Infected HepG2 Cells Research Articles

Construction and Identification of the RNAi Recombinant Lentiviral Vector Targeting Human DEPDC7 Gene.

Human DEP domain containing 7 (DEPDC7) gene was originally found expressing high in liver tissue and low in most other tissues, but its function was largely unknown. In this study, we construct an RNA interference (RNAi) recombinant lentiviral vector particle targeting DEPDC7 in order to knockdown its gene expression in human hepatocellular carcinoma cell line HepG2. We screened three RNAi sequences targeting DEPDC7 and a scramble sequence by the aid of short hairpin RNAs (shRNA) design tools. Then, these sequences were separately cloned into the pLV-H1-EF1α-puro vector to construct four lentiviral vectors (pshRNA-DEPDC7-NC, pshRNA-DEPDC7-RNAi1, pshRNA-DEPDC7-RNAi2 and pshRNA-DEPDC7-RNAi3). All of the recombinant plasmids were identified and confirmed by double digestion and DNA sequencing. After infecting HepG2 cells, the DEPDC7 mRNA and protein expression levels were examined by real-time PCR and western blot, respectively, and the gene expression was significantly down-regulated at both levels (P<0.01). Cell motility and invasiveness were detected by Matrigel migration and invasion assay, and the results revealed that migration and invasion of HepG2 cells were significantly increased (P<0.05). Our study showed successful construction of three lentiviral RNAi vectors targeting DEPDC7 gene and shRNA-mediated knockdown of DEPDC7 enable promotion of cell migration and invasion.

Tiger frog virus ORF080L protein interacts with LITAF and impairs EGF-induced EGFR degradation

Tiger frog virus (TFV) belongs to the genus Ranavirus, family Iridoviridae, and causes severe mortality in commercial cultures in China. TFV ORF080L is a gene homolog of lipopolysaccharide-induced TNF-α factor (LITAF), which is a regulator in endosome-to-lysosome trafficking through its function in the endosomal sorting complex required for transport machinery. The characteristics and biological roles of TFV ORF080L were identified. TFV ORF080L was predicted to encode an 84-amino acid peptide (VP080L). It had high-sequence identity with mammalian LITAF, but lacked the N-terminus of LITAF, which contains two PPXY motifs. Transcription and protein level analyses showed that TFV ORF080L was a late viral gene. Localization in the virons also showed that TFV VP080L was a viral structural protein. Immunofluorescence staining showed that TFV ORF080L was predominantly colocalized with plasma membrane and partly distributed with the late endosome in infected HepG2 cells. SiRNA-mediated TFV ORF080L silencing decreased viral reproduction. Moreover, TFV ORF080L interacted with human/zebrafish LITAF and impaired EGF-induced EGFR degradation, thereby indicating that TFV ORF080L played a role in endosome-to-lysosome trafficking. These findings suggested that TFV ORF080L might negate the function of cellular LITAF to impair endosomal sorting and trafficking. Results provide a clue to the link between the dysregulated endosomal trafficking and iridovirus pathogenesis.

Inactivation of Dengue and Yellow Fever viruses by heme, cobalt-protoporphyrin IX and tin-protoporphyrin IX.

To investigate the effect of heme, cobalt-protoporphyrin IX and tin-protoporphyrin IX (CoPPIX and SnPPIX), macrocyclic structures composed by a tetrapyrrole ring with a central metallic ion, on Dengue Virus (DENV) and Yellow Fever Virus (YFV) infection. Treatment of HepG2 cells with heme, CoPPIX and SnPPIX after DENV infection reduced infectious particles without affecting viral RNA contents in infected cells. The reduction of viral load occurs only with the direct contact of DENV with porphyrins, suggesting a direct effect on viral particles. Previously incubation of DENV and YFV with heme, CoPPIX and SnPPIX resulted in viral particles inactivation in a dose-dependent manner. Biliverdin, a noncyclical porphyrin, was unable to inactivate the viruses tested. Infection of HepG2 cells with porphyrin-pretreated DENV2 results in a reduced or abolished viral protein synthesis, RNA replication and cell death. Treatment of HepG2 or THP-1 cell lineage with heme or CoPPIX after DENV infection with a very low MOI resulted in a decreased DENV replication and protection from death. Heme, CoPPIX and SnPPIX possess a marked ability to inactivate DENV and YFV, impairing its ability to infect and induce cytopathic effects on target cells. These results open the possibility of therapeutic application of porphyrins or their use as models to design new antiviral drugs against DENV and YFV.

