German Cuadra and Dr. Bradford Berges, Department of Microbiology and Molecular Biology

Our main goal of this project was to document the existence of human macrophages in the brain of humanized mice. After the accomplishment of this goal, we planned to infect humanized mice with HIV and find out if these human macrophages support infection in mouse brain tissue. We hypothesize that humanized mice will have human macrophages engrafted in the brain and that these cells will become infected with HIV via normal pathways after introduction of virus via other routes, as in humans. By identifying these cells in humanized mice, we plan to develop a mechanism to study the relationship between HIV and neurological diseases in AIDS patients. Unfortunately, the identification of these cells became our most complicated step.

We haven’t made as much progress as we had wished to make at this date. So far we have not being able to present or publish our project. Mainly because of major delays related to our mice colony.

By March of this year, we had already dissected four mice brains and started to look for the human cells of interest with no significant positives results. We had our controls and a basic protocol that would lead us to the finding of human macrophages in humanized mice brains. We noticed in our experiments that the presence of human cells was extremely low and started to investigate why this was happening.

We found out that our mice were no longer achieving successful engraftment. After multiple analyses and experiments, we were able to find out that most of our mice were of the wrong genotype. As a consequence we ultimately had to eliminate our entire colony and start over again. All of this resulted in serious delays to my project (about 8 months were lost).

While we were waiting to have a successful colony that would really help our project, we make important advancement in other aspects of the investigation: producing HIV stocks for infection of humanized mice was one of them. Also, we perfected our technique for the perfusion of the mouse vascular system and staining and fixation of mice cells. We have also worked to develop FACS protocols to detect human macrophages in mouse brain tissue by introducing macrophages into mouse brain tissues post-extraction.

The next big challenge after the reestablishment of it has been the extraction and separation of cells from the mice brains. After reading several papers and techniques, we are still developing our own protocol for gradient extraction of macrophages and other cell types in mice brains using Percoll.

So far, we have performed the experiment four times, sacrificing and studying brain tissue from over ten different mice. It was the first experiment that gave us the best results because of the percentage of human engraftment in our mice.

We repeated our experiment two weeks ago, but we were not able to determine with exactness the existence of macrophages in mouse brain tissue. We are determining the definitive area and density where in these cells is our Percoll gradient. Once we determine this important information, we will be able to effectible characterize human macrophages in human mice brains.

Last week, we started a new experiment that will lead us to determine the location of human macrophages in the percoll gradient by using human macrophages as controls and examining all possible volume and densities in our sample.

We plan to continue working on this project until we can effectively determine the presence or absence of human HIV infected microphages in humanized mice tissue. We hope once we determine the location of these cells in the Percoll gradient we will be able to infect and probe our hypothesis.