media thickness were increased, and the normal internal elastic lamia structure and endothelial cell disappeared (P < 0.05) in the oxLDL and CRP/oxLDL/ß2GPI groups (P < 0.05). CRP/oxLDL/ß2GPI complex dramatically promoted infiltration of SMCs, macrophages, and T cells, improved the mRNA expression of ABCA1 and ABCG1, but reduced the mRNA expression of SR-BI and CD36 and increased the phosphorylation of p38MAPK and MKK3/6 (all P < 0.05). The highest expression levels of IL-1, IL-9, PF-4, bFGF, and IGF-II were detected in the CRP/oxLDL/ß2GPI group (P < 0.05). CONCLUSIONS: CRP/oxLDL/ß2GPI complex aggravated AS in diabetic BALB/c mice by increasing lipid uptake, the mechanism of which may be mediated by the p38MAPK signal pathway.

Glycemic control among patients in China with type 2 diabetes mellitus receiving oral drugs or injectables.

BACKGROUND: The prevalence of type 2 diabetes mellitus (T2DM) is increasing rapidly among Chinese adults, and limited data are available on T2DM management and the status of glycemic control in China. We assessed the efficacy of oral antidiabetes drugs (OADs), glucagon-like peptide-1 (GLP-1) receptor agonists, and insulin for treatment of T2DM across multiple regions in China. METHODS: This was a multicenter, cross-sectional survey of outpatients conducted in 606 hospitals across China. Data from all the patients were collected between April and June, 2011. RESULTS: A total of 238,639 patients were included in the survey. Eligible patients were treated with either OADs alone (n=157,212 [65.88%]), OADs plus insulin (n=80,973 [33.93%]), or OADs plus GLP-1 receptor agonists (n=454 [0.19%]). The OAD monotherapy, OAD + insulin, and OAD + GLP-1 receptor agonist groups had mean glycosylated hemoglobin (HbA1c) levels (±SD) of 7.67% (±1.58%), 8.21% (±1.91%), and 7.80% (±1.76%), respectively. Among those three groups, 34.63%, 26.21%, and 36.12% met the goal of HbA1c <7.0%, respectively. Mean HbA1c and achievement of A1c <7.0% was related to the duration of T2DM. CONCLUSIONS: Less than one third of the patients had achieved the goal of HbA1c <7.0%. Glycemic control decreased and insulin use increased with the duration of diabetes.

[Functional analysis of hepatitis B virus immune escape mutants with insertion mutations in the surface antigen].

OBJECTIVE: To evaluate the influence of insertion mutations occurring in the hydrophobic region, between amino acids 114 and 115, of the hepatitis B surface antigen (HBsAg) on viral antigenicity and replication. METHODS: Hepatitis B virus (HBV) DNA was obtained from patients with HBsAg-positive chronic hepatitis B (CHB) infection and subjected to sequence analysis and comparison to GenBank reference sequences for HBV genotype B (AB073826) and genotype C (AF286594). Insertion mutations detected in the HBsAg region were used to make recombinant expression plasmids via site-directed mutagenesis. After transfecting the recombinant HBsAg into Huh7 cells, the mutants' effects on viral antigenicity and replication were evaluated by chemiluminescence microparticle immunoassay (CMIA) and Southern blot hybridization, respectively. The viral antigenicity of each mutant was predicted by bioinformatic analysis, using the Jameson-Wolf method to predict the antigenic index, the Hopp-Woods method to predict hydrophilicity, the Emini method to predict the probability of a region lying of the protein's surface, and the Karplus-Schulz method to predict the flexibility of the protein backbone. RESULTS: Two CHB patients harbored HBV with insertion mutations in HBsAg: one with two (NT) and one with three (NTT) inserted amino acids between 114 and 115. The NTT recombinant HBsAg mutant showed no impact on viral replication and reacted weakly with anti-HBs in CMIA (P = 0.02). The antigen indices for the insertion of NTT were 1.00, -0.16, and 0.18, and insertion of the three amino acids affected the index values of five proximal amino acid sites (with an average increase of 0.13). The hydrophilic indices for the insertion of NTT were 0.2, -0.4, and -0.4, with no significant effect on the proximal amino acids. The insertion of the three amino acids changed both the surface probability (range: -0.55 to 2.97; affecting eight proximal amino acids) and the flexibility (range: -0.01 to 1.1; affecting five proximal amino acids). CONCLUSION: The NTT three amino acids insertion in the HBsAg open reading frame, between 114 and 115 of the wild-type sequence, detected in a CHB patient may play a role in HBV immune escape without influencing the viral replicative capacity.

Cardiac fibrosis and dysfunction in experimental diabetic cardiomyopathy are ameliorated by alpha-lipoic acid.

