FA 5 Progress Report WV-INBRE - Joan C. Edwards School of ...

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Program Director/Principal Investigator (Last, First, Middle): Rankin, Gary O 40that do not express. The current list of proteins that have been expressed are N474I, T304A andS209A. We are working on E300V currently and have expressed R307L which has an impact onthis project.The purification of the expressed protein is being carried out according to protocol received fromthe lab of Dr. Timothy S. Tracy. We have successfully purified the wild type form of the protein and2 of the expressed mutated forms. Purification is a week long process that takes approximately 8hours of steady work to reach a stopping point. The time factor and use of undergraduates at thispoint make it very difficult to manage schedules to be sure we can cover 8 hours in a day. Theaddition of a half time technician would help in this area.With the success of expression and now purification we are in the process of beginning our kineticanalysis. The kinetic analysis is a faster process and it is foreseeable to carry out 2 experiments aday. Leaving us with the task of increasing rate of purification so that kinetic analysis can continueat an acceptable rate.The molecular modeling part of the experiment has been completed in terms of looking atcorrelations of distances and hydrogen bonding. We are still trying to determine a viable way toaddress stacking. MM-PBSA has been run for the majority of our mutations in silico. We are in ahold to see if this data will correlate with the kinetics.We have been successful and an additional centrifuge, obtained from an equipment grant, will allowus to speed up our processes and finish the kinetics an d put out publications. We are submittingan area grant to the NIH before the February 25th deadline. We were close but after discussionswith my mentor decided we needed to change a few things to make it more competitive. My writingskills need improved and I am looking for a good grant writing workshop. The publications will beginto come forth upon completion of kinetics. I have had the unfortunate experience that although themodeling data is promising on its own it needs to be completed with actual kinetics data. This hasbeen mentioned to me at several of the meetings at which we presented posters.PROTECTION AGAINST RESEARCH RISKSN 1. Will human subjects be involved next year?N 2. Will vertebrate animals be used next year?N 3. Will recombinant DNA experiment(s) be conducted next year?N 4. Are there potential hazards to laboratory workers (carcinogens, pathogens, ionizing radiation, etc.)involved in the proposed research for next year? If yes, identify:N5. Will any of the research-risk categories,not involved next year, be involved future years? If yes, identify:PHS 2590 (Rev. 06/09)Continuation Format Page
Program Director/Principal Investigator (Last, First, Middle): Rankin, Gary O 41EFFECT OF STRESS ON PATHOGENESIS AND IMMUNITY DURING CHLAMYDIAGENITAL INFECTION (0039)TYPE:Research Subproject%IDeA $: 5.000% IDeA $: 174,247INVESTIGATOR, DEGREEBelay, Tesfaye PHDYu, Hongwei PHDDEPARTMENTBiological SciencesBiochemistry &MicrobiologyNON-HOST INSTITUTION: STATE,COUNTRYBluefield State College, Wv UsaTotal # human subjects expected for entire study: 0Total # human subjects enrolled to date: 0SUBPROJECT DESCRIPTIONGenital infection by Chlamydia trachomatis is the most common bacterial sexually transmitted disease (STD)worldwide. The fact that not all infected women develop chlamydia-induced sequelae would suggest thatcertain socio-biological factors such as stress status may contribute to how an infection evolves intocomplications. However, the effect of stress on the susceptibility, intensity and complication of chlamydialinfection are unknown. The central objective of this proposal is to utilize a reliable mouse model of coldstressand genital chlamydia infection that reproduces the pathological and immunological processes in humans toinvestigate the effect and mechanism(s) of stress-induced hormones on: (a) the susceptibility to and intensityof genital chlamydial infection; (b) the development of pathologies such as tubal inflammation and infertility;and (c) the host innate and acquired immune responses to Chlamydia. Our hypothesis is that cold-stressincreases the severity of chlamydia genital infection and development of complications by modulating theimmune response against Chlamydia. Based on our preliminary results, we predict that coldstressapplication leads to decreased resistance to chlamydia genital infection via induction of elevated stresshormones that are immunosuppressive. Aim #1 will continue analyzing the effect of cold water stress on thecytokine, chemokine and receptor dynamics in mice infected intravaginally with Chlamydia and assess theinfluence of cold-stress on the trafficking and distribution of immune cells in different regions of the genitaltract during genital chlamydial infection. Aim #2 will continue determining the effect of cold water stress onascending chlamydial infection and the histopathological changes of the genital tract of infected animals. Aim#3 will continue directly assessing the effect of cold-stress on Chlamydia-induced infertility by determiningthe effect on pregnancy and number of pups. Aim #4 will investigate and define the possible mechanism (s)how Chlamydia clearance is impaired due to the impact of releasing NE in our stressmouse model. The findings from this project may aid in developing an approach to reduce complications ofchlamydal genital infection and ultimately infertility in the host.SUBPROJECT PROGRESSAim #1: To assess the influence of stress on distribution pattern of immune cells in differentregions of the genital tract during Chlamydia trachomatis infectionThe purpose of this aim was to determine the localization of variety of immune cells with surfacemarkers during Chlamydia genital infection within the regions of the genital tract of mouse stressmodel using flow cytometry analysis. Cells were harvested by collagenase Type 1 treatment ofgenital tract of infected mice. Fluorescein-labeled monoclonal antibodies directed against murineCD3, CD4, CD8, CD54 (ICAM-l), CD71, CD 102 (ICAM-2), MadCAM-l, and NK were used to identifythe cell surface markers in the genital tract of mice. In general, the total number of immune cells instressed mice was reduced; however, no statistically significant difference between stressed andnon-stressed was obtained. Increased infiltration of leukocytes into the genital tract of stressed ornon-stressed infected mice was obtained. Further flow cytometry experiments are underway tolocalize neutrophils, lymphocytes and dendritic cells or adhesion molecules in the regions of thegenital tract of stressed mice during chlamydia infection. These data will aid to examine whetherthere is a direct or indirect correlation between immune cells and chlamydia load in the genital tractPHS 2590 (Rev. 06/09)Continuation Format Page
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