Robert Smolić

Positional effects and strand preference of RNA interference against hepatitis C virus target sequences

Summary.  The hepatitis C virus (HCV) 3′‐untranslated region (UTR) and negative‐strand RNA sequences contribute cis‐acting functions essential to viral RNA replication. Although efficient suppression of HCV replicon RNA in cell culture has been demonstrated with small interfering RNAs (siRNAs) directed against various sequences in the 5′ UTR and coding regions, data regarding siRNA targeting of the 3′ UTR have been lacking. Furthermore, it has not been definitively shown whether the active constructs, identified to date, exert their effect exclusively via suppression of the replicon positive strand, negative strand or some combination of both strands. In the present study, we assayed inhibitory activity of various siRNAs targeting the 3′ UTR by transient transfection in a subgenomic replicon cell culture model. A survey of 13 candidate target sites in the 3′‐UTR X sequence indicated a uniformly low activity of siRNA constructs against the steady‐state level of replicon. In contrast, the majority of these same siRNAs exhibited high activity against HCV X sequences of either polarity when these targets were presented in the context of a mammalian polymerase II mRNA transcript. Transfection of siRNAs directed against other regions of the replicon revealed differences in the magnitude of inhibitory effects against positive‐strand and negative‐strand target sites. Strand preference of siRNA activity was further demonstrated through the introduction of base‐pair‐destabilizing mutations that promote strand‐specific targeting. The results suggest that the HCV positive‐strand 5′ UTR and coding region are efficiently and directly targeted by siRNA, whereas the 3′ UTR and the entire negative strand are relatively resistant to RNA interference.

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.

Antioxidant Pre-Treatment Reduces the Toxic Effects of Oxalate on Renal Epithelial Cells in a Cell Culture Model of Urolithiasis

Urolithiasis is characterized by the formation and retention of solid crystals within the urinary tract. Kidney stones are mostly composed of calcium oxalate, which predominantly generates free radicals that are toxic to renal tubular cells. The aim of the study is to explore possible effects of antioxidant pre-treatment on inhibition of oxidative stress. Three cell lines were used as in vitro model of urolithiasis: MDCK I, MDCK II and LLC-PK1. Oxidative stress was induced by exposure of cells to sodium oxalate in concentration of 8 mM. In order to prevent oxidative stress, cells were pre-treated with three different concentrations of l-arginine and vitamin E. Oxidative stress was evaluated by determining the expression of superoxide dismutase (SOD), osteopontin (OPN), and by the concentration of glutathione (GSH). In all three cell lines, pre-treatment of antioxidants increased cell survival. Positive correlation of SOD and OPN expression as well as GSH concentration was observed in all groups of cells. Our results indicate that an antioxidant pre-treatment with l-arginine and vitamin E is able to hamper oxalate-induced oxidative stress in kidney epithelial cells and as such could play a role in prevention of urolithiasis.

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.

Primary Care Provider Counseling Practices about Adverse Drug Reactions and Interactions in Croatia

Background: Prescribing medications is one of the most common medical decisions that is made by primary care providers (PCPs). In the Republic of Croatia, PCPs hold a key position in prescribing and evaluating the medications that are provided for patients. Accordingly, providing advice for patients regarding the potential adverse drug reactions (ADRs) and drug-drug interactions (DDIs) is frequently the responsibility of the PCPs. The aim of the current study was to assess the knowledge, attitudes, and counseling practices of PCPs regarding drug interactions and adverse effects. Methods: After enrolling 195 PCPs that were selected at random, a survey was conducted while using an anonymous questionnaire that was created based on previously published studies, adjusted in a way that includes the most commonly prescribed medications in Croatia. Results: Of the 10 questions on knowledge about DDIs and ADRs, the median number of correct responses by PCPs was 5 (interquartile range 4 to 7). More than half of respondents (56%) agreed with the claim that knowledge of drug side effects facilitated their work in family medicine. Almost all of the respondents (92.8%) explained side effects and drug interactions to special groups of patients (pregnant women, elderly patients etc.). Conclusion: The results show a need for additional education in the field of drug prescribing. However, PCPs were aware of the importance of counseling practices about adverse drug reactions and interactions and counseling practices among special patients populations are satisfactory.

Association of Wnt Inhibitors, Bone Mineral Density and Lifestyle Parameters in Women with Breast Cancer Treated with Anastrozole Therapy

Aim: To determine the levels of Wnt inhibitors in patients treated with aromatase inhibitors (AIs) prior to therapy and to investigate their association with bone mineral density (BMD) and lifestyle parameters. Methods: 137 breast cancer patients were divided into a group treated with 1 mg of anastrozole and a group w/o anastrozole therapy. Serum concentrations of sclerostin and dickkopf1 (DKK1) were measured by ELISA. BMD was measured by dual-energy X-ray absorptiometry (DXA). Lifestyle factors were investigated by a self-reported questionnaire. Results: Sclerostin was significantly higher in the AI-treated group (31.8 pmol/L vs. 24.1 pmol/L; p < 0.001), whereas DKK1 was significantly lower in the AI-treated group (24.3 pmol/L vs. 26.02 pmol/L; p < 0.001). Total hip and femoral neck BMD were significantly lower in the AI-treated group. Conclusion: AI treatment was associated with increased levels of sclerostin and decreased levels of DKK1.

Models of Drug Induced Liver Injury (DILI) – Current Issues and Future Perspectives

Background: Drug-induced Liver Injury (DILI) is an important cause of acute liver failure cases in the United States, and remains a common cause of withdrawal of drugs in both preclinical and clinical phases. Methods: A structured search of bibliographic databases – Web of Science Core Collection, Scopus and Medline for peer-reviewed articles on models of DILI was performed. The reference lists of relevant studies was prepared and a citation search for the included studies was carried out. In addition, the characteristics of screened studies were described. Results: One hundred and six articles about the existing knowledge of appropriate models to study DILI in vitro and in vivo with special focus on hepatic cell models, variations of 3D co-cultures, animal models, databases and predictive modeling and translational biomarkers developed to understand the mechanisms and pathophysiology of DILI are described. Conclusion: Besides descriptions of current applications of existing modeling systems, associated advantages and limitations of each modeling system and future directions for research development are discussed as well.

Current Management and Novel Therapeutic Strategies to Combat Chronic Delta Hepatitis

HDV is an unusal, defective hepatotropic virus which causes severe acute hepatitis and most progressive chronic viral hepatitis. Despite the efforts in eradication of HDV, as its obligatory helper HBV, prevalence of HDV in developed countries remains stable and represents a relevant public health concern. Current conventional therapy of hepatitis delta is characterized with poor overall response, thus further investigations of novel treatment options are needed. Continuous research of virology and pathogenesis is necessary to provide fundamentals for development of novel approaches in treatment of HDV.

Pharmacogenetic selection of transplanted human hepatocytes in immunocompetent rats

To introduce a genetic survival advantage for transplanted human hepatocytes over host cells in rats.

Mn-SOD and Chronic Inflammation of Gastric Mucosa

The involvement of reactive oxygen species in the inflammatory tissue destruction is well known. Significant changes in the activity and expression of several isoforms of superoxide dismutase were observed in the human gastric disease. Mn-SOD attracted the attention of researchers because of its inducibility by oxidative stress. There is increasing evidence that oxidative stress plays a role in the progression of mucosal damage leading to gastric cancer. The evaluation of possible modulation of Mn-SOD activity during chronic inflammation of gastric mucosa could reveal whether its assessment is important to prevent the accumulation of gastric epithelial cell damage and thereby reduce the risk of gastric carcinoma.