Tumor control by human cytomegalovirus in a murine model of hepatocellular carcinoma

Although viruses can cause cancer, other studies reported the regression of human tumors upon viral infections. We investigated the cytoreductive potential of human cytomegalovirus (HCMV) in a murine model of human hepatocellular carcinoma (HCC) in severe-immunodeficient mice. Infection of HepG2 cells with HCMV resulted in the absence of tumor or in a limited tumor growth following injection of cells subcutaneously. By contrast all mice injected with uninfected HepG2 cells and with HepG2 cells infected with UV-treated HCMV did develop tumors without any significant restriction. Analysis of tumors indicated that in mice injected with HCMV-infected-HepG2 cells, but not in controls, a restricted cellular proliferation was observed parallel to a limited activation of the STAT3-cyclin D1 axis, decreased formation of colonies in soft agar, and activation of the intrinsic apoptotic pathway. We conclude that HCMV can provide antitumoral effects in a murine model of HCC which requires replicative virus at some stages that results in limitation of tumor cell proliferation and enhanced apoptosis mediated through the intrinsic caspase pathway.

Hepatitis c virus genotype 4 replication in the hepatocellular carcinoma cell line HepG2/C3A.

Background/Aims:The lack of a reliable cell culture system allowing persistent in vitro hepatitis C virus (HCV) propagation is still restraining the search for novel antiviral strategies. HepG2 cells transfection with HCV allows for viral replication. However, the replication is weak presumably because of HepG2 lack of miRNA-122, which is essential for viral replication. Other agents such as polyethylene glycol (PEG) and dimethyl sulfoxide (DMSO) have been shown to increase the efficiency of infection with other viruses. This study included comparison of HCV genotype 4 5′UTR and core RNA levels and HCV core protein expression at different time intervals in the absence or presence of PEG and/or DMSO postinfection.Materials and Methods:We used serum with native HCV particles in infecting HepG2 cells in vitro. HCV replication was assessed by reverse transcriptase polymerase chain reaction for detection of HCV RNA and immunofluorescence and flow cytometry for detection of HCV core protein.Results:HCV 5′UTR and core RNA expression was evident at different time intervals after viral infection, especially after cells were treated with PEG. HCV core protein was also evident at different time intervals using both immunofluorescence and flow cytometry. PEG, not DMSO, has increased the HCV core protein expression in the treated cells, similar to its effect on viral RNA expression.Conclusions:These expression profiles suggest that the current model of cultured HepG2 cells allows the study of HCV genotype 4 replication and different stages of the viral life cycle.

Interleukin-22-Induced Antimicrobial Phospholipase A2 Group IIA Mediates Protective Innate Immunity of Nonhematopoietic Cells against Listeria monocytogenes.

Listeria monocytogenes is a bacterial pathogen which establishes intracellular parasitism in various cells, including macrophages and nonhematopoietic cells, such as hepatocytes. It has been reported that several proinflammatory cytokines have pivotal roles in innate protection against L. monocytogenes infection. We found that a proinflammatory cytokine, interleukin 22 (IL-22), was expressed by CD3(+) CD4(+) T cells at an early stage of L. monocytogenes infection in mice. To assess the influence of IL-22 on L. monocytogenes infection in hepatocytes, cells of a human hepatocellular carcinoma line, HepG2, were treated with IL-22 before L. monocytogenes infection in vitro. Gene expression analysis of the IL-22-treated HepG2 cells identified phospholipase A2 group IIA (PLA2G2A) as an upregulated antimicrobial molecule. Addition of recombinant PLA2G2A to the HepG2 culture significantly suppressed L. monocytogenes infection. Culture supernatant of the IL-22-treated HepG2 cells contained bactericidal activity against L. monocytogenes, and the activity was abrogated by a specific PLA2G2A inhibitor, demonstrating that HepG2 cells secreted PLA2G2A, which killed extracellular L. monocytogenes. Furthermore, colocalization of PLA2G2A and L. monocytogenes was detected in the IL-22-treated infected HepG2 cells, which suggests involvement of PLA2G2A in the mechanism of intracellular killing of L. monocytogenes by HepG2 cells. These results suggest that IL-22 induced at an early stage of L. monocytogenes infection enhances innate immunity against L. monocytogenes in the liver by stimulating hepatocytes to produce an antimicrobial molecule, PLA2G2A.

