Gotowa bibliografia na temat „Predictive functional assay”

Heparin-induced thrombocytopenia (HIT) is a thrombocytopenia caused by heparin and mediated by an atypical immune mechanism leading to a paradoxical high thrombotic risk, associated with severe morbidity or death. The diagnosis of HIT combines a clinical scoring of pretest probability and laboratory testing. First-line routine tests are antigen binding assays detecting specific antibodies. The most sensitive of these tests have a high HIT-negative predictive value enabling HIT diagnosis to be ruled out when negative. However, HIT-positive predictive value is low, and a functional assay evaluating the pathogenicity of the antibodies should be performed to exclude false-positive results. In contrast to screening assays, functional assays are highly specific but technically challenging, and are thus performed in referral laboratories, where platelet activation is detected using radioactive serotonin (serotonin release assay, SRA) or visually (heparin-induced platelet activation, HIPA). Flow cytometry is a possible alternative. It is, however, currently not widely used, mostly because of the lack of standardization of the published assays. This article describes and discusses the standardization of a HIT flow cytometry assay (HIT-FCA) method, which subsequently led to the development and commercialization of a CE-marked assay (HIT Confirm®, Emosis, France) as a suitable rapid HIT functional test.

SummaryThe R506Q mutation (“Factor V Leiden”) is responsible for the resistance to activated Protein C (aPCR), that is evaluated by coagulation tests. Such tests cannot be used in patients with lupus anticoagulants (LAs), due to the interfering effect exerted by these antibodies on “in vitro” phospholipid-dependent coagulation tests. For this reason, assays have been developed to evaluate aPCR that are insensitive to the presence of LA antibodies. We evaluated two such coagulation tests in the plasma of 82 consecutive patients with LAs. By polymerase chain reaction 3 patients (3.6%) were found heterozygous for the R506Q mutation. aPCR was evaluated by two clotting assays, proposed to be “insensitive” to the presence of LAs: 1. aPCR-tissue factor-based assay, using Factor V deficient plasma and 1:40 diluted test plasma; 2. aPCR-dRVVT-based assay with highly concentrated phospholipids. Their interassay coefficient of variation was 28% and 6.2%, respectively. Compared to the polymerase chain reaction analysis, the 2 tests displayed the following characteristics: sensitivity 67% vs 100%, specificity 92% vs 96%, positive predictive value 25% vs 50%, negative predictive value 99% vs 100%, respectively. Among LA patients without the R506Q mutation, 5 scored positive in the aPCR-tissue factor-based assay, 2 in the aPCR-dRVVT-based assay and another one in both assays. Our findings suggest that the aPCRdRVVT-based test is more reliable and sensitive than the aPCR-tissue factor-based one to the R506Q mutation in patients with LAs. Both assays, when negative, make unlikely the presence of the R506Q mutation. Polymerase chain reaction analysis remains, however, to be performed when either test is positive.

Abstract Identifying functional variants underlying disease risk and adoption of personalized medicine are currently limited by the challenge of interpreting the functional consequences of genetic variants. Predicting the functional effects of disease-associated protein-coding variants is increasingly routine. Yet, the vast majority of risk variants are non-coding, and predicting the functional consequence and prioritizing variants for functional validation remains a major challenge. Here, we develop a deep learning model to accurately predict locus-specific signals from four epigenetic assays using only DNA sequence as input. Given the predicted epigenetic signal from DNA sequence for the reference and alternative alleles at a given locus, we generate a score of the predicted epigenetic consequences for 438 million variants observed in previous sequencing projects. These impact scores are assay-specific, are predictive of allele-specific transcription factor binding and are enriched for variants associated with gene expression and disease risk. Nucleotide-level functional consequence scores for non-coding variants can refine the mechanism of known functional variants, identify novel risk variants and prioritize downstream experiments.

