Week Eight

It's really strange, but somehow eight weeks have gone by. I can't believe I've been here for two months now! My constant adventures around the Boston/Cambridge area are going to come to an end...Here is a picture of the Charles. What a beautiful place!



Ellen has finished her time here last week, and Josh, Vanetta, Joe, and Megan have all been getting ready to head off for an annual conference in Australia at the end of this week. I helped Josh work on his presentation and slideshow that he'll be presenting next week, and later shadowed Carrie as she showed me how to do DNA extraction. We started with mRNA, made cDNA, then converted back to RNA and then DNA once again. Tuesday, we ran the PCR for this, and irradiated the mice for Wednesday's bone marrow transplant.

Journal Club was led by Josh this week, and I spent some time really trying to understand the article before the meeting. Surprisingly, I found that it was pretty straightforward, and that I really was understanding most of the data just reading it through myself. The article dealt with an immunostimulatory T cell Ig mucin (Tim)-1 specific antibody that deprograms Tregs. T-regs are a type of CD4+ helper T-cells that are resonsible for immunosuppression and anti-inflammation, which make them key players in tolerance studies as they do their best to prevent a graft-vs-host immune response. With the T-regs "deprogrammed" - or not completely functional in the presence of this 3B3 anti-Tim-1 mAb, transplant tolerance is not possible. I ended up presenting a couple of the figures, which was an initially uncomfortable but altogether rewarding experience. It's really hard to sit in front of PhDs and MDs and attempt explain a scientific figure, but I'm glad I did. Explaining the figures to others also helped me realize exactly how much of the material I had understood.

Reflecting back on everything, I can't even imagine how everything was at the lab eight weeks ago. Time has definitely moved fast. Science, and research in particular, has tremendous potential in so many different areas of study. To think that in a few years, we may have gotten much closer to finding different alternatives and better success rates for organ transplants, or cured autoimmune diseases, is an amazing thing. Although research will inevitably lead to a lot of failure, it also leads to great success, and with more breakthroughs that will follow in time to come, we are sure to progress in treatment and care of humans. Everything here starts in the mouse, but success at the smaller animal level is what leads to clinical trials and success in people. I will really miss coming here and learning something new every single day. I've realized that what I love most is talking about the science behind the lab work, and how that thought process can generate new hypotheses that we can look forward to testing and growing on. It was a wonderful experience that I'm very grateful for, and I can only hope to come back to later on. Thank you!

Week Seven

I started off the week with two final ELISAs, and the data definitely confirmed things from the Elispot and FACS. It's great to finally have this data ready, because it proves the validity of this experiment, even though the data itself is unexpected and contradicts previous findings. Because of this, we will have to closely re-examine the previous experiments dealing with the complement receptors.
Journal Club, which we've been attending regularly, was on an especially interesting topic. We studied an article which explores the mechanisms why females are more prone to autoimmune diseases than males. The journal's findings suggest that the XX chromosome complement is what actually confers greater susceptibility to autoimmune diseases, such as lupus, as compared to the XY chromosome complement. The experiments themselves used castration techniques to focus on the susceptibility of chromosome complements alone, without the added variability factor of sex hormones. The study was mostly in vivo, and done in mice models. A hypothetical conclusion that could be derived has to do with their data showing that there is a higher expression of IL-13Ra2 on XX than on XY. IL-13Ra2 is only expressed on the X chromosome, and it can downregulate Th2 responses (which are CD4+ helper T cells regulating antibody-mediated immunity, a major part of the immune system). Figuring out the mechanics of the puzzle may eventually lead to a cure for severely debilitating autoimmune diseases - that would be wonderful.
By the end of the week, I performed a mini-takedown myself. I harvested and mashed the spleen, counted the cells, diluted and resuspended, and titrated the antibodies to find the correct titer for use in future experiments.
Overall, it was a great week. Summer is going by so quickly - only one more week here!

Week Six

The ELISA has been coming out pretty well for the last two weeks. Here's a bit of background information about the uses of this assay in this particular experiment.

As I mentioned before, alpha-Gal (abbrev. Gal) is a sugar found on natural antibody producing B cells, binding onto the BSA molecule. Gal is found in mice and pigs, but not in humans, which makes the field of xenotransplantion (transplanting organs between different species) very difficult, as the presence of Gal on a transplanted organ can trigger an immune response because humans have anti-Gal antibodies. This is one of the many barriers to xenotransplantation. However, if the barriers can be overcome, xenotransplantation could open up possibilities of almost endless organ transplant options, especially from pigs to humans. This new source of organs could curb the waiting list for tranplants dramatically, and save millions of lives of people who are dying just waiting for an transplant option to open. To study this, we use Gal KO mice (that are abnormal in that they don't have any Gal) and then give them a bone marrow transplant from a normal Gal positive mouse. The ELISAs that I have been doing have been testing for anti-Gal antibodies - that is, an immune response to Gal. If the mice are tolerant, there is no immune response - therefore, there are no anti-Gal antibodies seen in the assay. These tolerant mice are chimeras.

Week Five

This week has been full of collecting data through the experiment known as the ELISA. ELISA stands for Enzyme-Linked ImmunoSorbent Assay. It's used for looking for antibodies. Here, the ELISA data can be used along with the Elispot data and the FACS data to prove that there is indeed an immune response taking place where they are supposed to.

