Increasing Scientific Confidence in Adverse Outcome Pathways: Application of Tailored Bradford-Hill Considerations for Evaluating Weight of Evidence.

Systematic consideration of scientific support is a critical element in developing and, ultimately, using adverse outcome pathways (AOPs) for various regulatory applications. Though weight of evidence (WoE) analysis has been proposed as a basis for assessment of the maturity and level of confidence in an AOP, methodologies and tools are still being formalized. The Organization for Economic Co-operation and Development (OECD) Users' Handbook Supplement to the Guidance Document for Developing and Assessing AOPs (OECD 2014a; hereafter referred to as the OECD AOP Handbook) provides tailored Bradford-Hill (BH) considerations for systematic assessment of confidence in a given AOP. These considerations include (1) biological plausibility and (2) empirical support (dose-response, temporality, and incidence) for Key Event Relationships (KERs), and (3) essentiality of key events (KEs). Here, we test the application of these tailored BH considerations and the guidance outlined in the OECD AOP Handbook using a number of case examples to increase experience in more transparently documenting rationales for assigned levels of confidence to KEs and KERs, and to promote consistency in evaluation within and across AOPs. The major lessons learned from experience are documented, and taken together with the case examples, should contribute to better common understanding of the nature and form of documentation required to increase confidence in the application of AOPs for specific uses. Based on the tailored BH considerations and defining questions, a prototype quantitative model for assessing the WoE of an AOP using tools of multi-criteria decision analysis (MCDA) is described. The applicability of the approach is also demonstrated using the case example aromatase inhibition leading to reproductive dysfunction in fish. Following the acquisition of additional experience in the development and assessment of AOPs, further refinement of parameterization of the model through expert elicitation is recommended. Overall, the application of quantitative WoE approaches hold promise to enhance the rigor, transparency and reproducibility for AOP WoE determinations and may play an important role in delineating areas where research would have the greatest impact on improving the overall confidence in the AOP.

Organotypic Modeling Platform for Adverse Outcome Pathways of Male Reproductive and Developmental Processes

Thesis (Master's)--University of Washington, 2016-06

Increased fibrinogen levels at diagnosis are associated with adverse outcome in patients with acute myeloid leukemia

Increased plasma fibrinogen levels are associated with shortened overall survival (OS) in some solid tumor types. In contrast, the prognostic significance of varying fibrinogen levels in acute myeloid leukemia (AML) at diagnosis is unknown. In this study, we assessed the prognostic significance of fibrinogen levels in AML patients. In a comprehensive retrospective single-center study, we determined the survival rates of 375 consecutive AML patients undergoing at least one cycle of intensive chemotherapy induction treatment. Patients were dichotomized between low (<4.1 g/L) and high fibrinogen levels (≥4.1 g/L) at diagnosis of AML before initiation of treatment. Subsequently, quartile ranges were applied to analyze the association of varying fibrinogen levels on survival. We observed that the rates of complete remission, early death, and admission to intensive care unit were equal in the low versus high fibrinogen group. However, OS was significantly better in the low fibrinogen group (27.3 vs 13.5 months; p = 0.0009) as well as progression-free survival (12.3 vs 7.8 months; p = 0.0076). This survival difference remained significant in the multivariate analysis (p = 0.003). Assessing quartiles of fibrinogen values, we further confirmed this observation. Our data suggest that high fibrinogen levels at diagnosis of AML are associated with unfavorable OS and progression-free survival but not with increased mortality during induction treatment.

