Silencing of the SR gene induces a decrease in the basal proliferative capacity of large cholangiocytes compared with large

mock-transfected cholangiocytes. In our evaluation of SR expression, we found a time-dependent increase in the expression of SR in large cholangiocytes during BDL compared with normal large cholangiocytes. This finding was consistent with previous studies showing that: (1) in the rodent liver SR is only expressed by large cholangiocytes,1, 4, 5, 9, 12 (2) SR expression is up-regulated following BDL ligation in large cholangiocytes,14, 17 and (3) the extent of secretin effects on cholangiocyte functions parallel with the duration of BDL.16 This finding parallels recent findings that mouse cholangiocytes share a similar heterogeneous high throughput screening assay profile

as rat cholangiocytes5 and freshly isolated and immortalized large mouse cholangiocytes are the only cell types to express the SR.5, 8, 14 In human, SR expression is present in the biliary tract in Pirfenidone normal bile ducts and ductules and the majority of cholangiocarcinomas, but is not present in hepatocytes or hepatocellular carcinoma.26, 27 Consistent with animal models of cholestasis, SR expression was up-regulated in ductular reactions in liver cirrhosis.27 In our in vivo model, the level of the reduction of cholangiocyte proliferation is consistent with the paradigm that cholangiocyte proliferation is regulated in autocrine and paracrine mechanisms by a number of stimulatory neurohormonal factors.18, 20, 28 In a knockout mouse model for α-calcitonin gene-related peptide, the lack of circulating α-calcitonin gene-related peptide also reduces biliary proliferation during BDL to a similar degree as the lack of SR,20 which indicates that the regulation of biliary

proliferation during extrahepatic cholestasis is multifactorial and a complex regulatory system.18, 20, 28 The trophic effects of secretin were dependent upon the activation of the cAMP/PKA/ERK1/2 signaling. The second messenger system, cAMP, is a key factor for the function of large cholangiocytes.1, 4, 7, 9, 13 Secretin stimulates bicarbonate secretion of large bile ducts through activation of cAMP-dependent CFTRCl−/HCO3− anion exchanger 2.1, 4, 7, 9, 13 Also, the activation of the cAMP/PKA/ERK1/2 Niclosamide pathway modulates cholangiocyte proliferation.12, 15, 18, 29 In fact, the direct stimulation of adenylyl cyclase activity by the chronic administration of forskolin stimulates normal cholangiocyte proliferation both in vivo and in vitro, which is associated with activation of the PKA/Src/MEK/ERK1/2 pathway.29 Maintenance of cAMP levels by forskolin administration prevents the impairment of cholangiocyte proliferation and enhancement of biliary apoptosis induced by vagotomy.30 Furthermore, Banales et al. have shown31 that cAMP stimulates cholangiocyte proliferation through two downstream effectors (i.e., PKA and Epacs) in an animal model of autosomal recessive polycystic kidney disease.

Mouse sera were assayed for HBsAg and capsid-associated HBV DNA at the indicated time points after injection. The SensoLyte FDP SEAP Reporter Gene Assay kit (AnaSpec, Fremont, CA) was used to detect SEAP activity in mouse sera. For immunofluorescence staining of the HBV core antigen, mouse livers were fixed with 4% formalin overnight, cryoprotected in 30% sucrose, and sectioned at a thickness of 10 μm, using Leica cryostat (Leica

Microsystems, Buffalo Grove, IL), and mounted on Superfrost glass slides (Thermo Fisher Scientific). Sections were incubated with the primary antibody (anti-HBc; US Biological) overnight, followed by incubation with the goat anti-rabbit secondary antibody conjugated with Alexa Fluor 568 (Invitrogen). Slides were subsequently counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Images were captured using a Zeiss LSM 510 Meta Confocal Microscope (Carl Zeiss GmbH, Jena, Germany). 5-Fluoracil For Western blot analysis of the HBV core protein, approximately 120 mg of the mouse liver was rinsed with cold buffer A (50 mM Tris-HCl, pH 7.0, 2 mM EDTA, and 150 mM NaCl) and homogenized in buffer B (50 mM Tris-HCl, pH 7.0, 10% glycerol, 5 mM MgCl2, 0.2 mM EDTA, Selleckchem MLN0128 1 mM dithiothreitol, and 1 × protease inhibitor cocktail). The homogenates were centrifuged at 15,000g