Abstract Implementing screening assays that identify functional and structural cardiotoxicity earlier in the drug development pipeline has the potential to improve safety and decrease the cost and time required to bring new drugs to market. In this study, a metabolic biomarker-based assay was developed that predicts the cardiotoxicity potential of a drug based on changes in the metabolism and viability of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM). Assay development and testing was conducted in 2 phases: (1) biomarker identification and (2) targeted assay development. In the first phase, metabolomic data from hiPSC-CM spent media following exposure to 66 drugs were used to identify biomarkers that identified both functional and structural cardiotoxicants. Four metabolites that represent different metabolic pathways (arachidonic acid, lactic acid, 2′-deoxycytidine, and thymidine) were identified as indicators of cardiotoxicity. In phase 2, a targeted, exposure-based biomarker assay was developed that measured these metabolites and hiPSC-CM viability across an 8-point concentration curve. Metabolite-specific predictive thresholds for identifying the cardiotoxicity potential of a drug were established and optimized for balanced accuracy or sensitivity. When predictive thresholds were optimized for balanced accuracy, the assay predicted the cardiotoxicity potential of 81 drugs with 86% balanced accuracy, 83% sensitivity, and 90% specificity. Alternatively, optimizing the thresholds for sensitivity yields a balanced accuracy of 85%, 90% sensitivity, and 79% specificity. This new hiPSC-CM-based assay provides a paradigm that can identify structural and functional cardiotoxic drugs that could be used in conjunction with other endpoints to provide a more comprehensive evaluation of a drug’s cardiotoxicity potential.

Abstract Background One of the resistance mechanisms of chronic myeloid leukemia (CML) is multidrug resistance (MDR) mediated by overexpression of P-glycoprotein (P-gp) and MDR related protein-1 (MRP1). The flow cytometric functional assay using efflux inhibitor cyclosporine and Rhodamine-123 (Rho-123 efflux assay) detects MDR functional activity. In this study, we performed Rho-123 efflux assay as well as MRP1 and P-gp expression assays by flow cytometry, and evaluated the performance of each test for the prediction of treatment failure in CML patients. Methods A total of 229 peripheral blood samples (19 diagnosis and 210 follow-up) from 94 CML patients treated with TKIs were enrolled. These samples were categorized into three subgroups [optimal response (N=120), suboptimal response (N=54), treatment failure (N=36)] defined by the response criteria to treatment with TKIs in CML patients (Best Pract Res Clin Haematol 2009;22:331-41), and ABL1 mutation analysis was performed in some patients (N=18) with treatment failure. In addition, cutoff values (mean + 1.65SD) for each test were determined from 35 normal controls. The Rho-123 efflux assay, MRP1 and P-gp expression assays were performed separately with all samples. In the Rho-123 efflux assay, the MDR functional activity was measured as the ratio of mean fluorescence intensity (mean channel fluorescence, mcf) with and without cyclosporine. In the P-gp and MRP1 expression assays, MRP1 and P-gp expression levels were expressed as the positivity (%) with each representative monoclonal antibody. The results were compared between patients with treatment response (optimal and suboptimal) and failure. The sensitivity/specificity/negative predictive values (NPV)/positive predictive values (PPV) of 3 test results were calculated using estimated cutoff values and compared. Results The cutoff values of Rhodamine-123 efflux assay, MRP1, and P-gp expression assays were calculated to be > 1.68, > 4.18%, and > 5.07%, respectively. The patients with treatment failure showed significantly higher MRP1 expression [mean 14.08% vs. 4.48%, P = 0.038 (diagnosis); 5.24% vs. 3.54%, P = 0.006 (follow-up)] and P-gp expression [12.27% vs. 3.41%, P = 0.027 (diagnosis); 5.25% vs. 3.48%, P = 0.005(follow-up)] than those with treatment response. However, there were no significant differences in the Rhodamine-123 efflux assay (P = 0.769 and 0.199 at diagnosis and follow-up) between 2 groups. Subsequent analysis revealed that 58.7% of follow-up patients with treatment failure show positive results in at least one test, and 53.3% of these patients with negative for the ABL1 kinase domain mutation also show positive results in at least one test. The sensitivity/specificity/NPV/PPV of 3 test results for the prediction of treatment failure were estimated as 22.9%/85.1%/84.6%/23.5% (Rhodamine-123 efflux assay), 37.1%/69.7%/83.9%/20.6% (MRP1 expression assay), and 31.4%/78.8%/84.4%/23.9% (P-gp expression assay). Conclusion MRP1 and P-gp expression were significantly higher in follow-up CML patients with treatment failure than those with treatment response. The patients who experienced treatment failure, had shown significantly higher MRP1 and P-gp expression at diagnosis than those who did not. Low sensitivity and PPV are inevitable for assays measuring MDR, because MDR is only one of mechanism that causes treatment failure. Our study revealed relatively high specificity and NPV in both MRP-1 and P-gp expression assay, and at least half of ABL1 mutation negative patients with treatment failure possessed positivity in MDR tests. Therefore, it can be speculated that the estimation of MRP-1 and P-gp expression levels can provide useful informations for the prediction of treatment failure in CML patients. Disclosures: No relevant conflicts of interest to declare.

