Immunology of viral disease, how to curb persistent infection

1. 1. Summaries 1.1. Preamble and extended abstract The present thesis dissertation addresses the question of antiviral immunity from the particular standpoint of the adaptive T cell-mediated immune response. The experimental work is presented in the form of three published articles (two experimental articles and one review article, see sections 4.1, 4.2 and 4.3 on pages 73, 81 and 91, respectively), describing advances both in our understanding of viral control by CD8 T lymphocytes, and in vaccine development against the Human Immunodeficiency Virus Type 1 (HIV-1). Because the articles focus on rather specialized areas of antiviral immunity, the article sections are preceded by a general introduction (section 3) on the immune system in general, and on four viruses that were addressed in the experimental work, namely HIV-1, Cytomegalovirus (CMV), Epstein Barr Virus (EBV) and Influenzavirus (Flu). This introduction section is aimed at providing a glimpse on viral molecular biology and immunity, to help the hypothetical non-expert reader proceeding into the experimental part. For this reason, each section is presented as individual entity and can be consulted separately. The four viruses described are of peculiar relevance to immunity because they induce an array of opposite host responses. Flu causes a self limiting disease after which the virus is eradicated. CMV and EBV cause pauci-symptomatic or asymptomatic diseases after which the viruses establish lifelong latency in the host cells, but are kept in check by immunity. Eventually, HIV-1 establishes both latency - by inserting its genome into the host cell chromosome - and proceeds in destroying the immune system in a poorly controlled fashion. Hence, understanding the fundamental differences between these kinds of viral host interactions might help develop new strategies to curb progressive diseases caused by viruses such as HIV-1. Publication #1: The first article (section 4.1, page 73) represents the main frame of my laboratory work. It analyses the ability of CD8 T lymphocytes recovered from viral-infected patients to secrete interferon γ (IFN-γ) alone or in conjunction with interleukin 2 (IL-2) when exposed in vitro to their cognate viral antigens. CD8 T cells are instrumental in controlling viral infection. They can identify infected cells by detecting viral antigens presented at the surface of the infected cells, and eliminate both the cell and its infecting virus by triggering apoptosis and/or lysis of the infected cell. Recognition of these antigens triggers the cognate CD8 cells to produce cytokines, including IFN-γ and IL-2, which in turn attract and activate other pro-inflammatory cells. IFN-γ triggers both intrinsic antiviral activity of the infected cells and distant activation of pro-inflammatory cells, which are important for the eradication of infection. IL-2 is essential for clonal expansion of the antigen (Ag)-specific CD8 T cell. Hence the existence of Ag-specific CD8 cells secreting both IFN-γand IL-2 should be beneficial for controlling infection. In this first work we determined the percentage of IFN-y/IL-2 double positive and single IFN-γsecreting CD8 T cells against antigens HIV-1, CMV, EBV and Flu in three groups of subjects: (i) HIV-1 infected patients progressing to disease (progressors), (ii) HIV-1-infected subjects not progressing to disease (long-term non progressors or LTNP), and (iii) HIV negative blood donors. The results disclosed a specific IFN-y/IL-2 double positive CD8 response in all subjects able to control infection. In other words, IFN-y/IL-2 double positive CD8 cells were present in virus-specific CD8 T cells against Flu, CMV and EBV as well against HIV-1 in LTNP. In contrast, progressors only had single IFN-γsecreting CD8 T cells. Hence, the ability to develop an IFN-y/IL-2 double positive response might be critical to control infection, independently of the nature of the virus. Additional experiments helped identify the developmental stage of the missing cells (using different markers such as CD45RA and CCR7) and showed a correlation between the absence of IL-2 secreting CD8 T cells and a failure in the proliferation capacity of virus-specific CD8 T cells. Addition of exogenous IL-2 could restore clonal expansion of HIV-1 specific CD8 T cells, at least in vitro. It could further been shown, that IL-2 secreting CD8 T cells are sufficient to support proliferation even in absence of CD4 help. However, the reason for the missing IFN-y/IL-2 double positive CD8 T cell response in HIV-1 progessors has yet to be determined. Publication #2: The second article (section 4.2, page 81) explores new strategies to trigger CD8 T cell immunity against specific HIV-1 proteins believed to be processed and exposed as "infection signal" at the surface of infected cells. Such signals consist of peptide fragments (8- 13 amino acids) originating from viral proteins and presented to CD8 T cells in the frame of particular cell surface molecules of the major histocompatibility complex class I* (MHC I). To mimic "natural" viral infection, the HIV-1 polyprotein Gagpolnef was inserted and expressed in either of two attenuated viruses i.e. vaccinia virus (MVA) or poxvirus (NYVAC). Mice were infected with these recombinant viruses and specific CD8 T cell response to Gagpolnef peptides was sought. Mice could indeed mount a CD8 T cell response against the HIV-1 antigens, indicating that the system worked, at least in this animal model. To further test whether peptides from Gagpolnef could also be presented in the frame of the human MHC class I proteins, a second round of experiments was performed in "humanized" transgenic mice expressing