Validation of Humanized Mouse Antibodies

Antibody therapy is being developed and tested as one of the most promising agents for treatment of various human diseases. As of March 2016, 350 antibody candidates are in clinical trials. Many of these antibodies have been taken from animals and “humanized” by genetic modification. Our experiment tests monoclonal antibodies that have been harvested from mouse hybridoma (spleen-derived) cells and cloned until the heavy and light chains of the antibody can be recognized by human cells. Because of this “humanization” procedure, basic antibody assays are needed to demonstrate that the binding, specificity and functional parameters of the antibodies are not lost during cloning. The purpose of this research is to perform this validation through assays. The antibodies are harvested from cell supernatants and purified using affinity chromatography. Then, the antibody fractions are tested for reactivity with human target protein PTP-Beta, via western blot and ELISA procedures. Cross-reactivity of the antibody is tested against human eta and cynomolgus beta proteins. The work presented in this poster describes results from one particular mouse antibody, R15, which has been humanized to functionally enhance endothelial survival. The goal is to generate a therapeutic antibody candidate that improves endothelium survival and stability

Welcome to the Excerpts in Pharmacy Research Journal

The editorial board at Cedarville University School of Pharmacy welcomes you to the inaugural issue of the Excerpts in Pharmacy Research Journal (ISSN 2374-4693)

Monoclonal Antibody Activity in Human Umbilical Endothelial Cells That Possess Opposing Growth Factor Signaling Receptors

In various clinical settings such a peripheral vascular disease and diabetes, patients can develop leaky blood vessels that leads to the extravasation of fluid in surrounding tissues, mainly in the lower limbs, ultimately resulting in edema and compromised blood flow. In an attempt to maintain vascular integrity and stability researchers have tried to modulate two key receptors on endothelial cells, Vascular Endothelial Growth Factor Receptor-2 (VEGFR2) and tunica internal endothelial cell kinase 2 (Tie2) receptor using various approaches, including ligand administration and small molecule inhibition of kinase activity on the intracellular part of Tie-2. Various strategies for a therapy include monoclonal antibodies (Mabs) that influence the aforementioned pathways. The current poster describes a monoclonal antibody that binds a cell surface target protein and indirectly modulates the Tie-2 receptor activity

Are Cell Death Proteins/Antigens Found on Interdigital Cells Dying During Limb Development Expressed in a Simple Organism Such As Tetrahymena?

Numerous studies have been published that describe the genes and proteins that control cell death in various biological systems including normal embryonic development and in disease such as cancer. We describe attempts to look at a possible conserved cell death antigen in the simple organism Tetrahymena, using a unique monoclonal antibody that recognizes only dying cells in the chick limb. The main impetus for the research is to answer the question; does the cell death process have key proteins that exist in the dying process that can be modulated prior to the completion of the cell death process? Using various stimuli to induce cell death in tetrahymena thermophila including staurosporine, hypoxia and other know cell death modulators, we describe the preliminary methods used to verify that cells across two species may express conserved cell death proteins at certain times during the death process. The goal is to demonstrate that normal interdigit cell death is an ideal system for isolating programmed cell death antigens and provides a way to identify common mediators/markers in other model systems such as tetrahymena thermophila

Biological Implications of Hydroxyapatite Coatings on 3D Printed Titanium Implants

This study sought to determine the growth of and viability of osteoblast cells on hydroxyapatite coatings of 3D-printed titanium implants. The experiment used twenty 3D-printed titanium disks each of which had a determined surface roughness. These disks were printed by Tangible Solutions, LLC (Fairborn, OH) and then sonicated. Ten of the disks were coated with hydroxyapatite through the process of electrodeposition using an open cell and a three lead potentiostat. Using the hydroxyapatite coated titanium disks and uncoated disks, two four-day growth trials were performed. Two trials used five control disks (uncoated titanium disks) and five coated disks each, making an n of 10 for the total experiment. The disks were each placed into the wells of a culture plate and each disk was seeded with 15,000 human osteoblast cells. After four days in the incubator, the cells were removed using trypsin and the counted using the CytoSmart Automated Hemocytometer. The cell count from each disk as well as the viability of the cells from each disk were recorded. Means comparison was performed using Tukey-Kramer method of analysis. Results from the cell count portion of the experiment showed that the mean of the hydroxyapatite group was not significantly greater than the control group (p=0.83). In addition, cell viability of the hydroxyapatite group was also not significantly different than the control group (p=0.31). This data was unexpected but may be due to a change in the surface roughness between the two groups caused by the hydroxyapatite coating decreasing the surface roughness. The surface roughness selected for the experiment was chosen due to it being the most ideal for osteoblast growth, but any less rough surfaces were shown to be less ideal for osteoblast cell growth. Future Experiments will remove the variable of surface roughness

Development of a Novel Aspirin Suppository Formulation and Evaluation of the Acetylation of COX-1 Via a HT-29/Caco-2 Cell Absorption Assay Used to Detect the Absorption of Aspirin Formulated With Various Bases and Excipients

