Peimisine ameliorates DSS-induced colitis by suppressing Jak-Stat activation and alleviating gut microbiota dysbiosis in mice.

OBJECTIVES: Inflammatory cytokine secretion and gut microbiota dysbiosis play crucial roles in ulcerative colitis. In this research, the protective effects of peimisine on colitis mice were investigated. METHODS: The protective effects were evaluated by the disease activity index, colonic length, hematoxylin-eosin, and AB/PAS Staining. The protective mechanisms were analyzed by ELISA, Western-blot, immunohistochemistry staining, immunofluorescence staining, and 16S rRNA gene analysis. KEY FINDINGS: The results showed that peimisine treatment could reduce the disease activity index, prevent colonic shortening, and alleviate colon tissue damage. Peimisine treatment also decreased the levels of MCP-1, IL-1ß, IL-6, IFN-γ, TNF-α and affected macrophage polarization and Th17/Treg cell balance by downregulating the expression of jak1/2, p-jak1/2, stat1/3, and p-stat1/3. Moreover, peimisine treatment significantly increased the abundances of beneficial microbes (e.g. Ruminococcaceae UCG-014 and Lachnospiraceae_NK4A136_group) and decreased the abundances of harmful microbes (e.g. Bacteroides and Escherichia). CONCLUSIONS: Peimisine can ameliorate colitis by inhibiting Jak-Stat signaling pathway, reversing gut microbiota alterations, suppressing macrophage M1 polarization, maintaining the Th17/Treg cell balance, and reducing sustained inflammatory cytokines-related inflammatory injury.

Inhibitory Effects of Esculetin on Liver Cancer Through Triggering NCOA4 Pathway-Mediation Ferritinophagy in vivo and in vitro.

Objective: To explore the effects of Esculetin on liver cancer and explore potential mechanisms of Esculetin-inducing cells death. Methods: Esculetin's effects on the proliferation, migration and apoptosis of HUH7 and HCCLM3 cells were detected by using CCK8, crystal violet staining, wound healing, TranswellTM and Annexin V-FITC/PI. Flow cytometry, fluorescence staining, Western blot, T-AOC, DPPH radical scavenging assay, hydroxyl radical's inhibitory capability and GSH test were used to examine the esculetin's effects on the ROS level, the oxidation-related substances and proteins' expression in hepatoma cells. In vivo experiment was performed by xenograft model. Ferrostatin-1 was used to determine the death way of hepatoma cells induced by esculetin. Live cell probe, Western blot, Fe2+ content, MDA, HE staining, Prussian blue staining and immunohistochemistry were used to examine the ferritinophagy-related phenomenon induced by esculetin in hepatoma cells. The relationship between esculetin and NCOA4-mediated ferritinophagy was confirmed through gene silence and overexpression, immunofluorescence staining and Western blot. Results: Esculetin suppressed the proliferation, migration and apoptosis of HUH7 and HCCLM3 cells significantly, influenced the oxidative stress level, altered the autophagy and iron metabolism levels in cells, and produced a ferritinophagy-related phenomena. Esculetin increased the levels of cellular lipid peroxidation and reactive oxygen species. In vivo, esculetin could decrease tumour volume, promote LC3 and NCOA4 expressions, suppresse hydroxyl radical's inhibiting capacity and GSH, increase Fe2+ and MDA levels, decrease antioxidant proteins expression in tumour tissue. In addition, Esculetin could also increase the iron deposition of tumour tissues, promote ferritinophagy, and induce tumours' ferroptosis. Conclusion: Esculetin has an inhibitory effect on liver cancer in vivo and in vitro through triggering NCOA4 pathway-mediation ferritinophagy.

Apoptotic and autophagic cell death induced in cervical cancer cells by a dual specific oncolytic adenovirus.

