Tissue microarray and immunohistochemistry as tools for evaluation of antibodies against Chlamydia-like bacteria.

Tissue microarray technology was used to establish immunohistochemistry protocols and to determine the specificity of new antisera against various Chlamydia-like bacteria for future use on formalin-fixed and paraffin-embedded tissues. The antisera exhibited strong reactivity against autologous antigen and closely related heterologous antigen, but no cross-reactivity with distantly related species.

Allergie aux venins d'hyménopteres: nouveautés diagnostiques et prise en charge [Hymenoptera venom allergy: new diagnostic tools and management].

Anaphylactic reactions to hymenoptera venoms are common and, in our latitudes, mainly concern wasps and bees. Recently, molecular biology techniques have contributed to identifying and to sequencing the major allergens of insect venoms and led to the production of recombinant allergens. Assays for specific IgE directed against these recombinant allergens have recently been made available in clinical practice. They provide considerable assistance in identifying the insect responsible for an anaphylactic reaction, in particular when standard tests are positive for both wasp and bee. This article focuses on these new laboratory tests and also reviews the management of patients experiencing an anaphylactic reaction after hymenoptera sting.

First insights into the transcriptome and development of new genomic tools of a widespread circum-Mediterranean tree species, Pinus halepensis Mill.

Aleppo pine (Pinus halepensis Mill.) is a relevant conifer species for studying adaptive responses to drought and fire regimes in the Mediterranean region. In this study, we performed Illumina next-generation sequencing of two phenotypically divergent Aleppo pine accessions with the aims of (i) characterizing the transcriptome through Illumina RNA-Seq on trees phenotypically divergent for adaptive traits linked to fire adaptation and drought, (ii) performing a functional annotation of the assembled transcriptome, (iii) identifying genes with accelerated evolutionary rates, (iv) studying the expression levels of the annotated genes and (v) developing gene-based markers for population genomic and association genetic studies. The assembled transcriptome consisted of 48,629 contigs and covered about 54.6 Mbp. The comparison of Aleppo pine transcripts to Picea sitchensis protein-coding sequences resulted in the detection of 34,014 SNPs across species, with a Ka /Ks average value of 0.216, suggesting that the majority of the assembled genes are under negative selection. Several genes were differentially expressed across the two pine accessions with contrasted phenotypes, including a glutathione-s-transferase, a cellulose synthase and a cobra-like protein. A large number of new markers (3334 amplifiable SSRs and 28,236 SNPs) have been identified which should facilitate future population genomics and association genetics in this species. A 384-SNP Oligo Pool Assay for genotyping with the Illumina VeraCode technology has been designed which showed an high overall SNP conversion rate (76.6%). Our results showed that Illumina next-generation sequencing is a valuable technology to obtain an extensive overview on whole transcriptomes of nonmodel species with large genomes.

Segmentation and grid generation for numerical simulations of vad connections with patient-specific data

Objectives: We are interested in the numerical simulation of the anastomotic region comprised between outflow canula of LVAD and the aorta. Segmenta¬tion, geometry reconstruction and grid generation from patient-specific data remain an issue because of the variable quality of DICOM images, in particular CT-scan (e.g. metallic noise of the device, non-aortic contrast phase). We pro¬pose a general framework to overcome this problem and create suitable grids for numerical simulations.Methods: Preliminary treatment of images is performed by reducing the level window and enhancing the contrast of the greyscale image using contrast-limited adaptive histogram equalization. A gradient anisotropic diffusion filter is applied to reduce the noise. Then, watershed segmentation algorithms and mathematical morphology filters allow reconstructing the patient geometry. This is done using the InsightToolKit library (www.itk.org). Finally the Vascular Model¬ing ToolKit (www.vmtk.org) and gmsh (www.geuz.org/gmsh) are used to create the meshes for the fluid (blood) and structure (arterial wall, outflow canula) and to a priori identify the boundary layers. The method is tested on five different patients with left ventricular assistance and who underwent a CT-scan exam.Results: This method produced good results in four patients. The anastomosis area is recovered and the generated grids are suitable for numerical simulations. In one patient the method failed to produce a good segmentation because of the small dimension of the aortic arch with respect to the image resolution.Conclusions: The described framework allows the use of data that could not be otherwise segmented by standard automatic segmentation tools. In particular the computational grids that have been generated are suitable for simulations that take into account fluid-structure interactions. Finally the presented method features a good reproducibility and fast application.

