Jordan Mabey and Dr. Brian Poole, Department of Microbiology and Molecular Biology

Introduction

B cells are specialized cells which participate in the normal immune response to an infection. As B cells encounter antigens in the body, they become activated and travel to what is known as a B cell follicle within a lymph node. At the outer follicle B cells combine with T cells and move to the inner follicle to become specialized immunity participating cells. Normally upon reaching the outer follicle, the B cell up-regulates Epstein-Barr virus induced molecule-2 (EBI2) which causes it to pause until the cell can complex with the proper T cell. Once this connection between the two cells has been established, EBI2 is down-regulated and the cell complex moves into the inner follicle (1).

Goal and Purpose of the Research

An infection by Epstein-Barr virus (EBV) causes the expression of EBI2 in B cells to increase 200 fold (2). The purpose of this increase is still undefined, but is crucial to a better understanding of the virus and how it affects its host. The goal of this project is to knock down expression of EBI2 in EBV infected cells and observe changes in the migration patterns of these cells through the B cell follicle.

Importance of the Project

EBV has been associated with various health complications such as mononucleosis (commonly known as mono), certain cancers and lupus. By understanding the way in which this virus infects humans, we will better be able to conceive and create therapeutic measures to help those who struggle with health complications related to infection by EBV.

The Research

The course of this research began well and the first steps went as was expected. The procedure had been carefully crafted and was based on prior experience in the lab. It was amazing to me though how many unforeseen obstacles became immediately apparent once the work began. Problems that we could not have anticipated impeded the work at every step, but we worked hard and got creative to overcome these obstacles.

This occasionally got discouraging and at one point I became frustrated with the work. I reworked the first quarter of the proposed procedure about 15 times and yet we could not successfully express our inserted portion of RNA. With every failure we re-analyzed what may have gone wrong, which in retrospect was a fantastic learning experience. The cycle of failing and retrying deepened my understanding of the project and the science behind our project such as a lecture never could. My understanding of lab procedures was greatly increased and my ability to imagine alternative possibilities improved.

The largest of these problems was in our insert. We had done research in published articles to specifically design our insert for the procedure. This design was complicated and required custom ordering segments of the insert from a company, then attempting to insert them into a plasmid. We recently have found a simpler way to knock down the expression of EBI2 in order to assess its effects in immune reactions. This new process needs to be researched further. Once beginning with a new insert the project has progressed much more rapidly. In the last month we have managed to see as much progress as in the past eight or nine months of attempting to insert the plasmid.

We expected to have publishable results by this point, but those hopes have been frustrated. The project has proved more difficult than we anticipated, but now those results seem to be near. Given our current progress we expect to have publishable results by summer time.

The ORCA process has been very rewarding for me. It has allowed me to pursue this project, and although we are still working for results our way forward looks very promising. I have found the thrill of discovery through this process and would highly recommend that everyone interested in research participate in the program. I am very grateful for the opportunity that ORCA has given me.

References

• João P. Pereira, Lisa M. Kelly, Ying Xu & Jason G. Cyster, EBI2 mediates B cell segregation between the outer and centre follicle, Nature International Weekly Journal of Science. Nature 460, 27 August 2009 2.
• Mette M. Rosenkilde, Tau Benned-Jensen, Helene Andersen, Peter J. Holst, Thomas N. Kledal, Hans R. Lüttichau, Jørgen K. Larsen, Jan P. Christensen and Thue W. Schwartz. Molecular Pharmacological Phenotyping of EBI2, Journal of Biological Chemistry. March 9, 2006