OBJECTIVE: Oncolytic adenoviruses are capable of exerting anticancer effects via a variety of mechanisms, including apoptosis and autophagy. In the present study, the dual-specific antitumor oncolytic adenovirus, Ad-Apoptin-hTERT-E1a (ATV), was used to infect cervical cancer cell lines to test its antitumor effects. METHODS: To explore the use of apoptin in tumor gene therapy, a recombinant adenovirus ATV expressing the apoptin protein was assessed to determine its lethal and growth-inhibitory effects on human cervical cancer cell line (HeLa) cells in vitro . Nonapoptotic autophagy of HeLa cells infected with ATV was assessed by examining the cell morphology, development of acidic vesicular organelles and the conversion of microtubule-associated protein 1 light chain 3 (LC3) from its cytoplasmic to autophagosomal membrane form. Using gene silencing (knockdown of LC3 and Belin-1), autophagy-associated molecules (e.g. ATG5, ATG12 and ULK1) were monitored by real-time PCR and western blot. RESULTS: A series of experiments demonstrated that ATV could significantly induce apoptosis and autophagy in cervical cancer cells, and provided evidence that ATV not only induced apoptosis but also autophagy and ATG5, ATG12 and ULK1 related pathways were not entirely dependent on LC3 and Beclin-1. CONCLUSION: These results indicate that ATV may have a potential application in tumor gene therapy.

Apoptin causes apoptosis in HepG-2 cells via Ca2+ imbalance and activation of the mitochondrial apoptotic pathway.

BACKGROUND: Apoptin is derived from the chicken anemia virus and exhibits specific cytotoxic effects against tumor cells. Herein, we found that Apoptin induced a strong and lasting endoplasmic reticulum (ER) stress response, Ca2+ imbalance, and triggered the mitochondrial apoptotic pathway. The aim of this study was to explore the mechanisms by which Apoptin exhibited anti-tumor effects in HepG-2 cells. METHODS: The intracellular levels of calcium (Ca2+ ) were induced by ER stress and determined by electron microscopy, flow cytometry, and fluorescence staining. The mitochondrial injury was determined by mitochondrial membrane potential and electron microscopy. Western blotting was used to investigate the levels of key proteins in ER stress and the apoptotic pathway in mitochondria. The relationship between Ca2+ levels and apoptosis in Apoptin-treated cells was analyzed using a Ca2+ chelator (BAPTA-AM), flow cytometry, and fluorescence staining. We also investigated the in vivo effects of Ca2+ imbalance on the mitochondrial apoptotic pathway using tumor tissues xenografted on nude mice. RESULTS: This study showed that Apoptin induced a strong and long- lasting ER stress and injury, which subsequently led to an imbalance of cellular Ca2+ levels, a reduction in the mitochondrial membrane potential, a significant extent image in the mitochondrial structure, and an increase in the expression levels of Smac/Diablo and Cyto-C. CONCLUSIONS: In summary, Apoptin induced apoptosis in HepG-2 cells via Ca2+ imbalance and activation of the mitochondrial apoptotic pathway. This study provided a new direction for antitumor research in Apoptin.

Effects of Copper(II) Oxide on the Co-Pyrolysis of Waste Polyester Enameled Wires and Poly(vinyl chloride).

The emission of chlorinated pollutants is one of the main problems when recovering copper (Cu) via pyrolysis from waste enameled wires. This is mainly attributed to other wastes which possess high poly(vinyl chloride) content, such as electrical wires and cables, which are often recycled together with enameled copper wires. In this research, to control the chlorinated pollutants, copper(II) oxide (CuO) was chosen and demonstrated to be an efficient dechlorinating agent, and CuO did not introduce any impurities that influence the quality of the recovered Cu. The pyrolysis and co-pyrolysis of polyester enameled wires, PVC, and CuO were investigated, and special attention was paid to chlorinated compounds in released pyrolytic products. In particular, the co-pyrolysis of this ternary mixture was studied for the first time, and some new pyrolysis behaviors were discovered. For example, the results of Py-GC/MS analyses showed that the addition of CuO removed about 75% of the chloro-organic products, the main types of which were chloroaromatic compounds rather than the more toxic chloroesters. Moreover, pyrolysis gases were collected and characterized via ion chromatography, and the results showed that the chlorine content in the pyrolysis gases decreased by about 71%. TG analysis indicated that CuO only minimally affected the pyrolysis of polyester paint. However, through the chlorine fixation effect, CuO influenced the dechlorination and dehydrochlorination of PVC, as well as secondary reactions between HCl and pyrolysis products of polyester paint, therefore changing the products and behaviors of co-pyrolysis. Mechanism of reducing chlorine-containing pollutants and reaction mechanism of forming typical pyrolysis products closely correlated to the effects of CuO were also proposed, providing theoretical guidance for the recycling of waste enameled wires.