Hepatitis B virus efficiently infects non-adherent hepatoma cells via human sodium taurocholate cotransporting polypeptide.

Sodium taurocholate cotransporting polypeptide (NTCP) has been reported as a functional receptor for hepatitis B virus (HBV) infection. However, HBV could not efficiently infect HepG2 cells expressing NTCP (NTCP-HepG2 cells) under adherent monolayer-cell conditions. In this study, NTCP was mainly detected in the basolateral membrane region, but not the apical site, of monolayer NTCP-HepG2 cells. We hypothesized that non-adherent cell conditions of infection would enhance HBV infectivity. Non-adherent NTCP-HepG2 cells were prepared by treatment with trypsin and EDTA, which did not degrade NTCP in the membrane fraction. HBV successfully infected NTCP-HepG2 cells at a viral dose 10 times lower in non-adherent phase than in adherent phase. Efficient infection of non-adherent NTCP-HepG2 cells with blood-borne or cell-culture-derived HBV was observed and was remarkably impaired in the presence of the myristoylated preS1 peptide. HBV could also efficiently infect HepaRG cells under non-adherent cell conditions. We screened several compounds using our culture system and identified proscillaridin A as a potent anti-HBV agent with an IC50 value of 7.2 nM. In conclusion, non-adherent host cell conditions of infection augmented HBV infectivity in an NTCP-dependent manner, thus providing a novel strategy to identify anti-HBV drugs and investigate the mechanism of HBV infection.

Circulating microRNA-196a as a candidate diagnostic biomarker for chronic hepatitis C.

Previous studies have demonstrated the inhibitory effect of microRNA (miR)-196a on hepatitis C virus (HCV) expression in human hepatocytes. However, the clinical implications of aberrant miR-196a expression and the application of circulating miR-196a in the diagnosis and management of chronic hepatitis C (CHC) require further investigation. The present study aimed to examine the possibility of using serum miR-196a as a biomarker for CHC. The Affymetrix miRNA array platform was used for miRNA expression profiling in adenovirus (Ad)-HCV core-infected (HepG2-HCV) and Ad-enhanced green fluorescence protein (EGFP)-infected HepG2 cells (HepG2-control). miR-196a downregulation and levels were analyzed using stem-loop reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis of the sera of 43 patients with CHC and 22 healthy controls. A total of six miRNAs were identified as significantly different (≥1.5 fold; P≤0.05) between the two groups. Of note, significant miR-196a downregulation was observed in HepG2-HCV as compared with HepG2-EGFP. Furthermore, as compared with that of the healthy control group, serum miR-196a was demonstrated to be significantly lower in patients with CHC. In addition, analysis of the receiver operating characteristic (ROC) curve for serum miR-196a revealed an area under the ROC curve of 0.849 (95% confidence interval, 0.756–0.941; P<0.001) with 81.8% sensitivity and 76.7% specificity in discriminating chronic HCV infection from healthy controls at a cut-off value of 6.115×10‒5, demonstrating significant diagnostic value for CHC. However, no correlation was identified between serum miR-196a and alanine aminotransferase, aspartate aminotransferase or HCV-RNA. In conclusion, the present study identified circulating miR-196a as a specific and noninvasive candidate biomarker for the diagnosis of CHC.

Different Responses in MMP/TIMP Expression of U937 and HepG2 Cells to Dengue Virus Infection.

