Fruit and vegetable derived polyphenols have been linked with many health benefits. In light of this, many consumers are seeking to increase their intake of polyphenols, with many turning to dietary supplements that contain concentrated doses of purified polyphenols. However, the safety of this consumption modality is not known, nor are the dose-dependent metabolic changes that may be present, especially when considering colonically generated phenolic metabolites. Using blueberry polyphenols as a model, we explored these phenomena in a rat model. Animals were dosed with blueberry polyphenols at levels up to 20 times what would be consumed in 1-2 servings of whole blueberries in an adult human. In the first study, animals were acutely dosed with blueberry polyphenols and urine and plasma pharmacokinetics measured. In the second study, animals were repeatedly dosed for 90d, with urinary metabolites monitored throughout the study and a complete necropsy performed following standard guidelines. In both studies, metabolite excretion patterns were similar: cinnamic acids accounted for a majority of the observed metabolites, followed by hippuric acids and then phenylpropionic acids (PPA). A dose-dependent shift in metabolite production was observed; as the dose increased, the relative amounts of PPA increased while hippuric acids decreased. No adverse or toxic effects were found, and, though there were several statistically significant differences in toxicological endpoints, all measured parameters remained in the normal range for these animals and thus were not deemed biologically significant. These results indicate that high doses of blueberry polyphenols, as may be present in dietary supplements, are safe for consumption. These results also demonstrate dose-dependent shifts in metabolism that may impact gut function and affect the health benefits derived from blueberry polyphenols.<br>

There is good pharmacological evidence that cannabinoids caused cellular changes by interacting with specific cannabinoid receptors (CBR) (Klein et al., 2000). To date, two CBRs have been identified in the human body, designated Cannabinoid Receptor 1 (CB1) and Cannabinoid Receptor 2 (CB2) (Begg et al., 2005). Endogenously occurring compounds with action at the CBRs also exist and they are called endocannabinoids. One of the four known endocannabinoids is anandamide (AEA). The endocannabinoid system, present in the human body, plays a significant role in altering the physiology of the immune system. Enhancement of this system’s anti-inflammatory effect could possibly present a vital therapeutic target for central and peripheral inflammatory disorders. A number of synthetic CB1 or CB2 specific antagonists have been developed including the highly specific CB1 receptor antagonist/reverse agonist named Rimonabant/ SR141716A. SR compounds are considered unique because these compounds not only inhibit the binding and function of cannabimimetic agents, but also act as inverse agonists. Activation of CB1 receptors produces inappropriate CNS side effects including psychoactivity, dependence and sedation (Clayton et al., 2002) whereas CB1 receptor antagonists/inverse agonists avoid or prevent these side effects. Taking the above information into consideration, Rimonabant has the potential to offer an effective long term treatment of chronic inflammatory disorders without the serious side effects of commonly used treatments. The main aim of this study is to investigate the in vitro effects of Rimonabant alone and in combination with anandamide on inflammatory associated cytokine production by human umbilical vein endothelial cells (HUVEC) and macrophage cultures. After careful consideration of the evidence stating that endothelial cells produce several important molecules vital to the inflammatory response of the body and the confirmation that CB1 receptor mRNA is generally present in endothelial cells, the use of HUVEC was deemed to be satisfactory for this study. The first phase of the study was dedicated to establishing the technique to isolate HUVEC from fresh human umbilical cord within the local laboratory and to maintain these in culture for further use during experimental procedures to test the effects of CB1 ligands. The isolation procedure, trypsinising, freezing away and thawing methods used during this experiment produced healthy HUVEC in sufficient numbers for further use. The next step was to determine the maximum in vitro concentrations at which Rimonabant and anandamide had insignificant cytotoxic effect on selected human cells and in doing so, determine suitable concentrations for further experimentation. Both compounds had a dose related anti-proliferative response when tested on HUVEC. The same dose related response was observed during the Rimonabant exposure to human lymphocytes, but no decrease in lymphocyte viability was observed when treated with anandamide at the concentrations tested. It is evident from the results that there was an almost ten times difference in the IC50 value of the two different products (14.3 μM for Rimonabant and 124.2 μM for anandamide) which was statistically significant. Flow cytometry was used to determine the effects of Rimonabant and anandamide on the surface expression of the CR3 complement receptor by human neutrophils. Neither Rimonabant nor anandamide significantly affect CR3 expression on the surface of freshly isolated human neutrophils and would exclude the CR3 expression pathways as a potential mechanism of action for the anti-inflammatory effects of these compounds. The in vitro effect of Rimonabant and anandamide alone and in combination on the production of cytokines by human macrophages and by HUVEC was determined. Anandamide was shown to inhibit the production of all the detectable cytokines (IL-8, IL-1β and IL-6 in both cell types and IL-10 and TNF-α in macrophages). Furthermore this inhibitory effect was attenuated by pre-treatment Rimonabant. These results would suggest that anandamide could induce anti-inflammatory effects observed in macrophages and HUVEC, through cannabinoid receptors. Rimonabant also inhibited the production of all the detectable cytokines following treatment with 0.5 μM and 3 μM respectively. The anti-inflammatory effects of anandamide were attenuated when combined with 1 μM of Rimonabant. Throughout the various cytokine responses, the dose-response relationship appeared to follow a bell-shaped dose-response. This occurrence proposes that Rimonabant displaces anandamide and blocks the anti-inflammatory effects of the agonist. Flow cytometry was used to determine the effects of Rimonabant and anandamide alone and in combination on the extracellular surface expression of ICAM-1 by HUVEC. Neither Rimonabant nor anandamide had any significant inhibitory effect on the expression of ICAM-1 by HUVEC. Considering the low levels for ICAM-1 expressed by the HUVEC during this experiment and the literature supporting more effective methods of activating the ICAM-1 gene and subsequent up-regulation of ICAM-1 proteins, TNF-α stimulation of HUVEC might produce a different result compared to IL-1β stimulation. The final phase of the project was to determine the effects of Rimonabant and anandamide on the adhesion of human neutrophils to HUVEC. There was no significant difference with relation to the neutrophil adhesion to HUVEC following the treatment with various combination concentrations of the compounds, and also no significant effect following treatment with either test compound individually. Although a specific mechanism of action for Rimonabant could not be uncovered during this study, there is evidence that several possible mechanisms can be excluded. The results support observations made by other researchers and the hypothesis that Rimonabant has anti-inflammatory effects. The results provide motivation for further experimentation to better understand these anti-inflammatory actions of Rimonabant. / Dissertation (MSc)--University of Pretoria, 2012. / Pharmacology / unrestricted