MAR elements as tools to increase protein production by CHO cells

One of the major hurdles of isolating stable, inducible or constitutive high-level producer cell lines is the time-consuming selection, analysis and characterization of the numerous clones required to identify one with the desired characteristics. Various boundary elements, matrix attachment regions, and locus control regions were screened for for their ability to augment the expression of heterologous genes in CHO and other cells. The 5'-matrix-attachment region (MAR) of the chicken lysozyme gene was found to significantly increase stable gene expression, in culture dishes and in bioreactors. These MAR elements can be easily combined with various existing expression systems, as they can be added in trans (i.e. on a separate plasmid) in co-transfections with previously constructed expression vectors. Using cell population analysis, we found that the use of the MAR increases the proportion of high-producing CHO cell clones, thus reducing the number of cell lines that need to be screened while increasing maximal productivity. Random cDNA cloning and sequencing indicated that over 12% of the ESTs correspond to the transgene. Thus, productivity is no longer limited by transcriptional events in such MAR-containing cell lines. The identification of small and more convenient active MAR portions will also be summarized. Finally, we will show examples of how MAR elements can be combined with short term expression to increase the simultaneous synthesis of many proteins in parallel by CHO cells. Overall, we conclude that the MAR sequence is a versatile tool to increase protein expression in short and long term production processes.

The Effect Of Induced Intraocular Straylight (cataract Simulation) On Sensitivity Measures Of Standard Automated Perimetry, Pulsar Perimetry And Moorfields Motion Displacement Test

Purpose: To investigate the effect of incremental increases in intraocular straylight on threshold measurements made by three modern forms of perimetry: Standard Automated Perimetry (SAP) using Octopus (Dynamic, G-Pattern), Pulsar Perimetry (PP) (TOP, 66 points) and the Moorfields Motion Displacement Test (MDT) (WEBS, 32 points).Methods: Four healthy young observers were recruited (mean age 26yrs [25yrs, 28yrs]), refractive correction [+2 D, -4.25D]). Five white opacity filters (WOF), each scattering light by different amounts were used to create incremental increases in intraocular straylight (IS). Resultant IS values were measured with each WOF and at baseline (no WOF) for each subject using a C-Quant Straylight Meter (Oculus, Wetzlar, Germany). A 25 yr old has an IS value of ~0.85 log(s). An increase of 40% in IS to 1.2log(s) corresponds to the physiological value of a 70yr old. Each WOFs created an increase in IS between 10-150% from baseline, ranging from effects similar to normal aging to those found with considerable cataract. Each subject underwent 6 test sessions over a 2-week period; each session consisted of the 3 perimetric tests using one of the five WOFs and baseline (both instrument and filter were randomised).Results: The reduction in sensitivity from baseline was calculated. A two-way ANOVA on mean change in threshold (where subjects were treated as rows in the block and each increment in fog filters was treated as column) was used to examine the effect of incremental increases in straylight. Both SAP (p<0.001) and Pulsar (p<0.001) were significantly affected by increases in straylight. The MDT (p=0.35) remained comparatively robust to increases in straylight.Conclusions: The Moorfields MDT measurement of threshold is robust to effects of additional straylight as compared to SAP and PP.

Tools in pharmacovigilance in psychiatry: therapeutic drug monitoring, pharmacogenetic tests and drug-drug interactions databases