A comparative study of the ability of recombinant oncolytic adenovirus, doxorubicin and tamoxifen to inhibit the proliferation of breast cancer cells.

In this study, we compared the inhibitory effects of recombinant oncolytic adenovirus (Ad-apoptin-hTERTp-E1a, Ad-VT) with that of doxorubicin (DOX), a first-line chemotherapy drug, and tamoxifen (TAM), an endocrine therapy drug, on the proliferation of breast cancer cells. We found that Ad-VT could effectively inhibit the proliferation of breast cancer cells (p < 0.01); the inhibition rate of Ad-VT on normal mammary epithelial MCF-10A cells was less than 20%. DOX can effectively inhibit the proliferation of breast cancer cells and also has a strong inhibitory effect on MCF-10A cells (p < 0.01). TAM also has a strong inhibitory effect on breast cancer cells, among which the oestrogen-dependent MCF-7 cell inhibition was stronger (p < 0.01), At higher concentrations, TAM also had a high rate of inhibition (>70%) on the proliferation of MCF-10A cells. We also found that both recombinant adenovirus and both drugs could successfully induce tumour cell apoptosis. Further Western blot results showed that the recombinant adenovirus killed breast cancer cells through the endogenous apoptotic pathway. Analysis of the nude mouse subcutaneous breast cancer model showed that Ad-VT significantly inhibited tumour growth (the luminescence rate of cancer cells was reduced by more than 90%) and improved the survival rate of tumour-bearing mice (p < 0.01). Compared with DOX and TAM, Ad-VT has a significant inhibitory effect on breast cancer cells, but almost no inhibitory effect on normal breast epithelial cells, and this inhibitory effect is mainly through the endogenous apoptotic pathway. These results indicate that Ad-VT has significant potential as a drug for the treatment of breast cancer.

Mucosal IgA response elicited by intranasal immunization of Lactobacillus plantarum expressing surface-displayed RBD protein of SARS-CoV-2.

Coronavirus Disease 2019 (COVID-19) caused by a novel betacoronavirus SARS-CoV-2 has been an ongoing global pandemic. Several vaccines have been developed to control the COVID-19, but the potential effectiveness of the mucosal vaccine remains to be documented. In this study, we constructed a recombinant L. plantarum LP18:RBD expressing the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein via the surface anchoring route. The amount of the RBD protein was maximally expressed under the culture condition with 200 ng/mL of inducer at 33 °C for 6 h. Further, we evaluated the immune response in mice via the intranasal administration of LP18:RBD. The results showed that the LP18:RBD significantly elicited RBD-specific mucosal IgA antibodies in respiratory tract and intestinal tract. The percentages of CD3 + CD4+ T cells in spleens of mice administrated with the LP18:RBD were also significantly increased. This indicated that LP18:RBD could induce a humoral immune response at the mucosa, and it could be used as a mucosal vaccine candidate against the SARS-CoV-2 infection. We provided the first experimental evidence that the recombinant L. plantarum LP18:RBD could initiate immune response in vivo, which implies that the mucosal immunization using recombinant LAB system could be a promising vaccination strategy to prevent the COVID-19 pandemic.

Imbalance Between Soluble and Membrane-Bound CD100 Regulates Monocytes Activity in Hepatitis B Virus-Associated Acute-on-Chronic Liver Failure.

