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Tjri: COrNCiL FO~rt TosACCo RE-'~UArtc•Ii-U.S.A., I.NC. Octcber 9, 1979 H KR 1 9 3Gu4 G SIT-- VISIT TO: A. Janoff, Ph.D., Stony Brook, Lew York 11794 Grant ho. 1143 "Further Studies on Suppression of D*otease-Inhibitimn by Cigarette SmoY,e" DATE OF VISIT: September 20, 1979 SITE-VISITORS: Drs. Ford and Stone Janoff has a Continuation Application (Ido. 1143A), oi' san?e title as above, which will be considered at the October 1979 SAB meeting. The following is a lfist of publications not previously reported to .:TR, and papers in press. NEW PU'BLICATIONS 1. Carp, H. and Janoff, A. Possible mechanisms of emphysema in smokers. In vitro suppression of serum elastase inhibitory capacity by fresh cigarette smoke and its prevention by antioxidants. Am. Rev. Respir. Pis., 118, 617 (1978). 2. Carp, H. and Janoffy A. In vitro suppression of serum elastase inhibitory capaci- ty by reactive oxygen species generated by phagocytosing palymorphonuclear levi:ocytes. J. Clin. Invest., 65:793 (1979). IN PRESS: 1. White, R,R,, Norby, D., Janoff, A. and Dearing, R. Partial purification and charac- terization of mouse peritoneal exudative macrophage elastase. Biochem. Biovhys. Acta., 2. (in press). Will come out soon. White, R.R., White, J. and JanoiY', A. Effects of cigarette smoke on elastase secre- tion by murine macrophages. J. Lab. C].in. Med., (in press). out 3. Janoff, A., White, R.R., Carp, H., Harel, S., Dearing, R, and Lee, D.K. Lung in3ury induced by leukocytic proteases. Am. J. Path., (in press). Next issue, b. Janoff, A., Carp, H., Lee, D.K. and Drew, R.T. Cigarette smoke inhalation decreases alpha 1-proteinase inhibitor activity in rat lung. Science, (submitted for publica- tion). - accepted (revised). (Copies of New Publications (1 and 2 and manuscript No. 4(now in press) are attached) The overall main goal of Janoff's work continues to be to study the role of leukocyte proteases neutrophil and macrophage elastases), of endogenous protease inhi- bitors, and of those environmental agents which may influence the interaction o.^ these factors (e : Ci arette smoke in the pathogenesis of pulmonary;emphysema.In his concept, emphysema results from an imbalance between proteases and anti-proteases.

T»r C'ot•NCtL Fc~1?TnBAC(•u Isc. - 2 - Ii i50110 ~) 6' 4 7 A side-effort concerned the possible regulation of A,l protcinase inhibitor (e1-Pi) synthesis. When d1Pi molecules are consumed in vivo by cafplexinc, ti-ith proteo- lytic enzymes, they are rapidly replaced by newly synthesized inhibitor molecules, pri- marily Prom the liver. T'othing is known, at present, about control of this important process. The possible role of pj,1Pi: Protease complexes or degradation fragments act as signal molecules for new 4.1Pi synthesis by liver cells. This has been studied both in human hepatocyte cultures and in the intact animal employing anti-r,--t d.L'i anti- bodies from rabbits. The results were negative and the studies have no: been abandoned. (As was noted at the Gordon Conference on Prot.?ases in June 1979, the 50,000 I:ti: 'frag- ment "lost' when vtl binds to eltstase, is now in question. The lnajoritv opinion holds that the fragment (which might have been the 'regulator' for dCl synthesis) is merely a 'fragment' of the imagination.) At any rate, the main effort is going well, and the inactiva,tion of 01,~ l.Di by cigarette smoke has moved to an in vivo phase. OL1Pi (active center at Net ) oxidati+on Leu. Gln. Ala. Isle. Pro. Met. Ser. Isle. Pro. Pro. Val -ysul.foxide of D:eth ir (Tnr) d.l-pi Janoff has employed rats exposed to cigarette smoke (Glaltor. Horizontal Machine See manuscript Jtio. 4(revised copy attached). This will apnear in Science back to back with a paper by Ron Crystal from NIH who has confirming data in hurhan smokers on inhibi- tion of d,].Pi. Janoff has studied acute smoke in a limited number of rats. Bronchopulnonary lavage fluid was assayed by Rocket electrophoresis vs. specific antibody (ab) to ft(.1Pi for total •%1 measured in terms of the ratio mg elastase inhibited: T1his is the elastase- inhibitory-capacity (EIC). mg e{,1Pi present In acutely smoked Sprague Dawley rats the following data have been found. ~ NOS. RATS NO. PUFFS EIC 9 3 0.42 = 0.05 lk 6 0.36 } o.06 16 Control 0.59 = 0.06 That it is a question of the oxidizing capacity of the smoke is indicated by the use of ozone-tolerant rats - where no difference in d(iPi activity was seen. Further, addition of a reducing agent to the lavage fluid from non-tolerant-smoked rats returned d.1Pi to full activity. In addition,.,tlPi is inhibited in the presence of specificaL]Pi antibody. Janoff has human data which suggest that amoking causes a 20~a decrease in EIC which appears to be compensated b$ an equivalent 20;- increase in seirum d(,lPi 'ie. increased synthesis in the liver).

Trtr Corxut. Fonr ToxACrO Isc~. -3- PROPOSAL FOR ItiiXT T'h'0 YEAR PERIOD Janoff will study two aspects: 1. Extension of present stuclips on ak1Pi, pa~.r ticularly in chronic states. 2. Iiew studies on low Mr7, acid stable broncha.al mucous inhibitor of elastase (Human chronic and rat acute sraokiaig studies) • P'?i - v_ rotect; against bronchitis. BtTi may be similar to HuSi (human seminal plasma inhibitor - and cervical). Has at least 3 methionine residues/mole and are not synthesi%ed by liver and not seen in serum. HuSi - inactivated by oxidants and cigarette smoke• Protectiorr by reducing agents (eg. thympl). Dog submaendibular inhibitor is also synthesized and present locally in the tissues. BhiPi still to be purified and antibodies raised before It can be used to determine if smoke inhalation leads to a reduction of its activity. A1P3 iiihibits PNII•J elastase/trypsin/pancreatic elastase, ;;hile E??i inhibits PMt: elastase/trypsin/but not pancreatic elastase. (The guestion was raised as to whether such data suggest that parcres.tic elastase may be the majoi protease involved in bronchitis?) In order to obtain sufficient FM for purification and a prodxction, Janof^ xill inject pilocarpine in rats to cause mucous gland goblet cell hyperplasic. OT}M CTR SUPPORT Jelnoff has a second gratdt no, 1259 entitled "Iinmuriologic Assay of Lunc Elastin Degradation". COP?CLUSION rec•zested. Important studies carried out by a competent researcher. 4iould support as (hOTE: David Johnson also studying low W, acid stable inhibitor).