human MHC molecules. The transgenic mice were also able to load Gagpolnef peptides on their human MHC molecule, and these cells could be detected and destroyed by Ag-specific CD8 T cells isolated from HIV-1-infected patients. Therefore, expressing Gagpolnef on attenuated recombinant viruses might represent a valid strategy for anti-HIV-1 immunization in human. Publication #3: This is a review paper (section 4.3, page 91) describing the immune response to CMV and newly developed methods to detect this cellular immune response. Some of it focuses on the detection of T cells by using in vitro manufactured tetramers. These consist of four MHC class I molecules linked together and loaded with the appropriate antigenic peptide. The tetramer can be labeled with a fluorochrome and analyzed with a fluorescence-activated cell sorter. Taken together, the work presented indicates that (i) an appropriate CD8 T cell response consisting of IFN-y/IL-2 double positive effectors, can potentially control viral infection, including HIV-1 infection, (ii) such a response might be triggered by recombinant viral vaccines, and (iii) CD8 T cell response can be monitored by a variety of techniques, including recently-developed MHC class I tetramers. 1. 2. Préambule et résumé élargi Le présent travail de thèse s'intéresse à l'immunité antivirale du point de vue particulier de la réponse adaptative des cellules T. Le travail expérimental est présenté sous la forme de trois articles publiés (2 articles expérimentaux et 1 article de revue, voir sections 4.1, 4.2 et 4.3, pages 58, 66 et 77, respectivement), décrivant des progrès dans la compréhension du contrôle de l'infection virale par les lymphocytes T CD8, ainsi que dans le développement de nouveaux vaccins contre le Virus d'Immunodéficience de Humaine de type 1 (VIH-1). En raison du caractère spécialisé de l'immunité antivirale de type cellulaire, les articles sont précédés par une introduction générale (section 3), dont le but est de pourvoir le lecteur non avisé avec des bases nécessaire à une meilleure appréhension du travail expérimental. Cette introduction présente les grandes lignes du système immunitaire, et décrit de façon générale les 4 virus utilisés dans le travail expérimental: à savoir le virus VIH-1, le Cytomégalovirus (CMV), le virus Epstein Barr (EBV) et le virus Influenza A (Flu). Toutes les sections sont présentées de façon individuelle et peuvent être consultées séparément. La description des 4 virus a une pertinence particulière quant à leur interaction avec le système immun. En effet, ils induisent une panoplie de réponses immunitaires s'étendant aux extrêmes de la réaction de l'hôte. Influenza A est à l'origine d'une maladie cytopathique aiguë, au décours de laquelle le virus est éradiqué par l'hôte. CMV et EBV sont classiquement à l'origine d'infections pauci-symptomatiques, voire asymptomatiques, après lesquelles les virus persistent de façon latente dans la cellule hôte. Cependant, ils restent sous le contrôle du système immun, qui peut prévenir une éventuelle réactivation. Enfin, VIH-1 s'établit à la fois en infection latente - par l'insertion de son génome dans le chromosome des cellules hôtes - et en infection productive et cytopathique, échappant au contrôle immunitaire et détruisant ses cellules cibles. La compréhension des différences fondamentales entre ces différents types d'interactions virus-hôte devraient faciliter le développement de nouvelles stratégies antivirales. Article 1: Le premier article (section 4.1 Page 58) représente l'objet principal de mon travail de laboratoire. Il analyse la capacité des lymphocytes T CD8 spécifiques de différent virus à sécréter de l'interféron gamma (IFN-y) et/ou de l'interleukine 2 (IL-2) après stimulation par leur antigène spécifique. Les cellules T CD8 jouent un rôle crucial dans le contrôle des infections virales. Elles identifient les cellules infectées en détectant des antigènes viraux présentés à la surface de ces mêmes cellules, et éliminent à la fois les cellules infectées et les virus qu'elles contiennent en induisant l'apoptose et/ou la lyse des cellules cibles. Parallèlement, l'identification de l'antigène par la cellule T CD8 la stimule à sécréter des cytokines. L'IFN-γen est un exemple. L'IFN-γ stimule les cellules infectées à développer une activé antivirale intrinsèque. De plus, il attire sur place d'autres cellules de l'inflammation, et active leur fonction d'éradication des pathogènes. L'IL-2 est un autre exemple. L'IL-2 est essentielle à l'expansion clonale des cellules T CD8 spécifiques à un virus donné. Elle est donc essentielle à augmenter le pool de lymphocytes antiviraux. En conséquence, la double capacité de sécréter de l'IFN-γ et de IL-2 pourrait être un avantage pour le contrôle antiviral par les cellules T CD8. Dans ce travail nous avons comparé les proportions de lymphocytes T CD8 doubles positifs (IFN-γ/IL-2) et simples positifs (IFN-γ) chez trois groupes de sujets: (i) des patients infectés par VIH-1 qui ne contrôlent pas l'infection (progresseurs), (ii) des patients infectés par VIH-1, mais contrôlant l'infection malgré l'absence de traitement ("long term non progressors" [LTNP]) et (iii) des donneurs de sang négatifs pour l'infection à VIH-1. Les résultats ont montré que les individus capables de contrôler une infection possédaient des cellules T CD8 doubles positifs (IFN-γ/IL-2), alors que les patients ne contrôlant pas l'infection procédaient prioritairement des CD8 simples positifs (IFN-γ). Spécifiquement, les lymphocytes T spécifiques pour Flu, CMV, EBV, et VII-1-1 chez les LTNP étaient tous IFN-γ/IL-2 doubles positifs. Au contraire, les lymphocytes T CD8 spécifique à VIH-1 étaient IFN-γ simples positifs chez les progresseurs. La