Abstract Activated protein C (APC) resistance is the most frequent hereditary defect associated with deep vein thrombosis (DVT). Over 80% of the APC resistance phenotypes can be explained by the Factor V Leiden (FVL) mutation. This defect is caused by a point mutation in the factor V gene resulting in a replacement of the amino acid Arg 506 by a Gln residue. For patients who are at risk of thrombosis or who have active thrombosis, it is of interest to be able to screen for the FVL mutation in a easy and reliable manner. There are two types of assays for the detection of FVL. The genotype is reliably detected by PCR technology, but this technology is not readily available to all laboratories nor is it inexpensive. The phenotypic expressions of the defect can be identified by plasma-based functional assays, of which several are commercially available. The functional Pefakit® APC-R FVL assay (Pentapharm; Basel, Switzerland) (APC-R) is a new plasma-based clotting assay developed to overcome the limitations of the current assays, including sensitivity and specificity. Additionally, the APC-R assay is more informative as results specify heterozygous / homozygous opposed to a report of normal / abnormal by all other assays. The APC-R assay is designed to specifically act at the level of the prothrombinase complex using a factor V dependent prothrombin activator isolated from the snake venom of the Notechis scutatus. We investigated the phenotypic and genotypic expressions of FVL in patients with cancer and acute symptomatic DVT and/or PE (n=67). Normal healthy volunteers (n=50) collected in-house served as controls. DNA isolated from buffy coats obtained from citrated anticoagulated blood was used to profile FVL using standard PCR probes. Citrated plasma was used to determine APC-R by the functional Pefakit® assay. Of the 67 patients profiled by molecular analysis, 5 (7%) were heterozygous and 1 (1%) was homozygous for the FVL defect. In the normal healthy volunteers, 3 (6%) were heterozygous and 1 (2%) was homozygous for FVL. Using the APC-R functional plasma-based assay, 4 cancer patients (6%) were heterozygous and 2 (3%) were homozygous. In the normal volunteers, 4 (8%) were heterozygous and 1 (2%). was homozygous with the APC-R assay. The APC-R functional plasma-based assay did not miss any FVL patient shown positive by PCR. The heterozygous cancer patients detected with the functional assay were detected as heterozygous with the PRC method; one patient detected as homozygous with the functional method was determined to be heterozygous by PCR. In the normal healthy volunteers, 1 volunteer that was heterozygous by the plasma based assay was negative by PCR. This study demonstrates that the prevalence of the FVL defect in cancer patients with thrombosis is similar to the normal healthy population. Although additional studies to validate the sensitivity/specificity of the APC-R assay and to establish its positive / negative predictive values are needed, the results of this first investigation suggest that the APC-R assay may provide more useful results than other commercially available assays for the detection of FVL. The results of this study showed a good correlation of the functional FVL. APC-R assay with the reference standard molecular PCR method. These data suggest a practical role of this new assay in the clinical laboratory diagnosis of FVL.

G-protein-coupled receptors (GPCR) participate in many disease pathways and represent the largest family of therapeutic targets. Thus, great investments are made to discover drugs modulating GPCR-mediated events. Among functional assays for screening GPCRs, the Transfluor® imaging assay is based on redistribution of cytosolic β-arrestin to an activated GPCR and has become widely used in high-content screening. However, assessing Transfluor® alone has limitations: relying on a single mechanistic step of β-arrestin redistribution during GPCR activation, providing no information on the stimulated GPCR's intracellular fate, and using only a single fluorescent color (green fluorescent protein). Taking full advantage of high-content imaging to screen approximately 2000 compounds, the authors multifplexed the Transfluor® assay with an immunofluorescence-based quantification of GPCR internalization. This approach identified and classified 377 compounds interfering with agonist-induced activation of the Transfluor ® assay, receptor internalization, or both. In addition, a subset of compounds was analyzed for their performance across imaging, cell-based calcium release (fluorometric imaging plate reader [FLIPR]), and biochemical receptor binding assays (scintillation proximity assay). This indicated that the imaging assays have even better predictive power for direct inhibition of receptor binding than the FLIPR assay. In conclusion, compounds inducing unique responses can suggest novel mechanisms of action and be used as tools to study GPCR activation and internalization. ( Journal of Biomolecular Screening 2008:449-455)