Disease severities following dengue virus (DV) infection are the result of increased vascular permeability leading to hypovolemic shock. Matrix metalloproteinases (MMPs) are believed to play a key role in promoting such severities. A previous study reported that supernatants of DV-infected dendritic cells (DCs), which contained high levels of MMP-2 and MMP-9, induced vascular leakage in a mouse model. In the present study, we investigated whether hepatocytes (HepG2) and monocytes (U937) could be additional sources of MMPs during DV infection. HepG2 and U937 cells were exposed to DV serotype 2 strain 16681. The secretion of MMP-2 and MMP-9 was detected using gelatin zymography. We found that DV infection in the HepG2 cells promoted MMP-2 production while that in the U937 cells promoted MMP-9 production. Semi-quantitative RT-PCR results also confirmed that DV infection in the HepG2 cells up-regulated the expression of MMP-2 mRNA, whereas that in the U937 cells enhanced the expression of MMP-9 mRNA. We monitored the expression of endogenous TIMP-1 and TIMP-2. DV infection induced TIMP-1 expression in the U937 cells. However, lower expression of TIMP-2 was observed in the infected HepG2 cells. We believed that following DV infection, monocytes and hepatocytes can act as MMP-9 and MMP-2 producers, respectively. Their responses could be attributed to the disturbance of TIMP expression by DV in different cell types.

Potential antiviral activities of camel, bovine, and human lactoperoxidases against hepatitis C virus genotype 4

Lactoperoxidases (LPOs) were assayed against hepatitis C virus (HCV) using PCR. Direct interaction of HCV with LPO neutralized the viral particles and prevented entry into cells. LPOs inhibited virus amplification in infected HepG2 cells with a relative activity of 100%.

Packaging, Amplification, and Appraisal of the Recombinant Tumor-Selective Type I Herpes Simplex Virus Carrying GALV.fus Gene

The aim of the study was to successfully construct three plasmids, which include the GALV.fus gene plasmid regulated by the herpes simplex virus type 1 (HSV-1) late expression gene-UL38 promoter and induced by HSV-1 (HSV-UL38P-GALV.fus), the cytomegalovirus promoter without tumor specificity (CMVP) GALV.fus plasmid (HSV-CMVP-GALV.fus), and the control plasmid in which the GALV.fus gene fragment was replaced by the enhanced green fluorescent protein (EGFP) gene fragment (HSV-CMVP-EGFP). The three constructed plasmids were all packaged and named as Synco-2, Synco-1, and Baco-1. The plasmids were amplified in coliform bacterium and transfected into Vero cells using lipofectamine. These recombinant HSV-1 were amplified in Vero cells and purified by conventional methods of cesium chloride, TCID50 method is used to measure virus titers. The total RNA was then extracted from the HepG2 cells transfected by Synco-1 and Synco-2, and the expression of GALV.fus mRNA was detected by RT-PCR. The three recombinant HSV-1 vectors were propagated in Vero cells and purified by cesium chloride density gradient centrifugation, titrated by TCID50 method, and packaged. The titers of Baco-1, Synco-1, and Synco-2 were 3 × 10(10), 1 × 10(11), and 4 × 10(10) pfu/ml. The GALV.fus gene was identified in the infected HepG2 cells by RT-PCR method.

Effectiveness of human, camel, bovine and sheep lactoferrin on the hepatitis C virus cellular infectivity: comparison study

PurposeThe prevalence of HCV infection has increased during recent years and the incidence reach 3% of the world's population, and in some countries like Egypt, may around 20%. The developments of effective and preventive agents are critical to control the current public health burden imposed by HCV infection. Lactoferrin in general and camel lactoferrin specifically has been shown to have a compatitive anti-viral activity against hepatitis C virus (HCV). The purpose of this study was to examine and compare the anti-infectivity of native human, camel, bovine and sheep lactoferrin on continuous of HCV infection in HepG2 cells.Material and methodsUsed Lfs were purified by Mono S 5/50 GL column and Superdex 200 5/150 column. The purified Lfs were evaluated in two ways; 1. the pre-infected cells were treated with the Lfs to inhibit intracellular replication at different concentrations and time intervals, 2. Lfs were directly incubated with the virus molecules then used to cells infection. The antiviral activity of the Lfs were determined using three techniques; 1. RT-nested PCR, 2. Real-time PCR and 3. Flowcytometric.ResultsHuman, camel, bovine and sheep lactoferrin could prevent the HCV entry into HepG2 cells by direct interaction with the virus instead of causing significant changes in the target cells. They were also able to inhibit virus amplification in HCV infected HepG2 cells. The highest anti-infectivity was demonstrated by the camel lactoferrin.ConclusioncLf has inhibitory effect on HCV (genotype 4a) higher than human, bovine and sheep lactoferrin.

