Pretreatment of Garlic Oil Extracts Hampers Epithelial Damage in Cell Culture Model of Peptic Ulcer Disease.

Background and Objectives: Peptic ulcer disease is a chronic disease affecting up to 10% of the world's population. Proton pump inhibitors, such as lansoprazole are the gold standard in the treatment of ulcer disease. However, various studies have shown the effectiveness of garlic oil extracts in the treatment of ulcer disease. A cellular model can be established in the human gastric cell line by sodium taurocholate. The aim of this study was to explore the effects of garlic oil extracts pretreatment and LPZ addition in the cell culture model of peptic ulcer disease by examining oxidative stress and F-actin distribution. Materials and Methods: Evaluation was performed by determination of glutathione and prostaglandin E2 concentrations by ELISA; human gastric cell line proliferation by cell counting; expression of ATP-binding cassette, sub-family G, member 2; nuclear factor kappa B subunit 2 by RT PCR; and F-actin cytoskeleton visualization by semi-quantification of Rhodamine Phalloidin stain. Results: Our results showed significant reduction of cell damage after sodium taurocholate incubation when the gastric cells were pretreated with lansoprazole (p < 0.001) and increasing concentrations of garlic oil extracts (p < 0.001). Pretreatment with lansoprazole and different concentrations of garlic oil extracts increased prostaglandin E2 and glutathione concentrations in the cell culture model of peptic ulcer disease (p < 0.001). Positive correlation of nuclear factor kappa B subunit 2 (p < 0.01) with lansoprazole and garlic oil extracts pretreatment was seen, while ATP-binding cassette, sub-family G, member 2 expression was not changed. Treatment with sodium taurocholate as oxidative stress on F actin structure was less pronounced, although the highest concentration of garlic oil extracts led to a statistically significant increase of total amount of F-actin (p < 0.001). Conclusions: Hence, pretreatment with garlic oil extracts had gastroprotective effect in the cell model of peptic ulcer disease. However, further experiments are needed to fully elucidate the mechanism of this protective role.

Targeted delivery of mitochondria to the liver in rats.

BACKGROUND AND AIM: Mitochondrial damage is commonly involved in liver injury. We have previously shown that normal mitochondria can be coated with a carrier protein to form complexes that are specifically taken up by liver cells in culture. The aim of the current study was to determine whether mitochondrial complexes could be specifically delivered to the livers of living rats by intravenous injection. METHODS: Mitochondria were harvested from fresh mouse liver, mixed with an asialoglycoprotein-based carrier, asialoorosomucoid-polylysine (AsOR-PL), and purified to form complexes. To facilitate the release of internalized mitochondria from endosomes, an endosomolytic peptide, listeriolysin O (LLO), was coupled to AsOR to form AsOR-LLO. Mitochondria alone, mitochondrial complexes with AsOR-PL, and mitochondrial complexes plus AsOR-LLO conjugate all containing the same number of mitochondria were injected intravenously. Animals were killed, and organs were removed and analyzed by quantitative polymerase chain reaction of mouse mitochondrial DNA, electron microscopy (EM), and in situ polymerase chain reaction and hybridization followed by immunohistochemical analyses. RESULTS: Calculations revealed that approximately 27% of the total injected mitochondria was detected in the liver, while less than 2% was found in spleen, and < 1% in lungs. Immunohistochemistry showed that mouse mitochondrial DNA staining was minimal with mitochondrial complexes alone, strong periportal with mitochondrial complexes co-injected with AsOR-LLO, and absent with mitochondria alone. CONCLUSIONS: Targetable mitochondrial complexes can be delivered to rat liver, and the efficiency of that process is greatly enhanced by co-injection of a targetable endosomal release agent, AsOR-LLO.

An Improved Method for Preparation of Uniform and Functional Mitochondria from Fresh Liver.

