Markers Of Replication Research Articles

A novel cell permeable DNA replication and repair marker

Proliferating Cell Nuclear Antigen (PCNA) is a key protein in DNA replication and repair. The dynamics of replication and repair in live cells is usually studied introducing translational fusions of PCNA. To obviate the need for transfection and bypass the problem of difficult to transfect and/or short lived cells, we have now developed a cell permeable replication and/or repair marker. The design of this marker has three essential molecular components: (1) an optimized artificial PCNA binding peptide; (2) a cell-penetrating peptide, derived from the HIV-1 Trans Activator of Transcription (TAT); (3) an in vivo cleavable linker, linking the two peptides. The resulting construct was taken up by human, hamster and mouse cells within minutes of addition to the media. Inside the cells, the cargo separated from the vector peptide and bound PCNA effectively. Both replication and repair sites could be directly labeled in live cells making it the first in vivo cell permeable peptide marker for these two fundamental cellular processes. Concurrently, we also introduced a quick peptide based PCNA staining method as an alternative to PCNA antibodies for immunofluorescence applications. In summary, we present here a versatile tool to instantaneously label repair and replication processes in fixed and live cells.

Sustained inhibition of hepatitis B virus replication in vivo using RNAi-activating lentiviruses.

Chronic infection with hepatitis B virus (HBV) puts individuals at high risk for complicating cirrhosis and liver cancer, but available treatment to counter the virus rarely eliminates infection. Although harnessing RNA interference (RNAi) to silence HBV genes has shown the potential, achieving efficient and durable silencing of viral genes remains an important goal. Here we report on the propagation of lentiviral vectors (LVs) that successfully deliver HBV-targeting RNAi activators to liver cells. Mono- and tricistronic artificial primary microRNAs (pri-miRs) derived from pri-miR-31, placed under transcriptional control of the liver-specific modified murine transthyretin (mTTR) promoter, caused efficient inhibition of HBV replication markers. The tricistronic cassette was capable of silencing a mutant viral target and the effects were observed without disrupting the function of an endogenous miR (miR-16). The mTTR promoter stably expressed a reporter transgene in mouse livers over a study period of 1 year. Good silencing of HBV genes, without evidence of toxicity, was demonstrated following intravenous injection of LVs into neonatal HBV transgenic mice. Collectively, these data indicate that LVs may achieve sustained inhibition of HBV replication that is appealing for their therapeutic use.

DNA replication in nurse cell polytene chromosomes of Drosophila melanogaster otu mutants.

Drosophila cell lines are used extensively to study replication timing, yet data about DNA replication in larval and adult tissues are extremely limited. To address this gap, we traced DNA replication in polytene chromosomes from nurse cells of Drosophila melanogaster otu mutants using bromodeoxyuridine incorporation. Importantly, nurse cells are of female germline origin, unlike the classical larval salivary glands, that are somatic. In contrast to salivary gland polytene chromosomes, where replication begins simultaneously across all puffs and interbands, replication in nurse cells is first observed at several specific chromosomal regions. For instance, in the chromosome 2L, these include the regions 31B-E and 37E and proximal parts of 34B and 35B, with the rest of the decondensed chromosomal regions joining replication process a little later. We observed that replication timing of pericentric heterochromatin in nurse cells was shifted from late S phase to early and mid stages. Curiously, chromosome 4 may represent a special domain of the genome, as it replicates on its own schedule which is uncoupled from the rest of the chromosomes. Finally, we report that SUUR protein, an established marker of late replication in salivary gland polytene chromosomes, does not always colocalize with late-replicating regions in nurse cells.

Endogenous versus exogenous markers of adult neurogenesis in canaries and other birds: advantages and disadvantages.