capacité de développer une réponse IFN-γ/IL-2 pourraient être primordiale pour le contrôle de l'infection, indépendamment de la nature du virus. En effet, il a été montré que l'absence de sécrétion d'IL2 par les lymphocytes T CD8 corrélait avec leur incapacité de proliférer. Dans nos mains, cette prolifération a pu être restaurée in vitro par l'adjonction exogène d'IL-2. Toutefois, la faisabilité de ce type de complémentation in vivo n'est pas claire. Des expériences additionnelles ont permis de préciser de stade de développement des lymphocytes doubles positifs et simples positifs par le biais des marqueurs CD45RA et CCR7. Il reste maintenant à comprendre pourquoi certains lymphocytes T CD8 spécifiques sont incapables à sécréter de l'IL-2. Article 2: Le deuxième article explore des nouvelles stratégies pour induire une immunité T CD8 spécifique aux protéines du VIH-1, qui sont édités et exposés à la surface des cellules infectées. Ces signaux consistent en fragments de peptide de 8-13 acide aminés provenant de protéines virales, et exposées à la surface des cellules infectées dans le cadre des molécules spécialisées d'histocompatibilité de classe I (en anglais "major histocompatibility class I" ou MHC I). Pour mimer une infection virale, la polyprotéine Gagpolnef du VIH-1 a été insérée et exprimée dans deux vecteurs viraux atténués, soit MVA (provenant de vaccinia virus) ou NYVAC (provenant d'un poxvirus). Ensuite des souris ont été infectées avec ces virus recombinants et la réponse T CD8 aux peptides issus de Gagpolnef a été étudiée. Les souris ont été capables de développer une réponse de type CD8 T contre ces antigènes du VIH-1. Pour tester si ces antigènes pouvaient aussi être présentés par dans le cadre de molécules MHC humaines, des expériences supplémentaires ont été faites avec des souris exprimant un MHC humain. Les résultats de ces manipulations ont montré que des cellules T CD8 spécifique aux protéines du VIH pouvaient être détectées. Ce travail ouvre de nouvelles options quant à l'utilisation des virus recombinants exprimant Gagpolnef comme stratégie vaccinale contre le virus VIH-I chez l'homme. Article 3: Ces revues décrivent la réponse immunitaire à CMV ainsi que des nouvelles méthodes pouvant servir à sa détection. Une partie du manuscrit décrit la détection de cellule T à l'aide de tétramères. Il s'agit de protéines chimériques composées de 4 quatre molécules MHC liées entre elles. Elles sont ensuite "chargées" avec le peptide antigénique approprié, et utilisée pour détecter les cellules T CD8 spécifiques à ce montage. Elles sont aussi marquées par un fluorochrome, qui permet une analyse avec un cytomètre de flux, et l'isolement ultime des CD8 d'intérêt. En résumé, le travail présenté dans cette thèse indique que (i) une réponse T CD8 appropriée - définie par la présence des cellules effectrices doublement positives pour l'IFN-γ et l'IL-2 - semble indispensable pour le contrôle des infections virales, y compris par le VIH-1, (ii) une telle réponse peut être induite par des vaccin viral recombinant, et (iii) la réponse T CD8 peut être analysée et suivie grâce à plusieurs techniques, incluant celle des tétramères de MHC class I. 1.3. Résumé pour un large public Le système immunitaire humain est composé de différents éléments (cellules, tissus et organes) qui participent aux défenses de l'organisme contre les pathogènes (bactéries, virus). Parmi ces cellules, les lymphocytes T CD8, également appelés cellules tueuses, jouent un rôle important dans la réponse immunitaire et le contrôle des infections virales. Les cellules T CD8 reconnaissent de manière spécifique des fragments de protéines virales qui sont exposés à la surface des cellules infectées par le virus. Suite à cette reconnaissance, les cellules T CD8 sont capables de détruire et d'éliminer ces cellules infectées, ainsi que les virus qu'elles contiennent. Dans le contexte d'une infection par le virus de l'immunodéficience humaine (VIH), le virus responsable du SIDA, il a pu être montré que la présence des cellules T CD8 est primordiale. En effet, en l'absence de ces cellules, les individus infectés par le VIH progressent plus rapidement vers le SIDA. Au cours de la vie, l'Homme est exposé à plusieurs virus. Mais à l'opposé du VIH, certains d'entre eux ne causent pas des maladies graves : par exemple le virus de la grippe (Influenza), le cytomégalovirus ou encore le virus d'Epstein-Barr. Certains de ces virus peuvent être contrôlés et éliminés de l'organisme (p. ex. le virus de la grippe), alors que d'autres ne sont que contrôlés par notre système immunitaire et restent présents en petite quantité dans le corps sans avoir d'effet sur notre santé. Le sujet de mon travail de thèse porte sur la compréhension du mécanisme de contrôle des infections virales par le système immunitaire : pourquoi certains virus peuvent être contrôlés ou même éliminés de l'organisme alors que d'autres, et notamment le VIH, ne le sont pas. Ce travail a permis de démontrer que les cellules T CD8 spécifiques du VIH ne sécrètent pas les mêmes substances, nécessaires au développement d'une réponse antivirale efficace, que les cellules T CD8 spécifiques des virus contrôlés (le virus de la grippe, le cytomégalovirus et le virus d'Epstein-Barr). Parallèlement nous avons également observé que les lymphocytes T CD8 spécifiques du VIH ne possèdent pas la capacité de se diviser. Ils sont ainsi incapables d'être présents en quantité suffisante pour assurer un combat efficace contre le virus du SIDA. La (les) différence(s) entre les cellules T CD8 spécifiques aux virus contrôlés (grippe, cytomégalovirus et Epstein-Barr) et au VIH pourront peut-être nous amener à comprendre comment restaurer une immunité efficace contre ce dernier.