Background and Aims: As the major energy source for mammalian cells, mitochondria have been the subject of numerous studies. However, the isolation and purification of healthy mitochondria, especially from fresh tissue, remains challenging. The most popular methods and kits involve various centrifugation steps which require substantial time and equipment but do not consistently provide pure preparations of functional mitochondria. The aim of this study was to determine whether methods could be devised to improve the purity and yield of functional mitochondria from fresh tissue. Methods: Fresh mouse liver was homogenized, and cells lysed. Particle size analysis, quantitation of mitochondrial DNA, mitochondrial oxygen consumption, and purity of mitochondria (by electron microscopy) were measured in samples after various purification steps and significant differences determined. Results: A two-step procedure consisting of centrifugation followed by filtration through 1.2µ and 0.8µ filters resulted in uniform mitochondrial preparations with diameters between 520-540 nm, and approximately 5-times more pure samples. The mitochondria thus obtained had oxygen consumption and sensitivities to mitochondrial inhibitors that were indistinguishable from those purified by centrifugation alone. Electron microscopy confirmed the presence of more uniform and 4-5 times greater concentrations of mitochondria compared to centrifugation alone. Conclusions: A two-step procedure consisting of sequential centrifugation followed by filtration is a rapid method for the production of highly purified, uniform and functional mitochondria.

Targeted transplantation of mitochondria to hepatocytes.

BACKGROUND: Mitochondrial defects in hepatocytes can result in liver dysfunction and death. Hepatocytes have cell-surface asialoglycoprotein receptors (AsGRs) which internalize AsGs within endosomes. The aim of this study was to determine whether mitochondria could be targeted to hepatocytes by AsGR-mediated endocytosis. MATERIALS AND METHODS: An AsG, AsOR, was linked to polylysine to create a conjugate, AsOR-PL, and complexed with healthy and functional mitochondria (defined by normal morphology, cytochrome c assays, and oxygen-consumption rates). Huh7 (AsGR+) and SK Hep1 (AsGR-) cells were treated with a mitochondrial toxin to form Huh7-Mito- and SK Hep1-Mito- cells, lacking detectable mitochondrial DNA. An endosomolytic peptide, LLO, was coupled to AsOR to form AsOR-LLO. A lysosomal inhibitor, amantadine, was used in mitochondria-uptake studies as a control for nonspecific endosomal release. RESULTS: Coincubation of complexed mitochondria and AsOR-LLO with Huh7-Mito- cells increased mitochondrial DNA to >9,700-fold over control at 7 days (P<0.001), and increased mitochondrial oxygen-consumption rates to >90% of control by 10 days. CONCLUSION: Rescue of mitochondria-damaged hepatocytes can be achieved by targeted uptake of normal mitochondria through receptor-mediated endocytosis.

Secondary Structural Elements of the HCV X-region Involved in Viral Replication.

BACKGROUND AND AIMS: The noncoding regions in the 3'-untranslated region (UTR) of the hepatitis C virus (HCV) genome contain secondary structures that are important for replication. The aim of this study was to identify detailed conformational elements of the X-region involved in HCV replication. METHODS: Ribonucleic acid (RNA) structural analogs X94, X12, and X12c were constructed to have identical conformation but 94%, 12%, and 0% sequence identity, respectively, to the X region of HCV genotype 2a. Effects of structural analogs on replication of HCV genotypes 1b and 2a HCV RNA were studied by quantitative reverse transcriptase polymerase chain reaction. RESULTS: In replicon BB7 cells, a constitutive replication model, HCV RNA levels decreased to 55%, 52%, 53%, and 54% after transfection with expression plasmids generating RNA structural analogs 5B-46, X-94, X-12, and X-12c, respectively (p<0.001 for all). In an HCV genotype 2a infection model, RNA analogs 5B-46, X-94, and X-12 in hepatic cells inhibited replication to 11%, 9%, and 12%, respectively. Because the X-12 analog was only 12% identical to the corresponding sequence of HCV genotype 2a, the sequence per se, or antisense effects were unlikely to be involved. CONCLUSIONS: The data suggest that conformation of secondary structures in 3'-UTR of HCV RNA genome is required for HCV replication. Stable expression of RNA analogs predicted to have identical stem-loop structures might inhibit HCV infection of hepatocytes in liver and may represent a novel approach to design anti-HCV agents.

Effects of short RNA structural analogues against hepatitis C virus genotypes 2, 3 and 4 in replicon cells.

