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INFOTAB ,.,.m.,,o.., To.o=o ,.,o..,.,o. 0..,.. Cenlre International d'lnformalion du febac MEMO To : From: Date : Re : See Distribution List Catharine Browne August 18, 1986 Reports on scientific meetings .° . -,, ~-:-~. ., ! !- "-- 22 ~uG m6 1 \ ..... • .............. 1 ~".-----" ........................... Please find enclosed Observers' reports on the following meetings: - "Second International Symposium on Pulmonary Emphysema and Proteolysis" (January 14-16, 1986), Pasadena, California - "American Occupational Health Conference" (April 27-May 2, 1986), Denver, Colorado - "Annual Meeting of the American Lung Association and American Thoracic Society" (May 11-14, 1986), Kansas City, Missouri. Best regards, Catharine Browne Analyst IS-892.86 "-4 C~ -~ kO Rue Montoyer 10 - Bte 2 B-l~0 Bms~ls Belgium Tel. 02/511.91.10 Telex 24218 F~ 0~513.~.43 TVA 419.7~.~ BATCo document for PFSFC 1 March 1999

DISTRIBUTION LIST Dr. F. Adlkofer Dr. H. Bentley Mr. P. Brown Dr. Colcucci Mrs. K. Comer Mr. J. Dollisson Dr. G. Felton Dr. H. Gaisch Dr. C. Green Mr. T. Haynes Mr. Lasse Hjern Mr. G.W. Moore Dr. C.W. Nystrom Mr. T. Osdene Ms. M. Pottorff Dr. R. Ralph Dr. D. Rowland Dr. G. Smith Dr. R. Thornton Dr. T. Vossenaar Dr. D. Westcott Dr. D. Woodman / 0 "-4 kO mO BATCo document for PFSFC 1 March 1999

Second International Symposium on Pulmonary Emphysema and Proteolysis (January 14-16, 1986) Pasadena, California, USA (An Observer's Report) There have been tremendous gains in scientific knowledge about pulmonary emphysema since the first symposium on the disease was held in 1971. At the same time, and not surprisingly, the ad- vances have raised further major questions that have yet to be answered. (Pulmonary emphysema involves the breakdown of the al- veoli, the tiny air sacs in the lungs, by enzyme degradation of elastic tissue; the alveolar air spaces are enlarged, reducing the surface area available for gas exchange.) Studies in the last 15 years have led to what is now clearly the most popular hypothesis of causation for most cases of the disease: the inactivation by substances in cigarette smoke of the so-called alpha-one protease inhibitor {aIPI), a protective screen in the lung, and the consequent degradation of the al- veolar tissues by proteolytic enzymes. The responsible agents in the smoke are believed by many to be primarily oxidants which, by inactivating aIPI_, tip the delicate aIPI-protease balance the wrong way, enabling the elastase to wreak its damage. This imbalance, some claim, causes perhaps 80-90% of the cases of emphysema that occur in a minority, per- haps 15%, of mostly male smokers (the numbers vary according to who's reporting). The remaining cases are those individuals who are seriously deficient in aIPI as a result of genetic factors. There are an estimated 100,000 such PiZZ phenotype cases in the United States; the genetic tendency for emphysema is so strong in these cases that they develop ~he dlsease regardless of anything else. These and other points were brought out at the symposium which attracted some 150-175 scientists from the U.S. and abroad, most- ly from Europe. Many of the attendees were present or former Council for Tobacco Fesearch grantees, and at least a dozen persons from the~e classifications were on ~he progra:. It's fair to say that a number of CTR grantees are among the handful of scientists wbo've made (and are continuing to make) major con- tributions to emphysema research. The symposium was organized by CHARLES MITTMAN o:" the City of Hope, Duarte, CA, who also organ- ized the 1971 ses:~icn. Sponsors included several drug companies which obviously a:-e working on drug treatments for emphysema and other serious lung diseases. O "-4 L~4 BATCo document for PFSFC 1 March 1999