CD100 is an important immune semaphorin that is a secreted and membrane bound protein involved in infectious diseases. However, CD100 expression profile and the regulation to innate immune system in hepatitis B virus (HBV)-associated acute-on-chronic liver failure (ACLF) was not previously reported. The aim of this study was to investigate CD100 level and modulatory function of CD100 to CD14+ monocytes in HBV-ACLF patients. Plasma-soluble CD100 (sCD100) level and membrane-bound CD100 (mCD100) expression on peripheral CD14+ monocytes was analyzed in HBV-ACLF patients. CD14+ monocytes-induced cytotoxicity and CD14+ monocytes-mediated T cell activation in response to CD100 stimulation was also assessed in direct and indirect contact coculture culture systems. HBV-ACLF patients had lower plasma sCD100 and higher mCD100 level on CD14+ monocytes compared with asymptomatic HBV carriers, chronic hepatitis B patients, and controls. CD14+ monocytes from HBV-ACLF patients induced limited target Huh7.5 cell death and secreted less interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and granzyme B in both direct and indirect contact coculture systems compared with controls. Recombinant sCD100 not only enhanced CD14+ monocytes-mediated Huh7.5 cell death and granzyme B secretion, but it also elevated CD14+ monocytes-induced IFN-γ/interleukin-17 production by CD4+ T cells as well as IFN-γ/TNF-α secretion by CD8+ T cells in HBV-ACLF patients. The current data indicated that severe inflammation induced sCD100/mCD100 imbalance to inactivate CD14+ monocytes response, which might be beneficial for the survival of HBV-ACLF patients.

Effects of Apoptin-Induced Endoplasmic Reticulum Stress on Lipid Metabolism, Migration, and Invasion of HepG-2 Cells.

In this study, we investigated the effects of Apoptin-induced endoplasmic reticulum (ER) stress on lipid metabolism, migration and invasion of HepG-2 cells, and preliminarily explored the relationship between endoplasmic reticulum stress, lipid metabolism, migration, and invasion. The effects of Apoptin on ER function and structure in HepG-2 cells were determined by flow cytometry, fluorescence staining and western blotting by assessing the expression levels of ER stress related proteins. The effects of Apoptin on HepG-2 cells' lipid metabolism were determined by western blot analysis of the expression levels of triglyceride, cholesterol, and lipid metabolism related enzymes. The effects of Apoptin on HepG-2 cells' migration and invasion were studied using migration and invasion assays and by Western-blot analysis of the expression of proteins involved in migration and invasion. The in vivo effects of endoplasmic reticulum stress on lipid metabolism, migration and invasion of HepG-2 cells were also investigated by immunohistochemistry analysis of tumor tissues from HepG2 cells xenografted nude mice models. Both in vitro and in vivo experiments showed that Apoptin can cause a strong and lasting ER stress response, damage ER functional structure, significantly change the expression levels of lipid metabolism related enzymes and reduce the migration and invasion abilities of HepG-2 cells. Apoptin can also affect HepG-2 cells' lipid metabolism through endoplasmic reticulum stress and the abnormal expression of enzymes closely related to tumor migration and invasion. These results also showed that lipid metabolism may be one of the main inducements that reduce HepG-2 cells' migration and invasion abilities.

Circ_0035483 Functions as a Tumor Promoter in Renal Cell Carcinoma via the miR-31-5p-Mediated HMGA1 Upregulation.

BACKGROUND: Renal cell carcinoma (RCC) that originates from the proximal renal tubules is the most common cancer of the human kidney. Increasing circRNA/miRNA/mRNA networks have been found in RCC regulation. This study will explore the regulatory relation of circular RNA (circRNA) circ_0035483, microRNA-31-5p (miR-31-5p) and high mobility group A1 (HMGA1). METHODS: The levels of circ_0035483, miR-31-5p and HMGA1 were measured by real-time polymerase chain reaction (qRT-PCR) or Western blot. Cell proliferation was determined using Cell Counting Kit-8 (CCK-8) and colony formation assays. Cell migration and invasion were assessed by transwell assay. HMGA1 and epithelial-mesenchymal transition (EMT)-related protein levels were quantified using Western blot. Glycolytic metabolism was evaluated by glucose consumption and lactate production. The interaction between targets was confirmed via dual-luciferase reporter, RNA immunoprecipitation (RIP) and RNA pull-down assays. In vivo experiment was performed through the establishment of xenograft models in mice. RESULTS: Circ_0035483 expression was upregulated in RCC tissues and cells. The inhibitory effects on RCC cell proliferation, migration, invasion, EMT and glycolysis were induced after circ_0035483 was downregulated. MiR-31-5p was identified as a target of circ_0035483 and miR-31-5p upregulation was related to the function of circ_0035483 knockdown in RCC cells. Additionally, miR-31-5p targeted HMGA1 and inhibited the malignant behaviors of RCC cells by negatively regulating HMGA1. Moreover, HMGA1 expression was regulated by circ_0035483 via targeting miR-31-5p. Circ_0035483 also affected tumor growth in vivo by relying on the miR-31-5p/HMGA1 axis. CONCLUSION: These findings clarified that the tumor-promoting function of circ_0035483 in RCC was partly achieved by regulating the miR-31-5p/HMGA1 axis.