HBx and HBs regulate RhoC expression by upregulating transcription factor Ets-1

The available evidence suggests that HBV proteins play an important role in the development of hepatocellular carcinoma (HCC). RhoC, a member of the Rho subfamily of the Ras superfamily of homologous genes, had been implicated in tumorigenesis and tumor progression. In a previous study, we demonstrated that HBx and HBs could up-regulate RhoC expression by enhancing its promoter activity. However, the specific mechanisms remain unclear. Here, we demonstrate that overexpression of Ets-1 results in upregulation of RhoC promoter activity and mRNA and protein levels. Expression of transcription factor Ets-1 was significantly higher in HepG2.2.15 cells than that in HepG2 cells. Meanwhile, infection of HepG2 cells with an HBV-adenovirus recombinant virus led to up-regulation of Ets-1. Of the four HBV proteins, HBx and HBs, could increase expression of Ets-1, which consequently contributed to the upregulation of RhoC. These findings might provide a novel insight into HBV-induced HCC metastasis.

Glucose regulates secretion of exogenously expressed insulin from HepG2 cells in vitro and in a mouse model of diabetes mellitus in vivo

Glucose-controlled insulin secretion is a key component of its regulation. Here, we examined whether liver cell secretion of insulin derived from an engineered construct can be regulated by glucose. Adenovirus constructs were designed to express proinsulin or mature insulin containing the conditional binding domain (CBD). This motif binds GRP78 (HSPA5), an endoplasmic reticulum (ER) protein that enables the chimeric hormone to enter into and stay within the ER until glucose regulates its release from the organelle. Infected HepG2 cells expressed proinsulin mRNA and the protein containing the CBD. Immunocytochemistry studies suggested that GRP78 and proinsulin appeared together in the ER of the cell. The amount of hormone released from infected cells varied directly with the ambient concentration of glucose in the media. Glucose-regulated release of the hormone from infected cells was rapid and sustained. Removal of glucose from the cells decreased release of the hormone. In streptozotocin-induced diabetic mice, when infected with adenovirus expressing mature insulin, glucose levels declined. Our data show that glucose regulates release of exogenously expressed insulin from the ER of liver cells. This approach may be useful in devising new ways to treat diabetes mellitus.

Preparation of Microspheres Encapsulating a Recombinant TIMP-1 Adenovirus and their Inhibition of Proliferation of Hepatocellular Carcinoma Cells

The study aim was to prepare poly-DL-lactide-poly (PELA) microspheres encapsulating recombinant tissue inhibitors of metalloproteinase-1 (TIMP-1) in an adenovirus to investigate its inhibition on the proliferation of hepatocellular carcinoma cells HepG2. Microspheres were prepared by encapsulating the recombinant TIMP-1 adenovirus into biodegradable PELA. The particle size, viral load, encapsulation efficiency and in-vitro release were measured. Microspheres were used to infect HepG2 cells, then infection efficiency was examined under a fluorescent microscope and ultrastructural changes assessed by TEM. Expression of TIMP-1 mRNA in HepG2 cells was examined by semi-quantitative RT-PCR and proliferation by MTT and cell growth curve assays. We successfully prepared microspheres encapsulating recombinant TIMP-1 adenovirus with a diameter of 1.965μm, an encapsulation efficiency of 60.0%, a viral load of 10.5?108/mg and approximate 60% of virus release within 120 h, the total releasing time of which was longer than 240 h. The microspheres were confirmed to be non-toxic with blank microspheres. Infected HepG2 cells could stably maintain in-vitro expression of TIMP-1, with significantly effects on biological behaviour. PELA microspheres encapsulating a recombinant TIMP-1 adenovirus can markedly inhibit the proliferation of HepG2 cells, which provides an experimental basis for polymer/chemistry-based gene therapy of hepatocellular carcinomas.