SummaryA rapid diagnostic work-up is required in patients with suspected heparin-induced thrombocytopenia (HIT). However, diagnosis of HIT is challenging due to a number of practical issues and methodological limitations. Many laboratory tests and a few clinical scoring systems are available but the individual characteristics and the diagnostic accuracy of these are hard to appraise. The 4Ts score is a well evaluated clinical assessment tool with the potential to rule out HIT in many patients. Still, it requires experience and is subject to a relevant inter-observer variability. Immunoassays such as enzyme-linked immunosorbent assays or recently developed rapid assays are able to exclude HIT in a number of patients. But, accuracy of immunoassays differs depending on type of assay, threshold, antibody specificity and even manufacturer. Due to a comparatively low positive predictive value, HIT cannot be confirmed by immunoassays alone. In addition, only some of them are immediately accessible, particularly in small laboratories. While functional assays such as the serotonin release assay (SRA) and the heparin-induced platelet activation assay (HIPA) are considered as gold standard for diagnosis of HIT, they require a highly specialised laboratory. In addition, some of them are not adequately evaluated. In clinical practice, we recommend an integrated diagnostic approach combining not only clinical assessment (the 4Ts score) but immunoassays and functional assays as well. We propose a clear diagnostic algorithm supporting clinical decision-making. Furthermore, we provide an overview of all current laboratory techniques for HIT and discuss diagnostic pathways and strategies to reduce diagnostic errors, and future perspectives.

Calcitonin gene-related peptide (CGRP) is a small neuropeptide and a potent vasodilator that is widely associated with chronic pain and migraine. An antibody that inhibits CGRP function would be a potential therapeutic for treatment of these disorders. Here we describe the isolation of highly potent antibodies to CGRP from phage and ribosome display libraries and characterization of their epitope, species cross-reactivity, kinetics, and functional activity. Homogenous time-resolved fluorescence (HTRF) binding assays identified antibodies with the desired species cross-reactivity from naïve libraries, and HTRF epitope competition assays were used to characterize and group scFv by epitope. The functional inhibition of CGRP and species cross-reactivity of purified scFv and antibodies were subsequently confirmed using cAMP assays. We show that epitope competition assays could be used as a surrogate for functional cell-based assays during affinity maturation, in combination with scFv off-rate ranking by biolayer interferometry (BLI). This is the first time it has been shown that off-rate ranking can be predictive of functional activity for anti-CGRP antibodies. Here we demonstrate how, by using just four simple assays, diverse panels of antibodies to CGRP can be identified. These assay formats have potential utility in the identification of antibodies to other therapeutic targets.

ABSTRACT The neuraminidase inhibitors (NAIs) zanamivir and oseltamivir are currently the only antiviral drugs effective for the treatment and prophylaxis of 2009 pandemic influenza A (H1N1) virus infections. The proven potential of these viruses to acquire NAI resistance during treatment emphasizes the need to assess their NAI susceptibility. The 50% inhibitory concentrations (IC50s) are known to vary depending on the neuraminidase inhibition (NI) test used; however, few side-by-side comparisons of different NI assays have been done. In the present study, a panel of 11 isolates representing 2009 seasonal and pandemic influenza H1N1 viruses, including oseltamivir-resistant H275Y variants, were tested in three functional NI assays: chemiluminescent (CL), fluorescent (FL), and colorimetric (CM). The sensitivities of the viruses to zanamivir, oseltamivir, and three investigational NAIs (peramivir, R-125489, and A-315675) were assessed. All isolates with the exception of H275Y variants were sensitive to all five NAIs by all three NI assays. The H275Y variants showed substantially elevated IC50s against oseltamivir and peramivir. The three NI assays generally yielded consistent results; thus, the choice of NI assay does not appear to affect conclusions based on drug susceptibility surveillance. Each assay, however, offers certain advantages compared to the others: the CL assay required less virus volume and the FL assay provided the greatest difference in the IC50s between the wild type and the variants, whereas the IC50s obtained from the CM assay may be the most predictive of the drug concentrations needed to inhibit enzyme activity in humans. It would be desirable to develop an NI assay which combines the advantages of all three currently available assays but which lacks their shortcomings.

