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Development of a robust and improved system for studying interactions between CCL20 and CCR6 using both recombinant and chemically synthesized rhesus macaque chemokines

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 (Unpublished)

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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.


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Details

Item Type: University of Pittsburgh ETD
Status: Unpublished
Creators/Authors:
CreatorsEmailPitt UsernameORCID
Klamar, Cynthia Rene`cynrenklamar@yahoo.com
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairReinhart, Todd Areinhar@pitt.eduREINHAR
Committee MemberMurphey-Corb, Michaelmcorb@pitt.eduMCORB
Committee MemberAyyavoo, Velpandivelpandi@pitt.eduVELPANDI
Date: 24 September 2010
Date Type: Completion
Defense Date: 21 July 2010
Approval Date: 24 September 2010
Submission Date: 26 July 2010
Access Restriction: 5 year -- Restrict access to University of Pittsburgh for a period of 5 years.
Institution: University of Pittsburgh
Schools and Programs: Graduate School of Public Health > Infectious Diseases and Microbiology
Degree: MS - Master of Science
Thesis Type: Master's Thesis
Refereed: Yes
Uncontrolled Keywords: CCL20; CCR6
Other ID: http://etd.library.pitt.edu/ETD/available/etd-07262010-122329/, etd-07262010-122329
Date Deposited: 10 Nov 2011 19:54
Last Modified: 15 Nov 2016 13:47
URI: http://d-scholarship.pitt.edu/id/eprint/8626

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