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THt: COUNcIL FOR TOBACCO RE SEARCH - U.S.A sss TrMin bFF1v'crE COPOffTTEE: NEw YORK. rr. Y: 10017 . Dr. Bing Dr. Cattell Dr. Jacobson Dr. Lynch _... _... . •: ....._ _. ~~~ ._.., .. .. .._ . ... - ..: D Med li M it K B F ,, . . z e er, r . ::~ ~. ; ' ` 2. Institution & New York University School of Medicine :, 550 First Avenue, New York, N.Y. 10016 4. Proposed Starting Date: pcCober,1, .1967 ~ :° 5. Anticipated Duration of this Specific Study: 3 years • 6. Brief Descripton of Objectives or Specific Aims: •'. The inference of tobacco combustion products, including nicotine and carbon Address: _.which intravascular coagulation is initiated under carefully controlled monoxide on blood coagulation will be studied in an experimental model in - condit..ons. : =• intravenous infusion of a sublethal dose of endotoxin in rabbits produces : It has been shown recently'in this laboratory that a single continuous _ glomerular fibrin deposition and renal cortical necrosis. Although the .histopathology of fibrin deposition is identical to that of the generalized ` . =: Shwartzman reaction, this experimental model differs from that of the generalized Shwartzman reaction where two injections of a sublethal dose of endotoxin are administered 24- hours apart. The difficulty of interpreting the overlapping effects of tcvo doses of endotoxin is avoided by the continuous intravenous infusion, which is also able to initiate fibrin deposition as a dose related phenomenon. The pathophysiology of disseminated fibrin deposition has not been fully elucidated. However, endotoxin has several distinct, direct and indirect effects on blood coagulation. Platelets .•. fall in a linear fashion, followed;by a subsequent decrease of the "consumable" coagulation factors including fibrinogen. Leukocytes decrease initially but increase after 4-5 hours during the course of the infusion. We have observed that the complement titer falls in concert with hemolysis and .• fibrin N. deposition. The thrombi in-the lung, liver and heart developed in an O irregular pattern in the early phases, that is 2-3 hours following the C initiation of the infusion lomer ot seen until fib l i d iti i ; g ~ u ar n on s n r epos approximately 6 hours after th n R l t t d t ti f , on. a e e s ar o ra en otoxin adminis WDA h td i l d ocvn • s u s re ate to glomerular fibrin deposition. The development of glomerular capillary necrosis is apparently dependent on the following: C5 (con tinued on next page) .: 7. Give a Brief Statement of your Vi/orking Hypothesis: Our initial observations indicate that the infusion of endotoxin in rabbits .' produces a reliable and reproducible method for disseminated intravascular (continued on next page)

. Question 6, continued; .2. the reactivity of the kidney andits ability to lyse fibrin 1. the amount of glomerular involved 1. the gaseous combustion product of tobacco in its entirety The study proposed is designed to examine the effect of: 3. the size of the dead vessels involved 2. nicotine . carbon monoxide ..- i... In both acute ad chronic experiments utilizing animals n : pretreated with a continuous intravenous infusion of doto i ._, : en x n. .coagulation as well as the initiation of the fibrinolytic system. =*:~ :.Such a model permits the investigation of a variety of substances ~ •` which might either protect the animal on the one hand or d) any combination of these effects. c) the failure of lysis of fibrin or delay of lysis of fibrin_ -b) the earlier initiation of coagulation or fibrin deposition. . } .. .:~ • . fibrin deposition ,~r? a) in the lowering of the total dose of endotoxin required for. e es n severa Y man was: Y th if t i l the combustion products of tobacco effect the coagulation system per:~g, =^x?. en o ox n on e o er I . potentiate the dilatarious effects of d t i th th f W`In addition, an effect not obvious in the untreated animal may `become manifest. .

