Yunbo Chen

Characterization of fecal microbial communities in patients with liver cirrhosis

Liver cirrhosis is the pathologic end stage of chronic liver disease. Increasing evidence suggests that gut flora is implicated in the pathogenesis of liver cirrhosis complications. The aim of this study was to characterize the fecal microbial community in patients with liver cirrhosis in comparison with healthy individuals. We recruited 36 patients with liver cirrhosis and 24 healthy controls. The fecal microbial communities was analyzed by way of 454 pyrosequencing of the 16S ribosomal RNA V3 region followed by real‐time quantitative polymerase chain reaction. Community‐wide changes of fecal microbiota in liver cirrhosis were observed compared with healthy controls. The proportion of phylum Bacteroidetes was significantly reduced (P = 0.008), whereas Proteobacteria and Fusobacteria were highly enriched in the cirrhosis group (P = 0.001 and 0.002, respectively). Enterobacteriaceae (P = 0.001), Veillonellaceae (P = 0.046), and Streptococcaceae (P = 0.001) were prevalent in patients with cirrhosis at the family level. A positive correlation was observed between Child‐Turcotte‐Pugh (CTP) score and Streptococcaceae (R = 0.386, P = 0.02). Lachnospiraceae decreased significantly in patients with cirrhosis (P = 0.004) and correlated negatively with CTP score (R = −0.49, P = 0.002). Using partial least square discriminate analysis, we identified 149 operational taxonomic units (OTUs) as key phylotypes that responded to cirrhosis, most of which were Lachnospiraceae (65 OTUs), Streptococcaceae (23 OTUs), and Veillonellaceae (21 OTUs). Conclusion: Fecal microbial communities are distinct in patients with cirrhosis compared with healthy individuals. The prevalence of potentially pathogenic bacteria, such as Enterobacteriaceae and Streptococcaceae, with the reduction of beneficial populations such as Lachnospiraceae in patients with cirrhosis may affect prognosis. (HEPATOLOGY 2011;)

Intestinal microbiota was assessed in Cirrhotic patients with Hepatitis B Virus infection intestinal microbiota of HBV Cirrhotic patients

To unravel the profile of intestinal microecological parameters in Chinese patients with asymptomatic carriage of hepatitis B virus (HBV), chronic hepatitis B, decompensated HBV cirrhosis, and health controls and to establish their correlation with liver disease progression, we performed quantitative PCR and immunological techniques to investigate fecal parameters, including population of fecal predominant bacteria and the abundance of some virulence genes derived from Escherichia coli, Bacteroides fragilis, Clostridium difficile, and Clostridium perfringens in fecal crude DNA and some immunological parameters in extracts of all fecal samples. Data analysis indicated that 16S rRNA gene copy numbers for Faecalibacterium prausnitzii, Enterococcus faecalis, Enterobacteriaceae, bifidobacteria, and lactic acid bacteria (Lactobacillus, Pediococcus, Leuconostoc, and Weissella) showed marked variation in the intestine of HBV cirrhotic patients. The Bifidobacteria/Enterobacteriaceae (B/E) ratio, which may indicate microbial colonization resistance of the bowel, was decreased significantly in turn from 1.15 ± 0.11 in healthy controls, 0.99 ± 0.09 in asymptomatic carriers, and 0.76 ± 0.08 in patients with chronic hepatitis B to 0.64 ± 0.09 in patients with decompensated HBV cirrhosis (for all, P < 0.01). This suggests that B/E ratio is useful for following the level of intestinal microecological disorder in the course of liver disease progression. The data for virulence gene abundance suggested increased diversity of virulence factors during liver disease progression. Fecal secretory IgA and tumor necrosis factor-α in decompensated HBV cirrhotic patients were present at higher levels than in other groups, which indicates that a complicated autoregulatory system tries to achieve a new intestinal microecological balance.