BACKGROUND: Alpha-lipoic acid (ALA), a naturally occurring compound, exerts powerful protective effects in various cardiovascular disease models. However, its role in protecting against diabetic cardiomyopathy (DCM) has not been elucidated. In this study, we have investigated the effects of ALA on cardiac dysfunction, mitochondrial oxidative stress (MOS), extracellular matrix (ECM) remodeling and interrelated signaling pathways in a diabetic rat model. METHODS: Diabetes was induced in rats by I.V. injection of streptozotocin (STZ) at 45 mg/kg. The animals were randomly divided into 4 groups: normal groups with or without ALA treatment, and diabetes groups with or without ALA treatment. All studies were carried out 11 weeks after induction of diabetes. Cardiac catheterization was performed to evaluate cardiac function. Mitochondrial oxidative biochemical parameters were measured by spectophotometeric assays. Extracellular matrix content (total collagen, type I and III collagen) was assessed by staining with Sirius Red. Gelatinolytic activity of Pro- and active matrix metalloproteinase-2 (MMP-2) levels were analyzed by a zymogram. Cardiac fibroblasts differentiation to myofibroblasts was evaluated by Western blot measuring smooth muscle actin (α-SMA) and transforming growth factor-ß (TGF-ß). Key components of underlying signaling pathways including the phosphorylation of c-Jun N-terminal kinase (JNK), p38 MAPK and ERK were also assayed by Western blot. RESULTS: DCM was successfully induced by the injection of STZ as evidenced by abnormal heart mass and cardiac function, as well as the imbalance of ECM homeostasis. After administration of ALA, left ventricular dysfunction greatly improved; interstitial fibrosis also notably ameliorated indicated by decreased collagen deposition, ECM synthesis as well as enhanced ECM degradation. To further assess the underlying mechanism of improved DCM by ALA, redox status and cardiac remodeling associated signaling pathway components were evaluated. It was shown that redox homeostasis was disturbed and MAPK signaling pathway components activated in STZ-induced DCM animals. While ALA treatment favorably shifted redox homeostasis and suppressed JNK and p38 MAPK activation. CONCLUSIONS: These results, coupled with the excellent safety and tolerability profile of ALA in humans, demonstrate that ALA may have therapeutic potential in the treatment of DCM by attenuating MOS, ECM remodeling and JNK, p38 MAPK activation.

Efficacy and safety comparison between liraglutide as add-on therapy to insulin and insulin dose-increase in Chinese subjects with poorly controlled type 2 diabetes and abdominal obesity.

OBJECTIVE: To assess the efficacy and safety of adding liraglutide to established insulin therapy in poorly controlled Chinese subjects with type 2 diabetes and abdominal obesity compared with increasing insulin dose. METHODS: A 12-week, randomized, parallel-group study was carried out. A total of 84 patients completed the trial who had been randomly assigned to either the liraglutide-added group or the insulin-increasing group while continuing current insulin based treatment. Insulin dose was reduced by 0-30% upon the initiation of liraglutide. Insulin doses were subsequently adjusted to optimized glycemic control. Glycosylated hemoglobin (HbA1c) values, blood glucose, total daily insulin dose, body weight, waist circumference, and the number of hypoglycemic events and adverse events were evaluated. RESULTS: At the end of study, the mean reduction in HbA1c between the liraglutide-added group and the insulin-increasing group was not significantly different (1.9% vs. 1.77%, p>0.05). However, the percentage of subjects reaching the composite endpoint of HbA1c ≤ 7.0% with no weight gain and no hypoglycemia, was significantly higher in the liraglutide-added group than in the insulin-increasing group (67% vs. 19%, p<0.001). Add-on liraglutide treatment significantly reduced mean body weight (5.62 kg, p<0.01), waist circumference (5.70 cm, p<0.01), body mass index (BMI) (1.93 kg/m2, p<0.01) and daily total insulin dose (dropped by 66%) during 12-week treatment period, while all of these significantly increased with insulin increasing treatment. Add-on liraglutide treated patients had lower rate of hypoglycemic events and greater insulin and oral antidiabetic drugs discontinuation. Gastrointestinal disorders were the most common adverse events in the liraglutide added treatment, but were transient. CONCLUSIONS: Addition of liraglutide to abdominally obese, insulin-treated patients led to improvement in glycemic control similar to that achieved by increasing insulin dosage, but with a lower daily dose of insulin and fewer hypoglycemic events. Adding liraglutide to insulin also induced a significant reduction in body weight and waist circumference. Liraglutide combined with insulin may be the best treatment option for poorly controlled type 2 diabetes and abdominal obesity.