OBJECTIVE: To determine whether computer-predicted short RNA structural analogues could inhibit hepatitis C virus (HCV) genotype 2a, 3a and 4a replication in cultured cells. METHODS: Short RNA sequences, X12, X12a and X12b, designed to be identical in secondary structure to the X region in the 3'-untranslated region (3'-UTR) of the HCV 1b genome, as well as shorter stem-loop components of X region, were inserted into a plasmid and transfected into separate Huh7.5 human hepatoma cells stably transfected with subgenomic replicons for genotypes 2a, 3a and 4a. All replicons included a firefly luciferase reporter gene. After 48 h of plasmid transfection, the inhibition of HCV replication was determined by HCV RNA isolation and quantification by real-time polymerase chain reaction and luciferase assays. RESULTS: All the secondary structural analogues to genotype 1b X region cross-inhibited genotype 2a, 3a and 4a replicons. The maximum inhibition by genotype 1b X region structural analogues was obtained against genotype 2a cells in which X12, X12a and X12b inhibited replication by 30%, 63% and 72%, respectively (P < 0.05 for all), compared to an unrelated hepatitis B viral analogue. CONCLUSIONS: Despite substantial sequence dissimilarity, HCV RNA genotype 1b X region analogues cross-inhibited the replication of HCV genotypes 2a, 3a and 4a. Particular conformations and not the sequence of the stem-loops of the X region are involved in HCV replication.

The Molecular and Structural Basis of HBV-resistance to Nucleos(t)ide Analogs.

Infection with hepatitis B virus (HBV) is a worldwide health problem. Chronic hepatitis B can lead to fibrosis, liver cirrhosis, and hepatocellular carcinoma (HCC). Management of the latter two conditions often requires liver transplantation. Treatment with conventional interferon or pegylated interferon alpha can clear the virus, but the rates are very low. The likelihood, however, of viral resistance to interferon is minimal. The main problems with this therapy are the frequency and severity of side effects. In contrast, nucleos(t)ide analogs (NAs) have significantly lower side effects, but require long term treatment as sustained virological response rates are extremely low. However, long term treatment with NAs increases the risk for the development of anti-viral drug resistance. Only by understanding the molecular basis of resistance and using agents with multiple sites of action can drugs be designed to optimally prevent the occurrence of HBV antiviral resistance.

Update on the Development of Anti-Viral Agents Against Hepatitis C.

Hepatitis C virus (HCV) infects nearly 170 million people worldwide and causes chronic hepatitis, cirrhosis, and hepatocellular carcinoma. The search for a drug regimen that maximizes efficacy and minimizes side effects is quickly evolving. This review will discuss a wide range of drug targets currently in all phases of development for the treatment of HCV. Direct data from agents in phase III/IV clinical trials will be presented, along with reported side-effect profiles. The mechanism of action of all treatments and resistance issues are highlighted. Special attention is given to available trial data supporting interferon-free treatment regimens. HCV has become an increasingly important public health concern, and it is important for physicians to stay up to date on the rapidly growing novel therapeutic options.

Pharmacogenetic selection of transplanted human hepatocytes in immunocompetent rats.

OBJECTIVE: To introduce a genetic survival advantage for transplanted human hepatocytes over host cells in rats. METHODS: Green fluorescent protein (GFP) was introduced into Huh-7 human hepatoma cells to create fluorescent GFP-Huh-7 cells. mRNA of CYP2E1, the enzyme that converts acetaminophen (APA) into hepatotoxic intermediates, was quantified by real-time polymerase chain reaction (PCR). The effects of APA on GFP-Huh-7 and control Huh-7 cells were determined in a cell culture. Immunological tolerance was induced by the injection of GFP-Huh-7 cells into fetal rats in utero. The GFP-Huh-7 cells were transplanted after birth of the rats into tolerant rats followed by APA treatment. Serum alanine aminotransferase (ALT) levels and liver histological data were obtained. GFP-Huh-7 cells were detected by quantitive PCR and microscopy. RESULTS: CYP2E1 mRNA levels in the GFP-Huh-7 cells were 2.7% of parental Huh-7 cells. In 1 mmol/L APA, parental Huh-7 cells decreased by 60% while GFP-Huh-7 cells increased to within 95% of untreated controls after 5 days. In rats in which GFP-Huh-7 cells were transplanted and treated with APA, serum ALT increased to a peak of 200 U/L on day 1 and returned to normal levels by day 3. Fluorescence microscopy of liver specimens from rats transplanted with GFP-Huh-7 cells showed substantial increases in GFP-Huh-7, but not Huh-7 cells by day 7 after APA treatment. Real-time PCR confirmed a 10-fold increase of GFP mRNA in APA-treated rats, but not in those without APA treatment. CONCLUSIONS: The difference in CYP2E1 gene expression between GFP-Huh-7 and rat hepatocytes provides a convenient means for the enrichment of transplanted human cells in rat liver.

