Tobacco Documents Online

. ......_. ._ . ... ~_~._ 'I"ttE f':n'-%rti. F„H Tc:x,crco Iir,sU.tirutt-L'.S.A.. 1Nc. Navember 3, 1981 HhD116508 NlQt'IORAtaDUM T0: Dr. S. C. Soarmrs and Staff FI4rM: D. H. Ford Si1BJnCr: Site Visit with Dr. G. Weinbaum and staff, Albert Einstein Nlsdical Center, Philadelphia, Pennsylvania - October 28, 1981 Grant No. 90113R7. "Bronchioalveolar Lavage of Human Smnkers and Non-Smokers: Studies on Cell Chemotaxis, fhzyme Release and Cellul,ar Ultrastructure" Dr. Weinbaum presented a well organized review of his total program, indi- cating the four majar areas of interest. 1. Is there a specific enzyme associated with the development of emphysema? 2. If so, how does it act to cause the specific damage? 3. What is the role of oxidants? Is there any protective me-hanism to reactivate oxidized :y.l-P? 4. Human studies. Weinbaian's group is nvar beginning to apply the information they have learned fran their dog model to humans. Ultimate goal is to develop an early recognition of developing emphysena before it attains a clinical state. The development of such a test is important since at this time there is no pul- nonary function test which will reveal the presence of a clini- cal condition before up to 40 to 50% of the lung tissue has been destroyed. Hopefully, then therapies might be developed to halt the progression of the disease before serious pathology occurs. This might be achieved by stimulating the bodies ocuk: defense me- chanisms (i.e., the newly discovered reductase for miethionine sulfoxide). REVIEW CF RFSULTS: 1. There is r.aa no question but that ertphysema develops as a result of release of a specific protease for elastin. The primary source of the enzyne is the azurophilic granule of the :PNAt, which must be released in the alveolar space to be effective. (Intravenous instillation off the enzyme had no effect on the lung tissue. )'i'hey have purified the enzyme from the azurophilic granules oin an affinity column. Using this pure enzyme, Weinbaun's group have been able to deromstxate a dose related developnent of emphysema in dogs. Some of Weinbaum's data suggests that a1l elastins are not alike and that there may be different elastases for the different elastins. Is attacking this pro- blcan immmologically? Conceivably, specific peptides from specific elastins may be identifiable as fragments in serum and thus differentiate between eyphysema and age changes in skin elastin, etc. .../..