Effect assessment of nanoparticles toxicity in the terrestrial compartment

Over 11 million tons of nanomaterials (NMs) have been produced in 2012 and predictions point the increase in production. Despite predictions and extended usage via consumer products and industry, the understanding of the potential impact of these materials on the environment is virtually absent. The main aim of this thesis is to understand how a selected group of nanomaterials (metal based particles) may impact soil invertebrates, with special focus on the mechanisms of response. Since a case-by-case Environmental Risk Assessment (ERA) of all the emerging contaminants (particularly NMs) is impossible, among others due to time and cost reasons, to gain understanding on the mechanism of action and response is very important to reach a common paradigm. Understanding the modes of action provides predictive characters in cross particle extrapolation. Besides, it also provides insight for the production of new and sustainable materials. Overall, the effects of the selected NMs (Copper and Silver, Titanium and Zirconium oxides) and the respective salt forms, were investigated at the gene expression (using high-throughput tools, microarray and qPCR technology), biochemical (using enzymatic assays for analysis of oxidative stress markers) and organism (survival and reproduction as in OECD test guidelines) levels, this using standard soil species (Enchytraeus albidus, Enchytraeus crypticus, Eisenia fetida). Gene expression analysis provided valuable information on the mechanisms affected by each of the NMs. The gene expression profile highlighted a (nano)material signature and the effect of the duration of exposure. The functional analyses integrated with the biochemical and organism data, revealed a good understanding power. The biochemical parameters (oxidative stress related) were distinct across the materials and also influenced by duration of exposure and concentration. The standardized organismal responses differed the least between the various materials. The overall outcome is that, in this context of NMs effect assessment, gene expression and enzymatic assays introduced a very important knowledge gap, which could not had been achieved by the standard organismal effects alone. A reoccurring issue with some metal based NMs is the possible dissolution and subsequent release of ions that then causes toxicity e.g. Cu-NPs or Ag-NPs release Cu2+ or Ag+. The oxidation state of the particles was investigated, although this was not the focus of the thesis. The study of fate, e.g. dissolution of NPs, is also only in its beginning and the appropriate techniques are currently being developed. The results showed a specific nanoparticle effect. The UV exposure with titanium dioxide nanoparticles increased its effect.

Clinical outcome and predictors for adverse events after transcatheter aortic valve implantation with the use of different devices and access routes

Background Transcatheter aortic valve implantation (TAVI) is a treatment option for high-risk patients with severe aortic stenosis. Previous reports focused on a single device or access site, whereas little is known of the combined use of different devices and access sites as selected by the heart team. The purpose of this study is to investigate clinical outcomes of TAVI using different devices and access sites. Methods A consecutive cohort of 200 patients underwent TAVI with the Medtronic CoreValve Revalving system (Medtronic Core Valve LLC, Irvine, CA; n = 130) or the Edwards SAPIEN valve (Edwards Lifesciences LLC, Irvine, CA; n = 70) implanted by either the transfemoral or transapical access route. Results Device success and procedure success were 99% and 95%, respectively, without differences between devices and access site. All-cause mortality was 7.5% at 30 days, with no differences between valve types or access sites. Using multivariable analysis, low body mass index (<20 kg/m2) (odds ratio [OR] 6.6, 95% CI 1.5-29.5) and previous stroke (OR 4.4, 95% CI 1.2-16.8) were independent risk factors for short-term mortality. The VARC-defined combined safety end point occurred in 18% of patients and was driven by major access site complications (8.0%), life-threatening bleeding (8.5%) or severe renal failure (4.5%). Transapical access emerged as independent predictor of adverse outcome for the Valve Academic Research Consortium–combined safety end point (OR 3.3, 95% CI 1.5-7.1). Conclusion A heart team–based selection of devices and access site among patients undergoing TAVI resulted in high device and procedural success. Low body mass index and history of previous stroke were independent predictors of mortality. Transapical access emerged as a risk factor for the Valve Academic Research Consortium–combined safety end point.

The MyD88+ phenotype is an adverse prognostic factor in epithelial ovarian cancer