Animal models for the study of hepatitis C virus infection and replication.

Hepatitis C virus (HCV) hepatitis, initially termed non-A, non-B hepatitis, has become one of the leading causes of cirrhosis and hepatocellular carcinoma worldwide. With the help of animal models, our understanding of the virus has grown substantially from the time of initial discovery. There is a paucity of available animal models for the study of HCV, mainly because of the selective susceptibility limited to humans and primates. Recent work has focused modification of animals to permit HCV entry, replication and transmission. In this review, we highlight the currently available models for the study of HCV including chimpanzees, tupaia, mouse and rat models. Discussion will include methods of model design as well as the advantages and disadvantages of each model. Particular focus is dedicated to knowledge of pathophysiologic mechanisms of HCV infection that have been elucidated through animal studies. Research within animal models is critically important to establish a complete understanding of HCV infection, which will ultimately form the basis for future treatments and prevention of disease.

Granulomatous liver diseases: a review.

Granulomas that consist of focal accumulations of macrophages are commonly found in the liver due to stimulation of the immune system by a number of agents. Manifestations are variable depending on whether the underlying cause is a systemic disease or a primary hepatic granulomatous reaction. This article describes the common causes, presentation, histopathology, and manifestations of granulomatous diseases as well as various diagnostic and management strategies.

Inhibition of hepatitis C virus replication by single-stranded RNA structural mimics.

AIM: To examine the effect of hepatitis C virus (HCV) structural mimics of regulatory regions of the genome on HCV replication. METHODS: HCV RNA structural mimics were constructed and tested in a HCV genotype 1b aBB7 replicon, and a Japanese fulminant hepatitis-1 (JFH-1) HCV genotype 2a infection model. All sequences were computer-predicted to adopt stem-loop structures identical to the corresponding elements in full-length viral RNA. Huh7.5 cells bearing the BB7 replicon or infected with JFH-1 virus were transfected with expression vectors generating HCV mimics and controls. Cellular HCV RNA and protein levels were quantified by real-time polymerase chain reaction and Western blotting, respectively. To evaluate possible antisense effects, complementary RNAs spanning a mimic were prepared. RESULTS: In the BB7 genotype 1b replicon system, mimics of the polymerase (NS-5B), X and BA regions inhibited replication by more than 90%, 50%, and 60%, respectively. In the JFH-1 genotype 2 infection system, mimics that were only 74% and 46% identical in sequence relative to the corresponding region in JFH-1 inhibited HCV replication by 91.5% and 91.2%, respectively, as effectively as a mimic with complete identity to HCV genotype 2a. The inhibitory effects were confirmed by NS3 protein levels. Antisense RNA molecules spanning the 74% identical mimic had no significant effects. CONCLUSION: HCV RNA structural mimics can inhibit HCV RNA replication in replicon and infectious HCV systems and do so independent of close sequence identity with the target.

Effects of PEG-interferon-alpha-2A on Schistosoma mansoni infection in mice.

In some regions of the world, co-existence of schistosomiasis and hepatitis C (HCV) infection is common. Because the morbidity in human schistosomiasis is primarily due to host cell-mediated immune response, it was of interest to determine the effects on Schistosoma mansoni infection of an immune stimulator used in the standard treatment of HCV infection. Schistosoma mansoni -infected mice were treated with PEG-interferon-alpha-2a (PEG-IFN-alpha) by subcutaneous injection. Groups 1, 2, and 3 received 0.2 microg, 0.6 microg, and 1 microg PEG-IFN-alpha/wk, respectively, while group 4 received saline. The total worm burden was lower in all treated groups, with a maximal reduction of 35% after 9 wk of treatment with 1 microg PEG-IFN-alpha. Interferon treatment also increased the proportion of single worms over pairs. Ova counts in intestine and liver, as well as the number of liver granulomas, were greatly decreased at all time points for all treated groups. PEG-IFN-alpha also had inhibitory effects on the size of granulomas after 4 wk of treatment. The results suggest that PEG-IFN-alpha may be worth investigating for the treatment of human schistosomiasis when standard oral agents cannot be used, or when rapid inhibition of granuloma formation may be a priority.