On Monday, I did an ELISA looking at the immune responses with the sera of a few different mice in different dilutions, so as to determine which of these seras and at which particular dilution is good to serve as a control for the next few ELISAs.

On Tuesday, the ELISA utilized the sera of mice that were part of the same takedown that yielded the Elispot and FACS data on the spleen and peritoneal cavity over the last two weeks. Wednesday was a repeat of the same ELISA to check that the results are correct. Thursday, I did an ELISA using the sera from the other 3 mice part of the same takedown. With a combination of all these results, we're going to lab meeting to present the data and look for ideas and input from the other lab fellows on what to do next.

Week Four

As the weeks go by, much more of the analysis that comes with the experimentation that is taking place here at the lab is making sense to me. Any time that a summer student has a question, especially if it is asked for clarity on a particular subject of the experiment being done, the fellows here are more than glad to sit down for a few minutes and review many more concepts of immunology and their associations and implications in this field of study. For example, Carrie has a new student this week, and I've been able to listen to a few of her explanations, from the basics of the immune system to the different methods to how exactly a gene is "knocked out". I've gradually been absorbing more and more information, and it definitely gets easier!

An example of a concept that I've begun to grasp - A knocked out gene means that although it is still present, it is no longer expressed. One way to knockout a gene is to use a method of homologous recombination, by basically providing another pathway (a vector) for the mouse genome to follow for a specific gene, which essentially makes the gene usually in this replaced spot no longer expressed. To target and quantify those genes that follow this process of homologous recombination - that is, the genes that forego their original path and successfully take up the vector - a neomycin resistance gene can be used in the vector. This way, you ensure which mouse genomes have taken up the vector, and use only these mice for breeding chimeras. Another method is transgenic mice, which is a less specifically targeted approach to this.

Why are gene knockouts important? By knocking out specific genes, a researcher can enlarge the possibilities of research on various topics almost endlessly.

This week for experiments, I've seen another Elispot done on the peritoneal cavity cells of the mice taken down last week. Tuesday, we went out to dinner in Charlestown with Mr. Palm, which was a lot of fun! I've also been helping map and catalog more of the in vivo imaging data which Ellen and Tosh are working on. And on Friday, I got to do my first assay by myself! It's called an Elisa, and I'll be doing many more of these for the next few weeks.

Week Three

This week has been great for lab results. Earlier in the week, Josh enlisted the help of Meredith to perform a takedown of ten mice, testing for differences between the results of chimeras, normal B6 mice, and a naive Gal KO mouse (in which the Gal antibodies commonly present in mice and pigs are knocked out). Tests were done using the spleen and the fluid from the peritoneal cavity of the mice. According to the hypotheses for this takedown, there should have been a loss of B cells that bind Gal in the FACS analysis, as well as a loss of Gal antibody secreting cells in the Elispot treatment. These were predicted in the Gal KO mouse as well as the control B6. Both the FACS and the Elispot yielded clear results. I've been helping with the centrifuging, cell counting, and FACS and flow-jo analyses on each part of the experiment this week, and I've been learning new techniques and protocols every day.

Another thing that came in this week was a newer version of the toxin product that needed to be tested before using it on mouse injections for bone marrow transplants and irradiation to induce chimerism. The product worked well, so it is safe to say that a switch can be made from the old product to the new one.

Meanwhile, Ellen and her postdoc Tosh have been continuing to conduct their research on tumor cells in mice, using the GFP (green fluorescent protein) and identifying it in the pictures taken through in vivo imaging down at a lab at the MGH main campus. Ellen and I have been helping to assemble the data maps into easily readable files on the computer.

Week Two

This week has gone by pretty fast. Yesterday, Josh did a bone marrow transplant on mice. When I arrived at the lab, Josh was starting a MACS T-Cell depletion. A T-cell depletion is when you add magnetic beads to the bone marrow cells in a resuspension, and pass this through a special filter inside of a big magnet. The magnetic beads that we put into the cell solution binded to the two types of T-cells - one is the receptor CD4 for the helper T-cells, and one is the receptor CD8 for cytotoxic T-cells. The magnet traps the binded t-cells at the top, so the rest of the cell solution that filters through has no T-cells in it. We then checked to make sure the experiment worked using a FACS analysis.

Josh, Tosh, Ellen and I also went to Journal Club, a meeting organized by Carrie to talk about scientific journals and their significance to the lab. This week's journal topic discussed chimeras, which are mice with accepted foreign bone marrow received from other mice. This foreign bone marrow is what makes the mice tolerant to later receiving foreign organ transplants as well. Tolerance is a big part of the studies that go on at the TBRC, and the article was a bit difficult to understand but pretty interesting in the end. We'll be attending Journal Club meetings once a week, to keep talking about other research being done outside of our lab and to better understand new breakthroughs.

I've learned that lab work comes in bursts. Sometimes there are many experiments going on at once, but other times the postdocs have paperwork or other tasks to get done around the lab. During my down time, I've been shadowing Ellen and her postdoc, Tosh. They have been working on something a bit different than Josh's bone marrow transplants - Tosh has been studying why grafts that are not tolerated by a recipient still show signs of tumor cells being killed. The experiments that I have watched for this study involve plasmid DNA purification, cutting the plasmids with certain restriction enzymes to insert a phosphorescent marker in these plasmids, and checking these cuts through gel electropheresis. These glowing plasmids will later be inserted in the tumor cells, where we'll be able to see the cellular interactions.