Studies on integrating kinematic design method with mechanical systems simulation techniques

Theultimate goal of any research in the mechanism/kinematic/design area may be called predictive design, ie the optimisation of mechanism proportions in the design stage without requiring extensive life and wear testing. This is an ambitious goal and can be realised through development and refinement of numerical (computational) technology in order to facilitate the design analysis and optimisation of complex mechanisms, mechanical components and systems. As a part of the systematic design methodology this thesis concentrates on kinematic synthesis (kinematic design and analysis) methods in the mechanism synthesis process. The main task of kinematic design is to find all possible solutions in the form of structural parameters to accomplish the desired requirements of motion. Main formulations of kinematic design can be broadly divided to exact synthesis and approximate synthesis formulations. The exact synthesis formulation is based in solving n linear or nonlinear equations in n variables and the solutions for the problem areget by adopting closed form classical or modern algebraic solution methods or using numerical solution methods based on the polynomial continuation or homotopy. The approximate synthesis formulations is based on minimising the approximation error by direct optimisation The main drawbacks of exact synthesis formulationare: (ia) limitations of number of design specifications and (iia) failure in handling design constraints- especially inequality constraints. The main drawbacks of approximate synthesis formulations are: (ib) it is difficult to choose a proper initial linkage and (iib) it is hard to find more than one solution. Recentformulations in solving the approximate synthesis problem adopts polynomial continuation providing several solutions, but it can not handle inequality const-raints. Based on the practical design needs the mixed exact-approximate position synthesis with two exact and an unlimited number of approximate positions has also been developed. The solutions space is presented as a ground pivot map but thepole between the exact positions cannot be selected as a ground pivot. In this thesis the exact synthesis problem of planar mechanism is solved by generating all possible solutions for the optimisation process ¿ including solutions in positive dimensional solution sets - within inequality constraints of structural parameters. Through the literature research it is first shown that the algebraic and numerical solution methods ¿ used in the research area of computational kinematics ¿ are capable of solving non-parametric algebraic systems of n equations inn variables and cannot handle the singularities associated with positive-dimensional solution sets. In this thesis the problem of positive-dimensional solutionsets is solved adopting the main principles from mathematical research area of algebraic geometry in solving parametric ( in the mathematical sense that all parameter values are considered ¿ including the degenerate cases ¿ for which the system is solvable ) algebraic systems of n equations and at least n+1 variables.Adopting the developed solution method in solving the dyadic equations in direct polynomial form in two- to three-precision-points it has been algebraically proved and numerically demonstrated that the map of the ground pivots is ambiguousand that the singularities associated with positive-dimensional solution sets can be solved. The positive-dimensional solution sets associated with the poles might contain physically meaningful solutions in the form of optimal defectfree mechanisms. Traditionally the mechanism optimisation of hydraulically driven boommechanisms is done at early state of the design process. This will result in optimal component design rather than optimal system level design. Modern mechanismoptimisation at system level demands integration of kinematic design methods with mechanical system simulation techniques. In this thesis a new kinematic design method for hydraulically driven boom mechanism is developed and integrated in mechanical system simulation techniques. The developed kinematic design method is based on the combinations of two-precision-point formulation and on optimisation ( with mathematical programming techniques or adopting optimisation methods based on probability and statistics ) of substructures using calculated criteria from the system level response of multidegree-of-freedom mechanisms. Eg. by adopting the mixed exact-approximate position synthesis in direct optimisation (using mathematical programming techniques) with two exact positions and an unlimitednumber of approximate positions the drawbacks of (ia)-(iib) has been cancelled.The design principles of the developed method are based on the design-tree -approach of the mechanical systems and the design method ¿ in principle ¿ is capable of capturing the interrelationship between kinematic and dynamic synthesis simultaneously when the developed kinematic design method is integrated with the mechanical system simulation techniques.

