All posts by Stephen Pelsue

Simulated Microgravity and its Impact on Phosphatidylinositol Composition and Plasma Membrane Dynamics in Leukocytes

Extended time in space has been shown to have impacts on the immune system; while studies vary on the extent of the impact it appears that extended time in space results in reduction in immune capacity.  With plans to return to the Moon and as well as a manned mission to Mars, a better understanding of the effect of zero gravity on the development and function of the immune system is paramount to the health of astronauts spending extended time in space.  While, true, zero gravity cannot be studied in Earth bound experiments, microgravity can be simulated through a rotating wall vessel (RWV) culture system.  Phosphatidylinositol (PI) and its phosphorylated derivatives play a critical role in maintaining cellular membranes as well as regulating cell signaling complexes.  We have determined that the regulation of PI-4 Kinase is critical for the normal regulation of leukocytes, particularly lymphocytes.  To better understand the cellular and molecular impact of microgravity on immune function we will evaluate simulated gravity on the cellular structures and dynamics related to PI composition in leukocyte cell lines.  This will be accomplished by he evaluation of organelle structure and function, PI composition, PI-4K and PI-5K activity, and stress responses (particularly ER stress) in leukocyte cell lines cultured in simulated microgravity.

Regulation of Phosphatidylinositol-4-Phosphate by By TTC7 and its role in Plasma Cell Differentiation and Autoantibody Production

Lupus is a chronic autoimmune disease where the immune defence system attacks the cells, tissues, and organ systems rather than protecting the body against harmful pathogens, such as viruses and bacteria.  More than 1.5 million Americans have lupus and over 90% are female, and women of color have two to three times higher risk of having Lupus. Antibodies produced by the immune system attack the skin, kidneys, heart, blood, brain, joints, and lungs and which is the leading cause of early onset kidney disease, cardiovascular disease, and stroke in women.  The key to better health is the early detection of Lupus and therapeutic management; however Lupus is difficult to diagnose and often goes undiagnosed or misdiagnosed, sometimes for years.

To better understand Lupus it is important to characterize the process of how and why the immune system attacks its own body.  The development of the immune system has several checks and balances to insure it is not harmful to its own body while protecting against harmful microorganisms. Antibodies are produced to detect and remove these harmful microorganisms. So why then do patients with Lupus produce antibodies that attack their own bodies?  This is the key question that will be addressed by this project.  A new idea will be explored that links a specific type of cellular stress with the uncoupling of the normal controls of the B lymphocytes—the cells that produce antibodies—and lead to the production of autoantibodies, the antibodies that attack one’s own body. Specific components of the cell membrane are required to maintain the normal function of the cells, this research will focus on one type of membrane component, phosphatidylinositol-4phosphate (PI4P for short), and the enzyme that produces PI4P and determine if defects in the regulation of this system are associated with inducing stress in B lymphocytes and allowing autoantibodies to be produced.

The project will use B Lymphocyte cell lines that can be easily cultured as a model of B lymphocytes in the body.  These cell lines will be manipulated to alter the regulation of one of the processes associated with the production of PI4P and measure 1) the level of a specific type of cellular stress and 2) the ability of these cells to produce antibodies.  This research will provide insight into a new model of autoantibody production and with a more detailed understanding of this process it is hoped that new diagnostic tools or therapies can be developed based on these findings.

“Opening” Up

There has been a great deal of discussion about the benefits of Open Science and Open Notebooks to the advancement of science, increase in exposure of an individuals work, and also the ability to collaborate with a much broader community. I have been an advocate for Open Science for quite some time, I have been talking about it with my undergraduate and graduate students working in my lab, I have added curriculum to my graduate courses that include discussions of Open Science and Open Access, but admittedly I have been slow to actually implement Open Science practices in my own laboratory.

I recently have come to a transition point in my lab, where my research focus has taken a slight turn, but consistent with my overall research interests, and I had complete turnover of students in my group.  I took this opportunity to reflect on lab operations and it was clear that I needed to practice what I preach. This reflection has lead me to develop a more “Open” approach to research practice.

After a few discussions about Open Notebooks I decided to partner with the Open Notebook Science Network to develop the Open Notebook/Open Science pelsuelab.org site that you are presently visiting (I would like to give special thanks to Brian Glanz for volunteering to build the site). I thought for my first blog entry I would take a moment to explain why I decided to “Open” up my lab.