In the investigation of radiation accidents, it is of great importance to estimate the dose absorbed by exposed persons in order to plan their therapy. Although occasionally in these situations physical dose measurements are possible, most often biological methods are required for dose estimation. The aim of this investigation was to assess the suitability of the cytokinesis blocked (CB) micronucleus assay as a biodosimetric method using lymphocytes irradiated in vivo. The approach adopted to achieve this was to estimate whole body doses by relating micronuclei yields in patients undergoing radiotherapy treatment with an in vitro radiation dose-response curve. These biologically derived estimates were then compared with the corresponding doses obtained by physical measurement and calculation. As a first approach a study was performed of the in vitro dose-response of gamma-ray induced micronuclei following cytokinesis-block in the lymphocytes of peripheral blood samples obtained from 4 healthy donors. The results indicated that the distribution of the induced micronuclei were overdispersed. Furthermore, a linear dose-response relationship was established when a curve was fitted to the data by an iteratively reweighted least squares method. By means of an analysis of covariance it was demonstrated that this result is in agreement with the dose-response relationships found by various other workers (Fenech et al., 1985; Fenech et al., 1986; Fenech et al., 1989; Balasem et al., 1992, and Slabbert, 1993). To assess the suitability and accuracy of dose assessment using the CB micronucleus assay for in vivo exposure of lymphocytes, blood samples obtained from 8 patients undergoing radiotherapy before, during and after treatment were examined. The physical doses of these patients were determined according to conventional radiation treatment plans and cumulative dose-volume histograms. The dose-volume histograms permitted calculation of integral doses and subsequently the estimate of equivalent whole-body doses. The results of the CB micronucleus assay applied to peripheral blood lymphocytes of 6 patients undergoing fractionated partial-body irradiation showed a dose-related increase in micronucleus frequency in each of the patients studied. This demonstrated that micronuclei analysis may serve as a quantitative biological measure of such exposures. The pooled data of these patients compared to the pooled data of the healthy donors show that there was no statistically significant difference between in vitro and in vivo results, however a slightly lower induced micronuclei frequency was observed after in vivo exposure. When the biological dose estimates for equivalent whole-body doses obtained from the in vitro dose response curve were compared with calculated physical doses, it was found that: biologically estimated dose = 0.936 physical dose. However, there was inadequate statistical evidence to discard the hypothesis that the gradient of the equation was equal to one. Therefore, the analysis of micronuclei induced in lymphocytes in vivo yields highly quantitative information on the equivalent whole-body dose. The negative binomial method was used for analysing the micronucleus data from two patients who received single, relatively larger tumour doses of 10 Gy each, with the objective to obtain estimates of the exposed body fraction and the dose to this fraction. The dose estimates to the irradiated volume were found to be within 30% of the physical tumour dose. The irradiated volume estimates seemed to be higher than the physically calculated volumes but by discarding the correction for the loss of cells due to interphase death the agreement was good between the physically and biologically determined integral doses. This study has revealed that the CB micronucleus assay appears to offer a reliable, consistent and relatively rapid biological method of whole body dose estimation. It is recognised that further corroborative work using the techniques described in this thesis is required for estimating localized exposure.