for 30 minutes twice to pellet the cell debris. Next, 150 μg of total proteins were analyzed in 15% SDS-PAGE, using the same protocol described above for HepG2 cell lysates. BLOCK-iT Pol II miR RNAi expression vectors (Invitrogen) were used to knock down the expression of KLF15 in mice. To analyze the expression level of KLF15 in miR RNAi-transfected hepatocytes, mice were anesthetized and their livers were perfused with collagenase 3 days after hydrodynamic

injection to obtain hepatocytes, which were subsequently sorted by flow cytometry to separate transfected (i.e., green fluorescent protein [GFP]-positive) hepatocytes from untransfected (i.e., GFP-negative) hepatocytes. To analyze the effect of KLF15 on viral gene expression, 10 μg of pAAV-HBV1.2 or pAAV-HBV1.2-CPm2 and 5 μg of pLive-SEAP were coinjected into mice through the tail veins. All of the plasmids used mafosfamide for hydrodynamic injection were prepared using the EndoFree plasmid preparation kit (Qiagen). The Student t test and Mann-Whitney U test were used to analyze data. A value of P < 0.05 was regarded as statistically significant. To identify host factors that can promote HBV gene expression, we initiated a yeast one-hybrid assay to screen for transcription factors that could bind the HBV major surface promoter. Multiple screens pulled out the previously identified NF-Y transcription factor, as well as a few members of the KLF family of transcription factors12 (T. Tan and T.S.B. Yen, unpublished data).

Interpretation of data comparisons, and subsequent predictions of virulence genes, are heavily dependent on the experimental design, and relate directly to the choice of the time point(s), choice of the reference sample(s) and reliance on data drawn from populations of cells. Single time-point analyses evidently do not provide the resolving power necessary to predict virulence determinants relevant to multistage pathogenetic processes, as evidenced by the requirement for glyoxylate cycle-encoding gene products,

acting at prepenetrative stages of infection, for virulence in M. grisea (Wang et al., 2003) Opaganib chemical structure and their apparently static levels of transcription (Table 2) in invasive hyphae. For comparative microarray analyses (including the find more choice of the comparator SAGE tag library in SAGE analytical approaches), the origin of the reference sample profoundly impacts on up- and downregulated genesets. It may, therefore, be naive to expect experiments using reference samples of diverse nutrient compositions (e.g. YPD, RPMI1640 and LIM) to result in similar gene expression profiles. A case in point is provided by a collective

impediment to fungal propagation in plant and animals: the lack of available iron, which is an essential cofactor for many cellular processes. Ustilago maydis, M. grisea and A. fumigatus use siderophores, a class of nonribosomal peptide synthase (NRPS)-dependent secondary metabolites, to scavenge ferric ion selectively through the formation of soluble chelation complexes (Schrettl et al., 2007; Bolker et al., 2008; Hof et al., 2009). Intra- and extracellular siderophores are required for full virulence in a pulmonary murine model of invasive aspergillosis (Schrettl et al., 2007), and accordingly, gene expression at siderophore biosynthetic gene clusters was induced in a similar murine model at 14-h postinfection, indicating that the response to iron limitation in the mammalian host is addressed at a very early stage of infection (McDonagh et al., 2008). Therefore,

concordance between transcriptional data and important Amino acid virulence determinants can be expected from this type of analysis. However, despite the observed similarity of gene expression profiles between A. fumigatus and C. neoformans, iron acquisition was not identified as an important component of the infecting C. neoformans transcriptome. This may, in part, be due to the use of an LIM comparator in the C. neoformans experimentation, which would undoubtedly occlude, at the transcriptional level, this aspect of pathogenic growth. While C. neoformans does not synthesize siderophores, iron acquisition is crucial for C. neoformans virulence (Jung et al., 2009). Thewes and colleagues also found gene expression that reflected iron limitation.

