Yurong Gu

Intestinal flora imbalance results in altered bacterial translocation and liver function in rats with experimental cirrhosis.

Background/purpose The intestinal microflora plays a major role in human health. Intestinal flora imbalances are seen in clinical settings, such as cirrhosis, in which bacterial translocation (BT) results in migration of bacteria or bacterial products from the intestinal lumen to extraintestinal sites. In this study, we explored the effect of alterations in gut flora on BT and liver function in cirrhotic rats. Methods In this study, we used a carbon tetrachloride-induced cirrhotic rat model to compare the abundance of major aerobic and anaerobic bacterial species in healthy and cirrhotic rats. We used antibiotic (norfloxacin) and different probiotic treatments to change the status of gut flora in the cirrhotic rats and evaluated BT, liver function, and endotoxemia in the different models. Results We found higher levels of Enterobacteriaceae in cirrhotic rats when compared with healthy rats. Bifidobacteria treatment resulted in lower levels of Enterobacteriaceae along with increased levels of Lactobacillus when compared with the normal saline group. Both Bifidobacteria and Enterococcus treatments resulted in lower endotoxin levels than in the normal saline group. Conclusion Gut flora imbalances in cirrhotic rats result in significant changes in BT and liver function in cirrhotic rats.

Amniotic-fluid-derived mesenchymal stem cells overexpressing interleukin-1 receptor antagonist improve fulminant hepatic failure.

Uncontrolled hepatic immunoactivation is regarded as the primary pathological mechanism of fulminant hepatic failure (FHF). The major acute-phase mediators associated with FHF, including IL-1β, IL-6, and TNF-α, impair the regeneration of liver cells and stem cell grafts. Amniotic-fluid–derived mesenchymal stem cells (AF-MSCs) have the capacity, under specific conditions, to differentiate into hepatocytes. Interleukin-1–receptor antagonist (IL-1Ra) plays an anti-inflammatory and anti-apoptotic role in acute and chronic inflammation, and has been used in many experimental and clinical applications. In the present study, we implanted IL-1Ra–expressing AF-MSCs into injured liver via the portal vein, using D-galactosamine–induced FHF in a rat model. IL-1Ra expression, hepatic injury, liver regeneration, cytokines (IL-1β, IL-6), and animal survival were assessed after cell transplantation. Our results showed that AF-MSCs over-expressing IL-1Ra prevented liver failure and reduced mortality in rats with FHF. These animals also exhibited improved liver function and increased survival rates after injection with these cells. Using green fluorescent protein as a marker, we demonstrated that the engrafted cells and their progeny were incorporated into injured livers and produced albumin. This study suggests that AF-MSCs genetically modified to over-express IL-1Ra can be implanted into the injured liver to provide a novel therapeutic approach to the treatment of FHF.

Dynamic Changes of Lipopolysaccharide Levels in Different Phases of Acute on Chronic Hepatitis B Liver Failure

Background High serum levels of lipopolysaccharide (LPS) with LPS-MD-2/TLR4 complex activated NF-kb and cytokine cause hepatic necrosis in animal models. We investigated the dynamic changes of LPS levels in patients with acute on chronic hepatitis B liver failure (ACHBLF). Methods We enrolled ACHBLF patients for a 12-week study. Patients’ LPS levels were measured along with 10 healthy controls. Patients on supportive care and recovered without intervention(s) were analyzed. Patients’ LPS levels during the disease progression phase, peak phase, and remission phase were compared with healthy controls. Results Among 30 patients enrolled, 25 who received interventions or expired during the study period were excluded from the analysis, five patients on supportive care who completed the study were analyzed. Significant abnormal distributions of LPS levels were observed in patients in different phases (0.0168±0.0101 in progression phase; 0.0960±0.0680 in peak phase; 0.0249±0.0365 in remission phase; and 0.0201±0.0146 in controls; respectively, p<0.05). The highest level of LPS was in the peak phase and significantly elevated when compared to controls (0.0201±0.0146 vs. 0.0960±0.0680, p = 0.007). There were no statistically significant differences in LPS levels between healthy controls and subjects in the progression phase or remission phase. Dynamic changes of LPS were correlated with MELD-Na in the progression phase (p = 0.01, R = 0.876) and in the peak phase (p = 0.000, R = −1.00). Conclusions Significant abnormal distributions of LPS levels were observed in ACHBLF with the highest level in the peak phase. The dynamic changes of LPS were correlated with disease severity and suggested LPS causing secondary hepatic injury.

