Treatment of chronic hepatitis C genotype 3 with Sofosbuvir-based therpy: a real-life study.

BACKGROUND AND AIMS: Recently, Sofosbuvir was launched in India at affordable cost. We conducted a real-life study to determine the efficacy and safety of Sofosbuvir plus Ribavirin, with and without peginterferon-alfa 2a, in patients with chronic hepatitis C (CHC) genotype 3, the commonest genotype in South Asia. METHODS: This study included data of CHC patients from 11 sites in northern India between March 2015 and December 2015 (n = 1203). Patients with CHC genotype 3 (n = 931), who were treated with either Sofosbuvir 400 mg plus weight-based Ribavirin, daily ×24 weeks (n = 432) (dual therapy), or Peginterferon-α2a 180 mcg weekly, Sofosbuvir 400 mg plus weight-based Ribavirin, daily ×12 weeks (n = 499) (triple therapy) were included for analysis. Primary outcome was the proportion of patients achieving sustained viral response at 12 weeks post-therapy. RESULTS: The overall SVR rates were 91 and 92% in the dual and triple therapy arms, respectively. The SVR rates in treatment experienced were 67 and 74% versus 93 and 96% in naïve patients, on the dual and triple therapy arms, respectively. The SVR rates of cirrhotics were 73 and 75% on the dual and triple treatment arms, respectively. The SVR rates were low in the experienced cirrhotic patients: 44% (dual therapy) and 58% (triple therapy). Common adverse events were fatigue, headache, and myalgia. CONCLUSION: Both dual and triple therapy regimes resulted in SVR rates of >95% in CHC genotype 3 who were naive non-cirrhotics. However, the SVR rates were low in treatment-experienced cirrhotics.

QTL x environment interactions in rice. I. heading date and plant height.

One hundred twenty six doubled-haploid (DH) rice lines were evaluated in nine diverse Asian environments to reveal the genetic basis of genotype x environment interactions (GEI) for plant height (PH) and heading date (HD). A subset of lines was also evaluated in four water-limited environments, where the environmental basis of G x E could be more precisely defined. Responses to the environments were resolved into individual QTL x environment interactions using replicated phenotyping and the mixed linear-model approach. A total of 37 main-effect QTLs and 29 epistatic QTLs were identified. On average, these QTLs were detectable in 56% of the environments. When detected in multiple environments, the main effects of most QTLs were consistent in direction but varied considerably in magnitude across environments. Some QTLs had opposite effects in different environments, particularly in water-limited environments, indicating that they responded to the environments differently. Inconsistent QTL detection across environments was due primarily to non- or weak-expression of the QTL, and in part to significant QTL x environment interaction effects in the opposite direction to QTL main effects, and to pronounced epistasis. QTL x environment interactions were trait- and gene-specific. The greater GEI for HD than for PH in rice were reflected by more environment-specific QTLs, greater frequency and magnitude of QTL x environment interaction effects, and more pronounced epistasis for HD than for PH. Our results demonstrated that QTL x environment interaction is an important property of many QTLs, even for highly heritable traits such as height and maturity. Information about QTL x environment interaction is essential if marker-assisted selection is to be applied to the manipulation of quantitative traits.

Identification of QTL for growth- and grain yield-related traits in rice across nine locations of Asia.

Rice double-haploid (DH) lines of an indica and japonica cross were grown at nine different locations across four countries in Asia. Genotype-by-environment (G x E) interaction analysis for 11 growth- and grain yield-related traits in nine locations was estimated by AMMI analysis. Maximum G x E interaction was exhibited for fertility percentage number of spikelets and grain yield. Plant height was least affected by environment, and the AMMI model explained a total of 76.2% of the interaction effect. Mean environment was computed by averaging the nine environments and subsequently analyzed with other environments to map quantitative trait loci (QTL). QTL controlling the 11 traits were detected by interval analysis using mapmaker/qtl. A threshold LOD of >/=3.20 was used to identify significant QTL. A total of 126 QTL were identified for the 11 traits across nine locations. Thirty-four QTL common in more than one environment were identified on ten chromosomes. A maximum of 44 QTL were detected for panicle length, and the maximum number of common QTL were detected for days to heading detected. A single locus for plant height (RZ730-RG810) had QTL common in all ten environments, confirming AMMI results that QTL for plant height were affected the least by environment, indicating the stability of the trait. Two QTL were detected for grain yield and 19 for thousand-grain weight in all DH lines. The number of QTL per trait per location ranged from zero to four. Clustering of the QTL for different traits at the same marker intervals was observed for plant height, panicle number, panicle length and spikelet number suggesting that pleiotropism and or tight linkage of different traits could be the possible reason for the congruence of several QTL. The many QTL detected by the same marker interval across environments indicate that QTL for most traits are stable and not essentially affected by environmental factors.

