Association of Helicobacter pylori Positivity With Risk of Disease and Mortality.

INTRODUCTION: Helicobacter pylori colonizes the human stomach. Infection causes chronic gastritis and increases the risk of gastroduodenal ulcer and gastric cancer. Its chronic colonization in the stomach triggers aberrant epithelial and inflammatory signals that are also associated with systemic alterations. METHODS: Using a PheWAS analysis in more than 8,000 participants in the community-based UK Biobank, we explored the association of H. pylori positivity with gastric and extragastric disease and mortality in a European country. RESULTS: Along with well-established gastric diseases, we dominantly found overrepresented cardiovascular, respiratory, and metabolic disorders. Using multivariate analysis, the overall mortality of H. pylori -positive participants was not altered, while the respiratory and Coronovirus 2019-associated mortality increased. Lipidomic analysis for H. pylori -positive participants revealed a dyslipidemic profile with reduced high-density lipoprotein cholesterol and omega-3 fatty acids, which may represent a causative link between infection, systemic inflammation, and disease. DISCUSSION: Our study of H. pylori positivity demonstrates that it plays an organ- and disease entity-specific role in the development of human disease and highlights the importance of further research into the systemic effects of H. pylori infection.

[Current Developments in Nutritional Therapy of Intensive Care Patients]. / Aktuelle Entwicklungen in der Ernährungstherapie von Intensivpatienten.

The goals of nutritional therapy for critically ill patients are the enteral or parenteral provision of calories, proteins, electrolytes, vitamins, trace elements and fluids. The metabolism of critically ill patients is characterized by a catabolic stress condition, which is caused by a systemic inflammatory response and is associated with an increased infection rate, occurrence of multiple organ failure and increased mortality.Traditionally, nutritional therapy has been considered as an "adjunctive" therapy for critically ill patients with the primary goal of providing energy to maintain muscle mass. Based on the scientific developments of recent years, it became evident that adequate nutritional therapy can favorably influence stress-related metabolic processes, reduce oxidative cell damage and modulate the immune response of the body. Important measures that can positively influence the clinical course of intensive care patients include early enteral nutrition, adequate intake of macro- and micronutrients, and blood glucose control.

Hsp72 protects against liver injury via attenuation of hepatocellular death, oxidative stress, and JNK signaling.

BACKGROUND & AIMS: Heat shock protein (Hsp) 72 is a molecular chaperone that has broad cytoprotective functions and is upregulated in response to stress. To determine its hepatic functions, we studied its expression in human liver disorders and its biological significance in newly generated transgenic animals. METHODS: Double transgenic mice overexpressing Hsp72 (gene Hspa1a) under the control of a tissue-specific tetracycline-inducible system (Hsp72-LAP mice) were produced. Acute liver injury was induced by a single injection of acetaminophen (APAP). Feeding with either a methionine choline-deficient (MCD; 8 weeks) or a 3,5-diethoxycarbonyl-1,4-dihydrocollidine-supplemented diet (DDC; 12 weeks) was used to induce lipotoxic injury and Mallory-Denk body (MDB) formation, respectively. Primary hepatocytes were treated with palmitic acid. RESULTS: Patients with non-alcoholic steatohepatitis and chronic hepatitis C infection displayed elevated HSP72 levels. These levels increased with the extent of hepatic inflammation and HSP72 expression was induced after treatment with either interleukin (IL)-1ß or IL-6. Hsp72-LAP mice exhibited robust, hepatocyte-specific Hsp72 overexpression. Primary hepatocytes from these animals were more resistant to isolation-induced stress and Hsp72-LAP mice displayed lower levels of hepatic injury in vivo. Mice overexpressing Hsp72 had fewer APAP protein adducts and were protected from oxidative stress and APAP-/MCD-induced cell death. Hsp72-LAP mice and/or hepatocytes displayed significantly attenuated Jnk activation. Overexpression of Hsp72 did not affect steatosis or the extent of MDB formation. CONCLUSIONS: Our results demonstrate that HSP72 induction occurs in human liver disease, thus, HSP72 represents an attractive therapeutic target owing to its broad hepatoprotective functions. LAY SUMMARY: HSP72 constitutes a stress-inducible, protective protein. Our data demonstrate that it is upregulated in patients with chronic hepatitis C and non-alcoholic steatohepatitis. Moreover, Hsp72-overexpressing mice are protected from various forms of liver stress.

