Evaluating intra-articular drug delivery for the treatment of osteoarthritis in a rat model.

Osteoarthritis (OA) is a degenerative joint disease that can result in joint pain, loss of joint function, and deleterious effects on activity levels and lifestyle habits. Current therapies for OA are largely aimed at symptomatic relief and may have limited effects on the underlying cascade of joint degradation. Local drug delivery strategies may provide for the development of more successful OA treatment outcomes that have potential to reduce local joint inflammation, reduce joint destruction, offer pain relief, and restore patient activity levels and joint function. As increasing interest turns toward intra-articular drug delivery routes, parallel interest has emerged in evaluating drug biodistribution, safety, and efficacy in preclinical models. Rodent models provide major advantages for the development of drug delivery strategies, chiefly because of lower cost, successful replication of human OA-like characteristics, rapid disease development, and small joint volumes that enable use of lower total drug amounts during protocol development. These models, however, also offer the potential to investigate the therapeutic effects of local drug therapy on animal behavior, including pain sensitivity thresholds and locomotion characteristics. Herein, we describe a translational paradigm for the evaluation of an intra-articular drug delivery strategy in a rat OA model. This model, a rat interleukin-1beta overexpression model, offers the ability to evaluate anti-interleukin-1 therapeutics for drug biodistribution, activity, and safety as well as the therapeutic relief of disease symptoms. Once the action against interleukin-1 is confirmed in vivo, the newly developed anti-inflammatory drug can be evaluated for evidence of disease-modifying effects in more complex preclinical models.

Functional Evaluation of Causal Mutations Identified in Human Genetic Studies

Human genetics has been experiencing a wave of genetic discoveries thanks to the development of several technologies, such as genome-wide association studies (GWAS), whole-exome sequencing, and whole genome sequencing. Despite the massive genetic discoveries of new variants associated with human diseases, several key challenges emerge following the genetic discovery. GWAS is known to be good at identifying the locus associated with the patient phenotype. However, the actually causal variants responsible for the phenotype are often elusive. Another challenge in human genetics is that even the causal mutations are already known, the underlying biological effect might remain largely ambiguous. Functional evaluation plays a key role to solve these key challenges in human genetics both to identify causal variants responsible for the phenotype, and to further develop the biological insights from the disease-causing mutations.

We adopted various methods to characterize the effects of variants identified in human genetic studies, including patient genetic and phenotypic data, RNA chemistry, molecular biology, virology, and multi-electrode array and primary neuronal culture systems. Chapter 1 is a broader introduction for the motivation and challenges for functional evaluation in human genetic studies, and the background of several genetics discoveries, such as hepatitis C treatment response, in which we performed functional characterization.

Chapter 2 focuses on the characterization of causal variants following the GWAS study for hepatitis C treatment response. We characterized a non-coding SNP (rs4803217) of IL28B (IFNL3) in high linkage disequilibrium (LD) with the discovery SNP identified in the GWAS. In this chapter, we used inter-disciplinary approaches to characterize rs4803217 on RNA structure, disease association, and protein translation.

Chapter 3 describes another avenue of functional characterization following GWAS focusing on the novel transcripts and proteins identified near the IL28B (IFNL3) locus. It has been recently speculated that this novel protein, which was named IFNL4, may affect the HCV treatment response and clearance. In this chapter, we used molecular biology, virology, and patient genetic and phenotypic data to further characterize and understand the biology of IFNL4. The efforts in chapter 2 and 3 provided new insights to the candidate causal variant(s) responsible for the GWAS for HCV treatment response, however, more evidence is still required to make claims for the exact causal roles of these variants for the GWAS association.

Chapter 4 aims to characterize a mutation already known to cause a disease (seizure) in a mouse model. We demonstrate the potential use of multi-electrode array (MEA) system for the functional characterization and drug testing on mutations found in neurological diseases, such as seizure. Functional characterization in neurological diseases is relatively challenging and available systematic tools are relatively limited. This chapter shows an exploratory research and example to establish a system for the broader use for functional characterization and translational opportunities for mutations found in neurological diseases.

Overall, this dissertation spans a range of challenges of functional evaluations in human genetics. It is expected that the functional characterization to understand human mutations will become more central in human genetics, because there are still many biological questions remaining to be answered after the explosion of human genetic discoveries. The recent advance in several technologies, including genome editing and pluripotent stem cells, is also expected to make new tools available for functional studies in human diseases.

Real-Time Decision Making and Aggressive Behavior in Youth: A Heuristic Model of Response Evaluation and Decision (RED).

Considerable scientific and intervention attention has been paid to judgment and decision-making systems associated with aggressive behavior in youth. However, most empirical studies have investigated social-cognitive correlates of stable child and adolescent aggressiveness, and less is known about real-time decision making to engage in aggressive behavior. A model of real-time decision making must incorporate both impulsive actions and rational thought. The present paper advances a process model (response evaluation and decision; RED) of real-time behavioral judgments and decision making in aggressive youths with mathematic representations that may be used to quantify response strength. These components are a heuristic to describe decision making, though it is doubtful that individuals always mentally complete these steps. RED represents an organization of social-cognitive operations believed to be active during the response decision step of social information processing. The model posits that RED processes can be circumvented through impulsive responding. This article provides a description and integration of thoughtful, rational decision making and nonrational impulsivity in aggressive behavioral interactions.

Emergent group level navigation: an agent-based evaluation of movement patterns in a folivorous primate.