Effect of Noncondensable Gases on Circulation of Primary Coolant in Nuclear Power Plants in Abnormal Situations

The present study focuses on two effects of the presence of a noncondensable gas on the thermal-hydraulic behavior of thecoolant of the primary circuit of a nuclear reactor in the VVER-440 geometry inabnormal situations. First, steam condensation with the presence of air was studied in the horizontal tubes of the steam generator (SG) of the PACTEL test facility. The French thermal-hydraulic CATHARE code was used to study the heat transfer between the primary and secondary side in conditions derived from preliminary experiments performed by VTT using PACTEL. In natural circulation and single-phase vapor conditions, the injection of a volume of air, equivalent to the totalvolume of the primary side of the SG at the entrance of the hot collector, did not stop the heat transfer from the primary to the secondary side. The calculated results indicate that air is located in the second half-length (from the mid-length of the tubes to the cold collector) in all the tubes of the steam generator The hot collector remained full of steam during the transient. Secondly, the potential release of the nitrogen gas dissolved in the water of the accumulators of the emergency core coolant system of the Loviisa nuclear power plant (NPP) was investigated. The author implemented a model of the dissolution and release ofnitrogen gas in the CATHARE code; the model created by the CATHARE developers. In collaboration with VTT, an analytical experiment was performed with some components of PACTEL to determine, in particular, the value of the release time constant of the nitrogen gas in the depressurization conditions representative of the small and intermediate break transients postulated for the Loviisa NPP. Such transients, with simplified operating procedures, were calculated using the modified CATHARE code for various values of the release time constant used in the dissolution and release model. For the small breaks, nitrogen gas is trapped in thecollectors of the SGs in rather large proportions. There, the levels oscillate until the actuation of the low-pressure injection pumps (LPIS) that refill the primary circuit. In the case of the intermediate breaks, most of the nitrogen gas is expelled at the break and almost no nitrogen gas is trapped in the SGs. In comparison with the cases calculated without taking into account the release of nitrogen gas, the start of the LPIS is delayed by between 1 and 1.75 h. Applicability of the obtained results to the real safety conditions must take into accountthe real operating procedures used in the nuclear power plant.

Risk factors for head and neck cancer in young adults: a pooled analysis in the INHANCE consortium.

BACKGROUND: Increasing incidence of head and neck cancer (HNC) in young adults has been reported. We aimed to compare the role of major risk factors and family history of cancer in HNC in young adults and older patients. METHODS: We pooled data from 25 case-control studies and conducted separate analyses for adults ≤45 years old ('young adults', 2010 cases and 4042 controls) and >45 years old ('older adults', 17 700 cases and 22 704 controls). Using logistic regression with studies treated as random effects, we estimated adjusted odds ratios (ORs) and 95% confidence intervals (CIs). RESULTS: The young group of cases had a higher proportion of oral tongue cancer (16.0% in women; 11.0% in men) and unspecified oral cavity / oropharynx cancer (16.2%; 11.1%) and a lower proportion of larynx cancer (12.1%; 16.6%) than older adult cases. The proportions of never smokers or never drinkers among female cases were higher than among male cases in both age groups. Positive associations with HNC and duration or pack-years of smoking and drinking were similar across age groups. However, the attributable fractions (AFs) for smoking and drinking were lower in young when compared with older adults (AFs for smoking in young women, older women, young men and older men, respectively, = 19.9% (95% CI = 9.8%, 27.9%), 48.9% (46.6%, 50.8%), 46.2% (38.5%, 52.5%), 64.3% (62.2%, 66.4%); AFs for drinking = 5.3% (-11.2%, 18.0%), 20.0% (14.5%, 25.0%), 21.5% (5.0%, 34.9%) and 50.4% (46.1%, 54.3%). A family history of early-onset cancer was associated with HNC risk in the young [OR = 2.27 (95% CI = 1.26, 4.10)], but not in the older adults [OR = 1.10 (0.91, 1.31)]. The attributable fraction for family history of early-onset cancer was 23.2% (8.60% to 31.4%) in young compared with 2.20% (-2.41%, 5.80%) in older adults. CONCLUSIONS: Differences in HNC aetiology according to age group may exist. The lower AF of cigarette smoking and alcohol drinking in young adults may be due to the reduced length of exposure due to the lower age. Other characteristics, such as those that are inherited, may play a more important role in HNC in young adults compared with older adults.

Impacts of upland open drains upon runoff generation: a numerical assessment of catchment-scale impacts