~e It was the kind of meeting where contradictions in findings were adm~itted by some researchers or seemed to become apparent in the data presented. It also was a meeting where several speakers talked about the role of smoke [or smoking) in emphysema, commen- ted that there still was controversy about this role, and noted that the mechanism(s) for smoke's role was unclear or unknown. What was clear is that there is considerable information about what can happen or what's possible in terms of emphysema causa- tion in smokers. What's much less clear is what is actually hap- pening, and how, and by what components of cigarette smoke. At least that's the way BENJAMIN BURROWS of the University of Arizona in Tucson put it as he tried to sum up the symposium as the final speaker. It was a difficult task for hlm, he said, not because he's a clinician, but because there are, in his view, so many important questions that still remain unanswered and so many areas which, despite the great amount of research that's been done, still have to be examined. He seemed to be saying: let's avoid the dogma and the flat assertions because emphysema is far too complex a disease for simple explanations that some people are offering. He also seemed to be saying that so much information about emphysema is being obtained that it's difficult to decide what's really valid or applicable and what's unimportant or inapplica- ble. There are "more mechanisms than we need to explain how smoking might result in emphysema," he said at one point, and one can make "elegant" theoretical frameworks for pathogenesis of the disease even when there are normal levels of circulatinE anti- protease. Confusion arises when, for example, lnvestlgators report that lung protective cells (macrophages) also produce destructive enzymes, that another type of cell (the mast cell) has been discovered to make elastase, that there appear to be several enzymes with different characteristics that can degrade lung elastin, and that the "garden variety" of emphysema (a phrase used by many speakers) occurs in only a small fraction of those who smoke cigarettes. Also, there may be several forms of at?I, perhaps as many as four, and it's not sure whether all have been fully identified or defined. Some speakers at the sym;osium talked about clinical testz for replacement therapy that may benefit t~e minority of emp~yse:5 victims who are deficient in aIPI because of hereditary factors. However, the efficacy of aIPI in the alveoli in the a~sence of significant circulating levels of the inhibitory agent (in t~e blood) is unknown. Yet there are efforts to use aIPI and also other "protective" agents, including vitamin C, in treating or ;reventing emphysema (other lung ~iseases may be treated as well). Some "new" findings of possible significance were disclosed at the symposium, but by and large the reports offered were, for the ~D L~n BATCo document for PFSFC 1 March 1999

o most part, reviews and/or summaries of work In progress. The highlights: I. Burrows, last on the program but certainly not least, did not endear himself to too many with some of his comments. Here- with a relevant selection: Emphysema in smokers is far more complex than that in the hereditary form of the disease (PiZZ phenotype). There's general agreement that emphysema in smokers is not dependent on a defi- ciency of circulating aIPI. There's no reason to think that in- travenous a]PI would be effective in preventing or treating the disease. There's evidence that smoking can influence the protease-anti- protease balance. Smoking elicits a cellular response leading to release of proteolytic materials; the cell-cell interactions are very complex, and new mediators and new consequences of these interactions are being discovered regularly. Simultaneously, smoking can inhibit certain antiprotease mechanisms and at least part of this seems related to oxidation. The lung's antiprotease mechanisms are extremely complex; some depend on the same cells that release proteolytic enzymes. Smok- ing also may interfere with elastin repair (a process that seems to occur normally in some experimental animals following induc- tion of what is sald to be emphysema or emphysema-like damage). There are too many potential mechanisms. Their relative impor- tance seems to vary from one animal model or vitro model to an- other, and their effect in man is often obscure. There is no te- llable animal model for smoke-induced emphysema and this makes it difficult to interpret the effects of smoking in many experimen- tal settings. Much of what has been learned appears applicable to inflamma- tory (lung) disease in general and in some cases to the genesis of lung fibrosis. It's sometimes difficult to place such studies in perspective in terms of human emphysema. Adding to the com- plexity are the possible contributions of microorganisms and their foreign substances. A defect cr deficit in some regu!a~ory mechanism could well ~e the the critical ~eterminant in smoker's emphysema, and our knowledge of such mechanisms is limited indeed. A popular theory is that there's a defect in the antioxldant capacity of suscep- tible smokers that is of major importance in the development of emphysema. Ultimately, determining a specific derangement or set of de- fects that are critical in emphysema in a presumed minority of smokers will depend on demonstrating an abnormality that's clearly related to the risk of developing the disease. A bit of mythology perpetuated at this conference needs to Oe mentioned; 14 BATCo document for PFSFC 1 March 1999