Although the existence of newborn neurons had originally been suggested, but not broadly accepted, based on studies in adult rodent brains, the presence of an active neurogenesis process in adult homoeothermic vertebrates was first firmly established in songbirds. Adult neurogenesis was initially studied with the tritiated thymidine technique, later replaced by the injection and detection of the marker of DNA replication 5-bromo-2'-deoxyuridine (BrdU). More recently, various endogenous markers were used to identify young neurons or cycling neuronal progenitors. We review here the respective advantages and pitfalls of these different approaches in birds, with specific reference to the microtubule-associated protein, doublecortin (DCX), that has been extensively used to identify young newly born neurons in adult brains. All these techniques of course have limitations. Exogenous markers label cells replicating their DNA only during a brief period and it is difficult to select injection doses that would exhaustively label all these cells without inducing DNA damage that will also result in some form of labeling during repair. On the other hand, specificity of endogenous markers is difficult to establish due to problems related to the specificity of antibodies (these problems can be, but are not always, addressed) and more importantly because it is difficult, if not impossible, to prove that a given marker exhaustively and specifically labels a given cell population. Despite these potential limitations, these endogenous markers and DCX staining in particular clearly represent a useful approach to the detailed study of neurogenesis especially when combined with other techniques such as BrdU.

Hypoxic Stress Facilitates Acute Activation and Chronic Downregulation of Fanconi Anemia Proteins

Hypoxia induces genomic instability through replication stress and dysregulation of vital DNA repair pathways. The Fanconi anemia (FA) proteins, FANCD2 and FANCI, are key members of a DNA repair pathway that responds to replicative stress, suggesting that they undergo regulation by hypoxic conditions. Here acute hypoxic stress activates the FA pathway via ubiquitination of FANCD2 and FANCI in an ATR-dependent manner. In addition, the presence of an intact FA pathway is required for preventing hypoxia-induced DNA damage measurable by the comet assay, limiting the accumulation of γH2AX (a marker of DNA damage or stalled replication), and protecting cells from hypoxia-induced apoptosis. Furthermore, prolonged hypoxia induces transcriptional repression of FANCD2 in a manner analogous to the hypoxic downregulation of BRCA1 and RAD51. Thus, hypoxia-induced FA pathway activation plays a key role in maintaining genome integrity and cell survival, while FA protein downregulation with prolonged hypoxia contributes to genomic instability. This work highlights the critical role of the FA pathway in response to hypoxic stress and identifies the pathway as a therapeutic target under hypoxic conditions.

Current methods of studying of the proliferative activity in experiment

Current methods of studying of the proliferative activity in experiment

Clinical utility of hepatitis C virus core antigen quantification in patients with chronic hepatitis C

BackgroundHepatitis C virus (HCV) core antigen has been proposed as a surrogate marker of HCV replication. HCV core antigen detection and quantification can thus theoretically be used instead of nucleic acid testing (NAT) to diagnose infection and manage antiviral therapy, with several advantages compared to HCV RNA assays. HCV core antigen can now be easily detected and quantified by means of a chemiluminescent microparticle immunoassay on the Abbott Architect device. ObjectiveThe aim of the present study was to evaluate the clinical performance of the Architect HCV Ag assay for the detection and quantification of HCV core antigen in patients with chronic HCV genotype 1–6 infections. ResultsThe Architect HCV Ag assay had a specificity of 100% (95%CI: 97.8–100%). HCV core antigen levels were not influenced by the HCV genotype. The lower limit of detection of 3fmol/L corresponded to approximately 1000IU/mL of HCV RNA, regardless of the real-time PCR assay used. HCV core antigen and HCV RNA levels correlated for HCV genotype 1–4 (r=0.89 and r=0.88 for the m2000 and the CAP/CTM v2.0 assay, respectively). ConclusionsThe Architect HCV Ag assay is highly specific and easy to perform. It represents a valuable screening, diagnostic and monitoring tool, especially in the era of new all-oral, interferon-free antiviral strategies that do not require high analytical sensitivity.

Hepatic steatosis is highly prevalent in hepatitis B patients and negatively associated with virological factors.