A rapid and reproducible on line automated technique to determine endothelial function.

OBJECTIVE: To evaluate clinically a new on line, automated technique to measure flow mediated dilatation (FMD) as a marker of endothelial function. DESIGN: Prospective study. PATIENTS: 12 healthy volunteers and 12 patients with significant, angiographically documented coronary artery disease. INTERVENTIONS: Brachial arteries were imaged using a standard vascular ultrasound system with a 5-12 MHz linear transducer. Arterial diameter was measured on line (in real time) by connecting the ultrasound system to a personal computer equipped with a frame grabber and artery wall detection software (VIA) specially developed by the authors' group. By using this new technique, FMD was measured following 4.5 minutes of ischaemia of the proximal forearm in all subjects on two separate days. RESULTS: The mean (SD) day to day variability in FMD measurements was 0.90 (0.48)%,which compares very favourably with current methods. The FMD measurement was available within seconds of completing the scan. CONCLUSIONS: Personal computer based automated techniques to assess FMD involve image acquisition and recording after which a second (off line) image interpretation session is required. The need for off line analysis makes current methods time consuming and increases the variability of measurement. This on line, automated analysis technique for FMD assessment reduces the variability and greatly increases the speed of measurement. Using this system may mean that fewer patients will be required in clinical trials assessing the effects of interventions on endothelial function. Adopting this method may also facilitate the screening of larger numbers of subjects for endothelial dysfunction.

Effect of raw wheat germ addition on the physical texture and objective color of a designer food (pan bread).

Pan bread formulations based on raw wheat germ, vital wheat gluten, and enzyme-active soybean flour were optimized with the objective of developing a phytochemical-enriched designer food product with superior nutritional and sensory qualities. The objective texture values (measured as compression force, g) indicated that the test bread with 10% wheat germ addition was comparable (299.9 g) to the control (210.1 g), but the compression force was significantly higher (415.4 g) at 20% wheat germ level. With 0.5% sodium stearoyl-2-lactylate (SSL), 30 ppm potassium bromate and 50 ppm ascorbic acid, the test breads with 10 and 20% wheat germ had compression force values of 313.8 g and 367.7 g, respectively. Comparing the CIE L*a* values, the test bread samples having up to 20% wheat germ were slightly darker in crumb color than the white flour control bread, but were significantly lighter than the whole wheat flour bread. However, the addition of wheat germ increased the yellow color of bread crumb as indicated by the higher b* values of 11.4, 16.4 and 21.4, for control, 10% and 20% wheat germ breads, respectively. The physical texture and objective color measurements can be used in evaluating the quality of a phytochemical-enriched designer food (pan bread). It can be concluded that wheat germ-enriched bread can be prepared by using white flour, 20% raw wheat germ, 0.5% SSL, 30 ppm potassium bromate and 50 ppm ascorbic acid to provide consumers with a functional food.

Papillary fibroelastoma of the left atrial appendage.

Cardiac papillary fibroelastomas are benign tumors that usually arise from the valvular endocardium. They are clinically important because of their propensity to embolize. We describe, to the best of our knowledge, the first reported case of a papillary fibroelastoma arising from the left atrial appendage, giving rise to multiple cerebral embolic events. The tumor was excised surgically, with no further embolic events.

PI3K inhibitors reverse the suppressive actions of insulin on CYP2E1 expression by activating stress-response pathways in primary rat hepatocytes.

Insulin-associated signaling pathways are critical in the regulation of hepatic physiology. Recent inhibitor-based studies have implicated a mechanistic role for phosphatidylinositol 3' kinase (PI3K) in the insulin-mediated suppression of CYP2E1 mRNA levels in hepatocytes. We investigated the dose dependence for this response and for the effects of insulin and extracellular matrix on PI3K signaling and CYP2E1 mRNA expression levels using a highly defined rat primary hepatocyte culture system. The PI3K inhibitors wortmannin and LY294002 stimulated stress-activated protein kinase/c-Jun NH(2)-terminal kinase (SAPK/JNK) and p38 mitogen-activated protein kinase (MAPK) phosphorylation in a rapid and concentration-dependent manner that paralleled the inhibition of protein kinase B (PKB) phosphorylation. Although PI3K inhibitors reversed the suppressive effects of insulin on CYP2E1 expression, these effects only occurred at concentrations well in excess of those required to achieve complete inhibition of PKB phosphorylation. These same concentrations produced cytotoxic responses as evidenced by perturbed cellular morphology and elevated release of lactate dehydrogenase. Wortmannin-mediated activation of the SAPK/JNK and p38 MAPK pathways also resulted in the mobilization of activator protein-1 complex to the nuclear compartment. We conclude that the suppression of CYP2E1 mRNA expression by insulin is not directly associated with PI3K-dependent pathway activation, but rather is linked to a cytotoxic response stemming from acute challenge with PI3K inhibitors.