Les carcinomes épidermoïdes (CE) des voies aéro-digestives supérieures (VADS) sont le 5ème cancer en France, et son pronostic est sombre avec une survie à 5 ans tous stades confondus de l’ordre de 40%. Les stratégies thérapeutiques reposent sur des associations des traitements (chirurgie, radiothérapie, chimiothérapie à base de platine) avec une lourde toxicité pour les patients. La résistance aux traitements est également une problématique majeure. Ces éléments soulignent l’importance d’une médecine personnalisée pour améliorer la prise en charge des patients atteints d’un CE des VADS et augmenter l’efficacité des traitements tout en réduisant le risque de toxicité de la prise en charge globale. Dans ce contexte, l’utilisation de tests fonctionnels sur des modèles ex vivo, comme les tumoroïdes, pourrait être une solution pour répondre à ces problématiques. Les objectifs de cette thèse étaient d’établir des lignées de tumoroïdes des CE des VADS, de vérifier leur pertinence en comparant leurs caractéristiques histologiques et moléculaires avec les tumeurs d’origine, de les exposer à des thérapies conventionnelles pour étudier la corrélation avec la réponse clinique du patient et de les exposer à des thérapies innovantes pour explorer de nouvelles possibilités thérapeutiques. Nous avons établi 24 lignées de tumoroïdes de CE des VADS à long terme avec une amélioration des conditions de culture permettant d’atteindre un taux d’établissement de 26%. Les tumoroïdes avaient les même les caractéristiques histologiques de CE que les tumeurs appariées et avaient une expression des marqueurs tumoraux comparable. La réponse des tumoroïdes au cisplatine et à la radiothérapie a pu être évaluée et la réponse au cisplatine était corrélée au pronostic du patient. Les tumoroïdes ont été exposé à des thérapies innovantes, notamment des ions carbone, permettant de montrer l’intérêt de ces modèles dans l’étude des particules lourdes et la meilleure efficacité biologique des ions carbone par rapport aux rayons X. Notre travail a donc permis de consolider les arguments en faveur de l’utilisation des tumoroïdes de VADS comme modèle pertinent en cancérologie, que ce sont à des fins de recherche préclinique ou à des fins de médecine personnalisée
Head and Neck Squamous Cell Carcinoma (HNSCC) is the 5th most common cancer in France, and has a poor prognosis with a 5-year survival rate for all stages combined around 40%. Therapeutic strategies are based on combinations of treatments (surgery, radiotherapy, platinum-based chemotherapy), with high toxicity for patients. Resistance to treatment is also a major problem. These factors underline the importance of personalized medicine in improving the management of patients with HNSCC, by increasing the efficiency of treatments while reducing the risk of toxicity. In this context, the use of functional tests on ex vivo models, such as patient-derived tumor organoids (PDTO), could be a solution to address these issues. The aims of this thesis were to establish HNSCC PDTO lines, validate their relevance by comparing their histological and molecular characteristics with the original tumors, expose them to conventional therapies to study correlation with patient clinical response, and expose them to innovative therapies to explore new therapeutic possibilities. We established 24 long-term HNSCC PDTO lines achieving a success rate of establishment of 26%. The PDTO had the same histological features of SCC as the matched tumors and had matched tumor marker expression. The response of PDTO to cisplatin and radiotherapy had been assessed, and response to cisplatin correlated with patient prognosis. The PDTO were exposed to innovative therapies, in particular carbon ions, demonstrating the interest of these models in the study of heavy particles and the greater biological efficacy of carbon ions over X-rays. Our work has thus consolidated the arguments in favor of using HNSCC PDTO as a relevant model in oncology, whether for preclinical research or personalized medicine