Changes of Fecal Bifidobacterium Species in Adult Patients with Hepatitis B Virus-Induced Chronic Liver Disease

The beneficial effects of Bifidobacteria on health have been widely accepted. Patients with chronic liver disease have varying degrees of intestinal microflora imbalance with a decrease of total Bifidobacterial counts. Since different properties have been attributed to different Bifidobacterium species and there is no information available for the detailed changes in the genus Bifidobacterium in patients with chronic liver disease heretofore, it is meaningful to investigate the structure of this bacterium at the species level in these patients. The aim of this study was to characterize the composition of intestinal Bifidobacterium in patients with hepatitis B virus-induced chronic liver disease. Nested-PCR-based denaturing gradient gel electrophoresis (PCR-DGGE), clone library, and real-time quantitative PCR were performed on the fecal samples of 16 patients with chronic hepatitis B (CHB patients), 16 patients with hepatitis B virus-related cirrhosis (HBV cirrhotics), and 15 healthy subjects (Controls). Though there was no significant difference in the diversity among the three groups (P = 0.196), Bifidobacterium dentium seems to be specifically enhanced in patients as the PCR-DGGE profiles showed, which was further validated by clone library and real-time quantitative PCR. In contrast to the B. dentium, Bifidobacteriumcatenulatum/Bifidobacterium pseudocatenulatum were detected less frequently in the predominant profile and by quantitative PCR in HBV cirrhotics than in the controls, and the level of this species was also significantly different between these two groups (P = 0.023). Although having no quantitative difference among the three groups, Bifidobacterium longum was less commonly detected in HBV cirrhotics than in CHB patients and Controls by quantitative PCR (P = 0.011). Thus, the composition of intestinal Bifidobacterium was deeply altered in CHB and HBV cirrhotic patients with a shift from beneficial species to opportunistic pathogens. The results provide further insights into the dysbiosis of the intestinal microbiota in patients with hepatitis B virus-induced chronic liver disease and might potentially serve as guidance for the probiotics interventions of these diseases.

Phenotypes and clinical significance of circulating CD4+CD25+ regulatory T cells (Tregs) in patients with acute-on-chronic liver failure (ACLF)

BackgroundCD4+CD25+ regulatory T cells (Tregs) play an important role in maintaining immunological tolerance to self and foreign antigens. T cell receptors (TCR) reflect the composition and function of T cells. It is not universally agreed that there is a relationship between CD4+CD25+ Treg frequency and the severity of acute-on-chronic liver failure (ACLF). The repertoire of TCR beta chain variable (TCRBV) regions of peripheral Tregs in ACLF patients is not well understood.MethodsHuman PBMCs were separated and sorted into CD4+CD25+ Treg subsets using density gradient centrifugation and magnetic activated cell sorting (MACS). The CD4+CD25high Treg frequency in peripheral blood of ACLF and chronic hepatitis B (CHB) patients was measured by flow cytometry. The molecular profiles of TCRBV CDR3 were determined using gene melting spectral pattern (GMSP) analysis. TCRBV gene families were cloned and sequenced when the GMSP profiles showed a single-peak.ResultsCD4+CD25high Treg prevalence in peripheral blood of ACLF patients is increased significantly compared to healthy donors (HDs) (P < 0.01) and CHB patients (P < 0.01). The prevalence of CD4+CD25high Tregs in ACLF or CHB patients is positively correlated with HBV DNA load. The TCRBV11, BV13.1, BV18, BV20 are the most prevalent TCRBV in CD4+CD25+ Tregs in ACLF and CHB patients. In addition, the CDR3 motifs were relatively conserved in these four TCRBV gene families.ConclusionsThe CD4+CD25high Tregs prevalence in peripheral blood is indicative of disease severity in ACLF or CHB patients. The relatively conserved TCRBV20 CDR3 motif “TGTGHSPLH” and TCRBV11 CDR3 motif “VYNEQ” may be used in helping diagnosis and treat patients with ACLF.