5 In contrast Wang et al. found that a higher BMI predicted absence of symptoms.6 Men and older patients have been thought to have a higher pain threshold12,13 but clearly we need to understand the pathogenesis of symptoms in GERD better. The classical explanation of evocation of heartburn is that it is caused by the contact of acid on the nerve endings in the lower esophagus.

Patients with erosive reflux esophagitis would then intuitively experience more pain than those with non-erosive reflux disease (NERD). This, however, has not been the case and NERD patients may in fact experience more severe symptoms than those with erosive disease.14 It is clear therefore that apart from the degree of acid exposure, various other putative PD0325901 supplier mechanisms

are plausible. Esophageal mucosal sensitivity, prolonged or abnormal esophageal contraction and psychological factors have all been shown to play a role.15–17 The role of esophageal sensitivity in the pathogenesis of symptoms is intriguing. In a recent study from our group, we identified a group of patients with asymptomatic esophagitis who did not report “heartburn” with acid perfusion, which we labeled as having a “hyposensitive” esophagus.18 What are the clinical implications of silent GERD? The highest prevalence of asymptomatic GERD is in patients with extra-esophageal manifestations of GERD. In patients with refractory asthma and chronic cough associated with GERD, it Decitabine has been noted that 25–75% do not have classical symptoms of GERD.19,20 For these groups of patients, the presence of underlying GERD should be suspected and investigated. Proton-pump

inhibitors could be empirically prescribed and this is a common clinical practice.19 Similarly, asymptomatic GPX6 GERD is also common in children, with unexplained pneumonia and recurrent asthma. These children should also be investigated and treated for GERD where appropriate.21 We do not understand yet the natural history of silent esophagitis. While the majority of cases are of milder grades, do we know whether they will evolve to more severe grades and Barrett’s esophagus without treatment? If so, this would be a cause for concern, given that 25% of Barrett’s esophagus and 40% of all esophageal adenocarcinomas occur in patients without, or with only minimal, prior reflux symptoms.2,22 This group of patients with silent GERD and erosive esophagitis clearly needs further, in-depth study and long-term follow up. “
“Lee WM, Hynan LS, Rossaro L, Fontana RJ, Stravitz RT, Larson AM, et al.; Acute Liver Failure Study Group. Intravenous N-acetylcysteine improves transplant-free survival in early stage non-acetaminophen acute liver failure. Gastroenterology 2009;137:856–886. (Reprinted with permission.) BACKGROUND & AIMS: N-acetylcysteine (NAC), an antidote for acetaminophen poisoning, might benefit patients with non-acetaminophen-related acute liver failure.

We collected data including of liver function, blood-lipids, fasting blood-glucose (FBG), HOMA-IR and liver ultrasound, then explored the distribution of blood-lipids and its relation to degree of fatty liver, hepatic CT, BMI (body mass index). Results: The blood-lipids distribution of NAFLD showed high level of TG. The degree of fatty liver was positive correlation

with BMI, c-Met inhibitor course of disease (P < 0.05). The levels of FBG, HOMA-IR, TC, APO-B, NON-HDL-C were increased gradually with the degree of fatty liver getting higher, on the contrary, the level of LP (α) was negative correlation with it (P < 0.05). The levels of HDL-C, LDL, APO-A1, TG had no obvious difference among the degree of fatty liver (P > 0.05). The level of ALT was positive correlation with degree of fatty liver, BMI and HOMA-IR, and was negative correlation with age, course of disease, LP (α) (P < 0.05), but there was no difference in FBG, other blood-lipids. selleck chemicals We found no connection between blood-lipids with BMI layered.(P > 0.05). Conclusion: Blood-lipids of NAFLD showed high level of TG. The level of TC, APO-B, NON-HDL-C were increased gradually with the degree of fatty liver getting higher, and there was no connection between ALT, BMI and blood-lipids. Early treatment of NAFLD is important to prevent blood-lipids disorders.