The prognosis of epithelial ovarian cancer is poor in part due to the high frequency of chemoresistance. Recent evidence points to the Toll-like receptor-4 (TLR4), and particularly its adaptor protein MyD88, as one potential mediator of this resistance. This study aims to provide further evidence that MyD88 positive cancer cells are clinically significant, stem-like and reproducibly detectable for the purposes of prognostic stratification. Expression of TLR4 and MyD88 was assessed immunohistochemically in 198 paraffin-embedded ovarian tissues and in an embryonal carcinoma model of cancer stemness. In parallel, expression of TLR4 and MyD88 mRNA and regulatory microRNAs (miR-21 and miR-146a) was assessed, as well as in a series of chemosensitive and resistant cancer cells lines. Functional analysis of the pathway was assessed in chemoresistant SKOV-3 ovarian cancer cells. TLR4 and MyD88 expression can be reproducibly assessed via immunohistochemistry using a semi-quantitative scoring system. TLR4 expression was present in all ovarian epithelium (normal and neoplastic), whereas MyD88 was restricted to neoplastic cells, independent of tumour grade and associated with reduced progression-free and overall survival, in an immunohistological specific subset of serous carcinomas, p<0.05. MiR-21 and miR-146a expression was significantly increased in MyD88 negative cancers (p<0.05), indicating their participation in regulation. Significant alterations in MyD88 mRNA expression were observed between chemosensitive and chemoresistant cells and tissue. Knockdown of TLR4 in SKOV-3 ovarian cells recovered chemosensitivity. Knockdown of MyD88 alone did not. MyD88 expression was down-regulated in differentiated embryonal carcinoma (NTera2) cells, supporting the MyD88+ cancer stem cell hypothesis. Our findings demonstrate that expression of MyD88 is associated with significantly reduced patient survival and altered microRNA levels and suggest an intact/functioning TLR4/MyD88 pathway is required for acquisition of the chemoresistant phenotype. Ex vivo manipulation of ovarian cancer stem cell (CSC) differentiation can decrease MyD88 expression, providing a potentially valuable CSC model for ovarian cancer.

The impact of frailty and polypharmacy on adverse outcomes in older inpatients

Background Older people are at significant risk of adverse outcomes as a result of changes in physiology, frailty, co-morbidity and polypharmacy.1 Timely identification of high-risk patients may facilitate the optimization of medication and reduce the incidence of adverse outcomes. The aims of this study were to evaluate in older inpatients the relationships between risk factors, including frailty and polypharmacy, and adverse health outcomes. Methods This is a prospective study of 1418 patients, aged 70 and older, admitted to general medical units in 11 acute care hospitals across Australia. The interRAI Acute Care (interRAI AC) assessment tool was used for data collection. Frailty status was measured using a Frailty Index (FI), adding each individual’s deficits and dividing by the total number of deficits considered. Adverse health outcomes included falls in hospital, delirium, in-hospital functional and cognitive decline, discharge to a higher level of care and inpatient mortality. Results Patients had a mean age 81 ± 6.8 years with a median length of hospital stay of 6 days (interquartile range 4 to 11 days); 701 (50%) experienced at least one adverse outcome. Polypharmacy (5-9 drugs per day) was observed in almost half of the study population (n=695, 49%) and hyper-polypharmacy (≥10 drugs) observed in about one-third of patients (n=490, 34.6%). Cognitive impairment was shown to be associated with the lower rate of prescribing. FI had a significant association with all adverse outcomes studied (p = <0.05). In contrast, no association was observed between polypharmacy categories and adverse outcomes except for those on 10 or more drugs where they were more likely to be discharged to a higher level of care (p= 0.014). Conclusions Among older inpatients, frailty status was a significant predictor of adverse outcomes. Lower rates of prescribing to patients with cognitive impairment may underpin the lack of an association between polypharmacy and adverse outcomes in this cohort. References: 1. Olsson IN, Runnamo R, Engfeldt P. Medication quality and quality of life in the elderly, a cohort study.Health Qual Life Outcomes.2011;9:95