Anti-tumorigenic and anti-angiogenic effects of natural conifer Abies sibirica terpenoids in vivo and in vitro.

AIM: The natural terpenoid compound was explored in vitro and in vivo to investigate the anti-HCC properties. METHODS: For our study we used Abisilin®- a novel natural pharmacological terpenoid compound extracted and purified from coniferous Pinaceae trees. Anti-tumorigenic properties of different concentrations of Abisilin® were tested on murine hepatoma Hepa 1-6 cell lines. The analysis of proliferation and apoptosis was performed using immunofluorescence microscopy, FACS and qPCR. As an in vivo approach, we tested Abisilin® (400mg/kg/day, 14 days, orally) in xenograft mouse models of liver cancer and investigated tumor growth, proliferation, apoptosis and angiogenesis by means of Western blotting, immunofluorescence microscopy and qPCR. RESULTS: Application of Abisilin® for 24h at a dosage ranging from 0.03 to 0.045mg significantly reduced the number of viable Hepa 1-6 cells and induced apoptotic cell death with microscopic evidence of changes in cell morphology, and positive TUNEL, cleaved caspase 3 and Annexin V/Propidium Iodide (PI) stainings. Furthermore, treatment with Abisilin® strongly inhibited proliferation, impaired mitosis and prompted cell cycle arrest by down-regulation of the Cyclin D1, E1 and A2 expression levels. In Hepa 1-6 xenograft in vivo model, Abisilin® considerably decreased the xenograft tumor size and tumor volume. Consistently with in vitro Abisilin® administration elicited apoptosis and inhibit proliferation in the xenograft tumor. We also found that Abisilin® remarkably decrease microvessel density, diminished tumor angiogenesis and reduced expression of ICAM-1. Moreover, the expression of pAMPK, a cellular energy sensor, was up-regulated after Abisilin® application. CONCLUSIONS: Anti-proliferative, pro-apoptotic activity and anti-angiogenic potential of natural conifer terpenoids might turn these compounds into an attractive drug candidate for combination therapy against liver cancer.

Hepcidin knockout mice spontaneously develop chronic pancreatitis owing to cytoplasmic iron overload in acinar cells.

Iron is both an essential and a potentially toxic element, and its systemic homeostasis is controlled by the iron hormone hepcidin. Hepcidin binds to the cellular iron exporter ferroportin, causes its degradation, and thereby diminishes iron uptake from the intestine and the release of iron from macrophages. Given that hepcidin-resistant ferroportin mutant mice show exocrine pancreas dysfunction, we analysed pancreata of aging hepcidin knockout (KO) mice. Hepcidin and Hfe KO mice were compared with wild-type (WT) mice kept on standard or iron-rich diets. Twelve-month-old hepcidin KO mice were subjected to daily minihepcidin PR73 treatment for 1 week. Six-month-old hepcidin KO mice showed cytoplasmic acinar iron overload and mild pancreatitis, together with elevated expression of the iron uptake mediators DMT1 and Zip14. Acinar atrophy, massive macrophage infiltration, fatty changes and pancreas fibrosis were noted in 1-year-old hepcidin KO mice. As an underlying mechanism, 6-month-old hepcidin KO mice showed increased pancreatic oxidative stress, with elevated DNA damage, apoptosis and activated nuclear factor-κB (NF-κB) signalling. Neither iron overload nor pancreatic damage was observed in WT mice fed iron-rich diet or in Hfe KO mice. Minihepcidin application to hepcidin KO mice led to an improvement in general health status and to iron redistribution from acinar cells to macrophages. It also resulted in decreased NF-κB activation and reduced DNA damage. In conclusion, loss of hepcidin signalling in mice leads to iron overload-induced chronic pancreatitis that is not seen in situations with less severe iron accumulation. The observed tissue injury can be reversed by hepcidin supplementation. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Analysis of drug-related problems in three departments of a German University hospital.