Liver myofibroblasts up-regulate monocyte CD163 expression via PGE2 during hepatitis B induced liver failure

BackgroundAlthough patients with liver failure exhibit a generalized inflammatory-imbalance status, substantial evidence indicates that this immunosuppressive or anti-inflammatory state may be deleterious. Increased expression of CD163 (known to be involved in several anti-inflammatory functions of the immune system) in patients with liver failure is significantly correlated with a fatal outcome. However, little is known of the regulatory mechanisms that influence the expression of CD163.MethodsWe assessed the expression of CD163 on monocytes from both circulating cells and the liver tissues of patients with hepatitis B induced liver failure using flow cytometry and isolated the myofibroblasts from diseased livers. The ability of human liver myofibroblasts to regulate CD163 expression on monocytes was studied in vitro.ResultsWe showed that CD163+ monocytes were enriched primarily in diseased livers and that they were associated with liver myofibroblasts in the same area. Accordingly, liver myofibroblasts were significantly superior to normal skin fibroblasts in inducing the expression of CD163 on monocytes in vitro. Moreover, we found that liver myofibroblasts triggered the activation of monocytes by secreting PGE2. Inhibition of PGE2 production in liver myofibroblasts using NS-398 markedly reduced CD163 expression in vitro.ConclusionThese results suggest that liver myofibroblasts play a direct role in regulating the expression of CD163 on monocytes in human liver tissues and thereby may regulate monocyte function during hepatitis B induced liver failure.

HBV core promoter mutations and AKT upregulate S-phase kinase-associated protein 2 to promote postoperative hepatocellular carcinoma progression

Mutations in the hepatitis B virus (HBV) core promoter (CP) have been shown to be associated with hepatocellular carcinoma (HCC). The CP region overlaps HBV X gene, which activates AKT to regulate hepatocyte survival. However, the cooperation between these two cascades in HCC progression remains poorly understood. Here, we assayed virological factors and AKT expression in liver tissues from 56 HCC patients with better prognoses (BHCC, ≥5-year survival) and 58 with poor prognoses (PHCC, <5-year survival) after partial liver resection. Results showed double mutation A1762T/G1764A (TA) combined with other mutation(s) (TACO) in HBV genome and phosphorylated AKT (pAKT) were more common in PHCC than BHCC. TACO and pAKT levels correlated with proliferation and microvascularization but inversely correlated with apoptosis in HCC samples. These were more pronounced when TACO and pAKT co-expressed. Levels of p21 and p27 were decreased in TACO or pAKT overexpressing HCC due to SKP2 upregulation. Levels of E2F1 and both mRNA and protein of SKP2 were increased in TACO expressing HCC. Levels of 4EBP1/2 decreased and SKP2 mRNA level remained constant in pAKT-overexpressing HCC. Therefore, TACO and AKT are two independent predictors of postoperative survival in HCC. Their co-target, SKP2 may be a diagnostic or therapeutic marker.

Veritable antiviral capacity of natural killer cells in chronic HBV infection: an argument for an earlier anti-virus treatment

BackgroundThere is limited information on innate immunity, especially natural killer (NK) cell function, in different chronic hepatitis B (CHB) stages. Therefore, we examined whether the clinical staging strategy accurately reflects veritable NK cell immunity.MethodsA total of 237 eligible CHB patients and 22 healthy controls were enrolled in our study. Demographic and clinical data were collected, and the CHB phases (immune active-IA, immune tolerant phase-IT, inactive CHB-IC, and grey zone-GZ) were classified according to the latest American Association for the Study of Liver Disease guidelines. Peripheral blood mononuclear cells from patients and healthy controls were tested for NK cell frequency, phenotype and function using flow cytometry.ResultsA significant decrease in activating receptor NKp44 and NKp46 expression and significant increase of exhaustion molecule Tim-3 expression were observed in NK cells from CHB patients. Reduced cytokine secretion and preserved or elevated cytotoxic function were also observed. Patients in the IT group exhibited comparable cytokine secretion and cytolytic capacity as age-matched IA patients. NK cell anti-viral functions were preserved in GZ patients. Some of the NK cell function in patients who were excluded from treatment by the current treatment guidelines was less compromised than patients who qualified for treatment.ConclusionOur findings provide evidence of veritable NK cell immunity during different natural history phases in treatment-naïve patients with chronic HBV Infection. Chronic HBV infection hindered NK cell function in CHB patients. However, the presumed IT and GZ statuses of CHB patients based on the clinical parameters may not accurately reflect the inner immune status of these patients and should be reconsidered. Some patients excluded from treatment by the current treatment guidelines may be able to be selected as candidates for treatment.