Shallow upland drains, grips, have been hypothesized as responsible for increased downstream flow magnitudes. Observations provide counterfactual evidence, often relating to the difficulty of inferring conclusions from statistical correlation and paired catchment comparisons, and the complexity of designing field experiments to test grip impacts at the catchment scale. Drainage should provide drier antecedent moisture conditions, providing more storage at the start of an event; however, grips have higher flow velocities than overland flow, thus potentially delivering flow more rapidly to the drainage network. We develop and apply a model for assessing the impacts of grips on flow hydrographs. The model was calibrated on the gripped case, and then the gripped case was compared with the intact case by removing all grips. This comparison showed that even given parameter uncertainty, the intact case had significantly higher flood peaks and lower baseflows, mirroring field observations of the hydrological response of intact peat. The simulations suggest that this is because delivery effects may not translate into catchment-scale impacts for three reasons. First, in our case, the proportions of flow path lengths that were hillslope were not changed significantly by gripping. Second, the structure of the grip network as compared with the structure of the drainage basin mitigated against grip-related increases in the concentration of runoff in the drainage network, although it did marginally reduce the mean timing of that concentration at the catchment outlet. Third, the effect of the latter upon downstream flow magnitudes can only be assessed by reference to the peak timing of other tributary basins, emphasizing that drain effects are both relative and scale dependent. However, given the importance of hillslope flow paths, we show that if upland drainage causes significant changes in surface roughness on hillslopes, then critical and important feedbacks may impact upon the speed of hydrological response. Copyright (c) 2012 John Wiley & Sons, Ltd.

Reporting of methodologic information on trial registries for quality assessment: a study of trial records retrieved from the WHO search portal

Background: Although randomized clinical trials (RCTs) are considered the gold standard of evidence, their reporting is often suboptimal. Trial registries have the potential to contribute important methodologic information for critical appraisal of study results. Methods and Findings: The objective of the study was to evaluate the reporting of key methodologic study characteristics in trial registries. We identified a random sample (n = 265) of actively recruiting RCTs using the World Health Organization International Clinical Trials Registry Platform (ICTRP) search portal in 2008. We assessed the reporting of relevant domains from the Cochrane Collaboration’s ‘Risk of bias’ tool and other key methodological aspects. Our primary outcomes were the proportion of registry records with adequate reporting of random sequence generation, allocation concealment, blinding, and trial outcomes. Two reviewers independently assessed each record. Weighted overall proportions in the ICTRP search portal for adequate reporting of sequence generation, allocation concealment, blinding (including and excluding open label RCT) and primary outcomes were 5.7% (95% CI 3.0–8.4%), 1.4% (0–2.8%), 41% (35–47%), 8.4% (4.1–13%), and 66% (60–72%), respectively. The proportion of adequately reported RCTs was higher for registries that used specific methodological fields for describing methods of randomization and allocation concealment compared to registries that did not. Concerning other key methodological aspects, weighted overall proportions of RCTs with adequately reported items were as follows: eligibility criteria (81%), secondary outcomes (46%), harm (5%) follow-up duration (62%), description of the interventions (53%) and sample size calculation (1%). Conclusions: Trial registries currently contain limited methodologic information about registered RCTs. In order to permit adequate critical appraisal of trial results reported in journals and registries, trial registries should consider requesting details on key RCT methods to complement journal publications. Full protocols remain the most comprehensive source of methodologic information and should be made publicly available.

Identifying the index lesion with template prostate mapping biopsies.

PURPOSE: The natural history of prostate cancer might be driven by the index lesion. We determined the percent of men in whom the index lesion could be defined using transperineal template prostate mapping biopsies. MATERIALS AND METHODS: Included in study were consecutive men undergoing transperineal template prostate mapping biopsies with biopsies grouped into 20 zones. Men with clinically significant disease in only 1 prostate area were considered to have an identifiable index lesion. We evaluated the impact of using 2 definitions of clinically significant disease (Gleason grade pattern 4 and/or lesion volume 0.5 cc or greater) and 2 clustering rules (stringent and tolerant) to define the index lesion. RESULTS: Included in study were 391 men with a median age of 62 years (IQR 58-67) and a median prostate specific antigen of 6.9 ng/ml (IQR 4.8-10.0). Of the men 269 (69%) were previously diagnosed with prostate cancer. By deploying a median of 1.2 cores per ml (IQR 0.9-1.7) cancer was diagnosed in 82.9% of the men (324 of 391) with a median of 6 positive cores (IQR 2-9), a median maximum cancer core length of 5 mm (IQR 3-8) and a total cancer core length per zone of 7 mm (IQR 3-13). Insignificant disease was found in 26.3% to 42.9% of cases. When a stringent spatial relationship was used to define individual lesions, 44.4% to 54.6% of patients had 1 index lesion and 12.7% to 19.1% had more than 1 area with clinically significant disease. These proportions changed to 46.6% to 59.2% and 10.5% to 14.5%, respectively, when less stringent spatial clustering was applied. CONCLUSIONS: Transperineal template prostate mapping biopsies enable the index lesion to be localized in most men with clinically significant disease. This information may be important to select appropriate candidates for targeted therapy and to plan a tailored treatment strategy in men undergoing radical therapy.