E. DeloitsofExparimentalDesianandProced~res:.fAtfach5en.,..,t.v.....a While the basis of the investigation will be the infusion o$ rabbits with a con.tinuous dose of endot 2 o 0 8 1 = Plan 1: In the study of the acute effects of tobacco combustion products, animals will be infused with endotoxin and ex o d' t xin ( - 0 mg/Kg/hour) for periods of 6-14'hours, several distinct experimental plans will b f 1 ; o nicotine, carbon l p se t monoxide and tobacco smoke Either si . mu aneously o r immediately prior to ~ -= -ttie-infusion of endbtoxin animals will be t di s u ed with rest t : sia:; peco: ~;Wa) the dose range of endotoxin in controls as comparedto treatd 4 e 3 l ., an ma s « tt; b) the time lag,required for lomerular fibrin deposition, and -c) ariy change in the g ~~;f. parameters to be studied by serial blood sampling. ,~'.*:~ In the second plan endot ox l in wi l be infused for a period of 10-14 G'h i CfiQuK$3nfold•owing which, one kidney will be surgically removed (control) . _--_ The_animals.will then be exposed to tobacco combustion products for an additional 10-14 hours and sacrificed. The kidney so obtained will be --oompared•to that of the control kidney with respect to-the depos3stion of - ;. fibrin•in glomerular capillaries. In plan 3 the animals will be exposed to-tobacco combustion products over extended periods of time and subsequently infused with•endotoxin. The ''do'se"rarige and- parameters of coagulation will be studied in, comparison with that of the controls. (contindt) ue on nex page 9. Physical Faci(ties Available (Where Other 1han,Adminiatering-Organization Indicate Geograpfiicaf location)' -'A fu11y equipped laboratory for the study of blood eoagu3ation and,the fibrinoiytic enzyme system,with~standardized techniques as well as histological and histopathological techniques ' i l b f C• 10. Adddwnal$equirements:. Smoking machine ( at proposal of Dr. Kuschner, Department of Pathology): `: C~11. Biographicol4ketcNes of all principal and professionol personnel (append) check appended:pages 12: List ofpublications: (Five most recentos pertinent) (append) check appended pages ` s avai a le. Thas.laboratory is located in Bellevue Hospitali as part of the Department of Obstetrics.and;Gynecology.

Methods: Among the methods emp-loyed will be frozen and permanent tissue =1ung, heart, pituitary as well as kidney. Immunofluorescent methods demonstration of fibrin. Among the organs studied will be the liver, sections utilizing conventional histochemical techniques for the .to substantiate the specificity of fibrin deposition. Histochemical :employing anti rabbit fibrinogen.produced= in the rat will be utilized ':; identification.of tissue activator will be studied utilizing the . The coagulation system will be studied from the following points: . ,fibrin films and,suitable tissue sections (Todd). c) urinary output, d) body temperature, e) blood pRand pC02, f) leucocytes., g) hemolysis. (Johnson et al.) fibrin plates heated and unheated (Astrup). : Biological parameters: a) arterial pressure, b) osmolarity, :4:.:..'Study of the fibrinolytic enzyme system: a) euglobulin lysis ° time, b)plasminogen, c) plasmin inhibitor and kinase inhibitor factor VIII levels (TGT) : 3- thromboelastogram (Hartert) . 1- platelet enumeration (phase contrast) .2- fibrinogen (Ratnaff and Menzie), factor V (on phase method) and Question 11: ' D. 1955). Assistant and Associate Professor, University of Tuebingen.1:956- ;i Research Council 1954},Dozent Obs & Gyn, University of Giessen (Med. Sci.::':; Internship and residency in Obstetrics & Gynecology, University of Giesseml948-1954. Max Plank Institute of Radiobiology (Trainee German -Principle Investigator: Fritz K. Beller, M.D., age 43. MD Marburg 1948, -1961. Professor of Obstetrics & Gynecology, Tuebingen 1961.- Visiting Associate Professor, NYU School of Medicine, Dept. Ob-Gyn, ..1961-1963. Associate Professor, Dept. of Ob-Gyn 1963-1967. Professor .of Ob-Gyn, NYU School of Medicine, September 1, 1967. Career Scientist •,!~1.i; .•:- German Specialty Board, 1957. Diplomate American Board or Obstetrics or the Health Research Council of the City of New York 1961 to present. .,;04, : and Gynecology 1967. State Board New York 1963. NYU Chapter XS. U.S. Citizenship; 1966. . ., . ., ., g ..Amer Soc Exp Pathol Intern Colle e Pathol New York Acad. Sci . =.~,,.. .f,. Societies: FACOG, FRSM (London) Soc. Gynecol. Investigation, Co-Investigator: Fred Gorstein, M.D „ age 37, MD NYU School of Med icine 1955. `` Intern Bellevue Hospital 1956-1957. Assistant Resident Pathology, Bellevue Hospital 1957-1960. Fellowships: PHS Training Fellow Pathology 1957-19b0, PIiS Post doctoral Fellow Pathology 1960-1961. Career Scientist of the Health Research Council of the City of New'York 1963 - present. Instructor Pathology, NYU School of Medicine 1960-1963. Assistant Professor 1963-1967. Associate Professor, Sept. 1,1967. Diplomate American Board;of Pathology 1961,1967. Societies: N.Y. Acad. Sci., Harvey Soc., Citizen; U.S. - 100354T022

