Protein crystals in adenovirus type 5-infected cells: requirements for intranuclear crystallogenesis, structural and functional analysis

Intranuclear crystalline inclusions have been observed in the nucleus of epithelial cells infected with Adenovirus serotype 5 (Ad5) at late steps of the virus life cycle. Using immuno-electron microscopy and confocal microscopy of cells infected with various Ad5 recombinants modified in their penton base or fiber domains, we found that these inclusions represented crystals of penton capsomers, the heteromeric capsid protein formed of penton base and fiber subunits. The occurrence of protein crystals within the nucleus of infected cells required the integrity of the fiber knob and part of the shaft domain. In the knob domain, the region overlapping residues 489–492 in the FG loop was found to be essential for crystal formation. In the shaft, a large deletion of repeats 4 to 16 had no detrimental effect on crystal inclusions, whereas deletion of repeats 8 to 21 abolished crystal formation without altering the level of fiber protein expression. This suggested a crucial role of the five penultimate repeats in the crystallisation process. Chimeric pentons made of Ad5 penton base and fiber domains from different serotypes were analyzed with respect to crystal formation. No crystal was found when fiber consisted of shaft (S) from Ad5 and knob (K) from Ad3 (heterotypic S5-K3 fiber), but occurred with homotypic S3K3 fiber. However, less regular crystals were observed with homotypic S35-K35 fiber. TB5, a monoclonal antibody directed against the Ad5 fiber knob was found by immunofluorescence microscopy to react with high efficiency with the intranuclear protein crystals in situ. Data obtained with Ad fiber mutants indicated that the absence of crystalline inclusions correlated with a lower infectivity and/or lower yields of virus progeny, suggesting that the protein crystals might be involved in virion assembly. Thus, we propose that TB5 staining of Ad-infected 293 cells can be used as a prognostic assay for the viability and productivity of fiber-modified Ad5 vectors

A922 Sequential measurement of 1 hour creatinine clearance (1-CRCL) in critically ill patients at risk of acute kidney injury (AKI)

Meeting abstrac

Impact assessment of the European Clinical Trials Directive: a longitudinal, prospective, observational study analyzing patterns and trends in clinical drug trial applications submitted since 2001 to regulatory agencies in six EU countries

<p>Abstract</p> <p>Background</p> <p>Shifts in clinical trial application rates over time indicate if the attractiveness of a country or region for the conduct of clinical trials is growing or decreasing. The purpose of this observational study was to track changes in drug trial application patterns across several EU countries in order to analyze the medium-term impact of the EU Clinical Trials Directive 2001/20/EC on the conduct of drug trials.</p> <p>Methods</p> <p>Rates of Clinical Trial Applications (CTA) for studies with medicinal products in those six countries in the EU, which authorize on average more than 500 trials per year, were analyzed. Publicly available figures on the number of annually submitted CTA, the distribution of trials per phase and the type of sponsorship were tracked; missing data were provided by national drug agencies.</p> <p>Results</p> <p>Since 2001, the number of CTA in Italy and Spain increased significantly (5.0 and 2.5% average annual growth). For Italy, the gain was driven by a strong increase of applications from academic trial sponsors; Spain's growth was due to a rise in trials run by commercial sponsors. The Netherlands, Germany, France and the UK saw a decline (1.9, 2.3, 3.0 and 5.3% average annual diminution; significant (<it>P </it>< 0.05) except for Germany) in clinical drug trials. The decrease in the UK was caused by a sharp fall in academic trial activities. Across the six analyzed countries, no EU-wide trial-phase-specific patterns or trends were observed.</p> <p>Conclusions</p> <p>The EU Clinical Trials Directive 2001/20/EC did not achieve the harmonization of clinical trial requirements across Europe. Rather, it resulted in the leveling of clinical trial activities caused by a continuing decrease in CTA rates in the Netherlands, Germany, France and the UK. Southern European countries, Italy and Spain, benefited to some extent from policy changes introduced by the Directive. In Italy's case, national funding measures helped to considerably promote the conduct of non-commercial trials. On the other hand, the EU Directive-driven transition from liberal policy environments, based on non-explicit trial approval through notifications, towards red-taped processes of trial authorization, contributed to the decreases in trial numbers in Germany and the UK. In the latter case, national research governance concerns had a share in the country's marked decline. However, different EU member states successfully developed best practices, which a new European legislation should take into consideration to resume Europe's attractiveness and international competitiveness for the conduct of clinical trials.</p

