New CFRI Research Targets CF Defect Site

By Lorin Fink, Summer 1994

CFRI is so dedicated to finding a cure or a control for cystic fibrosis (CF) that it has made a commitment to funding a new cell-secretion study-- despite the current focus in CF research on genetic solutions. While genetic research dominates newspaper headlines, physiological studies provide the foundation for pursuing a genetic solution for CF. The current cell-secretion study by Dr. Madireddi Reddy of the University of California at Riverside represents an investment in basic physiological CF research that CFRI thinks will pay big dividends in finding a control and cure for the disease.

What Basic Research Will Accomplish

The benefits of approaching the CF problem through research into cellular physiology, according to Dr. Reddy, are two-fold. By understanding the specific cell type in which the CF process occurs--and the exact locations within those cells where, for example, ion transport through chloride channels is or is not functioning--researchers can pharmaceutically intervene to control the problem. In the longer term, identifying CF on a cellular level may help gene therapists to target and optimize Cystic Fibrosis Transmembrane Regulating protein (CFTR) gene therapy. (With CFTR therapy, gene therapists insert normal CFTR genes into cells that have defective or non-existent CFTR genes, thereby creating a "heterozygote" or "carrier" cell that functions at least somewhat normally.)

Because this experiment will attempt to answer one of the most fundamental questions facing CF researchers, CFRI has recently funded Dr. Reddy's $50,000 study, "New Secretory Mechanisms of Fluid Secretion from Identified Secretory Cells in Cystic Fibrosis." In this study, Dr. Reddy hopes to be the first CF researcher to identify exactly the specific cell type where the CF defect is occurring in secretory coil cells. This issue is germane to the targeting aspect of any therapy. Each internal organ, whether it is the lungs, intestines, pancreas, or other exocrine glands, is comprised of hundreds of different cell types. Finding the exact cells that are causing the problem within these cell systems will then help therapists focus their efforts and thus speed up the process of controlling cystic fibrosis.

The Study's Methodology

Because many CF problems occur in the exocrine gland cells in various organ systems, exact cell-typing of clear and dark cells may bring a greater understanding of the causes of CF. Dr. Reddy's lab will biopsy and study the secretory coils in the sweat glands of people who do not have CF. The secretory coils are being studied because of their simple makeup of only three cell types: myoepithelial, clear, and dark cells. The clear and dark cells in the sweat gland secretory coil function similarly to cells in exocrine glands and other locations where the CF defect occurs. Many researchers hypothesize that the clear cell is a "fluid-secreting" cell and the dark cell is a macromolecule (i.e., enzyme or other solid molecule) secreting cell.

To identify the clear and dark cells, Dr. Reddy will first stimulate an isolated normal secretory coil cell with a chemical agent. After determining that there is secretion based upon electrical measurement, Dr. Reddy will then mark that particular cell with a fluorescent dye. He can then observe under the microscope cross-sections of the dye-stained cell to determine whether it is clear or dark. Likewise, if Dr. Reddy isolates a cell that does not respond to chemical stimulation, he will then identify it under the microscope as either dark or clear.

This basic cell-typing and property identification has been technically impossible to accomplish until recently. In 1994, Dr. Reddy's lab was first able to identify the electrical signature of the myoepithelial cell of the sweat gland secretory coil. Musculature, dirt, and macromolecules have all damaged microelectrodes as researchers tried working on cells measuring only .3 microns. The specific micromanipulation techniques developed by Dr. Reddy and his lab will now enable him to first measure the electrical signature of the dark and clear cells and at the same time morphologically link that signature to the exact cell type.

The next step in the research project involves identifying specific properties of these newly typed or identified cells. The key question is, "Are there different or alternate chloride channels in these cells where other chemical agents can stimulate secretion?" The properties of the unaffected secretory cells will then be compared to the properties of the CF-affected secretory coil cells. Once this is accomplished, researchers can then demonstrate where the defect is occurring in the CF secretory coil cells and, hopefully, find alternate pathways for proper fluid secretion, thus controlling the thick mucus that causes havoc in CF patients.

Goals Of The Research

In summary, Dr. Reddy hopes to achieve three goals through his experiment. First, he will work to identify which cells are fluid-secreting and macromolecule-secreting by connecting electrical signatures to dye-stained images of the cells. He will then identify the different properties of these normal, unaffected secretory coil cells; in so doing, he will establish a physiological and morphological control for two essential secreting cell types.

Second, he plans to test the effects of a variety of secretion-stimulating chemicals to induce secretion in the cell types. He hopes that alternative pathways will be discovered in the normal cells that might be used to circumvent the defective CF pathway.

Finally, Dr. Reddy wants to conduct the same experiments with CF secretory coil cells and compare the results. With this comparative data, he should be able to make some conclusive statements as to where the CF defect is occurring in the cell and what alternate pharmacological stimulating agents may alter the CF condition in the sweat gland secretory cell.

After Secretory Coil Cells?

Once Dr. Reddy has successfully completed the secretory cell experiments, he can then move on to other more critical cell types, like the sub-mucosal serous cells in the lungs. These serous cells have been hypothesized to be the site of the excessive mucus production in the lungs of people who have CF. If the models of the fluid- secreting and macromolecule-secreting cells of the secretory coils can be applied to this and other cell types, then perhaps CF researchers will be very close to understanding where the CF defect is occurring, and what alternate pathways in those cells could be exploited to circumvent the CF cellular defect.

Dr. Manreddi Reddy of University of California at Riverside is one of the world's leading technicians in the micromanipulation of CF cells that measure .3 microns in size. His laboratory experiments will now try to match electrical signatures and morphological structures to determine in which cells the CF defect is occurring.

"I'm enthusiastic about gene therapy research but I'm also realistic enough to know we need a great deal more fundamental physiological research and cell biological research before gene therapy will work," said Dr. Jeffrey Wine of Dr. Reddy's research. Dr. Wine is a Professor at the Stanford University Cystic Fibrosis Research Laboratory.

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