4 • the mythical 15% or 20% of smokers who supposedly are sensitive Co getting emphysema. Clearly, a spectrum of risk is involved• We're really talking about those who will get the disease so early in life that it's disabling before other competing risks arise. There are a variety of attractive theories related to the question of what kind of smoker may be most at risk. The solution will depend on the ability to recognize emphysema before its crippling effects become evident. Once the disease is present, the problems in Interpreting bronchoalveolar lavage (BAL) fluids become greater. A major advance would be the ability to recognize when elastin breakdown begins to occur. There's more to chronic obstructive pulmonary disease than simple destruction of lung elastin. Emphysema is almost univer- sally present in middle-aged male smokers who die of airflow obstruction. But emphysema is not the sole cause of obstruction. Indeed, the exact place of emphysema in the total evolution of chronic disabling airflow obstruction remains controversial. 2. AARON JANOFF, long-time emphysema researcher at the State University of New York, Stony Brook, reported he's undertaking a new study that will use immunochemical techniques to detect and perhaps localize oxidized aIPI in smokers rather than measure average values in BAL. He'll use tissues from baboons and mono- clonal antibodies that specifically recognize oxidized aIPi. It's been reported that oxidants can affect lung tissue directly, Janoff said. This information, plus the contention that oxidants affect the protease-antiprotease balance, makes clear that there's still a controversy about the effect of smoking in aIPl function. There's some evidence that smoking, either directly or indi- rectly, can affect the function of antielastases in the lung so as to permit the elastases to degrade the tissue. This, "if true" in humans, may in turn be strongly influenced by antioxidants in the lung. Therefore, it will be important to see whether varia- tions in the function of these antioxidants (such as ceruloplas- min, transferrin, etc.) can be assessed to determine susceptibil- ity to emphysema. Jazoff went on to speculate a~cut a "new idea" on which ne's working -- the ~otential role cf mast cells (and basopnils} in smokers' emphysema. (Hast cells are connective tissue cells whose -specific physiologic function is unknown; basophi!s, whic~ can be derived from mast cells, contain vasoactive substances such as histamine and serotonin). The human alveolar wall contains many mast cells -- over 300 per square millimeter, Jaroff estimated -- and they directly abut collagen and elastln fibers in the alveolar wall. It's been re- ported that two kinds of ~nzyme -- neutrophll elastase and cath- epsln G [a relatively wea~ elastase) -- have been found in purl- "-4 kO rko L21 cy% BATCo document for PFSFC 1 March 1999

5. fled human lung mast cells and basophil cells. His laboratory has confirmed the presence of small amounts of neutrophil elastase in basophlls. (Neutrophils are granular leukocytes, or white blood cells.) He's also found that cigarette smoke can "degranulate" baso- phils (i.e., make the cells secrete what's in their granules); the cells released histamine. There's one paper in the literature that has reported smoke also degranulates lung mast cells ob- tained from subhuman primates. Janoff said he's done a study of 8 smokers and 10 nonsmokers and found significantly more histamine in BAt fluids from the former than in fluids from the latter. Therefore, the mast cell is another potential source for lung damage in smokers. Basic compounds such as amines can trigger mast cell activation and secretion, and many such compounds exist in cigarette smoke. Or it may be that certain smokers who are sensitized to antigens in smoke will have mast cells that will undergo such a reaction. The mast cells may secrete radicals or proteases; they may release substances that could attract other cells to the site, cells that secrete destructive enzymes. There were lots of "mays" as Janoff admitted there was no hard data to suppor~ his comments; he was just speculating. Later, during discussion, Janoff made these comments: Many investigators have reported experiments in which It was found that aIPI can be inactivated directly by oxidants in ciga- rette smoke or indirectly by oxidants released from smoke-stimu- lated phagocytes. Whether this happens in man is presently con- troversial• In fact, different groups have reported different findings about smoke and its effects on aIFl. 3. WILLIAM PRYOR of Louisiana State University in Baton Rouge, another veteran emphysema scientist, set out to review the free radical chemistry of cigarette smoke and the oxidation of aIPI. The outcome, however, was a long, often rambling discourse that seemed to touch on every conceivable aspect of the disease and his own research. Some of the voluminous data he presented appeared to be new or very recent, particularly thosec.=~o-~.n~- with protecting a1?Z against inactivation. Among his comments: The inactivation of alP! in humans originally was attributed " to so-called reactive oxygen species [hydrogen peroxide, super- oxide anion, hydroxyl radicals) that derived from certain smoke- stimulated cells in t~e lungs [polymorphonuclear cells and alv~- olaf macrophages). Hcxever, it has been learned that smoke can directly inactivate aIPI in vitro, suggesting the presence of oxidants in the smoke. Additionally, reactive oxygen substances in the lung and smoke components such as nitrogen oxides could somehow combine to pr:duce an inactivating agen; (or agents). ====am r~o L/I BATCo document for PFSFC 1 March 1999

