Multispectral Optical Tweezers for Biochemical Fingerprinting of CD9-Positive Exosome Subpopulations.

Extracellular vesicles (EVs), including exosomes, are circulating nanoscale particles heavily implicated in cell signaling and can be isolated in vast numbers from human biofluids. Study of their molecular profiling and materials properties is currently underway for purposes of describing a variety of biological functions and diseases. However, the large, and as yet largely unquantified, variety of EV subpopulations differing in composition, size, and likely function necessitates characterization schemes capable of measuring single vesicles. Here we describe the first application of multispectral optical tweezers (MS-OTs) to single vesicles for molecular fingerprinting of EV subpopulations. This versatile imaging platform allows for sensitive measurement of Raman chemical composition (e.g., variation in protein, lipid, cholesterol, nucleic acids), coupled with discrimination by fluorescence markers. For exosomes isolated by ultracentrifugation, we use MS-OTs to interrogate the CD9-positive subpopulations via antibody fluorescence labeling and Raman spectra measurement. We report that the CD9-positive exosome subset exhibits reduced component concentration per vesicle and reduced chemical heterogeneity compared to the total purified EV population. We observed that specific vesicle subpopulations are present across exosomes isolated from cell culture supernatant of several clonal varieties of mesenchymal stromal cells and also from plasma and ascites isolated from human ovarian cancer patients.

Increased expression of P-glycoprotein and doxorubicin chemoresistance of metastatic breast cancer is regulated by miR-298.

MicroRNAs (miRNAs) are short, noncoding RNA molecules that regulate the expression of a number of genes involved in cancer; therefore, they offer great diagnostic and therapeutic targets. We have developed doxorubicin-resistant and -sensitive metastatic human breast cancer cell lines (MDA-MB-231) to study the chemoresistant mechanisms regulated by miRNAs. We found that doxorubicin localized exclusively to the cytoplasm and was unable to reach the nuclei of resistant tumor cells because of the increased nuclear expression of MDR1/P-glycoprotein (P-gp). An miRNA array between doxorubicin-sensitive and -resistant breast cancer cells showed that reduced expression of miR-298 in doxorubicin-resistant human breast cancer cells was associated with increased expression of P-gp. In a transient transfection experiment, miR-298 directly bound to the MDR1 3' untranslated region and regulated the expression of firefly luciferase reporter in a dose-dependent manner. Overexpression of miR-298 down-regulated P-gp expression, increasing nuclear accumulation of doxorubicin and cytotoxicity in doxorubicin-resistant breast cancer cells. Furthermore, down-regulation of miR-298 increased P-gp expression and induced doxorubicin resistance in sensitive breast cancer cells. In summary, these results suggest that miR-298 directly modulates P-gp expression and is associated with the chemoresistant mechanisms of metastatic human breast cancer. Therefore, miR-298 has diagnostic and therapeutic potential for predicting doxorubicin chemoresistance in human breast cancer.

Inhibition of hepatitis C virus replication by intracellular delivery of multiple siRNAs by nanosomes.

Sustained antiviral responses of chronic hepatitis C virus (HCV) infection have improved recently by the use of direct-acting antiviral agents along with interferon (IFN)-α and ribavirin. However, the emergence of drug-resistant variants is expected to be a major problem. We describe here a novel combinatorial small interfering RNA (siRNA) nanosome-based antiviral approach to clear HCV infection. Multiple siRNAs targeted to the highly conserved 5'-untranslated region (UTR) of the HCV genome were synthesized and encapsulated into lipid nanoparticles called nanosomes. We show that siRNA can be repeatedly delivered to 100% of cells in culture using nanosomes without toxicity. Six siRNAs dramatically reduced HCV replication in both the replicon and infectious cell culture model. Repeated treatments with two siRNAs were better than a single siRNA treatment in minimizing the development of an escape mutant, resulting in rapid inhibition of viral replication. Systemic administration of combinatorial siRNA-nanosomes is well tolerated in BALB/c mice without liver injury or histological toxicity. As a proof-of-principle, we showed that systemic injections of siRNA nanosomes significantly reduced HCV replication in a liver tumor-xenotransplant mouse model of HCV. Our results indicate that systemic delivery of combinatorial siRNA nanosomes can be used to minimize the development of escape mutants and inhibition of HCV infection.