, . . . '*Senior technician: Peter Mitchell, age 25, B.S. Cornell University 1963 Worked in this laboratory since 1963. Continued school at NYU at night. uestion 11: continued: M.S. expected, 1968. Citizen; .f~~ • ;-"•Technieian: Helge Bornhausen; age 33 German Gymnasium Sehool for f.Medical Technicians Mainz 1955-1957. Joined this laboratory in 1966. 4 'C; .-itizen•Germany. :Immigrant Visa. uestion 12: ; 1) Beller,F.K, and Graeff,H.: Deposition of glomerular fibrin in the .. _.3) Beller, F.K., Debrovner,Ch.H. and Douglas,G.W,: Potentiation of -,rabbit after infusion with endotoxin. Nature, in press. il 2) Beller,F.K., Mitchell,P. and Gorstein,F.: Fibrin deposition in the Haemorrh. 17:427, 1967. ~ :-rabbit kidney produced by protease inhibitors. Thrombos. Diathes. the lethal effect of endotoxin by heter•ologous plasma. J. exper. system in newbornes. Amer. J. Obstetr. Gynec. 96:977, 1966. Beller,F.K., Douglas,G.W. and Epstein,M.D,: The fibrinolytic enzyme .Med. 118:245, 1963. . 5) Beller,F.K. and Porges,R.: Blood coagulation and fibrinolytic enzyme studies during cyclical and continuous application of progestational agents. Amer. J. Obstetr. Gynec. 97•448, 1967.

R: REDACTED MATERIAL C A. Solaries (P,ersonnel by.names): Professional Fritz K. Beller, M.D. Fred Gorstein, M.D. (co-investigator) Fringe Benefits 'Technical 1 technician 1 senior technician Fringe Benefits L Consumable Supplies (list by.categories) Glassware Laboratory reagents (chemicals iimnune sera, endotoxins, etc.) C. Other Expenses (itemize) Laboratory animals D. Permanent Equipment (itemize) Thromboelastogram Water bath - Deep freeze unit 2 ovens: slide file cabinet E. Overhead (15% of A + 6fC)1 2601._ 26391 Estimated Future Requirementsc =• Salaries Consumable Suppl. Other Expenses Permanent Equip Overhead Totbil Year2• -.f61GF'- 1500 3000 Year3 r16600i=' 1500 3000. 1/ is understood lhotltHe opplicant'and institutionaliolficern in applying for. a grant have read and found acceptable the Council's "Statement.oF Policy Contaihing Conditions and Terms Under WhicH Pro,~ectlGranfs Are Alade." 500 i.3100• 242_67_ 500`); 3195 .'• 249,95! Signature DK.~..ar.rrol"0R9-3200 ext. 2732 -Telbphone Signature 4 016 i Ih -• i Jr .. ,iU.w ~ e ~. t w . Arms Norg ' rong, M.D. D ixec to r Telephone 0 ~ 4_ `_

i a r~ n OtfMr Sources of Finanetal5upport I Lhtlinanetal support for ns~arch from all sources, lncluding own (nstitution, forthit and/orrnlatod retwrch pro)ec1h, 5Z04,PSE00T i B I Source