Outcome of HIV-exposed uninfected children undergoing surgery

<p>Abstract</p> <p>Background</p> <p>HIV-exposed uninfected (HIVe) children are a rapidly growing population that may be at an increased risk of illness compared to HIV-unexposed children (HIVn). The aim of this study was to investigate the morbidity and mortality of HIVe compared to both HIVn and HIV-infected (HIVi) children after a general surgical procedure.</p> <p>Methods</p> <p>A prospective study of children less than 60 months of age undergoing general surgery at a paediatric referral hospital from July 2004 to July 2008 inclusive. Children underwent age-definitive HIV testing and were followed up post operatively for the development of complications, length of stay and mortality.</p> <p>Results</p> <p>Three hundred and eighty children were enrolled; 4 died and 11 were lost to follow up prior to HIV testing, thus 365 children were included. Of these, 38(10.4%) were HIVe, 245(67.1%) were HIVn and 82(22.5%) were HIVi children.</p> <p>The overall mortality was low, with 2(5.2%) deaths in the HIVe group, 0 in the HIVn group and 6(7.3%) in the HIVi group (p = 0.0003). HIVe had a longer stay than HIVn children (3 (2-7) vs. 2 (1-4) days p = 0.02). There was no significant difference in length of stay between the HIVe and HIVi groups. HIVe children had a higher rate of complications compared to HIVn children, (9 (23.7%) vs. 14(5.7%) (RR 3.8(2.1-7) p < 0.0001) but a similar rate of complications compared to HIVi children 34 (41.5%) (RR = 0.6 (0.3-1.1) p = 0.06).</p> <p>Conclusion</p> <p>HIVe children have a higher risk of developing complications and mortality after surgery compared to HIVn children. However, the risk of complications is lower than that of HIVi children.</p

Brevenal Inhibits Pacific Ciguatoxin-1B-Induced Neurosecretion from Bovine Chromaffin Cells

Ciguatoxins and brevetoxins are neurotoxic cyclic polyether compounds produced by dinoflagellates, which are responsible for ciguatera and neurotoxic shellfish poisoning (NSP) respectively. Recently, brevenal, a natural compound was found to specifically inhibit brevetoxin action and to have a beneficial effect in NSP. Considering that brevetoxin and ciguatoxin specifically activate voltage-sensitive Na+ channels through the same binding site, brevenal has therefore a good potential for the treatment of ciguatera. Pacific ciguatoxin-1B (P-CTX-1B) activates voltage-sensitive Na+ channels and promotes an increase in neurotransmitter release believed to underpin the symptoms associated with ciguatera. However, the mechanism through which slow Na+ influx promotes neurosecretion is not fully understood. In the present study, we used chromaffin cells as a model to reconstitute the sequence of events culminating in ciguatoxin-evoked neurosecretion. We show that P-CTX-1B induces a tetrodotoxin-sensitive rise in intracellular Na+, closely followed by an increase in cytosolic Ca2+ responsible for promoting SNARE-dependent catecholamine secretion. Our results reveal that brevenal and β-naphtoyl-brevetoxin prevent P-CTX-1B secretagogue activity without affecting nicotine or barium-induced catecholamine secretion. Brevenal is therefore a potent inhibitor of ciguatoxin-induced neurotoxic effect and a potential treatment for ciguatera

Suppression of HIV-Specific and Allogeneic T Cell Activation by Human Regulatory T Cells Is Dependent on the Strength of Signals

Regulatory T cells (Tregs) suppress immune responses against both self and non-self antigens. Tregs require activation through the T cell receptor (TCR) and IL-2 to exert their suppressive functions. However, how strength of TCR signals modulate the potency of Treg-mediated suppression of antigen-specific T cell activation remain unclear. We found that both strength of TCR signals and ratios of Tregs to target cells, either through superantigen, allogeneic antigens or HIV-specific peptides, modified the suppressive ability of Tregs. While human Tregs were able to mediate suppression in the presence of only autologous antigen-presenting cells, this was much less efficient as compared to when Tregs were activated by allogeneic dendritic cells. In another physiologically relevant system, we show that the strength of peptide stimulation, high frequency of responder CD8+ T cells or presence of high IL-2 can override the suppression of HIV-specific CD8+ T cells by Tregs. These findings suggest that ratios and TCR activation of human Tregs, are important parameters to overcome robust immune responses to pathogens or allogeneic antigens. Modulating the strength of T cell signals and selective enhancement or depletion of antigen-specific Tregs thus may have implications for designing potent vaccines and regulating immune responses during allogeneic transplantation and chronic infections