Increased expression of P-glycoprotein is associated with doxorubicin chemoresistance in the metastatic 4T1 breast cancer model.

Development of drug resistance is one of the major causes of breast cancer treatment failure. The goal of this study was to understand the chemoresistance mechanism using the highly metastatic 4T1 breast cancer model, which emulates stage IV breast cancer in humans. The metastatic 4T1 breast cancer cell line treated with either doxorubicin or 5-FU showed a concentration-dependent reduced cell proliferation, with induced G2-phase growth arrest (doxorubicin) or G1-phase growth arrest (5-FU). Doxorubicin treatment partially suppressed the multiorgan metastasis of 4T1 breast cancer cells in the lung, heart, liver, and bone, compared with either 5-FU or cyclophosphamide. We isolated and characterized 4T1 breast cancer cells from doxorubicin-resistant metastatic tumors (cell line 4T1-R). Multiorgan metastasis of drug-resistant 4T1 breast tumors was totally resistant to doxorubicin treatment. Our results indicate that doxorubicin is localized exclusively in the cytoplasm of resistant 4T1 breast cancer cells and that it cannot reach the nucleus because of increased nuclear expression of P-glycoprotein. Pretreatment of doxorubicin-resistant 4T1-R breast cancer cells with verapamil, a general inhibitor of P-glycoprotein, increased nuclear translocation of doxorubicin and cellular cytotoxicity. Thus, impaired nuclear translocation of doxorubicin due to increased expression of P-glycoprotein is associated with doxorubicin resistance of highly metastatic 4T1 breast cancer.

Free fatty acids induce ER stress and block antiviral activity of interferon alpha against hepatitis C virus in cell culture.

BACKGROUND: Hepatic steatosis is recognized as a major risk factor for liver disease progression and impaired response to interferon based therapy in chronic hepatitis C (CHC) patients. The mechanism of response to interferon-alpha (IFN-α) therapy under the condition of hepatic steatosis is unexplored. We investigated the effect of hepatocellular steatosis on hepatitis C virus (HCV) replication and IFN-α antiviral response in a cell culture model. METHODS: Sub-genomic replicon (S3-GFP) and HCV infected Huh-7.5 cells were cultured with a mixture of saturated (palmitate) and unsaturated (oleate) long-chain free fatty acids (FFA). Intracytoplasmic fat accumulation in these cells was visualized by Nile red staining and electron microscopy then quantified by microfluorometry. The effect of FFA treatment on HCV replication and IFN-α antiviral response was measured by flow cytometric analysis, Renilla luciferase activity, and real-time RT-PCR. RESULTS: FFA treatment induced dose dependent hepatocellular steatosis and lipid droplet accumulation in the HCV replicon cells was confirmed by Nile red staining, microfluorometry, and by electron microscopy. Intracellular fat accumulation supports replication more in the persistently HCV infected culture than in the sub-genomic replicon (S3-GFP) cell line. FFA treatment also partially blocked IFN-α response and viral clearance by reducing the phosphorylation of Stat1 and Stat2 dependent IFN-ß promoter activation. We show that FFA treatment induces endoplasmic reticulum (ER) stress response and down regulates the IFNAR1 chain of the type I IFN receptor leading to defective Jak-Stat signaling and impaired antiviral response. CONCLUSION: These results suggest that intracellular fat accumulation in HCV cell culture induces ER stress, defective Jak-Stat signaling, and attenuates the antiviral response, thus providing an explanation to the clinical observation regarding how hepatocellular steatosis influences IFN-α response in CHC.

Mechanism of HCV's resistance to IFN-α in cell culture involves expression of functional IFN-α receptor 1.