On regeneration and collateral sprouting to hind limb digits after sciatic nerve injury in the rat

This study examines the proportions of regenerative and collateral sprouting to the skin after peripheral nerve injury. Methods: In the first experimental paradigm, primary afferent neurones were pre-labelled with Diamidino Yellow (DY), injected in digit 3, followed by sciatic nerve section and repair. After three months of regeneration, digit 3 was re-injected with Fast Blue (FB) to label regernating cells. Fluoro-Gold (FG) was applied to the femoral (FEM) and musculocutaneous (MC) nervers four days later to quantify their contribution to the innveration. In the second experimental paradigm, sciatic nerve was first sectioned and repaired. Three months later, the sciatic was resected, and digit 3 injected with FB. After four more days, FEM and MC were resected and FG injected in all digits. Results: Neurones in dorsal root ganglion (DRG) L5 had a higher rate of correct reinnervation of digit 3 (44-72%) than neurones in DRG L4 (14-44%). Like in control cases, only occasional axons were traced from the FEM and MC. In the second experiment, only occasional labelled neurones appeared. Conclusions: The results indicate differences in the capacity for correct peripheral sensory reinnvervation between segmental levels and that in this model collateral sprouting was practically non-existent compared to regenerative sprouting.

Diagnosis of the nutrient compositional space of fruit crops

Tissue analysis is a useful tool for the nutrient management of fruit orchards. The mineral composition of diagnostic tissues expressed as nutrient concentration on a dry weight basis has long been used to assess the status of 'pure' nutrients. When nutrients are mixed and interact in plant tissues, their proportions or concentrations change relatively to each other as a result of synergism, antagonism, or neutrality, hence producing resonance within the closed space of tissue composition. Ternary diagrams and nutrient ratios are early representations of interacting nutrients in the compositional space. Dual and multiple interactions were integrated by the Diagnosis and Recommendation Integrated System (DRIS) into nutrient indexes and by Compositional Nutrient Diagnosis into centered log ratios (CND-clr). DRIS has some computational flaws such as using a dry matter index that is not a part as well as nutrient products (e.g. NxCa) instead of ratios. DRIS and CND-clr integrate all possible nutrient interactions without defining an ad hoc interactive model. They diagnose D components while D-1 could be diagnosed in the D-compositional Hilbert space. The isometric log ratio (ilr) coordinates overcome these problems using orthonormal binary nutrient partitions instead of dual ratios. In this study, it is presented a nutrient interactive model as well as computation methods for DRIS and CND-clr and CND-ilr coordinates (CND-ilr) using leaf analytical data from an experimental apple orchard in Southwestern Quebec, Canada. It was computed the Aitchison and Mahalanobis distances across ilr coordinates as measures of nutrient imbalance. The effect of changing nutrient concentrations on ilr coordinates are simulated to identify the ones contributing the most to nutrient imbalance.

Excess winter deaths caused by cardiovascular diseases are associated with both mild winter temperature and socio-economic inequalities in the U.S.

Mortality from cardiovascular diseases (CVD) exhibits seasonal variation. For example, 30% more deaths occurred in winter compared to summer in a multicountry study [1]. The effect of cold temperature on several CVD risk factors and on seasonal influenza infection may partially underlie this seasonal variation [2] and [3]. However an unexplained paradox has been observed: seasonality in CVD mortality is larger in temperate mid-latitude countries (e.g. Portugal) than in colder northern countries (e.g. Scandinavian countries) [1]. This paradox has also been previously observed in Europe for overall mortality, and it may relate to uneven proportions between countries of people who are unable to adequately protect themselves against cold due to low socio-economic status (SES), e.g. inadequate clothing, housing insulation and heating systems [4] and [5]. We hypothesized that the seasonal variability in CVD mortality is larger in low socio-economic U.S. states experiencing mild winters compared to high socio-economic states experiencing cold winters.

Occurrence of inorganic arsenic in edible Shiitake (Lentinula edodes) products

The present study reports arsenic speciation analysis in edible Shiitake (Lentinula edodes) products. The study focused on the extraction, and accurate quantification of inorganic arsenic (iAs), the most toxic form of arsenic, which was selectively separated and determined using anion exchange LC-ICPMS. A wide variety of edible Shiitake products (fresh mushrooms, food supplements, canned and dehydrated) were purchased and analysed. A cultivated Shiitake grown under controlled conditions was also analysed. The extraction method showed satisfactory extraction efficiencies (>90%) and column recoveries (>85%) for all samples. Arsenic speciation revealed that iAs was the major As compound up to 1.38 mg As per kg dm (with a mean percentage of 84% of the total arsenic) and other organoarsenicals were found as minor species. Shiitake products had high proportions of iAs and therefore should not be ignored as potential contributors to dietary iAs exposure in populations with a high intake of Shiitake products.

Guavira emergence and seedling production with substrates containing organic compost and soil under different screen environments

This study aimed to evaluate different proportions of organic compost and soil as a substrate for the guavira emergence and seedling formation under different protected environments, in the high Pantanal region of the state of Mato Grosso do Sul. The seeds were placed in polyethylene bags (15 x 25 centimeters) filled with four percentages of organic compost (0%, 20%, 80%, and 100% of total volume) mixed with soil. These substrates were tested in agro-nurseries covered with black screen and 50% thermo-reflecting shade cloths. The substrate with 20% soil and 80% organic compost and the black screen shade cloth promote the best performance in the seedling production.