Dual-color bioluminescent assay using infected HepG2 cells sheds new light on Chlamydia pneumoniae and human cytomegalovirus effects on human cholesterol 7α-hydroxylase (CYP7A1) transcription

Chlamydia pneumoniae and human cytomegalovirus (HCMV) are intracellular pathogens able to infect hepatocytes, causing an increase in serum triglycerides and cholesterol levels due to the production of inflammatory cytokines. We investigated whether these pathogens could interfere with cholesterol metabolism by affecting activity of hepatic cholesterol 7α-hydroxylase (CYP7A1) promoter. CYP7A1 is the rate-limiting enzyme responsible for conversion of cholesterol to bile acids, which represents the main route of cholesterol catabolism. A straightforward dual-reporter bioluminescent assay was developed to simultaneously monitor CYP7A1 transcriptional regulation and cell viability in infected human hepatoblastoma HepG2 cells. C. pneumoniae and HCMV infection significantly decreased CYP7A1 promoter activity in a dose-dependent manner, with maximal inhibitions of 33±10% and 32±4%, respectively, at a multiplicity of infection of 1. To support in vitro experiments, serum cholesterol, high-density lipoprotein (HDL) cholesterol, triglycerides and glucose levels were also measured in Balb/c mice infected with C. pneumoniae. Serum cholesterol and triglycerides also increased in infected mice compared with controls. Although further investigation is required, this work presents the first experimental evidence that C. pneumoniae and HCMV inhibit CYP7A1 gene transcription in the cultured human hepatoblastoma cell line.

HepG2 cells infected with Klebsiella pneumoniae show DNA laddering at apoptotic and necrotic stages

Virulent Klebasiella pneumoniae (KP) inflicts severe liver abscesses in infected patients. This study investigated how the bacterial infection affected cell survival at the molecular level, in a cultured cell model. A strain of KP highly virulent in mice was isolated from a patient with liver abscess, and was used to infect HepG2 cells. The infected cells were examined for their viability, DNA fragmentation, and proteins involved in apoptosis or necrosis. We found that the infection decreased the viability of HepG2 cells at 4hours (h) to 12h post infection (pi). DNA ladders appeared 6-16h pi and flow cytometry analysis showed apoptosis at 3-5h pi, secondary necrosis at 6-9h pi and primary necrosis at 8-9h pi. Cleavages of Caspase 7, Caspase 9, α-Fodrin, and PARP were evident at 2-4h pi. At 7h pi, we observed the following: increased nuclear AIF, the release from mitochondria of cytosolic Apaf-1 and Cyt c, increased DFF40 expression, decreased DFF45, decreased BcL-xL and the release of Endo G from mitochondria to nucleus. Cellular ATP concentration decreased at 4-8h pi, accompanied by increased Calpain-2 expression. In summary, infected HepG2 cells underwent apoptosis early after infection and progressed to secondary necrosis and primary necrosis. Nuclear fragmentation corresponded to Caspase 7 activation and the appearance of Endo G and DFF40 in the nucleus, with a concomitant decrease in DFF45. Mitochondrial release of Cyt c together with activation of Caspase 9 and Apaf-1 in cytosol was also observed. Early-hour cleavage of poly(ATP-ribose) polymerase (PARP) followed by the later activation of PARP corresponded to the appearance of DNA laddering, and the depletion of cellular ATP was associated with the appearance of necrosis.

