Systematic review of influenza resistance to the neuraminidase inhibitors

<p>Abstract</p> <p>Background</p> <p>Antivirals play a critical role in the prevention and the management of influenza. One class of antivirals, neuraminidase inhibitors (NAIs), is effective against all human influenza viruses. Currently there are two NAI drugs which are licensed worldwide: oseltamivir (Tamiflu^®) and zanamivir (Relenza^®); and two drugs which have received recent approval in Japan: peramivir and laninamivir. Until recently, the prevalence of antiviral resistance has been relatively low. However, almost all seasonal H1N1 strains that circulated in 2008-09 were resistant to oseltamivir whereas about 1% of tested 2009 pandemic H1N1 viruses were found to be resistant to oseltamivir. To date, no studies have demonstrated widespread resistance to zanamivir. It seems likely that the literature on antiviral resistance associated with oseltamivir as well as zanamivir is now sufficiently comprehensive to warrant a systematic review.</p> <p>The primary objectives were to systematically review the literature to determine the incidence of resistance to oseltamivir, zanamivir, and peramivir in different population groups as well as assess the clinical consequences of antiviral resistance.</p> <p>Methods</p> <p>We searched MEDLINE and EMBASE without language restrictions in September 2010 to identify studies reporting incidence of resistance to oseltamivir, zanamivir, and peramivir. We used forest plots and meta-analysis of incidence of antiviral resistance associated with the three NAIs. Subgroup analyses were done across a number of population groups. Meta-analysis was also performed to evaluate associations between antiviral resistance and clinical complications and symptoms.</p> <p>Results</p> <p>We identified 19 studies reporting incidence of antiviral resistance. Meta-analysis of 15 studies yielded a pooled incidence rate for oseltamivir resistance of 2.6% (95%CI 0.7% to 5.5%). The incidence rate for all zanamivir resistance studies was 0%. Only one study measured incidence of antiviral resistance among subjects given peramivir and was reported to be 0%. Subgroup analyses detected higher incidence rates among influenza A patients, especially for H1N1 subtype influenza. Considerable heterogeneity between studies precluded definite inferences about subgroup results for immunocompromised patients, in-patients, and children. A meta-analysis of 4 studies reporting association between oseltamivir-resistance and pneumonia yielded a statistically significant risk ratio of 4.2 (95% CI 1.3 to 13.1, p = 0.02). Oseltamivir-resistance was not statistically significantly associated with other clinical complications and symptoms.</p> <p>Conclusion</p> <p>Our results demonstrate that that a substantial number of patients may become oseltamivir-resistant as a result of oseltamivir use, and that oseltamivir resistance may be significantly associated with pneumonia. In contrast, zanamivir resistance has been rarely reported to date.</p

Mitogen-activated protein kinases are involved in hepatocanalicular dysfunction and cholestasis induced by oxidative stress

