Eligibility and Safety of Triple Therapy for Hepatitis C: Lessons Learned from the First Experience in a Real World Setting

Background

HCV protease inhibitors (PIs) boceprevir and telaprevir in combination with PEG-Interferon alfa and Ribavirin (P/R) is the new standard of care in the treatment of chronic HCV genotype 1 (GT1) infection. However, not every HCV GT1 infected patient is eligible for P/R/PI therapy. Furthermore phase III studies did not necessarily reflect real world as patients with advanced liver disease or comorbidities were underrepresented. The aim of our study was to analyze the eligibility and safety of P/R/PI treatment in a real world setting of a tertiary referral center.

Methods

All consecutive HCV GT1 infected patients who were referred to our hepatitis treatment unit between June and November 2011 were included. Patients were evaluated for P/R/PI according to their individual risk/benefit ratio based on 4 factors: Treatment-associated safety concerns, chance for SVR, treatment urgency and nonmedical patient related reasons. On treatment data were analyzed until week 12.

Results

208 patients were included (F3/F4 64%, mean platelet count 169/nl, 40% treatment-naïve). Treatment was not initiated in 103 patients most frequently due to safety concerns. 19 patients were treated in phase II/III trials or by local centers and a triple therapy concept was initiated at our unit in 86 patients. Hospitalization was required in 16 patients; one patient died due to a gastrointestinal infection possibly related to treatment. A platelet count of <110/nl was associated with hospitalization as well as treatment failure. Overall, 128 patients were either not eligible for therapy or experienced a treatment failure at week 12.

Conclusions

P/R/PI therapies are complex, time-consuming and sometimes dangerous in a real world setting, especially in patients with advanced liver disease. A careful patient selection plays a crucial role to improve safety of PI based therapies. A significant number of patients are not eligible for P/R/PI, emphasizing the need for alternative therapeutic options.     

Reduction of apoptosis and preservation of mitochondrial integrity under ischemia/reperfusion injury is mediated by estrogen receptor β

Background

Estrogen improves cardiac recovery after ischemia/reperfusion (I/R) by yet incompletely understood mechanisms. Mitochondria play a crucial role in I/R injury through cytochrome c-dependent apoptosis activation. We tested the hypothesis that 17β-estradiol (E2) as well as a specific ERβ agonist improve cardiac recovery through estrogen receptor (ER)β-mediated mechanisms by reducing mitochondria-induced apoptosis and preserving mitochondrial integrity.

Methods

We randomized ovariectomized C57BL/6N mice 24h before I/R to pre-treatment with E2 or a specific ERβ agonist (ERβA). Isolated hearts were perfused for 20min prior to 30min global ischemia followed by 40min reperfusion.

Results

Compared with controls, ERβA and E2 treated groups showed a significant improvement in cardiac recovery, i.e. an increase in left ventricular developed pressure, dP/dtmax and dP/dtmin. ERβA and E2 pre-treatment led to a significant reduction in apoptosis with decreased cytochrome c release from the mitochondria and increased mitochondrial levels of anti-apoptotic Bcl2 and ACAA2. Protein levels of mitochondrial translocase inner membrane (TIM23) and mitochondrial complex I of respiratory chain were increased by ERβA and E2 pre-treatment. Furthermore, we found a significant increase of myosin light chain 2 (MLC2) phosphorylation together with ERK1/2 activation in E2, but not in ERβA treated groups.

Conclusions

Activation of ERβ is essential for the improvement of cardiac recovery after I/R through the inhibition of apoptosis and preservation of mitochondrial integrity and can be a achieved by a specific ERβ agonist. Furthermore, E2 modulates MLC2 activation after I/R independent of ERβ.

     

Hindcasting Historical Breeding Conditions for an Endangered Salamander in Ephemeral Wetlands of the Southeastern USA: Implications of Climate Change