The mechanisms underlying the Hepatitis C virus (HCV) resistance to interferon alpha (IFN-α) are not fully understood. We used IFN-α resistant HCV replicon cell lines and an infectious HCV cell culture system to elucidate the mechanisms of IFN-α resistance in cell culture. The IFN-α resistance mechanism of the replicon cells were addressed by a complementation study that utilized the full-length plasmid clones of IFN-α receptor 1 (IFNAR1), IFN-α receptor 2 (IFNAR2), Jak1, Tyk2, Stat1, Stat2 and the ISRE-luciferase reporter plasmid. We demonstrated that the expression of the full-length IFNAR1 clone alone restored the defective Jak-Stat signaling as well as Stat1, Stat2 and Stat3 phosphorylation, nuclear translocation and antiviral response against HCV in all IFN-α resistant cell lines (R-15, R-17 and R-24) used in this study. Moreover RT-PCR, Southern blotting and DNA sequence analysis revealed that the cells from both R-15 and R-24 series of IFN-α resistant cells have 58 amino acid deletions in the extracellular sub domain 1 (SD1) of IFNAR1. In addition, cells from the R-17 series have 50 amino acids deletion in the sub domain 4 (SD4) of IFNAR1 protein leading to impaired activation of Tyk2 kinase. Using an infectious HCV cell culture model we show here that viral replication in the infected Huh-7 cells is relatively resistant to exogenous IFN-α. HCV infection itself induces defective Jak-Stat signaling and impairs Stat1 and Stat2 phosphorylation by down regulation of the cell surface expression of IFNAR1 through the endoplasmic reticulum (ER) stress mechanisms. The results of this study suggest that expression of cell surface IFNAR1 is critical for the response of HCV to exogenous IFN-α.

Intracellular expression of IRF9 Stat fusion protein overcomes the defective Jak-Stat signaling and inhibits HCV RNA replication.

Interferon alpha (IFN-α) binds to a cell surface receptor that activates the Jak-Stat signaling pathway. A critical component of this pathway is the translocation of interferon stimulated gene factor 3 (a complex of three proteins Stat1, Stat2 and IRF9) to the nucleus to activate antiviral genes. A stable sub-genomic replicon cell line resistant to IFN-α was developed in which the nuclear translocation of Stat1 and Stat2 proteins was prevented due to the lack of phosphorylation; whereas the nuclear translocation of IRF9 protein was not affected. In this study, we sought to overcome defective Jak-Stat signaling and to induce an antiviral state in the IFN-α resistant replicon cell line by developing a chimera IRF9 protein fused with the trans activating domain (TAD) of either a Stat1 (IRF9-S1C) or Stat2 (IRF9-S2C) protein. We show here that intracellular expression of fusion proteins using the plasmid constructs of either IRF9-S1C or IRF9-S2C, in the IFN-α resistant cells, resulted in an increase in Interferon Stimulated Response Element (ISRE) luciferase promoter activity and significantly induced HLA-1 surface expression. Moreover, we show that transient transfection of IRF9-S1C or IRF9-S2C plasmid constructs into IFN-α resistant replicon cells containing sub-genomic HCV1b and HCV2a viruses resulted in an inhibition of viral replication and viral protein expression independent of IFN-α treatment. The results of this study indicate that the recombinant fusion proteins of IRF9-S1C, IRF9-S2C alone, or in combination, have potent antiviral properties against the HCV in an IFN-α resistant cell line with a defective Jak-Stat signaling.

Impaired antiviral activity of interferon alpha against hepatitis C virus 2a in Huh-7 cells with a defective Jak-Stat pathway.