Prevalence of carbapenem-hydrolyzing class D β-lactamase genes in Acinetobacter spp. isolates in China

In order to assess the prevalence of carbapenem-hydrolyzing class D β-lactamase genes in Acinetobacter spp. isolates in China, we conducted a polymerase chain reaction (PCR)-based surveillance of OXA-type β-lactamase gene clusters for a total of 2,880 Acinetobacter spp. isolates collected from 23 Chinese provinces. All isolates were tested for susceptibility to 12 antimicrobial agents and showed high rates of resistance to all these agents except minocycline. We also found that the vast majority of carbapenem-resistant Acinetobacter spp. were OXA-23-like-producing isolates, predominantly Acinetobacter baumannii isolates. Besides, blaOXA-58-like and blaOXA-24-like genes were detected in 32 and 11 isolates, respectively, involving many provinces throughout China. Furthermore, these two carbapenem-resistance determinants were located on transferable plasmids in most cases, indicating an emerging threat for both OXA-58-like- and OXA-24-like-producing Acinetobacter spp. isolates in China. Interestingly, a novel homologue of the blaOXA-143 gene was identified in a susceptible Acinetobacter pittii isolate. Overall, these observations suggest that the blaOXA-23-harboring A. baumannii isolates are the most frequent carbapenem-resistant Acinetobacter spp. in China, and the blaOXA-24-like and blaOXA-58-like genes have emerged as potential threats of hospital outbreaks of multidrug-resistant Acinetobacter spp.

Establishment and Characterization of Immortalized Porcine Hepatocytes for the Study of Hepatocyte Xenotransplantation

BACKGROUND In light of the critical shortage of donor livers, xenogeneic sources offer the best alternative to human hepatocytes for the treatment of acute liver failure. This study investigated whether a combination of simian virus 40 large T antigen (SV40 LT) and human telomerase catalytic subunit (hTERT) genes could immortalize primary porcine hepatocytes that could reverse acute liver failure (ALF) in rats. METHODS We cotransfected SV40 LT and hTERT genes into primary porcine hepatocytes to examine the features of the transfected cell lines. We characterized the potentially therapeutic effect of immortalized porcine hepatocytes in a rat model of ALF induced by 90% hepatectomy. RESULTS An immortalized porcine hepatocyte cell line, HepLi, was expanded by >250 passages. HepLi cells maintained the defining characteristics of primary porcine hepatocytes, including porcine albumin secretion, urea production, and diazepam metabolism. Intrasplenic transplantation of HepLi cells significantly improved liver function, and significantly prolonging the survival of rats with ALF. CONCLUSIONS Cotransfection of SV40 LT and hTERT immortalized primary porcine hepatocytes without tumorigenicity in vitro. The Immortalized porcine hepatocytes served as a potential cell resource for xenotransplantation.

Correlation between plasma amino acid profiles and the various stages of hepatitis B infection

The amino acid metabolism in patients with hepatitis B virus (HBV) infection is significantly changed. In this study, we analyzed the relationship between the amino acid profiles and varying clinical stages of HBV infection, and investigated their significance. The plasma amino acid concentrations in 115 patients with HBV infection and 32 healthy donors were detected and analyzed, and the main indicators of liver function were measured. Correlation analysis was performed between the amino acid profiles (Fischer’s ratio, branched-chain amino acid to tyrosine ratio [BTR]) and the key indicators of liver function in patients with HBV infection. Fisher’s ratio and the BTR of patients with HBV infection was found to differ from that of the healthy controls, and was also found to significantly correlate with the stage of HBV infection. Changes in the BTR were closely related to the level of key indicators of liver function, and a significant relationship was detected between the Fischer’s ratio and the BTR (r = 0.928, p < 0.001). These results suggest that Fischer’s ratio and the BTR can indirectly reflect the degree of liver cell injury. Determining and tracking the plasma amino acid profiles could, therefore, be used for the diagnosis, treatment selection, and prognosis of patients with varying stages of HBV infection.