Baculovirus vectors repress phenobarbital-mediated gene induction and stimulate cytokine expression in primary cultures of rat hepatocytes.

Baculovirus transfection strategies have proven successful at transferring foreign DNA into hepatoma cells and primary hepatocytes. When testing the utility of these methodologies in cultured hepatocytes, we discovered that the presence of baculovirus disrupts the phenobarbital (PB) gene induction process, a potent transcriptional activation event characteristic of highly differentiated hepatocytes, and repressed expression of the albumin gene. In concert with previous reports from our laboratory demonstrating that increased cAMP levels can completely repress the induction of specific cytochrome P450 (CYP) genes, cAMP concentrations and PKA activities were measured in the primary hepatocytes subsequent to baculovirus exposure. However, neither parameter was affected by the presence of the virus. To evaluate whether immune response modulation was triggered by baculovirus exposure, RNase protection assays were performed and demonstrated that baculovirus infection activates TNF-alpha, IL-1alpha and IL-1beta expression in the primary hepatocyte cultures. Immunocytochemical experiments indicated that the production of cytokines was likely due to the presence of small numbers of Kupffer cells present in the culture populations. Exogenously added TNF-alpha was also effective in repressing PB induction, consistent with other reports indicating that inflammatory cytokines are capable of suppressing expression of biotransformation enzyme systems. Comparative studies demonstrated the specificity of these effects since exposures of hepatocytes to adenoviral vectors did not result in down-regulation of hepatic gene responsiveness. These results indicate that baculovirus vectors enhance the expression of inflammatory cytokines in primary hepatocyte cultures, raising concerns as to whether these properties will compromise the use of baculovirus vectors for study of cytochrome P450 gene regulation, as well as for liver-directed gene therapy in humans.

An alleged poisoning with methanol and formaldehyde.

It was alleged that a defendant added an unspecified amount of undyed formalin solution, containing formaldehyde and methanol, to the victim's bottle of ice and drinking water. The medical report indicated that except for a slight elevation of total creatine kinase, all other chemistry profiles were within normal ranges. The elevation of creatine kinase suggested muscle injury and inflammation; however, the significance of this elevation was not clear. Toxicological evaluations were made by conducting risk assessments. Based upon the medical report and risk assessments, the following conclusions were made: The calculated exposure doses of methanol and formaldehyde were too low to cause appreciable adverse effects; however, formaldehyde may have irritated the gastrointestinal tract causing smooth muscle and mucosal inflammation. The doses of methanol and formaldehyde were too low to cause death. The exposure scenario (a single oral exposure to formaldehyde) would not likely increase the cancer risk in the victim. The risk assessments provided resulted in a reduction in charge from attempted murder to felony.

Insulin-mediated modulation of cytochrome P450 gene induction profiles in primary rat hepatocyte cultures.

In this investigation, we examined the effects of insulin on gene induction responsiveness in primary rat hepatocytes. Cells were cultured for 72 hours either in the absence or presence of 1 microM insulin and then exposed to increasing concentrations of phenobarbital (PB; 0.01-3.5 mM). Culturing in the absence of insulin produced 1.5-2-fold increases in the induction magnitude of CYP2B1 and CYP2B2 mRNA expression resulting from PB exposures, without altering the bell-shaped dose-response curve characteristic of this agent. However, for the CYP3A1 gene, insulin removal led to a pronounced shift in both the PB-induction magnitude and dose-response relationships of the induction response, with higher levels of CYP3A1 expression resulting from exposures to lower concentrations of inducer. Insulin removal also reduced the time required to attain maximal induction of CYP2B1/2 and CYP3A1 gene expression. The insulin effects were not specific for PB induction, as insulin deprivation similarly enhanced both dexamethasone- and beta-naphthoflavone-inducible CYP3A1 and CYP1A1 expression profiles, respectively. In contrast, the level of albumin mRNA expression was reduced considerably in cells deprived of insulin. We conclude that insulin is an important regulator of inducible and liver-specific gene expression in primary rat hepatocytes.