Les stratégies de traitement biologiques ciblant le TNF-α se sont avérées efficaces pour réduire l'inflammation et les symptômes cliniques dans plusieurs maladies inflammatoires chroniques et sont maintenant couramment utilisées pour les patients qui ne répondent pas aux AINS au cours de la spondyloarthrite (SpA). Cependant, 30 à 40% des patients ne répondent pas aux anti-TNF, et il est actuellement impossible de prédire la réponse des patients à ces biomédicaments. Pour améliorer les résultats cliniques, nous avons besoin d’une part d’une meilleure compréhension des mécanismes d’action des anti-TNF sur le système immunitaire, et d’autre part de biomarqueurs permettant de prédire la réponse à ces biomédicaments afin de guider la décision thérapeutique. Mon projet de doctorat a porté sur deux objectifs complémentaires: (i) l'objectif principal était de progresser dans notre compréhension des mécanismes pathogéniques impliqués dans la SpA axiale et de définir de quelle façon les anti-TNF-α affectent les réponses immunitaires des patients, (ii) de développer des biomarqueurs pour prédire la réponse thérapeutique aux inhibiteurs du TNF. En collaboration avec l'équipe du Pr. Dougados à l'Hôpital Cochin, nous avons recruté deux cohortes indépendantes de patients SpA ayant une maladie active et pour lesquels nous avons collecté des échantillons de sang avant l'initiation du traitement par anti-TNF puis 1 semaine et 3 mois après le début du traitement. Les réponses immunitaires de ces patients ont été analysées à l'aide de tests hautement standardisés réalisés ex-vivo sur sang circulant. Ces tests "TruCulture" se présentent sous forme de seringues, dans lesquelles 1 ml de sang total est mis à incuber avec un stimulus spécifique ; 20 stimuli différents ont été testé et validé avant et après traitement dans les deux cohortes de patients. Nous avons observé une réduction très significative de la sécrétion de IL-1ra, IL-1β, IL-8, and MIP-1β en réponse à des stimuli microbiens et à des agonistes des TLR dans les échantillons de sang prélevés 7 jours et/ou 3 mois après le début du traitement. Pour identifier les bases moléculaires de l’action des inhibiteurs du TNF nous avons analysé l'expression des gènes dans ces différentes conditions de stimulation. L'analyse bioinformatique quantitative de l'expression des gènes (QuSAGE) a révélé que les gènes les plus modulés par le traitement anti-TNF étaient NF-KB et les gènes cibles de NF-kB, y compris le TNF lui-même et l’IL1B. Nos données suggèrent que les inhibiteurs du TNF agissent principalement en perturbant une boucle autorégulatrice pilotée par NF-kB. Afin d'identifier les signatures immunologiques de réponse aux anti-TNF avant le début du traitement, nous avons corrélé les réponses immunitaires chez les patients analysés au temps 0 à la réponse thérapeutique aux anti-TNF mesurée à 3 mois. Nos résultats suggèrent que les patients atteints de SpA et exprimant des niveaux inférieurs de PAX5 et des niveaux supérieurs de SPP1 en réponse à la stimulation avec SEB avant l'initiation de la thérapie anti-TNF ont les meilleures réponses thérapeutiques. Notre recherche montre que les tests TruCulture sont un outil efficace pour étudier les fonctions immunitaires chez les patients atteints de SpA et que les effets du traitement anti-TNF peuvent être mesurés lorsque les cellules immunitaires sont stimulées. En terme de recherche translationnelle, nous avons identifié des molécules qui pourront être utilisés comme biomarqueurs pour aider les cliniciens à prédire les réponses thérapeutiques aux traitements anti TNF
The introduction of anti-TNF therapy has proven effective to reduce inflammation and clinical symptoms in several chronic inflammatory diseases. However, 30-40% of patients do not respond to TNF blockers and it is currently not possible to predict responsiveness of patients to anti-TNF therapy. Furthermore, their impact on the immune system is incompletely understood. The goals of my PhD project were (i) to define the impact of anti-TNF therapy on immune responses to microbial challenges and stimuli targeting specific immune pathways in spondyloarthritis (SpA) patients, and (ii) to identify immunological correlates associated with therapeutic responses to TNF-blockers.Using a set of whole-blood, syringe-based assays to perform ex vivo stimulation while preserving physiological cellular interactions (TruCulture assays), we have performed a pilot study in SpA patients and investigated immune responses to 20 different stimuli before and 3 months after initiation of anti-TNF therapy. These findings were validated in a replication cohort, also assessing the effects of anti-TNF agents after only one week of treatment. We observed a highly significant reduction of the secretion of IL-1ra, IL-1β, IL-8 and MIP-1β in response to selected stimuli after 3 months of treatment compared to the baseline. Interestingly, these changes were already detectable after a single injection of an anti-TNF agent. To gain insight into the molecular mechanism of TNF blockers, we profiled gene expression in the stimulation cultures from all patients. Quantitative set analysis for gene expression (QuSAGE) revealed that the gene modules most affected by anti-TNF therapy are NF-kB transcription factors and inhibitors and NF-kB target genes, including TNF itself and IL1B. Our data suggest that TNF-blockers primarily act by disrupting an autoregulatory loop driven by NF-kB. We also tested whether there is a correlation between the responses of immune cells to specific stimuli and the clinical response to TNF-blockers. The decision tree model that we trained and validated suggests that SpA patients who expressed lower levels of PAX5 and higher levels of SPP1 in response to SEB stimulation before initiation of anti-TNF therapy had the best therapeutic responses. Our study shows that TruCulture assays are an efficient and robust tool to monitor immune functions in SpA patients and that the effects of anti-TNF therapy can be measured when immune cells are challenged, but not at steady state. Our data also indicate that analyzing immune responses in patients before therapy is a promising strategy to develop biomarkers for prediction of therapeutic responses to TNF-blockers