The interaction between hepatitis B virus (HBV) infection and hepatic steatosis remains unclear. We aimed to explore the trend of prevalence of hepatic steatosis and its relationship with virological factors in HBV infected patients. Consecutive untreated patients with chronic HBV infection at Shunde Hospital between 2002 and 2011 were included. Quantification of HBV replication markers was performed by enzyme immunoassay, real-time polymerase chain reaction assay and immunohistochemical staining. Hepatic steatosis was defined as at least 5% hepatocytes affected. A total of 3,212 patients (2,574 men) with a mean age of 32 ± 9.3 years were analyzed. Serological testing showed detectable HBsAg in all, HBeAg in 63.8% and HBV DNA in 78.4% of patients. Liver biopsies demonstrated HBsAg- and HBcAg-positive immunostaining in 96.6 and 71% patients, respectively. Hepatic steatosis was present in 554 (17.3%) patients, with annual prevalence increased over time from 8.2 to 31.8% (trend analysis, x (2) = 51.657, P < 0.001). Compared to patients without steatosis, the percentages of serum HBeAg-positive and detectable HBV DNA, and intrahepatic HBsAg- and HBcAg-positive staining were decreased in steatosis patients (all P < 0.001). Adjusted for age and gender, intrahepatic HBsAg-positive staining remained as an independent factor associated with lower risk of steatosis (adjusted odds ratio 0.90, 95% confidence interval 0.835, 0.971) in multivariate analysis. Hepatic steatosis in HBV infected patients has been raging over the past decade, and it is negatively associated with intrahepatic expression of HBsAg. Lifestyle intervention may be needed to halt the onset of steatosis in chronic HBV infection.

Identification of genetic variants associated with capecitabine-induced hand–foot syndrome through integration of patient and cell line genomic analyses

A primary challenge in identifying replicable pharmacogenomic markers from clinical genomewide association study (GWAS) trials in oncology is the difficulty in performing a second large clinical trial with the same drugs and dosage regimen. We sought to overcome this challenge by incorporating GWAS results from cell-based studies using the same chemotherapy as a clinical cohort. In this study, we test whether the overlap between genetic variants identified in a preclinical study and a clinical study on capecitabine is more than expected by chance. A GWAS of capecitabine-induced cytotoxicity was performed in 164 lymphoblastoid cell lines derived from the CEU HapMap population and compared with a GWAS of hand-foot syndrome (HFS), the most frequent capecitabine-induced adverse drug reaction, in Spanish breast and colorectal cancer patients (n=160) treated with capecitabine. We observed an overlap of 16 single nucleotide polymorphisms associated with capecitabine-induced cytotoxicity (P<0.001) in lymphoblastoid cell lines and HFS (P<0.05) in patients, which is a greater overlap than expected by chance (genotype-phenotype permutation empirical P=0.015). Ten tag single nucleotide polymorphisms, which cover the overlap loci, were genotyped in a second patient cohort (n=85) and one of them, rs9936750, was associated with capecitabine-induced HFS (P=0.0076). The enrichment results imply that cellular models of capecitabine-induced cytotoxicity may capture components of the underlying polygenic architecture of related toxicities in patients.

Levels of hepatitis B virus replicative intermediate in serum samples of chronic hepatitis B patients