In adaptive design clinical trials, an endpoint at the final analysis that takes a long time to observe is not feasible to be used for making decisions at the interim analysis. For example, overall survival (OS) in oncology trials usually cannot be used to make interim decisions. However, biomarkers correlated to the final clinical endpoint can be used. Hence, considerable interest has been drawn towards the biomarker informed adaptive clinical trial designs. Shun et al. (2008) proposed a "biomarker informed two-stage winner design" with 2 active treatment arms and a control arm, and proposed a normal approximation method to preserve type I error. However, their method cannot be extended to designs with more than 2 active treatment arms. In this dissertation, we propose a novel statistical approach for biomarker informed two-stage winner design that can accommodate multiple active arms and control type I error. We further propose another biomarker informed adaptive design called "biomarker informed add-arm design for unimodal response". This design utilizes existing knowledge about the shape of dose-response relationship to optimize the procedure of selecting best candidate treatment for a larger trial. The key element of the proposed design is that, some inferior treatments do not need to be explored and the design is shown to be more efficient than biomarker informed two-stage winner design mathematically. Another important component in the study of biomarker informed adaptive designs is to model the relationship between the two endpoints. The conventional approach uses a one-level correlation model, which might be inappropriate if there is no solid historical knowledge of the two endpoints. A two-level correlation model is developed in this dissertation. In the new model a new variable that describes the mean level correlation is developed, so that the uncertainty of the historical knowledge could be more accurately reflected. We use this new model to study the "biomarker informed two-stage winner design" and the "biomarker informed add-arm design for unimodal response". We show the new proposed model performs better than conventional model via simulations. The concordance of inference based on biomarker and primary endpoint is further studied in a real case.

Indiana University-Purdue University Indianapolis (IUPUI) / A semi-parametric approach incorporates parametric and nonparametric functions in the model and is very useful in situations when a fully parametric model is inadequate. The objective of this dissertation is to extend statistical methodology employing the semi-parametric modeling approach to analyze data in health science research areas. This dissertation has three parts. The first part discusses the modeling of the dose-response relationship with correlated data by introducing overall drug effects in addition to the deviation of each subject-specific curve from the population average. Here, a penalized spline regression method that allows modeling of the smooth dose-response relationship is applied to data in studies monitoring malaria drug resistance through the ex-vivo assays.The second part of the dissertation extends the SiZer map, which is an exploratory and a powerful visualization tool, to detect underlying significant features (increase, decrease, or no change) of the curve at various smoothing levels. Here, Penalized Spline Significant Zero Crossings of Derivatives (PS-SiZer), using a penalized spline regression, is introduced to investigate significant features in correlated data arising from longitudinal settings. The third part of the dissertation applies the proposed PS-SiZer methodology to analyze HIV data. The durability of significant weight change over a period is explored from the PS-SiZer visualization. PS-SiZer is a graphical tool for exploring structures in curves by mapping areas where rate of change is significantly increasing, decreasing, or does not change. PS-SiZer maps provide information about the significant rate of weigh change that occurs in two ART regimens at various level of smoothing. A penalized spline regression model at an optimum smoothing level is applied to obtain an estimated first-time point where weight no longer increases for different treatment regimens.