The utilization of in vitro tests with a tiered testing strategy for detection of mild ocular irritants can reduce the use of animals for testing, provide mechanistic data on toxic effects, and reduce the uncertainty associated with dose selection for clinical trials. The first section of this thesis describes how in vitro methods can be used to improve the prediction of the toxicity of chemicals and ophthalmic products. The proper utilization of in vitro methods can accurately predict toxic threshold levels and reduce animal use in product development. Sections two, three and four describe the development of new sensitive in vitro methods for predicting ocular toxicity. Maintaining the barrier function of the cornea is critical for the prevention of the penetration of infections microorganisms and irritating chemicals into the eye. Chapter 2 describes the development of a method for assessing the effects of chemicals on tight junctions using a human corneal epithelial and canine kidney epithelial cell line. In Chapter 3 a method that uses a primary organ culture for assessing single instillation and multiple instillation toxic effects is described. The ScanTox system was shown to be an ideal system to monitor the toxic effects over time as multiple readings can be taken of treated bovine lenses using the nondestructive method of assessing for the lens optical quality. Confirmations of toxic effects were made with the utilization of the viability dye alamarBlue. Chapter 4 describes the development of sensitive in vitro assays for detecting ocular toxicity by measuring the effects of chemicals on the mitochondrial integrity of bovine cornea, bovine lens epithelium and corneal epithelial cells, using fluorescent dyes. The goal of this research was to develop an in vitro test battery that can be used to accurately predict the ocular toxicity of new chemicals and ophthalmic formulations. By comparing the toxicity seen in vivo animals and humans with the toxicity response in these new in vitro methods, it was demonstrated that these in vitro methods can be utilized in a tiered testing strategy in the development of new chemicals and ophthalmic formulations.

AbstractTorsade de Pointes (TdP) is a type of ventricular tachycardia that can occur as a side effect of several medications. The Comprehensive in vitro Proarrhythmia Assay (CiPA) is a novel testing paradigm that utilizes single cell electrophysiological simulations to predict TdP risk for drugs that could potentially be used clinically. However, the effects on mechanical performance and mechano-electrical feedback are neglected. Here, we demonstrate that including electromechanical simulations in CiPA testing can provide additional insights into the predicted drug-induced TdP risk. In this work, we analyzed six drugs, namely flecainide, ibutilide, metronidazole, mexiletine, quinidine and ranolazine. We compared previously classified risks (low, intermediate, high) with our fully coupled electromechanical simulation results based upon the action potential, the electromechanical window, and the maximum active tension [1]. For ranolazine and metronidazole the predicted risk changed from low to intermediate and intermediate to high, respectively. For the latter, while electrophysiological markers indicated a low risk, the active tension decreased by 58% which can result in a loss of heart function. Therefore, adding mechanics to CiPA testing results in an altered prediction of drug-related TdP risk.

Abstract Despite the pervasiveness of anxiety and trauma-related disorders, there is no mechanistic explanation for their causes, and the search is ongoing for therapeutic interventions that are effective and do not cause undesirable side effects. Given the evolutionarily advantageous function of anxiety and fear responses, there are multiple redundant circuits. However, the robustness that this redundancy provides also represents a challenge for targeted anxiolytic therapies to penetrate the multiple layers of circuitry. To find new targets, preclinical models are required to delve into the neural underpinnings of anxiety. The most utilized preclinical models for anxiety have historically been demonstrated to have various degrees of face, predictive, and construct validity but only assess a limited range of behaviors. Broader use of multiple anxiety assays combined with unsupervised machine learning and discovery of behavioral motifs using computer vision tools will allow researchers to parse anxiety phenotyping into greater granularity.

Glomerular filtration rate (GFR) is considered the best parameter for the assessment of renal function, being usually determined on the basis of urine or plasma clearance of exogenous renal markers. The common methodology is invasive, time consuming and cumbersome, with multiple blood and/or urine sampling and following laboratory assays required. The method detailed here allows to transcutaneously determine the renal function in awake animals, in a non-invasive and efficient manner by using an electronic device which detects the fluorescence emitted through the skin from the renal marker FITC-Sinistrin. A crucial target has been to improve the fixation of the device, which is dependent on the skin structure. For validation, the technique has been compared with the classical clearance method, and its robustness has been demonstrated in healthy and diseased murine models. Moreover, the method allows sequential measurements in the same individual. Thus progression and recovery of renal failure can be followed. Therefore, its future application in humans would allow an accurate and appropriate prediction and monitoring of patients with established kidney disease over time. Furthermore, it will be possible to observe those patients under other pathological conditions with associated risk of developing renal problems.