Mutation in fucose synthesis gene ofKlebsiella pneumoniaeaffects capsule composition and virulence in mice

The emerging pathogenicity of Klebsiella pneumoniae (KP) is evident by the increasing number of clinical cases of liver abscess (LA) due to KP infection. A unique property of KP is its thick mucoid capsule. The bacterial capsule has been found to contain fucose in KP strains causing LA but not in those causing urinary tract infections. The products of the gmd and wcaG genes are responsible for converting mannose to fucose in KP. A KP strain, KpL1, which is known to have a high death rate in infected mice, was mutated by inserting an apramycin-resistance gene into the gmd. The mutant expressed genes upstream and downstream of gmd, but not gmd itself, as determined by reverse transcriptase polymerase chain reaction. The DNA mapping confirmed the disruption of the gmd gene. This mutant decreased its ability to kill infected mice and showed decreased virulence in infected HepG2 cells. Compared with wild-type KpL1, the gmd mutant lost fucose in capsular polysaccharides, increased biofilm formation and interacted more readily with macrophages. The mutant displayed morphological changes with long filament forms and less uniform sizes. The mutation also converted the serotype from K1 of wild-type to K2 and weak K3. The results indicate that disruption of the fucose synthesis gene affected the pathophysiology of this bacterium and may be related to the virulence of this KpL1 strain.

Oyster Mushroom Laccase Inhibits Hepatitis C Virus Entry into Peripheral Blood Cells and Hepatoma Cells

There is no protective vaccine or effective drug against hepatitis C virus (HCV). Sustained virological response to INF/ribavirin treatment regimen has an efficiency of about 50%. Many patients worldwide have used traditional medicines and herbal medicine in particular. A laccase has been purified from oyster mushroom (Pleurotus ostreatus) to homogeneity by DEAE Affi-gel blue gel, CM-Sephadex G-50 and Sephadex G-100. The molecular weight of the laccase was about 58 kDa in SDS-PAGE. The optimum pH and temperature of the laccase activity were pH 4.0 and 60 degrees C, respectively. The activity of the enzyme increased steadily from 20 to 40 degrees C, then very slowly from 40 degrees to 60 degrees C, while the enzyme activity decreased to 9% at 90 degrees C. The activity of the laccase changed gradually over the pH range 2.0-4.0. However, the enzyme activity was totally abrogated at the pH 8 and above. Incubation of peripheral blood cells PBCs and hepatoma HepG2 cells with laccase which were then infected with HCV did not protect the cells from HCV attack and entry, while direct interaction between HCV and the laccase at the concentrations of 2.0 and 2.5 mg/ml led to a complete inhibition of virus entry after seven days of incubation. Meantime, the laccase at the concentrations of 1.0 and 1.5 mg/ml did not display any blocking activity. The potential activity of the laccase on intracellular HCV replication in infected HepG2 cells has been examined. The laccase was capable of inhibiting HCV replication at the concentrations of 1.25 and 1.5 mg/ml after first dose of treatment for four days and at the concentrations of 0.75, 1.0, 1.25 and 1.5 mg/ml after the second dose of treatment for another four days.

Schistosoma mansoni soluble egg antigens enhance HCV replication in mammalian cells

This work demonstrates successful propagation of HCV in HepG2 and human blood cells as well as viral shedding into their culture media. The influence of Schistosoma mansoni crude soluble egg antigens (SEA) on the rate of viral propagation in both mammalian cells was also monitored. HepG2 cells were inoculated with HCV viremic human sera and some wells were exposed to HCV infection in presence of SEA. Cells were harvested for RT-PCR and Western blotting analysis. HepG2 media was collected for HCV ELISA. Blood samples from HCV-infected humans were cultured in the presence and absence of SEA. Media were collected at different time points post culturing and subjected to HCV ELISA. The ELISA concentration of HCV antigens were generally higher in media of infected HepG2 cells compared to media of control cells at all time intervals post infection. Western blots showed reactivity to immunogenic peptides of different molecular weights in lysate of infected HepG2 cells that were not evidenced in uninfected cells. In presence of SEA, RT-PCR results revealed earlier detection of viral RNA in infected HepG2 cells compared to in absence of such bilharzial antigen. Also, ELISA results revealed higher levels of detected HCV antigens in media of both infected HepG2 and blood cells cocultured with S. mansoni SEA compared to that of cultured infected cells in absence of the parasite antigens. HepG2 cells as well as whole blood cultures maintain HCV replication. Furthermore, SEA has the potential to enhance HCV propagation.