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TAr COUNCIL FOR TOBACCO RESEARCH-U.S.A., INC. A September 19, 1985 TO: Dr. S. C. Sommers and Staff FROM: D. H. Ford RE: Site visit with Dr.,Re$gro and Staff, University of Colorado, Denver, CO. Grant #1322ARI, "Basic Mechanisms of Lung Injury From Oxygen Radicals" Site Visitors: Drs. D. H. Ford and H. McAllister OVERVIEW Dr. Repine reviewed the major concept of the program concerning the role of oxygen radicals in the causation of lung disease. This is well covered by his last progress report, which indicates an important role for PMNs in ARDS. His total research group now totals about 30 individuals, but only 10, besides himself, are involved in the CTR program. In Repine's view, the following schematic illustrates the relationship between PMNs and ARDS: Chemotaxins (from macrophages) prostaglandins + metabolites Hem ynamic endothelial ~ changes ~ permiability Other intermediates (Kinins Histamine) Dr. Repine and his group have made a major effort to quantitate the levels of oxygen radicals released and the degree of damage caused by PMNs over the last 4 to 5 years,with CTR support. During this period they (as well as others) have clearly established a relationship between macrophages, PMNs and free radicals + protease and seem to be attaining the point in this series of investigations where their initial goals will have essentially been reached. Only the role of the antichemotaxin from macrophages (Dr. Harada) and the role of RBCs (Dr. Beehler) as carriers of free radical scavengers appear as interesting goals yet to be resolved. The following briefly reviews the studies of individuals group members: Dr. Natalie Parker: DMSO has been used to measure the levels of hydroxyl radical and DMTU to measure peroxide, both in vitro and in vivo,in a perfused lung model as well as in rats. Since-each scavenger is specific for a particular reactant, they can be used to measure either one independently. Further, the re- action between DMTU and H202 forms a product which can be measured quantitatively. 4

SITE VISIT DR. REPINE - GRANT 11322AR1 P. 2 Dr. Ruth Harada: Dr. Harada has observed that while activated macrophages release a chemotaxin for PMNs, unactivated macrophages release a negative taxin which prevents recruitment of PMNs and their adherence to endothelial membranes. While Dr. Harada has characterized this compound as a small molecular weight lipid soluble molecule, she has been unable to identify it further. She has recently been joined in her project by a biochemist, Dr. J. Gerens, who Repine hopes will provide the needed expertise to characterize the ligand and then help determine its role with the chemotaxin in regulating PMN movement. Mr. Tom Eyen (2nd year medical student): He is using bleomycin instead of hyperoxia to activate macrophages and cause toxic damage in the lung. The mechanism by which bleomycin produces its damage in lung appears to be by the release of H202, since the damage is blocked by DMTU, but not by DMSO. Lung damage in this model is assessed by the increase in lactate dehydrogenase (LDH). Dr. Ruth MacDonald (Assistant Professor of Pediatrics): Dr. MacDonald has a 5-year NIH fellowship which provides her salary. Her study confirms the concept that 02 metabolites cause lung damage according to the scheme: Phorbol myristate acetate (PMA) + PMNs -> 02 metabolites ~ increase in thromboxane (TXB2), lung weight (edema) and pulmonary artery pressure. This is accomplished using an isolated gerfuse lung preparation. The increas, in TXB2 is blocked by aspirin and indomethicin, while DMTU blocks the effect of the PMA in activating the PMNs to release 02 radicals. Mr. Bruce Baird (3rd year medical student): Mr. Baird's observation supports the reports of others that free radicals derived from oxygen inactivate the anti- proteases, thus permitting uninhibited activity of PMN proteases on elastin and other proteins in an isolated perfused lung model. As perfusate levels of H202 increase, levels of antiprotease activity decrease. This was accompanied by an increase in lung weight (edema) and an increase in pulmonary artery pressure. The question was raised and discussed as to what degree the raise in pulmonary artery pressure could be due to an effect of oxygen radicals on ligands of the intrinsic peptidergic neuronal system of the lung, which appear to play a role in regulating vascular diameter (Polak and Bloom). In this model the presence of oxygen radicals appears to exacerbate the damaging effect of PMN derived protease. Dr. Carl White (Assistant Professor of Pediatrics): Dr. White also has a 5-year grant for salary support from NIH. He has been working with polyethylene glycol (PEG) and found that when both SOD and catalase are conjugated to PEG and administered together, they block lung injury caused by oxygen radicals,due to the increased circulating half-life of the enzymes. Further,while in this con- jugated form, there is no interference with immune functions. Pleural effusion is decreased and the oxidation of GSH by 02 to GSSH is also reduced. However, the two conjugates do not promote an additional synthesis of GSH. Miss Karen Toth (3rd year medical student): Miss Toth has been evaluating the role of RBCs in preventing ARDS by their contained antioxidants (GSH, catalase and hemoglobin). Her studies show that it is the GSH which functions as the

r SITE VISIT DR. REPINE - GRANT 41322ARL Page 3 effective antioxidant. In rats exposed to smoke, GSH increased 36% in RBCs while catalase increased only 13%. GSH peroxidase did not increase at all. The same was observed in humans who smoke. Further, RBCs from smokers protected endo- thelial cells (as measured by LDH release) more than RBCs from non-smokers. Dr. Connie Beehler (a pulmonary specialist post doctoral fellow): Her study deals with acute lung disease caused by hyperoxia in rats. Hyperoxia increased RBC GSH in both adult and neonatal rats. (Hyperoxia is used with the rat model because it produces a lung injury similar to the ARDS seen in man). Repine feels that the increase in RBC GSH may be used as an index of degree of lung injury, though this remains to be determined. COMMENT This program now has 11 active participants. However, most of them, except Repine, are part-time, being students or having clinical responsibilities. Thus, it is conceivable that the minimal progress which has occurred since our last visit is due to the other responsibilities of the group. Nevertheless, in consideration of the large group concerned with this program, we were disappointed in their failure to make appreciable progress in the program beyond where they were last year. Some areas of investigation (i.e., the dual role of the macrophage in attracting or preventing attraction of PMNs to a site and the role of RBCs in protecting against free radicals) are essentially unchanged from the time of our visit a year ago. Thus, while this group has been productive in publishing papers, it appears to progress very slowly toward the stated goals and to be almost endlessly designing 'studies which confirm that free radicals are a major cause of damage to lung tissue. Thus, while we would recommend continued support through the final year of their current 3-year cycle, we would hesitate to consider future support beyond that time unless they have clearly attained most of their goals and defined a new and exciting attainable program for the future. DHF/HMcA '4b