BACKGROUND: During the last decades, pharmaceutical care services have been developed and implemented to optimize drug therapies and ensure medication safety. To investigate the need for pharmaceutical care services, drug-related problems can be measured. OBJECTIVE: Thus, the aim of this study was to analyse number, type and occurrence of drug-related problems in different clinical departments. SETTING: A pharmaceutical care service was established on general wards in Urology, Neurology and Gastroenterology at the University Hospital RWTH Aachen, Germany. METHOD: For each of a total of 306 patients, a pharmacist conducted an extended medication history, performed medication reconciliation, conducted medication safety checks and if drug-related problems were discovered, gave valid recommendations to the attending healthcare team. Drug-related problems were classified using the APS-Doc system. For statistical analyses, SAS(®) 9.1.3, SAS Institute, Cary NC, USA was applied. The project was approved by the local ethics committee. MAIN OUTCOME MEASURE: Type, occurrence and frequency of DRP in different medical departments. RESULTS: On average, 2.3 drug-related problems per patient were documented for all three departments. Drug-related problems were found in each category of the APS-Doc system. The most pronounced drug-related problems found were drug-drug interactions (34.6 %). 37 % of the identified drug-related problems occurred before hospital admission, 27 % during transitional care, and 36 % on the ward. Subgroup analysis revealed specific drug-related problem patterns for each clinical department. The number of drug-related problems was found to be associated with the number of drugs and age. CONCLUSION: Drug-related problems frequently occur in all investigated clinical departments. A holistic pharmaceutical care service could be an option to address this issue. In case of limited resources, individual drug-related problem patterns can be used as a basis for a tailored pharmaceutical care service. As number of drugs and age have been shown to be significant risk factors, it is crucial that the healthcare team including the pharmacist pays special attention to elderly patients and those with polymedication.

Loss of keratin 19 favours the development of cholestatic liver disease through decreased ductular reaction.

Keratins (K) are cytoprotective proteins and keratin mutations predispose to the development of multiple human diseases. K19 represents the most widely used marker of biliary and hepatic progenitor cells as well as a marker of ductular reaction that constitutes the basic regenerative response to chronic liver injury. In the present study, we investigated the role of K19 in biliary and hepatic progenitor cells and its importance for ductular reaction. K19 wild-type (WT) and knockout (KO) mice were fed: (a) 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC); (b) cholic acid (CA); (c) a choline-deficient, ethionine-supplemented (CDE) diet; or (d) were subjected to common bile duct ligation (CBDL). The bile composition, liver damage, bile duct proliferation, oval cell content and biliary fibrosis were analysed. In untreated animals, loss of K19 led to redistribution of the K network in biliary epithelial cells (BECs) but to no obvious biliary phenotype. After DDC feeding, K19 KO mice exhibited (compared to WTs): (a) increased cholestasis; (b) less pronounced ductular reaction with reduced ductular proliferation and fewer oval cells; (c) impaired Notch 2 signalling in BECs; (d) lower biliary fibrosis score and biliary bicarbonate concentration. An attenuated oval cell proliferation in K19 KOs was also found after feeding with the CDE diet. K19 KOs subjected to CBDL displayed lower BEC proliferation, oval cell content and less prominent Notch 2 signal. K19 deficiency did not change the extent of CA- or CBDL-induced liver injury and fibrosis. Our results demonstrate that K19 plays an important role in the ductular reaction and might be of importance in multiple chronic liver disorders that frequently display a ductular reaction.

Caspase 8 differentially controls hepatocytes and non-parenchymal liver cells during chronic cholestatic liver injury in mice.

BACKGROUND & AIMS: Receptor mediated cell death through the activation of caspases has been identified as an important mechanism to control life and death in various tissues and is thus crucial for the maintenance of liver tissue homeostasis. Here we investigated how caspase 8 (Casp8) differentially regulates immune-mediated liver injury and regeneration in distinct liver cell types during chronic liver injury. METHODS: Conditional knockout mice with hepatocellular (Casp8(Δhepa)) and ubiquitous deletion of Casp8 (Casp8(ΔMx)) were used in models of cholestatic hepatitis [(DDC (3,5-diethoxycarbonyl-1,4-dihydrocollidine) treatment, bile duct ligation (BDL) and choline deficient diet with ethionine supplementation (CDE)]. RESULTS: Mice with a hepatocellular deletion of Casp8 (Casp8(Δhepa)) were protected after DDC-treatment. Animals with a ubiquitous conditional Casp8 knockout (Casp8(ΔMx)) displayed a significantly enhanced liver injury in various models of cholestatic liver injury. This was associated with higher transaminases, bilirubin levels and finally more liver fibrosis. However, caspase 3 (Casp3) activity was reduced in both knockout strains, suggesting additionally mechanisms contributing to the phenotype. Casp8(ΔMx) mice displayed a stronger infiltration of mononuclear immune cells and more proliferation of liver-parenchymal cells in periportal areas. Further analysis confirmed that these infiltrating immune cells are resistant against extrinsic apoptosis. Bone-marrow-transplantation (BMT) experiments demonstrated that Casp8-deficient bone marrow derived cells are responsible for increased liver injury in DDC fed mice. CONCLUSIONS: Our results demonstrate that cell-type specific differences in apoptosis resistance mediated by Casp8 deletion are of significant relevance for the outcome of chronic liver injury.