Transcription Factor MafB Suppresses Type I Interferon Production by CD14+ Monocytes in Patients With Chronic Hepatitis C.

Transcription factor MafB regulates differentiation and activity of monocytes/macrophage and is associated with the development of atherosclerosis and cancers. However, the role of MafB in modulation of CD14+ monocytes in chronic viral hepatitis was not fully elucidated. Thus, the aim of current study was to investigate the immunoregulatory function of MafB to type I interferon (IFN) secretion by CD14+ monocytes and its contribution to pathogenesis of chronic hepatitis C virus (HCV) infection. A total of 29 chronic hepatitis C patients and 21 healthy individuals were enrolled. Serum IFN-α1 and IFN-ß was measured by ELISA, while MafB mRNA and protein expression were assessed by real-time PCR and Western blot. MafB siRNA or MafB expression plasmid was transfected into purified CD14+ monocytes to suppress or increase MafB expression. The function of MafB siRNA transfected CD14+ monocytes to HCV in cell culture (HCVcc)-infected Huh7.5 cells or CD4+ T cells was also investigated in direct and indirect contact co-culture system. Serum IFN-α1 and IFN-ß was robustly reduced in chronic hepatitis C patients. By contrast, MafB was notably elevated in chronic hepatitis C patients and negatively correlated with serum IFN-α1. Overexpression of MafB reduced the IFN-α1 production by CD14+ monocytes from healthy individuals. However, MafB inhibition elevated IFN-α1 secretion by CD14+ monocytes and interferon regulatory factor 3 phosphorylation in chronic hepatitis C. MafB inhibition also promoted CD14+ monocytes-induced viral clearance in HCVcc-infected Huh7.5 cells by up-regulation of IFN-α1 and IFN-ß without increasingly destroying hepatocytes, however, did not affect CD14+ monocytes-induced CD4+ T cells differentiation in chronic hepatitis C patients. The current data revealed that overexpression of MafB in chronic hepatitis C patients might suppress type I IFN production by CD14+ monocytes, leading to the viral persistence. MafB might be a potential therapeutic target for treatment of chronic hepatitis C.

Notch signaling suppresses CD14+ monocytes cells activity in patients with chronic hepatitis C.

Hepatitis C virus (HCV) infection always leads to chronic hepatitis via dysregulation of host immunity. Notch signaling also modulates the response of monocytes/macrophages. Thus, we aimed to investigate the regulatory role of Notch signaling to CD14+ monocytes. Forty patients with chronic hepatitis C and twenty normal controls (NC) were enrolled. CD14+ monocytes and CD4+ T cells were purified from peripheral bloods. Notch receptors' mRNA expression in CD14+ monocytes was semi-quantified by real-time PCR. Cytokine production by CD14+ monocytes in response to γ-secretase inhibitor (GSI) was investigated by ELISA. GSI-induced CD14+ monocytes activity to HCV clearance in Huh7.5 cells and to CD4+ T cell differentiation was also assessed in direct and indirect contact co-culture system. Notch1 mRNA relative level was approximately 10-fold elevated in CD14+ monocytes from chronic hepatitis C patients when compared with NC. GSI stimulation resulted in enhanced cytokines production by CD14+ monocytes from chronic hepatitis C patients. GSI-stimulated CD14+ monocytes from chronic hepatitis C patients induced suppression of HCV RNA replication in both direct and indirect contact co-culture system of CD14+ monocytes and HCVcc-infected Huh7.5 cells, and this process was accompanied by elevation of interferon-γ production but not increased target cell death. Moreover, GSI stimulation also enhanced CD14+ monocytes-induced Th1 and Th17 cells activation, and this process required direct cell-to-cell contact. Effective antiviral therapy down-regulated Notch1 mRNA expression and promoted cytokine production by CD14+ monocytes from chronic hepatitis C. Current data revealed an important immunoregulatory property of Notch signaling to CD14+ monocytes in chronic HCV infection.