Physiological and pathophysiological regulation of cytochrome P450.

This article is a report on a symposium sponsored by the American Society for Pharmacology and Experimental Therapeutics and held at the April 1998 Experimental Biology '98 meeting in San Francisco. The presentations focused on the mechanisms of regulation of cytochrome P450 gene expression by developmental factors and by hormones and cytokines, as well as on the interplay between physiological and chemical regulation. Approaches and systems used to address these questions included conditional gene knockouts in mice, primary hepatocyte cultures, immunofluorescence imaging of cells, and cell lines stably expressing reporter gene constructs.

Invasive cervical cancer in human immunodeficiency virus-infected and uninfected hospital patients.

OBJECTIVE: To compare the prevalence of invasive cervical cancer in women with, and in women without, human immunodeficiency virus (HIV) infection, so as to evaluate the inclusion of invasive cervical cancer in the AIDS surveillance case definition. METHODS: The Sentinel Hospital Surveillance System for HIV Infection collected data and serum specimens that remained after clinical testing of persons who received inpatient or outpatient care at 14 hospitals with high HIV prevalence. We analyzed data on invasive cervical cancer obtained from medical record review and HIV serostatus from white, black, and Hispanic women in the age groups 20-34, 35-44, and 45-54 years. RESULTS: In 1994 and 1995, 2684 (6.6%) of the 40,524 women sampled were HIV infected. Of the HIV-positive women, 28 had invasive cervical cancer (10.4 per 1000 women) and of the HIV-negative women, 236 had invasive cervical cancer (6.2 per 1000 women, relative risk [RR] 1.7, 95% confidence interval [CI] 1.1, 2.5). The prevalence of invasive cervical cancer was higher for HIV-positive than for HIV-negative black women aged 20-34 (RR 3.8; CI 1.7, 8.5) and Hispanic women aged 20-34 (RR 7.3; CI 1.4, 37.1) and 35-44 (RR 3.9; CI 1.1, 14.7) years. Twenty-six of the 28 cases of invasive cervical cancer in HIV-positive women were in women known to be HIV-positive during admission. CONCLUSION: The prevalence of invasive cervical cancer was higher for women who were HIV positive than for women who were HIV negative. This lends support to the inclusion of invasive cervical cancer in the revision of the surveillance case definition for AIDS in 1993.

Differential induction of cytochrome P450 gene expression by 4n-alkyl-methylenedioxybenzenes in primary rat hepatocyte cultures.

A well-characterized primary rat hepatocyte culture system was used to examine induction patterns of cytochrome 450 gene expression by a series of 4-n-alkyl-methylenedioxybenzene (MDBs) derivatives. Hepatocytes were treated for 24, 48, or 72 hours with 0-500 microM of the MDB compounds, and total cellular RNA and protein from each treatment was evaluated by hybridization and immunochemical techniques. Exposure to MDB congeners possessing increasing 4-n-alkyl side-chain length (C0-C8) resulted in dose- and structure-dependent activation of CYP2B1, 2B2, 3A1, 1A1, and 1A2 gene expression. At equivalent 100 microM concentrations, the C6 and C8 MDB congeners were more effective than the prototypical inducer phenobarbital (PB) with respect to induction potency of CYP2B1, CYP2B2, and CYP3A1 gene expression. In contrast to PB, longer side-chain-substituted MDBs effectively induced CYP1A1 and CYP1A2 gene expression, in addition to the CYP2B and CYP3A genes. At equivalent molar concentrations, the catechol derivative of C6-MDB was ineffective in its ability to induce CYP gene expression, indicating the importance of the intact methylenedioxy bridge in the induction mechanism. Levels of MDB-inducible CYP2B1 and CYP2B2 mRNA were highly correlated with CYP2B1/2 apoprotein levels, ascertained by immunoblot analysis of cultured hepatocyte S9 fractions. Compared with results from previous in vivo analysis (12), the current data indicate that pharmacodynamic factors may influence MDB induction profiles and that differences in MDB effects on CYP gene expression result depending on distinct structure-activity relationships.

Effects of chemical inducers on human microsomal epoxide hydrolase in primary hepatocyte cultures.