As the baby-boomer population ages, hospitalization rates will rise, increasing the number of patients who are NPO. Research indicates that aspirin use also increases with advanced age. With the increased prevalence of this demographic, there continues to be a growing need for alternative dosage forms for aspirin administration. A common and limited-risk alternative is rectally administered aspirin. However, there appears to be only one commercially available aspirin suppository and it has yielded erratic results as shown in previous research. Aspirin is considered a pro-drug; once it is inside the body, the acidic environment cleaves the aspirin molecule down to salicylic acid, its active form. Rectal cells may not provide an acidic environment needed to cleave the aspirin molecule into salicylic acid, thereby inhibiting the absorption and rate of onset of the drug. With this thought in mind and with the erratic results from the literature, the aim of this study, to be completed by summer of 2015, is to create a novel aspirin suppository. The study will be a prospective preclinical in-vitro design conducted in the Cedarville University Pharmaceutical Sciences lab. The samples will include two colonic adenocarcinoma cell lines, Caco-2 and HT-29. A standard curve will be developed as a baseline by using a 12(S)-HETE ELISA Assay using purified 12(S)-HETE. The two cell lines will be cultured, then incubated. Aspirin will be added to the samples and incubated again for 30 minutes. After incubation, medium samples will be taken and the same ELISA Assay will be performed on the results. The cell line that yields the most consistent results will be selected. The various aspirin formulations will be tested on this cell line in the same fashion. The ELISA assay will be performed and the concentration of 12(S)-HETE will be determined, plotted, and compared to the standard curve. A repeated-measures ANOVA will then be performed to analyze statistical significance

A Pharmacogenomic and Protein Analysis of Human Lacrimal Fluid in Varying Age Groups

Proteins are large biological molecules located within all cells. They are considered the basic functional components of cells that allow them to operate appropriately. Genes consist of both DNA and RNA, and are the cellular components that code for the proteins. A biomarker is any cellular component that is an indication of a biological state. Therefore, genetic and protein biomarkers are specific genes and proteins, respectively, present in cells that indicate a specific biological state of a cell. Identification of proteins and genetic biomarkers in relative quantities has been found to reflect various disease states and age groups in humans. Comparisons of possible techniques for collecting lacrimal fluids from human subjects which could potentially be utilized in the design of the study

Protein Analysis of Human Lacrimal Fluid in Varying Age Groups

Purpose: The objective of this research project was to identify proteins secreted from human lacrimal fluids onto the extra-ocular surface of the eye that could be later used to predict eye health, disease, and age-related changes. The identification of specific lacrimal proteins in relative quantities and patterns in younger versus older patients may reflect both ocular and extra-ocular disease states. Methods: This observational study collected samples of lacrimal fluid from 20 subjects between the ages of 18 and 25 years and 20 subjects over the age of 50 years with the use of Schirmer strips. The protein composition of these lacrimal fluid samples was then analyzed to determine specific proteins that evidenced unique patterns among the subject populations. Results: The protein concentrations between the two age groups (n = 40) was significantly higher in the younger patient group (1408.3 ug/mL versus 1152.5 ug/mL, p = 0.03). No consistent qualitative differences in the protein bands were observed between the two different patient age groups. However, excising and analyzing the outlying protein bands revealed unique proteins within the older patient group (aldehyde dehydrogenase and serotransferrin precursor). Preliminary attempts were made to determine the presence of proteins in lacrimal fluid that may originate from cells lining the ducts and blood vessels associated with the ocular environment. Conclusion: These preliminary results in age related differences in eye lacrimal fluid will contribute to future research endeavors in order to determine which specific proteins were increased or decreased quantitatively in the younger population, if any, and what role they might have in eye health, disease, and age-related changes

The Different Artificial Sweeteners and Their Effects on Endothelial Cell/Blood Vessel Health: Possible Implications for Ringing in the Ear

Abstract: Background: Tinnitus, a condition whose remarkable symptom is ringing in the ear (RIE), is a problem plaguing people all around the world in varying degrees of severity, though it is most common and severe in older populations. Literature is lacking on its etiology. Therefore, it is difficult to diagnose and treat. Several possible components could play a role in the development of tinnitus including neurological, physiological, traumatic, dietary and vascular factors. No factor has yet been definitively linked to the development of tinnitus. Vascular health can be significantly impacted by diet- especially in regard to sugars. As artificial sweeteners are used widely in the American diet, they may play a significant role in vascular health. Objective: This project aims to investigate a possible connection between artificial sugars’ impact on vascular health and complaints of RIE among age groups through a patient survey and laboratory experiments. Methods: A survey assessing individuals’ demographic information, subjective severity of RIE and reported artificial sweetener consumption will be distributed to audiologists’ offices in major Ohio cities, via Qualtrics, where patients will complete them. Data collected will be analyzed for interrelationships among sugar intake, age and severity of RIE. In the laboratory, an ELISA assay will evaluate the effects of artificial sweeteners on endothelial cells- the same cells that comprise blood vessels- through quantifying stimulation of the Tie-2 survival and angiogenesis pathway via the cellular messenger pAKT

3D Printed PLA Scaffolds to Promote Healing of Large Bone Defects

One challenge modern medicine faces is the ability to repair large bone defects and stimulate healing. Small defects typically heal naturally, but large bone defects do not and current solutions are to replace the missing tissue with biologically inert materials such as titanium. This limits the amount of bone healing as the defect is not repaired but rather replaced. The focus of our research is to develop a method of using 3D printing to create biodegradable scaffolds which promote bone in-growth and replacement. To accomplish this we used poly lactic acid (PLA) filament and a desktop 3D printer. To promote bone healing and provide mechanical support our team investigated different design methodologies to provide a scaffold of customizable stiffness while allowing cell attachment and in-growth. Our team used CAD modeling to create unique architecture design systems which we analyzed for stiffness using Finite Element Analysis (FEA). We developed a unit cell method of scaffold construction that allowed for customized stiffness of irregular shapes. We 3D printed our designs using a desktop 3D printer and verified our stiffness through mechanical tension and compression testing. We investigated cell viability of the scaffolds by immersing test specimens in culturing media and fibroblast cells. Fibroblast cells are from the same lineage as osteoblast cells but are much faster growing, allowing for more efficient testing. Specimens were left in the media for one week then a total cell count was performed. Scaffold designs were then evaluated based on stiffness and cell viability. We have produced several different viable models with appropriate stiffness for human trabecular bone and good cellular adhesion