Accurate prediction of burst pressure in line pipes is critical for their safety design and operation. Different equations for predicting burst pressure of line pipes have been proposed over the years, but broad agreements between the prediction equations did not exist. To this end, the present authors recently developed a new multi-axial plastic yield theory that is referred to as Average Shear Stress Yield (ASSY) theory [6]. Based on this theory, a theoretical closed-form solution for predicting burst pressure was proposed as a function of the pipe diameter, thickness, ultimate tensile stress and strain hardening exponent. The results showed that the ASSY-based burst pressure solution predicts generally the average of experimental data, and gives the best prediction among different models in a comparison of over 100 full-size burst tests for different line pipe geometries and grades. This conclusion is consistent with the observation by Zimmerman et al. [7]. On the other hand, Law at al. [1–3] recently proposed a so-called CIS (cylindrical instability stress) model that can implicitly predict the burst pressure of line pipes, and claimed that the CIS model is the best one for predicting burst pressure. To clarify the argument and to determine a truly accurate prediction equation, this paper will reevaluate the available models of burst pressure using various experimental data used by Law et al. and others. Detailed comparisons and discussions on the predictions of burst pressure with the experimental results are performed.

In Vitro models are being used as a bridge between animal and human studies. Being able to reproduce specific tissue-like structures, functions and responses in a way that is more physiologically relevant allows for huge advantages for tissue engineering, pharmaco-toxicology and food research. These systems are not designed to be directly implanted into patients, but can be used to study human tissue physiology and pathophysiology in vitro. In vitro models are based on human cells, which can capture the responses of the human body, particularly those that are species specific. Models of tissues and organs can give enhanced predictive power, particularly for large-scale screening assays and to understand complex disease pathology.

In soil solarization, moist soil is covered with a transparent plastic film, resulting in passive solar heating which inactivates soil-borne pathogen/weed propagules. Although solarization is an effective alternative to soil fumigation and chemical pesticide application, it is not widely used due to its long duration, which coincides with the growing season of some crops, thereby causing a loss of income. The basis of this project was that solarization of amended soil would be utilized more widely if growers could adopt the practice without losing production. In this research we examined three factors expected to contribute to greater utilization of solarization: 1) investigation of techniques that increase soil temperature, thereby reducing the time required for solarization; 2) development and validation of predictive soil heating models to enable informed decisions regarding soil and solarization management that accommodate the crop production cycle, and 3) elucidation of the contributions of microbial activity and microbial community structure to soil heating during solarization. Laboratory studies and a field trial were performed to determine heat generation in soil amended with compost during solarization. Respiration was measured in amended soil samples prior to and following solarization as a function of soil depth. Additionally, phytotoxicity was estimated through measurement of germination and early growth of lettuce seedlings in greenhouse assays, and samples were subjected to 16S ribosomal RNA gene sequencing to characterize microbial communities. Amendment of soil with 10% (g/g) compost containing 16.9 mg CO2/g dry weight organic carbon resulted in soil temperatures that were 2oC to 4oC higher than soil alone. Approximately 85% of total organic carbon within the amended soil was exhausted during 22 days of solarization. There was no significant difference in residual respiration with soil depth down to 17.4 cm. Although freshly amended soil proved highly inhibitory to lettuce seed germination and seedling growth, phytotoxicity was not detected in solarized amended soil after 22 days of field solarization. The sequencing data obtained from field samples revealed similar microbial species richness and evenness in both solarized amended and non-amended soil. However, amendment led to enrichment of a community different from that of non-amended soil after solarization. Moreover, community structure varied by soil depth in solarized soil. Coupled with temperature data from soil during solarization, community data highlighted how thermal gradients in soil influence community structure and indicated microorganisms that may contribute to increased soil heating during solarization. Reliable predictive tools are necessary to characterize the solarization process and to minimize the opportunity cost incurred by farmers due to growing season abbreviation, however, current models do not accurately predict temperatures for soils with internal heat generation associated with the microbial breakdown of the soil amendment. To address the need for a more robust model, a first-order source term was developed to model the internal heat source during amended soil solarization. This source term was then incorporated into an existing “soil only” model and validated against data collected from amended soil field trials. The expanded model outperformed both the existing stable-soil model and a constant source term model, predicting daily peak temperatures to within 0.1°C during the critical first week of solarization. Overall the results suggest that amendment of soil with compost prior to solarization may be of value in agricultural soil disinfestations operations, however additional work is needed to determine the effects of soil type and organic matter source on efficacy. Furthermore, models can be developed to predict soil temperature during solarization, however, additional work is needed to couple heat transfer models with pathogen and weed inactivation models to better estimate solarization duration necessary for disinfestation.