Long-term Outcome of DSM-IV Major Depressive Disorder in Psychiatric Care

Much of what we know regarding the long-term course and outcome of major depressive disorder (MDD) is based on studies of mostly inpatient tertiary level cohorts and samples predating the era of the current antidepressants and the use of maintenance therapies. In addition, there is a lack of studies investigating the comprehensive significance of comorbid axis I and II disorders on the outcome of MDD. The present study forms a part of the Vantaa Depression Study (VDS), a regionally representative prospective and naturalistic cohort study of 269 secondary-level care psychiatric out- and inpatients (aged 20-59) with a new episode of DSM-IV MDD, and followed-up up to five years (n=182) with a life-chart and semistructured interviews. The aim was to investigate the long-term outcome of MDD and risk factors for poor recovery, recurrences, suicidal attempts and diagnostic switch to bipolar disorder, and the association of a family history of different psychiatric disorders on the outcome. The effects of comorbid disorders together with various other predictors from different domains on the outcome were comprehensively investigated. According to this study, the long-term outcome of MDD appears to be more variable when its outcome is investigated among modern, community-treated, secondary-care outpatients compared to previous mostly inpatient studies. MDD was also highly recurrent in these settings, but the recurrent episodes seemed shorter, and the outcome was unlikely to be uniformly chronic. Higher severity of MDD predicted significantly the number of recurrences and longer time spent ill. In addition, longer episode duration, comorbid dysthymic disorder, cluster C personality disorders and social phobia predicted a worse outcome. The incidence rate of suicide attempts varied robustly de¬pending on the level of depression, being 21-fold during major depressive episodes (MDEs), and 4-fold during partial remission compared to periods of full remission. Although a history of previous attempts and poor social support also indicated risk, time spent depressed was the central factor determining overall long-term risk. Switch to bipolar disorder occurred mainly to type II, earlier to type I, and more gradually over time to type II. Higher severity of MDD, comorbid social phobia, obsessive compulsive disorder, and cluster B personality disorder features predicted the diagnostic switch. The majority of patients were also likely to have positive family histories not exclusively of mood, but also of other mental disorders. Having a positive family history of severe mental disorders was likely to be clinically associated with a significantly more adverse outcome.

Hemodynamics and outcome of septic shock

Septic shock is a common killer in intensive care units (ICU). The most crucial issue concerning the outcome is the early and aggressive start of treatment aimed at normalization of hemodynamics and the early start of antibiotics during the very first hours. The optimal targets of hemodynamic treatment, or impact of hemodynamic treatment on survival after first resuscitation period are less known. The objective of this study was to evaluate different aspects of the hemodynamic pattern in septic shock with special attention to prediction of outcome. In particular components of early treatment and monitoring in the ICU were assessed. A total of 401 patients, 218 with septic shock and 192 with severe sepsis or septic shock were included in the study. The patients were treated in 24 Finnish ICUs during 1999-2005. 295 of the patients were included in the Finnish national epidemiologic Finnsepsis study. We found that the most important hemodynamic variables concerning the outcome were the mean arterial pressures (MAP) and lactate during the first six hours in ICU and the MAP and mixed venous oxygen saturation (SvO2) under 70% during first 48 hours. The MAP levels under 65 mmHg and SvO2 below 70% were the best predictive thresholds. Also the high central venous pressure (CVP) correlated to adverse outcome. We assessed the correlation and agreement of SvO2 and mean central venous oxygen saturation (ScvO2) in septic shock during first day in ICU. The mean SvO2 was below ScvO2 during early sepsis. Bias of difference was 4.2% (95% limits of agreement 8.1% to 16.5%) by Bland-Altman analysis. The difference between saturation values correlated significantly to cardiac index and oxygen delivery. Thus, the ScvO2 can not be used as a substitute of SvO2 in hemodynamic monitoring in ICU. Several biomarkers have been investigated for their ability to help in diagnosis or outcome prediction in sepsis. We assessed the predictive value of N-terminal pro brain natriuretic peptide (NT-proBNP) on mortality in severe sepsis or septic shock. The NT-proBNP levels were significantly higher in hospital nonsurvivors. The NT-proBNP 72 hrs after inclusion was independent predictor of hospital mortality. The acute cardiac load contributed to NTproBNP values at admission, but renal failure was the main confounding factor later. The accuracy of NT-proBNP, however, was not sufficient for clinical decision-making concerning the outcome prediction. The delays in start of treatment are associated to poorer prognosis in sepsis. We assessed how the early treatment guidelines were adopted, and what was the impact of early treatment on mortality in septic shock in Finland. We found that the early treatment was not optimal in Finnish hospitals and this reflected to mortality. A delayed initiation of antimicrobial agents was especially associated with unfavorable outcome.

