Public Health Relevance: The United States has 80 million employees with chronic pain resulting in annual losses of 61.2 billion dollars due to pain-related productive time lost. In addition, pain-related depression and inactivity reduce the quality of life. The development of effective analgesics is therefore important from a public health perspective. In this dissertation, the natural properties of herpes simplex virus (HSV-1) vectors are exploited to (i) develop an HSV-1 vector-based selection system that can potentially identify natural or chemical inhibitors of chronic pain and (ii) to test HSV-1 vector-expressed dominant negative PKCε (DNP) as a strategy to treat chronic pain.
The vanilloid/capsaicin receptor (TRPV1) is a pro-nociceptive calcium ion channel that is upregulated in chronic pain. This occurs partly due to protein kinase C epsilon (PKCε)−mediated receptor phosphorylation. An HSV-1 vector expressing TRPV1 (vTT) was engineered and vTT-expressed TRPV1 functionality was confirmed.Treatment of vTT-infected cells with capsaicin or resiniferatoxin caused concentration-dependent Ca+2 influx, leading to cell-death and a dramatic reduction in infectious particle yield. TRPV1 antagonists, ruthenium red and SB-366791 reversed agonist-induced cell-death and rescued vTT growth, providing a basis for selection. Selection for antagonists was modeled using a mixed infection of vTT and vHG (capsaicin resistant control vector) and virus passage in the presence capsaicin. These experiments demonstrated that a single control vector particle was readily isolated from a population of 10^5 vTT particles. This approach can be used to identify antagonists from chemical or gene libraries and offers advantages of (i) a platform assay applicable to other ion channels and (ii) adaptability to high throughput formats.
Dominant negative PKCε (DNP) was engineered into HSV-1 to create the vector, vHDNP. Following functional confirmation of vHDNP in U2OS, Vero cells and neurons, cobalt uptake showed a reduction of capsaicin sensitive vHDNP-transduced neurons. Electrophysiology confirmed this and also demonstrated a knockdown of TRPV1-PKCε coupling in nociceptive neurons. In-vivo studies of noxious heat-induced nocisponsive behavior in vHDNP-inoculated rats showed a subtle inhibition of withdrawal responses when compared with controls. In conclusion, HSV-1 expressed dominant negative PKCε is a viable strategy to specifically inhibit TRPV1 function in order to treat chronic pain.
| Identifer | oai:union.ndltd.org:PITT/oai:PITTETD:etd-07302006-164356 |
| Date | 25 September 2006 |
| Creators | Srinivasan, Rahul |
| Contributors | Donald B DeFranco, Joseph C Glorioso, Robert E Ferrell, Saleem A Khan |
| 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-07302006-164356/ |
| Rights | unrestricted, 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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