Host susceptibility to schistosomes: effect of host sera on cell proliferation of Schistosoma mansoni schistosomula.

To determine effects of the sera on cell proliferation, schistosomula of Schistosoma mansoni (20-days-old) were incubated in medium containing fetal calf serum plus hamster (highly susceptible host) portal or peripheral venous serum, or rat (poorly susceptible host) portal or peripheral venous serum in the presence of bromodeoxyuridine (BrdU). Compared with schistosomula cultured in presence of control medium containing fetal calf serum alone, BrdU labeling indices (BLIs) were increased by 39% in the presence of portal, but not in peripheral, serum of hamsters. In contrast, no significant differences were observed in the BLIs in rat portal, or peripheral, sera or in control media. In vivo BrdU labeling results revealed that there was no detectable cell proliferation in S. mansoni schistosomula (6 days old) in the lungs. However, cell proliferation was detected in schistosomula beginning at 17 days. The results indicated that portal venous serum from a highly susceptible host, but not from a poorly susceptible host, stimulated schistosome cell proliferation in vitro. The timing of the increase in cell proliferation in terms of development corresponded to liver portal-mesenteric localization of schistosomula. Together, the data support the conclusion that in susceptible hosts, portal serum may play a role in schistosome cell proliferation, possibly resulting in termination of schistosome migration. This may explain the colocalization of adults, and the known organ selectivity of disease.

Human uridine-cytidine kinase phosphorylation of ribavirin: a convenient method for activation of ribavirin for conjugation to proteins.

Ribavirin is a synthetic nucleoside analog that is used for the treatment of hepatitis C virus (HCV) infection. Its primary toxicity is hemolytic anemia, which sometimes necessitates dose reduction or discontinuation of therapy. Selective delivery of ribavirin into liver cells would be desirable to enhance its antiviral activity and avoid systemic side effects. One approach to liver-specific targeting is conjugation of the ribavirin with asialoglycoprotein that is taken up specifically by liver cells. Human uridine-cytidine kinase-1 (UCK-1) was used for ribavirin phosphorylation to its monophosphate form. 1-Ethyl-3-diisopropylaminocarbodiimide (EDC) was used as a coupling agent. The best results were obtained using direct conjugation protocol with a molar ratio of 6.5 ribavirin monophosphate (RMP) molecules per one asialoorosomucoid (AsOR) molecule. Our findings show that ribavirin is a potential substrate of UCK-1, and RMP formed could be chemically coupled to AsOR to form a conjugate for liver specific targeting.

A novel G418 conjugate results in targeted selection of genetically protected hepatocytes without bystander toxicity.

G418, an aminoglycoside neomycin analogue, is an antimicrobial agent that interferes with protein synthesis and has been used extensively for selection of mammalian cell lines that possess neomycin resistance (NR). It is potent and nonspecific in its effects that occur through tight binding to ribosomal elements. Because of the potent intracellular effect, we wondered whether G418 could be used to select a specific cell type based on receptor-mediated endocytosis. The objective of this study was to target G418 specifically to liver cells via asialoglycoprotein receptors (AsGR) which are known to be highly selective for these cells. A novel G418 conjugate was synthesized chemically by coupling G418 to a galactose-terminating carrier protein, asialoorosomucoid (AsOR), in a molar ratio of 5:1. AsOR-G418 conjugates inhibited viability of AsGR (+) cells by 84.3%, while inhibition in AsGR (-) cells was only by 19%. In AsGR (+) cells, stably transfected with a NR gene, the conjugate decreased viability by less than 9%. Furthermore, incubation of conjugate in cocultures of AsGR (+), and AsGR (-) cells did not result in the loss of viability of neighboring AsGR (-) cells. Our data demonstrate for the first time that G418 can be covalently bound to AsOR to form a conjugate for hepatocyte-specific targeting and toxicity. AsOR-G418 conjugates may be useful tools for genetic manipulation of human liver cells in the presence of nonhepatic cells.