Improving outcome of childhood bacterial meningitis by simplified treatment : Experience from Angola

Background Acute bacterial meningitis (BM) continues to be an important cause of childhood mortality and morbidity, especially in developing countries. Prognostic scales and the identification of risk factors for adverse outcome both aid in assessing disease severity. New antimicrobial agents or adjunctive treatments - except for oral glycerol - have essentially failed to improve BM prognosis. A retrospective observational analysis found paracetamol beneficial in adult bacteraemic patients, and some experts recommend slow β-lactam infusion. We examined these treatments in a prospective, double-blind, placebo-controlled clinical trial. Patients and methods A retrospective analysis included 555 children treated for BM in 2004 in the infectious disease ward of the Paediatric Hospital of Luanda, Angola. Our prospective study randomised 723 children into four groups, to receive a combination of cefotaxime infusion or boluses every 6 hours for the first 24 hours and oral paracetamol or placebo for 48 hours. The primary endpoints were 1) death or severe neurological sequelae (SeNeSe), and 2) deafness. Results In the retrospective study, the mortality of children with blood transfusion was 23% (30 of 128) vs. without blood transfusion 39% (109 of 282; p=0.004). In the prospective study, 272 (38%) of the children died. Of those 451 surviving, 68 (15%) showed SeNeSe, and 12% (45 of 374) were deaf. Whereas no difference between treatment groups was observable in primary endpoints, the early mortality in the infusion-paracetamol group was lower, with the difference (Fisher s exact test) from the other groups at 24, 48, and 72 hours being significant (p=0.041, 0.0005, and 0.005, respectively). Prognostic factors for adverse outcomes were impaired consciousness, dyspnoea, seizures, delayed presentation, and absence of electricity at home (Simple Luanda Scale, SLS); the Bayesian Luanda Scale (BLS) also included abnormally low or high blood glucose. Conclusions New studies concerning the possible beneficial effect of blood transfusion, and concerning longer treatment with cefotaxime infusion and oral paracetamol, and a study to validate our simple prognostic scales are warranted.

The effects of chemicals in isopods : a multi-organizational evaluation

The global aim of this thesis was to evaluate and assess the effects of a pesticide (dimethoate) and a metal (nickel), as model chemicals, within different organization levels, starting at the detoxification pathways (enzymatic biomarkers) and energy costs associated (energy content quantification, energy consumption and CEA) along with the physiological alterations at the individual and population level (mortality), leading to a metabolomic analysis (using liquid 1H-NMR) and finally a gene expression analysis (transcriptome and RT-qPCR analysis). To better understand potential variations in response to stressors, abiotic factors were also assessed in terrestrial isopods such as temperature, soil moisture and UV radiation. The evaluation performed using biochemical biomarkers and energy related parameters showed that increases in temperature might negatively affect the organisms by generating oxidative stress. It also showed that this species is acclimated to environments with low soil moisture, and that in high moisture scenarios there was a short gap between the optimal and adverse conditions that led to increased mortality. As for UV-R, doses nowadays present have shown to induce significant negative impact on these organisms. The long-term exposure to dimethoate showed that besides the neurotoxicity resulting from acetylcholinesterase inhibition, this stressor also caused oxidative stress. This effect was observed for both concentrations used (recommended field dose application and a below EC50 value) and that its combination with different temperatures (20ºC and 25ºC) showed different response patterns. It was also observed that dimethoate’s degradation rate in soils was higher in the presence of isopods. In a similar study performed with nickel, oxidative stress was also observed. But, in the case of this stressor exposure, organisms showed a strategy where the energetic costs necessary for detoxification (biomarkers) seemed to be compensated by positive alterations in the energy related parameters. In this work we presented for the first time a metabolomic profile of terrestrial isopods exposed to stressors (dimethoate and niquel), since until the moment only a previous study was performed on a metabolomic evaluation in nonexposed isopods. In the first part of the study we identify 24 new metabolites that had not been described previously. On the second part of the study a metabolomic profile variation of abstract non-exposed organism throughout the exposure was presented and finally the metabolomic profile of organisms exposed to dimethoate and nickel. The exposure to nickel suggested alteration in growth, moult, haemocyanin and glutathione synthesis, energy pathways and in osmoregulation. As for the exposure to dimethoate alterations in osmoregulation, energy pathways, moult and neurotransmission were also suggested. In this work it was also presented the first full body transcriptome of a terrestrial isopod from the species Porcellionides pruinosus, which will complement the scarce information available for this group of organisms. This transcriptome also served as base for a RNA-Seq and a RT-qPCR analysis. The results of the RNA-Seq analysis performed in organisms exposed to nickel showed that this stressor negatively impacted at the genetic and epigenetic levels, in the trafficking, storage and elimination of metals, generates oxidative stress, inducing neurotoxicity and also affecting reproduction. These results were confirmed through RT-qPCR. As for the impact of dimethoate on these organisms it was only accessed through RT-qPCR and showed oxidative stress, an impact in neurotransmission, in epigenetic markers, DNA repair and cell cycle impairment. This study allowed the design of an Adverse Outcome Pathway draft that can be used further on for legislative purposes.