BACKGROUND: The sustained virological response to interferon-alpha (IFN-alpha) in individuals infected with hepatitis C virus (HCV) genotype 1 is only 50%, but is about 80% in patients infected with genotype 2-6 viruses. The molecular mechanisms explaining the differences in IFN-alpha responsiveness between HCV 1 and other genotypes have not been elucidated. RESULTS: Virus and host cellular factors contributing to IFN responsiveness were analyzed using a green fluorescence protein (GFP) based replication system of HCV 2a and Huh-7 cell clones that either possesses or lack a functional Jak-Stat pathway. The GFP gene was inserted into the C-terminal non-structural protein 5A of HCV 2a full-length and sub-genomic clones. Both HCV clones replicated to a high level in Huh-7 cells and could be visualized by either fluorescence microscopy or flow cytometric analysis. Huh-7 cells transfected with the GFP tagged HCV 2a genome produced infectious virus particles and the replication of fluorescence virus particles was demonstrated in naïve Huh-7.5 cells after infection. IFN-alpha effectively inhibited the replication of full-length as well as sub-genomic HCV 2a clones in Huh-7 cells with a functional Jak-Stat pathway. However, the antiviral effect of IFN-alpha against HCV 2a virus was not observed in Huh-7 cell clones with a defect in Jak-Stat signaling. HCV infection or replication did not alter IFN-alpha induced Stat phosphorylation or ISRE promoter-luciferase activity in both the sensitive and resistant Huh-7 cell clones. CONCLUSIONS: The cellular Jak-Stat pathway is critical for a successful IFN-alpha antiviral response against HCV 2a. HCV infection or replication did not alter signaling by the Jak-Stat pathway. GFP labeled JFH1 2a replicon based stable cell lines with IFN sensitive and IFN resistant phenotypes can be used to develop new strategies to overcome IFN-resistance against hepatitis C.

Intracytoplasmic stable expression of IgG1 antibody targeting NS3 helicase inhibits replication of highly efficient hepatitis C Virus 2a clone.

BACKGROUND: Hepatitis C virus (HCV) infection is a major public health problem with more than 170 million cases of chronic infections worldwide. There is no protective vaccine currently available for HCV, therefore the development of novel strategy to prevent chronic infection is important. We reported earlier that a recombinant human antibody clone blocks viral NS3 helicase activity and inhibits replication of HCV 1b virus. This study was performed further to explore the mechanism of action of this recombinant antibody and to determine whether or not this antibody inhibits replication and infectivity of a highly efficient JFH1 HCV 2a virus clone. RESULTS: The antiviral effect of intracellular expressed antibody against the HCV 2a virus strain was examined using a full-length green fluorescence protein (GFP) labeled infectious cell culture system. For this purpose, a Huh-7.5 cell line stably expressing the NS3 helicase gene specific IgG1 antibody was prepared. Replication of full-length HCV-GFP chimera RNA and negative-strand RNA was strongly inhibited in Huh-7.5 cells stably expressing NS3 antibody but not in the cells expressing an unrelated control antibody. Huh-7.5 cells stably expressing NS3 helicase antibody effectively suppressed infectious virus production after natural infection and the level of HCV in the cell free supernatant remained undetectable after first passage. In contrast, Huh-7.5 cells stably expressing an control antibody against influenza virus had no effect on virus production and high-levels of infectious HCV were detected in culture supernatants over four rounds of infectivity assay. A recombinant adenovirus based expression system was used to demonstrate that Huh-7.5 replicon cell line expressing the intracellular antibody strongly inhibited the replication of HCV-GFP RNA. CONCLUSION: Recombinant human anti-HCV NS3 antibody clone inhibits replication of HCV 2a virus and infectious virus production. Intracellular expression of this recombinant antibody offers a potential antiviral strategy to inhibit intracellular HCV replication and production.

Reduced expression of Jak-1 and Tyk-2 proteins leads to interferon resistance in hepatitis C virus replicon.

BACKGROUND: Alpha interferon in combination with ribavirin is the standard therapy for hepatitis C virus infection. Unfortunately, a significant number of patients fail to eradicate their infection with this regimen. The mechanisms of IFN-resistance are unclear. The aim of this study was to determine the contribution of host cell factors to the mechanisms of interferon resistance using replicon cell lines. RESULTS: HCV replicons with high and low activation of the IFN-promoter were cultured for a prolonged period of time in the presence of interferon-alpha (IFN-alpha2b). Stable replicon cell lines with resistant phenotype were isolated and characterized by their ability to continue viral replication in the presence of IFN-alpha. Interferon resistant cell colonies developed only in replicons having lower activation of the IFN promoter and no resistant colonies arose from replicons that exhibit higher activation of the IFN promoter. Individual cell clones were isolated and nine IFN resistant cell lines were established. HCV RNA and protein levels in these cells were not altered by IFN- alpha2b. Reduced signaling and IFN-resistant phenotype was found in all Huh-7 cell lines even after eliminating HCV, suggesting that cellular factors are involved. Resistant phenotype in the replicons is not due to lack of interferon receptor expression. All the cell lines show defect in the JAK-STAT signaling and phosphorylation of STAT 1 and STAT 2 proteins were strongly inhibited due to reduced expression of Tyk2 and Jak-1 protein. CONCLUSION: This in vitro study provides evidence that altered expression of the Jak-Stat signaling proteins can cause IFN resistance using HCV replicon cell clones.

