Ping-Zhong Wang

Overexpression of Toll-like receptor 2/4 on monocytes modulates the activities of CD4(+)CD25(+) regulatory T cells in chronic hepatitis B virus infection.

The significance of TLR expression and Tregs in HBV infection has not been clearly described. In this report, flow cytometry was performed to assess TLR2/4 expression on monocytes and circulating CD4(+)CD25(+)CD127(low/-) Tregs frequency of 16 acute hepatitis B (AHB), 42 chronic hepatitis B (CHB), 22 asymptomatic HBV carriers (AsC), and 20 normal controls (NC). We found that TLR2 and TLR4 were overexpressed on CD14(+) monocytes in HBV-infected patients as compared with NCs. Upregulation of TLR2 in NCs and TLR4 in CHBs was observed following HBeAg incubation. However, TLR2 and TLR4 expression decreased after HBcAg stimulation. The difference in the proportion of Tregs between NCs and CHBs was significant. Both Pam3Csk4 (TLR2 agonist)- and lipopolysaccharide (TLR4 agonist)-activated CD4(+)CD25(+) Tregs showed enhanced suppression function in CHBs. These results suggest that overexpression of TLR2 and TLR4 may modulate the suppressive function of Tregs, which contribute to the immunotolerance of chronic HBV infection.

Circulating Toll-like receptor (TLR) 2, TLR4, and regulatory T cells in patients with chronic hepatitis C.

Wang J‐P, Zhang Y, Wei X, Li J, Nan X‐P, Yu H‐T, Li Y, Wang P‐Z, Bai X‐F. Circulating Toll‐like receptor (TLR) 2, TLR4, and regulatory T cells in patients with chronic hepatitis C. APMIS 2010; 118: 261–70.

Hantaan virus induces toll-like receptor 4 expression, leading to enhanced production of beta interferon, interleukin-6 and tumor necrosis factor-alpha

Hantaan virus (HTNV) infects endothelial cells and is associated with increased vascular permeability during hemorrhagic fever with renal syndrome (HFRS). The pattern of increased vascular permeability is mediated by immune response. Therefore, it is necessary to characterize the mechanism of HTNV involvement in the hosts innate immune. In this study, the expression of five toll-like receptors (TLRs) was analyzed in Endothelial vein cells (EVC-304) following HTNV infection in vitro. TLR4 showed an altered expression after HTNV infection. HTNV infection significantly increased IFN-beta, IL-6 and TNF-alpha secretion from EVC-304 cells, particularly after lipopolysaccharide stimulation. The increased IFN-beta, IL-6 and TNF-alpha production was mediated by TLR4 induction, since the introduction of the small interfering RNA against TLR4 specifically inhibited the HTNV-induced cytokine production. In conclusion, HTNV infection directly induces TLR4 expression and thereby enhanced production of IFN-beta, IL-6 and TNF-alpha, which may contribute to the hosts innate immune response.

Inhibition of connective tissue growth factor suppresses hepatic stellate cell activation in vitro and prevents liver fibrosis in vivo

Activation of hepatic stellate cells (HSC) represents a critical event in fibrosis, and connective tissue growth factor (CTGF) plays a profibrotic activity and a key factor in the pathogenesis of tissue fibrosis. The current study aimed to determine whether lentivirus-mediated short hairpin RNA (shRNA)–targeted CTGF downregulates the CTGF expression and furthermore whether it suppresses the activation and proliferation of HSC in vitro and prevents liver fibrosis in vivo. HSC-T6 cells were treated with recombinant lentivirus carrying CTGF siRNA. Real-time PCR, Western blotting, MTT, and flow cytometry were performed to investigate the activation and proliferation of HSC-T6 cells in response to CTGF silence. CCl4-induced rats were received lentivirus containing CTGF siRNA by intraportal vein injection. Levels of liver fibrosis were assessed by biochemical and histopathologic examinations. Recombinant lentivirus containing CTGF siRNA could effectively and specifically downregulate the expression of CTGF in both HSC-T6 cells and CCl4-induced rats with liver fibrosis. Blockade of CTGF resulted in significant inhibition of HSC activation and proliferation with decrease in TIMPs, MMP2, MMP9, and collagen I, as well as increase in cells in S phase. Silencing CTGF expression with siRNA prevented liver fibrosis in CCl4-induced rat model. These findings indicated that CTGF plays a key role in the pathogenesis of liver fibrosis and lentiviral-mediated CTGF siRNA has the potential to be an effective treatment for liver fibrosis.