- 2 - i•111: On-.cn. roir Towrco R,aL.tR<•»-U.ti..1., I.c. H ISO1 1 65081 2. Dr. Damiano has devel.oped a technique for quantitating,the number of granules iri a PAIlV, present or released. Can also be applied to phagocytosis of particles by macrophages. Original procedure was set up and tested using glass microspheres by light microseopy, utilizing a sterological method. E!y making counts of the number of hits or intersects whi.ch a series of lines of specific length make when overlaid or, a photecnicrograph, one can calculate volumie of cell, or specific intraoellular organelles as well as number of organelles. Similar data can also be used to determine oell manbrane surface area. The procedure was then applied to PNIIrIs and macrophages cultured for +.hree hours in presence of srroke (or no ssmke). 9roke fran one cigarette decreased PM viability somewhat, wfi.ile smoke fxxn ten cigarettes decreased viability 70%. Smoke frun three ciga- rettes induced a release of azurophilic granules, causing a 90% decrease in the elastase eont.ained/cell. Viability of rnacrophages was not effected by smoke, but siroke frorn four cigarettes caused a maximal release of macrophage elastase. This phase required two steps, since the macrophage elastase had to be synthe- sized before it could be released, as it is not conveniently stored in granules. The total elastase per macrophage is much less than for a M, but initially, 95% of the free cells present are macrophages; so the earliest assault on the lung tissue may be induced by the nacrophages. Damiano has applied his sterological procedures to FIN photaanicrographs and cniantitated the loss of granules from PNNs, granules being identified by my- eloper.oxidase. Loss of granules/cell in lavaged cells fran 3nflamed lungs is appro.~tely 70%. Biochemical measu_ements indicated a 40% drop in cellular elastase on basis of hydrolysis of elastin. Sequence of events: lung injury _4 inflamnation -~ attraction of PANs and macrophages ._~ macrophages release a chenioattractant for PNIlVs PMNs flood into alveolar spaces ~~ release elastase ___.~hydrolysis of elastin. Damiano is also now workimg on a project involving cloned DNA for elastin synthesis to determine the sites of elastin synthesis in cells. Hopes to determine if the message to synthesize new elastin in sites of emphysenla is a faulty messageo as suggested by Foster. Dr. Weinbaum discussed at some length what is essentially a new area: that is the presence of reductase, which was initially described by Weisbach and Brot Moche) in E. coli. This enzyme, which appears to require the presence of some other reducing agent to function has been found in the cytoplasm of PNIlVs as well as in a number of mannallian tissues (brain, lung, lymph nodes, liver, spleen, skeletal muscle and kidney.) It appears to act, accoxt3ing to Abrams, to reduce the oxidized methionine in the 4Nl Pi molecule, but is ineffective against oxidized nethionine not incorporated in sane sort of peptide or protein chain. This enzyme, now knoum to be present in lung, may serve as another defense against elastase degradation of elastin. Cbnceivably, the non-smokers who develop arphy- sFina and who possess the 1+1 genetic state may be deficient in this enzyme. (`c,l: versely, smokers who do not develop e9rphysema may have an exceptionally aci-.ive reductase oomponent in their system. Weinbaum and his group are eollaborating with the Roche group and with Janofff on this project. Early studies show that this enzyne, called methionine-sulfoxide peptide reductase (I~'s15O-PEPTIUE REDUC- TASE) is effective in reducing the eo:idized methionine of ~. 1-Pi after exposure to the oxidant, chloramine-T, in vitro and requires the presence of the reduc- tant dithiothreotol. Preliminary studies with patients indicate the PNAis in the lavage fluid fxran smokers with emphysema show a 50% loss of activity of this en- zyme, while PMVs from smokers without esphysema had normal levels of the reduc- tase. Similar data was obtained fran biopsied lung tissue. Question: Where is the enzyme found in lung tissue, how is it released and is it itself effected by oxidants?

-3- Tirn; Couxcir. Fun ToB,.cco RPaI.Aiecn-U.S.:1.. Is" N X211 6508, Z HUmN S'UDIFS: Another aspect di the'human study program is to apply the technique which was successful in dogs for measuring elastin peptides in serum. So far they have changed over fram the hemagglutination assay which oould successfully recognize elastin fragments in serum at a level of 400ng to an, ELISA assay, which can detect as little as 10-15 ng of elastin fragments. Hopefully, this assay may be able to distinguish between pulrrcronary, skin and vascular elastin, etc., which sone workers believe are not identical to each other. So far, using the ELISA assay, Weinbaum has noted that elastin fragment levels in serum of srrokers increase about 50%, about 70% in OOPD patients and about 50% in ARDS pa- tients. Note, 50% of smokers show no increase in elastin fragtrents. Also, smo- kers did not have free elastase in lavage fluid (may be bound to elastin). Of the normall smokers so far surveyed, 50% had levels of elastin of 50ng, which is essentially normal. Has also noted in the serum of non-srrokers, a slight increase in elastin fracgnents with age. Plans to use this observation to pursue a longituclinal study wherein a patient.population of smkers and non-srrokers will be follawed for se- veral years. Hopes to derpmi*+e if patients with high levels of elastin frag- ments show deterioration of elastin at a faster rate than those showing no sign of disease. REPR=: Six reprints of previously iureceived reports were provide.d the visitors (these have been turned over to D. Cohen). All acknowledge CPR. CCMMIDNT: This omtinues to be a multifaceted clinical/basic program directed tosaard obtaining an understanding of the basic mechanisns involved in emphysema. The prelir.ii.nary dog model appears to have provided many of the answers to the basic questions. weinbaum is nbw atte~ting to deterr~ine haa well the hypothe- ses developed from the dog apply to the human. So far, they seen to apply very well. Early work with the new reductase suggests that it may well be a part of the system in lung which protects elastin fresn elastase. The quantitation stu- dies by IM and IIM have pxrovided several insights into the changes occurring in PMs in relation to release of elastase. The program oontinues to be well or- ganized and directed. The changes in emphasis which have occurred appear logi- cal and likely to be productive. There is no doubt but that this is an excellent productive program which oontinues to merit support. Donald H. Fbrd DHF:am