Key Word(s): 1. non-alcoholic; 2. fatty liver; 3. blood-lipids; 4. characteristic; Presenting Author: WAH KHEONG CHAN Additional Authors: NORHAZINA BAHAR, HAMIZAH RAZLAN, ANUSHYA VIJAYANANTHAN, PAVAI STHANESHWAR, KHEAN LEE GOH Corresponding Author: WAH KHEONG CHAN Affiliations: University Ureohydrolase of Malaya Objective: There is till date no study on the prevalence of NAFLD among young adults in Malaysia. Whether the prevalence of NAFLD is different among young adults of different ethnic origin is unknown. Methods: This was a cross-sectional study on students pursuing their tertiary education at the Faculty of Medicine, University of Malaya. Demographic and anthropometric data and relevant clinical and laboratory data were obtained using a standard protocol. Diagnosis

of NAFLD was by trans-abdominal ultrasonography and following exclusion of significant alcohol intake and other causes of chronic liver disease. Results: Data for 472 subjects were analyzed (mean age 23.2 ± 2.4 years old, 40.5% men). The racial distribution was: Chinese 53.6%, Malay 30.3%, Indian 15.5% and others 0.6%. The prevalence of NAFLD was 8.1% (38/472). Subjects with NAFLD were older, had greater BMI and WC, and recorded higher SBP and DBP. They had higher FBS, serum TG and LDL levels and lower serum HDL level. Serum ALP, ALT, AST and GGT levels were higher in subjects with NAFLD. All subjects who had NAFLD had insulin resistance. The prevalence of NAFLD was significantly higher among males compared to females (17.9 % vs. 3.3 %, p < 0.001).

17,18 A 5-year study of LAM treatment in 74 HBeAg-positive patients was reported by Yuen et al.19 They concluded that serum HBV DNA < 2000 IU/mL at week 4 and < 800 IU/mL at week 16 were associated with a favorable response (HBV DNA < 400 IU/mL, HBeAg seroconversion, normal ALT) Selleck PS341 and no drug resistance at year 5 (Table 1). Hadziyannis et al.20 also reported a study involving 156 HBeAg-negative patients treated with LAM and demonstrated that undetectable HBV DNA at 3 months and 6 months had a positive predictive value of 93% and 72% for maintained response for 2 and > 4 years, respectively. Regarding drug-resistant HBV variants prediction, in a study involving

150 Asian HBeAg-positive patients during a median LAM treatment period of 30 months, it was demonstrated that drug resistance developed in 8%, 13%, 32% and 63% of patients with week 24 serum HBV DNA < 40 IU/mL,

< 200 IU/mL, < 2000 IU/mL Dorsomorphin solubility dmso and > 2000 IU/mL, respectively (Table 2).21 In the LAM-controlled Ldt trial, the 1-year LAM resistance rate was 3%, 10%, 15% and 17% in HBeAg-positive patients and 2%, 20%, 38% and 50% in HBeAg-negative patients with serum HBV DNA levels ≤ 60, 60–< 200, 200–< 2000 and ≧ 2000 IU/mL at week 24, respectively (Table 2).17 The antiviral potency of ADV is lowest among the five NA. It can reduce serum HBV DNA levels by 3–4 log10. Although the antiviral potency is low, the genetic barrier is higher than LAM and Ldt. The HBeAg seroconversion rate in HBeAg-positive patients during ADV therapy increased along with prolonged treatment (12% at 1 year; 29% at 2 years; 43% at 3 years) with gradually cumulative drug-resistant rates in treatment naïve patients (0% at 1 year; 3% at 2 years; 11% at 3 years; 18% at 4 years; 29% at 5 years).22 In the many ADV-controlled Ldt trial, 45 patients were enrolled into the ADV monotherapy group, with whom undetectable HBV DNA (< 200 IU/mL) at week 24 was also associated with better week 52 response to ADV therapy (undetectable HBV DNA in