Extending body space in immersive virtual reality: a very long arm illusion

Recent studies have shown that a fake body part can be incorporated into human body representation through synchronous multisensory stimulation on the fake and corresponding real body part- the most famous example being the Rubber Hand Illusion. However, the extent to which gross asymmetries in the fake body can be assimilated remains unknown. Participants experienced, through a head-tracked stereo head-mounted display a virtual body coincident with their real body. There were 5 conditions in a between-groups experiment, with 10 participants per condition. In all conditions there was visuo-motor congruence between the real and virtual dominant arm. In an Incongruent condition (I), where the virtual arm length was equal to the real length, there was visuo-tactile incongruence. In four Congruent conditions there was visuo-tactile congruence, but the virtual arm lengths were either equal to (C1), double (C2), triple (C3) or quadruple (C4) the real ones. Questionnaire scores and defensive withdrawal movements in response to a threat showed that the overall level of ownership was high in both C1 and I, and there was no significant difference between these conditions. Additionally, participants experienced ownership over the virtual arm up to three times the length of the real one, and less strongly at four times the length. The illusion did decline, however, with the length of the virtual arm. In the C2-C4 conditions although a measure of proprioceptive drift positively correlated with virtual arm length, there was no correlation between the drift and ownership of the virtual arm, suggesting different underlying mechanisms between ownership and drift. Overall, these findings extend and enrich previous results that multisensory and sensorimotor information can reconstruct our perception of the body shape, size and symmetry even when this is not consistent with normal body proportions.

Biologisen jätevedenpuhdistuksen ilmastuksen tehostaminen

Työssä tutkittiin, mikä on kustannustehokkain tapa lisätä ilmastuskapasiteettia ja selvitettiin, saostaako vesilaitokselta flotaatioaltaasta poistettava ferrisulfaattisakka jäteveden fosforia esiselkeyttimessä. Työssä määritettiin teoreettinen hapentarve ilmastusaltaassa maksimikuormituksen aikana ja hapensiirtonopeus jäteveteen olemassa olevalla laitteistolla. Hapensiirtonopeuden funktion korjauskertoimet  ja  sekä kokonaishapensiirtokerroin määritettiin laboratorio-olosuhteissa. Lisäksi laskettiin kuinka paljon nykyisten ilmastimien kautta saadaan happea jäteveteen liukenemaan ja verrattiin tätä lukua teoreettiseen hapentarpeeseen. Jälkimmäistä menettelytapaa käyttäen arvioitiin lisähapen tarve. Tulokseksi saatiin, että maksimi BOD-kuormituksen aikana happea tarvitsee saada jäteveteen liuotettua 5200 m³/d lisää. Ilmastuskapasiteetin lisäyksen kustannuslaskelmassa verrattiin neljää vaihtoehtoa: puhdasta happea liuotettuna happimattojen kautta tai OKI-ilmastimen kautta, onsite-happilaitoksen puhdasta happea liuotettuna OKI-ilmastimen kautta tai ilman happea liuotettuna jäteveteen EDI-ilmastimien kautta. Laskelmat osoittivat uusien EDI-ilmastimien hankinnan olevan edullisin vaihtoehto pitkällä aikavälillä. Vaikka EDI-ilmastimien investointikustannukset ovat korkeat, tulevat ne käyttökustannuksiltaan kaikkein edullisimmaksi. Vesilaitokselta tulevan rautasuolasakan fosforia saostavaa vaikutusta tutkittiin laboratoriossa astiakokein. Näytteet otettiin vesilaitokselta jätevesilaitokselle menevästä rautasuolasakasta sekä esiselkeyttimen kirkasteesta. Näitä eri suhteissa keskenään sekoittamalla saatiin selville, että rautasuolasakka ja sakan mukana tuleva reagoimaton rautasuola saostavat liukoista ja kokonaisfosforia esiselkeytyksessä.

Chemical, physico-chemical and sensory characterization of mixed açai (Euterpe oleracea) and cocoa´s honey (Theobroma cacao) jellies

Four formulations of mixed açaí (Euterpe oleracea) (A) and cocoa´s honey (Theobroma cacao) (CH) jellies were prepared according to the following proportions: T1 (40% A:60% CH), T2 (50% A:50% CH), T3 (60% A: 40% CH) and T4 (100% A - control). All formulations were prepared using a rate 60:40 (w/w) of sucrose and pulp, plus 0.5% pectin and the products reached to average of 65% soluble solids content. The jellies were analyzed by chemical and physicochemical (titratable acidity, pH, soluble solid content, dry matter, total protein, lipids, vitamin C and calories) and sensory characteristics; also were evaluated levels of P, K, Ca, Mg, Fe, Zn, Cu and Mn. It was used a hedonic scale of 7 points to evaluate the attributes: overall impression, spreadability, brightness, flavor, texture and color, and also was verified the purchase intention score. The titratable acidity and pH ranged from 0.46 to 0.64% and 3.35 to 3.64, respectively, that are within the range found at most fruit jellies. The soluble solids content ranged between 65.2 and 65.5 ºBrix. The sensory acceptance results showed that all treatments (T1, T2, T3 and T4) presented means of sensory attributes above 4, demonstrating good acceptance of the product, but the treatment T1 presented the higher scores for the evaluated attributes. Cocoa´s honey added a positive influence on the attributes of color, texture and spreadability.