SUMMARYIn order to increase drug safety we must better understand how medication interacts with the body of our patients and this knowledge should be made easily available for the clinicians prescribing the medication. This thesis contributes to how the knowledge of some drug properties can increase and how to make information readily accessible for the medical professionals. Furthermore it investigates the use of Therapeutic drug monitoring, drug interaction databases and pharmacogenetic tests in pharmacovigilance.Two pharmacogenetic studies in the naturalistic setting of psychiatric in-patients clinics have been performed; one with the antidepressant mirtazapine, the other with the antipsychotic clozapine. Forty-five depressed patients have been treated with mirtazapine and were followed for 8 weeks. The therapeutic effect was as seen in other previous studies. Enantioselective analyses could confirm an influence of age, gender and smoking in the pharmacokinetics of mirtazapine; it showed a significant influence of the CYP2D6 genotype on the antidepressant effective S-enantiomer, and for the first time an influence of the CYP2B6 genotype on the plasma concentrations of the 8-OH metabolite was found. The CYP2B6*/*6 genotype was associated to better treatment response. A detailed hypothesis of the metabolic pathways of mirtazapine is proposed. In the second pharmacogenetic study, analyses of 75 schizophrenic patients treated with clozapine showed the influence of CYP450 and ABCB1 genotypes on its pharmacokinetics. For the first time we could demonstrate an in vivo effect of the CYP2C19 genotype and an influence of P-glycoprotein on the plasma concentrations of clozapine. Further we confirmed in vivo the prominent role of CYP1A2 in the metabolism of clozapine.Identifying risk factors for the occurrence of serious adverse drug reactions (SADR) would allow a more individualized and safer drug therapy. SADR are rare events and therefore difficult to study. We tested the feasibility of a nested matched case-control study to examine the influence of high drug plasma levels and CYP2D6 genotypes on the risk to experience an SADR. In our sample we compared 62 SADR cases with 82 controls; both groups were psychiatric patients from the in-patient clinic Königsfelden. Drug plasma levels of >120% of the upper recommended references could be identified as a risk factor with a statistically significant odds ratio of 3.5, a similar trend could be seen for CYP2D6 poor metaboliser. Although a matched case-control design seems a valid method, 100% matching is not easy to perform in a relative small cohort of one in-patient clinic. However, a nested case-control study is feasible.On the base of the experience gained in the AMSP+ study and the fact that we have today only sparse data indicating that routine drug plasma concentration monitoring and/or pharmacogenetic testing in psychiatry are justified to minimize the risk for ADR, we developed a test algorithm named "TDM plus" (TDM plus interaction checks plus pharmacogenetic testing).Pharmacovigilance programs such as the AMSP project (AMSP = Arzneimittelsicherheit in der Psychiatrie) survey psychiatric in-patients in order to collect SADR and to detect new safety signals. Case reports of such SADR are, although anecdotal, valuable to illustrate rare clinical events and sometimes confirm theoretical assumptions of e.g. drug interactions. Seven pharmacovigilance case reports are summarized in this thesis.To provide clinicians with meaningful information on the risk of drug combinations, during the course of this thesis the internet based drug interaction program mediQ.ch (in German) has been developed. Risk estimation is based on published clinical and pharmacological information of single drugs and alimentary products, including adverse drug reaction profiles. Information on risk factors such as renal and hepatic insufficiency and specific genotypes are given. More than 20'000 drug pairs have been described in detail. Over 2000 substances with their metabolic and transport pathways are included and all information is referenced with links to the published scientific literature or other information sources. Medical professionals of more than 100 hospitals and 300 individual practitioners do consult mediQ.ch regularly. Validations with comparisons to other drug interaction programs show good results.Finally, therapeutic drug monitoring, drug interaction programs and pharmacogenetic tests are helpful tools in pharmacovigilance and should, in absence of sufficient routine tests supporting data, be used as proposed in our TDM plus algorithm.RESUMEPour améliorer la sécurité d'emploi des médicaments il est important de mieux comprendre leurs interactions dans le corps des patients. Ensuite le clinicien qui prescrit une pharmacothérapie doit avoir un accès simple à ces informations. Entre autres, cette thèse contribue à mieux connaître les caractéristiques pharmacocinétiques