Qiyi Zhao

Promotion of Hepatic Differentiation of Bone Marrow Mesenchymal Stem Cells on Decellularized Cell-Deposited Extracellular Matrix

Interactions between stem cells and extracellular matrix (ECM) are requisite for inducing lineage-specific differentiation and maintaining biological functions of mesenchymal stem cells by providing a composite set of chemical and structural signals. Here we investigated if cell-deposited ECM mimicked in vivo livers stem cell microenvironment and facilitated hepatogenic maturation. Decellularization process preserved the fibrillar microstructure and a mix of matrix proteins in cell-deposited ECM, such as type I collagen, type III collagen, fibronectin, and laminin that were identical to those found in native liver. Compared with the cells on tissue culture polystyrene (TCPS), bone marrow mesenchymal stem cells (BM-MSCs) cultured on cell-deposited ECM showed a spindle-like shape, a robust proliferative capacity, and a suppressed level of intracellular reactive oxygen species, accompanied with upregulation of two superoxide dismutases. Hepatocyte-like cells differentiated from BM-MSCs on ECM were determined with a more intensive staining of glycogen storage, an elevated level of urea biosynthesis, and higher expressions of hepatocyte-specific genes in contrast to those on TCPS. These results demonstrate that cell-deposited ECM can be an effective method to facilitate hepatic maturation of BM-MSCs and promote stem-cell-based liver regenerative medicine.

Peginterferon-α2a combined with response-guided short-term lamivudine improves response rate in hepatitis B e antigen-positive hepatitis B patients: a pilot study.

Aims The hepatitis B e antigen (HBeAg) seroconversion rate in HBeAg-positive chronic hepatitis B patients treated with peginterferon-&agr;2a (peg-IFN &agr;2a) is still low (about 30%). The aim of this study was to find a new combination therapy of peg-IFN &agr;2a with lamivudine to improve the efficacy in HBeAg-positive chronic hepatitis B patients. Patients and methods All patients started with peg-IFN &agr;2a treatment at a dose of 135 &mgr;g/week. If the concentration of hepatitis B virus (HBV) DNA was greater than 1.0×104 copies/ml and if the patient was positive for HBeAg at 12 weeks of treatment, lamivudine was included into the treatment for 12 weeks. Thereafter, the patients continued on peg-IFN &agr;2a alone for the full 52-week treatment course. Results Thirty-two patients were recruited, and eight of them achieved HBV DNA concentrations of less than 1.0×104 copies/ml or HBeAg loss at 12 weeks of treatment when lamivudine was not administered (group A). The other 24 patients received additional lamivudine, started from 12 weeks of treatment for 12 weeks (group B). At the end of treatment (EOT), in the peg-IFN &agr;2a monotherapy group (group A), eight patients (100%) had HBV DNA loss, six patients (75%) achieved HBeAg seroconversion, and eight patients (100%) achieved alanine aminotransferase (ALT) normalization. This level of response was sustained for 24 weeks after treatment in all patients with an early response. In the peg-IFN &agr;2a combined short-term lamivudine group (group B), 12 patients (50%) had HBV DNA loss, nine patients (38%) achieved HBeAg seroconversion, one patient (4%) achieved hepatitis B surface antigen loss, and 15 patients (63%) achieved ALT normalization at EOT. One patient had an HBV DNA rebound and an HBeAg reversion 24 weeks after treatment. The total HBV DNA loss rate, HBeAg seroconversion rate, hepatitis B surface antigen loss rate, and ALT normalization rate in all patients were 59, 47, 3, and 69%, respectively, at EOT and were 56, 44, 3, and 69% 24 weeks after treatment, respectively. Conclusion This study indicates that the response-guided approach resulted in an overall HBeAg seroconversion rate of 47% at EOT and 44% 24 weeks after treatment. This promising strategy to increase response rates with peg-IFN &agr;2a warrants further investigation.