In previous studies, we showed that the pro-oxidant model agent tert-butyl hydroperoxide (tBuOOH) induces alterations in hepatocanalicular secretory function by activating Ca2+-dependent protein kinase C isoforms (cPKC), via F-actin disorganization followed by endocytic internalization of canalicular transporters relevant to bile formation (Mrp2, Bsep). Since mitogen-activated protein kinases (MAPKs) may be downstream effectors of cPKC, we investigated here the involvement of the MAPKs of the ERK1/2, JNK1/2, and p38MAPK types in these deleterious effects. tBuOOH (100 µM, 15 min) increased the proportion of the active, phosphorylated forms of ERK1/2, JNK1/2, and p38MAPK, and panspecific PKC inhibition with bisindolylmaleimide-1 (100 nM) or selective cPKC inhibition with Gö6976 (1 μM) prevented the latter two events. In isolated rat hepatocyte couplets, tBuOOH (100 µM, 15 min) decreased the canalicular vacuolar accumulation of the fluorescent Bsep and Mrp2 substrates, cholylglycylamido fluorescein, and glutathione-methylfluorescein, respectively, and selective inhibitors of ERK1/2 (PD098059), JNK1/2 (SP600125), and p38MAPK (SB203580) partially prevented these alterations. In in situ perfused rat livers, these three MAPK inhibitors prevented tBuOOH (75 µM)-induced impairment of bile flow and the decrease in the biliary output of the Bsep and Mrp2 substrates, taurocholate, and dinitrophenyl-S-glutathione, respectively. The changes in Bsep/Mrp2 and F-actin localization induced by tBuOOH, as assessed by (immuno)fluorescence staining followed by analysis of confocal images, were prevented total or partially by the MAPK inhibitors. We concluded that MAPKs of the ERK1/2, JNK1/2, and p38MAPK types are all involved in cholestasis induced by oxidative stress, by promoting F-actin rearrangement and further endocytic internalization of canalicular transporters critical for bile formation.Fil: Toledo, Flavia D. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental (IFISE-CONICET); Argentina.Fil: Basiglio, Cecilia Lorena. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental (IFISE-CONICET); Argentina.Fil: Barosso, Ismael R. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental (IFISE-CONICET); Argentina.Fil: Boaglio, Andrea C. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental (IFISE-CONICET); Argentina.Fil: Zucchetti, Andrés E. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental (IFISE-CONICET); Argentina.Fil: Sanchez Pozzi, Enrique J. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental (IFISE-CONICET); Argentina.Fil: Roma, Marcelo Gabriel. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental (IFISE-CONICET); Argentina

Security and privacy in massively-multiplayer online games and social and corporate virtual worlds

2007 was the year of online gaming fraud - with malicious programs that specifically target online games and virtual worlds increasing by 145% and the emergence of over 30,000 new programs aimed at stealing online game passwords. Such malware is invariably aimed at the theft of virtual property accumulated in a user’s account and its sale for real money. With 217 million regular users of MMO/VWs (Massively Multiplayer Online Games and Virtual Worlds) and real-money sales of virtual objects estimated at nearly US\$ 2 billion worldwide at the end of 2007, this is a serious issue. The failure to recognise the importance of protecting the real-money value locked up in this grey-zone of the economy is leading to an exponential increase in attacks targeting online MMO/VWs. Another important area of risk is the disclosure of private data. MMO/VWs are commonly perceived as being completely separate from the real lives of their users and therefore immune to privacy risks. In reality, representing yourself as an avatar is little different from using any other form of online persona. The inclusion of IRC and VOIP channels, along with the false sense of security created by MMO/VWs, leads to significantly increased disclosures of private data such as location and personal characteristics. The main body of this report describes in detail these risks and others, including in-game access-control vulnerabilities, scripting vulnerabilities, denial of service, spam and threats to minors, before making a number of recommendations on how to remedy them

Schematic representation of the signalling events involved in E₂17G-induced cholestasis by endocytic internalization and further retention of canalicular transporters relevant to bile formation (Bsep, Mrp2).

<p>p38, acting downstream of cPKC, triggers endocytic internalization of the apical carriers presumably towards apical early endosomes (AEE), the first intracellular endosomal compartment receiving internalized proteins from the apical membrane, in a microtubule-independent manner (solid arrow). These transporters traffic to, and accumulate into, apical recycling endosomes (ARE), from where they can be retargeted to the apical membrane during the recovery of the cholestatic process, in a microtubule-dependent manner (dashed arrows). Activation of the PI3K/Akt/ERK1/2 signalling pathway halts this latter process, thus explaining the increased colocalization of Bsep/Mrp2 with Rab11a, an ARE marker. This prolongs the cholestatic effect of E₂17G by impeding the fast, spontaneous retargeting of intracellular transporters that would lead to a rapid recovery from the cholestatic injury.</p

Effect of inhibition of p38 or ERK1/2, and coinhibition of cPKC-ERK1/2, PI3K-p38, or p38-ERK1/2 on E₂17G-induced retrieval of Bsep and Mrp2 in IRHCs.