. Several laboratories have reported ~he inactivation of aIPl by aqueous extracts of cigarette smoke and have claimed the results were due to free radicals. However, tree radicals in gas-phase smoke are very short-lived in solution [much less than one second). His laboratory examined'the effect of bubbling gas-phase smoke directly through buffered solutions of aIPl, a situation that closely mimics human smoking. In this experiment, the alPI was found to lose its protective effect, called elastate inhibitory capacity (EIC), slowly over a period of several days or for as long as the protein remained in contact with the extract. On the other hand, the direct exposure of aIPI to cigarette smoke causes an initial rapid loss of EIC; the aIPl then undergoes the same slow, continuous loss of EIC that follows exposure to aqueous extracts. It seems that the fast inactivation of aIPl exposed directly to fresh gas-phase smoke is caused by agents in the smoke that are short-lived in aqueous solutions. On the other hand, the slow inactivation must be caused by agents that are stable in aqueous solutions for several days. The free radicals in gas-phase smoke have apparent lifetimes of many minutes, yet their structures are like those of substan- ces known to have lifetimes of milliseconds or less. This paradox is offset by the proposal that the free radicals in gas-phase smoke are continuously produced (by reactions similar to those that occur in smog). In this mechanism, nitric oxide, which is present in smoke up to 300 micrograms/cigarette, is oxidized to nitrogen dioxide (NO2); the NO2 then reacts with other reactive smoke components to continually produce free radicals. A mixture of NO2 and isoprene (a basic building unit of a certain kind of hydrocarbon) in air, at levels like those in cigarette smoke, continuously produces a mixture of free radicals quite similar to tna~ in the smoke. Also, this synthetic gas-phase smoke oxidizes aIPI in the same process as does authentic cigarette smoke. Though it's not known which smoke component(s) inactivate aIPI, work began to find compounds that might provide protection. Tests showed that ascorbate {vitamin C) might have value in protecting smokers against loss of aIPl. Another agent found to be effective in protecting aIPi against smoke-induceO loss of EIC w~s glutathione [a substance :hat ac~ =~ the respiratory carrier ¢f oxygen). Later in the meeting, Pryor said he formerly used Kentucky reference cigarettes in his work because the source of his original support "forbade" the use of commercial cigarettes. He recently studied commercial cigarettes as well as those used by the National Cancer Institute in its "safe cigarette study" (directed by GIO GORI) and found that the free radicals in the tar differed from those in the Kentucky cigarettes. He said that the smoke of low-tar cigarettes has greater oxidizing capability than that of high-tar cigarettes. Low-tar CD -..j kO rk~ cxD BATCo document for PFSFC 1 March 1999