Neuronal nicotinic acetylcholine receptors (nAChRs) are expressed in both the periperhal and central nervous systems, and are involved in pre-, post-, and non-synaptic control of neuronal activation. In the brain, these receptors play an important role in a variety of physiological processes such as cognition, development, learning, and memory formation. Malfunction of these receptors have been implicated in neurodegenerative diseases like Alzheimer's disease (AD), schizophrenia, and Parkinson's disease. To date, 17 different nAChR subunits, including α2-α7 and β2-β4, have been cloned that can form homo- and/or hetero-pentameric ionotropic receptors. The unique combinations of subunit pentamers manifest in distinct functional receptors. Using single-cell real-time quantitative RT-PCR, we identified the individual expression rates and co-expression rates of the different nAChR subunits in rat CA1 hippocampal interneurons in efforts to characterize functional receptors involved in learning and memory. The two-way combination of subunits with highest expression in hippocampal interneurons was α3β2. Moreover, this combination was expressed in ratios near 1:3 or 3:1 α3 to β2 respectively. To investigate the functionality of α3β2 receptors in different stoichiometries, we injected human α3 and rat β2 subunit mRNA in 1:3, 1:1, and 3:1 ratios into Xenopus laevis oocytes for expression. Two-electrode voltage clamp was then performed with the application of different concentrations of ACh to produce full dose-response curves and channel kinetics data. Distinct α3β2 functional channels were identified from the different expression ratios based on significant differences in channel kinetics (i.e.- peak current rise times, peak current decay times, steady state current in forced desensitization) Dose-response curves produced no significant difference in EC50 values in the different expression groups. However, there was a trend to greater agonist sensitivity with increased α3 expression relative to β2. α3β2 receptors were further characterized through forced desensitization of the receptors and generation of IV plots. The findings from this study elucidate the neuronal nAChR subunit combinations that form functional channels in hippocampal interneurons.

Academic learning time (ALT) has long had the theoretical underpinnings sufficient to claim a causal relationship with academic achievement, but to this point empirical evidence has been lacking. This dearth of evidence has existed primarily due to difficulties associated with operationalizing ALT in traditional educational settings. Recent advancements in computer-based instruction provide an unprecedented opportunity to model ALT and to test the underlying theory. A widely-used computer-based early-reading curriculum was operationalized using Berliner's model of ALT (Berliner, 1991). This curriculum was then mapped to a computer-based assessment to determine an appropriate method of quantifying early-reading ALT. Software limitations required that ALT be quantified as a summative measure. Data were collected from 1,347 prekindergarteners and were analyzed using a dose-response approach that associated usage of the curriculum with a generalized variable of early-reading achievement. Gains across four early-reading skills were demonstrated via linear regression to be predicted by minutes of usage (Adj. R2 = .078). A sample optimized to test the hypothesis showed a stronger correlation (Adj. R2 = .096). Time spent using the Free Play version of the curriculum did not uniquely predict additional variance. Similarly, gains on reading skills that were not taught explicitly by the curriculum were not predicted by overall usage. These three results were interpreted as supporting the ALT learning model. Post-hoc analyses were performed on curriculum-usage compliance and on within-curriculum progress, both of which were statistically significant when added to the basic dose-response model. Multiple exploratory best-fit models were constructed. The strongest accounted for just under 20% of the overall variance (Adj. R2 = .186). Effect sizes were in the medium-to-large range for the entire sample (D = 0.71) with significant improvement for the optimized sample (D = 1.26). Children in the optimized sample who used the program over 20% more than recommended had even stronger gains (D = 1.67). The ability to remotely and accurately quantify interaction with a computer-based curriculum and assessment in the home defines a new vista in ALT research.