Targeting Tumor-Associated Exosomes with Integrin-Binding Peptides.

All cells expel a variety of nano-sized extracellular vesicles (EVs), including exosomes, with composition reflecting the cells' biological state. Cancer pathology is dramatically mediated by EV trafficking via key proteins, lipids, metabolites, and microRNAs. Recent proteomics evidence suggests that tumor-associated exosomes exhibit distinct expression of certain membrane proteins, rendering those proteins as attractive targets for diagnostic or therapeutic application. Yet, it is not currently feasible to distinguish circulating EVs in complex biofluids according to their tissue of origin or state of disease. Here we demonstrate peptide binding to tumor-associated EVs via overexpressed membrane protein. We find that SKOV-3 ovarian tumor cells and their released EVs express α3ß1 integrin, which can be targeted by our in-house cyclic nonapeptide, LXY30. After measuring bulk SKOV-3 EV association with LXY30 by flow cytometry, Raman spectral analysis of laser-trapped single exosomes with LXY30-dialkyne conjugate enabled us to differentiate cancer-associated exosomes from non-cancer exosomes. Furthermore, we introduce the foundation for a highly specific detection platform for tumor-EVs in solution with biosensor surface-immobilized LXY30. LXY30 not only exhibits high specificity and affinity to α3ß1 integrin-expressing EVs, but also reduces EV uptake into SKOV-3 parent cells, demonstrating the possibility for therapeutic application.

HCV-hepatocellular carcinoma: new findings and hope for effective treatment.

We present here a comprehensive review of the current literature plus our own findings about in vivo and in vitro analysis of hepatitis C virus (HCV) infection, viral pathogenesis, mechanisms of interferon action, interferon resistance, and development of new therapeutics. Chronic HCV infection is a major risk factor for the development of human hepatocellular carcinoma. Standard therapy for chronic HCV infection is the combination of interferon alpha and ribavirin. A significant number of chronic HCV patients who cannot get rid of the virus infection by interferon therapy experience long-term inflammation of the liver and scarring of liver tissue. Patients who develop cirrhosis usually have increased risk of developing liver cancer. The molecular details of why some patients do not respond to standard interferon therapy are not known. Availability of HCV cell culture model has increased our understanding on the antiviral action of interferon alpha and mechanisms of interferon resistance. Interferons alpha, beta, and gamma each inhibit replication of HCV, and the antiviral action of interferon is targeted to the highly conserved 5'UTR used by the virus to translate protein by internal ribosome entry site mechanism. Studies from different laboratories including ours suggest that HCV replication in selected clones of cells can escape interferon action. Both viral and host factors appear to be involved in the mechanisms of interferon resistance against HCV. Since interferon therapy is not effective in all chronic hepatitis C patients, alternative therapeutic strategies are needed to treat chronic hepatitis C patients not responding to interferon therapy. We also reviewed the recent development of new alternative therapeutic strategies for chronic hepatitis C, which may be available in clinical use within the next decade. There is hope that these new agents along with interferon will prevent the occurrence of hepatocellular carcinoma due to chronic persistent hepatitis C virus infection. This review is not inclusive of all important scientific publications due to space limitation.

3D plasmonic nanobowl platform for the study of exosomes in solution.

Thin silver film coated nanobowl Surface Enhanced Raman Spectroscopy (SERS) substrates are used to capture exosomes in solution for SERS measurements that can provide biochemical analysis of intact and ruptured exosomes. Exosomes derived via Total Exosome Isolation Reagent (TEIR) as well as ultracentrifugation (UC) from the SKOV3 cell line were analyzed. Spectra of exosomes derived via TEIR are dominated by a signal characteristic for the TEIR kit that needs to be subtracted for all measurements. Differences in SERS spectra recorded at different times during the drying of the exosome solution are statistically analyzed with Principal Component Analysis (PCA). At the beginning of the drying process, SERS spectra of exosomes exhibit peaks characteristic for both lipids and proteins. Later on during the drying process, new SERS peaks develop, suggesting that the initially intact exosome ruptures over time. This time-dependent evolution of SERS peaks enables analysis of exosomal membrane contents and the contents inside the exosomes.

