Research Experience Statement

14 June 2010
This is the research that I have been doing for the past 2 years. Read it if you are interested or just skip it.

For nearly two years, I worked in Dr. Gregory Burton’s laboratory at Brigham Young University. His studies focus on understanding the molecular interactions between follicular dendritic cells (FDCs) and HIV. Specifically, we examined the contributions of two FDC receptors, CD32 (FcγR) and CD21 (CR2), that played significant roles in the trapping and long-term maintenance of HIV.

My first project was molecular cloning of HIV. We received samples of lymph nodes from HIV infected patients. I isolated the HIV virons and the RNA, and performed reverse transcription. After making cDNA, I PCR amplified the products, inserted the amplimers into vectors, and inserted them into bacteria. I then sequenced the inserts in the vectors and performed phylogenetic analysis of the HIV genome. This work showed that the HIV genome from one lymph node is not the same as another, and that the genome in the same lymph node is very similar. These findings were consistent with past research. This project was used to train me in basic laboratory techniques, and double-check the findings for a paper that is still in the process of being published.

My second project examined different pathways of FDC activation, and if FDCs up-regulate or down-regulate CD32 and CD21 through the different pathways. I isolated FDCs from human tonsillar tissue through positive selection using a FACS machine. To activate the cells, I incubated the FDCs with lipopolysaccharide (LPS), antibodies, complement, immune complexes, immune complexes+complement, neutralizing HIV antibody immune complexes, or neutralizing HIV antibody immune complexes+complement. After the incubation, I extracted the RNA from FDCs, performed reverse transcription, and then analyzed the cDNA with real time PCR. I found that each of these known immune system activators up-regulated CD32 and CD21 at differing degrees. During this project, I also incubated FDCs with alpha-1 antitrypsin. We found that alpha-1 antitrypsin deactivates FDCs, but we did not know why it deactivated these cells. These findings focused our group’s research on determining the intracellular pathways of activation through CD32 and CD21.

My third project focused on discovering the proteins associated with CD21 on FDCs. I hypothesized that CD21 on FDCs is associated with similar proteins as CD21 on B cells, meaning that the B cell co-receptor (CD21/CD19/CD81) is on FDCs. The B cell co-receptor could then activate FDCs in the same or a similar intracellular signaling pathway as B cells. The experimental protocol that I designed was to use fluorescently labeled antibodies to CD21, CD19, and CD81, and then use FRET analysis to determine if the proteins are associated with one another. If they were associated with one another, we would then perform protein cross-linkings and immunoprecipitations to determine the intracellular pathway of activation, and establish if it was the same or similar to the B cell co-receptor activation pathway. I grew hybridomas for CD21, CD19, and CD81, isolated the antibodies, and fluorescently labeled them. Then I isolated B cells from peripheral blood mononuclear cells (PBMCs) through positive selection using a MACS machine, and I isolated FDCs with the same methods as my second project. After separation of the cells, I incubated them with the fluorescently labeled antibodies, used a confocal microscope to perform FRET, and analyzed the results. When I left the laboratory, I had performed some preliminary trials without the α-CD81 antibody, because we were waiting for the α-CD81 antibody to be produced by another principle investigator. Despite missing the α-CD81 antibody, the preliminary results were promising.

My fourth project studied the role of FDCs in activating CD4+ T cells with a latent HIV infection. Studies have shown that FDCs and FDC supernatant can activate latent T cells, but the mechanism is not known. In efforts to determine the mechanism, I isolated CD4+ T cells from PBMCs through positive selection using a MACS machine. After activating the growth signal with IL-2 in the isolated cells, I would infect the cells with an HIV variant. The HIV variant has a faulty envelope gene that can be used to infect the cell, but its progeny can never bud off the cell. If the infection were not latent, the cell would die from viral overload. However, if the infection were latent, the CD4+ T cells would survive. After the latent infection was established, I induced viral protein formation with differing amounts of PHA+Ionomycin or IL-2+IL-7, measured p24 levels within the cells with α-p24 antibodies, and created dose curves comparing activator versus viral expression. These dose curves were then compared to induction of viral protein formation when the latent T cells were incubated with FDCs and FDC supernatant. When I left the lab, we were continuing to induce latent T cells with FDCs and PHA+Ionomycin or IL-2+IL-7, and we were performing mass spectrometry on the FDC supernatant to determine the protein that is causing latent T cells to form HIV particles.

Currently, I am working in Dr. Jeffery Gildersleeve’s laboratory at the National Institutes of Health, specifically in the National Cancer Institute for a year long post-baccalaureate. His research focuses on developing carbohydrate microarrays to assist in analyzing cellular markers, tumors, and vaccine efficacy. I am working on developing a microarray to test epitopes and specificity of antibodies formed by the HIV/AIDS vaccine. Developing this microarray would give researchers a rapid determination of the efficacy of the HIV/AIDS vaccine in the different stage trials of FDA approval.


Ned said...

Wow, that's pretty cool. I couldn't tell someone else much about it, but it kind of made sense as I read it on an abstract level. It would be interesting if you would translate this, or summarize it in plain English. Probably not too fun for you since the vocabulary enables you to convey complex ideas without having to explain as you go.

By the way, are you still swimming?

Mister Curie said...

Sounds like some awesome research. I loved Dr. Burton's Immunology class when I was at BYU. Good luck with your research at the NIH.

A Gay Mormon Boy said...

Good luck on these apps. I'm preparing mine right now and it's difficult, but somehow reassuring.

You might have studied with one of my good friends at BYU. He's at Southwestern now.