Pharmacological inhibition of the chemokine CCL2 (MCP-1) diminishes liver macrophage infiltration and steatohepatitis in chronic hepatic injury.

OBJECTIVE: Monocyte chemoattractant protein-1 (MCP-1, CCL2), the primary ligand for chemokine receptor C-C chemokine receptor 2 (CCR2), is increased in livers of patients with non-alcoholic steatohepatitis (NASH) and murine models of steatohepatitis and fibrosis. It was recently shown that monocyte/macrophage infiltration into the liver upon injury is critically regulated by the CCL2/CCR2 axis and is functionally important for perpetuating hepatic inflammation and fibrogenesis. The structured L-enantiomeric RNA oligonucleotide mNOX-E36 (a so-called Spiegelmer) potently binds and inhibits murine MCP-1. Pharmacological inhibition of MCP-1 with mNOX-E36 was investigated in two murine models of chronic liver diseases. METHODS: Pharmacological inhibition of MCP-1 by thrice-weekly mNOX-E36 subcutaneously was tested in murine models of acute or chronic carbon tetrachloride (CCl(4))- and methionine-choline-deficient (MCD) diet-induced chronic hepatic injury in vivo. RESULTS: Antagonising MCP-1 by mNOX-E36 efficiently inhibited murine monocyte chemotaxis in vitro as well as migration of Gr1(+) (Ly6C(+)) blood monocytes into the liver upon acute toxic injury in vivo. In murine models of CCl(4)- and MCD diet-induced hepatic injury, the infiltration of macrophages into the liver was significantly decreased in anti-MCP-1-treated mice as found by fluorescence-activated cell sorting (FACS) analysis and immunohistochemistry. In line with lower levels of intrahepatic macrophages, proinflammatory cytokines (tumour necrosis factor α, interferon γ and interleukin 6) were significantly reduced in liver tissue. Overall fibrosis progression over 6 (CCl(4)) or 8 weeks (MCD diet) was not significantly altered by anti-MCP-1 treatment. However, upon MCD diet challenge a lower level of fatty liver degeneration (histology score, Oil red O staining, hepatic triglyceride content, lipogenesis genes) was detected in mNOX-E36-treated animals. mNOX-E36 also ameliorated hepatic steatosis upon therapeutic administration. CONCLUSIONS: These results demonstrate the successful pharmacological inhibition of hepatic monocyte/macrophage infiltration by blocking MCP-1 during chronic liver damage in two in vivo models. The associated ameliorated steatosis development suggests that inhibition of MCP-1 is an interesting novel approach for pharmacological treatment in liver inflammation and steatohepatitis.

Serotonin receptor type 3 antagonists improve obesity-associated fatty liver disease in mice.

