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Investigations of Filarial Nematode Motility, Response to Drug Treatment, and Pathology Western Michigan University ScholarWorks at WMU Dissertations Graduate College 8-2015 Investigations of Filarial Nematode Motility, Response to Drug Treatment, and Pathology Charles Nutting Western Michigan University, [email protected] Follow this and additional works at: https://scholarworks.wmich.edu/dissertations Part of the Biochemistry Commons, Biology Commons, and the Pathogenic Microbiology Commons Recommended Citation Nutting, Charles, "Investigations of Filarial Nematode Motility, Response to Drug Treatment, and Pathology" (2015). Dissertations. 745. https://scholarworks.wmich.edu/dissertations/745 This Dissertation-Open Access is brought to you for free and open access by the Graduate College at ScholarWorks at WMU. It has been accepted for inclusion in Dissertations by an authorized administrator of ScholarWorks at WMU. For more information, please contact [email protected]. INVESTIGATIONS OF FILARIAL NEMATODE MOTILITY, RESPONSE TO DRUG TREATMENT, AND PATHOLOGY by Charles S. Nutting A dissertation submitted to the Graduate College in partial fulfillment of the requirements for the degree of Doctor of Philosophy Biological Sciences Western Michigan University August 2015 Doctoral Committee: Rob Eversole, Ph.D., Chair Charles Mackenzie, Ph.D. Pamela Hoppe, Ph.D. Charles Ide, Ph.D. INVESTIGATIONS OF FILARIAL NEMATODE MOTILITY, RESPONSE TO DRUG TREATMENT, AND PATHOLOGY Charles S. Nutting, Ph.D. Western Michigan University, 2015 More than a billion people live at risk of chronic diseases caused by parasitic filarial nematodes. These diseases: lymphatic filariasis, onchocerciasis, and loaisis cause significant morbidity, degrading the health, quality of life, and economic productivity of those who suffer from them. Though treatable, there is no cure to rid those infected of adult parasites. The parasites can modulate the immune system and live for 10-15 years. Testing of compounds against filarial nematodes is complicated due to a lack of an objective platform on which to analyze in vitro treatments. There is no published, immunocompetent laboratory model for lymphatic filariasis. This work addresses each of those areas. Imatinib mesylate is shown to have macrofilaricidal potential, killing adult male and female Brugia pahangi worms in-vitro. Histological analysis of rats infected with Brugia pahangi show the same lymphangectasia and inflammatory cellular response seen in humans. The WiggleTron, a light scatter based detection system, can detect and quantify parasite motion; analysis of that motion can be correlated with worm health to study the in-vitro effects of potential anthelminthic drugs. Finally, this work will show the relative uniqueness of secreted microRNAs and discuss their potential as novel therapeutic agents. Copyright by Charles S. Nutting 2015 ACKNOWLEDGEMENTS My success at WMU would not have been possible without the support of others. Rob Eversole, thank you for your leadership, mentorship, and friendship far beyond this research. Your guidance has helped me become a better scientist, a better problem solver, a better teacher, and a better human (although it seems the monkey will always exert some control). I am, and will continue to be, grateful for all that you’ve done for me. Charles Mackenzie, thank you for providing me with opportunities to be a part of research all over the world. Taking the time to include me in your efforts studying and combatting filarial diseases has been invaluable to my training and perspective. Pam Hoppe and Charles Ide, thank you for your critical review of my work. Your comments and insights helped me bridge the gaps from individual experiments to a complete story. Kevin Blair thank you, without your brilliance none of my WT work would be possible. Karim Essani, thank you for giving me my first opportunity to work in a lab and beginning my first real science education. I had no idea what I was doing. The previous statement is now somewhat less accurate thanks to your efforts. Finally, Rebecca, having a partner who is wholeheartedly committed to the idea that I should pursue something that truly makes me happy, regardless of the privations that you have to suffer, is an incredible thing. I can’t write all that you’ve done to support this work. I’m overcome with emotion even thinking about it; Thank you. Charles S. Nutting ii TABLE OF CONTENTS ACKNOWLEDGEMENTS ............................................................................................................. ii LIST OF TABLES ......................................................................................................................... vii LIST OF FIGURES ...................................................................................................................... viii CHAPTER I. INTRODUCTION ........................................................................................................................ 1 Nematode Biology ..................................................................................................... 1 Assessing Nematode Motion ..................................................................................... 2 Light Scattering Detection ......................................................................................... 3 Parasitism Involving Nematodes ............................................................................... 4 Lymphatic Filariasis ................................................................................................... 4 Onchocerciasis ......................................................................................................... 20 Loaisis ...................................................................................................................... 29 Remaining Areas of Inquiry..................................................................................... 34 2. RAT LYMPHATIC PATHOLOGY .......................................................................................... 36 Evaluation of Lymphatic Pathology ........................................................................ 36 Previous Work ......................................................................................................... 36 Slide Preparation ...................................................................................................... 37 Nova Red and Hematoxylin Staining ...................................................................... 37 iii Table of Contents - continued CHAPTER Grading Scale ........................................................................................................... 38 Immunohistochemistry Results ................................................................................ 38 Grading Lymphatic Change ..................................................................................... 42 Statistics ................................................................................................................... 50 Discussion ................................................................................................................ 50 Treatment of Infected Rats with Potential Macrofilariacidal Agents ...................... 54 Animals .................................................................................................................... 54 Parasites ................................................................................................................... 54 Infection ................................................................................................................... 55 Monitoring Patency .................................................................................................. 55 Failure of Infection .................................................................................................. 55 Confirmation of Protocol ......................................................................................... 56 Discussion ................................................................................................................ 56 3. DETECTION OF FILARIAL NEMATODE MOTION AND DRUG SCREENING .............. 58 Introduction .............................................................................................................. 58 Motion Sensing Equipment...................................................................................... 59 Measurement and Analysis ...................................................................................... 61 iv Table of Contents - continued CHAPTER Initial Analysis of Nematode Motion ...................................................................... 62 Parasites ................................................................................................................... 63 Initial Drug Testing .................................................................................................. 65 Statistics ................................................................................................................... 67 Results ...................................................................................................................... 67 Detection of Motion ................................................................................................. 67 Estimation of Worm Number................................................................................... 72 Analysis of Worm Health ........................................................................................ 73 Discussion ...............................................................................................................
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