Elevated vascular endothelial growth factor levels induce hyperpermeability of endothelial cells in hantavirus infection.

Objective: To investigate the role of vascular endothelial growth factor (VEGF) in haemorrhagic fever with renal syndrome (HFRS). Methods: VEGF, soluble VEGF receptor (sVEGFR)-2, angiopoietin (Ang)-1, tumour necrosis factor (TNF)-α and interferon (IFN)-γ levels were measured in serum samples from 68 patients with HFRS. Cultured human umbilical vein endothelial cells (HUEVCs) were infected by Hantaan virus (HTNV) and/or stimulated with recombinant VEGF; dextran permeability of the cells was determined. Claudin-1 and vascular endothelial (VE)-cadherin levels were determined by real-time reverse transcription-polymerase chain reaction and Western blot analyses. Results: Serum VEGF, TNF-α and IFN-γ levels were significantly elevated, whereas sVEGFR2 and Ang-1 levels were reduced, during the acute phase of HFRS. In vitro cell permeability was unaffected by HTNV infection or VEGF stimulation alone, but the combination of HTNV infection and VEGF treatment significantly increased the permeability of endothelial cell monolayers in a time-dependent manner. Claudin-1 and VE-cadherin were downregulated at both the mRNA and protein level by combined HTNV infection and VEGF stimulation. Conclusions: Elevated VEGF induced by HTNV infection may play an important role in the vascular hyperpermeability that is characteristic of HFRS.

Imbalance of regulatory T cells and T helper type 17 cells in patients with chronic hepatitis C.

Pegylated interferon and ribavirin combination therapy is known to be effective in suppressing viral replication in 50–60% of hepatitis C virus (HCV) ‐infected patients. However, HCV‐infected patients often exhibit varied responses to therapy. Therefore, the identification of immunological markers associated with the clinical outcomes of antiviral treatment is critical for improvement of therapeutic options. In this study, we aimed to investigate the ratio of CD4+ CD25+ FoxP3+ regulatory T (Treg) cells to interleukin‐17A (IL‐17A) ‐producing T helper type 17 (Th17) cells, and its association with clinical outcomes in response to anti‐HCV treatment. In all, 114 patients with HCV infection received pegylated interferon‐α2a and ribavirin therapy for 48 weeks, and the frequency of Treg cells and Th17 cells as well as the levels of secreted cytokines were longitudinally analysed by flow cytometry and ELISA. Treg cell proportions and IL‐10 production were significantly elevated in HCV‐infected patients, especially for HCV genotype 1b. However, the frequency of Th17 cells as well as the secretion of IL‐17, IL‐22 and IL‐23 did not reveal notable difference between HCV infections and healthy individuals. Inhibition of HCV replication was accompanied by a reduction in Treg cells, but little influence on Th17 cells, which led to a significant decrease in Treg : Th17 ratios. Skewed Treg : Th17 ratios existed in chronic hepatitis C. HCV RNA load is closely associated with Treg : Th17 ratios during pegylated interferon‐α2a and ribavirin treatment in HCV‐infected patients. The imbalance of Treg cells to Th17 cells might play an important role in persistent HCV infection.

Calcineurin promotes proliferation, migration, and invasion of small cell lung cancer.