Diverse correlations between fibrosis-related factors and liver stiffness measurement by transient elastography in chronic hepatitis B

Background Several fibrosis-related factors influence liver stiffness measurements (LSM); however, these changes have not been investigated in the context of the various disease stages of chronic hepatitis B (CHB). Aim The aim of this study was to assess the correlations between fibrosis-related factors and LSM in different disease stages of CHB. Patients and methods Patients with mild CHB (n=305) and cirrhotic hepatitis B (cirrhotic HB) (n=137) were compared with determine the relationship between LSM and fibrosis-related factors including parameters of liver inflammation [aminotransferase (ALT), aspartate transaminase (AST), total bilirubin (Tbil)], albumin, globulin, peripheral blood cells (neutrophil granulocytes, red blood cells, platelets), abdominal ultrasound B-scan parameters including right liver thickness, portal vein inradium, diameter of spleen (DS), thickness of spleen (TS), and splenic vein inradium (SV). Results In patients with mild CHB, LSM was correlated strongly with ALT (r=0.3534, P<0.0001), AST (r=0.3976, P<0.0001), and ALT+AST (r=0.3760, P<0.0001). LSM was correlated closely with Tbil (r=0.2237, P<0.0001), albumin (r=−0.3126, P<0.0001), albumin/globulin (r=−0.3086, P<0.0001), SV (r=0.3317, P<0.0001), DS (r=0.4157, P<0.0001), and spleen volume (DS×TS) (r=−0.4399, P<0.0001). Red blood cells were correlated negatively with LSM in both mild CHB and cirrhotic HB patients (r=−0.1981, P=0.0203; r=−0.1593, P=0.0053). LSM was not correlated with age, peripheral blood cell parameters, right liver thickness, portal vein inradium, or TS in mild CHB or cirrhosis HB patients. However, in patients with cirrhotic HB, LSM values were not correlated significantly with other fibrosis-related factors, except for Tbil (r=0.2272, P=0.0076). Conclusion Our findings suggest that the magnitude of these correlations differs significantly between mild CHB and cirrhotic HB patients.

A Practical Model Evaluating Antiviral Cytokines by Natural Killer Cells in Treatment Naïve Patients with Chronic Hepatitis B Virus Infection

Natural killer (NK) cells play a major role in anti-viral immunity as first line defense during hepatitis B infection, particularly in untreated patients whose T cells functions are profoundly impaired. Cytokine interferon (IFN)-γ and tumor necrosis factor (TNF)-α produced by NK cells are important anti-viral factors. However, there is lack of a quantifiable model to evaluate cytokine responses by NK cells. In this study, almost half of the patients (47.9%) beyond treatment criteria had high cytokine activity, although it was lower than those recommended for antiviral therapy (78.2%). Moreover, we developed a model that low levels of HBsAg, HBcAb, and albumin and high fibrosis values predicted strong antiviral cytokine production by NK cells. Based on the cut-off score (0.361) obtained from the multivariable model, patients with 67%, 8%, 92%, and 74% in immune-active (IA), immune-tolerant (IT), immune-inactive (IC), and grey zone (GZ), respectively, showed active antiviral cytokines produced by NK cells. These results suggest that those who possess activated cytokine responses beyond the current treatment criteria may have potential implications for the timing of antiviral therapy to achieve better virus control.

Synergistic effect of farnesyl transferase inhibitor lonafarnib combined with chemotherapeutic agents against the growth of hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC) is a common and deadly cancer worldwide and is often refractory to chemotherapy due to the development of multidrug resistance. Lonafarnib is an orally active and potent non-peptidomimetic inhibitor of farnesyl transferase. Here, using in vitro HCC cell models, we demonstrated that lonafarnib inhibited tumor proliferation and reduced the activity of mitogen-activated protein kinases pathways. In addition, lonafarnib caused G1 to S phase arrest through the downregulation of Cyclin D1, CDK6 and SKP2, while it induced cellular apoptosis by promoting the cleavage and activation of Caspase-3 and PARP. When combined with doxorubicin and sorafenib, lonafarnib was able to increase the sensitivity of HCC cells to chemotherapy. Furthermore, we also constructed ABCB1-overexpressing HCC cells and found that lonafarnib decreased chemoresistance by inhibiting ABCB1-mediated drug efflux activity. These results suggest that lonafarnib may be a promising synergistic agent for improving the treatment of drug-resistant HCC.