The hydroperiod of ephemeral wetlands is often the most important characteristic determining amphibian breeding success, especially for species with long development times. In mesic and wet pine flatwoods of the southeastern United States, ephemeral wetlands were a common landscape feature. Reticulated flatwoods salamanders (Ambystoma bishopi), a federally endangered species, depend exclusively on ephemeral wetlands and require at least 11 weeks to successfully metamorphose into terrestrial adults. We empirically modeled hydroperiod of 17 A. bishopi breeding wetlands by combining downscaled historical climate-model data with a recent 9-year record (2006–2014) of observed water levels. Empirical models were subsequently used to reconstruct wetland hydrologic conditions from 1896–2014 using the downscaled historical climate datasets. Reconstructed hydroperiods for the 17 wetlands were highly variable through time but were frequently unfavorable for A. bishopi reproduction (e.g., only 61% of years, using a conservative estimate of development time [12 weeks], were conducive to larval development and metamorphosis). Using change-point analysis, we identified significant shifts in average hydroperiod over the last century in all 17 wetlands. Mean hydroperiods were shorter in recent years than at any other point since 1896, and thus less suitable for A. bishopi reproduction. We suggest that climate change will continue to impact the reproductive success of flatwoods salamanders and other ephemeral wetland breeders by reducing the number of years these wetlands have suitable hydroperiods. Consequently, we emphasize the importance of conservation and management for mitigating other forms of habitat degradation, especially maintenance of high quality breeding sites where reproduction can occur during appropriate environmental conditions.     

Break the pattern: breakpoints in beta diversity of vertebrates are general across clades and suggest common historical causes

The use of correlative analyses might be insufficient to understand the processes that control biodiversity, because the variables accounting for different hypotheses (e.g. current climate, past climate change, post‐glacial dispersal limitation) are mutually correlated. We suggest here that, in order to gain insight, it could be useful to search for latitudinal thresholds that could provide information about qualitative changes in the way biodiversity varies in space. Such tipping points could inform about higher‐level processes that are not reflected in correlative analyses. We test whether similar breakpoints in latitudinal beta‐diversity patterns exist for different vertebrate groups with diverse life histories and dispersal abilities. In birds, bats and non‐volant mammals we find breakpoints similar to those of amphibians. Differences in species composition are mainly due to species replacement from the equator to the breakpoint, but are dominated by nested species losses from the breakpoint to higher latitudes. Thus, marked thresholds discriminate two world regions where different processes appear to drive biodiversity.     

Adenosine arrests breast cancer cell motility by A3 receptor stimulation

In neutrophils, adenosine triphosphate (ATP) release and autocrine purinergic signaling regulate coordinated cell motility during chemotaxis. Here, we studied whether similar mechanisms regulate the motility of breast cancer cells. While neutrophils and benign human mammary epithelial cells (HMEC) form a single leading edge, MDA-MB-231 breast cancer cells possess multiple leading edges enriched with A3 adenosine receptors. Compared to HMEC, MDA-MB-231 cells overexpress the ectonucleotidases ENPP1 and CD73, which convert extracellular ATP released by the cells to adenosine that stimulates A3 receptors and promotes cell migration with frequent directional changes. However, exogenous adenosine added to breast cancer cells or the A3 receptor agonist IB-MECA dose-dependently arrested cell motility by simultaneous stimulation of multiple leading edges, doubling cell surface areas and significantly reducing migration velocity by up to 75 %. We conclude that MDA-MB-231 cells, HMEC, and neutrophils differ in the purinergic signaling mechanisms that regulate their motility patterns and that the subcellular distribution of A3 adenosine receptors in MDA-MB-231 breast cancer cells contributes to dysfunctional cell motility. These findings imply that purinergic signaling mechanisms may be potential therapeutic targets to interfere with the motility of breast cancer cells in order to reduce the spread of cancer cells and the risk of metastasis.     

Quantitative structure analysis of genetic diversity among spring bread wheats (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) from different geographical regions

Genetic diversity in spring bread wheat (T.␣aestivum L.) was studied in a total of 69 accessions. For this purpose, 52 microsatellite (SSR) markers were used and a total of 406 alleles were detected, of which 182 (44.8%) occurred at a frequency of <5% (rare alleles). The number of alleles per locus ranged from 2 to 14 with an average of 7.81. The largest number of alleles per locus occurred in the B genome (8.65) as␣compared to the A (8.43) and D (5.93) genomes, respectively. The polymorphism index content (PIC) value varied from 0.24 to 0.89 with an average of 0.68. The highest PIC for all accessions was found in the B␣genome (0.71) as compared to the A (0.68) and D␣genomes (0.63). Genetic distance-based method (standard UPGMA clustering) and a model-based method (structure analysis) were used for cluster analysis. The two methods led to analogical results. Analysis of molecular variance (AMOVA) showed that 80.6% of the total variation could be explained by the variance within the geographical groups. In comparison to the diversity detected for all accessions (H _e = 0.68), genetic diversity among European spring bread wheats was H _e = 0.65. A comparatively higher diversity was observed between wheat varieties from Southern European countries (Austria/Switzerland, Portugal/Spain) corresponding to those from other regions.     