A key turning point in my thinking about Open Access was listening to a presentation by Dr. Michael B. Eisen, and comment he later made on Twitter:

This implied to me that the future of publishing might just be the ability to post any piece of work and its value will be defined based on the content and how it is received and reviewed by experts in the field, if not the broader scientific community at large. This struck me as a naïve view of scientific publishing, but in fact the more I thought about it I realized it was a very advanced approach.  We could actually just provide our methods, data, and results and open up a discussion about its meaning and value rather than presenting it as a complete and defined story as we try to do in peer reviewed journals. While this model does open an individual to greater criticism and exposure—not to mention being the target of anti-science crackpots—but more importantly it could lead to greater discussion and collaboration within the scientific community at large.

I decided to take this idea to heart and not only commit myself to future publishing in Open Access sources (I am presently developing my first manuscripts to submit to Open Access Journals), but also to  Open Up the entire lab and move towards an Open Notebook, such that with the push of the enter button the progress in the lab would be open to all that are interested.

The more I thought about it, I also thought this would benefit my students as well.  I have tried to set up a process that will streamline their notebook recording, as well as my ability to review progress and issues for more immediate input. I also think that more thought might be given to the notebook knowing that others will be looking at their entries. I also envision that they will be able to more readily  convert their notebook into manuscript or thesis formats to increase the efficiency of publishing in my lab (something that has been an issue for me as I am typically the bottleneck in getting manuscripts organized and submitted). They would be able to demonstrate to potential graduate or medical programs, employers, etc.  their specific accomplishments, not just a listing of technical abilities on a resume or a manuscript in a c.v., but the actual data and analysis.

While we have just begun this journey I invite you to check back as often as you wish to see what’s happening in the Pelsue Lab.

The Pelsue Lab is now “Open”.

Cell Mask Staining

Project: 

 

Purpose:

To stain plasma membrane and use a live stain for nucleus

Experimental Design:

 

To prepare Cell Mask:
Official Protocol calls for 1 mL of stain per coverslip- needs to be diluted.
Dilution:
Stock= 5 mg/mL
Want 2.5 ug/mL
Add 2.5 uL of stain to 5 mL of PBS
Stain cells with Cell Mask for 5 min.

To prepare Hoescht:
Want 1 ug/mL in PBS
Stock= 5 ug/mL
Add 1 uL of stain to 5 mL of PBS
Stain cells with Hoescht for 30 min according to Pelsue, stained for 5 min this time

Harvest Cells- took 5 mL of cells
Add stain to cells + media
Spin off stain
Wash with PBS
Transfer to eppindorf tubes
Fix cells

Do PBS wash twice
Pour off all but approx. 50 uL of PBS and flick to resuspend
Add 150 uL of Paraformaldehyde (2-4% for WEHI)
Put in fridge under foil for 10 minutes
Spin at 1500G to 2000G for 3 minutes and check formation of pellet- spin more accordingly
Remove Paraformaldehyde (put in its own waste)
Add 200 uL of PBS
Spin (still at higher G) for 5 minutes
Pour off all but approx. 50 uL again and flick to resuspend
Use 1 drop Promega gold mounting medium (1 drop=10uL)
Add 10 uL of cells to well with mounting medium in it
Mix and spread around well
Add coverslip and nail polish it down
Allow to set- check in 2 days and again in 6

Results:

Both the plasma membrane and nucleus are stained.

Notes:

Important to do cell count beforehand

Sample Storage Location:

 

File Locations:

 

 

IL-4 Expression in fsn Mice

A and B: Gross structure of splenic longitudinal sections showing IL-4 production (blue) and CD3 positive cells (red) at 40X magnification. A, WT 8-week section showing red-stained T cell zones and non-detectable IL-4. B, 8-week section of Ttc7fsn spleen divided into 4 parts showing dramatic enlargement of organ size and disintegration of distinct T cell zones. IL-4 expression was detectable throughout spleen, but especially in follicular areas as indicated by arrows. 40X magnification. C and D: IL-4 expression in 8-week spleen visualized with laser scanning confocal microscopy using a 20x objective. Spleen sections were stained for IL-4 (green), CD3+ T cells (red) and IgM+ B cells (blue). Ttc7fsnspleen (D) shows an increase in IL-4-positive intensely stained T cells (yellow) as compared to WT littermate (C). In addition, T cells are located outside of the follicle in the B-cell-rich zone consistent with the location of pStat6 positive B cells (figure 3). All staining was repeated three times in three separate animals with similar results.

Activation of Stat6 in Ttc7-fsn B cells

These are some figures from my lab examining B cells from the Autoimmune prone flaky skin mutant mice.

Purified splenic B cells were unstimulated (0) or stimulated with IL-4 for 15 and 30 minutes, then stained for Stat6 (red) and the nucleus (green) and visualized with confocal microscopy using a 60x objective. Co-localization of Stat6 with the nucleus, denoting activation, is yellow. Ttc7fsn B cells show more yellow, activated cells than normal even without stimulation, suggesting pre-activation in vivo.