Decreased CD4+CD25+CD127dim/- Regulatory T Cells and T Helper 17 Cell Responsiveness to Toll-Like Receptor 2 in Chronic Hepatitis C Patients with Daclatasvir Plus Asunaprevir Therapy.

Direct-acting antivirals (DAAs) not only rapidly inhibited hepatitis C virus (HCV) replication but also modulated innate and adaptive immune response in chronic hepatitis C patients. However, the regulatory activity of DAAs to Toll-like receptor 2 (TLR2) stimulation on CD4+CD25+CD127dim/- regulatory T cells (Tregs) and T helper (Th) 17 cells was not completely understood. In the present study, a total of 23 patients with chronic HCV genotype 1b infection were enrolled, and blood samples were collected at baseline (treatment naive), end of therapy (EOT), and 12 weeks after EOT (SVR12) with daclatasvir plus asunaprevir therapy. TLR2 expression on Tregs and Th17 cells was measured by flow cytometry. Cellular proliferation, cytokine production, and suppressive activity were also tested in purified CD4+CD25+CD127dim/- Tregs in response to the stimulation of Pam3Csk4, an agonist of TLR2. Inhibition of HCV RNA by daclatasvir and asunaprevir did not affect either percentage of Tregs/Th17 cells or TLR2 expression on Tregs/Th17 cells. Pam3Csk4 stimulation also did not influence either cellular proliferation or Tregs/Th17 proportion at each time point. Stimulation with Pam3Csk4 only enhanced the suppressive function and interleukin (IL)-35 production by Tregs purified from baseline, but not those from EOT or SVR12. Similarly, Pam3Csk4 stimulation only elevated Th17 cell frequency of CD4+ T cells from baseline, but not those from EOT or SVR12. Moreover, daclatasvir and asunaprevir therapy did not promote TLR2-induced shift of Tregs toward Th17-like phenotype and function. These data suggested that daclatasvir plus asunaprevir therapy resulted in the decreased responsiveness of Tregs/Th17 cells to TLR2 stimulation in chronic hepatitis C patients, which might provide a novel mechanism underlying DAA-induced immunoregulation.

Interleukin-7 Regulates T Follicular Helper Cell Function in Patients with Chronic Hepatitis C.

Signaling through interleukin (IL)-7 is essential and required for development, differentiation, proliferation, and homeostasis of T cells. However, the role of IL-7 in regulation of CD4+ T cells in chronic viral infections was not fully elucidated. Thus, the aim of the current study was to investigate the immunomodulatory activity of IL-7 to T follicular helper (Tfh) cells and its contribution to pathogenesis of chronic HCV, hepatitis C virus (HCV) infection. A total of 47 patients with chronic hepatitis C and 19 normal controls were enrolled. Serum IL-7 and proportion of Tfh cells was measured. The regulatory function of IL-7 to Tfh cells was also investigated in CD4+ T cells and CD4+ T/HCVcc-infected Huh7.5 cell cocultured system. Serum IL-7 concentration was significantly downregulated in patients with chronic hepatitis C, and was negatively correlated with HCV RNA level. Tfh frequency and Tfh-associated cytokines (IL-21 and IL-6) were also reduced in chronic HCV-infected patients. Moreover, recombinant IL-7 stimulation elevated proportion of Tfh cells and IL-21/IL-6 secretion in both HCV-specific and nonspecific manners. Furthermore, IL-7-treated CD4+ T cells exhibited elevated antiviral activities without killing infected hepatocytes, which presented as inhibition of HCV RNA, induction of antiviral proteins, and promotion of cytokine production (especially IL-21) in cocultured system. This process might be dependent on IL-6 secretion. The current data revealed that IL-7 regulated HCV-specific and nonspecific activated Tfh cells, which might contribute to viral clearance. IL-7 could be a potential therapeutic agent for the treatment of chronic hepatitis C.