de deux médicaments. Elle examine aussi l'utilisation de trois outils en pharmacovigilance : le monitorage thérapeutique des taux plasmatiques des médicaments (« therapeutic drug monitoring »), un programme informatisé d'estimation du risque de combinaisons médicamenteuses, et enfin des tests pharmacogénétiques.Deux études cliniques pharmacogénétiques ont été conduites dans le cadre habituel de clinique psychiatrique : l'une avec la mirtazapine (antidépresseur), l'autre avec la clozapine (antipsychotique). On a traité 45 patients dépressifs avec de la mirtazapine pendant 8 semaines. L'effet thérapeutique était semblable à celui des études précédentes. Nous avons confirmé l'influence de l'âge et du sexe sur la pharmacocinétique de la mirtazapine et la différence dans les concentrations plasmatiques entre fumeurs et non-fumeurs. Au moyen d'analyses énantiomères sélectives, nous avons pu montrer une influence significative du génotype CYP2D6 sur l'énantiomère S+, principalement responsable de l'effet antidépresseur. Pour la première fois, nous avons trouvé une influence du génotype CYP2B6 sur les taux plasmatiques de la 8-OH-mirtazapine. Par ailleurs, le génotype CYP2B6*6/*6 était associé à une meilleure réponse thérapeutique. Une hypothèse sur les voies métaboliques détaillées de la mirtazapine est proposée. Dans la deuxième étude, 75 patients schizophrènes traités avec de la clozapine ont été examinés pour étudier l'influence des génotypes des iso-enzymes CYP450 et de la protéine de transport ABCB1 sur la pharmacocinétique de cet antipsychotique. Pour la première fois, on a montré in vivo un effet des génotypes CYP2C19 et ABCB1 sur les taux plasmatiques de la clozapine. L'importance du CYP1A2 dans le métabolisme de la clozapine a été confirmée.L'identification de facteurs de risques dans la survenue d'effets secondaire graves permettrait une thérapie plus individualisée et plus sûre. Les effets secondaires graves sont rares. Dans une étude de faisabilité (« nested matched case-control design » = étude avec appariement) nous avons comparé des patients avec effets secondaires graves à des patients-contrôles prenant le même type de médicaments mais sans effets secondaires graves. Des taux plasmatiques supérieurs à 120% de la valeur de référence haute sont associés à un risque avec « odds ratio » significatif de 3.5. Une tendance similaire est apparue pour le génotype du CYP2D6. Le « nested matched case-control design » semble une méthode valide qui présente cependant une difficulté : trouver des patients-contrôles dans le cadre d'une seule clinique psychiatrique. Par contre la conduite d'une « nested case-control study » sans appariement est recommandable.Sur la base de notre expérience de l'étude AMSP+ et le fait que nous disposons que de peux de données justifiant des monitorings de taux plasmatiques et/ou de tests pharmacogénétiques de routine, nous avons développé un test algorithme nommé « TDMplus » (TDM + vérification d'interactions médicamenteuses + tests pharmacogénétique).Des programmes de pharmacovigilances comme celui de l'AMSP (Arzneimittelsicherheit in der Psychiatrie = pharmacovigilance en psychiatrie) collectent les effets secondaires graves chez les patients psychiatriques hospitalisés pour identifier des signaux d'alertes. La publication de certains de ces cas même anecdotiques est précieuse. Elle décrit des événements rares et quelques fois une hypothèse sur le potentiel d'une interaction médicamenteuse peut ainsi être confirmée. Sept publications de cas sont résumées ici.Dans le cadre de cette thèse, on a développé un programme informatisé sur internet (en allemand) - mediQ.ch - pour estimer le potentiel de risques d'une interaction médicamenteuse afin d'offrir en ligne ces informations utiles aux cliniciens. Les estimations de risques sont fondées sur des informations cliniques (y compris les profils d'effets secondaires) et pharmacologiques pour chaque médicament ou substance combinés. Le programme donne aussi des informations sur les facteurs de risques comme l'insuffisance rénale et hépatique et certains génotypes. Actuellement il décrit en détail les interactions potentielles de plus de 20'000 paires de médicaments, et celles de 2000 substances actives avec leurs voies de métabolisation et de transport. Chaque information mentionne sa source d'origine; un lien hypertexte permet d'y accéder. Le programme mediQ.ch est régulièrement consulté par les cliniciens de 100 hôpitaux et par 300 praticiens indépendants. Les premières validations et comparaisons avec d'autres programmes sur les interactions médicamenteuses montrent de bons résultats.En conclusion : le monitorage thérapeutique des médicaments, les programmes informatisés contenant l'information sur le potentiel d'interaction médicamenteuse et les tests pharmacogénétiques sont de précieux outils en pharmacovigilance. Nous proposons de les utiliser en respectant l'algorithme « TDM plus » que nous avons développé.