90% vs 25%, HBeAg seroconversion in 50% vs 9% and ALT normalization in 90% vs 83%) (Table 1).23 In another study, Gallego et al. treated 42 patients (88% HBeAg-negative) with ADV for more than 12 months; they also showed that 77% of patients having an HBV DNA reduction ≧ 4 log10 IU/mL at week 24 achieved undetectable HBV DNA at month 12 as compared with 5% of the patients with less HBV DNA reduction (Table 1).24 Furthermore, Locarnini et al.25 demonstrated that patients treated with ADV and achieving HBV DNA < 200 IU/mL at week 48 showed an ADV resistance rate of 4% compared with 26% and 67% of those with corresponding HBV DNA levels of 200–2 × 105 and > 2 × 105 IU/mL, respectively (Table 2). Additionally, Hadziyannis et al.

17,18 A 5-year study of LAM treatment in 74 HBeAg-positive patients was reported by Yuen et al.19 They concluded that serum HBV DNA < 2000 IU/mL at week 4 and < 800 IU/mL at week 16 were associated with a favorable response (HBV DNA < 400 IU/mL, HBeAg seroconversion, normal ALT) Wnt inhibitor and no drug resistance at year 5 (Table 1). Hadziyannis et al.20 also reported a study involving 156 HBeAg-negative patients treated with LAM and demonstrated that undetectable HBV DNA at 3 months and 6 months had a positive predictive value of 93% and 72% for maintained response for 2 and > 4 years, respectively. Regarding drug-resistant HBV variants prediction, in a study involving

150 Asian HBeAg-positive patients during a median LAM treatment period of 30 months, it was demonstrated that drug resistance developed in 8%, 13%, 32% and 63% of patients with week 24 serum HBV DNA < 40 IU/mL,

< 200 IU/mL, < 2000 IU/mL GPCR Compound Library clinical trial and > 2000 IU/mL, respectively (Table 2).21 In the LAM-controlled Ldt trial, the 1-year LAM resistance rate was 3%, 10%, 15% and 17% in HBeAg-positive patients and 2%, 20%, 38% and 50% in HBeAg-negative patients with serum HBV DNA levels ≤ 60, 60–< 200, 200–< 2000 and ≧ 2000 IU/mL at week 24, respectively (Table 2).17 The antiviral potency of ADV is lowest among the five NA. It can reduce serum HBV DNA levels by 3–4 log10. Although the antiviral potency is low, the genetic barrier is higher than LAM and Ldt. The HBeAg seroconversion rate in HBeAg-positive patients during ADV therapy increased along with prolonged treatment (12% at 1 year; 29% at 2 years; 43% at 3 years) with gradually cumulative drug-resistant rates in treatment naïve patients (0% at 1 year; 3% at 2 years; 11% at 3 years; 18% at 4 years; 29% at 5 years).22 In the Cell Cycle inhibitor ADV-controlled Ldt trial, 45 patients were enrolled into the ADV monotherapy group, with whom undetectable HBV DNA (< 200 IU/mL) at week 24 was also associated with better week 52 response to ADV therapy (undetectable HBV DNA in

90% vs 25%, HBeAg seroconversion in 50% vs 9% and ALT normalization in 90% vs 83%) (Table 1).23 In another study, Gallego et al. treated 42 patients (88% HBeAg-negative) with ADV for more than 12 months; they also showed that 77% of patients having an HBV DNA reduction ≧ 4 log10 IU/mL at week 24 achieved undetectable HBV DNA at month 12 as compared with 5% of the patients with less HBV DNA reduction (Table 1).24 Furthermore, Locarnini et al.25 demonstrated that patients treated with ADV and achieving HBV DNA < 200 IU/mL at week 48 showed an ADV resistance rate of 4% compared with 26% and 67% of those with corresponding HBV DNA levels of 200–2 × 105 and > 2 × 105 IU/mL, respectively (Table 2). Additionally, Hadziyannis et al.