Single exosome study reveals subpopulations distributed among cell lines with variability related to membrane content.

Current analysis of exosomes focuses primarily on bulk analysis, where exosome-to-exosome variability cannot be assessed. In this study, we used Raman spectroscopy to study the chemical composition of single exosomes. We measured spectra of individual exosomes from 8 cell lines. Cell-line-averaged spectra varied considerably, reflecting the variation in total exosomal protein, lipid, genetic, and cytosolic content. Unexpectedly, single exosomes isolated from the same cell type also exhibited high spectral variability. Subsequent spectral analysis revealed clustering of single exosomes into 4 distinct groups that were not cell-line specific. Each group contained exosomes from multiple cell lines, and most cell lines had exosomes in multiple groups. The differences between these groups are related to chemical differences primarily due to differing membrane composition. Through a principal components analysis, we identified that the major sources of spectral variation among the exosomes were in cholesterol content, relative expression of phospholipids to cholesterol, and surface protein expression. For example, exosomes derived from cancerous versus non-cancerous cell lines can be largely separated based on their relative expression of cholesterol and phospholipids. We are the first to indicate that exosome subpopulations are shared among cell types, suggesting distributed exosome functionality. The origins of these differences are likely related to the specific role of extracellular vesicle subpopulations in both normal cell function and carcinogenesis, and they may provide diagnostic potential at the single exosome level.

IFN-λ Inhibits MiR-122 Transcription through a Stat3-HNF4α Inflammatory Feedback Loop in an IFN-α Resistant HCV Cell Culture System.

BACKGROUND: HCV replication in persistently infected cell culture remains resistant to IFN-α/RBV combination treatment, whereas IFN-λ1 induces viral clearance. The antiviral mechanisms by which IFN-λ1 induces sustained HCV clearance have not been determined. AIM: To investigate the mechanisms by which IFN-λ clears HCV replication in an HCV cell culture model. METHODS: IFN-α sensitive (S3-GFP) and resistant (R4-GFP) cells were treated with equivalent concentrations of either IFN-α or IFN-λ. The relative antiviral effects of IFN-α and IFN-λ1 were compared by measuring the HCV replication, quantification of HCV-GFP expression by flow cytometry, and viral RNA levels by real time RT-PCR. Activation of Jak-Stat signaling, interferon stimulated gene (ISG) expression, and miRNA-122 transcription in S3-GFP and R4-GFP cells were examined. RESULTS: We have shown that IFN-λ1 induces HCV clearance in IFN-α resistant and sensitive replicon cell lines in a dose dependent manner through Jak-Stat signaling, and induces STAT 1 and STAT 2 activation, ISRE-luciferase promoter activation and ISG expression. Stat 3 activation is also involved in IFN-λ1 induced antiviral activity in HCV cell culture. IFN-λ1 induced Stat 3 phosphorylation reduces the expression of hepatocyte nuclear factor 4 alpha (HNF4α) through miR-24 in R4-GFP cells. Reduced expression of HNF4α is associated with decreased expression of miR-122 resulting in an anti-HCV effect. Northern blot analysis confirms that IFN-λ1 reduces miR-122 levels in R4-GFP cells. Our results indicate that IFN-λ1 activates the Stat 3-HNF4α feedback inflammatory loop to inhibit miR-122 transcription in HCV cell culture. CONCLUSIONS: In addition to the classical Jak-Stat antiviral signaling pathway, IFN-λ1 inhibits HCV replication through the suppression of miRNA-122 transcription via an inflammatory Stat 3-HNF4α feedback loop. Inflammatory feedback circuits activated by IFNs during chronic inflammation expose non-responders to the risk of hepatocellular carcinoma.