Potential role of RNAi in the treatment of HCV infection.

Chronic HCV infection is a leading cause of chronic hepatitis and its sequelae, liver cirrhosis and hepatocellular carcinoma. Current therapeutic options are limited, associated with significant adverse effects and costly. Accordingly, there is strong impetus to develop novel therapeutic strategies that act through alternate mechanisms. RNAi has been widely used for the analysis of gene function and represents a potentially promising approach for the treatment of HCV infection. siRNAs are short RNA duplexes approximately 21 nts long. When introduced into mammalian cells, siRNA can silence specific gene expression. Although efficient suppression of HCV replicon RNA in cell culture has been demonstrated with siRNAs, there is much work to be done to improve delivery, limit off-target effects and minimize development of virus resistance. Here, we review the use of RNAi as a tool to inhibit HCV gene expression and discuss the potential advantages and obstacles for this new potential therapeutic approach against HCV infection.

siRNA-resistance in treated HCV replicon cells is correlated with the development of specific HCV mutations.

RNA interference (RNAi) has been extremely effective against hepatitis C viral (HCV) gene expression in short-term cell culture. Our aim was to determine whether long-term RNAi might result in HCV-resistant mutants. Huh7 HCV subgenomic replicon cells were transfected with short interfering RNAs (siRNAs). HCV-RNA was quantified by real-time RT-PCR, and HCV NS5A levels were assayed by Western blots using specific antibody. Treatment with HCV-siRNA resulted in a 50% inhibition of HCV-RNA levels compared with pretreatment levels after 4 weeks (P < 0.05). HCV-RNA returned to 85% of pretreatment levels after cessation of HCV-siRNA treatment. Sequencing of the HCV-siRNA target and upstream region was performed on 10 colonies from subcloning using PCR products, each before, during and after siRNA treatment. All colonies except one from HCV-siRNA-treated cells during and after treatment had mutations. There were no mutations in the HCV-siRNA target region following control HBV-siRNA treatment. Subcloned replicon cells containing the point mutations in the target region were found to be resistant to HCV-siRNA inhibitory effects. In conclusion, even after 4 weeks of treatment of replicon cells with HCV-siRNA, HCV-RNA and HCV-NS5A protein expression could not be completely eliminated. HCV replicons isolated during or after treatment were associated with mutations in the siRNA target region, while controls were not.

Potential applications of siRNA in hepatitis C virus therapy.

Small interfering RNAs (siRNAs) are short RNA duplexes approximately 21 nucleotides long. When introduced into mammalian cells, siRNA can silence specific gene expression. Hepatitis C virus (HCV) replicates in the cytoplasm of liver cells without integration into the host genome. Because the HCV genome is a single-stranded RNA that functions both as a messenger RNA and as a viral replication template, destruction of HCV RNA could eliminate not only virally directed protein synthesis, but also viral replication. It has been demonstrated that siRNAs interfere with HCV gene expression and replication, and this review will describe the use of RNAi as a tool to inhibit HCV gene expression.

Novel delivery methods for treatment of viral hepatitis: an update.

Viral hepatitis represents the most common cause of chronic liver disease worldwide. Currently approved therapies for chronic hepatitis B include IFN, an immune modulator, and nucleoside analogues lamivudine and adefovir. For chronic hepatitis C, a combination of pegylated IFN-alpha and ribavirin represents the standard treatment. However, currently available treatments for both these viruses are effective only in a limited number of patients, are costly, prolonged, associated with significant side effects and require a substantial commitment from the patients and healthcare providers. A number of novel antiviral treatments, together with strategies to enhance the response to current therapies, are being explored at present. For all new therapies, as well as for improving existing treatments, selective delivery of medications into liver cells would be desirable to enhance antiviral activity and avoid systemic side effects. New achievements in the field of drug and gene delivery against chronic hepatitis to the liver are reviewed here.