Human microsomal epoxide hydrolase (mEH; EC 3.3.2.3) is an important biotransformation enzyme and potential risk determinant for pathologies such as cancer and teratogenesis. Currently, the effects of chemical exposures on human mEH gene expression are largely unknown, but they may constitute a unique modifier of disease susceptibility. To examine this issue, we exposed cultures of primary human hepatocytes isolated from seven donors to prototypic chemical inducers [such as phenobarbital (PB), polyaromatic hydrocarbons, dexamethasone, butylated hydroxyanisole, and ciprofibrate]. Basal levels of mEH RNA and protein were detected readily in untreated cells. Chemical treatment of cultured hepatocytes resulted in variable mEH RNA and protein expression, but, in general, only modest modulatory effects were detected following these exposures. The maximum increase in mEH RNA expression observed was approximately 3.5-fold following Arochlor 1254 exposure. Immunochemical levels of mEH protein were quantified for all treatment groups in three cultures and demonstrated less overall variation and, in general, a lack of concordance with corresponding mEH RNA levels. Cytochrome P450 (CYP) 1A2 and 3A mRNA levels were measured before and following exposure to beta-naphthaflavone and PB, respectively, to permit independent evaluation of hepatocyte inducer responsiveness. Substantial increases in RNA expression levels for both the CYP1A2 and CYP3A genes demonstrated that the hepatocyte cultures were robust and highly responsive to inducer treatment. These results indicate that the mEH gene in human hepatocytes is only modestly responsive to chemical exposures.

Artemisinin population pharmacokinetics in children and adults with uncomplicated falciparum malaria.

AIMS: To investigate the pharmacokinetics of the antimalarial artemisinin in the field setting using sparsely collected data. METHODS: Artemisinin concentrations were determined by h.p.l.c. in a total of 107 capillary plasma samples collected on the first day and in 33 samples on the last day of a 5-day oral artemisinin regimen of 10 mg kg(-1) day(-1) in 23 paediatric (aged 2-12 years) and 31 adult (aged 16-45 years) Vietnamese patients with uncomplicated falciparum malaria. The population model was developed using NONMEM, incorporating interoccasion variability and accounting for a systematic change in artemisinin pharmacokinetics with time, modelled as a change in oral bioavailability. RESULTS: Clinical efficacy, in terms of parasite clearance and fever subsidence times, was comparable between children and adults. A one-compartment model with separate pharmacokinetic estimates for children and adults was found best to describe the disposition of artemisinin after oral administration. The population estimates for artemisinin clearance and distribution volume, respectively, were 432 1 h(-1) and 16001 for adults and 14.41 h(-1) kg(-1) and 37.91 kg(-1) for children, with an intersubject variability (collectively for both age groups) of 45% and 104%, respectively. The oral bioavailability was estimated to decrease from Day 1 to Day 5 by a factor of 6.9, a value found to be similar for children and adults. CONCLUSIONS: Artemisinin pharmacokinetic data was successfully derived in both paediatric and adult patients using 2-3 capillary blood samples taken in conjunction with parasitaemia monitoring. This study's findings advocated the dosing of artemisinin to children according to bodyweight and to adults according to a standard dose.

Protein synthesis inhibitors exhibit a nonspecific effect on phenobarbital-inducible cytochome P450 gene expression in primary rat hepatocytes.

Previous investigations have indicated that de novo protein synthesis is a critical requirement for phenobarbital (PB) induction. We reexamined this issue in PB-responsive primary rat hepatocyte cultures using a broader array of protein synthesis inhibitors and experimental end points. Anisomycin, cycloheximide, emetine, puromycin, and puromycin aminonucleoside, a negative analog, were evaluated for their respective effects on protein synthesis and the PB-induction process. All of the inhibitors effectively repressed de novo protein synthesis in the cells in a concentration-dependent manner. However, anisomycin only minimally effected PB induction, ascertained though the measure of CYP2B1, CYP2B2, and CYP3A1 mRNA levels. The inactive agent, puromycin aminonucleoside, produced marked repression of the PB-induction response. Results from further experiments demonstrated that these protein synthesis inhibitors stimulated rapid and differential phosphorylation of the stress-activated protein kinase/c-Jun kinase (SAPK/JNK) pathway, indicating nonselective actions on cellular processes. Puromycin aminonucleoside was without effect on these pathways, despite its efficacy as an inhibitor of PB induction. These results demonstrate that de novo protein synthesis is not a requirement for PB induction, nor is activation of the SAPK/JNK kinase cascade responsible for down-regulating PB responsiveness in primary hepatocytes.