Mode measurement of few-mode fibers by mode-frequency mapping.

Since few-mode fibers (FMFs) have great potential as the new transmission media for optical communications, the ability to distinguish different fiber modes is essential. Most of the traditional schemes do not yield phase information, or are limited by beam size and mechanical requirements. Here, a method is presented to analyze the mode distribution of FMFs. The fiber modes are mapped to different frequencies by using dynamic spatial phase masks. The complex amplitudes at these frequencies indicate the amplitudes and phases of the fiber modes. The method can extract not only the amplitude distribution, but also the phase distribution of the fiber modes, and no other assisted light is needed.

Notch Signaling Regulates Circulating T Helper 22 Cells in Patients with Chronic Hepatitis C.

Notch signaling enhanced the response of interleukin (IL)-22-producing CD4+ T cells that were defined as T helper 22 (Th22) cells, and Notch-aryl hydrocarbon receptor (AhR)-IL-22 axis fine-tuned inflammatory response. Previous studies have demonstrated that both Notch signaling and Th22 cells took part in the pathogenesis of chronic hepatitis C virus (HCV) infection. Thus, in this study, we aimed at examining the regulatory role of Notch signaling in Th22 cells in HCV infection. A total of 59 patients with chronic hepatitis C and 22 normal controls (NCs) were enrolled in this study. The percentage of Th22 cells and mRNA expression of related transcriptional factors and cytokines were analyzed in response to γ-secretase inhibitor. Th22 cell frequency was significantly elevated in chronic hepatitis C in comparison with that in NCs. Inhibition of Notch signaling downregulated HCV-specific Th22 cells and IL-22 production, which was accompanied by the reduction of AhR and modulatory cytokines (IL-6 and tumor necrosis factor-α). Moreover, the suppression of Notch signaling also decreased the IL-22-mediated antimicrobial response in both normal and HCV-infected HepG2 cells/Huh7.5 cells. This process was also accompanied by the depression of signal transducers and activators of transcription 3 signaling. In conclusion, the current results suggested that Notch signaling acted as a critical pathway in determining the response to IL-22 in chronic hepatitis C. Thus, Notch-Th22 axis might be considered a new therapeutic target for HCV-infected patients.

Toll-Like Receptor 2 Modulates the Balance of Regulatory T Cells and T Helper 17 Cells in Chronic Hepatitis C.

Regulatory T cells (Tregs) and interleukin-17-producing T helper (Th17) cells were mutually antagonistic in the pathogenesis of chronic hepatitis C virus (HCV) infection. However, the regulation of imbalance between Tregs and Th17 cells was poorly understood in HCV infection. A recent report revealed the immunomodulatory role of Toll-like receptor (TLR) 2 in regulating the balance of Tregs/Th17 functions in multiple sclerosis. Thus, the aim of the current study was to assess the effect of TLR2 stimulation on the suppressive function of Tregs and Th17 differentiation in chronic hepatitis C. A total of 65 patients with chronic hepatitis C receiving pegylated interferon-a2a and ribavirin therapy for 48 weeks, as well as 20 of normal controls (NCs) were enrolled. Cellular proliferation and cytokine production was tested in purified CD4(+)CD25(+)CD127(dim/-) Tregs in response to the stimulation of Pam3Csk4, an agonist of TLR2. In treatment-naive patients, Tregs, but not Th17 cells, from chronic hepatitis C patients expressed higher levels of TLR2 compared with NCs. Stimulation with Pam3Csk4 enhanced the suppressive function of Tregs and production of IL-10 in chronic hepatitis C more than in NCs. However, TLR2 stimulation did not promote Th17 differentiation of Tregs in chronic hepatitis C patients. Moreover, effective anti-HCV therapy resulted in the induction of IL-17-secreting phenotypic shift of Tregs without loss of inhibitive function upon TLR2 stimulation. These data provided a novel mechanism underlying modulating the balance of Tregs/Th17 cells in chronic hepatitis C. HCV infection shifted Tregs/Th17 cells through TLR2 stimulation by inducing Tregs to produce IL-10 and enhancing inhibitive function of effector T cells, resulting in viral persistence.