A novel role for calcineurin (Cn) has been reported recently regarding the oncogenic potential in pancreatic and colorectal cancer. The aim of this study was to investigate the putative causal role calcineurin could play in the development of lung cancer with bone metastases. We found that CnAα, an isoform of calcineurin, was significantly overexpressed in lung cancer tissues with bone metastasis as compared to tumors with non-bone metastases as investigated by RT-PCR. Strong nuclear staining of tumor cells was observed in small cell lung cancer tissues with bone metastasis. Conversely, cytoplasmic staining of tumor cells was observed in small cell lung cancer tissues with non-bone metastasis. Western blots of nuclear proteins from lung cancer tissues indicated that CnAα was highly expressed in lung cancer tissues with bone metastases, but not in those with non-bone metastases. In vitro, it was demonstrated that the CnAα gene obviously promoted cell proliferation and inhibited cell apotosis. The CnAα gene affected the cell cycle and promoted G1➝S transition in SBC-3 cells. Transfection with the CnAα gene promoted cell migration and invasion. These results indicated that CnAα may affect the biological behavior of the human small cell lung cancer cell line SBC-3 in vitro and may be a candidate tumor promotor gene for developing bone metastases.

In Vitro Hepatic Differentiation of Mesenchymal Stem Cells from Human Fetal Bone Marrow

We examined whether human fetal mesenchymal stem cells (FMSCs) derived from fetal bone marrow were able to differentiate into functional hepatocyte-like cells in vitro The surface phenotype of FMSCs was characterized by flow cytometry. To induce hepatic differentiation of FMSCs, we added hepatocyte growth factor, basic fibroblast growth factor and oncostatin M into the cell culture medium. After 21 days of hepatocyte induction, FMSCs expressed the hepatocyte-specific markers, α-fetoprotein and cytokeratin 18, as demonstrated by immunofluorescence staining. Differentiated FMSCs also demonstrated in vitro functions characteristic of liver cells, including albumin production, urea secretion and glycogen storage. In conclusion, fetal bone marrow-derived FMSCs are able to differentiate into functional hepatocyte-like cells and may serve as a source of cells for liver disease therapy.

Expression of L protein of Hantaan virus 84FLi strain and its application for recovery of minigenomes

Hantaan virus (HTNV) is an Old World hantavirus associated with hemorrhagic fever with renal syndrome (HFRS). To visualize the localization of the L protein of HTNV strain 84FLi within cells, a fusion protein composed of enhanced green fluorescent protein and L protein, EGFP‐L, was expressed in Vero cells. The 273 KDa expressed fusion protein of EGFP‐L localized in the perinuclear region. We also described the development of a reverse genetics system for HTNV strain 84FLi. The RNA polymerase I (pol I)‐mediated transcription system was used to generate artificial viral RNA genome segments (minigenomes), which contained the chloramphenicol acetyltransferase (CAT) reporter gene in antisense (virus RNA) or sense (virus‐complementary RNA) orientation flanked by the noncoding regions of HTNV 84FLi L segment. CAT could be detected in cells after transfection, indicating the successful encapsidation, transcription and replication of the pol I‐derived minigenomes. The passaged transfer of CAT demonstrates that recombinant virus containing packaged pol I‐derived minigenomes has been produced. This system may be helpful in studying the gene function and pathogenesis of HTNV.

Hantaan Virus Triggers TLR4-Dependent Innate Immune Responses

The innate immune response induced by Hantavirus is responsible for endothelial cell dysfunction and viral pathogenicity. Recent studies demonstrate that TLR4 expression is upregulated and mediates the secretion of several cytokines in Hantaan virus (HTNV)-infected endothelial cells. To examine viral interactions with host endothelial cells and characterize the innate antiviral responses associated with Toll-like receptors, we selected TLR4 as the target molecule to investigate anti-hantavirus immunity. TLR4 mRNA-silenced EVC-304 (EVC-304 TLR4-) cells and EVC-304 cells were used to investigate signaling molecules downstream of TLR4. The expression of the adaptor protein TRIF was higher in HTNV-infected EVC-304 cells than in EVC-304 TLR4- cells. However, there was no apparent difference in the expression of MyD88 in either cell line. The transcription factors for NF-κB and IRF-3 were translocated from the cytoplasm into the nucleus in HTNV-infected EVC-304 cells, but not in HTNV-infected EVC-304 TLR4- cells. Our results demonstrate that TLR4 may play an important role in the antiviral immunity of the host against HTNV infection through an MyD88-independent signaling pathway.