Lactobacillus fermentum ZYL0401 Attenuates Lipopolysaccharide-Induced Hepatic TNF-α Expression and Liver Injury via an IL-10- and PGE2-EP4-Dependent Mechanism

Lipopolysaccharide (LPS) has essential role in the pathogenesis of D-galactosamine-sensitized animal models and alcoholic liver diseases of humans, by stimulating release of pro-inflammatory mediators that cause hepatic damage and intestinal barrier impairment. Oral pretreatment of probiotics has been shown to attenuate LPS-induced hepatic injury, but it is unclear whether the effect is direct or due to improvement in the intestinal barrier. The present study tested the hypothesis that pretreatment with probiotics enables the liver to withstand directly LPS-induced hepatic injury and inflammation. In a mouse model of LPS-induced hepatic injury, the levels of hepatic tumor necrosis factor-alpha (TNF-α) and serum alanine aminotransferase (ALT) of mice with depleted intestinal commensal bacteria were not significantly different from that of the control models. Pre-feeding mice for 10 days with Lactobacillus fermentum ZYL0401 (LF41), significantly alleviated LPS-induced hepatic TNF-α expression and liver damage. After LF41 pretreatment, mice had dramatically more L.fermentum-specific DNA in the ileum, significantly higher levels of ileal cyclooxygenase (COX)-2 and interleukin 10 (IL-10) and hepatic prostaglandin E2 (PGE2). However, hepatic COX-1, COX-2, and IL-10 protein levels were not changed after the pretreatment. There were also higher hepatic IL-10 protein levels after LPS challenge in LF41-pretreaed mice than in the control mice. Attenuation of hepatic TNF-α was mediated via the PGE2/E prostanoid 4 (EP4) pathway, and serum ALT levels were attenuated in an IL-10-dependent manner. A COX-2 blockade abolished the increase in hepatic PGE2 and IL-10 associated with LF41. In LF41-pretreated mice, a blockade of IL-10 caused COX-2-dependent promotion of hepatic PGE2, without affecting hepatic COX-2levels. In LF41-pretreated mice, COX2 prevented enhancing TNF-α expression in both hepatic mononuclear cells and the ileum, and averted TNF-α-mediated increase in intestinal permeability. Together, we demonstrated that LF41 pre-feeding enabled the liver to alleviate LPS-induced hepatic TNF-α expression and injury via a PGE2-EP4- and IL-10-dependent mechanism.

Rapid Large-Scale Culturing of Microencapsulated Hepatocytes: A Promising Approach for Cell-Based Hepatic Support

INTRODUCTION The efficacy of any bioartificial liver device requires both rapid production and proper bioactivity of the cells for the bioreactor. The goal of this study was to observe the effect of spinner speed and cell density on the proliferation of microencapsulated immortalized human hepatocytes (HepLL) and human hepatoma (HepG2) cells. MATERIALS AND METHODS Alginate-chitosan microcapsulated HepG2 and HepLL cells were randomly divided into 2 groups, and each group was further divided into 8 subgroups according to embedded cell density and spinner speed. The growth, metabolism, and functions of the encapsulated cells in each group were evaluated. RESULTS In each group, the cell number, ammonium removal, albumin synthesis, and diazepam clearance increased significantly with the spinner speed, whereas embedded cell density had no impact. Albumin synthesis, removal of ammonium, and diazepam clearance were significantly higher in the microencapsulated HepLL groups than in HepG2 cells at any time point, without any significant difference in cell numbers. CONCLUSIONS Spinner culture significantly promoted microencapsulated HepLL and HepG2 cell bioactivity. Wrapped cells had optimal function on day 10 in rolling culture groups. These data show that HepLL cells would be a promising candidate for cell-based liver support therapy.

Osteopontin expression and relation to streptococcal disease severity in mice

Abstract Osteopontin (OPN) is a phosphorylated glycoprotein that has been implicated in a number of infectious diseases. However, the role of OPN in Streptococcus pyogenes infection is unknown. To investigate whether OPN is involved in S. pyogenes infection, we first examined the plasma OPN levels after local injection of S. pyogenes. OPN expression was significantly increased at 2 h post-infection and increased thereafter. A correlation was found between plasma OPN levels and the development of S. pyogenes infection. The plasma OPN level in severe S. pyogenes infection was higher than during a normal infection. Levels of OPN were found to correlate with the severity of S. pyogenes infection. We also found that OPN production was suppressed by interleukin-6 and enhanced by tumour necrosis factor-alpha in immunocompetent cells. Collectively, these findings demonstrate that the OPN level may provide clues to the severity of S. pyogenes infection in the early phase of the infection.