Expression profile and kinetics of cytokines and chemokines in patients with chronic hepatitis C.

Cytokines and chemokines play an important role in defense against viral infection and modulating immune response. However, expression prolife of serum cytokines and chemokines, which were associated with the outcome of patients in response to anti-HCV treatment have not been fully elucidated. The current study aimed to determine the expression pattern of cytokines and chemokines in chronic HCV infection and their association with outcome in response to therapy. Seventy-two patients with HCV infection were enrolled, and fifty-one received peg-interferon α-2a and ribavirin therapy for 48 weeks. Thirty-nine cytokines and chemokines were analyzed by Luminex 200 and ELISA. In comparison to healthy individuals, production of IL-8 and IL-10 were increased in chronic hepatitis C patients. In contrast, IFN-γ, IL-7, and IL-15 were remarkably decreased, especially in HCV genotype 1b infection. HCV RNA load is closely associated with IL-10 and IL-15 expressions, and inhibition of HCV replication was accompanied by reduction in IL-10 and elevation in IL-7 and IL-15. Skewed cytokines and chemokines expression existed in chronic HCV infection, and might play an important role in persistent HCV infection. Exploiting the expression pattern of cytokines and chemokines may help to develop a better understanding of the pathogenesis of chronic HCV infection.

Therapeutic efficacy of an hTERT promoter-driven oncolytic adenovirus that expresses apoptin in gastric carcinoma.

The efficacy and specificity of treatment are the major challenges for cancer gene therapy. Oncolytic virotherapy is an attractive drug delivery platform of cancer gene therapy. Previous studies have determined that apoptin is a p53-independent, Bcl-2-insensitive apoptotic protein that has the ability to induce apoptosis specifically in tumor cells. In this study, we show that the administration of a dual cancer-specific oncolytic adenovirus construct, Ad-hTERT-E1a-apoptin [in which the adenovirus early region 1a (E1a) gene is driven by the cancer-specific promoter of human telomerase reverse transcriptase (hTERT) and that expresses apoptin simultaneously], suppresses tumor growth in gastric carcinoma cells in vitro and reduces the tumor burden in vivo in xenografted nude mice. The observation that infection with the Ad-hTERT-E1a-apoptin construct significantly inhibited the growth of gastric cancer cells and protected normal human gastric epithelium from growth inhibition confirmed the induction of cancer cell-selective adenovirus replication, growth inhibition and apoptosis by this therapeutic approach. In vivo assays were performed using BALB/c nude mice that had established primary tumors. Subcutaneous primary tumor volume was reduced not only in the intratumoral injection group but also in the systemic delivery mice following treatment with Ad-hTERT-E1a-apoptin. Furthermore, treatment of primary models with Ad-hTERT-E1a-apoptin increased the mouse survival time. These data reinforce previous research and highlight the potential therapeutic application of Ad-hTERT-E1a-apoptin for the treatment of neoplastic diseases in clinical trials.

[Establishment of a fluorescent real-time quantitative RT-PCR assay for detection of genotype 4 hepatitis E virus in swine stools].

The primers and probes for the Real-time RT-PCR were designed based on the multiple sequence (swine and humans HEV strains) alignments of the ORF3 region of genotype 4 HEV. A rapid, sensitive and stable TaqMan Real-time RT-PCR assay was established, and its specificity and sensitivity were assessed, and comparison of the Real-time RT-PCR with conventional and nested RT-PCR was performed. The results found that the crossing points showed linearly proportional to the logarithm of the input copy number. The correlation coefficient (R2) and the slope value of the standard curves with plasmid DNA were 0.994 and -3.312, respectively. The efficiency (E) of the PCR was 100%. Coefficients of variation values of the different diluted plasmid DNA were low in the same or different repeated experimental group. In addition, the assay was able to correctly detect genotype 4 HEV RNA from swine fecal samples. The sensitivity of established assay was 100-fold higher than that of conventional RT-PCR and 10-fold higher than nested RT-PCR.