Scientific Abstract

Proposal No.   IBD-0090R
Principal Investigator:  Otto Phanstiel, Ph.D.
Applicant Organization:   University of Central Florida (Orlando, U.S.A.)
Project Title:  Development of polyamine conjugates as sensitive probes to explore inflammatory bowel disease processes
Period of Award:  May 1, 2004 – February 28, 2007

The study will evaluate the possibility of high polyamine transport activity as a causative agent in inflammatory bowel disease (IBD).  Average polyamine levels will be determined in tissue, macrophages and monocytes obtained from both IBD (n=15) and healthy subjects (n=15). Several polyamine probes will be synthesized and used to assess polyamine uptake in isolated macrophages and monocytes from IBD subjects and healthy individuals.  These probes have a high affinity for the polyamine transporter (PAT) and can be used to rank the polyamine uptake rates in different samples by direct microscopy measurements or by high performance liquid chromatography (HPLC) analyses of cell lysates.  This approach is innovative as it uses novel fluorescently tagged polyamines to assess PAT activity in IBD-patient samples.

The proposed work is significant as the role of polyamines in inflammatory bowel disease is unknown and the study will quantify, for the first time, polyamine uptake in IBD macrophages and monocytes.

Short-term outcomes include the development of a sensitive fluorescent probe that has high affinity for intestinal cells with highly active polyamine transporters.  Long-term outcomes include a convenient assay, wherein microbiopsies or isolated macrophages from IBD patients could be incubated with the special probe and screened for high PAT activity.  In addition, polyamine transport inhibitors could provide a possible treatment for IBD by inhibiting the uptake of these polyamines.

The study will also attempt to isolate the mammalian polyamine transporter protein (PAT) by using a custom-built, affinity column that has a high-affinity immobilized-PAT substrate covalently bound to a Sepharose column.  By passing cell lysates from IBD tissue down this column, we hope to isolate the PAT protein responsible for polyamine transport into cells from IBD-afflicted tissue.  Since all cells are known to contain polyamines, characterizing the PAT protein will have implications for IBD, cancer and other polyamine-dependent diseases