SFlt-1/PlGF ratio as a prognostic marker of adverse outcomes in women with early-onset preeclampsia

Soluble fms-like tyrosine kinase 1 (sFlt-1) is an anti-angiogenic factor released in higher amounts by preeclamptic placentas and it has been implicated in the endothelial dysfunction observed in the disease. In this study we evaluated if circulating sFlt-1/PlGF ratio is useful to predict adverse outcomes in women with early-onset preeclampsia. This is a cohort study of 88 preeclamptic women with singleton pregnancies at ≤35 weeks of gestation. According to definitions used, adverse outcomes occurred in 46.5% (N = 43) of the patients. The median sFlt1/PlGF ratio (25th-75th centile) for all patients evaluated was of 42.26 (13.1-226.1). The median sFlt-1/PlGF ratio among women who had any adverse outcome (N = 43) versus no adverse outcomes (N = 45) was of 227.6 (80.3-346.1) versus 14.4 (3.35-30.0), (P < 0.0001). According to our analyses a sFlt-1/PlGF ratio cut-point of ≥85 gave a sensitivity of 74.0% and specificity of 97.0%. The positive predictive value and the negative predictive value were 96.0% and 80.0%, respectively. The median sFlt-1/PlGF ratio (25th-75th centile) for patients who delivered within <7 days was 260.0 (127.7-404.7) as compared to 14.4 (3.35-34.97) for those patients who delivered within two weeks or more (P < 0.0001). Our results suggest that sFlt-1/PlGF ratio is a promising marker for adverse outcomes in women with early-onset preeclampsia. © 2013 International Society for the Study of Hypertension in Pregnancy. Published by Elsevier B.V. All rights reserved.

Predittori precoci di outcome neurologico nei neonati affetti da Encefalopatia Ipossico-Ischemica sottoposti a trattamento ipotermico. Studio combinato Elettroencefalogramma ad integrazione di ampiezza e Spettroscopia nel vicino infrarosso

Background: Brain cooling (BC) represents the elective treatment in asphyxiated newborns. Amplitude Integrated Electroencephalography (aEEG) and Near Infrared Spectroscopy (NIRS) monitoring may help to evaluate changes in cerebral electrical activity and cerebral hemodynamics during hypothermia. Objectives: To evaluate the prognostic value of aEEG time course and NIRS data in asphyxiated cooled infants. Methods: 12 term neonates admitted to our NICU with moderate-severe Hypoxic-Ischemic Encephalopathy (HIE) underwent selective BC. aEEG and NIRS monitoring were started as soon as possible and maintained during the whole hypothermic treatment. Follow-up was scheduled at regular intervals; adverse outcome was defined as death, cerebral palsy (CP) or global quotient < 88.7 at Griffiths’ Scale. Results: 2/12 infants died, 2 developed CP, 1 was normal at 6 months of age and then lost at follow-up and 7 showed a normal outcome at least at 1 year of age. The aEEG background pattern at 24 hours of life was abnormal in 10 newborns; only 4 of them developed an adverse outcome, whereas the 2 infants with a normal aEEG developed normally. In infants with adverse outcome NIRS showed a higher Tissue Oxygenation Index (TOI) than those with normal outcome (80.0±10.5% vs 66.9±7.0%, p=0.057; 79.7±9.4% vs 67.1±7.9%, p=0.034; 80.2±8.8% vs 71.6±5.9%, p=0.069 at 6, 12 and 24 hours of life, respectively). Conclusions: The aEEG background pattern at 24 hours of life loses its positive predictive value after BC implementation; TOI could be useful to predict early on infants that may benefit from other innovative therapies.