Mutational analysis of cytochrome b at the ubiquinol oxidation site of yeast complex III

The cytochrome bc1 complex is a dimeric enzyme of the inner mitochondrial membrane that links electron transfer from ubiquinol to cytochrome c by a protonmotive Q cycle mechanism in which ubiquinol is oxidized at one center in the enzyme, referred to as center P, and ubiquinone is rereduced at a second center, referred to as center N. To better understand the mechanism of ubiquinol oxidation, we have examined catalytic activities and pre-steady-state reduction kinetics of yeast cytochrome bc1 complexes with mutations in cytochrome b that we expected would affect oxidation of ubiquinol. We mutated two residues thought to be involved in proton conduction linked to ubiquinol oxidation, Tyr132 and Glu272, and two residues proposed to be involved in docking ubiquinol into the center P pocket, Phe129 and Tyr279. Substitution of Phe129 by lysine or arginine yielded a respiration-deficient phenotype and lipid-dependent catalytic activity. Increased bypass reactions were detectable for both variants, with F129K showing the more severe effects. Substitution with lysine leads to a disturbed coordination of a b heme as deduced from changes in the midpoint potential and the EPR signature. Removal of the aromatic side chain in position Tyr279 lowers the catalytic activity accompanied by a low level of bypass reactions. Pre-steady-state kinetics of the enzymes modified at Glu272 and Tyr132 confirmed the importance of their functional groups for electron transfer. Altered center N kinetics and activation of ubiquinol oxidation by binding of cytochrome c in the Y132F and E272D enzymes indicate long range effects of these mutations.     

Analysis of intraviral protein-protein interactions of the SARS coronavirus ORFeome

The severe acute respiratory syndrome coronavirus (SARS-CoV) genome is predicted to encode 14 functional open reading frames, leading to the expression of up to 30 structural and non-structural protein products. The functions of a large number of viral ORFs are poorly understood or unknown. In order to gain more insight into functions and modes of action and interaction of the different proteins, we cloned the viral ORFeome and performed a genome-wide analysis for intraviral protein interactions and for intracellular localization. 900 pairwise interactions were tested by yeast-two-hybrid matrix analysis, and more than 65 positive non-redundant interactions, including six self interactions, were identified. About 38% of interactions were subsequently confirmed by CoIP in mammalian cells. Nsp2, nsp8 and ORF9b showed a wide range of interactions with other viral proteins. Nsp8 interacts with replicase proteins nsp2, nsp5, nsp6, nsp7, nsp8, nsp9, nsp12, nsp13 and nsp14, indicating a crucial role as a major player within the replication complex machinery. It was shown by others that nsp8 is essential for viral replication in vitro, whereas nsp2 is not. We show that also accessory protein ORF9b does not play a pivotal role for viral replication, as it can be deleted from the virus displaying normal plaque sizes and growth characteristics in Vero cells. However, it can be expected to be important for the virus-host interplay and for pathogenicity, due to its large number of interactions, by enhancing the global stability of the SARS proteome network, or play some unrealized role in regulating protein-protein interactions. The interactions identified provide valuable material for future studies.     

Reproductive characteristics of wild female Phayre's leaf monkeys

Understanding female reproductive characteristics is important for assessing fertility, interpreting female behavior, and designing appropriate conservation and captive management plans. In primate species lacking morphological signs of receptivity, such as most colobines, determination of reproductive parameters depends on the analysis of reproductive hormones. Here, we use fecal hormone analysis to characterize cycle patterns (N=6 females) and gestation length (N=7 females) in a group of wild Phayre's leaf monkeys (Trachypithecus phayrei crepusculus) in Phu Khieo Wildlife Sanctuary, Thailand. We found that both fecal estrogen (fE) and progestin (fP) levels showed clear biological patterns indicative of ovulation and conception. However, because fP patterns were inadequate in determining the end of the luteal phase, we used fE rather than fP patterns to delineate menstrual cycle parameters. We found a mean cycle length of 28.4 days (N=10), with follicular and luteal phases of 15.4 (N=10) and 12.5 days (N=14), respectively. On average, females underwent 3.57 (N=7) cycles until conception. Average gestation length was 205.3 days (N=7), with fE levels increasing over the course of pregnancy. Overall, the reproductive characteristics found for Phayre's leaf monkeys were consistent with results for other colobine species, suggesting that fecal hormone monitoring, particularly for fE metabolites, can provide useful reproductive information for this species. Am. J. Primatol. 72:1073–1081, 2010. © 2010 Wiley‐Liss, Inc.