Tumoral indoleamine 2, 3-dioxygenase 1 is regulated by monocytes and T lymphocytes collaboration in hepatocellular carcinoma

Indoleamine 2, 3-Dioxygenase 1 (IDO1) in cancer cells plays a critical role in tumor immunosuppression. However, the precise mechanisms regulating tumoral IDO1 expression in tumor milieus remain unclear. Here, we reported that IDO1 expression in tumor cells of hepatocelluar carcinomas (HCC), displayed a discrete rather than uniform pattern. In vitro culture, human hepatoma cell lines did not constitutively express IDO1. Interestingly, co-culture with peripheral blood mononuclear cells (PBMC) significantly induced and maintained IDO1 expression in these tumor cells, predominantly through IFN-γ. Mechanistically, we showed that IDO1 expression in tumor cells was only induced when co-cultured with both T lymphocytes and monocytes. Moreover, the cooperation between T lymphocytes and monocytes played an indispensable role on the tumoral IDO1 expression in immunocompromised mice. Taken together, our data supported the notion that IDO1 expression in tumor cells might serve as a counter-regulatory mechanism regulated by immune system, and provided new insights into the collaborative action of different inflammatory cells in tumor immunosuppression.

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.

BTLA identifies dysfunctional PD-1-expressing CD4(+) T cells in human hepatocellular carcinoma.

ABSTRACT Although immunotherapy targeting programmed cell death 1/programmed cell death ligand 1 (PD-1/PD-L1) pathway is being applied in clinic, the response outcomes are heterogeneous, suggesting existences of distinctive subsets within PD-1-expressing T cells that react differently to PD-1/PD-L1 blockade. However, markers to demarcate these subsets in human cancers remain unclear. Here, we found that both PD-1 and B and T lymphocyte attenuator (BTLA) were significantly upregulated on CD4+ T cells from tumor compared with those from paired non-tumor liver in hepatocellular carcinoma (HCC) patients. Interestingly, over 85% BTLA+ CD4+ T cells were PD-1-expressing cells and represented about 50% PD-1+ CD4+ T cells in tumors, and that level of BTLA+PD-1+ tumor CD4+ T cells were selectively associated with advanced stage HCC. BTLA+ identified highly dysfunctional PD-1-expressing CD4+ T cell subset, whereas BTLA− defined PD-1+ CD4+ T cells undergoing activation in HCC. Importantly, blockade of PD-L1 could restore the ability of IFNγ/TNF-α production in BTLA+PD-1+ tumor CD4+ T cells but partially suppressed the activation of BTLA−PD-1+ CD4+ T cells. Moreover, we provided evidence that BTLA signals also participated in suppressing CD4+ T cell function in HCC. In conclusion, BTLA could identify distinct function of PD-1 expressing CD4+ T cells in human cancer, which might not only advance our understanding of inhibitory receptor blockade, but also provide new targets for clinical predictors of response to these immunotherapies.

Intrahepatic NK cells function suppressed in advanced liver fibrosis

Although numerous epidemiological studies indicate that hepatitis B virus‐related liver fibrosis (HBV‐LF), particularly cirrhosis, represents the main risk factor for liver cancer development, the mechanisms determining the persistence of fibrosis and liver cancer pathogenesis are still poorly defined. Few studies have investigated the status of NK cells during different stages of HBV‐LF.

Peritumoral stromal neutrophils are essential for c-Met-elicited metastasis in human hepatocellular carcinoma

ABSTRACT Inflammation is a component of tumor progression mechanisms. Neutrophils are a common inflammatory infiltrate in many tumors, but their regulation and functions in neoplasia are not understood. Here, we showed, in detailed studies of c-Met molecule in 225 untreated patients with hepatocellular carcinoma (HCC), that high infiltration of neutrophils in HCC tissues determined malignant cell c-Met-associated clinical outcome of patients. High infiltration of neutrophils in HCCs determined malignant cell c-Met-associated clinical outcome of patients. Neutrophils were enriched predominantly in invading tumor edge of HCCs; the accumulated neutrophils were the major source of c-Met ligand HGF in HCCs. Exposure to HCC environments resulted in neutrophil activation and the following HGF production. Inhibiting the activities of Erk1/2, p38, and NF-κB, but not the phosphorylation of AKT or JNK, successfully attenuated the neutrophil HGF production induced by HCC environments. Further investigation revealed that GM-CSF was an important determinant in malignant cell-elicited neutrophil HGF production in vitro and in vivo. Moreover, we demonstrated that tumor neutrophils, via HGF/c-Met interaction, actively enhanced the metastasis of malignant cells in vitro and in vivo. These data provide direct evidence supporting the critical role of neutrophils in human tumor progression and reveal a fine-tuned collaborative action between cancer cells and immune cells in tumor milieu, which reroutes the immune activation into a tumor-promoting direction.