Human resources: the Cinderella of health sector reform in Latin America

Human resources are the most important assets of any health system, and health workforce problems have for decades limited the efficiency and quality of Latin America health systems. World Bank-led reforms aimed at increasing equity, efficiency, quality of care and user satisfaction did not attempt to resolve the human resources problems that had been identified in multiple health sector assessments. However, the two most important reform policies – decentralization and privatization – have had a negative impact on the conditions of employment and prompted opposition from organized professionals and unions. In several countries of the region, the workforce became the most important obstacle to successful reform. This article is based on fieldwork and a review of the literature. It discusses the reasons that led health workers to oppose reform; the institutional and legal constraints to implementing reform as originally designed; the mismatch between the types of personnel needed for reform and the availability of professionals; the deficiencies of the reform implementation process; and the regulatory weaknesses of the region. The discussion presents workforce strategies that the reforms could have included to achieve the intended goals, and the need to take into account the values and political realities of the countries. The authors suggest that autochthonous solutions are more likely to succeed than solutions imported from the outside

Stability of Yellow Fever Virus under Recombinatory Pressure as Compared with Chikungunya Virus

Recombination is a mechanism whereby positive sense single stranded RNA viruses exchange segments of genetic information. Recent phylogenetic analyses of naturally occurring recombinant flaviviruses have raised concerns regarding the potential for the emergence of virulent recombinants either post-vaccination or following co-infection with two distinct wild-type viruses. To characterize the conditions and sequences that favor RNA arthropod-borne virus recombination we constructed yellow fever virus (YFV) 17D recombinant crosses containing complementary deletions in the envelope protein coding sequence. These constructs were designed to strongly favor recombination, and the detection conditions were optimized to achieve high sensitivity recovery of putative recombinants. Full length recombinant YFV 17D virus was never detected under any of the experimental conditions examined, despite achieving estimated YFV replicon co-infection levels of ∼2.4×106 in BHK-21 (vertebrate) cells and ∼1.05×105 in C710 (arthropod) cells. Additionally YFV 17D superinfection resistance was observed in vertebrate and arthropod cells harboring a primary infection with wild-type YFV Asibi strain. Furthermore recombination potential was also evaluated using similarly designed chikungunya virus (CHIKV) replicons towards validation of this strategy for recombination detection. Non-homologus recombination was observed for CHIKV within the structural gene coding sequence resulting in an in-frame duplication of capsid and E3 gene. Based on these data, it is concluded that even in the unlikely event of a high level acute co-infection of two distinct YFV genomes in an arthropod or vertebrate host, the generation of viable flavivirus recombinants is extremely unlikely

FOXP3 Expression Is Upregulated in CD4+T Cells in Progressive HIV-1 Infection and Is a Marker of Disease Severity

Understanding the role of different classes of T cells during HIV infection is critical to determining which responses correlate with protective immunity. To date, it is unclear whether alterations in regulatory T cell (Treg) function are contributory to progression of HIV infection.FOXP3 expression was measured by both qRT-PCR and by flow cytometry in HIV-infected individuals and uninfected controls together with expression of CD25, GITR and CTLA-4. Cultured peripheral blood mononuclear cells were stimulated with anti-CD3 and cell proliferation was assessed by CFSE dilution.HIV infected individuals had significantly higher frequencies of CD4(+)FOXP3(+) T cells (median of 8.11%; range 1.33%-26.27%) than healthy controls (median 3.72%; range 1.3-7.5%; P = 0.002), despite having lower absolute counts of CD4(+)FOXP3(+) T cells. There was a significant positive correlation between the frequency of CD4(+)FOXP3(+) T cells and viral load (rho = 0.593 P = 0.003) and a significant negative correlation with CD4 count (rho = -0.423 P = 0.044). 48% of our patients had CD4 counts below 200 cells/microl and these patients showed a marked elevation of FOXP3 percentage (median 10% range 4.07%-26.27%). Assessing the mechanism of increased FOXP3 frequency, we found that the high FOXP3 levels noted in HIV infected individuals dropped rapidly in unstimulated culture conditions but could be restimulated by T cell receptor stimulation. This suggests that the high FOXP3 expression in HIV infected patients is likely due to FOXP3 upregulation by individual CD4(+) T cells following antigenic or other stimulation.FOXP3 expression in the CD4(+) T cell population is a marker of severity of HIV infection and a potential prognostic marker of disease progression