Decision-support tools to manage drug incompatibilities: evaluation by nurses

Introduction Preventing drug incompatibilities has a high impact onthe safety of drug therapy. Although there are no internationalguidelines to manage drug incompatibilities, different decision-supporttools such as handbooks, cross-tables and databases are available.In a previous study, two decision-support tools have been pre-selectedby pharmacists as fitting nurses' needs on the wards1. The objective ofthis study was to have these both tools evaluated by nurses todetermine which would be the most suitable for their daily practice.Materials & Methods Evaluated tools were:1. Cross-table of drug pairs (http://files.chuv.ch/internet-docs/pha/medicaments/pha_phatab_compatibilitessip.pdf)2. Colour-table (a colour for each drug according to the pH: red =acid; blue = basic; yellow = neutral; black = to be infused alone)2Tools were assessed by 48 nurses in 5 units (PICU, adult andgeriatric intensive care, surgery, onco-hematology) using a standardizedform1. The scientific accuracy of the tools was evaluated bydetermining the compatibility of five drugs pairs (rate of correctanswers according to the Trissel's Handbook on Injectable Drugs,chi-square test). Their ergonomics, design, reliability and applicabilitywere estimated using visual analogue scales (VAS 0-10; 0 =null, 10 = excellent). Results are expressed as the median and interquartilerange (IQR) for 25% and 75% (Wilcoxon rank sum test).Results The rate of correct answers was above 90% for both tools(cross-table 96.2% vs colour-table 92.5%, p[0.05).The ergonomics and the applicability were higher for the crosstable[7.1 (IQR25 4.0, IQR75 8.0) vs 5.0 (IQR25 2.7, IQR75 7.0), p =0.025 resp. 8.3 (IQR25 7.4, IQR75 9.2) vs 7.6 (IQR25 5.9, IQR75 8.8)p = 0.047].The design of the colour-table was judged better [4.6 (IQR25 2.9,IQR75 7.1) vs 7.1 (IQR25 5.4, IQR75 8.4) p = 0.002].No difference was observed in terms of reliability [7.3 (IQR25 6.5,IQR75 8.4) vs 6.7 (IQR25 5.0, IQR758.6) p[0.05].The cross-table was globally preferred by 65% of the nurses (27%colour-table, 8% undetermined) and 68% would like to have thisdecision-support tool available for their daily practice.Discussion & Conclusion Both tools showed the same accuracy toassess drug compatibility. In terms of ergonomics and applicabilitythe cross-table was better than the colour-table, and was preferred bythe nurses for their daily practice. The cross-table will be implementedin our hospital as decision-support tool to help nurses tomanage drug incompatibilities.

Disaster Risk, Livelihoods and Natural Barriers, Strengthening Decision-Making Tools for Disaster Risk Reduction: A case study from northern Pakistan

The goal of this interdisciplinary study is to better understand the land use factors that increase vulnerability of mountain areas in northern Pakistan. The study will identify and analyse the damages and losses caused by the October 2005 earthquake in two areas of the same valley: one "low-risk" watershed with sound natural resources management, the other, "high-risk" in an ecologically degraded watershed. Secondly, the study will examine natural and man-made causes of secondary hazards in the study area, especially landslides; and third it will evaluate the cost of the earthquake damage in the study areas on the livelihoods of local communities and the sub-regional economy. There are few interdisciplinary studies to have correlated community land use practices, resources management, and disaster risk reduction in high-risk mountain areas. By better understanding these linkages, development- humanitarian- and donor agencies focused on disaster reduction can improve their risk reduction programs for mountainous regions.

Orthotropic active strain models for the numerical simulation of cardiac biomechanics

An active strain formulation for orthotropic constitutive laws arising in cardiac mechanics modeling is introduced and studied. The passive mechanical properties of the tissue are described by the Holzapfel-Ogden relation. In the active strain formulation, the Euler-Lagrange equations for minimizing the total energy are written in terms of active and passive deformation factors, where the active part is assumed to depend, at the cell level, on the electrodynamics and on the specific orientation of the cardiac cells. The well-posedness of the linear system derived from a generic Newton iteration of the original problem is analyzed and different mechanical activation functions are considered. In addition, the active strain formulation is compared with the classical active stress formulation from both numerical and modeling perspectives. Taylor-Hood and MINI finite elements are employed to discretize the mechanical problem. The results of several numerical experiments show that the proposed formulation is mathematically consistent and is able to represent the main key features of the phenomenon, while allowing savings in computational costs.

The differentiation of fibre- and drug type Cannabis seedlings by gas chromatography/mass spectrometry and chemometric tools

Cannabis cultivation in order to produce drugs is forbidden in Switzerland. Thus, law enforcement authorities regularly ask forensic laboratories to determinate cannabis plant's chemotype from seized material in order to ascertain that the plantation is legal or not. As required by the EU official analysis protocol the THC rate of cannabis is measured from the flowers at maturity. When laboratories are confronted to seedlings, they have to lead the plant to maturity, meaning a time consuming and costly procedure. This study investigated the discrimination of fibre type from drug type Cannabis seedlings by analysing the compounds found in their leaves and using chemometrics tools. 11 legal varieties allowed by the Swiss Federal Office for Agriculture and 13 illegal ones were greenhouse grown and analysed using a gas chromatograph interfaced with a mass spectrometer. Compounds that show high discrimination capabilities in the seedlings have been identified and a support vector machines (SVMs) analysis was used to classify the cannabis samples. The overall set of samples shows a classification rate above 99% with false positive rates less than 2%. This model allows then discrimination between fibre and drug type Cannabis at an early stage of growth. Therefore it is not necessary to wait plants' maturity to quantify their amount of THC in order to determine their chemotype. This procedure could be used for the control of legal (fibre type) and illegal (drug type) Cannabis production.