Hepatitis B virus (HBV) cccDNA levels is an absolute marker of HBV replication in the liver of HBV infected patients. This study aimed to quantify the HBV cccDNA levels in sera and liver tissue samples of treatment naïve patients with chronic hepatitis B. Eighty one chronic hepatitis B (CHB) treatment naïve patients were enrolled from January 2009 to June 2011. Total HBV DNA and HBV cccDNA levels were quantified using sensitive real time PCR assay. The mean age of recruited patients was 34 ± 11.5 years. Fifty four (66.7%) patients were HBeAg negative. Liver tissue samples were available from 2 HBeAg positive and 21 HBeAg negative CHB patients. The amount of total intrahepatic HBV DNA ranged from 0.09 to 1508.92 copies/cell. The median intrahepatic HBV cccDNA was 0.31 and 0.20 copies/cell in HBeAg positive and HBeAg negative cases, respectively. Serum HBV cccDNA was detectable in 85.2 % HBeAg positive and 48.1% HBeAg negative CHB patients. Median serum HBV cccDNA was 46,000 and 26,350 copies/mL in HBeAg positive and HBeAg negative subjects, respectively. There was a significant positive correlation between the levels of intrahepatic total HBV DNA and intrahepatic HBV cccDNA (r = 0.533, p = 0.009). A positive correlation was also seen between serum HBV cccDNA levels and serum HBV DNA levels (r = 0.871, p < 0.001). It was concluded that serum HBV cccDNA could be detectable in higher proportion of HBeAg positive patients compared to HBeAg negative patients. Moreover, the median level of serum HBV cccDNA was significantly higher in HBeAg positive patients in contrast to HBeAg negative subjects.

Role of metabolic syndrome and antiretroviral therapy in adiponectin levels and oxidative stress in HIV-1 infected patients

ObjectiveHIV-1 infection is accompanied by severe metabolic and immune dysfunction. The aim of this study was to evaluate the role of metabolic syndrome (MetS) and antiretroviral therapy (ART) utilization on the adiponectin levels and oxidative stress in patients infected with HIV-1. MethodsWe allocated 285 patients into four groups: group 1: patients without MetS who were not using ART; group 2: patients without MetS using ART; group 3: patients with MetS who were not using ART; and group 4: patients with MetS using ART. Biochemical, immunologic, and oxidative stress parameters were measured. ResultsGroup 4 exhibited higher lipoperoxides when compared with group 1 (P < 0.0001) and higher advanced oxidation protein products (AOPP) compared with group 2 or group 1 (P < 0.0001). Group 3 also presented higher AOPP than group 2 (P < 0.05). Group 4 showed lower adiponectin levels compared with groups 1 or 2 (P < 0.0001). Similarly, group 3 presented lower adiponectin levels compared with group 2 (P < 0.05) or group 1 (P < 0.0001). Multivariate analysis showed that both an increase in AOPP and a decrease in total radical-trapping antioxidant parameter/uric acid were independently associated with MetS in HIV-1 patients. Regarding immunologic markers of HIV-1 disease progression and viral replication, group 4 exhibited significantly higher CD45+, CD3+, and CD4+ T cells count compared with group 2 (P < 0.01). ConclusionHIV-1–infected patients with MetS exhibited hypoadiponectinemia and increased oxidative stress, and these findings were not influenced by ART use. The findings of the present study allow the suggestion that MetS and inflammation might be mainly responsible for the aforementioned features. More studies are needed to verify whether drugs or food, which yield increased adiponectinemia and decreased oxidative stress, could reduce cardiovascular risk in HIV-infected patients.

Macrophages are required for dendritic cell uptake of respiratory syncytial virus from an infected epithelium.

We have previously shown that the respiratory syncytial virus [RSV] can productively infect monocyte derived dendritic cells [MoDC] and remain dormant within the same cells for prolonged periods. It is therefore possible that infected dendritic cells act as a reservoir within the airways of individuals between annual epidemics. In the present study we explored the possibility that sub-epithelial DCs can be infected with RSV from differentiated bronchial epithelium and that in turn RSV from DCs can infect the epithelium. A dual co-culture model was established in which a differentiated primary airway epithelium on an Air Liquid Interface (ALI) was cultured on a transwell insert and MoDCs were subsequently added to the basolateral membrane of the insert. Further experiments were undertaken using a triple co-culture model in which in which macrophages were added to the apical surface of the differentiated epithelium. A modified RSV [rr-RSV] expressing a red fluorescent protein marker of replication was used to infect either the MoDCs or the differentiated epithelium and infection of the reciprocal cell type was assessed using confocal microscopy. Our data shows that primary epithelium became infected when rr-RSV infected MoDCs were introduced onto the basal surface of the transwell insert. MoDCs located beneath the epithelium did not become infected with virus from infected epithelial cells in the dual co-culture model. However when macrophages were present on the apical surface of the primary epithelium infection of the basal MoDCs occurred. Our data suggests that RSV infected dendritic cells readily transmit infection to epithelial cells even when they are located beneath the basal layer. However macrophages appear to be necessary for the transmission of infection from epithelial cells to basal dendritic cells.

