Discipline: Biological Sciences
Mariana DuPont - Dillard University
Co-Author(s): Arnold H. Zea, Louisiana State University Health Sciences Center, New Orleans, LA
Tuberculosis is one of the leading causes of death today in America. It is cause by a bacterium called Mycobacterium Tuberculosis (Mtb). Mtb is an intracellular pathogen able to survive and multiply within macrophages. It is transmitted from person to person via droplets from the throat and lungs of people with the active respiratory disease. It remains unclear whether the L-Arginine-Nitric Oxide pathway has a role in humans infected with Mtb. The laboratory has been trying to infect human macrophages with Mtb without success; we thought the problem could be optimization of the cultures. Therefore my goal of this summer research was to find the optimal conditions necessary to make the cells susceptible to mycobacterial infection. My hypothesis is to establish the optimal conditions for TH-P1 human cells to be sensitive to arginase induction. This was done by culturing both half million and a million cells for 24, 48, and 72 hours using 50ng of phorbol myristate acetate (PMA) to allow the cells to attach in media containing 2βmercaptoethanol (BME) as recommended by the company. I tested the cells, in four different condition using 8Bromoadenosine 3′, 5’-cyclic monophosphate (8-Bromo-cAMP) at 10 µM as an activator for the TH-P1 cells to determine the best condition for arginase induction. Arginase activity and nitric oxide production was tested by enzymatic assay and Greiss assay respectively. Which was done in a 10 week period. My results showed that million cells cultured for 48 hours in the presence of PMA and absence of BME adhered perfectly. We also observed that after PMA treatment the cells needed to be rested for 48 hours, following stimulation or infection. Under these conditions we were able to induce arginase up to 5 fold. The most important finding was that the presence of BME was inhibiting arginase production. These results are important since the use of murine cell lines to study mycocacterial infection do not correlate well what we observe in humans. For effect of translational research in tuberculosis research this finding can be very important.
Funder Acknowledgement(s): Funded by NSF - #1359140
Faculty Advisor: Ruby Broadway,