The chemokine CCL20 is thought to be an integral part of the communication between the innate and adaptive arms of the immune system, due to expression of the cognate receptor, CCR6, on immature dendritic cells and on memory T cells and B cells. Interest in this particular chemokine/chemokine receptor interaction has grown over time and more recently due to roles in SIV infection, mucosal immunology, and vaccinology. The need to further study the CCL20/CCR6 interactions is bolstered by our laboratorys previous findings of increased expression of CCL20 in acutely SIV infected lymph nodes and the increased expression of CCL20 in response to PAMPs in cells of lymphatic vessels. This thesis aims to develop and improve a system for studying the interaction between CCL20 and CCR6. I have found that the recombinant expression system utilized to obtain macaque chemokines provided highly pure fusion proteins. However, cleavage of the fusion protein into macaque CCL20 has been inefficient. Rhesus macaque CCL20 chemically synthesized using regioselective cyclization was highly biologically active using the chemotaxis assay and stable cell lines expressing CCR6. Chemotactic inhibition studies identified five compounds that inhibited CCL20 induced chemotaxis. The surfactant, GML, did not inhibit CCL20 induced migration. The anti-inflammatory botanicals, EGCG and gallotannin, both inhibited CCL20-driven migration at high concentrations. The three CCR6 extracellular loop mimetic peptides also partially inhibited CCL20 induced migration at high concentrations. In conclusion, I have utilized both a recombinant protein expression system and regioselective cyclization peptide synthesis to obtain bioactive, nonhuman primate chemokines. I have also successfully developed an in vitro system to study CCL20 induced migration, and have identified a number of botanical and biochemical elements that inhibit CCL20-induced migration. The public health significance of this study is related to the fact that vaccine efficacy may be affected by anti-inflammatory compounds that inhibit CCL20 mediated chemotaxis. Another way in which public health could be affected by this study is in using the anti-inflammatory compounds studied to treat chronic inflammatory conditions in which the pathology of the disease is related to up-regulation of CCL20 and CCR6.
| Identifer | oai:union.ndltd.org:PITT/oai:PITTETD:etd-07262010-122329 |
| Date | 24 September 2010 |
| Creators | Klamar, Cynthia Rene` |
| Contributors | Michael Murphey-Corb, PhD, Velpandi Ayyavoo, PhD, Todd A Reinhart, ScD |
| Publisher | University of Pittsburgh |
| Source Sets | University of Pittsburgh |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.pitt.edu/ETD/available/etd-07262010-122329/ |
| Rights | restricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Pittsburgh or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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