Coronavirus Replicase-Reporter Fusions Provide Quantitative Analysis of Replication and Replication Complex Formation

The replication of coronaviruses occurs in association with multiple virus-induced membrane structures that evolve during the course of infection; however, the dynamics of this process remain poorly understood. Previous studies of coronavirus replication complex organization and protein interactions have utilized protein overexpression studies and immunofluorescence of fixed cells. Additionally, live-imaging studies of coronavirus replicase proteins have used fluorescent reporter molecules fused to replicase proteins, but expressed from nonnative locations, mostly late-transcribed subgenomic mRNAs, in the presence or absence of the native protein. Thus, the timing and targeting of native replicase proteins expressed in real time from native locations in the genome remain unknown. In this study, we tested whether reporter molecules could be expressed from the replicase polyprotein of murine hepatitis virus as fusions with nonstructural protein 2 or 3 and whether such reporters could define the targeting and activity of replicase proteins during infection. We demonstrate that the fusion of green fluorescent protein and firefly luciferase with either nonstructural protein 2 or 3 is tolerated and that these reporter-replicase fusions can be used to quantitate replication complex formation and virus replication. The results show that the replicase gene has flexibility to accommodate a foreign gene addition and can be used directly to study replicase complex formation and evolution during infection as well as to provide highly sensitive and specific markers for protein translation and genome replication. Coronaviruses are a family of enveloped, positive-sense RNA viruses that are important agents of disease, including severe acute respiratory syndrome coronavirus and Middle East respiratory syndrome coronavirus. Replication is associated with multiple virus-induced membrane structures that evolve during infection; however, the dynamics of this process remain poorly understood. In this study, we tested whether reporter molecules expressed from native locations within the replicase polyprotein of murine hepatitis virus as fusions with nonstructural proteins could define the expression and targeting of replicase proteins during infection in live cells. We demonstrate that the replicase gene tolerates the introduction of green fluorescent protein or firefly luciferase as fusions with replicase proteins. These viruses allow early quantitation of virus replication as well as real-time measurement of replication complexes.

ENOS-Dependent Antisenscence Effect of a Calcium Channel Blocker in Human Endothelial Cells

Senescence of vascular endothelial cells is an important contributor to the pathogenesis of age-associated vascular disorders such as atherosclerosis. We investigated the effects of antihypertensive agents on high glucose-induced cellular senescence in human umbilical venous endothelial cells (HUVECs). Exposure of HUVECs to high glucose (22 mM) for 3 days increased senescence-associated- β-galactosidase (SA-β-gal) activity, a senescence marker, and decreased telomerase activity, a replicative senescence marker. The calcium channel blocker nifedipine, but not the β1-adrenergic blocking agent atenolol or the angiotensin-converting enzyme inhibitor perindopril, reduced SA-β-gal positive cells and prevented a decrease in telomerase activity in a high-glucose environment. This beneficial effect of nifedipine was associated with reduced reactive oxygen species (ROS) and increased endothelial nitric oxide synthase (eNOS) activity. Thus, nifedipine prevented high glucose-induced ROS generation and increased basal eNOS phosphorylation level at Ser-1177. Treatment with N G-nitro-L-arginine (L-NAME) and transfection of small interfering RNA (siRNA) targeting eNOS eliminated the anti-senscence effect of nifedipine. These results demonstrate that nifedipine can prevent endothelial cell senescence in an eNOS-dependent manner. The anti-senescence action of nifedipine may represent a novel mechanism by which it protects against atherosclerosis.