An albumin, collagen IV, and longitudinal diameter of spleen scoring system superior to APRI for assessing liver fibrosis in chronic hepatitis B patients

OBJECTIVES The aim of this study was to screen the non-invasive indexes correlated with liver fibrosis and establish a scoring system for the diagnosis of liver fibrosis in hepatitis B patients. METHODS Data of 34 non-invasive indexes were collected for 208 hepatitis B patients. Correlation analysis and stepwise discriminant analysis was used to screen out indexes useful for the diagnosis of liver fibrosis. Finally, a scoring system composed of indexes screened out by stepwise discriminant analysis was established for the assessment of liver fibrosis. RESULTS Twenty-one indexes correlating with liver fibrosis were screened out by correlation analysis; hyaluronic acid had the highest r-value, 0.456. A scoring system including albumin, collagen IV, and the longitudinal diameter of the spleen was established. The areas under the receiver operating characteristic curves (AUC) for this scoring system and the aspartate aminotransferase to platelet ratio index (APRI) in differentiating S3-4 from S0-2 were 0.79 (95% confidence interval (CI) 0.72-0.85) and 0.27 (95% CI 0.18-0.35), respectively. With a cut-off value of <3, the presence of significant fibrosis (S3-4) could be excluded by this scoring system with a negative predictive value of 86.1% and sensitivity of 86.8%. With a cut-off of >6, the presence of S3-4 fibrosis could be correctly identified with a positive predictive value of 73.6% and specificity of 87.6%. Using this scoring system, 53.4% of patients could be classified correctly and avoid liver biopsy. CONCLUSIONS The scoring system provides a simpler method to identify significant fibrosis (S3-4) in chronic hepatitis B patients.

Liver myofibroblasts from hepatitis B related liver failure patients may regulate natural killer cell function via PGE2

BackgroundNatural killer (NK) cells are abundant in the liver and constitute a major innate immune component that contributes to immune-mediated liver injury. However, few studies have investigated the phenotypes and functions of NK cells involved in hepatitis B related liver failure (LF), and the precise mechanism underlying NK cell regulation is not fully understood.MethodsWe detected the percentage and function of peripheral NK cells both in hepatitis B related LF patients and healthy volunteers by flow cytometry and isolated the liver myofibroblasts (LMFs) from hepatitis B related LF livers. To determine the possible effects of LMFs on NK cells, mixed cell cultures were established in vitro.ResultsWe found a down-regulated percentage of peripheral NK cells in hepatitis B related LF patients, and their NK cells also displayed decreased activated natural cytotoxicity receptors (NCRs) and cytokine production. In a co-culture model, LMFs sharply attenuated IL-2-induced NK cell triggering receptors, cytotoxicity, and cytokine production. The inhibitory effect of LMFs on NK cells correlated with their ability to produce prostaglandin (PG) E2.ConclusionThese data suggest that LMFs may protect against immune-mediated liver injury in hepatitis B related LF patients by inhibiting NK cell function via PGE2.

The paradoxical changes of membrane and soluble herpes virus entry mediator in hepatocellular carcinoma patients

The herpes virus entry mediator (HVEM) network has become new directions in targeting novel checkpoint inhibitors for cancer therapy. However, the changes of membrane‐bound HVEM (mHVEM) and soluble HVEM (sHVEM) in hepatocellular carcinoma (HCC) are not fully understood. This study aims to study the changes of mHVEM and sHVEM in HCC patients.