Development and evaluation of a quantitative competitive reverse transcription polymerase chain reaction (RT-PCR) for hepatitis C virus RNA in serum using transcribed thio-RNA as internal control.

A method for quantitation of hepatitis C virus (HCV) RNA was developed based on competitive reverse transcription polymerase chain reaction (RT-PCR) using in vitro transcribed mutated thio-RNA as a competitor template. The thio-RNA is more resistant to RNAse and is stable over a year. This assay was compared with the commercially available Roche Amplicor HCV Monitor assay V 2.0 and real time PCR using SYBR green 1 dye method. A total of 18 pre-therapy serum samples from chronic hepatitis C cases were tested in parallel by the three assays. All samples could be quantitated using the in-house competitive RT-PCR and real time PCR and there was a significant correlation in the virus titer (P<0.05). However, 8 (44%) samples could not be quantified by Amplicor HCV Monitor assay, which has a lower detection range (10(2) to 10(5.5) copies/ml). The in-house method of competitive RT-PCR showed a detection range of 10(3) to 10(10) copies/ml. In the patients the mean viral titer was found to be (9.66+/-9.3)x10(6) copies/ml. Ten (55%) of the samples, assessed by the Amplicor HCV Monitor assay showed a mean viral titre of (1.13+/-0.75)x10(6) copies/ml, which was lower than the other two tests. The competitive PCR method and real time PCR could amplify all prevalent genotypes. This in-house quantitative competitive RT-PCR method is simple, cheap, reproducible and useful for estimation of HCV RNA load.

Impaired expression of type I and type II interferon receptors in HCV-associated chronic liver disease and liver cirrhosis.

PURPOSE: Chronic Hepatitis C Virus (HCV)-infected patients with liver cirrhosis (LC) respond poorly to interferon-alpha (IFN-α) and ribavirin (RBV) combination therapy, but the reason for this is unclear. We previously reported that HCV-infection induces endoplasmic reticulum (ER) stress and autophagy response that selectively down regulates the type I IFN-α receptor-1 (IFNAR1) and RBV transporters (CNT1 and ENT1), leading to IFN-α/RBV resistance. The goal of this study is to verify whether an increase in ER stress and autophagy response is also associated with the reduced expression of IFNAR1 and RBV transporters in chronic HCV-infected patients. METHODS: Primary human hepatocytes (PHH) were infected with cell culture grown HCV particles (JFH-ΔV3-Rluc). HCV replication was confirmed by the detection of viral RNA by RT-qPCR and HCV-core protein by Western blotting. The ER stress and autophagy response and expression of IFN receptors and RBV transporters in HCV infected PHH and liver tissues derived from patients were measured by Western blotting. RESULT: HCV infection of PHH showed impaired expression of IFNAR1, IFNγR1 (Type II IFN receptor) and RBV transporters but not IL10Rß (Type III IFN-λ receptor). ER stress markers (BiP, IRE1α and peIF2α) and autophagy response (LC3II, Beclin 1 and ATG5) were induced in HCV infected chronic liver disease (CLD) and LC patients. Liver biopsies (CLD) show a 50% reduced expression of IFNAR1 and RBV transporters. Furthermore, the expression of IFNAR1 and RBV transporters was impaired in almost all LC patients. CONCLUSION: HCV infection induces ER stress and autophagy response in infected PHH and chronically infected liver tissues. The expression of IFNAR1, IFNγR1 and RBV transporters were significantly impaired in CLD and cirrhotic livers. Our study provides a potential explanation for the reduced response rate of IFN-α and RBV combination therapy in HCV infected patients with liver cirrhosis.

Interferon and ribavirin combination treatment synergistically inhibit HCV internal ribosome entry site mediated translation at the level of polyribosome formation.