Galvanic vestibular stimulation impairs cell proliferation and neurogenesis in the rat hippocampus but not spatial memory

Galvanic vestibular stimulation (GVS) is a method of activating the peripheral vestibular system using direct current that is widely employed in clinical neurological testing. Since movement is recognized to stimulate hippocampal neurogenesis and movement is impossible without activation of the vestibular system, we speculated that activating the vestibular system in rats while minimizing movement, by delivering GVS under anesthesia, would affect hippocampal cell proliferation and neurogenesis, and spatial memory. Compared with the sham control group, the number of cells incorporating the DNA replication marker, bromodeoxyuridine (BrdU), was significantly reduced in the bilateral hippocampi in both the cathode left-anode right and cathode right-anode left stimulation groups (P ≤ 0.0001). The majority of the BrdU(+ve) cells co-expressed Ki-67, a marker for the S phase of the cell cycle, suggesting that these BrdU(+ve) cells were still in the cell cycle; however, there was no significant difference in the degree of co-labeling between the two stimulation groups. Single labeling for doublecortin (DCX), a marker of immature neurons, showed that while there was no significant difference between the different groups in the number of DCX(+ve) cells in the right dentate gryus, in the left dentate gyrus there was a significant decrease in the cathode left-anode right group compared with the sham controls (P ≤ 0.03). Nonetheless, when animals were tested in place recognition, object exploration and Morris water maze tasks, there were no significant differences between the GVS groups and the sham controls. These results suggest that GVS can have striking effects on cell proliferation and possibly neurogenesis in the hippocampus, without affecting spatial memory.

The transient HIV remission in the Mississippi baby: why is this good news?

As researchers, clinicians and community advocates gathered for the 20th International AIDS Society meeting in Melbourne in July of this year, the announcement of resumption of viremia in the Mississippi baby came as an unwelcome surprise. The case had previously stirred excitement that a cure was possible without the extreme measure of bone marrow transplantation as in the Berlin patient [ 1 ]. When this was no longer true, conversations about the end of our quest for a HIV cure ensued. (Published: 20 November 2014) Ananworanich J and Robb ML. Journal of the International AIDS Society 2014, 17 :19859 http://www.jiasociety.org/index.php/jias/article/view/19859 | http://dx.doi.org/10.7448/IAS.17.1.19859

Urine cytology as a screening tool for BK virus nephropathy: preliminary findings in 350 samples

Background BK virus allograft nephropathy (BKVAN) is caused by reactivation of BK polyomavirus and often leads to graft failure due to unsuccessful treatment. Screening and early detection with surrogate markers of viral replication are critical to guide preventative strategies. Method We prospectively evaluated the performance of urinary decoy cells, identified in ThinPrep processed specimens, and SV40T immunochemistry, performed on cell block preparations, to predict BKVAN (defined by reference histology standard, n = 350), in comparison with quantitative viraemia in kidney transplant recipients undergoing routine screening. The decoy cells were then quantified per 10 HPF. Results Decoy cells occurred in 14.8% of samples, with a sensitivity of 71.4%, specificity of 87.7%, positive predictive value (PPV) of 22.2% and negative predictive value (NPV) of 98.6% to predict BKVAN. Quantification of decoy cells improved the PPV to 53.8% (≥10cells/10 HPF), comparable to quantitative viraemia of 52.2% (≥104copies/mL). SV40T staining of urinary cell blocks provided 100% sensitivity and 91.5% specificity, however insufficient material hampered cell block preparation in many samples. Combinations of decoy cell and viraemia in a diagnostic matrix further improved BKVAN prediction. Conclusion Our preliminary findings show both quantified decoy cells and viraemia are acceptable surrogate markers of early BK viral replication for screening of kidney transplant recipients.