<p>The upper panels show representative confocal immunofluorescence images of the localization of Bsep and Mrp2 in DMSO-treated (control) or E₂17G (200 µM)-treated IRHCs, with or without the p38 inhibitor SB203580 (SB; 1 µM) or the ERK1/2 inhibitor PD98059 (PD; 5 µM), in combination or not with the cPKC inhibitor Gö6976 (Gö; 1 µM) or PI3K inhibitor wortmanin (WM; 100 nM). The lower panels show the densitometric analysis of the fluorescence intensity along a line (8 µm) perpendicular to the center of the canalicular vacuole (from +4 to −4 µm). The statistical analysis of the profiles of fluorescence showed a significant change in the E₂17G-treated group (P<0.05; number of analyzed canalicular vacuoles >10), but this reverted to normal in the E₂17G-SB, E₂17G-PD, E₂17G-PD-SB, E₂17G-Gö-PD and E₂17G-WM-SB groups for Bsep and Mrp2.</p

Effect of E₂17G on colocalization of Rab11a with Mrp2 or Bsep in IRHCs.

<p>Immunofluorescence confocal images showing staining of Mrp2 or Bsep (green) and Rab11a (red) in IRHCs treated with DMSO (control), the ERK1/2 inhibitor PD98059 (PD; 5 µM), the p38 inhibitor SB203580 (SB; 1 µM), or both inhibitors together. Colocalization of Rab11a with Mrp2 or Bsep in the E₂17G-treated group is indicated by orange-yellow fluorescence in merged images. Insets depict F-actin staining, which was used to demarcate the limits of the canalicular vacuoles.</p

Effect of the inhibition of p38, ERK1/2 and JNK1/2, or the coinhibition of cPKC-ERK1/2, PI3K-p38, or p38-ERK1/2, on E₂17G-induced impairment of the canalicular accumulation of the Bsep and Mrp2 fluorescent substrates in IRHCs.

<p>IRHCs were incubated with E₂17G (200 µM, 20 min) (or DMSO in controls), with or without pretreatment for 15 min with the JNK1/2 inhibitor SP600125 (1 µM), the ERK1/2 inhibitor PD98059 (PD; 5 µM), and/or the p38 inhibitor SB203580 (SB; 1 µM), together or not with the cPKC inhibitor Gö6976 (Gö; 1 µM) or PI3K inhibitor wortmanin (WM; 100 nM). Canalicular accumulation CGamF (Bsep substrate, panel A) and GS-MF (Mrp2 substrate, panel B) was determined as the percentage of couplets displaying visible fluorescence in their canalicular vacuoles from a total of at least 200 couplets per preparation. The results are expressed as percentages of the control group and are shown as mean ± SEM (<i>n</i> = 3–4). *P<0.05 <i>vs.</i> E₂17G, and ^#P<0.05 <i>vs.</i> E₂17G-WM, E₂17G-Gö, E₂17G-PD or E₂17G-SB.</p

E₂17G activates the MAPK signalling pathway.

<p><i>(A)</i> left panel: representative Western blottings of phospho (p)-p38, p-ERK1/2, p-JNK1/2 and total forms of all these MAPK types were obtained from whole cellular lysates of primary-cultured rat hepatocytes incubated with E₂17G (200 µM) for 10 to 60 min, or with E₂17G (200 µM) for 20 min in cells pretreated with the PI3K inhibitor wortmanin (WM, 100 nM) or with the cPKC inhibitor Gö6976 (Gö, 1 µM) for 15 min. <i>A</i> (right panel), and <i>B</i> and <i>C panels</i> show phosphorylation status of all MAPK types evaluated (calculated as the p-MAPK to total MAPK ratio for each experimental condition). An arbitrary value of 100 was assigned to the band of highest densitometric intensity in every Western blot before the ratio was calculated. The results are shown as mean ± SEM (<i>n</i> = 5). *P<0.05 <i>vs.</i> control (cells treated only with DMSO), and ^#P<0.05 <i>vs.</i> E₂17G (20 min).</p