7. cigarettes could more likely cause emphysema-like damage than high-tar cigarettes, but while this is a tenable hypothesis, it has not been adequately studied. 4. ROBERT STOCKLEY at the General ~ospital in Birmingham, England, has been studying antielastases in BAL samples from emphysema patients who smoked• He reported that oxidized {or inactivated) aIPl (he called it alpha-one-antitrypsin, aIAT, a similar substance) was detected in lung secretions from both smokers and exsmokers. He also found a few more inhibitors in the secretions and said there might even be other suc~ substances present. There may well be inhibitors "we can't quantify." 5. R.T. ABBOUD of Vancouver, Canada, described two studies he did with human subjects to evaluate the effects of cigarette smoke on elastase levels and antielastase activity in the lungs as indicated by BAL. In the first study, to determine the acute effects of smoking, he compared BAL samples from healthy smokers who had two lavages in sequence. The second compared lavages from the lung's upper and lower lodes after smoking in order to evaluate factors that might predispose smokers to develop upper lobe emphysema. There was no significant aIPI inactivation in the first study, but there was an increase in elastase levels immediately after smoking. In the second study, aIPI levels and activity were sim- ilar in both lobes, but the ratio of elastase to alPI was sig- nificantly higher in the upper lobe after smoking than in the lower lobe; this indicates that there was a greater release of e!astase relative to aIPl in the upper lobe than in the lower lode. Abboud concluded that smoking induces the release of elastase in the bronchoalveolar lining. Though the elastase was inhibited in the lavage fluid by the excess of aIPI present, studies by others suggest that elastase released close to elastin fibers may retain activity and thereby play a role in the development of emphysema in smokers. 6. How dc neutrop~!Is miErste (or pass) through lung connec- ti0.'e "i~cue~ HS~ERT ~=,nu~,7- he ~; ..... ""_~.c cf Z ational Jewish Cen:£r of Izmunology and 5espiratory Medicine in Denver thinks he has the answer. First, work was done with human neutrcphils in chamzers containing elastin-embedded filters. The neutrophil e!astase degraded the elastin despite the use of aIPI, but faile~ to do so in the presence of another inhibitor, chloromethyl ketone (CHK, a 0 widely studied oligopeptide consisting of a few amino acids) CHK "-4 • L,4 also blocked migration. Un BATCo document for PFSFC 1 March 1999

. Next, Sandhaus used beige mice, a strain with neutrophils deficient in elastase. Intratracheal instillation of a chemo- attractant peptlde called C5a failed to attract neutrophils to the lungs; however, the same treatment in the parent strain, C57BI/6 mice, led to an over-abundance of neutrophils. The results suggest that neutrophil passage through elastin- rich tissue requires the action of the cell's potent elastase and that elastin degradation accompanies the migration even in the presence of aIPI. 7. JEROME CANTOR of Columbia University in New York came with what he called an important message derived from his research on the resynthesis of elastin and its possible relation to emphy- sema: In addition to the protease-antiprotease balance in the lung, one should also consider the balance between elastin degradation and resynthesls. Anything that might impair resynthesis, such as cigarette smoke, may tip the balance and thereby augment damage to the lung interstitium. Cantor and his colleagues have been studying elastin resyn- thesis in different kinds of animal models of lung injury, in- cluding elastase-induced emphysema and fibrosis induced by bleo- mycin, an anticancer drug. 8. The role of human alveolar macrophages (HAM) in elastolytic lung disease remains unclear, according to R.J. ALBIN of Wash- ington University Medical Center, St. Louis. One reason for the situation is that HAMs, at least in vitro, seem to release an elastase inhibitor at the same time they release an elastase (a metalloproteinase elastase in this particular research). The inhibitor, called TIMP for tissue inhibitor of metalloproteinase, masks any elastase activity. The concomitant release of TIMP provides one explanation for the difficulty in consistently demonstrating elastase activity. TIMP actually is secreted by a variety of cells, among them fibroblasts, macrophages, osteoblasts, and smooth muscle cells. Some of these cells, if recruited or attracted to the sites of inflammation in the lung, are able ~o join in degrading cer~aln tissue~ of the so-called extrac£11ular =a~rix. 9. RAYMOND BRIDGES of the University of Kentucky, Louisville," studied the relationship between smoking and the development of obstructive lung disease (OLD) in 170 relatively young make smokers (average age 37 years) and 170 age-matched male nonsmokers. Both groups were checked in regard to blood leukocyte count::, neutrophil enzyme activities, and serum antiprotease levels and activity. Smoking-associated changes in these variables were then examined to determine their relationship to pulmonary function. 0 -..j L~4 0 BATCo document for PFSFC 1 March 1999