. .'tr..~t.. •. •t . . - . . . . • . , . , .. . . -_-- ~ , .. Additional information to Grant reqL-:!st; Dr.Beller. . - L . ~~;'- . . . ... ~ . . .. . . The principle parameter for the evaluation of smoke andismoke substances are primarily not paramiters of the coagulation and fibrinolytic enzyme -system in peripheral blood. They will be assayed but are of secondary i.nterest, The primary interest will be focused on biological parameters in 33CtW--X organ systems, especially the kidney after disseminated coagulation is produced by endotoxin infusion. The experimental design is best appreciated, from the following scheme. ... .:~., _ Removal of Removal of 1 kidney 2nd;kidney ."I. Pretreatment phase:Smoke or smoke substances will be applied to animals ;:'acute or ehronically before endotoxin infusion is induced. •t.Parameters: 1) Assaying the concentration of endotoxin needed to produce fibrin deposition with and without smoke substances. 2) Measuring the time 9 of phase II until fibrin deposition occurs. Assays and methods: 1) histologic sections and 2) histochemical sec#ions of the kidney, p'ituitary, liver and .luno. The kidney removed in different time lags in between 4-14 hours is Urine particularly suitable. 3) Renal excretion study. 4) ?0M osmolarity. 5) Venous and-artea.•ial pH. 6) Leucocytes. 7) Coagulation-assays. 1003547026 II. Infusion phase: Smoke and smoke substances will be applied' duririo phase II together with-the endotoxin infusion. Parameters and assays as under I. III. Postinfusion phase: Smoke and'smoke substances will be applied in phase III after terminating the endotoxin infusion.. 1) Histolooical sections and Development of fibrin deposition Reactive-thrombolysis .Endotoxin infusion for 14.hours Recovery phase

:.histochtmical sections in the second•kidney will be compared to the kid~ie ~ . . - . . - =_ . .. . . . . . . . . . .t,~ _.:~:;~, ; h~: ~ on us (con ro ) This experiment will b d .eone after termination of the inf i ' t l .. . . . . •. •:•.' . .. :,f;e~.tW in order to evaluate inh3bition or activation of the reactive thrombolytic =Y~ ::recoveryy p ase. Additional assays as under 1. h ; ssay 1) Histologicall sectionin thekidney: 100 glomeruli will be counte . ...... d s 'A ~ ~ n-each section and the number of glomerula involved with fibrin depositioli I am-enclosing a detailed procedure of the method. See also, Beller et al.,1961 •: expressed in /. 2) Fibrin layer according to Todd (J. Bacteriol..,78:281.) . rges ,.,.. : and 1967. The coagulation assays are described in Beller and Po l967 $ ;. 3) Activator assay on tissue extracts measured on fibrin plates (Astrup and; ',_~ . ... . .... Muellertz, Lit, in Albrechtsen,O.K.: Fibrinolysis in the org'anism... Acta .. - • , .•.