Obesity is a major cause for nonalcoholic fatty liver disease (NAFLD). Previous studies suggested that alterations in intestinal motility and permeability contribute to the development of NAFLD. Serotonin and serotonin receptor type 3 (5-HT(3)R) are key factors in the regulation of intestinal motility and permeability. Therefore, we studied the effect of the 5-HT(3)R antagonists tropisetron and palonosetron on the development of NAFLD in leptin-deficient obese mice. Four-week-old ob/ob mice and lean controls were treated for 6 weeks orally with tropisetron or palonosetron at 0.2 mg/kg per day. We determined markers of liver damage and inflammation, portal endotoxin levels, and duodenal concentrations of serotonin, serotonin-reuptake transporter (SERT), occludin, and claudin-1. Tropisetron treatment significantly reduced liver fat content (-29%), liver inflammation (-56%), and liver cell necrosis (-59%) in ob/ob mice. The beneficial effects of tropisetron were accompanied by a decrease in plasma alanine aminotransferase and portal vein plasma endotoxin levels, an attenuation of enhanced MyD88 and tumor necrosis factor-α mRNA expression in the liver, and an increase of tight junction proteins in the duodenum. Tropisetron treatment also caused a reduction of elevated serotonin levels and an increase of SERT in the duodenum of ob/ob mice. Palonosetron had similar effects as tropisetron with regard to the reduction of liver fat and other parameters. Tropisetron and palonosetron are effective in attenuating NAFLD in a genetic mouse model of obesity. The effect involves the intestinal nervous system, resulting in a reduction of endotoxin influx into the liver and subsequently of liver inflammation and fat accumulation.

Nor-ursodeoxycholic acid reverses hepatocyte-specific nemo-dependent steatohepatitis.

BACKGROUND: Hepatocyte-specific NEMO/NF-κB deleted mice (NEMO(Δhepa)) develop spontaneous non-alcoholic steatohepatitis (NASH). Free fatty acids and bile acids promote DR5 expression. TRAIL/NK cell-mediated activation of TRAIL-R2/DR5 plays an important role during acute injury in NEMO(Δhepa) mice. AIM: To inhibit the progression of NASH in the absence of hepatocyte-NEMO/NF-kB signaling. METHODS: NEMOf/f and NEMO(Δhepa) mice were fed with a low-fat diet, and with two anticholestatic diets; UDCA and NorUDCA. The impact of these treatments on the progression of NASH was evaluated. RESULTS: We show that high expression of DR5 in livers from NEMO(Δhepa) mice is accompanied by an abundant presence of bile acids (BAs), misregulation of BA transporters and significant alteration of lipid metabolism-related genes. Additionally, mice lacking NEMO in hepatocytes spontaneously showed ductular response at young age. Unexpectedly, feeding of NEMO(Δhepa) mice with low-fat diet failed to improve chronic liver injury. Conversely, anti-cholestatic treatment with nor-ursodeoxycholic acid (NorUDCA), but not with ursodeoxycholic acid (UDCA), led to a significant attenuation of liver damage in NEMO(Δhepa) mice. The strong therapeutic effect of NorUDCA relied on a significant downregulation of LXR-dependent lipogenesis and the normalisation of BA metabolism through mechanisms involving cross-talk between Cyp7a1 and SHP. This was associated with the significant improvement of liver histology, NEMO(Δhepa)/NorUDCA-treated mice showed lower apoptosis and reduced CyclinD1 expression, indicating attenuation of the compensatory proliferative response to hepatocellular damage. Finally, fibrosis and ductular reaction markers were significantly reduced in NorUDCA-treated NEMO(Δhepa) mice. CONCLUSIONS: Overall, our work demonstrates the contribution of bile acids metabolism to the progression of NASH in the absence of hepatocyte-NF-kB through mechanisms involving DR5-apoptosis, inflammation and fibrosis. Our work suggests a potential therapeutic effect of NorUDCA in attenuating the progression of NASH.

Lack of interleukin-6/glycoprotein 130/signal transducers and activators of transcription-3 signaling in hepatocytes predisposes to liver steatosis and injury in mice.

