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THE COUNCIL FOR TOBACCO RESEARCH-U.S.A., INC. November 26, 1985 MEMORANDUM: TO: Dr. S.C. Sommers and Staff From: D.H. Ford and D. Stone Re: Site visit with Dr. George Weinbaiiia and staff at the Graduate Hospital, Philadelphia, PA, Nov. 21, 1985 Grant No. 1604R2 entitled "The role of peptide methionine sulfoxide reductase in human lungs: A possible defense against protein oxidation and elastin degradation in smokers." Goal: To determine if the various isozymes of Met (0)-peptide reductase play an important role in reducing inactive oCl-PI to the active form to permit it to inactivate elastase and thus prevent elastin degradation. Observation: This has been a very productive program during the year and a half which has elapsed since our last visit, as evidenced by the attached list of publications (10 since our last visit about a year ago). Progress has been masle in a number of areas as briefly summarized below. Results: I. Use of antibodies to localize elastase and elastin in tissue by EM immunocytochemistry. Dr. Damiano has developed techniques which have permitted him to observe that elastase protein appears first in the ER, in PMN cells. It then migrates to the Golgi apparatus and then is released into vesicles which fuse with the plasma membrane to extrude elastase by exocytosis into the extracellular space, where it was observed to adhere to elastin, in a patchy manner. In human lung tissue from patients with emphysema he has observed interstitial cells (of unknown origin, possibly, derived from fibroblasts) to contain material which is immunologically positive as elastase, which follows the same organelle course as in the PMN cell, and is released into the extracellular spaces. Quantitative studies on EM sections demonstrated in 8 patients (had lung resections because of tumors) that the degree of development of emphysema was positively correlated with the amount of elastase shown to be in association with the elastin. II Animal studies performed with rats and with dogs exposed to smoke or treated with chloramine-T These studies confirm the observations that smoke exposure recruits PMNs to the lung. As PMNs pass through the interstitial tissue they degranulate and the elastase appears to then associate with the elastin.

-2- Some elastase released is taken by macrophages, thus adding to the indigenous macrophage elastase, which appears to be unaffected by oc 1-PI. How much the macrophage elastase contributes to the breakdown of elastin is unknown, however. No2 exposure also recruits PMNs. Under these circumstances the PMNs also lose their elastase, but it is not known where. Chloramine -T given intravenously also causes elastin degradation, because it interferes with normal activity of al-PI to inactivate elastase. Once elastase degradation of elastin has started, by whatever means, elastin degradation appears to continue even though the causative factors may no longer be operative. Do some of the peptide fragments of elastin produced by degradation act as signals to further recruitment of PMNS or is macrophage elastin now involved? A number of other studies were reported which cumulatively implicate the PMN as a major contributer to the release of elastase in lung tissue with the subsequent degradation of elastin. Weinbuam et al are improving their assays for detecting additional peptide fragments of elastin in plasma, Since urinary desmosine assays are not a specific or quantitative index of pulmonary elastin breakdown, they are also refining their EM, immunocytochemistry to quantitate the degree of association of released elastase with elastin in relation to exposure to toxic agents. III Met (0)-peptide reductase: A few years ago,when this enzyme was intitially described by Abrams and others, it was thought that it might comtribute to the emphysema picture by serving to reactivate the oxidized methionine molecule formed when « 1-PI is exposed to oxidants. Although the reductase will reduce oxidized oCl-PI in vitro, it now appears that it probably has little or no effect in vivo in a smoke exposed animal, since it too is inactivated by exposure to smoke products. Thus, while this seemed to be an interesting possible protective mechanism normally present in tissue, it now seems unlikely that it may contribute significantly to prevent elastase activity. Synthetic peptide: A synthetic peptide composed of 19 amino acids with a sequence similur to those from the carboxyl terminus of troRoelastin has been indentified. Antibodies to it cross react~elastin peptides released after degradation. Is this a unique peptide sequence normally found in blood after elastin degradation by elastase? If so, could it be used to indentify individuals early on in the development of emphysema? The gene sequence for this peptide has been determined. Is it found only in subjects who develop emphysema? Future aims: Determine in man if the amount of elastin bound elastase increases with the severity of disease.

-3- Intitial studies with the 8 tumor patients suggest a postive correlation. In the dog model, determine how long it takes after smoke exposure for the levels of elastase in the lung (in alveolar washes and associated with elastin as determined by EM immunocytochemistry) to return to normal in relation to how long it takes for the level of circulating elastin peptides to return to normal. Comment: This is a highly integrated morphological and biochemical program concerned with the causation of emphysema. Progress from year to year is consistently high. Thus, they are developing a quantitative morphological picture of the release of elastase containing granules from PMNs and the association of the enzyme with elastin. This is being correlated with assays of ciculating elastin peptides which will be able to be used to quantitate the degrees of elastase attack on elastin as well as to determine the severity of lung function in patients. Preliminary studies suggest that there may be unique amino acid active center sequences in elastin that are more readily attacked by elastase. While there are many other laboratory groups investigating this problem, The Weinbuam group has to be rated among the best. As such it merit our continued support. DHF/DS DHF/mla