PURPOSE: Although chronic hepatitis C virus (HCV) infection has been treated with the combination of interferon alpha (IFN-α) and ribavirin (RBV) for over a decade, the mechanism of antiviral synergy is not well understood. We aimed to determine the synergistic antiviral mechanisms of IFN-α and RBV combination treatment using HCV cell culture. METHODS: The antiviral efficacy of IFN-α, RBV alone and in combination was quantitatively measured using HCV infected and replicon cell culture. Direct antiviral activity of these two drugs at the level of HCV internal ribosome entry site (IRES) mediated translation in Huh-7 cell culture was investigated. The synergistic antiviral effect of IFN-α and RBV combination treatment was verified using both the CalcuSyn Software and MacSynergy Software. RESULTS: RBV combination with IFN-α efficiently inhibits HCV replication cell culture. Our results demonstrate that IFN-α, interferon lambda (IFN-λ) and RBV each inhibit the expression of HCV IRES-GFP and that they have a minimal effect on the expression of GFP in which the translation is not IRES dependent. The combination treatments of RBV along with IFN-α or IFN-λ were highly synergistic with combination indexes <1. We show that IFN-α treatment induce levels of PKR and eIF2α phosphorylation that prevented ribosome loading of the HCV IRES-GFP mRNA. Silencing of PKR expression in Huh-7 cells prevented the inhibitory effect of IFN-α on HCV IRES-GFP expression. RBV also blocked polyribosome loading of HCV-IRES mRNA through the inhibition of cellular IMPDH activity, and induced PKR and eIF2α phosphorylation. Knockdown of PKR or IMPDH prevented RBV induced HCV IRES-GFP translation. CONCLUSIONS: We demonstrated both IFN-α and RBV inhibit HCV IRES through prevention of polyribosome formation. The combination of IFN-α and RBV treatment synergistically inhibits HCV IRES translation via using two different mechanisms involving PKR activation and depletion of intracellular guanosine pool through inhibition of IMPDH.

Development and optimization of nanosomal formulations for siRNA delivery to the liver.

The objective of this study is to develop an effective siRNA delivery system for successful delivery to the liver for the treatment of HCV. Nanosize liposomes (nanosomes) have been prepared using a mixture of cholesterol and DOTAP. A functional siRNA was encapsulated into nanosomes following condensation with protamine sulfate. The delivery of siRNA was optimized in an in vitro cell culture system. The efficacy of the formulations was evaluated by measuring functional gene silencing and cytotoxicity. Encapsulation of siRNA ≥ 7.4 nM resulted in successful delivery of siRNA to nearly 100% of cells. The formulations containing lipid-to-siRNA ratio ≥ 10.56:1 instantly cleared approximately 85% of HCV while maintaining cell viability at about 90%. The formulations were sonicated to further reduce the particle size. The size of these formulations was decreased up to 100 nm. However, there were no significant changes observed in zeta potential, or in siRNA encapsulation and integrity following sonication. The sonicated formulations also showed higher liver hepatocytes deposition and gene silencing properties. This study therefore provides a novel approach of siRNA delivery to liver hepatocytes, which can also be applied to treat HCV in chronic liver diseases.

Cellular delivery of PEGylated PLGA nanoparticles.

OBJECTIVES: The objective of this study was to investigate the efficiency of uptake of PEGylated polylactide-co-gycolide (PLGA) nanoparticles by breast cancer cells. METHODS: Nanoparticles of PLGA containing various amounts of polyethylene glycol (PEG, 5%-15%) were prepared using a double emulsion solvent evaporation method. The nanoparticles were loaded with coumarin-6 (C6) as a fluorescence marker. The particles were characterized for surface morphology, particle size, zeta potential, and for cellular uptake by 4T1 murine breast cancer cells. KEY FINDINGS: Irrespective of the amount of PEG, all formulations yielded smooth spherical particles. However, a comparison of the particle size of various formulations showed bimodal distribution of particles. Each formulation was later passed through a 1.2 µm filter to obtain target size particles (114-335 nm) with zeta potentials ranging from -2.8 mV to -26.2 mV. While PLGA-PEG di-block (15% PEG) formulation showed significantly higher 4T1 cellular uptake than all other formulations, there was no statistical difference in cellular uptake among PLGA, PLGA-PEG-PLGA tri-block (10% PEG), PLGA-PEG di-block (5% PEG) and PLGA-PEG di-block (10% PEG) nanoparticles. CONCLUSION: These preliminary findings indicated that the nanoparticle formulation prepared with 15% PEGylated PLGA showed maximum cellular uptake due to it having the smallest particle size and lowest zeta potential.