Results: We examined 405 Alzheimer’s disease and with peptic ulcer diseases and H pylori eradication therapy cases and 405 controls. Compared with the group with no use of H pylori eradication therapy, the adjusted ORs were 0.62 (95% CI = 0.37–0.71). Conclusion: The results of

this study suggest that H pylori eradication may reduce the risk of Alzheimer’s disease. Key Word(s): 1. Helicobacter pylori; 2. Alzheimer’s disease; Presenting Author: BOR-SHYANG SHEU Additional Authors: YU-CHING TSAI, CHUN-TAI WU, YEN-LIN WANG, WEI-LUN CHANG, HSUI-CHI CHENG, HSIAO-BAI YANG Corresponding Author: BOR-SHYANG SHEU Affiliations: National Cheng Kung University Hospital; Private Chung Hwa Medical Technology University Objective: Intestinal metaplasia (IM) has overexpressions Doramapimod concentration of COX-2. Short-term 8-week celecoxib, a selective COX-2 inhibitor, exerts a preliminary hint to improve regression in part for persistent IM after Helicobacter pylori eradication. This study further validated whether or not a prolonged duration of celecoxib of up to 1 year can be safe and effective. Methods: One hundred and forty patients, with persistent IM after H. pylori eradication for 1 year, were included with half of them receiving celecoxib 200 mg/day for 12 months

and the other half BYL719 in vivo serving as controls. Each patient received serial checkups of blood creatinine levels every 4 months. After the 1-year follow-up, panendoscopy was repeated to assess the IM regression. The serial

gastric specimens, taken before and after celecoxib therapy, were immunochemically selleck kinase inhibitor stained for COX-2. Results: The intention-to-treat (ITT) and per-protocol (PP) analyses to the rates of IM regression were higher in the celecoxib group than in the controls (ITT: 44.3% [31/70] vs 14.3% [10/70], p < .001; and PP: 51.7% [31/60] vs 16.1% [10/62], p < .001). All enrolled patients had no renal impairment during follow-up. Even in the patients without IM regression, the mean IM scores and COX-2 expressions were significantly more decreased in the celecoxib group than in the controls (p < .005). Conclusion: One year 200-mg celecoxib daily be safely administered to improve the regression or prevent the progression of persistent IM after H. pylori eradication. Key Word(s): 1. H. pylori; 2. celecoxib; 3. metaplasia; Presenting Author: DENG-CHYANG WU Additional Authors: CHUN-YI HUANG, YUAN-CHIEH YANG, HUANG-MING HU, CHAO-HUNG KUO, YEOU-LIH HUANG, FU-CHEN KUO, WEN-MING WANG Corresponding Author: WEN-MING WANG Affiliations: Kaohsiung Municipal Hsiao-Kang Hospital; Kaohsiung Medical University Hospital; E-Da Hospital, I-Shou University; Kaohsiung Municipal Ta-Tung Hospital Objective: Helicobacter pylori (H.

“
“We report our technical success and complication rates in treating posterior circulation aneurysms at sites other than the basilar apex, superior cerebellar artery origin, or the posterior inferior cerebellar artery origin via endovascular embolization or sacrifice. We retrospectively reviewed case records for patients undergoing coil embolization of atypical find more posterior circulation aneurysms from January 2003 to December 2007. Thirty-two aneurysms in 32 patients were treated. Twenty-one patients (65%) presented with a subarachnoid hemorrhage. Twenty-two aneurysms

were treated with coiling alone, 9 with stent-assisted coiling, and 1 with a combination of Onyx plus stent-assisted coiling. Twelve aneurysms were treated with vessel sacrifice. Immediately post procedure, 27/32 aneurysms (84%) were considered successfully treated, resulting in either vessel sacrifice, complete obliteration, or minimal neck remnant.