Technique: (Mitchell, Weiss z ` ~ Prepartif fibi fil .aon ornm: :'bufftr is added to a te--t tube containing 10 ml of fibrinogen solution. . , . .- . ..' , . . ... _ , Bavine fibrinogen highly contaminated 14 ftithaelis Veronal buffer' (pH a 7.35) to a concentration of 300 mg to ~ ~ •tTg per 10 cc. 20 units of Parke.Davis Topical thrornbin in 1 cc of atb i itell euesnvered gnt:y to mix the soutions and is then poured uniforml 1.• . =:.onto an 8 cm x 14 cm piece of wettable cellophane (dialysis paper) wh.ich ;has been saturated with the Veronal buffer and placed ori a perfectly level :"~.surface. The solution is permitted to clot at room temperature and is then placed in a refrigerated (4°C) wet chamber for at least one half hour. 2 x 3 cm pieces of the film-may then be cut fro,n.the 8 x-14 cm piece using a sfliconized surgical scissor. These sections are inverted on clean micro- '' seo slidi tht bbbl f :~pees usng care soa nouesorm under the film. The cellophane is then peeled off leavin; a 2 mm fibrin film on th lid Th e s e . ese ~ films are stable, wheorefrigerated, for three days. Activator inhibitors ~-=may be incorporated into the films prior to the addition of thrombin. If .:.-::. =...:. _- ': ... .: . . . . . . 1the film# ~ to be heated to destroy the plasminogen (S6°C,/3(1 min.), ~.., . . . . : : ., . `~` steps should be carried out in a wet chamber after the ` = ' `' ` • •....:. .. :- .. . . ;> .,._..... ~ o thelid n se. ': ,: Tissue activator assay: film slides with a precooled forceps. If many filras are to be run, they are placed in. the refrigerated wet eli;3mber and t1h,eir incul:atior.s all comnense V • 1003547028 The tissue used in this.assay must be received fresh, and immediately quick frozen onto a cryostat block. In this ste-te.the tissue is stable for several days if it is sealed in parafilm to prevent drying. The tissue sections are then cut 8 microns thick and placed on the fibrin . , .~;

incubation is carried out in a moist 37°C incubator of time. A control slide (0 min=••incubation) is prepared for and to aid in interpretation of themsults. Incubations of 1) rinse slides in tap water bath 2) place in acid hematoxylin 90 sec. (or until red-blue color results 5) rinse in water • 6) 50% ethyl alcohol x 15 min. 7) 70•/G ethyl alcohol x 15 min. 8) 95% ethyl alcohol x 30 min. 9)10(/, ethyl alcohol x 45 min. 10) eosin Y x 20 sec. 11) wash in 100°; ethyl alcohol three times 12) 50% ethyl alcohol - 50% xylol 13) 100% xylol x 20 min. x 20 min. 14) Trim excess fibrim film from the slides with sharp knife and cover with synthetic mounting medium and a cover slip. The entire area under the cover slip must be filled with mountir.g meditnn to prevent drlrinh of the film. 'T};e sVes are •icw CoCi'JIIP.Int; S T-a 6 fe ) QuJ &_ILC~y 6,Z SyoreY; ~r_ot- The slides are best observecd witlh, a lm: pnwer stercoscopic micruscope. Lysed zones will first appear as depressedi ar.eps under the tissue section. 3) rinse in water 4) develop in dilute ammonia water until blue -*.Sei.vely inactive tissues have been carried :~r~ods of up to 24 hours. The reaction - 4 LCl% €ormaline-saline solution. : .~_ . ~: ..• . • .~. . ~ .' .' Staining procedure: 9

. . . . . . .:'~:'~J; may be made with reference to the time of incubation necessary : ~Semi-qeiontitative comparisons of activator activity in different ; ~ Sli~.fi iubated fc~r tonger pe : ~ds will have holes through the entire ri . . ..._ . . . .. . .. .. . . . "• -- • . .x, ..} fibrin film at areas under the tissue that correspond to the earlier, . . • #• - .. . _ . • ... . <; n ftresaions.-.Lysi:i an hea'ted_•films or those containing activator ,~ :~inhibitors ( e.q. K_x 10'~'i~AEA) is indicative of proteolytic activi in the tissc.e not due to plasminogen activator. ,.to procl~sce a certain observed amount of lysis. However, these comparisons mwat be made only between slides which are prepared with the same batch ". of fibrinogen on one film and with simultaneous incubations. It is preferable to use one batch of fibrinogen in which the plaminogen contamination is known (Ear assay in the true sense of the definition since the plasmin spreads. The Gh3~e~,=`-~xk~-: The method is not a histochemical localization is therefore dependent on a proper incubation time. Excessive-incubation of the slides results in autolysis of the entire tissue seetion:'';~:` 1