18 F-fluorothymidine uptake in follicular lymphoma and error-prone DNA repair

BackgroundWe observed a disproportional 18 F-fluorothymidine (F-FLT) uptake in follicular lymphoma (FL) relative to its low cell proliferation. We tested the hypothesis that the ‘excess’ uptake of 18 F-FLT in FL is related to error-prone DNA repair and investigated whether this also contributes to 18 F-FLT uptake in diffuse large B cell lymphoma (DLBCL).MethodsWe performed immunohistochemical stainings to assess the pure DNA replication marker MIB-1 as well as markers of both DNA replication and repair like PCNA, TK-1 and RPA1 on lymph node biopsies of 27 FLs and 35 DLBCLs. In 7 FL and 15 DLBCL patients, 18 F-FLT-PET had been performed.Results18 F-FLT uptake was lower in FL than in DLBCL (median SUVmax 5.7 vs. 8.9, p = 0,004), but the ratio of 18 F-FLT-SUVmax to percentage of MIB-1 positive cells was significantly higher in FL compared with DLBCL (p = 0.001). The median percentage of MIB-1 positive cells was 10% (range, 10% to 20%) in FL and 70% (40% to 80%) in DLBCL. In contrast, the median percentages of PCNA, TK-1 and RPA1 positive cells were 90% (range, 80 to 100), 90% (80 to 100) and 100% (80 to 100) in FL versus 90% (60 to 100), 90% (60 to 100) and 100% (80 to 100) in DLBCL, respectively.ConclusionsThis is the first demonstration of a striking discordance between 18 F-FLT uptake in FL and tumour cell proliferation. High expression of DNA replication and repair markers compared with the pure proliferation marker MIB-1 in FL suggests that this discordance might be due to error-prone DNA repair. While DNA repair-related 18 F-FLT uptake considerably contributes to 18 F-FLT uptake in FL, its contribution to 18 F-FLT uptake in highly proliferative DLBCL is small. This apparently high contribution of DNA repair to the 18 F-FLT signal in FL may hamper studies where 18 F-FLT is used to assess response to cytostatic therapy or to distinguish between FL and transformed lymphoma.

Sensitivity of drug-resistant mutants of hepatitis B virus to poly-IC.

The long-term benefits of antiviral treatment are limited by the resistance of hepatitis B virus (HBV). However, the effect of interferon (IFN)α treatment on drug-resistant HBVs is so far unknown. We, therefore, investigated the effects of IFN-α inducer poly-IC on the replication of HBV mutants resistant to drugs such as lamivudine (LAM), adefovir dipivoxil (ADV) and entecavir (ETV) in mice. HBV DNA and HBV DNA intermediate (RI) were employed as markers of the virus replication and 2',5'-oligoadenylate synthase (OAS) mRNA as a marker of IFN-α/β induction. Poly-IC inhibited wtHBV replication and increased levels of OAS mRNA. Compared to the wt virus, the capacity of virus replication was reduced in most LAMr and ETVr mutants except those with mutations rtM(204V+L180M+V173L), and was similary in the ADVr mutants except rt(A121V+N236T). The virus replication was reduced after poly-IC treatment with LAMr and ADVr mutants similary to the wt virus. In contrast, ETVr mutants were resistant to the poly-IC treatment. In conclusion, the capacity of HBV replication and the sensitivity to IFN therapy are influenced by drug-resistant mutations. The IFN therapy may effectively inhibit HBV replication in particular in patients with LAMr or ADVr mutations but not in patients with ETVr mutations.