UNLABELLED: A deregulated cytokine balance is involved in triggering the sequence from steatosis to nonalcoholic steatohepatitis, ultimately leading to liver fibrosis and cancer. To better define the role of proinflammatory interleukin-6 (IL-6)-type cytokines in hepatocytes we investigated the role of IL-6 and its shared receptor, glycoprotein 130 (gp130), in a mouse model of steatohepatitis. IL-6(-/-) mice were fed a choline-deficient, ethionine-supplemented (CDE) diet. Conditional gp130 knockout and knockin mice were used to achieve hepatocyte-specific deletion of gp130 (gp130(Deltahepa)), gp130-dependent rat sarcoma (Ras)-(gp130(DeltahepaRas)), and signal transducers and activators of transcription (STAT)-(gp130(DeltahepaSTAT)) activation. CDE-treated IL-6(-/-) mice showed a significant hepatic steatosis at 2 weeks after feeding. The mice rapidly developed elevated fasting blood glucose, insulin serum levels, and transaminases. To better define IL-6-dependent intracellular pathways, specifically in hepatocytes, we next treated gp130(Deltahepa) mice with a CDE diet. These animals also developed a marked steatosis with hyperglycemia and displayed elevated insulin serum levels. Additionally, gp130(Deltahepa) animals showed an imbalanced inflammatory response with increased hepatic tumor necrosis factor-alpha and decreased adiponectin messenger RNA levels. Dissecting the hepatocyte-specific gp130-dependent pathways revealed a similar disease phenotype in gp130(DeltahepaSTAT) mice, whereas gp130(DeltahepaRas) animals were protected. In CDE-treated mice lack of gp130-STAT3 signaling was associated with immune-cell-infiltration, jun kinase-activation, a blunted acute-phase-response, and elevated transaminases. Furthermore, gp130(Deltahepa) and gp130(DeltahepaSTAT) mice showed beginning signs of liver fibrosis compared to gp130(DeltahepaRas) mice and controls. CONCLUSION: During CDE treatment mice lacking IL-6 and gp130-STAT signaling in hepatocytes are prone to hepatic metabolic changes and inflammation. This ultimately leads to progressive steatohepatitis with signs of liver remodeling. Thus, the presented model allows one to further dissect the role of IL-6/gp130-type signaling in hepatocytes during fatty liver degeneration to define new therapeutic targets in metabolic liver diseases.

Elevation of Nepsilon-(carboxymethyl)lysine-modified advanced glycation end products in chronic liver disease is an indicator of liver cirrhosis.

OBJECTIVES: Progression of liver fibrosis to cirrhosis is a dire consequence of chronic liver diseases (CLD). Nepsilon-(carboxymethyl)lysine (CML)-modified advanced glycation end products (AGEs) in patients with CLD could reflect the degree of severity of the disease. DESIGN AND METHODS: In 110 patients with CLD and 124 healthy controls, CML serum levels and their diagnostic sensitivity and specificity were determined and compared to hyaluronan (HA). RESULTS: Serum levels of CML were significantly affected by the stage of liver cirrhosis and were closely associated with liver function capacity. CML correlated positively with HA (r = 0.639, P < 0.0001). In ROC analysis, the diagnostic sensitivity and specificity in distinguishing healthy controls from liver disease patients for CML (AUC 0.908; 95%-CI 0.863-0.942, cut-off 640 ng/mL, sensitivity 74.5% and specificity 97.6%) resembled HA (AUC 0.948; 95%-CI 0.907-0.974; cut-off 50 ng/mL, sensitivity 80.7% and specificity 97.9%). The combination of CML and HA shows an AUC of 0.932; 95%-CI 0.888-0.962; sensitivity 82.6%; and specificity 95.8%. CONCLUSIONS: Our data suggest that serum levels of CML could provide a supplementary diagnostic marker for advanced stages of liver cirrhosis. However, the quality of interaction needs further investigation.

Inhibition of hepatitis B virus replication in vivo by nucleoside analogues and siRNA.

BACKGROUND & AIMS: Hepatitis B virus (HBV) causes acute and chronic infections that may result in severe liver diseases. Animal models to study new treatment options in vivo have several drawbacks. Therefore, we were interested to establish a new small animal model in which HBV replication and especially new treatment options can be studied easily. METHODS: Naked DNA of an HBV replication competent vector was transferred via tail vein into NMRI mice. HBV replication was studied in serum and liver of the animals. HBV replication was modulated by treatment through siRNA and nucleoside analogues. RESULTS: Tail vein transfer of a HBV replication competent construct resulted in expression of HBV-specific transcripts in the liver, and up to 10% of hepatocytes became HBc- and HBsAg-positive. HBeAg, HBsAg, and viral DNA could be detected in the serum of the animals, followed by the induction of HBV-specific cellular immune responses. Nucleoside treatment of the mice resulted in reduced polymerase activity in the liver. Additionally, siRNA transfer in the animals led to a significant reduction of HBsAg and/or eventually HBeAg expression, which was dependent on the localization of the complementary sequence in the HBV genome. CONCLUSIONS: We have established a mouse model to study HBV replication and to investigate new and existing treatment approaches in vivo. Interestingly, siRNA seems a promising innovative treatment option to inhibit specifically HBV replication in vivo.