Hormesis is a nonlinear dose-response characterized by biological responses at low doses that are opposite to those observed at higher doses. Studies and review articles on hormesis are being published at an increasing rate by researchers from diverse disciplines and debate has emerged over the role hormesis in risk assessment. As a result, a survey was conducted to assess toxicologists and risk assessors knowledge and attitudes about the hormesis dose response. Study goals were to: 1) ascertain attitudes towards hormesis and other dose-response models, 2) identify whether acceptance or rejection of hormesis is based on knowledge of hormesis, predisposing values, or demographic characteristics, and 3) evaluate potential for response bias. The survey consisted of 44 questions pre-tested by 25 toxicologists and risk assessors. The survey was distributed via email to the membership of the Society of Toxicology and the Society for Risk Analysis, 9,500 potential respondents. The overall response rate was 17% (n= 1,463) with a completion rate over 87%. Major findings were that 50% of respondents indicated sufficient data exist to support the view hormesis occurs across a wide range of species and endpoints, 59% indicated evaluating potential benefits due to hormesis should be included in risk assessments, and 65% are in favor of modifying hazard assessment protocols to identify the presence of hormesis. Respondent characteristics such as: years of experience, society membership, education, residence, employment (excluding government and pharmaceutical companies), and political, economic or social views had little influence on opinion. One of the largest positive influences was experience with hormesis based on actual research; 79% of subjects who reported observing hormesis commonly in their studies agreed hormesis is broadly generalizable. The influence of non-response bias was evaluated through several internal and external measures. Despite a lower than hoped for response rate, but because of robust external validity measures, it is concluded that respondents’ opinions are likely a reasonable representation of the societies of which they are members. Because this is a baseline survey, a follow-up survey is in order. Future survey design should separately evaluate the science of dose-response from the regulatory approach to risk assessment.

Objective: Diet lifestyle can influence the risk of endometriosis. Therefore, we conducted a systematicmeta-analysis to investigate the association between dairy products and the risk of endometriosis. Besides, we performed a dose-responsemeta-analysis to evaluate the amount of dairy intake affecting the risk of endometriosis. Methods: Relevant studies were searched from Pubmed, Embase databases, Cochrane Library, and Web of Science from the inception to November 6th, 2020. Also, the dose-response meta-analysis was conducted. All the pooled results were performed by risk ratios (RRs). Results: Finally, seven high-quality studies were included in the present meta-analysis. Total dairy intake was inversely associated with the risk of endometriosis, and the risk of endometriosis tended to decrease with a decrease in the risk of endometriosis when dairy products intake was over 21 servings/week (RR 0.87, 95% CI 0.76–1.00; pnon−linearity = 0.04). Similarly, people who consumed more than 18 servings of high-fat dairy products per week had a reduced risk of endometriosis (RR 0.86, 95% CI 0.76–0.96). When stratified-analyses were conducted based on specific dairy product categories, it indicated that people with high cheese intake might have a reduced risk of endometriosis (RR 0.86, 95%CI 0.74–1.00). Other specific dairy products such as whole milk (RR 0.90, 95% CI 0.72–1.12), reduced-fat/skim milk (RR 0.83, 95% CI 0.50–1.73), ice cream (RR 0.83, 95% CI 0.50–1.73), and yogurt (RR 0.83, 95% CI 0.62–1.11) have not shown significant evidence of an association with the risk of endometriosis. However, there is a higher risk of endometriosis in the females with high butter intake compared to females with low butter intake (1.27, 95% CI 1.03–1.55). Conclusions: Overall, dairy products intake was associated with a reduction in endometriosis, with significant effects when the average daily intake 3 servings. When analyzed according to the specific type of dairy product, it was shown that females with higher high-fat dairy and cheese intake might have a reduced risk of endometriosis. However, high butter intake might be associated to the increased risk of endometriosis. More future studies are needed to validate and add to this finding.

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