Phyllanthus for chronic hepatitis B virus infection

Chronic hepatitis B virus (HBV) infection is a common disease worldwide. In 2011, CDC received 39,636 reports of chronic HBV infection, with many additional people likely unaware of their disease due to its asymptomatic nature.1.www.cdc.gov/hepatitisStatistics/2011Surveillance/Commentary.htm.Google Scholar The three-dose schedule HBV vaccine is 95% effective in preventing HBV infection and provides immunity for at least 20 years. Many supplements such as milk thistle are used for liver disease treatment. Lesser known plants such as Phyllanthus species are used for hepatic disorders as part of traditional Chinese medicine. In vitro studies reported that Phyllanthus species contain lignans possessing cytotoxic activity, which suppresses HBV antigen secretion. Two systematic reviews have evaluated Phyllanthus in HBV treatment. A 2001 review of Phyllanthus treatment in 22 randomized trials reported that the plant may be beneficial in clearing HBV markers.2.J Viral Hepatitis. 2001; 8: 358-366Crossref PubMed Scopus (75) Google Scholar In addition, liver function tests were normalized without serious adverse effects from the plant. The studies used extracts of the whole plant Phyllanthus; only one trial indicated the specific plant species used (P. urinaris). Trials used multiple dosage forms: Phyllanthus granules were either extracted and encapsulated as an oral tablet or used to create a decoction as a tea. Studies had small sample sizes and a limited description of randomization and allocation procedures. This introduces bias and questionable statistical power, leading to questions about the significance of the findings. The Jadad scale was used to assess study quality, and only five were ranked high quality.Evidence has suggested a potential benefit of Phyllanthus extracts when used with antiviral therapy A 2010 systematic review of the effectiveness of Phyllanthus species in chronic HBV infection evaluated data on 1,326 patients of varying ages in 16 different trials.3.Cochrane Database Syst Rev. 2011 doi:10.1002/14651858.CD008960.pub2.Google Scholar Trials used multiple species—P. amarus, P. urinaria (Chamber bitter), and P. niruri—and preparations prepared in the same manner as in the 2001 review. This systematic review used data collection and analysis methodology based on the Cochrane Handbook of Systematic Reviews of Interventions. Fifteen trials compared Phyllanthus in combination with standard antiviral treatment versus standard antiviral therapy alone. Phyllanthus significantly reduced serum HBV DNA (a marker of efficacy) as well as HBeAg (a marker of viral replication) when combined with antiviral therapy. The summary concluded that based on current evidence, Phyllanthus may be beneficial when combined with standard antiviral therapy but has no benefit alone. Most trials in the 2010 review are at high risk for bias due to methods of allocation and blinding, incomplete outcome data, selective outcome reporting, and other factors. The duration of follow-up after treatment was a major limitation. One trial measured outcomes for 2 weeks, another for 24 weeks, and another for 2 years. Available studies did not provide data on the effect of Phyllanthus on morbidity and mortality. Fewer than 2% of participants experienced adverse events, which included light nausea, decreased appetite, and stomachache.HIGHLIGHTS■HBV infection is best prevented by avoiding shared needles and unsafe sexual practices, in addition to being vaccinated against HBV.■In vitro studies reported that Phyllanthus species contain lignans possessing cytotoxic activity, which suppresses HBV antigen secretion. ■HBV infection is best prevented by avoiding shared needles and unsafe sexual practices, in addition to being vaccinated against HBV.■In vitro studies reported that Phyllanthus species contain lignans possessing cytotoxic activity, which suppresses HBV antigen secretion. Chronic HBV infection is a serious condition that may result in liver cancer or cirrhosis. HBV infection is best prevented by avoiding shared needles and unsafe sexual practices, in addition to being vaccinated against HBV. Dietary supplements are not a replacement for maintaining good liver health practices, including moderation of alcohol use. Phyllanthus species have been promoted for their potential liver benefits. Evidence has suggested a potential benefit of Phyllanthus extracts when used with antiviral therapy. Extracts may contain different species in varied strengths. Most Phyllanthus supplements recommend one capsule with a meal two to three times daily. As always, patients should purchase supplements from reputable sources.