eprintid: 8626 rev_number: 5 userid: 6 dir: disk0/00/00/86/26 datestamp: 2011-11-10 19:54:07 lastmod: 2016-11-15 13:47:01 status_changed: 2011-11-10 19:54:07 type: thesis_degree metadata_visibility: show contact_email: cynrenklamar@yahoo.com item_issues_count: 0 eprint_status: archive creators_name: Klamar, Cynthia Rene` creators_email: cynrenklamar@yahoo.com title: Development of a robust and improved system for studying interactions between CCL20 and CCR6 using both recombinant and chemically synthesized rhesus macaque chemokines ispublished: unpub divisions: sch_gsph_infectiousdiseasesmicrobiology full_text_status: public keywords: CCL20; CCR6 abstract: 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 laboratory's 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. date: 2010-09-24 date_type: completed institution: University of Pittsburgh refereed: TRUE etdcommittee_type: committee_chair etdcommittee_type: committee_member etdcommittee_type: committee_member etdcommittee_name: Reinhart, Todd A etdcommittee_name: Murphey-Corb, Michael etdcommittee_name: Ayyavoo, Velpandi etdcommittee_email: reinhar@pitt.edu etdcommittee_email: mcorb@pitt.edu etdcommittee_email: velpandi@pitt.edu etdcommittee_id: REINHAR etdcommittee_id: MCORB etdcommittee_id: VELPANDI etd_defense_date: 2010-07-21 etd_approval_date: 2010-09-24 etd_submission_date: 2010-07-26 etd_access_restriction: 5_year etd_patent_pending: FALSE assigned_doi: doi:10.5195/pitt.etd.2011.8626 thesis_type: thesis degree: MS committee: Todd A Reinhart, ScD (reinhar@pitt.edu) - Committee Chair committee: Michael Murphey-Corb, PhD (mcorb@pitt.edu) - Committee Member committee: Velpandi Ayyavoo, PhD (velpandi@pitt.edu) - Committee Member etdurn: etd-07262010-122329 other_id: http://etd.library.pitt.edu/ETD/available/etd-07262010-122329/ other_id: etd-07262010-122329 citation: Klamar, Cynthia Rene` (2010) Development of a robust and improved system for studying interactions between CCL20 and CCR6 using both recombinant and chemically synthesized rhesus macaque chemokines. Master's Thesis, University of Pittsburgh. (Unpublished) document_url: http://d-scholarship-dev.library.pitt.edu/8626/1/Cynthia_R_Klamar_8.11.10.2.pdf