Sixteen of 19 patients (84%) were considered successfully treated at a mean angiographic follow up of 8 months. The procedural morbidity and mortality was 15% and 6% respectively. Endovascular embolization remains selleck a viable and durable method of treatment for atypical posterior circulation aneurysms. “
“Conventional non-invasive angiographic techniques for evaluating cerebral Arteriovenous Malformations (cAVMs) after embolization treatment are limited by their inability to acquire time-resolved images. We describe the use of dynamic contrast-enhanced magnetic resonance angiography (MRA) in the evaluation of residual arteriovenous shunting in cAVMs following Onyx embolization. Six subjects who underwent multimodal MR imaging including dynamic MRA after different

stages of endovascular treatment with Onyx were included. CHIR-99021 cost Each MRA was assessed for the presence of residual arteriovenous shunting. The results were compared with digital subtraction angiography (DSA). Mean age was 41 years (range, 25–63) and the mean maximum AVM diameter was 5.3 cm (range, 4.7–6.0). Fourteen dynamic MRA were performed using a 1.5 T scanner. Arteriovenous shunting was detected in thirteen of fourteen patients by both dynamic MRA and DSA, with complete agreement between the two techniques. The only MRA without detectable residual arteriovenous shunting was for a subject who had complete treatment with no residual cAVM as confirmed by the DSA images. Dynamic contrast-enhanced MRA is a promising non-invasive modality in identifying residual arteriovenous shunting after different stages of AVM embolization, achieving 100% agreement in this small study. Embolization with Onyx caused no significant image artifact. “
“The exact origin and process of development of cerebral cavernous malformations (CCMs) is currently unknown.

A central laboratory (Covance Central Laboratory Services S.A., Geneva, Switzerland, and Covance Central Laboratory Services, Inc.,

Indianapolis, IN) evaluated all laboratory samples. HBV DNA was quantified using the LY2606368 supplier Roche COBAS TaqMan HBV test (Roche Diagnostics, Indianapolis, IN), which provides fully automated real-time PCR HBV viral load quantitation in serum and plasma. Analysis of ALT was performed using a Roche Modular Analyzer, which was calibrated daily. The pol/RT domain of the HBV polymerase region (amino acids 1-344) was sequenced in patients with HBV DNA ≥400 copies/mL at week 72, patients who discontinued from the study early with HBV DNA ≥400 copies/mL after 24 weeks of click here treatment, and patients who experienced virologic breakthrough.

Genotypic analysis was conducted by DDL Diagnostic Laboratories (Rijswijk, the Netherlands). Briefly, DNA was isolated from 200 mL of serum using the Roche MagNA Pure instrument, and the HBV pol/RT domain was amplified via PCR and nested PCR using the Expand high-fidelity PCR kit (Roche Molecular Systems). Di-deoxy sequencing of the amplified product was conducted using the ABI Big Dye terminator cycle sequencing kit employing a selection of forward and reverse primers, and analysis of the raw sequence data used ABI Seqscape software. Virologic breakthrough was defined as HBV DNA measurements of ≥400 copies/mL (after an earlier value <400 copies/mL) or a 10-fold increase in HBV DNA levels over 4��8C the patient’s lowest value. When conserved site changes were identified and/or when patients experienced virologic breakthrough, the HBV pol/RT was isolated from patients’ serum for phenotypic sensitivity testing to inhibition by tenofovir DF.10 If the conserved site change of interest

occurred as a mixture with wild-type virus, then a clone containing the appropriate amino acid substitution was tested. Average values for 50% of effective concentration obtained for the post-baseline sample were compared with those obtained for the patient’s baseline isolate to determine the fold-change to tenofovir DF. Safety assessments included patient signs and symptoms as well as radiographic and laboratory findings. The primary safety endpoint was the cumulative incidence of at least a 6% decrease from baseline in lumbar spine bone mineral density (BMD) through week 72. Any clinical manifestation of hepatic decompensation or worsening hepatic function was considered a serious adverse event; this included any case of serum ALT that was more than twice the baseline level and more than 10 times the ULN, regardless of the presence of symptoms.