The foraging activity of many organisms reveal strategic movement patterns, showing efficient use of spatially distributed resources. The underlying mechanisms behind these movement patterns, such as the use of spatial memory, are topics of considerable debate. To augment existing evidence of spatial memory use in primates, we generated movement patterns from simulated primate agents with simple sensory and behavioral capabilities. We developed agents representing various hypotheses of memory use, and compared the movement patterns of simulated groups to those of an observed group of red colobus monkeys (Procolobus rufomitratus), testing for: the effects of memory type (Euclidian or landmark based), amount of memory retention, and the effects of social rules in making foraging choices at the scale of the group (independent or leader led). Our results indicate that red colobus movement patterns fit best with simulated groups that have landmark based memory and a follow the leader foraging strategy. Comparisons between simulated agents revealed that social rules had the greatest impact on a group's step length, whereas the type of memory had the highest impact on a group's path tortuosity and cohesion. Using simulation studies as experimental trials to test theories of spatial memory use allows the development of insight into the behavioral mechanisms behind animal movement, developing case-specific results, as well as general results informing how changes to perception and behavior influence movement patterns.

Evaluation of an epithelial plasticity biomarker panel in men with localized prostate cancer.

BACKGROUND: Given the potential importance of epithelial plasticity (EP) to cancer metastasis, we sought to investigate biomarkers related to EP in men with localized prostate cancer (PC) for the association with time to PSA recurrence and other clinical outcomes after surgery. METHODS: Men with localized PC treated with radical prostatectomy at the Durham VA Medical Center and whose prostatectomy tissues were included in a tissue microarray (TMA) linked to long-term outcomes. We performed immunohistochemical studies using validated antibodies against E-cadherin and Ki-67 and mesenchymal biomarkers including N-cadherin, vimentin, SNAIL, ZEB1 and TWIST. Association studies were conducted for each biomarker with baseline clinical/pathologic characteristics an risk of PSA recurrence over time. RESULTS: Two hundred and five men contributed TMA tissue and had long-term follow-up (median 11 years). Forty-three percent had PSA recurrence; three died of PC. The majority had high E-cadherin expression (86%); 14% had low/absent E-cadherin expression. N-cadherin was rarely expressed (<4%) and we were unable to identify an E-to-N-cadherin switch as independently prognostic. No associations with clinical risk group, PSA recurrence or Gleason sum were noted for SNAIL, ZEB1, vimentin or TWIST, despite heterogeneous expression between patients. We observed an association of higher Ki-67 expression with Gleason sum (P=0.043), National Comprehensive Cancer Network risk (P=0.013) and PSA recurrence (hazard ratio 1.07, P=0.016). CONCLUSIONS: The expression of EP biomarkers in this cohort of men with a low risk of PC-specific mortality was not associated with aggressive features or PSA relapse after surgery.

Radiolabeled inhibitors as probes for imaging mutant IDH1 expression in gliomas: Synthesis and preliminary evaluation of labeled butyl-phenyl sulfonamide analogs.

INTRODUCTION: Malignant gliomas frequently harbor mutations in the isocitrate dehydrogenase 1 (IDH1) gene. Studies suggest that IDH mutation contributes to tumor pathogenesis through mechanisms that are mediated by the neomorphic metabolite of the mutant IDH1 enzyme, 2-hydroxyglutarate (2-HG). The aim of this work was to synthesize and evaluate radiolabeled compounds that bind to the mutant IDH1 enzyme with the goal of enabling noninvasive imaging of mutant IDH1 expression in gliomas by positron emission tomography (PET). METHODS: A small library of nonradioactive analogs were designed and synthesized based on the chemical structure of reported butyl-phenyl sulfonamide inhibitors of mutant IDH1. Enzyme inhibition assays were conducted using purified mutant IDH1 enzyme, IDH1-R132H, to determine the IC50 and the maximal inhibitory efficiency of the synthesized compounds. Selected compounds, 1 and 4, were labeled with radioiodine ((125)I) and/or (18)F using bromo- and phenol precursors, respectively. In vivo behavior of the labeled inhibitors was studied by conducting tissue distribution studies with [(125)I]1 in normal mice. Cell uptake studies were conducted using an isogenic astrocytoma cell line that carried a native IDH1-R132H mutation to evaluate the potential uptake of the labeled inhibitors in IDH1-mutated tumor cells. RESULTS: Enzyme inhibition assays showed good inhibitory potency for compounds that have iodine or a fluoroethoxy substituent at the ortho position of the phenyl ring in compounds 1 and 4 with IC50 values of 1.7 μM and 2.3 μM, respectively. Compounds 1 and 4 inhibited mutant IDH1 activity and decreased the production of 2-HG in an IDH1-mutated astrocytoma cell line. Radiolabeling of 1 and 4 was achieved with an average radiochemical yield of 56.6 ± 20.1% for [(125)I]1 (n = 4) and 67.5 ± 6.6% for [(18)F]4 (n = 3). [(125)I]1 exhibited favorable biodistribution characteristics in normal mice, with rapid clearance from the blood and elimination via the hepatobiliary system by 4 h after injection. The uptake of [(125)I]1 in tumor cells positive for IDH1-R132H was significantly higher compared to isogenic WT-IDH1 controls, with a maximal uptake ratio of 1.67 at 3 h post injection. Co-incubation of the labeled inhibitors with the corresponding nonradioactive analogs, and decreasing the normal concentrations of FBS (10%) in the incubation media substantially increased the uptake of the labeled inhibitors in both the IDH1-mutant and WT-IDH1 tumor cell lines, suggesting significant non-specific binding of the synthesized labeled butyl-phenyl sulfonamide inhibitors. CONCLUSIONS: These data demonstrate the feasibility of developing radiolabeled probes for the mutant IDH1 enzyme based on enzyme inhibitors. Further optimization of the labeled inhibitors by modifying the chemical structure to decrease the lipophilicity and to increase potency may yield compounds with improved characteristics as probes for imaging mutant IDH1 expression in tumors.